PCT
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`wo 99/62914
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`(51) International Patent Classification 6 :
`C07F 5/02, A61K 31/69
`
`Al
`
`(11) International Publication Number:
`
`(43) International Publication Date:
`
`9 December 1999 (09.12.99)
`
`(21) International Application Number:
`
`PCT/US99/10777
`
`(22) International Filing Date:
`
`14 May 1999 (14.05.99)
`
`(30) Priority Data:
`60/088,540
`
`5 June 1998 (05.06.98)
`
`us
`
`(71) Applicant: POINT THERAPEUTICS, INC. [US/US]; 75 Knee(cid:173)
`land Street, Boston, MA 02111 (US).
`
`(72) Inventor: WALLNER, Barbara, P.; 64 Arrowhead Road,
`Weston, MA 02193 (US).
`
`(74) Agent: PLUMER, Elizabeth, R.; Wolf, Greenfield & Sacks,
`P.C., 600 Atlantic Avenue, Boston, MA 02210 (US).
`
`(81) Designated States: AE, AL, AM, AT, AU, AZ, BA, BB, BG,
`BR, BY, CA, CH, CN, CU, CZ, DE, DK, 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, UZ, VN, YU, ZA, ZW,
`ARIPO patent (GH, GM, KE, LS, MW, SD, SL, SZ, UG,
`ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`TM), European patent (AT, BE, CH, CY, DE, DK, ES, FI,
`FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent
`(BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE,
`SN, TD, TG).
`
`Published
`With international search report.
`
`(54) Title: CYCLIC BOROPROLINE COMPOUNDS
`
`(57) Abstract
`
`Substantially pure preparations of eyelid boroProline compounds that bind, in cyclic or linear form, to CD26 are provided. Methods
`for using the cyclic compounds to stimulate the activation and/or proliferation of immune cells to achieve preselected normal in vivo levels
`of these cells also are provided. Evidence of the oral bioavailability and activity of a preferred cyclic compound, valine-prolineboronic
`acid (ValboroPro), also is provided.
`
`AstraZeneca Exhibit 2155
`Mylan v. AstraZeneca
`IPR2015-01340
`
`Page 1 of 52
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`AL
`AM
`AT
`AU
`AZ
`BA
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`cu
`cz
`DE
`DK
`EE
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Paso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`COte d'Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People's
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`sz
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`us
`uz
`VN
`YU
`zw
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`VietNam
`Yugoslavia
`Zimbabwe
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`CYCLIC BOROPROLINE COMPOUNDS
`
`Related Application
`
`This application claims priority under Title 35 §119(e), ofUnited States Provisional
`
`5 Application No. 60/088,540, filed June 5, 1998, and entitled "CYCLIC BOROPROLINE
`
`COMPOUNDS," the entire contents of which are incorporated herein by reference.
`
`The Field Of The Invention
`
`This invention relates to substantially pure forms of cyclic boroProline compounds
`
`that bind, in cyclic or linear form, to CD26. The invention also relates to methods for using
`
`10
`
`these compounds to stimulate the activation and/or proliferation of CD26-bearing cells to
`
`mobilize hematopoietic progenitor cells to spleen and periphery.
`
`Background Of The Invention
`
`CD26, a type II transmembrane protein, is expressed on the cell surface of a number
`
`of cell types, including lymphocytes (Marguet, D. et al., Advances in Neuroirnrnunol. 3:209-
`
`15 215 (1993)), hematopoietic cells (Vivier, I. et al., J Irnmunol. 147:447-454 (1991); Bristol, et
`
`al., J Irnrnunol. 149:367 (1992)), thymocytes (Dang, N.H. et al., J Irnrnunol. 147:2825-2832
`
`(1991), Tanaka, T. et al., J Irnrnunol. 149:481-486 (1992), Darmoul, D. et al., J Biol. Chern.
