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Calorimetry Studies of Chlorpromazine Hydrochloride in Solution Yoshihito David Saito, Shandiz Tehrani, Mariko M. Okamoto, Henry H. Chang, and Phoebe Dea* Department of Chemistry and Biochemistry, Occidental College, Los Angeles, California 90041 Received September 30, 1999. In Final Form: May 11, 2000 The self-association of the tranquilizer chlorpromazine hydrochloride in aqueous solution was studied. Using isothermal titration calorimetry, the critical micelle concentration and solvent-micelle interactions were evaluated as a function of temperature, ionic strength, and pH. The enthalpy of demicellization, ∆Hdemic, was directly measured from the experimental enthalpy values, which showed that the spontaneous demicellization of chlorpromazine hydrochloride is a highly endothermic process under all experimental conditions studied. The thermodynamic parameters were subsequently calculated from the temperature dependent thermograms. At 25 °C, the critical micelle concentration was determined to be 3.2 mM at pH 6.5 with a ∆Hdemic of 12.5 kJ/mol, a ∆Gdemic of 24.3 kJ/mol, and a T∆Sdemic of -10.1 kJ/mol. Our results suggest that the enthalpy-entropy compensation theory may be applied to these micelles. In addition, using differential scanning calorimetry, a broad endothermic demicellization peak was observed in postmicelle solutions during heating scans at approximately 45 °C. Cooling scans revealed a very broad peak centered around 40 °C that corresponds to the heat of micellization.
Introduction The pharmacological group of tranquilizers based on the phenothiazine ring system has been shown to be effective in the treatment of psychotropic disorders.1 One of the most widely studied phenothiazine derivatives is chlorpromazine hydrochloride (CPZ). CPZ has been shown to interact with various proteins such as dopamine (D2) receptors2,3 and serum proteins such as albumin.4 In addition, CPZ has been shown to perturb lipid bilayers and consequently their permeabilities.5,6 CPZ is often used as a model drug for the investigations of interactions between drug and biological systems. In particular, preferential recognition of micellar CPZ over monomeric CPZ has been suggested with albumin,4 synthetic membranes,5,7 and brain tubulin.8 Intuitively, the conformation of any molecule would play a large role in its interaction with highly selective proteins. The inherent difficulty is the detection of micelle versus monomer binding with membrane-bound proteins. As a result, significant emphasis has been placed on elucidating the physical properties of the CPZ micellization process. Due to its amphiphilicity, CPZ forms micelles in solution which would have an effect on the nature of its interactions in biological systems. A delicate balance lies between the stabilization of amphiphiles by micelle formation and the degree of steric repulsion that is encountered. This balance manifests itself in the size and shape of the micelle. Typically, micelle shapes vary from spherical to ellipsoidal * Corresponding author. (1) Ballus, C. Schizophr. Res. 1997, 28, 247. (2) Seeman, P.; Tallerico, T. Mol. Psychiatry 1998, 3, 123. (3) Snyder, A. P.; Feinberg, S. H. Proc. Natl. Acad. Sci. 1975, 72, 1899. (4) Hogg, P. J.; Winzor, D. J. Biochem. Pharmacol. 1984, 33, 1998. (5) Joshi, U. M.; Kodavanti, P. R.; Coudert, B.; Dwyer, T. M.; Mehendale, H. J. Pharmacol. Exp. Ther. 1988, 246, 150. (6) Frenzel, J.; Arnold, K.; Nuhn, P. Biochim. Biophys. Acta 1978, 507, 185. (7) Joshi, U. M.; Rao, P.; Kodavanti, S.; Lockard, V. G.; Mehendale, H. M. Biochim. Biophys. Acta. 1989, 1004, 309. (8) Cann, J. R.; Nichol, L. W.; Winzor, D. J. Mol. Pharmacol. 1981, 20, 244.
