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Demonstration of the Metabolic Pathway Responsible for Nevirapine-Induced Skin Rash Jie Chen, Baskar M. Mannargudi, Ling Xu,† and Jack Uetrecht* Faculty of Pharmacy, UniVersity of Toronto, Toronto, Ontario, Canada ReceiVed May 14, 2008
The reverse transcriptase inhibitor, nevirapine (NVP), causes skin rashes and hepatotoxicity. We used a rat model to determine if the rash is caused by the parent drug or a reactive metabolite. By manipulation of metabolic pathways and testing analogues, we eliminated all but one pathway, 12-hydroxylation, which involves the oxidation of an exocyclic methyl group, as being responsible for the rash. Treatment with 12-OH-NVP caused a rash, and an analogue in which the methyl hydrogens were replaced by deuterium to inhibit the 12-OH pathway did not cause a rash; however, quite unexpectedly, blood levels of the deuterated analogue were very low. This is due to partitioning of the benzylic free radial intermediate between oxygen rebound to form 12-OH-NVP and loss of another hydrogen atom to form a reactive quinone methide, which inactivates P450. Cotreatment with the P450 inhibitor, 1-aminobenzotriazole, led to comparable levels of NVP and the deuterated analogue, and the deuterated analogue still caused a lower rash incidence. These data clearly point to the 12-hydroxy pathway being responsible for NVP skin rash. We propose that the hepatotoxicity of NVP in humans is due to the quinone methide formed by P450 in the liver, while the skin rash may be due to the quinone methide formed in the skin by sulfation of 12-OH metabolite followed by loss of sulfate. This is the first example in which a valid animal model of an idiosyncratic drug reaction was used to determine the metabolic pathway responsible for the reaction. Introduction A fundamental question about idiosyncratic drug reactions (IDRs) is whether the parent drug or a reactive metabolite is responsible for the reaction. Although there is circumstantial evidence to suggest that reactive metabolites are responsible for most IDRs, it is impossible to rigorously test this hypothesis without a valid model in which there is a clinically evident IDR (1). In addition, many drugs form several reactive metabolites, and without being able to determine which reactive metabolite is responsible, it is impossible to know what types of reactive metabolites are most important to avoid (2). For example, conjugates of carboxylic acidssglucuronides and Co-A esterss covalently bind to proteins and have been proposed to be responsible for many IDRs (3); yet, it is not clear if carboxylic acids are “structural alerts” that need to be avoided. The nevirapine (NVP)-induced skin rash in rats is a model that can be used to address these questions. NVP, a non-nucleoside reverse transcriptase inhibitor, is associated with a relatively high incidence of life-threatening skin rash and liver toxicity in humans (4). Generally, the rash occurs more frequently in females than males, and the incidence is higher when used prophylactically than in patients with AIDS and a low CD4+ T cell count. Treatment of rats with NVP also leads to a skin rash that is strain-, sex-, and dose-dependent. The characteristics of the skin rash in rats are similar to those of the rash that occurs in humans (5). These characteristics include a delay in onset of a few weeks but a very rapid onset on rechallenge. In rats, this sensitivity can be transferred to naïve animals with spleen cells from sensitized rats, which provides * To whom correspondence should be addressed. Tel: 416-978-8939. E-mail:
[email protected]. † Current address: Millennium, Cambridge, MA 02139.
compelling evidence that this is an immune-mediated reaction. In both humans and rats, CD4+ T cell depletion is protective, while, at least in rats, CD8+ T cell depletion, if anything, makes the rash worse (6). On the basis of this and other characteristics, we believe that the basic mechanism of the rash is similar in rats and humans. Although it is likely that there are significant differences in the mechanism of different IDRs, this model provides a unique opportunity to study the mechanism of an IDR in detail. As indicated above, one basic question that can be answered with this model is whether the rash is due to a reactive metabolite and, if so, which reactive metabolite. There are many potential reactive metabolites of NVP. In both humans and rats, the major metabolic pathways of NVP involve 2-, 3-, and 12-hydroxylation (7, 8) (Figure 1). The 2and 3-positions are para to a nitrogen, and further oxidation could lead to quinoneimine type reactive metabolites, while 12OH-NVP has the potential to be sulfated followed by loss of sulfate to form a reactive quinone methide. In addition, 12OH-NVP is further metabolized to 4-COOH-NVP, which when conjugated with a good leaving group such as glucuronide or coenzyme A, has the potential to bind to protein (3). Furthermore, one-electron oxidation of the cyclopropylamine by a peroxidase in the skin such as prostaglandin synthase would lead to opening of the ring with formation of a carbon free radical (9). Although not a major route of clearance, this free radical could lead to significant local covalent binding. In addition, although NVP itself is not chemically reactive, by the p-i hypothesis proposed by Pichler, it might bind directly to the MHC/TCR complex in a reversible manner and induce an immune response (10). In this study, NVP analogues and P450 inhibitors were used in vivo to manipulate the metabolic pathways, and the skin rash incidence was correlated with the blood levels of the parent drug and urinary levels of the
10.1021/tx800177k CCC: $40.75 2008 American Chemical Society Published on Web 08/26/2008
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Figure 1. Biotransformation pathways and the possible reactive metabolites of NVP.