`
`267:4824-4833 (1992)), intestinal brush border membrane, endothelial cells, fibroblasts, and
`
`stromal cells. Cell surface associated CD26 is a sialoglycoprotein, with most of its mass on
`
`20
`
`the outside of the cell.
`
`CD26 has been best characterized on peripheral T cells where it functions as a potent
`
`costimulatory signal for T cell activation. Its surface expression is up regulated upon T cell
`
`activation (Dong, R.P. et al., Cell9:153-162 (1996), Torimoto, Y. et al., J Irnrnunol.
`
`147:2514 (1991), Mittrucker, H-W. et al., Eur. J Irnrnun. 25:295-297 (1995), Hafler, D.A. et
`
`25
`
`al, J Irnrnunol. 142:2590-2596 (1989), Dang, N.H. et al., J Imrnunol. 144:409 (1990)).
`
`CD26 has also been identified in rodents as an important regulatory surface receptor in
`
`hematopoiesis and lymphoid development (Vivier, I. et al., J Imrnunol. 147:447-454 (1991 )).
`
`The primary structure of CD26 is highly conserved between species (Ogata, S. et al., J Biol.
`
`Chern. 264:3596-3601 (1998)). In humans, CD26 reportedly is involved in the regulation of
`
`30
`
`thymocyte activation, differentiation and maturation (Dang, N.H. et al., J Imrnunol.
`
`147:2825-2832 (1991); Kameoka, J. et al., Blood 85:1132-1137 (1995)).
`
`CD26 has an enzymatic activity that is identical to that of Dipeptidyl Peptidase IV
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`-
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`(DPP-IV), a serine type exopeptidase with high substrate specificity. It cleaves N-terminal
`
`dipeptides from proteins if the penultimate amino acid is proline, or in some cases alanine
`
`(Fleischer, B.lmmunol. Today 15:180 (1994)).
`
`A class of low molecular weight synthetic monomeric molecules with high affinity for
`
`5 CD26 have previously been developed and characterized (G.R. Flentke, et al. Inhibition of
`
`dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors
`
`to examine the role ofDP-IV in T-cell function, PNAS (USA) 88, 1556-1559 (1991); W.G.
`
`Gutheil and W.W. Bachovchin. Separation ofL-Pro-DL-boroPro into Its Component
`
`Diastereomers and Kinetic Analysis of Their Inhibition ofDipeptidyl Peptidase IV. A New
`
`10 Method for the Analysis of Slow, Tight-Binding Inhibition, Biochemistry 32, 8723-8731
`
`(1993)). These molecules have been shown to be potent and specific synthetic inhibitors for
`
`CD26's associated DP IV proteinase activity.
`
`Representative monomeric structures of these transition-state-analog-based inhibitors,
`
`Xaa-boroPro, include Pro-boroPro, Ala-boroPro, Val-boroPro, and Lys-boroPro. BoroPro
`
`15
`
`refers to the analog of proline in which the carboxylate group (COOH) is replaced with a
`
`boronyl group [B(OH)2]. Pro-boroPro, the most thoroughly characterized of these inhibitors
`
`has a Ki of 16 picomolar (pM) (W.G. Gutheil and W.W. Bachovchin. Separation ofL-Pro(cid:173)
`
`DL-boroPro into Its Component Diastereomers and Kinetic Analysis of Their Inhibition of
`
`Dipeptidyl Peptidase IV. A New Method for the Analysis of Slow, Tight-Binding Inhibition,
`
`20 Biochemistry 32, 8723-8731 (1993)). Val-boroPro has even a higher affinity, with a Ki of 1.6
`
`pM (W.G. Gutheil and W.W. Bachovchin. Supra; R.J. Snow, et al. Studies on Proline
`
`boronic Acid Dipeptide Inhibitors ofDipeptidyl Peptidase IV: Identification of a Cyclic
`
`Species Containing a B-N Bond, JAm. Chern. Soc. 116, 10860-10869 (1994)). Thus, these
`Xaa-boroPro inhibitors are about 1 o+6 fold more potent than the next best known inhibitors.
