Effect of tetrachlorodibenzo-p-dioxin (TCDD) on the glucuronidation of retinoic acid in the rat.

Administration of a single oral dose (10 micrograms/kg) of tetrachlorodibenzo-p-dioxin (TCDD) caused a 33% decrease in retinyl esters in the livers of male rats, but a 13-fold increase in retinyl esters in the kidney and a 3-fold increase in serum retinol. Liver and kidney microsomal uridine diphosphoglucuronosyltransferase (UDPGT) activity toward all-trans-retinoic acid was increased 3.7- and 2.6-fold, respectively, ten days following exposure to TCDD. Verification of the in vitro formation of [3H]retinoyl beta-glucuronide (RG) was by cochromatography with authenic RG on reversed phase high pressure liquid chromatography (HPLC), identification of retinoic acid as the hydrolysis product after beta-glucuronidase treatment, and the characterization of the all-trans-retinoyl glucuronide by negative fragment mass spectroscopy, fast atom bobardment. We conclude that increased retinoic acid glucuronidation may be a contributing factor to the hepatic depletion of vitamin A and the increased excretion of vitamin A metabolites following TCDD exposure.

Glucuronidation of all-trans-retinoic acid in liposomal membranes.

Retinoyl beta-D-glucuronide is a biologically active metabolite of retinoic acid. The kinetics of UDP-glucuronosyltransferase-catalyzed biosynthesis of retinoyl beta-D-glucuronide was examined in rat liver and intestinal native microsomes incubated with [3H retinoic acid incorporated into liposomes. The product was identified by cochromatography with authentic all-trans retinoyl beta-D-glucuronide, by hydrolysis with beta-D-glucuronidase, and by mass spectrometry. In vitamin A-sufficient rats the apparent Km values for all-trans-retinoic acid were 173 microM and 125 microM, and the apparent Vmax, 62 and 41 pmol/min per mg, for small intestinal and liver microsomes, respectively. In vitamin A-deficient rats repleted with all-trans-retinyl acetate, the apparent Km (91 microM) and Vmax (53 pmol/min per mg) for intestinal microsomes were in range of those of vitamin A-sufficient rats. The similarities in the kinetic parameters for UDP-glucuronosyltransferase in small intestinal mucosa and liver suggest that the reactions are catalyzed by the same enzyme. In vitamin A-deficient rats given a large amount all-trans-retinoic acid (1.2 mmol/day for 3 days) the apparent Km was 105 microM and Vmax, 127 pmol/min per mg of intestinal microsomal protein. We conclude that the kinetics of intestinal retinoic acid glucuronidation are not characteristic of simple detoxification reactions. Retinoyl glucuronide may be important in mediating retinoic acid metabolism and function.

Antagonism of the osteoclast vitronectin receptor with an orally active nonpeptide inhibitor prevents cancellous bone loss in the ovariectomized rat.

An orally active, nonpeptide Arg-Gly-Asp (RGD) mimetic alpha(v)beta3 antagonist, (S)-3-Oxo-8-[2-[6-(methylamino)-pyridin-2-yl]-1-ethoxy]-2-(2,2,2-trifluoroethyl)-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetic acid (compound 1), has been generated, which prevented net bone loss and inhibited cancellous bone turnover in vivo. The compound binds alpha(v)beta3 and the closely related integrin alpha(v)beta5 with low nanomolar affinity but binds only weakly to the related integrins alpha(IIb)beta3, and alpha5beta1. Compound 1 inhibited alpha(v)beta3-mediated cell adhesion with an IC50 = 3 nM. More importantly, the compound inhibited human osteoclast-mediated bone resorption in vitro with an IC50 = 11 nM. In vivo, compound 1 inhibited bone resorption in a dose-dependent fashion, in the acute thyroparathyroidectomized (TPTX) rat model of bone resorption with a circulating EC50 approximately 20 microM. When dosed orally at 30 mg/kg twice a day (b.i.d.) in the chronic ovariectomy (OVX)-induced rat model of osteopenia, compound 1 also prevented bone loss. At doses ranging from 3 to 30 mg/kg b.i.d., compound 1 partially prevented the OVX-induced increase in urinary deoxypyridinoline. In addition, the compound prevented the OVX-induced reduction in cancellous bone volume (BV), trabecular number (Tb.N), and trabecular thickness (Tb.Th), as assessed by quantitative microcomputerized tomography (microCT) and static histomorphometry. Furthermore, both the 10-mg/kg and 30-mg/kg doses of compound prevented the OVX-induced increase in bone turnover, as measured by percent osteoid perimeter (%O.Pm). Together, these data indicate that the alpha(v)beta3 antagonist compound 1 inhibits OVX-induced bone loss. Mechanistically, compound 1 prevents bone loss in vivo by inhibiting osteoclast-mediated bone resorption, ultimately preventing cancellous bone turnover.

