Br. J. Pharmac. (1974), 50, 383-392

ANTI-INFLAMMATORY PROPERTYOF 401 (MCD-PEPTIDE), A PEPTIDEFROM THE VENOM OF THE BEE Apis mellifera(L.)

JENNIFER M. HANSON, J. MORLEY & C. SORIA-HERRERADivision of Immunology,The Kennedy Institute of Rheumatology, Bute Gardens, Hammersmith, London W6 7DW

1 Peptide 401, a potent mast cell degranulating factor from bee venom, substantiallyinhibited the oedema provoked by subplantar injection of carrageenin or intra-articularinjection of turpentine in the rat. The ED50 of 401 was c. 0.1 mg/kg. The anti-inflammatoryeffect was assessed by measurement of the increased 125 I-albumin content of an injected sitein comparison with an uninjected contralateral site.2 Peptide 401 also suppressed the increased vascular permeability due to intradermal injectionof various smooth muscle spasmogens (histamine, bradykinin, 5-hydroxytryptamine (5-HT),and prostaglandins).3 Other comparable mast cell degranulating agents (48/80 and melittin) showed littleevidence of anti-inflammatory activity when tested at comparable dosage on turpentine arthritisand carrageenin oedema.4 The anti-inflammatory effects were not abolished by pretreatment with mepyramine andmethysergide, which abolished the increased vascular permeability produced by local injectionof 40 1.5 The anti-inflammatory action of 401 was not affected by regional denervation orpretreatment with phenoxybenzamine, and was reduced but not abolished by adrenalectomy.6 Measurement of skin temperature, fractional extraction of 86Rb and blood flow in perfusedmesentery gave no evidence that the anti-inflammatory action of 401 was due to reduced tissueperfusion.7 It is concluded that 401 may exert its anti-inflammatory action directly by making thevascular endothelium anergic to phlogistic stimuli.

Introduction

The venom of the honey bee Apis mellifera (L.) isa complex mixture of pharmacologically andbiochemically active agents. Investigations into thecomposition of the venom were first reported byLanger (1897) and more recently it has beenshown by Neumann & Habermann (1954) thatmost of the biological activity was due to thepeptides and proteins in the venom.

The components of the venom can be separatedby dialysis into two fractions. The non-dialysablefraction contains the enzymes, phospholipase Aand hyaluronidase together with other proteinsnot known to have enzymatic activity. The dialy-sable fraction contains low molecular weight pep-tides, histamine, inorganic ions and otherunidentified compounds. Early work on theseparation of the components of the venom bychromatography and electrophoresis was carried

25

out by Habermann & Reiz (1965b) and the majorconstituents have now been purified.

Three basic low molecular weight peptides havebeen described. Melittin, by weight the principalconstituent of the venom (c. 30%) is a peptide of26 amino acid residues, which has a high surfaceactivity and is a potent haemolysin (Habermann &Jentsch, 1966). Apamin (c. 2% of the venom)contains 18 amino acid residues and is a neuro-toxin producing motor abnormalities associatedwith actions upon the central nervous system(Habermann & Reiz, 1 965a). The third peptide (c.1% of the venom), first isolated by Breithaupt &Habermann (1968), contains 22 amino acidresidues and was described as a mast cell degranu-lating (MCD)-peptide. The primary sequence ofthis peptide was reported by Haux (1969) and is inagreement with that found by Vernon, Hanson &

384 JENNIFER M. HANSON, J. MORLEY & C. SORIA-HERRERA

Brimblecombe (1969), who also determined theposition of the two disulphide bridges in thepeptide which they called 401 (Figure 1). Billing-ham, Morley, Hanson, Shipolini & Vernon (1973)observed that the peptide showed marked anti-inflammatory activity in the rat againstcarrageenin-induced oedema and that it suppresseddeveloping and established adjuvant arthritis.

The present study has confirmed the anti-inflammatory property of this peptide and hasattempted to establish its mechanism of action. Apreliminary account of this work was presented tothe Society (Hanson, Morley & Soria-Herrera,1972).

Methods

Iodination

Rat serum albumin was labelled with 125I by theiodine monochloride method (Macfarlane, 1958).The labelled albumin was separated from unboundiodine by column chromatography (G 10 Sepha-dex). Aliquots were stored at -200 C.

