Use of chromatographicR m values as a possible approach to calculation of the absorption rate constants for some related drugs

A possible correlation between the logarithm of the absorption rate constants (k_aof closely chemically related drugs consistent with the pH-partition theory and their R_m values in partition chromatograms is reported. The theoretical basis for the existence of this relationship is briefly outlined. On the basis of the data reported in the available literature for gastric absorption rate constants of several barbiturates and their R_m values in seven paper Chromatographic partition systems, correlations ranging from good to excellent were found.     

Simplified methods for the evaluation of the parameters of the time course of plasma concentration in the one-compartment body model with first-order invasion and first-order drug elimination including methods for ascertaining when such rate constants are equal

The many limitations in determining the pharmacokinetic parameters of firstorder invasion of, and elimination from, the onecompartment body model by the method of residuals or by feathering Ct data can be minimized by applying the simplified methods outlined herein. Comparisons of the apparent volumes of distribution, V, calculated on the premises that the Bateman Function represents k_a>k_e or its converse, k_e>k_a,i.e., flip-flop, can permit a proper choice of the correct version. Estimation of k_e can be obtained by regression of (A₀/V)/C(oncentration) on AUC₁/ Cwhere A₀/Vis estimable from knowledge of C_max and t_max since .The ratio of the magnitude of the rate constant of invasion to that of elimination, m=k_a/k_e,is related to k_et_max by the expression k_et_max=ln m/ (m–1)for all possible values of m.A table for the determination of m from values of k_et_max is given. When bioavailability, =A₀/Dose,is known or complete, k_e and Vcan be determined from the respective ordinate and abscissa of the intersection of and Cl(clearance)/k_e,both plotted against arbitrary k_e values. The two functions may not intersect at low values of mdue to errored C-t values but the k_e value when the two curves are closest (k_min)may approximate k_e.The intersections of and k_eAUCT (AUC_trap)plotted against variable k_e values (Method A) provide estimates of k_e from their abscissa values and A/Vfrom their ordinate values when is unknown. Method B appears to give more reliable estimates of k_e at the k_min of the difference plotted against k_e.Since k_min of this plot is 1/t_max when m=1,the identity of the mas unity underlying the C-t data is indicated when either k_mint_max is approximately unity or k_min ispractically synonymous with 1/t_max.This was clearly shown when 12 constructed m=1,C-t cases with 10% random error were evaluated by Method B. Better estimates were effected by all procedures when the raw C-t data were smoothed.We regretfully announce that Dr. Edward Garrett passed away on October 25, 1993, after an extended illness.     

Nimodipine nanocrystals for oral bioavailability improvement: Role of mesenteric lymph transport in the oral absorption

Purpose

We had conducted a comprehensive study on preparation, characterization and pharmacokinetics of nimodipine nanocrystals for oral administration previously, and nimodipine nanocrystals displayed lower dissolution profiles but higher bioavailability than Nimotop^®. In this study, we aimed at elucidating the reasons of unfavorable in vitro in vivo correlation for NMD nanocrystals and Nimotop^® with a hypothesis that special oral absorption mechanism was involved in the absorption of nimodipine nanocrystals.

Methods

Investigations of oral absorption mechanism of the nanocrystals were performed on everted gut sac models, lymphatically (mesenteric lymph duct) cannulated SD rats, Caco-2 cell monolayers and chylomicron flow blocking rats, respectively.

Results

The permeability of nanocrystals in duodenum, ileum and colon was not superior to that of Nimotop^®, suggestive of special absorption mechanisms involved. Exudates of nanocrystals from enterocytes were detected in mesenteric lymphatic fluids using a transmission electron microscope, and the bioavailability was only about half of the control after the mesenteric lymph was blocked. The nanocrystals were taken up by enterocytes via macropinocytosis and caveolin-mediated endocytosis pathways.

Conclusions

It was impossible to establish a favorable in vitro in vivo correlation for NMD nanocrystals and Nimotop^®, because portions of the nanocrystals underwent macropinocytosis and caveolin-mediated endocytosis by enterocytes as intact nanocrystal forms, then bypassed the liver first-pass metabolism.     

