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Successful Efavirenz Dose Reduction in HIV Type 1-Infected Individuals with Cytochrome P450 2B6 *6 and *26
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Clinical Infectious Diseases Nov 1 2007;45:1230-1237
Hiroyuki Gatanaga,1 Tsunefusa Hayashida,1 Kiyoto Tsuchiya,1 Munehiro Yoshino,2 Takeshi Kuwahara,2 Hiroki Tsukada,4 Katsuya Fujimoto,5 Isao Sato,6 Mikio Ueda,7 Masahide Horiba,8 Motohiro Hamaguchi,9 Masahiro Yamamoto,10 Noboru Takata,11 Akiro Kimura,12 Takao Koike,5 Fumitake Gejyo,4 Shuzo Matsushita,13 Takuma Shirasaka,3 Satoshi Kimura,1 and Shinichi Oka1
1AIDS Clinical Center, International Medical Center of Japan, Tokyo, 2Department of Pharmacy and 3AIDS Medical Center, Osaka National Hospital, Osaka, 4Division of Clinical Nephrology, Rheumatology, Respiratory Medicine and Infection Control and Prevention, Niigata University Graduate School of Medical and Dental Sciences, Niigata, 5Department of Medicine II, Hokkaido University Graduate School of Medicine, Sapporo, 6Sendai Medical Center, Sendai, 7Department of Hematology and Immunology, Ishikawa Prefecture Central Hospital, Kanazawa, 8Department of Respiratory Medicine, Higashi Saitama Hospital, Hasuda, 9Nagoya Medical Center, Nagoya, 10Kyushu Medical Center, Fukuoka, 11Division of Blood Transfusion Services, 12Department of Hematology, Hiroshima University, Hiroshima, and 13Division of Clinical Retrovirology and Infectious Diseases, Center for AIDS Research, Kumamoto University, Kumamoto, Japan
"....EFV dose reduction and initiation of EFV treatment at reduced dose is possible with therapeutic anti-HIV-1 potency retained in CYP2B6 *6/*6 homozygotes and *6/*26 heterozygotes, which could relieve the patients of the EFV-associated CNS symptoms. It may also decrease the risk of development of EFV-resistant HIV-1 after mandatory treatment discontinuation, such as abdominal surgery [23], and reduce the treatment cost, an important issue in developing countries [24]. After dose reduction, however, careful monitoring is necessary until larger studies confirm the safety of reduced dose in such specific genotype carriers."
ABSTRACT
Background. Efavirenz (EFV) is metabolized primarily by cytochrome P450 2B6 (CYP2B6), and high plasma concentrations of the drug are associated with a G→T polymorphism at position 516 (516G→T) of CYP2B6 and frequent central nervous system (CNS)-related side effects. Here, we tested the feasibility of genotype-based dose reduction of EFV.
Methods. CYP2B6 genotypes were determined in 456 human immunodeficiency virus type 1 (HIV-1)-infected patients who were receiving EFV treatment or were scheduled to receive EFV-containing treatment. EFV dose was reduced in CYP2B6 516G→T carriers who had high plasma EFV concentrations while receiving the standard dosage (600 mg). EFV-naive homozygous CYP2B6 516G→T carriers were treated with low-dose EFV. In both groups, the dose was further reduced when plasma EFV concentration remained high.
Results. CYP2B6 516G→T was identified in the *6 allele (found in 17.9% of our subjects) and a novel allele, *26 (found in 1.3% of our patients). All EFV-treated CYP2B6 *6/*6 and *6/*26 carriers had extremely high plasma EFV concentrations (>6000 ng/mL) while receiving the standard dosage. EFV dose was reduced to 400 mg for 11 patients and to 200 mg for 7 patients with persistently suppressed HIV-1 loads. EFV-containing treatment was initiated at 400 mg in 4 CYP2B6 *6/*6 carriers and one *6/*26 carrier. Two of them still had a high plasma EFV concentration while receiving that dose, and the dose was further reduced to 200 mg, with successful HIV-1 suppression. CNS-related symptoms improved with dose reduction in 10 of the 14 patients, although some had not been aware of the symptoms at initial dosage.