Inflammatory stimuli

Joint oedema was provoked by injection of0.01 ml turpentine oil into the synovial cavity ofthe right knee joint with a micrometer syringe(AGLA, Burroughs Wellcome). Carrageeninoedema was provoked by sub-plantar injection of0.1 ml of a solution of carrageenin (1% in 0.9%w/v NaCl solution) into the right hind foot. In allexperiments the contralateral uninjected site wasused as the control, the responses being expressedas a paired difference. Intradermal injections weremade in a volume of 0.1 ml into abdominal flankskin.

Vascular permeability measurements

Wistar strain rats (150-250g) received an intra-venous injection of 0.5 ml of 125 I-labelled ratserum albumin (c. 10 ,Ci/mg, 2.5 mg/ml) mixedwith Evans blue dye (2%). Injection was via thesublingual vein under light ether anaesthesia.Immediately following this intravenous injection,the appropriate inflammatory agent was injected.Anti-inflammatory agents were administered byintraperitoneal, subcutaneous or intravenous injec-tion either at the same time as, or at appropriateperiods before the application of the inflammatoryagent and the isotope injection. After an intervalof 1 h for skin tests and 4 h for joint or footlesions, rats were anaesthetized and 1 ml samples

Fig. 1 Structure of peptide 401.

of blood collected by cardiac puncture. In turpen-tine oedema experiments, the entire knee jointregion of each hind limb was removed by a wadpunch (diameter 23 mm) and in carrageeninoedema experiments the hackle joint was severedon both hind feet. For measurement of skinlesions, the flank and abdominal skin was re-moved, spread evenly and discs of skin (diameter20 mm) encompassing each skin test site were cutout with a wad punch.

Samples were placed in counting vials andcounted in an automatic 7-spectrometer. For eachspecimen, division of the specimen count by thecorresponding blood count (after background sub-tractions) gave the specimen albumin content interms of an equivalent blood volume. This makesallowance for differences in the dilution of theisotope caused by differences in the blood volumesof the animals and in individual intravenousinjection volumes. For the uninjected skin or joint,the specimen count reflects both intravascularalbumin and albumin accumulated as a conse-quence of normal permeability. For tissues bearinginflammatory lesions the count contains a corres-ponding component plus a count due to thealbumin which has extravasated during the periodof increased vascular permeability. The increasedextravasation of albumin was therefore calculatedby subtraction of the figure for the uninjected sitefrom that of the contralateral injected site.

Blood pressure

In rats and guinea-pigs, blood pressure wasrecorded under nembutal anaesthesia from thecommon carotid artery with a Statham straingauge pressure transducer. Test materials wereadministered via a cannula in the femoral vein. Inthe rat, blood pressure was also recorded in therestrained conscious animal. In these animals onecarotid was cannulated under phenobarbitoneanaesthesia 1-2 h before the subcutaneous injec-tion of 401.

ANTI-INFLAMMATORY PEPTIDE FROM BEE VENOM 385

Mast cell degranulation

Rat areolar tissue spreads were incubated for15 min in Tyrode solution containing the varioustest compounds. Degranulation was assessed bystaining the tissue with toluidine blue (1% w/v)and allocating each observed cell a score (0 to 4)according to the extent of degranulation and thecumulative total for 100 cells was expressed as apercentage of maximal degranulation.

Denervation

Rats were anaesthetized with ether and a 1 cmportion of the right sciatic nerve was removedabove the knee. Animals were used two days afternerve section.

A drenalectomy

Adrenals were removed from the rats under etheranaesthesia. Thereafter, these animals received0.4% sodium chloride in their drinking water.Animals were used six and nine days after adrena-lectomy.

Fractional extraction of 8Rubidium

Animals received an intravenous injection of 0.9%w/v NaCl solution (saline) containing Rubidiumvia the sublingual vein (under light ether anaes-thesia) and were killed by cervical dislocation 60 safter the injection. Samples of various tissues werecollected, counted and weighed.

Isolated perfused mesentery

The superior mesenteric artery of the rat wascannulated following cervical dislocation and themesenteric veins were cut. Mesenteric tissue wasperfused with a roller pump with an albuminenriched Tyrode solution (Wade & Beilin, 1970),perfusion pressure being recorded by a Stathamstrain gauge transducer. Close arterial injections ofdrugs were made via a length of pressure tubingimmediately proximal to the cannula.

Skin temperature recording

Skin temperature was measured with a disc shapedthermistor (S.T.C.) taped to a depilated area ofskin. Temperature was monitored on a flat bedpen recorder (Telsec 700).

