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Physicochemical Characterization and Membrane Binding Properties of Camptothecin
Physicochemical Characterization and Membrane Binding Properties of Camptothecin BILGE SELVI, SANJAY PATEL, MICHALAKIS SAVVA Division of Pharmaceutical Sciences, Arnold & Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, 75 Dekalb Avenue, Brooklyn, New York 11201
Received 26 October 2007; revised 6 December 2007; accepted 10 December 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21314
ABSTRACT: The intrinsic solubility of the CPT-lactone free base in acidic pH was determined to be 3.44 and 5.11 mM at 22 and 378C, respectively. The equilibrium solubility of the drug was found to increase with increasing temperature and decreasing pH. The enhanced solubility of the drug at very low pH is attributed to the protonation of the nitrogen atom in the ring B and the increased solvency of the highly concentrated acidic media. The logarithmic value of the intrinsic partition coefficient P of the free base CPT-lactone form was estimated to be 1.65, characteristic of a molecule suitable for absorption. Association constants Kf of the drug for bilayers composed of the zwitterionic (DOPC) and the negatively charged (DOPG) were determined at acidic pH by fluorescence anisotropy to be 35.4 4.5 M1 and 93.1 11.0 M1, respectively, indicative of the CPT tendency to preferentially bind to negatively charged membranes. The results indicate that at highly acidic media, CPT is positively charged and exists at its stable lactone form of increased solubility and capacity to bind to negatively charged membranes. These features could be used to invent novel formulation strategies to optimize the antitumor activity of CPT. ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:4379–4390, 2008
Keywords: camptothecin; hydrolysis; fluorescence spectroscopy; pH-dependent partition coefficient; anisotropy
INTRODUCTION Although camptothecin (CPT), a promising antitumor agent that exerts its effect through the inhibition of topoisomerase I,1–3 was discovered in the mid sixties, the therapeutic potential of this agent has not yet been achieved due to its poor solubility and the facile conversion of the active lactone form into a less active open-ring carboxylate species at physiological pH.4–6 Various types of drug delivery systems such as liposomes, polymeric micelles, bioconjugates and microspheres have been developed in order to Correspondence to: Michalakis Savva (Telephone: 718-4881471; Fax: 718-780-4586; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 4379–4390 (2008) ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association
improve the pharmacokinetics and unleash the full therapeutic potential of CPT and other insoluble analogs.7–9 Stabilization of CPT in its lactone form has seen a limited success using carrier systems, while the efficient drug loading of CPT-lactone form from aqueous molecular dispersions is still a challenge.10,11 Thus, CPT loading into colloidal carriers requires the use of toxic organic solvents due to its very low solubility and instability in aqueous media.12 Detailed molecular characterization of CPTlactone form will provide insight and new ideas to fabricating novel suitable CPT delivery systems with optimum therapeutic action.13 Although the problems of low solubility and pH-dependent hydrolysis of CPT-lactone form are mentioned in almost all the published scientific reports, to our knowledge, no comprehensive, quantitative
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and kinetic studies of CPT temperature and pHdependence solubility and stability have ever appeared in the literature.14 The present work is focused on a detailed investigation of the physicochemical properties and the membrane binding properties of the stable lactone form of CPT in acidic media. A thermodynamic analysis of the hydrolysis kinetics of CPT into the carboxylate form at physiological pH is also presented.
MATERIALS AND METHODS Materials Camptothecin powder (99%) was purchased from LC Laboratories (Woburn, MA). Hydrochloric acid 1.0 N solution was obtained from VWR Scientific Products (Bridgeport, NJ). 1-Octanol (HPLC grade, 99%) and triethylamine (99%) were purchased from Sigma-Aldrich (St. Louis, MO). 1,2Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DOPG) were purchased from AVANTI Polar Lipids, Inc. (Alabaster, AL).
Methods Fluorescence Instrumentation Experiments were conducted on a Cary Eclipse Fluorescence spectrophotometer (Varian, Inc., Victoria, Australia) equipped with motorized polarizers and thermoelectric temperaturecontrolled (0.028C) 4-window cuvette holder. Steady-state fluorescence measurements were recorded using excitation and emission wavelengths of 400 and 518 nm, respectively, with excitation and emission slit widths adjusted at 5 nm. Anisotropy values were automatically calculated by the Cary Eclipse software (version 1.1) according to the equation r ¼ ðIVV GIVH Þ= ðIVV þ 2GIVH Þ, where IVV is the intensity of the fluorescence measured with excitation and emission polarizers in the vertical position and, IVH is the intensity measured with the emission polarizer rotated at 908. The G factor accounts for the depolarization of fluorescence due to instrumental sources. HPLC Instrumentation The lactone and the carboxylate form of CPT, during the hydrolysis studies, were separated
on a C18-column (Aligent, Zorbax, 4.6 mm 250 mm, 5 mm particle size) and their concentrations were determined using a Waters HPLC system (Milford, MA) equipped with a 717 plus autosampler, a 474 scanning fluorescence detector, an interface module and a single 515 pump, at excitation and emission wavelengths of 370 and 438 nm, respectively. The mobile phase employed, consisted of 72% water, 27% acetonitrile, and 1% triethylamine buffer which was formed by mixing 7.2 mL triethylamine with 720 mL water and adjusting the pH to 5.5 with acetic acid. The flow rate was adjusted at 1 mL/min and injection volumes were either 10 or 20 mL. The total run time was 15 min and the retention times for carboxylate and lactone were 4.07 and 11.85 min, respectively.15,16 Hydrolysis Studies Equilibrium Hydrolysis. Solutions of constant ionic strength (20 mM) were prepared at various pH by mixing appropriate volumes of 0.02 N NaHPO4 with 0.02 N citric acid. Standard curves of CPT-Lactone and Carboxylate species were prepared at different pH using CPT concentration below its maximum solubility as follows: 200 mL of either CPT-Lactone or CPT-Carboxylate were mixed from corresponding stock solutions prepared at pH 3 and pH 10, respectively, with 800 mL of ice-cold organic solvent (33:67 MeOH/ AcCN). Mixing of CPT aqueous solution with organic solvent was necessary to prevent any species interconvertion during the chromatographic analysis (the hydrolysis at pH 8.18 for lactone could not be prevented using this method; see HPLC Method Validation Section below). Subsequently, 800 mL of the aforementioned solution was mixed with 100 mL of buffer of appropriate pH. The solutions were quickly mixed with mobile phase (3:1 v/v) and immediately injected into the HPLC for analysis. It is very important to emphasize that this method can only stop the hydrolysis or the ring closure reactions for only 20–30 min in strongly acidic or alkaline media. Thus, for the preparation of standard curves and for the hydrolysis kinetic studies, samples were injected immediately in the HPLC after their mixing with the mobile phase. Samples subjected to equilibrium hydrolysis studies were prepared as follows: aliquots from stock CPT solutions in Chloroform (7.125 mg/mL) were transferred to scintillation vials and organic solvent was removed under vacuum overnight.
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The dry residues were dissolved in 5 mL phosphate-citrate buffer of varying pH to a final CPT concentration of 28.5 ng/mL and kept in a thermostated orbital shaker-type water bath for 5 days at 258C to reach equilibrium. At the end of the 5th day samples were diluted with 800 mL of 33:67 MeOH/AcCN v/v as described previously and taken for HPLC analysis. All samples were prepared in triplicate. Peak integration (AUC) was performed with the Water Millennium software (version 3.20). The concentrations of lactone and carboxylate form were individually assessed by multiplying samples fluorescence intensity with the slopes of standard curves that were prepared at corresponding pH values. The data were fitted to the following equation, f ¼A
B 1 þ eðCÞðpHDÞ
addition of the mobile phase and PBS pH 7.2. The reversal in the mixing order of solvents effectively prevented hydrolysis and ring-closure reactions during the time period of sample analysis. The molar fractions of lactone (X) were plotted against time (t) and the curves were fitted to the following equation, X ¼ Xmin þ ðX0 Xmin ÞeðktÞ
(2)
The adjustable parameter Xmin represents the molar fraction of lactone form at equilibrium whereas, X0 Xmin is the difference between the fractional concentration at zero time and at equilibrium. HPLC Method Validation
(1)
where f is either the fraction of CPT-lactone or of carboxylate salt in each sample. A, B, C, and D are the adjustable parameters of the equation determined from a nonlinear fitting of the experimental data in an iterative fashion using the PSI-plotTM (Poly Software International, version 7, New York). Initial estimates for A, B were AUCmax and (AUCmax AUCmin), respectively, where AUCmax is the maximum fluorescence or average of the upper third and AUCmin represent the minimum fluorescence value or the average of the lower third of the fluorescence values of data points. The parameter D represents the pH at which 50% of CPT is present in its lactone form. Initial value of D was taken as the average of the pH values of the cross-over area (AUCmax/2). Best fit parameters were assessed within a 95% confidence interval. Hydrolysis Kinetics. For the hydrolysis kinetics experiments, samples were prepared by diluting 100 mL of a stock solution of CPT in DMSO (0.165 mg/mL) into 50 mL PBS solution pH 7.2 thermally equilibrated at the particular temperature of the study with the aid of a water circulator. Aliquots (200 mL) were withdrawn at various time intervals and immediately mixed with 400 mL icy-cold methanol/acetonitrile (33:67% v/v) solution in a glass test tube to make a final CPT concentration of 0.11 mg/mL. Samples were temporarily stored on dry ice or immediately injected (10 mL injection volume) into the HPLC. Experiments were conducted at 25, 29, 33, and 378C. Standard curves were prepared by diluting first the CPT-lactone or CPT-carboxylate in organic solvent followed by DOI 10.1002/jps
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Retention characteristics and peak quality of CPT-lactone and carboxylate remained unaffected of pH, undoubtedly due to low ionic strength and large volumes of organic solvent/mobile phase involved in the assay. The correlation coefficients of all standard curves, was 0.99 or higher. Irregardless of buffer pH, the slopes of the linear fits were found to be similar (CPT-lactone slope is slightly higher than that of CPT-carboxylate) with only a 2% and 2.5% deviation from the average value of the Lactone and Carboxylate species, respectively (results not shown). The method was further validated with a statistical t-test that was applied when both species were present, to ensure that the mass of both species adds up to the original CPT quantity. All results reported in this study were determined to be within a 95% confidence level or higher. Under the conditions used to construct the standard curves, hydrolysis of the lactone could not be prevented at pH 8.18, thus, quantification of this species was determined by difference based on the standard curve of the CPT-carboxylate. pKa Studies Aliquots of CPT in chloroform containing 2.4 mg of drug were transferred with the aid of a glass syringe to 21 mm 70 mm borosilicate glass vials (Kimble, VWR Scientific Products) and the organic solvent was removed under high vacuum. To that, 5 mL of test solution, ranging in pH from 0.698 to 3.65, were added at room temperature, under constant stirring conditions to ensure complete dissolution of drug. The acidity of the samples was adjusted with concentrated HCl within the pH range studied. Steady-state
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fluorescence measurements were recorded as described in Fluorescence Instrumentation Section and the data were fitted to Eq. (3), within a 95% confidence interval. The pH-dependent variable, F, is the sample’s measured steady-state fluorescence whereas the adjustable parameter D represents the CPT dissociation constant, pKa. F ¼A
B 1þ
10ðCÞðpHDÞ
(3)
The pH values plotted in the figures are the calculated ones based on the final HCl concentration in the samples and not the measured ones, although an excellent correlation was found between the two for pH values higher than 1.5.
