The disposition of saquinavir in normal and P-glycoprotein deficient mice, rats, and in cultured cells.

Protease inhibitors are very effective in treating patients infected with HIV. However, many drugs in this class penetrate poorly into the central nervous system (CNS) and may permit this site to be a sanctuary from which resistant virus can emerge. Previous studies have shown that the protease inhibitor saquinavir (SQV) interacts with the multidrug transport system, P-glycoprotein (P-gp), expressed in epithelial cells in the gut mucosa and at the blood-brain barrier, and thus might affect both the oral absorption and the penetration of SQV into the CNS. To determine whether SQV is a substrate for P-gp, its uptake was determined in cancer cells, which do (Dx5) and do not (MES-SA) express P-gp. The distribution of SQV between brain tissue and plasma was also investigated in rats and in normal and P-gp-deficient mdr1a(-/-) mice. The distribution ratio of SQV in plasma:brain:cerebrospinal fluid was approximately 100:10:0.2 in rats. The accumulation of SQV was enhanced in MES-SA cells (P-gp-negative) versus Dx5 cells (P-gp-positive). Bolus i.v. injection of [(14)C]SQV (2 and 5 mg/kg) into mdr1a(-/-) and normal mice (n = 3 or 4) resulted in 3-fold higher radioactivity in brains from mdr1a(-/-) mice. Similarly, oral administration of [(14)C]SQV (500 mg/kg) resulted in a 5-fold increase in systemic exposure and a 10-fold increase in brain levels in mdr1a(-/-) mice. These data demonstrate that saquinavir is a substrate for P-gp and that this transport system may play a role in limiting oral absorption and CNS exposure to this protease inhibitor.

Interactions of HIV protease inhibitors with a human organic cation transporter in a mammalian expression system.

Recently, we cloned a human organic cation transporter, hOCT1, which is expressed primarily in the liver. hOCT1 plays an important role in the cellular uptake and elimination of various xenobiotics including therapeutically important drugs. HIV protease inhibitors are a new class of therapeutic agents. The purpose of this study was to elucidate the interactions of HIV protease inhibitors with hOCT1 and to determine whether hOCT1 is involved in the elimination of these compounds. We studied the interactions of HIV protease inhibitors with hOCT1 in a transiently transfected human cell line, HeLa. Uptake studies were carried out 40 h post-transfection using the radiolabeled model organic cation, [(14)C]tetraethylammonium (TEA), under different experimental conditions. In cis-inhibition studies, all of the HIV protease inhibitors tested, i.e., indinavir (IC(50) of 62 microM), nelfinavir (IC(50) of 22 microM), ritonavir (IC(50) of 5.2 microM), and saquinavir (IC(50) of 8.3 microM) inhibited TEA uptake in HeLa cells expressing hOCT1. However, none of the HIV protease inhibitors trans-stimulated [(14)C]TEA uptake, suggesting that they are poorly translocated by hOCT1. Nelfinavir, ritonavir, and saquinavir demonstrated an apparent "trans-inhibition" effect. No enhanced uptake of [(14)C]saquinavir was observed in hOCT1 DNA-transfected cells versus empty vector-transfected cells. These data suggest that HIV protease inhibitors are potent inhibitors, but poor substrates, of hOCT1. Some HIV protease inhibitors may potently inhibit the uptake and elimination of cationic drugs that are substrates for hOCT1, leading to potential drug-drug interactions. Other transporters, e.g., MDR1 and MRP1, in HIV-targeted cells may control the intracellular concentrations of HIV protease inhibitors.

Interaction of anti-HIV protease inhibitors with the multidrug transporter P-glycoprotein (P-gp) in human cultured cells.

The anti-HIV protease inhibitors represent a new class of agents for treatment of HIV infection. Saquinavir, ritonavir, indinavir, and nelfinavir are the first drugs approved in this class and significantly reduce HIV RNA copy number with minimal adverse effects. They are all substrates of cytochrome P450 3A4, and are incompletely bioavailable. The drug transporting protein, P-glycoprotein (P-gp), which is highly expressed in the intestinal mucosa, could be responsible for the low oral bioavailability of these and other drugs which are substrates for this transporter. To determine whether these protease inhibitors are modulators of P-gp, we studied them in cell lines which do and do not express P-gp. Saquinavir, ritonavir and nelfinavir significantly inhibited the efflux of [3H]paclitaxel and [3H]vinblastine in P-gp-positive cells, resulting in an increase in intracellular accumulation of these drugs. However, similar concentrations of indinavir did not affect the accumulation of these anticancer agents. In photoaffinity labeling studies, saquinavir and ritonavir displaced [3H]azidopine, a substrate for P-gp, in a dose-dependent manner. These data suggest that saquinavir, ritonavir, and nelfinavir are inhibitors and possibly substrates of P-gp. Because saquinavir has a low bioavailability, its interaction with P-gp may be involved in limiting its absorption.

