A novel sustained release drug–resin complex-based microbeads of ciprofloxacin HCl

Abstract

Objective A novel multiparticulate system for the gastro-mucoadhesive delivery of ciprofloxacin HCl (CFN) was developed with the help of ion-exchange resin to deal with urinary tract (UT) infections effectively. Materials and methods An optimized complex (resinate) of CFN with sodium polystyrene sulfonate USP resin was prepared and entrapped within microbeads of sodium alginate and pectin. The developed systems were evaluated for drug entrapment efficiency, percentage of mucoadhesion and in vitro release patterns in simulated gastric fluid (pH 1.2). Results and discussion The interaction of the resin complex and polycation via alginate was consequently supported the formation of polyelectrolyte complex membrane. The in vitro drug release studies demonstrate that formulation without drug–resin complex (NRB) released the drug more swiftly than formulation containing drug–resin complex (DRC). This controlled release pattern of drug, resin complex containing microbeads was owed to complexation between drug and resin. Conclusion Preliminary results from the study suggested that this drug–resin complex-entrapped microbeads can be used to incorporate other antibiotic drugs and could be effective against UT infection. Such developed formulation could be subjected to in vivo studies in future in order to prove their efficacy for such type of infections.

Keywords:

• ciprofloxacin HCl
• microbeads
• sodium polystyrene sulfonate USP resin
• sodium alginate
• pectin

Introduction

A range of antimicrobial agents have been employed for the management of different pathological conditions. Ciprofloxacin HCl (CFN) prescribed for urinary tract (UT) infections is a broad-spectrum antibacterial agent of fluoroquinolones category with DNA gyrase inhibitory action. It is acid stable (pH 3.5–5) with a short half-life (3–4 h) and plasma protein binding up to 20–35% resulting into remarkably high concentration biologically. These properties of CFN make it to be used for shorter courses with repeated dosing intervals (William and Petri 2011). High dosing frequency (250–150 mg, BD), the low bioavailability (50–60%) due to high first-pass metabolism and elimination half-life of 3–4 h are also restricted the effectiveness of this antibacterial agent. It is absorbed from upper gastrointestinal (GI) tract and most of the drug is excreted via urine as unabsorbed form. Hence, it can be envisaged that increased residence time at the absorption site can enhance the absorption and bioavailability of CFN. Therefore, sustained release beads could contribute toward improved absorption and enhanced bioavailability of drugs as a result of prolonged retention in the GI tract, or specific targeting of drugs to the absorption site (Tadros 2010).

Sodium polystyrene sulfonate (SPS) USP (Amberlite IRP69) resin is an insoluble, strongly acidic, sodium form, cation exchange resin. It is suitable for both as an active ingredient and as a carrier for basic (cationic) drugs. Controlled or sustained release properties can also be imparted to oral dosage formulations through the formation of resin–drug complexes and with compatible coating technologies. The cationic water soluble drugs, i.e. chlorpheniramine maleate, pseudoephedrine HCl and propranolol HCl were bound to a cation-exchange resin, i.e. Amberlite IRP69 and microencapsulated with an aqueous solvent evaporation method (Sriwongjanya and Bodmeier 1997).

Previously our research group developed repaglinide–cholestyramine complex-loaded ethylcellulose microspheres to gastric mucosa for effective management of type 2 diabetes mellitus (Jain et al. 2014). Kulkarni et al. (2011) reported diltiazem HCl–indion 254 complex-loaded microcapsule of gellan gum and egg albumin. Madgulkar et al. (2009) developed release modulated beads of losartan potassium complexed with cholestyramine. Cation exchange resins, i.e. Tulsion 344, and Amberlite IR 120 are effectively bind with the CFN owed to its hydrochloride form, which easily bind with the –SO₃H group of resin (Upadhye et al. 2008). The aim of the present study was to develop sustained release drug–resin based multi-particulate system of CFN for the effective treatment of UT infections.

