The present invention features truncated MCH analogs active at the MCH receptor. The truncated MCH analogs are optionally modified peptide derivatives of mammalian MCH. The analogs can bind to the MCH receptor and, preferably, bring about signal transduction. MCH analogs have a variety of different uses including being used as a research tool and being used therapeutically.

SUMMARY OF THE INVENTION

The present invention features truncated MCH analogs active at the MCH receptor. The truncated MCH analogs are optionally modified peptide derivatives of mammalian MCH. The analogs can bind to the MCH receptor and, preferably, bring about signal transduction. MCH analogs have a variety of different uses including being used as a research tool and being used therapeutically.

Thus, a first aspect of the present invention describes a truncated MCH analog. The truncated MCH analog is an optionally modified peptide having the structure -- see Original Patent.

Another aspect of the present invention describes a method of screening for a compound able to bind a MCH receptor. The method comprises the step of measuring the ability of the compound to effect binding of a truncated MCH analog to either the MCH receptor, a fragment of the receptor comprising a MCH binding site, a polypeptide comprising such a fragment, or a derivative of the polypeptide.

Another aspect of the present invention describes a method for increasing weight in a subject. The method comprises the step of administering to the subject an effective amount of a truncated MCH analog to produce a weight increase.

Another aspect of the present invention describes a method for increasing appetite in a subject. The method comprises the step of administering to the subject an effective amount of a truncated MCH analog to produce an appetite increase.

Another aspect of the present invention describes a method for measuring the ability of a compound to decrease weight or appetite in a subject. The method comprising the steps of:

a) administering to the subject an effective amount of a truncated MCH analog to produce a weight increase or appetite increase,

b) administering the compound to the subject, and

c) measuring the change in weight or appetite of the subject.

Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Truncated MCH analogs contain about 10 to about 17 groups that are amino acids or amino acid derivatives. Using the present application as a guide truncated MCH analogs can be produced having significant MCH receptor activity, and in some cases having activity equal to or better than naturally occurring mammalian MCH. The smaller size of truncated MCH analogs offers advantages over longer-length MCH such as ease of synthesis and/or increased solubility in physiological buffers.

The MCH receptor is a G-protein coupled receptor that appears to be able to couple to Gi and Gq. Several references describe a receptor that is indicated to be a MCH receptor. (Chambers, et al., 1999. Nature 400, 261 265; Saito, et al., 1999. Nature 400, 265 269; Baichner, et al., 1999. FEBS Letters 457:522 524; and Shimomura, et al., 1999. Biochemical and Biophysical Research Communications 261, 622 626. These references are not admitted to be prior art to the claimed invention.)

The nucleic acid encoding for different variants of a MCH receptor is provided for by SEQ. ID. NOS. 1 3. The encoded amino acid sequences of the variants are provided by SEQ. ID. NOS. 4 6. The variants differ from each other by the presence of additional amino acids at the N-terminal. One or more of these variants may be a physiological MCH receptor.

Significant MCH activity is preferably at least about 50%, at least about 75%, at least about 90%, or at least about 95%, the activity of mammalian MCH as determined by a binding assay or MCH receptor activity assay. Examples of such assays are provided below.

MCH analogs have a variety of different uses including being used as a research tool and being used therapeutically. Research tool applications generally involve the use of a truncated MCH analog and the presence of a MCH receptor or fragment thereof. The MCH receptor can be present in different environments such as a mammalian subject, a whole cell and membrane fragments. Examples of research tool applications of truncated MCH analogs include screening for compounds active at the MCH receptor, determining the presence of the MCH receptor in a sample or preparation, examining the role or effect of MCH, and examining the role or effect of MCH antagonists.

Truncated MCH analogs can be used to screen for both MCH agonists and MCH antagonists. Screening for MCH agonists can be performed, for example, by using a truncated MCH analog in a competition experiment with test compounds. Screening for MCH antagonists can be performed, for example, by using a truncated MCH analog to produce MCH receptor activity and then measuring the ability of a compound to alter MCH receptor activity.

Truncated MCH analogs can be administered to a subject. A "subject" refers to a mammal including, for example, a human, a rat, a mouse, or a farm animal. Reference to subject does not necessarily indicate the presence of a disease or disorder. The term subject includes, for example, mammals being dosed with a truncated MCH analog as part of an experiment, mammals being treated to help alleviate a disease or disorder, and mammals being treated prophylactically to retard or prevent the onset of a disease or disorder.

MCH agonists can be used to achieve a beneficial effect in a subject. For example, a MCH agonist can be used to facilitate a weight gain, maintenance of weight and/or an appetite increase. Such effects are particularly useful for a patient having a disease or disorder, or under going a treatment, accompanied by weight loss. Examples of diseases or disorders accompanied by weight loss include anorexia, AIDS, wasting, cachexia, and frail elderly. Examples of treatments accompanied by weight loss include chemotherapy, radiation therapy, and dialysis.

