WO1999019295A1 - Compositions and methods for inhibiting arginase activity - Google Patents

Compositions and methods for inhibiting arginase activity Download PDF

Info

Publication number
WO1999019295A1
WO1999019295A1 PCT/US1998/021430 US9821430W WO9919295A1 WO 1999019295 A1 WO1999019295 A1 WO 1999019295A1 US 9821430 W US9821430 W US 9821430W WO 9919295 A1 WO9919295 A1 WO 9919295A1
Authority
WO
WIPO (PCT)
Prior art keywords
arginase
compound
hooc
smooth muscle
arginine
Prior art date
Application number
PCT/US1998/021430
Other languages
French (fr)
Inventor
David W. Christianson
Ricky Baggio
Daniel Elbaum
Original Assignee
Trustees Of The University Of Pennsylvania
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Trustees Of The University Of Pennsylvania filed Critical Trustees Of The University Of Pennsylvania
Priority to EP98952229A priority Critical patent/EP1049660A1/en
Priority to AU97979/98A priority patent/AU9797998A/en
Priority to CA002305703A priority patent/CA2305703A1/en
Publication of WO1999019295A1 publication Critical patent/WO1999019295A1/en
Priority to US09/545,737 priority patent/US6387890B1/en
Priority to US10/053,939 priority patent/US6723710B2/en
Priority to US10/661,965 priority patent/US20040063666A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/69—Boron compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/025—Boronic and borinic acid compounds

