Preparation and in Vitro Evaluation of Self- Micro Emulsifying Drug Delivery System Containing Roxithromycin

Roxithromycin is a semi-synthetic macrolide antibiotic used to treat respiratory tract, urinary and soft tissue infections.it is benefical in topical delivery too, as they help in improved skin retention and penetration. Roxithromycin have been useful in enhancing therapeutic efficacy against most skin diseases.

Roxithromycin is a derivative of erythromycin and  share a 14 membered lactone ring. They show side effects such as; diarrhea, nausea, headache and abdominal pain. Roxithromycin is a BCS Class IV drug  with bioavailability around 50% and it is eliminated with a half-life of 10h.Roxithromycin exhibits poor aqueous solubility and a low dissolution rate from pure drug formulations . Presence  of  food and interaction with other drugs accelerate issues regarding absorption .

Lipid based drug delivery  systems are useful in such cases. Lipid based drug delivery systems are known as formulations that use lipids as main component to offer specific drug action. In such a scenario, importance is given to lipid - based drug delivery systems like self-microemulsifying drug delivery system (SMEDDS). They offer excellent  advantages  including like spontaneity, high solubilization capacity, stability,  self-preserving  properties etc.

SMEEDS are isotropic mixtures  of  oil,  surfactants  and  cosurfactants  which produce fine oil in water microemulsion upon mild agitation and further dilution . SMEDDS can localize and target drug activity in the GI tract by optimizing the drug system and accordingly reducing drug dose.They give outstanding solution for improving delivery problems of most drugs.

Increasing solubility and absorption are the challenges accepted in this research study.  The aim of this research is to improve absorption and bioavailability of roxithromycin by development and evaluation of the required SMEDDS drug system.

1. MATERIALS AND METHODS

Roxithromycin have been obtained from Yarrow chem products (Mumbai) and tween 20 from burogyne burdiges and Co (Mumbai). Castor oil was obtained from isochem laboratories (kochi) and PEG 400 from finar limited (Ahmedabad).All buffers and reagents used were of analytical grade.

1. 1. PREFORMULATION STUDY

Preformulation studies give importance to  physicochemical properties of the  drug which will influence the performance of drug delivery system. The  initial step is creating a stable, safe and effective dosage form focusing to discover drug's kinetic nature and its compatibility nature.

1.2 ANALYTICAL METHOD

DETERMINATION OF UV ΛMAX

The pure drug of Roxithromycin was determined by UV spectroscopy and λmax was found to be 242nm.

STANDARD CALIBRATION CURVE OF ROXITHROMYCIN

Roxithromycin in phosphate buffer pH 7 was analyzed by UV spectroscopy and λ max was 242 nm. The absorbance of roxithromycin having concentration range in 5,10,15,20,25 μg/ml phosphate buffer pH 7 was determined.

1.3 SOLUBILITY STUDY

The solubility of roxithromycin in various oils, lipophilic  and hydrophilic surfactants were determined by shake flask method.  Drug have been  added to 250 ml conical flasks containing 100 ml of different oils and surfactants. Then shaken using rotary shaker. Then samples were filtered through Whatman filter Paper of grade 1. Absorbance was measured at 242 nm with UV spectroscopy technique.

1.4 CONSTRUCTION OF PSEUDO-TERNARY PHASE DIAGRAM

Ternary phase diagrams were used to evaluate concentration range of  various components and Pseudo-ternary phase diagrams were constructed using water titration method.  The Plots were created with oil, surfactant, and co-surfactant in different ratios (1:1, 1:2, 1:3, and 1:4, 4:1). The mixtures was diluted by adding water drop by drop under magnetic stirring and data is used to construct ternary plots with a ternary plot maker.

1.5 DRUG-EXCIPIENT COMPATIBILITY

FTIR spectroscopy is used to check drug and excipient compatibility and its spectra of the roxithromycin, Castor oil, Tween 20 , PEG 400 and their physical mixtures has been recorded and  peaks of  their individual  components and respective mixtures are plotted to check incompatibility.

