Selective HCN1 block as a strategy to control oxaliplatin-induced neuropathy.

Chemotherapy-Induced Peripheral Neuropathy (CIPN) is the most frequent adverse effect of pharmacological cancer treatments. The occurrence of neuropathy prevents the administration of fully-effective drug regimen, affects negatively the quality of life of patients, and may lead to therapy discontinuation. CIPN is currently treated with anticonvulsants, antidepressants, opioids and non-opioid analgesics, all of which are flawed by insufficient anti-hyperalgesic efficacy or addictive potential. Understandably, developing new drugs targeting CIPN-specific pathogenic mechanisms would dramatically improve efficacy and tolerability of anti-neuropathic therapies. Neuropathies are associated to aberrant excitability of DRG neurons due to the alteration in the expression or function of a variety of ion channels. In this regard, Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are overexpressed in inflammatory and neuropathic pain states, and HCN blockers have been shown to reduce neuronal excitability and to ameliorate painful states in animal models. However, HCN channels are critical in cardiac action potential, and HCN blockers used so far in pre-clinical models do not discriminate between cardiac and non-cardiac HCN isoforms. In this work, we show an HCN current gain of function in DRG neurons from oxaliplatin-treated rats. Biochemically, we observed a downregulation of HCN2 expression and an upregulation of the HCN regulatory beta-subunit MirP1. Finally, we report the efficacy of the selective HCN1 inhibitor MEL57A in reducing hyperalgesia and allodynia in oxaliplatin-treated rats without cardiac effects. In conclusion, this study strengthens the evidence for a disease-specific role of HCN1 in CIPN, and proposes HCN1-selective inhibitors as new-generation pain medications with the desired efficacy and safety profile.

Design, synthesis and preliminary evaluation of a series of histone deacetylase inhibitors carrying a benzodiazepine ring.

A series of new histone deacetylase inhibitors were designed and synthesized based on hybridization between SAHA or oxamflatin and 5-phenyl-1,4-benzodiazepines. The compounds were tested for their enzyme inhibitory activity on HeLa nuclear extracts, and on human recombinant HDAC1 and HDAC6. Antiproliferative activity was tested on different cancer cells types, while proapoptotic activity was primarily tested on NB4 cells. The compounds showed IC50 values similar to those of SAHA. Compound (S)-8 displayed interesting activity against hematological and solid malignancies.

Influence of ring size on the cognition-enhancing activity of DM235 and MN19, two potent nootropic drugs.

A series of analogs of DM235 and MN19, characterized by rings with different size, have been prepared and evaluated for their nootropic activity in the mouse passive-avoidance test. It was found that the optimal ring size for the analogs of DM235, showing endocyclic both amidic groups, is 6 or 7 atoms. For the compounds structurally related to MN19, carrying an exocyclic amide group, the piperidine ring is the moiety which gives the most interesting compounds.

The new low-toxic histone deacetylase inhibitor S-(2) induces apoptosis in various acute myeloid leukaemia cells.

Histone deacetylase inhibitors (HDACi) induce tumour cell cycle arrest and/or apoptosis, and some of them are currently used in cancer therapy. Recently, we described a series of powerful HDACi characterized by a 1,4-benzodiazepine (BDZ) ring hybridized with a linear alkyl chain bearing a hydroxamate function as Zn(++)--chelating group. Here, we explored the anti-leukaemic properties of three novel hybrids, namely the chiral compounds (S)-2 and (R)-2, and their non-chiral analogue 4, which were first comparatively tested in promyelocytic NB4 cells. (S)-2 and partially 4--but not (R)-2--caused G0/G1 cell-cycle arrest by up-regulating cyclin G2 and p21 expression and down-regulating cyclin D2 expression, and also apoptosis as assessed by cell morphology and cytofluorimetric assay, histone H2AX phosphorylation and PARP cleavage. Notably, these events were partly prevented by an anti-oxidant. Moreover, novel HDACi prompted p53 and α-tubulin acetylation and, consistently, inhibited HDAC1 and 6 activity. The rank order of potency was (S)-2 > 4 > (R)-2, reflecting that of other biological assays and addressing (S)-2 as the most effective compound capable of triggering apoptosis in various acute myeloid leukaemia (AML) cell lines and blasts from patients with different AML subtypes. Importantly, (S)-2 was safe in mice (up to 150 mg/kg/week) as determined by liver, spleen, kidney and bone marrow histopathology; and displayed negligible affinity for peripheral/central BDZ-receptors. Overall, the BDZ-hydroxamate (S)-2 showed to be a low-toxic HDACi with powerful anti-proliferative and pro-apototic activities towards different cultured and primary AML cells, and therefore of clinical interest to support conventional anti-leukaemic therapy.

