INTRODUCTION:

Hypertension, commonly referred to as “high blood pressure”, is a medical condition where the pressure is chronically elevated is one of the commonly found diseases, affecting most of the populations in the world. So, for treating hypertension effectively is main criterion of study. For treating hypertension, commonly used drugs include ACE inhibitors, Alpha Blockers, Beta Blockers, Calcium Channel Blocker, Diuretics and combination of any of these categories in immediate action required.

The objective of this study was to develop Ramipril pellets. Trial and error method was used for the development of formulation of ramipril pellets. With reservoir type coated pellet dosage forms, the polymeric coating must be able to withstand the compression force; it can deform, but should not rupture.

Without sufficient elasticity of the film, the coating could rupture during compression and the extended release properties would be lost. In addition, the bead core should also have some degree of plasticity, which can accommodate changes in shape and deformation during Tableting⁵. So, MCC can be used for the core material for drug coating. But directly drug coating on MCC will create compatibility problem or stress to the ramipril, so, film coating on MCC will overcome this problem and also give the elasticity to the drug.

Ramipril (marketed as Tritace® or Altace®) is an angiotensin-converting enzyme (ACE) inhibitor, used to treat hypertension and congestive heart failure. ACE inhibitors lower the production of angiotensin II, therefore relaxing arterial muscles while at the same time enlarging the arteries, allowing the heart to pump blood more easily, and increasing blood flow due to more blood being pumped into and through bigger passageways.

MATERIALS AND METHODS:

Materials

Ramipril, was obtained as kind gift sample. Hypromellose, Micro crystalline cellulose were also obtained as a gift sample from reputed and well known pharmaceutical industry. Other solvents and reagents used were of analytical grade.

Formulation of Ramipril Pellets

STEP: I: Seal coating on MCC:

Composition for seal coating on MCC:

Composition of pellets decided by the simple trial, in which 50 mg of pellets contain the 5 mg of pellets were tried to produce. A result of that will be nearer to 5 mg of ramipril. So, that formulation was used for next optimization of polymer coating. Here polymer used for seal coating was hypromellose which is used as film former in 5 – 15% concentration. So, here trial starting with the 5% concentration of polymer for seal coating. And for glidant talc was used in min concentration and seal coating was done on MCC as a core material⁶. Seal coating on MCC was done by Fluid bed processor (GLATT GPCG machine).

*Table: Formulation of seal coating on MCC.*
Ingredients	Qty. for 2500 tablets
Ingredients	B.No. P 007*	B.No. P 008*	B.No. P 009*
Microcrystalline cellulose	102	100	98
Hypromellose 2910 (6 CPS)	5.1425 (5 %)	7.1425 (7%)	9.1425 (9%)
Talcum	0.715	0.715	0.715
Purified Water	74.49	100.0	125.50
Total	107.8575	107.8575	107.8575
*Quantity in gm

Coating suspension preparation:

Dissolve Hypromellose 2910 (6cps) USP in Purified water under mechanical stirring till clear solution obtained. Then, Add Talcum USP-NF into above solution. Filter solution though 100# S.S. screen. Use this suspension for coating.

Seal coating on Celphere CP 102:

Composition for seal coating on Celphere CP 102

Celphere CP 102 is the biggest Size granular grade of MCC⁸. Celphere CP 102 is the particles of same particle size and spherical in shape. So film coating on this material will be good and also have the great plasticity than other grade of MCC. So, Seal coating optimization will be done by using Celphere CP 102 in place of MCC and other ingredients for the coating will be taken same as in Last trial.

*Table: Formulation for seal coating on Celphere CP 102.*
Ingredients	Qty. for 2500 tablets
Ingredients	B.No. P 10*	B.No. P 11*	B.No. P 12*
Microcrystalline cellulose (Celphere CP 102)	102	100	98
Hypromellose (6 CPS)	5.1425 (5%)	7.1425 (7%)	9.1425 (9%)
Talcum	0.715	0.715	0.715
Purified Water	74.49	100.0	125.50
Total	107.8575	107.8575	107.8575
*Quantity in gm

STEP: II: Drug coating on seal coated pellets.