In vitro metabolism of theophylline by rat and human liver tissue.

1. The metabolism of theophylline was studied in the recently developed, precision-cut, liver slice system. Metabolism of theophylline by rat and human liver slices increased over a 6 h incubation period. 2. Rat liver slices produced 1,3-dimethyluric acid, 1-methyluric acid, and uric acid. Human liver slices produced the same metabolite, as well as 3-methylxanthine. 3. Liver slices and microsomes obtained from the Aroclor-1254 pretreated rats metabolized theophylline at a 10-15-fold greater rate than those obtained from control rats. 4. When theophylline was administered orally to the control and Aroclor-1254 pretreated F-344 rats, the same metabolites were present in the urine as were produced in both in vitro systems. Much less theophylline and more metabolites were found in the urine of the Aroclor-1254 pretreated rat. 5. This study demonstrates that precision-cut slices maintained in the roller culture system can serve as a reliable in vitro method to assess the metabolism of xenobiotics.

Reduced ability of rat preantral ovarian follicles to metabolize 4-vinyl-1-cyclohexene diepoxide in vitro.

4-Vinylcyclohexene diepoxide (1-epoxyethyl-3,4-epoxycyclohexane, VCD), an industrial chemical, is a potential health hazard because it destroys oocytes in small preantral follicles in rats. We proposed that VCD destroys oocytes in these follicles because of their reduced capacity to detoxify VCD (convert VCD to tetrol, 4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane). Ovaries, livers, and adrenal glands were collected from immature and mature Fischer 344 rats. Tissues were dissociated and ovarian tissue was separated into distinct follicular fractions. Tissues were incubated with [14C]VCD and media were assayed for [14C]tetrol by HPLC. In immature rats, conversion of VCD to tetrol in large preantral follicles and hepatocytes was 1.5-fold greater than in small preantral follicles and 4-fold greater than in ovarian interstitial cells (p < 0.05). In adults, conversion of VCD to tetrol in large preantral follicles and hepatocytes was, respectively, 3- and 10-fold greater than in small preantral follicles and interstitial cells (p < 0.05). Compared with immature rats, all tissues from adult rats converted more VCD to tetrol (p < 0.05). These data demonstrate that interstitial cells and small preantral follicles from adult and immature rats have a reduced capacity to convert VCD to tetrol compared to large preantral follicles and liver cells. This may explain their increased susceptibility to VCD-induced ovotoxicity. Furthermore, adult rats may be less susceptible to VCD-induced ovotoxicity than immature rats.

Identification of metabolites of octamethylcyclotetrasiloxane (D(4)) in rat urine.

Octamethylcyclotetrasiloxane (D(4)) is an industrial chemical of significant commercial importance. In this study, its major urinary metabolites were identified. The urine samples described here were collected from male and female Fischer rats (F-344) administered [(14)C]D(4) i.v. The metabolite profile was obtained using an HPLC system equipped with a radioisotope detector. HPLC analysis was performed on a C18 column, using an acetonitrile/water mobile phase. The HPLC radiochromatogram revealed two major and at least five minor metabolites. The two major metabolites, constituting 75 to 85% of the total radioactivity, were identified as dimethylsilanediol [Me(2)Si(OH)(2)] and methylsilanetriol [MeSi(OH)(3)]. Formation of MeSi(OH)(3) clearly established demethylation at the silicon-methyl bonds of D(4). No parent D(4) was present in urine. The minor metabolites identified were tetramethyldisiloxane-1,3-diol [Me(2)Si(OH)-O-Si(OH)Me(2)], hexamethyltrisiloxane-1,5-diol [Me(2)Si(OH)-OSiMe(2)-OSi(OH)Me(2)], trimethyldisiloxane-1,3,3-triol [MeSi(OH)(2)-O-Si(OH)Me(2)], dimethyldisiloxane-1,1,3,3-tetrol [MeSi(OH)(2)-O-Si(OH)(2)Me], and dimethyldisiloxane-1,1,1,3,3-pentol [Si(OH)(3)-O-Si(OH)(2)Me]. The structural assignments were based on gas chromatography-mass spectrometry analysis of the tetrahydrofuran metabolite extracts, which were derivatized using bis(trimethylsiloxy)triflouroacetamide, a trimethylsilylating agent. The structures were confirmed by synthesizing (14)C-labeled standards and comparing their HPLC radiochromatograms with the corresponding components in the rat urine. GC-MS spectral comparisons of the trimethylsilylated derivatized standards and urinary components also were made to further confirm their identities. Finally, several of the urinary metabolites were fractionated using HPLC, and GC-MS comparisons were again made for positive structural identification. The pathways for metabolite formation are not yet understood, but a mechanistic hypothesis has been proposed to account for the various metabolites observed thus far.

