Pharmacokinetic and Pharmacodynamic Characterization of an Oral Lysophosphatidic Acid Type 1 Receptor-Selective Antagonist

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1 /11/ $20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 336, No. 3 Copyright 2011 by The American Society for Pharmacology and Experimental Therapeutics / JPET 336: , 2011 Printed in U.S.A. Pharmacokinetic and Pharmacodynamic Characterization of an Oral Lysophosphatidic Acid Type 1 Receptor-Selective Antagonist J. S. Swaney, C. Chapman, L. D. Correa, K. J. Stebbins, A. R. Broadhead, G. Bain, A. M. Santini, J. Darlington, C. D. King, C. S. Baccei, C. Lee, T. A. Parr, J. R. Roppe, T. J. Seiders, J. Ziff, P. Prasit, J. H. Hutchinson, J. F. Evans, and D. S. Lorrain Amira Pharmaceuticals, San Diego, California Received October 7, 2010; accepted December 14, 2010 Introduction Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that signals through a family of six known G proteincoupled receptors, designated LPA 1 6 (Yanagida et al., 2007; Choi et al., 2010). As its name indicates, LPA 1 was the first LPA receptor to be identified, and LPA 1 expression has since been detected in a variety of tissues (Anliker and Chun, 2004). Via coupling to the downstream G proteins, G i,g q, and G 12/13, LPA 1 regulates a wide range of cellular functions, including proliferation, migration, survival, and differentiation (Moolenaar, 1999; Contos et al., 2000). Gene deletion studies in mice have shown that LPA 1 plays a prominent Article, publication date, and citation information can be found at doi: /jpet ABSTRACT Lysophosphatidic acid (LPA) is a bioactive phospholipid that signals through a family of at least six G protein-coupled receptors designated LPA 1 6. LPA type 1 receptor (LPA 1 ) exhibits widespread tissue distribution and regulates a variety of physiological and pathological cellular functions. Here, we evaluated the in vitro pharmacology, pharmacokinetic, and pharmacodynamic properties of the LPA 1 -selective antagonist AM095 (sodium, {4 -[3-meth- yl-4-((r)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl- 4-yl}-acetate) and assessed the effects of AM095 in rodent models of lung and kidney fibrosis and dermal wound healing. In vitro, AM095 was a potent LPA 1 receptor antagonist because it inhibited GTP S binding to Chinese hamster ovary (CHO) cell membranes overexpressing recombinant human or mouse LPA 1 with IC 50 values of 0.98 and 0.73 M, respectively, and exhibited no LPA 1 agonism. In functional assays, AM095 inhibited LPAdriven chemotaxis of CHO cells overexpressing mouse LPA 1 (IC nm) and human A2058 melanoma cells (IC nm). In vivo, we demonstrated that AM095: 1) had high oral bioavailability and a moderate half-life and was well tolerated at the doses tested in rats and dogs after oral and intravenous dosing, 2) dose-dependently reduced LPA-stimulated histamine release, 3) attenuated bleomycin-induced increases in collagen, protein, and inflammatory cell infiltration in bronchalveolar lavage fluid, and 4) decreased kidney fibrosis in a mouse unilateral ureteral obstruction model. Despite its antifibrotic activity, AM095 had no effect on normal wound healing after incisional and excisional wounding in rats. These data demonstrate that AM095 is an LPA 1 receptor antagonist with good oral exposure and antifibrotic activity in rodent models. physiological role in embryonic development (Yang et al., 2002). However, increasing in vitro and in vivo evidence also demonstrates a pathophysiological role for LPA 1 in lung and kidney fibrosis (Pradère et al., 2007; Tager et al., 2008; Swaney et al., 2010), neuropathic pain (Inoue et al., 2004), and some cancers (Shida et al., 2003; Boucharaba et al., 2006). In patients with idiopathic pulmonary fibrosis, LPA levels are elevated in bronchoalveolar lavage (BALF), and LPA 1 is highly expressed on lung fibroblasts isolated from idiopathic pulmonary fibrosis BALF (Tager et al., 2008). In animals, pulmonary vascular leakage and fibrosis were also attenuated in LPA 1 knockout mice after bleomycin lung injury (Tager et al., 2008). We recently demonstrated that an LPA 1 antagonist reduced lung injury, vascular leakage, inflammation, and fibrosis and decreased BALF concentrations of profibrotic and proinflammatory mediators in the mouse bleo- ABBREVIATIONS: LPA, lysophosphatidic acid; LPA 1, LPA type 1 receptor; hlpa 1, human LPA 1 ; mlpa 1, mouse LPA 1 ; AM095, sodium, {4 -[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetate; BALF, bronchoalveolar lavage fluid; CHO, Chinese hamster ovary; UUO, unilateral ureteral obstruction; C max, maximum plasma concentration; ANOVA, analysis of variance; AM966, (4 -{4-[(R)-1- (2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid; Ki16425 (Debio-0719), 3-[[[4-[4-[[[1-(2-chlorophenyl)ethoxy]carbonyl]amino]-3-methyl-5-isoxazoly]phenyl]methyl]thio]-propanoic acid; VPC12249, (S)-phosphoric acid mono-[3-(4-benzyloxy-phenyl)-2-octadec-9-enoylamino-propyl] ester. 693