KATP channel openers: tissue selectivity of original 3-alkylaminopyrido- and 3-alkylaminobenzothiadiazine 1,1-dioxides

The present study was designed to further evaluate the biological effects and tissue selectivity of new 3-alkylaminobenzo- and 3-alkylaminopyridothiadiazine 1,1-dioxides bearing identical branched alkylamino chains at the 3-position. These original compounds were compared with their parent molecules; namely the K(ATP) channel openers diazoxide and pinacidil. All tested 3-alkylamino-substituted derivatives provoked a marked, concentration-dependent inhibition of the glucose-induced insulin secretion from rat pancreatic islets. The 3-alkylaminopyridothiadiazine 1,1-dioxides evoked a weak vasorelaxant activity whilst their 7-halo-substituted benzothiadiazine counterparts elicited pronounced, concentration-dependent, relaxations of rat aortic rings. The myorelaxant effect of the different drugs was tightly correlated with their capacity to increase (86)Rb outflow ((42)K substitute) from prelabelled and perifused rat aortic rings. EC(50)/IC(50) ratios (vascular/pancreatic) revealed a pronounced selectivity of the 3-alkylaminopyridothiadiazine 1,1-dioxides towards the pancreatic endocrine tissue. Structure-activity relationships suggest that, besides the requirement of an electronegative pole at the 7-position of the heterocycle, a minimal steric hindrance confers an optimal insulin-secreting cell selectivity. Lastly, radioisotopic, electrophysiological and pharmacological investigations indicate that the marked vasorelaxant properties of the 3-alkylaminobenzothiadiazine 1,1-dioxides are related to the activation of smooth muscle K(ATP) channels.     

In vitro metabolism of cyclosporine A by human kidney CYP3A5

The objectives of this study were to characterize and compare the metabolic profile of cyclosporine A (CsA) catalyzed by CYP3A4, CYP3A5 and human kidney and liver microsomes, and to evaluate the impact of the CYP3A5 polymorphism on product formation from parent drug and its primary metabolites. Three primary CsA metabolites (AM1, AM9 and AM4N) were produced by heterologously expressed CYP3A4. In contrast, only AM9 was formed by CYP3A5. Substrate inhibition was observed for the formation of AM1 and AM9 by CYP3A4, and for the formation of AM9 by CYP3A5. Microsomes isolated from human kidney produced only AM9 and the rate of product formation (2 and 20 microM CsA) was positively associated with the detection of CYP3A5 protein and presence of the CYP3A5*1 allele in 4 of the 20 kidneys tested. A kinetic experiment with the most active CYP3A5*1-positive renal microsomal preparation yielded an apparent Km (15.5 microM) similar to that of CYP3A5 (11.3 microM). Ketoconazole (200 nM) inhibited renal AM9 formation by 22-55% over a CsA concentration range of 2-45 microM. Using liver microsomes paired with similar CYP3A4 content and different CYP3A5 genotypes, the formation of AM9 was two-fold higher in CYP3A5*1/*3 livers, compared to CYP3A5*3/*3 livers. AM19 and AM1c9, two of the major secondary metabolites of CsA, were produced by CsA, AM1 and AM1c when incubated with CYP3A4, CYP3A5, kidney microsomes from CYP3A5*1/*3 donors and all liver microsomes. Also, the formation of AM19 and AM1c9 was higher from incubations with liver and kidney microsomes with a CYP3A5*1/*3 genotype, compared to those with a CYP3A5*3/*3 genotype. Together, the data demonstrate that CYP3A5 may contribute to the formation of primary and secondary metabolites of CsA, particularly in kidneys carrying the wild-type CYP3A5*1 allele.     