Definitions

  • the field of the invention is enzyme inhibitors.
  • arginase regulates cellular concentrations of arginine and omithine, which are used for biosynthetic reactions (Yip et al., 1972, Biochem. J. 127:893-899).
  • Arginine is used, by way of example, in the synthesis of nitric oxide.
  • arginase activity is reciprocally coordinated with the activity of the enzyme, nitric oxide synthase.
  • Reciprocal coordination of the activities of arginase and nitric oxide (NO) synthase modulates NO-dependent cytotoxicity (Corraliza et al., 1995, Biochem. Biophys. Res. Commun. 206:667-673; Daghigh et al., 1994, Biochem. Biophys. Res. Commun. 202:174-180; Chenais et al, 1993, Biochem. Biophys. Res. Commun. 196:1558-1565; Klatt et al., 1993, J. Biol. Chem. 268:14781-14787; Keller et al., 1991, Cell. Immunol.
  • Rat liver arginase is a trimeric metalloenzyme which contains a binuclear manganese cluster in the active site of each subunit. This binuclear cluster is required for maximal catalytic activity (Reczkowski et al., 1992, J.
  • arginase catalyzes the divalent cation-dependent hydrolysis of L-arginine to form L-ornithine and urea.
  • the enzyme is currently known to serve three important functions: the production of urea, the production of omithine, and regulation of substrate arginine levels for nitric oxide synthase (Jenkinson et al.,
  • Urea production provides a mechanism to excrete nitrogen in the form of a highly soluble, non-toxic compound, thus avoiding the potentially dangerous consequences of high ammonia levels.
  • L-omithine is a precursor for the biosynthesis of polyamines, spermine and spermidine, which play important roles in cell proliferation and differentiation.
  • arginase modulates the production of nitric oxide by regulating the levels of arginine present within tissues.
  • Toxicol. 35:1-27; Yamaguchi et al., 1992, Eur. J. Biochem., 204:547-552; Pufahl et al, 1995, Biochemistry 34:1930-1941), is an endogenous arginase inhibitor (Chenais et al., 1993, Biochem. Biophys. Res. Commun., 196:1558-1565; Buga et al., 1996, Am. J. Physiol. Heart Circ. Physiol.
  • an arginase inhibitor such as L-OH-Arg may not be selective since it also serves as a NO synthase substrate (Pufahl et al., 1992, Biochemistry 31 :6822-6828; Furchgott, 1995, Annu. Rev. Pharmacol. Toxicol. 25:1- 27; Pufahl et al, 1995, Biochemistry 34:1930-1941; Chemais et al., 1993, Biochem. Biophys. Res. Commun. 196:1558-1565; Boucher et al., 1994, Biochem. Biophys. Res.
  • the invention include a composition comprising the formula HOOC-CH(NH 2 )-Y 1 -Y 2 -Y3-Y 4 -B(OH)2 wherein each of Y j , Y 2 , Y3, and Y4 is selected from the group consisting of CH 2 , S, O NH, and N-alkyl, except Y 2 is not S when each of Y j , Y3, and Y4 is CH ,.
  • the composition has the formula HOOC-CH(NH 2 )-CH 2 -CH 2 -CH 2 -CH 2 -B(OH) 2 .
  • the composition of the invention further comprises a pharmaceutically acceptable carrier.
  • a composition comprising a pharmaceutical acceptable carrier and a compound having the formula
  • the compound has a formula selected from the group consisting of
  • the invention further includes a method of inhibiting arginase.
  • the method comprising contacting the arginase with a composition having the formula
  • each of Y j , Y 2 , Y3, and Y4 is selected from the group consisting of CH , S, O NH, and N-alkyl.
  • the compound has a formula selected from the group consisting of
  • the arginase may be a yeast arginase or a mammalian arginase.
  • the arginase is human arginase.
  • Also included in the invention is a method of inhibiting arginase in a mammal.
  • the method comprises administering to the mammal a composition comprising a pharmaceutically acceptable carrier and a compound having the formula HOOC-CH(NH 2 )-Y r Y 2 -Y 3 -Y 4 -B(OH) 2 wherein each of Y ⁇ , Y 2 , Y3, and Y4 is selected from the group consisting of CH 2 , S, O NH, and N-alkyl.
  • the compound has a formula selected from the group consisting of
  • the mammal is a human.
  • a tissue in the human has an abnormally high level of arginase activity.
  • a tissue in the human has an abnormally low level of nitric oxide synthase activity. Also included in the invention is a method of treating a disease in a human. The method comprises administering to the human a composition comprising a pharmaceutically acceptable carrier and a compound having the formula
  • each of Y j , Y 2 , Y3, and Y4 is selected from the group consisting of CH 2 , S, O NH, and N-alkyl, and wherein the disease is selected from the group consisting of a disease associated with an abnormally low level of nitric oxide synthase activity in a tissue of the human and a disease associated with an abnormally high level of arginase activity in a tissue of the human.
  • the compound has a formula selected from the group consisting of
  • the disease is selected from the group consisting of heart disease, systemic hypertension, pulmonary hypertension, erectile dysfunction, autoimmune encephalomyelitis, chronic renal failure and cerebral vasospasm.
  • the method comprises administering to the mammal a composition comprising a pharmaceutically acceptable carrier and a compound having the formula
  • Y j , Y 2 , Y3, and Y4 is selected from the group consisting of CH 2 , S, O NH, and N-alkyl.
  • the compound has a formula selected from the group consisting of
  • the smooth muscle is selected from the group consisting of gasterointestinal smooth muscle, anal sphincter smooth muscle, esophageal sphincter muscle, corpus cavemosum, sphincter of Oddi, arterial smooth muscle, heart smooth muscle, pulmonary smooth muscle, kidney smooth muscle, uterine smooth muscle, vaginal smooth muscle, cervical smooth muscle, placental smooth muscle, and ocular smooth muscle.
  • the invention includes a method of making a compound having the formula
  • HOOC-CH(NH 2 )-CH 2 -CH 2 -CH 2 -CH 2 -B(OH) 2 the method comprising contacting a molecule of the tert-butyl ester of 2(S)-N-(tert- butyloxycarbonyl)-6-[(l S,2S,3R,5S)-(+)-pinanedioxaboranyl]-hexanoic acid in an organic solvent with BCI3.
  • the organic solvent is CH 2 C1 2 .
  • Also included in the invention is a method of making a compound having the formula HOOC-CH(NH 2 )-CH 2 -CH 2 -CH 2 -CH 2 -B(OH) 2 .
  • the method comprises the steps of
  • HOOC-CH(NH 2 )-CH 2 -CH 2 -CH 2 -CH 2 -B(OH) 2 Also included in the invention is a method of identifying an arginase inhibitor antagonist, the method comprising the steps of
  • FIG. 1 is a diagram which illustrates the topology of an arginase monomer. Relative locations of metal ligands are indicated by solid circles.
  • Figure 2 is an image depicting a ribbon plot of an arginase trimer.
  • the Mn ⁇ -Mn ⁇ cluster in the active site of each monomer is represented by a pair of spheres.
  • Figure 3 is a pair of images which depict omit maps of the binuclear manganese cluster of arginase.
  • the maps were calculated using Fourier coefficients
  • the hydrogen bond between O ⁇ 2 of Mn ligand Asp 128 and bridging solvent, which is represented by a sphere, is indicated by a dashed line.
  • Panel B the manganese coordination interactions are depicted, with coordination bond lengths indicated in A.
  • Figure 4 is an image which depicts a model of arginine binding to the active site of arginase.
  • a salt link between Glu-277 and the substrate guanidinium group may orient the substrate for nucleophilic attack by the metal-bridging solvent molecule. Salt links are indicated by dotted lines. His-141 may serve as a catalytic proton shuttle.
  • Figure 5 is an image which illustrates a proposed mechanism of arginase-catalyzed arginine hydrolysis by a metal-activated solvent molecule complexed with the Mn 94- -Mn 9+ cluster in the active site of arginase.
  • the ⁇ -amino and ⁇ -carboxylate groups of the arginine molecule are omitted for clarity.
  • Figure 6 is an image which illustrates the synthetic scheme described herein for the production of ABHA.
  • Figure 7 is an image which depicts an ORTEP representation of the molecular structure of ABHA, using 30% probability thermal ellipsoids.
  • Figure 8, comprising Panels A and B, is a pair of images which illustrate the similarity between the interaction of arginase with the proposed tetrahedral intermediate of arginase-catalyzed arginine hydrolysis, depicted in Panel A, and the interaction of arginase with the proposed the proposed tetrahedral conformation of hydrated ABHA, depicted in Panel B.
  • Figure 9 is an image which depicts an omit electron density map of the arginine-ornithine-borate complex, contoured at 3 ⁇ . Refined atomic coordinates are superimposed. One oxygen atom of the tetrahedral borate anion bridges the binuclear manganese cluster. The precise conformation of omithine ( ⁇ 5A away from borate) is ambiguous, due to the low resolution of the electron density map.
  • FIG. 10 is a diagram of Scheme 1 depicting the chemical structures of compounds 7 (ABHA), 12, 15 (BEC), and 16.
  • Figure 11(a) is a scheme illustrating the reciprocal coordination of NO synthase and arginase activities.
  • Figure 11(b) is a scheme of the arginase mechanism proposed by
  • Figure 12 is a graph depicting the Eadie-Hofstee plot for the new chromogenic substrate, l-nitro-3-guanidinobenzene (18), where v Q is observed velocity and (S) is substrate concentration.
  • Figure 13 comprising panels (a), (b), (c) and (d) is a quartet of graphs depicting plots of v 0 /v as a function of (I) indicate reversible inhibition only if linear. Only the trihydroxysilane, S-(2-trihydroxysilylethyl)-L-cysteine, yields a linear plot (Panel a).
  • HEET R L Figure 14 comprising Panels (a), (b) and (c) is a trio of graphs depicting plots of v/v Q versus (I)/(E Q ) for all the boronic acid inhibitors. Plots of v/v 0 versus (I)/(E Q ) typically are linear for inhibitors that behave as inactivators. Extrapolation of linear v/v Q versus (I)/(E Q ) plots to the (I)/(E Q ) intercept gives the turnover number for the inactivator. However, the (I)/(E Q ) intercept from v/v Q versus
  • Figure 15 comprising Panels A and B, is two graphs depicting the titration of arginase by 7 in 100 ⁇ M MnCl 2 , 50 mM bicine (pH 8.5) at 25 °C.
  • Panel A contains the raw data obtained by titration of 0.0358 mM arginase with 30 x 2.5 ⁇ L injections of 1.5 mM 7.
  • Panel B the area under each peak was integrated and plotted against [7]/[arginase].
  • the solid line represents the best fit of the experimental data using non-linear least squares fitting, indicating a stoichiometry (n) of 1.07 moles of bound 7 per mole of arginase monomer, an association constant (K a ) of 8.89 x 10"
  • Figure 16 comprising Panels A and B, is two graphs depicting the titration of arginase by 15 in 100 ⁇ M MnCl 2 , 50 mM bicine (pH 8.5) at 25 °C.
  • Panel A depicts the raw data obtained by titration of 0.0358 mM arginase with 40 x 2.5 ⁇ L injections of 1.5 mM 15.
  • Panel B the area under each peak was integrated and plotted against [15]/[arginase].
  • the solid line represents the best fit of the experimental data using non-linear least squares fitting, indicating a stoichiometry (n) of 0.964 moles of bound 15 per mole of arginase, an association constant (K a ) of 4.50 x lO ⁇ M "1 , and an enthalpy change ( ⁇ H) of -12.75 kcal/mol.
  • Figure 17 depicts the proposed binding mode for arginase inhibitors 7, 12, and 15.
  • the metal-bridging hydroxide ion of the native arginase likely attacks the trigonal planar boronic acid to form the tetrahedral boronate anion.
  • Figure 18 is a graph depicting the effect of exogenous arginase before and after application of the arginase inhibitor N ⁇ -hydroxy-L-Arginine (L-HO-Arg), on the IAS relaxation by different frequencies of electrical field stimulation (EFS). Note the significant suppression of the IAS relaxation by arginase alone and its reversal by L-HO-Arg.
  • Figure 19 is a graph depicting the effect of exogenous arginase treatment, before and after application of the arginase inhibitor 2 (S)-Amino-6- boronohexanoic Acid (ABHA), on the IAS smooth muscle relaxation by different frequencies of EFS. Note the significant suppression of the IAS relaxation by treatment with arginase alone, and its reversal by ABHA.
  • S arginase inhibitor 2
  • ABHA arginase inhibitor 2
  • Figure 20 is a graph depicting the percent maximal fall in IAS tension by EFS before and after treatment of the tissue with the NO synthase inhibitor L-NNA or L-NNA plus different concentrations of L-HO-Arg.
  • the L-NNA- attenuated IAS relaxation was reversed by L-HO-Arg in a concentration-dependent manner.
  • L-HO-Arg (3 x 10 M) caused complete reversal of the suppressed IAS relaxation.
  • Figure 23 is a graph depicting the influence of L-HO-Arg on the NANC nerve-mediated IAS relaxation produced by EFS. Note that L-HO-Arg caused a significant augmentation of the EFS-induced relaxation of the IAS in a concentration- dependent manner and this was evident only in the lower frequencies of EFS (p ⁇ 0.05).
  • Figure 24 is a graph depicting the influence of 2 (S)-Amino-6- boronohexanoic Acid (ABHA) on the NANC nerve-mediated IAS relaxation. Note that ABHA caused a significant augmentation of the IAS smooth muscle relaxation caused by the lower frequencies of EFS, in a concentration-dependent manner (p ⁇ 0.05).
  • ABHA 2 (S)-Amino-6- boronohexanoic Acid
  • Figure 25 is a graph depicting the basal arginase activity of the tissue homogenates of the liver, internal anal sphincter (IAS), rectum and brain.
  • the basal arginase activity for the nonhepatic tissues was found to be -10° of that in the liver.
  • the basal arginase activity in the IAS smooth muscle was found to be nearly four fold higher than in the adjoining region of the rectum, and the brain.
  • Figure 26 is a graph depicting the effect of N ⁇ -hydroxy-L-arginine on the arginase activity found in hepatic and nonhepatic tissues. N ⁇ -hydroxy-L-arginine was found to be ⁇ 10 times more potent in inhibiting the liver arginase activity than the arginase activities from the nonhepatic tissues.
  • Figure 27 is a graph depicting a comparison of the effect of 2 (S)- Amino-6-boronohexanoic Acid (ABHA) on the hepatic versus the nonhepatic tissues of the I AS, rectum and brain.
  • ABHA 2 (S)- Amino-6-boronohexanoic Acid
  • Figure 28 depicts the influence of the NO synthase inhibitor L-NNA, N ⁇ -hydroxy-L-arginine (L-HO-Arg) and the combination of L-NNA plus L-HO-Arg on the arginase activity of the liver ( Figure 28 A), internal anal sphincter (IAS) ( Figure 28B), rectum (Figure 28C) and brain ( Figure 28D).
  • L-NNA had no significant effect on either the basal arginase activity or on the L-HO-Arg-attenuated arginase activity in any of the tissues examined.
  • the data suggest that the differential inhibitory effects of L-HO-Arg in these tissues do not result from variable interactions of this NO synthase substrate with the endogenous NO synthase.
  • the invention is based upon the discover ' of compounds which inhibit the activity of arginase. These compounds, which are heretofore not known to inhibit this enzyme, are therefore useful for a variety of applications in medicine and in research.
  • compositions and methods for inhibiting the activity of arginase including, but not limited to, yeast and mammalian arginase, are provided.
  • Inhibition of mammalian arginase activity using the boronic acid-based arginine analog 2(S)- amino-6-boronohexanoic acid (ABHA) is demonstrated herein, as is inhibition of arginase using the boronic acid based arginine analog S-(2-boronoethyl)-L-cysteine (BEC).
  • compositions of the invention may be used to inhibit arginase activity in vitro or in vivo, for example, in a human.
  • the compositions of the invention may also be used to treat a disease characterized either by abnormally high arginase activity in a tissue of a mammal or by abnormally low nitric oxide synthase activity in a tissue of the mammal, preferably a human.
  • the composition of the invention comprises a compound having the formula
  • each of Y j , Y 2 , Y3, and Y4 is selected from the group consisting of CH 2 , S, O NH, and N-alkyl, except Y 2 is not S when each of Y1 , Y3, and Y4 is CH 2 .
  • the composition has the formula HOOC-CH(NH 2 )-CH 2 -CH 2 -CH 2 -CH 2 -B(OH) 2 .
  • This compound is 2(S)-amino-6-boronohexanoic acid (ABHA).
  • ABHA is also referred to herein as compound 7.
  • Data presented herein confirm that ABHA is a potent inhibitor of arginase activity.
  • the conformation of the hydrated form of ABHA resembles a transition state intermediate postulated to be formed during the arginine hydrolysis reaction catalyzed by arginase.
  • composition of the invention further comprises a pharmaceutically acceptable carrier.
  • composition comprising a pharmaceutical acceptable carrier and a compound having the formula HOOC-CH(NH 2 )-Y , -Y 2 -Y 3 -Y 4 -B(OH) 2 wherein each of Y ⁇ , Y 2 , Y3, and Y4 is selected from the group consisting of CH 2 , S, O NH, and N-alkyl.
  • compositions which also include a pharmaceutically acceptable carrier, include ABHA and a compound having the formula HOOC-CH(NH 2 )-CH 2 -S-CH 2 -CH 2 -B(OH) 2 ,
  • This compound is S-(2-boronoethyl)-L-cysteine (BEC).
  • BEC is also referred to herein as compound 15.
  • Any physiologically acceptable anion may be used as a counterion when the composition of the invention is prepared in the form of a salt.
  • Physiologically acceptable anions are well known in the art.
  • the composition of the invention may be a hydrochloride salt of ABHA or BEC.
  • the invention provides a method of inhibiting arginase comprising providing a composition of the invention to an arginase enzyme, preferably a yeast arginase enzyme or a mammalian arginase enzyme. When the enzyme is a mammalian arginase, the arginase is preferably a human arginase enzyme.
  • an arginase inhibitor by an arginase inhibitor as used herein, is meant a reduction in the level of arginase activity in the presence of an inhibitor, compared with the level of arginase activity in the absence of the inhibitor.
  • Arginase may be inhibited in yeast by contacting the yeast with the composition of the invention. Inhibition of arginase in yeast serves to minimize urea production during fermentation of alcoholic beverages.
  • the compositions of the invention are useful as anti- fungicides in agriculturally or otherwise economically important plant life.
  • the composition of the invention may be administered to the plant by spraying or other meand well known in the art of plant biology.
  • Arginase activity may be inhibited in a mammal, for example, by administering a pharmaceutical composition comprising the composition of the invention to the mammal.
  • the composition of the invention may be used to treat a disease in a mammal, wherein the disease is associated with expression of an abnormally high level of arginase activity in a tissue of the mammal. Because NO synthase activity is regulated in a reciprocal fashion with respect to arginase activity in mammals, more particularly humans, the composition of the invention may be used to treat a disease in a mammal, wherein the disease is associated with expression of an abnormally low level of NO synthase activity in a tissue of the mammal.
  • arginase and NO synthase Since the reciprocal interaction of arginase and NO synthase has implications for the function of smooth muscle as described in further detail herein in Example 4, the use of the compounds described herein for the regulation of smooth muscle activity in an animal is also contemplated in the invention.
  • arginase activity is meant a level of arginase activity which is exceeds the level found in normal tissue, which normal tissue does not exhibit an arginase related disease phenotype.
  • abnormally low level of NO synthase activity is meant a level of NO synthase activity which is lower than that found in normal tissue, which normal tissue does not exhibit an NO synthase related disease state.
  • an increase in arginase activity has been associated with the pathophysiology of a number of conditions including an impairment in nonadrenergic and noncholinergic (NANC) nerve-mediated relaxation of the gastrointestinal smooth muscle.
  • NANC nonadrenergic and noncholinergic
  • a method of enhancing smooth muscle relaxation comprising contacting the smooth muscle with an arginase inhibitor.
  • the smooth muscle is preferably within the body of an animal and the arginase inhibitor is preferably ABHA or BEC.
  • the type of smooth muscle to be relaxed includes, but is not limited to, gasterointestinal smooth muscle, anal sphincter smooth muscle, esophageal sphincter muscle, corpus cavemosum, sphincter of Oddi, arterial smooth muscle, heart smooth muscle, pulmonary smooth muscle, kidney smooth muscle, uterine smooth muscle, vaginal smooth muscle, cervical smooth muscle, placental smooth muscle, and ocular smooth muscle.
  • the type of gastrointestinal smooth muscle includes, but is not limited to, the internal anal sphincter muscle.
  • the invention includes a method of treating an arginase-related disease in an animal.
  • the animal is a human.
  • Diseases which are associated with either an abnormally high level of arginase activity in a tissue of a mammal or an abnormally low level of nitric oxide synthase activity in a tissue of the mammal are known in the art.
  • Diseases to be treated using the compositions of the invention include, but are not limited to, heart disease, systemic hypertension, pulmonary hypertension, erectile dysfunction, autoimmune encephalomyelitis, chronic renal failure and cerebral vasospasm.
  • the composition of the invention is administered to the mammal, and preferably comprises a pharmaceutically acceptable carrier as described in further detail herein.
  • the invention also contemplates the use of an arginase inhibitor in an in vitro arginase inhibition/smooth muscle functional assay, for the purpose of identifying compounds which affect smooth muscle function.
  • Compounds so identified are considered to be candidate arginase inhibitor antagonists, in that, as described in further detail below, these compounds are identified by their ability to counteract the ABHA or BEC mediated inhibition of arginase activity.
  • Example 4 an assay for smooth muscle activity using the internal anal sphincter muscle and one on the preferred arginase inhibitors of the invention ABHA.
  • strips of the internal anal sphincter muscle obtained from adult opossums are induced to relax by nonadrenergic and noncholinergic (NANC) nerve-mediated relaxation using electrical field stimulation (EFS); relaxation is reversed by contacting the muscle strips with arginase; and, reversal of relaxation is accomplished by contacting the muscle with an arginase inhibitor.
  • NANC nonadrenergic and noncholinergic
  • EFS electrical field stimulation
  • the muscle strips are then subsequently contacted with a test compound and the effect of the test compound on subsequent reversal of muscle relaxation is then assessed. Any significant reversal of the relaxation state of the muscle in the presence of the test compared with the relaxation state of the muscle in the absence of the test compound is an indication that the test compound is a candidate arginase inhibitor antagonist.
  • arginase inhibitor antagonist a compound which inhibits the inhibition of arginase by an arginase inhibitor.
  • the invention encompasses the preparation and use of pharmaceutical compositions comprising an arginase inhibitor as an active ingredient.
  • a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these.
  • Administration of one of these pharmaceutical compositions to a subject is useful for inhibiting arginase activity and thereby treating a disease or disorder associated with arginase enzyme activity, as described elsewhere in the present disclosure.
  • the active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
  • the term "pharmaceutically acceptable carrier” means a chemical composition with which the active ingredient may be combined and which, following the combination, can be used to administer the active ingredient to a subject.
  • the term "physiologically acceptable" ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.
  • the formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
  • compositions suitable for administration to humans are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation.
  • Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs, birds including commercially relevant birds such as chickens, ducks, geese, and turkeys, fish including farm-raised fish and aquarium fish, and crustaceans such as farm-raised shellfish.
  • compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration.
  • Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
  • a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • compositions of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.
  • additional agents include anti-emetics and scavengers such as cyanide and cyanate scavengers.
  • Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.
  • a formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared, packaged, or sold in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient.
  • Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion.
  • an "oily" liquid is one which comprises a carbon- containing liquid molecule and which exhibits a less polar character than water.
  • a tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients.
  • Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent.
  • Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture.
  • compositions used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents.
  • Known dispersing agents include, but are not limited to, potato starch and sodium starch glycollate.
  • Known surface active agents include, but are not limited to, sodium lauryl sulphate.
  • Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate.
  • Known granulating and disintegrating agents include, but are not limited to, com starch and alginic acid.
  • binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose.
  • Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.
  • Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient.
  • a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets.
  • tablets may be coated using methods described in U.S. Patents numbers 4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlled release tablets.
  • Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically elegant and palatable preparation.
  • Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
  • an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
  • Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin.
  • Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.
  • Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.
  • Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle.
  • Aqueous vehicles include, for example, water and isotonic saline.
  • Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents.
  • Oily suspensions may further comprise a thickening agent.
  • suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose.
  • Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively).
  • Known emulsifying agents include, but are not limited to, lecithin and acacia.
  • Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid.
  • Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.
  • Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.
  • Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent.
  • Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent.
  • Aqueous solvents include, for example, water and isotonic saline.
  • Oily solvents include, for example, almond oil, oily esters, ethyl alcohol. vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.
  • a pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in- water emulsion or a water-in-oil emulsion.
  • the oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these.
  • Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate.
  • These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for rectal administration.
  • a composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation.
  • Suppository formulations may be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e. about 20 °C) and which is liquid at the rectal temperature of the subject (i.e. about 37°C in a healthy human).
  • Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides.
  • Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.
  • Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier.
  • enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject.
  • Enema preparations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for vaginal administration.
  • a composition may be in the form of, for example, a suppository, an impregnated or coated vaginally-insertable material such as a tampon, a douche preparation, or a solution for vaginal irrigation.
  • Douche preparations or solutions for vaginal irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier.
  • douche preparations may be administered using, and may be packaged within, a delivery device adapted to the vaginal anatomy of the subject.
  • Douche preparations may further comprise various additional ingredients including, but not limited to, antioxidants, antibiotics, antifungal agents, and preservatives.
  • parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, so long as the arginase inhibitor compound is not administered systemically.
  • Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.
  • Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • a suitable vehicle e.g. sterile pyrogen-free water
  • compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions.
  • Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent.
  • Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity.
  • a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers.
  • Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low- boiling propellant in a sealed container.
  • such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers.
  • Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
  • Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition.
  • the propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient).
  • compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension.
  • Such formulations may be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization or atomization device.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate.
  • the droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 nanometers.
  • the formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition of the invention.
  • Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered in the manner in which snuff is taken i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nares.
  • Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration.
  • Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein.
  • formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient.
  • Such powdered, aerosolized, or aerosolized formulations, when dispersed preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for ophthalmic administration.
  • Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier.
  • Such drops may further comprise buffering agents, salts, or one or more other of the additional ingredients described herein.
  • Other ophthalmalmically- administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form or in a liposomal preparation.
  • additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.
  • compositions of the invention are known in the art and described, for example in Genaro, ed., 1985, Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton, PA, which is incorporated herein by reference.
  • dosages of the arginase inhibitor which may be administered to an animal, preferably a human, range in amount from 1 ⁇ g to about 100 g per killogram of body weight of the animal. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration. Preferrably, the dosage of the compound will vary from about 1 mg to about 10 g per killogram of body weight of the animal. More preferably, the dosage will vary from about 10 mg to about 1 g per killogram of body weight of the animal.
  • the compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even lees frequently, such as once every several months or evenonce a year or less.
  • the frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.
  • Arginase is one of the very few enzymes that requires a spin-coupled Mn 9 ⁇ + -Mir 9-+ cluster for catalytic activity in vitro and in vivo (Reczkowski et al., 1992,
  • the overall folding pattern of the arginase monomer indicates that the protein belongs to the ⁇ / ⁇ protein class.
  • the arginase monomer has a globular structure, having approximate dimensions of 40 * 50 x 50 A.
  • One side of the active- site cleft is partially defined by the central eight-stranded ⁇ -sheet, and the metal binding site is located on one edge of the ⁇ -sheet.
  • Metal ligands are located immediately adjacent to helix C, to strand 4, and to strand 7, as depicted in Figure 1.
  • the image in Figure 2 was generated with MOLSCRIPT (Kraulis et al., 1991, J. Appl. Crystallogr. 24:946-950) and Raster 3D (Bacon et al., 1988, J. Mol. Graph. 6:219-200; Merrit et al.. 1994. Acta Crystallogr. D50:869-873).
  • the binuclear manganese cluster is located at the base of an approximately 15 A-deep active site cleft in each monomer, as depicted in Figure 4.
  • the metal ion that is more deeply situated in the active site cleft is designated Mn + and is coordinated by His-101 (N ⁇ ), Asp-124 (O ⁇ l), Asp-128 (O ⁇ l), Asp-232 (O ⁇ l) and a solvent molecule.
  • the coordination of Mn ⁇ + by arginase has square pyramidal geometry.
  • a solvent molecule bridges Mn ⁇ and MIL " and also donates a hydrogen bond to Asp-128, the O ⁇ 2-O separation distance being about 2.8 A.
  • the second metal ion is designated MIL + and is coordinated by His- 126 (N ⁇ ), Asp- 124 (O ⁇ 2), Asp-232 (O ⁇ l), Asp-234 (bidentate O ⁇ l and O ⁇ 2).
  • the coordination of Mn + by arginase and the bridging solvent molecule has a octahedral geometry which is distorted, owing to the bidentate coordination of Asp-234.
  • the separation between Mn ⁇ and MIL + _S about 3.3 A. All metal ligands except Asp- 128 make hydrogen-bond interactions with other protein residues, and these interactions contribute to the stability of the metal binding site (Christianson et al, 1989, J. Am. Chem. Soc. 111 :6412-6419).
  • the carboxylate side chain of Asp-232 is a monodentate bridging ligand, with O ⁇ l coordinated to both Mn ⁇ + and MIL + with anti- and syrc-coordination stereochemistry, respectively (Rardin et al., 1991, New J. Chem. 15:417-430).
  • the O ⁇ 2 oxygen of Asp-232 is about 3.8 A away from M ⁇ .
  • the solvent molecule bridges both manganese ions symmetrically, with the separation distance of the O atom of the solvent from both of Mn ⁇ and MIL + being about 2.4 A.
  • the arginase structure is the first atomic resolution structure determined for a functional metalloenzyme that has a specific catalytic and physiological requirement for two Mn + ions.
  • Arginase does not use transition metals promiscuously for catalysis, unlike other metalloenzymes, in which Mn + and, for instance, Mg 9+ or other metal ions are interchangeable, albeit with some effect on catalytic activity.
  • the identity and oxidation state of the manganese cluster of arginase has been conclusively established by electron paramagnetic resonance spectroscopy (Reczkowski et al., 1992, J. Am. Chem. Soc. 114:10992-10994).
  • the catalytic metal requirement of arginase is rooted in the prefened geometry of manganese coordination, which properly orients the metal-bridging solvent molecule, such that it arginase hydrolysis is catalyzed. Because the metal-bridging solvent molecule must satisfy the coordination preferences of two manganese ions simultaneously, the position of the solvent molecule, and therefore its optimal catalytic activity, is highly sensitive to substitution of one or both Mn 9 ⁇ + ions with a different metal ion. Coordination of the solvent molecule to two metal ions, rather than one, enhances the dependence of optimal catalytic activity on proper metal composition of arginase. Only two other polar residues are found in the immediate vicinity of the active site of arginase, namely Glu-277 and His-141.
  • Glu-277 is located deep in the active-site cleft, about 4.5 A away from Mn 2+ .
  • a roughly 20° rotation about ⁇ 1 of this side chain yields an ideal salt link with the substrate guanidinium group.
  • this salt link positions the electrophilic guanidinium carbon of the substrate directly over the metal-bridging solvent molecule, which is likely to be a nucleophilic hydroxide ion in the active catalyst, as depicted in Figures 4 and 5. It is unlikely that the deprotonated substrate guanidinium group binds directly to the metal(s) because of its high pK a value, namely 13.5.
  • the side chain of His-141 is located about midway between the top and the bottom of the active-site cleft.
  • His-141 ⁇ Asn arginase When asparagine substituted in place of histidine at amino acid position 141 of arginase (His-141 ⁇ Asn arginase), the enzyme retains roughly ten percent of its wild type activity (Cavalli et al., 1994, Biochemistry 33:10652-10657). Because its location is only about 4.2 A from the metal-bridging solvent molecule, it is possible that His-141 acts a proton shuttle in arginase catalysis, mediating proton transfer to and from bulk solvent.
  • Direct proton transfer with bulk solvent may be possible in the absence of His-141 , which could account for the significant residual catalytic activity of His-141- ⁇ -Asn arginase.
  • a proton shuttle function for His-141 of arginase is analogous to that recently reviewed for His-64 of the zinc metalloenzyme carbonic anhydrase II (Christianson et al., 1996, Ace. Chem. Res. 29:331-339).
  • charged amino acid residues are located on opposite sides of the active-site cleft, near the surface of the arginase trimer. The presence of these charged residues may contribute to the extraordinar specificity of substrate recognition by arginase.
  • FIG. 5 A model of arginine hydrolysis by arginase is illustrated in Figure 5.
  • the ionization of metal-bound water probably reflects the apparent p__ " a value of 7.9 observed in the pH rate profile of the enzyme (Kuhn et al., 1991, Arch. Biochem. Biophys. 286:217-221).
  • Asp-128 stabilizes the metal-bridging hydroxide ion with a hydrogen bond, while the hydroxide ion performs a nucleophilic attack at the guanidinium carbon of arginine.
  • the resulting tetrahedral intermediate collapses upon proton transfer to the amino group of omithine. This proton transfer is probably mediated by Asp-128.
  • His-141 shuttles a proton from bulk solvent to the ⁇ -amino group of omithine prior to dissociation of omithine from arginase.
  • a water molecule displaces urea, resulting in dissociation of urea from arginase.
  • Metal coordination facilitates the ionization of this water molecule to regenerate a nucleophilic hydroxide ion. Proton transfer from the water molecule to bulk solvent may be mediated by His- 141.
  • Example 2fS VAmino-6-Boronohexanoic Acid (ABHA is an Effective Inhibitor of Arginase Using structural data described in Example 1 , an inhibitor of arginase activity was designed which resembles the proposed transition state intermediate of the arginine hydrolysis reaction catalyzed by arginase. The inhibitor was tested and it was determined to be a potent inhibitor of arginase activity.
  • the tetrahedral borate anion is a modest inhibitor of Mn 2+ -arginase, a critical metalloenzyme of mammalian nitrogen metabolism.
  • the crystal structure of the arginase-ornithine-borate complex, as described herein, reveals a net displacement of the solvent molecule bridging the binuclear manganese cluster by a borate oxygen atom in the native enzyme active site. This binding mode is reminiscent of the tetrahedral intermediate proposed for arginase-catalyzed arginine hydrolysis, as described in Example 1 herein.
  • ABHA a boronic acid-based arginine isostere appears to bind to arginase as the tetrahedral boronate anion and mimic the conformation of the tetrahedral arginine-hydrolysis intermediate.
  • ABHA was synthesized and the ability of ABHA to inhibit arginase- catalyzed arginine hydrolysis was evaluated.
  • ABHA is one of the most potent reversible inhibitors of arginase described to date, having an IC ⁇ Q value of 0.8 micromolar. Complete kinetic characterization of ABHA was complicated by nonlinearity of unknown origin, there being no evidence for slow-binding behavior. Competition binding experiments using N-hydroxyarginine indicate that K ⁇ for ABHA was less than or equal to 0.1 micromolar.
  • ABHA metal- bridging solvent molecule observed in the native enzyme is displaced by an oxygen atom of the tetrahedral boronic acid anion.
  • the boronic acid moiety of ABHA effects inhibition of the enzyme by displacing the metal -bridging solvent molecule in a similar manner.
  • TLC Thin layer chromatography
  • Merck Merck & Co., West Point, PA
  • silica gel 60 F 2 ⁇ 4 glass plates Compounds with tert- butyloxycarbonylamino groups or free amino groups were visualized on TLC plates by applying a ninhydrin solution (comprising 0.1% (w/v) ninhydrin in 95% (v/v) n- butanol, 4.5% (v/v) water, 0.5% (v/v) glacial acetic acid) to the plates and then heating the plates until color evolved.
  • a ninhydrin solution comprising 0.1% (w/v) ninhydrin in 95% (v/v) n- butanol, 4.5% (v/v) water, 0.5% (v/v) glacial acetic acid
  • Arginase activity was assessed using the 14 C-guanidino)-arginine assay of Ruegg et al. (1980, Anal. Biochem. 102:206-212), as described (Reczkowski et al., 1994, Arch. Biochem. Biophys. 312:31-37).
  • Compound 5 had the following properties H- NMR (CDC1 3 ) ⁇ 4.95 (d, IH), 4.20 (d, IH), 4.1 (m, IH), 2.40-2.15 (m, 2H), 2.05 (m, IH), 1.95-1.65 (m, 4H), 1.60 (s, 3H), 1.42 (2s, 18H), 1.35 (apparent s, 4H), 1.25 (s, 3H), 1.05 (d, IH), 0.80 (m, 5H).
  • the molecular mass calculated for C 25 H 44 NO 6 B + H is 466.33.
  • the molecular mass of Compound 5 observed using ZAB-E CI+/MS mass spectrometry was 466.25. Synthesis of 2CS .-amino-6-boronohexanoic acid, hydrochloride salt
  • Table 2 lists cell information, data collection parameters, and refinement data used to determine the structure of ABHA.
  • Final positional and equivalent isotropic thermal parameters are given in Table 3.
  • Anisotropic thermal parameters are in Table 4.
  • Tables 5 and 6 list bond distances and bond angles.
  • a representation of the molecular structure of ABHA, made using the ORTEP program, is depicted in Figure 7.
  • anisotropic displacement parameter is: exp[-2 ⁇ 2 (a* 2 U ⁇ h 2 + b* 2 U 22 k 2 + c* 2 U 33 l 2 + 2b*c*U 23 kl + 2a*c*U 13 hl +
  • Arginine hydrolysis involves a metal-activated solvent molecule that symmetrically bridges the Mn -Mn 2+ ion pair in the native enzyme.
  • the reaction coordinate of hydrolysis is postulated to proceed through a tetrahedral intermediate resulting from nucleophilic attack of the metal-bridging hydroxide ion at the guanidinium carbon of arginine, as depicted in Figure 8, Panel A (Kanyo et al, 1996, Nature 383:554-557).
  • Omithine and borate are known to be relatively weak inhibitors of arginase activity. The simultaneous presence of both omithine and borate inhibits arginase activity more that the presence of either compound alone.
  • the electron-deficient boron atom of a boronic acid promotes the addition to the boron atom of a suitable nucleophile, such as a protein-bound nucleophile or a solvent molecule, yielding a stable anionic tetrahedral species.
  • ABHA 2(S)-amino-6- boronohexanoic acid
  • the tetrahedral borate anion is a modest, noncompetitive inhibitor of arginase, having a K j value of 1.0 millimolar and a ⁇ value of 0.26 millimolar. Inhibition of arginase activity by borate is even more pronounced in the presence of omithine, which is a competitive inhibitor of arginase, having a K j value of 1.0 millimolar (Pace et al., 1981 Biochem. Biophys. Acta 658:410-412; Reczkowski et al., 1994, Arch. Biochem. Biophys. 312:31-37; Khangulov et al, 1995, Biochemistry 34:2015-2025). Measurement of the inhibitory potential of arginase inhibitors yielded an IC5 Q value of 80 micromolar for N-hydroxyarginine and an IC ⁇ value of 0.8 micromolar for ABHA.
  • the enzymes of arginine catabolism have been the subject of increasingly intense research interest.
  • the critical macrophage enzymes arginase and nitric oxide (NO) synthase.
  • Arginase catalyzes the hydrolysis of L-arginine to form L-ornithine and urea (Christianson, 1997, Prog. Biophys. Molec. Biol. 67:217-252)
  • NO synthase catalyzes the oxidation of L-arginine to form citrulline and NO (Griffith et al., 1995, Annu. Rev. Physiol. 57:707-736).
  • N°-Hydroxy-L-arginine is an intermediate in the NO synthase reaction (Pufahl et al., 1992, Biochemistry 31:6822-6828; Stuehr et al., 1991, J. Biol. Chem.
  • 2(S)-amino-6-boronohexanoic acid (7) exhibited substantial nonlinearity in kinetic replots (Example 2), and a full description of its binding kinetics has been reported herein.
  • TLC Thin layer chromatography
  • 2(S)-N-(tert-butyloxycarbonyl)-aspartic acid tert-butyl ester (1) was purchased from Bachem.
  • Triethylamine and DMSO (dimethyl sulfoxide) were purchased from Fisher. All other reagents were purchased from Aldrich and used without further purification.
  • 2fSVamino-6-boronohexanoic acid (1) The synthesis and preliminary evaluation of this boronic-based analogue is described in Example 2 herein.
  • the Swem oxidation was completed by the addition of 0.726 mL (14.6 mmol) of triethylamine.
  • the mixture was stirred for 5 min. at -78 °C and then warmed to room temperature.
  • the CH C1 was evaporated in vacuo.
  • the crude material was taken up in THF and the precipitant triethylamine salt was removed by filtration.
  • the material was purified by column chromatography on silica gel (with 6:1 hexane:ethyl acetate as an eluent). The product was obtained in 69% yield (980 mg).
  • Recombinant rat liver arginase was purified as described previously (Cavalli et al., 1994, Biochemistry 33:10652-10657). The concentrations of enzyme stock solutions were determined from the absorbance at 280 nm using an extinction coefficient of 1.09 mL mg "1 cm-1 (Schimke, 1970, Methods Enzymol. 17a:313-317). The activity of wild-type arginase was monitored spectrophotometrically on a Pharmacia Biotech UltroSpec 2000. Assays were performed in 100 ⁇ M MnCl 2 , 50 mM bicine-NaOH (pH 9.0).
  • the compound l-guanidino-3 -nitrobenzene (18) proved to be a new substrate for arginase hydrolysis, producing products urea and the chromogen w-nitroaniline.
  • a stock solution of 200 mM 18 in DMSO was prepared. A period of 15 minutes was allowed for 18 to equilibrate with bicine buffer prior to assay.
  • Dialysis experiments of arginase with the arginase-7 and arginase-15 complex were performed in following way. Thirty microliters of arginase at a concentration of 11.2 mg/mL were preincubated for thirty minutes with an appropriate concentration of 7 ( 5 ⁇ L of 100 ⁇ M stock solution). For 15, 300 ⁇ L of arginase at a concentration of 0.938 mg/mL were preincubated for thirty minutes with a sufficient concentration of 15 (30 ⁇ L of 200 ⁇ M stock solution) to suppress all detectable enzymatic activity. Preincubates were then added to bicine buffer with 4 mM 18. The final volume of the assay reaction solution was 1 mL.
  • the inhibition of arginase by 7 and 15 was also evaluated using a modified version of the radioactive assay by R ⁇ egg and Russell (R ⁇ egg et al., 1980, Anal. Biochem. 102:206-212). Assays were performed in 100 ⁇ M MnCl 2 , 100 mM ches-NaOH (pH 9.0). The reactions were initiated by the addition of 5 ⁇ L of a ⁇ 1 unit mL enzyme solution to 45 ⁇ L of reaction mixture containing ches buffer, 1.4 mM L-arginine, -5.0 x 10 4 cpm of L-[guanidino- C]arginine, and variable concentrations of the appropriate inhibitor.
  • the contents of the sample cell were titrated with 30-40 aliquots (2.5 ⁇ L each) of inhibitor (an initial 1 ⁇ L injection was made, but not used in data analysis). After each injection, the heat change was measured and converted to the conesponding enthalpy value.
  • the reaction mixture was continuously stined at 400 rpm during titration. Control experiments were carried out by titrating the inhibitor into the buffer solution under identical experimental conditions. Data analysis was performed using Origin software provided with the instrument. The calorimetric data are presented with the background titrations subtracted from the experimental data. The amount of heat produced per injection was calculated by integration of the area under each peak.
  • k cat 15,000 ⁇ 1200 min. " 1 ) (Cavalli et al., 1994, Biochemistry 33:10652-10657). Despite its low k cat value, substrate 18 provided a continuous chromogenic assay with no apparent background and high precision. Furthermore, the synthesis of 18 required only two straightforward steps, which facilitates the rapid production of large quantities of this compound.
  • boronic acid inhibitors 7 and 15 were essentially equipotent (within experimental enor), with compound 7 showing a marginally lower tn_, seu(j0 .
  • the rip Seucj0 for boronic acid 12 was two orders of magnitude higher than the DSeucj0 values for 7 and 15. Thus, inhibitor 12 was ⁇ 400-fold less potent than 7 or 15.
  • silanetriol 16 has a fixed tetrahedral configuration, and only 16 yielded a linear plot of v Q /v versus (I) (indicative of reversible inhibition).
  • Silanetriol inhibitors have recently been developed against ⁇ -lactamase (Curley et al., 1997, J. Am. Chem. Soc. 119:1529-1538).
  • silanetriols are significantly poorer inhibitors than their boronic acid analogues.
  • Silanetriols have additional complications as enzyme inhibitors due to their generally poorer solubility and greater tendency to dimerize (Knight et al.,1989, J. Chem. Soc, Dalton Trans.275-281; McNeil et al., 1980, J. Am. Chem. Soc. 102:1859-1865).
  • IAS intemal anal sphincter
  • the composition of the Krebs' solution was as follows (mM): NaCl, 118.07; KC1 4.69; CaCl 2 , 2.52; MgSO 1.16; NaH 2 PO 1.01; NaHCO 3 , 25 and glucose (Pufahl et al., 1995, Biochemistry 34:1930-1941; Chenais et al., 1993, Biochem. Biophys. Res. Commun. 196:1558-1565).
  • the anal canal was cleaned of the extraneous connective tissue and the blood vessels. Following this, the anal canal was opened flat by an incision along the longitudinal axis, and was pinned flat with the mucosal side facing up.
  • the IAS smooth muscle strips prepared as described above were mounted onto the thermostatically controlled 2 ml muscle baths (37°C) containing oxygenated (95% O 2 and 5% CO 2 ) Krebs' solution.
  • One end of the muscle strip was fixed to the bottom of the muscle bath with a tissue holder to the stand and the other end was attached to a isometric force transducer (model FT03; Grass Instruments Co., Quincy, MA) for the measurement of isometric tension.
  • the smooth muscle tension was recorded on a Dynograph recorder (model R411 ; Beckman Instruments, Schiller Park, IL).
  • EFS was delivered via a pair of platinum wires, from a Grass stimulator (Model S88; Grass Instruments Co., Quincy, MA) connected in series to a Med-Lab Stimu-Splitter II (Med-Lab Instruments, Loveland, CO).
  • the Stimusplitter was used to amplify and measure the stimulus intensity using the optimal stimulus parameters for the neural stimulation (12V, 0.5 ms pulse duration, 200-400 mA, 4 sec train) at varying frequencies of 0.5 to 20 Hz.
  • These parameters of EFS are known to cause relaxation of the IAS smooth muscle via the selective activation of NANC myenteric neurons.
  • Neurally-mediated relaxation of the IAS smooth muscle strips was quantified in response to different frequencies of EFS. All the experiments were performed in the presence of atropine (1 x 10 " " M) and guanethidine (3 x 10 " " M).
  • Tissue samples of the opossum intemal and sphincter muscle (I AS), the adjoining rectal tissue, the liver, and the brain were homogenized with an ultratunax tissue homogenizer (Tekamr, Cincinnati, OH) in 10 mM Tris-HCl, 150 mM KC1, 25 mM MnCl 2 , pH7.4.
  • the resulting homogenates were then dialyzed overnight against 10 mM Tris-HCl, 150 mM KC1, 25 mM MnCl 2 , pH 7.4.
  • the dialyzed homogenates were centrifuged to remove insoluble material and concentrated with Amicon Centricon 30 microconcentrators to give stock protein concentrations of 2.4 mg/ml for the IAS smooth muscle, 3.6 mg/ml for the rectal smooth muscle, 3 mg/ml for the brain, and 17.5 mg/ml for the liver. Protein concentrations were estimated with the Pierce Coomassie Protein Reagent using bovine serum albumin as a standard.
  • Arginase Assay The arginase activity of tissue homogenates was evaluated using the radioactive L-[guanidino 14 C]arginine assay of R ⁇ egg and Russell (1980, Anal. Biochem. 102:206-212).
  • Assays were performed in 100 mM CHES-NaOH 0.1 mM MnCl 2 at pH 9.0. The reactions were initiated by the addition of 5 ⁇ l of tissue homogenate to 45 ⁇ l of reaction mixture that contained the CHES buffer, the appropriate concentration of arginine (0.5 to 5 mM) and - 5.0 x 10 4 cpm of L-
  • Bovine liver arginase, N ⁇ -hydroxy-L-arginine (L-HO-Arg), N ⁇ -nitro- L-arginine (L-NNA), N ⁇ -nitro-D-arginine methyl ester (D-NNA), L-arginine hydrochloride, D-arginine, D-omithine and atropine sulfate were obtained from Sigma Chemical Co., St. Louis, MO. Guanethidine monosulfate was from Ciba
  • Ethylenediamine tetraacetic acid (EDTA) tetrasodium salt was from Fisher Scientific Co., Fair Lawn, NJ 2(S)-amino-6-boronohexanoic Acid (ABHA) was synthesized as described in Example 2.
  • the responses to EFS and other relaxants were expressed as the percent of maximal relaxation caused by 5 mM EDTA.
  • the results are expressed as means ⁇ S.E. Statistical significance between different groups were determined using t-test and a p value smaller than 0.05 was considered significant.
  • the IAS relaxations in response to 0.5, 1 and 2 Hz of EFS in control experiments were 31.0 ⁇ 0.7, 55.9 ⁇ 3.0 and 67.7 ⁇ 3.9%, respectively.
  • the pretreatment of the tissues with L-HO-Arg (1 x 10 "4 M) before the addition of arginase antagonized the effect of arginase in attenuating the EFS-induced IAS relaxation.
  • L-HO-arg was found to be the most potent in inhibiting arginase activity in the liver (Figure 26).
  • the IC ⁇ Q values for the inhibition of arginase activity in liver, IAS, rectum and brain homogenates by L- HO-arg were 2.4, 25, 42 and 40 ⁇ M respectively.
  • liver arginase activity was approximately 10-20 fold more sensitive to inhibition by L-HO-Arg than the arginase activities in the other tissues.
  • the ability of ABHA to inhibit arginase activity in the tissues was in striking contrast to the inhibition observed with L-HO-Arg.
  • ABHA was found to be the most potent in inhibiting arginase activity in brain and rectum, followed by IAS and liver; the conesponding IC ⁇ Q values were 0.05, 0.05, 0.10 and 0.44 ⁇ M for the arginase activities in brain, rectum, IAS and liver, respectively ( Figure 27).
  • Inhibition constants for ABHA were estimated by titrating the inhibitor into assay mixtures containing an arginine concentration fixed at the K ⁇ . value, and assuming competitive inhibition. These experiments yielded estimated K j values of 0.018, 0.026, 0.05 and 0.19 ⁇ M for the arginase activities in brain, rectum, IAS and liver, respectively.
  • the estimated K j for ABHA inhibition of the liver enzyme is in good agreement with the K ⁇ of 0.11 ⁇ M determined by titration calorimetry. Influence of the NO synthase Inhibitor L-N-Nitro- Arginase (L-NNA, and L-NNA Plus the Arginase Inhibitors on the arginase Activity in Different Tissues
  • L-HO-Arg is not only an intermediate in the biosynthesis of NO, but is also a substrate for NO synthase.
  • the tissue variations in the inhibition of arginase activities by L-HO-Arg could be due to depletion of added L-HO-Arg by conversion to NO.
  • the effects of L-HO-Arg on the arginase activities in the different tissue homogenates were determined in the presence of the NO synthase inhibitor L-NNA.
  • L-NNA at 80 ⁇ M had no effect on the activities of the various arginases in the presence of L-HO-Arg ( Figure 28), indicating that depletion of added L-HO-Arg by the action of NO synthase was not a concern in these experiments.
  • Type I arginase is found predominantly in mammalian liver and red blood cells, while the type II enzyme is thought to be expressed in macrophages, kidney and endothelial cells. Although the expression patterns for the arginase in opossum rectum, brain, IAS and liver are not known, the differential effects of ABHA and N ⁇ -hydroxy- L-arginine on the arginase activities in these tissues are consistent with type II enzyme expression in the non-hepatic tissues.
  • N Q -hydroxy-L-arginine against hepatic arginase as compared to the non-hepatic tissue extracts might be related to the ability of N ⁇ - hydroxy-L-arginine to serve as a substrate for NO synthase.
  • added N ⁇ -hydroxy-L-arginine would be rapidly converted to NO and citrulline, lowering the effective concentration of N ⁇ -hydroxy-L- arginine and lowering arginase inhibition.
  • inhibition studies with N ⁇ -hydroxy-L-arginine were repeated in the presence of L-NNA, a known inhibitor of NO synthase.
  • the functional data in the IAS also support the concept that ABHA is more selective in inhibiting arginase as compared to L-HO-Arg. This was evident from the IAS studies in the presence of the NO synthase inhibitor L-NNA. As discussed in introduction, L-HO-Arg may also serve as an NO synthase substrate. The experiments were performed to examine the influence of L-HO-Arg on the NANC relaxation of the IAS that was attenuated by the NO synthase inhibitor. The effects of L-HO-Arg and ABHA in reversing the attenuation of the NANC relaxation, were compared with L- arginine, an authentic substrate for NO synthase.
  • L-HO-Arg caused the reversal of L-NNA-attenuated NANC relaxation of the IAS with a potency comparable to L-arginine.
  • ABHA had no effect on the IAS relaxation suppressed by the NO synthase inhibitor.
  • L-HO-Arg may in part be a substrate for NO synthase and that it may be less selective than ABHA in inhibiting arginase.
  • NO synthase pathway is the predominant pathway responsible for the NANC relaxation of the IAS (Rattan and Chakder, 1992, Am. J. Physiol. Gastrointest. Liver Physiol. 262:G107-G112; Rattan et al., 1992, Gastroenterology 103:43-50)
  • the augmentation of the IAS relaxation is speculated to be due to the upregulation of the NO synthase pathway by an increase in the tissue levels of L-arginine.
  • exogenously administered L-arginine in the normal tissues has no significant effect on the NANC relaxation of the IAS unless the tissues are made L-arginine deficient (Chakder and Rattan, 1997, J. Pharmacol. Exp.
  • exogenous L-arginine In the basal state, in the normal tissues, exogenous L-arginine has no significant effect on either the basal IAS tone or the NANC relaxation (Chakder and Rattan, 1997, J. Pharmacol. Exp. Ther. 282:378-384). Conversely, in the L-arginine deficient tissues, exogenous L-arginine causes a significant fall in the basal IAS tone and reversal of the impaired NANC relaxation in the IAS smooth muscle ((Chakder and Rattan, 1997, J. Pharmacol. Exp. Ther. 282:378-384; Rattan and Chakder, 1997, Gastroenterology 112:1250-1259).
  • the effect of exogenous L- arginine in the L-arginine deficient tissues is due to an increase in the uptake of the amino acid leading to an augmentation of the NANC relaxation via the upregulation of NO synthase pathway.
  • Such mechanisms may not be operative in the normal tissues due to the normal state of equilibrium of L-arginine levels at the cellular levels.
  • the augmentation of the NANC relaxation in the presence of arginase inhibitors may be due to the increase in the intracellular L-arginine levels as proposed in other nongastrointestinal tissues (Jenkinson et al., 1996, Comp. Biochem. Physiol. 114b: 107- 132; Buga et al., 1996, Am. J. Physiol. Heart Circ. Physiol.
  • L-HO-Arg acts at both the levels, as an arginase inhibitor and as a substrate for the NO synthase.
  • ABHA is selective for arginase inhibition only. Rattan and Chakder (1992, Am. J. Physiol. Gastrointest. Liver Physiol. 262:G107-G112), Rattan et al. (1992, Gastroenterology 103:43-50), Chakder and Rattan (1993, Am. J. Physiol. Gastrointest. Liver Physiol.
  • Arginase inhibitors therefore may have significant role in the pathophysiology and potential therapy in a number of disease conditions.
  • the newly described arginase inhibitor ABHA may play a novel role in the identification of isozyme specific arginase pathways and their role as therapeutic potentials for the specific control of the hemodynamic effects associated with the unregulated arginase activity.
  • the disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Compositions and methods for inhibiting arginase activity, including arginase activity in a mammal, are provided. Methods of making the compositions of the invention are also provided as are methods of using the compositions therapeutically.

