Boropeptide Inhibitors Of Enteropeptidase And Their Uses In Treatment Of Obesity, Overweight And/or Diseases Associated With An Abnormal Fat Metabolism

Abstract

The present invention relates to novel compounds, particularly derivatives of boroarginine, boroornithine and borolysine that selectively modulate, regulate, and/or inhibit enteropeptidase. The invention also relates to compositions, particularly pharmaceutical compositions, as well as methods to treat excess weight, obesity and diseases associated with an abnormal fat metabolism.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to novel non-absorbable oligopeptides based on boroanalogs of amino acids incorporating a protonatable function on their side chain, such as boroarginine, borolysine, boroornithine, and related compounds, that selectively modulate, regulate, and/or inhibit enteropeptidase. These compounds are to used individually, in combination or in association with other known compounds for the treatment of excess weight, obesity and diseases associated with an abnormal fat metabolism.

BACKGROUND AND RELATED PRIOR ART

[0002] Obesity is a multi-faceted chronic condition and is the most prevalent nutritional problem in the United States today. Obesity, a condition caused by an excess of energy intake as compared to energy expenditure, contributes to the pathogenesis of hypertension, type II or non-insulin dependent diabetes mellitus, hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, heart disease, pancreatitis, and such common forms of cancer such as breast cancer, prostate cancer, uterine cancer and colon cancer.

[0003] At present, only a limited number of drugs for treating obesity are commercially available. Unfortunately, while some of these drugs may bring short-term relief to the patient, a long-term successful treatment has not yet been achieved. Exemplary methods of treating obesity are also disclosed in U.S. Pat. Nos. 3,867,539; 4,446,138; 4,588,724; 4,745,122; 5,019,594; 5,300,298; 5,403,851; 5,567,714; 5,573,774; 5,578,613; and 5,900,411.

[0004] One of the presently available drugs for treating obesity, developed by Hoffman-LaRoche, is an inhibitor of pancreatic lipase (PL). Pancreatic lipase is responsible for the degradation of triglycerides to monoglycerides. However, it has been associated with side-effects such as severe diarrhea resulting in absorption inhibition of only one specific fraction of fatty acids and, has been known to induce allergic reactions. Treatment with PL inhibitors is thus highly disadvantageous and may even expose the treated subject to life-threatening risks.

[0005] Recently, it has been suggested that fat absorption may be reduced by inhibiting the activity of the microsomal triglyceride-transfer protein (MTP), which is involved in the formation and secretion of very light density lipoproteins (VLDL) and chylomicrons. Sharp et al., [Nature (1993) 365:65-69] and Wetterau et al., [Science (1994) 282:751-754,] demonstrated that the mtp gene is responsible for abetalipoproteinemia disease. U.S. Pat. Nos. 6,066,650, 6,121,283 and 6,369,075 describe compositions that include MTP inhibitors, which are aimed at treating various conditions associated with excessive fat absorption. However, patients treated with MTP inhibitors suffer major side effects including hepatic steatosis, which are attributed to reduced MTP activity in both intestine and liver. This is not surprising since people naturally deficient for MTP activity were shown to develop fatty livers [Kane and Havel (1989); Disorders of the biogenesis and secretion of lipoproteins containing the apolipoprotein B. pp. 1139-1164 in: "The metabolic basis of inherited disease" (Scrivers et al., eds.). McGraw-Hill, New York]. In fact, the company Brystol Myers Squibb, that developed MTP inhibitors for the treatment of obesity, has recently decided to abandon this target, due to this fatty liver side effect.

[0006] The presently known targets for the treatment of obesity and related disorders can be divided into four main classes: (i) appetite blockers, which include for example the NPY (neuropeptide Y); (ii) satiety stimulators, which include, for example, the product of the ob, db and agouti genes; (iii) energy or fatty acid burning agents, which include the UCPs (Uncoupling Proteins); and (iv) fat absorption inhibitors such as those acting on PL and MTP in the intestine, described above.

[0007] As discussed herein, the use of these targets is highly limited by their redundancy, their multiple targeting and/or their lack of tissue specificity.

[0008] There is thus a widely recognized need for, and it would be highly advantageous to have compositions and methods for treating obesity and related diseases and disorders devoid of the above limitations.

[0009] Serine proteases are involved in a large number of important physiological processes. Selective inhibition of a given serine protease is one of the strategies for the treatment of pathological conditions associated with the activity or overactivity of these serine proteases. Below is a non-exhaustive list of serine protease inhibitors disclosed in the literature: [0010] phosphorus-based inhibitors such as the diisopropylphosphofuloridate (DFP) (Jansen et al., (1952) Adv. Enzymol. 13: 321-343) or diphenyl phosphonate ester analogues; [0011] fluorine-containing serine proteases, such as trifluoromethyl ketones (TFMKs); [0012] peptide-based aldehydes, chloromethyl ketones, fluoromethyl ketones, dimethyl sulphonium salts, .alpha.-keto-acids and amides, .alpha.-keto esters and .alpha.-keto-aldehydes (glyoxals); [0013] natural products such as the cyclotheonamides, derived from the Japanese marine sponge Theonella sp.; [0014] molecules based on heterocyclic structure; [0015] N-hydroxysuccimide heterocycles and related compounds; [0016] isocoumarins such as 3,4-dichloroisocoumarin; [0017] .beta. lactam-based inhibitors; [0018] metal-potentiated compounds; [0019] aprotinin (Trasylol.RTM.), used to reduce bleeding; and [0020] serpins (serine protease inhibitors) such as antithrombin and .alpha.-1-antitrypsin having a role in coagulation/thrombosis and emphysema/A1AT respectively.

[0021] However, few compounds have been described as serine protease inhibitors with a specific and selective inhibition of a unique target. Moreover, no compounds have been disclosed or suggested, to selectively and specifically inhibit the enteropeptidase, and to be used in the treatment of obesity, excess weight or diseases associated with an abnormal fat metabolism.

[0022] Enteropeptidase is a serine protease situated on the surface of epithelial intestinal cells (enterocytes) (Lancet. 1969 Apr. 19; 1(7599):812-3; Am J Physiol Gastrointest Liver Physiol. 2003 December; 285(6):G1235-41; Proc Soc Exp Biol Med. 1994 June; 206(2):114-8; Ciba Found Symp. 1979 January 16-18; (70):169-87; Lancet. 1982 Aug. 28; 2(8296):504). The substrate of enteropeptidase is trypsinogen, a precursor to trypsin. Enteropeptidase converts trypsinogen into a molecule of trypsin. In turn, trypsin, which is also a serine protease, converts the precursors of a series of digestive enzymes, such as procarboxypeptidases A and B, chymotrypsinogen, pancreatic prolipase and pro-elastase, into the active forms of the enzymes (carboxypeptidases A and B, chymotrypsin, pancreatic lipase and elastase). The latter active forms of such digestive enzymes are required for the processing and ultimate absorption of protein and fat matter in the gastrointestinal (GI) tract.

[0023] Because enteropeptidase is located in the intestinal lumen, inhibition of this enzyme requires that the compounds selectively inhibit enteropeptidase without interfering with circulating serine proteases, such as thrombin, kalikrein, and the like.

[0024] Thus, there is a need for compounds to treat obesity, excess overweight as well as diseases associated with an abnormal fat metabolism, on a long term basis that have a specific target.

[0025] It is an object of the present invention to provide compounds that inhibit enteropeptidase, and more particular that selectively inhibit enteropeptidase. In particular, these compounds are non-absorbable i.e., they do not pass from the intestine into the blood.

[0026] It is another object of the present invention to provide compounds that are derivatives of boroanalogs of amino acids incorporating a protonatable functionality on their side chain, such as borolysine, boroornithine, boroarginine and the like, and are strong, non-absorbable inhibitors of enteropeptidase.

[0027] Yet another object of the invention, are compositions, especially pharmaceutical compositions, comprising at least one of the compounds disclosed in the present invention.

[0028] It is also another object of the invention to provide methods to treat obesity, excess weight or diseases associated with an abnormal fat metabolism, comprising administrating, to a mammal in need thereof, at least one of the compounds disclosed in the present invention or a composition described in the present invention.

[0029] Yet another object of the present invention is the use of at least one of the compounds or of the composition disclosed herein, for the treatment of obesity, excess weight and diseases associated with an abnormal fat metabolism. A compound or a composition of the invention for use in the treatment of obesity, excess weight and diseases associated with an abnormal fat metabolism, is also provided.

[0030] These and other objects are achieved by the present invention as evidence by the summary of the invention, description of the preferred embodiments and the claims.

SUMMARY OF THE INVENTION

[0031] In one aspect, the invention relates to a compound having the following formula (I):

##STR00001##

[0032] wherein [0033] B represents a boron atom; [0034] W is a nitrogen-containing functionality group, sustaining a positive charge either through protonation or quaternization, this group being selected from:

[0035] (i) an amino group of the formula:

##STR00002## [0036] wherein G.sup.1 and G.sup.2 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms;

[0037] (ii) a quaternary ammonium group of formula:

##STR00003## [0038] wherein G.sup.1, G.sup.2 and G.sup.3 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms; or

[0039] (iii) a guanidine group of formula:

##STR00004## [0040] wherein G.sup.1, G.sup.2, G.sup.3, G.sup.4 and G.sup.5 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates one or more of the guanidine N atoms and that contains a total of up to 15 atoms; [0041] X is a linker unit having the formula (CX.sup.1X.sup.2).sub.p, wherein 1<p<10, and wherein X.sup.1 and X.sup.2 are, independently, H, or linear or branched alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring residue containing from 3 to 10 atoms; [0042] Y is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms; [0043] Z is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms, or is a linear or branched or cyclic alkyl group containing from 1 to 15 carbon atoms; [0044] R1 is chosen among: [0045] (i) an aminoacyl residue derived from an amino acid. [0046] (ii) a derivative of the foregoing aminoacyl residue in which the amino group is additionally acylated, or sulfonylated, or phosphorylated to form an amide, or peptide, or sulfonamide, or phosphoramide bond; [0047] (iii) an acyl group of general formula R'--CO, wherein R' is: [0048] a. a linear, branched or cyclic alkyl group that contains from 1 to 10 C atoms; [0049] b. a saturated heterocyclic ring incorporating up to 20 atoms chosen from C, O, N, and S atoms; [0050] c. an aryl group selected from phenyl or a substituted variant thereof bearing any combination, at any one ring position, of one or more substituents selected from halogen, alkyl groups containing from 1 to 10 carbon atoms, trifluoromethyl and alkoxy groups; or [0051] d. a heteroaryl group; [0052] R.sup.2 is either H, or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms, or an OR group wherein R may be H or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms.

[0053] In another aspect, the invention also concerns a compound having the formula

##STR00005##

[0054] II:

[0055] wherein [0056] B represents a boron atom; [0057] W is a nitrogen-containing functionality group, sustaining a positive charge either through protonation or quaternization, this group being selected from:

[0058] (i) an amino group of the formula:

##STR00006## [0059] wherein G.sup.1 and G.sup.2 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms;

[0060] (ii) a quaternary ammonium group of formula:

##STR00007## [0061] wherein G.sup.1, G.sup.2 and G.sup.3 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms; or

[0062] (iii) a guanidine group of formula:

##STR00008##

[0063] wherein G.sup.1, G.sup.2, G.sup.3, G.sup.4 and G.sup.5 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates one or more of the guanidine N atoms and that contains a total of up to 15 atoms; [0064] X is a linker unit having the formula (CX.sup.1X.sup.2).sub.p, wherein 1<p<10, and wherein X.sup.1 and X.sup.2 are, independently, H, or linear or branched alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring residue containing from 3 to 10 atoms; [0065] Y is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms; [0066] Z is an OH, or an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms, or is a linear or branched or cyclic alkyl group containing from 1 to 15 carbon atoms; [0067] R.sup.2 is either H, or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms; or an OR group, wherein R may be H or a or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms; and [0068] "aa" refers to any amino acyl residue or derivative thereof, and n is at least two.

[0069] In a yet another aspect, the invention is directed to the following compounds having formula (III) or (IV):

##STR00009##

[0070] wherein: [0071] B represents a boron atom;

[0072] Y is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms; [0073] Z is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms, or is a linear or branched or cyclic alkyl group containing from 1 to 15 carbon atoms; [0074] R.sup.2 is either H, or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms or an OR group wherein R may be H or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms; [0075] G.sup.1, G.sup.2 and G.sup.3 or G.sup.1 and G.sup.2 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates one or more of the guanidine N atoms and that contains a total of up to 15 atoms; [0076] "aa" refers to any amino acid residue or derivative thereof, and n is at least two; and [0077] the index, m, equals 3 or 4.

[0078] In another aspect, the invention also concerns a pharmaceutical composition comprising at least one compound as described above.

[0079] In a last aspect, the invention also refers to a method comprising administering to a mammal in need of said treatment, at least one compound or a composition of the invention for treating a patient having obesity, having excess weight or suffering from abnormal fat metabolism.

BRIEF DESCRIPTION OF THE DRAWINGS

[0080] FIG. 1 is a graph showing the activity of the enteropeptidase according to increasing concentrations of compound OBE 1999.

[0081] FIG. 2 is a graph showing the activity of the enteropeptidase according to increasing concentrations of compound OBE 2000.

[0082] FIG. 3 is a graph showing the activity of the enteropeptidase according to increasing concentrations of compound OBE 2001.

[0083] FIG. 4 is a graph showing the activity of the enteropeptidase according to increasing concentrations of compound OBE 2002.

[0084] FIG. 5 is a Table showing the IC 50 (in nM) of enteropeptidase, various serine proteases and other enzyme families, in the presence of compounds OBE 1999, OBE 2000, OBE 2001 or OBE 2002.

[0085] FIG. 6 is the nucleotide and protein sequences of the human enteropeptidase (PRSS7). The first line indicates the nucleotide sequence, grouped by codons; the second line indicates the amino acid sequence corresponding to the above codons with the three-letter code. The first codon of translation and the stop codon are shown in bold. Numbering of the nucleic acid is at the right end of the first line, and numbering of the amino acids is indicated under amino acid residue (third line).

[0086] FIG. 7 represents examples of processes to synthesize the compounds of the invention. (A): example of a process for the synthesis of the Acetyl-Ala-Phe-BoroLysine (*pin: pinandiol group); (B and C): first example of a process for the synthesis of the Acetyl-Ala-Phe-BoroArginine; (D): second example of a process for the synthesis of the Acetyl-Ala-Phe-BoroArginine; this second process also enables the synthesis of the Acetyl-Ala-Phe-BoroOrnithine (compound 8), since Acetyl-Ala-Phe-BoroOrnithine is an intermediate compound in the synthesis of Acetyl-Ala-Phe-BoroArginine by this process; (E): synthesis of the Acetyl-Ala-Phe-OH group, to be inserted in step 10 of the process in FIG. 7C or in step 7 of the process in FIG. 7D.

[0087] FIG. 8 is a graph illustrating the follow up in days of the weight (in grams) of mice to having received water (vehicle) as compared to mice having received OBE2001 at a concentration of 40 mg/kg/day (B).

[0088] FIG. 9 is a graph illustrating the follow up in minutes of the triglycerides present in the plasma of mice having received water (solvent), one dosage of OBE2001 (OBE 25 mg/kg) or two dosages of OBE2001 (OBE 25+25 mg/kg).

[0089] FIG. 10 is a graph illustrating the follow up in hours of labelled proteins in plasma of mice having received water (solvent), one dosage of OBE2001 (OBE 5 mg/kg) or two dosages of OBE2001 (OBE 50 mg/kg).

[0090] FIG. 11 is a schematic representation of the process used to obtain a constitutive enteropeptidase knockout (KO) allele in mice, using the deletion of the exons 23-28 of the Prss7 gene.

[0091] FIG. 12 is a schematic representation of a vector comprising the constitutive enteropeptidase knockout (KO) allele, as well as the 17,480 by nucleotide sequence of this vector.

[0092] FIG. 13 is the AfIII restriction map of the wild type enteropeptidase allele (above) and the knockout (KO) enteropeptidase allele (below).

[0093] FIG. 14 is a Southern Blot obtained with genomic DNA from embryonic stem (ES) cells successfully transformed with the enteropeptidase KO allele (A-D2 and A-E8) and obtained with the genomic DNA of non transformed cells (WT).

[0094] FIG. 15 is a blot in which PCR fragments have been run, from a simultaneous amplification with primers 1260.sub.--1 and 1260.sub.--2, and 1472.sub.--23 and 1472.sub.--24. Genomic DNA has been extracted from heterologous mice (115695, 115702, 115706, 115707 and 115708) or wild type mice (115696 or 115705). 585 bp (c): control fragment; 412 by (cony): KO allele.

[0095] FIG. 16 is a blot in which PCR fragments, obtained from a simultaneous amplification with primer 1472.sub.--23 and primer PRRS7 WT, and beta actin forward and beta actin backward primers, have been run. Genomic DNA has been extracted from heterologous mice (115755 and 115648) or KO homologous pups (1A, 2A, 3A, 4A and 5A); 533 bp (c): WT enteropeptidase allele; 300 bp: control allele (actin).

[0096] FIG. 17 is a photograph showing a mouse homozygous for the enteropeptidase knockout allele (Ho) and a mouse heterozygous for the enteropeptidase knockout allele (He), both 7 days old.

