November 2004, Volume 54, Issue 11

Original Article

To Evaluate and Compare the effects of First Generation Anti-Histamine (Chlorpheniramine Maleate) and Second Generation Anti-Histamine (Loratadine) on Isolated Trachea of Rabbit

G. A. Jalbani  ( Liaquat University Hospital, Hyderabad/Jamshoro1 )
K. Aamir  ( Therapeutics, BMSI, Jinnah Postgraduate Medical Centre2, Karachi )
A.M.Shaikh  ( Chandka Medical College3,4, Larkana )
M.A. Unar  ( Chandka Medical College3,4, Larkana )
R. Ashraf  ( Fatima )
F. M. Soomro6  ( PNS Rahat, Karsaz6, Karachi. )

Abstract

Objective: The incidence of respiratory allergy has increased gradually over the past several years and current estimates suggest that allergic rhinitis affects approximately 20% of the population. Large scales population surveys indicate that upto 38% of patients with rhinitis have asthma. The allergic response in the airways is an important pathogensis to cause bronchoconstriction owing to increased responsiveness of tracheo bronchial tree to various stimuli and also causes the release of histamine and other chemical mediators from mast cells. Histamine has been shown to be an important mediator of an allergic reaction in both the upper and lower respiratory airways. Chlorpheniramine maleate is a stable, most potent, sedative first generation anti-histamine and is effective in the treatment of allergic disorders. Loratadine is a highly potent, non-sedating, long acting tricyclic, second generation anti-histamine. It is indicated in allergic rhinits, chronic idiopathic urticaria and allergic bronchial asthma. The purpose of study was to evaluate the antagonistic effects of chlorpheniramine maleate and loratadine on histamine induced contractions in isolated trachea of rabbit and also to compare the effects of first generation anti-histamine (chlorpheniramine maleate and second generation anti-histamine Loratadine).

Methods: In this study twenty-four experiments were performed on isolated trachea of rabbit, in the presence of selected standard concentration of histamine dihydrochloride, antagonistic effects of various concentrations of chlorpheniramine maleate (10-18 to 10-3 gm/ml) and loratadine from concentrations 10-18 to 10-3 gm/ml were recorded by Polygraph Model 7B in terms of rate and amplitude.

Results: Chlorpheniramine maleate showed non-significant antagonistic effect from concentrations 10-18 to 10-3gm/ml in case of rate and 10-18 to 10-8 gm/ml in case of amplitude. Significant response showed from concentrations 10-8 to 10-3 gm/ml in case of rate (P<0.001) and 10-7 to 10-3 gm/ml in case of amplitude (P<0.001)while, loratadine showed non-significant response from concentrations10-18 to 10-12 gm/ml in case of rate and from concentration 10-18 to 10-14gm/ml in case of amplitude. Significant response observed from concentrations 10-11 to 10-3 gm/ml in case of rate and 10-13 to 10-3 gm/ml in case of amplitude.

Conclusion: It was concluded that chlorpheniramine maleate antagonized the histamine induced contractions 80.65% at concentration 10-3 gm/ml in case of amplitude and 11.35% at concentration 10-3 gm/ml in case of rate and loratadine 76.82% in case of amplitude and 10.59% in case of rate (JPMA 54:556;2004).

Introduction

Histamine is generally considered as the principal mediator of acute inflammatory process and allergic and anaphylactic reaction1, in both the upper and lower respiratory airways.2 It is the natural candidate as mast cell mediator3, also has an important role in gastric acid secretion and function as neurotransmitter and neuromodulator.4 It is found in all tissues, but high amount is found in lung, skin and gastrointestinal tract, high concentration in mast cells or basophils.5 At least three storage sites of histamine are found.

i)   In granules of mast cells and basophils, where it is bound with heparin and cannot exert its effects or may be metabolized.6

ii)  In the mucosal layer of the gastrointestinal tract.

iii) A third fraction is held in the hypothalamus and area postrema of brain.

