Influence of enterohepatic recycling on the time course of brain-to-blood partitioning of valproic acid in rats.

A widely used metric of substrate exposure in brain is the brain-to-serum partition coefficient (K(p,brain); C(brain)/C(serum)), most appropriately determined at distribution equilibrium between brain tissue and serum. In some cases, C(brain)/C(serum) can peak and then decrease, as opposed to monotonically increasing to a plateau, precluding accurate estimation of partitioning. This "overshoot" has been observed with compounds that undergo enterohepatic recycling (ER), such as valproic acid (VPA). Previous simulation experiments identified a relationship between overshoot in the C(brain)/C(serum) versus time profile and distribution into a peripheral "compartment" (e.g., the ER loop). This study was conducted to evaluate model predictions of that relationship. Initial experiments tested the ability of activated charcoal, antibiotics, or Mrp2 deficiency to impair VPA ER in rats, thereby limiting the apparent volume of distribution associated with ER. Mrp2 deficiency (significantly) and antibiotics (moderately) interrupted VPA ER. Subsequently, brain partitioning was evaluated in the presence versus absence of ER modulation. Although overshoot was not eliminated completely, deconvolution revealed that overshoot was reduced in Mrp2-deficient and antibiotic-treated rats. Consistent with model predictions, overshoot was higher after antibiotic treatment (moderate ER interruption) than in Mrp2 deficiency (substantial ER interruption). Steady-state K(p,brain) was unaffected by experimental manipulation, also consistent with model predictions. These data support the hypothesis that C(brain)/C(serum) may overshoot K(p,brain) based on the extent of peripheral sequestration. Consideration of this information, particularly for compounds that undergo significant extravascular distribution, may be necessary to avoid erroneous estimation of K(p,brain).

Mechanisms underlying differences in systemic exposure of structurally similar active metabolites: comparison of two preclinical hepatic models.

Selection of in vitro models that accurately characterize metabolite systemic and hepatobiliary exposure remains a challenge in drug development. In the present study, mechanisms underlying differences in systemic exposure of two active metabolites, furamidine and 2,5-bis (5-amidino)-2-pyridyl furan (CPD-0801), were examined using two hepatic models from rats: isolated perfused livers (IPLs) and sandwich-cultured hepatocytes (SCH). Pafuramidine, a prodrug of furamidine, and 2,5-bis [5-(N-methoxyamidino)-2-pyridyl] furan (CPD-0868), a prodrug of CPD-0801, were selected for investigation because CPD-0801 exhibits greater systemic exposure than furamidine, despite remarkable structural similarity between these two active metabolites. In both IPLs and SCH, the extent of conversion of CPD-0868 to CPD-0801 was consistently higher than that of pafuramidine to furamidine over time (at most 2.5-fold); area under the curve (AUC) of CPD-0801 in IPL perfusate and SCH medium was at least 7-fold higher than that of furamidine. Pharmacokinetic modeling revealed that the rate constant for basolateral (liver to blood) net efflux (k(A_net efflux)) of total formed CPD-0801 (bound + unbound) was 6-fold higher than that of furamidine. Hepatic accumulation of both active metabolites was extensive (>95% of total formed); the hepatic unbound fraction (f(u,L)) of CPD-0801 was 5-fold higher than that of furamidine (1.6 versus 0.3%). Incorporation of f(u,L) into the pharmacokinetic model resulted in comparable k(A_net efflux,u) between furamidine and CPD-0801. In conclusion, intrahepatic binding markedly influenced the disposition of these active metabolites. A higher f(u,L) explained, in part, the enhanced perfusate AUC of CPD-0801 compared with furamidine in IPLs. SCH predicted the disposition of prodrug/metabolite in IPLs.

The influence of distributional kinetics into a peripheral compartment on the pharmacokinetics of substrate partitioning between blood and brain tissue.

