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Hydrolysis of Oxaliplatin—Evaluation of the Acid Dissociation Constant for the Oxalato Monodentate Complex
NOTE Hydrolysis of Oxaliplatin—Evaluation of the Acid Dissociation Constant for the Oxalato Monodentate Complex ELIN JERREMALM,1 STAFFAN EKSBORG,1,2 HANS EHRSSON1,3 1
Karolinska Pharmacy, Karolinska Hospital, SE-171 76 Stockholm, Sweden
2
Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden
3
Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden
Received 10 July 2002; revised 10 September 2002; accepted 10 September 2002
ABSTRACT: Alkaline hydrolysis of the platinum anticancer drug oxaliplatin gives the oxalato monodentate complex and the dihydrated oxaliplatin complex in two consecutive steps. The acid dissociation constant for the oxalato monodentate intermediate was determined by a kinetic approach. The pKa value was estimated as 7.23. The monodentate intermediate is assumed to rapidly react with endogenous compounds, resulting in a continuous conversion of oxaliplatin via the monodentate form. ß 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:436–438, 2003
Keywords:
kinetics; pKa; rate constant
INTRODUCTION Oxaliplatin (Figure 1), a novel platinum anticancer drug with activity in colorectal tumors, is thought to exert the cytotoxic effect by interaction with DNA.1 However, the in vitro reactivity with DNA is low.2 Oxaliplatin undergoes extensive biotransformation,1 resulting in a terminal halflife of 14 min in man.3 In plasma ultrafiltrate from cancer patients, biotransformation products with, for examples, chloride, water, methionine, and glutathione have been identified.1,4 In aqueous solutions, the oxalato ligand of oxaliplatin is detached in two consecutive steps, forming the oxalato monodentate complex and the dihydrated oxaliplatin complex (DOC) (Figure 1).5 The ring-opening step has a half-life of 16 min and the loss of the oxalato ligand occurs with a half-life of 92 min at 378C. In this study, the acid dissociation constant for the oxalato monodentate Correspondence to: Hans Ehrsson (Telephone: 46-851775326; Fax: 46-8-307346; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 92, 436–438 (2003) ß 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association
436
complex and the rate constant for the ring-closing step (k1) have been evaluated at 378C.
EXPERIMENTAL SECTION Materials Oxaliplatin was a gift from Sanofi-Synthelabo (Malvern, PA). All chemicals were obtained from commercial suppliers, of analytical grade, and used without further purification. Methods The deprotonated monodentate complex (A, Figure 1) was isolated by the following procedure. A mixture of the intermediate and DOC was formed from oxaliplatin (1.5 103 M) in 0.05 M sodium hydroxide, using incubation times of 2– 3 min at 708C. The monodentate complex was separated from DOC, and fractions were collected using a liquid chromatographic system with a Hypercarb S column (Thermo Hypersil-Keystone, Runcorn, UK) and a mobile phase containing 10% v/v methanol in 0.02 M sodium hydroxide.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 2, FEBRUARY 2003
OXALIPLATIN INTERMEDIATE ACID DISSOCIATION CONSTANT
Figure 1. Hydrolysis of oxaliplatin. k1 is the rate constant for the ring opening of the oxalato ring, k1 is the rate constant for the ring-closing reaction to oxaliplatin, ka is the acid dissociation constant, and k2 is the rate constant for the formation of DOC from the monodentate intermediate.
Photometric detection at 225 nm was employed. A chromatogram and detailed apparatus information are given in Jerremalm et al.5 The rate constants for the formation of oxaliplatin (k1, Figure 1) from the isolated monodentate complex were evaluated photometrically on a Spectronic Genesys 5 (Thermo Spectronic, Rochester, NY) and calculated by nonlinear estimation of the exponential increase of the absorbance plotted against time. HEPES buffer (0.025 M, pH 2.6, 1.00 mL) was mixed with 1.00 mL of the isolated monodentate complex, resulting in a pH of 4. The change in absorbance of oxaliplatin was measured every 1.5–4 s (254 nm) at temperatures ranging from 7.8 to 25.58C, against a blank (pH 4) consisting of 10% v/v methanol in 0.02 M sodium hydroxide (1.00 mL) and HEPES buffer (0.025 M, pH 2.6, 1.00 mL). The measurements were continued for 1–2.5 min, until the absorbance had reached a plateau. Extended least squares (ELS) nonlinear regression6 of the Arrhenius equation (k ¼ AeEa/RT) was used to fit a line to the rate constants versus temperature data, and k1 at 378C was calculated by extrapolation.
