Prostate effect in dogs with the aldosterone receptor blocker eplerenone.

Eplerenone (Inspra) is an aldosterone receptor antagonist approved for the treatment of hypertension and heart failure after a myocardial infarction. In vitro receptor binding and transactivation studies showed eplerenone had high selectivity for the mineralocorticoid receptor over other steroid receptors (glucocorticoid, androgen, and progesterone). The most sensitive off-target effect of orally administered eplerenone preclinically was prostate atrophy in dogs. Dose-related prostate atrophy was observed at eplerenone dosages ≥15 mg/kg/day for 13 weeks or longer. The no observed adverse effect level (NOAEL) for the prostate effect in dogs was 5 mg/kg/day. The maximal effect was seen by 13 weeks and the atrophy was reversible even after 1 year of daily treatment. An additional study demonstrated dogs with eplerenone-induced prostate atrophy (confirmed by intrarectal ultrasound) had slightly decreased semen volume but no compound-related effects on libido, semen protein content, sperm motility, daily sperm production, or epididymal sperm transit time. Four possible mechanisms for prostate effect were investigated: (1) inhibition of testosterone synthesis and secretion; (2) inhibition of 5α-reductase, the enzyme within the prostate that converts testosterone into the more active growth factor dihydrotestosterone (DHT); (3) competitive antagonism of the androgen receptor; and (4) inhibition of 5α-reductase or competitive antagonism of the androgen receptor by aldosterone, which increased in dogs treated with eplerenone. Data from these studies supported blockade of androgen receptors at suprapharmacological concentrations of eplerenone. Another mineralocorticoid blocker, spironolactone, had greater antiandrogenic activity than eplerenone both in vivo and in vitro, and it has well known clinically significant antiandrogenic effects in humans, whereas eplerenone does not.

Skin permeability and pharmacokinetics of diclofenac epolamine administered by dermal patch in Yorkshire-Landrace pigs.

PURPOSE: This study compared the pharmacokinetic profile, and systemic and local absorption of diclofenac, following dermal patch application and oral administration in Yorkshire-Landrace pigs. PATIENTS AND METHODS: Twelve anesthetized, female, Yorkshire-Landrace pigs were randomized to receive either the dermal patch (FLECTOR(®) patch, 10 × 14 cm; Alpharma Pharmaceuticals, a subsidiary of Pfizer Inc, New York, NY) or 50 mg oral diclofenac (Voltaren(®); Novartis, East Hanover, NJ). Tissue (skin area of 2 × 2 cm and underlying muscles approximately 2-3 cm in depth) and blood (10 mL) samples were collected at timed intervals up to 11.5 hours after initial patch application or oral administration. The concentrations of diclofenac in plasma, skin, and muscle samples were analyzed using validated ultra performance liquid chromatography tandem mass spectrometric methods. RESULTS: Peak systemic exposure of diclofenac was very low by dermal application compared with oral administration (maximum concentration [C(max)] values of 3.5 vs 9640 ng/mL, respectively). Absorption of diclofenac into underlying muscles beneath the dermal patch was sustained, and followed apparently zero-order kinetics, with the skin serving as a depot with elevated concentrations of diclofenac. Concentrations of diclofenac in muscles beneath the patch application site were similar to corresponding tissues after oral administration (C(max) values of 879 and 1160 ng/mL, respectively). In contrast to the wide tissue distribution of diclofenac after oral administration, dermal patch application resulted in high concentrations of diclofenac only on the treated skin and immediate tissue underneath the patch. Low concentrations of diclofenac were observed in the skin and muscles collected from untreated areas contralateral to the site of dermal patch application. CONCLUSION: Dermal patch application resulted in low systemic absorption and high tissue penetration of diclofenac compared with oral administration.

Increased serum enzyme levels associated with kupffer cell reduction with no signs of hepatic or skeletal muscle injury.

Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that is responsible for the survival and proliferation of monocytes and the differentiation of monocytes into macrophages, including Kupffer cells (KCs) in the liver. KCs play an important role in the clearance of several serum enzymes, including aspartate aminotransferase and creatine kinase, that are typically elevated as a result of liver or skeletal muscle injury. We used three distinct animal models to investigate the hypothesis that increases in the levels of serum enzymes can be the result of decreases in KCs in the apparent absence of hepatic or skeletal muscle injury. Specifically, neutralizing M-CSF activity via a novel human monoclonal antibody reduced the CD14(+)CD16(+) monocyte population, depleted KCs, and increased aspartate aminotransferase and creatine kinase serum enzyme levels in cynomolgus macaques. In addition, the treatment of rats with clodronate liposomes depleted KCs and led to increased serum enzyme levels, again without evidence of tissue injury. Finally, in the osteopetrotic (Csf1(op)/Csf1(op)) mice lacking functional M-CSF and having reduced levels of KCs, the levels of serum enzymes are higher than in wild-type littermates. Together, these findings support a mechanism for increases in serum enzyme levels through M-CSF regulation of tissue macrophage homeostasis without concomitant histopathological changes in either the hepatic or skeletal system.

PF-03475952: a potent and neutralizing fully human anti-CD44 antibody for therapeutic applications in inflammatory diseases.

