Comparison of a New Intranasal Naloxone Formulation to Intramuscular Naloxone: Results from Hypothesis-generating Small Clinical Studies.

Easy-to-use naloxone formulations are needed to help address the opioid overdose epidemic. The pharmacokinetics of i.v., i.m., and a new i.n. naloxone formulation (2 mg) were compared in six healthy volunteers. Relative to i.m. naloxone, geometric mean (90% confidence interval [CI]) absolute bioavailability of i.n. naloxone was modestly lower (55%; 90% CI, 43-70% vs. 41%; 90% CI, 27-62%), whereas average (±SE) mean absorption time was substantially shorter (74 ± 8.8 vs. 6.7 ± 4.9 min). The opioid-attenuating effects of i.n. naloxone were compared with i.m. naloxone (2 mg) after administration of oral alfentanil (4 mg) to a separate group of six healthy volunteers pretreated with 240 mL of water or grapefruit juice. The i.m. and i.n. naloxone attenuated miosis by similar extents after water (40 ± 15 vs. 41 ± 21 h*%) and grapefruit juice (49 ± 18 vs. 50 ± 22 h*%) pretreatment. Results merit further testing of this new naloxone formulation.

Nonstationary disposition of valproic acid during prolonged intravenous infusion: contributions of unbound clearance and protein binding.

Circadian variations in disposition have been observed for a variety of agents, including anticonvulsants. Valproic acid (VPA), an anticonvulsant used to control generalized and partial seizures, has exhibited diurnal oscillations in steady-state concentrations during long-term administration to humans and non-human primates. The present study was conducted to assess potential diurnal changes in the disposition of VPA during prolonged i.v. infusion in rats. Animals, maintained on a strict 12-h per day light cycle, were equipped with venous cannulae and an arterial microdialysis probe. VPA was administered as a 50-mg/kg loading dose followed by a 42 mg/kg/h infusion for 70 h. Blood and microdialysate samples were obtained at timed intervals after establishment of steady-state throughout two complete light/dark cycles; and total (serum) and unbound (microdialysate) VPA was determined by gas chromatography. Modest oscillations (6-7 h period) in total and unbound VPA were observed; clearance and binding parameters were not different between light and dark periods. However, unbound clearance increased, and unbound fraction decreased, with time over the course of the infusion. These results suggest that time-dependent changes in VPA disposition occur in rats, although oscillations in steady-state concentrations do not appear to be diurnal in nature.

Effect of mdr1a P-glycoprotein gene disruption, gender, and substrate concentration on brain uptake of selected compounds.

PURPOSE: This study assessed the influence of mdr1a P-glycoprotein (P-gp) gene disruption, gender and concentration on initial brain uptake clearance (Clup) of morphine, quinidine and verapamil. METHODS: Clup of radiolabeled substrates was determined in P-gp-competent and deficient [mdr1a(-/-)] mice by in situ brain perfusion. Brain:plasma distribution of substrates after i.v. administration was determined in both strains. RESULTS: Genetic disruption of mdr1a P-gp resulted in 1.3-, 6.6- and 14-fold increases in Clup for morphine, verapamil and quinidine, respectively. With the exception of small differences for verapamil, gender did not affect Clup. Saturable transport of verapamil and quinidine was observed only in P-gp-competent mice, with apparent IC50 values for efflux of 8.6 +/- 2.3 microM and 36 +/- 2 microM, respectively. Verapamil Clup was approximately 50% higher in mdr1a(+/-) vs. mdr1a(+/+) mice; no such difference was observed for quinidine. In P-gp-competent mice, uptake of verapamil and quinidine was unaffected by organic vehicles. Plasma decreased VER Clup to a greater extent in the presence of P-gp. The influence of P-gp in situ was lower than, but correlated with, the effect in vivo. CONCLUSIONS: P-gp decreases Clup of morphine, verapamil and quinidine in situ with little or no influence of gender, but this effect cannot fully account for the effects of P-gp in vivo. P-gp is the only saturable transport mechanism for verapamil and quinidine at the murine blood-brain barrier. The influence of protein binding on Clup may be enhanced by P-gp-mediated efflux.

Pharmacokinetic and pharmacodynamic implications of P-glycoprotein modulation.

