Chicken intestine inflammation model for predictive in vitro screening of Anti-inflammatory activity

 

U. S. Jijith1,2, S. Jayakumari1*

1School of Pharmaceutical Sciences, Vels Institute of Sciences, Technology and Advanced Studies (VISTAS), Pallavaram, Chennai-600117, India

2College of Pharmaceutical Sciences, Govt. Medical College, Kozhikode-673008, Kerala, India

*Corresponding Author E-mail: nisajayaa@gmail.com

 

ABSTRACT:

Objective: A key goal in pharmacological screening is a good understanding of the in vivo performance. So development of in vitro evaluations that could be correlated to in vivo performance would be commendable. The purpose of this research was to develop easily predictable in vitro method for the evaluation of anti-inflammatory activity. Further, this research work was also aimed to establish an in vitro - in vivo correlation of anti-inflammatory activity. Methods: Kappa-carrageenan was used to induce inflammation on the chicken intestine. We proposed a new apparatus which we name ‘Jijith-Jayakumari apparatus’ for the in vitro evaluation of anti-inflammatory agents. Diclofenac sodium was used as the model drug. Results and Conclusion: The developed in vitro method using chicken intestine was worth for screening ant-inflammatory activities. A good correlation was obtained between in vitro and in vivo data. Thus, an IVIVC was established for anti-inflammatory activity.

 

KEYWORDS: Jijith-Jayakumari apparatus, Kappa-Carrageenan, Diclofenac sodium, Chicken intestine.

 

 


1. INTRODUCTION:

Development of predictable in vitro evaluation or screening techniques that could be used as surrogate for animal studies could be worthy in the present scenario. The challenge for pharmacologist always will be correlate in vitro data with in vivo findings. The development of new convenient in vitro and in vivo methods will be worthy during the establishment of in vitro and in vivo correlation of anti-inflammatory activities. These in vitro techniques could replace complex and inconclusive animal studies1. These also avoid ethical issues in animal handling experimentation. Evaluation of anti-inflammatory activity is widely reported2-7. A correlation of in vitro activity with a pharmacodynamics screening method in animals could be much beneficial8.

 

Rat paw edema method is commonly employed for the screening of anti-inflammatory drugs by using plethysmometer. This tool involved in the measurement of small volume changes and this method is widely employed in the pharmacological screening of anti-inflammatory activity9-18.  The use of this method suffers the disadvantages of animal usage and ethical issues. In this context, we hypothesized an in vitro system which would avoid such disadvantages. We proposed a new apparatus which we name ‘Jijith-Jayakumari apparatus’ for the in vitro evaluation of anti-inflammatory agents. Moreover, we term the method as ‘Jijith-Jayakumari method’ for the in vitro evaluation of anti-inflammatory agents. A good number of methods have been suggested for determination of rat paw edema during in vivo evaluation of anti-inflammatory agents19-21. The anti-inflammatory activity of the standard drug, diclofenac sodium was evaluated on adult Sprague Dawley rats by carrageenan induced rat paw edema test.

 

2. MATERIALS AND METHODS:

2.1. Materials:

Kappa-carrageenan was from SD Fine Chemicals, Mumbai, India. Diclofenac sodium was purchased from Yarrow Chem Products, Mumbai, India. All other chemicals and reagents used were of analytical grade.

 

2.2. Development and validation of in vitro method:

2.2.1. Design of the Jijith-Jayakumari apparatus:

The apparatus  in its basic model has a round bottomed flask of around 250 mL capacity with a long neck and grounded opening. The second part consists of a Westergren tube inserted in inverted position into a rubber cork in such a way that the readings are visible from the outside of the apparatus. The low diameter and accurate graduations in the Westergren tube makes it a perfect choice for the apparatus. Provisions for aerations and stirring could be provided in its advanced version.

 

2.2.2. Validation of the Jijith-Jayakumari apparatus:

Study was carried out to demonstrate that swelling of chicken intestine takes place when incubated with carrageenan solution.