2 694 Swaney et al. mycin model (Swaney et al., 2010). In the kidney Pradère et al. (2007) showed that renal tubulointerstitial fibrosis was reduced in LPA 1 -deficient mice by an LPA 1/3 antagonist after unilateral ureteral obstruction (UUO). In addition, the LPA 1/3 antagonist decreased macrophage and myofibroblast markers in kidney tissue and inhibited mrna expression of the profibrotic markers collagen type III, transforming growth factor, and connective tissue growth factor (Pradère et al., 2007). Increased signaling through LPA 1 is believed to regulate neuropathic pain, whereby LPA 1 -dependent activation of the Rho/Rho kinase pathway promotes dorsal root demyelination and associated behavioral allodynia and hyperalgesia (Inoue et al., 2004). Regarding the role of LPA 1 in cancer, LPA 1 expression was increased in several breast cancer cell lines and advanced-stage breast cancers (Li et al., 2009). Moreover, LPA stimulated pancreatic cell migration and increased proliferation of malignant pleural mesothelioma cells, and these effects were inhibited by an LPA 1/3 antagonist (Yamada et al., 2008; Komachi et al., 2009). It was recently shown that LPA released from tumor cells acts as a paracrine stimulator of cell differentiation, tumor growth, and angiogenesis via an LPA 1 - dependent signaling mechanism (Jeon et al., 2010). Therefore, we propose that LPA 1 antagonism may be efficacious in treating a variety of fibrotic, inflammatory, and proliferative diseases. AM095 (sodium, {4 -[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetate) is a selective, orally bioavailable LPA 1 antagonist that is currently in preclinical development for lung and skin fibrosis. Based on mouse calcium flux assays, AM095 has similar LPA 1 potency to that of our recently published LPA 1 antagonist, (4 -{4-[(R)-1-(2-chloro-phenyl)- ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)- acetic acid (AM966) (Swaney et al., 2010). However, AM095 is more than 200-fold more selective for LPA 1 compared with LPA 3, whereas AM966 has only 9-fold greater selectivity for LPA 1. The selectivity profile of AM095 and antifibrotic activity in a mouse skin fibrosis model have been described by Castelino et al. (2010)). Here, we characterize the in vitro pharmacology and in vivo pharmacokinetic and pharmacodynamic properties of AM095 and demonstrate its efficacy in rodent models of LPA-stimulated histamine release, bleomycin-induced lung fibrosis, and UUO-driven kidney injury. Studies were also conducted to address the toxicological implications of antagonizing LPA 1 in the setting of normal wound healing. Materials and Methods Drugs AM095 is a novel LPA 1 receptor antagonist developed by Amira Pharmaceuticals (Fig. 1). AM966 was synthesized as described previously (Swaney et al., 2010), and Debio-0719 [formerly Ki16425 [3-[[[4-[4-[[[1-(2-chlorophenyl)ethoxy]carbonyl]amino]-3- methyl-5-isoxazoly]phenyl]methyl]thio]-propanoic acid]] was synthesized based on previously published structural information (Ohta et al., 2003). In Vitro Potency Screening and Counter Screening Cell Lines. Chinese hamster ovary (CHO)/FlpIn cells from Invitrogen (Carlsbad, CA) were generated that stably express human LPA 1 (hlpa 1 /CHO) or mouse LPA 1 (mlpa 1 /CHO). A2058 human melanoma cells were obtained from the American Type Culture Collection (Manassas, VA.). The hlpa 1 /CHO and mlpa 1 /CHO stable cell lines were grown in Ham s F12 medium plus Glutamax Fig. 1. Structure of AM095. supplemented with 10% fetal bovine serum, penicillin/streptomycin, and 1 mg/ml hygromycin B. The A2058 cells were grown in Dulbecco s modified Eagle s medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. All lines were cultured at 37 C in 5% CO 2. [ 35 S]-GTP S Binding Assay. Assays were conducted using both hlpa 1 /CHO and mlpa 1 /CHO cells. A cell pellet of hlpa 1 /CHO or mlpa 1 /CHO cells was resuspended in 20 ml of ice-cold membrane buffer containing 10 mm HEPES, ph 7.4, 1 mm dithiothreitol, and protease inhibitors (Roche Applied Science, Indianapolis, IN.). Cells were sonicated, and the cell lysate was centrifuged at 2000 rpm for 10 min at 4 C. The supernatant was further