P-glycoprotein (P-gp) is a cell membrane-associated protein that transports a variety of drug substrates. Although P-gp has been studied extensively as a mediator of multidrug resistance in cancer, only recently has the role of P-gp expressed in normal tissues as a determinant of drug pharmacokinetics and pharmacodynamics been examined. P-glycoprotein is present in organ systems that influence drug absorption (intestine), distribution to site of action (central nervous system and leukocytes), and elimination (liver and kidney), as well as several other tissues. Many marketed drugs inhibit P-gp function, and several compounds are under development as P-gp inhibitors. Similarly, numerous drugs can induce P-gp expression. While P-gp induction does not have a therapeutic role, P-gp inhibition is an attractive therapeutic approach to reverse multidrug resistance. Clinicians should recognize that P-gp induction or inhibition may have a substantial effect on the pharmacokinetics and pharmacodynamics of concomitantly administered drugs that are substrates for this transporter.

Probenecid-associated alterations in valproate glucuronide hepatobiliary disposition: mechanistic assessment using mathematical modeling.

The complexity of processes associated with the hepatobiliary disposition of xenobiotics may require a multiexperimental approach, including pharmacokinetic modeling, to assess mechanisms of drug interactions. The objective of this study was to examine the disposition of valproate glucuronide (VG) in the rat isolated perfused liver (IPL), and to determine the mechanisms of interaction with probenecid (PRB). Livers were isolated and perfused with standard techniques, and valproate (VPA) (20 mg) was administered in the absence and presence of PRB (approximately 75 microg/ml). Concentrations of VPA and VG in perfusate and bile were determined at timed intervals. In the absence of PRB, total recovery of VPA and VG in perfusate and bile was approximately 80%; PRB significantly increased this recovery to approximately 100%, suggesting a decrease in oxidative VPA metabolism. Similarly, pharmacokinetic modeling of the IPL data indicated that PRB competitively inhibited formation of oxidative VPA metabolites. PRB also significantly inhibited formation, biliary excretion, and sinusoidal egress of VG. These observations suggest a competitive interaction between PRB and VG for transport across the canalicular and sinusoidal membranes. Despite PRB-associated impairment of VG formation, mathematical modeling of the data revealed that hepatocyte VG concentrations were increased by PRB, presumably due to simultaneous inhibition of VG biliary excretion and sinusoidal egress by PRB. These results demonstrate the utility of pharmacokinetic modeling in elucidating the mechanisms of alteration in the hepatobiliary disposition of xenobiotics.

Uptake and efflux of the peptidic delta-opioid receptor agonist.

P-glycoprotein (P-gp) and organic anion transporting polypeptides (Oatp) are expressed at the blood-brain barrier (BBB). There is little functional evidence for Oatp-mediated transport at the BBB. The peptidic delta opioid-receptor agonist [D-penicillamine(2,5)]-enkephalin (DPDPE) is a substrate of mdr1a P-gp and Oatp2. The present study evaluated the influence of these transporters on brain uptake of DPDPE by in situ perfusion in mice. Brain uptake was increased approximately 12-fold in mice lacking P-gp in the BBB, but the P-gp inhibitor dexverapamil did not increase uptake in P-gp-competent mice. In P-gp-deficient mice, DPDPE uptake was saturable (K(m) approximately 24 mM), and was inhibited by dexverapamil and the Oatp2 substrates digoxin, estradiol-17beta-glucuronide and fexofenadine. These results confirm P-gp-mediated efflux of DPDPE, and suggest functional uptake transport of DPDPE by Oatp, at the murine BBB.

Pharmacokinetics and pharmacodynamics of 7-nitroindazole, a selective nitric oxide synthase inhibitor, in the rat hippocampus.

