PYY analogs
阅读说明：本技术 Pyy类似物 (PYY analogs ) 是由 S·R·布鲁姆 于 2018-12-04 设计创作，主要内容包括：通过用Ala23、Glu23、Lys23、Gln23或AIB23取代Ser23而不同于天然人PYY的PYY类似物。另外的任选特征包含在另外位置的取代、天然人PYY的Tyr1残基的丧失和C端的酰胺化。适合于用作用于治疗和预防病症,特别是糖尿病和肥胖症的药物。(PYY analogues that differ from native human PYY by substitution of Ser23 with Ala23, Glu23, Lys23, Gln23 or AIB 23. Further optional features include substitution at further positions, loss of Tyr1 residue of native human PYY and amidation of the C-terminus. Suitable for use as a medicament for the treatment and prevention of disorders, in particular diabetes and obesity.)
1. A PYY analogue which differs from the native human PYY sequence in the following ways (Tyr1 Pro2 Ile3Lys4 Pro5 Glu6 Ala7 Pro8 Gly9 Glu10 Asp11Ala12 Ser13 Pro14Glu15 Glu16 Leu17Asn18 Arg19 Tyr20 Tyr21Ala22 Ser23 Leu24Arg25 His26Tyr27 Leu28 Asn29 Leu30Val31 Thr32 Arg33 Gln34 Arg35 Tyr 36; SEQ ID NO: 1):
ser23 substituted by Ala23, Glu23, Lys23, Gln23 or AIB23 (preferably Ala23)
And the PYY analogue may further differ from the native human PYY sequence in one or more of the following further aspects:
the Tyr1 is deleted,
one or more of residues 2 to 9, 13 to 16, 20, 21, 24, 25 or 26 are subject to conservative substitution,
glu10 substituted by Ala10, Lys10 or Gln10
Asp11 substituted by Gly11
Ala12 substituted by AIB12
Leu17 substitution by Ile17 or AIB17
Asn18 substituted by Leu18, AIB18 or Ala18
Arg19 substituted by His19 or Lys19
Ala22 substitution by Val22 or Ile22
Tyr27 substituted by Phe27
Leu30 substituted by His30
Val31 substituted by Leu31
And wherein the C-terminal residue optionally terminates in a primary amide (-C (O) NH)2) Radicals replacing carboxylic acid groups (-CO)2H)；
Or a derivative of the compound; or a salt of said compound or said derivative.
2. An analogue of PYY as claimed in claim 1, wherein
Tyr1 deletion
Residues 2 to 9, 13 to 16, 20, 21, 24, 25, 26, 28, 29, 32, 33, 34 and 35 are unsubstituted.
3. An analogue of PYY as claimed in claim 1 or claim 2, wherein
Glu10 unsubstituted or substituted by Lys10 or Gln10
Asp11 substituted by Gly11
Residue 12 is unsubstituted
Leu17 is unsubstituted
Asn18 substituted by Leu18, AIB18 or Ala18
Arg19 substituted by His19
Ala22 is unsubstituted
Ser23 substituted by Ala23, Glu23, Ala23 or AIB23
Tyr27 substituted by Phe27
Leu30 substituted by His30
Val31 is unsubstituted or substituted with Leu 31.
4. An analogue of PYY as claimed in claim 1 or 2, wherein
Glu10 is unsubstituted
Asp11 substituted by Gly11
Residue 12 is unsubstituted
Leu17 is unsubstituted
Asn18 unsubstituted or substituted by Leu18
Arg19 is His19
Ala22 is unsubstituted
Replacement of Ser23 by Ala23
Tyr27 substituted by Phe27
Leu30 substituted by His30
Val31 is unsubstituted or substituted with Leu 31.
5. An analogue of PYY as claimed in claim 1 or claim 2, wherein
Asp11 is substituted with Gly 11.
6. An analogue of PYY as claimed in claim 1, 2 or 5, wherein
Asn18 is substituted with AIB18, Ala18 or Leu18, preferably with Leu 18.
7. An analogue of PYY as claimed in claim 1, 2,5 or 6 wherein
Leu30 was substituted with His 30.
8. An analogue of PYY as claimed in claim 1, 2,5, 6 or 7 wherein
Val31 is unsubstituted.
9. An analogue of PYY as claimed in claim 1, 2,5, 6, 7 or 8 wherein
Tyr1 was deleted.
10. An analogue of PYY as claimed in claim 1, 2,5, 6, 7, 8 or 9 wherein the C-terminal residue terminates in a primary amide group (-C (O) NH)2) In place of carboxylic acid groups (-CO)2H)。
11. An analogue of PYY as claimed in claim 1, 2,5, 6, 7, 8 or 9 wherein
Ser23 substituted with Glu 23.
12. An analogue of PYY as claimed in claim 1, 2,5, 6, 7, 8 or 9 wherein
Ser23 substituted by Ala 23.
13. An analogue of PYY as claimed in claim 1, 2,5, 6, 7, 8 or 9 wherein
Ser23 was substituted with AIB 23.
14. An analogue of PYY as claimed in claim 1, 2,5, 6, 7, 8 or 9 wherein
Ser23 was substituted with Gln 23.
15. An analogue of PYY as claimed in claim 1, selected from the group consisting of: y1319, Y1371, Y1372, Y1419, Y1421, Y1518, Y1528, Y1558, Y1568, Y1572, Y1579, Y1553, and Y1581.
16. An analogue of PYY as claimed in claim 1, having the sequence Pro-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Gly-Ala-Ser-Pro-Glu-Glu-Leu-Leu-His-Tyr-Tyr-Ala-Ala-Leu-Arg-His-Phe-Leu-Asn-His-Val-Thr-Arg-Gln-Arg-Tyr-NH2Compound Y1419 (SEQ ID NO:23) or a salt or solvate thereof.
17. A pharmaceutical composition comprising an analogue of PYY according to any one of claims 1 to 16 and a pharmaceutically acceptable carrier and optionally an additional therapeutic agent.
18. The pharmaceutical composition of claim 17, comprising an additional therapeutic agent that is a GLP-1 analog or a glucagon analog, or a derivative of either thereof.
19. The pharmaceutical composition of claim 17 or 18, comprising Zn2+Ions.
20. An analogue of PYY as claimed in any one of claims 1 to 16 or a pharmaceutical composition as claimed in claim 17, 18 or 19 for use as a medicament.
21. An analogue of PYY according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 17, 18 or 19 for use as a medicament for the prevention or treatment of a disorder of energy metabolism such as diabetes and/or obesity or for the prevention of loss of pancreatic islet function or for restoring pancreatic islet function.
22. An analogue of PYY as claimed in any one of claims 1 to 16 or a pharmaceutical composition as claimed in claim 17, 18 or 19 for use as a medicament for preventing diabetes or obesity in a subject.
23. A method of treating or preventing a disease or disorder or other undesired physiological state in a subject, the method comprising administering to the subject a therapeutically acceptable/effective amount of an analogue of PYY according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 17, 18 or 19.
24. A method of preventing or treating a disorder of energy metabolism such as diabetes and/or obesity, preventing loss of pancreatic islet function and/or restoring pancreatic islet function, reducing appetite, reducing food intake and/or reducing calorie intake in a subject, the method comprising administering to the subject a therapeutically effective amount of a PYY analogue according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 17, 18 or 19.
25. A method of preventing or treating diabetes and/or obesity in a subject, the method comprising administering to the subject a therapeutically effective amount of an analogue of PYY according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 17, 18 or 19.
26. An analogue of PYY or a pharmaceutical composition for use according to any one of claims 20 to 22 or a method according to any one of claims 23 to 25, wherein the subject is overweight, obese and/or suffering from diabetes.
27. An analogue or pharmaceutical composition of PYY for use according to any one of claims 20 to 22 or a method according to any one of claims 23 to 25, wherein the analogue or pharmaceutical composition of PYY is administered parenterally.
28. An analogue or pharmaceutical composition of PYY for use according to any one of claims 20 to 22 or a method according to any one of claims 23 to 25, wherein the analogue or pharmaceutical composition of PYY is administered subcutaneously.
29. A method of inducing weight loss or preventing weight gain in a subject for cosmetic purposes, the method comprising administering to the subject an effective amount of an analogue of PYY according to any one of claims 1 to 16 or a composition according to any one of claims 17 to 19.
Technical Field
The present application relates to peptide YY (PYY) analogs that can be used, alone or in combination with other agents (particularly GLP-1 analogs), for the treatment of conditions such as diabetes and obesity.
Background
According to the National Health and nutrition Survey (NHANES, 2011 to 2012), over two thirds of the united states of america have overweight or obesity in adults. In the united states, 78% of men and 74% of women are overweight or obese by
The cause of obesity is complex and multifactorial. There is increasing evidence that obesity is not a simple problem of self-control, but rather a complex disorder involving appetite regulation and energy metabolism. In addition, obesity is associated with a variety of conditions that are associated with increased morbidity and mortality in a population. Although the etiology of obesity has not been clearly determined, genetic, metabolic, biochemical, cultural, and psychosocial factors are thought to play a role. Generally, obesity has been described as a condition in which body fat is so excessive that an individual is at health risk.
There is strong evidence that obesity is associated with increased morbidity and mortality. The risk of diseases, such as cardiovascular disease risk and
While diet and exercise provide a simple method for reducing weight gain, overweight and obese individuals often do not have sufficient control over these factors to effectively lose weight. Drug therapy may be used; the food and Drug Administration (the food Administration) has approved several weight loss drugs that can be used as part of a comprehensive weight loss program. However, many of these drugs have serious adverse side effects. Bariatric surgery is an option in carefully selected clinically severely obese patients when minimally invasive approaches have failed and the patient is at high risk for obesity-related morbidity or mortality. However, these treatments are high risk and are only suitable for a limited number of patients. Not only are obese subjects desirous of losing weight. A person weighing within the proposed range (e.g., in the upper portion of the proposed range) may wish to lose their weight to bring them closer to the ideal weight. Thus, there remains a need for agents that can be used to achieve weight loss in overweight and obese subjects.
PYY is a 36 amino acid peptide produced by L cells of the intestinal tract, of which it is found in the highest concentration in the large intestine and rectum. Two endogenous forms of PYY and PYY 3-36 are released into the circulation. PYY 3-36 is further generated by cleavage of the Tyr-Pro amino terminal residue of PYY with dipeptidyl peptidase IV (DPP-IV). PYY 3-36 binds to the Y2 receptor of the Y family of receptors (De Silva and Bloom, journal of intestinal Liver (Gut Liver), 2012,6, p 10-20). Studies have shown that peripheral administration of PYY 3-36 to rodents and humans results in significant inhibition of food intake, leading to the promise that PYY analogs may be used to treat conditions such as obesity (see, e.g., Batterham et al, Nature, 2002,418, p 650-654; Batterham et al, new england journal of medicine, 2003,349, p 941-948).
