- Email: [email protected]
The effect of antihistaminics on plasmin activity
THE
EFFECT
OF ANTIHISTAIKINICS
RNA RIPSTEIN SOICHER,
M.D.,
D.
MONTREAL,
PATRICIA QUEBEC,
ON PLASMIN SMITH,
ALSc.,
AND
ACTIVITY” BRAM
ROSE, M.D.,
CANADA
A
CTIVATION of blood proteolytic enzyme has been considered to bc of major importance in the mechanism of histamine release in the living organism.4, 17,1% This theory is based on a rat,her large and diversified body of expcrimental evidence. Peptone, which causes the discharge of histamine from cells both in vitr021, 22,24 and in vivo,14s lQl2o also brings about the release of an active proteolytic enzyme.23 Soybean trypsin inhibitor inactivates plasmin, and also inhibits the release of histamine by ‘peptone from rabbit cells. If it is injected into animals before the administration of peptone there is a decrease in the amount of plasmin liberated, and the animals survive for a significantly longer time.20 Blood proteolytic enzyme may also be activated by the addition of specific antigen to sensitized serum.24 Anaphylactic shock is associated with free proteolytic enzyme in the blood stream3 along with a decreased antitryptic power of the serum.2j 3 In those animals which are “antianaphylactic” the antiproteolytic activity is higher than in those which succumb.z The addition of trypsin to the perfusing fluid of guinea pig lung has been shown to result in the release of histamine into the perfusate, while intravenous trypsin leads to an increase in t,he blood histamine level two to five minutes following injection.16 This is accompanied by a fall in blood pressure and a fall in the histamine content of the liver. Parenteral injection of either trypsin or plasmin results in a shocklike syndrome not unlike that seen after intravenous histamine.:2 I3 However, pretreatment of animals with Benadryl did not prevent the fall in blood pressure seen after plasmin. Furthermore, the increased capillary permeability induced by trypsin is also not altered by treatment with Benadryl or Neoantergan8 With the exception of the two last-mentioned observations a close association, if not a causal relationship, is suggested between the activation of plasmin and the release of histamine. It has been postulated that at least part of the effect of the antihistaminic compounds may bc due to an inhibitory action on blood proteolytic enzyme.‘; Some of the antihistamines inhibit certain other enzymes, although there does not seem to be any relationship between antienzyme and antihistaminic potency. Pyribenzamine and Antistine have been shown to inhibit hyaluronidase”, I1 while Antistine and other imidazoline derivatives inhibit cholinesterase, diamine oxidase, polyphenol oxidase, and adrenaline oxidase.‘? Benadryl, in common From the McGill University Clinic, Royal Victoria Hospital. Keceived for publication Nov. 17, 1962. *Aided by a grant from the Canadian National Research Council. tion wits supported by a grant from the Ciba Co., Montreal, through D. L. Menzies.
106
Part the
of the investigacourtesy of Mr.
SOICHER
ET
AL.
ANTIHISTAMINICS
:
AND
PLASMIN
ACTIVITY
107
with other compounds with a diphenyl nucleus, and some sulfonamides and Dibenaminc have a significant. inhibiting effect on the activity of cytochrome oxidase.’ The present paper is concerned with experiments designed to study the effect in vitro of various antihistamines on an active preparation of bovine plasmin (Loomis’ fibrinolysin) .w These effects were compared to those of two known active plasmin inhibitors, namely Loomis’ antifibrinolysin (beef antiplasmin) * and pancreatic trypsin inhibit,or.t6
STANDARD TIME-CONCENTRATION OBTAINED USING LOOMIS’
LYSlS 00
TIME
OF
STANDARD
2’00
FIBRIN
500 Fig.
CURVE ‘FIBRINOLYSIN’
CLOT
(SEC)
1000
1.
No significant inhibition of plasmin activity by any of the antihistaminic compounds studied was noted, except at extremely high concentrations. In fact, in two instances, a very slight enhancing effect on fibrinolysis was noted at low concentration levels. Experimental.-Plasmin was assayed by means of the estimation of the lysis time of a standard fibrin clot. This is carried out in the following manner : Mich.
*Supplied
through
the
courtesy
of
Dr.
