Formulation Development and Evaluation of Capsule Containing Fluclaxacillin Sodium and Amoxicillin Trihydrate with Enteric Coated Granules of Lactic Acid Bacillus

Gopal Rao M.¹, M. A. Amutha Gnana Arasi^1*, S. Jayaprakash², M. Arun Kumar³ and P. Kavitha⁴

¹Department of Pharmaceutics, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, 395, Sarojini Naidu Road, New Sidhapudur, Coimbatore-641044, Tamilnadu, India.

²Department of Pharmaceutics, K.M. College of Pharmacy, Uthangudi, Madurai, Tamilnadu, India.

³Executive, Formulation Development Department, Medopharm Pvt. Ltd., Kayirambedu, Chennai, Tamilnadu, India.

⁴Department of Pharmaceutics, K.K College of Pharmacy, Gerugambakam, Chennai.

ABSTRACT:

This work presents the preparation of capsule formulation containing ethyl cellulose coated Flucloxacillin sodium, Amoxicillin trihydrate with enteric coated Lactic acid bacillus granules. Preformulation studies were carried out for active ingredients and excipients using DSC, FTIR and exposing them into different temperature. Preparation of enteric coated Lactic acid bacillus granules was optimized using two enteric polymers cellulose acetate phthalate and hydoxy propyl methyl cellulose acetate phthalate (HP-55) using enteric test and microbial assay. Optimized one was utilized in the capsule formulation. Formulation was optimized by changing the excipients. Flow properties of the formulation were evaluated then they were filled into hard gelatin capsules by hand operated capsule filling machine. The capsules were evaluated for their thickness, weight variation, Net content and disintegration. In-vitro drug release and drug content analysis were also carried out by RP-HPLC method using isocratic and gradient elution method. Microbial assay method is used for the spore count of Lactic acid bacillus. The packaging optimization also carried out by packing the capsules in three different packaging systems and by conducting the accelerated stability testing studies. In conclusion the capsule formulation in strip packing is stable in accelerated condition for a period of two months.

KEYWORDS: Antibiotics; Probiotic; Enteric coating; Capsule

INTRODUCTION:

Solid oral dosage form is one of the most preferred and extensively used dosage forms and occupies an important place in the drug delivery system armamentarium. The capsule provides a tasteless, odorless delivery system that does not require a secondary coating step. So drugs having an unpleasant odor and taste like antibiotics can be filled in capsule¹. Flucloxacillin sodium is a narrow spectrum β-lactam antibiotic used to treat infections caused by susceptible Gram-positive bacteria^2,3. Flucloxacillin has relatively poor activity against non-β-lactamase producing bacteria including streptococcus pyogenes, therefore empirical therapy for significant cellulitis often involves dual therapy to cover both staphylococci and streptococci, using either Penicillin or Ampicillin in addition to Flucloxacillin^4,5. So Amoxicillin trihydrate was combined with Flucloxacillin sodium. When the composition of the intestinal flora is changed, following therapeutic administration of antibiotics, various disorders may result, such as, nausea, vomiting, colitis and diarrhea⁶. Diarrhea is the common side effect seen in patients administered with antibiotics. To avoid this Lactic acid bacillus was added with Flucloxacillin and Amoxicillin. Acidophilus lactobacillus has the ability to adhere to the intestinal epithelia.

As Lactic acid bacillus is less gastric resistant and it grows well in the pH above 5.0, it is enteric coated. Production of enteric coated Lactic acid bacillus tablet which may be placed in the capsule containing Flucloxacillin and Amoxicillin involves long processing and some amount of Lactic acid bacillus spores were wasted and destroyed in the tablet compression and coating machines. So in the present study enteric coated granules of Lactic acid bacillus were prepared, mixed with the other active ingredients and filled in capsule. Accelerated stability study was conducted for the optimized formulation.

MATERIALS AND METHODS:

Materials:

The following chemicals were obtained from different sources and used as received.

Flucloxacillin sodium, Amoxicillin trihydrate, Lactic acid bacillus, Hydroxy propyl methyl cellulose phthalate and Cetyl alcohol were purchased from Penam Laboratories Ltd; Haryana, DSM Anti-infective India Ltd, Deepak Cellulose Pvt Ltd, Mumbai, Unisankyo Ltd, Hyderabad and Godrej Industries Ltd, Gujarat respectively. All other materials were commercially purchased and used as such. Reagents used for analytical work of HPLC grade and analytical grade.

