Selective T-type Calcium Channel Blocker Research Articles

Estradiol enhances T-type calcium channel activation in human myometrium telocytes.

Uterine peristalsis is essential for gamete transport and embryo implantation. It shares the characteristics of spontaneity, rhythmicity, and directivity with gastrointestinal peristalsis. Telocytes, the "interstitial Cajal-like cells" outside the digestive canal, are also located in the uterus and may act as pacemakers. To investigate the possible origin and regulatory mechanism of periodic uterine peristalsis in the human menstrual cycle, telocytes in the myometrium were studied to determine the effect of estradiol on T-type calcium channel regulation. In this study, biopsies of the human myometrium were obtained for cell culture, and double-labeling immunofluorescence screening was used to identify telocytes and T-type calcium channel expression. Intracellular calcium signal measurements and patch-clamp recordings were used to investigate the role of T-type calcium channels in regulating calcium currents with or without estradiol. Our study demonstrates that telocytes exist in the human uterus and express T-type calcium channels. The intracellular Ca2+ fluorescence intensity marked by Fluo-4AM was dramatically decreased by NNC 55-0396, a highly selective T-type calcium channel blocker, but enhanced by estradiol. T-type calcium current amplitude increased in telocytes incubated with estradiol in a dose-dependent manner compared to the control group. In conclusion, our study demonstrated that telocytes exist in the human myometrium, expressing T-type calcium channels and estradiol-enhanced T-type calcium currents, which may be a reasonable explanation for the origin of uterine peristalsis. The role of telocytes in the human uterus as pacemakers and message transfer stations in uterine peristalsis may be worth further investigation.

Crystal structure determination and computational studies of 1,4-dihydropyridine derivatives as selective T-type calcium channel blockers

Different types of calcium channels are crucial regulators of many key physiological functions throughout the body. Therefore, calcium channel modulators are accepted as precious molecules for the therapeutic intervention of various pathologies ranging from cardiovascular to neurological diseases. 1,4-dihydropyridines (DHPs) primarily target L-type calcium channel (Cav1.2) for the treatment of hypertension and occupy a central position among all calcium channel blockers. Subsequently, T-type calcium channel Cav3.2 isoform has been established to play a significant role in chronic pain conditions. Despite the growing interest and identification of many compounds with Cav3.2 blocking activities, no molecule has passed the clinical trials and approved for the treatment of pain by now. Our group has recently identified two DHP-based molecules (HM8 and MD20) that selectively blocked T-type over L-type calcium channel. As these compounds hold great therapeutic value as potential drug candidates, we carried out further structural and computational analyses to gain insights into their various properties. Initially, the three-dimensional structure of HM8 was resolved by single-crystal X-ray analysis. The computational analysis encompassed density functional theory (DFT) and molecular dynamics (MD) simulations. DFT calculations were used to identify local reactivity properties and pharmaceutical stability, while time-dependent DFT calculations were used to simulate the UV/Vis spectra and identify the molecular parts principally responsible for the light absorption. MD simulations were used to understand the influence of water on studied molecules, and also to identify the substances that could be potentially used as excipients in pharmaceutical formulations based on HM8 and MD20.

Unsuccessful story of ABT-639 in neuropathic pain in different phases of clinical trials

Neuropathic pain is a chronic pain state caused by injury of the nervous system, and there are various conditions associated with it such as diabetic neuropathy, postherpetic neuralgia, cancer pain, trigeminal neuralgia, cervical radiculopathy, and anticancer and anti-HIV-induced neuropathic pain. The pathophysiological role of voltage-gated T-type calcium channels in neuropathic pain has been well documented. ABT-639 is a peripherally acting, selective T-type calcium channel blocker, and it is shown to efficiently reduce nociceptive and neuropathic pain in rats in multiple models. However, three clinical studies exploring the role of single dose or multiple doses of ABT-639 (100 mg) in diabetic neuropathy have not found significant pain-attenuating actions. The present review discusses the unsuccessful clinical trials of peripherally acting ABT-639 in patients with diabetic neuropathy.

Discovery of a Potent, Selective T-type Calcium Channel Blocker as a Drug Candidate for the Treatment of Generalized Epilepsies.