centrifuged at 25,000 rpm for 70 min at 4 C. The membrane pellet was resuspended in 5 ml of ice-cold membrane buffer and homogenized using a Potter-Elvehjem tissue grinder (BellCo Glass, Vineland, NJ). Final protein concentration was determined using the Bradford Protein Assay Kit (Bio- Rad Laboratories, Hercules, CA.). Known amounts of AM095 (diluted in dimethyl sulfoxide) or vehicle (dimethyl sulfoxide) were added to 25 to 40 g of hlpa 1 /CHO or mlpa 1 /CHO membranes and 0.1 nm [ 35 S]- GTP S (PerkinElmer Life and Analytical Sciences, Waltham, MA) in buffer (50 mm HEPES, 0.1 mm NaCl, 10 mm MgCl 2,50 g/ml saponin, ph 7.5) containing 0.2% fatty acid-free human serum albumin (Sigma- Aldrich, St. Louis, MO) and 5 M GDP. To test for LPA 1 antagonist activity, the ability of AM095 to inhibit GTP S binding stimulated by 900 nm LPA (18:1; Avanti Polar Lipids, Alabaster, AL) was measured. Alternatively, to test for agonist effects, the ability of AM095 to stimulate GTP S binding in the absence of LPA was measured. Reactions were incubated for 30 min at 30 C, before harvesting membranes onto glass filter binding plates (UniFilter GF/B; PerkinElmer Life and Analytical Sciences) and washing three times with cold buffer containing 50 mm HEPES, ph 7.4, 100 mm NaCl, 10 mm MgCl 2 using a Brandel (Gaithersburg, MD) 96-tip cell harvester. Plates were dried and then cpm were evaluated by using a Packard TopCount NXT microplate scintillation counter (PerkinElmer Life and Analytical Sciences). Chemotaxis Assay. Studies were conducted using A2058 and mlpa 1 /CHO cells as described previously (Swaney et al., 2010). In Vivo Animals. For pharmacokinetic studies, male Sprague-Dawley rats with surgically implanted jugular vein catheters ( g)

3 were purchased from Charles River Laboratories, Inc.(Wilmington, MA). Female CD-1 mice (Harlan; g) were used for histamine release assays. For wound healing studies male rats ( g; noncatheterized) were purchased from Harlan. For bleomycin and UUO studies, female C57BL/6 mice (Harlan; g) were used. Rats (one per cage) and mice (four per cage) were given free access to food and water and allowed to acclimate for at least 7 days before each study. Animals were housed in accordance with the guidelines outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animal protocols were approved by the Amira Pharmaceuticals Institutional Animal Care and Use Committee. Pharmacokinetic Studies. In all studies, animals were fasted 15 to 24 h before dosing. For rats, AM095 was administered intraveneously at a dose of 2 mg/kg in 0.9% saline given as a 1 ml/kg bolus injection into the jugular vein. To determine oral exposure, AM095 was administered as a solution in 0.5% methylcellulose via an oral gavage at a dose of 10 mg/kg in a volume of 3 ml/kg. Blood samples (approximately 300 l of total blood) were taken from each rat via the jugular vein catheter at times up to 24 h postdose (10 11 samples per animal) in potassium EDTA tubes. After each sampling, the catheter was flushed with an equivalent volume of saline. Plasma samples, prepared by centrifugation of whole blood, were stored frozen ( 80 C) before analysis. AM095 was dosed intravenously at 2 mg/kg and orally at 5 mg/kg to male beagle dogs (n 3). Plasma samples were collected and analyzed for AM095 concentration by liquid chromatography/mass spectrometry as described previously (Swaney et al., 2010). Histamine Release. For time-course and dose-response studies, mice received intravenous LPA challenge (3 300 g/mouse in 0.1% fatty acid-free bovine serum albumin) and were then sacrificed at the indicated time points to determine plasma histamine concentrations. For the antagonist studies, mice received AM095 (1 30 mg/kg p.o.) in a volume of 10 ml/kg 2 h before the intravenous LPA (300 g/mouse) challenge. Histamine concentrations in the plasma were determined by enzyme immunoassay (VWR Scientific, West Chester, PA). Drug concentrations in plasma were determined by mass spectrometry. The EC 50 was determined by nonlinear regression of plotting the plasma concentrations versus percentage of inhibition of histamine. Bleomycin Model. Studies were conducted as described previously (Swaney et al., 2010). Ureteral Obstruction Model. Mice underwent UUO or sham surgery to the left kidney. In brief, a longitudinal, upper left incision was performed to expose the left kidney. The renal artery was located and a 6/0 silk thread was passed between the artery and the ureter. The thread was looped around the ureter and knotted three times insuring full ligation of ureter. The kidney