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Research ArticleArticle

Selective Biotransformation of the Human Immunodeficiency Virus Protease Inhibitor Saquinavir by Human Small-Intestinal Cytochrome P4503A4

Potential Contribution to High First-Pass Metabolism

Michael E. Fitzsimmons and Jerry M. Collins
Drug Metabolism and Disposition February 1997, 25 (2) 256-266;

Potential Contribution to High First-Pass Metabolism

Abstract

Saquinavir is a HIV1 protease inhibitor used in the treatment of patients with acquired immunodeficiency syndrome, but its use is limited by low oral bioavailability. The potential of human intestinal tissue to metabolize saquinavir was assessed in 17 different human small-intestinal microsomal preparations. Saquinavir was metabolized by human small-intestinal microsomes to numerous mono- and dihydroxylated species with KM values of 0.3–0.5 μM. The major metabolites M-2 and M-7 were single hydroxylations on the octahydro-2-(1H)-isoquinolinyl and (1,1-dimethylethyl)amino groups, respectively. Ketoconazole and troleandomycin, selective inhibitors of cytochrome P4503A4 (CYP3A4), were potent inhibitors for all oxidative metabolites of saquinavir. The cytochrome P450-selective inhibitors furafylline, fluvoxamine, sulfaphenazole, mephenytoin, quinidine, and chlorzoxazone had little inhibitory effect. All saquinavir metabolites were highly correlated with testosterone 6β-hydroxylation and with each other. Human hepatic microsomes and recombinant CYP3A4 oxidized saquinavir to the same metabolic profile observed with human small-intestinal microsomes. Indinavir, a potent HIV protease inhibitor and a substrate for human hepatic CYP3A4, was a comparatively poor substrate for human intestinal microsomes and inhibited the oxidative metabolism of saquinavir to all metabolites with a Ki of 0.2 μM. In addition, saquinavir inhibited the human, small-intestinal, microsomal CYP3A4-dependent detoxication pathway of terfenadine to its alcohol metabolite with aKi value of 0.7 μM. These data indicate that saquinavir is metabolized by human intestinal CYP3A4, that this metabolism may contribute to its poor oral bioavailability, and that combination therapy with indinavir or other protease inhibitors may attenuate its low relative bioavailability.

Saquinavir (Ro 31-8959), a potent HIV-1 and HIV-2 protease inhibitor with an IC90 of 20 nM, has been approved for use in the treatment of patients with acquired immunodeficiency syndrome. Saquinavir is effective in reducing viral load, well tolerated, and, at the doses administered, not limited by adverse side effects (1). The antiviral activity of saquinavir is attenuated, however, by poor oral bioavailability and by the potential for viral resistance (2, 3). HIV-1 mutant strains that contain two or three amino acid exchanges display a 40- to 50-fold increase in saquinavir IC90. Mutant strains for HIV-2 protease have not been identified.

Historically, first-pass effect (low bioavailability) has been attributed to hepatic clearance. Numerous data indicate, however, that extrahepatic tissues, specifically the intestinal mucosa, have considerable amounts of drug-metabolizing enzymes that may contribute to the metabolism of orally administered xenobiotics. UDP-glucuronosyltransferase (4), sulfotransferase (5, 6),N-acetyltransferase (7), and cytochrome P450 activities (8,9) have been detected in human small-intestinal tissue. Of the cytochrome P450 enzymes detected in the small intestine, CYP3A4 is the most abundant, with highest concentrations in the duodenum and with decreasing concentrations descending toward the colon. CYP1A, CYP2C, and CYP2D families have also been detected in human small intestine, although at substantially lower concentrations. Cyclosporin A and midazolam are orally administered pharmaceutical agents that are metabolized by human small-intestinal CYP3A4 (10-13). Drug interactions in the intestine between rifampin or ketoconazole and cyclosporin A or midazolam have been demonstrated (14-16).

The objectives of the present research were to determine the potential of human hepatic and small-intestinal microsomes to metabolize saquinavir, to identify the enzyme systems involved in its biotransformation, and to assess in vitro potential metabolic interactions. The results presented herein show that saquinavir is oxidized by both human hepatic and small-intestinal microsomes to multiple metabolites, that CYP3A4 is the predominant enzyme involved in its biotransformation, and that drug interactions of saquinavir with indinavir or terfenadine at the level of the small intestine may be significant. Based on the present data, the high rate of intestinal oxidation demonstrated in vitro suggests that the small intestine may play a critical role in the extensive first-pass metabolism of saquinavir and, therefore, in its low relative bioavailability. Combination therapy with saquinavir and indinavir may attenuate the high first-pass effect, increase its oral bioavailability, and result in greater efficacy of HIV protease inhibition.

