The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins.

Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (Kd) determined were 1.08±0.49 and 0.0086±0.0006µM, respectively, consistent with previously reported values. Furthermore, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy.

{Beta}2-adrenergic receptor agonists inhibit the proliferation of 1321N1 astrocytoma cells.

Astrocytomas and glioblastomas have been particularly difficult to treat and refractory to chemotherapy. However, significant evidence has been presented that demonstrates a decrease in astrocytoma cell proliferation subsequent to an increase in cAMP levels. The 1321N1 astrocytoma cell line, as well as other astrocytomas and glioblastomas, expresses ß(2)-adrenergic receptors (ß(2)-ARs) that are coupled to G(s) activation and consequent cAMP production. Experiments were conducted to determine whether the ß(2)-AR agonist (R,R')-fenoterol and other ß(2)-AR agonists could attenuate mitogenesis and, if so, by what mechanism. Receptor binding studies were conducted to characterize ß(2)-AR found in 1321N1 and U118 cell membranes. In addition, cells were incubated with (R,R')-fenoterol and analogs to determine their ability to stimulate intracellular cAMP accumulation and inhibit [(3)H]thymidine incorporation into the cells. 1321N1 cells contain significant levels of ß(2)-AR as determined by receptor binding. (R,R')-fenoterol and other ß(2)-AR agonists, as well as forskolin, stimulated cAMP accumulation in a dose-dependent manner. Accumulation of cAMP induced a decrease in [(3)H]thymidine incorporation. There was a correlation between concentration required to stimulate cAMP accumulation and inhibit [(3)H]thymidine incorporation. U118 cells have a reduced number of ß(2)-ARs and a concomitant reduction in the ability of ß(2)-AR agonists to inhibit cell proliferation. These studies demonstrate the efficacy of ß(2)-AR agonists for inhibition of growth of the astrocytoma cell lines. Because a significant portion of brain tumors contain ß(2)-ARs to a greater extent than whole brain, (R,R')-fenoterol, or some analog, may be useful in the treatment of brain tumors after biopsy to determine ß(2)-AR expression.

Development and characterization of immobilized cannabinoid receptor (CB1/CB2) open tubular column for on-line screening.

Cannabinoid receptors, CB1 and CB2, are therapeutic targets in the treatment of anxiety, obesity, movement disorders, glaucoma, and pain. We have developed an on-line screening method for CB1 and CB2 ligands, where cellular membrane fragments of a chronic myelogenous leukemia cell line, KU-812, were immobilized onto the surface of an open tubular (OT) capillary to create a CB1/CB2-OT column. The binding activities of the immobilized CB1/CB2 receptors were established using frontal affinity chromatographic techniques. This is the first report that confirms the presence of functional CB1 and CB2 receptors on KU-812 cells. The data from this study confirm that the CB1/CB2-OT column can be used to determine the binding affinities (K(i) values) for a single compound and to screen individual compounds or a mixture of multiple compounds. The CB1/CB2-OT column was also used to screen a botanical matrix, Zanthoxylum clava-herculis, where preliminary results suggest the presence of a high-affinity phytocannabinoid.

Stereoselective binding of chiral ligands to single nucleotide polymorphisms of the human organic cation transporter-1 determined using cellular membrane affinity chromatography.

Membranes from stably transfected cell lines that express two point mutations of the human organic cation transporter-1 (hOCT1), R488M and G465R, have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form two cellular membrane affinity chromatography (CMAC) columns, CMAC(hOCT1(G465R)) and CMAC(hOCT1(R488M)). Columns were created using both stationary phases, and frontal displacement chromatography experiments were conducted using [(3)H] MMP(+) (1-methyl-4-phenylpyridinium) as the marker ligand and various displacers, including the single enantiomers of verapamil, fenoterol, and isoproterenol. The chromatographic data obtained were used to refine a previously developed pharmacophore for hOCT1.

Pharmacokinetics and metabolism of (R,R)-methoxyfenoterol in rat.