Methods:

Preformulation studies:

Preformulation studies on pure active ingredients and on final formulation were carried out by Fourier Transform Infrared (FTIR) Spectrometer (Perkin Elmer spectrum100) and by Differential Scanning Colorimeter (DSC) (Shimadzu DSC-60) and exposing the pure material and their combinations with excipients into different temperatures.

Formulation of enteric coated Lactic acid bacillus granules:

Enteric coating of Lactic acid bacillus using Cellulose acetate phthalate:⁶

Cellulose acetate phthalate was dissolved in acetone; it was stirred properly to get a clear solution. The dibutyl phthalate, titanium dioxide and talc were added to the above solution to obtain a white paste. Then Lactic acid bacillus was added to the above paste to get a semisolid mass and it was passed through sieve no 20.The granules were collected and then were air dried. The granules were weighed and were subjected to evaluation.

Enteric coating of Lactic acid bacillus using hydroxy propyl methyl cellulose phthalate (HP-55):⁶

Cetyl alcohol was dissolved in acetone. Hydroxy propyl methyl cellulose phthalate (HP-55) was added and dissolved to get a clear solution. Titanium dioxide and talc were added to the above solution to obtain a white paste. Then the above procedure was followed.

Evaluation of enteric coated Lactic Acid Bacillus granules:

Flow characteristics of granules and loss on drying were determined using Tap density tester (USP) (Electrolab ETD-1020) and Moisture balance (OhausMB-45) respectively.

Test for gastric resistance on enteric coated Lactic acid bacillus spores:⁶

The enteric test or gastric resistance test was carried out using the six vessel Dissolution tester (Electrolab TDL-08L) as described by USP XX11 Ed. (Page 1580), operating at 37⁰C and 60 rpm in 750 ml of 0.1 N HCL.1 ml of the sample was taken at the end of 2 hours and it was transferred into a sterile petridish. About 15 ml of glucose agar medium which was previously sterilized, melted and cooled to 45⁰C was added to the petridish. One more plate was treated as negative control. The plates were incubated at 37⁰C for 72 hours. After 72 hours the plates were checked for the existence of the colonies. If colonies were absent the granules passes the test for gastric resistance and vice versa. If the granule passes the test then the bacterial count was carried out by the microbiological assay method.

Microbial assay of enteric coated Lactic acid bacillus:

Validated in house method was developed for the estimation of Lactic acid bacillus spore count. About 600 lacs equivalent Lactic acid bacillus spores sample powder was weighed accurately; it was transferred into a sterile flask. 80 ml of sterile normal saline solution (0.9%) was added and was transferred into a sterile mixer. The flask was rinsed with 20 ml of sterile normal solution (0.9%) and was transferred in to the same mixer. The contents were mixed at about 12000-15000 RPM for 5-7 minutes and it was diluted further stepwise through a series of test tubes containing 9 ml of sterile blank solution, by an appropriate decimal dilution method given under.

Decimal dilution:

600 lacs equivalent sample → 100.0ml → 1.0ml → 10.0ml →1.0ml → 10.0ml →1.0ml → 10.0ml →1.0ml → 10.0ml →1.0ml → 10.0ml (solution A)

The test tube with the diluted solution was allowed to stand in a water bath at 75⁰C for 30 minutes (Heat shock) and was cooled immediately to about 45⁰C. Each 1.0 ml of the heat shock solution was transferred into a sterile petri dish. One more sterile plate was treated as negative control about 15 ml of glucose yeast extract agar medium which was previously sterilized, melted and cooled to 45 C, was added to each of the two petridishes. The plates were incubated at 37⁰C for 72 hours and the number of colonies in each plate was counted. The average value of colonies was found and the viable Lactic acid bacillus spores in the sample were calculated using the following formula.

100 10 10 10 10

Total spore count= Ac X --- X ----- X ------- X ---- X--- X weight taken

1 1 1 1 1

Where, Ac - Average number of colonies observed

Total spore count was calculated and was divided by 10,00,000. The results obtained were spores in million.