was returned to abdomen, the abdominal muscle was sutured, and the skin was closed with staples. The contralateral (right) kidney served as an uninjured control. AM095 (30 mg/kg) or vehicle (water) was given 1 to 4 h before UUO and then b.i.d. thereafter by oral gavage. After 8 days, mice were euthanized using inhaled CO 2, and the kidneys were harvested and cut in half for histopathological and biochemical analysis of fibrosis. To assess fibrosis, half of the kidney was fixed in 10% neutral buffered formalin and stained using Masson s trichrome. The other half of the kidney was frozen at 80 C for subsequent biochemical analysis of collagen content. Collagen Analysis. Soluble collagen concentrations in BALF and kidney tissue were assessed using the Sircol soluble collagen assay kit (Biocolor Ltd., Carrickfergus, UK) according to manufacturer s recommendations. For BALF, 50 l was analyzed according to the manufacturer s recommendations. Data are expressed as micrograms of collagen per milliliter of BALF. For tissue collagen concentrations, kidney tissue was thawed and 1 ml of RIPA lysis buffer (490 ml of phosphate-buffered saline, 5 ml of Nonidet P40, 2.5 g of deoxycholic acid, and 5 ml of 10% SDS containing protease inhibitor) was added to each sample. Tissue was homogenized for 1 min using a Polytron tissue homogenizer (Brinkmann Instruments, Westbury, NY). The homogenate was sonicated at 40 W for 45 to 60 s and Pharmacokinetic Characterization of LPA 1 Antagonist 695 centrifuged at 10,000g for 10 min to remove insoluble debris. Kidney collagen concentrations were expressed as microgram of collagen per milligram of protein. Rat Dermal Wound Healing. Animals were weighed and dosed orally with AM095 (30 mg/kg) or vehicle (water) 1 h before wounding and then daily dosing for 14 days. Based on AM095 drug exposure rats (see Fig. 3A), once daily was used. A single incisional wound and a single exicisional wound were made on the back of each animal while under isoflurane anesthesia. For incisional wounds, a surgical scalpel was used to create a 1-cm-long, full-thickness incision in the dorsal skin of the animal. The incision was placed parallel to the midline along the dorsal skin of the animal. For excisional wounds, an 8-mm, full-thickness skin biopsy punch was made on the back of each animal opposite to the site of the incision. Animals were recovered from the anesthesia and housed individually during the recovery period. The wounds were allowed to heal without further treatment other than test compound or vehicle delivered orally as noted above. Immediately after wounding (day 0) and at specific days afterward a photograph was taken of each wound while animals were under isoflurane anesthesia. Images were digitally analyzed to measure wound healing. At the end of the study (day 14) animals were euthanized with CO 2 inhalation. Wound closure was determined for each subject on days 1, 3, 7, and 14 for rats relative to day 0. Statistical Analyses. All values are expressed as the means S.E.M. Statistical analysis was performed by use of Student s t test for two-group analysis or one-way ANOVA for multiple group comparisons followed by Newman-Keuls post hoc comparisons (Prism; GraphPad Software Inc., San Diego, CA). Statistical significance was set at p Results In Vitro GTP S Binding and Chemotaxis. AM095 was recently described as being a potent and selective LPA 1 receptor antagonist as determined by whole-cell, calcium flux-based assays (Castelino et al., 2010). We further profiled AM095 using both a GTP S binding assay and a cellular chemotaxis assay. AM095 inhibited GTP S binding to human and mouse LPA 1 in a dose-dependent manner, resulting in average IC 50 values of and M, respectively (Fig. 2A). As shown in Fig. 2B, the IC 50 of AM095 in the human LPA 1 GTP S binding assay was comparable with that of our previously published compound AM966 (IC M) and the Debio-0719 compound (IC M) (Swaney et al., 2010). AM095 was tested for agonist activity by incubating increasing concentrations of AM095 ( M) with membranes expressing human LPA 1 and measuring GTP S binding (Fig. 2C). AM095 showed no agonist activity at the LPA 1 receptor at any concentration tested, whereas increasing concentrations of the endogenous ligand, LPA, showed dose-dependent stimulation of GTP S binding with an average EC 50 of 38 nm. To examine the nature of the antagonism at the LPA 1 receptor, AM095 was tested for its ability to antagonize LPA-stimulated GTP S binding over a range of LPA concentrations. Increasing concentrations of AM095 caused a rightward shift in the LPA concentration response curves, lowered the B max, and increased the IC 50. Based on the lowering of the B max in the LPA concentration response curves, AM095 functions as an insurmountable antagonist at the LPA 1 receptor (Fig. 2D). To assess the potency of AM095 in an intact cell functional assay, we measured the effects of AM095 on LPAstimulated chemotaxis using both recombinant and native

4 696 Swaney et al. cells. AM095 inhibited LPA 1 -driven chemotaxis of both human A2058 melanoma cells (IC M; n 7) and mouse LPA 1 /CHO cells (IC M; n 3). In Vivo Pharmacokinetics. The plasma concentration time curves for AM095 in male Sprague-Dawley rats and male beagle dogs after both intravenous and oral administration are shown in Fig. 3. After oral (10 mg/kg) dosing in rats, Fig. 3. Pharmacokinetic profile of AM095 in vivo. A, AM095 plasma concentration curves for male Sprague-Dawley rats after a single oral (PO; 10 mg/kg) or intravenous (IV; 2 mg/kg) dose of AM095. B, plasma concentration curves for beagle dogs after a single oral (5 mg/kg) or intravenous dose (2 mg/kg) of AM095. Fig. 2. [ 35 S]-GTP S binding assay. A, dose-dependent antagonism of LPA-induced GTP S binding to human and mouse LPA 1 -expressing membranes. Human or mouse LPA 1 -containing membranes were incubated with increasing concentrations of AM095 ( M) in the presence of 900 nm LPA and then assayed for inhibition of LPA-stimulated GTP S binding to the membranes. B, human LPA 1 -containing membranes were incubated with increasing concentrations of AM095, AM966, or Debio-0719 in the presence of 900 nm LPA and then assayed for inhibition of LPA-stimulated GTP S binding to the membranes. C, assessment of AM095-mediated agonism of the LPA 1 receptor. Human LPA 1 -containing membranes were incubated with increasing concentrations of LPA ( M) or AM095 ( M) and then assayed for stimulation of GTP S binding. The basal level of GTP S binding averaged cpm and increased to cpm after the addition of LPA. Data are expressed as the percentage increase or decrease compared with control and represent the mean S.E.M. of four to seven separate experiments. D, GTP S binding (cpm) by human LPA 1 -containing membranes showing LPA concentration response curves with increasing concentrations of AM095 (12.5 nm 25 M). AM095 plasma concentrations peaked at 2 h with a C max of 41 M, thereafter decreasing to 10 nm by 24 h. After intravenous (2 mg/kg) dosing, a C max of 12 M was observed within 15 min, which also decreased to approximately 10 nm by 24 h, yielding a t 1/2 of 1.79 h (Fig. 3A). In dogs, a single oral dose of 5 mg/kg yielded a peak plasma concentration of 21 M within 15 min of dosing, which then decreased to 10 nm by 24 h. In contrast, an intravenous dose of 2 mg/kg resulted in a C max of 11 M within 15 min and decreased to 15 nm by 8 h, yielding a t 1/2 of 1.5 h (Fig. 3B). The oral exposure was high for both rats and dogs with a oral bioavailability 100. The detailed pharmacokinetic parameters for AM095 are shown in Table 1. AM095 Inhibits LPA-Stimulated Histamine Release in Mice. Previous studies have shown that histamine release is increased after intravenous LPA administration and this effect is inhibited by a mixed LPA 1/3 antagonist (Hashimoto et al., 2006). Here, we examined the specific role of LPA 1 in histamine release and used the histamine assay as an in vivo pharmacodynamic readout to confirm the LPA 1 -selective action of AM095. As shown in Fig. 4A, LPA stimulated histamine release in a dose-dependent manner, resulting in a nearly 14-fold stimulation at the highest concentration tested (300 g/mouse). AM095 dose-dependently inhibited histamine release with an ED mg/kg and a maximal reduction of 80% at a dose of 30 mg/kg (Fig. 4B). By plotting the percentage inhibition of histamine release versus AM095 plasma concentrations for each individual animal and assuming a maximum response of 80% we generated an EC 50 of 1.5 M (Fig. 4E). This value is in good agreement with the mouse GTP S and mlpa 1 /CHO cell chemotaxis IC 50 values of 0.8 M. AM095 Reduces Lung Collagen, Vascular Leakage, and Inflammation after Bleomycin-Induced Lung Injury. We recently demonstrated that AM966, an LPA 1 receptor antagonist, inhibits fibrosis, inflammation, and vascular leakage at multiple time points after bleomycin-induced lung injury in mice (Swaney et al., 2010). However, based on the improved mouse LPA 1 selectivity of AM095 compared with

