Ovarian sex steroid-dependent plasticity of nociceptin/orphanin FQ and opioid modulation of spinal dynorphin release.

Pregnancy and its hormonal simulation via 17beta-estradiol (E(2)) and progesterone (P) are associated with spinal opioid antinociception, primarily driven by augmented dynorphin/kappa-opioid activity. This study addresses the ovarian sex steroid-activated mechanism(s) that underlie this activation using an ex vivo spinal cord preparation. In lumbar spinal cord obtained from control animals, exogenous kappa- or delta-opioid agonists (but not mu), as well as nociceptin (orphanin FQ; N/OFQ), dose dependently inhibit the stimulated release of dynorphin. Consistent with these observations, stimulated dynorphin release is enhanced following selective blockade of opioid or N/OFQ receptors, indicating that their endogenous ligands are negative modulators of dynorphin release. In lumbar spinal cord obtained from ovariectomized animals exposed to pregnancy blood levels of E(2)/P, basal and stimulated rates of dynorphin release increase approximately 2-fold. Moreover, evoked dynorphin release is no longer negatively modulated by kappa- or delta-opioid agonists or N/OFQ. Interestingly, in these preparations, release can be facilitated by delta-opioid receptor activation, and neither spinal opioid nor N/OFQ receptor blockade enhances evoked dynorphin release. Consistent with these observations, guanosine-5'-O-3-[(35)S]-thio triphosphate binding analyses indicate a reduction in functional N/OFQ receptors. These data indicate that at least part of the E(2)/P-induced augmented activity of lumbar dynorphin neurons results from their disinhibition via the removal of negative opioid and N/OFQ modulation. These results underscore the plasticity of spinal opioid and N/OFQ systems and their dependence on the ovarian sex steroid milieu. Ovarian sex steroid-activated antinociception reveals mechanisms that enable sustained opioid activation without concomitant tolerance formation.

Characterization of opiates, neuroleptics, and synthetic analogs at ORL1 and opioid receptors.

Nociceptin/orphanin FQ (N/OFQ) was recently identified as the endogenous ligand for the opioid-receptor like (ORL1) receptor. Although the ORL1 receptor shows sequence homology with the opioid receptors, the nociceptin/ORL1 ligand-receptor system has very distinct pharmacological actions compared to the opioid receptor system. Recently, several small-molecule ORLI receptor ligands were reported by pharmaceutical companies. Most of these ligands had close structural similarities with known neuroleptics and opiates. In this study, we screened several available neuroleptics and opiates for their binding affinity and functional activity at ORL1 and the opioid receptors. We also synthesized several analogs of known opiates with modified piperidine N-substituents in order to characterize the ORL1 receptor ligand binding pocket. Substitution with the large, lipophilic cyclooctylmethyl moiety increased ORL1 receptor affinity and decreased mu receptor affinity and efficacy in the fentanyl series of ligands but had a different effect in the oripavine class of opiate ligands. Our results indicate that opiates and neuroleptics may be good starting points for ORL1 receptor ligand design, and the selectivity may be modulated by appropriate structural modifications.

1,2,4-Benzotriazine 1,4-dioxides. An important class of hypoxic cytotoxins with antitumor activity.

Tirapazamine (1,2,4-benzotriazin-3-amine 1,4-dioxide, SR 4233, WIN 59075) is the lead compound representing this class of anticancer drugs. It is also the first compound to be introduced in the clinic as a pure bioreductive cytotoxic agent. Tirapazamine represents a completely novel approach to the treatment of solid tumors and has generated considerable interest, with research being carried out on all aspects of the its anticancer activity. Phase III trials of tirapazamine in combination with cisplatin (cDDP) have recently been concluded, and phase II trials of triapazamine in combination with irradiation are presently being performed. We developed a drug discovery program into this class of compounds designed to produce derivatives with improved in vivo activity against solid tumors. Based on the hypothesis that these compounds require bioreductive activation for antitumor activity, the research was primarily directed at producing analogues with greater electron affinity and improved aqueous solubility. The in vitro and in vivo data for a variety of structural analogues clearly show that 1,2,4-benzotriazine 1,4-dioxides have considerable potential as anticancer agents. When their activity is compared directly with the activity observed for triapazamine, the most promising series of analogues appears to be the 3-alkyl-substituted derivatives, especially the 3-ethyl- and 3-(2'-methoxyethyl)-derivatives, SR 4895 and SR 4941 respectively.