 

Four samples were selected for the study:

(a)  Chicken intestine with 5 mL 100 mg/mL of aqueous solution of carrageenan

(b)  Chicken intestine with diclofenac sodium (50 mg) pre-treatment (30 min) and carrageenan

(c)  Chicken intestine alone

(d)  5 mL of 100 mg/mL aqueous solution of carrageenan without chicken intestine

(e)  With the physiological buffer solution alone

 

Freshly isolated chicken intestine was collected and kept under aeration in physiological buffer. The inside of the chicken intestine was thoroughly cleaned using physiological  buffer and was cut in 45cm long pieces. The study was carried out with samples a-e as mentioned above. In the case of sample ‘a’, into the chicken intestine, 5mL of 100mg/mL solution of carrageenan was added. The intestine was tied at both ends. The tied intestine was kept completely immersed in physiological buffer solution filled in a round bottomed flask. A rubber cork with a Westergren tube inserted into it in an inverted position was firmly fitted on the grounded opening of the round bottomed flask. The rubber corked was fixed firmly in such a way that not air leaks existed between the grounded surface and rubber tube and between the Westergren tube and the rubber tube. In the case of sample ‘b’, pre-treatment with 50mg diclofenac sodium was carried out for 30 min, before addition of 5mL of 100mg/mL solution of carrageenan. In the case of sample ‘c’, the intestine was filled with 5mL of physiological buffer solution.

 

Optimization of the Jijith-Jayakumari method:

During the preliminary studies, it was noted that it is necessary to fix or optimize the time duration or time intervals and total time for the evaluation of the anti-inflammatory agents. The selected periods should be better for monitoring the difference in the results.

 

2.2.3. In vitro evaluation of anti-inflammatory activity of diclofenac sodium using Jijith-Jayakumari apparatus:

Freshly isolated chicken intestine was collected and kept under aeration in physiological buffer. The inside of the chicken intestine was thoroughly cleaned using physiological  buffer and was cut in 45cm long pieces. Into the chicken intestine in groups 2-4, diclofenac sodium in amounts mentioned in treatment table (Table 1) was added and incubated for 30 minutes. After 30 min of incubation with the drug, 5mL of of 100 mg/mL solution of carrageenan was added into. Immediately, the sample intestine was tied at both ends. The tied intestine was kept completely immersed in physiological buffer solution filled in a round bottomed flask. A rubber cork with a Westergren tube inserted into it in an inverted position was firmly fitted on the grounded opening of the round bottomed flask. The rubber corked was fixed firmly in such a way that not air leaks existed between the grounded surface and rubber tube and between the Westergren tube and the rubber tube. All these steps were done immediately without any significant time delay. The reading of the fluid level in the Westergren tube was noted at time intervals of 0 1, 2, 3, 4 and 5 h.

 

Table 1: Treatment plan for the in vitro evaluation of anti-inflammatory activity

Group

Treatment

1

Control with carrageenan alone

2

Diclofenac sodium, 5 mg

3

Diclofenac sodium, 10 mg

4

Diclofenac sodium, 20 mg

 

Level of the fluid was measured using the developed apparatus at predetermined time intervals of 0, 1, 2, 3, 4 and 5 h after carrageenan addition.

 

2.3. In vivo screening technique of anti-inflammatory activity:

The anti-inflammatory activity of the standard drug, diclofenac sodium, was evaluated on adult Sprague Dawley rats by carrageenan induced rat paw edema test. The protocol for animal studies was approved (Proposal No. 977/165/JSCP/1686/IAEC/008, dated 23.06.2018) by the Institutional Animal Ethics Committee (IAEC) of JamiaSalafiya Pharmacy College, Pulikkal, Malappuram, Kerala, Pin: 673637 (1686/PO/a/13/ CPCSEA, 15/03/2013).

 

The rats were maintained on pellet diet and water ad libitum.  Animals were housed in propylene cages with 4 per cage with dust free rice husk as bedding material under laboratory conditions with controlled environment of temperature 30oC±2oC.  They were fed adlibitum with rodents chow and free access to drinking water.  The treatment plan is given in Table 2.

