A Novel Approach in Development of Diffusion Cell for In-Vitro Diffusion Study

Mahajan N M1*, Manmode AS2 and Sakarkar DM3

1G.S.P.S.Institute of Pharmacy, Kaulkhed, Akola (M.S.) India

2Astron Research Limited, Ahemadabad, India

3S.N. Institute of Pharmacy, Pusad, Dist.: Yavatmal (M.S.) India

*Corresponding Author E-Mail: nmmahajan78@gmail.com

 

ABSTRACT:

In the present study, a new design for diffusion cell was developed for in-vitro transdermal permeation. The cell consists of an inlet compartment, a donor compartment and a receptor compartment by increasing depth of an inlet compartment. This cell maintains sink condition easily as compared to static Franz diffusion cell. The performance of the cells was tested with permeation experiment using Nimesulide as a model drug for permeation through membrane. Diffusion study was carried out for the period of eight hours. Newer design was given better performance as compared to Franz Diffusion Cell. The manual sampling of the Franz cell requires constant attention during experiment. Air bubbles are easily formed in the receptor compartment while withdrawing samples. Entrapment of air bubbles was avoided in newer design by increasing depth of inlet compartment. The depth of the inlet compartment was important to avoid air bubbles entrapment in the receptor compartment in the newer design. Newer designs avoid the entrapment of air bubbles in the receptor compartment and maintain sink condition. Different transdermal gels were prepared using various gel bases. The polymers selected were Na CMC, MC and HPMC. In vitro permeation study was performed using rat skin. Formulated gel containing methyl cellulose found to be more stable. Methyl cellulose containing Nimesulide was found to better in all aspects compared to other gel formulations and this was comparable to the marketed gel.

 

KEY WORDS: Diffusion Cell, Transdermal Permeation, Nimesulide, Gel.

 


INTRODUCTION:

During the past few years, skin has been shown to be suitable delivery route for drugs formulated in transdermal therapeutic systems1. The permeation of chemicals through the skin can be measured by in-vivo and in-vitro techniques. In-vitro techniques are generally used due to simple experimental conditions. Franz diffusion cell is widely used static design for studying in-vitro permeation. This design has several disadvantages such as formation of air bubbles, laborious and large variation between experiments2, 3. The main aim of the designs small diffusion area, small dead volume, air bubble free, variable rinsing of the receptor compartment, flexible and reproducible collection of receptor solution, and adequate temperature control. Experiments were performed with Nimesulide as a model drug permeating through rat skin. Nimesulide is a Non-steroidal anti-inflammatory drug (NSAID), which acts by selectively inhibiting the cyclooxygenase-2 (COX-2) 4. It showed better anti-inflammatory and analgesic activity in clinical trials 4, 5, 6.

 

Nimesulide is generally well tolerated in short term treatments, but in case of long term treatments which require higher doses (200 mg or greater per day) as in the treatment of osteoarthritis, the incidence of side effects was greater 7. In the present investigation we attempted to formulate the stable TDDS of Nimesulide and the development of newer design for in-vitro transdermal permeation and their comparative study over Franz diffusion cell.

 

MATERIALS AND METHODS:

Gel containing Nimesulide purchased from market. Nimesulide, sodium Carboxy Methyl Cellulose (Na CMC), Hydroxy Propyl Methyl Cellulose (HPMC 100 cps) and methyl cellulose were obtained as a gift sample from Themis Laboratories Ltd. Mumbai. All other ingredients were of analytical grade and were used as procured.

 

Preparation of transdermal gels:

Preliminary batches were prepared by varying the proportion pf Na CMC / MC / HPMC and concentration other Excipients were kept constant. Each material was weighed accurately and added as per the steps given below.

Step 1. The polymer Na CMC / MC / HPMC was suspended with trituration in a part of distilled water containing dissolved methyl paraben and allowed to swell overnight.

 

Step 2. Nimesulide and propyl paraben was dissolved in a mixture of propylene glycol, cetiol and cetomacrogol 1000 (the later two were melted first at 700).