Development of CNS-targeted agents often focuses on identifying compounds with "good" CNS exposure (brain-to-blood partitioning >1). Some compounds undergoing enterohepatic recycling (ER) evidence a partition coefficient, K (p,brain) (expressed as C (brain) /C (plasma)), that exceeds and then decreases to (i.e., overshoots) a plateau (distribution equilibrium) value, rather than increasing monotonically to this value. This study tested the hypothesis that overshoot in K (p,brain) is due to substrate residence in a peripheral compartment. Simulations were based on a 3-compartment model with distributional clearances between central and brain (CL (br)) and central and peripheral (CL (d)) compartments and irreversible clearance from the central compartment (CL). Parameters were varied to investigate the relationship between overshoot and peripheral compartment volume (V (p)), and how this relationship was modulated by other model parameters. Overshoot magnitude and duration were characterized as peak C (brain)/C (plasma) relative to the plateau value (%OS) and time to reach plateau (TRP). Except for systems with high CL (d), increasing V (p) increased TRP and %OS. Increasing brain (V (br)) or central (V (c)) distribution volumes eliminated V (p)-related OS. Parallel increases in all clearances shortened TRP, but did not alter %OS. Increasing either CL or CL (d) individually increased %OS related to V (p), while increasing CL (br) decreased %OS. Under realistic peripheral distribution scenarios, C (brain)/C (plasma) may overshoot substantially K (p,brain) at distribution equilibrium. This observation suggests potential for erroneous assessment of brain disposition, particularly for compounds which exhibit a large apparent V (p), and emphasizes the need for complete understanding of distributional kinetics when evaluating brain uptake.

Fexofenadine brain exposure and the influence of blood-brain barrier P-glycoprotein after fexofenadine and terfenadine administration.

P-glycoprotein (P-gp) plays an important role in determining net brain uptake of fexofenadine. Initial in vivo experiments with 24-h subcutaneous osmotic minipump administration demonstrated that fexofenadine brain penetration was 48-fold higher in mdr1a(-/-) mice than in mdr1a(+/+) mice. In contrast, the P-gp efflux ratio at the blood-brain barrier (BBB) for fexofenadine was only approximately 4 using an in situ brain perfusion technique. Pharmacokinetic modeling based on the experimental results indicated that the apparent fexofenadine P-gp efflux ratio is time-dependent due to low passive permeability at the BBB. Fexofenadine brain penetration after terfenadine administration was approximately 25- to 27-fold higher than after fexofenadine administration in both mdr1a(+/+) and mdr1a(-/-) mice, consistent with terfenadine metabolism to fexofenadine in murine brain tissue. The fexofenadine formation rate after terfenadine in situ brain perfusion was comparable with that in a 2-h brain tissue homogenate in vitro incubation. The fexofenadine formation rate increased approximately 5-fold during a 2-h brain tissue homogenate incubation with hydroxyl-terfenadine, suggesting that the hydroxylation of terfenadine is the rate-limiting step in fexofenadine formation. Moreover, regional brain metabolism seems to be an important factor in terfenadine brain disposition and, consequently, fexofenadine brain exposure. Taken together, these results indicate that the fexofenadine BBB P-gp efflux ratio has been underestimated previously due to the lack of complete equilibration of fexofenadine across the blood-brain interface under typical experimental paradigms.

Relationship between drug/metabolite exposure and impairment of excretory transport function.

The quantitative impact of excretory transport modulation on the systemic exposure to xenobiotics and derived metabolites is poorly understood. This article presents fundamental relationships between exposure and loss of a specific excretory process that contributes to overall clearance. The mathematical relationships presented herein were explored on the basis of hepatic excretory data for polar metabolites formed in the livers of various transporter-deficient rodents. Experimental data and theoretical relationships indicated that the fold change in exposure is governed by the relationship, 1/(1 - f(e)), where f(e) is the fraction excreted by a particular transport protein. Loss of function of a transport pathway associated with f(e) < 0.5 will have minor consequences (<2-fold) on exposure, but exposure will increase exponentially in response to loss of function of transport pathways with f(e) > 0.5. These mathematical relationships may be extended to other organs, such as the intestine and kidney, as well as to systemic drug exposure. Finally, the relationship between exposure and f(e) is not only applicable to complete loss of function of a transport pathway but also can be extended to partial inhibition scenarios by modifying the equation with the ratio of the inhibitor concentration and inhibition constant.