Materials

401 and melittin were pure peptides isolated frombee venom (Rodopa, Bulgaria) (Hanson, Shipolini& Vernon, unpublished method).

125 Iwas carrier-free iodine IMS-4, and 86Rbwas rubidium chloride RGS-I P (RadiochemicalCentre, Amersham); rat serum albumin (Sigma);Evans blue (Gurr); turpentine (Boots); carrageenin(Viscarin, Marine Colloids Inc.,); indomethacin(MSD); phenylbutazone (Geigy); bradykinin(Sandoz); 48/80 (Weilcome Trust); sodium sali-cylate (B.D.H.); prednisolone acetate (Roussel);adrenocorticotrophic hormone (ACTH) (Armour);phenoxybenzamine hydrochloride (Smith, Kline &French); dexamethasone sodium phosphate(Merck Sharp & Dohme Ltd); mepyramine maleate(May & Baker), and methysergide bimaleate(Sandoz).

Concentrations of histamine acid phosphate(B.D.H.) and 5-hydroxytryptamine oxalate (5-HT)(Sigma) are expressed as base.

Prostaglandins El and F20 were a gift of DrJ.F. Pike (Upjohn). Serum kallikrein was preparedfrom guinea-pig serum by the method of Davies &Lowe (1963).

Results

Effect of 401 on turpentine and carrageeninoedema

Intra-articular injection of 0.01 ml of turpentine inthe rat causes a severe inflammatory response withan initial oedematous phase occurring in the firstfew hours. In this oedematous phase, the increasein vascular permeability of the synovial andneighbouring vessels gave a substantial accumula-tion of extravascular albumin. The time course ofthis accumulation was determined by giving anintravenous injection of 125 I-albumin to animalsat 0, 1, 2, 3 and 5 h after intra-articular turpentineinjection and measuring the extravasated albumin1 h later (Figure 2a).

Subplantar injection of carrageenin in the hindfoot of the rat also causes a severe oedema and thetime course of this accumulation of extravascularalbumin was determined in a similar manner(Figure 2b). Turpentine oedema was also assessedby measuring the joint swelling with a gauge and incarrageenin oedema the paw volume was measuredby weighing the hind feet. Figure 2 shows that theresults obtained by these methods were compar-able and that isotope accumulation provided asensitive method for measurement of these inflam-matory reactions. The measurements of isotopeaccumulation show that the response is biphasic inboth tests and that a period of 4 h includes bothphases of the response. Accordingly in both testsystems an intravenous pulse of 1 5I-albumin of4 h duration was used to measure the inflamma-tory reaction.

386 JENNIFER M. HANSON, J. MORLEY & C. SORIA-HERRERA

Q6

a)2 0.4

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a

a98 KEE0.8

0~~~~~~~~~~.

Uo- 0-4 ~~~~~~0.4

02 02

0 1 2 3 4 5 6 0 1 2 3 4 5 6Time (h) Time (h)

Fig. 2 Time course of inflammatory reaction in (a)turpentine oedema; (s) 1251-albumin accumulation interms of equivalent blood volume (ml); (e) increasedjoint circumference (mm); (b) carrageenin oedema; (o)1251-albumin accumulation in terms of equivalentblood volume (ml); (o) increased weight of foot (g).Histograms show the hourly accumulation of12 5I -albumin from which the cumulative extravasationwas calculated in terms of equivalent blood volume(ml). Observations represent the mean of four animals+ sd.

Peptide 401 given subcutaneously immediatelybefore intra-articular turpentine injection sup-pressed the oedema in a dose-related manner witha maximal effect at c. 1 mg/kg (Figure 3a). Thisdose reduced albumin accumulation by about85%. There was a similar suppression of theinflammatory response caused by subplantar injec-tion of carrageenin and a 80% reduction inalbumin accumulation was obtained with a dose of1 mg/kg (Figure 3b).