Figure 3. (A) NVP blood levels in different strains and sexes of rats treated with NVP (150 mg/kg/day, n ) 4 each group) in food. (B) Incidence of skin rash with these treatments.
Figure 2. Representative LC-MS-MS chromatographs of NVP, its four major metabolites, and the internal standard using MRM. The M + 1 and fragment ions for each chromatogram are shown in parentheses. 2-OH-NVP and 3-OH-NVP share some fragments, but these two metabolites are separated by retention time.
metabolites. The enzyme inhibitor used was 1-aminobenzotriazole (ABT), which is oxidized to benzyne that inhibits P450 nonspecifically (11, 12). More specific inhibitors such as
ketoconazole were not helpful presumably because the metabolic pathways are mediated by a combination of P450s (13). The analogues included substitution of the cyclopropyl group by an ethyl group (ethyl-NVP), 12-OH-NVP, and 12-trideutero-NVP (DNVP, the methyl hydrogens have been replaced by deuterium). The 12-OH-NVP metabolite has the potential to be activated by sulfation followed by loss of sulfate to form a quinone methide. If breaking the carbon hydrogen bond of the methyl group to form 12-OH-NVP is the rate-limiting step, substitution of the hydrogen atoms by deuterium should significantly decrease the formation of 12-OH-NVP (deuterium isotope effect), but it should have virtually no other effects on the properties of the drug.
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Figure 4. (A) NVP blood levels in female BN rats (n ) 4 except the last group where n ) 2) treated with NVP 150 mg/kg/day, NVP 50 mg/kg/day, NVP 50 mg/kg/day + ABT 50 mg/kg/day, or ABT 50 mg/ kg/day only. NVP was in food; ABT was given by gavage. (B) Incidence of skin rash with these treatments.
Materials and Methods Chemicals. NVP, ethyl-NVP (a NVP analogue where the cyclopropyl group has been replaced by an ethyl group), 2-OHNVP, and 3-OH-NVP were kindly supplied by BoehringerIngelheim Pharmaceuticals, Inc. (Ridgefield, CT). ABT, β-glucuronidase type IX-A (G7396), and other chemicals were obtained from Sigma (Oakville, ON). Analytical. 1H and 13C NMR spectra were determined at 300 MHz. NVP blood levels were determined by HPLC as described previously (6). Solid-phase extraction (SPE) was performed for many experiments with a Strata C18-E (55 µm, 70A) 200 mg SPE column (Phenomenex, Torrance, CA). Quantification of NVP and Its Metabolites. Urine (50 µL; from a 24 h urine sample) was mixed with 100 µL of the internal standard (ethyl-NVP, 27.5 µg/mL in HPLC mobile phase) and 10 µL of β-glucuronidase (92593 U/mL) in 100 mM KH2PO4 buffer, pH 6.8. This mixture was incubated overnight at 37 °C and then concentrated with SPE and analyzed by LC/MS. The HPLC column was an Ultracarb C18 30 mm × 2.0 mm, 5 µm column (Phenomenex). A gradient elution with a flow rate of 0.2 mL/min was used in which mobile phase A consisted of water (with 1% acetic acid and 2 mM ammonium acetate) and mobile phase B was acetonitrile (with 1% acetic acid and 2 mM ammonium acetate). Mass spectrometry was carried out using a PE Sciex API 3000 quadrupole system with an electrospray ionization source (Sciex, Concord, ON). The ion pairs for multiple reaction monitoring (MRM) Q1/Q3 were as follows: 267.0/226.1 (NVP), 283.1/161.0 (2-OH-NVP), 283.1/ 223.1 (12-OH-NVP), 