CypExpress 3A4 Catalyzed Conversion of Testosterone (TE) to 6β- Hydroxytestosterone (HT)

Transcription

1 TM CASE STUDY CypExpress 3A4 Catalyzed Conversion of to Shuvendu Das, 1 Enrique Martez, 2 and Mani Subramanian 1 1 Center for Biocatalysis and Bioprocessg, University of Iowa 2 Oxford Biomedical Research, Inc. 215 Oxford Biomedical Research, Inc.

2 Introduction: These studies were performed usg the FDA- recommended substrate Testosterone to evaluate the utility of CypExpress3A4 for drug metabolism and disposition studies. Introduction: These studies were performed usg the FDA- recommended substrate Introduction: Testosterone These studies to evaluate were performed the utility usg of the CypExpress3A4 FDA- recommended for drug metabolism substrate Dextromethorphan and disposition to studies. evaluate the utility of CypExpress2D6 for drug metabolism and disposition studies. Metabolite Identification 24- well microplates: 25-5 μm, 2-1 mg/ml CypExpress3A4, 5 mm KPi buffer ph 7.5, 3 C, 6 rpm microplate shaker, Reaction volume 2 μl, Reaction time 2 to 4 h Metabolite Identification 24- well microplates: 25-5 μm, 2-1 mg/ml CypExpress3A4, Metabolite Identification 5 mm KPi 24- well buffer microplates: ph 7.5, 3 C, 5 6 1, rpm μm DOM, microplate 2-1 shaker, Reaction mg/ml CypExpress2D6, volume 2 μl, 5 Reaction mm KPi buffer time ph 2 7.5, to 4 3 C, h 2-6 rpm microplate shaker, Reaction volume 2 μl, Reaction time 3 to 4 h or as specified. Pilot- scale reactions were performed a total volume of 1. ml a 2x15 mm Pilot- scale reactions were performed a total volume of 1. ml a 2x15 mm glass glass tube tube usg the the followg followg fal fal concentrations: concentrations: Substrate = 5 2 to 1, 5 μm TE DOM. (5 µm for scale- up). Unless otherwise CypExpress 2D6 specified, = 1 mg/ml TE was added to the reaction mixture as a concentrated solution DMSO). Buffer = 5 mm KPi ph 7.5 CypExpress NADP+, G6P = 3A4 NONE = 1 ADDED mg/ml for 1 st cycle; see details for additional cycles Buffer G6PDH = and 5 Mg++ 1 = mm NONE KPi, ADDED. or Tris- HCl Contaed (as specified CypExpress tables), system. ph 7.5 The 2 x 15 mm reaction tube was placed a tilted position on a shaker platform at 3 C, G6PDH and agitated and Mg++ by rotation = NONE at 225 ADDED. rpm for Contaed 3. h. CypExpress system. Pilot- scale reactions were performed a total volume of 1. ml a 2x15 mm glass tube usg the followg fal concentrations: Substrate = 2 to 5 μm TE (5 µm for scale- up). Unless otherwise specified, TE was added to the reaction mixture as a concentrated solution DMSO). CypExpress 3A4 = 1 mg/ml Buffer = 5 1 mm KPi, or Tris- HCl (as specified tables), ph 7.5 G6PDH and Mg++ = NONE ADDED. Contaed CypExpress system. The 2 x 15 mm reaction tube was placed a tilted position on a shaker platform at 3 C, and agitated by rotation at 225 rpm for 3. h. The 2 x 15 mm reaction tube was placed a tilted position on a shaker platform at 3 C, A. The and entire agitated suspension by rotation was extracted at 225 rpm and subjected for 3. h. to HPLC (figure 1). Or At the end of the reaction period, either B. CypExpress2D6 was pelleted by centrifugation at 14, x g. A. The a. entire The supernatant suspension from was the extracted first cycle and was subjected analyzed by to HPLC. HPLC (figure 1). Or which the entire suspension was extracted and subjected to HPLC. B. CypExpress3A4 was pelleted by centrifugation at 14, x g. 215 Oxford which Biomedical the Research, entire suspension Inc. All Rights was Reserved. extracted and subjected Page to 2 HPLC. A. The entire suspension was extracted and subjected to HPLC (figure 1). Or B. CypExpress3A4 was pelleted by centrifugation at 14, x g. which the entire suspension was extracted and subjected to HPLC. 215 Oxford Biomedical Research, Inc. All Rights Reserved. Page 2

