# Patent

## Title
Treatment of diseases

## Abstract

The invention provides (i) a method of treating metabolic syndrome in an animal, (ii) a method of suppressing the appetite of an animal, (iii) a method of treating obesity in an animal, (iv) a method of reducing the weight of an animal in need thereof, (v) a method of reducing a blood lipid level in an animal in need thereof, (vi) a method of treating non-alcoholic steatohepatitis in an animal, and (vii) a method of inhibiting adipogenesis. The methods comprise administering an effective amount of an active ingredient, wherein the active ingredient comprises a diketopiperazine, a prodrug of a diketopiperazine or a pharmaceutically-acceptable salt of either of them to the animal. The invention also provides a kit comprising a container holding a diketopiperazine, a prodrug of a diketopiperazine or a pharmaceutically-acceptable salt of either of them; and instructions for administration. The diketopiperazines have the formula given in the application.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a divisional application of U.S. patent application Ser. No. 13/227,109, filed Sep. 7, 2011, which claims the benefit of U.S. Provisional Patent Application Ser. No. 61/512,151, filed Jul. 27, 2011, and U.S. Provisional Patent Application Ser. No. 61/380,426, filed Sep. 7, 2010, the complete disclosures of which are incorporated herein by reference.


FIELD OF THE INVENTION
The invention relates to a treatment for metabolic syndrome comprising administering to an animal in need thereof an effective amount of a diketopiperazine of formula I set forth below, a prodrug of a diketopiperazine of formula I or a pharmaceutically-acceptable salt of either of them. The invention further relates to a method of suppressing the appetite of an animal, a method of treating obesity in an animal, a method of reducing the weight of an animal in need thereof, a method of reducing a blood lipid level in an animal in need thereof, a method of treating non-alcoholic steatohepatitis in an animal, and a method of inhibiting adipogenesis in an animal. These methods also comprise administering an effective amount of a diketopiperazine of formula I, a prodrug of a diketopiperazine of formula I or a pharmaceutically-acceptable salt of either of them to the animal. The invention also relates to a kit comprising a container holding a diketopiperazine of formula I, a prodrug of a diketopiperazine of formula I or a pharmaceutically-acceptable salt of either of them; and instructions for administration of the diketopiperazine, the prodrug or the pharmaceutically-acceptable salt according to a method of the invention.
BACKGROUND
Metabolic syndrome is a complex of risk factors for cardiovascular disease and type 2 diabetes. The most widely recognized of the risk factors are atherogenic dyslipidemia, elevated blood pressure and elevated plasma glucose. In addition, patients with these characteristics commonly manifest a prothrombotic state and a proinflammatory state. Atherogenic dyslipidemia consists of an aggregation of lipoprotein abnormalities that includes elevated serum triglyceride and apolipoprotein B, increased small low-density lipoprotein particles and a reduced level of high-density lipoprotein cholesterol. Most patients with metabolic syndrome also have abdominal (central) obesity and insulin resistance. For general background on metabolic syndrome factors and diagnosis, see Alberti et al., Circulation, 120:1640-1645 (2009).
Metabolic syndrome is common and has a rising prevalence worldwide, which relates largely to increasing obesity and sedentary lifestyles. Patients with the metabolic syndrome are at twice the risk of developing cardiovascular disease over the next five to ten years as individuals without the syndrome. The risk over a lifetime undoubtedly is even higher. Furthermore, the metabolic syndrome confers a five-fold increase in risk for type 2 diabetes mellitus. Metabolic syndrome is now both a public health and a clinical problem, and there is clearly a need for new and effective treatments for metabolic syndrome.
SUMMARY OF THE INVENTION
The invention provides such a treatment. In particular, the invention provides a method of treating metabolic syndrome in an animal. The method comprises administering to the animal an effective amount of an active ingredient, wherein the active ingredient comprises a diketopiperazine of formula I given below, a prodrug of a diketopiperazine of formula I or a pharmaceutically-acceptable salt of either of them.
The invention also provides a method of suppressing the appetite of an animal, a method of treating obesity in an animal, a method of reducing the weight of an animal in need thereof, a method of reducing a blood lipid level in an animal in need thereof, a method of treating non-alcoholic steatohepatitis in an animal, and a method of inhibiting adipogenesis in an animal. These methods also comprise administering an effective amount of an active ingredient, wherein the active ingredient comprises a diketopiperazine of formula I, a prodrug of a diketopiperazine of formula I or a pharmaceutically-acceptable salt of either of them to the animal.
The invention further provides a kit comprising a container holding a diketopiperazine of formula I, a prodrug of a diketopiperazine of formula I or a pharmaceutically-acceptable salt of either of them; and instructions for administration of the diketopiperazine, the prodrug or the pharmaceutically-acceptable salt according to a method of the invention.
The diketopiperazines useful in the invention have the following formula I:




wherein:

R1 and R2, which may be the same or different, each is:

    
    
        (a) a side chain of an amino acid, wherein the amino acid is glycine, alanine, valine, norvaline, α-aminoisobutyric acid, 2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine, isoleucine, norleucine, serine, homoserine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, homoarginine, citrulline, phenylalanine, p-aminophenylalanine, tyrosine, tryptophan, thyroxine, cysteine, homocysteine, methionine, penicillamine or ornithine; or
        (b) R1 is —CH2—CH2—CH2— or —CH2—CH(OH)—CH2— and together with the adjacent ring nitrogen forms proline or hydroxyproline and/or R2 is —CH2—CH2—CH2— or —CH2—CH(OH)—CH2— and together with the adjacent ring nitrogen forms proline or hydroxyproline; or
        (b) a derivative of a side chain of an amino acid, wherein the amino acid is one of those recited in (a), and the derivatized side chain has:
        
            (i) an —NH2 group replaced by an —NHR3 or —N(R3)2 group, wherein each R3 may independently be a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl;
            (ii) an —OH group replaced by an —O—PO3H2 or —OR3 group, wherein each R3 may independently be a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl;
            (iii) a —COOH group replaced by a —COOR3 group, wherein each R3 may independently be a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl;
            (iv) a —COOH group replaced by a —CON(R4)2 group, wherein each R4 may independently be H or a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl;
            (v) an —SH group replaced by —S—S—CH2—CH(NH2)—COOH or —S—S—CH2—CH2—CH(NH2)—COOH;
            (vi) a —CH2— group replaced by a —CH(NH2)— or a —CH(OH)— group;
            (vii) a —CH3 group replaced by a —CH2—NH2 or a —CH2—OH group; and/or
            (viii) an H which is attached to a carbon atom replaced by a halogen;
            provided, however, that when R1 is the side chain of histidine or a derivative of the side chain of histidine, then R2 cannot be proline or hydroxyproline, and that when R2 is the side chain of histidine or a derivative of the side chain of histidine, then R1 cannot be proline or hydroxyproline.
        
        
    
    





BRIEF DESCRIPTION OF THE DRAWING
FIG. 1: Effect of DA-DKP on adipogenesis. Statistical significance versus untreated controls is indicated by an asterisk (*).