PURPOSE: This study was conducted to assess the pharmacokinetics and pharmacodynamics of 7-nitroindazole (7-NI), a selective inhibitor of neuronal nitric oxide synthase (NOS). METHODS: Male Sprague-Dawley rats were equipped with peritoneal/ venous cannulae and a microdialysis probe in the hippocampal cortex. Rats received 7-NI in peanut oil (25 mg/kg) ip every 2 h for 14 h or peanut oil alone. Blood samples were obtained at timed intervals for serum 7-NI; brain tissue microdialysate for determination of extracellular 7-NI and NO was obtained every 20 min. A pharmacokinetic-pharmacodynamic model was constructed to evaluate the effects of 7-NI on NOS activity. RESULTS: Consistent with previous reports. NOS activity in controls evidenced circadian variation. These cyclic changes in NO production were incorporated into the model of 7-NI effects on NOS. 7-NI produced a rapid (within 2 h) decrease in hippocampal NO. Under the conditions of this experiment, 7-NI produced an approximately 50% decrease in hippocampal NO, which was sustained during 7-NI administration. The decrease in NOS activity by 7-NI was concentration-dependent with an apparent IC50 of approximately 17 microg/ml. CONCLUSIONS: Multiple ip injections of 7-NI result in a predictable, sustained decrease in NO production in the hippocampus. The pharmacokinetic-pharmacodynamic model developed allows design of dosing regimens that can produce designated changes in brain NO content, facilitating use of 7-NI to probe the pharmacological implications of NO in the central nervous system.

Microdialysis and drug delivery to the eye.

The eye presents unique challenges in both the development of tools for elucidating drug disposition as well as for the development of modes of drug delivery for treatment of ocular diseases. In this paper, we present a discussion of the anatomical and physiological characteristics and limitations present in the eye for microdialysis sampling of endogenous substrates and xenobiotics. To date, over twenty papers describing microdialysis approaches for assessment of ocular drug delivery and endogenous substrate characterization have been published. Although the majority of papers describe sampling of vitreous humor, recent efforts have been directed towards ocular anterior segment sampling using microdialysis. With this approach, an appreciable reduction in animal use has been realized. In addition, simultaneous examination of administered drug and endogenous substrates modulated by the drug is possible with this approach, facilitating construction of ocular pharmacokinetic/pharmacodynamic relationships through use of relevant surrogate markers.

Probenecid-associated alterations in valproic acid pharmacokinetics in rats: can in vivo disposition of valproate glucuronide be predicted from in vitro formation data?

Previous investigations have suggested that probenecid (PRB) alters the in vivo disposition of valproic acid (VPA), perhaps by inhibiting hepatic formation of valproate glucuronide (VG). Because VPA and PRB bind moderately to plasma proteins, protein binding also is a potential locus of interaction. The purpose of this investigation was to determine whether in vitro systems could accurately predict PRB-associated perturbations in the hepatobiliary disposition of VPA and VG in vivo. VPA and PRB were coadministered to rats for 60 min at various infusion rates to examine steady-state VPA disposition. PRB did not alter the binding of VPA in serum or hepatic cytosol. However, PRB decreased the apparent intrinsic clearance of VPA (1.81 +/- 0.58 versus 1.23 +/- 0.23 ml/min; P =.025) by competitively inhibiting VPA elimination. In a separate study, rat hepatic S9 fractions were incubated with VPA (7.2-721 microg/ml) and PRB (0-2850 microg/ml). VG formation (V(max) = 0.80 +/- 0.06 microg/min/mg of protein; K(m) = 173 +/- 28.8 microg/ml) was impaired by PRB in a competitive manner (K(i) = 876 +/- 559 microg/ml), consistent with the in vivo data. Despite inhibition of phase II metabolism of VPA to VG by PRB, the VG biliary excretion rate at similar unbound VPA concentrations in hepatic cytosol was not lower in PRB-treated rats. These results indicate that VG disposition in the presence of PRB cannot be predicted accurately based solely on in vitro inhibition of glucuronidation and emphasize the complexity of processes associated with the hepatobiliary system.

Morphine antinociception is enhanced in mdr1a gene-deficient mice.

