JP2002523365A - Regulation of substrate activity - Google Patents

Abstract

  (57) [Summary] Methods for modulating the activity of a substrate in vivo are useful for the treatment of medical disorders such as inflammation, arteriosclerosis and angiogenesis. The method includes administering an effective amount of a DPP-IV inhibitor to a patient in need of such treatment.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION The present invention relates to inhibitors of dipeptidyl peptidase IV used to control substrate activity. As used herein, the term "substrate" refers to chemokines, cytokines, and biological peptides that are substrates for DPP-IV. The invention also relates to the use of a DPP-IV inhibitor in the treatment of a medical disorder that may result from the inactivation of a substrate associated with the medical disorder. As used herein, dipeptidyl peptidase IV is alternatively described as "DPP-IV,""DP-IV," and "CD26." CD26
Is an ectoenzyme having the same activity as DPP-IV.

DPP-IV is a serine-type exopeptidase with high substrate specificity that cleaves N-terminal dipeptides from proteins when the penultimate amino acid is proline or, in some cases, alanine ( Fleischer, B .;
Immunol. Today 15: 180 (1994)).

A class of low molecular weight synthetic monomer molecules with high affinity for CD26 has been previously developed and characterized (GR Flentke et al., Xaa-boro).
Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Pro dipeptide and use of these inhibitors to test the role of DP-IV in T cell function, PNAS (USA) 88, 1556-1559 (1991);
W. G. FIG. Gutheil and W.C. W. Bachovchin, L-Pro-D
Separation of the components of L-boroPro into diastereomers and kinetic analysis of their dipeptidyl peptidase IV inhibition: a novel method for the analysis of slow, tightly coupled inhibition, Biochemistry 32, 8723-8731 (1993)). These molecules have been shown to be potent and specific synthetic inhibitors of DP-IV protease activity associated with CD26.

[0004] Representative monomer structures of inhibitors based on these transition state analogs, X
aa-boroPro (where Xaa is an amino acid residue) is designated as Pro-bo
roPro, Ala-boroPro, Val-boroPro, and Lys
-Including boroPro. BoroPro refers to an analog of proline in which the carboxylic acid group (COOH) is substituted with a boronyl group [B (OH) ₂ ]. The most thoroughly characterized Pro-boroPro of these inhibitors is 16
It has a Ki molar concentration (pM) of Ki (WG Gutheil and WW
Bachovchin, supra). Val-boroPro has an even higher affinity, a Ki of 1.6 pM (WG Gutheil and WW Bach).
ovchin, supra; J. Snow et al., Studies on Proline Boronic Acid Dipeptide Inhibitors of Dipeptidyl Peptidase IV: Identification of Cyclic Species Containing BN Bonds, J. Am. Am. Chem. Soc. 116, 10860-10869 (1
994)). Thus, these Xaa-boroPro inhibitors are about 10 ^{+ 6} times more potent than the second best known inhibitor.

[0005] US Pat. Nos. 4,935,493 (Bachovchin'493) and 5,462,928 (Bacho), both of which are incorporated herein by reference.
vchin'928) are protease inhibitors and transition state analogs (
('493 patent) and methods of treating transplant rejection, arthritis, or systemic lupus erythematosus (SLE) in patients by administering potent inhibitors of the catalytic activity of the soluble aminopeptidase activity of dipeptidyl peptidase type IV. (GR Frentke et al., Supra).

[0006] Chemokines, or chemoattractant cytokines, are a family of small proteins with conserved cysteine motifs. These small proteins are involved in a wide range of disease states, such as acute and chronic inflammatory processes, angiogenesis, leukocyte infiltration, regulation of cell proliferation and maturation, hematopoiesis, viral replication, and other immunomodulatory functions I have. Chemokines are expressed by many different cells and have distinct but overlapping cellular targets.

[0007] There are two groups of chemokines defined by their structural characteristics: the CXC group and the CC group. Further, C-chemokines and CX3C
-Chemokines have been identified. Members of the CXC group, including SDF-1 and IL-8, mainly attract neutrophils, whereas the CC group acts on monocytes and granulocytes. The CC group comprises human monocyte chemoattractant protein 1 (MCP-1) and RA
Includes molecules such as NTES. MCP-1 and RANTES are potent direct mediators of histamine release by human basophils. Both groups of chemokines are involved in lymphocyte migration to sites of inflammation.

[0008] Most of the chemokine receptors are transmembrane molecules belonging to the G protein-coupled receptor family. Many of these receptors bind to guanine nucleotide binding proteins and transmit cell signals. Chemokine and receptor expression is up-regulated during the inflammatory response and cell activation. Chemokines have been shown to be involved in many physiological conditions through binding to their respective receptors. For example, CX such as interleukin-8
Group C chemokines can stimulate angiogenesis. On the other hand, platelet factor-4, growth-associated oncogene-β (GRO-β) and interferon-γ-inducible protein-10 (IP-10) inhibit endothelial cell proliferation and angiogenesis. Interleukin-8 stimulates endothelial cell proliferation and chemotaxis in vitro and appears to be the major inducer of angiogenesis induced by macrophages.
The activity of these chemokines have been shown to be dependent on the NH ₂ -terminal amino acid sequence (Streiter et, J.Biol.Chem.270; 27348-2
7357). Another CXC chemokine, SDF-1, is active in recruiting and recruiting hematopoietic cells from the bone marrow and attracting monocytes and lymphocytes. In addition, it inhibits the interaction between HIV-1 and CXCR-4, thereby
Inhibits IV-1 cell infection. Like other chemokines in this group, the amino-terminal sequence regulates its activity (Shioda, T. et al., PNAS, 95; 6).
331-6336).

[0009] Chemokine receptors have not only been shown to act as receptors for chemokines, but have also recently been identified as receptors for various microorganisms and the HIV-1 virus. For example, the Duffy blood group antigen, the chemokine receptor on erythrocytes, is a receptor for the malaria parasite Plasmodium vivax, and the platelet-activating receptor is the Stereptococcus pne
umonia is a receptor.

