CypExpress 2D6 Catalyzed Conversion of Dextromethorphan (DOM) to Dextrorphan (DOH)

Transcription

1 TM CASE STUDY CypExpress 2D6 Catalyzed Conversion of Dextromethorphan (DOM) to Dextrorphan (DOH) Shuvendu Das and Mani Subramanian Center for Biocatalysis and Bioprocessing, University of Iowa 2015 Oxford Biomedical Research, Inc.

2 These studies were performed using the FDA- recommended Introduction: These studies were performed using the FDA- recommended substrate Dextromethorphan to evaluate the utility of CypExpress2D6 for drug substrate Dextromethorphan to evaluate the utility of CypExpress2D6 for drug metabolism metabolism and and disposition studies. Reaction Conditions: Metabolite Identification in 24- well microplates: 500 1,000 μm DOM, mg/ml CypExpress2D6, 50 mm KPi buffer ph 7.5, 30 C, rpm in microplate shaker, Reaction volume 200 μl, Reaction time 3 to 4 h or as specified. Pilot- scale reactions were performed in a total volume of 1.0 ml in a 20x150 mm glass tube using the following final concentrations: Substrate = 500 to 1,000 μm DOM. CypExpress 2D6 = 100 mg/ml Buffer = 50 mm KPi ph 7.5 NADP+, G6P = NONE ADDED for 1 st cycle; see details for additional cycles G6PDH and Mg++ = NONE ADDED. Contained in CypExpress system. The 20 x 150 mm reaction tube was placed in a tilted position on a shaker platform at 30 C, and agitated by rotation at 225 rpm for 3.0 h. At the end of the reaction period, either A. The entire suspension was extracted and subjected to HPLC (figure 1). A. The entire suspension was extracted and subjected to HPLC (figure 1). Or Or B. CypExpress2D6 was pelleted by centrifugation at 14,000 x g. B. CypExpress2D6 a. The supernatant was pelleted from by the centrifugation first cycle was at 14,000 analyzed x g. by HPLC. a. b. The The supernatant pellet was from resuspended the first cycle in fresh was analyzed buffer containing by HPLC. G6P, but no b. The additional pellet was substrate, resuspended and incubated fresh for buffer a second containing reaction G6P, cycle. but no After additional which the substrate, entire suspension and incubated was extracted for a second and reaction subjected cycle. to HPLC. After which the entire suspension was extracted and subjected to HPLC. Page Oxford Biomedical Research, Inc. All Rights Reserved. Page 2

3 Stability of CypExpress2D6 These studies stored were performed at - 80 C: CypExpress using the FDA- recommended is a novel in vitro P450 substrate catalytic Dextromethorphan system with multiple to components evaluate the (recombinant utility of CypExpress2D6 human 2D6, for drug metabolism recombinant and human disposition NADP studies. oxidoreductase, cofactors, G6PDH, and antioxidants in an eukaryotic matrix). Hence, it was important to fully characterize various aspects of the system and its activity. The stability upon storage under various conditions was evaluated. Not unsurprisingly, it proved very stable when stored as a dry powder at - 80 C (figure 1): Although not recommended, we have also observed that dry CypExpress powder can be stored at room temperature for up to 3 weeks, and that CypExpress suspensions in buffer can be frozen and thawed up to four times without demonstrable loss of activity. Dose- dependent conversion of DOM to DOH by CypExpress2D6 in 24 well microplates: Investigations into P450- mediated metabolism of new chemical entities, including determination of the metabolism (if any) catalyzed by specific P450 isoforms and the identification of the metabolite(s) produced is typically performed CypExpress in for microplates such applications followed was by investigated HPLC- MS analysis. using CypExpress2D6 The suitability (100 of CypExpress mg/ml) conversion for such of applications DOM to DOH was in 50 investigated mm phosphate using buffer CypExpress2D6 ph 7.5. The (100 production of DOH from 500 µm DOM by different concentrations of mg/ml) conversion of DOM to DOH in 50 mm phosphate buffer ph 7.5. The CypExpress2D6 at varying incubation times (30 C) was examined (figure 2). production of DOH from 500 µm DOM by different concentrations of Although the reaction rate slows with time, the greatest product yield was obtained CypExpress2D6 using 100 mg/ml at of varying CypExpress2D6 incubation for times 3-4 hrs. (30 C) This is was a convenient examined time (figure for high- 2). Although throughput the microplate reaction rate studies. slows with time, the greatest product yield was obtained using 100 mg/ml of CypExpress2D6 for 3-4 hrs. This is a convenient time for high- throughput microplate studies Oxford Biomedical Research, Inc. All Rights Reserved. Page 3 Page 3

4 CypExpress 2D6 2D6 Catalyzed Conversion of of Dextromethorphan (DOM) to Dextrorphan (DOH) These studies were performed using the FDA- recommended substrate Dextromethorphan to evaluate the utility of CypExpress2D6 for drug Determination of Km and Vmax of CypExpress2D6 for DOM: Km and Vmax values were obtained for CypExpress2D6 (100 mg/ml) incubated for 4 hrs at 30 C with with DOM ranging from 50 1,00 µm. A Lineweaver- Burke plot of the results thus obtained is presented in figure 3, and gave a Km of 304 µm and a Vmax of 714 µm/hr/gram of CypExpress. However, as shown in Figure 2 and Table 1 (below), at 100 mg/ml CypExpress 2D6, the reaction slows significantly after 2 hr Oxford Biomedical Research, Inc. All Rights Reserved. Page 4 Page 4