`
`25
`
`United States Patent Nos. 4,935,493 (Bachovchin '493) and 5,462,928 (Bachovchin
`
`'928), both of which are incorporated herein by reference, disclose protease inhibitors and
`
`transition state analogs (the '493 patent) and methods for treating transplant rejection in a
`
`patient, arthritis, or systemic lupus erythematosis (SLE) by administering a potent inhibitor of
`
`the catalytic activity of soluble amino peptidase activity of dipeptidyl peptidase type IV (DP-
`
`30
`
`IV; (G.R. Flentke, et al. Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro
`
`dipeptides and use of these inhibitors to examine the role ofDP-IV in T-cell function, PNAS
`
`(USA) 88, 1556-1559 (1991 )).
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`PCT published application WO 98/00439 (Multivalent Compounds for Crosslinking
`
`Receptors and Uses Thereof) reports that in aqueous solution at all pH values, a boroProline(cid:173)
`
`type CD26 inhibitor exists as a slowly equilibrating mixture of two conformations: an open
`
`chain structure which is inhibitory (active species), and a cyclic structure which is non-
`
`5
`
`inhibitory (inactive species). The open, active, inhibitory chain species is favored at low pH
`
`while the cyclized structure is favored at high pH. In view of the foregoing, the WO
`
`98/00439 proposes preventing peptide conformational changes, e.g., intermolecular
`
`cyclization, by constructing a bivalent or multivalent compound containing an olefin group to
`
`form novel CD26 inhibitors. According to WO 98/00439, "if cyclization can be blocked, the
`
`1 0
`
`inventors predict that the bioavailability of the compounds taught herein can be increased by
`
`approximately 100- 1000 fold".
`
`Summary Of The Invention
`
`The invention is based upon a variety of surprising and unexpected findings. It has
`
`been discovered, unexpectedly, that boro-Pro compounds of the type described in U.S.
`
`15 4,935,493 (Bachovchin '493) in cyclic form can be orally administered to a subject for
`
`treating the same types of conditions for which the linear molecules are useful. It is believed
`
`that the cyclic boro-Pro compounds undergo a transformation reaction under acidic
`
`conditions in vivo (e.g., stomach) to form a linear reaction product that is capable of
`
`selectively binding to CD26 (DP-IV). Thus, according to this aspect, the methods and
`
`20
`
`compositions of the invention are directed to a novel pharmaceutical prodrug, namely, cyclic
`
`boro-Proline compounds, for oral administration. Novel compositions containing the
`
`substantially pure cyclic boroProline compounds of the invention, in solution or dry form,
`
`also are provided.
`
`It is believed that the cyclic compounds of the invention are biologically active in
`
`25
`
`cyclic form, as well as in linear form. Accordingly, the invention also embraces methods and
`
`compositions in which the cyclic compounds are administered to a subject or otherwise used
`
`in vitro (e.g., screening assays for selection of competitive molecules) in which the cyclic
`
`compound is not first subjected to conditions to induce conversion to the linear form. Thus,
`
`the cyclic compounds can be administered in oral form (whereby they may or may not be
`
`30
`
`substantially converted to a linear form in the acidic conditions of the stomach), as well as in
`
`parenteral form with, or without, prior treatment to convert to the linear form.
`
`The agents useful according to the invention are the cyclic forms of the compounds of
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`Formula I (shown in figure 4), including all isomeric forms of this compound (discussed in
`
`more detail below. Referring to the Formula I compound, each X 1 and X2 is, independently, a
`hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous
`
`solution at physiological pH; and X represents an amino acid or a peptide which mimics the
`
`5
`
`site of a substrate recognized by a post prolyl cleaving enzyme. In the preferred
`
`embodiments, the amino acids are L-amino acid residues (for glycine there is no such
`
`distinction); preferably, the C bonded to B is in the L-configuration. By "the C bonded to B
`
`is in the L-configuration" is meant that the absolute configuration of the C is like that of an L(cid:173)
`
`amino acid.