In these experiments, the inflammatory res-ponse was calculated as the increase in 125albumin content of the injected site (knee or foot)over that observed for the contralateral uninjectedsite. Thus an agent causing an increase in the125I-albumin content of the uninjected site (e.g.by systemic inflammatory action) might cause an

a0.81

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025Dose (rng/kg)

0.5 1.0 2.0

Fig. 3 Suppression of increased vascular permeabilityby increasing doses of 401 in (a) turpentine oedema,and (b) carrageenin oedema. 1251-albumin accumula-tion 4 h after subcutaneous injection of 401 (c) orsaline (o), and in rats treated with mepyramine (2.5mg/kg) and methysergide (2.5 mg/kg) immediatelybefore intravenous injection of 401 (a) or saline (o).Points represent the mean increased accumulation (interms of equivalent blood volume ± s.d.) at theinjected site compared with the contralateral un-injected site (paired difference) for groups of fiveanimals.

apparent reduction in the observed differencewithout having suppressed the inflammatory res-ponse at the injection site. There is in factevidence of some systemic inflammatory action of401 at higher doses (0.5-2 mg/kg) causing anincrease in both albumin accumulation and weightof the uninjected foot (Figure 4). The magnitudeof this systemic effect in carrageenin oedema wassmall when compared with the inflammatoryresponse to turpentine or carrageenin. The anti-inflammatory action of 401 was accordinglyaccentuated at higher dosage. However, itremained clearly demonstrable at doses

-

ANTI-INFLAMMATORY PEPTIDE FROM BEE VENOM 387

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Dose (mg/kg)

Fig. 4 Suppression of increased vascular permeability in carrageenin oedema: (.) 125 I-albumin content of theright (injected) foot; (o) '25I-albumin content of the left (uninjected) foot; (a) weight of the right (injected)foot; (o) weight of the left (uninjected) foot. Points represent the mean of five animals ± s.d.

(0.025-0.25 mg/kg) in which 401 had no systemiceffect on the contralateral foot. Use of intravenousvital dye (Evans Blue) gave visual confirmationthat 401 reduced the extravasation of albumin atthe site of injection of carrageenin or turpentine.The potent anti-inflammatory action of 401 wasvery striking and compared well with the effects ofthe established anti-inflammatory agents; mepy-ramine, indomethacin, phenylbutazone, salicylate,

ACTH, prednisolone and dexamethasone. None ofthese inhibited turpentine oedema to the sameextent at the doses indicated (Table 1).

Mechanism of the anti-inflammatory action ofpeptide 401

(1) Role of mast cell degranulation. In additionto exhibiting anti-inflammatory action, 401 itself

Table 1 Suppression of turpentine arthritis

Treatment

Saline(no intra-articular turpentine)Saline

Non-steroidal anti-inflammatory drugs:MepyraminendomethacinPhenylbutazoneSodium salicylate

Steroidal anti-inflammatory drugs:ACTHPredn isoloneDexamethasone

401

Dose Response *(mg/kg) (ml blood equivalent)

- -0.01 ± 0.05 (n = 9)- 0.57 ± 0.19 (n = 22)

2 0.55 ± 0.29 (n = 7)5 0.31±0.21(n=15)

50 0.25±0.17(n=5)500 0.24 ± 0.06 (n = 11)

(1 unit)1

0.025

0.47 ± 0.09 (n = 5)0.39 ± 0.06 (n = 3)0.34 ± 0.01 (n = 4)

2 0.08 ± 0.04 (n = 10)

* Means response ± s.d. (n = no. of animals). Increased '251-albumin content of the right (injected) knee in

comparison with the left (uninjected) knee.Administration of drugs: mepyramine maleate was given intravenously 5 min before intra-articular turpentineinjection; indomethacin, phenylbutazone and sodium salicylate were given by intraperitoneal injection 15 minbefore turpentine injection: ACTH, prednisolone and dexamethasone were given by intraperitoneal injection 3 hbefore turpentine injection and 401 was given subcutaneously at the time of turpentine injection.

% Inhibitionof '25I-alb.

accumulation

4465758

183340

86

388 JENNIFER M. HANSON, J. MORLEY & C. SORIA-HERRERA

aOA4 0.15

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P / ~~~~~0.05- (1

0.001 0.01 0.1 1.0 Uninjected 0.08 04 2 10Dose (pg) skin

. Fig. 5 125 I-albumin accumulation following intradermal injection of 401 (-) and melittin (o) in (a) the rat, and(b) the guinea-pig. Points represent the mean accumulation in the skin test sites ± s.d. (a) for groups of fourobservations (six animals); (b) for groups of 16 observations (four animals).