PURPOSE: Previous studies have suggested that P-glycoprotein (P-gp) modulates opioid antinociception for selected mu-and delta-agonists. This study was undertaken to assess morphine antinociception in mice lacking the mdr1a gene for expression of P-gp in the CNS. METHODS: Morphine (n = 4-5/group) was administered as a single s.c. dose to mdr1a(-/-) mice (3-5 mg/kg) or wild-type FVB controls (8-10 mg/kg). Tail-flick response to radiant heat, expressed as percent of maximum response (%MPR), was used to determine the antinociceptive effect of morphine. Concentrations in serum, brain tissue, and spinal cord samples obtained immediately after the tail-flick test were determined by HPLC with fluorescence detection. Parallel experiments with R(+)-verapamil, a chemical inhibitor of P-gp, also were performed to further investigate the effect of P-gp on morphine-associated antinociception. RESULTS: Morphine-associated antinociception was increased significantly in the mdr1a(-/-) mice. The ED50 for morphine was > 2-fold lower in mdr1a(-/-) (3.8+/-0.2 mg/kg) compared to FVB (8.8+/-0.2 mg/kg) mice. However, the EC50 derived from the brain tissue was similar between the two mouse strains (295 ng/g vs. 371 ng/g). Pretreatment with R(+)-verapamil produced changes similar to those observed in gene-deficient mice. P-gp does not appear to affect morphine distribution between spinal cord and blood, as the spinal cord:serum morphine concentration ratio was similar between gene-deficient and wild-type mice (0.47+/-0.03 vs. 0.56+/-0.04, p>0.05). CONCLUSIONS: The results of this study are consistent with the hypothesis that P-gp attenuates the antinociceptive action of morphine by limiting the brain:blood partitioning of the opioid.

Pharmacodynamics of beta-blocker modulation of aqueous humor production.

A conscious rabbit model with microdialysis sampling of endogenous aqueous humor ascorbate was developed in order to assess the pharmacodynamics of beta-blocker modulation of aqueous humor production. CMA/20 microdialysis probes were implanted in the anterior chamber of each eye of rabbits (n = 6). After a 2 week recovery period, an i.v. bolus of 14C-ascorbate (20 microCi) was administered. Blood samples and aqueous humor microdialysis probe effluent were collected and analysed for endogenous and 14C-ascorbate to estimate the basal rate of ascorbate blood to aqueous humor secretion (Ro). After a 1 hr washout, each rabbit received a series of three doses of 3H-propranolol (750-3000 microg, 16.5 microCi mg(-1)) every 60 min into the lower cul-de-sac of each eye. Probe effluent was analysed for endogenous ascorbate and 3H-propranolol; ascorbate and propranolol in the iris/ciliary body, vitreous and aqueous was determined at the end of the experiment. Nonlinear least-squares regression analysis of the concentration-time profiles for aqueous humor ascorbate was performed to estimate the change in aqueous humor flow. The average basal aqueous humor ascorbate secretion rate was approximately 48/microg hr(-1). Propranolol (1500 microg) produced significant increases in aqueous humor ascorbate, this observation is consistent with a reduction in aqueous humor production (approximately 47%). Analysis of intraocular tissue ascorbate indicated that propranolol inhibited ascorbate secretion at the 3000 microg dose, the highest dose examined in this study; this inhibition was not observed at the 750 microg or 1500 microg doses. Changes in aqueous humor production precipitated by the administration of beta-adrenergic antagonists can be estimated by measuring changes in aqueous humor ascorbate concentrations in the conscious rabbit. Microdialysis sampling of aqueous humor for endogenous ascorbate provides a relevant analytic tool to estimate modulatory effects of anti-glaucoma drugs on aqueous humor production.

Assessment of ascorbate ocular disposition in the conscious rabbit: an approach using the microdialysis technique.

PURPOSE: This study was performed to evaluate the transport kinetics of ascorbate in aqueous humor of conscious rabbits. METHODS: Following the development of a spectrophotometric assay for ascorbate in serum, aqueous humor and microdialysate, and preliminary studies of ascorbate systemic disposition in the rabbit, microdialysis probes were placed into the anterior chamber of one eye, and the posterior chamber of the contralateral eye, of four New Zealand white rabbits. After a one-month recovery period, conscious rabbits were placed in restraining devices, and marginal ear veins were cannulated for repeat blood sampling and ascorbate administration. A tracer i.v. bolus of (14)C-ascorbate, followed by stepwise increasing i.v. infusions of unlabelled ascorbate, was administered. Estimates of basal ascorbate transport into aqueous were determined by analysis of tracer ( 14)C-ascorbate in microdialysis probe effluent and serum. Kinetic modeling was employed to assess ascorbate disposition during infusion. RESULTS: Systemic disposition of exogenously administered ascorbate was well characterized by a two-compartment model. Kinetic modeling returned physiologically realistic volumes for the posterior chamber, and reliable estimates governing ascorbate flux into, between, and from the posterior and anterior chambers. CONCLUSIONS: In vivo assessment of ascorbate kinetics in aqueous humor and blood of the rabbit was facilitated by the microdialysis technique. Contrary to reports in the literature, ascorbate saturable uptake from blood to aqueous was not observed at physiologic blood concentrations ( approximately 11 to 30 mg/L).