`
`10
`
`Peptides that mimic the substrate binding site of the post-prolyl cleaving enzyme DP
`
`IV (also referred to herein as "CD 26") are described in U.S. Patent No. 4,935,493
`
`("Bachovchin '493") and U.S. 5,462,928 ("Bachovchin '928").
`
`The open chain (linear form) to cyclic form reaction involves a trans to cis
`
`isomerization of the proline and the formation of a new N-B bond. Accordingly, "cyclic
`
`15
`
`form" refers to the cyclized structure of the compounds of formula I that are the boron
`
`analogs of a diketopiperazine. This transformation is illustrated in figure 3.
`
`By "substantially pure" it is meant that the cyclic compounds of the invention
`
`represent at least about 90% by weight of the composition. In the more preferred
`
`embodiments, the cyclic boroProline compound represents at least 98% by weight of the
`
`20
`
`composition.
`
`In certain embodiments, the cyclic boroProline compound represents a percentage by
`
`weight of the composition selected from the group consisting of at least 5%, at least 10%, at
`
`least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at
`
`least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least
`
`25 99.5%.
`
`In these and other embodiments, the compositions contain a linear boroProline
`
`compound that represents a percentage by weight of the composition selected from the group
`
`consisting of a percentage that is less than 95%, less than 90%, less than 80%, less than 70%,
`
`less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%,
`
`30
`
`less than 5%, less than 4%, less than 3%, less than 2%, less than 1.0 %, and less than 0.5.
`
`In a particularly preferred embodiment of the invention, the cyclic boroProline
`
`compound is a Val-boroProline compound. A "Val-boroProline compound" refers to a
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`compound of formula I in which the carboxy terminal boroProline is covalently coupled via a
`
`peptide linkage in accordance with standard peptide chemistry to a valine amino acid residue.
`
`A preferred embodiment is the cyclic form ofVal-boroPro, which can be in the following
`
`forms: L-Val-S-boroPro, L-Val-R-boroPro, D-Val-S-boroPro, and D-Val-R-boroPro. More
`
`5
`
`preferably, the compound is L-Val-S-boroPro or L-Val-R-boroPro.
`
`According to yet another aspect of the invention, pharmaceutical compositions and
`
`methods for manufacturing such compositions are provided. The pharmaceutical
`
`compositions of the invention contain: (1) a pharmaceutically acceptable carrier; and (2) one
`
`or more of the cyclic boroProline compounds of the invention. Preferably, the
`
`1 0 pharmaceutical compositions are formulated for oral administration; however, lyophilized
`
`forms of the cyclic compounds are also provided for oral or parenteral administration. Such
`
`lyophilized or otherwise dried forms of the compounds of the invention may be reconstituted
`
`in a buffer of appropriate pH prior to administration. It is believed that oral formations
`
`containing the cyclic boroProline compounds of the invention undergo a conformation
`
`15
`
`change from cyclic to linear form following administration, namely, when the compounds are
`
`subjected to the acidic pH conditions of the digestive system. For this reason, the preferred
`
`oral formations are tablets, capsules, or other solid forms which do not include an enteric
`
`coating. The method for manufacturing a pharmaceutical composition involves placing a
`
`cyclic boroProline compound of the invention in a pharmaceutically acceptable carrier and,
`
`20 optionally, formulating the cyclic compound into a tablet or other form that is suitable for
`
`oral administration.