Preclinical development and current status of the fluorinated 2-nitroimidazole hypoxia probe N-(2-hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide (SR 4554, CRC 94/17): a non-invasive diagnostic probe for the measurement of tumor hypoxia by magnetic resonance spectroscopy and imaging, and by positron emission tomography.

Hypoxia occurs to a variable extent in a vast majority of rodent and human solid tumors. It results from an inadequate and disorganized tumor vasculature, and hence an impaired oxygen delivery. A probe for the non-invasive detection of tumor hypoxia could find important utility in the selection of patients for therapy with bioreductive agents, anti-angiogenic/anti-vascular therapies and hypoxia-targeted gene therapy. In addition, tumor hypoxia has been shown to predict for treatment outcome following radio- or chemotherapy in human cancers, the underlying mechanism for which may involve hypoxia driving genetic instability and resulting tumor progression. Beyond oncology, utility can also be envisaged in stroke, ischemic heart disease, peripheral vascular disease, arthritis and other disorders. Design, validation, preclinical development and current status of a fluorinated 2-nitroimidazole, N-(2-hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-l-imidazolyl) acetamide (SR 4554, CRC 94/17), which has been rationally designed for the measurement of tumor hypoxia by magnetic resonance spectroscopy (MRS) and imaging (MRI), are reviewed. Application in positron emission tomography (PET) detection is also proposed. Design goals were: (i) a nitro group with appropriate redox potential for selective reduction and binding in hypoxic tumor cells; (ii) hydrophilic/hydrogen bonding character in the side chain to limit nervous tissue penetration and prevent neurotoxicity; and (iii) three equivalent fluorine atoms to enhance MRS/MRI detection, located in a metabolically stable position. Reduction of SR 4554 by mouse liver microsomes was dependent on oxygen content, with a half-maximal inhibition at 0.48 +/- 0.06%. SR 4554 underwent nitroreduction by hypoxic but not oxic tumor cells in vitro and electron energy loss spectroscopic analysis showed selective retention in the hypoxic regions of multicellular tumor spheroids. Pharmacokinetic design goals were met. In particular, low brain tissue concentrations were seen in contrast to excellent tumor levels, as measured by high performance liquid chromatography. The extent of this restricted entry to brain tumor was surprising given the overall octanol/water partition coefficient and was attributed to the hydrophilic/hydrogen bonding character of the side chain. Quantitative MRS was used to assess the retention of 19F signal in murine tumors and human tumor xenografts. The 19F retention index (FRI; ratio of 19F signal levels at 6 h relative to that at 45 min) ranged from 0.5 to 1.0 and 0.2 to 0.9 for murine tumors and human xenografts respectively. The correlation between SR 4554 retention and pO2 was not a linear one, but when FRI was > 0.5, the % pO2 < or = 5 mmHg was always > 60%, indicating that high FRI was associated with low levels of oxygenation. Finally, whole body 19F-MRI in mice demonstrated that SR 4554 and related metabolites localized mainly in tumor, liver and bladder regions. A selective MRS signal was readily detectable in tumors at doses at least 7-fold lower than those likely to cause toxicity in mice. We conclude that proof of principle is established for the use of SR 4554 as a non-invasive MRS/MRI probe for the detection of tumor hypoxia. Based on these promising studies, SR 4554 has been selected for clinical development.

Preclinical evaluation of the fluorinated 2-nitroimidazole N-(2-hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide (SR-4554) as a probe for the measurement of tumor hypoxia.

A novel probe, N-(2-hydroxy-3,3,3,-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide (SR-4554), has been used to detect tumor hypoxia noninvasively by 19F magnetic resonance spectroscopy (19F MRS). The compound was designed to undergo a hypoxia-dependent, one-electron reduction to metabolites that are selectively retained in tumors and has attractive pharmacokinetic, toxicological, and detection sensitivity properties. As a prelude to clinical studies, we report here for the first time on the ability to detect a MR signal following SR-4554 administration in various transplantable tumors and describe validation studies, consisting of a correlation between signal retention and radiobiological hypoxic fraction, and the effects of modulating the degree of hypoxia by hydralazine and carbogen breathing. SR-4554 was absorbed and then eliminated from EMT6 tumors with a half-life of 51 min following an injection of 180 mg/kg i.p. of SR-4554. Using a quantitative 19F MRS technique, the 19F retention index (19FRI; 19F signal level at 6 h/45 min) was determined for four commonly used murine tumors (EMT6, SCCVII, KHT, and RIF-1). The retention of high tumor concentrations of fluorinated probe at 6 h, despite the much lower (20-fold) concentration of parent SR-4554 detected by high-performance liquid chromatography, was consistent with the involvement of one or more nitroreduced metabolites and suggested that 19F MRS might give a quantitative measure of tumor hypoxia. In these murine tumors, 19FRI correlated with the reported radiobiological hypoxic fraction of the tumors (r = 0.988; P = 0.01). In addition, changes in tumor microenvironment were detected by 19F MRS. An increase in hypoxia induced by hydralazine treatment of RIF-1 tumor-bearing mice was associated with a 2.4-fold increase in 19FRI compared to untreated controls. In contrast, carbogen breathing by C3H mammary tumor-bearing mice produced a 6-fold decrease in the 19FRI compared to air-breathing mice. The data presented support the preclinical and clinical development of SR-4554 as a noninvasive probe for tumor hypoxia.