[0097] FIG. 18 is the nucleotide and protein sequences of human trypsin.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

[0098] As used herein the term "protecting group" means a chemical group used to modify the compound of the invention, in one of its functional groups, in order to obtain in a subsequent chemical reaction and to avoid unwanted reactions. Examples of protecting groups are as follows: [0099] Alcohol protecting groups: Acetyl (Ac), .beta.-Methoxyethoxymethyl ether (MEM), Methoxymethyl ether (MOM), p-Methoxybenzyl ether (PMB), Methylthiomethyl ether, Pivaloyl (Piv), Tetrahydropyran (THP), Silyl ether (most popular ones include trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), and triisopropylsilyl (TIPS) ethers) and methyl ethers; [0100] Amine protecting groups: Acetyl (Ac), Benzoyl (Bz), Pivaloyl (Pv) Methanesulfonyl (Ms), Benzenesulfonyl (PhSO.sub.2), para-Toluenesulfonyl (Ts), Phosphoryl [(HO).sub.2P(O)], Dibenzyloxyphosphoryl [(BnO).sub.2P(O)], Benzyloxy-methanephosphonyl [(CH.sub.3)(BnO)P(O)], Carbobenzyloxy (Cbz) group, tert-Butyloxycarbonyl (BOC) group, 9-Fluorenylmethyloxycarbonyl (FMOC) group, Benzyl (Bn) group and p-methoxyphenyl (PMP) group; [0101] Carbonyl protecting groups: Acetals and Ketals, Acylals and Dithianes; and [0102] Carboxylic acid protecting groups: Methyl esters, Benzyl esters, tert-Butyl esters and Silyl esters.

[0103] "Chemoselectivity" as used herein means the preferential outcome of one instance of a generalized reaction over a set of other plausible reactions.

[0104] "An amino acid residue" is defined in the present invention as one of the following 21 amino acid acyl residues: glycine, alanine, valine, leucine, isoleucin, phenylalanine, tyrosine, tryptophan, methionine, cystine, cysteine, serine, threonine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine and proline.

[0105] A "derivative of an amino acid residue" means an amino acid residue as defined in the previous paragraph, for which the amino group is additionally acylated, or sulfonylated, or phosphorylated to form an amide, or carbamate, or urea, or N-substituted urea, or peptide, or sulfonamide, or phosphoramide bond. Additionally, the amino group may be further substituted with a small alkyl group containing from 1 to 5 C atoms. Particular derivatives of amino acids are exemplified by, but not limited to, the following representative structures, (i)-(iii), wherein the wavy line represents the bond that connects these structures to the other part of the molecule:

TABLE-US-00001 ##STR00010## R.sup.1 R.sup.2 R.sup.3 name CH.sub.3 CH.sub.3 H N-acetylalanyl CH.sub.3 CH.sub.3 CH.sub.3 N-methyl-N-acetylalanyl CH.sub.3 (CH.sub.2).sub.2COOH H N-acetylglutamyl CH.sub.3 CH--C.sub.6H.sub.5 H N-acetylphenylalanyl (CH.sub.3).sub.3C CH.sub.3 CH.sub.3 N-methyl-N-pivaloylalanyl C.sub.6H.sub.5 (CH.sub.2)COOH H N-benzoylglutamyl 4-Cl--C.sub.6H.sub.4 (CH.sub.2)COOH H N-(4-chlorobenzoyl)glutamyl OCH.sub.2C.sub.6H.sub.5 CH.sub.3 H N-(carbobenzyloxy)alanyl NH.sub.2 CH--C.sub.6H.sub.5 H N-(carbamoyl)phenylalanyl N(CH.sub.2CH.sub.3).sub.2 CH.sub.3 H N-(diethylcarbamoyl)alanyl ##STR00011## R.sup.1 R.sup.2 R.sup.3 name CH.sub.3 CH.sub.3 H N-methanesulfonylalanyl CH.sub.3 CH.sub.3 CH.sub.3 N-methyl-N-methanesulfonylalanyl 4-CH.sub.3--C.sub.6H.sub.4 (CH.sub.2).sub.2COOH H N--p-toluenesulfonylglutamyl CH.sub.3 CH--C.sub.6H.sub.5 H N-methansulfonylphenylalanyl C.sub.6H.sub.5 CH.sub.3 CH.sub.3 N-methyl-N-benzenesulfonylalanyl ##STR00012## R.sup.1 R.sup.2 R.sup.3 R.sup.4 name OH OH CH.sub.3 H N-phosphorylalanyl OCH.sub.2C.sub.6H.sub.4 OCH.sub.2C.sub.6H.sub.4 CH.sub.3 H N-(dibenzyloxyphosphoryl) alanyl OCH.sub.2C.sub.6H.sub.4 OCH.sub.2C.sub.6H.sub.4 CH.sub.3 CH.sub.3 N-methyl-N- (dibenzyloxyphosphoryl)alanyl CH.sub.3 OH CH.sub.3 H N-(methanephosphonyl)alanyl

[0106] The expression "pharmaceutically acceptable salt" means an acid salt or a basic salt that is suitable or compatible with the treatment of the subject.

[0107] By "inhibition of the activity of the enteropeptidase", is meant that a decrease of 50% of the in vitro activity of the enteropeptidase is obtained, with a concentration of the compound of the invention that is less than 10 .mu.M, less than 1 .mu.M, less than 100 nM, less than 10 nM or less than 1 nM, for 1 nM of enteropeptidase. Said concentration can be determined as described in details in point 2.1. below, and particularly using Np Tosyl Gly Pro Arg pNa as a substrate.

[0108] The expression "specific inhibition of the trypsin-like serine protease" refers to the inhibition of proteases from the serine protease family and more particularly to serine protease of the trypsin-like subtype only. In contrast, the activity of other proteases such as cysteine peptidases, aspartate peptidases, metallo-proteases, lipases and/or glucosidases are not altered by the compound of the invention. In another embodiment, in combination with the previous one, the activity of chymotrypsin-like serine proteases is not altered by compound of the invention.

[0109] The expression "selective inhibition of the enteropeptidase" refers to the inhibition of the enteropeptidase only (in vivo), whereas the activity of other proteases from the same subtype (trypsin-like subtype) are not altered by the compounds of the invention. This distinction between the specificity and selectivity has been rendered possible by the fact that the compounds of the invention are non-absorbable and therefore the inhibition is limited to the enteropeptidase whose location is intestinal.

[0110] The determination of the IC.sub.50 of the compounds of the invention on enteropeptidase or on other serine protease may be tested in vitro as described in the examples below.

[0111] The term "treatment" as used herein refers not only to the loss of weight of the mammal following the administration of at least one compound or the composition of the invention, but also to the maintenance of the weight such that there is no weight increase.

[0112] The term "mammal" encompasses any of various warm-blooded vertebrate animals of the class Mammalia, including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young.

[0113] "Pharmaceutically acceptable vehicles or carriers" encompass any substance that enables the formulation of the compounds of the invention within a composition. A vehicle is any substance or combination of substance physiologically acceptable i.e., appropriate for its use in a composition in contact with a mammal, and thus non-toxic. Examples of such vehicles are phosphate buffered saline solutions, distilled water, emulsions such as an oil/water emulsions, various types of wetting agents sterile solutions and the like.

[0114] "Obesity" is in general defined in human by at least 20% over the average weight for the person's age, sex and height. Obesity is defined by a body mass index (BMI=kg/m.sup.2) over 30. Obesity can also be defined by absolute waist circumference (>102 cm in men and >88 cm in women) or Waist-to-hip ratio (WHR) (WHR more than 0.7 for women and more than 0.9 for men). "Excessive weight" is defined by a BMI that is comprised between 25 and 29.9.

[0115] More specifically, in a first embodiment, the present invention relates to compounds having an inhibitory activity on the enzymatic activity of enteropeptidase. In another aspect, compounds are provided that have a selective inhibitory activity on the enzymatic activity of enteropeptidase.

[0116] The compounds of the invention have the following formula I:

##STR00013##

[0117] wherein: [0118] B represents a boron atom; [0119] W is a nitrogen-containing functionality group, sustaining a positive charge either through protonation or quaternization, this group being selected from:

[0120] (i) an amino group having the formula:

##STR00014## [0121] wherein G.sup.1 and G.sup.2 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms.

[0122] (ii) a quaternary ammonium group having the formula:

##STR00015## [0123] wherein G.sup.1, G.sup.2 and G.sup.3 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms;

[0124] (iii) a guanidine group having the formula:

##STR00016## [0125] is wherein G.sup.1, G.sup.2, G.sup.3, G.sup.4 and G.sup.5 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates one or more of the guanidine N atoms and that contains a total of up to 15 atoms; [0126] X is a linker unit having the formula (CX.sup.1X.sup.2).sub.p, wherein 1<p<10, and wherein X.sup.1 and X.sup.2 are, independently, H, or linear or branched alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring residue containing from 3 to 10 atoms. [0127] Y is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms; [0128] Z is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms, or is a linear or branched or cyclic alkyl group containing from 1 to 15 carbon atoms; [0129] R1 is selected from: [0130] (i) an aminoacyl residue derived from an amino acid. [0131] (ii) a derivative of the foregoing aminoacyl residue in which the amino group is additionally acylated, or sulfonylated, or phosphorylated to form an amide, or peptide, or sulfonamide, or phosphoramide bond; [0132] (iii) an acyl group of general formula R'--CO, wherein R' is: [0133] a. a linear, branched or cyclic alkyl group that contains from 1 to 10 C atoms; [0134] b. a saturated heterocyclic ring incorporating up to 20 atoms chosen from C, O, N, and S atoms; [0135] c. an aryl group selected from phenyl or a substituted variant thereof bearing any combination, at any one ring position, of one or more substituents selected from halogen, alkyl groups containing from 1 to 10 to carbon atoms, trifluoromethyl and alkoxy groups; or [0136] d. a heteroaryl group.

[0137] For compounds of formula (I) above, examples of a heteroaryl group (R.sup.1) is a 2,3, or 4-pyridyl group, which may additionally bear any combination of one or more substituents such as halogen, alkyl groups containing from 1 to 10 carbon atoms, trifluoromethyl, alkoxy, alkylthio, alkylsulfonyl, etc. Similarly, examples of a five-membered ring aromatic heterocyclic group (R.sup.1) are 2-thienyl, 3-thienyl, 2-thiazolyl, 4-thiazolyl and 5-thiazolyl groups. These groups may additionally bear any combination of one or more substituents such as halogen, an alkyl group containing from 1 to 10 carbon atoms, trifluoromethyl, alkoxy, alkylthio, alkylsulfonyl, etc.

[0138] In another embodiment, the present invention relates to the compound having the formula II:

##STR00017##

[0139] wherein [0140] B represents a boron atom; [0141] W is a nitrogen-containing functionality group, sustaining a positive charge either through protonation or quaternization, this group being selected from:

[0142] (i) an amino group having the formula:

##STR00018## [0143] wherein G.sup.1 and G.sup.2 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms;

[0144] (ii) a quaternary ammonium group having the formula:

##STR00019## [0145] wherein G.sup.1, G.sup.2 and G.sup.3 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 to atoms;

[0146] (iii) a guanidine group having the formula:

##STR00020## [0147] wherein G.sup.1, G.sup.2, G.sup.3, G.sup.4 and G.sup.5 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates one or more of the guanidine N atoms and that contains a total of up to 15 atoms; [0148] X is a linker unit having the formula (CX.sup.1X.sup.2).sub.p, wherein 1<p<10, and wherein X.sup.1 and X.sup.2 are, independently, H, or linear or branched alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring residue containing from 3 to 10 atoms; [0149] Y is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms; [0150] Z is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms, or is a linear or branched or cyclic alkyl group containing from 1 to 15 carbon atoms; [0151] R.sup.2 is either H, or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms, or an OR group wherein R may be H or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms; and [0152] "aa" refers to any amino acyl residue or derivative thereof, and n is at least two, or in another embodiment n is between 2 and 20, in another embodiment between 2 and 15, in another embodiment between 2 and 10, and in another embodiment between 2 and 5.

[0153] With respect to the compounds of formula (II), in another aspect, n is 2, 3 or 4. The at least two amino acid residues are selected among the following: Ala, Arg, Asn, Asp, Cys, Cystine, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.

[0154] In the case where n=2, a combination of amino acid residues is Tyr and Pro, Ala and Phe, Arg and Glu, Glu and Gly, Trp and Glu, Thr and Pro, and Leu and Leu.

[0155] In the case where n=3, a combination of amino acid residues is Phe, Arg and Val, Arg, Cys and Thr, Gly, Cys and Pro, Gly, Cys and Asn and Lys, Gly and Asp.

[0156] In yet another embodiment, the amino group of the first N-terminal amino acid residue bears a protecting group selected from the group of acyl, sulfonyl, or phosphoryl, as specified on page 10, and it may further bear an alkyl group containing from 1 to 5 C atoms, as indicated in the table that appears on page 11.

[0157] In yet another embodiment, the amino group of the first N-terminal amino acid residue can be derivatized with an acyl, sulfonyl, or phosphoryl group to form an amide, or carbamate, or urea, or N-substituted urea, or peptide, or sulfonamide, or phosphoramide bond, and it may be further substituted with a small alkyl group containing from 1 to 5 C atoms, as indicated in the table that appears on page 11.

[0158] With respect to the compounds of formula (I) or (II) as defined above:

[0159] When G.sup.1 to G.sup.5 are, independently, branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms, these 15 atoms include any permutation of carbon, oxygen, nitrogen, and sulfur atoms,

[0160] Examples of ring residues for the amino group (W) are pyrrolidine, piperidine, morpholine, thiomorpholine, piperazine, and N-methylpiperazine. Representative embodiments are as follows:

##STR00021##

[0161] Examples of G.sup.1 and G.sup.2 for the amino group (W) are the following: hydrogen, methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, phenyl, etc. Representative embodiments are as follows:

##STR00022##

[0162] Examples of ring residues for the quaternary ammonium group (W) are pyrrolidine, piperidine, morpholine, thiomorpholine, piperazine, and N-methylpiperazine. Representative embodiments are as follows:

##STR00023##

[0163] Examples of G.sup.1, G.sup.2 and G.sup.3 for the quaternary ammonium group (W) are the following: methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, phenyl, etc, Representative embodiments are as follows:

##STR00024##

[0164] Examples of ring residues for the guanidine group (W) are pyrrolidine, piperidine, morpholine thiomorpholine, piperazine, N-methylpiperazine, and tetrahydropyrimidine. In addition, G.sup.2 may also be a lone pair of electrons. Representative embodiments are as follows.

##STR00025##

[0165] Examples of G.sup.1, G.sup.2, G.sup.3, G.sup.4 and G.sup.5 for the guanidine group (W) are the following: methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, phenyl, etc, In addition, G.sup.2 may also be a lone pair of electrons. Representative embodiments are as follows:

##STR00026##

[0166] Examples of ring residues contained in X are cyclopropane, cylobutane, cyclopentane, cyclohexane or phenyl. Examples of X are shown in the following representative embodiments, where the wavy lines represent the bonds that connect X to the W part and the bore-containing part:

##STR00027##

[0167] In yet another embodiment, Y and/or Z are OH or OR. When R is a cyclic alkyl group, this group optionally incorporates one or more heteroatoms such as N, O, S and halogen.

[0168] Examples of such Y and Z groups are shown in the following representative embodiments, where the wavy lines represent the bond connecting the boron atom to the other part of the molecule:

##STR00028##

[0169] In yet another embodiment, Y and Z are branches of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms. Examples of such embodiments are shown below. The wavy line represents the bond connecting the B atom to the other part of the molecule:

##STR00029##

[0170] In yet another embodiment, Z is a linear or branched or cyclic alkyl group containing from 1 to 15 carbon atoms. Optionally, the linear or branched alkyl group for Z is substituted with one or more heteroatoms such as N, O, S and halogen. Examples of such Z groups are shown in the following representative embodiments, where the wavy lines represent the bond connecting Z to the other part of the molecule:

##STR00030##

[0171] In yet another embodiment, the W--X--C--N(C.dbd.O)--R.sup.2 part of the compound having formula I or II (structure bearing the boron atom) is an amino acid residue positively charged. An amino acid residue positively charged may be selected among arginine, lysine, ornithine or a derivative of one of these three amino acid residues as defined above.

[0172] In yet another aspect, the present invention relates to a compound based on boroarginine and having the following formula III:

##STR00031##

[0173] wherein [0174] B represents a boron atom; [0175] Y is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms; [0176] Z is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms, or is a linear or branched or cyclic alkyl group containing from 1 to 15 carbon atoms; [0177] R.sup.2 is either H, or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms, or an OR group wherein R may be H or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms; [0178] G.sup.1, G.sup.2 and G.sup.3 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms; and [0179] "aa" refers to any amino acyl residue or derivative thereof, and n is at least two.

[0180] Another embodiment of a compound based on boroarginine is a compound of formula IV, wherein at least one of R.sup.2, G.sup.1, G.sup.2 and G.sup.3 is H. Alternatively or in combination with the previous embodiment, at least one of R.sup.2, G.sup.1, G.sup.2 and G.sup.3 is a protecting group as defined above.

[0181] In another embodiment of a compound based on boroarginine is a compound of formula III, wherein G.sup.1, G.sup.2 and G.sup.3 are H and having the following formula:

##STR00032##

[0182] A compound based on boroarginine is a compound of formula III, wherein R.sup.2, G.sup.1, G.sup.2 and G.sup.3 are H and having the following formula:

##STR00033##

[0183] In yet another embodiment, the compound of the invention is based on borolysine or boroornithine and has the following formula IV:

##STR00034##

[0184] wherein [0185] B represents a boron atom; [0186] Y is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms; [0187] Z is an OH, or an is OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms, or is a linear or branched or cyclic alkyl group containing from 1 to 15 carbon atoms; [0188] R.sup.2 is either H, or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms, or an OR group wherein R may be H or a or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms; [0189] G.sup.1 and G.sup.2 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms; [0190] "aa" refers to any amino acid residue or derivative thereof, and n is at least two; and [0191] m equals to 3 or 4.