[0010] Many chemokines, such as RANTES, MIP-1, and SDF-1, or cytokines, such as IL-2, or peptides, such as GLP-1, GLP-2, and substance P, are involved in DPP-IV Substrate. DPP-IV is
If the penultimate amino acid is proline, cleave the peptide at the NH ₂ terminus. Some cytokines and chemokines have the conserved sequence NH ₂ X-Pro
-X and was shown to be a substrate for DPP-IV. DPP-IV is expressed on the surface of T cells and macrophages. Although not clear relationship to its immune function CD26 protease activity, however, several NH cytokine ₂ - cleavage by CD26 dipeptides, been shown to alter the receptor specificity and / or functional activity I have.

[0011] Cytokines known to be potential substrates for DPP-IV are G-
CSF, erythropoietin, IL-1β, IL-2, IL-3, IL-6, IL
-11, TNF-β, and GM-CSF.

[0012] In addition to cytokines and chemokines, many biologically active peptides have at their amino terminus the amino acid sequence Xaa-, which functions as a substrate for DPP-IV.
Pro-Xaa. Among these are the glucagon-like peptides, GLP-1 and GLP-2. GLP-1 is involved in insulin release and glucose uptake, and cleavage by DPP-IV causes inactivation of its activity. Inhibition of DPP-IV prolongs the activation state of the peptide and represents a therapeutic indicator of a DPP-IV inhibitor. GLP-2 peptides are involved in intestinal growth and nutrient uptake, and the increased activity of GLP-2 increases nutrient uptake in patients with intestinal disease.

[0013] In addition to the foregoing, CD26 is known to be highly expressed in hepatosplenic T-cell lymphoma and has a DPP-IV activity, ie, the ability of CD26 to bind to extracellular matrix collagen fibronectin. May be part of the pathogenic mechanism utilized by neoplastic cells (Ruiz et al., Cytometer
y 1998, 34: 30-35).

[0014] DPP-IV and DPP-II levels are known to be significantly increased in gingival exudates of periodontitis patients. These two increased levels of proteases
It appears to be associated with increased adhesion loss. The collagen-CD26 interaction is considered to be part of the pathological observation.

[0015] PCT published application WO 95/11689 describes DPP- for inhibiting CD26.
Disclosed are the use of IV inhibitors, thereby inhibiting the entry of HIV into cells having CD26. These inhibitors have the general formula X-Pro-Y-boroP
It is a tetrapeptide having ro. Where X and Y are selected from any amino acids. Although dipeptides are also disclosed, the references state that dipeptides are unstable and that tetrapeptides are preferred. Inhibitors are used to treat pre-symptomatic HIV-infected patients, rather than by neutralizing the virus, but by inhibiting the entry of the virus into cells. References are:
The effect of DPP-IV inhibition on chemokine activity is not disclosed.

SUMMARY OF THE INVENTION The present invention provides a method for the treatment of a medical disorder in a patient mediated by the activity of a substrate. According to the method, the pharmaceutical composition is administered to the patient in an amount effective to inhibit DPP-IV activity. This pharmaceutical composition contains, as an active ingredient, a compound represented by the general formula PR. Where P is a targeting molecule that binds to DPP-IV and R is a reactive group that reacts with a functional group of DPP-IV, preferably a reactive center of DPP-IV. Preferably, the active ingredient is a compound containing an amino group covalently linked to the alpha aminoboronic acid analog of proline ("boroPr
The term "o" is used herein to refer to an analog in which the carboxyl group of proline has been replaced with a B (OH) ₂ group. Where (OH) ₂ represents two hydroxyl groups and B represents boron). The active ingredient is therefore Xaa-boroPr
o. Here, Xaa is an amino acid residue. Most preferably, the active ingredient is Val-boroPro. Where boroPro at the carboxyl end
The line connects to the valine amino acid residue via a peptide bond by standard peptide chemistry.

[0017] This active ingredient is a DPP-IV substrate.
It acts to suppress activity and increases the bioavailability of chemokines that are active in vivo. The net effect is, among others, inflammation, angiogenesis, arteriosclerosis,
Clear therapeutic benefits to patients for certain disease states, such as bowel disease, diabetes, anorexia, and antitumor activity. It could not be expected based on current knowledge about DPP-IV activity or inhibition.

Substrates applicable to the methods of the invention include those that share a conserved NH ₂ —X-Pro sequence, where X is any amino acid or short peptide. These D
The substrate for PP-IV, and its activity, changes upon cleavage of the N-terminal sequence. The altered substrate may theoretically have either enhanced or attenuated activity. However, they are most typically deactivated to some extent. Particular examples of chemokines inactivated by DPP-IV include, but are not limited to, SDF-1, RANTES, MIP-3. Inhibition of DPP-IV activity inhibits the digestion of chemokines, cytokines and growth factors. The substrates of the present invention have been implicated in various disease states, including inflammation, atherosclerosis, angiogenesis, and antitumor activity. Inhibition of DPP-IV by the active compounds of the present invention is believed to cause (increase) the bioavailability of the substrate to enhance the response to medical trauma.

Pharmaceutical compositions typically contain the active compound (preferably Xaa-boroPro).
And most preferably Val-boroPro), and a pharmaceutically acceptable carrier. Pharmaceutical compositions can also include various adjuvants, enhancers, cytokines, and the like, as known to those of skill in the art. Patient dosage is typically 0.001
The range is from mg / kg to 100 mg / kg.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of certain compounds to inhibit DPP-IV activity when properly administered to a subject. As used herein, the term "subject" is intended to mean humans, non-human primates, dogs, cats, sheep, goats, horses, cows, pigs, and rodents. Preferably, the subject is a human patient undergoing medical treatment.

