Membrane-ordering effects of barbiturates on pure phospholipid model membranes

Intramolecular excimer formation of 1,3-di(1-pyrenyl)propane (Py-3-Py) and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) were used to investigate the effects of barbiturates on the fluidity of model membranes of phosphatidylcholine (SPMVPC), phosphatidylserine (SPMVPS), and phosphatidylinositol (SPMVPI) fractions of synaptosomal plasma membrane vesicles (SPMV) isolated from bovine cerebral cortex. In a dose-dependent manner, barbiturates decreased the excimer to monomer fluorescence intensity ratio (I′/I) of Py-3-Py and increased the anisotropy(r), rotational relaxation time(φ), limiting anisotropy(r_∞), and order parameter (S) of DPH in SPMVPC, SPMVPS and SPMVPI. This indicates that barbiturates decreased both the lateral and rotational diffusion of the probes in SPMVPC, SPMVPS and SPMVPI. The relative potencies of barbiturates in ordering the membranes were in the order: pentobarbital>hexobarbital>amobarbital>phenobarbital. This order correlates well with the anesthetic potencies of barbiturates and the potencies for enhancement of γ-aminobutyric acid-stimulated chloride uptake. Thus, it is strongly suggested that a close relationship might exist between the membrane-ordering effects of barbiturates and the chloride fluxes across SPMV.     

Effects of dopamine-hci on structural parameters of bovine brain membranes

Fluorescence probes located in different membrane regions were used to evaluate the effect of dopamine.HCl on the structural parameters (transbilayer lateral mobility, annular lipid fluidity, protein distribution, and thickness of the lipid bilayer) of synaptosomal plasma membrane vesicles (SPMV), which were obtained from the bovine cerebral cortex. An experimental procedure was used based on selective quenching of 1,3-di(1-pyrenyl)propane (Py-3-Py) by trinitrophenyl groups, and radiationless energy transfer from the tryptophan of membrane proteins to Py-3-Py and energy transfer from Py-3-Py monomers to 1-anilinonaphthalene-8-sulfonic acid (ANS) was also utilized. Dopamine.HCl increased both the bulk lateral mobility and annular lipid fluidity, and it had a greater fluidizing effect on the inner monolayer than on the outer monolayer. Furthermore, the drug had a clustering effect on membrane proteins.     

The Effect of Lidocaine · HCl on the Fluidity of Native and Model Membrane Lipid Bilayers

The purpose of this study is to investigated the mechanism of pharmacological action of local anesthetic and provide the basic information about the development of new effective local anesthetics. Fluorescent probe techniques were used to evaluate the effect of lidocaine·HCl on the physical properties (transbilayer asymmetric lateral and rotational mobility, annular lipid fluidity and protein distribution) of synaptosomal plasma membrane vesicles (SPMV) isolated from bovine cerebral cortex, and liposomes of total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from the SPMV. An experimental procedure was used based on selective quenching of 1,3-di(1-pyrenyl)propane (Py-3-Py) and 1,6-diphenyl-1,3,5-hexatriene (DPH) by trinitrophenyl groups, and radiationless energy transfer from the tryptophans of membrane proteins to Py-3-Py. Lidocaine·HCl increased the bulk lateral and rotational mobility of neuronal and model membrane lipid bilayes, and had a greater fluidizing effect on the inner monolayer than the outer monolayer. Lidocaine·HCl increased annular lipid fluidity in SPMV lipid bilayers. It also caused membrane proteins to cluster. The most important finding of this study is that there is far greater increase in annular lipid fluidity than that in lateral and rotational mobilities by lidocaine·HCl. Lidocaine·HCl alters the stereo or dynamics of the proteins in the lipid bilayers by combining with lipids, especially with the annular lipids. In conclusion, the present data suggest that lidocaine, in addition to its direct interaction with proteins, concurrently interacts with membrane lipids, fluidizing the membrane, and thus inducing conformational changes of proteins known to be intimately associated with membrane lipid.     

Pharmacokinetics and distribution of 6-mercaptopurine administered intravenously in children with lymphoblastic leukaemia

