UU9[l-95561979510-l l5'.'—] lt34$[l2.O[l.‘U
`DRUG METAB(JLlSM AND DISPOSITION
`Copyright (C: 1997 by The Atnerican Society for Phannacology and Experimental Therapeutics
`
`Vol. 25, No. 10
`Pritireil in U.S.A.
`
`BIOACTIVATION OF THE ANTICANCER AGENT CPT-11 TO SN-38 BY HUMAN HEPATIC
`
`MICROSOMAL CARBOXYLESTERASES AND THE IN VITRO ASSESSMENT OF
`
`POTENTIAL DRUG INTERACTIONS
`
`J. GREG SLATFER, PING SU,1 JAMES P. SAMS, LARRY J. SCHAAF, AND LARRY C. WIENKERS
`
`Drug Metabolism Research (J.G.S., P. 8., J.P.S., L. C. W.) and Clinical Pharmacokinetics (L.J.S.), Pharmacia and Upjohn
`
`(Received March 28, 1997; accepted June 5, 1997)
`
`ABSTRACT:
`
`Human hepatic micrcsomes were used to investigate the carboxy-
`lesterase-mediated bioactivation of CPT-11 to the active metabo-
`
`lite. SN-38. SN-38 formation velocity was determined by HPLC over
`a concentration range of 025-200 ).iM CPT-11. Biphasic Eadie
`Hofstee plots were observed in seven donors, suggesting that two
`isoforms catalyzed the reaction. Analysis by nonlinear least
`squares regression gave KM estimates of 129-164 p.M with a Vmax
`of 5.3-1? pmollmglmin for the low affinity isoforrn. The high affinity
`isoform had KM estimates of 1.4-3.9 f.l.M with Vmx of 1.2-2.6 pmoll
`mg/min. The low KM carboxylesterase may be the main contributor
`to SN-38 formation at clinically relevant hepatic concentrations of
`CPT-11.
`Using standard incubation conditions, the effects of potential
`inhibitors of carboxylesterase-mediated CPT-11 hydrolysis were
`evaluated at concentrations 2 21 piM. Positive controls bis-nitro-
`phenylphosphate (BNPP) and physostigmine decreased CPT-11
`hydrolysis to 1.3-3.3% and 23% of control values, respectively.
`Caffeine, acetylsalicylic acid, coumarin, cisplatin, ethanol, dexa-
`methasone, 5-fluorouracil, loperamide, and prochlorperazine had
`no statistically significant effect on CPT-11 hydrolysis. Small de-
`
`creases were observed with metoclopramide (91% of control),
`acetaminophen (93% of control), probenecid (87% of control), and
`fluoride (91% of control). Of the compounds tested above, based
`on these in vitro data, only the potent inhibitors of carboxy|ester-
`ase (BNPP, physostigmine) have the potential to inhibit CPT-11
`bioactivation if administered concurrently.
`The carboxylesterase-mediated hydrolysis of at-naphthyl acetate %
`(I1-NA} was used to determine whether CPT-11 was an inhibitor of ;
`hydrolysis of high turnover substrates of carboxylesterases. lnhi-3
`bition of a-NA hydrolysis by CPT-11 was determined relative too.
`positive controls BNPP and NaF. Incubation with rnicrosornes pre- p.
`treated with CPT-11 (80—440 p.M) decreased u:-naphthol formationg
`to approximately 80% of control at (I-NA concentrations of 50-8003
`p.M. The inhibitors BNPP (360 am) and NaF (500 am) inhibited 8
`cu-naphthol formation to 9-10% of control and to 14-20% of con-
`S]BllJ
`trol, respectively. Therefore, CPT-11-sensitive carboxylesterase -9‘
`isoforms may account for only 20% of total or-NA hydrolases. Thus,0°
`‘E
`CPT-11 is unlikely to significantly inhibit high turnover, nonseIec- "’
`tive substrates of carboxylesterases.
`
`E0|LlAt\OC[
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`
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`Slwmortadsv
`
`for the assessment of CPT-I l bioactivation by carboxylesterases was 3
`
`lrinotecan (CPT-11,2 Camptosar, Pharmacia & Upjohn, Kalama-
`200, MI) is a carboxylesterase-labile, carbamate prodrug of the anti-
`neoplastic topoisomerase 1 inhibitor SN-38 (I-3). CPT-11 and SN-38
`exist
`in a pH- and protein-dependent equilibrium between active
`lactone and inactive hydroxy acid anion forms (4). Bioactivation of
`CPT-11 by microsomal carboxylesterases (E.C. 3.1.1.1) occurs pri-
`marily in the liver (5, 6) and has been studied previously using
`purified human carboxylesterase (7, 8). In this study, an in vitra model
`
`A preliminary account of this work was given at the 4th International ISSX
`Meeting, Abstracts #88, 218 and 219. Seattle WA, Aug 27-31, 1995.
`‘ Present address: Department of Biopharmaceutical Sciences, University of
`California at San Francisco.