We report here the discovery and pharmacological characterization of N-(1-benzyl-1H-pyrazol-3-yl)-2-phenylacetamide derivatives as potent, selective, brain-penetrating T-type calcium channel blockers. Optimization focused mainly on solubility, brain penetration, and the search for an aminopyrazole metabolite that would be negative in an Ames test. This resulted in the preparation and complete characterization of compound 66b (ACT-709478), which has been selected as a clinical candidate.

Blocking of T-Type Calcium Channels by TTA-A2 Reveals a Conservative Binding Site for State-Dependent Antagonists

T-type calcium channels, or CaV3 channels, are involved in several cell functions, and they can generate low-threshold spikes that lead to burst firing and oscillatory behavior in various types of neurons. In addition, CaV3 channels are also implicated in a number of pathophysiological processes such as epilepsy, sleep disorders, hypertension, neuropathic pain and cancer. However, until recently there were no potent and selective antagonists of CaV3 channels that facilitate further investigation of T-type channel function in neurological diseases and to validate these channels as efficient drug targets. Recently, a series of potent and selective T-type calcium channel blockers (T-type antagonists or TTAs) have been reported in several studies. Here, we describe the identification of one aminoacid that is determinant for the molecular coupling of TTA-A2 ([2-(4-cyclopropylphenyl)-N-((1R)-1-{5-[(2,2,2-trifluoroethyl)oxo]pyridin-2-yl}ethyl)acetamide]) on the CaV3.1 recombinant human T-type calcium channel. By using a combination of molecular modeling, docking studies, mutagenesis and patch-clamp experiments of CaV3.1 channels expressed in HEK-293 cells, we have collected experimental evidence that supports the existence of a conservative binding site for TTA-A2 and state-dependent antagonists similar to TTA-A2, e.g., mibefradil. Blocking potency of TTA-A2 was reduced by two thirds in CaV3.1 mutants, where the identified aminoacid was replaced for alanine, proline, or arginine, among others. Sequence alignment of the three human CaV3 channels suggest that the binding site is conserved in the CaV3 subfamily, but not in the L-type CaV1.2 channel. In addition, our data also provides further evidence that TTA-Q4, a positive allosteric modulator of TTA-A2 functional inhibition, does not share the same binding site with the latter. Our results contribute to the knowledge for the rational design of T-type channel antagonists based on the structure of the TTA-A2 binding site.Supported by CONACYT-MEXICO 167790.

NIMG-35RESULTS OF EXPLORATORY 18F-FLT PET IMAGING IN TEN RECURRENT HIGH-GRADE GLIOMA PATIENTS TREATED WITH THE SELECTIVE T-TYPE CALCIUM CHANNEL BLOCKER MIBEFRADIL FOLLOWED BY TEMOZOLOMIDE (ABTC TRIAL 1101)

BACKGROUND: Mibefradil (MIB) is a selective T-type calcium channel blocker that has shown preclinical impact on cell cycle activity, specifically arrest at G1/S in tumor cells, suggesting a potential chemo-sensitizing effect if given prior to chemotherapy. In our study, MIB was administered orally QID x 7 days prior to standard temozolomide (150-200 mg/m2 x 5/28d) in recurrent high-grade glioma (HGG) patients. 18F-FLT PET was used as an exploratory imaging biomarker to assess influence of MIB on overall tracer accumulation in tumor. METHODS: 18F-FLT PET was performed in 10 patients treated at the maximum tolerated dose of MIB (87.5 mg po QID) at 5 different institutions. Prior to MIB administration, 2 PET scans were performed as a double baseline to assess repeatability of PET measurements. A third scan was performed on day 7 of MIB treatment to assess 18F-FLT uptake for response. Tumor standardized uptake values (SUVs) were measured at a central laboratory by automatically positioning a 1mL sphere at the region of highest uptake (SUVpeak). RESULTS: The double baseline data indicated 95% limits of repeatability of 18.5% (6.7% within-patient coefficient of variation) for SUVpeak. Two of the 10 patients demonstrated a drop in SUVpeak after 7 days of MIB that exceeded these limits (-47% and -67%). No significant change between double baseline and day 7 of MIB was seen in the remaining 8 patients. CONCLUSIONS: In this first 18F-FLT PET imaging study within a multicenter HGG trial, 18F-FLT PET was found to be feasible and highly repeatable. The results suggest influence of MIB on tumor accumulation of the radiotracer, possibly in part due to alterations in cell cycle activity in a subset of patients, but the precise mechanism remains to be determined as tracer delivery/transport effects may also be contributing to the decline in signal observed in the two patients.