Species  : Adult Sprague Dawley rats

Weight  : 150-200 g

Gender  : Either sex

 

Table 2: Treatment plan for the pharmacodynamics evaluation of anti-inflammatory activity

Group

No. of rats

Treatment

1

6

Control with carrageenan alone

2

6

Diclofenac sodium, 5 mg/kg, orally

3

6

Diclofenac sodium, 10 mg/kg, orally

4

6

Diclofenac sodium, 20 mg/kg, orally

 

Animals of groups 2-4 were pretreated with oral diclofenac sodium half an hour before injection of carrageenan. Carrageenan solution in 0.9% saline(0.1 mlof 1%w/v) was injected into the planter aponeurosis of the right hind paw of each rat. Volume of the paw was measured using the developed apparatus22 at predetermined time intervals of 1, 2 and 3 h after carrageenan administration.

 

2.4. In vitro–in vivo correlation:

The in vitro in vivo correlation (IVIVC) was carried out using the obtained in vitro and in vivo data. The in vitro data shows a continuous increase in the volume. At the same time, in vivo data shows a time dependent change in paw edema. Thus it was finally decided to select the data at time points of 1,2 and 3 hours.

 

3. RESULTS AND DISCUSSION:

3.1. Development and validation of in vitro method:

3.1.1. Design of the Jijith-Jayakumari apparatus:

The observed results were promising from preliminary evaluation using Jijith-Jayakumari apparatus. The results are shown in Figure 1.

 

·       Sample (a) was studied to check whether the isolated chicken intestine responds to the carrageenan solution resulting in its swelling. 

·       Sample (b) was carried out to confirm that there is an inhibition of volume increase due the effect of an anti-inflammatory drug. This is an important study sample as the objective of the screening of an anti-inflammatory agent is to monitor its efficiency in inhibition of inflammatory response.

·       Sample (c) was carried out to check whether the intestine alone will cause any volume increase due to tissue degradation or some other possible reasons.

·       Sample (d) was used to evaluate the possibility of error due to the swelling of carrageenan alone. Carrageenan is a hydrophilic polymer which has swelling ability in aqueous medium. In this situation, it was required that we should be able to prove that any significant increase in the volume is due to the swelling of intestine and not due to carrageenan. Here, the carrageenan solution, in the same volume and in the same strength used for sample (a) was used.

·       Sample (e) was carried out to evaluate whether there is any volume increase due to physiological solution alone.


 

Figure 1:  (a) Photograph of developed apparatus with and without intestine and (b) Photograph of swollen intestine after treatment with carrageenan

 

Figure 2: Preliminary evaluation using Jijith-Jayakumari apparatus  (a) Chicken intestine with 5 mL of 100 mg/mL solution of carrageenan  (b) Chicken intestine with diclofenac sodium (50 mg) pre-treatment and carrageenan (c) Chicken intestine alone (d) 5 mL of 100 mg/mL solution of carrageenan without chicken intestine (e) With the physiological buffer solution alone

 


The observed results were promising for further studies. It was very clear that the volume of sample with carrageenan treated chicken intestine show a drastic increase in volume (Figure 2). The study also inferred that pre-treatment of intestine with 50 mg of diclofenac sodium drastically reduces the volume change, thus simulating an in vivo response. We expected some interference due to degradation of the intestine and consequent volume expansion. But, fortunately, there was no such effect. There was no significant volume expansion due to swelling of carrageenan. The swelling of carrageenan at the concentration used might be unable to interfere with the result. This was a very important observation in our preliminary study. The possibility of gas formation and subsequent volume expansion due to any carbonate or bicarbonate present in the physiological solution was also eliminated as the buffer alone sample does not produce any volume expansion.

 

Optimization of the Jijith-Jayakumari method:

From the preliminary data it was decided to monitor the volume level till 5 hours after carrageenan addition.

 

3.1.2. In vitro evaluation of anti-inflammatory activity of diclofenac sodium using Jijith-Jayakumari apparatus:

The percentage volume increase and percentage inhibition of volume increase in each group were calculated using equations by developed us (Eqn. 1 and 2).                            