`2 Abbreviations used are: CPT, camptothecin; CPT-11. irinotecan hydrochlo-
`ride trihydrate; EtOH, ethanol; ASA, acetylsalicylic acid; HMB, hydroxymercu-
`ribenzote; CMB. chloromercuribenzoate; HPLC, high performance liquid chroma-
`tography; KM, Michaelis constant; 5-FU, 5-fluorouracil; MeOH, methanol; BNPP,
`bis-nitrophenylphosphateg a—NA, alpha naphthyl acetate; SN-38, active antineo-
`plastic metabolite of CPT-1 1; DMSO, dirnethylsulfoxide; UPACS, Upjohn Physical
`and Analytical Chemistry System; V. reaction velocity; QC, quality control stan-
`dard; [S], substrate concentration.
`
`Send reprint requests to: Dr. J. Greg Slalter, Drug Metabolism Research,
`Pharmacia and Upjohn Co., 301 Henrietta St., Kalamazoo MI 4900?. E-mail:
`john.g.s|atter@am.pnu.com.
`
`developed using human liver microsomes. Michaelis-Menten kineticg
`constants were determined and a new high affinity isoform was;
`discovered.
`5
`Since CPT-11 will be used clinically with a variety of other drugsfw
`an in vitro screen for potential drug interactions involving CPT-I13
`bioactivation was developed.
`Some chemicals were chosen based on possible clinical use and
`have no known carboxylesterase-inhibiting ability (e.g. 5-FU, ethanol
`(EtOH), caffeine, acetaminophen, coumarin, dexamethasone, cispla-
`tin, and probenecid). Others were chosen based on previously de-
`scribed effects on carboxylesterases (e.g. ASA (9), fluoride ( l0),
`HMB, CMB (6), physostigmine (6,
`I 1), bis-nitrophenylphosphate
`(BNPP) (7), metoclopramide (I2), loperamide (8), and prochlorpera-
`zine (13)). To avoid misleading conclusions about the possible clinical
`significance of in virro experiments conducted at high inhibitor con-
`centrations, statistically significant results were discussed in the con-
`text of clinically relevant concentrations of inhibitor.
`As a carbamate, CPT-l l is a relatively poor substrate for carboxy-
`lesterases. This was proposed to be a result of slow decarbamylation
`ofthe serine esteratic site, inferring that CPT-1 l is a slowly reversible
`competitive inhibitor, possibly also with an allosteric inhibitory effect
`on other substrates at a modulator site of the enzyme (8). Therefore,
`CPT-ll could act as an inhibitor of endogenous or xenobiotic high
`1157
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`Patent Owner, UCB Pharma GmbH — Exhibit 2059 - 0001
`
`

`
`1158
`
`SLATFER ET AL.
`
`turnover substrates of carboxylesterases. To investigate this, CPT—ll
`was tested as an inhibitor of mierosomal earboxylesterase-mediated
`hydrolysis of the model substrate, cr-naphthyl acetate (ar-NA) (14,15).
`
`Methods
`
`Reagents and Materials. lrinotecan hydrochloride trihydrate (CPT-l 1) and
`SN-38, were supplied by Pharmacia and Upjohn,
`Inc. Camptothecin and
`inhibitors were obtained from Sigma or Aldrich (St. Lo11is, MO). Transplant
`quality human liver tissue was obtained through the lntemational Institute for
`the Advancement of Medicine (Exton, PA). Microsomes were prepared by
`standard methods (16) and stored in 0.25 M sucrose at —70°C. Protein was
`determined by the Bicinchoninic Acid (BCA) assay (Pierce Chemical, Rock-
`ford, IL) and standardized relative to bovine serum albumin.
`Kinetics and Inhibitors of CPT-ll Bioaetivation.
`lrm.'n'm.'.v'rms, Kinetic
`parameters were determined using CPT-11 that was at equilibrium in pH 7.4
`phosphate buffer. Under these conditions the lactone/hydroxy acid anion ratio
`for CPT-I 1 was fixed at 87/13 (4). Kinetic studies were done in 2 ml
`Eppendorf Safe-lock polypropylene tubes (Brinkman, Westbury, NY) tubes
`using human hepatic microsomes and CPT-1 l. incubated in a linal volume of
`0.25 mL sodium phosphate (0.1 M, pH 7.4) at 37°C, with 80 rpm mixing.
`CPT—l1 solutions were pre-incubated for 5 min and incubations were initiated
`by the addition of pre-warmed microsomes. Reactions were quenched and
`analyzed for SN-38 content by HPLC as described under sample preparation.
`All concentrations refer to total (lactone plus hydroxy acid anion) forms of
`CPT-ll and SN-38. Incubation times (5-60 Inin) and protein concentrations
`(0.125-1.8 mg/ml) were tested to identify conditions that resulted in quanti-
`fiable and linear formation of SN—38.
`Defemnfnaffnn r2f‘Mfr'ham'f.s“-Marten kinetic‘ ,1'J(1‘I’(1‘f?1(’l'(’I’S‘. lncubations were
`done in duplicate using microsomal protein (1 mg/ml) and CPT-1 l (0.25-200
`,u.M, 17 concentrations), incubated for 15 min, A 50 ;.;.M CPT-ll control in
`phosphate buffer (without microsomes) and a boiled microsome control were
`r11n with each experiment. The hydrolysis of CPT-ll
`in phosphate buffer.
`in
`the absence ofmicrosomal protein. and in the presence of boiled microsomal
`protein, was negligible over the incubation time course: however, the CPT-1 1
`stock solution contained a small amount of SN-33 (approxilmilely 0,04%].