Na(+)-dependent purine nucleoside transporter from human kidney: cloning and functional characterization.

Many purine nucleosides and their analogs are actively transported in the kidney. Using homology cloning strategies and reverse transcriptase-polymerase chain reactions, we isolated a cDNA encoding a Na(+)-dependent nucleoside transporter, hSPNT1, from human kidney. Functional expression in Xenopus laevis oocytes identified hSPNT1 as a Na(+)-dependent nucleoside transporter that selectively transports purine nucleosides but also transports uridine. The Michaelis constant (K(m)) of uridine (80 microM) in interacting with hSPNT1 was substantially higher than that of inosine (4.5 microM). hSPNT1 (658 amino acids) is 81% identical to the previously cloned rat Na(+)-nucleoside transporter, SPNT, but differs markedly from SPNT in terms of its primary structure in the NH2 terminus. In addition, an Alu repetitive element (approximately 282 bp) is present in the 3'-untranslated region of the hSPNT1 cDNA. Northern analysis revealed that multiple transcripts of hSPNT1 are widely distributed in human tissues including human kidney. In contrast, rat SPNT transcripts are absent in kidney and highly localized to liver and intestine. The hSPNT1 gene was localized to chromosome 15. This is the first demonstration of a purine nucleoside transporter in human kidney.

Mechanisms of nucleobase transport in rabbit choroid plexus. Evidence for a Na(+)-dependent nucleobase transporter with broad substrate selectivity.

The overall goal of this study was to determine the mechanisms by which nucleobases are transported in the choroid plexus. Choroid plexus tissue slices were obtained from the lateral ventricles of rabbit brains and depleted of ATP with 2,4-dinitrophenol. In the presence of an initial inwardly directed Na+ gradient, hypoxanthine accumulated in the tissue slices against a concentration gradient. Na(+)-stimulated hypoxanthine uptake was saturable with a Km of 31.1 +/- 9.71 microM and a Vmax of 2.69 +/- 0.941 nmol/g/s (mean +/- S.E.). Na(+)-stimulated hypoxanthine uptake was inhibited by (100) microM naturally occurring purine and pyrimidine nucleobases (adenine, cytosine, guanine, hypoxanthine, thymine, uracil, and xanthine) as well as by the nucleoside analog, dideoxyadenosine. The stoichiometric coupling ratio between Na+ and hypoxanthine was 1.7:1. The data demonstrate the presence of a novel Na(+)-dependent nucleobase transporter in the choroid plexus, which is distinct from the previously described Na(+)-nucleoside transporter in choroid plexus and from Na(+)-dependent nucleobase transporters in other tissues in terms of its kinetics, substrate selectivity, and Na(+)-nucleobase stoichiometry. This transporter may play a role in the targeting of both salvageable nucleobases and therapeutic nucleoside analogs to the central nervous system.

The effect of N-ethylmaleimide on the Na(+)-dependent nucleoside transporter (N3) in rabbit choroid plexus.