`
`According to another aspect of the invention, methods are provided for modulating
`
`immune system function. The compounds of the invention are administered to subjects in
`
`need of immune system modulation in amounts effective to modulate immune system
`
`25
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`function. Modulation of immune system function includes, but is not limited to, increasing
`
`immune function such as by stimulating proliferation and specific immune function of
`
`immune cells (e.g., CD26-bearing cells) to produce a prophylactic or therapeutic result
`
`relating to infectious disease, cancer, and the like. Specific conditions that may be treated
`
`according to the invention are deemed specific independent aspects of the invention and are
`
`30 described in detail in the examples. Exemplary conditions that can be treated by
`
`administering the compounds ofthe invention include: HIV infection; neoplasms (wherein
`
`the lymphocytes are cytolytic or helper T cells to attack the neoplasm); side effects of
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`chemotherapy or radiation therapy (e.g., resulting from a depletion of cells of the immune
`
`system such as a depletion of cells derived from lymphoid, erythroid and/or myeloid
`
`lineages); kidney failure (e.g., resulting in depletion of cells of the immune system); bone
`
`marrow disorders resulting in immunodeficiency; autoimmunity; and immunodeficiency
`
`5
`
`(e.g., resulting from depletion of cells of the immune system).
`
`More particularly, the compounds of the invention are useful for the stimulation of
`
`proliferation, differentiation and mobilization of lymphocytes and hematopoietic cells, as well
`
`as for stimulation of cytokine production by stromal cells such as IL-6, IL-11, and G-CSF,
`
`and for stabilization or activation of cytokines which are substrates for CD26/DPP-IV
`
`10 protease activity (i.e., the cytokines terminate in the amino acid sequence XaaProline,
`
`wherein Xaa is an amino acid, and wherein the peptide sequence is subject to cleavage by
`
`CD26/DDP-IV). Accordingly, the invention is useful whenever it is desirable to stimulate
`
`the proliferation or differentiation of, or to mobilize, such immune cells, or to stimulate,
`
`stabilize or activate cytokine production. Mobilization of hematopoietic cells is characterized
`15 by the enrichment of early progenitor cells in the bone marrow and the recruitment of these
`
`cells to the periphery in response to a mobilization agent (e.g. G-CSF, GM-CSF, etc.). The
`
`agents useful according to the invention can be used to treat lymphocyte and hematopoietic
`
`cell deficiencies or to restore hematopoietic and mature blood cell count in subjects with such
`
`deficiencies. Such agents also may be used in connection with hematopoietic cell transplants,
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`20
`
`such as bone marrow or peripheral blood transplants, when used to replenish or create an
`
`immune system in a subject. The agents further can be used as an immune booster. The
`
`agents also are useful in vitro in connection with the culturing of cells for therapeutic and
`
`research uses.
`
`The methods for stimulating activation or proliferation of human lymphocytes,
`
`25 hematopoietic cells, or stromal cells. The method involves contacting the lymphocytes,
`
`hematopoietic cells and/or stromal cells, in vivo or in vitro, with an activation or
`
`proliferation-inducing concentration of one or more cyclic boroProline compounds of the
`
`invention. In certain preferred embodiments, contacting is carried out by orally administering
`
`the compound to a human patient in need of such treatment, i.e., the patient is diagnosed as
`
`30 having an adverse medical condition characterized by inadequate lymphocyte or
`
`hematopoietic cell activation or concentration. Parenteral administration alternatively can be
`
`used to practice the method of treatment on a subject. As used herein, subject means humans,
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`- nonhuman primates, dogs, cats, sheep, goats, horses, cows, pigs and rodents.
`
`The compounds of the invention can be administered alone, or in combination with
`
`additional agents for treating the condition, e.g., a different agent which stimulates activation
`
`or proliferation of said lymphocytes, hematopoietic cells and/or stromal cells. Contacting the
`
`5
`
`lymphocytes with the compounds of the invention can be performed in vitro or in vivo.
`
`As used herein, compound of the invention means the compounds described above as
`
`well as salts thereof.
`
`These and other aspects of the invention will be described in greater detail below.
`
`All patents, patent applications, references and other documents that are identified in
`
`1 0
`
`this patent application are incorporated in their entirety herein by reference.
`
`By "amino acid" is meant to include imino acid.