Objective: The pharmacokinetics of 6-mercaptopurine, including cerebrospinal-fluid (CSF) distribution, and the erythrocyte 6-thioguanine nucleotide concentrations were determined in children randomised to receive intravenous mercaptopurine for acute lymphoblastic leukaemia (ALL), according to the EORTC protocol ALL n°58881. Results: After 1 month of oral treatment at a dose of 50 mg · m⁻² · day⁻¹, the pharmacokinetic parameters were determined after the first i.v. administration of 1 g · m⁻² (bolus dose of 0.2 g · m⁻² followed by an 8-h infusion of 0.8 g · m⁻²) in 11 patients: systemic clearance was 23.02 l · h⁻¹, volume of distribution was 0.75 l · kg⁻¹, and elimination half-life was 1.64 h. The erythrocyte thioguanine concentrations were measured in the same 11 patients and increased significantly between the beginning and the end of infusion (10 pmol × 10⁸ packed RBC) or within 24 h of infusion (223 pmol × 10⁸ packed RBC). The CSF concentration was 3.78 μmol · l⁻¹, 1–6 h after the beginning of infusion (n=28) and the CSF to plasma ratio was 0.15 (n=16). In patients receiving the oral dose of 50–165 mg · m⁻² · day⁻¹ of 6-mercaptopurine, CSF concentrations were below 0.18 μmol · l⁻¹, 1–24 h after drug intake (n=67), and the CSF to plasma ratio was not calculated. Conclusion: Following the i.v. administration of 6-mercaptopurine, we observed high CSF concentrations of 6-mercaptopurine and an acute increase of erythrocyte thioguanine nucleotide concentrations. The clinical trial (EORTC protocol ALL n°5881), comparing the oral and i.v. administrations of mercaptopurine, will demonstrate if the i.v. administration reduces the incidence of CNS relapses. Received: 15 August 1996 / Accepted in revised form: 8 April 1997     

Urotensin II acutely increases myocardial length and distensibility: potential implications for diastolic function and ventricular remodeling

Urotensin II (U-II) is a cyclic peptide that may be involved in cardiovascular dysfunction. In the present study, the acute effects of U-II on diastolic properties of the myocardium were investigated. Increasing concentrations of U-II (10⁻⁸ to 10⁻⁶ M) were added to rabbit papillary muscles in the absence (n = 15) or presence of: (1) damaged endocardial endothelium (EE; n = 9); (2) U-II receptor antagonist, urantide (10⁻⁵ M; n = 7); (3) nitric oxide (NO) synthase inhibitor, N^G-Nitro-l-Arginine (10⁻⁵ M; n = 9); (4) cyclooxygenase inhibitor, indomethacin (10⁻⁵ M; n = 8); (5) NO synthase and cyclooxygenase inhibitors, N^G-Nitro-l-Arginine (10⁻⁵ M) and indomethacin (10⁻⁵ M), respectively, (n = 8); or (6) protein kinase C (PKC) inhibitor, chelerythrine (10⁻⁵ M; n = 9). Passive length–tension relations were constructed before and after a single concentration of U-II (10⁻⁶ M; n = 3). U-II concentration dependently decreased inotropy and increased resting muscle length (RL). At 10⁻⁶ M, active tension decreased 13.8 ± 5.4%, and RL increased to 1.007 ± 0.001 L/L _max. Correcting RL to its initial value resulted in an 18.1 ± 3.0% decrease in resting tension, indicating decreased muscle stiffness, which was also suggested by the down and rightward shift of the passive length–tension relation. This effect remained unaffected by EE damage and PKC inhibition. In contrast, the presence of urantide and NO inhibition abolished the effects of U-II on myocardial stiffness, while cyclooxygenase inhibition significantly attenuated them. U-II decreases myocardial stiffness, an effect that is mediated by the urotensin-II receptor, NO, and prostaglandins. This represents a novel mechanism of acute neurohumoral modulation of diastolic function, suggesting that U-II is an important regulator of cardiac filling. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Presented in part at the American Heart Association Scientific Sessions conference, 2006, in Chicago, Illinois.     

Pharmacokinetics of cloxacillin in patients undergoing hip or knee replacement

Objective: The pharmacokinetics of cloxacillin was investigated in 14 men and 24 women undergoing cemented hip (n = 19; age range 56–90) or knee replacement surgery (n = 19; age range 51–84) for osteoarthritis. Cloxacillin 1 g was given intravenously as a bolus dose at the induction of anesthesia, and plasma samples and urine were collected for 6 h. Drug levels were determined using HPLC. Results: Preoperative serum creatinine levels were 84 μmol · l⁻¹ in hip patients and 72 μmol · l⁻¹ in knee patients. The calculated values for creatinine clearance were 63 and 85 ml · min⁻¹ · 1.73 m⁻², respectively. Total clearance of cloxacillin was 134 ml · min⁻¹ · 1.73 m⁻² in eighteen evaluated patients undergoing hip replacement, and 162 ml · min⁻¹ · 1.73 m⁻² in eighteen patients undergoing knee surgery. Renal clearance was 72 and 79 ml · min⁻¹ · 1.73 m⁻², respectively. Non-renal clearance was 57 ml · min⁻¹ · 1.73 m⁻² in hip patients and 77 ml · min⁻¹ · 1.73 m⁻² in knee patients. Renal clearance of cloxacillin correlated with the estimated creatinine clearance (r = 0.652). Although women received higher doses than men (median 2.02 vs 2.32 mmol · 1.73 m⁻²), there were no sex differences in clearance corrected for body surface area. Conclusion: Total clearance of cloxacillin was lower in patients undergoing hip replacement than in patients undergoing replacement of the knee, but there was no difference between men and women. Received: 7 May 1996 / Accepted in revised form: 15 October 1996     

