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.

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.

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.

Effect of prior morphine-3-glucuronide exposure on morphine disposition and antinociception.

Morphine-3-glucuronide (M3G), the primary metabolite of morphine in humans and rats, has been reported to antagonize morphine-induced pharmacologic effects. The present experiment was conducted to evaluate the effect of prior systemic M3G exposure on morphine disposition and antinociceptive response in male Sprague-Dawley rats. Saline (N = 6), low dose M3G (0.15 mg/hr, N = 7), or high dose M3G (0.30 mg/hr, N = 6) was infused for 720 min prior to the administration of morphine by i.v. bolus (2 mg/kg). Tail-flick latencies in response to hot water (50 degrees) were assessed prior to and for 180 min after the morphine test dose. M3G exposure had no significant effect on morphine pharmacokinetics, although a disproportionate increase in M3G concentrations was observed following the morphine i.v. bolus dose in rats infused with high dose M3G. Morphine-induced antinociception, expressed as the percent of maximum response (%MPR), was maximum 15 min after morphine administration and returned to baseline by 180 min. A pharmacokinetic-pharmacodynamic model was constructed to relate tail-flick latencies to morphine serum concentrations. In saline-exposed rats, the antinociceptive response to morphine was characterized by a sigmoidal Emax model, with an EC50 of 328 ng/mL, a Hill coefficient (gamma) of 4.5, and a half-life for the offset of pharmacologic effect of 11 min. No statistically significant differences in the intensity or duration of morphine-induced response were detected between saline- and M3G-exposed animals. These results suggest that systemic formation of M3G is unlikely to contribute significantly to the development of tolerance to morphine antinociception.

Protein binding and hepatobiliary distribution of valproic acid and valproate glucuronide in rats.

The protein binding and hepatobiliary distribution of valproic acid (VPA) and its glucuronide conjugate (V-G) were examined in rats with a combination of in vitro and ex vivo protocols. VPA was moderately bound to proteins in both serum and hepatic cytosol, and the degree of binding was lower ex vivo than in vitro. V-G, which was more highly bound than VPA ex vivo in serum, may have displaced the parent drug from its binding sites when VPA was administered in vivo. Examination of ex vivo hepatic subcellular distribution revealed that VPA localization tended to be high in cytosol and low in the microsomal fraction; V-G appeared to be distributed evenly throughout the cell although V-G concentrations within the liver were very low. The steady-state elimination rate of VPA did not increase proportionately with increasing steady-state concentrations of unbound VPA in serum, consistent with saturable systemic elimination of the drug. In contrast, steady-state VPA elimination was related linearly to unbound cytosolic VPA concentrations. Moreover, a nonlinear relationship between the unbound concentrations of VPA in hepatic cytosol and serum was observed, consistent with saturable distribution of the unbound drug between the two compartments in vivo. These observations suggest that the nonlinear elimination of VPA in rats may be due to concentration-dependent penetration of the drug into the liver as opposed to saturable biotransformation.

Evidence for reversible sequestration of morphine in rat liver.

The residence of morphine in the systemic circulation is prolonged despite a high systemic clearance, suggestive of significant extravascular sequestration. The present study was conducted to test the hypothesis that morphine binds significantly in tissues, and that the liver plays an important role in morphine binding. [14C]Morphine was administered to male Sprague-Dawley rats 55 min before unlabeled morphine or saline. Blood 14C increased immediately after injection of unlabeled morphine; the area under the blood concentration-time curve (AUC) for 14C increased approximately 2-fold after morphine compared with saline injection. Residual radioactivity in the liver was lower in morphine-treated rats than in controls, suggesting that unlabeled drug displaced [14C]morphine (or a metabolite) from binding sites. To examine this phenomenon more directly, a recirculating isolated perfused liver system was employed. [14C]Morphine was added to the perfusate reservoir 15 min before unlabeled morphine or saline; perfusate and bile samples were collected for 120 min. Upon termination of perfusion, the liver was fractionated to identify the hepatic subcellular fraction(s) in which morphine was sequestered. The perfusate AUC for [14C]morphine was increased approximately 2-fold in response to unlabeled drug, consistent with the in vivo experiment. Morphine was associated preferentially with the cytosolic fraction, and [14C]morphine in all relevant fractions was reduced after administration of unlabeled morphine. In contrast, unlabeled drug had no influence on derived [14C]morphine-3-beta,D-glucuronide. These data are consistent with significant, reversible binding of morphine in hepatic tissue.

