IMPROVEMENT OF BIOAVAILABILITY OF CEFUROXIME AXETIL ORAL SUSPENSION BY INCLUSION COMPLEXATION METHOD

RENJU PRABHAKARAN1, JANAKIRAMAN K.2, JYOTI HARINDRAN3

1Sance Laboratories Pvt Ltd, Kottayam, Kerala, India 686573, 2Department of Pharmacy, Annamalai University, Annamalai Nagar, Tamilnadu, India 608002, 3Department of Pharmaceutical Sciences, Mahatma Gandhi University, Kottayam, Kerala, India 686631
Email: [email protected]

Received: 28 Dec 2015 Revised and Accepted: 03 Feb 2016


ABSTRACT

Objective: Cefuroxime axetil, a prodrug of Cefuroxime, is a poorly water soluble drug, thus it has got only limited solubility and dissolution rate in gastric fluids. Also, the bioavailability of Cefuroxime axetil oral suspension is only 40-45% when compared to the 60% bioavailability of tablets. The objective of this study was to develop an oral suspension of Cefuroxime axetil with improved oral bioavailability by inclusion complexation method using Hydroxypropyl beta cyclodextrin [HP-Beta-cylcodextrin]

Methods: The complexation of Cefuroxime axetil and HP-Beta cyclodextrin was carried out at 1:1, 1:2, 1:2.5 and 1:3 ratios respectively. The prepared suspensions were evaluated for various parameters like pH, viscosity, re-dispersibility, pourability, and assay and in-vitro dissolution profile. A leading marketed product and the optimized formulation were evaluated for the pharmacokinetic parameters like Cmax, AUC0-t, AUC0-8 and Tmax in healthy adult male rabbits.

Results: Considering the in-vitro dissolution profile, formulation with 1:2.5 ratios of Cefuroxime axetil and HP-Beta cyclodextrin was selected as the optimized formulation. The Cmax of Optimized formulation and Marketed product were 148±1.26ng/ml and 126±1.52 ng/ml respectively, and the AUC0-t of Optimized formulation and Marketed product were 989±16.42 ng. h/ml and 613±24.26 ng. h/ml respectively, which shows a significant improvement in the bioavailability of optimized formulation

Conclusion: From the results obtained it can be observed that there is a significant improvement in the bioavailability of optimized formulation compared to the marketed product. This demonstrates that the inclusion complexation method with HP-Beta cyclodextrin can significantly improve the oral bioavailability of Cefuroxime axetil.

Keywords: Cefuroxime axetil, Bioavailability, Beta cyclodextrin, Inclusion complexation


INTRODUCTION

Cefuroxime axetil is a prodrug of Cefuroxime, which is a second-generation cephalosporin antibiotic with activity against wide range of organisms. The bactericidal activity of cefuroxime axetil is due to Cefuroxime’s binding to essential target proteins and the resultant inhibition of bacterial cell wall synthesis. After oral administration, Cefuroxime axetil is absorbed and then rapidly hydrolyzed by the nonspecific esterases which is distributed in the intestinal mucosa and portal blood, and ultimately transformed into the pharmacologically active Cefuroxime [1-3].

Cefuroxime axetil is a poorly water soluble drug; thus, it has got only limited solubility and dissolution rate in gastric fluids. The bioavailability of oral suspension is only 40-45% when compared to the 60% bioavailability of tablets. Therefore, the oral suspension and tablets cannot be substituted each other on mg/mg basis [4-6].

In this study attempt has been made to improve the oral bioavailability of Cefuroxime axetil oral suspension 125 mg by inclusion complexation method using HP-Beta cyclodextrin. Cyclodextrins are having a ‘bucket-like’ structure, which allows them to accommodate guest molecules within its cavity, so forming an inclusion complexation, which helps in improving the solubility, oral bioavailability, and stability of a variety of drugs [7-15]. In this study, HP-beta-cyclodextrin is selected due to its improved water solubility and safety compared to other cyclodextrins [16]. A leading marketed sample is used in this study as a competitor product, which has used spray drying technology with Stearic acid to mask the taste [17, 18]. A comparative evaluation of prepared oral suspensions and marketed product has been conducted for various in-vitro and in-vivo parameters.