3 CypExpress 3A4 Conversion of Testosterone to 6β- Hydroxytestosterone Conversion of TE to HT a sgle reaction cycle: CypExpress 3A4 Introduction: (1 mg/ml) These catalyzed studies conversion were performed of 2 usg μm the FDA- recommended to substrate 6β- Hydroxytestosterone Stability Testosterone of CypExpress2D6 to (HT) evaluate stored a sgle at the - 8 C: utility 3 hr. CypExpress reaction of CypExpress3A4 cycle is a novel 5 vitro for μm drug TE, P45 5 mm metabolism KPi catalytic of ph 7.5 and system at disposition 3 C with was multiple vestigated studies. components by (recombant RP- HPLC after human extraction 2D6, of the entire reaction recombant mixture human with HPLC NADP Mobile oxidoreductase, Phase contag cofactors, 79% G6PDH, (v/v) and methanol, antioxidants 1% (v/v) acetic an acid, eukaryotic and 2% matrix). (v/v) Hence, water it was 1:1 important (v/v) ratio. to The fully results characterize are shown various figure 1 which aspects the of the black system le and represents its activity. the The HT stability standard, upon the storage light under blue various le represents results conditions obtaed was usg evaluated. a vector Not unsurprisgly, control (no 3A4), it proved and the very red stable le when represents stored results obtaed as a dry usg powder CypExpress3A4. at - 8 C (figure 1): 1 SPD-M1Avp-24 nm 1uM 6-betaOHTest stand 1uLd Isolation RM 1uL GO Cont 1uL 1 6β- Hydroxytestosterone 8 (HT) 7 7 Metabolite Identification 24- well microplates: 25-5 μm, 2-1 mg/ml 6 6 CypExpress3A4, 5 mm KPi buffer ph 7.5, 3 C, 6 rpm microplate shaker, 5 5 Reaction volume 2 μl, Reaction time 2 to 4 h TE mau mau Pilot- scale reactions were performed a total volume of 1. ml a 2x mm HT glass tube usg the followg fal concentrations: 2 Flow- Thru Substrate = 2 to 5 μm TE (5 µm for scale- up). Unless otherwise 1 1 Although not recommended, specified, we TE have was also added observed to the that reaction dry CypExpress mixture powder as a can be stored at room concentrated temperature solution for up to 3 weeks, DMSO). and that CypExpress suspensions buffer can be frozen and thawed Mutes up to four times without demonstrable CypExpress loss 3A4 of activity. = 1 mg/ml Mutes Buffer = 5 1 mm KPi, or Tris- HCl (as specified tables), ph 7.5 Dose- dependent conversion of DOM to DOH by CypExpress2D6 24 well Yield of HT from TE a sgle reaction NADP+, G6P = NONE ADDED for 1 st cycle: Usg the conditions stated microplates: Investigations to P45- mediated cycle; metabolism see details of new chemical for additional cycles entities, above cludg (5 µm determation TE, 1 mg/ml of the CypExpress3A4, metabolism (if any) 3 C, catalyzed 3 hr., 5 by mm specific Pi ph 7.5), P45 the isoforms G6PDH yield of and HT the Mg++ a identification sgle = NONE reaction ADDED. of the cycle metabolite(s) Contaed was: produced CypExpress is typically system. performed microplates followed by HPLC- MS analysis. The suitability of 87.9 µm = 17.6% conversion The CypExpress 2 x 15 for mm such reaction applications tube was vestigated placed usg a tilted CypExpress2D6 position on (1 a shaker platform at mg/ml) 3 C, and conversion agitated of by DOM rotation to DOH at mm rpm phosphate for 3. buffer h. ph 7.5. The production Comparison of DOH of from the 5 catalytic µm DOM efficiency by different and concentrations product purify of for pre- washed At CypExpress2D6 the vs. end unwashed of the at reaction varyg CypExpress3A4: cubation period, either times All conditions (3 C) was were examed identical (figure to those 2). the first Although the reaction rate slows with time, the greatest product yield was obtaed set of experiments, except that a parallel experiment the CypExpress3A4 was usg A. 1 The mg/ml entire of suspension CypExpress2D6 was for extracted 3-4 hrs. This and is subjected a convenient to HPLC time for (figure high- 1). pre- washed by (a) suspendg reaction buffer and allowed to stand for throughput microplate studies. various Or periods of time (see graph and chart) followed by (b) centrifugation at 14, x g, (c) removg the supernatant, and (d) resuspendg the B. CypExpress3A4 fresh was reaction pelleted buffer(g6p)? by centrifugation. Results at 14, comparg x g. the HPLC profiles a. and The the supernatant catalytic efficiency from the are first presented cycle was Figure analyzed 2 and by HPLC. Table Oxford which Biomedical the Research, entire suspension Inc. All Rights was Reserved. extracted and subjected Page to 3 HPLC. 215 Oxford Biomedical Research, Inc. All Rights Reserved. Page mau