Materials and Methods

Saquinavir was obtained from Hoffmann-LaRoche (Nutley, NJ). Indinavir was obtained from Merck Research Laboratories (Rahway, NJ). Terfenadine alcohol metabolite and terfenadine carboxylate were purchased from Ultrafine Chemicals (Manchester, UK). Furafylline and amitriptyline were purchased from Research Biochemicals International (Natick, MA). Cyclosporin A and mephenytoin were purchased from the United States Pharmacopeial Convention (Rockville, MD). Sulfaphenazole, chlorzoxazone, and fluvoxamine were obtained from reference stocks at the U.S. Food and Drug Administration. Troleandomycin, ketoconazole, midazolam, quinidine sulfate, 7,8-benzoflavone, quercetin, terfenadine, phenylmethylsulfonyl fluoride, NADP, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase were purchased from Sigma Chemical Co. (St. Louis, MO). Homogenization buffer (0.154 M KCl, 0.1 M Na+ and K+ phosphate, and 1 mM EDTA; pH 7.4), reaction buffer (0.1 M Na+ and K+ phosphate, 1 mM EDTA, 5.0 mM MgCl2; pH 7.4), and mucosal buffer (0.154 M KCl, 0.1 M Na+ and K+ phosphate, 10 mM dithiothreitol, 0.1 μM pepstatin A, 5 mM EDTA, and 0.25 mM phenylmethylsulfonyl fluoride; pH 7.4) were purchased from Quality Biological, Inc. (Gaithersburg, MD). Note:Phenylmethylsulfonyl fluoride is unstable in aqueous solutions over prolonged periods; therefore, 20 ml of 0.25 M phenylmethylsulfonyl fluoride in ethanol was added to 20 liters of mucosal buffer immediately before use. The hepatosome test kit was purchased from Human Biologics, Inc. (Phoenix, AZ). Human recombinant cytochrome P450 microsomes were purchased from Gentest Corporation (Woburn, MA). Prism HPLC columns and Prism Javelin precolumns were purchased from Keystone Scientific, Inc. (Bellafonte, PA).

Tissue Procurement and Preparation.

Human liver and small intestine, medically unsuitable for organ transplantation, were obtained under the auspices of the Washington Regional Transplantation Consortium (Washington, DC). Hepatic microsomes were prepared from frozen human liver samples HL 19, 20, 27, and 29, as described previously (17). Microsomal protein concentration was determined with a Bio-Rad protein assay kit (Hercules, CA).

Small intestines were placed on ice, the lumens of the small intestines were perfused with 5 liters of ice-cold mucosal buffer, and the small intestines were transported to the laboratory on ice. The lumens of the small intestines were perfused with an additional 15 liters of ice-cold mucosal buffer, the small intestines were cut into 30-cm sections, the sections were cut longitudinally and placed flat, the mucosal layer was isolated by scraping, and the mucosal tissue from the whole small intestine was pooled and stored in 250-ml polypropylene centrifuge tubes at −80°C. Mucosal tissue was isolated and frozen within 3 hr of blood flow termination to the organ. Small-intestinal microsomes were prepared from the frozen, human mucosal tissue by the following procedure; all steps were performed at 4°C. Human mucosal tissue (∼500 ml) was diluted with ∼500 ml of homogenization buffer and homogenized with a dounce, the homogenate was placed into 250-ml centrifuge bottles and centrifuged at 13,500g for 20 min, and the supernatant was decanted and saved. The pellet was suspended with an additional 500 ml of homogenization buffer, the suspension was homogenized with a dounce, and the homogenate was centrifuged at 13,500g for 20 min. Supernatants from the 13,500gcentrifugations were combined and centrifuged again at 13,500g for 20 min. The mitochondrial pellet was discarded, and the supernatant was centrifuged at 105,000g for 60 min. The supernatant was decanted, the pellet was suspended in reaction buffer, and the homogenate was centrifuged again at 105,000gfor 60 min. The microsomal pellet was suspended in reaction buffer and aliquoted into 1-ml fractions. Protein concentration was determined with a Bio-Rad protein assay kit.

Microsomal Incubations with Saquinavir.

Human hepatic and small-intestinal microsomes were diluted with reaction buffer to give a final protein concentration of ∼0.2 and 0.05 mg/ml, respectively, unless otherwise noted. Microsomes from the human B-lymphoblastoid cell line AHH-1TK+/− that contained cDNAs for specific cytochrome P450 enzymes were diluted with reaction buffer to give a final protein concentration of 0.1 mg/ml. All microsomal suspensions (0.99 ml) were preincubated with a NADPH-generating system (1 mM NADP+, 10 mM glucose-6-phosphate, and 1 unit/ml yeast glucose-6-phosphate dehydrogenase) for 3 min at 37°C, and the reactions were started by the addition of 10 μl of a 100-fold concentrated solution of saquinavir in 1:1 acetonitrile:water. Incubation reactions were stopped at the appropriate time by adding 1 ml of ice-cold acetonitrile to the microsomal suspension and then storing the reaction tubes on ice.

