Facilitation of enkephalins catabolism inhibitor-induced antinociception by drugs classically used in pain management.

The aim of this study was to investigate the facilitatory effects of subanalgesic or low doses of different drugs (acetylsalicylic acid, ibuprofen and morphine) on the antinociceptive responses induced by the endogenous opioid peptides, enkephalins, protected from their catabolism by the dual enkephalin-degrading enzymes inhibitor RB101. According to the analgesic profile of the three studied compounds different antinociceptive assays were used: the hot plate and formalin tests in mice, and the tail flick and paw pressure tests on inflamed paws in rats and polyarthritic rats. Facilitatory effects of subanalgesic doses of acetylsalicylic acid and ibuprofen on RB101-induced antinociceptive responses were observed in the early and late phases of the formalin test, respectively. In the hot plate, tail flick and paw pressure tests, the dose-dependent analgesic effects of RB101 were strongly potentiated by subanalgesic doses of morphine (0.5 mg/kg), while in these tests, acetylsalicylic acid and ibuprofen were unable to modify the RB101-induced antinociceptive responses. The synergism in antinociceptive effects observed with the combination of RB101 and morphine supported by isobolographic analysis, may have interesting clinical implications, considering both the lack of opiate drawbacks observed with RB101 and the high potentiation of its antinociceptive effects with very low doses of morphine.

Effects of the potent analgesic enkephalin-catabolizing enzyme inhibitors RB101 and kelatorphan on respiration.

We investigated whether the enkephalin-catabolizing enzyme inhibitors RB101 and kelatorphan, which have been shown to be potent analgesics, depress respiration as do opioid analgesics. Ventilation was measured in cats and rodents by the barometric method, in the awake state and during anesthesia. Tissue distribution of the inhibitors was either generalized (RB101, 40-160 mg/kg i.p.), largely restricted by the blood-brain barrier to the periphery (kelatorphan, 0.7-20 mg/kg i.v.), or restricted to the brainstem (i.c.v. injection of RB101 in the fourth ventricle). RB101 did not affect ventilation in any condition tested, and large doses of kelatorphan produced a naloxone-reversible increase in ventilation and breathing frequency. Thus endogenous opioids released during conditions of normal ventilation do not exert any depressant neuromodulatory effect on this function, even when their extracellular concentrations are increased by peptidase inhibitors. The differential effect of these inhibitors on ventilation and nociception is discussed. We conclude that kelatorphan and RB101 are devoid of respiratory-depressant effects and might be interesting pharmacological alternatives to morphine and other opioid agonists.

Aminopeptidase A, which generates one of the main effector peptides of the brain renin-angiotensin system, angiotensin III, has a key role in central control of arterial blood pressure.

Overactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several experimental animal models. We have recently reported that, in the murine brain RAS, angiotensin II (AngII) is converted by aminopeptidase A (APA) into angiotensin III (AngIII),which is itself degraded by aminopeptidase N (APN), both peptides being equipotent to increase vasopressin release and arterial blood pressure when injected by the intracerebroventricular (i.c.v.) route. Because AngII is converted in vivo into AngIII, the exact nature of the active peptide is not precisely known. To delineate their respective roles in the central control of cardiovascular functions, specific and selective APA and APN inhibitors are needed to block the metabolic pathways of AngII and AngIII respectively. In the absence of such compounds for APA, we first explored the organization of the APA active site by site-directed mutagenesis. This led us to propose a molecular mechanism of action for APA similar to that proposed for the bacterial enzyme thermolysin deduced from X-ray diffraction studies. Secondly, we developed a specific and selective APA inhibitor, compound EC33 [(S)-3-amino-4-mercaptobutylsulphonic acid], as well as a potent and selective APN inhibitor, PC18 (2-amino-4-methylsulphonylbutane thiol). With these new tools we examined the respective roles of AngII and AngIII in the central control of arterial blood pressure. A central blockade of APA with the APA inhibitor EC33 suppressed the pressor effect of exogenous AngII, suggesting that brain AngII must be converted into AngIII to increase arterial blood pressure. Furthermore, EC33, injected alone i.c.v. but not intravenously, caused a dose-dependent decrease in arterial blood pressure by blocking the formation of brain AngIII but not systemic AngIII. This is corroborated by the fact that the selective APN inhibitor PC18 administered alone via the i.c.v. route increased arterial blood pressure. This pressor response was blocked by prior treatment with the angiotensin type 1 receptor antagonist losartan, showing that blocking the action of APN on AngIII metabolism leads to an increase in endogenous AngIII levels, resulting in arterial blood pressure increase through an interaction with angiotensin type 1 receptors. These results demonstrate that AngIII is a major effector peptide of the brain RAS, exerting a tonic stimulatory control over arterial blood pressure. Thus APA, the enzyme responsible for the formation of brain AngIII, represents a potential central therapeutic target that justifies the development of APA inhibitors, crossing the blood-brain barrier, as central anti-hypertensive agents.