4 CypExpress 3A4 Conversion of Testosterone to 6β- Hydroxytestosterone Figure 2. HPLC analysis of TE - > HT catalyzed by unwashed CypExpress3A4 (light Introduction: blue) and CypExpress3A4 These studies followg were performed washg usg 5 (dark the red), FDA- recommended 1 (dark blue), 15 substrate (dark green) Testosterone or 2 m (light to evaluate green). the utility of CypExpress3A4 for drug SPD-M1Avp-24 nm metabolism and disposition studies. h wash 1uL New 1 MP 1uL 5m wash 1uL 1m wash 1uL 15m wash 1uL 2m wash 1uL 6 h wash 1uL PM.dat new 1 mp 1ul pm.dat 5m wash 1ul pm.dat 1m wash 1ul pm.dat 15m wash 1ul pm.dat 2m wash 1ul pm.dat mau Metabolite Identification 24- well microplates: 25-5 μm, 2-1 mg/ml 15 Determation 15 of Km and Vmax of CypExpress2D6 for DOM: Km and Vmax values CypExpress3A4, 5 mm KPi buffer ph 7.5, 3 C, 6 rpm microplate shaker, were obtaed 1 for CypExpress2D6 (1 mg/ml) cubated for 4 hrs at 3 C with 1 Reaction with DOM volume rangg 2 from μl, 5 Reaction 1, µm. A time Leweaver- Burke 2 to 4 h plot of the results thus 5 5 obtaed is presented figure 3, and gave a Km of 34 µm and a Vmax of 714 Pilot- scale reactions were performed a total volume of 1. ml a 2x15 mm µm/hr/gram of CypExpress. However, as shown Figure 2 and Table 1 (below), at glass 1 tube mg/ml 1.75 usg CypExpress 2. the 2.25 followg 2.5 2D6, the fal 3.25 reaction 3.5 concentrations: 3.75 slows 4. significantly after hr Mutes Substrate = 2 to 5 μm TE (5 µm for scale- up). Unless otherwise specified, TE was added to the reaction mixture as a concentrated solution DMSO). CypExpress 3A4 wash μm of TE Reaction hrs μm of HT* CypExpress 3A4 = 1 mg/ml Buffer = 5 None 1 mm KPi, or 5 Tris- HCl (as specified 3 tables), ph m G6PDH and Mg++ = NONE ADDED. Contaed CypExpress system. 1 m The 2 x 15 mm reaction tube was placed a tilted position on a shaker platform at 3 C, and agitated by rotation at 225 rpm for 3. h. 15 m m A. The entire suspension was extracted and subjected to HPLC (figure 1). CONCLUSION: Or Pre- washg CypExpress reduces the size of the flow- thru peak comprised of very hydrophilic components, but does not significantly alter the B. catalytic CypExpress3A4 efficiency of was CypExpress. pelleted by centrifugation at 14, x g. 215 Oxford which Biomedical the Research, entire suspension Inc. All Rights was Reserved. extracted and subjected Page to 4 HPLC. 215 Oxford Biomedical Research, Inc. All Rights Reserved. Page mau

5 Applications of CypExpress for P45 profilg & metabolite identification: Introduction: Investigations These to P45- mediated studies were metabolism performed of usg new the chemical FDA- recommended entities, substrate cludg The data Testosterone presented determation Table to of evaluate 1 the also metabolism demonstrate the utility (if the any) of reproducibility CypExpress3A4 catalyzed by of specific these for drug P45 metabolism isoforms microplate and studies. and the disposition identification studies. of the metabolite(s) produced is typically performed microplates followed by HPLC- MS analysis. The suitability of CypExpress for such applications was vestigated usg CypExpress3A4 (1 mg/ml) conversion CypExpress of 2D6 TE to HT μm of 5 DOM mm phosphate Reaction hrs buffer ph μm 7.5. of DOH The conversion of various concentrations of TE to HT a 2 µl reaction volume 24 well microplates 1 mg/ml cubated with 25 shakg for 3 2 hr at 3 C is 54.6 shown ± 1.7 Table 2: Sample 1 mg/ml TE (μm) 5 Reaction 2 Time (h) 67.1 ± 2.2 HT (μm) Testosterone 1 (TE) mg/ml ± mg/ml ± 1. Metabolite 3 Identification well microplates: μm, mg/ml CypExpress3A4, 4 5 mm 25 KPi buffer ph 7.5, 3 C, 3. 