Intestinal absorption of novel-dipeptide prodrugs of saquinavir in rats

Saquinavir (SQV) was the first human immuno-virus-1 (HIV-1) protease inhibitor approved by FDA. However, P-glycoprotein (P-gp), an efflux pump limits its oral and brain bioavailabilities. The objective of this study is to investigate whether prodrug modification of SQV to dipeptide prodrugs Valine-Valine-Saquinavir (Val-Val-SQV) and Glycine-Valine-Saquinavir (Gly-Val-SQV) targeting intestinal peptide transporter can enhance intestinal permeability of SQV by circumventing P-gp mediated efflux. Single pass intestinal perfusion experiments in rat jejunum were performed to calculate the absorption rate constant and intestinal permeability of SQV, Val-Val-SQV and Gly-Val-SQV. Equimolar concentration (25 microM) of SQV, Val-Val-SQV and Gly-Val-SQV were employed in the perfusion studies. Perfusion experiments were also carried out in the presence of cyclosporine (10 microM) and glycyl-sarcosine (20 mM). Absorption rate constants in rat jejunum (ka) for SQV, Val-Val-SQV and Gly-Val-SQV were found to be 14.1+/-3.4x10(-3), 65.8+/-4.3x10(-3), and 25.6+/-5.7x10(-3) min(-1), respectively. Enhanced absorption of Val-Val-SQV and Gly-Val-SQV relative to SQV can be attributed to their translocation by the peptide transporter in the jejunum. Significant permeability enhancement of SQV across rat jejunum was observed in the presence of cyclosporine 10 microM (P-gp inhibitor). However, permeability of Val-Val-SQV was unchanged in the presence of cyclosporine suggesting lack of any interaction of the prodrug with efflux pump. Intestinal absorption of Val-Val-SQV was significantly inhibited in the presence of gly-sar indicating the involvement of peptide transporter in intestinal absorption. In conclusion, peptide transporter targeted prodrug modification of P-gp substrates could lead to shielding of these drug molecules from efflux pumps.     

Kinetics of di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate in blood and of DEHP metabolites in urine of male volunteers after single ingestion of ring-deuterated DEHP

The plasticizer di(2-ethylhexyl) phthalate (DEHP) is suspected to induce antiandrogenic effects in men via its metabolite mono(2-ethylhexyl) phthalate (MEHP). However, there is only little information on the kinetic behavior of DEHP and its metabolites in humans. The toxikokinetics of DEHP was investigated in four male volunteers (28-61 y) who ingested a single dose (645 ± 20 μg/kg body weight) of ring-deuterated DEHP (DEHP-D₄). Concentrations of DEHP-D₄, of free ring-deuterated MEHP (MEHP-D₄), and the sum of free and glucuronidated MEHP-D₄ were measured in blood for up to 24 h; amounts of the monoesters MEHP-D₄, ring-deuterated mono(2-ethyl-5-hydroxyhexyl) phthalate and ring-deuterated mono(2-ethyl-5-oxohexyl) phthalate were determined in urine for up to 46 h after ingestion. The bioavailability of DEHP-D₄ was surprisingly high with an area under the concentration-time curve until 24 h (AUC) amounting to 50% of that of free MEHP-D₄. The AUC of free MEHP-D₄ normalized to DEHP-D₄ dose and body weight (AUC/D) was 2.1 and 8.1 times, that of DEHP-D₄ even 50 and 100 times higher than the corresponding AUC/D values obtained earlier in rat and marmoset, respectively. Time courses of the compounds in blood and urine of the volunteers oscillated widely. Terminal elimination half-lives were short (4.3-6.6 h). Total amounts of metabolites in 22-h urine are correlated linearly with the AUC of free MEHP-D₄ in blood, the parameter regarded as relevant for risk assessment.     

The thermodynamic dissociation constants of four non-steroidal anti-inflammatory drugs by the least-squares nonlinear regression of multiwavelength spectrophotometric pH-titration data