where
Papp ¼
½Cu o ½Cu o ¼ ½Ctotal w ½Cu w þ ½Ci w
(4)
Inverse values of the partition coefficient were then plotted against the calculated hydrogen concentrations, and initial estimates of the intrinsic partition coefficient P of the undissociated form of CPT-lactone and pKa, were obtained from the intercept and slope of the line, respectively, as described by Eq. (5). 1 1 1 ¼ þ ½H þ Papp P PKa
(5)
(6)
Recognize that Eq. (5) as derived from Eqs. (4) and (6), assumes that the ionized form of the drug is not soluble in the octanol phase. To obtain an estimate of the partition coefficient of the ionized form of the drug, P0 , the initial estimates of P obtained from Eq. (5), were used to fit the experimental values of apparent partition coefficient, Papp plotted against pH using Eqs. (7– 10), Papp ¼
Octanol–Water Partition Studies The octanol–water partition coefficient of CPT was determined by the shake-flask method. Briefly, aliquots from stock solutions (1.2 mM) that were prepared at different pH values using HCl, were vigorously mixed with 1-octanol (1:1 v/v ratio, 6 mL each phase) in 15 mL polypropylenemade conical tubes. Samples were allowed to equilibrate for more than 12 h at room temperature, after which time a needle attached to a 2 mL syringe was inserted through the wall of the polypropylene tube and the aqueous layers were transferred to a 10 mm 75 mm borosilicate glass tube (pyrex, Corning, Inc., NY) for content analysis using fluorescence spectroscopy as described in Fluorescence Instrumentation Section. The concentration of CPT in the aqueous phase was measured before (initial) and after (final) mixing it with octanol. The concentration of CPT in octanol was determined from the difference of the initial and final aqueous drug concentrations. The apparent partition coefficient Papp, of CPT was first determined from the concentrations of CPT in the two phases using:
½Cu o ½Cu w
P¼
P 1þ
½Hþ Ka
þ
P0 1 þ ½HKþa
(7)
where Papp ¼
½Cu o þ ½Ci o ½Cu w þ ½Ci w
P0 ¼
½Ci o ½Ci w
(8)
(9)
where, [Ci]w and [Ci]o are the concentrations of the ionized species of the drug in the aqueous and organic phase, respectively.17 Dividing all terms of Eq. (7) by 1 þ ½HKþa and performing some algebra yields, Ka Ka Papp 1 þ þ ¼ P þ P (10) ½H ½H þ
Solubility Studies Excess amount of powder CPT (10 mg) was suspended in 50 mL of dissolution media, adjusted to various pH using HCl as described in the previous Methods Section, in Kontes glassjacketed beakers thermostated with a water circulator (VWR brand, Model 1162, PolyScience, IL). The jacketed beakers were covered at all times with paraffin film to minimize solvent evaporation and the suspensions were kept under constant agitation using a magnetic stirrer. At specified time intervals, 5 mL of suspension were withdrawn from the beaker using a tuberculin syringe and immediately filtered through a sterile mixed-cellulose ester filter (25 mm diameter, 0.22 mm pore size, Whatman1, VWR Scientific Products), while simultaneously replenishing the reaction with fresh solution to maintain sink conditions. The concentrations of CPT in each
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filtered sample were determined from standard curves as described before. CPT concentrations in solution were plotted against the calculated hydronium concentration and data were fitted to Eq. (11) using linear regression analysis. The intrinsic solubility of the CPT free base [CPT] and the dissociation constant Ka, were determined from the intercept and slope of Eq. (11), respectively. CT ¼ ½CPT þ ½CPT
½H þ Ka
(11)
In a series of separate experiments the nonspecific binding of CPT-lactone on the filter was determined at variable concentrations and pH. The % nonspecific binding of the drug was found to be pH-dependent, that is, 12.3 3.3, 8.4 3.2, 4.5 2.8, and 1.6 0.4 at pH 3, 2, 1, and 0, respectively. It is important to understand that this experiment can only validate the dissolution studies qualitatively simply because the dissolution studies involve filtration of excess powder along with dissolved drug. Thus, the results shown in Figure 6 are uncorrected for possible binding of the drug on the filter. Membrane Binding Studies Concentrated DOPG and DOPC lipid dispersions (100 mg/mL) were prepared in 12 mm 100 mm borosilicate glass tubes at pH 1.5 adjusted with HCl (31.6 mM). Hydration of the lipid dispersions was effected first at 308C (Gyrotory model G76, New Brunswick Scientific, Inc., NJ) for 30 min with periodic 5–6 min vortexing, followed by a 5–7 min sonication using a bath-type sonicator, to obtain vesicles characterized by a 100–150 nm average particle size as measured by a Malvern Zetasizer Nano system (Malvern Instruments, Inc., Southborough, MA). Binding isotherms were generated by titrating drug solution (2.36 mM, pH 1.5) with increasing amounts of lipid dispersions. Sample equilibration times between lipid additions were 2–3 min. All anisotropy measurements were carried out at 258C as described in the Fluorescence instrumentation Section. Anisotropy values plotted are uncorrected for the contribution of scattered light (<2%) which was evaluated at various lipid concentrations in the absence of CPT.18–20 The binding constant Kf, was determined by fitting the anisotropy values to the hyperbolic Eq. (12) by the method of nonlinear least-square DOI 10.1002/jps
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analysis using the Psi-PlotTM version 7.01. Dr ¼
Drmax ½L Kd þ ½L
(12)
Dr ¼ rr0, and Drmax ¼ rmaxr0 where r0, r and rmax are the anisotropy values of CPT in the absence, at an intermediate and ‘‘saturated’’ concentrations (see Results and Discussion Section) of DOPC and DOPG vesicles. The dissociation constant, Kd is equal to 1/Kf and [L] denotes the lipid concentration.21–24
RESULTS AND DISCUSSION The fitted results depicted in Figure 1 suggest that below pH 4.65 the lactone structure is the stable form of CPT, whereas above pH 8.65 CPT is present exclusively at its open-ring carboxylate form. It was estimated from Eq. (1) that 50% of the CPT is present as lactone at pH 6.65, while at pH 7.2 and T ¼ 258C the equilibrium concentration of CPT was found to be 22.91% 1.3%, which is a little bit lower than the equilibrium hydrolysis concentrations reported by others.25,26 The spectroscopic properties of CPT at a pH range within which the lactone form was identified to be stable were next investigated. In accord with other reports, decreasing the pH of the solution from 3.4 to 1 resulted in a reduction of the absorption intensity at 370 nm (Fig. 2, left panel).16,27 At pH lower than 1, a shift of the excitation lmax from 370 to longer wavelengths (400–430 nm) was observed. Interestingly, the isosbestic point at around 450 nm shown in Figure 2 (right panel) could only be seen upon excitation at 400 nm, for excitation at lmax 370 nm of the acidic CPT solutions would result in spectra characterized with emission maximum wavelength centered at 430 nm. Spectral alterations were weak and unreliable and no new isosbestic points corresponding to other titratable groups were detected upon increasing the pH from 3 to 5 (not shown). Nonlinear fitting of the pH-dependent fluorescence intensity of CPT to Eq. (3) enabled an estimated pKa1 value of 1.2 (Fig. 3), suggesting that the isosbestic point observed in Figure 2 is representative of the presence of only two species, that is, unprotonated and protonated CPT at the B ring.28 Interestingly, the value of pKa determined from spectra recorded upon excitation and emission at 370 and 430 nm, was 1.79 (not shown).
