Irving Innerfield

Bradykinin antagonism following oral protease therapy.

Summary Orally given proteolytic enzymes appear to antagonize 3 bradykinin physiologic responses: hypotension, smooth muscle contraction and enhanced capillary permeability. Whether these observations relate to anti-inflammatory activity of orally given proteolytic enzymes requires further study.

Analgesic and anti-inflammatory effects of orally given streptokinase.

According to Lewis(1), the nonapeptide bradykinin is an important mediator of the acute inflammatory reaction. It is, therefore, of interest that orally given proteases were recently shown to antagonize 3 pharmacologic actions of bradykinin: vasodilatation, vascular permeability and smooth muscle contraction(2). The objects of the present investigation were: first, to assess the action of orally given streptokinase against the fourth pharmacologic action of bradykinin, namely, pain production; and second, to appraise the anti-inflammatory action of orally given streptokinase in a controlled animal and clinical experiment. Methods. 1. Analgesia study. The cantharidin blister technic of Armstrong et al. (3) was slightly modified. This study comprised 30 apparently healthy students and technicians. A band-aid containing 2 drops of tincture cantharidin was applied to the skin of the flexor surface of the forearm the evening before the day of the experiment. When the band-aid was removed the next morning, the area showed a blister with a diameter of about 1.5 to 2 cm. Blisters remained relatively unchanged for 24–48 hours. Intact blisters were opened and the separated epidermis cut away exposing the blister base, which was bathed in Ringers solution warmed to 37°C. Before the application of bradykinin, the bathing solution was removed and the area dried with filter paper. 0.2 ml bradykinin (0.1 μg) was applied to the dry blister base and the following information recorded: 1. Length of time for pain to develop. 2. Duration of pain. 3. Intensity of pain. This factor was evaluated by each subject on a 1 to 4 + basis. Ten control subjects received placebo tablets; 10 treated subjects received tablets containing streptokinase 10,000 units; 10 treated subjects received aspirin, 5 grains. Control and experimental groups received one tablet every 15 minutes for one hour following pretreatment control determinations.

Effect of orally administered proteolytic enzymes on carbon tetrachloride induced granuloma pouch.

Summary Two host responses developed in rats 24 hours after CCI4 injections sub-cutaneously: (1) tense, ballooned-out granu-lomas; (2) dense leucocytic invasion, edema and necrosis of cutaneous muscle. These responses were inhibited, or significantly modified, in rats receiving SK-P or trypsin orally. Epsilon aminocaproic acid blocked the anti-inflammatory and granuloma-inhibiting effects of trypsin or SK-P. Papain, bromelain, chy-motrypsin and Aspergillus protease were inactive. The important implications of the data are: (1) that orally administered trypsin or SK-P is active in modifying the inflammatory response to CC14, and (2) that the action of these substances is related to their enzymatic activity.

Tissue Plasminogen in Normal and Inflamed Human Gingiva

The purpose of this investigation was to determine the level of plasminogen in normal and inflamed human gingival tissue. Plasminogen, a naturally occurring serum globulin, is the precursor of the fibrinolytic enzyme, plasmin, and has been found in gingival crevicular fluid (G. T. GUSTAFSSON and I. M. NILSSON, Proc Soc Exp Biol and Med 106:277-280, 1961). It is converted into plasmin by a variety of bacterial, tissue, and serum activators. With inflammatory insult, the basement membrane of the endothelium permits plasma proteins to enter the injured area. Under normal conditions, the clotting mechanism is activated and fibrin is deposited in the pores of the basement membrane, sealing off the injured area. In the presence of plasmin in high concentration, fibrin is dissolved, and the unsealed pores allow the spread of local edema. It has been shown, however, that plasmin, in suitably low concentration, activates prothrombin into thrombin, thereby promoting fibrin deposition and the localization and subsidence of tissue inflammation and edema (I. INNERFIELD, G. R. ROWLEY, P. ZWEIL, and J. PORPORA, Thromb Diath Haemorrh, 18:447-455, 1967). The gingival tissue obtained for study was divided into four histologic and clinical categories: Group 1, normal; Group 2, mild chronic inflammation; Group 3, moderate chronic inflammation; and Group 4, severe chronic inflammation. After excision, the gingiva was washed, a section was taken for histologic study, and the remainder of the tissue was weighed and homogenized. To determine the amount of both plasminogen and plasmin, the following method was used. A control mixture containing 0.6 ml of 0.07 M saline-0.06 M phosphate buffer pH 7.5, 0.2 ml of bovine fibrinogen,* and 0.1 ml of streptokinaset (1,000 units/0.1 ml) was clotted with 0.1 ml of bovine thrombint (1,000 NIH units/vial). The noncontrol tubes contained, in addition, 0.1 ml of gingival homogenate. To test for plasmin alone, 0.1 ml of the same

Modification of kinin activity in injured tissue by oral kinases and proteases.

The anti-inflammatory action of orally given proteases has been documented, in the main, by uncontrolled clinical observations(1). In a recent animal study, however, bradykinin blocking effects followed orally given kinases and proteases(2). In view of the current interest in bradykinin as the biochemical mediator of the inflammatory reaction(3), the present study was undertaken to appraise any relationship between the anti-inflammatory activity of orally given enzymes(4,5) and kinin antagonism. Materials and methods. This study comprised 48 male hamsters. A standard inflammatory stimulus was applied to the everted cheek pouch of the hamster. This stimulus consisted of a 2 minute immersion of the everted pouch in water pre-heated and maintained at 45°C. Animals were anesthetized with Nembutal (50–80 mg/kg) intraperitoneally. Twenty-four animals served as controls and received placebo tablets, orally, every 6 hours for 24 hours. The remaining 24 hamsters were divided into 3 groups of 8: one group received 30,000 units of Streptokinase∗ plus human plasma every 6 hours; the second group received 100 mg papain† every 6 hours; the third group received 400 casein units fungal protease‡ every 6 hours. All medications were suspended in saline and administered orally via stomach tube. The treatment period covered the 24 hours following injury. At the end of the treatment period a biopsy specimen was obtained for histologic examination and a strip of pouch tissue was excised. Tissue strips weighing 375 mg ± 5 mg were used for assay. The tissue was immediately homogenized in a Waring Blendor and each homogenate was eluted into a bath containing an intact rat uterus suspended in 17 ml Tyrodes solution at 37°C. 2 Bromolysergic acid diethylamide (BOL) 10–20 ng was added to the bath and allowed to stay in contact with the tissue 10–15 minutes before each sample was tested. Results and discussion. The results are summarized in Table I. Addition of bradykinin to the bath caused slow, sustained, uterine contractions. In 21 of the 24 control hamsters, pouch homogenates produced slow, sustained uterine contractions strongly resembling those following the addition of bradykinin. In these experiments the action of serotonin was excluded by the use of BOL.

INHIBITION OF THE GENERALIZED SHWARTZMAN REACTION IN THE PREGNANT GOLDEN HAMSTER BY ORAL STREPTOKINASE.

Abstract The generalized Shwartzman reaction developed in pregnant hamsters following single intraperitoneal doses of colchicine. In the present study we were able to confirm the simplicity and reproducibility of Galtons method for provoking disseminated renal glomerular capillary thrombosis and cortical necrosis in the hamster. It is of further interest that, in this study, oral doses of streptokinase appeared to inhibit the colchicine induced Shwartzman reaction.