Yutaka Kitano

Protective Effects of Kampo Medicines and Baicalin against Intestinal Toxicity of a New Anticancer Camptothecin Derivative, Irinotecan Hydrochloride (CPT-11), in Rats

In clinical use, irinotecan hydrochloride (CPT‐11; 7‐ethyl‐10‐[4‐(piperidmo)‐l‐piperidino]carbonyl‐oxycamptothecin), a novel antitumor agent, causes a relatively high incidence of severe forms of diarrhea. We investigated whether baicalin, an inhibitor of β‐glucuronidase, which deconjugates the glucuronide of the active metabolite of CPT‐11, SN‐38 (7‐ethyl‐10‐hydorxycamptothecin), and Japanese herbal medicines (Kampo medicines) which contain baicalin can ameliorate CPT‐11‐induced intestinal toxicity in rats. CPT‐11 (60 mg/kg i.v. once daily for 4 consecutive days) induced intestinal toxicity characterized by diarrhea, loss of body weight, anorexia and disruption of intestinal epithelium. Treatment with baicalin (25 mg/kg p.o. twice daily) or Kampo medicines (TJ‐14 and TJ‐114; 1 g/kg p.o. twice daily) from the day before to 4 or 10 days after the start of CPT‐11 administration resulted in significantly decreased weight loss, improved anorexia and a delayed onset of diarrheal symptoms. Histological examination revealed that Kampo medicine‐treated animals had less damage to the intestinal epithelium and that damage was repaired more rapidly than in control rats. These results suggest that the prophylactic use of Kampo medicines (TJ‐14 and TJ‐114) may be of value against CPT‐11‐induced intestinal toxicity.

Increasing-current electroshock seizure test: A new method for assessment of anti- and pro-convulsant activities of drugs in mice

We developed the increasing-current electroshock seizure (ICES) test, a new method for assessment of anti- and pro-convulsant activities of drugs in mice. In this method, a single train of pulses (square wave, 5 msec, 20 Hz) of linearly increasing intensity from 5 to 30 mA (increment of 0.1 mA/0.1 sec, i.e., 5-30 mA in 25 sec) was applied via ear electrodes. The current at which tonic hindlimb extension occurred was recorded as the seizure threshold. Thus, this method allows determination of the seizure threshold current for individual animals. Carbamazepine, phenytoin, valproate, phenobarbital, diazepam, and morphine all increased the seizure threshold current in a dose-dependent manner, whereas ethosuximide was not effective. The seizure threshold current decreased after treatment with reserpine, chlorpromazine, aminophylline, strychnine, pentylenetetrazol, bicuculline, picrotoxin, and ethyl-beta-carboline-3-carboxylate (beta-CCE). These results indicate that the ICES test, like the maximal electroshock seizure test, is a model of grand mal-type seizure and is useful for evaluation of both the anti- and pro-convulsant activities of drugs.

Enhancement of immobility in mouse forced swimming test by treatment with human interferon

We investigated the depression induced by human interferons using the forced swimming test in mice. Intravenous (i.v.) administration of interferon-alpha s (natural interferon-alpha, recombinant interferon-alpha-2a and recombinant interferon-alpha-2b, 600-60000 IU/kg) increased the immobility time in the forced swimming test in a dose-dependent manner, but natural interferon-beta and recombinant interferon-gamma-1a did not affect the immobility time. The increase in the immobility time induced by recombinant interferon-alpha-2b peaked at 15 min after dosing. Administration of recombinant interferon-alpha-2b (6000 IU/kg, i.v.) once daily for 7 consecutive days increased the immobility time, but natural interferon-beta and recombinant interferon-gamma-la did not. Recombinant interferon-alpha-2b in combination with the anti-depressants imipramine (10 mg/kg, i.p.) and mianserin (20 mg/kg, i.p.) did not increase the immobility time. These results suggest that interferon-alpha has a greater potential for inducing depression than interferon-beta and -gamma, and that anti-depressants are effective against interferon-alpha-induced depression.

