Use of ghrelin splice variant for treating cachexia and/or anorexia and/or anorexia-cachexia and/or malnutrition and/or lipodystrophy and/or muscle wasting and/or appetite-stimulation (2024)

U.S. patent number 7,981,860 [Application Number 11/716,137] was granted by the patent office on 2011-07-19 for use of ghrelin splice variant for treating cachexia and/or anorexia and/or anorexia-cachexia and/or malnutrition and/or lipodystrophy and/or muscle wasting and/or appetite-stimulation.Invention is credited to Liat Mintz.

Use of ghrelin splice variant for treating cachexia and/or anorexiaand/or anorexia-cachexia and/or malnutrition and/or lipodystrophyand/or muscle wasting and/or appetite-stimulation

Abstract

The present disclosure relates, in one aspect, to use of ghrelinsplice variant or an analogue thereof for the preparation of amedicament for one or more of: treatment and/or prevention of lossof body weight and body fat, prophylaxis or treatment of cachexia,stimulation of appetite, stimulation of food intake, stimulation ofweight gain, or increasing body fat mass, or increasing body leanmass. Another aspect relates to the use of a ghrelin splicevariant-like compound for the preparation of a medicament for theprophylaxis or treatment of cancer cachexia in an individual inneed of such treatment. Another aspect relates to the use of aghrelin splice variant-like compound for the preparation of amedicament for prophylaxis or treatment of cachexia in anindividual by administering a subcutaneous dosage of saidmedicament to the individual. A further aspect relates to the useof a ghrelin splice variant-like compound or a pharmaceuticallyacceptable salt thereof for the preparation of a medicament forstimulation of appetite in an individual by administering asubcutaneous dosage of said medicament to the individual. A furtheraspect relates to a number of new ghrelin splice variant-likecompounds and uses thereof, as well as to pharmaceuticalcompositions and medical packaging comprising the new ghrelinsplice variant-like compounds.

Prior Publication Data

Related U.S. Patent Documents

References Cited [Referenced By]

U.S. Patent Documents

Foreign Patent Documents

Other References

Primary Examiner: Kemmerer; Elizabeth C
Assistant Examiner: Ballard; Kimberly A

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional ApplicationNo. 60/781,860, filed Mar. 13, 2006, incorporated herein byreference in its entirety.

Claims

I claim:

1. An isolated ghrelin splice variant-like compound having theformula Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally presentprotecting group; each X1 is independently selected from anaturally occurring amino acid and a synthetic amino acid; X2 isselected from a naturally occurring amino acid and a syntheticamino acid, said amino acid being modified with a bulky hydrophobicgroup; each X3 is independently selected from a naturally occurringamino acid and a synthetic amino acid, wherein one or more of X1and X3 optionally may be modified with a bulky hydrophobic group;Z2 is an optionally present protecting group; m is an integer inthe range of from 1-10; n is an integer in the range of from 4-92;provided that the compound according to formulaZ1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length, has at least95% homology to SEQ ID NO:1, and stimulates the appetite, weightgain, or a combination thereof of a subject when administeredthereto.

2. The isolated ghrelin splice variant-like compound of claim 1,wherein the compound according to formula Z1-(X1)m-(X2)-(X3)n-Z2has at least 98% homology to SEQ ID NO:1.

3. The isolated ghrelin splice variant-like compound of claim 1,wherein the compound is 22-29 amino acids in length.

4. The isolated ghrelin splice variant-like compound of claim 1,wherein the bulky hydrophobic group is an acyl group or a fattyacid group.

5. The isolated ghrelin splice variant-like compound of claim 4,wherein the acyl group is a C.sub.1-C.sub.35 acyl group.

6. The isolated ghrelin splice variant-like compound of claim 5,wherein the acyl group is a C.sub.7-C.sub.12 acyl group.

7. The isolated ghrelin splice variant-like compound of claim 1,wherein the compound has the formula Z1-Gly-(X1)m-(X2)-(X3)n-Z2,Z1-Gly-Ser-(X2)-(X3)n-Z2, or Z1-Gly-(X2)-(X3)n-Z2.

8. The isolated ghrelin splice variant-like compound of claim 1,wherein (X1)m is Gly, Gly-Ser, Gly-Cys, Gly-Lys, Gly-Asp, Gly-Glu,Gly-Arg, Gly-His, Gly-Asn, Gly-Gln, Gly-Thr, or Gly-Tyr.

9. The isolated ghrelin splice variant-like compound of claim 1,wherein (X2) is modified Ser, modified Cys, modified Asp, modifiedLys, modified Trp, modified Phe, modified Ile, or modified Leu.

10. The isolated ghrelin splice variant-like compound of claim 1,wherein (X3)n comprises a fragment of SEQ ID NO:6.

11. The isolated ghrelin splice variant-like compound of claim 1,wherein Z1 is selected from the group consisting ofC.sub.1-C.sub.10 alkyl, C.sub.1-C.sub.10 substituted alkyl,C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 substituted alkenyl,aryl, C.sub.1-C.sub.6 alkyl aryl,C(O)--(CH.sub.2)--(C.sub.1-C.sub.6 alkyl)-COOH,C(O)--(C.sub.1-C.sub.6 alkyl), C(O)-aryl,C(O)--O--(C.sub.1-C.sub.6-alkyl), and C(O)--O-aryl.

12. The isolated ghrelin splice variant-like compound of claim 1,wherein Z2 is selected from the group consisting of amide,methylamide, and ethylamide.

13. The isolated ghrelin splice variant-like compound of claim 1which binds to the growth hormone secretagogue (GHS) receptor GHS-R1a.

14. The isolated ghrelin splice variant-like compound of claim 13,wherein the compound has an EC50 potency on the GHS-R1a of lessthan 500 nM.

15. The isolated ghrelin splice variant-like compound of claim 13,wherein the compound has a dissociation constant of less than 500nM.

16. The isolated ghrelin splice variant-like compound of claim 1,wherein the compound has at least about 50% of the functionalactivity of ghrelin.

17. The isolated ghrelin splice variant-like compound of claim 16,wherein functional activity is activation of Gq/G11, accumulationof inositol phosphate, mobilization of calcium from intracellularstores, activation or deactivation of MAP kinases, NF.kappa.Btranslocation, CRE driven gene transcription, binding of arrestinto ghrelin receptor, or a combination thereof.

18. The isolated ghrelin splice variant-like compound of claim 1,wherein the compound is conjugated to a polymer molecule.

19. The isolated ghrelin splice variant-like compound of claim 18,wherein the polymer molecule is selected from the group consistingof polyalkylene oxide, polyalkylene glycol, poly-vinyl alcohol,poly-carboxylate, poly-(vinylpyrolidone), polyethylene-co-maleicacid anhydride, polystyrene-co-maleic acid anhydride, anddextran.

20. The isolated ghrelin splice variant-like compound of claim 1,wherein the compound is modified with a chemically reactivegroup.

21. The isolated ghrelin splice variant-like compound of claim 20,wherein the chemically reactive group is selected from the groupconsisting of N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide,maleimide-benzoyl-succinimide, gamma-maleimido-butyryloxysuccinimide ester, and maleimidopropionic acid.

22. A pharmaceutical composition comprising the isolated ghrelinsplice variant like-compound of claim 1.

23. The pharmaceutical composition of claim 22, further comprisinga pharmaceutically acceptable carrier, a vehicle, an excipient, atransport molecule, a wetting agent, emulsifying agent, pHbuffering agent, or a combination thereof.

24. The pharmaceutical composition of claim 22, comprising amixture of at least two different ghrelin splice variant-likecompounds.

25. A kit for administering a ghrelin splice variant-like compoundcomprising: (a) a dosage form comprising a pharmaceuticallyacceptable amount of (1) the isolated ghrelin splice variant-likecompound of claims 1; and (2) optionally, a ghrelin splice variant;and (b) optionally, instructions for administering (a).

Description

FIELD OF INVENTION

The present disclosure relates to compounds for treating orpreventing cachexia and/or lipodystrophy and/or muscle wasting andconditions related thereto.

BACKGROUND OF INVENTION

Ghrelin is a bioactive peptide that induces food intake, bodyweight gain, and adiposity in rodents (Tschop M. et al., Nature407:908-13 (2000); Wren A. M. et al., Diabetes 50:2540-47 (2001)).Acute administration of ghrelin induces food intake in healthy menand women (Wren A. M. et al., J. Clin. Endocrinol. Metab.86:5992-95 (2001); Druce M. R. et al., Int. J. Obes. Relat. Metab.Disord. 29:1130-36 (2005)) as well as in cancer patients withanorexia (Neary N. M. et al., J. Clin. Endocrinol. Metab.89:2832-36 (2004)). Repeated administration of ghrelin increaseslean body mass, body weight and food intake in cachectic patientswith Chronic Obstructive Pulmonary Disease (COPD) (Nagaya N. etal., Chest 128:1187-93 (2005)) and improved muscle wasting inpatients with chronic heart failure (Nagaya N. et al., Circulation110:3674-79 (2004)). A similar effect was also shown in a mousemodel (Hanada T. et al., Biochem. Biophys. Res. Commun. 301:275-79(2003)).

Tumor growth is associated with profound metabolic andneurochemical alterations, which can lead to the onset of theanorexia-cachexia syndrome. Anorexia is defined as the loss of thedesire to eat, while cachexia results from progressive wasting ofskeletal muscle mass and to a lesser extent adipose tissue,occurring even before weight loss becomes apparent. Canceranorexia-cachexia syndrome is highly prevalent among cancerpatients, has a large impact on morbidity and mortality, andimpinges on patient quality of life. However, its clinicalrelevance is frequently overlooked, and treatments are usually onlyattempted during advanced stages of the disease (Laviano A. et al.,Nat. Clin. Pract. Oncol. 3:158-65 (2005)).

Ghrelin is secreted in the pre-meal situation starting 1-2 hoursbefore the meal resulting in a sharp, short-lived surge in plasmalevels of ghrelin before the meal and lasting a short while afterinitiation of the meal. Since ghrelin is the only knownperipherally-produced orexigenic (appetite-promoting) substance, itis believed that the increase in plasma levels of ghrelin iscrucial for the initiation of the meal.

In its role as a key initiator of appetite, ghrelin, released fromthe endocrine cells in the mucosa of the gastrointestinal (GI)tract, may act both locally as a paracrine substance and centrallyas a hormone, as discussed infra in the section related to cancercachexia.

The GHRL (ghrelin) gene encodes a variety of products resultingfrom alternatively spliced transcripts, various types of cleavageof the prepropeptide, and various post-translational modifications(Kojima M. & Kangawa M., Physiol. Rev. 85:495-522 (2005); ZhangJ. V. et al., Science 310:996-99 (2005)). In addition, differentdegradation products are produced by various tissues (De Vriese C.et al., Endocrinology 145:4997-5005 (2004)). Some of these GHRLproducts are described herein.

Ghrelin is a 28 amino acid peptide bearing an n-octanoyl side chainon the third serine, resulting from the cleavage of signal andpropeptide from the 117 amino acid preproghrelin and an acylationevent. The acylated N-terminus of ghrelin is essential for theendocrine functions (Kojima M. et al., Nature 402:656-60 (1999);Bednarek M. A. et al., J. Med. Chem. 43:4370-76 (2000)). Des-acylghrelin, which lacks the endocrine functions, was shown to have anantagonistic effect to that of ghrelin on glucose output in vitro(Gauna C. et al., J. Clin. Endocrinol. Metab. 89:5035-42 (2004)).An alternatively-spliced ghrelin mRNA encodes a 116 amino acidprepropeptide that is further processed to a Des-Gln14-ghrelin anda 27 amino acid processed peptide (Hosoda H. et al., J. Biol. Chem.275:21995-22000 (2000)). Another peptide, Obestatin, is cleavedfrom the preproghrelin and has no sequence overlap with processedghrelin peptide. This peptide was shown to have some antagonisticeffect to acylated ghrelin, inhibiting food intake and body weightgain (Zhang J. V. et al., Science 310:996-99 (2005)). Yet anotherpeptide, the 66 amino acid C-terminus of the preproghrelin, mayalso be functional (Pemberton C. et al., Biochem. Biophys. Res.Comm. 310:567-73 (2003)). A variety of isoforms, including isoformsencoded by different splice variants, are known for other proteins,e.g. for vascular endothelial growth factor (VEGF), where differentisoforms share roles as angiogenesis, while differing in some othercharacteristics, such as binding affinity (Neufeld G. et al., FASEBJ. 13:9-22 1999). Thus, the variety of products of the GHRL genemay reflect a similarly complex control of the endocrine andparacrine action of the ghrelin isoforms.

Previously, administration of ghrelin by continuous infusions of 5pmol/kg/min doses for 270 minutes was shown to increase food intakein healthy humans (Wren A. M. et al., J. Clin. Endocrinol. Metab.86:5992-95 (2001)). It was also shown that infusion of ghrelin for90 minutes could increase food intake by 30% in cancer cachexiapatients (Abstract P09, Digestive Hormones, Appetite and EnergyBalance, Baylis and Starling meeting, London, June 2003). Recently,it was shown that subcutaneous injection of 3.6 nmol/kg acylatedghrelin prior to a meal, thereby ensuring a close mimic of thenatural pre-meal situation, increased energy intake by 27%. Ghrelinalso appeared to enhance the perceived palatability of the foodoffered (Druce M. R. et al., Int. J. Obes. Relat. Metab. Disord.29:1130-36 (2005)).

These studies demonstrate that parenteral administration of ghrelincan increase appetite in both normal subjects and in patients withloss of appetite. Furthermore, Applicant has found that it ispossible to obtain a significant effect of body weight gain and asignificant increase in food consumption with a novel ghrelinsplice variant (see co-owned, co-pending published U.S. PatentApplication No. 2005/0059015, incorporated herein by reference)when administered to a subject, in particular when administeredsubcutaneously prior to a meal, thereby ensuring a close mimic ofthe natural pre-meal situation. Applicant's novel ghrelin splicevariant effect of weight gain is mainly on lean mass mass whileghrelin's effect on weight gain is mainly on fat mass.

SUMMARY OF THE INVENTION

One aspect is a ghrelin splice variant-like compound having theformula Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally presentprotecting group; each X1 is independently selected from anaturally occurring amino acid and a synthetic amino acid; X2 isselected from a naturally occurring amino acid and a syntheticamino acid, said amino acid being modified with a bulky hydrophobicgroup; each X3 is independently selected from a naturally occurringamino acid and a synthetic amino acid, wherein one or more of X1and X3 optionally may be modified with a bulky hydrophobic group;Z2 is an optionally present protecting group; m is an integer inthe range of from 1-10; n is an integer in the range of from 4-92;provided that the compound according to formulaZ1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has atleast 80% homology to SEQ ID NO:1.

Another aspect is a pharmaceutical composition comprising theghrelin splice variant-like compound having the formulaZ1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally presentprotecting group; each X1 is independently selected from anaturally occurring amino acid and a synthetic amino acid; X2 isselected from a naturally occurring amino acid and a syntheticamino acid, said amino acid being modified with a bulky hydrophobicgroup; each X3 is independently selected from a naturally occurringamino acid and a synthetic amino acid, wherein one or more of X1and X3 optionally may be modified with a bulky hydrophobic group;Z2 is an optionally present protecting group; m is an integer inthe range of from 1-10; n is an integer in the range of from 4-92;provided that the compound according to formulaZ1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has atleast 80% homology to SEQ ID NO:1.

A further aspect is a method of treating cachexia comprisingadministering to a mammal in need thereof a pharmaceuticallyacceptable amount of (a) ghrelin splice variant; (b) a ghrelinsplice variant-like compound having the formulaZ1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally presentprotecting group; each X1 is independently selected from anaturally occurring amino acid and a synthetic amino acid; X2 isselected from a naturally occurring amino acid and a syntheticamino acid, said amino acid being modified with a bulky hydrophobicgroup; each X3 is independently selected from a naturally occurringamino acid and a synthetic amino acid, wherein one or more of X1and X3 optionally may be modified with a bulky hydrophobic group;Z2 is an optionally present protecting group; m is an integer inthe range of from 1-10; n is an integer in the range of from 4-92;provided that the compound according to formulaZ1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has atleast 80% homology to SEQ ID NO:1; or (c) a mixture thereof.

An additional aspect is a method for preventing cachexia comprisingadministering to a mammal in need thereof a pharmaceuticallyacceptable amount of (a) ghrelin splice variant; (b) a ghrelinsplice variant-like compound having the formulaZ1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally presentprotecting group; each X1 is independently selected from anaturally occurring amino acid and a synthetic amino acid; X2 isselected from a naturally occurring amino acid and a syntheticamino acid, said amino acid being modified with a bulky hydrophobicgroup; each X3 is independently selected from a naturally occurringamino acid and a synthetic amino acid, wherein one or more of X1and X3 optionally may be modified with a bulky hydrophobic group;Z2 is an optionally present protecting group; m is an integer inthe range of from 1-10; n is an integer in the range of from 4-92;provided that the compound according to formulaZ1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has atleast 80% homology to SEQ ID NO:1; or (c) a mixture thereof.

A further aspect is a method for the stimulation of appetite, foodintake, and/or weigh gain comprising administering to a mammal inneed thereof a pharmaceutically acceptable amount of (a) ghrelinsplice variant; (b) a ghrelin splice variant-like compound havingthe formula Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionallypresent protecting group; each X1 is independently selected from anaturally occurring amino acid and a synthetic amino acid; X2 isselected from a naturally occurring amino acid and a syntheticamino acid, said amino acid being modified with a bulky hydrophobicgroup; each X3 is independently selected from a naturally occurringamino acid and a synthetic amino acid, wherein one or more of X1and X3 optionally may be modified with a bulky hydrophobic group;Z2 is an optionally present protecting group; m is an integer inthe range of from 1-10; n is an integer in the range of from 4-92;provided that the compound according to formulaZ1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has atleast 80% homology to SEQ ID NO:1; or (c) a mixture thereof.

Another aspect is a method for treating lipodystrophy comprisingadministering to a mammal in need thereof a pharmaceuticallyacceptable amount of (a) ghrelin splice variant; (b) a ghrelinsplice variant-like compound having the formulaZ1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally presentprotecting group; each X1 is independently selected from anaturally occurring amino acid and a synthetic amino acid; X2 isselected from a naturally occurring amino acid and a syntheticamino acid, said amino acid being modified with a bulky hydrophobicgroup; each X3 is independently selected from a naturally occurringamino acid and a synthetic amino acid, wherein one or more of X1and X3 optionally may be modified with a bulky hydrophobic group;Z2 is an optionally present protecting group; m is an integer inthe range of from 1-10; n is an integer in the range of from 4-92;provided that the compound according to formulaZ1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has atleast 80% homology to SEQ ID NO:1; or (c) a mixture thereof.

A further aspect is a method for preventing lipodystrophycomprising administering to a mammal in need thereof apharmaceutically acceptable amount of (a) ghrelin splice variant;(b) a ghrelin splice variant-like compound having the formulaZ1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally presentprotecting group; each X1 is independently selected from anaturally occurring amino acid and a synthetic amino acid; X2 isselected from a naturally occurring amino acid and a syntheticamino acid, said amino acid being modified with a bulky hydrophobicgroup; each X3 is independently selected from a naturally occurringamino acid and a synthetic amino acid, wherein one or more of X1and X3 optionally may be modified with a bulky hydrophobic group;Z2 is an optionally present protecting group; m is an integer inthe range of from 1-10; n is an integer in the range of from 4-92;provided that the compound according to formulaZ1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has atleast 80% homology to SEQ ID NO:1; or (c) a mixture thereof.

Another aspect is a method for treating muscle wasting comprisingadministering to a mammal in need thereof a pharmaceuticallyacceptable amount of (a) ghrelin splice variant; (b) a ghrelinsplice variant-like compound having the formulaZ1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally presentprotecting group; each X1 is independently selected from anaturally occurring amino acid and a synthetic amino acid; X2 isselected from a naturally occurring amino acid and a syntheticamino acid, said amino acid being modified with a bulky hydrophobicgroup; each X3 is independently selected from a naturally occurringamino acid and a synthetic amino acid, wherein one or more of X1and X3 optionally may be modified with a bulky hydrophobic group;Z2 is an optionally present protecting group; m is an integer inthe range of from 1-10; n is an integer in the range of from 4-92;provided that the compound according to formulaZ1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has atleast 80% homology to SEQ ID NO:1; or (c) a mixture thereof.

A further aspect is a method for preventing muscle wastingcomprising administering to a mammal in need thereof apharmaceutically acceptable amount of (a) ghrelin splice variant;(b) a ghrelin splice variant-like compound having the formulaZ1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally presentprotecting group; each X1 is independently selected from anaturally occurring amino acid and a synthetic amino acid; X2 isselected from a naturally occurring amino acid and a syntheticamino acid, said amino acid being modified with a bulky hydrophobicgroup; each X3 is independently selected from a naturally occurringamino acid and a synthetic amino acid, wherein one or more of X1and X3 optionally may be modified with a bulky hydrophobic group;Z2 is an optionally present protecting group; m is an integer inthe range of from 1-10; n is an integer in the range of from 4-92;provided that the compound according to formulaZ1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has atleast 80% homology to SEQ ID NO:1; or (c) a mixture thereof.

An additional aspect is a method of treating cachexia and/oranorexia and/or anorexia-cachexia and/or malnutrition and/orlipodystrophy and/or appetite-stimulation comprising administeringto a mammal in need thereof a pharmaceutically acceptable amount ofa secretagogue comprising (a) ghrelin splice variant; (b) a ghrelinsplice variant-like compound having the formulaZ1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally presentprotecting group; each X1 is independently selected from anaturally occurring amino acid and a synthetic amino acid; X2 isselected from a naturally occurring amino acid and a syntheticamino acid, said amino acid being modified with a bulky hydrophobicgroup; each X3 is independently selected from a naturally occurringamino acid and a synthetic amino acid, wherein one or more of X1and X3 optionally may be modified with a bulky hydrophobic group;Z2 is an optionally present protecting group; m is an integer inthe range of from 1-10; n is an integer in the range of from 4-92;provided that the compound according to formulaZ1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has atleast 80% homology to SEQ ID NO:1; or (c) a mixture thereof;wherein treatment is selected from the group consisting ofprophylaxis or treatment of cachexia, prophylaxis or treatment oflipodystrophy, stimulation of appetite, stimulation of food intake,stimulation of weight gain, increasing body fat mass, increasinglean body mass, or a combination thereof.

A further aspect is a kit for administering ghrelin splice variantor a ghrelin splice variant-like compound comprising (a) a dosageform comprising a pharmaceutically acceptable amount of (1) ghrelinsplice variant; (2) a ghrelin splice variant-like compound havingthe formula Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionallypresent protecting group; each X1 is independently selected from anaturally occurring amino acid and a synthetic amino acid; X2 isselected from a naturally occurring amino acid and a syntheticamino acid, said amino acid being modified with a bulky hydrophobicgroup; each X3 is independently selected from a naturally occurringamino acid and a synthetic amino acid, wherein one or more of X1and X3 optionally may be modified with a bulky hydrophobic group;Z2 is an optionally present protecting group; m is an integer inthe range of from 1-10; n is an integer in the range of from 4-92;provided that the compound according to formulaZ1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has atleast 80% homology to SEQ ID NO:1; or (3) a mixture thereof; and(b) optionally, instructions for administering (a).

Another aspect is for a method of producing a ghrelin splicevariant-like compound, said method comprising: (a) providing a cDNAcomprising a polynucleotide sequence encoding a ghrelin splicevariant-like compound as described above; (b) inserting said cDNAin an expression vector such that the cDNA is operably linked to apromoter; (c) introducing said expression vector into a host cellwhereby said host cell produces said ghrelin splice variant-likecompound; and (d) optionally recovering the ghrelin splicevariant-like compound produced in step (c).

Other objects and advantages will become apparent to those skilledin the art upon reference to the detailed description thathereinafter follows.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 represents human prepro ghrelin splice variant afterthe signaling sequence.

SEQ ID NO:2 represents 22 amino acid human ghrelin splicevariant.

SEQ ID NO:3 represents 24 amino acid human ghrelin splicevariant.

SEQ ID NO:4 represents 24 amino acid modified human ghrelin splicevariant.

SEQ ID NO:5 represents 29 amino acid human ghrelin splicevariant.

SEQ ID NO:6 represents a fragment of full-length human ghrelinsplice variant.

SEQ ID NO:7 represents mouse prepro ghrelin splice variant afterthe signaling sequence.

SEQ ID NO:8 represents rat prepro ghrelin splice variant after thesignaling sequence.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a line graph showing cumulative body weight gain ofacylated ghrelin splice variant-treated (SEQ ID NOs: 2 and 5) 129Svmice compared to vehicle-treated controls. Acylated ghrelin splicevariant induces body weight gain in male wild-type mice (n=10 pergroup, P=0.0001). In mice treated once daily for two weeks withacylated ghrelin splice variant (0.8 mg/kg, subcutaneously),cumulative weight gain of the SEQ ID NO:2 group was 3.4 times morethan the vehicle-injected control animals. Cumulative weight gainof the acylated SEQ ID NO:5 group was 2 times more than thevehicle-injected control animals. FIG. 1B is a line graph showingcumulative food consumption of acylated ghrelin splicevariant-treated (SEQ ID NOs: 2 and 5) 129Sv mice compared tovehicle-treated controls. Acylated ghrelin splice variant treatmentincreased food consumption in wild-type mice. Mice treated oncedaily for two weeks with acylated ghrelin splice variant (0.8mg/kg, subcutaneously) ate 13% more than the vehicle-injectedcontrol animals (n=10 per group, P=0.004).

FIG. 2A is a line graph showing cumulative body weight gain ofacylated ghrelin splice variant-treated (SEQ ID NOs: 2 and 4) 129Svmice and of unacylated SEQ ID NO:5 compared to vehicle-treatedcontrols. Acylated ghrelin splice variants induces body weight gainin male wild-type mice (n=8 per group, P=0.0001). In mice treatedonce daily for seven days with acylated or un-acylated ghrelinsplice variant (7.2 mg/kg, subcutaneously), cumulative weight gainof the SEQ ID NO:2 and SEQ ID NO:4 group was 2.2 times more thanthe vehicle-injected control animals. Cumulative weight gain of theun-acylated SEQ ID NO:5 group was 25% less than thevehicle-injected control animals. FIG. 2B is a line graph showingcumulative food consumption of acylated ghrelin splicevariant-treated (SEQ ID NOs: 2 and 4) 129Sv mice and of unacylatedSEQ ID NO:5 compared to vehicle-treated controls. Acylated ghrelinsplice variants treatment increased food consumption in wild-typemice. Mice treated once daily for seven days with acylated ghrelinsplice variants (7.2 mg/kg, subcutaneously) ate 18% more than thevehicle-injected control animals (n=8 per group). Mice treated oncedaily for seven days with SEQ ID NO:5 (7.2 mg/kg, subcutaneously)ate 2% less than the vehicle-injected control animals (n=8 pergroup).

FIG. 3 is a bar graph showing serum growth hormone concentrationafter subcutaneous administration of acylated ghrelin splicevariant (SEQ ID NOs: 2, 4, 5) and unacylated SEQ ID NO:5 comparedto vehicle-administered controls. Effect of saline or acylatedghrelin splice variant (0.8 mg/kg, subcutaneously) on plasma growthhormone at 10 minutes and 20 minutes post injection in wild typemice (n=5 per group) is shown.

FIG. 4 is a bar graph showing the change in body composition ofacylated ghrelin splice variant-treated (SEQ ID NOs: 2 and 4) 129Svmice and unacylated SEQ ID NO:5 treated mice compared to vehicleand ghrelin-treated controls. Effect of seven days dailysubcutaneous treatment with saline, ghrelin or acylated ghrelinsplice variant (7.2 mg/kg, subcutaneously) on fat and lean bodymass as measured by NMR is shown.