Breast cancer resistance protein interacts with various compounds in vitro, but plays a minor role in substrate efflux at the blood-brain barrier.

Expression of breast cancer resistance protein (Bcrp) at the blood-brain barrier (BBB) has been revealed recently. To investigate comprehensively the potential role of Bcrp at the murine BBB, a chemically diverse set of model compounds (cimetidine, alfuzosin, dipyridamole, and LY2228820) was evaluated using a multiexperimental design. Bcrp1 stably transfected MDCKII cell monolayer transport studies demonstrated that each compound had affinity for Bcrp and that polarized transport by Bcrp was abolished completely by the Bcrp inhibitor chrysin. However, none of the compounds differed in brain uptake between Bcrp wild-type and knockout mice under either an in situ brain perfusion or a 24-h subcutaneous osmotic minipump continuous infusion experimental paradigm. In addition, alfuzosin and dipyridamole were shown to undergo transport by P-glycoprotein (P-gp) in an MDCKII-MDR1 cell monolayer model. Alfuzosin brain uptake was 4-fold higher in mdr1a(-/-) mice than in mdr1a(+/+) mice in in situ and in vivo studies, demonstrating for the first time that it undergoes P-gp-mediated efflux at the BBB. In contrast, P-gp had no effect on dipyridamole brain penetration in situ or in vivo. In fact, in situ BBB permeability of these solutes appeared to be primarily dependent on their lipophilicity in the absence of efflux transport, and in situ brain uptake clearance correlated with the intrinsic transcellular passive permeability from in vitro transport and cellular accumulation studies. In summary, Bcrp mediates in vitro transport of various compounds, but seems to play a minimal role at the BBB in vivo.

Sex-dependent disposition of acetaminophen sulfate and glucuronide in the in situ perfused mouse liver.

Breast cancer resistance protein (BCRP, ABCG2) is expressed in the hepatic canalicular membrane and mediates biliary excretion of xenobiotics including sulfate and glucuronide metabolites of some compounds. Hepatic Bcrp expression is sex-dependent, with higher expression in male mice. The hypothesis that sex-dependent Bcrp expression influences the hepatobiliary disposition of phase II metabolites was tested in the present study using acetaminophen (APAP) and the generated APAP glucuronide (AG) and sulfate (AS) metabolites in single-pass in situ perfused livers from male and female wild-type and Abcg(-/-) (Bcrp-deficient) mice. Pharmacokinetic modeling was used to estimate parameters governing the hepatobiliary disposition of APAP, AG, and AS. In wild-type mice, the biliary excretion rate constant was 2.5- and 7-fold higher in males than in females for AS and AG, respectively, reflecting male-predominant Bcrp expression. Sex-dependent differences in AG biliary excretion were not observed in Bcrp-deficient mice, and AS biliary excretion was negligible. Interestingly, sex-dependent basolateral excretion of AG (higher in males) and AS (higher in females) was noted in wild-type mice with a similar trend in Bcrp-deficient mouse livers, reflecting an increased rate constant for AG formation in male and AS formation in female mouse livers. In addition, the rate constant for AS basolateral excretion was increased significantly in female mouse livers compared with that in male mouse livers. It is interesting to note that multidrug resistance-associated protein 4 was higher in female than in male mouse livers. In conclusion, sex-dependent differences in conjugation and transporter expression result in profound differences in the hepatobiliary disposition of AG and AS in male and female mouse livers.

Pharmacokinetic analysis of trichloroethylene metabolism in male B6C3F1 mice: Formation and disposition of trichloroacetic acid, dichloroacetic acid, S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine.

