The Thermodynamic parameters of Chlorpromazine hydrochloride partitioning into Dimyrstoylphosphatidylcholine liposomes
Farah Hamad Farah
Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences,
Ajman University, Ajman P O Box 346, United Arab Emirates.
*Corresponding Author E-mail: f.hamad@ajman.ac.ae
ABSTRACT:
This study investigates the influence of wide range of concentrations of the model cationic, surface-active drug chlorpromazine hydrochloride (CPZ-HCl) partitioning into dimyrstoylphosphatidylcholine (DMPC) liposomes in phosphate buffer (pH 6.0) as a function of temperature, both below and above the phase transition temperature (Tc) of DMPC, pertinent to calculate various thermodynamic parameters using the Van’t Hoff equation. The partitioning of CPZ-HCl into DMPC liposomes was found to be concentration dependent at temperature both below and above the phase transition temperature (Tc). The temperature dependence of the equilibrium partition coefficient (K) over the temperature range 5o-40oC permitted the calculation of free energy (ΔG (W/L)), enthalpy (ΔH (W/L)) and entropy (ΔS (W/L)) of partitioning using the Van’t Hoff equation. ΔG (W/L) values for all concentrations studied were negative indicating that the partitioning of CPZ-HCl into DMPC liposomes over the temperature range of 5-40oC is predominantly entropically controlled. A linear relationship was observed between ΔS (W/L) and ΔH (W/L) both below and above the Tc. However a relatively poor correlations were observed between ΔG (W/L) and ΔH(W/L) as well as between ΔG(W/L) and ΔS(W/L). ΔG (W/L) values for all CPZ-HCl concentrations studied were negative indicating that the partitioning of CPZ-HCl into DMPC liposomes over the temperature range of 5o-40oC is predominantly entropically controlled. Log K values were observed to increase linearly as a function of log molar CPZ-HCl at concentrations known to cause anesthesia, reflecting a possible relationship between the drug anesthetic potency in-vivo and its ability to partition into phospholipid bilayers.
KEYWORDS: Partitioning, CPZ-HCl, DMPC liposomes, free energy, enthalpy, entropy.
INTRODUCTION:
Numerous effects of CPZ on cellular systems have been reported that most likely originate from its interactions with biological membranes. The well-known protective effect of CPZ on red cell osmolysis clearly involves CPZ-plasma membrane interactions, and depends on the membrane’s content of linoleate.[8] A recent study on human erythrocytes have shown that high concentrations of CPZ induce the formation of stomatocytes leading to erythrocyte hemolysis via interacting with the inner layer of erythrocyte membrane hence increasing membrane permeability.[9] The partitioning of a series of phenothizines into dimyrstoylphosphatidylcholine liposomes showed that higher partitioning was observed above the Tc and a linear relationship between the enthalpy and the entropy of partitioning was found and the partitioning was observed to be entropically controlled.[10] In this article, the partitioning of a wide range of CPZ-HCl concentrations between DMPC liposomes/phosphate buffer (pH 6.0) was examined. The partitioning was determined as a function of temperature to calculate the thermodynamic parameters using the Van’t Hoff equation. The partitioning of CPZ-HCl at concentrations known to cause anesthesia into DMPC liposomes was also examined.
MATERIALS AND METHODS:
Materials:
Synthetic dimyrstoylphosphatidylcholine (not less than 98% pure) and chlorpromazine hydrochloride were purchased from Sigma Co. Chloroform (Analar grade), sodium dihydrogen phosphate and disodium hydrogen phosphate were purchased from BDH.
Methods:
Preparation of liposomes for partitioning experiments:
An accurately weighed phospholipid or phospholipid and cholesterol mixture was dissolved in chloroform (10 g/ml) in a 50ml quick-fit round bottom flask. The chloroform was removed by rotary evaporation (Rotavapor R100. Buchi, Switzerland) at 10oC above the Tc of DMPC. CPZ-HCl at different concentrations in phosphate buffer (pH 6.0) or phosphate buffer (pH 7.4) was added to the dry phospholipid film. The flask was then swirled using a vortex mixer to form multilayer liposomes of a final phospholipid concentration of a1mg/ml. The liposomes were equilibrated at a constant speed in a water bath kept at the corresponding temperature for 48 hr protected from light. Partitioning was examined over the temperature range 5o-40oC. The equilibrium partition coefficient (K) was determined by removing aliquots of the equilibrated liposomes and separating them by centrifugation (MSE Prepspin 50 ultracentrifuge) adjusted at 50,000rpm for one hour at the corresponding temperature. The supernatant was assayed spectrophotometrically using (CE 5095, Double Beam Spectrophotometer, Cecil Co., Cambridge) for the free CPZ-HCl content at λ=252nm in phosphate buffer (pH 6.0) or phosphate buffer (pH 7.4), using a standard curve of CPZ-HCl to convert absorbance values to concentration. The amount of CPZ-HCl associated with liposomes was found by difference and K calculated. Each experiment was repeated three times (n=3).