Composition for drug coating on seal coated pellets:

The best batch from seal coated pellets (B.No. P 11) was taken for drug coating formula optimization. For drug coating, drug Ramipril along with Binder Hypromellose were taken. Here polymer concentration had been taken from 2% to 6% because Hypromellose has been used as binder for in this range of concentration. Here binder was used because of drug particles can stick to the seal coated pellets and make a uniform drug coating on seal coated pellets and appropriate amount of drug to be contained in to selected quantity of pellets.

*Table: Formulations for drug coating on seal coated pellets.*
Ingredients	Qty. for 2500 tablets
Ingredients	B.No. P 13*	B.No. P 14*	B.No. P 15*
Polymer coated pellets (7%)^a	107.43	107.43	107.43
Ramipril^b	13.8	13.8	13.8
Hypromellose 2910 (6 CPS)^b	2.29 (2%)	4.591 (4%)	6.88 (6%)
Purified Water^b	149.82	171.25	192.56
Total	123.52	125.821	128.11
*Quantity in gm

^a0.42 gm of Polymer coated Pellets of step –I is not considered due to process loss.

^b Quantity of item No 2,3 and 4 depends on the yield of polymer coated pellets.

STEP: III: Film coating on drug coated pellets.

Composition for film coating on drug coated pellets^{10- 12}.

Drug coated pellets of B. No. P 14 was taken for film coating. Here formulation for film coating on drug coated pellets was varied by different concentration of polymer used as a film former.

For film coating, Polymer concentration started from 7%, because in seal coating 7% polymer concentration shows good results. But, pellets after drug coating are bigger in size than Celphere CP 102, so, theoretically amount of polymer required is high than seal coating. So, for film coating polymer concentration taken was in range of 7% to 9%.

Talc was added for reducing the static charge in to pellets. By adding talc in to spraying solution, evaporate during spraying and stick to the pellets and remove the static charge of pellets during spraying and drying¹³.

Evaluation parameters of pellets⁷

Step I

Appearance: Pellets were evaluated for its appearance and uniform film coating on core material.

Particle size distribution study: particle size distribution study was performed by vibrating sieve shaker.

Step II

Appearance: Pellets were evaluated for its appearance and uniform Drug coating on Seal coated pellets.

Particle size distribution study: particle size distribution study of pellets was performed by using different size sieves on vibrating sieve shaker for 5 minutes at 1.5 amplitude.

% Assay: % Assay of drug coated pellets was performed for checking the quantity of drug entrapped into selected quantity of pellets. % Assay was performed by HPLC method as described in 5.1.3.1.1.

Step III

Appearance: Pellets were evaluated for its appearance and uniform Drug coating on Seal coated pellets.

Particle size distribution study: particle size distribution study was performed by vibrating sieve shaker.

% Assay and % CU: % Assay of drug coated pellets was performed for checking the quantity of drug entrapped, and performed by Method as described in 5.1.3.1.1.

In vitro dissolution study: in vitro dissolution study was conducted by incorporated pellets in to its dosage form (tablet). Final formulation of pellets is tablet. So, for tablet of ramipril pellet, placebo formulation for tablet was prepared which is shown in table. In this formulation all quantity was taken by utilizing all excipients used in innovator formulation. This tablet dosage form put in to dissolution apparatus (USP Type I, Basket) and takes the sample after specified time interval and measure the concentration release in time profile by HPLC parameters.

*Table: Composition of Placebo tablets formulation*
Ingredients	Qty. for all batch*
Ramipril Pellets 10% w/w eq. to 5 mg Ramipril	50.00
MCC	70.00
Mannitol (Pearlitol SD 200)	70.00
SSG	06.00
Talcum	01.00
Mg Stearate	02.00
Aerosil (colloidal silicone dioxide)	01.00
Total	200.00
* Quantity in mg.

Accelerated stability study:

Placebo Tablets formulated with ramipril pellets were subjected to the Accelerated Stability studies in Aluminum / Aluminum pouch pack as aluminum strip is considered the best protecting packaging material. As the dosage form is formulated for delivery to stomach, no change should occur in its %dissolution profile and related impurities. Ramipril is very sensitive to light, moisture and any physical or chemical stress, so by that it’s degrade in to ramipril – DKP product by cyclization. So, after study, no degradation of ramipril will be obtained. For study tablets prepared from pellets and only pellets of B. No. P 19 were packed in aluminum pouch and in vial respectively, charged for accelerated stability study at 40⁰ C and 75% RH for 3 months in a chamber. Stability study of pellets were performed by, first checking the initial parameters of pellets Then put it in specified condition for 1 month. After 1 month check for all parameters. If it shows satisfactory results then continue the test for next month and continue for 3 months.