Trichloroethylene (TCE) is a well-known carcinogen in rodents and concerns exist regarding its potential carcinogenicity in humans. Oxidative metabolites of TCE, such as dichloroacetic acid (DCA) and trichloroacetic acid (TCA), are thought to be hepatotoxic and carcinogenic in mice. The reactive products of glutathione conjugation, such as S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and S-(1,2-dichlorovinyl) glutathione (DCVG), are associated with renal toxicity in rats. Recently, we developed a new analytical method for simultaneous assessment of these TCE metabolites in small-volume biological samples. Since important gaps remain in our understanding of the pharmacokinetics of TCE and its metabolites, we studied a time-course of DCA, TCA, DCVG and DCVG formation and elimination after a single oral dose of 2100 mg/kg TCE in male B6C3F1 mice. Based on systemic concentration-time data, we constructed multi-compartment models to explore the kinetic properties of the formation and disposition of TCE metabolites, as well as the source of DCA formation. We conclude that TCE-oxide is the most likely source of DCA. According to the best-fit model, bioavailability of oral TCE was approximately 74%, and the half-life and clearance of each metabolite in the mouse were as follows: DCA: 0.6 h, 0.081 ml/h; TCA: 12 h, 3.80 ml/h; DCVG: 1.4 h, 16.8 ml/h; DCVC: 1.2 h, 176 ml/h. In B6C3F1 mice, oxidative metabolites are formed in much greater quantities (approximately 3600 fold difference) than glutathione-conjugative metabolites. In addition, DCA is produced to a very limited extent relative to TCA, while most of DCVG is converted into DCVC. These pharmacokinetic studies provide insight into the kinetic properties of four key biomarkers of TCE toxicity in the mouse, representing novel information that can be used in risk assessment.

Assessment of blood-brain barrier permeability using the in situ mouse brain perfusion technique.

PURPOSE: To assess the blood-brain barrier (BBB) permeability of 12 clinically-used drugs in mdr1a(+/+) and mdr1a(-/-) mice, and investigate the influence of lipophilicity, nonspecific brain tissue binding, and P-gp-mediated efflux on the rate of brain uptake. METHODS: The BBB partition coefficient (PS) was determined using the in situ mouse brain perfusion technique. The net brain uptake for 12 compounds, and the time course of brain uptake for selected compounds ranging in BBB equilibration kinetics from rapidly-equilibrating (e.g., alfentanil, sufentanil) to slowly-equilibrating (fexofenadine), was determined and compared. RESULTS: There was a sigmoidal relationship in mdr1a(-/-) mice between the log-PS and clogD(7.4) in the range of 0-5. The brain uptake clearance was a function of both permeability and blood flow rate. The brain unbound fraction was inversely proportional to lipophilicity. Alfentanil achieved brain equilibrium approximately 4,000-fold faster than fexofenadine, based on the magnitude of PSxfu,brain. CONCLUSIONS: In situ brain perfusion is a useful technique to determine BBB permeability. Lipophilicity, ionization state, molecular weight and polar surface area are all important determinants for brain penetration. The time to blood-to-brain equilibrium varies widely for different compounds, and is determined by a multiplicity of pharmacokinetic factors.

Coordinated nuclear receptor regulation of the efflux transporter, Mrp2, and the phase-II metabolizing enzyme, GSTpi, at the blood-brain barrier.

Xenobiotic efflux pumps at the blood-brain barrier are critical modulators of central nervous system pharmacotherapy. We previously found expression of the ligand-activated nuclear receptor, pregnane X receptor (PXR), in rat brain capillaries, and showed increased expression and transport activity of the drug efflux transporter, P-glycoprotein, in capillaries exposed to PXR ligands (pregnenolone-16alpha-carbonitrile (PCN) and dexamethasone) in vitro and in vivo. Here, we show increased protein expression and transport activity of another efflux pump, multidrug resistance-associated protein isoform 2 (Mrp2), in rat brain capillaries after in vitro and in vivo exposure to PCN and dexamethasone. The phase-II drug-metabolizing enzyme, glutathione S-transferase-pi (GSTpi), was found to be expressed in brain capillaries, where it colocalized to a large extent with Mrp2 at the endothelial cell luminal plasma membrane. Like Mrp2, GSTpi protein expression increased with PXR activation. Colocalization and coordinated upregulation suggest functional coupling of the metabolizing enzyme and efflux transporter. These findings indicate that, as in hepatocytes, brain capillaries possess a regulatory network consisting of nuclear receptors, metabolizing enzymes, and efflux transporters, which modulate blood-brain barrier function.