DETAILED DESCRIPTION OF THE PRESENTLY-PREFERRED EMBODIMENTS OF THE INVENTION
The invention provides (i) a method of treating metabolic syndrome in an animal, (ii) a method of suppressing the appetite of an animal, (iii) a method of treating obesity in an animal, (iv) a method of reducing the weight of an animal in need thereof, such as an obese or overweight animal, (v) a method of reducing a blood lipid level in an animal in need thereof, and (vi) a method of treating non-alcoholic steatohepatitis in an animal.
The methods of the invention comprise administering an effective amount of an active ingredient, wherein the active ingredient comprises a diketopiperazine, a prodrug of a diketopiperazine or a pharmaceutically-acceptable salt of either of them to the animal, wherein the diketopiperazine has the following formula I:




wherein:

R1 and R2, which may be the same or different, each is:

    
    
        (a) a side chain of an amino acid, wherein the amino acid is glycine, alanine, valine, norvaline, α-aminoisobutyric acid, 2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine, isoleucine, norleucine, serine, homoserine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, homoarginine, citrulline, phenylalanine, p-aminophenylalanine, tyrosine, tryptophan, thyroxine, cysteine, homocysteine, methionine, penicillamine or ornithine; or
        (b) R1 is —CH2—CH2—CH2— or —CH2—CH(OH)—CH2— and together with the adjacent ring nitrogen forms proline or hydroxyproline and/or R2 is —CH2—CH2—CH2— or —CH2—CH(OH)—CH2— and together with the adjacent ring nitrogen forms proline or hydroxyproline; or
        (b) a derivative of a side chain of an amino acid, wherein the amino acid is one of those recited in (a), and the derivatized side chain has:
        
            (i) an —NH2 group replaced by an —NHR3 or —N(R3)2 group, wherein each R3 may independently be a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl;
            (ii) an —OH group replaced by an —O—PO3H2 or —OR3 group, wherein each R3 may independently be a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl;
            (iii) a —COOH group replaced by a —COOR3 group, wherein each R3 may independently be a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl;
            (iv) a —COOH group replaced by a —CON(R4)2 group, wherein each R4 may independently be H or a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl;
            (v) an —SH group replaced by —S—S—CH2—CH(NH2)—COOH or —S—S—CH2—CH2—CH(NH2)—COOH;
            (vi) a —CH2— group replaced by a —CH(NH2)— or a —CH(OH)— group;
            (vii) a —CH3 group replaced by a —CH2—NH2 or a —CH2—OH group; and/or
            (viii) an H which is attached to a carbon atom replaced by a halogen;
            provided, however, that when R1 is the side chain of histidine or a derivative of the side chain of histidine, then R2 cannot be proline or hydroxyproline, and that when R2 is the side chain of histidine or a derivative of the side chain of histidine, then R1 cannot be proline or hydroxyproline.
        
        
    
    


By “replaced” is meant that, with reference to the formula of an amino acid side chain, the specified group is replaced by the other specified group. For instance, the formula of the isoleucine side chain is —CH(CH3)—CH2—CH3. If the terminal —CH3 group is replaced with a —CH2—OH group, then the formula of the resulting derivatized isoleucine side chain would be —CH(CH3)—CH2—CH2—OH. As another example, the formula of the alanine side chain is —CH3. If one of the hydrogen atoms is replaced by a chlorine atom, then the resulting derivatized alanine side chain would be —CH2—Cl. Note that the side chain of glycine is —H and, if this H is replaced by a chlorine (or other halogen) atom, the resulting side chain will —Cl, with the chlorine atom attached to the ring carbon (e.g., R1=—Cl)
By “side chain” of an amino acid is meant that portion of the amino acid attached to the common




backbone of all of the amino acids listed above. For instance, the side chain of glycine is —H, the side chain of alanine is —CH3, and the side chain of serine is —CH2OH.