T-type calcium channels contribute to calcium disturbances in brain during hyponatremia

Disturbance of calcium homeostasis is implicated in the normal process of aging and brain pathology prevalent in the elderly such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis. Previous studies demonstrated that applying a hyponatremic iso-osmotic (low-NaCl) artificial cerebrospinal fluid (ACSF) to rodent hippocampus causes extracellular calcium to rapidly decrease. Restoring normonatremia after low-NaCl treatment causes a rapid increase in extracellular calcium that overshoots baseline. This study examined the amplitude, timing, and mechanism of these surprising calcium changes. We also tested whether hyponatremia increased calcium entry into brain cells or calcium binding to chondroitin sulfate (CS), a negatively charged constituent of the extracellular matrix (ECM) that may be occupied by sodium during normonatremia. We report three major findings. First we show that CS does not contribute to extracellular calcium changes during low-NaCl treatments. Second, we show that the time to minimum extracellular calcium during low-NaCl treatment is significantly shorter than the time to maximum extracellular calcium in recovery from low-NaCl treatment. Third, we show that the decrease in extracellular calcium observed during hyponatremia is attenuated by ML 218, a highly selective T-type calcium channel blocker. Together these data suggest that calcium rapidly enters cells at the onset of low-NaCl treatment and is extruded from cells when normonatremia is restored. Calcium binding to CS does not significantly contribute to calcium changes in brain during hyponatremia. Differences in timing suggest that extracellular calcium changes during and in recovery from hyponatremia occur by distinct mechanisms or by a multistep process. Finally, partial block of extracellular calcium influx by ML 218 suggests that T-type channels are involved in calcium entering cells during hyponatremia. Given the high prevalence of hyponatremia among elderly patients and the growing understanding of calcium's role in multiple neurologic pathologies, this study promotes a novel approach for studying and potentially preventing the effects of hyponatremia on calcium dysregulation in brain tissue.

Timed-sequential therapy with mibefradil and temozolomide in patients with recurrent high-grade gliomas: A phase I Adult Brain Tumor Consortium study.

2033 Background: Mibefradil (MIB) is a selective T-type calcium channel blocker that had previously been approved for treatment of hypertension. MIB has shown significant preclinical activity in ma...

Z944: a first in class T-type calcium channel modulator for the treatment of pain.

Zalicus, Inc. is a small, publicly-traded biotech company in Cambridge, Massachusetts. The potential target for neuropathic pain lies in the T-type calcium channels, members of the low-voltage activated channel family. By opening at relatively negative membrane depolarizations, T-type channels can act as modulators of the threshold of the action potential by increasing the calcium current, leading to further depolarization of the cell and activation of other calcium channels, and ultimately, sodium and potassium channels. This is a target that is widely expressed in both the peripheral as well as the central nervous system (Zamponi et al., 2009). It is known that T-type calcium channel blockade can modulate pain behavior and disrupting this mechanism can reduce pain in neuropathic, inflammatory, and visceral pain models. There is evidence that the calcium channel protein itself as well as the current that is delivered by this channel is increased in certain pain models (Marger et al., 2011). Another important feature of this target is its central action. There are three subtypes of the T-type calcium channel, denoted Cav3.1, 3.2, and 3.3. They are expressed in different places in the thalamus, the cerebellum, and other parts of the brain. It is thought that they participate in the regulation of connectivity or crosstalk between the sensory nervous system and the cortex by promoting thalamic neuron burst firing and network oscillations important for relay and interpretation of signals to and by the cortex. A number of central nervous system (CNS) diseases have been linked to aberrant thalamocortical oscillations (thalamocortical dysrhythmia), and there is evidence that T-type calcium channel mutants can play a role in hyperexcitability disorders, for example, epilepsy syndromes. Some approved anticonvulsants also hit the T-type channel as part of their polypharmacology. Zalicus' approach has been to discover and develop a small molecule blocker of T-type calcium channels as a potential therapeutic for pain. The medication, Z944, is a novel, oral, state-dependent, selective T-type calcium channel blocker. This blocker has >150-fold selectivity vs. non-T-type voltage-gated ion channels and has been shown to inhibit native neuronal T-type currents at high potency. The blockade of Cav3.2 by Z944 is more pronounced during high-frequency firing and may provide some advantage to the molecule by selectively targeting a pathological state of the channel (Short et al., 2013). A phase 1, double-blind, placebo-controlled study to determine the safety, tolerability, and pharmacokinetics of single and multiple ascending doses of oral Z944 in healthy male subjects in the fed and fasted states using an immediate release formulation showed that the medication was safe and well tolerated. Mild and expected CNS side effects were observed; no serious adverse events were reported. A phase 1b clinical study utilized a state-of-the-art experimental clinical model measuring Laser-Evoked Potentials (LEP) from skin irritated following topical application of capsaicin (modeling neuropathic pain signaling and central sensitization) or exposure to UV light (modeling inflammatory pain signaling). Healthy volunteers received 20, 40, and 80 mg doses split in four administrations every 2 h vs. placebo. The data showed that Z944 reduced peak-to-peak amplitudes of LEPs from both capsaicin and UV irritated skin models and reduced subjective Visual Analog Scale (VAS) pain scores in both skin types. The noted adverse events were generally mild to moderate and primarily central nervous system related. No serious adverse events occurred in this study. On the basis of these findings, several modified release formulations of Z944 are currently being advanced in clinical development for pain indications.