(R,R)-fenoterol (Fen), a beta(2)-adrenoceptor agonist, is under clinical investigation in the treatment of congestive heart disease. The pharmacokinetics and metabolism of the 4-methoxyphenyl derivative of (R,R)-Fen, (R,R)-MFen, have been determined following intravenous and oral administration to the rat and compared with corresponding results obtained with (R,R)-Fen. Results from the study suggest that (R,R)-MFen can offer pharmacokinetic and metabolic advantages in comparison to an earlier (R,R)-Fen. The oral administration revealed that the net exposure of (R,R)-MFen was about three-fold higher than that of (R,R)-Fen (7.2 versus 2.3 min x nmol ml(-1)), while intravenous administration proved that the clearance was significantly reduced, 48 versus 146 ml min(-1) kg(-1), the T(1/2) was significantly longer, 152.9 versus 108.9 min, and the area under the curve (AUC) was significantly increased, 300 versus 119 min x nmol ml(-1). (R,R)-MFen was primarily cleared by glucuronidation associated with significant presystemic glucuronidation of the compound. After intravenous and oral administration of (R,R)-MFen, (R,R)-Fen and (R,R)-Fen-G were detected in the urine samples indicating that (R,R)-MFen was O-demethylated and subsequently conjugated to (R,R)-Fen-G. The total (R,R)-Fen and (R,R)-Fen-G as a percentage of the dose after intravenous administration was 3.6%, while after oral administration was 0.3%, indicating that only a small fraction of the drug escaped presystemic glucuronidation and was available for O-demethylation. The glucuronidation pattern was confirmed by the results from in vitro studies where incubation of (R,R)-MFen with rat hepatocytes produced (R,R)-MFen-G, (R,R)-Fen and (R,R)-Fen-G, while incubation with rat intestinal microsomes only resulted in the formation of (R,R)-MFen-G.

Characterization of a multiple ligand-gated ion channel cellular membrane affinity chromatography column and identification of endogenously expressed receptors in astrocytoma cell lines.

Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [(3)H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) and heteromeric nicotinic acetylcholine receptors (alpha(x)beta(y) nAChRs), which was confirmed by the addition of subtype-specific inhibitors, alpha-bungarotoxin (alpha7 nAChR) and kappa-bungarotoxin (alpha(x)beta(y) nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), gamma-aminobutyric acid (GABA(A)) and N-methyl-D-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABA(A) receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors.

Ligand and protein fishing with heat shock protein 90 coated magnetic beads.

Heat shock protein 90alpha (Hsp90alpha) is a molecular chaperone that has been targeted for the development of new anticancer therapies. To date, co-immunoprecipitation (IP) has been primarily used to identify novel client proteins. We now report an alternative approach in which Hsp90alpha has been immobilized onto the surface of silica-based magnetic beads. The beads were used to isolate known Hsp90alpha ligands from a mixture containing ligands and nonligands. In addition, they were also used to isolated proteins from a mixture of proteins, as well as a cellular extract. The results indicate that the Hsp90alpha coated magnetic beads can be used to "fish" from complex chemical and biological mixtures for new lead drug candidates and client proteins.

Determination and modelling of stereoselective interactions of ligands with drug transporters: a key dimension in the understanding of drug disposition.

1. Stereochemistry is an important dimension in pharmacology and plays a role in every aspect of the pharmacological fate of chiral xenobiotics. This includes small molecule-drug transporter binding. 2. This paper reviews the reported stereoselectivities of substrate and inhibitor interactions with P-glycoprotein and the organic cation transporter obtained using standard functional and binding studies, as well as data obtained from online cellular membrane affinity chromatography studies. 3. The use of stereochemical data in quantitative structure-activity relationship (QSAR) and pharmacophore modelling is also addressed as is the effect of ignoring the fact that small molecule-drug transporter interactions take place in three-dimensional and asymmetric space.

Pharmacophore modelling of stereoselective binding to the human organic cation transporter (hOCT1).

