Table 1: Formulation of Dry syrup

Sr. No.	Ingredients	Quantity
01.	RMP	150mg/5 ml
02.	INH	75mg/5ml
03.	Xanthan Gum	0.1%
04.	Sucrose (200#)	40%
05.	Methyl and propyl paraben (9:1)	0.2%
06.	Ascorbic acid	0.25%
07.	Colloidal Silicon dioxide (Aerosil-200)	1%
08	Sodium Saccharine	0.1%
09	Dry Flavour (Orange)	1%

The above formulation would be reconstituted either with freshly boiled and cooled water or with Oil (MCT (3.8ml/5mL) + 0.1% tocopherol + 0.1%Tween 80)

Table 2: Formulation of Oil based Suspension

Sr. No.	Ingredients	Quantity
01.	RMP	150mg/5 ml
02.	INH	75 mg/5ml
03.	Sucrose (200#)	2gm/5mL
04.	Tocopherol (Vitamin-E)	0.1%
05	Tween-80	0.1%
06	Sodium Saccharin	0.1%
07	Flavour orange (oil soluble)	0.2%
08	Medium chain Triglycerides (MCT)	Q.S to make 100 ml

It has been reported that RMP in the presence of INH under aqueous conditions as a suspension undergoes very rapid decomposition. This study was reported for the suspension of RMP, INH and pyrazinamide in which the formulations of the drugs alone and their combinations were stored at 4, 24 and 40 °C for 28 days⁴. The suspensions had a pH range between 4.05 and 6.10. Differential extents of decomposition were observed between suspensions of single drugs and mixtures of two and three drugs. Degradation, as high as 98% in the presence of INH has been reported within 28 days.

With the above background it was thought to develop RMP and INH product through different alternatives of administering the drugs; as suspension. Different types of suspension formulations were thought to be made like dry reconstitutable suspension (constituted with oil/water as vehicle) and ready to use oil based suspension which would be made with a view to minimize the degradation of RMP in a combined dosage form with INH. Simple formulations were thought to be made in each category. These would be then evaluated for various physicochemical properties and more importantly stability studies.

2.0 EXPERIMENTAL:

2.1 Materials:

RMP (USP), INH (IP) and known degradation products of RMP were gifted by Lupin Ltd, Pune, India. All formulation ingredients were locally purchased. All other solvents and reagents used were of analytical grade.

2.2 Formulation of RMP and INH suspensions:

Three different types of suspension formulations [Dry (reconstituted with oil/water) and oil based] were made with a view to minimize the degradation of rifampicin in combined dosage forms. The dry syrup formulation would be reconstituted either with freshly boiled and cooled water or with Oil [Medium chain triglycerides (MCT)+ tocopherol (Vitamin E) + Tween 80] at the time of administration. Tween 80 would ensure proper wetting of the hydrophobic drug, RMP and would also ensure dissolution in the GI media.

The formulae of dry and oily formulations are given in Tables 1and 2. These formulations were made to ascertain that in which type of formulations this drug combination is more stable. For the preparation of ‘dry syrup’ all ingredients were taken in geometric proportions in a mortar pestle and were lightly triturated and this blend was then filled in bottles.

For the ‘ready to use oily suspension’, weighed quantity of RMP was taken in a beaker. Sucrose was very finely triturated (passed through 200#) along with sodium saccharin. To it INH was added with light trituration and was then mixed with the above. Tween 80 and tocopherol were dissolved in MCT (Oil) and added to the prepared dry mixture with stirring using a mechanical stirrer. Finally flavor was added to this suspension.

Table 3: Sedimentation Volume (S.V.R.) of reconstituted suspension

Sr. No.	Time (days)	S.V.R.
Sr. No.	Time (days)	Oily	Aqueous
1	0	1	1
2	4	0.62	0.91
3	8	0.45	0.85
4	12	0.42	0.84
5	14	0.41	0.81

Table No 4: Flow properties of dry suspension

Sr. No.	Parameters	Values
01.	Bulk Density( g/cc)	0.55
02.	Tapped Density (g/cc)	0.70
03.	Carr’s Index (%)	21.42
04.	Angle of repose (⁰)	32

Table 5: Assay of RMP

Time/condition	Oily Suspension		Dry Syrup
	RMP	INH	RMP	INH
0 day	100.4	99.71	99.89	99.81
1 month 40⁰C/75%RH	---	---	97.02	98.51
3 months 40⁰C/75%RH	---	---	96.41	97.41
1 month 2-8⁰C	97.60	98.21	----	----
3months 30⁰C/65%RH	93.31	96.52	98.52	98.11