A peripherally acting, selective T-type calcium channel blocker, ABT-639, effectively reduces nociceptive and neuropathic pain in rats

Activation of T-type Ca2+ channels contributes to nociceptive signaling by facilitating action potential bursting and modulation of membrane potentials during periods of neuronal hyperexcitability. The role of T-type Ca2+ channels in chronic pain is supported by gene knockdown studies showing that decreased Cav3.2 channel expression results in the loss of low voltage-activated (LVA) currents in dorsal root ganglion (DRG) neurons and attenuation of neuropathic pain in the chronic constriction injury (CCI) model. ABT-639 is a novel, peripherally acting, selective T-type Ca2+ channel blocker. ABT-639 blocks recombinant human T-type (Cav3.2) Ca2+ channels in a voltage-dependent fashion (IC50=2μM) and attenuates LVA currents in rat DRG neurons (IC50=8μM). ABT-639 was significantly less active at other Ca2+ channels (e.g. Cav1.2 and Cav2.2) (IC50>30μM). ABT-639 has high oral bioavailability (%F=73), low protein binding (88.9%) and a low brain:plasma ratio (0.05:1) in rodents. Following oral administration ABT-639 produced dose-dependent antinociception in a rat model of knee joint pain (ED50=2mg/kg, p.o.). ABT-639 (10–100mg/kg, p.o.) also increased tactile allodynia thresholds in multiple models of neuropathic pain (e.g. spinal nerve ligation, CCI, and vincristine-induced, and capsaicin secondary hypersensitivity). ABT-639 did not attenuate hyperalgesia in inflammatory pain models induced by complete Freund's adjuvant or carrageenan. At higher doses (e.g. 100 - 300mg/kg) ABT-639 did not significantly alter hemodynamic or psychomotor function. The antinociceptive profile of ABT-639 provides novel insights into the role of peripheral T-type (Cav3.2) channels in chronic pain states.

Sequential therapy with the selective T-type calcium channel blocker mibefradil and temozolomide in patients with recurrent high-grade gliomas: An Adult Brain Tumor Consortium phase I study (ABTC1101).