The pharmacokinetics, bioavailability and biodistribution in mice of a rationally designed 2-nitroimidazole hypoxia probe SR-4554.

N-(2-Hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide (SR-4554) is a fluorinated 2-nitroimidazole which has been rationally designed as a non-invasive probe for tumor hypoxia. The key selection criteria for this molecule were low central nervous system penetration and toxicity, high metabolic stability other than nitroreduction, good tumor uptake and high sensitivity for detection by magnetic resonance spectroscopy. As part of the pre-clinical development strategy, pharmacokinetic, bioavailability and biodistribution studies were performed in mice. Pharmacokinetic studies in mice demonstrated that SR-4554 was rapidly absorbed into plasma following i.p. administration and eliminated with a half-life of 42 min, similar to other 2-nitroimidazoles. By comparing the areas under the concentration-time curve (AUC), the tumor exposure towards SR-4554 was on average 84% of the value obtained for the plasma exposure. SR-4554 penetrated tumor tissue extremely well but, in contrast to misonidazole and certain other fluorinated analogues, its distribution into brain tissue was poor (AUCbrain/AUCplasma = 0.07), suggesting potentially lower toxicity in spite of its higher lipophilicity (P = 0.43 versus 0.63, respectively). The bioavailability of SR-4554 from i.p. and p.o. routes was 100 and 96% respectively. In non-tumor-bearing mice, SR-4554 was excreted mainly as unchanged drug. The percentage of the injected i.p. dose of SR-4554 excreted unchanged in the urine over 24 h was 68 +/- 8%. Neither SR-4554 nor its metabolites were detected in mouse feces. We propose that these favorable pharmacokinetic properties of SR-4554 are due to the hydrophilic character and hydrogen-bonding capability of the amide and hydroxyl functions in the compound.

Development and validation of a solid-phase extraction and high-performance liquid chromatographic assay for a novel fluorinated 2-nitroimidazole hypoxia probe (SR-4554) in Balb/c mouse plasma.

N-(2-Hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide, a novel 2-nitroimidazole, is currently being developed as a non-invasive probe for tumour hypoxia. A sensitive (minimum quantifiable level = 25 ng/ml; C.V. = 6.01%) and selective assay has, therefore, been developed for the analysis of this compound in mouse plasma. The assay employed a solid-phase extraction followed by a rapid (10 min) HPLC analysis with UV-photodiode-array detection. No drug-related metabolites were observed in plasma when mice were treated with 180 mg/kg of the drug. The assay has proved to be suitable for studying the plasma pharmacokinetics of this fluorinated 2-nitroimidazole in mice.

Quaternary salts of 2-[(hydroxyimino)methyl]imidazole. 4. Effect of various side-chain substituents on therapeutic activity against anticholinesterase intoxication.

A series of quaternary salt derivatives of 2-[(hydroxyimino)methyl]-1-methylimidazole incorporating various side chains bearing ether, silyl, nitrile, ester, halogen, nitro, sulfone, amino, or aminosulfonyl substituents was prepared and evaluated in vivo for the treatment of anticholinesterase intoxication. Test results in the mouse revealed that the type and location of the side-chain substituent both have a significant influence on the toxicity and antidotal effectiveness of the compounds. Some of the more active examples represent the most potent therapeutics to date against intoxication by the powerful cholinesterase inhibitors soman and tabun. Significantly, the antidotal effectiveness of the compounds was not dependent on the inhibiting agent nor was there any correlation between in vivo efficacy and in vitro reactivation of ethyl (4-nitrophenyl)methylphosphonate inhibited human acetylcholinesterase. These observation suggested that the main mode of antidotal protection by the compounds is something other than enzyme reactivation.