5 Pharmacokinetic Characterization of LPA 1 Antagonist 697 TABLE 1 Pharmacokinetic parameters for AM095 in fasted male Sprague-Dawley rats and beagle dogs All values represent the mean for each parameter. The S.E.M. is provided for all studies involving n 3 animals/group. %F is a ratio (calculated using dose-adjusted AUC oral/the dose-adjusted AUC intravenous) and therefore does not have a standard error value. Parameter Rat Dog Dose Route Intravenous (n 2) Oral (n 2) Intravenous (n 3) Oral (n 3) Dose (mg/kg) Area under the curve (h g/ml) Plasma clearance (ml/min/kg) Volume of distribution at steady state (L/kg) t 1/2 (hr) %F Plasma concentration at 5 min ( g/ml) C max ( g/ml) Time to maximum plasma concentration (h) TABLE 2 Fibrosis, vascular leakage, and inflammatory markers in BALF 7 days after bleomycin-induced lung injury Data represent the mean S.E.M. AM095 Dose BALF Collagen BALF Protein AM966, we repeated the 7-day bleomycin studies using AM095. We found that AM095 significantly (p 0.05) decreased BALF collagen and protein at doses of 30 and 60 mg/kg, resulting in an ED 50 of approximately 10 mg/kg for both endpoints (Table 2). Furthermore, significant reductions in both macrophage and lymphocyte infiltration were observed in response to AM095 treatment. These findings confirm that the antifibrotic activity in the lung is related to LPA 1 antagonism and further validate the use of a selective LPA 1 receptor antagonist in this setting. AM095 Reduces Kidney Fibrosis in a Mouse UUO Model. To determine whether inhibition of fibrosis by AM095 would generalize to other tissues, mice underwent UUO and kidney fibrosis was measured after treatment with AM095 (30 mg/kg) for 8 days. Trichrome staining revealed increases in fibrotic tissue in obstructed (Fig. 5Ab) compared with unobstructed (Fig. 5Aa) kidneys of vehicle-treated mice. Fibrosis was decreased in AM095-treated mice (Fig. 5Ad) compared with obstructed mice receiving vehicle treatment Inflammatory Cells in BALF Fig. 4. AM095 inhibits LPA-stimulated histamine release. A, dose-dependent effects of LPA (3 300 g/mouse i.v.) on plasma histamine concentration in mice. B, dose-dependent inhibition of LPA (300 g/mouse i.v.)-stimulated histamine release by AM095 (1 30 mg/kg p.o.) 2 min after LPA challenge. All bar graph data represent the mean S.E.M. of n 3 6 mice per group., p 0.05,, p versus vehicle; ###, p versus LPA alone using one-way ANOVA with Newman-Keuls post test. C, data from B were replotted to generate a curve showing percentage of inhibition of histamine release versus plasma AM095 concentration. Total MØ Polymorphonuclear Leukocytes Lymphocytes mg/kg g/ml * ** * ** ** * * * P 0.05; ** P 0.01 vs. 0 (BLM only) using an unpaired t test. (Fig. 5Ab). No significant changes in collagen deposition were seen in mice receiving AM095 alone (Fig. 5Ac). These findings were confirmed after biochemical measurement of total collagen in kidney tissue (Fig. 5B). AM095 Does Not Affect Normal Wound Healing. LPA is recognized as a modulator of dermal wound healing (Demoyer et al., 2000; Balazs et al., 2001), and LPA receptormediated regulation of fibroblast function is believed to play a role in wound repair (Watterson et al., 2007). Therefore, we investigated the effects of AM095 in rat models of full-thickness incisional and excisional dermal wounding. As shown in Fig. 6, once-daily oral administration of AM095 (30 mg/kg) did not affect the extent or rate of wound closure after either incisional or excisional wounding in rats. Discussion LPA is a pleiotropic lysophospholipid that has gained increasing recognition for its ability to regulate both physiolog-

6 698 Swaney et al. Fig. 5. AM095 reduces kidney fibrosis after unilateral ureteral obstruction. A, representative histopathological images of mice that underwent unilateral ureteral obstruction (b and d) or sham surgery (a and c) and were then treated with vehicle (a and b) or 30 mg/kg AM095 (c and d), orally twice a day for 8 days. Fibrotic tissue is indicated by blue staining of tissue using trichrome. B, mice were then euthanized and the kidneys were harvested for biochemical analysis of fibrosis using the Sircol collagen assay. Total collagen data were expressed as micrograms of collagen per milligram of protein. All values are means S.E.M. of 10 mice/group., p versus unobstructed vehicle; ##, p 0.01 versus obstructed vehicle using two-way ANOVA with Bonferroni post-test. ical and pathological processes. Among the six G proteincoupled receptors that LPA is known to activate (Yanagida et al., 2009; Choi et al., 2010), LPA 1 is one of the most notable. LPA 1 is a member of the endothelial differentiation gene family (Hecht et al., 1996; An et al., 1997) and exhibits widespread tissue distribution with high levels of expression in the brain, heart, small intestine, kidney, ovary, testis, prostate, colon, thymus, and pancreas (Yang et al., 2002; Anliker and Chun, 2004). Furthermore, increased LPA 1 signaling has been implicated in a number of disease processes (Shida et al., 2003; Inoue et al., 2004; Boucharaba et al., 2006; Murph et al., 2008; Pradère et al., 2008; Tager et al., 2008), making this receptor a novel target for therapeutic intervention. However, there are currently no selective LPA 1 antagonists being used clinically. Here, we present pharmacokinetic and pharmacodynamic data for AM095, an orally bioavailable, LPA 1 -selective antagonist that is currently under preclinical investigation for the treatment of pathologies that are driven by LPA 1 activation. As a primary in vitro screen, AM095 was evaluated for its ability to inhibit LPA 1 activation by its endogenous ligand, LPA. Inhibition of LPA 1 activation was measured as a decrease in the LPA-stimulated association of GTP with the receptor/g-protein complex. This assay has previously been used to assess the potency of nonselective LPA receptor antagonists (Ohta et al., 2003). The average IC 50 values for the AM095-mediated inhibition of LPA-stimulated GTP S binding to membranes expressing human or mouse LPA 1 were 0.98 and 0.73 M, respectively. It is noteworthy that AM095 did not demonstrate any agonist effects on LPA 1 at concentrations as high as 30 M. There was a significant level of basal GTP S binding in the absence of exogenous LPA, and this basal level of activity was also inhibited by AM095. At this point, it is unclear whether the basal activity reflects the presence of endogenous LPA in the membrane preparation or is LPA-independent. Studies to address this question are ongoing. It is noteworthy that the nature of antagonism of AM095 differs from the LPA mimetic, (S)-phosphoric acid mono-[3-(4-benzyloxy-phenyl)-2-octadec-9-enoylaminopropyl] ester (VPC12249), in that LPA-stimulated GTP S binding was insurmountable in the presence of AM095 (Heise et al., 2001). It is noteworthy that we also observed insurmountable antagonism with Ki16425 in the GTP S assay and insurmountable antagonism with AM095 and Ki16425 in an LPA 1 -mediated calcium flux assay (data not shown). There are several proposed mechanisms for insurmountable antagonism that include (but are not limited to) allosteric binding, slow dissociation of antagonist-receptor complexes, irreversible binding, or antagonist-mediated conformational changes in the receptors rendering them refractory to agonist stimulation. We are currently performing experiments to further understand the nature of this insurmountable antagonism. Studies have shown that LPA stimulates the migration or chemotaxis of multiple human cancer cell lines through an LPA 1 receptor-dependent mechanism and that this process critically depends on G i -coupled Rac1 activation (Hama et al., 2004). Moreover, G i coupling mediates LPA-stimulated chemotaxis of CHO cells recombinantly expressing the human LPA 1 receptor (Sugimoto et al., 2006). Therefore, as a secondary functional readout, we measured the ability of AM095 to inhibit LPA-driven cell chemotaxis. AM095 inhibited chemotaxis of human A2058 melanoma cells and mouse LPA 1 / CHO cells, providing further confirmation of the in vitro potency and the functional antagonism of LPA 1 by AM095. This is of particular importance in light of previous findings that showed that an LPA 1/3 antagonist (Ki16425) blocked migration of various cancer cells in vitro (Hama et al., 2004) and increased LPA 1 activation and expression potentiated the metastasis of cancer cells to bone (Boucharaba et al., 2004, 2006). The pharmacokinetic profile of AM095 was assessed in male Sprague-Dawley rats and male beagle dogs (see Fig. 3 and Table 2). AM095 was well absorbed, had a moderate half-life, and was well tolerated after oral dosing in both species. We observed some variation in the time to maximum plasma concentration between species after oral dosing. However, the time to peak plasma concentration can vary because of fasted/fed conditions, stress, transit time, and other factors and was not directly investigated in these studies. As a pharmacodynamic readout of LPA 1 antagonism, we examined the ability of AM095 to inhibit histamine release in mice after an intravenous administration of LPA. Hashimoto et al. (2006) have previously shown that LPA-stimulated histamine release in mice depends on LPA 1 and/or LPA 3.