to counteract the steric repulsion of nonpolar groups. However, a micellar model was proposed for CPZ based on 1H NMR, where the phenothiazine rings overlap in a staircase-like stack, with the ring nitrogen of one molecule interacting with the aromatic ring of a contiguous molecule via a charge-transfer mechanism.9 Two staircase-like stacks may interact hydrophobically to complete the micelle. Chromatography and NMR chemical shifts determined over a wide concentration range of CPZ suggested a micellization process according to stepwise association.10,11 Premicelles have been reported by Attwood et al. on the basis of proton chemical shift data.11 It is commonly accepted that CPZ micelles proceed through a rapid stepwise association, in which micellization is marked by a gradual increase in size.12 However, the quantification of ∆Hmic becomes increasingly difficult, since all individual enthalpies must be detected and accounted for. Since micellization is a reversible process, the heat of demicellization (∆Hdemic) would correlate directly to the overall ∆Hmic. The demicellization of aggregates, in which a micelle begins to break up into its constituent monomers relatively quickly, is much more abrupt. The corresponding enthalpy of demicellization is much greater than the individual enthalpies of a stepwise aggregation, following a pseudo-first-order phase transition. The thermodynamics of micellization has been previously investigated through ab initio and various experimental methods.13,14 In this study, the self-association of (9) Dea, P. K.; Keyzer, H. In Modern Bioelectrochemistry; Gutman, F., Keyzer, H., Eds.; Plenum Press: New York, 1986; p 481. (10) Funasaki, N.; Hada, S.; Paiement, J. J. Phys. Chem. 1991, 95, 4131. (11) Attwood, D.; Waigh, R.; Blundell, R.; Bloor, D.; Thevand, A.; Boitard, E.; Dubes, J. P.; Tachiore, H. Magn. Reson. Chem. 1994, 32, 468. (12) Moroi, Y. Micelles Theoretical and Applied Aspects; Plenum Press: New York, 1992; Chapter 4. (13) Attwood, D.; Boitard, E.; Dubes, J.-P.; Tachoire, H. J. Phys. Chem. B 1997, 101, 9586. (14) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes, 2nd ed.; J. Wiley & Sons: New York, 1980; Chapters 6 and 7.
10.1021/la991297l CCC: $19.00 © 2000 American Chemical Society Published on Web 06/22/2000
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CPZ was studied first using isothermal titration calorimetry. The advantage of using titration calorimetry for micelle studies and subsequent thermodynamic evaluation is that the enthalpy values can be directly measured rather than measured through intrinsic micellization properties. It has been shown in other systems, such as octyl glucoside and sodium dodecyl sulfate, that the entropy of micelle formation approaches zero at elevated temperatures and that enthalpy effects are what ultimately drive micelle formation.15 Furthermore, the Gibbs free energy does not change over an extended temperature range. Whether or not this enthalpy-entropy compensation theory can be applied to CPZ is of particular interest, since the charge repulsion and the unique micelle shape that the phenothiazine moiety induces may play a role in determining whether the micelle formation in this system is primarily an enthalpy-driven mechanism. The effect of changing conditions, such as pH16,17and ionic strength,18-20 on the critical micelle concentration (cmc) has been previously reported. We have extended the changing experimental conditions and evaluated the cmc values. The thermodynamics of micellization was correlated with the possible structure of the CPZ micelle. In addition, differential scanning calorimetry experiments were carried out to observe directly for the first time both the micellization and demicellization processes in CPZ.
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Figure 1. Typical ITC thermogram of CPZ in 0.1 M phosphate buffer at 25 °C.