1.6 FORMULATION AND PREPARATION

Roxithromycin was gradually mixed with castor oil under constant stirring. Here, Surfactant system was prepared by mixing selected surfactant and co-surfactant in the required quantities. Further mixture of the drug and oil phase has been added to the prepared surfactant system with constant stirring using magnet stirrer. Stirring was performed with magnet stirrer until the uniform mixture were formed. Then, mixture were kept at 25 °C and formulations of SMEDDS of roxithromycin  was prepared, kept at suitable storage  conditions and were examined on different parameters.

Table no 1-Formulation

Formulations F1-F5 of SMEDDS of roxithromycin and each containing an equivalent to one dose  was added to 10 ml volumetric flask containing water and stirred well. Solution has been diluted and scanned using  UV spectrophotometer at 242 nm.

1.8 DETERMINATION OF SELF-EMULSIFICATION TIME AND CLOUD POINT

A USP type II apparatus were  used to determine the self-emulsification time of the developed formulations and 500 ml of water have been  placed in  dissolution flask and equilibrated at 37°C. 1mlof formulation  added to  flask with stirring at 50 rpm. The time required to form a homogenous dispersion were determined as the self-emulsification time . The emulsion becomes cloudy , as temperature rises and  temperature at which cloudiness is observed is checked and this is known as the cloud point. 1ml of every formulation  diluted with water and heated with water bath. The temperature has been slowly raised, and the point at which cloudiness was found has been evaluated and checked.

1.9 RHEOLOGICAL PROPERTIES DETERMINATION

Viscosity studies are important to ensure physical stability during storage and  viscosity was determined using a Brookfield viscometer with spindle 4 at 50 rpm.

1.10. ENTRAPMENT EFFICIENCY

Percentage entrapment efficiency was determined by taking 1 ml of SMEDDS formulation and  diluted with phosphate buffer (pH 7)  and filtering done with  Whatman filter paper of grade 1. Drug content was evaluated using  UV spectrophotometer at 242 nm.

1.11  IN-VITRO RELEASE STUDY

Dissolution studies were performed using   USP type II apparatus and  release nature studied  by conducting experiment  at paddle speed of 50 rpm in 900ml of pH 7 phosphate buffer at temperature around 37±0.2°C. 2ml solution  withdrawn from medium at time intervals of 0, 5, 15, 30, 45, and 60 minutes. The absorbance was measured at 242 nm and percentage drug release evaluated.

1.12.   DETERMINATION OF PARTICLE SIZE

Particle size analysis and zeta potential determination done using Malvern Zeta Sizer instrument. Zeta potential is -2.55 mV  and average particle diameter was  195.5 nm with polydispersity index 0.257.

1.13 STATISTICAL ANALYSIS

One way analysis of variance (ANOVA) was applied for comparison of results and to demonstrate graphically  influence of every  factor on the specific response by  indicating  different levels of factor and parameter. The contour and response surface plots were generated using Design expert software(Stat-ease, 13). All  data measured and evaluated was averages of minimum of triplicate measurement and values are expressed as ±standard deviation .

2. RESULT AND DISCUSSION

2.1 ANALYTICAL METHOD

The drug was measured in UV region (200-400) nm by using phosphate buffer pH 7 to find out wavelength of maximum absorption (λ max). The λ max was found to be 242 nm and standard calibration curve of roxithromycin were formed  at the above wavelength. Standard calibration curve of roxithromycin were determined using phosphate buffer pH 7 and absorbance against concentration  plot at 242nm have been graphically drawn.

Fig no 1  - Standard calibration curve

Fig no 2-  Absorption maxima of roxithromycin

 2.2 PHYSICO-CHEMICAL PROPERTIES OF ROXITHROMYCIN

It was found that  roxithromycin  is White to off-white in  colour ,odourless  and  tasteless in nature. The melting point was determined by capillary tube method and it was found to be 125 ± 1.09°C.