Description

COMPOSITIONS AND METHODS FOR INHIBITING ARGINASE ACTIVITY
FIELD OF THE INVENTION The field of the invention is enzyme inhibitors.
BACKGROUND OF THE INVENTION
Each individual excretes roughly ten kilograms of urea per year, as a result of the hydrolysis of arginine in the final cytosolic step of the urea cycle (Krebs et al., 1932, Hoppe-Seyler's Z. Physiol. Chem. 210:33-66). The activity of the liver enzyme, arginase, permits disposal of nitrogenous wastes which result from protein catabolism (Herzfeld et al., 1976, Biochem. J. 153:469-478). In tissues which lack a complete complement of the enzymes which catalyze the reactions of the urea cycle, arginase regulates cellular concentrations of arginine and omithine, which are used for biosynthetic reactions (Yip et al., 1972, Biochem. J. 127:893-899). Arginine is used, by way of example, in the synthesis of nitric oxide. In macrophages, arginase activity is reciprocally coordinated with the activity of the enzyme, nitric oxide synthase.
Reciprocal coordination of the activities of arginase and nitric oxide (NO) synthase modulates NO-dependent cytotoxicity (Corraliza et al., 1995, Biochem. Biophys. Res. Commun. 206:667-673; Daghigh et al., 1994, Biochem. Biophys. Res. Commun. 202:174-180; Chenais et al, 1993, Biochem. Biophys. Res. Commun. 196:1558-1565; Klatt et al., 1993, J. Biol. Chem. 268:14781-14787; Keller et al., 1991, Cell. Immunol.
134:249-256; Albina et al., 1995, J. Immunol. 155:4391-4396).
Synthesis and evaluation of nonreactive arginine analogues for use as enzyme inhibitors or receptor antagonists is a rapidly growing area of medicinal chemical research (Griffith et al., 1995, Annu. Rev. Physiol. 57:707-736; Gross et al, 1990, Biochem. Biophys. Res. Commun. 170:96-103; Hibbs et al., 1987, J. Immunol. 138:550-565; Lambert et al., 1991, Life Sci. 48:69-75; Olken et al., 1992, J. Med. Chem. 35:1137-1144; Feldman et al., 1993, J. Med. Chem. 36:491-496; Narayanan et al., 1994, FASEB J. 8:A360; Narayanan et al., 1994, J. Med. Chem. 37:885-887; Moore et al., 1994, J. Med. Chem. 37:3886-3888; Moynihan et al., 1994, J. Chem. Soc. Perkin Trans.769-771; Robertson et al., 1995, J. Bioorganic Chem. 23:144-151).
To date, the X-ray crystal structure of one of the enzymes of mammalian arginine catabolism, namely rat liver arginase, is available (Kanyo et al., 1996, Nature 383:554-557). Rat liver arginase is a trimeric metalloenzyme which contains a binuclear manganese cluster in the active site of each subunit. This binuclear cluster is required for maximal catalytic activity (Reczkowski et al., 1992, J.
Am. Chem. Soc. 114:10992-10994).
As noted herein, arginase catalyzes the divalent cation-dependent hydrolysis of L-arginine to form L-ornithine and urea. The enzyme is currently known to serve three important functions: the production of urea, the production of omithine, and regulation of substrate arginine levels for nitric oxide synthase (Jenkinson et al.,
1996, Comp. Biochem. Physiol. 114B:107-132; Kanyo et al, 1996, Nature 383:554- 557; Christianson, 1997, Prog. Biophys. Molec. Biol. 67:217-252). Urea production provides a mechanism to excrete nitrogen in the form of a highly soluble, non-toxic compound, thus avoiding the potentially dangerous consequences of high ammonia levels. L-omithine is a precursor for the biosynthesis of polyamines, spermine and spermidine, which play important roles in cell proliferation and differentiation. Finally, arginase modulates the production of nitric oxide by regulating the levels of arginine present within tissues.
Since both NO synthase and arginase compete for the same substrate, the possibility of reciprocal regulation of both arginine metabolic pathways has recently been explored (Modelell et al., 1995, Eur. J. Immunol. 25:1101-1104; Wang et al., 1995, Biochem. Biophys. Res. Commun. 210:1009-1016). Furthermore, Nω- hydroxy-L-arginine (L-HO-Arg), an intermediate in the NO synthase reaction (Pufahl et al, 1992, Biochemistry 31 :6822-6828; Klau et al, 1993, J. Biol. Chem. 268:14781- 14787; Furchgom, 1995, Annu. Rev. Pharmacol. Toxicol., 35:1-27; Yamaguchi et al., 1992, Eur. J. Biochem., 204:547-552; Pufahl et al, 1995, Biochemistry 34:1930-1941), is an endogenous arginase inhibitor (Chenais et al., 1993, Biochem. Biophys. Res. Commun., 196:1558-1565; Buga et al., 1996, Am. J. Physiol. Heart Circ. Physiol.
271 :H1988-H-19998; Daghigh et al, 1994, Biochem. Biophys. Res. Commun. 202:174-180; Boucher et al., 1994, Biochem. Biophys. Res. Commun. 203:1614-1621). The phenomenon of reciprocal regulation between arginase and NO synthase has only been examined recently (Chakder and Rattan, 1997, J. Pharmacol. Exp. Ther. 282:378- 384; Langle et al., 1997, Transplantation 63:1225-1233; Langle et al., 1995,
Transplantation 59:1542-1549). In the internal anal sphincter (IAS), it was shown that the exogenous administration of arginase causes an attenuation of the NO synthase- mediated nonadrenergic and noncholinergic (NANC) nerve-mediated relaxation (Chakder and Rattan, 1997, J. Pharmacol. Exp. Ther. 282:378-384). An excess of arginase has recently been associated with a number of pathological conditions that include gastric cancer (Wu et al., 1992, Life Sci. 51 :1355- 1361; Leu and Wang, 1992, Cancer 70:733-736; Straus et al., 1992, Clin. Chim. Acta 210:5-12; Ikemoto et al, 1993, Clin. Chem. 39:794-799; Wu et al., 1994, Dig. Dis. Sci. 39:1107-1112), certain forms of liver injury (Ikemoto et al., 1993, Clin. Chem. 39:794- 799), and pulmonary hypertension following the orthotopic liver transplantation
(Langle et al, 1997, Transplantation 63:1225-1233; Langle et al., 1995, Transplantation 59:1542-1549). Furthermore, high levels of arginase may cause impairment in NANC-mediated relaxation of the IAS (Chakder and Rattan, 1997, J. Pharmacol. Esp. Ther. 282:378-384). Previous studies have demonstrated that arginase pretreatment causes a significant suppression of the NANC nerve-mediated relaxation of the IAS (Chakder and Rattan 1997, J. Pharmacol. Exp. Ther. 282:378-384) that is mediated primarily via the L-arginine-NO synthase pathway (Rattan and Chakder, 1992, Am. J. Physiol. Gastrointest. Liver Physiol. 262:G107-G112; Rattan and Chakder, 1992, Gastroenterology 103:43-50). The impairment in NANC relaxation by excess arginase may be related to L-arginine depletion (Wang et al., 1995, Eur. J. Immunol. 25:1101-1104). Furthermore, the suppressed relaxation could be restored by the arginase inhibitor L-HO-Arg. It is possible, therefore, that patients with certain conditions associated with an increase in arginase activity may stand to benefit from treatment with arginase inhibitors. However, an arginase inhibitor such as L-OH-Arg may not be selective since it also serves as a NO synthase substrate (Pufahl et al., 1992, Biochemistry 31 :6822-6828; Furchgott, 1995, Annu. Rev. Pharmacol. Toxicol. 25:1- 27; Pufahl et al, 1995, Biochemistry 34:1930-1941; Chemais et al., 1993, Biochem. Biophys. Res. Commun. 196:1558-1565; Boucher et al., 1994, Biochem. Biophys. Res.
Commun. 203:1614-1621; Griffith and Stuehr, 1995, Annu. Rev. Physiol. 57:707-736). Because of this, the exact role of arginase in pathophysiology and the potential therapeutic actions of arginase inhibitors remains undetermined.
A need remains for inhibitors of arginase activity, which are useful for treating diseases or disorders characterized either by abnormally high arginase activity in a tissue of a mammal or by abnormally low nitric oxide synthase activity in a tissue of the mammal.
SUMMARY OF THE INVENTION The invention include a composition comprising the formula HOOC-CH(NH2)-Y1-Y2-Y3-Y4-B(OH)2 wherein each of Yj, Y2, Y3, and Y4 is selected from the group consisting of CH2, S, O NH, and N-alkyl, except Y2 is not S when each of Yj, Y3, and Y4 is CH ,. In one aspect, the composition has the formula HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2. In a preferred embodiment, the composition of the invention further comprises a pharmaceutically acceptable carrier. Also included in the invention is a composition comprising a pharmaceutical acceptable carrier and a compound having the formula
HOOC-CH(NH2)-YrY2-Y3-Y4-B(OH)2 wherein each of Y^, Y2, Y3, and Y4 is selected from the group consisting of CH2, S, O NH, and N-alkyl.
In one aspect, the compound has a formula selected from the group consisting of
HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2 and HOOC-CH(NH2)-CH2-S-CH2-CH2-B(OH)2. The invention further includes a method of inhibiting arginase. The method comprising contacting the arginase with a composition having the formula
HOOC-CH(NH2)-Y1-Y2-Y3-Y4-B(OH)2 wherein each of Yj, Y2, Y3, and Y4 is selected from the group consisting of CH , S, O NH, and N-alkyl. In one aspect, the compound has a formula selected from the group consisting of
HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2 and
HOOC-CH(NH2)-CH2-S-CH2-CH2-B(OH)2. In preferred embodiments, the arginase may be a yeast arginase or a mammalian arginase. When the arginase is mammalian arginase, the arginase is human arginase.
Also included in the invention is a method of inhibiting arginase in a mammal. The method comprises administering to the mammal a composition comprising a pharmaceutically acceptable carrier and a compound having the formula HOOC-CH(NH2)-YrY2-Y3-Y4-B(OH)2 wherein each of Y^, Y2, Y3, and Y4 is selected from the group consisting of CH2, S, O NH, and N-alkyl. In one aspect, the compound has a formula selected from the group consisting of
HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2 and
HOOC-CH(NH2)-CH2-S-CH2-CH2-B(OH)2. In a preferred embodiment, the mammal is a human.
In another preferred embodiment, a tissue in the human has an abnormally high level of arginase activity.
In yet another preferred embodiment, a tissue in the human has an abnormally low level of nitric oxide synthase activity. Also included in the invention is a method of treating a disease in a human. The method comprises administering to the human a composition comprising a pharmaceutically acceptable carrier and a compound having the formula
HOOC-CH(NH2)-Y1-Y2-Y3-Y4-B(OH)2 wherein each of Yj, Y2, Y3, and Y4 is selected from the group consisting of CH2, S, O NH, and N-alkyl, and wherein the disease is selected from the group consisting of a disease associated with an abnormally low level of nitric oxide synthase activity in a tissue of the human and a disease associated with an abnormally high level of arginase activity in a tissue of the human.
In one aspect, the compound has a formula selected from the group consisting of
HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2 and
HOOC-CH(NH2)-CH2-S-CH2-CH2-B(OH)2. In another aspect, the disease is selected from the group consisting of heart disease, systemic hypertension, pulmonary hypertension, erectile dysfunction, autoimmune encephalomyelitis, chronic renal failure and cerebral vasospasm.
Further included in the invention is a method of relaxing smooth muscle in a mammal. The method comprises administering to the mammal a composition comprising a pharmaceutically acceptable carrier and a compound having the formula
HOOC-CH(NH2)-Yι -Y2-Y3-Y4-B(OH)2 wherein each of Yj, Y2, Y3, and Y4 is selected from the group consisting of CH2, S, O NH, and N-alkyl.
In one aspect, the compound has a formula selected from the group consisting of
HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2 and HOOC-CH(NH2)-CH2-S-CH2-CH2-B(OH)2. In another aspect, the smooth muscle is selected from the group consisting of gasterointestinal smooth muscle, anal sphincter smooth muscle, esophageal sphincter muscle, corpus cavemosum, sphincter of Oddi, arterial smooth muscle, heart smooth muscle, pulmonary smooth muscle, kidney smooth muscle, uterine smooth muscle, vaginal smooth muscle, cervical smooth muscle, placental smooth muscle, and ocular smooth muscle.
In addition, the invention includes a method of making a compound having the formula
HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2 , the method comprising contacting a molecule of the tert-butyl ester of 2(S)-N-(tert- butyloxycarbonyl)-6-[(l S,2S,3R,5S)-(+)-pinanedioxaboranyl]-hexanoic acid in an organic solvent with BCI3.
In a preferred embodiment, the organic solvent is CH2C12. Also included in the invention is a method of making a compound having the formula HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2 .
The method comprises the steps of
(a) mixing a solution of the tert-butyl ester of 2(S)-N-(tert- butyloxycarbonyl)-glutamic acid in tetrohydrofuran with triethylamine and ethyl chloroformate to produce a first mixture, removing the resulting triethylammonium hydrochloride salt by filtration, and treating the remaining mixture with an aqueous solution of sodium borohydride to provide a first compound, wherein the first compound is the tert-butyl ester of 2(S)-N-(tert-butyloxycarbonyl)-5-hydroxypentanoic acid;
(b) subjecting the first compound to Swern oxidation to produce a second compound;
(c) subjecting the second compound to a Wittig reaction in the presence of triphenylphosphonium methylide to produce a third compound; (d) mixing a solution of BH3 with the third compound in the presence of tetrahydrofuran to produce a second mixture;
(e) adding (lS,2S,3R,5S)-(+)-pinanediol to the second mixture to produce a fourth compound, wherein the fourth compound is the tert-butyl ester of 2(S)-N-(tert-butyloxycarbonyl)-6-[(lS,2S,3R,5S)-(+)-pinanedioxaboranyl]-hexanoic acid; and
(f) mixing the fourth compound with BCI3 in the presence of CH2C12 to produce a compound having the formula
HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2 . Also included in the invention is a method of identifying an arginase inhibitor antagonist, the method comprising the steps of
(a) inducing relaxation of a muscle in vitro;
(b) reversing the relaxation by contacting the muscle with arginase;
(c) adding an arginase inhibitor to the muscle so reversed to renew relaxation of the muscle in the presence or absence of a test compound; and (d) measuring the level of renewed relaxation of the muscle, wherein a lower level of renewed relaxation of the muscle in the presence of the test compound, compared with the level of renewed relaxation of the muscle in the absence of the test compound, is an indication that the test compound is an arginase inhibitor antagonist. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram which illustrates the topology of an arginase monomer. Relative locations of metal ligands are indicated by solid circles.
Figure 2 is an image depicting a ribbon plot of an arginase trimer. The Mn^ -Mn^ cluster in the active site of each monomer is represented by a pair of spheres.
Figure 3, comprising Panels A and B, is a pair of images which depict omit maps of the binuclear manganese cluster of arginase. The maps were calculated using Fourier coefficients |FJ - |FC| and phases derived from the final model, less the atoms of metal ligands or metal ions Mn^and MIL . In Panel A, the hydrogen bond between Oδ2 of Mn ligand Asp 128 and bridging solvent, which is represented by a sphere, is indicated by a dashed line. In Panel B, the manganese coordination interactions are depicted, with coordination bond lengths indicated in A.
Figure 4 is an image which depicts a model of arginine binding to the active site of arginase. A salt link between Glu-277 and the substrate guanidinium group may orient the substrate for nucleophilic attack by the metal-bridging solvent molecule. Salt links are indicated by dotted lines. His-141 may serve as a catalytic proton shuttle.
Figure 5 is an image which illustrates a proposed mechanism of arginase-catalyzed arginine hydrolysis by a metal-activated solvent molecule complexed with the Mn 94- -Mn 9+ cluster in the active site of arginase. The α-amino and α-carboxylate groups of the arginine molecule are omitted for clarity.
Figure 6 is an image which illustrates the synthetic scheme described herein for the production of ABHA. Figure 7 is an image which depicts an ORTEP representation of the molecular structure of ABHA, using 30% probability thermal ellipsoids. Figure 8, comprising Panels A and B, is a pair of images which illustrate the similarity between the interaction of arginase with the proposed tetrahedral intermediate of arginase-catalyzed arginine hydrolysis, depicted in Panel A, and the interaction of arginase with the proposed the proposed tetrahedral conformation of hydrated ABHA, depicted in Panel B.
Figure 9 is an image which depicts an omit electron density map of the arginine-ornithine-borate complex, contoured at 3σ. Refined atomic coordinates are superimposed. One oxygen atom of the tetrahedral borate anion bridges the binuclear manganese cluster. The precise conformation of omithine (~5A away from borate) is ambiguous, due to the low resolution of the electron density map.
Figure 10 is a diagram of Scheme 1 depicting the chemical structures of compounds 7 (ABHA), 12, 15 (BEC), and 16.
Figure 11(a) is a scheme illustrating the reciprocal coordination of NO synthase and arginase activities. Figure 11(b) is a scheme of the arginase mechanism proposed by
Kanyo et al. (1996, Nature 383:554-557).
Figure 12 is a graph depicting the Eadie-Hofstee plot for the new chromogenic substrate, l-nitro-3-guanidinobenzene (18), where vQ is observed velocity and (S) is substrate concentration. Figure 13, comprising panels (a), (b), (c) and (d) is a quartet of graphs depicting plots of v0/v as a function of (I) indicate reversible inhibition only if linear. Only the trihydroxysilane, S-(2-trihydroxysilylethyl)-L-cysteine, yields a linear plot (Panel a). The boronic acid-based arginine analogues 2(S)-amino-6-boronohexanoic acid (Panel b), S-(2-boronoethyl)-L-cysteine (Panel c), and 2(S)-amino-5-boronopentanoic acid (Panel d) did not yield linear plots, indicative of irreversible inhibition. However, dialysis experiments with 2(S)-amino-6-borono-hexanoic acid indicated that it was a reversible inhibitor.
- 10 -
HEET R L Figure 14 comprising Panels (a), (b) and (c) is a trio of graphs depicting plots of v/vQ versus (I)/(EQ) for all the boronic acid inhibitors. Plots of v/v0 versus (I)/(EQ) typically are linear for inhibitors that behave as inactivators. Extrapolation of linear v/vQ versus (I)/(EQ) plots to the (I)/(EQ) intercept gives the turnover number for the inactivator. However, the (I)/(EQ) intercept from v/vQ versus
(I)/(EQ) plots gave rise to a constant, designated the trivial name pseudo-turnover number. tnpSeud0- Smaller tn_seucj0 values indicated better inhibition. Within experimental error, boronic acid inhibitors 2(S)-amino-6-borono-hexanoic acid and S-(2-boronoethyl)-L-cysteine were essentially equipotent (Panel a) and (Panel b), respectively). The tn_seu(j0 for boronic acid 2(S)-amino-5-boronopentanoic acid
(Panel c) was two orders of magnitude higher than the values for the other
Figure imgf000013_0001
two boronates. The first two boronates were ~400-fold more potent than 2(S)-amino-5-boronopentanoic acid.
Figure 15 comprising Panels A and B, is two graphs depicting the titration of arginase by 7 in 100 μM MnCl2, 50 mM bicine (pH 8.5) at 25 °C. Panel A contains the raw data obtained by titration of 0.0358 mM arginase with 30 x 2.5 μL injections of 1.5 mM 7. In Panel B the area under each peak was integrated and plotted against [7]/[arginase]. The solid line represents the best fit of the experimental data using non-linear least squares fitting, indicating a stoichiometry (n) of 1.07 moles of bound 7 per mole of arginase monomer, an association constant (Ka) of 8.89 x 10"
M , and an enthalpy change (ΔH) of -12.97 kcal/mol.
Figure 16 comprising Panels A and B, is two graphs depicting the titration of arginase by 15 in 100 μM MnCl2, 50 mM bicine (pH 8.5) at 25 °C. Panel A depicts the raw data obtained by titration of 0.0358 mM arginase with 40 x 2.5 μL injections of 1.5 mM 15. In Panel B the area under each peak was integrated and plotted against [15]/[arginase]. The solid line represents the best fit of the experimental data using non-linear least squares fitting, indicating a stoichiometry (n) of 0.964 moles of bound 15 per mole of arginase, an association constant (Ka) of 4.50 x lO^ M"1 , and an enthalpy change (ΔH) of -12.75 kcal/mol.
Figure 17 depicts the proposed binding mode for arginase inhibitors 7, 12, and 15. The metal-bridging hydroxide ion of the native arginase likely attacks the trigonal planar boronic acid to form the tetrahedral boronate anion.
Figure 18 is a graph depicting the effect of exogenous arginase before and after application of the arginase inhibitor Nω-hydroxy-L-Arginine (L-HO-Arg), on the IAS relaxation by different frequencies of electrical field stimulation (EFS). Note the significant suppression of the IAS relaxation by arginase alone and its reversal by L-HO-Arg.
Figure 19 is a graph depicting the effect of exogenous arginase treatment, before and after application of the arginase inhibitor 2 (S)-Amino-6- boronohexanoic Acid (ABHA), on the IAS smooth muscle relaxation by different frequencies of EFS. Note the significant suppression of the IAS relaxation by treatment with arginase alone, and its reversal by ABHA.
Figure 20 is a graph depicting the percent maximal fall in IAS tension by EFS before and after treatment of the tissue with the NO synthase inhibitor L-NNA or L-NNA plus different concentrations of L-HO-Arg. L-NNA caused a marked attenuation of the EFS-induced IAS relaxation (p < 0.05; n = 5). The L-NNA- attenuated IAS relaxation was reversed by L-HO-Arg in a concentration-dependent manner. L-HO-Arg (3 x 10 M) caused complete reversal of the suppressed IAS relaxation.
Figure 21 is a graph depicting the percent maximal fall in IAS tension produced by EFS, before and after application of the NO synthase inhibitor L-NNA or L-NNA plus different concentrations of L-arginine. Note that L-arginine caused a significant and concentration-dependent reversal of the suppressed IAS relaxation and that this effect was somewhat similar to that of L-HO-Arg (p < 0.05; n = 7). Figure 22 is a graph depicting the percent maximal fall in IAS tension produced by EFS before and after application of the NO synthase inhibitor L-NNA or L-NNA plus different concentrations of 2 (S)-Amino-6-boronohexanoic Acid (ABHA). Note that unlike L-HO-Arg, ABHA had no effect on the L-NNA-suppressed IAS relaxation (p < 0.05; n = 4).
Figure 23 is a graph depicting the influence of L-HO-Arg on the NANC nerve-mediated IAS relaxation produced by EFS. Note that L-HO-Arg caused a significant augmentation of the EFS-induced relaxation of the IAS in a concentration- dependent manner and this was evident only in the lower frequencies of EFS (p < 0.05).
Figure 24 is a graph depicting the influence of 2 (S)-Amino-6- boronohexanoic Acid (ABHA) on the NANC nerve-mediated IAS relaxation. Note that ABHA caused a significant augmentation of the IAS smooth muscle relaxation caused by the lower frequencies of EFS, in a concentration-dependent manner (p < 0.05).
Figure 25 is a graph depicting the basal arginase activity of the tissue homogenates of the liver, internal anal sphincter (IAS), rectum and brain. The basal arginase activity for the nonhepatic tissues was found to be -10° of that in the liver. Interestingly, among the nonhepatic tissues, the basal arginase activity in the IAS smooth muscle was found to be nearly four fold higher than in the adjoining region of the rectum, and the brain.
Figure 26 is a graph depicting the effect of Nω-hydroxy-L-arginine on the arginase activity found in hepatic and nonhepatic tissues. Nω-hydroxy-L-arginine was found to be ~ 10 times more potent in inhibiting the liver arginase activity than the arginase activities from the nonhepatic tissues.
Figure 27 is a graph depicting a comparison of the effect of 2 (S)- Amino-6-boronohexanoic Acid (ABHA) on the hepatic versus the nonhepatic tissues of the I AS, rectum and brain. In contrast to the effects seen with Nω-hydroxy L-arginine, ABHA was found to be more potent in the nonhepatic than the hepatic arginase activities.
Figure 28, comprising parts A-D, depicts the influence of the NO synthase inhibitor L-NNA, Nω-hydroxy-L-arginine (L-HO-Arg) and the combination of L-NNA plus L-HO-Arg on the arginase activity of the liver (Figure 28 A), internal anal sphincter (IAS) (Figure 28B), rectum (Figure 28C) and brain (Figure 28D). The data illustrate that L-NNA had no significant effect on either the basal arginase activity or on the L-HO-Arg-attenuated arginase activity in any of the tissues examined. The data suggest that the differential inhibitory effects of L-HO-Arg in these tissues do not result from variable interactions of this NO synthase substrate with the endogenous NO synthase.
DETAILED DESCRIPTION The invention is based upon the discover ' of compounds which inhibit the activity of arginase. These compounds, which are heretofore not known to inhibit this enzyme, are therefore useful for a variety of applications in medicine and in research.
Compositions and methods for inhibiting the activity of arginase including, but not limited to, yeast and mammalian arginase, are provided. Inhibition of mammalian arginase activity using the boronic acid-based arginine analog 2(S)- amino-6-boronohexanoic acid (ABHA) is demonstrated herein, as is inhibition of arginase using the boronic acid based arginine analog S-(2-boronoethyl)-L-cysteine (BEC).
The compositions of the invention may be used to inhibit arginase activity in vitro or in vivo, for example, in a human. The compositions of the invention may also be used to treat a disease characterized either by abnormally high arginase activity in a tissue of a mammal or by abnormally low nitric oxide synthase activity in a tissue of the mammal, preferably a human. The composition of the invention comprises a compound having the formula
HOOC-CH(NH2)-Y ι -Y2-Y3-Y4-B(OH)2 wherein each of Yj, Y2, Y3, and Y4 is selected from the group consisting of CH2, S, O NH, and N-alkyl, except Y2 is not S when each of Y1 , Y3, and Y4 is CH2.
In one aspect, the composition has the formula HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2. This compound is 2(S)-amino-6-boronohexanoic acid (ABHA). ABHA is also referred to herein as compound 7. Data presented herein confirm that ABHA is a potent inhibitor of arginase activity. The conformation of the hydrated form of ABHA resembles a transition state intermediate postulated to be formed during the arginine hydrolysis reaction catalyzed by arginase. The tetrahedral structure around the boron atom of the hydrated compound of the invention, as depicted in Figure 8, Panel B, closely resembles the tetrahedral intermediate formed by hydroxyl ion nucleophilic attack at the guanidinium carbon of arginine.
In a preferred embodiment, the composition of the invention further comprises a pharmaceutically acceptable carrier.
Also included in the invention is a composition comprising a pharmaceutical acceptable carrier and a compound having the formula HOOC-CH(NH2)-Y , -Y2-Y3-Y4-B(OH)2 wherein each of Y^, Y2, Y3, and Y4 is selected from the group consisting of CH2, S, O NH, and N-alkyl.
Preferred compositions which also include a pharmaceutically acceptable carrier, include ABHA and a compound having the formula HOOC-CH(NH2)-CH2-S-CH2-CH2-B(OH)2 ,
This compound is S-(2-boronoethyl)-L-cysteine (BEC). BEC is also referred to herein as compound 15. Any physiologically acceptable anion may be used as a counterion when the composition of the invention is prepared in the form of a salt. Physiologically acceptable anions are well known in the art. For example, the composition of the invention may be a hydrochloride salt of ABHA or BEC. The invention provides a method of inhibiting arginase comprising providing a composition of the invention to an arginase enzyme, preferably a yeast arginase enzyme or a mammalian arginase enzyme. When the enzyme is a mammalian arginase, the arginase is preferably a human arginase enzyme.
By the term "inhibition of arginase" by an arginase inhibitor as used herein, is meant a reduction in the level of arginase activity in the presence of an inhibitor, compared with the level of arginase activity in the absence of the inhibitor. Arginase may be inhibited in yeast by contacting the yeast with the composition of the invention. Inhibition of arginase in yeast serves to minimize urea production during fermentation of alcoholic beverages. In addition, the compositions of the invention are useful as anti- fungicides in agriculturally or otherwise economically important plant life. The composition of the invention may be administered to the plant by spraying or other meand well known in the art of plant biology.
Arginase activity may be inhibited in a mammal, for example, by administering a pharmaceutical composition comprising the composition of the invention to the mammal.
Thus, the composition of the invention may be used to treat a disease in a mammal, wherein the disease is associated with expression of an abnormally high level of arginase activity in a tissue of the mammal. Because NO synthase activity is regulated in a reciprocal fashion with respect to arginase activity in mammals, more particularly humans, the composition of the invention may be used to treat a disease in a mammal, wherein the disease is associated with expression of an abnormally low level of NO synthase activity in a tissue of the mammal. Since the reciprocal interaction of arginase and NO synthase has implications for the function of smooth muscle as described in further detail herein in Example 4, the use of the compounds described herein for the regulation of smooth muscle activity in an animal is also contemplated in the invention. By the term "abnormally high level of arginase activity" as used herein, is meant a level of arginase activity which is exceeds the level found in normal tissue, which normal tissue does not exhibit an arginase related disease phenotype.
By the term "abnormally low level of NO synthase activity" as used herein, is meant a level of NO synthase activity which is lower than that found in normal tissue, which normal tissue does not exhibit an NO synthase related disease state.
As noted herein, an increase in arginase activity has been associated with the pathophysiology of a number of conditions including an impairment in nonadrenergic and noncholinergic (NANC) nerve-mediated relaxation of the gastrointestinal smooth muscle. The use of an arginase inhibitor is predicted to rectify this condition.
Thus, included in the invention is a method of enhancing smooth muscle relaxation comprising contacting the smooth muscle with an arginase inhibitor. The smooth muscle is preferably within the body of an animal and the arginase inhibitor is preferably ABHA or BEC. The type of smooth muscle to be relaxed includes, but is not limited to, gasterointestinal smooth muscle, anal sphincter smooth muscle, esophageal sphincter muscle, corpus cavemosum, sphincter of Oddi, arterial smooth muscle, heart smooth muscle, pulmonary smooth muscle, kidney smooth muscle, uterine smooth muscle, vaginal smooth muscle, cervical smooth muscle, placental smooth muscle, and ocular smooth muscle. When the smooth muscle is gastrointestinal smooth muscle, the type of gastrointestinal smooth muscle includes, but is not limited to, the internal anal sphincter muscle. When the smooth muscle in within the body of the animal, the invention includes a method of treating an arginase-related disease in an animal. In a preferred embodiment, the animal is a human.
Diseases which are associated with either an abnormally high level of arginase activity in a tissue of a mammal or an abnormally low level of nitric oxide synthase activity in a tissue of the mammal are known in the art. Diseases to be treated using the compositions of the invention include, but are not limited to, heart disease, systemic hypertension, pulmonary hypertension, erectile dysfunction, autoimmune encephalomyelitis, chronic renal failure and cerebral vasospasm. To treat an arginase-related disease in a mammal, the composition of the invention is administered to the mammal, and preferably comprises a pharmaceutically acceptable carrier as described in further detail herein.
The invention also contemplates the use of an arginase inhibitor in an in vitro arginase inhibition/smooth muscle functional assay, for the purpose of identifying compounds which affect smooth muscle function. Compounds so identified are considered to be candidate arginase inhibitor antagonists, in that, as described in further detail below, these compounds are identified by their ability to counteract the ABHA or BEC mediated inhibition of arginase activity. For example, there is described herein in Example 4 an assay for smooth muscle activity using the internal anal sphincter muscle and one on the preferred arginase inhibitors of the invention ABHA. In this assay, strips of the internal anal sphincter muscle obtained from adult opossums are induced to relax by nonadrenergic and noncholinergic (NANC) nerve-mediated relaxation using electrical field stimulation (EFS); relaxation is reversed by contacting the muscle strips with arginase; and, reversal of relaxation is accomplished by contacting the muscle with an arginase inhibitor. To identify an arginase inhibitor antagonist, the muscle strips are then subsequently contacted with a test compound and the effect of the test compound on subsequent reversal of muscle relaxation is then assessed. Any significant reversal of the relaxation state of the muscle in the presence of the test compared with the relaxation state of the muscle in the absence of the test compound is an indication that the test compound is a candidate arginase inhibitor antagonist.