PC18, a specific aminopeptidase N inhibitor, induces vasopressin release by increasing the half-life of brain angiotensin III.

Angiotensin III (AngIII), which is metabolized in vivo by aminopeptidase N (APN), was previously shown to be one of the main effector peptides of the brain renin-angiotensin system (RAS) in the control of vasopressin release. Recently, a potent APN inhibitor, PC18 (2-amino-4-methylsulfonyl butane thiol, methionine thiol), has been developed. In this study, we first checked the in vitro selectivity of PC18 towards APN, aminopeptidase A (APA) and aminopeptidase B (APB), three zinc metalloproteases with significant identity between their amino acid sequences. The Ki values of this compound on APN were found to be in the nanomolar range (Ki = 8.0 +/- 1.7 nM) but it was 2,150 and 125 times less active on APA and APB, respectively. Secondly, we evaluated in vivo the effect of brain APN inhibition with PC18 on the inactivation of brain AngIII and on vasopressin secretion in mice. For this purpose, mice received [3H]AngII intracerebroventricularly in the presence or absence of the APN inhibitor PC18 (30 microg). At different times after the injection, [3H]AngIII levels were evaluated from hypothalamus homogenates after separation by cation-exchange chromatography. PC18 induced an accumulation of [3H]AngIII, increasing its half-life 3.9 times as compared with control values. In addition, the effect of PC18 on vasopressin release was studied in mice. PC18 (10-100 microgram) was injected intracerebroventricularly, and plasma vasopressin levels were estimated by radioimmunoassay. PC18 increased vasopressin levels in a dose-dependent manner. The maximal increase in vasopressin release (+220%) is observed for a dose of PC18 of 100 microgram and was inhibited 75% by the coadministration of the AngII receptor antagonist (Sar1-Ala8)-AngII (0.5 microgram). These results indicate that in vivo, in the mouse brain, APN inhibition by PC18 increases the half-life of endogenous AngIII, resulting in an enhanced vasopressin release.

Evidence by site-directed mutagenesis that arginine 203 of thermolysin and arginine 717 of neprilysin (neutral endopeptidase) play equivalent critical roles in substrate hydrolysis and inhibitor binding.

Neprilysin (neutral endopeptidase-24.11, EC 3.4.24.11) is a mammalian zinc-endopeptidase involved in the degradation of biologically active peptides. Although no atomic structure is available for this enzyme, site-directed mutagenesis studies have shown that its active site resembles closely that of the bacterial zinc-endopeptidase, thermolysin (EC 3.4.24.27). One active site residue of thermolysin, Arg-203, is involved in inhibitor binding by forming hydrogen bonds with the carbonyl group of a residue in the P1 position and also participates in a hydrogen bond network involving Asp-170. Sequence alignment data shows that Arg-717 of neprilysin could play a similar role to Arg-203 of thermolysin. This was investigated by site-directed mutagenesis with Arg-203 of thermolysin and Arg-717 of neprilysin being replaced by methionine residues. This led, in both cases, to decreases in kcat/Km values, of 122-fold for neprilysin and 2300-fold for thermolysin, essentially due to changes in kcat. The Ki values of several inhibitors were also increased for the mutated enzymes. In addition, the replacement of Asp-170 of thermolysin by Ala residue resulted in a decrease in kcat/Km of 220-fold. The results, coupled with a molecular modeling study, suggest that Arg-717 of neprilysin corresponds to Arg-203 of thermolysin and that in both enzymes a hydrogen bond network exists, involving His-142, Asp-170, and Arg-203 in thermolysin and His-583, Asp-650, and Arg-717 in neprilysin, which is crucial for hydrolytic activity.