Conclusions. Genotype-based EFV dose reduction is feasible in CYP2B6 *6/*6 and *6/*26 carriers, which can reduce EFV-associated CNS symptoms.
BACKGROUND
Efavirenz (EFV) is an important anti-HIV-1 agent in current combination treatment and is usually prescribed at a fixed dosage of 600 mg once daily [1, 2]. The plasma concentration of EFV varies widely in individuals, and the prevalence of CNS symptoms is higher in those with high concentrations [3]. EFV is metabolized mainly by cytochrome P450 2B6 (CYP2B6), and its concentration was reported to be associated with the CYP2B6 516G→T genetic polymorphism [4-8]. Previously, we reported that all Japanese patients with the 516TT genotype had extremely high EFV concentrations (>6000 ng/mL), without exception [4]. However, other studies reported some exceptional cases of subjects with the 516TT genotype with normal concentrations, although most of the 516TT carriers had high concentrations [5-8]. The difference between our data and those of others may reflect polymorphisms other than 516G→T in CYP2B6. If this is the case, analysis of other polymorphisms and determination of the CYP2B6 haplotype may be helpful in predicting EFV plasma levels. In the present study, we determined the CYP2B6 haplotype of 456 HIV-1-infected patients and analyzed its relationship with EFV concentration in 111 of them. Furthermore, we reduced the EFV dose in 12 patients whose EFV concentrations had been high while receiving the standard dosage. We also used reduced doses of EFV in 5 EFV-naive patients in whom EFV concentration was predicted to become extremely high while receiving the standard dosage, on the basis of CYP2B6 haplotype determination.
SUBJECTS, MATERIALS, AND METHODS
Patients. This analysis included 60 previously reported HIV-1-infected individuals at the International Medical Center of Japan (IMCJ) [4] and another group of 396 HIV-1-infected patients who were receiving treatment of the standard dosage (600 mg once daily) of EFV or were scheduled to begin receiving EFV-containing treatment at the following 11 hospitals in Japan: Hokkaido University (Sapporo), Sendai Medical Center (Sendai), Niigata University (Niigata), Higashi Saitama Hospital (Hasuda), IMCJ (Tokyo), Ishikawa Prefecture Central Hospital (Kanazawa), Nagoya Medical Center (Nagoya), Osaka National Hospital (Osaka), Hiroshima University (Hiroshima), Kyushu Medical Center (Fukuoka), and Kumamoto University (Kumamoto). The ethics committee of each hospital approved this study, and each participant gave written informed consent.
CYP2B6 genotype. DNA samples were extracted from peripheral blood specimens obtained from participants, and genotyping of CYP2B6 64C→T (rs8192709), 415A→G (rs12721655), 499CG (rs3826711), 516GT (rs3745274), 777CA (rs number not available), 785A→G (rs2279343), 1375A→G (rs number not available), and 1459C→T (rs3211371) was performed by allele-specific fluorogenic 5 nuclease chain reaction assay with predesigned primers and TaqMan MGB probes (TaqMan SNP Genotyping Assay; Applied Biosystems) or previously published primers and MGB probes [4]. In subjects confirmed to carry 499C→G, all 9 exons of the CYP2B6 gene were amplified with previously published primers [9], and their DNA sequences were directly determined. For haplotype analysis of the CYP2B6 allele, PCR amplification of the genomic region (3130 bp) containing exons 4 and 5 was performed using sense primer 5'-AACTGTACTCACTCCCAGAGT-3' and antisense primer 5'-CTCCCTCTGTCTTTCATTCTGT-3'. The amplified PCR product was subjected to subcloning, and the DNA sequence of each clone was determined. For genotyping of CYP2B6 983TC (rs28399499), new primers and probes were designed as follows: forward primer, 5'-GCCTGAAATGCCTCTTTAAAATGAGATTC-3'; reverse primer, 5'-GCGATGTGGGCCAATCAC-3'; VIC probe for 983T, 5'-CTGTTCAATCTCCC-3'; and FAM probe for 983C, 5'-CTGTTCAGTCTCCC-3'. The obtained genotyping results of CYP2B6 983T→C for >10 patients were confirmed by direct sequencing of exons 7 and 8 with use of primers published elsewhere [9].