Development of an in situ mouse brain perfusion model and its application to mdr1a P-glycoprotein-deficient mice.

An in situ mouse brain perfusion model predictive of passive and carrier-mediated transport across the blood-brain barrier (BBB) was developed and applied to mdr1a P-glycoprotein (Pgp)-deficient mice [mdr1a(-/-)]. Cerebral flow was estimated from diazepam uptake. Physical integrity of the BBB was assessed with sucrose/inulin spaces; functional integrity was assessed with glucose uptake, which was saturable with a Km of approximately 17 mmol/L and Vmax of 310 mmol x 100 g(-1) x min(-1). Brain uptake of a Pgp substrate (colchicine) was significantly enhanced (two- to fourfold) in mdr1a(-/-) mice. These data suggest that the model is applicable to elucidating the effects of efflux transporters, including Pgp, on brain uptake.

Increased brain P-glycoprotein in morphine tolerant rats.

The objective of this study was to determine whether chronic morphine exposure increased P-glycoprotein in rat brain. Male Sprague-Dawley rats were treated with morphine, saline, or dexamethasone for 5 days. On day 6, antinociceptive effect was measured to evaluate the extent of functional tolerance to morphine. Brain P-glycoprotein was detected by Western blot analysis of whole brain homogenate. Morphine- and dexamethasone-treated rats exhibited decreased antinociceptive response when compared to saline-treated controls. Brain P-glycoprotein was approximately 2-fold higher in morphine-treated rats compared to saline controls based on Western blot analysis. Chronic morphine exposure appears to increase P-glycoprotein in rat brain. P-glycoprotein induction may enhance morphine efflux from the brain, thus reducing morphine's pharmacologic activity. Induction of P-glycoprotein may be one mechanism involved in the development of morphine tolerance.

Pharmacokinetics and protein binding of the selective neuronal nitric oxide synthase inhibitor 7-nitroindazole.

Utilization of nitric oxide (NO) synthase (NOS) inhibitors to probe the role of NO in various central nervous system processes requires use of an inhibitor selective for neuronal NOS, and is facilitated by knowledge of the pharmacokinetics of the inhibitor. The present project was undertaken to elucidate the disposition of the selective neuronal NOS inhibitor 7-nitroindazole (7-NI). A simple, specific HPLC assay was developed with requisite sensitivity to quantitate 7-NI in serum after administration of pharmacologically relevant doses. Further experiments were performed to assess the effects of administered dose on 7-NI disposition. 7-NI displayed marked nonlinearity, consistent with saturable elimination, when administered by ip injection in peanut oil. The nonlinearity was related to total dose, but not to the concentration of 7-NI in the vehicle. Binding of 7-NI in rat serum was concentration-independent and does not contribute to the nonlinearity. Various formulations for iv administration of this water-insoluble compound were evaluated; the optimal vehicle, from the standpoint of 7-NI solubility, appeared to inhibit the clearance of 7-NI from the systemic circulation. Considering the nonlinear disposition of 7-NI, knowledge of the pharmacokinetics of this inhibitor is requisite to designing administration protocols to achieve the desired magnitude and duration of NOS inhibition.

P-glycoprotein-mediated transport of morphine in brain capillary endothelial cells.