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Figure 1. Equilibrium hydrolysis of CPT-lactone (&) to carboxylate ( ) as a function of pH, at ambient temperatures. The pH was adjusted to different values while keeping the ionic strength constant using combinations of sodium monophosphate/sodium citrate salt solution, as described in the Methods Section.
Obviously, the less specific absorption of the whole CPT molecule at the high energy wavelength, as opposed to the specific absorption of the quinoline moiety at the low energy wavelength, coupled with the fact that the 430 nm emission intensity
Figure 2. Fluorescence excitation (left panel) and emission spectra (right panel) of CPT at various pH, obtained at ambient temperature. The pH of CPT solutions (1.37 106 M) was adjusted with HCl. Values of pH below 1.4 were calculated as described in the Materials and Methods Section. Arrows show spectra alteration upon increasing pH while numerical values from 1 to 8 denote spectra obtained with solutions of low-tohigh pH in the following order: 0.1, 0.25, 0.47, 0.65, 0.95, 1.25, 1.65, 2.05, respectively. Emission spectra were recorded with excitation at 400 nm. All the bands are associated with an isosbestic point for excitation and emission spectra at approximately 380 and 450 nm, respectively.
Figure 3. pH-dependent fluorescence intensity of 1.37 106 M CPT. The values shown in the abscissa represent the calculated pH values. Experiments were conducted at ambient temperature with excitation and emission wavelengths set at 400 and 518 nm, respectively. Experimental values are the average fluorescence intensity readings obtained from two independent samples. Data were fitted to Eq. (3) using a nonlinear method (broken line) as described in the Materials and Methods Section. The value of the parameter C was determined to be 1.022 in accordance with the ionization theory.
of the protonated form is closed to zero, as compared to the 518 nm emission intensity which becomes maximum when the molecule is fully protonated and zero when the molecule is present as a free base, is the reason for this discrepancy in the values of pKa. Thus, the true pKa value is the one determined at excitation wavelength 400 nm. Strel’tsov et al.27 were the first to report the presence of a second dissociation constant at pH 3.6 associated with the protonation of the ring D of topotecan. In their work, they have commented that the increased basicity of the quinoline moiety of CPT molecules as compared to topotecan could actually make it very difficult to detect weak spectral changes associated with the titration of the N4 atom. The quantum mechanical calculations of Sanna et al.29 have addressed the issue of topotecan tautomerism at moderately acidic pH 5 but unfortunately did not provide any information about the protonation of the N4 atom at highly acidic pH. Although we were not able to detect a second isosbestic point indicative of the protonation of the ‘‘bridged’’ nitrogen of the D ring, we have cautiously proceeded by performing octanol–water partition experiments within 0.5 pH units from the pKa1. The results of
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these experiments as a function of pH are shown in Figure 4A. Neglecting possible partition of the protonated form of CPT in octanol, the data were fitted to Eq. (5) as described in the Methods Section, and afforded a true or intrinsic partition coefficient of the unprotonated CPT-lactone form of 65 and a Ka1 equal to 0.0676 (pKa1 ¼ 1.17). The partition experiments were subsequently performed at a wider pH range and the results were now fitted to Eq. (10), which takes into account possible partition of the protonated form of CPT into the octanol phase. Figure 4B, shows that the data were fitted ‘‘satisfactorily’’ (data at pH < 0 lie below the fitted line) using linear regression analysis with P equal to 52 and P0 equal to 3, thereby, denoting the amazing transformation of a hydrophobic molecule into the protonated hydrophilic species at pH lower than the pKa1. On the other hand, at pH > 2 only 2.25% of the drug is present in aqueous phase (log P ¼ 1.65) signifying the hydrophobic nature of the CPT molecule suitable for absorption and easy permeation into mammalian cells.30–33 For comparative purposes, the log P of the CPT-Carboxylate species was determined from octanol/water (pH 10) to be 2.49.18 The values of the true partition coefficients were then fitted on the logarithmic version of Eq. (7) and the simulated data were plotted along with the experimental ones for comparison (Fig. 4C). At extremely low pH (pH < 0; HCl > 1 M) the results deviate significantly from the ones that were simulated using the CPT pH-dependent dissociation theory in aqueous media. That is, the curve described by experimental results failed to change concavity at or below the pKa value (inflection point). It is to be noted that this behavior was independent of ionic strength, that is, it could not be reproduced by the addition of neutral electrolyte. Thus, the increased solubility of CPT can only be explained by the increased solvency of the highly concen-
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trated acidic water toward the protonated CPTlactone salt. As mentioned before, we were not able to detect any protonation of the D ring (pKa2 ¼ 3.6). As shown in Figures 4C, the apparent partition coefficient remained constant at its highest values from pH 2–3.5, suggesting that CPT does not carry a positive charge at pH close to pKa2, but it may rather exists as an enol, as shown in Figure 5, in agreement with the work of others.27,34 In accord with the octanol–water partitioning data, the equilibrium solubility of CPT in water increased with decreasing pH. The effect of the temperature on the solubility of CPT-lactone at acidic pH was also studied. It was found that increasing the temperature from 22 to 378C, resulted on average in 40% increase of the solubility at all pH tested (Fig. 6A). The effect of temperature on CPT dissolution as shown in Figure 6B also implies that the process is endothermic. The dissolution rate constant estimated from a nonlinear regression analysis of the data was highly variable and failed to show any temperature dependence. Nevertheless, it was found to be in the range of 3.3 104 s1 to 1.5 103 s1. Preliminary thermodynamic analysis of the dissolution process of CPT solutions at pH > 1 and temperatures 24, 31, and 378C, suggested that the process is indeed endothermic with an Energy of activation estimated to be about 1–10 kJ/mol (not shown). The low energy values accompanied by the quite high dissolution rate constants which are in turn very similar to diffusion coefficient values suggest that dilute solutions of CPT behave ideally and that the process of dissolution is diffusion controlled. A more extensive analysis is though required to substantiate these preliminary conclusions. To gain more insight into the process of dissolution of CPT in acidified water, the equilibrium solubility data were plotted against the
Figure 4. (A) Inverse measured partition coefficients of CPT are plotted against hydrogen ion concentration. The acidity of the solutions was adjusted as described in the Materials and Methods Section within 0.5 pH units from the pKa value. The data points were fitted (solid line) using Eq. (5), with a correlation coefficient equal to 0.9964. The intercept and the slope of the linear fit were 0.0153 (P ¼ 65) and 0.2262 (pKa ¼ 1.17), respectively. Each data point is the average standard deviation of two samples. (B) Apparent partition coefficients fitted into a line (R2 ¼ 0.9941) using Eq. (10). The slope and the intercept of the line were 52.359 and 2.963 respectively. Notice that Papp at pH < 0.3 lie below the regression line. (C) Measured apparent partition coefficients (filled diamonds) compared with simulated results (broken line) using the logarithmic version of Eq. (7). DOI 10.1002/jps
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Figure 4. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 10, OCTOBER 2008
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Figure 5. Protonation and plausible tautomerism of camptothecin in acidic pH.
hydronium concentration (Fig. 6B). The intrinsic solubility of the CPT-lactone free base at 228C was estimated from the intercept of the line using Eq. (11) to be equal to 1.14 mg/mL (3.44 106 M). As expected, the intrinsic solubility of the lactone form of CPT is much lower than the published values of 2.5 mg/mL in water35 and 3.8 mg/mL in isotonic NaCl36 at physiological pH, where CPT is present predominantly as a carboxylate salt. The pKa1 of the CPT was determined from the slope to be 1.11, in agreement with the calculated value of the direct titration and octanol–water partition studies. Knowledge of the intrinsic solubility of the CPT free base, enabled the simulations shown in Figure 6C, using a different version of Eq. (11), CT ¼ ½CPT ð1 þ 10pKa pH Þ. Interestingly, the solubility of CPT can be accurately predicted by the Henderson–Hasselbalch equation only for solutions of pH > 0. At pH 0 (1 M HCl) the solubility of CPT-lactone has increased 2.5 times from the anticipitated value, that is, from 17.8 to 45.4 mg/L. No degradation of the molecule was observed at this extremely low pH (not shown). This unexpected behavior, that was also observed during the partition experiments (Fig. 4C), is due to the combined effect of the full protonation of the CPTlactone at the B ring and the increased solvency of the highly concentrated acidic water. Obtaining an estimate of the true solubility of the salt form of CPT using a modified form of Eq. (11), CT ¼ ½CPT H þ ð1 þ 10pHpKa Þ, was not possible because of the continuously changing polarity of the solvent. The important aspect of these studies is that one could use this increased solvency of the concentrated acidic media ( 1 M HCl) to safely prepare a highly concentrated molecular dispersion of CPT-lactone and use it to load colloidal carriers without having to take recourse to the use of toxic organic solvents. The equilibrium binding constant of camptothecin and other camptothecin analogs to lipid bilayers at physiological pH was studied in the DOI 10.1002/jps
past by several investigators,23,24 but never at acidic pH. Careful inspection of the adsorption isotherms of CPT present in solutions of pH 1.5 to small unilamellar liposomes composed of DOPC and DOPG unequivocally pointed out a higher affinity of the CPT for the DOPG (Fig. 7). It is important to recognize that in order to keep the population of the fluorescence species constant, we have varied the lipid concentration while the concentration of CPT was kept constant throughout the experiment. Thus, saturation of all membrane binding sites will most likely occur during the first addition. More specifically, the molar ratio of lipid to CPT was 567/1 upon the first addition and 22973/1 at the end of the experiment. Considering an average liposome size of 120 nm, the amount of CPT associated with each individual liposome was calculated to be 228 and 6 for the lower and higher lipid concentration, respectively.37 The data were accurately fitted with a 95% confidence interval to a hyperbola using Eq. (12) to yield an estimated Kf for DOPC and DOPG equal to 35.4 4.5 M1 and 93.1 11.0 M1, respectively. The binding of CPT-lactone to larger vesicles at acidic pH was more efficient for both lipids, with DOPG to DOPC binding constants ratio equal to 2.7 (Kf(DOPC) ¼ 63 M1; Kf(DOPG) ¼ 173 M1; not shown). The cross-over area of the adsorption isotherms is reproducible irregardless of liposome size. The exact origin of it was not investigated herein, but it should not be confused with a low binding affinity with a concomitant high CPT binding capacity of the DOPC since as mentioned above, saturation of the bilayer sites to CPT should occur during the first addition and not during the last ones. Nonetheless, the higher anisotropy values observed at low concentrations of CPT with DOPC liposomes can certainly be interpreted as a tighter association of CPT with DOPC perhaps due to a deeper penetration of CPT within the switterionic DOPC bilayer as compared to negatively charged DOPG lamellae. Burke and coworkers have shown that at pH 7.4
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Figure 7. Representative adsorption isotherms of CPT to DOPG (~) and DOPC (^) small unilamellar liposomes at pH 1.5 and constant temperature at 258C. Data points presented are the mean and standard deviations obtained from triplicate experiments. Results fitted using Eq. (12), are also shown (broken line).
