Efficient validated method of UPLC-MS/MS to determine curcumin in rat plasma and ovarium

Curcumin, a lipophilic polyphenol derived from the roots of Curcuma longa. Recently, it has been widely investigatedas a therapeutic agent for cancer. Thus, there is a growing interest in measuring curcumin concentrations in theplasma and other target tissues in relevant animal models. We developed and validated a simple, fast, and reliablemethod for quantifying curcumin in biological matrices by Ultra Performance Liquid Chromatography (UPLC)-MassSpectrometry (MS)/MS. The liquid chromatography system is using rapid separation on Acquity UPLC®BEH C18with gradient mobile phase contained formic acid and acetonitrile. Prior to detection, curcumin and internal standard(IS) were ionized using electrospray ionization positive source and the ions were monitored at m/z 369 → 177 and 260→ 183 for curcumin and IS, respectively. The calibration curve was linear (r ≥ 0.99) over the concentration range of1–50 ng/ml and 1–30 ng/ml for rat plasma and for ovary homogenate, respectively. The lower limit of quantificationwas 1 ng/ml. The mean accuracy ranged from 98.9% to 103.2% and 98% to 108.9%, while the coefficient of variation(CV) values of precision in rat plasma were below 11.92% and 10.47% for within and between run, respectively. Inrat ovary homogenate, the mean concentration and CV of within run accuracy and precision were 95.53%–109.78%and 3.34%–9.14%, respectively. The developed method was used to quantify curcumin in rat plasma and ovary afteran oral gavage. In conclusion, the developed and validated method should be useful for quantification of curcuminaccurately and precisely in plasma and target organs from relevant animal models of human diseases.

Influence of primaquine and ritonavir interaction on CYP3A4 mRNA expression in HepG2 cell culture.

Background: Concomitant treatment with antimalaria and antiretroviral drug is a new challenge in the management of malaria and HIV co-infection. Primaquine is a substrate and also an inhibitor of CYP3A4, while ritonavir is a substrate, an inhibitor, and also an inducer for CYP3A4. The objective of this study is to measure the CYP3A4 mRNA expression in HepG2 cell culture induced by primaquine and ritonavir co-treatment. Methods: For the initial study HepG2 cells were treated with 30, 40, 50 uM of primaquine; 2, 10, 20 uM ritonavir; DMSO ≤0.1 % for negative control; or 20 uM rifampicin for positive control. While for the co-treatment study the cells were treated with 40 uM primaquine+10 uM ritonavir; DMSO ≤0.1 %; or 20 uM rifampicin for 72 hours. The cells were harvested using trypsin–EDTA and total RNA was extracted using the Tripure isolation reagent. After determining the quantity of RNA spectrophotometrically, CYP3A4 mRNA expression was quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). Results: The expression of CYP3A4 mRNA was up-regulated (1.22 fold over control) in HepG2 cells co-treated with primaquine and ritonavir. These data suggest that the induction effect of ritonavir was more dominant than the inhibitory effect of primaquine. Conclusion: Concomitant administration of primaquine and ritonavir result in up-regulation of CYP3A4 mRNA expression in vitro.

Primaquine decreased plasma concentration of ritonavir: single- and repeated-dose study in Sprague Dawley rats.

Background: The present study was aimed to explore the effects of ritonavir and primaquine combination given as a singledose or repeated-dose compared to ritonavir alone on ritonavir plasma concentration in the rats. Methods: In single-dose study, 30 male Spraque Dawley rats were randomly allocated to receive ritonavir 20 mg/kg BW or ritonavir 20 mg/kg BW + primaquine 1.2 mg/kg BW or ritonavir 20 mg/kg BW + ketokonazole 10 mg/kg BW. Ketokonazole was used as positive control of ritonavir metabolism inhibitor. In the repeated-dose study, thirty Spraque Dawley male rats were randomly allocated to receive ritonavir 20 mg/kg BW/day or ritonavir 20 mg/kg BW/day + primaquine 1.2 mg/kg BW/day or ritonavir 20 mg/kg BW/day + rifampicin 100 mg/kg BW/day. Rifampicin was used as a positive control of ritonavir metabolism inducer. Results: In the single-dose study, ketokonazole increased the area under the plasma concentration (AUC) of ritonavir (↑114.8%, p< 0.05), while primaquine tended to decrease the AUC of ritonavir (↓ 32.6%, p> 0.05). Repeated-dose study showed that rifampicin decreases the AUC of ritonavir (↓ 42.8%, p< 0.001), and primaquine decreased the AUC of ritonavir plasma concentration (↓ 46.6%, p< 0.001). Conclusion: Concomitant administration of primaquine and ritonavir decreases the AUC of ritonavir. This effect may result in the insufficient concentration of ritonavir as anti-HIV, which may lead to treatment failure with ritonavir.

Incretin-based therapies for type 2 diabetes mellitus in Asian patients: Analysis of clinical trials.

Aim To review the effi cacy and safety data on incretin-based therapies currently available (exenatide, liraglutide, sitagliptin, vildagliptin) for the treatment of type 2 diabetes mellitus in Asian population. Methods We conducted Medline search of all relevant randomized clinical trials of incretin-based therapies for type 2 diabetes mellitus in Asian populations. Data pertinent to the effi cacy and safety of GLP-1 mimetics and DPP-4 inhibitors were extracted and used. Results We found 14 randomized controlled trials of incretin based-therapy which included 3567 type 2 diabetes mellitus in Asian population (Japanese, Chinese, Korean, Indian). It was shown that incretin-based therapies improved HbA1c at higher extent (up to -1.42% in exenatide 10 mcg bid, -1.85% for liraglutide 0.9 mg qd, -1.4% for sitagliptin 100 mg and -1.4% for vildagliptin 50 mg bid) compared to the effects observed in studies with Caucasian population, with comparable safety profi le. Conclusion The effi cacy of incretin-based therapies in Asian patients improved glycemic parameters in a higher magnitude on some glycemic parameters compared with those in Caucasian population. These results indicate that incretin-based therapies may be more effective in Asian population than in Caucasian.

The effect of lycopene on the total cytochrome P450, CYP1A2 and CYP2E1.

Aim Some carotenoids such as canthaxantin, astaxanthin and beta apo-8’-carotenal were reported to have modulatory effect on the cytochrome P450. The present study was conducted to investigate the effects of lycopene, a nonprovitamin A carotenoid, on microsomal cytochrome P450, CYP1A2 and CYP2E1. Methods Total cytochrome P450 levels, CYP1A2 and CYP2E1-catalyzed reactions (acetanilide 4-hydroxylation and p-nitrophenol hydroxylation) were studied in the liver microsomes of male Sprague Dawley rats. Microsomes were prepared using differential centrifugation combined with calcium aggregation method. Lycopene was orally administered in the dosages of 0, 25, 50 or 100 mg/kgBW/day for 14 days in a repeated fashion. Data were analyzed using ANOVA test. Results Total cytochrome P450 level and acetanilide 4-hydroxylase activity were unaffected by any of the treatments. The CYP2E1 probe enzyme (p-nitrophenol hydroxylase) was signifi cantly reduced by repeated administration of 100 mg/kgBW/day lycopene (7.88 + 2.04 vs 12.26 + 2.77 n mol/min/mg prot). Conclusion The present results suggest that lycopene does not affect the total cytochrome P450 or CYP1A2 activity but it inhibits the activity of CYP2E1 (p-nitrophenol hydroxylase) in the rat.