Involvement of central opioid systems in human interferon-α induced immobility in the mouse forced swimming test

We investigated the mechanism by which human interferon‐α (IFN‐α) increases the immobility time in a forced swimming test, an animal model of depression. Central administration of IFN‐α (0.05–50 IU per mouse, i.cist.) increased the immobility time in the forced swimming test in mice in a dose‐dependent manner. Neither IFN‐β nor ‐γ possessed any effect under the same experimental conditions. Pre‐treatment with an opioid receptor antagonist, naloxone (1 mg kg−1, s.c.) inhibited the prolonged immobility time induced by IFN‐α (60 KIU kg−1, i.v. or 50 IU per mouse. i.cist.). Peripheral administration of naloxone methiodide (1 mg kg−1, s.c.), which does not pass the blood–brain barrier, failed to block the effect of IFN‐α, while intracisternal administration of naloxone methiodide (1 nmol per mouse) completely blocked. The effect of IFN‐α was inhibited by a μ1‐specific opioid receptor antagonist, naloxonazine (35 mg kg−1, s.c.) and a μ1/μ2 receptor antagonist, β‐FNA (40 mg kg−1, s.c.). A selective δ‐opioid receptor antagonist, naltrindole (3 mg kg−1, s.c.) and a κ‐opioid receptor antagonist, nor‐binaltorphimine (20 mg kg−1, s.c.), both failed to inhibit the increasing effect of IFN‐α. These results suggest that the activator of the central opioid receptors of the μ1‐subtype might be related to the prolonged immobility time of IFN‐α, but δ and κ‐opioid receptors most likely are not involved.

Human interferon-α induces immobility in the mouse forced swimming test: involvement of the opioid system

In a previous study, we indicated that human interferon (IFN)-alpha (IFN-alpha, 6 x 10(4) IU/kg, i.v.), but not human IFN-beta or -gamma, prolonged the immobility time of the forced swimming test in mice. In this study, we investigated the mechanism of the effect of human IFN-alpha. None of the mouse IFNs tested (IFN-alpha/beta, IFN-beta, and IFN-gamma, 3 x 10(5) U/kg, i.v.) changed the immobility time or the spontaneous locomotor activity in mice. Indomethacin (10 mg/kg, s.c.), a cyclooxygenase inhibitor, did not affect the increase in the immobility time induced by human IFN-alpha (6 x 10(4) IU/kg, i.v.). However, naloxone (1 mg/kg, s.c.), an opioid receptor antagonist, blocked the increasing caused by human IFN-alpha in the forced swimming test. These results suggest that the increase in the immobility time caused by human IFN-alpha in the forced swimming test might be mediated through opioid receptors, but not mouse IFN receptors.

Anticonvulsant and neuroprotective effects of the novel nootropic agent nefiracetam on kainic acid-induced seizures in rats

Nefiracetam is a novel pyrrolidone-type nootropic agent, and it has been reported to possess a potential for antiepileptic therapy as well as cognition-enhancing effects. We investigated the anticonvulsant and neuroprotective effects of nefiracetam in kainic acid-induced seizures of rats, compared with levetiracetam and standard antiepileptic drugs. Subcutaneous injection of kainic acid (10 mg/kg) induced typical behavioral seizures such as wet dog shakes and limbic seizures and histopathological changes in the hippocampus (degeneration and loss of pyramidal cells in CA1 to CA4 areas). Nefiracetam (25, 50 and 100 mg/kg po) had no effect on the behavioral seizures and dose-dependently inhibited the hippocampal damage. In contrast, levetiracetam, a pyrrolidone-type antiepileptic drug, inhibited neither. Valproic acid and ethosuximide prevented the hippocampal damage without attenuating the behavioral seizures as nefiracetam. Zonisamide and phenytoin did not inhibit the behavioral seizures, while zonisamide enhanced the hippocampal damage and phenytoin increased the lethality rate. Carbamazepine inhibited the behavioral seizures at 50 mg/kg and enhanced that at 100 mg/kg, and it completely inhibited the hippocampal damage at both doses. We have previously reported that anticonvulsant spectrum of nefiracetam paralleled that of zonisamide, phenytoin or carbamazepine in standard screening models. However, the pharmacological profile of nefiracetam was closer to valproic acid or ethosuximide than that of zonisamide, phenytoin or carbamazepine in this study. These results suggest that anticonvulsant spectrum and mechanism of nefiracetam are distinct from those of standard antiepileptic drugs, and nefiracetam possesses a neuroprotective effect that is unrelated to seizure inhibition.