INTRODUCTION: CD44 is a cell adhesion molecule believed to play a critical role in T cell and monocyte infiltration in the inflammatory process. The reduction of CD44 expression or its ability to properly interact with its key ligand, hyaluronic acid (HA), inhibits migration and subsequent activation of cells within sites of inflammation. CD44-deficient mice exhibit decreased disease in a mouse arthritis model. METHODS: Accordingly, we developed PF-03475952, a fully human IgG2 anti-CD44 monoclonal antibody (mAb). RESULTS: Binding of PF-03475952 to CD44 inhibits binding of HA and induces loss of CD44 from the cell surface. PF-03475952 also passed a series of safety pharmacology assays designed to assess the risk of the mAb to bind Fc gamma receptors, stimulate cytokine release from human whole blood, and stimulate cytokine release from peripheral blood mononuclear cells (PBMC) using plate-bound antibodies. The latter assay was designed specifically to evaluate the risk of cytokine storm that had been observed with TGN1412 (immunostimulatory CD28 superagonist mAb). PF-003475952 exhibits high-affinity binding to both human and cynomolgus monkey CD44, but does not cross-react with rodent CD44. Thus, a rat anti-mouse CD44 mAb was used to demonstrate a dose-dependent decrease of disease in mouse collagen-induced arthritis. Importantly, efficacy was correlated with >50% loss of cell surface CD44 on circulating cells. Loss of CD44 expression on CD3+ lymphocytes was monitored following a single dose of PF-03475952 in cynomolgus monkeys as a pharmacodynamic marker. The recovery of CD44 expression was found to be dose-dependent. PF-03475952 doses of 1, 10, and 100 mg/kg reduced CD44 expression below 50% for 218, 373, and >504 hours, respectively. CONCLUSION: Targeting of CD44 is a unique mechanism of action in the treatment of inflammatory diseases and is expected to reduce joint damage induced by inflammatory mediators, resulting in disease modification in inflammatory diseases such as rheumatoid arthritis.

Increased connective tissue extracellular matrix in the op/op model of osteopetrosis.

Mice that are homozygous for the recessive osteopetrosis spontaneous mutation (op/op) develop severe osteopetrosis due to a defect in the production of macrophage colony-stimulating factor (M-CSF) and a deficiency in monocyte-derived osteoclasts. Our study describes a novel soft tissue finding in an osteopetrosis (B6C3Fe a/a-Csf1(op)/J) mouse model. Tissues were obtained from B6C3Fe a/a-Csf1(op)/J mice and age-matched wild-type mice, processed for hematoxylin and eosin sections, and comprehensive light microscopic tissue evaluation was performed. Mutant mice had characteristic traits of op/op deficiency including missing incisors and domed skulls. Histologically, the bone marrow cavity was effaced by interweaving thick bony trabeculae consistent with osteopetrosis. An increase in a finely granular, basophilic interstitial extracellular matrix (ECM) was observed in the subcutaneous connective tissue of the op/op mice when compared with controls. Histochemically, the ECM was negative with periodic acid Schiff and stained dark blue with alcian blue at a pH of 2.5, indicating that it is composed primarily of nonsulfated glycosaminoglycans (GAGs). This work suggests an increased ECM that is composed mainly of GAGs located in the subcutaneous tissue in op/op mice. This increase in ECM may be related to altered matrix production or turnover because of changes in M-CSF production.

Evaluation of the effects of subchronic oral administration of n-butyl maleate in Sprague-Dawley rats.

n-Butyl maleate, also referred to as monobutyl maleate, is an ester of maleic acid, which is used as a counterion in the pharmaceutical industry. While substantial published data exist on short-term treatment, maleic acid-induced renal toxicity in the rat, no toxicity data are available on the monobutyl ester. This study evaluated the oral subchronic nephrotoxicity potential of n-butyl maleate administered to Sprague-Dawley rats (10/males and females/group) at doses of 0 (vehicle control), 10, 30, or 60 mg/kg/d for 2 wk. Statistically significant elevations in organ weights were noted in males at 60 mg/kg/d and included: (a) increases in absolute heart, kidney, and liver weights; (b) increased liver to body weight ratios; and (c) increased heart, kidney, liver, spleen, and epididymides to brain weight ratios. In females, statistically significant increases in organ weights were limited to increases in adrenal to brain weights at > or = 10 mg/kg/d, kidney to brain weights at > or = 30 mg/kg/d, and kidney to body weight and liver to brain weight ratios at 60 mg/kg/d. There were no macroscopic or microscopic pathology changes observed in any of the tissues examined. Importantly, light microscopic examination of the kidney was unremarkable at the end of the 2-wk dosing period with n-butyl maleate. Although lacking a histopathological correlate, resultant increases in organ weights at 60 mg/kg/d might be considered indicative of an adverse effect. However, renal perturbation induced by n-butyl maleate was mild in comparison to maleic acid-induced renal toxicity, which manifested as impaired tubular resorption and necrosis of the proximal tubules at doses > or = 60 mg/kg/d. The no-observed-adverse-effect level (NOAEL) for the study was 30 mg/kg/d.