Comparison of Enhanced Solubility Profile Analysis of Thermodynamic Parameters and Pharmacokinetic Profile Related to Tamoxifen Citrate Solubilisation
Laboni Mondal, Biswajit Mukherjee*, Shreyasi Chakraborty, Sanchari Bhattacharya, Iman Ehsan, Soma Sengupta and Murari M Pal
Department of Pharmaceutical Technology, Jadavpur University, India
Submission: June 14, 2018; Published: July 09, 2018
*Corresponding author: Biswajit Mukherjee, Department of Pharmaceutical Technology, Jadavpur University, Kolkata, West Bengal, India.
How to cite this article: Mondal L, Mukherjee B,Shreyasi C, Sanchari B, Iman E, et al. Comparison of Enhanced Solubility Profiles Analysis of Thermodynamic Parameters and Pharmacokinetic Profile Related to Tamoxifen Citrate Solubilisation. Nov Appro Drug Des Dev 2018; 3(5) : 555624. DOI: 10.19080/NAPDD.2018.03.555624
The aim of this study was to investigate the improvement of solubility of a poorly water soluble drug tamoxifen citrate (TC) by various methods such as cosolvency, micellisation, and complexation. Cosolvents (ethanol, polyethylene glycol-400), surfactants [polyoxyethylene sorbitan monooleate (Tween-80), poloxamer-407 and poloxamer-188], and cyclodextrins [β-cyclodextrin (BCD) and hydroxypropyl-β-cyclodextrin (HPBCD)] were used as solubilizing agents in this study. Solubility improvement approaches showed variable degrees of solubility improvement of TC. Among the solubilizing agents used, the modified β-cyclodextrin was found to be the most effective. The solubility of TC was enhanced to 6.31 mmolL-1 in water (about 7.1 fold solubility improvement) using 0.05% m/v hydroxy propyl-β-cyclodextrin. Different thermodynamic parameters, enthalpy and entropy, were analyzed for solubility enhancement of TC with different cyclodextrins which showed enthalpy not the entropy was the driving force for TC solubilisation. The less positive enthalpy of BCD complexation than HPBCD complexation signifies the higher solubilising efficacy of HPBCD. Pharmacokinetic study was performed using HPBCD as solubility enhancer at its optimized concentration which also resulted in improved bioavailability when compared to the bioavailability obtained with free tamoxifen.
Tamoxifen citrate (TC) is an antiestrogenic drug and is first choice treatment of breast cancer in both pre- and post-menopausal women. The antiestrogenic effects may be related to its ability to compete with estrogen for binding sites in target tissues such as breast . Chemically, TC is the isomer of a triphenylethylene derivative. The chemical name is (Z) 2-[4-(1,2-diphenyl-1-butenyl) phenoxy]-N,N-dimethylethanamine-2-hydorxy-1,2,3-propane tricarboxylate. Following a single oral dose of 20 mg tamoxifen, an average peak plasma concentration of 40 μg L-1(range 35 to 45 ng ml-1) occurred in 4-7 h after dosing [2,3] and this indicates poor bioavailability of the drug. Poorly water-soluble drugs often provide limited bioavailability if dissolution is the rate-limiting step in overall oral absorption process . Since TC is poorly soluble in water (equilibrium solubility inwater at 37 °C is 0.5 mg ml-1) , it is ,therefore, important to improve its solubility to ameliorate its bioavailability .
Although there has been enormous amount of research works performed using different techniques of solubilisation,
yet a comparative profile is very scarce. In this study, the effect of different solubilisation approaches such as micellar solubilisation, complexation by cyclodextrins and cosolvency on the aqueous solubility of TC has been presented in a comparative approach. An attempt has been made to provide an insight into the mechanism of solubilisation of TC particularly by complexation based on analysing thermodynamic parameters, since complexation was found to be the most successful approach among the methods tried.
In each of the different approaches (cosolvency, micellisation,
and complexation) of solubilisation, solubility of TC was
determined by placing an excess amount of TC (10 mg) in water (5
ml), in different test tubes containing increasing concentrations
(Table1) of various cosolvents, surfactants and the complexation
agents so that the total volume in each case remained to 5 ml.