It occurs in plants as well as in animal tissues. Also occurs as a component of venoms and secretions from insect sting. 7 It is formed by decarboxylation of the amino acid L-histadine. In mammalian tissues this reaction is catalyzed by the enzyme histadine decarboxylase. After formation it is either stored or rapidly inactivated contractions of smooth muscles by histamine is usually associated with depolarization and increased adenosine phosphate discharge. Histamine also contracts isolated fragments of smooth muscles of cell membrane. In tracheal muscle an increase in cAMP was detected.

The anti-histamines are the classic H1 receptors mediated response blockers and competitively block the receptor mediated response of a target tissue. 8 They are divided into first and second generation anti-histamines. 9 The main distinguishing points between first and second generation anti-histamines are that first generation drugs are widely distributed throughout the body and are more likely to block autonomic receptors and enter the central nervous system readily while the second generation drugs are less lipid soluble and enter the central nervous system with difficulty or not at al, so they show less sedative and antichlorinergic effects. 10

Chlorpheniramine Maleate

It is a stable, most potent, first generation, sedative anti-histamine belongs to alkylamine group and acts by reversible competitive antagonism at H1 receptors. 11 It is widely distributed in the body, including passage into the central nerve system. It is used in the treatment of urticaria, allergic rhinitis, angioedema, conjunctivitis, and in pruritic skin disorders. It is a common ingredient of cough and cold preparations.

Loratadine

It is highly potent2, non-sedative and long acting tricyclic11 second generation anti-histamine 12 with selective competitive peripheral histamine H1 receptor antagonistic activity 13 , belonging to the piperidine group and structurally related to azatadine. 14 It is less lipophilic, has no central nervous system activity and is essentially free of sedation. 15 It is proven to be effective in the treatment of seasonal allergic rhinitis, chronic idopathic urticaria and allergic bronchial asthma. 16

Study Design

The purpose of study was to evaluate the antagonistic effects of chlorpheniramine maleate and loratadine on histamine induced contractions of isolated trachea of rabbit and also to compare the effects of first generation anti-histamine (chlorpheniramine maleate) and second generation anti-histamine (loratadine).

All experimental works were carried out in the Department of Pharmacology and Therapeutics, Basic Medical Sciences Institute, Jinnah Postgraduate Medical Centre, Karachi.

Material and Methods

Preparation of Serial Dilution of Drugs

Serial dilutions were made by taking 1 ml of drug (1 ml/ml) and adding 9 ml of distilled water to make the ratio 1:9. In this way serial dilutions of different drugs were prepared from concentrations 10-3 to 10-18 gm/ml.

Nutrition Solution

In this vitro project Kreb's bicarbonate solution was used for the perfusion of isolated tracheal tissues. 17 For the preparation of 5 liters of Kreb's bicarbonate solution, following quantities of ingredients were used: sodium chloride 34.50 gm, sodium bicarbonate 10.50 gm, D-glucose 10.00 gm, sodium dihydrophosphate 0.60 gm, potassium chloride 1.85 gm, magnesium chloride 0.23 gm, distilled water 5000 ml.

Preparation and Isolation of Tracheal Smooth Muscle

Twenty-four healthy adult rabbits male and female (non-pregnant), approximately 2 kg weight were selected and used for the present study. 18 The animals were sacrificed, trachea was removed and transferred to petri dish containing aerated (oxygenated) Kreb's bicarbonate solution, where it was cleaned of extraneous tissues. 19 A chain of tracheal section was made by cutting several rings of cartilages and tying them together loosely in such a way that muscles of two rings were at 180° to each other. 20 Chain was suspended vertically in an inner organ bath containing 20 ml Kreb's bicarbonate solution with the help of tissue holder and connected to the Polygraph with the help of force transducer. 21 The nutritional solution was continuously aerated 10-12 bubbles per minute and temperature was maintained at 37°C. The preparations were allowed to equilibrate in Kreb's bicarbonate solution for 90 minutes under an initial lead of 2 gm.

Bath solutions were changed after every 15 minutes. The drugs were added in small quantities (1 ml) at each interval to inner organ bath from lower concentration 10-18 gm/ml to higher concentration 10-3 gm/ml according to experimental protocol and responses from each dilution were recorded on Grass Polygraph under resting tension of 1 gm. 22

Methodology

First of all spontaneous contractions of tracheal smooth muscles were recorded as a baseline. In group I, tissues were challenged with serial dilutions 10-18 to 10-3 gm/ml of histamine dihydrochloride and responses were recorded. From those responses, standard concentration of histamine dihydrochloride was selected, which had produced maximum response.

