Identification and pharmacological characterization of a novel inhibitor of autotaxin in rodent models of joint pain.

OBJECTIVE: Autotaxin is a secreted lysophospholipase that mediates the conversion of lysophosphatidyl choline (LPC) to lysophosphatidic acid (LPA), a bioactive lipid mediator. Autotaxin levels in plasma and synovial fluid correlate with disease severity in patients with knee osteoarthritis (OA). The goal of this study was to develop and characterize a novel small molecule inhibitor of autotaxin to inhibit LPA production in vivo and determine its efficacy in animal models of musculoskeletal pain. DESIGN: Compound libraries were screened using an LPC coupled enzyme assay that measures the amount of choline released from LPC by the action of autotaxin. Hits from this assay were tested in a plasma assay to assess inhibition of endogenous plasma autotaxin and subsequently tested for their ability to lower plasma LPA levels upon oral dosing of rats. The best compounds were then tested in animal models of musculoskeletal pain. RESULTS: Compound screening led to the identification of compounds with nanomolar potency for inhibition of autotaxin activity. Studies in rats demonstrated a good correlation between compound exposure levels and a decrease in LPA levels in plasma. The leading molecule (compound-1) resulted in a dose dependent decrease in joint pain in the mono-sodium iodoacetate (MIA) and meniscal tear models and a decrease in bone fracture pain in the osteotomy model in rats. CONCLUSION: We have identified and characterized a novel small molecule inhibitor of autotaxin and demonstrated its efficacy in animal models of musculoskeletal pain. The inhibitor has the potential to serve as an analgesic for human OA and bone fracture.

Reversal of resistance in multidrug resistance protein (MRP1)-overexpressing cells by LY329146.

The benzothiophene LY329146 reverses the drug resistance phenotype in multidrug resistance protein (MRP1)-overexpressing cells when dosed in combination with MRP1-associated oncolytics doxorubicin and vincristine. Additionally, LY329146 inhibited MRP1-mediated uptake of the MRP1 substrate LTC4 into membrane vesicles prepared from MRP1-overexpressing cells.

4,5-Diaryloxazole inhibitors of cyclooxygenase-2 (COX-2).

A series of methysulfonyl or sulfonamido substituted 4,5-diaryloxazole were prepared and evaluated for their ability to inhibit the inducible form of cyclooxygenase (COX-2) in vitro and in vivo. Several unique substitution patterns were identified that led to potent and selective inhibitors of COX-2. In general, 2-trifluoromethly-4,5-diaryloxazoles substituted with a methylsulfonyl or sulfonamido group were particularly potent inhibitors. One of the more potent compounds with a selectivity for COX-2 of about 800 fold was 4b (SC-299). SC-299, a highly fluorescent molecule, may be useful for spectroscopic studies on preferential inhibitor binding to COX-2.

Liquid chromatography/nuclear magnetic resonance spectroscopy and liquid chromatography/mass spectrometry identification of novel metabolites of the multidrug resistance modulator LY335979 in rat bile and human liver microsomal incubations.

Compound LY335979 is a P-glycoprotein inhibitor currently entering phase I clinical trials for potential reversal of multidrug resistance to cancer chemotherapy. In early exploratory studies, LY335979 was found to be rapidly transformed in incubations with liver microsomes from rats, dogs, monkeys, and humans. Although the parent compound was completely metabolized, no prominent metabolite peaks were observed. One peak did appear early in the time course, but it did not increase over time. In another preliminary experiment, rats were treated iv with [3H]LY335979 (prepared for pharmacology studies), and urine and bile fractions were collected. Analysis of the urine by reverse-phase HPLC with UV and radioactivity detection revealed that almost all of the material eluted with the solvent front. More than half the radioactivity in bile was accounted for by two peaks eluting earlier than the parent compound (the rest eluted at the solvent front). With both bile and the incubations with microsomes, initial attempts to isolate metabolites were not successful. There was also evidence in both systems of products derived from cleavage of LY335979 (by both further metabolism and degradation). LC/NMR was thus used to analyze materials directly in their respective matrices. An N-oxide metabolite (LY389551) formed by oxidation of the quinoline nitrogen was identified in the microsomal incubations; in bile, three glucuronide metabolites were identified, all of which were conjugates of products formed by oxidation of the quinoline ring of LY335979. There have been few reports in the literature of LC/NMR analysis of bile, which is a more complex matrix than either urine or microsomal suspensions. However, the HPLC techniques developed in this work for the HPLC/UV and LC/MS analyses of LY335979 metabolites in the microsomal matrix and in bile proved readily adaptable for LC/NMR. Using a 500-MHz instrument, basic 1H NMR spectra could be obtained in 2-3 hr with approximately 100 ng of material in the LC/NMR microprobe. With approximately 1.5 microg of material injected onto the column, 1H-1H correlation spectroscopy spectra could be acquired overnight. Along with LC/MS data, the LC/NMR technique facilitated direct identification of a number of metabolites of LY335979 at a point at which their identification by traditional methods would not have been pursued.

Pharmacological characterization of LY335979: a potent cyclopropyldibenzosuberane modulator of P-glycoprotein.

The above data indicate that LY335979 displays the following characteristics of an 'ideal modulator' of Pgp-mediated multidrug resistance: high affinity binding to Pgp, high potency for in vitro reversal of drug resistance, high therapeutic index (activity was demonstrated at doses ranging from 1-30 mg/kg) observed in in vivo antitumor efficacy experiments, and a lack of pharmacokinetic interactions that alter the plasma concentration of coadministered oncolytic agents. These desirable features strongly suggest that LY335979 is an exciting new clinical agent to test the hypothesis that inhibition of P-glycoprotein activity will result in reversal of multidrug resistance in human tumors.

Studies on the mechanism of phosphatidylinositol 3-kinase inhibition by wortmannin and related analogs.

Wortmannin, a fungal metabolite, was identified as a potent inhibitor (IC50 = 4.2 nM) of phosphatidylinositol 3-kinase (PI 3-kinase). Due to the importance of PI 3-kinase in several intracellular signaling pathways, structure-activities studies on wortmannin analogs were performed in an effort to understand the structural requirements necessary for PI 3-kinase inhibition. Since wortmannin is an irreversible inhibitor of PI 3-kinase, it was postulated that covalent attachment at the electrophilic C-21 site was a possible mode of action for PI 3-kinase inhibition. We have prepared various wortmannin analogs which address the possibility of this mechanism. Of particular interest are compounds which affect the C-21 position of wortaminnin either sterically or electronically. Our results support the conclusion that nucleophilic addition by the kinase onto the C-21 position of wortmannin is required for inhibition of PI 3-kinase by wortmannin analogs. Additionally, we have prepared several D-ring analogs of wortmannin, and their activities are reported herein. We conclude that the wortmannin D ring is an important recognition site since modifications have such a dramatic effect on inhibitor potency. Finally, the identification of 17beta-hydroxywortmannin represents the first reported subnanomolar inhibitor of PI 3-kinase. These studies, along with in vivo antitumor experiments, suggest that the mechanism of PI 3-kinase inhibition correlates to the associated toxicity observed with wortmannin-based inhibitors of PI 3-kinase.