283.1/214.0 (3-OH-NVP), 297.1/210.1 (4COOH-NVP), 270.1/229.3 (DNVP), and 255.1/227.2 (ethyl-NVP) (Figure 2). Standard curves prepared with 2-OH-NVP (0.43-102.9 µg/mL), 3-OH-NVP (0.36-86.8 µg/mL), 12-OH-NVP (0.38-91.0 µg/mL), 4-COOH-NVP (0.26-61.8 µg/mL), NVP (0.74-176.9 µg/ mL), and DNVP (0.86-206.4 µg/mL) had R2 values of >0.99. Detection of the Sulfate of 12-OH-NVP in the Urine and Bile. Bile and urine samples were first concentrated with a SPE column. The methanol eluant was collected and separated with an off-line sample preparation by using the HPLC system described above. Mass spectrometry of fractions was carried out using a LCPacking’s Ultimate Micropump and a Qstar XL Q-TOF mass spectrometer with a nanoelectrospray ionization source in the negative ion mode. The samples were loaded onto the nanotip and transferred into the ion source with 50% methanol with a flow rate of 200 nL/min. Animal Care. Rats (150-175 g) were obtained from Charles River (Montreal, QC) or Harlan (Indianapolis, IN) and doubly
Figure 5. Urinary excretion of (A) 2-OH-NVP, (B) 12-OH-NVP, (C) 3-OH-NVP, and (D) 4-COOH-NVP in female BN rats treated with NVP 50 mg/kg/day or NVP 50 mg/kg/day + ABT 50 mg/kg/day (n ) 4 each group).
housed with a 12:12 h light:dark cycle at 22 °C. After a 1 week acclimatization period, the rats were given standard rat meal (2018, Agribrands, Leis Pet Distributing Inc., ON) for a week during which food intake was monitored. All of the animal studies were conducted in accordance with the guidelines of the Canadian Council on Animal Care. NVP-sensitized animals refers to rats that were treated with NVP at a dose of 150 mg/kg/day in food until they developed a skin rash, and then, the drug was stopped for at least 4 weeks until the rash resolved. If NVP was put in food, the drug was first mixed with a small amount of food, then mixed with bulk food, and thoroughly mixed; samples were taken from different locations of the batch and analyzed to ensure a homogeneous mixture. The amount of NVP in food was adjusted based on the average food intake during the acclimatization period. Blood samples were taken in the morning (9-10 a.m.), and also, food intake was monitored 2-3 times a week during the treatment to further quantify NVP exposure. The initial dose of NVP was lower because the rats did not like the taste of NVP, but in a short period of time, food intake returned close to that during the acclimatization. When NVP was given by gavage or s.c. in the morning, the blood samples were collected ∼8 h after dosing. ABT was dissolved in water (20 mg/ mL) and administered by gavage at 3-5 p.m. Methylcellulose (0.5% in water) was used to suspend NVP or its analogues for gavage or s.c. Various other modes of NVP administration were tried, but because of the combination of very low solubility, especially in water, and the acute toxicity associated with the high peak levels resulting from once a day oral gavage, dosing in food or the slow
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Figure 6. (A) NVP blood levels in male BN rats (n ) 4 each group, except the last group where n ) 2) with NVP 150 mg/kg/day, NVP 150 mg/kg/day + ABT 30 mg/kg/day, or ABT 30 mg/kg/day. NVP was put in food; ABT was given by gavage. (B) Incidence of skin rash with the above treatments.
