Application of cosolvency and cocrystallization approach to enhance acyclovir solubility

Abstract

Acyclovir (ACY) is a commonly used antiviral drug with limited aqueous solubility and poor permeability. It is administered via oral and intravenous routes. The aim of this work is to employ cocrystallization and cosolvency method to improve the solubility of orally administered dosage forms, including tablet and oral solution. Cocrystallization is carried out through the solvent evaporation method using different types of coformers including formic acid (FA), tartaric acid (TA), and ascorbic acid (AA). The physical states of the obtained samples were characterized by differential scanning calorimeter (DSC), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FT-IR). The solubility of the developed samples were evaluated and compared with those of the parent drug. DSC and XRD findings indicate that ACY-AA and ACY-TA may be classified as cocrystals. Physicochemical characterization of ACY-FA did not demonstrate any change with respect to the parent drug. ACY-AA had the highest increase in drug solubility and created almost 3 folds enhancement. In another approach, cosolvency was employed to study the solubilization enhancement of 6 different binary solvent mixtures at 298.15 K. PEG 400 + water at 0.5:0.5 demonstrated a twofold enhancement in drug solubility. Both cocrystallization and cosolvency method seem effective in enhancing drug solubility and have the potential to be employed for the development of oral drug delivery systems including tablets and oral suspensions. The findings of this work suggest that ACY solubility can be enhanced by proper choice of the coformer and cosolvent. The result of this finding is beneficial for the development of acyclovir dosage forms.

Graphical abstract

Background

Acyclovir (ACY) is a commonly used antiviral drug with incomplete absorption in the gastrointestinal tract and low oral bioavailability of 10–30%. This drug has a limited solubility of 1.6 mgmL⁻¹ in water and therefore its biological activity depends on its dissolution rate and intestinal permeability. Due to its limited solubility, ACY is administered in high oral doses (200–1000 mg three to four times a day) in the form of tablet, oral suspension, and powder for infusion [1, 2].

There are various methods that help to enhance drug solubility. Some of these include salt formation, micellization, cosolvency, and the application of cocrystallization. Cosolvency is a widely used method to help enhance drug solubility. Application of a cosolvent helps to reduce surface tension and polarity of the aqueous system. This can disrupt the structure of the water molecule and help enhance drug solubility. This method is convenient and inexpensive however there may be potential cosolvent toxicity and the possibility of drug precipitation upon dilution in the dissolution media [3, 4]. Cocrystallization with the application of an appropriate coformer can enhance the physicochemical properties of the API with poor solubility and bioavailabillity. This method can offer many advantages including coformer diversity and stability of the obtained product due to hydrogen bonding in the crystal structure. One of the major challenges with this method is limitation in scalability and prediction of cocrystallization [5, 6]. The obtained cocrystals are usually characterized through various methods. Fourier transform infrared spectroscopy (FTIR) is used as a tool to identify changes in the molecular structure of the developed compound mainly through the presence of hydrogen bonding. X-ray diffraction (XRD) is a precise method used for the accurate detection of co-crystals. Differential scanning calorimeter (DSC) can be utilized to screen cocrystals through changes in the endothermic and exothermic peaks in the co-crystals in comparison with pure components [7].

Various coformers have been employed for the development of ACY cocrystals [2, 8–11]. Carboxylic acids are recognized as safe and efficient coformers which can greatly impact drug solubility in water. The presence of charges and the chemical nature of the carboxylic acid functional group can influence drug lipophilicity, solubility, and cell permeation [12].

Ascorbic acid (AA) is a vinylogous carboxylic acid with a vinyl group positioned between the carboxyl and hydroxyl groups. This functional group has the possibility to form a resonance through a conjugated system [13]. This water-soluble molecule has been used for the cocrystallization of zwitterion structures. Co-administration of AA with furosemide has demonstrated significant enhancement in the oral bioavailability of furosemide [14, 15].Tartaric acid (TA) is a widely used coformer applied in drug cocrystallization [16]. Formic acid has also been used to enhance characteristics of drug crystals [17, 18].