RESULTS AND DISCUSSION:

Step I

(a)Appearance and Particle size distribution study:

Appearance of all batches pellets was not spherical, and not formed. Only fines or powders of MCC seen. From the comparisons of P.S.D. of different batch shown that in 5% of film coating of polymer, pellets are not formed, and powder was seen only. In 7% polymer film coating, pellets are formed but it was very small in size. In 9% coating aggregation of particle was seen because of higher concentration of polymer and core material is as small for coating. So, from the data of P.S.D. shown that pellets were not formed properly in MCC used as a base material for coating. So, Granular grade of MCC (Celphere CP 102) was tried for proper pellets for next trial. P.S.D data shown in table.

*Table:* *Comparative P.S.D study of Batch P 007, P008, P009.*
Sieve no.	% wt retain of Pellets
Sieve no.	B.No. P 007	B.No. P 008	B.No. P 009
20	0	0	0
40	0	0	0
60	1.8	2.1	4.5
80	4.2	5.6	12.5
100	11.4	16.8	14.8
Above 100 (Base)	82.6	75.5	68.2

Figure: Comparative P.S.D study of Batch P 007, P008, P009.

(b)By using Celphere cp 102, pellets were in spherical shape, and properly form. From the comparisons of P.S.D. of different batch shown that in 7% polymer film coating, pellets are formed and it is very regularly distributed in 60# sieves than other two concentrations. And also the 7% film coating is sufficient for no interaction of drug with MCC and also for elasticity to pellets than other two concentrations of polymers. Pellets of this batch were not breaking easily which, show good film formation than other two batches.

In 5% film coating, fines of powder seen very high than other two batches. Batch P 10 shown the small quantity of pellets formation than other two batches and also the pellets were break freely means film coating was not properly done.

In 9% film coating aggregation of pellets was seen and pellets were stick each other and somewhat slugging observed. So, for next experiment of drug coating on polymer coated pellets will be done on Batch no P 11 (7% polymer coating) pellets. P.S.D data of different batches shown in table.

*Table:* *Comparative P.S.D study of Batch P 10, P 11, P12.*
Sieve no.	% wt retain of Pellets
Sieve no.	B.No. P 10	B.No. P 11	B.No. P 12
20	0	0	0
40	0.32	12.2	21.6
60	53.6	74.9	72.1
80	23.7	6.3	4.1
100	15.2	2.4	0.9
Above 100 (Base)	7.18	4.2	1.3

Figure: Comparative P.S.D study of Batch P 10, P 11, P12.

Step II

Appearance and Particle size distribution study:

Drug coating on the seal coated pellets with different proportion of binder concentration was evaluated. Three different concentration of binder was taken for drug coating. In B. No. P 13, P 14, P 15 hypromellose concentration was used 2%, 4%, 6% respectively. But by appearance of all three batch pellets, B. No. P 14 pellets were seen uniform in shape and size. And also the pellets of B. No. P14 had good flow than other batches.

P.S.D. study of all three batches shown that drug coated pellets was retained highest on 40# sieve in B.No. P 14 than other two batches. And also fines were very less observed in B. No. P 14 than in B. No. P 13 because in B. No. P 13 less concentration of binder was used. Aggregation of pellets was also less in B. No. P 14 than B.No. P 15 because in B. No. P 15 Binder concentration was higher than other batches.

So, by appearance and Particle size it was concluded that B. No. P 14 was used for next film coating on drug coated pellets. P.S.D data of all three batches was shown in table.

*Table:* *Comparative P.S.D study of Batch P 13, P 14, P 15.*
Sieve no.	% wt retain of Pellets
Sieve no.	B.No. P 13	B.No. P 14	B.No. P 15
20	0	0	0.86
30	0.2	2.3	8.94
40	41.7	73.8	71.9
60	32.2	20.4	14.2
80	15.4	1	2.1
100	3.8	0.7	0.8
Above 100 (Base)	6.7	1.8	1.2

Figure: Comparative P.S.D study of Batch P 13, P 14, P 15.