437
The observed rate constants (kobs) for the reversion of the monodentate complex to oxaliplatin in the pH range 8.3–8.8 (buffered with 0.025 M HEPES) were determined by liquid chromatography and evaluated by nonlinear regression on the exponential association of the areas under the chromatographic peaks plotted against time. Isolated monodentate fractions (1.00 mL) were mixed with an appropriate amount (0.80–1.00 mL) of 0.025 M HEPES at 378C. Samples (75 mL) were taken out every 30 or 60 s and quenched with ice cold 0.1 M sodium hydroxide (85 mL). The chromatographic system already described was used, but the mobile phase was changed to 60% v/v methanol in 0.1 M sodium hydroxide and oxaliplatin was detected at 254 nm until the peak area had reached a plateau (6–40 min). An Orion pH meter model 525 A with a pH electrode AquaPro (Orion Research, Inc., Beverly, MA) was used. The acid dissociation constant was calculated by ELS nonlinear regression, using k1 at 378C as a constant and kobs and ah (activity of hydrogen ions) as variables. The maximal fraction of the monodentate intermediate present at physiological pH was calculated using k1, k2, (from ref 5) k1, and the acid dissociation constant of the monodentate intermediate.
RESULTS AND DISCUSSION The mixed acid dissociation constant (ka) is defined by eq. 1: ka ¼
ah ½A ½HA
ð1Þ
where ah is the activity of hydrogen ions, [A] is the concentration of the deprotonated form, and [HA] is the concentration of the protonated form (Figure 1). The monodentate complex is supposed to be highly reactive only when present in its protonated form (in analogy with the monoaquated complex of cisplatin, pKa ¼ 6.56).7 The fraction of the monodentate intermediate available for ring closing to oxaliplatin can be expressed by: ½HA ½HA 1 ¼ ¼ ½HA þ ½A ½HA þ ½HAka 1 þ aka a h
ð2Þ
h
The observed rate constant (kobs) for the ringclosing reaction equals the rate constant for the ring closing of the protonated intermediate (k1) times the fraction of the monodentate intermediate present in the protonated form (eq. 2), which gives: JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 2, FEBRUARY 2003
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JERREMALM, EKSBORG, AND EHRSSON
Figure 2. Rate constants for the ring closing (k1) of the monodentate oxalato complex at 7.8–25.58C. The 95% confidence intervals are within the symbols. The solid line is given by ELS nonlinear regression analysis of the Arrhenius equation.
kobs ¼
k1 1þ
ka ah
ð3Þ
The value of k1 was evaluated at pH 4 between 7.8 and 25.58C (Figure 2). At this pH, at least 2 pH units below the expected pKa value, ah >> ka and thus kobs ¼ k1 (eq. 3). There is little risk of protonation of the oxalato ligand (pKa1 for oxalic acid ¼ 1.3). The absorbance of the monodentate intermediate did not interfere with the evaluation of k1 at the wavelength used (254 nm). DOC will not be formed during the analysis because the time spans used are extremely short compared with the rate of formation of DOC from the intermediate.5 Because of the rapid reaction at higher temperatures, the rate constant at 378C was determined as 14.6 0.7 min1 (mean SD) by extrapolation using the Arrhenius equation. The values of kobs for the reversion of the isolated monohydroxy form (A) to oxaliplatin at pH 8.3–8.8 are displayed in Figure 3. The experimental pH could not be >8.8 because the rate of
Figure 3. Observed rate constants (kobs) for the ringclosing reaction, with 95% confidence intervals, versus hydrogen ion activity (ah) at 378C. The solid line was calculated with eq. 3 by ELS nonlinear regression analysis, with k1 ¼ 14.6 min1. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 2, FEBRUARY 2003
formation of oxaliplatin was affected both by the degradation of the intermediate to DOC and hydrolysis of the formed oxaliplatin back to the intermediate. The pH could not be <8.3 because of the fast rate of the reaction. The acid dissociation constant was determined to 5.9 108, with a coefficient of variation of 3%, which gives pKa ¼ 7.23. At pH 7.4, a small fraction (maximally 0.7%) of oxaliplatin will exist as the monodentate intermediate. However, it could rapidly react with endogenous compounds, resulting in a continuous conversion of oxaliplatin via the monodentate form.