Selective pharmacological inhibition of the pacemaker channel isoforms (HCN1-4) as new possible therapeutical targets.

The pacemaker channel isoforms are encoded by the hyperpolarization-activated and cyclic nucleotide-gated (HCN) gene family and are responsible for diverse cellular functions including regulation of spontaneous activity in sino-atrial node cells and control of excitability in different types of neurons. Four channel isoforms exist (HCN1-HCN4). The hyperpolarization-activated cardiac pacemaker current (I(f)) has an important role in the generation of the diastolic depolarization in the sino-atrial node, while its neuronal equivalent (I(h)) is an important contributor to determination of resting membrane potential, and plays an important role in neuronal functions such as synaptic transmission, motor learning and generation of thalamic rhythms. Ivabradine is a novel, heart rate-lowering drug which inhibits the pacemaker (I(f)) current in the heart with high selectivity and with minimal effect on haemodynamic parameters. Ivabradine is beneficial in patients with chronic stable angina pectoris equally to beta receptor blocker and calcium channel antagonist drugs. There is increasing interest to apply this drug in other fields of cardiology such as heart failure, myocardial infarction, cardiac arrhyhtmias. Heart rate reduction might improve clinical outcomes in heart failure. HCN upregulation presumably contributes to increased (I(f)) and may play a role in ventricular and atrial arrhythmogenesis in heart failure. In the nervous system the HCN channels received attention in the research areas of neuropathic pain, epilepsy and understanding the mechanism of action of volatile anaesthetics. This article delineates that the pharmacological modulation of cardiac and neuronal HCN channels can serve current or future drug therapy and introduces some recently investigated HCN channel inhibitor compounds being potential candidates for development.

Design, synthesis and preliminary biological evaluation of new hydroxamate histone deacetylase inhibitors as potential antileukemic agents.

This study concerns the synthesis of new histone deacetylase inhibitors (HDACi) characterized by a 1,4-benzodiazepine ring used as the cap, joined through an amide function or a triple bond as connection units, to a linear alkyl chain bearing the hydroxamate function as Zn2+-chelating group. Biological tests performed in human acute promyelocytic leukemia NB4 cells showed that new hybrids can induce histone H3/H4 acetylation, growth arrest, and also apoptosis. Notably, chiral compounds exhibit stereoselective activity.

Pharmacological characterization of DM232 (unifiram) and DM235 (sunifiram), new potent cognition enhancers.

DM232 (unifiram) and DM235 (sunifiram) are potent cognition-enhancers, which are four order of magnitude more potent than piracetam. These compounds, although not showing affinity in binding studies for the most important central receptors or channels, are able to prevent amnesia induced by modulation of several neurotransmission systems. These compounds are able to increase the release of acetylcholine from rat cerebral cortex, and, as far as unifiram is concerned, to increase the amplitude of fEPSP in rat hippocampal slices. In vitro experiments, performed on hippocampal slices, also supported the hypothesis of a role of the AMPA receptors for the cognition-enhancing properties of unifiram and sunifiram.

WEB-2086 and WEB-2170 trigger apoptosis in both ATRA-sensitive and -resistant promyelocytic leukemia cells and greatly enhance ATRA differentiation potential.

PAF-receptor antagonists WEB-2086 and WEB-2170 (WEBs) have been previously shown to induce differentiation in murine and human leukemia cells. The present study describes the apoptotic-differentiative effect of WEBs in all-trans-retinoic acid (ATRA)-sensitive (NB4) and -resistant (NB4-007-6 and NB4-MR4) acute promyelocytic leukemia (APL) cell lines as well as blasts from patients with t(15;17) APL. NB4 cells exposed to 0.5-1 mM WEBs underwent striking growth arrest and massive apoptosis without appreciable differentiation; IC50 values after 3-day treatment of NB4 were 0.4 and 0.25 mM for WEB-2086 and WEB-2170, respectively. WEBs induced apoptosis also in the two ATRA-resistant NB4-007-6 and NB4-MR4 cell lines and in blasts from patients with t(15;17) APL. Moreover, subapoptotic WEBs acted synergistically with low-dose (0.025-0.05 microM) ATRA; this allowed to increase ATRA differentiation potential up to 40-fold and to improve both number and intensity of NBT-positive NB4 cells at definitely higher levels than with 1 muM ATRA alone. The powerful antiproliferative-apoptotic activities of WEBs in vitro on ATRA-sensitive, ATRA-resistant APL cells and blasts from patients with APL as well as drug capabilities to enhance ATRA differentiation potential suggested that these agents also due to their recognized tolerability in vivo might improve, alone or in combination, clinical treatment of APL.