The mixed dissociation constants of four non-steroidal anti-inflammatory drugs (NSAIDs) ibuprofen, diclofenac sodium, flurbiprofen and ketoprofen at various ionic strengths I of range 0.003-0.155, and at temperatures of 25 degrees C and 37 degrees C, were determined with the use of two different multiwavelength and multivariate treatments of spectral data, SPECFIT/32 and SQUAD(84) nonlinear regression analyses and INDICES factor analysis. The factor analysis in the INDICES program predicts the correct number of components, and even the presence of minor ones, when the data quality is high and the instrumental error is known. The thermodynamic dissociation constant pK(a)(T) was estimated by nonlinear regression of (pK(a), I) data at 25 degrees C and 37 degrees C. Goodness-of-fit tests for various regression diagnostics enabled the reliability of the parameter estimates found to be proven. PALLAS, MARVIN, SPARC, ACD/pK(a) and Pharma Algorithms predict pK(a) being based on the structural formulae of drug compounds in agreement with the experimental value. The best agreement seems to be between the ACD/pK(a) program and experimentally found values and with SPARC. PALLAS and MARVIN predicted pK(a,pred) values with larger bias errors in comparison with the experimental value for all four drugs.     

Fasting and nonfasting blood ethanol concentrations following repeated oral administration of ethanol to one adult male subject

Whole capillary blood ethanol concentrations following the oral administration of a 30-ml dose of absolute alcohol (0.32 g/kg absolute alcohol) and 45-ml doses of 95% alcohol (0.45 g/kg absolute alcohol) under varying test meal conditions to an adult male volunteer were measured. Ten different test conditions were two fasting (30 ml and 45 ml), three liquid test meals (45 ml), and five solid test meals (45 ml). The terminal concentration-time data were simultaneously fitted to a modification of the integrated form of the Michaelis-Menten equation. The parametersV _m andK _m of the Michaelis-Menten equation obtained in this fitting were very close to the average values of these parameters following the constant-rate intravenous infusion of ethanol to six adult male volunteers in another study. The area under the concentration-time curves following the liquid and solid food test meals decreased with an increase in the size of the meal. The reduction in area caused by food was the result of two factors: (a) decrease in efficiency of absorption (average 55%) and (b) slowing of the absorption rate coupled with the operation of Michaelis-Menten elimination kinetics (average 45%).This work was supported by Grant No. 1R01AA00683-01A1 from the National Institute on Alcohol Abuse and Alcoholism of the Alcohol, Drug Abuse, and Mental Health Administration, Rockville, Maryland.     

Comparison of a genomic and a multiplex cell imaging approach for the detection of phospholipidosis

Phospholipidosis (PLD) is a topic of concern in drug development because it may be associated with toxicological consequences. The aim of the study was to determine the best method to screen proprietary compounds with regard to their potential to induce PLD. Two in vitro approaches, a genomic method previously evaluated in our laboratory and a fluorescent cell based approach to detect PLD were compared using HepG2 cells. The same set of reference compounds (15 PLD inducing, 7 non-PLD inducing and 4 additional compounds) were used to compare both approaches. The same sensitivity (15/15) and similar specificity (7/7 versus, 6/7 for the genomic approach) were obtained. In addition, 11 proprietary compounds were tested in 4-day exploratory rat toxicity studies as well as in both in vitro approaches. Two of the 11 compounds induced alveolar foamy macrophages and lung vacuolization in vivo and were considered as PLD inducers. Sensitivity (2/2) and specificity (7/9) were better with the fluorescent method than with the genomic approach (1/2 and 3/9, respectively). In conclusion, compared to the genomic approach, the fluorescent method is the test of choice for screening compounds at an early stage of drug development. Indeed, the fluorescent method is more adapted to medium throughput, detects the positive reference compounds at lower (8/15) or equal (7/15) concentrations, allows multiplexing and is associated with higher sensitivity and specificity to predict lung foamy macrophages and vacuolization in vivo. Nevertheless, to confirm our conclusion, it would be necessary to compare the predictivity of both in vitro approaches by using a wide range of reference and proprietary compounds, with information on their potential to induce PLD under in vivo conditions.     