For chemical inhibition studies, inhibitors were dissolved in HPLC grade ethanol as 100-fold concentrated solutions, and 10 μl of these solutions was added to 0.98 ml of the microsomal suspension. Human small-intestinal or hepatic microsomes were preincubated with inhibitor and a NADPH-generating system for 5 min at 37°C. Reactions were started by the addition of 10 μl of a 0.1 mM saquinavir solution in 1:1 acetonitrile:water to 0.99 ml of the inhibitor/microsomal solutions. Microsomal incubations were stopped after 15 min as noted. Inhibition experiments with mechanism-based inactivators were conducted essentially as noted, except microsomal suspensions were preincubated with the inhibitor for 15 min and a NADPH-generating system before the addition of saquinavir. The final organic concentration in the microsomal suspensions was 1.5% (v/v), and all control incubations contained the same organic concentration but without inhibitors.

Human Small-Intestinal Microsomal Incubations with Indinavir (MK-639).

Human small-intestinal microsomes (HG30) were diluted with reaction buffer to give a final protein concentration of 0.5 mg/ml. Microsomal suspensions were preincubated with a NADPH-generating system for 5 min at 37°C, and reactions were started by the addition of 10 μl of a 0.5 mM indinavir solution in 1:1 acetonitrile:water to 0.99 ml of the microsomal solutions. Incubations were stopped after 1 hr by the addition of 1 ml of ice-cold acetonitrile. The final organic concentration in the microsomal suspensions was 0.5% (v/v).

Saquinavir Enzyme Kinetics and Ki Determination for Ketoconazole and Indinavir.

For kinetic analyses and chemical Ki determinations, human small-intestinal microsomal protein was diluted with reaction buffer to achieve a final protein concentration of ∼0.012 mg/ml and a final volume of 2 ml. After a 3-min preincubation with a NADPH-generating system, microsomal suspensions were incubated at 37°C with 0.1–1.5 μM saquinavir for 5 min. ApparentKM and Vmax constants were determined with a Lineweaver-Burk plot. Kinetic constants were also determined for saquinavir in the presence of either ketoconazole or indinavir. Saquinavir (0.1–1.5 μM final concentration) was added to preincubated, small-intestinal, microsomal suspensions that contained either ketoconazole (0.05–0.3 μM) or indinavir (0.25–1.5 μM). Inhibition constants for both ketoconazole and indinavir were determined by plotting the slopes of the Lineweaver-Burk plotsvs. inhibitor concentrations (18).

Drug Interaction Between Saquinavir and Terfenadine in Human Small-Intestinal Microsomes.

Human small-intestinal microsomes (HG30; 0.05 mg protein/ml) were preincubated with a NADPH-generating system and with 0–10 μM saquinavir for 3 min at 37°C. Reactions were started by the addition of 10 μl of 100-fold concentrated solutions of terfenadine in ethanol to give final incubation concentrations of 0.3–10 μM. The final organic concentration in the microsomal suspensions was 1.5% (v/v), and all control incubations contained the same organic concentration but without inhibitors.

Correlation Analyses.

The formation of saquinavir metabolites was correlated with cytochrome P450-selective marker activities in human hepatic microsomes. Hepatic microsomes from 10 individual liver donors and one pooled microsomal suspension (Human Biologics, Inc.) were incubated with saquinavir, as noted previously; the final concentration of saquinavir was 5 μM, and the incubation time was 5 min.

Sample Preparation and Instrumental Analyses.

Internal standard (5 ml of 0.05 μM amitriptyline · HCl in acetonitrile) was added to microsomal incubation mixtures, protein was removed by centrifugation, and samples were dried with a Savant speed-vac (Farmington, NY). Samples were dissolved in 200 μl of acetonitrile:water (1:1), 2 ml of acetonitrile was added to the solutions, precipitate was removed by centrifugation, and the samples were dried with a speed-vac. The final residue was dissolved in 12 μl of 10% acetonitrile/water (v/v), and 10 μl of these solutions was analyzed with a Hewlett-Packard 1050 HPLC system coupled to a Keystone Prism column (2.0 × 150 mm; 5 μm particle size) fitted with a Keystone Prism precolumn. Samples were eluted with water (A) and 0.1% (v/v) formic acid in acetonitrile (B) as follows: the initial eluant profile was held at 95% A for 5 min at 0.2 ml/min; B was increased linearly to 65% over 30 min, and then both B and the flow rate increased linearly to 85% and 0.25 ml/min, respectively, over 10 min; B and the flow rate were decreased linearly to 5% and 0.2 ml/min, respectively, over 5 min; the column was equilibrated with 95% A for 15 min. The absorbance of the eluate was monitored at 240 nm, and UV absorption spectra were recorded at 210–450 nm. In some cases,M-4 and M-5 coeluted and could not be quantitated. Saquinavir concentrations were determined with standard curves of authentic standard. Metabolite concentrations were calculated with saquinavir standard curves based on the assumption that the extinction coefficients for the metabolites were identical with that of saquinavir. This assumption seems valid based on the following data.1) Hydroxylation of saquinavir was limited to nonconjugated and nonaromatic regions of the molecule (see mass spectral data forM-2, M-3, and M-7) and, therefore, molar absorptivity should not be altered; and 2) UV absorption spectra for M-2, M-3, andM-7 were identical with that of saquinavir.