By “hydrophobic” is meant a side chain or side chain derivative that is uncharged at physiological pH and is repelled by an aqueous solution.
By “neutral” is meant a side chain or side chain derivative that is uncharged at physiological pH.
By “basic” is meant a side chain or side chain derivative that is positively charged at physiological pH.
By “acidic” is meant a side chain or side chain derivative that is negatively charged at physiological pH.
By “alkyl” is meant a saturated straight-chain or branched hydrocarbon containing 1-10 carbon atoms, preferably 1-6, carbon atoms. “Lower alkyl” means a saturated straight-chain or branched hydrocarbon containing 1-6 carbon atoms.
By “cycloalkyl” is meant a saturated cyclic hydrocarbon containing at least one ring, each ring containing at least three carbon atoms. Preferably, the cycloalkyl contains one ring of 4-8 carbon atoms.
By “heterocycloalkyl” is meant a cycloalkyl having one or more of the ring carbon atoms of at least one of the rings replaced by an O, S and/or N.
By “aryl” is meant an aromatic group having at least one aromatic ring (e.g., phenyl).
By “alkylaryl” is meant a lower alkyl having an H replaced by an aryl (e.g., —CH2—C6H5 or —CH3CH(C6H5)CH3).
By “arylalkyl” is meant an aryl having an H replaced by a lower alkyl (e.g., —C6H4—CH3).
By “heteroaryl” is meant an aryl having one or more of the ring carbon atoms of at least one of the rings replaced by an O, S and/or N.
By “substituted” is meant that the moiety is substituted with one or more substituents selected from the following group: —OH, NH2, —SH, —COOH and/or a halogen atom.
By “halogen” is meant chlorine, fluorine, bromine or iodine. Preferred is chlorine or bromine.
Most preferred for use in the invention are diketopiperazines of formula I wherein R1, R2 or both is the side chain of aspartic acid or glutamic acid or a derivative of such a side chain wherein the —COOH group is replaced by a —COOR3 group or a —CON(R4)2 group, wherein R3 and R4 are defined above. Of this group of compounds, preferred are diketopiperazines comprising the side chains of aspartic acid and alanine (Asp-Ala DKP or DA-DKP), the side chains of glutamic acid and alanine (Glu-Ala DKP or EA-DKP), the side chains of tyrosine and aspartic acid (Tyr-Asp DKP or YD-DKP), the side chains of tyrosine and glutamic acid (Tyr-Glu DKP or YE-DKP) and derivatives of the aspartic acid or glutamic acid side chains of these four diketopiperazines wherein the —COOH group is replaced by a —COOR3 group or a —CON(R4)2 group, wherein R3 and R4 are defined above. Most preferred is DA-DKP.
Also preferred are diketopiperazines wherein R1, R2 or both are hydrophobic side chains or hydrophobic side chain derivatives. By “hydrophobic side chain derivative” is meant that the derivatized side chain is hydrophobic. In particular, preferred are diketopiperzines wherein R1 and/or R2, which may be the same or different, each is the side chain of glycine, alanine, valine, norvaline, α-aminobutyric acid, leucine, isoleucine, norleucine, methionine, phenylalanine, tryptophan or tyrosine. Of this group, preferred are diketopiperzines wherein R1 and/or R2, which may be the same or different, each is the side chain of glycine, alanine, valine, norvaline, α-aminobutyric acid, leucine, isoleucine, norleucine, methionine or tyrosine, more preferably alanine, valine, norvaline, α-aminobutyric acid, leucine, isoleucine or norleucine. Generally, proline is not preferred, although it is hydrophobic.
Additional preferred diketopiperazines are those wherein R1, R2 or both side chains are neutral side chains or neutral side chain derivatives. By “neutral side chain derivative” is meant that the derivatized side chain is neutral. In particular, preferred are diketopiperzines wherein R1 and/or R2, which may be the same or different, each is the side chain of asparagine, glutamine, serine, homoserine, threonine, tyrosine, cysteine or methionine. Of this group, preferred are diketopiperzines wherein R1 and/or R2, which may be the same or different, each is the side chain of asparagine, glutamine, serine or threonine.
Also preferred are diketopiperazines wherein R1, R2 or both are basic side chains or basic side chain derivatives. By “basic side chain derivative” is meant that the derivatized side chain is basic. In particular, preferred are diketopiperzines wherein R1 and/or R2, which may be the same or different, each is the side chain of citrulline, 2,4-diaminobutryic acid, 2,3-diaminobutyric acid, lysine, hydroxylysine, arginine, homoarginine, citrulline, p-aminophenylalanine, or ornithine. Of this group, preferred are diketopiperzines wherein R1 and/or R2, which may be the same or different, each is the side chain of citrulline, 2,4-diaminobutryic acid, 2,3-diaminobutyric acid, lysine, arginine, homoarginine or p-aminophenylalanine. Generally, histidine is not preferred, although it is basic.
Further preferred diketopiperazines are those wherein R1, R2 or both is the side chain of methionine, the side chain of arginine or a derivative of these side chains. Most preferred of this group is a diketopiperazine wherein R1 is the side chain of methionine and R2 is the side chain of arginine (Met-Arg DKP or MR-DKP).
Highly preferred for use herein is DA-DKP. Patients with metabolic syndrome can be treated by administering an effective amount of a DA-DKP, a prodrug of a DA-DKP or a pharmaceutically-acceptable salt of either of them. DA-DKP has multiple activities that should make it a particularly effective treatment for metabolic syndrome.
First, DA-DKP has been found to suppress the appetite. DA-DKP has also been found to inhibit the differentiation of preadipocytes into adipocytes. Accordingly, it is expected to be beneficial in the treatment of metabolic syndrome by contributing to weight loss and the treatment of obesity. It can also be used as a treatment for obesity and for weight loss in general, not just the obesity and excess weight associated with metabolic syndrome.
In addition, DA-DKP has also been found to reduce levels of lipids (cholesterol, triglycerides, low-density lipoprotein and high-density lipoprotein) in blood. For this reason, it is expected to have beneficial effects on the dyslipidemia which is one of the risk factors of metabolic syndrome.
Another pathological condition associated with the metabolic syndrome is the development of non-alcoholic steatohepatitis (NASH) in obese patients. NASH is characterized by an accumulation of lipid molecules in the liver accompanied by severe inflammation, fibrosis, and insulin/leptin resistance. In view of the foregoing activities of DA-DKP, it is expected to be beneficial in the treatment of NASH, whether associated with metabolic syndrome or independent of metabolic syndrome.
Further, DA-DKP has beneficial effects on the vascular endothelial barrier, including the ability to inhibit vascular hyperpermeability. Accordingly, it is expected to be an effective treatment for those exhibiting early signs of, or with a predisposition to, type 2 diabetes, such as those diagnosed with metabolic syndrome, to reduce, delay or even potentially prevent the vascular complications of diabetes. See Applicant's co-pending provisional application No. 61/380,404, filed Sep. 7, 2010.
Finally, DA-DKP is known to inhibit the aggregation of platelets, to be anti-inflammatory and to inhibit the activation of T-cells. See U.S. Pat. Nos. 6,555,543 and 7,732,403. Accordingly DA-DKP is expected to be useful in combating the prothrombotic and proinflammatory states observed in metabolic syndrome.
Methods of making diketopiperazines are well known in the art, and these methods may be employed to synthesize the diketopiperazines of the invention. See, e.g., U.S. Pat. Nos. 4,694,081, 5,817,751, 5,990,112, 5,932,579 and 6,555,543, US Patent Application Publication Number 2004/0024180, PCT applications WO 96/00391 and WO 97/48685, and Smith et al., Bioorg. Med. Chem. Letters, 8, 2369-2374 (1998), the complete disclosures of which are incorporated herein by reference.
For instance, diketopiperazines can be prepared by first synthesizing dipeptides. The dipeptides can be synthesized by methods well known in the art using L-amino acids, D-amino acids or a combination of D- and L-amino acids. Preferred are solid-phase peptide synthetic methods. Of course, dipeptides are also available commercially from numerous sources, including Sigma-Aldrich, St. Louis, Mo. (primarily custom synthesis), Phoenix Pharmaceuticals, Inc., Belmont, Calif. (custom synthesis), Fisher Scientific (custom synthesis) and Advanced ChemTech, Louisville, Ky.
Once the dipeptide is synthesized or purchased, it is cyclized to form a diketopiperazine. This can be accomplished by a variety of techniques.
For example, U.S. Patent Application Publication Number 2004/0024180 describes a method of cyclizing dipeptides. Briefly, the dipeptide is heated in an organic solvent while removing water by distillation. Preferably, the organic solvent is a low-boiling azeotrope with water, such as acetonitrile, allyl alcohol, benzene, benzyl alcohol, n-butanol, 2-butanol, t-butanol, acetic acid butylester, carbon tetrachloride, chlorobenzene chloroform, cyclohexane, 1,2-dichlorethane, diethylacetal, dimethylacetal, acetic acid ethylester, heptane, methylisobutylketone, 3-pentanol, toluene and xylene. The temperature depends on the reaction speed at which the cyclization takes place and on the type of azeotroping agent used. The reaction is preferably carried out at 50-200° C., more preferably 80-150° C. The pH range in which cyclization takes place can be easily determine by the person skilled in the art. It will advantageously be 2-9, preferably 3-7.
When one or both of the amino acids of the dipeptide has, or is derivatized to have, a carboxyl group on its side chain (e.g., aspartic acid or glutamic acid), the dipeptide is preferably cyclized as described in U.S. Pat. No. 6,555,543. Briefly, the dipeptide, with the side-chain carboxyl still protected, is heated under neutral conditions. Typically, the dipeptide will be heated at from about 80° C. to about 180° C., preferably at about 120° C. The solvent will be a neutral solvent. For instance, the solvent may comprise an alcohol (such as butanol, methanol, ethanol, and higher alcohols, but not phenol) and an azeotropic co-solvent (such as toluene, benzene, or xylene). Preferably, the alcohol is butan-2-ol, and the azeotropic co-solvent is toluene. The heating is continued until the reaction is complete, and such times can be determined empirically. Typically, the dipeptide will be cyclized by refluxing it for about 8-24 hours, preferably about 18 hours. Finally, the protecting group is removed from the diketopiperazine. In doing so, the use of strong acids (mineral acids, such as sulfuric or hydrochloric acids), strong bases (alkaline bases, such as potassium hydroxide or sodium hydroxide), and strong reducing agents (e.g., lithium aluminum hydride) should be avoided, in order to maintain the chirality of the final compound.
Dipeptides made on solid phase resins can be cyclized and released from the resin in one step. See, e.g., U.S. Pat. No. 5,817,751. For instance, the resin having an N-alkylated dipeptide attached is suspended in toluene or toluene/ethanol in the presence of acetic acid (e.g., 1%) or triethylamine (e.g., 4%). Typically, basic cyclization conditions are preferred for their faster cyclization times.
To prepare diketopiperazines wherein the amino acid side chains are derivatized, amino acid derivatives can be used in the synthesis of the dipeptides, the dipeptides can be derivatized and/or the diketopiperazines can be derivatized, as is known in the art. See, e.g., those references cited above.