[0192] In a embodiment, the compound is based on borolysine (m equals 4), and has the following formula (V)

##STR00035##

[0193] wherein [0194] B represents a boron atom; [0195] Y is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms; [0196] Z is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms, or is a linear or branched or cyclic alkyl group containing from 1 to 15 carbon atoms; [0197] R.sup.2 is either H, or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms, or an OR group wherein R may be H or a or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms; [0198] G.sup.1 and G.sup.2 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms; and [0199] "aa" refers to any amino acid residue or derivative thereof, and n is at least two.

[0200] In another embodiment, the compound is based on boroornithine (m equals 3), and has the following formula (VI)

##STR00036##

[0201] wherein [0202] B represents a boron atom; [0203] Y is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms; [0204] Z is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms, or is a linear or branched or cyclic alkyl group containing from 1 to 15 carbon atoms; [0205] R.sup.2 is either H, or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms, or an OR group wherein R may be H or a or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms; [0206] G.sup.1 and G.sup.2 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms; and [0207] "aa" refers to any amino acid residue or derivative thereof, and n is at least two.

[0208] Another compound is a compound of formula IV, V or VI, wherein at least one of R.sup.2, G.sup.1 and G.sup.2 is H. Alternatively or in combination with the previous embodiment, at least one of R.sup.2, G.sup.1 and G.sup.2 is a protecting group as defined above.

[0209] Other compounds based either on formula IV, V or VI are compound wherein G.sup.1 and G.sup.2 are H and have the following formula:

##STR00037##

[0210] In another embodiment of a compound based on a compound of formula IV, V or VI, wherein R.sup.2, G.sup.1 and G.sup.2 are H and have the following formula:

##STR00038##

[0211] In yet another aspect, the compounds based on boroarginine (based on formula IV), on borolysine (based on formula V) or on boroornithine (based on formula VI), disclosed above, are also characterized by at least one of the following: [0212] at least one of Y or Z is OH. In an embodiment, Y and Z are OH. [0213] n is at least 2, and in another aspect is between 2 and 20, in another embodiment between 2 and 15, in another embodiment between 2 and 10, and in another embodiment between 2 and 5. In yet another aspect equals 2, 3, 4 or 5. When n equals 2, the resulting molecule is called a triboropeptide; when n equals 3, the resulting molecule is called a tetraboropeptide. The particular amino acid residues or derivative thereof that may constitute the triboropeptides or tetraboropeptides of the invention as well as the particular combination of amino acids residues have already been disclosed above for the definition of the compound of formula II, and apply in the same manner for the present triboropeptides and tetraboropeptides; and [0214] the first N-terminal amino acid residue of the n amino acids bears a protecting group as defined above. In another embodiment, the protecting group is linked to the free NH.sub.2 group of the first N-terminal amino acid.

[0215] Examples of the compounds of formula I indicated are the following triboropetides and tetraboropetides: Tyr-Pro-BoroArg, Ala-Phe-BoroArg, Arg-Glu-BoroArg, Glu-Gly-BoroArg, Trp-Glu-BoroArg, Thr-Pro-BoroArg, Leu-Leu-BoroArg, Phe-Arg-Val-BoroArg, Arg-Cys-Thr-BoroArg, Gly-Cys-Pro-BoroArg, Gly-Pro-Cys-BoroArg, Gly-Cys-Asn-BoroArg and Lys-Gly-Asp-BoroArg.

[0216] Other compounds are Ala-Phe-BoroArg, Ac-Ala-Phe-BoroArg, Glu-Gly-BoroArg and Ac-Glu-Gly-BoroArg, as disclosed respectively in formulas VII, VIII, IX and X.

##STR00039##

[0217] All the compounds of the invention disclosed in the present application are under a free base form or are pharmaceutically acceptable salts thereof.

[0218] In contrast, the compounds of the invention are not at least one of the following: [0219] Ac-(D,L)Phe-boroArg-C.sub.10H.sub.16.BSA (benzene sulfonic acid), Ac-Phe-boroOrn-C.sub.10H.sub.16.BSA, Ac-Phe boroArg-C.sub.10H.sub.16.HCl, H-(D)Phe-Pro-boroIrg-C.sub.10H.sub.16.HBr.HCl, Boc-(D)Phe-Pro-boroIrg-C.sub.10H.sub.16. HBr, Ac-Phe-boroIrg-C.sub.10H.sub.16.HBr, Ac-Ala-Lys(Boc)-boroOrn-C.sub.10H.sub.16.BSA, Ac-Ala-Lys(Boc)-boroIrg-C.sub.10H.sub.16.H Br, Boc-(D)Phe-Pro-boroArg-C.sub.10H.sub.16.BSA, Boc-(D)Phe-Phe-BoroIrg-C.sub.10H.sub.16.HBr, H-(D)Phe-Pro-boroArg-C.sub.10H.sub.16.HCl, Boc-(D)Phe-Phe-boroOrn-C.sub.10H.sub.16.BSA, Boc-(D)Phe-Phe-boroArg-C.sub.10H.sub.16.BSA, Ac-Ala-Lys(Boc)-boroArg-C.sub.10H.sub.16.BSA, Ac-(D)Phe-Pro-boroArg-C.sub.10H.sub.16.HCl, Ac-(D)Phe-Pro-boroArg-OH.HCl, Boc-Leu-Gly-Leu-Ala-boroIrg-C.sub.10H.sub.16.HBr, Boc-Leu-Gly-Leu-Ala-boroOrn-C.sub.10H.sub.16.BSA, Boc-Leu-Gly-Leu-Ala-boroArg-C.sub.10H.sub.16.BSA, Bz-Pro-Phe-boroOrn-C.sub.10H.sub.16.BSA, Bz-Pro-Phe-boroArg-C.sub.10H.sub.16.BSA, Boc-Ala-Phe-(D,L)boroIrg-C.sub.10H.sub.12.HBr, Bz-Glu(OBu)-Gly-boroIrg-C.sub.10H.sub.16.HBr, Bz-Glu-Gly-boroArg-C.sub.10H.sub.16.BSA, Bz-Glu(OBu)-Gly-boroOrg-C.sub.10H.sub.16.BSA, Bz-Glu(OBu)-Gly-boroArg-C.sub.10H.sub.16.BSA, Bz-Pro-Phe-boroIrg-C.sub.10H.sub.16.HBr, Z-Phe-Gly-Gly-boroIrg-C.sub.10H.sub.16.HBr, Boc-Ala-Phe-(D,L)borohomoIrg-C.sub.10H.sub.12.HBr, Bz-Pro-Phe-boroArg-OH.HCl, Bz-Pro-Phe-boroArg-F, H-(D)Phe-Pro-boroArg-C.sub.10H.sub.16.2HCl, H-(D)Phe-Phe-boroArg-C.sub.10H.sub.16.2HCl, Ac-Ala-Lys-boroArg-C.sub.10H.sub.16.2HCl, H-Leu-Gly-Leu-Ala-boroArg-C.sub.10H.sub.16.HCl. BSA, Boc-Ala-Phe-(D,L)boroLys-C.sub.10H.sub.12.HCl, H-Ala-Phe-(D,L)boroLys-C.sub.10H.sub.12.2HCl, Boc-(D)Val-Leu-boroLys-C.sub.10H.sub.12.HCl, Ac-Phe-boroLys-C.sub.10H.sub.12.HCl, Bz-Glu-Gly-boroArg-C.sub.10H.sub.16.BSA, H-(D)Phe-Phe-boroIrg-C.sub.10H.sub.16.2HBr, H-Leu-Gly-Leu-Ala-boroIrg-C.sub.10H.sub.16.2HBr, H-Ala-Phe-(D,L)boroIrg-C.sub.10H.sub.12.2HBr, Bz-Glu-Gly-boroIrg-C.sub.10H.sub.16.H Br, H-Ala-Phe-(D,L)boroHomoIrg-C.sub.10H.sub.12.2H Br, Ac-Ala-Lys-boroIrg-C.sub.10H.sub.16.2HBr, Bz-boroIrg-C.sub.10H.sub.12.HBr, Bz-boroOrn-C.sub.10H.sub.12.BSA, Bz-boroArg-C.sub.10H.sub.12.BSA, Ac-Leu-Thr(OBu)-boroOrn-C.sub.10H.sub.16.BSA, Ac-Leu-Thr(OBu)boroArg-C.sub.10H.sub.16.BSA, Ac-Leu-Thr-boroArg-C.sub.10H.sub.16.BSA, Ac-Lys(Boc)-Pro-boroOrn-C.sub.10H.sub.16.BSA, Ac-Lys(Boc)-Pro-boroArg-C.sub.10H.sub.16.BSA, Ac-Lys-Pro-boroArg-C.sub.10H.sub.16.BSA, Ac-Ala-Glu(OBu)-boroOrn-C.sub.10H.sub.16.BSA, Ac-Ala-Glu(OBu)-boroArg-C.sub.10H.sub.16.BSA, Ac-Ala-Glu-boroArg-C.sub.10H.sub.16.BSA, Boc-Val-Val-boroLys-C.sub.10H.sub.12.BSA, H-Val-Val-boroLys-C.sub.10H.sub.12.BSA.TFA, Boc-(D)Phe-Phe-boroLys-C.sub.10H.sub.12.BSA, H-(D)Phe-Phe-boroLys-C.sub.10H.sub.12.BSA.TFA, Boc-Glu-Phe-boroLys-C.sub.10H.sub.12.BSA and PyroGlu-Phe-boroLys-C.sub.10H.sub.12.BSA, disclosed in U.S. Pat. No. 5,187,157; [0220] Ac-boroArg-OH.HCl, disclosed in Lebarbier et al.; (1998) Biorganic and Medicial Chemistry letters 8: 2573-2576;

[0221] Ac-(D)-Phe-Pro-boroArg-OH, disclosed in Quan et al.; (1997) Biorganic and Medicial Chemistry letters 7(13): 1595-1600; [0222] Ac-Ala-Lys-boroArg-OH.sub.2, disclosed in Holyoak et al. (2003); Biochemistry 42: 6709-6718. [0223] Pro-Phe-BoroArg-OH, disclosed in Stadnicki et al. (1998); The FASEB Journal 12: 325-333. [0224] Bz-Nle-Lys-Lys-boroArg-OH.sub.2, disclosed in Yien et al. (2006); Biorganic and Medicial Chemistry letters 16: 36-39. [0225] H-Phe-Pro-BoroArg, disclosed in Kettner et al. (1990); The Journal of Biological Chemistry 265(30): 18289-18297. [0226] Ac-Arg-Glu-Lys-boroArg pinanediol, disclosed in Komiyama et al. (2005); to Antimicrobial Agents and Chemotherapy 49(9): 3875-3882. [0227] (BOC)-Ala-Val-Lys-boronate, disclosed in Katz et al. (1995); Biochemistry 34: 8264-8280.

[0228] The molecules of the invention are synthesized using well-know processes. As an example, FIG. 7 discloses various processes to synthesize any compound of the present invention. The examples disclosed relates to the synthesis of a borolysine tripeptide (7A), a bororarginine tripeptide and a boroornithine tripeptide (7B to 7E). These examples may be easily applied to synthesize other compounds disclosed herein. Especially, the person skilled in the art may replace the nature and number of amino acid residues to link to the boroarginine, borolysine or boroornithine moiety.

[0229] Besides their structural feature, the compounds of the invention, and especially the tri or tetraboropeptides, have the capacity to specifically inhibit the activity of serine proteases and especially the activity of trypsin-like subtype serine proteases. In another embodiment, the compounds of the invention, and especially the tri or tetraboropeptides, have the capacity to selectively inhibit, the activity of enteropeptidase, especially mammalian enteropeptidases, and more especially human enteropeptidase. The sequence of the human enteropeptidase is disclosed in FIG. 6.

[0230] In a second embodiment, the invention relates to a composition comprising one or more than one compound of the invention, especially 2, 3 or 4 compounds.

[0231] Such composition can take the form of a pharmaceutical composition which can be formulated using pharmaceutically acceptable carriers well known in the art, in suitable dosages.

[0232] In a particular embodiment, the composition further comprises a pharmaceutically suitable excipient or carrier and/or vehicle, when used for enteral or oral administration.

[0233] Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like.

[0234] Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize, wheat, rice, or potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0235] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0236] Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

[0237] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0238] In addition to the active ingredients, these pharmaceutical compositions may also contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

[0239] In an embodiment of the invention, the compounds of the present invention may be administered in combination with at least one other drug, to achieve enhanced effects, for example with other drugs targeting enzymes different from the enteropeptidase. As an example, the compounds of the invention may be combined with drug(s) able to inhibit the adsorption or the metabolism of triglycerides.

[0240] In a third embodiment, the present invention relates to the use of any compound as defined above, and particularly any compound of the formula I to VI for the treatment of obesity, excess weight and diseases associated with an abnormal fat metabolism. Therefore, the compounds of the invention, particularly the compounds of the formula I to VI for use as drug, particularly for use in the treatment of obesity, excess weight and/or diseases associated with an abnormal fat metabolism, are part of the invention, as well as the use of a compound or a composition of the invention for the manufacture of a drug to treat obesity, excess weight and/or diseases associated with an abnormal fat metabolism.

[0241] The invention is also directed to a method to treat a mammal having obesity, having excess weight and/or suffering from diseases associated with an abnormal fat metabolism, comprising administrating at least one compound or a composition of the invention to a mammal in need thereof.

[0242] In an embodiment, any compound of the invention may be used to decrease the in vivo absorption of proteins. In another embodiment, any compound of the invention may be used to decrease the in vivo absorption of triglycerides. In a further embodiment, any compound of the invention may be used to decrease the in vivo absorption of proteins and the in vivo absorption of triglycerides.

[0243] In a further aspect, independently or in combination with the use of the compounds of the invention in absorption decrease, any compound of the invention may be used to decrease the food intake, i.e.; to decrease the appetite (appetite blocker).

[0244] The compound or the composition of the invention may be used in dosage ranging from 10 mg to 10 g per day, or from 100 mg to 1 g per day, one or several times daily. The amount of compound(s) or the composition of the present invention may be administered in dosages according to the severity of the obesity, the amount of excess weight, the age of the mammal and/or the general health of the mammal.

[0245] The compounds or compositions of the invention are suitable for treating various forms of obesity and in particular obesity resulting from environmental causes (excessive nutrient intake and/or a sedentary lifestyle), resulting from genetic alterations (such as FTO gene), resulting from medical illness (such as hypothyroidism, Cushing's syndrome, growth hormone deficiency), resulting from smoking cessation, resulting from medications (such as steroids, atypical antipsychotics or some fertility medication), and resulting from neurological disorders.

[0246] The treatment by at least one the compounds or by the compositions of the invention of diseases associated with an abnormal fat metabolism is also contemplated. Such diseases are the following: gout disease (metabolic arthritis), type II diabetes, hyperglycemia, hyperinsulinemia, elevated blood levels of fatty acids or glycerol, syndrome X, diabetic complications, dysmetabolic syndrome and related diseases, hypercholesterolemia, atherosclerosis, hypertension, pancreatitis, hypertriglyceridemia, hyperlipidemia, stroke, coronary heart diseases, peripheral vascular diseases, peripheral arterial diseases, vascular syndromes, cholesterol-related disorders (e.g., LDL-pattern B and LDL-pattern L) and drug-induced lipodystrophy.

[0247] The invention also concerns an animal model in which the enteropeptidase gene has been inactivated. This model encompasses a mammal model (non-human) or a murine model (such as a mouse). By "inactivated", is meant that an enteropeptidase gene incorporated in this model encodes a protein having less than 50% enteropeptidase activity as compared to the wild type enteropeptidase. In one embodiment, the inactivated enteropeptidase has less than 40% enteropeptidase activity as compared to the wild type enteropeptidase. In another embodiment, the enteropeptidase of the animal model has less than 30% enteropeptidase activity as compared to the wild type enteropeptidase. In yet another embodiment, the enteropeptidase of the animal model has less than 20% or less than 10% enteropeptidase activity as compared to the wild type enteropeptidase. In still another embodiment, the inactivated enteropeptidase has no peptidase activity at all. In another aspect, the percentage of enteropeptidase activity is defined according to the conversion of trypsinogen into trypsin.

[0248] The animal model having an inactivated enteropeptidase gene may be obtained by any conventional techniques known to the person skilled in the art to obtain a knockout (KO) animal model, such as by inserting one or more amino acid substitution(s) affecting the peptidase activity of the enteropeptidase or by deleting one or several exons of the enteropeptidase gene. When the animal model is a rodent, such as a mouse, mutations may be, for example, a deletion of exons 23-28 of the prss7 gene i.e., the murine counterpart of human enteropeptidase.

[0249] The invention is also directed to the use of this animal model in drug development, for example in the screening of drugs or molecules having effects on weight. Therefore, the model can be used in a process to determine the effect of a drug or a molecule on weight comprising (a) administrating a drug to the model and (b) measuring the weight of the animal. The effect of the drug is determined by comparing the weight of the animal administered with said drug with control animals (which are administered with known molecules or with a placebo).

[0250] The expression "effect on weight" means that the drug or molecule is able to increase the weight of the animal model as compared to a placebo, or in contrast to decrease the weight of the animal model as compared to a placebo. The effect of the molecule may be observed under a low, normal or high caloric regimen.

[0251] The administration of the drug or the molecule to the animal model may be carried out orally intravenously, intraperitoneally, intramuscularly, intraarterially or by sustained release systems.

[0252] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention.