increases vascular permeability as would beexpected from its potency as a mast cell degranu-lating agent (Breithaupt & Habermann, 1968). Itproduces increased vascular permeability followingintradermal injection in both the rat and theguinea-pig. The inflammatory dose-response rela-tionships (10 ng-100 /ug/ml) are shown in Figures5a and b. Since the local concentration occurringon subcutaneous injection of 401 in anti-inflammatory studies exceeded these doses, thepossibility existed that the anti-inflammatoryactivity of 401 might in some way be associated

with its own local inflammatory actions. Melittin,another peptide from bee venom which also causesmast cell degranulation, hypotension (Table 2) andincreased vascular permeability (Fig. 5a), and thepotent mast cell degranulating synthetic com-pound 48/80 (Table 2) were therefore comparedwith 401 for their ability to suppress increasedvascular permeability. Melittin (2 mg/kg) showedno significant inhibition of turpentine oedema nordid it reduce responses evoked by intradermalinjection of histamine, 5-HT or bradykinin. Incontrast, comparable doses of 401 caused marked

Table 2 Comparison of 401, Melittin and 48/80

Material

Test Dose

Mast cell degranulation(values represent% degranulation)

Blood pressuredepression in mmHg(values representsingle observations)

Inhibition ofturpentine oedemaED,O (mg/kg)

1 0 ,g/ml10.10.010.0010

79725219

5

5 _4 48,322 30,241 18

0.07 >2.0

* 401 and 48/80 reported to be equipotent (w/w) on rat blood pressure (Breithaupt & Habermann, 1968).

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nottested *

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ANTI-INFLAMMATORY PEPTIDE FROM BEE VENOM 389

Sal. 401 401 480 4880 MelMep+ Mep+Meth Meth

I

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0.61

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Sal. 401 401Mep+Meth

Fig. 6 The effect of mepyramine and methysergide on the anti-inflammatory action of 401 in turpentineoedema. (a) Increased accumulation of 125 I-albumin following intra-articular injection of turpentine in animalspretreated as follows: subcutaneous saline (sal); subcutaneous 401 (1 mg/kg) (401); subcutaneous 401 (1 mg/kg)following systemic mepyramine (2.5 mg/kg) and methysergide (2.5 mg/kg) (401, Mep+ Meth); subcutaneous48/80 (1 mg/kg) (48/80); subcutaneous 48/80 (1 mg/kg) following systemic mepyramine (2.5 mg/kg) andmethysergide (2.5 mg/kg) (48/80, Mep + Meth); and subcutaneous melittin (2 mg/kg) (Mel). Columns representmean of five animals ± sd. (b) 125 I-albumin content of right (injected) knee (-) and left (uninjected) knee (o).Points represent mean of six animals ± sd.

inhibition in these tests. Similarly, 48/80(1 mg/kg) had no anti-inflammatory action com-parable with 401 in turpentine oedema (Table 2)nor did it suppress the increased vascular perme-ability caused by intradermal histamine, 5-HT andbradykinin. These results demonstrate that theanti-inflammatory action of 401 cannot be simplyattributed to its mast cell degranulating propertyor its inflammatory actions. This conclusion isstrengthened by the observation that, in theguinea-pig, neither 401 nor melittin suppressed theincrease in vascular permeability in turpentinearthritis, hypersensitivity arthritis and skin reac-tions of allergic inflammation at doses up to2 mg/kg, despite the fact that both peptides causean inflammatory response in this species(Figure Sb).

Pretreatment of the rat with the specificantagonists, mepyramine maleate (2.5 mg/kg) andmethysergide bimaleate (2.5 mg/kg) fully sup-pressed skin reactions to histamine (0.5 mg) and5-HT (0.2 mg) but not bradykinin (0.3 Mg) andcaused only slight reduction of responses tointra-articular turpentine or subplantar carra-geenin. This pretreatment had little effect on theability of peptide 401 to reduce the inflammatoryresponse to subplantar carrageenin (Figure 3). Inthese 401-treated animals the use of mepyramineand methysergide caused a reduction in the countof both injected and uninjected feet (Fig. 6), aneffect presumably due to antagonism of systemi-cally released histamine and 5-HT. Whilst this

pretreatment was without effect on the anti-inflammatory action of 401, it did cause somereduction in the anti-inflammatory activity of48/80. Because of its potent mast cell degranu-lating action, 401 is relatively toxic on intravenousinjection. However, pretreatment with mepy-ramine and methysergide permitted intravenousadministration of 401 and in such experiments theanti-inflammatory potency of 401 was comparablewith that observed following subcutaneous injec-tion (Figure 3).