DETAILED DESCRIPTION OF THE INVENTION

Applicants specifically incorporate the entire content of all citedreferences in this disclosure. Further, when an amount,concentration, or other value or parameter is given as either arange, preferred range, or a list of upper preferable values andlower preferable values, this is to be understood as specificallydisclosing all ranges formed from any pair of any upper range limitor preferred value and any lower range limit or preferred value,regardless of whether ranges are separately disclosed. Where arange of numerical values is recited herein, unless otherwisestated, the range is intended to include the endpoints thereof, andall integers and fractions within the range. It is not intendedthat the scope of the invention be limited to the specific valuesrecited when defining a range.

In the context of this disclosure, a number of terms shall beutilized.

"Affinity" as used herein means the strength of binding betweenreceptors and their ligands, for example between an antibody andits antigen.

"Amino acid residue" as used herein means an amino acid formed uponchemical digestion (hydrolysis) of a polypeptide at its peptidelinkages. The amino acid residues described herein are preferablyin the "L" isomeric form. However, the amino acid encompasses everyamino acid such as L-amino acid, D-amino acid, alpha-amino acid,beta-amino acid, gamma-amino acid, natural amino acid and syntheticamino acid or the like as long as the desired functional propertyis retained by the polypeptide. NH.sub.2 refers to the free aminogroup present at the amino terminus of a polypeptide. COOH refersto the free carboxy group present at the carboxy terminus of apolypeptide. Standard polypeptide abbreviations for amino acidresidues are shown in Table 1.

TABLE-US-00001 TABLE 1 1-Letter Code 3- Letter Code Amino Acid AAla Alanine B Asx, Asn, and/or Asp Aspartic Acid and/or AsparagineC Cys Cysteine D Asp Aspartic Acid E Glu Glutamic Acid F PhePhenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K LysLysine L Leu Leucine M Met Methionine N Asn Asparagine P ProProline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr ThreonineV Val Valine W Trp Tryptophan X Xaa Unknown or Other Y Tyr TyrosineZ Glx, Gln, and/or Glu Glutamic Acid and/or glutamine -- Dpr2,3-diaminopropionic acid

It should be noted that all amino acid residue sequencesrepresented herein by formula have a left-to-right orientation inthe conventional direction of amino terminus to carboxy terminus.In addition, the phrase "amino acid residue" is broadly defined toinclude the amino acids listed in the Table 1 and modified andnon-naturally occurring amino acids. Furthermore, it should benoted that a dash at the beginning or end of an amino acid residuesequence indicates a peptide bond to a further sequence of one ormore amino acid residues or a covalent bond to an amino-terminalgroup such as NH.sub.2 or acetyl or to a carboxy-terminal groupsuch as COOH.

"Anti-neoplastic treatment" as used herein means treatment aimed athalting or reducing abnormal tissue growth (such as a neoplasm) inan individual. Examples of such treatment include cancer therapies,such as radiotherapy or chemotherapy.

"Appetite" in relation to an individual is assessed by measuringthe amount of food ingested and by assessing the individual'sdesire to eat. Herein, appetite (i.e., hunger) is typicallyassessed with a short questionnaire given to individuals on arandom basis several times a week. Typically, subjects rate theirhunger, preoccupation with food, and desire to eat greaterquantities and different types of food by answering the questionsusing analogue scales ranging from 1 (not at all) to 5(extremely).

"Body Mass Index" or "BMI" is a measure of an individual's heightto weight ratio. BMI is determined by calculating weight inkilograms divided by the square of height in meters. The BMI"normal" range is 18.5-25.

"Body fat mass" can be measured, e.g., by the fat fold technique.In the fat fold technique, a pincer-type caliper is used to measuresubcutaneous fat by determining skin fold thickness atrepresentative sites on the body. These skin fold measurements arethen used to compute body fat by either adding the scores from thevarious measurements, and using this value as an indication of therelative degree of fatness among individuals, or by using themeasurements in mathematical equations that have been developed topredict percent body fat (Fogelholm M. & van Marken LichtenbeltW., Eur. J. Clin. Nutr. 51:495-503 (1997)).

"Concentration equivalent" as used herein means an equivalentdosage of a ghrelin splice variant-like compound having in vitroand/or in vivo the same response as evaluated from adosage-response curve of the ghrelin splice variant.

"Dissociation constant" or "Kd" is a measure describing thestrength of binding (or affinity or avidity) between receptors andtheir ligands, for example an antibody and its antigen. The smallerthe Kd, the stronger the binding.

A "fusion polypeptide" is a polypeptide comprised of at least twopolypeptides and a linking sequence to operatively link the twopolypeptides into one continuous polypeptide. The two polypeptideslinked in a fusion polypeptide are typically derived from twoindependent sources, and therefore a fusion polypeptide comprisestwo linked polypeptides not normally found linked in nature.

"Human ghrelin" as used herein is a polypeptide having the aminoacid sequence as set forth in GenBank.RTM. Accession No.NP.sub.--057446 or Swiss-Prot Identifier GHRL_HUMAN. Human ghrelinpreprotein has 117 amino acids. This preprotein undergoes thefollowing post-translational processing. The signal peptide (aminoacids 1-23) is removed and the remaining 94 amino acids are cleavedby a protease to provide a mature 28 amino acid ghrelin (aminoacids 24-51) or a mature 27 amino acid ghrelin (amino acids 24-50)and a mature 23 amino acid obestatin (amino acids 76-98). The 27 or28 amino acid mature ghrelin peptides can be further modified atthe serine at position 26 in the preprotein by either an O-octanoylgroup or an O-decanoyl group. The obestatin mature peptide can befurther modified at the lysine at position 98 of the preprotein byan amide group. An additional ghrelin preprotein is known, whichlacks the glutamine at position 37 of the preprotein.

"Ghrelin splice variant" is a polypeptide having the amino acidsequence as set forth in SEQ ID NO:1 or any peptide of 15 aminoacids or more from SEQ ID NO:1 with or without post translationalmodification, or any SEQ ID NO:1 homologs as set forth in SEQ IDNO:7 or SEQ ID NO:8, and/or any peptide of 15 amino acids or morefrom SEQ ID NO:7 or SEQ ID NO:8 with or without post translationalmodification.

"Ghrelin splice variant-like compound" as used herein refers to anycompound which mimics the function of ghrelin splice variant, inparticular human ghrelin splice variant, particularly in terms ofthe ghrelin splice variant functions leading to the desiredtherapeutic effects described herein, such as stimulation ofappetite and/or treatment and/or prophylaxis of cachexia and isdefined by the Formula I: Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is anoptionally present protecting group; each X1 is independentlyselected from a naturally occurring amino acid and a syntheticamino acid; X2 is selected from a naturally occurring amino acidand a synthetic amino acid, said amino acid being modified with abulky hydrophobic group; each X3 is independently selected from anaturally occurring amino acid and a synthetic amino acid, whereinone or more of X1 and X3 optionally may be modified with a bulkyhydrophobic group; Z2 is an optionally present protecting group; mis an integer in the range of from 1-10; n is an integer in therange of from 4-92; provided that the compound according to formulaZ1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has atleast 80% (or, in alternative embodiments, 85%, 90%, 93%, 95%, 97%,98%, 99%, 100%) homology to SEQ ID NO:1. In a preferred embodiment,the ghrelin splice variant-like compound is 22-29 amino acids inlength.

"Immunologically distinct" refers to the ability to distinguishbetween two polypeptides on the ability of an antibody tospecifically bind one of the polypeptides and not specifically bindthe other polypeptide.

An "individual" is an animal or human susceptible to a condition,in particular a cachectic condition as defined herein. In preferredembodiments, the individual is a mammal, including human, andnon-human mammals such as dogs, cats, pigs, cows, sheep, goats,horses, rats, and mice. In the most preferred embodiment, theindividual is a human.

"Isolated" is used to describe the various ghrelin splicevariant-like compounds, i.e. polypeptides and nucleotides disclosedherein, that have been identified and separated and/or recoveredfrom a component of its natural environment. Contaminant componentsof a natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the ghrelinsplice variant-like compounds will be purified.

"Modified amino acid" as used herein is an amino acid wherein anarbitrary group thereof is chemically modified.

"Non-standard amino acid" as used herein is an amino acid that doesnot belong to the standard 20 amino acids. Non-standard amino acidsare usually formed through chemical modifications to standard aminoacids. They can also be formed naturally as an intermediatecomponent of a metabolic pathway or by microorganisms and/orplants.

"Monoclonal Antibody", in its various grammatical forms, refers toa population of antibody molecules that contains only one speciesof antibody combining site capable of immunoreacting with aparticular antigen.

A "non-acylated ghrelin splice variant-like compound" is a ghrelinsplice variant-like compound, as defined herein, which does notcontain an acyl group attached to any of its constituent aminoacids.

"Palliative treatment" is a treatment which relieves or sooths thesymptoms of a disease or disorder but without a curing effect.

"Polyclonal antibodies" are a mixture of antibody moleculesrecognizing a specific given antigen; hence, polyclonal antibodiesmay recognize different epitopes within said antigen.

"Polypeptide" refers to a molecule comprising amino acid residueswhich do not contain linkages other than amide linkages betweenadjacent amino acid residues.

"Processed ghrelin" means the 27 or 28 amino acid residue, acylatedghrelin (the post-translational product of cleavage and acylationof 116 and 117 amino acid residues long preproghrelin, respectively(e.g., SWISS-PROT Q9UBU3 GHRL_HUMAN)).

A "receptor" is a molecule, such as a protein, glycoprotein and thelike, that can specifically (non-randomly) bind to anothermolecule.

A "secretagogue" is a substance stimulating growth hormone release,such as ghrelin or a ghrelin-like compound. A secretagogueaccording to the present disclosure may for example be selectedfrom L-692-429 and L-692-585 (benzoelactam compounds; availablefrom Merck & Co, Inc., Whitehouse Station, N.J.), MK677(spiroindaner; available from Merck) G-7203, G-7039, G-7502(isonipecotic acid peptidomimetic; available from Genentech, Inc.,South San Francisco, Calif.), NN703 (Novo Nordisk Inc., Princeton,N.J.), or ipamorelin. In particular, the secretagogue is a ghrelinsplice variant-like compound, including a 29 amino acid humanghrelin splice variant, a 24 amino acid human ghrelin splicevariant, or a 22 amino acid human ghrelin splice variant (e.g., SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5). The growthhormone secretagogue may in one embodiment be non-acylated, forinstance a non-acylated form of ghrelin splice variant or anon-acylated ghrelin splice variant-like compound.

A "surfactant molecule" is a molecule comprising a hydrophobic partand a hydrophilic part; i.e. a molecule capable of being present inthe interphase between a lipophilic phase and a hydrophilicphase.

Indications

The present disclosure relates to the use of a secretagogue, suchas a ghrelin splice variant-like compound, in the treatment orprophylaxis of conditions, e.g., relating to pathological weightloss or lean and/or fat mass loss including a) prophylaxis ortreatment of cachexia, and/or b) prophylaxis or treatment oflipodystrophy, and/or c) stimulation of appetite, and/or d)stimulation of food intake, and/or e) stimulation of weight gain,and/or f) increase of body fat mass, and/or g) increase of bodylean mass. In particular, the present disclosure relates to thetreatment or prophylaxis of cachexia and/or the stimulation ofappetite, stimulation of palatability, increasing quality of life,most preferably the prophylaxis or treatment of cachexia.

Cachexia

Cachexia is one of the most distressing and devastating symptoms ofseveral severe diseases, such as cancer, robbing individuals oftheir energy, sense of well-being, quality of life and increasingtheir dependence on others. Cachexia often accompanies malignanciesof the pancreas, stomach, esophagus, lung, and intestines.

The foremost sign of cachexia is weight loss, not only of fattytissue, but also of muscle tissue and even bone. This non-fattytissue is also known as "lean body mass". In addition, there isloss of appetite (anorexia), weakness (asthenia), and a drop inhemoglobin level (anemia).

Treatment of cachexia is not simply a matter of eating more. Evenif the individual wants to eat, even if the individual tries toeat, even if the individual is given nutrients through a stomachtube or intravenously, the condition will normally not bereversed.

Recent research has revealed that the condition is now regarded aspart of a body's reaction to the presence of the underlyingdisease. (Laviano A. et al., Nat. Clin. Pract. Oncol. 2:158-65(2005)). Recent research also indicates that, in some cases, tumorsthemselves produce substances that induce cachexia. (Esper D. H.& Harb W. A., Nutr. Clin. Pract. 20:369-76 (2005)).

Cachexia, or wasting, as it may also be called, is seen withseveral diseases, such as AIDS, cancer, post hip fracture, chronicheart failure, chronic lung disease such as COLD (chronicobstructive lung disease) and COPD (chronic obstructive pulmonarydisease), liver cirrhosis, renal failure, autoimmune diseases suchas rheumatoid arthritis and systemic lupus, sepsis, tuberculosis,cystic fibrosis, Crohn's Disease, and severe infection.Furthermore, wasting is also seen in aging.

Although cachexia represents the complex metabolic syndrome that isseen in such patients, it is commonly recognized as a progressiveweight loss with depletion of host reserves of adipose tissue andskeletal muscle.

Cancer Cachexia

The core of cancer cachexia syndrome relates to the problem ofprogressive tumor growth and the catabolic side effects ofconventional anti-neoplastic therapy. These two phenomena give riseto alterations in the neuro-endocrine system, to the production ofa variety of pro-inflammatory cytokines, and to the release ofcancer-specific cachectic factors. In turn, these mediators cause areduction in food intake, abnormality in the metabolism, or acombination of these two.

Cancer cachexia is reported to occur in about half of all cancerpatients and is associated with more than 20 percent of cancerdeaths (Tisdale M. J., Nat. Rev. Cancer 2:862-71 (2002)). Thecondition often occurs during advanced cancer, in particular whenmetastatic tumors are present in the body. Cachexia is also morecommon in children and elderly patients. Specific cancers are alsoconsistently identified where the frequency of cancer cachexia isparticularly high: upper GI cancers (including: pancreas, stomach,esophagus, and liver) (Bruera E., Br. Med. J. 315:1219-22 (1997);Palesty J. A. et al., Dig. Dis. 21:198-213 (2003)); lung cancer, inparticular small cell lung cancer; head and neck cancer; colorectalcancer; other solid tumors (Bruera E., Br. Med. J. 315:1219-22(1997)). IWL (involuntary weight loss) has been associated with anapproximate 50% drop in survival and decreased tolerance of cancertherapy (Laviano A. et al., Nat. Clin. Pract. Oncol. 2:158-65(2005)). Cancer sites associated with the greatest risks for weightloss are those affecting the aerodigestive tract (lung, head andneck, and esophagus) and the gastrointestinal system, especiallypancreas, stomach, and liver. Furthermore, at the moment ofdiagnosis, 80% of all patients with cancer in the upper GI tractand 60% of all patients with lung cancer have already experiencedsubstantial weight loss (Bruera E., Br. Med. J. 315:1219-22(1997)). On average, the prevalence of cachexia increases from 50%to more than 80% percent before death and, in more than 20% of thepatients, cachexia is the main cause of death (Bruera E., Br. Med.J. 315:1219-22 (1997)).

Detection of Cancer Cachexia

Nutritional state is evaluated with a combination of clinicalassessment, anthropometric tests (body weight, skin fold thicknessand mid arm circumference) and imaging (DEXA scan, MR scan, CT scanand bioelectric impedance measuring). Cachexia is generallysuspected if an involuntary weight loss of greater than 5% of thepremorbid weight is observed within a six-month period--especiallywhen combined with muscle wasting.

The most commonly used laboratory parameter is serum albumin. It ishowever an unspecific parameter. Other markers are proteins with ashort half life; transferrin and transthyretin have also beenused.

Other markers of cachexia are IGF-1, IGFBP-3, ALP (alkalinephosphatase), and testosterone.

Relationship Between Cancer and Cancer Cachexia

Cancer may cause cachexia through a variety of mechanisms,including induction of anorexia and/or increase or change ofmetabolism, as described below:

Anorexia

Energy intake has been shown to be substantially reduced amongweight-losing cancer patients. Cancer patients may frequentlysuffer from physical obstruction of the GI tract, pain, depression,constipation, malabsorption, debility or the side effects oftreatment (such as, e.g., treatments with opiates, radiotherapy orchemotherapy), which all may decrease food intake (Barber M. D. etal., Surg. Oncol. 8:133-41 (1999)). Cancer-associated hypercalcemiamay also induce nausea, vomiting and appetite loss.

However, there remains a large number of patients with cancer inwhom there is no obvious clinical cause of reduced food intake.

The central mechanism of cancer-induced anorexia and cachexia iscomplex and includes many different cytokines, hormones and otherfactors produced by the cancer cells.

Leptin

In normal physiological situations, leptin plays an important rolein triggering the adaptive response to starvation, since weightloss causes leptin levels to fall in proportion to the loss of bodyfat. However, in cancer patients, an increased level of cytokines(e.g., IL-1, IL-6, TNF-.alpha., INF-.gamma.) produced by the cancercells may stimulate the expression and/or the release of leptin.Another possible mechanism of the cytokines is that they mimic thehypothalamic effect of excessive negative feedback signaling fromleptin, leading to the prevention of the normal compensatorymechanism regarding food intake and body weight.

NPY (Neuropeptide Y)

The hypothalamic NPY system is one of the key neural pathwaysdisrupted in anorexia and cancer induced by IL-1 or othercytokines. The cytokines decrease the sensitivity for NPY. It wasshown that NPY is a growth-regulatory factor for neuroendocrinetumors, acting both by autocrine activation of tumor cellproliferation or apoptosis and by angiogenesis (Kitlinska J et al.,Cancer Res. 65:1719-28 (2005)). In addition, Y1 and Y2 receptorshave been found to be expressed in breast carcinomas, adrenal glandand related tumors, renal cell carcinomas, and ovarian cancers inboth tumor cells and tumor-associated blood vessels. Their broadexpression in cancer cells allows them to mediate NPY effects ontumor cell proliferation and tumoral blood supply (for a review seeKorner M & Reubi J C, Peptides 28:419-25 (2007)).

Melanocortins

Aberrant melanocortin signaling may be a contributing factor inboth anorexia and cachexia. Despite marked loss of body weightwhich would normally be expected to down-regulate the anorexigenicmelanocortin signaling system as a way to conserve energy stores,the melanocortin system remains active during cancer-inducedcachexia. Central melanocortin blockade by AgRP (Agouti-relatedpeptide) or other antagonists reversed anorexia and cachexia in theanimal models, suggesting a pathogenic role of this system (WisseB. E. et al., Ann. N.Y. Acad. Sci. 994:275-81 (2003)). In addition,recent experiments show that blockade of melanocortin signalingusing antagonists to the melanocortin MC(4) receptor attenuatesdisease-associated anorexia and wasting in rodent models of cancerand renal failure (DeBoer M D & Marks D L, Nat. Clin. Pract.Endocrinol. Metab. 2:459-66 (2006)).

Metabolism

Hyper metabolism is defined as an elevation of the resting energyexpenditure (REE) and is a cardinal feature of cachexia. Totalenergy expenditure involves REE (approximately 70%) and voluntaryenergy expenditure (approximately 25%) and energy expenditure indigestion (5%). Voluntary energy expenditure may be decreased incachexia which may manifest clinically as apathy, fatigue anddepression.

The orexigenic and the anorexigenic signals are known torespectively decrease and increase sympathetic nervous activity,which regulate REE by activating thermogenesis in brown adiposetissue in rodents and possibly in muscle in humans, throughinduction of the mitochondrial uncoupling proteins (UCP) (Alvarez Ret al., J. Biol. Chem. 270:5666-73 (1995)). It has been suggestedthat activation of UCP in muscle and in white adipose tissue bycytokines might be one of the molecular mechanisms underlying theincrease in the heat production and muscle wasting (Inui A., CACancer J. Clin. 52:72-91 (2002); Fearon K. C. & Moses A. G.,Int. J. Cardiol. 85:73-81 (2002)).

Altered nutrition metabolism has also been described in patientswith cancer. Solid tumors produce large amounts of lactate, whichis converted back into glucose through a process that uses largeamounts of ATP and is very energy inefficient, thus furtherincreasing the energy expenditure. Furthermore, tumor-derived lipidmobilizing factor (LMF) has been shown to act directly onadipocytes and cause increased lipolysis, leading to release offree fatty acid and glycerol (Islam-Ali B. et al., Br. J. Cancer85:758-63 (2001)), and attenuating free radical toxicity in thetumor cells (Sanders P M & Tisdale M J, Br. J. Cancer90:1274-78 (2004)).

It has also been suggested that the increased level of cytokinesmay induce muscle protein catabolism indirectly by affecting themuscle repair processes (Islam-Ali B. et al., Br. J. Cancer85:758-63 (2001)).

The Rationale for Using Secretagogues in the Treatment of CancerCachexia

Without being bound by theory, the rationale for the treatment witha secretagogue, in particular a ghrelin splice variant-likecompound is based on the following: ghrelin splice variant releasedfrom the endocrine cells in the mucosa of the GI tract may act bothlocally as a paracrine substance and centrally as a hormone.Locally, ghrelin splice variant may act as an initiator of afferentactivity in, for example, afferent vagal neurons. Such neurons willrelay the ghrelin splice variant stimulus to centers in the CNSsuch as the nucleus tractus solitarirus (NTS) which furthercommunicate with appetite and energy homeostasis regulatory centerssuch as the paraventricular nucleus and arcuate nucleus in thehypothalamus. As a hormone, ghrelin splice variant is believed toact on central appetite regulating POMC (proopiomelanocortin) andNPY/AgRP neurons, which express ghrelin splice variantreceptors.

Recently it has been described that ghrelin is transported acrossthe blood brain barrier (Banks W. A. et al., J. Pharmacol. Exp.Ther. 302:822-27 (2002)). It is important to note that, at thecentral appetite regulatory center, for example at the NPY/AgRPneurons--i.e., the first level neurons in the stimulatory branch ofappetite control--ghrelin acting through stimulatory ghrelinreceptors is the only stimulatory input known from the periphery.All other known hormones and neurotransmitters, e.g., leptin,insulin, PYY3-36, a-MSH, etc., act as inhibitors on the NPY/AgRPneurons in this important "appetite gate-keeping" center. Since theNPY system is down-regulated during cancer-induced cachexia,ghrelin stimulation of this system may be able to normalize thecondition. Similarly, the melanocortin that is active duringcancer-induced cachexia may be inhibited by ghrelin and ghrelinsplice variant through stimulation of AgRP.

Increase in ghrelin concentration has also been shown to increaseACTH (adrenocorticotropic hormone) with a resulting increase incortisol level. This action may have important beneficialimplications for the treatment of cachexia, as cortisol decreasesthe level of cytokines (e.g., IL-1, IL-6, TNF-a, IFN-a).Administration of glucocorticoids is already widely used in thepalliative setting for symptoms associated with cancer (Inui A., CACancer J. Clin. 52:72-91 (2002)). Furthermore, it has been shownthat ICV injection of ghrelin decreases core body temperature inrodents, which indicates a decrease in the REE (Lawrence C. B. etal., Endocrinology 143:155-62 (2002)). Again, without being boundby theory, it is expected that ghrelin splice variant, similar towild-type ghrelin, will revert the increase in REE, which is animportant feature of cachexia as described above.

The secretagogue, in particular a ghrelin splice variant-likecompound, may be administered using any suitable regimen, takinginto account the knowledge of the expected cancer progress as wellas the anti-neoplastic therapy regime.

In one embodiment, it is envisaged that, according to the presentdisclosure, a secretagogue can be administered to any individualsuffering from any cancer type, regardless of etiology, tosuccessfully treat, reduce, or prevent cancer cachexia.

Thus, in one preferred embodiment, the treatment of an individualwith a secretagogue, such as ghrelin splice variant or a ghrelinsplice variant-like compound, is for the treatment or prevention ofcancer cachexia caused by, for example, one or more of thefollowing cancer types: Acute Lymphoblastic Leukemia, Acute MyeloidLeukemia, Adrenocortical Carcinoma, AIDS-Related Cancers,AIDS-Related Lymphoma, Anal Cancer, Astrocytoma, ChildhoodCerebella Astrocytoma, Childhood Cerebral Basal Cell Carcinoma,Extrahepatic Bile Duct Cancer, Bladder Cancer, Bone Cancer,Osteosarcoma/Malignant Fibrous Histiocytoma, Brain Stem Glioma,Brain Tumor, Breast Cancer, Male Bronchial Adenomas/Carcinoids,Burkitt's Lymphoma, Carcinoid Tumor, Carcinoma of Unknown Primary,Central Nervous System Lymphoma, Primary CerebralAstrocytoma/Malignant Glioma, Cervical Cancer, Childhood Cancers,Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, ChronicMyeloproliferative Disorders, Colon Cancer, Cutaneous T-CellLymphoma, Endometrial Cancer, Ependymoma, Childhood EsophagealCancer, Ewing's Family of Tumors, Extracranial Germ Cell Tumor,Childhood Extragonadal Germ Cell Tumor, Eye Cancer, IntraocularMelanoma Eye Cancer, Retinoblastoma Gallbladder Cancer, Gastric(Stomach) Cancer, Gastrointestinal Carcinoid Tumor, GestationalTrophoblastic Tumor, Glioma, Hairy Cell Leukemia, Head and NeckCancer, Hepatocellular (Liver) Cancer, Hodgkin's Lymphoma,Hypopharyngeal Cancer, Hypothalamic and Visual Pathway Glioma,Intraocular Melanoma Islet Cell Carcinoma (Endocrine Pancreas),Kaposi's Sarcoma Kidney (Renal Cell) Cancer, Laryngeal Cancer, Lipand Oral Cavity Cancer, Lung Cancer, Non-Small Cell Lung Cancer,Small Cell Lymphoma, AIDS-Related Lymphoma, Cutaneous T-CellLymphoma, Non-Hodgkin's Macroglobulinemia, Waldenstrom's MalignantFibrous Histiocytoma of Bone/Osteosarcoma, Medulloblastoma,Childhood Melanoma Merkel Cell Carcinoma, Mesothelioma, AdultMalignant Mesothelioma, Childhood Metastatic Squamous Neck Cancerwith Occult Primary Multiple Endocrine Neoplasia Syndrome,Childhood Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides,Myelodysplastic Syndromes, Myelodysplastic/MyeloproliferativeDiseases, Myeloma, Multiple Chronic Myeloproliferative Disorders,Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer,Nasopharyngeal Cancer, Childhood Neuroblastoma, OropharyngealCancer, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Cancer, Childhood Ovarian Epithelial Cancer, Ovarian GermCell Tumor, Ovarian Low Malignant Potential Tumor, PancreaticCancer, Paranasal Sinus and Nasal Cavity Cancer, ParathyroidCancer, Senile Cancer, Pheochromocytoma, Pineoblastoma andSupratentorial Primitive Neuroectodermal Tumors, ChildhoodPituitary Tumor, Pleuropulmonary Blastoma, Prostate Cancer, RenalPelvis and Ureter Cancer, Transitional Cell Cancer Retinoblastoma,Rhabdomyosarcoma, Childhood Salivary Gland Cancer, Adult-Onset SoftTissue Sarcoma, Sarcoma, Childhood Uterine Sarcoma, SezarySyndrome, Skin Cancer (Non-Melanoma), Skin Carcinoma, Merkel CellSmall Intestine Cancer, Supratentorial Primitive NeuroectodermalTumors, Childhood Cutaneous T-Cell Lymphoma, Testicular Cancer,Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional CellCancer of the Renal Pelvis and Ureter, Trophoblastic Tumor,Gestational Ureter and Renal Pelvis Cancer, Transitional CellCancer, Urethral Cancer, Endometrial Uterine Cancer, UterineSarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Childhood Waidenstrom's Macroglobulinemia, Wilms' Tumor.

As discussed above, cancer cachexia may be due to a catabolicdisorder, e.g. a hypermetabolic state as described above, eitherresulting from the progressive tumor growth or from the catabolicside effects of the anti-cancer therapy. However, the cancercachexia may also be due to an anorectic disorder, such as is thecase when the individual suffering from the cancer has no appetiteor the position of the tumor reduces or prevents food intake.

Accordingly, one embodiment is for the treatment or prevention witha secretagogue, such as a ghrelin splice variant-like compound, ofcancer cachexia caused by a catabolic disorder. This isparticularly suitable when the cancer is a GI tract cancer,especially upper GI tract cancer (it is to be understood hereinthat the term "upper GI tract cancer" also encompasses pancreaticcancer), lung cancer (in particular small cell lung cancer), and/orliver cancer (it is to be understood herein that the term "livercancer" also encompasses metastatic cancer processes in theliver).