After there is evidence that PYY is able to restore impaired insulin and glucagon secretion in
WO2011/092473 and WO2012/101413 (Imperial innovations limited) disclose certain PYY analogs. However, there is still a need for further compounds with suitable properties such that they are effective as therapeutic agents for the treatment or prevention of disorders of energy metabolism (such as obesity and/or diabetes).
Disclosure of Invention
In a first aspect, the invention provides a PYY analogue which differs from the native human PYY sequence in the following ways (Tyr1 Pro2 Ile3Lys4 Pro5 Glu6 Ala7 Pro8 Gly9 Glu10 Asp11Ala12 Ser13 Pro14Glu15 Glu16 Leu17Asn18 Arg19 Tyr20 Tyr21Ala22 Ser23 Leu24Arg25 His26Tyr27 Leu28 Asn29 Leu30Val31 Thr32 Arg33
-Ser23 substituted by Ala23, Glu23, Lys23, Gln23 or AIB23 (preferably Ala23)
And the PYY analogue may further differ from the native human PYY sequence in one or more of the following further aspects:
-Tyr1 is deleted,
-one or more of
-Glu10 substitution by Ala10, Lys10 or Gln10
Asp11 substituted by Gly11
-Ala12 substituted by AIB12
-Leu17 substitution by Ile17 or AIB17
Substitution of Asn18 by Leu18, AIB18 or Ala18
-Arg19 substituted by His19 or Lys19
-Ala22 substitution by Val22 or Ile22
-Tyr27 substituted by Phe27
-Leu30 substituted by His30
-Val31 substituted by Leu31
And wherein the C-terminal residue optionally terminates in a primary amide (-C (O) NH)2) Radicals replacing carboxylic acid groups (-CO)2H)；
Or a derivative of the compound; or a salt of said compound or said derivative.
The present invention is based on the following findings: PYY analogs in which specific amino acid residues are deleted and/or substituted can also be administered to a subject to cause a reduction in food intake, a reduction in calorie intake, a reduction in appetite, and a change in energy metabolism. In many cases, PYY analogues of the invention show improved potency and/or longer duration of action and/or fewer side effects compared to the native PYY.
The PYY analogues of the invention are also particularly suitable for use in combination therapy with agonists of the GLP-1 receptor. This is because PYY and GLP-1 analogs have widely compatible and similar chemical compositions that make them suitable for combined formulation, so they can be conveniently administered in a single injection. In addition, PYY analogs and GLP-1 analogs suppress appetite by different and separate mechanisms, and thus patients receiving combination therapy are less prone to 'escape' the desired drug effect than treatment with either agent alone. Finally, the different mechanisms of action allow for additive or synergistic effects on appetite suppression, thereby making the therapy more effective.
In another aspect, the invention provides a pharmaceutical composition comprising an analogue of PYY according to the invention and a pharmaceutically acceptable carrier and optionally an additional therapeutic agent.
In another aspect, the invention provides a PYY analogue according to the invention or a pharmaceutical composition comprising said PYY analogue for use as a medicament. The PYY analogue or pharmaceutical composition finds use in the prevention or treatment of an energy metabolism disorder such as diabetes and/or obesity, in the prevention of loss of pancreatic islet function and/or in restoring pancreatic islet function in a subject.
In another aspect, the invention provides the use of an analogue of PYY according to the invention for the manufacture of a medicament for the prevention or treatment of an energy metabolism disorder, such as diabetes and/or obesity, for the prevention of loss of pancreatic islet function and/or for restoring pancreatic islet function in a subject.
In another aspect, the invention provides a method of treating or preventing a disease or disorder or other undesired physiological state in a subject, the method comprising administering to the subject a therapeutically effective amount of an analogue of PYY according to the invention or a pharmaceutical composition comprising the PYY analogue. The PYY analogue or pharmaceutical composition finds use in a method for preventing or treating an energy metabolism disorder, such as diabetes and/or obesity, preventing loss of pancreatic islet function and/or restoring pancreatic islet function, reducing appetite, reducing food intake and/or reducing calorie intake in a subject.
In another aspect, the invention also provides a method of inducing weight loss or preventing weight gain in a subject for cosmetic purposes, the method comprising administering to the subject an effective amount of a PYY analogue according to the invention or a composition comprising the PYY analogue.
Drawings
FIG. 1 is a table listing the amino acid sequences of some PYY analogs relevant to a particularly preferred embodiment of the present invention. The naturally occurring sequence of human PYY is included for reference. The first column entitled "REF" shows the SEQ ID NO used in the appended sequence Listing. Natural human PYY is
Figure 2 shows the results of the in vitro receptor potency assay described in the examples.
Figure 3 shows the results of in vivo experiments (n-5 to 8) demonstrating the efficacy of compound Y242 as described in the examples.
Figure 4 shows the results of in vivo experiments (n-7 to 8) demonstrating the efficacy of compound Y1419 as described in the examples.
Figure 5 shows the results of in vivo experiments demonstrating the efficacy of other compounds of the invention.
Figure 6 shows the results of in vitro receptor potency experiments described in the examples.
Sequence listing
The amino acid sequences listed in this application are shown using standard letter abbreviations for amino acids. The specific sequences given herein relate to specific preferred embodiments of the invention. This application includes a machine readable sequence listing in which a PYY analogue is assigned the same SEQ ID NO identifier as the example number given in figure 1.
Definition of
To facilitate a review of the various embodiments of the present disclosure, the following explanation of specific terms is provided:
animals: living multicellular vertebrate organisms, including classes such as mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes human and livestock subjects.
Appetite: a natural desire or craving for food. In one embodiment, appetite is measured by a survey for assessing the desire for food. Increased appetite usually results in increased eating behavior.
Appetite suppressants: reducing the desirable compounds for food. Commercially available appetite suppressants include, but are not limited to, amfepramone (diethylpropion), phentermine, mazindol, phenylpropanolamine, dexfenfluramine, and fluoxetine.
Body Mass Index (BMI): a mathematical formula for measuring body mass, sometimes referred to as the ketley Index (Quetelet's Index). By dividing the weight (in kg) by the height2(with rice)2In units) to calculate BMI. The currently accepted standard for "normal" men and women is that the BMI is 20-24.9kg/m2. In one embodiment, greater than 25kg/m2Can be used to identify obese subjects. The BMI corresponding to the I-class obesity is 25-29.9kg/m2. BMI for class II obesity is 30-40kg/m2(ii) a And class III obesity corresponds to a BMI greater than 40kg/m2(Jequier, J.Clin.Nutr., USA 45:1035-47, 1987). The ideal weight will vary by species and individual based on height, build, skeletal structure and gender.
Conservative substitutions: the amino acid residue is replaced in the polypeptide with another, biologically similar residue. The term "conservative variation" also encompasses the replacement of a parent amino acid with a substituted amino acid (i.e., an amino acid in which one or more atoms are replaced with another atom or group), provided that the polypeptide retains its activity or provided that an antibody raised against the substituted polypeptide also immunoreacts with the unsubstituted polypeptide. Typical but non-limiting conservative substitutions are substitutions for one another between the aliphatic amino acids Ala, Val, Leu and Ile; exchange of the hydroxyl-containing residue Ser with Thr; interchange of the acidic residue Asp with Glu; interchange between amide-containing residues Asn and gin; interchange of the basic residues Lys and Arg; exchange of aromatic residues Phe and Tyr; and the interchange of the small-form amino acids Ala, Ser, Thr, Met and Gly. Additional conservative substitutions involve the replacement of an amino acid with another amino acid of similar steric or steric configuration, e.g., Asn for Asp or Gln for Glu.
Non-limiting examples of conservative amino acid substitutions
Non-conservative substitutions: an amino acid residue is replaced in a polypeptide by another residue that is biologically non-similar. For example, the amino acid residue is replaced with another residue having a substantially different charge, a substantially different hydrophobicity, or a substantially different steric or steric configuration.
Diabetes mellitus: because of the endogenous deficiency of insulin and/or the deficiency in insulin sensitivity, cells are unable to transport endogenous glucose across their cell membrane. Diabetes is a chronic syndrome in which carbohydrate, protein and fat metabolism is impaired due to insufficient insulin secretion or insulin resistance in target tissues. It occurs in two main forms: insulin-dependent diabetes mellitus (IDDM, type I) and non-insulin-dependent diabetes mellitus (NIDDM, type II), the etiology, pathology, genetics, age of onset and treatment of the two major forms being different.
Both major forms of diabetes are characterized by an inability to deliver a certain amount of insulin and have the precise timing required for controlling glucose homeostasis. Type I diabetes or Insulin Dependent Diabetes Mellitus (IDDM) is caused by β cell destruction, resulting in insufficient endogenous insulin levels. Type II diabetes or non-insulin dependent diabetes mellitus is caused by a deficiency in the sensitivity of the human body to insulin and a relative lack of insulin production.
Food intake: the amount of food consumed by the individual. Food intake can be measured by volume or weight. For example, the food intake may be the total amount of food consumed by the individual. Alternatively, the food intake may be the amount of protein, fat, carbohydrate, cholesterol, vitamins, minerals or any other food component of the individual. "protein intake" refers to the amount of protein consumed by an individual. Similarly, "fat intake," "carbohydrate intake," "cholesterol intake," "vitamin intake," and "mineral intake" refer to the amount of protein, fat, carbohydrate, cholesterol, vitamin, or mineral consumed by an individual.
Normal daily diet: average food intake of individuals of a given species. The normal daily diet may be expressed in terms of calorie intake, protein intake, carbohydrate intake, and/or fat intake. The normal daily diet of humans typically includes the following: about 2,000, about 2,400, or about 2,800 to significantly more calories. In addition, the normal daily diet of humans typically comprises from about 12g to about 45g of protein, from about 120g to about 610g of carbohydrate, and from about 11g to about 90g of fat. The reduced calorie diet will not exceed about 85%, and preferably not exceed about 70%, of the normal caloric intake of a human subject.
In animals, caloric and nutritional requirements vary depending on the species and size of the animal. For example, in cats, the total caloric intake per pound and the percentage distribution of protein, carbohydrate, and fat varies with the age and reproductive status of the cat. However, the general guideline for cats is 40 calories/pound/day (18.2 calories/kilogram/day). About 30% to about 40% should be protein, about 7% to about 10% should be from carbohydrate, and about 50% to about 62.5% should be derived from fat intake. One skilled in the art can readily identify the normal daily diet of an individual of any species.
