Gillian M. Anlezark

The bioactivation of 5-(aziridin-1-yl)2,4-dinitrobenzamide (CB1954). II: A comparison of an Escherichia coli nitroreductase and walker DT diaphorase

A nitroreductase enzyme that has been isolated from Escherichia coli B is capable of bioactivating CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] to a cytotoxic agent, a property shared with the mammalian enzyme Walker DT diaphorase [NAD(P)H dehydrogenase (quinone), EC 1.6.99.2] as isolated from Walker cells. In contrast to Walker DT diaphorase, which can only reduce the 4-nitro group of CB1954, the E. coli nitroreductase can reduce either (but not both) nitro groups of CB1954 to the corresponding hydroxylamino species. The two hydroxylamino species are formed in equal proportions and at the same rates. CB1954 is reduced much more rapidly by the E. coli nitroreductase than by Walker DT diaphorase. If the reduction of CB1954 was carried out in the presence of V79 cells (which are insensitive to CB1954) a large cytotoxic effect was evident. This cytotoxicity was only observed under conditions in which the E. coli nitroreductase or Walker DT diaphorase reduced the drug. It is proposed that E. coli B nitroreductase would be a suitable enzyme for antibody-directed enzyme prodrug therapy (ADEPT) in combination with CB1954.

Convulsant and anticonvulsant actions in DBA/2 mice of compounds blocking the reuptake of GABA

Compounds blocking the uptake of GABA into neurons or glia have been injected intracerebroventricularly (icv) or intraperitoneally (ip) in DBA/2 mice, age 21-28 days. Protection against audiogenic seizures was seen 30 min after the icv injection of (+)-2,4-diaminobutyric acid (0.5-2.0 mumoles), (+/-)-nipecotic acid (1.6-3.2 mumoles), (+)-ethyl nipecotate (0.4-0.8 mumoles), (-)-piperazic acid (4 mumoles) and putrescine (2 mumoles) or the ip injection of (+)-2,4-diaminobutyric acid (4-8 mmoles/kg and (+)-ethyl nipecotate (0.24-0.32 mmoles/kg). Of these ethyl nipecotate and nipecotic acid were the most effective anticonvulsants icv, but nipecotic acid was ineffective ip. Limb myoclonus and other epileptic manifestations (rearing, wild running, tonic clonic seizures) occurred in the absence of auditory stimulation after (+)-2,4-diaminobutyric acid (0.5-2.0 mumoles), (+/-)-cis-3-aminocyclohexane carboxylic acid (3.2-6.4 mumoles) and putrescine (2 mumoles). beta-Alanine (2-4 mumoles, icv) depressed respiration but did not protect against audiogenic seizures or induce myoclonus.

Bioactivation of dinitrobenzamide mustards by an E. coli B nitroreductase

A nitroreductase isolated and purified from Escherichia coli B has been demonstrated to have potential applications in ADEPT (antibody-directed enzyme prodrug therapy) by its ability in vitro to reduce dinitrobenzamides (e.g. 5-aziridinyl 2,4-dinitrobenzamide, CB 1954 and its bischloroethylamino analogue, SN 23862) to form cytotoxic derivatives. In contrast to CB 1954, in which either nitro group is reducible to the corresponding hydroxylamine, SN 23862 is reduced by the nitroreductase to form only the 2-hydroxylamine. This hydroxylamine can react with S-acetylthiocholine to form a species capable of producing interstrand crosslinks in naked DNA. In terms of ADEPT, SN 23862 has a potential advantage over CB 1954 in that it is not reduced by mammalian DT diaphorases. Therefore, a series of compounds related to SN 23862 has been synthesized, and evaluated as potential prodrugs both by determination of kinetic parameters and by ratio of IC50 against UV4 cells when incubated in the presence of prodrug, with and without the E. coli enzyme and cofactor (NADH). Results from the two studies were generally in good agreement in that compounds showing no increase in cytotoxicity in presence of enzyme and cofactor were not substrates for the enzyme. None of the analogues were activated by DT diaphorase isolated from Walker 256 carcinoma cells. For those compounds which were substrates for the E. coli nitroreductase, there was a positive correlation between kcat and IC50 ratio. Two compounds showed advantageous properties: SN 25261 (with a dihydroxypropylcarboxamide ring substituent) which has a more than 10-fold greater aqueous solubility than SN 23862 whilst retaining similar kinetic characteristics and cytotoxic potency; and SN 25084, where a change in the position of the carboxamide group relative to the mustard resulted in an increased cytotoxicity ratio and kcat compared with SN 23862 (IC50 ratios 214 and 135; kcat values of 75 and 26.4 sec-1, respectively). An analogue (SN 25507) incorporating both these structural changes had an enhanced kcat of 576 sec-1. This study elucidates some of the structural requirements of the enzyme and aids identification of further directions in the search for suitable prodrugs for an ADEPT nitroreductase system.