In group II, tissues were challenged with serial dilutions 10-18 to 10-3 gm/ml of chlorapheniramine maleate in the presence of selected standard concentration of histamine dihydrochloride and responses were recorded for each dilution, respectively.

In group III, tissues were challenged with serial dilutions 10-18 to 10-3 gm/ml of loratadine in the presence of selected standard concentration of histamine dihydrochloride and responses were recorded for each dilution, respectively. After taking response of each concentration the tissues were washed and giving rest for 3 minutes before applying the next concentration.

Observations and Results

Effects of Histamine on Isolated Trachea of Rabbit (Group I)

In this group, eight experiments were performed and eight rabbits were used in each group. First of all spontaneous contractions of tracheal smooth muscles were recorded as a baseline. Then responses of different concentrations of histamine from 10-18 to 10-3 gm/ml were recorded. The results obtained in these experiments were expressed in terms of rate and amplitude (mm). The difference between the mean values of these two data showed that histamine dose dependently increased the amplitude 11.38 mm at maximum concentration that was 10-3 gm/ml and increased the rate 26.99% as shown in Table 1(a) and 1(b). Statistically: The increase in rate at the concentrations 10-18 to 10-8 gm/ml was non-significant, while at the concentrations 10-7 to 10-3 gm/ml, showed the significant response (P<0.001).

On the other hand, the increase in amplitude at the concentrations 10-18 to 10-9 gm/ml was non-significant, while at the concentrations 10-8 to 10-3 gm/ml showed the significant response (P<0.001).

Effects of Chlorpheniramine Maleate on Histamine Induced Contractions in Isolated Trachea of Rabbit (Group II)

In this group, eight experiments were performed and eight rabbits were used in each group. After recording the baseline, selected standard concentration of histamine dihydrochloride was used to record the agonistic effects. Then different concentrations from 10-18 to 10-3 gm/ml of chlorpheniramine maleate were used to record the antagonistic effects. The difference between mean values of these two data showed that chlorpheniramine dose dependently antagonized the rate and amplitude 11.35% and 80.65%, respectively a maximum concentration that was 10-3 gm/ml as shown in Figure 1(a) and 1(b).

Statistically the antagonism in rate by chlorpheniramine maleate from concentrations 10-18 to 10-9 gm/ml were non-significant, while from concentrations 10-8 to 10-3 gm/ml, it was significant (P<0.001). The antagonism in amplitude by chlorpheniramine maleate from concentrations 10-18 to 10-8 gm/ml were non-significant, while from10-7 to 10-3 gm/ml it was significant (P<0.001).

Effects of Loratadine on Histamine Induced Contractions in Isolated Trachea of Rabbit (Group III)

In this group, eight experiments were performed and eight rabbits were used in each group. First of all spontaneous contractions of tracheal smooth muscles were recorded as a baseline. Then responses of different concentrations of histamine from 10-18 to 10-3 gm/ml were recorded. The results obtained in these experiments were expressed in terms of rate and amplitude (mm). The

Table 1(a). Effects of histamine on isolated trachea of rabbit in group 1 (amplitude in mm).
Drug Baseline Histamine Baseline to P value
concentration Mean SEM Mean SEM histamine (%)  
10-18 1.00 0.00 1.00 0.00 0.00 NS
10-17 1.00 0.00 1.00 0.00 0.00 NS
10-16 1.00 0.00 1.00 0.00 0.00 NS
10-15 1.00 0.00 1.00 0.00 0.00 NS
10-14 1.00 0.00 1.00 0.00 0.00 NS
10-13 1.00 0.00 1.00 0.00 0.00 NS
10-12 1.00 0.00 1.00 0.00 0.00 NS
10-11 1.00 0.00 1.00 0.00 0.00 NS
10-10 1.00 0.00 1.00 0.00 0.00 NS
10-9 1.00 0.00 1.00 0.00 0.00 NS
10-8 1.13 0.13 2.25 0.16 99.01 0.001
10-7 1.38 0.18 3.25 0.16 135.50 0.001
10-6 2.00 0.00 4.50 0.19 125.00 0.001
10-5 2.00 0.00 6.75 0.25 237.50 0.001
10-4 2.00 0.00 10.25 0.37 412.50 0.001
10-3 2.13 0.13 11.38 0.38 434.22 0.001
            SEM = Standard error of mean
NS = Non-significant.
gm/ml = Gram per milliliter