Figure 6. (A) Comparison of the pH-dependent solubility of CPT-lactone at temperature 228C (^) and 378C (&). (B) Plot of total solubility of CPT versus [Hþ] at 228C. The intercept and slope of the linear fit (R2 ¼ 0.995) were 6.77E-08 and 8.6E-07, respectively. (C) Measured solubility at 228C (*) plotted in comparison with simulated results (dashed line) using Eq. (11). The mole fractions were calculated using ððC VÞ=ðC V þ ðmH2 O =MH2 O ÞÞÞ where, V is the volume of the dissolution media equal to 50 mL, C is the molar concentration of CPT, and MH2 O is equal to 18.0153. The data presented are the average values obtained from two independent experiments performed at different times.
camptothecin adsorbed similarly to DMPG and DMPC small unilamelar liposomes whereas the positively charged topotecan exhibited a strong binding affinity for the negatively charged DMPG liposomes.26 Our preformulation studies suggest that at pH 1.5, CPT-lactone binds preferentially to negatively charged liposomes, in a fashion similar to the positively charged topotecan at physiological pH. More extensive formulation studies to evaluate the stability of the DOPG associated/encapsulated CPT are currently underway. A kinetic evaluation of the temperature-dependent rate of conversion of CPT-lactone to CPTcarboxylate at physiological pH is displayed in Figure 8, panels A–B, while other fitted parameters and relaxation times of the hydrolysis reaction as a function of temperature are summarized in Table 1.28,38,39 The half-life of camptothecin in PBS pH 7.2 was determined to be 18.3 8.7 min, in agreement with other published reports.24,25 The raw data fitted to Eq. (2) provided values of rates of hydrolysis, which were in turn used to construct an Arrhenius plot (Fig. 8B). From the slope of the Arrhenius equation the energy of activation of the reaction Ea, was determined to be 114.3 33.4 kJ/mol. This value was then used to predict the kinetics of CPT hydrolysis at lower temperatures. It was calculated that the half-life t½ of the reaction at pH 7.2 and 108C is 5.1 days whereas the time required for 1% of CPT-lactone to get hydrolyzed
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Figure 8. (A) Representative plot of thermodynamic evaluation of the rate of lactone ring opening for CPT in PBS buffer at pH 7.2 0.05. Molar fractions of CPTlactone were plotted as a function of time at four different temperatures (*) 378C, (&) 338C, (~) 298C, and (^) 258C (n ¼ 3). Dashed lines represent the fitted parameters as described in Materials and Methods Section. (B) The plot of ln k against 1/T is a straight line indicative that the hydrolysis reaction follows the behavior described by the Arrhenius equation. Data are the average of two independent experiments (n ¼ 2 and n ¼ 3). Table 1. Summary of the Kinetic parameters of Camptothecin in PBS Buffer pH 7.2 Ea (kJ/mol) ¼ 114.3 33.4; A (min1) ¼ 4.15 1021 5.86 1021; %CPT-lactone eq ¼ 19.9 2.8 T (8C) 25 29 33 37 10
Relaxation Time, t (min) 145.0 21.6 92.3 1.8 51.7 8.46 23.5 10.7 7.4 (days)a
a Calculated from the fitted parameters using an Arrhenius equation and the 1st order exponential Eq. (3).
to CPT-carboxylate (t99%) was determined to be equal to 1.8 h, thus allowing a sufficient time to safely handle CPT-lactone at low temperatures. DOI 10.1002/jps
1. Wall ME, Wani MC. 1991. Chemistry and antitumor activities of camptothecins. In: Potmesil M, Kohn KW, editors. DNA topoisomerases in cancer. New York: Oxford University Press. pp 93–102. 2. Muggia FM. 1995. Twenty years later: Review of clinical trials with camptothecin sodium (NSC100880). In: Potmesil M, Pinedo H, editors. Camptothecins: New anticancer agents. Boca Raton: CRC Press. p 44. 3. O’Leary J, Muggian FM. 1998. Camptothecins: A review of their development and schedules of administration. Eur J Cancer 34:1500–1508. 4. Gottlieb JA, Guarino AM, Call JB, Block JB. 1970. Preliminary pharmacological and clinical evaluation of camptothecin sodium (NCS 100880). Cancer Chemother Rep 54:461–470. 5. Wani MC, Ronman PE, Lindley JT, Wall ME. 1980. Plant antitumor agents. 18. Synthesis and biological activity of camptothecin analogs. J Med Chem 23:554–560. 6. Jaxel C, Kohn KW, Wani MC, Wall ME, Pommier Y. 1989. Structure-activity study of the actions of camptothecin derivatives on mammalian topoisomerase I: Evidence for a specific receptor site and a relation to antitumor activity. Cancer Res 49:1465–1469. 7. Tyner MK, Schiffman SR, Giannelis PE. 2003. Nanobiohybrids as delivery vehicle for camptothecin. J Control Release 95:501–514. 8. Cortesi R, Esposito E, Maietti A, Menegatti E, Nastruzzi C. 1997. Formulation study for the antitumor drug camptothecin: Liposomes, micellar solutions and a microemulsion. Int J Pharm 159:95–103. 9. Koo MO, Rubinstein I, Onyuksel HJ. 2006. Camptothecin in sterically stabilized phospholipid micelles: A novel nanomedicine. Nanosci Nanotechnol 6:2996–3000. 10. Burke TG, Gao X. 1994. Stabilization of topotecan in low pH liposomes composed of distearoylphosphatidylcholine. J Pharm Sci 7:967–969. 11. Bom D, Curran DP, Zhang J, Zimmer SG, Bevins R, Kruszewski S, Howe JN, Bingcang A, Latus LJ, Burke TG. 2001. The highly lipophilic DNA topoisomerase I inhibitor DB-67 displays elevated lactone levels in human blood and potent anticancer activity. J Control Release 74:325–333. 12. Hatefi A, Amsden B. 2002. Camptothecin delivery methods. Pharm Res 19:1389–1399. 13. Glabermen U, Rabinowitz I, Verschraegen CF. 2005. Alternative administration of camptothecin analogues. Expert Opin Drug Deliv 2:323–333. 14. Slichenmyer WJ, Rowingsky EK, Donehower RC, Kaufmann SH. 1993. The current status of camptothecin analogues as antitumor agents. J Natl Cancer Inst 85:271–291.
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15. Warner DL, Burke TG. 1997. Simple and versatile high-performance liquid chromatographic method for the simultaneous quantitation of the lactone and carboxylate forms of camptothecin anticancer drugs. J Chromatogr B Biomed Sci Appl 691:161– 171. 16. Nabiev I, Fleury F, Kudelina I, Pommier Y, Charton F, Riou JF, Alix AJP, Manfait M. 1998. Spectroscopic and biochemical characterisation of selfaggregates formed by antitumor drugs of the camptothecin family: Their possible role in the unique mode of drug action. Biochem Pharmacol 55:1163– 1174. 17. Kim JH, Lee SK, Lim JL, Shin HJ, Hong C. 2002. Preformulation studies of a novel camptothecin anticancer agent, CKD-602: Physicochemical characterization and hydrolytic equilibrium kinetics. Int J Pharm 239:207–211. 18. Peikov V, Ugwu S, Parmar M, Zhang A, Ahmad I. 2005. pH-dependent association of SN-38 with lipid bilayers of a novel liposomal formulation. Int J Pharm 299:92–99. 19. Shinitzky M, Dianoux AC, Gitler C, Weber G. 1971. Microviscosity and order in the hydrocarbon region of micelles and membranes determined with fluorescent probe. I. Synthetic micelles. Biochemistry 10:2106–2113. 20. Lentz BR, Moore BM, Barrow DA. 1979. Lightscattering effects in the measurement of membrane microviscosity with diphenylhexatriene. Biophys J 25:489–494. 21. Burke TG, Tritton TR. 1985. Structural basis of anthracycline selectivity for unilamellar phosphatidylcholine vesicles: An equilibrium binding stud. Biochemistry 24:1768–1776. 22. Burke TG, Staubus AE, Mishra AK. 1992. Liposomal stabilization of camptothecin’s lactone ring. J Am Chem Soc 114:8318–8319. 23. Mi Z, Burke GT. 1994. Differential interactions of camptothecin lactone and carboxylate forms with human blood components. Biochemistry 33:10325– 10336. 24. Burke TG, Mishra KA, Wani MC, Wall ME. 1993. Lipid bilayer partitioning and stability of camptothecin drugs. Biochemistry 32:5352–5364. 25. Chourpa I, Millot JM, Sockalingum GD, Riou JF, Manfait M. 1998. Kinetics of lactone hydrolysis in antitumor drugs of camptothecin series as studied by fluorescence spectroscopy. Biochim Biophys Acta 1379:353–366. 26. Saetern AM, Flaten GE, Brand M. 2004. A method to determine the incorporation capacity of camptothecin liposomes. AAPS Pharm Sci Tech 5:40. 27. Strel’tsov SA, Grokhovskii SL, Kudelina IA, Oleinikov VA, Zhuze AL. 2001. Interaction of topotecan,
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DNA topoisomerase I inhibitor, with doublestranded polydeoxyribonucleotides 1. Topotecan dimerization in solution. Mol Biol 35:432–441. Fassberg J, Stella JV. 1992. A kinetic and mechanictic study of the hydrolysis of camptothecin and some analogues. J Pharm Sci 81:676–684. Sanna N, Chillemi G, Grandi A, Castelli S, Desideri A, Barone VJ. 2005. New hints on the pH-driven tautomeric equilibria of the topotecan anticancer drug in aqueous solutions from an integrated spectroscopic and quantum-mechanical approach. Am Chem Soc 127:15429–15436. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. 1997. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 23:3–25. Yalkowsky SH, Valvani SC. 1981. Correlation of water solubility with octanol-water partition coefficient: A response. J Pharm Sci 70:1177–1178. Yalkowsky SH. 1999. Solubility and solubilization in aqueous media, New York, New York. Yalkowsky SH, Valvani SC, Roseman TJ. 1983. Solubility and partitioning VI: Octanol solubility and octanol-water partition coefficients. J Pharm Sci 72:866–870. Solntsev KM, Sullivan EN, Tolbert LM, Ashkenazi S, Leidderman P, Huppert D. 2004. Excited-state proton transfer reactions of 10-hydroxycamptothecin. J Am Chem Soc 126:12701–12708. Kingsbury W, Boehm J, Jakas D, Holden K, Hecht S, Gallgher G, Caranfa M, McCabe F, Faucette L, Johnson R, Hertzberg R. 1991. Synthesis of water soluble (aminoaklyl) camptothecin analogues: Inhibition of topoisomerase I and antitumor activity. J Med Chem 34:98–107. Warnecke A, Kratz F. 2003. Derivatives of camptothecin as albumin-binding prodrugs: Synthesis and antitumor efficacy. Bioconjugate Chem 14: 377–387. Enoch GH, Strittmatter P. 1979. Formation and properties of 1000-A-diameter, single-bilayer phospholipid vesicles. Proc Natl Acad Sci USA 76:145– 149. Opanasopit P, Ngawhirunpat T, Chaidedgumjorn A, Rojanarata T, Apirakaramwong A, Phongying S, Choochottiros C, Chirachanchai S. 2006. Incorporation of camptothecin into N-phthaloyl chitosan-gmPEG self-assembly micellar system. Eur J Pharm Biopharm 64:269–276. Opanasopit P, Yokoyama M, Watanabe M, Kawano K, Maitani Y, Okano T. 2005. Influence of serum and albumins from different species on stability of camptothecin loaded micelles. J Control Release 104:313–321.
DOI 10.1002/jps
Received 26 October 2007; revised 6 December 2007; accepted 10 December 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21314
ABSTRACT: The intrinsic solubility of the CPT-lactone free base in acidic pH was determined to be 3.44 and 5.11 mM at 22 and 378C, respectively. The equilibrium solubility of the drug was found to increase with increasing temperature and decreasing pH. The enhanced solubility of the drug at very low pH is attributed to the protonation of the nitrogen atom in the ring B and the increased solvency of the highly concentrated acidic media. The logarithmic value of the intrinsic partition coefficient P of the free base CPT-lactone form was estimated to be 1.65, characteristic of a molecule suitable for absorption. Association constants Kf of the drug for bilayers composed of the zwitterionic (DOPC) and the negatively charged (DOPG) were determined at acidic pH by fluorescence anisotropy to be 35.4 4.5 M1 and 93.1 11.0 M1, respectively, indicative of the CPT tendency to preferentially bind to negatively charged membranes. The results indicate that at highly acidic media, CPT is positively charged and exists at its stable lactone form of increased solubility and capacity to bind to negatively charged membranes. These features could be used to invent novel formulation strategies to optimize the antitumor activity of CPT. ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:4379–4390, 2008
Keywords: camptothecin; hydrolysis; fluorescence spectroscopy; pH-dependent partition coefficient; anisotropy
INTRODUCTION Although camptothecin (CPT), a promising antitumor agent that exerts its effect through the inhibition of topoisomerase I,1–3 was discovered in the mid sixties, the therapeutic potential of this agent has not yet been achieved due to its poor solubility and the facile conversion of the active lactone form into a less active open-ring carboxylate species at physiological pH.4–6 Various types of drug delivery systems such as liposomes, polymeric micelles, bioconjugates and microspheres have been developed in order to Correspondence to: Michalakis Savva (Telephone: 718-4881471; Fax: 718-780-4586; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 4379–4390 (2008) ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association
improve the pharmacokinetics and unleash the full therapeutic potential of CPT and other insoluble analogs.7–9 Stabilization of CPT in its lactone form has seen a limited success using carrier systems, while the efficient drug loading of CPT-lactone form from aqueous molecular dispersions is still a challenge.10,11 Thus, CPT loading into colloidal carriers requires the use of toxic organic solvents due to its very low solubility and instability in aqueous media.12 Detailed molecular characterization of CPTlactone form will provide insight and new ideas to fabricating novel suitable CPT delivery systems with optimum therapeutic action.13 Although the problems of low solubility and pH-dependent hydrolysis of CPT-lactone form are mentioned in almost all the published scientific reports, to our knowledge, no comprehensive, quantitative
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and kinetic studies of CPT temperature and pHdependence solubility and stability have ever appeared in the literature.14 The present work is focused on a detailed investigation of the physicochemical properties and the membrane binding properties of the stable lactone form of CPT in acidic media. A thermodynamic analysis of the hydrolysis kinetics of CPT into the carboxylate form at physiological pH is also presented.
MATERIALS AND METHODS Materials Camptothecin powder (99%) was purchased from LC Laboratories (Woburn, MA). Hydrochloric acid 1.0 N solution was obtained from VWR Scientific Products (Bridgeport, NJ). 1-Octanol (HPLC grade, 99%) and triethylamine (99%) were purchased from Sigma-Aldrich (St. Louis, MO). 1,2Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DOPG) were purchased from AVANTI Polar Lipids, Inc. (Alabaster, AL).
Methods Fluorescence Instrumentation Experiments were conducted on a Cary Eclipse Fluorescence spectrophotometer (Varian, Inc., Victoria, Australia) equipped with motorized polarizers and thermoelectric temperaturecontrolled (0.028C) 4-window cuvette holder. Steady-state fluorescence measurements were recorded using excitation and emission wavelengths of 400 and 518 nm, respectively, with excitation and emission slit widths adjusted at 5 nm. Anisotropy values were automatically calculated by the Cary Eclipse software (version 1.1) according to the equation r ¼ ðIVV GIVH Þ= ðIVV þ 2GIVH Þ, where IVV is the intensity of the fluorescence measured with excitation and emission polarizers in the vertical position and, IVH is the intensity measured with the emission polarizer rotated at 908. The G factor accounts for the depolarization of fluorescence due to instrumental sources. HPLC Instrumentation The lactone and the carboxylate form of CPT, during the hydrolysis studies, were separated
on a C18-column (Aligent, Zorbax, 4.6 mm 250 mm, 5 mm particle size) and their concentrations were determined using a Waters HPLC system (Milford, MA) equipped with a 717 plus autosampler, a 474 scanning fluorescence detector, an interface module and a single 515 pump, at excitation and emission wavelengths of 370 and 438 nm, respectively. The mobile phase employed, consisted of 72% water, 27% acetonitrile, and 1% triethylamine buffer which was formed by mixing 7.2 mL triethylamine with 720 mL water and adjusting the pH to 5.5 with acetic acid. The flow rate was adjusted at 1 mL/min and injection volumes were either 10 or 20 mL. The total run time was 15 min and the retention times for carboxylate and lactone were 4.07 and 11.85 min, respectively.15,16 Hydrolysis Studies Equilibrium Hydrolysis. Solutions of constant ionic strength (20 mM) were prepared at various pH by mixing appropriate volumes of 0.02 N NaHPO4 with 0.02 N citric acid. Standard curves of CPT-Lactone and Carboxylate species were prepared at different pH using CPT concentration below its maximum solubility as follows: 200 mL of either CPT-Lactone or CPT-Carboxylate were mixed from corresponding stock solutions prepared at pH 3 and pH 10, respectively, with 800 mL of ice-cold organic solvent (33:67 MeOH/ AcCN). Mixing of CPT aqueous solution with organic solvent was necessary to prevent any species interconvertion during the chromatographic analysis (the hydrolysis at pH 8.18 for lactone could not be prevented using this method; see HPLC Method Validation Section below). Subsequently, 800 mL of the aforementioned solution was mixed with 100 mL of buffer of appropriate pH. The solutions were quickly mixed with mobile phase (3:1 v/v) and immediately injected into the HPLC for analysis. It is very important to emphasize that this method can only stop the hydrolysis or the ring closure reactions for only 20–30 min in strongly acidic or alkaline media. Thus, for the preparation of standard curves and for the hydrolysis kinetic studies, samples were injected immediately in the HPLC after their mixing with the mobile phase. Samples subjected to equilibrium hydrolysis studies were prepared as follows: aliquots from stock CPT solutions in Chloroform (7.125 mg/mL) were transferred to scintillation vials and organic solvent was removed under vacuum overnight.