`
`Definitions
`
`By "boroPro" is meant an alpha-amino boronic acid analog of proline bonded to an
`
`amino acid to form a dipeptide with boroPro as the C-terminal residue. "BoroPro" is used to
`
`15 designate such an analog having the carboxyl group of proline replaced with a B(OH)2 group,
`where (OH)2 represents two hydroxyl groups and B represents boron.
`By Xaa is meant any amino acid residue, e.g., a lysine residue, a valine residue.
`
`"CD26 ligand" is any protein, glycoprotein, lipoprotein or polypeptide that binds to
`
`the T cell receptor CD26 and may provide a stimulatory or inhibitory signal.
`
`20
`
`CD26, Dipeptidyl Peptidase IV (DP IV or DPPIV) and dipeptidyl aminopeptidase IV
`
`are used interchangeably. CD26 is a postproline cleaving enzyme with a specificity for
`
`removing Xaa-Pro (where Xaa represents any amino acid) dipeptides from the amino
`
`terminus of polypeptides.
`
`By alpha-carbon of an amino acid is the one to which the carboxylic acid group is
`
`25
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`attached. All naturally occurring amino acids are alpha-amino acids or alpha-imino, which
`
`means that the amino and carboxylic acid groups are both attached to the same carbon atom.
`
`Each amino acid can be thought of as a single carbon atom (the alpha carbon, C) to which
`
`there is attached one carboxyl group, one amino group, a side chain denoted Rand a
`
`30
`
`hydrogen, wherein: "R" is a side chain; "NH2" is the alpha amino group; the first carbon (C)
`attached to the NH2 group having a hydrogen (H) and an R group attached is the alpha
`carbon; and the carbon double bonded to an oxygen and a hydroxyl group (OH) is the alpha
`
`carboxyl group.
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`The NH2 and COOH groups are used to covalently couple amino acids to one another.
`
`The hydroxyl group (OH) of one amino acid on the carboxyl end and the hydrogen (H) on the
`
`N terminus are removed (H20) when two amino acids are linked together. To form a protein,
`
`the amino group of one amino acid reacts with the carboxyl group of another by the
`
`5
`
`elimination of water; the resulting chemical bond is called a peptide bond.
`
`By "peptides" is meant a small molecule, e.g., usually containing less than 50 amino
`
`acid residues, which do not generally possess a well-defined three-dimensional structure.
`
`Pharmaceutical preparations and modes of administration are described herein.
`
`Other features and advantages of the invention will be apparent from the following
`
`10
`
`detailed description, and from the claims.
`
`Brief Description Of The Drawings
`
`Figure 1 shows the oral bioavailability of cyclic PT -100 and the HCl and methane
`
`sulfonate salts of PT -1 00;
`
`Figure 2 shows the effect of cyclic PT1 00 and the Chl and methane sulfonate salts of
`
`15 PT -1 00 on the regeneration of neutrophils in cyclophosphamide treated mice;
`
`Figure 3 shows the structures ofthe open and cyclized forms ofXaa-boroPro
`
`inhibitors (conformational equilibrium of Xaa-boroProline inhibitors).
`
`Figure 4 shows the structure of Formula I.
`
`Figure 5 shows the reaction scheme for the proposed boronoproline synthetic route.
`
`20
`
`Figure 6 shows the reaction scheme for the N-BOC-(S)-Val-(R)-boroPro-
`
`(1 S,2S,3R,5S)-pinanediol ester, IP068.
`
`Figure 7 shows the reaction scheme for the H2N-(S)-Val-(R)-boroPro-(l S,2S,3R,5S)(cid:173)
`
`pinanediol ester, HCl IP069.
`
`Figure 8 shows the reaction scheme for the Cyclo-(S)-Val-(R)-boroPro, IP070.
`
`25
`
`Figure 9 shows the reaction scheme for the H2N-(S)-Val-(R)-boroPro-OH,HCl,FP020.
`
`Detailed Description
`
`The invention is based upon a variety of surprising and unexpected findings. It has
`
`been discovered, unexpectedly, that boro-Pro compounds of the type described in U.S.