[0021] It has now been discovered that inhibition of DPP-IV activity results in changes in certain substrates involved in various medical disorders. Since the substrate of the present invention is a substrate for DPP-IV, it is believed that DPP-IV acts on the substrate in vivo to cleave the N-terminus of the two sequences where the penultimate amino acid is proline. Alternatively, another peptidase, such as peptide A or N, cleaves the terminal sequence to Xaa-Pro-X
The aa portion may be exposed. This cleavage can result in a change in the receptor specificity or functionality of the substrate, and typically results in a change in the activity of the substrate. This can directly result in the patient's response to a particular medical disorder or trauma. For example, SDF-1,
Certain chemokines such as MIP-1 and RANTES are known to act as attractants for lymphocytes, monocytes and the like. Cleavage of these chemokines by DPP-IV alters its activity and recruits lymphocytes to sites of inflammation,
Affects hematopoiesis or immune function. DPP-IV inhibitor is DPP-IV
By either competitive interaction with or binding to the active site
It acts to inhibit IV from cleaving chemokines.

Active Compounds Compounds useful in the present invention inhibit DPP-IV and include, but are not limited to, compounds encompassed by the formula PR. Where P represents a targeting molecule that binds to DPP-IV, and R is a functional group of DPP-IV, preferably DPP-IV.
It represents a reactive group that reacts with the reaction center of IV. P has the formula: D～A containing DPP-IV binding molecules encompassed by ₁ -A ₂ -A ₃ -A _4, can be any molecule that binds to DPP-IV. Wherein D is independently selected from the group consisting of NH and NH ₂ ,
Where N represents any isotopes of nitrogen, where H represents all isotopes of hydrogen, "~" is independently selected from the group consisting of a single bond and a double bond; A ₁ is C,
Selected from the group consisting of CX, and N, wherein C represents any isotope of carbon, X represents any atom that forms a single bond with carbon; A ₂ , A ₃ , and A ₄ each represent CX Moieties, CXZ moieties, CZ moieties, NX moieties, and O, wherein X and Z are any atom forming a single bond with C or N, and any molecule forming a double bond Independently selected from the group consisting of and wherein O represents any isotope of oxygen.

Compounds useful according to the present invention include the following alternative structures, referred to as Group I or Group II.

Group I has the following structure:

[0025]

Embedded image Where H represents hydrogen; C represents carbon; O represents oxygen; N represents nitrogen;
R is each independently selected from the group consisting of R groups of amino acids including proline; X
Represents any atom forming a single bond having carbon (including hydrogen and halogen); Y represents the following:

[0026]

Embedded image And R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , and R ₈ are each independently:
Does not significantly interfere with the site-specific recognition of inhibitory compounds by DPP-IV, and DP
A group that allows a complex to be formed with P-IV. Preferably,
R ₁ -R ₈ are H, where H each represents a hydrogen atom; and q and p are integers that vary independently between 0 and 4, inclusive.

Alternatively, Group I has the following structure:

[0028]

Embedded image Wherein n is between 0 and 3 inclusive, and G ₂ and G ₃ are each independently H or C ₁ -C ₃ (1-3 carbon atoms) alkyl , G ₁ is NH ₃ (
H ₃ represents three hydrogens) or

[0029]

Embedded image (H ₂ represents two hydrogens), or G ₁ is NG ₄ , wherein G ₄ is

[0030]

Embedded image Where G ₅ and G ₆ can be NH, H or C ₁ -C ₃ alkyl or C ₁ -C ₃ alkenyl, wherein one or more carbons have been replaced by nitrogen. G ₁ carries a charge, and G ₁ and G ₂ do not form a covalent ring structure at pH 7.0.

Group I may also have the following structure:

[0032]

Embedded image Wherein one or two of the groups a, b, c, d, e and f are N and the remainder is C, and S ₁ -S ₆ are each independently H Or C ₁ -C ₃ alkyl. Group I may also include an unsaturated 5-membered ring having two nitrogen atoms, for example, an imidazole ring.

Group II has the following structure:

[0034]

Embedded image Where T is a group of the formula:

[0035]

Embedded image Wherein D ₁ and D ₂ are each independently a hydroxyl group or a group that can be hydrolyzed to a hydroxyl group in an aqueous solution at physiological pH; or T is a group of the formula:

[0036]

Embedded image Wherein G is any of H, F or an alkyl group containing 1-20 carbon atoms and optionally a heteroatom which may be N, S or O; or T is a phosphonate group of the formula:

[0037]

Embedded image Wherein each J is independently any combination of any number of C, H, O or N atoms, or O-alkyl, N-alkyl or alkyl, wherein O-alkyl, N -Alkyl or alkyl each contains 1 to 20 carbon atoms and optionally contains a heteroatom which can be N, S or O;

[0038]

Embedded image Wherein R is an alkyl or aryl group and may be substituted or unsubstituted and is an alpha ketoester; or

[0039]

Embedded image T can generally form a complex with the catalytic site of DPP-IV.

Y is:

[0041]

Embedded image And R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , and R ₈ each independently do not significantly interfere with the site-specific recognition of the inhibitory compound by DPP-IV. And a group that enables it to form a complex with DPP-IV. Preferably, R _1-
R ₈ is H. X is any number of C, H, O, S, or N atoms in any combination, including any amino acid or organic molecule, and m can vary from 0 to 20.

In a preferred embodiment, T is a boronic acid group, a phosphonate group, a cyano group, or a trifluoroalkyl ketone group; R ₁ -R ₈ are each H; R ₁ and R ₂ are each H and Y are each is CH ₂ -CH _2; R are each selected from R groups independently proline and alanine; inhibition compound least 10 ^-9 M
Has a binding or dissociation constant for DPP-IV of 10 ^-8 M or even 10 ^-7 M; and D1 and D2 are each independently F, or D1 and D2 are both 1-20 carbon atoms; And optionally a ring containing a heteroatom which can be N, S or O. These compounds are described in U.S. Patent No. 5,462,928, which is hereby incorporated by reference.