Infantile Pain Episodes Associated with Novel Nav1.9 Mutations in Familial Episodic Pain Syndrome in Japanese Families.

Painful peripheral neuropathy has been correlated with various voltage-gated sodium channel mutations in sensory neurons. Recently Nav1.9, a voltage-gated sodium channel subtype, has been established as a genetic influence for certain peripheral pain syndromes. In this study, we performed a genetic study in six unrelated multigenerational Japanese families with episodic pain syndrome. Affected participants (n = 23) were characterized by infantile recurrent pain episodes with spontaneous mitigation around adolescence. This unique phenotype was inherited in an autosomal-dominant mode. Linkage analysis was performed for two families with 12 affected and nine unaffected members, and a single locus was identified on 3p22 (LOD score 4.32). Exome analysis (n = 14) was performed for affected and unaffected members in these two families and an additional family. Two missense variants were identified: R222H and R222S in SCN11A. Next, we generated a knock-in mouse model harboring one of the mutations (R222S). Behavioral tests (Hargreaves test and cold plate test) using R222S and wild-type C57BL/6 (WT) mice, young (8–9 weeks old; n = 10–12 for each group) and mature (36–38 weeks old; n = 5–6 for each group), showed that R222S mice were significantly (p < 0.05) more hypersensitive to hot and cold stimuli than WT mice. Electrophysiological studies using dorsal root ganglion neurons from 8–9-week-old mice showed no significant difference in resting membrane potential, but input impedance and firing frequency of evoked action potentials were significantly increased in R222S mice compared with WT mice. However, there was no significant difference among Nav1.9 (WT, R222S, and R222H)-overexpressing ND7/23 cell lines. These results suggest that our novel mutation is a gain-of-function mutation that causes infantile familial episodic pain. The mouse model developed here will be useful for drug screening for familial episodic pain syndrome associated with SCN11A mutations.

Pharmacological, pharmacokinetics and safety profiles of DS-5565, a novel α2δ ligand

OBJECTIVE: To clarify the characteristics of DS-5565 as a novel α 2 δ ligand , we conducted the experiments using pregabalin (PGB) as a reference. BACKGROUND: DS-5565 is an analgesic drug that binds to the α 2 δ subunit (α 2 δ-1 and α 2 δ-2) of voltage-dependent Ca 2+ channels. The α 2 δ-1 is the main target for the analgesic effect of α 2 δ ligands. The contribution of the α 2 δ-2 to the CNS side effects of α 2 δ ligands still remains to be elucidated. DESIGN/METHODS: The binding affinity and dissociation rate were investigated with rat α 2 δ-1 and α 2 δ-2 transfected cells. The analgesic effect was investigated with von Frey test in streptozotocin (STZ)-induced diabetic rats. The CNS side effects were investigated with rota-rod performance (RR) and locomotor activity (LA) in rats.The plasma drug concentration was measured by LC-MS/MS. RESULTS: The binding affinities of DS-5565 for α 2 δ-1 and α 2 δ-2 were comparable to those of PGB. Interestingly DS-5565 showed a slower dissociation rate from α 2 δ-1 than α 2 δ-2, in particular α 2 δ-1 compared to PGB. DS-5565 showed potent and sustained analgesic effects and the ED 50 was ca 2.5 mg/kg (ED 50 for PGB: 29.3 mg/kg). The plasma concentration of DS-5565 in the STZ rats was about 65-fold less than PGB. DS-5565 inhibited RR (ID 50 : 9.4 mg/kg) and LA (ID 50 : 43.9 mg/kg) and the ratios ID 50 /ED 50 (CNS safety margin) were ca 3.8 in RR and ca 18 in LA. The ratios for PGB were 0.4 and 3.9, respectively. CONCLUSIONS: DS-5565 has superior analgesic effects with a wider CNS safety margin relative to PGB. These profiles of DS-5565 are possibly due to its unique binding characteristics to α 2 δ-1 and α 2 δ-2. Disclosure: Dr. Yokoyama has received personal compensation for activities with Daiichi Pharmaceutical Corp. as an employee. Dr. Arakawa has received personal compensation for activities with Daiichi Pharmaceutical Corporation as an employee. Dr. Domon has received personal compensation for activities with Daiichi Pharmaceutical Corp. as an employee. Dr. Matsuda has received personal compensation for activities with Daiichi Pharmaceutical Corp. Dr. Inoue has received personal compensation for activities with Daiichi Pharmaceutical Corp. as an employee. Dr. Kitano has received personal compensation for activities with Daiichi Pharmaceutical Corp. Dr. Takahashi has received personal compensation for activities with Daiichi Pharmaceutical Corp. as an employee. Dr. Yamamura has received personal compensation for activities with Daiichi Sankyo. Dr. has received personal compensation for activities with Daiichi Pharmaceutical Corp. as an employee.