E.
C. I~oomis,
Wupplied
through
the
courtesy
of
Dr.
L.
A.
Kazal,
Parke, Sharpe
Davis & Dohme,
&
Company, Inc.
Detroit,
108
THE
.JGURNAL
OF
ALLERGY
Matmeria~ls.Borate buffer pH 7.7.’ Fibrinogen-Armour Bovine Plasma Protein Fr. I 60.55 per cent, clottabh~. Five-tenths per cent solution in borate buffer. Thrombin-Parke Davis Topical Thrombin IO units/ml. in borate buffer. Plasmin-Loomis’ fibrinolysin dissolved in borate buffer. All assays carried out bet,ween one-half and two hours aft)er solut,ion of the enzyme. Five-tenths ml. of 0.5 per cent fibrinogen are placed in a small culture tube with a flanged top. To this is added 1. ml. of enzyme solution as a st,op watch is started. Thromhin solution, 0.2 ml., is then added from a burette and the test tube gently shaken until the stop watch shows that thirty seconds have elapsed since the addition of the enzyme. The tube is t,hen suspended on the rack of a t.ransparent constant temperature water bath kept at 37O C. and the contents observed for clot lysis. Thr end point, is ta.ken when five bubbles trapped in t,hc clot have risen t,o the meniscils. Ten to twelve determinations can bc carrictl 0111cwncurrent1.v. I,oomis unit system has been adhered t,o tZhroughont, although his Assam twhniqiic is somewhat, different. Fig. 1 shows the standard tiIne-(.oncerltratic,n
) COMPOUND
CONCEN‘I-RATION (ohr. %)
( - 1
PLASMIN CONCENTRATION
LYSIS TIME OF CT,OT
1
RESIDUAL PLASMIN CONC.
PER C!F,NT CHANGE __-
0.0123
1.82 U/ml.
0.025
1.82
0.0125 0.025
1.82
0.0125 0.025 0.25 0.5
1.82
286.5
1.x
+4.04 -8.4 -20.3
Mnltergml
0.0 12.7 0 . O”5 2. 0.25
1.8’ 1.82 1.7
216.0 212.5 L’T4.0
2.02 2.0:: I .4::
+11.16 +11.2 -16.47
Antistine TIydrochlori~le
0.1 I).2
1 .G
“X.5 259.0
1.6 1.5;
Auktine
0.1 0.2
1 .(i5 I.6
1.61 I .%I
0.05 0.25 0.3
I .(iS
1.67
1 .(i5 1.fi5
l.ti4 I.52
I’yrrolxzote
Hulfilte
l’yribenzaminc Hydrochloride Yyribenzamine (Strate Antifibrinolysin
Pancreatic Inhibitor
Trypsin
1.82
247.3 SW. 231.2.0
1 .fi8 IJ/ml.
222.0 223.0
1.96 1.05
+7.7
1.7 1.91
4.6
1.82
1.7 1.7
l.ti.5
1.55
0.05
1 .G
0.25 0.5
1.65
248.3 “44.5
1 .G
248.0
0.0125 0.025 0.125 n:J5d
1.R”
299.0 270.5 fi82.0
0.0125 0 . cl“5 0.0fi3.5 0. 125
1X 1.7
1.7 1x 1.8” 1.71.;
1.85
1,45(i.O 248.5 2 0 3 .5 420 0 liso:o
1.67 1.71
-7.7 +1.64 +7.15
-Xl 5 4.85 -.2..$0 -6.66 ~I.01 -O.fi i.RS
t(1.01 I, .I> +.,.I,., cl .13:! 4o.s -50.5 -‘il).ij -9 1.s 4.23 l;j.Z!) -5ti.4 -so. 1
SOICHER
ET
AL.