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.

A pharmacokinetic model of inhaled methanol in humans and comparison to methanol disposition in mice and rats.

We estimated kinetic parameters associated with methanol disposition in humans from data reported in the literature. Michaelis-Menten elimination parameters (Vmax = 115 mg/L/hr; Km = 460 mg/L) were selected for input into a semi-physiologic pharmacokinetic model. We used reported literature values for blood or urine methanol concentrations in humans and nonhuman primates after methanol inhalation as input to an inhalation disposition model that evaluated the absorption of methanol, expressed as the fraction of inhaled methanol concentration that was absorbed (phi). Values of phi for nonexercising subjects typically varied between 0.64 and 0.75; 0.80 was observed to be a reasonable upper boundary for fractional absorption. Absorption efficiency in exercising subjects was lower than that in resting individuals. Incorporation of the kinetic parameters and phi into a pharmacokinetic model of human exposure to methanol, compared to a similar analysis in rodents, indicated that following an 8-hr exposure to 5000 ppm of methanol vapor, blood methanol concentrations in the mouse would be 13- to 18-fold higher than in humans exposed to the same methanol vapor concentration; blood methanol concentrations in the rat under similar conditions would be 5-fold higher than in humans. These results demonstrate the importance in the risk assessment for methanol of basing extrapolations from rodents to humans on actual blood concentrations rather than on methanol vapor exposure concentrations.

Acute valproic acid overdose. Clinical course and pharmacokinetic disposition of valproic acid and metabolites.

Acute toxicity in the setting of valproic acid (valproate sodium) overdose is in most cases benign and readily reversible. However, serious toxicity has been reported. We present a case of accidental acute valproic acid overdose in a 26-month-old female, in whom serious neurological, metabolic, haematological and respiratory sequelae occurred. The major toxicity observed was delayed cerebral oedema. We also present data not previously reported, which describes the pharmacokinetic disposition of valproic acid and several of its metabolites during the course of this acute overdose. A comparison of an enzyme immunoassay and gas liquid chromatographic methodologies for measuring valproic acid in this setting is also presented. It appears that the 2-EN-valproic acid metabolite plays a role in the neurological toxicity.

Accumulation and washout kinetics of valproic acid and its active metabolites.

There is growing evidence that the metabolites of valproic acid (VPA) may be pharmacologically active and could contribute to both the therapeutic and toxic effects of the drug. The accumulation and washout kinetics of VPA and its oxidative metabolites were, therefore, examined in five healthy volunteers. Valproic acid (250-mg capsules) was administered bid for 15 days. Blood samples were obtained periodically during the 15 days of drug administration and for seven days following termination of treatment. Urine was also collected over the final dosing interval. Steady-state serum concentrations of VPA were achieved within three to four days of treatment. The accumulation of all metabolites in serum lagged behind that of the parent compound, with the mono-desaturated metabolites accumulating more slowly than the hydroxylated species. Furthermore, the apparent washout half-life of each metabolite was longer than the elimination half-life of VPA. In general, the unsaturated metabolites were eliminated more slowly than the hydroxylated metabolites. The serum and urinary metabolite profiles of VPA observed in the healthy volunteers were comparable with those reported for epileptic patients. The differences in the disposition kinetics of VPA and of its potentially active metabolites may explain the previously observed dissociation between the pharmacokinetics and pharmacodynamics of the drug in epileptic patients.

Pharmacokinetics and effect of food on the bioavailability of orally administered venlafaxine.