Plasma EFV concentration. Samples of peripheral blood were collected during a daytime office visit (9-16 h after the patient took EFV) from the patients who had received EFV treatment at 600-mg dose at bedtime for >4 weeks. EFV concentration was measured by the reverse-phase high-performance liquid chromatography (HPLC) method [10]. For cases of EFV-dose reduction, plasma concentration was measured >2 weeks after the change in EFV dose. Differences in EFV concentrations between groups were examined for statistical significance with Student's t test. A P value <.05 denoted the presence of a statistically significant difference.
RESULTS
Novel CYP2B6 allele. The CYP2B6 genotype was analyzed in 456 HIV-1-infected patients, including 442 Japanese, 8 other Asians, and 6 others. During the analysis, we noticed that some patients had the CYP2B6 499C→G polymorphism, substituting Ala for Pro at the 167th amino acid, which is already registered in the SNP Database, although the CYP2B6 allele containing 499G had not been determined yet. TaqMan Genotyping Assay indicated that CYP2B6 449G was heterozygous with 499C in 12 individuals (2.6%), who were all Japanese (table 1). Direct sequencing of all the exons confirmed the results of TaqMan Genotyping Assay and showed that 8 subjects had 516GT, 785AG, and 1375AA genotypes; 3 had 516TT, 785GG, and 1375AA genotypes; and 1 had 516GT, 785AG, and 1375AG genotypes without any other mutation. Subcloning analysis of the PCR products confirmed that 499G always coexisted in the same allele with 516T and 785G (figure 1). Therefore, it was concluded that the novel haplotype containing 499C→G had 2 other single-nucleotide polymorphisms (SNPs): 516G→T and 785A→G. We formally registered this novel allele with the Human Cytochrome P450 Allele Nomenclature Committee, and it was designated "CYP2B6 *26" (http://www.cypalleles.ki.se/). With use of this nomenclature, the CYP2B6 haplotype of the twelve 499C→G carriers were identified as eight *1/*26 heterozygotes, three *6/*26 heterozygotes, and one *23/*26 heterozygote (table 1). The allelic frequency of *26 was 1.3% in our study participants.
CYP2B6 haplotype determination. In 456 HIV-1-infected individuals, we determined the genotypes of 9 SNP positions (64C→T, 415A→G, 499C→G, 516G→T, 777C→A, 785A→G, 983T→C, 1375A→G, and 1459C→T) in CYP2B6 (table 1). No CYP2B6 genetic polymorphism was detected in 211 patients, and their haplotype was determined to be *1/*1. The haplotypes of single-SNP carriers with 64CT, 785AG, 1375AG, and 1459CT were determined to be *1/*2, *1/*4, *1/*23, and *1/*5, respectively. Those of homozygous polymorphism carriers with 785GG only, 1459TT only, and both 516TT and 785GG were determined to be *4/*4, *5/*5, and *6/*6, respectively. When the fact that *2 is the only allele harboring 64C→T is considered, patients with 64CT and 785AG; 64CT and 1459CT; and 64CT, 516GT, and 785AG were identified as *2/*4, *2/*5, and *2/*6 heterozygotes, respectively. Patients with both 516GT and 785GG genotypes but without other polymorphisms were determined to have *4/*6 heterozygotes. There were 104 patients (22.8%), including 101 Japanese, who held both 516GT and 785AG genotypes without other polymorphisms. There were 2 possible haplotypes, *1/*6 and *4/*9, in this genotypic pattern. When the fact that *9 had not been reported in Japanese subjects was considered [11], we found that all 101 Japanese were *1/*6 heterozygotes. Haplotype analysis by subcloning of PCR products described above was performed in the 3 others, and their haplotype was determined as *1/*6. One Japanese patient had 516GT, 785AG, and 1459CT genotypes without other polymorphisms, and there were 2 possible haplotypes, *1/*7 and *5/*6, in this genotypic pattern. Because *7 had not been reported in Japanese subjects [11], the haplotype in this patient was determined to be *5/*6. Overall, the allelic frequency of *6 was 17.9% in our study participants. The 415A→G, 777C→A, and 983T→C polymorphisms, which are the determinants of *8, *3, and *18, respectively, were not observed in our subjects.
CYP2B6 and EFV concentration. We determined the CYP2B6 haplotype in 251 patients at IMCJ and in 205 patients at the other 10 hospitals. Of the 251 genotype-analyzed patients at IMCJ, 101 were being treated or were beginning treatment with a standard dose of EFV during this study period (figure 2). Plasma EFV concentrations were measured in all 101 patients, including sixty-seven 516GG holders, twenty-eight 516GT holders, and six 516TT holders. To clarify the effect of the 516TT genotype, EFV concentration was also measured in ten 516TT holders undergoing treatment with the standard dose of EFV at other hospitals. The mean concentration (±SD) of EFV in all patients was 3740 ± 2800 ng/mL. When divided by the genotype of position 516, striking discreteness was observed (figure 3). All (95% CI 91.1%-100%) of the 16 carriers of 516TT genotype, including fourteen *6/*6 carriers and two *6/*26 carriers, had extremely high EFV concentrations (>6000 ng/mL). Their mean concentrations (9500 ± 2580 ng/mL) were many orders of magnitude higher than those of the other genotype carriers (P < 10-4). There was no significant difference in EFV concentration between *6/*6 carriers and *6/*26 carriers. On the other hand, there were only 2 patients who had such high EFV concentrations among the other genotype carriers. One was a *1/*6 carrier (7140 ng/mL), and the other was a *1/*26 carrier (9710 ng/mL). Direct sequencing of all CYP2B6 exons showed no polymorphism other than 499C→G, 516G→T, and 785AG in these individuals. The mean concentrations of EFV of the twenty-eight 516GT carriers, including twenty-five *6-heterozygotes (3320 ± 1240 ng/mL; P < 10-4) and three *26-heterozygotes (5470 ± 3840 ng/mL; P < 10-4), were significantly higher than those of the sixty-seven 516GG genotype carriers (2450 ± 770 ng/mL). None (95% CI 0%-0.1%) of the 516GG carriers had a high EFV concentration (>6000 ng/mL). Considered together, it was concluded that high plasma EFV concentrations were associated with CYP2B6 *6 and *26 and that CYP2B6 *6/*6 and *6/*26 carriers had extremely high plasma EFV concentrations at standard dosage, without exception.
EFV dose reduction from 600 mg. To determine whether the EFV dose can be reduced in patients who have a high concentration while receiving the standard dose, a dose-reduction protocol was applied in 12 patients with high plasma concentrations (>6000 ng/mL [range, 6170-14,690 ng/mL]), including one *1/*26 heterozygote, nine *6/*6 homozygotes, and two *6/*26 heterozygotes. Before the dose reduction, plasma HIV-1 load was undetectable (<50 copies/mL) in all patients for >1 month with treatment of a standard antiretroviral regimen containing 600 mg of EFV. In these 12 patients, the EFV dose was reduced from 600 to 400 mg in 11 subjects and was further reduced to 200 mg in 7 of them who consented to further reduction. The plasma EFV concentrations decreased by approximately one-third (36%-46%), to 3720-6160 ng/mL, with dose reduction from 600 to 400 mg in 10 of 11 subjects, and further decreased by approximately one-half (51%-59%), to 1620-2960 ng/mL, with reduction from 400 to 200 mg in 6 of 7 subjects (figure 4). In one patient who had a markedly high EFV concentration (14,690 ng/mL) at the standard 600-mg dose, however, the concentration decreased unexpectedly by 69%, to 4500 ng/mL, with the reduction to 400 mg and further decreased by 82%, to 790 ng/mL, lower than the recommended range (>1000 ng/mL) [1], with the reduction from 400 to 200 mg. Therefore, the dose was increased in this patient back to 400 mg. In another patient who had reported severe dizziness during treatment with the standard dose (600 mg), the dose was reduced immediately to 200 mg at the patient's request. The plasma EFV concentration was also markedly high in this patient (14,360 ng/mL) during treatment with the standard dosage. However, it decreased by 83%, to 2410 ng/mL, with the dose reduction to 200 mg. Consequently, the final EFV dose was 400 mg in 5 subjects and 200 mg in 7 subjects. The determined dosage for each patient was continued for >6 months (the longest was 26 months for a patient who received the 200-mg dose), and the plasma HIV-1 load was continuously undetectable in all patients.
