TM
CASE STUDY CypExpress™ 2C19 Catalyzed Conversion of Mephenytoin (MP) to 4-­‐Hydroxymephenytoin (HMP) Shuvendu Das1 and Mani Subramanian1 1Center for Biocatalysis and Bioprocessing, University of Iowa ©2014 Oxford Biomedical Research, Inc. 10#PYt0YGPSE.*tUFMtGBYtIQQUXXXPYGPSECJPNFEDPN
CypExpress™ 2C19 Mephenytoin ephenytoin S-­‐4-­‐Hydroxymepheny
CypExpress™ 2C19 CConversion onversion oof f M
to tSo -­‐4-­‐Hydroxymephenyto
in toin CypExpress™ 2D6 C atalyzed Conversion of Dextromethorphan (DOM) to Dextrorphan (DOH) Introduction: These studies w
were ere p
sing the FDA-­‐recommended Introduction: T hese studies performed erformed uu
sing the FDA-­‐recommended substrate M
ephenytoin t
o e
valuate t
he u
tility o
f C
ypExpress2C19 for fdor rug Introduction: These studies w
ere p
erformed u
sing t
he F
DA-­‐recommended substrate Mephenytoin to evaluate the utility of CypExpress2C19 drug metabolism a
nd d
isposition s
tudies. substrate D
extromethorphan t
o e
valuate t
he u
tility o
f C
ypExpress2D6 f
or d
rug metabolism and disposition studies. metabolism and disposition studies. O
N
O
O
N
O
CYP2C19
CYP2C19
NH
NH
S-­‐Mephenytoin (MP) O
S-­‐Mephenytoin (MP) Reaction Conditions: N
O
O
O
N NH
NH
OH OH S-­‐4-­‐Hydroxymephenytoin (HMP)
S-­‐4-­‐Hydroxymep
henytoin (HMP)
Reaction Conditions: Pilot-­‐scale reactions were performed in a total volume of 1.0 Identification in 24-­‐well microplates: 500 – 1,000 μM DOM, 20-­‐100 Metabolite mL i
n a
2
0x150 m
m g
lass t
ube u
sing t
he f
ollowing f
inal c
oncentrations: mg/mL CC
ypExpress2D6, 50 mM KPi buffer pH 7.5, 30°C, 200-­‐600 rpm in microplate Reaction onditions: P
ilot-­‐scale reactions were performed in a total volume of 1.0 shaker, RSubstrate eaction volume 2o 00 μL, μ M Reaction time 3 tfo or as specified. =
2
0 t
500 MP (500 µM or 4s hcale-­‐up). Unless otherwise mL in a 20x150 mm glass tube using the following final concentrations: reactions specified, MP in waas added to the as Pilot-­‐scale were performed total volume of r1eaction .0 mL in m
a ixture 20x150 mam Substrate =
2
0 t
o 5
00 μ
M M
P (
500 µ
M f
or concentrated s
cale-­‐up). U
nless s
olution o
therwise i
n D
MSO glass tube using the following final concentrations: CypExpress M
P was added to the reaction mixture as a 2specified, C19 = 100 mD
g/mL Substrate = 500 to 1,000 μM OM. concentrated solution in DMSO Buffer = 50 – 1=00 mM KPi, or Tris-­‐HCl (as specified in tables), pH 7.5 CypExpress 2D6 100 mg/mL CypExpress 2C19 = 100 mg/mL Buffer = 50 mM =K P
pH 7.5 NADP+, G6P NiONE ADDED for 1st cycle; see details for additional cycles Buffer = G5a6P 0 nd –= 1
m=M KPi, ofAor r DDED. T1stris-­‐HCl as details specified in tables),
pH 7.5 NADP+, N00 ONE A N
DDED cycle; s(ee additional csycles G6PDH M
g++ ONE Contained in Cfor ypExpress™ ystem. NADP+, 6P NONE ADDED for 1st cycle; see details fsor G6PDH aGnd M=g++ = NONE ADDED. Contained in C
ypExpress™ ystem. additional cycles The 20 x 150 mm reaction tube was placed in a tilted position on a shaker platform G6PDH nd Mg++ NONE DDED. ontained system. at 320°C, nd m
aagitated by rt=ube otation t laced 225 irn pm for 3p.0 h. in oCn ypExpress™ The 0 x 1a50 m reaction was apA
aC
tilted osition a shaker platform 30°C, and agitated by rotation at 225 rpm for 3.0 h. at 20 x 150 mm reaction tube was p
The laced in a tilted position on a shaker platform At the end of the reaction period, either At the end of the reaction period, either at 3