Obesity: conditions in which excess body fat may place a person at Health risk (see Barlow and Dietz, journal of Pediatrics (Pediatrics) 102: E29,1998; National institutes of Health (National institutes of Health), National Institute of Heart, Lung, and hematology (NHLBI), obesity research(Obes.Res.) 6 (journal of 2):51S-209S, 1998). Excess body fat is the result of an imbalance between energy intake and energy consumption. For example, Body Mass Index (BMI) may be used to assess obesity. In one common practice, the BMI is 25.0kg/m2To 29.9kg/m2Is overweight and has a BMI of 30kg/m2Or more is obesity.
In another convention, waist circumference is used to assess obesity. In this convention, a waist circumference of 102cm or more is considered to be obese in men, and 89cm or more is considered to be obese in women. There is strong evidence that obesity affects both morbidity and mortality in individuals. For example, obese individuals suffer from heart disease; non-insulin dependent (type 2) diabetes; hypertension; stroke; cancer (e.g., endometrial, breast, prostate, and colon cancer); dyslipidemia; gallbladder diseases; sleep apnea syndrome; reduced fertility and an increased risk of other diseases such as osteoarthritis (see Lyznicki et al, physicians in the United states (am. fam. Phys.), (63: 2185,2001).
Overweight: an individual whose weight exceeds its ideal weight. An overweight individual may be obese, but not necessarily obese. For example, an overweight individual is any individual for whom it is desirable to reduce their weight. In one convention, overweight individuals have a BMI of 25.0kg/m2To 29.9kg/m2Of (a).
Pegylated and pegylated: a process of reacting a poly (alkylene glycol), preferably an activated poly (alkylene glycol), to form a covalent bond. Promoters such as amino acids (e.g., lysine) may be used. Although "pegylation" is typically carried out using poly (ethylene glycol) or a derivative thereof, such as methoxypoly (ethylene glycol), the term is not limited herein to the use of methoxypoly (ethylene glycol) but also encompasses the use of any other useful poly (alkylene glycol), such as poly (propylene glycol).
pI: pI is an abbreviation for isoelectric point. An alternative abbreviation sometimes used is IEP. Which is the pH at which a particular molecule does not carry a net charge. At pH below its pI, the protein or peptide carries a net positive charge. At pH above its pI, the protein or peptide carries a net negative charge. Proteins and peptides can be separated according to their isoelectric points using a technique known as isoelectric focusing, which is an electrophoretic method that utilizes a pH gradient contained within a polyacrylamide gel.
Peptide yy (pyy): the term PYY as used herein refers to the peptide YY polypeptide, a hormone secreted into the blood by cells arranged in the lower small intestine (ileum) and colon. The naturally occurring wild-type PYY sequence of each species is shown in table 1.
Table 1: PYY sequences of various species
Peripheral administration: administration outside of the central nervous system. Peripheral administration does not involve direct administration to the brain. Peripheral administration includes, but is not limited to, intravascular, intramuscular, subcutaneous, inhalation, oral, rectal, transdermal, or intranasal administration.
Polypeptide: wherein the monomers are polymers of amino acid residues linked together by amide bonds. When the amino acid is an α -amino acid, an L-optical isomer or a D-optical isomer may be used, with the L-isomer being preferred. The term "polypeptide" or "protein" as used herein encompasses any amino acid sequence and includes modified sequences such as glycoproteins. The term "polypeptide" specifically encompasses naturally occurring proteins as well as recombinantly or synthetically produced proteins. The term "polypeptide fragment" refers to a portion of a polypeptide, such as a fragment that exhibits at least one useful sequence on binding to a receptor. The term "functional fragment of a polypeptide" refers to all fragments of a polypeptide that retain the activity of the polypeptide. Biologically functional peptides may also comprise fusion proteins in which a peptide of interest has been fused to another peptide that does not diminish its desired activity.
Subcutaneous administration: subcutaneous administration is the application of a substance to the subcutaneous fat layer found between the dermis and the underlying tissue of the skin. Subcutaneous administration may be by injection using, for example, a hypodermic needle fitted onto a syringe or "pen" type injection device. Other methods of administration, such as microneedles, may be used. Injection with a hypodermic needle typically involves a degree of pain on behalf of the recipient. Such pain can be masked by the use of a local anesthetic or analgesic. However, a common method for reducing perceived injection pain is to distract the subject only immediately before and during the injection. Pain can be minimized by using a relatively small gauge hypodermic needle, by injecting a relatively small volume of substance, and by avoiding overly acidic or basic compositions that can cause a subject to experience a "stinging" sensation at the injection site. Compositions having a pH between 4 and 10 are generally considered quite comfortable.
A therapeutically effective amount of: a dose sufficient to prevent the progression of the condition or cause the regression of the condition, or a dose capable of alleviating a sign or symptom of the condition, or a dose capable of achieving a desired result. In several embodiments, a therapeutically effective amount of a compound of the present invention is an amount sufficient to inhibit or prevent weight gain, or to reduce appetite, or to reduce calorie intake or food intake or to increase energy expenditure.
Detailed Description
According to a first aspect of the invention, there is provided a PYY analogue which differs from the native human PYY sequence in the following ways (Tyr1 Pro2 Ile 56 3Lys4 Pro5 Glu6 Ala7 Pro8 Gly9 Glu10 Asp11Ala12
-Ser23 substituted by Ala23, Glu23, Lys23, Gln23 or AIB23 (preferably Ala23)
And the PYY analogue may further differ from the native human PYY sequence in one or more of the following further aspects:
-Tyr1 is deleted,
-one or more of
-Glu10 substitution by Ala10, Lys10 or Gln10
Asp11 substituted by Gly11
-Ala12 substituted by AIB12
-Leu17 substitution by Ile17 or AIB17
-Asn18 substituted by AIB18, Ala18 or Leu18 (preferably Leu18)
-Arg19 substituted by His19 or Lys19 (preferably His19)
-Ala22 substitution by Val22 or Ile22
-Tyr27 substituted by Phe27
-Leu30 substituted by His30
-Val31 substituted by Leu31
And wherein the C-terminal residue optionally terminates in a primary amide (-C (O) NH)2) Radicals replacing carboxylic acid groups (-CO)2H)；
Or a derivative of the compound; or a salt of said compound or said derivative.
The amino acid sequence of formula (I) above is shown with the N-terminus at the top left and the C-terminus at the bottom right. Unless otherwise indicated, the amino acid residues in the sequence of formula (I) are L-amino acids.
In a preferred embodiment, Tyr1 is absent. Removal of Tyr1 increases the selectivity of PYY analogues for the Y2 receptor over the Y1 receptor, and agonism of the Y2 receptor is responsible for the inhibition of feeding.
In a preferred embodiment,
In another preferred embodiment,
In another embodiment, Tyr1 is deleted,
In another embodiment, Tyr1 is deleted,
In another particularly preferred embodiment, Tyr1 is deleted and
According to one embodiment
Deletion of-
According to a particularly preferred embodiment:
deletion of-
-
According to one embodiment:
-Glu10 substitution by Lys10 or Gln10
Asp11 substituted by Gly11
-Leu17 is unsubstituted
-Asn18 substituted by AIB18, Ala18 or Leu18
-Arg19 substituted by His19
-Ala22 is unsubstituted
-Ser23 substituted by Glu23, Ala23, AIB23 or Gln23
-Tyr27 is substituted by Phe27
-Leu30 substituted by His30
-Val31 substituted with
According to one embodiment:
deletion of-
-Glu10 substitution by Lys10 or Gln10
Asp11 substituted by Gly11
-Leu17 is unsubstituted
-Asn18 substituted by AIB18, Ala18 or Leu18
-Arg19 substituted by His19
-Ala22 is unsubstituted
-Ser23 substituted by Glu23, Ala23, AIB23 or Gln23
-Tyr27 is substituted by Phe27
-Leu30 substituted by His30
-Val31 substituted with
According to one embodiment:
deletion of-
-Glu10 substitution by Lys10 or Gln10
Asp11 substituted by Gly11
-Asn18 substituted by AIB18, Ala18 or Leu18
-Arg19 substituted by His19
-Ala22 is unsubstituted
-Ser23 substituted by Glu23, Ala23, AIB23 or Gln23
-Tyr27 is substituted by Phe27
-Leu30 substituted by His30
-Val31 substituted with
According to one embodiment:
deletion of-
-Glu10 substitution by Lys10 or Gln10
Asp11 substituted by Gly11
-Asn18 substituted by AIB18, Ala18 or Leu18
-Arg19 substituted by His19
-Ala22 is unsubstituted
-Ser23 substituted by Glu23, Ala23, AIB23 or Gln23
-Tyr27 is substituted by Phe27
-Leu30 substituted by His30
-Val31 substituted by Leu31
The C-terminal residue terminating in a primary amide (-C (O) NH)2) Radicals replacing carboxylic acid groups (-CO)2H)。
According to one embodiment:
glu10 is unsubstituted
Asp11 substituted by Gly11
-Leu17 is unsubstituted
-Asn18 is unsubstituted
Arg19 is His19
-Ala22 is unsubstituted
-Ser23 substituted by Ala23
-Tyr27 substituted by Phe27
-Leu30 substituted by His30
-Val31 substituted by Leu31
The C-terminal residue terminating in a primary amide (-C (O) NH)2) Radicals replacing carboxylic acid groups (-CO)2H)。
According to one embodiment:
deletion of-
Glu10 is unsubstituted
Asp11 substituted by Gly11
-Leu17 is unsubstituted
-Asn18 is unsubstituted
Arg19 is His19
-Ala22 is unsubstituted
-Ser23 substituted by Ala23
-Tyr27 substituted by Phe27
-Leu30 substituted by His30
-Val31 substituted by Leu31
The C-terminal residue terminating in a primary amide (-C (O) NH)2) Radicals replacing carboxylic acid groups (-CO)2H) (ii) a Optionally, all of
In some embodiments, Asp11 is substituted with
In some embodiments, Asn18 is substituted with AIB18, Ala18, or Leu18, preferably with
According to one embodiment:
-Glu10 substitution by Lys10 or Gln10
Asp11 substituted by Gly11
-Leu17 is unsubstituted
-Asn18 substituted by Leu18
-Arg19 substituted by His19
-Ala22 is unsubstituted
-Ser23 substituted by Glu23, Ala23, AIB23 or Gln23
-Tyr27 is substituted by Phe27
-Leu30 substituted by His30
-Val31 substituted with
According to one embodiment:
deletion of-
-Glu10 substitution by Lys10 or Gln10
Asp11 substituted by Gly11
-Leu17 is unsubstituted
-Asn18 substituted by Leu18
-Arg19 substituted by His19
-Ala22 is unsubstituted
-Ser23 substituted by Glu23, Ala23, AIB23 or Gln23
-Tyr27 is substituted by Phe27
-Leu30 substituted by His30
-Val31 substituted with
According to one embodiment:
deletion of-
-Glu10 substitution by Lys10 or Gln10
Asp11 substituted by Gly11
-Asn18 substituted by Leu18
-Arg19 substituted by His19
-Ala22 is unsubstituted
-Ser23 substituted by Glu23, Ala23, AIB23 or Gln23
-Tyr27 is substituted by Phe27
-Leu30 substituted by His30
-Val31 substituted with
According to one embodiment:
deletion of-
-Glu10 substitution by Lys10 or Gln10
Asp11 substituted by Gly11
-Asn18 substituted by Leu18
-Arg19 substituted by His19
-Ala22 is unsubstituted
-Ser23 substituted by Glu23, Ala23, AIB23 or Gln23
-Tyr27 is substituted by Phe27
-Leu30 substituted by His30
-Val31 substituted by Leu31
The C-terminal residue terminating in a primary amide (-C (O) NH)2) Radicals replacing carboxylic acid groups (-CO)2H)。
According to one embodiment:
glu10 is unsubstituted
Asp11 substituted by Gly11
-Leu17 is unsubstituted
-Asn18 substituted by Leu18
Arg19 is His19
-Ala22 is unsubstituted
-Ser23 substituted by Ala23
-Tyr27 substituted by Phe27
-Leu30 substituted by His30
-Val31 substituted by Leu31
The C-terminal residue terminating in a primary amide (-C (O) NH)2) Radicals replacing carboxylic acid groups (-CO)2H)。
According to one embodiment:
deletion of-
Glu10 is unsubstituted
Asp11 substituted by Gly11
-Leu17 is unsubstituted
-Asn18 substituted by Leu18
Arg19 is His19
-Ala22 is unsubstituted
-Ser23 substituted by Ala23
-Tyr27 substituted by Phe27
-Leu30 substituted by His30
-Val31 substituted by Leu31
The C-terminal residue terminating in a primary amide (-C (O) NH)2) Radicals replacing carboxylic acid groups (-CO)2H)
-optionally, all of
According to one embodiment:
deletion of-
Asp11 substituted by Gly11
-Asn18 substituted by Leu18
-Arg19 substituted by His19
-Ser23 substituted by Ala23
-Tyr27 is substituted by Phe27
-Leu30 substituted by His30
-Val31 substituted by Leu31
The C-terminal residue terminating in a primary amide (-C (O) NH)2) Radicals replacing carboxylic acid groups (-CO)2H)。
According to a preferred embodiment, at least 9 of the following 10 criteria apply:
1-
2-
3-
4-
5-
6-
Substitution of 7-
8-
9-
10-C terminal residue terminating in a primary amide (-C (O) NH)2) Radicals replacing carboxylic acid groups (-CO)2H)。
The present invention contemplates the above embodiments, wherein at least
According to some embodiments, Leu30 is substituted with His 30.