ACKNOWLEDGMENTS Nadia Merclin and colleagues at the Department of Pharmacy, BMC, Uppsala, Sweden, and Inger Wallin and Gareth Getvoldsen at the Karolinska Pharmacy are gratefully acknowledged.
REFERENCES 1. Graham MA, Lockwood GF, Greenslade D, Brienza S, Bayssas M, Gamelin E. 2000. Clinical pharmacokinetics of oxaliplatin: A critical review. Clin Cancer Res 6:1205–1218. 2. Butour JL, Mazard AM, Macquet JP. 1985. Kinetics of the reaction of cis-platinum compounds with DNA in vitro. Biochem Biophys Res Commun 133:347–353. 3. Ehrsson H, Wallin I, Yachnin J. 2002. Pharmacokinetics of oxaliplatin in man—correlation between in vivo clearance and degradation rate in whole blood. Presented at the 12th ICACT, Paris, France. Abstract. 4. Allain P, Heudi O, Cailleux A, Le Bouil A, Larra F, Boisdron-Celle M, Gamelin E. 2000. Early biotransformations of oxaliplatin after its intravenous administration to cancer patients. Drug Metab Dispos 28:1379–1384. 5. Jerremalm E, Videhult P, Alvelius G, Griffiths WJ, Bergman T, Eksborg S, Ehrsson H. 2002. Alkaline hydrolysis of oxaliplatin—isolation and identification of the oxalato monodentate intermediate. J Pharm Sci, in press. 6. Peck CC, Beal SL, Sheiner LB, Nichols AI. 1984. Extended least squares nonlinear regression: a possible solution to the ‘‘choice of weights’’ problem in analysis of individual pharmacokinetic data. J Pharmacokinet Biopharm 12:545–558. 7. Andersson A, Hedenmalm H, Elfsson B, Ehrsson H. 1994. Determination of the acid dissociation constant for cis-diammineaquachloroplatinum(II) ion. A hydrolysis product of cisplatin. J Pharm Sci 83: 859–862.
Karolinska Pharmacy, Karolinska Hospital, SE-171 76 Stockholm, Sweden
2
Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden
3
Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden
Received 10 July 2002; revised 10 September 2002; accepted 10 September 2002
ABSTRACT: Alkaline hydrolysis of the platinum anticancer drug oxaliplatin gives the oxalato monodentate complex and the dihydrated oxaliplatin complex in two consecutive steps. The acid dissociation constant for the oxalato monodentate intermediate was determined by a kinetic approach. The pKa value was estimated as 7.23. The monodentate intermediate is assumed to rapidly react with endogenous compounds, resulting in a continuous conversion of oxaliplatin via the monodentate form. ß 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:436–438, 2003
Keywords:
kinetics; pKa; rate constant
INTRODUCTION Oxaliplatin (Figure 1), a novel platinum anticancer drug with activity in colorectal tumors, is thought to exert the cytotoxic effect by interaction with DNA.1 However, the in vitro reactivity with DNA is low.2 Oxaliplatin undergoes extensive biotransformation,1 resulting in a terminal halflife of 14 min in man.3 In plasma ultrafiltrate from cancer patients, biotransformation products with, for examples, chloride, water, methionine, and glutathione have been identified.1,4 In aqueous solutions, the oxalato ligand of oxaliplatin is detached in two consecutive steps, forming the oxalato monodentate complex and the dihydrated oxaliplatin complex (DOC) (Figure 1).5 The ring-opening step has a half-life of 16 min and the loss of the oxalato ligand occurs with a half-life of 92 min at 378C. In this study, the acid dissociation constant for the oxalato monodentate Correspondence to: Hans Ehrsson (Telephone: 46-851775326; Fax: 46-8-307346; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 92, 436–438 (2003) ß 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association
436
complex and the rate constant for the ring-closing step (k1) have been evaluated at 378C.