Other methods of cyclizing dipeptides and of making diketopiperazines are known in the art and can be used in the preparation of diketopiperazines useful in the practice of the invention. See, e.g., those references listed above. In addition, many diketopiperazines suitable for use in the present invention can be made from proteins and peptides as described in U.S. Pat. No. 7,732,403, the complete disclosure of which is incorporated herein by reference. Further, diketopiperazines for use in the practice of the invention can be obtained commercially from, e.g., Syngene, India and Hemmo Pharmaceuticals Pvt. Ltd, India (both custom synthesis).
The diketopiperazines of the invention include all possible stereoisomers than can be obtained by varying the configuration of the individual chiral centers, axes or surfaces. In other words, the diketopiperazines include all possible diastereomers, as well as all optical isomers (enantiomers).
“Prodrug” means any compound which releases an active parent drug (a diketopiperazine in this case) in vivo when such prodrug is administered to an animal. Prodrugs of diketopiperazines include derivatives that may be cleaved in vivo to generate the diketopiperazine. Examples of diketopiperazine prodrugs include esters.
The physiologically-acceptable salts of the diketopiperazines and prodrugs of the invention may also be used in the practice of the invention. Physiologically-acceptable salts include conventional non-toxic salts, such as salts derived from inorganic acids (such as hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, and the like), organic acids (such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, glutamic, aspartic, benzoic, salicylic, oxalic, ascorbic acid, and the like) or bases (such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation or organic cations derived from N,N-dibenzylethylenediamine, D-glucosamine, or ethylenediamine). The salts are prepared in a conventional manner, e.g., by neutralizing the free base form of the compound with an acid.
As noted above, a diketopiperazine of formula I, a prodrug of a diketopiperazine of formula I or a pharmaceutically-acceptable salt of either one of them can be used in the methods of the invention. To do so, the diketopiperazine, prodrug or pharmaceutically-acceptable salt is administered to an animal in need of treatment. Preferably, the animal is a mammal, such as a rabbit, goat, dog, cat, horse or human. Most preferably, the animal is a human.
A diketopiperazine of formula I, a prodrug of a diketopiperazine of formula I or a pharmaceutically-acceptable salt of either one of them is used in the present invention as an active ingredient. “Active ingredient” is used herein to mean a compound having therapeutic, pharmaceutical or pharmacological activity, and particularly the therapeutic, pharmaceutical or pharmacological activities described herein. The diketopiperazine, prodrug or salt of either one of them is not used in the present invention as a carrier or as part of a carrier system of a pharmaceutical composition as described in, e.g., U.S. Pat. Nos. 5,976,569, 6,099,856, 7,276,534 and PCT applications WO 96/10396, WO 2006/023943, WO 2007/098500, WO 2007/121411 and WO 2010/102148.
Effective dosage forms, modes of administration and dosage amounts for the compounds of the invention (i.e., a diketopiperazine of formula I, a prodrug of a diketopiperazine of formula I or a pharmaceutically-acceptable salt of either one of them) may be determined empirically using the guidance provided herein. It is understood by those skilled in the art that the dosage amount will vary with the particular disease or condition to be treated, the severity of the disease or condition, the route(s) of administration, the duration of the treatment, the identity of any other drugs being administered to the animal, the age, size and species of the animal, and like factors known in the medical and veterinary arts. In general, a suitable daily dose of a compound of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. However, the daily dosage will be determined by an attending physician or veterinarian within the scope of sound medical judgment. If desired, the effective daily dose may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day. Administration of the compound should be continued until an acceptable response is achieved.
In particular, an effective dosage amount of a diketopiperazine of formula I will be from 10 ng/kg/day to 225 mg/kg/day, preferably from 500 ng/kg/day to 150 mg/kg/day, most preferably from 1 mg/kg/day to 30 mg/kg/day. When given orally to an adult human, the dose will preferably be from about 1 mg/day to about 10 g/day, more preferably the dose will be from about 60 mg/day to about 6 g/day, most preferably the dose will be from about 100 mg/day to about 1200 mg/day, preferably given in several doses.
The compounds of the present invention (i.e., a diketopiperazine of formula I, prodrugs thereof and pharmaceutically-acceptable salts of either of them) may be administered to an animal patient for therapy by any suitable route of administration, including orally, nasally, parenterally (e.g., intravenously, intraperitoneally, subcutaneously or intramuscularly), transdermally, intraocularly and topically (including buccally and sublingually). Generally preferred is oral administration for any disease or condition treatable according to the invention.
While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). The pharmaceutical compositions of the invention comprise a compound or compounds of the invention as an active ingredient in admixture with one or more pharmaceutically-acceptable carriers and, optionally, with one or more other compounds, drugs or other materials. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the animal. Pharmaceutically-acceptable carriers are well known in the art. Regardless of the route of administration selected, the compounds of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington's Pharmaceutical Sciences. 
Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules or as a solution or a suspension in an aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil liquid emulsions, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), and the like, each containing a predetermined amount of a compound or compounds of the present invention as an active ingredient. A compound or compounds of the present invention may also be administered as bolus, electuary or paste.
In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient (i.e., a diketopiperazine of formula I, a prodrug of a diketopiperazine of formula I, a pharmaceutically-acceptable salt of either one of them, or combinations of the foregoing) is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monosterate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form.
Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions, in addition to the active ingredient, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
The invention also provides pharmaceutical products suitable for treatment of the eye. Such pharmaceutical products include pharmaceutical compositions, devices and implants (which may be compositions or devices).
Pharmaceutical formulations (compositions) for intraocular injection of a compound or compounds of the invention into the eyeball include solutions, emulsions, suspensions, particles, capsules, microspheres, liposomes, matrices, etc. See, e.g., U.S. Pat. No. 6,060,463, U.S. Patent Application Publication No. 2005/0101582, and PCT application WO 2004/043480, the complete disclosures of which are incorporated herein by reference. For instance, a pharmaceutical formulation for intraocular injection may comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, suspensions or emulsions, which may contain antioxidants, buffers, suspending agents, thickening agents or viscosity-enhancing agents (such as a hyaluronic acid polymer). Examples of suitable aqueous and nonaqueous carriers include water, saline (preferably 0.9%), dextrose in water (preferably 5%), buffers, dimethylsulfoxide, alcohols and polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like). These compositions may also contain adjuvants such as wetting agents and emulsifying agents and dispersing agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as polymers and gelatin. Injectable depot forms can be made by incorporating the drug into microcapsules or microspheres made of biodegradable polymers such as polylactide-polyglycolide. Examples of other biodegradable polymers include poly(orthoesters), poly(glycolic) acid, poly(lactic) acid, polycaprolactone and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes (composed of the usual ingredients, such as dipalmitoyl phosphatidylcholine) or microemulsions which are compatible with eye tissue. Depending on the ratio of drug to polymer or lipid, the nature of the particular polymer or lipid components, the type of liposome employed, and whether the microcapsules or microspheres are coated or uncoated, the rate of drug release from microcapsules, microspheres and liposomes can be controlled.
The compounds of the invention can also be administered surgically as an ocular implant. For instance, a reservoir container having a diffusible wall of polyvinyl alcohol or polyvinyl acetate and containing a compound or compounds of the invention can be implanted in or on the sclera. As another example, a compound or compounds of the invention can be incorporated into a polymeric matrix made of a polymer, such as polycaprolactone, poly(glycolic) acid, poly(lactic) acid, poly(anhydride), or a lipid, such as sebacic acid, and may be implanted on the sclera or in the eye. This is usually accomplished with the animal receiving a topical or local anesthetic and using a small incision made behind the cornea. The matrix is then inserted through the incision and sutured to the sclera.
The compounds of the invention can also be administered topically to the eye, and a preferred embodiment of the invention is a topical pharmaceutical composition suitable for application to the eye. Topical pharmaceutical compositions suitable for application to the eye include solutions, suspensions, dispersions, drops, gels, hydrogels and ointments. See, e.g., U.S. Pat. No. 5,407,926 and PCT applications WO 2004/058289, WO 01/30337 and WO 01/68053, the complete disclosures of all of which are incorporated herein by reference.
Topical formulations suitable for application to the eye comprise one or more compounds of the invention in an aqueous or nonaqueous base. The topical formulations can also include absorption enhancers, permeation enhancers, thickening agents, viscosity enhancers, agents for adjusting and/or maintaining the pH, agents to adjust the osmotic pressure, preservatives, surfactants, buffers, salts (preferably sodium chloride), suspending agents, dispersing agents, solubilizing agents, stabilizers and/or tonicity agents. Topical formulations suitable for application to the eye will preferably comprise an absorption or permeation enhancer to promote absorption or permeation of the compound or compounds of the invention into the eye and/or a thickening agent or viscosity enhancer that is capable of increasing the residence time of a compound or compounds of the invention in the eye. See PCT applications WO 2004/058289, WO 01/30337 and WO 01/68053. Exemplary absorption/permeation enhancers include methysulfonylmethane, alone or in combination with dimethylsulfoxide, carboxylic acids and surfactants. Exemplary thickening agents and viscosity enhancers include dextrans, polyethylene glycols, polyvinylpyrrolidone, polysaccharide gels, Gelrite®, cellulosic polymers (such as hydroxypropyl methylcellulose), carboxyl-containing polymers (such as polymers or copolymers of acrylic acid), polyvinyl alcohol and hyaluronic acid or a salt thereof.
Liquid dosage forms (e.g., solutions, suspensions, dispersions and drops) suitable for treatment of the eye can be prepared, for example, by dissolving, dispersing, suspending, etc. a compound or compounds of the invention in a vehicle, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like, to form a solution, dispersion or suspension. If desired, the pharmaceutical formulation may also contain minor amounts of non-toxic auxillary substances, such as wetting or emulsifying agents, pH buffering agents and the like, for example sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.
Aqueous solutions and suspensions suitable for treatment of the eye can include, in addition to a compound or compounds of the invention, preservatives, surfactants, buffers, salts (preferably sodium chloride), tonicity agents and water. If suspensions are used, the particle sizes should be less than 10 μm to minimize eye irritation. If solutions or suspensions are used, the amount delivered to the eye should not exceed 50 μl to avoid excessive spillage from the eye.
Colloidal suspensions suitable for treatment of the eye are generally formed from microparticles (i.e., microspheres, nanospheres, microcapsules or nanocapsules, where microspheres and nanospheres are generally monolithic particles of a polymer matrix in which the formulation is trapped, adsorbed, or otherwise contained, while with microcapsules and nanocapsules the formulation is actually encapsulated). The upper limit for the size of these microparticles is about 5μ, to about 10μ.
Ophthalmic ointments suitable for treatment of the eye include a compound or compounds of the invention in an appropriate base, such as mineral oil, liquid lanolin, white petrolatum, a combination of two or all three of the foregoing, or polyethylene-mineral oil gel. A preservative may optionally be included.
Ophthalmic gels suitable for treatment of the eye include a compound or compounds of the invention suspended in a hydrophilic base, such as Carpobol-940 or a combination of ethanol, water and propylene glycol (e.g., in a ratio of 40:40:20). A gelling agent, such as hydroxylethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose or ammoniated glycyrrhizinate, is used. A preservative and/or a tonicity agent may optionally be included.
Hydrogels suitable for treatment of the eye are formed by incorporation of a swellable, gel-forming polymer, such as those listed above as thickening agents or viscosity enhancers, except that a formulation referred to in the art as a “hydrogel” typically has a higher viscosity than a formulation referred to as a “thickened” solution or suspension. In contrast to such preformed hydrogels, a formulation may also be prepared so to form a hydrogel in situ following application to the eye. Such gels are liquid at room temperature but gel at higher temperatures (and thus are termed “thermoreversible” hydrogels), such as when placed in contact with body fluids. Biocompatible polymers that impart this property include acrylic acid polymers and copolymers, N-isopropylacrylamide derivatives and ABA block copolymers of ethylene oxide and propylene oxide (conventionally referred to as “poloxamers” and available under the Pluronic® tradename from BASF-Wayndotte).
Preferred dispersions are liposomal, in which case the formulation is enclosed within liposomes (microscopic vesicles composed of alternating aqueous compartments and lipid bilayers).
Eye drops can be formulated with an aqueous or nonaqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Drops can be delivered by means of a simple eye dropper-capped bottle or by means of a plastic bottle adapted to deliver liquid contents dropwise by means of a specially shaped closure.
The compounds of the invention can also be applied topically by means of drug-impregnated solid carrier that is inserted into the eye. Drug release is generally effected by dissolution or bioerosion of the polymer, osmosis, or combinations thereof. Several matrix-type delivery systems can be used. Such systems include hydrophilic soft contact lenses impregnated or soaked with the desired compound of the invention, as well as biodegradable or soluble devices that need not be removed after placement in the eye. These soluble ocular inserts can be composed of any degradable substance that can be tolerated by the eye and that is compatible with the compound of the invention that is to be administered. Such substances include, but are not limited to, poly(vinyl alcohol), polymers and copolymers of polyacrylamide, ethylacrylate and vinylpyrrolidone, as well as cross-linked polypeptides or polysaccharides, such as chitin.
Dosage forms for the other types of topical administration (i.e., not to the eye) or for transdermal administration of compounds of the invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active ingredient may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any buffers, or propellants which may be required. The ointments, pastes, creams and gels may contain, in addition to the active ingredient, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to the active ingredient, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. Transdermal patches have the added advantage of providing controlled delivery of compounds of the invention to the body. Such dosage forms can be made by dissolving, dispersing or otherwise incorporating one or more compounds of the invention in a proper medium, such as an elastomeric matrix material. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel. A drug-impregnated solid carrier (e.g., a dressing) can also be used for topical administration.
Pharmaceutical formulations include those suitable for administration by inhalation or insufflation or for nasal administration. For administration to the upper (nasal) or lower respiratory tract by inhalation, the compounds of the invention are conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as 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 to deliver a metered amount.
Alternatively, for administration by inhalation or insufflation, the composition may take the form of a dry powder, for example, a powder mix of one or more compounds of the invention and a suitable powder base, such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator, insufflator or a metered-dose inhaler.
For intranasal administration, compounds of the invention may be administered by means of nose drops or a liquid spray, such as by means of a plastic bottle atomizer or metered-dose inhaler. Liquid sprays are conveniently delivered from pressurized packs. Typical of atomizers are the Mistometer (Wintrop) and Medihaler (Riker).
Nose drops may be formulated with an aqueous or nonaqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Drops can be delivered by means of a simple eye dropper-capped bottle or by means of a plastic bottle adapted to deliver liquid contents dropwise by means of a specially shaped closure.
Pharmaceutical compositions of this invention suitable for parenteral administrations comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Also, drug-coated stents may be used.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monosterate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug is accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.
The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.
A diketopiperazine of formula I, a prodrug of a diketopiperazine of formula I or a pharmaceutically-acceptable salt of either one of them, may be given alone in the methods of the invention. Alternatively, the diketopiperazine, prodrug or salt may be given in combination with each other and/or in combination with one or more other treatments or drugs suitable for treating the disease or condition. For instance, the diketopiperazine, the prodrug or the salt can be administered prior to, in conjunction with (including simultaneously with), or after another treatment or drug. In the case of another drug, the drug and the diketopiperazine, prodrug or salt, may be administered in separate pharmaceutical compositions or as part of the same pharmaceutical composition.
The invention also provides kits. The kit comprises a container holding a diketopiperazine of formula I, a prodrug thereof or a pharmaceutically-acceptable salt of either of them. The kit may further comprise one or more additional containers each holding one or more other drugs suitable for use in the methods of the invention. Suitable containers include vials, bottles (including a bottle with a dropper or a squeeze bottle), blister packs, inhalers, jars, nebulizers, packets (e.g., made of foil, plastic, paper, cellophane or another material), syringes and tubes. The kit will also contain instructions for administration of the diketopiperazine, prodrug or salt and, optionally, the one or more other drugs suitable for use in the methods of the invention. The instructions may, for instance, be printed on the packaging holding the container(s), may be printed on a label attached to the kit or the container(s), or may be printed on a separate sheet of paper that is included in or with the kit. The packaging holding the container(s) may be, for instance, a box, or the container(s) may wrapped in, for instance, plastic shrink wrap. The kit may also contain other materials which are known in the art and which may be desirable from a commercial and user standpoint. The kit may, for instance, contain diet and/or exercise information.
As used herein, “obese” or “obesity” means a medical condition in which excess body fat has accumulated to the extent that it may have an adverse effect on health, leading to reduced life expectancy and/or increased health problems. Obesity increases the likelihood of various diseases, particularly heart disease, type 2 diabetes, breathing difficulties during sleep, certain types of cancer and osteoarthritis. According to the Centers For Disease Control And Prevention (CDC), a body mass index (BMI) of 30 or higher defines adult humans as obese, and a BMI of from 25.0 to 29.9 defines adult humans as overweight. BMI is calculated by dividing the subject's mass by the square of his or her height (e.g., (pounds×703)/inches2). “Obesity” in cats and dogs is defined as a body weight that is greater than 20% above optimal body weight.
“Treat,” “treating” or “treatment” is used herein to mean to reduce (wholly or partially) the symptoms, duration or severity of a disease or condition, including curing the disease, or to prevent the disease or condition.
“Inhibiting, “inhibit” and similar terms are used herein to mean to reduce, delay or prevent.
As used herein, “a” or “an” means one or more.
As used herein, “comprises” and “comprising” include within their scope all narrower terms, such as “consisting essentially of” and “consisting of” as alternative embodiments of the present invention characterized herein by “comprises” or “comprising”. In regard to use of “consisting essentially of”, this phrase limits the scope of a claim to the specified steps and materials and those that do not materially affect the basic and novel characteristics of the invention disclosed herein. The basic and novel characteristics of the invention can be treatment of metabolic syndrome, suppression of the appetite of an animal in need thereof, treatment of obesity in an animal, reduction of the weight of an animal in need thereof, reduction of a blood lipid level in an animal in need thereof, treatment of non-alcoholic steatohepatitis in an animal, inhibition of adipogenesis in an animal, or a combination of any of the foregoing.
Additional objects, advantages and novel features of the present invention will become apparent to those skilled in the art by consideration of the following non-limiting examples.
EXAMPLES
Example 1
Protocol


