Indian Journal of Ex perimenta l Bio logy Vol. 39, September 200 I , pp. 871 -877

Antiallergic/antiasthmatic effect of novel antiallergic hexapeptide-95/220 in various experimental models

Rashmi Singh, Amar Nath & P P Gupta

Di vision of Pharmacology

and

M Shukla, S K Khare & 8 Kundu

Di vision of Biopolymer, Central Drug Research Institute, Lucknow 226 001 , India

Received /8 August 2000; revised 20 February 2001

The effects of newly sy nthesized anti allergic hexapeptide 95/220 was investigated on various a llerg ic and asthmat ic test mode ls. Thi s newly developed peptide was found to be more potent than c linically used drug disodium cromoglycate (DSCG). Hexapeptide 95/220 inhibited immedi ate hypersensiti vity reac tions such as pass ive cutaneous anaphylaxis (PCA) and mast cell degranulation in rats, ant igen-induced bronchoconstricti on in acti vely sensitized guinea pigs in dose dependent manner like DSCG. Anti gen-induced contraction of guinea pi g ileum was also markedly inhibited by thi s newly developed hexapeptide in the same fashion as ketoti fen and DSCG did but at comparati ve ly lower dose. Egg albumin-induced hi stamine release was also blocked by this hexapeptide from chopped lung ti ssues of sensitized guinea pigs. These results sugges t that hexapeptide' 95/220 has potent inhibitory effect on immedi ate hypersensitivity reacti ons thereby inhibiting mediator release from mast cell. Moreover, this newly synthesized peptide is orally acti ve and effective at lower doses as compared to standard drugs.

The number of deaths from asthma is ri sing and these alarming increases are occurring despite a marked increase in prescribed asthma therapy 1.2. The ti ssue injury within the airway wall in asthma is due to the influence of chemica l mediato rs an smg from infiltrating infl ammatory and res ident cell types3

. For many years mast cells have been known to play a central role in the pathogenesis of asthma. The traditional understanding has been that these cells are activated as a result of the interaction of allergen with IgE coated mast cells4

·5

. Thi s in turn releases a series of chemical medi ators including histamine6

·7

,

leukotrienes (LTs)8, platelet acti vating fac tor (PAF)9

,

thromboxane A210 and prostagl andin (PG) D2

11 have significant roles in airway infl ammati on responses such as airway eosinophili a, late asthmatic response and airway hyperresponsiveness as well as in im mediate hypersensiti vity reac tion such as bronchi al contraction and airway pl asma extravasation .

Novel therapeutic approaches based on new mechanisms of action are being developed with more emphas is on the preventi on and cure of d isease. In this direction, various peptides, immunosuppressants, neuropeptide antagoni sts IL-l inhi bi tors , blockers

Correspondent au thor: Fax No.: 9 1-0522-223405; Email: root@cscdri.ren.ni c .in

inhibiting binding of IgE to mast cell s have been recently reported 12

. Inhibitors of IgE and its high affinity receptor Fe£ R 1, appears to be an interesting approach for blocking allergic response.

From therapeutic point of view, lgE-Fc fragment would be able to prevent activation of the mast ce lls by interfering with the binding of the receptor and in terms about the early events that follow triggering of receptors. Although various pharmacologically active agents have been investigated as anti allergic/­antiasthmatic agents but drug of choice todate remains limited up to symptomatic relief. Newly synthesized hexapeptide (Ala-Giy-Gly-Asp-Gly-Lys) have been found to have potent antia llerg ic/antiasthmatic effects in rats and guinea pigs among the series of other oligopeptides screened for developing as anti allergic and antiasthmatic drug.

Materials and Methods Animals-Male Sprague-Dawley (S.D.) rats (120-

150 g) and male Engli sh albino guinea pigs (325-375 g) were provided from animal house of Central Drug Research Institute, Lucknow.

Chemicals-Egg albumin (Grade V), compound 48/80, o-phthaldialhyde, toluidine blue and RPMI-1640 were purchased from Sigma Chemi cal Co., USA.

872 INDIAN J EXP BIOL, SEPTEMBER 200I

Among drugs, disodium cromoglycate was manufactured by Ralli s India Ltd., Mumbai and Ketotifen and cetirizine was obtained from Micro Labs Ltd. , Bangalore. Novel anti a llerg ic oligopeptide which is investigated in the present study was sy nthesized in Medicinal Chemistry Divi sion, Central Drug Research Institute, Lucknow. These drugs were dissolved in 0.9% saline for oral admin istration.