By the term "arginase inhibitor antagonist" as used herein, is meant a compound which inhibits the inhibition of arginase by an arginase inhibitor.
The invention encompasses the preparation and use of pharmaceutical compositions comprising an arginase inhibitor as an active ingredient. Such a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. Administration of one of these pharmaceutical compositions to a subject is useful for inhibiting arginase activity and thereby treating a disease or disorder associated with arginase enzyme activity, as described elsewhere in the present disclosure. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition with which the active ingredient may be combined and which, following the combination, can be used to administer the active ingredient to a subject.
As used herein, the term "physiologically acceptable" ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered. The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs, birds including commercially relevant birds such as chickens, ducks, geese, and turkeys, fish including farm-raised fish and aquarium fish, and crustaceans such as farm-raised shellfish.
Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.
In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents. Particularly contemplated additional agents include anti-emetics and scavengers such as cyanide and cyanate scavengers.
Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology. A formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared, packaged, or sold in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion.
As used herein, an "oily" liquid is one which comprises a carbon- containing liquid molecule and which exhibits a less polar character than water.
A tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycollate. Known surface active agents include, but are not limited to, sodium lauryl sulphate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, com starch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.
Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in U.S. Patents numbers 4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically elegant and palatable preparation. Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.
Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.
Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.
Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol. vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations. A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in- water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for rectal administration. Such a composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation.
Suppository formulations may be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e. about 20 °C) and which is liquid at the rectal temperature of the subject (i.e. about 37°C in a healthy human). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides. Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives. Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject. Enema preparations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for vaginal administration. Such a composition may be in the form of, for example, a suppository, an impregnated or coated vaginally-insertable material such as a tampon, a douche preparation, or a solution for vaginal irrigation.
Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e. such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying. Douche preparations or solutions for vaginal irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, douche preparations may be administered using, and may be packaged within, a delivery device adapted to the vaginal anatomy of the subject. Douche preparations may further comprise various additional ingredients including, but not limited to, antioxidants, antibiotics, antifungal agents, and preservatives.
As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, so long as the arginase inhibitor compound is not administered systemically. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.
Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low- boiling propellant in a sealed container. Preferably, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form. Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient).
Pharmaceutical compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations may be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 nanometers. The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition of the invention.
Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered in the manner in which snuff is taken i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nares.
Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may further comprise one or more of the additional ingredients described herein.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein. A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. Such drops may further comprise buffering agents, salts, or one or more other of the additional ingredients described herein. Other ophthalmalmically- administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form or in a liposomal preparation.
As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed., 1985, Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton, PA, which is incorporated herein by reference.
Typically dosages of the arginase inhibitor which may be administered to an animal, preferably a human, range in amount from 1 μg to about 100 g per killogram of body weight of the animal. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration. Preferrably, the dosage of the compound will vary from about 1 mg to about 10 g per killogram of body weight of the animal. More preferably, the dosage will vary from about 10 mg to about 1 g per killogram of body weight of the animal.
The compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even lees frequently, such as once every several months or evenonce a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.
The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these examples but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Example 1. Structure of a Unique Binuclear Manganese Cluster in Arginase
The molecular structure of and surrounding the pair of manganese atoms of the arginase monomer was determined by X-ray diffraction and other methods. These data suggest the mechanism by which arginine hydrolysis is catalyzed by arginase and permit the design of arginase inhibitors which mimic the transition state intermediates of the reaction, as described in Example 2. The materials and methods used in the experiments presented in this Example are now described.
The crystallization and preliminary X-ray diffraction analysis of rat liver arginase has been reported (Kanyo et al., 1992, J. Mol. Biol. 224:1175-1177). Arginase crystals diffract to 2.1 A resolution and belong to space group R32, having hexagonal unit-cell dimensions of a = b = 88.5 A, and c = 106.2 A, and having one 105 kilodalton trimer in the asymmetric unit. Phase determination by multiple isomorphous replacement was hindered by chronic non-isomorphism between native and heavy-atom derivative crystals, as well as non-isomorphism among native crystals themselves. The c-axis length typically ranged from 104 A to 115 A (Kanyo et al., 1992, J. Mol. Biol. 224:1175-1177).
Diffraction data were collected at room temperature on an R-AXIS lie image plate area detector. Data reduction was performed by MOSFLM (Nyborg et al., 1977, In: The Rotation Method in Crystallography. Arndt et al., eds, North-Holland, Amsterdam, 139-152) and CCP4 (Collaborative Computational Project No. 4, 1994,
Acta Crystallogr. D50:760-763). For phasing, initial heavy atom positions were determined in difference Patterson maps and refined with the program, PHASES (Burey et al., 1990, Am. Crystallogr. Assoc. Mtg. Prog. Abstr. 18:73). Heavy-atom binding indicated that the non-crystallographic symmetry (NCS) axis of the trimer was tilted ~ 9° away from a normal to the a-b plane. The model was fit into an electron- density map calculated with solvent-flattened NCS-averaged phases at 3.0 A resolution. Subsequent refinement and rebuilding of the native model was done with X-PLOR (Brunger et al, 1987, Science 235:458-460) and O (Jones et al., 1991, Acta. Crystallogr. A47:l 10-119), respectively. Group B factors were refined, and a bulk solvent correction was applied. In the final stages of refinement, the quality of the model was improved by gradually releasing NCS constraints into appropriately weighted restraints as judged by Rfree. Refinement statistics are recorded in Table 1. the final protein model has excellent stereochemistry with only Gln-64 adopting a disallowed φ/φ conformation. This residue is located in a type II' β-turn between strand 2 and helix B and is characterized by clear and unambiguous electron density.
The results of the experiments presented in this Example are now described. Arginase is one of the very few enzymes that requires a spin-coupled Mn 9^+ -Mir 9-+ cluster for catalytic activity in vitro and in vivo (Reczkowski et al., 1992,
J. Am. Chem. Soc. 114:10992-10994). The 2.1 A-resolution crystal structure of trimeric rat liver arginase reveals that this unique metal cluster resides at the bottom of an active-site cleft that is about 15 A deep (Kanyo et al., 1992, J. Mol. Biol. 224:1175- 1177). Analysis of the crystal structure of arginase indicates that arginine hydrolysis involves a metal-activated solvent molecule which symmetrically bridges the two Mn 9^+ ions.
Table 1. Data collection and refinement statistics.
Figure imgf000036_0001
cPhasing power = (Fj.)/E, where (Fj.) is the root-mean-square heavy atom structure factor and E is the residual lack of closure ercor.
15 dp R _ =_ I |FQ I - |FC I |/∑|FQ |, where R and Rfree are calculated using the working and free reflection sets, respectively. The free reflections, representing 5% of the total, were held aside throughout refinement.
The overall folding pattern of the arginase monomer indicates that the protein belongs to the α/β protein class. The arginase monomer has a globular structure, having approximate dimensions of 40 * 50 x 50 A. One side of the active- site cleft is partially defined by the central eight-stranded β-sheet, and the metal binding site is located on one edge of the β-sheet. Metal ligands are located immediately adjacent to helix C, to strand 4, and to strand 7, as depicted in Figure 1. The arginase trimer, depicted in Figure 2, has a relative molecular mass of about 105,000 (Mr = 105 kilodaltons) and excludes about 2,080 A2 of monomer surface area from solvent at each monomer-monomer interface. A majority of intermonomer contact is mediated by a novel 'S'-shaped oligomerization motif at the carboxyl terminus, as depicted in Figures 1 and 2, and the conformation of this segment is stabilized by numerous intermonomer van der Waals interactions, hydrogen bonds, and salt links. The image in Figure 2 was generated with MOLSCRIPT (Kraulis et al., 1991, J. Appl. Crystallogr. 24:946-950) and Raster 3D (Bacon et al., 1988, J. Mol. Graph. 6:219-200; Merrit et al.. 1994. Acta Crystallogr. D50:869-873).
The binuclear manganese cluster is located at the base of an approximately 15 A-deep active site cleft in each monomer, as depicted in Figure 4. The metal ion that is more deeply situated in the active site cleft is designated Mn +and is coordinated by His-101 (Nδ), Asp-124 (Oδl), Asp-128 (Oδl), Asp-232 (Oδl) and a solvent molecule. The coordination of Mn^+ by arginase has square pyramidal geometry. A solvent molecule bridges Mn^and MIL" and also donates a hydrogen bond to Asp-128, the Oδ2-O separation distance being about 2.8 A. The second metal ion is designated MIL+ and is coordinated by His- 126 (Nδ), Asp- 124 (Oδ2), Asp-232 (Oδl), Asp-234 (bidentate Oδl and Oδ2). The coordination of Mn +by arginase and the bridging solvent molecule has a octahedral geometry which is distorted, owing to the bidentate coordination of Asp-234. The separation between Mn^and MIL+_S about 3.3 A. All metal ligands except Asp- 128 make hydrogen-bond interactions with other protein residues, and these interactions contribute to the stability of the metal binding site (Christianson et al, 1989, J. Am. Chem. Soc. 111 :6412-6419).
Three different types of metal bridging ligands facilitate the observed spin coupling between Mn +and Mn!+ (Reczkowski et al., 1992, J. Am. Chem. Soc. 114:10992-10994). The carboxylate side chain of Asp-124 is a syn-syn bidentate bridging ligand, with Oδl coordinated to Mn +and Oδ2 coordinated to Mn +(Rardin et al., 1991, New J. Chem. 15:417-430). The carboxylate side chain of Asp-232 is a monodentate bridging ligand, with Oδl coordinated to both Mn^+and MIL+ with anti- and syrc-coordination stereochemistry, respectively (Rardin et al., 1991, New J. Chem. 15:417-430). The Oδ2 oxygen of Asp-232 is about 3.8 A away from Mτζ. The solvent molecule bridges both manganese ions symmetrically, with the separation distance of the O atom of the solvent from both of Mn^and MIL+ being about 2.4 A.
The arginase structure is the first atomic resolution structure determined for a functional metalloenzyme that has a specific catalytic and physiological requirement for two Mn + ions. Arginase does not use transition metals promiscuously for catalysis, unlike other metalloenzymes, in which Mn + and, for instance, Mg 9+ or other metal ions are interchangeable, albeit with some effect on catalytic activity. The identity and oxidation state of the manganese cluster of arginase has been conclusively established by electron paramagnetic resonance spectroscopy (Reczkowski et al., 1992, J. Am. Chem. Soc. 114:10992-10994). The catalytic metal requirement of arginase is rooted in the prefened geometry of manganese coordination, which properly orients the metal-bridging solvent molecule, such that it arginase hydrolysis is catalyzed. Because the metal-bridging solvent molecule must satisfy the coordination preferences of two manganese ions simultaneously, the position of the solvent molecule, and therefore its optimal catalytic activity, is highly sensitive to substitution of one or both Mn 9^+ ions with a different metal ion. Coordination of the solvent molecule to two metal ions, rather than one, enhances the dependence of optimal catalytic activity on proper metal composition of arginase. Only two other polar residues are found in the immediate vicinity of the active site of arginase, namely Glu-277 and His-141. Glu-277 is located deep in the active-site cleft, about 4.5 A away from Mn2+. A roughly 20° rotation about χ1 of this side chain yields an ideal salt link with the substrate guanidinium group. Moreover, this salt link positions the electrophilic guanidinium carbon of the substrate directly over the metal-bridging solvent molecule, which is likely to be a nucleophilic hydroxide ion in the active catalyst, as depicted in Figures 4 and 5. It is unlikely that the deprotonated substrate guanidinium group binds directly to the metal(s) because of its high pKa value, namely 13.5. Furthermore, site-directed mutagenesis studies indicate that substrate Km values are not perturbed by variations in the metal cluster, including metal depletion. This indicates that a substrate-metal interaction does not occur in the Michaelis complex (Cavalli et al., 1994, Biochemistry 33:10652-10657).
The side chain of His-141 is located about midway between the top and the bottom of the active-site cleft. When asparagine substituted in place of histidine at amino acid position 141 of arginase (His-141 →Asn arginase), the enzyme retains roughly ten percent of its wild type activity (Cavalli et al., 1994, Biochemistry 33:10652-10657). Because its location is only about 4.2 A from the metal-bridging solvent molecule, it is possible that His-141 acts a proton shuttle in arginase catalysis, mediating proton transfer to and from bulk solvent. Direct proton transfer with bulk solvent may be possible in the absence of His-141 , which could account for the significant residual catalytic activity of His-141-→-Asn arginase. A proton shuttle function for His-141 of arginase is analogous to that recently reviewed for His-64 of the zinc metalloenzyme carbonic anhydrase II (Christianson et al., 1996, Ace. Chem. Res. 29:331-339). As depicted in Figure 3, charged amino acid residues are located on opposite sides of the active-site cleft, near the surface of the arginase trimer. The presence of these charged residues may contribute to the exquisite specificity of substrate recognition by arginase. Structure-activity relationships using arginine analogues indicate that electrostatic interactions of arginase with the α-substituents of the substrate are critical for catalysis. Deletion of the α-carboxylate group or the α- amino group of arginine results in 10 9 -105 -fold reductions in k^/K^ (Reczkowski et al., 1994, Arch. Biochem. Biophys. 312:31-37). Inspection of the arginase active site indicates that the positively charged side chain of Arg-21 interacts with the negatively charged α-carboxylate group of the substrate, and that the negatively charged side chain of Asp-181 interacts with the positively charged α-amino group of the substrate. A model of arginine binding to the active site of arginase is illustrated in Figure 4.
A model of arginine hydrolysis by arginase is illustrated in Figure 5. The ionization of metal-bound water probably reflects the apparent p__" a value of 7.9 observed in the pH rate profile of the enzyme (Kuhn et al., 1991, Arch. Biochem. Biophys. 286:217-221). In the first step of the hydrolytic mechanism, Asp-128 stabilizes the metal-bridging hydroxide ion with a hydrogen bond, while the hydroxide ion performs a nucleophilic attack at the guanidinium carbon of arginine. The resulting tetrahedral intermediate collapses upon proton transfer to the amino group of omithine. This proton transfer is probably mediated by Asp-128. Subsequently, His-141 shuttles a proton from bulk solvent to the ε-amino group of omithine prior to dissociation of omithine from arginase. Upon omithine dissociation, a water molecule displaces urea, resulting in dissociation of urea from arginase. Metal coordination facilitates the ionization of this water molecule to regenerate a nucleophilic hydroxide ion. Proton transfer from the water molecule to bulk solvent may be mediated by His- 141.
It is instructive to consider the chemical function of arginase not only within the context of mammalian nitrogen metabolism, but also within the greater context of the biosphere. In the nitrogen cycle, two binuclear metalloenzymes of different tertiary structure are prominent, namely Mn -arginase and Ni2+-urease. The former enzyme provides for the abundant release of urea into the environment by mammals, and the latter enzyme allows the use of this urea as a nitrogen source by bacteria, fungi, and plants. The recent determination of the crystal structure of Ni2+-urease obtained from the bacterium Klebsiella aerogenes (Jabri et al, 1995, Science 268:998- 1004) reveals some interesting parallels between these two enzymes which have evolved to catalyze urea chemistry. Both arginase and urease comprise a binuclear transition metal cluster, and both enzymes contain roughly similar constellations of catalytically important carboxylate and imidazole groups within their respective active sites. Interestingly, Mn2+-substituted urease exhibits catalytic activity, albeit only 2% of that of the native Ni +-urease (Park et al., 1996, Biochemistry 35:5345-5352). Preliminary results with Ni2+-substituted arginase reveal no measurable catalytic activity. Optimal catalytic activity in each system seems to have evolved with high selectivity for one particular binuclear metal cluster of specific composition and structure.
Example 2. 2fS VAmino-6-Boronohexanoic Acid (ABHA is an Effective Inhibitor of Arginase Using structural data described in Example 1 , an inhibitor of arginase activity was designed which resembles the proposed transition state intermediate of the arginine hydrolysis reaction catalyzed by arginase. The inhibitor was tested and it was determined to be a potent inhibitor of arginase activity.
The tetrahedral borate anion is a modest inhibitor of Mn2+ -arginase, a critical metalloenzyme of mammalian nitrogen metabolism. The crystal structure of the arginase-ornithine-borate complex, as described herein, reveals a net displacement of the solvent molecule bridging the binuclear manganese cluster by a borate oxygen atom in the native enzyme active site. This binding mode is reminiscent of the tetrahedral intermediate proposed for arginase-catalyzed arginine hydrolysis, as described in Example 1 herein. ABHA, a boronic acid-based arginine isostere appears to bind to arginase as the tetrahedral boronate anion and mimic the conformation of the tetrahedral arginine-hydrolysis intermediate. ABHA was synthesized and the ability of ABHA to inhibit arginase- catalyzed arginine hydrolysis was evaluated. ABHA is one of the most potent reversible inhibitors of arginase described to date, having an IC^Q value of 0.8 micromolar. Complete kinetic characterization of ABHA was complicated by nonlinearity of unknown origin, there being no evidence for slow-binding behavior. Competition binding experiments using N-hydroxyarginine indicate that K^ for ABHA was less than or equal to 0.1 micromolar.
Based on analysis of the crystal structure of the arginase-ornithine- borate complex, a binding model for ABHA was postulated, in which the metal- bridging solvent molecule observed in the native enzyme is displaced by an oxygen atom of the tetrahedral boronic acid anion. Presumably, the boronic acid moiety of ABHA effects inhibition of the enzyme by displacing the metal -bridging solvent molecule in a similar manner.
The materials and methods used in the experiments presented in this Example are now described.
Thin layer chromatography (TLC) was performed on Merck (Merck & Co., West Point, PA) silica gel 60 F2^4 glass plates. Compounds with tert- butyloxycarbonylamino groups or free amino groups were visualized on TLC plates by applying a ninhydrin solution (comprising 0.1% (w/v) ninhydrin in 95% (v/v) n- butanol, 4.5% (v/v) water, 0.5% (v/v) glacial acetic acid) to the plates and then heating the plates until color evolved.
Column chromatography was performed using a column packed with Merck (Merck & Co., West Point, PA) silica gel 60 (230-240 mesh ASTM) under positive nitrogen pressure. Melting points were determined visually in an open capillary on a
Thomas Hoover capillary melting point apparatus.
*H-NMR and B-NMR spectra were measured using a Bruker AC- 250 (250 MHZ) NMR spectrometer and a Bruker AC-200 (200 MHZ) NMR spectrometer, respectively. Chemical shifts were expressed in parts per million (ppm) and referenced either CDCI3 or CD3OD.
Arginase activity was assessed using the 14C-guanidino)-arginine assay of Ruegg et al. (1980, Anal. Biochem. 102:206-212), as described (Reczkowski et al., 1994, Arch. Biochem. Biophys. 312:31-37). In initial experiments, IC^Q values for ABHA and N-hydroxyarginine (Kj = 42 micromolar; Daghigh et al., 1994, Biochem. Biophys. Res. Commun. 202:174-180) were compared. Assays were performed in 100 millimolar CHES, pH 9.0, containing 1 millimolar arginine. 2(S)-N-(tert-butyloxycarbonyl)-glutamic acid tert-butyl ester (compound 1 in Figure 6) was obtained from Sigma Chemical Company (St. Louis, MO). Triethylamine and DMSO (dimethyl sulfoxide) were obtained from Fisher Scientific (Malvem, PA). All other reagents were obtained from Aldrich Chemical Co. (St. Louis, MO).
The chemical synthetic scheme used to make ABHA is illustrated in Figure 6.
Synthesis of 2f S VN-ftert-butyloxycarbonylVS-hydroxypentanoic acid. tert-butyl ester (Compound 2 in Figure 6 ,
To a -5°C solution of 2.339 grams (7.71 millimoles) 2(S)-N-(tert- butyloxycarbonyl)-glutamic acid tert-butyl ester in 70 milliliters of tetrahydrofuran (THF) was added 7.80 milliliters (77.1 millimoles) triethylamine and 7.37 milliliters (77.1 millimoles) ethyl chloroformate. The mixture was stirred at -5°C for ten minutes. The precipitant triethylamine salt was quickly removed by filtration. The filtrate was added to a suspension of sodium borohydride in 10 milliliters of water and stirred at room temperature overnight. The resulting crude product was purified by column chromatography on silica gel, using 2:1 hexane:ethyl acetate as an eluent, to yield 2(S)-N-(tert-butyloxycarbonyl)-5-hydroxypentanoic acid, tert-butyl ester ("Compound 2").
Synthesis of Compound 3 in Figure 6 Compound 3 was made by Swem oxidation of Compound 2, as described (Mancuso et al., 1978, J. Org. Chem. 43:2480-2482), and was used directly without purification.
Synthesis of Compound 4 in Figure 6 Compound 4 was made by subjecting crude Compound 3 to a Wittig reaction as described (Bowden, 1975, Synthesis 784) using triphenylphosphonium methylide. Compound 3, 1.151 grams (4.01 millimoles), was dissolved in THF and was added directly to the triphenylphosphonium methylide solution. The Wittig reaction was stirred and allowed to proceed for fifteen minutes at -78 °C and then ovemight at room temperature. Compound 4 was purified by separating the Wittig reaction products using a silica gel column using 10:1 hexane:ethyl acetate as an eluent, and then repeating the separation to improve purity. Following concentration in vacuo, 450 mg (22% over two steps) of product as a colorless oil was obtained. TLC using 3:1 hexane: ethyl acetate as the mobile phase indicated a single spot at R =0.60. Compound 4 had the following properties: H-NMR (CDC13) δ 5.80 (m, 1H), 5.00 (m, 3H), 4.15 (m, 1H), 2.05 (m, 2H), 1.85 (m, 1H), 1.65 (m, 1H), 1.45 (s, 9H), 1.40 (s, 9H).
Synthesis of 2(SVN -f ter/-butyloxycarbonyn-6-fπS.2S.3R.5SVf+V pinanedioxaboranyl]-hexanoic acid, tert-butyl ester ("Compound 5 in Figure 6)
To a stirred solution of 6.05 milliliters (6.05 millimoles) of 1 molar BH3»THF in 60 milliliters THF at 0°C was added 345 milligrams (1.21 millimoles) of Compound 4. The reaction mixture was stirred for two hours at room temperature and monitored for the disappearance of Compound 4 by performing TLC, using 3 : 1 hexane:ethyl acetate as the mobile phase. Excess unreacted borane was quenched by the slow addition of methanol until gas evolution, as indicated by bubbling, ceased. The solvent was evaporated in vacuo. The crude material was taken up in CH2C1 . Excess (lS,2S,3R,5S)-(+)-pinanediol (1 gram, 5.87 millimoles) was added to the solution of crude material in CH2C12. Esterification with (+)-pinanediol was allowed to proceed at room temperature for two hours. Compound 5 was purified by column chromatography using silica gel, using 25:1 hexane:ethyl acetate as an eluent. Compound 5 was obtained as an oil in 31% yield (174 milligrams) following concentration in vacuo. TLC, using 10:1 hexane:ethyl acetate as the mobile phase, indicated a single spot at
Figure imgf000045_0001
0.15. Compound 5 had the following properties H- NMR (CDC13) δ 4.95 (d, IH), 4.20 (d, IH), 4.1 (m, IH), 2.40-2.15 (m, 2H), 2.05 (m, IH), 1.95-1.65 (m, 4H), 1.60 (s, 3H), 1.42 (2s, 18H), 1.35 (apparent s, 4H), 1.25 (s, 3H), 1.05 (d, IH), 0.80 (m, 5H). The molecular mass calculated for C25H44NO6B + H is 466.33. The molecular mass of Compound 5 observed using ZAB-E CI+/MS mass spectrometry was 466.25. Synthesis of 2CS .-amino-6-boronohexanoic acid, hydrochloride salt
(ABHA: Compound 6 in Figure 6)
To a stirred solution of 174 milligrams of Compound 5 in CH2C12 at -78 °C was slowly added 1.49 milliliters of 1 molar BCI3 (1.49 millimoles) in CH2C12. The reaction mixture was stined for fifteen minutes at -78 °C and then for thirty minutes at 0°C. The solvent CH2C12 and excess BCI3 were evaporated by passing a flow of nitrogen gas over the mixture. Continued drying with N2 gas afforded crude product as an orange solid. Addition of acetonitrile removed most of the color from the crude product. Vacuum filtration followed by several washings with acetonitrile yielded a pale pink-colored solid which, upon air-drying, had a faint discoloration. Acetone was added to the pale pink-colored solid. Vacuum filtration followed by several washings with acetone removed all discoloration from the solid to yield 33 milligrams (42% yield) of pure ABHA as a white solid. TLC, using 80:10:10 n- butanol: acetic acid: water as the mobile phase, indicated a single spot at Rf = 0.15. ABHA had a melting point between 148 and 150°C, dec. >130°, and the following properties: !H-NMR (CD3OD) δ 3.95 (t, IH), 1.90 (m, 2H), 1.45 (m, 4H), 0.82 (m, 2H); πB NMR (CD3OD) δ 18.86 (s).
Crystallization and X-ray crystal structure determination of ABHA In a 3.7 milliliter screw-cap glass vial, 11 milligrams of ABHA were dissolved in 0.200 milliliters of ethanol. The screw-cap on the glass vial was not tightened, in order to allow for the slow evaporation of ethanol. The vial was placed in a fume hood for two days. Upon inspection, all solvent had evaporated; and ABHA had crystallized as needles and plates. A section of one of the plates proved suitable for crystallographic studies. Larger crystals were grown by the slow evaporation of 11 milligrams ABHA dissolved in 0.200 milliliters of water over a period of five days.
ABHA, which has the molecular formula
Figure imgf000046_0001
crystallizes in the orthorhombic space group P2^2 ^2 -^ (systematic absences hOO: l=odd; OkO: k=odd; 001: l=odd), with a=9.914(2)A, b=20.213(2)A, c=5.1801(6)A, V=1038.0(2)A3, Z=4 and dcajc=1.353 grams per cubic centimeter. X-ray intensity data were collected using a Rigaku R-AXIS lie area detector employing graphite-monochromated Mo-Kα radiation (λ=0.71069 A) at a temperature of 295 °K. Indexing was performed from a series of 1 ° oscillation images, with exposures of thirty minutes per frame. A hemisphere of data was collected using 8 ° oscillation angles, with exposures of sixty minutes per frame and a crystal-to-detector distance of 82 millimeters. Oscillation images were processed, using the bioteX program, to produce a listing of unaveraged
F 9^ and (Y 9Δ) values which were provided to a Silicon Graphics Indigo R4000 computer for further processing and structure solution, using the teXsan program. A total of 4539 reflections were measured over the ranges: 5.76 < 2θ < 50.70°, -10 < h <
11 , -21 < k < 24, -6 < 1 < 6, yielding 1838 unique reflections (Rint = 0.0590). The intensity data were conected for Lorentz and polarization effects, but not for absorption.
The structure was solved by direct methods, using the SIR92 program. Refinement was by full matrix least squares, based on F2 using the SHELXL-93 program. All reflections were used during refinement. Values of F2 that were experimentally negative were replaced by F2 = 0. The weighting scheme used was w=l/[σ2(F0 2) + 0.0537P2 + 2.4010P], where P = (FQ 2 + 2FC 2) 3. Non-hydrogen atoms were refined anisotropically, and hydrogen atoms were refined according to a "riding" model in which the positions of the hydrogen atoms are reidealized before each least squares cycle by applying the coordinate shifts of the atom to which each hydrogen is attached. Refinement converged to Rj=0.0768 and wR2=0.1627 for 1580 reflections for which F > 4σ(F) and R^O.0905, wR2=0.1756 and GOF = 1.106 for all 1838 unique, non-zero reflections and 122 variables. The maximum Δ/σ in the final cycle of least squares was 0.002 and the two most prominent peaks in the final difference Fourier were +0.351 and -0.514 e/A .
Crystal Structure of the Arginine-Ornithine-B orate Complex Crystals of rat liver arginase were prepared as described (Kanyo et al.,
1992, J. Mol., Biol. 224:1175-1177) and transfened to a buffer solution comprising 10 millimolar omithine and 10 millimolar sodium borate. X-ray diffraction data to 3.0 A resolution were collected and processed as described herein in Example 1. 28,047 total reflections, and 13,114 unique reflections (9-3 A) were used in refinement, which was 74% complete, with R^^g = 0.062.
The atomic coordinates of native rat liver arginase, as described herein in Example 1, served as the starting model for refinement of the structure of the arginase-omithine-borate complex using the X-PLOR program (Brunger et al., 1987, Science 235:458-460). Refinement of the structure of the complex converged smoothly to a final crystallographic R factor of 0.190 for 9 - 3 A data (Rfree =0.301 ), with root-mean-square deviations from ideal bond lengths and angles of 0.013 A and 1.6°, respectively.
Other methods which were used but not described herein are well known and within the competence of one of ordinary skill in the art of chemical synthesis and molecular biology.