Systemic in vitro and in vivo evaluation for determining the feasibility of making an amorphous solid dispersion of a B-Raf (rapidly accelerated fibrosarcoma) inhibitor

It is well acknowledged that oral bioavailability of a drug candidate is often influenced by factors such as the permeability, physico-chemical properties, and metabolism of the drug. Among the physico-chemical properties, solubility and dissolution rate are considered the most critical factors affecting the oral bioavailability of a compound G–F is a potent and selective B-Raf inhibitor with poor solubility and adsorption is limited by solubility at high doses. In order to overcome this issue using a spray-dried amorphous dispersion (SDD) formulation was evaluated. A combination of theoretical solubility prediction and in vitro dissolution, were used to predict the in vivo exposure of G–F. The predicted value was found to have good agreement with the in vivo exposure from dosing the crystalline and amorphous form of G–F.     

Polymorphisms of human cytochrome P450 2C9 and the functional relevance

Human cytochrome P450 2C9 (CYP2C9) accounts for ∼20% of hepatic total CYP content and metabolizes ∼15% clinical drugs such as phenytoin, S-warfarin, tolbutamide, losartan, and many nonsteroidal anti-inflammatory agents (NSAIDs). CYP2C9 is highly polymorphic, with at least 33 variants of CYP2C9 (*1B through *34) being identified so far. CYP2C9*2 is frequent among Caucasians with ∼1% of the population being homozygous carriers and 22% are heterozygous. The corresponding figures for the CYP2C9*3 allele are 0.4% and 15%, respectively. There are a number of clinical studies addressing the impact of CYP2C9 polymorphisms on the clearance and/or therapeutic response of therapeutic drugs. These studies have highlighted the importance of the CYP2C9*2 and *3 alleles as a determining factor for drug clearance and drug response. The CYP2C9 polymorphisms are relevant for the efficacy and adverse effects of numerous NSAIDs, sulfonylurea antidiabetic drugs and, most critically, oral anticoagulants belonging to the class of vitamin K epoxide reductase inhibitors. Warfarin has served as a practical example of how pharmacogenetics can be utilized to achieve maximum efficacy and minimum toxicity. For many of these drugs, a clear gene–dose and gene–effect relationship has been observed in patients. In this regard, CYP2C9 alleles can be considered as a useful biomarker in monitoring drug response and adverse effects. Genetic testing of CYP2C9 is expected to play a role in predicting drug clearance and conducting individualized pharmacotherapy. However, prospective clinical studies with large samples are warranted to establish gene–dose and gene–effect relationships for CYP2C9 and its substrate drugs.     

Effect of green tea on pharmacokinetics of 5-fluorouracil in rats and pharmacodynamics in human cell lines in vitro

Tea drinking is widely practiced in the world and has recently increased among cancer patients. However, the effects of concurrent consumption of tea on the bioavailability and the net therapeutic potential of co-administered chemical drugs are not clear. In this study, the effects of green tea on the pharmacokinetics of 5-fluorouracil (5-FU) in rats and the pharmacodynamics in human cell lines in vitro were studied. The pharmacokinetic experiment indicated that there was an approximately 151% increase in the maximum plasma concentration (C_max) and an approximately 425% increase in the area under the plasma concentration curve (AUC) of 5-FU in the green tea-treated group compared with the control group. Green tea consumption increased the plasma concentration of 5-FU. In addition, the pharmacodynamics experiment showed that at the moderate dose level (equivalent to <6 cups daily in human), neither fresh green tea extract nor (−)-epigallocatechin-3-gallate (EGCG) showed significant additive effects on the cytotoxicity of 5-FU in human cell lines. The results showed that it is crucial to perform therapeutic drug monitoring (TDM) when the cancer patients have a habit of drinking green tea.     

Novel strategies for microdose studies using non-radiolabeled compounds

Microdose studies using non-radiolabeled compounds enable assessment of the clinical pharmacokinetics of drug candidates in humans without the need to synthesize radiolabeled compounds. We have demonstrated that the quantification limits of many drugs measured by LC-MS/MS are low enough to allow estimation of their pharmacokinetic parameters following administration of a microdose. Our previous microdose studies with LC-MS/MS demonstrated the linear pharmacokinetics of fexofenadine between microdoses and therapeutic doses. We also obtained time profiles of plasma concentrations of nicardipine and its multiple metabolites following administration of a microdose. A significant advantage of using non-radiolabeled compounds is the ability to perform cassette microdose studies. By administering multiple drug candidates to the same subject, we can select compounds with appropriate pharmacokinetic properties simultaneously. We can also clarify major factors dominating the pharmacokinetics of drug candidates by cocktail microdosing of the test compounds and probe substrates with or without specific inhibitors for enzymes/transporters.     

