Utilización de hidrolasas en la preparación de fármacos e intermedios homoquirales / Hydrolases use in the preparation of drugs and homochiral intermediates

La exquisita regio y estereoselectividad que presentan los biocatalizadores,amén de la buena sostenibilidad inherente a su empleo,permiten la realización de protocolos sintéticos difícilmente alcanzablespor las metodologías clásicas, a menos que se lleven a cabocostosos procesos de protección y desprotección. En este trabajo serevisan algunos ejemplos en los cuales las hidrolasas (las enzimas másempleadas dentro del ámbito de las Biotransformaciones) están implicadascomo biocatalizadores para la obtención del eutómero (esteroisómeroactivo, que presenta la actividad terapéutica deseada) biende diferentes fármacos quirales, o bien de precursores a través de loscuales se puedan sintetizar. Así, se comentarán distintos tipos de biotransformacionespara la obtención de compuestos con diferentesactividades: antivirales, anticancerosos, antihipertensivos, antiinflamatorios,etc, haciendo hincapié en la versatilidad y comodidad delempleo de los biocatalizadores en los pasos sintéticos descritos(AU)

The excellent regio and steroselectivity of biocatalysts, combinedwith their environmental friendly behaviour, make possible to carryout under biocatalytical conditions many processes which, conductedon strictly classical methodologies, would demand expensive andtedious protection and de-protection steps. In this work we reviewsome examples in which hydrolases (the most useful enzymes in theBiotransformations field) catalyse different reactions for synthesizingonly the therapeutically essential stereoisomer of differenthomochiral building blocks for drugs. Thus, processes leading toantiviral, anticancer, antihypertensive or antiinflammatory drugs,along with many others, are described, remarking the versatility andutility of the biocatalysts in the above-mentioned processes(AU)

Tetrodotoxin block of single germitrine-activated sodium channels in cultured rat cardiac cells.

1. The open time of single Na+ channels in excised (outside-out) patches from cultured late-fetal rat ventricular myocytes was prolonged to several minutes by germitrine (0.5 mM) in order to analyse tetrodotoxin (TTX) blocking kinetics. 2. The germitrine modification appeared during depolarizing pulses that activated normal Na+ channels. Following repolarization to -100 mV, the modified Na+ channel remained activated for 136 +/- 186 s (mean +/- S.D., n = 54) with an open-channel current amplitude of -0.5 pA. The predominant open state with a mean open time of 0.13 s was interrupted by brief closing events lasting for milliseconds. Replacing extracellular Na+ by Cs+ decreased the current amplitude to -0.1 pA. 3. Extracellular superfusion with TTX (3 x 10(-7) M) of a single germitrine-activated Na+ channel induced full channel closures lasting seconds (blocked events) separated by channel reopenings (unblocked events) that were indistinguishable in terms of amplitude and gating kinetics from the germitrine-activated state in the absence of TTX. 4. Cumulative probability histograms of blocked and unblocked events (n greater than 140) collected during long-lasting germitrine modifications at 10(-7) and 3 x 10(-7) M-TTX are well described by single exponentials. The 3-fold increase in [TTX] decreased the time constant of the unblocked state, tau o, from 11.9 to 4.7 s, while the time constant of the blocked state, tau c, was not significantly altered from 8.6 to 9.7 s. A microscopic association rate constant of 7.7 x 10(5) M-1 s-1, dissociation rate constant of 0.11 s-1, and equilibrium dissociation constant of 1.4 x 10(-7) M (at -100 mV) were calculated (20 degrees C). 5. Increasing [TTX] to 10(-5) M decreased tau o to 86 ms. This argues against the existence of a slower conformational step interposed between the binding of TTX to an open channel and the resultant channel closure. 6. Setting the membrane potential to -50 or 0 mV subsequent to a germitrine modification at -100 mV did not significantly alter TTX (3 x 10(-7) M) blocking kinetics: tau o was 6.7 s at -50 mV and 5.2 s at 0 mV; tau c was 8.9 and 8.1 s, respectively. 7. These results suggest that blocked events correspond to the random times that a TTX molecule resides on the Na+ channel before it dissociates, and unblocked events correspond to the random waiting times of an unoccupied channel before it binds another toxin molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Reversal of dantrolene sodium-induced depression of skeletal muscle in the cat.