EXPERIMENTAL SECTION Materials Oxaliplatin was a gift from Sanofi-Synthelabo (Malvern, PA). All chemicals were obtained from commercial suppliers, of analytical grade, and used without further purification. Methods The deprotonated monodentate complex (A, Figure 1) was isolated by the following procedure. A mixture of the intermediate and DOC was formed from oxaliplatin (1.5 103 M) in 0.05 M sodium hydroxide, using incubation times of 2– 3 min at 708C. The monodentate complex was separated from DOC, and fractions were collected using a liquid chromatographic system with a Hypercarb S column (Thermo Hypersil-Keystone, Runcorn, UK) and a mobile phase containing 10% v/v methanol in 0.02 M sodium hydroxide.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 2, FEBRUARY 2003
OXALIPLATIN INTERMEDIATE ACID DISSOCIATION CONSTANT
Figure 1. Hydrolysis of oxaliplatin. k1 is the rate constant for the ring opening of the oxalato ring, k1 is the rate constant for the ring-closing reaction to oxaliplatin, ka is the acid dissociation constant, and k2 is the rate constant for the formation of DOC from the monodentate intermediate.
Photometric detection at 225 nm was employed. A chromatogram and detailed apparatus information are given in Jerremalm et al.5 The rate constants for the formation of oxaliplatin (k1, Figure 1) from the isolated monodentate complex were evaluated photometrically on a Spectronic Genesys 5 (Thermo Spectronic, Rochester, NY) and calculated by nonlinear estimation of the exponential increase of the absorbance plotted against time. HEPES buffer (0.025 M, pH 2.6, 1.00 mL) was mixed with 1.00 mL of the isolated monodentate complex, resulting in a pH of 4. The change in absorbance of oxaliplatin was measured every 1.5–4 s (254 nm) at temperatures ranging from 7.8 to 25.58C, against a blank (pH 4) consisting of 10% v/v methanol in 0.02 M sodium hydroxide (1.00 mL) and HEPES buffer (0.025 M, pH 2.6, 1.00 mL). The measurements were continued for 1–2.5 min, until the absorbance had reached a plateau. Extended least squares (ELS) nonlinear regression6 of the Arrhenius equation (k ¼ AeEa/RT) was used to fit a line to the rate constants versus temperature data, and k1 at 378C was calculated by extrapolation.
437
The observed rate constants (kobs) for the reversion of the monodentate complex to oxaliplatin in the pH range 8.3–8.8 (buffered with 0.025 M HEPES) were determined by liquid chromatography and evaluated by nonlinear regression on the exponential association of the areas under the chromatographic peaks plotted against time. Isolated monodentate fractions (1.00 mL) were mixed with an appropriate amount (0.80–1.00 mL) of 0.025 M HEPES at 378C. Samples (75 mL) were taken out every 30 or 60 s and quenched with ice cold 0.1 M sodium hydroxide (85 mL). The chromatographic system already described was used, but the mobile phase was changed to 60% v/v methanol in 0.1 M sodium hydroxide and oxaliplatin was detected at 254 nm until the peak area had reached a plateau (6–40 min). An Orion pH meter model 525 A with a pH electrode AquaPro (Orion Research, Inc., Beverly, MA) was used. The acid dissociation constant was calculated by ELS nonlinear regression, using k1 at 378C as a constant and kobs and ah (activity of hydrogen ions) as variables. The maximal fraction of the monodentate intermediate present at physiological pH was calculated using k1, k2, (from ref 5) k1, and the acid dissociation constant of the monodentate intermediate.
RESULTS AND DISCUSSION The mixed acid dissociation constant (ka) is defined by eq. 1: ka ¼
ah ½A ½HA
ð1Þ
where ah is the activity of hydrogen ions, [A] is the concentration of the deprotonated form, and [HA] is the concentration of the protonated form (Figure 1). The monodentate complex is supposed to be highly reactive only when present in its protonated form (in analogy with the monoaquated complex of cisplatin, pKa ¼ 6.56).7 The fraction of the monodentate intermediate available for ring closing to oxaliplatin can be expressed by: ½HA ½HA 1 ¼ ¼ ½HA þ ½A ½HA þ ½HAka 1 þ aka a h
ð2Þ
h
The observed rate constant (kobs) for the ringclosing reaction equals the rate constant for the ring closing of the protonated intermediate (k1) times the fraction of the monodentate intermediate present in the protonated form (eq. 2), which gives: JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 2, FEBRUARY 2003
438
JERREMALM, EKSBORG, AND EHRSSON
Figure 2. Rate constants for the ring closing (k1) of the monodentate oxalato complex at 7.8–25.58C. The 95% confidence intervals are within the symbols. The solid line is given by ELS nonlinear regression analysis of the Arrhenius equation.