:
ANTIHISTAMINICS
ASD
PLASMIN
ACTIVITY
109
curve relating the plasmin concentration to the lysis time of the standard fibrin clot. All substances whose antiplasmin activity were studied were also dissolved in borate buffer. Certain antihistaminic compounds such as Thephorin could not be included because of insolubility in t,he buffer solution. The higher concentrations of certain other antihistamines were omitted for the same reason. Equal volumes of ant,ihistaminic or antiproteolytic sub&ances and enzyme were incubated together and concentrations are expressed as those present in the resulting mixture. Incubation was carried out for one hour at 25’ C., and tbc solution assayed for residual proteolytic activity. The control was treated in Ihe same way. The results are expressed as percentage change from the value of the cont,rol, and are shown in Table I. The minus sign denotes inhibition while the plus sign denotes increase in act,ivity over that of the control. Percentage changes less than 10 per cent cannot be considered of significance since one rnust allow fol a variation of fifteen seconds on duplicate readings of the lysis time of the standard fibrin clot. A series of experiments such as just described requires the use of several controls, since there may be sorne spontaneous falling off of enzyme activity on incubation. This explains the fact that there is slight variation in the control enzyme value, since a new reading was made for each group of estimations. The values, however, do not differ more than 70 per cent. In t,he case of plasmin inhibition with serum dilutions one can vary the enzyme concentrat,ion fairl? widely with no significant change in the percentage inhibition in a given periocl of time. It is felt, therefore, that the slight variations in the control levels do not invalidate the results obtained. Results.-The only antihistaminics which caused more than a 10 per cent inhibition of the control enzyme activity were Neoantergan at a concentration of 0.5 Gm. per cent and multergan* at 0.25 Gm. per cent. Both these concentrations are grossly in excess of the levels which might be expected in blood or tissues. Rlultergan at concentrations of 0.125 and 0.25 Cm. per cent showed 11.15 and 11.2 per cent increase in the activity of the control, but this change is not far outside the allowable experimental error. Antifibrinolysin and pancreatic trypsin inhibitor in the same concentrations rcsultcd in very marked inhibition of plasmin activity. Normal human serum, in a dilution of one part to twenty-five of buffer (final dilution l/50) and untler the same experimental conditions as just described, caused an inhibition of 40 to SO per cent of the control enzyme level. DISCUSSION
Although the antihistaminics produce other pharmacologic effects to varying extents, their chief activity, interest, and use center around their ability to counteract histamine. That they might play a role in preventing its liberation *Multergan is an antihistamine with marked anticholinergic with the formula [ (Phenothiazinyl -10. -1 propyl l-2) J-N-trimethyl It is a dibenzoparathiazine derivative and was cle~lo~erl from
properties. It is R.P. 3554 ammonium methyl sulfate. Phenergan (R.P. 3277).
110
THE
JOURNAL
0~
ALLERGY
as well is an interesting possibility. The results described in this paper do not preclude this entirely, since the drugs might act in such a way as to prevent the in vivo activation of plasminogen by a serum or tissue kinase, or influence the liberation of the kinase itself. However, once the plasmin has been activated. the present experiments indicate that 111~antihistamines fail to exert any direct antifibrinolytic effect, at least in vitro.
1. The literature relating the release of histamine to the activation of blood luoteolytic enzyme (plasmin) is reviewed briefly. 2. Evidence is cited concerning inhibitory effects of antihistaminic compounds on various enzyme systems. 3. Experiments are described in which eight different antihistaminic compounds in varying concentrations were incubated with a known amount of active bovine plasma proteolytic enzyme (Loomis’ fibrinolysin) and the effects compared to those of equal concentrations of antifibrinolysin (antiplasmin) and pancreatic trypsin inhibitor, and normal human serum. 4. There was no significant inhibition of plasmin in vitro by any of the antihistamines studied. Antifibrinolysin, pancreatic trypsin inhibitor, and dilutions of human serum led to marked inhibition of the enzyme. 5. The antihistaminics probably do not prevent the liberation of histamine by the action of blood plasmin. The possibility that they may influence the activation of plasminogen has not been ruled out by these experiments. courtesy
Neoantergan of Mr.
1. Abood, 2. 3. 4. 5. 6. 7. 5. 9. 10. 11. 13. 13. 14. 15.
and multergan W. L. Jeffrey.
were
supplied
by
I’oulenc
Limited,
Montreal,
through
the
L. G., and Gerard, R. W.: A New Class of Cytochrome Inhibitors, Am. J. Physiol. 167: 763, 1951. Burdon, K. L.: Changes in Antitryptic Power of the Blood Associated With Anaphylaxis in Guinea Pigs, Proc. Sot. Exper. Biol. & Med. 49: 24, 1942. Cliffton, E. E.: Variations in the Proteolytic and the Antiproteolytic Reactions of Serum. Effect of Disease, Trauma, X-ray, Anaphylactic Shock, ACTH, and Cortisone, J. Lab. & Clin. Med. 39: 105, 1952. Feldberg, W.: Histamine and Anaphylaxis, Ann. Rev. Physiol. 3: 671, 1941. Guest, M. M., Murphy, R. C., Bodnar, S. R., Ware, A. G., and Seegers, W. H.: Physiological Effects of a Plasma Protein : Blood Pressure,, Leucocyte Concentration, Smooth Muscle and Cardiac Muscle Activity, Am. J. Physiol. 150: 471, 1947. Kazal, L. A., Spicer, D. S., and Brahinsky, R. A.: Isolation of a Trypsin InhibitorAnticoagulant Protein From Pancreas, J. Am. Chem. Sot. 70: 3034, 1948. Lewis, J. H., and Ferguson, J. H.: Studies on Proteolytic Enzyme System of the Blood. I. Inhibition of Fibrinolysis, J. Clin. Investigation 29: 486, 1951. The Pharmacology of Benadryl and the Specificity of Antihistamine Drugs, Loew, E. R.: Ann. New York Acad. SC. 50: 1142, 1950. Fibrinolysin; Nomenclature, Unit, Assay, Loomis, E. C., George, C., and R,yder, A.: Preparation and Properties, Arch. Biochem. 12: 1, 1947. The Activity of Pyribenzamine and Related Compounds With Special Mayer, R. L.: Reference to Their Mode of Action, Ann. New York Acad. SC. 50: 1127, 1950. Influence of Pyribenzamine and Antistine Upon the Action Mayer, R. L., and Kull, F. C.: of Hyaluronidase, Proc. Sot. Exper. Biol. & Med. 66: 392, 1947. Antistine and Related Imidazolines, Ann. New York Acad. SC. 50: 1161, 1950. Meier, R.: Renal and Hepatic Injury in Trypsin Shock, Proc. SOC. E. D.: Mirsky, A., and Freis, Exper. Biol. & Med. 57: 278, 1944. The Coagulation of the Blood, Medicine 17: 381, 1938. Nolf, P.: Personal communication. Pellerat, A. T.:
SOICHER
ET
AL. :
ANTIHISTAMINICS
AND
PLASMIN
ACTIVITY
111
16
Ramirez de Arellano, M., Lawton, A. H., and Dragstedt, C. A.: Liberation of Histamine by Trypsin, Proc. Sot. Exper. Biol. & Med. 43: 360, 1940. 17. Rocha E Silva. M.: L’Histamine des Tissus. Mecanisme de sa Liberation. J. de phvsiol. - ” 39: 401, iQ47. 18. Rocha E Silva, M.: The Role Played by Leucocytes and Platelets in Shock, Ann. New York Acad. SC. 56: 1045, 1050. 19. Roeha E Silva, M., and Teixeira, R. M.: Role Played by Leucocytes! Platelets and Plasma Trypsin in Peptone Shock in the Dog, Proc. Soe. Exper. Biol. & Med. 61: 276.
20. 21. 22. 23. 24.
1946.
Scroggik: A.-E., Jaques, L. B., and Rocha E Silva, M.: Activation of Serum Protease in Peptone Shock, Proc. Soe. Exper. Biol. & Med. 66: 326, 1947. Ungar, G.: The Inhibition of Histamine Release by a Pituitary Adrenal Mechanism, J. Physiol. 103: 333, 1944. Ungar, G.: Release of Proteolytic Enzyme in Anaphylactic and Peptone Shock in Vitro, Lancet 1: 708, 1947. Ungar, G., and Damgaard, E.: Studies on the Fibrinolysin-Antifibrinolysin System in Serum, J. Exper. Med. 93: 89, 1951. Ungar, Cr., and Mist, S. H.: Observations on the Release of Serum Fibrinolysin by Sperific Antigen, Peptone and Certain Polysaceharides, J. Exper. Med. 90: 39, 1949.
EFFECT
OF ANTIHISTAIKINICS
RNA RIPSTEIN SOICHER,
M.D.,
D.
MONTREAL,
PATRICIA QUEBEC,
ON PLASMIN SMITH,
ALSc.,
AND
ACTIVITY” BRAM
ROSE, M.D.,
CANADA
A
CTIVATION of blood proteolytic enzyme has been considered to bc of major importance in the mechanism of histamine release in the living organism.4, 17,1% This theory is based on a rat,her large and diversified body of expcrimental evidence. Peptone, which causes the discharge of histamine from cells both in vitr021, 22,24 and in vivo,14s lQl2o also brings about the release of an active proteolytic enzyme.23 Soybean trypsin inhibitor inactivates plasmin, and also inhibits the release of histamine by ‘peptone from rabbit cells. If it is injected into animals before the administration of peptone there is a decrease in the amount of plasmin liberated, and the animals survive for a significantly longer time.20 Blood proteolytic enzyme may also be activated by the addition of specific antigen to sensitized serum.24 Anaphylactic shock is associated with free proteolytic enzyme in the blood stream3 along with a decreased antitryptic power of the serum.2j 3 In those animals which are “antianaphylactic” the antiproteolytic activity is higher than in those which succumb.z The addition of trypsin to the perfusing fluid of guinea pig lung has been shown to result in the release of histamine into the perfusate, while intravenous trypsin leads to an increase in t,he blood histamine level two to five minutes following injection.16 This is accompanied by a fall in blood pressure and a fall in the histamine content of the liver. Parenteral injection of either trypsin or plasmin results in a shocklike syndrome not unlike that seen after intravenous histamine.:2 I3 However, pretreatment of animals with Benadryl did not prevent the fall in blood pressure seen after plasmin. Furthermore, the increased capillary permeability induced by trypsin is also not altered by treatment with Benadryl or Neoantergan8 With the exception of the two last-mentioned observations a close association, if not a causal relationship, is suggested between the activation of plasmin and the release of histamine. It has been postulated that at least part of the effect of the antihistaminic compounds may bc due to an inhibitory action on blood proteolytic enzyme.‘; Some of the antihistamines inhibit certain other enzymes, although there does not seem to be any relationship between antienzyme and antihistaminic potency. Pyribenzamine and Antistine have been shown to inhibit hyaluronidase”, I1 while Antistine and other imidazoline derivatives inhibit cholinesterase, diamine oxidase, polyphenol oxidase, and adrenaline oxidase.‘? Benadryl, in common From the McGill University Clinic, Royal Victoria Hospital. Keceived for publication Nov. 17, 1962. *Aided by a grant from the Canadian National Research Council. tion wits supported by a grant from the Ciba Co., Montreal, through D. L. Menzies.
106
Part the
of the investigacourtesy of Mr.
SOICHER
ET
AL.
ANTIHISTAMINICS
:
AND
PLASMIN
ACTIVITY
107
with other compounds with a diphenyl nucleus, and some sulfonamides and Dibenaminc have a significant. inhibiting effect on the activity of cytochrome oxidase.’ The present paper is concerned with experiments designed to study the effect in vitro of various antihistamines on an active preparation of bovine plasmin (Loomis’ fibrinolysin) .w These effects were compared to those of two known active plasmin inhibitors, namely Loomis’ antifibrinolysin (beef antiplasmin) * and pancreatic trypsin inhibit,or.t6
STANDARD TIME-CONCENTRATION OBTAINED USING LOOMIS’
LYSlS 00
TIME
OF
STANDARD
2’00
FIBRIN
500 Fig.
CURVE ‘FIBRINOLYSIN’
CLOT
(SEC)
1000
1.
No significant inhibition of plasmin activity by any of the antihistaminic compounds studied was noted, except at extremely high concentrations. In fact, in two instances, a very slight enhancing effect on fibrinolysis was noted at low concentration levels. Experimental.-Plasmin was assayed by means of the estimation of the lysis time of a standard fibrin clot. This is carried out in the following manner : Mich.
*Supplied
through
the
courtesy
of
Dr.
E.