6 rpm microplate 83.3 shaker, CONCLUSION: CypExpress is well suited for microplate reactions. NOTE: for Reaction longer volume 2 μl, Reaction time 2 to 4 h 5 reaction times 5 it is important to restrict evaporation, 3. e.g. by performg a humidified environment. Pilot- scale 6 reactions were 1 performed a total 3. volume of 1. ml a 2x15 mm glass Scale- up tube usg conversion the followg of DOM fal to DOH concentrations: a sgle reaction cycle: The rate of CONCLUSION: conversion 5 Substrate CypExpress μm DOM to DOH = 2 to 5 is well by μm TE suited CypExpress (5 for µm microplate 2D6(1 for scale- up). reactions. mg/ml) at Unless NOTE: 3 C otherwise for a of a longer 2 ml reaction reaction times volume specified, it was is important vestigated TE was to restrict by RP- HPLC added to evaporation, after extraction the reaction e.g. mixture by of performg entire as a reaction mixture with HPLC Mobile Phase contag 79% (v/v) methanol, 1% (v/v) a humidified environment. acetic acid, and 2% (v/v) concentrated water 1:1 solution (v/v) ratio. DMSO). The results are shown figure Stability 4: CypExpress of CypExpress3A4 3A4 = 1 stored mg/ml at - 8 C: CypExpress is a novel vitro P45 catalytic system with multiple components (recombant human 3A4, recombant Buffer = human 5 1 NADP mm oxidoreductase, KPi, or Tris- HCl cofactors, (as specified G6PDH, and tables), antioxidants ph an eukaryotic matrix). Hence, it was important NADP+, G6P = NONE ADDED for 1 st to fully characterize various cycle; see details for additional cycles aspects of the system 12 and its activity. The stability upon storage under various conditions G6PDH was and evaluated. Mg++ = Not NONE unsurprisgly, ADDED. Contaed it proved very CypExpress stable when system. stored 1 as a dry powder at - 8 C (figure 3): The 2 x 15 mm 8 reaction tube was placed a tilted position on a shaker platform at 3 C, and agitated by rotation at 225 rpm for 3. h. 6 At the end of the reaction 4 period, either Dextrorphan (µm) A. The entire 2 suspension was extracted and subjected to HPLC (figure 1). Or B. CypExpress3A4 was pelleted Time by centrifugation (hr) at 14, x g. 215 Oxford which Biomedical the Research, entire suspension Inc. All Rights was Reserved. extracted and subjected Page to 5 HPLC. 215 Oxford Biomedical Research, Inc. All Rights Reserved. Page 5

6 Although not recommended, we have also observed that dry CypExpress powder Introduction: can be stored at These room studies temperature were performed up to 3 weeks, usg and the that FDA- recommended CypExpress substrate The suspensions HPLC Testosterone profiles buffer of aliquots can to be evaluate of frozen the reaction the and utility thawed mixture of up CypExpress3A4 taken to four at times these without time for tervals drug metabolism are demonstrable presented and loss Figure disposition of activity. 5: studies. Effects of solvents: Testosterone and many pharmaceutically active substances are poorly soluble aqueous solutions, so they are typically added to vitro P45 reaction systems as a concentrated solution a water- miscible solvent. The general guidance has been that the fal concentration of that solvent the to vitro P45 reaction system should be kept below 1%. For CypExpress, we vestigated the effect of the solvent used to dissolve TE on the conversion of TE to HT. The results obtaed are shown figure 4: 14 Metabolite 12 Identification 24- well microplates: 25-5 μm, 2-1 mg/ml CypExpress3A4, 5 mm KPi buffer ph 7.5, 3 C, 6 rpm microplate shaker, Reaction 1.4% volume 2 μl, Reaction time 2 to 4 h.8% Pilot- scale 8 reactions were 1.7% performed a total volume of 1. ml a 2x15 mm glass tube usg the followg 2.5% fal concentrations: Figure Substrate 6 zooms 6 = on 2 the to peak 5 obtaed μm TE (5 for DOH µm production for scale- up). Unless otherwise specified, TE was added to the reaction mixture as a 4 concentrated solution DMSO). 