According to some embodiments, Val31 is unsubstituted.
According to certain embodiments, the naturally occurring Pro residues at
-Ser23 substituted by Ala23, Glu23, Lys23, Gln23 or AIB23 (preferably Ala23)
Such compounds may further differ from the native human PYY sequence in one or more of the following respects:
deletion of-Tyr
-one or
-Glu10 substitution by Ala10, Lys10 or Gln10
Asp11 substituted by Gly11
-Ala12 substituted by AIB12
-Leu17 substitution by Ile17 or AIB17
-Asn18 substituted by AIB18, Ala18 or Leu18 (preferably Leu18)
-Arg19 substituted by His19 or Lys19 (preferably His19)
-Ala22 substitution by Val22 or Ile22
-Tyr27 substituted by Phe27
-Leu30 substituted by His30
-Val31 substituted by Leu31
And wherein the C-terminal residue optionally terminates in a primary amide (-C (O) NH)2) Radicals replacing carboxylic acid groups (-CO)2H)；
Or a derivative of the compound; or a salt of said compound or said derivative.
According to all embodiments, it is preferred that the C-terminal residue terminates in a primary amide group (-C (O) NH)2) In place of carboxylic acid groups (-CO)2H) In that respect The loss of amide group results in a significant reduction in the agonistic effect of the Y2 receptor.
According to all examples, Ser23 was substituted with an alternative amino acid residue. Although it has been previously disclosed that the substitution at
According to some embodiments, Ser23 is substituted with Glu23, Ala23, AIB23, or
Preferred specific sequences include those disclosed herein and in particular those identified as Y1319, Y1371, Y1372, Y1419, Y1421, Y1518, Y1528, Y1558, Y1568, Y1572, Y1579, Y1553 or Y1581 (and in particular Y1419). The invention encompasses as preferred sequences the sequences corresponding to Y1319, Y1371, Y1372, Y1419, Y1421, Y1518, Y1528, Y1558, Y1568, Y1572, Y1579, Y1553 or Y1581 (and in particular Y1419) which are substituted 1 or 2 times (e.g. have been conservatively substituted) and which retain the activity of the sequence on which they are based (e.g. at least 20%, 50% or 70% of the activity in one of the assays disclosed herein).
According to a further aspect of the invention there is provided a pharmaceutical composition comprising an analogue of PYY according to the invention and a pharmaceutically acceptable carrier and optionally a further therapeutic agent.
The second therapeutic agent can be an anti-obesity agent, an appetite suppressant, or an anti-diabetic agent. Preferably, the therapeutic agent is a GLP-1 analog or a glucagon analog or a derivative of either thereof.
According to the further discussion belowIn certain embodiments, the composition comprises Zn2+Ions.
According to a further aspect of the invention there is provided a PYY analogue according to the invention or a pharmaceutical composition according to the invention for use as a medicament.
According to certain embodiments, the medicament is for the prevention or treatment of disorders of energy metabolism such as diabetes and/or obesity or for the prevention of loss of pancreatic islet function or for restoring pancreatic islet function.
According to certain embodiments, the medicament is for preventing diabetes or obesity in a subject.
According to a further aspect of the invention there is provided a method of treating or preventing a disease or disorder or other undesired physiological state in a subject, the method comprising administering to the subject a therapeutically acceptable/effective amount of a PYY analogue according to the invention or a pharmaceutical composition according to the invention.
According to a further aspect of the invention there is provided a method of preventing or treating a disorder of energy metabolism such as diabetes and/or obesity, preventing loss of pancreatic islet function and/or restoring pancreatic islet function, reducing appetite, reducing food intake and/or reducing calorie intake in a subject, the method comprising administering to the subject a therapeutically effective amount of a PYY analogue according to the invention or a pharmaceutical composition according to the invention according to the claims.
According to a further aspect of the invention there is provided a method of preventing or treating diabetes and/or obesity in a subject, the method comprising administering to the subject a therapeutically effective amount of a PYY analogue according to the invention or a pharmaceutical composition according to the invention.
With respect to all aspects of the invention, the subject may be overweight, obese and/or suffering from diabetes. Alternatively, the subject may have a healthy weight.
The PYY analogue or pharmaceutical composition according to the invention is administered parenterally. Thus, it may be provided in a format suitable for such administration. For example, it may be provided in an injection device.
Preferably, the PYY analogue or pharmaceutical composition is administered subcutaneously and may be provided in a format suitable for such administration.
According to a further aspect of the invention there is provided a method of inducing weight loss or preventing weight gain in a subject for cosmetic purposes, the method comprising administering to the subject an effective amount of an analogue of PYY according to the invention. In such cases, the subject may be obese, overweight, or have a healthy weight according to some preferred embodiments.
The peptide hormone analogs of the invention may be produced by recombinant methods well known in the art, or alternatively, the peptide hormone analogs may be produced by synthetic methods also well known in the art.
Compared to human PYY, PYY analogues according to the invention preferably have a more sustained effect or a stronger effect on decreasing food intake. Preferably, it has a food intake reducing effect that is at least as strong as but more durable than native human PYY. The increased duration of appetite suppression is particularly important to avoid the effect known as "escape". A short duration appetite suppressant may reduce appetite or the time taken for a meal and subjects will typically eat less food during that meal. However, if the appetite suppressant is subsequently metabolized or otherwise removed from the subject's circulation, the subject may regain his "normal" appetite at the next meal. Given that the subject had eaten a small meal at a previous meal time, the subject may actually have an increased appetite at the second meal. If the subject feels satisfied with his appetite, the total food intake of the two meals may not be less than that in the absence of the appetite suppressant. That is, the subject may have "escaped" the effect of the appetite suppressant. "escape" may be reduced by the use of additional doses of appetite suppressant or by the use of appetite suppressants that act longer in duration. If the subject's appetite decreases for a longer period of time, the extent to which it can compensate for the deficiency of one meal in the next meal will decrease, and this is because there is a practical limit to the total capacity of a particular meal.
Preferably, the PYY analogues of the invention are selective for the Y2 receptor. That is, the PYY analog binds Y2 with higher affinity than other receptors such as Y1, Y3, Y4, Y5, and Y6. Those receptors are recognized based on binding affinity, pharmacology, and sequence. Most, if not all, receptors are G protein-coupled receptors. The Y1 receptor is generally thought to be postsynaptic and to mediate many of the known effects of neuropeptide Y peripherally. Initially, this receptor was described as having a weaker affinity for the C-terminal fragment of neuropeptide Y (e.g., fragments 13-36), but interacting with full-length neuropeptide Y and peptide YY with equal affinity (see PCT publication WO 93/09227).
Pharmacologically, the Y2 receptor differs from Y1 by showing affinity for the C-terminal fragment of neuropeptide Y. Although 3-36 fragments of neuropeptide Y and peptide YY provide improved affinity and selectivity, the Y2 receptor is most often distinguished by the affinity of neuropeptide Y (13-36). (see Dumont et al, Society for neuroscience abstracts (19: 726,1993)). Signaling through the Y1 and Y2 receptors is coupled with inhibition of adenylate cyclase. Binding to the Y2 receptor was also found to reduce intracellular levels of calcium in synapses by selectively inhibiting N-type calcium channels. In addition, the Y2 receptor shows differential coupling to second messengers like the Y1 receptor (see U.S. patent No. 6,355,478). The Y2 receptor is found in various brain regions including the hippocampus, lateral substantia nigra, thalamus, hypothalamus, and brainstem. Human, murine, monkey and rat Y2 receptors have been cloned (see, e.g., U.S. patent No. 6,420,352 and U.S. patent No. 6,355,478). Preferably, the PYY analogue of the invention binds Y2 with at least two, at least five or at least ten times greater affinity than Y1, Y3, Y4, Y5 and/or Y6.
The PYY analogues according to the invention preferably have a low or no net ionic charge in solution (i.e. in solution near physiological conditions as found e.g. in interstitial fluid or plasma, e.g. at pH7.4), e.g. preferably the PYY analogues of the invention have a net ionic charge of +1, 0 or-1 at pH 7.4. For example, a PYY analog having two acidic groups and two basic groups will have a net ionic charge of 0. Acidic groups with pKa's in water of less than 6.4 are believed to contribute an ionic charge of-1 at pH 7.4. Basic groups with pKa greater than 8.4 are believed to contribute an ionic charge of +1 at pH 7.4. It is hypothesized that the low net ionic charge (and in particular the absence of net ionic charge under in vivo conditions) limits the in vivo solubility of PYY analogues, and this results in slower absorption and thus prolonged presence in the circulation following subcutaneous administration of high concentrations of peptide.
The PYY analogues according to the invention contain histidine as in the native PYY at
By way of further explanation, histidine is a unique amino acid that is uncharged at pH7.4 (i.e., under physiological conditions in the circulation or subcutaneously after administration). However, since the pI of the NH side chain of histidine is about 6.0, it is fully charged at pH 5 (or lower). According to certain preferred embodiments, PYY analogues according to the invention have low or no total charge at physiological pH (pH7.4) and are preferably formulated as part of a composition having a pH of about pH 5 (e.g. pH 4.5 to pH 6.0-pH below about pH4 or 5 may be undesirable for injectable compositions as it may increase pain sensation at the injection site) to show histidine ionisation and preferably a total net change at such lower pH. The increased number of charged residues increases the solubility of the injectable composition in the vial and thus allows for a small volume injection of a relatively concentrated peptide solution. However, after subcutaneous injection, the analogue is exposed to physiological pH at which the number of ionized residues and in particular the number of ionized histidine residues decreases and thus solubility decreases. This caused the peptide to precipitate subcutaneously.
The presence of a His residue enhances this effect.
According to certain preferred embodiments, the PYY analogues according to the invention have a combination of the following preferred features:
1) peptide sequences that have no or low net ionic charge (e.g., +1, 0, or-1) at pH7.4 and may generally have relatively few charged groups and hydrophilic groups to reduce intrinsic solubility.
2) There are many histidines that produce a net positive charge at
3) Suitable for subcutaneous administration in low volumes and high concentrations, exceeding the solubility constant at pH7.4 but not exceeding the solubility constant at
In addition to histidine being a particularly advantageous amino acid residue for causing this differential pH-dependent solubility effect, the differential solubility of peptides containing histidine residues is greatly enhanced if formulated with zinc ions. This is because zinc ions will bind to uncharged histidine residues in aqueous solution. It is believed that zinc ions can simultaneously bind up to 4 uncharged histidines. This allows the zinc to coordinate with histidine residues in several individual peptide molecules and thus weakly cross-link the peptide molecules with other similar peptide molecules, resulting in a decrease in solubility. However, the zinc ion is not bound to the charged histidine. Thus, in a composition containing zinc ions, the histidine-containing peptide will crosslink at pH7.4 but not at pH 5.0 through weak ionic bonds. Thus, the presence of His residues bound to zinc ions enhanced peptide precipitation following subcutaneous injection, but did not affect solubility in vials or syringes prior to administration. This means that peptides with an overall pI of about 7 will have no charged residues at about neutral pH and include histidine residues in formulations comprising zinc ions are advantageously soluble in vials or syringes, but precipitate subcutaneously after administration. Thus, pH7 neutral peptides with histidine in formulations comprising zinc ions are advantageously soluble in vials and syringes, but precipitate subcutaneously after administration. Furthermore, the zinc enhanced precipitation is gradually reversible, as the concentration of zinc ions will decrease after injection as the zinc ions are gradually flushed out of the injection site. Thus, a delay in subcutaneous absorption was observed, with better pharmacokinetics without loss of bioavailability. The absorption rate of a given histidine-containing neutral peptide can be controlled by the amount of zinc added.
The introduction of at least one further histidine residue preferably results in the PYY analogues of the invention having at least one occurrence of two histidine residues separated from each other by 1 to 3 intervening amino acid residues (a pair of histidine residues). This spacing appears to be optimal for a single zinc ion associated with both histidine residues in pairs in aqueous solution. In an advantageous embodiment of the invention, the amino acid residues at
Preferably, the analogue according to the invention has a total pI between 6.5 and 8.5. This means that at physiological pH (e.g., pH7.4) the analog total charge is low or insignificant (e.g., as mentioned above, preferably the PYY analog of the invention has a net ionic charge of +1, 0, or-1 at pH 7.4). The total pI of the molecule may be calculated using techniques well known to those skilled in the art, or alternatively, may be determined experimentally by using isoelectric focusing.
In order to fully exploit this effect, the inventors have found that a combination of the following features is particularly preferred.
1) Peptide sequences having a low or no net ionic charge (e.g., +1, 0, or-1) at pH 7.4.
2) There are three histidines that produce a net positive charge at
3) Has a high solubility at
4) There are zinc ions that produce crosslinking of uncharged histidine residues with adjacent molecules at pH7.4 but do not crosslink with charged histidine at pH or about
It was found that analogues with His residues at
Thus, according to certain preferred embodiments, there is provided a PYY analogue which differs from the native PYY sequence in the following respects:
-Ser23 substituted by Ala23, Glu23, Lys23, Gln23 or AIB23 (preferably Ala23)
-Arg19 substituted by His19
-Leu30 substituted by His30
Such compounds may further differ from the native human PYY sequence in one or more of the following respects:
deletion of-Tyr
-one or more of
-Glu10 substitution by Ala10, Lys10 or Gln10
Asp11 substituted by Gly11
-Ala12 substituted by AIB12
-Leu17 substitution by Ile17 or AIB17
-Asn18 substituted by AIB18, Ala18 or Leu18 (preferably Leu18)
-Ala22 substitution by Val22 or Ile22
-Tyr27 substituted by Phe27
-Val31 substituted by Leu31
And wherein the C-terminal residue optionally terminates in a primary amide (-C (O) NH)2) Radicals replacing carboxylic acid groups (-CO)2H) (ii) a Or a derivative of the compound; or a salt of said compound or said derivative.
Preferably,
Derivatives of the same
The PYY analogs of the invention may be derivatives modified by well-known methods including amidation, glycosylation, carbamylation, acylation, e.g., acetylation, sulfation, phosphorylation, cyclization, lipidation, pegylation, and fusion with another peptide or protein to form a fusion protein. The PYY analogues of the invention may be modified at random positions within the molecule or at predetermined positions within the molecule and may comprise one, two, three or more linked chemical moieties.
The PYY analogues of the invention may be fusion proteins, wherein the analogue is fused to another protein or polypeptide (fusion partner) using recombinant methods known in the art. Alternatively, such fusion proteins may be synthesized by any known synthetic method. Any suitable peptide or protein may be used as a fusion partner (e.g., serum albumin, carbonic anhydrase, glutathione-S-transferase, thioredoxin, or the like). Preferred fusion partners will not have undesirable biological activity in vivo. Such fusion proteins can be prepared by linking the carboxy terminus of the fusion partner to the amino terminus of the PYY analog or vice versa. Optionally, a cleavable linker may be used to link the PYY analog to the fusion partner. The resulting cleavable fusion protein may be cleaved in vivo, allowing the release of the active form of the compound of the invention. Examples of such cleavable linkers include, but are not limited to, linkers D-D-D-D-Y, G-P-R, A-G-G and H-P-F-H-L, which can be cleaved by enterokinase, thrombin, ubiquitin lyase, and renin, respectively. See, for example, U.S. patent No. 6,410,707, the contents of which are incorporated herein by reference. According to certain embodiments of all aspects of the invention, the PYY analogue is not a fusion protein.
The PYY analogues of the invention may be physiologically functional derivatives. The term "physiologically functional derivative" is used herein to denote a chemical derivative of the PYY analogue of the invention which has the same physiological function as the corresponding unmodified PYY analogue of the invention. For example, a physiologically functional derivative may be converted in vivo to a PYY analogue of the invention. Examples of physiologically functional derivatives according to the present invention include esters, amides and carbamates; esters and amides are preferred.
Pharmaceutically acceptable esters and amides of the compounds of the invention may include C attached at an appropriate site (e.g., at an acid group)1-20Alkyl-, C2-20Alkenyl-, C5-10aryl-C5-10ar-C1-20Alkyl-or amino acid-esters or-amides. Examples of suitable moieties are hydrophobic substituents having from 4 to 26 carbon atoms, preferably from 5 to 19 carbon atoms. Suitable lipid groups include, but are not limited to, the following: lauroyl (C)12H23) Palm base (C)15H31) Oleyl (C)15H29) Stearyl group (C)17H35) Cholate; and deoxycholate.
U.S. patent No. 5,936,092; U.S. patent No. 6,093,692; and U.S. Pat. No. 6,225,445 discloses a method for lipidating a thiol-group-containing compound with a fatty acid derivative. Fatty acid derivatives of the compounds of the invention including compounds of the invention linked to fatty acids by disulfide bonds may be used to deliver the compounds of the invention to neuronal cells and tissues. Lipidation significantly increases the absorption of the compound and prolongs blood and tissue retention of the compound relative to the absorption rate of the corresponding non-lipidated compound. Furthermore, the disulfide bonds in lipidated derivatives are relatively unstable in cells and thus facilitate intracellular release of the molecule from the fatty acid moiety. Suitable lipid-containing moieties are hydrophobic substituents having from 4 to 26 carbon atoms, preferably from 5 to 19 carbon atoms. Suitable lipid groups include, but are not limited to, the following: palm base (C)15H31) Oleyl (C)15H29) Stearyl group (C)17H35) Cholate; and deoxycholate.
Cyclization methods include cyclization by formation of disulfide bridges and head-to-tail cyclization using cyclization resins. Due to its conformational constraints, the cyclized peptide can have enhanced stability, including increased resistance to enzymatic degradation. Cyclization may be particularly advantageous when the cyclized peptide contains an N-terminal cysteine group. Suitable cyclized peptides include monomeric and dimeric head-to-tail cyclized structures. The cyclized peptide may comprise one or more additional residues, in particular additional cysteines incorporated for the purpose of forming disulfide bonds or side chains incorporated for the purpose of resin-based cyclization.
The PYY analogs of the invention can be pegylated structures of PYY analogs. The pegylated compounds of the invention can provide additional advantages such as increased solubility, stability and circulation time of the polypeptide or reduced immunogenicity (see U.S. Pat. No. 4,179,337).
The chemical moiety used to derivatize the compounds of the present invention may also be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, and the like. The polymer moiety used to derivatize the compounds of the present invention may be of any molecular weight, and may be branched or unbranched. For ease of handling and preparation, the preferred molecular weight of the polyethylene glycol used to derivatize the compounds of the present invention is from about l kDa to about 100kDa, the term "about" indicating that in the preparation of polyethylene glycol, some molecules have a molecular weight greater than the specified molecular weight and some are less than the specified molecular weight. Other molecular weight polymers may be used depending on the desired treatment regimen, such as the duration of sustained release desired, the effect on bioactivity (if any), ease of handling, degree or absence of antigenicity, and other known effects of polyethylene glycol on therapeutic proteins or the like. For example, the average molecular weight of the polyethylene glycol may be about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
In one embodiment, the PYY analogue of the invention is not a derivative.
Salts of the PYY analogues of the invention suitable for use in medicine are salts in which the counter-ion is pharmaceutically acceptable. However, salts with non-pharmaceutically acceptable counterions are within the scope of the invention, e.g., as intermediates in the preparation of the PYY analogues of the invention and pharmaceutically acceptable salts and/or derivatives thereof.
Suitable salts according to the invention comprise salts formed from organic or inorganic acids or bases. Pharmaceutically acceptable acid addition salts include those formed from: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, citric acid, tartaric acid, acetic acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, trifluoroacetic acid, succinic acid, perchloric acid, fumaric acid, maleic acid, glycolic acid, lactic acid, salicylic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and isethionic acid.
Pharmaceutically acceptable salts formed from bases include ammonium salts; alkali metal salts such as potassium and sodium salts; alkaline earth metal salts such as calcium and magnesium salts; and salts formed with organic bases such as dicyclohexylamine and N-methyl-D-glucamine.
Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they react or from which they precipitate or crystallize. These complexes are known as "solvates". For example, complexes with water are referred to as "hydrates". Solvates (e.g., hydrates) may exist when a drug substance is incorporated into a solvent (e.g., water) in stoichiometric or non-stoichiometric amounts in the crystal lattice. Since hydrates can be encountered at any stage of the drug manufacturing process or during storage of the drug substance or dosage form, drug substances are routinely screened for the presence of hydrates. Solvates are described in the following documents: byrn et al, Pharmaceutical research 12(7),1995,954-954 and Water-Insoluble Drug formulations (Water-Insoluble Drug Formulation), 2 nd edition, R.Liu, CRC Press, page 553, which are incorporated herein by reference. Thus, it will be appreciated by those skilled in the art that the PYY analogues of the invention, as well as derivatives and/or salts thereof, may therefore be present in the form of solvates. Solvates of the PYY analogues of the invention suitable for use in medicine are those wherein the relevant solvent is a pharmaceutically acceptable solvate. For example, hydrates are examples of pharmaceutically acceptable solvates.
Conditions are as follows:
the invention also provides a PYY analogue according to the invention or a pharmaceutical composition comprising said PYY analogue for use as a medicament. The PYY analogs and pharmaceutical compositions find use in the treatment and/or prevention of conditions such as diabetes and obesity. The PYY analogs and pharmaceutical compositions comprising the PYY analogs also find use in reducing appetite in a subject, reducing food intake in a subject, and/or reducing calorie intake in a subject.
The invention also provides the use of an analogue of PYY according to the invention for the manufacture of a medicament for the prevention or treatment of diabetes and/or obesity. The invention also provides the use of an analogue of PYY according to the invention for the manufacture of a medicament for reducing appetite, reducing food intake and/or reducing calorie intake in a subject.
The invention also provides a method of treating or preventing a disease or disorder or other undesired physiological state in a subject, the method comprising administering to the subject a therapeutically effective amount of an analogue of PYY according to the invention or a pharmaceutical composition comprising the PYY analogue.
The invention also provides a method of preventing or treating diabetes and/or obesity, reducing appetite, reducing food intake and/or reducing calorie intake in a subject, the method comprising administering to the subject a therapeutically effective amount of a PYY analogue according to the invention or a pharmaceutical composition comprising the PYY analogue.
In one embodiment, the PYY analog or pharmaceutical composition is administered parenterally. In one embodiment, the PYY analog or pharmaceutical composition is administered subcutaneously. In one embodiment, the PYY analog or pharmaceutical composition is administered intravenously, intramuscularly, intranasally, transdermally, or sublingually.
A subject to whom a PYY analogue according to the invention or a pharmaceutical composition comprising the PYY analogue may be administered may be overweight, e.g. the subject may be obese. Alternatively or additionally, the subject may be diabetic, e.g. have insulin resistance or glucose intolerance or both. The subject may have diabetes, for example, the subject may have type II diabetes. The subject may be overweight, e.g., obese, and suffering from diabetes, e.g., type II diabetes. Alternatively, the subject may have type I diabetes.
The PYY analogs of the present invention are believed to protect islets of langerhans cells, particularly beta cells, when challenged with a toxin (e.g., streptozotocin), pathogen, or autoimmune response, allowing them to retain their normal physiological functions, such as the ability to secrete insulin in response to an appropriate stimulus. The PYY analogs of the invention are also believed to be effective in restoring or rescuing pancreatic islet function, and in particular beta cell function, following a deterioration in physiological function resulting from exposure to a toxin, pathogen, or autoimmune response. Functional recovery may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the function exhibited prior to deterioration. Accordingly, the present invention also provides a PYY analogue of the invention or a pharmaceutical composition comprising the PYY analogue for use in preventing loss of pancreatic islet function (e.g. beta cell function) and/or restoring pancreatic islet function (e.g. beta cell function). The invention further provides the use of an analogue of PYY of the invention for the manufacture of a medicament for preventing loss of pancreatic islet function (e.g. beta cell function) and/or for restoring pancreatic islet function (e.g. beta cell function). The invention further provides a method of preventing loss of pancreatic islet function (e.g. beta cell function) and/or restoring pancreatic islet function (e.g. beta cell function) in a subject, the method comprising administering to the subject an effective amount of a PYY analogue of the invention or a pharmaceutical composition comprising the PYY analogue.
The islet protective properties of the PYY analogs of the present invention make them useful for administration in combination with additional therapeutic agents that have side-effect islet toxicity. An example of such a therapeutic agent is streptozotocin. Thus, the present invention also provides a PYY analogue according to the invention in combination with a further therapeutic agent having islet toxicity as a side effect. The invention also provides a pharmaceutical composition comprising an analogue of PYY according to the invention and a further therapeutic agent having islet toxicity as a side effect, and a pharmaceutically acceptable carrier.
Additionally or alternatively, the subject may have or may be at risk of having a condition in which obesity or overweight is a risk factor. Such conditions include, but are not limited to, cardiovascular diseases such as hypertension, atherosclerosis, congestive heart failure and dyslipidemia; stroke; gallbladder diseases; osteoarthritis; sleep apnea syndrome; reproductive disorders, such as polycystic ovary syndrome; cancers, such as breast, prostate, colon, endometrial, renal, and esophageal cancers; varicose veins; acanthosis nigricans; eczema; exercise intolerance; insulin resistance; hypertensive hypercholesterolemia; cholelithiasis; osteoarthritis; orthopedic injuries; insulin resistance, e.g.,
Other obesity-related conditions include depression, anxiety, panic attacks, migraine, PMS, chronic pain states, fibromyalgia, insomnia, impulsivity, obsessive-compulsive disorder, and myoclonus. In addition, obesity is a recognized risk factor for increased incidence of general anesthesia complications. (see, e.g., Kopelman, Nature 404:635-43, 2000). Generally, obesity reduces longevity and carries a serious risk of co-morbidity as listed above.
Other diseases or disorders associated with obesity are birth defects, maternal obesity associated with increased incidence of neural tube defects, Carpal Tunnel Syndrome (CTS); chronic Venous Insufficiency (CVI); sleepiness during the day; deep Vein Thrombosis (DVT); end-stage renal disease (ESRD); gout; heat syndrome; impaired immune response; impaired respiratory function; infertility; liver disease; lower back pain; obstetric and gynecological complications; pancreatitis; and abdominal hernias; acanthosis nigricans; endocrine abnormalities; chronic hypoxia and hypercapnia; dermatological effects; elephantiasis; gastroesophageal reflux; heel spurs and inflammation; edema of the lower limbs; breast hypertrophy, which causes a number of problems such as breast tightening pain, skin damage, cervical pain, long-term off-flavors and infections in the underlying skin folds; large anterior abdominal wall masses, such as abdominal cellulitis with frequent cellulitis, which hampers walking, causes frequent infections, bad odors, difficulty dressing, lower back pain; musculoskeletal diseases; pseudoencephaloma (or benign intracranial hypertension) and sliding hiatal hernia.
The invention further provides a method for increasing energy expenditure in a subject. The method comprises peripherally administering to the subject a therapeutically effective amount of a PYY analog of the invention, thereby altering energy expenditure, for example. Energy is burned in all physiological processes. By adjusting the efficiency of these processes or changing the number and nature of the processes that are occurring, the body can directly change the rate of energy expenditure. For example, during digestion, the body consumes energy by passing food through the gut and digesting food, and within cells, the efficiency of cellular metabolism can be altered to produce more or less heat.
In one aspect, the method of the present invention involves manipulating arcuate circuitry that cooperatively alters food intake and conversely alters energy expenditure. Energy expenditure is the result of cellular metabolism, protein synthesis, metabolic rate, and calorie utilization. Thus, in this aspect of the invention, administration of the PYY analogue according to the invention results in increased energy expenditure and decreased calorie utilization.
The invention also provides a method for improving lipid analysis in a subject, the method comprising administering to the subject a PYY analogue according to the invention or a pharmaceutical composition comprising the PYY analogue. The invention also provides a method for alleviating a condition or disorder which may be alleviated by reduced nutrient availability, the method comprising administering to a subject a PYY analogue according to the invention or a pharmaceutical composition comprising the PYY analogue.
Appetite may be measured by any method known to those skilled in the art. For example, appetite reduction may be assessed by psychological assessment. For example, administration of a compound of the invention results in a perceived change in hunger, satiety and/or satiation. Starvation may be assessed by any method known to those skilled in the art. For example, hunger is assessed using psychology, such as by using questionnaires (such as, but not limited to, Visual Analog Scoring (VAS) questionnaires) to assess hunger and sensory perception. In one specific non-limiting example, hunger is assessed by answering questions related to: the desire for food, beverages, expected food consumption, nausea, and the perception of odor or taste.