0°C, and agitated by rotation at 225 rpm for 3.0 h. A. The entire suspension was extracted subjected to H(figure PLC (figure A. The entire suspension was extracted and asnd ubjected to HPLC 1). 1). At the end o f the reaction period, either Or Or A. The entire suspension was bey xtracted and ast ubjected B. CypExpress2D6 was pelleted centrifugation 14,000 x gt. o HPLC (figure 1). B. CypExpress2C19 was pelleted by centrifugation at 6,000 x g. a. The supernatant from the first cycle was analyzed by HPLC. supernatant from the first cycle was caontaining nalyzed bGy 6P, HPLC. he pellet – was resuspended in fresh buffer but no Or b.a. TThe substrate, nd incubated for a second reaction cycle. GA6P, fter but no b. additional The pellet – was raesuspended in fresh buffer containing which t
he e
ntire s
uspension w
as e
xtracted a
nd s
ubjected t
o H
PLC. additional ubstrate, and incubated for a second reaction B. CypExpress2C19 wsas pelleted by centrifugation at 6,000 x g. cycle. After which the entire suspension was extracted and subjected to HPLC. a. The supernatant from the first cycle was analyzed by HPLC. b. T he pellet – was resuspended in fresh buffer containing G6P, but no additional substrate, and incubated for a second reaction cycle. After © 2015 Oxford B iomedical Research, Inc. All Rights Reserved. Page 2 which the entire suspension as eRxtracted to Page HPLC. Biomedical Research, Inc. All Rw
ights eserved. and subjected 2 © 2015 Oxford CypExpress™ 2C19 Conversion of Mephenytoin to S-­‐4-­‐Hydroxymephenytoin CypExpress™ 2C19 Conversion of Mephenytoin to S-­‐4-­‐Hydroxymephenytoin CypExpress™ 2D6 Catalyzed Conversion of Dextromethorphan (DOM) to Dextrorphan (DOH) Conversion of MP to HMP in a single reaction cycle: Introduction: These studies were performed using the FCypExpress™ 2C19 DA-­‐recommended (100 mg/mL) catalyzed conversion of 200 μM Mephenytoin Stability of CypExpress2D6 stored att he -­‐80°C: CypExpress is a novel in vitro P450 substrate Mephenytoin to evaluate utility of CypExpress2C19 for d(MP) rug to S-­‐4-­‐Hydroxymephenytoin (HMP) in a single 3 hr. reaction cycle 2iD6, n 500 μM MP, in 50 catalytic system with multiple components (recombinant human metabolism and disposition studies. recombinant human NADP oxidoreductase, ofactors, G6PDH, antioxidants mM KPi of pH 7 .5 at 30°C was investigated bcy RP-­‐HPLC after aend xtraction of the entire in an eukaryotic matrix). Hence, it was important to fully characterize various reaction mixture with HPLC Mobile Phase containing 15% (v/v) Methanol, 84.5% Oof the aspects system its Aacetic ctivity. The Tshe tability upon storage various (v/v) Water, and 0.5% a(nd v/v) acid. results are shown uinder n figure 1O
. O
conditions was evaluated. Not unsurprisingly, it proved very stable when stored N a dry powder at -­‐80°C (figure 1): as N
CYP2C19
NH
O
S-­‐Mephenytoin (MP) NH
OH S-­‐4-­‐Hydroxymephenytoin (HMP)
Reaction Conditions: 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 = 20 to 500 μM MP (500 µM for scale-­‐up). Unless otherwise specified, MP was added to the reaction mixture as a concentrated solution in DMSO Although not recommended, we have also observed that dry CypExpress powder can be stored at r2oom for up to 3 weeks, and that CypExpress CypExpress C19 temperature = 100 mg/mL suspensions in buffer can be frozen and thawed up to four times without Buffer = 50 – 1o00 mM KPi, or Tris-­‐HCl (as specified in tables), pH 7.5 demonstrable loss f activity. NADP+, G6P = NONE ADDED for 1st cycle; see details for additional cycles Dose-­‐dependent conversion of iD
to DOH by CypExpress2D6 in 2the 4 wcell Yield of MPH from MP n OM a single reaction cycle: Using onditions stated microplates: I
nvestigations i
nto P
450-­‐mediated m
etabolism o
f n