Cooperative exosite-dependent cleavage of synaptobrevin by tetanus toxin light chain.

The light chain (L chain) of tetanus neurotoxin (TeNT) has been shown to have been endowed with zinc endopeptidase activity, selectively directed toward the Gln76-Phe77 bond of synaptobrevin, a vesicle-associated membrane protein (VAMP) critically involved in neuroexocytosis. In previous reports, truncations at the NH2 and COOH terminus of synaptobrevin have shown that the sequence 39-88 of synaptobrevin is the minimum substrate of TeNT, suggesting either the requirement of a well defined three-dimensional structure of synaptobrevin or a role in the mechanism of substrate hydrolysis for residues distal from the cleavage site. In this study, the addition of NH2- and COOH-terminal peptides of synaptobrevin, S 27-55 (S1) and S 82-93 (S2), to the synaptobrevin fragment S 56-81 allowed the cleavage of this latter peptide by TeNT to occur. This appears to result from an activation process mediated by the simultaneous binding of S1 and S2 with complementary sites present on TeNT as shown by surface plasmon resonance experiments and the determination of kinetic constants. All these results favor an exosite-controlled hydrolysis of synaptobrevin by TeNT, probably involving a conformational change of the toxin. This could account for the high degree of substrate specificity of TeNT and, probably, botulinum neurotoxins.

Identification and characterization of various cholecystokinin B receptor mRNA forms in rat brain tissue and partial determination of the cholecystokinin B receptor gene structure.

Using the powerful method of DNA amplification, we have been able to isolate several cholecystokinin B (CCKB) receptor (CCKB-R) mRNA forms from rat brain tissue, allowing the detection of a truncated mRNA species and the determination of the CCKB-R gene structure. Unspliced precursor mRNA and the mature form were identified in the cerebral cortex, hypothalamus, and hippocampus in apparently differing proportions according to the region examined, suggesting that the expression of the CCKB-R could be modulated at a posttranscriptional level. In the case of the cerebellum, only a completely unspliced mRNA form was detected, in agreement with previous studies in which CCKB ligand binding sites were not observed. In contrast, a truncated CCKB-R mRNA, lacking 250 bp, was detected in all the studied brain regions except for the cerebellum. This mRNA, for which a cellular function has not been assigned, potentially encodes a protein consisting of 168 amino acids.

Antinociceptive response induced by mixed inhibitors of enkephalin catabolism in peripheral inflammation.

RB101 (N-((R,S)-2-benzyl-3[(S)(2-amino-4-methylthio)butyl dithio]-1-ox-opropyl)-L-phenylalanine benzyl ester) is a recently developed full inhibitor of the enkephalin-catabolizing enzymes able to cross the blood-brain barrier, whereas RB38A ((R)-3-(N-hydroxycarboxamido-2-benzylpropanoyl)-L-phenylalanine) is as potent as RB101 but almost unable to enter the brain. In this study, we have investigated the effects of systemic administration of morphine, RB101 and RB38A on nociception induced by pressure on inflamed peripheral tissues. Antinociceptive test was performed between 4 and 5 days after injection into the rat left hindpaw of Freund's complete adjuvant to produce localized inflammation. Morphine (1, 2 and 4 mg/kg, i.v.) induced antinociception in inflamed paws at all the doses used, and only at the highest dose in non-inflamed paws. RB101 (10 and 20 mg/kg, i.v.) induced an antinociceptive response only in the inflamed paws. RB38A, also induced an antinociceptive effect in the inflamed paws, but only at the highest dose (20 mg/kg, i.v.). The responses induced by morphine and the inhibitors of enkephalin catabolism were antagonized by the systemic administration of naloxone (1 mg/kg) or methylnaloxonium (2 mg/kg) which acts essentially outside the brain. Central injection (i.c.v.) of methylnaloxonium (2 micrograms) blocked the effect of morphine only in non-inflamed paws, and slightly decreased the response induced by RB101 on inflamed paws. These results indicate that the endogenous opioid peptides, probably enkephalins, are important in the peripheral control of nociception from inflamed tissues.