Effects of thiopentone/suxamethonium on intraocular pressure after pretreatment with alfentanil

Summary The effects of pretreatment with alfentanil on intraocular pressure (IOP) were investigated in 40 patients undergoing ophthalmic surgery. Patients were randomly allocated to two study groups. Group 1 patients (n=20) received alfentanil 15 μg · kg⁻¹, vecuronium 0.01 mg · kg⁻¹, thiopentone 3-4 mg · kg⁻¹, and suxamethonium 1 mg · kg⁻¹ for anaesthetic induction, whereas patients in group 2 (n = 20) received vecuronium 0.01 mg · kg⁻¹, thiopentone 3–4 mg · kg⁻¹, and suxamethonium 1 mg · kg⁻¹. A total of seven measurements of intraocular pressure were taken in each patient, starting before premedication and ending after extubation of the trachea. In group 2 patients, there was an increase in IOP after endotracheal incubation. In group 1 patients, a decrease in IOP occurred which was related to the decrease in arterial blood pressure. We conclude that alfentanil pretreatment can prevent the increase in IOP following suxamethonium administration.     

Extrahepatic glucuronidation of propofol in man: possible contribution of gut wall and kidney

Objective: Results from clinical pharmacokinetic studies of propofol indicate that this i.v. anaesthetic agent may undergo significant extrahepatic glucuronidation. We have investigated whether glucuronidation of propofol takes place in the kidney and/or the gut wall. First, propofol concentrations were measured in arterial (radial artery) and portal venous blood of 12 cirrhotic patients with trans internal jugular porto-systemic shunting (TIPSS). Results: In 7 of the 12 patients arterial propofol concentrations were higher than portal venous concentrations. In the remaining patients, propofol concentrations were higher in the portal vein than the radial artery. Since an additional study in 5 patients anaesthetized with propofol while undergoing cholecystectomy showed propofol and an acid-labile conjugate of it in bile, it is difficult to interpret the results in patients with TIPSS due to the possibility of enterohepatic cycling. Next, in vitro studies with human liver (n = 5), kidney (n = 5) and small intestinal (n = 5) microsomes showed that all three tissues were capable of forming propofol glucuronide. V_max for propofol glucuronidation was approximately 3 to 3.5 times higher in kidney (5.56 nmol ⋅ min⁻¹⋅ mg⁻¹ protein) than liver (1.80 nmol ⋅ min⁻¹⋅ mg⁻¹ protein) and small intestine (1.61 nmol ⋅ min⁻¹⋅ mg⁻¹ protein). Conclusion: Based on these in vitro results, it is concluded that extrahepatic glucuronidation in the small intestine and especially in the kidney may contribute to the overall glucuronidation of propofol in man. Received: 28 July 1995/Accepted in revised form: 30 October 1995     

Multidrug resistance modulation in vivo: The effect of cyclosporin A alone or with dexverapamil on idarubicin pharmacokinetics in acute leukemia

Objective: To determine the effect of the coadministration of the multidrug resistance (MDR) modulators cyclosporin A (CyA) alone or plus dexverapamil (D-Ver) on idarubicin (IDA) pharmacokinetics in patients with acute leukemia. Methods: Pharmacokinetic studies were performed in 27 patients with a diagnosis of acute myelogenous leukemia (AML), who were being treated with a combination chemotherapy regimen including idarubicin and cytarabine for the induction of a first remission (n = 14), or of a second remission (n = 7), or for remission consolidation (n = 6). Of these 27 patients, nine were coadministered CyA and seven were coadministered CyA plus D-Ver as MDR modulators. Blood was sampled at appropriate intervals after each of the three IDA daily administrations. IDA and idarubicinol (IDAOL) were assayed by HPLC. Pharmacokinetic evaluations were performed by means of a two-compartment open model with zero-order absorption and first-order elimination using the WinNonlin pharmacokinetic software package. Results: CyA markedly increased the area under the concentration time-curve (AUC) of both IDA [558.26 (197.25) μg · h · l⁻¹ vs 315.44 (158.28) μg · h · l⁻¹; P < 0.01] and IDAOL [2896.60 (736.38) μg · h · l⁻¹ vs 1028.49 (603.95) μg · h · l⁻¹; P < 0.001] when coadministered as a single modulator, due to a lower total body clearance (CL) [83.51 (52.44) l · h⁻¹ · m⁻² vs 139.65 (69.45) l · h⁻¹ · m⁻²; NS]. When patients received two MDR modulators simultaneously (D-Ver plus CyA), IDA exposure was essentially the same as in those of the no inhibitor group [331.29 (95.49) μg · h · l⁻¹ vs 315.44 (158.28) μg · h · l⁻¹; NS], whereas the IDAOL total body exposure was greater than in the no inhibitor group [2030.32 (401.11) μg · h · l⁻¹ vs 1028.49 (603.95) μg · h · l⁻¹; P < 0.01], even if less than in patients receiving CyA as a single MDR modulator (IDA + CyA group) [AUC 2030.32 (401.11) μg · h · l⁻¹ vs 2896.60 (736.38) μg · h · l⁻¹; P < 0.05], suggesting an antagonistic effect against those of CyA on IDA and IDAOL elimination and/or an unpredictable redistribution. The main pharmacokinetic parameters of IDA, such as CL and volume of distribution at steady state (Vd_ss), were remarkably affected by the coadministration of CyA or CyA plus D-Ver, but no statistically significant difference was noted because of IDA pharmacokinetic interpatient variation. Conclusion: The results show that CyA alone at a dose of 10 mg · kg⁻¹ daily significantly increased systemic body exposure to both IDA and IDAOL in acute leukemia, and suggest that these pharmacokinetic effects were at least partially decreased when D-Ver was coadministered with CyA. Our findings raise important questions concerning the need for a dosage adjustment of IDA when MDR modulators are coadministered. Received: 2 June 1998 / Accepted in revised form: 3 December 1998     