Formulation of capsule:

Initially capsule was formulated just passing Amoxicillin trihydrate and Flucloxaciilin sodium; Talc and Colloidal silicon dioxide through sieve no 30. To this enteric coated Lactic acid bacillus was added, lubricated and mixed and filled into 0E (Elongated) hard gelatin capsules using hand operated capsule filling machine (Pam Pac 300) having tamping pin diameter - 6 mm and the tamping pin Length 12 mm.In next trials Flucloxacillin sodium was coated with hydroxy propyl methyl cellulose and ethyl cellulose respectively by wet granulation method then the above procedure was repeated.

Evaluation of capsules:

Capsules were evaluated for their physical appearance, weight variation, net content and disintegration test. The disintegration test was performed as per B.P specifications⁷.

RP-HPLC method:

Validated Reverse phase HPLC in house developed method was used for the estimation of Flucloxacillin sodium, Amoxicillin trihydrate. A mixture of volume of 50 volumes of buffer and 50 volumes of water was prepared as mobile phase A. Buffer was prepared by dissolving monobasic potassium phosphate was dissolved in water to make 1000 ml of solution, and was adjusted with dilute sodium hydroxide to a pH of 5.0 + 0.1. A mixture of 50 volumes of buffer, 10 volumes of water and 40 volumes of acetonitrile was prepared as mobile phase B. Mixture of 50:46:4 of buffer, water and acetonitrile was prepared as diluents.

Column (Octa Decyl Silane, 150 x 4.6mm, 5μm (Luna, C₁₈ (2) or equivalent), UV detector (225nm) were used in the analysis. Flow rate was 1.5 ml per minute, Injection volume was 20μl.The HPLC analysis was conducted at ambient temperature.

Isocratic elution was started with mobile phase with the ratio A: B of 90:10 for three minutes and the linear gradient elution was started to reach a mobile phase A: B of 25:75 over a period of three minutes. The chromatography was continued with the mobile phase A:B of 25:75 for eight minutes, then changed to original concentration in two minutes and the column was equilibrated for four minutes with the mobile phase which was chosen originally.(Total time 20 minutes).

In-vitro release studies:

Dissolution profile:

In-vitro drug release of Flucloxacillin sodium and Amoxicillin trihydrate were determined using Dissolution tester (Electrolab, TDL-08L) (paddle type with 100 rpm for 30 minutes in water) and the drug present in the sample were analyzed by HPLC method. 900 ml of water was taken in the beaker and it was equilibrated to 37±0.5⁰C_. One capsule was placed in the apparatus and the apparatus was immediately operated at a rate of 100 RPM. At every 5 minutes, 5 ml of the sample was withdrawn from the beaker and it was replaced with the water. The sampling process was continued for 30 minutes. The sample withdrawn from the dissolution apparatus was filtered using Whatman filter paper 1.5 ml of the filtrate was transferred to a clean dry flask and 10.0 ml of the diluents was added. It was mixed and was filtered through 0.45µ membrane filter. The filtrates were injected separately. The amount of active ingredients dissolved was determined in mg by using the following formula

Test area Standard weight 15 Purity of standard

Amount =------------- X ---------------- X ------- X ----------------- X 900

Standard 500 5 100

DISSOLUTION:

After fixing the dissolution time from the dissolution profile data⁸, the dissolution process was repeated for 6 capsules. Each one of 6 capsules was placed in 6 beakers of dissolution apparatus. After 30 minutes the samples were withdrawn from all the beakers and were analyzed by HPLC method. The dissolution time was determined by taking the average of dissolution times of six individual capsules.

Drug content analysis

Amount of Flucloxacillin and Amoxicillin present in a capsule was analyzed by the above said RP-HPLC method. 20 capsules were weighed and the contents were powdered. About 100.0 mg of Amoxicillin equivalent sample powder was weighed and transferred to a 100 ml volumetric flask. About 80 ml of water was added. It was shaken for 10 minutes, was sonicated for 5 minutes and made up to the volume with the same. 5 ml of the above solution was transferred in to a 50 ml volumetric flask and made up to the volume with diluent. It was filtered through 0.45µ membrane.

The test solution was injected into the chromatographic column. The content of active ingredients in a capsule of average weight using following formula

A_t W_S Purity of standard

Content =-------- X -------- X ---------------------------- X 2 X Ta

A_S T_S 100

Where, A_t-Area of Amoxicillin or Flucloxacillin peak in standard solution; A_s-Average area of corresponding peaks in test solution chromatograms; W_s-Weight of corresponding working standard in mg; T_s-Sample weight taken in mg; T_a-Average net content of a capsule in mg.