AMPA-receptor activation is involved in the antiamnesic effect of DM 232 (unifiram) and DM 235 (sunifiram).

DM 232 and DM 235 are novel antiamnesic compounds structurally related to ampakines. The involvement of AMPA receptors in the mechanism of action of DM 232 and DM 235 was, therefore, investigated in vivo and in vitro. Both compounds (0.1 mg/kg(-1) i.p.) were able to reverse the amnesia induced by the AMPA receptor antagonist NBQX (30 mg/kg(-1) i.p.) in the mouse passive avoidance test. At the effective doses, the investigated compounds did not impair motor coordination, as revealed by the rota rod test, nor modify spontaneous motility and inspection activity, as revealed by the hole board test. DM 232 and DM 235 reversed the antagonism induced by kynurenic acid of the NMDA-mediated release of [(3)H]NA in the kynurenate test performed in rat hippocampal slices. This effect was abolished by NBQX. DM 232 increases, in a concentration dependent manner, excitatory synaptic transmission in the rat hippocampus in vitro. These results suggest that DM 232 and DM 235 act as cognition enhancers through the activation of the AMPA-mediated neurotransmission system.

DM235 (sunifiram): a novel nootropic with potential as a cognitive enhancer.

DM235 (sunifiram), a new compound structurally related to piracetam, prevented the amnesia induced by scopolamine (1.5 mg kg(-1) i.p.), after intraperitoneal (0.001-0.1 mg kg(-1)) or oral (0.01-0.1 mg kg(-1)) administration, as shown by a passive avoidance test in mice. The antiamnesic effect of DM235 was comparable to that of well-known nootropic drugs such as piracetam (30-100 mg kg(-1) i.p.), aniracetam (100 mg kg(-1) p.o.) or rolipram (30 mg kg(-1) p.o.). DM235 also prevented mecamylamine (20 mg kg(-1) i.p.)-, baclofen (2 mg kg(-1) i.p.)- and clonidine (0.125 mg kg(-1) i.p.)-induced amnesia in the same test. In the Morris water maze test with rats, scopolamine (0.8 mg kg(-1) i.p.) inhibited the reduction of escape latency in both acquisition and retention/retraining tests. DM235 (0.1 mg kg(-1) i.p.), 20 min before each daily acquisition training, prevented the scopolamine-induced memory impairment. DM235 (1 mg kg(-1) i.p.) also reduced the duration of pentobarbitone-induced hypnosis in mice without modifying the induction time of hypnosis. At the highest effective doses, the investigated compound neither impaired motor coordination (rota-rod test), nor modified spontaneous motility and inspection activity (Animex and hole board tests). These results indicate that DM235, a compound structurally related to piracetam, is a novel nootropic endowed with the capability to prevent cognitive deficits at very low doses. Indeed, its potency is about 1,000 times higher than that of the most active piracetam-like compounds.

Structure-affinity relationships of a unique nicotinic ligand: N(1)-dimethyl-N(4)-phenylpiperazinium iodide (DMPP).

DMPP is a well-known nicotinic agonist that does not fit any proposed pharmacophore for nicotinic binding and represents a unique ligand among the hundreds of nicotinic agonists studied in the past decades. A systematic modulation of the chemical structure of DMPP, aimed to establish its structure-affinity relationships, is reported. The research has allowed to identify molecules such as 11c, 13c, 14c, and 28c, with affinities for alpha(4)beta(2) receptors in the low nanomolar range, some 2 orders of magnitude lower than the lead compound. The agonistic properties of the most interesting compounds have been assessed by measuring their analgesic activity on mice (hot-plate test). Another result of the research was the identification of DMPP analogues, such as 3a (K(i) = 90 nM) and 14b (K(i) = 180 nM), that maintain affinity for the central nicotinic receptor when the ammonium function is changed into an aminic one and are therefore possible leads for drug development in neurodegenerative diseases.