 

Change in volume, V= Vt-V0            ----------- (1)

% Inhibition = [(Vc - VS)t / (Vc)t] × 100----------- (2)

 

Where, Vo is the initial volume level (at t=0 h) of the carrageenan addition, Vt the volume level at time ‘t’ after the carrageenan addition. (Vt - Vo ) represents the change in volume readings (V). Vc is the change in volume reading of the carrageenan added control. Similarly, Vs is the change in volume reading of the drug pre-treated sample before carrageenan addition. (Vc-Vs )t represents the difference in change in volume reading between the carrageenan added control and the drug pre-treated sample. (Vc)t is the change in volume reading of the carrageenan added control at time ‘t’. Using these equations, the percentage inhibition of volume increase by the diclofenac sodium was determined with respect to that of the carrageenan added control. The obtained results are shown in Figure 3.

 

Figure 3: Column chart for % inhibition of volume increase (Diclo = Diclofenac sodium)

3.2. In vivo screening technique of anti-inflammatory activity:

The percentage edema (percentage paw volume increase) and percentage inhibition of edema in each group were calculated using following equations (Eqn. 3 and 4)23-25.

 

% Edema = [(Vt - Vo)/ Vo] × 100      --------------------- (3)

 

% Inhibition = [(Ec - Et)/ Ec] × 100   --------------------- (4)

 

where Vo is the mean paw volume before the carrageenan injection, Vt the mean paw volume after the carrageenan injection at time ‘t’, Ec the % edema of the control and Et is the % edema of the treated group at time ‘t’. The percentage inhibition of rat paw edema by the diclofenac sodium was determined with respect to that of the control. The observed results are shown in Figure 4.

 

Figure 4: Column chart for % inhibition of paw edema of rats (Diclo = Diclofenac sodium)

 

3.3. In vitro–in vivo correlation:

Figure 5 shows the IVIVC plots. The IVIVC plot for time interval of 1 showed least correlation with an R2 value of 0.838. The IVIVC plot at 2 h was better than that obtained at 1 h. It was with an R2 value of 0.949. Interestingly, the IVIVC plot at 3 h shows excellent correlation between in vitro and in vivo data with a phenomenal R2 value of 0.999. On a close examination and comparison of the present in vitro data for 2 h and 3 h, it was observed that the in vitro data at 2 h better distinguished the effect of drug dose than that at 3 h. The mean values of in vitro data were clearly different for diclofenac sodium doses for 2 h.

 

Finally, it was concluded that the in vitro and in vivo data at the time period of 2 h could be used for correlation and prediction of in vivo response. Thus a correlation between in vitro and in vivo data was established. From the obtained plot we can easily predict the volume of paw edema from the in vitro data.

 

Figure 5: IVIVC plots of data at 1,2, and 3 h

 

4. SUMMARY AND CONCLUSIONS:

A new apparatus was developed and new method has been demonstrated for in vitro evaluation of anti-inflammatory activity. The developed in vitro apparatus and method was useful in evaluating the anti-inflammatory activity of diclofenac sodium. The in vivo response of the selected dose of diclofenac was evaluated in rats by paw edema method. A good correlation was obtained between in vitro and in vivo data. Thus, an IVIVC was established for anti-inflammatory activity. From the obtained plot we can easily predict the volume of paw edema from the in vitro data.

 

5. AUTHOR CONTRIBUTIONS:

USJ and SJ conceived the study, designed the experiments, analyzed and integrated the results, and wrote the manuscript.

 

6. COMPLIANCE WITH ETHICAL STANDARDS:

The protocol for animal studies was approved (Proposal No. 977/165/JSCP/1686/IAEC/008, dated 23.06.2018) by the Institutional Animal Ethics Committee (IAEC) of Jamia Salafiya Pharmacy College, Pulikkal, Malappuram, Kerala, Pin: 673637 (1686/PO/a/13/ CPCSEA, 15/03/2013).

 

7. CONFLICT OF INTEREST:

USJ has made two patent applications (Indian Patent Application Nos. 201941009319 and 201941009320, dated 11th March 2019) related to this work.

 

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Received on 17.06.2019            Modified on 02.08.2019

Accepted on 18.09.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(6): 2843-2848.

DOI: 10.5958/0974-360X.2020.00506.5