HUBUNGAN VARIASI GEN CYTOCHROME P450 2B6 (CYP2B6) DENGAN FARMAKOKINETIKA DIHIDROARTEMISININ (DHA) SETELAH PEMBERIAN ARTEMISININ DOSIS TUNGGAL SECARA ORAL PADA ETNIS TIMOR DI KABUPATEN TIMOR TENGAH SELATAN

LINAWATI HANANTA, Prof. Dr. Mustofa, M.Kes., Apt.; Dr. Med. dr. Indwiani Astuti.; dr. Ahmad Hamim Sadewa, Ph.D

2018 | Disertasi | DOKTOR ILMU KEDOKTERAN DAN KESEHATAN

Latar belakang: Profil farmakokinetika antimalaria yang dihubungkan dengan variasi gen CYP2B6 hingga saat ini masih sangat terbatas. Beberapa penelitian melaporkan resistensi artemisinin (ART) dan derivatnya. Salah satu faktor penyebabnya adalah dosis sub-terapeutik yang berkaitan farmakokinetika ART dan derivatnya. Sitokrom P450 adalah enzim yang bertanggungjawab terhadap metabolisme ART dan metabolit aktif dihidroartemisin (DHA), terutama adalah gen CYP2B6. Sehingga variasi gen CYP2B6 diduga mempengaruhi metabolisme ART pada individu. Penelitian ini menganalisis hubungan variasi gen CYP2B6 dengan farmakokinetika DHA pada etnis Timor yang tinggal di wilayah endemik malaria. Metode: Penelitian dilakukan dua tahap, yang pertama dengan metode cross- sectional. dilakukan analisis gen CYP2B6. Total 555 sampel dari Kab. TTS dengan kriteria inklusi; usia >15 tahun, Hb 10 gr/dL, fungsi hati dan ginjal harus batas normal. Tahap-1 identifikasi variasi gen CYP2B6 ( 4, 9 dan 6) dilakukan dengan PCR-RFLP (protokol dari Promega, Madison, USA) di Laboratorium FKIK Universitas Katolik Indonesia Atma Jaya. Data dianalisis bivariat, multivariat dan uji t dengan p<=0,05 dengan 95% Confident Interval (CI). Tahap-2 uji farmakokinetika dilakukan pada 10 subjek dengan pemberian ART 500 mg per oral pada ketiga kelompok: mutan homozigot, wild type, dan heterozigot dengan sembilan kali pengambilan darah dalam 12 jam. Selanjutnya duukur kadar DHA dalam plasma menggunakan LCMS/MS dan dianalisis dengan modul Saemix versi 2.1 dan non-linier mixed effect model (nlme). Hasil: Total sampel yang berhasil dianalisis PCR-RFLP; 81% (451/555) 4, 93% (516/555) 9, dan 78% (432/555) 6. Prevalensi gen CYP2B6 6 yang mutasi sebesar 38,4%,, tidak mutasi 61,6% dan alel 6 homozigot 18,8%. Pada CYP2B6 4, genotip terbanyak ditemukan A>G sebesar 51,7%, A>A (wild-type) 26,8% dan G>G 21,5%. Selanjutnya CYP2B6 9 terbesar yaitu 41,7% dengan gonotip G>T, berturut-turut T>T, G>G (wild-type) adalah 34,5%, 23,8%. Hasil analisis profil farmakokinetika dengan sparse data menggunakan model satu kompartemen terbuka, nilai rerata Km 3,8 +-3,3 l/jam; Tmaks 1,7 +-1,4 jam; Cmaks 83,9 +-31,8.10mg/L; AUC 262,1 +-158,4.10-3mg.jam/L; Vd 3,2 +-1,7.10.L; CL5 +-3,5.103.L/jam; k eliminasi 2,9 +-3,5 l/jam dan T1/2 1,4 +-1,4 jam. Pada parameter farmakokinetika terdapat perbedaan bermakna antara kelompok varian gen mutanhomozigot dengan wild-type yaitu Tmaks, Cmaks, dan AUC pada mutan homozigot lebih besar; CL, k lebih rendah dan T1/2 lebih panjang dibanding wild type (p<=0,05). Kesimpulan: Variasi gen CYP2B6 pada etnis Timor tinggi dengan variasi gen CYP2B6 6 sebesar 18,8% dan profil farmakokinetika DHA (Tmaks, Cmaks, AUC, CL, k dan T1/2) terdapat hubungan secara bermakna antara variasi gen CYP2B6 homozigot dengan wild-type.