Antinociceptive effect of systemic PC 12, a prodrug mixed inhibitor of enkephalin-degrading enzymes, in normal and arthritic rats.

The antinociceptive effects of various i.v. doses (2.5, 3.75, 5, 10, 15 and 20 mg/kg) of a prodrug mixed inhibitor of enkephalin degrading enzymes, PC 12, were tested in normal and Freund's adjuvant-induced arthritic rats, using vocalization thresholds to paw pressure as a nociceptive test. In normal animals, PC 12 produced a dose-dependent antinociceptive effect over the 2.5-15 mg/kg range, but the time-course of the response was shorter with the dose of 20 mg/kg. In arthritic rats, PC 12 had no significant effect at 2.5 mg/kg, but induced a highly significant dose-dependent antinociceptive action with 3.5 and 5 mg/kg, which decreased with the highest doses. The antinociceptive effect of PC 12 (10 mg/kg i.v.) was prevented by naloxone (0.5 mg/kg i.v.) in the two categories of rats, providing evidence for the involvement of opioid receptors. These results are discussed in relation with the increased level of endogenous opioid substances in the spinal dorsal horn of arthritic rats and the nociceptive test used.

Modulation of opioid antinociception by CCK at the supraspinal level: evidence of regulatory mechanisms between CCK and enkephalin systems in the control of pain.

1. Much evidence in the literature supports the idea that cholecystokinin (CCK) interacts with opioids in pain mechanisms. In this work, we have investigated the supraspinal interactions between enkephalins and CCK, using the hot plate test in mice. 2. Intracerebroventricular (i.c.v.) administration of BDNL (a mixed CCKA/CCKB agonist) induced dose-dependent antinociceptive responses on both paw lick and jump responses. In contrast, using the same test, the i.c.v. injection of BC 264 (a selective CCKB agonist) induced a hyperalgesic effect, which was restricted to paw licking and occurred only at a high dose of 2.5 nmol. 3. In addition, i.c.v. administration of BDNL potentiated the antinociceptive effects of the mixed inhibitor of enkephalin degrading enzymes, RB 101 and of the mu-agonist, DAMGO, while BC 264 reduced these effects. 4. Furthermore, at a dose where it interacts selectively with delta-opioid receptors, the opioid agonist BUBU reversed the hyperalgesic responses of BC 264 (2.5 nmol) but was unable to modify the effects induced by BDNL. 5. Taken together, these results suggest the existence of regulatory mechanisms between CCK and enkephalin systems in the control of pain. These regulatory loops could enhance the antinociceptive effects of morphine allowing the opiate doses used to be reduced and thus, possibly, the side-effects to be minimized.

Unlike morphine the endogenous enkephalins protected by RB101 are unable to establish a conditioned place preference in mice.