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The dry residues were dissolved in 5 mL phosphate-citrate buffer of varying pH to a final CPT concentration of 28.5 ng/mL and kept in a thermostated orbital shaker-type water bath for 5 days at 258C to reach equilibrium. At the end of the 5th day samples were diluted with 800 mL of 33:67 MeOH/AcCN v/v as described previously and taken for HPLC analysis. All samples were prepared in triplicate. Peak integration (AUC) was performed with the Water Millennium software (version 3.20). The concentrations of lactone and carboxylate form were individually assessed by multiplying samples fluorescence intensity with the slopes of standard curves that were prepared at corresponding pH values. The data were fitted to the following equation, f ¼A
B 1 þ eðCÞðpHDÞ
addition of the mobile phase and PBS pH 7.2. The reversal in the mixing order of solvents effectively prevented hydrolysis and ring-closure reactions during the time period of sample analysis. The molar fractions of lactone (X) were plotted against time (t) and the curves were fitted to the following equation, X ¼ Xmin þ ðX0 Xmin ÞeðktÞ
(2)
The adjustable parameter Xmin represents the molar fraction of lactone form at equilibrium whereas, X0 Xmin is the difference between the fractional concentration at zero time and at equilibrium. HPLC Method Validation
(1)
where f is either the fraction of CPT-lactone or of carboxylate salt in each sample. A, B, C, and D are the adjustable parameters of the equation determined from a nonlinear fitting of the experimental data in an iterative fashion using the PSI-plotTM (Poly Software International, version 7, New York). Initial estimates for A, B were AUCmax and (AUCmax AUCmin), respectively, where AUCmax is the maximum fluorescence or average of the upper third and AUCmin represent the minimum fluorescence value or the average of the lower third of the fluorescence values of data points. The parameter D represents the pH at which 50% of CPT is present in its lactone form. Initial value of D was taken as the average of the pH values of the cross-over area (AUCmax/2). Best fit parameters were assessed within a 95% confidence interval. Hydrolysis Kinetics. For the hydrolysis kinetics experiments, samples were prepared by diluting 100 mL of a stock solution of CPT in DMSO (0.165 mg/mL) into 50 mL PBS solution pH 7.2 thermally equilibrated at the particular temperature of the study with the aid of a water circulator. Aliquots (200 mL) were withdrawn at various time intervals and immediately mixed with 400 mL icy-cold methanol/acetonitrile (33:67% v/v) solution in a glass test tube to make a final CPT concentration of 0.11 mg/mL. Samples were temporarily stored on dry ice or immediately injected (10 mL injection volume) into the HPLC. Experiments were conducted at 25, 29, 33, and 378C. Standard curves were prepared by diluting first the CPT-lactone or CPT-carboxylate in organic solvent followed by DOI 10.1002/jps
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Retention characteristics and peak quality of CPT-lactone and carboxylate remained unaffected of pH, undoubtedly due to low ionic strength and large volumes of organic solvent/mobile phase involved in the assay. The correlation coefficients of all standard curves, was 0.99 or higher. Irregardless of buffer pH, the slopes of the linear fits were found to be similar (CPT-lactone slope is slightly higher than that of CPT-carboxylate) with only a 2% and 2.5% deviation from the average value of the Lactone and Carboxylate species, respectively (results not shown). The method was further validated with a statistical t-test that was applied when both species were present, to ensure that the mass of both species adds up to the original CPT quantity. All results reported in this study were determined to be within a 95% confidence level or higher. Under the conditions used to construct the standard curves, hydrolysis of the lactone could not be prevented at pH 8.18, thus, quantification of this species was determined by difference based on the standard curve of the CPT-carboxylate. pKa Studies Aliquots of CPT in chloroform containing 2.4 mg of drug were transferred with the aid of a glass syringe to 21 mm 70 mm borosilicate glass vials (Kimble, VWR Scientific Products) and the organic solvent was removed under high vacuum. To that, 5 mL of test solution, ranging in pH from 0.698 to 3.65, were added at room temperature, under constant stirring conditions to ensure complete dissolution of drug. The acidity of the samples was adjusted with concentrated HCl within the pH range studied. Steady-state
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fluorescence measurements were recorded as described in Fluorescence Instrumentation Section and the data were fitted to Eq. (3), within a 95% confidence interval. The pH-dependent variable, F, is the sample’s measured steady-state fluorescence whereas the adjustable parameter D represents the CPT dissociation constant, pKa. F ¼A
B 1þ
10ðCÞðpHDÞ
(3)
The pH values plotted in the figures are the calculated ones based on the final HCl concentration in the samples and not the measured ones, although an excellent correlation was found between the two for pH values higher than 1.5.
where
Papp ¼
½Cu o ½Cu o ¼ ½Ctotal w ½Cu w þ ½Ci w
(4)
Inverse values of the partition coefficient were then plotted against the calculated hydrogen concentrations, and initial estimates of the intrinsic partition coefficient P of the undissociated form of CPT-lactone and pKa, were obtained from the intercept and slope of the line, respectively, as described by Eq. (5). 1 1 1 ¼ þ ½H þ Papp P PKa
(5)
(6)
Recognize that Eq. (5) as derived from Eqs. (4) and (6), assumes that the ionized form of the drug is not soluble in the octanol phase. To obtain an estimate of the partition coefficient of the ionized form of the drug, P0 , the initial estimates of P obtained from Eq. (5), were used to fit the experimental values of apparent partition coefficient, Papp plotted against pH using Eqs. (7– 10), Papp ¼
Octanol–Water Partition Studies The octanol–water partition coefficient of CPT was determined by the shake-flask method. Briefly, aliquots from stock solutions (1.2 mM) that were prepared at different pH values using HCl, were vigorously mixed with 1-octanol (1:1 v/v ratio, 6 mL each phase) in 15 mL polypropylenemade conical tubes. Samples were allowed to equilibrate for more than 12 h at room temperature, after which time a needle attached to a 2 mL syringe was inserted through the wall of the polypropylene tube and the aqueous layers were transferred to a 10 mm 75 mm borosilicate glass tube (pyrex, Corning, Inc., NY) for content analysis using fluorescence spectroscopy as described in Fluorescence Instrumentation Section. The concentration of CPT in the aqueous phase was measured before (initial) and after (final) mixing it with octanol. The concentration of CPT in octanol was determined from the difference of the initial and final aqueous drug concentrations. The apparent partition coefficient Papp, of CPT was first determined from the concentrations of CPT in the two phases using:
½Cu o ½Cu w
P¼
P 1þ
½Hþ Ka
þ
P0 1 þ ½HKþa
(7)
where Papp ¼
½Cu o þ ½Ci o ½Cu w þ ½Ci w
P0 ¼
½Ci o ½Ci w
(8)
(9)
where, [Ci]w and [Ci]o are the concentrations of the ionized species of the drug in the aqueous and organic phase, respectively.17 Dividing all terms of Eq. (7) by 1 þ ½HKþa and performing some algebra yields, Ka Ka Papp 1 þ þ ¼ P þ P (10) ½H ½H þ
Solubility Studies Excess amount of powder CPT (10 mg) was suspended in 50 mL of dissolution media, adjusted to various pH using HCl as described in the previous Methods Section, in Kontes glassjacketed beakers thermostated with a water circulator (VWR brand, Model 1162, PolyScience, IL). The jacketed beakers were covered at all times with paraffin film to minimize solvent evaporation and the suspensions were kept under constant agitation using a magnetic stirrer. At specified time intervals, 5 mL of suspension were withdrawn from the beaker using a tuberculin syringe and immediately filtered through a sterile mixed-cellulose ester filter (25 mm diameter, 0.22 mm pore size, Whatman1, VWR Scientific Products), while simultaneously replenishing the reaction with fresh solution to maintain sink conditions. The concentrations of CPT in each
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filtered sample were determined from standard curves as described before. CPT concentrations in solution were plotted against the calculated hydronium concentration and data were fitted to Eq. (11) using linear regression analysis. The intrinsic solubility of the CPT free base [CPT] and the dissociation constant Ka, were determined from the intercept and slope of Eq. (11), respectively. CT ¼ ½CPT þ ½CPT
½H þ Ka
(11)
In a series of separate experiments the nonspecific binding of CPT-lactone on the filter was determined at variable concentrations and pH. The % nonspecific binding of the drug was found to be pH-dependent, that is, 12.3 3.3, 8.4 3.2, 4.5 2.8, and 1.6 0.4 at pH 3, 2, 1, and 0, respectively. It is important to understand that this experiment can only validate the dissolution studies qualitatively simply because the dissolution studies involve filtration of excess powder along with dissolved drug. Thus, the results shown in Figure 6 are uncorrected for possible binding of the drug on the filter. Membrane Binding Studies Concentrated DOPG and DOPC lipid dispersions (100 mg/mL) were prepared in 12 mm 100 mm borosilicate glass tubes at pH 1.5 adjusted with HCl (31.6 mM). Hydration of the lipid dispersions was effected first at 308C (Gyrotory model G76, New Brunswick Scientific, Inc., NJ) for 30 min with periodic 5–6 min vortexing, followed by a 5–7 min sonication using a bath-type sonicator, to obtain vesicles characterized by a 100–150 nm average particle size as measured by a Malvern Zetasizer Nano system (Malvern Instruments, Inc., Southborough, MA). Binding isotherms were generated by titrating drug solution (2.36 mM, pH 1.5) with increasing amounts of lipid dispersions. Sample equilibration times between lipid additions were 2–3 min. All anisotropy measurements were carried out at 258C as described in the Fluorescence instrumentation Section. Anisotropy values plotted are uncorrected for the contribution of scattered light (<2%) which was evaluated at various lipid concentrations in the absence of CPT.18–20 The binding constant Kf, was determined by fitting the anisotropy values to the hyperbolic Eq. (12) by the method of nonlinear least-square DOI 10.1002/jps