Three sets of sample vials were prepared for each particular
solubilising agent. The test tubes were shaken mechanically in
a shaker water bath at 37°C for 48 h. At equilibrium (after 2
days, as preliminary studies showed that this period of time was
sufficient to ensure saturation at 37°C), aliquots were removed,
centrifuged for 10 min at 10000 rpm. After proper dilution
with water the samples were analyzed spectrophotometrically
at 275 nm using Shimadzu UV/Vis spectrophotometer (Japan)
 taking appropriate blank solution. The cosolvents used were
ethanol, poly (ethylene glycol) 400 and glycerine. The surfactants
were polyoxyethylene sorbitan monooleate (Tween 80),
poloxamer-407 and poloxamer-188. The complexation ligands
were β-cyclodextrin (BCD) and hydroxypropyl-β-cyclodextrin
Thermodynamic analysis was performed by measuring the
solubility measurement with BCD and HPBCD concentration
(0.05% m/v to 0.5% m/v) at different temperatures (300, 310
and 320 K) (Tables 2&3). Rest of the procedure was same as the
phase solubility study. Gibbs and van’t Hoff equations were used
to estimate the thermodynamic parameters, enthalpy (ΔHo),
entropy (ΔSo) and Gibbs free energy (ΔGo).
The general form of van’t Hoff equation for calculation of
ln K = ΔSo/R -ΔHo/RT
log K= -(ΔHo/ 2.303R) 1/T + ΔSo/ 2.303R
The Gibbs equation gives the values of (ΔHo) and (ΔSo) and
therefore, the values of (ΔGo) were calculated in each case from
ΔGo= ΔHo – T ΔSo
For a plot of ln K versus 1/T, slope = –ΔHo/R and intercept =
ΔSo/R were calculated.
Where, K (equilibrium constant) represents either S (drug
inherent solubility) or K1:1 (equilibrium constant considering
1:1 complex formation). The values of S were initially estimated
from the phase solubility diagrams) .
Swiss albino mice (either sex, 25-30 g) were purchased from
registered breeders, and were given normal standard diet with
tap water ad libitum. Animals were kept under a 12 hrs light
dark cycle. The animals were maintained in this condition for
at least one week prior to the experiment. All experiments were
conducted as per the guidelines of the animal ethics committee
(AEC), Jadavpur University, Kolkata.
Animals were divided into two groups (10 animals in
each group) and fasting condition for at least 24 h prior to the
experiment. Animals from the free drug group (FD) were given
tamoxifen citrate at 10 mg/kg oral dose and animals of the test
group (CD) were given HPBCD (as a solubility enhancer at the
concentration of 0.5% w/v) along with TC in an equivalent oral
dose of 10 mg/kg. mixing in water. After oral dosing mice were
anaesthetized with diethyl ether. Blood samples were collected
by heart puncture technique at various time points from 0.25 h,
0.5 h, 1h, 2 h, 4 h, 8h and 24 h. Blood samples were centrifuged
at 6000 rpm for 5 min and plasma were collected and stored at
-80°C until further study by LC-MS/ MS.
Working stocks of TC were prepared by serial dilution in
HPLC grade methanol. Working stocks and blank plasma were
spiked to prepare calibration control (CC) and quality control
(QC) samples. Liquid liquid extraction (LLE) technique was used
for the extraction of CC, QC and test samples.
The plasma concentrations of TC in both the groups were
determined by LC-MS/MS assay using a method described by Choi
and Kang . At first, 0.05 ml butyl paraben (IS) of concentration
8μg/ml in methanol was added to 0.2 ml acetonitrile and 0.2 ml
of plasma sample. This mixture was vortexed and centrifuged
at 13,000 rpm for 10 min and 0.05 ml of the supernatant was
loaded to LC-MS/MS (LC: Shimadzu Model 20AC, MS: AB-SCIEX,
Model: API 4000, Software: Analyst 1.6) for analysis. Plasma data
were plotted against time and PK parameters were determined
by WinNonlin software (Certara,UK).
Figure 1 shows straight lines in semi-logarithmic plot of TC
solubility vs. volume fractions of the experimental cosolvents.
Solubility study of TC with different concentrations of the
cosolvents, ethanol and PEG-400 at 37°C showed that efficiency
of ethanol (15% V/V) as cosolvent was higher (6.10-fold) than
that of 15% PEG-400 (5.62-fold) compared to the inherent
solubility of TC in water.
Cosolvents are widely used in pharmaceutical industry for
solubilisation purpose. They work by reducing hydrogen bond
density of aqueous system and create a less polar environment
in bulk . This results in more solubilisation of sparingly
soluble or less soluble drug molecules. Cosolvents generally
possess non-polar regions which do not interact strongly with
water and they decrease the capability of water molecules to
squeeze out non-polar solutes from the aqueous system .
