Because of the presence of an assymetric carbone atom, IQB-9302 is a mixture of two stereoisomers L (-)- IQB-9302 and D-(+)-9302. Both enantiomers have been obtained and purified by recrystallization of salts of optically active tartaric acid and investigated for their pharmacological activity as local anesthetics and for their systemic and cardiac toxicity. 
 

Local Anesthetic activity    a) Infiltration anesthesia:  100 ml of  0.05, 0.1 and 0.5% of solutions of racemic IQB-9302 and its L- and D-enantiomers were compared with bupivacaine given intradermally to male guinea-pig.  Skin reflex inhibition over a 6 h period was evaluated to determine the relative potency of test compounds.  Although the results of this study did not reveal a clear advantage of one of the enantiomers over another, the data show that IQB-9302.HCl was more potent and had longer duration of action than bupivacaine

Corneal reflex was measured after application of 30 ml racemic, L- and D-IQB-9302l and bupivacaine directly to the corneal surface of male guinea-pigs. In this test, L-IQB-9302.HCl and bupivacaine showed dose dependent long lasting equivalent anesthetic effects. Racemic IQB-9203 was less active although due to the acidic pH one out of three animals did not respond to anesthesia and therefore its activity is probably underestimated. 
 

Once again, racemic, L- and D-IQB-9302 were compared with bupivacaine on the blockade of sciatic nerve of anesthetized rats. All test substances were very active on this test. On the basis of AUC inhibition of muscular activity vs time, racemic IQB-9302 and L-IQB-9302 were more active than bupivacaine, although due to the small number of animals (n= 6) differences were not statistically significant. D-IQB-9302 was less active. 
 

Conclusion: From this series of tests, it can be concluded that IQB-9302 (both racemic and L-IQB-9302) exhibited a potent and long lasting anesthetic effect at least as potent as bupivacaine. No significant differences were shown in the respective activity of L- and racemic-IQB-9302. D-IQB-9302 was the less potent of test compounds 

d) Acute toxicity in rats: 

Intravenous DL₅₀ of L-, D- and racemic-IQB-9302 were determined in Wistar rats according to the standard protocol.  
All test substances showed the same acute toxicity (range 4.4-5.7 mg/kg). Interestingly, female rats were more sensitive than male rats (ranges 4.4-4.8 mg/kg  and 5.2-5.4 mg/kg, respectively) 
 

e) Effects of cardiac conduction: 

Bupivacaine  and L-, D- and racemic IQB-9302, 20 mM were tested for impairment of electrical conduction in isolated rat ventricle. Tissues were stimulated with a AgCl/Ag electrode set at 1 MHz and electrical responses were measured with a glass electrode. Standard electrophysiological parameters were recorded. 

Bupivacaine induced total conduction blockade of the tissue in 80% of experiments. IQB-9302 (both racemic, L- and D-) induced blockade only in 40% of the tissues. In these experiments electrical activity was fully recovered by increasing threshold voltage.  

Amplitude of action potentials was significantly reduced by bupivacaine and IQB-9302 in comparison with the control level. Maximum reduction of 40% was observed at 15 min with bupivacaine, whereas racemic IQB-9302 only reduced amplitude by 20% over all the perfusion period. 
Activity on this parameter activity was: 

Delay of action potentials (i.e. impairment of ventricular conduction) increased significantly in comparison with control levels. Activity was: 

CONCLUSION:  No differences were observed in the systemic intravenous toxicity of IQB-9302 (both racemic and L- and D-isomers) and bupivacaine in rats. 
As far as effects on ventricular conduction are concerned, bupivacaine was significantly more toxic than racemic and L-IQB-9302. Total blockades were more frequent in tissues perfused with bupivacaine and both amplitude and delay of action potentials were signicantly more affected by bupivacaine 

CONCLUSION OF THE DEFINITION PHASE: Although no significant differences were observed as far as anesthetic effects are concerned between bupivacaine and IQB-9302, a trend to a deeper anesthesia and longer duration of action was definitively observed for L- and racemic IQB-9302. No differences in systemic toxicity were found between bupivacaine y IQB-9302, but impairment of cardiac conduction was significantly less for the later.  
Further pharmacological and clinical studies are needed to confirm the efficacy and safety of racemic IQB-9302 in comparison with bupivacaine.