Effect of albumin on the biliary clearance of compounds in sandwich-cultured rat hepatocytes.

The purpose of the present study was to evaluate the effects of bovine serum albumin (BSA) and essentially fatty acid-free BSA (BSA-FAF) on the biliary clearance of compounds in sandwich-cultured rat hepatocytes. Unbound fraction, biliary excretion index (BEI), and unbound intrinsic biliary clearance (intrinsic Clbiliary') were determined for digoxin, pravastatin, and taurocholate in the absence or presence of BSA or BSA-FAF. BSA had little effect on the BEI or intrinsic Clbiliary' of these compounds. Surprisingly, BSA-FAF decreased both BEI and intrinsic Clbiliary' for digoxin and pravastatin, which represent low and moderately bound compounds, respectively. The BEI and intrinsic Clbiliary' of taurocholate, a highly bound compound, were not altered significantly by BSA-FAF. Neither BSA nor BSA-FAF had a discernable effect on the bile canalicular networks based on carboxydichlorofluorescein retention. Neither the addition of physiological concentrations of calcium nor the addition of fatty acids to BSA-FAF was able to restore the BEI or intrinsic Clbiliary' of the model compounds to similar values in the absence or presence of BSA. Careful consideration is warranted when selecting the type of BSA for addition to in vitro systems such as sandwich-cultured rat hepatocytes.

Neuronal nitric oxide modulates morphine antinociceptive tolerance by enhancing constitutive activity of the mu-opioid receptor.

NO is a key mediator of morphine antinociceptive tolerance. This work was conducted to evaluate the specific effects of NO on mu-opioid receptor activity. To investigate the effects of morphine- and L-arginine (the NO precursor)-induced increases in NO, five groups of rats were treated with saline, l-arginine (100-, 300-, or 500-mg/kg/h), or morphine 3-mg/kg/h for 8h on Day 1; brain tissue was collected on Day 2. To evaluate the effects of additional increases in NO on morphine-induced alterations of the mu-opioid receptor, six groups of rats were treated with 8-h intravenous infusions for two consecutive days as per the following scheme (Day 1:Day 2): saline:saline (control); saline:morphine 3-mg/kg/h (tolerant); L-arginine 500-mg/kg/h:saline (NO control); L-arginine 100-mg/kg/h:morphine 3-mg/kg/h; L-arginine 300-mg/kg/h:morphine 3-mg/kg/h; and L-arginine 500-mg/kg/h:morphine 3-mg/kg/h (supertolerant). Brain tissue was collected at the end of Day 2. The time course of effects on morphine-induced receptor alterations due to increased NO also was evaluated. Brain tissue was analyzed for changes in radioligand (agonist and antagonist) binding and [(35)S]GTPgammaS binding (agonist and antagonist). In the absence of agonist exposure, NO produced an alteration in the mu-opioid receptor that increased receptor activity. In the presence of agonist, NO increased constitutive activation of the mu-opioid receptor and reduced the ability of a selective mu-opioid agonist to activate the mu-opioid G-protein-coupled receptor; these molecular effects occurred in a time course consistent with the development of antinociceptive tolerance. This work establishes important NO-induced alterations in mu-opioid receptor functionality, which directly lead to the development of opioid antinociceptive tolerance.

Nasal drug administration: potential for targeted central nervous system delivery.