The present study was conducted with peptide 95/220 which was synthesized by solid phase method and purified to more than 98% column followed by di verse phase HPLC.

PCA in rats-Egg albumin (EA) was used as an tigen. Rat an ti serum was prepared by sensitizing them on 1st, 3rd and 5th day. On lOth day of sensitization, serum was separated from the blood taken from the orbital plexus of rats and 1: I 0 dilution of serum containing IgE type of antibodies was used for sensitization.

Male SO rats were sensitized intradermally on the ir shaved backs with 0.1 ml of antiserum containing lgE. After 48 hrs. of sensitization, test agent (0.5 to 7.5 mg/kg) and standard drug (50 mg/kg) were g iven orally and intraperitoneal routes respectively . One hour later, 1% egg albumin along with 0.5% Evan's blue (0.25 ml each) were administered intravenously . After half to one hour, the inhibitory effects of test drugs were measured as the percent reduction in dye leakage area and compared with that of vehicle treated animals 13

.

Mast cell stabilising activity-Compound 48/80, a condensation product of p-methoxy-N-methylphenyl­amine was used as potent histamine releaser in the non-immunological type of mas t cell degranulation.

]n the present investigation, normal saline ( 10 ml) was injected into the peritoneal cavity of SO rats. After gentle massage, the peritoneal fluid was collected and transferred into the siliconised - test tubes containing 10 ml of RMP I-1640 (pH 7.2-7.4).

The test pesptides (0.1 to 0.5 mg/kg p.o. x 5 days) and standard (10 mg/kg, ip x 5 days) drugs were given to rats according to experimental protocol, prior to collection of mast cells. Mast cells were washed thrice by centrifugation at low speed (400-500 rpm) discarding the supernatant and taking the pellet of mast cells in the medium.

Jn non-immunological type of mast ce ll degranulation, mast cell s from normal and treated groups were recovered, purified and were incubated with compound 48/80 (I J..lg/ml ) at 37°C for 10 min.

After incubation, mast cells were stained with toluidine blue (0.1%) and per cent protection against degranulation was counted under a high power microscope (x450) 14

•

Active anaphylaxis (Schultz-Dale study)- Male guinea pigs were sensitized with egg albumin (10 mg/kg, ip) on days 1 and 3. On day 14, animals were sacrificed and ileum was surgically removed under aseptic condition and ileal strips were intensively washed with Tyrode's solution and put on an Schultz­Dale apparatus and incubated there at 37°C in Tyrode's solution into which air was bubbled. Motility of the segment of the ileum was recorded by Grass Model 7 Polygraph. As soon as the spontaneous motility (amplitude, frequency and tone of contractions) reached a constant level, tissue viability was tested by histamine hydrochloride (0.25 J..lg/ml ). Antigen (10 J..lg/ml) was added to the bath (capacity 20 ml) and responses were recorded for 90 seconds. Various concentrations of test drugs were added to the organ bath 10 min before the add it ion of antigen (EA). The inhibitory effects of test drugs were expressed as the percent reduction in ileal contraction compared with that measured when the ileum was treated with the EA only (control) 15

·16

•

Allergic asthma and histamine aerosol test-Male guinea pigs were sensitized with an tigen (EA, 50 mg/ml, im) on 1st, 3rd and 5th day . After 4 weeks of sent1st1zation test peptide (5 mg/kg, po) was administered 1 hr prior to challenge. Then the sensitized and normal animals were kept in the aerosol chamber and an atomised mist. of antigen (EA, 5%) and histamine (2%) were separately sprayed by nebulizer under pressure at 280 mm of Hg with the help of compressor. Guinea pigs were pass ively sensitized for EA and normal guinea pigs for histamine are employed in these tests. Animal s exposed to broncho-constrictor aerosol behave in a characteristic manner and show progressive signs of difficulty in breathing and anoxic convulsions. As soon as preconvulsive breathing commences, the animals are p 1 aced in fresh air and the time is noted. Protection afforded by the drug is calculated as (l-T 1/T2xl00) where T 1 is a mean of control preconvulsion time and T 2 1s post-treatment convulsion time 17

