Richard M. Shepard

CP-481,715, a Potent and Selective CCR1 Antagonist with Potential Therapeutic Implications for Inflammatory Diseases

The chemokines CCL3 and CCL5, as well as their shared receptor CCR1, are believed to play a role in the pathogenesis of several inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and transplant rejection. In this study we describe the pharmacological properties of a novel small molecular weight CCR1 antagonist, CP-481,715 (quinoxaline-2-carboxylic acid [4(R)-carbamoyl-1(S)-(3-fluorobenzyl)-2(S),7-dihydroxy-7-methyloctyl]amide). Radiolabeled binding studies indicate that CP-481,715 binds to human CCR1 with a Kd of 9.2 nm and displaces 125I-labeled CCL3 from CCR1-transfected cells with an IC50 of 74 nm. CP-481,715 lacks intrinsic agonist activity but fully blocks the ability of CCL3 and CCL5 to stimulate receptor signaling (guanosine 5′-O-(thiotriphosphate) incorporation; IC50 = 210 nm), calcium mobilization (IC50 = 71 nm), monocyte chemotaxis (IC50 = 55 nm), and matrix metalloproteinase 9 release (IC50 = 54 nm). CP-481,715 retains activity in human whole blood, inhibiting CCL3-induced CD11b up-regulation and actin polymerization (IC50 = 165 and 57 nm, respectively) on monocytes. Furthermore, it behaves as a competitive and reversible antagonist. CP-481,715 is >100-fold selective for CCR1 as compared with a panel of G-protein-coupled receptors including related chemokine receptors. Evidence for its potential use in human disease is suggested by its ability to inhibit 90% of the monocyte chemotactic activity present in 11/15 rheumatoid arthritis synovial fluid samples. These data illustrate that CP-481,715 is a potent and selective antagonist for CCR1 with therapeutic potential for rheumatoid arthritis and other inflammatory diseases.

The Human Specific CCR1 Antagonist CP-481,715 Inhibits Cell Infiltration and Inflammatory Responses in Human CCR1 Transgenic Mice

We previously described the in vitro characteristics of the potent and selective CCR1 antagonist, CP-481,715. In addition to being selective for CCR1 vs other chemokine receptors, CP-481,715 is also specific for human CCR1 (hCCR1), preventing its evaluation in classical animal models. To address this, we generated mice whereby murine CCR1 was replaced by hCCR1 (knockin) and used these animals to assess the anti-inflammatory properties of CP-481,715. Cells isolated from hCCR1 knockin mice were shown to express hCCR1 and migrate in response to both murine CCR1 and hCCR1 ligands. Furthermore, this migration is inhibited by CP-481,715 at dose levels comparable to those obtained with human cells. In animal models of cell infiltration, CP-481,715 inhibited CCL3-induced neutrophil infiltration into skin or into an air pouch with an ED50 of 0.2 mg/kg. CP-481,715 did not inhibit cell infiltration in wild-type animals expressing murine CCR1. In a more generalized model of inflammation, delayed-type hypersensitivity, CP-481,715 significantly inhibited footpad swelling and decreased the amount of IFN-γ and IL-2 produced by isolated spleen cells from sensitized animals. It did not, however, induce tolerance to a subsequent challenge. These studies illustrate the utility of hCCR1 knockin animals to assess the activity of human specific CCR1 antagonists; demonstrate the ability of the CCR1 antagonist CP-481,715 to inhibit cell infiltration, inflammation, and Th1 cytokine responses in these animals; and suggest that CP-481,715 may be useful to modulate inflammatory responses in human disease.

Novel quinoline derivatives as inhibitors of bacterial DNA gyrase and topoisomerase IV.

A structurally novel set of inhibitors of bacterial type II topoisomerases with potent in vitro and in vivo antibacterial activity was developed. Dual-targeting ability, hERG inhibition, and pharmacokinetic properties were also assessed.

Concentration of azithromycin in human prostatic tissue

Prostatic tissue was obtained from 36 patients at two study locations and assayed for azithromycin by HPLC or bioassay. The mean concentration of azithromycin in human prostatic tissue (2.54 µg/ml) 14 h after 500 mg oral dosing (two 250 mg doses 12 h apart) was much greater than plasma concentrations (≤ 0.1 µg/ml). Azithromycin was slowly eliminated from prostatic tissue (half-life 60 h) and a mean concentration of 0.62 µg/ml remained 137 h after dosing.