Muscle relaxant and neurotoxic activities of intrathecal baclofen in rats

Intrathecal baclofen therapy by the continuous intrathecal infusion of baclofen has been shown to be an effective treatment for spasticity in patients with spinal cord injury, cerebral palsy, traumatic brain injury, multiple sclerosis and other disorders. To demonstrate the efficacy and safety of intrathecal baclofen therapy, we investigated the muscle relaxant and neurotoxic activities of intrathecal baclofen in rats, compared with intravenous baclofen. Intrathecal and intravenous administration of baclofen dose-dependently inhibited the anemic decerebrate rigidity with ED(50) values of 0.31microg/animal (=1.1-1.3microg/kg) and 0.43mg/kg, respectively. Intrathecal administration of baclofen induced no noticeable changes in a spontaneous electroencephalogram at 30microg/animal. Intravenous administration of baclofen induced an abnormal electroencephalogram with flat waves in all the animals and the no-observed-effect level was estimated to be 5mg/kg. In some animals, intravenous administration of baclofen induced sporadic spikes or sharp waves with background flat waves, indicating inhibitory and excitatory effects on the central nervous system. In conclusion, intrathecal administration of baclofen dose-dependently inhibited anemic decerebrate rigidity in rats and the effective dose was more than 300 times lower than that of intravenous baclofen. The safety margin of intrathecal baclofen was greater than that of intravenous baclofen (> or =97 versus 12). These results suggest that intrathecal baclofen therapy is superior to systemic baclofen therapy in both efficacy and safety.

Binding Characteristics and Analgesic Effects of Mirogabalin, a Novel Ligand for the α2δ Subunit of Voltage-Gated Calcium Channels

Mirogabalin ([(1R,5S,6S)-6-(aminomethyl)-3-ethylbicyclo[3.2.0]hept-3-en-6-yl]acetic acid), a novel ligand for the α2δ subunit of voltage-gated calcium channels, is being developed to treat pain associated with diabetic peripheral neuropathy and postherpetic neuralgia. In the present study, we investigated the in vitro binding characteristics and in vivo analgesic effects of mirogabalin compared with those of pregabalin, a standard α2δ ligand. Mirogabalin showed potent and selective binding affinities for the α2δ subunits, while having no effects on 186 off-target proteins. Similar to pregabalin, mirogabalin did not show clear subtype selectivity (α2δ-1 vs. α2δ-2) or species differences (human vs. rat). However, in contrast to pregabalin, mirogabalin showed greater binding affinities for human α2δ-1, human α2δ-2, rat α2δ-1, and rat α2δ-2 subunits; further, it had a slower dissociation rate for the α2δ-1 subunit than the α2δ-2 subunit. Additionally, in experimental neuropathic pain models, partial sciatic nerve ligation rats and streptozotocin-induced diabetic rats, mirogabalin showed more potent and longer lasting analgesic effects. In safety pharmacological evaluations, mirogabalin and pregabalin inhibited rota-rod performance and locomotor activity in rats; however, the safety indices of mirogabalin were superior to those of pregabalin. In conclusion, mirogabalin shows potent and selective binding affinities for the human and rat α2δ subunits, and slower dissociation rates for the α2δ-1 subunit than the α2δ-2 subunit. It shows potent and long-lasting analgesic effects in rat models of neuropathic pain, and wider safety margins for side effects of the central nervous system. These properties of mirogabalin can be associated with its unique binding characteristics.