Influence of the SLCO1B1*1b and *5 haplotypes on pravastatin’s cholesterol lowering capabilities and basal sterol serum levels

We previously showed that variant SLCO1B1 haplotype *1b (A388G) accelerates and that *5 (T521C) delays hepatocellular uptake of the HMG-CoA reductase inhibitor pravastatin [Mwinyi et al. (2004): Clin Pharmacol Ther 75:415–421]. In the present study we checked for differential effects of variant SLCO1B1 haplotypes on hepatocellular cholesterol synthesis. We analyzed the serum levels of cholesterol, lathosterol, and campesterol in healthy white males which had been grouped on the basis of their SLCO1B1 haplotype: *1a (n=10), *1b (n=10), and *5 (n=8). The subjects received a single oral dose of 40 mg pravastatin. Cholesterol and lathosterol levels were lower in all subjects following pravastatin intake for up to 24. Median levels 6 h post-dosing of lathosterol decreased in each SLCO1B1 haplotype group in the rank order of *1b (−0.11 mg dl^–1; min–max: −0.20 to −0.04; p=0.005) > *1a (−0.09 mg dl^–1; min–max: −0.22 to −0.05; p=0.005) > *5 (−0.07 mg dl^–1; min–max: −0.17 to −0.05; p=0.012). Lathosterol median-change values were significantly greater in haplotype *1b than in haplotype *5 individuals (p=0.041, non-adjusted), which was congruent with the extent of mean changes in lathosterol-to-cholesterol ratios, although the latter did not reach statistical significance. Post-treatment serum levels of campesterol were not affected by SLCO1B1 haplotype. Interestingly, sterol basal serum levels tended to be highest in *1b carriers, followed by those in *1a and *5 individuals, with significant differences in lathosterol concentrations between the *1b and *5 (p=0.041, non-adjusted) haplotype group. Our findings suggest an association of SLCO1B1*1b and *5 haplotypes to pravastatin’s inhibition of the hepatocellular HMG-CoA reductase. Furthermore, SLCO1B1 haplotypes seem to play a role in basal cholesterol homeostasis.     

Involvement of multiple cytochrome P450 isoforms in naproxen O-demethylation

Objective: A series of studies was undertaken to determine the cytochrome P450 isoform(s) involved in naproxen demethylation and whether this included the same isoforms reported to be involved in the metabolism of other NSAIDs. Methods: (S)-Naproxen was incubated with human liver microsomes in the presence of a NADPH-generating system and the formation of desmethylnaproxen was measured by high-performance liquid chromatography (HPLC). To further clarify the specific isoforms involved, experiments were conducted with preparations expressing only a single P450 isoform (vaccinia virus-expressed cells and microsomes derived from a lymphoblastoid cell line, each transfected with specific P450 cDNAs) as well as inhibition studies using human liver microsomes and putative specific P450 inhibitors. Results: In human liver microsomes (n=7), desmethylnaproxen formation was observed with a mean k_M of 92 (21) μmol · l⁻¹, V_max of 538 pmol · min⁻¹ · mg⁻¹ protein and C_int2 (reflective of a second binding site) of 0.36 μl · min⁻¹ · mg⁻¹ protein. This C_int2 term was added since Eadie-Scatchard analysis suggested the involvement of more than one enzyme. Studies using putative specific P450 inhibitors demonstrated inhibition of this␣reaction by sulfaphenazole, (apparent Ki= 1.6 μmol · l⁻¹), warfarin (apparent Ki=27 μmol · l⁻¹), piroxicam (apparent Ki=23 μmol · l⁻¹) and tolbutamide (apparent Ki=128 μmol · l⁻¹). No effect was observed when α-naphthoflavone and troleandomycin were employed as inhibitors, but reaction with furafylline produced, on average, a maximum inhibition of 23%. At a naproxen concentration of 150 μmol · l⁻¹, formation of desmethylnaproxen was observed in cells expressing P450 1A2, 2C8, 2C9 and its allelic variant 2C9R144C. To further characterize these reactions, saturation kinetics experiments were conducted for the P450s 1A2, 2C8 and 2C9. The k_M and V_max for P450 1A2 were 189.5 μmol · l⁻¹ and 7.3 pmol · min⁻¹ · pmol⁻¹ P450, respectively. Likewise, estimates of k_M and V_max for P450 2C9 were 340.5 μmol · l⁻¹ and 41.4 pmol · min⁻¹ · pmol⁻¹ P450, respectively. Reliable estimates of k_M and V_max could not be made for P450 2C8 due to the nonsaturable nature of the process over the concentration range studied. Conclusion: Multiple cytochrome P450 isoforms (P450 1A2, 2C8 and 2C9) appear to be involved in naproxen demethylation, although 2C9 appears to be the predominant form. Received: 16 September 1996 / Accepted in revised form: 20 December 1996     