PYY analogues of the invention may be used for weight management and treatment (e.g. reduction or prevention) of obesity, in particular any one or more of the following: prevention and reduction of weight increase; inducing and promoting weight loss; and reducing obesity as measured by body mass index. The PYY analogues of the invention may be used to control any one or more of appetite, satiety and hunger, in particular any one or more of the following: reduce, suppress and suppress appetite; inducing, increasing, enhancing and promoting satiety and satiety; and reducing, suppressing and suppressing hunger and hunger sensation. The PYY analogues of the invention may be used to maintain any one or more of a desired body weight, a desired body mass index, a desired appearance and good health. Thus, the invention also provides a method of inducing weight loss or preventing weight gain in a subject for cosmetic purposes, the method comprising administering to the subject an effective amount of a PYY analogue according to the invention or a composition comprising the PYY analogue.
The subject may be a subject for whom weight loss is desired, for example, female and male subjects for whom it is desired to change their appearance. The subject may desire to reduce hunger, for example the subject may be a person participating in a lengthy task requiring a high degree of concentration, such as an active soldier, an air traffic controller or a truck driver on a long haul route, etc.
The invention may also be used to treat, prevent, ameliorate or ameliorate a condition or disorder caused, complicated or exacerbated by relatively high nutrient availability. The term "condition or disorder that can be alleviated by reducing caloric (or nutrient) utilization" is used herein to denote any condition or disorder in a subject that is caused, complicated, or exacerbated by relatively high nutrient utilization, or that can be alleviated by reducing nutrient utilization (e.g., by reducing food intake). Subjects with insulin resistance, glucose intolerance, or any form of diabetes (e.g.,
The present invention relates to the treatment of metabolic disorders, such as disorders of energy metabolism. Such disorders include conditions or disorders associated with increased caloric intake, including but not limited to insulin resistance, glucose intolerance, obesity, diabetes (including
According to the invention PYY analogues are preferably used for the treatment of humans. However, while the compounds of the invention will generally be used to treat a human subject, the compounds may also be used to treat similar or identical conditions in the following animals: other vertebrates (e.g., other primates); farm animals (e.g., pigs, cattle, and poultry); sports animals (e.g., horses); companion animals (e.g., dogs and cats).
Composition comprising a metal oxide and a metal oxide
While it is possible to administer the active ingredient alone, it is preferred that the active ingredient be present in a pharmaceutical formulation or composition. Thus, the invention also provides a pharmaceutical composition comprising an analogue of PYY according to the invention and a pharmaceutically acceptable carrier and optionally other therapeutic ingredients. The pharmaceutical compositions of the present invention may take the form of pharmaceutical formulations as described below.
The pharmaceutical formulation according to the present invention comprises a pharmaceutical formulation suitable for administration of: orally taking; parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular); inhalation (containing fine particle dust or mist that can be generated by various types of metered dose pressurized aerosols, nebulizers, or insufflators); rectal and topical (including dermal, transdermal, transmucosal, buccal, sublingual and intraocular), although the most suitable route may depend, for example, on the condition and disorder of the recipient.
The formulations may be conveniently presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods comprise the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired formulation.
Formulations of the present invention suitable for oral administration may be presented as discrete units, such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; in the form of powder or granules; as a solution or suspension in an aqueous liquid or a non-aqueous liquid; or in the form of an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. Various pharmaceutically acceptable carriers and formulations thereof are described in the discussion of standard formulations, e.g., Remington's Pharmaceutical Sciences, e.g., e.w. martin. See also Wang, Y.J. and Hanson, M.A., Journal of Parenteral science and Technology, technical report No. 10, supplement 42:2S, 1988.
Tablets may be prepared by compression or moulding, optionally with one or more accessory ingredients. Can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form (e.g., powder or granules), optionally mixed with a binder, lubricant, inert diluent, lubricant, surfactant or dispersant. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein. The compounds of the invention may be administered, for example, in a form suitable for immediate release or extended release. Immediate or extended release may be achieved by the use of suitable pharmaceutical compositions comprising the compounds of the invention, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps. The compounds of the invention may also be administered via liposomes.
Preferably, the composition according to the invention is suitable for subcutaneous administration, e.g. by injection. According to certain embodiments, the composition may contain metal ions such as copper, iron, aluminum, zinc, nickel, or cobalt ions. The presence of such ions may limit solubility and thus delay absorption from the subcutaneous site of administration into the circulatory system. In a particularly preferred embodiment, the composition contains zinc ions. The zinc ions may be present in any suitable concentration, for example in a molar ratio to the peptide molecule of 10:1 to 1:10, 8:1 to 1:8, 5:1 to 1:5, 4:1 to 1:4, 3:1 to 1:3, 2:1 to 1:2 or 1: 1. In one embodiment, the pH of the pharmaceutical composition is less than 5 and the pharmaceutical composition includes zinc ions.
Exemplary compositions for oral administration comprise suspensions which may contain, for example, microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweetening or flavoring agents as are known in the art; and immediate release tablets which may contain, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and/or lactose and/or other excipients, binders, fillers, disintegrants, diluents and lubricants as are known in the art. The PYY analogues of the invention, or variants, derivatives, salts or solvates thereof, may also be delivered orally by sublingual and/or buccal administration. Molded tablets, compressed tablets or freeze-dried tablets are exemplary forms that may be used. Exemplary compositions include compositions in which one or more compounds of the invention are formulated with a fast dissolving diluent such as mannitol, lactose, sucrose, and/or cyclodextrin. Such formulations may also contain high molecular weight excipients such as cellulose (avicel) or polyethylene glycol (PEG). Such formulations may also include excipients to aid mucosal adhesion, such as hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), sodium carboxymethyl cellulose (SCMC), maleic anhydride copolymers (e.g., Gantrez), and agents to control release, such as polypropylene copolymers (e.g., carbopol 934). Lubricants, glidants, flavoring agents, coloring agents and stabilizers may also be added for ease of manufacture and use.
Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes of the formulation that provide isotonicity with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Exemplary compositions for parenteral administration include injectable solutions or suspensions which may contain, for example, suitable non-toxic parenterally acceptable diluents or solvents (e.g., mannitol, 1, 3-butanediol, water, ringer's solution, isotonic sodium chloride solution); or other suitable dispersing or wetting agents and suspending agents including synthetic mono-or diglycerides and fatty acids including oleic acid or Cremaphor. The aqueous carrier can be, for example, an isotonic buffer solution having a pH of about 3.0 to about 8.0, preferably a pH of about 3.5 to about 7.4 (e.g., 3.5 to 6.0, such as 3.5 to about 5.0). Useful buffers include sodium citrate-citric acid and sodium phosphate-phosphoric acid and sodium acetate/acetic acid buffers. The composition preferably does not contain oxidizing agents and other compounds known to be detrimental to PYY and related molecules. Excipients which may be included are, for example, other proteins, such as human serum albumin or plasma formulations. If desired, the pharmaceutical compositions may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate.
In one embodiment, the pharmaceutical composition is present in a syringe or other administration device for subcutaneous administration to a human.
Exemplary compositions for nasal aerosol or inhalation administration comprise solutions in saline which may contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other solubilizing or dispersing agents as are known in the art. Conveniently, in compositions for nasal aerosol or inhalation administration, the compounds of the invention may be delivered in the form of an aerosol spray presentation from a pressurised pack or nebuliser, with the use of a suitable propellant (e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve for delivering a metered amount. Capsules and cartridges (e.g., of gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. In one specific non-limiting example, the compounds of the present invention are administered as an aerosol from a metered dose valve through an aerosol adapter (also referred to as an actuator). Optionally, a stabilizer and/or porous particles for deep lung delivery (see, e.g., U.S. patent No. 6,447,743).
Formulations for rectal administration may be presented as a retention enema or suppository with, for example, cocoa butter, synthetic glycerides or polyethylene glycols. Such carriers are typically solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.
Formulations for topical administration in the mouth (e.g., buccally or sublingually) include lozenges comprising the active ingredient in a flavored base (such as sucrose and acacia or tragacanth) and pastilles comprising the active ingredient in a base (such as gelatin and glycerin or sucrose and acacia). An exemplary composition for topical application comprises a topical carrier, such as liquid paraffin and ointment base of polyethylene (Plastibase) (mineral oil gelled with polyethylene).
A preferred unit dose formulation is one containing an effective dose of the PYY analog, or an appropriate portion thereof, as previously described.
It will be appreciated that, with respect to the type of formulation in question, in addition to the ingredients specifically mentioned above, the formulations of the present invention may contain other agents conventional in the art, for example, formulations suitable for oral administration may contain flavoring agents.
The PYY analogues of the invention are also suitable for administration as a sustained-release system. Suitable examples of sustained-release systems of the present invention comprise suitable polymeric materials, such as a semi-permeable polymer matrix in the form of a shaped article (e.g., a film or a microcapsule); suitable hydrophobic materials, such as an emulsion in an acceptable oil; or an ion exchange resin; and sparingly soluble derivatives of the compounds of the invention, e.g., sparingly soluble salts. Sustained-release systems may be administered as follows: orally taking; a rectum; parenteral administration; in the brain pool; in the vagina; intraperitoneal administration; topically (e.g., as a powder, ointment, gel, drop, or transdermal patch); transbuccal; or as an oral or nasal spray.
Formulations for administration may be suitably formulated to give controlled release of the compounds of the invention. For example, the pharmaceutical composition may be in the form of particles comprising one or more of a biodegradable polymer, a polysaccharide gelling and/or bioadhesive polymer, an amphiphilic polymer, an agent capable of modifying the interfacial properties of the compound particles of formula (I). These compositions exhibit certain biocompatible characteristics that allow for the controlled release of the active substance. See U.S. patent No. 5,700,486.
The PYY analogs of the invention may be delivered by pump (see Langer, supra; Sefton, CRC bio-pharmaceutical engineering review (CRC crit.ref.biomed.eng.) 14:201,1987; Buchwald et al, Surgery (Surgery) 88:507,1980; Saudek et al, new england journal of medicine 321:574,1989) or by continuous subcutaneous injection, e.g., using a micropump. Intravenous solution bags may also be used. A key factor in selecting an appropriate dosage is the result obtained, as measured by a reduction in the ratio of total body weight or fat mass to muscle mass or by other criteria for measuring control or prevention of obesity or obesity-related conditions as deemed appropriate by the practitioner. Other controlled release systems are discussed in Langer's review (Science 249: 1527-. In another aspect of the disclosure, the compounds of the invention are delivered by implantable pumps, such as described in the following U.S. patents: U.S. Pat. nos. 6,436,091; U.S. patent No. 5,939,380; U.S. patent No. 5,993,414.
Implantable drug infusion devices are used to provide a constant and long-term dose or infusion of a drug or any other therapeutic agent to a patient. Essentially, such devices can be classified as active or passive. The compounds of the invention may be formulated as depot preparations. Such long acting depot formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, a compound may be formulated with: suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil); or an ion exchange resin; or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.