Venlafaxine is a unique antidepressant currently under evaluation for treatment of various affective disorders. The pharmacokinetics and relative bioavailability of venlafaxine were evaluated in healthy volunteers after oral administration. The bioavailability of 50 mg of venlafaxine as a tablet relative to a solution was determined in a two-period randomized crossover study. The rate of absorption from the gastrointestinal tract was assessed by the time to peak plasma concentration (tmax), a model-dependent calculation of the first-order absorption rate constant, and a model-independent calculation of mean residence time. The extent of absorption was assessed by peak plasma concentration (Cmax) and area under the concentration-time curve (AUC). No statistically significant differences were observed between the two formulations for either the rate or extent of absorption. Similarly, systemic concentrations of the active O-demethylated metabolite did not significantly differ after administration of the two venlafaxine formulations. AUC ratios indicated that the relative bioavailabilities of the parent drug, and formulation of metabolite were approximately 98% and 92%, respectively, for the tablet versus the solution. A separate study was conducted to examine the influence of food on venlafaxine absorption from the 50-mg tablet. A standard, medium-fat breakfast eaten immediately before drug administration delayed the tmax of venlafaxine but did not affect Cmax or AUC. Therefore the tablet formulation of venlafaxine is bioequivalent to the oral solution, and the presence of food appears to decrease the rate but not the extent of absorption of venlafaxine from the tablet formulation.

Comparison of two cyclosporine formulations in healthy volunteers: bioequivalence of the new Sang-35 formulation and Neoral.

This study was conducted to establish bioequivalence between a newly developed oral cyclosporine formulation, Sang-35 (SangStat Medical Corp., Menlo Park, CA), and the microemulsion formulation Neoral (Novartis Pharmaceuticals, East Hanover, NJ). In a randomized, open-label, two-way crossover study, 36 fasted, healthy male volunteers received a single 500-mg cyclosporine dose formulated either as Sang-35 or Neoral. Mean are under the concentration-time curve to infinity (AUC0-infinity) for Sang-35 was 13,900 microg x hr/L compared with 14,000 microg x hr/L for Neoral, with a 90% confidence interval (CI) of 96% to 103% for the geometric mean ratio of the two formulations. Mean maximum concentration (Cmax) was 1,690 microg/L for Sang-35 and 1,700 microg/L for Neoral, with a 90% CI of 96% to 103%. Geometric mean ratios for both AUC0-infinity and Cmax were within the acceptance criteria for bioequivalence (80-125%). Additional studies showed no differences between Sang-35 and Neoral after high-fat meals (n = 19), in female volunteers (n = 25) and in black volunteers (n = 7). It is concluded that single doses of the oral cyclosporine formulations Sang-35 and Neoral are bioequivalent in healthy fasted subjects, after high-fat meals, in women, and in blacks.

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.

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.

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.

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.

Enterohepatic recirculation and renal metabolism of morphine in the rat.

Morphine (2.5 mg/kg) was administered iv to intact (I), bile duct-cannulated (BC), and bile duct-cannulated--renal-ligated (BC-RL) rats (n = 4 per group) to investigate the extent of enterohepatic recirculation and renal metabolism of the drug. A decrease in the serum area under the concentration-time curve (AUC) was observed for the BC in comparison with I rats. From these AUC values, it was determined that approximately 16% of the administered dose was subject to enterohepatic recirculation. In addition, a statistically significant (p less than 0.05) decrease in the systemic clearance of morphine was observed in the BC-RL rats compared with the BC animals (55.2 +/- 17.2 versus 31.4 +/- 8.5 mL/min/kg). This decrement in systemic clearance appeared to be the result of a significant decrease in the formation clearance of morphine glucuronide after ligation of the renal pedicles (23.2 +/- 4.8 versus 10.9 +/- 5.0 mL/min/kg). Renal metabolic clearance was calculated as 15.7 mL/min/kg, accounting for 28.5% of the systemic clearance of morphine. Hepatic clearance (31.4 +/- 8.5 mL/min/kg) accounted for 56.8% of total systemic clearance.

Specific assay for the quantitation of indocyanine green in rat plasma using high-performance liquid chromatography with fluorescence detection.