EXAMPLES

A. In Vitro Experiments

[0253] 1. Materials

[0254] 1.1. Buffer: [0255] TN: Tris 50 mM pH=7.5 NaCl 150 mM (Tris: Euromedex Ref 26-128-3097; NaCl: Euromedex Ref 1112); [0256] TCN: Tris 50 mM pH=7.5 NaCl 150 mM CaCl.sub.2 10 mM; [0257] TCNB: Tris 50 mM pH=7.5 NaCl 150 mM, CaCl.sub.2 10 mM, 0.05% Brij 35 (Brij: sigma Ref B4184); [0258] Tris 25 mM pH 8; [0259] Pancreatic lipase Buffer: 25 mM Tris pH 9.2-0.1 mM CaCl.sub.2-20 mM sodium; [0260] Deoxycholate; [0261] Thermolysine (Sigma, ref T7902); [0262] Phosphoramidon disodium salt (Sigma, ref R7385); [0263] Acetic Acid (Sigma ref A0808)

[0264] 1.2. Plates [0265] Microplate 384 small volume, clear (Greiner, ref 784101) [0266] Microplate 384 flat bottom black (Corning, ref 3573) [0267] Plate 96 black Nunc (VWR, ref 13634.01) [0268] Plate 96-1/2 area (Corning, Ref 3695) [0269] Microplate 96 well 800 .mu.l unifilter (Whatman, Ref 7700-1804)

[0270] 1.3. Compounds

[0271] Table I below lists four compounds of the invention (triboropeptides) and their names.

TABLE-US-00002 TABLE I Name Compound/Composition Protection Obe 1999 Ala-Phe-BoroArg 10 mM DMSO No Obe 2000 Glu-Gly-BoroArg 10 mM DMSO No Obe 2001 Acetyl-Ala-Phe-BoroArg 10 mM DMSO Yes (Acetyl on the first Ala) Obe 2002 Acetyl-Glu-Gly-BoroArg 10 mM DMSO Yes (Acetyl of the first Glu)

[0272] 1.4. Enzymes:

[0273] Table II below lists the enzymes tested for inhibition by the compounds of the invention, as well as the suppliers and the associated commercial references.

TABLE-US-00003 TABLE II Enzyme Supplier, reference Recombinant human enteropeptidase RD system, ref 1585SE Trypsin from human pancreas SIGMA, ref T6424 Thrombin from human plasma SIGMA, ref T1063 Kallikrein from human plasma SIGMA, ref K2638 Plasmin from human plasma SIGMA, ref P1867 Elastase Calbiochem, ref 324682 Chymotrypsin Sigma, ref C8946 DPPIV (Dipeptidyl peptidase IV) RD System, ref 1180-SE Recombinant human Carboxypeptidase A1 RD System, ref 2856-ZN Recombinant human Carboxypeptidase B1 RD System, ref 2897-ZN Alpha Amylase from human pancreas Sigma, ref A9972 Lipase Sigma, ref L0382

[0274] 1.5. Substrates:

[0275] Table III below lists the substrates used for testing the inhibition of the above enzymes by the compounds of the invention, as well as the suppliers and the associated commercial references.

TABLE-US-00004 TABLE III Corresponding Substrate Supplier, reference enzyme N-p-Tosyl-Gly-Pro-Arg- SIGMA, ref T1637 Enteropeptidase, pNa Trypsin and Thrombin H-D-Pro-Phe-Arg-pNa Chromogenix, ref S-2302 Kallikrein D-Ile-Phe-Lys-pNa SIGMA, ref I6886 Plasmin N-Succinyl-Ala-Ala-Pro- Sigma, ref S7388 Chymotrypsin Phe-pNA Suc-Ala-Ala-Pro-Abu-pNA Calbiochem, ref 324699 Elastase Ala-Pro-7-amido-4- Calbiochem, ref 125510 DPPIV trifluoromethylcoumarin N-(4-Methoxyphenylazo- Bachem, ref M2245 Carboxy- formyl)-Phe-OH potassium peptidase A1 salt N-(4-Methoxyphenylazo- Bachem, ref M2525 Carboxy- formyl)-Arg-OH HCl peptidase B1 6,8-difluoro-methylum- InVitrogen, ref D12200 Lipase belliferyl octanoate (DIFMu) Starch Azur Sigma, ref S7629 Alpha Amylase

[0276] 2. Protocols

[0277] 2.1. Enteropeptidase Assay

[0278] Activation of the Enteropeptidase

[0279] A mix of enteropeptidase at 58.8 nM (final) and thermolysine at 1.58 ng/l (final) in TCN buffer was prepared; the mix was incubated at 37.degree. C. during 30 minutes for activation. Phosphoramidon (10 .mu.M final) was added to stop the activation by thermolysine.

[0280] Measurement of Enteropeptidase Activity without Inhibitor (Positive Control)

[0281] In 17 .mu.l of TCN, 1 .mu.l of active enzyme (2.9 nm final) and 2 .mu.l Np Tosyl Gly Pro Arg pNa (1 mM final) were mixed, just before reading. The Absorbance was measured at 405 nm on EnVision (Perkin Elmer).

[0282] Inhibition in 384 Well Plate Small Volume (20 .mu.l Final)

[0283] In 15 .mu.l of TCN, 1 .mu.l of active enzyme and 2 .mu.l of Inhibitor (compound of the invention) at different concentrations were mixed, and incubated at RT (room temperature) for 30 minutes; 2 .mu.l Np Tosyl Gly Pro Arg pNa (1 mM final) was added just before reading. The Absorbance was measured at 405 nm on EnVision (Perkin Elmer).

[0284] 2.2. Thrombin, Trypsin, Kallikrein, Plasmin, Chymotrypsin and Elastase Assay

[0285] Table IV below lists the final concentrations of the tested enzymes and the corresponding substrates, in the determination of the IC.sub.50 without inhibitor (positive control) and in inhibition protocols:

TABLE-US-00005 TABLE IV Enzyme Susbstrate Trypsin 10 nM N-p-Tosyl-Gly-Pro-Arg-pNa 1 mM Thrombin 10 nM N-p-Tosyl-Gly-Pro-Arg-pNa 0.75 mM Plasmin 50 nM D-Ile-Phe-Lys-pNa 1 mM Kallikrein 10 nM H-D-Pro-Phe-Arg-pNa 1 mM Chymotrypsin 50 nM N-Succinyl-Ala-Ala-Pro-Phe-pNA 0.5 mM Elastase 25 nM Suc-Ala-Ala-Pro-Abu-pNA 0.5 mM

[0286] Measurement without Inhibitor (Positive Control)

[0287] In 17 .mu.l of TN, 1 .mu.l of enzyme and 2 .mu.l of substrate was mixed, just before reading; The Absorbance was measured at 405 nm on EnVision (Perkin Elmer).

[0288] Inhibition in 384 Well Plate Small Volume (20 .mu.l Final)

[0289] In 15 .mu.l of TN, 1 .mu.l of enzyme and 2 .mu.l of Inhibitor (compound of the invention) at different concentrations were mixed and incubated at RT for 30 minutes; 2 .mu.l of substrate was added, just before reading; The Absorbance was measured at 405 nm on EnVision (Perkin Elmer).

[0290] 2.3 Carboxypeptidase A1 and B1 Assay

[0291] Activation of Carboxypeptidase A1

[0292] In 20 .mu.l of TCNB, Carboxypeptidase A1 (100 .mu.g/ml final) and trypsin (1 .mu.g/ml final) were mixed and incubated at RT for 2 hours.

[0293] Activation of Carboxypeptidase B1

[0294] In 20 .mu.l of TCNB, Carboxypeptidase B1 (100 .mu.g/ml final) and trypsin (1 .mu.g/ml final) were mixed and incubated at RT for 1 hour.

[0295] Table V below lists the final concentrations of the tested enzymes and the corresponding substrate, in the determination of the IC.sub.50 without inhibitor (positive control) and in inhibition protocols:

TABLE-US-00006 TABLE V Enzyme Susbstrate Carboxypeptidase A1, 20 nM N-(4-Methoxyphenylazoformyl)-Phe-OH, potassium salt 100 .mu.M Carboxypeptidase B1, 20 nM N-(4-Methoxyphenylazoformyl)-Arg-OH, HCl 100 .mu.M

[0296] Measurement without Inhibitor (Positive Control)

[0297] In 17 .mu.l of TN, 1 .mu.l of activated enzyme and 2 .mu.l substrate were mixed, just before reading; The Absorbance was measured at 355 nm on EnVision (Perkin Elmer).

[0298] Inhibition in 384 Well Plate Small Volume (20 .mu.l Final)

[0299] In 15 .mu.l of TN, 1 .mu.l of activated enzyme and 2 .mu.l of Inhibitor (compounds of the invention) at different concentrations were mixed, and incubated at RT for 30 minutes; 2 .mu.l of substrate was added, just before reading; The Absorbance was measured at 355 nm on EnVision (Perkin Elmer).

[0300] 2.4. DPPIV Assay

[0301] Measurement without Inhibitor (Positive Control)

[0302] In 17 .mu.l of Tris 25 mM pH 8, 1 .mu.l of enzyme (1 nM final) and 2 .mu.l of substrate (Ala-Pro-7-amido-4-trifluoromethylcoumarin, at 100 .mu.M final) were mixed, just before reading. The Absorbance was measured on EnVision (Perkin Elmer) (Excitation 400 nm/Emission 505 nm).

[0303] Inhibition in 384 Well Plate Black (20 .mu.l Final)

[0304] In 15 .mu.l of Tris 25 mM pH 8, 1 .mu.l of enzyme (1 nM final) and 2 .mu.l of Inhibitor (compounds of the invention) at different concentrations were mixed, and incubated at RT for 30 minutes. 2 .mu.l of substrate (Ala-Pro-7-amido-4-trifluoromethylcoumarin, at 100 .mu.M final) was added, just before reading. The absorbance was measured on EnVision (Perkin Elmer) (Excitation 400 nm/Emission 505 nm).

[0305] 2.5. Pancreatic Amylase Assay

[0306] Measurement without Inhibitor (Positive Control)

[0307] In 50 .mu.l, 5 .mu.l Amylase (at a starting concentration of 0.25 mg/ml) and 45 .mu.l of Starch Azur (at a starting concentration of 2% in 20 mM NaH2PO4/50 mM Na Cl pH 7) were mixed and incubated 1 h at 37.degree. C. with shaking. 20 .mu.l Acetic Acid Solution (starting concentration of 2.75 M) was added, and the suspension filtered. The absorbance was measured in microplate 96 well unifilter Whatman at 595 nm.

[0308] Inhibition in 96 Well Plate Black

[0309] In 35.5 .mu.l buffer (NaH.sub.2PO.sub.4/50 mM NaCl pH 7-37.degree. C.), 5 .mu.l of amylase (0.25 mg/ml) and 5 .mu.l of Inhibitor (compounds of the invention) at different concentrations were mixed and incubated 30 minutes at RT. 4.5 .mu.l of substrate (Starch Azur 20% in buffer) were added and incubated 1 hour at 37.degree. C. under shaking. Then, 20 .mu.l of Acetic Acid Solution (2.75 M) were added and the suspension was filtered in a microplate 96 well unifilter Whatman. Absorbance was read at 595 nm on EnVision (PerkinElmer).

[0310] 2.6. Lipase Assay

[0311] Measurement without Inhibitor (Positive Control)

[0312] In 85 .mu.l of lipase buffer, 5 .mu.l of lipase (56 U/ml final) and 10 .mu.l of substrate (DiFMu at 10 .mu.M final) were mixed, just before reading. The florescence was measured on EnVision (Perkin Elmer) (Excitation 358 nm/Emission 452 nm).

[0313] Inhibition in 96 Well Plate Black (20 .mu.l Final)

[0314] In 80 .mu.l of lipase buffer, 5 .mu.l of lipase (56 U/ml final) and 5 .mu.l of Inhibitor (compounds of the invention) at different concentrations were mixed, and incubated at RT for 30 minutes. Add 10 .mu.l of substrate (DiFMu at 10 .mu.M final) just before reading. The fluorescence was measured on EnVision (Perkin Elmer) (Excitation 358 nm/Emission 452 nm).

[0315] 3. Results

[0316] As shown in FIGS. 1 to 4, the 4 compounds (OBE1999 to OBE2000), belonging to the boropeptide family, are particularly efficient in vitro against enteropeptidase and inhibit the activity of enteropeptidase, with a high IC50 inhibition constant, at a nanomolar range from 7 to 63 nM. Moreover, these four compounds are specific of serine proteases, sub-type trypsin-like, and in contrast do not inhibit chymotrypsin-like serine proteases, metallo-proteases or glucosidases (FIG. 5).

[0317] More specifically, OBE1999 is a very good inhibitor of enteropeptidase (IC50 of 33 nM). However, it also inhibits with a good IC50 (7.5 to 29.8 nM) other enzymes such as trypsin, kallikrein and plasmin. Since the enteropeptidase is specifically located in the luminal intestine and this compound is non-absorbable, this compound is an excellent molecule to selectively inhibit the enteropeptidase, and is thus not expected to inhibit in vivo other serine protease (whose location is non-intestinal). As far OBE2001 is concerned, this compound shares the same profile as the one of OBE 1999 and therefore fulfils the same specificity and selectivity criteria.

[0318] OBE2000 is more specific for enteropeptidase and trypsin than the compounds OBE1999 and OBE2001, with low value of IC50 for enteropeptidase, and high value of IC50 not only for thrombin but also for plasmin (7100 nM) and kalikrein (260 nM). OBE2002 presents the same profile as OBE2000, except for the low IC50 value against kallikrein and plasmin as compared to OBE2000.

[0319] Consequently, these results also clearly show that all tested compounds have a remarkable efficiency in the in vitro inhibition of enteropeptidase, and can be to considered as promising molecules for future in vivo experiments in animals and/or in humans. These compounds have been shown to fulfil at least two requirements necessary for treatment: the specificity of inhibition to trypsin-like subtype serine proteases, and the selectivity of the inhibition for the enteropeptidase.

B. In Vivo Experiments

[0320] 1. Effect of OBE2001 on Weight

[0321] The aim of this first in vivo experiment is to determine the anti-obesity effect of OBE2001 in a model of obesity induced by a hyperlipidic diet in mice.

[0322] OBE2001 (Acetyl-Ala-Phe-BoroArg) was provided under salt form (molecular weight of 568.3 g/mol). Swiss male mice, 4 weeks of age, were divided into 2 groups of 10 animals each, receiving the following:

TABLE-US-00007 Group number Diet Dosing 1 Hight fat, ad libitum Vehicle (water) 2 Hight fat, ad libitum OBE2001 (40 mg/kg/day)

[0323] Administration of OBE2001 was carried out as a solution in water. Both OBE2001 or vehicle (water used a negative control) were administrated to the animals daily by the oral route in an administration of 5 ml/kg body weight.

[0324] Concerning the feeding of the animals, before the experiments (acclimation period), normal diet (reference No. 824050 SDSIDIETEX food) was available ad libitum during the acclimation period. 7 days before the beginning of the study, a hypercaloric High Fat diet (45% proteins, 4.73 Kcal/g; reference 824053 SDSIDIETEX) was given to groups 1 and 2.

[0325] Animals were weighed daily from at D-8 (8 days before the beginning of the experiment) then from D-5 to the end of the study. From D1 (first day of the experiment), food was given ad libitum.

[0326] Results

[0327] The initial body weight (in g) of mice of groups 1 and 2 at D-1 (one day prior to the experiment) as well as the gain or loss of weight according to the initial weight (for D1 to D13) are summarized in the following table and represented on FIG. 8.

TABLE-US-00008 Group D - 1 D 1 D 2 D 3 D 4 D 5 D 6 D 7 1 Mean 28.3 -2.8 -0.3 1.0 2.3 2.7 3.2 3.9 SEM 0.7 0.2 0.1 0.2 0.3 0.4 0.4 0.5 2 Mean 28.5 -2.7 -1.2 -0.8 0.2 0.7 1.0 0.9 SEM 0.7 0.2 0.2 0.3 0.5 0.6 0.9 1.0 p NS NS NS NS * * * * Group D 8 D 9 D 10 D 11 D 12 D 13 1 Mean 4.4 4.7 5.1 5.4 5.9 6.3 SEM 0.6 0.7 0.7 0.8 0.8 0.8 2 Mean 1.3 1.0 1.1 0.5 1.8 2.0 SEM 1.3 1.3 1.4 1.5 1.0 1.1 p ** ** ** ** ** ** Mean: mean weight of the 10 mice per group; SEM: standard error of the mean; NS: non significant difference between the two groups (p > 0.05); * p .ltoreq. 0.05; ** p .ltoreq. 0.01

[0328] As shown in FIG. 8, this experiment showed that the weight of mice having received the OBE2001 compound is significantly lower than the weight of the control group mice. This difference is significant from day 4 and highly significant from day 8 (see Table). Consequently, the administration of OBE 2001 in mice results in the decrease of the weight as soon as day 4.

[0329] 2. Effect of OBE2001 on Triglyceride Absorption

[0330] The scope of this second in vivo experiment was to analyze the effects of the OBE2001 compound on triglycerides absorption. To that purpose, mice were injected with OBE2001, before and/or after gavage with a solution enriched with cholesterol and free fatty acids (clinoleic at 20%).

[0331] In this study, 7 week old Swiss CD1 male mice were weighed and randomized for body weight; 3 groups of 5 mice were then constituted of: [0332] Group 1 (solvent): control group. Vehicle (H.sub.2O+2% DMSO) at t-5 min+solution A (clinoleic 20%+cholesterol) at to. [0333] Group 2 (OBE 25+25): OBE2001 25 mg/kg (before and after gavage). OBE2001 25 mg/kg (solution B) at t-5 min+OBE2001 25 mg/kg in solution A (solution C) at t0. [0334] Group 3 (OBE 25): OBE2001 25 mg/kg (before gavage). Vehicle (H.sub.2O+2% DMSO) at t-5 min+OBE2001 25 mg/kg in solution A (solution C) at 0.

[0335] 30 minutes before treatment (with solution A, B or C) blood was collected by retro orbital puncture after a slight anesthesia (Isoflurane), to determine basal (t0) triglycerides levels. All solutions were then administered by oral gavage at t-5 (vehicle or solution B) or t0 (solution A or C). After administration of the solution A or C (containing fatty acids+cholesterol), blood was collected again by retro orbital puncture after a slight anesthesia (Isoflurane) at time 60, 120, 180, 300 and 360 minutes for measurement of triglycerides levels. At time 360 minutes, mice were sacrificed.