Materials and methods

Materials

Ciprofloxacin hydrochloride was obtained as a gift sample from M/s Akums Drugs and Pharmaceutical Ltd., Haridwar, India. SPS USP resin (Tulsion 344®) was obtained as a gift sample from Thermex Ltd., Pune, India. Calcium chloride, sodium alginate and pectin were purchased from CDH Laboratory Pvt. Ltd., Mumbai, India. All other chemicals used were of analytical grade.

Purification and activation of resin

SPS resin (5 g) was washed in triplicate with 50 ml of deionized water in a flask followed by successive washing with methanol (50 ml) and deionized water to remove organic and color impurities. Each washing period lasted 1 h and was performed with continuous stirring. Consequently, the resin was treated with 60 ml of 2 M NaOH and 60 ml of 2 M HCl, respectively, for activation followed by washing after each treatment with deionized water. The resin in hydrogen/acid form was washed with deionized water until elute was neutral. The activated resin was dried in a hot air oven at 50 °C for 12 h (Cuna et al. 2001).

Preparation of drug–resin complex

A batch process (Kulkarni et al. 2011; Pisal et al. 2004) was employed for the preparation of drug–resin complexes (DRC) using different ratios of SPS resin (Table 1). For each batch of drug–resin ratio, required quantity of previously activated resin was added to 20 ml of deionized water followed by stirring on a magnetic stirrer (REMI, Mumbai, India) at 200 rpm for 30 min. Aqueous solution of CFN was added and stirred continuously for 4 h. After stirring, each batch was filtered through Whatman filter paper (#41) and washed with deionized water to remove uncomplexed drug. The complexes were dried overnight in a hot-air oven at 40 °C and then stored in tightly closed desiccator. Simultaneously, the filtrate containing unbound drug was analyzed spectrophotometrically by UV spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan).

Table 1. Ratio of sodium polystyrene sulfonate resin to drug for drug–resin complex.

S. No.	Assigned code	Drug–resin
1	DRC-A	1:1
2	DRC-B	1:1.5
3	DRC-C	1:2
4	DRC-D	1:3
5	DRC-E	1:4

DRC, drug–resin complex.

The ion exchange drug–resin combination which showed best result in drug binding efficiency as well as consume comparatively less resin was selected as optimum drug–resin complex for the preparation of sustained release beads (data not shown).

Preparation of sodium alginate–pectin microbeads

The DRC loaded alginate–pectin beads were prepared by an ionotropic gelation method (Assifaoui et al. 2013). For this, different concentration of sodium alginate and pectin was dissolved in distilled water (Table 2). These polymeric solutions were mixed and stirred with continuous heating. The DRC was mixed uniformly with the resultant alginate–pectin mixture with stirring. The resultant dispersion was added to 100 ml of cross-linking agent, i.e. 5% w/v calcium chloride (CaCl₂) solution in a drop-wise manner via a needle (21G). The gelled beads were formed instantaneously and allowed to cure for 10 min in the CaCl₂ solution. The beads were collected by filtration and washed with deionized water. Finally, the beads were dried in oven at 37 °C for 24 h. A batch of beads containing only drug, i.e. non-resin beads (NRB), was prepared using the same method. The amount of DRC (optimized) was kept constant in all formulations and only polymer ratio was altered.

Table 2. Batch specification of beads.

Batch	Code	DRC:polymer	Polymer ratio (alginate:pectin)	Drug:polymer
Batch-I	NRB1	–	2.5:2.5	1:1
	BDRC-A1	1:1	2.5:2.5	–
	BDRC-B1	1:1	3:2	–
	BDRC-C1	1:1	4:1	–
	BDRC-D1	1:1	1:4	–
	BDRC-E1	1:1	2:3	–
Batch-II	NRB2	–	2.5:2.5	0.5:1
	BDRC-A2	0.5:1	2.5:2.5	–
	BDRC-B2	0.5:1	3:2	–
	BDRC-C2	0.5:1	4:1	–
	BDRC-D2	0.5:1	1:4	–
	BDRC-E2	0.5:1	2:3	–

DRC, drug–resin complex; NRB, non-resin beads; BDRC, beads containing drug–resin complex.