Discovery, synthesis and SAR of azinyl- and azolylbenzamides antagonists of the P2X7 receptor

The discovery, of a series of 2-Cl-5-heteroaryl-benzamide antagonists of the P2X(7) receptor via parallel medicinal chemistry is described. Initial analogs suffered from poor metabolic stability and low Vd(ss). Multi parametric optimization led to identification of pyrazole 39 as a viable lead with excellent potency and oral bioavailability. Further attempts to improve the low Vd(ss) of 39 via introduction of amines led to analogs 40 and 41 which maintained the favorable pharmacology profile of 39 and improved Vd(ss) after iv dosing. But these analogs suffered from poor oral absorption, probably driven by poor permeability.

Discovery of Inhibitors of Human Renin with High Oral Bioavailability

Knowledge of the sequence of a bioactive protein (angiotensinogen) and the availability of a natural product inhibitor lead (pepstatin) were the starting point for discovery of potent penta- and hexapeptide renin inhibitors. Study of the metabolism and disposition of these substances forced the discovery of simpler inhibitors leading to the discovery of oral activity in Terlakiren (22). Modification of physical properties led to the synthesis of aminopiperidine 30, which was identified by oral efficacy profiling. Structural modification to give enzymatic stability produced the bioavailable benzylsuccinate inhibitor 34. Its bioactive monomethylamine metabolite (35, CP-108,671) was subsequently found to have uniformly high oral bioavailability and activity in various species including primates.

The Use of Explant Lens Culture to Assess Cataractogenic Potential

Abstract: Explanted cultures of crystalline lenses have been used to investigate mechanisms of xenobiotic‐induced cataract formation. However, very few studies have utilized mechanistic information to predict the cataractogenic potential of structurally diverse xenobiotics. The present investigation outlines how visual assessment of lens clarity, biochemical endpoints of toxicity, and mechanisms of lenticular opacity formation can be used to select compounds with a lower probability of causing cataract formation in vivo. The rat lens explant culture system has been used to screen thiazolidinediones against ciglitazone for their direct cataractogenic potential in vitro. The two compounds that were selected as development candidates (englitazone and darglitazone) did not produce cataracts in rats exposed daily for 3 months. The culture system has also been used to illustrate that the lens is capable of metabolizing compounds to reactive intermediates. In this example, the toxicity of S‐(1,2‐dichlorovinyl)‐L‐cysteine (DCVC), a model cataractogen, was attenuated by inhibiting lenticular cysteine conjugate β‐lyase metabolism using aminooxyacetic acid. Finally, this model was used retrospectively to investigate the cataractogenic potential of CJ‐12,918 and CJ‐13,454 in rats. These compounds showed differences in the incidence of cataract formation in vivo based on differences in hepatic metabolism and penetration of parent drug and metabolites into the lens. The rank order of cataractogenic potential in vitro correlated better with in vivo results when an induced S9 microsomal fraction was added to the culture media. However, the model did not correctly predict the cataractogenic potential of ZD2138, a structurally similar compound. These studies illustrate the use of explant culture to assess mechanisms of cataract formation and outline its use and limitations for predicting cataractogenic potential in vivo.

Pharmacokinetics of Levonantradol in Laboratory Animals and Man

Abstract: Levonantradol exhibits potent analgetic activity in standard animal tests and appears not to act directly at opiate receptors. It is currently in clinical trials as an analgesic and antiemetic. The pharmacokinetic behavior of levonantradol, administered orally or intramuscularly, has been investigated in man at dose levels of 0.25 to 3.0 mg and in animals at 0.03 to 10 mg/kg, using a specific HPLC/electrochemical assay. Only desacetyllevonantradol is detected in plasma, due to rapid in vivo deacetylation of levonantradol. In man, at the various doses tested, plasma concentrations of desacetyllevonantradol range from < 5 to 51 ng/ml. Comparable plasma levels and clinical activity occur at nearly identical doses after oral and intramuscular dosing, suggesting good oral absorption in man. In animals, desacetyllevonantradol has a time of peak concentration of 1 to 2 hours and an elimination half‐life of 1 to 2 hours. In animals, a tenfold higher oral dose is needed to achieve the same plasma drug levels as that afteran intramuscular dose, suggesting extensive first‐pass metabolism following oral admininstration. Little or no evidence for drug accumulation and sex differences in plasma concentrations of desacetyllevonantradol is noted during chronic intramuscular dosing in laboratory animals.