The anticonvulsant action of the (-)- and (+)-enantiomers of propranolol.

m e threshold for electrically-or chemically-induced seizures in rodents can be raised by 8-adrenoceptor antagonists, such as propranolol (Murmann et al 1966; Yeoh & Wolf 1968). An exacerbation of chemically-induced seizures by (f)-propranolol has also been reported (Kilian & Frey 1973; Madan & Barar 1974). A convulsant action of ,9-adrenoceptor antagonists is consistent with the evidence for ,9-adrenoceptors in the cerebellum, cerebal cortex and hypothalamus (Conway et a1 1978) at which noradrenaline has an inhibitory action (Bevan et al 1977). However, propranolol and some other 8-adrenoceptor antagonists are also potent 5-HT antagonists as judged by both behavioural tests (Green & Grahame-Smith 1976; Weinstock et al 1977) and in vitro membrane studies (Middlemiss et al 1977). Drugs acting on 5-hydroxytryptaminergic mechanisms can readily modify seizure thresholds (Meldrum 1978). Propranolol also possesses membrane stabilizing properties as indicated by a local anaesthetic or quinidine-like action on nerve and heart cell membrane, decreasing excitability and prolonging refractoriness (Basset & Hoffman 1971; Hellenbrecht et al 1973). This stabilizing effect is probably related to inhibition of calcium binding at a plasma membrane site (Feldman & Weinhold 1977). Some central effects of ,6-blockers (or their dextroenantiomers) can be attributed to a membrane stabilizing effect on brain-stem neurons (Bousquet et al 1978). Whereas the effects on ,6adrenoceptors and 5-hydroxytryptaminergic receptors show a high degree of stereospecificity, the (4enantiomer being 10-100 times as potent as the (+)enantiomer (Green & Grahame-Smith 1976; Weinstock et al 1977; Conway et al 1978), the membrane stabilizing effect by the two enantiomers is shown to be approximately equal (Bassett & Hoffman 1971 ; Bousquet et a1 1978). To clarify the role of the different actions of (f)propranolol on seizure threshold we have studied the effect of (+)and (-)-propranolol on seizure responses induced in DBA/2 mice by loud auditory stimulation. Groups of 9-13 DBA/2 mice (bred in the laboratory from suppliers stock) were used at 19-26 days old (weight range 7-13 g). The enantiomers of propranolol were dissolved in 0.9% NaCl solution to give a maximum volume equivalent to 1% of the body weight of a mouse, and were injected intraperitoneally, 45 min before testing.

Dopamine agonists and audiogenic seizures: the relationship between protection against seizures and changes in monoamine metabolism.

Abstract Apomorphine (1 and 10 mg/kg), (-) N - n -propylnorapomorphine, (NPA, 0.1 mg/kg) ergot alkaloids (ergocornine, 2 mg/kg; bromocryptine, 10 mg/kg; d -lysergic acid diethylamide, LSD, 9 mg/kg; ergometrine, 4 and 10 mg/kg), and quipazine (50 mg/kg) protect DBA/2 mice against sound-induced seizures, although LSD, ergometrine, and quipazine do not prevent the initial wild running seizure response. The order of potency for prevention of the clonic phase of the seizure response is NPA > ergocornine ⩾ apomorphine > bromocryptine > LSD ⩾ ergometrine > quipazine. Apomorphine (10 mg/kg) increases dopamine (+ 72%) and noradrenaline ( + 91%) content after 15 min. Apomorphine, NPA and the ergot alkaloids reduce cerebral concentration of homovanillic acid (HVA) (by 31–73 per cent), but with a longer time course than the seizure protection (maximal reduction after 1–3 hr). After LSD, ergometrine and quipazine a fall in 5-hydroxyindoleacetic acid content (by 35–48 per cent) occurs before the fall in HVA content. All the drugs except quipazine lower body temperature (maximum decrease 6.9° after apomorphine 10 mg/kg). The greatest decrease in temperature occurs at the time of maximal protection against seizures. A protective effect of the ergot alkaloids (LSD and ergocornine) was still apparent when the fall in temperature was prevented by external heating. The anti-epileptic action of ergocornine, bromocryptine and the apomorphine derivatives is associated with stimulation of receptors pharmacologically equivalent to the DAe receptors of Cools et al ., 1976 (i.e. activated by dopamine and apomorphine, blocked by haloperidol).