Through this study, we aim to provide further information on the solubility of ACY as a model API by employing two commonly used methods for solubility enhancement including cosolvency and cocrystallization method. The effect of carboxylic acid group on the solubility of ACY was evaluated. Three coformers were used; (1) formic acid (FA) as a simple carboxylic acid, (2) tartaric acid (TA) a dicarboxylic acid, and (3) ascorbic acid (AA) as a vinylogous carboxylic acid for the formation of ACY cocrystals. Cocrystals were developed using the solvent evaporation method and the physical states of the obtained samples were characterized using DSC, XRD, and FTIR. The solubility of the obtained samples was evaluated and compared with those of pure drug.

In another approach cosolvency was employed to study the solubilization of ACY in 6 different binary solvent mixtures commonly used in the pharmaceutical industry including water + ethanol, water + methanol, water + acetone, water + polyethylene glycol 400, ethanol + methanol and acetone + polyethylene glycol 400 at 298.15 K. Experimental solubility data were correlated using the Jouyban-Acree and modified Wilson model.

Results and discussion

FTIR analysis

The IR spectra of ACY and the obtained compounds are shown in Fig. 1. The IR spectrum of ACY shows the primary and secondary amine bands in the region of 3476 cm⁻¹ and 3432 cm⁻¹. The C = O carbonyl and amide group band is seen at 1733 cm⁻¹. Two bands at 1540 cm⁻¹ and 1485 cm⁻¹ are related to the O–H group and the amide group is seen at 1599 cm⁻¹ [2, 19, 20].

Fig. 1.

FT-IR spectrum of drug, coformer and obtained samples at the drug:coformer ratio 1:3

The spectra of AA show a C = O stretching of the lactone ring at 1754 cm⁻¹ and the C = C stretching is seen at 1672 cm⁻¹. The C–O–C and –OH stretching is seen at 1274 cm⁻¹ and 1322 cm⁻¹ respectively. O–H stretching due to the presence of moisture in the sample is seen in the region of 3216 cm⁻¹ to 3526 cm⁻¹ [21, 22]. Two distinct peaks were observed for TA at 3410 cm⁻¹ and 3337 cm⁻¹ which corresponds to the presence of O–H group. C = O stretching of TA is observed at 1741 cm⁻¹ [23, 24].

Changes in the C = O and N–H bending frequency of ACY is observed for both ACY-AA and ACY-TA which may explain the possibility of bond formation between the drug and the coformer. The functional groups of ACY-FA did not show any shift with respect to ACY at 1733 cm⁻¹and 1599 cm⁻¹ which may reflect less/no interaction between ACY and FA (Fig. 1).

FTIR spectroscopy may be used as a tool to study co-crystallization through hydrogen bonding, however its accuracy is subject to interference from the drug and the coformer. XRD pattern and DSC thermogram may help to effectively identify and confirm the formation of cocystals [7].

DSC analysis

Figure 2 represents the DSC thermograms of the drug, coformer and the obtained samples. For pure drug, an intense endothermic peak corresponding to its melting point is seen at 256.77 °C. A less visible broad endotherm is seen at 117.04 °C which is associated with dehydration and water release [2]. TA and AA melting point is seen at 172 °C and 192 °C, respectively. The results are supported with previous literature [25, 26].

Fig. 2.

DSC thermograms of pure components and obtained samples at the drug:coformer ratio 1:3

An endothermic peak at 140.92 °C and 174.84 °C is seen for ACY-TA and ACY-AA respectively. A decrease in melting point may indicate that the new crystals have a melting point below that of pure drug or the coformer. Other endothermic peaks may represent the presence of polymorphs [2]. The broader melting peak observed for ACY-AA and ACY-TA samples indicates that the complete melting of the cocrystals happened over a wider temperature range due to the high coformer concentration [20]. The melting point of the ACY-FA is seen at 255.70 °C which is very close to the melting point of the pure drug. The similarity in the melting point observed with this sample and the parent ACY may indicate that FA did not form any cocrystal with the drug.

XRD analysis

Among various methods available on characterization of developed compounds, XRD can provide reliable results needed to confirm cocrystallization [7]. The XRD pattern of all pure samples and the resulting compounds are shown in Fig. 3a. The residual solids in each of the solvent media were also found to be in its crystalline state (Fig. 3b). The diffractogram of pure ACY shows main peaks at 2Ɵ values of 7.05, 10.55, 16.1, 21.07, 26.19, and 29.29. Similar observations were reported earlier [24, 27]. The diffraction patterns of ACY-TA and ACY-AA are different from those of the pure components which may confirm the formation of a new crystal lattice. The XRD pattern of ACY-FA is a summation of the diffractogram patterns of the pure components and the sharp peak for ACY at 2Ɵ 7.09 is also detected in ACY-FA. This can indicate that a new solid phase may not have been formed. All samples reflect sharp peaks indicating the crystalline nature of the samples however the peak intensities have declined.