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analysis using the Psi-PlotTM version 7.01. Dr ¼
Drmax ½L Kd þ ½L
(12)
Dr ¼ rr0, and Drmax ¼ rmaxr0 where r0, r and rmax are the anisotropy values of CPT in the absence, at an intermediate and ‘‘saturated’’ concentrations (see Results and Discussion Section) of DOPC and DOPG vesicles. The dissociation constant, Kd is equal to 1/Kf and [L] denotes the lipid concentration.21–24
RESULTS AND DISCUSSION The fitted results depicted in Figure 1 suggest that below pH 4.65 the lactone structure is the stable form of CPT, whereas above pH 8.65 CPT is present exclusively at its open-ring carboxylate form. It was estimated from Eq. (1) that 50% of the CPT is present as lactone at pH 6.65, while at pH 7.2 and T ¼ 258C the equilibrium concentration of CPT was found to be 22.91% 1.3%, which is a little bit lower than the equilibrium hydrolysis concentrations reported by others.25,26 The spectroscopic properties of CPT at a pH range within which the lactone form was identified to be stable were next investigated. In accord with other reports, decreasing the pH of the solution from 3.4 to 1 resulted in a reduction of the absorption intensity at 370 nm (Fig. 2, left panel).16,27 At pH lower than 1, a shift of the excitation lmax from 370 to longer wavelengths (400–430 nm) was observed. Interestingly, the isosbestic point at around 450 nm shown in Figure 2 (right panel) could only be seen upon excitation at 400 nm, for excitation at lmax 370 nm of the acidic CPT solutions would result in spectra characterized with emission maximum wavelength centered at 430 nm. Spectral alterations were weak and unreliable and no new isosbestic points corresponding to other titratable groups were detected upon increasing the pH from 3 to 5 (not shown). Nonlinear fitting of the pH-dependent fluorescence intensity of CPT to Eq. (3) enabled an estimated pKa1 value of 1.2 (Fig. 3), suggesting that the isosbestic point observed in Figure 2 is representative of the presence of only two species, that is, unprotonated and protonated CPT at the B ring.28 Interestingly, the value of pKa determined from spectra recorded upon excitation and emission at 370 and 430 nm, was 1.79 (not shown).
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Figure 1. Equilibrium hydrolysis of CPT-lactone (&) to carboxylate ( ) as a function of pH, at ambient temperatures. The pH was adjusted to different values while keeping the ionic strength constant using combinations of sodium monophosphate/sodium citrate salt solution, as described in the Methods Section.
Obviously, the less specific absorption of the whole CPT molecule at the high energy wavelength, as opposed to the specific absorption of the quinoline moiety at the low energy wavelength, coupled with the fact that the 430 nm emission intensity
Figure 2. Fluorescence excitation (left panel) and emission spectra (right panel) of CPT at various pH, obtained at ambient temperature. The pH of CPT solutions (1.37 106 M) was adjusted with HCl. Values of pH below 1.4 were calculated as described in the Materials and Methods Section. Arrows show spectra alteration upon increasing pH while numerical values from 1 to 8 denote spectra obtained with solutions of low-tohigh pH in the following order: 0.1, 0.25, 0.47, 0.65, 0.95, 1.25, 1.65, 2.05, respectively. Emission spectra were recorded with excitation at 400 nm. All the bands are associated with an isosbestic point for excitation and emission spectra at approximately 380 and 450 nm, respectively.
Figure 3. pH-dependent fluorescence intensity of 1.37 106 M CPT. The values shown in the abscissa represent the calculated pH values. Experiments were conducted at ambient temperature with excitation and emission wavelengths set at 400 and 518 nm, respectively. Experimental values are the average fluorescence intensity readings obtained from two independent samples. Data were fitted to Eq. (3) using a nonlinear method (broken line) as described in the Materials and Methods Section. The value of the parameter C was determined to be 1.022 in accordance with the ionization theory.
of the protonated form is closed to zero, as compared to the 518 nm emission intensity which becomes maximum when the molecule is fully protonated and zero when the molecule is present as a free base, is the reason for this discrepancy in the values of pKa. Thus, the true pKa value is the one determined at excitation wavelength 400 nm. Strel’tsov et al.27 were the first to report the presence of a second dissociation constant at pH 3.6 associated with the protonation of the ring D of topotecan. In their work, they have commented that the increased basicity of the quinoline moiety of CPT molecules as compared to topotecan could actually make it very difficult to detect weak spectral changes associated with the titration of the N4 atom. The quantum mechanical calculations of Sanna et al.29 have addressed the issue of topotecan tautomerism at moderately acidic pH 5 but unfortunately did not provide any information about the protonation of the N4 atom at highly acidic pH. Although we were not able to detect a second isosbestic point indicative of the protonation of the ‘‘bridged’’ nitrogen of the D ring, we have cautiously proceeded by performing octanol–water partition experiments within 0.5 pH units from the pKa1. The results of
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these experiments as a function of pH are shown in Figure 4A. Neglecting possible partition of the protonated form of CPT in octanol, the data were fitted to Eq. (5) as described in the Methods Section, and afforded a true or intrinsic partition coefficient of the unprotonated CPT-lactone form of 65 and a Ka1 equal to 0.0676 (pKa1 ¼ 1.17). The partition experiments were subsequently performed at a wider pH range and the results were now fitted to Eq. (10), which takes into account possible partition of the protonated form of CPT into the octanol phase. Figure 4B, shows that the data were fitted ‘‘satisfactorily’’ (data at pH < 0 lie below the fitted line) using linear regression analysis with P equal to 52 and P0 equal to 3, thereby, denoting the amazing transformation of a hydrophobic molecule into the protonated hydrophilic species at pH lower than the pKa1. On the other hand, at pH > 2 only 2.25% of the drug is present in aqueous phase (log P ¼ 1.65) signifying the hydrophobic nature of the CPT molecule suitable for absorption and easy permeation into mammalian cells.30–33 For comparative purposes, the log P of the CPT-Carboxylate species was determined from octanol/water (pH 10) to be 2.49.18 The values of the true partition coefficients were then fitted on the logarithmic version of Eq. (7) and the simulated data were plotted along with the experimental ones for comparison (Fig. 4C). At extremely low pH (pH < 0; HCl > 1 M) the results deviate significantly from the ones that were simulated using the CPT pH-dependent dissociation theory in aqueous media. That is, the curve described by experimental results failed to change concavity at or below the pKa value (inflection point). It is to be noted that this behavior was independent of ionic strength, that is, it could not be reproduced by the addition of neutral electrolyte. Thus, the increased solubility of CPT can only be explained by the increased solvency of the highly concen-
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trated acidic water toward the protonated CPTlactone salt. As mentioned before, we were not able to detect any protonation of the D ring (pKa2 ¼ 3.6). As shown in Figures 4C, the apparent partition coefficient remained constant at its highest values from pH 2–3.5, suggesting that CPT does not carry a positive charge at pH close to pKa2, but it may rather exists as an enol, as shown in Figure 5, in agreement with the work of others.27,34 In accord with the octanol–water partitioning data, the equilibrium solubility of CPT in water increased with decreasing pH. The effect of the temperature on the solubility of CPT-lactone at acidic pH was also studied. It was found that increasing the temperature from 22 to 378C, resulted on average in 40% increase of the solubility at all pH tested (Fig. 6A). The effect of temperature on CPT dissolution as shown in Figure 6B also implies that the process is endothermic. The dissolution rate constant estimated from a nonlinear regression analysis of the data was highly variable and failed to show any temperature dependence. Nevertheless, it was found to be in the range of 3.3 104 s1 to 1.5 103 s1. Preliminary thermodynamic analysis of the dissolution process of CPT solutions at pH > 1 and temperatures 24, 31, and 378C, suggested that the process is indeed endothermic with an Energy of activation estimated to be about 1–10 kJ/mol (not shown). The low energy values accompanied by the quite high dissolution rate constants which are in turn very similar to diffusion coefficient values suggest that dilute solutions of CPT behave ideally and that the process of dissolution is diffusion controlled. A more extensive analysis is though required to substantiate these preliminary conclusions. To gain more insight into the process of dissolution of CPT in acidified water, the equilibrium solubility data were plotted against the
Figure 4. (A) Inverse measured partition coefficients of CPT are plotted against hydrogen ion concentration. The acidity of the solutions was adjusted as described in the Materials and Methods Section within 0.5 pH units from the pKa value. The data points were fitted (solid line) using Eq. (5), with a correlation coefficient equal to 0.9964. The intercept and the slope of the linear fit were 0.0153 (P ¼ 65) and 0.2262 (pKa ¼ 1.17), respectively. Each data point is the average standard deviation of two samples. (B) Apparent partition coefficients fitted into a line (R2 ¼ 0.9941) using Eq. (10). The slope and the intercept of the line were 52.359 and 2.963 respectively. Notice that Papp at pH < 0.3 lie below the regression line. (C) Measured apparent partition coefficients (filled diamonds) compared with simulated results (broken line) using the logarithmic version of Eq. (7). DOI 10.1002/jps
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Figure 4. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 10, OCTOBER 2008
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Figure 5. Protonation and plausible tautomerism of camptothecin in acidic pH.