•

Release of histamine from lung tissues-Egg albumin (2 mg/ml) and Freund's complete adjuvant were thoroughly mixed at the ratio of I: 1 by volume, and the resultant emul sion was used for sensitization. Male guinea pigs were actively sensi tized by

SINGH era/. : ANTIALLERGIC ACTIVITY OF HEXAPEPTIDE 95/220 873

intradermal injection of I ml of the emulsion on the shaved dorsal sulfaces. After three weeks of sensitization, the animals were exsanguinated, and the lungs were perfused with 20 ml of Ca2

+ free Tyrode's solution and then removed. The lungs were then chopped into fragments. The chopped lung tissues (200 mg wet weight) were placed in tubes with 2 ml of ice-cold Ca2

+ free Tyrode's solution and kept on ice. The reaction tubes containing the lung tissues were supplemented with CaCI2 ( 1.8 mM) and incubated for 10 min at 37°C. After that the lung

tissues were incubated with test drugs ( 1-500 jlM/ml) or vehicle for 60 min.and then again with EA (2 mg/ml). After 15 min, the reaction was stopped by filtration of the medium through nylon mesh (100

jlm). Histamine in the medium was determined fl uorometricall y 18

•

Results Protection to passive cutaneous anaphylaxis­

Peptide 95/220 exhibited dose-dependent inhibition of passive cutaneous anaphylaxis in rats when administered by oral route thereby showing antiallergic activity and its activity was compared with standard drug DSCG.

DSCG exhibited 78% anti -PCA actiVIty by intraperitoneal route at 50 mg/kg whereas peptide 95/220 exhibi ted 45 to 95% anti-PCA activity in dose-dependent manner by oral routes and ED50 value was 1.0 mg/kg (Fig.l).

Mast cell stabilization-Peptide 95/220 also exhibited mast cell stabi li sing acttvtty when peritoneal mast cells were stimulated to degranulate by compound 48/80, a potent histamine releaser. Mast cell stabi li zing activity of test peptide was also assessed by comparing with DSCG, a potent known mast cell stabi li zer. Peptide inhibited the degranulation in dose-dependent manner and maximal activity (83%) was shown at 7.5 mg/kg in divided doses of 1.5 mg/kg for 5 days (Fig. 2) . DSCG at 50 mg/kg ip (10 mg/kg for 5 days) showed 68% protection of mast cells.

Effect on active anaphylaxis (Schultz-Dale test) in guinea pigs- Peptide 95/220 and the reference drugs inhibited antigen-induced contraction of isolated guinea pig ileum in concentrat ion dependent manner.

Peptide 95/220 showed maximum protection to contract ion of ileal strip at 5 jlg/ml (72%) and blocked the contraction (20-72%) of ileal tissue in dose-dependent manner (1-5 jlg/ml) .

Protection to antigen-induced contraction of guinea pig ileum produced by peptides was compared with standard drug DSCG, ketotifen and cetirizine which also blocked the contraction in dose-dependent manner (Figs 3-6) .

DSCG protected antigen-induced contraction of sensitized guinea pig ileum and maximum protection

was exhibited at 6 jlg/ml which blocked contraction up to 72% whereas ketotifen and cetirizine exhibited 93% and 95% protection at very low doses, i.e. 2.5 and 3 jlg/ml respectively .

Effect on bronchodilator test-Bronchodi Ia tory activity of peptide 95/220 has been evaluated in both sensitized and normal guinea pigs in antigen and histamine-induced aerosol test at higher dose (5.0 mg/kg, po) and its activity have been compared with standard drug DSCG ( administered intraperitoneal ly at 50 mg/kg) and cetirizine (1.0 mg/kg, po) . Antihi stamjnic activity have also been evaluated for peptides and DSCG and not for cetirizine because it is a known antihistamine drug (Table 1 ).

Effect on antigen induced histamine-Peptide 95/220 inhibited antigen-induced release of histamin~ from chopped lung tissues of actively sensitized guinea pigs in a concentration dependent manner, and

maximum inhibition was observed at 10 jlM. Ketotifen showed least inhibition as compared to DSCG and peptide 95/220. The inhibitory effect of DSCG was observed from 100-500 11M whereas

100

90

.. 80

~ 70

':; 60

c 50 .2 40

~ 30 £ 20

"' 10

,.---.-...