Materials and Methods 1. Chemicals and Sample Preparation. Deionized water was further purified with the Millipore Milli-Q Plus water purifier (Bedford, MA) in all experiments. Chlorpromazine hydrochloride was purchased from Sigma Chemical Co. and used without further purification. Sodium chloride and monobasic and dibasic sodium phosphate used in preparing buffer solutions were purchased from J. T. Baker. Unless noted otherwise, aqueous buffered samples were prepared in 0.1 M phosphate solution buffered at pH 6.5. Stock solutions of CPZ were prepared, stored in a refrigerator, and diluted to desired concentrations before use. In addition, all CPZ samples were wrapped in aluminum foil and kept under nitrogen to prevent free radical formation. Before each run, samples were purged with nitrogen. 2. Isothermal Titration Calorimetry. All experiments were carried out using the model 4209 titration calorimeter from Calorimetry Sciences Corporation (Provo, UT). A 250 µL injection syringe was used for all studies. Titrant concentrations of CPZ were chosen so as to exceed the estimated critical micelle concentrations during the duration of the experiment. Typical CPZ concentrations in the titration syringe were between 0.05 and 0.10 M. Typically, 30 × 8 µL injections of concentrated CPZ solution were made into a reaction vessel (1.30 mL) with a 200 s interval between each injection. The reaction vessel was filled with the corresponding solvent for all runs. All runs were allowed to equilibrate for a minimum of 4 h and stirred at a rate of 300 rpm to ensure the stability of the experiment. Data analyses were performed using ITC Data Works and Microsoft Excel. 3. Differential Scanning Calorimetry. DSC scans were made using a Calorimetry Sciences, Inc., Multicell DSC H-T Model 4100 at a scan rate of 10 °C/h. Samples were equilibrated at 20 °C for 30 min before the first heating scan to 85 °C and were subsequently cooled to 20 °C. The thermograms were analyzed and the peaks integrated using software provided by Calorimetry Sciences Corporation. (15) Paula, S.; Sues, W.; Tuchtenhagen, J.; Blume, A. J. Phys. Chem. 1995, 99, 11742. (16) Wajnberg, E.; Tabak, M.; Nussenzveig, P. A.; Lopes, C. M. B.; Louro, S. R. W. Biochim. Biophys. Acta 1988, 944, 185. (17) Attwood, D.; Natarajan, R. J. Pharm. Pharmacol. 1981, 33, 136. (18) Attwood, D.; Blundell, R.; Mosquera, V. J. Colloid Interface Sci. 1993, 157, 50. (19) Attwood, D.; Mosquera, V.; Rey, C.; Vasquez, E. J. Chem. Soc., Faraday Trans. 1991, 87, 2971. (20) Attwood, D. J. Chem. Soc., Faraday Trans. 1 1983, 79, 2669.
Figure 2. Graph of the molar enthalpy value for each titration as a function of total CPZ concentration in the reaction vessel. The first derivative plot shown below can be used to determine the cmc.
Results and Discussion Isothermal Titration Calorimetry. A typical experimental titration curve obtained from the dilution of a micellar solution of CPZ into a buffered aqueous solution at 25 °C is shown in Figure 1. Each peak on the thermogram is the heat flow resulting from the titration of a concentrated solution of CPZ into the reaction vessel. Upon dilution, at vessel concentrations below the cmc, micelles will dissociate, resulting in a heat flow. Initial injections of CPZ resulted in large heat flows due primarily to this phenomenon. Once the concentration of CPZ in the reaction vessel exceeds the cmc, the heat flow is reduced significantly and the reaction enthalpies approach a constant value. These reduced enthalpies are the result of dilution of micelles in the reaction vessel. Integration of each heat pulse over time gives the molar enthalpy values which are graphed as a function of total CPZ concentration in Figure 2. Upon extrapolation of the upper and lower portions of the curve, the difference between the pre-cmc and post-cmc integrated enthalpies gives a measure of the enthalpy of demicellization. This corresponds to 12.5 kJ/mol at 25 °C. Additionally, the cmc can be determined by plotting the first derivative of this curve.
Calorimetry Studies of Chlorpromazine Hydrochloride
Figure 3. First-derivative plots of the molar enthalpy value of each titration as a function of total CPZ concentration at different temperatures, increasing from left to right: 9.5, 21.6, 28.7, 33.6, and 40 °C.