The effect of the irreversible sulfhydryl-modifying agent, N-ethylmaleimide (NEM), on the transport of purine and pyrimidine nucleosides in choroid plexus was examined. [3H]thymidine and [3H]guanosine were used as model compounds to determine the effect of NEM on the Na(+)-dependent nucleoside transporter (N3) in rabbit choroid plexus tissue slices that were ATP-depleted with 2,4 dinitrophenol. Thymidine uptake in choroid plexus tissue slices preincubated with NEM was irreversibly inhibited in a concentration- (IC50 = 0.18 mM) and time-dependent fashion. NEM treatment also reduced the Na(+)-dependent uptake of other purine and pyrimidine nucleosides to a similar extent. In addition, an amine-selective modifying reagent, phenylisothiocyanate, had no effect on Na(+)-dependent thymidine uptake. Treatment of choroid plexus tissue slices with other sulfhydryl-modifying agents, including 4,4-dithiodipyridine, a reagent specific for cysteine residues, reduced the Na(+)-dependent uptake of thymidine. Preincubation of choroid plexus slices with NEM (2.5 mM) increased the Km of guanosine (control 64.5 +/- 5.7 microM; treated 120 +/- 23 microM) whereas the Vmax was unaffected (control 4.7 +/- 1 nmol/g/sec; treated 4.03 +/- 0.26 nmol/g/sec). These data suggest that covalent modification of sulfhydryl groups reduces the Na(+)-dependent uptake of nucleosides in choroid plexus slices by decreasing the affinity of nucleosides for the transporter.

Chemical, enzymatic, and human enantioselective S-oxygenation of cimetidine.

The S-oxygenation of cimetidine was investigated using achiral chemical and chiral chemical and enzymatic S-oxygenation procedures. The products of the reactions were thoroughly characterized by spectral, chiroptical, chromatographic, and stereochemical correlation methods. S-Oxygenation by the Kagan method or in the presence of pig liver microsomes or pig liver flavin-containing monooxygenase (FMO) (form I) all gave essentially identical enantioselectivity: the average enantiomeric excess was -13.4% and the stereopreference was for formation of (+)-cimetidine S-oxide in a ratio of (+)56.7%:(-)43.3%. The profile of immunoreactivity and the effect of metabolism inhibitors on cimetidine S-oxide formation in the presence of pig liver microsomes were consistent with a role of FMO (form I) in enantioselective (+)-cimetidine S-oxide formation. Administration of cimetidine to seven healthy male volunteers provided pharmacokinetic parameters for cimetidine and cimetidine S-oxide that were typical of those for previously reported studies. The urinary cimetidine S-oxide was isolated and the stereopreference was for formation of (-)-cimetidine S-oxide in a ratio of (+)25.5%:(-)74.5%. In good agreement with the enantiomeric enrichment values observed for the adult human urinary metabolite, the relative configuration of cimetidine S-oxide formed in adult human liver microsomes was (+)-15.8%:(-)-84.2%. Because the enantioselectivity and profile of immunoreactivity and the effect of metabolism inhibitors on cimetidine S-oxygenation in adult human liver microsomes are consistent with a role of FMO (form II) in cimetidine S-oxide formation and because the enantioselectivity of cimetidine S-oxide observed in adult humans is similar, we conclude that in vivo, cimetidine is S-oxygenated principally by FMO (form II).

Interaction of nucleoside analogues with the sodium-nucleoside transport system in brush border membrane vesicles from human kidney.

The therapeutic efficacy of nucleosides and nucleoside analogues as antitumor, antiviral, antiparasitic, and antiarrhythmic agents has been well documented. Pharmacokinetic studies suggest that many of these compounds are actively transported in the kidney. The goal of this study was to determine if therapeutically relevant nucleosides or analogues interact with the recently characterized Na(+)-driven nucleoside transport system of the brush border membrane of the human kidney. Brush border membrane vesicles (BBMV) were prepared from human kidney by divalent cation precipitation and differential centrifugation. The initial Na(+)-driven 3H-uridine uptake into vesicles was determined by rapid filtration. The effect of several naturally occurring nucleosides (cytidine, thymidine, adenosine), a pyrimidine base (uracil), a nucleotide (UMP), and several synthetic nucleoside analogues [zidovudine (AZT), cytarabine (Ara-C), and dideoxycytidine (ddC)] on Na(+)-uridine transport was determined. At a concentration of 100 microM the naturally occurring nucleosides, uracil, and UMP significantly inhibited Na(+)-uridine transport, whereas the three synthetic nucleoside analogues did not. Adenosine competitively inhibited Na(+)-uridine uptake with a Ki of 26.4 microM (determined by constructing a Dixon plot). These data suggest that naturally occurring nucleosides are substrates of the Na(+)-nucleoside transport system in the renal brush border membrane, whereas synthetic nucleoside analogues with modifications on the ribose ring are not. The Ki of adenosine is higher than clinically observed concentrations and suggests that the system may play a physiologic role in the disposition of this nucleoside.