% Assay:

*Table: 6.1.11 % Assay of Batch P 13, P 14, P 15.*
B. NO.	Avg. %Assay of Ramipril in 50 mg of pellets
P 13	94.1
P 14	99.6
P 15	106.8

From the P.S.D data and % Assay data, it was concluded that B. No. P 14 shows good drug entrapment and appearance of pellets with 4% polymer used as a binder. So, for next film coating on drug coated pellets were done using B. No P 14 pellets

Step III

Appearance and particle size distribution study:

From the appearance and P.S.D. study of pellets, batch no. P 18, P 19 shown good pellets. In Batch P 16 and P 17, film coating on pellets was not completely done. In 7 and 7.5% concentration polymer film coating on pellets was observed not proper and also plasticity wasn’t observed in pellets. Most of the film coated pellets have to retain on the30 and 40# sieve with minimum fines. This criterion was only observed in B. No. P 18, P 19, P 20. But in Batch P 20 aggregation of pellets was observed in little amount and sticking of pellets occurred to the each other. So from appearance and P.S.D study, it was concluded that B. No. P 18 and P 19 show good pellets in comparisons with other batches. P.S.D data of all batches are shown in table.

*Table: P.S.D. Study of B. NO. P 16, P 17, P 18, P 19, P 20.*
Sieve no.	% wt retain of Pellets
Sieve no.	P 16*	P 17*	P 18*	P 19*	P 20*
20	0.7	0.6	1.8	5.8	10.1
30	42.8	50.8	55.1	66.1	71.8
40	35.8	30.1	30.7	24.3	15.8
60	12.2	7.6	4.8	1.3	1.4
80	2.5	4.1	4.1	0.7	0.2
100	3.3	3.8	2.2	1.1	0
Above 100 (Base)	2.7	3	1.3	0.7	0.7

Figure: P.S.D. Study of B. NO. P 16, P 17, P 18, P 19, P 20.

% Assay and Content Uniformity:.

% Assay and CU of all batches was acceptable and came in to the range, except batch P 16. %assay and % CU results of all batches shown that all batch contain equivalent amount of ramipril within range. But, Batch P 19 shows the comparable and exact result of % assay and %CU. So, from the results it was concluded that Batch P 19 was good for film coating. Assay results of all batches are shown in table.

*Table: % Assayand CU of B. No. P 16, P 17, P 18, P 19, P 20.*
B. NO.	Avg. % Assay	Avg. % CU
P 16	105.1	106.8
P 17	103.3	104.3
P 18	101.8	102.8
P 19	99.8	100.2
P 20	98.0	98.4

In-vitro Dissolution study:

Because of final formulation of pellets will be tablet, dissolution of pellets was carried out by converting it in to tablet dosage form. In-vitro dissolution data of tablet was compared with the release profile of innovator dosage form. From the dissolution data, B. No. P 16 and P 17 show very fast release of ramipril. This may be because of rupture of pellets during compression and this rupture of pellets also may cause the stability problem of ramipril. While, B. No. P 18 and P 19 shows good and comparable results with the innovator dosage form. And B. No. P 20 show little retardation of release than reference product. So, Batch P 19 show good release than other batches and put for accelerated stability study.

So, tablet prepared by pellets and only pellets of B. No. P 19 was subjected for stability study. So after stability study, it can be confidently concluded that B. NO. P 19 shows good result than other batches and also used for next tablet preparation of pellets.

Similarity factor:

Methods of comparing in vitro dissolution profile data are critical in the development in sustained release dosage formulations. Test and reference usually compared by: 1.) Graphical comparison: which is shown in figure 2.) Mathematical methods which compare the entire dissolution profile for test and reference.

The similarity factor, F₂ given by SUPAC guidelines for modified release dosage form was used as a basis to compare dissolution profiles. The dissolution profiles are considered to be similar when f₂ is between 50 and 100. The similarity factor F₂ is logarithmic reciprocal square roort transformations of one plus the average mean squared (the avg. sum of squares) differences of drug % dissolved between the test and reference products over all time points. The dissolution profile of products were compared using F₂ is calculated by following formula.