    
    
        1. Two human male volunteers and one human female volunteer (ages 61, 50 and 30 respectively) were administered 100 mg of DA-DKP (packed in a gelatin capsule) orally on an empty stomach after overnight fasting. The DA-DKP was composed of L-aspartic acid and L-alanine.
        2. Venous blood samples were drawn into heparinized and EDTA vacutainers at baseline (time zero), 2 hours (hrs), 4 hrs and 24 hrs after the ingestion of the DA-DKP.
        3. Blood samples were analyzed for a complete blood count (CBC) and a biochemical survey, including plasma lipids at baseline and at the 24 hrs time points. Plasma was also used for measurement of the level of DA-DKP by a liquid chromatography mass spectrometer (LCMS) method (protocol and results given below).
        4. Peripheral blood monocytes (PBMC) from heparinized bloods were extracted by Ficoll gradient method and analyzed by flow cytometry. The protocol and results are given below.
        5. Urines were collected for 24 hrs and volumes were recorded. DA-DKP levels were also measured by LCMS.
        6. Saliva at 2 hrs was tested for the presence of DA-DKP by LCMS.

Summary of Results:

        1. CBC's were unchanged and remained within the normal range.
        2. BUN, glucose electrolytes, creatinine, liver function tests, total protein, albumin and calcium were unchanged and within the normal range at 24 hours post ingestion.
        3. Lipids decreased across the board for all three subjects at 24 hours post ingestion (see Table 1).
        4. DA-DKP plasma levels peaked at about 2 hrs and it was not detectable at 24 hrs post ingestion (see Table 2).
        5. Urine levels of DA-DKP were calculated (see Table 2).
        6. Saliva DA-DKP levels were calculated (see Table 2)
        7. PBMC results are described below.
        8. Effects noticed by subjects:
        
            a. Significant loss of appetite (lasting 8-9 hrs)—3/3 subjects
            b. Slight dry mouth—2/3 subjects
            c. Thick saliva—1/3 subjects
            d. Analgesic effect—1/3 subjects (others did not have pain)*
            e. Laxative effect—1/3 subjects
            f. Slight drowsiness—1/3 subjects

* Analgesic Effect:

        
        
    
    


One subject (61 years old) had left knee and left Sacroiliac (SI) joint pain. Approximately 1-2 hrs after ingestion of the DA-DKP, the knee pain was resolved completely and the SI joint pain was reduced by about 50%. The pain originates probably from inflammation caused by sarcoidosis and was 6-7 on a scale of 1-10 before treatment. The effect lasted for about 4 hrs.