Development of safety profile evaluating pharmacokinetics, pharmacodynamics and toxicity of a combination of pioglitazone and olmesartan medoxomil in Wistar albino rats

Pioglitazone (PIO), an antidiabetic drug and olmesartan medoxomil (OLM), an antihypertensive drug were administered orally alone and in combination to Wistar albino rats for evaluation of pharmacokinetics, pharmacodynamics and repeated dose 28-day oral toxicity of individual drugs and their combination. Pharmacokinetic study was performed by orally administering PIO and OLM at single dose of 3 and 2mg/kg, respectively alone and in combination analyzing the plasma samples using LC-MS/MS. Antidiabetic activity evaluation was done in type-2 diabetes mellitus induced animals at same dose level as in pharmacokinetic study daily for 30 days. PIO and/or OLM were administered orally to animals at daily doses of 50, 100 and 150 mg/kg for 28 days for toxicity study. There was no significant alteration in the pharmacokinetic parameters of either drug indicating absence of any pharmacokinetic interaction when co-administered. Positive pharmacodynamic interaction between PIO and OLM was established in this study. Two drugs in combination showed no evidence of potentiation of 28-day repeated dose toxicity in animals. Again, drugs, alone and in combination, caused only minor changes in clinical-laboratory tests and histopathological change was not found in the experiment performed. In conclusion, PIO and OLM combination can primarily be stated as safe in terms of present toxicity and pharmacokinetics findings.     

Improvement of a capillary electrophoresis/frontal analysis (CE/FA) method for determining binding constants: Discussion on relevant parameters

Drug–plasma protein interactions have a significant impact on both pharmacokinetics (i.e., absorption, distribution, metabolism, and excretion) and pharmacodynamics (pharmacological effects). Therefore, it is of high interest to evaluate this binding during the drug development process. Capillary electrophoresis (CE) is an interesting analytical tool for drug–protein binding characterization because it consumes a relatively low amount of reagents and enables assays that can be carried out under near-physiological conditions. The most interesting mode of CE for the study of biomolecular interactions is CE/frontal analysis (CE/FA). However, some confusion in how to conduct CE/FA experiments has emerged in the literature. The present study examines, using research into drug–albumin interactions as an example, the most important steps to take into consideration when building up new CE/FA binding assays. These include the following: choosing the buffer and applied voltage; evaluating protein adsorption onto the capillary wall; choosing the injection volume; choosing the drug and protein concentrations; and, finally, verifying the co-migration of the protein and drug–protein complex. The experimental part of the present report can serve as a checklist for developing the key parameters that need to be addressed for successful and reliable interaction studies. In a second time, short-end injection was used to enhance throughput. The strengths of the binding constants (K_a) for nine selected drugs (basic, neutral, and acidic substances) to albumin, which is the most important plasma protein, were from log K_a 2.9 to 5.4. These values were compared to those obtained with validated methods and good agreement was achieved.     

Enhancement of muscle blood cell flux and pO2 by cromakalim (BRL 34915) and other compounds enhancing membrane K+ conductance,but not by Ca2+ antagonists or hydralazine,in an animal model of occlusive arterial disease

