A role for P450s in antinociceptive drug action () requires that P450 inhibitors behave as antagonists
of analgesic drugs. However, the presently-observed antinociceptive effects of large doses of the P450 blockers miconazole, sulconazole and ketoconazole () are the opposite
of this hypothesis. Earlier studies described the antinociceptive properties of the P450 inhibitor proadifen [12
], but the antinociceptive properties of other P450 inhibitors have not been previously reported. Although these drugs are used clinically, they do not penetrate the blood-brain barrier [10
]; this barrier was circumvented presently by icv drug administration. Despite the extensive literature on miconazole-like P450 inhibitors (Suppl. Table 1
), the affinities of these drugs for some receptors () have not been previously reported. The ability of naltrexone to antagonize azole-induced antinociception (, observed earlier with proadifen [12
]) may be relevant to the opioid receptor affinities for some of these drugs (), but this requires further study.
In contrast to the results with high doses of P450 inhibitors (250 – 500 nmol, ), the prediction that these drugs should inhibit improgan antinociception was verified with lower doses (, Figs. , ). Miconazole is a potent, versatile inhibitor of many P450 isoforms (Suppl. Table 1
), and this drug has been used to show biological roles for P450 epoxygenases [46
]. However, since miconazole has many non-P450 actions (Suppl. Table 1
), the inhibition of improgan antinociception by the more selective inhibitor fluconazole ([49
] & Suppl. Table 1
) considerably strengthens the P450 hypothesis, and may help to identify the analgesia-relevant P450 isoforms.
The finding that miconazole behaved as an improgan antagonist with a 300-fold greater potency than that of CC12 (, ) prompted the design of MW06-25, a CC12 isomer more closely resembling the structure of miconazole. MW06-25 has an N-substituted side chain, a feature found in miconazole, but not in CC12 (). Like CC12 and miconazole, MW06-25 is a potent P450 inhibitor (). Unlike these other drugs, however, MW06-25 lacks affinity for the H3 and other receptors (), thereby demonstrating an enhanced P450 selectivity. The potent antagonism of improgan antinociception by MW06-25 () also supports the P450 hypothesis.
Many P450s (e.g. CYP4A2/3 [45
], CYP4X1 [42
], and members of the 2B, 2C, and 2J families [18
]) can convert AA to EETs (). But P450s can also metabolize EETs [50
], and can hydroxylate AA to several hydroxyeicosatetraenoic acids [50
]. All of these reactions can be inhibited by the heme-binding, miconazole-like P450 inhibitors, and all should be impaired in the Cprlow
mice. To further distinguish epoxygenase vs non-epoxygenase P450 mechanisms, we used MS-PPOH (), a selective P450 epoxygenase blocker [1
]. In vivo brain studies with MS-PPOH are limited [36
], but the extraordinarily potent inhibition of improgan antinociception by this compound () strongly implicates a brain epoxygenase pathway in improgan action. The weak activity of MS-PPOH on our prototype P450 (CYP2C19, ) confirms that the relevant P450 epoxygenase has not been found.
Since P450s are associated with drug metabolism, the inhibition of drug action in vivo by a P450 inhibitor can mean that drug metabolism is required to produce an active metabolite. A well known example is codeine, which is activated by conversion to morphine by hepatic CYP2D [3
]. Although brain P450s can metabolize drugs [23
], the presently-observed inhibition of improgan antinociception by P450 blockers is not likely to be due to a drug-drug metabolic interaction for the following reasons: 1) All drugs were administered directly into the brain, circumventing hepatic metabolism. 2) The rapid onset and short duration of antinociceptive activity following icv improgan (onset within 5 – 10 min, rapid decline thereafter, ) is not commensurate with the expected time course of brain metabolic activation. The activity of a brain-injected pro-drug would not be immediate, but instead should develop gradually over 30 to 60 min (e.g. see [24
]). 3) The P450 blocker CC12 not only inhibits the antinociceptive activity of improgan, but also the effects of morphine (which is metabolized in the rat by glucuronidation, not by P450 oxidation [19
]), and the effects of a cannabinoid [16
]. Thus, the presently-observed effects of P450 inhibitors on improgan action are most likely to represent a pharmacodynamic (vs pharmacokinetic) drug – drug interaction ().