Extraction of octamethylcyclotetrasiloxane and its metabolites from biological matrices.

Octamethylcyclotetrasiloxane (D4) is an industrial chemical of significant commercial importance. It is a key ingredient in a variety of product formulations and a critical intermediate in the production of silicone polymers. As part of the pharmacokinetic investigation of its disposition and metabolism, an efficient extraction methodology has been developed to recover both the parent D4 and its metabolites from various biological matrices, including blood, plasma, urine, feces, liver, lung, and fat. Of the common organic solvents evaluated (hexane, methylene chloride, diethylether, chloroform, methylisobutylketone, etc.), tetrahydrofuran (THF) was demonstrated to be an excellent choice for extracting both D4 and its metabolites. Test samples were either spiked with 14C-D4 or obtained from animals administered 14C-D4 via various exposure routes (inhalation, i.v., etc.) and then subjected to THF extraction. Recoveries of D4 and its unbound metabolites from plasma were essentially quantitative in three extractions. Even in a single extraction, the recoveries were > 90%. Similar results were obtained for other matrices as well. Extraction efficiencies were measured as follows: lung, 98.2 +/- 0.3%; liver, 95.4 +/- 0.4%; fat, 99.4 +/- 0.8%; urine, 98.1 +/- 0.2%; feces, 94.1 +/- 0.6%. HPLC profiles obtained before and after extraction were essentially identical, further confirming the suitability of THF as an extractant.

SB-239063, a potent and selective inhibitor of p38 map kinase: preclinical pharmacokinetics and species-specific reversible isomerization.

PURPOSE: A series of studies was conducted to evaluate the preclinical pharmacokinetics of SB-239063 (trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl)-5-[(2-methoxy)pyrimidin-4-yl] imidazole), a potent and selective p38 MAP kinase inhibitor. METHODS: SB-239063 was administered both i.v. and p.o. in the rat, dog, cynomolgus monkey, and rhesus monkey, with standard pharmacokinetic parameters generated from the concentration vs. time data. RESULTS: Initial rat studies suggested possible nonlinear disposition, however, assay refinement revealed an in vivo trans-cis isomerization of SB-239063 to a metabolite with nearly identical chromatographic and mass spectral properties. SB-239063 exhibited low to moderate clearance and good bioavailability in the rat and dog, but poor bioavailability in the cynomolgus monkey. Substantial in vivo trans-cis isomerization occurred in the rat and cynomolgus monkey, but occurred to a far lesser extent in the dog. The isomerization reaction was reversible, with a recycled fraction of 0.20 and 0.0003 in the rat and cynomolgus monkey, respectively. In the rhesus monkey, bioavailability was also poor. but no in vivo isomerization was observed. Conclusions. These studies demonstrate the necessity of exercising vigilance in conducting high-throughput analytical method development, and the importance of using a variety of preclinical species when evaluating the disposition of new drug candidates.

SB-242235, a selective inhibitor of p38 mitogen-activated protein kinase. I: preclinical pharmacokinetics.