The mixed inhibitor prodrug, RB101, was used to study the motivational properties of the endogenous opioid peptides, the enkephalins. In the conditioned place preference test, which is commonly used to investigate the reinforcing properties of drugs, mice alternately treated with morphine (3 mg/kg i.p.) on the initially non-preferred compartment and with saline on the preferred one, for four place pairings, spent more time in the drug-associated compartment. This shift in place preference after the conditioning procedure was not found after treatment with RB101 (80 mg/kg i.p.). Administration of naloxone (1 mg/kg s.c.) after the conditioning phase increased the preference for the drug-associated compartment of mice treated with 6 mg/kg (i.p.) of morphine. This illustrates the negative motivational properties of morphine withdrawal or the establishment of psychic dependence on the drug. In contrast, no modification of preference was observed after injection of naloxone in animals treated with a high dose of RB101 (160 mg/kg i.p.). The failure to establish conditioned place preference by inhibiting endogenous enkephalin metabolism, and the lack of development of psychic dependence after RB101 administration demonstrate for the first time the interest of mixed inhibitors of enkephalin-degrading enzymes as potent new non-addictive analgesics.

Centrally administered cholecystokinin suppresses feeding through a peripheral-type receptor mechanism.

Agonists and antagonists selective for the brain-type [cholecystokinin (CCK)-B] and the peripheral-type (CCK-A) CCK receptor were used to localize the site(s) of action at which CCK inhibits food consumption. BC 264, a highly selective CCK-B receptor agonist, did not decrease consumption of a palatable meal when administered either i.p. or into the lateral ventricles of the brain, whereas CCK decreased feeding when administered i.p. at the same doses. CCK decreased feeding when administered i.v.t. at a high dose, 5 micrograms. L-364,718, an antagonist selective for the CCK-A receptor, blocked completely the action of centrally administered CCK, whereas L-365,260, a selective CCK-B receptor antagonist, had no effect on the ability of centrally administered CCK to inhibit feeding. To estimate the quantity of i.v.t. administered CCK which reached the periphery, a tracer of radiolabeled [3H]p-CCK8 ([3H]CCK octapeptide sulfate), combined with unlabeled pCCK8 (5 micrograms) was administered i.c.t. Thirty minutes after administration, intact radiolabeled pCCK8 was extracted from the plasma and measured in the blood in nanomolar concentrations, exceeding the amounts of CCK octapeptide sulfate reported previously to be present in the plasma after a meal. Intraventricularly administered CCK thus appears to reduce feeding in the rat through a mechanism involving a CCK-A receptor subtype in the periphery.

Affinity states of rat brain opioid receptors in different tissue preparations.

The binding of [3H]Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol ([3H]DAGO) and [3H]Tyr-D-Thr-Gly-Phe-Leu-Thr ([3H]DTLET), selective agonists for mu- and delta-opioid binding sites, respectively, has been investigated using different rat brain tissue preparations and buffer systems. The results were compared with the binding of the ligands to crude membrane fractions in Tris-HCl, the most commonly used preparation for binding studies. In both rat brain membranes and intact cells, Krebs-HEPES induced a decrease in the affinities of [3H]DAGO and [3H]DTLET, but little modification was observed when 20-microns tissue slices were used, whatever the brain area studied. The dissociation rate of [3H]DTLET was clearly dependent on the tissue preparation used, because the koff value of this ligand in Krebs-HEPES was 2.5-fold higher in membrane fractions than that measured in intact cells. The kinetic dissociation constant of [3H]DTLET in membrane fractions in Krebs-HEPES was 6.5-fold greater than that measured in Tris-HCl. In intact cells, the koff value for [3H]DTLET was lower than that found in membrane fractions in Krebs-HEPES and similar to that observed in membrane preparations in Tris-HCl supplemented with 30 mM NaCl. These data suggest (a) that the koff constant of [3H]DTLET was regulated by the ionic environment of the delta-opioid receptor, which is clearly dependent on the preservation of cellular structure, and (b) that opioid receptors could exist under different states that are regulated, in part, by the intracellular Na+ concentration.

Lack of significant changes in mu, delta opioid binding sites and neutral endopeptidase EC 3.4.24.11 in the brain and spinal cord of arthritic rats.

The possible changes in neutral endopeptidase EC 3.4.24.11 ("enkephalinase", NEP), mu and delta opioid binding sites, were investigated using in vitro quantitative radioautography in various regions of the central nervous system of the Freund's adjuvant-induced arthritic rat, a model of chronic pain. Enkephalinase was labeled by a specific tritiated inhibitor, [3H]N-[(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]glycine ([3H]HACBO-Gly), while mu and delta opioid binding sites were selectively labelled with [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol ([3H]DAGO) and [3H]Tyr-D-Thr-Gly-Phe-Leu-Thr ([3H]DTLFT), respectively. As compared to controls, no significant modifications were found in NEP, mu or delta binding sites at both supraspinal and spinal levels of arthritic rats. These results suggest that the enhanced efficiency of exogenous opioids or endogenous enkephalins, reported to occur in this model of chronic inflammatory pain, are not directly related to changes in mu and delta opioid binding sites or steady state levels of NEP.

Bisintercalation of ditercalinium into a d(CpGpApTpCpG)2 minihelix: a 1H- and 31P-NMR study.

The structure of the complex formed in aqueous solution between ditercalinium, a bisintercalating drug, and the self-complementary hexanucleotide d(CpGpApTpCpG)2 is investigated by 400-MHz 1H-nmr and 162-MHz 31P-nmr. Whatever the drug to helix ratio, ditercalinium occurred in the bound form, whereas free and complexed hexanucleotide are in slow exchange. This allows unambiguous resonance assignment through two-dimensional chemical exchange experiments. The strong upfield shifts measured on most aromatic protons on both drug and bases as well as on DNA imino protons are consistent with bisintercalation of the dimer. Nuclear Overhauser effects observed between drug and nucleotide protons give a defined geometry for complexation, and suggest a DNA conformational change upon drug binding.

Distribution of CCK mRNA in particular regions (hippocampus, periaqueductal grey and thalamus) of the rat by in situ hybridization.

Cholecystokinin (CCK) mRNA was detected by in situ hybridization at high magnification in some rat brain regions where CCK octapeptide (CCK-8) is thought to produce its pharmacological effects. The labeling of the dentate gyrus and the sparse but intensively stained cells found in the CA1 layer, stratum radiatum and hilus could correspond to interneurons involved in hippocampal neural activity, in agreement with excitatory responses induced by local injection of CCK-8. The intense labeling of the Edinger-Westphal nucleus and more generally the presence of CCK mRNA in the periaqueductal gray and thalamus ventrobasal nuclei could account for the various effects of CCK in pain transmission.

In vivo 31P MRS in new antineoplastic agents evaluation on experimental tumor models.

Two new antineoplastic agents, a nitrosourea and a DNA-bis-intercalator have been studied in vivo by 31P magnetic resonance spectroscopy on a rat glioma and Walker carcinoma. On rat glioma, spectra are modified when the tumor is treated by the nitrosourea, showing the depletion of high-energy phosphates. On Walker carcinoma both drugs delay the tumor evolution to necrosis, showing important levels of high-energy phosphates on NMR spectra. There appears to be a great dependence upon energy metabolism during chemotherapy, depending on the nature and physiology of the observed tumor.

Conformational studies of d(m5CpGpm5CpG) and d(CpGpCpG) by 1H and 31P NMR.

Exhaustive conformational studies of d(CpG)2 and d(m5CpG)2, two convenient targets for DNA bisintercalating drugs, have been carried out by 1H and 31P NMR in low salt concentration and in the presence of 30% ethanol. Unambiguous 31P assignments of the B for are obtained with low-power heteronuclear decoupling experiments, while 31P assignments in the Z form are obtained by two-dimensional homonuclear chemical exchange experiments. The 31P chemical shifts and 3JH3'P coupling constants studied at various temperatures in methylated and non-methylated tetranucleotides, are interpreted as resulting from conformational differences between the compounds. These features are corroborated by homonuclear proton nuclear Overhauser effect experiments showing the steric role of the 5-methylcytosine in the induction of an alternating B form in d(m5CpG)2.