[0336] Results: The following values were obtained at different times after administration of the solution:

TABLE-US-00009 % of basal value Time (min) 0 60 120 180 300 360 Group 1 100.00 227.30 298.72 203.76 109.67 89.41 Group 2 100.00 243.39 202.34 127.81 62.35 56.71 Group 3 100.00 293.17 269.24 126.45 70.22 60.92

[0337] Oral gavage with a solution of clinoleic at 20% led to an increase in triglycerides blood levels in all study groups the three first hours. Mice treated with OBE2001 at 25 mg/kg in group 2 and 3 show a decrease in triglyceride absorption as compared to the control group 1 (FIG. 9).

[0338] Moreover, for each group, the area under the curve was calculated according to the values of the above Table.

TABLE-US-00010 Area under the curve % of decrease Group 1 65453 Group 2/group 1 26% Group 2 48559 Group 3/group 1 14% Group 3 56273

[0339] This calculation demonstrated that the overall triglycerides absorption is reduced by 26% in group 2 as compared to group 1, and of 14% in group 3 as compared to group 1. Consequently, these results demonstrate that OBE2001 is able to inhibit the enteropeptidase and reduces significantly the absorption of triglycerides in treated animals.

[0340] 3. Effect of OBE2001 on Protein Absorption

[0341] This third in vivo experiment was designed to obtain information on the absorption of .sup.14C-proteins in absence or in presence of OBE2001 at two different doses (5 mg/Kg or 50 mg/Kg) after an oral administration to male Swiss mice.

[0342] 15 male Swiss (CD-1) mice, 5 weeks-old and having a mean body weight of 0.021.+-.0.002 kg were divided in 3 groups: [0343] Control Group (G1; solvent) containing 5 mice, received water at 5 mL/Kg at t-5 min and .sup.14C-proteins at 10 mL/Kg at T=0 min; [0344] Group 2 (G2) containing 5 mice (OBE 5 mg/Kg), received OBE2001 at 5 mg/Kg at t-5 min and .sup.14C-proteins at 10 mL/Kg at T=0 min; [0345] Group 3 (G3) containing 5.sup.2 mice (OBE 50 mg/Kg), received OBE2001 at 50 mg/Kg at t-5 min and .sup.14C-proteins at 10 mL/Kg at T=0 min.

[0346] A preparation containing 5 ml of Clinoleic 20 (Baxter Ref DDB9500) with 1 g of glucose (Sigma; ref:G8270) and 1 g of casein (Sigma; ref: C3400) was prepared. Water was added until the solution reached 10 ml. After concentration of the radioactive solution containing the .sup.14C proteins (GE Healthcare; ref: CFA626) (.times.10), 200 .mu.l of this radioactive solution was added to 2.8 ml of the preparation, in order to reach a concentration of ca 3.3 .mu.Ci/ml. The animals were starved before administration of this radioactive preparation. The radioactive preparation was orally administered by intragastric gavage with a single dosing (ca 33.3 .mu.Ci/kg) in a volume of ca 10 ml/kg of body weight.

[0347] OBE2001 was provided in a mixture of two salts (HCl and TFA) with a 90% purity. OBE2001 was orally administered by intragastric gavage with a single dosing of 5 mg/Kg for G2 and 50 mg/Kg for G3 in a volume of ca 5 mL/kg body-weight.

[0348] Plasma aliquots were removed and placed into 6 ml pre-weighed plastic vials, at t+15, t+30, t+60, t+120 and t+240 min. About 4 ml Pico-fluor 40 was added and after shaking, the total radioactivity contained within the different samples was determined by liquid scintillation using a Packard 1900 CA spectrometer equipped with an external standard system (spectral analysis). A quenching curve for calibration purposes was set up using a .sup.14C quenched set supplied by Packard Instruments.

[0349] Liquid scintillation (LS) Counting was carried out with 2 sigma=2%, and for a maximum duration of 5 minutes (according to the method created in the 1900CA.RTM. software). Dpm (disintegration per minute) values of less than twice the background level of blank biological medium were reported as BLQ (Below the Limit of Quantitation).

[0350] Results:

[0351] During this animal experiment, no sign of morbidity or mortality occurred, and no particular effect related to dosage regimen was observed.

[0352] A Cmax of 0.9%.+-.0.27 administered dose/g occurring at 15 min was observed for group 1 (control), while the Cmax for the treated groups tended to be lower. Indeed, for treated groups (5 mg/kg and 50 mg/kg), the Cmax were 0.58%.+-.0.54, and 0.54%.+-.0.24 of administered dose/g respectively. The kinetic data tended to a plateau at concentrations from 1 h to 4 h post-administration reaching respectively 0.5% administered dose/g for group 1 and 0.4% administered dose/g for groups 2 and 3 (FIG. 10).

[0353] The AUC mean for group 1 was 2.214.+-.0.1875% dose.h.g-1. AUC mean for two others groups was respectively 1.841.+-.0.6402% dose.h.g-1 and 1.718.+-.0.4366% dose.h.g-1.

[0354] This result demonstrates that protein absorption is greatly decreased in mice to having received OBE2001 as compared to control mice, in the first 30 minutes following protein administration, and that this decrease is maintained during at least 4 hours.

C. Generation of a Constitutive Knock Out (KO) Mice for Enteropeptidase

[0355] In order to check that enteropeptidase is a correct target protein in counteracting obesity, a knockout (KO) mouse mimicking the enteropeptidase deficiency has been produced to first validate the target and for use as an animal model for drug development.

[0356] The generation of Constitutive Knock Out mice consists of the following steps:

[0357] 1. Targeting Vector Design and Construction;

[0358] 2. Targeted C57BL/6 embryonic stem (ES) cells;

[0359] 3. Generation of heterozygous mice for the Constitutive Knock Out;

[0360] 4. Generation of homozygous mice for the Constitutive Knock Out.

[0361] 1. Targeting Vector Design and Construction

[0362] The murine counterpart of human enteropeptidase is called Prss7 and is located on mouse chromosome 16.

[0363] In order to inactivate the enteropeptidase gene, exons 23-28, which encode the catalytic domain, have been replaced by an FRT-flanked NeoR cassette. The constitutive KO allele (exons 1-22) encodes a C-terminally truncated protein which lacks the catalytic domain. The NeoR cassette has been excised through in vivo Flp-mediated recombination. Interference with the expression of the tail-to-tail positioned gene (Chodl) through this modification of the Prss7 gene cannot be excluded completely. The replacement of exons 23-28 (approximately 15 kb) with a FRT-Neo cassette (approximately 2 kb) decreases the efficiency of homologous recombination. A review of the KO allele construction is disclosed on FIG. 11.

[0364] This targeting strategy allowed the construction of a vector comprising a constitutive enteropeptidase KO allele (sequence and scheme depicted in FIG. 12).

[0365] 2. Targeted C57BL/6 Embryonic Stem (ES) Cells

[0366] In a second step, C57BL/6N embryonic stem (ES) cells were transfected by the vector comprising the constitutive enteropeptidase KO allele by electroporation. The transformed cells were selected for their resistance to G418 and gancyclovir. Up to 251 individual ES clones were picked and 2 positive ES clones were obtained (A-D2 and A-E8). In this step, all ES Cells (ESCs) were cultured on multi-drug resistant embryonic feeder cells without antibiotics and were regularly monitored by PCR and luminometric assays for absence of contaminating bacterias and mycoplasma. Quality control (QC) also included karyotyping (chromosome count) of all expanded ES clones and lines (for an example of a publication reporting the generation of KO mice, see Roberds et al. Human molecular Genetics, 2001, Vol 10, No 12, 1317-1324).

[0367] Before being injected into blastocysts, ES cell clones were extensively validated by Southern Blot Analysis with various probes. Therefore, genomic DNA from WT cells and A-D2 or A-E8 clones was digested with AfIII, run on a gel and the transfer membrane hybridized with one of these probes, probe 5e2 (sequence: GCCGCACTATTTGCAGCATG) (FIG. 13). The deletion of exons 23-28 of Prss7 resulted in an AfIII fragment of 11.7 kb (from position 5373 to position 17180) instead of the wild type (WT) allele of 18.8 kb (from position 5373 to position 24211).

[0368] As shown in FIG. 14, AD-2 and A-E8 clones have a wild type allele (18.8 kb) as well as a KO allele (11.7 kb). In contrast, wild type (WT) cells have two WT (non deleted) alleles (18.8 kb).

[0369] These results confirm that the AD-2 and A-E8 clones have successfully incorporated the KO allele.

[0370] 3. Generation of Heterozygous Mice for the Constitutive Knock Out

[0371] In a third step, 2 ES cell clones were injected into diploid host blastocysts, and the injected blastocyst transferred in pseudopregnant recipients (microoperation under specific pathogen free (SPF) conditions). Therefore, heterozygote (>50%) coat color chimeras were generated and breeded to heterozygosity.

[0372] To control the success of this third step, offspring were genotyped by the following PCR protocol. Genomic DNA, extracted from mouse tail, was amplified simultaneously with a first set of primers (primer 1472.sub.--23 with sequence: CGTTACTATCCATCACCTAAGC and primer 1472.sub.--24 with sequence GGGAATTCAGCTGTGTCTGAAC) corresponding to the enteropeptidase KO allele, and a second set of primers (primer 1260.sub.--1: GAGACTCTGGCTACTCATCC and primer 1260.sub.--2: CCTTCAGCAAGAGCTGGGGAC) corresponding to an internal control. The size of the enteropeptidase KO allele was 412 bp (cony), and the size of the internal control was 585 bp (c).

[0373] As expected, the amplification of genomic DNA from WT mice (115696 and 115705) gave a unique band at 585 bp. In contrast, the amplification of genomic DNA from heterozygous mice (115695, 115702, 115706, 115707 and 115708) gave two bands, one for the control allele (585 bp) and one for the KO allele (412 bp) (FIG. 15).

[0374] 4. Generation of Homozygote Pups for Enteropeptidase

[0375] In a last step, homozygote pups for enteropeptidase KO were generated. Enteropeptidase KO heterozygotes were crossed and 46 pups were obtained. Among the newborns, 30% (14 pups) were homozygote for the enteropeptidase KO, 50% were heterozygotes (22 pups) and 20% were wild type (homozygote dominant for the enteropeptidase gene).

[0376] The presence of the KO enteropeptidase in a homozygous strain was checked by PCR analysis, from genomic DNA, extracted from mouse tail. Genomic DNA was simultaneously amplified with a first set of primers (primer 1472.sub.--23 with sequence: CGTTACTATCCATCACCTAAGC and primer PRRS7 with sequence ATCAAGGAATCTTGGGAGCA) corresponding to the WT enteropeptidase allele, and a second set of primers (actin forward primer: CGGAACCGCYCATTGGC and actin backward primer: ACCCACACTGTGCCCATCTA) corresponding to the actin control. The size of the enteropeptidase allele was 533 bp, and the size of the actin control was 300 bp.

[0377] As can be seen from FIG. 16, the amplified fragments from heterozygous mice, in lanes named 115755 and 115648, present two bands, one corresponding to the actin control (300 bp) and one to the WT enteropeptidase allele (533 bp). In contrast, fragments amplified from newborns 1A, 2A, 3A, 4A and 5A has a size of 300 bp and thus correspond to the sole actin control, demonstrating that they lack a WT enteropeptidase allele.

[0378] 5. Phenotypic Observations

[0379] Newborns, obtained from the crossing of enteropeptidase knockout (KO) heterozygotes, were grown during 7 days and their size compared.

[0380] As can be shown in FIG. 17, the mouse homozygous for the enteropeptidase KO (Ho) is twice smaller than the mouse heterozygous for the enteropeptidase KO (He).

[0381] These results demonstrated that enteropeptidase is a good target to fight obesity, since its complete specific inhibition results in a significant size decrease.