LC/MS analyses of saquinavir and metabolites M-1 toM-7 were conducted with a Hewlett-Packard 1050 HPLC system coupled to a Finnigan TSQ-7000 mass spectrometer (San Jose, CA) operating in the positive-ion ESI mode. The HPLC system, column, and gradient profile were the same as noted previously. The operating conditions for the mass spectrometer in the ESI/Q3MS scan mode were as follows: ESI spray voltage, 4.5 kV; capillary temperature, 250°C; sheath gas, 65 psi; auxiliary gas, 5 psi; scan range, 150–1000m/z; scan rate, 1.5 sec; and electron multiplier voltage, 1,250 V. Conditions for LC/MS/MS analyses in the daughter scan mode were the same as noted for the ESI/Q3MS scan mode with the following additions: collision gas, argon; collision gas pressure, 1 mTorr; and collision cell offset voltage, −60 eV.

For HPLC analysis of terfenadine and terfenadine metabolites, preparation of microsomal incubations was the same as that for saquinavir sample preparation. The final residue was dissolved first in 40 μl of 1:1 acetonitrile:water and then 60 μl of water was added to achieve a final volume of 100 μl; 10 μl of the final solution was injected for HPLC analysis. HPLC analyses were conducted with the same HPLC system, column, and mobile phases noted previously, but with the following gradient profile (time in min:% of water/% of 0.1% formic acid in acetonitrile:flow rate in ml/min): 0:90/10:0.1; 2:90/10:0.1; 3.5:90/10:0.2; 4:90/10:0.25; 5:90/10:0.25; 30:45/55:0.25; 32:2/98:0.25; 33:2/98:0.4; 39.8:2/98:0.4; 40:2/98:0.25; 45:90/10:0.25; 45:90/10:0.2; 50:90/10:0.2; and 52:90/10/:0.1. Fluorescence of the eluate was recorded with a Hewlett-Packard 1046 fluorescence detector with an excitation wavelength of 228 nm and an emission wavelength of 291 nm. Terfenadine metabolite concentrations were determined with standard curves of authentic alcohol standard.

Results

Biotransformation of Saquinavir by Human Small-Intestinal and Human Hepatic Microsomes.

Incubations with either human small-intestinal or human hepatic microsomes showed that saquinavir was oxidized to multiple metabolites and that the qualitative pattern of metabolite formation was the same for both tissues. HPLC analyses showed the formation of two major metabolites and five minor metabolites from both hepatic and small-intestinal microsomal incubation mixtures (fig. 1,A and B, respectively); metabolites were labeledM-1 through M-7 based on their order of elution. For both small-intestinal and hepatic microsomes, saquinavir biotransformation was limited to microsomal fractions, was protein- and NADPH-dependent, and was essentially time-linear for ∼5 min (fig.2). The deviation from linearity at longer time periods probably results from depletion of parent compound and from competitive inhibition by its metabolites, specifically M-7, which is oxidized to the dihydroxylated metabolite M-1.

Figure 1
Figure 1

HPLC chromatograms of microsomal-derived saquinavir metabolites.

Incubation mixtures contained either human small-intestinal microsomes (A), human hepatic microsomes (B), or human recombinant CYP3A4 (C), a NADPH-generating system, and 5 μM saquinavir. M-1 through M-7 indicate microsomal-derived saquinavir metabolites. Retention time for saquinavir ≈ 25 min. ISD denotes internal standard peak (tR ≈ 21.5 min). The absorbance of the eluate was recorded at 240 nm.

Figure 2
Figure 2

Time course for saquinavir biotransformation with human small-intestinal microsomes.

Microsomal suspensions (HG30) were diluted to ∼0.02 mg/ml with reaction buffer, warmed at 37°C in the presence of a NADPH-generating system, and then incubated with 1 μM saquinavir for the times shown. Total is the concentration of saquinavir plus the concentrations ofM-2 and M-7. Data are presented as means ± SD (N = 3).