Another embodiment is for the treatment or prevention with asecretagogue, such as a ghrelin splice variant-like compound, ofcancer cachexia caused by an anorectic disorder.

Yet another embodiment is for the treatment or prevention with asecretagogue, such as a ghrelin splice variant-like compound, ofcancer cachexia independent of how the cancer has induced thecachexia, as well as for cachexia caused by a combination of thecatabolic disorder and the anorectic disorder.

In a preferred embodiment, a secretagogue, such as ghrelin splicevariant or a ghrelin splice variant-like compound, is used in thetreatment or prevention of cancer cachexia caused by a solidtumor.

Another sub-group of cancers are those with anorexia caused bydysregulation of the central appetite regulatory center inhypothalamus, where other possible reasons to eat less areexcluded.

In particular individuals in terminal cancer states where furthercancer treatment is impossible, ghrelin splice variant treatment asa palliative treatment to increase food intake, improve digestion,and improve metabolism could prove beneficial. Accordingly, anotheraspect relates to the palliative treatment to increase food intake,improve digestion, and improve metabolism in an individual in needthereof, such as wherein said individual is suffering fromadvanced-stage cancer, particularly terminal cancer.

In accordance with the above, the compounds disclosed herein areparticularly suitable for treating or preventing cachexia in anindividual suffering from the following aerodigestive tract cancerforms: pancreatic cancer; cancer of the upper GI tract, such asstomach cancer and/or esophagus cancer; head and neck cancer, inparticular cancer of the thyroid or cancer of the salivary glands;and lung cancer, in particular small lung cell cancer.

In another preferred embodiment, a secretagogue, such as ghrelinsplice variant or a ghrelin splice variant-like compound, is usedin the treatment or prevention of cancer cachexia caused by lowerGI cancer, such as colorectal cancers, in particular by coloncancer.

In another preferred embodiment, a secretagogue, such as ghrelinsplice variant or a ghrelin splice variant-like compound, is usedin the treatment or prevention of cancer cachexia caused by anendocrine cancer, i.e. a cancer in an endocrine organ of anindividual's body.

The compounds disclosed herein are also useful for treatingindividuals suffering from cancer of the ovaries or breastcancer.

In a further preferred embodiment, a secretagogue, such as ghrelinsplice variant or a ghrelin splice variant-like compound, is usedin the treatment or prevention of cancer cachexia caused in wholeor in part by anti-cancer treatment, such as chemotherapy orradiotherapy or combinations thereof.

In one preferred embodiment, the individual treated for cancercachexia is elderly, such as 60-120 years old, such as 70-120 yearsold, such as 80-120 years old, such as 90-120 years old. Equallypreferable are embodiments where said individual is a child, suchas from 0-20 years old, such as 0-15 years old, such as 0-10 yearsold, such as 0-5 years old, such as 0-1 years old, such as anewborn child less than 2 months old.

In one embodiment, it is preferred that the secretagogue, such as aghrelin splice variant-like compound, is administeredprophylactically for preventing a cachectic state. In thisembodiment, the treatment may be started before any anti-neoplastictreatment initiates. The secretagogue may be administeredcontinuously during the anti-neoplastic treatment, or it may beadministered at intervals, for example between periods withanti-neoplastic therapy. By administering during and in particularbetween the periods of anti-neoplastic therapy, the risk that thetreated individual acquires infections and/or other complicationsmay be reduced due to better health conditions of theindividual.

Treatment of cancer cachexia using a secretagogue, such as ghrelinsplice variant or a ghrelin splice variant-like compound, may beachieved using any administration method known in the art.Preferably, treatment may be achieved using any of theadministration methods described herein, more preferably usingintravenous or subcutaneous administration, most preferably usingsubcutaneous administration methods.

Lipodystrophy

In another aspect, the present disclosure relates to use of asecretagogue, such as ghrelin splice variant or a ghrelin splicevariant-like compound as defined above, for the treatment of alipodystrophic syndrome, or for the manufacture of a medicament forthe treatment of a lipodystrophic syndrome. Lipodystrophicsyndromes encompass a heterogeneous group of rare disorderscharacterized by partial or generalized loss of adipose tissuedepots (Garg A., Am. J. Med. 108:143-52 (2000)). There are severaldifferent types of lipodystrophies, and the degree of fat loss mayvary from very small depressed areas to near complete absence ofadipose tissue. Some patients may have only cosmetic problems,while others may also have severe metabolic complications such asdyslipidemia, hepatic steatosis, and severe insulin resistance(Reitman M. L. et al., Trends Endocrinol. Metab. 11:410-16 (2000)).These disorders can either be inherited (familial or geneticlipodystrophies) or can occur secondary to various types ofillnesses or drugs (acquired lipodystrophies). Inheritedlipodystrophies are caused by mutations in a gene.

Several genes responsible for different types of inheritedlipodystrophies have been identified. These include AGPAT2(1-acylglycerol-3-phosphate-O-acyltransferase 2), BSCL2(Berardinelli-Seip congenital lipodystrophy 2), in CongenitalGeneralized Lipodystrophy (CGL), Lamin A/C (LMNA) gene in FamilialPartial Lipodystrophy Dunnigan variety (Familial partiallipodystrophy), and PPARG (peroxisome proliferator-activatedreceptor gamma) gene in familial partial lipodystrophy. Severalother candidate genes are currently under investigation for othervarieties of inherited lipodystrophies.

Acquired lipodystrophies are, for example, HAART (highly activeantiretroviral therapy)/HIV-induced Lipodystrophy in HIV-infectedpatients (LD-HIV), Acquired Generalized Lipodystrophy (AGL),Acquired Partial Lipodystrophy (APL), and localized lipodystrophy.Acquired lipodystrophies do not have a direct genetic basis.Rather, many mechanisms may be involved. One such mechanism may bean autoimmune response that destroys normal fat cells (Misra A. etal., Medicine (Baltimore) 83:18-34 (2004)).

HAART/HIV-induced Lipodystrophy has become the most common acquiredform of generalized Lipodystrophy. The overall incidence of thesephysical abnormalities is about 50% after 12-18 month of therapywith protease inhibitors. The difference between the presentreports range from 18% to 83% percent due to confounding factorssuch as type and duration of the retroviral therapy. It has beensuggested that the lipodystrophy syndrome associated with proteaseinhibitors may be due to partial analogy between lipid andadipocyte regulatory proteins and the catalytic site of HIV-1protease to which the protease inhibitors bind (Carr A. et al.,Lancet 351:1881-83 (1998)).

Localized lipodystrophies are defined as a localized loss ofsubcutaneous fat from small areas or from parts of a limb. Theremay be single or multiple lesions, characterized by depressed areascorresponding to the loss of subcutaneous fat. In some cases, itmay be associated with tender, painful nodules in the skin.Usually, it occurs in diabetic patients at the site of insulininjections. In some patients, fat loss occurs from areas wherepressure is applied frequently (for example, pressing a thighagainst a make-up table).

The pathogenesis of lipodystrophy is largely unknown. However,accumulating evidence points at a mitochondrial defect as one ofthe factors for an increased induction of apoptosis in theadipocytes. (Misra A. et al., Medicine (Baltimore) 83:18-34(2004)). Several proteins encoded by HIV-1 trigger apoptosis byinducing permeabilization of the mitochondrial membrane. Severalnucleoside analogs used clinically in the treatment of HIV-1inhibit the replication of mitochondrial DNA (mtDNA) and/orincrease the frequency of mtDNA mutations. Both of these factorsmay cause severe mitochondriopathy and contribute to lipodystrophy.A treatment that could inhibit the apoptosis of the adipocytescould be a very useful treatment of and especially prevention forthe development of lipodystrophy, in particular in the HIV/HAARTinduced from.

The metabolic consequences of lipodystrophy are highly importantfor the general health and the survival. The fact that insulinresistance and the consequent progression to diabetes can resultfrom either obesity or lipodystrophy reflects the crucial role ofadipose tissue in carbohydrate and lipid metabolism. In the absenceof adequate adipocyte capacity, excess calories cannot be divertedto their normal storage depot; instead they accumulate as increasedtriglyceride stores in liver, in skeletal and cardiac muscle, andin pancreatic .beta. cells. This extra-adipose lipid accumulation,through as-yet unclear means, is associated with impaired insulinaction and, often, diabetes.

In addition to their passive role as storage depots, normaladipocytes secrete a number of peptides ("adipokines") that mayinfluence insulin sensitivity and/or energy balance (Kahn B. B.& Flier J. S., J. Clin. Invest. 106:473-81 (2000); Berg A. H.et al., Trends Endocrinol. Metab. 13:84-89 (2002)). These includepotential insulin sensitizers, such as leptin and Acrp30 (alsoknown as adiponectin), and insulin antagonists, includingTNF-.alpha., IL-6, and possibly resistin. The insulin resistance oflipodystrophy may therefore be the result of disturbed lipid fluxesand/or abnormalities of adipokine secretion.

Therapy with rhGH has been reported to cause reduction in the sizeof "buffalo hump" and truncal fat in a small number of patients.However, fat loss and lipid abnormalities did not improve and bloodglucose control worsened (Lo J. C. et al., J. Clin. Endocrinol.Metab. 86:3480-87 (2001)).

Treatment of lipodystrophy using a secretagogue, such as ghrelinsplice variant or a ghrelin splice variant-like compound, may beachieved using any administration method known in the art.Preferably, treatment may be achieved using any of theadministration methods described herein, more preferably usingintravenous or subcutaneous administration, most preferably usingsubcutaneous administration methods.

Quality of Life

In all embodiments, it is preferred that the treatment methodand/or pharmaceutical compositions and/or compounds of the presentdisclosure are capable of affording the individual thus treated animproved Quality of Life (QOL), for example as is evidenced byimproved appetite and/or body weight and/or nutritional statusand/or physical function. Thus, one aspect relates to improvementsof Quality of Life using a secretagogue, such as ghrelin splicevariant or a ghrelin splice variant-like compound as definedabove.

In another embodiment, said improvement in an individual's QOL isassessed using a "Quality of Life" questionnaire, as is known toone skilled in the art.

Two validated Quality of Life surveys preferred for use inassessing improved QOL as caused by the administration of thecompounds disclosed herein are as follows: (i) the Medical OutcomesStudy Short-Form Health Survey (SF-36) and (ii) the EORTC QLQ-C30(+3) questionnaire. SF-36 contains 36 questions that assess eightaspects of the patient's QOL: physical functioning (PF),role-physical functioning (RP), bodily pain (BP), general health(GH), vitality (VT), social functioning (SF), role emotionalfunctioning (RE), and mental health (MH). According to the manualand interpretation guide, responses to questions within scales aresummed and linearly transformed to scale scores that range from 0(representing poor health status) to 100 (representing optimalhealth status). The Swedish version has been validated andnormative data have been presented for the general Swedishpopulation (Sullivan M. et al., "Halsoenkat: Svensk Manual ochTolkningsguide" (SF-36 Health Survey, Swedish Manual andInterpretation Guide), Goteborg, Sahigrenska University Hospital,1994).

EORTC QLQ-C30 (version 3.0) is a 30 item core questionnaireintended for assessment of QOL among patients (developed by theEORTC Quality of Life Study group), see the National Institutes ofHealth website and see, for example, a specimen of EORTC QLQ-C30(version 3.0, available on the EORTC website and incorporatedherein by reference). The first version has been validated incancer patients and reference data from general populations havebeen published. The questionnaire comprises five functional scales:physical functioning (five questions), role functioning (twoquestions), emotional functioning (four questions), cognitivefunctioning (two questions) and social functioning (two questions).There are three symptom scales: fatigue (three questions), nauseaand vomiting (two questions), and pain (two questions). There aresix single items on dyspnoea, insomnia, loss of appetite,constipation, diarrhea and financial difficulties. Two globalquestions are asked about the patient's health status and overallQOL. All scales and single-item measures range in score from 0 to100. A high score for the functioning scales and the global healthstatus and QOL represents a high level of functioning/health statusand QOL. A high score for the symptom/item scales represents a highlevel of symptoms/problems. The QOL scores can be calculatedaccording to the EORTC QLQ-C 30 scoring manual.

Preferred questionnaires for assessing a patient's improved qualityof life after treatment with one or more secretagogue compounds aregiven in Example 9, infra.

In preferred embodiments, treatment of a patient with condition(s)described herein results in a significant improvement in thepatient's QOL. Preferably, the treatment results in a significantincrease in QOL as measured using any method for testing the QOLincluding, but not limited to, the above mentioned questionnaires,e.g. an increase in the QOL score(s), or a composite QOL score, asappropriate for the individual measuring tool, or a decrease inscore(s) related to the symptoms and/or problems, respectively.

This increase or decrease, respectively, is preferably 1% above thescore obtained prior to initiation of the treatment, morepreferably 2% above, even more preferred 5%, such as 10%, even morepreferred 20%, 50% or 75% above the pre-treatment score. In anotherembodiment, the treatment results in measurable increases in QOLscore such that the score after treatment is equal to the averagescore found in a comparable healthy subject pool, or close to sucha "normal" score, i.e. more than 50% of the score, even morepreferably 60% of the score, or more preferably 75% of the score.Further, in another embodiment, the treatment results in a decreasein the score(s) related to the symptoms and/or problems of at least1%, more preferably 3%, even more preferably 5% or more preferred10%, 20%, 30% or 50% of the score(s) prior to initiation oftreatment. These increases or reductions, respectively, may referto one, several, or all of the aspects of the individual QOLmeasuring tool, or a composite score when appropriate.

Stimulation of Appetite, Food Intake, Weight Gain, Increase of BodyLean Mass, Increase of Body Fat Mass

As discussed above, facilitating a weight gain or facilitatingmaintenance of weight, in particular in individuals suffering froma pathological weight loss, is not only a matter of stimulatingappetite and/or food intake but rather also a correction of theimbalance between energy intake and energy consumption, i.e. totalbody metabolism. However, some individuals will still benefit fromstimulation of appetite, particularly those individuals for whom apathological process has led to a lowered appetite, which willnaturally lead to an unhealthy weight loss. Thus, one aspectrelates to the stimulation of appetite by administering asecretagogue, such as a ghrelin splice variant-like compound. Thestimulation of appetite may be measured using for instance a visualanalog scale for measuring appetite, feeling of hunger or satietylevel. In a preferred embodiment, the stimulation is at least 5%compared to prior to the treatment, such as 10% higher, morepreferably 20% higher or even more preferably 30%, 40% or 50%higher.

Stimulation of appetite does not necessarily lead to an increase infood intake, and accordingly, the present disclosure furtherrelates to another aspect: the stimulation of food intake byadministering a secretagogue, such as a ghrelin splice variant-likecompound.

Food intake can be measured using a multitude of techniquesincluding self-reporting using, e.g., diaries or questionnaires,measurements of calorie-intake from a buffet meal, using weighingof food prior to ingestion, or weighing and analysis of pairedquantities of food. Food intake may be measured on a meal basis, adaily basis, a weekly basis or a monthly basis. In a preferredembodiment, the treatment results in a 1% increase in food intake,such as an increase of 2%, more preferably 3%, or 5% or 7%, andeven more preferred 10% above average food intake prior toinitiation of treatment. In another embodiment, the treatment leadsto increase in calorie intake irrespective of changes in foodintake, since amount of food ingested may not be directly relatedto the ingested calorie intake, as the various food items such asfat, carbohydrates and proteins contain different amounts ofcalories per amount food. In a preferred embodiment, the treatmentresults in a 1% increase in calorie intake, such as an increase of2%, more preferably 3%, or 5% or 7%, and even more preferred 10% incalorie intake.

Another aspect relates to stimulation of weight gain or maintaininga stable body-weight by administering a ghrelin splice variant-likecompound.

Preferably, the secretagogue, such as a ghrelin splice variant-likecompound, is useful for stimulating food intake and weight gain.More preferably the secretagogue, such as a ghrelin splicevariant-like compound, is useful for stimulating weight gain or formaintaining stable body weight.

As discussed below, it is preferred that the secretagogue, such asa ghrelin splice variant-like compound, is administered prior to ameal, such as within 180 minutes prior to a meal, such as within150 minutes prior to a meal, such as within 120 minutes prior to ameal, such as within 100 minutes prior to a meal, such as within 80minutes prior to a meal, such as within 60 minutes prior to a meal,such as within 45 minutes prior to a meal, such as within 30minutes prior to a meal, such as within 15 minutes prior to a meal.Furthermore, it is preferred that the ghrelin splice variant-likecompound is administered subcutaneously.

Furthermore, a secretagogue, such as a ghrelin splice variant-likecompound may be administered to facilitate maintenance of physicalfunctioning and/or facilitate recovery of physical function, forexample in individuals recovering from major surgeries, such asinsertion of a hip prosthesis, amputations, bone fractures, andopen heart surgery.

In particular, a secretagogue, such as a ghrelin splicevariant-like compound, is useful for treatment of underweightsubjects, or for preventing loss of weight to a stage ofunderweight. Underweight subjects include those having a bodyweight about 3%, 5% or less, 10% or less, 20% or less, or 30% orless, than the lower end of "normal" weight range or BMI. "Normal"weight ranges are well known in the art and take into accountfactors such as a patient age, height, and body type. Furthermore,the compounds disclosed herein are suitable for treating patientswho have experienced an involuntary weight-loss prior tocommencement of treatment, such as a weight-loss of 1% per month,2% or less per month, or 5% for less per 6 months.

Increasing weight or appetite can be useful for a patient having adisease or undergoing treatment that affects weight or appetite. Inaddition, for example, farm animals such as pigs, cows and chickenscan be treated to gain weight.

An increase in the body fat mass of an individual can be readilyassessed by the skilled person using a number of known techniques.One embodiment relates to an increase in body fat mass without theindividual gaining weight overall. A preferred embodiment leads toan increase in body fat of 2% compared to prior to the initiationof treatment, more preferably 4%, such as 5%, and 8% and 10%, evenmore preferably 20% or 40% above pre-treatment values.

Further conditions of an individual capable of being treatable inaccordance with the present disclosure are bulimia nervosa,anorexia, male erectile dysfunction, female sexual dysfunction,amelioration of ischemic nerve or muscle damage, as well assystemic lupus erythematosus.

Subcutaneous Administration

It is important to note that ghrelin and ghrelin splice variantactivate the GHS receptors and additional yet to be identifiedreceptors. These receptors are found on GH producing cells, in thehypothalamic centers for appetite control and in a number ofadditional places in the organism. In the CNS, these receptors aretuned to receiving signals from local ghrelin splicevariant-containing neurons. Peripherally-secreted orartificially-administered ghrelin splice variants will reach suchsites and will pass the blood brain barrier specifically activatingthe appropriate receptors and triggering a specific pathway.However, currently available "so-called" GH secretagogues, whichare small organic compounds such as MK-0677 (Merck), generallytargeted to bind the GHS receptor will pass the blood brain barrierand also reach these sites, activating various GHS receptor relatedpathways and consequently having the danger of causing unwantedside effects such as dizziness, nausea, falling, elevated fastingserum glucose and insulin, and blurred vision. Thus, such compoundswhich do have the advantage of being, for example, orally activewill not be optimal for mimicking the natural pre-meal,appetite-inducing surge of ghrelin splice variant, since they willactivate non-specifically all GHS receptor related pathways in thebody. In contrast, by using the natural peptide, ghrelin splicevariant itself, or homologues thereof, and administering itperipherally--as in a preferred embodiment--it is ensured that onlythe relevant, appetite-regulating ghrelin splice variant receptorsand pathways are reached and stimulated.

Any parenteral administration form that will ensure that theghrelin splice variant receptors which normally are the target forperipherally-produced ghrelin splice variant in the pre-mealsituation will be exposed to sufficient levels of the bioactiveform of ghrelin splice variant to ensure robust and appropriateappetite stimulation, without causing desensitization of thesystem, may be part of an embodiment of the present disclosure.However, taking into consideration that the individuals to betreated possibly will have to receive treatment for a longerperiod, such as weeks or months, it is preferred that theadministration form is well-suited thereto. Accordingly, it ispreferred that the secretagogue, such as a ghrelin splicevariant-like compound, is administered subcutaneously in an amountallowing sufficient levels of the bioactive form of ghrelin splicevariant, i.e. the acylated form, to reach the receptors prior tothe forthcoming meal.

The present disclosure preferably deals with methods foradministering a secretagogue, such as ghrelin splice variant, in away which mimics the physiologically pre-meal situation as closelyas possible yet provides patients in need of increased food intake,for example fragile elderly, post-operative patients, and/orpatients with lost appetite as part of cachexia for exampleprecipitated by cancer, cardiac disease, etc., with a sufficientextra stimulatory input to their appetite-regulating ghrelin splicevariant receptors, which normally are reached by ghrelin splicevariant in the pre-meal situation.

Bolus Administration

Furthermore, from a molecular pharmacological point-of-view, it isimportant to note that it has been found that the ghrelin receptor,and therefore ghrelin splice variant receptor, normally is exposedto short-lived surges in ghrelin concentration. The GHS-R 1areceptor (growth hormone secretagogue receptor 1a) belongs to theclass of G protein coupled receptors or 7.TM. receptors, which uponcontinued exposure to an agonist will be desensitized,internalized, and down-regulated. These mechanisms, which areinherent to the overall signal transduction system, involveprocesses such as receptor phosphorylation (which, in itself,decreases the affinity of the receptor for the agonist) and bindingof inhibitory proteins such as arrestin (which sterically block thebinding of signal transduction molecules such as G proteins).Another part of the agonist-mediated desensitization process isreceptor internalization (i.e. physical removal of the receptorfrom the cell surface where it could bind the agonist) as well asreceptor down regulation (i.e. decreased production/expression ofthe receptor). Receptor internalization could, after short-livedexposure of the receptor to agonist, be followed by are-sensitization process, where the receptor is dephosphorylatedand recycled to the cell surface to be used again. Without beingbound by theory, it is believed that, upon prolonged stimulationwhich would occur for example during a long-lasting continuousinfusion of the agonist, the receptor down-regulation processensures that the target cell is adjusted in its signal transductionsystem to this situation.

Optimal Administration

The present disclosure also provides a procedure for an optimaladministration of ghrelin splice variant to patients in order toobtain a maximal response and to avoid, for example,desensitization mechanisms.

Accordingly, one aspect relates to administration of asecretagogue, such as a ghrelin splice variant-like compound, inboluses, preferably a bolus prior to each main meal. It has beenfound, in contrary to the prolonged administration processes in theprior art, that a bolus administration leads not only tostimulation of appetite, but also to stimulation of feed intake andmore important to stimulation of weight gain. Without being boundby theory, it is believed that pre-meal subcutaneous injection,intravenous injection, or short-term infusions of appropriate dosesof a secretagogue, such as ghrelin splice variant or a ghrelinsplice variant-like compound, will ensure that a robust stimulationof appetite-inducing ghrelin splice variant receptors will beobtained with minimal constraint to, e.g., the mobility of thepatient. Thus, for example, patients with hip fractures can, in thepost-operative situation, be treated in the pre-meal period and, ifrequired during the meal as such, be free to move around andparticipate in the important post-operative physicotherapeuticregimens. In one preferred embodiment, a secretagogue such asghrelin splice variant or a ghrelin splice variant-like compound isadministered as a bolus in an amount equivalent to 10 .mu.g per kgbody weight.

Ghrelin Splice Variant-Like Compound

Any secretagogue, such as ghrelin splice variant or a ghrelinsplice variant-like compound, may be used in the presentlydisclosed methods. One preferred type of ghrelin splicevariant-like compound described herein is a compound comprising astructure defined by Formula I: Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1is an optionally present protecting group; each X1 is independentlyselected from a naturally occurring amino acid and a syntheticamino acid; X2 is selected from a naturally occurring amino acidand a synthetic amino acid, said amino acid being modified with abulky hydrophobic group; each X3 is independently selected from anaturally occurring amino acid and a synthetic amino acid, whereinone or more of X1 and X3 optionally may be modified with a bulkyhydrophobic group; Z2 is an optionally present protecting group; mis an integer in the range of from 1-10; n is an integer in therange of from 4-92; provided that the compound according to formulaZ1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has atleast 80% (or, in alternative embodiments, 85%, 90%, 93%, 95%, 97%,98%, 99%, 100%) homology to SEQ ID NO:1. In a preferred embodiment,the ghrelin splice variant-like compound is 22-29 amino acids inlength.

Accordingly, the term "secretagogue" includes the naturallyoccurring 29 amino acid human ghrelin splice variant, the aminoacid sequence of which is shown in SEQ ID NO:5, as well as thenaturally occurring 22 amino acid human ghrelin splice variant, theamino acid sequence of which is shown in SEQ ID NO:2, and thenaturally occurring 24 amino acid human ghrelin splice variant, thesequence of which is shown in SEQ ID NO:3.

The present disclosure includes diastereomers as well as theirracemic and resolved enantiomerically pure forms. Secretagogues cancontain D-amino acids, L-amino acids, alpha-amino acid, beta-aminoacid, gamma-amino acid, natural amino acid and/or synthetic aminoacid or the like or a combination thereof. Preferably, amino acidspresent in a ghrelin splice variant-like compound are theL-enantiomers.

The ghrelin splice variant-like compound preferably comprises anamino acid modified with a bulky hydrophobic group. The number ofamino acids N-terminal to the modified amino acid is preferablywithin the range of from 1-9. Accordingly, m is preferably aninteger in the range of from 1-9, such as of from 1-8, such as offrom 1-7, such as of from 1-6, such as of from 1-5, such as of from1-4, such as of from 1-3, such as of from 1-2, such as 2.

It is more preferred that the number of amino acids N-terminally tothe modified amino acid is low, such as of from 1-3, such as offrom 1-2. Most preferably 2 amino acids are positioned N-terminalto the modified amino acid.

In a preferred embodiment, (X1)m has a Gly residue in theN-terminal part of the sequence. Accordingly, in preferredembodiment, (X1)m is selected from the sequences: Gly, Gly-Ser,Gly-Cys, Gly-Lys, Gly-Asp, Gly-Glu, Gly-Arg, Gly-His, Gly-Asn,Gly-Gln, Gly-Thr, and Gly-Tyr. More preferably (X1)m is selectedfrom Gly-Ser, and Gly-Cys; most preferably (X1)m is Gly-Ser.

In other words, in a preferred embodiment the ghrelin splicevariant-like compound is selected from Z1-Gly-(X1)m-(X2)-(X3)n-Z2(Formula II), Z1-Gly-Ser-(X2)-(X3)n-Z2 (Formula II), andZ1-Gly-(X2)-(X3)n-Z2 (Formula IV). More preferably, the ghrelinsplice variant-like compound has Formula III.

As described above, X2 may be any amino acid modified with a bulkyhydrophobic group. In particular, X2 is selected from the groupconsisting of modified Ser, modified Cys, modified Asp, modifiedLys, modified Trp, modified Phe, modified Ile, and modified Leu.More preferably, X2 is selected from the group consisting ofmodified Ser, modified Cys, and modified Lys; most preferably X2 ismodified Ser.

Furthermore, (X1)m-(X2) is preferably Gly-Xaa-Ser* or Gly-Xaa-Cys*,wherein Xaa is any amino acid, more preferably (X1)m-(X2) isGly-Ser-Ser* or Gly-Ser-Cys*, wherein * indicates that the aminoacid residue is modified with a bulky hydrophobic group.

(X3)n preferably comprises a sequence which is a fragment ofghrelin splice variant, such as human ghrelin splice variant.Accordingly, (X3)n preferably comprises a fragment of the followingsequence (SEQ ID NO:6): Phe Leu Ser Pro Glu His Gln Arg Val Gln ValArg Pro Pro His Lys Ala Pro His Val Val Pro Ala Leu Pro Leu Ser AsnGln Leu Cys Asp Leu Glu Gln Gln Arg His Leu Trp Ala Ser Val Phe SerGln Ser Thr Lys Asp Ser Gly Ser Asp Leu Thr Val Ser Gly Arg Thr TrpGly Leu Arg Val Leu Asn Arg Leu Phe Pro Pro Ser Ser Arg Glu Arg SerArg Arg Ser His Gln Pro Ser Cys Ser Pro Glu Leu.