Fig. 3.

a XRD pattern of drug, coformer and the obtained samples, b X-RD pattern of residual solids in the solubility medium at the drug:coformer ratio 1:3

Solubility of ACY cocrystals

Determination of the aqueous solubility of ACY cocrystals was conducted in deionized water and the results are shown in Fig. 4. API solubility was improved in the order: ACY-AA > ACY-TA > ACY-FA. The findings of this work are consistent with the results of the DSC, XRD and FTIR. The aqueous solubility of ACY in distilled water was found to be 1.774 mg.mL⁻¹. Similar values have been reported in the literature [28].

Fig. 4.

Solubility of ACY cocrystals in water with respect to drug: coformer ratio, dotted line represents intrinsic solubility of ACY

As the ratio of coformer increased, the drug solubility of the compound increased. Maximum drug solubility is seen at the drug: coformer ratio of 1:5. This may be due to the change in media pH which may help to enhance drug solubility. ACY is amphoteric and has both acidic (pK_a1 = 2.27; pK_a2 = 9.25) and nitrogen basic sites (pK_b1 = 0.4, pK_b2 = -0.5). Formic acid has a pK_a value of 3.75 [28, 29] and AA and, TA have a pk_a1 of 4.2 and 2.9 respectively [30, 31]. Therefore this drug can both be deprotonated and protonated to form anion or cation [1].

Solubility results indicate that AA had the highest increase in drug solubility which may indicate that vinylogous carboxylic acid is more likely to interact with ACY molecule to form a stable crystalline structure relative to the dibasic carboxylic acid (TA). FA was not able to form cocrystals with the drug molecule. Cocrystals are mostly formed through hydrogen bonding [12].

The solubility enhancement may be due to the hydrogen bond between ACY and the coformer. This increases the internal energy of the structure and enhances thermodynamic properties which lead to higher solubility [2]. Figure 5 depicts the chemical structure of drug and the coformers and the groups involved in the bond formation are shown in the dashed line. ACY can interact with the coformers through its three hydrogen bond donor and 4 hydrogen bond acceptor sites [28].

Fig. 5.

Chemical structure of drug and the coformers and the groups involved in bond are shown in dashed line

The experimental results shown in Fig. 4 indicate that ACY-FA 1:1 was not able to enhance solubility relative to the un-processed drug, however at a higher ratio, enhanced solubility was observed. This may be due to the formation of acidic environment, which may increase drug solubility. Tartaric acid can act as an acidifier and induce drug supersaturation by lowering pH of the gastric media [32]. Ascorbic acid can reduce gastric pH in hypochlorohydric patients and reacidify gastric fluid [33]. The results of this study confirm that addition of each of these coformers was able to decrease pH of the dissolution media (water). The results of this study show that pH of the dissolution media containing pure ACY was around 7.8. Addition of the developed compounds to the dissolution media showed significant reduction in pH of the dissolution media. ACY-FA and ACY-AA in the entire drug: coformer ratio demonstrated a pH ≥ 2.5 while ACY-TA demonstrated pH ≤ 2. This can demonstrate that lowering pH can facilitate drug solubility but is not the sole reason for enhanced solubility in the developed compounds as ACY-AA was able to create higher solubility enhancement. AA has been used as a coformer for the cocrystallization of several zwitterions structures leading to carboxylate-hydroxyl supramolecular heterosynthons [34]. The possibility of structural interaction between AA and drug molecule may help to enhance its solubility. ACY in its cationic and anionic form can have different solubility in water, thus by employing the proper choice of the counter ion its solubility can be enhanced [1]. Cocrystallization of betulinic acid with AA not only enhanced the anticancer activity of betulinic acid but also increased its solubility [34]. The oral bioavailability of itraconazole increased when it was co-administered with AA [35]. Some coformers such as AA may help to increase drug bioavailability through gastrointestinal absorption. Therefore AA can be applied as an appropriate coformer and a promising tool for utilization in the formulation of BCS class IV drugs with limited solubility and permeability.