1. SB-242235 (1-(4-piperidinyl)-4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl) imidazole) is a potent and selective p38 MAP kinase inhibitor that may be an effective therapy for cytokine-mediated diseases such as autoimmune or inflammatory diseases. The present studies were conducted to evaluate the pharmacokinetics of SB-242235 in several preclinical species, including rat, dog and monkey. 2. SB-242235 demonstrates generally favourable pharmacokinetic properties in all species examined. Systemic plasma clearance was high in rat, but in the non-rodent species SB-242235 demonstrated low to moderate clearance with plasma half-lives > 4h. Oral bioavailability in each preclinical species was high. In rat and monkey, SB-242235 demonstrated non-linear elimination kinetics that manifested as a decrease in clearance with increasing dose and apparent oral bioavailability > 100% at high oral doses. Furthermore, SB-242235 displayed concentration-dependent plasma protein binding over a concentration range of 1000-10,000 ng ml(-1). 3. In conclusion, SB-242235 demonstrates high oral bioavailability across the major preclinical species, and may thus be a useful tool compound for investigation of the role of p38 inhibition in various disease states. However, the observations of non-linear protein binding and disposition also suggest the need for caution in the design of and data interpretation from such studies.

Preclinical pharmacokinetics and interspecies scaling of a novel vitronectin receptor antagonist.

Allometric scaling may be used in drug development to predict the pharmacokinetics of xenobiotics in humans from animal data. Although allometry may be successful for compounds that are excreted unchanged or that are oxidatively metabolized (with corrections for metabolic capacity), it has been more challenging for compounds excreted primarily as conjugates in bile. (S)-10, 11-Dihydro-3-[3-(pyridin-2-ylamino)-1-propyloxy]-5H-dibenzo[ a, d]cycloheptene-10-acetic acid (SB-265123) is a novel alphavbeta3 ("vitronectin receptor") antagonist. In this study, the in vivo pharmacokinetics and in vitro plasma protein binding of SB-265123 were examined in four species: mice, rats, dogs, and monkeys. In monkeys and dogs, SB-265123 exhibited moderate clearance, whereas low clearance (<20% hepatic blood flow) was observed in the rat, and high clearance (>70% hepatic blood flow) was seen in the mouse. The concentration-time profiles indicated the possibility of enterohepatic recirculation; subsequent studies in bile duct-cannulated rats demonstrated extensive biliary excretion of an acyl-glucuronide of SB-265123. In allometric scaling to predict the disposition of SB-265123 in humans, various standard correction factors were applied, including protein binding, maximum lifespan potential, and brain weight; each failed to produce adequate interspecies scaling of clearance (r(2) < 0.72). Consequently, a novel correction factor incorporating bile flow and microsomal UDP-glucuronosyltransferase activity in each species was applied, demonstrating substantial improvement in the correlation of the allometric plot (r(2) = 0.96). This study demonstrates a novel allometric correction that may be applicable to compounds that undergo conjugation and biliary excretion.

Design and characterization of orally active Arg-Gly-Asp peptidomimetic vitronectin receptor antagonist SB 265123 for prevention of bone loss in osteoporosis.

The Arg-Gly-Asp (RGD)-binding integrin alpha(V)beta(3) is highly expressed on osteoclasts and has been proposed to mediate cell-matrix adhesion required for osteoclast-mediated bone resorption. Antagonism of this receptor should prevent stable osteoclast adhesion and thereby inhibit bone resorption. We have generated an orally bioavailable, nonpeptide RGD mimetic alpha(v)beta(3) antagonist, SB 265123, which prevents bone loss in vivo when dosed by oral administration. SB 265123 binds alpha(v)beta(3) and the closely related integrin alpha(v)beta(5) with high affinity (K(i) = 3.5 and 1.3 nM, respectively), but binds only weakly to the related RGD-binding integrins alpha(IIb)beta(3) (K(i) >1 microM) and alpha(5)beta(1) (K(i) >1 microM). The compound inhibits alpha(v)beta(3)-mediated cell adhesion with an IC(50) = 60 nM and more importantly, inhibits human osteoclast-mediated bone resorption in vitro with an IC(50) = 48 nM. In vivo, SB 265123 completely blocks bone resorption in a thyroparathyroidectomized rat model of acute bone resorption when dosed at 2.5 mg/kg/h by continuous i.v. infusion. When dosed orally with 3 to 30 mg/kg b.i.d. , in the ovariectomy-induced rat model of osteoporosis, SB 265123 prevents bone resorption in a dose-dependent fashion. This is the first report of an orally active alpha(v)beta(3) antagonist that is effective at inhibiting bone resorption when dosed in a pharmaceutically acceptable fashion. Such a molecule may provide a novel therapeutic agent for the treatment of postmenopausal osteoporosis.