In a preferred embodiment, the length of the ghrelin splicevariant-like compound is substantially similar to the length ofprocessed human ghrelin, i.e. 29 amino acids. Accordingly, n ispreferably an integer in the range of from 4 to 25, such as of from4 to 24, such as of from 4 to 22, such as of from 4 to 15, such asof from 4 to 10, such as of from 10 to 25, such as of from 10 to24, such as of from 15 to 25, such as of from 15 to 24. Mostpreferably, a ghrelin splice variant-like compound is the 29 aminoacid human ghrelin splice variant, the amino acid sequence of whichis shown in SEQ ID NO:5; is the 22 amino acid human ghrelin splicevariant, the amino acid sequence of which is shown in SEQ ID NO:2;is the 24 amino acid human ghrelin splice variant, the amino acidsequence of which is shown in SEQ ID NO:3; or is the 24 amino acidhuman ghrelin splice variant having a 2,3-diaminopropionic acid(Dpr) residue in the third position, the amino acid sequence ofwhich is shown in SEQ ID NO:4.

Functionality

The secretagogues described herein are active at the receptor forGHS as described above, i.e. the receptor GHS-R 1a. The compoundscan bind to GHS-R 1a, and preferably, stimulate receptor activity.In some embodiments, the compounds can bind other receptors and,optionally, stimulate their activity.

Receptor activity can be measured using different techniques suchas detecting a change in the intracellular conformation of thereceptor, in the G-protein coupled activities, and/or in theintracellular messengers. One simple measure of the ability of aghrelin splice variant-like compound to activate the ghrelin splicevariant receptor is to measure its EC50, i.e. the dose at which thecompound is able to activate the signaling of the receptor to halfof the maximal effect of the compound. When measuring, e.g., EC50,the receptor can either be expressed endogenously on primary cellcultures, for example pituitary cells, or heterologously expressedon cells transfected with the ghrelin receptor. Whole cell assaysor assays using membranes prepared from either of these cell typescan be used depending on the type of assay.

As the receptor is generally believed to be primarily coupled tothe Gq signaling pathway, any suitable assay which monitorsactivity in the Gq/G11 signaling pathway can be used, for example:1) An assay measuring the activation of Gq/G11 performed, forexample, by measurement of GTPgS binding combined with, e.g.,anti-G-alpha-q or -11 antibody precipitation in order to increasethe signal to noise ratio. This assay may also detect coupling toother G-proteins than Gq/11. 2) An assay which measures theactivity of phospholipase C (PLC), one of the first down-streameffector molecules in the pathway, for example by measuring theaccumulation of inositol phosphate which is one of the products ofPLC. 3) More down stream in the signaling cascade is themobilization of calcium from the intracellular stores, which can bemeasured by any method known to one of ordinary skill in the art.4) Even more down stream, signaling molecules such as the activityof different kinds of MAP kinases (p38, jun, etc.), NF.kappa.Btranslocation and CRE driven gene transcription may also bemeasured. 5) Binding of fluorescently-tagged arrestin to theactivated ghrelin receptor.

Examples of suitable protocols for use in determining secretagoguefunctionality are given in Example 4, infra.

In one embodiment, the binding of a compound to the receptor GHS-R1a can be measured by the use of the assay described hereinabove.

A ghrelin splice variant-like compound preferably has at leastabout 50%, at least about 60%, at least about 70%, at least about80%, or at least about 90%, functional activity relative to the 28amino acid human wild-type ghrelin as determined using an assaydescribed herein above, and/or an EC50 greater than about 1,000,greater than about 100, or greater than about 50, or greater thanabout 10. Greater refers to potency and thus indicates a lesseramount is needed to achieve binding inhibition.

In one embodiment, the compound has potency (EC50) on the GHS-R 1aof less than 500 nM. In another embodiment the compound has apotency (EC50) on the GHS-R 1a of less than 100 nM, such as lessthan 80 nM, such as less than 60 nM, such as less than 40 nM, suchas less than 20 nM, such as less than 10 nM, such as less than 5nM, such as less than 1 nM, such as less than 0.5 nM, such as lessthan 0.1 nM, such as less than 0.05 nM, such as less than 0.01nM.

In a further embodiment, the dissociation constant (Kd) of thecompound is less than 500 nM. In a still further embodiment thedissociation constant (Kd) of the ligand is less than 100 nM, suchas less than 80 nM, such as less than 60 nM, such as less than 40nM, such as less than 20 nM, such as less than 10 nM, such as lessthan 5 nM, such as less than 1 nM, such as less than 0.5 nM, suchas less than 0.1 nM, such as less than 0.05 nM, such as less than0.01 nM.

Binding assays can be performed using recombinantly-producedreceptor polypeptides present in different environments. Suchenvironments include, for example, cell extracts and purified cellextracts containing the receptor polypeptide expressed fromrecombinant nucleic acid or naturally occurring nucleic acid, andalso include, for example, the use of a purified GHS receptorpolypeptide produced by recombinant means or from naturallyoccurring nucleic acid which is introduced into a differentenvironment.

Using a recombinantly expressed GHS receptor offers severaladvantages, such as the ability to express the receptor in adefined cell system, so that a response to a compound at thereceptor can more readily be differentiated from responses at otherreceptors. For example, the receptor can be expressed in a cellline such as HEK 293, COS 7, and CHO not normally expressing thereceptor by an expression vector, wherein the same cell linewithout the expression vector can act as a control.

Identity and Homology

The term "identity" or "homology" shall be construed to mean thepercentage of amino acid residues in the candidate sequence thatare identical with the residue of a corresponding sequence to whichit is compared, after aligning the sequences and introducing gaps,if necessary to achieve the maximum percent identity for the entiresequence, and not considering any conservative substitutions aspart of the sequence identity. Neither N- or C-terminal extensionsnor insertions shall be construed as reducing identity or homology.Methods and computer programs for the alignment are well known inthe art. Sequence identity may be measured using sequence analysissoftware (e.g., Sequence Analysis Software Package, GeneticsComputer Group, University of Wisconsin Biotechnology Center,Madison, Wis.). This software matches similar sequences byassigning degrees of homology to various substitutions, deletions,and other modifications.

A ghrelin splice variant homologue of one or more of the sequencesspecified herein may vary in one or more amino acids as compared tothe sequences defined, but is capable of performing the samefunction, i.e. a homologue may be envisaged as a functionalequivalent of a predetermined sequence.

A ghrelin splice variant homologue is preferably a ghrelin splicevariant-like compound as defined above.

As described above, a homologue of any of the predeterminedsequences herein may be defined as i) homologues comprising anamino acid sequence capable of being recognized by an antibody,said antibody also recognizing the 22 amino acid and/or the 24amino acid human ghrelin splice variant and/or the 29 amino acidhuman ghrelin splice variant, preferably the acylated 22 amino acidand/or the 24 amino acid human ghrelin splice variant and/or the 29amino acid human ghrelin splice variant, and/or ii) homologuescomprising an amino acid sequence capable of binding selectively toGHS-R 1a, and/or iii) homologues having a substantially similar orhigher binding affinity to GHS-R 1a than the 22 amino acid and/orthe 24 amino acid human ghrelin splice variant and/or the 29 aminoacid human ghrelin splice variant, preferably the acylated 22 aminoacid and/or the 24 amino acid human ghrelin splice variant and/orthe 29 amino acid human ghrelin splice variant. (The 22 amino acidand/or the 24 amino acid and/or the 29 amino acid human ghrelinsplice variant can have the sequence shown in SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, or SEQ ID NO:5, and, when acylated, is acylatedat position 3.)

The antibodies used herein may be antibodies binding the N-terminalregion of ghrelin splice variant or the C-terminal region ofghrelin splice variant, preferably the N-terminal region. Theantibodies may be antibodies as described in Ariyasu H. et al.,Endocrinology 143:3341-50 (2002).

Exemplary homologues comprise one or more conservative amino acidsubstitutions including one or more conservative amino acidsubstitutions within the same group of predetermined amino acids,or a plurality of conservative amino acid substitutions, whereineach conservative substitution is generated by substitution withina different group of predetermined amino acids. Homologues may thuscomprise conservative substitutions independent of one another,wherein at least one glycine (Gly) of said homologue is substitutedwith an amino acid selected from the group of amino acidsconsisting of Ala, Val, Leu, and Ile, and independently thereof,homologues, wherein at least one of said alanines (Ala) of saidhomologue thereof is substituted with an amino acid selected fromthe group of amino acids consisting of Gly, Val, Leu, and Ile; and,independently thereof, homologues wherein at least one valine (Val)of said homologue thereof is substituted with an amino acidselected from the group of amino acids consisting of Gly, Ala, Leu,and Iie; and, independently thereof, homologues wherein at leastone of said leucines (Leu) of said homologue thereof is substitutedwith an amino acid selected from the group of amino acidsconsisting of Gly, Ala, Val, and Iie; and, independently thereof,homologues wherein at least one isoleucine (Iie) of said homologuesthereof is substituted with an amino acid selected from the groupof amino acids consisting of Gly, Ala, Val and Leu; and,independently thereof, homologues wherein at least one of saidaspartic acids (Asp) of said homologue thereof is substituted withan amino acid selected from the group of amino acids consisting ofGlu, Asn, and Gln; and, independently thereof, homologues whereinat least one of said phenylalanines (Phe) of said homologuesthereof is substituted with an amino acid selected from the groupof amino acids consisting of Tyr, Trp, His, and Pro, and preferablyselected from the group of amino acids consisting of Tyr and Trp;and, independently thereof, homologues wherein at least one of saidtyrosines (Tyr) of said homologues thereof is substituted with anamino acid selected from the group of amino acids consisting ofPhe, Trp, His, and Pro, preferably an amino acid selected from thegroup of amino acids consisting of Phe and Trp; and, independentlythereof, homologues wherein at least one of said arginines (Arg) ofsaid fragment is substituted with an amino acid selected from thegroup of amino acids consisting of Lys and His; and, independentlythereof, homologues wherein at least one lysine (Lys) of saidhomologues thereof is substituted with an amino acid selected fromthe group of amino acids consisting of Arg and His; and,independently thereof, homologues wherein at least one of saidasparagines (Asn) of said homologues thereof is substituted with anamino acid selected from the group of amino acids consisting ofAsp, Glu, and Gln; and, independently thereof, homologues whereinat least one glutamine (Gln) of said homologues thereof issubstituted with an amino acid selected from the group of aminoacids consisting of Asp, Glu, and Asn; and, independently thereof,homologues wherein at least one proline (Pro) of said homologuesthereof is substituted with an amino acid selected from the groupof amino acids consisting of Phe, Tyr, Trp, and His; and,independently thereof, homologues wherein at least one of saidcysteines (Cys) of said homologues thereof is substituted with anamino acid selected from the group of amino acids consisting ofAsp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr, and Tyr.

Conservative substitutions may be introduced in any position of apreferred predetermined sequence. It may however also be desirableto introduce non-conservative substitutions, particularly, but notlimited to, a non-conservative substitution in any one or morepositions.

A non-conservative substitution leading to the formation of afunctionally equivalent homologue of the sequences herein would forexample i) differ substantially in polarity, for example a residuewith a non-polar side chain (Ala, Leu, Pro, Trp, Val, Ile, Gly,Leu, Phe or Met) substituted for a residue with a polar side chainsuch as Ser, Thr, Cys, Tyr, Asn, or Gln or a charged amino acidsuch as Asp, Glu, Arg, or Lys, or substituting a charged or a polarresidue for a non-polar one; and/or ii) differ substantially in itseffect on polypeptide backbone orientation such as substitution ofor for Pro or Gly by another residue; and/or iii) differsubstantially in electric charge, for example substitution of anegatively charged residue such as Glu or Asp for a positivelycharged residue such as Lys, His or Arg (and vice versa); and/oriv) differ substantially in steric bulk, for example substitutionof a bulky residue such as His, Trp, Phe or Tyr for one having aminor side chain, e.g. Ala, Gly or Ser (and vice versa).

Substitution of amino acids may in one embodiment be made basedupon their hydrophobicity and hydrophilicity values and therelative similarity of the amino acid side-chain substituents,including charge, size, and the like. Exemplary amino acidsubstitutions which take various of the foregoing characteristicsinto consideration are well known to those of skill in the art andinclude, for example, arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine,leucine, and isoleucine.

In a preferred embodiment, the binding domain comprises a homologuehaving an amino acid sequence at least 60% homologous to SEQ IDNO:1. More preferably the homology is at least 65%, such as atleast 70% homologous, such as at least 75% homologous, such as atleast 80% homologous, such as at least 85% homologous, such as atleast 90% homologous, such as at least 95% homologous, such as atleast 98% homologous to SEQ ID NO:1. In a more preferredembodiment, the percentages mentioned above relate to the identityof the sequence of a homologue as compared to SEQ ID NO:1.Homologues to SEQ ID NO:1 may be 22 amino acid human ghrelin splicevariant (SEQ ID NO:2), 24 amino acid human ghrelin splice variant(SEQ ID NO:3), or 29 amino acid human ghrelin splice variant (SEQID NO:5). Other homologues are the variants described in EP 1 197496 (Kangawa), incorporated herein by reference.

Bulky Hydrophobic Group

The bulky hydrophobic group of secretagogues disclosed herein isany bulky hydrophobic group capable of providing the des-acylated28 amino acid human wild-type ghrelin, or an analogue thereof, withbinding affinity to GHS-R 1a and or GS-R-1b. Any suitable aminoacid may be modified with any suitable bulky hydrophobic group. Ina preferred embodiment, a Ser residue (preferably, amino acidnumber 3 in the ghrelin splice variant amino acid chain) ismodified with the bulky hydrophobic group. When the amino acidbeing modified contains, e.g., --OH, --SH, --NH or --NH.sub.2 as asubstituent group in a side chain thereof, a group formed byacylating such a substituent group is preferred. The mode oflinkage may thus be selected from the group consisting of ester,ether, thioester, thioether, amide and carbamide. For example, ifthe modified amino acid is serine, threonine, tyrosine oroxyproline, the amino acid has a hydroxyl group in the side chain.If the modified amino acid is cysteine, the amino acid has amercapto group in the side chain. If the modified amino acid islysine, arginine, histidine, tryptophan, proline or oxyproline, ithas an amino group or imino group in the side chain.

The hydroxyl group, mercapto group, amino group and imino groupdescribed above may thus be chemically modified. That is, thehydroxyl group or mercapto group may be, for example, etherized,esterified, thioetherified or thioesterified. The imino group maybe, for example, iminoetherified, iminothioetherified or alkylated.The amino group may be, for example, amidated, thioamidated orcarbamidated. Further, the mercapto group may be, for example,disulfidated; the imino group may be, for example, amidated orthioamidated; and the amino group may be, for example, alkylated orthiocarbamidated.

In a preferred embodiment, the modified amino acid is Ser coupledthrough an ester linkage to the hydrophobic group or, in anotherpreferred embodiment, the modified amino acid is Dpr coupledthrough an amide linkage to the hydrophobic group.

The hydrophobic group may be any group with a saturated orunsaturated alkyl or acyl group containing one or more carbonatoms. In one embodiment, the bulky hydrophobic group is an acylgroup, including groups formed by removing a hydroxyl group from anorganic carboxylic acid, organic sulfonic acid or organicphosphoric acid. The organic carboxylic acid includes, e.g., fattyacids, and the number of carbon atoms thereof is preferably 1 to35. In the organic sulfonic acid or organic phosphoric acid, thenumber of carbon atoms thereof is preferably 1 to 35.

Accordingly, the acyl group is preferably selected from aC.sub.1-C.sub.35 acyl group, such as a C.sub.1-C.sub.20 acyl group,such as a C.sub.1-C.sub.15 acyl group, such as a C.sub.6-C.sub.15acyl group, such as a C.sub.6-C.sub.12 acyl group, such as aC.sub.8-C.sub.12 acyl group. More preferably, the acyl group isselected from the group consisting of C.sub.7 acyl group, C.sub.8acyl group, C.sub.9 acyl group, C.sub.10 acyl group, C.sub.11 acylgroup, and C.sub.12 acyl group. Such acyl group may be formed fromoctanoic acid (preferably caprylic acid), decanoic acid (preferablycapric acid), or dodecanoic acid (preferably lauric acid), as wellas monoene or polyene fatty acids thereof.

Furthermore, the modified amino acid may be any amino acid whereina group is modified as described in EP 1 197 496 (Kangawa), whichis hereby incorporated by reference.

Protecting Group

The ghrelin splice variant-like compound according to the presentdisclosure may comprise a protecting group at the N-terminus or theC-terminus or at both. A protecting group covalently joined to theN-terminal amino group reduces the reactivity of the amino terminusunder in vivo conditions. Amino protecting groups include, forexample, C.sub.1-C.sub.10 alkyl, C.sub.1-C.sub.10 substitutedalkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 substitutedalkenyl, aryl, C.sub.1-C.sub.6 alkyl aryl,C(O)--(CH.sub.2)--(C.sub.1-C.sub.6 alkyl)-COOH,C(O)--(C.sub.1-C.sub.6 alkyl), C(O)-aryl,C(O)--O--(C.sub.1-C.sub.6-alkyl), or C(O)--O-aryl. Preferably, theamino terminus protecting group is acetyl, propyl, succinyl,benzyl, benzyloxycarbonyl or t-butyloxycarbonyl.

A protecting group covalently joined to the C-terminal carboxygroup reduces the reactivity of the carboxy terminus under in vivoconditions. The carboxy terminus protecting group is preferablyattached to the a-carbonyl group of the last amino acid. Carboxyterminus protecting groups include, for example, amide,methylamide, and ethylamide.

Conjugates

The secretagogue, such as a ghrelin splice variant-like compound,may be provided in the form of a secretagogue conjugate, i.e. amolecule comprising the secretagogue conjugated to another entity.The other entity may be any substance that is capable of conferringimproved properties to the secretagogue, e.g. in terms of improvedstability, half-life, etc. Examples of suitable entities aredescribed in the following. For example, the secretagogue may beconjugated to a peptide, such as a peptide having effect onnociceptin receptor ORL1. In one embodiment, the conjugate is aconjugate of ghrelin splice variant or a derivative or homologuethereof and a peptide having effect on ORL1, e.g. the peptideAc-RYY (RK) (WI) RK)-NH.sub.2, where the brackets show allowablevariation of amino acid residues. Examples of other suitablepeptides are found in Published U.S. Patent Application Nos.2003/040472 and 2002/004483, and U.S. Pat. No. 5,869,046, each ofwhich is incorporated herein by reference.

In another embodiment, a secretagogue, such as ghrelin splicevariant or a ghrelin splice variant-like compound, is conjugated toa polymer molecule. The polymer molecule may be any suitablepolymer molecule, such as a natural or synthetic polymer, typicallywith a molecular weight in the range of about 1-100 kDa, such asabout 3-20 kDa, such as 5-10 kDa. The polymer is attached to areactive group present on the secretagogue, e.g. an amine group ora thiol group. Examples of suitable polymer molecules includepolymer molecules selected from the group consisting ofpolyalkylene oxide (PAO), including polyalkylene glycol (PAG), suchas linear or branched polyethylene glycol (PEG) and polypropyleneglycol (PPG); poly-vinyl alcohol (PVA); poly-carboxylate;poly-(vinylpyrolidone); polyethylene-co-maleic acid anhydride;polystyrene-co-maleic acid anhydride; and dextran, includingcarboxymethyl-dextran. Preferably, the polymer molecule is a PEGmolecule, in particular a monofunctional PEG, such asmethoxypolyethylene glycol (mPEG). Suitable activated PEG moleculesare available from, for example, Nektar Therapeutics Inc.(Huntsville, Ala.) or Valentis, Inc. (Burlingame, Cal.).Alternatively, the polymer molecules can be activated byconventional methods known in the art, e.g., as disclosed in WO90/13540, incorporated herein by reference. Specific examples ofactivated PEG polymers include the following linear PEGs: NHS-PEG(e.g., SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG,and SCM-PEG), NOR-PEG, BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG,CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, andbranched PEGs such as PEG2-NHS and those disclosed in U.S. Pat.Nos. 5,932,462 and 5,643,575, both of which are incorporated hereinby reference.

The PEGylation (i.e., conjugation of the secretagogue polypeptideand the activated polymer molecule) is conducted in accordance withestablished procedures, e.g., as described in the followingreferences (which also describe suitable methods for activation ofpolymer molecules): R. F. Taylor, (1991), "Protein Immobilization.Fundamentals and Applications", Marcel Dekker, N.Y.; S. S. Wong,(1992), "Chemistry of Protein Conjugation and Crosslinking", CRCPress, Boca Raton; G. T. Hermanson et al., (1993), "ImmobilizedAffinity Ligand Techniques", Academic Press, N.Y.).

It is also contemplated according to the present disclosure tocouple the polymer molecules to the secretagogue through a linker.Suitable linkers are well known to the skilled person. A preferredexample is cyanuric chloride (Abuchowski A. et al., J. Biol. Chem.252:3578-81 (1977); U.S. Pat. No. 4,179,337; Shafer et al., J.Polym. Sci. Polym. Chem. Ed. 24:375-78 (1986)).

In yet another embodiment, the secretagogue is conjugated to anoligosaccharide molecule, such as dextran, glycan, transferrin,etc. Such conjugation may be achieved in accordance withestablished technologies, e.g. those available from NeoseTechnologies, Inc. (Horsham, Pa.).

In yet another embodiment, the secretagogue is conjugated to an Fcregion of an IgG molecule, typically in the form of a fusionprotein. For instance, a salvage receptor binding epitope of the Fcregion of an IgG (i.e. the Fc portion of an immunoglobulin of theisotype IgG) is incorporated into the secretagogue so as toincrease its circulatory half-life, but so as not to lose itsbiological activity. This can take place by any means, such as bymutation of the appropriate region in the secretagogue to mimic theFc region or by incorporating the epitope into a peptide tag thatis then fused to the secretagogue at either end or in the middle orby DNA or peptide synthesis.

The salvage receptor binding epitope is any suitable such epitopeas known to the person skilled in the art, and its nature willdepend, e.g., on the type of secretagogue being modified. Theepitope is introduced into the secretagogue such that thebiological activity of the secretagogue is maintained, i.e., theepitope does not adversely affect the conformation of thesecretagogue or affect its binding to ligands that confers itsbiological activity.

Alternatively to providing the secretagogue in the form of aconjugate, the secretagogue may be modified to include suitablereactive groups, whereby the thus modified secretagogue is capableof forming a conjugate in vivo (after having been administered toan individual) through covalent bonding with available reactivefunctionalities on blood components. The present disclosure alsorelates to such modified secretagogues, and methods for their use.Also, the present disclosure relates to conjugates formed in vitrobetween a modified secretagogue as described above and a bloodcomponent. The conjugates formed in accordance with this embodimentare contemplated to have an increased in vivo half life as comparedto the corresponding non-modified secretagogue.

In accordance with this embodiment, the secretagogue is modifiedwith a chemically reactive group (reactive entity). The reactiveentity may, e.g., be selected from the wide variety of activecarboxyl groups, particularly esters, where the hydroxyl moiety isphysiologically acceptable. Such groups may be selected from thegroup consisting of N-hydroxysuccinimide (NHS),N-hydroxy-sulfosuccinimide (sulfo-NHS),maleimide-benzoyl-succinimide (MBS), gamma-maleimido-butyryloxysuccinimide ester (GMBS) and maleimidopropionic acid (MPA). Theprincipal targets for this group of entities are primary amines onthe blood component. Another group of active entities isconstituted by a maleimido-containing group such as MPA andgamma-maleimide-butrylamide (GMBA). Such groups react with thiolgroups present on the blood component (Lee V. H. L. in "Peptide andProtein Drug Delivery", New York, N.Y., M. Dekker, 1990).

The blood component with which the modified secretagogue isdesigned to react may be any blood component having an availabletarget group, e.g. an amine or a thiol group, and which is suitableas a carrier for binding the modified secretagogue in vivo andthereby extend the circulating half-life thereof. Examples of suchblood components are serum albumin and IgG.

As mentioned above, the covalent bonding of a modified secretagogueto a blood component may be achieved in vivo by administration ofthe modified secretagogue directly to the patient. Theadministration may be done in any suitable form, such as in theform of a bolus or introduced slowly over time by infusion usingmetered flow or the like. Alternatively, the secretagogue/bloodcomponent conjugate may also be prepared ex vivo by combining bloodwith the modified secretagogue, allowing covalent bonding of themodified secretagogue to reactive functionalities on bloodcomponents and then returning or administering the conjugated bloodto the individual.

Moreover, the above may also be accomplished by first purifying anindividual blood component or limited number of components, such asred blood cells, immunoglobulins, serum albumin, or the like, andcombining the component or components ex vivo with the chemicallyreactive secretagogues.

Production of Ghrelin Splice Variant-Like Compounds

Ghrelin splice variant-like compounds can be produced usingtechniques well known in the art. For example, a polypeptide regionof a ghrelin splice variant-like compound can be chemically orbiochemical synthesized and modified. Techniques for chemicalsynthesis of polypeptides are well known in the art (see, e.g., LeeV. H. L. in "Peptide and Protein Drug Delivery", New York, N.Y., M.Dekker, 1990). Examples of techniques for biochemical synthesisinvolving the introduction of a nucleic acid into a cell andexpression of nucleic acids are provided in Ausubel F. M. et al.,"Current Protocols in Molecular Biology", John Wiley, 1987-1998,and Sambrook J. et al., "Molecular Cloning, A Laboratory Manual",2d Edition, Cold Spring Harbor Laboratory Press, 1989, each ofwhich is incorporated herein by reference. Another exemplarytechnique, described in U.S. Pat. No. 5,304,489, incorporatedherein by reference, is the use of a transgenic mammals havingmammary gland-targeted mutations which result in the production andsecretion of synthesized ghrelin splice variant-like compound inthe milk of the transgenic mammal.

The ghrelin splice variant-like compounds can also be recombinantlyproduced using routine expression methods known in the art. Thepolynucleotide encoding the desired ghrelin splice variant-likecompound is operably linked to a promoter into an expression vectorsuitable for any convenient host. Both eukaryotic and prokaryotichost systems are used in forming recombinant ghrelin splicevariant-like compounds. The ghrelin splice variant-like compound isthen isolated from lysed cells or from the culture medium andpurified to the extent needed for its intended use.

Consequently, a further embodiment is for a method of producing aghrelin splice variant-like compound, said method comprising thesteps of: (a) providing a cDNA comprising a polynucleotide sequenceencoding a ghrelin splice variant-like compound; (b) inserting saidcDNA in an expression vector such that the cDNA is operably linkedto a promoter; and (c) introducing said expression vector into ahost cell whereby said host cell produces said ghrelin splicevariant-like compound.

In one aspect of this embodiment, the method further comprises thestep of recovering the ghrelin splice variant-like compoundproduced in step (c). Another embodiment is a ghrelin splicevariant-like compound obtainable by the method described in thepreceding paragraph. The expression vector is any of the mammalian,yeast, insect, or bacterial expression systems known in the art.Commercially available vectors and expression systems are availablefrom a variety of suppliers including Genetics Institute(Cambridge, Mass.), Stratagene (La Jolla, Calif.), Promega(Madison, Wis.), and Invitrogen (San Diego, Calif.). If desired, toenhance expression and facilitate proper protein folding, the codoncontext and codon pairing of the sequence is optimized for theparticular expression organism in which the expression vector isintroduced, as explained in U.S. Pat. No. 5,082,767, whichdisclosure is hereby incorporated by reference in its entirety.

In another embodiment, it is often advantageous to add to therecombinant polynucleotide additional nucleotide sequence(s) whichcodes for secretory or leader sequences, pro-sequences, sequenceswhich aid in purification, such as multiple histidine residues, oran additional sequence for stability during recombinantproduction.