EFV initiation at 400-mg dose. Our analysis showed that CYP2B6 *6/*6 and *6/*26 carriers had extremely high EFV concentrations, without exception (figure 3), and that dose reduction was possible in patients with high EFV concentration with retention of therapeutically effective anti-HIV-1 activity (figure 4). In the next phase of our study, we used an antiretroviral regimen containing a reduced dose (400 mg) of EFV in 5 EFV-naive patients (four *6/*6 homozygotes and one *6/*26 heterozygote). Before the introduction of low-dose EFV-containing regimen, the plasma HIV-1 loads had been undetectable during receipt of the previous protease inhibitor-containing regimen in all 5 patients. Their EFV concentrations were 4080-9450 ng/mL, and all such concentrations (95% CI, 99.5%-100%) were therapeutically adequate (>1000 ng/mL) at the 400-mg dose (figure 5). One *6/*6 homozygote developed severe dizziness, necessitating discontinuation of EFV-treatment at day 16. His EFV concentration was 5430 ng/mL. In one *6/*26 heterozygote, severe thrombocytopenia emerged, probably because of overdosage of rifabutin prescribed for the treatment of coinfection with Mycobacterium intracellulare, and EFV treatment was stopped at day 15. The EFV concentration was 5770 ng/mL. Two of the remaining 3 patients still had extremely high EFV concentrations (6760 and 9450 ng/mL) at the 400-mg dose, and their dose was subsequently reduced to 200 mg. The plasma EFV concentrations decreased to 2690 and 3660 ng/mL (i.e., by 60% and 61%, respectively). Consequently, 2 subjects discontinued the EFV-containing regimen, and 3 subjects continued low-dose EFV-containing regimen (400 mg for 1 patient and 200 mg for 2 patients). The low-dose regimen was continued for >6 months, and the plasma HIV-1 load was persistently undetectable in all 3 patients.
Improvement of CNS symptoms. As described above, the EFV dose was reduced from 600 to 400 and 200 mg as the final dose in 5 and 7 subjects, respectively (figure 4), and it was decreased from 400 mg as the initial dose to 200 mg for 2 other subjects (figure 5). To delineate the changes in CNS symptoms associated with the decrease in EFV concentration, a questionnaire survey of these 14 patients was conducted regarding 6 items: dizziness, strange dreams, depression, irritability, concentration problems, and sleep difficulty. More than 1 month after the dose had been reduced to the lowest dose, the patients were asked to judge the 6 CNS symptoms above at initial and final doses, with use of a 5-grade system ("none," "slight," "sometimes," "often," and "always"). Ten (71%) of the 14 patients had some of the aforementioned CNS symptoms during treatment with the initial dose (table 2). The most common symptom was dizziness (57%), followed by strange dreams (50%). Interestingly, all the symptoms improved after dose reduction in the 10 patients. Furthermore, dizziness and concentration problems disappeared during treatment with the final dose in one-half of the patients, although strange dreams and sleep difficulty were still reported by all the patients who had those difficulties at the initial dose. Finally, when the patients were asked whether they wanted to reincrease EFV to the previous dose, all 10 patients with CNS symptoms at the initial dose answered "no" (9 answered "absolutely no").