Skin penetration enhancers are widely used to increase drug permeation across skin [36]. AA and its esters have demonstrated enhancement in drug permeation across human excised skin [37–39]. Carboxylic acids have been used in topical formulations for their wound healing and penetration enhancing effects. FA can accelerate wound healing in diabetic wound models and has shown a significant effect in the treatment of warts [40, 41]. TA has been employed for the development of biodegradable glue for skin closure. It has shown significant improvement in the penetration and absorption of drug through the transmucosal pathway [42, 43]. Application of these coformers can not only enhance ACY solubility but can also increase drug permeation across biological barriers. When used in topical ACY formulation, these coformers may also enhance the wound healing properties of ACY for Herpes simplex infection.

Drug solubility in binary solvent mixtures

The relationship between the solubility of ACY and binary solvent composition is reflected in Table 1. ACY solubility in pure water is 1.744 mgmL⁻¹. The highest drug solubility is seen in acetone (w₁ = 0.4) + water with a total drug solubility of 4.72 mgmL⁻¹. Acetone is widely used for crystallization or extraction of various drug molecules. Purification of valacyclovir showed maximum efficiency in acetone + water solvent system [44].

Table 1.

Experimental mass fraction solubility (C_m) ACY in the binary solvent mixtures at 298.2 K.^*

W1	Mass solubility (Cm) mg.mL−1
	Methanol (W1) + Water	Acetone (W1) + Water	PEG** 400 (W1) + Water	Ethanol (W1) + water	Ethanol (W1) + Methanol	Acetone (W1) + PEG 400
0.000	1.774	1.774	1.774	1.774	0.739	2.090
0.100	1.945	2.871	2.103	2.152	0.695	2.346
0.200	2.131	3.954	2.533	2.582	0.630	2.682
0.300	2.343	4.506	2.959	3.026	0.570	2.963
0.400	2.467	4.716	3.381	3.325	0.520	3.230
0.500	2.322	4.401	3.837	3.374	0.486	3.364
0.600	2.080	3.919	3.543	2.653	0.452	3.170
0.700	1.768	3.323	3.110	1.924	0.410	2.910
0.800	1.451	2.812	2.762	1.230	0.365	2.586
0.900	1.103	2.194	2.415	0.765	0.312	2.199
1.000	0.739	1.735	2.090	0.120	0.120	1.735

^*The relative standard uncertainty for temperature is 0.05 K. The relative standard uncertainty of the mass fraction of cosolvent in the binary solvent mixtures (w₁) is 0.0001

^**PEG represents poly ethylene glycol

Results of the present work (Table 1) demonstrated that PEG + water in a ratio of 0.5:0.5 was able to provide a twofold increase in drug solubility. PEGs present great opportunities for the development of oral solutions of poorly water soluble drugs. Digoxin, danazol, and ibuprofen have shown higher gastric absorption rates and oral bioavailability when formulated as PEG 400 oral solution [45].

Other solvent mixtures listed in Table 1 have also increased the ACY solubility but to a smaller degree. All binary solvents, with the exception of ethanol + methanol system, show a similar trend and a sigmoidal pattern in drug solubility relative to the solvent’s mass fraction. Maximum drug solubility is mostly observed at a cosolvents mass fraction of 0.4–0.5. In ethanol + methanol mixture, solubility increases as a function of ethanol composition and reaches maximum solubility in neat ethanol. Knowledge of solubility profile patterns can help to optimize drug solubility in cosolvent ratio. It is known that drug solubility reaches its maximum value in solvents with similar polarity and intermolecular forces. Drug solubility pattern depends on the polarity of the binary solvent mixture and in some drugs the pattern is linear while in others it is sigmoidal [8, 46].

Mathematical representation of experimental data

Two cosolvency models were used to correlate and back-calculate the generated solubility data in the respective solvent systems. Computed parameters as well as the MRD% values are reported in Tables 2 and 3. MRD% value for back-calculation of ACY with the CNIBS/R-K model is 1.6% for methanol + water system, 0.7% for acetone + water system, 4.0% for PEG 400 + water system, 9.2% for ethanol + water system, 4.6% for ethanol + methanol system, and 0.8% for acetone + PEG 400 system. Moreover, MRD% values for back-calculation of ACY with the modified Wilson model is 1.7% for methanol + water system, 1.0% for acetone + water system, 4.2% for PEG 400 + water system, 7.2% for ethanol + water system, 9.3% for ethanol + methanol system, and 1.5% for acetone + PEG 400 system. As the calculated values for MRDs% in all investigated binary systems are lower than 10%, it can be concluded that these models are the reliable methods for the solubility prediction of ACY in the investigated systems which is considered a useful tool in pharmaceutical industries.