Metabolites M-1 to M-7 possessed UV absorption spectra similar to that of saquinavir. Metabolites M-2,M-3, and M-7 were identified by LC/MS/MS as monohydroxylated products due to hydroxylation on the octahydro-2-(1H)-isoquinolinyl (M-2 andM-3) and (1,1-dimethylethyl)amino (M-7) groups (fig. 3). Figure 4 shows the fragmentation pattern for saquinavir and the m/z ratios and relative ion abundances for saquinavir, M-2, M-3, and M-7. Metabolites M-4, M-5, andM-6 were identified as monohydroxylated products and showed molecular ions of m/z 687, but the site of oxidation for these metabolites was not elucidated. Sequential oxidation ofM-7 gave the dihydroxylated metabolite M-1(molecular ion of m/z 703).

Figure 3
Figure 3

Proposed metabolic pathways of saquinavir in human small-intestinal microsomes.

Figure 4
Figure 4

Mass spectral fragmentation pattern for saquinavir and mass-to-charge ratios and relative ion abundances for saquinavir, M-2, M-3, and M-7.

ND indicates m/z ion abundances of <5%.    

Because M-7 was oxidized to M-1 and because saquinavir oxidation rate decreases with longer incubation times, microsomal incubations for kinetic analyses were conducted with low protein concentrations and short incubation times to maintain a high saquinavir to M-7 ratio and, therefore, to limit the inhibitory effect of saquinavir metabolites. Kinetic analyses showed that human, small-intestinal, microsomal oxidation of saquinavir toM-2 and M-7 followed Michaelis-Menten kinetics, and Lineweaver-Burk plots showed apparent KM values that ranged from 0.3 to 0.5 μM. ApparentVmax values for M-2 andM-7 were 1.33 and 2.63 nmol/min/mg of HG30 microsomal protein, respectively.

Biotransformation of Indinavir by Human Small-Intestinal Microsomes.

Incubation with human small-intestinal microsomes showed that indinavir was metabolized to multiple metabolites. HPLC analyses showed the formation of two major metabolites and at least five minor metabolites (fig. 5). Metabolites labeled with an asterisk have similar electronic absorption spectra compared with that of indinavir. Indinavir biotransformation was limited to microsomal fractions and was NADPH-dependent. No further analyses were performed to identify the metabolites or to determine enzyme kinetic values.

Figure 5
Figure 5

HPLC chromatogram of an incubation mixture that contained indinavir and human small-intestinal microsomes.

Incubation mixtures contained 0.5 mg/ml HG30 microsomes, a NADPH-generating system, and 5 μM indinavir (MK-639). Incubations were conducted at 37°C for 1 hr. *Probable metabolite that showed similar electronic absorption spectra compared with that of indinavir. The absorbance of the eluate was recorded at 260 nm.

Effect of Selective Cytochrome P450 Inhibitors on Saquinavir Biotransformation.

Ketoconazole at concentrations of 1 or 3 μM inhibited the formation of all saquinavir metabolites from human small-intestinal microsomes HG19 and HG30 by >95% (table 1). Kinetic analysis showed that ketoconazole was a mixed-type inhibitor with aKi value of 0.02 μM (fig. 6). Ketoconazole had a similar inhibitory effect on saquinavir biotransformation with human hepatic microsomes HL19 (data not shown). In addition, troleandomycin was an effective inhibitor of saquinavir metabolism (table 1). Human intestinal CYP3A substrates cyclosporin A and midazolam were effective inhibitors of the oxidative metabolism of saquinavir to all metabolites.

View this table:
Table 1

Effect of cytochrome P450-selective inhibitors on saquinavir oxidation in human small-intestinal microsomes

Figure 6
Figure 6

Lineweaver-Burk plots for the formation ofM-7 in the presence of different fixed concentrations of ketoconazole.

HG30 microsomes were incubated with 0.1–1.5 μM saquinavir and with either 0, 0.05, 0.1, or 0.2 μM ketoconazole in the presence of a NADPH-generating system at 37°C for 5 min. The inset plots the slopes of the 1/v vs. 1/s lines against ketoconazole concentrations; the Ki value was estimated from the X-axis intercept. K = ketoconazole.

Preincubation of various flavonoids with small-intestinal microsomes had mixed inhibitory effects on the oxidation of saquinavir. 7,8-Benzoflavone (1 μM) inhibited the formation of M-1through M-6, but stimulated the oxidation of saquinavir toM-7. At higher, nonselective concentrations of 7,8-benzoflavone, M-7 formation was also inhibited (table1). Quercetin had a larger inhibitory effect on M-2 than onM-7 at all concentrations but did not stimulate the formation of M-7. The other cytochrome P450 substrates or inhibitors furafylline (CYP1A2), fluvoxamine (CYP1A2), sulfaphenazole (CYP2C9), mephenytoin (CYP2C19), quinidine (CYP2D6), and chlorzoxazone (CYP2E1) had no inhibitory effect (table 1).