TPS2105 Background: Despite recent advances in their treatment, high-grade gliomas (HGG) carry a dismal prognosis. Treatment options at recurrence are particularly limited and a re-challenge with temozolomide (TMZ) frequently appears as the most appropriate systemic treatment option in patients whose progression occurred off TMZ. This study investigates the sequential treatment of mibefradil (MIB), a selective Cav3 calcium channel blocker, and standard dose TMZ. Preclinical data showed that Cav3 inhibitors such as MIB can slow tumor growth without significant effect on normal tissues and can induce cell cycle arrest in cancer cells at the G1/S-phase checkpoint. We hypothesize that withdrawal of MIB could synchronize and release cells into S-phase, thereby potentiating the cytotoxic effect of TMZ. Methods: This trial is an open-label, multicenter phase I study, structured into a dose escalation phase to determine the maximum tolerated dose of MIB (MTD; Primary Objective) and an expansion cohort of 10 patients at the MTD level. Adults with recurrent HGG (WHO grade 3 or 4) who have previously received standard adjuvant therapy with radiation (RT) and TMZ (last dose ≥ 3 months prior to enrollment) and who have not received other cytotoxic therapy (except for carmustine wafers) are eligible. Patients receive oral MIB for 7 days on a 4 x/day (QID) schedule, followed by standard dose TMZ at 150-200 mg/m2 for 5 days each 28-day cycle. Dose finding uses a modified 3+3 design, starting at MIB 25 mg po QID (100 mg/day) with dose increases of 25 mg/dose per dose level. The target dose-limiting toxicity (DLT) rate is ≤ 33%. Secondary Objectives: (1) safety and adverse event analysis (incl. cardiac monitoring during cycle 1), (2) pharmacokinetic profile of MIB, (3) response assessment (RANO criteria), and (4) assessment of the potential effect of MIB on tumor DNA synthesis as determined by fluorothymidine positron emission tomography (FLT PET; extension cohort). Enrollment status as of January 2013: cohort 1 completed without DLT; cohort 2 enrolling at MIB 200 mg/day. Clinical trial information: NCT01480050.

Topical application of disodium isostearyl 2-O-L-ascorbyl phosphate, an amphiphilic ascorbic acid derivative, reduces neuropathic hyperalgesia in rats

Ca(v) 3.2 T-type calcium channels, targeted by H(2) S, are involved in neuropathic hyperalgesia in rats and ascorbic acid inhibits Ca(v) 3.2 channels. Therefore, we evaluated the effects of intraplantar (i.pl.) administration of ascorbic acid or topical application of disodium isostearyl 2-O-L-ascorbyl phosphate (DI-VCP), a skin-permeable ascorbate derivative on hyperalgesia induced by NaHS, an H(2) S donor, and on neuropathic hyperalgesia. In rats mechanical hyperalgesia was evoked by i.pl. NaHS, and neuropathic hyperalgesia was induced by L5 spinal nerve cutting (L5SNC) or by repeated administration of paclitaxel, an anti-cancer drug. Dermal ascorbic acid levels were determined colorimetrically. The NaHS-evoked Ca(v) 3.2 channel-dependent hyperalgesia was inhibited by co-administered ascorbic acid. Topical application of DI-VCP, but not ascorbic acid, prevented the NaHS-evoked hyperalgesia, and also increased dermal ascorbic acid levels. Neuropathic hyperalgesia induced by L5SNC or paclitaxel was reversed by i.pl. NNC 55-0396, a selective T-type calcium channel blocker, ascorbic acid or DI-VCP, and by topical DI-VCP, but not by topical ascorbic acid. The effects of i.pl. ascorbic acid and topical DI-VCP in the paclitaxel-treated rats were characterized by the faster onset and greater magnitude, compared with their effects in the L5SNC rats. Dermal ascorbic acid levels in the hindpaw significantly decreased after paclitaxel treatment, but not L5SNC, which was reversed by topical DI-VCP. Ascorbic acid, known to inhibit Ca(v) 3.2 channels, suppressed neuropathic hyperalgesia. DI-VCP ointment for topical application may be of benefit in the treatment of neuropathic pain.

In vivo evaluation of oral anti-tumoral effect of 3,4-dihydroquinazoline derivative on solid tumor

An extension of our previously reported 3,4-dihydroquinazoline derivative is investigated. Oral anti-tumoral activity of 3,4-dihydroquinazoline derivative (KYS05090) as potent and selective T-type calcium channel blocker was in vivo evaluated against A549 xenograft in BALB/c(nu/nu) nude mice. The rate of tumor volume increment in mouse model with KYS05090-treated group was remarkably slower than that of control group. With respect to tumor weight, it exhibited 60% and 67% tumor growth inhibition through oral administration of 1 and 5mg/kg of bodyweight, respectively, compared to control and was more potent than paclitaxel (53%). In addition, KYS05090 (10 and 50mg/kg, po) was found to have a marked analgesic effect in acetic acid-induced writhing test, whereas it did not show any effect on hot plate test.