Partitioning experiments:
The following experiments were carried out:
(i) The partitioning of CPZ-HCl over the sub-critical micelle concentration range of 5.6×10-6M to 8.44×10-3M, between DMPC liposomes/phosphate buffer (pH 6.0) over a temperature range of 5o-40oC from which the free energy (ΔG (W/L)), enthalpy (ΔH (W/L)) and entropy (ΔS (W/L)) of partitioning were calculated.
(ii) The partitioning of CPZ-HCl at concentration range (1×10-5-2×10-4) known to cause anesthesia between DMPC liposomes/phosphate buffer (pH 7.4) at 37oC.
Determination of the equilibrium partition coefficient (K), enthalpy (∆H (W/L)), free energy (∆G (W/L)) and entropy (∆S (W/L)) of partitioning.
The equilibrium partition coefficient (K):
The equilibrium partition coefficient (K) for CPZ-HCl between DMPC liposomes/phosphate buffer was calculated from the following equation:
K=Concentration of CPZ-HCl in the liposomal membrane phase (mg/ml) ×103 (1)
Concentration of CPZ-HCl in the phosphate buffer phase (mg/ml)
A factor of 103 was introduced to correct for differences in the phase volumes of the two phases.
The enthalpy (∆H (W/L)) of partitioning:
The temperature dependence of K is given by the following Van’t Hoff equation:
Ln K=constant- ∆H (W/L) (2)
RT
Where ∆H (W/L) is the enthalpy of partitioning, T is the absolute temperature and R is the gas constant (8.3143 Jmol-1K-1). ∆H (W/L) can be found from the slope of ln K vis T-1, where the slope is equal to ∆H(W/L)/R.
The free energy (∆G (W/L)) of partitioning
The free energy (∆G (W/L)) of partitioning is given by:
∆G (W/L) = -R T ln K (3)
The entropy (∆S (W/L)) of partitioning
Once ∆H (W/L) and ∆G(W/L) are known, then ∆S(W/L) can be calculated from the following equation:
∆S(W/L) = ∆H(W/L) - ∆G(W/L) (4)
T
Statistical analysis:
Statistical analysis of the results was performed using one-way analysis of variances to determine the significance of differences between groups. All values obtained and plotted in different graphs were the mean of three experiments (n= 3) with mean ± SD shown as error bars.
RESULTS AND DISCUSSION:
The concentration dependence of the equilibrium partition coefficient (K) for CPZ-HCl into DMPC liposomes was determined over the concentration range of 5×10-6 to 8.44×10-3 M as a function of temperature both below and above the phase transition temperature (Tc). K increases at low concentrations, but decreases above 2.8 ×10-4 M as a function of temperature below and above the phase transition temperature (Tc) (figure 1).
Fig. 1: Equilibrium partition coefficient (K) as a function of CPZ-HCl concentration in phosphate buffer (pH 6.0) at different temperatures.
The values obtained were the mean of three experiments (n= 3). Mean ± SD are shown as error bars.
Similar results have been reported for the partitioning of CPZ-HCl into egg phosphatidylcholine.[11] The increase in K at low CPZ-HCl concentrations indicate that the ionized form of the drug is adsorbed at the surface of the liposomal bilayers, only a relatively small fraction of the available drug may be associated with the hydrophobic interior of the bilayer. This increase may reflect a non-uniform distribution of the drug within the bilayer into a “CPZ-HCl-rich” and a “CPZ-HCl-poor” region, whereby some sites may be available for the binding of more CPZ-HCl molecules. The decrease in K at higher CPZ-HCl concentrations has been explained based on a possible pre-micellar aggregation, where the formation of these aggregates in the aqueous phase could reduce the concentration of CPZ-HCl monomers available for partitioning[12]. In addition, similar study showed that the partitioning of CPZ-HCl between red cells/buffer decreases at higher concentrations.[13] Figure 1 shows that K values increase with increasing temperature both below and above the Tc of DMPC liposomes. There is a distinct break point at or near 23oC, which corresponds to the Tc of DMPC liposomes (figure 2). The Tc is due to change in the phase of DMPC liposomes from the gel crystalline state to the liquid crystalline state.[14]. K values are also higher above the Tc compared with values below Tc. This is consistent with a similar study. [15] In addition, it was observed that the interaction of CPZ with DMPC liposomes had inherent effects on the Tc and hence, fluidity of DMPC phospholipid membranes.[16] Figure 2 shows Ln K as a function of T-1, from the slope of which, ∆H (W/L) has been calculated.