More preferred compounds are of the formula:

[0044]

Embedded image Wherein D ₁ and D ₂ are each independently a hydroxyl group or a group that can be hydrolyzed to a hydroxyl group in an aqueous solution at physiological pH; and wherein X mimics a site on the substrate that is recognized and cleaved by DPP-IV. A targeting molecule, and preferably it is an amino acid, imino acid, or a peptide that mimics the site of the substrate recognized by postproline degrading enzyme; C is attached to B in an L-shaped configuration; The bonds between C and between Y and N are peptide bonds. The expression "C is attached to B in the L configuration" means that the absolute configuration of C is similar to that of the L amino acid.

The active Xaa-boroPro compounds of the present invention may have a cleaved chain (linear) or cyclic form. The linear form can be converted to the cyclic form by the trans to cis isomerization of proline and the formation of new NB bonds. Therefore, "cyclic type" is
Refers to the cyclized structure of the compound described in the above formula which is a boron analog of diketopiperazine.

In a particularly preferred embodiment of the invention, the active compound is a Xaa-boroPro compound, and even more preferably a Val-boroPro compound. "Val-boroPro compound" refers to a compound as defined in the above formula wherein the carboxy-terminal boroPro is covalently linked to the valine amino acid residue via a peptide bond by standard peptide chemistry. In a most preferred embodiment, the compounds of the invention are Val-boroPro (by the manufacturer's nomenclature "
PT-100 ").

Preferred active compounds have a targeting moiety that is a peptide that mimics the substrate binding site of DPP-IV. Peptide analogs and non-peptide or peptidomimetics can also be used as targeting moieties. Such molecules can be rationally designed based on the known sequence of the DPP-IV substrate, and can be identified using combinatorial chemistry and screening assays, such as those described below.

The development of phage display and chemical combinatorial libraries allows the selection of synthetic compounds that mimic the substrate binding site of proteases such as DPP-IV. Such libraries can be obtained by assaying for protease cleavage activity in the presence or absence of putative phage display library molecules or combinatorial library molecules, and by analyzing the molecule for its natural substrate or substrate analog (e.g., By determining whether or not it inhibits cleavage of the chromophore substrate analog), which is easily detectable by spectrophotometric assays, it can be screened to identify non-natural putative targeting molecules. Those phage library and / or combinatorial library molecules that exhibit protease inhibition can then be covalently linked to the reactive groups R disclosed herein, and again these new molecules selectively bind to proteases (E.g., by repeating the screening assay described above). In this manner, a simple, high-throughput screening assay is provided to identify non-naturally-targeted moieties of the invention.

Substrates The substrates of the present invention are substrates that share a conserved NH ₂ -X-Pro sequence (where X is any amino acid or short peptide) at the NH ₂ end of the molecule. Substrates having this structural configuration act as substrates for DPP-IV. The inhibitory effect of the compounds on DPP-IV acts to increase the bioavailability of the substrate in the subject, which in turn produces a positive biological effect. For example, increased bioavailability of a selected substrate can produce increased immune and anti-inflammatory functions in a subject. Suitable substrates are known in the art, but to date no one has associated the use of DPP-IV inhibitors with a positive medical effect on their condition mediated by the presence and activity of the substrate.

Representative substrates that are the subject of the present invention are described more fully below. However, as a matter of course, the substrate is not other specific with NH ₂ -X-Pro sequence conserved also include, but are not particularly described, it is recognized that within the scope of the present invention.

SDF-1, or stromal cell-derived factor 1, is a CXC chemokine that contains a proline residue at the second position from the N-terminus of the molecule. SDF-1 acts on lymphocytes and monocytes but not on neutrophils in vitro. And a potent mononuclear cell attractant that is effective in vivo. SDF-1 is expressed in a wide range of tissues, which may aid in the treatment of arteriosclerosis and may also have anti-HIV activity.

MIP-1 (macrophage inflammatory protein-1) is a lymphocyte attractant required for immune and inflammatory responses.

RANTES (regulated on activation, nor
mal T-cell expressed and secreted)
It regulates integrin adhesion and has also been implicated in inflammatory diseases.

GLP-1 (glucagon-like peptide-1) is known to stimulate insulin secretion. This effect is limited in vivo due to the rapid degradation of GLP-1 by DPP-IV. Inhibition of DPP-IV with the active compounds of the invention can enhance its insulinotropic effects and provide assistance in the treatment of diabetes.

GLP-2 (glucagon-like peptide-2) is known to stimulate small intestinal growth through the induction of intestinal epithelial proliferation. GLP-2 is also in vivo in DPP-
Inactivated by IV. Inhibition of DPP-IV may increase the capacity for digestion and absorption of nutrients in vivo and may provide treatment for AIDS, anemia, and anorexia. GLP-2 may be therapeutically useful for enhancing mucosal regeneration in patients with intestinal disorders. Inhibition of DPP-IV is demonstrated by enterostatin and desangine-enterostost in rat jejunum.
Promotes absorption of atin.

G-CSF (granulocyte-colony stimulating factor) is a growth factor for hematopoietic cells such as neutrophils.

EPO (erythropoietin) is a red blood cell growth factor.

[0058] IL-6 (interleukin-6) is a hematopoietic and lymphocyte growth factor.

[0059] IL-11 (interleukin-11) is a lymphokine.

[0060] IL-8 (interleukin-8) is a hematopoietic growth factor, an angiogenic cytokine.

[0061] Substance P is a neuropeptide and hematopoietic growth factor.

Other suitable chemokines are substance P, which has vasoactive properties, monomeric fibrin which causes blood clotting, fibronectin which can promote hepatocyte binding and enhance liver regeneration, B cells A type I, II, III, and IV collagen that partially regulates the movement of many effector cells, including T cells, through the endothelial barrier. Including.

Similarly, other medical disorders that can be treated by the methods of the present invention include allergies,
Includes angiogenesis, cardiac development, anti-tumor response, liver disease, and organ neovascularization.