Table 2.

Parameters of CNIBS/R-K model and MRD% for prediction of ACY solubility in the investigated binary systems at 298.2 K

Cosolvency system	J0	J1	J2	MRD%
Methanol + water	2.783	0.613	0a	1.6
Acetone + water	3.698	− 1.747	0.629	0.7
PEG 400 + water	2.324	0a	0a	4.0
Ethanol + water	7.549	4.885	3.927	9.2
Ethanol + methanol	1.816	3.019	3.946	4.6
Acetone + PEG 400	2.196	0.398	-0.484	0.8

^aNot statistically significant (p > 0.05)

Table 3.

Parameters of the modified Wilson and MRD%for prediction of ACY solubility in the investigated binary mixtures at 298.2 K

Cosolvency system	λ12	λ21	MRD%
Methanol + water	1.673	1.105	1.7
Acetone + water	1.046	2.674	1.0
PEG 400 + water	1.354	1.372	4.2
Ethanol + water	3.916	0.977	7.2
Ethanol + methanol	5.575	0.179	9.3
Acetone + PEG 400	1.575	1.111	1.5

Materials and method

Materials

ACY purity of 97% was obtained from Temad Pharmaceutical (Tehran, Iran). Formic acid (FA), L-tartaric acid (TA), glacial acetic acid (99.9%), methanol, acetone, polyethylene glycol 400, and absolute ethanol all with analytical grade were obtained from Merck (Darmstadt, Germany). Ascorbic acid (AA) was obtained from Osveh Pharmaceutical (Tehran, Iran). Double distilled water was used for all experiments.

Preparation of cocrystals

Cocrystals were prepared with the three different coformers including AA, TA, and FA in a drug:coformer molar ratios of 1:1, 1:3, and 1:5 by the solvent evaporation method obtained from previous study. Briefly, the drug and the coformer were separately dissolved in glacial acetic acid prior to mixing. The mixture was placed overnight on magnetic stirrer to allow solvent evaporation. Finally, the obtained samples were milled and passed through a 60 sieve and stored in desiccators for further studies [9, 47, 48].

Fourier transform infrared spectroscopy (FTIR)

The possible interactions between the drug and the coformer was acquired on a Perkin Elmer (Spectrum Two) FTIR at room temperature in the wavelength region 450–4000 cm⁻¹. Prior analysis, samples were mixed with KBr powder (spectroscopic grade) in a mortar and compressed into a 13 mm disc using a hydraulic press (1000 psi) under the pressure of 30 tons for 3 min using a hydraulic press.

Differential scanning calorimetry (DSC)

The DSC thermograms of each sample was obtained using a differential scanning calorimeter (Mettler Toledo DSC 25, Switzerland). Calibration of the instrument was carried out using indium melting point. Samples were weighed and placed in sealed aluminium crucibles and scanned with a heating rate of 10 °C.min⁻¹ through a temperature range of 0–300 °C under nitrogen gas flow. An empty holder was employed as a reference.

X-ray diffraction (XRD)

The diffraction pattern of the native drug, the coformers, and the resulting compounds were determined by PANalytical X’Pert Pro operating at a voltage of 40 kV and current of 40 mV. Diffraction measurements were recorded at room temperature with a step size: 0,020°. The 2θ scanning was carried out between 5° and 40°.

Solubility studies of cocrystals

In vitro dissolution studies were conducted using the shake-flask method at room temperature and atmospheric pressure [49]. An excess amount of the drug or the obtained sample was added in a flask of distilled water at 298.15 K placed on a shaker-incubator (Behdad, Iran). Samples were left to equilibrate for 24 h prior to centrifugation at 13,000 rpm (Eppendorf 5452 Minispin, Germany). Samples were filtered (0.45 μm, BioFil, China) and diluted before quantification. ACY content was determined at a wavelength of 251 nm spectrophotometrically using a UV- spectrophotometer (Cecil CE 7200, 7000 series, UK). All samples were diluted using distilled water prior to quantification [2].