Introduction of a polynucleotide encoding a ghrelin splicevariant-like compound into a host cell can be effected by calciumphosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation,transduction, infection, or other methods. Such methods aredescribed in many standard laboratory manuals, such as Davis etal., (1986) Basic Methods in Molecular Biology, ed., ElsevierPress, NY, which disclosure is hereby incorporated by reference inits entirety. It is specifically contemplated that the ghrelinsplice variant-like compounds disclosed herein may in fact beexpressed by a host cell lacking a recombinant vector or naturallyproduced by a cell.

Ghrelin splice variant-like compounds can be recovered and purifiedfrom recombinant cell cultures by well-known methods includingdifferential extraction, ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography (see, for example,"Methods in Enzymology: Aqueous Two-Phase Systems", Walter H et al.(eds.), Academic Press (1993), incorporated herein by reference,for a variety of methods for purifying proteins). In oneembodiment, high performance liquid chromatography ("HPLC") isemployed for purification. A recombinantly produced version of aghrelin splice variant-like compound can be substantially purifiedusing techniques described herein or otherwise known in the art,such as, for example, by the one-step method described in Smith& Johnson, Gene 67:31 40 (1988), which disclosure is herebyincorporated by reference in its entirety. Ghrelin splicevariant-like compounds also can be purified from recombinantsources using antibodies directed against the ghrelin splicevariant-like compounds, such as those described herein, in methodswhich are well known in the art of protein purification.

In one embodiment, the recombinantly expressed ghrelin splicevariant-like compound is purified using standardimmunochromatography techniques. In such procedures, a solutioncontaining the ghrelin splice variant-like compound of interest,such as the culture medium or a cell extract, is applied to acolumn having antibodies against the ghrelin splice variant-likecompound attached to the chromatography matrix. The recombinantghrelin splice variant-like compound is allowed to bind theimmunochromatography column. Thereafter, the column is washed toremove non-specifically bound proteins. The specifically boundsecreted ghrelin splice variant-like compound is then released fromthe column and recovered using standard techniques.

Depending upon the host employed in a recombinant productionprocedure, the ghrelin splice variant-like compounds may beglycosylated or may be non-glycosylated. In addition, polypeptidesof the invention may also include an initial modified methionineresidue, in some cases as a result of host-mediated processes.Thus, it is well known in the art that the N-terminal methionineencoded by the translation initiation codon generally is removedwith high efficiency from any protein after translation in alleukaryotic cells. While the N-terminal methionine on most proteinsalso is efficiently removed in most prokaryotes, for some proteins,this prokaryotic removal process is inefficient, depending on thenature of the amino acid to which the N-terminal methionine iscovalently linked.

Pharmaceutical Compositions

While it is possible for the compounds or salts of the presentdisclosure to be administered as the raw chemical, it is preferredto present them in the form of a pharmaceutical composition.Accordingly, one aspect relates to a pharmaceutical compositioncomprising a ghrelin splice variant-like compound as defined inFormula I.

Another embodiment relates to a pharmaceutical compositioncomprising a mixture of at least two different ghrelin splicevariant-like compounds, such as a mixture of a ghrelin splicevariant-like compound acylated with a C.sub.8 acyl and a ghrelinsplice variant-like compound acylated with a C.sub.10 acyl. Withoutbeing bound by theory, it is believed that such a mixture will havea longer half-life in plasma.

In yet another embodiment, the pharmaceutical composition comprisesacylated ghrelin splice variant-like compounds, optionallycompounds having different acyl chain lengths preferably selectedfrom the group consisting of C.sub.7 acyl group, C.sub.9 acylgroup, and C.sub.11 acyl group, optionally in combination with adesacylated ghrelin splice variant-like compound.

Another aspect relates to a pharmaceutical composition comprisingany secretagogue, such as any ghrelin splice variant-like compoundas defined above or a pharmaceutically acceptable salt thereof andpharmaceutical acceptable carriers, vehicles and/or excipients;said composition further comprising transport molecules. Thetransport molecules are primarily added in order to increase thehalf-life of the acylated compound, preventing prematuredes-acylation, since the des-acylated ghrelin splice variant mightnot be active at the GHS-R 1a.

Transport molecules act by having incorporated into or anchored toit a compound disclosed herein. Any suitable transport moleculeknown to the skilled person may be used. Examples of transportmolecules are those described in the conjugate section, supra.Other preferred examples are liposomes, micelles, and/ormicrospheres.

Conventional liposomes are typically composed of phospholipids(neutral or negatively charged) and/or cholesterol. The liposomesare vesicular structures based on lipid bilayer surrounding aqueouscompartments. They can vary in their physio-chemical propertiessuch as size, lipid composition, surface charge and number, andfluidity of the phospholipids bilayer. The most frequently usedlipids for liposome formation are:1,2-Dilauroyl-sn-Glycero-3-Phosphocholine (DLPC),1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC),1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC),1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC),1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC),1,2-Dimyristoyl-sn-Glycero-3-Phosphoethanolamine (DMPE),1,2-Dipaimitoyl-sn-Glycero-3-Phosphoethanolamine (DPPE),1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE),1,2-Dimyristoyl-sn-Glycero-3-Phosphate (Monosodium Salt) (DMPA),1,2-Dipalmitoyl-sn-Glycero-3-Phosphate (Monosodium Salt) (DPPA),1,2-Dioleoyl-sn-Glycero-3-Phosphate (Monosodium Salt) (DOPA),1,2-Dimyristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (SodiumSalt) (DMPG),1,2-Dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (SodiumSalt) (DPPG), 1,2-Dioleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)](Sodium Salt) (DOPG),1,2-Dimyristoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium Salt)(DMPS), 1,2-Dipalmitoyl-sn-Glycero-3-[Phospho-L-Serine] (SodiumSalt) (DPPS), 1,2-Dioleoyl-sn-Glycero-3-[Phospho-L-Serine] (SodiumSalt) (DOPS),1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(glutaryl) (SodiumSalt) and 1,1',2,2'-Tetramyristoyl Cardiolipin (Ammonium Salt).Formulations composed of DPPC in combination with other lipid ormodifiers of liposomes are preferred, e.g., in combination withcholesterol and/or phosphatidylcholine.

Long-circulating liposomes are characterized by their ability toextravasate at body sites where the permeability of the vascularwall is increased. A preferred way to produce long circulatingliposomes is to attach hydrophilic polymer polyethylene glycol(PEG) covalently to the outer surface of the liposome. Some of thepreferred lipids are:1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000] (Ammonium Salt),1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-5000] (Ammonium Salt),1,2-Dioleoyl-3-Trimethylammonium-Propane (Chloride Salt)(DOTAP).

Possible lipids applicable for liposomes are supplied by AvantiPolar Lipids, Inc., (Alabaster, Ala.). Additionally, the liposomesuspension may include lipid-protective agents which protect lipidsagainst free-radical and lipid-peroxidative damages on storage.Lipophilic free-radical quenchers, such as alpha-tocopherol andwater-soluble iron-specific chelators, such as ferrioxianine, arepreferred.

A variety of methods are available for preparing liposomes, asdescribed in, e.g., Szoka F. & Papahadjopolous D., Ann. Rev.Biophys. Bioeng. 9:467-508 (1980); U.S. Pat. Nos. 4,235,871,4,501,728 and 4,837,028; all of which are incorporated herein byreference. Another method produces multilamellar vesicles ofheterogeneous sizes. In this method, the vesicle-forming lipids aredissolved in a suitable organic solvent or solvent system and driedunder vacuum or an inert gas to form a thin lipid film. If desired,the film may be redissolved in a suitable solvent, such as tertiarybutanol, and then lyophilized to form a more homogeneous lipidmixture which is in a more easily hydrated powder-like form. Thisfilm is covered with an aqueous solution of the targeted drug andthe targeting component and allowed to hydrate, typically over a15-60 minute period with agitation. The size distribution of theresulting multilamellar vesicles can be shifted toward smallersizes by hydrating the lipids under more vigorous agitationconditions or by adding solubilizing detergents such asdeoxycholate.

Micelles are formed by surfactants (molecules that contain ahydrophobic portion and one or more ionic or otherwise stronglyhydrophilic groups) in aqueous solution. As the concentration of asolid surfactant increases, its monolayers adsorbed at theair/water or glass/water interface become so tightly packed thatfurther occupancy requires excessive compression of the surfactantmolecules already in the two monolayers. Further increments in theamount of dissolved surfactant beyond that concentration causeamounts equivalent to the new molecules to aggregate into micelles.This process begins at a characteristic concentration called"critical micelle concentration".

The shape of micelles formed in dilute surfactant solutions isapproximately spherical. The polar head groups of the surfactantmolecules are arranged in an outer spherical shell whereas theirhydrocarbon chains are oriented toward the center, forming aspherical core for the micelle. The hydrocarbon chains are randomlycoiled and entangled and the micellar interior has a nonpolar,liquid-like character. In the micelles of polyoxyethylated nonionicdetergents, the polyoxyethlene moieties are oriented outward andpermeated by water. This arrangement is energetically favorablesince the hydrophilic head groups are in contact with water and thehydrocarbon moieties are removed from the aqueous medium and partlyshielded from contact with water by the polar head groups. Thehydrocarbon tails of the surfactant molecules, located in theinterior of the micelle, interact with one another by weak van derWaals forces.

The size of a micelle or its aggregation number is governed largelyby geometric factors. The radius of the hydrocarbon core cannotexceed the length of the extended hydrocarbon chain of thesurfactant molecule. Therefore, increasing the chain length orascending homologous series increases the aggregation number ofspherical micelles. If the surfactant concentration is increasedbeyond a few percent and if electrolytes are added (in the case ofionic surfactants) or the temperature is raised (in the case ofnonionic surfactants), the micelles increase in size. Under theseconditions, the micelles are too large to remain spherical andbecome ellipsoidal, cylindrical or finally lamellar in shape.

Common surfactants well known to one of skill in the art can beused in the micelles of the present disclosure. Suitablesurfactants include sodium laureate, sodium oleate, sodium laurylsulfate, octaoxyethylene glycol monododecyl ether, octoxynol 9 andPLURONIC.RTM. F-127 (BASF Corp., Florham Park, N.J.). Preferredsurfactants are nonionic polyoxyethylene and polyoxypropylenedetergents compatible with intravenous injection such as,TWEEN.RTM.80, PLURONIC.RTM.D F-68, n-octyl-beta-D-glucopyranoside,and the like. In addition, phospholipids, such as those describedfor use in the production of liposomes, may also be used formicelle formation.

In another preferred embodiment, the compounds disclosed herein areformulated as described in the literature for an administrationroute selected from: buccal delivery, sublingual delivery,transdermal delivery, inhalation and needle-free injection, such asusing the methods developed by Powderjet.

For inhalation, the compounds disclosed herein can be formulatedusing methods known to those skilled in the art, for example anaerosol, dry powder or solubilized such as in microdroplets,preferably in a device intended for such delivery (such ascommercially available from Aradigm Corp. (Hayward, Cal.),Alkermes, Inc. (Cambridge, Mass.), or Nektar Therapeutics (SanCarlos, Cal.)).

Administration

Suitable dosing regimens for the various compounds and methods ofthe present disclosure are preferably determined taking intoaccount factors well known in the art including, e.g., type ofsubject being dosed; age, weight, sex and medical condition of thesubject; the route of administration; the renal and hepaticfunction of the subject; the desired effect; and the particularcompound employed. Preferably, the composition will comprise about0.5% to 75% by weight of a secretagogue disclosed herein, with theremainder consisting of suitable pharmaceutical excipients.

Optimal precision in achieving concentrations of drug within therange that yields efficacy without toxicity requires a regimenbased on the kinetics of the drug's availability to target sites.This involves a consideration of the distribution, equilibrium, andelimination of a drug.

As described above, in one aspect, the secretagogue, such asghrelin splice variant or a ghrelin splice variant-like compound,is administered subcutaneously.

In another aspect, the secretagogue, such as ghrelin splice variantor a ghrelin splice variant-like compound, is administered as apremeal bolus, wherein the administration form may be any suitableparenteral form. In a preferred embodiment, the secretagogue, suchas ghrelin splice variant or a ghrelin splice variant-likecompound, is administered subcutaneously in a premeal bolus.

The secretagogue, such as ghrelin splice variant or a ghrelinsplice variant-like compound, can also be administered during ameal as a bolus. The mode of administration during a meal includessubcutaneous administration, such as a subcutaneously administeredbolus.

Pharmaceutical compositions for parenteral administration includesterile aqueous and non-aqueous injectable solutions, dispersions,suspensions or emulsions, as well as sterile powders to bereconstituted in sterile injectable solutions or dispersions priorto use. Other suitable administration forms include suppositories,sprays, ointments, creams, gels, inhalants, dermal patches,implants, pills, tablets, lozenges and capsules.

A typical dosage is in a concentration equivalent to from 10 ng to10 mg ghrelin splice variant per kg bodyweight. The concentrationsand amounts herein are given in equivalents of amount ghrelinsplice variant, wherein the ghrelin splice variant is a 22 aminoacid human ghrelin splice variant (SEQ ID NO:2) and/or a 29 aminoacid human ghrelin splice variant (SEQ ID NO:5) and/or a 24 aminoacid human ghrelin splice variant (SEQ ID NO:3) and/or a 24 aminoacid human ghrelin splice variant having a Dpr residue at the thirdposition (SEQ ID NO:4). Equivalents may be tested as described inthe section entitled "Functionality", above.

In a preferred embodiment, the medicament is administered in aconcentration equivalent to from 0.1 .mu.g to 1 mg ghrelin splicevariant per kg bodyweight, such as from 0.5 .mu.g to 0.5 mg ghrelinsplice variant per kg bodyweight, such as from 1.0 .mu.g to 0.1 mgghrelin splice variant per kg bodyweight, such as from 1.0 .mu.g to50 .mu.g ghrelin splice variant per kg bodyweight, such as from 1.0.mu.g to 10 .mu.g ghrelin splice variant per kg bodyweight.

As described above, the secretagogue, such as ghrelin splicevariant or a ghrelin splice variant-like compound, is preferablyadministered as a bolus. Accordingly, in one embodiment themedicament is administered as a bolus prior to a meal, said boluscomprising an amount of the secretagogue or a salt thereofequivalent to from 0.3 .mu.g to 600 mg ghrelin splice variant. Morepreferably, the medicament is administered as a bolus prior to ameal, said bolus comprising an amount of the secretagogue or a saltthereof equivalent to from 2.0 .mu.g to 200 mg ghrelin splicevariant, such as from 5.0 .mu.g to 100 mg ghrelin splice variant,such as from 10 .mu.g to 50 mg ghrelin splice variant, such as from10 .mu.g to 5 mg ghrelin splice variant, such as from 10 .mu.g to1.0 mg ghrelin splice variant.

It should be noted that the normal ghrelin splice variant-likeresponse which occurs before a meal is a short-lived surge inplasma concentrations of ghrelin splice variant and that, due tothe relatively short half life of the peptide, an intravenousinjection of ghrelin splice variant will ensure that a similarshort-lived peak on ghrelin splice variant concentrations can beobtained. The administration route must ensure that thenon-degraded, bioactive form of the peptide will be the dominatingform in the circulation, which will reach and stimulate the ghrelinsplice variant receptors.

Thus, in order to obtain the maximum effect of the medicament, itis preferably administered from one to three times daily, eachadministration being within 45 minutes of a meal, such as within 30minutes of a meal, such as within 25 minutes of a meal, such aswithin 20 minutes of a meal, such as within 15 minutes of a meal,such as within 10 minutes of a meal, such as within 5 minutes of ameal. More preferably, the medicament is administered prior to eachmain meal, such as administered three times daily.

Compounds disclosed herein may also be formulated for nasaladministration. The solutions or suspensions are applied directlyto the nasal cavity by conventional means, for example with adropper, pipette or spray. The compositions may be provided in asingle or multidose form. In the latter case of a dropper orpipette, this may be achieved by the patient administering anappropriate, predetermined volume of the solution or suspension. Inthe case of a spray, this may be achieved for example by means of ametering atomizing spray pump.

The compounds disclosed herein may be formulated for aerosoladministration, particularly to the respiratory tract and includingintranasal administration. The compound will generally have a smallparticle size, for example of the order of 5 microns or less. Sucha particle size may be obtained by means known in the art, forexample by micronization. The active ingredient is provided in apressurized pack with a suitable propellant such as ahydrofluoroalkane (HFA) for example hydrofluoroalkane-134a andhydrofluoroalkane-227, carbon dioxide or other suitable gas. Theaerosol may conveniently also contain a surfactant such aslecithin. The dose of drug may be controlled by a metered valve.Alternatively, the active ingredients may be provided in a form ofa dry powder, for example a powder mix of the compound in asuitable powder base such as lactose, starch, starch derivativessuch as hydroxypropylmethyl cellulose and polyvinylpyrrolidine(PVP). The powder carrier will form a gel in the nasal cavity. Thepowder composition may be presented in unit dose form for examplein capsules or cartridges of, e.g., gelatin or blister packs fromwhich the powder may be administered by means of an inhaler.

Compositions administered by aerosols may be prepared, for example,as solutions in saline, employing benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,employing fluorocarbons, and/or employing other solubilizing ordispersing agents.

Compounds disclosed herein may also be formulated foradministration by injection pen in a similar way as for cartridgedgrowth hormone (GH) or Insulin. The cartridge contains compoundsdisclosed herein in solvents. The pen, which is basically a needle,syringe and vial in one piece, is operated by a turning movementand allows different doses to be administrated. This device offerssimplicity, convenience, and enhanced safety features for compoundsdelivery. It provides a simple device design, few administrationsteps and one-step dial-back dose knob. Such injection pen can beobtained by means known in art. For example, several manufacturersoffer drug developers injection pens to be used with the drugdevelopers compounds (BD--Medical--Pharmaceutical Systems, Inc.;Owen Mumford Inc. etc.).

Compositions for Oral Administration

Those secretagogue types capable of remaining biologically activein an individual after oral administration (such as, e.g., smallmolecules and short peptides) can be formulated in a wide range oforal administration dosage forms. The pharmaceutical compositionsand dosage forms may comprise the compounds disclosed herein ortheir pharmaceutically acceptable salt or crystal forms thereof asthe active component.

The pharmaceutical acceptable carriers can be either solid orliquid. Solid form preparations include powders, tablets, pills,capsules, cachets, suppositories, and dispersible granules. A solidcarrier can be one or more substances which may also act asdiluents, flavoring agents, solubilizers, lubricants, suspendingagents, binders, preservatives, wetting agents, tabletdisintegrating agents, or an encapsulating material.

For oral administration, such excipients include, e.g.,pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, talcum, cellulose, glucose, gelatin,sucrose, magnesium carbonate, and the like.

In powders, the carrier is a finely divided solid which is amixture with the finely divided active component. In tablets, theactive component is mixed with the carrier having the necessarybinding capacity in suitable proportions and compacted in the shapeand size desired. The powders and tablets preferably containingfrom one to about seventy percent of the active compound. Suitablecarriers are magnesium carbonate, magnesium stearate, talc, sugar,lactose, pectin, dextrin, starch, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose, a low melting wax,cocoa butter, and the like. The term "preparation" is intended toinclude a composition comprising an active compound disclosedherein with encapsulating material as carrier providing a capsulein which the active component, with or without carriers, issurrounded by a carrier, which is in association with it.Similarly, cachets and lozenges are included.

Tablets, powders, capsules, pills, cachets, and lozenges can be assolid forms suitable for oral administration.

Drops may comprise sterile or nonsterile aqueous or oil solutionsor suspensions, and may be prepared by dissolving the activeingredient in a suitable aqueous solution, optionally including abactericidal and/or fungicidal agent and/or any other suitablepreservative, and optionally including a surface active agent. Theresulting solution may then be clarified by filtration, transferredto a suitable container which is then sealed and sterilized byautoclaving or maintaining at 98-100.degree. C. for half an hour.Alternatively, the solution may be sterilized by filtration andtransferred to the container aseptically. Examples of bactericidaland fungicidal agents suitable for inclusion in the drops arephenylmercuric nitrate or acetate (0.002%), benzalkonium chloride(0.01%) and chlorhexidine acetate (0.01%). Suitable solvents forthe preparation of an oily solution include glycerol, dilutedalcohol and propylene glycol.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions,and emulsions. These preparations may contain, in addition to theactive component, colorants, flavors, stabilizers, buffers,artificial and natural sweeteners, dispersants, thickeners,solubilizing agents, and the like.

Other forms suitable for oral administration include liquid formpreparations including emulsions, syrups, elixirs, aqueoussolutions, aqueous suspensions, toothpaste, gel dentifrice, chewinggum, or solid form preparations which are intended to be convertedshortly before use to liquid form preparations. Emulsions may beprepared in solutions in aqueous propylene glycol solutions or maycontain emulsifying agents such as lecithin, sorbitan monooleate,or acacia. Aqueous solutions can be prepared by dissolving theactive component in water and adding suitable colorants, flavors,stabilizing and thickening agents. Aqueous suspensions can beprepared by dispersing the finely divided active component in waterwith viscous material, such as natural or synthetic gums, resins,methylcellulose, sodium carboxymethylcellulose, and other wellknown suspending agents. Solid form preparations include solutions,suspensions, and emulsions, and may contain, in addition to theactive component, colorants, flavors, stabilizers, buffers,artificial and natural sweeteners, dispersants, thickeners,solubilizing agents, and the like.

Compositions for Parenteral Administration

The compounds disclosed herein may be formulated for parenteraladministration (e.g., by injection, for example bolus injection orcontinuous infusion) and may be presented in unit dose form inampules, pre-filled syringes, small volume infusion or inmulti-dose containers with an added preservative. The compositionsmay take such forms as suspensions, solutions, or emulsions in oilyor aqueous vehicles, for example solutions in aqueous polyethyleneglycol. Examples of oily or nonaqueous carriers, diluents, solventsor vehicles include propylene glycol, polyethylene glycol,vegetable oils (e.g., olive oil), and injectable organic esters(e.g., ethyl oleate), and may contain formulatory agents such aspreserving, wetting, emulsifying or suspending, stabilizing and/ordispersing agents. Alternatively, the active ingredient may be inpowder form, obtained by aseptic isolation of sterile solid or bylyophilization from solution for constitution before use with asuitable vehicle, e.g., sterile, pyrogen-free water. Aqueoussolutions should be suitably buffered if necessary, and the liquiddiluent first rendered isotonic with sufficient saline or glucose.The aqueous solutions are particularly suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. Thesterile aqueous media employed are all readily available bystandard techniques known to those skilled in the art.

Solutions of ghrelin splice variant or a ghrelin splicevariant-like compound or pharmaceutical acceptable salt thereof(and for example antigenic epitopes and protease inhibitors) can beprepared in water or saline, and optionally mixed with a nontoxicsurfactant. Compositions for intravenous or intra-arterialadministration may include sterile aqueous solutions that may alsocontain buffers, liposomes, diluents and other suitableadditives.

Oils useful in parenteral compositions include petroleum, animal,vegetable, or synthetic oils. Specific examples of oils useful insuch compositions include peanut, soybean, sesame, cottonseed,corn, olive, petrolatum, and mineral. Suitable fatty acids for usein parenteral compositions include oleic acid, stearic acid, andisostearic acid. Ethyl oleate and isopropyl myristate are examplesof suitable fatty acid esters.

Suitable soaps for use in parenteral compositions include fattyalkali metal, ammonium, and triethanolamine salts, and suitabledetergents include (a) cationic detergents such as, for example,dimethyl dialkyl ammonium halides, and alkyl pyridinium halides;(b) anionic detergents such as, for example, alkyl, aryl, andolefin sulfonates, alkyl, olefin, ether, and monoglyceridesulfates, and sulfosuccinates; (c) nonionic detergents such as, forexample, fatty amine oxides, fatty acid alkanolamides, andpolyoxyethylenepolypropylene copolymers; (d) amphoteric detergentssuch as, for example, alkyl-beta-aminopropionates, and2-alkyl-imidazoline quaternary ammonium salts; and (e) mixturesthereof.

The parenteral compositions typically will contain from about 0.5to about 25% by weight of the active ingredient in solution.Preservatives and buffers may be used. In order to minimize oreliminate irritation at the site of injection, such compositionsmay contain one or more nonionic surfactants having ahydrophile-lipophile balance (HLB) of from about 12 to about 17.The quantity of surfactant in such compositions will typicallyrange from about 5 to about 15% by weight. Suitable surfactantsinclude polyethylene sorbitan fatty acid esters, such as sorbitanmonooleate and the high molecular weight adducts of ethylene oxidewith a hydrophobic base, formed by the condensation of propyleneoxide with propylene glycol. The parenteral compositions can bepresented in unit-dose or multi-dose sealed containers, such asampules and vials, and can be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid excipient, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensionscan be prepared from sterile powders, granules, and tablets of thekind previously described.

The pharmaceutical dosage forms suitable for injection or infusioncan include sterile aqueous solutions or dispersions comprising theactive ingredient that are adapted for administration byencapsulation in liposomes. In all cases, the ultimate dosage formmust be sterile, fluid and stable under the conditions ofmanufacture and storage.

Sterile injectable solutions are prepared by incorporating ghrelinsplice variant or a ghrelin splice variant-like compound orpharmaceutical acceptable salt thereof in the required amount inthe appropriate solvent with various of the other ingredientsenumerated above, as required, followed by, e.g., filtersterilization.

Compositions for Topical Administration

The compounds disclosed herein can also be delivered topically.Regions for topical administration include the skin surface andalso mucous membrane tissues of the rectum, nose, mouth, andthroat. Compositions for topical administration via the skin andmucous membranes should not give rise to signs of irritation, suchas swelling or redness.

The topical composition may include a pharmaceutical acceptablecarrier adapted for topical administration. Thus, the compositionmay take the form of, for example, a suspension, solution,ointment, lotion, cream, foam, aerosol, spray, suppository,implant, inhalant, tablet, capsule, dry powder, syrup, balm orlozenge. Methods for preparing such compositions are well known inthe pharmaceutical industry.

The compounds disclosed herein may be formulated for topicaladministration to the epidermis as ointments, creams or lotions, oras a transdermal patch. Ointments and creams may, for example, beformulated with an aqueous or oily base with the addition ofsuitable thickening and/or gelling agents. Lotions may beformulated with an aqueous or oily base and will in general alsocontaining one or more emulsifying agents, stabilizing agents,dispersing agents, suspending agents, thickening agents, orcoloring agents. Compositions suitable for topical administrationin the mouth include lozenges comprising active agents in aflavored base, usually sucrose and acacia or tragacanth; pastillescomprising the active ingredient in an inert base such as gelatinand glycerin or sucrose and acacia; and mouthwashes comprising theactive ingredient in a suitable liquid carrier.

Creams, ointments or pastes according to the present disclosure aresemi-solid compositions for external application comprising theactive ingredient. They may be made by mixing the active ingredientin finely-divided or powdered form, alone or in solution orsuspension in an aqueous or non-aqueous fluid, with the aid ofsuitable machinery, with a greasy or non-greasy base. The base maycomprise hydrocarbons such as hard, soft or liquid paraffin,glycerol, beeswax, a metallic soap; a mucilage; an oil of naturalorigin such as almond, corn, arachis, castor or olive oil; wool fator its derivatives; or a fatty acid such as steric or oleic acidtogether with an alcohol such as propylene glycol or a macrogel.The composition may incorporate any suitable surface active agentsuch as an anionic, cationic or non-ionic surfactant such as asorbitan ester or a polyoxyethylene derivative thereof. Suspendingagents such as natural gums, cellulose derivatives or inorganicmaterials such as silicaceous silicas, and other ingredients suchas lanolin, may also be included.

Lotions according to the present disclosure include those suitablefor application to the skin or eye. An eye lotion may comprise asterile aqueous solution optionally containing a bactericide andmay be prepared by methods similar to those for the preparation ofdrops. Lotions or liniments for application to the skin may alsoinclude an agent to hasten drying and to cool the skin, such as analcohol or acetone, and/or a moisturizer such as glycerol or an oilsuch as castor oil or arachis oil.

The compounds described herein can be administered transdermally.Transdermal administration typically involves the delivery of apharmaceutical agent for percutaneous passage of the drug into thesystemic circulation of the patient. The skin sites includeanatomic regions for transdermally administering the drug andinclude the forearm, abdomen, chest, back, buttock, mastoidal area,and the like.

