Pierre Berryer

Dehydrophenylalanyl analogs of bradykinin: synthesis and biological activities.

Abstract We have synthesized three analogs of the potent vasodilator peptide bradykinin, ArgProProGlyPhe SerProPheArg (BK), containing dehydrophenylalanine (Δ z Phe) in place of the phenylalanyl residues at positions 5 and/or 8. The analogs, [Δ z Phe 5 ]BK, [Δ z Phe 8 ]BK, and [Δ z Phe 5,8 ]BK, were assayed for their effects on isolated smooth muscle tissues and on the systemic arterial blood pressure of rats. In these assays [Δ z Phe 5 ]BK showed considerably high biological activities, particularly in terms of its blood pressure-lowering effects, being over 23 times more potent than BK when given intravenously. [Δ z Phe 8 ]BK was less potent than BK and [Δ z Phe 5,8 ]BK had effects comparable to those of BK. All three synthetic analogs appear to be more resistant than BK to enzymic degradation during passage through the pulmonary vascular bed.

Slow Tight Binding Inhibitors of Angiotensin Converting Enzyme

We have found that apparent Ki values of some, but not all, carboxyalkyl-dipeptide inhibitors of angiotensin converting enzyme decrease as a function of incubation time. The most potent of the ACE inhibitors tested so far is RAC-X-65 (N-[1(S)-carboxy-3-carboxanilidiopropyl]-L-Ala-L-Pro). When RAC-X-65 is not preincubated with human serum ACE (2.4 X 10(-11) M), the apparent Ki value is 4.4 X 10(-10) M. Preincubation of RAC-X-65 with ACE for 15 min before addition of substrate yields an apparent Ki of 4.1 X 10(-11) M. a 90 min preincubation of the inhibitor with ACE yields an apparent Ki of 1.2 X 10(-11) M, i.e., the reaction of the inhibitor with enzyme is virtually stoichiometric. The enzyme:inhibitor complex is poorly separated by molecular sieve chromatography or by dilution. That such tightly bound complexes are formed in vivo is suggested by the following results: The intravenous ED50 (anesthetized rats) of RAC-X-65 is 9.43 nmol/kg, and the time for half recovery (t1/2) of responsiveness to i.v. angiotensin I, 120 ng/kg, following a cumulative dose of 240 nmol/kg of the inhibitor is 165 min. For comparison, the i.v. ED50 of captopril is 105 nmol/kg, and its t1/2 following a cumulative dose of 240 nmol/kg is 16 min. Implied is the possibility that slow tight binding inhibitors of ACE may be used in a 1 pill per day regimen for the treatment of hypertension.

A radioassay for aminoacylproline hydrolase (aminopeptidase P) activity

A radioassay was developed in which aminoacylproline hydrolase acts on Arg-Pro-Pro-[3H]benzylamide to yield arginine plus Pro-Pro-[3H]benzylamide. By stopping the reaction with base (0.1 M NaOH), the radioactive product is deprotonated to an organophilic form and is separable from the hydrophilic substrate by extraction of the alkaline aqueous solution with an organic solvent. When scintillants are included in the organic solvent, the enzyme:substrate reaction, extraction and quantification of Pro-Pro-[3H]benzylamide can all be conducted using a single liquid scintillation vial. Thus, aminoacylproline hydrolase activity is measured in terms of the rate of release of Pro-Pro-[3H]benzylamide. The substrate is obtainable at greater than 20 Ci/mmol, which enables its use under conditions of first-order enzyme kinetics. Conditions of near-zero order kinetics are readily attained by adding unlabeled substrate (Km 0.7 microM). The substrate is highly reactive (a 1:2000 dilution of guinea pig plasma hydrolyzed greater than 10% of the substrate during a 10 min incubation at 37 degrees C) and specific in that it is not degraded by leucine aminopeptidase, aminopeptidase A or N, dipeptidyl peptidase IV nor prolyl endopeptidase. The assay was used to measure aminoacylproline hydrolase specific activities in tissues of rat and guinea pig. Activity was found in virtually all major tissues of both species, and some guinea pig tissues (e.g. kidney and plasma) were found to be notably rich sources of the enzyme.

Pulmonary Anaphylaxis and the Kallikrein-Kinin System

The kallikrein-kinin system has been thought to participate in the pathogenesis of anaphylaxis. Kallikrein, released from lungs, has been postulated to contribute to cardiovascular collapse. Further to test the hypothesis, we examined for the occurrence of a kallikrein-like enzyme in guinea pig lungs and examined for release of such an enzyme by isolated, perfused lungs of guinea pig sensitized to and challenged with egg albumin. In addition, we treated guinea pigs with the bradykinin potentiating agents, BPP9a and SQ 14,225. In parallel experiments, we examined for effects of non-steroidal anti-inflammatory agents on the supposition that prostaglandin-related substances may mediate or modulate actions of kinins during anaphylaxis. A plasma kallikrein-like enzyme was found in lung homogenates and occurred in concentrations greater than that of plasma itself. Similarly, a store of kininogen occurs in lungs. However, using a sensitive radioassay for kallikrein-like enzymes, we were unable to confirm that antigenic challenge of sensitized lungs causes the release of enzyme into pulmonary venous effluent. Further, we were unable to modify the acute course of anaphylaxis by pretreatment of guinea pigs with bradykinin potentiating agents. However, indomethacin and aspirin at 20-40 mg/kg were found to greatly increase the severity of pulmonary anaphylaxis in terms of increased resistance to ventilation and increased numbers of lung hemorrhages. Paradoxically, aspirin or sodium salicylate at 80-100 mg/kg prevents the characteristic rise of insufflation pressure and the formation of lung hemorrhages.

Preparation of Intrinsically-Labelled Kinins

As part of a program to prepare bradykinin (H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH) labelled at high specific radioactivities, we have synthesized three analogs for dehalogenation in tritium gas: [4-Br-Phe5]-bradykinin (BK), [4-Br-Phe8]-BK and [4-Br-Phe5,8]-BK. The analogs were synthesized by the Merrifield solid-phase method and were purified by molecular sieve and partition chromatography. The analogs themselves possess biological activity (as assayed for effects on mean arterial blood pressure and isolated rat uterus). [4-Br-Phe8]-BK was 1.5 to 3 times as active as bradykinin. [4-Br-Phe5,8]-BK was approx. 22% as active as BK and [4-Br-Phe5]-BK was approx. 18% as active. [4-Br-Phe5]-BK was submitted to catalytic dehalogenation with 10% Pd/C and 5% Rh/CaCO3 in H2O and DMF (1:1) plus 10 Ci of 3H2. [4-3H-Phe5]-BK was obtained at 6.7 Ci/mmole in an overall yield of 15%. [4-3H-Phe8]-BK was prepared similarly to yield an intrinsically-labelled peptide with a specific radioactivity of 21 Ci/mmole.

PULMONARY MICROVASCULAR OCCLUSION IN AGGREGATE ANAPHYLAXIS

The acute respiratory distress syndrome (ARDS) is a common late stage manifestation of several different kinds of severe lung injury (1). A priori, there is little reason to believe that the early pathogenesis of, e.g., sepsis-related lung injury is identical, or even similar, to the early pathogenesis of lung injury caused by aspiration of gastric contents, pneumonia or microthromboembolism even though all causes of serious diffuse lung injury may lead to a common clinical presentation. Thus, it is timely to examine separately the multiple causes of lung injury with the goal of developing appropriate means of treatment of each so as to prevent progression of the lung injuries to frank ARDS.