Drug Content analysis of Lactic Acid Bacillus (Microbial Method)

Estimation of lactic acid bacillus spore count was carried out as described above in microbial assay methods of lactic acid bacillus spores section.

The average value of colonies was found and the viable Lactic acid bacillus spores in a capsule of average net content was calculated using the following formula.

100 10 10 10 10 10 Ta

Content =Ac X ------ X ------- X ------ X ------ X ------ X ------- X ----

1 1 1 1 1 1 T_W

Where, Ac - Average number of colonies observed; Tw -Weight of sample taken in mg; Ta - Average net content of a capsule in mg; Total spores were calculated and was divided by 1000,000. The results obtained were spores in million.

Accelerated stability studies

The optimized formulation (F5) was prepared for 2 kg. To know the suitable packaging system for the formulation F5 the capsules were packed in different packing systems like strip, blister and tropack packaging systems. The packed capsules were subjected to accelerated stability studies (45⁰ C/75% RH) for a period of two months and the samples were evaluated for its physical appearance, weight variation, net content, disintegration, dissolution and drug content analysis at the interval of one month.

RESULTS AND DISCUSSION:

Drug-excipients compatibility studies

Drug and excipients were subjected to a drug-excipient compatibility studies as the mixture shown to have no color change and lumping except Sodium carboxy methyl cellulose.

FTIR Analysis:

FTIR spectrum of Flucloxacillin sodium and Amoxicillin trihydrate (pure drug) and spectrum of F5 (Figure 3) were compared. There were no major changes in the position of the spectrum. So formulation F5 has no interaction with added excipients.

Figure 1. In vitro release of F5

Thermal analysis:

In Figure 2. The Tg values of the pure drugs situated within the Tg values of capsule revealed that there was no major interaction between the pure drug and excipients used in the formulation.

Figure 2. DSC analysis of F5

Powder flow characteristics:

The Carr’s index of pure drugs were observed high, confirming that the drug has poor flow properties and compressibility⁹.To improve flow properties, the formulations were prepared by wet granulation method. In the wet granulation method mainly Flucloxacillin sodium was coated with hydroxy propyl methyl cellulose or ethyl cellulose. Ethyl cellulose coating was effective to improve the flow as well as to reduce hygroscopic nature of Flucloxacillin¹⁰ without affecting the dissolution. The Carr’s index of the formulation F1, F2, F3, F4 and F5 were 42.1, 26.5, 18.18, 19.27 and 16.87 respectively. The flow property was initially extremely poor (F1) and it was observed as good flow property in F5.

Figure 3. FTIR spectrum of formulation F5

Formulation Quantity in Gram
Ingredients in Gram	LB 1		LB 2	LB 3	LB 4	LB 5	LB 6	LB 7	LB 8	LB 9
Lactic Acid Bacillus	50		100	100	100	100	100	100	100	100
CAP^b	4.5		7	5	-	-	-	-	-	-
HPMCP^c	-		-	-	5	7	10	12	15	14
Cetyl Alcohol	-		-	-	0.9	1	1.5	2	3	2.5
Dibutyl Phthalate	1.5		2.5	2	-	-	-	-	-	-
Titanium Dioxide	0.5		1	1	1	1	1.5	1.5	2	2
Talc	0.5		1	1	1	1	1.5	1.5	2	2
Acetone(ml)	45		92	90	85	85	87	88	92	90
Evaluation
Gr test^d	P	P		F^f	F	F	P	P	P	P
Assay (million spores)	-	22.5		-	-	-	13.5	22.5	72	81

Table 1. Formulation and evaluation of enteric coated lactic acid bacillus granules

CAP^b -Cellulose Acetate Phthalate, HPMCP^c - Hydroxy Propyl Methyl Cellulose Phthalate (HP 55), Gr test^d -Gastric Resistance Test

