Role of efflux transporters in the absorption, distribution and elimination in rodents of a novel PDE4 inhibitor, CHF6001.

CHF6001 is a new and potent PDE4 inhibitor for the treatment of human lung diseases, designed for topical administration by inhalation. In preclinical assessment CHF6001 appeared safe and devoid of emetic effect, which is typical side effect of PDE4 inhibitors in humans. CHF6001 absorption, distribution and excretion were evaluated in rats by PO and IV administration of [14C]CHF6001; additionally the role of transporters was investigated by using transfected cells expressing either human transporters or MDR1 and BCRP KO mice. [14C]CHF6001 intravenously administered as bolus distributed in all the tissues (with very low levels in brain and fetus) and it was mainly eliminated in bile. Following oral administration [14C]CHF6001 about half of the dose was absorbed through the gut. In vitro, CHF6001 was a substrate of human membrane transporters MDR1 and BCRP. In wild and BCRP KO mice CHF6001 was not detectable in brain, whereas it was measurable in Mdr1a/b KO mice. Therefore, in animal species Mdr1a/b plays a significant role in CHF6001 disposition, limiting its distribution into brain and contributing to the safety profile observed in preclinical evaluation. This behavior was confirmed by the results of the first human studies, where CHF6001 was safe and with no emetic effect at all the evaluated doses.

4-Biphenylaldehyde and 9-anthraldehyde: two fluorescent substrates for determining P450 enzyme activities in rat and human.

1. 4-Biphenylaldehyde (4-BA) and 9-anthraldehyde (9-AA) were examined as substrates for cytochrome P450 (CYPs) enzymes in rat and human. Both aldehydes were oxidized by CYPs to fluorescent carboxylic acids, which can be assayed with a high sensitivity by an easy fluorimetric method. 2. With liver microsomes from control and induced rats, the oxidation of both 9-AA and 4-BA followed simple Michaelis-Menten kinetics. Only microsomes from rats pretreated with phenobarbital (a strong inducer of P4502B1/2) could increase (about threefold) the oxidation rates (V(max)) of both aldehydes above the control values, which were 6.7+/-1.1 and 3.3+/-0.6 nmol min(-1) mg(-1) protein for 4-BA and 9-AA, respectively. On the other hand, the (K)(m)'s, which were similar for both aldehydes (about 25 micro M), did not change significantly with any inducer. The use of purified rat CYP1A1, 2E1, 2B1 and 2C11 in a reconstituted system showed that only 2B1 and 2C11 could oxidize both substrates with a high turnover. 3. In human liver microsomes, the oxidation rates of these aldehydes (1.6+/-0.2 and 0.42+/-0.1 nmol min(-1) mg(-1) protein for 4-BA and 9-AA, respectively) were lower than those of rat but with similar K(m)'s(20-26 microm). 4. The oxidation of these aldehydes was also determined with cDNA-expressed CYP1A1, 1A2, 2A6, 2B6, 2C9, 2D6, 2E1 and 3A4 and with a characterized bank of 14 human liver microsomes. In a reconstituted system, only CYP2B6, 2A6, 3A4 and with a lower turnover 2C9 oxidized both substrates. 5. Among the CYP marker activities of the 14 human samples, good correlations were only observed between CYP3A-dependent 6 beta-testosterone hydroxylase and the oxidation of 4-BA (r = 0.74) or 9-AA (r = 0.80) and between the oxidation of 4-BA versus 9-AA (r = 0.74). Weak correlations were also found between the 2B6-linked S-mephenytoin N- demethylase and the oxidation of 4-BA (r = 0.58) or 9-AA (r = 0.65). 6. Inhibition experiments revealed that the oxidation of these aldehydes was inhibited by ketoconazole, 8-methoxypsoralene and sulphophenazole, selective inhibitors for P4503A6, 2A6 and 2C9, respectively. 7. In summary, based on the use of cDNA-expressed CYPs, correlation analysis and chemical inhibition, the metabolism in human liver microsomes of these aldehydes appears primarily catalysed by CYP3A, although CYP2A6, 2B6 and 2C9 may play a role. 9-AA and particularly 4-BA, owing to the high rate of its metabolism, may be two novel useful fluorescent probe substrates for assaying CYP activities in various species.

Effects of REMs and PGOs on visual evoked responses during paradoxical sleep of cats.

The amplitude modifications of VERs from paired geniculate stimuli during paradoxical sleep were studied in five cats with chronically implanted electrodes, in the presence of REMs and during PGOs without REMs. In both cases the C5 component of evoked potentials was significantly enhanced with respect to the controls. This facilitation affecting presumably only the postsynaptic component of the response seems linked to signals reaching the visual cortex through other than geniculo-striate pathways. The disinhibition of the second VER appears to be due to intracortical mechanisms, and is not specifically related to phasic events of sleep, such as we previously described during light conditioning and in the absence of eye movements. It is proposed that this effect may be a more general phenomenon caused by changes in cortical activation during eye movement and conditioning.

[Changes in the monosynaptic reflex during wakefulness and sleep of children with cerebral paralysis]. / Considerazioni sulle variazioni del riflesso monosinaptico nella veglia e nel sonno di bambini affetti da paralisi cerebrale.

Changes of the H reflex during sleep were studied in 13 children with cerebral palsy (8 with spastic tetraplegia, 2 with a mixed form of cerebral palsy without spasticity, 3 with hypotonic diplegia or tetraplegia). These modifications were compared with those of 5 normal children of the same age. During repeated night recordings, responses in the calf muscle elicited by electrical stimuli to the posterior tibial nerve were studied at the same time as the EEG, EOG and EMG of the mental muscles. The results show that:--in normal children the max H reflex progressively decreases in amplitude from wakefulness to REM sleep; -- in spastic patients there is only a slight decrease in the H reflex in NREM sleep and no significant change in REM sleep; the amplitude of the H reflex is always greater than that in the control group; -- in the dystonic and hypotonic group the results obtained are similar to those of the control group. From these results one may draw the conclusion that in spastic patients as opposed to the control, dystonic and hypotonic groups, normal balance between the function of supraspinal systems regulating the amplitude of the spinal reflexes is alterated probably through the scarce functionality of the supraspinal inhibitory structures.