The inhibitory dose-response curves for the P450 epoxygenase blockers on improgan antinociception are complicated by biphasic (and even triphasic) actions of some of these drugs (). If, as suggested by these curves, different brain P450s play opposing roles in pain modulation, then a particular spectrum of action of a single inhibitor on these enzymes could produce a specfic pattern of biphasic effects. This ‘dual P450’ idea seems consistent with the hypothesis that EETs mediate improgan antinociception (), since P450s can perform both EET biosynthesis and EET metabolism [50
]. Alternatively, off-target actions could account for the improgan-enhancing effects of these drugs, since many P450 inhibitors have side effects on ion channels and other enzymes (Suppl. Table 1
). The biphasic actions of the more selective agents MS-PPOH and fluconazole may not support this possibility. Further studies with these compounds (e.g. in P450-deficient animal models) will help to clarify these issues.
Genetic approaches to the study of P450 in mammals have been hampered by the large number of P450 genes (over 120 in the mouse [28
]). In contrast, CPR, an electron provider required for microsomal P450 and heme oxygenase function, is encoded by a single gene [37
]. However, germ-line, whole-body inactivation of Cpr
produced 100% embryonic lethality [30
], preventing assessment of P450 functions in adult knockouts. Wu and colleagues [47
] developed a transgenic mouse model (Cprlow
) which surmounts these obstacles. In these mice, embryonic insertion of the neo
gene into Cpr
led to widespread reductions in CPR and P450 activities, with limited embryonic lethality and a 90% reduction in brain CPR activity [47
]. As predicted, improgan antinociception was significantly reduced in Cprlow
as compared with wild-type mice (). The finding that improgan responses were reduced
but not eliminated
mice may be due to the fact that P450 activity is reduced, but not eliminated in this mouse model; a small amount of Cpr
expression is retained in these animals [47
]. The robust antagonism of improgan by CC12 in control mice, and the virtual elimination of this effect in Cprlow
mice show that CC12’s anti-analgesic activity in normal subjects depends on brain P450 activity. The fact that Cprlow
mice not only have a deficiency in P450 activity, but also in heme oxygenase activity (another CPR-requiring enzyme [47
]), suggests an alternative, non-P450 explanation for the present results. However, heme oxygenase is not likely to account for changes in improgan or CC12 effects in these mice, since miconazole and fluconazole (which block improgan antinociception) do not inhibit the brain form of heme oxygenase (Suppl. Table 1
The presently-established P450 epoxygenase requirement for improgan antinociception provides significant progress toward understanding this drug’s mechanism of action. The present model () suggests that improgan activates an unknown site that is distinct from known opioid, cannabinoid or histamine receptors [9
]. Endocannabinoid release is proposed to follow receptor activation, yet precede a P450-mediated step. Improgan-evoked release of endocannabinoids has not been directly shown, but the model is based on 1) blockade of improgan antinociception by the CB1
antagonist rimonabant [9
], 2) lack of rimonabant-sensitive improgan responses in CB1
-deficient mice [17
], 3) reduced improgan antinociception in cannabinoid-tolerant mice [26
], 4) lack of improgan affinity for known cannabinoid receptors [9
], and 5) inhibition of cannabinoid antinociception by the P450 inhibitor CC12 [16
]. In addition, recent in vivo studies of RVM neuronal activity found that CC12 reverses some (but not all) of improgan’s actions [11
]. These results support the hypothesis that a P450-based mechanism follows activation of the improgan receptor. The alternative hypothesis (that CC12 is an improgan receptor blocker) predicts that CC12 should block all improgan actions, but none of the effects of opioids or cannabinoids; this is not what was found. Many elements of the proposed model () remain untested.
The present findings also broaden the significance of the recently-discovered P450 epoxygenase mechanism for opioid analgesia [4
]. For example, dose-response curves with P450 inhibitors showed both antinociceptive () as well as anti-analgesic () actions The former could lead to novel analgesic drug development. In addition, two kinds of P450-deficient mutant mice (Cprlow
, , and brain-Cpr
- null, [4
] ) have now been shown to lack normal responses to analgesic drugs (improgan and morphine, respectively). The present studies are also the first to show that the anti-analgesic activity of P450 inhibitors in control subjects is not observable in P450-deficient mice (). Finally, taken with other recent results, the present findings show that both non-opioid (improgan) and opioid (morphine) analgesic drugs use P450 epoxygenase mechanisms, suggesting a point of convergence for these two kinds of signaling (). Further studies are needed to investigate this hypothesis, and to identify the relevant P450 isoforms, their cellular localization(s), and the products of their activity.