Inhibition of the enkephalin-metabolizing enzymes by the first systemically active mixed inhibitor prodrug RB 101 induces potent analgesic responses in mice and rats.

N-([(R,S)-2-benzyl-3[(S)(2-amino-4-methylthio)butyl dithio]-1-oxopropyl)-L-phenylalanine benzyl ester (RB101) is the first systemically active prodrug generating through a biologically dependent cleavage of the disulfide bond the potent (S)2-amino-1-mercapto-4-methylthio butane (aminopeptidase N) (IC50 = 11 nM) and N-[(R,S)-2-mercapto-methyl-1-oxo-3-phenylpropyl]-L-phenylalanine (neutral endopeptidase) (IC50 = 2 nM) inhibitors (aminopeptidase N). RB101 easily crosses the blood-brain barrier, as shown by the observed complete inhibition of cerebral endopeptidase 24.11 after i.v. injection in mice. The prodrug induces strong, dose-dependent antinociceptive responses in mice after i.v., i.p. or s.c. administration, in the hot plate (ED50 = 9 mg/kg) and phenylbenzoquinone-induced writhing (ED50-3.25 mg/kg) tests in mice, which are currently used in analgesics screening. RB101 is also active in the tail-flick and tail-electric stimulation tests in rats. In contrast, under disulfide forms, the above selective aminopeptidase N or endopeptidase 24.11 inhibitors are inactive after i.v. administration and their association 3 times less potent than RB101 alone. In all the tests used, the pain-alleviating effect of RB101 was suppressed by naloxone, but, except for the tail-flick and the motor response to tail-electric stimulation, not by the delta-selective antagonist naltrindole. The preferential involvement of mu opioid receptors in the analgesic effects of endogenous enkephalins, whose extracellular levels are increased by the two RB101-generated inhibitors, is suggested by the similar apparent pA2 values for RB101-naloxone (pA2: 7.53 +/- 0.046) and DAMGO (mu-selective ligand)-naloxone (pA2: 7.38 +/- 0.049).(ABSTRACT TRUNCATED AT 250 WORDS)