05

-,-

7 5

Peptide 951220 (mgllcg, po)

Fig. I- Dose-dependent anti-PCA activity of peptide 95/220 (n = 5 m each group; mean of 3 experiments)

01 0 2 0 s I 5

Pr.pl irte 9!5 1220 (myncg x 5 do.y~l

Fig. 2-Dose-dependent inh ibiti on (29-83%) of mast cell degranu lation by peptide 95/220 by oral route. (n = 5 in each group; mean of 3 experiments)

874 INDIAN J EXP BIOL, SEPTEMBER 200 1

c

A I t

95/22 0 EA 2 5)Jgjm

B

j\ ~

95/220 EA l .)Jg/ ml

D

Fig. 3-Schultz-Dale study in sensitized gu inea pig ileum. Arrow (1') shows administration of egg albumin (EA). A-control response; B-0, dose-dependent blockade (20-72%) of EA-induced cant rnr l inn in thP. nrr~rnrf' nf twnt iriP Q'i/??0 ( 1- 'i 11 o/mll

/\

~ CON IIlQl 1

E•

c ~

DSCG

1• P9jmt [A

DSCG

nscG

l _,ugjrnl [tr.

0

~

DSCG t 3,.ugjml [h

DSCG

5 , IHJ/ml E A

Fig. 4-Schultz-Dale study in sensitized guinea pig ileum. Arrow (i) shows administration o f egg albumin (EA) A-control response, B-G dose-dependent inhibiti on ( 17-72%) of EA-induced contraction in the presence of standard drug DSCG.

peptide 95/220 exhibited equal potency at 10 )..lM, (Fig. 7) .

Discussion In the present experimental studies, the effects of

newly developed antia llerg ic peptide 95./220 on immediate hypersensitivity reaction were inves tigated usmg various experimental models and a trway

A

~ 1'

EA 10_.ugjm1

B

Y~~~u

i Ketotifen -t· fA

(l_.ug jmt)

c

~ D

·~'I'WwJUIL!l!J, ,,iW ......

t Keto lifen + EA

2 JJ9/ ml

i k e to t1fen t- [ 11

2 Sygjml

Fig. 5-Sensitized guinea pi g il eum showing EA indued contracti ons A-control response, B-D dose-dependent blockade ( 14-93%) of EA-induced contraction in the presence of standard drug Ketotifcn.

t EA

( 10,JJ9/ mt)

c

~ t

EA + Ce tir iz ine

2 .)J9 / ml

t Ell

+ Ce tir i zine ( l )Jgjml)

D

~-------t

EA + Cetirizine

( 3 .)Jg/ rnt )

Fig. 6-Sensitized guinea pig ileum showing cellnztne dose­dependent blockade (B-D) with cetirizine ( l -3 ~-tg/ml , 30-95%). (EA) A-control response.

responses. Degranulation of mast cells when exposed to antigen, has been taken as the criterion of positive anaphylaxis. It was found that oral admi ni stration of 95/220 was effective in inhibiting immediate hypersensitivity at very low dose as compared to standard drug DSCG which is effective by intraperitoneal route at much higher dose.

Peptide 95/220 showed substantially higher potency in inhibiting the PCA reaction in rats than did DSCG. The inhibitory effect of 95/220 on antigen-

SINGH et a/.: ANTIALLERGIC ACTIVITY OF HEXAPEPTIDE 95/220 875

70

.. "' 60 .. .. ~ .. 50 " E .. 40 v; :c 0 30 .

" ~ 20 .. :0 :c ·= 10 . :!'-

0 - --··--- --·--------------- -------- ·----- -

5 10 100 250 500

Concentration uM

Fig. 7- Effect of peptide 95/220 (+ ), DSCG <• l and Ketotifen <• l on antigen released hi stamine from chopped lung tissues of sensitized guinea pigs.

Table I - Bronchodilator study - Aeroso l test

Compound Dose %_Protec tion (mg/kg) Route Hi stamine Egg

albumin

95/220 5.0 po 17.10 74.00

DSCG 50.0 ip 17. 10 72.10

Cet iri zi ne 1.0 po * 81.0

*Cetirizine is known antihistam inic so its antihistam inic activity was not determined in our laboratory. (n = 5 in each group)

induced bronchoconstriction in guinea pigs was more potent than DSCG at much higher dose and less or equally potent to cetirizine.