Thiopurine S-methyltransferase activity in human erythrocytes: a new HPLC method using 6-thioguanine as substrate

Objectives: To develop a non-radioactive assay to measure thiopurine S-methyltransferase (TPMT) activity. The assay was used to study the distribution of TPMT activity in a healthy German population. Methods: The assay is based on the conversion of 6-thioguanine (6-TG) to 6-methylthioguanine (6-MTG) using non-radiolabelled S-adenosyl-l-methionine (SAM) as the methyl donor. At the end of the incubation period (60 min) 6-MTG is extracted into chloroform/2-propanol and quantitated by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection at Ex 315 nm and Em 390 nm. Results and discussion: The method is rapid, sensitive and reproducible, with an interassay CV of 6.7% (quality control sample with TPMT activity of 43 nmol 6-MTG · g⁻¹ Hb · h⁻¹) and thus suitable for routine monitoring of TPMT activity. The TPMT activity of 219 healthy German blood donors showed the known trimodal distribution with a range from 1.3 to 68.3 nmol 6-MTG · g⁻¹ Hb · h⁻¹ with a median value of 38.8 nmol 6-MTG · g⁻¹ Hb · h⁻¹. When the cut-off value for intermediate to high activity was set at 23.5 nmol 6-MTG · g⁻¹ Hb · h⁻¹, 14.1% belonged to the group with intermediate and 83.6% to the group with high TPMT activity. Five individuals had a very low TPMT activity of <2 nmol 6-MTG · g⁻¹ Hb · h⁻¹. Genetic analysis revealed that these persons were found either homozygote for the variant allele *3A (n=3) or they were compound heterozygotes for the variant alleles *3A/*3C (n=2). With these alleles for low TPMT activity they would run an increased risk of myelosuppression in case of treatment with standard doses of thiopurine drugs. Received: 30 September 1997 / Accepted in revised form: 17 January 1998     

CYP2C19 genotypes and omeprazole metabolism after single and repeated dosing when combined with clarithromycin

Objectives: Omeprazole is metabolized mainly by CYP2C19 which has two major mutations (CYP2C19*2 in exon5 and CYP2C19*3 in exon4) associated with the poor metabolizer (PM) phenotype. The aim of this study was to examine the relationship between genetic polymorphism of CYP2C19 and metabolism of omeprazole administrated as a single dose or as repeated-doses, which were in both cases co-administered with clarithromycin. Methods: Twelve healthy Japanese subjects were typed for CYP2C19 polymorphism. In the single-dose study, plasma levels of omeprazole and its metabolites were measured for 24 h after administration of 20 mg omeprazole and 400 mg clarithromycin to six healthy Japanese subjects. In the repeated-dose study, plasma levels of omeprazole and its metabolites were measured after repeated oral administration of 20 mg omeprazole and 400 mg clarithromycin twice daily for 6 days and then after 20 mg omeprazole and 400 mg clarithromycin once on the 7th day to the other 6 healthy Japanese subjects. Results: In the single-dose study, the areas under the plasma concentration-versus-time curve (AUCs) of omeprazole of homozygotes for the wild-type allele (*1/*1 n = 2), heterozygotes (n = 3) for the CYP2C19*2 (*1/*2) or for the CYP2C19*3 (*1/*3) and heterozygote (n = 1) for the two defects (*2/*3) were on average 450, 1007 and 6710 ng · h⁻¹ · ml⁻¹, respectively. The ratios of AUCs of omeprazole/5-hydroxyomeprazole for *1/*1, *1/*2 or *1/*3 and *2/*3 were 1, 2 and 30, respectively. In the repeated-dose study, the AUCs of omeprazole for *1/*1, *1/*2 or *1/*3 and *2/*3 were 4041 (n = 2), 3149 (n = 3) and 6684 (n = 1) ng · h⁻¹ · ml⁻¹, respectively. The ratios of AUCs of omeprazole/5-hydroxyomeprazole for *1/*1, *1/*2 or *1/*3 and *2/*3 were 7, 11 and 30, respectively. In the repeated-dose study, the AUC of omeprazole of *1/*1 genotypes was nine-fold higher, that of *1/*2 and *1/*3 genotypes was three-fold higher, and the C_max value of omeprazole was three-fold higher compared with subjects with the same genotype in the single-dose study. However, there were few differences in the AUC and C_max of omeprazole between the *2/*3 genotype in the single-dose study and the homozygote for the CYP2C19*2 (*2/*2) in the repeated-dose study. Conclusion: Subjects with *1/*1, *1/*2 and *1/*3 genotypes in the repeated-dose study had lower CYP2C19 activity than subjects of the same genotype in the single-dose study. The difference in omeprazole metabolism between subjects with different genotypes observed on day 1 seemed to disappear after 7 days of repeated-dose administration. Received: 5 June 1998 / Accepted in revised form: 27 October 1998     