Cell accumulation, transendothelial permeability, and efflux studies were conducted in bovine brain capillary endothelial cells (BBCECs) to assess the role of P-glycoprotein (P-gp) in the blood-brain barrier (BBB) transport of morphine in the presence and absence of P-gp inhibitors. Cellular accumulation of morphine and rhodamine 123 was enhanced by the addition of the P-gp inhibitors N-{4-[2-(1,2,3,4-tetrahydro-6,7dimethoxy-2-isoquinolinyl)-ethyl]-phenyl}-9,10-dihydro-5-methoxy-9- carboxamide (GF120918), verapamil, and cyclosporin A. Positive (rhodamine 123) and negative (sucrose and propranolol) controls for P-gp transport also were assessed. Morphine glucuronidation was not detected, and no alterations in the accumulation of propranolol or sucrose were observed. Transendothelial permeability studies of morphine and rhodamine 123 demonstrated vectorial transport. The basolateral to apical (B:A) fluxes of morphine (50 microM) and rhodamine (1 microM) were approximately 50 and 100% higher than the fluxes from the apical to the basolateral direction (A:B), respectively. Decreasing the extracellular concentration of morphine to 0.1 microM resulted in a 120% difference between the B:A and A:B permeabilities. The addition of GF120918 abolished any significant directionality in transport rates across the endothelial cells. Efflux studies showed that the loss of morphine from BBCECs was temperature- and energy-dependent and was reduced in the presence of P-gp inhibitors. These observations indicate that morphine is transported by P-gp out of the brain capillary endothelium and that the BBB permeability of morphine may be altered in the presence of P-gp inhibitors.

Microdialysis evaluation of the ocular pharmacokinetics of propranolol in the conscious rabbit.

PURPOSE: This study was conducted to assess the effects of anesthesia and aqueous humor protein concentrations on ocular disposition of propranolol. METHODS: Rabbits were anesthetized and a microdialysis probe was inserted into the anterior chamber of one eye; the contralateral eye served as a control. At timed intervals after probe placement, a 100-microl sample of aqueous humor was aspirated from each eye to determine protein concentration. In vitro protein binding parameters were used to simulate the impact of protein concentration on propranolol disposition. To assess the influence of anesthesia, probes were implanted in the anterior chamber of each eye. After >5-day stabilization, conscious and anesthetized rabbits (n = 3/group) received a 200-microg topical dose of [3H]DL-propranolol in each eye; propranolol was assayed in probe effluent. RESULTS: Changes in aqueous humor protein concentrations were observed following probe insertion. Simulations demonstrated that the unbound propranolol AUC (approximately 2.4-fold) in aqueous humor should be reduced due to protein influx. Intraocular propranolol exposure in anesthetized rabbits was approximately 8-fold higher than in conscious rabbits, and approximately 1.9-fold higher than in rabbits without a post-surgical recovery period. CONCLUSIONS: Anesthesia and time-dependent aqueous humor protein concentrations may alter ocular pharmacokinetics, and must be taken into account in the design of microdialysis experiments.

Effects of a potent and specific P-glycoprotein inhibitor on the blood-brain barrier distribution and antinociceptive effect of morphine in the rat.

Previous data suggest that the analgesic effect of morphine may be modulated by P-glycoprotein (P-gp) inhibition. The effects of the P-gp inhibitor GF120918 on brain distribution and antinociceptive effects of morphine were examined in a rat cerebral microdialysis model. Pretreatment with GF120918 increased both the area under the concentration-time curve of unbound morphine in brain extracellular fluid (ECF) and morphine-associated antinociception. The area under the concentration-time curve ratio for unbound morphine in brain ECF versus unbound morphine in blood was significantly higher in GF120918-treated rats compared with control rats (1.21 +/- 0.34 versus 0.47 +/- 0.05, respectively; p <.05). Modulation of morphine brain-blood distribution was confirmed by quantitating brain tissue morphine in a separate group of rats; GF120918 increased the brain tissue:serum concentration ratio approximately 3-fold. The half-life of unbound morphine in brain ECF was approximately 3-fold longer in GF120918-treated rats compared with controls (p <.05). The fraction unbound of morphine in whole blood was not altered significantly in the presence of GF120918 (0.651 +/- 0.039) as compared with controls (0.662 +/- 0.035). Concentrations of unbound morphine-3-glucuronide in blood and brain ECF were increased in GF120918-treated rats versus controls. An integrated pharmacokinetic/pharmacodynamic model was developed to characterize the unbound blood and brain ECF morphine concentration profiles and concentration-effect relationships. The results of this study indicate that alteration of morphine antinociception by a potent P-gp inhibitor appears to be mediated at the level of the blood-brain barrier.