BACKGROUND AND PURPOSE: The human organic cation transporter-1 (hOCT1) is a polyspecific transporter that plays a role in drug distribution, metabolism and excretion. Previous studies have demonstrated that hOCT1 binding can be stereoselective, but the mechanism for stereochemical recognition has not been described. The purpose of this study was to develop a pharmacophore model to describe stereoselective binding to hOCT1. EXPERIMENTAL APPROACH: A set of 22 compounds including 8 pairs of enantiomers and five pairs of diastereomers was used to develop a pharmacophore model. The pharmacophore modeling was carried out using Catalyst version 4.11 and HypoGen and was based upon the correlation of the structures and activities (K(i) values) of the compounds used in the study. KEY RESULTS: The resulting model contained a positive ion, hydrophobic and two hydrogen-bond acceptor interaction sites. The relative enantioselectivity of 8/8 enantiomeric pairs and diastereoselectivity of 5/5 diastereomers was described by mapping to a combination of at least 3 of the 4 functional feature sites of the model. CONCLUSIONS AND IMPLICATIONS: The pharmacophore model describes stereoselective interactions with hOCT1 at one of the binding sites on the molecule.

Efficacy, toxicity, pharmacokinetics, and in vitro metabolism of the enantiomers of ifosfamide in mice.

The enantiomers of the anticancer drug ifosfamide, (+)-(R)-IFF and (-)-(S)-IFF, were prepared from the racemic compound rac-IFF using enantioselective liquid chromatographic techniques. The efficacy, toxicity, and pharmacokinetics of the individual enantiomers and rac-IFF were studied in mice. The results of the studies indicate that there were no statistically significant differences between the efficacy of (+)-(R)-IFF, (-)-(S)-IFF, and rac-IFF against childhood rhabdomyosarcoma (HxRh28) maintained in vivo as a xenograft in immune-deprived female CBA/CaJ mice. Similar results were found in toxicity and pharmacokinetic studies conducted in non-tumor-bearing female CBA/CaJ mice. The production of two major metabolites, aldoifosfamide and isophosphoramide mustard, by mice hepatic microsomes from non-tumor-bearing female CBA/CaJ mice was also investigated. There were no statistically significant differences in the calculated kinetic parameters, Vmax and Km, of the production of these two metabolites when the separate enantiomers or the racemic mixture were used as substrate.

Efficacy and toxicity of ifosfamide stereoisomers in an in vivo rat mammary carcinoma model.

Ifosfamide (IFF) is a nitrogen mustard with significant activity against a number of tumors. Since it is a chiral molecule, it has been suggested that enantioselective metabolism could result in different efficacy and toxicity profiles for (R)- and (S)-ifosfamide. Both experimental animal and clinical data suggest that N-dechloroethyl metabolites of (S)-IFF are more significantly associated with neurological toxicity, which may limit therapeutic use of IFF. We have used purified ifosfamide enantiomers to examine the pharmacokinetics; spectrum of toxicity including lethality, weight loss, and myelosuppression; and antitumor effects of the mixture compared to each of the purified enantiomers. In the MatB mammary carcinoma grown in female Fischer rats we demonstrated that the antitumor efficacy appears to be the same for (R)-IFF and (S)-IFF, while the (R)-IFF has greater myelotoxicity. Pharmacokinetic analysis of plasma concentration-time confirms that the (R)-IFF is metabolized to a greater extent than (S)-IFF via the activation pathway. These data suggest that purified (R)-IFF may be an effective way to delivery active cytotoxic drug while limiting the generation of neurotoxic metabolites.

Influence of dose of [6RS]leucovorin on reduced folate pools and 5-fluorouracil-mediated thymidylate synthase inhibition in human colon adenocarcinoma xenografts.

Using preclinical models of human colon adenocarcinomas in immune-deprived mice, the influence of dose of [6RS]leucovorin ([6RS]LV, 20 to 1000 mg/m2) administered by 24-h i.v. infusion was determined on the following parameters: (a) plasma concentrations of the active [6S] and inactive [6R] isomers of [6RS]LV and the biologically active diastereoisomer of 5-methyltetrahydrolate (5-CH3-H4PteGlu); (b) expansion of intratumor pools of 5,10-methylenetetrahydrofolates (CH2-H4PteGlun) and tetrahydrofolates (H4PteGlun), that may influence the binding of 5-fluorodeoxyuridylate to thymidylate synthase; (c) the distribution of polyglutamate forms of CH2-H4PteGlun and H4PteGlun; and (d) (5-fluorouracil (FUra)-mediated thymidylate synthase inhibition in Hx-ELC2, HxGC3, HxVRC5, and HxHC1 tumors. Folypolyglutamate synthetase activities were also determined in each line. Linear increases in plasma concentrations of [6R]LV, [6S]LV, and 5-CH3-H4-PteGlu were determined over the complete range of [6RS]LV doses examined. However, in neoplastic tissues three patterns of biochemical modulation by [6RS]LV were evident. (a) In HxELC2 and HxVRC5 tumors, pools of CH2-H4PteGlun and H4PteGlun were elevated in proportion to the dose of [6RS]LV between dose levels of 50 and 200 mg/m2. Subsequent expansion of these pools continued that was disproportionate to the dose of [6RS]LV until no further increase was observed beyond 800 mg/m2 [6RS]LV, at which point pools were maximally expanded by 4- to 4.5-fold. The extent of retardation of recovery of thymidylate synthase activity increased as the dose of [6RS]LV was increased in both tumors, when FUra (15 or 50 mg/kg), was administered by i.v. bolus injection 3 h into the 24-h infusion of [6RS]LV. This was related to the increase in predominance of CH2-H4PteGlu2-5 with increasing dose of [6RS]LV. (b) For HxHC1 tumors, little expansion of CH2-H4PteGlun and H4PteGlun pools (maximum, 137% of control) was detected at the highest dose levels of [6RS]LV, and no significant modulation of FUra-inhibited thymidylate synthase activity was detected, even at 1000 mg/m2 [6RS] LV. CH2-H4PteGlu5 remained similar or decreased as the dose of [6RS] LV was increased. (c) For line HxGC3, pools of CH2-H4PteGlun and H4PteGlun increased gradually from 169% of control at 20 mg/m2 [6RS] LV to 233% of control at 1000 mg/m2 [6RS]LV, and were intermediate between the expansion observed in HxHC1 in comparison to HxELC2 and HxVRC5 tumors. CH2-H4PteGlu3-5 were elevated at low dose levels of [6RS]LV.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between dose rate of [6RS]Leucovorin administration, plasma concentrations of reduced folates, and pools of 5,10-methylenetetrahydrofolates and tetrahydrofolates in human colon adenocarcinoma xenografts.

