CA1322160C - Therapeutic use of enkephalinase - Google Patents

Abstract

Abstract of the Disclosure A method and therapeutic composition for the treatment of pathological disorders associated with endogenous peptides by the administration of enkephalinase or derivatives thereof.

LG8x396.mhg

Description

DOCKET 393 Pl ~3221 60 THERAPEUTIC VSE OF ENKEPHALINASE

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Thi~ l~ven~ion, i~i part, was ~ade with government ~upport und~r Grsint #~I24136 with the ~aton~1 Institu~e~ of ~ealth and the Un~versity of California. ~he Go~ern~ent has ~ert~in rights in part of thi~ in~e~tlcn.

The presen~ invention Tel~teS to the treatment of patholo~ical cond~ ns assoc~ted ~ith: v~rious endogenous pept$des. In~:partic~lar, th~ ~ventlo~ relate~ to the use of : o~kephalinase ~E.C. 3.4.24.11~ and novel ~crms thereof in ~ucih ~r~-tment.
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Varlous endogenous~peptides have beeD di~iicovered which appear acti~e ~in ~ar~ous physlologi~al sy~tems. For example, ~wo pentapeptldes, referred to a~ enkephalins, wes~ extracted fro~ the brain. The ~ffects ~:~f e~kephalin~ incl~de analgesi~, therm~regulat~on,~ tranquiliz~tion, gastrointestin&l functlon and increa~ing appetite. ~ntil the present ~n~entlon, the sole physiological acti~ity of enkephalinase w~s thought ts be the L~8x396.~h~ .
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l3221 60 cleavage of enkephalins in the central ner~ous system (Schwartz, J.C. et ~1., Tr~nds Pharmacol. Sci. S: 472-476 11985]), ~hich to date is not ~nown to be associated with any path~logical dlsorder.
Because cuch ~ct~vity wDuld result in ~n enhancement of pain, scientific research has focused on inh~biting enkephalin~se. This in~ention est~blishes therapeutic uses ~f ~nkephalinase for the first time.

Another endogenous psptide, angiotensin II, is presumed to lV be an etiologic agent in the pathological condit$on of renal hypertension. Br~dykin~n ~nd kallidin have be~n associ~ed ~ith other pathologicsl conditions ~uch as ~cu~e inflsmmation ~ssociated witb burns, rheumatoid ~rthrltis, edema, earcinoid ~ndrome, pancr~atitis, ~igraine he~dache, reac~ions after tr~nsfusion with plasma products, aller~ic diseases, endotoxic shock and anaphylaceic ~hock.

Another clsss of endo~enous peptides are the ~achykinins ~ich oehare 60me of the eame physiological' ~ctivity. The tachykinins include substance P, eledoisin, neurokinin A and B, physalaemin ~nd kassinin. Subs~ance P has been ~hown to be ~ssociated with smooth ~uscle contraction, neurotransmission, pain, co~gh, exocrlne secreti~n, v~sodilation, increased vascular per~ability, i~cre~sed e &erence of lcukocytes to ~enules, ~5 ~timulation of polymorph~nuclear leukocyte~, ~acrophages, T
lymphocytes, and degranula~ion of ~ast cells. Endogenous peptides such as bombeein have been found to b~ present in endocrine cells in normal lungs (Cutz ~ Al., Experientia ~7, 765-767 [1981]), released from carcino$d tumors, ~nd implica~ed in the associated cut~neous ~lushes, t~langiect~si~, disrrhea snd bronchocGnscriction.

Bombesin functions as ~ grow~h f~ctor for ~1rw~y epithelial celle (Uilley et ~1 ., Exp. Cell Res . ;~, 245-248 [1984] ) tmd f~r LC~x39~.mhg : ` ::

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. ~ 3 ~322~60 human small-eell lun~ cancer (cuttieta~ F. et ~1., Nature 316, 823-826 11985]). Substance P ~nd bombesin, apart ~rom their tumor-associated e~fects, have been shown to contract the pulm~nary artery ~nd the airways. In ligh~ of ~he observAt~ons ~f the instant invention these endogenous peptides msy ~ediate various pathophysiologic 6tstes includin~ bronchi~l ~sthma and hypoxic pul~onary vAsooonstriction. Some peptides ~re chemotactic, e.g.
eosinophil chemotactic f~ctor, C3a and 6ubstance P. They are ~enerated ~t the slce of i~flammation and at~ract various i~unological cells including neutrophils to the ~ite. Flnally, other peptid~s such as cholecystokinin, somatostat1n, oxytocin and caerul~n have p~ent effects on ~arious tissues which may give rise to other pathological disorders.

Enkephalinase has been purifiet fr~m kidney (Kerr, H.A. and Kenny, A.J. Bi~chem. J. ~ 477-488 [1974], G~ff~rd, J. et 1., Biochemistry ~, 3265-3271 ~1983] and Malfroy, B. and Schwartz, J.C., Life ~ 1, 1745-~748 [1982]), intest~ne (Danielsen, E.M.
et ~1., Biochem. J. 191, 545-548 [1980]), pituit~ry ~Orlowski, M.
~nd Uilk, S. Biochemistry 20: 4942-4945 11981]), br~ln (Relton, J.~. et al., Biochem. J. ~: 755-762 [1983]~ and lymph nodes (Bowes, N.A. and Kenny, A.J., Biochem. J. 2~: 801-810 [1986]~.
Enkephal~nase has been deteeted in m~ny peripher~l organs (Llorens, C. ~nd Sch~artz, J.C., Eur. J. Phan~ac~ , 113-116 (1981~ ~nd in human neutrophils ~Connelly, J.C. ~ al., Proc. ~a~l. Ac~d.
Sci.[USA] ~ 737-8741 [1985]). The distribution of enkephalinase in the brain closely parallels th~t of the enkeph~lins ~Llorens, C.
.e~ 1., J. ~eurochem. ~2: 1081-1089 [1982]). The observatisns of the instant invent~on es~ablish that enkephalinase is als~ present in those peripheral ~isgues ~nd cells that respond to endogenous peptides. ~nkephal~nase is ~ ~embr~ne-bound glycoprotein with ~ubun~t Nr values in the range of 87,000 to 94,000. Yari~tion in the Mr ~alues i~ attrlbuted to di~ferences in the extent and p~ttern of glycosylatlon.

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The substrate specfffcity of enkephalinase has been fitudied using the enzyme from rRt and hu~an kidney. Malfroy, B
snd Schwartz, J.C., J. Biol. Che~. ~ 14365-14370 (1984); GAfford et al., Blochemistry ~: 3265-3271 (1983~; ~nd Pozs~ay, M. et ~l., Biochemistry 25: 1292-1299 (1986). These studies ind~ ate that enkephallnase preferenti~lly hydrolyzes peptide bonds co~prising ~he amlno group uf a hydrophobic residue, ~hows ~ marked pref~rence or shor~ peptides, ~nd is ~os~ efffcient when i~ acts as a dipeptidyl carboxypeptidase releas~ng a carboxy terminal dipeptide.
~nkephalinase, whioh had been found in cerebral synaptic ~embr~nes, efficlently ~l~aves the Gly3-Phe4 ~mide bond o~ enkephalins (Malfroy, B. e~ ~l-, Nature (Lond.~ 523-526 11978~.
Enkephalinase has ~lso been found to cleave the heptapeptide (Net5)~nk~phalin-Arg6-Phe7 (Sch~artz, 3.C. ~ fil., ln Prooeedings Int~rn~tiQnal Union of Pharmacology 9~h Congress of Pharmacolo~y, ~: ~d. by J.F. Mitchell et al., 277-283, ~cMillan Press Ltd., London, ~1984~) ~s well as a varlety of other neuropeptides, such ~s cholecystokinin (Zuz~l, K.A. e~ al., Neuroscience 15: 149-158 [1985~), substance 2 (Hvrsthemke, B. et al. Bioch~m. Biophys. Res.
Com~. l2~: 72~-733 [19843), neurotensin (~hecler et 1., 1983), an~iotensin I ~nd ang~otensin II (Matsas t ~1., Bioche~ J. ~
433 [1984] and Gafford ~ iochemistry ~ : 3265 [1983~), klnins, e.g. bradykinin (Gafford, J.T. ~ ~1., Biochemlstry 2~:
3265-327l 11983]), oxytocin (John~on et l. ~ 1984), and ~o~a~ostatin (Mu~ford, R.A. ~t ~l-- Proc. Natl. Acad. Sci. [USA]
78:6623-6627 11981]). ~hile enkephalin~se Is o~pable of hytrolyzing many biGlogic~l peptides ~n Yi~Q (KPnny, A.J. Trends in Blochem. Sci. 11: ~0-42 ~19R6]), ln Yi~ ~nkephalinase has to dste only been ~mplicsted in the hydrolysis of e~dogenou~
enkephalins when released in ~ra~n (Schwartz, J.~. .et ~l., Life Sciences 29: 1715-1740 [1981] ~nd leco~te, J.M. t ~1., J.
Pharmacol. Exp. Ther. ~: 937-946 [19863). Althou~h the le~els of enkephalinase in blood ~re normally ~ery low ~ConDelly et al.

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) enkephalinase was found to be pr~sent ln high levels in the seru~ from pati~nt~ with adul~ respiratory di tress fiyndrome (Connelly et l. Supra). Enkephalin~se ~le~ves the chemotactic tripeptide i~et-Leu-Phe. Id. It was ~lso obsçrved that neutrophils from ~onors who ~moked had enkephalinss~ ~ctivites about twice that of nons~okers. Idr Enkephalinase has al~o been found in high levels in the micro~ill~ of human plccent~e (30hnson, A.R. ~t al., Peptldes ~: 789-796 ~19e4~).

The presene invention iB based on t~e novel obser~stions that ~pec~fic inhibitsrs of enkephalinase, thiorphan, leucine-thiorphan ~nd phosphora~idon, potentiate ~irway ~ucus ~ecretion and ~mooth musclo contractlon induced by endogenous peptides, e.g.
substance P and other tachykinins, and kinins ~uch as bradykinin.
The ~nvention is also ~aeed on the novel ob6ervation that ~ ~iVQ
enXephalin~se inhibits ~ubstance P-induced lncreases in ~ascular permesbility. Enkephalinase i~ known to cleave substance P into two fr~g~ents observed to be ineffect~ve in ~timulating ~ucus ~ecreeion and/or 6~00th ~uscle con~raction. 5he'invention is also based on the observation that enkephalinase digests chemotactic ~olecules and thus oay inhibit ~he attraction of various infl~mmatory cells lncluding neutrophil~ to the ~ite of ~n~u~y.

~n ob~ect of the prPs~nt ln~ention 1~ to provide a ther~peut1c compcsition for ehe ~reat~en~ of pa~hclo~ical conditions in which endogenous peptides may be in~olved.
Specifically, enkephalinase ~ay be used ~s a therapeutic agent ~o overcome ~d~erse effects of ~ubstance P or other neuropeptides.
~ore ~pecifically, enkephalinase ~ay be used to reduce peptide-mediated ~NCUS ~ecretlon and bronchoconstric~ion in ~he Airway c~nsequent to ~Rrious dise~ses e.g. ss~h~a, chronlc bronchitis, cy~tic fibrosis, and viral infec~ions. Enkeph~linase may also be used as a therspeutic agent in the treatment of vario~s tumors, e.g. carcinoid tw~ors ~nd small cell carcinoma of ~h~ lung. Yet ~C8x396.~hg ., . . ~ ~
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finother ~bject of this inventi~n is the use of enkephali~ase derivatives in the treetment of ~rious patholo~ical disorders medisted by certain endogenous peptides. Other peptide-lnduced disorders ~ay erise in ~he gastrointestinal, vlsual, ~rinary, circulatory, reproductlve systems ~nd ~oints. The cytoplasmic and/or tr~nsmembrane deleted or ~ubs~ituted enkephalinase may be used in the treatment of v~riou~ pathologic~l disorders med~ted by cert~in endogenous peptides.

~y~ar~ of the Invention The present invention i~ basad on the novel observations that the effects ~f entogenvus peptides 6uch as 6ubstaDce P and other ~achykinins, and/or bradykinin on ~ucus secretion and 6mooth ~uscle contr~ction in ~irway5 ~re potentiated by pecific enkephalinase inhibitors. ~he inve~tion i~ dir~cted to the ~dministration of therapeutic compositlons compri~in~ ~nkephalinase or -deriv~i~es ~h~reof for ~he treatment of certain pathologic~l disorders ~ediated by varlous endogenous peptides, for example, bronchoconstriction, airway hypersecretion, acu~e inflammation or hyperlmmune responses, systemic hypertension, coug~, $nfertility or cancer.

~ief Description of the Drawi ~s FIG. 1. Concentration-dependence of 6ubst~nce P (SP)-induced ~ecretion (mean ~ SE), ~asured as release of 35so4-labeled ~acro~olecules ~6ulfate 1ux~. Tissues from 6 ferrets were incubated ~ith SP ~t ~he concentr~tions indlcs~ed, and ~he chan~e in sulfate flux for each tissue was calc~lated by subtrActlng the flux of bound S04 for the ~mple collected ~ di~tely before adding 5P from ~he flux o~ bound S04 for the sample collec~ed either 15 or 30 min sfter adding SP, ~hlchever was ~re~ter. The aver3ge change in basellnP 6ecretion ~B) o~er the 15 min prior to ~dding SP ~ shown for co~parison (B). SP

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~ -7-1322~0 -stimulated the relesse of 35S04 labeled ~acro~oleeules in ~ dose dependent fashion.

FIG. 2. Effects of ~rs~ments of 6ubstance P (SP) on culfate flux from two segments from 8 ~erret. Tlssues ~ere lncubated in chambers with 35S34 on the luminnl ~lde, and after 3h, fr~g~ents of SP were added to the ~ubmucosal sides of the chambers. Left: C-terminal fr~ment, SP 6~ 10-5M) st~mulated ~ecretion. Right: N-terminal fr~ent, SP 1-9 (10-5N) had no ~l~nific~nt effec~ on ~ecretlon.

FI~. 3. Effect ~f proteinase inhibitor6 on ~ubstance P (SP)-induced change ~n ~ulfa~e flux ~rom one ti~sue from ~
ferre~. Left: oontrol~t~s ue, ~ncu~ted wi~h 35so4 ~nd exposed to SP ~lO~ Righ~: ti66ue pr~-treated with the combinstion of 9 proteinase inhibitors described in the text ~9 INHIB) potentiated the ~ecretory response to SP (10-6~).

FIG. 4. Effect~ of protei~se lnhlbi~ors on s~bstance P (SP)-lnduced ch~nge ~n ~ulf~te flux (~ean + SE) ~ro~ tl~sues from ferre~:trachea6.~ Open bar~: respon~e to SP ~10-6M) in ccntrol tl~sues ~om each ~roup. ~atched bars:
respon e to SP 10-~ ln eissues pre-trea~ed with 9 pr~teinase ~nhibitors (9 INHIB; 10 ~g/~l), phosphor~midon ~10-5M; PHOSP~, thivrphan (10-4H; THIOR), captopril (lO-4~; CAPTO), ~epr~tide ~10-4~; TEPRO) or other i~hibitors (OTHERS) lncludin~ leupeptin, ~pro~onin, bacitracin, bovine erum Rlbu~in (~ach inhibitor, 10 ~/ml), or bestatin (10-5M). *: p~O.D5. Only phosphor~idon and ~hiorphan (enkephalinase inhlb~tors) potentl~ted the ~ec~etogogue effects o~ SP.