MCH antagonists can also be used to achieve a beneficial effect in a patient. For example, a MCH antagonist can be used to facilitate weight loss, appetite decrease, weight maintenance, cancer (e.g., colon or breast) treatment, pain reduction, stress reduction and/or treatment of sexual dysfunction.

Truncated MCH Analogs

A truncated MCH analog is an optionally modified peptide having the structure -- see Original Patent.

The present invention is meant to comprehend diastereomers as well as their racemic and resolved enantiomerically pure forms. Truncated MCH analogs can contain D-amino acids, L-amino acids or a combination thereof. Preferably, amino acids present in a truncated MCH analog are the L-enantiomer.

In different embodiments, MCH analogs contain a preferred (or more preferred) group at one or more different locations. More preferred embodiments contain preferred (or more preferred) groups in each of the different locations.

A protecting group covalently joined to the N-terminal amino group reduces the reactivity of the amino terminus under in vivo conditions. Amino protecting groups include optionally substituted --C.sub.1-10 alkyl, optionally substituted --C.sub.2-10 alkenyl, optionally substituted aryl, --C.sub.1-6 alkyl optionally substituted aryl, --C(O)--(CH.sub.2).sub.1-6--COOH, --C(O)--C.sub.1-6 alkyl, --C(O)-optionally substituted aryl, --C(O)--O--C.sub.1-6 alkyl, or --C(O)--O-optionally substituted aryl. Preferably, the amino terminus protecting group is acetyl, propyl, succinyl, benzyl, benzyloxycarbonyl or t-butyloxycarbonyl.

A protecting group covalently joined to the C-terminal carboxy group reduces the reactivity of the carboxy terminus under in vivo conditions. The carboxy terminus protecting group is preferably attached to the .alpha.-carbonyl group of the last amino acid. Carboxy terminus protecting groups include amide, methylamide, and ethylamide.

"Alkyl" refers to carbon atoms joined by carbon-carbon single bonds. The alkyl hydrocarbon group may be straight-chain or contain one or more branches or cyclic groups. Preferably, the alkyl group is 1 to 4 carbons in length. Examples of alkyl include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, and t-butyl. Alkyl substituents are selected from the group consisting of halogen (preferably --F or --Cl) --OH, --CN, --SH, --NH.sub.2, --NO.sub.2, --C.sub.1-2 alkyl substituted with 1 to 6 halogens (preferably --F or --Cl, more preferably --F), --CF.sub.3, --OCH.sub.3, or --OCF.sub.3.

"Alkenyl" refers to a hydrocarbon group containing one or more carbon-carbon double bonds. The alkenyl hydrocarbon group may be straight-chain or contain one or more branches or cyclic groups. Preferably, the alkenyl group is 2 to 4 carbons in length. Alkenyl substituents are selected from the group consisting of halogen (preferably --F or --Cl), --OH, --CN, --SH, --NH.sub.2, --NO.sub.2, --C.sub.1-2 alkyl substituted with 1 to 5 halogens (preferably --F or --Cl, more preferably --F), --CF.sub.3, --OCH.sub.3, or --OCF.sub.3.

"Aryl" refers to an optionally substituted aromatic group with at least one ring having a conjugated pi-electron system, containing up to two conjugated or fused ring systems. Aryl includes carbocyclic aryl, heterocyclic aryl and biaryl groups. Preferably, the aryl is a 5 or 6 membered ring, more preferably benzyl. Aryl substituents are selected from the group consisting of --Cl.sub.1-4 alkyl, --C.sub.1-4 alkoxy, halogen (preferably --F or --Cl), --OH, --CN, --SH, --NH.sub.2, --NO.sub.2, --C.sub.1-2 alkyl substituted with 1 to 5 halogens (preferably --F or --Cl, more preferably --F), --CF.sub.3, or --OCF.sub.3.

A labeled derivative indicates the alteration of a substituent with a detectable label. Examples of detectable labels include luminescent, enzymatic, and radioactive labels. A preferred radiolabel is .sup.125I. Both the type of label and the position of the label can effect MCH activity. Labels should be selected so as not to substantially alter the activity of the truncated MCH analog at the MCH receptor. The effect of a particular label on MCH activity can be determined using assays measuring MCH activity and/or binding.

In naturally occurring full length MCH, alteration of the tyrosine at position 13 by labeling with .sup.125I substantially effects MCH activity. (Drozdz, et al., 1995. FEBS letters 359, 199 202.) .sup.125I labeled analogs of full length mammalian MCH having substantial activity can be produced, for example, by replacing the tyrosine at position 13 with a different group, then replacing valine at position 19 with tyrosine, and labeling the tyrosine. Examples of such analogs include [.sup.125I][Phe.sup.13, Try.sup.19]-MCH and (D-(p-benzoylphenylalanine).sup.13, tyrosine.sup.19)-MCH. (Drozdz, et al., FEBS letters 359, 199 202, 1995; and Drozdz, et al., J. Peptide Sci. 5, 234 242, 1999.)

