Combination of dabrafenib with irinotecan might trigger off a higher frequency of adverse events by pharmacokinetic interaction

Dabrafenib and irinotecan are two drugs that can be utilized to treat melanoma. A previous in vivo study has shown that dabrafenib enhances the antitumor activity of irinotecan in a xenograft model with unclear mechanism. This study aims to investigate the inhibition of dabrafenib on SN-38 (the active metabolite of irinotecan) glucuronidation using human liver microsomes (HLMs) and recombinant human UGT1A1, trying to elucidate the possible mechanism underlying the synergistic effect. Our data indicated that dabrafenib noncompetitively inhibited SN-38 glucuronidation in pooled HLMs and recombinant UGT1A1 with a K i,u value was 12.43 ± 0.28 and 2.64 ± 0.27 μM, respectively. Based on the in vitro K i,u value and estimation of kinetic parameters, dabrafenib administered at 150 mg twice daily may result in about 17%-81.9 % increase in the area under the curve (AUC) of SN-38 in vivo . Moreover, the ratios of [ I ] gut / K i,u are 0.70 and 3.32 in HLMs and recombinant UGT1A1, respectively, indicating a high risk of drug-drug interactions (DDIs) when dabrafenib was used in combination with irinotecan. Our study provides a basis for further development and optimization of this combination in clinical research.

incidence. Specific oncogenes mutation, such as the BRAF mutation, as an important underlying cause of the melanoma, is present in approximately 60% of melanoma patients and has been developed as a therapeutic target [1][2][3]. Dabrafenib is a potent and selective ATP-competitive inhibitor for the BRAF kinase and has been approved for the treatment of unresectable or metastatic BRAF V600-mutat melanoma [4]. As a selective single agent, dabrafenib shows limited clinical activity. Therefore, several combination therapy studies of dabrafenib have been evaluated recently to improve the efficacy in melanoma patients [5][6][7]. Pharmacokinetic studies have shown that dabrafenib is metabolized by cytochrome P450 (CYP) 2C8 and CYP3A4, and is also an inducer of CYP3A4, CYP2B6, and CYP2Cs, implying possibility of drug-drug interactions (DDIs). However, only a few studies have described the potential for DDIs of dabrafenib [8,9].
Irinotecan, a commonly used chemotherapeutic agent, has been recognized in the treatment of cancers including melanoma [10]. SN-38 is the pharmacologically active metabolite of irinotecan and is mainly cleared by the hepatic UDPglucuronosyltransferase 1A1 (UGT1A1) to SN-38 glucuronide (SN-38G), with weaker contributions of UGT1A3, 1A6, 1A7 and 1A9 [11]. The inhibition of UGT1A1 in vivo may increase systemic exposure to SN-38, which is responsible for the irinotecaninduced severe diarrhea and neutropenia [12]. Several tyrosine kinase inhibitors (TKIs), i.e., gefitinib, sunitinib and pazopanib, have been investigated in the combination with irinotecan and may cause clinical toxicity of irinotecan by inhibiting UGT1A1 [13][14][15].
An in vivo study has indicated that the combination of dabrafenib plus irinotecan can 4 result in increased anti-tumor activity in a xenograft model [16], suggesting a pharmacokinetic or pharmacodynamic interaction. Considering that dabrafenib has been proven to be a potent inhibitor of UGT1A1 [17], the combination of dabrafenib and irinotecan may produce DDIs by inhibiting UGT1A1-mediated SN-38 glucuronidation.
In this study, we investigated the effects of dabrafenib on SN-38 glucuronidation using human liver microsomes (HLMs) and recombinant human UGT1A1. The area under the plasma concentration-time curve (AUC) ratio was used to estimate the likely magnitude of drug interactions between dabrafenib and SN-38.

Chemicals
Dabrafenib (purity>99%) was purchased from Selleck Chemicals (Houston, USA). SN-38 and its glucuronide SN-38G, 7-hydroxycoumarin and UDPGA (trisodium salt) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Alamethicin and Tris-HCl were acquired from Aladdin Industrial Corporation (Shanghai, China). All other reagents were of the highest grade and obtained from commercial sources.

SN-38 glucuronidation inhibition assay
SN-38 glucuronidation inhibition assay was conducted by using a previously published method [18], with minor modifications.
Nonspecific binding was accounted for when inhibitor constants (Ki,u) were calculated from in vitro inhibition studies. The fraction unbound of inhibitor (fumic) was calculated at a microsome concentration using Eq. (8) [25]. the presence and absence of dabrafenib. This ratio was calculated using Eq. (9) for drugs with negligible renal clearance [26].
Ki,u is unbound inhibitor constant; [I] is the concentration of dabrafenib at UGT1A1 catalytic site ( [I]max and [I]gut).

Inhibition of SN-38 glucuronidation by dabrafenib in pooled HLMs and recombinant UGT1A1
To

Inhibition kinetic analysis in pooled HLMs and recombinant UGT1A1
Inhibition kinetic analysis was performed to further characterize the inhibition type and the inhibition constant (Ki) of dabrafenib against SN-38 glucuronidation. The representative Lineweaver-Burk plots (Fig. 1B, 2B) and Dixon plots (Fig. 1C, 2C

Potential for dabrafenib induced UGT1A1-mediated DDIs
To determine whether dabrafenib can cause significant DDIs when combined with irinotecan by inhibiting UGT1A1-mediated SN-38 glucuronidation, the inhibition potential in vivo was evaluated by estimating the alteration of AUC of SN-38 using the Ki,u values obtained in the present study, [I]

Discussion
A previous in vivo study has shown synergistic effect of dabrafenib and irinotecan, but the mechanism is unclear [16]. Our results confirm that dabrafenib is a potent inhibitor of UGT1A1 and shows a concentration-dependent inhibition of SN-38 glucuronidation in HLMs and recombinant human UGT1A1, indicating potential dabrafenib-irinotecan interaction.
Fatal adverse events (up to 5.3%) have been reported among people receiving irinotecan monotherapy [29]. Exposure to SN-38, expressed as AUC of SN-38, is considered to be associated with the dose-limiting toxicities of irinotecan. The duration and severity of neutropenia were significantly correlated to prolonged systemic SN-38 exposure [30,31], and a higher incidence of severe diarrhea has been found to be correlated with a higher biliary index (the AUC of irinotecan multiplied by the ratio of SN-38 to SN-38G AUC) [32][33][34]. UGT1A1 is the main enzyme involved in the metabolic clearance of SN-38, and the prevalence of severe toxicity in cancer patients receiving irinotecan monotherapy can be well predicted by UGT1A1 genetic variation [35]. Both UGT1A1*6 and *28 polymorphisms, with approximately 70% reduction of UGT1A1 activity [36,37], have been shown to be associated with the increased systemic exposure to SN-38 in patients homozygous for these variants and increased clinical irinotecan-associated toxicity [38][39][40][41]. Considering that the UGT1A1 activity, the exposure level of SN-38 and irinotecan toxicity are closely related clinically, our results can help to optimize the dosage of the combination of dabrafenib and irinotecan, 11 thereby improving efficiency and reducing toxicity.
In vitro-in vivo extrapolation has become a common method for evaluating potential DDIs in drug development [42]. As reported previously [25], However, many other factors, including uptake and secretory transporters, comorbid conditions and dietary factors, may also influence the pharmacokinetic data [42]. It is necessary to conduct further pharmacokinetic study to acquire more accurate knowledge of potential DDIs associated with the combination of dabrafenib and irinotecan.
In conclusion, the present study reveals that dabrafenib has a significant inhibitory

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of interest
The authors declare that there is no conflict of interests regarding the publication of