Sequence CWU 1

1

1113696DNAArtificial SequenceHuman enteropeptidase 1accagacagt tcttaaatta gcaagccttc aaaaccaaaa atg ggg tcg aaa aga 55 Met Gly Ser Lys Arg 1 5ggc ata tct tct agg cat cat tct ctc agc tcc tat gaa atc atg ttt 103Gly Ile Ser Ser Arg His His Ser Leu Ser Ser Tyr Glu Ile Met Phe 10 15 20gca gct ctc ttt gcc ata ttg gta gtg ctc tgt gct gga tta att gca 151Ala Ala Leu Phe Ala Ile Leu Val Val Leu Cys Ala Gly Leu Ile Ala 25 30 35gta tcc tgc ctg aca atc aag gaa tcc caa cga ggt gca gca ctt gga 199Val Ser Cys Leu Thr Ile Lys Glu Ser Gln Arg Gly Ala Ala Leu Gly 40 45 50cag agt cat gaa gcc aga gcg aca ttt aaa ata aca tcc gga gtt aca 247Gln Ser His Glu Ala Arg Ala Thr Phe Lys Ile Thr Ser Gly Val Thr 55 60 65tat aat cct aat ttg caa gac aaa ctc tca gtg gat ttc aaa gtt ctt 295Tyr Asn Pro Asn Leu Gln Asp Lys Leu Ser Val Asp Phe Lys Val Leu70 75 80 85gct ttt gac ctt cag caa atg ata gat gag atc ttt cta tca agc aat 343Ala Phe Asp Leu Gln Gln Met Ile Asp Glu Ile Phe Leu Ser Ser Asn 90 95 100ctg aag aat gaa tat aag aac tca aga gtt tta caa ttt gaa aat ggc 391Leu Lys Asn Glu Tyr Lys Asn Ser Arg Val Leu Gln Phe Glu Asn Gly 105 110 115agc att ata gtc gta ttt gac ctt ttc ttt gcc cag tgg gtg tca gat 439Ser Ile Ile Val Val Phe Asp Leu Phe Phe Ala Gln Trp Val Ser Asp 120 125 130caa aat gta aaa gaa gaa ctg att caa ggc ctt gaa gca aat aaa tcc 487Gln Asn Val Lys Glu Glu Leu Ile Gln Gly Leu Glu Ala Asn Lys Ser 135 140 145agc caa ctg gtc act ttc cat att gat ttg aac agc gtt gat atc cta 535Ser Gln Leu Val Thr Phe His Ile Asp Leu Asn Ser Val Asp Ile Leu150 155 160 165gac aag cta aca acc acc agt cat ctg gca act cca gga aat gtc tca 583Asp Lys Leu Thr Thr Thr Ser His Leu Ala Thr Pro Gly Asn Val Ser 170 175 180ata gag tgc ctg cct ggt tca agt cct tgt act gat gct cta acg tgt 631Ile Glu Cys Leu Pro Gly Ser Ser Pro Cys Thr Asp Ala Leu Thr Cys 185 190 195ata aaa gct gat tta ttt tgt gat gga gaa gta aac tgt cca gat ggt 679Ile Lys Ala Asp Leu Phe Cys Asp Gly Glu Val Asn Cys Pro Asp Gly 200 205 210tct gac gaa gac aat aaa atg tgt gcc aca gtt tgt gat gga aga ttt 727Ser Asp Glu Asp Asn Lys Met Cys Ala Thr Val Cys Asp Gly Arg Phe 215 220 225ttg tta act gga tca tct ggg tct ttc cag gct act cat tat cca aaa 775Leu Leu Thr Gly Ser Ser Gly Ser Phe Gln Ala Thr His Tyr Pro Lys230 235 240 245cct tct gaa aca agt gtt gtc tgc cag tgg atc ata cgt gta aac caa 823Pro Ser Glu Thr Ser Val Val Cys Gln Trp Ile Ile Arg Val Asn Gln 250 255 260gga ctt tcc att aaa ctg agc ttc gat gat ttt aat aca tat tat aca 871Gly Leu Ser Ile Lys Leu Ser Phe Asp Asp Phe Asn Thr Tyr Tyr Thr 265 270 275gat ata tta gat att tat gaa ggt gta gga tca agc aag att tta aga 919Asp Ile Leu Asp Ile Tyr Glu Gly Val Gly Ser Ser Lys Ile Leu Arg 280 285 290gct tct att tgg gaa act aat cct ggc aca ata aga att ttt tcc aac 967Ala Ser Ile Trp Glu Thr Asn Pro Gly Thr Ile Arg Ile Phe Ser Asn 295 300 305caa gtt act gcc acc ttt ctt ata gaa tct gat gaa agt gat tat gtt 1015Gln Val Thr Ala Thr Phe Leu Ile Glu Ser Asp Glu Ser Asp Tyr Val310 315 320 325ggc ttt aat gca aca tat act gca ttt aac agc agt gag ctt aat aat 1063Gly Phe Asn Ala Thr Tyr Thr Ala Phe Asn Ser Ser Glu Leu Asn Asn 330 335 340tat gag aaa att aat tgt aac ttt gag gat ggc ttt tgt ttc tgg gtc 1111Tyr Glu Lys Ile Asn Cys Asn Phe Glu Asp Gly Phe Cys Phe Trp Val 345 350 355cag gat cta aat gat gat aat gaa tgg gaa agg att cag gga agc acc 1159Gln Asp Leu Asn Asp Asp Asn Glu Trp Glu Arg Ile Gln Gly Ser Thr 360 365 370ttt tct cct ttt act gga ccc aat ttt gac cac act ttt ggc aat gct 1207Phe Ser Pro Phe Thr Gly Pro Asn Phe Asp His Thr Phe Gly Asn Ala 375 380 385tca gga ttt tac att tct acc cca act gga cca gga ggg aga caa gaa 1255Ser Gly Phe Tyr Ile Ser Thr Pro Thr Gly Pro Gly Gly Arg Gln Glu390 395 400 405cga gtg ggg ctt tta agc ctc cct ttg gac ccc act ttg gag cca gct 1303Arg Val Gly Leu Leu Ser Leu Pro Leu Asp Pro Thr Leu Glu Pro Ala 410 415 420tgc ctt agt ttc tgg tat cat atg tat ggt gaa aat gtc cat aaa tta 1351Cys Leu Ser Phe Trp Tyr His Met Tyr Gly Glu Asn Val His Lys Leu 425 430 435agc att aat atc agc aat gac caa aat atg gag aag aca gtt ttc caa 1399Ser Ile Asn Ile Ser Asn Asp Gln Asn Met Glu Lys Thr Val Phe Gln 440 445 450aag gaa gga aat tat gga gac aat tgg aat tat gga caa gta acc cta 1447Lys Glu Gly Asn Tyr Gly Asp Asn Trp Asn Tyr Gly Gln Val Thr Leu 455 460 465aat gaa aca gtt aaa ttt aag gtt gct ttt aat gct ttt aaa aac aag 1495Asn Glu Thr Val Lys Phe Lys Val Ala Phe Asn Ala Phe Lys Asn Lys470 475 480 485atc ctg agt gat att gcg ttg gat gac att agc cta aca tat ggg att 1543Ile Leu Ser Asp Ile Ala Leu Asp Asp Ile Ser Leu Thr Tyr Gly Ile 490 495 500tgc aat ggg agt ctt tat cca gaa cca act ttg gtg cca act cct cca 1591Cys Asn Gly Ser Leu Tyr Pro Glu Pro Thr Leu Val Pro Thr Pro Pro 505 510 515cca gaa ctt cct acg gac tgt gga gga cct ttt gag ctg tgg gag cca 1639Pro Glu Leu Pro Thr Asp Cys Gly Gly Pro Phe Glu Leu Trp Glu Pro 520 525 530aat aca aca ttc agt tct acg aac ttt cca aac agc tac cct aat ctg 1687Asn Thr Thr Phe Ser Ser Thr Asn Phe Pro Asn Ser Tyr Pro Asn Leu 535 540 545gct ttc tgt gtt tgg att tta aat gca caa aaa gga aag aat ata caa 1735Ala Phe Cys Val Trp Ile Leu Asn Ala Gln Lys Gly Lys Asn Ile Gln550 555 560 565ctt cat ttt caa gaa ttt gac tta gaa aat att aac gat gta gtt gaa 1783Leu His Phe Gln Glu Phe Asp Leu Glu Asn Ile Asn Asp Val Val Glu 570 575 580ata aga gat ggt gaa gaa gct gat tcc ttg ctc tta gct gtg tac aca 1831Ile Arg Asp Gly Glu Glu Ala Asp Ser Leu Leu Leu Ala Val Tyr Thr 585 590 595ggg cct ggc cca gta aag gat gtg ttc tct acc acc aac aga atg act 1879Gly Pro Gly Pro Val Lys Asp Val Phe Ser Thr Thr Asn Arg Met Thr 600 605 610gtg ctt ctc atc act aac gat gtg ttg gca aga gga ggg ttt aaa gca 1927Val Leu Leu Ile Thr Asn Asp Val Leu Ala Arg Gly Gly Phe Lys Ala 615 620 625aac ttt act act ggc tat cac ttg ggg att cca gag cca tgc aag gca 1975Asn Phe Thr Thr Gly Tyr His Leu Gly Ile Pro Glu Pro Cys Lys Ala630 635 640 645gac cat ttt caa tgt aaa aat gga gag tgt gtt cca ctg gtg aat ctc 2023Asp His Phe Gln Cys Lys Asn Gly Glu Cys Val Pro Leu Val Asn Leu 650 655 660tgt gac ggt cat ctg cac tgt gag gat ggc tca gat gaa gca gat tgt 2071Cys Asp Gly His Leu His Cys Glu Asp Gly Ser Asp Glu Ala Asp Cys 665 670 675gtg cgt ttt ttc aat ggc aca acg aac aac aat ggt tta gtg cgg ttc 2119Val Arg Phe Phe Asn Gly Thr Thr Asn Asn Asn Gly Leu Val Arg Phe 680 685 690aga atc cag agc ata tgg cat aca gct tgt gct gag aac tgg acc acc 2167Arg Ile Gln Ser Ile Trp His Thr Ala Cys Ala Glu Asn Trp Thr Thr 695 700 705cag att tca aat gat gtt tgt caa ctg ctg gga cta ggg agt gga aac 2215Gln Ile Ser Asn Asp Val Cys Gln Leu Leu Gly Leu Gly Ser Gly Asn710 715 720 725tca tca aag cca atc ttc tct acc gat ggt gga cca ttt gtc aaa tta 2263Ser Ser Lys Pro Ile Phe Ser Thr Asp Gly Gly Pro Phe Val Lys Leu 730 735 740aac aca gca cct gat ggc cac tta ata cta aca ccc agt caa cag tgt 2311Asn Thr Ala Pro Asp Gly His Leu Ile Leu Thr Pro Ser Gln Gln Cys 745 750 755tta cag gat tcc ttg att cgg tta cag tgt aac cat aaa tct tgt gga 2359Leu Gln Asp Ser Leu Ile Arg Leu Gln Cys Asn His Lys Ser Cys Gly 760 765 770aaa aaa ctg gca gct caa gac atc acc cca aag att gtt gga gga agt 2407Lys Lys Leu Ala Ala Gln Asp Ile Thr Pro Lys Ile Val Gly Gly Ser 775 780 785aat gcc aaa gaa ggg gcc tgg ccc tgg gtt gtg ggt ctg tat tat ggc 2455Asn Ala Lys Glu Gly Ala Trp Pro Trp Val Val Gly Leu Tyr Tyr Gly790 795 800 805ggc cga ctg ctc tgc ggc gca tct ctc gtc agc agt gac tgg ctg gtg 2503Gly Arg Leu Leu Cys Gly Ala Ser Leu Val Ser Ser Asp Trp Leu Val 810 815 820tcc gcc gca cac tgc gtg tat ggg aga aac tta gag cca tcc aag tgg 2551Ser Ala Ala His Cys Val Tyr Gly Arg Asn Leu Glu Pro Ser Lys Trp 825 830 835aca gca atc cta ggc ctg cat atg aaa tca aat ctg acc tct cct caa 2599Thr Ala Ile Leu Gly Leu His Met Lys Ser Asn Leu Thr Ser Pro Gln 840 845 850aca gtc cct cga tta ata gat gaa att gtc ata aac cct cat tac aat 2647Thr Val Pro Arg Leu Ile Asp Glu Ile Val Ile Asn Pro His Tyr Asn 855 860 865agg cga aga aag gac aac gac att gcc atg atg cat ctg gaa ttt aaa 2695Arg Arg Arg Lys Asp Asn Asp Ile Ala Met Met His Leu Glu Phe Lys870 875 880 885gtg aat tac aca gat tac ata caa cct att tgt tta ccg gaa gaa aat 2743Val Asn Tyr Thr Asp Tyr Ile Gln Pro Ile Cys Leu Pro Glu Glu Asn 890 895 900caa gtt ttt cct cca gga aga aat tgt tct att gct ggt tgg ggg acg 2791Gln Val Phe Pro Pro Gly Arg Asn Cys Ser Ile Ala Gly Trp Gly Thr 905 910 915gtt gta tat caa ggt act act gca aac ata ttg caa gaa gct gat gtt 2839Val Val Tyr Gln Gly Thr Thr Ala Asn Ile Leu Gln Glu Ala Asp Val 920 925 930cct ctt cta tca aat gag aga tgc caa cag cag atg cca gaa tat aac 2887Pro Leu Leu Ser Asn Glu Arg Cys Gln Gln Gln Met Pro Glu Tyr Asn 935 940 945att act gaa aat atg ata tgt gca ggc tat gaa gaa gga gga ata gat 2935Ile Thr Glu Asn Met Ile Cys Ala Gly Tyr Glu Glu Gly Gly Ile Asp950 955 960 965tct tgt cag ggg gat tca gga gga cca tta atg tgc caa gaa aac aac 2983Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Met Cys Gln Glu Asn Asn 970 975 980agg tgg ttc ctt gct ggt gtg acc tca ttt gga tac aag tgt gcc ctg 3031Arg Trp Phe Leu Ala Gly Val Thr Ser Phe Gly Tyr Lys Cys Ala Leu 985 990 995cct aat cgc ccc gga gtg tat gcc agg gtc tca agg ttt acc gaa 3076Pro Asn Arg Pro Gly Val Tyr Ala Arg Val Ser Arg Phe Thr Glu 1000 1005 1010tgg ata caa agt ttt cta cat tag cgcatttctt aaactaaaca ggaaagtcgc 3130Trp Ile Gln Ser Phe Leu His 1015attattttcc cattctactc tagaaagcat ggaaattaag tgtttcgtac aaaaatttta 3190aaaagttacc aaaggttttt attcttacct atgtcaatga aatgctaggg ggccagggaa 3250acaaaatttt aaaaataata aaattcacca tagcaataca gaataacttt aaaataccat 3310taaatacatt tgtatttcat tgtgaacagg tatttcttca cagatctcat ttttaaaatt 3370cttaatgatt atttttatta cttactgttg tttaaaggga tgttatttta aagcatatac 3430catacactta agaaatttga gcagaattta aaaaagaaag aaaataaatt gtttttccca 3490aagtatgtca ctgttggaaa taaactgcca taaattttct agttccagtt tagtttgctg 3550ctattagcag aaactcaatt gtttctctgt cttttctatc aaaattttca acatatgcat 3610aaccttagta ttttcccaac caatagaaac tatttattgt aagcttatgt cacaggcctg 3670gactaaattg attttacgtt cctctt 369621019PRTArtificial SequenceSynthetic Construct 2Met Gly Ser Lys Arg Gly Ile Ser Ser Arg His His Ser Leu Ser Ser1 5 10 15Tyr Glu Ile Met Phe Ala Ala Leu Phe Ala Ile Leu Val Val Leu Cys 20 25 30Ala Gly Leu Ile Ala Val Ser Cys Leu Thr Ile Lys Glu Ser Gln Arg 35 40 45Gly Ala Ala Leu Gly Gln Ser His Glu Ala Arg Ala Thr Phe Lys Ile 50 55 60Thr Ser Gly Val Thr Tyr Asn Pro Asn Leu Gln Asp Lys Leu Ser Val65 70 75 80Asp Phe Lys Val Leu Ala Phe Asp Leu Gln Gln Met Ile Asp Glu Ile 85 90 95Phe Leu Ser Ser Asn Leu Lys Asn Glu Tyr Lys Asn Ser Arg Val Leu 100 105 110Gln Phe Glu Asn Gly Ser Ile Ile Val Val Phe Asp Leu Phe Phe Ala 115 120 125Gln Trp Val Ser Asp Gln Asn Val Lys Glu Glu Leu Ile Gln Gly Leu 130 135 140Glu Ala Asn Lys Ser Ser Gln Leu Val Thr Phe His Ile Asp Leu Asn145 150 155 160Ser Val Asp Ile Leu Asp Lys Leu Thr Thr Thr Ser His Leu Ala Thr 165 170 175Pro Gly Asn Val Ser Ile Glu Cys Leu Pro Gly Ser Ser Pro Cys Thr 180 185 190Asp Ala Leu Thr Cys Ile Lys Ala Asp Leu Phe Cys Asp Gly Glu Val 195 200 205Asn Cys Pro Asp Gly Ser Asp Glu Asp Asn Lys Met Cys Ala Thr Val 210 215 220Cys Asp Gly Arg Phe Leu Leu Thr Gly Ser Ser Gly Ser Phe Gln Ala225 230 235 240Thr His Tyr Pro Lys Pro Ser Glu Thr Ser Val Val Cys Gln Trp Ile 245 250 255Ile Arg Val Asn Gln Gly Leu Ser Ile Lys Leu Ser Phe Asp Asp Phe 260 265 270Asn Thr Tyr Tyr Thr Asp Ile Leu Asp Ile Tyr Glu Gly Val Gly Ser 275 280 285Ser Lys Ile Leu Arg Ala Ser Ile Trp Glu Thr Asn Pro Gly Thr Ile 290 295 300Arg Ile Phe Ser Asn Gln Val Thr Ala Thr Phe Leu Ile Glu Ser Asp305 310 315 320Glu Ser Asp Tyr Val Gly Phe Asn Ala Thr Tyr Thr Ala Phe Asn Ser 325 330 335Ser Glu Leu Asn Asn Tyr Glu Lys Ile Asn Cys Asn Phe Glu Asp Gly 340 345 350Phe Cys Phe Trp Val Gln Asp Leu Asn Asp Asp Asn Glu Trp Glu Arg 355 360 365Ile Gln Gly Ser Thr Phe Ser Pro Phe Thr Gly Pro Asn Phe Asp His 370 375 380Thr Phe Gly Asn Ala Ser Gly Phe Tyr Ile Ser Thr Pro Thr Gly Pro385 390 395 400Gly Gly Arg Gln Glu Arg Val Gly Leu Leu Ser Leu Pro Leu Asp Pro 405 410 415Thr Leu Glu Pro Ala Cys Leu Ser Phe Trp Tyr His Met Tyr Gly Glu 420 425 430Asn Val His Lys Leu Ser Ile Asn Ile Ser Asn Asp Gln Asn Met Glu 435 440 445Lys Thr Val Phe Gln Lys Glu Gly Asn Tyr Gly Asp Asn Trp Asn Tyr 450 455 460Gly Gln Val Thr Leu Asn Glu Thr Val Lys Phe Lys Val Ala Phe Asn465 470 475 480Ala Phe Lys Asn Lys Ile Leu Ser Asp Ile Ala Leu Asp Asp Ile Ser 485 490 495Leu Thr Tyr Gly Ile Cys Asn Gly Ser Leu Tyr Pro Glu Pro Thr Leu 500 505 510Val Pro Thr Pro Pro Pro Glu Leu Pro Thr Asp Cys Gly Gly Pro Phe 515 520 525Glu Leu Trp Glu Pro Asn Thr Thr Phe Ser Ser Thr Asn Phe Pro Asn 530 535 540Ser Tyr Pro Asn Leu Ala Phe Cys Val Trp Ile Leu Asn Ala Gln Lys545 550 555 560Gly Lys Asn Ile Gln Leu His Phe Gln Glu Phe Asp Leu Glu Asn Ile 565 570 575Asn Asp Val Val Glu Ile Arg Asp Gly Glu Glu Ala Asp Ser Leu Leu 580 585 590Leu Ala Val Tyr Thr Gly Pro Gly Pro Val Lys Asp Val Phe Ser Thr 595 600 605Thr Asn Arg Met Thr Val Leu Leu Ile Thr Asn Asp Val Leu Ala Arg 610 615 620Gly Gly Phe Lys Ala Asn Phe Thr Thr Gly Tyr His Leu Gly Ile Pro625 630 635 640Glu Pro Cys Lys Ala Asp His Phe Gln Cys Lys Asn Gly Glu Cys Val 645 650 655Pro Leu Val Asn Leu Cys Asp Gly His Leu His Cys Glu Asp Gly Ser 660 665 670Asp Glu Ala Asp Cys Val Arg Phe Phe Asn Gly Thr Thr Asn Asn Asn 675 680 685Gly Leu Val Arg Phe Arg Ile Gln Ser Ile Trp His Thr Ala Cys Ala 690 695