- INH ready to use oily suspension and dry syrup

2.3 Evaluation of suspensions:

The dry syrups were analysed as such in the dry form for all powder characteristics including flow properties and densities. The bulk density (B.D), tapped density (T.D), Carr’s index (TD-B.D/TD× 100) and angle of repose (θ= tan^-1 b/h) was determined for dry suspension formulations by the standard methods. The reconstituted formulations/ oily suspension were analyzed for sedimentation rate and were also subjected to dissolution studies. The sedimentation rate was observed for 14 days (after reconstitution) by using the formula as given below:

F = Vu/Vo

Where F= sedimentation rate

Vu=Volume of sediment at time ‘t’

Vo= Volume of sediment at time ‘t=0’

2.4 Drug content for RMP/INH suspension and RBN/INH suspension:

Assay of the all the three types of suspensions was performed on zero day for dry syrup and for the reconstituted suspension/s using HPLC (Shimadzu) for both RMP and INH as per USP⁵.

Table 6: Assay of RMP- INH reconstituted dry syrup

Time/condition	Aqueous based reconstituted Suspension		Oil based reconstituted suspension
	RMP	INH	RMP	INH
0 day	98.51	98.01	99.53	98.64
7 days (2-8⁰C)	81.23	90.41	99.31	98.11
14 days (2-8⁰C)	61.53	86.21	98.62	97.23
7 days RT	---	---	98.68	98.53
14 days RT	---	---	98.51	97.89

2.5 Stability testing:

2.5.1 Accelerated stability testing for drug content and other parameters:

The dry syrup and the oil based suspensions were subjected to accelerated stability testing. The dry syrups were placed at 30⁰C/65% RH and 40⁰C and 75% RH and tested for all parameters as described in section 2.3 up to three months (1 and 3 months). After reconstitution (Oil/water) the suspensions were kept for 14 days in refrigerator and at room temperature and checked for assay. The drug content was determined and stability was also checked during the accelerated stability testing period in the dry form as well as after reconstitution (14 days) by HPLC as per USP.

2.6 Dissolution of suspensions:

Based on the stability and assay of the suspensions it was observed that dry syrup, reconstituted with oil had maximum stability for the RMP/INH combination. Only this suspension was subjected to dissolution studies.

2.6.1 Dissolution Conditions:

Media: 0.1N HCl , 900 mL, RPM: 50, Time Point: 10, 20, 30, 45 Mins. Temp: 37±0.5ºC

Apparatus: USP Type II (Paddle) Electrolab Pvt.Ltd., India, TDT-08L.

2.6.2 Standard Preparation:

150 mg RMP and 75 mg of INH along with 5 mL diluent was diluted to 100 mL with methanol. This solution was then suitably diluted further with methanol to get a final concentration of 50 mcg/mL.

2.6.3 Test Preparation:

Suspension equivalent to 150 mg RMP (166.66 µg/mL) and 75 mg INH (83.33 µg/mL) was added in 900 ml dissolution media, volume withdrawn at each sampling point was 10 mL. 3 mL of this sample was diluted up to 10 mL with methanol to get final concentration of 50 µg/mL.

3.0 RESULTS AND DISCUSSION:

3.1 Formulation of suspensions:

Although most common, reconstitutable suspension dosage form uses water as a vehicle, but reconstitution with oil also can be an alternative for drugs with poor aqueous stability. For ciprofloxacin suspension (dry syrup) available in the US market, oil is recommended for reconstitution. The same concept was used for RMP and INH suspension because of known inherent degradation of RMP under aqueous environment in the presence of INH. This would be a viable alternative for an aqueous suspension which will enable better patient compliance and in turn improve therapy outcome in pediatric tuberculosis.