MATERIALS AND METHODS

Materials

Cefuroxime axetil for the study was procured from Covalent Laboratories private limited, Hyderabad, India. HP-Beta cyclodextrin was purchased from Signet Chemical Corporation Pvt ltd, India (mfg. by: Roquette). Sucrose was received from EID Parry Ltd, India; Xanthan was procured from Deosen, China. Acesulfame potassium was procured from Ningbo Hi-Tech Biochemicals Co-Ltd China. Aspartame was received from NutraSweet, China. Tutti fruity flavor and Peppermint flavor were procured from Firmenich, Switzerland.

Methods

Preparation of cefuroxime axetil: HP-beta-cyclodextrin complex by inclusion complexation method

Cefuroxime axetil and HP-Beta cyclodextrin were taken at 1:1, 1:2, 1:2.5 and 1:3 combinations at molecular weight ratio (shown in table-1).


Table 1: Combinations of cefuroxime axetil and HP-beta cyclodextrin for complexation

Ingredients

Ratio (mg/5 ml)

1:1

1:2

1:2.5

1:3

Cefuroxime axetil

158.250

158.250

158.250

158.250

HP-Beta-cylcodextrin

477.915

955.830

1194.790

1433.750

Total weight

636.165

1114.080

1353.040

1592.000


Accurately weighed Cefuroxime axetil and HP-Beta cyclodextrin were sifted through #30 mesh and mixed together to get a uniform blend. The resulting mixture was slowly added to purified water in a beaker under stirring using mechanical stirrer. The stirring process continued for six hours to get thick slurry of Cefuroxime axetil and HP-Beta-cylcodextrin complex.

The slurry was transferred to a tray and dried in hot air oven at 45 °C until the complex is adequately dried. The dried complex was passed through #60 mesh and mixed thoroughly. The resulted Cefuroxine axetil: HP-Beta-cylcodextrin complex in different ratios were used for further processing to make dry suspension.

Preparation of taste masked dry suspension of cefuroxime axetil 125 mg/5 ml

Cefuroxime axetil taste masked dry suspension was formulated by mixing Cefuroxime axetil: HP Beta-cylcodextrin complex along with other inactive ingredients as shown in table 2. Formulations were prepared with each combination of Cefuroxime axetil: HP Beta-cylcodextrin complexes (1:1, 1:2, 1:2.5 and 1:3).


Table 2: Formula for cefuroxime axetil taste masked dry suspension 125 mg/5 ml

S. No.

Ingredients

Ratio (mg/5 ml)

1:1

1:2

1:2.5

1:3

1

Cefuroxime axetil: HP Beta-cylcodextrin complex

636.165

1176.468

1454.518

1634.984

2

Sucrose (#40 mesh grade)

1691.876

1286.649

1078.111

942.762

3

Sucrose (#80 mesh grade)

563.959

428.883

359.371

314.254

4

Acesulfame K

25.000

25.000

25.000

25.000

5

Aspartame

30.000

30.000

30.000

30.000

6

Xanthan gum

8.000

8.000

8.000

8.000

7

Tutty friuty premium flavour

35.000

35.000

35.000

35.000

8

Peppermint premium flavour

10.000

10.000

10.000

10.000

 

Average weight:

3000.000

3000.000

3000.000

3000.000

Cefuroxime axetil: HP-Beta-cylcodextrin complex, Sucrose (#80 mesh), Acesulfame K, Aspartame, Xanthan gum, Tutti fruity flavor and Peppermint premium flavor were sifted through mesh # 40 and mixed together. Sucrose (# 40 mesh) was sifted through mesh #30 and added to above blend and mixed well. 18g of the blend was filled in 30 ml High-density polyethylene [HDPE] bottle and closed with HDPE cap. Each bottle needs to be reconstituted with water before administration to make the oral suspension.


Evaluation of cefuroxime axetil oral suspension 125 mg/5 ml

Physiochemical properties of suspension

The physiochemical properties of suspension like color, pH, dispersibility, Viscosity, Assay and pourability were evaluated.

In-vitro dissolution studies

In-vitro dissolution of all the combinations and market sample were tested using ELECTROLAB dissolution apparatus as per the method specified in United States Pharmacopoeia [USP]. 900 ml of pH 7.0 Phosphate buffer was used as dissolution medium with USP apparatus 2 (Paddle), at 50 rotations per minute [rpm]. The temperature of the medium was maintained at 37±0.5 °C. The dry suspension was reconstituted with water and a quantity equivalent to 125 mg of Cefuroxime axetil were used for the dissolution study. During the dissolution study, 5 ml samples were withdrawn at 10 min, 20 min and 30 min intervals. The samples were filtered through 0.22 µm filter, and the concentration of Cefuroxime axetil in the filtrate was tested using a spectrophotometer. The limit for dissolution as per USP is not less than 60% (Q) in 30 min.