Enhanced antinociception of the model opioid peptide [D-penicillamine] enkephalin by P-glycoprotein modulation.

PURPOSE: This study was conducted to examine the influence of P-glycoprotein (P-gp) modulation on the pharmacodynamics of the model opioid peptide DPDPE. METHODS: Mice (n = 5-7/group) were pretreated with a single oral dose of the P-gp inhibitor GF120918 (25 or 250 mg/kg) or vehicle. 3H-DPDPE (10 mg/kg) or saline was administered 2.5 hr after pretreatment. Antinociception was determined, and blood and brain tissue were obtained, 10 min after DPDPE administration. RESULTS: A significant difference (p < 0.001) in DPDPE-associated antinociception was observed among mice pretreated with a 25- (83 +/- 16% MPR) or 250- (95 +/- 5% MPR) mg/kg dose of GF120918 in comparison to mice pretreated with vehicle (24 +/- 14% MPR) or mice receiving GF120918 without DPDPE (12 +/- 8% MPR). A significant difference (p < 0.01) in brain tissue DPDPE concentration also was observed among treatment groups [25 +/- 6 ng/g (vehicle), 37 +/- 11 ng/g (25 mg/kg GF120918), 70 +/- 8 ng/g (250 mg/kg GF120918)]. In contrast, blood DPDPE concentrations were not statistically different between groups (678 +/- 66, 677 +/- 130, and 818 +/- 236 ng/ml for vehicle, GF120918 [25 mg/kg], and GF120918 [250 mg/kg], respectively). A single 100-mg/kg i.p. dose of (+)verapamil increased the brain:blood DPDPE concentration ratio by approximately 70% relative to saline-treated control mice (0.139 +/- 0.021 vs. 0.0814 +/- 0.0130, p < 0.01), a change in partitioning similar to that observed with the low dose of GF120918. These data provide further support for a P-gp-based mechanism of brain:blood DPDPE distribution. CONCLUSIONS: The present study demonstrates that GF120918 modulates blood-brain disposition and antinociception of DPDPE. Coadministration of a P-gp inhibitor with DPDPE may improve the pharmacologic activity of this opioid peptide.

Dose-specific production of chlorinated quinone and semiquinone adducts in rodent livers following administration of pentachlorophenol.

Production of chlorinated quinoid metabolites was investigated in the livers of Sprague-Dawley rats and B6C3F1 mice following single oral administration of pentachlorophenol (PCP) (0-40 mg/kg body weight) and in male Fischer 344 rats, following chronic ingestion of PCP at 1,000 ppm in the diet for 6 months (equivalent to 60 mg PCP/kg body weight/day). Analyses of the rates of adduction in the livers of Sprague-Dawley rats and B6C3F1 mice suggested that the production of tetrachloro-1,2-benzosemiquinone (Cl4-1,2-SQ) adducts was proportionally greater at low doses of PCP (less than 4-10 mg/kg body weight) and was 40-fold greater in rats than in mice. Production of tetrachloro-1,4-benzoquinone (Cl4-1,4-BQ) adducts, on the other hand, was proportionally greater at high doses of PCP [greater than 60-230 mg/kg body weight] and was 2- to 11-fold greater in mice than in rats over the entire range of dosages. A mathematical model employed these data to predict the rates of daily adduct production and steady state levels of PCP-derived quinone and semiquinone adducts in rats and mice. To evaluate predictions of the model, levels of PCP-derived adducts at steady state were investigated in the livers of male Fischer 344 rats chronically ingesting 60 mg PCP/kg body weight/day. Levels of total Cl4-1,4-BQ-derived adducts in liver cytosolic proteins (Cp) (22.0 nmol/g) and in liver nuclear proteins (Np) (3.07 nmol/g) were comparable to those of model predictions (15.0 and 3.02 nmol/g for Cp and Np, respectively). Overall, these results suggest that species differences in the metabolism of PCP to semiquinones and quinones were, in part, responsible for the production of liver tumors in mice but not rats in chronic bioassays.