kobs ¼
k1 1þ
ka ah
ð3Þ
The value of k1 was evaluated at pH 4 between 7.8 and 25.58C (Figure 2). At this pH, at least 2 pH units below the expected pKa value, ah >> ka and thus kobs ¼ k1 (eq. 3). There is little risk of protonation of the oxalato ligand (pKa1 for oxalic acid ¼ 1.3). The absorbance of the monodentate intermediate did not interfere with the evaluation of k1 at the wavelength used (254 nm). DOC will not be formed during the analysis because the time spans used are extremely short compared with the rate of formation of DOC from the intermediate.5 Because of the rapid reaction at higher temperatures, the rate constant at 378C was determined as 14.6 0.7 min1 (mean SD) by extrapolation using the Arrhenius equation. The values of kobs for the reversion of the isolated monohydroxy form (A) to oxaliplatin at pH 8.3–8.8 are displayed in Figure 3. The experimental pH could not be >8.8 because the rate of
Figure 3. Observed rate constants (kobs) for the ringclosing reaction, with 95% confidence intervals, versus hydrogen ion activity (ah) at 378C. The solid line was calculated with eq. 3 by ELS nonlinear regression analysis, with k1 ¼ 14.6 min1. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 2, FEBRUARY 2003
formation of oxaliplatin was affected both by the degradation of the intermediate to DOC and hydrolysis of the formed oxaliplatin back to the intermediate. The pH could not be <8.3 because of the fast rate of the reaction. The acid dissociation constant was determined to 5.9 108, with a coefficient of variation of 3%, which gives pKa ¼ 7.23. At pH 7.4, a small fraction (maximally 0.7%) of oxaliplatin will exist as the monodentate intermediate. However, it could rapidly react with endogenous compounds, resulting in a continuous conversion of oxaliplatin via the monodentate form.
ACKNOWLEDGMENTS Nadia Merclin and colleagues at the Department of Pharmacy, BMC, Uppsala, Sweden, and Inger Wallin and Gareth Getvoldsen at the Karolinska Pharmacy are gratefully acknowledged.
REFERENCES 1. Graham MA, Lockwood GF, Greenslade D, Brienza S, Bayssas M, Gamelin E. 2000. Clinical pharmacokinetics of oxaliplatin: A critical review. Clin Cancer Res 6:1205–1218. 2. Butour JL, Mazard AM, Macquet JP. 1985. Kinetics of the reaction of cis-platinum compounds with DNA in vitro. Biochem Biophys Res Commun 133:347–353. 3. Ehrsson H, Wallin I, Yachnin J. 2002. Pharmacokinetics of oxaliplatin in man—correlation between in vivo clearance and degradation rate in whole blood. Presented at the 12th ICACT, Paris, France. Abstract. 4. Allain P, Heudi O, Cailleux A, Le Bouil A, Larra F, Boisdron-Celle M, Gamelin E. 2000. Early biotransformations of oxaliplatin after its intravenous administration to cancer patients. Drug Metab Dispos 28:1379–1384. 5. Jerremalm E, Videhult P, Alvelius G, Griffiths WJ, Bergman T, Eksborg S, Ehrsson H. 2002. Alkaline hydrolysis of oxaliplatin—isolation and identification of the oxalato monodentate intermediate. J Pharm Sci, in press. 6. Peck CC, Beal SL, Sheiner LB, Nichols AI. 1984. Extended least squares nonlinear regression: a possible solution to the ‘‘choice of weights’’ problem in analysis of individual pharmacokinetic data. J Pharmacokinet Biopharm 12:545–558. 7. Andersson A, Hedenmalm H, Elfsson B, Ehrsson H. 1994. Determination of the acid dissociation constant for cis-diammineaquachloroplatinum(II) ion. A hydrolysis product of cisplatin. J Pharm Sci 83: 859–862.