`Levels of the SN-38 impurity at other substrate concentrations were calculated
`by linear interpolation of a calibration line passing through the experimentally-
`determined 50 ,u.M blank data point and the origin. Total SN-38 formation in
`the presence of microsomes was corrected for the SN-38 impurity at each
`substrate concentration. Substrate depletion in kinetic studies was maximally
`10% at 0.25 ;.LM CPT-11.
`irihibitors on SN-38_jiJrma:tirm.
`Determirmtion of the r.j}_'fer.'t o_fpoter1tiai
`lncuhations were done in triplicate using pooled human liver microsomes, as
`described in Inctiharfons, at a CPT-11 concentration of 10 ,uM. Microsomes
`(25 111] were mixed with potential inhibitors (0-25 ptl) and phosphate b11ffer
`(100-125 ,u.l) and pre-incubated for 5 min. Inhibitor concentrations were 2 21
`,uM and were chosen to represent clinically attainable concentrations. with the
`exception of loperamide, where the 25 ;.LM test concentration was approxi-
`mately 1000-fold higher than clinically relevant plasma levels.
`lncubations
`were initiated by the addition of prewarmed CPT—11 solution (100 ytl). In the
`final incubation. DMSO content was 0,0016" 9 and MeO|I content was 0% or
`1%. Reactions were quenched and analyzed as described under sample prep-
`aration. SN-38 formation was measured as nginl/15 min.
`Inhibition was
`expressed as per cent of control.
`HPLC and rinafyticsal standard preparation. A Perkin-Elmer [SS-200 auto-
`injector and PE 410 pump were used (Perkin Elmer, Norwalk, CT). Chroma-
`tography was done on a Zorbax SB-C8 column (4.6 X 250 mm, 5 ptm particle
`size, Mac Mod Analytical lnc., Chadds Ford, PA) with a Brownlee Newguard
`RPS guard column using a mobile phase of 74:26, v:v triethylamine buffer:
`acetonitrile at a flow rate of 1 mL.’min. Detection was done using a Waters
`Model 474 fluorescence detector (Millipore Corp., Milford, MA). Calibration
`standards (0-I0 concentrations) were prepared in duplicate in phosphate
`buffer to cover a concentration range ofapproximately l to -4000 ng/ml SN—38.
`Quality control (QC) standards were prepared in duplicate from separately
`prepared stock solutions at nominal SN-38 concentrations of 23, 76, and 1900
`ng/ml. The stability ofstock and control solutions in MeOH and in phosphate
`buffer ill 4°C (storage conditions) were proven by HPLC analysis after storage
`
`periods of0, 5, 22, 28, and 41 days. SN—38 recovery from microsomes relative
`to phosphate buffer was determined to be complete by comparing triplicate
`incubations ofmicrosome suspensions spiked with 4, 90, 300, and 1900 ng/ml
`SN—38 with triplicate QC samples prepared identically in phosphate buffer.
`Srrmpic ,-Jre,r1r.'mr."rm. A 250 ,u.l aliquot of incubation sample. calibration
`standard or QC sample was mixed with 500 ,ul quench solution (acetonitrilef
`acetic acid/methanol (95.6/4.0/0.4. w/w/w) containing 24 riyrnl eamptothecin
`(CPT) internal standard). The samples were heated in a water bath at 37°C for
`20 1nin to convert SN—38 to the lactone for1n. Samples were mixed with 1.0 ml
`of 50 mM, pH 42 triethylamine/acetate buffer. Precipitated protein was
`separated by centrifiigation at 14,000 rpm for 2 min. A 50 ,ul aliquot of
`supernatant was analyzed by HPLC. Retention times were sensitive to small
`changes in mobile phase composition. The detector was programmed to change
`wavelengths and detector gain. From 0-10 min the excitation and emission
`wavelengths were 372 and 425, respectively (gain 10, measures CPT-l 1).
`From 10-14 min the excitation and emission wavelengths were 372 and 535,
`respectively (gain 100, measures SN-38). From 14 min onward the excitation
`and emission wavelengths were 372 and 425, respectively (gain I00, measures
`CPT).
`Dam c'r;.I'.I'em'rm mm’ umi.{i'.s'r'.s'. Data were collected and processed using the U
`UPACS (Upjohn) chromatography system, Version 5.2. Quantitation was done;
`using peak height ratios of the analyte to internal standard. Calibration curves E-
`were determined by using linear regression 1hrough-the-origin best fit with ;.g_
`lfconcentration weighting factor. Unknown SN-38 concentrations in incuba-8
`tion solutions were determined by comparison of the peak height ratios with E‘
`the calibration curve. Correction factors for
`the SN-351 impurity at eachE
`concentrationftime datapoint were calculated by subtracting background 5‘
`SN—38 from the total SN—38 formed in the presence of microsomes. Unknown if
`concentrations were determined as corrected (carboxylestcrasc-mediated)3
`ng/ml SN—38 produced in 15 min and were converted to units of velocityil-,
`(pmol/mg/min) using a formula weight of 4104 g/Mol for SN-38 monohy-
`91.1.1110
`drate.