(2) Specificity of action. The inflammatory res-ponses in the rat to intra-articular injection ofturpentine and subplantar carrageenin are com-plex, possibly involving sequential release of anumber of endogenous agents (Di Rosa, Giroud &Willoughby, 1971). Drugs which increased thecalibre and permeability of blood vessels by directaction on the vascular wall were therefore used.Peptide 401 at a dose of 1 mg/kg injected sub-cutaneously totally abolished dye accumulationfollowing intradermal injection of bradykinin(0.2 pg), prostaglandin E1 (0.5 Mg), serum kalli-krein (100 Ag), histamine (0.3 Ag), 5-HT (0.1 Mug)and 48/80 (0.025 Mg). An interval of 1 h or morebetween the subcutaneous injection of 401 andthe subsequent intradermal injections was neces-sary for demonstration of the anti-inflammatoryeffect. However, when injected intravenously, 401(200 Mg/kg) was effective immediately and abo-lished the leakage of blue dye produced by

1pb

01

390 JENNIFER M. HANSON, J. MORLEY & C. SORIA-HERRERA

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No treatment

1E.1

2A 2B 2CSal 401 401Adrenalectomy

3A 3B1"4A 4BSal 401 Sal 401

DeervationIPhenoxybenzamine

Fig. 7 The effect of adrenalectomy (2), denervation (3) or pretreatment with phenoxybenzamine (10 mg/kg)(4),on the anti-inflammatory activity of 401. Increased accumulation of '251-albumin after intra-articularinjection of turpentine into knee joint following treatment with saline 1A, 2A, 3A and 4A or 401 (1 mg/kg) 1 B,2B and C, 3B and 4B. Adrenalectomy was performed six (2A and 2B) and nine (2A and 2C) days before theexperiments. Columns represent the mean ± s.d. from five animals.

intradermal injections of bradykinin (0.5 pg), hist-amine (0.8 Ag) and 5-HT (0.2 Mg). This suggestedthat the delay required on subcutaneous injectioncould be attributed to slow absorption from theinjection site.(3) Contribution to endogenous anti-inflammatorysystems. The irritant action of 401 raised thepossibility that its injection initiated the acutephase of shock (Florey, 1962) and thereby acti-vated endogenous anti-inflammatory mechanisms,particularly the sympathetic and adrenal systems.However, pretreatment of the animals with mepy-ramine and methysergide prevented the increasedvascular permeability on subcutaneous injection of401 without substantially affecting its anti-inflammatory activity. The possible contributionof neural mechanisms to the anti-inflammatoryactivity was investigated by studying the effect of401 on turpentine oedema in denervated limbs.This treatment had no significant effect on theanti-inflammatory action of 401 (Figure 7). Pre-treatment of the animals with phenoxybenzamine(10 Mg/kg) an agent which specifically blocksa-adrenoceptors, also had little effect on theanti-inflammatory activity of 401 (Figure 7).

The effect of 401 on turpentine oedema wasalso measured in adrenalectomized animals six andnine days after operation. Although there wassome reduction of the anti-inflammatory activityof 401 (Fig. 7) it is clear that the release of adrenalcortico-steroids cannot account for a large part ofthis anti-inflammatory action.

The release of prostaglandin F20a was con-sidered as a possible mechanism in view of itsreported anti-inflammatory action in this species(Willoughby, 1968). However, in doses up to50 Mug/kg any apparent anti-inflammatory action ofprostaglandin F2CI could be accounted for by theincreased count in the contralateral knee. Theseexperiments show that the anti-inflammatoryaction of 401 cannot merely be attributed to thestimulation of adrenergic or corticosteroid factorsor release of prostaglandin F2a-

(4) Vasomotor activity. Subcutaneous injectionof 401 at a dose level at which anti-inflammatoryactivity is maximal in turpentine arthritis andcarrageenin oedema resulted in a sustained fall inblood pressure apparent after 1 h and persistingfor several hours (Figure 8). This hypotensiveresponse is reduced by pretreatment with mepy-ramine (2.5 mg/kg) and methysergide (2.5 mg/kg)which allows intravenous injection of 401. Thispretreatment does not affect the anti-inflamma-tory activity of 401 (Fig. 3) suggesting that hypo-tension is not responsible for anti-inflammatoryactivity. Production of an anti-inflammatory effectby selective regional vasoconstriction remains apossibility although the inability of even largedoses of 401 (up to 1 mg) to cause detectablevasoconstriction in the isolated perfused mesen-tery suggests that such vasoconstriction wouldhave to be indirect. No evidence that 401 causedselective vasoconstriction in skin was obtained

ANTI-INFLAMMATORY PEPTIDE FROM BEE VENOM

p- -10 1 2 3 4 5 6

Time (h)

Fig. 8 (a) Arterial blood pressures followingFlank skin temperature in the same experiment.

either by measurement of skin temperature over

several hours following subcutaneous injection of401 (Fig. 8) or by measurement, 2 h after sub-cutaneous injection of 401, of the fractionalextraction of 86Rb in skin tissue.