Brain Uptake of Nonsteroidal Anti-Inflammatory Drugs: Ibuprofen, Flurbiprofen, and Indomethacin

Purpose To determine the roles of blood–brain barrier (BBB) transport and plasma protein binding in brain uptake of nonsteroidal anti-inflammatory drugs (NSAIDs)—ibuprofen, flurbiprofen, and indomethacin. Methods Brain uptake was measured using in situ rat brain perfusion technique. Results [¹⁴C]Ibuprofen, [³H]flurbiprofen, and [¹⁴C]indomethacin were rapidly taken up into the brain in the absence of plasma protein with BBB permeability–surface area products (PS_u) to free drug of (2.63 ± 0.11) × 10⁻², (1.60 ± 0.08) × 10⁻², and (0.64 ± 0.05) × 10⁻² mL s⁻¹ g⁻¹ (n = 9–11), respectively. BBB [¹⁴C]ibuprofen uptake was inhibited by unlabeled ibuprofen (K_m = 0.85 ± 0.02 mM, V_max = 13.5 ± 0.4 nmol s⁻¹ g⁻¹) and indomethacin, but not by pyruvate, probenecid, digoxin, or valproate. No evidence was found for saturable BBB uptake of [³H]flurbiprofen or [¹⁴C]indomethacin. Initial brain uptake for all three NSAIDs was reduced by the addition of albumin to the perfusion buffer. The magnitude of the brain uptake reduction correlated with the NSAID free fraction in the perfusate. Conclusions Free ibuprofen, flurbiprofen, and indomethacin rapidly cross the BBB, with ibuprofen exhibiting a saturable component of transport. Plasma protein binding limits brain NSAID uptake by reducing the free fraction of NSAID in the circulation.     

Atrial natriuretic factor in patients with acromegaly

Summary In acromegaly the plasma volume is chronically elevated and it returns to normal when the disease is successfully treated. To define the role of ANF in such a chronic disorder of extracellular fluid volume homeostasis the plasma level was assayed in 37 acromegalic patients with active or inactive (successfully treated) disease. Five patients were studied before and after therapy. The effects of acute change in sodium-fluid status on plasma ANF levels was examined in 7 active and 4 inactive acromegalic patients and in 7 healthy subjects. As compared to 14 patients with inactive acromegaly, 23 patients with active acromegaly had an expanded plasma volume (n=12; 50.1 vs 37.6 ml · kg⁻¹ BW) and an increased blood concentration of growth hormone (n=23; 22.5 vs 2.1 ng · ml⁻¹). Plasma ANF concentrations in active and inactive acromegalic patients (33.2 and 26.6 pg · ml⁻¹, respectively) did not differ significantly from one another or from the level in the controls (26.9 pg · ml⁻¹). In those patients there was no correlation between plasma volume and ANF level. Infusion of 21 isotonic saline in 2 h led to a similar, significant increase in ANF levels in active (from 26.2 to 72.4 pg · ml⁻¹) and in inactive acromegalic patients (from 33.6 to 96.7 pg · ml⁻¹) as well as in healthy subjects (from 21 to 70.6 pg · ml⁻¹). Successful treatment reduced the plasma volume (from 49.2 to 35.8 ml · kg⁻¹ BW) and growth hormone level (from 10.1 to 2.6 pg · ml⁻¹), while the ANF level remained unchanged (from 33.8 to 35.5 pg · ml⁻¹). In the patients with active or inactive acromegaly and cardiac failure the plasma ANF level was markedly increased (89.9 and 66.1 pg · ml⁻¹, respectively) as compared to all the other groups. It is concluded that ANF levels are not elevated in chronically volume expanded acromegalic patients. In the present patients ANF was released in response to acute volume expansion, therefore it may be important in the cardiovascular and renal response to acute changes in extracellular fluid volume.     