L~Bx396.~hg : .. . . - .

FIG. 5. Effects of incressin~ concentrations of the enXephalinase inhibitor thiorphan on fiubstance P (SP)-lnduced ~ulfate flux from tr~cheal ~egments from 6 ferret~ (oe~n + SE).
Filled bars: fncre~se in sulfate flux dur~ng the 15 min prior to adding drugs. Stippled ~rs: increase in sulfste flux sfter adding ~hiorphan in th~ concentrations ~ndicated. Ha~ched bars: increase in ~ulf~te flux induced by SP (10-5M). *: pC0.05; compared ~o the spontaneous increase in sulfate flux; n~6. **:
p<0.05, compared ~o the response to SP in control tissues; n-6. Thio~phan potentiated the SP-induced effecte on 8ecretion in a dose-dep~ndent fashicn.

FIG. 6. Effects sf t~chyk~nins ~nd of the enkephslinase inhlbi~or, phosph3rsmidon, on rele~se of 35SO4-labeled macr~molecules from ferret tr~cheas. Filled bar: Change in sulfate flu~ during a 15 min baseline period (BL) ln the absence ~f drugs. Open bars: Response~ to the ~achykinins, substance P (SP), neur~kinin A (~K-A), ~eurok~nin B (NK-B), physsl~emin (PHYS), eledoisin (ELED), and kassinin (KA5S) (each drug, 10-5 M). H~tched bars: Responses to ~chykinins ~fter pretreatin~ tissues w~h phosphora~idon (10-5 ~). *: p~0.05 compared t~
baseline **: p<0.05 compared ~o the response ~o the same tachyklnins ln the ~bsence ~f phGsphoramidon.
~h~sphoremidon potentia~ed the secretogogue effects of e~ch tachykinin.

FIG. 7. Ef$ects of trypsin cn enkephalinase ~ctivity and s~bstance P-induced ~ucus secretion ln 3 ~errets. Left:
Enkephalinase ~ct~vity in lung hom~gen~e~ ~xpressed as the degradation of (3H-Tyrl, DAla2~ LeuS)enkephalin.
Ri~ht: Filled bsr: chan~e ln ~ucus secre~n ~urin~ a 15 min b~seline period in the ~bsence of drug~. Open bar:

L~x396.mhg ~: ~ . . , , :
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Response to ~ubst~nce P (SP). Stippled bar: Change in ~uc~s ~ecre~ion sfter adding tryps~n. Hatched bar:
Change in ~ucus ~erretion lnd~ced by SP in tissues pretreated with trypsin. Data expressed ~s ~ean ~ SEM.
Trypsin decreases enkephalinase activity and potentlates ~ecretion induced by ~ubstance P.

FIG. S. Effect6 of ~ubstance P (SP) and of th~ enkephalinase ~nhibitor lou-thiorphan on Active tension in l~olated segm~nts of ferret tr~cheal s~ooth ~uscle. Results are reported as ~esn + S~M o~ 12 ferrets SSP ~ 10-6 M) or 6 ferrets (SP 2 5 x 1~-6 ~). Significsnt differences from corr~sp~nding ~ontrol ~lues re ~ndicated by: * p <
0.05; ** p ~ 0.~1; *** - p ~ 0.001. Substance P alone (open 6quareB) lncr~a~ed tension, but only ~t c~ncen~rations of 5 ~ 10-6 N ~nd higher. L~u-thiorphan ~solld di~monds) caused ~ shift in the dGse-response curve to lower concener~tions of SP.
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FIG. 9. ~ffects o~ receptor ~nt~g~ni~ts on actiYe tensi~n produced by substaw e P ~10-6 ~) in the presence of the enkephalinase inhib~tor leu-thi~rphAn ~10-5 M) in 4 solated ~egmen~ of tracheal ~oosth ~uscle in ferrets.
Each poin~ is the me~n + SE of ehe decre~ses in tension pr~uced by ~aoh ~ntagonist compared to the corresponding control responses to SP:plu~ leu-thiorphan~ Significant differences fro~ csntrol values are ~ndicated by: * - <
O.05; *** p < 0.01. The ~uscarinio antagonist, atroplne (10-5 N), the SP ~ntagonis~, (DPro2,DTrp7~9)SP
(10-5 ~) and ~ co~bination of bQth drugs decreAsed SP
plus leu-thiorphan-induced contr~ctions ~ignific~ntly;
the effect of ~he ~P antag~n~st was gre~ter than tha~ of atropine.

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FIG. 10. Effects of subst~nce (SP~ plus the enXeph~linase inhibitor 10u-th~orphan on ~ctive tension produced by electr-cal field ~timulation (5 Hz) in isol~t~d se~ments of tracheal ~mooth muscle in ferrets. D~ta are expressed as percent of conerol responses to el~ctrical field ~timulation without ~dded druss and are reported ~s m~An + SE (N 10 ~t - 10-5 M; n - 4 ~t 10-4 ~). Signlficant differences fro~ SP alone or SP plus leu-th~orphan ~re indicated by: * - p ~ 0.05; *** - p C 0.001. Substance P
~lone augmented contractile responses to electrical field Rtimulation, and this augmentation was potentiated by leu-thio~han.

FIG. ll. Effoots of ~he enkep~allna~e lnhibltor leu thiorph~n and of the 6ubs~ance P ~SP) Rnta~oni~t, (DPro2,DTrp7~9)SP on ~ct~ve eensiOn protuced by electricsl field stimulation S5 H2) in lsolated ~egments of tracheal ~mooth muscle in ferr~ts. Data ~re expressed as percent of control responses ~o eleotrlc~l field st~mulat~on without add~d drugs end are reported ~s ~e~n + SE (n - 5). Significant di$f~rences from control ~alues ~re indic~ed by~ *** ~ p < O.Ql. Leu-thiorphan aug~ented contr~otions produced by electr~c~l ield stlmulat1on, ~nd ~hi~ augmentation was inhlbited by the SP ~ntagonl~t ~10-5 M).

FIG. 12. ~ffçrt of ~ubstance P (SP) (~riangles~, neurokinin A (NK-A) (circles) and nouroklnin B (NK-B) (squ~res), and of the cnkephalinase lnhibitor, leu-thiorph~n, on actlve tension in isolated ~egments of ferre~ tra~heal smooth ~uscle. D~t~ expresged as ~ peroent of responses tD
acetylchol~ne (10-3 ~). Resul~s are reported as mean +
SE of 10 ferrets (10-6 ~) snd 5 ferrets (10-5 M) Si~nific~n~ dlffeF~nces bet~een contraceions ~ith and ~i~hout leu-th~orp~an (10-5 M) for each ~achykinin were LC8x396.mh~

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obtAined. Leu-thiorph~n (Qolid ~ymbols) caused a ~hift in the ~ose-response curves to lower concen~rations.
Although NK-A was ~ore p~tent than NK-B ~n the ab~ence of leu-thiorphan, there were no ~ignific~nt dlf~erences ln ~uscle contr&ction be~ween NK-A and NK-B ~n the presence of leu-thiorphan ~10-5 M).

FIG. 13. Effect of increasin~ concentratio~s of the enkephslinase inhibitor leu-thlorphan on ~cti~e tension induced by 6ubstance P (SP~, neurokinln A ~N~-A), ~nd neurokinin B
~NK-B) in ~solated ~egments of tracheal ~mooth ~us~le in ~errets. Data e~pre sed ~s pgrGent vf sesponses to seetylcholine (10-3 ~) 4nd reported ~s ~ean ~ ~E (n-5 a~
10-5 H; n-3 at 3~10-5 N) Leu-thlorpha~ potentiated ontractions ~nduced by ~ach t~chykin~n ~n ~ dose-d~pendent f~shion.

FI&. 14. Effect of substance P (SP), neurokinin A (NK-A), and neurokin~n ~ (NK-B) ~nd of the tachykinin rec~ptor ~ntagonist, (DPro2,DTrp7~g)SP, on act~ve tension produced by electric~l field ztimul~tion (5 H~) in isola~ed ~e~ents of tracheal 6~00~h ~uscle in ferre~s. Da~a ~re Oxpressed 85 percent of control responses to ~lectr~c~l ~ield ~imula~ion without ~dd~d dru~s. ~ach t~chykinin poten~isted olec~rically-~ndu~ed ~ntraetion. SP had the most potent ~fect. The tachyk1nin receptor antagonist inh~bited this pc~entiation.

FIS. 15. Effect of incre~s~ng ~oncentratioas ~i` bradykinin ~A) or lys-bradykiain SB) ~nd of the enkephalina~e inhibitor, leu-thiorphan (10'5H) on act~e tension in i~ola~ed ~sgments of erret tr~ch~al ~mooth ~uscle. Da~a are expressed ~s ~ percent of response~ t~ ac~tylch~l~ne (10-3~). R~sults report~d ~s ~ean ~ SE. Signlfican~

lG8x396.~hg . . ; ,, :-, , ,. :~ ,: , -~ 322~
differences between contrsctions with and without leu-thiorphan are indicat~d by *-p<0.05. ~radykinin ~nd lys-~radykinin increased acti~e ten610n in a dose-dependen~
f~shion. Leu-thiorphan c~used 9 6hift in .both dose-response curves to lower concentr~tioDs.

FIG. 16. Effect o$ increaQ~ng conc~ntratio~s of 6ubst~nc~ P and of the ~nkep~liDase inhibitor leu-thiorphan (10-5M), on active ~ension in isolatet longitudi~al ~egments of ileal ~ooth muscle in ferrets. Substance P ~lone (op~n circles) caused incre~sed ten~ion in ~ do~e-dependent f~shlon. The enkephslinas2 ~nh~bitor, leu-th~orphan ~solld circles), potentiated t~e subs~nce P-induced con~ractions.
' FIG. 17~-b. Fi~ures 17~ and 17b hereinafter referred to as Fig.
17. Amino ~cld ~e~uence Df rat enk~phalinase.

FIG. 18a-b. Figures l~a and 18b herein~fter r~ferred to ~s Fig.
18. Amino aci~ ~equence of hu~an enkephalinase.

.
~nkeph-linase prefer~ntislly hydroIyzss peptide b~nds ~o~prislng the ~mino group o~ ~ hydrophoblc residue, ~howing ~
~arked preference for short pep~ldes. The use of enkeph~linase as a therapeutic ~gent for pathologic~l conditions i5 esta~lished by the ~bservatiDns of the ~nstant invention ~sing as examples, the air~ay responses and effects on capill~ry per~eabllity resulting from release o~: endogenous peptldes; ~ch ~s 6ubstance P or bradykinin. Enkephallnase, aleo known as neutral ~ndopep~idase or kidn~y brush border Deutral proteinase ~E.C. 3.4.24.11, recommended n~me of the En2yme Co~miæsion), ~nd derivatlve~ theroof ~ay be used as a therapeutic ~gent ~n the~ trestment of ~ho3e Yarious LC8x396.~hg . : -;
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p~ehological conditions. The ~xperiments are descr~bed in detail below.

ndogenous peptldes ~uch as enkeph~l~ns, ang~ot~nsin I and angiotensin II, cholecystokinin, tachyX~nins e.g. ~ubstance P, neurok~nin A or B, phys~lae~in, eledoisin, k~ssinin, kinins, e.g.
br~dykinin, lys-br~dykinin (k~llidin), other peptides, such as neurotensin, oxytocin, somatostat$n, bo~besin ~nd ~hemotactic f~ctors, for ex~mple oosinophil chemotac~lc factors, have been i~plicated in vasious physlological and pat~ologic~l oonditions.
Ex~ples of effects ~re outaneous fLushes, telangiec~sia, diarrhea and bronchoconstriction resulting from the rele~se of ~arious peptides ~uoh as, ~ubstance P, br~dyklnin ~nd bombesin from carc~noid tu~ors.
Circulating angistensin I i8 converted by ~ngiotensln-~onvert~ng enzyme ~ACE~ eo ~nglotensln-II, ~ potent vasoconstrlc~o~. Sys~emic hype~tension ~y ~rlfie from the effects of ~ngio~ensin II. Curren~ therapy for 6ysremic hypertenslon includes prevention o$ the conver~ion of ~ngiotensin I to ~ioten~in II by ~nhibiti~g ACE. Cough is a ~ide effect ~f ~his treatfflen~ w~th AC~ inhibitors snd 1~ proposed to be ~ue to ~he inhibition o~ bre&kdown of cough-provokin~ peptides (e.g. 9 bradykini~). Enkephallnase degrades &nglote~sin I and II ~nd thus may 6erve ~ an antihypertens~ve ~gent. Enkephalin~se, by clea~ing ~ou~h-provok~ng peptides 6uch ~s bradykinin should elim~nate cough ~.~ a ~ide effect of snt~hypertensive therapy. ~nother patholo~lcal condition i6 tachykinin-~edi~ed ~ucus hypersecre~ion from su~murosal glands of the tr~chea following irritation o~ the airway epithelium, for example, by an ~llergen. In ~dd~tion to ~ucus ~ecretion there is likely to occur: ~nfl~m~tion, lncreased capillary permeability, neutrophil ~hemotaxis, ede~a and bronchial smooth ~uscle contr~ction (broncho onstric~lon). In ehe skin, elevated levels of tachykin~ns or kinins protuce eequelae of LC8x39S.~hg . .

1~22~ ~

sy~ptoms broadly referred to ~s derm~tit~s, including pain, itching, redness and heat ~nd blistering. T~chykininc releassd in the gastroineestinal and ~rlnary systems h~ve b0en ~mplicated in 6ecretion e.g. ~alivation from the parotid gland, ~Qoth ~uscle ~ontraction of the ileu~ ~nd esophagus, effects on frequency of urination, 6timulation of secretion of water and electrolytes from ~he ~e~unum ~nd pancreat$c exocrine ~ecretion. Tachykinins also promote embryonic implantation.

0 ThR d~ta set forth in this speciflcation establish that ~nkephalin~se within the ~irWBy degrade~ subst~nce P ~nd other endogenous peptid~s to ~nsctive ~etabolit2s. Thi6 in~cti~ation is ~ ~echanism for ~it~h~ng the effects of endogenous peptides on DUCUS secretion ~nd bron~hoconstriction. This is ~ased on the ~bservation th~e thiorphan and phosphor~midon, speci~ic inhibitors of enkephalinase, potenti~ted the secretory ~nd contractile rssponsss to ~ubstance P ~nd vther pepeides in a ~oncen~ration-dependent fashion. The inh~bi~ors of enkephalinase were ~hown to ha~e no direct effect on ~ucus ~ecreelon or ~scle contraction.
The effects of the enkephalinase inhibitors snd ~f exogenous t~chykinins on ~ucus 6ecretion were observed when ~d~inis~ered to ehe ~u~mucosal ~urface of ~racheal tissue. Inhlbitors ~f other enzyme ~ystems did not slter endogenous peptlde-induced secreeion.
For ~x~mple, ~nglotensin converting enzyme (ACE) 16 kn~wn to be present ln the lun~ ~nd to degrade ~ubst~nce P. Howe~er, ~pecific inhibitors of ACE d~d not potentlate substsnce P-induced secretion or bronchoconstriction. S~ilarly, ~nhlbitors of serine proteases, pr~teases which are secreted from Yarious cells (including neutrophils and ~st cells~ did not potentiAte substance P-~edi~ted mucus secretion from ~racheal tissue. Leu-thiorphsn w~s ~lso observed to potentiate ~b~tance P-induced ~maoth muscle contraction of lleal tissue, indicating that enkephalln~se present in ~astr~intestinal ~ile~l) tlssu~ inhibits ~ubseanoe P-lnduced effects. The enkephalinase inhibieor, le~-th~orp~an, ~as also L~8x396.~hg : : :
,: : . :
:~
: . , : . ~

. ~ -15-` `` 1322160 observed to inhibit bradyklnin and kallidin (lys-~radykinin) ~nduced c~ooth muscle contraction of re~pir~tory tis~ue. This demonstrates that ~ndogenous enkephalin~se ir~activ~tes klnins, as well as eachykinins. Thus enkephalinase ~ppears to h~ve ~ role in modulating endogenous peptide ~ediated ~ucus ~ecretion and/or smooth ~uscle contraction in the ~irway. Enkephalinase ln~ected intravPnously W8S shown to lnhibit substAnce P-induced extravasation Df dye in rats. This proYldes direct evidence tha~
enkephalinase edministered to ~he body c~n prevent pep~ide-induced effects. Administration of ~nkeph~linase or der~vRtives ~her~of by aerosol would spply the tharapeutic agent locally to the ~rachea.