In preferred embodiments the optionally modified peptide has the structure -- see Original Patent.

In different embodiments the truncated MCH analog is a peptide of SEQ. ID. NOS. 7, 8, 9, or 10, a labeled derivative of said peptide or a pharmaceutically acceptable salt of said peptide or of said labeled derivative. SEQ. ID. NOS. 7 12 are made up of L-amino acids and have the following sequences -- see Original Patent.

In additional embodiments the peptide has a sequence selected from the group consisting of SEQ. ID. NOs. 7, 8, 10, 15, 24, 25, 27, 28, 30 49, 51, 52, 56, 57, 61, 62, 63, 65 67, 69 72, and 77, is a labeled derivative of said peptide or a pharmaceutically acceptable salt of said peptide or of said labeled derivative. Preferred sequences are those with an IC.sub.50 less than 0.3 nM, preferably less than 0.1 nM; and/or those having a % activation greater than about 90%, preferably greater than 100%. Examples of preferred sequences are provided in Example 4, Tables 1 7.

Truncated MCH analogs can be produced using techniques well known in the art. For example, a polypeptide region of a truncated MCH analog can be chemically or biochemically synthesized and, if desired modified to produce a blocked N-terminus and/or blocked C-terminus. Techniques for chemical synthesis of polypeptides are well known in the art. (See e.g., Vincent, in Peptide and Protein Drug Delivery, New York, N.Y., Dekker, 1990.) Examples of techniques for biochemical synthesis involving the introduction of a nucleic acid into a cell and expression of nucleic acids are provided in Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987 1998, and Sambrook, et al., in Molecular Cloning, A Laboratory Manual, 2.sup.nd Edition, Cold Spring Harbor Laboratory Press, 1989.

MCH Receptor Binding Assay

Assays measuring the ability of a compound to bind a MCH receptor employ a MCH receptor, a fragment of the receptor comprising a MCH binding site, a polypeptide comprising such a fragment, or a derivative of the polypeptide. Preferably, the assay uses the MCH receptor or a fragment thereof.

A polypeptide comprising a MCH receptor fragment that binds MCH can also contain one or more polypeptide regions not found in a MCH receptor. A derivative of such a polypeptide comprises a MCH receptor fragment that binds MCH along with one or more non-peptide components.

The MCH receptor amino acid sequence involved in MCH binding can be readily identified using labeled MCH or truncated MCH analogs and different receptor fragments. Different strategies can be employed to select fragments to be tested to narrow down the binding region. Examples of such strategies include testing consecutive fragments about 15 amino acids in length starting at the N-terminus, and testing longer length fragments. If longer length fragments are tested, a fragment binding MCH can be subdivided to further locate the MCH binding region. Fragments used for binding studies can be generated using recombinant nucleic acid techniques.

Binding assays can be performed using individual compounds or preparations containing different numbers of compounds. A preparation containing different numbers of compounds having the ability to bind to the MCH receptor can be divided into smaller groups of compounds that can be tested to identify the compound(s) binding to the MCH receptor. In an embodiment of the present invention a test preparation containing at least 10 compounds is used in a binding assay.

Binding assays can be performed using recombinantly produced MCH receptor polypeptides present in different environments. Such environments include, for example, cell extracts and purified cell extracts containing the MCH receptor polypeptide expressed from recombinant nucleic acid or naturally occurring nucleic acid; and also include, for example, the use of a purified MCH receptor poly,peptide produced by recombinant means or from naturally occurring nucleic acid which is introduced into a different environment.

Screening for MCH Receptor Active Compounds

Screening for MCH active compounds is facilitated using a recombinantly expressed MCH receptor. Using recombinantly expressed MCH receptor polypeptides offers several advantages such as the ability to express the receptor in a defined cell system so that response to MCH receptor active compounds can more readily be differentiated from responses to other receptors. For example, the MCH receptor can be expressed in a cell line such as HEK 293, COS 7, and CHO not normally expressing the receptor by an expression vector, wherein the same cell line without the expression vector can act as a control.

Screening for MCH receptor active compounds is facilitated through the use of a truncated MCH analog in the assay. The use of a truncated MCH analog in a screening assay provides for MCH receptor activity. The effect of test compounds on such activity can be measured to identify, for example, allosteric modulators and antagonists. Additionally, such assays can be used to identify agonists.