Dantrolene sodium, a muscle relaxant, does not have a clinically useful antagonist. The present study was undertaken to test the efficacies of germine monoacetate, 4-aminopyridine, neostigmine, and calcium chloride as possible agents for the reversal of the direct skeletal muscle depression produced by dantrolene sodium in the cat anesthetized with alpha-chloralose. Depression of the indirectly elicited twitch responses (0.1 Hz) of the tibialis anterior muscle by 25, 50, 75 and 84 per cent was produced by dantrolene, 0.16, 0.36, 0.88 and 1.13 mg/kg respectively; spontaneous recovery of twitch tension during the subsequent 30 min was negligible. After the 30-min observation period had elapsed, one of the reversal agents under study was given (iv) in divided doses at intervals of 10 min (five cats for each agent). Germine monoacetate (2 X 0.5 mg/kg) immediately reversed the dantrolene-induced twitch depression, with an over-shoot that persisted for an hour. 4-Aminopyridine (4 X 0.5 mg/kg) caused a steady but incomplete reversal to 17 per cent depression, 30 min after the last dose. Neostigmine (4 X 0.04 mg/kg) caused an immediate, but transient, reversal of the twitch depression, with overshoot. Calcium chloride (4 X 10 mg/kg) was without effect. It is concluded that germine monoacetate is the drug of choice for reversal of the muscle depression produced by dantrolene sodium in the cat.

Sarcolemmal sodium permeability and contractile force of guinea pig papillary muscle: effects of germitrine.

The action potential of guinea pig papillary muscle exposed to the ceveratrum alkaloid germitrine (2 mugM) is followed by a long-lasting after-depolarization (maximal amplitude, 8 mV; half-time of decay, 32 seconds; total duration, approximately 75 seconds). This after-depolarization interrupts the terminal phase of repolarization. During repetitive stimulation (0.1-1.0 Hz; 80 nM germitrine) the after-depolarizations that follow consecutive action potentials are summed, causing persistent depolarization of up to 10 mV. The after-depolarization is reversibly abolished by tetrodotoxin (TTX). Test contractions evoked at various times during or after the germitrine-induced after-depolarization reveal a phase during which the ability of the muscle to develop force is transiently increased. This positive inotropic influence reaches its maximum 1 minute after the conditioning stimulus and thereafter decays with a half-time 4.8 times longer than the half-time of decay of the after depolarization. It is reversibly abolished by TTX and augmented by dihydro-ouabain (DHO). We conclude: Germitrine induces an after-depolarization by prolonging dramatically the Na permeability component which is mediated by the fast Na channels and normally restricted to the first few milliseconds of the action potential. The germitrine-induced selective and persistent increase of sarcolemmal sodium permeability (PNA) causes a positive inotropic effect, probably because intracellularly accumulating Na ions exchange for extracellular Ca ions.

The effects of protoveratrine and germines on the release of acetylcholine from the Auerbach plexus of the guinea-pig ileum.

The effect of protoveratrine A and germine-3-acetate (GMA) on the release mechanism of acetylcholine from the nerve terminals of the Auerbach plexus in the longitudinal muscle layer of the guinea-pig ileum was studied. Protoveratrine A, GMA and germine potentiated neuroeffector transmission in Auerbach's plexus in the longitudinal muscle preparation provided the neurons were stimulated at low frequencies (less than 10 Hz). Protoveratrine A and GMA enhanced the release of acetylcholine from the nerve terminals during the resting period and at low frequency of stimulation (less than 10 Hz). At continous stimulation with high frequency they were ineffective (greater than 10 Hz). When trains of 20 stimuli, with a pulse interval of 0.1 s (10 Hz) were repeatedly applied with intervals of 0.1 to 20 s between trains, the effect of GMA to increase ACh release depended on the length of the train interval; the longer the resting period between trains the higher was the output of ACh. This fact indicates that the release of ACh increased primarily during the resting periods following single stimuli or trains. The effect of GMA on ACh release proved to be highly temperature-dependent: in the presence of GMA Q10 increasing from 3.25 to 4.92. A high Ca concentration, removal of Mg or lowering of the Na concentration abolished the effect of GMA to enhance ACh release.