DISCUSSION
In this study, we identified a novel CYP2B6 allele, *26, which includes 499C→G, 516G→T, and 785A→G in 12 Japanese patients, and we showed that, without exception, all *6/*6 and *6/*26 carriers, all holding 516TT, had extremely high plasma EFV concentrations while receiving the standard dose (600 mg) [4]. In other reports, however, there were some exceptional subjects with 516TT who had normal concentrations of EFV, and the discreteness of the EFV concentration with the position 516 genotype was not as clear as it was in our patients [5-8]. This difference may be because some of the 516TT carriers had other CYP2B6 alleles, such as *7 (containing 516G→T, 785A→G, and 1459C→T), *9 (containing 516G→T only), and *13 (containing 415A→G, 516G→T, and 785A→G). Those alleles could not be found in our subjects, and their effects on EFV concentration were not well described. Because numerous additional CYP2B6 variants with impact on expression and/or function were recently reported [12-18], correct determination of CYP2B6 haplotype seems indispensable for prediction of EFV plasma levels.
We reduced the EFV dose in 12 patients whose plasma EFV concentrations were extremely high while receiving the standard dose, and we initiated EFV treatment at a 400-mg dose in 5 EFV-naive *6/*6 and *6/*26 carriers. In most patients, the plasma EFV concentration decreased proportionally with the dose-reduction ratio. In 2 subjects, however, the concentrations decreased much more than expected, given the dose reduction ratio. Both of these patients had markedly high concentrations at standard dosage. Hasse et al. [19] reported a patient with excessively high plasma EFV concentration at standard dose, which decreased to one-thirtieth following dose reduction from 600 to 200 mg. Long-term exposure to such excessively high concentrations may induce CYP2B6 enzymatic expression in the liver, which could result in an unexpectedly large decrease in plasma EFV concentration by dose reduction if deinduction of the enzyme takes several weeks. At the 400-mg dose, the plasma concentrations of EFV were therapeutically adequate in all the treated *6/*6 and *6/*26 carriers in this study. Regarding the reduced dose, it is noteworthy that a phase II study during EFV development supported the use of a lower dose [20]. The same study indicated that the 600-mg dose of EFV is associated with a high rate of adverse events that could lead to discontinuation, which suggests that the lower dose of 400 mg may be almost as effective without the high discontinuation rate. In the present study, associated with the dose-reduction regimen, a significant number of patients experienced improvement of CNS symptoms, which was unexpected on the basis of previous reports [5, 21, 22]. Interestingly, some of these patients did not appreciate their clinical state and considered themselves to have no CNS-related symptoms during the standard-dose treatment. However, after the dose reduction, they reassessed the status and evaluated symptoms during the treatment with the standard dose as associated with CNS symptoms and indicated that the reduced dose of EFV relieved them of such symptoms. Because EFV-treated patients often stick to the regimen, previous reports of symptom questionnaires conducted during the standard treatment might have underestimated the EFV-associated CNS symptoms [5, 21, 22]. However, this finding might be confounded by placebo effect, because the patients were told that their EFV levels were high while receiving the initial dose and decreased throughout the dose-reduction protocol. Because of this possible placebo effect, a double-blind, placebo-controlled study would best address this question.
EFV dose reduction and initiation of EFV treatment at reduced dose is possible with therapeutic anti-HIV-1 potency retained in CYP2B6 *6/*6 homozygotes and *6/*26 heterozygotes, which could relieve the patients of the EFV-associated CNS symptoms. It may also decrease the risk of development of EFV-resistant HIV-1 after mandatory treatment discontinuation, such as abdominal surgery [23], and reduce the treatment cost, an important issue in developing countries [24]. After dose reduction, however, careful monitoring is necessary until larger studies confirm the safety of reduced dose in such specific genotype carriers.
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