[0064] Substrates known and unknown and capable of acting as substrates for DPP-IV, not specifically disclosed herein, are considered to be entirely within the scope of the present invention.

Formulation of Pharmaceutical Compositions The compounds of the invention or compositions thereof may be administered alone, or in combination with each other, or in combination with other therapeutic agents. For example, treatment with one or more compounds of the present invention may be combined with more traditional treatments for treating medical disorders, or with other cytokines to enhance the success of the treatment.

When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions. Such preparations include salts, buffers,
Preservatives, compatible carriers, and optionally other therapeutic agents may conventionally be included. When used in medicine, the salts are pharmaceutically acceptable, but the non-pharmaceutically acceptable salts are conveniently used to prepare the pharmaceutically acceptable salts. It is not excluded from the scope of the present invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: Hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid and the like. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts. The pharmaceutical composition may also optionally include a suitable preservative such as benzalkonium chloride; chlorobutanol; parabens; and thimerosal. Oral,
Formulations of carriers suitable for subcutaneous, intravenous, intramuscular administration, etc.
Pharmaceutical Sciences, Mack Publics
ing Co. , Easton, PA. Routes of Administration Various routes of administration are available for treating a subject. The particular mode of delivery chosen will, of course, depend on the particular compound chosen, the severity of the condition being treated and the dosage required for therapeutic efficacy. The method of the present invention generally comprises
It may be performed using any mode of administration that is medically acceptable. This means any manner of producing an effective level of the active compound without producing adverse clinically unacceptable effects. Such modes of administration include oral, rectal, topical, nasal, interdermal and parenteral routes.

Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the invention. Other compositions include suspensions in aqueous or non-aqueous liquids such as syrups, elixirs or emulsions. Oral preparations may include an enteric coating.

The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, or infusion. Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the compound, which is preferably isotonic with the blood of the recipient. This aqueous preparation can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. This sterile injectable preparation also
It can be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed. This includes synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Intravenous or intramuscular routes are not particularly suitable for long-term treatment and prevention. But,
These are preferred in emergency situations. Oral administration is preferred for prophylaxis and other treatments due to the ease of the patient and the dosing regimen.

Pharmaceutical compositions can be conveniently administered in unit dosage form and can be prepared by any of the methods well-known in the art of pharmacy. The methods include the step of bringing the compound of the invention into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the compound into association with a liquid carrier, a finely divided solid carrier or both, and then, if necessary, shaping the product. .

Other delivery systems may include timed, delayed or sustained release delivery systems. Such a system may avoid repeated administrations of the compounds described above, increasing convenience to subjects and physicians. Many types of release delivery systems are available;
And it is known to those skilled in the art. These include polymer-based systems (eg, poly (lactide-glycolide)) copolyoxalate, polycaprolactone, polyesteramide, polyorthoester, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the above polymers containing drugs are described, for example, in US Pat. No. 5,075,109. Delivery systems also include non-polymeric systems, including: sterol-containing lipids such as cholesterol, cholesterol esters and fatty acids or neutral lipids such as monoglycerides, diglycerides and triglycerides; hydrogel release systems; stick(
peptide-based systems; wax coatings; compressed tablets with conventional binders and excipients; partially melted implants and the like. Specific examples include, but are not limited to: (a) perishable systems, wherein
This compound is disclosed in U.S. Pat. Nos. 4,452,775 and 4,667,014;
No. 4,748,034 and 5,239,660), and (b) a diffusion system wherein the active compound is controlled U.S. Pat. Nos. 3,832,253 and 3,854 at speeds
480). In addition, pump-based hardware delivery systems may be used, some of which are adapted for implantation.

The use of a long-term sustained release implant is particularly suitable for treatment of chronic conditions. As used herein, "prolonged release" means that the implant is constructed and arranged to deliver the active ingredient at therapeutic levels for at least 10 days, more preferably 60 days. Long-term sustained release implants are well known to those skilled in the art and include some of the sustained release systems described above.

[0072] The compounds described herein are administered in effective amounts. An effective amount is a dosage of the compound that is sufficient to provide a medically desired result. The effective amount will depend on the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent treatment, if any, the particular route of administration, and the like. It varies according to factors, which are within the knowledge and experience of health industry workers.
An effective amount to stimulate a desired immune response can also be measured, including, for example, altering immune function in a subject (eg, increasing a B cell response, increasing a cytotoxic T cell response or delaying infection). , Stopping or preventing). An effective amount for treating an autoimmune disease or allergic disorder is an amount sufficient to attenuate or inhibit the immune or allergic response associated with the disorder so as to slow or stop the onset or progression of the disorder. is there.
As used in the claims, "inhibition" includes all of the above. Similarly, an effective amount for treating an immune system disorder will delay or arrest all symptoms associated with the immune system disorder in order to prevent, delay the progression or stop the progression of the immune system disorder. Is the amount to get. In general, it is preferred that the highest dose be used, that is, the highest safe dose according to mild medical judgment.

Generally, doses of active compound will range from about 0.001 mg / kg / day to 1000 mg / kg.
kg / day. A dose range of 0.01 to 100 mg / kg / day is appropriate, and is expected to be preferably oral and from one to several administrations per day.
Smaller doses result from other forms of administration, such as intravenous administration.
The initial dose at which the response in the subject is applied is insufficient and higher doses (or more efficient, higher doses due to a different and more localized route of delivery) are tolerated to the extent that patient tolerance allows. Can be used. Multiple doses per day are intended to achieve appropriate systemic levels of the compound.

All patents, references and other references mentioned in this patent application are hereby incorporated by reference in their entirety.

The following examples are for illustrative purposes only and are not intended to limit the invention in any way. Val- which is a particularly preferred compound of the present invention
boroPro ("PT-100") is used in the examples.