The results obtained in the experiments presented in this Example are now described. Table 2 lists cell information, data collection parameters, and refinement data used to determine the structure of ABHA. Final positional and equivalent isotropic thermal parameters are given in Table 3. Anisotropic thermal parameters are in Table 4. Tables 5 and 6 list bond distances and bond angles. A representation of the molecular structure of ABHA, made using the ORTEP program, is depicted in Figure 7.
Table 2. Summary of Structure Determination of ABHA
Formula: C6H15BNO4Cl
Formula weight: 211.45
Crystal class: Orthorhombic
Space group: P212121 (#19)
4
Cell constants: a, A 9.914(2) b, A 20.213(2) c, A 5.1801(6) v, A3 1038.0(2) μ, centimeters 3.52 crystal size, millimeters 0.15 x 0.05 0.005
Dcaιc, grams per cubic centimeter 1.353
F(000) 448
Radiation: Mo-Ko (λ=0.71069A)
2Θ range 5.76-50.70° hkl collected: -lO ≤ h ≤ 11; -21 ≤ k ≤ 24; -6 ≤ 1 ≤ 6
No. reflections measured: 4539
No. unique reflections: 1838 (11^=0.0590)
No. observed reflections 1580 (F>4σ)
No. reflections used in refinement 1838
No. parameters 122
R indices (F>4σ) R^O.0768 wR2=0.1627
R indices (all data) R^O.0905 wR2=0.1756
GOF 1.106
Final Difference Peaks, e/A3 +0.351, -0.514
Table 3. Refined Positional Parameters for ABHA
Atom X y z Ueq, A2
Cl 0.5824(2) 0.58210(8) 0.7367(3) 0.0481(4)
01 0.5795(5) 0.8844(2) 0.4197(8) 0.0503(11)
HI 0.614(3) 0.898(3) 0.286(8) 0.075
02 0.4088(5) 0.9503(2) 0.2973(7) 0.0462(11)
03 0.3464(5) 0.5523(2) 1.3516(8) 0.0451(11)
H3 0.394(6) 0.5711(6) 1.457(8) 0.068
04 0.2110(5) 0.5741(2) 0.9914(8) 0.0428(10)
H4 0.206(6) 0.5337(4) 1.002(9) 0.064
Nl 0.2768(5) 0.9342(2) 0.7515(9) 0.0377(11)
Hla 0.238(3) 0.923(2) 0.900(5) 0.056
Hlb 0.218(2) 0.929(2) 0.623(5) 0.056
Hlc 0.3019(8) 0.9765(3) 0.759(9) 0.056
Cl 0.4592(6) 0.9135(3) 0.4522(10) 0.0351(13)
C2 0.3974(6) 0.8919(2) 0.7051(10) 0.0336(13)
H2 0.4628(6) 0.8988(2) 0.8444(10) 0.045
C3 0.3579(7) 0.8183(2) 0.6964(10) 0.043(7)
H3a 0.2711(7) 0.8142(2) 0.6113(10) 0.057
H3b 0.4237(7) 0.7946(2) 0.5930(10) 0.057
C4 0.3491(8) 0.7856(3) 0.9608(11) 0.043(2)
H4a 0.2794(8) 0.8072(3) 1.0617(11) 0.058
H4b 0.4342(8) 0.7912(3) 1.0506(11) 0.058 Table 4. Refined Thermal Parameters Cϋ's) for ABHA
Atom Ul l u22 U33 u23 U13 u12
Cl 0.0505(9) 0.0562(8) 0.0377(7) -0.0026(8) -0.0056(8) 0.0058(7)
01 0.044(3) 0.062(3) 0.044(3) 0.013(2) 0.013(2) 0.015(2)
02 0.053(3) 0.048(2) 0.037(2) 0.010(2) 0.007(2) 0.002(2)
03 0.053(3) 0.035(2) 0.047(3) 0.008(2) -0.013(2) -0.003(2)
04 0.055(3) 0.033(2) 0.041(2) -0.001(2) -0.012(2) -0.003(2)
Nl 0.044(3) 0.037(2) 0.032(2) 0.003(2) 0.006(2) -0.003(2)
Cl 0.046(3) 0.033(3) 0.027(3) 0.000(3) 0.003(2) -0.005(3)
C2 0.044(3) 0.032(2) 0.025(3) 0.000(2) 0.004(3) 0.004(2)
C3 0.070(4) 0.028(3) 0.030(3) 0.004(2) 0.004(3) -0.003(3)
C4 0.072(4) 0.027(3) 0.032(3) 0.004(2) 0.001(3) -0.002(3)
C5 0.074(5) 0.034(3) 0.038(3) 0.006(3) -0.007(3) -0.004(3)
C6 0.057(4) 0.033(3) 0.040(4) 0.003(3) -0.004(3) -0.007(3)
Bl 0.047(4) 0.029(3) 0.033(3) 0.002(3) -0.003(3) -0.001(3)
The form of the anisotropic displacement parameter is: exp[-2π2(a*2Uπh2 + b*2U22k2 + c*2U33l2 + 2b*c*U23kl + 2a*c*U13hl +
2a*b*U12hk)].
Table 5. Bond Distances in ABHA
10
Figure imgf000052_0001
Table 6. Bond Angles in ABHA
15 Bond Angle, Bond Angle,
Pair degrees Pair degrees
O2-C1-O1 123.9(5) C4-C3-C2 114.1(5)
O2-C1-C2 126.1(5) C3-C4-C5 111.7(5)
O1-C1-C2 110.0(5) C6-C5-C4 114.8(5)
20 N1-C2-C1 107.4(4) C5-C6-B1 116.5(5)
N1-C2-C3 110.8(5) O3-B1-O4 116.9(5)
C1-C2-C3 111.0(4) O3-B1-C6 123.7(5)
O4-B1-C6 119.3(5)
Arginine hydrolysis involves a metal-activated solvent molecule that symmetrically bridges the Mn -Mn2+ ion pair in the native enzyme. The reaction coordinate of hydrolysis is postulated to proceed through a tetrahedral intermediate resulting from nucleophilic attack of the metal-bridging hydroxide ion at the guanidinium carbon of arginine, as depicted in Figure 8, Panel A (Kanyo et al, 1996, Nature 383:554-557). Omithine and borate are known to be relatively weak inhibitors of arginase activity. The simultaneous presence of both omithine and borate inhibits arginase activity more that the presence of either compound alone. In order to understand the mode of inhibition, the X-ray crystal structure of the ternary arginase- ornithine-borate complex was determined. The tetrahedral borate anion mimics binding interactions postulated for the tetrahedral transition states in the physiological arginine-hydrolysis reaction, as depicted in Figure 8, Panel A (Kanyo et al., 1996, Nature 383:554-557). The high affinity of ABHA for arginase apparently results from the structural similarity between the hydrated form of ABHA, as depicted in Figure 8, Panel B, and the proposed tetrahedral intermediate and flanking transition states for arginase-catalyzed arginine hydrolysis, as depicted in Figure 8. Panel A.
Crystal Structure of the Arginase-Ornithine-B orate Complex The crystal structure of the arginase-ornithine-borate complex revealed the net displacement of the manganese-bridging solvent molecule of the native enzyme by an oxygen of the tetrahedral borate anion, as depicted in Figure 9. No other structural changes are observed in the manganese coordination polyhedra, and the average metal-metal separation distance is 3.5A, as predicted from EPR studies (Khangulov et al., 1995, Biochemistry 34:2015-2025). The average metal-metal separation is 3.3 A in the native enzyme (Kanyo et al., 1996, Nature 383:554-557).
Although the low resolution of this structure determination precludes a definitive conclusion on the binding conformation of omithine, an interaction between the α-carboxylate group of omithine and the side chain of Arg-21 is evident in two of the three arginase subunits. The interaction between Arg-21 and the carboxylate group is consistent with the structure-based proposal for arginase-substrate recognition, as described herein in Example 1. Using the crystal structure of the complex determined by this experiment, a boronic acid-based arginase analog, ABHA, was designed.
Evaluation of the Inhibitory Potential of 2(SVAmino-6- Boronohexanoic Acid f ABHA^ Boronic acids are effective aminopeptidase and serine protease inhibitors because they presumably bind as tetrahedral transition state analogues (Baker et al., 1980, Fed. Proc, Fed. Am. Soc. Exp. Biol. 39:1686; Matteson et al., 1981, J. Am. Chem. Soc. 103:5241-5242; Baker et al., 1983, Biochemistry 22:2098-2103; Kettner et al., 1984, J. Biol. Chem. 259:15106-15114; Shenvi, 1986, Biochemistry 25:1286-1291). The electron-deficient boron atom of a boronic acid promotes the addition to the boron atom of a suitable nucleophile, such as a protein-bound nucleophile or a solvent molecule, yielding a stable anionic tetrahedral species. Based on the structure of the ternary arginase-ornithine-borate complex described herein, it was postulated that the boronic acid analogue of arginine, 2(S)-amino-6- boronohexanoic acid (ABHA), would bind avidly to arginase as the hydrated anion to mimic the tetrahedral intermediate and its flanking transition states (see Figure 8). ABHA is the first example of a boronic acid-based arginine isostere.
Determination of the crystal structure of ABHA confirmed the presence of the expected trigonal planar geometry of the boronic acid moiety of ABHA.
The tetrahedral borate anion is a modest, noncompetitive inhibitor of arginase, having a Kj value of 1.0 millimolar and a ^ value of 0.26 millimolar. Inhibition of arginase activity by borate is even more pronounced in the presence of omithine, which is a competitive inhibitor of arginase, having a Kj value of 1.0 millimolar (Pace et al., 1981 Biochem. Biophys. Acta 658:410-412; Reczkowski et al., 1994, Arch. Biochem. Biophys. 312:31-37; Khangulov et al, 1995, Biochemistry 34:2015-2025). Measurement of the inhibitory potential of arginase inhibitors yielded an IC5Q value of 80 micromolar for N-hydroxyarginine and an IC^ value of 0.8 micromolar for ABHA.
A more complete kinetic analysis of inhibition of arginase by ABHA was complicated by nonlinearity of kinetic replots. The origin of this nonlinearity is not clear, since ABHA is a reversible inhibitor that shows no evidence of slow-binding behavior. Additional evidence for high affinity of arginase for ABHA was derived from competition binding experiments using ABHA and N-hydroxyarginine, as monitored by fluorescence spectroscopy. Addition of N-hydroxyarginine to a solution of arginase results in a significant decrease in intrinsic protein fluorescence at 327 nanometers. Addition of a saturating concentration of ABHA to the arginase-N- hydroxyarginine complex restores the fluorescence of the enzyme to that observed for the enzyme alone. Addition of ABHA alone to arginase does not result in significant changes in protein fluorescence. These experiments indicate that K^ < 0.1 micromolar for ABHA at pH 7.5 and at pH 9.0. ABHA is one of the most potent inhibitors of Mn2+ 2-arginase reported to date. Previously reported inhibitors include various free amino acids (millimolar Kj values), N-hydroxyarginine (Kj = 42 micromolar), N- hydroxyindospicine (Kj = 20 micromolar), N-hydroxylysine (Kj = 4 micromolar), and N-hydroxy-nor-arginine (Kj = 0.5 micromolar; Daghigh et al, 1994, Biochem. Biophys. Res. Commun. 202:174-180; Hunter et al, 1946, J. Biol. Chem. 157:427-446; Boucher et al., 1994, Biochem. Biophys. Res. Commun. 203:1614-1621; Custot et al., 1996, J. Biol. Inorg. Chem. 1 :73-82; Custot et al., 1997, J. Am. Chem. Soc. 119:4086- 4087). It is known that the closer the structural analogy between an inhibitor and the catalytic transition state, the tighter the inhibitor is expected to bind (Pauling, 1946, Chem. Eng. News 24:1375-1377; Wolfenden, 1969, Nature 223:704-705; Wolfenden, 1976, Annu. Rev. Biophys. 5:271-306). It appears that the high affinity of arginase for ABHA arises from the fact that the hydrated form of ABHA is the closest structural analogue of the tetrahedral intermediate of the arginase-catalyzed arginine hydrolysis reaction, and its flanking transition states, generated to date. Example 3: Additional Boronic Acid-Based Arginine Analogues Inhibit Arginase with High Affinities and Unusual Binding Kinetics Boronic acid-based and trihydroxysilyl-based transition state analogue inhibitors for arginase have been designed, synthesized, and evaluated. Initial characterization of the inhibitory potency of these compounds was achieved with a new chromogenic substrate, l-nitro-3-guanidinobenzene (18). Surprisingly, only the trihydroxysilyl-based inhibitor, S-(2-trihydroxysilylethyl) -cysteine (16), yielded linear kinetics with a modest Kj of 420 μM (assuming competitive inhibition). Due to nonlinearity of kinetic replots, the two most potent boronic acid-based inhibitors, 2(S)-amino-6-boronohexanoic acid (7) and S-(2-boronoethyl)-L-cysteine (15), were further characterized by assay with [ C-guanidino]-L-arginine and by the technique of titration calorimetry. The K^ values obtained by titration calorimetry for 7 and 15 were 0.11 μM and 2.22 μM, respectively.
The enzymes of arginine catabolism have been the subject of increasingly intense research interest. Of particular interest are the critical macrophage enzymes, arginase and nitric oxide (NO) synthase. Arginase catalyzes the hydrolysis of L-arginine to form L-ornithine and urea (Christianson, 1997, Prog. Biophys. Molec. Biol. 67:217-252), and NO synthase catalyzes the oxidation of L-arginine to form citrulline and NO (Griffith et al., 1995, Annu. Rev. Physiol. 57:707-736). Intriguingly, these two enzymes are reciprocally regulated at the level of transcription (Modolell et al., 1995, Eur. J. Immunol. 25:1101-1104; Wang, et al., 1995, Biochem. Biophys. Res. Commun. 210:1009-1016) and at the level of catalytic activity (Conaliza et al., 1995, Biochem. Biophys. Res. Commun. 206:667-673). N°-Hydroxy-L-arginine is an intermediate in the NO synthase reaction (Pufahl et al., 1992, Biochemistry 31:6822-6828; Stuehr et al., 1991, J. Biol. Chem. 266;6259-6263; Klatt et al., 1993, J. Biol. Chem. 268:14781-14787; Campos et al., 1995, J. Biol. Chem. 270:1721-1728; Pufahl et al., Biochemistry 34:1930-1941); significant concentrations of N^-hydroxy-L-arginine appear to dissociate from NO synthase to serve as an endogenous competitive inhibitor of arginase with Kj = 42 μM (Chenais et al., 1993, Biochem. Biophys. Res. Commun. 196:1558-1565; Buga et al., 1996, Amer. J. Physiol. 271 :H1988-H1998; Daghigh et al., 1994, Biochem. Biophys. Res. Commun.
202:174-180; Boucher et al, 1994, Biochem. Biophys. Res. Commun.203;1614-16210. Since arginase can regulate NO synthase activity by depleting cellular concentrations of arginine (Griffith et al., 1995, Annu. Rev. Physiol. 57:707-736), the two enzymes are reciprocally coordinated at the level of enzyme activity (Figure 11). Selective inhibition of arginase or NO synthase might therefore result in beneficial physiological effects due to the modulation of macrophage function.
In the present Example, various boronic acid-based arginine analogues were tested for their ability to be selective inhibitors of arginase. The binuclear manganese cluster of arginase functions to activate a bridging hydroxide ion for attack at the guanidinium group of the substrate arginine (Reczkowski et al., 1992, J. Am. Chem. Soc. 114:10992-10994; Kanyo et al., 1996, Nature 383:554-557). A key tetrahedral intermediate results (Figure 11), and boronic acid-based inhibitor 2(S)-amino-6-boronohexanoic acid (7) was designed to mimic this intermediate and its flanking transition states (Example 2). Specifically, the electron-deficient boron atom of the boronic acid moiety is sufficiently electrophilic to facilitate the addition of a catalytic nucleophile, such as a protein atom or a solvent molecule. Similar design strategies have resulted in potent boronic acid-based inhibitors of serine proteases (Matteson et al., 1981, J. Am. Chem. Soc. 103:5241-5242; Kettner et al., 1984, J. Biol. Chem. 259: 15106-15114) and aminopeptidases (Shenvi 1986, Biochemistry 25:1286-1291). Accordingly, 2(S)-amino-6-boronohexanoic acid (7) is one of the most potent boronic acid-based inhibitors of arginase known to date with IC^Q = 0.8 μM, yet it exhibits no inhibitory activity against NO synthase (Example 2). Surprisingly, 2(S)-amino-6-boronohexanoic acid (7) exhibited substantial nonlinearity in kinetic replots (Example 2), and a full description of its binding kinetics has been reported herein. These unusual binding kinetics may arise from the unique chemistry of the electron-deficient boronic acid moiety. In order to complement this work and further probe the unusual chemistry of boronic acid-based analogues of arginine, we also report the synthesis and kinetic evaluation of 2(S)-amino-5-boronopentanoic acid (12), and the kinetic evaluation of S-(2-boronoethyl)-L-cysteine (15) (the synthesis of 15 is previously reported; Matteson et al, 1964, J. Med Chem. 7:640-643.). Additionally, the synthesis and kinetic evaluation of a trihydroxysilane-based arginine analogue,
S-(2-trihydroxysilylethyl)-cysteine (16), as well as a new chromogenic substrate which allows for a continuous assay of arginase activity is reported herein.
The Materials and Methods used in this Example are now described. General Methods Thin layer chromatography (TLC) was performed on E. Merck silica gel 60 F254 glass plates. Compounds with tert-butyloxycarbonylamino groups or free amino groups were visualized on TLC plates by first dipping the plates in a ninhydrin solution (0.1% ninhydrin in 95% n-butanol, 4.5% water, 0.5% glacial acetic acid) and then heating the plates until color evolved. Column chromatography was performed on E. Merck silica gel 60 (230-240 mesh ASTM) under positive nitrogen pressure. -NMR and 1 ^-NMR spectra were recorded on a Bruker AC-250 (250 MHZ) NMR and on a Bruker AC-200 (200 MHZ) NMR, respectively ( H chemical shifts are referenced to CHCI3, HDO, or DMSO, and B chemical shifts are referenced to BF3 diethyl ether. Electrospray mass spectrometry was conducted by personnel in the Mass Spectrometry Center, University of Pennsylvania.
2(S)-N-(tert-butyloxycarbonyl)-aspartic acid tert-butyl ester (1) was purchased from Bachem. Triethylamine and DMSO (dimethyl sulfoxide) were purchased from Fisher. All other reagents were purchased from Aldrich and used without further purification. 2fSVamino-6-boronohexanoic acid (1) The synthesis and preliminary evaluation of this boronic-based analogue is described in Example 2 herein.
2rS>-N-ftgrt-butyloxycarbonylV4-hydroxypentanoic acid. tert-butyl ester (& >
To a -5°C solution of 2.90 g (10.0 mmol) of 2(S)-N-(tert-butyloxycarbonyl)-aspartic acid, tert-butyl ester in 70 mL of THF was added 2.79 mL (20.0 mmol) triethylamine and 1.92 mL (20.0 mmol) ethyl chloroformate. The mixture was stirred at -5°C for five minutes. The precipitant triethylamine salt was quickly removed by filtration. The filtrate was added to a suspension of sodium borohydride in 5 mL of water and stined at room temperature overnight. The resulting crude product was purified by column chromatography on silica gel (with 5:1 hexane:ethyl acetate as an eluent). Following concentration in vacuo, 1.44 g of product was obtained (52% yield). TLC (1 :1 hexane:ethyl acetate) indicated a single spot (Rf = 0.41). !H-ΝMR (CDC13): δ 1.45 (s, 18H), 2.15 (m, 2H), 3.70 (m, 2H), 4.40 (m, IH), 5.40 (d, IH).
2(S -N-("tert-butyloxycarbonylV4-oxobutanoic acid, tert-butyl ester (9) To a solution of 1.83 mL (20.9 mmol) of oxalyl chloride in 30 mL CH2C12 at -78 °C was added 2.97 mL (41.9 mmol) of DMSO. The solution was stined at -78°C for 5 min. A solution of 1.44 g (5.24 mmol) of 8 dissolved in 10 mL of CH2C12 was prepared, added by canula to the pre-formed Swem reactant, and stined at -78 °C for 15 min. The Swem oxidation was completed by the addition of 0.726 mL (14.6 mmol) of triethylamine. The mixture was stirred for 5 min. at -78 °C and then warmed to room temperature. The CH C1 was evaporated in vacuo. The crude material was taken up in THF and the precipitant triethylamine salt was removed by filtration. The material was purified by column chromatography on silica gel (with 6:1 hexane:ethyl acetate as an eluent). The product was obtained in 69% yield (980 mg). TLC (1 :1 hexane:ethyl acetate) and staining with ninhydrin indicated a single spot (Rf = 0.65) following concentration in vacuo. -NMR (CDCI3): δ 1.45 (s, 18H), 3.0 (t, 2H), 4.45 (m, IH), 5.35 (d, IH), 9.75 (s, IH).
2CSVN-('tert-butyloxycarbonylVpent-4-enoic acid, tert-butyl ester CIO') The Wittig salt triphenylphosphonium methylide was prepared by the addition of 5.11 g (14.3 mmol) of potassium tert-butoxide to a partially dissolved solution of 1.61 g (14.3 mmol) methyltriphenylphosphonium bromide in 100 mL of THF at 0°C. The mixture was stined at 0°C for 10 min. and then at room temperature for 90 min. The mixture turned yellow and was cooled to -78 °C. The aldehyde 9, 0.980 g (3.59 mmol) dissolved in THF was added directly to the triphenylphosphonium methylide solution. The Wittig reaction was stined and allowed to proceed for 5 min. at -78 °C and then ovemight at room temperature. The olefin 10 was purified by column chromatography on silica gel (with 20:1 hexane:ethyl acetate as an eluent). Following concentration in vacuo, 632 mg of product was obtained (65% yield). TLC (3:1 hexane:ethyl acetate) indicated a single spot (Rf = 0.56). *H-ΝMR (CDCI3): δ 1.40 (s, 9H), 1.45 (s, 9H), 2.50 (m, 2H). 4.25 (m, IH), 5.1 (s, IH), 5.15 (d, 2H), 5.6-5.8 (m, IH).
2fSVN -rtert-butyloxycarbonylV5-[('lS.2S.3R.5SV('+Vpinanedioxaboranyl]-pentanoic acid. tert-butyl ester (11). To a 0°C chilled, stined solution of 11.6 mL (11.6 mmol) of 1 M BH3.THF in 10 mL THF was added 632 mg (2.33 mmol) of olefin 10. The reaction mixture was stined for 15 min. at 0°C and then 2 h at room temperature. Excess unreacted borane was quenched by the slow addition of methanol until gaseous evolution as indicated by bubbling ceased. The solvent was evaporated in vacuo. The crude material was taken up in CH2C12. Excess (lS,2S,3R,5S)-(+)-pinanediol (0.793 g, 4.66 mmol) was added to the solution of crude material in CH2C12. Esterification with (+)-pinanediol was allowed to proceed ovemight at room temperature. Compound 11 was purified by column chromatography on silica gel (with 20:1 hexane:ethyl acetate as an eluent). The product was obtained in 22% yield (226 mg) following concentration in vacuo. TLC (3:1 hexane: ethyl acetate) indicated a single spot (Rf = 0.58). -NMR (CDC13): δ 0.80 (m, 5H), 1.05 (d, 2H), 1.25 (s, 3H), 1.35 (s, 3H), 1.40-1.50 (m, 20H), 1.6-1.95 (m, 3H), 2.05 (t, IH), 2.10-2.35 (m, 2H), 4.15 (m, IH), 4.25 (d, IH), 5.0 (d, 2H). 2fS)-amino-5-boronopentanoic acid, hydrochloride salt (\2)
To a -78 °C chilled, stirred solution of 226 mg of 11 in CH2C12 was slowly added 2.00 mL of 1 M BCI3 in CH2C12 (2.00 mmol). The reaction mixture was stined for 15 min. at -78°C and then for 30 min. at 0°C. The solvent CH2C12 with excess BCI3 were evaporated by allowing a flow of nitrogen gas to pass over the mixture.
The product material was taken up in acetonitrile and collected by vacuum filtration. Repeated washings with acetonitrile and then with diethyl ether gave a crude hygroscopic solid. The solid was taken up in a minimum volume of ethanol. Precipitation of product was induced by the addition of acetone. The highly hygroscopic precipitate was collected and removed from the filter paper by the addition of ethanol. The ethanol filtrate was collected. Ethanol was evaporated in vacuo to give 16 mg of pure product (16% yield). TLC (80:10:10 «-butanol:acetic acid:water) indicated a single spot (Rf = 0.096). *H-NMR (D2O): δ 0.85 (t, 2H), 1.55 (m, 2H), 1.95 (t, 2H), 3.95 (t, IH). S-f2-boronoethylVL-cvsteine. hydrochloride salt (T51
This compound was synthesized as previously described (Matteson et al., 1964, J. Med Chem. 7;640-643; Matteson, 1960, J. Am. Chem. Soc. 82: 4228-4233). The compound was originally synthesized and tested as a water-soluble candidate for boron neutron-capture therapy (Matteson et al, 1964, J. Med Chem. 7:640-643). TLC (80: 10: 10 H-butanol: acetic acid:water) of the product indicated a single spot (Rf = 0.44). H-NMR (D2O): δ 0.95 (t, 2H), 2.50 (t, 2H), 2.75-3.05 (m, 2H), 3.72 (m, IH). nB NMR (D2O) δ 31.33 (s).
S- -trihydroxysilylethyl VL-cysteine Cl 6) A solution consisting of 3.96 g (32.6 mmol) L-cysteine in 50 mL water and 5 mL (32.6 mmol) of vinyl trimethoxysilane in 40 mL methanol was refluxed at 80°C under a nitrogen atmosphere. At time intervals of 0, 5, and 6 hours, 32 mg portions of azobisisobutyronitrile were added to the solution. The solution was refluxed for a total of 8.5 hours at 80°C, then stined overnight at room temperature. The solvents water and methanol were removed in vacuo. The residue was taken up in a minimal volume of water, acidified with 1 mL of concentrated HCl, and then suspended in excess acetone. After a precipitate settled, the acidic solvent was decanted and discarded. This wash with acid and acetone was repeated two more times in order to remove all unreacted starting material. The final precipitate was a crystalline, glass-like solid free of contamination from unreacted L-cysteine, which was present in the filtrate. The product yield was 12% (1.032 g). H-NMR (D2O): δ 0.90 (brs, IH), 2.55 (brs, 2H), 2.95 (brs, 2H), 3.90 (brs, IH), 5.85 (brs, 3H). Electrospray mass spectroscopy indicated a strong tendency to dimerize via the silanetriol moiety. Calculated C5H14NO5SSi+, 228.04; found: 406.9, 428.8, and 444.8.
1 -nitro-3-CN.N -bis tert-butyloxycarbonyπguanidino)benzene (\1) To a stirred solution of 238 mg (1.72 mmol) w-nitroaniline and 500 mg (1.72 mmol) l,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea in CH2C12 was added 438 mg (2.58 mmol) of silver nitrate (Bergeron et al., 1987, J. Org. Chem. 52:1700-1703; Natsugari et al., U.S. Patent No. 4,851,422 July 25, 1989; Morimoto et al., U.S. Patent No. 4,876,251 October 24, 1989). A white precipitate indicative of silver methylsulfide formed after one hour. The mixture was stined ovemight at room temperature. Compound 17 was purified by column chromatography on silica gel (with 10:1 hexane: ethyl acetate as an eluent). The product was obtained in 67% yield (439 mg) following concentration in vacuo. TLC (3:1 hexane:ethyl acetate) indicated a single spot (Rf = 0.43). *H-NMR (CDC13): δ 1.50 (s, 9H), 1.60 (s, 9H), 7.50 (t, IH), 7.95 (d, IH), 8.05 (d, IH), 8.5 (s, IH), 10.6 (s, IH), 11.6 (s, IH). l-nitro-3-guanidinobenzene. hydrochloride salt (18) To a stined solution of 17 (238 mg, 0.492 mmol) in CH2C12 was added 1.97 mL of 1 M BC13 (1.97 mmol) in CH2C1? After 30 min, excess BC13 and CH2C12 were blown off with a stream of nitrogen gas. The material was washed with CH2C12. The product was obtained as a white powder in 62% yield. TLC (4:1 :1 /ι-butanol:acetic acid:water) indicated a single spot that was also UV active (Rf = 0.47). *H-NMR (DMSO): δ 7.6-7.8 (m, 3H), 8.0-8.15 (m, IH), 10.25 (brs.).
Enzyme Assays
Recombinant rat liver arginase was purified as described previously (Cavalli et al., 1994, Biochemistry 33:10652-10657). The concentrations of enzyme stock solutions were determined from the absorbance at 280 nm using an extinction coefficient of 1.09 mL mg"1 cm-1 (Schimke, 1970, Methods Enzymol. 17a:313-317). The activity of wild-type arginase was monitored spectrophotometrically on a Pharmacia Biotech UltroSpec 2000. Assays were performed in 100 μM MnCl2, 50 mM bicine-NaOH (pH 9.0). The compound l-guanidino-3 -nitrobenzene (18) proved to be a new substrate for arginase hydrolysis, producing products urea and the chromogen w-nitroaniline. A stock solution of 200 mM 18 in DMSO was prepared. A period of 15 minutes was allowed for 18 to equilibrate with bicine buffer prior to assay.
Reaction velocities were measured at 372 nm, where the liberated product,
-5 1 1 w-nitroaniline, has an extinction coefficient of 1.28 ¥ 10J _Vf cm" . For Kj j and kcat determinations, 40 μL of 0.448 mg/mL arginase was added to a cuvette containing bicine buffer and the appropriate concentration of 18 (0.8 - 2.3 mM) for a final volume of 1 mL. Eadie-Hofstee plots were used to analyze the data.
Compounds 7, 12, 15, and 16 (Figure 10, Scheme 1) were assayed using 2 mM 18 as the assay substrate. The concentrations of 7 and 15 were varied in the range 0.5-9.0 μM, those of 12 and 16 were varied in the range 200-1000 μM.
When activity as a function of enzyme concentration was assayed, the concentration of arginase trimer was varied in the range 1.1-6.4 μM. For the boronic acid inhibitors, reciprocal plots of inverse velocity as a function of inhibitor concentration were nonlinear. For the trihydroxysilane inhibitor, reciprocal plots of inverse velocity as a function of inhibitor concentration were linear.
Dialysis experiments of arginase with the arginase-7 and arginase-15 complex were performed in following way. Thirty microliters of arginase at a concentration of 11.2 mg/mL were preincubated for thirty minutes with an appropriate concentration of 7 ( 5 μL of 100 μM stock solution). For 15, 300 μL of arginase at a concentration of 0.938 mg/mL were preincubated for thirty minutes with a sufficient concentration of 15 (30 μL of 200 μM stock solution) to suppress all detectable enzymatic activity. Preincubates were then added to bicine buffer with 4 mM 18. The final volume of the assay reaction solution was 1 mL.
The inhibition of arginase by 7 and 15 was also evaluated using a modified version of the radioactive assay by Rϋegg and Russell (Rϋegg et al., 1980, Anal. Biochem. 102:206-212). Assays were performed in 100 μM MnCl2, 100 mM ches-NaOH (pH 9.0). The reactions were initiated by the addition of 5 μL of a ~ 1 unit mL enzyme solution to 45 μL of reaction mixture containing ches buffer, 1.4 mM L-arginine, -5.0 x 104 cpm of L-[guanidino- C]arginine, and variable concentrations of the appropriate inhibitor. After a 5-15 minute reaction time, 200 μL of a stop solution containing 7 M urea, 10 mM L-arginine, and 0.25 M acetic acid (pH 4.5) was added to the reaction (arginase has essentially no activity under these conditions). [14C]-Urea was separated from unreacted L-[guanidino- C]arginine by treatment with 200 μL of a 1:1 (vol/vol) slurry of Dowex 50 W-X8 in water, and quantitated by adding 200 μL of the supernatant from the Dowex treatment to 3 mL of Liquiscint (National Diagnostics) for liquid scintillation counting in a Beckman LS5000CE counter. Isothermal Titration Calorimetry
All calorimetry experiments were conducted on a MCS isothermal calorimeter from MicroCal, Inc. (Northampton, MA). Arginase was exhaustively dialyzed against 100 μM MnCl2, 50 mM bicine-NaOH (pH 8.5). Inhibitor was dissolved at a concentration of 1.5 mM in an aliquot of the same buffer. Prior to the titration experiment, samples were degassed under vacuum for 5 min. The sample cell (effective volume 1.366 mL) was overfilled with 1.8 mL of arginase at a concentration of 0.0358 mM and the reference cell was filled with water. The contents of the sample cell were titrated with 30-40 aliquots (2.5 μL each) of inhibitor (an initial 1 μL injection was made, but not used in data analysis). After each injection, the heat change was measured and converted to the conesponding enthalpy value. The reaction mixture was continuously stined at 400 rpm during titration. Control experiments were carried out by titrating the inhibitor into the buffer solution under identical experimental conditions. Data analysis was performed using Origin software provided with the instrument. The calorimetric data are presented with the background titrations subtracted from the experimental data. The amount of heat produced per injection was calculated by integration of the area under each peak. The data were fit to the following equation, q = VAU [E]t K [L] / l + K [L], where q is the heat evolved during the course of the reaction, "is the cell volume, ΔH is the binding enthalpy per mole of ligand, [E]t is the total enzyme concentration, K is the binding constant, and [L] is inhibitor concentration (Fisher et al., 1995, Methods Enzymol. 1995 259:194-221; Wiseman et al, 1989, Anal. Biochem. 179:131-137). The Results of the experiments presented in this Example are now described.
The K c and kcat values for arginase-catalyzed hydrolysis of the new chromogenic assay substrate, l-nitro-3-guanidinobenzene (18), were 1.6 ± 0.2 mM and 0.09 ± 0.02 min , respectively (Figure 2), based on a monomer molecular mass of 35 kDa. Within experimental enor, the Kj^ value for 18 was identical to that reported for arginine itself (K^ =1.4 ± 0.3 mM) ( Fisher et al., 1995, Methods Enzymol. 259:194-221; Wiseman et al., Anal. Biochem. 179:131-137). The kcat value of 18 was five orders of magnitude lower than that reported for arginine (kcat = 15,000 ± 1200 min." 1) (Cavalli et al., 1994, Biochemistry 33:10652-10657). Despite its low kcat value, substrate 18 provided a continuous chromogenic assay with no apparent background and high precision. Furthermore, the synthesis of 18 required only two straightforward steps, which facilitates the rapid production of large quantities of this compound.
For fully competitive, noncompetitive, and uncompetitive inhibitors,
Figure imgf000066_0001
as a function of (I), should be linear (Todhunter, 1979, Methods Enzymol. 63:383-411; where vQ is enzyme velocity in the absence of inhibitor, v is enzyme velocity in the presence of inhibitor, and (I) is the inhibitor concentration, (EQ) is total enzyme concentration). For example, this was the case for the trihydroxysilane inhibitor 16, which is a fully reversible inhibitor with a modest Kj of 420 ± 30 μM (assuming competitive inhibition; Figure 13a). However, for boronic acid inhibitors 7, 12, and 15, plots of vQ/v as a function of (I) were nonlinear and concave upward (Figures 13b- 13d). Plots of v/vQ versus (I) at constant (EQ) yielded a linear relationship for the boronic acid inhibitors. Likewise, plots of v/vQ as a function of 1/(EQ) at constant (I) (4 μM 2(S)-amino-6-boronohexanoic acid) were linear. These data lead to the observation that v/vQ as a function of (I)/(EQ) was linear for all the boronic acid inhibitors (Figure 14). Significantly, this relationship was consistent with enzyme inactivation (Silverman, 1988, In: Mechanism-based Enzyme Inactivation: Chemistry and Enzymology ; Vol. I; CRC Press, Inc.: Boca Raton, Florida, pp 22-23). Paradoxically, the boronic acid inhibitors gave no indication of inactivation or time-dependent inhibition, and dialysis experiments indicated complete and virtually instantaneous reversible inhibition when enzyme and inhibitor were incubated for short time periods (30 min), in that dialysis experiments with boronic acid inhibitor 7 exhibited completely instantaneous reversible inhibition when arginase and 7 preincubated for 30 minutes were diluted and assayed. Upon dilution the concentration of 7 went from 14.3 μM to 0.5 μM; the concentration of arginase was diluted from 274 μM to 9.6 μM. The observed inhibited velocity for the diluted and assayed preincubate was 0.46 μM min. A control experiment of arginase at 9.6 μM assayed with 0.5 μM 7 yielded an inhibited velocity of 0.47 μM/min.