`in phosphateg
`in Me()H or
`l’(1i'ffl'(1t‘frJI1. Standards of CPT-ll
`Ar1m'_vii::af
`buffer. prepared in polystyrene centrifuge tubes and stored at 4°C. were stable”?
`over a period of4l clays. Because of a limited supply of human microsomes, 33;
`
`W1.10S[Pl.LIf'l0['
`
`standards and QC samples prepared in phosphate buffer were used to quantify 3
`microsomal concentrations of SN-38. Recoveries of SN-38 from microsomes E
`spiked in triplicate with SN-38 at concentrations of4, 90, 300, and 1900 ngfml ._]
`were approximately 100% in both microsomes and phosphate buffer. Corre-
`lation coefficients (r2) for linear standard curves in kinetic assays were 2
`0.’-J98. QC data were typically within : 15%. The assay lower limit of
`quantitation (LLOQ) was 1.1-2.4 ng/ml SN-38.
`Curvefitting metliocis. KM and 1/,,,._,,‘ were determined by nonlinear least
`squares regression using Systat Version 5.2.1 for Macintosh (Version 7,0].fi
`SN-38 formation Vl)(.’.".S“£t'.S“ [S] curves were fit for a two enzyme system with
`l/V3 weighting. or-NA hydrolysis V ve.v'.s'u.s' [S] curves were best fit by a single 5
`enzyme Michaelis-Menten equation.
`um
`D£’tE’FM1fR(ttf0R rJ_f'i'nhr'hi'I0r potency. Data were analyzed using Microsofig
`Excel Version 4.0a (Microsoft Corp. Redmond, WA). Means and standard
`deviations of uncorrected SN-38 production in the presence and absence of
`inhibitors were compared with a Student’s t test. Mean SN—38 production was
`corrected for mean SN-38 impurity measured in the presence of heat denatured
`(boiled) microsomes and SN-38 production was expressed as per cent of
`enzyme-catalyzed control. When MeOH was necessary as an aid to solution for
`inhibitors. an additional control containing an equal amount ofMeOH was r11n.
`The earboxylesterase inhibitor BNPP and a phosphate buffer control
`(no
`protein] were used as positive control and blank, respectively.
`
`Kinetics of or-naphthyl acetate hydrolysis and inhibition by CPT-ll
`
`An assay for the carboxylesterase-inediated formation of at-naphthol from
`oi-NA was developed based on the formation of an azo dye from the reaction
`of fast blue RR (a diazonium salt, Sigma, St. Louis, MO) with or-naphthol (17).
`Enzyme kinetics were detennined using modified literature methods (14, 15)
`on a THERMOmax Microplate Reader (Molecular Devices, Sunnyvale, CA)
`(18). Optical Density (O.D,) was measured at 450 nm. lnitial velocities were
`recorded in units ofmO.D./min and were corrected for background hydrolysis
`in buffer, determined simultaneously. CPT-1 1 (80-440 ,uM) was tested as an
`inhibitor of or-NA hydrolysis. Control carboxylesterase-mediated hydrolysis
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2059 - 0002
`
`

`
`BIOACTIVATION OF CPT-11 BY CARBOXYLESTERASES
`
`1 159
`
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`was detennined on the same plate, simultaneous with each inhibitor concen-
`tration. since preineubation effected the control kinetics. Results were ex-
`pressed as per cent of control sample initial Velocity (mO.D./min}.
`Reagent Preparation. BNPP and NaF were prepared in 0.1 M. pH 7.4
`phosphate butfer. Stock solutions of or-naphthol
`for standard curves were
`prepared in acetone. Final acetone concentration in the standard curve wells
`was 3.3%. Stock CPT-ll solutions were prepared at 360 y.M in phosphate!
`MeOH or at 80. 140. and 440 pLM in phosphate/dimethylsulfoxide (DMSO) at
`90/10 wfw. The co-solvent concentration in each incubation well was 0.33%.
`Controls containing an equal amount of eo—solvent were prepared. Stock er-NA
`solutions were prepared in acetone/phosphate at 50/50 (W/w). The final acetone
`concentration of 1.7% in all wells does not effect enzyme activity (14). Fast
`Blue RR was prepared fresh in phosphate buffer at 0.25% V/v for each
`experiment and was filtered prior to use.
`Preliminary Experiments. Low protein concentrations and shon reaction
`times at 23°C and substrate concentrations of 2 40 ;.tM were needed to avoid
`substrate depletion in excess of 15-20%.
`At (v—NA concentrations 2 40 [.LlVl product formation was linear for 3-4
`min. Estimates of initial velocity (mO.D.fmin) were determined using 13 data
`points acquired over 2 min.
`lnitial velocity measured over 2 min was linear
`from 1-10 ,u.g/rnl microsomal protein. S11bseq11ent experiments were con-
`ducted at 5 ,ug/ml protein. Calibration curves of O.D. ver.s'u.s' concentration
`(,uM) for the or-naphthol (prodtlell/Llytt complex were linear (r2 2 0,98) over
`the concentration range 0-150 ;.tM. Kinetic experiments were conducted in
`quadruplicate in two male (donors 10M and 24M) and three female (donors
`14F. 17F. and 20F) donor rnierosomes over an H:-NA concentration range of
`5-800 ,i.LM. Means of quadruplicate experiments had percent coefficient of
`variation values of typically less than 15% for each concentration 2 40 ,uM.