Summary The effect of Ca²⁺ antagonists, hydralazine and agents which enhance membrane K⁺ conductance (cromakalim, pinacidil and nicorandil) in smooth muscle cells, was compared on normal and hypoxic skeletal muscle blood cell flux and pO₂. The K⁺ conductance enhancers and verapamil, diltiazem and nifedipine increased blood cell flux in normally perfused muscle. At equieffective blood pressure lowering dosages, the Ca²⁺ antagonists produced greater increases than the K⁺ channel openers. Hydralazine did not elevate blood cell flux in the normal muscle. In hypoxic skeletal muscle, the K⁺ conductance enhancers produced a marked increase in blood cell flux and in tissue oxygen tension, indicating that they had increased the nutritive blood flow in the muscle. The Ca²⁺ antagonists and hydralazine either did not change hypoxic muscle blood cell flux and pO₂ or reduced them. The dissimilarity in the activity of the compounds may be due to differences in their site of action in the vascular bed. Ca²⁺ antagonists and hydralazine are known to reduce arteriolar vessel resistance and do not increase blood flow in hypoxic skeletal muscle. The positive effect of cromakalim, pinacidil and nicorandil may be due to relaxant activity on larger arterial blood vessels including collaterals. This effect could be related to their ability to enhance membrane K²⁺ conductance in vascular smooth muscle cells.Send offprint requests to: D. Angersbach     

Mechanisms of olfactory toxicity of the herbicide 2,6-dichlorobenzonitrile: Essential roles of CYP2A5 and target-tissue metabolic activation

The herbicide 2,6-dichlorobenzonitril (DCBN) is a potent and tissue-specific toxicant to the olfactory mucosa (OM). The toxicity of DCBN is mediated by cytochrome P450 (P450)-catalyzed bioactivation; however, it is not known whether target-tissue metabolic activation is essential for toxicity. CYP2A5, expressed abundantly in both liver and OM, was previously found to be one of the P450 enzymes active in DCBN bioactivation in vitro. The aims of this study were to determine the role of CYP2A5 in DCBN toxicity in vivo, by comparing the extents of DCBN toxicity between Cyp2a5-null and wild-type (WT) mice, and to determine whether hepatic microsomal P450 enzymes (including CYP2A5) are essential for the DCBN toxicity, by comparing the extents of DCBN toxicity between liver-Cpr-null (LCN) mice, which have little P450 activity in hepatocytes, and WT mice. We show that the loss of CYP2A5 expression did not alter systemic clearance of DCBN (at 25 mg/kg); but it did inhibit DCBN-induced non-protein thiol depletion and cytotoxicity in the OM. Thus, CYP2A5 plays an essential role in mediating DCBN toxicity in the OM. In contrast to the results seen in the Cyp2a5-null mice, the rates of systemic DCBN clearance were substantially reduced, while the extents of DCBN-induced nasal toxicity were increased, rather than decreased, in the LCN mice, compared to WT mice. Therefore, hepatic P450 enzymes, although essential for DCBN clearance, are not necessary for DCBN-induced OM toxicity. Our findings form the basis for a mechanism-based approach to assessing the potential risks of DCBN nasal toxicity in humans.     

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

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.     

5-Fluorouracil catabolism to 5-fluoro-5,6-dihydrouracil is reduced by acute liver impairment in mice

This study investigated the effect of acute liver damage on the inactivation of 5-fluorouracil (5-FU) to its main catabolite 5-fluoro-5,6-dihydrouracil (5-FUH2) in mice. Plasma pharmacokinetics of 5-FU and 5-FUH2 in mice receiving 5-FU (10, 30, and 90 mg/kg) were compared to those in mice pretreated with carbon tetrachloride and receiving the same 5-FU doses. Carbon tetrachloride-induced hepatic damage was histopathologically examined under light microscopy and serum transaminases and dihydropyrimidine dehydrogenase activities were also measured. Liver histopathology and elevated aminotransferase activity levels confirmed the presence of liver damage. 5-FU C(max) and AUC both increased up to 71% in mice with liver damage. This was reflected by decreased 5-FUH2 production, since 5-FUH2 C(max) and AUC levels decreased up to 47% and 61%, respectively. Metabolic ratios between 5-FUH2 and 5-FU AUCs were considerably decreased as well, further suggesting that liver damage caused a reduction in 5-FU catabolism. DPD activity was not altered in damaged livers. The present results indicate that 5-FU disposition in mice could be profoundly altered in the presence of severe liver impairment, potentially leading to enhanced anabolic activation of 5-FU. This effect seems to be ascribed to a reduction of viable hepatocytes, rather than to an inactivation of DPD activity.