`
`30
`
`4,935,493 (Bachovchin '493) in cyclicform can be orally administered to a subject for
`
`treating the same types of conditions for which the linear molecules are useful. It is
`
`postulated that the cyclic boro-Pro compounds undergo a transformation reaction under acidic
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`- conditions in vivo (e.g., stomach) to form a linear reaction product that is capable of
`
`selectively binding to CD26 (DP-IV). These unexpected results have important therapeutic
`
`and experimental research implications. Thus, the methods and compositions of the
`
`invention are directed to a novel pharmaceutical prodrug, namely, cyclic bora-Proline
`
`5
`
`compounds, for oral administration. The invention also embraces lyophilized forms of the
`
`cyclic boroProline compounds that can be reconstituted in an acidic buffer prior to use to
`
`form linear boro-Proline compounds for use in accordance with the methods of the invention.
`
`Novel compositions containing the substantially pure cyclic boroProline compounds of the
`
`invention, in solution or dry form, also are provided.
`
`1 0
`
`The agents useful according to the invention are the cyclic forms of the compounds of
`
`Formula I (as shown in figure 4), wherein each X1 and X2 is, independently, a hydroxyl group
`
`or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at
`
`physiological pH; and X represents an amino acid or a peptide which mimics the site of a
`
`substrate recognized by a post prolyl cleaving enzyme. In the preferred embodiments, the
`
`15
`
`amino acids are L-amino acid residues (for glycine there is no such distinction); preferably,
`
`the C bonded to B is in the L-configuration. By "the C bonded to B is in the L-configuration"
`
`is meant that the absolute configuration of the Cis like that of an L-amino acid.
`
`Thus, the
`
`20
`
`group has the same relationship to the C as the --COOH group of an L-amino acid has to its
`
`a carbon. In some embodiments, X is valine, alanine, or proline residues and the inhibitor,
`
`preferably, is L-Val-L-boroPro, L-Ala-L-boroPro; or L-Pro-L-boroPro, or any isomer ofthe
`
`foregoing, respectively.
`
`As used herein, compound of the invention means the compounds described above as
`
`25 well as salts thereof.
`
`Peptides which reportedly have utility for inhibiting post-prolyl cleaving enzymes and
`
`which, if coupled to a reactive group, form a covalent complex with a functional group in the
`
`reactive site of a post-prolyl cleaving enzyme are described in U.S. Patent No. 4,935,493,
`
`"Protease Inhibitors", issued to Bachovchin et al. ("Bachovchin '493"); U.S. 5,462,928,
`
`30
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`"Inhibitors ofDipeptidyl-aminopeptidase Type IV", issued to Bachovchin et al. ("Bachovchin
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`'928"); "Proline Derivatives and Compositions for Their Use as Inhibitors ofHIV Protease",
`
`issued to Hanko et al., ("Hanko '604"); PCT/US92/09845, "Method for Making a
`
`Prolineboronate Ester", and its U.S. priority applications (USSN 07/796,148 and 07/936,198),
`
`Applicant Boehringer Ingelheim Pharmaceuticals, Inc. ("Boehringer"); and
`
`5 PCT/GB94/02615, "DP-IV-Serine Protease Inhibitors", Applicant Ferring V.V. ("Ferring").
`
`The cyclic boroProline compounds of the invention undergo a conformational change
`
`to a linear form ("linear boroProline compounds") that mimics the substrate binding site of
`
`the post-prolyl cleaving enzyme DP IV (also referred to herein as "CD 26"). DP IV is a post(cid:173)
`
`prolyl cleaving enzyme with a specificity for removing Xaa-Pro (where Xaa represents any
`
`1 0
`
`amino acid) dipeptides from the amino terminus of a polypeptide substrate. Representative
`
`structures oftransition-state analog-based inhibitors Xaa-boroPro, include Val-BoroPro, Lys(cid:173)
`
`BoroPro, Pro-BoroPro and Ala-BoroPro in which "boroPro" refers to the analog of proline in
`
`which the carboxylate group (COOH) is replaced with a boronyl group [B(OH)2].