P^e-Passes the Gastric resistance test, F^f-Fails the gastric resistance test

Table 2: Formulation of capsule

Ingredients	Quantity required for a capsule
Ingredients	F 1 mg	F 2 mg	F 3 mg	F 4 mg	F 5 mg	Stability batch of f5 (g)
Granulation
Flucloxacillin sodium	272.5 (p^a)	272.5 (c^b)	272.5 (c^b)	272.5 (c^b)	272.5 (c^b)	855.57 (c^b)
Aerosil	-	-	-	-	2.5	7.85
Hpmc	-	-	5	-	-	-
Ethyl cellulose	-	-	-	5	2.5	15.70
Methylene Chloride	-	-	Q.s	Q.s	Q.s	225ml
Blending
Amoxicillin Trihydrate	287.5 (p^a)	287.5 (c^b)	287.5 (c)	287.5 (c)	287.5 (c)	902.63
Aerosil	5	5	5	5	2.5	7.85
Talc	6	6	6	6	6	18.84
Enteric coated Lb granules (320% overage)	56	56	56	56	56	175.82
Lubrication
Magnesium Stearate	5	5	5	5	5	15.70

p^a-Powder form, c^b-Compacted form

Table 3. Accelerated stability analysis

M	In vitro release						Drug content
	Flucloxacillin			Amoxicillin			Flucloxacillin			Amoxicillin			LB^a
	S^b	T^c	B^d	S^b	T^c	B^d	S^b	T^c	B^d	S^b	T^c	B^d	S^b	T^c	B^d
0	98.9	98.9	98.9	99.4	99.4	99.4	100.4	100.4	100.4	100.6	100.6	100.6	329	329	329
1	98.6	97	40	99	98.5	96	98	91.6	38.4	94.3	94.6	88.8	300	285	188
2	97.4	85.3	38.5	95.4	93.2	87.5	96.9	90.13	35.4	94.6	80.6	80.4	263	188	150

S^b^- capsules in strip packing; T^C- capsules in tropack packing; B^d- capsules in blister packing

Accelerated stability studies:

In accelerated stability studies the physical appearance, weight variation, net content, disintegration time of capsules did not show much variation. Dissolution values of Flucloxacillin for the capsules in the strip and tropack were within the limit. The capsules in blister did not comply with the BP limit. Dissolution values of Amoxicillin for the capsules of all the packs were within the British Pharmacopieal limit⁷.

Drug content analysis:

At the end of a month the drug content analysis showed that the percentage amount of Flucloxacillin in capsules in strip was within the British Pharmacopieal limit (92.5-110)⁷. The capsules in other two packs did not comply with the limit.

At the end of a month the percentage amount of Amoxicillin of capsules in all packs except the blister pack were within the British Pharmacopieal limits (92.5-110%)⁷. At the end of two months the percentage amount of Flucloxacillin and Amoxicillin of capsules in strip pack were within the British Pharmacopieal limit. The capsules in other two packs did not comply with the limit.

Microbiological assay showed that at the end of a month and at the end of two months the spore count of Lactic acid bacillus was within the label claim (80 million spores) in all the three packs. But the spore count is greater in capsules packed strip pack than the other two packs. These details were shown in Table 3.

CONCLUSION:

The capsules containing Amoxicillin trihydrate, Flucloxacillin sodium and enteric coated Lactic acid bacillus granules were prepared. Flucloxacillin sodium granules were prepared by wet granulation method using Ethyl cellulose (0.9% of the drug) .Enteric coating of Lactic acid bacillus was done with enteric polymer Hydroxy methyl cellulose polymer(14%) in LB9. LB9 has sufficient gastric resistance capacity and has required Lactic acid bacillus spore count in the microbiological assay. LB9 was considered to be the optimized trial for enteric coating of Lactic acid bacillus. It was used in the capsule formulation.F5 was considered to be the optimized formulation with the desired drug release and active ingredient.F5 has shown desired dissolution. The stability studies of optimized formulation F5 at 40⁰C /75 RH for two months showed more variation in drug content analysis of capsules in blister and tropack, but the drug content analysis in the capsules packed in strip did not show much variation and were within the pharmacopeia limits. Strip packing system was selected as optimized packing for the formulation F5.

The FTIR and DSC analysis showed that the drug and the excipients are compatible. Thus the F5 formulation in strip pack has achieved the goal in delivering active ingredients in capsules as solid oral delivery system with maximum potency.

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Received on 13.04.2010

Accepted on 30.05.2010

Research Journal of Pharmaceutical Dosage Forms and Technology. 2(3): May-June 2010, 241-246