Sulfonamides are bacteriostatic agents which are systematically used in the treatment of bacterial infections. SMZ, a derivative of sulfonamide, inhibits the synthesis of folic acid, which is an important metabolite of bacteria’s DNA synthesis (Martindale, 1993; Raja et al., 2009). SMZ is absorbed from gastro-intestinal area; however, its absorption and bioavailability are limited with its dissolution rate, due to its low solubility like all the other sulfonamide groups. The aim of the study was to prepare  the  inclusion  complexes  of  SMZ  using  CD_s to enhance the solubility, dissolution rate and oral bioavailability.

CD_s which are made up of 6D (+) glucopyranose units at minimum, with α 1-4 glycoside bonds and formed as a result of degradation of starch by the help of glucosyltransferase (CGT) enzyme, are cyclic oligosaccharides with hydrophobic cavity and hydrophilic outer surface (1).CD_s can be defined as enzymatically modified starch made of glucopyranose units (Szejtli, 1991a). The commonly  available  CD_s  are  α-, β-  and  γ-cyclodextrins, which consist of six, seven and eight glycopyranose units, respectively (Gabriel et al., 2016; Uekama and Otagiri, 1987). The more the glucose units, the bigger the dimensions of CD_s. CD_s are molecules with a polar hydrophilic outside, and an apolar hydrophobic cavity, which provides a guest-host relation to hydrophobic drugs in hydophilic medium which is called “inclusion complex” (Brewster and Loftsson, 2007; Jambhekar and Breen, 2016).

Some CD_s contain 9 and more glucose units, but they are not used since it is difficult to obtain them in pure form and they have low complex forming capacity. Those containing less than 6 units of glucose, on the other hand, cannot be prepared for steric reasons (Bekers et al., 1991; Fromming and Szejtli, 1994).

In these complexes, the drug (guest molecule) is entrapped in the cavity of the cyclodextrin (CD) (host). After the complexation, some physical properties of the guest molecule can be changed. For instance, the strength against oxidation, hydrolysis and photochemical reactions can increase, the evaporation speed of volatile substances can significantly decrease. Also when poor soluble drugs are in complex form, the solubility increases. For this reason, in order to enhance the water solubility of poor soluble substances, complex forming with CD’s are widely used (Duchene and Woussidjewe, 1990; Sharma and Baldi, 2016; Sinha et al., 2007; Szejtli, 1988).

The stoichiometries of active material:cyclodextrin complexes and the numerical values of their stability or binding constants are frequently obtained the from the plots of drug solubility against cyclodextrin concentration. This phase-solubility technique was first developed by Higuchi and Connors (1965). Phase-solubility diagrams are categorized into A and B types. A type phase-solubility profiles are obtained when the solubility of the active material increases with an increase in cyclodextrin concentration. The A-type profile is further subdivided into three profiles. The A_L profile indicates that there is a linear increase in solubility as a function of cyclodextrin concentration; the A_P profile indicates an isotherm, wherein the curve deviates from linearity in a positive manner, suggesting that a ligand or a solubilizer is proportionally more effective at higher concentrations. Conversely, the A_N type relation indicates a negative deviation from linearity, which means that the cyclodextrin is proportionally less effective at higher concentration (Jambhekar and Breen, 2016).

In general, the water-soluble CD_s form A-type phase solubility profiles, whereas the less soluble natural CD_s generally form B-type profiles. Type B phase-solubility profiles indicate the formation of a complex with naturally occurring CD_s, in particular β-CD (Brewster and Loftsson, 2007).

It is accepted that water soluble polymers or non-ionic surfactants have significant effects on the formation of the inclusion compounds (Hirlekar et  al.,  2009;  Loftsson et al., 1991, 1994, 1996). The effects of commonly used water soluble polymers, polyethylene glycol 4000 (PEG4000), polyethylene glycol 10000 (PEG10000), polyethylene glycol 20000 (PEG20000), hydroxypropyl methylcellulose (HPMC) and the non-ionic surfactants polysorbate 20 (PS20), polysorbate 40 (PS40), and polysorbate 60 (PS60) on the dissolution rate and solubility of SMZ from the inclusion complexes were also investigated.

In this study, the solid dispersions were prepared by coprecipitation method (Dhanaraju et al., 1998; Elgindy et al., 2011; Özdemir and Erkin, 2012; Polonosamy and Khonam, 2011; Patel and Rajput, 2009). The rates of release of the active material from the complexes were determined from dissolution studies using USP XXII paddle method (USP XXII, 1990).