`In a particularly preferred embodiment of the invention, the cyclic boroProline
`
`15
`
`compound is a Val-boroProline compound. A "Val-boroProline compound" refers to a
`
`compound of formula I in which the carboxy terminal boroProline is covalently coupled via a
`
`peptide linkage in accordance with standard peptide chemistry to a valine amino acid residue.
`
`The valine amino acid, optionally, is further coupled via a peptide linkage to additional amino
`
`acid residues, provided that the additional amino acid residues do not inhibit the ability of the
`
`20 Val-boroProline compound to bind to CD26. In a most preferred embodiment, the compound
`
`of the invention is Val-boroPro (also referred to as "PT -1 00"). Because of the chiral carbon
`
`atoms present on the amino acid residues and on the carbon attached to the boron atom, Val(cid:173)
`
`boroPro can exist in multiple isomeric forms: (a) L-Val-S-boroPro, (b) L-Val-R-boroPro, (c)
`
`D-Val-S-boroPro, and (d) D-Val-R-boroPro. More preferably, the compound is L-Val-S-
`
`25 boroPro or L-Val-R-boroPro. In an analogous manner, the other cyclic boroProline
`
`compounds of the invention can exist in multiple isomeric forms; however, in general, the
`
`forms in which each amino acid chiral center has an "L-" configuration and the boroPro is in
`
`the R or S configuration are the preferred forms of the compounds.
`
`Throughout this application, conventional terminology is used to designate the
`
`30
`
`isomers as described below and in appropriate text books known to those of ordinary skill in
`
`the art. (See, e.g., Principles in Biochemistry, editor A.L. Lehninger, page 99-100, Worth
`
`Publishers, Inc. (1982) New York, NY; Organic Chemistry, Morrison and Boyd, 3rd Edition,
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`Chap. 4, Allyn and Bacon, Inc., Boston, MA (1978); See also. Patent Cooperation Treaty
`
`published application W093/10127, application no. PCTIUS92/09845).
`
`All amino acids, with the exception of glycine, contain an asymmetric or chiral carbon
`
`and may contain more than one chiral carbon atom. The asymmetric ex carbon atom of the
`
`5
`
`amino acid is referred to as a chiral center and can occur in two different isomeric forms.
`
`These forms are identical in all chemical and physical properties with one exception, the
`
`direction in which they can cause the rotation of plane-polarized light. These amino acids are
`
`referred to as being "optically active, i.e., the amino acids can rotate the plane-polarized light
`
`in one direction or the other.
`
`1 0
`
`The four different substituent groups attached to the ex carbon can occupy two
`
`different arrangements in space. These arrangements are not super imposable mirror images
`
`of each other and are referred to as optical isomers, enantiomers, or stereo isomers. A
`
`solution of one stereo isomer of a given amino acid will rotate plane polarized light to the left
`
`and is called the levorotatory isomer [designated (-)]; the other stereo isomer for the amino
`
`15
`
`acid will rotate plane polarized light to the same extent but to the right and is called
`
`dextrorotatory isomer [designated(+)].
`
`A more systematic method for classifying and naming stereo isomers is the absolute
`
`configuration of the four different substituents in the tetrahedryin around the asymmetric
`
`carbon atom (e.g., the ex carbon atom). To establish this system, a reference compound was
`
`20
`
`selected (glyceraldehyde), which is the smallest sugar to have an asymmetric carbon atom.
`
`By convention in the art, the two stereo isomers of glyceraldehyde are designated Land D.
`
`Their absolute configurations have been established by x-ray analysis. The designations,
`
`Land D, also have been assigned to the amino acids by reference to the absolute configuration
`
`of glyceraldehyde. Thus, the stereo isomers