ew chemical G6PDH and MPg++ = NmONE ADDED. Contained in CypExpress™ above (200 µM H, 100 g/mL CypExpress2C19, 30°C, 4 hr., 50 mM Psi ystem. pH 7.5), entities, including determination of the metabolism (if any) catalyzed by specific the average yield of MPH in a single reaction cycle were: P450 isoforms and the identification of the metabolite(s) produced is typically The 2
0 x 150 mm reaction tube w
as placed ain a tilted psosition on performed in microplates followed HPLC-­‐MS nalysis. The uitability of a shaker platform 42.76 bµy M = 8.6% conversion at CypExpress 30°C, a
nd a
gitated b
y r
otation a
t 2
25 r
pm f
or 3
.0 h
. for such applications was investigated using CypExpress2D6 (100 mg/mL) conversion of DOM to DOH in 50 mM phosphate buffer pH 7.5. The DOH from 500 M DOM by different concentrations of for pre-­‐washed At production the Comparison o
f the catalytic fficiency and product purify end of otf he reaction pµeriod, eeither CypExpress2D6 at varying incubation times was ew
xamined (figure vs. unwashed CypExpress2C19: All (c30°C) onditions ere identical to 2t). hose in the Although roeaction r
ate s
lows w
ith t
ime, t
he g
reatest p
roduct y
ield w
as btained A. first The sthe eet ntire s
uspension was etxtracted and subjected to tHhe PLC (figure 1w
). as f experiments, except hat in a parallel experiment CoypExpress using 1
00 m
g/mL o
f C
ypExpress2D6 f
or 3
-­‐4 h
rs. T
his i
s a
c
onvenient t
ime f
or pre-­‐washed by (a) suspending in reaction buffer, (b) centrifugation aht igh-­‐
6,000 x g, throughput microplate tudies. (c) removing the ssupernatant, and (d) resuspending the CypExpress2C19 in Or fresh reaction buffer . Results comparing the HPLC profiles and the catalytic are presented Figure 2b y and 3 and in Table a1t . 6 ,000 x g. B. efficiency CypExpress2C19 was ipn elleted centrifugation a. The supernatant from the first cycle was analyzed by HPLC. b. The pellet – was resuspended in fresh buffer containing G6P, but no additional ubstrate, and incubated for a second reaction cycle. After Rsesearch, © 2015 Oxford B iomedical Inc. All Rights Reserved. Page 3 which the entire suspension was extracted and subjected to HPLC. © 2015 Oxford Biomedical Research, Inc. All Rights Reserved. Page 3 CypExpress™ 2C19 Conversion of Mephenytoin to S-­‐4-­‐Hydroxymephenytoin CypExpress™ 2C19 Conversion of Mephenytoin to S-­‐4-­‐Hydroxymephenytoin CypExpress™ 2D6 Catalyzed Conversion of Dextromethorphan (DOM) to Dextrorphan (DOH) Figure 2. HPLC analysis of Mw
P ere -­‐> HpMP catalyzed by washed vs. unwashed Introduction: These studies erformed using the FDA-­‐recommended CypExpress2C19 substrate Mephenytoin to evaluate the utility of CypExpress2C19 for drug metabolism and disposition studies. O
N
O
NH
O
CYP2C19
N
O
NH
OH S-­‐4-­‐Hydroxymep
henytoin (HMP)
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 Reaction Cronditions: P0 ilot-­‐scale were performed a total with DOM anging from 5
– 1,00 µM. rAeactions Lineweaver-­‐Burke plot of the irn esults thus volume of 1.0 obtained is presented n figure 3, aund gave a Kfm of 304 µM and caoncentrations: Vmax of 714 mL in a 20x150 mm gilass tube sing the ollowing final µM/hr/gram of CypExpress. However, as shown in Figure 2 and Table 1 (below), at = 20 to 500 μM MP (500 µM for scale-­‐up). otherwise 100 mSubstrate g/mL CypExpress 2D6, the reaction slows significantly after 2U hnless r. specified, MP was added to the reaction mixture as a S-­‐Mephenytoin (MP) concentrated solution in D
MSO The HMP product p
eak is more clearly visible by “zooming” the HPLC to the first 7 min as shown in 2figure . 00 mg/mL CypExpress C19 =3 1
