| PHARMACOLOGY
8.1. ANESTHETIC EFFECTS OF IQB-9302.HCl The anesthetic effects of IQB-9302.HCl were studied by three standard tests (i.e. infiltration anesthesia in the guinea-pig, palpebral anesthesia in the rabbit and the guinea-pig and the sciatic nerve block in the rat) in comparison witn mepivacaine (see screening phase) and with bupivacaine (See comparison of effects of racemic, L and D-IQB-9302). Results of these tests have been described elsewhere and demonstrated a long lasting effect at least equivalent to that of bupivacaine. Sensory and motor blocking effects of epidural IQB-9302 and bupivacaine were studied under blind randomised conditions in 12 conscious beagle dogs. Groups of 6 dogs with a chronically implanted epidural catheter received in three experiments 3 ml of one of the following concentrations of IQB-9302 or bupivacaine : 0.25%, 0.50% and 0.75%. A venous catheter was used to obtain blood samples for plasma levels determination. Consecutive dosing was done at least at 48 hour intervals. After the third treatment with either drug, animals were sacrificed for anatomopathological examination of the epidural space. Responses to sensory nociceptive stimulus (pinching the toes with forceps) and to non nociceptive stimulus (pinching dermatomes) and motor blockade (gait and ability to stand on the hind limbs) were observed before and 5, 10, 15, 30, 45, 60, 75, 90, 105 and 120 minutes and at 30 minute intervals thereafter. Samples of blood were withdrawn inmediately after observation of anesthetic effects. a)Sensory
blockade:
Dermatomes pinching: blockade of the dermatome response was maximum in the most caudal dermatome y minimum in the most craneal dermatome. Effect was always observed 5 minutes after administration of both compounds and duration was maximum for IQB-9302 0.75% and minimum for bupivacaine 0.25%. b)
Motor blockade: complete motor blockade was similar for both drugs
at identical concentrations and was dose-dependent. Maximum blockade was
observed after bupivacaine 0.75% (397 ±
76 minutes); at this concentration, duration of motor blockade induced
by IQB-9305 was 282 ± 39 minutes (p<0.05). Recovery of
motor blockade was more rapid for IQB-9302 (as indicated by the slope of
the terminal part of the curves shown in figure
3)
Anatomopathological examinations: No macroscopic nor microscopic alterations were observed after either drug. Conclusion: This blind randomized study in the beagle dog demonstrated that IQB-9302 induces sensory and motor blockades similar to those induced by bupivacaine. Nevertheless, the sensory response induced by IQB-9302 0.5% is similar to that induced by bupivacaine 0.75% and sensory response induced by IQB-9302 0.25% is simiilar to that produced by bupivacaine 0.50%. Moreover, IQB-9302 presented two definitive advantages over bupivacaine:
8.2. MOLECULAR PHARMACOLOGY and ELECTROPHYSIOLOGY The effects of the R(+) and S(-)
enantiomers of IQB-9302 on hKv1.5 channels stably expressed in Ltk- cells
were analyzed by using the whole-cell configuration of the patch-clamp
technique. R(+) and S(-)IQB-9302 inhibited hKv1.5 current with KD
values of 18.6±1.5 mM
(n=22) and 58.6±4.0 mM
(n=29), respectively. This block was voltage dependent consistent with
values of electrical distance (d) of 0.17±0.02;
n=12 and 0.18±0.02; n=10, for R(+)
and S(-)IQB-9302, respectively. Consistent with an open channel block mechanism,
both, the R(+) and S(-) enantiomers slowed the time course of the tail
currents, inducing a "crossover" phenomenon. All these results suggest
that the length of the substituent at the tertiary amine in the molecule
is an structural determinant of the degree of stereoselective block (Preliminary
report)
These values are smaller than those
of R(+) Bupivacaine (KD
= 4.1 mM)
and racemic Bupivacaine (8.1 mM)
(Valenzuela, C. et al.,
1998) and might explain, at least partially, the lesser "cardiotoxic"
effects of IQB-9302 seen in the isolated rat ventricle (see Effects
on cardiac conduction)
b) Effects on sodium channels Whole-cell
patch-clamp techniques were used to directly examine the kinetics of
The blocking
effect was fast and reversed after wash-out,
with a t
of 32.7±3.7s. INa
was blocked by IQB-9302 in a voltage-dependent manner,
the effect being greater at depolarising membrane potentials. IQB-9302
seems to facilitate
the inactivation of voltage-dependent sodium channels, producing ahyperpolarizing
shift in the inactivation curve of INa.
c) Effects on voltage-dependent Ca2+ channels and neuronal nicotinic receptor ion channels. Potential blocking effects of IQB-9302 and bupivacaine on voltage-dependent calcium channels and nicotinic acetylcholine receptor-associated channels were studied by determining the 45Ca uptake into bovine chromaffin cells induced by K+ depolarisation or by stimulation of nicotinic receptors with the agonist DMPP (dimethylphenyl-piperazinium iodide). Both IQB-9302 and bupivacaine exhibited an effect as blockers of voltage-dependent Ca2+ channels at concentrations of 100 mM to 1 mM, concentrations that are used for local application of these anesthetics but that will never be reached sistemically. IC50 were 310 mM and 110 mM for IQB-9302 and bupivacaine, respectively. For nicotinic stimulation, the blockade
is obtained at concentrations 10 times lower than for K+ (IC50
= 9,11 mM
for IQB-9302 and IC50
= 0,32 mM
por bupivacaine). Side effects derived from nicotinic receptor blockade
of sympathetic ganglia should not be found clinically after local applications
of these compounds. It is worth noting that IQB-9302 blocked nicotinic
receptors with an IC50
30-fold
lower than bupivacaine. This is an important difference that might have
safety implications for the compounds. In the case of systemic accidental
injection the hypotension derived from ganglionic blockade should be much
less with IQB-9302 than with bupivacaine.
Additionally a comprehensive panel of experiments have been planned to dilucidate the mechanism of action of IQB-9302.HCl. Most of these studies will be performed at the Instituto Teófilo Hernando (Faculty of Medicine, University of Madrid) or at C.S.I.C (Consejo Superior de Investigaciones Científicas)
8.3.1. Effects of IQB-9302 on basal and evoked contractions of isolated rat aorta Effects of IQB-9302.HCl and bupivacaine.HCl on isolated rat aorta have been compared in the following experiments 1) Precontracted
with 35 mM KCl
IQB-9302 and bupivacaine showed little effect on the basal tone of isolated rat aorta at concentrations ranging from 10-7 to 10-5 M. At 10-4 bupivacaine increased basal tone, whereas no effects were elicited by IQB-9302 (figure 2) At concentrations ranging from 10-7
to 10-5 M, small
effects were shown by both drugs on contractions evoked by phenylephrine
or KCl 35 mM. Higher concentrations of IQB-9302 and bupivacaine induced
a similar and significant vasorelaxant effect (figure
3 and figure 4)
8.3.2. Effects of IQB-9302 on hemodynamics of anesthesized rats Heart rate and MAP (mean arterial blood pressure) were measured in anesthetized rats after i.v. injections of 0.3, 1 and 3 mg/kg of IQB-9302 or bupivacaine. Both drugs increased MAP with no significant change in heart rate. Although differences were not statistically significant, IQB-9302 exhibited a somewhat more potent pressor response. It can be concluded that capillary vasodilator effect of IQB-9302 does not exceed this effect of bupivacaine (Preliminary report) An extensive set of experiments have been planned to further characterize the cardiovascular effects of IQB-9302. Some of them are:
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