C. I~oomis,
Wupplied
through
the
courtesy
of
Dr.
L.
A.
Kazal,
Parke, Sharpe
Davis & Dohme,
&
Company, Inc.
Detroit,
108
THE
.JGURNAL
OF
ALLERGY
Matmeria~ls.Borate buffer pH 7.7.’ Fibrinogen-Armour Bovine Plasma Protein Fr. I 60.55 per cent, clottabh~. Five-tenths per cent solution in borate buffer. Thrombin-Parke Davis Topical Thrombin IO units/ml. in borate buffer. Plasmin-Loomis’ fibrinolysin dissolved in borate buffer. All assays carried out bet,ween one-half and two hours aft)er solut,ion of the enzyme. Five-tenths ml. of 0.5 per cent fibrinogen are placed in a small culture tube with a flanged top. To this is added 1. ml. of enzyme solution as a st,op watch is started. Thromhin solution, 0.2 ml., is then added from a burette and the test tube gently shaken until the stop watch shows that thirty seconds have elapsed since the addition of the enzyme. The tube is t,hen suspended on the rack of a t.ransparent constant temperature water bath kept at 37O C. and the contents observed for clot lysis. Thr end point, is ta.ken when five bubbles trapped in t,hc clot have risen t,o the meniscils. Ten to twelve determinations can bc carrictl 0111cwncurrent1.v. I,oomis unit system has been adhered t,o tZhroughont, although his Assam twhniqiic is somewhat, different. Fig. 1 shows the standard tiIne-(.oncerltratic,n
) COMPOUND
CONCEN‘I-RATION (ohr. %)
( - 1
PLASMIN CONCENTRATION
LYSIS TIME OF CT,OT
1
RESIDUAL PLASMIN CONC.
PER C!F,NT CHANGE __-
0.0123
1.82 U/ml.
0.025
1.82
0.0125 0.025
1.82
0.0125 0.025 0.25 0.5
1.82
286.5
1.x
+4.04 -8.4 -20.3
Mnltergml
0.0 12.7 0 . O”5 2. 0.25
1.8’ 1.82 1.7
216.0 212.5 L’T4.0
2.02 2.0:: I .4::
+11.16 +11.2 -16.47
Antistine TIydrochlori~le
0.1 I).2
1 .G
“X.5 259.0
1.6 1.5;
Auktine
0.1 0.2
1 .(i5 I.6
1.61 I .%I
0.05 0.25 0.3
I .(iS
1.67
1 .(i5 1.fi5
l.ti4 I.52
I’yrrolxzote
Hulfilte
l’yribenzaminc Hydrochloride Yyribenzamine (Strate Antifibrinolysin
Pancreatic Inhibitor
Trypsin
1.82
247.3 SW. 231.2.0
1 .fi8 IJ/ml.
222.0 223.0
1.96 1.05
+7.7
1.7 1.91
4.6
1.82
1.7 1.7
l.ti.5
1.55
0.05
1 .G
0.25 0.5
1.65
248.3 “44.5
1 .G
248.0
0.0125 0.025 0.125 n:J5d
1.R”
299.0 270.5 fi82.0
0.0125 0 . cl“5 0.0fi3.5 0. 125
1X 1.7
1.7 1x 1.8” 1.71.;
1.85
1,45(i.O 248.5 2 0 3 .5 420 0 liso:o
1.67 1.71
-7.7 +1.64 +7.15
-Xl 5 4.85 -.2..$0 -6.66 ~I.01 -O.fi i.RS
t(1.01 I, .I> +.,.I,., cl .13:! 4o.s -50.5 -‘il).ij -9 1.s 4.23 l;j.Z!) -5ti.4 -so. 1
SOICHER
ET
AL.