Pharmacokinetic study in rabbits

The bioavailability evaluation of optimized formulation and marketed product was carried out at Albino Research and Training Institute, Hyderabad, India with approval from the Committee for the Purpose of Control And Supervision of Experiments on Animals (CPCSEA) for the study with registration no: 1722/ RO/ ERe/S/13/CPCSEA.

Animals and study design

Six healthy adult male rabbits (Weighed: 1.5-2 Kg, aged: 8-10 mo) were enrolled in the study. Rabbits fasted for 12 h with free access to water before the study started. A single dose two-way crossover design study was conducted on rabbits. There was a washout period of one week between the two doses. The rabbits were divided into two groups

Protocol of study

Administration of the two products (optimized formulation and Marketed product) to the animals was carried by means of a two-way crossover design. The subjects were randomly divided into two equal groups and assigned to one of the two sequences of administration. Each animal received a single dose at a time.

Sampling procedure

The blood samples were collected from the ear marginal vein of the rabbits, which were held in wooden cages, in heparinized glass centrifuge tubes with the aid of sterilized disposable plastic syringes just before and at 1, 2, 4, 6, 8, 10, 12, 15, 18, 21 and 24h after the drug administration. The blood samples were centrifuged at 3000 rpm for 10 min to separate the plasma for analysis

Quantitative drug analysis

The concentration of drug in plasma was determined by HPLC technique with ultraviolet detection at 279 nm. Estimation of drug concentration was carried out by interpolating the peak area of best formulation on a calibration curve of spiked the blank plasma over the range assayed

Pharmacokinetic analysis

The pharmacokinetic characteristics such as Cmax (ng/ml), Tmax (h), Kel (h-1), t½el (h), Vd (ng/ml), AUC0-24 (ng. h/ml), AUC0-8 (ng. h/ml), AUMC0-24 (ng. h2/ml), AUMC0-8 (ng. h2/ml), MRT0-24 (h) and MRT0-8 (h) of drugs were determined from the plasma concentration-time profile. The maximum plasma concentration (Cmax) and time to reach maximum plasma concentration (Tmax) were obtained directly from the plasma concentration-time data. The area under the plasma concentration-time curve up to the last time (t) showing a measurable concentration (Ct) of the analyte (AUC0-t) was determined by applying the linear trapezoidal rule. The apparent elimination rate constant (Kel) was calculated by the log-linear regression of the data points of describing a terminal log-linear decaying phase. The AUC0-8 values (express the magnitude of absorption) were determined by adding the quotient of *Ct and the appropriate kel to the corresponding AUC0-t, which is,

AUC0-∞ = AUC0-t+*Ct/Kel

Where *Ct is the last detectable plasma concentration.

The sampling period covered more than 96% of the total AUCs for both reference and test. The apparent elimination half-life (t1/2) of drug and in plasma was calculated by using the following equation,

t½ = (ln 2)/Kel

The ratio of Cmax/AUC0-8 was also computed and used as a measure for the rate of absorption.

Statistical analysis

All values are expressed as the mean±standard deviation (SD). The pharmacokinetic parameters obtained by following a single dose administration of the marketed product and the optimized formulation to normal Rabbits were compared using paired ‘t’ test, considering a probability of P<0.05 to be significant.

Bioavailability and statistical analysis were performed according to the Food and Drug Administration (FDA) guidelines by using a software Bear v2.5.3

RESULTS

Physiochemical properties of suspension

Physiochemical properties of reconstituted suspension were tested as part of quality control tests, the results of which are shown in table 3.