 

Table 1 (b). Effects of histamine on isolated trachea of rabbit in group 1 (rate).
Drug Baseline Histamine Baseline to P value
concentration Mean SEM Mean SEM histamine (%)  
10-18 28.50 0.42 29.38 0.46 0.03 NS
10-17 28.50 0.42 29.50 0.42 3.50 NS
10-16 28.75 0.45 29.75 0.53 3.47 NS
10-15 29.25 0.53 30.13 0.52 0.88 NS
10-14 29.50 0.42 30.50 0.42 3.38 NS
10-13 30.25 0.45 31.00 0.42 2.47 NS
10-12 30.50 0.42 31.50 0.42 3.27 NS
10-11 31.00 0.46 32.13 0.40 3.64 NS
10-10 31.38 0.46 32.38 0.46 3.18 NS
10-9 31.88 0.52 32.88 0.52 3.13 NS
10-8 32.25 0.53 33.75 0.59 4.65 NS
10-7 32.88 0.55 36.63 0.46 11.40 0.001
10-6 33.63 0.53 39.13 0.67 16.35 0.001
10-5 34.50 0.60 41.25 0.90 19.56 0.001
10-4 34.75 0.49 42.75 0.77 23.02 0.001
10-3 35.63 0.56 45.25 0.84 26.99 0.001
SEM = Standard error of mean
NS = Non-significant.
gm/ml = Gram per mililiter

[(0)]

difference between the mean values of these two data showed that loratadine dose dependently antagonized the

[(1)]

rate and amplitude 10.59% and 76.82%, respectively as shown in Figure 2(a) and 2(b). Statistically: The antagonism in rate at the concentrations of 10-18 to 10-12 gm/ml was non-significant, while the concentrations 10-11 to 10-3 gm/ml showed the significant response (P<0.001).

The antagonism in amplitude at the concentrations 10-18 to 10-14 gm/ml was non-significant, while at the concentrations 10-13 to 10-3 gm/ml showed significant response (P<0.001).

[(2)]

 

[(3)]

 

Discussion

In the present in vitro study, we have observed the effects of first generation anti-histamine (chlorpheniramine maleate) and second generation anti-histamine (loratadine), on histamine induced contractions of tracheal smooth muscles. In this study histamine produced a prominent action on the smooth muscles of the tracheal tissues of the rabbit characterized by contractions and both the anti-histamines showed significant antagonistic effects, but the effects of chlorpheniramine maleate were more than the loratadine. This study is according to in vivo study done by Druce et al 12 in allergic rhinits who has compared the symptomatic effects of brompheniramine (belongs to same group as chlorpheniramine maleate) and loratadine. He found that brompheniramine was superior than loratadine in clinical efficacy. Still there is no in vitro study available on loratadine to observe its effects on the respiratory tract. More work on this drug is necessary.

References

1. Gozsy B, Kato L. Investigations into the mechanism of action of antihistamines. Ind J Med Res 1961;49:595-601.

2. Corren J, Harris AG, Aaronson D, et al. Efficacy and safety of loratadine plus pseudoephedrine in patients with seasonal allergic rhinitis and mild asthma. J Allergy Clin Immunol 1997;100:781-8.

3. Roquet A, Dahlen B, Kumlin M, et al. Combined antagonism of leukotrienes of allergen-induced early and late phase airway obstruction in asthmatics. Am J Resp Crit Care Med 1997;155:1856-63.