Correlation Analyses of Saquinavir Biotransformation.

Because the oxidation of saquinavir was qualitatively the same in both human hepatic and human small-intestinal microsomes (fig. 1,A and B) and because enzyme-selective specific activities of cytochromes P450 in human intestinal microsomes were not known, the rates of saquinavir metabolite formation were correlated with human liver microsomes with known cytochrome P450-specific activities. The rate of formation of saquinavir metabolitesM-2 and M-7 was strongly correlated with 6β-hydroxytestosterone formation (r2 = 0.92 and 0.87, respectively; p < 0.001). Correlations between saquinavir metabolite formation and other marker enzyme activities were generally low (table 2). Modest correlations were observed, however, with coumarin 7-hydroxylation (r2 ≈ 0.4), tolbutamide methylhydroxylation (r2 ≈ 0.5), and lauric acid ω-hydroxylation (r2 ≈ 0.4). Metabolites M-4,M-5, and M-6 could not be quantitated with human liver microsomes and, therefore, correlation with known cytochrome P450-specific activities could not be determined.

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Table 2

Correlation between rate of formation of saquinavir metabolites with cytochrome P450 marker activities

Biotransformation of saquinavir was studied in 17 different human small-intestinal microsomes. Figure 7 shows the variance in the formation of M-2 and M-7. Variance for all saquinavir metabolites was similar to that for M-2 andM-7, and correlations between the rate of formation for all metabolites was high (table 3).

Figure 7
Figure 7

Rate of M-2 and M-7formation in a panel of human small-intestinal microsomes.

Microsomal suspensions were diluted to ∼0.05 mg/ml with reaction buffer, warmed at 37°C in the presence of a NADPH-generating system, and then incubated with 5 μM saquinavir for 15 min.

View this table:
Table 3

Correlation between rates of saquinavir metabolite formation in human small-intestinal microsomes

Human Recombinant Cytochrome P450-Dependent Biotransformation of Saquinavir.

Human recombinant CYP3A4 oxidized saquinavir to a qualitatively similar metabolic profile that was observed with both human small-intestinal and human hepatic microsomes (fig. 1C). Ketoconazole (3 μM) effectively inhibited the formation of all recombinant CYP3A4-generated metabolites (data not shown). Incubation with recombinant CYP2D6 showed the formation of an unidentified metabolite; human recombinant CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, and 2E1 did not oxidize saquinavir (data not shown).

In Vitro Small-Intestinal Drug Interaction between the HIV Protease Inhibitors Indinavir and Saquinavir.

Formation of saquinavir metabolites M-1 to M-7 by human small-intestinal microsomes was inhibited by coincubation with indinavir. Kinetic analyses showed that indinavir was a competitive inhibitor of saquinavir oxidation and that indinavir had aKi value of ∼0.2 μM for the formation of saquinavir metabolites M-2 and M-7 (fig.8).

Figure 8
Figure 8

Lineweaver-Burk plots for the formation of saquinavir metabolite M-7 in the presence of different fixed concentrations of indinavir.

Human small-intestinal microsomes (HG30; 0.01 mg/ml) were incubated with 0.1–1.5 μM saquinavir and with either 0, 0.25, 0.5, 1.0, or 1.5 μM indinavir in the presence of a NADPH-generating system at 37°C for 5 min. The inset plots the slopes of the Lineweaver-Burk plots against indinavir concentrations; Ki values were estimated from the X-axis intercepts. I = indinavir.

In Vitro Small-Intestinal Drug Interaction between Terfenadine and Saquinavir.

Detoxication of the antihistamine terfenadine to its pharmacologically active, nontoxic alcohol metabolite is catalyzed by human hepatic CYP3A4 (19-21). Human small-intestinal microsomes catalyzed the oxidation of terfenadine to its alcohol metabolite. Saquinavir was a potent inhibitor of this detoxication pathway, and saquinavir showed aKi value of 0.7 μM (fig. 9).

Figure 9
Figure 9

Lineweaver-Burk plots for the formation of terfenadine alcohol in the presence of different fixed concentrations of saquinavir.

HG30 microsomes were incubated with 0.3–5 μM terfenadine and with either 0, 0.3, 1, 3, or 10 μM saquinavir in the presence of a NADPH-generating system at 37°C for 15 min. The insetplots the slopes of the Lineweaver-Burk plots against saquinavir concentrations; the Ki value was estimated from the X-axis intercept. S = saquinavir.