For ineversible inhibitors, the slope of linear v/vQ versus (I)/(E0) plots represented a reciprocal tumover number: moles of enzyme inactivated per moles of inactivator. The reciprocal slopes derived from v/vQ versus (I)/(EQ ) plots gave rise to a constant designated the "pseudo-turnover number", tnpSeu(j0, since the boronic acids were behaving reversibly and not as inactivators. The npSeucj0 provides a useful means for comparing the relative inhibitor potency (Table 7). When assayed with 18, boronic acid inhibitors 7 and 15 were essentially equipotent (within experimental enor), with compound 7 showing a marginally lower tn_,seu(j0. The ripSeucj0 for boronic acid 12 was two orders of magnitude higher than the DSeucj0 values for 7 and 15. Thus, inhibitor 12 was ~400-fold less potent than 7 or 15.
Table 7: Kinetic and Thermodynamic Data for Boronic Acid Inhibitors
Figure imgf000068_0001
Determined by assay with substrate 18. b Determined by titration calorimetry.
Apparent K values for 7 and 15 were obtained using the radioactive [14C-guanidino]-L-arginine assay of Ruegg et al. 1980, Anal. Biochem. 102:206-212; Table 7). At low concentrations of 7 and 15, linear plots of v v as a function of (I) yielded Kj values of 0.1 μM and 0.7 μM for 7 and 15, respectively (assuming competitive inhibition). It appeared that 7 was significantly more potent than 15 against the natural substrate, L-arginine. Qualitative differences in the results of inhibitor assays using L-arginine or 18 as an assay substrate may arise from structural differences between these two substrates: substrate 18 lacks an amino acid moiety and therefore might be less sensitive to displacement by the amino acid moiety of 7 or 15.
The titration calorimetry data for 7 and 15 reveal three important insights on the inhibition of arginase by boronic acids (Table 7; Figures 15 and 16). First, ΔHtø jncjjng values for 7 and 15 to the arginase monomer were nearly identical (-12.97 kcal/mol and -12.75 kcal/mol, respectively), suggesting a similar association mechanism. Second, the stoichiometry of inhibitor binding was 1.07 and 0.96 per monomer for 7 and 15, respectively. Finally K^ values (K^ = l/__) of 0.11 μM and 2.22 μM were obtained for 7 and 15, respectively. Interestingly, the results of kinetic assays agree well with titration calorimetry data for 7, with Kj ~ K^ . However different assays yielded different results for 15, i.e., K.- and K^ values disagree by nearly an order of magnitude. Unlike Kj , K^ is obtained at thermodynamic equilibrium. Prior to thermodynamic equilibrium, 15 may perhaps undergo a conformational change for which Kj, a kinetic parameter, is less sensitive.
Do Boronic-Acid Based Arginine Analogues Bind to Arginase as Mimics of the Transition State? Boronic acids 7, 12, and 15 are expected to bind to arginase as the tetrahedral boronate anions, thereby mimicking the proposed tetrahedral intermediate and its flanking transition states for arginine hydrolysis (Figure 17). This type of transition state analogue exploits the electron-deficient nature of boron, which facilitates the addition of a nucleophilic solvent molecule to generate the boronate anion. In solution, the neutral trigonal planar boronic acid is in a pH dependent equilibrium with the tetrahedral boronate anion. At alkaline pH values [i.e., high (HO")], the tetrahedral species predominates (Anderson et al., 1964, J. Phys. Chem. 68:1128-1132); this is most likely the case for inhibitors binding to the arginase active site, where the local concentration of metal -bridging hydroxide is high. By way of analogy, the tetrahedral borate anion is a noncompetitive inhibitor of arginase with a KJJ = 0.26 mM and a Kjg = 0.98 mM, and binds to the active site by displacing the metal-bridging hydroxide ion (Example 2; Pace et al., 1981, Biochim. Biophys. Acta 658:410-412; Reczkowski, R.S. Ph.D. Thesis, Temple University School of Medicine, 1990). It should be noted that (a) the binding mode of borate, (b) the high affinities of boronic acid-based arginine analogues, and (c) the relative invariance of substrate K^ values with perturbation of the binuclear manganese cluster (Cavalli et al., 1994, Biochemistry 33:10652-10657) do not support a recently proposed arginase mechanism involving direct arginine-manganese coordination (Khangulov et al., 1998, Biochemistry 37:8539-8550); instead, these data continue to be consistent with the mechanism proposed by Kanyo et al. (1996, Nature 383:554-557).
In contrast with the boronic acid-based arginine analogues, silanetriol 16 has a fixed tetrahedral configuration, and only 16 yielded a linear plot of vQ/v versus (I) (indicative of reversible inhibition). However, arginase inhibition by 16 is modest: assuming competitive inhibition, Kj = 420 μM. Silanetriol inhibitors have recently been developed against β-lactamase (Curley et al., 1997, J. Am. Chem. Soc. 119:1529-1538). Here, too, silanetriols are significantly poorer inhibitors than their boronic acid analogues. Silanetriols have additional complications as enzyme inhibitors due to their generally poorer solubility and greater tendency to dimerize (Knight et al.,1989, J. Chem. Soc, Dalton Trans.275-281; McNeil et al., 1980, J. Am. Chem. Soc. 102:1859-1865).
Compounds 7 and 15 are among the most potent arginase inhibitors reported to date, and the use of the new chromogenic substrate 18 facilitates the rapid screening of these inhibitors. Titration calorimetry yields precise K^ values of 0.11 μM and 2.22 μM for 7 and 15, respectively. The unrivaled potency and selectivity of 7 for arginase make this compound ideal for probing the reciprocal functions of arginase and nitric oxide synthase, e.g., in the regulation of nitric oxide-induced smooth muscle relaxation as described in Example 4. Example 4: Biochemical and Functional Profile of Arginase Inhibitors
An increase in arginase activity has been associated with the pathophysiology of a number of conditions including an impairment in nonadrenergic and noncholinergic (NANC) nerve-mediated relaxation of the gastrointestinal smooth muscle. It was hypothesized that an arginase inhibitor may rectify this condition. The effects of a newly designed arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABHA) with the cunently available Nω-hydroxy-L-arginine (L-HO-Arg) on the NANC nerve-mediated I AS smooth muscle relaxation and the arginase activity in the IAS and other tissues was compared in this Example.
Arginase caused an attenuation of the IAS smooth muscle relaxations by NANC nerve stimulation that was restored by the arginase inhibitors. Interestingly, L-HO-Arg but not ABHA caused does-dependent and complete reversal of Nω-nitro-L- arginine (L-NNA)-suppressed IAS relaxation that was similar to that seen with L- arginine. Both ABHA and L-HO-Arg caused an augmentation of NANC nerve- mediated relaxation of the IAS. In the IAS, ABHA was found to be -250 times more potent than L-HO-Arg in inhibiting the arginase activity. L-HO-Arg was found to be 10 to 18 times more potent in inhibiting the arginase activity in the liver as compared to that in nonhepatic tissues. It was therefore concluded that arginase plays a significant role in the regulation of NO synthase-mediated NANC relaxation in the IAS. The advent of new and selective arginase inhibitors therefore plays a significant role in the discrimination of arginase isozymes and have important pathophysiological and therapeutic implications in gastrointestinal motility disorders. The purpose of the present investigation was to test a newly designed and selective arginase inhibitor, 2(S)-amino-6-boronohexanoic acid (ABHA) (Example 2, compound 7) for its effectiveness in the physiologically relevant system. The effectiveness of ABHA on the arginase activity of the I AS, rectum, brain and liver was also examined. The Materials and Methods used in the experiments presented in this
Example are now described.
Functional Studies Preparation of Smooth Muscle Strips
Studies were performed on circular smooth muscle strips of the intemal anal sphincter (IAS) obtained from adult opossums (Didelphis virginiana) of either sex following pentobarbital anesthesia (40 mg/kg; i.p.) and subsequent exsanguination. The entire anal canal was isolated carefully using the sharp dissection and transfened to a dissecting tray containing oxygenated (95% O2 plus 5% CO2) Krebs' solution. The composition of the Krebs' solution was as follows (mM): NaCl, 118.07; KC1 4.69; CaCl2, 2.52; MgSO 1.16; NaH2PO 1.01; NaHCO3, 25 and glucose (Pufahl et al., 1995, Biochemistry 34:1930-1941; Chenais et al., 1993, Biochem. Biophys. Res. Commun. 196:1558-1565). The anal canal was cleaned of the extraneous connective tissue and the blood vessels. Following this, the anal canal was opened flat by an incision along the longitudinal axis, and was pinned flat with the mucosal side facing up. The mucosa along with the submucosa was removed by a sharp dissection. Circular smooth muscle strips were obtained from the whole circumference of the anal canal and divided into two equal strips (- 1 x 8 mm). Both ends of the muscle strips were secured with silk sutures (4-0: Ethicon Inc., Sommerville, NJ) and used for the measurement of isometric tension. Measurement of Isometric Tension
The IAS smooth muscle strips prepared as described above were mounted onto the thermostatically controlled 2 ml muscle baths (37°C) containing oxygenated (95% O2 and 5% CO2) Krebs' solution. One end of the muscle strip was fixed to the bottom of the muscle bath with a tissue holder to the stand and the other end was attached to a isometric force transducer (model FT03; Grass Instruments Co., Quincy, MA) for the measurement of isometric tension. The smooth muscle tension was recorded on a Dynograph recorder (model R411 ; Beckman Instruments, Schiller Park, IL). After an equilibration period of 1 hour with intermittent washings, the optimal length (LQ) and the baseline of the resting tension of each smooth muscle strip was determined as described previously (Mourami and Rattan, 1988, Am. J. Physiol. 255:G571-G578). Only those smooth muscle strips that developed spontaneous and steady tension and relaxed in response to electrical field stimulation (EFS) were used. Nonadrenergic Noncholinergic (NANC .■ Nerve Stimulation with
Electrical Field Stimulation (EFS .■
EFS was delivered via a pair of platinum wires, from a Grass stimulator (Model S88; Grass Instruments Co., Quincy, MA) connected in series to a Med-Lab Stimu-Splitter II (Med-Lab Instruments, Loveland, CO). The Stimusplitter was used to amplify and measure the stimulus intensity using the optimal stimulus parameters for the neural stimulation (12V, 0.5 ms pulse duration, 200-400 mA, 4 sec train) at varying frequencies of 0.5 to 20 Hz. These parameters of EFS are known to cause relaxation of the IAS smooth muscle via the selective activation of NANC myenteric neurons. Neurally-mediated relaxation of the IAS smooth muscle strips was quantified in response to different frequencies of EFS. All the experiments were performed in the presence of atropine (1 x 10"" M) and guanethidine (3 x 10""M).
Drug Responses
To determine the influence of arginase on the NANC nerve-mediated relaxation of the IAS, the effects of different doses of arginase on the relaxation were first determined. Arginase in the dose of 30 u/ml was found to be the most effective in causing the attenuation of relaxation. The effectiveness of the arginase inhibitors (L- OH-Arg and ABHA) on the arginase-induced attenuation of the EFS-induced IAS relaxation was then tested. The optimal doses of arginase and L-OH- Arg in the IAS have been reported before (Chakder and Rattan, 1997, J. Pharmacol. Exp. Ther. 282:378-384). In order to determine the selectivity of the arginase inhibitors in the IAS, their effects on the suppressed IAS relaxation by the NO synthase inhibitor (L- NNA (3 x 10-5 M) were tested. This was then compared with the reversal by the NO synthase substrate L-arginine in different concentrations. To examine the physiological relevance of the arginase inhibitors in the IAS relaxation, the influence of different concentrations of arginase inhibitors on the EFS-induced IAS relaxation was examined. At the end of each experiment, the smooth muscle strips were treated with 5 mM EDTA to establish the maximal relaxation (Biancani et al., 1985, Gastroenterology 89:867-874). Each smooth muscle strip served as its own control. Synthesis of the Arginase Inhibitor. 2(SVAmino-6-boronohexanoic Acid (ABHA^) The details of the synthesis of ABHA, a boronic acid-based arginine isostere has been described in Example 2 herein. ABHA is the most potent inhibitor of Mn2+ 2_ arginase reported to date, with an estimated binding constant of 0.1 μM (Example 2 herein). In the hydrated form, the boronic acid moiety of ABHA is believed to mimic the putative tetrahedral intermediate formed during arginine hydrolysis, and thus may serve as a transition state analog. Tissue Preparation
Tissue samples of the opossum intemal and sphincter muscle (I AS), the adjoining rectal tissue, the liver, and the brain were homogenized with an ultratunax tissue homogenizer (Tekamr, Cincinnati, OH) in 10 mM Tris-HCl, 150 mM KC1, 25 mM MnCl2, pH7.4. The resulting homogenates were then dialyzed overnight against 10 mM Tris-HCl, 150 mM KC1, 25 mM MnCl2, pH 7.4. The dialyzed homogenates were centrifuged to remove insoluble material and concentrated with Amicon Centricon 30 microconcentrators to give stock protein concentrations of 2.4 mg/ml for the IAS smooth muscle, 3.6 mg/ml for the rectal smooth muscle, 3 mg/ml for the brain, and 17.5 mg/ml for the liver. Protein concentrations were estimated with the Pierce Coomassie Protein Reagent using bovine serum albumin as a standard. Arginase Assay The arginase activity of tissue homogenates was evaluated using the radioactive L-[guanidino14C]arginine assay of Rϋegg and Russell (1980, Anal. Biochem. 102:206-212). Assays were performed in 100 mM CHES-NaOH 0.1 mM MnCl2 at pH 9.0. The reactions were initiated by the addition of 5 μl of tissue homogenate to 45 μl of reaction mixture that contained the CHES buffer, the appropriate concentration of arginine (0.5 to 5 mM) and - 5.0 x 104 cpm of L-
[guanidino- 4C]arginine. The IAS, rectal and brain homogenates were incubated with the assay mixture at room temperature for one hour, while the liver sample was incubated for 5 min. The reactions were stopped by the addition of 200 μl of a solution containing 0.25 M acetic acid/7 M urea and 10 mM arginase at pH 4.5. Arginase has essentially no activity at the low pH of the stop solution. [ C] Urea was separated from unreacted L-[guanidino- 4C]arginine by treatment with 200 μl of 1 :1 v/v slurry of Dowex 50W-X8 in water, and quantitated by adding 200 μl of the supernatant from the Dowex treatment to 3 ml of Liquiscint (National Diagnostics) for liquid scintillation counting in a Beckman LS 5000CE counter. The data were analyzed using double-reciprocal plots of the initial velocity measurements; standard enors were determined by regression analysis. Arginase Inhibition Studies Assays in the presence of the inhibitors Nω-hydroxy-L-arginine (1 to 100 μM) and 2 (S)-amino-6-boronohexanoic acid (0.05 to 100 μM) were performed in a fashion similar to the one described above. The amino acid D-ornithine served as a control in these experiments, since this amino acid, unlike L-omithine, is not an inhibitor of arginase.
Drugs and Chemicals
Bovine liver arginase, Nω-hydroxy-L-arginine (L-HO-Arg), Nω-nitro- L-arginine (L-NNA), Nω-nitro-D-arginine methyl ester (D-NNA), L-arginine hydrochloride, D-arginine, D-omithine and atropine sulfate were obtained from Sigma Chemical Co., St. Louis, MO. Guanethidine monosulfate was from Ciba
Pharmaceuticals (Summit, NJ). Ethylenediamine tetraacetic acid (EDTA) tetrasodium salt was from Fisher Scientific Co., Fair Lawn, NJ 2(S)-amino-6-boronohexanoic Acid (ABHA) was synthesized as described in Example 2.
L-[guanidino- CJArginine (specific activity 2.5 GBq mmol" 1) was from NEN/Dupont.
All chemicals used were of the highest purity available. Solutions of all the chemicals were prepared in Krebs' solution fresh on the day of the experiment. Data analysis
The responses to EFS and other relaxants were expressed as the percent of maximal relaxation caused by 5 mM EDTA. The results are expressed as means ± S.E. Statistical significance between different groups were determined using t-test and a p value smaller than 0.05 was considered significant.
The Results of the experiments presented in this Example are now described. Influence of Exogenous Administration of Arginase Before and After the Arginase Inhibitors Nωhydroxy-L-arginine (L-HO-Arg .< and 2 (5VAmino-6- boronohexanoic Acid (ABHA.- on the NANC Nerve-mediated Relaxation of the IAS First, the effects of different concentrations of arginase on the IAS relaxation by the NANC nerve stimulation was determined. Arginase in the dose of 30 units/ml was found to be optimal in causing the attenuation of the NANC nerve mediated relaxation of the IAS. In the experiments used to examine the influence of L- HO-Arg, the IAS relaxations in response to 0.5, 1 and 2 Hz of EFS in control experiments were 31.0 ± 0.7, 55.9 ± 3.0 and 67.7 ± 3.9%, respectively. Following the arginase pretreatment, the IAS relaxation in response to EFS was significantly suppressed and these values in response to 0.5, 1 and 2 Hz EFS were 12.1 ± 2.5, 28.2 ± 7.3 and 36.6 ± 7.4%, respectively (Figure 18; p<0.05; n = 5). The pretreatment of the tissues with L-HO-Arg (1 x 10"4 M) before the addition of arginase antagonized the effect of arginase in attenuating the EFS-induced IAS relaxation.
The effect of ABHA in antagonizing the arginase-suppressed IAS relaxations was similar to that of L-HO-Arg (Figure 19; p <0.05; n = 5).
Influence of Arginase Inhibitors on the NANC Nerve-Mediated IAS Relaxation in the Presence of the NO synthase Inhibitor L-NNA
It is well known that the NO synthase inhibitor L-NNA causes a marked suppression of IAS relaxation by NANC nerve stimulation. In order to test the selectivity of the arginase inhibitors in the IAS, their effects on the IAS relaxation suppressed by the NO synthase inhibitor L-NNA (3 x 10"" M) were examined. The results obtained were then compared with the reversal of the NANC relaxation of the IAS by the NO synthase substrate L-arginine at different concentrations. Interestingly, the L-NNA-suppressed IAS relaxation was completely reversed by L-HO-Arg (Figure 20). In control experiments, the fall in the basal IAS tension in response to 0.5, 1, 2 and 5 Hz EFS was 26.7 ± 3.6, 46.4 ± 4.0, 64.1 ± 3.0 and 74.8 ± 3.9% respectively. L- NNA caused a significant attenuation of the IAS relaxation to 0 ± 0, 0.6 ± 0.6, 1.8 ± 1.1 and 5.4 ± 2.1%, respectively (p < 0.05; n = 5). The NANC nerve-mediated IAS relaxation in the presence of L-NNA plus L-HO-Arg (3 x 10" M) was indistinguishable from that of control values (p < 0.05; n = 5). In this regard, L-HO-Arg was nearly as potent as L-arginine in causing the reversal (Figure 21).
Interestingly, and in contrast to L-HO-Arg, the newly synthesized arginase inhibitor ABHA (3 x 10 M) failed to cause any reversal of L-NNA- suppressed IAS relaxation (Figure 22; p < 0.05; n = 4).
Influence of the Arginase Inhibitors on IAS Relaxation caused bv NANC Nerve Stimulation
In order to determine the physiological significance of arginase in the gastrointestinal smooth muscle, the effects of arginase inhibitors on the NANC nerve- mediated IAS relaxation were examined. Interestingly, both L-HO-Arg (Figure 23) and ABHA (Figure 24) caused significant and concentration-dependent augmentation of the NANC nerve-mediated IAS relaxation by EFS. This was particularly evident at the lower frequencies of EFS. In control experiments for these series of studies, the fall in the IAS tension with 0.5 and 1 Hz EFS before and after L-H)-Arg (3 x 10"4M) was 35.5 ± 7.0 and 54.7 ± 7.0, and 57.4 ± 4.9, 68.8 ± 4.9%, respectively (p < 0.05; n = 4). Similar data were obtained in experiments with ABHA: 29.3 ± 5.7, 49.6 ± 3.9 and 56.5 ± 7.0, 73.6 ± 3.5%, respectively of the fall in the basal IAS tension before and after the selective arginase inhibitor (1 x 10"4M) (p < 0.05; n = 4).
Basal Levels of Arginase Activity in Different Tissues The comparison of the basal arginase activity of different tissues is given in Figure 25. Among the tissues examined, the liver was found to contain the highest levels of arginase activity (7,400 nmols/min/mg protein), consistent with the role of this tissue in nitrogen metabolism and urea synthesis. Among the nonhepatic tissues tested, the IAS was found to contain the highest levels of arginase activity (7.8 nmols/min/mg protein), while the rectum and brain had lower levels (1.7 nols/min/mg protein). The K-j. values for each of these enzymes were similar, ranging from 1.0 to 1.9 mM. These K^ values are comparable to those for the native and recombinant rat liver enzymes (Cavaili et al., 1994, Biochemistry 33:10652-10657).
Influence of the Arginase Inhibitors Nω-Hvdroxy L-arginine (L-HO- Arg) and 2 (SVAmino-ό-boronohexanoic Acid (ABHA') on the Basal Arginase Activity in Different Tissues
Among different tissues investigated, L-HO-arg was found to be the most potent in inhibiting arginase activity in the liver (Figure 26). The IC^Q values for the inhibition of arginase activity in liver, IAS, rectum and brain homogenates by L- HO-arg were 2.4, 25, 42 and 40 μM respectively. Thus, liver arginase activity was approximately 10-20 fold more sensitive to inhibition by L-HO-Arg than the arginase activities in the other tissues. The ability of ABHA to inhibit arginase activity in the tissues was in striking contrast to the inhibition observed with L-HO-Arg. ABHA was found to be the most potent in inhibiting arginase activity in brain and rectum, followed by IAS and liver; the conesponding IC^Q values were 0.05, 0.05, 0.10 and 0.44 μM for the arginase activities in brain, rectum, IAS and liver, respectively (Figure 27). Inhibition constants for ABHA were estimated by titrating the inhibitor into assay mixtures containing an arginine concentration fixed at the K^. value, and assuming competitive inhibition. These experiments yielded estimated Kj values of 0.018, 0.026, 0.05 and 0.19 μM for the arginase activities in brain, rectum, IAS and liver, respectively. The estimated Kj for ABHA inhibition of the liver enzyme is in good agreement with the K^ of 0.11 μM determined by titration calorimetry. Influence of the NO synthase Inhibitor L-N-Nitro- Arginase (L-NNA, and L-NNA Plus the Arginase Inhibitors on the arginase Activity in Different Tissues
It is well known that L-HO-Arg is not only an intermediate in the biosynthesis of NO, but is also a substrate for NO synthase. Thus, it is possible that the tissue variations in the inhibition of arginase activities by L-HO-Arg could be due to depletion of added L-HO-Arg by conversion to NO. In order to test this possibility, the effects of L-HO-Arg on the arginase activities in the different tissue homogenates were determined in the presence of the NO synthase inhibitor L-NNA. L-NNA at 80 μM, had no effect on the activities of the various arginases in the presence of L-HO-Arg (Figure 28), indicating that depletion of added L-HO-Arg by the action of NO synthase was not a concern in these experiments. Furthermore, the results indicate that the tissue-specific variations in arginase inhibition by L-HO-Arg are likely to result from inherent differences in the arginases expressed in these tissues. The present studies demonstrate that the newly synthesized arginase inhibitor 2 (S)-Amino-6-boronohexanoic Acid (ABHA) is a potent, tissue-selective inhibitor of arginase. The data establish that ABHA was 5, 250, 840, and 800 times more potent than Nω-hydroxy-L-arginine in inhibiting the arginase activity in liver, intemal anal sphincter (IAS), rectum, and brain homogenates, respectively. Among all the tissues examined, ABHA was more potent in inhibiting brain, rectum, and IAS arginase activity than the liver, with estimated Kj values of 0.018, 0.026, and 0.05 μM, respectively, assuming competitive inhibition. Although complete inhibition patterns were not determined, previous studies have shown that ABHA can displace the competitive inhibitor Nω-hydroxy-L-arginine from the rat liver enzyme (Example 2). In contrast to the inhibition results with ABHA, Nω-hydroxy-L-arginine was more potent in inhibiting arginase activity in liver homogenates than in the other tissues (Figure 26).
Two isozymes of arginase have been described in mammals, the hepatic (type I) arginase and non-hepatic type (type II) arginase (Jenkinson et al., 1996, Comp. Biochem. Physiol. 114B:107-132; Buga et al, 1996, Am. J. Physiol. Heart Circ. Physiol. 271: H1988-H1998; Daghigh et al., 1994, Biochem. Biophys. Res. Commun. 202:174-180; Boucher et al., 1994, Biochem. Biophys. Res. Commun. 203:1614-1621; Hecker et al, 1995, FEBS Lett. 359:251-254; Gotoh et al, 1996, FEBS Lett. 395:119- 122). Type I arginase is found predominantly in mammalian liver and red blood cells, while the type II enzyme is thought to be expressed in macrophages, kidney and endothelial cells. Although the expression patterns for the arginase in opossum rectum, brain, IAS and liver are not known, the differential effects of ABHA and Nω-hydroxy- L-arginine on the arginase activities in these tissues are consistent with type II enzyme expression in the non-hepatic tissues.
The higher potency of NQ-hydroxy-L-arginine against hepatic arginase, as compared to the non-hepatic tissue extracts might be related to the ability of Nω- hydroxy-L-arginine to serve as a substrate for NO synthase. Thus, in those tissues expressing high levels of NO synthase, added Nω-hydroxy-L-arginine would be rapidly converted to NO and citrulline, lowering the effective concentration of Nω-hydroxy-L- arginine and lowering arginase inhibition. To assess this possibility, inhibition studies with Nω-hydroxy-L-arginine were repeated in the presence of L-NNA, a known inhibitor of NO synthase. Control experiments established that L-NNA had no effect on the arginase activities of the various tissues. The combination of L-NNA and Nω- hydroxy-L-arginine was no more effective than Nω-hydroxy -L-arginine alone in inhibiting the arginase activities, indicating that the differential inhibition of the arginase activities in liver, brain, rectum and IAS is not due to NO synthase depletion of Nω-hydroxy-L-arginine, and is therefore likely to reflect differences in affinity of the arginase for the inhibitor.
The functional data in the IAS also support the concept that ABHA is more selective in inhibiting arginase as compared to L-HO-Arg. This was evident from the IAS studies in the presence of the NO synthase inhibitor L-NNA. As discussed in introduction, L-HO-Arg may also serve as an NO synthase substrate. The experiments were performed to examine the influence of L-HO-Arg on the NANC relaxation of the IAS that was attenuated by the NO synthase inhibitor. The effects of L-HO-Arg and ABHA in reversing the attenuation of the NANC relaxation, were compared with L- arginine, an authentic substrate for NO synthase. Interestingly, L-HO-Arg caused the reversal of L-NNA-attenuated NANC relaxation of the IAS with a potency comparable to L-arginine. ABHA on the other hand had no effect on the IAS relaxation suppressed by the NO synthase inhibitor. This suggests that L-HO-Arg may in part be a substrate for NO synthase and that it may be less selective than ABHA in inhibiting arginase. Before the present study, there has been only limited information on the physiological relevance of arginase in the NANC relaxation in the gastrointestinal smooth muscle. The present data showed that the arginase inhibitors, (L-HO-Arg and ABHA), caused an augmentation of the NANC relaxation of the IAS. Since NO synthase pathway is the predominant pathway responsible for the NANC relaxation of the IAS (Rattan and Chakder, 1992, Am. J. Physiol. Gastrointest. Liver Physiol. 262:G107-G112; Rattan et al., 1992, Gastroenterology 103:43-50), the augmentation of the IAS relaxation is speculated to be due to the upregulation of the NO synthase pathway by an increase in the tissue levels of L-arginine. Interestingly, it has previously been established that exogenously administered L-arginine in the normal tissues has no significant effect on the NANC relaxation of the IAS unless the tissues are made L-arginine deficient (Chakder and Rattan, 1997, J. Pharmacol. Exp. Ther. 282:378-384). In the basal state, in the normal tissues, exogenous L-arginine has no significant effect on either the basal IAS tone or the NANC relaxation (Chakder and Rattan, 1997, J. Pharmacol. Exp. Ther. 282:378-384). Conversely, in the L-arginine deficient tissues, exogenous L-arginine causes a significant fall in the basal IAS tone and reversal of the impaired NANC relaxation in the IAS smooth muscle ((Chakder and Rattan, 1997, J. Pharmacol. Exp. Ther. 282:378-384; Rattan and Chakder, 1997, Gastroenterology 112:1250-1259). It is possible that the effect of exogenous L- arginine in the L-arginine deficient tissues is due to an increase in the uptake of the amino acid leading to an augmentation of the NANC relaxation via the upregulation of NO synthase pathway. Such mechanisms may not be operative in the normal tissues due to the normal state of equilibrium of L-arginine levels at the cellular levels. The augmentation of the NANC relaxation in the presence of arginase inhibitors may be due to the increase in the intracellular L-arginine levels as proposed in other nongastrointestinal tissues (Jenkinson et al., 1996, Comp. Biochem. Physiol. 114b: 107- 132; Buga et al., 1996, Am. J. Physiol. Heart Circ. Physiol. 271:H1988-H1998; Boucher et al., 1994, Biochem. Biophys. Res. Commun. 203:1614-1621; Hecker et al., 1995, FEBS Lett. 359:251-254). Surprisingly, in contrast to the potencies in the arginase inhibitory activity in the IAS, where ABHA was found to be -250 more potent than L-HO-Arg, in the smooth muscle NANC relaxation experiments, the potencies of these two agents were approximately similar in augmenting the NANC relaxation. There is a plausible explanation for these findings. In the functional data, the smooth muscle relaxation is the final outcome of the multiple pathways that involve not only arginase but also NO synthase. The net IAS smooth muscle relaxation in response to NANC nerve stimulation in the presence of inhibitors is the result of their interaction with different pathways available to them. L-HO-Arg acts at both the levels, as an arginase inhibitor and as a substrate for the NO synthase. ABHA, on the other hand, is selective for arginase inhibition only. Rattan and Chakder (1992, Am. J. Physiol. Gastrointest. Liver Physiol. 262:G107-G112), Rattan et al. (1992, Gastroenterology 103:43-50), Chakder and Rattan (1993, Am. J. Physiol. Gastrointest. Liver Physiol. 264:G7-G12) and others (Tottruo et al, 1992, Gastroenterology 102:409-415; O'Kelly et al., 1993, Gut 34:689-693) have shown that the NO synthase pathway is the predominant pathway for the NANC nerve-mediated smooth muscle relaxation. Therefore, the augmentation of the NANC relaxation in the IAS by L-HO-Arg may be due to the summation of its effects in causing an increase in the intracellular concentrations of L- arginine, as an NO synthase substrate plus the arginase inhibition. It is reasonable therefore that in the final analysis, the potencies of the ABHA and L-HO-Arg in causing an augmentation of the IAS smooth muscle relaxation did not turn out to be significantly different. In the enzymatic assays, the comparison is straightforward since one is only examining their influence on the arginase activity. The increase in the levels of arginase has been associated with a number of pathological conditions including gastric cancer (Wu et al, 1992, Life Sci. 51:1355-1361; Leu and Wang, 1992, Cancer 70:733-736; Straus et al, 1992, Clin. Chim. Acta 210:5-12; Ikemoto et al., 1993, Clin. Chem. 39:794-799; Wu et al, 1994, Dig. Dis. Sci 39:1107-1112). Additionally, elevated arginase levels following human orthotopic liver transplantation have been shown to cause pulmonary hypertension and reduced hepatic blood flow (Langle et al., 1997, Transplantation 63:1225-1233; Langle et al., 1995, Transplantation 59:1542-1549). Higher blood levels of arginase have been found in patients with various tumors (Wu et al., 1992, Life Sci. 51 :1355-1361; Leu and Wang, 1992, Cancer 70:733-736; Straus et al., 1992, Clin. Chim. Acta 210:5-12; Wu et al. 1994, Dig. Dis. Sci., 39:1107-1112; Paranuli and Singh, 1996, Cancer Lett., 107:249-256) or in certain forms of hepatic injury (Ikemoto et al., 1993, Clin. Chem. 39:794-799). Arginase inhibitors therefore may have significant role in the pathophysiology and potential therapy in a number of disease conditions.
In addition to its therapeutic potentials, the newly described arginase inhibitor ABHA may play a novel role in the identification of isozyme specific arginase pathways and their role as therapeutic potentials for the specific control of the hemodynamic effects associated with the unregulated arginase activity. The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.
While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