A relationship between the total drug solubility (Dtot) and cosolvent concentration (C) in a drug-cosolvent-solvent mixture
has been described by using the equation, log Dtot = log Du+
σC [12,13] where, Du and σ are drugs solubility in water and
cosolvent solubilisation power, respectively. The value of σ is
inversely correlated with the polarities of both the solute and
the cosolvent. The more non-polar the solvent and the solute, the
larger is the σ value .
Figure1 shows straight lines in semi-logarithmic plot of TC
solubility vs volume fractions of the experimental cosolvents.
The findings suggest exponential increase in TC solubility with
the increasing concentration of the cosolvent, ethanol and PEG-
400. Both ethanol and PEG-400 obey 1st
order solubilisation kinetic.
For a single non-polar solute, cosolvent solubilisation power
‘σ’ depends only on cosolvent polarity . Table 1 indicates
that solubility enhancement of TC follows the cosolvent order
as: EtOH (σ: 0.036) > PEG-400 (σ: 0.025). The less polar is the
cosolvent, the more effective it is at disrupting hydrogen bonding
interactions in water molecules . In the present study,
more efficient improvement of solubility of TC by ethanol may
be because ethanol is the less polar solvent than PEG-400 .
Dtot- total drug solubility in a mixed solvent and cosolvent concentration (C). Du- drug solubility in water. BCD: β-cyclodextrin; HPBCD: hydroxyl
Figure 2 shows the effects of poloxamer-407, poloxamer-188
and Tween 80 on solubility profiles of TC, respectively which
indicates that TC solubility was enhanced in the surfactant
order as: poloxamer-407 (5.36-fold) > Tween80 (5.30-fold) >
Micelle formation is one of the important mechanisms to
solubilise solutes. Incorporation of solute molecules to the
micelles depends on the degree of non-polarity of solutes and
their micellar partitioning performances .The more nonpolar
the solute, the more likely it is to be incorporated near
the nonpolar core or center of micelles . Researchers have
described a relationship of micellar surfactant concentration
and solubility of solute (drug) [15,16]. Total drug solubility
(Dtot) depends on inherent solubility (Du) and concentration of
micellar surfactant (S) (i.e., the total surfactant concentration
minus the critical micellar concentration) and is presented by
Dtot= Du + κ D uS, where κ is micellar partition coefficient.
Product of κ and Du reflects number of surfactant molecules
required to solubilise one solute molecule .
Table 1 indicates that TC solubility was enhanced in the
following sequences: poloxamer-407 (κ: 2.28) > Tween-80 (κ:
0.97) > poloxamer-188 (κ: 0.25). Poloxamer-407 was found to
improve the solubility of TC maximally among the surfactants
tested. Due to higher micellar partitioning, more non-polar TC
molecules were incorporated in the poloxamer-407 micelles.
Figure 3 shows a linear relationship between TC solubility
with different concentration of BCD and HPBCD used to solubilize
TC. HPBCD showed higher solubilisation of TC than natural BCD.
The solubility improvement of TC was about 5.8-fold with 0.5%
m/v BCD and 7.1-fold with 0.5% m/v HPBCD compared to the
original solubility of TC in water.
The solubility of TC with different cyclodextrins has been
described using the following equation, Dtot = Du + KDuL, where
L is the total ligand concentration, and K is the complexation
constant/ solubilisation capacity of the drug-ligand complex.
Dtot and Du have been described earlier. Table 1 shows that the
solubilisation capacity K1:1 of HPBCD (0.68) is slightly greater
than that of natural BCD (0.48). The modified β-cyclodextrins
have been widely used and reportedly have higher solubilisation
capacity than natural BCD for most drugs [17,18]. Complexation
constant or solubilising capacity, K, depends on the geometry
and polarity of the solute molecules and compatibility between
the solute and the cyclodextrin cavity .
Except for differences in size, the overall geometries of the
cyclodextrins are similar. Each having a torus equals to the
length of the appropriate number of glycosides. The derivatized
cyclodextrins are characterized by the nature, position and
degree of the substituents and there they differ in the available
sites. Size and structure of the molecules are important for
formation of inclusion compounds. Structurally smaller drug
insertion (as compared to CD cavity) is not energetically
favored and for appropriately larger solutes, they can fill most
of the CD cavity and form the more stable complexes. Thus, TC
solubilisation capacity of HPBCD is higher by forming HPBCDTC
inclusion complex than that of BCD. Again, equilibrium
analysis of drug cyclodextrin complex within the experimental
concentration range (data not shown) was found to be of 1:1
stoichiometry which has been reported to happen with a low
ligand concentration, as at higher ligand concentration higher
order complexes are formed . In conclusion, HPBCD is a
better complexation ligand for TC than natural BCD.