 

Step 3. Contents of step 1 and step 2 were mixed thoroughly to produce emulsion. pH of the mixture of step 3 was adjusted to 7.0 with addition of diethyl amine to affect gelation. The gel was then filled in collapsible tubes and labeled.

 

Table 1: Formulations of Various Nimesulide Gels

Ingredients

MC gel

Na CMC gel

HPMC gel

Nimesulide

1.0

1.0

1.0

Propylene glycol

15.0

15.0

15.0

Cetiol

2.0

2.0

2.0

Dethyl amine

q.s.

q.s.

q.s.

HPMC

--

--

20.0

Na CMC

--

20.0

--

MC

20.0

--

--

Cetomacrogol 1000

2.0

2.0

2.0

Methyl paraben

0.1

0.1

0.1

Propyl paraben

0.015

0.015

0.015

Distilled water up to

100.0

100.0

100.0

 

Preparation of rat skin:

Male Wister rat was used in the present study. Dorsal fur was removed with mechanical hair clipper and depilatory cream was wiped with distilled water. The animal was sacrificed after 24hours by cervical dislocation at the neck and a dorsal section of the skin was excised. The adhering tissues and other visceral debris were scrapped off the skin by a blunt knife and skin was prepared for in vitro permeation studies after conditioning according to Hadgraft et al.8 The skin thickness was 550 microns and had a surface area of 5 sq. cm.

 

In vitro release study with Franz diffusion cell:

The skin was so placed on the Franz diffusion cell that the epidermal surface was exposed to gel where as dermal surface faced the permeation fluid9. The rat skin was clamped in place on the diffusion cell and gel was applied uniformly on the epidermal surface of the skin. One gram of gel equivalent to 10mg Nimesulide was placed on the skin located on the donor area for evaluating the drug permeation rate and extent The Phosphate buffer saline of pH 7.2, volume 10ml and the temperature 37 10 was used as the permeation fluid. The fluid was flushed to the dermal surface of the rat skin and was stirred magnetically at 50rpm. 2.0ml of aliquots were withdrawn through sampling port of receptor compartment at 0, 1, 2, 3, 4, 5, 6, 7 & 8 hours after the commencement of experiment. Withdrawn aliquots were replaced with an equal volume of fresh buffer. The collected samples were analyzed spectrometrically (Shimadzu UV 1601 Japan) at 395nm after suitable dilution.

Fig. 1.

 

Fig. 2.

 

In vitro release study with newer cell design:

Three newer cells were specially designed. All the cells (Fig.2, 3, & 4) consisted of an inlet compartment (A), a donor compartment (B) and a receptor compartment(C). The cell was made from acrylic sheet. A rat skin was placed in to a receptor compartment that was filled with PBS buffer. O- ring (E) was use to position skin membrane between donor and receptor compartment. The donor compartment was screwed on to receptor compartment. The receptor solution entered the cell via the inlet compartment (A), flowed through inlet channel (F) to the receptor compartment (C) and left the cell via outlet channel (G) to sample collector, there was speed control knob arrangement. The entire cell is very compact with a contact area of 0.51cm.2. The flow rate was adjusted with 9ml/2min. The 2ml sample was withdrawn from sample collector at 0, 1, 2, 3, 4, 5, 6, 7 & 8 hours. The experiment was performed for prepared gel and marketed gel.

 

Table 2: Viscosity of Various Nimesulide Gel

Sr. No.

Gel

Viscosity in Centipoises (cps)

1

MC

8490

2

Na CMC

12350

3

HPMC

7852

4

Marketed (Std)

8210

 

Evaluation of Nimesulide gel:

Known amount of prepared gel was dissolved in phosphate buffer pH 7.2, suitably diluted and absorbance was measured at 395nm by spectrometer (Shimadzu UV 1601 Japan).

 

Fig. 3.