Where R_t and T_t are the cumulative percentage dissolved at each of the selected n time points of the reference and test product respectively. The factor f2 measures the closeness between the two profiles. FDA has set a public standard of f2 value between 50 – 100 to indicate similarity between two dissolution profiles.

*Table: Comparative F₂ value for dissolution of different batches.*
B.No.	P 16	P 17	P 18	P 19	P 20
F₂Value (%)	61.40	66.49	76.62	81.36	60.09

From the above comparisons of F₂ value, all batches shown similarity with the reference product release. B. No. P 18 and P 19 show very good similarity with the reference product. But, in both of them B. No. P 19 shows 81.36 % similarity with reference product which is greater than B. No P 18 (76.62%). So, from the all above observation it was decided, B. No. P 19 was used for next tablet formulation development of Ramipril / HCTZ tablets.

Accelerated stability study:

Stability study of tablet containing pellets and only pellets of Batch No. P 19 was performed for 3 months at 40⁰C / 75% RH condition. Stability study of B. NO P 19 was performed for mainly reason of its degradation. Tablet of pellets was subjected to study for in alu / alu pouch and only pellets of B. no. P 19 Was subjected for study in open and closed vial both at 40⁰ C and 75% RH for 3 months in a chamber

The results of Comparisons of dissolution profile after 3 months with initial were shown in table and in figure shown below.

*Table: Comparative dissolution profile of B. No. P 19 and after 3 month stability study sample.*
Time (min)	% Cumulative drug release
Time (min)	P 19 *	After 3 month study
0	0	0
5	76.4	73.8
10	93.1	89.6
15	96.5	92.9
30	98.7	94.8
45	99.8	97.1
60	100.1	98.4
Similarity factor (f₂)	75.91%

Figure: Comparative dissolution profile of B. No. P 19 and after 3 month stability study sample.

CONCLUSION:

For making stable Ramipril pellets, three steps procedure developed in which first film coating on Celphere CP 102, then Drug coating on Seal coated pellets, then Film coating on drug coated pellets.

In step I, Seal coating on Celphere CP 102 was first done on MCC but in that case very fines form because of no support for coating. So, pellets were not form properly in using MCC as a core material. By using of Celphere CP 102, support for coating is obtained and pellets were form in spherical shape. Here seal coating was tried with 3 different polymer concentrations. But in 7% polymer concentration, pellet size was proper and also the plasticity also achieved than 5% concentration. And in next 9% concentration aggregation of pellets was occurred so, for next drug coating 7 % coated pellets were taken.

In step II, drug coating was done. Here a main criterion was to obtained proper assay of pellets with proper drug loading. Only drug solution spraying was not possible so, drug suspension with 3 different concentrations (2%, 4%, and 6%) of polymer as a binder was used for spraying. In 2% concentration of polymer drug loading was not achieved properly and also the fines of powder were seen. In 4% polymer drug loading was completely achieved and also the pellets were in spherical shape and free flowing. In 6% polymer drug loading is achieved but main problem of little aggregation obtained. So for next film coating 4% concentration polmer used drug coated pellets were taken.

Film coating on drug coated pellets was started with 7% concentration to 9% because 7% concentration was best optimized during seal coating. But here after drug coating pellets were little big in size than Celphere CP 102, so may require high concentration of polymer. Film coating was done using 5 different concentrations (7%, 7.5%, 8%, 8.5%, and 9%). In 7%, and 7.5%polymer concentration film coated pellets were not formed properly and also fines was observed and not proper film coating was seen and in 9% polymer concentration film coated aggregation as shown in seal coating was seen but in 8% and 8.5% polymer concentration film coated pellets were in proper shape, also achieved assay, and also show comparable dissolution profile with the reference product. But by similarity factor F₂ 8.5% polymer concentration film coated pellets were shown 81.36% similarity with reference product release than other concentrations. And also the stability study of 8.5% polymer concentration film coated pellets shown no degradation of Ramipril at 40⁰ C and 75% RH condition after 3 month. So, this film coated pellets were taken for next optimization of tablet dosage form.