Fig. 2: Ln K of different CPZ-HCl concentrations in DMPC liposomes/phosphate buffer (pH 6.0) as a function of absolute temperature.
*Tc corresponds to the phase transition temperature. The values obtained were the mean of three experiments (n= 3). Mean ± SD are shown as error bars.
Table 1: Enthalpies (∆H (W/L)), free energies (∆G (W/L)) and entropies (∆S (W/L)) of partitioning of CPZ-HCl between DMPC liposomes/phosphate buffer (pH 6.0) below and above the phase transition temperature (Tc)
|
CPZ-HCl (M) |
Below Tc |
Above Tc |
|||||
|
∆H(W/L) (kJ.mol-1) |
∆G(W/L) (kJ.mol-1) |
∆S(W/L) (J.mol-1K-1) |
∆H(W/L) (k J.mol-1) |
∆G(W/L) (kJ.mol-1) |
∆S(W/L) (J.mol-1K-1) |
∆H(W/L) (kJ.mol-1) |
|
|
5.6×10-6 |
42.49±2.9 |
-13.94±1.3 |
199.29±5.8 |
4.47±0.6 |
-17.04±1.5 |
70.38±2.4 |
42.49±2.9 |
|
2.8×10-5 |
46.48±3.1 |
-16.66± 1.6 |
222.92±6.6 |
14.10±1.2 |
-19.94±1.9 |
111.74±3.4 |
46.48±3.1 |
|
5.6×10-5 |
39.58±2.7 |
-16.85±1.4 |
199.0±5.6 |
22.30±1.4 |
-20.47±2.0 |
139.9±4.7 |
39.58±2.7 |
|
1.4×10-4 |
29.10±2.1 |
-17.10±1.8 |
163.18±4.9 |
22.0±1.09 |
-20.40±1.9 |
139.0±4.5 |
29.10±2.1 |
|
5.6×10-4 |
36.55±2.4 |
-15.09±1,6 |
189.75±5.2 |
19.37±0.9 |
-20.74±2.2 |
131.57±3.9 |
36.55±2.4 |
|
2.8×10-3 |
91.86±4.2 |
-12.42±1.3 |
377.70±9.5 |
7.33±0.42 |
-19.82±1.6 |
88.57±2.9 |
91.86±4.2 |
|
4.22×10-3 |
71.09±3.9 |
-12.42±1.5 |
294.93±7.9 |
13.47±0.81 |
-16.33±1.3 |
97.82±3.1 |
71.09±3.9 |
|
8.44×10-3 |
110.08±4.6 |
-11.28±1.4 |
429.63±10.2 |
6.40±0.33 |
-15.61±0.9 |
71.63±2.7 |
110.08±4.6 |
∆H (W/L), ∆G (W/L) and ∆S (W/L) values, shown in Table 1 are the mean values of three experiments (n=3) with mean ± SD, for the temperature ranges studied below and above the Tc.
From table 1, it is apparent that ∆H (W/L) and ∆S (W/L) values are considerably higher below the Tc for all concentrations of CPZ-HCl studied. This reflects that CPZ-HCl causes perturbation of DMPC liposomes membrane structure on transference from the aqueous phase to a liquid crystalline state of the membrane than to the corresponding gel crystalline state of the membrane. Both ∆H (W/L) and ∆S (W/L) are positive and ∆S (W/L) increases with increasing ∆H (W/L), whereas ∆G (W/L) is negative (table 1). Below the Tc, ∆H (W/L) and ∆S (W/L) values showed no concentration dependence, whereas ∆G (W/L) values increase towards negativity at lower concentrations and decrease at higher CPZ-HCl concentrations (table 1). Above the Tc, ∆H (W/L), ∆S (W/L) and ∆G (W/L) were observed to increase at lower concentrations and decrease at higher CPZ-HCl concentrations (table 1). A linear relationship was observed between ∆H (W/L) and ∆S (W/L) both below and above the Tc (Fig 3).
