Corresponding Author:
Neelam Seedher
Department of Chemistry, Panjab University, Chandigarh-160 014, India
E-mail: [email protected]
Date of Submission 21 April 2008
Date of Revision 14 August 2008
Date of Acceptance 10 February 2009
Indian J Pharm Sci, 2009, 71 (1): 82-87

Abstract

Solubility enhancement of antimicrobial drug enrofloxacin has been studied using a series of co-solvents and surfactants. Aqueous solubility of enrofloxacin could be increased up to 26 times. Co-solvents alone produced only small increase in solubility. However, the combined effect of co-solvents and buffer was synergistic and a large increase in solubility could be attained. Ionic surfactants were found to be much better solubilizing agents than non-ionic surfactant. Amongst ionic surfactants, solubility was found to be very high in anionic surfactant, sodium dodecylsulphate as compared to the cationic surfactant, cetyltrimethylammonium bromide. Up to 3.8 mg/ml of enrofloxacin could be dissolved in sodium dodecylsulphate. Mechanism of solubilization has been proposed and surfactant solubilization parameters have been calculated.

Keywords

Enrofloxacin, solubility, co-solvents, surfactants, sodium dodecylsulphate

The development of fluoroquinolone class of antibiotics has been a major breakthrough in the treatment of bacterial infections. These medications are active against many bacterial types including pseudomonas and are not associated with serious side effects that plagued the aminoglycoside group. Enrofloxacin, a broad spectrum fluoroquinolone antibiotic, which has shown efficacy for veterinary use [1]. Some physico-chemical properties of enrofloxacin have been reported [2].

Enrofloxacin is beset with the disadvantage of poor aqueous solubility. The very poor aqueous solubility and wettability of the drug gives rise to difficulties in the design of pharmaceutical formulations and leads to variable bioavailability. The pH-solubility profile of enrofloxacin has been reported by Lizondo et al. [2]. However, there appear to be no reports on the enhancement of solubility of enrofloxacin. The use of co-solvents is a highly effective technique to enhance the solubility of poorly-soluble drugs [3-5]. In the present study an attempt has been made to increase the solubility of enrofloxacin using a series of co-solvents and surfactants.

Pure enrofloxacin was obtained as a gift sample from Ranbaxy Laboratories Ltd. India. All solvents used were of analytical grade. Water used was double distilled in all glass apparatus. Ultraviolet absorption spectrophotometric technique was employed for the estimation of enrofloxacin. Due to limited solubility of enrofloxacin in water/buffer, 0.02 N sodium hydroxide was used as solvent. Extinction coefficient of drug, determined at 271 nm, was used to estimate the unknown drug concentration.

For the determination of solubility, excess of drug was placed in contact with 5 ml of solvent in sealed conical flasks. The flasks were maintained at 250 and the contents were stirred on a magnetic stirrer for the required time period (3½/24 h). The solution was centrifuged and the supernatant was filtered through 0.45 µm filter. The absorbance of clear solution was determined at λmax of the drug after suitable dilution with the appropriate solvent. The concentration of drug was determined from Beer Lambert law using extinction coefficients, determined in the relevant solvent. The solubility was calculated by multiplying the drug concentration, so obtained, by the appropriate dilution factor. The reported data are an average of three determinations.

The solubility of enrofloxacin in water, 0.1 M phosphate buffer (PB, pH 7.4), 10-40% concentration of co-solvents: polyethylene glycol 400 (PEG 400), propylene glycol (PG), glycerol and ethanol, 25 and 50 mM micellar concentrations (total surfactant concentration; CMC) of a cationic (cetyl trimethylammonium bromide, CTAB), anionic (sodium dodecylsulfate, SDS) and non-ionic (polyoxyethylene (20) sorbitan monooleate, Tween 80) surfactant and 1:1 and 2:1 PEG 8000:drug solid dispersions, prepared by solvent evaporation method [6], was determined at 250. Water as well as PB (pH 7.4) medium was used for solubility determination in each case.

Enrofloxacin is an amphoteric drug with pKa1= 5.94, corresponding to carboxyl group and pKa2= 8.70, corresponding to basic piperazinyl group and the isoelectric pH= 7.32. The lipophilicity, measured as octanol-aqueous buffer partition coefficient (log D) is 3.48 at pH 7.00 pH 7.002 [2]. Thus at near neutral pH drug exists in zwitterionic form and has high lipohilicity. Aqueous solubility of enrofloxacin in water at 250 was found to be 146 µg/ml and therefore, the drug can be categorized as poorly soluble. The solubility was seen to increase from 146 to 182 µg/ ml by using 0.1 M PB (pH 7.4) as solvent.