Buffer = 50 – 100 mM KPi, or Tris-­‐HCl (as specified in tables), pH 7.5 NADP+, G6P = NONE ADDED for 1st 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 1). A. The entire suspension was extracted and subjected to HPLC (figure Or B. CypExpress2C19 was pelleted by centrifugation at 6,000 x g. a. The supernatant from the first cycle was analyzed by HPLC. o b. The pellet – was resuspended in fresh buffer containing G6P, but n
additional ubstrate, and incubated for a second reaction cycle. After Rsesearch, © 2015 Oxford Biomedical Inc. All Rights Reserved. Page 4 which the entire suspension was extracted and subjected to HPLC. © 2015 Oxford Biomedical Research, Inc. All Rights Reserved. Page 4 CypExpress™ 2C19 Conversion of Mephenytoin to S-­‐4-­‐Hydroxymephenytoin CypExpress™ 2C19 Conversion of Mephenytoin to S-­‐4-­‐Hydroxymephenytoin CypExpress™ 2D6 Catalyzed Conversion of Dextromethorphan (DOM) to Dextrorphan (DOH) Introduction: These studies were performed using the FDA-­‐recommended The data M
pCypExpress resented in T2able also dμM emonstrate the these C19 of M
Reaction hrs of μM off or HMP substrate ephenytoin to e1valuate the uP tility orf eproducibility CypExpress2C19 drug microplate s
tudies. metabolism and disposition studies. N
O
CypExpress 2D6 O Washed Unwashed 100 mg/mL NH
μM of 500 DOM Reaction 4 hrs CYP2C19
500 250 2 4 μM of D45.33* OH O
54.6 ±N
136.07 .7 NH
*The integration of the peak area may not be a2 ccurate due 67.1 to broad O
100 mg/mL 500 ± 2.2 peak area. CONCLUSION: Pre-­‐washing CypExpress reduces f the ±f low-­‐thru peak 100 mg/mL 250 4 the size o64.5 0.0 comprised of very hydrophilic components, but does not significantly alter the OH catalytic 100 efficiency mg/mL of CypExpress. 500 4 78.5 ± 1.0 S-­‐4-­‐Hydroxymep
henytoin (HMP)
S-­‐Mephenytoin (
MP) Effect of buffer on the production of HMP from MP: Other than the buffer, all CONCLUSION: CypExpress wtell suited for fm
icroplate eactions. NOTE: for conditions were identical is to hose in the irst set of erxperiments. The CypExpress Reaction Conditions: Pit ilot-­‐scale reactions weere p
erformed i
n a
t
otal olume longer r
eaction t
imes i
s i
mportant t
o r
estrict vaporation, e
.g. b
y p
erforming in 2C19 catalyzed reaction for this substrate proceeds equally well in 50 mvM Tris of 1.0 mL in a a
0x150 m
m g
lass t
h 2
umidified environment. ube u
sing t
he f
ollowing f
inal c
oncentrations: or Phosphate. Scale-­‐up conversion DOH in a µ single cycle: The rate of Substrate = 20 tof o 5DOM 00 μto M M
P (500 M for sreaction cale-­‐up). Unless otherwise 500 to DOH by CypExpress™ g/mL) am
t 3ixture 0°C in aa s oHMP f aa conversion μM DOM specified, MP was added 2tD6(100 o the rm
eaction Buffer MP (
μM) Reaction T
ime (
h) 200 mL reaction volume was investigated by RP-­‐HPLC after extraction of the entire concentrated solution in DMSO (μM) reaction mixture with HPLC Mobile Phase containing 79% (v/v) methanol, 1% (v/v) acetic acid, and 20% (v/v) water in 1:1 (v/v) ratio. The results are shown in figure CypExpress Tris-­‐HCl, 50 m2
M C19 = 100 mg/mL 500 4.0 41.54 4: Buffer =1 500 0 –m 1M 00 mM KPi, or 500 Tris-­‐HCl (as specified 7.5 Tris-­‐HCl, 4.0 in tables), pH 33.15 NADP+, G140
6P = NONE ADDED for 1st cycle; see details KPi, 50 mM 500 4.0 for additional 42.76 cycles G6PDH and KPi ,100 m
M 500 Contained in 4.0 32.11 CypExpress™ system. 120Mg++ = NONE ADDED. Dextrorphan (µM)
100
The 20 x 150 mm CONCLUSIONS: 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. 80
• Increasing buffer strength did not increase the yield of HMP; rather it decreased the yield At the end of the r60
eaction period, either •
Tris-­‐HCl b40