`Thereafter.
`in inhibitor studies.
`incubations were conducted in duplicate in
`sodium phosphate buffer (pH 7.4, 0.1M]. When MeOH or DMSO were used
`an aid to solution for C‘PT—l 1. control experiments were also eondueted in
`parallel with the appropriate concentration of co—solvent.
`In all experiments.
`duplicate controls were run simultaneously for each substrate concentration on
`the same plate.
`Incubation Conditions. ln a 96 well microplate. each well contained 260
`[.Ll buffer‘. 20 pal microsomes (0.075 mymL] or buffer‘. 10 at last blue RR (0.25
`mg/ml). and 10 ,ul substrate or buffer. The total volume was 300 ptl. Substrate
`was added last using a multi-channel pipette. Immediate automatic mixing was
`employed and ineubations were kondllcled with continuous assessment of
`optical density within the thermostatted compartment of the plate reader.
`Preincubations with inhibitors were done inside the plate reader.
`Kinetics of a-Naphthyl Acetate Hydrolysis. Substrate concentrations of0.
`S, 10, 20, -40, 60, 80, 100, 200, 400, 600, and 800 ].r.Nl (12 concentrations/lane]
`were incubated in quadruplicate on the same plate with a matching quadru-
`plicate set of enzyme-free controls. Substrate depletion. calculated from the
`cr—naphthol standard curve, was maximally 17"’/u at 2 min at 60 p'.M a—NA for
`donor 20F. and was 13% or less in all other donors at ¢r—NA 2 40 j.)'.lVl. Data
`were fit to a single enzyme Michaelis Menten equation using substrate con-
`centrations ol'4(l-(10 pLM to (100-800 pLM.
`l’m__“ data were compared to l’mM
`data for low and high KM isoforms ofthe CPT-1 1 hydrolyzing earboxylesterase
`by linear regression comparing donors 10M, 24M, 17F, and 20F.
`Inhibition by CPT-ll. Mierosomes from donor 17 were preincubated with
`CPT-ll (360 ,ul\/1). or BNPP (360 ptM). or NaF (500 ,ul\/1) for 10 min before
`the addition of (x—NA (0-120 pLM, 8 eoneentrationsfplate lane) to start the
`reaction. The experiment was repeated with CPT-ll (80. 140. and 440 ,u.M)
`preincubated for l h at 23°C over cx—NA concentrations of0-800 ,uLM.
`Results
`
`Hydrolysis Kinetics of CPT-ll in the Presence of Human Liver
`Microsomal Carhoxylesterases
`
`0 ' _' '
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`Retention Time (Min)
`1, Strpw'r'r:Ipr:.~‘w.’ IIPLF t':'1rrmmrogrmns .w'1r:n'."ng S'i\z'—3rS’_frJJ'r:r:':1'F11’ :
`donor 10M m1'Cro.rome.r (1 mg/mL protein,
`.75 min) at CPT-ll‘
`S
`L'o.'rL'er1t."r.ttio.'rs o_fU.5 (.l'r)wr:r tmr.‘r:), 5 (.s'r.*r.‘w1c[tmL'e), 25 (t.l'rr'rr[ r‘rrtL'e), mic! Q.
`1'00 MM (Ii,-rpm‘ .'rm‘c=).
`2
`
`Fin,
`
`U.]110f]9ClS17.'
`
`low substrate concentrations and to adequately differentiate enzy1ne-
`catalyzed SN-38 formation frotn the SN-38 impurity.
`Kinetic Results for SN-38 Formation. HPLC chromatogramsg
`showing the effect of CPT-ll concentration on the fonnation of'g
`Sl\l—38 by rnierosomes are shown in fig. 1. In microsomes from SCVCl"1U:;
`different liver donors, Eadie Hofstec plots were clearly biphasic over;
`0.25-200 ,uM CPT-ll (data not shown). In accord with the linear%
`plots, V ver‘.ru.r [S] data, tnodeled using nonlinear least squares regres— E
`sion analysis. were best fit by a two enzyme equation. KM and 1/um E‘
`estimates are presented in table 1. A representative Eadie Hofstee plotg
`for the pooled microsomes, comparing titted with actual data is shown
`in lig. 2. The actual and theoretical curves were superimposed. The?
`calculated KM and I/Hm for each earboxylesterase isoform are indi-E
`vidually represented in the satne figure by the calculated linear plots;
`for each isoform.
`5
`Data for all donors (N = 7) gave a well defined apparent KM of“N
`1.4-3 .9 p.M for the low KM isoform and 129-164 ,uM for the high KM 3
`isoform. The Vmux/KM ofthe low KM isoform was higher than the high U‘
`KM enzyme.
`A histogram comparing the relative contribution of the low and
`high KM CPT-l l carboxylesterases to the total production ofSN-38 is
`shown 1’¢’r.'ms CPT-ll concentration in fig. 3. The low KM enzyme is
`the dominant source of SN-38 at CPT-l 1 concentrations up to 20 ,uM.
`Thus, at physiologically relevant hepatic concentrations of Cl’T—l 1,
`the low KM isoform will be the predominant enzyme responsible for
`CPT-ll bioactivation.