[6RS]Leucovorin (5-formyltetrahydrofolate; 5-CHO-H4PteGlu) administered in different regimens in combination with 5-fluorouracil (FUra) has increased the response rates to FUra in patients with colon adenocarcinoma. Using preclinical models of human colon adenocarcinomas as xenografts in immune-deprived mice, the effect of the rate of administration of racemic [6RS]leucovorin on the concentration-time profile of reduced folates in plasma, size of intratumor pools of 5,10-methylenetetrahydrofolates (CH2-H4PteGlun) and tetrahydrofolates (H4PteGlun), and the distribution of their polyglutamate species have been examined. Bolus injection i.v., or 4-h or 24-h infusion of [6RS]leucovorin (500 mg/m2) yielded similar concentration profiles of the biologically active [6S] and inactive [6R] isomers of 5-CHO-H4-PteGlu and 5-methyltetrahydrofolate (5-CH3-H4PteGlu) in mouse plasma to those previously reported in humans, but with more rapid elimination half-lives (t1/2 = 11 to 16 min, 23 to 41 min, and 30 to 35 min, respectively). Thus, reduced folates remained elevated in plasma during the period of [6RS]leucovorin administration. In HxELC2 and HxGC3 tumors, pools of CH2-H4PteGlun and H4PteGlun were increased from 350% to 700% of control, but only during [6RS]leucovorin infusion. Intracellular levels subsequently declined rapidly, similar to the loss of reduced folates from plasma. Increasing the rate of [6RS]leucovorin delivery by decreasing the time for administration from a 24-h to a 4-h infusion did not further increase the intratumor pools of CH2-H4PteGlun and H4PteGlun, suggesting saturation in the cellular metabolism of [6RS]leucovorin. In HxGC3 tumors, CH2-H4PteGlu4-5 were elevated more rapidly than in line HxELC2, which accumulated predominantly a shorter chain length species following i.v. bolus injection. During the 4-h infusion schedule, di- and triglutamate species in particular accumulated in both tumors with no elevation in CH2-H4PteGlu5 until the infusion was discontinued, when this species increased as the shorter chain length forms were declining. However, during the 24-h infusion of [6RS]leucovorin, CH2-H4PteGlu3-5 were elevated in tumors. Since these species have been reported to increase the binding affinity of [6-3H]5-fluorodeoxyuridine monophosphate ([6-3H]FdUMP) to thymidylate synthase, and intratumor pools of CH2-H4PteGlun and H4PteGlun were elevated during the 24-h infusion of [6RS]leucovorin, this was considered to be the preferred schedule for administration.(ABSTRACT TRUNCATED AT 400 WORDS)

Development and characterization of the α3ß4α5 nicotinic receptor cellular membrane affinity chromatography column and its application for on line screening of plant extracts.

The α3ß4α5 nAChR has been recently shown to be a useful target for smoking cessation pharmacotherapies. Herein, we report on the development and characterization of the α3ß4α5 nicotinic receptor column by frontal displacement chromatography. The binding affinity of the nicotine and minor alkaloids found in tobacco smoke condensates were determined for both the α3ß4 and α3ß4α5 nicotinic receptors. It was demonstrated that while no subtype selectivity was observed for nicotine and nornicotine, anabasine was selective for the α3ß4α5 nicotinic receptor. The non-competitive inhibitor binding site was also studied and it was demonstrated while mecamylamine was not selective between subtypes, buproprion showed subtype selectivity for the α3ß4 nicotinic receptor. The application of this methodology to complex mixtures was then carried out by screening aqueous-alcoholic solutions of targeted plant extracts, including Lycopodium clavatum L. (Lycopodiaceae) and Trigonella foenum graecum L. (Fabaceae) against both the α3ß4 and α3ß4α5 nAChRs.

Subchronic administration of (R,S)-ketamine induces ketamine ring hydroxylation in Wistar rats.

Subchronic administration of (R,S)-ketamine, (R,S)-Ket, is used in the treatment of neuropathic pain, in particular Complex Regional Pain Syndrome, but the effect of this protocol on the metabolism of (R,S)-Ket is unknown. In this study, daily administration of a low dose of (R,S)-Ket for 14-days to Wistar rats was conducted to determine the impact of sub-chronic dosing on the pharmacokinetics of (R,S)-Ket and its major metabolites. The data indicate that, relative to a single administration of (R,S)-Ket, subchronic administration resulted in increased clearance of (R,S)-Ket and the N-demethylated metabolite norketamine measured as elimination half-life (t1/2) and decreased plasma concentrations of these compounds. Subchronic administration produced a slight decrease in t1/2 and an increase in plasma concentration of the major metabolite, (2S,6S;2R,6R)-hydroxynorketamine, and produced significant increases in the plasma concentrations of the (2S,6R;2R,6S)-hydroxynorketamine and (2S,4R;2R,4S)-hydroxynorketamine metabolites. The metabolism of (R,S)-Ket predominately occurs via two microsomal enzyme-mediated pathways: (R,S)-Ket⇒(R,S)-norketamine⇒(2S,6S;2R,6R)-hydroxynorketamine and (2S,4R;2R,4S)-hydroxynorketamine and the (R,S)-Ket⇒(2S,6R;2R,6S)-hydroxyketamine⇒(2S,6R;2R,6S)-hydroxynorketamine and (2S,6S;2R,6R)-hydroxynorketamine. The results indicate that the activity of both metabolic pathways are increased by subchronic administration of (R,S)-Ket producing new metabolite patterns and potential differences in clinical effects.