uffer is equally efficient for the CYP2C19 catalyzed bioconversion A. The entire uspension of M
P to HsMP compared o KePxtracted wtas and subjected to HPLC (figure 1). i 20
Or 0
-0.5
0.5
1.5
2.5
3.5
4.5
B. CypExpress2C19 was pelleted b
y c
entrifugation a
t 6
,000 x g. Time (hr)
HPLC. a. The supernatant from the first cycle was analyzed by he pellet b. T
– was resuspended in fresh buffer containing G6P, but no additional ubstrate, and incubated for a second reaction cycle. After iomedical Rsesearch, © 2015 Oxford B
Inc. All Rights Reserved. Page 5 which the entire suspension was extracted and subjected to HPLC. © 2015 Oxford Biomedical Research, Inc. All Rights Reserved. Page 5 CypExpress™ 2C19 Conversion of Mephenytoin to S-­‐4-­‐Hydroxymephenytoin CypExpress™ 2C19 Conversion of Mephenytoin to S-­‐4-­‐Hydroxymephenytoin CypExpress™ 2D6 Catalyzed Conversion of Dextromethorphan (DOM) to Dextrorphan (DOH) Increased ields studies for the w
production of HMP from MP by CypExpress in Introduction: Tyhese ere performed using the FDA-­‐recommended multiple reaction tco ycles: substrate Mephenytoin evaluate the utility of CypExpress2C19 for drug Rationale: S
tudies o
f t
he s
tability The HPLC profiles of aliquots of the reaction mixture taken at these time intervals metabolism and disposition studies. and activity of CypExpress systems expressing resented riecombinant n Figure 5: human P450 enzymes have shown that: are pvarious • Significant CypExpress activity is retained after a single reaction cycle O
O
• CypExpress typically adsorbs large quantities of relatively hydrophobic substrates from the reaction mixture, but releases most Nof the more N
NH
hydrophobic product(s) into the buffer. NH
• Low speed centrifugation after one reaction cycle O
pellets CypExpress O
containing significant quantities of unreacted substrate as well as some product. • Typically, the total product yield is greater for multi-­‐cycle reactions vs. OH longer incubations for a single cycle. This may be due to inhibition of S-­‐4-­‐Hydroxymep
henytoin S-­‐Mephenytoin (MP) some P450 reactions by substrate or product in long incubation periods. (HMP)
• Additional reaction cycles convert substantial amounts of the retained Reaction Conditions: substrate t o roduct – trhereby increasing overall metabolite yield. Ppilot-­‐scale eactions were performed in a total volume of 1.0 mL in aConditions: 20x150 mM
m ulti-­‐cycle glass tube u
sing t
he f
ollowing f
inal c
oncentrations: production of HMP from MP by CypExpress2C19. The reactions conditions were generally identical to those in the first set of Substrate = 20 to 500 μM MP (500 µM for scale-­‐up). Unless otherwise experiments. Initial MP concentrations of 200 µM and 500 µM were used at o specified, MeP was added to the reaction mixture as determine relative catalytic fficiency as a function of substrate concentration. Figure 6 zooms in on tconcentrated solution in DMSO peak 4o btained p
roduction HPLC profile: In he Figure below, tfor he DbOH lack curve is the analysis of a sample of the CypExpress a2fter supernatant cycle 1. Tm
he g/mL blue curve is the results obtained following C19 = 100 extraction of the entire suspension following the second cycle. CYP2C19
Buffer = 50 – 100 mM KPi, or Tris-­‐HCl (as specified in tables), pH 7.5 NADP+, G6P = NONE ADDED for 1st 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). Or B. CypExpress2C19 was pelleted by centrifugation at 6,000 x g. a. The supernatant from the first cycle was analyzed by HPLC. b. The pellet – was resuspended in fresh buffer containing G6P, but no additional substrate, and incubated for a second reaction cycle. After which the entire suspension was extracted and subjected to HPLC. © 2015 Oxford Biomedical Research, Inc. All Rights Reserved. © 2015 Oxford Biomedical Research, Inc. All Rights Reserved. Page 6 Page 6 CypExpress™ C19 Conversion of ephenytoin Mephenytoin to S-­‐4-­‐Hydroxymephenytoin CypExpress™ 22