Discussion

Several data indicate that saquinavir is metabolized by one cytochrome P450 enzyme or multiple closely related and regulated enzymes. First, kinetic analyses of saquinavir oxidation toM-2 and M-7 showed identicalKM values and monophasic kinetics over a selected concentration range (0.1–1.5 μM). Second, despite the large variance in M-2 and M-7 formation between intestinal microsomal preparations, the metabolic profiles were the same and the ratio of M-2 to M-7 was constant (fig. 7). Moreover, the rate of formation of metabolites in multiple microsomal preparations was highly correlated with each other (table3). Contribution of multiple cytochrome P450 enzymes to the oxidation of saquinavir would be expected to give dissimilar metabolic profiles and ratios between microsomal samples, as well as reduced correlations between all metabolites. Only enzymes that are highly homologous and coregulated would give analogous metabolic profiles and ratios. Third, the qualitatively identical metabolic profiles from incubations of saquinavir with human hepatic microsomes, human small-intestinal microsomes, and recombinant CYP3A4 are indicative of a common cytochrome P450 enzyme (fig. 1). These data are suggestive of the CYP3A family because it is one of the few cytochrome P450 enzymes expressed both in liver and small intestine capable of metabolizing substrates to numerous metabolites (22-25) and because recombinant CYP3A4 was exclusive of all recombinant enzymes studied in metabolizing saquinavir to any oxidative product. These data do not preclude, however, the potential involvement of CYP3A5, which is present in ∼25% of the population (26).

The substantive inhibition of saquinavir oxidation by low concentrations of ketoconazole and troleandomycin is solid evidence for the involvement of the CYP3A family. Midazolam and cyclosporin A, substrates of human intestinal CYP3A4 (10-13), were also effective in decreasing the biotransformation of saquinavir (table 1). A role for other cytochrome P450 families in the oxidation of saquinavir could not be demonstrated. Furafylline and fluvoxamine, inhibitors of CYP1A2 (27-29), had no inhibitory effect on the formation of any saquinavir metabolite. In addition, the cytochrome P450-selective inhibitors sulfaphenazole, mephenytoin, quinidine, and chlorzoxazone for CYP2C9 (30), CYP2C19 (31), CYP2D6 (32, 33), and CYP2E1 (34), respectively, were ineffective in inhibiting the formation of any saquinavir metabolite (table 1). The modest inhibitory effect observed with higher concentrations of quinidine is consonant with previous studies that showed that quinidine at high concentrations inhibits CYP3A4 in addition to CYP2D6 (35, 36).

The inhibition of M-1 through M-6 by 7,8-benzoflavone, an inhibitor of CYP1A (37, 38), cannot be attributed to the inhibition of CYP1A because furafylline and fluvoxamine had no substantive effect on the metabolism of saquinavir. Rather, the activation and regioselectivity of M-7 formation by 7,8-benzoflavone (table 2) is in agreement with previous studies that show that 7,8-benzoflavone is an activator of CYP3A4 activity (24). Metabolism of benzo(a)pyrene and dapsone is stimulated by 7,8-benzoflavone (25, 39) and is attributed either to the involvement of different enzymes in the CYP3A family or to conformational changes at the active site. Shou et al. (22) presented evidence that 7,8-benzoflavone and phenanthrene bind simultaneously to the active site of CYP3A4, that the simultaneous binding of both substrates did not alter the affinity of each substrate for the enzyme, and that the rates of metabolism of both substrates were changed. The stimulation and regioselectivity of M-7 formation caused by coincubation with 7,8-benzoflavone may be the result of a couple of factors. First, binding of 7,8-benzoflavone to the active site may prevent saquinavir from binding in different orientations in the active site other than that required for the formation of M-7 and would, therefore, result in the loss of competing reactions. Second, although not mutually exclusive, 7,8-benzoflavone may exclude water from the active site and may, therefore, enhance catalytic activity by preventing the loss of activated oxygen to the production H2O2.

A role for CYP3A4 in the oxidation of saquinavir was also shown by correlation analyses. Testosterone 6β-hydroxylation, a known marker activity for CYP3A4, showed the highest correlation with saquinavir metabolite formation, whereas marker activities for CYP1A2, CYP2C19, CYP2D6, and CYP2E1 showed no significant correlation (table 2). Modest correlation was also observed between saquinavir metabolite formation and tolbutamide methylhydroxylation, indicating the possible involvement of CYP2C9 in human hepatic microsomes. The involvement of CYP2C9 in the intestinal biotransformation of saquinavir, however, is probably negligible because sulfaphenazole had no inhibitory effect on the metabolism of saquinavir (table 1), because of the relatively low intestinal concentrations of CYP2C9 (11), and because recombinant CYP2C9 did not metabolize saquinavir. Correlations with coumarin 7-hydroxylation and lauric acid 12-hydroxylation were relatively low, and the contribution of CYP2A6 and CYP4A1 to the metabolism of saquinavir is probably insignificant because little evidence has been shown for expression of these enzymes in the gastrointestinal tract. Although correlation analyses indicate a predominant role for CYP3A4, these data emphasize the potential differences between hepatic and small-intestinal metabolism.