Interactions of racemic mefloquine and its enantiomers with P-glycoprotein in an immortalised rat brain capillary endothelial cell line, GPNT.

The brain distribution of the enantiomers of the antimalarial drug mefloquine is stereoselective according to the species. This stereoselectivity may be related to species-specific differences in the properties of some membrane-bound transport proteins, such as P-glycoprotein (P-gp). The interactions of racemic mefloquine and its individual enantiomers with the P-glycoprotein efflux transport system have been analysed in immortalised rat brain capillary endothelial GPNT cells. Parallel studies were carried out for comparison in human colon carcinoma Caco-2 cells. The cellular accumulation of the P-glycoprotein substrate, [(3)H]vinblastine, was significantly increased both in GPNT cells and in Caco-2 cells when treated with racemic mefloquine and the individual enantiomers. In GPNT cells, the (+)-stereoisomer of mefloquine was up to 8-fold more effective than its antipode in increasing cellular accumulation of [(3)H]vinblastine, while in Caco-2 cells, both enantiomers were equally effective. These results suggest that racemic mefloquine and its enantiomers are effective inhibitors of P-gp. Furthermore, a stereoselective P-glycoprotein inhibition is observed in rat cells but not in human cells. The efflux of [(14)C]mefloquine from GPNT cells was decreased when the cells were incubated with the P-gp modulators, verapamil, cyclosporin A or chlorpromazine, suggesting that MQ could be a P-gp substrate.

Development and characterization of an immobilized human organic cation transporter based liquid chromatographic stationary phase.

Membranes from a stably transfected cell line that expresses the human organic cation 1 transporter (hOCT1) have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form the hOCT1(+)-IAM stationary phase. Membranes from the parent cell line that does not express the hOCT1 were also immobilized to create the hOCT1(-)-IAM stationary phase. Columns were created using both stationary phases, and frontal displacement chromatography experiments were conducted using [(3)H]-methyl phenyl pyridinium ([(3)H]-MPP(+)) as the marker ligand and MPP(+), verapamil, quinidine, quinine, nicotine, dopamine and vinblastin as the displacers. The K(d) values calculated from the chromatographic studies correlated with previously reported K(i) values (r(2)=0.9987; p<0.001). The data indicate that the hOCT1(+)-IAM column can be used for the on-line determination of binding affinities to the hOCT1 and that these affinities are comparable to those obtained using cellular uptake studies. In addition, the chromatographic method was able to identify a previously undetected high affinity binding site for MPP(+) and to determine that hOCT1 bound (R)-verapamil to a greater extent than (S)-verapamil.

Verapamil stereoisomers during racemic verapamil administration: effects of aging and comparisons to administration of individual stereoisomers.