The use of co-solvents is a simple and effective technique, widely used to enhance the solubility of poorly soluble drugs [3-5]. Initially the time period for drug dissolution was kept 3½ h (210 min) for all the solvents. The amount of drug dissolved and the initial dissolution rate in water, phosphate buffer and 10-40% concentration of co-solvents in water as well as 0.1 M PB (pH 7.4) is given in Table 1. All the co-solvents were found to increase the dissolution rate of drug and the amount of drug dissolved increased with increase in the concentration of the co-solvent in each case. The dissolution rate was found to vary from 20 µg/ml/h in water to 125 µg/ml/h in the presence of co-solvents; the values were highest in ethanol followed by glycerol and PG. It is also seen that for each solvent, the dissolution rate improved considerably when water was replaced by PB (pH 7.4) for the preparation of co-solvent solutions.

Solvent Amount dissolved*
(µg/ml)
Initial dissolution rate
(µg/ml/hr)
Water Buffer Water  Buffer
Water 70.002     - 20.001     -
PB (7.4)     - 107.46     - 30.703
10% PEG 400 78.819 194.074 22.52 55.45
20% PEG 400 88.287 246.154 25.225 70.33
40% PEG 400 100.475 494.502 28.707 141.286
10% PG 107.012 135.105 30.575 38.602
20% PG 132.654 256.385 37.901 73.253
40% PG 325.991 1203.703 93.141 343.915
10% Glycerol 102.555 170.552 29.302 48.729
20% Glycerol 156.062 201.992 44.589 57.712
40% Glycerol 364.849 753.176 104.243 215.193
10% Ethanol 166.09 343.803 47.454 98.229
20% Ethanol 213.423 568.357 60.978 162.387
40% Ethanol 439.821 1300.383 125.663 371.358
25 mM CTAB** 300.184 612.907 85.767 175.116
50 mM CTAB** 458.186 930.737 130.991 265.925
25 mM SDS** 2517.373 2852.572 719.249 815.021
50 mM SDS** 3314.075 3478.288 946.878 993.796
25 mM Tween-80** 173.668 197.781 49.619 56.508
50 mM Tween-80** 312.696 320.678 89.342 91.622

Table 1: Amount dissolved and initial dissolution rate for enrofloxacin in various co-solvents at 25°

Since the amount of drug dissolved was small at 10% co-solvent concentration, twenty four hour solubility data was obtained only for 20 and 40% concentration of co-solvent in water as well as phosphate buffer medium. Results are given in Table 2. In aqueous medium, the solubility was found to increase from about 146 µg/ml in water to about 439 µg/ml in the presence of co-solvents. Again highest solubility was obtained in ethanol followed by glycerol and PG. Although large solubility enhancement was observed, the maximum solubility was much less than l mg/ml.

Solvent Solubility (µg/ml)* Solubility Ratio
Water Buffer
Water 145.868     -    -    -  -    -
PB (7.4)     - 181.916    -    -    - 1.247
20% PEG 400 162.009 300.175 1.111 1.65 2.058 1.854
40% PEG 400 188.799 555.725 1.294 3.055 3.81 2.943
20% PG 210.214 465.737 1.441 2.56 3.193 2.216
40% PG 399.349 1216.786 2.738 6.689 8.342 3.047
20% Glycerol 179.289 370.402 1.229 2.036 2.539 2.067
40% Glycerol 428.791 755.153 2.939 4.151 5.177 1.761
20% Ethanol 214.541 578.212 1.471 3.178 3.964 2.695
40% Ethanol 492.771 1699.342 3.378 9.341 11.65 3.448
25 mM CTAB** 324.813 723.902 2.227 3.979 4.963 2.229
50 mM CTAB** 426.783 1050.439 2.926 5.774 7.201 2.461
25 mM SDS** 2598.398 2914.401 21.73 16.02 19.979 1.122
50 mM SDS** 3385.296 3793.154 23.208 20.851 26.004 1.121
25 mM Tween-80** 217.547 232.969 1.491 1.281 1.597 1.07
50mM Tween-80** 342.67 351.786 2.349 1.934 2.412 1.027

Table 2: Solubility and solubility ratios for enrofloxacin in various solvents at 25°

Since the amount of drug dissolved was small at 10% co-solvent concentration, twenty four hour solubility data was obtained only for 20 and 40% concentration of co-solvent in water as well as phosphate buffer medium. Results are given in Table 2. In aqueous medium, the solubility was found to increase from about 146 μg/ml in water to about 439 μg/ml in the presence of co-solvents. Again highest solubility was obtained in ethanol followed by glycerol and PG. Although large solubility enhancement was observed, the maximum solubility was much less than l mg/ml.

Since the solubility of enrofloxacin in PB (pH 7.4) was higher than that in water, it was thought of interest to study the combined effect of co-solvents and buffer. For this purpose solubility was determined in the presence of 20 and 40% co-solvent solutions prepared in PB (pH 7.4). Solubility in PB (pH 7.4) in the absence and presence of 20 and 40% cosolvent solutions is given in Table 2. The presence of co-solvent as well as buffer produced a very large increase in solubility in all cases.

Solubility enhancement has been expressed in terms of solubility ratios, Sco-sol(w)/Sw, Sco-sol(b)/Sb, Sco-sol(b)/Sw and Sco-sol(b)/Sco-sol(w), where Sco-sol(w) and Sw are the solubility of drug in the presence and absence of co-solvent, respectively and Sco-sol(b) and Sb are the corresponding values in buffer. Various solubility ratios in different solvents have also been recorded in Table 2. Sco-sol(w)/Sw ratio, which represents the enhancement of aqueous solubility in the presence of co-solvent in water, was found to vary from 1.111 to 3.378. However, when the co-solvent solutions were prepared in buffer, the corresponding enhancement in buffer solubility (Sco-sol(b)/Sb ratio) was 3.055 to 9.341 times. The total solubility enhancement due to the combined effect of co-solvent and buffer, expressed as solubility ratio Sco-sol(b)/Sw, was found to be in the range 3.810 to 11.650. Significant solubility enhancement could, therefore, be achieved. Enrofloxacin could be dissolved up to about 1.7 and 1.2 mg/ml in ethanol and PG, respectively corresponding to 11.65 and 8.33 times enhancement of aqueous solubility of enrofloxacin.

Higher pH of buffer can cause only a small increase in solubility since enrofloxacin is amphoteric [2]. The ratio of drug solubility in buffer and water (Sb/Sw) is only 1.247. For a given co-solvent, the ratio of solubility in buffer and water medium (Sco-sol(b)/Sco-sol(w) ratio in Table 2), was found to be in the range 1.761 to 3.448 only. Thus large increase in solubility when the co-solvent solutions are prepared in buffer, cannot be due to increase in pH or dissolution of drug by buffer components. The presence of buffer and co-solvents has synergistic effect. The efficiency of the co-solvents increases in the presence of buffer components. This result is important especially since the buffer used is biocompatible and at near neutral pH. The only explanation which can be offered for the dramatic increase in the solubility when co-solvent solutions are prepared in buffer is given below.

The solubilization efficiency of a solvent is a function of the relative magnitudes of the various solute-solute, solute-solvent and solvent-solvent interactions. Reduction in solute-solute and solvent-solvent interactions should increase the solubilization efficiency. The co-solvents used in the present work have a tendency to form inter- and intra-molecular hydrogen bonds. Higher pH of buffer and increase in the ionic strength due to the presence of buffer components should decrease the ability of co-solvent molecules to form inter- and intra-molecular hydrogen bonds resulting in decrease in solvent-solvent interactions and consequent increase in solubility.

For a given solvent system, the solubilization power gives a quantitative estimate of the solubilization potential of the co-solvent [7]. The solubilization power (Φ) of various co-solvents was determined using equation, log Smix= log S+ΦVco-sol, where Smix and S are the solubilities of drug in solvent mixture and pure solvent, respectively. Vco-sol is the volume fraction of the co-solvent and Φ is defined as the solubilization power of the co-solvent. The Φ values for various solvents are given in Table 3. The order of solubilization potential was found to vary as ethanol>glycerol>PG>PEG 400 in water and ethanol>PG>glycerol>PEG 400 in buffer. Dielectric constants of the solvents show that the polarity of the solvent varies as glycerol>PG>ethanol>PEG 400 (Table 3). It appears that the polarity of co-solvent is not the only factor affecting solubility of drug. PEG 400 is not a good solvent for enrofloxacin in spite of it being least polar. It appears that the ability of solvent to form hydrogen bonds with the hetero-atoms in the drug molecule is another important factor governing the solubility of drug. Similar findings have also been reported by Seedher and Bhatia [8]. Since the drug exists in zwitterionic form at neutral pH, hydrophobic interactions with the non-polar part of PEG are also not significant.

Solvent Dielectric constant   of co-solvent Solubilization  
power (φ) Buffer
PEG 400 12.4 0.2801 1.2125
Ethanol 24.3 1.3218 2.4263
PG 32 1.0938 2.0635
Glycerol 42.5 1.1707 1.5458

Table 3: Solubilization power for various co-solvents in water and buffer

Since the solubility enhancement using PEG as co-solvent was least, the use of 1:1 and 2:1 PEG 8000:drug solid dispersions was also tried. Due to the formation of a sticky mass, which was difficult to dry, solid dispersions could not be prepared using PEG 400. The solubility of solid dispersions in water and PB (pH 7.4) was found to be 233 and 486 μg/ml, respectively. Thus aqueous solubility increased from 146 to 233 μg/ml, about 1.6 times. The total increase in aqueous solubility due to the combined effect of solid dispersion and buffer (146 to 486 μg/ml) was 3.33 times.

Surfactants are known to solubilize poorly-soluble drugs at concentrations above CMC [9,10]. Solubility of enrofloxacin in CTAB, SDS and Tween 80 at 25 and 50 mM micellar concentration in water and PB (pH 7.4) is given in Table 2. Tween 80 was not found to be a good solvent for enrofloxacin. Amongst ionic surfactants, SDS was found to be a much better solvent as compared to the cationic surfactant, CTAB. Solubility was found to be exceptionally high in SDS. About 3.4 and 3.8 mg/ ml of enrofloxacin could be dissolved in water and buffer medium, respectively at 50 mM micellar concentration of SDS.

A very high solubility of drug in SDS shows that the non-polar part of the molecule is solubilized into the micellar interior while positively charged piperazinyl groups are in the outer core, decreasing the repulsive forces between the head groups of the surfactant molecules, thereby decreasing CMC, increasing aggregation number and volume of micelles and increasing solubilization. Much lower solubility in CTAB showed that the orientation of solubilized molecules is such that the negatively charged carboxyl groups do not take part in solubilization. Tween 80 also enhanced the solubility of drug but the solubilization power was lower than ionic surfactants. In non-ionic surfactant, the drug appears to be located preferentially in the palisade layer and is stabilized by formation of hydrogen bonds with polyoxyethylene groups in the surfactant. This is possible since the drug molecule contains seven hydrogen bond acceptors and one hydrogen bond donor.

Thermodynamically the solubilization can be considered as partitioning of the drug between micellar and aqueous phase, and the standard free energy of solubilization (ΔGS0) can be represented by the expression ΔGS0 = -RT ln K, where R is the gas constant, T is the absolute temperature and K is the partition coefficient of drug between micellar and aqueous phase [11]. The micelle-water partition coefficient (K) is the ratio of the drug concentration in the micelle to the drug concentration in water for a particular surfactant concentration and is given by K= (Stot- Sw)/Sw. K and ΔGS0 values for various surfactants are given in Table 4. Surfactant solubilization parameters are found to be very low for Tween 80 and exceptionally high for SDS and thus SDS is an excellent solvent for enrofloxacin.

Solvent* Surfactant    Solubilization   parameters
K ΔGS0(kJmol-1)
Water Buffer Water Buffer
25 mM CTAB 1.227 3.963 -0.507 -3.412
50 mM CTAB 1926 6.201 -1.624 -4.522
25 mM SDS 16.813 18.979 -6.993 -7.294
50 mM SDS 22.208 25.004 -7.683 -7.977
25 mM Tween-80 0.491 0.597 1.762 1.278
50 mM Tween-80 1.349 1.412 -0.742 -0.855

Table 4: Solubilization parameters for various surfactants

Since the commonly used dose of enrofloxacin is 5.7 mg, the minimum solubility of drug required for 10 ml, 5 ml and 2 ml doses are 570, 1140 and 2850 μg/ ml, respectively. Solvents with drug solubility greater than that required for 10 ml, 5 ml and 2 ml doses are given in Table 5. The data can be useful for the development of parenteral formulations of this drug.

Solvents with drug solubility greater than that required for
10 ml dose 5 ml dose 2 ml dose
40% glycerol in buffer, 40% PG in buffer, 25 mM SDS in buffer,
20% ethanol in buffer, 40% ethanol in buffer, 50 mM SDS in water and buffer
25 & 50 mM CTAB in buffer, 25 mM SDS in buffer,  
40% PG in buffer, 50 mM SDS in water and buffer  
40% ethanol in buffer,    
25 mM SDS in buffer,    
50 mM SDS in water and buffer    

Table 5: Solvents with drug solubility greater than that required for minimum dose

References