MCH receptor activity can be measured using different techniques such as detecting a change in the intracellular conformation of the MCH receptor, Gi or Gq activity, and/or intracellular messengers. Gi activity can be measured using techniques well known in the art such as a melonaphore assay, assays measuring cAMP production, inhibition of cAMP accumulation, and binding of .sup.35S-GTP. cAMP can be measured using different techniques such as radioimmunoassay and indirectly by cAMP responsive gene reporter proteins.

Gq activity can be measured using techniques such as those measuring intracellular Ca.sup.2+. Examples of techniques well known in the art that can be employed to measure Ca.sup.2+ include the use of dyes such as Fura-2 and the use of Ca.sup.2+-bioluminescent sensitive reporter proteins such as aequorin. An example of a cell line employing aequorin to measure G-protein activity is HEK293/aeq17. (Button, et al., 1993. Cell Calcium 14, 663 671, and Feighner, et al., 1999. Science 284, 2184 2188, both of which are hereby incorporated by reference herein.)

Chimeric receptors containing a MCH binding region functionally coupled to a G protein can also be used to measure MCH receptor activity. A chimeric MCH receptor contains an N-terminal extracellular domain; a transmembrane domain made up of transmembrane regions, extracellular loop regions, and intracellular loop regions; and an intracellular carboxy terminus. Techniques for producing chimeric receptors and measuring G protein coupled responses are provided for in, for example, International Application Number WO 97/05252, and U.S. Pat. No. 5,264,565, both of which are hereby incorporated by reference herein.

Weight or Appetite Alteration

Truncated MCH analogs can be used in methods to increase or maintain weight and/or appetite in a subject. Such methods can be used, for example, as part of an experimental protocol examining the effects of MCH antagonists, to achieve a beneficial effect in a subject and/or to further examine the physiological effects of MCH.

Experimental protocols examining the effects of MCH antagonists can be performed, for example, by using a sufficient amount of a truncated MCH analog to produce a weight or appetite increase in a subject and then examining the effect of a test compound. Changes in weight and appetite can be measured using techniques well known in the art.

Increasing weight or appetite can be useful for maintaining weight or producing a weight or appetite gain in an under weight subject, or in a patient having a disease or undergoing treatment that effects weight or appetite. In addition, for example, farm animals such as pigs, cows and chickens can be treated to gain weight.

Under weight subjects include those having a body weight about 10% or less, 20% or less, or 30% or less, than the lower end of a "normal" weight range or Body Mass Index ("BMI"). "Normal" weight ranges are well known in the art and take into account factors such as a patient age, height, and body type.

BMI measures your height/weight ratio. It is determined by calculating weight in kilograms divided by the square of height in meters. The BMI "normal" range is 19 22.

Administration

Truncated MCH analogs can be formulated and administered to a subject using the guidance provided herein along with techniques well known in the art. The preferred route of administration ensures that an effective amount of compound reaches the target. Guidelines for pharmaceutical administration in general are provided in, for example, Remington's Pharmaceutical Sciences 18.sup.th Edition, Ed. Gennaro, Mack Publishing, 1990, and Modern Pharmaceutics 2.sup.nd Edition, Eds. Banker and Rhodes, Marcel Dekker, Inc., 1990, both of which are hereby incorporated by reference herein.

Truncated MCH analogs can be prepared as acidic or basic salts. Pharmaceutically acceptable salts (in the form of water- or oil-soluble or dispersible products) include conventional non-toxic salts or the quaternary ammonium salts that are formed, e.g., from inorganic or organic acids or bases. Examples of such salts include acid addition salts such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate; and base salts such as ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine and lysine.

Truncated MCH analogs can be administered using different routes including oral, nasal, by injection, transdermal, and transmucosally. Active ingredients to be administered orally as a suspension can be prepared according to techniques well known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavoring agents. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants.

Truncated MCH analogs may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form. When administered by injection, the injectable solution or suspension may be formulated using suitable non-toxic, parenterally-acceptable diluents or solvents, such as Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

Suitable dosing regimens are preferably determined taking into factors well known in the art including type of subject being dosed; age, weight, sex and medical condition of the subject; the route of administration; the renal and hepatic function of the subject; the desired effect; and the particular compound employed.

Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug. The daily dose for a subject is expected to be between 0.01 and 1,000 mg per subject per day.

Truncated MCH analogs can be provided in kit. Such a kit typically contains an active compound in dosage forms for administration. A dosage form contains a sufficient amount of active compound such that a weight or appetite increase can be obtained when administered to a subject during regular intervals, such as 1 to 6 times a day, during the course of 1 or more days. Preferably, a kit contains instructions indicating the use of the dosage form for weight or appetite increase and the amount of dosage form to be taken over a specified time period.

 

Claim 1 of 18 Claims

1. An optionally substituted peptide consisting of the structure -- see Original Patent.

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