`
`Effects of Potential Inhibitors of CPT-ll Bioactivation
`
`Incubation Conditions. In pilot studies, heat denaturation (boiled
`microsomes) and the organophosphate esterase inhibitor. BNPP re-
`duced SN—33 production by human hepatic mic1'oso1nes to zero,
`providing evidence for the involvement of esterase enzymes. An
`optimal protein concentration of 1 mgfml and incubation time of 15
`min were chosen frotn the linear‘ regions of SN—38 production. These
`conditions were necessary to surpass the assay limit of quantitation at
`
`Results of four separate inhibition experiments are compared with
`their respective control values in table 2. Inhibition was expressed as
`per cent ofcont1'ol. Controls containing an equal amount of 1 "0 Me()H
`were run when MeOH was used to dissolve inhibitors. MeOH de-
`
`creased activity by 3.5, 6.6, and 18.2% in experiments 2, 3, and 4,
`respectively. Organic solvent effects on carboxylesterase activity have
`been documented previously (19).
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2059 - 0003
`
`

`
`1160
`
`SLATFER ET AL.
`
`TABLE 1
`
`r[ri1ri_/ow‘ r:rm'i (f!J.'I!J." wer'r:_/ii‘ i':_i-'
`.l'i'i*r:." tl1.it‘I'!J.S'!Jtl1t‘.S'. 1'' HS‘.
`i':_i-' n'iimimi
`Siriirimi.';i= of1'1/1'iL':'iriei'i.§—Mr:m‘eri Ri'.'iei‘r'L' pri."riiiir:1r:.".s'_/or S.'V—3cS'_/o."iim1ir)ri_/.5'rmi (_'i”i"—."i'
`iiriii.l."iimi' i'e§:i'e,i‘.\‘!riii m o‘ 2 wi:i*iiie il/fir':'in'm'f.\‘-Meiirwi
`(’(_l'll(i‘l'fi).f1 m rihrriiii K,, mm’ l’,mH (’,\‘ffItTiI1(’.\‘
`Low K,” Isoform
`High K,.,1sot'0rin
`V
`_
`V
`_
`uiiiiiilfiiigfiiiiii)
`lpiiiolfiiigfiiiin)
`2.04 I 0.30
`14.8 I 2.0
`2.46 I 0.56
`12.7 I 1.5
`1.21 I 0.20
`5.30 I 1.3
`2.41I0.23
`15.1 I4.6
`2.03 I 0.64
`11.0 I 4.9
`1.37 I 0.27
`(3.75 I 1.2
`2.60 I 0.21
`8.14 I 1.4
`2.61 I 0.39
`16.6 I 2.3
`219 I 0.52
`10.5 I 29
`2.1] I 0.82
`10.8 I 5.8
`
`L""“fK"'
`0.86
`1.63
`0.44
`1.72
`1.26 I 0.71
`0.35
`0.84
`0.89
`069 I 030
`0.98 I 0.58
`
`K“ (MM)
`146 I 43
`129 I 45
`164 I 83
`150 I73
`148 I 120
`150 I ()6
`150 I 54
`150 I 46
`150 I 97
`149 I 154
`
`l""“‘fK"”
`0.10
`0.10
`0.03
`0.10
`0.08 I 0.04
`0.04
`0.05
`0.11
`007 I 004
`0.07 I 0.04
`
`DoiiorfSex
`
`Pooled
`10 M
`15 M
`24 M
`Mean I S.D.
`9 F
`17 F
`20 F
`Mean I SD,
`Grand mean I S.D.
`
`K“ (MM)
`2.4 I 0.4
`1.5 I 0.8
`2.7 I 0.5
`l.4I0.2
`1.9 I 1.0
`3.9 I 0.8
`1.4 I 0.2
`2.9 I 0.5
`2.7 I 0,9
`2.3 I 1.4
`
`M. male: F. female: SD calculated
`
`root of sum of squared deviations/N.
`
`12
`
`9
`
`B
`
`1: Low Km Enzyme Actlvlly
`Hlgli Km Enzyme Activity
`-w Enzvmemv
`
`
`
`e
`5 E
`1:
`E .3
`E 5
`en '5”
`"'1 E
`5 E
`3
`
`—---——jV_—--—-
`‘
`::.".‘;::::::::::::a:i.:i:i";.::;:i:::,:'::.i
`:
`o Cnrri|ii.ilerFllledCPT-1H-lydralFl3eAi‘1tvty
`I Actual er-r-11 Hyrimiaae Acllvty
`..,_. -
`
`
`
`u
`
`u
`
`I
`.
`fl
`O .
`.
`,,
`,__,__,___‘__,..__._, ..__,_,
`0.4
`0.0
`WCPT-11 Concentration [ii M]
`
`o
`_,'_
`
`_n ‘!____.
`1.0
`
`0.0
`
`1"
`
`3
`
`5
`
`3.
`';
`E
`03
`‘—“E-
`S E
`E?
`— E
`ET 4
`3
`>
`
`0'
`ii:
`.'
`' H
`: C)
`. 5
`0
`'
`- 'i
`.
`.
`‘
`2;-.. ,'_
`j
`_
`0.1 .
`II
`0.0
`
`i
`
`I
`
`'
`
`'
`
`‘
`
`_.
`
`.‘
`0.2
`
`.