TABLE 1







(% Change in Lipids after 24 Hours)












Cholesterol
LDL
HDL
Triglyceride















1 (61 years)
−9.2%
 −10%
 −12%
 −2.1%


2 (30 years)
−3.0%
−7.9%
  −7%
 +0.5%


3 (50 years)*
−1.3%
−6.6%
−6.7%
+28.1% (had large






fatty meal the night






before)





*The 50 year-old male has Gilbert disease






DA-DKP LCMS Measurement

Plasma or urines were passed through a <5 Kd filter and 50 μl of the flow-through fractions were injected into high performance liquid chromatography (HPLC, 2975 system, Waters, Mass.) coupled to a mass spectrometer (LCT-TOF, Micromass, UK) and DA-DKP was quantified using a strong anion exchange column (Spherisorb, S5 SAX 250 mm×4.0 mm, Waters) and a ternary mobile phase consisting of water, methanol and 200 mM Ammonium Formate (Sigma Aldrich, Mo.), pH 5.4, at 0.5 ml/min flow rate in a gradient as shown in Table A.










TABLE A








200 mM Ammonium


Time (min)
Water (%)
Methanol (%)
Formate pH 5.4 (%)


















0
25
40
35


10
10
40
50


15
10
40
50


15.01
25
40
35


20
25
40
35









The output of the HPLC was split 1:20 (v:v) and injected into the mass spectrometer using negative electrospray ionization (−ESI MS) with a scan range of 80 to 1000 m/z, cone voltage of 30 eV, source temperature of 100° C., and gas temperature of 300° C. DA-DKP was measured by monitoring the mass 185 in time, which corresponds to DA-DKP minus a single proton (—H+). DA-DKP elutes at 10.4 minutes and was quantified by integrating the area under the curve. The area was compared to a standard curve derived from synthetic DA-DKP standards (DMI Synthesis, Newport Wales, UK) of known concentrations (2000 ng/ml, 1000 ng/ml, 400 ng/ml, 200 ng/ml, 100 ng/ml, 20 ng/ml). The calibration curve was found to fit very well in this range using a quadratic curve on log axes with an R2 of 0.997.
The results are presented in Table 2 below. The results are for plasma unless otherwise noted in Table 2.







TABLE 2







(DA-DKP in μM)


















0
15 min
30 min
60 min
120 min
240 min
360 min
24 hrs
Urine 24 hrs
Saliva 2 hrs





















1 (61
0
0.14
0.35
0.84
1.68
NA
0.61
0.25
6.98
NA


years








(2150 cc)


old)


2 (30
0.07
NA
NA
NA
0.4
0.34
NA
0.14
3.52
0.29


years








(3500 cc)


old)


3 (50
0.1
NA
NA
NA
0.54
0.25
NA
0.09
4.25
0.22


years








(2000 cc)


old)