Transdermal delivery is accomplished by exposing a source of theactive compound to a patient's skin for an extended period of time.Transdermal patches have the added advantage of providingcontrolled delivery of a compound complex to the body (seeTransdermal Drug Delivery: Developmental Issues and ResearchInitiatives, Hadgraft and Guy (eds.), Marcel Dekker, Inc., (1989);Controlled Drug Delivery: Fundamentals and Applications, Robinsonand Lee (eds.), Marcel Dekker Inc., (1987); and TransdermalDelivery of Drugs, Vols. 1-3, Kydonieus and Berner (eds.), CRCPress, (1987)). Such dosage forms can be made by dissolving,dispersing, or otherwise incorporating a compound disclosed hereinin a proper medium, such as an elastomeric matrix material.Absorption enhancers can also be used to increase the flux of thecompound across the skin. The rate of such flux can be controlledby either providing a rate-controlling membrane or dispersing thecompound in a polymer matrix or gel.

A variety of types of transdermal patches will find use in themethods described herein. For example, a simple adhesive patch canbe prepared from a backing material and an acrylate adhesive. Theactive compound and any enhancer are formulated into the adhesivecasting solution and allowed to mix thoroughly. The solution iscast directly onto the backing material and the casting solvent isevaporated in an oven, leaving an adhesive film. The release linercan be attached to complete the system.

Alternatively, a polyurethane matrix patch can be employed todeliver a compound disclosed herein. The layers of this patchcomprise a backing, a polyurethane drug/enhancer matrix, amembrane, an adhesive, and a release liner. The polyurethane matrixis prepared using a room temperature curing polyurethaneprepolymer. Addition of water, alcohol, and complex to theprepolymer results in the formation of a tacky firm elastomer thatcan be directly cast only the backing material.

A further embodiment will utilize a hydrogel matrix patch.Typically, the hydrogel matrix will comprise alcohol, water, drug,and several hydrophilic polymers. This hydrogel matrix can beincorporated into a transdermal patch between the backing and theadhesive layer.

A liquid reservoir patch will also find use in the methodsdescribed herein. This patch comprises an impermeable orsemipermeable, heat sealable backing material, a heat sealablemembrane, an acrylate based pressure sensitive skin adhesive, and asiliconized release liner. The backing is heat sealed to themembrane to form a reservoir which can then be filled with asolution of the complex, enhancers, gelling agent, and otherexcipients.

Foam matrix patches are similar in design and components to theliquid reservoir system, except that the gelled pharmaceuticalagent-chemical modifier solution is constrained in a thin foamlayer, typically a polyurethane. This foam layer is situatedbetween the backing and the membrane which have been heat sealed atthe periphery of the patch.

For passive delivery systems, the rate of release is typicallycontrolled by a membrane placed between the reservoir and the skin,by diffusion from a monolithic device, or by the skin itselfserving as a rate-controlling barrier in the delivery system (SeeU.S. Pat. Nos. 4,816,258; 4,927,408; 4,904,475; 4,588,580,4,788,062; and the like, all of which are incorporated herein byreference). The rate of drug delivery will be dependent, in part,upon the nature of the membrane. For example, the rate of drugdelivery across membranes within the body is generally higher thanacross dermal barriers. The rate at which the active compound isdelivered from the device to the membrane is most advantageouslycontrolled by the use of rate-limiting membranes which are placedbetween the reservoir and the skin. Assuming that the skin issufficiently permeable to the active compound (i.e., absorptionthrough the skin is greater than the rate of passage through themembrane), the membrane will serve to control the dosage rateexperienced by the patient.

Suitable permeable membrane materials may be selected based on thedesired degree of permeability, the nature of the active compound,and the mechanical considerations related to constructing thedevice. Exemplary permeable membrane materials include a widevariety of natural and synthetic polymers, such aspolydimethylsiloxanes (silicone rubbers), ethylenevinylacetatecopolymer (EVA), polyurethanes, polyurethane-polyether copolymers,polyethylenes, polyamides, polyvinylchlorides (PVC),polypropylenes, polycarbonates, polytetrafluoroethylenes (PTFE),cellulosic materials, e.g., cellulose triacetate and cellulosenitrate/acetate, and hydrogels, e.g., 2-hydroxyethylmethacrylate(HEMA).

Other items may be contained in the device, such as otherconventional components of therapeutic products, depending upon thedesired device characteristics. For example, the compositionsdisclosed herein may also include one or more preservatives orbacteriostatic agents, e.g., methyl hydroxybenzoate, propylhydroxybenzoate, chlorocresol, benzalkonium chlorides, and thelike. These pharmaceutical compositions also can contain otheractive ingredients such as antimicrobial agents, particularlyantibiotics, anesthetics, analgesics, and antipruritic agents.

Compositions for Administration as Suppositories

The compounds disclosed herein may be formulated for administrationas suppositories. A typical suppository is produced by providing alow melting wax, such as a mixture of fatty acid glycerides orcocoa butter, that is first melted and the active component isdispersed homogeneously therein, for example, by stirring. Themolten homogeneous mixture is then poured into convenient sizedmolds, allowed to cool, and to solidify.

The active compound may be formulated into a suppositorycomprising, for example, about 0.5% to about 50% of a compounddisclosed herein, disposed in a polyethylene glycol (PEG) carrier(e.g., PEG 1000 [96%] and PEG 4000 [4%]).

Formulation

A preferred aspect contemplates pharmaceutical compositions usefulfor practicing the therapeutic methods described herein.Pharmaceutical compositions can contain a physiologically tolerablecarrier together with at least one species of a secretagogue, suchas ghrelin splice variant or a ghrelin splice variant-like compoundas described herein, dissolved or dispersed therein as an activeingredient. In a preferred embodiment, the pharmaceuticalcomposition is not immunogenic when administered to a humanindividual for therapeutic purposes, unless that purpose is toinduce an immune response.

One aspect relates to a pharmaceutical composition comprising atleast one secretagogue, such as ghrelin splice variant or a ghrelinsplice variant-like compound as defined above in Formula I. In apreferred embodiment, the pharmaceutical composition comprises atleast two different ghrelin splice variant-like compounds asdefined above in Formula I in order to increase the effect of thetreatment. The difference may for example be compounds havingdifferent acylations as discussed above.

As used herein, the terms "pharmaceutically acceptable","physiologically tolerable" and grammatical variations thereof, asthey refer to compositions, carriers, diluents and reagents, areused interchangeably and represent that the materials are capableof administration to or upon a human without the production ofundesirable physiological effects such as nausea, dizziness,gastric upset and the like.

The preparation of a pharmacological composition that containsactive ingredients dissolved or dispersed therein is wellunderstood in the art. Typically, such compositions are prepared assterile injectables either as liquid solutions or suspensions,aqueous or non-aqueous; however, solid forms suitable for solution,or suspensions, in liquid prior to use can also be prepared. Thepreparation can also be emulsified.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the activeingredient and in amounts suitable for use in the therapeuticmethods described herein. Suitable excipients are, for example,water, saline, dextrose, glycerol, ethanol or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents and the like which enhancethe effectiveness of the active ingredient. It is preferred thatthe formulation has a pH within the range of 3.5-8, such as in therange 4.5-7.5, such as in the range 5.5-7, such as in the range6-7.5, most preferably around 7.3. However, as is understood by oneskilled in the art, the pH range may be adjusted according to theindividual treated and the administration procedure. For example,certain secretagogues, such as ghrelin splice variant and ghrelinsplice variant homologs, may be easily stabilized at a lower pH;so, in another preferred embodiment, the formulation has a pHwithin the range 3.5-7, such as 4-6, such as 5-6, such as 5.3-5.7,such as 5.5.

Pharmaceutical compositions disclosed herein can includepharmaceutically acceptable salts of the compounds therein. Thesesalts will be ones which are acceptable in their application to apharmaceutical use, meaning that the salt will retain thebiological activity of the parent compound and the salt will nothave untoward or deleterious effects in its application and use intreating diseases.

Pharmaceutically acceptable salts are prepared in a standardmanner. If the parent compound is a base, it is treated with anexcess of an organic or inorganic acid in a suitable solvent. Ifthe parent compound is an acid, it is treated with an inorganic ororganic base in a suitable solvent.

The compounds disclosed herein may be administered in the form ofan alkali metal or earth alkali metal salt thereof, concurrently,simultaneously, or together with a pharmaceutically acceptablecarrier or diluent, especially and preferably in the form of apharmaceutical composition thereof, whether by, e.g., oral, rectal,or parenteral (including subcutaneous) route, in an effectiveamount.

Examples of pharmaceutical acceptable acid addition salts for usein the present inventive pharmaceutical composition include thosederived from mineral acids, such as, e.g., hydrochloric,hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids,and organic acids, such as, e.g., tartaric, acetic, citric, malic,lactic, fumaric, benzoic, glycolic, gluconic, succinic,p-toluenesulphonic, and arylsulphonic acids.

Other suitable pharmaceutically acceptable salts include the acidaddition salts (formed with the free amino groups of thepolypeptide). Other examples of salts include pharmaceuticallyacceptable acid addition salts, pharmaceutically acceptable metalsalts, ammonium salts and alkylated ammonium salts. Acid additionsalts include salts of inorganic acids as well as organic acids.Representative examples of suitable inorganic acids includehydrochloric, hydrobromic, hydriodic, phosphoric, sulfuric andnitric acids and the like. Representative examples of suitableorganic acids include formic, acetic, trichloroacetic,trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric,glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric,pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic,tartaric, ascorbic, pamoic, bismethylene salicylic,ethanedisulfonic, gluconic, citraconic, aspartic, stearic,palmitic, ethylenediaminetetraacetic (EDTA), p-aminobenzoic,glutamic, benzenesulfonic, and p-toluenesulfonic acids and thelike. Further examples of pharmaceutically acceptable inorganic ororganic acid addition salts include the pharmaceutical acceptablesalts listed in Berge S. M. et al., J. Pharm. Sci. 66:1-19 (1977),which is incorporated herein by reference. Examples of metal saltsinclude lithium, sodium, potassium and magnesium salts and thelike.

Examples of ammonium and alkylated ammonium salts include ammonium,methylammonium, dimethylammonium, trimethylammonium, ethylammonium,hydroxyethylammonium, diethylammonium, butylammonium andtetramethylammonium salts and the like.

Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,procaine and the like.

Also included within the scope of compounds or pharmaceuticalacceptable acid addition salts thereof in the context of thepresent disclosure are any hydrates (hydrated forms) thereof.

For parenteral administration, solutions of the present compoundsin sterile aqueous solution, aqueous propylene glycol or sesame orpeanut oil may be employed. Such aqueous solutions should besuitably buffered if necessary, and the liquid diluent firstrendered isotonic with sufficient saline or glucose. The aqueoussolutions are particularly suitable for intravenous, intramuscular,subcutaneous and intraperitoneal administration. The sterileaqueous media employed are all readily available by standardtechniques known to those skilled in the art.

Liquid compositions can also contain liquid phases in addition toand to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, organicesters such as ethyl oleate, and water-oil emulsions.

Suitable pharmaceutical carriers include inert solid diluents orfillers, sterile aqueous solution and various organic solvents.Examples of solid carriers are lactose, terra alba, sucrose,cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesiumstearate, stearic acid or lower alkyl ethers of cellulose. Examplesof liquid carriers are syrup, peanut oil, olive oil, phospholipids,fatty acids, fatty acid amines, polyoxyethylene or water. Nasalaerosol or inhalation formulations may be prepared, for example, assolutions in saline, employing benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,employing fluorocarbons, and/or employing other solubilizing ordispersing agents.

The pharmaceutical compositions formed by combining the compoundsdisclosed herein and the pharmaceutical acceptable carriers arethen readily administered in a variety of dosage forms suitable forthe disclosed routes of administration. The formulations mayconveniently be presented in unit dosage form by methods known inthe art of pharmacy.

In a preferred embodiment, the formulation comprises thesecretagogue or a salt thereof as a lyophilisate, and theformulation further comprises a solvent, said lyophilisate and saidsolvent being in separate compartments until administration. Inanother embodiment, the formulation is a solution of thesecretagogue or a salt thereof. In either embodiment, the solventmay be any suitable solvent, such as those described herein, andpreferably the solvent is saline.

Another aspect relates to a method for preparing a medicament orpharmaceutical composition comprising a compound disclosed herein,the method comprising admixing at least one ghrelin splicevariant-like compound, as defined above in Formula I, with aphysiologically acceptable carrier. A further aspect relates to apharmaceutical composition comprising, as an active ingredient, acompound as defined above in Formula I or a pharmaceuticallyacceptable salt thereof together with a pharmaceutically-acceptablecarrier. Accordingly, the formulation may further include thetransport molecules as described above.

Combination Treatments

In a further aspect, the present compounds may be administered incombination with additional pharmacologically-active substances orother pharmacologically-active material and/or may be administeredin combination with another therapeutic method. By the phrase "incombination with another substance(s) and/or therapeutic method(s)"is meant herein that said another substance(s) and/or therapeuticmethod(s) is administered to the individual thus treated before,during (including concurrently with) and/or after treatment of anindividual with a secretagogue. In all cases of combinationtreatment described herein, the combination may be in the form ofkit-in-part systems, wherein the combined active substances may beused for simultaneous, sequential or separate administration. Inall cases, it is preferred that any of the herein-mentionedmedicaments are administered in pharmaceutically effective amounts,i.e. an administration involving a total amount of each activecomponent of the medicament or pharmaceutical composition or methodthat is sufficient to show a meaningful patient benefit.

In the following sections, combination therapies for use inpreferred embodiments are grouped as follows:

1) Combinations wherein all active ingredients areappetite-regulating agents or in other ways useful for treatingcachexia and/or lipodystrophy.

The secretagogue(s) according to the present disclosure can beadministered in combination with other appetite-regulating agents,including more than one type of growth hormone secretagogue, suchas another ghrelin splice variant-like compound, such as a ghrelinsplice variant-like compound comprising a structure defined byFormula I, described herein. Other secretagogues suitable forcombination administration with another secretagogue compound areany of the secretagogue compounds described herein. In onepreferred embodiment, ghrelin splice variant (most preferably humanghrelin splice variant) is administered in combination with adifferent, ghrelin splice variant-like compound--this combinationis envisaged to enhance and/or prolong the effect of thesecretagogues on the ghrelin receptor. In a similar way, severaldifferent secretagogues may be administered to an individual toincrease efficacy on the ghrelin receptor, such as greater than 2different secretagogue types, such as 3, such as 4, such as 5, suchas 6, such as 7, such as greater than 8 different secretagoguetypes. The secretagogue according to the present disclosure, suchas ghrelin splice variant or a ghrelin splice variant-likecompound(s) can also be administered in combination with apharmaceutically effective amount of a growth hormone, includinghGH.

In one preferred embodiment, the secretagogue, such as ghrelinsplice variant or a ghrelin splice variant-like compound, may beadministered in combination with IGF-1, IGFBP-3, or ALP, preferablywith IGF-1. The rationale behind this combination treatment is toincrease the level of IGF-1, IGFBP-3, and/or ALP found to be low incachectic individuals.

In a further embodiment, the secretagogues, such as ghrelin splicevariant or a ghrelin splice variant-like compound, may beadministered in combination with compounds known to stimulateappetite, such as ghrelin, melanocortin receptor antagonists,neuropeptide Y receptor agonists including agonists selective forindividual subtypes of the neuropeptide Y receptors, leptin orleptin receptor agonists, cannabinoids including marijuana andmarijuana derivatives, antipsychotics, especially atypicalantipsychotics such as sertindole, Sufpirid, Clozapine,Risperidone, Quetiapin, Amisulpride, Ziprasidon, andOlanzapine.

2) Combinations of the secretagogue, such as ghrelin splice variantor a ghrelin splice variant-like compound, with an ingredient ortherapy active against a disease causing or being associated withthe disease or condition treated with the secretagogue, such asghrelin splice variant or a ghrelin splice variant-likecompound.

Particularly in relation to cancer cachexia, administration of asecretagogue, such as a ghrelin splice variant-like compound, maybe performed in combination with any anti-cancer therapy, includingantineoplastic chemotherapy, radiotherapy and surgical treatment.In particular, it is used in combination with chemotherapy andradiotherapy. Thus, one embodiment relates to a method of treatingcancer comprising administering an effective amount of radiotherapyand an effective amount of a secretagogue, such as a ghrelin splicevariant-like compound according to the present disclosure. Thetreatment with the secretagogue, such as a ghrelin splicevariant-like compound, may be started before the radiotherapytreatment initiates. It may be administered continuously during theradiotherapy or it may be administered at intervals, for examplebetween periods with radiotherapy therapy.

Another embodiment relates to a method of treating cancercomprising administering an effective amount of antineoplasticchemotherapy and an effective amount of a secretagogue, such as aghrelin splice variant-like compound according to the presentdisclosure. The treatment with the secretagogue, such as a ghrelinsplice variant-like compound, may be started before thechemotherapy treatment initiates. It may be administeredcontinuously during the chemotherapy, or it may be administered atintervals, for example between periods with chemotherapytherapy.

Furthermore, the combination treatment may be co-formulations ofthe secretagogue, such as a ghrelin splice variant-like compound,and the antineoplastic chemotherapy.

A secretagogue according to the present disclosure, such as ghrelinsplice variant or a ghrelin splice variant-like compound, may alsobe administered in combination with a pharmaceutically effectiveamount of glucocorticoid steroids and prokinetic treatment as wellas other treatment used in cancer therapy. Thus, in anotherpreferred embodiment, a secretagogue according to the presentdisclosure, such as ghrelin splice variant or a ghrelin splicevariant-like compound, is administered in combination with apharmaceutical effective amount of one or more of: progestationaldrugs, such as megastrol and/or cyproheptadines (and/or other 5-HTreceptor antagonists); and/or branched chain amino acids; and/oroxandralin; and/or anti-TNF-.alpha. agents, such as infliximab,etanercept, or adalimumab; and/or testosterone; and/or a "cocktail"comprising immunonutrition drugs, antioxidants and COX2 inhibitors;and/or cannabinoids; and/or eicosapentaenoic acid; and/ormelatonin; and/or thalidomide; and/or a .beta.2 adrenergic drug;most preferably for the treatment of cachexia, such as cancercachexia.

In yet another embodiment, the secretagogue, such as a ghrelinsplice variant-like compound, is administered in combination withanti-inflammatory compounds, preferably an NSAID, such asindomethacin, and COX1 inhibitors or COX2 inhibitors; and/oranti-TNF-.alpha. compounds such as infliximab, etanercept, oradalimumab. Another combination may be with erythropoietin/EPO.Another combination can be with angiotensin II lowering agents,such as Vitor. Another combination can be with selective androgenreceptor modulator(s). Another combination may be with one or moreof leptin, agonists of the renin-angiotensin system, opioidreceptor agonists or peroxisome proliferator-activated receptorgamma agonists.

In relation to treatment of lipodystrophy, another embodimentrelates to a treatment wherein a secretagogue, such as ghrelinsplice variant, more preferably a ghrelin splice variant-likecompound, is administered in combination with a lipodystrophytreatment, such as one or more of the treatments or compoundsdescribed herein suitable for treating a lipodystrophicsyndrome.

Thus, other pharmacologically active substances that may beadministered in combination with said secretagogue, such as aghrelin splice variant-like compound, in the methods of the presentdisclosure comprise: (a) Leptin: Leptin has been shown to have apositive effect on the metabolic abnormalities associated withlipodystrophy (Oral E. A. et al., J. Clin. Endocrinol. Metab.91:621-28 (2006)). This treatment has proven to be beneficial bothto those patients that suffer from a low plasma level of leptin andto those that have a normal level. (b) Peroxisomeproliferator-activated receptor (PPAR-.gamma.) agonists:PPAR-.gamma. has in several studies been demonstrated to beimportant for adipocyte metabolism and metabolic syndrome, and itis proposed that PPAR-.gamma. agonists will decrease the symptomsof lipodystrophy (Semple R. K. et al., J. Clin. Invest. 116:581-89(2006)). (c) Agonists of the renin-angiotensin system: It has beenshown that treatment with HAART increases the activity of ACE inthe T-cells, which means that agonists of the renin-angiotensinsystem may improve HAART induced lipodystrophy (Hegele R. A. &Leff T., J. Clin. Invest. 114:163-65 (2004)). (d) Opioid receptorantagonists: Opioid receptor antagonists, such as Naloxone andNaltrexone, have been shown to prolong the period of time fromprotease inhibitor treatment to development of the first symptomsof lipodystrophy (AIDS Patient Care STDS 14:283 (2000)). (e)Des-acyl ghrelin splice variant: Ghrelin splice variant incombination with des-acyl ghrelin splice variant has been found todecrease insulin resistance, which is an important feature of thelipodystrophy syndrome (Koutkia P. et al., Am. J. Physiol.Endocrinol. Metab. 286:E296-303 (2004)). (f) Adiponectin andanti-diabetic treatment including other compounds for the treatmentand/or prevention of insulin resistance and diseases whereininsulin resistance is the pathophysiological mechanism. (g) Therapywith rhGH has been reported to cause reduction in the size of"buffalo hump", truncal fat and to increase the lean body mass in asmall number of patients (Lo J. C. et al., J. Clin. Endocrinol.Metab. 86:3480-87 (2001)). However, fat loss and lipidabnormalities did not improve and blood glucose control worsened.Examples of syndromes treated with hGH include HIV, AIDS andcancer. Without being bound by theory, it is believed thattreatment with ghrelin splice variant or a analog thereof wouldmaintain and/or increase body fat in patients being treated withhGH, thereby effectively counteracting or at least reducinglipodystrophy caused by hGH. Thus, one preferred embodiment relatesto use of ghrelin splice variant or ghrelin splice variant-likecompound in combination with a growth hormone, preferably inindividuals suffering from HIV or AIDS and/or cancer cachexia. Saidtreatment with ghrelin splice variant or an analog thereof may beprior to, and/or during and/or after the individual is subjected totreatment with a growth hormone. Said growth hormone is preferablyhGH. (h) Treatment with combinations of different secretagogues asdescribed above under group 1), supra. 3) Combinations of thesecretagogue, such as ghrelin splice variant or a ghrelin splicevariant-like compound, with an ingredient active or therapy againstsymptoms associated with the disease or condition treated with thesecretagogue, such as ghrelin splice variant or a ghrelin splicevariant-like compound.

One aspect relates to combination treatment, wherein one of theingredients in the combination is used for treating symptoms orconditions that may be encountered in individuals suffering fromcachexia. Thus, uses and combination treatments involvingadministration of a secretagogue, such as the ghrelin splicevariant-like compound according to the present disclosure, can alsoinvolve treatment in combination with one or more of a) prophylaxisand/or alleviation and/or treatment of a clinical depression, whichcombination treatment further comprises administering anantidepressant, a prodrug thereof, or a pharmaceutical acceptablesalt of said antidepressant or said prodrug. In the abovecombination treatment, the antidepressant is preferably anorepinephrine reuptake inhibitor (NERI), a selective serotoninreuptake inhibitor (SSRI), a monoamine oxidase inhibitor (MAO), acombined NERI/SSRI, or an atypical antidepressant, a prodrug ofsaid antidepressant or a pharmaceutically acceptable salt of saidantidepressant or said prodrug. Preferred antidepressants are SSRI,a prodrug thereof or a pharmaceutical acceptable salt of said SSRIor said prodrug. The SSRI is preferably citalopram, escitalopram,femoxetine, fluoxetine, fluvoxamine, indalpine, indeloxazine,milnacipran, paroxetine, sertraline, sibutramine or zimeldine, aprodrug of said SSRI or a pharmaceutical acceptable salt of saidSSRI or said prodrug. Of the above, citalopram and escitalopram, aprodrug or a pharmaceutical acceptable salt thereof, are preferredin certain embodiments of combination treatments. b) prophylaxisand/or alleviation and/or treatment of an emetic condition,including nausea and vomiting, which combination treatment furthercomprises administering an antiemetic agent, a prodrug thereof, ora pharmaceutically acceptable salt of said antiemetic agent or saidprodrug. Preferred antiemetic agents used in combination treatmentsaccording to the present disclosure include meclizinehydrochloride, prochlorperazine, promethazine, trimethobenzamidehydrochloride and ondansetron hydrochloride. In particular, emesismay be caused by cancer, either due to the anti-cancer treatment ordue to the cancer disease as such. c) prophylaxis and/oralleviation and/or treatment of a psychotic condition, whichcombination treatment further comprises administering anantipsychotic agent, a prodrug thereof or a pharmaceuticalacceptable salt of said antipsychotic agent or said prodrug.Preferred antipsychotic agents used in combination treatments inaccordance with the present disclosure include chlorpromazine,haloperidol, clozapine, loxapine, molindone hydrochloride,thiothixene, olanzapine, ziprasidone, ziprasidone hydrochloride,prochlorperazine, perphenazine, trifluoperazine hydrochloride andrisperidone. d) prophylaxis and/or alleviation and/or treatment ofanxiety, which combination treatment further comprisesadministering an antianxiety agent, a prodrug thereof or apharmaceutically acceptable salt of said antianxiety agent or saidprodrug. Preferred antianxiety agents used in combinationtreatments in accordance with the present disclosure includealprazolam, clonazepam, lorazepam, oxazepam, chlordiazepoxidehydrochloride, diazepam, buspirone hydrochloride, doxepinhydrochloride, hydroxyzine pamoate and clonazepam.

Of course, combinations of the above groups (1-3) are also withinthe scope of this disclosure.

Medical Packaging

The compounds disclosed herein may be administered alone or incombination with pharmaceutically acceptable carriers orexcipients, in either single or multiple doses.

The formulations may conveniently be presented in unit dosage formby methods known to those skilled in the art.

It is preferred that the compounds according to the presentdisclosure are provided in a kit. Such a kit typically contains anactive compound in dosage forms for administration. A dosage formcontains a sufficient amount of active compound such that adesirable effect can be obtained when administered to a subject,preferably prior to at least one meal a day, more preferably priorto each main meal, such as three times a day, during the course of1 or more days. Thus, it is preferred that the medical packagingcomprises an amount of dosage units corresponding to the relevantdosage regimen. Accordingly, in one embodiment, the medicalpackaging comprises a pharmaceutical composition comprising acompound as defined above or a pharmaceutically acceptable saltthereof and pharmaceutically acceptable carriers, vehicles and/orexcipients, said packaging having from 7 to 21 dosage units, ormultiple thereof, thereby having dosage units for one week ofadministration or several weeks of administration.

In one embodiment, the medical packaging is for administration oncedaily in a week, and comprises 7 dosage units, in anotherembodiment the medical packaging is for administration twice daily,and comprises 14 dosage units. In yet another more preferredembodiment, the medical packaging is for administration three timesdaily, and comprises 21 dosage units.

The dosage units are as defined above, i.e. a dosage unitpreferably comprises an amount of the ghrelin splice variant-likecompound or a salt thereof equivalent to from 0.3 .mu.g to 600 mgghrelin splice variant, such as of from 2.0 .mu.g to 200 mg ghrelinsplice variant, such as from 5.0 .mu.g to 100 mg ghrelin splicevariant, such as from 10 .mu.g to 50 mg ghrelin splice variant,such as from 10 .mu.g to 5 mg ghrelin splice variant, such as from10 .mu.g to 1.0 mg ghrelin splice variant.

The medical packaging may be in any suitable form for parenteral,in particular subcutaneous, administration. In a preferredembodiment, the packaging is in the form of a cartridge, such as acartridge for an injection pen, the injection pen being such as aninjection pen known from insulin treatment or from hGHtreatment.

When the medical packaging comprises more than one dosage unit, itis preferred that the medical packaging is provided with amechanism to adjust each administration to one dosage unitonly.

Preferably, a kit contains instructions indicating the use of thedosage form to achieve a desirable affect and the amount of dosageform to be taken over a specified time period. Accordingly, in oneembodiment the medical packaging comprises instructions foradministering the pharmaceutical composition. In particular saidinstructions may include instructions referring to administrationof said pharmaceutical composition either during a meal, orpreferably at the most 45 minutes prior to a meal, such as at themost 30 minutes prior to a meal, such as at the most 25 minutesprior to a meal, such as at the most 20 minutes prior to a meal,such as at the most 15 minutes prior to a meal, such as at the most10 minutes prior to a meal, such as at the most 5 minutes prior toa meal.