Figure 1-3: Cross-Section of Three Newer Designs

A = inlet compartment; B = donor compartment; C = receptor compartment; D = membrane; E = O-ring; F = inlet channel; G = outlet channel; H = outlet tube; J = sample collector; I, K = speed control knob.

 

Table 3: Release Pattern of Gel through Franz Diffusion Cell

Sr.

No.

Time

(Hrs.)

HPMC Gel

Na CMC Gel

MC Gel

Marketed Gel

1

0

0

0

0

0

2

1

2.52

3.25

8.52

12.24

3

2

5.23

6.22

15.24

20.45

4

3

10.23

11.02

22.36

28.24

5

4

14.23

16.35

27.14

32.26

6

5

18.54

21.34

33.64

39.65

7

6

21.03

27.35

39.25

45.21

8

7

25.26

30.25

44.25

51.24

9

8

29.31

33.21

49.25

58.25

 

Measurement of Viscosity:

Viscosity was determined using a Brookfield viscometer. In the present study, we selected the rate of shear (G) of 6rpm. The formulated gel and the marketed gel were sheared at 6rpm (constant rate of shear) for 5min. the shear stress, (F) i.e. dial reading was recorded for each formulation. From this, viscosity was calculated for the purpose of comparison.

 

Stability study of Nimesulide gel:

The gels were formulated according to formula (containing 1% w/w of the drug). The gel samples were filled in collapsible tube. Each gel preparations were then kept at different temperature conditions like; ambient temperature (temperature in the working area) 250 to 280C, 8 10 C (refrigerator temperature), 37 20C temperature (in the incubator) for three months. The following parameters were studied; colour, consistency, drug content and degradation rate constant.

 

RESULTS AND DISCUSSION:

The objective in designing newer cell was to develop an alternative for the Franz diffusion cell. The manual sampling of the Franz cells requires constant attention and is often limited to the normal laboratory hour that means a less accurate fitting of the curve. Also air bubbles are easily formed in the receptor compartment while withdrawing samples 9, 10. Initially, the entrapment of air bubbles were also problem for the two design (fig.1 and fig.2) but could be avoided by improving the depth of the inlet compartment of design (fig.3) of the cells. Magnetic mixing is of the receptor compartment was not necessary because the flow rate of buffer through the newer design cell (fig. 3) was sufficient to remove the permeated drug. However, with Franz cells magnetic stirring is crucial to provide a homogenous receptor solution 11.

 

Figure 4: Comparative Release of Nimesulide from Different Gel Bases through Franz Diffusion Cell

 

Table 4: Release Pattern through Newer Cell Design

Sr. No.

Time

(Hrs.)

HPMC Gel

Na CMC Gel

MC Gel

Marketed

Gel

1

0

0

0

0

0

2

1

3.12

4.12

9.25

13.25

3

2

6.23

7.56

15.24

19.56

4

3

11.12

9.12

23.65

28.45

5

4

15.37

13.59

29.65

33.69

6

5

19.57

16.58

33.29

39.65

7

6

22.65

20.12

38.49

44.56

8

7

25.39

23.98

45.28

53.21

9

8

30.58

32.58

51.24

60.28

 

The temperature of an in vitro system should be controlled to maintain a target temperature and minimize variation in experimental condition. With Franz diffusion cells, temperature control of receptor solution is maintained with a water jacket. However, the temperature of the donor compartment is not thermostatic and is therefore subject a variation in environmental temperature 12. Our present experiments with newer design (fig.1, fig.2 and fig.3) were performed to focus on design and were not temperature controlled. The experiments were performed at room temperature (around 250 to 280). The volume and flow rate through the receptor can be varied and the collection of the receptor solution is reproducible and can be adapted to the needs.