5 The data presented These in studies Table were 1 also performed demonstrate using the reproducibility the FDA- recommended of these substrate microplate Dextromethorphan studies. to evaluate the utility of CypExpress2D6 for drug CypExpress 2D6 μm of DOM Reaction hrs μm of DOH 100 mg/ml ± mg/ml ± mg/ml ± mg/ml ± 1.0 CONCLUSION: CypExpress is well suited for microplate reactions. NOTE: for CONCLUSION: longer reaction CypExpress times it is important is well suited to restrict for microplate evaporation, reactions. e.g. by NOTE: performing in longer a humidified reaction environment. times it is important to restrict evaporation, e.g. by performing in a humidified environment. The rate of Scale- up conversion conversion 500 μm DOM of to DOM DOHto by DOH CypExpress in a single 2D6(100 reaction mg/ml) cycle: at 30 C The in rate a of of a conversion 200 ml reaction 500 μm volume DOM was to DOH investigated by CypExpress by RP- HPLC 2D6(100 after mg/ml) extraction at 30 C of the in entire a of a 200 reaction ml reaction mixture volume with HPLC was Mobile investigated Phase containing by RP- HPLC 79% after (v/v) extraction methanol, of the 1% entire (v/v) reaction acetic acid, mixture and 20% with (v/v) HPLC water Mobile in Phase 1:1 (v/v) containing ratio. The 79% results (v/v) are methanol, shown 1% in figure (v/v) acetic 4: acid, and 20% (v/v) water in 1:1 (v/v) ratio. The results are shown in figure 4: 140 Dextrorphan (µm) Time (hr) 2015 Oxford Biomedical Research, Inc. All Rights Reserved. Page 5 Page 5

6 The HPLC profiles These of studies aliquots were of the performed reaction using mixture the taken FDA- recommended at these time intervals substrate are presented Dextromethorphan in Figure 5: to evaluate the utility of CypExpress2D6 for drug Figure 6 zooms in on the peak obtained for DOH production 2015 Oxford Biomedical Research, Inc. All Rights Reserved. Page 6 Page 6

7 Figure 7 zooms These in on studies the time- dependent were performed diminution using the of FDA- recommended the peak obtained for the substrate substrate Dextromethorphan DOM to evaluate the utility of CypExpress2D6 for drug Yield of DOH from DOM in a single reaction cycle: Using the conditions stated above (500 µm DOM, 100 mg/ml CypExpress2D6, 30 C, 3 hr., 50 mm Pi ph 7.5), the yield of 5.65 DOM in a single reaction cycle (extracted reaction suspension). If the CypExpress is centrifuged, the quantity of DOH recoverable from the supernatant fraction was 3.0 mg. Increased yield for the production of DOH from DOM by CypExpress2D6 in multiple reaction cycles: Rationale: Studies of the stability and activity of CypExpress systems expressing various recombinant human P450 enzymes have shown that: CypExpress Significant typically CypExpress adsorbs activity large quantities is retained of relatively after a single hydrophobic reaction cycle substrates from the reaction mixture, but releases most of the more CypExpress typically adsorbs large quantities of relatively hydrophobic hydrophobic product(s) into the buffer. substrates from the reaction mixture, but releases most of the more Low speed centrifugation after one reaction cycle pellets CypExpress, which hydrophobic product(s) into the buffer. contains significant quantities of unreacted substrate and some product. Low speed centrifugation after one reaction cycle pellets CypExpress, which Typically, the total product yield is greater for multi- cycle reactions vs. longer contains incubations significant for a single quantities cycle. of unreacted substrate and some product. Additional Typically, reaction the total cycles product convert yield substantial is greater amounts for multi- cycle of the retained reactions vs. substrate longer to incubations product thereby for a single increasing cycle. overall metabolite yield. Additional reaction cycles convert substantial amounts of the retained substrate to product thereby increasing overall metabolite yield Oxford Biomedical Research, Inc. All Rights Reserved. Page 7 Page 7

8 Scale- up production These studies of DOH were in a second performed catalytic using cycle: the A FDA- recommended 200 ml reaction was substrate performed Dextromethorphan using a DOM concentration to evaluate of the 500 utility µm. After of CypExpress2D6 a 4 hr. first reaction for drug metabolism cycle, the and CypExpress disposition was pelleted, studies. and the supernatant harvested. The pelleted CypExpress was stored overnight at 4 C, then resuspended in fresh buffer containing G6P but no additional NADP+ or DOM substrate. This suspension was incubated at 30 C and aliquots withdrawn at different time intervals to follow the time course of the reaction by HPLC. The results obtained (figure 8) supports: a) The retention by CypExpress of significant quantities of unreacted DOM substrate b) Retention of significant amounts of the DOH product c) Retained catalytic activity of the resuspended CypExpress (see insert), d) However, unlike some other CypExpress systems (see 3A4/Testosterone case study) production of some additional DOH from the retained DOM was relatively low Dextrorphan (µm) Dextrorphan (µm) Time (hr) Time (hr) 2015 Oxford Biomedical Research, Inc. All Rights Reserved. Page 8 Page 8

9 Figure 9: HPLC These analysis studies of the were samples performed withdrawn using at the specified FDA- recommended times from the substrate second Dextromethorphan CypExpress2D6 reaction to evaluate cycle. the utility of CypExpress2D6 for drug Discussion: CypExpress2D6 retains activity after several hours. However, relatively little DOH was synthesized from the DOM retained by the CypExpress2D6 after an initial reaction cycle. Simple extraction of the pellet yields about the same amount of DOH as was released into the supernatant during the initial reaction cycle. The total yield obtained from 27 mg of DOM substrate in two reaction cycles was 13.2 mg, or ~ 50% conversion of the substrate in this 200 ml reaction mixture Sigma-Aldrich Co. LLC. All rights reserved. SIGMA-ALDRICH is a trademark of Sigma-Aldrich Co. LLC, registered in the US and other countries. Page 9