`
`0
`
`0.2
`
`0.5
`
`1.0
`
`_
`2.0
`
`‘
`15.0
`
`:
`100.0
`
`150.0
`
`200.0
`
`40.0
`20.0
`i0.0
`4.0
`CPT-11 Concentration [ii M]
`FIG. 3. Hfsiogmni s.lirJn=r'iig the crilrtifrirefl rc.’ri.*.I'vc crJnrri'hii.'r'rJns rJ_f'c(m'i
`e.s'i‘er'(i.s'e i'.s'rg/r‘J."iii to Hit: mm.’ [J."!J(fl!t‘1.fr)!1 rJ_/'S.'V—3cS'
`(1.1
`i'm'r'r:ri.s'i'.'ig
`r‘om'eii.'m.'.imi.i- of (“PT-H.
`
`UC
`E
`
`E‘
`&
`a
`,-:_p
`C
`E
`E"
`CL.
`1%‘
`‘=1
`.1‘:
`O
`5
`-'3-__—_
`
`.
`(E
`91
`53
`E*-l
`E:
`
`E‘
`
`FIG. 2. Emt'i'c= ilri/,i'rm* plrii g(’!1(’I'EI1(’(frfilffril-IWig iinm'r'm=c.'r r'iii'i’r{/irrfiii: ri/'1’
`versus [S] daia_fiJr prided nii'crrJsrJnies to (.5 2 eiizynie Mic'h(m'i.9-Menreii
`eqiiurirm.
`
`max
`Linear ll‘llC1']‘J()lill1lil‘l!-i for czich isofoim. based on nonlinear K” and V
`estimates. illustrate graphically the correct intercept and slope for the individ-
`ual isoforins. KM and 1”m,_u estimates are in table 1.
`
`As expected, BNPP at both 25 and 100 ,uM reduced CPT—ll
`hydrolysis to l.3—1.(i% of control values. Pliysostiginiiie, a known
`inhibitor of butyryleholinesterases (E.C. 3.1.1.8. closely related to
`carboxylesterases) showed significant (77% decrease, (P < 0.05))
`inhibition ofCPT—l 1 hydrolysis, in accord with previous data (6,
`1 1).
`Probenecid (25 ,u.M) showed a significant (13%, P < 0.01) inhibition
`ofCPT-11 hydrolysis, while 5-FU (25 ,uM) had no significant inhib-
`itory effect (2%, P < 0.1). Sodium fluoride (NaF), a known carboxy—
`lesterase inhibitor (10, 20) inhibited hydrolysis signilieantly (P <
`0.01) by 9% at 25 ,uM. 14% at 100 ,uM. and 35% at 481 ,uM.
`Aggressive therapy with loperairiide is used to treat CPT—11—i1i—
`duced delayed diarrhea (21). A small decrease (7.5%)
`in SN-38
`formation owing to loperainide was observed; however. the difference
`was not significantly different from control values. Ethanol at 25 and
`100 p.M had no significant effect on CPT—ll hydrolysis. Two iner-
`curibenzoate inhibitors of A-estcrases (HMB and CMB) also had no
`significant effect. Caffeine, ASA, and couiriarin all showed no sig-
`nificant effect on CPT—ll hydrolysis. Small, but statistically sigiiifi—
`cant. decreases were observed with metoelopramide (9.1% decrease.
`
`the high
`At clinically relevant hepatic concentrations of below 20 pth/1.
`aftinity, low KM isoform is the dominant contributor to C1"l'-1 1 bioactivation.
`
`J.
`P < 0.01) and acetaminophen (7% decrease, P < 0.05). Dexameth-g
`asone, cisplatin, and prochlorperazine were without significant effect:
`with 3.4%, 5.6%. and 7.0% di1Terences from control values. resnec- -
`tively.
`
`Z1ll-“"1/\l
`Effect of CPT—ll on at-Naphthyl Acetate. ii High Turnover Substrate of"
`S103
`Human Liver Carboxylesterase
`
`Fig. 4 shows initial velocity versiis substrate concentration plots
`over 40-800 p.M for microsomes from live donors. Data were lit by
`nonlinear regression to a single enzyme Miehaelis Menten equation
`giving corrected correlation coefficient (R2) values of0.94—0.98. KM
`estimates for or-NA hydrolysis were 115 I 20.8 itM (range 906-146
`,u.M. CV = 18%). 17”,“ was estimated at 1.64 I 0.45 ,u.Mol/mg/min
`(range 1.1-2.6 ,uMol/mg/min, CV : 27.3%). Linear
`regression
`(forced through the origin) of 1/1"” for or-NA hydrolysis Vi’.‘l".\'ti‘.\' 1/1"”
`for low and high K.” isoforms of the CPT—ll hydrolase afforded
`correlation coefficients of 0.94 with the CPT—1 1 high KM isoform and
`a lower correlation coefficient of0.82 for the CPT-1 1 low KM isoforin
`in five donors. The narrow range of I/mi“ for the CPT—ll
`low KM
`isoforin and the limited number of donors (N = 4, range 2.41-2.61),
`may be responsible for the lower correlation coefficient. A lai'gei'
`sample size would be needed to conclude that CPT—ll and as-N/\
`carboxylesterase activities are correlated.