 

Sulfamethoxazole (SMZ) was obtained from Fako Medical (Turkey); in addition, the following were used in the study: β-cyclodextrin (β-CD), hydroxypropyl-β-cyclodextrin (HPβ-CD), γ-cyclodextrin (γ-CD) (Fluka), polyethylene glycol 4000 (PEG4000), polyethylene glycol 10000 (PEG10000), polyethylene glycol 20000 (PEG20000), hydroxypropyl methylcellulose (HPMC) polysorbate 20 (PS20), polysorbate 40 (PS40) and polysorbate 60 (PS60) (Merck). All other chemicals were of analytical grade.

Apparatus

UV spectrophotometer (Shimadzu UV 1202, Japan), IR spectrophotometer (Jasco FT/IR 420, Japan), differential scanning calorimetry (DSC) (Netzsch Geratebau DSC 204, Germany) and dissolution rate apparatus (Aymes, Turkey) were used.

Phase-solubility studies                

Solubility measurements were performed by the method of Higuchi and solutions containing various concentrations of CD_s ranging from 2 × 10^-3 to 15 × 10^-3 M (the solubility of β-CD is 16.3 × 10^-3 M) were shaken with 100 mg of SMZ in sealed flasks in a thermostate water bath at a constant temperature of 37°C (Loftsson and Duchene, 2007). After an equilibrium was attained (approximately after three days), aliquots were with drawn and filtered through 0.45 μm filters. A portion of the filtrate was then diluted with water and analyzed spectrophotometrically. The solubility constant and the ratio of SMZ:CDs in the complexes were calculated from the phase solubility diagram (Connors and Mollica, 1966; Higuchi and Connors, 1965).

The solubilizing effect of water-soluble polymers and non-ionic surfactants was also investigated with phase-solubility studies. For this purpose, an 100 mg active material was added to aqueous solutions containing 0 to 15% of CD_s and 0.50% (w/v) of water soluble polymers (PEG4000, PEG10000, PEG20000 and HPMC) and non-ionic surfactants (PS20, PS40 and PS60). After equilibration, which took at least three days took place at room temperature, the suspensions were filtered through at 0.45 μm membrane fitler and analyzed by UV spectrophotometer. The type of complexation and the apparent stability constants (K_c) of the drug : cyclodextrin complexes were calculated from the slope of the phase solubility diagrams (Loftsson et al., 1991, 1994, 1996).

Solubility studies

The cationic form of SMZ is the dominating form at pH below 1.5, while the nonionized form dominates between pH 2.0 and 6.0 and the anionic form dominates pH above 7.0. Although CD_s are able to solubilize both nonionized and the anionic forms of the drug, they have a small solubilizing effect on the cationic form. After taking these results into consideration, the pH of the mediums to be used for solubility and the dissolution studies were adopted to be either pH 4.5 or 7.0.

Excess amount of active material (more than it can be dissolved) was added to a closed flask with either pH 4.5 or 7.0 and then mixed in a magnetic mixer at 37°C. Thereafter, the liquid phase was filtered through 0.45 μm filters and the amount of active material in the solution was determined. Solubility of the active material was calculated by the point measured during the formation of the equilibrium status.

Preparation of inclusion compounds

Coprecipitation method given below was used to prepare SMZ’s inclusion compounds with CD_s.

Coprecipitation method

SMZ was dissolved in acetone and added to the aqueous solution of CD at 1:1 molar ratio. The solvent was allowed to evaporate and then dried under vacuum at 40°C for 24 h and sieved to obtain the optimum particle size (Dhanaraju et al., 1998).

The solid complexes including solubility enhancer such as water-soluble polymers (PEG4000, PEG10000, PEG20000, HPMC) and non-ionic surfactants (PS20, PS40, PS60) were also prepared with the same method.

Characterization of complexes

The infrared spectroscopy (IR) of SMZ, β-CD and the inclusion complexes were measured with KBr discs at 400 to 4000 cm^-1. The amount of active material in each sample was kept constant in each measurement. Differential scanning calorimetry (DSC) was performed using a scanning rate of 10°C/min on a Netzsch Geratebau DSC 204. Samples were heated in a sealed aluminum pans from 50 to 300°C.

Dissolution rate studies

Active material release from all the inclusion compounds prepared were performed by using the USP XXII paddle method at 50 rpm in 37°C for three hours (USP XXII, 1990). It was done in 900 ml and at pH 4.5 and 7.0 mediums. Samples taken at certain intervals were measured spectrophotometrically.