Since micellization is not a true first-order phase transition, the critical micelle concentration occurs over a small concentration range. The maximum heat change, dq/ d[CPZ], observed at 3.2 mM in Figure 2 represents the point at which the greatest degree of monomer to micelle transition is occurring. Figure 3 shows the plot of dq/ d[CPZ] versus CPZ concentration over a range of temperatures. The magnitude of dq/d[CPZ] is directly proportional to the rate at which the monomer to micelle transition occurs. The value of dq/d[CPZ] approaches infinity for a true first-order transition. It has been suggested by Paula et al. that cmc values need to be reevaluated with respect to the critical micellar concentration range due to the deviation from the firstorder phase transition seen in surfactant systems such as octyl glucoside.15 Although Attwood has reported that CPZ follows a stepwise association model where premicelles slowly increase in size to micelles, it was shown that the association constant was high with K > 11 L mol-1.18 Using ITC, the phase transition from a monomeric to micellar solution can be qualitatively studied. In particular, it is important to address this issue, since the thermodynamic formulation in this paper is contingent upon this system exhibiting a relatively rapid phase transition. Assuming that ∆Hdemic is the greatest at the cmc, the magnitude of dq/d[CPZ] at the cmc is a good measure of the degree to which micellization exhibits a true first-order transition as well as the extent to which the CPZ micelle transition deviates from a first-order phase transition. As shown in Figure 3, dq/d[CPZ] decreases steadily with increasing temperature, indicating that, with increasing temperatures, the monomer-to-micelle transition range steadily increases. Thus, as the temperature is increased, the micellization of CPZ moves from a first-order to a pseudofirst-order phase transition. On the basis of the first-orderlike transition that CPZ undergoes, the use of the mass action model, which assumes a first-order transition, is justified over the temperature range studied. When the ITC experiments were performed over a wide range of temperatures, the temperature dependence of the cmc in buffered solution was found to be linear from 5 to 35 °C. These results are summarized in Figure 4. With increasing temperature, the critical micelle concentration of CPZ increases. The overwhelming factor in this increase in the cmc is intramolecular interactions, such as the flipping of the phenothiazine rings in the CPZ micelles. This increase in intramolecular phenomena provides a qualitative explanation for the decrease in micellar stability and consequently the increase in the cmc with increasing temperature. At temperatures above
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Figure 4. Critical micelle concentration of CPZ at various temperatures with a second cmc appearing at temperatures above 39 °C.
Figure 5. ITC thermogram of CPZ at 39 °C.
39 °C, the profile of the thermogram changed dramatically. This is shown in Figure 5. These thermograms are characterized by the initial titrations resulting in small exothermic enthalpies followed by increasingly exothermic enthalpies and ending in small dilution enthalpies. By integrating the thermograms as previously shown in Figure 2, two inflection points were observed, presumably corresponding to two distinct micellar phase transitions. The first cmc had a very small temperature dependence while the second cmc grew as temperatures were increased (see Figure 4). In addition, the demicellization enthalpies are considerably higher than the values measured at lower temperatures. These observations suggest a sudden change in the structural features of the CPZ micelles at higher temperatures. The appearance of a second cmc at higher temperatures has not been previously reported. The initial titrations of thermograms containing two cmc’s (Figure 5) yield enthalpies that are similar in magnitude to those of normal thermograms obtained at 25 °C (see Figure 1), suggesting that the same process of demicellization is occurring over this concentration range. This concentration range is from 0 to 3 mM, and the heats observed do not change with increasing temperature. At concentrations > 3 mM the titrations yield significantly higher heats. The first cmc’s in these thermograms have an exothermic ∆H typically around -26 kJ/mol, approximately twice that of the endothermic ∆Hdemic values found in normal thermograms. The second cmc occurs when the CPZ concentration approaches 5 mM, and it has a ∆Hdemic of about 39 kJ/mol at 39 °C. The final titrations yield heats comparable to those of thermograms obtained at lower temperatures, suggesting that the same process of micelle dilution is occurring after the second cmc. The net difference in ∆H between the two different cmc points is approximately 13 kJ/mol, equal in sign and magnitude to ∆Hdemic values observed at room temperature. This suggests that the net process of monomer to
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Figure 6. Solubility, 0, and cmc, 4, of CPZ at various pH levels. Table 1. Critical Micelle Concentration of CPZ as a Function of Ionc Strength at 10 °C and 30 °C [NaCl], mM
cmc(10 °C), mM
cmc(30 °C), mM
10 100 250 500
2.63 1.62 1.32 1.32
3.81 3.02 2.23 2.01
Figure 7. 7. Thermodynamics of CPZ demicellization in aqueous solution: b, ∆Gdemic, free energy of demicellization; 9, ∆Hdemic, enthalpy of demicellization; 2, T∆Sdemic, where ∆Sdemic is the entropy of demicellization. The open symbols represent the corresponding thermodynamics parameters of CPZ in 0.1 M phosphate buffer, pH 6.5.
