Mechanism of indinavir-induced hyperbilirubinemia

Abstract

Indinavir is a viral protease inhibitor used for the treatment of HIV infection. Unconjugated hyperbilirubinemia develops in up to 25% of patients receiving indinavir, prompting drug discontinuation and further clinical evaluation in some instances. We postulated that this side-effect is due to indinavir-mediated impairment of bilirubin UDP-glucuronosyltransferase (UGT) activity and would be most pronounced in individuals with reduced hepatic enzyme levels, as occurs in approximately 10% of the population manifesting Gilbert's syndrome. This hypothesis was tested in vitro, in the Gunn rat model of UGT deficiency, and in HIV-infected patients with and without the Gilbert's polymorphism. Indinavir was found to competitively inhibit UGT enzymatic activity (K(I) = 183 microM) while concomitantly inducing hepatic bilirubin UGT mRNA and protein expression. Although oral indinavir increased plasma bilirubin levels in wild-type and heterozygous Gunn rats, the mean rise was significantly greater in the latter group of animals. Similarly, serum bilirubin increased by a mean of 0.34 mg/dl in indinavir-treated HIV patients lacking the Gilbert's polymorphism versus 1.45 mg/dl in those who were either heterozygous or homozygous for the mutant allele. Whereas saquinavir also competitively inhibits UGT activity, this drug has not been associated with hyperbilirubinemia, most likely because of the higher K(I) (360 microM) and substantially lower therapeutic levels as compared with indinavir. Taken together, these findings indicate that elevations in serum-unconjugated bilirubin associated with indinavir treatment result from direct inhibition of bilirubin-conjugating activity.

Figure 1

Bilirubin conjugation by rat liver microsomes. (Upper) Time course for the generation of bilirubin glucuronides following the addition of 20 μM (■) or 50 μM (●) unconjugated bilirubin to a suspension of rat liver microsomes (2 mg/ml protein). Each point reflects the mean (±SD) of three experiments and is corrected for baseline absorbance. (Lower) The initial rate of bilirubin glucuronide production in the presence (■) or absence (●) of 500 μM indinavir is plotted against the concentration of unconjugated bilirubin (UCB).

Figure 2

Reciprocal plots of bilirubin UGT activity. (Left) A reciprocal plot of the initial velocity of bilirubin glucuronide formation versus the concentration of unconjugated bilirubin (UCB) measured in the absence (●) or presence of 250 μM (▴) or 500 μM (■) indinavir. Points reflect the mean (±SD) of three experiments and are fit to linear functions (solid lines). Values for the K_m (12.8 ± 1.0 μM, ±SE) and V_max (655 ± 22 pmol glucuronide × mg protein⁻¹ × min⁻¹) of bilirubin UGT were determined from the fit of data obtained in the absence of indinavir to the expression: y = 1/V_max + K_m/V_max. (Right) Identical experimental conditions were used in an analysis of the effect of 500 μM saquinavir (■) on bilirubin UGT activity (K_I = 360 ± 11 μM). In the absence of saquinavir (●), K_m and V_max values were 12.9 ± 0.9 μM and 654 ± 20 pmol × mg protein⁻¹ × min⁻¹, respectively.

Figure 3

Effect of indinavir on UGT expression in rat hepatoma cells. Monolayers of H35 rat hepatoma cells were grown for 18 h in the presence of indinavir (0–50 μM). Cells were harvested, and 25 μg of total cellular RNA were subjected to Northern blotting with a murine cDNA probe specific for exon 1 of UGT1A1 (Left) and with a nonspecific UGT probe (Center). Blots were stripped and rehybridized with cDNA for ubiquitin to control for loading (Lower). (Right) H35 cells were harvested following an 18-h incubation in the absence (−) or presence (+) of 50 μM indinavir, and the microsomal fraction (30 μg protein) was subjected to SDS/PAGE on a 10% gel. The final lane contains rat liver microsomes (m) as control. Western blotting was performed with rabbit antiserum (1:2,000 dilution), and a 55-kDa band corresponding to bilirubin UGT was identified.

Figure 4

Effect of indinavir on plasma bilirubin levels in Wistar and heterozygous Gunn rats. (Upper) The concentration of total bilirubin in the plasma of wild-type (+/+) Wistar (n = 6, dark circles) and heterozygous (j/+) Gunn (n = 6, gray circles) rats before (pre) and after (post) receiving four doses of indinavir (240 mg/kg) every 8 h via gavage. Mean plasma bilirubin levels increased from 0.050 ± 0.002 to 0.070 ± 0.003 mg/dl (±SE) in +/+ rodents (P < 0.0001) and from 0.054 ± 0.002 to 0.096 ± 0.007 mg/dl (P = 0.0007) in j/+ animals. (Lower) the change in the plasma bilirubin concentration, stratified by genotype, is plotted for each animal. The lines reflect mean values for the +/+ (0.018 ± 0.004 mg/dl) and j/+ (0.043 ± 0.008 mg/dl) rats (P = 0.02).

Figure 5

Hepatic UGT expression in indinavir-treated and untreated rats. Total RNA was isolated from the livers of untreated (−) heterozygous Gunn (j/+) and wild-type Wistar (+/+) rats and from animals treated with four doses of indinavir (240 mg/kg) every 8 h via gavage (+). Northern blotting (25 μg of RNA) was performed by using a murine cDNA probe specific for exon 1 of UGT1A1 (Upper Left) and a nonspecific UGT probe (Upper Right). (Lower) 500 μg of whole liver homogenate (Left) and 50 μg of isolated hepatic microsomes (Right) from indinavir-treated (+) and untreated (−) rats was subjected to Western blotting with rabbit antiserum (1:500 dilution).

Figure 6

Genotyping of UGT1A1 promoter polymorphisms. DNA samples from five separate HIV-infected patients were amplified by PCR using primers specific for the UGT1A1 promoter region and subjected to electrophoresis on a 12% acrylamide gel. The presence of the A(TA)₆TAA allele results in a 71-bp PCR product, whereas the A(TA)₇TAA allele yields a 73-bp fragment. Genotypes are assigned as follows: 6/6, homozygous for the wild-type A(TA)₆TAA allele; 7/7, homozygous for the A(TA)₇TAA Gilbert's allele; 6/7, heterozygous for the A(TA)₆TAA and A(TA)₇TAA alleles.

Figure 7

Influence of the Gilbert's polymorphism on serum bilirubin levels in HIV-infected patients treated with indinavir. (Upper) Serum total bilirubin concentrations for individual patients with UGT1A1 promoter genotype 6/6 (dark circles), 6/7 (gray squares), and 7/7 (gray triangles) before (pre) and after (post) receiving a minimum of 4 weeks of oral indinavir. (Lower) The change in serum bilirubin concentration for each patient following indinavir treatment is stratified for the absence (6/6, dark circles) or presence (6/7, dark squares; or 7/7, gray triangles) of the Gilbert's mutation. Mean values for each group (solid lines) were 0.34 ± 0.29 mg/dl and 1.45 ± 0.99 mg/dl (±SD), respectively (P = 0.030).