Throughout the application, and in particular in each example, particular embodiments have been described and illustrated. It should be understood that any of the reactive groups disclosed herein can be substituted for a particular reactive group (eg, a boron group) described in the examples. Example 1 General Synthesis of Active Compounds The synthesis of the boroPro compounds of the present invention is described in Bachovchin '493. Generally, this preparation technique involves linear peptide coupling chemistry. Standard peptide coupling chemistry methods and procedures used in the present invention are readily available. Examples of books using these methods include, but are not limited to, the following references (incorporated herein by reference): D. Bailey, An Introduction to
Peptide Chemistry, Publisher: John Wiley & S
ons, 1990; Miklos Bodansky, Peptide Che
mistry, A Practical Textbook, Publisher: Spring
ger-Verlag, 1988; Miklos Bodansky, Prin.
chips of Peptide Synthesis, "Reactive
entity and Structure Concepts in Organi
c Chemistry, "Vol. 16, Publishing: Springer-Verla.
g, 1984; and Miklos Bodansky, Principle
s of Peptide Synthesis, "Reactivity a
nd Structure Concepts in Organic Che
mistry, "Volume 21, Publishing: Springer-Verlag, 1984.
.

The compounds of the present invention can be prepared using H-boro as taught in WO 98/00439.
One can start with the synthesis of Pro. The use of H-boroPro is for illustrative purposes only and is not intended to limit the scope of the present invention.

According to WO 98/00439, H-boroPro was developed previously,
And the described synthetic route (GR Flentke, et al., "Inhibiti
on of dipeptidyl aminopeptidase IV (D
P-IV) by Xaa-boroPro dipeptides and u
se of these inhibitors to exam th
e role of DP-IV in T-cell function, "
PNAS (U.S.A.) 88,1556-1559 (1991); also described in U.S. Patent No. 5,462,928). Or,
H-boroPro may be generated by a new procedure (Kelly, T.A.
Et al., "The effective synthesis and simpl.
e resolution of a proline boronate e
star suite for enzyme inhibition
studies, "Tetrahedron 49, 1009-1016 (19
93)). Both of these synthetic routes have been reported to produce racemic H-boroPro pinanediol.

According to WO 98/00439, stereochemically pure Z-Lys-boro
The L, L and L, D diastereomers of Pro are first converted to the racemic H-bo
roPro is converted to an optically active block protecting group ((IS, 2S, 3R, 5S)-+
-Pinanediol isomer) and subsequent coupling of isotopically pure L-boroPro and D-boroPro with the stereochemically pure L-isomer of lysine (US Patent No. 5,462,928). Alternatively, racemic H-boroPro is converted to L-Lys
And the resulting diastereomer Z-Lys-bo
The roPro-diester has been described previously for the diastereomers of its components L, D and L, L and reverse phase HPLC has been described previously for the diastereomer Pro-boroPro (WG Gutheil and WW Bachovchin, "S
evolution of L-Pro-DL-boroPro into I
ts Component Diastereomers and Kinet
ic Analysis of Their Inhibition of D
ipeptidyl Peptidase IV. A New Method
for the Analysis of Slow, Tight-Bind
ing Inhibition, "Biochemistry 32, 872
3-8731 (1993)) to give Lys-b
The diastereomers of L, L and L, D of oroPro were prepared. . Example 2: Synthesis of Active Cyclic Compounds In aqueous solution at all pH values, the inhibitor slowly equilibrates in two conformations (open-chain structure (linear boroproline compound. (Active species)) and cyclic structures (cyclic boroproline compounds, which are inert)). This cyclic structure is preferred at neutral pH, whereas the inhibitory species of the opening activity is preferred at low pH. The reaction is completely reversible: the chain at this opening is low pH
Become dominant in The reaction from the open chain to the cyclic species involves the trans to cis isomerization of proline and the formation of a new NB bond. This cyclized structure is the boron analog of diketopiperazine, which is a product often found in peptide chemistry. Cyclization releases one equivalent of H +, which explains the need for a base in cyclic reactions and an acid in open reactions. The cyclic structure is very stable in high pH aqueous solutions.

Prolonged incubation at high pH was not possible with the tested Xaa-boroPro
It does not result in complete loss of DPP IV inhibitory activity for any of the compounds.
This observation was the first evidence that the activity inhibitor was in a conformational equilibrium with the non-inhibiting species rather than undergoing irreversible inactivation. The half-life for reconstitution of cyclic structures into open-chain species is surprisingly low. Thus, it was concluded that the loss of inhibitory activity in aqueous solution was due to a pH dependent conformational equilibrium rather than a degradation reaction.

The fact that the inhibitory activity does not go to zero for any of the Xaa-boroPro inhibitors after prolonged incubation is combined with the fact that the reverse reaction (ie, the cyclic to open chain) is slow. It was suggested that measuring the apparent Ki at equilibrium and that it should be possible to determine the equilibrium constant for conformational equilibrium by comparing it to the true Ki. [Cyclic] form: [open] form ratio at neutral pH, Pro-boro
156: 1 for Pro and 1 for Val-boroPro.
130: 1 (WG Gutheil and WWB).
achovchin, Separation of L-Pro-DL-bor
oPro into Its Component Diastereomer
s and Kinetic Analysis of Their Inhi
bition of Dipeptidyl Peptidase IV. A
New Method for the Analysis of Slow,
Tight-Binding Inhibition, Biochemistr
y 32, 8723-8731 (1993)). This results in less than 1% Pro-boroPro and less than 0.1% Val-boroP at equilibrium at pH 7.0.
ro means that it is present as an inhibitory species of the open chain. Nevertheless,
Under these conditions, these inhibitors were 2.5 nM and 1.8, respectively.
It behaves as if it has a Ki of nM. This apparent Ki of the "fully inactivated" species is still substantially (about 1000-fold) better than that of the other inhibitors of DPP-IV reported so far.