Inhibition of T-type calcium channels and hydrogen sulfide-forming enzyme reverses paclitaxel-evoked neuropathic hyperalgesia in rats

Hydrogen sulfide (H₂S), a gasotransmitter, facilitates pain sensation by targeting Ca(v)3.2 T-type calcium channels. The H₂S/Ca(v)3.2 pathway appears to play a role in the maintenance of surgically evoked neuropathic pain. Given evidence that chemotherapy-induced neuropathic pain is blocked by ethosuximide, known to block T-type calcium channels, we examined if more selective T-type calcium channel blockers and also inhibitors of cystathionine-γ-lyase (CSE), a major H₂S-forming enzyme in the peripheral tissue, are capable of reversing the neuropathic pain evoked by paclitaxel, an anti-cancer drug. It was first demonstrated that T-type calcium channel blockers, NNC 55-0396, known to inhibit Ca(v)3.1, and mibefradil inhibited T-type currents in Ca(v)3.2-transfected HEK293 cells. Repeated systemic administration of paclitaxel caused delayed development of mechanical hyperalgesia, which was reversed by single intraplantar administration of NNC 55-0396 or mibefradil, and by silencing of Ca(v)3.2 by antisense oligodeoxynucleotides. Systemic administration of dl-propargylglycine and β-cyanoalanine, irreversible and reversible inhibitors of CSE, respectively, also abolished the established neuropathic hyperalgesia. In the paclitaxel-treated rats, upregulation of Ca(v)3.2 and CSE at protein levels was not detected in the dorsal root ganglia (DRG), spinal cord or peripheral tissues including the hindpaws, whereas H(2)S content in hindpaw tissues was significantly elevated. Together, our study demonstrates the effectiveness of NNC 55-0396 in inhibiting Ca(v)3.2, and then suggests that paclitaxel-evoked neuropathic pain might involve the enhanced activity of T-type calcium channels and/or CSE in rats, but not upregulation of Ca(v)3.2 and CSE at protein levels, differing from the previous evidence for the neuropathic pain model induced by spinal nerve cutting in which Ca(v)3.2 was dramatically upregulated in DRG.

Effect of Efonidipine on TGF-β1–Induced Cardiac Fibrosis Through Smad2-Dependent Pathway in Rat Cardiac Fibroblasts

Transforming growth factor beta-1 (TGF-β1) plays a critical role in progression of cardiac fibrosis, which may involve intracellular calcium change. We examined effects of efonidipine, a dual T-type and L-type calcium channel blocker (CCB), on TGF-β1-induced fibrotic changes in neonatal rat cardiac fibroblast. T-type and L-type calcium channel mRNAs were highly expressed in cultured cardiac fibroblasts. TGF-β1 (5 ng/mL) significantly increased Smad2 phosphorylation and [(3)H]-leucine incorporation, which were attenuated by pretreatment with efonidipine (10 µM). Neither R(-)efonidipine (10 µM), selective T-type CCB, nor nifedipine (10 µM), selective L-type CCB, efficaciously inhibited both TGF-β1-induced Smad2 phosphorylation and [(3)H]-leucine incorporation. However, both were markedly attenuated by combination of R(-)efonidipine and nifedipine, EDTA, or calcium-free medium. Pretreatment with Smad2 siRNA significantly attenuated [(3)H]-leucine incorporation induced by TGF-β1. These data suggest that efonidipine elicits inhibitory effects on TGF-β1- and Smad2-dependent protein synthesis through both T-type and L-type calcium channel-blocking actions in cardiac fibroblasts.

Antitumor activity of 3,4-dihydroquinazoline dihydrochloride in A549 xenograft nude mice

In the previous article we have reported that 3,4-dihydroquinazoline 1 is a potent and selective T-type calcium channel blocker that exhibited strong anti-cancer activity in vitro. Compound 1·2HCl was further in vivo evaluated against A549 xenograft in BALB/c nude mice, which exhibited 49% tumor-weight inhibition through intravenous administration of 2mg/kg of body weight and was more potent than doxorubicin. Moreover, compound 1·2HCl has an oral bioavailability of 98% with LD50 values of 693mg/kg (po route) and 40.0mg/kg (iv route) of body weight. In addition, its efficient scale-up synthetic method was developed.