Method for Monitoring the Effect of Treatment with Ghrelin SpliceVariant and/or a Ghrelin Splice Variant-Like Compound

Another aspect relates to a method for monitoring the effect of theadministration of a secretagogue, such as the ghrelin splicevariant-like compounds disclosed herein, in a method of the presentdisclosure, comprising measuring one or more markers, in particularmarkers, selected from GH, IGF-1, IGFBP-3, ALP (acidic labeled),thyroid hormones, sex hormones, and albumin; more preferablyselected from IGF-1, IGFBP-3, ALP (acidic labeled); even morepreferably IGF-1. These markers are all low in cachetic patientsand are expected to increase after treatment with ghrelin splicevariant. Other markers that are expected to increase aftertreatment with ghrelin splice variant are the blood GH level andthe body weight. In addition, the body composition is expected tochange, and the lean body mass is expected to increase. The bodycomposition changes can be assessed by using MRI or NMR.

Thus, one embodiment relates to a method for monitoring the effectof any of the treatments of an individual with a secretagoguedescribed herein, said method comprising measuring the blood levelof said individual of one or more of: (i) IGF-1 and/or (ii) IGFBP-3and/or (iii) ALP and/or (iv) one or more thyroid hormones and/or(v) one or more sex hormones and/or (vi) albumin or, morepreferably, one or more of: (i) IGF-1 and/or (ii) IGFBP-3 and/or(iii) ALP and/or (iv) GH and/or (v) body weight and/or (vi) bodycomposition.

Methods for measuring substances in the blood level of anindividual are well known in the art. As an example, an isolatedblood sample may be tested by methods such as Western blot or byenzyme-linked assay (ELISA).

EXAMPLES

The present disclosure is further defined in the followingExamples. It should be understood that these Examples, whileindicating preferred embodiments, are given by way of illustrationonly. From the above discussion and these Examples, one skilled inthe art can ascertain the preferred features of this disclosure,and without departing from the spirit and scope thereof, can makevarious changes and modifications to adapt it to various uses andconditions.

Examples 2, 5, 6, 7, 8, and 9 are working examples. Examples 1, 3,4, 10, and 11 are prophetic examples.

Example 1

Competition Binding Assays

Transfected COS-7 cells are transferred to culture plates one dayafter transfection at a density of 1.times.10.sup.5 cells per wellaiming at 5-8% binding of the radioactive ligand. Two days aftertransfection, competition binding experiments are performed for 3hours at 4.degree. C. using 25 .mu.M of [.sup.125I]Ghrelin (GEHealthcare, Piscataway, N.J., USA). Binding assays are performed in0.5 ml of a 50 mM Hepes buffer, pH 7.4, supplemented with 1 mMCaCl.sub.2, 5 mM MgCl.sub.2, and 0.1% (w/v) bovine serum albumin,40 .mu.g/ml bacitracin. Non-specific binding is determined as thebinding in the presence of 1 micromole of unlabeled ghrelin splicevariant. Cells are washed twice in 0.5 ml of ice-cold buffer and0.5-1 ml of lysis buffer (8 M Urea, 2% NP40 in 3 M acetic acid) isadded and the bound radioactivity is counted. Determinations aremade in duplicate.

Example 2

Synthetic Production of Ghrelin Splice Variant-Like Compound

Amino acid derivatives and synthesis reagents can be obtained fromcommercial sources. Peptide chain extension can be performed usingApplied Biosystem 433A synthesizer produced by Perkin Elmer, and aprotected peptide derivative-resin can be constructed by the Boc orFmoc method. The protected peptide resin obtained by the Boc methodis deprotected with anhydrous hydrogen fluoride (HF) in thepresence of p-cresol thereby releasing the peptide, which is thenpurified. The protected peptide resin obtained by the Fmoc methodis deprotected with trifluoroacetic acid (TFA) or dilute TFAcontaining various scavengers, and the released peptide ispurified. Purification is performed in reversed phase HPLC on a C4or C18 column. The purity of the purified product can be confirmedby reverse phase HPLC, and its structure can be confirmed by aminoacid composition analysis and mass spectrometry.

Peptides disclosed herein can be produced by a conventional peptidesynthesis method. Specifically, synthesis of acylated or alkylatedpeptides is exemplified below.

Abbreviations: "HMP resin" means 4-hydroxymethyl-phenoxymethylresin; "Fmoc amide resin" means4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamido-ethylresin; "PAM resin" means phenylacetoamidomethyl resin; "HBTU" means2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate; "TBTU" means2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate; "HOBt" means 1-hydroxybenzotriazole; "DCC" meansdicyclohexylcarbodiimide; "DIPCI" means diisopropylcarbodiimide;"TFA" means trifluoroacetic acid; "DIPEA" meansdiisopropylethylamine; "TIPS" means triisopropylsilane; "Fmoc"means fluorenylmethoxycarbonyl; "Boc" means t-butyloxycarbonyl;"Trt" means trityl; "Bu" means t-butyl; "Pmc" means2,2,5,7,8-pentamethylchroman-6-sulfonyl; "Prl" means propionyl;"PhPrl" means phenylpropionyl; "Bzl" means benzyl; "Bom" meansbenzyloxymethyl; "Tos" means toluenesulfonyl; "Cl--Z" means2-chloro-benzyloxycarbonyl; "Pis" means 2-phenylisopropyl; "Mtt"means 4-methyltrityl; "DMF" means N,N-dimethylformamide; "NMP"means N-methylpyrrolidone; "DMAP" means 4-dimethylaminopyridine;"HOSu" means N-hydroxysucciniimide; "Adod" means 2-aminododecanoicacid; "Aib" means 2-aminoisobutylic acid; "Ape" means5-aminopentanoic acid; "Cha" means cyclohexylalanine; "Dap" means2,3-diaminopropionic acid; "NaI" means naphtylalanine; "Nie" meansnorleucine.

Protecting amino acids which can be used in synthesis Fmoc method:Boc-Gly, Fmoc-Gly, Fmoc-Ser (Bu), Fmoc-Ser (Trt), Fmoc-Glu (OBu),Fmoc-His (Boc), Fmoc-Gln (Trt), Fmoc-Arg (Pmc), Fmoc-Lys (Boc),Fmoc-Pro, Fmoc-Leu, Fmoc-Ala, Fmoc-Val, Fmoc-Phe, Fmoc-Phe,Fmoc-Ser (n-C.sub.8H.sub.17), Fmoc-Ser (n-C.sub.8H.sub.17),Fmoc-Cys (n-C.sub.8H.sub.17), Fmoc-Asp (OP is), Fmoc-Ser (Bzl),Fmoc-Cys (Trt), Fmoc-Dap (Octanoyl), Fmoc-2-Nal, Fmoc-2-Nal,Fmoc-Nle, Fmoc-Lys (Mtt), Fmoc-Aib-OH, Fmoc-Asp(O--C.sub.7H.sub.15). Boc method: Boc-Gly, Boc-Ser (Bzl), Boc-Ser(Ac), Boc-Ser (Prl), Boc-Glu (OBzl), Boc-His (Bom), Boc-Gln,Boc-Arg (Tos), Boc-Lys (Cl--Z), Boc-Pro, Boc-Leu, Boc-Ala, Boc-Val,Boc-Phe, Boc-Cys (n-C.sub.8H.sub.17), Boc-Ape, Boc-Ser(n-C.sub.8H.sub.17)

Units used: (a) Analytical HPLC system Unit: Shimadzu LC-10ASystem; Column: YMC PROTEIN-RP (4.6 mm.times.150 mm); Columntemperature: 40.degree. C.; Eluent: A linear gradient of from 0 to50% acetonitrile for 20 minutes in 0.1% trifluoroacetic acid; Flowrate: 1 mL/min; Detection: UV (210 nm); Injection volume: 10 to 100mu l. (b) Preparative HPLC system Unit: Waters 600 MultisolventDelivery System; Columns: YMC-Pack-ODS-A (5 mu m, 20 mm.times.250mm) YMC-Pack-PROTEIN-RP (5 mu m, C4, 10 mm.times.250 mm) YMC-PackPROTEIN-RP (5 mu m, C4, 20 mm.times.250 mm) YMC PROTEIN-RP (4.6mm.times.150 mm); Eluent: A suitable linear gradient ofacetonitrile concentration in 0.1% trifluoroacetic acid; Flow rate:10 mL/min. (for columns of an inner diameter of 20 mm), 3 mL/min.(for the column of an inner diameter of 10 mm), 1 mL/min. (for thecolumn of an inner diameter of 4.6 mm); Detection: 210 nm, 260 nm;Injection: 10 to 2000 mu 1 (2000 mu l or more was injected via apump) (c) Mass spectrometer Unit: Finnigan MAT TSQ700; Ion source:ESI; Detection ion mode: Positive Spray; Voltage: 4.5 kV; Capillarytemperature: 250.degree. C.; Mobile phase: A mixture of 0.2% aceticacid and methanol (1:1); Flow rate: 0.2 mL/min; Scan range: m/z 300to 1,500 (d) Analysis of amino acid sequence Unit: AppliedBiosystem 477A, 492 model sequencer manufactured by Perkin Elmer(e) Analysis of amino acid composition Unit: L-8500 model aminoacid analyzer manufactured by Hitachi, Co., Ltd.; Sample: Unlessotherwise specified, the sample is hydrolyzed with 6 M HCl at110.degree. C. for 24 hours in a sealed tube. Example of Synthesisof a Derivative having Acyl Serine (Fmoc Method, Carboxyl-TerminalAmide Derivatives) Ghrelin Splice Variant

GSS (CO--C.sub.7H.sub.15) FLSPEHQRVQVRPPHKAPH Fmoc-His(Pmc)-HMP-resin (403 mg, 0.25 mmol, ABI Co., Ltd.) is treated with20% piperazine for 20 minutes and subjected repeatedly tointroduction of Fmoc-amino acid by HBTU/HOBt and elimination ofFmoc by piperazine sequentially to construct Fmoc-Ser (Bu)-Ser(Trt)-Phe-Leu-Ser (tBu)-Pro-Glu (OBu)-His (Boc)-Gln (Trt)-Arg(Pmc)-Val-Gln-Val (Trt)-Arg (Pmc)-Pro-Pro-His (Boc)-Lys (Boc)-Ala(Boc)-Pro (Boc)-Pro-His (Pmc)-resin. After Boc-Gly is finallyintroduced by DCC/HOBt, the resulting protected peptide resin (1.3g) is treated with 1% TFA-5% TIPS-methylene chloride solution (15mL) for 30 minutes.

The peptide resin is filtrated, washed several times with methylenechloride (30 mL), and washed with 5% DI EA (10 mL) and then withmethylene chloride (30 mL). The resulting de-Trt peptide resin(about 1.3 g) is swollen with NMP (10 mL), and octanoic acid (144.2mg, 1.0 mmol) and DIPCI (126.2 mg, 1.0 mmol) are added thereto inthe presence of DMAP (61.1 mg, 0.5 mmol) and allowed to react for 8hours. The resin is recovered by filtration and washed with NMP andthen with methylene chloride, followed by drying under vacuum togive about 1.2 g protected peptide resin wherein the side chain ofthird serine is octanoylated. To this product is added ade-protecting reagent (10 mL) consisting of 88% TFA-5% phenol-2%TIPS-5% H.sub.2O, and the mixture is stirred at room temperaturefor 2 hours. The resin is removed by filtration, and the filtrateis concentrated followed by adding ether to the resulting residuesto form precipitates. The precipitates are recovered by filtrationand dried to give about 550 mg crude peptide. 200 mg of thisproduct is dissolved in 10 mL water and applied to YMC-PackPROTEIN-RP (C4, 20 mm.times.250 mm) and eluted with a lineargradient (flow rate: 10 mL/min.) for 60 minutes of from 0 to 54%acetonitrile in 0.1% trifluoroacetic acid. The desired fractionsare collected and lyophilized to give about 120 mg of the desiredproduct.

Example of Synthesis of a Derivative having Acyl Serine (FmocMethod, Carboxyl-Terminal Amide Compounds) Ghrelin Splice Variant(1-22)-NH.sub.2

GSS (CO--C.sub.7H.sub.15) FLSPEHQRVQVRPPHKAPH-NH.sub.2Fmoc-amide-resin (403 mg, 0.25 mmol, ABI Co., Ltd.) is treated with20% piperazine for 20 minutes and subjected repeatedly tointroduction of Fmoc-amino acid by HBTU/HOBt and elimination ofFmoc by piperazine sequentially to construct Fmoc-Ser (Bu)-Ser(Trt)-Phe-Leu-Ser (Bu)-Pro-Glu (OBu)-His (Boc)-Gln (Trt)-Arg(Pmc)-Val-Gln-Val (Trt)-Arg (Pmc)-Pro-Pro-His (Boc)-Lys (Boc)-Ala(Boc)-Pro (Boc)-Pro-His (Boc)-resin. After Boc-Gly is finallyintroduced by DCC/HOBt, the resulting protected peptide resin(about 550 mg) is treated with 1% TFA-5% TIPS-methylene chloridesolution (10 mL) for 30 minutes. The peptide resin is recovered byfiltration, washed several times with methylene chloride (30 mL),and washed with 5% DIEA (10 mL) and then with methylene chloride(30 mL). The resulting de-Trt peptide resin (about 750 mg) isswollen with NMP (10 mL), and octanoic acid (144.2 mg, 1.0 mmol)and DIPCI (126.2 mg, 1 mmol) are added thereto in the presence ofDMAP (61.1 mg, 0.5 mmol) and allowed to react for 4 hours. Theresin is recovered by filtration and washed with NMP and then withmethylene chloride, followed by drying under vacuum to give about800 mg protected peptide resin wherein the side chain of thirdserine is octanoylated. TFA (10 mL) is added to this product andstirred at room temperature for 30 minutes. The resin is removed byfiltration, and the filtrate is then concentrated followed byadding ether to the resulting residues to form precipitates. Theprecipitates are recovered by filtration and dried to give about250 mg crude peptide. About 200 mg of this product is dissolved in10 mL of 30% aqueous acetic acid and applied to YMC-Pack PROTEIN-RP(C4, 20 mm.times.250 mm) and eluted with a linear gradient (flowrate: 10 mL/min.) for 60 minutes of from 0 to 54% acetonitrile in0.1% trifluoroacetic acid. The desired fractions are collected andthen lyophilized to give about 150 mg of the desired product.

Example of Synthesis of a Derivative Having Acyl Serine (BocMethod) [Ser3 (Propionyl)]-Ghrelin Splice Variant (1-22)

GSS (CO--CH.sub.2CH.sub.3) FLSPEHQRVQVRPPHKAPH protected ghrelinsplice variant resin (4-22) is constructed from Boc-His (Tos)-Pamresin (0.75 g, 0.5 mmol) by Boc chemistry, and Boc-Ser(CO--CH.sub.2CH.sub.3)--OH, Boc-Ser (Bzl)-OH and Boc-Gly-OH arecondensed with a half (1.4 g) of the resin. The resulting resin,1.5 g, is then treated with a mixture of HF and p-cresol (8.5mL:1.5 mL) at 0.degree. C. for 1 hour, and the HF is evaporated.Ether is added to the residues, whereby 671 mg crude peptide isobtained. This sample is then dissolved in 50% acetic acid (AcOH)and applied to a preparative column YMC-Pack-ODS-A (5 mu m, 20mm.times.250 mm) and eluted at a rate of 10 mL/min by a gradient offrom 0 to 95% acetonitrile concentration in 0.1% TFA solution for75 minutes. Those fractions containing the desired product arelyophilized to give approximately 135.8 mg crude peptide. A part(0.5 mg) of this product is applied to YMC-A-302 column (C18, 4.6mm.times.150 mm) and eluted at a flow rate of 1 mL/min. by agradient of from 15 to 19% concentration acetonitrile. Thispurification procedure is then repeated and the desired fractionsare combined to give approximately 0.41 mg of the desiredproduct.

Other compounds according to the present disclosure can be producedlikewise.

The above method was used to synthesize acylated and non-acylatedSEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:5.

Example 3

A Randomized, Single Center, Four-Period Crossover Trial toInvestigate the Absolute Bioavailability of IntravenouslyAdministered Ghrelin Splice Variant and Subcutaneously AdministeredGhrelin Splice Variant at Three Different Single Doses in HealthySubjects

Objectives:

Primary: To investigate the absolute bioavailability of threedifferent doses of ghrelin splice variant administered as singleintravenous and subcutaneous doses. Secondary: 1) To investigatethe dose linearity (dose proportionality) of the ascending doses.2) To investigate and compare the pharmacodynamic profiles betweenthe treatments. 3) To assess the safety and local tolerability.Trial Design: A randomized, single center, unbalanced block design,four-period crossover trial to investigate the absolutebioavailability between intravenously administered ghrelin splicevariant and subcutaneously administered ghrelin splice variant atthree different single doses in healthy subjects. Three doses willbe used for each way of administration: low, medium and high. Toreduce the number of dosings to each individual and hence reducethe length of the trial, each subject will only receive four dosesof the total of six doses, i.e. two dose levels administered asintravenous and subcutaneous, respectively. The unbalanced blockdesign will ensure that all three-dose levels will be covered inthis way although not all subjects will receive all dose levels. Asufficient washout period will be placed between the individualdosing periods. Endpoints: Pharmacokinetics of ghrelin splicevariant: AUC.sub.0-t, AUC, C.sub.max, t.sub.max, t, C.sub.l/f,V.sub.z/f, C.sub.l, V.sub.z, t.sub.1/z MRT Pharmacodynamics: GH:AUC, C.sub.max and t.sub.max Cardiac output, assessment of hunger,food/energy intake, degree of pleasure related to food intake, bodymass, energy expenditure, DEXA. Safety: Safety and localtolerability will be assessed throughout the study by clinicalevaluations (physical examination and vital signs),electrocardiography and laboratory tests (hematology and clinicalchemistry). Trial population and power calculation: Healthy malesubjects, aged 18-45 years with a body mass index (BMI) of 19-26kg/m.sup.2 (both inclusive).

The primary objective of this study is to investigate the absolutebioavailability of ghrelin splice variant administered viaintravenous and subcutaneous administration. An unbalanced blockdesign will be applied to reduce the trial period time and reducethe number of dosings per subject. The number of subjects needed toperform a statistical analysis of absolute bioavailability per doselevels as well as an analysis of dose linearity between doses willbe calculated based on existing literature data.

Trial products: ghrelin splice variant for intravenous andsubcutaneous administration.

Example 4

Functional Tests on the Ghrelin Receptor

Transfections and tissue culture-COS-7 cells are grown inDulbecco's modified Eagle's medium 1885 supplemented with 10% fetalcalf serum, 2 mM glutamine and 0.01 mg/ml gentamicin. Cells aretransfected using calcium phosphate precipitation method withchloroquine addition as previously described (Holst B. et al., Mol.Pharmacol. 53:166-175 (1998)). For gene dose experiments, variableamounts of DNA are used. The amount of cDNA (20 .mu.g/75 cm.sup.2)resulting in maximal signaling is used for dose response curves.HEK-293 cells are grown in D-MEM, Dulbecco's modified Eagle'smedium 31966 with high glucose supplemented with 10% fetal calfserum, 2 mM glutamine and 0.01 mg/ml gentamicin. Cells aretransfected with Lipofectamine.TM. 2000 (Invitrogen Corp.,Carlsbad, Cal.).

Phosphatidylinositol turnover: One day after transfection, COS-7cells are incubated for 24 hours with 5 .mu.Ci of [3H]-myo-inositol(GE Healthcare, Piscataway, N.J.) in 1 ml medium supplemented with10% fetal calf serum, 2 mM glutamine and 0.01 mg/ml gentamicin perwell. Cells are washed twice in buffer, 20 mM HEPES, pH 7.4,supplemented with 140 mM NaCl, 5 mM KCl, 1 mM MgSO.sub.4, 1 mMCaCl.sub.2, 10 mM glucose, 0.05% (w/v) bovine serum; and areincubated in 0.5 ml buffer supplemented with 10 mM LiCl at37.degree. C. for 30 min. After stimulation with variousconcentrations of peptide for 45 min at 37.degree. C., cells areextracted with 10% ice-cold perchloric acid followed by incubationon ice for 30 min. The resulting supernatants are neutralized withKOH in HEPES buffer, and the generated [3H]-inositol phosphate ispurified on Bio-Rad AG 1-X8 anion-exchange resin (Bio-RadLaboratories, Hercules, Cal.) as per manufacturer's instructions.Determinations are made in duplicates. CRE, SRE and NF-.kappa.-Breporter assay: HEK293 cells (30,000 cells/well) seeded in 96-wellplates are transiently transfected. In case of the CRE reporterassay, the cells are transfected with a mixture of pFA2-CREB andpFR-Luc reporter plasmid (PathDetect CREB trans-Reporting System,Stratagene, La Jolla, Cal.) or SRE-Luc (PathDetect SRECis-Reporting System, Stratagene, La Jolla, Cal.) and the indicatedamounts of receptor DNA. Following transfection, cells aremaintained in low serum (2.5%) throughout the experiments and aretreated with the respective inhibitor of intracellular signalingpathways. One day after transfection, cells are treated with therespective ligands in an assay volume of 100 .mu.l medium for 5hrs. The assay is terminated by washing the cells twice with PBSand addition of 100 .mu.l Luciferase.RTM. assay reagent(LucLite.RTM., PerkinElmer, Inc., Wellesley, Mass.). Luminescenceis measured in a TopCounter (Top Count NETT, Packard InstrumentCo., Meriden, Conn.) for 5 sec. Luminescence values are given asrelative light units (RLU). MAP Kinase assay: COS 7 cells (seedingdensity 150,000 cells/well) are transfected in the assay plates.Two days after transfection, the indicated concentration of ligandare added to assay medium without any serum and incubated for 10min at 37.degree. C. The reaction is stopped by removing the mediumand two washing steps with ice cold PBS. The cells are lysed insample buffer and separated on 10% SDS-PAGE according to Laemmli U.K., Nature 227:680-85 (1970). Proteins are transferred ontonitrocellulose and Western blot analysis carried out using a 1:5000dilution of mouse monoclonal antiphospho-ERK1/2 antibody (SantaCruz Biotechnology, Inc., Santa Cruz, Cal.). Total ERK protein isdetermined using a 1:10000 dilution of anti-ERK antibody (SantaCruz Biotechnology, Inc., Santa Cruz, Cal.). Blots are probed withanti-mouse horseradish peroxidase-conjugated secondary antibodies,visualized using enhanced chemiluminescence reagent (GE Healthcare,Piscataway, N.J.) and quantified by densiometric analysis. ERK1/2phosphorylation is normalized according to the loading of proteinby expressing the data as a ratio of phosphoERK1/2 over totalERK1/2. Results are expressed as percentage of the value obtainedin non-stimulated mock transfected cells.

Example 5

Efficacy of Subcutaneous Administration of Acylated Ghrelin SpliceVariant on Weight Gain and Food Consumption

Acylated ghrelin splice variant (20 .mu.g (FIGS. 1A and 1B) or 180.mu.g (FIGS. 2A and 2B)) or the Vehicle (1.6% mannitol) wereadministered once daily for 14 successive days (FIGS. 1A and 1B) or7 successive days (FIGS. 2A and 2B), via the subcutaneous (SC)route, to groups comprising n=10 (FIGS. 1A and 1B) or n=8 (FIGS. 2Aand 2B) 129Sv male mice. No mortality occurred in any of theanimals throughout the entire study period. No clinical signs wereobserved in any of the animals throughout the entire study period.All animals were subjected to terminal bleeding, under CO.sub.2anesthesia, immediately prior to euthanasia. Terminal bloodcollection was performed serially as per animal number, and not asper group.

Biochemistry: Blood for biochemistry analysis was collected intonon-coated pre-labeled tubes. The tubes were pre-labeled andcontained the following information: Study number, group number,animal number and date. Following clotting, the blood from eachanimal was centrifuged, and the serum was collected into twopre-labeled tubes and submitted for analysis as follows: Serum, 250.mu.l, was kept at 2-8.degree. C. until analysis. The samples weresubjected to the following listed tests using a Hitachi 917 system:Creatinine, Total bilirubin, Glucose, Triglycerides, Cholesterol,HDL, LDL, Total protein, Globulin, Albumin, Urea, Potassium,Phosphorus, Calcium, Sodium, Chloride, sGOT, sGPT, ALP. Urinalysis:Urine was collected into pre-labeled tubes (as above) from allanimals (where possible) prior and/or after euthanasia. For allsurviving animals, urine collection was performed serially as peranimal number, and not as per group. An attempt was made to attainthe maximal amount as possible to perform the tests listed below.Urinalysis is performed using a commercial test stick (Bayer,Multistix.RTM. 10SG) applied to urine sample and evaluating thefollowing parameters: glucose, ketone, pH value, leukocytes, blood,density, nitrite, bilirubin, urobilinogen and protein. NecropsyProcedures and Macroscopic Examination: All animals were subjectedto a full detailed necropsy. For all surviving animals, necropsywas performed serially as per animal number, and not as per group,immediately following the scheduled terminal bleeding. At necropsy,a thorough examination is made and any abnormality or grosspathological changes in tissues and/or organs are observed andrecorded. Organ/Tissue Collection: The following organs andtissues: Brain, Liver, Kidney, Stomach, Pancreas, Lungs, Spleen,Heart, Epididymal WAT, Retroperitoneal WAT, Interscapular BAT wereexcised and weighed wet as soon as possible after excision andremoval of the attached fat and other connective tissues. Allorgans from one animal were collected into one container,pre-labeled with the following information: Study number, groupnumber, animal number and date. Body composition assessment: On thefirst and the last day of treatment NMR was used in order toanalyze the change in either fat or lean body mass (FIG. 4).Results: The cumulative body weight gain of the acylated ghrelinsplice variant-treated 129Sv mice was significantly higher(p=6E.sup.-05) than the Vehicle-treated controls (2.2 g and 0.7 g,respectively). See FIG. 1A. The cumulative food consumption of theacylated ghrelin splice variant-treated 129Sv mice wassignificantly higher (p=0.04) than the Vehicle-treated controls(53.2 g and 47.21 g, respectively). See FIG. 1B. No mortalityincidence and no evidence as to obvious clinical signs in reactionto treatment were noted among any of the test animals throughoutthe entire study period. Based on the above study results, humanacylated ghrelin splice variant significantly promotes body weightgain and food consumption.

Example 6

Effect of Subcutaneous Administration of Acylated Ghrelin SpiceVariant on GH Release

Acylated ghrelin splice variants (20 .mu.g) or the Vehicle (1.6%mannitol) were administered by a subcutaneous bolus injection(Corresponding to 0.3 .mu.mol/kg) to 5 mice each. Blood was sampled10 and 20 min after injection. Serum samples were stored at-70.degree. C. and analyzed by the DSL-10-72100 ACTIVE.RTM.Mouse/Rat Growth Hormone ELISA kit (Diagnostic SystemsLaboratories, Inc., Webster, Tex.).

Results: The serum growth hormone concentration 10 minutes aftersubcutaneous administration of acylated ghrelin splice variant orthe Vehicle was 2-14-fold higher in the ghrelin splice variantgroup in comparison to the vehicle group (see FIG. 3).

Example 7

Pharmacokinetics of Acylated Ghrelin Splice Variant Range Finderinto Rat

Subcutaneous administration of ghrelin splice variants wasperformed at three dose levels of 0.5, 2.5 and 10 mg/kgcorresponding to concentrations of 0.1, 0.5 and 2 mg/ml,respectively, and at a constant volume dosage of 5 ml/kg.Intravenous injection was performed at one dose level of 0.5 mg/kgcorresponding to a concentration of 0.1 mg/ml and a constant volumedosage of 5 ml/kg as well. The study comprised 9 male and 9 femaleSprague-Dawley.TM. (SD.TM.) rats per dose level and route ofadministration.