Aging decreases elimination of racemic verapamil but reports vary regarding effects of aging on clearance of individual verapamil enantiomers. To determine effects of aging on elimination of S- and R-verapamil, racemic verapamil was infused to steady-state concentrations of approximately 30, 60, and 120 ng/ml in 27 healthy subjects ranging in age from 23 to 81 years (young, 20 to 39 years; middle aged, 40 to 59 years; old, 60 to 81 years), and enantiomer concentrations were measured at each steady-state and after infusions. S-Verapamil clearance was greater than R-verapamil clearance in all age groups (p < 0.001), and aging decreased S-verapamil (p < 0.05) and R-verapamil (p < 0.008) clearance (average +/- SD, S-verapamil clearance was 14.3 +/- 4.7, 13.4 +/- 5.2, and 11.7 +/- 5.2 ml/min/kg; R-verapamil clearance was 6.5 +/- 3.3, 5.6 +/- 2.8, and 4.5 +/- 1.6 ml/min/kg in young, middle-aged, and older subjects, respectively). Enantiomer clearance was not effected by verapamil concentration. A trend toward an age effect on elimination half-lives was seen (S-verapamil half-life, 281 +/- 116 versus 234 +/- 89 minutes in elderly versus young; R-verapamil half-life, 253 +/- 56 versus 199 +/- 58 minutes in elderly versus young, p = 0.08). R- but not S-verapamil clearance during multistage infusions of racemic verapamil was lower than previously reported clearance after single intravenous enantiomer doses (p < 0.0001). In summary, aging decreases clearance of both S- and R-verapamil during steady-state intravenous dosing of racemic verapamil with preserved stereoselective clearance of verapamil with aging.

N-acetylation among HIV-positive patients and patients with AIDS: when is fast, fast and slow, slow?

BACKGROUND: The discrepancy between genotype and expressed phenotype of the polymorphic N-acetyltransferase (NAT2) has been suggested by separate genotypic and phenotypic studies in populations with human immunodeficiency virus (HIV). Only one study has examined both genotype and phenotype in the same population, and no discrepancies were observed. METHODS: In a cross-sectional study, 105 HIV-positive patients and patients with acquired immunodeficiency syndrome (AIDS) were phenotyped for NAT2 activity with use of caffeine as an in vivo probe; 50 of these patients were also genotyped by restriction mapping and allele-specific amplification. In a longitudinal study, 23 patients were phenotyped at least twice during the 2-year study. RESULTS: The distribution of the NAT2 phenotype among the 105 patients was unimodal and skewed toward slow acetylators as opposed to the bimodal distribution observed in healthy white populations. The genotype distribution was 26:24 slow:fast. There were 18 discrepancies between genotype and phenotype: 12 slow acetylators with fast genotypes and six fast acetylators with slow genotypes. No drug-related effects on NAT2 activity were apparent, but the role of disease progression was evident. Among the slow acetylators whose genotype was fast, the incidence of AIDS was higher (six of 12) than that among the fast acetylators whose genotype was fast (two of 14). Among patients phenotyped more than once (mean time between samples, 10.4 months) changes in phenotype from fast to slow were associated with progression of HIV infection. CONCLUSIONS: Disease progression in HIV infection and AIDS may alter expression of the NAT2 gene. The genotype and the phenotype are not interchangeable measurements. In the HIV population, to know the genotype is useful only if the phenotype is also known and vice versa.

Verapamil decreases lymphocyte protein kinase C activity in humans.

To determine if clinically used doses of the calcium antagonist verapamil measurably alter intracellular transduction mechanisms associated with the phosphatidylinositol pathway, lymphocyte protein kinase C activity was determined in subjects in a drug-free state, after 1 week of verapamil treatment (120 mg three times daily) and after a second week of verapamil treatment (240 mg sustained-release preparation once daily). Nine healthy male volunteers were studied and in these subjects baseline protein kinase C activity (mean +/- SEM; 5.07 +/- 0.76 pmol/microgram protein/min) tended to decrease after 1 week (3.50 +/- 0.20 pmol/micrograms protein/min) and was significantly decreased after 2 weeks (3.14 +/- 0.27 pmol/micrograms protein/min; p < 0.05 from baseline) of verapamil treatment. These data indicate that verapamil, at usual clinical doses, decreases protein kinase C activity in a marker tissue, the circulating lymphocyte. If protein kinase C activity in this tissue is a surrogate for other verapamil target tissues, such as vascular smooth muscle and heart muscle, these findings may provide insight into the in vivo mechanism by which verapamil decreases protein synthesis, limits cell growth, and reverses cellular hypertrophy in these tissues.