700Glu Asn Trp Thr Thr Gln Ile Ser Asn Asp Val Cys Gln Leu Leu Gly705 710 715 720Leu Gly Ser Gly Asn Ser Ser Lys Pro Ile Phe Ser Thr Asp Gly Gly 725 730 735Pro Phe Val Lys Leu Asn Thr Ala Pro Asp Gly His Leu Ile Leu Thr 740 745 750Pro Ser Gln Gln Cys Leu Gln Asp Ser Leu Ile Arg Leu Gln Cys Asn 755 760 765His Lys Ser Cys Gly Lys Lys Leu Ala Ala Gln Asp Ile Thr Pro Lys 770 775 780Ile Val Gly Gly Ser Asn Ala Lys Glu Gly Ala Trp Pro Trp Val Val785 790 795 800Gly Leu Tyr Tyr Gly Gly Arg Leu Leu Cys Gly Ala Ser Leu Val Ser 805 810 815Ser Asp Trp Leu Val Ser Ala Ala His Cys Val Tyr Gly Arg Asn Leu 820 825 830Glu Pro Ser Lys Trp Thr Ala Ile Leu Gly Leu His Met Lys Ser Asn 835 840 845Leu Thr Ser Pro Gln Thr Val Pro Arg Leu Ile Asp Glu Ile Val Ile 850 855 860Asn Pro His Tyr Asn Arg Arg Arg Lys Asp Asn Asp Ile Ala Met Met865 870 875 880His Leu Glu Phe Lys Val Asn Tyr Thr Asp Tyr Ile Gln Pro Ile Cys 885 890 895Leu Pro Glu Glu Asn Gln Val Phe Pro Pro Gly Arg Asn Cys Ser Ile 900 905 910Ala Gly Trp Gly Thr Val Val Tyr Gln Gly Thr Thr Ala Asn Ile Leu 915 920 925Gln Glu Ala Asp Val Pro Leu Leu Ser Asn Glu Arg Cys Gln Gln Gln 930 935 940Met Pro Glu Tyr Asn Ile Thr Glu Asn Met Ile Cys Ala Gly Tyr Glu945 950 955 960Glu Gly Gly Ile Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Met 965 970 975Cys Gln Glu Asn Asn Arg Trp Phe Leu Ala Gly Val Thr Ser Phe Gly 980 985 990Tyr Lys Cys Ala Leu Pro Asn Arg Pro Gly Val Tyr Ala Arg Val Ser 995 1000 1005Arg Phe Thr Glu Trp Ile Gln Ser Phe Leu His 1010 1015317480DNAArtificial SequenceVector 3cggccgcccc tgtcagttgc cgggatgagc ttaagtcctt tgtccatcat tcttagatcc 60tccccttttc ctttcttcca ggaaagcaag gagacacttc atcaaagact ccactccatt 120atcatctgag tgccttgcag aaagtttaaa ttccaatctg gtacacatgg cgggttctct 180cattccatac catttccaaa ataactctcc ttcccttaac tagtagaaca ttttttagtg 240aatgattaag cacaattgag gccacatgga tatcttaact acttttctta ccatttaaaa 300tcccttcctc ttcataaact ttgcctgact tactttctct tcatgctgga tatacctggt 360aacattatgt tgatcttatt gattagaagg aatatccata gaatgtgtaa tttggaattt 420tatttatcac cacaacactc aaattgatat gaagcaagtg tttttgcatc ataatgctca 480aggctttaga agtagatctt tttgtcttta aagagttctg caaatcgaca ggatggtgga 540atgttgcaaa ggtgtatctg tgagtggggg ccaaaagcta ttaggggttg aactgtggga 600accttgggcc cacataagga ctaaacatag ggggctgtgg atagaccaag tgaaagatta 660tttccaggaa acgaaggcaa agaaatgcag atcttgacat ttgatcttgg ggatacttac 720aggcactttt accgtagcca gaacaatgac tactatggga aagaacaaac tgaatcccag 780gcacaatccc ataagggcct aggtctcgtt ctagggaatg gaacttacat gtcagaccta 840tcttggagga agaactcaca agcccccgag tccgtgaatg tgcatctgcc ttggacatac 900attgattcga gagagaggca caatgaacct ttcagtacac aaaggactta aatccagttt 960aggaagacta aaggaaaaaa aaaaacaata gatggttact tgcacctagg actggagcat 1020gtgtttggga acgattggac actaattgta tcaacaatta tatcagagtg cccataaagc 1080gagattcaga aacctggcca aagaaaacaa ggtttattgg gggagggggg aatgggaggt 1140tttagtactc tgctgggcac tgttgcacat gcgtgtttga gctctatggt tagtgaaaag 1200atacaaatat gcaaatatgc ttgtctgttc tcaacactta taagaactgt ttgccatttt 1260tacatcttgg aataatctag actttttttt tgtaggaatg gaaagtattc tcaggaggga 1320agaagaactc ataatatctg tctgactgtc tccatttgga ccgagacaca tttagtttga 1380tacgactcaa gaactgaatt tatttcttct tatgtttttt ttccagtaaa ggaagatcaa 1440aaactagccc gaatcagtct acactgtgga tttcaaaaag cacgaggaag gaaagtggca 1500tggaggtata ataatgaact ggcattgttc tggaaaaata aaatagtttt gtaatcgttg 1560atcaatgtaa ggaatggcat catcaccttg gaatggcttg aaatctcaaa ggatctgcaa 1620gatggactgt aagctccccc ttgaggcaaa tgtcaaaata atttttagaa gtttatagtt 1680tcacttacaa catatgctga gtgagacctg gttactctcc taaaggatac acctaaattt 1740catgtaaaag aaatggacca tgcggatata atttctgtgg accaatctgg aatacctgtg 1800ttgggagtag ttactcttgc tctctttctt ttttcatctt cataaatttt gatctatgtc 1860atgtaatata aattgtattt caatttacgg tgtgcagaaa cattctacct ttgcaaaagt 1920gtttgatgaa aatagagtgt tctgatattt atatttatga catttgattt ttacatatat 1980agtgttttca ttaaaaactg aattcacaca gacacaaaga aatccttgca tagggaaaac 2040atttctttgg taggatagtt catgtttgtt ctgtttcttt gctttgggtt cgataaagtt 2100taggaagtat cttcagaaat aagaaactat ttcattgact gtgctgttaa tctgctgaaa 2160cactttactt agaggcaaga actgtctaac ttcaattgtg caagacacgt gccttacaat 2220tattcttaat ttaaaattaa acagattttt gtaataaagt atctttaata atacttgtat 2280agacaccaat agagttaacc ttgaaaaaaa tggcatctgc agtacaaatc acaggtcttc 2340acatgttgcc tatagaattg caaatagctc cccagagtgt caggaatcaa tgtggctcct 2400ccctttcaat caaacagagt aactatttga aggctttgga gtcagacagt tgctgtttgg 2460ctgaggttct cctaactgaa tttagcctgt ataatatata aagctcaatg gtttccagca 2520gtgtgatgct aagcagaaca cactgatatt aatgattaaa agaagaagaa gaagaaaaac 2580aaaactacaa atcacatcat ttaatgggcc agggactcga ggttgctgat gtttgcttat 2640ctttgatgag tgttgtttgt ggttgtgaac tgtggcttcc ttctgtctga tggagtttcc 2700tcaacacgta gcacttgttc ttctcaagag agttgtaact attttgtctt caaatgtccc 2760tgtgtcactt ttagccaaat aaagagttct tgtttctgaa gaatgatctt tggtgttgtc 2820tacattctta agggaccaag gcccagatgt gtttcatcat tacctgagga tgaaatcaaa 2880cattgcatgt gaagagccca gacagcggca gatctgaagg aacagggatg tttcttcttt 2940ctctgtcagt cttgttcagt tagacagtcc ttaagtagac ttggtttttc caaacaagag 3000gcaaaaatgg gctctcagat cttttcctgc tgccttgaga ctgaatggac aactgcattc 3060taagctcagc tgcccccact tagctccatc cttttgggtc aaatgggagg ccagctgttt 3120ggatctatgt tttgcctgta tgccaactcc actcgtttct ccatttagct atccagaact 3180ctaacagctg aaggatggaa gcacaaactg aaaggggaag gctggtgttt cttttattat 3240caaggtcagg aaagcatcca gtgttaatgg caaactccat ctgtgacaag ggtgggaaaa 3300caattctctg tcctcttaaa agatggtgat gataccatct ttgaaaaggg tttgtaaagt 3360tttagctggc ccttgcaaaa ccggtacatg atgtaactgc tctgtatttt tcagaacata 3420gtgttaatat atgttgggaa atgatccatt caagtattca caaacaaacc acacactcgc 3480ttcttcttct tattttccct tctaggttga ggttattggc aaggttttcc tgagaagagt 3540gtgcagagac ctttgattat tagtgtcagt gcttgtggga acatgcagtc ccctatcatc 3600tgggcgaatc ttcttttatt gaaatgtttt ccatgctctg actgtcttca gttagaacat 3660gattgtctcg aaacctctgt cctccatttt cagggtcact gtcacctgta gagttgtctc 3720tagtgtctct taaacgcatc gaattgtaat tatcccatct aaagcatagc tcaagctttc 3780tcccttcccc aaagcaaata ttagcagttg cttttttaaa aaaaattttt aaatacaact 3840catttgaacc ctattcattc ttgcattgga ttaattcatg aaagaaaaat gaaaacaatg 3900aatgtgttta tgttgatgaa atatgtctgt gtcagtgtta gtgtgtgtgt gtgtgtgtgt 3960gtgtgtgtgt gtgtgtgtgt atgtatttgt gtcaagttag catggatttc agcagattct 4020gtgtgataaa ctgaactgac atttgcattt tctctggaaa taatccaaga gattaaagaa 4080gtggagcaaa ttaatttatt acacattttt tgcctttagt ggggaattca gctgtgtctg 4140aacaaatttt tcacatgttc aggatagtct ctaaactgta caattttccg cggctcgagc 4200ctaggggtaa ccgaagttcc tatactttct agagaatagg aacttcggaa taggaacttc 4260ttataatcta gaactagtgg atcgatccac gattcgaggg cccctgcagg tcaattctac 4320cgggtagggg aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg 4380ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg 4440cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc 4500aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca 4560cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt 4620tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg 4680ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc 4740tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct 4800tcctcatctc cgggcctttc gacctgcagc caatatggga tcggccattg aacaagatgg 4860attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca 4920acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt 4980tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg 5040gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga 5100agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca 5160ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct 5220tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac 5280tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc 5340gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt 5400gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt 5460catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg 5520tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat 5580cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagg 5640ggatcgatcc gctgtaagtc tgcagaaatt gatgatctat taaacaataa agatgtccac 5700taaaatggaa gtttttcctg tcatactttg ttaagaaggg tgagaacaga gtacctacat 5760tttgaatgga aggattggag ctacgggggt gggggtgggg tgggattaga taaatgcctg 5820ctctttactg aaggctcttt actattgctt tatgataatg tttcatagtt ggatatcata 5880atttaaacaa gcaaaaccaa attaagggcc agctcattcc tcccactcat gatctataga 5940tctatagatc tctcgtggga tcattgtttt tctcttgatt cccactttgt ggttctaagt 6000actgtggttt ccaaatgtgt cagtttcata gcctgaagaa cgagatcagc agcctctgtt 6060ccacatacac ttcattctca gtattgtttt gccaagttct aattccatca gaagctgact 6120ctagatcctg caggaattaa ttcatatgaa gttcctatac tttctagaga ataggaactt 6180cggaatagga acttcaaaat gtcgcggcgc gccatgtctt gtggtttcct gaagactgtg 6240agaaatgttt gcttttgatt acttttatca cgaattattt tgatttcagc aaacatggat 6300ttcactactt tctcattcct agggaatatg tttcaattac tctagtctat atctaaaacc 6360acttacagtg ctaaaccctg tgttcaacat ggtctctttc aatcaagtac ttgtgacaca 6420tcacggttgt aaatcctgaa ttttgggctt aggtgatgga tagtaacgca aattgattat 6480ttttcttctc tacttttttt ttctttttgg tgaatagata acacatgcta atgccaggct 6540gtaattttta agcaatatta gcatatgact tttctttctc tccttactaa gtagaaaact 6600tttacttaaa gaaacagtct ttctgtagtc tggtactact cttgggcaaa gcatggctta 6660cgtggagaaa agaactgaac gctgcaagta atcttgggaa ttaaattaat ttctgaagga 6720tcagcggaaa ggtaacgcat gctttgttct tgccacaaca agagatggtt cactccggac 6780tcagtgagat tgcctagtgc tcccgagatt ggtaataact gaagctaatg aactgctaat 6840gtatggattt accctttaat acttttagac tatgattggc catttggtga cagacgtagt 6900agaaacaaac tgtaagaaaa agatgaactg atgaagaccc gcttccagtc tcatctactt 6960cagggctgtt cagtgacaag cccttctatg aggctgtgaa attcattctg gaaagcaaat 7020gggcttacgc tggtaaaaga ggcagacatg tgcgtagcga ctataaaata acgactacgc 7080gtaaactact gaaatcacat cgcctatagt tttgaaattt ggtttttcca aagccaggaa 7140aacttacatt aaaaaataaa attgtttacg aggttttata caatttgacc agcagagggc 7200aatgttacac caggaatcat aacactgcca ctattctgcc tctgcggata tcccacacat 7260taaatatttg tatatgtgtg cccacacata gaattaaatg ctctacactt atgtacttac 7320ttaaatgacg tgtgcatgtt gcttgagtac tcttactttg gaaaaaggat aaaagaaaaa 7380aaaaaaacac aaaacaaagc tatgtgctca ctaaaagcaa ctccatagtt ttagaacacg 7440aatggtaaag cttatttaaa aagaacgtac gacattctat tatcaacatt ctcttttaat 7500atataagaca acttcaatag aaatataaga aaatgtactt tgtgaatgat cagtatttct 7560atatacctct ctataaaatg cctcgaaaat ctcaattaat ttcccttttg atgctcacgt 7620gtgtttgaaa gaaaagtagg ctacaggatg cattgtagga tactttcaag tctcacaaat 7680gcgcttttca aatctacagt gctcattaat aaaaatccat attcagaatg gctcctggga 7740cagtgttact ggccttagaa agggcaaaca gtgcttaaat atttttaatc aagcacataa 7800gcagttaata gctcattgcc atctcacaag aaatttcaca ttttctccga taacttcttc 7860cttatttttg ttacagcttt tagaagaagg ataccctcca aagcactatt tttccatctt 7920acactaaatg ccacaattat ataacattaa ttaactaagg gactgaataa atcaatatgt 7980ctcaattttg cctgctcagt ataatagggc caattaaaga aaggattgaa gacatttagc 8040taactggctt ttttggtgta aatgagcata aagggtacat ctatataaag tctttctttt 8100ttcaactacc atgaatataa gtataatcgt ggcactaata ttagttgaaa ctgtcacctg 8160actcctacaa ctttgtagca gatgctcagc tgggtcttca tgtgggtccc ctgacaattg 8220tagcaggcac tgtctctgaa tctattgcct gaacctggat ccccttcctc tagctgaact 8280gccttgttgg gcctcagtgg aaggggtgca cttagtcctg caacagtttg atctgcttgg 8340gcaggttggt acccggatgt gtgtgaggcc ttcctatctc aggagggata gttggggagg 8400ggctggggag gatgagcctg ggaggagagg agggagggtg ctgcaatcag ggatgtaaag 8460tgaataaata aattaagaaa aaagaaaaaa aaggaaataa tacggttttt ctctgaaaga 8520ggcattcaat taatttgaat ggctttaaac tttgttttct ctttcttact aatatttaca 8580tctaggaaac ctgaataggt agaattctca gcataccgga gaaccatggg ctctcagaac 8640gccttattag aaaggaagtt gtgaaacctg tgtgaaaact ttcaggtgga ttggctcaaa 8700cccactggga tcgctatctg tcaacactga cagacagtgc taaaccccaa gattctctcg 8760cagatactaa atgtagagct tacaaatttg ttttattgct tgagctttgg ggagtgctga 8820ctttcctgag cagtggaata agctgatgct gttttaacgt caagtctgtt aagtaaagtg 8880tactggtaac aggatgctaa ctgttaagag tgcaattctg atgtctcttt gcagtcattt 8940ctcatcccga gtttatctgt agtgggaata tgagattcag aggccaattt cctcagtctc 9000aaacaaaaat cactactaat gtctaaatag gagaaattac ataatatact atgacagaat 9060ttttgaatag aggaaggatg ctgattttta atacttcaca aaaaagggta taagagaaga 9120tctagcttaa aatagcaaaa aaaagataca atataggact aattatatta tttttaaatt 9180atattcatat ttgtttgcta agaaagataa ccccttgatt ctcacgtaac cccttgtctt 9240ttgatggttg tctgaggcgt atttatccta tttattctaa aacttacaat ttattttcat 9300gatatttaga gctttagagt atgagactaa gaacagtatt atggtatatc atcttcctct 9360ctgtatgtca taacagcaat ggcttaaaac agtaaattag actgtcttat acaaatgaag 9420atcaaacaat cctatcagga aaaaacaaac ccatatgttt gttgacatta cacaaataaa 9480agccaggcag tggtggcaca tacctttaat cccagcactt gggaggcaga ggcaggcgga 9540tttctgagtt tgagaccagc ctggtctaca gagtgagttc caggacagcc agggctacac 9600agagaaaccc tgtctcaaaa caaacaaaaa gcaagcaagc aagcaagcaa gcaagcaacc 9660aacaagcaaa agaaattgaa aaaaaatttc aaatgtatgt ggtcaatgtg tggtggcact 9720gacacattaa tggctctctc tctctctctc tctctctctc tctctctctc tctatatata 9780tatatatata tatatatata tatatatata tatatatata tatatatata tatatatatt 9840cctatgtacc aaataaaaat aaaacatcct ggcacccacc taggagttag tatcaagctt 9900ccattagggg cttggtttac tctcacaaat ggtccacctc cggtagatga tattggcatt 9960gatgagtttg cactcctaag gtagaataaa cgacatatga acaggagata aaattaccaa 10020cattaatgct acattttcat tattggatga gacataattg gaagtaaatt atgttaatac 10080ttgggaaaag tggagggcag agaaactggg caggatctat tgtataaggg aagaatccat 10140tttcaataga tataatatca aacaatatta tagaccgtta gcaaaattat tacttaggac 10200attaaatatt gtcagttttt gggaaagcct ttagtacacg tttgaatatc gtacattata 10260ttacaaggtc gctacacatt tctctcgagg atctgatttc tgttctgttc tgttttcccc 10320ttttcaatgc tgttgagagg atgtgaatag tttaatggat cagctcatat aaaatttgaa 10380ttaagtttga atgagaaaaa taaatacacc gattataatt taagcctttc tttaaggagg 10440tataactcat ctttcaagaa aatcaaatcg cctaagaact acacagagta gttctagctt 10500tagtagttcg gagtttagct ttctagtttt ttttttgctt ttgttatctc attaaattta 10560aagtcaaagc agtcccaaat ccaaggacga agacaagcgg ttgctccgag ggcctccata 10620acctccatct gcagttgcca actggtgagc caccgcaggg tgacaaaaga cacctccatg 10680taacacagca tccaatcctc taggctacaa cctgtggcat ctgcaccatc tgctgtgagg 10740cttagtgaag gtttctctta attaatttta cttttatttt aaaatttaac taaatttagt 10800taaattaaaa tctgaccctt tttaaaataa ttcttttact caatcaatat cttagtatta 10860atataaaact atgcctttaa atgtgaaagt cagtgggctg aacctgcaaa ggggaaaagt 10920gatggttaaa agtgatacaa tatgcatgtg ggaaatcgtc agcagttaaa tcaccatcac 10980catcaccatc atcatcacca tcaccatcac catcatcacc atcaccatca ccaccctcac 11040cctcaccctc accctcacac tcaccctcac cctcaccctc accatcatca ttaccatcac 11100cacgtttgcc tctggattct aggtatatta attttactgt tctctatggt tgttaggaga 11160caatgtaacc tctagctact gaaataacaa gttgagtgtg cttgttgcct ctccagaata 11220tttaatgtct ctcctggatg atagcaaaca ataacaatgt gtttaggtgc acgggaaatg 11280gaccaggagc acccaaaatt cttatgagca gtgaaaagaa aaaacaaatg ttcaagaggt 11340agttcctctc ctttccaaag ccttcgtcag acttagcctt tatttgctgc cttccagcca 11400ctgaacatta tgttgggaaa atagatgctc tgtctaatgt cccaatattt ggtatatttc 11460acttccactt gataagcaaa caaatgcaat gaaagaacac aaagccatta atcttccttt 11520aacaaaatat caaatagatt ttccgaatca aagagaagtc ttgctgttgt tatttttgga 11580tttgatacta tcttctccaa ccacatgagc agaagagtta tttcatttaa atagctctat 11640gcaatgtatt tgctgaagct taggggaaaa aaaggggggg ggcagaagtg gatgtagaag 11700cttagggatg cttgagtcat agaaacttct accgatataa actagtcaca ggtgaagaac 11760cacacatttt ttgtgagagc aaatgtagat caaggcctct atgttttatc agggtgtggc 11820ttctggtaga tcgcatcaac gcacccaggt aaagtatcag gcctcctgaa tgttggcttc 11880aggcttcagt tctggaattg gttagtgcac ctgcatgccg ggggcgcggg gtggggtggg 11940gtaaagtcca cagtgatgat tactctctaa tctctcttca gacagcgttc ttctcttgtt 12000tactcgagtt acagttcctt attctgccta cggtttgcag ccttggagca cttttctacg 12060tagctctcta agtctcttat ctttcaggag acattgtcac agtgctctgt caacagtagc 12120ggcggtcgct ccagaagatt aaaaattaaa acgataaact acccagagcc actttagatc 12180aaatgggatt ggaaagtcag gttgcccatc cagcgtgcta cagtaagaaa cctttcaaat 12240gatcctaata tttgctatag aaaaggaaac gaggtcgggc cggccaagct taaggaattc 12300gctagcatgc atgttaacgg atccttaatt aatgtacagg gtcccgttta aacagtaacg 12360ctagggataa cagggtaata taatcgagct gcaggattcg agggccccgg caggtcaatt 12420ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc 12480gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg 12540taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct 12600agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt 12660agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg 12720cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg 12780aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag 12840gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc 12900ctcttcctca tctccgggcc tttcgacctg cagccaatgc accgtccttg ccatcatggc 12960ctcgtacccc ggccatcaac acgcgtctgc gttcgaccag gctgcgcgtt ctcgcggcca 13020tagcaaccga cgtacggcgt tgcgccctcg ccggcagcaa gaagccacgg aagtccgccc 13080ggagcagaaa atgcccacgc tactgcgggt