REFERENCES:

1. Sheikh Shafiq, Faiyaz Shakeel, Sushma Talegaonkar, Farhan J. Ahmad, Roop K. Khar, Mushir Ali, Development and bioavailability assessment of Ramipril nanoemulsion formulation European Journal of Pharmaceutics and Biopharmaceutics, Article in Press, Corrected Proof.

2. Koytchev R, Ozalp Y, Erenmemisoglu A, van der Meer MJ, Alpan RS, Effect of the combination of lisinopril and hydrochlorothiazide on the bioequivalence of tablet formulations, Arzneimittelforschung. 2004 Sep; 54(9a):605-10.

3. Li CL, Martini LG, Ford JL, Roberts M, The use of Hypromellose in oral drug delivery, J Pharm Pharmacol. 2005 May; 57 (5):533-46.

4. Stabilized Individually Coated Ramipril Particles, Compositions and Methods, Wo2006050533, 2006-05-11.

5. Stabilized Ramipril compositions and methods of making, WO2006052968, 2006-05-18.

6. http://www.nbent.com/details.htm

7. http://en.wikipedia.org/wiki/microcrystalline_cellulose

8. http://www.ceolus.com/eng/product/celphere/index.html

9. Ketan A. Mehta, M. Serpil Kislalioglu, Wantanee Phuapradit, A. Waseem Malick and Navnit H. Shah, Effect of formulation and process variables on porosity parameters and release rates from a multi unit erosion matrix of a poorly soluble drug, Journal of Controlled Release, Volume 63, Issues 1-2, 3 January 2000, Pages 201-211.

10. Cole G, Hogan J, Aulton M. Pharmaceutical coating technology. Taylor and Francis, London, 1995.

11. Armstrong NA. Tableting. In M.E. Aulton (ed.), Pharmaceutics: the science of dosage form design. Churchill Livingstone, New York, 1997.

12. Ragnarsson G, Johansson MOJ. Coated drug cores in multiple unit preparations: Influence of particle size. Drug Dev. Ind. Pharm. 1988;14:2285,2297.

13. Raymand C Rowe, Paul J Sheskey, Paul J Weller, “Handbook of Pharmaceutical Exipients”, 4th edition, publish by Pharmaceutical Press, 641-643.

14. Laicher A, Lorck CA, Tobin J, Stanilaus F. Process optimization of pellet coating and drying using fluid-bed production units. Pharm. Tech. Eur. 1994;8:41,48.

15. El-Mahrouk GM, Al-Meshal MA, Al-Anagary AA, Mahrous GM. Preparation and evaluation of sustained-release indomethacin nonpareil seeds. Drug. Dev. Ind. Pharm. 1993;19: 1903,1916.

16. Hosny EA, El-Mahrouk GM, Gouda MW. Formulation and in vitro and in vivo availability of diclofenac sodium enteric-coated beads. Drug. Dev. Ind. Pharm. 1998;24: 661-666, PubMed.

Received on 06.08.2012 Accepted on 27.08.2012

Asian J. Pharm. Tech. 2(3): July-Sept. 2012; Page 87-93

*Table: Formulations for film coating on drug coated pellets.*
Ingredients	Qty. for 2500 tablets
Ingredients	P 16*	P 17*	P 18*	P 19*	P 20*
Drug coated pellets (4%)^a	124.91	124.91	124.91	124.91	124.91
Hypromellose 2910 (6 CPS)^b	8.74 (7%)	9.36 (7.5%)	9.99 (8%)	10.61 (8.5%)	11.24 (9%)
Talc^b	0.878	0.940	1.00	1.066	1.129
Purified Water^b	123.39	132.14	140.99	149.8	158.59
Total	134.52	135.21	135.9	136.58	137.27
*Quantity in gm

*Table: Comparative dissolution profile of B. No. P16, P17, P18, P19, P20.*
Time (min)	ref Ramipril	P 16*	P 17*	P 18*	P 19*	P 20*
0	0	0	0	0	0	0
5	80.8	95.6	92.3	87.7	76.4	72.3
10	95.8	99.7	99.2	97.9	93.1	85.2
15	98.5	100.1	100	99.6	96.5	91.4
30	99.8	100.4	100.1	99.9	98.7	95.1
45	100.1	100.6	100.1	100.1	99.8	97.2
60	100	100.6	100.3	100.2	100.1	98.1