hydronium concentration (Fig. 6B). The intrinsic solubility of the CPT-lactone free base at 228C was estimated from the intercept of the line using Eq. (11) to be equal to 1.14 mg/mL (3.44 106 M). As expected, the intrinsic solubility of the lactone form of CPT is much lower than the published values of 2.5 mg/mL in water35 and 3.8 mg/mL in isotonic NaCl36 at physiological pH, where CPT is present predominantly as a carboxylate salt. The pKa1 of the CPT was determined from the slope to be 1.11, in agreement with the calculated value of the direct titration and octanol–water partition studies. Knowledge of the intrinsic solubility of the CPT free base, enabled the simulations shown in Figure 6C, using a different version of Eq. (11), CT ¼ ½CPT ð1 þ 10pKa pH Þ. Interestingly, the solubility of CPT can be accurately predicted by the Henderson–Hasselbalch equation only for solutions of pH > 0. At pH 0 (1 M HCl) the solubility of CPT-lactone has increased 2.5 times from the anticipitated value, that is, from 17.8 to 45.4 mg/L. No degradation of the molecule was observed at this extremely low pH (not shown). This unexpected behavior, that was also observed during the partition experiments (Fig. 4C), is due to the combined effect of the full protonation of the CPTlactone at the B ring and the increased solvency of the highly concentrated acidic water. Obtaining an estimate of the true solubility of the salt form of CPT using a modified form of Eq. (11), CT ¼ ½CPT H þ ð1 þ 10pHpKa Þ, was not possible because of the continuously changing polarity of the solvent. The important aspect of these studies is that one could use this increased solvency of the concentrated acidic media ( 1 M HCl) to safely prepare a highly concentrated molecular dispersion of CPT-lactone and use it to load colloidal carriers without having to take recourse to the use of toxic organic solvents. The equilibrium binding constant of camptothecin and other camptothecin analogs to lipid bilayers at physiological pH was studied in the DOI 10.1002/jps
past by several investigators,23,24 but never at acidic pH. Careful inspection of the adsorption isotherms of CPT present in solutions of pH 1.5 to small unilamellar liposomes composed of DOPC and DOPG unequivocally pointed out a higher affinity of the CPT for the DOPG (Fig. 7). It is important to recognize that in order to keep the population of the fluorescence species constant, we have varied the lipid concentration while the concentration of CPT was kept constant throughout the experiment. Thus, saturation of all membrane binding sites will most likely occur during the first addition. More specifically, the molar ratio of lipid to CPT was 567/1 upon the first addition and 22973/1 at the end of the experiment. Considering an average liposome size of 120 nm, the amount of CPT associated with each individual liposome was calculated to be 228 and 6 for the lower and higher lipid concentration, respectively.37 The data were accurately fitted with a 95% confidence interval to a hyperbola using Eq. (12) to yield an estimated Kf for DOPC and DOPG equal to 35.4 4.5 M1 and 93.1 11.0 M1, respectively. The binding of CPT-lactone to larger vesicles at acidic pH was more efficient for both lipids, with DOPG to DOPC binding constants ratio equal to 2.7 (Kf(DOPC) ¼ 63 M1; Kf(DOPG) ¼ 173 M1; not shown). The cross-over area of the adsorption isotherms is reproducible irregardless of liposome size. The exact origin of it was not investigated herein, but it should not be confused with a low binding affinity with a concomitant high CPT binding capacity of the DOPC since as mentioned above, saturation of the bilayer sites to CPT should occur during the first addition and not during the last ones. Nonetheless, the higher anisotropy values observed at low concentrations of CPT with DOPC liposomes can certainly be interpreted as a tighter association of CPT with DOPC perhaps due to a deeper penetration of CPT within the switterionic DOPC bilayer as compared to negatively charged DOPG lamellae. Burke and coworkers have shown that at pH 7.4
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Figure 7. Representative adsorption isotherms of CPT to DOPG (~) and DOPC (^) small unilamellar liposomes at pH 1.5 and constant temperature at 258C. Data points presented are the mean and standard deviations obtained from triplicate experiments. Results fitted using Eq. (12), are also shown (broken line).
Figure 6. (A) Comparison of the pH-dependent solubility of CPT-lactone at temperature 228C (^) and 378C (&). (B) Plot of total solubility of CPT versus [Hþ] at 228C. The intercept and slope of the linear fit (R2 ¼ 0.995) were 6.77E-08 and 8.6E-07, respectively. (C) Measured solubility at 228C (*) plotted in comparison with simulated results (dashed line) using Eq. (11). The mole fractions were calculated using ððC VÞ=ðC V þ ðmH2 O =MH2 O ÞÞÞ where, V is the volume of the dissolution media equal to 50 mL, C is the molar concentration of CPT, and MH2 O is equal to 18.0153. The data presented are the average values obtained from two independent experiments performed at different times.
camptothecin adsorbed similarly to DMPG and DMPC small unilamelar liposomes whereas the positively charged topotecan exhibited a strong binding affinity for the negatively charged DMPG liposomes.26 Our preformulation studies suggest that at pH 1.5, CPT-lactone binds preferentially to negatively charged liposomes, in a fashion similar to the positively charged topotecan at physiological pH. More extensive formulation studies to evaluate the stability of the DOPG associated/encapsulated CPT are currently underway. A kinetic evaluation of the temperature-dependent rate of conversion of CPT-lactone to CPTcarboxylate at physiological pH is displayed in Figure 8, panels A–B, while other fitted parameters and relaxation times of the hydrolysis reaction as a function of temperature are summarized in Table 1.28,38,39 The half-life of camptothecin in PBS pH 7.2 was determined to be 18.3 8.7 min, in agreement with other published reports.24,25 The raw data fitted to Eq. (2) provided values of rates of hydrolysis, which were in turn used to construct an Arrhenius plot (Fig. 8B). From the slope of the Arrhenius equation the energy of activation of the reaction Ea, was determined to be 114.3 33.4 kJ/mol. This value was then used to predict the kinetics of CPT hydrolysis at lower temperatures. It was calculated that the half-life t½ of the reaction at pH 7.2 and 108C is 5.1 days whereas the time required for 1% of CPT-lactone to get hydrolyzed
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REFERENCES
Figure 8. (A) Representative plot of thermodynamic evaluation of the rate of lactone ring opening for CPT in PBS buffer at pH 7.2 0.05. Molar fractions of CPTlactone were plotted as a function of time at four different temperatures (*) 378C, (&) 338C, (~) 298C, and (^) 258C (n ¼ 3). Dashed lines represent the fitted parameters as described in Materials and Methods Section. (B) The plot of ln k against 1/T is a straight line indicative that the hydrolysis reaction follows the behavior described by the Arrhenius equation. Data are the average of two independent experiments (n ¼ 2 and n ¼ 3). Table 1. Summary of the Kinetic parameters of Camptothecin in PBS Buffer pH 7.2 Ea (kJ/mol) ¼ 114.3 33.4; A (min1) ¼ 4.15 1021 5.86 1021; %CPT-lactone eq ¼ 19.9 2.8 T (8C) 25 29 33 37 10
Relaxation Time, t (min) 145.0 21.6 92.3 1.8 51.7 8.46 23.5 10.7 7.4 (days)a
a Calculated from the fitted parameters using an Arrhenius equation and the 1st order exponential Eq. (3).
to CPT-carboxylate (t99%) was determined to be equal to 1.8 h, thus allowing a sufficient time to safely handle CPT-lactone at low temperatures. DOI 10.1002/jps
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