6β-Hydroxytestosterone (μm) CypExpress 2 3A4 = 1 mg/ml Buffer = 5 1 mm KPi, or Tris- HCl (as specified tables), ph 7.5 NADP+, G6P = Methanol NONE ADDED for 1 st DMF cycle; see details for DMSO additional cycles CONCLUSIONS: G6PDH and As Mg++ has been = NONE reported ADDED. for other Contaed vitro P45 CypExpress catalytic systems, system. the use of methanol as a solvent should be avoided. However, at least for this The 3A4 2 system x 15 with mm reaction TE as substrate, tube was CypExpress placed is a activity tilted position is mimally on a hibited shaker platform by at concentrations 3 C, and agitated of DMSO by rotation and DMF at up 225 to at rpm least for 2.5%. 3. h. At Effect the end of buffer of the reaction the production period, either of HT from TE: The production of HT Tris and Phosphate buffers was compared (Table 3): A. The entire suspension was extracted and subjected to HPLC (figure 1). HT Buffer Or TE (μm) Reaction Time (h) (μm) KPi, B. 1 CypExpress3A4 mm was pelleted 5 by centrifugation 3. at 14, x g Tris- HCl, b. 1 The mm pellet was resuspended 5 fresh 3. buffer contag G6P, but no 215 Oxford which Biomedical the Research, entire suspension Inc. All Rights was Reserved. extracted and subjected Page to 6 HPLC. 215 Oxford Biomedical Research, Inc. All Rights Reserved. Page 6

7 CypExpress CypExpress 3A4 3A4 Conversion Conversion of Testosterone of Testosterone to 6β- Hydroxytestosterone to 6β- Hydroxytestosterone CONCLUSION: Although high levels of conversion of TE to HT were obtaed Introduction: reactions catalyzed These by studies CypExpress3A4 were performed both buffers, usg the for this FDA- recommended isoform and this Figure substrate 7 zooms Testosterone the conversion the time- dependent to Tris- HCl evaluate buffer the dimution utility is more of efficient of CypExpress3A4 the peak than obtaed the same for for drug reaction the metabolism substrate performed DOM and KPi disposition studies. Increased yields for the production of HT from TE by CypExpress3A4 multiple reaction cycles: Rationale: Studies of the stability and activity of CypExpress systems expressg various recombant human P45 enzymes have shown that: Significant CypExpress activity is retaed after a sgle reaction cycle CypExpress typically adsorbs large quantities of relatively hydrophobic Testosterone substrates from (TE) the reaction mixture, but releases most of the more hydrophobic product(s) to the buffer. Reaction Low speed Conditions: centrifugation after one reaction cycle pellets CypExpress, which Metabolite contas Identification significant quantities 24- well of unreacted microplates: substrate 25-5 and μm, some 2-1 product. mg/ml CypExpress3A4, Typically, the 5 total mm product KPi buffer yield ph is greater 7.5, 3 C, for 6 multi- cycle rpm reactions microplate vs. shaker, Reaction longer volume cubations 2 μl, for Reaction a sgle cycle. time 2 to 4 h Additional reaction cycles convert substantial amounts of the retaed Pilot- scale reactions were performed a total volume of 1. ml a 2x15 mm substrate to product thereby creasg overall metabolite yield. glass tube usg the followg fal concentrations: Sgle cycle scale- up synthesis of TE: A 2 ml reaction was performed usg a Yield TE concentration of Substrate DOH from = DOM of 2 25 to 5 µm a sgle and μm reaction withdrawg TE (5 cycle: µm aliquots Usg for scale- up). the at conditions different Unless stated time otherwise tervals above (5 to follow µm DOM, the time 1 mg/ml course specified, of CypExpress2D6, the TE reaction. was added HPLC 3 C, 3 analysis to hr., the 5 reaction mm of the Pi ph production mixture 7.5), the yield as of a HT of from 5.65 TE DOM (figure a sgle 5) dicates concentrated reaction that cycle the (extracted solution reaction reaction rate DMSO). slows suspension). appreciably If the after ~ 2hr. CypExpress is centrifuged, the quantity of DOH recoverable from the supernatant