As used hereln, enkephalinase or ~nkephalinase derivatiYes refers to pro~eins which ~re enzym~tically actlve or are ~unologically~ cross-~esctlve with enzy~atically active enkephal~nage. ~n2ym~tically functional enkephalinase is c~pable sf cleaving the Gly3-Phe4 a~ide bond of 3H-~DAla2, Leu5)enkephalin in ~n ~ssa~ ~s described ~y Llorens et ~1. (1982).

Enkephal~nase or enkephalinase derlv~ti~es ray be prepared using previously described ~ethods of purific~tion, see e.g.
~alfroy ~nd Schwartz, J. Biol. Chem. ~ 14365-14370 (1984) or by recombinant ~eans as described in oopending Canadian Patent , Appllcation S.N. 555,183 filed December 23,1987.

Included within the gcope of enXephalinase as ~hat ~erm ~s used herein are enkephalinase having native glycosylation and ~he amino acid sequences of rat and human enkephalinsse ~s ~et forth in Figs. 17 or 18, analogous enkephalinases from other anim~l species 6uch ~s bovine, porcine and the llke, deglycosylated or unglycosylated derivstives of cuch enkephalinases, amino acid ~equence varlants of ~nkephallnase ~nd ~ ylE~-generAted oovalent deri~fitives of enkephal~nases. All of these for~s ~f enkephalinase are enzymatically ~ctive.

LC8x39~.~hg . . . .
- . . . .
: ~, . . . .
.~ . - . ~ :
: ~:
: ., : .
: .. ,.. ~ ,, ~' '~ ,, -lS-- " ~ 3~2~

Amino acid ~equence variants of 2n~ephal~n~se f~ll lnto one or more oi three cl~sAes ~ubstitutional, in6er~10nal or deletional varian~s. A~ino acid ~equence ~riants are characterized by the predeter~ined na~ure of the vari~tion, feature that set~ ~hem spar~ from naturally occurring allelic or ~nterspecies v~rl~tion of the enkephslinase ~mino sclt ~equence.
The v~riants typically exhibit the 0ame qualitative bi~logical activ~ ey ~s the naturally^occurring analogue, ~lthough ~ariants 0 al80 are elec~ed to modify the eh&r~cteristics of enkep~alinase as will be more fully described below.

Amino acid substitut~ons sre typic~lly of ~ingle residues;
lnsertions usually ~ill be on the order of ~bDut from i to 10 ~mino ~cid residues; ~nd delé~ions will r~nge ~bout from 1 to 30 reaidues. Deletions os ~nsertisns prefer~bly ~re ~de in Adjacent palrs, i.e. a deletion of 2 residue~ or insertlon of 2 residues.
Substitut~on~, deletions, lnsertions or any co~bin~tion thereof may be combined ~o ~rrive ~t A flnsl deriv~tive.
Substitutional varinnt~ are those in ~hioh at least one residue in the Fig. 17 or 18 ~equences hss been rem0Yed ~nd dlfferent residue inserted in ~ts place. Such ~ubstitutions gener~lly ~re made in ~ccordance with the follo~ing T~ble 1 ~hen i~
i~ desired to finely modulst~ the char~cteristics of enkephal~nase.

LC8x396.~h~

, :
::.
- . . :: . ~ : :
:: : , .:~:
`

:'; - ~ - :

-17~3~,6~ .

~la cer Arg lys Asn gln; his Asp glu Cys ser Gln ~sn Glu ~sp Gly pro Hi ~sn; gln lle leu; Y81 Leu ile; val Lys arg; Esln; glu Met lgu; ile Phe met; l~u; tyr S~r thr Thr ser Trp~tyr Tyr trp; phe Val ile; leu S~bst~ntlal changes ln function or l~unologlcal ldentity are ~ade by ~elec~in~ s~bsti~utlons that ~re less ¢onservative than tho6e ~n Table 1, i.e., ~elocting re~idues tha~ differ more si~n~fic~ntly in their ~ffect on maintaining (a~ the atructure of the polypeptide backbone in ~he ere~ of the ~ubs~i~ution, for example as a sheet ~r helic~Ll confonnation, (b) the charge or hydrophol~icity of ~he ~olecule ~t th~ tArget 6i~e or (c) the b~lk of ~he side chain. The substitutions which in general ~re expected ~o produce the greatest change~ in enkephal~nase propest~es will be ~o~e in which ~a) ~ hydrophilic residue, e.g. ~eryl or threonyl, ls ~ubstitu~ed for (or by~ ~ hydr~phobic resl~uæ, ~.~. leucyl, is~leucyl, phenylalanyl, ~lyl or ~lanyl; ~b) ~ cy6~elne or proline 1~8x396.Dih~

::

:' ; ~ ." ' . . ,.,.: .

"
" ,~

221~

is substituted for (or by) nny other residue; (c) ~ residue having an electropositi~e ~lde chaln, e.g., lysyl, argi~yl, or hlstidyl, ~s subs~tuted for ~or by) ~n electronegAtiYe rexidue, e.g., glutamyl or ~spartyl; or (d) a residue hav~n ~ ~ulky 6ide chain, e.~., phenylalanine, is ~ubstituted for (or by) on~ not hav~ng a side chain, e.g., ~lycine.

A ma~or class of substitutiQnal or deletional ~ariants are those ~nvolving the transmembrane and/or cy~opl~s~ic regions of enkeph~linase. The cy~oplas~c domain of enkephalinase ~ the sequence of ~mino acid rasidues commencing at either of two ~ethionine residues shown ln Fig. 17 (Met~8 or Ne~~ l ) and continu~ng for approxlmately 21-24 ~ddltioDal res~d~es. In the ra~
~nd human ~quence the Pro-Lys-Pro-Lys Lys-Lys domsin is believed to ~erve ~s a ~top transfer sequenc2; the confor~ational bends introduced by the prolyl resldues and the electropositive character provided by the lysyl resldues act together with the ~ransmembrane reglon described below, to bar ~ransfer of enkephal1nase through the cell ~embr~ne. ,~
The tr~nsme~brane region of ~k~phallnase i5 located ln the r~ ~equence a~ abaut res~dues 21-44 ~where Asp is ~1 as ~ho~n in Fig. 17), ~nd {n the human ~quence ~ the ~nalogous loca~ion.
~his seg~on ~ ~ highly hydrophobie domain that is the proper slze to 6pan the lip~d bilayer of the cellular ~embsane. It i~ believed to function ~n eoncert with the cytopla6mic do~ain to anchor onkephalinæse in the oell me~br~ne.

Deletion or substitution of either or both of the 3Q cytoplasmic and ~rans~e~brane domains ~111 reduce the lipld affinity of ~he protein ~n~ l~prvve -its water ~olubil~y SD
detergents wlll not be required to ~alntain ~nkephal~nase ln ~ueous solution. Deletion of the cytoplasmic domain ~l~ne, while retalning the transmembrane sequence, will produce enkephalinase LC8x39~.~hg : :

1322~60 ~hich would be ~olubilized with detergent but whlch offers therspeutic adv~nt~ges. The cytoplnsmic domsin-deleted enkephalinase will be more likely to insert into ~e~branes when ad~Inistered ~s a therapeutic ~gent, thereby targetlng its activity to the immediate extr~cellular ~ilieu ~n which it is ordinarily active, and would ~mprove its ~or~ula~ion in 6alve or lip~somal composit~ons containing hydrophobio micelles. Pref~rably, the cytoplasmic or transmembrane do~ins ~re deleted, rather than ~ubst~tu~ed, to avoid ~he introduct~on cf pote~tially im~uno~enic ~pitopes.

For use in the invent~on hereln, enkeph~linsse deriv~ives thereof ~y be formulated into ~ither an ~n~eo~s~le or toplcal prepnratIon. Paren~eral formul~tions ~re known and ~re 6uitable for u~e in the invention, prefer~bly for io~ or 1.~.
~dministrat~on. Intramuscul~r or 1.~. ~hernpeutlc preparations may - preferably ~nclude transmembrane and oytoplasm~c-transmembrane deleted enkephalinase deriv~tive. The for~ula~ions c~nta~n ther~peutically effec~ve ~ou~t6 of enkephalin~se or derivat~ves thereof, ~re either ~erile liquid ~olutlons, liquid ~uspensions or lyophilized versions nnd ~pt~on~lly contain ~tabilizers or exciplent6. Lynphil~zed co~positions ar~ reconstituted ~lth ~uitable diluen~R ~e.8.) ~ater ~or in~ec~ion, saline and the like ~ a level of ~bout from .001 mg/kg to 25 ~g/kg where the biologie~1 activ~ty ~ 10 nanomol~s/~g/min ~en ~ssyed at 25DC in 50 ~M, pH 7.4 HEPES buffer cont~ining 0.1% T~een 20 uslng (3H-DAla2, LeuS) enkephalin as ~ubstrAte. Typically, the lyophilized compositions containing enkephali~ase ~ill be ~dministered in a rsnge of from ~bout .01 me/kg to ~bout 20 mg/kg ~f the treated ~nimal. Preferably, the lyophilized composi~ions containin~, enkephalinsse will be atministered in a range o~ from about 0.1 mg/kg to sbout 10 mg/kg of the treated anim~l.

*Trade-mark I~8x396.~hg :` :

:
` , ' ~' : ''' . ~ ' ' ~, ' .

1322~

Enkeph~linase is formulAted into topic~l preparations for loc~l therApy by inoluding a ther~peutlcally effecti~e ooncentr~tion of enkeph~linase in a dermatologic~l Yehicle. Such topical preparat~ons ~ay prefer~bly include the cytoplasmic doma~n deleted enkephalinasç derivative. The amount of enkeph~linase to be administered, snd the enkephalin~e eoncentration in the topical for~ul~tions, will depend upon the vehlole ~elected, the clinical condition of the patlen~, ~he enkephallna~e u~ed ~nd the s~ability of enkephal~nase in the formulation. Thus, the physician will necessArily employ the ~ppropriate prep~retion oontaining ~he appropriate conoen~ration of enkephalin~se in the for~ul~tion, as well ~s the ~ount of for~ulation ~dministered dependin~ upon clinioal experlence ~th the patient in que~ion or wi~h ~imilar p~tien~s. The concen~r~ion of enkephal~n~se or topical for~ul~tions is ~n ~he r~nge of gr~ater than nbout from 0.1 ~g/ml to abou~ 25 m~J~l. Typioally, ~he eonc~ntrstion of ~nkephalinase for topic~l ~Dr~ul~tions is ln the range of greater thAn ab~ut from 1 ~g/ml tD abou~ 20 ~/ml. Solid dispers~ons of enkephalinase as well as ~olubilized preparations can be used. .Thus, the precise conoentration to be u~ed in ~he vehicle ~ill be ~ub~c~ ~o ~odest experlmental ~anipulation in ~rder ~o opti~ize the therapeutio response. Gre~ter ~han ~bout 10 ~g ~nkephalinRse/100 gr~ms of ~eh~cle ~ay be u~eful ~ith 1~ w~ hydrogel vehicles ln the tre~t~ent of ~kin ~nfl~mati~n. Sultable vehicle~, ln additlon to gels, are oil-ln-w~ter or ~a~er-ln-oil ~mulsions ~sing ~ineral oils, petrolatum and th~ like.

EnkephRlinase optlon~lly is ~dminlstered topic~lly by the u~e of a tr~nsder~al ther~peutic ~ystem (Barry, 1983, 3U ~ atolo~al For~ulations, p. 181 ~nd lltrature cited therein~.
Preferr~d topical prepar~tions ~uld ~o~prise enkephalinase including the cytoplasmic-transm~mbrane domainq. ~ost pre erred topical preparations would comprise enkephalinase lecking the cytopl~smlc-d~ain. While such topical delivery ~y~tems ha~e been LC8x396.~hg ~ ,:

:L3221~
~esigned l~rgely for trsnsder~al admlnistr~tlon ~f low ~lecular wei~ht dru~s, by deflnltlon they ~r capabl~ of p~rcutaneous delivery. They ~ay be re~dily ~dapted to ~dmlni~trat~on ~f enkephalinase or derlvatives thereof and ~oc~ted ther3peu~ic protsins by appropriate ~election of the r~te-sontrolllng ~icroporous membrAne.

Topical preparations of enkephalinase either for ~ystemic or local deli~ery may be employed and m~y contAin excip~ents as descrlbed ~bove for pArenteral ~dminlstration and other excipients used in ~ topical preparation such as cosolven~s, surfactants, oils, humect~nts, emolllents, preRer~ative.s, 6tabilizers and ~ntioxidant~. A~y phAr~cologically acceptable buffer ~y be used ~.g. ~ri~ or pho~phate buffers. The eopic~l formul~tlons ~ay also opeionally include ~ne or ~ore ~gents ~ariously ~ermed enhancers, surf~ctan~s, ~ccelerants, ~dsorption pro~oters or penetration enh~ncer~, ~uch ~s ~n agent for enhancing percutaneous penetration of the enkephalinase or other agents. Such agents should desirably possess co~e or ~11 o$ the following features as~would be known to the ordlnarily skllled ~r~isan: be phaD acologically iner~, non-promotive of body fluid or elec~rolyte loss, ~ompatible with enkephalinaqe ~non-inac~ivatl~g), and c~pable of formulation into cresms, gels or other topical deli~ery sys~ems as desired.

EnkephAlinase m~y also ~e ~dministered by ~erosol to achieve local~zed delivery to the lun~s. ~his ~S accomplished by - preparlng an ~queous ~erosol, 1~PGSOma1 prepara~ion or solid particles containing enkephslinase or derivat~ves thereof.
Ordinarily, ~n ~queous aerosol is ~ade by formulatin~ an aqueous solution or ~uspension g~ ~nkeph~linase to~ether ~ith convention~l pher~aceutic~lly accept~ble c~rriers snd ~tabilizers. The carriers ~nd ~tabil~zers w~ll ~ary depending upon ~he require~ents for the particul~r enkephelina~e, but typic~lly i~cl~de nonionic ~urfect~nt~ ~Tweens, Pluronics, or polyethylene glycol), ~nnocuous ~x396.mh~

13221~0 proteins like seru~ slbumin, sorbitan esters, oleic sc$d, lecithln, amlno acids ~uch as glycine, buffers, 6alts, ou~ar~ or ~ugar alcohols. The formulations ~lso can lncl~de ~ucol~tlc ~gents as well ~s bronchodilating agents. The for~ul~t~ons ~111 pe sterile.
Aerosols generally will be prepared from i60~0nlc 6clutlons. The particles optionally include norm~l lung surfactants.

~erosols may be formed o~ p~rticles ~n aqueous or nonaqueous (e.g., fluorocarbon propellant~ 6uspension. uch psrticleg include, for exsmple, intr~molecular aggregates of enkephalinase or derivstives ehereof or liposom~l or mlcrocapsular-entrapped enkeph~linase ~r derivative~ thereof. The ~erosols should be free of lung lrritsnts, l.e. subs~ance~ whic~ cause ncute bronchoconstriction, co~hing, pul~onary edema or t~ssue destruction. However, nonirrltating ~bsorption enhancing agents ~re sui~able for use herein. Sonlc nebulizers prefer~bly are used in preparing ~erosol~. Sonic nebulizers ~inimiæe ~xposing the enkephalinase or derivatlves ~hereof to 6hear, which can result in degradat$on of enkephalinase.
~nkephalinase msy be ~dministered ~y~te~cally, r~ther ~han topically, by in~ection i,=., xubcut~neously or into v~scular ~paces, partlcularly ~nto the ~oints, e.g. lntraart~cular in~ec~ion ~t ~ dosage of ~reater ~han ~bout 1 ~g~cc ~oint fluid/day. The dose w~ll be dependent ~pon the properties oi the enkephAlinase employed, e.g. ~ts ~cti~ity and biologlcal half-life, the concentr~tion of enkephalinase ~n the formulation, the site and rate ~f dosage, the clinical tolersnce of the patient involved, the pathological condition afflicting ~he patient and the l~ke as is well wlthin the skill of ~he physiclan.

The enkephal~nase of the present Inven~lon ~ay be ~d~inl~tered in 801uti~n. Preferably, enkephalinase lacking ~he transmembrane domain or the cytopl~smic ~nd transmembrane domains LC8x3g6.~ g ~, :, ,;. .,.,: ~ .:

- : : : :: : : - .. :
-: .,, : : :
: . - :... : . .
: ::: :,, ` . ::':;: : ~ :
:: . . :
.

~3~2~6~

would be used for adm1ni~tration in solution. The pH of the 801ution should be in ~he r~nge of pH S to 9.5, prefer~bly pH 6.5 to 7.5. Tha enkephalina~e or derivativ~s thereof ~hould be ln a solution havin~ a suit~ble ph~rmac~utically ~cceptable buffer such as phosphate, tris ~hydroxymethyl) s~inomethane-HCl or c~trate and the like. Buffer concen~r~t~ons ~hould b2 in the r~nge of 1 to 10 ~M. ~he s~lution of ~nkeph~l~n~se m~y also contsin ~ æslt, such RS
sodiu~ chloride or potassium chlor We in ~ c~ncentr~tion of 50 to 150 mM. ~n effective a~ount of a st~bilizin~ ~gent such as ~n albumin, ~ globulin, ~ ~elatin, a protamine or a ~alt of pro~amine ~ay ~lso be lncluded and may be ~dded to ~ ~olution oontaining enkephalinAse ~r to ~he compositlon from which ~he ~olution ls pr~pared.

Systemic ~dministrAtion of ~nkephalinase is wade dally, ge~erally by intramuscular injection, ~lthough intr~va~culsr infusion is acceptable. Administrstlon may also be intr~nasal or by other nonparenteral routes. Enkephal~nase ~ay filso be ~dministered ~i~ ~irrosphere6, liposomes or other ~icroparticulate delive~y ~ystems pl~ced in certain t~ssues lncluding blood.
Topic~l prepara~ons ~re appl~ed daily direc~ly ~o ~he ~kin or ~s ~ucosa ~nd then preferably occluded, i.e. pro~ectet by overl~yin~
band~ge, polyolefin ~$1~ ~r other barrier impermeable to the ~opical preparation.
The ~ethod is illustrat~d by way of the following examples, whlch ~re not to be construed as li~iting the invention.

L~8x3~6.mh~

~1 322~6~
~AMPLE 1 ~e~sure~ent_o~_~ubst~nce ~

The content of ~ubstance P-like in~unore~cti~i~y in ferret trachea was deter~lned by ~incin~ the tracheA ln 10 times the tissue weight of 2N ~cetic acid, extr~cting overnight, centrifuging the ti~sue fr~gments, and applying the ~upernatant to C-18 column (Sep-Pak; Waters ~nd Associ~tes) that had been ~quilibr~ted with wa~er c3ntainin& 0.1% trifluoroace~ic acid (TFA) and pre-cycled w~th 0.2 ~1 of o. 5 ~g/ml poly-L-~ysine (Sigma) ~n 0.1~ TFA. The Sep-Pak ~s then washed sequenei~lly with a ~tep gradient consis~ing ~f 4.0 ml each ~f 0, 20~ 40 and 60~ meth~nol ln 0.1~ ~FA in watert ~i~h the ~ub~t~nce P ~luting ~t 60~ methanol.
The ~eehsnol was ~vapor~d under N2. snd the Namples were then lyophili~ed and reconstituted ~n ~ssay buffer before be~g s~sayed.
The assay b~ffer consisted of 0.5~ 2-meroaptoeth~nol, 0.25~ bovine sçrum albumin (BSA), 0.03~ N~N3 in 0.05 M ~aP04 buffer at a pH of 7.4. S~mples of ~edi~m obta~ned directly from U5sing ch~mber ~nd ~he tissue extracts ~ere assayed :~3lng ~ 'pre-equil~bration radioi~unoassay. Separstion of free radiolabeled substance P from an~ibndy-b~und radiol~beled substance P w~s ach$eved using Dextran T7O-GOa~ed charcoal (Pharmacia).

Radiolabeled substance P ~as ~de by coupling substance P
to ~olton-Hunter re~gent (Bolton, A.E. ~nd Hun~er, ~.M., Blochem.
J. 1~: 529-539 [1973]) ~nd ~hen purifying the coupled product u~lng HPLC (Hewlett P~ckard ~odel 1090). The reaction mlxture w~s ~pplled to a 10 ~m C-18 reverse phase HPLC column (Waters ~nd Associ~tes~ and ~as elu~ed isocratically ~rom the colu~n a~ a flow rate ~f 1 ~l~in using 55~ metha~ol and .045~ TFA in ~ter. The no-derivatized 6ubs~ance P was r~dioiodinated using chlor~mi~e-T
(HcConahey, P. snd Dixon, R., ~ethods ~n Enzy~olo&y ~Q: 210-213 [1980]). The resction mlxture was ~pplied to ~ Sep-Pak C-18 LCBx396 . ~g , , ~ .

~ -25-~21~
column, And the iodinated Bolton-Hunter der~ative of ~ubstance P
~as eluted with 60~ ~ethanol.

Rabbits were immunized with a ~ixture o~ Freund's complete ~d~uvant, buffered saline ~nd substence P thAt had been coupled to purified ~S~ using glutaraldehyde. Substsnce P was c~nJugated by d~ssol~in~ 25 ~e ~onomerlc ~SA ~nd 5 mg ~ubstance P
in 1.0 mL of 0.1~ NaP04 buffer ~t ~ pH of 6.8, and then, with constan~ ~tlrring, slowly added 0.1 mL o~ 0.54 glu~araldehyde in lQ ~aP04 buffer. The ~ixture ~as incubat~d for ~t least 2 hourc, a~t~r which time, ~he reaction ~as quenched by adding l.Q ~L of 0.1 (NH3)2C03. After F'tirring for an ~dditional 30 min~tes, the ~1xture ~ exhauQtiYely dlalyzed ~gainst dlstilled H20, lyophilized, ~nd weighed, 5erum was harvestad, lyophilized, snd frozen. Serum was recons~ituted in dise~lled wa~er for rsd~oi~unoassays. ThR
subs~ance P antiserum was tested for cro~s reactivlty ~gainst othes peptides includlng bradykinin, ly~yl-br~dy~inin, ~asoac~e intestinal peptlde, ~omato~tatin, ~hulecy~tokinin-8, gastrin, bombesin and neuro~en~in (Pen~nsula LQbs, Belmont, CA).
Addit~onally, we tested ~everal proteinase inhibitors ~ec below~, the N-terminal ~ragment of ~ubst~nce P, ~ubstanc~ Pl g, ~nd ~he C-termlnal fragment, fiubs~ance P6 11 ~Peninsula Labs).
~erret tr~cheal segments contained an aver~ge of 0.58 O.230 pmoles of substance P-like lmmunoreacti~ity per g~. we~
wei~ht (n-3), sugge~ting that substance P i~ presen~ in the air~ays of ferre~.

~x396.~hg ,- . .' : - : ~

,, ~ . - .
. : , ~ . ~.

::

~322~ ~

~X~SPLE

~ucu~ secretion fro~ tracheal seg~entC o~ 36 ferrets was measured using methods described earlier (Borson, D.B. et ~., J.
Appl. Physiol. 57: 457-466 [19~41). Briefly, sfter anesthetizing adult ~ale ferre~s weighing 1-2 kg ~ith sodium pentobarbitsl (45-60 ~g/kg, ip), the trachea was removed ~nd wa~hed ln ~edium 199JEarles HC03 bubbled with 95~ ~ ~ 5% C2 at 38-C. Tracheal 6egments were ~ounted in pl~stic Ussing-type chamberæ connected to perfusion chs~bers ~ade of 6ilicon~zed ~ass tMRA Inc., Clea~water, Florida). After exposing bo~h 6~des to ~edium 19~, 0.167 mCi ~a2 35so~ ~as ~dded to he ~ub~ucosal ~ide. A~ lS winute int~rv~l~, the medium w~ drained from ~h~ lu~inal ~ide and replaced ~ieh fresh ~nlRbeled ~edium. Each ~a~ple was plsced in dlalys~s tubing (Spectropore no. D1615-1; MW cutoff: 12~14,000) and exhau~tively dialyzed wi~h ~he res~ of the samples from that axperiment ~gainst a~ least 6 change6 of distilled water (4 liters each) c~nt~ining excess unlabeled S04 and ~odium ~zide (10 ~g/L) to ~0 preven~ bacterial degr&dation of the ~acro~olecules. After dialysis, the bound rAdioactivity of each ~&mple ~as determlned usin~ ~ Beckm~n In~trument~ beta co~nter ~del LS 75Q0).
Molecular weight profil~s of the ~ater~ls secreted were detenmined by irst desslting s~mples using ~ Sephadex G-S0 col~mn, then concen~rating carbohydr~e-con~aining ~lecules on DEA$ Sephacel, ~nd determining apparent ~olecular size under denaturin~ cond~ions (4 M g~anidine, lOmM 2-nercaptoethAnol, 0.1% Triton X-100) using a Sephaross CL-6B column. Pre~ious ~tudies de~onstr~ted that ~naly ls ~f snmples using dialysl~ and column chromatography yielded qusntit2~i~ely similar result~ (Borson et ~1.. J. Appl.
Physiol. ~7:457-466 [1984]~.

Ch~racteristics of ~he rssponse to substance ~ lDcluding ~he tims cour~e, ~oncentrat~on dependence, ~nd reproduc~bility were *Trade-mark LC8x396.nhg ~^ .

` -27-~322~
studied. Tracheal segments from B ferr~t~ were incub~ted in the presence of radiolabel f or 3 hours to which ~er~ ~dded different concentrations of ~ubstance P (10-9M to 10-5M; Peninsula Labs, Belm~nt, CA). Reproducibility of the response to substance P ~as established by ndding 10-5M substance P twice, firct sfter 3 hours, ~nd ~gain ~fter 4 hours of incub&tion. All treatments were r~ndomized with regard to the site of 6~mpling in the trache~ to elimin~te systematic bias.

After ~ountin~ tissues in the chn~bers ~nd Adding r~diolabel, the r~te of secretion of 35S~4-macromolecules into the luminal s$de of the ti6sues ~ncreRsed, and by two ~ three hcurs, ~eeretion ~as ~ncreasln~ in an ~pproxim~tely line~r f~sh$cn. After thr~e hours of labelin~, tissues ~ecret~d at ~n ~ver~ge rate of 111.8 ~ 15.6 pmol bound S04/c~2Jh. At ~his time, ~he average ~ncrease ~n flux of bv~nd S04 per 6&mpl8 lnter~al was 11.7 ~ 2.5 p~ol bound S04/cm2/h (10.5~ ~f baseline). When ~dded to the ~ub~uco~al ~ides of tissues, 6ubst~nce P increased secretion wlthin 15 min, after which time, secretion retu~ned tow~rds b~seline, ~nd finally re~ched the values obt~ined by e~tending th~ pre-stimulus baseline forw~rd in time. Gel per~eatlon ohro~atDgraphy ~ndicsted thst 85~ of the radiolabeled materials ~ecreted in response to ~ubstance P h~d ~olecul~r ~eights ~n exce~s of 106, ~uggesting ~hat ~st ~ere likely to be ~ucin~ ~r prot~oglycans. Substance P
~ncra~sed flux of b~und S04 in a concentration-depsndent f~shion, with a threshold of 10-9 M ~nd a respons0 eO 10-5 ~ subseance P of 156.4 ~ 26.1 pmol bound S04/cm2/h or 155 ~ 42~ ~bove the pre~
sti~ulus baseline ~Fi8. 1). The ~ubst~nc~ P-induced secreti~n was sign~ficantly gre~ter than the increase due to time alone for 811 concentr~tions of ~ubst~nce P greater than lO-9 M (p<0.05; n~6 esch). P~r~hermore, repeat~d ~timulaeion c~used repe~tsd respcnses. The ~econd response ~s ~n aver~ge of 71.4 ~ 24.2% of the first response (p>0.1; n~4). R~movi~g ~he sub~t~nce P from the ~edlum ~ft~r the f~rse ~timulus l~cre~sed ehe magnleude of ~he L~8x396.~h~

.

.~ , ~22~6~
~econd response 61ightly to 91.1 + 4~.34 of the first resp~nse, but this WAS not significantly different from ~he second resp~nse ~f the tissues that ~ere continuously exposed to ~ubstanc~ P (p>0.~;
nr4 eaj.

The C-terminal frag~ent, ~ubstance P6 11 (10-5M) caused a potent releas~ of bound SO4, whereas the ~-ten~lnal fragment, substance Pl g, did not ~369.4 + 18.3 V6 33.7 1 24.7 p~ol bound S04/cm2/h; pC0.05; n-4 es) (e.g., Fig. 2). The response to ~ubstance P6.11 was ~gnific~ntly Breater than the response to ~ubstance P~ t the ~ame concentration (156.4 ~ 26.1 pmol bound S04/c~2/h; p~0.301; n~4). Secreti'Dn $nduced by substanca Pl,g was n~t different from the ~pontsn~ous change in baseline flux ~n the~e tissues due to t~me ~l~ne (-18.4 + 16.8 p~ ound S04/cm2/h;
p>0.1; n~4).