Discussion

These results demonstrate that peptide 401 is a

potent anti-inflammatory agent in the rat. It isable to reduce or abolish responses to intradermalinjection of chemically defined agents such as

histamine, 5-HT, bradykinin and prostaglandins, as

well as responses to more complex stimuli such as

local injection of carrageenin or turpentine, inwhich several mediators are involved (Di Rosa etal., 1971). In addition to these actions, peptide401 suppresses the development of adjuvant arth-ritis and reduces the severity of primary andsecondary lesions in established adjuvant arthritis(Billingham et al., 1973).

In addition to its anti-inflammatory action, 401is a potent mast cell degranulating agent in vivo

subcutaneous injection of 401 (2 mg/kg) in restrained rat. (b)

and in vitro and this property could account forthe hypotensive and vascular permeability effectsobserved on intravenous or subcutaneous injec-tion. However, it is not possible to attribute theanti-inflammatory action of 401 merely to vaso-

active amine release following mast cell degranula-tion since other mast cell degranulating agentswith a different (e.g. melittin) or similar (e.g.48/80) mechanism of action (Breithaupt & Haber-mann, 1968) do not exhibit comparable anti-inflammatory activity in the tests employed in thisinvestigation. Also, pretreatment of rats withmepyramine and methysergide to antagonize theactions of histamine and 5-HT reduced the sys-temic hypotension and abolished the local inflam-mation produced by 401 without loss of anti-inflammatory activity; the slight reduction inanti-inflammatory activity against carrageenin andturpentine oedema being consistent with specificantagonism of histamine and 5-HT release in theseinflammatory reactions. 401 is without anti-inflammatory action in guinea-pigs, although itcauses increased vascular permeability on intra-dermal or subcutaneous injection. It is therefore

pZ3

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391

392 JENNIFER M. HANSON, J. MORLEY & C. SORIA-HERRERA

unlikely that the anti-inflammatory action of 401depends upon its irritant properties. Nonetheless,it remains possible that the anti-inflammatoryproperty of 401 may be in some way related tomast cell degranulation or other tissue damagesince large doses of prostaglandin El (1 mg/day)reduce the severity of adjuvant arthritis (Zurier &Ballas, 1973) and alkylpseudothioureas reduceinflammatory reactions to a wide range of stimuli(Ercoli, Arbona & Tabernero, 1971).

The lack of specificity of action of 401 andmore especially its ability to suppress responses tointradermal injection of smooth muscle spas-mogens, indicate that 401 does not depend upondepletion of substrates (e.g. kininogen, comple-ment) for its mode of action. Whilst adrena-lectomy caused some reduction in the potency of401, denervation and pretreatment with phenoxy-benzamine had little effect. It can be concluded,therefore, that alterations in cortico-steroid releaseor vasomotor activity can only make minor contri-butions to the anti-inflammatory action of 401. Amodification of prostaglandin synthesis has notbeen excluded, although for such a mechanism of

action to operate, it is necessary to presupposethat skin reactions to histamine, bradykinin and5-HT depend upon coincident prostaglandinsynthesis, at present an unlikely assumption inview of the inability of non-steroidal anti-inflammatory drugs to suppress all of theseresponses in rats (see Collier, 1969).

Gross measurements of plasma protein extra-vasation did not differentiate between the effectsof altered transmural pressure or altered vessel wallarea (e.g. due to vasoconstriction or vasodilatationin the microvascular bed) and altered vascularpermeability itself. Nevertheless, persistence of theanti-inflammatory property of 401 in mepy-ramine- and methysergide-treated animals, to-gether with the inability of 401 treatment toaffect fractional extractions of 86Rb by skin andthe absence of any vasoconstrictor activity of 401,suggest that peptide 401 acts directly on the vesselwall to produce anergy to agents causing increasedvascular permeability.

We acknowledge general support from The Arthritis &Rheumatism Council.

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(Received August 24, 1973)