fraction CypExpress was 3. mg. The 3A4 = 1 mg/ml Increased Buffer yield = 5 for the 1 production mm KPi, or of Tris- HCl DOH from (as DOM specified by CypExpress2D6 tables), ph 7.5 multiple reaction cycles: Rationale: Studies of the stability and activity of CypExpress systems expressg various G6PDH recombant and Mg++ human = NONE P45 ADDED. enzymes Contaed have shown that: CypExpress system. Significant CypExpress activity is retaed after a sgle reaction cycle The 2 x 15 mm reaction tube was placed a tilted position on a shaker platform CypExpress typically adsorbs large quantities of relatively hydrophobic at 3 C, substrates and agitated from by the rotation reaction at mixture, 225 rpm but releases for 3. most h. of the more hydrophobic product(s) to the buffer. yield Low speed centrifugation after one reaction cycle pellets CypExpress, which of A. HT The contas entire significant suspension quantities was extracted of unreacted and substrate subjected and some to HPLC product. (figure 1). Typically, the total product yield is greater for multi- cycle reactions vs. Or longer cubations for a sgle cycle. Additional reaction cycles convert substantial amounts of the retaed B. CypExpress3A4 substrate to product was thereby pelleted creasg by centrifugation overall metabolite at 14, yield. x g. obtaed a. at The supernatant from the first cycle was analyzed by HPLC. different b. time The tervals pellet this was experiment resuspended is shown fresh Figure buffer 6 contag below. G6P, but no 215 Oxford which Biomedical the Research, entire suspension Inc. All Rights was Reserved. extracted and subjected Page to 7 HPLC. 215 Oxford Biomedical Research, Inc. All Rights Reserved. Page 7

8 CypExpress CypExpress 3A4 3A4 Conversion Conversion of Testosterone of Testosterone to 6β- Hydroxytestosterone to 6β- Hydroxytestosterone Introduction: These 8 studies were performed usg the FDA- recommended substrate Testosterone 7 to evaluate the utility of CypExpress3A4 for drug metabolism and disposition 6 studies Time (hr) Metabolite Scale- up production Identification of TE 24- well a second microplates: catalytic cycle: 25-5 A 2 ml μm, reaction 2-1 was mg/ml performed usg a TE concentration of 25 µm. After a 3 hr. first reaction cycle, CypExpress3A4, 5 mm KPi buffer ph 7.5, 3 C, 6 rpm microplate shaker, the CypExpress was pelleted, and the supernatant harvested. The pelleted Reaction volume 2 μl, Reaction time 2 to 4 h CypExpress was stored overnight at 4C, then resuspended fresh buffer Pilot- scale contag reactions G6P but no were additional performed NADP+ a or total TE substrate. volume of This 1. suspension ml a 2x15 was mm cubated at 3 C and aliquots withdrawn at different time tervals to follow glass tube usg the followg fal concentrations: the time course of the reaction by HPLC. The results obtaed (figure 6) support the production Substrate of = substantial 2 to 5 μm additional TE (5 TE µm for for at least scale- up). an additional Unless 2.5 otherwise hr. specified, TE was added to the reaction mixture as a concentrated solution DMSO). 6β-Hydroxytestosterone (µm) CypExpress 3A4 = 1 mg/ml Buffer = 5 1 mm KPi, or Tris- HCl (as specified tables), ph 7.5 G6PDH and Mg++ = NONE ADDED. Contaed CypExpress system. The 2 x 15 mm reaction tube was placed a tilted position on a shaker platform at 3 C, and agitated by rotation at 225 rpm for 3. h. A. The entire suspension was extracted and subjected to HPLC (figure 1). Or Discussion: CypExpress3A4 retas activity after several hours and the yields of metabolite B. CypExpress3A4 scale- up can was be pelleted significantly by centrifugation creased by multiple at 14, reaction x g. cycles. which the entire suspension was extracted and subjected to HPLC. 215 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA-ALDRICH is a trademark of Sigma-Aldrich Co. LLC, registered the US and other countries. 215 Oxford Biomedical Research, Inc. All Rights Reserved. Page 8