E~ll~l , ~ffect of Proteinase_Inhibit~ Q~
Substance P-Induced Mucus ~_cretipn In ~he next çries of e~perlments, the effects of b~ ~ubstance P ~et~bolism on ~ecretory responses to ~ubstance P ~s stutied. Different enzymes cleave 6ubst~nce P ~ differ~nt ~ites.
The active portion of the peptide, the C-terminal fra~ment~ causes ~ecretory act~Yity. Enkephalin~se was sho~n to cle~ve substAnce P
~o the inactlve fragmsnt ubstance Pl g.
. , .
The effects o~ ~ndogenous prot~inases were ~tudied by incubating at least ~wo tissues fro~ ~ach of 4 ferrets for 30 minutes in ~edlum for 2.5 hours ~nd adding a co~bination of nine (9) proteinase inhib~ors to the ~ubmucosal ~lde of each ti6sue (10 ~/mL each). ~he inhibitors u~ed lncluded leupeptin, ~ntipaln, pepstatin A, thiorph~n, 6ubstance Pl g (Growcott, J.~. ~nd T~rpey, A.~. ~u~. J. Ph~r~ 107-109 [19823) ~nd the ~ngiotensin LC8x396.~h~

,: '" ; ::

~ -29-13~2~ ~
converting en~yme inhibitor, tepr~tide (Peninsul~ Labs), ~protonin, BSA, ~nd bsc~tracin (Slgma). Thirty minutes ~fter ~ddition of inhibitor, ~ubst~nce P (10-6M) w~s adde~ to the ~bmucoæal side.
In the ~bsence of proteinase inhibltors, this ~onceDtration of subs~ance P csused consistent ~n~ submaxi~al effects. In 60me o the tissue~, the combination Df inhibitors 6ti~ulated ~ecretion, and ~h~n present, the effect re~ched a ~aximu~ ~ithin 30 ~n after ~dding the 1nhibitors. The mean responses to substance P in the presence of inhibitors was compared wi~h the ~ean re~ponses of two con~rol ~egments from each of the ~a~e ~nlmals. Subsequent experi~ents tested the ef~e~ts o~ the ~ndividuAl proteinsse inhibitors, th~orphan, teprotids, phosphoramidon (Sigma), ~ptopril ~Squibb Phar~aceuticals, Inc.~, tepro~ide, bo~ine ~erum, albumin, bacitracin, leupeptln, ~pr~onin, ~n~ bestat~n (Sigma), an inhibitor ~f an ~minopeptidase ln brain that degrades enkephalins (Chaillet, P. çt ~1., Eur. J. Pharmacol. 86: 329-336 11983]).

The concentration-dependenoe of enkephalin~se inhibition ~as explored by adding thiorph~n in different'concentra~ions to differen~ t~ssues. TD determ~ne whether the secretory effect of 6ubstance P ~ighe be ~ediated Indirectly v~a ~he release of endogen~us enkephalins, we tested the effects of ~et-enkeph~lin ~Peninsula Labs) on ~ecretory respon es.

Ue calcul~ted the fl~x of ~ound SO4 ~JS0~) ~ro~ the cpm in the dialyzed ~amples ~ccord$n~ to the equ~tion modified from (Corr~les, R.J. ç~ ~1., J. ~ppl. Phy6iol. ~: 1076-1082 11985]):
J 2 ~opm r~ov-rot/15 ~s~ Y ~ lall y 106 pm~l oold ~ /5ml~) 3t) ~iO4 (pme~l SO~tclD Ib) - -- _ 0.5 6~ x cpm 1~ 5.0 IDL o~ hDt-sl~ diu~
Changes in 1u~ due to drugs were o~lcul~ted by s~btrActing the flux during the 15 ~in period prior to ~dding dru~s from the flux observed for that tissue sither 1~ or 30 ~in after addi~g dru~s, which ever ~as hi~her. Hean fl~xes or ch~nges in fluxes for e~ch conditlon were co~p~red wleh each other by one-LC~x396 .~hg , : : :, :
.

1 322~ 60 w~y ~nalysis Df var~ance. ~ew~an-Keuls ~est for multiple comparisons was used to determine dlfferences between groups (Zar, J.H., ~Multiple Comparisons" ln ~5 J~L~hL~L~ Y I- [Prentice Hall, Englewood Cliffs, N.J. 1974]~. !

When added to the ~ubmucosal ~ides sf the t~ssues, the combitl~tion of 9 proteinase inhibitors lncreased the flux of bound S04 into the lu~en ~llghtly, but not ~lgnificantly (45.8 + 28.2 p~ol bound SQ4/cm2/h) co~par~d ~o the 1ncrease due to time alone (12.3 + 6.2 pmol bound S04~cm2/h). After ~ddinB subst~nce P to tha submucosal 8 i des of these tl~sues, the flux of bound S0~
~ncre~sed by ~n average of 383.5 ~ 14B pmol bound S04~cm2/h, or 438.6 ~ 105.2% of the respoD6s to ~ubst~nce P of ~ehe control ~i~sues from the ~ame ~n~m~ls ~83.7 ~ 21.4 p~ol bDund S04/c~2/h;
p<0,05; ~w4~ (Figs. 3 and 4). The incr0a~ed re~pon~e to ~ubstance P ~as nGt due to additl~e effects of the ~nhib~tors ~nd ~ubstance P because ~he 5um of the indlvidual effects, 83.7 pmol bound S04~c~2/h (gubstance P) plus 45.8 p~ol bound S04/cm2/h (inhibitor6) ~ 12~.5 p~sl bound 504/cm2/h, or~only 34~ of the response to ~ubstance P ~n the pre~enc~ ~f the inhib~tors (383.5 pmol bound S04/cm2/h).
~,s Of the intividual inhibitors studied, only thiorphan ~nd phosphor~midon, inhibi~ors of ~nkephalinase, poten~ated 6ubstance P-indu~d se~retlDn (Figs. 4 ~nt 53. Uhen ~dded to the ~ubmucosQl ~de of the tis~ues at 10'4M, thiorphan by it~elf ~ncreased the flux of bound S04 into the lumen ~y an avera~e of 57 ~ 17.B pmol bound S04/cm2/h. Thi8 was ~i~nificantly ~res~r ~han the ~pont~n~o~s incresse in flux in the ~ame tissues before ~ddin~
th~orph~n (20.5 6.3 pmol bound S04/cm2/h; p<0.05; n 6). At lower concentratlons, thiorphan did not 6timul~e 6ecre~ion signific~ntly. Howe~er, thiorph~n potentiat~d the ~ecretory response to substance P In a concentration-dependene fsshion, ~ith a threshold of approxlmately 10'8~ (Fig. 5). In tissues pre-Le~x396.~h~

:: . . ..

: . : - ~ :
,:
:` :

1 3~1 60 tre~ted with thiorphan (10-4M), 6ubst~nce P increased flux of bound S04 by an aver~e of 268.0 ~ 5B.0 pmol~c~2jh (502 ~ 1474 of control; p<0.05, n 6). Thi8 svera~e response ~BS ~ignificantly greater than that oP control tissues from the ~me ~ni~als (79.2 ~
13.0 pmGl bound S04/c~/h; p~0.05; n-6). The lncrease in substance P-induced secretion was not ~n ~dditive effect of ehiorphan and substance P: ~he sum of the individua~ eff~cts, 79.2 pmol bound S04~cm2/h (substance P) plus 57.0 pmol bound S04/cm2/h (thiorphan), or 136.2 pmol b~und S04/cm2fh, was only 51~ Df ~he response to 6ubs~nce P in the presence Df thiorphan (~68.0 p~ol bDund S04/c~2fh). In tis~ues treated with pho ph~r~midon (10-5M), ~ubstance P (lO'bM) lncreased flux by ~n sversge ~f 357 + 91.9 pmol bound S04/c~2Jh, wh~ch WBS 6ign~fic~ntly greater than ~he responses of the control tissues from ehe ~ame 8ni~als (62 . 3 24.8 pmol bound S04~cm2/h; p~0.05; n,4 ea).

In contrast to inhibitors ~f enkephalinase, inhibitors of other protein~ses did not potentiate ~ubstance P-induc~d ~ecretion (Fi~. 4). Thus, capt3pr~1 ~nd teproti~e, inhibitors of kininase II (angiotensin convertlng enzy~e), potenti~ted subs~ance P-induced ~ecre~ion 61ightly ~by 44 ~ 27~, and 49 ~ 39~, respectively), but the increases ~ere not cign~fioantly different fro~ responses ~f the ~ontrol ti~ues from the 6ame an$mals (p>0.2; nr4 ea). Similarly, leupeptin, ~p~oton~n, bacltracin, BSA, and bes~atin fail~d to p~enti~te Eubs~nce P-induc~d ~cretlon.

Experl~ents were oarri2d ~ut to Investlg~te ~hether the secretory response to substance P mlght be ~ediated by ~ndogenously-released enkephalins. Met-enkephalin did not ~imulate secretlon 6ignlficantly. Af~er ~dding ~et~onkephalin (10-4M) ~he change in flu~ of bound S04 was 6.7 ~ 4.2 pm~l~cm2/h, ~hich was no~ different fro~ the change in flux in ~ontrol ~issues at the ~ Yalen~ ti~e of 7.0 ~ 1.5 pmol bound S04/cm2/h (p>0.5;

L~8x396.~h~

, . .

,. . , ~ .
~ ,': '~
: , : ;: , ~, .
: - ~
: -1 3223.~) nY4). These studies indioate that ankephalin~se pre6ent in airway tissue inhiblts subst~nce P-induced secre~ion.

Effects ~f ~nkeshalInase Inhibitors on Secretorv Responses tD ~chvkinins In this n2xt 68r1 es of experlments, the effects of different tachykinins and the enkephalinase Inhibitor, phosphoram~don were compAred. Tissues from erre~ tracheas were moun~ed in ch~mbers ~nd l~beled ~sing prevlcusly described ~ethods (Ex~mple 2). T~ssues ~ere then ~xposed to one of ehe tachykinins ~ubs~nce P (SP), neuro~nin ~ (~R-~, neurokinin~ ~ ~NK~
~ledoi~in ~ELED), physalaemin (PHYS), ~r ~as8~nin ~R~SS~. E~ch ta~hykinin wa ~inistered ~t lO'SM ln the absenc~ or presence of pho~phoraLidon (10-5M, 30 min; ~lg~a). ~he release of h1gh m~lecular wei~ht 35so4 ~as determined as described above in Exa~ple 2. The ~ata is shown in Fi~. 6. In the absence of phosphora~idon, ~o~t ~f the t~chykinins 6timul~tcd ecre~i~n with ~n osder of pot~ncy;
qubs~ance P> physalaemin~ele~olsln-Xass~nin ~ ~eurokin~n A.
(p ~ 0.05, n-5 each~. Neurokinin ~j: in ~he ~bsence of phosphoramid~, was ineffectiYe. However, ~n ti~8ue8 pretreated h phosphoramidon; ~eeretory responses to e~ch tachykinin were : 25 ~gnificantly i~creased (P ~ 0.05, n-5 e~ch). Fu~ther~ore, in the presence of phosphoramidon, the order of potency was al~ered:
~ub~Sr,nc- P~llourolc~nln A~phy~ mln~ dol-~n-h~ nlD~n3uro}1n~n ~.

In the absence of enkephalinase inhibition, the effect of ~ubstAnce~P was greater than thst of neurok~nin A. After enkephalinase inhibition, the efec~s of ~b~t~nce P and neurokinin A were the ssme. These result6 ~ugge~t that enkephalinase cle~ves neurokinin A with greater ef~ci~ncy than it clo~ves ~ubstance P. They also indicate ~hae ~nkephalinase LC~x39~.~hg - ,:. -- ,. . , : .:
: .: ~ ::,: : :,:: : . , : ~
::.: .:: .: . : :: :-.: :: ~ : . ; ::
::-, :,:, . -- :.~: : :
:- .. ~ - . . - :

, ~ :: , - : . , ~322~
present In ~irway tissue inhibit~ secreti~n induced by vari~us tachykinins.

c~lizat~on an~ s~ ~L~ L;I~
EnkePh~linase ln Ain~ys In ehis ~ries of experiment~, the tissue locatio~s of onkephalinase were determined. Enkephallnas~ was purified from th~ erret kidn~y to nesr homogenei~y~using publ~sh~d ~ethods (~lfroy and Schwartz, Supra~. Enkephallnase sctiYity ~n membrane fractions derived from the ~agu~ nerve, tr~cheal ~pithelium, ~ub~ucn6a, ~usole 9 lungs, ~nd ~i~neya of ferre~ ~as.determined.
Ferre~s ~ere:anestheti~d, ~nd ~he vagl, tr~che~, lungs, a~d kidneys removed. The tracheal ~pithel~um, ~ubmucosa, ~nd muscle ~ere separa~ed from eaeh other by incuba~ing the trashe~ in Ca~+-free mediu~ f~r 15 ~inuteq, after w~ich time, the ~pl~helium was easily removed. The ~usc~e ~as then ~eparated from the gland-containing submucosa. Each tissue ~as ~inced and ~hen homogenized ~n 50mN HEPES buffer ~pH 7.4) using ~ PolytrnD homogenizer.

: ~ Enkephal~n~se Ct~Yity ~as ~easured by deter~ining ~he r~tes ~f Gle~Vage of (3H-Tyrl, DAla~ u5)enkephalin (Research Produ~s Internation~ by ~embrane ~ractions ~r purified ~nkephalis4se. Fifty ~1 of Dem~rsne frsctl~n ~5 incuba~ed for 40 minutes at room temper~ture wlth 50 ~l cf buffer containlng t3H-Tyrl, DAla~,Leu5~enkephalin (20nM), after ~hich tl~e the re~ction ~as q~enched by ~dding 50 ~l of 2N HCl.

æoven~y~five ~1 ~f ~his ~ol~tion ~as appll@d to oolu~ns co~taining polystyrene be~ds (Poropsk-Q~; the char~cteri~tic ~etabollte, 3H-Tyr-DAl~-Gly, eluted ~ith ~ster, ~nd the radioactivity determined. The protein concentr~ions ~ere determined by the Bradford procedure eBradford, ~.~. An~lytical lCB~396.~hg :: .:. : ~ . . :
:: : , : - ;, :'. ::

` ` 1322~
Biochem. 7~:248-254 11976]), ~nd results of ~nkephalinase ~ctlvities were expressed ~s f~oles substrate cleaYed p~r ~in per ~g protein. ~esults are ~hown ln T~ble 2.

TA~I~ 2 lncera~ ns ~l~h Leu Th~or~han and Subs~r~tes . Ti~s~e~
Ep~he- Sub-Y~ l~n~ mU~s~ t~lÇ L~ ~i ~r~
nke halln~e 614690 1226 520 540 7~19*
e~ Y~ + 116~ L87 + 211 ~ 155 ~ 70 KI

leu-~hiorphan (nM) 2.1 8.7 2.8 2.1 2,8 3,3 (D~ e~ M~ 53.2 20.0 30.6 32.4 24.1 ~2.0 Subs~ance P (~N) 5 6 5.3 3.2 3.6 Neur~kinin A (~) 6.~ 9.7 4.4 5.2 3.0 6.1 * ~ean ~f triplicate de~er~inat~on~fro~ one experi~ent.
~ ** ~ata expressed ~s fmoles/m~n/mg:proteln.
30: - - ~

: . . : Esch tissue :clea~ed the substrate,~with the hi~hest activity in the ~irway present ln the~:submucosa. Lucine-th{orphan (leu-thiorphan, ~n e~kephalinase inhibitor) inhlblted ~ubstrate cleavage ~lth affinlty con5tant5 ~KI) in the nanomolar ran~e for each tissue. In cDntrast with leu-thiorphan, other proteinase or peptidase inh~bltors (10-5M):, dld not inhibit substrate clea~age by any tissue. These inhibitors included: captopril, ~n inhibitor of an~iotensin co~verting ~nzyme; bestatin, an inh~bitor of ~minopeptidsses; aprotonin, an inhibitor of ~eri~e protelnases;
or, leupeptin, an inhlbitor of: serine or thiol proteinases.

: .
~ LCBx396.~hg ~

, - -. , . -. . ,- ,., .

, ~ . .. " - . , ~ : ~
::: . ~ :: , . . .

~322160 Therefore, ~ubstrate cle&v~ge is due excl~si~ely to the action of enkephal~nase. The pept~des, ~DA1~2,Leu5)enkephalin, ~ub~tance P, and neurokin~n A ~l~o inhib$~ed ~ubstrate cle~vage w1th ~ffini~y constants (KI) th~t, for c~ch substr~t~, ~ere the ~me Por all tissues (see T~ble 2). The affinity of ~nkephslinase for substance P and for neurokinin ~ Is approximately t~n times ~he affinity of enkephalinase for (DAl~2,Leu5)enkephal1n. ~ecAuse the affin~ty con~tan~s for subst&nce P and for nçurokinln A are the ~me, the differences in ~ctiviti~q of the~e peptlde~ on ~ucus 8ecre~ion SExa~ple 4) ~nd ~uscl~ contr~cti~n (Ex2mple 8) are probably due to differeno~s ln turnover numbers ~Kc~t) ~nd not to differences 1n affini~es of enkephalinase for the ~ubstrstes.

ffect_of Try~s~ on ~nkeph~lin~se ~ctiv~t~ ~d Substance P-induced Mucus Secreti~n In thls series of experlment~, the effects of prototype extracellular proteinase on enkephal~n'~se and ~ubstanoe P-~ediated ~ucus seoretion were atudied. The r~tionale for these s~udies ls ~hat ~ ~ariety of proteinase~ ~re rele~sed from h different cells ~nd tissues ~e.g. neutrophil elast~s~, ~lkaline prote1nase, ~ast eall t~ypt~6e ~nd chym~se), ~nd if ~h~se protelnases dçstroy enkephali~ase, then alterations in peptide-indu~ed ~ecretion ~hould be observed. This hypothesi6 was tested using trypsin (10-11 to 10-5~; 15 min) ~ pro~otype serine proteinase. The efect of trypsin on enkephalinase activity of lung homogenates measured as degradation of (3H-Tyr-DAla2-Leu)enkephalin, and mucus ~ecretion from tr~cheas of 3 ferrets w~s determined.

Trypsin incub~tion (15 ~nutes) decre~sod enkephalinase activi~y in homogenates of lung~ (Fig. 7) in a concentraeion-dependent fashion, ~ieh ~ threshold ~bo~e 10-11~ and a ~aximal LCBx396.~h~

: , ~:, . . . ..

. ~. :. : ;
: ~

~ - : : :

.~ -36-3L322~

effect at 10-5M. Addi~ionally, trypsin (10 5M, 30 ~in) by itself d~d no~ increase mucus ~ecretion ~ore than the incr~e due to time alone. ~owever, trypsin increased the ~ecretory response to substsnce P. Thus, BS i6 the cas~ for enkephalln~se lnhib~tors (e.g $hiorphan), the decrease in enkephalina~e.~cti~lty c~used by trypsin is associated with increased ~ecretory responses to 6ubstance P. Therefore, it i~ likely th~t o~her pro~inases (e.g.
those from inflammatory cells, mast cells, neutrophlls, ~tc.) also might ~lter peptide-~nduced re~ponse6 indir~ctly, by regulating ~he ~mount or sc~vity of ~nkephalinase in the tlssue.

~LZ
Effect of_prote i ase In~bitors on Sub~ance P~ d Twenty-sev~n ferret~ ~ere ~nes~he~zed ~ith pentobarbital godiu~ (45-60 ~g~kg, i.p.), ~nd the trachea ~ere rcm~ved. Tr~nsverse rings (8 ~ long) ~ere cut from the ~raohea and moun~sd in glass chamber6 filled ~Ith 14 ~1 o~ Krebs-Henseleit 601ution of the ~ollowing composit~on (in ~MolestL): ~aCl 118, KCl 5.9, CaC12 2.5, ~gSO4 1.2, N~H2PO4 1.2, NaHCO3 25.5, glucose 5.6, 0.1~ bovine ~eru~ ~lbumln, ~nd pe~cillin-~trepto~yc~n (100 Unl~s/ml). The solu~ion ~a8 ~aint~ined ~t 37'C and was ~er~ted con~inu~usly ~n a ~ixture ~ 95~ 02-5~ C02~ which produced ~ p~ of 7.4. Six ~r~ch~al rings were ~tudi~d conourrsntly.

The trache~l rings were connected eo ~tr~in gauges (Gr~ss FT03) for cont~nuous recording of ~sometric tenslon, ~nd the rings were placed between two rectangular pl~tlnu~ electrodes ~6 x 40 ~m~ for electrioa~ field ~tlmulst~on. The rings were initially ~tre~ched ~o a tension of 20 g for 30 seconds ~nd ~ere then allowed to eq~ilibrate for 1 hour while restin~ eo~sion was ad~usted to 4 g (Skoogh~ B-E. et a~.v J. Appl. Phy~lol. ~: 253-257 119821). During ~quilibr~tion, ~he ~edium ~as chv~ed every LC8x396.~hg , :, ., :
, :: :

~ 3.~2l6a 15 minutes. Prelimlnary studies 6howed that maxlm~l re6ponses to electrical field stimulation ~biphasic, pul~e durat~on 0.5 ~s; 20 V for 20 s, frequency, 20 Nz) were obtained w~th 4 g of resting tension. Id.
s The responsiveness to ~ubstance P (uslng concentratlons rang~ng from 10-8 ~ to 10-5 M) and to leu-th~orphan and cumul~tive dose-response curves to substance P ~Peninsula Labs~ were obtained. Each ucceeding concentr~t~on ~f 6ubs~ance P was added after contraction re~ched a plateau. After completion of ehe first dose-response curYe to ~ubstance P, leu-th~orphan (lG-5 M, Squibb Pharmaceutieal) wa~ added to the organ bath. Follo~i~g 8 15 minute incubation, ~ ~cond dose-re~pon6e curve to;~ubstance P
w~s obtained.
The ~-terminal fr~g~ent, ~ubstance Pl g was 6~udied because enkephalinase cleaves substance P betwee~ the 9 cnd 10 po~itions ~t~as, R. ~ ~l.. Proc. ~atl. Acad. Sci. [~SA] ~0:
3111-3115 11983]), generatlng that fragment. (10-5 ~ ~eninsula Lab~). E~periment~ were ~l~o ~arried out wi~h (DPro2, DTrp7~9)-~bs~ance P (10-5 ~ (Peninsula Lab~ ubse~nce P ~n~agonis~
6~ ~Hakanson, R. et ~1..... Br. J~ PhAr~ac. 77: 697-700 [19821) by ~dding the ~ntagonis~ after the r~sponse to substance P (lQ-6 M) and leu-thiorph~n (10-5 M) resched a pl~teau.
Substance P was dl~solved ln 0.1 H ~cetic ~cid, and leu-th~orph~n was dis~lved ~ 14 e~hanol to glve ~tock ~olutions of approximately 10-3 ~. These drugs were 6tored at -25-C, and ~l~quots were tha~ed and diluted in Krebs-~en~ele~t ~clut~on for ~ach experiment.

Data were expressed ns ~e~n ~ SE. For the dG~e-response curves to ~ubst~nce P, the ~eans between ~wo gro~ps ~t e~ch ~oncentration wers ~ompnred by ~ pair~d t test. F~r the ~tudies LC8x396.~hg 1. : `

:- : :

: `

33~

Df elec~rical field stimulation, responses were co~p~red by one way ~nalysi- of varlance and Newman-Keuls ~ult~ple range test.
Signif~cance was accepted ~t P < 0.05.

Substance P alone cau~ed ~n incre~sed ~uscle tension, ~ut only ~t ooncentrntions of 5 x 10-6 N or greater (~ig. 8~.
Additivn of leu-thiorp~n (10-5 M) slone had no ~ignlfic~nt effect on resting tension, but it shiftet the dose~response relatlonship to subst~nce P to lower cvncentr~tlons by ~pprox~mately one lo~
unit (Fig. 8). In oontrast to ~ubst~nce P, the N-ter~inal fr~gment substanGe Pl g ~10-5 M), had no gignificant efect on resting ~enslon (n - 3).
;

Ihe contr~ction produced by substance ~ (10-6 M) ~n ~he presence of leu-thlorphan ~as deoreased by the ~ubstance P
~ntagonist (DPro2,DTrp7~9)substRnce P (10-5 N) ~36.0 ~ lO.0~ of control3. The subs~ance P antagonist d~creased ~ubst~nce P-induced contractions signifioantly more than did a~ropine ~lone (p < 0.01) (Fig 9).
~lectric~l field sti~ulation-lnduced coff~ractlon ~n the presence of sugstance P (5 x 10-ll Mj ~nd either c~ptopril (Squibb ~h~r~ace~tic~ lO-S M), bestatin ~Sigma) (10-5~ or leupeptin (Peninsula LAbs) (10-5 M) was measured to determine ~hether inhibieion of angi~tensin converting enzyme (A~E), aminopeptid~ses, and serine ~r ~hiol proteinases were resp~nslble f~r potentistlng responses to electric~l field stimulation.
Incre~sing concentra~lons of leu-ehiorph~n (10-11 ~o 10-4 ~) ~ere ~dded ~nd the ~e~ muli repeat~d ~o deter~ine whether leu-thiorphan ~odulated electrical field stimulation-induced contractions. The effects of leu-thi~rphan ~n electric~lly ~nduced contr~c~i~n was studled uslng five additional fe~re~s. After deter~lning control response~ to olectrioal field ~ti~ulation (S ~z), l~u-~hiorphan ~10-5 M; 15 ~in~ was ~dded, ~he ~ti~ulus repeated, L~8~39~.mhg -.

- - : ' (DPro~,DTrp7~9)substance P, the 6ubstance P nntagonist (10-5 M; 15 min; Peninsula L3bs) was ~ddPd, and the 6timulus wa~ repeated, Substance P alone, even in ~ery low cow entra~$ons (5 x 10-11 M), augmented the responses to electric~l field stimulation (~ig, 10). Subst~nee P (10-11 or 10-1 M), reproducibly ~ugmented electr~c~l field ~timulstion-induced contr~ction, with no Eignificant tachyphyl~xis of the effect, ~en after 5 responses (n 3). Leu-thiorphan ~t concentrations up to 10-4 M dld not alter resting tension in ~ny experlment. However, addition of increas~ng concentrations o~ ~eu-~hiorphan produced dose-relsted increases $n the r~sponses to ~leotrical fleld 6timulat~0n, with a threshold ~f approxi~Ately 10-9 ~ ~nd a ~axi~um effect ~t 10-5 M
(Fig. 10~.
L~u-thiorphan potentiat~d the re~ponse t~ electrical field stimulation in ~ concentratlon-dependent fashion 5Fig. 11).
(DPro2,DTrp7~9)substance P (10-5 N) ~i~nifi~antly inhibited the incre~se in the response to electrical field stlmulatiGn lnduced by l~u-thiorphan (101.6 ~ compared to 118 ~ 1.7~ in the pressnce of leu-thiorphan; p > 0.01; n ~ 5).
~, In contr~s~ to the e~fec~s o leu-thiorp~an, none o~ the other peptidase 1nhibitors used potentlated the response to electr~cal field sti~ulat1on in the presence of gu~st~nce P (5 x 10-11 M) (p > 0.5; n 3).

These studies es~ablish that enkephalinase is present in ~{rway 3uscl0 ~nd nerves, and th~ it decresses substance P-induced effec~s, including bronchoconstrirtion and potcn~ia~on of neurotransm~ssion to ~ir~ay smooth ~uscle.

Enkephalinase is involved in decreasin~ ~ubst~nce P-~ntuced effeets because leu-thiorphan augmented substanoe P-LC~x396.mhg ., .. : :

:
: :~ :

induced effects, wher~as other proteinase and peptidas~ inhibitors such as captopril, an inhibitor of angiotensln convertin~ enzy~e (Turner, A.J. et ~1.. B~ochem. Phsrmacol. ~: 1347-13S6 [1985]~, be~ta~cin, ~n ir~ibitor of ullinop~ptldase6, or l~upeptin, an $nhibitor of serine or thiol proteinases, wers withou~ effec~.

EXAMPI~ 8 ~f fect oiE PrQtei~a~
Induced Contra~tlon o~ ~irwa~z ~mooth ~usçle The methods ~sed to ~xcise tracheal tissue, ~ount ~nd electric~lly ~timul~te the ti~ue were tdentic~l; to thos~
~escrlbed ~bove. The responses to tachykinins ~nd to leu-th~orphan, cumulat~e doae-response curves to sub~tance P
(Peninsula Labs), ~eurokinin A (Peninsul~ Labs) ~nd neurokinin B
(Peninsula Lab~ were obtained us~ng ~OnCentrBtiOns r~nging fro~
10-11 ~ to 10-5 M in ~he pre6ence and ~bsence of leu-thiorphan (10-5 M, 15 ~in; Squibb Pharmaceut~cal). 'Each succeeding concent~ation of tachykinin w~s ~dded ~fter ~h~ previous ~ontractlon had reached a pla~eau.

Electrical field ~timulation- ~duced contr~ctions were measured in the pr¢sence of substance P (10-1 M), neurokinin A
~10-1 ~) ~nd neurokinin ~ ~10'1 M), combined ~ith either captopril (10-5 M; 15 ~in; Squibb Pharmaceutical), bes~atin (10-5 M); lS ~in; Sigma) or leupeptin (10-5 M; 15 min; Peninsula Labs) to determine whether inhibition of an~iotensin conv~rtin~ enzyme (ACE), ~minopeptidases, or serine or ~hiol proteinases were responsible or potentiating responses ~o electrical field ~timulation.

Dat~ ~ere expressed ~s ~ean + SE. For the dose-response curves ~o tachy~1nlns ~he ~eans between two ~roups ~t each LC8x396.~h~

~, - i' .

.:

- ~3~21~

concentra~ion were ~nalyzed by an unpaired t ~est. For the study ~f electrical field 6t~mulation, responses w~re ~:o~parad by one ~ay sn~lysls of vari~nce ant ~ewman-Keul6 ~ultiple range ~cest.
~ignific~nce was accep~ed at p<O.OS.

Substance P, neurokinin A ~nd ~eurokinin B el~ne c~used ~ooth muscle contraction, but only ~t concentrRtion~ ~f 10'6 M or ~reater for neurokinin A and 10-5 M for substance P and neurokinin B ~Fig. 12). Contraction3 induced by t~chykinins were compsred at 10 6 ~ ~nd 10-5 M, indicatlng ~ r~nk order of potency:
neurokinin A > subst~nce P ~ neurokinin fi~
nlthough 6tatisticslly 8~ gn~firant differences were obtained only bet~een neuroXinin A and ~ubstance P ~p<0.05) or neurDkinin ~ ~nd neurDkinin B (p~0 . 01 ) at 10 ~ ~ M, ~nd b~t~een s~eurokinin A and ~euroklnin B ~p<O.Olj ~t 10-5 M. Addition of leu-~hiorphan (10-5 M~ ~lone had ~o significant effec~ on restin~ tension, but shifted the dose-response cur~es to 6ubstance P, neurokinin A and Deurokinin B to lower concentra~ions by approxim~tely one-log un~t for substance P, two-log units for neurokin~n ~A, ~nd three-log units for neurokinin B (Fig. 1~). A rank order oP potency of t~chykinins ~n the presence of leu-~h~orplh~n ~as:
S neurokinin ~-neurokinln ~ ~ substance P.
Leu-~hi~rphan po~en~i~ted dhe responses to tachykinins ln e concentration-dependen~ fashion, wi~h a ~hreshold ~f approximately 10-7 M and ~ ~axi~um effect ~t 10-5 M (Fig. 13). In contrast to the effects of leu-thiorphan, none of ~he other peptidase inhibitors u~ed po~entiated the response to electrical f~eld stimulation in the pre~ence of substance P, neurokinin A or neuro~in~n B ~10-7 M).
3~ ~
5ubstance P, neurokinin A ~nd neurokinln B each potentlat~d the responses to electrical field ~ti~ulation in a c~ncentration-dependent fashion. Substance P po~entl~ted the r~sponse to eleetr~c~l ~ield ~ti~ulation ~i~nif~cantly ~ore than LG8x39~.~hg .. ...
:: : : . :, . ;

:

- :::: . :. :
:- :: :: :~ :.
: - : : ' : ~. , `:

~3~2~

neurokinin A and neurokinin B. (DPro2, VTrp7~9)substance P ~10-5 ~, n spacific tachykinin ~ntagonist) inhibited ~ubstance P-, ~nd neuroklnin A-, n~d Deurokinin B-induced potentiatlng responses to ~lectrical field stlmulation (Fig. 14).

These studie6 establish that sub~tance P, neurok~nin A
and neurokinin B csuse cmooth mu~cle contrsctio~ and they potenti~te neurotran~mi~æion to ~irway ~mooth ~uæcle with differene potencies. Enkephalinas~ pre~ent in airways i~ ~n important inhibitor o~ tachykinin-induced effects. Th8 mechanis~
by ~hich leu-thiorph&n potentiates t~chykinin-induced effects ~s ~ost li~ely by preventing de~r~d~t~on of the peptides by ~nkephalinase. ~he ~ensitlv$ty to hydrolysi~ o diff~rent ~achykinins by enkephalina6e ~ay be ehe expl~nation of the chsnge in rsnk orter ~f potency ln taehykinin-induced effects.

The ~ubstance P antagonist, (DPro2 DTrp7~9~substance P
inhibited electrical field ~ti~ul~tion-induced contraction, ~ug~esting that the effects of tachykinins ~re ~ediated via tachykinin receptors because this antagonlst is selective for ~achykinins ~Leander, S.R. 8~ ature ~2~: 467-469 [1981]).

~s 6hown ~bove, the t~chykinin-induoed effect3 in ferret trachea may be mediated by enkephalinase because leu-thiorphan aug~ented tachykinin-induced effects, whereas other inhibitors of proteinsses ~nd peptidases did not. Those lnhib~tors described ~bove include: captopril, ~n ~nhibitor af angiotensin convertin~
enzyme; bestatin, an inhibitor of aminopeptidases; and leupeptin, ~n inhibi~or of ~erlne or thiol prot~inases. Furthermore, ~he effects of leu-thiorphan on tachykinin-i~d~ced contractions ~ere concentration-dependent (Fig. 13), sugges~ing th~t ~cti~ity of ondogenous enkephalinuse is closely related to tschykinln-induced ~ffect6.

LC8x39S.~h~

:
: :
. :

~ 3 ~

E~
Effects of Enke~hal~ase InhibitoEs~2~ Bra~y~ni Induced Airway Smoo~h~ 5b~ 5~

The ~ethods used were the ~me ~ thos~ d~scribed ln Example 7. Bradykin~n (Sig~8) ~nd lys-bradykinisl (kallidin;
Sigma) c~used contraction in ~ dose-dependent f~shion (10-11 to lQ'5 M) (Fig. 15). In the ~b~ence o~ enkephalinase inhibi~ors, bradyklniD ~as more pote~ than ly~-~radykinin. Leu-thiorphan (10-6 M) ~hifted the dose-r2sponse curves to lower concentrations by 1 to 1.5 log ~nit~. In the presence o~ Leu-thiorphan, the coner~stile effect~ of br~dyklnin ~nd lys-brAdykinin were equally pote~t. This 6tudy de~onstrate~ tha~ onksphalin~se presen~ in ~irway ~issue decre~e~ the ~ffect cf ~inin-induce~ smooth ~uscle cDntraction.

~ffe~ts of ~nkephal~nase Inhlbitors ~n_Sub~tance P-Induced eal Smooth Muscle Contracti~ ' Using ~ethods ~imilar ~o those ~ith ferrç~ 8ir~y g~oo~h ~uscle, described ~n ~xample 7, longltudin~ al Rmooth ~uscle ~s examined. In she ~ontrol c~te, ln the ~bsence of enkephalin~se ibhibitors, sub~tance P esu~ed tonic c~ntraction in a dose dspendent f~sh~on (Fig. 16). Leu-thiorphan ~10-5 H~
~hifted the dose response curve ~o lower concentrations by 1 log unit. This study d~monstra~es that enkephalinase present in gastrointestinal (ileal) tissue decrcases ~he effec~ of ~ubstanre P-indured smooth ~uscle contraction.

LC8x3~6.Dhg .: :. ~ ,. ~ .
.. .::: .. - :.. .

~322~

~b~mot~ctlç~ y The nor~al ~unctions of ma~ure ~eutrophils ~re ch~motaxis, phagocytosis, mierobicidal ~ction, ~nd digestion of foreign ~aterlAl. Chemotactie ~actors ~re generated at the ~ite Df lnflammation which attract various immunological cells including neutroph$l~ to that ~ite. The ~echani~m undexlying the chemotac~ic attraction of n~utroph~ls to the inflamm~tory ~ite is ~ot f~lly understood. Enkephalinase has been implicated in ~he ~echanls~. Connelly, J-C- ~ , Proc. N~tl. Acad. Sci. (USA) ~2. 8737-B741 (19B5). In c~rtain c~se6 of hyperi~une respon~es ~bnonm~l influx of ~eutrophil6 ~nd ~h~r ~une cells ~ay cause ~ddlt~onal tissue d~R~e.
Enkephalinase has been found to be bound to the cell ~e~brsne of human neutrophils. Gonnelly, et l., ~y~r~. Membrane bound enkephalinase from neutroph1ls cleaves the chemotactic pep~ide fMet-Leu-Phe. (Id.) Neutrophil degranula~ion ~nd chemota~s requ~re cles~ge of chemotactic peptides (S~i~h, R. et ed. ProcF Fed. A~. Soc. Exp. Biol. 44, 576 [1985]) a~d Aswanikum~r, S. et 1., Proc. ~atl. Acad. Sci. SUSA) ~, 2439-2442 [l976}). Thus, it ha~ bèen sug~ested that neutrophil membrane bound enkephalin~se may be ~ssoci~ted with the c~emot~ct~c 8ignal by elea~ing fMet-Leu-Phe in the ~mmediate v~cinity Df the neutrophil receptor. ~his degr~datl~n wo~tld control the local concentration of the chemotactlc peptide.

~n ~ssay was used tG te8t the ~ffects ~f ~nkephalinase on ~eutrophil chenotaxis.A commercially available chemotactic assay (Chemotaxis Kit, Neuroprobe, Cabin John, ~) was.used and modified as described below.
Neutrophils.were isolated.by sedimentation over dextran from peripheral-~blood:of human~donoxs. A sample of neutrophils is placed over a 5 m filter in a chemotaxis chamber containing aliquots of test material. Three to six replicates lC8x396.~hg , ' `
~` : " ' .~, `

:: . - :-: .

: :. . ~ , .~.: . . . : ..

. ~ -45-` `` 132216~
were run for each ~est for 1 hr. at 37~C. The number of migrating neutrophils ln ~ach ch~mber i8 then ~Qunted. Ihe chem~taet~c pGtential i8 ~valuated by the number o~ cell6 ln fl~e selec~ed unit areas. A c~mmer~lally ~vail~ble che~ot~xis kit, ~europrobe, Cabin John, Md. was used. Yarious 6peclfic lnhibit~rs of enkephalinase were used to deter~ne the role of enkephalinase in ~he~o~axi~ of neucr~phils. Chemot~ctlc ~cti~ity is reported 2S
the total number of neutrophils observed in five fields of the kit ~embrane under lOOX ~agnificatlQn. Thus, the lArger the number the more chemot~ctic was a p~rti ular test composit~on.

.
(~ control) Formyl Met-Leu-Phe l~H 100 For~yl Het-L~u-Phe ~ Thiorphan lO~M 29 + 19 (n 5) F~r~yl ~et-Leu-Phe ~ Phosphorsmidon lO~M 65 ~n - 1) T~us, neutrophil enkephalinase ~odulates chemotactic activ~ty.

E~
Enkephalinase Cleava~o~ ~o~besin Bombesin ~nd bombesin-like peptides have been ~hown to .. functioD as growth factors for airway epithelial eells (~illey et) and ~n human oe~ cell lung c~ncer (SCLC) (Cuttitta ~ ~1.. N~ture ~ 823-826 11~851). It was ~h~w~ that a monocl~nal antibody to b~mbesin inhiblted ~he in ~ ~rowth of SCLC cells ln ~ice. Id.

Bombesin (10-4M) and (Leu5)enkeph~1in (10-4M) ~ere ~dded ~o pur~fied r~t kidney enkephalinase (50 n~ in 150 ul 50 ~M, pH7.4 ~PES buffer containing 0.02% Triton X-100?. Af~er 30 minutes ~t *Trade-mark ~x396.~hg ~ Y~

L3~2~6~
37-C, 50 ~1 2N HCl wa~ ~ddet and the incubation ~ediu~ was - ~n~ly~ed by HPLC (C18 ~Bond~pak column, 30 minute line~r gradient from 0 to 75~ Acetonitrite in 0.1~ trifluGroacetic ~cid). ~hile 15% of the (Leu5) enkephalin ~as found degraded, ~s ~uc~ ~6 804 of ehe bombesin was hydrolyzed. Bombesin ~ppears to be ~ good substrate for enkephalinase. ~dminlstration of therapeutically effective ~mounts of enkephalinase ~y retard the growth of a tumor reql~ir~ng bombesin fos cellul~r proliferation.

E~l~l~
In Vivo ~tudies of th~_~f~ects of Enke~ha~inas~

This ~tu~y demonstrates that ~ubst~nce P in~ection ~ncrease~ ~h~ extravasatlon of Evans Blue dye. Pretreatment of animal with enkephalinase retuced those effects.

Male Long E~ans rats wer used for this study. Each rat ~250 gm) was anestheti~ed by ~n~ectin~ ~odium ~ethylhe~abarbital (75 mg/kg, lp~, ~nd placed ~n a 6upine positlon. Venou cutdowns in the ~ugular or femorsl veins ~re ~ade for in~ravascular in~ections.

Extravasation of Evans Blue dye ~as measured to study c~anges ln Y~scular permeability. After intra~ascular in~ectlon, the dye mixes rapidly in the ~ascular system where it binds to ~rum ~l~umin, thereby creaeing a high molecular w~i~ht dye-protein complex. The dye-protein complex remains in the vascul~r syste~ unless blood vessel per~eabil~ty was increased.
Permeability changes were ~onitored by in~ecting Evans Blue dye ~o~u~$~n ~.250 ~1 of ~ 30 mg~ml solution ln s~line) into the ~enous circulation. A~ter 1 m{nute, ~ubstance P ~1 ~g/kg~ in 6~11ne was in~eceed~ Five minutes later, the ~ni~al w~s perfused with ~ fixAtiqe solution consisting of 0.05 M citrA~e buffer (pH
*Trade-mark LC8x396.~hg - - :

~: - ~ . . . :
- . ~

: ~ : :. :

13221~
3.5) containing 13 paraform21dehyde for 2 minutes. The ~cidic 1xative prevents the Evans Blue from dif~uslng out of the tlssues. After fixation, the ~in of the feet ~nd no~e was dissected free from the underlying connectiYe tisgue, ~eighed, and placed in 2.0 ml of formamide (50C, 24 hours) to ~xtrsct the dye.
Ihe ~mount of dye extracted fr~m each tlssue was ~etermined by ~easuring the absorbance at 620 nm ~nd ~omparing the results with standard curve for known concentra~ions vf dye. Th~ Evans Blue ~ontent is exprsssed ~s ng dye/gm ~issue wet weight.
Preliminary experi~ent~ ~ere desi~ned to dete D in~ the dose of substance P that c~used r~producible, ~odest resp~nses.
Experi~ents ~howed that 1 ~g/k~ ~as a ~atisfa~ory does.
Subsequent ~xperiments were designed to de~ermlne ~hether enkephalinase inhibits respon~es to aubstance P.

Enkephal~n~se ~s purified from rat kidneys using published ~ethods (~alfroy and Schwartz, J. Biol. Chem. ~1984]) ~nd was concentr~ted to 1 ~g~ml buff~r for use. The buffer consis~ed ~f 5 ~N HEPES (pH 7.4) containing 0.1~ Trito~ X-100 Pnd 500 m~ ~ethyl-~-D-glucopyra~os~de. The effect of enkeph~linase on the responses to s~bstance P ~aæ ~tudled by ~n~ection of the en~yme (100 ~g i.~.) per ~nlmAl 15 ~inutes pri~r ~o inJection eubstance P.
Results of these experiments are shown in Table 4.
These studies demonstrate that ~ubstance P increased v~scular permeability in the ~kin of the n~se and paws. Studies showed thst 1.0 y~kg of 6ubstance P (intravenous) increased the permeabillty ~o Ev~ns Blue dye, And that ~nkephalinase (100 ~g~
inh~bited the Y~Qcular resp~nses in the fikin in throe of four experiments to subst~nce P.

LC8x396.~h~

-: :

~ -48-1322~

~ABLE 4 Effect Qf Enkephalinase Qn V~scular Per~ea~LLiy , S Experiment Control Enkephalinase ~ Control nu~ber ~ se Experiment #R-4 18.3 + 1.12* 12.1 66.2 Experi~ent #R-6 58.4 30.0 51.3 Experi~ent ~R-4 9.4 + 2.7 11.1 118 Experiment #R-6 11.9 3.6 29.9 * Evans Blue Content in ng/g~ vet weight E~ ~
In Vivo Effects of Enke~h~linase on A~r~low Resistance The effect~ of eDk0phalinase on alrway 6mooth muscle y$~Q, i~ studied using pr~v~ou~ly publi~hed ~ethods (Holtz~an e~
4~ ~1. Am. Re~. Respirat. Disea6e l~I: 686 [1983~ lrflo~
re~i~tance i6 ~ea~ured in ~nes~hetlzed ~ni~als (sodium psntobarbital, 30 ~g/kg, lp, ~r chlor~lose, 40 to 60 mg/kg, iv).
An endotr~che~l t~be is ~serS~d ~nto the upp~s tr~ch~a and the ~n$~al is vent~lsted ~ccording to lt~ ~ize using ~ const~nt volu~e ventilator. L~rge ~nimals ~uch ~s dogs require tid~l ~olumes of 10 ~l/kg a~ a frequency of 30 breath~ per minute. Esophageal pres~ure ~s measured using ~ balloon ~theeer ~nserted into ~he middle of ~he ~sopha~us. The tr~nspulmonary pr~ssure ~8 the difference in pressures be~ween the ondotr~cheal Sube ~nd the esophageal c~theter. Airflow r~tes ~re ~easured ~ith ~ ~ensitive pneu~otachograph, ~nd the ~irflow resistance i8 c~lcul~ted by ~ethod of electr~cal ~ubtraction.