C19 Conversion of M
to S-­‐4-­‐Hydroxymephenytoin Yield: The HMP yields obtained following the first and tshe econd cycles are shown Introduction: These studies were performed using FDA-­‐recommended in t
able 2
. substrate Mephenytoin to evaluate the utility of CypExpress2C19 for drug metabolism and disposition studies. Reaction Reaction Sample μM of MP μM of HMP hrs. O
O
C-­‐1 Supernatant NC-­‐1 Supernatant NH
C-­‐2 Cell Suspension C-­‐2 Cell Suspension 200 500 200 in Cycle-­‐1 No addition in C-­‐2 500 in Cycle-­‐1 No addition in C-­‐2 CYP2C19
4 4 4 4 O
N
O
40.83 43.64 NH
53.07** 95.53** OH *After Cycle-­‐1, cell pellet was stored at 4°C over night before start of Cycle-­‐2 S-­‐4-­‐Hydroxymep
of an aliquot following the 2nd cycle confirmed that majority of henytoin (HMP)
S-­‐Mephenytoin (MP) **Centrifugation nd
the HMP following the 2 cycle was present in the supernatant. Summary: C onditions: For 200 µM P, two reaction cycles effected 47% conversion HMP. of 1.0 Reaction PM
ilot-­‐scale reactions were performed in a total to volume mL in a 20x150 m5m ube using the cfollowing final concentrations: For 00 gµlass M MtP, two reaction ycles effected 28% conversion to HMP. Substrate = 2s0 to 500 μincrease M MP (500 µM yfields or scale-­‐up). therwise The apparently very ignificant in HMP from 500 Uµnless M MP o
in a second reaction cycle s uggest specified, MP was caoncentrations dded to the rm
eaction mixture as a that high substrate ay inhibit this reaction. To confirm solution in DiMSO the results concentrated obtained in this experiment, t was repeated in duplicate. The results are shown in table 3. CypExpress 2C19 = 100 mg/mL Buffer = 50 – 100 mM KPi, or Tris-­‐HCl (as Reaction specified in tables),
7.5 μM of MP μM of pHH MP Reaction Sample hrs. NADP+, G6P = NONE ADDED for 1st cycle; see details for additional cycles C-­‐1 Supernatant 500 4 36.07 G6PDH and Mg++ = NONE ADDED. Contained in CypExpress™ system. C-­‐1 Supernatant 500 4 31.77 500 The 2
0 xC 1
50 mm reaction tube win as Cpycle-­‐1 laced in a tilted 4 position on a94.75 shaker platform C-­‐2 ell Suspension No addition in C-­‐2 at 30°C, and agitated by rotation at 225 rpm for 3.0 h. 500 in Cycle-­‐1 C-­‐2 Cell Suspension 4 77.84 No addition in C-­‐2 At the end of the reaction period, either Discussion: The data presented support the adsorbtion of MP during the first A. The entire suspension was extracted and subjected to HPLC (figure 1). reaction cycle and, after centrifugation to remove unabsorbed MP and the HMP produced during the first cycle, CypExpress 2C19 catalyzes significant additional Or of HMP in a second cycle. These data are consistent with the inhibition of production CypExpress2C19 at high concentrations of the substrate MP. B. CypExpress2C19 was pelleted by centrifugation at 6,000 x g. a. The supernatant from the first cycle was analyzed by HPLC. b. The pellet – was resuspended in fresh buffer containing G6P, but no additional substrate, and incubated for a second reaction cycle. After the eRntire suspension was extracted and to HPLC. © 2015 Oxford which Biomedical esearch, Inc. All Rights Reserved. subjected Page 7 © 2015 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA-ALDRICH is a trademark of Sigma-Aldrich Co. LLC, registered in the US and other countries.