:
ANTIHISTAMINICS
ASD
PLASMIN
ACTIVITY
109
curve relating the plasmin concentration to the lysis time of the standard fibrin clot. All substances whose antiplasmin activity were studied were also dissolved in borate buffer. Certain antihistaminic compounds such as Thephorin could not be included because of insolubility in t,he buffer solution. The higher concentrations of certain other antihistamines were omitted for the same reason. Equal volumes of ant,ihistaminic or antiproteolytic sub&ances and enzyme were incubated together and concentrations are expressed as those present in the resulting mixture. Incubation was carried out for one hour at 25’ C., and tbc solution assayed for residual proteolytic activity. The control was treated in Ihe same way. The results are expressed as percentage change from the value of the cont,rol, and are shown in Table I. The minus sign denotes inhibition while the plus sign denotes increase in act,ivity over that of the control. Percentage changes less than 10 per cent cannot be considered of significance since one rnust allow fol a variation of fifteen seconds on duplicate readings of the lysis time of the standard fibrin clot. A series of experiments such as just described requires the use of several controls, since there may be sorne spontaneous falling off of enzyme activity on incubation. This explains the fact that there is slight variation in the control enzyme value, since a new reading was made for each group of estimations. The values, however, do not differ more than 70 per cent. In t,he case of plasmin inhibition with serum dilutions one can vary the enzyme concentrat,ion fairl? widely with no significant change in the percentage inhibition in a given periocl of time. It is felt, therefore, that the slight variations in the control levels do not invalidate the results obtained. Results.-The only antihistaminics which caused more than a 10 per cent inhibition of the control enzyme activity were Neoantergan at a concentration of 0.5 Gm. per cent and multergan* at 0.25 Gm. per cent. Both these concentrations are grossly in excess of the levels which might be expected in blood or tissues. Rlultergan at concentrations of 0.125 and 0.25 Cm. per cent showed 11.15 and 11.2 per cent increase in the activity of the control, but this change is not far outside the allowable experimental error. Antifibrinolysin and pancreatic trypsin inhibitor in the same concentrations rcsultcd in very marked inhibition of plasmin activity. Normal human serum, in a dilution of one part to twenty-five of buffer (final dilution l/50) and untler the same experimental conditions as just described, caused an inhibition of 40 to SO per cent of the control enzyme level. DISCUSSION
Although the antihistaminics produce other pharmacologic effects to varying extents, their chief activity, interest, and use center around their ability to counteract histamine. That they might play a role in preventing its liberation *Multergan is an antihistamine with marked anticholinergic with the formula [ (Phenothiazinyl -10. -1 propyl l-2) J-N-trimethyl It is a dibenzoparathiazine derivative and was cle~lo~erl from
properties. It is R.P. 3554 ammonium methyl sulfate. Phenergan (R.P. 3277).
110
THE
JOURNAL
0~
ALLERGY
as well is an interesting possibility. The results described in this paper do not preclude this entirely, since the drugs might act in such a way as to prevent the in vivo activation of plasminogen by a serum or tissue kinase, or influence the liberation of the kinase itself. However, once the plasmin has been activated. the present experiments indicate that 111~antihistamines fail to exert any direct antifibrinolytic effect, at least in vitro.
1. The literature relating the release of histamine to the activation of blood luoteolytic enzyme (plasmin) is reviewed briefly. 2. Evidence is cited concerning inhibitory effects of antihistaminic compounds on various enzyme systems. 3. Experiments are described in which eight different antihistaminic compounds in varying concentrations were incubated with a known amount of active bovine plasma proteolytic enzyme (Loomis’ fibrinolysin) and the effects compared to those of equal concentrations of antifibrinolysin (antiplasmin) and pancreatic trypsin inhibitor, and normal human serum. 4. There was no significant inhibition of plasmin in vitro by any of the antihistamines studied. Antifibrinolysin, pancreatic trypsin inhibitor, and dilutions of human serum led to marked inhibition of the enzyme. 5. The antihistaminics probably do not prevent the liberation of histamine by the action of blood plasmin. The possibility that they may influence the activation of plasminogen has not been ruled out by these experiments. courtesy
Neoantergan of Mr.