Structure-activity relationships in 2,2-diphenyl-2-ethylthioacetic acid esters: unexpected agonistic activity in a series of muscarinic antagonists.

As a continuation of previous research on anticholinergic drugs derived from 2,2-diphenyl-2-ethylthioacetic acid, several 5,5-diphenyl-5-ethylthio-2-pentynamines (2-11) were synthetised and their antimuscarinic activity on M(1-4) receptor subtypes was evaluated by functional tests and binding experiments. One of the compounds obtained showed unexpected agonistic activity in functional experiments on M(2) receptors. Since the compound carried a phenylpiperazine moiety, other similar compounds (12-17) were prepared and found to be endowed with similar behaviour. These ligands, although possessing the bulky structure characterising muscarinic antagonists, display agonistic activity at M(2) subtypes while, as expected, behaving as antagonists on M(3) and M(4) subtypes. On M(1) subtypes, they show agonistic activity which, however, is not blocked by atropine. The peculiar pharmacological profile of these compounds is of interest for studying muscarinic receptor subtypes.

Molecular simplification of 1,4-diazabicyclo[4.3.0]nonan-9-ones gives piperazine derivatives that maintain high nootropic activity.

Several 4-substituted 1-acylpiperazines, obtained by molecular simplification of 4-substituted 1,4-diazabicyclo[4.3.0]nonan-9-ones, have been synthesized and tested in vivo on the mouse passive avoidance test, to evaluate their nootropic activity. The results show that, apparently, an N-acylpiperazine group can mimic the 2-pyrrolidinone ring of 1,4-diazabicyclo[4.3.0]nonan-9-one, as the compounds of the new series maintain high nootropic activity. Moreover molecular simplification produces more clear-cut structure-activity relationships with respect to the parent series. The mechanism of action also appears to be similar in the two series. In fact, although the molecular mechanism remains to be elucidated, the most potent compound of each class (DM232 and 13, DM235) is able to increase acetylcholine release in rat brain. Piperazine derivatives represent a new class of nootropic drugs with an in vivo pharmacological profile very similar to that of piracetam, showing much higher potency with respect to the reference compound. Among the compounds studied, 13 (DM235) shows outstanding potency, being active at a dose of 0.001 mg kg(-1) sc.

Design, synthesis, and preliminary pharmacological evaluation of 1, 4-diazabicyclo[4.3.0]nonan-9-ones as a new class of highly potent nootropic agents.

Several 4-substituted 1,4-diazabicyclo[4.3.0]nonan-9-ones have been synthesized and tested in vivo on mouse passive avoidance test, to evaluate their nootropic activity. The results show that they represent a new class of nootropic drugs with a pharmacological profile very similar to that of piracetam, showing much higher potency with respect to the reference. Among the compounds studied, 7 (DM 232) shows outstanding potency, being active at the dose of 0. 001 mg kg(-1) sc.

Synthesis and binding properties of photoactivable biotin-conjugated verapamil derivatives for the study of P-170 glycoprotein.

The design and synthesis of two photoactivable biotin-labeled analogues of verapamil (6 and 7) is reported. Preliminary evaluation of the biological profile of 6 (EDP 137) and 7 (EDP 141) shows that they have comparable affinities to that of verapamil for P-170, the protein responsible for multidrug resistance (MDR). Since both appear to bind irreversibly to the protein and the presence of biotin in their structure makes them easily detectable by avidin, they promise to be of great help in studying the protein and its mechanism of action.

Binding profiles of a series of 2-arylpropionic acid esters on cloned human muscarinic receptor subtypes (m1-m5) and their relationship to nootropic activity.

The muscarinic binding profile of a series of 2-arylpropionic acid esters on cloned human muscarinic receptor subtypes (m1-m5) was determined to investigate whether there is a correlation between pharmacological activity and muscarinic receptor subtype selectivity. Among the tested compounds, 1, 7 and 9 showed the highest affinity for the m2 and m4 receptors. Compounds 1, 7 and 9 show good affinity for m4 receptors (pKi = 7.87; 7.73 and 7.10, respectively) and are able to discriminate 10-60 fold between m4/m1, m4/m3, and m4/m5 subtypes. Conversely, these compounds are able only to weakly discriminate between m4/m2. Compounds 1 (50-300 micrograms kg-1 i.p.) and 7 (1-10 micrograms kg-1 i.p.), injected 20 min before the training session, are able to prevent the amnesia induced by dicyclomine (2 mg kg-1 i.p.) in the mouse passive-avoidance test. Compounds 1 and 7, at the highest antiamnesic doses, do not modify motor coordination and spontaneous motility as evaluated by the rota-rod test and Animex apparatus experiments.