Synthesis and Biological Evaluation of Isophthalamide Derivatives as T-type Calcium Channel Blockers

E-mail: ys4049@kist.re.kr Received July 29, 2008Key Words : T-type calcium channel blocker, Neuropathic pain, IsophthalamideSince mibefradil, a selective T-type calcium channel blocker,was withdrawn from the market in 1998 due to drug-druginteraction, there have been efforts to discover novel T-typecalcium blockers.

T-type calcium channels mediate rebound firing in intact deep cerebellar neurons

Neurons of the deep cerebellar nuclei (DCN) form the main output of the cerebellar circuitry and thus play an important role in cerebellar motor coordination. A prominent biophysical property observed in rat DCN neurons is rebound firing; a brief but strong hyperpolarizing input transiently increases their firing rate to much higher levels compared with that prior to the inhibitory input. Low-threshold T-type voltage-gated calcium channels have been suspected for a long time to be responsible for this phenomenon, but direct pharmacological evidence in support of this proposition is lacking. Even though a multitude of functional roles has been assigned to rebound firing in DCN neurons, their prevalence under physiological conditions is in question. Studies aimed at delineating the physiological role of rebound firing are hampered by the lack of a good pharmacological blocker. Here we show that mibefradil, a compound that blocks T-type calcium channels, potently blocks rebound firing in DCN neurons. In whole-cell experiments both mibefradil and NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride]. a more selective T-type calcium channel blocker, effectively blocked rebound firing produced by direct current injection. Thus, mibefradil and other T-type channel modulators may prove to be invaluable tools for elucidating the functional importance of DCN rebound firing in cerebellar computation.

3D pharmacophore based virtual screening of T-type calcium channel blockers

Virtual screening of the commercial databases was done by using a three dimensional pharmacophore previously developed for T-type calcium channel blockers using CATALYST™ program. Biological evaluation of 25 selected virtual hits resulted in the discovery of a highly potent compound VH04 with IC 50 value of 0.10 μM, eight times as potent as the known selective T-type calcium channel blocker, mibefradil. Search for similar compounds yielded several hits with micro-molar IC 50 values and high T-type calcium channel selectivity. Based on the structure of the virtual hits, small molecule libraries with novel scaffolds were designed, synthesis and biological evaluation of which are currently in progress. This result shows a successful example of ligand based drug discovery of potent T-type calcium channel blockers.

Ethosuximide reverses paclitaxel- and vincristine-induced painful peripheral neuropathy

Paclitaxel (Taxol®) is one of the most effective and frequently used chemotherapeutics for the treatment of solid tumours. However, paclitaxel produces peripheral neurotoxicity with patients reporting sensory abnormalities and neuropathic pain during and often persisting after paclitaxel therapy. The mechanisms underlying this dose-limiting side effect are currently unknown and there are no validated drugs for its prevention or control. Male Sprague–Dawley rats received four intraperitoneal (i.p.) injections on alternate days of 2 mg/kg paclitaxel. Behavioural assessment using von Frey filaments and acetone showed that such paclitaxel treatment induced a pronounced mechanical and cold allodynia/hyperalgesia. Thus these studies aim to test potential analgesics on established paclitaxel-induced pain. Paclitaxel-induced pain appears to be relatively resistant to opioid therapy i.p. 4 mg/kg morphine was ineffective and i.p. 8 mg/kg morphine only elicited up to a 50% reversal of mechanical allodynia/hyperalgesia. Interestingly, a maximally tolerated dose (i.p. 0.2 mg/kg) of the potent NMDA receptor antagonist MK-801 produced no significant reversal of the mechanical allodynia/hyperalgesia suggesting that NMDA receptors have little role in paclitaxel-induced pain. Ethosuximide (i.p. 450 mg/kg) an anti-epileptic and relatively selective T-type calcium channel blocker elicited a near complete reversal of mechanical allodynia/hyperalgesia. Repetitive dosing with ethosuximide (i.p. 100 or 300 mg/kg daily for 3 days) showed a dose-related consistent reversal of mechanical allodynia/hyperalgesia with no evidence of tolerance. Ethosuximide (i.p. 300 mg/kg) also reversed paclitaxel-induced cold allodynia and vincristine-induced mechanical allodynia/hyperalgesia. These data suggest that T-type calcium channels may play a role in chemotherapy-induced neuropathy and moreover identify ethosuximide as a new potential treatment for chemotherapy-induced pain.