1. Abood, 2. 3. 4. 5. 6. 7. 5. 9. 10. 11. 13. 13. 14. 15.
and multergan W. L. Jeffrey.
were
supplied
by
I’oulenc
Limited,
Montreal,
through
the
L. G., and Gerard, R. W.: A New Class of Cytochrome Inhibitors, Am. J. Physiol. 167: 763, 1951. Burdon, K. L.: Changes in Antitryptic Power of the Blood Associated With Anaphylaxis in Guinea Pigs, Proc. Sot. Exper. Biol. & Med. 49: 24, 1942. Cliffton, E. E.: Variations in the Proteolytic and the Antiproteolytic Reactions of Serum. Effect of Disease, Trauma, X-ray, Anaphylactic Shock, ACTH, and Cortisone, J. Lab. & Clin. Med. 39: 105, 1952. Feldberg, W.: Histamine and Anaphylaxis, Ann. Rev. Physiol. 3: 671, 1941. Guest, M. M., Murphy, R. C., Bodnar, S. R., Ware, A. G., and Seegers, W. H.: Physiological Effects of a Plasma Protein : Blood Pressure,, Leucocyte Concentration, Smooth Muscle and Cardiac Muscle Activity, Am. J. Physiol. 150: 471, 1947. Kazal, L. A., Spicer, D. S., and Brahinsky, R. A.: Isolation of a Trypsin InhibitorAnticoagulant Protein From Pancreas, J. Am. Chem. Sot. 70: 3034, 1948. Lewis, J. H., and Ferguson, J. H.: Studies on Proteolytic Enzyme System of the Blood. I. Inhibition of Fibrinolysis, J. Clin. Investigation 29: 486, 1951. The Pharmacology of Benadryl and the Specificity of Antihistamine Drugs, Loew, E. R.: Ann. New York Acad. SC. 50: 1142, 1950. Fibrinolysin; Nomenclature, Unit, Assay, Loomis, E. C., George, C., and R,yder, A.: Preparation and Properties, Arch. Biochem. 12: 1, 1947. The Activity of Pyribenzamine and Related Compounds With Special Mayer, R. L.: Reference to Their Mode of Action, Ann. New York Acad. SC. 50: 1127, 1950. Influence of Pyribenzamine and Antistine Upon the Action Mayer, R. L., and Kull, F. C.: of Hyaluronidase, Proc. Sot. Exper. Biol. & Med. 66: 392, 1947. Antistine and Related Imidazolines, Ann. New York Acad. SC. 50: 1161, 1950. Meier, R.: Renal and Hepatic Injury in Trypsin Shock, Proc. SOC. E. D.: Mirsky, A., and Freis, Exper. Biol. & Med. 57: 278, 1944. The Coagulation of the Blood, Medicine 17: 381, 1938. Nolf, P.: Personal communication. Pellerat, A. T.:
SOICHER
ET
AL. :
ANTIHISTAMINICS
AND
PLASMIN
ACTIVITY
111
16
Ramirez de Arellano, M., Lawton, A. H., and Dragstedt, C. A.: Liberation of Histamine by Trypsin, Proc. Sot. Exper. Biol. & Med. 43: 360, 1940. 17. Rocha E Silva. M.: L’Histamine des Tissus. Mecanisme de sa Liberation. J. de phvsiol. - ” 39: 401, iQ47. 18. Rocha E Silva, M.: The Role Played by Leucocytes and Platelets in Shock, Ann. New York Acad. SC. 56: 1045, 1050. 19. Roeha E Silva, M., and Teixeira, R. M.: Role Played by Leucocytes! Platelets and Plasma Trypsin in Peptone Shock in the Dog, Proc. Soe. Exper. Biol. & Med. 61: 276.
20. 21. 22. 23. 24.
1946.
Scroggik: A.-E., Jaques, L. B., and Rocha E Silva, M.: Activation of Serum Protease in Peptone Shock, Proc. Soe. Exper. Biol. & Med. 66: 326, 1947. Ungar, G.: The Inhibition of Histamine Release by a Pituitary Adrenal Mechanism, J. Physiol. 103: 333, 1944. Ungar, G.: Release of Proteolytic Enzyme in Anaphylactic and Peptone Shock in Vitro, Lancet 1: 708, 1947. Ungar, G., and Damgaard, E.: Studies on the Fibrinolysin-Antifibrinolysin System in Serum, J. Exper. Med. 93: 89, 1951. Ungar, Cr., and Mist, S. H.: Observations on the Release of Serum Fibrinolysin by Sperific Antigen, Peptone and Certain Polysaceharides, J. Exper. Med. 90: 39, 1949.