In rats chronically treated with clozapine, tyrosine depletion attenuates the clozapine-induced in vivo increase in prefrontal cortex dopamine and norepinephrine levels

We previously reported that depletion of brain tyrosine attenuated the acute clozapine (CLZ)-induced increase in medial prefrontal cortex (MPFC) dopamine (DA) levels. This effect was now examined after chronic CLZ treatment. Male rats received CLZ (10 mg kg⁻¹ day⁻¹) in drinking water for 21 days. On day 18, a cannula was stereotaxically implanted over the MPFC. A microdialysis probe was inserted on day 20. On day 21 after a stable baseline was reached, rats received an acute injection of vehicle (VEH) or a tyrosine- and phenylalanine-free mixture of neutral amino acid [NAA(−)] (total 1 g kg⁻¹, i.p., two injections, 1 h apart) followed by CLZ (10 mg kg⁻¹, i.p.) or VEH. Basal tyrosine or norepinephrine (NE) levels were not different between the groups, but basal DA was higher in the group treated chronically with CLZ (p<0.05). Acute CLZ (10 mg kg⁻¹, i.p.) increased MPFC DA and NE levels to 370% and 510% of baseline, respectively, and similarly in rats chronically pretreated with CLZ or VEH. NAA(−) did not affect basal MPFC DA or NE levels but significantly attenuated acute CLZ-induced DA (220% of baseline) and NE (330% of baseline) levels (p<0.01) in rats pretreated chronically with CLZ or with VEH. These data demonstrate that even after chronic CLZ administration, the acute CLZ-induced increases in MPFC DA and NE levels depend on the availability of brain tyrosine. Judicious manipulation of brain tyrosine levels may provide a useful probe as well as a mechanism for enhancing psychotropic drug actions.     

The oxidative metabolism of metoprolol in human liver microsomes: inhibition by the selective serotonin reuptake inhibitors

Objective: Biotransformation of metoprolol to α-hydroxymetoprolol (HM) and O-demethylmetoprolol (ODM) is mediated by CYP2D6. The selective serotonin reuptake inhibitors (SSRIs) are known to inhibit CYP2D6. The aim was to study in vitro the potential inhibitory effect of SSRIs on metoprolol biotransformation. Methods: Using microsomes from two human livers, biotransformation of metoprolol to α-hydroxymetoprolol (HM) and O-demethylmetoprolol (ODM) as a function of the concentrations of the SSRIs and of some of their metabolites was studied. Results: The kinetics of the formation of both metabolites are best described by a biphasic enzyme model. The estimated values of V_max and k_M for the high affinity site are for the α-hydroxylation in human liver HL-1 32 pmol mg⁻¹ min⁻¹ and 75 μmol · l⁻¹ respectively, and in human liver HL-9 39 pmol mg⁻¹ · min⁻¹ and 70 μmol · l⁻¹ respectively; for the O-demethylation in HL-1 131 pmol mg⁻¹ min⁻¹ and 95 μmol · l⁻¹ respectively, and in HL-9 145 pmol mg⁻¹ min⁻¹ and 94 μmol · l⁻¹ respectively. Quinidine is for both pathways a potent inhibitor of the high-affinity site, with K_i values ranging from 0.03 to 0.18 μmol · l⁻¹. Fluoxetine, norfluoxetine and paroxetine are likewise potent inhibitors, with K_i values ranging from 0.30 to 2.1 μmol · l⁻¹ fluvoxamine, sertraline, desmethylsertraline, citalopram and desmethylcitalopram are less potent inhibitors, with K_i values above 10 μmol · l⁻¹. Conclusion: The rank order of the SSRIs for inhibition of metoprolol metabolism is comparable to that reported in the literature for other CYP2D6 substrates, with fluoxetine, norfluoxetine and paroxetine being the most potent. These findings need further investigation to determine their clinical relevance. Received: 24 October 1997 / Accepted: 17 January 1998     

Glucose Partition Coefficient and Diffusivity in the Lower Skin Layers

Purpose This work aims to estimate the diffusivity and partitioning of glucose in the dermis and the viable epidermis of human skin. Methods The partition coefficient of glucose between phosphate-buffered saline and dermis, tape-stripped epidermis (TSE), stratum corneum (SC), and split-thickness skin, was measured in vitro using human cadaver skin. Glucose permeability across dermis and tape-stripped split-thickness skin (TSS) was measured using side-by-side diffusion cells. Glucose desorption from TSE and human epidermal membrane (HEM) was measured. All measurements were conducted at 32°C. Results The partition coefficient for glucose [mean ± SD (no. of samples)] was 0.65 ± 0.09 (n = 25) for dermis, 0.81 ± 0.06 (n = 10) for TSE, and 0.53 ± 0.12 (n = 9) for SC. Glucose diffusivity in dermis was calculated to be 2.64 ± 0.42 × 10⁻⁶ cm²/s (n = 14). Glucose diffusivities in the viable epidermis estimated from TSS permeation, TSE desorption, and HEM desorption were 0.075 ± 0.050 × 10⁻⁶ cm²/s (n = 5), 0.037 ± 0.018 × 10⁻⁶ cm²/s (n = 4), and 1.0 ± 0.6 × 10⁻⁶ cm²/s (n = 4), respectively. Conclusion The tissue/buffer partition coefficient of glucose in all skin layers was found to be less than unity, suggestive of excluded volumes in each layer. Glucose diffusivity in human dermis was found to be one third of its value in water, indicative of hindered diffusion related to the structural components of the tissue. A substantially lower value for glucose diffusivity in viable epidermis is suggested.     