Bleeding sampling design was confined to 9 bleeding time points foreach dose level: pre-dosing, 5, 15, 30, 60 and 90 min, 3, 5 and 24hrs, post-dosing. Each group was divided into 3 sub-groups, eachbeing assigned 3 specific bleeding time points, in order to receive3 individual samples/time point/group (total of 27 individualsamples/group). Mean group body weight values at initiation of thestudy were similar among all groups and did not exceed .+-.20% ofthe mean weight per gender. Whole blood samples were kept on icefrom the time of blood collection until the time of centrifugation.The obtained plasma samples were flash frozen in liquid nitrogenand kept on dry ice until removal to -70.degree. C.

Concentrations of the Test Item in plasma were determined byLC/MS/MS (liquid chromatography/mass spectrometry/massspectrometry). Pharmacokinetic analysis of Ghrelin Splice Variantwas based on mean plasma concentration time profiles for each dosegroup as obtained by Non-compartmental Pharmacokinetic analysis,generated by the use of the computer software: "PK Solutions 2.0"(Summit Research Services, CO, USA).

Pharmacokinetic analysis for the SC route showed thatAUC.sub.0-.infin. values were similar for males and femalesadministered the low dose (6.1 and 5.2 mcgmin/ml, respectively) andmid dose (18.8 and 20.8 mcgmin/ml, respectively). At the high dose,AUC values of the females were substantially lower (49.1 mcgmin/ml)than that of males (79.2 mcgmin/ml). T.sub.max occurred at 5minutes post-dosing for all dose groups. T.sub.1/2 values rangedfrom 17.4 to 26.4 minutes for male rats and 10.7 to 28.9 minutesfor female rats.

Example 8

Effect of Single Dose Acute Subcutaneous Administration of AcylatedGhrelin Splice Variant on Toxicity in Rats

Acylated ghrelin splice variant (2.5; 15 and 75 .mu.g) or theVehicle (saline) were administered once via the subcutaneous (SC)route, to groups comprising n=6 Sprague-Dawley.TM. (SD.TM.) rats.No mortality occurred in any of the animals throughout the entirestudy period. No clinical signs were observed in any of the animalsthroughout the entire study period. All animals were subjected toterminal bleeding, under CO.sub.2 anesthesia, immediately prior toeuthanasia.

Clinical Signs:

Animals were observed individually after dosing at least onceduring the first 30 minutes, periodically during the first 24hours, with special attention given during the first 4 hours, andclinical signs are recorded. Thereafter, animals were inspected andclinical signs were recorded once daily for a total of 14 days.Observations included changes in skin, fur, eyes, mucous membranes,occurrence of secretions and excretions (e.g. diarrhea) andautonomic activity (e.g. lacrimation, salivation, piloerection,pupil size, unusual respiratory pattern). Changes in gait, postureand response to handling, as well as the presence of bizarrebehavior, tremors, convulsions, sleep and coma were also included.Body Weight: Determination of individual body weights of animalswas made shortly before ghrelin splice variant administration (Day0), 2, 7 and 14 days following dosing. Fasted body weightmeasurements were taken just prior to necropsy. Clinical Pathology:Hematology, biochemistry and coagulation parameters listed downwere determined from all surviving animals prior to the scheduledeuthanasia. Hematology: Blood samples were obtained followingovernight food deprivation. Blood samples (at least 500 .mu.l ofwhole blood) were collected into pre labeled EDTA coated tubescontain the following information: Study number, group number,animal number and date. The samples were kept until analysis at2-8.degree. C. Hematology parameters that are tested using ADVIA120 Hematology System (Beyer) are: WBC, RBC, HGB, HCT, MCV, MCH,MCHC, Platelets, differential count. Reticulocytes were countedmanually. Biochemistry: Blood for biochemistry analysis wascollected into non-coated pre-labeled tubes. The tubes werepre-labeled and contained the following information: Study number,group number, animal number and date. Following clotting, the bloodfrom each animal was centrifuged, and 300 .mu.l serum was collectedinto two pre-labeled tubes and submitted for analysis while kept at2-8.degree. C. until analysis. The samples were subjected to thefollowing listed tests using a HITACHI MODULAR P-800 system:Creatinine, Calcium, Glucose, Cholesterol, Total Protein, Globulin,LDH, Potassium, Aspartate, Aminotransferase (AST), CPK, Phosphorus,Urea, Albumin, Total Bilirubin, Alanine, Aminotransferase (ALT),Sodium, .gamma.-Glutamyl Transpeptidase (GGT), Chloride,Triglycerides, High Density Lipoprotein (HDL), Low DensityLipoprotein (LDL), Alkaline Phosphatase (ALP). Coagulationparameters: Blood for coagulation analysis was collected byretro-orbital sinus bleeding under CO.sub.2 anesthesia intotri-sodium citrate coated tubes. Following completion of bloodcollection, all blood and serum samples were kept at 2-8.degree. C.until further analysis. The samples were subjected to the followinglisted tests using the Sysmex CA-1500 system: PT, APTT. Urinalysis:Individual samples of voided urine were collected from all animals(if applicable), prior to the scheduled study termination or priorto sacrifice in case of removal from the study for animal welfarereasons, by either pressing the abdominal area over the bladder orby collecting voided urine, otherwise, directly from the bladder bycystocentesis. Analysis was performed using a commercial test kit(Bayer Multistix.RTM. 10 SG) applied to void urine sample andevaluating the following parameters: glucose, ketone, pH,leukocyte, blood, density, nitrite, bilirubin, urobilinogen andprotein. Necropsy Procedures and Macroscopic Examination: Allanimals were subjected to gross necropsy following the scheduledeuthanasia. At necropsy, all animals were subjected to thoroughexamination, including the external surface of the body, allorifices, cranial, thoracic and abdominal cavities and theircontents. Any abnormality or gross pathological changes in tissuesand/or organs are observed and recorded. It is noted if a grosspathological abnormality occurs on the external body surface thatis located at or near the injection site. The following tissuesand/or organs including those with macroscopic abnormalities arepreserved in 4% formaldehyde solution: Adrenals, Aorta thoracic,Brain, Ceccum, Colon, Duodenum, Epididymis, Eyes, Harderian glands,Heart, Femur and knee joint, Ileum, Jejunum, Kidneys, Lachrymalglands, Liver, Lungs, Lymph nodes--superficial cervical, Lymphnodes--mesenteric, Mammary gland+skin, Oesophagus, Optic nerves,Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands,Sciatic nerve, Seminal vesicles, Skeletal muscle (thigh),Skin--injection site, Spleen, Spinal Cord (cervical, thoracic,lumbar), Sternum (bone marrow), Stomach, Testes, Thymus, Thyroid(with parathyroid if applicable), Tongue, Trachea, Urinary bladder,Uterus with cervix, Vagina. Organ/Tissue Weighing and Organ/TissueFixation: Organ/Tissue weighing and fixation was performed for allanimals. The organs listed above were weighed wet as soon aspossible after dissection and removal of the attached fat andconnective tissues (in the case of paired organs, the individualweights were determined but presented as the mean organ weight).All organs/tissues were then fixed and preserved in 4% formaldehydesolution (excluding the eyes, optic nerves and harderian glandswhich are fixed in Davidson's solution) for at least 48-hr fixationperiod prior tissues' delivery. In addition, any otherorgans/tissues with gross macroscopic changes were preserved aswell in 4% formaldehyde solution. The organs/tissues per animal arepacked in plastic containers in which each container is identifiedby the Study No., Group No., Animal No. & Date of necropsy.Bone marrow is obtained from a single femur, moistened by a foetalbovine serum to allow an appropriate smearing and smeared onto aclean and labeled glass slide by the use of a second glass slide.Thereafter, slides are left in the open air to dry and dipped inMethanol for about 5 minutes for appropriate fixation. At least 2slides/animal are prepared. Results: No mortality incidence and noevidence as to obvious clinical signs in reaction to treatment werenoted among any of the test animals throughout the entire studyperiod.

Example 9

Effect of Single Dose Acute Subcutaneous Administration of AcylatedGhrelin Splice Variant on Toxicity and Toxicokinetics inMinipigs

Aimed towards establishing a maximum tolerated dose (MTD)representing the highest dose not causing unacceptable toxicity ora maximum feasible dose (MFP), 2 minipigs Siwine/HsdScr:Sinclair ofa preliminary trial group are administered incremental doses up toa maximum of 75 mg/kg acylated ghrelin splice variant. Based on theeffects observed in the preliminary trial an appropriately selectedhighest single dose is selected and administered to main studyanimals via the subcutaneous (SC) route, to groups comprising n=2minipigs. On day 1 (day of dosing) blood samples for bioanalyticalassays are collected at a total of 9 time points: `0`-pre-testbaseline, 5, 15, 30, 60, 90 min, 3, 5 and 24 hours post-dosing.Data evaluation of standard PK parameters (C.sub.max, T.sub.max,T.sub.1/2, AUC) are obtained. The mini pigs are further observedfor an additional 14-day observation period.

All pharmacokinetics parameters were obtained and no mortalityoccurred in any of the animals throughout the entire study period.No clinical signs were observed in any of the animals throughoutthe entire study period. All animals were subjected to terminalbleeding, under CO.sub.2 anesthesia, immediately prior toeuthanasia.

Clinical Signs:

Animals are observed individually after dosing at least once duringthe first 30 minutes, periodically during the first 24 hours, withspecial attention given during the first 4 hours, and clinicalsigns are recorded. Thereafter, animals are inspected and clinicalsigns are recorded once daily for a total of 14 days. Observationsinclude changes in skin, fur, eyes, mucous membranes, occurrence ofsecretions and excretions (e.g. diarrhea) and autonomic activity(e.g. lacrimation, salivation, piloerection, pupil size, unusualrespiratory pattern). Changes in gait, posture and response tohandling, as well as the presence of bizarre behavior, tremors,convulsions, sleep and coma are also included. Body Weight:Determination of individual body weights of animals is made shortlybefore Test Item administration (Day 0), 2, 7 and 14 days followingdosing. Fasted body weight measurements are taken just prior tonecropsy. In case of decedents, body weight is determined as closeas possible to death. Clinical Pathology: Hematology, biochemistryand coagulation parameters listed down are determined from allsurviving animals prior to the scheduled euthanasia. Hematology:Blood samples are obtained following overnight food deprivation.Blood samples (at least 500 .mu.l of whole blood) are collectedinto pre labeled EDTA coated tubes contain the followinginformation: Study number, group number, animal number and date.The samples are kept until delivery and analysis at 2-8.degree. C.Hematology parameters that are tested using ADVIA 120 HematologySystem (Beyer) are: WBC, RBC, HGB, HCT, MCV, MCH, MCHC, Platelets,differential count. Reticulocytes are counted manually.Biochemistry: Blood for biochemistry analysis is collected intonon-coated pre-labeled tubes. The tubes are pre-labeled andcontained the following information: Study number, group number,animal number and date. Following clotting, the blood from eachanimal is centrifuged, and at least 300 .mu.l serum is collectedinto two pre-labeled tubes and submitted for analysis while kept at2-8.degree. C. until analysis. The samples are subjected to thefollowing listed tests using HITACHI MODULAR P-800 system:Creatinine, Calcium, Glucose, Cholesterol, Total Protein, Globulin,LDH, Potassium, Aspartate, Aminotransferase (AST), CPK, Phosphorus,Urea, Albumin, Total Bilirubin, Alanine, Aminotransferase (ALT),Sodium, .gamma.-Glutamyl Transpeptidase (GGT), Chloride,Triglycerides, High Density Lipoprotein (HDL), Low DensityLipoprotein (LDL), Alkaline Phosphatase (ALP). Coagulationparameters: Blood for coagulation analysis is collected byretro-orbital sinus bleeding under CO.sub.2 anesthesia intotri-sodium citrate coated tubes. Following completion of bloodcollection, all blood and serum samples are kept at 2-8.degree. C.until further analysis. The samples are subjected to the followinglisted tests using Sysmex CA-1500 system: PT, APTT. Urinalysis:Individual samples of voided urine are collected from all animals(if applicable), prior to the scheduled study termination or priorto sacrifice in case of removal from the study for animal welfarereasons, by either pressing the abdominal area over the bladder orby collecting voided urine, otherwise, directly from the bladder bycystocentesis. Analysis is performed using a commercial test kit(Bayer Multistix.RTM. 10 SG) applied to void urine sample andevaluating the following parameters: glucose, ketone, pH,leukocyte, blood, density, nitrite, bilirubin, urobilinogen andprotein. Necropsy Procedures and Macroscopic Examination: Allanimals are subjected to gross necropsy following the scheduledeuthanasia. At necropsy, all animals are subjected to thoroughexamination, including the external surface of the body, allorifices, cranial, thoracic and abdominal cavities and theircontents. Any abnormality or gross pathological changes in tissuesand/or organs are observed and recorded. It is noted if a grosspathological abnormality occurs on the external body surface thatis located at or near the injection site. The following tissuesand/or organs including those with macroscopic abnormalities arepreserved in 4% formaldehyde solution: Adrenals, Aorta thoracic,Brain, Ceccum, Colon, Duodenum, Epididymis, Eyes, Harderian glands,Heart, Femur and knee joint, Ileum, Jejunum, Kidneys, Lachrymalglands, Liver, Lungs, Lymph nodes--superficial cervical, Lymphnodes--mesenteric, Mammary gland+skin, Oesophagus, Optic nerves,Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands,Sciatic nerve, Seminal vesicles, Skeletal muscle (thigh),Skin--injection site, Spleen, Spinal Cord (cervical, thoracic,lumbar), Sternum (bone marrow), Stomach, Testes, Thymus, Thyroid(with parathyroid if applicable), Tongue, Trachea, Urinary bladder,Uterus with cervix, Vagina. Organ/Tissue Weighing and Organ/TissueFixation: Organ/Tissue weighing and fixation are performed for allanimals. The organs listed above are weighed wet as soon aspossible after dissection and removal of the attached fat andconnective tissues (in the case of paired organs, the individualweights are determined but presented as the mean organ weight). Allorgans/tissues are then fixed and preserved in 4% formaldehydesolution (excluding the eyes, optic nerves and harderian glandswhich are fixed in Davidson's solution) for at least 48-hr fixationperiod prior tissues delivery. In addition, any otherorgans/tissues with gross macroscopic changes are preserved as wellin 4% formaldehyde solution. The organs/tissues per animal arepacked in plastic containers in which each container is identifiedby the Study No., Group No., Animal No. & Date of necropsy.Bone marrow is obtained from a single femur, moistened by a foetalbovine serum to allow an appropriate smearing and smeared onto aclean and labeled glass slide by the use of a second glass slide.Thereafter, slides are left in the open air to dry and dipped inMethanol for about 5 minutes for appropriate fixation. At least 2slides/animal are prepared. Results: No mortality incidence and noevidence as to obvious clinical signs in reaction to treatment werenoted among any of the test animals throughout the entire studyperiod.

Example 10

Example of 7, 10, 28 Day or 3, 6, 9 Months Repeat Dose SubcutaneousAdministration of Acylated Ghrelin Splice Variant inRats/Dogs/Minipigs

Acylated ghrelin splice variant in various doses (0.2, 1.0, and 5.mu.mol/kg, all in 100 .mu.l vehicle) or the Vehicle (in saline)are administered once daily for various lengths of successive days(7, 10, 28 days or 3, 6, 9 months), via the subcutaneous (SC)route, to groups comprising n=10 rats or beagle dogs or humansubjects with cachexia. Food intake, weight gain and spontaneouslocomotor activity are measured for 20 h after the injection.

Example 11

Examples of Diaries/Questionnaires Assessing Patient Quality ofLife

A) EORTC QLQ-C30 (Aaronson et al., J. Natl. Cancer Inst. 85: 365-76(1993)), see the National Institutes of Health website and see, forexample, a specimen of EORTC QLQ-C30 (version 3.0), available onthe EORTC website and incorporated herein by reference.

We are interested in information regarding you and your health.Please answer the follow questions by ticking off the number thatapplies best to you. There are no "right" or "wrong" answers. Thisinformation will be treated with confidentiality.

Questions: See specimen of EORTC QLQ-C30 (version 3.0), availableon the EORTC website and incorporated herein by reference).

Example 12

Examples of Suitable Formulations for Preparing PharmaceuticalCompositions for Use in the Present Disclosure

4% Mannitol Solution is prepared by dissolving D-Mannitol in Waterfor Injection to achieve final concentration of 40 mg/ml. GhrelinSplice Variant Stock Solution is prepared by dissolving ghrelinsplice variant in TFA salt or in Acetate (5 mg) in 10 ml of 4%Mannitol solution, divided into aliquots and kept frozen(-70.degree. C.) until the time of use.

Ghrelin Splice Variant Dosing Solution: On each day of dosing, therequired amount of each test item is thawed and diluted withPhysiological Saline to a concentration of 0.2 mg/ml.

Control Item Dosing Solution: Prepared on each day of dosing, bydiluting 4% Mannitol Solution with Physiological Saline at the sameratio as the Ghrelin Splice Variant Dosing Solution.

Patients: Patients with documented cancer cachexia and documentedcancer cachexia with significant weight-loss in the precedingperiod and reduced appetite. The cachexia may be caused by any typeof cancer, including, e.g., esophageal, lung, breast, gastric,pancreatic, neurological and urinary tract, bone, hematological,reproductive tract, exocrine gland, endocrine gland, multipleendocrinological neoplasms, testicular, prostate, nephrological,skin, thyroid, liver, and colon. Efficacy of ghrelin splice variantaction will be assessed according to clinical assessments: (1)Acute Food intake: Dietician-assessed food intake during theinfusion. (2) Chronic Food intake: A daily report of the amount offood consumed during the day, and assessment of the pleasantnessrelated to the food intake. This will be validated by urinenitrogen excretion, based on 4 day diet diary. (3) Body-weight:Standard and calibrated scale will be used at the clinic. (4)Resting energy expenditure (REE) is a very important measurement,since it is affected both by the state of the disease and the bodysize. (5) Exercise test: Actigraph is used according to standardprotocol described on the Actigraph website. (6) Health-related QOLusing standard forms as described supra. (7) Para-clinicalassessments: (i) Nitrogen excretion in the urine: 24 h urinecollection should be used as validation of the reported foodintake. (ii) Plasma glucose, plasma FFA, plasma triglycerides,plasma glycerol and plasma amino acids: Plasma substrates measuredto ensure the reported food intake is in accordance with theabsorbed amount of food intake. (iii) Lean body mass and fat massassessed by TSF thickness and mid-arm circumference as ameasurement of body composition. (iv) Total body fat (and fat freemass) will be assessed DEXA scan, using software 1.31 for the lunarDPX-L (Scanexport Medical, Helsingborg, Sweden). (v) Plasma Leptin:Leptin is produced by and secreted from the fat cell. The plasmalevel of leptin gives an estimate of the total fat cell burden.(vi) Plasma Ghrelin: The basal ghrelin level tends to be increasedin cachectic patients. (vii) Plasma-GH: In previous studies, GH hasbeen measured as a control for the effect of ghrelin administration(Enomoto M. et al., Clin. Sci. (Lond). 105:431-35 (2003)). (viii)IGF-1: A single determination of IGF-I summarizes 24 h of GHsecretion. This has been demonstrated in healthy volunteers wherelevels of circulating IGF-I have been shown to correlate withspontaneous GH secretion (Rose S. R. et al., N. Engl. J. Med.319:201-07 (1988)). IGF-I may also increase independently of GHincrease by improved nutritional status. (ix) IGFBP-3: One of thecarrier proteins for IGF-I. It increases in parallel with IGF-I butwith a slower response rate. (x) Albumin: Is an indicator ofnutritional status. (xi) Prealbumin: Indicator of nutritionalstatus, with quicker response to alterations than albumin. (xii)Cortisol: Ghrelin administration has been shown to increase theserum cortisol level (Broglio F. et al., J. Clin. Endocrinol.Metab. 88:1537-42 (2003)). Corticosteroids have been shown to havea significant anti-nausea effect and to improve asthenia and paincontrol, which may be beneficial for the cachectic cancer patient.However, cortisol has never been shown to increase weight incachectic cancer patients. (xiii) CRP and ESR: Acute phase proteinsand ESR are often good indicators of systemic inflammation relatedto the cancer process (Inui A., C A Cancer J. Clin. 52:72-91(2002)).

Example 13

Treatment of Patients with Cancer-Related Anorexia/Cachexia

Patients with advanced cancer suffering from the anorexia/cachexiasyndrome (ACS), such as patients with any type of advances,incurable cancer, are believed to benefit from the presentdisclosure in terms of improved quality of life, increasedappetite, increased food intake, maintenance or gain of weight,food pleasantness, and/or fat deposition.

Patients will receive subcutaneous administration of 10 .mu.g/kgdose of ghrelin splice variant and placebo. The protocol will startat 08.00 hours after an overnight fast. A 22-gauge catheter will beinserted into an antecubital vein for blood sampling. After anequilibration period of 30 min, ghrelin splice variant (10.mu.g/kg) or placebo (0.9% saline) will be administeredsubcutaneously.

Investigational treatment: Ghrelin splice variant will be availablein GMP-quality in prepared vials of 10 .mu.g/kg from BACHEM AG,Switzerland or NeoMPS Inc., USA. Placebo consists of normal saline(or the vehicle used to dissolve study substance), which will beprovided by a hospital pharmacy. Ghrelin splice variant isdissolved in saline, and a dose of 10 .mu.g/kg ghrelin splicevariant will be administered to the patient. Assessments ofefficacy: (1) Eating related symptoms: assessed using an adaptedversion of the "Functional Assessment of Appetite and CachexiaTherapy" (FAACT) questionnaire; the EORTC-QLQ-30 Anorexia/Cachexiaquestionnaire (see, for example, a specimen of EORTC QLQ-C30(version 3.0), available on the EORTC website and incorporatedherein by reference); the NCCTG-Anorexia/Cachexia questionnaire(see the National Institutes of Health website) and/or the EdmontonSymptom assessment scale (see Bruera E et al., J. Palliat. Care7:6-9 (1991)). (2) Quality of life: will be assessed using theEORTC-QLQ-C30 questionnaire (see example 9). (3) Nutritional intakeand food preferences: food intake measurement will be by percentagecalculation of food products consumed at each meal by the patient,the clinical dietician will assess the food preferences as part oftheir routine assessments. (4) Food pleasantness: will be assessedafter lunch using visual analogue scales, following establishedanchors. (5) Perceived appetite, hunger, nausea and satiety: willbe assessed in the morning, before infusion, and before and afterlunch using visual analogue scale, following established anchors.Applicant will also apply a shortened ad hoc taste questionnaire.(6) Growth hormone (GH): since GH reflects directly the biologicalfunction of ghrelin, with a rapid increase of GH after ghrelininjections, Applicant will also monitor GH levels at the same timepoints as ghrelin. A standard ghrelin assay will be used. (7) Bodycomposition: body compositions will be assessed by BMI,bioimpedance analysis and dual photon absorptiometry/dual energyx-ray absorptiometry (DEXA). (8) Albumin and transferrin levelswill be determined as parameters for nutritional status. (9)Cardiovascular autonomic function: for the screening of autonomicdisorders, a 20 minute Holter EKG will be performed, and the SDNNvalue determined. (10) Mediators of the primary anorexia/cachexiasyndrome: mediators of the proinflammatory reaction (CRP, IL-6,TNF-.alpha.), the activated metabolism (free fatty acids,triglycerides, insulin, glucose, leptin), the gut-brain axis(ghrelin), and the somatotrophic axis (IGF-1, free testosterone)will be determined as baseline in the first week. A urine samplewill be reserved for assessment of proteolysis-inducing factor(PIF), a mediator of the paraneoplastic anorexia/cachexiasyndrome.

SEQUENCE LISTINGS

1

8194PRTHomo sapiens 1Gly Ser Ser Phe Leu Ser Pro Glu His Gln ArgVal Gln Val Arg Pro1 5 10 15Pro His Lys Ala Pro His Val Val Pro AlaLeu Pro Leu Ser Asn Gln 20 25 30Leu Cys Asp Leu Glu Gln Gln Arg HisLeu Trp Ala Ser Val Phe Ser 35 40 45Gln Ser Thr Lys Asp Ser Gly SerAsp Leu Thr Val Ser Gly Arg Thr 50 55 60Trp Gly Leu Arg Val Leu AsnGln Leu Phe Pro Pro Ser Ser Arg Glu65 70 75 80Arg Ser Arg Arg SerHis Gln Pro Ser Cys Ser Pro Glu Leu 85 90222PRTHomo sapiens 2GlySer Ser Phe Leu Ser Pro Glu His Gln Arg Val Gln Val Arg Pro1 5 1015Pro His Lys Ala Pro His 20324PRTHomo sapiens 3Gly Ser Ser Phe LeuSer Pro Glu His Gln Arg Val Gln Val Arg Pro1 5 10 15Pro His Lys AlaPro His Val Val 20424PRTHomo sapiensMISC_FEATURE(3)..(3)Xaa is Dpr4Gly Ser Xaa Phe Leu Ser Pro Glu His Gln Arg Val Gln Val Arg Pro1 510 15Pro His Lys Ala Pro His Val Val 20529PRTHomo sapiens 5Gly SerSer Phe Leu Ser Pro Glu His Gln Arg Val Gln Val Arg Pro1 5 10 15ProHis Lys Ala Pro His Val Val Pro Ala Leu Pro Leu 20 25691PRTHomosapiens 6Phe Leu Ser Pro Glu His Gln Arg Val Gln Val Arg Pro ProHis Lys1 5 10 15Ala Pro His Val Val Pro Ala Leu Pro Leu Ser Asn GlnLeu Cys Asp 20 25 30Leu Glu Gln Gln Arg His Leu Trp Ala Ser Val PheSer Gln Ser Thr 35 40 45Lys Asp Ser Gly Ser Asp Leu Thr Val Ser GlyArg Thr Trp Gly Leu 50 55 60Arg Val Leu Asn Gln Leu Phe Pro Pro SerSer Arg Glu Arg Ser Arg65 70 75 80Arg Ser His Gln Pro Ser Cys SerPro Glu Leu 85 907181PRTMus musculus 7Gly Ser Ser Phe Leu Ser ProGlu His Gln Lys Ala Gln Val Ser Gln1 5 10 15Ser Val Ser Leu Ser ProHis Ile Tyr Pro Asp Leu Cys Val Cys Val 20 25 30Arg Glu Arg Glu ArgGlu Pro Ser Phe Pro Phe Gln Gln Arg Lys Glu 35 40 45Ser Lys Lys ProPro Ala Lys Leu Gln Pro Arg Ala Leu Glu Gly Trp 50 55 60Leu His ProGlu Asp Arg Gly Gln Ala Glu Glu Thr Glu Glu Glu Leu65 70 75 80GluIle Arg Val Cys Thr Gln Ala Pro Ala Cys Ser Tyr Asn Ser Lys 85 9095Gly Val Gly Val Trp Arg Val Ser His Met Leu Ala Phe Gln Ala Thr100 105 110Gln Gly Leu Glu Ser Ser Thr Asn Ser Ser Thr Arg Gly SerGlu Ser 115 120 125Pro Ser Gln Glu Val Thr Val Ser Arg Val Ala ArgGlu Gln Gln Thr 130 135 140Cys Ala Gln Lys Thr Lys Gln Ile Glu GlySer Gln Glu Pro Gly Ser145 150 155 160Thr Asp Gly Tyr Arg Asn ArgArg Lys Pro Cys Leu Ser Gln Asp Leu 165 170 175Ser Gly Leu Pro Trp180890PRTRattus norvegicus 8Gly Ser Ser Phe Leu Ser Pro Glu His GlnLys Ala Gln Val Ser Leu1 5 10 15Ser Pro Gln Val Pro His Leu Ser TrpSer Val Val Cys Ser Phe Pro 20 25 30Phe Gln Gln Arg Lys Glu Ser LysLys Pro Pro Ala Lys Leu Gln Pro 35 40 45Arg Ala Leu Glu Gly Trp LeuHis Pro Glu Asp Arg Gly Gln Ala Glu 50 55 60Glu Ala Glu Glu Glu LeuGlu Ile Arg Val Gly Pro Arg Ala Pro Ala65 70 75 80Tyr Ser Cys AsnSer Lys Gly Phe Gly Val 85 90

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