A rapid and sensitive reversed-phase HPLC assay employing fluorescence detection was developed for quantitating indocyanine green (ICG) in rat plasma. Sample preparation entailed precipitation of plasma proteins with acetonitrile prior to injection on the column. The assay was linear from 0.4 to 200 micrograms/mL, with a detection limit of 3 ng on column. The plasma concentration-time profile of ICG was characterized by this HPLC method and compared with the traditional spectrophotometric assay following iv bolus and iv infusion administration of 5 mg/kg of ICG to rats. Concentrations of ICG obtained using the spectrophotometric assay were consistently higher than those determined by HPLC. In animals receiving ICG by infusion, the maximum difference between the two assays was observed 1 min post-infusion and became negligible by 5 min post-infusion. The calculated pharmacokinetic parameters for ICG, systemic clearance and apparent volume of distribution, were higher using the HPLC assay as compared with the spectrophotometric procedure. The data suggest that a biotransformation or degradation product of ICG is formed in the rat and interferes with the determination of ICG by the spectrophotometric assay. Since the HPLC assay is specific for the parent dye, it is suggested that this assay method be used when determining pharmacokinetic parameters of ICG in rats.

Effect of renal failure and bis(2-ethylhexyl) phthalate pretreatment on the disposition and metabolism of antipyrine in the rat.

Renal failure patients undergoing hemodialysis are regularly exposed to phthalate plasticizers leached from dialysis tubings. Previous studies have shown that antipyrine is eliminated more rapidly in chronic renal failure patients compared with normal individuals. Therefore, the effect of bis(2-ethylhexyl) phthalate on the metabolism of antipyrine was investigated in normal and renal failure rats. In normal animals, the elimination kinetics of an intravenous dose of antipyrine (20 mg/kg) was determined before and after 14 days of peroral treatment with 2 mL/kg/d of bis(2-ethylhexyl) phthalate. The plasma clearance of antipyrine increased markedly after bis(2-ethylhexyl) phthalate treatment. There was a corresponding decrease in the elimination half-life of antipyrine, whereas the apparent volume of distribution was not affected. Both liver weight and hepatic cytochrome P450 content increased following exposure to bis(2-ethylhexyl) phthalate, indicating the induction of hepatic microsomal enzymes. The fractional urinary recovery of the N-demethyl, 4-hydroxy, and 3-hydroxymethyl metabolites of antipyrine was not altered, suggesting that all three oxidative pathways were induced to the same extent. Renal failure alone did not affect the elimination kinetics of antipyrine. However, antipyrine clearance was induced to a greater extent by bis(2-ethylhexyl) phthalate treatment in the renal failure rats as compared with the control animals. The potential for phthalate plasticizers to alter hepatic drug metabolism in hemodialysis patients should be considered.

Anticonvulsant pharmacodynamics and disposition of triazolam in rats.

Triazolam (TZ) is a triazolobenzodiazepine used in the treatment of insomnia that possesses significant anticonvulsant properties. Despite the widespread use of this drug, detailed pharmacokinetic-pharmacodynamic information is lacking, especially with respect to inhibition of seizure activity. TZ disposition has been described previously by methods with limited specificity, and the concentration-anticonvulsant effect relationship has not been characterized. The current studies were undertaken to examine TZ disposition with a specific HPLC method, and to evaluate the relationship between anticonvulsant effect and concentration in Sprague-Dawley rats. TZ pharmacokinetics were characterized after bolus or infusion administration; in a separate experiment, TZ pharmacodynamics were assessed with pentylenetetrazol-induced seizures. The systemic disposition of TZ could be described with a two-compartment model; systemic clearance ranged from 2.45 to 5.30 L/h/ kg, steady-state volume of distribution ranged from 2.10 to 4.02 L/kg, and mean residence time ranged from 47 to 65 min. The concentration-effect relationship was well described by a simple Emax model: Emax, expressed as the ratio of post-TZ to pre-TZ threshold convulsant doses of pentylenetetrazol, was 9.9 +/- 0.7, and the EC50 values were 10.0 +/- 4.6 ng/mL and 34.8 +/- 9.0 ng/g in serum and whole brain tissue, respectively. Under single-dose conditions, TZ is a very potent anticonvulsant in the rat pentylenetetrazol seizure model.