PBMC Protocol and Results

Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood, and 2,000,000 cells were cultured in DMEM medium with 10% human AB serum plus 2% (final concentration) of anti-T-cell receptor complex antibody OKT3. A separate culture without OKT3 antibody was used as the NIL control. After culture overnight at 37° C. cells were washed, incubated with an anti-CD69 antibody, fixed and analyzed on the flow cytometer for CD69 expression. CD69 is an early activation antigen indicative of leukocyte immunocompetence. Results (for a single individual) indicated that CD69 expression on CD45RA and CD45RO cells was decreased after ingestion of DA-DKP from that before DA-DKP intake (time zero) with the greatest effect seen at 4 hours after DA-DKP ingestion. Dual histogram analysis indicated that the major effect was seen on the CD45RO (or previously activated “memory”) subset of T-cells (4 hours, OKT3 Stim. panel).
Discussion
The main effects observed after oral administration of DA-DKP were the lack of appetite (which could lead to weight loss, obesity being a cardinal feature of the metabolic syndrome) and the decrease in blood cholesterol, triglycerides, LDL and HDL (dyslipidemia being another feature of the metabolic syndrome). Also, the prevention of activation of previously activated T cells in response to an anti-CD3 stimulation, but not of the naïve T cells, is an important finding (inflammation being implicated in the metabolic syndrome). These together with in vitro data showing the inhibition of the differentiation of preadipocytes into adipocytes by DA-DKP (see Example 2) and the beneficial effects of DA-DKP on the vascular endothelial barrier (see co-pending provisional application No. 61/380,404, filed Sep. 7, 2010), support the idea of using DA-DKP as a therapeutic for the metabolic syndrome.
Example 2
A. Introduction
Adipogenesis is a multi-step process that involves the conversion of stem cells into fat-storing cells termed adipocytes. Adipose tissue mass enlarges and ultimately obesity ensues when energy intake chronically exceeds energy expenditure (1) (the numbers in parentheses refer to the references listed at the end of this Example 2). At the cellular level, this balance shift manifests itself through mature adipocytes accommodating excess energy through enhanced triacylglycerol storage (2). When a critical size threshold is reached, it is hypothesized that overloaded adipocytes signal preadipocytes to differentiate into new adipocytes to store the excess energy (3). Not surprisingly, obese animals have an increased amount of, and larger, adipocytes compared with lean animals (4). Any factor that inhibits cellular differentiation into the adipocyte lineage could be medically useful in treating obesity. However, a complete understanding of the adipogenic pathway, both inhibitory and stimulatory pathways, is necessary since the modulation of adipose tissue mass may have both advantageous and deleterious health effects (5).
Recently, due to increased obesity research, adipose tissue is now recognized as an endocrine organ due to its hormonal role in the regulation of metabolism, energy intake, and fat storage (6). Adipose tissue accomplishes fat regulation by secreting >50 proteins, termed adipokines, that act in an autocrine, paracrine, and endocrine fashion to control various metabolic functions (6). The most studied adipokines are adiponectin and leptin which decrease serum free fatty acid, glucose, and triacylglycerol concentrations by insulin sensitivity enhancement (7), and influence food intake by the hypothalamus (8), respectively. Additionally, pro-inflammatory cytokines such as TNFα and IL-6 play an important role in normal and pathological adipogenesis (2). After receptor binding by adipokines and/or cytokines, various intercellular molecular pathways important to adipogenesis can become activated or inhibited. Activation of mitogen activated protein kinases (MAPK) has an important role in adipocyte differentiation, and deregulation of MAPK can contribute to obesity (9).
B. Materials and Methods
Cell Culture
Subcutaneous preadipocytes (Zen-Bio, Inc., Research Triangle Park, N.C.) were cultured to near confluency at 5% CO2, 37° C. in Preadipocyte Growth Medium-2 (PGM-2, Lonza, Walkersville, Md.) containing 10% fetal bovine serum (FBS), L-glutamine, and GA-1000. Cells were harvested using trypsin/EDTA (Lonza) and initially seeded in 96-well black/clear bottom plates at 3,000 cells/well. Cells were allowed to form a confluent monolayer for 7 days in culture.
Cell Dosing and Differentiation
7-day old medium was aspirated and cells were dosed with DA-DKP (doses used in PGM-2 media: 25, 50, or 100 μM) in triplicate on two plates. The DA-DKP was composed of L-aspartic acid and L-alanine. PGM-2 differentiation media (containing insulin, dexamethasone, indomethacin, and isobutyl-methylxanthine; Lonza) was added to one plate, while PGM-2 media was added to the other. Plates were then placed back in culture and allowed to incubate for 14 days.
Adipogenesis Assay
After the 14 day incubation period, culture media were collected from all wells and stored at −20° C. for IL-6 ELISA (see below). Wells were rinsed once with 1×PBS (Lonza). 50 μL M-PER lysis solution (Thermo Scientific, Rockford, Ill.) were added to all wells, and complete cell lysis was achieved by mixing each well. 150 μL 1×PBS was added to each well. 5 μL AdipoRedTM Reagent (Lonza) was added to each well, and the plate was mixed immediately by gentle tapping. After 10 minutes, fluorescence was measured on a plate reader set at λEX=485 nm and λEM=572 nm.
IL-6 ELISA
Enzyme-linked immunosorbent assay (ELISA) matched-pair antibodies for interleukin-6 (IL-6) were obtained from Thermo Scientific (Rockford, Ill.). IL-6 was analyzed by ELISA and performed according to the manufacturer's instructions. A 1:10 dilution of each supernatant was measured in duplicate using an IL-6 standard (Thermo Scientific, Rockford, Ill.).
Data Analysis
The triplicate fluorescent values for each treatment were averaged and subtracted from the average media only (i.e. no cells) wells. For the differentiated wells, a % change was calculated by subtracting the average fluorescent value for the differentiated cells from the undifferentiated cells for each treatment group. This result was then subtracted from the difference between the differentiated and undifferentiated control groups (i.e. cells only with no treatment). Finally, this result was then divided by the difference between the differentiated and undifferentiated control groups to give a % change. All data are expressed as ±SD. Statistical significance is reported when p<0.05 based on the Student t-test (Microsoft Excel).
C. Results
DA-DKP caused an increase in adipogenesis at the highest dose (100 μM) while the lowest dose (25 μM) caused a significant decrease in adipogenesis (see FIG. 1). Interestingly, DA-DKP had no significant effect on IL-6 levels after the 2 week differentiation period (data not shown).
D. Discussion
Obesity has been described as the epidemic of the 21st century due to an increase in the prevalence of obesity as well as an earlier age of onset (6). This results in a major public health issue since obesity increases the risk of several chronic diseases such as diabetes, cardiovascular complications, arthritis, asthma, and certain cancers (10). Naturally, the search for a therapeutic agent that reverses the deleterious effects of obesity is the subject of a considerable amount of current research efforts. In this study, DA-DKP was effective at preventing adipogenesis (as measured by total intracellular triglyceride concentrations).
A potential cellular pathway target of DA-DKP is the family of mitogen activated protein kinases (MAPK). MAPK are serine/threonine kinase that include extracellular signal-regulated kinases (ERK), c-Jun amino-terminal kinases (JNK), and p38. MAPKs are important in regulating both normal and pathological adipogenesis (9). Previously, the inhibitory effect of DA-DKP on MAPK activity in T-lymphocytes has been shown (12). It is possible that DA-DKP inhibits adipogenesis by MAPK inhibition, but specific inhibition of MAPK isoforms such as ERK1 and JNK1 seems promising in preventing pathological adipogenesis without interference to other essential MAPK functions (9).
Another important pathway involved in cellular differentiation including adipogenesis involves the cytoskeleton. Disruption of the actin cytoskeleton, forced cell rounding, microtubule disruption, and stress fiber formation are all observed cytoskeletal changes during adipogenesis (11). DA-DKP has been shown to stabilize the cytoskeletal network in endothelial cells (see co-pending provisional application No. 61/380,404, filed Sep. 7, 2010).
DA-DKP is a naturally occurring molecule produced by an unknown mechanism involving the cleavage of the first two amino acids of human serum albumin and subsequent cyclization of the cleaved dipeptide (13). Additionally, DA-DKP has strong anti-inflammatory characteristics in immune cell models (12, 13). Interestingly, the pro-inflammatory pathway plays an important role in adipogenesis (6). In deregulated adipose tissue seen in obesity and diabetes, both local and systemic inflammation as well as insulin resistance and ectopic lipid accumulation are observed (14). Therefore, the anti-inflammatory tendencies of DA-DKP could be useful in combating the degree of inflammation seen in obese patients. However, no effect of DA-DKP on IL-6 levels was seen at the end of the differentiation period, suggesting that DA-DKP does not interfere with the pathway that regulates IL-6 levels during adipogenesis. This does not rule out the possibility of using DA-DKP as an anti-inflammatory in already obese patients.
Recently, a clustering of cardiovascular risk factors that include diabetes, obesity, dyslipidemia, and hypertension have been grouped together and termed the metabolic syndrome (15). It has been estimated that adults with the metabolic syndrome are twice as likely to develop cardiovascular disease compared to those without the syndrome (16). Clearly, decreases in abdominal obesity lead to substantial improvements in the metabolic risk profile resulting in a reduced risk of cardiovascular disease (17). Another pathological condition associated with the metabolic syndrome is the development of non-alcoholic steatohepatitis (NASH) in obese patients. NASH is characterized by an accumulation of lipid molecules in the liver accompanied by severe inflammation, fibrosis, and insulin/leptin resistance (18). Therefore, therapeutics that regulate further adipogenesis and/or decrease inflammation in adipose tissue could decrease the increased cardiovascular risk associated with the metabolic syndrome and be an effective treatment for NASH.
The increased prevalence of obesity in Western countries has lead to increases in various co-morbidities such as cardiovascular disease, hypertension, and diabetes. Searching for a therapeutic that increases fat metabolism and/or decreases fat storage has been the goal of many nutritional scientists. Here, evidence is presented of the anti-adipogenic effects of a compound that potentially targets a key regulator of adipogenesis (MAPK), stabilizes the cytoskeleton, and decreases inflammation, making it an attractive potential treatment for metabolic syndrome and a treatment to reduce obesity.
E. References for Example 2


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## Claims

1. A method of treating non-alcoholic steatohepatitis in an animal comprising administering to the animal in need thereof an effective amount of an active ingredient, wherein the active ingredient comprises a diketopiperazine, a prodrug of a diketopiperazine or a pharmaceutically-acceptable salt of either of them, wherein the diketopiperazine has the formula:




wherein:
R1 is the side chain of aspartic acid, and R2 is the side chain of alanine.

2. The method of claim 1 wherein the diketopiperazine, prodrug or pharmaceutically-acceptable salt is administered orally.

3. The method of claim 1 wherein the animal is a human.