Characterization of DRC and microbeads

Drug loading in SPS resin

Sample of DRC (50 mg) was slurried with an excess of 10% NaCl solution until the drug was totally displaced from the resin. Sample of this supernatant was taken, diluted and drug concentration was measured by UV-visible spectrophotometer at 277 nm.

Micromeritic properties

The particle size and shape of drug, resin, DRC and formulations were determined by a light photo microscope. The beads were also characterized for true density, tapped density, compressibility index and porosity. These properties were calculated as follows:

All the experiments were performed in triplicate.

Percentage yield and drug entrapment efficiency

The prepared beads were collected by filtration, and dried at 37 °C in the oven for 24 h. Percentage yield was calculated using the following formula:

Drug entrapment efficiency (DEE) was calculated by a previously suggested method (Abd El-Ghaffar et al. 2012). Accurately weighed 50 mg of beads from each batch were taken separately and placed in 20 ml of phosphate buffer saline (PBS, pH 7.4) for 1 h. The swelled beads were crushed using pestle and mortar and placed in 10% NaCl solution and kept for 24 h with occasional shaking at 37 ± 0.5 °C. After the stipulated time, the dispersion was stirred at 500 rpm for 20 min using a magnetic stirrer to extract the complete drug. Then, the polymeric dispersion was centrifuged at 4000 rpm for 30 min to remove the polymer debris and resin, clear solution was collected, diluted and analyzed by a UV spectrophotometer at 277 nm for drug content. All experiments were performed in triplicate. The DEE of beads was calculated using the following formula:

Infrared spectroscopy

IR spectroscopy of drug, resin and DRC was performed on Fourier Transform infrared spectrophotometer (FT-IR 8400S, Shimadzu). The sample and KBr were mixed properly in the ratio 95:5 and placed on the sample holder. The spectra were scanned in the range of 4000–400 cm⁻¹.

Differential scanning calorimetry

A differential scanning calorimeter (DSC) (DSC-60, Shimadzu) equipped with an intracooler and a refrigerated cooling system was used to analyze the thermal behavior of drug, resin and DRC in hermetically sealed flat aluminum crucible, with temperature ranging from 50 to 600 °C. Heating/cooling rate was 10 °C/min. Nitrogen was purged at 30 and 50 ml/min through a cooling unit.

Swelling studies

The swelling property of the alginate–pectin beads was determined in simulated gastric fluid (SGF, pH 1.2) (Anal and Stevens 2005). Accurately weighed dry beads (50 mg) were placed in a petridish containing 20 ml of SGF at 37 °C and allowed to swell. The swollen beads were periodically removed and weighed. The wet weight of the swollen beads was determined by blotting them with filter paper to remove moisture adhering to the surface, immediately followed by weighing on an electronic balance. All experiments were done in triplicate. The percentage of swelling of the beads was calculated from the formula:

In vitro drug release study, statistical treatment and model fitting of data

The in vitro release of CFN from NRB and DRC containing beads was studied for a period of 2 h for each dissolution medium such as SGF (pH 1.2), KCl–HCl buffer (pH 4.5), PBS (pH 6.8) and PBS (pH 7.4) in 900 ml at 37 ± 0.5 °C using USP-I basket type dissolution apparatus (Jyoti Scientific Laboratories, Gwalior, India) at rotation speed of 100 rpm. Aliquots were taken at predetermined intervals and replenished immediately with the same volume of fresh media. Aliquots were passed through Whatman filter paper (#41) before spectrophotometric analysis. Same procedure was repeated for DRC and marketed tablet formulation of CFN (CIFRAN OD, 1000 mg, Ranbaxy, Gurgaon, India).

Dunnett’s multiple comparison test was performed to determine differences in in vitro drug release of CFN from SPS resin-based microbead formulations and marketed formulation of CFN (CIFRAN). Calculations were performed with the GraphPad-Instat Software Program, La Jolla, CA.

Five kinetic models including the zero-order, first-order, Higuchi matrix, Peppas–Korsmeyer and Hixson–Crowell release equations were applied to interpret the in vitro release data in order to find out the best fit equation (Jain et al. 2014).

Results and Discussion

Purification and activation of resin

Resin purification is desired to remove impurities associated with industrial scale production or during storage or handling of resin. SPS resin was treated with methanol to solubilize the impurities, which can be easily separated by filtration. Furthermore, it was washed with deionized water for complete clearance of impurities. The activation process exposed the exchangeable groups of resin for rapid drug exchange and hence higher drug binding was achieved. The highest drug binding efficiency with resin was attained when both acid–alkali treatments were given for activation. Acidic group of resin was activated with hydrochloric acid (2 M) and basic group was activated with sodium hydroxide (2 M).

Preparation of drug-resin complex

The drug was loaded to SPS resin by batch process (Pisal et al. 2004). CFN loading involved replacement of Na⁺ of SPS resin with Cl⁻ of drug. SPS, a strong cationic exchange resin, can exchange its sodium cation with cationic drug and produce SPS–ciprofloxacin complex and sodium chloride. Schematic presentation of complex formation is shown in Figure 1.

Figure 1. Schematic diagram of chemical reaction for drug resin complex.

Process of drug loading onto ion-exchange resin

Initially the binding sites in the SPS resin are all occupied by counter-ions (sodium ion). Once exposed to a drug solution, the counter-ions on the surface are displaced by drug ions and carried away by the well-stirred bulk liquid. The adsorbed drug ions further diffuse inwards due to a concentration gradient, and exchange with counter-ions. The free counter-ions diffuse to the surface of the resin owing to a concentration gradient, weaker affinity to the polymer and larger diffusion coefficient than drug ions (Wagh and Pawar 2012). The counter-ions arriving at the surface exchange with drug ions in the liquid adjacent to the SPS resin and then removed. This ion exchange–diffusion–removal process proceeds until equilibrium is established between drug concentration in the SPS resin and in the liquid. CFN loading in SPS was increased with increasing the concentration of resin. Drug–resin ratio 1:1 was selected due to lesser amount of resin involved and good percentage of drug loading was obtained as compared with 1:1.5, 1:2, 1:3 and 1:4 drug–resin ratio. Drug–resin ratio 1:1 gives optimum loading (80.75%) and considered as optimized drug–resin complex and was further used for the preparation of beads.

Preparation of DRC-loaded microbeads

Sustained release beads of CFN were prepared using alginate and pectin polymers, as these polymers easily gelled in the presence of cross-linking agent. An aqueous solution of sodium alginate and pectin containing DRC was dropped into CaCl₂ solutions and gelled spheres were formed instantaneously. Intermolecular cross-links were formed between the Ca²⁺ and COOH⁻ of the pectin molecules and Ca²⁺ and Na⁺ of alginate molecules (Matricardi et al. 2008). Schematic diagram of preparation of bead is shown in Figure 2.

Figure 2. Schematic diagram of preparation of bead by an ionotropic gelation method.

Characterization of DRC and microbeads

Micromeritic properties

Drug, resin, DRC and prepared beads were subjected for image analysis using light photo microscope. The microscopic image of drug and SPS resin showed their crystalline form (Figure 3A and B). The increased size of DRC as shown in Figure 3(C) clearly indicates drug and resin complexation. Also, the image of bead illustrates the spherical shape (Figure 3D). The average particle size of optimized DRC (i.e. DRC-A) was found to be 19.53 ± 4.24 μm, which was slightly more than the usual particle size of pure resin (i.e. 11.25 ± 2.50 μm) indicating the presence of drug in it. The particle size of the prepared formulations, i.e. batch-I and batch-II ranged from 1.127 ± 0.13 to 1.452 ± 0.08 mm and 1.123 ± 0.12 to 1.427 ± 0.08 mm, respectively (Table 3). By keeping all factors constant, the bead size was found to increase with the increase in the concentration of pectin (Kulkarni et al. 2011). However, as the amount of resinate increases, the size of beads increases because resinate might have occupied the interstitial space between polymer segment. This aspect was in agreement with the previously published results (Halder et al. 2005). The tapped density of batch-I and batch-II was found to be in the range from 1.04 ± 0.52 to 1.19 ± 0.48 g/cm³ and 1.06 ± 0.63 to 1.22 ± 0.39 g/cm³, respectively. The true density of batch-I and batch-II was calculated between 1.25 ± 0.31 and 1.61 ± 0.64 g/cm³ and 1.25 ± 0.38 and 1.51 ± 0.64 g/cm³, respectively. The tapped density and true density of formulations was found to be more than the density of gastric fluids (1.04 g/cm³), which indicate that beads lack the floating ability. Porosity of batch-I and batch-II was found to be 14.96 ± 0.57–29.53 ± 0.41% and 13.12 ± 0.47–27.69 ± 0.49%, respectively, which indicates the porous nature of beads (Table 3). The percentage of compressibility index values of batch-I ranged between 10.63 ± 0.56% and 13.63 ± 0.27% and batch-II from 11.63 ± 0.46% to 14.90 ± 0.69%, suggesting good flow characteristics.

Figure 3. Light microscopic photographs at 10×. (A) CFN, (B) SPS resin, (C) Drug–resin complex and (D) microbead.

Table 3. Micromeritic studies of the prepared formulations.

Batch	Formulation code	Particle size (mm)	Tapped density (g/cm^3)	True density (g/cm^3)	Compressibility index (%)	Porosity (%)	Yield (%)	Drug entrapment efficiency (%)
Batch-I	NRB1	1.13 ± 0.13	1.19 ± 0.48	1.43 ± 0.28	12.04 ± 0.19	16.67 ± 0.51	91.90 ± 1.03	76.24 ± 1.02
	BDRC-A1	1.35 ± 0.09	1.04 ± 0.52	1.28 ± 0.49	13.06 ± 0.78	18.80 ± 0.25	93.50 ± 1.12	78.83 ± 1.36
	BDRC-B1	1.31 ± 0.11	1.09 ± 0.61	1.35 ± 0.58	12.34 ± 0.64	19.61 ± 0.59	95.70 ± 1.02	79.74 ± 0.65
	BDRC-C1	1.20 ± 0.12	1.25 ± 0.37	1.47 ± 0.29	11.00 ± 0.27	14.97 ± 0.32	97.50 ± 1.34	80.74 ± 1.25
	BDRC-D1	1.45 ± 0.08	1.14 ± 0.49	1.61 ± 0.64	13.63 ± 0.27	29.53 ± 0.41	90.60 ± 1.27	71.55 ± 1.18
	BDRC-E1	1.39 ± 0.09	1.06 ± 0.57	1.25 ± 0.31	10.63 ± 0.26	14.96 ± 0.57	89.20 ± 1.17	74.97 ± 1.11
Batch-II	NRB2	1.12 ± 0.12	1.11 ± 0.46	1.31 ± 0.31	12.77 ± 0.19	15.64 ± 0.57	89.13 ± 1.12	77.02 ± 1.36
	BDRC-A2	1.32 ± 0.07	1.16 ± 0.57	1.39 ± 0.57	12.90 ± 0.69	16.29 ± 0.31	93.73 ± 1.09	78.43 ± 1.16
	BDRC-B2	1.30 ± 0.14	1.06 ± 0.63	1.47 ± 0.48	13.89 ± 0.54	27.69 ± 0.49	94.17 ± 1.36	79.38 ± 1.48
	BDRC-C2	1.19 ± 0.11	1.16 ± 0.47	1.35 ± 0.71	14.90 ± 0.69	14.01 ± 0.62	96.10 ± 1.19	80.78 ± 0.99
	BDRC-D2	1.43 ± 0.08	1.22 ± 0.39	1.51 ± 0.64	12.07 ± 0.59	19.55 ± 0.41	92.80 ± 1.28	72.39 ± 1.69
	BDRC-E2	1.34 ± 0.13	1.09 ± 0.29	1.25 ± 0.38	11.63 ± 0.46	13.12 ± 0.47	90.53 ± 1.06	73.45 ± 1.04

DRC, drug–resin complex; NRB, non-resin beads; BDRC, beads contain drug–resin complex. Data are the average of values (mean ± SD) (n = 3).

Differential scanning calorimetry

DSC thermogram of pure CFN showed two peaks, one endothermic peak at 151.10 °C and one exothermic peak at 304.17 °C corresponding to loss of water and melting of pure drug, respectively (Figure 4A). These results were in agreement with the previously published results (Dillen et al. 2004). DSC curve of SPS resin (Figure 4B) showed one exothermic peak at 471.35 °C corresponding to melting of resin. Thermal analysis of DRC shows an exothermic peak at 412.03 °C corresponding to resin and absence of drug peak indicates its complex formation with resin (Figure 4C).

Figure 4. DSC thermograms of (A) CFN, (B) Tulsion 344 resin, (C) drug–resin complex (DRC-A).

Infrared spectroscopy

The obtained IR spectrum of CFN showed all prominent peaks at 3379.29 cm⁻¹ due to carboxylic acid (–OH stretching), 3533.59 cm⁻¹ due to cyclohexane (N–H stretching), 2924.09 cm⁻¹ due to cyclopropane (C–H stretching), 1627.92 cm⁻¹ due to aromatic (C = C stretching), 1710 cm⁻¹ due to cyclohexanone (C = O stretching), 1273.02 cm⁻¹ due to aromatic halide (C–F stretching) and 3093.82 cm⁻¹ due to aromatic (C–H stretching), confirming identification of the drug (Figure 5A). Furthermore, resin identification was also confirmed via IR spectrum and determined the prominent characteristic peaks at 833.25 cm⁻¹ due to aliphatic (C–H stretching), 1627.92 cm⁻¹ due to aromatic (C = C stretching), 3062.96 cm⁻¹ due to aromatic (C–H stretching) and 1411.89 cm⁻¹ due to sulfonate group (S = O stretching) (Figure 5B). Finally, drug–resin complex formation was confirmed by analyzing characteristic peaks of ciprofloxacin and resin in the spectrum, i.e. 3425.58 cm⁻¹ due to carboxylic acid (O–H stretching), 3545.67 cm⁻¹ due to cyclohexane (N–H stretching), 2924.09 cm⁻¹ due to cyclopropane (C–H stretching), 1627.92 cm⁻¹ due to aromatic (C = C stretching), 1705.07 cm⁻¹ due to cyclohexanone (C = O stretching), 1273.02 cm⁻¹ due to aromatic halide (C–F stretching) and 3032.10 cm⁻¹ due to aromatic (C–H stretching). Furthermore, conjugation between sulfonate group of Tulsion resin 344 and nitrogen of piperazine ring of ciprofloxacin was confirmed by analyzing characteristic peaks of sulfonyl group at 1334.74 cm⁻¹ due to asymmetric (S = O stretching) and 894.97 cm⁻¹ due to (N–S stretching) of drug–resin complex (Figure 5C).

Figure 5. IR spectra of (A) CFN, (B) Tulsion 344 and (C) drug–resin complex.

Percentage yield and DEE

Percentage yield of the prepared formulations, i.e. batch-I and batch-II was ranged from 89.2 ± 1.45% to 97.50 ± 2.04% and 89.13 ± 2.12% to 96.10 ± 2.36%, respectively, showing no regular trend as well as no significant change with increase in polymer content (Table 3). DEE of the batch-I and batch-II was ranged from 71.55 ± 1.41% to 80.74 ± 1.25% and 72.39 ± 1.89% to 80.78 ± 0.99%, respectively.

The decrease in initial alginate concentration decreased the DEE, as decrease in initial alginate concentration provides lesser number of binding sites of alginate for Ca²⁺ ions resulting in the formation of a less compact gel membrane, which, in turn, increases influx of Ca²⁺ ions leading to decrease the encapsulation of particles in beads as reported by Halder et al. (2005). It is evident with observations of this study that entrapment efficiency decreases with increase in pectin concentration in combination of alginate (Table 3).

Dynamic swelling study

The swelling of beads depends upon the extent of cross-linking. The swelling of beads decreases with an increase in the amount of sodium alginate, which may be due to the formation of stiffer network and also increases with increase in pectin concentration. At low cross-linking density, the polymer network is loose with more hydrodynamic free volume and can absorb more of the solvent resulting in higher swelling. Swelling property of the formulations (batch-I and batch-II) followed the order: BDRC-D₁ > BDRC-E₁ > NRB₁ ≥ BDRC-A₁ > BDRC-B₁ > BDRC-C₁ and BDRC- D₂ > BDRC-E₂ > NRB₂ ≥ BDRC-A₂ > BDRC-B₂ > BDRC-C₂, respectively (Figures 6 and 7). These results were in agreement with the findings of Halder et al. (2005).

Figure 6. Swelling properties of various formulations (Batch-I). NRB, non-resin beads; BDRC, Beads contain drug–resin complex.

Figure 7. Swelling properties of various formulations (Batch-II). NRB, non-resin beads; BDRC, beads contain drug–resin complex.

In vitro drug release study

It was observed that the optimized drug–resin complex, i.e. DRC-A released entire drug within 3.5 h (data not shown). Results clearly demonstrated that formulation without DRC (NRB₁ and NRB₂) release the drug more rapidly as compared to the formulation containing DRC, which released the drug in more sustained manner due to complex formation between drug and resin. The microbeads containing optimized drug–resin complex were shown controlled release behavior of CFN. The release pattern obtained from the prepared formulations (batch-I and batch-II) followed an order, i.e. DRC-A > NRB₁ > BDRC-D₁ > BDRC-E₁ > BDRC-A₁ > BDRC-B₁ > BDRC-C₁ and DRC-A > NRB₂ > BDRC-D₂ > BDRC-E₂ > BDRC-A₂ > BDRC-B₂ > C-C₂. The pattern also provides an idea about the effect of polymer content on drug release, i.e. more sodium alginate polymer, lesser was the drug release and more pectin concentration, higher was the drug release.

Drug release from the marketed product of CFN (CIFRAN OD) was found to be almost 100% within 3.5 h. Formulation BDRC-C₁ (60.05 ± 0.47%) from batch-I and BDRC-C₂ (60.99 ± 0.28%) from batch-II showed more sustained release behavior in 8 h study period than optimized drug–resin complex, marketed formulation of drug and other formulations (Figures 8 and 9).

Figure 8. In vitro drug release profile of CFN from various formulations (Batch-I). NRB, non-resin beads; BDRC, beads contain drug–resin complex; CIFRAN, marketed formulation of CFN. Values are mean ± SD (n = 3).

Figure 9. In vitro drug release profile of CFN from various formulations (Batch-II). NRB, non-resin beads; BDRC, beads contain drug–resin complex; CIFRAN, marketed formulation of CFN. Values are mean ± SD (n = 3).

When the data of CIFRAN OD (marketed product) were compared with all formulations, i.e. batch-I and -II containing DRC-A by one-way ANOVA (Dunnett multiple comparison) test, the in vitro drug release in simulated GI fluids from formulations was found to be significantly different (p < 0.01) (Tables 4 and 5).

Table 4. One-way ANOVA (Dunnett multiple comparison) test for in vitro drug release of CFN in GI fluid.

Comparison	Mean difference	q	p Value
CIFRAN vs. BDRC-A1	48.335	6.708**	p < 0.01
CIFRAN vs. BDRC-B1	51.276	7.116**	p < 0.01
CIFRAN vs. BDRC-C1	54.043	7.500**	p < 0.01
CIFRAN vs. BDRC-D1	42.779	5.936**	p < 0.01
CIFRAN vs. BDRC-E1	45.273	6.283**	p < 0.01

CIFRAN, marketed formulation of CFN; BDRC, beads containing drug–resin complex. ANOVA indicates analysis of variance; q, parameter obtained with p when performing ANOVA;

**very significant.

Table 5. One-way ANOVA (Dunnett multiple comparison) test for in vitro drug release of CFN in GI fluid.

Comparison	Mean difference	q	p Value
CIFRAN vs. BDRC-A2	49.096	6.872**	p < 0.01
CIFRAN vs. BDRC-B2	50.722	7.100**	p < 0.01
CIFRAN vs. BDRC-C2	54.012	7.560**	p < 0.01
CIFRAN vs. BDRC-D2	43.047	6.025**	p < 0.01
CIFRAN vs. BDRC-E2	45.686	6.395**	p < 0.01

CIFRAN, marketed formulation of CFN; BDRC, beads containing drug–resin complex. ANOVA indicates analysis of variance; q, parameter obtained with p when performing ANOVA;

**very significant.

The release mechanism of CFN from these formulations was also evaluated on the basis of theoretical dissolution equations including zero-order, first-order, Higuchi matrix, Peppas–Korsmeyer and Hixon–Crowell kinetic models. During the drug release study, it was found that NRB₁ and NRB₂ have maximum correlation coefficient (r² values), i.e. 0.9947 and 0.9957, respectively (Table 6), for zero-order model. Thus, the drug release from NRB₁ and NRB₂ does not depend upon the concentration. In case of BDRC-C₁ and BDRC-C₂, maximum correlation coefficient (r² values), i.e. 0.9936 and 0.9905, respectively, and n values 0.3232 and 0.3040 (n < 0.45), respectively, for Peppas–Korsmeyer model were calculated and it provides an idea about fickian diffusion of drug from these formulations. Thus, BDRC-C₁ and BDRC-C₂ were followed Peppas–Korsmeyer model for drug release.

Both selected formulations, i.e. BDRC-C₁ and BDRC-C₂ from batch-I and batch-II, respectively were shown almost similar type of controlled release behavior and thus may be selected for further studies.

Table 6. Release kinetics of formulation (Batch-I and Batch-II).

	Zero order	First order	Higuchi matrix	Korsemeyer– Peppas		Hixson– Crowell
	Formulation code	r^2	r^2	r^2	r^2	n	r^2
NRB1	0.9947	0.8858	0.9546	0.9862	0.2177	0.9066
BDRC-A1	0.9809	0.9277	0.9166	0.9968	0.288	0.8983
BDRC-B1	0.9837	0.9443	0.9212	0.9934	0.3055	0.9042
BDRC-C1	0.9857	0.9568	0.8252	0.9936	0.3232	0.9080
BDRC-D1	0.9874	0.9578	0.9489	0.9941	0.2749	0.9436
BDRC-E1	0.9906	0.9509	0.9415	0.9919	0.2838	0.9388
NRB2	0.9957	0.8951	0.9537	0.9883	0.2252	0.9151
BDRC-A2	0.9787	0.9274	0.9105	0.9979	0.2933	0.8989
BDRC-B2	0.9790	0.9347	0.9121	0.9971	0.2972	0.8902
BDRC-C2	0.9858	0.9522	0.9296	0.9905	0.304	0.9001
BDRC-D2	0.9866	0.9544	0.9438	0.9898	0.2603	0.9055
BDRC-E2	0.9909	0.9494	0.9399	0.9915	0.2644	0.9140

NRB, non-resin beads; BDRC, beads contain drug–resin complex. r², correlation coefficient.

Conclusion

Incorporation of SPS as a complex with CFN in the beads proved to be effective method to achieve the better sustained release behavior. The drug was released from BDRC by diffusion followed by erosion mechanisms through Fick’s law of diffusion. It was concluded that the method used for the preparation of microbeads was simple, reproducible and provides good yield. Prepared formulation showed better controlled release behavior when compared to marketed product of CFN. These developed formulations could be subjected to in vivo studies in order to design a viable formulation for better treatment against UT infections.

Declaration of interest

The authors declare no conflict of interest. Neeraj Prajapati gratefully acknowledges University Grants Commission, New Delhi, India, for awarding the Junior Research Fellowship during his PG program.