L~8x396.~hg :. : , . , , :
~: : , ~ , ~ -49-~322~
Tachykinins such ~s ~ubstance P ere delivered by ~erosol, intratracheal ~nstilla~ion, or intr~venously. A dose of mediator pept~de thse inor~ase~ nirflow r~si6t~nce by ~pproximately t~o times i8 used for ~ubsequent studi~6 0f ~lrway responsiveneas. A oomparlson of the respon6e to the peptide ~gonist in the absence and presence 9f different do6es of ~nkeph~lins~e is made.

E~MPLE 15 Me~surement of Inflammato~y_5~ esponsesl n VIvo Neutrophll ch~mot~xls, pres~nce of ~sinophils And other lnfla~matory cells, ~nd ~st cell degran~l~t~on 1~ Y~Q. is ~eas~red in biopsy spec~m~ns from ~rway ~nd other tissues.
~issues ~r~ t~ken fro~ con~rol ~ni~ls, ~ni~als trea~ed wlth ~ubstance P or other ~ndogenous pep~ldes mediator6, ~nd from ~nl~als pretre~ted with ~nk~phslinase before ~r ~fter exposing Ani~als t~ endogenous peptide~. Speci~ens are ~$xed in 10%
buffer~d formalin, i~bedded in p~raff~n, ~nd 3 ~ceions 4 E~ thick ~re obtained from each t~sue. Section~ are st~ined ~ith hematoxylin-eosin ~ollowed by ~aph~hol ~S-D chloroacetate osterase. The number of neutrophils or other cells are de~er~ined for ~ach ~ec~ion fr~m each biopsy ~pecimen t~ ~ssess airway infl~m~ation. Cell ¢ounts are ~ade ~t 630~. The volume of opitheliu~ or other t~sue ~s determined ~slng a digiti~er (~odel 614B; ~alos Inc.) to obtain the ax~a ~nd the thickness of the section (4 ~), and data are expressed ~s the number of cells per volume of t~ssue.

LC8x396.~h~

, ' ' ~ ! `` ` ~

-so-1322~
~XAMPLE 16 Effects of Enkephalinass on ~ b~g~l The contraction of the lower ~oph~geal ephincter in anesthetized ~ni~ls is ~easured using ~odific~tions vf the ~thods publl~hed pre~lvusly (Reynolds et ~1-. Am. J. Physiol.
[Gastrointestinsl Physiology] 246: 346 11984]). A catheter syste~
consisting of two tubes, one for recording pressure ~nd ~nfusion of fluid or drugs, ~nd another for recovery of the fluid in the esophagu6 ls ~nserted into the ~opha~s, ~nd ~ater ~s pu~ped 5flo~ r~t~: approxi~ately 0.75 ml/~ln) ehrough the catheter. The ~luid ls contin~ously withdra~n from the es~phagus ~a aq ~o ~eep the ~olu~ of fluid ln the esophagus eons~ant, snd the ~sophagus patent. By fixing the l~wer ond of the e~ophagus to pre~ent shortenlng of the loneitudinal ~uscles only circular ~uscular contractions are recorded. The pressure rec~rding c~theter is a~t~ched to a transducer (e.g., Sta~h~ ~odel P23) snd pressure is recorded on a ch~rt recorder.
~XAMPLE 11 Effect of ~nke~hallna~s_5n~2 p~ertenslon ln ~ s Ele~ated arterl~} pressure or hypertensio~ ~ y be caused by renal dlsease. One type of ~enal hypertensi~n is due to activation of the ren1n-angiotensin syste~. Renin, a proeeolytic enzyme prcduced by the kidney, converts angiotensinogen to the decapeptide, angiotensin I, ~hich ln turn is converted to angiotensin II. ~ngiotensin II i6 R potent pressor compound and ~erts this pressor effect directly on arteriolar ~ooth ~uscle.
Enkephalinase clesves angioten~in I, ~hus pre~en~ing its eonversion to ~ng~otensin II and ~hus ~s likely to be ~n efective therspeut~c ~n the treat~nt of renal hypertens~on.

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The effects of enkephalinase ~n renal hypertension is stud~ed using the foll~ing pr~cedure; one renal artery is ~arrowed pr~ducing high concentra~ions of renin followed by hi~h levels of ~ngiotensln man~fested in systemic hypertension.

Systemic blood pressure i6 ~onitored by fi~rglcally placing ~ catheter in a femoral artery ~nd bringing the catheter out of the body via the back. ~hi~ allows the ~ccurate monitoring of blood pressure in unanesthetized And unrestr~ined anim~ls. The effects o~ ~nkephalin~se and enkephalinase lnhibitors on the arterial blood pressure, the blood levels of renin, angiotensin I
snd anglotensin II i8 ~nitored.

~ LE_ ffect Qf Enke~_linase on Substnnce_~

~ubstanoe P-like biological ~cti~ity or'~unoreac~ivi~y h~s besn found in Yari~uS parts of the eye, ~ncluding the cornea ~nd li~bus, irls, cili~ry body, ~nd ohoroid. (Ston~, R.A. ~nd Ku~yama, Y., Arch. Ophthal~ol. ~03:1207 ~1985]). Substance P-l~e acti~ity has been observ~d in the retina of vario~s anim~ls lncluding the chick~n, pigeon, rat, ~uinea pig, rabbit, dog, cow and ~ankey (e.g. St~ernsohant~, J. t 1. J. Neuroohemistry ~B:1323-1328 11982J; Unger, W.G. ~ 1., Exp. Eye Res. 12:367-377 [lg~l])-The func~lon o~ ~ubstance P in the eye is not est~bllshed. It h~s been euggested that substance P may ~ediate ~ntidromic vasodilation ~nd neurugenic pl~sma ex~r~s~ion ~s part of the inflam~atory response to tr~uma in the ~ye (Hol~dahl, G. ~ 1. Science ~1~:1029 [1981]).

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The role of substance P in the retina, iridi~l ~mooth Quscle~, ocular eirculation ~nd the blood-~queou6 bsrrier, ~ntrsocul~r pressure, for~ation ~nd Dutflow of aqueous humor h~s b~en reviewed. (St~ernschantz, J. in EharmacologY_Q~ ~e Eve ed.
Sears, M.L. [Springer-Verlag, 1979~). Substsnce P has been shown to have marked effects on the sphincter muscle of the iris (Nandahl, A. ~nd Blll, A. Act~. Physiol. Sc~nd. lQ2:26 11980]), It has also been shown th~t the contr~otile respons¢ to nerve stimul~tion of the rnbbit iris sphin~er could be antagoni~ed by ~
0 8~bS~anCe P 8n~10g. 5hese results sugges~ that ~ubstance P or a closely related peptlde ls ~pecifically ~n~olved in this response.
~Leander, J. Acta Phys~ol. Scand. ~ lR5-193 [1981]). In ~i~Q
pupillary block ~nd increased lntra-ophthalmio pressure ~coo~panying intense ~nd ~ustained mi~sis has b~en ~bssrved following ~cute in~ury. (Al-Chadyan, ~. $~ ~1............. Invest.
Ophthal~ol. Vis. Sci. 18:361-365 11979]; St~ernschantz, J. et ~
In~es~. Ophthalmol. Vis. Scl. ~0:53-60 [1981]). Ihus ~ubstance P
~ay be involved in the sustained ~nd intense ~io~ls observed during catar~ct ~urgery or in respons2 to acute ~h~ury to the eye.
In~ectlon of high doses (2 1 ~g) of substance P into ~he ~nterior oha~ber of the eye induoed an increase ln intraophthalmic pressure. (Mandahl, A. ~nd Bill~ ~., Acta Ph~siol. Scand. 112:331-338 [1981~; Stjernschant?, J. e~ ~1., Invest. Ophthamol. Vis. Sci.
~0:53-60 [1~81]). L~w doses (< 10 ng) induce ~ioses but no ~ppreci~ble $ncrease ~n ~ntr~ophthalmic pressure. (Nishiy~na, A.
et al., Jpn. J. Ophthal. ~5:362-369 l1981~).

S~n~e enkephalinase cleaves ~ubstance P, ~dmlnistration of enkephalin~se ei~her prior to, d~ring ~r i~media~ely following eatarsot ~urgery is likely to prevent the int~nse ~io~is ~nd lncreased ophthalmic pressure. The effects ~f ~nkephalinase on the inten e ~osis ~nd increased ophth~lmic pre~re ~ay be ~tudled using various opthalm~c ~odels in rabbits ~uch as the LC~396.mhg : .. : : :; : :

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aphakic rabbit nodel ~or ~tudylng ~he effects o~ topic~lly ~pplied dru~s ~Nirate, D.J. ç~ l., Curr.Eye Res. 1[8~:491-493 ll9Bl] and Anderson, J.A. et Ql., Arch. Opth~lmol. lQQ[4]:642-645 [1982]).
Contraction of the pupil snd of the iris sphincter ~Nscle ~s well S as intraocular pressure and drug ~bsorption to sreas of the eye is ~onitored during surgery with and without administration of enkephalinase.

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Claims (66)

1. A pharmaceutical preparation comprising an enkephalinase or a functional derivative thereof and a pharmaceutically acceptable carrier.

2. The preparation of claim 1 wherein the enkephalinase derivative is a cytoplasmic domain-deleted enkephalinase.

3. The preparation of claim 2 wherein the carrier is a salve for epidermal application.

4. The preparation of claim 1 wherein the enkephalinase derivative is a transmembrane domain-deleted enkephalinase.

5. Enkephalinase and functional derivatives thereof for pharmaceutical use in treating peptide-induced disorders.

6. The use of an enkephalinase or a functional derivative thereof in the preparation of a medicament for the treatment of disorders associated with endogenous peptides susceptible to cleavage by enkephalinase.

7. The use of claim 6 wherein the endogenous peptides are tachykinins.

8. The use of claim 7 wherein the endogenous peptide is substance P.

9. The use of claim 7 wherein the endogenous peptide is neurokinin A.

10. The use of claim 7 wherein the endogenous peptide is neurokinin B.

7299/sza

11. The use of claim 7 wherein the endogenous peptide is physalaemin.

12. The use of claim 7 wherein the endogenous peptide is kassinin.

13. The use of claim 7 wherein the endogenous peptide is eledoisin.

14. The use of claim 6 wherein the endogenous peptide is a chemotactic peptide.

15. The use of claim 6 wherein the endogenous peptide is angiotensin I.

16. The use of claim 6 wherein the endogenous peptide is bombesin.

17. The use of claim 6 wherein the endogenous peptides are kinins.

18. The use of claim 17 wherein the endogenous peptide is bradykinin.

19. The use of claim 17 wherein the endogenous peptide is kallidin.

20. The use of claim 6 wherein the pathological disorder is inflammation.

21. The use of claim 6 wherein the pathological disorder is allergic dermatitis.

22. The use of claim 7 wherein the pathological disorder is allergic dermatitis.

7299/sza

23. The use of claim 8 wherein the pathological disorder is allergic dermatitis.

24. The use of claim 9 wherein the pathological disorder is allergic dermatitis.

25. The use of claim 10 wherein the pathological disorder is allergic dermatitis.

26. The use of claim 6 wherein the pathological disorder is an airway disease.

27. The use of claim 26 wherein the airway disease is asthma.

28. The use of claim 6 wherein the pathological disorder is a cough associated with tachykinins and/or kinins.

29. The use of claim 6 wherein the pathological disorder is a tumor.

30. The use of claim 29 wherein the pathological disorder is associated with carcinoid tumor.

31. The use of claim 29 wherein the tumor is small cell lung cancer.

32. The use of claim 6 wherein the pathological disorder is renal hypertension.

33. The use of any one of claims 6 to 32 wherein the enkephalinase lacks the cytoplasmic and transmembrane domain.

34. The use of any one of claims 6 to 32 wherein the enkephalinase lacks the cytoplasmic domain.

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35. The use of any one of claims 6 to 32 wherein the enkephalinase lacks the transmembrane domain.

36. The use of any one of claims 6 to 32 wherein the enkephalinase is unaccompanied by associated native glycosylation.

37. The use of claim 33 wherein the enkephalinase is unaccompanied by associated native glycosylation.

38. The use of claim 34 wherein the enkephalinase is unaccompanied by associated native glycosylation.

39. The use of claim 35 wherein the enkephalinase is unaccompanied by associated native glycosylation.

40. The use of any one of claims 6 to 32 or 37 to 39 wherein the medicament is for intramuscular administration.

41. The use of claim 33 wherein the medicament is for intramuscular administration.

42. The use of claim 34 wherein the medicament is for intramuscular administration.

43. The use of claim 35 wherein the medicament is for intramuscular administration.

44. The use of claim 36 wherein the medicament is for intramuscular administration.

45. The use of any one of claims 6 to 32 or 37 to 39 wherein the medicament is for intravenous administration.

46. The use of claim 33 wherein the medicament is for intravenous administration.

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47. The use of claim 34 wherein the medicament is for intravenous administration.

48. The use of claim 35 wherein the medicament is for intravenous administration.

49. The use of claim 36 wherein the medicament is for intravenous administration.

50. The use of any one of claims 6 to 32 or 37 to 39 wherein the medicament is for administration by intrapulmonary inhalation.

51. The use of claim 33 wherein the medicament is for administration by intrapulmonary inhalation.

52. The use of claim 34 wherein the medicament is for administration by intrapulmonary inhalation.

53. The use of claim 35 wherein the medicament is for administration by intrapulmonary inhalation.

54. The use of claim 36 wherein the medicament is for administration by intrapulmonary inhalation.

55. The use of any one of claims 6 to 32 or 37 to 39 wherein the medicament is for topical administration.

56. The use of claim 33 wherein the medicament is for topical administration.

57. The use of claim 34 wherein the medicament is for topical administration.

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58. The use of claim 35 wherein the medicament is for topical administration.

59. The use of claim 36 wherein the medicament is for topical administration.

60. The use of any one of claims 6 to 32 or 37 to 39 wherein the medicament is for administration of enkephalinase in a therapeutically effective dose of from about 0.001 mg/kg to about 25 mg/kg.

61. The use of claim 33 wherein the medicament is for administration of enkephalinase in a therapeutically effective dose of from about 0.001 mg/kg to about 25 mg/kg.

62. The use of claim 34 wherein the medicament is for administration of enkephalinase in a therapeutically effective dose of from about 0.001 mg/kg to about 25 mg/kg.

63. The use of claim 35 wherein the medicament is for administration of enkephalinase in a therapeutically effective dose of from about 0.001 mg/kg to about 25 mg/kg.

64. The use of claim 36 wherein the medicament is for administration of enkephalinase in a therapeutically effective dose of from about 0.001 mg/kg to about 25 mg/kg.

65. The use of claim 55 wherein the medicament is for administration of enkephalinase in a therapeutically effective dose of from about 0.01 mg/kg to about 20 mg/kg.

66. The use of any one of claims 56 to 59 wherein the medicament is for administration of enkephalinase in a therapeutically effective dose of from about 0.01 mg/kg to about 20 mg/kg.

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