Figure1: Dissolution of Rifampicin and Isoniazid from reconstituted oily suspension

Two kinds of suspension formulations of RMP and INH combination were prepared as per formula shown in Table 1 and 2 by a very simple procedure. The oily and dry formulations were made to determine the maximum stability of RMP and INH combination. Since RMP is prone to oxidation, antioxidants were added in each formulation⁶. The choice of antioxidants was made according to the nature of formulations. Tocopherol (Vitamin E) was added as an antioxidant in oil based suspension/oil used for reconstitution of dry syrup because it is oil miscible and hence can provide more effective protection of drug as well as fatty acids presents in oily vehicle. Ascorbic acid was the antioxidant of choice for dry as well as reconstituted aqueous suspension because it can act by different mechanisms namely preferential oxidation as well as can bring about reduction. As well as it is water soluble so it can provide effective protection even after reconstitution of the suspension. In case of dry suspension, methyl and propyl paraben were added as preservatives because this formulation contained a natural gum which is prone to microbial degradation. Formulations containing antimicrobial agents are generally devoid of preservative but since rifamycin class of antibiotics are active against a selective class of microbes so a need was felt to add anti microbial preservatives in this formulation.

3.2 Evaluation of suspension:

The prepared formulations were evaluated for sedimentation volume and the result for the same is shown in Table 3. For the dry suspension the flow related properties of the dry powder were also evaluated and their results are shown in table 4.

The sedimentation volume results show that the sedimentation rate for suspension was slow and the cake if any was easily redispersible. The dry syrup reconstituted with water settled slowly as compared to the one which was reconstituted with oil. However due to the presence of aerosil there was no hard cake formation. The result of flow property of dry suspension reveals adequate flow characters for the dry powder for suspension.

3.3 Drug content analysis by HPLC:

The results of assay for RMP and INH (based on HPLC) on zero day for all products as well as after storage under various conditions is listed in table 5 and 6.

The dry syrup was reconstituted using water and in the second study it was reconstituted with oil. Both the reconstituted products were stored at 2-8⁰C under refrigeration, while the product which was reconstituted with oil was also kept at room temperature to study the assay of RMP and INH. The data is depicted in table 6. All the assays were carried out using HPLC method as per USP.

3.4 Dissolution of reconstituted suspension (oil based):

As it is reported in literature that fat reduces the absorption of RMP⁷ it was thought necessary to carry out the dissolution of the oil based reconstitutable suspension to check that whether there is any problem with the dissolution of RMP or INH (table 7, figure1). The drug content was determined by HPLC as per USP. The dissolution studies revealed that the oil based product shows rapid and complete dissolution of RMP and INH clearly indicating that the oily vehicle has not impacted the dissolution adversely. This could be because of the use of surfactant, namely tween 80 and presence of hydrophilic components like sugar.

4.0 CONCLUSION:

Based on the stability data following conclusions can be drawn

1. Readymade suspension of combination drug even using oily vehicle may not be feasible option for long term stability.

2. Dry syrup which can be reconstituted with oil (MCT oil) would be best option for having a uniform stable product for pediatric administration

3. Based on the stability data of reconstituted product it could be concluded that if the reconstituted product is stored at 2-8⁰c then it can be used minimum up to one month whereas if it is stored at room temperature it can be consumed minimum up to 14 days.

4. The dissolution studies revealed that the oil based product shows rapid and complete dissolution of both the drugs.

5. The sedimentation volume study revealed that reconstituted suspension does not have caking tendency and are easily redispersed.

5.0 REFERENCES:

1. Fixed‐Dose Combinations for HIV / AIDS, tuberculosis and malaria, report of a meeting held from 16‐18 December 2003, World Health Organisation, Geneva.

2. Shishoo CJ et al. Stability of rifampicin in dissolution medium in presence of isoniazid, Int. J. Pharm. 1999; 190: 109-123.

3. Singh S et al. The reason for an increase in decomposition of rifampicin in the presence of isoniazid under acid conditions, Pharm. Pharmacol. Commun. 2000a; 6: 405-410.

4. Seifart HI, Parkin DP and Donald PR. Stability of Isoniazid, Rifampicin and Pyrazinamide in suspensions used for the treatment of tuberculosis in children, Pediatr. Infect. Dis. 1991; 10: 827–831.

5. USP 27-NF 22, The United States Pharmacopoeial Convention, Rockville, MD, 2004, pp 1729.

6. Singh S et al, Behaviour of uptake of moisture by drugs and excipients under accelerated conditions of temperature and humidity in the absence and the presence of light. 1. Pure anti-tuberculosis drugs and their combinations. Int. J. Pharm. 2002; 245: 37– 44.

7. . Purohit SD et al, Dietary constituents and rifampicin absorption, Tubercle 68 (1987) 151-152.

Received on 08.09.2009

Accepted on 15.10.2009

Research Journal of Pharmaceutical Dosage Forms and Technology. 1(3): Nov. – Dec. 2009, 05-12