7 Pharmacokinetic Characterization of LPA 1 Antagonist 699 Fig. 6. AM095 has no effect on normal wound healing. A and B, representative images of dermal wounds on day 0 and 14 days after incisional (A) and excisional (B) wounding in male Sprague-Dawley rats. Rats were dosed orally with vehicle (water) or AM095 (30 mg/kg) 1 h before wound formation and then once daily for 13 days after wound formation. C and E, the time-dependent effects on wound closure were determined for each subject on days 1, 3, 7, and 14. D and F, data are expressed as the percentage wound closure from days 1 to 14. All data represent the mean S.E.M. of five rats/group. AM095 inhibited histamine release in a dose-dependent manner, resulting in an 82% reduction at the 30 mg/kg (b.i.d.) dose. The lack of a 100% reduction by AM095 probably reflects the contribution of other LPA receptors (e.g., LPA 3 ) to the LPA-induced release of histamine. In summary, these findings demonstrate that LPA-stimulated histamine release is mediated primarily through LPA 1 and, in vivo, 30 mg/kg AM095 shows maximal functional antagonism of LPA 1. We recently established that an LPA 1 antagonist (AM966) decreases pulmonary vascular leakage, inflammation, and fibrosis at multiple time points in a mouse bleomycin model (Swaney et al., 2010). However, AM966 has only 9-fold greater selectivity for LPA 1 versus LPA 3 in mice, whereas AM095 is over 200-fold more selective for LPA 1 (Castelino et al., 2010; Swaney et al., 2010). For this reason, we investigated the effects of AM095 on the development of lung fibrosis in a 7-day bleomycin model. By repeating the 7-day bleomycin studies using AM095 we were able to confirm that the reduction in vascular leakage, inflammation, and fibrosis observed in the AM966 studies (Swaney et al., 2010) was, indeed, related to antagonism of LPA 1 and not LPA 3. Consistent with our previous work, AM095 (1 60 mg/kg) dosedependently decreased BALF collagen and protein concentrations and reduced inflammatory cell infiltration in the lung after bleomycin insult. To explore the efficacy of AM095 to inhibit fibrosis in multiple tissues, we conducted subsequent studies in the UUO kidney fibrosis model. Pradère et al. (2007) showed previously that LPA 1 was a mediator of kidney fibrosis in mice. The antifibrotic effects of AM095 in the kidney were consistent with those observed in the lung, confirming the efficacy of AM095 to reduce fibrosis in multiple organs. A common toxicological concern with antifibrotic agents is whether, via their mode of action, patients will exhibit a delay in normal wound healing or develop nonhealing wounds as a result of their antifibrotic therapy. Studies have shown that topical LPA promotes dermal wound healing (Demoyer et al., 2000; Balazs et al., 2001) and LPA is recognized for its ability to modulate fibroblast function during tissue repair (Watterson et al., 2007). To address concerns regarding the potentially negative effects of AM095 on normal wound healing, we conducted incisional and excisional wounding studies in rats. It is noteworthy that therapeutic doses of AM095 had no effect on wound healing rate or the magnitude of wound closure in rats. Therefore, it seems that

8 700 Swaney et al. perturbing the basal state of LPA 1 activation does not affect the normal wound healing process. In summary, AM095 is a potent, orally bioavailable LPA 1 receptor antagonist with favorable pharmacokinetic properties in rodents and dogs and good pharmacokinetic and pharmacodynamic properties and demonstrated efficacy in two rodent models of tissue fibrosis. Based on the potential role of LPA 1 in cancer, neuropathic pain, and tissue fibrosis, AM095 has exciting potential for treating a range of clinical diseases/ disorders. Authorship Contributions Participated in research design: Swaney, Stebbins, and Lorrain. Conducted experiments: Swaney, Chapman, Correa, Stebbins, Broadhead, Bain, Santini, Darlington, Ziff, and Lorrain. Contributed new reagents or analytic tools: Parr, Roppe, and Seiders. Performed data analysis: Swaney, Stebbins, Bain, King, Baccei, Lee, and Lorrain. 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Biochim Biophys Acta 1582: Address correspondence to. Dr. James Swaney, Amira Pharmaceuticals, 9535 Waples St., San Diego CA james.swaney@amirapharm.com