Associations between CYP2E1 promoter polymorphisms and plasma 1,3-dimethyluric acid/theophylline ratios

Objective Theophylline is metabolized to 1,3-dimethyluric acid (1,3-DMU), 3-methylxanthine, and 1-methylxanthine by CYP1A2 and partly by CYP2E1. Because 1,3-DMU is the major metabolite of theophylline, the 1,3-DMU/theophylline ratio is viewed as a good indicator of theophylline metabolic clearance. Here, we investigated the associations between 1,3-DMU/theophylline ratios and genetic polymorphisms of CYP2E1 and CYP1A2.Methods Polymerase chain reaction (PCR) and direct sequencing or PCR-restriction fragment length polymorphism (RFLP) were performed to analyze CYP2E1 and CYP1A2 promoter polymorphisms in 62 Korean asthma patients. Plasma theophylline and 1,3-DMU levels were measured by liquid chromatography-tandem mass spectrometry.Results Eleven polymorphisms including Ins₉₆, −1566 T>A, −1515 T>G, −1414 C>T, −1295 G>C, −1055 C>T, −1027 T>C, −930 A>G, −807 T>C, −352 A>G, and −333 T>A were detected in the 5′ flanking region of the CYP2E1 gene (numbering according to GenBank Accession number NT_017795). Of these, five single nucleotide polymorphisms (SNPs) (−1566 T>A, −1295 G>C, −1055 C>T, −1027 T>C, and −807 T>C) were closely linked. Another three polymorphisms (Ins_96, −930 A>G, and −352 A>G) and two polymorphisms (−1515 T>G and −333 T>A) were also closely linked. The five closely linked polymorphisms were associated with significantly different 1,3-DMU/theophylline ratios between heterozygotes plus homozygotes of a rare allele (n=23, 0.0368±0.0171) and common allelic homozygotes (n=39, 0.0533±0.0343) (p=0.024 by Mann-Whitney U test). In the CYP1A2 gene, the −2964G>A polymorphisms exhibited a significant difference in 1,3-DMU/theophylline levels between heterozygotes plus homozygotes of a rare allele (n=30, 0.0406±0.0272) and homozygotes of a common allele (n=32, 0.0534±0.0316) (p=0.032).Conclusion We confirm that hydroxylation at the 8 position of theophylline (1,3-DMU) is significantly affected by genetic polymorphism in CYP2E1 in addition to CYP1A2.     

Dose-dependent influence of buspirone on the activities of selective serotonin reuptake inhibitors in the mouse forced swimming test

Recent clinical data suggest that buspirone may enhance the efficacy and/or reduce the latency to therapeutic effect of selective serotonin reuptake inhibitors (SSRIs) in unipolar major depressive disorder. The present study, using the mouse forced swimming test, was performed to investigate further the mechanisms involved in the potential antidepressant-enhancing effects of buspirone. Prior administration of buspirone (0.06 mg kg⁻¹, IP) significantly enhanced the anti-immobility effects of subactive doses of fluvoxamine (4 mg kg⁻¹, IP; P < 0.01), paroxetine (4 mg kg⁻¹, IP; P < 0.01), citalopram (4 mg kg⁻¹, IP; P < 0.01) and sertraline (2 mg kg⁻¹, IP; P < 0.01) in the forced swimming test. However, pretreatment with buspirone did not induce antidepressant-like effects when tested in combination with fluoxetine (4 mg kg⁻¹, IP). Each antidepressant tested reduced immobility time in the forced swimming test [citalopram (16 mg kg⁻¹, IP; P < 0.01), fluoxetine (32 mg kg⁻¹, IP; P < 0.01), fluvoxamine (32 mg kg⁻¹, IP; P < 0.01), paroxetine (16 mg kg⁻¹, IP; P < 0.01) and sertraline (16 mg kg⁻¹, IP; P < 0.01)]. Pretreatment with buspirone (0.5 mg kg⁻¹, IP), or its major metabolite 1-PP (0.5 mg kg⁻¹, IP), attenuated all SSRI-induced anti-immobility effects (P < 0.01). Concomitant studies of locomotor activity ruled out any stimulant or sedative effects of the interactions. The results of the present study suggested that low dose buspirone enhanced the activity of subactive doses of SSRIs in the mouse forced swimming test, probably via an action at 5-HT_1A receptors. On the other hand, a high dose of buspirone attenuated the antidepressant-like effects of active doses of these drugs, possibly via the generation of an active metabolite (1-PP) acting at alpha₂-adrenoreceptors. Received: 19 October 1997 / Final version: 16 December 1997