A therapeutically effective amount of a PYY analog of the invention may be administered in a single pulsed dose, in a bolus dose, or in a pulsed dose over time. Thus, in a pulsed dose, there is provided a bolus administration of a PYY analogue of the invention, followed by a period of time in which no compound of the invention is administered to the subject, followed by a second bolus administration. In specific non-limiting examples, pulsed doses of the compounds of the invention are administered during the course of a day, the course of a week, or the course of a month.
The invention also provides a PYY analogue according to the invention for administration simultaneously, sequentially or separately with an additional therapeutic agent. The invention also provides a pharmaceutical composition comprising an analogue of PYY according to the invention and a further therapeutic agent. Examples of additional therapeutic agents include additional appetite suppressants, food intake reducing agents, blood glucose reducing agents, or blood lipid altering agents. Specific non-limiting examples of additional appetite suppressants include amfepramone (diethylpropion), phentermine, mazindol and phenylpropanolamine, fenfluramine, dexfenfluramine, and fluoxetine. As mentioned above, the PYY analogue of the invention may be administered simultaneously with the additional appetite suppressant or may be administered sequentially or separately. In one embodiment, the compounds of the present invention are formulated and administered in a single dose with an appetite suppressant.
The PYY analogs of the invention may be administered whenever an effect is desired, such as suppressing appetite, reducing food intake, or reducing calorie intake, or whenever a slightly earlier time than the effect is desired (such as, but not limited to, about 10 minutes, about 15 minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about 120 minutes before the time that the effect is desired).
A therapeutically effective amount of a PYY analog of the invention will depend upon the molecule utilized, the subject being treated, the severity and type of the disease, and the mode and route of administration. For example, a therapeutically effective amount of a PYY analog of the invention can vary from about 0.01 milligrams per kilogram (kg) of body weight to about 1 gram per kg of body weight, e.g., from about 0.1 μ g to about 20mg per kg of body weight, e.g., from about 1 μ g to about 5mg per kg of body weight or from about 5 μ g to about 1mg per kg of body weight.
In one embodiment of the invention, the PYY analogue of the invention may be administered to the subject in the range of 5 to 1000nmol per kg body weight, for example in the range of 10 to 750nmol per kg body weight, for example in the range of 20 to 500nmol per kg body weight, in particular in the range of 30 to 240nmol per kg body weight. For a 75kg subject, such doses correspond to the following doses: 375nmol to 75 μmol, for example 750nmol to 56.25 μmol, for example 1.5 to 37.5 μmol, in particular 2.25 to 18 μmol.
In an alternative embodiment, a PYY analog of the invention may be administered to a subject at 0.5 to 135 picomoles (pmol) per kilogram body weight, for example 5 to 100 picomoles (pmol) per kilogram body weight, for example 10 to 90 picomoles (pmol) per kilogram body weight, for example about 72pmol per kilogram body weight. In a specific non-limiting example, the PYY analogs of the invention are administered in a dosage of about 1nmol or more, 2nmol or more, or 5nmol or more. In this example, the PYY analogue of the invention is typically dosed at a dose of no more than 100nmols, e.g. 90nmols or less, 80nmols or less, 70nmols or less, 60nmols or less, 50nmols or less, 40nmols or less, 30nmols or less, 20nmols or less, 10 nmols. For example, a dosage range may include any combination of any of the lower specified dosages and any of the upper specified dosages. Thus, examples of non-limiting dosage ranges for the compounds of the invention are in the range of 1 to 100nmols, 2 to 90mols, 5 to 80 nmols.
In a specific non-limiting example, about 1 to about 50nmol, e.g., about 2 to about 20nmol, e.g., about 10nmol of a PYY analog of the invention is administered by subcutaneous injection. The exact dosage will be readily determined by those skilled in the art based on the potency of the particular PYY analog employed, the route of delivery of the PYY analog, and the age, weight, sex, and physiological condition of the subject.
Suitable doses of the PYY analogues of the invention also comprise doses which result in a reduced calorie intake, a reduced food intake or a reduced appetite or an increased energy expenditure equivalent to a reduced calorie intake, a reduced food intake or a reduced appetite or an increased energy expenditure caused by normal postprandial levels of PYY. Examples of doses include, but are not limited to, doses that produce a demonstrated effect when the serum level of PYY is from about 40pM to about 60pM, or from about 40pM to about 45pM, or about 43 pM.
The dosages discussed above may be administered, for example, once a day, twice a day, three times a day, or four times a day. Alternatively, the dose may be administered once every 2, 3 or 4 days. In slow release formulations containing zinc, administration of the dose once every 3,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days is possible. According to certain embodiments, the dose may be administered once shortly before each meal.
Specific sequences of the invention
According to certain embodiments of the invention, the PYY analogue has an amino acid sequence given in one of the specific sequences shown in figure 1.
The invention is illustrated by the following non-limiting examples.
Examples of the invention
The material and the method are as follows:
peptide synthesis
The peptides were prepared by standard automated fluorenylmethyloxycarbonyl (Fmoc) Solid Phase Peptide Synthesis (SPPS) method. On tricyclic amide linking resinsAnd (4) peptide synthesis. The amino acids were attached using the Fmoc strategy. Each amino acid is added sequentially from the C-terminus to the N-terminus. Peptide coupling was mediated by the reagent TBTU. Cleavage of the peptide from the resin was achieved with trifluoroacetic acid in the presence of a scavenger. Obtaining native PYY 3-36NH as previously described2(WO 03/026591); de novo synthesis using tricyclic amide resins and Fmoc chemistry is also possible.
Peptides were purified by reverse phase HPLC. Full quality control was performed on all purified peptides and peptide purity was shown to be greater than 95% by HPLC in two buffer systems. Amino acid analysis after acid hydrolysis confirmed the amino acid composition. MALDI-MS shows the expected molecular ions.
Binding study
Isolation of overexpressed human Y2 receptor (NPYR200000, Missouri S) by osmotic lysis and differential centrifugation&T cDNA resource center)) as described in Morgan et al (journal of Neuroendocrinol 1996.8283-290). Receptor binding assays were performed as described by Druce et al (2009 Endocrinology 150(4)712-22), except that the buffer used was 0.02M HEPES ph7.4, 5mM CaCl2、1mM MgCl21% bovine serum albumin, 0.1mM statin A, 0.2mM PMSF, 10. mu.M phosphorylmethadone, as radiolabel125I-PYY1-36And in addition to the human Y2 receptor used.
In vitro receptor potency studies
Will be provided with
Pro-Ile-His-Pro-His-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-His-Tyr-Tyr-Ala-Ala-Leu-Arg-His-Tyr-Leu-Asn-His-Val-Thr-Arg-Gln-Arg-Tyr-NH2(SEQ ID NO:44)。
Animal(s) production
Male Wistar rats (Charles river Ltd, Margate, UK) were used for animal experiments
Rat feeding study
Rats were housed individually in IVC cages. Animals were randomized into treatment groups, with stratification by body weight. All peptide solutions were freshly prepared immediately prior to administration. The vehicle used for all studies was 5% v/v water and 95% NaCl (0.9% w/v). The peptide and vehicle were administered by subcutaneous injection at a dose of 25 or 50nM/Kg body weight. Animals were injected every 24 hours for 6 days and the study was continued for an additional 24 hours, for a total of 7 days from the first injection to the end of the study. During the study period, the animals had free access to food and water. Animals were weighed at the end of the study and total weight loss (if any) was recorded.
Results
Fig. 1a discloses a number of specific sequences encompassed by the scope of the present invention in all its aspects. Each of these sequences is a specific embodiment of the present invention. The numbers assigned to each sequence correspond to the "Y" numbers mentioned elsewhere herein (i.e., the rows beginning with "1419" are understood to disclose a sequence of analog numbers "Y1419"). Figure 1a also discloses in the first row for reference the naturally occurring human PYY sequence.
The following table provides cells at hY2 CHO-K1Receptor potency data for the exemplary peptide of figure 1 at overexpressed human neuropeptide Y receptor Y2. Ten thousand cells per well (96-well plate) were resuspended in medium containing 0.2mM IBMX and 0.02mM forskolin (stimulating cAMP production) and at a range of concentrationsPeptides were tested in-house for 30 minutes. The reaction was stopped by lysis of the cells and after 60 minutes cAMP was quantified using the Cisbio cAMP dynamic 2 kit. The values provided are exemplary compounds EC50And PYY2-36EC50Ratio of (e.g. for "to PYY)2-36Average ratio of ± SEM column, value 0.5 would indicate that the concentration of exemplary compound required to inhibit 50% of maximal cAMP release is the desired PYY2-36And a value of 2 would indicate that the concentration of the exemplary compound required to inhibit 50% of maximum cAMP release is
Peptides
And PYY2-36Average ratio of + -SEM
Y242
10.0±0.69
Y1276
0.6±0.06
Y1319
0.6±0.09
Y1371
0.9±0.06
Y1372
1.1±0.16
Y1377
1.0±0.07
Y1379
0.9±0.09
Y1419
0.8±0.16
Y1421
0.9±0.09
Y1431
0.9±0.07
Y1447
1.4±0.31
Y1448
1.7±0.46
Y1450
1.1±0.20
Y1477
0.9±0.06
Y1489
1.0±0.06
Y1490
0.9±0.28
Y1518
1.7±0.13
Y1528
0.5±0.08
Y1553
0.8±0.25
Y1558
0.6±0.05
Figure 2 shows the different abilities of compound Y242 and compound Y1419 to inhibit the production of forskolin. It can be seen that 1419 is a more potent inhibitor than the previous generation of preferred compounds.
Figures 3 and 4 show the results of a rat feeding experiment comparing the activity of Y242 (figure 3) and Y1419 (figure 4) in overnight fasted male wistar rats that have received a single subcutaneous injection of control saline or peptide in saline. Food intake and body weight were monitored over the next 6 days and values shown were mean +/-SEM. It can be seen that although comparative compound Y242 produced the desired reduction in food intake and weight loss in the first 48 hours, this reduction did not last for a longer period of time (indicating that the comparative compound, although effective, required re-administration after one or two days). Compound Y1419 performed much better and a single initial dose was still viable 160 hours after administration, indicating that compound is promising for use as a weekly or less frequently administered drug.
Figure 5 shows the results of a feeding experiment in which fasted (24 hours) male wista rats (charles river) received a single subcutaneous injection of saline or an exemplary peptide of the invention (100 nmol/kg). Total food intake from administration to 48 hours after administration and body weight change from before fasting to 48 hours after administration were measured.
Figure 6 shows the ability of various indicated compounds of the invention to inhibit the production of forskolin. The data for PYY2-36 is also included as a comparison.