Table 3: Physiochemical properties of suspension

S. No.

Tests

Formulations

1:1

1:2

1:2.5

1:3

1

Color

White

White

White

White

2

pH (Limit: 3.5 to 7)

5.98

6.01

5.92

6.02

3

Viscosity

319cps

340cps

395cps

410cps

4

Redispersibility

Easy

Easy

Easy

Easy

5

Pourability

Easily Pourable

Easily Pourable

Easily Pourable

Easily Pourable

6

Assay (Limit: 90 to 110 %)

97.85 %

96.89 %

99.50 %

97.68 %

Cefuroxime axetil dry suspension was reconstituted with an adequate quantity of water. The color of the suspensions was observed to be white. The pH of all the formulations was within the specified limit of 3.5 to 7 as in USP. Adequate viscosity was observed in all the formulations, providing sufficient stability and pourability of suspension. All the formulations were easy to redisperse with water by shaking by hand for some time. All the suspensions were easily pourable making it easy to dispense. The Assay of all the formulations was meeting the specified limit of 90 to 110 % as per USP.


In-vitro dissolution studies

In-vitro dissolution of all the combinations and market sample were tested using ELECTROLAB dissolution apparatus as per the method specified in United States Pharmacopoeia. The results of in-vitro dissolution studies are given in fig. 1.



Fig. 1: In-vitro dissolution studies-All values are represented as mean, n=6

From the dissolution studies, it was found that all the formulations and marketed sample were meeting the dissolution criteria of not less than 60 % (Q) in 30 min. Among these formulation 1:2.5 seemed to have better release pattern than the marketed sample.

Pharmacokinetic study

The pharmacokinetic evaluation of optimized formulation and marketed product was carried out using a single dose, two-way crossover design study on six healthy male rabbits. The Plasma drug concentrations at different time intervals for optimized formulation and Marketed Product are presented in fig. 2 and the major pharmacokinetic parameters are presented in table 4.



Fig. 2: Plasma drug concentrations at different time intervals for optimized formulation and marketed product

Various pharmacokinetic parameters were calculated during the study, from which the Cmax of optimized formulation and marketed product were found to be 148±1.26ng/ml and 126±1.52 ng/ml respectively, and the AUC0-t optimized formulation and marketed product were 989±16.42 ng. h/ml and 613±24.26 ng. h/ml respectively, which shows a significant improvement in the bioavailability of optimized formulation, compared to the marketed product.


Table 4: Comparison of pharmacokinetic parameters of optimized formulation and Marketed Product

Parameters

Optimized formulation

Marketed product

Cmax(ng/ml)

148±1.26

126±1.52

AUC0-t(ng. h/ml)

989±16.42

613±24.26

AUC0-8 (ng. h/ml)

1225±38.54

1004±35.14

Tmax (h)

6.00±1.23

4.50±0.24

t1/2 (h)

3.05±0.519

1.56±0.01

Kel (h-1)

2.807±0.11

2.189±0.33

Each value is mean±SEM of 6 rabbits in each group


DISCUSSION

In this study attempt has been made to improve the oral bioavailability of Cefuroxime axetil oral suspension 125 mg by inclusion complexation method using HP-Beta-cylcodextrin. The optimized formulation is better in taste, dissolution and bioavailability compared to the marketed product. Many studies are available for the improvement of bioavailability of Cefuroxime axetil with plain Beta-cyclodextrin [1, 2]. In this study, HP-Beta-cylcodextrin is selected instead of plain Beta-cyclodextrin due to its improved water solubility and safety profile [16]. So it is an additional advantage to the patients to have tastier, efficient and safer medicine. Some other studies are available for masking the bitter taste of Cefuroxime axetil, like spray drying technique with Stearic acid, complexation with ion exchange resins, etc, but non of them claim to have improved the bioavailability of oral suspension[18].

CONCLUSION

An oral suspension of Cefuroxime axetil was successfully developed by inclusion complexation method using HP-Beta-cylcodextrin. All the formulations developed were subjected to various quality control tests including physiochemical parameters and in-vitro dissolution, where all the formulations were meeting the quality parameters. Among the four formulations prepared formulation with 1:2.5 ratios of Cefuroxime axetil and HP-Beta-cylcodextrin showed improved dissolution compared to the marketed product. So formulation with 1:2.5 ratios was selected as the optimized formulation. The optimized formulation and the marketed product were subjected to pharmacokinetic study using healthy male rabbits, which the results shows a significant improvement in the bioavailability of optimized formulation compared to the marketed product. This demonstrates that the inclusion complexation method with HP-Beta-cylcodextrin can significantly improve the oral bioavailability of Cefuroxime axetil.

ACKNOWLEDGEMENT

The authors are thankful to the management of Sance Laboratories Pvt Ltd for providing the necessary facilities to carry out this work.

CONFLICT OF INTERESTS

Declare none

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