The inventors believe that the cyclic compounds of the present invention have the ability to specifically bind to CD26. Thus, the present inventors have determined that the biological function of the compounds of the invention allows oral administration and conformational changes (eg, linearization) of the cyclic compounds of the invention to occur in vivo (eg, , Under acidic conditions of the stomach).

Thus, where linearization is required, linearization can be achieved in vivo, and thus therapeutic concentrations in the systemic circulation can be generated in situ, thus reducing the biological activity of the compounds of the invention. It is believed that it can increase by about 100-1000 times. In addition, the cyclic boroproline compounds of the invention, in lyophilized or solid form, are believed to have improved shelf-life properties, thereby contributing to the further utility of the compounds of the invention.

Each of the compounds prepared as described above can be purified to homogeneity using HPLC, and their identities are confirmed by NMR spectroscopy, amino acid composition or mass spectrometry when deemed necessary. Can be done.

The cyclic compounds of the present invention can be converted to the linear form by adjusting the pH to an acidic pH (eg, the pH range is 1-3), and the CD26 proteinase activity of the linear boroproline compound is reduced. Inhibition capacity can be determined using conventional enzyme assays (examples are provided below). In addition, the immunomodulatory effects of the compounds of the present invention may be determined by in vivo experiments using animal models and in vitro experiments using cell culture methods (these methods are considered by one skilled in the art to be predictive indicators of in vivo activity). .).

Example 3 Evaluation of Functional Activity The compounds of the present invention have at least the following properties: (I) the binding site is DPP
-IV active site; and (ii) exhibits species cross-specificity.

Assays used to assess functional activity include: DPP-IV activity, oral and subcutaneous bioavailability assays,
These are described below.

Example 4: Measurement of standard DPP-IV activity Assays for measuring DPP-IV activity can be performed on compounds of the present invention. Interaction of small peptidomimetic inhibitors with DPP-IV and CD
A method has been developed for quantitatively measuring the interaction of H.26 with larger ligands (eg, HIV Tat protein) (WG Gutheil and WW Bachovchin. Separation of L-Pro-).
DL-boroPro into Its Component Diaste
reomers and Kinetic Analysis of Thei
r Inhibition of Dipeptidyl Peptidase
IV. A New Method for the Analysis of
Slow, Tight-Binding Inhibition, Bioch
mistry 32, 8723-8731 (1993); Gutheil, W.
. G. FIG. , And W.C. , B. W. Kinlsq, A Matlab Program
m for Fitting Kinetics Data with Num
electrically integrated Rate Equations a
nd Its Application to the Analysis o
f Slow, Tight Binding Data, Analytical
Biochemistry 223, 13-20 (1994); Gutheil
, W.S. G. FIG. , Et al., HIV-1 Tat Bindings to DP IV (CD2
6): A possible Mechanism for Tat's Im
Munosuppressive Activity, Proc. Natl. A
cad. Sci. U. S. A. 91, 6594-6598 (1994)). These methods involve the chromogenic substrate Ala-Pro-p-nitroanilide (AppNA) and the fluorescent substrate Ala-Pro-7-amino-4-trifluoromethylcoumarin (
(AP-AFC). AppNA and AP-AFC are commercially available (eg, Enzyme Systems Products, Dublin,
CA).

Example 5 Measurement of Oral Bioavailability An in vitro assay for serum DPP-IV activity was developed. This can be used as a surrogate marker to determine the bioavailability of PT-100 in serum after subcutaneous or oral administration in mice. This assay is based on the ability of PT-100 to inhibit serum DPP-IV protease activity in a dose-dependent manner.

As shown in FIG. 1, mice were given PT-100 at the indicated dose by gavage gavage. Blood samples were taken two hours after administration and the serum synthetic substrate DPP-
Determine IV activity. DPP-IV activity is measured in a fluorometric assay using 7-amino-4-trifluoromethylcoumarin (AFC) AlaPro (15). All inhibition of serum DPP-IV activity was inhibited by 2-5 μg of PT-10.
Achieved at zero dose. Dose less than 2 μg results in a dose-dependent decrease in DPP-IV inhibition.

Although the present invention has been described with reference to particular embodiments, it should be understood that many modifications and changes may be made by those skilled in the art without departing from the spirit of the invention. Such modifications, changes and equivalents are intended to fall within the scope of the appended claims.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows PT-100 (Val-boroPro) and HC of PT-100.
Figure 2 shows the bioavailability of L and methanesulfonate.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 29/00 A61P 31/00 31/00 31/12 31/12 35/00 35/00 37/08 37 / 08 43/00 105 43/00 105 111 111 C12N 9/99 // C12N 9/99 A61K 37/02 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR , GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD) , TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT , AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX , NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZWF Terms (reference) 4C084 AA02 AA17 BA01 BA08 BA14 BA15 BA16 BA17 BA23 BA31 CA59 DA58 DA59 MA52 NA14 ZA36 ZA45 ZA66 ZA75 ZB11 ZB13 ZB21 ZB26 ZB32 ZB33

Claims (21)