Nasal administration as a means of delivering therapeutic agents preferentially to the brain has gained significant recent interest. While some substrates appear to be delivered directly to the brain via this route, the mechanisms governing overall brain uptake and exposure remain unclear. Some substrates utilize the olfactory nerve tract and gain direct access to the brain, thus bypassing the blood-brain barrier (BBB). However, most agents of pharmacologic interest likely gain access to the brain via the olfactory epithelium, which represents a more direct route of uptake. While the traditional BBB is not present at the interface between nasal epithelium and brain, P-glycoprotein (and potentially other barrier transporters) is expressed at this interface. In addition, work in this laboratory has demonstrated that P-glycoprotein throughout the brain can be modulated with nasal administration of appropriate inhibitors. The potential for targeted central nervous system delivery via this route is discussed.

Drug transport at the blood-brain barrier and the choroid plexus.

The blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) represent the main interfaces between the central nervous system (CNS) and the peripheral circulation. Drug exposure to the CNS is dependent on a variety of factors, including the physical barrier presented by the BBB and the BCSFB and the affinity of the substrate for specific transport systems located at both of these interfaces. It is the aggregate effect of these factors that ultimately determines the total CNS exposure, and thus pharmacological efficacy, of a drug or drug candidate. This review discusses the anatomical and biochemical barriers presented to solute access to the CNS. In particular, the important role played by various efflux transporters in the overall barrier function is considered in detail, as current literature suggests that efflux transport likely represents a key determinant of overall CNS exposure for many substrates. Finally, it is important to consider not only the net delivery of the agent to the CNS, but also the ability of the agent to access the relevant target site within the CNS. Potential approaches to increasing both net CNS and target-site exposure, when such exposure is dictated by efflux transport, are considered.

The development of morphine antinociceptive tolerance in nitric oxide synthase-deficient mice.

UNLABELLED: Elevations in nitric oxide (NO) have been implicated in the development of morphine antinociceptive tolerance. This study was conducted to establish the role of specific isoforms of NO synthase (NOS) in morphine tolerance development using genetically modified mice. METHODS: Three groups of mice (endothelial NOS [eNOS]-deficient, neuronal NOS [nNOS]-deficient, and NOS-competent) were used in this experiment. On Day 1, the analgesic response (radiant heat tail-flick) to a challenge dose of morphine (4 mg/kg) was determined over 3 hr. Tolerance was induced on Days 1-5 by administering morphine subcutaneously (10 mg/kg) or L-arginine, a NO precursor, intraperitoneally (200 mg/kg), twice daily. Analgesic response to the challenge dose was determined again on Day 6. RESULTS: Following sustained morphine administration, nNOS-deficient mice exhibited less tolerance development when compared to the control group, although measurable tolerance still occurred. Mice deficient in eNOS evidenced a degree of tolerance similar to that of control. Prolonged L-arginine administration produced significant functional tolerance to morphine in NOS-competent and eNOS-deficient mice. The loss of morphine responsivity after L-arginine administration was similar to that after morphine pretreatment. L-Arginine did not affect the antinociceptive response to morphine in mice deficient in nNOS, suggesting that the small degree of morphine-induced tolerance in this group occurs through an alternate pathway. CONCLUSIONS: These data demonstrate the pivotal role of the neuronal isoform of NOS in development of morphine antinociceptive tolerance. Furthermore, tolerance development appears to be predominantly a NO-mediated process, but likely is augmented by a secondary (non-NO) pathway.

Variable modulation of opioid brain uptake by P-glycoprotein in mice.