Cosolvency

ACY solubility in binary solvent mixture was evaluated using the Higuchi shake-flask method [48]. Various mass fractions of binary solvent mixtures were weighed using an electronic balance (Satorius Scientific Instrument model: BSA224S; standard uncertainty 1 × 10^–4 g) with an accuracy of 1 × 10^–5 g. An access amount of ACY was placed in various mass fractions of the binary solvent mixtures for 24 h at 298.15 K. After equilibrium, suspensions were centrifuged, filtered, and diluted prior to quantification via spectrophotometric method. The experimental solubility of ACY was studied in various mass fractions of six solvent mixture including acetone (w₁) + water, methanol (w₁) + water, polyethylene glycol 400 (w₁) + water, ethanol (w₁) + water, ethanol (w₁) + methanol, acetone (w₁) + polyethylene glycol 400. All experiments were carried out in triplicates at 298.15 K.

Computational modelling

A multiple linear cosolvency model including Jouyban-Acree, a non-linear model, and the modified Wilson model were used to correlate/predict ACY solubility. Model accuracy was evaluated through the mean relative deviation (MRD%) equation:

$$MRD\%=\frac{100}{N}\sum \left(\frac{CalculatedValue-ObservedValue}{\mathit{ObservedValue}}\right)$$ 1

where N is the number of data points.

The Jouyban-Acree model

The Jouyban-Acree model relates the solubility data to both temperature and solvent composition as [50]:

$$ln{C}_{m,T}=w_{1}ln{C}_{1,T}+w_{2}ln{C}_{2,T}+\frac{w_1.w_2}{T}\sum _{i=0}^2{J}_i.{(w_1-w_2)}^i$$ 2

In the isothermal condition, this model can be reduced to the combined nearly ideal binary solvent/Redlich–Kister (CNIBS/R-K) model which correlates solubility data in the different solvent composition at any given temperature by incorporating solute solubility in each of the mono-solvents [51] according to Eq. (3):

$$ln{C}_m=w_1.ln{C}_1+w_2.ln{C}_2+w_1.w_2\sum _{i=0}^2{J}_i.{\left(w_1-w_2\right)}^i$$ 3

C₁ and C₂ are considered as the solubility in the mono solvents 1 and 2, w₁ and w₂ are the mass fraction of solvents 1 and 2 in the absence of solute and J_i denotes the model coefficient obtained through linear regression.

The modified Wilson model

The modified Wilson model correlates the solubility data at various solvent mixtures in a given temperature using [19]:

$$-ln{C}_m=1-\frac{w_1\left[1+ln\left(C_1\right)\right]}{w_1+w_2{\lambda }_{12}}-\frac{w_2\left[1+ln\left(C_2\right)\right]}{w_1{\lambda }_{21}+w_2}$$ 4

where λ₁₂ and λ₂₁ are model parameters obtained by the non-linear least square analysis.

Conclusion

Acyclovir (ACY) is a commonly used antiviral drug with limited aqueous solubility and intestinal permeability. The current research has focused on the cosolvency and cocrystallization method to increase ACY solubility.

Cocrystallization of ACY through the application of various coformers including FA, TA, and AA was carried out. Superior enhancement in saturation solubility of ACY was achieved through the cocrystallization method using AA. The obtained samples have the potential to be used in the development of oral tablets. Solubility and permeability of ACY can be simultaneously enhanced via the cocrystallization approach using these coformers.

Six binary solvent mixtures were studied for the solubility enhancement of ACY. The highest drug solubility was found in acetone + water and PEG 400 + water mixtures. These solvent mixtures have the potential to be used for drug crystallization using aqueous mixture of acetone and for the development of oral ACY solution using PEG 400 aqueous mixture.

The finding of this work suggests that based on the type of dosage form needed, ACY solubility can be enhanced by the proper choice of the coformer and cosolvent. Both cocrystallization and cosolvency method seem effective in enhancing drug solubility and they have the potential to be employed for the development of oral drug delivery systems including tablet and suspension.

Author contributions

E. A.-K: Investigation, Methodology, A. J.: Conceptualization, Writing-Reviewing and Editing, A.F.A.: Supervision, Data curation, Draft preparation.

Funding

This work is financially supported by Urmia University of Medical Sciences grant No. 1397- 2455. IR.UMSU.REC.1397.359.

Data availability

The data of the current study is available upon reasonable request from the corresponding author.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interest

The authors declare no competing interests.