Further, Peptide 95/220 exhibited protection to mast cell degranulation when induced by a potent histamine releaser compound 48/80, thereby acting as mast cell stabiliser. However, till Naguchi's peptide it was controversial whether the pentapeptide synthesized by Hamburger21 is able to compete with IgE in binding to mast cells and basophils to impair antigen-induced histamine release from mast cells or inhibit PCA reaction.

Nio et al. 19'20 carried out the sy nthesis of some

peptide fragments and these peptides were assayed for their capacity to inhibit PCA in vitro. The results suggested that an octapeptide corresponding to 345-352 in the human lgE molecule may be an IgE binding site. 1t exhibited significant inhibiti on of PCA, probably by occupying the Fe epsilon receptor sites on the cells.

Anti-PCA actiVIty of peptide 95/220 was investigated by administering it through

intraperitoneal route and its act iVIty was compared with DSCG . Peptide 95/220 exhibited better anti-PCA activity by ip route as compared to oral route. The main advantage of peptide over clinically used cromoglycate is that it is at least 50 times more potent dose per dose than DSCG.

Further it is postulated that anti-anaphylactic effect of DSCG is due to its ability to inhibit the release of mediators from mast cells . This inhibition of mast cell degranulation and medi ator release has been demonstrated in animal studies in response to both immunological and non- immunological stimuli 21 and in humans following allergen stimulation22

.

In active anaphylaxis (Schultz-Dale study) peptide 95/220 inhibited antigen-induced contraction of ileal ti ssue and its activity was compared with standard drugs DSCG, ketotifen and cetirizine. Peptide 95/220 and DSCG were found to be equipotent. They have inhibited egg albumin-induced contraction at same dose whereas ketotifen and cetirizine have blocked the contraction at lower doses as compared to DSCG and peptide 95/220. These results also indicate that peptide also possess mast cell stabilizing activity as does DSCG.

In general, experiments for evaluating bronchodilatory activity of peptide was also compared with DSCG and cetirizine possessed bronchodilatory activity approximately in same range by oral rou te unlike DSCG which was given by intraperitoneal route and much higher dose range. Peptide as well as DSCG do not have anti-histaminic activity as these have shown only 17% inhibition to bronchoconstriction when normal guinea pigs were exposed to histamine spray through nebuliser.

876 INDIAN J EXP BIOL, SEPTEMB ER 200 1

Whereas known antihistaminic drug cetmzme also inhibited antigen-induced bronchoconstriction in sensitized guinea pigs . These results indicate that peptide 95/220 like DSCG have ability to stabilise mast cell membrane by competitively binding with mast cell membrane receptors but do not have abi lity to antagoni se the effec t of released medi ators. Unlike clinically used drug DSCG, this peptide is orally active at much lower dose.

Peptide 95/220 markedly inhibited immedi ate hypersensitivity reactions such as antigen- induced cutaneous reacti on, mast cell degranulat ion induced by non-immunologica l stimuli (Compound 48/80), Schultz-Dale phenomenon and bronchoconstri ction when given orally. These effec ts of 95/220 may be beneficial for the treatment of allergic inflammatory di seases such as bronchial asthma, all ergic rhin iti s and allergic dermatiti s in human. Peptide 95/220 and DSCG inhibited hi stamine release from the chopped lung tissues of acti vely sensiti zed guinea pigs wh ich showed their anti allergic activity. Many Histamine antagoni st compounds such as epinastin 23 and ketotifen24 have been found to suppress antigen or cal cium ionophore induced release of chemi cal medi ators. It has been suggested that the inhibitory effects of the anti allergics on chemical medi ator release could be ascribed to the inhibition of cellular Ca2

+ uptakc25·26 and the enhancement of the cellular

cyclic AMP level27·28

. It is necessary to exam ine the effects of peptide 95/220 on the second messengers to clarify the mechanism of chemi cal medi ator release inhibition.

Thus, our studies provide the first experimental evidence for the presence of anti allergic activity in small peptides by oral route and open a new avenue for future explorati on. Therefore, small eptides comprised of 5-10 amino acid residues which can interact with airways immune response may be of signi ficant interest as lead molecules fo r realising the long awaited goal of preventive therapy of asthma. With this in mind detailed studi es with clinically established immunomodulatory peptides may lead to better therapeutic modaliti es against all ergic diseases .

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SINGH eta/ .: ANTIALLERGIC ACTIVITY OF HEXAPEPTIDE 95/220 877

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