 

Phase solubility diagram

Data obtained from the phase solubility studies were used to calculate the apparent stability constant (Kc) according to the following equation:

 

K_c = S_t – S_o / [ S_o (L_t – ( S_t + S_o ) ]                                  (1)

S_t = total concentration of dissolved SMZ; S_o = molar solubility of SMZ in the presence of cyclodextrin; L_t = total cyclodextrin concentration.

A B_s type solubility graph was obtained with β-CD. The stoichiometric ratio of the complex determined from the descending part of the diagram was found to be 1:1 (SMZ:β-CD) and the stability constant was found as 122.3 M^-1 (Figure 1). A type phase-solubility profiles are obtained when the solubility of the active material increases with an increase in cyclodextrin concentration. A_L type solubility graph was obtained by chemically modified HPβ-CD and the stability constant was too low (39 M^-1) (Figure 2). No significant increase in solubility of SMZ was observed with γ-CD and it was concluded that the inclusion complex was not formed (Figure 3). However, high stability constants of the complexes indicate high stability, very high values complicate the solubility of the drug. Comparing the stability constants, it was concluded that the β-CD was the best complex producing CD for SMZ. Thus, in all the remaining studies, only β-CD was used as host molecule (Duchene and Woussidjewe, 1990).

 

 

 

Solubility studies

At pH 4.5 (Figures 4 and 5) and pH 7.0 (Figures 6 and 7), the solubility of the active material was enhanced in the presence of water soluble polymers (PEG4000, PEG10000 and PEG20000) and non-ionic surfactants (PS20, PS40 and PS60) (Table 1). No significant increase in the solubility of active material with HPMC was observed. Hence, this result is not given in Table 1. As can be seen in Figure 5, a higher increase in solubility was observed when PS20 was used rather than PS40 and PS60.

 

 

 

 

The characterization of inclusion compound formation

Infrared spectrophotometers (IR) and differential scanning calorimetry (DSC) were used to prove the formation of inclusion complexes between the active material and β-CD. The characteristic bonds of SMZ at 1599 (-SO₂NH) and 1306 (-SO₂) which were observed by IR, seemed to disappear in the inclusion compounds or become modified significantly (Figure 8).

 

 

The observed decreases in the intensities of the characteristic bonds of SMZ may be due to its restriction within the β-CD cavity.

Figure 9 shows the thermogram of SMZ, β-CD  and the inclusion complexes obtained from DSC measurements. The β-CD displayed no peaks in the temperature range between 50 and 300°C, while the SMZ exhibited its characteristic endothermic peak associated with the melting point of the drug around 171°C. Although, inclusion complexes showed broad peaks, the peak of the SMZ disappeared especially in the presence of PEG20000 and PS20 without distinct phase transition around this temperature.

 

 

The SMZ:β-CD coprepicipitated complex showed a less intense peak. The absence of the peak in DSC curves may be considered as a strong indication of the inclusion formation of the drug into the β-CD cavity.

Studies on dissolution rate

The studies conducted at pH 4.5 conditions, in which active material exists in non-ionic form, demonstrated an increase in dissolution rates of complexes as compared to pure active drug rates. On the other hand, adding water soluble polymers (PEG4000, PEG10000 and PEG20000) to the formulation by 0.50% (w/v) increased the drug release as compared to the pure drug. 4.7% of pure SMZ were dissolved within 30 min, whereas the measured values for the formulations including water soluble polymers coded as β-CD:SMZ, β-CD:SMZ:PEG4000,    β-CD:SMZ:PEG10000      and     β-CD:SMZ:PEG20000 were 38.0, 22.5, 38.0 and 41.7%, respectively (Figure 10).  It was  found  that, as the  PEG molecule weight increases, so does the active material release. The  effect  of non-ionic surfactants (PS20, PS40 and PS60) with the concentration of 0.5% (w/v) were also examined with dissolution tests. The active material released at the end of the three hours were 39.1, 42.9, 44.1%, and 37.3% from β-CD: SMZ, β-CD:SMZ:PS20, β- CD:SMZ:PS40, and β-CD:SMZ:PS60 respectively. To conclude,  the    presence    of    these   substances   was observed to have a somehow significant effect on the active material release (Figure 11).

 

 

 

 

 

 

 

 

 

As a result of this study, it may be concluded that the solubility and the dissolution rate of SMZ was significantly enhanced by the complex formation with β-CD. The water soluble polymers and non-ionic surface active substances added to the solid dispersions increased the solubility and dissolution rate of active material-CD complexes at either pH 4.5 or 7.0.

 

The authors declare that there is no conflict of interest.