micelle that occurs at higher temperatures is the same as that at lower temperatures. It is likely, however, that another type of CPZ aggregate is formed between 3 and 5 mM, that is, between the first and second cmc values. Consequently, at 5 mM, this CPZ aggregate is breaking down into the typical CPZ micelles formed at lower temperatures. More studies need to be carried out to further elucidate these observations. The cmc was also measured as a function of pH to study the effect of changing the environment on the micelle stability. These results are summarized in Figure 6. The cmc was found to increase from 3.1 mM at pH 6.7 to 7.4 mM at pH 5.5. Our observed trends are in good agreement with those reported by Wajnberg et al. using stearic acid spin label solubility measurements.15 The differences in cmc determinations using different techniques have long been a subject of debate. Furthermore, the cmc can be directly correlated to the solubility of CPZ in solution, experimentally determined from UV-vis spectroscopy. It has been previously hypothesized by Wajnberg et al. that the stability of CPZ micelles is largely due to the change in the polar head charge.15 Figure 6 shows that as the pH of the solution is increased, the solubility decreases and hence the cmc of CPZ is lowered. With decreasing pH, the ratio of the uncharged to charged CPZ decreases so that, at low pH, the repulsive force between the charged polar head groups would prevent micellization. Consequently, the dependence of cmc on pH follows closely that of its solubility. The cmc of CPZ was also determined as a function of ionic strength. Our results are summarized in Table 1, where the concentration of added NaCl was varied from 10 to 500 mM. As the mass action model predicts for ionic surfactants, the cmc decreases with increasing ionic strength. In the mass action model, an equilibrium between A and B exists:
n[A] + n[B] a [AB]n
(1)
where A is the amphiphile, B is the counterion, and n is the mean aggregation number. The presence of a closely associated counterion is required in ionic surfactants to avoid charge-charge repulsions in micelles. The addition of electrolytes drives the equilibrium toward micelle formation because of interaction with cationic CPZ. Our
Figure 8. Heating (top) and cooling (bottom) thermograms of a 0.01 M CPZ solution in 0.1 M phosphate buffer, pH 6.5.
results are consistent with studies by Attwood et al., who reported that, with increasing ionic strength, the derived values of the CPZ-counterion interaction decreased.18 The effect of ionic strength on the critical micelle concentration follows a similar trend at both 10 and 30 °C. In general, raising the temperature increases the cmc, while increases in ionic strength and pH lower the cmc. These cmc values are very sensitive to experimental conditions. This variance is reflected in the range of cmc values reported in the literature on CPZ. Thermodynamic Parameters. As explained earlier, the enthalpy of demicellization, ∆Hdemic, was determined for each solution by extrapolating the pre-cmc and postcmc integrated enthalpies and measuring the difference between these two extrapolated lines. The ∆Hdemic for CPZ as a function of temperature is summarized in Figure 7 in both unbuffered and buffered solutions. Despite the large difference in the cmc values between the two systems, both exhibit similar trends in the enthalpy changes with temperature. There is a downward trend in ∆Hdemic, suggesting that demicellization becomes enthalpically favored at higher temperatures. This is consistent with the fact that there is increased inter- and intramolecular motion at higher temperatures, destabilizing the micelle. Since enthalpy is largely a result of bond-making and bond-breaking processes, increasing the temperature helps to destabilize the interactions that support micelle formation, namely the π-π bond overlap and the hydrophobic interactions of the phenothiazine moiety.