The interaction between saquinavir and indinavir and between saquinavir and terfenadine provide additional evidence that CYP3A4 is involved in the intestinal biotransformation of saquinavir. Indinavir, a potent HIV protease inhibitor, is a substrate for human hepatic CYP3A4, with an apparent KM value of ∼2 μM (40). Despite the fact that indinavir has a relatively high affinity for CYP3A4, the turnover rate is relatively low, and higher small-intestinal protein concentrations were required to observe significant metabolism of indinavir (fig. 5). The high affinity-low turnover of indinavir with CYP3A4 and the low Ki value of indinavir on the metabolism of saquinavir suggests that combination therapy with these two protease inhibitors may improve the low relative bioavailability of saquinavir. Combination therapy between saquinavir and ritonavir, another HIV protease inhibitor and CYP3A4 substrate, increased saquinavir blood concentrations and antiviral activity in humans (41).

In addition to the interaction observed with indinavir, an interaction was observed between the antihistamine terfenadine and saquinavir. Oxidation of terfenadine to its alcohol metabolite is catalyzed by CYP3A4 and constitutes a detoxication pathway (19-21). Drug interactions that result in the inhibition of terfenadine oxidation have been shown to increase the risk of torsades de pointes, a potentially lethal cardiac arrhythmia (42-44). The lowKi value of saquinavir (fig. 9) accentuates the potential risks associated with multidrug therapy with CYP3A4 substrates.

Collectively, data presented provide evidence that saquinavir is a substrate for CYP3A4 and that saquinavir is extensively metabolized by human small-intestinal microsomes. The inhibitory effect of the cytochrome P450-selective inhibitors ketoconazole and troleandomycin, the inhibitory effect of CYP3A4 substrates cyclosporin A and midazolam, the correlation with CYP3A4 marker activity testosterone 6β-hydroxylation, the biotransformation of saquinavir by recombinant CYP3A4, and the inhibition of the CYP3A4 substrate terfenadine indicate that CYP3A4 is the principal enzyme involved in the intestinal biotransformation of saquinavir.

Data also show that indinavir is a potent inhibitor of saquinavir oxidation, suggesting the potential for increased bioavailability of saquinavir when used in combination with other HIV protease inhibitors. An increase in bioavailability as a result of combination therapy with multiple protease inhibitors may enhance the antiviral activity of this class of compounds, as well as decrease the risk of viral resistance. Although the data presented herein conclusively show that human intestinal tissue, specifically CYP3A4, has the capability to metabolize saquinavir, the contribution of the intestinal tract to the overall first-pass metabolism and to the low bioavailability of saquinavir remains to be determined. Experiments with perfused sections of human intestine, the use of intestinal model systems, and furtherin vivo studies may indicate the role of the gastrointestinal tract in the first-pass metabolism of saquinavir, identify additional phase I or phase II reactions that may be involved at the higher saquinavir concentrations that would be expected in the gastrointestinal tract after a standard oral dose, and highlight the potential role of the intestinal mucosa in the clearance of orally administered xenobiotics.

Acknowledgments

We thank Hoffmann-LaRoche for their contribution of saquinavir and Merck Research Laboratories for their contribution of indinavir; J. W. Harris and A. Rahman for their assistance in acquiring and preparing small intestine; and E. Eidbo from the Washington Regional Transplant Consortium for his professionalism and commitment to research.

Footnotes

  • Send reprint requests to: Dr. Michael E. Fitzsimmons, U.S. Food and Drug Administration, Division of Clinical Pharmacology Research, 4 Research Court, Room 311, Rockville, MD 20850.

  • Abbreviations used are::
    HIV
    human immunodeficiency virus
    IC90
    inhibitory concentration of 90%
    CYP
    cytochrome P450
    HL
    human liver
    HG
    human small intestine
    ESI
    electrospray ionization
    • Received September 12, 1996.
    • Accepted November 19, 1996.

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Selective Biotransformation of the Human Immunodeficiency Virus Protease Inhibitor Saquinavir by Human Small-Intestinal Cytochrome P4503A4

Michael E. Fitzsimmons and Jerry M. Collins
Drug Metabolism and Disposition February 1, 1997, 25 (2) 256-266;
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