Design, synthesis, and in vitro activity of catamphiphilic reverters of multidrug resistance: discovery of a selective, highly efficacious chemosensitizer with potency in the nanomolar range.

On the basis of the results obtained in previous research, three series of compounds (A-C), derived from verapamil, were designed and synthesized to obtain drugs able to revert multidrug resistance (MDR), an acquired resistance that frequently impairs cancer chemotherapy. The ability of the obtained compounds to revert MDR was evaluated on anthracycline-resistant erythroleukemia K 562 cells, measuring the uptake of THP-adriamycin (pirarubicin) by continuous spectrofluorometric monitoring of the decrease of the fluorescence signal of the anthracycline at 590 nm (lambdaex = 480 nm), after incubation with cells. Cardiovascular activity, which is responsible for unwanted side effects, was also evaluated. The results obtained show that many of the compounds studied are potent reverters of MDR and are endowed with reduced cardiovascular activity. One of the compounds (7, MM36) presents a pharmacological profile (unprecedented nanomolar potency, high reversal of MDR, low cardiovascular activity) that makes it a promising drug candidate to treat MDR and a useful tool for studying P-glycoprotein.

Hybridized and isosteric analogues of N1-acetyl-N4-dimethyl-piperazinium iodide (ADMP) and N1-phenyl-N4-dimethyl-piperazinium iodide (DMPP) with central nicotinic action.

A series of piperazine derivatives, obtained by hybridization of N1-acetyl-N4-dimethyl-piperazinium iodide (1, ADMP) and N1-phenyl-N4-dimethyl-piperazinium iodide (3, DMPP) or of the corresponding tertiary bases (2, 4) with arecoline (5) and arecolone (6) or by isosteric substitution of the phenyl ring of DMPP, has been synthesized. Hybridization afforded compounds that, both as tertiary bases and as iodomethylates, have no affinity for the nicotinic receptor. On the contrary, isosteric substitution gave compounds that maintain affinity for the receptor; among them, two tertiary bases (37, 38), show affinity in the nanomolar range for the nicotinic receptor. The pharmacological profile of these isomeric compounds is quite interesting as they present differences in their peripheral and central effects, suggesting that they interact with different subtypes of the nicotinic receptor.

Further structure-activity relationships in the series of tropanyl esters endowed with potent antinociceptive activity.

Several analogs of the alpha-tropanyl esters of 2-(4-chlorophenoxy)butyric acid (SM21) and 2-phenylthiobutyric acid (SM32), endowed with potent antinociceptive and cognition enhancing activity, were synthesized, aimed at obtaining more potent and safe drug candidates. Variation of the acyl moiety (4-11), as well as the conformational restriction of atropine to give the alpha-tropanyl ester of 2,3-dihydrobenzofurane-3-carboxylic acid (18), practically abolished activity. In the case of 18, the antimuscarinic activity was also severely affected by the conformation restrain. On the contrary, conformational restriction of phenoxybutyric and phenylthiobutyric acid derivatives to give the alpha-tropanyl ester of 2,3-dihydro-benzofurane-2-carboxylic acid and 2,3-dihydro-benzothiophene-2-carboxylic acid (12-17), afforded potent analgesic drugs that unfortunately were too toxic to be reliable drug candidates. A series of related esters of benzofurane-3-carboxylic acid (20-27) and benzothiophene-3-carboxylic acid (28) were also studied and found to be potent but toxic analgesics.

SAR studies on the potent and selective muscarinic antagonist 2-ethylthio-2,2-diphenylacetic acid N,N-diethylaminoethyl ester.

Molecular modification of the potent and selective muscarinic antagonist 2-ethylthio-2,2-diphenylacetic acid N,N-diethylaminoethyl ester was performed in order to identify M2 selective antagonists able to cross the blood brain barrier and potentially useful in the treatment of Alzheimer's disease. Modifications included substitution or hydrogenation of one of the phenyl rings as well as their incorporation in a tricyclic system. In general the changes introduced were detrimental for both affinity and selectivity. Only a modest M2 selectivity is present in some compounds that, on the other hand, carry a quaternary ammonium group which precludes their penetration into the brain.