 

The results of the in vitro release study from different gels with Franz diffusion cells and with newer design cell is depicted in Fig. 4, fig.5. The cumulative release of drug during the study for 8hours obtained from Franz diffusion cells and newer design cell was found to be similar. It can be predict that the newer design cell (fig.3) is suitable to study in vitro release characteristics of the drug in the gel formulation. The release pattern of drug from the marketed gel was found to better than from prepared gels. The marketed gel contain 66% of alcohol may enhanced the solubility of the drug. In prepared gel methyl cellulose gel showed better compared to Na CMC gel and HPMC gel.. The tendency of film formation is also high as compared to Na CMC gel and HPMC gel in compared to MC gel, which might be a reason for poor release.

 

The viscosity of various formulated Nimesulide gels and also the marketed gel was measured using a Brookfield viscometer. Viscosity of different formulations was presented in Table 2. Except for the marketed gel, the fading of the color was observed for other gels. This was prominent in case of HPMC gel. The consistency of all gels was found to be same especially ambient and 80 temperature, but at 370 there was slight decrease in consistency of HPMC gel after two month.

 

Nimesulide was found to be more stable in the MC gel when compared to Na CMC and HPMC gel, which is evidence by the degradation rate constant (K) value at different temperature. The K values from MC gel at ambient, refrigerator and incubator temperature were; 0.0227, 0.0145 and 0.0421 per day respectively. Similarly K values for Na CMC gel were, 0.0289, 0.0251 and 0.0521 and for HPMC gel, 0.0351, 0.0361 and 0.0685 per day respectively.

 

The stability of gel formulated made with HPMC was found to be unsatisfactory. After one month, the gel consistency had decreased (visual examination), as oozing of interpenetrate liquid. The reason may be that, the gel structure was not formed or there may be some incompatibility of the HPMC with Nimesulide or with other Excipients in the formulation, the reason for which was not traced out.

 

CONCLUSION:

In conclusion, the third design of the cell is compact with a contact area of 0.51cm2 and a total volume of 84ml. The size and depth of the inlet compartment was important to avoid air bubbles entrapment in the receptor compartment. Third design avoids the entrapment of air bubbles in the receptor compartment and maintains sink condition. MC gel containing Nimesulide was found to be better in all aspect when compared to other formulations and it was comparable marketed gel, which contains 66% alcohol. Moreover, the prepared gels were not containing any permeation enhancers resulted in many tissue irritation and skin sensitization reactions. The newer design is found suitable and alternative to Franz diffusion cell in near future.

Figure 5: Comparative Release of Nimesulide from Different Gel Bases through Newer Design of Diffusion Cell.

 

ACKNOWLEDGEMENT:

All the authors are highly obliged to Leben Labs. Pvt. Ltd., Akola(M.S.) for providing the gift sample of Nimesulide. We also acknowledge S.N. Institute of Pharmacy, Pusad, Dist.- Yavatmal(M.S.) for providing us the necessary chemicals and laboratory for the experimentations to carried out.

REFERENCES:

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2.       Sanghvi P.P., Collins C.C. Comparision of Diffusion Studies of Hydrocortisone Franz Cell and the Enhancer Cell, Drug Dev. Ind. Pharm. 1993; 19, 1573.

3.       Clowes H.M., Scott R.C. Heylings J. R., Skin Absorption: Flow through or static diffusion cells, Toxic in-vitro . 1994; 8, 827.

4.       Davis, R. and Brodgen, N. R. Drugs. 1994; 48, 431.

5.       Dresier, R. L. and Riebenfeld, D. Drugs. 1993; 46, 191.

6.       Winter, C. A., Risley, E. A. and Nuss, G. W. Proc. Soc. Exp. Biol. Med. 1962; 111, 544.

7.       Hadgraft, J., Int. J. Pharm. 1996; 135, 1.

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9.       Frantz, S. W., Methods for skin absorption CRC press, Florida, 1990; 35.

10.     Chien, Y. W., ln; Tojo, K., Transdermal Controlzled System Medications, Marcel Dekker, New York, 1987; 127.

 

 

 

 

Received on . .2008 Modified on . .2008

Accepted on . .2008 RJPT All right reserved

Research J. Pharm. and Tech.2 (2): April.-June.2009; Page 315-318