ttatatagac ggtccccacg ggatggggaa 13140aaccaccacc acgcaactgc tggtggccct gggttcgcgc gacgatatcg tctacgtacc 13200cgagccgatg acttactggc gggtgctggg ggcttccgag acaatcgcga acatctacac 13260cacacaacac cgcctcgacc agggtgagat atcggccggg gacgcggcgg tggtaatgac 13320aagcgcccag ataacaatgg gcatgcctta tgccgtgacc gacgccgttc tggctcctca 13380tatcgggggg gaggctggga gctcacatgc cccgcccccg gccctcaccc tcatcttcga 13440ccgccatccc atcgccgccc tcctgtgcta cccggccgcg cggtacctta tgggcagcat 13500gaccccccag gccgtgctgg cgttcgtggc cctcatcccg ccgaccttgc ccggcaccaa 13560catcgtgctt ggggcccttc cggaggacag acacatcgac cgcctggcca aacgccagcg 13620ccccggcgag cggctggacc tggctatgct ggctgcgatt cgccgcgttt acgggctact 13680tgccaatacg gtgcggtatc tgcagtgcgg cgggtcgtgg cgggaggact ggggacagct 13740ttcggggacg gccgtgccgc cccagggtgc cgagccccag agcaacgcgg gcccacgacc 13800ccatatcggg gacacgttat ttaccctgtt tcgggccccc gagttgctgg cccccaacgg 13860cgacctgtat aacgtgtttg cctgggcctt ggacgtcttg gccaaacgcc tccgttccat 13920gcacgtcttt atcctggatt acgaccaatc gcccgccggc tgccgggacg ccctgctgca 13980acttacctcc gggatggtcc agacccacgt caccaccccc ggctccatac cgacgatatg 14040cgacctggcg cgcacgtttg cccgggagat gggggaggct aactgagggg atcgatccgt 14100cctgtaagtc tgcagaaatt gatgatctat taaacaataa agatgtccac taaaatggaa 14160gtttttcctg tcatactttg ttaagaaggg tgagaacaga gtacctacat tttgaatgga 14220aggattggag ctacgggggt gggggtgggg tgggattaga taaatgcctg ctctttactg 14280aaggctcttt actattgctt tatgataatg tttcatagtt ggatatcata atttaaacaa 14340gcaaaaccaa attaagggcc agctcattcc tcccactcat gatctataga tctatagatc 14400tctcgtggga tcattgtttt tctcttgatt cccactttgt ggttctaagt actgtggttt 14460ccaaatgtgt cagtttcata gcctgaagaa cgagatcagc agcctctgtt ccacatacac 14520ttcattctca gtattgtttt gccaagttct aattccatca gaagctgact ctaggccgga 14580cgcccgggcg accggccgag ctccaattcg ccctatagtg agtcgtatta caattcactg 14640gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 14700gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 14760tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc 14820gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc 14880gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 14940ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa 15000aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 15060cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 15120ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat 15180tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg 15240cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 15300tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 15360aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 15420ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 15480ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 15540tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 15600tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 15660actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 15720gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 15780acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 15840gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 15900acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 15960gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 16020ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 16080gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 16140cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 16200agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 16260catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 16320tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 16380cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 16440gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 16500taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc 16560ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 16620tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 16680ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 16740cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 16800agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 16860gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 16920atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 16980gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 17040gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 17100ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 17160cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 17220cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 17280acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 17340cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 17400accatgatta cgccaagctc gaaattaacc ctcactaaag ggaacaaaag ctgtcgagat 17460ctagatatcg atggccatag 17480420DNAArtificial SequenceProbe 5e2 4gccgcactat ttgcagcatg 20522DNAArtificial SequencePrimer 1472_23 5cgttactatc catcacctaa gc 22622DNAArtificial SequencePrimer 1472_24 6gggaattcag ctgtgtctga ac 22720DNAArtificial SequencePrimer 1260_1 7gagactctgg ctactcatcc 20821DNAArtificial SequencePrimer 1260_2 8ccttcagcaa gagctgggga c 21920DNAArtificial SequencePrimer PRRS7 9atcaaggaat cttgggagca 201017DNAArtificial SequenceActin forward primer 10cggaaccgcy cattggc 171120DNAArtificial SequenceActin backward primer 11acccacactg tgcccatcta 20

Claims

1. A compound having the following formula (I): ##STR00040## wherein B represents a boron atom; W is a nitrogen-containing functionality group, sustaining a positive charge either through protonation or quaternization, this group being selected from: (i) an amino group of the formula: ##STR00041## wherein G.sup.1 and G.sup.2 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms; (ii) a quaternary ammonium group of formula: ##STR00042## wherein G.sup.1, G.sup.2 and G.sup.3 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms; or (iii) a guanidine group of formula: ##STR00043## wherein G.sup.1, G.sup.2, G.sup.3, G.sup.4 and G.sup.5 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates one or more of the guanidine N atoms and that contains a total of up to 15 atoms; X is a linker unit having the formula (CX.sup.1X.sup.2).sub.p, wherein 1<p<10, and wherein X.sup.1 and X.sup.2 are, independently, H, or linear or branched alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring residue containing from 3 to 10 atoms; Y is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms; Z is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms, or is a linear or branched or cyclic alkyl group containing from 1 to 15 carbon atoms; R1 is chosen among: (i) an aminoacyl residue derived from an amino acid. (ii) a derivative of the aminoacyl residue of option (i), in which the amino group is additionally acylated, or sulfonylated, or phosphorylated to form an amide, or peptide, or sulfonamide, or phosphoramide bond; (iii) an acyl group of general formula R'--CO, wherein R' is: a. a linear, branched or cyclic alkyl group that contains from 1 to 10 C atoms; b. a saturated heterocyclic ring incorporating up to 20 atoms chosen from C, O, N, and S atoms; c. an aryl group selected from phenyl or a substituted variant thereof bearing any combination, at any one ring position, of one or more substituents selected from halogen, alkyl groups containing from 1 to 10 carbon atoms, trifluoromethyl and alkoxy groups; or d. a heteroaryl group; R.sup.2 is either H, or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms, or an OR group wherein R may be H or a or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms.

2. The compound according to claim 1, wherein groups G.sup.1 and G.sup.2 of the amino group of W are selected, independently, from the group consisting of: ##STR00044##

3. The compound according to claim 1, wherein groups G.sup.1, G.sup.2 and G.sup.3 of the quaternary ammonium group of W are selected, independently, from the group consisting of: ##STR00045##

4. The compound according to claim 1, wherein groups G.sup.1, G.sup.2, G.sup.3, G.sup.4 and G.sup.5 of the guanidine group of W are selected, independently, from the group consisting of: ##STR00046##

5. The compound according to claim 1, wherein group X is selected from the group consisting of: ##STR00047##

6. The compound according to claim 1, wherein group Y is selected from the group consisting of: ##STR00048##

7. The compound according to claim 1, wherein group Z is selected from the group consisting of: ##STR00049##

8. The compound according to claim 1, having the formula II: ##STR00050## wherein B represents a boron atom. W is a nitrogen-containing functionality group, sustaining a positive charge either through protonation or quaternization, this group being selected from: (i) an amino group of the formula: ##STR00051## wherein G.sup.1 and G.sup.2 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms; (ii) a quaternary ammonium group of formula: ##STR00052## wherein G.sup.1, G.sup.2 and G.sup.3 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms; or (iii) a guanidine group of formula: ##STR00053## wherein G.sup.1, G.sup.2, G.sup.3, G.sup.4 and G.sup.5 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates one or more of the guanidine N atoms and that contains a total of up to 15 atoms; X is a linker unit having the formula (CX.sup.1X.sup.2).sub.p, wherein 1<p<10, and wherein X.sup.1 and X.sup.2 are, independently, H, or linear or branched alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring residue containing from 3 to 10 atoms; Y is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms; Z is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms, or is a linear or branched or cyclic alkyl group containing from 1 to 15 carbon atoms; R.sup.2 is either H, or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms, or an OR group wherein R may be H or a or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms; and "aa" refers to any amino acid residue or derivative thereof, and n is at least two.

9. The compound according to claim 1, wherein the W--X--C--N(C.dbd.O)--R.sup.2 is an amino acid positively charged.

10. The compound according to claim 9, wherein said positively charged amino acid is arginine, lysine, ornithine or a derivative thereof.

11. The compound according claim 8, having the following formula III: ##STR00054## wherein B represents a boron atom; Y is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms; Z is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms, or is a linear or branched or cyclic alkyl group containing from 1 to 15 carbon atoms; R.sup.2 is either H, or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms, or an OR group wherein R may be H or a or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms; G.sup.1, G.sup.2 and G.sup.3 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates one or more of the guanidine N atoms and that contains a total of up to 15 atoms "aa" refers to any amino acid residue or derivative thereof, and n is at least two.

12. The compound according to claim 11, wherein at least one of R.sup.2, G.sup.1, G.sup.2 and G.sup.3 is H.

13. The compound according to claim 12, wherein G.sup.1, G.sup.2 and G.sup.3 is H.

14. The compound according to claim 12, wherein R.sup.2, G.sup.1, G.sup.2 and G.sup.3 are H.

15. The compound according to claim 8, having the following formula IV: ##STR00055## wherein B represents a boron atom; Y is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms; Z is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms, or is a linear or branched or cyclic alkyl group containing from 1 to 15 carbon atoms; R.sup.2 is either H, or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms or an OR group, wherein R may be H or a or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms; G.sup.1, G.sup.2 and G.sup.3 or G.sup.1 and G.sup.2 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates one or more of the guanidine N atoms and that contains a total of up to 15 atoms; "aa" refers to any amino acid residue or derivative thereof, and n is at least two; and m equals 3 or 4.

16. A compound according to claim 15 having the following formula V: ##STR00056## wherein B represents a boron atom; Y is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms; Z is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms, or is a linear or branched or cyclic alkyl group containing from 1 to 15 carbon atoms; R.sup.2 is either H, or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms, or an OR group wherein R may be H or a or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms; G.sup.1 and G.sup.2 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms; and "aa" refers to any amino acid residue or derivative thereof, and n is at least two.

17. A compound according to claim 8, having the following formula VI: ##STR00057## wherein B represents a boron atom; Y is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms; Z is an OH, or is an OR group wherein R is a linear, branched or cyclic alkyl group incorporating from 1 to 10 carbon atoms, or is a branch of a homocyclic or heterocyclic structure, which incorporates the B atom and which contains up to 20 atoms chosen from C, O, N, and S atoms, or is a linear or branched or cyclic alkyl group containing from 1 to 15 carbon atoms; R.sup.2 is either H, or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms, or an OR group wherein R may be H or a or a linear or branched or cyclic alkyl group containing from 1 to 10 carbon atoms; G.sup.1 and G.sup.2 are, independently, H, or linear or branched or cyclic alkyl groups containing from 1 to 10 carbon atoms, or branches of a ring system that incorporates the N atom and that contains a total of up to 15 atoms; and "aa" refers to any amino acid residue or derivative thereof, and n is at least two.

18. The compound according to claim 15, wherein at least one of R.sup.2, G.sup.1 and G.sup.2 is H.

19. The compound according to claim 15, wherein G.sup.1 and G.sup.2 are H.

20. The compound according to claim 15, wherein R.sup.2, G.sup.1 and G.sup.2 are H.

21. The compound according to claim 8, wherein n is comprised between 2 and 5.

22. The compound according to claim 21, wherein n equals 2, 3 or 4.

23. The compound according to claim 21, wherein n equals 2.

24. The compound according to claim 23, wherein said two amino acids residues are Tyr and Pro, Ala and Phe, Arg and Glu, Glu and Gly, Trp and Glu, Thr and Pro or Leu and Leu.

25. The compound according to claim 8, wherein n equals 3.

26. The compound according to claim 25, wherein said three amino acids residues are Phe, Arg and Val, Arg, Cys and Thr, Gly, Cys and Pro, Gly, Cys and Asn or Lys, Gly, Asp.

27. The compound according to claim 8, wherein the amino group of the first N-terminal amino acid residue bears a protecting group selected from the group consisting of acyl, sulfonyl or phosphoryl.

28. The compound according to claim 8, wherein the amino group of the first N-terminal amino acid residue is derivatized with an acyl, sulfonyl, or phosphoryl group to form an amide, or carbamate, or urea, or N-substituted urea, or peptide, or sulfonamide, or phosphoramide bond.

29. The compound according to claim 1, wherein at least one of Y or Z is OH.

30. The compound according to claim 1, wherein Y and Z are OH.

31. The compound according to claim 1, which is Glu-Gly-BoroArg, Ala-Phe-BoroArg, Ac-Glu-Gly-BoroArg and Ac-Ala-Phe-BoroArg.

32. The compound according to claim 1, which inhibits the enzymatic activity of the enteropeptidase.

33. The compound according to claim 32, wherein the enteropeptidase activity inhibition is selective over other serine proteases.

34. The compound according to claim 1, which is non-absorbable.

35. The compound according to claim 1, which is in a free base form or is a pharmaceutically acceptable salt thereof.

36. A composition comprising at least one compound according to claim 1.

37. The composition according to claim 36, further comprising at least one pharmaceutically acceptable carrier.

38. The composition according to claim 36, formulated for oral or enteral administration.

39. The composition according to claim 36, further comprising at least one other drug.

40. A method for treating a mammal having obesity, comprising administrating, in said patient in need of said treatment, at least one compound according to claim 1 or a composition according to claim 36.

41. The method according to claim 40, wherein the mammal is a human being.

42. The method according to claim 41, wherein obesity results from environmental causes, from genetic alterations, from medical illness, from smoking cessation, from medications or from neurological disorders.

43. A method for treating a mammal having excess weight, comprising administrating, to said mammal in need of said treatment, at least one compound according to claim 1 or a composition according to claim 36.

44. A method for treating a mammal suffering from abnormal fat metabolism comprising administrating, to said mammal in need of said treatment, at least one compound according to claim 1 or a composition according to claim 36.

45. The method according to claim 44, wherein the mammal is a human being.

46. The method according to claim 44, wherein said abnormal fat metabolism disease is selected from the group consisting of: gout disease, type II diabetes, hyperglycemia, hyperinsulinemia, elevated blood levels of fatty acids or glycerol, syndrome X, diabetic complications, dysmetabolic syndrome and related diseases, hypercholesterolemia, atherosclerosis, hypertension, pancreatitis, hypertriglyceridemia, hyperlipidemia, stroke, coronary heart diseases, peripheral vascular diseases, peripheral arterial diseases, vascular syndromes, cholesterol-related disorders or drug-induced lipodystrophy.

47. (canceled)

48. The compound according to claim 17, wherein at least one of R.sup.2, G.sup.1 and G.sup.2 is H.

49. The compound according to claim 17, wherein G.sup.1 and G.sup.2 are H.

50. The compound according to claim 17, wherein R.sup.2, G.sup.1 and G.sup.2 are H.

51. The compound according to claim 8, wherein at least one of Y or Z is OH.

52. The compound according to claim 8, wherein Y and Z are OH.

53. A composition comprising at least one compound according to claim 8.

54. A method for treating a mammal having obesity, comprising administrating, in said patient in need of said treatment, at least one compound according to claim 8 or a composition according to claim 53.

55. A method for treating a mammal having excess weight, comprising administrating, to said mammal in need of said treatment, at least one compound according to claim 8 or a composition according to claim 53.

56. A method for treating a mammal suffering from abnormal fat metabolism comprising administrating, to said mammal in need of said treatment, at least one compound according to claim 8 or a composition according to claim 53.