Thermodynamic parameters calculated are shown in Table 1.
It shows the effect of increasing experimental temperatures and
concentrations of both the cyclodextrins on TC solubilisation.
The solubility of TC was increased with both the conditions.
TC solubility was characterized by a negative ΔG°, indicative
of spontaneous dissolution and positive ΔH° indicative of
endothermic dissolution . Van der Waal interactions,
hydrogen bonding, hydrophobic interactions, release of highenergy
water molecules from the cavity of cyclodextrin and release
of strain energy in the ring of cyclodextrin structure etc. are the
known driving forces for the formation of cyclodextrin inclusion
complexes with foreign molecules . These interactions cause
conformational changes in cyclodextrin structure, dissolvation
to complex stability and drug solubility. Breakdown of water
structure around a solute creates a higher positive ΔS° and a
positive ΔH° known to be governed by hydrophobic interaction
. In the present study dissolution thermodynamics of TC
in aqueous BCD and HPBCD were characterized by a positive
ΔH°(Figures 4&5), indicative of endothermic dissolution .
Reports suggests that complex formation with BCD and
HPBCD yields negative or positive ΔH°and negative or positive
ΔS° . In our study both BCD and HPBCD complex formations
resulted in positive ΔH° as well as positive ΔS°. Breakdown of
water structure around TC creates a large positive ΔS° and a
positive ΔH° (Tables 2&3), apparently governed by hydrophobic
interactions . Positive ΔS° for TC may be attributed to
transfer of TC from polar aqueous medium to nonpolar cavities
of CDs . Positive ΔH° indicates endothermic dissolution
thermodynamics of TC in aqueous BCD and HPBCD. In this
experiment, the enthalpy difference (ΔH°) decreased with the
increasing concentrations of BCD and HPBCD.
This is favourable for a thermodynamic process to happen and
in this case, enthalpy was the driving force for complexation of
TC with BCD and HPBCD. The entropy difference (ΔSo) decreased
with (Tables 2&3) increasing BCD and HPBCD concentrations.
The entropy was not the driving force for complexation of TC
with CDs. With respect to the HPBCD, the complexation of
natural BCD with TC is characterized by less positive enthalpy,
the contribution from which the solubility improvement profile
of TC with BCD is lesser than HPBCD.
The study result of pharmacokinetic assay by LC-MS/MS was
represented by the plasma concentration-time profile (Figure
6).The pharmacokinetic parameters of tamoxifen as a free drug
(FD) and along with a solubility enhancer HPBCD (HCD) (Table
4) revealed that Area under the curve (AUC) and maximum
concentration reached (Cmax) for tamoxifen was much higher in
case of CD than FD which indicated that cyclodextrin had a clear
effect on improving the bioavailability of tamoxifen
Mean±SD (n=6), AUC; area under the plasma concentration time curve from 0 h to infinity, Cmax; peak concentration, Tmax; time to reach the peak
concentration, MRT; mean residence time; CL; total body clearance. HCD; hydroxy propyl β cyclodextrin added to tamoxifen, FD; tamoxifen as
a free drug.
On other hand, clearance rate was higher for FD in
comparison with HCD which may be due to the faster elimination
of tamoxifen when administered alone (FD) than with a solubility
enhancer (HCD). After in vivo administration, free tamoxifen due
to its poor solubility was absorbed less but eliminated quickly
after absorption. On the other hand, tamoxifen-HPBCD complex
was absorbed faster and distributed in the system with a slower
The water solubility of TC was increased 7.1-fold in the
presence of 0.5% m/v HPBCD, compared to 0.5% m/v natural
BCD (increase 5.8-fold). Thermodynamic parameters derived
from TC solubility in the presence of various concentrations of
BCD and HPBCD at several temperatures reveal that the solubility
of TC increased with an increase in temperature. Besides, TCBCD
complex formation was a characteristic of a very strong
hydrophobic interaction. Furthermore, pharmacokinetic study
was also representing the similar observation with higher
Area under the plasma concentration-time curve and peak
plasma concentrations of TC, when administered along with
HPBCD. However, clinical trial will be needed to conclude the
simultaneous oral administration of cyclodextrin along with
tamoxifen to enhance the bioavailability in human.
We acknowledge the funding agency University Grants
Commissions, Government of India and Indian Council of
Medical Research (ICMR), Grant number: Nan/BMS -45/6/2013
for providing the necessary grants for the study.