2.3 SOLUBILITY PROFILE

Solubility studies were carried out in various oils, lipophilic surfactants and hydrophilic surfactants and the result are showed in following tables.

Table no 2– solubility profile in oils

Table no 3-solubility profile in surfactant

2.4 PLOT OF PSEUDO TERNARY PHASE DIAGRAMS

Phase diagrams of  system containing castor oil as  oil phase, tween 20 as  surfactant, and PEG 400 as the co-surfactant was constructed at S:Co-s ratios of 1:1, 1:2, 1:3, 1:4, and 1:5 to determine the existence of microemulsion regions. As concentration of  co-surfactant elevates, the microemulsion region diminishes, so concentration of  surfactant increases and microemulsion region increases as required. The concentration of  surfactant have significant effect on  microemulsion region.

Fig no 3 -  Ternary plots of Tween 20 : PEG 400  in ratios 1:1,1:2,1:3,1:4,4:1 as per order

2.5 DRUG-EXCIPIENT COMPATIBILITY STUDIES

Drug identification were done using FT-IR studies and suitable  peaks obtained. There were no appearance of new peaks and no absence of any interfering peaks in  FT-IR spectra of drug compared to  mixtures of the drug and polymers and indicating no drug-excipient incompatibility.

Fig no 4 - FTIR peaks of Roxithromycin, castor oil,PEG 400 ,Tween 20 and their physical mixture respectively.

2.6 DRUG CONTENT

Fig 5  - Drug content

The drug content of 5 formulations were  found to be between 90.5% to 98.1%, . Percentage drug content of all SMEDDS formulations  were within acceptable limit.

2.7 DETERMINATION OF SELF EMULSIFICATION TIME AND CLOUD POINT

The self-emulsification time of all formulations i.e., were  less than 200 -seconds implicating, that they  form microemulsion rapidly using speed of 50 rpm during formulation. Emulsions were unstable at higher temperatures  and two phases tend to separate on heating. The cloud point were above 50°C for every formulation, indicating their stability during processing and storage.

2.8 RHEOLOGICAL PROPERTIES DETERMINATION

The formulation with the highest drug content i.e F9 exhibited viscosity 15-31 cps compared to other formulations which is a proper  indicator of stability .

Fig no 6 – viscosity profile

2.9 ENTRAPMENT EFFICIENCY

The percentage entrapment efficiency of 5 formulations was found to be 82.12%- 97.75%

Fig no 7 -Entrapment efficiency

The in-vitro drug release of 5 formulations was done with  USP type II apparatus in phosphate buffer pH 7.98.83%±0.37 of drug was released fromF9formulation within 60 minutes compared to other  formulation

2.9 PARTICLE SIZE ANALYSIS AND ZETA POTENTIAL DETERMINATION

Particle size analysis and zeta potential determination done using Malvern Zeta Sizer instrument. Zeta potential is -2.55 mV  and average particle diameter was  195.5 nm with polydispersity index 0.257.

Fig no 8 -  Particle size analysis and zeta potential determination

2.10 OPTIMIZATION BY DESIGN EXPERT SOFTWARE

Optimization of the above formulations was done using design expert software. Here, Fit summary and Anova for quadratic model for drug content and in vitro drug release studies is given for better understanding. 3-D response surface plot for effect of concentration of castor oil, PEG 400, Tween 20 is also provided for further analysis. Desirability function is used here in optimization techniques and they measures how well combined goals for all responses were used. The numerical optimization tool provided with necessary  optimal solutions and area of optimized formulation were also evaluated and  they confirm validity of obtained optimal formulation, experiment was carried out in triplicate measurements.

Table No 4 - Fit Summary of Drug Content

Fig no-3-D response surface plot for effect of concentration of castor oil, PEG 400, Tween 20 on drug  content

Table no  5 - Anova for quadratic model in vitro drug release

Fig no -    3-D  response surface plot for effect of concentration of castor oil, PEG 400, Tween 20 on in vitro drug  release