Fig. 3: The entropy of partitioning as a function of the enthalpy of partitioning of CPZ-HCl between DMPC liposomes/phosphate buffer (pH 6.0) both below and above the phase transition temperature (Tc).
*R=the regression line correlation coefficient. The values obtained were the mean of three experiments (n= 3). Mean ± SD are shown as error bars.
From table 1 and figure 3, it is apparent that enthalpy and entropy values are higher below the Tc. This reflects that CPZ-HCl causes perturbation of DMPC liposomes membrane on transference from the aqueous phase to a liquid crystalline state of the membrane. Relatively poor relationships between ∆G(W/L) and ∆S (W/L) (figure 4) as well as between ∆G(W/L) and ∆H(W/L) were observed both below and above the Tc (figure 5); with correlation coefficients (r) of 0.794 and 0.809 below and above the Tc respectively for ∆G(W/L) against ∆S (W/L) and 0.781 and 0.696 below and above the Tc respectively for ∆G(W/L) against ∆H(W/L).
Fig. 4: The entropy of partitioning (∆S (W/L)) as a function of the free energy of partitioning (∆G (W/L)) for CPZ-HCl between DMPC liposomes/phosphate buffer (pH 6.0) both belowand above the phase transition temperature (Tc).
*R=the regression line correlation coefficient. The values obtained were the mean of three experiments (n= 3). Mean ± SD are shown as error bars.
Fig. 5: The enthalpy of partitioning (∆H (W/L)) as a function of the free energy of partitioning (∆G (W/L)) for CPZ-HCl between DMPC liposomes/phosphate buffer (pH 6.0) both below and above the phase transition temperature (Tc).
*R=the regression line correlation coefficient. The values obtained were the mean of three experiments (n= 3). Mean ± SD are shown as error bars.
Since ∆G (W/L) values for all concentrations were negative (table 1), it can be concluded in general that the partitioning of CPZ-HCl into DMPC liposomes over the temperature range of 5o to 40oC is non-spontaneous and entropically controlled, since the partitioning is said to be either enthalpy-dominated or entropy-dominated depending on whether ∆G (W/L) > 0 or < 0 respectively.[10] A significant entropy contribution was also observed for partition of CPZ to the neutral lipid membranes.[17] Similar thermodynamic study of fluoxetine (a cationic amphiphile) partitioning into dipalmotylphospatidylcholine liposomes revealed that the partitioning process is entropically controlled.[18] In addition, the thermodynamics study of some phenomena similar to partitioning like complex formation between chromium III and carbohydrazone (a cationic amphiphile)[19] as well as micelle formation of cetyl pyridinium pyridiym chloride (a cationic amphiphile) in sodium chloride solution were observed to be entropically controlled.[20]
Fig. 6: Log equilibrium partition coefficient of CPZ-HCl between DMPC liposomes/phosphate buffer (pH 7.4) as a function of log molar CPZ-HCl at concentrations known to cause anesthesia at 37oC. *R=the regression line correlation coefficient. The values obtained were the mean of three experiments (n= 3). Mean ± SD are shown as error bars.
A linear relationship was found between log K and log molar CPZ-HCl concentrations (figure 6). This may reflect the possible relationship between the drug anesthetic potency in-vivo and its ability to partition into phospholipid bilayers, which exhibit striking resemblance to biological membranes.
CONCLUSION:
The partitioning of CPZ-HCl into DMPC liposomes was found to be concentration dependent at temperature both below and above the Tc. A linear relationship was found between ∆H (W/L) and ∆S (W/L) and a relatively poor correlation was observed between ∆G (W/L) and ∆S (W/L) as well as between ∆G (W/L) and ∆H (W/L) below and above the Tc. The partitioning of CPZ-HCl into DMPC liposomes over the temperature range studied was found to be non-spontaneous and entropically controlled. Log K values were observed to increase linearly as a function of log molar CPZ-HCl at concentrations known to cause anesthesia reflecting a possible relationship between the drug anesthetic potency in-vivo and its ability to partition into phospholipid bilayers.
ACKNOWLEDGEMENT:
The author is grateful to the college of pharmacy, Ajman University for the provision of research facilities.
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Received on 16.01.2020 Modified on 07.03.2020
Accepted on 21.04.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2020; 13(12):5716-5720.
DOI: 10.5958/0974-360X.2020.00995.6