The efflux transporter P-glycoprotein (P-gp) is an important component of the blood-brain barrier (BBB) that limits accumulation of many compounds in brain. Some opioids have been shown to interact with P-gp in vitro and in vivo. Genetic or chemical disruption of P-gp has been shown to enhance the antinociceptive and/or toxic effects of some opioids, although the extent of this phenomenon has yet to be understood. The purpose of this study was to assess quantitatively the influence of mdr1a P-gp on initial brain uptake of chemically diverse opioids in mice. The brain uptake of opioids selective for the mu (fentanyl, loperamide, meperidine, methadone, and morphine), delta (deltorphin II, DPDPE, naltrindole, SNC 121) and kappa (bremazocine and U-69593) receptor subtypes was determined in P-gp-competent (wild-type) and P-gp-deficient [mdr1a(-/-)] mice with an in situ brain perfusion model. BBB permeability of the opioids varied by several orders of magnitude in both mouse strains. The difference in brain uptake between P-gp-competent and P-gp-deficient mice ranged from no detectable effect (meperidine) to >/=8-fold increase in uptake (DPDPE, loperamide, and SNC 121). In addition, loperamide efflux at the BBB was inhibited by quinidine. These results demonstrate that P-gp modulation of opioid brain uptake varies substantially within this class of compounds, regardless of receptor subtype. P-gp-mediated efflux of opioids at the BBB may influence the onset, magnitude, and duration of analgesic response. The variable influence of P-gp on opioid brain distribution may be an important issue in the context of pharmacologic pain control and drug interactions.

Contribution of morphine-6-glucuronide to antinociception following intravenous administration of morphine to healthy volunteers.

This study was performed to develop an integrated pharmacokinetic-pharmacodynamic model for estimating the contribution of morphine-6-glucuronide (M6G) to morphine-associated antinociception in humans. Healthy volunteers (n = 8) received 10 mg of morphine sulfate as a 5-minute i.v. infusion. A Contact Thermode heat probe was placed on the volar forearm to elicitpain. Thermal threshold, defined as the temperature at which pain was first perceived, was measured at fixed time intervals over 8 hours. Serum concentrations of morphine and M6G were determined by LC/MS. Concentration- and effect-time data were analyzed by stepwise nonlinear least-squares regression. The pharmacodynamic parameter estimates were recovered with a linear effect-compartment model and were used to assess the contribution of M6G to morphine-associated analgesia. The estimates (mean +/- SEM) for morphine total clearance and steady-state volume of distribution were 1.0 +/- 0.07 L/h/kg and 1.6 +/- 0.1 L/kg, respectively. The AUC ratio of M6G to morphine was 0.73 +/- 0.06. The contribution of M6G to analgesia ranged from < 0.1% to 66% and was inversely related to the overall effect elicited by the morphine dose (r2 = 0.776). Differences in gender were observed where the contribution (mean +/- SEM) of M6G to analgesia was 32% +/- 19% in males (n = 3) and 13% +/- 8% in females (n = 5). These results suggest that as the overall effect of morphine increases, the fractional contribution of M6G declines and the contribution of M6G to analgesia may differ between males and females. Alterations in the M6G/morphine system may have clinically significant pharmacodynamic consequences.

Pharmacodynamics of morphine-induced neuronal nitric oxide production and antinociceptive tolerance development.

Elevated nitric oxide (NO) production has been implicated in the development of morphine antinociceptive tolerance. This study was conducted to establish the temporal relationship between morphine-induced increases in neuronal NO and loss of pharmacologic activity. Five groups of rats equipped with microdialysis probes in the jugular vein and hippocampus received an intravenous infusion of saline or morphine (0.3, 1, 2, or 3 mg/kg/h) for 8 h. Morphine concentrations in the blood and hippocampal microdialysate were determined by LC/MS-MS; NO production was quantified with an amperometric sensor implanted in the contralateral hippocampus. Antinociceptive effect was monitored at selected time points during and following infusion by electrical stimulation vocalization. The data were fit with a pharmacokinetic/pharmacodynamic model to obtain parameters governing morphine disposition, stimulation of NO production, antinociception, and antinociceptive tolerance development. An additional three groups of rats were pretreated with l-arginine, the NO precursor (100, 300, or 500 mg/kg/h for 8 h), to elevate NO concentrations prior to morphine infusion. Morphine administration resulted in a dose-dependent increase in NO production; the time course of altered NO production coincided with the development of antinociceptive tolerance. l-arginine pretreatment initially enhanced morphine-induced analgesia early in the morphine infusion. However, this NO-associated increase in opioid response dissipated rapidly due to a dominant NO-induced loss of antinociception. Pharmacodynamic modeling suggested that this latter effect was consistent with a hyperalgesic response. These data define a strong, time-dependent relationship between morphine-induced stimulation of NO production and tolerance development, identify specific NO-induced alterations in nociceptive processing after morphine administration, and indicate that NO is a key mediator of antinociceptive tolerance development.