What is claimed is:
1. A composition comprising the formula
HOOC-CH(NH2)-YrY2-Y3-Y4-B(OH)2 wherein each of Y , Y2, Y3, and Y is selected from the group consisting of CH2, S, O NH, and N-alkyl, except Y2 is not S when each of Y ^ , Y3, and Y4 is CH2.
2. The composition of claim 1 having the formula HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2.
3. The composition of claim 1, further comprising a pharmaceutically acceptable canier.
4. A composition comprising a pharmaceutical acceptable carrier and a compound having the formula
HOOC-CH(NH2)-YrY2-Y3-Y4-B(OH)2 wherein each of Yj, Y2, Y3, and Y4 is selected from the group consisting of CH2, S, O NH, and N-alkyl.
5. The composition of claim 4, wherein said compound has a formula selected from the group consisting of
HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2 and HOOC-CH(NH2)-CH2-S-CH2-CH2-B(OH)2.
6. A method of inhibiting arginase, said method comprising contacting said arginase with a composition having the formula
HOOC-CH(NH2)-Y1-Y2-Y3-Y4-B(OH)2 wherein each of Yj, Y2, Y3, and Y4 is selected from the group consisting of CH2, S, O NH, and N-alkyl.
7. The method of claim 6, wherein said arginase is yeast arginase.
8. The method of claim 6, wherein said compound has a formula selected from the group consisting of
HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2 and HOOC-CH(NH2)-CH2-S-CH2-CH2-B(OH)2.
9. The method of claim 6, wherein said arginase is mammalian arginase.
10. The method of claim 9, wherein said arginase is human arginase.
11. A method of inhibiting arginase in a mammal, said method comprising administering to said mammal a composition comprising a pharmaceutically acceptable carrier and a compound having the formula
HOOC-CH(NH2)-Y1-Y2-Y3-Y4-B(OH)2 wherein each of Yj, Y2, Y3, and Y4 is selected from the group consisting of CH , S, O NH, and N-alkyl.
12. The method of claim 11 , wherein said compound has a formula selected from the group consisting of
HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2 and HOOC-CH(NH2)-CH2-S-CH2-CH2-B(OH)2.
13. The method of claim 11, wherein said mammal is a human.
14. The method of claim 13, wherein a tissue in said human has an abnormally high level of arginase activity.
15. The method of claim 13, wherein a tissue in said human has an abnormally low level of nitric oxide synthase activity.
16. A method of treating a disease in a human, said method comprising administering to said human a composition comprising a pharmaceutically acceptable carrier and a compound having the formula
HOOC-CH NH^-Yj -Y2-Y3-Y4-B(OH)2 wherein each of Yj, Y2, Y3, and Y4 is selected from the group consisting of CH2, S, O NH, and N-alkyl, and wherein said disease is selected from the group consisting of a disease associated with an abnormally low level of nitric oxide synthase activity in a tissue of said human and a disease associated with an abnormally high level of arginase activity in a tissue of said human.
17. The method of claim 16, wherein said compound has a formula selected from the group consisting of
HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2 and HOOC-CH(NH2)-CH2-S-CH2-CH2-B(OH)2.
18. The method of claim 16, wherein said disease is selected from the group consisting of heart disease, systemic hypertension, pulmonary hypertension, erectile dysfunction, autoimmune encephalomyelitis, chronic renal failure and cerebral vasospasm.
19. A method of relaxing smooth muscle in a mammal, said method comprising administering to said mammal a composition comprising a pharmaceutically acceptable carrier and a compound having the formula
HOOC-CH(NH2)-Y1-Y2-Y3-Y4-B(OH)2 wherein each of Yj, Y2, Y3, and Y4 is selected from the group consisting of CH2, S, O NH, and N-alkyl.
20. The method of claim 19, wherein said compound has a formula selected from the group consisting of
HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2 and HOOC-CH(NH2)-CH2-S-CH2-CH2-B(OH)2.
21. The method of claim 19, wherein said smooth muscle is selected from the group consisting of gasterointestinal smooth muscle, anal sphincter smooth muscle, esophageal sphincter muscle, corpus cavemosum, sphincter of Oddi, arterial smooth muscle, heart smooth muscle, pulmonary smooth muscle, kidney smooth muscle, uterine smooth muscle, vaginal smooth muscle, cervical smooth muscle, placental smooth muscle, and ocular smooth muscle.
22. A method of making a compound having the formula HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2 , said method comprising contacting a molecule of the tert-butyl ester of 2(S)-N-(tert- butyloxycarbonyl)-6-[(l S,2S,3R,5S)-(+)-pinanedioxaboranyl]-hexanoic acid in an organic solvent with BCI3.
23. The method of claim 22, wherein said organic solvent is CH2C12.
24. A method of making a compound having the formula HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2 , said method comprising the steps of
(a) mixing a solution of the tert-butyl ester of 2(S)-N-(tert- butyloxycarbonyl)-glutamic acid in tetrohydrofuran with triethylamine and ethyl chloroformate to produce a first mixture, removing the resulting triethylammonium hydrochloride salt by filtration, and treating the remaining mixture with an aqueous solution of sodium borohydride to provide a first compound, wherein said first compound is the tert-butyl ester of 2(S)-N-(tert-butyloxycarbonyl)-5-hydroxypentanoic acid;
(b) subjecting said first compound to Swem oxidation to produce a second compound;
(c) subjecting said second compound to a Wittig reaction in the presence of triphenylphosphonium methylide to produce a third compound; (d) mixing a solution of BH3 with said third compound in the presence of tetrahydrofuran to produce a second mixture;
(e) adding (lS,2S,3R,5S)-(+)-pinanediol to said second mixture to produce a fourth compound, wherein said fourth compound is the tert-butyl ester of 2(S)-N-(tert-butyloxycarbonyl)-6-[(lS,2S,3R,5S)-(+)-pinanedioxaboranyl]-hexanoic acid; and
(f) mixing said fourth compound with BCI3 in the presence of CH2C12 to produce a compound having the formula
HOOC-CH(NH2)-CH2-CH2-CH2-CH2-B(OH)2 .
25. A method of identifying an arginase inhibitor antagonist, said method comprising the steps of
(a) inducing relaxation of a muscle in vitro;
(b) reversing said relaxation by contacting said muscle with arginase; (c) adding an arginase inhibitor to said muscle so reversed to renew relaxation of said muscle in the presence or absence of a test compound; and
(d) measuring the level of renewed relaxation of said muscle, wherein a lower level of renewed relaxation of said muscle in the presence of said test compound, compared with the level of renewed relaxation of said muscle in the absence of said test compound, is an indication that said test compound is an arginase inhibitor antagonist.
PCT/US1998/021430 1997-10-10 1998-10-09 Compositions and methods for inhibiting arginase activity WO1999019295A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
EP98952229A EP1049660A1 (en) 1997-10-10 1998-10-09 Compositions and methods for inhibiting arginase activity
AU97979/98A AU9797998A (en) 1997-10-10 1998-10-09 Compositions and methods for inhibiting arginase activity
CA002305703A CA2305703A1 (en) 1997-10-10 1998-10-09 Compositions and methods for inhibiting arginase activity
US09/545,737 US6387890B1 (en) 1997-10-10 2000-04-10 Compositions and methods for inhibiting arginase activity
US10/053,939 US6723710B2 (en) 1997-10-10 2002-01-23 Compositions for inhibiting arginase activity
US10/661,965 US20040063666A1 (en) 1998-10-09 2003-09-12 Compositions for inhibiting arginase activity