`At or—l\lA concentrations of 0 to 100 itM, hydrolysis was almost
`completely inhibited by positive control carboxylesterase inhibitors.
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2059 - 0004
`
`

`
`BIOACTIVATION OF CPT-11 BY CARBOXYLESTERASES
`
`1161
`
`‘I60
`
`140
`
`-[go
`
`+ Dune! ITF
`~—I— DcrIm1E11‘.ll
`-5- D-1iIar2'4M
`-as-Donor 14F
`
`8Jo's[nLuno_l19ds19.'p
`
`0
`
`200
`
`400
`
`600
`
`E00
`
`9
`[51 (“M1
`s_
`Flt‘-. 4. E[ir'.'c‘.' rgfiu-rm,r1.Trrl1_i-‘.1 rwcrrrrc c‘m1c‘en'rr(rrr'rm (40—R.90 pol/I) on the
`E-
`i.-zitiuf vc:'m.‘.'.'.t_v u_j'.l2unmr1 fiver n1i'cru.s'un1c cu1m'yzed, curlJcJ.ryle.s‘1cru.re-
`§
`.um.’r'um.’_fiu-rimiimi rgf‘ (II-11'!!!)/I.'.ll'rJ1l.
`"1
`,
`.
`,
`Data shown are mean I SD for simultaneous quadruplicate determinations E:
`on a 96 well microplate. Results tor each well are corrected tor any measurable 5
`background hydrolysis, determined simultaneously in enzymc—l'rcc phosphate Q
`buffer. Mean data in m0. Djrnin were converted using a standard curve tog
`max"
`units otipinolfing/min for calculation ot‘K,,, and V
`
`Discussion
`CPT-ll Bioactivation
`
`The Enzymology of Carboxylesterases. To develop a clinically-
`relevant perspective on the bioactivation of CPT-ll, and to under—§=:;
`V
`stand the implications ofcarboxylestcrase inhibition on antincoplastic U3
`activity,
`it
`is necessary to understand the basic chemistry of thefi
`enzyme class. The mechanism of hydrolysis by serine hydrolases such :
`as the carboxylesterases is well understood from kinetic studies orig
`substrates and inhibitors (19). However,
`the molecular biology ofa
`carboxylesterase enzymes is only now being elucidated,
`in part be-E
`cause of technical difficulties inherent
`in subtle differences in se-5
`quencc, structure. and substrate and inhibitor specificity (22. 23). Twog
`highly similar human liver microsomal carboxylesterases were re-EL
`cently sequenced and expressed by Kroetz et at’.
`(22), and theser,
`authors conclude, based on substrate diversity.
`that additional ear-ug
`boxylesterases must exist. Kettennan et ai. have purified and charac- 3
`terized mid and low Pi human hepatic carboxylesterases and have
`proposed, based on kinetic comparison ol'dilTcrcnt donors, that poly-
`morphism occurs within each purified carboxylesterase (24. 25).
`Therefore, it is likely that more than one carboxylesterase isoform
`may contribute to CPT-1 1 bioactivation.
`CPT-ll Hydrolysis and Antitumor Activity. Although CPT-ll
`has weak antineoplastic activity of its own, a l04—fold increase in
`cytotoxicity in vitro is realized when SN-38 is released by carboxy-
`lesterases in rat serum (26). The conversion of CPT-ll to SN-38 has
`been studied in a wide variety of tissues, cell
`lines, and purified
`enzyme preparations in vitrr) (5— 8,
`1 1, 27). The sensitivity of prolif-
`crating tissues or cell lines to the cytotoxic effects of CPT-1 1 may be
`related their carboxylesterase levels (27). Some studies indicate that
`tumor levels ofca1'boxyleste1'ases are reduced relative to peritumo1'a1
`normal tissue (28).
`A New CPT-ll Hydrolase. Our observation of biphasic CPT-ll
`kinetics in human liver‘ microsomes suggests at least two carboxyles—
`terase isoforms are present in our human microsomal preparation. We
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2059 - 0005
`
`4— Dollar zur T:"
`
`c:
`E 100 --
`
`DE
`
`E‘
`83
`>
`
`so —
`
`60
`
`40 --
`
`20
`
`0
`
`TABLE 2
`
`Inhibitor
`
`r'r1.l'tr'n'Ji'1w‘.s' on 1.11:‘ r.‘rtr‘.’JrJ.\jv.l'r:si‘m‘rt.sr:—mm'iuJeri
`771:‘ r.j,_/]r':r.‘1 rg/i vm‘r'rm.s' pr)i‘m11i'm'
`,r1.v'r)cfim‘i'rin r1f'SN-3r5'Vfl‘rJm (“PT-H
`Mean SN—38 Mean "A. of Mean % of
`tonncd
`Control
`[,‘ontro|
`Exlmrli
`mam (ng/ml1‘l5 min)
`Ifuncorr.)
`(boiled Corr.)
`1
`17.2 I 0.1
`100
`100
`1
`16.8 + 0.1
`97.8
`97.6
`1
`15.8 I 0.2
`91.9
`91.0"
`1
`15.1 I 0.1
`87.6
`86.3"
`1
`H8IOJ
`$7
`saw
`1
`1.9 I 0.1
`10.8
`1.3"
`1
`1.9 I 0.1
`11.