[Claims] 1. A method for treating a medical disorder in a subject mediated by a change in the activity of a substrate, comprising: administering to the subject an effective amount of a compound having the formula PR. Process, where:
P represents a target moiety that binds to DPP-IV, and R represents a reactive group that reacts with the reactive center of DPP-IV, the amount of which interferes with chemokine changes by inhibiting DPP-IV activity. A process that is sufficient to 2. The method of claim 1, wherein said compound has the following formula: Wherein H represents hydrogen; C represents carbon; O represents oxygen; N represents nitrogen; and R is each independently selected from the group consisting of R groups of amino acids including proline. X represents any atom forming a single bond having carbon; Y represents: And R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , and R ₈ are each independently:
Does not significantly interfere with the site-specific recognition of inhibitory compounds by DPP-IV, and DP
H is a group that allows a complex to be formed with P-IV, H represents a bond or hydrogen, respectively; and q and p vary independently between 0 and 4, inclusive. The method, which is an integer. 3. The method of claim 1, wherein said compound has the following formula: Wherein n is between 0 and 3 inclusive, G ₂ and G ₃ are each independently H or C ₁ -C ₃ alkyl, and G ₁ is NH ₃ Or Or G ₁ is NG ₄ , wherein G ₄ is , And the where, G ₅ and G ₆ are, NH, is H or one or more C ₁ -C ₃ carbon atoms is substituted with nitrogen alkyl or C ₁ -C ₃ alkenyl obtained; G _1, the charge And G ₁ and G ₂ do not form a covalent ring structure at pH 7.0. 4. The method of claim 1, wherein said compound has the following formula:Wherein one or two of the groups a, b, c, d, e and f are
N and the rest are C and S₁~ S₆Are each independently H or C ₁ -C_ThreeThe method is an alkyl. 5. The method of claim 1, wherein said compound has two nitrogen atoms.
The method is an unsaturated 5-membered ring having one or more rings. 6. The method of claim 5, wherein said compound is an imidazole ring. 7. The method according to claim 1, wherein the compound has the following formula: Wherein T is a group of the following formula: Where D ₁ and D ₂ are each independently a hydroxyl group or a physiological pH
A group that can be hydrolyzed to a hydroxyl group in an aqueous solution of the formula: Wherein G is H, F or any alkyl group containing a heteroatom which may be 1 to 20 carbon atoms and optionally N, S or O; a phosphonate group of the following formula: Wherein each J is independently any combination of any number of C, H, O or N atoms, or is O-alkyl, N-alkyl or alkyl, wherein O-alkyl, N- Alkyl or alkyl each contains 1 to 20 carbon atoms and optionally contains a heteroatom which may be N, S or O; groups of the formula: A group of the following formula: Wherein R is an alkyl or aryl group and may be substituted or unsubstituted and is an alpha ketoester; or a group of the following formula: Wherein Y is a group of the following formula: Wherein each R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ is H; X is any number of C, H, O, S or N And m can vary between 0 and 20. 8. The method according to claim 7, wherein T is a boronic acid group, a phosphone group.
R, a cyano group, or a trifluoroalkyl ketone group;₁And R_Two Are each H and Y is each CH_Two-CH_TwoR is each independently
Selected from the R group of lorin and alanine; ^-9 M has a binding or dissociation constant of DPP-IV; and D1 and D2 are
Each independently is F or D1 and D2 together form 1-20 carbons
A ring comprising atoms and optionally a heteroatom which may be N, S or O. 9. The method according to claim 7, wherein the compound has the following formula: Wherein D1 and D2 are each a hydroxyl group; wherein X is an amino acid; and C is attached to B in the L configuration. 10. The compound according to claim 9, wherein said compound is Val-boroPro.
The method described in. 11. The method of claim 9, wherein said compound is cyclic Xaa-boroPro. 12. The method according to claim 12, wherein the substrate is SDF-1, RANTES, MIP-1, M
IP-3, GLP-2, G-CSF, EPO, IL-6, IL-11, IL-8
The method of claim 1, wherein the method is selected from the group consisting of: substance P, fibronectin and monomeric fibrin. 13. The medical condition may include atherosclerosis, allergy, inflammation, angiogenesis, cardiac development, neoplasia, tumor, cancer, liver disease, intestinal disease, organ vascularization, microbial infection and viral infection. The method of claim 1, wherein the method is selected from the group consisting of: 14. The method of claim 1, wherein said compound is given to a subject by oral administration. 15. The method of claim 1, wherein said compound is given to a subject by parenteral administration. 16. The method of claim 1, wherein said effective amount ranges from 0.01 mg / kg per day to 100 mg / kg per day. 17. A pharmaceutical composition for treating a medical disorder in a subject, mediated by chemokine inactivation, comprising: a pharmaceutically acceptable carrier; and an effective amount of the following formula: Including compounds having: Where T is a group of the following formula: Where D ₁ and D ₂ are each independently a hydroxyl group or a physiological pH
A group that can be hydrolyzed to a hydroxyl group in an aqueous solution of the formula: Wherein G is H, F or any alkyl group containing 1 to 20 carbon atoms and optionally a heteroatom which may be N, S or O; a phosphonate group of the following formula: Wherein each J is independently any combination of any number of C, H, O or N atoms, or O-alkyl, N-alkyl or alkyl, wherein O-alkyl, N- Alkyl or alkyl each contains 1 to 20 carbon atoms and optionally contains a heteroatom which can be N, S or O; radicals of the formula: A group of the following formula: Wherein R is an alkyl or aryl group and may be substituted or unsubstituted and is an alpha ketoester; or a group of the following formula: Wherein Y is a group of the following formula: Wherein each R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ is H; B is boron; X is any number of C, H , O, S or N atoms; and m can vary between 0 and 20. 18. The pharmaceutical composition according to claim 17, wherein T is a boronic acid group, a phosphonate group, a cyano group, or a trifluoroalkyl ketone group;
R ₁ and R ₂ are each H, and Y is each CH ₂ —CH ₂ ;
Independently selected from the R groups of proline and alanine; the inhibitory compound has a binding or dissociation constant of DPP-IV of at least 10 ^-9 M; and D1
And D2 are each independently F or a ring wherein D1 and D2 together comprise 1-20 carbon atoms and optionally a heteroatom which may be N, S or O. Composition. 19. A pharmaceutical composition for treating a medical disorder in a subject, mediated by chemokine inactivation, comprising: a pharmaceutically acceptable carrier; and an effective amount of the following formula: Embedded image Wherein D1 and D2 are each a hydroxyl group; wherein X is an amino acid; and C binds to B in the L configuration, comprising an effective amount of the compound. Composition. 20. The compound of claim 1, wherein said compound is Val-boroPro.
10. The pharmaceutical composition according to 9. 21. The pharmaceutical composition according to claim 19, wherein said compound is cyclic Xaa-boroPro.