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US6160797P 1997-10-10 1997-10-10
US60/061,607 1997-10-10

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US09/545,737 Continuation-In-Part US6387890B1 (en) 1997-10-10 2000-04-10 Compositions and methods for inhibiting arginase activity

Publications (1)

Publication Number Publication Date
WO1999019295A1 true WO1999019295A1 (en) 1999-04-22

Family

ID=22036891

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/021430 WO1999019295A1 (en) 1997-10-10 1998-10-09 Compositions and methods for inhibiting arginase activity

Country Status (5)

Country Link
US (2) US6387890B1 (en)
EP (1) EP1049660A1 (en)
AU (1) AU9797998A (en)
CA (1) CA2305703A1 (en)
WO (1) WO1999019295A1 (en)

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6277884B1 (en) 1998-06-01 2001-08-21 Nitromed, Inc. Treatment of sexual dysfunction with N-hydroxyguanidine compounds
US6436997B1 (en) 1998-06-01 2002-08-20 Nitromed, Inc. Endogenous nitric oxide synthesis under conditions of low oxygen tension
WO2004073623A2 (en) 2003-02-14 2004-09-02 Children's Hospital & Research Center At Oakland Treatment of conditions associated with decreased nitric oxide bioavailability, including elevated arginase conditions
US20080021097A1 (en) * 2006-07-21 2008-01-24 Schmitz Harold H Arginase levels/activity
US7648840B2 (en) 2004-12-01 2010-01-19 Children's Hospital & Research Center At Oakland Diagnosis of conditions associated with decreased arginine bioavailability
WO2011133653A1 (en) * 2010-04-22 2011-10-27 Mars, Incorporated Inhibitors of arginase and their therapeutic applications
EP2389352A2 (en) * 2009-01-26 2011-11-30 The Trustees Of The University Of Pennsylvania Arginase inhibitors and methods of use
WO2012058065A1 (en) * 2010-10-26 2012-05-03 Mars Incorporated Boronates as arginase inhibitors
FR2973234A1 (en) * 2011-03-31 2012-10-05 Oreal Use of arginase inhibitor as an agent for slowing down hair loss, eyebrows or eyelashes, and for preventing and treating hair loss of natural origin
US9200011B2 (en) 2012-04-18 2015-12-01 Mars, Incorporated Ring constrained analogs as arginase inhibitors
US9266908B2 (en) 2011-10-19 2016-02-23 Mars, Incorporated Inhibitors of arginase and their therapeutic applications
WO2016108707A1 (en) 2014-12-29 2016-07-07 OncoArendi Therapeutics Sp. z o.o. Arginase inhibitors and their therapeutic applications
WO2017191130A2 (en) 2016-05-04 2017-11-09 Oncoarendi Therapeutics Sa Arginase inhibitors and their therapeutic applications
WO2018089490A1 (en) 2016-11-08 2018-05-17 Calithera Biosciences, Inc. Arginase inhibitor combination therapies
JP2018083833A (en) * 2010-12-31 2018-05-31 アストラゼネカ・ユーケイ・リミテッドＡｓｔｒａＺｅｎｅｃａ ＵＫ Ｌｉｍｉｔｅｄ Arginase inhibitors and methods of use thereof
WO2018119440A1 (en) 2016-12-22 2018-06-28 Calithera Biosciences, Inc. Compositions and methods for inhibiting arginase activity
US10065974B2 (en) 2015-10-30 2018-09-04 Calithera Biosciences, Inc. Compositions and methods for inhibiting arginase activity
US10143699B2 (en) 2015-06-23 2018-12-04 Calithera Biosciences, Inc. Compositions and methods for inhibiting arginase activity
WO2019186497A1 (en) 2018-03-29 2019-10-03 Oncoarendi Therapeutics S.A. Dipeptide piperidine derivatives
US10494339B2 (en) 2017-05-12 2019-12-03 Calithera Biosciences, Inc. Method of preparing (3R,4S)-3-acetamido-4-allyl-N-(tert-butyl)pyrrolidine-3-carboxamide
WO2022137156A1 (en) 2020-12-22 2022-06-30 Oncoarendi Therapeutics S.A. Arginase inhibitors and methods of use thereof
US12054500B2 (en) 2018-03-05 2024-08-06 Arcus Biosciences, Inc. Arginase inhibitors

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6511425B1 (en) * 1997-05-30 2003-01-28 Aerocrine Ab Method for the diagnosis of food intolerance
ATE420656T1 (en) * 2000-05-05 2009-01-15 Univ City COMPOSITIONS FOR STIMULATING REGENERATION AND RESTORATION OF THE NERVOUS SYSTEM BY REGULATING ARGINASE 1 AND POLYAMINE SYNTHESIS
US20040057926A1 (en) * 2002-03-12 2004-03-25 Lsu Medical Center Modulation of the immune response through the manipulation of arginine levels
WO2007005620A2 (en) * 2005-07-01 2007-01-11 The Johns Hopkins University Arginase ii: a target treatment of aging heart and heart failure
US8367412B2 (en) * 2007-02-23 2013-02-05 Kwansei Gakuin Educational Foundation Protein crystallizing agent and method of crystallizing protein therewith
WO2010075863A1 (en) * 2008-12-29 2010-07-08 University Of Tartu (Tartu Ülikool) Arginase inhibitors for the treatment of depression
KR101770981B1 (en) 2009-10-30 2017-08-24 가부시키가이샤 한도오따이 에네루기 켄큐쇼 Logic circuit and semiconductor device
KR20190093705A (en) 2009-11-27 2019-08-09 가부시키가이샤 한도오따이 에네루기 켄큐쇼 Semiconductor device and method for manufacturing the same
DE102010025663A1 (en) 2010-06-30 2012-01-05 Karl-Heinz Glüsenkamp Novel beta-aminoaldehyde derivatives, processes for their preparation and their chemical use as reactive intermediates
WO2012153697A1 (en) 2011-05-06 2012-11-15 Semiconductor Energy Laboratory Co., Ltd. Semiconductor memory device
TWI568181B (en) 2011-05-06 2017-01-21 半導體能源研究所股份有限公司 Logic circuit and semiconductor device
WO2012158518A1 (en) * 2011-05-13 2012-11-22 The Penn State Research Foundation Treatment of renal injury
TWI536502B (en) 2011-05-13 2016-06-01 半導體能源研究所股份有限公司 Memory circuit and electronic device
JP5951351B2 (en) 2011-05-20 2016-07-13 株式会社半導体エネルギー研究所 Adder and full adder
US9135182B2 (en) 2012-06-01 2015-09-15 Semiconductor Energy Laboratory Co., Ltd. Central processing unit and driving method thereof
CL2014002403A1 (en) * 2014-09-11 2015-01-09 Univ Pontificia Catolica Chile Method for enantioselective preparation and purification of 2 (s) -amino-6-boronohexanoic acid (abh) from the dissolution of bpb-ni-gly in tetrahydrofuran, neutralization and subsequent extraction with dichloromethane, dissolution in a C1-C5 alcohol , cooling to achieve bpb precipitation, obtaining abh and its subsequent purification.
US9956240B2 (en) 2014-11-21 2018-05-01 University Of South Carolina Therapeutic monosaccharide-based inhibitors of hexokinase and glucokinase for parasitic diseases, along with methods of their formation and use
US10682359B2 (en) 2017-03-31 2020-06-16 University Of South Carolina Inhibitors of glucose kinases, along with methods of their formation and use
US11059842B2 (en) 2019-04-29 2021-07-13 University Of South Carolina Monosaccharide amine and 3-nitro-2-phenyl-2H-chromene based inhibitors of glucose kinases
US11555047B2 (en) 2019-10-31 2023-01-17 University Of South Carolina One-step synthesis of phosphate-based inhibitors and applications thereof
WO2023283332A1 (en) * 2021-07-07 2023-01-12 Emory University Uses of arginase inhibitors for managing kidney disease and cardiovascular conditions
CN114195724A (en) * 2021-12-16 2022-03-18 深圳市祥根生物医药有限公司 Preparation method of brimonidine tartrate impurity E

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4256108A (en) 1977-04-07 1981-03-17 Alza Corporation Microporous-semipermeable laminated osmotic system
US4160452A (en) 1977-04-07 1979-07-10 Alza Corporation Osmotic system having laminated wall comprising semipermeable lamina and microporous lamina
US4265874A (en) 1980-04-25 1981-05-05 Alza Corporation Method of delivering drug with aid of effervescent activity generated in environment of use
US4477391A (en) * 1981-08-14 1984-10-16 Collins James F Amino acid isomers, their production and their medicinal use
EP0219923B1 (en) 1985-04-30 1992-09-02 Takeda Chemical Industries, Ltd. Antibiotic derivatives, their production and use
JP2531183B2 (en) 1986-06-17 1996-09-04 武田薬品工業株式会社 Tricyclic penam compound, production method and use thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE STN CAPLUS 1 January 1900 (1900-01-01), BAGGIO R, ET AL.: "THE DESIGN OF A TRANSITION STATE ANALOG INHIBITOR, 2(S) -AMINO-6-BORONOHEXANOIC ACID", XP002915706 *
DATABASE STN CAPLUS 1 January 1900 (1900-01-01), DENNIEL V, ET AL.: "HYDROBORATION OF VINYLGLYCINE AND ALLYLGLYCINE AS A ROUTE TO BORON-DERIVATIVES OF .ALPHA.-AMINO ACIDS", XP002915707 *

Cited By (62)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6436997B1 (en) 1998-06-01 2002-08-20 Nitromed, Inc. Endogenous nitric oxide synthesis under conditions of low oxygen tension
US6277884B1 (en) 1998-06-01 2001-08-21 Nitromed, Inc. Treatment of sexual dysfunction with N-hydroxyguanidine compounds
EP2397128A1 (en) * 2003-02-14 2011-12-21 Children's Hospital & Research Center at Oakland Use of L-arginine optionally combined with magnesium for the treatment of sickle cell disease and/or pulmonary hypertension
WO2004073623A2 (en) 2003-02-14 2004-09-02 Children's Hospital & Research Center At Oakland Treatment of conditions associated with decreased nitric oxide bioavailability, including elevated arginase conditions
EP1596854A2 (en) * 2003-02-14 2005-11-23 Children's Hospital &amp; Research Center at Oakland Treatment of conditions associated with decreased nitric oxide bioavailability, including elevated arginase conditions
EP1596854A4 (en) * 2003-02-14 2007-06-06 Childrens Hosp & Res Ct Oak Treatment of conditions associated with decreased nitric oxide bioavailability, including elevated arginase conditions
US7651846B2 (en) 2003-02-14 2010-01-26 Children's Hospital & Research Center At Oakland Treatment and diagnosis of conditions associated with elevated arginase activity
US7648840B2 (en) 2004-12-01 2010-01-19 Children's Hospital & Research Center At Oakland Diagnosis of conditions associated with decreased arginine bioavailability
US8309320B2 (en) 2004-12-01 2012-11-13 Children's Hospital & Research Center At Oakland Diagnosis of conditions associated with decreased arginine bioavailability
US20080021097A1 (en) * 2006-07-21 2008-01-24 Schmitz Harold H Arginase levels/activity
EP2389352A4 (en) * 2009-01-26 2013-02-20 Univ Pennsylvania Arginase inhibitors and methods of use
JP2015205891A (en) * 2009-01-26 2015-11-19 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア arginase inhibitors and methods of use
JP2012515799A (en) * 2009-01-26 2012-07-12 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア Arginase inhibitors and methods of use
US10118936B2 (en) 2009-01-26 2018-11-06 The Trustees Of The University Of Pennsylvania Arginase inhibitors and methods of use
EP2389352A2 (en) * 2009-01-26 2011-11-30 The Trustees Of The University Of Pennsylvania Arginase inhibitors and methods of use
JP2013525364A (en) * 2010-04-22 2013-06-20 マーズ インコーポレイテッド Arginase inhibitors and their therapeutic use
WO2011133653A1 (en) * 2010-04-22 2011-10-27 Mars, Incorporated Inhibitors of arginase and their therapeutic applications
KR101931822B1 (en) * 2010-04-22 2018-12-24 마아즈, 인코오포레이티드 Inhibitors of arginase and their therapeutic applications
US9994594B2 (en) 2010-04-22 2018-06-12 Mars, Incorporated Inhibitors of arginase and their therapeutic applications
US9040703B2 (en) 2010-04-22 2015-05-26 Mars, Incorporated Inhibitors of arginase and their therapeutic applications
US10538537B2 (en) 2010-04-22 2020-01-21 Mars, Incorporated Inhibitors of arginase and their therapeutic applications
AU2011242794B2 (en) * 2010-04-22 2016-11-24 Mars, Incorporated Inhibitors of arginase and their therapeutic applications
RU2586219C2 (en) * 2010-04-22 2016-06-10 Марс, Инкорпорейтед Arginase inhibitors and therapeutic use thereof
CN106008569A (en) * 2010-10-26 2016-10-12 马尔斯公司 Boronates as arginase inhibitors, compositions and applications thereof
WO2012058065A1 (en) * 2010-10-26 2012-05-03 Mars Incorporated Boronates as arginase inhibitors
CN103249737A (en) * 2010-10-26 2013-08-14 马尔斯公司 Boronates as arginase inhibitors
EP3034509A1 (en) * 2010-10-26 2016-06-22 Mars, Incorporated Arginase inhibitors as therapeutics
US11389464B2 (en) 2010-10-26 2022-07-19 Mars, Incorporated Arginase inhibitors as therapeutics
US9440995B2 (en) 2010-10-26 2016-09-13 Mars, Incorporated Arginase inhibitors as therapeutics
US9233985B2 (en) 2010-10-26 2016-01-12 Mars, Incorporated Arginase inhibitors as therapeutics
US10098902B2 (en) 2010-10-26 2018-10-16 Mars, Incorporated Arginase inhibitors as therapeutics
EP3719024A1 (en) * 2010-10-26 2020-10-07 Mars, Incorporated Arginase inhibitors as therapeutics
US10603330B2 (en) 2010-10-26 2020-03-31 Mars, Incorporated Arginase inhibitors as therapeutics
CN103249737B (en) * 2010-10-26 2016-06-08 马尔斯公司 As the borate of arginase inhibitor
JP2013542223A (en) * 2010-10-26 2013-11-21 マーズ インコーポレイテッド Boronates as arginase inhibitors
JP2018083833A (en) * 2010-12-31 2018-05-31 アストラゼネカ・ユーケイ・リミテッドＡｓｔｒａＺｅｎｅｃａ ＵＫ Ｌｉｍｉｔｅｄ Arginase inhibitors and methods of use thereof
FR2973234A1 (en) * 2011-03-31 2012-10-05 Oreal Use of arginase inhibitor as an agent for slowing down hair loss, eyebrows or eyelashes, and for preventing and treating hair loss of natural origin
US9266908B2 (en) 2011-10-19 2016-02-23 Mars, Incorporated Inhibitors of arginase and their therapeutic applications
US9200011B2 (en) 2012-04-18 2015-12-01 Mars, Incorporated Ring constrained analogs as arginase inhibitors
US10329310B2 (en) 2014-12-29 2019-06-25 Onco Arendi Therapeutics S.A. Arginase inhibitors and their therapeutic applications
WO2016108707A1 (en) 2014-12-29 2016-07-07 OncoArendi Therapeutics Sp. z o.o. Arginase inhibitors and their therapeutic applications
US10398714B2 (en) 2015-06-23 2019-09-03 Calithera Biosciences, Inc. Compositions and methods for inhibiting arginase activity
US10143699B2 (en) 2015-06-23 2018-12-04 Calithera Biosciences, Inc. Compositions and methods for inhibiting arginase activity
US10905701B2 (en) 2015-06-23 2021-02-02 Calithera Biosciences, Inc. Compositions and methods for inhibiting arginase activity
US10851118B2 (en) 2015-10-30 2020-12-01 Calithera Biosciences, Inc. Compositions and methods for inhibiting arginase activity
US10065974B2 (en) 2015-10-30 2018-09-04 Calithera Biosciences, Inc. Compositions and methods for inhibiting arginase activity
US10844080B2 (en) 2015-10-30 2020-11-24 Calithera Biosciences, Inc. Compositions and methods for inhibiting arginase activity
JP2019518726A (en) * 2016-05-04 2019-07-04 オンコアレンディ セラピューティクス エス．エー． Arginase inhibitors and their therapeutic applications
WO2017191130A2 (en) 2016-05-04 2017-11-09 Oncoarendi Therapeutics Sa Arginase inhibitors and their therapeutic applications
US11291674B2 (en) 2016-11-08 2022-04-05 Calithera Biosciences, Inc. Arginase inhibitor combination therapies
WO2018089490A1 (en) 2016-11-08 2018-05-17 Calithera Biosciences, Inc. Arginase inhibitor combination therapies
EP3842442A1 (en) 2016-12-22 2021-06-30 Calithera Biosciences, Inc. Compositions and methods for inhibiting arginase activity
WO2018119440A1 (en) 2016-12-22 2018-06-28 Calithera Biosciences, Inc. Compositions and methods for inhibiting arginase activity
US11021495B2 (en) 2016-12-22 2021-06-01 Calithera Biosciences, Inc. Compositions and methods for inhibiting arginase activity
US10287303B2 (en) 2016-12-22 2019-05-14 Calithera Biosciences, Inc. Compositions and methods for inhibiting arginase activity
US12054501B2 (en) 2016-12-22 2024-08-06 Precision Pharmaceuticals, Inc. Compositions and methods for inhibiting arginase activity
US10906872B2 (en) 2017-05-12 2021-02-02 Calithera Biosciences, Inc. Method of preparing (3R,4S)-3-acetamido-4-allyl-n-(tert-butyl)pyrrolidine-3-carboxamide
US10494339B2 (en) 2017-05-12 2019-12-03 Calithera Biosciences, Inc. Method of preparing (3R,4S)-3-acetamido-4-allyl-N-(tert-butyl)pyrrolidine-3-carboxamide
US11370754B2 (en) 2017-05-12 2022-06-28 Calithera Biosciences, Inc. Method of preparing (3R,4S)-3-acetamido-4-allyl-n-(tert-butyl)pyrrolidine-3-carboxamide
US12054500B2 (en) 2018-03-05 2024-08-06 Arcus Biosciences, Inc. Arginase inhibitors
WO2019186497A1 (en) 2018-03-29 2019-10-03 Oncoarendi Therapeutics S.A. Dipeptide piperidine derivatives
WO2022137156A1 (en) 2020-12-22 2022-06-30 Oncoarendi Therapeutics S.A. Arginase inhibitors and methods of use thereof

Also Published As

Publication number Publication date
US20030036529A1 (en) 2003-02-20
EP1049660A1 (en) 2000-11-08
US6387890B1 (en) 2002-05-14
US6723710B2 (en) 2004-04-20
CA2305703A1 (en) 1999-04-22
AU9797998A (en) 1999-05-03

Similar Documents

Publication Publication Date Title
WO1999019295A1 (en) Compositions and methods for inhibiting arginase activity
US7468383B2 (en) Proteasome inhibitors and methods of using the same
Straeter et al. Two-metal ion mechanism of bovine lens leucine aminopeptidase: active site solvent structure and binding mode of L-leucinal, a gem-diolate transition state analog, by X-ray crystallography
US7915236B2 (en) Proteasome inhibitors and methods of using the same
De Simone et al. Carbonic anhydrase inhibitors. Zonisamide is an effective inhibitor of the cytosolic isozyme II and mitochondrial isozyme V: solution and X-ray crystallographic studies
Temperini et al. Carbonic anhydrase activators: X-ray crystal structure of the adduct of human isozyme II with L-histidine as a platform for the design of stronger activators
US7223745B2 (en) Proteasome inhibitors and methods of using the same
JP6152167B2 (en) Ring-restricted analogs as arginase inhibitors
EP1791535B1 (en) Novel tellurium compounds and their use as immunomodulators
Cama et al. Inhibitor coordination interactions in the binuclear manganese cluster of arginase
US20170014434A1 (en) Small Molecule HSP70 Inhibitors
US20040063666A1 (en) Compositions for inhibiting arginase activity
US20130338107A1 (en) Inhibitors of fatty acid amide hydrolase
US8748172B2 (en) Cell modeling of heme deficiency using ferrochelatase mutations
US8802064B2 (en) Inhibitors of fatty acid amide hydrolase

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 97979/98

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2305703

Country of ref document: CA

Ref country code: CA

Ref document number: 2305703

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 09545737

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 1998952229

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1998952229

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1998952229

Country of ref document: EP