Background: The antimalarial pharmacokinetics profile associated with CYP2B6 gene variation is still very limited. Several studies have reported artemisinin (ART) resistance and its derivatives. One of the contributing factors is sub- therapeutic doses related to ART pharmacokinetics and its derivatives. Cytochrome P450 is an enzyme responsible for the metabolism of ART and the active metabolite dihydroartemisinin (DHA), mainly the CYP2B6 gene. Thus, the CYP2B6 gene variation is thought to affect the metabolism of ART in individuals. This study analyzes the relationship of CYP2B6 gene variation with DHA pharmacokinetics to ethnic Timor who live in malaria-endemic areas. Methods: The study was conducted in two stages, the first with cross-sectional method. CYP2B6 gene analysis was performed. Total samples were 555 from Kab. TTS with inclusion criteria; age >15 years, Hb 10 gr/dL, liver and kidney function should be normal. Phase-1 identification of CYP2B6 gene variations ( 4, 9 and 6) was performed with PCR-RFLP (protocol from Promega, Madison, USA) at FKIK Laboratory of Atma Jaya Catholic University of Indonesia. Data were analyzed bivariate, multivariate and t-test with p<=0,05 with 95% Confident Interval (CI). Pharmacokinetics phases were performed on 10 subjects with ART 500 mg orally in all three groups: homozygous, wild-type, and heterozygous mutants with nine blood samples were collected in 12 hours. Then we measured DHA levels in plasma using LCMS / MS and analyzed with Saemix module version 2.1 and non-linear mixed effect model (nlme). Results: Total samples successfully analyzed by PCR-RFLP; 81% (451/555) for CYP2B6 4, 93% (516/555) for 9, and 78% (432/555) for 6. The prevalence of CYP2B6 6 gene with a mutation of 38.4%, no mutation 61.6% and allele 6 homozygous 18.8%. In CYP2B6 4, most genotypes were found A>G of 51.7%, A>A (wild-type) 26.8% and G>G 21.5%. The next largest CYP2B6 9 was 41.7% with gonotype G>T, respectively T>T, G>G (wild-type) was 34.5%, 23.8%. The result of pharmacokinetics profile analysis with sparse data using open one compartment model, average value Km 3.8 +- 3.3 l/hr; Tmax 1.7 +- 1.4 hours; Cmax 83,9 +- 31,8.10-3 mg / L; AUC 262.1 +- 158,4.10-3mg.jam / L; Vd 3.2 +- 1.7.103.L; CL 5 +- 3.5.103.L/hr; elimination k 2.9 +-3.5 l/h and T1/2 1.4 +- 1.4 h. In the pharmacokinetics parameter there were significant differences between the variant groups of homozygous mutants and wild-type genes, Tmax, Cmax, and AUC in larger homozygous mutants; CL, k is lower and T1/2 is longer than wildtype (p<=0,05). Conclusion: CYP2B6 gene variation in high ethnic Timor with CYP2B6 6 gene were 18.8% and DHA pharmacokinetics profile (Tmax, Cmax, AUC, CL, k and T1/2) there was a significant relationship between variation of homozygous CYP2B6 genes with wild-type.

Kata Kunci : dihidroartemisinin, farmakogenetik artemisinin, etnis Timor , CYP2B6, dihydroartemisinin, artemisinin pharmacogenetic, Timor ethnic,