4. Morrow JD, Roberts LJ, Brow N, et al. Autacoids; drug therapy of inflammation In: Goodman and Gilman's Pharmacological basis of therapeutics. 10th ed. New York: McGraw Hill 2001, pp 211-12.

5. Katzung BG. Histamine serotonin and the ergolalkaloids: basic and clinical pharmacology 8th ed. New York: McGraw-Hill 2001, pp 272-91.

6. Estelle F, Simons R. A double-blind, single-dose, crossover comparison of cetirizine, terfenadine, loratadine, astemizol and chlorpheniramine versus placebo: Suppressive effects on histamine-induced wheals and flares during 24 hours in normal subject. J Allergy Clin Immunol 1990;540-7.

7. Lippincott's, autocoids and Autocoid antagonists. In: Harvey RA, Champe PC (eds.). Lippincott's illustrated reviews 2nd edition. Philadelphia 2000; 419-28.

8. Kay GG, Berman B, Mockoviak SH, et al. Initial and steady-state effects of diphenhydramine and loratadine on sedation, cognition, mood and psychomotor performance. Arch Intern Med 1997;157:2350-6.

9. Nicholson AN. Therapeutics: antihistamines and sedation. Lancet 1983;2:211-12.

10. Sheffer AL, Samuels LL. Cetirizine: antiallergic therapy beyond traditional H1 antihistamines. J Allergy Clin Immunol 1990;86:1040-6.

11. Therapeutic drugs (Cetirizine). 2nd ed. 1999;1:C152-C156.

12. Druce HM, Thoden WR, Mure P, et al. Brompheniramine, loratadine and placebo in allergic rhinitis: a placebo-controlled comparative clinical trial. J Clin Pharmacol 1998;38:382-9.

13. Carpio JD, Kabbash L, Turenne Y, et al. Efficacy and safety of loratadine (10 mg once daily), terfenadine (10 mg twice daily) and placebo in the treatment of seasonal allergic rhinitis.J Allergy Clin Immunol 1989;84:741-6.

14. Gutkowski A, Bedard P, Carpio JD, et al. Comparison of the efficacy and safety of loratadine, terfenadine and placebo in the treatment of seasonal allergic rhinitis. J Allergy Clin Immunol 1988;81:902-7.

15. Reicin Alise, White R, Weinstein SF, et al. Montelukast, a leukotriene receptor antagonist in combination with loratadine, a histamine receptor antagonist, in the treatment of chronic asthma. Arch Intern Med 2000;160:2481-8.

16. Day JH, Briscoe M, Widlitz MD. Cetirizine, loratadine, or placebo in subjects with seasonal allergic rhinits: Effects after controlled ragweed pollen challenge in an environmental exposure unit. J Allergy Clin Immunol 1998;101:638-45.

17. Compton MR, Hillery SJ, Kerr SJ, et al. Different immunological sensitizing regimens and airway reponsiveness in vitro to contractile agonists in guinea pigs. Eur J Pharmacol 1988;158:129-34.

18. Bristow M, Sherrod TR, Green RD. Analysis of Beta receptor drug interactions in isolated rabbit atrium, aorta, stomach and trachea. J Pharmacol Exp Therap 1970;171:52-9.

19. Drazen JM, Schneider MW. Comparative responses of tracheal spirals and parenchymal strips to histamine and carbachol in vitro. J Clin Invest 1977; 61:1441-7.

20. Blattner R, Lassen HG, Dehhert H, et al. Experiments on isolated smooth muscle preparations. HSE Biological Techniques 1978. Hugosachs Electroni KKG. Germany.

21. Diamantis CW, Sofia RD. Modulation of in vitro anaphylaxis of guinea-pig isolated tracheal segments by azelastine, inhibitors of arachidonic acid metabolism and selected anti-allergic drugs. Pharac 1986;87:443-8.

22. Brink C, Duncan PG, Douglas JS. The response and sensitivity to histamine of respiratory tissues from normal and ovalbumin-sensitized guinea pigs: Effects of cyclo-oxygenase and lipoxygenase inhibition. J Pharmacol Exp Therap 1981;217:592-601

Journal of the Pakistan Medical Association has agreed to receive and publish manuscripts in accordance with the principles of the following committees: