Abacavir/lamivudine/zidovudine as a combined formulation tablet: bioequivalence compared with each component administered concurrently and the effect of food on absorption.

A single-center, open-label, three-way crossover study was conducted in 24 healthy subjects to assess (1) the bioequivalence of a combined abacavir 300 mg/lamivudine 150 mg/zidovudine 300 mg (A/L/Z) combination tablet relative to the separate brand-name components administered simultaneously and (2) the effect of food on the bioavailability of the drugs from the combination tablet. The subjects were randomly assigned to receive each of the following three treatments, separated by a 2-day washout period: one A/L/Z combination tablet after an overnight fast, one abacavir 300 mg tablet + one lamivudine 150 mg tablet + one zidovudine 300 mg tablet sequentially after an overnight fast, or one A/L/Z combination tablet 5 minutes after completing a standardized high-fat breakfast (67 g fat, 58 g carbohydrate, and 33 g protein). Serial blood samples were collected up to 24 hours postdose for determination of abacavir, lamivudine, and zidovudine serum concentrations. Standard pharmacokinetic parameters were estimated. Treatments were considered bioequivalent if 90% confidence intervals (CI) for geometric least squares (GLS) mean ratios for abacavir, lamivudine, and zidovudine area under the serum concentration-time curve (AUC(infinity)) and maximum observed serum concentration (Cmax) fell entirely within 0.80 to 1.25 for log-transformed parameters. The combined A/L/Z tablet was bioequivalent in the extent (AUC) and rate of absorption (Cmax and time of Cmax [tmax]) to the individual brand-name drug components administered concurrently under fasted conditions. GLS ratios and 90% CI for AUC(infinity) and Cmax were 0.99 (0.96, 1.03) and 1.00 (0.90, 1.11), respectively, for abacavir; 0.95 (0.91, 0.99) and 0.90 (0.84, 0.99), respectively, for lamivudine; and 0.95 (0.89, 1.02) and 0.96 (0.80, 1.15), respectively, for zidovudine. The extent of absorption of abacavir, lamivudine, and zidovudine from the combination tablet was not altered by administration with meals, indicating that this formulation may be administered with or without food. However, food slowed the rate of absorption, delayed the tmax, and reduced the Cmax of abacavir, lamivudine, and zidovudine. These changes, which were consistent with those observed with the individual reference formulations when administered with food, were not considered clinically important. All formulations were well tolerated underfasted and fed conditions.

A prodrug approach to the design of cRaf1 kinase inhibitors with improved cellular activity.

Earlier we reported potent cRaf1 kinase inhibitors with a key acidic phenol pharmacophore that had, at best, adequate cellular efficacy. To improve the cellular potency, phenol isosteres and prodrugs were investigated. Many phenol isosteres were synthesized and tested, but failed to provide adequate enzyme potency. A prodrug approach resulted in a 2- to 17-fold improvement over the parent compound in cell-based efficacy.

Transport of 5-fluorouracil and uracil into human erythrocytes.

The transport of 5-fluorouracil (5-FU) and uracil into human erythrocytes has been investigated under initial velocity conditions with an "inhibitor-stop" assay using a cold papaverine solution to terminate influx. At 37 degrees and pH 7.3, 5-FU influx was nonconcentrative; was partially inhibited by adenine, hypoxanthine, thymine, and uracil; and was insensitive to inhibition by nucleosides or inhibitors of nucleoside transport. Inhibition of the influx of 5-FU or uracil by adenine (3.0 mM) did not increase when other pyrimidines or inhibitors of nucleoside transport were combined with adenine. 5-FU and uracil exhibited similar saturable (Km approximately 4 mM, Vmax approximately 500 pmol/sec/5 microL cells) and nonsaturable (rate constant approximately 80 pmol/sec/mM/5 microL cells) components of influx. 5-FU, uracil, adenine, and hypoxanthine were competitive inhibitors of each other's influx with Ki values matching their respective Km values for influx. We conclude that 5-FU and uracil enter human erythrocytes at similar rates via both nonfacilitated diffusion and the same carrier that transports adenine and hypoxanthine.

Membrane permeation mechanisms of 2',3'-dideoxynucleosides.

The mechanism of membrane permeation of several 2',3'-dideoxynucleosides was investigated at 37 degrees with human erythrocytes using an "inhibitor-stop" assay. Transport (per 5 microL cells) via the nucleoside and nucleobase carriers was assessed by inhibition of influx with dilazep and adenine, respectively. Mechanisms of cellular entry were highly individualized: 2',3'-dideoxyadenosine and 3'-deoxythymidin-2'-ene via nonfacilitated diffusion, with high rates; 2',3'-dideoxyguanosine mainly via the nucleobase carrier (Km = 390 microM, Vmax = 32 pmol/sec); 2',3'-dideoxyinosine by both nucleobase (Km = 850 microM, Vmax = 2.7 pmol/sec) and nucleoside (Km = 7.4 mM, Vmax = 16 pmol/sec) carriers, with a low rate of nonfacilitated diffusion; and 2',3'-dideoxycytidine, equally by the nucleoside carrier (Km = 23 mM, Vmax = 65 pmol/sec) and by nonfacilitated diffusion, with a low rate. These results demonstrate that the nucleobase carrier plays an important role in the influx of two of these dideoxynucleotides and that nonfacilitated diffusion is not necessarily the chief mode of membrane permeation of this class of drugs.

Inhibition of thymidine transport by 3'-azido-3'-deoxythymidine and its metabolites.

3'-Azido-3'-deoxythymidine (AZT) competitively inhibited the transport of thymidine (Km = 0.23 mM) into human erythrocytes with a Ki of 1.0 mM at 37 degrees C. The principal human metabolite of AZT in plasma, the 5'-glucuronide (GAZT), was a weak inhibitor of the nucleoside transporter (< 20% inhibition of the influx of 1.0 microM thymidine by 10 microM GAZT). The minor AZT metabolite, 3'-amino-3'-deoxythymidine (AMT), competitively inhibited thymidine transport with a Ki of 9.1 mM. The influx of AMT into human erythrocytes was found to be a saturable process (Km = 12 mM) that was largely inhibited by dilazep, thus indicating that AMT influx occurs via the nucleoside transporter. High extracellular concentrations of AZT may contribute to the synergistic cytotoxicity of AZT plus either 5-fluorouracil or methotrexate by inhibiting thymidine transport into cancer cells whose de novo biosynthesis of dTMP is impaired pharmacologically or by inhibiting efflux of 2'-deoxy-5-fluorouridine and/or 2'-deoxyuridine from these cells.

Enantiomeric selectivity of carbovir transport.

Carbovir (9-[4 alpha-(hydroxymethyl)cyclopent-2-ene-1 alpha-yl]guanine) (CBV) is a carbocyclic analogue of 2',3'-dideoxyguanosine that exhibits potent and selective in vitro activity against human immunodeficiency virus. Antiviral activity is associated with only the (-)-enantiomer. The transport characteristics of both (-)-CBV and (+)-CBV were investigated in human erythrocytes at 37 degrees C using a papaverine-stop assay. The influx of both enantiomers appeared saturable and was inhibited greater than 90% by a combination of adenine (a low Km permeant of the nucleobase carrier) and dilazep (a potent inhibitor of nucleoside transport). The influx of (-)-CBV and (+)-CBV proceeded primarily via the nucleobase carrier with Vmax (picomoles/second/5 microliters of cells)/Km (millimolar) values of 17/0.12 and 140/1.9, respectively. To a lesser extent, the influx of (-)-CBV and (+)-CBV also occurred via the nucleoside transporter. Although both compounds exhibited a similar low affinity for this latter carrier (Km approximately 2 mM), the Vmax for (-)-CBV influx was approximately 4-fold higher than the Vmax for (+)-CBV influx. We conclude that both CBV enantiomers enter human erythrocytes by two transporters that are enantiomerically selective.

Membrane permeation characteristics of 5'-modified thymidine analogs.

The membrane permeation characteristics of 5'-deoxythymidine (5'-ddThd) and 5'-azido-5'-deoxythymidine (5'-N3-5'-ddThd) were investigated in human erythrocytes, with an inhibitor-stop assay, at 20 degrees. Uptake of both nucleoside analogs occurred without metabolism, was nonconcentrative, and was partially inhibited by nucleosides or inhibitors of nucleoside transport at micromolar permeant concentrations. At higher permeant concentrations (greater than 1.0 mM), the influx rate of each analog was linearly dependent on concentration and insensitive to inhibition by nucleosides, inhibitors of nucleoside transport, and nucleobases. Kinetic analyses using nonlinear regression revealed that a saturable component of 5'-ddThd influx (Km = 200 microM) was competitively inhibited by thymidine (dThd) (Ki = 86 microM) or 5-iodo-2'-deoxyuridine (Ki = 84 microM). Similarly, a saturable component of 5'-N3-5'-ddThd influx (Km = 220 microM) was competitively inhibited by 2-chloroadenosine (Ki = 18 microM). The Ki values for these nucleoside inhibitors were similar to their reported Km values as permeants of the nucleoside transporter. Both 5'-ddThd and 5'-N3-5'-ddThd competitively inhibited the influx of dThd (Km = 60 microM), with similar Ki values (150 and 200 microM, respectively). We conclude that these two 5'-modified dThd analogs enter human erythrocytes both by nonfacilitated diffusion and by the nucleoside transporter. The absence of the 5'-hydroxyl group of dThd (5'-ddThd) resulted in a large increase in the octanol/buffer partition coefficient, in an ability to permeate human erythrocytes by nonfacilitated diffusion, and in a 3-fold diminished binding to the nucleoside transporter. The 5'-azido group (5'-N3-5'-ddThd) resulted in an additional 1.4-fold increase in the octanol/buffer partition coefficient and in a 2-fold increase in the rate of nonfacilitated diffusion.

Desciclovir permeation of the human erythrocyte membrane by nonfacilitated diffusion.

The mechanism of transport of desciclovir (DCV)--a structural analogue and prodrug of acyclovir (ACV) which provides an improved oral bioavailability of ACV--was investigated in human erythrocytes with a "papaverine-stop" assay. DCV influx was nonconcentrative, linearly dependent on DCV concentration (0.9 microM to 15 mM), insensitive (less than or equal to 20% inhibition) to nucleobases, nucleosides, or potent inhibitors of nucleoside transport, and occurred without permeant metabolism. However, DCV was a weak competitive inhibitor of the influx of adenine (Ki = 1.3 mM) and of 5-iodo-2'-deoxyuridine (Ki = 2.9 mM). permeants of the erythrocyte nucleobase and nucleoside carriers, respectively. This indicates that DCV has an affinity for both of these transporters, even though it appears not to be an effective permeant. We conclude that, in contrast to ACV which enters human erythrocytes primarily via the nucleobase carrier, DCV permeates these cells chiefly (greater than or equal to 80%) by nonfacilitated diffusion. This mechanistic difference in transport between ACV and DCV is attributed to differences in their desolvation energies and suggests an explanation for the differences in the oral bioavailability of ACV which is observed after the administration of these two "acyclic nucleosides."

Ganciclovir permeation of the human erythrocyte membrane.

The membrane permeation of ganciclovir (DHPG)--a structural analogue of acyclovir (ACV) with activity against cytomegalovirus--was investigated in human erythrocytes at 37 degrees with an "inhibitor-stop" assay. DHPG influx was nonconcentrative, occurred without permeant metabolism, and was rate-saturable. While substantial inhibition of the influx of 13 microM DHPG occurred only in the presence of permeants of the purine nucleobase carrier, nucleosides and inhibitors of nucleoside transport markedly inhibited DHPG influx at higher DHPG concentrations (greater than or equal to 200 microM). Adenine and dilazep (a potent inhibitor of the nucleoside carrier) each inhibited the influx of DHPG only partially; when present together, however, they inhibited DHPG permeation completely. DHPG permeation via the purine nucleobase carrier (Km = 0.89 mM) was characterized by assessing influx in the presence of 1.0 microM dilazep. Adenine and ACV were shown to competitively inhibit this process, while DHPG (Ki = 0.90 mM) was found to competitively inhibit adenine influx. DHPG influx via the nucleoside carrier (Km = 14 mM) was characterized by assessing influx in the presence of 2 mM adenine. DHPG (Ki = 10 mM) also appeared to competitively inhibit the influx of 5-iodo-2'-deoxyuridine. These results indicate that DHPG permeates the human erythrocyte membrane primarily by the purine nucleobase carrier and secondarily by the nucleoside transporter.

2',3'-Dideoxythymidine permeation of the human erythrocyte membrane by nonfacilitated diffusion.

The influx of 2',3'-dideoxythymidine into human erythrocytes was characterized to gain insight into the molecular properties of 3'-azido-3'-deoxythymidine which allow this latter nucleoside analog to permeate cell membranes by nonfacilitated diffusion (J. Biol. Chem. 262, 5748-5754 (1987]. The influx of 2',3'-dideoxythymidine was (1) nonconcentrative, (2) a linear function of permeant concentration (0.05 to 12 mM), and (3) insensitive to potent inhibitors of nucleoside transport and to permeants of either the nucleoside or nucleobase transporter. It is concluded that 2',3'-dideoxythymidine, like 3'-azido-3'-deoxythymidine, permeates the human erythrocyte membrane predominantly by nonfacilitated diffusion. This unusual characteristic of these two nucleoside analogs is attributed both to their lack of a 3'-hydroxyl moiety, a structural determinant which appears to be important for transport by the nucleoside carrier, and to their relatively high partition coefficients (greater than or equal to 0.2).

Purine nucleobase transport in human erythrocytes. Reinvestigation with a novel "inhibitor-stop" assay.

A novel "inhibitor-stop" method for the determination of initial rates of purine nucleobase transport in human erythrocytes has been developed, based on the addition of seven assay volumes of cold 19 mM papaverine to terminate influx. In view of our finding that the initial velocities of adenine, guanine, and hypoxanthine influx into human erythrocytes were linear for only 4-6 s at 37 degrees C, the present method has been used to reexamine the kinetics of purine nucleobase transport in these cells. Initial influx rates of all three purine nucleobases were shown to be the result of concurrent facilitated and nonfacilitated diffusion. The nonfacilitated influx rates could be estimated either from the linear concentration dependence of nucleobase influx at high concentrations of permeant or from residual influx rates which were not inhibited by the presence of co-permeants. Appropriate corrections for nonfacilitated diffusion were made to the influx rates observed at low nucleobase concentrations. Kinetic analyses indicated that adenine (Km = 13 +/- 1 microM, n = 7), guanine (Km = 37 +/- 2 microM, n = 5), and hypoxanthine (Km = 180 +/- 12 microM, n = 6) were mutually competitive substrates for transport. The Ki values obtained with each nucleobase as an inhibitor of the influx of the other nucleobases were similar to their respective Km values for influx. Furthermore, the transport of the purine nucleobases was not inhibited by nucleosides (uridine, inosine) or by inhibitors of nucleoside transport (6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, dilazep, dipyridamole). It is concluded that all three purine nucleobases share a common facilitated transport system in human erythrocytes which is functionally distinct from the nucleoside transporter.

Acyclovir transport into human erythrocytes.

The mechanism of transport of the antiviral agent acyclovir (ACV) into human erythrocytes has been investigated. Initial velocities of ACV influx were determined with an "inhibitor-stop" assay that used papaverine to inhibit ACV influx rapidly and completely. ACV influx was nonconcentrative and appeared to be rate-saturable with a Km of 260 +/- 20 microM (n = 8). However, two lines of evidence indicate that ACV permeates the erythrocyte membrane by means other than the nucleoside transport system: 1) potent inhibitors (1.0 microM) of nucleoside transport (dipyridamole, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and dilazep) had little (less than 8% inhibition) or no effect upon the influx of 5.0 microM ACV; and 2) a 100-fold molar excess of several purine and pyrimidine nucleosides had no inhibitory effect upon the influx of 1.0 microM ACV. However, ACV transport was inhibited competitively by adenine (Ki = 9.5 microM), guanine (Ki = 25 microM), and hypoxanthine (Ki = 180 microM). Conversely, ACV was a competitive inhibitor (Ki = 240-280 microM) of the transport of adenine (Km = 13 microM), guanine (Km = 37 microM), and hypoxanthine (Km = 180 microM). Desciclovir and ganciclovir, two compounds related structurally to ACV, were also found to be competitive inhibitors of acyclovir influx (Ki = 1.7 and 1.5 mM, respectively). These results indicate that ACV enters human erythrocytes chiefly via the same nucleobase carrier that transports adenine, guanine, and hypoxanthine.

3'-azido-3'-deoxythymidine. An unusual nucleoside analogue that permeates the membrane of human erythrocytes and lymphocytes by nonfacilitated diffusion.

The demonstrated in vitro and in vivo activity of 3'-azido-3'-deoxythymidine (N3dThd) against the infectivity and the cytopathic effect of human immunodeficiency virus has prompted an investigation of the mechanism by which this nucleoside analogue permeates the cell membrane. As with the transport of thymidine, the influx of N3dThd into human erythrocytes and lymphocytes was nonconcentrative during short incubation times (less than 5 min) which did not allow significant metabolism of this nucleoside. However, in contrast with thymidine transport, the initial velocity of N3dThd influx was strictly a linear function of nucleoside concentration (0.5-10 mM), without evidence of saturability; insensitive to micromolar concentrations of potent inhibitors of nucleoside transport (dipyridamole, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and dilazep); insensitive to a 1000-fold excess of other nucleosides (thymidine, uridine, 2-chloroadenosine); and relatively insensitive to temperature, with Q10 values (37-27 degrees C) of 1.4 and 2.7 for N3dThd and thymidine, respectively, determined in erythrocytes. Although the above results indicate that N3dThd permeates the cell membrane chiefly by nonfacilitated diffusion and not via the nucleoside transporter, millimolar concentrations of this nucleoside analogue were observed to inhibit both zero-trans influx of thymidine and efflux of thymidine from [3H]thymidine-loaded erythrocytes. The partition coefficients (1-octanol:0.1 M sodium phosphate, pH 7.0) of N3dThd and thymidine were determined to be 1.26 and 0.064, respectively. The unusual ability of N3dThd to diffuse across cell membranes independently of the nucleoside transport system may be attributed to the considerable lipophilicity imparted to this molecule by the replacement of the 3'-hydroxyl group of thymidine with an azido moiety.

An assay for inhibitors of nucleoside transport based upon the use of 5-[125I]iodo-2'-deoxyuridine as permeant.

5-[125I]Iodo-2'-deoxyuridine (IdUrd) has been shown to serve as a permeant for the nucleoside transport system of human erythrocytes and to be matabolically inert in these cells. Linear initial velocities were obtained at 20 degrees C for 125IdUrd transport, yielding a Km of 73 +/- 18 microM (n = 6). Low-affinity inhibitors of 125IdUrd transport, such as adenosine (Ki = 32 +/- 2 microM, n = 2), could be characterized by Michaelis-Menten kinetics. However, high-affinity inhibitors, such as 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, caused nonlinear initial velocities when added to the cells simultaneously with 125IdUrd. Conditions were defined (viz., 20-min pretreatment of cells with test compound followed by 5.0-min incubation with 1.0 microM 125IdUrd, all at 20 degrees C) whereby high-affinity inhibitors of IdUrd transport can be identified and evaluated according to their 50% inhibitory concentrations. The use of 125IdUrd as permeant greatly expedites the testing of compounds as inhibitors of nucleoside transport by allowing the cell pellets generated in these assays to be monitored directly in a gamma spectrometer, thereby circumventing the solubilization and decolorization of cell pellets required by assays that use 3H- or 14C-labeled nucleoside permeants.