Pharmacokinetics and pharmacodynamics of L-arginine in rats: a model of stimulated neuronal nitric oxide synthesis.

Nitric oxide (NO) is believed to be involved in a variety of central nervous system (CNS) functions, including opioid responsivity. Elucidation of the role of NO in the CNS requires the ability to elevate systematically neuronal NO concentrations in vivo. This study was conducted to assess the pharmacokinetics of L-arginine, a NO precursor, and to relate the disposition of this amino acid to the pharmacodynamic endpoint of neuronal NO production. L-Arginine (250-, 500-, or 1000-mg/kg/h) or saline was infused intravenously for 6 h to rats. L-Arginine was quantified in brain and blood (after in vivo microdialysis) with high-performance liquid chromatography. NO was quantified simultaneously with a sensitive and specific amperometric sensor placed in the hippocampus. The data were fit with a comprehensive pharmacokinetic-pharmacodynamic (PK/PD) model to obtain parameters governing the systemic disposition of L-arginine, the uptake of L-arginine into the brain, and subsequent NO production. Exogenous administration of L-arginine resulted in incremental elevations in hippocampal NO, with a approximately 33, 48, and approximately 50% increase from control for the 250-, 500-, and 1000-mg/kg/h L-arginine treated rats, respectively. The PK/PD model, which incorporated known characteristics of the system (saturable uptake of L-arginine into brain; NO production governed by circadian changes in enzyme activity) was capable of describing accurately the observed data. The model developed herein will be invaluable in characterizing the numerous roles of NO in the CNS.

Use of an electrochemical nitric oxide sensor to detect neuronal nitric oxide production in conscious, unrestrained rats.

INTRODUCTION: Amperometric sensors that directly measure nitric oxide (NO) are readily employed in pharmacologic research. While several of these sensors have been developed, none has been investigated for use in conscious, freely moving animals. An approach was developed and validated for real-time quantitation of neuronal NO production in rats without restricting locomotor activity or other potentially useful behavioral endpoints. METHODS: Male Sprague-Dawley rats were equipped with a femoral vein or intraperitoneal cannula. A guide cannula and an amperometric NO sensor were placed in the left and right hippocampus, respectively. Following recovery, rats received a 6-h intravenous infusion of saline, L-arginine (an NO precursor; 250 or 500 mg/kg/h), or incremental intraperitoneal 7-nitroindazole (an NO synthase inhibitor; 200-mg/kg loading dose and 100 mg/kg every 2 h). The sensor recorded NO production continuously and microdialysis samples were collected incrementally throughout the experiment. Griess assay analysis of microdialysate samples was compared to sensor readings in vivo. In vitro degradation of an NO donor also was used to validate sensor performance. RESULTS: Exogenous administration of L-arginine resulted in incremental increases in the neuronal NO signal. A reduction in NO production was observed during administration of 7-nitroindazole, a selective neuronal NO synthase inhibitor. A significant correlation was observed in vitro between the Griess assay analysis, an indirect analytical approach, and the NO sensor readings. The lack of a strong correlation between these measures in vivo is consistent with the indirect nature of the Griess assay. DISCUSSION: The current approach allows real-time determination of neuronal NO production in unrestrained rats. This model will be invaluable in evaluating pharmacologic issues regarding brain tissue NO synthesis, assessing brain NO synthase as a molecular target, and establishing the effects of pharmacologic agents on neuronal NO production.