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In addition to ∆Hdemic, other thermodynamic parameters can be determined using phase change thermodynamics. This treatment is valid only if micellization is truly representative of a phase change. Since it is known that the process of micellization is primarily a pseudo-phase transition, the degree to which micellization follows a true phase transition can be visualized. For CPZ, the monomer to micelle phase transition was small enough that firstorder phase transition thermodynamics can be applied. The Gibbs free energy formulation for micellizing systems must take into consideration the contribution of monomers as well as micelles to the free energy of the system. Since it is known that, at equilibrium, all components of a polydispersed solution must be in equilibrium, the Gibbs free energy of micellization can be defined by14
∆Gmic ) RT ln Xw + RT ln γ - (RT/m) ln(Xmic/m) (2) where Xw is the mole fraction of monomers at the cmc, γ is the activity coefficient at that concentration, and m is the mean number of monomers required to make up the micelle. According to Funasaki et al.,10 the mean aggregate numbers for CPZ micelles approach 40 under most conditions and the activity coefficient approaches unity at millimolar concentrations. Therefore, the ∆Gmic can be simplified to
∆Gmic ) RT ln Xw ) -RT ln(cmc′)
(3)
where Xw can be replaced by cmc′, the critical micelle concentration in mole fraction terms. Once ∆Gdemic is determined, ∆Sdemic can be readily calculated using the classical Gibbs energy relationship
∆Gdemic ) ∆Hdemic - T∆Sdemic
(4)
The entropy of demicellization, ∆Sdemic, was found to be negative at all temperatures evaluated. This is due to the solvent effect commonly found in surfactant solutions. The monomeric form of CPZ has a highly ordered solvent cage around its hydrophobic moiety and a solvent shell and counterion associated with the hydrophilic dimethylamine moiety. The formation of a CPZ micelle presents a decrease in order because the solvent cage formed around the phenothiazine disperses, resulting in a large decrease in order. The ∆Sdemic also decreased with increasing temperature. As seen in Figure 7, it follows a similar trend to that for the ∆Hdemic. At higher temperatures, demicellization proceeds with a greater gain in order (higher negative ∆Sdemic) than at lower temperatures because the transition
from micelle to monomer presents a greater reordering of the solvent cage around the phenothiazine moiety. Hence, micellization is entropically favored at higher temperatures. ∆Gdemic values do not change significantly with changes in temperature. Taking into consideration that micellization is entropically favored yet enthalpically unfavored at higher temperatures, the ∆Gdemic does not vary much, as a consequence of these two factors balancing one another. This is as predicted by the enthalpy-entropy compensation theory reported by Paula et al.,15 where the ∆Hdemic and ∆Sdemic vary in such a way that the ∆Gdemic does not change with temperature. Differential Scanning Calorimetry Studies. On the basis of the cmc and thermodynamics values obtained in the ITC experiments, differential scanning calorimetry experiments were carried out on CPZ micelles in phosphate buffer to directly study the processes involved in the micellization and demicellization processes. A typical heating and cooling scan is shown in Figure 8. As a 0.01 M sample of CPZ in phosphate buffer is heated, an endothermic peak occurs at around 45 °C, indicating that demicellization has occurred. The shape of the peak qualitatively expresses the kinetics of the demicellization process. The initial sharpness observed at the onset of the transition peak is consistent with the ITC data in which the micelle begins to break up into its constituent monomers relatively quickly. The enthalpy of demicellization corresponds to 9.1 ( 0.5 kJ/mol, a value slightly less than that obtained by ITC. Upon cooling, the relatively broad peak ranging from 46 to 36 °C reveals a ∆Hmic value of -9.3 ( 1.5 kJ/mol, approximately equal to but opposite in sign to ∆Hdemic. The larger standard deviation may be attributed to the broadness of the peak. The kinetics of micellization is much slower than that of demicellization, providing direct evidence and support for the stepwise aggregation theory in the micellization process. The relatively smaller heats of micellization are recorded as the micelle packs slowly to form the final supramolecule. These DSC results represent the first direct observation of the micellization process and confirm that the micellization and demicellization processes in CPZ are reversible with slower kinetics for micellization. Acknowledgment. This work was supported in part by grants from the National Science Foundation (NSFREU), CUR-AIURP, the ACS Petroleum Research Fund (#31300-B4), the Beckman Foundation, and the Howard Hughes Medical Institute through the Undergraduate Biological Sciences Education Program. LA991297L