absorption from a subcutaneous injection of a NVP suspension appeared to be the optimal methods of administration. Preliminary experiments using more specific P450 inhibitors (ketoconazole, 40 mg/kg/day, or ethylxanthate, 25 mg/kg/day) did not result in significantly different metabolite ratios or changes in rash incidence, presumably because multiple P450 enzymes were involved in the oxidation of NVP, and so, this avenue was abandoned. Synthesis of 12-OH-NVP Was Carried out by Using the Method of Grozinger (14) with a Yield of 19%. 1H NMR (DMSO-d6): δ 0.37-0.41 (m, 2H), 0.87-0.90 (m, 2H), 3.61-3.66 (m, 1H), 4.53 (d, J ) 15.9 Hz, 1H), 4.75 (d, J ) 15.6 Hz, 1H), 5.54 (bs, 1H), 7.19 (dd, J ) 4.8, 7.5 Hz, 1H), 7.25 (d, J ) 5.1 Hz, 1H), 8.01 (dd, J ) 2.1, 7.8 Hz, 1H), 8.19 (d, J ) 5.1 Hz, 1H), 8.51 (dd, J ) 1.8, 4.8 Hz,1H), 9.70 (bs, 1H). ESI-MS 283 (MH+). Synthesis of 4-COOH-NVP Was Carried out by Using the Method of Grozinger (15) with a Yield of 27%. 1H NMR (DMSO-d6 + D2O): δ 0.49-0.58 (m, 2H), 0.97-0.99 (m, 2H), 3.55-3.59 (m, 1H), 7.29-7.33 (m, 1H), 7.58-7.62 (m, 1H), 8.18-8.21 (m, 2H), 8.48-8.52 (m,1H). ESI-MS 297 (MH+). Synthesis of DNVP. To a flame-dried flask was added NVP (0.10 g, 0.37 mmol), followed by potassium tert-butoxide (0.08 g, 0.74 mmol) and DMSO-d6 (2.4 mL, 34.28 mmol), and the mixture was refluxed at 140 °C under argon for 48 h. The reaction mixture was diluted with cold water (10 mL) and extracted with ethyl acetate (20 mL). The ethyl acetate layer was then washed with brine (20 mL × 2), dried over anhydrous sodium sulfate, and concentrated to yield crude product, which was column purified using ethyl acetate to yield 0.09 g of product as a yellow solid in 97% yield. 1 H NMR (CDCl3): δ 0.31-0.41 (m, 2H), 0.83-0.90 (m, 2H), 3.60-3.64 (m, 1H), 7.06 (d, J ) 4.8 Hz, 1H), 7.19 (dd, J ) 4.8, 7.5 Hz, 1H), 8.01 (dd, J ) 2.1, 6.6 Hz, 1H), 8.08 (d, J ) 4.8 Hz, 1H), 8.50 (dd, J ) 1.8, 4.8 Hz, 1H), 9.90 (bs, 1H). ESI-MS 270 (MH+), ratio of the peak heights of 267:268:269:270 was 0:0.007: 0.124:0.869, which indicated that synthesized DNVP contained only traces of NVP. Synthesis of the Sulfates of 12-OH-NVP and 3-OH-NVP. 12OH-NVP (1.0 mmol) was mixed with a sulfur trioxide pyridine complex (1.1 equiv) in DMF (1.0 mL) at 0 °C (16, 17). The mixture was stirred overnight and then purified by SPE. 3-OH-NVP (1.0 mmol) was mixed with sulfur trioxide-dimethylformamide complex (DMF-SO3, 1.1 equiv) in DMF (1.0 mL) in the presence of pyridine and nitrogen at 0 °C. The mixture was stirred overnight and then concentrated with a SPE column. Incubation of Rat Liver Microsomes with NVP or DNVP. NVP or DNVP at a concentration of 25 µg/mL was incubated with liver microsomes prepared from male BN rats (100 µL, 2.5 mg of
Figure 7. Urinary excretion of (A) 2-OH-NVP, (B) 12-OH-NVP, (C) 3-OH-NVP, and (D) 4-COOH-NVP in male BN rats treated with NVP 150 mg/kg/day or NVP 150 mg/kg/day + ABT 30 mg/kg/day (n ) 4 each group).
protein) and NADPH (10 µL, 1 mM) in 50 mM phosphate buffer, pH 7.4, containing 20% glycerol and 0.4% KCl. After 0.25, 0.5, 0.75, or 1 h, 50 µL of acetonitrile was added to quench the reaction as well as 10 µL of internal standard (ethyl-NVP, 27 µg/mL). After centrifugation, the supernatant was concentrated by SPE and analyzed by LC/MS.
Results Treatment of Different Strains/Sexes of Animals with NVP. Female Brown Norway (BN) rats, female Lewis rats, and male BN rats were treated with NVP; the resulting incidence of skin rash and the blood levels were different. Female BN rats had the highest blood levels (∼30-40 µg/mL) and the highest incidence of skin rash (100%); the male BN rats had the lowest NVP blood levels (
