Jeffry L. Vaught

Topiramate: Preclinical Evaluation of a Structurally Novel Anticonvulsant

Summary: Topiramate [TPM, 2,3:4,5‐his‐O‐(1‐methyl‐ethylidene)‐β‐D‐fructopyranose sulfamate] (RWJ‐17021‐000, formerly McN‐4853) is a structurally novel antiepileptic drug (AED). The preclinical anticonvulsant profile suggests that TPM acts primarily by blocking the spread of seizures. TPM was highly effective in the maximal electroshock (MES) seizure test in rats and mice. Activity was evident 0.5. h after oral administration and lasted at least 16 h. The ED50 values 4 h after oral dosing were 13.5 and 40.9 mg/kg in rats and mice, respectively. TPM blocked pentylenetetrazol (PTZ)‐induced clonic seizures at high doses in mice (ED50= 1,030 mg/kg orally, p.o.). With motor incoordination and loss of righting reflex used as indicators of neurologic impairment, the neuroprotective index (TD50/MES ED50) for TPM was equivalent or superior to that of several approved AEDs. In mice pretreated with SKF‐525A (a P450 enzyme inhibitor), the anticonvulsant potency was either increased or unaffected when TPM was tested 0.5, 1, or 2 h after i.p. administration, suggesting that TPM rather than a metabolite was the active agent. In mice pretreated with reserpine or tetrabenazine, the activity of TPM in the MES test was markedly reduced. TPM was inactive in a variety of receptor binding, neurotransmitter uptake, and ion channel tests. TPM weakly inhibited erythrocyte carbonic anhydrase (CA) activity. However, the anticonvulsant activity of TPM appears to differ mechanistically from that of acetazolamide.

Cep-1347 (KT7515), a semisynthetic inhibitor of the mixed lineage kinase family.

CEP-1347 (KT7515) promotes neuronal survival at dosages that inhibit activation of the c-Jun amino-terminal kinases (JNKs) in primary embryonic cultures and differentiated PC12 cells after trophic withdrawal and in mice treated with 1-methyl-4-phenyl tetrahydropyridine. In an effort to identify molecular target(s) of CEP-1347 in the JNK cascade, JNK1 and known upstream regulators of JNK1 were co-expressed in Cos-7 cells to determine whether CEP-1347 could modulate JNK1 activation. CEP-1347 blocked JNK1 activation induced by members of the mixed lineage kinase (MLK) family (MLK3, MLK2, MLK1, dual leucine zipper kinase, and leucine zipper kinase). The response was selective because CEP-1347 did not inhibit JNK1 activation in cells induced by kinases independent of the MLK cascade. CEP-1347 inhibition of recombinant MLK members in vitro was competitive with ATP, resulting in IC50values ranging from 23 to 51 nm, comparable to inhibitory potencies observed in intact cells. In addition, overexpression of MLK3 led to death in Chinese hamster ovary cells, and CEP-1347 blocked this death at doses comparable to those that inhibited MLK3 kinase activity. These results identify MLKs as targets of CEP-1347 in the JNK signaling cascade and demonstrate that CEP-1347 can block MLK-induced cell death.

Insulin-like Growth Factor-I: Potential for Treatment of Motor Neuronal Disorders

Motor neuronal disorders, such as the loss of spinal cord motor neurons in amyotrophic lateral sclerosis or the degeneration of spinal cord motor neuron axons in certain peripheral neuropathies, present a unique opportunity for therapeutic intervention with neurotrophic proteins. Normally, such proteins do not cross the blood-brain barrier, but spinal cord motor neuron axons and nerve terminals lie outside the barrier and thus may be targeted by systemic administration of protein growth factors. Insulin-like growth factor-I (IGF-I) receptors are present in the spinal cord, and, like members of the neurotrophin receptor family, IGF-I receptors mediate signal transduction via a tyrosine kinase domain. IGF-I was found to prevent the loss of choline acetyltransferase activity in embryonic spinal cord cultures, as well as to reduce the programmed cell death of motor neurons in vivo during normal development or following axotomy or spinal transection. Consistent with earlier reports that IGF-I enhances motor neuronal sprouting in vivo, subcutaneous administration of IGF-I increases muscle endplate size in rats. Subcutaneous injections of IGF-I also accelerate functional recovery following sciatic nerve crush in mice, as well as attenuate the peripheral motor neuropathy induced by chronic administration of the cancer chemotherapeutic agent vincristine in mice. Doses of IGF-I that accelerate recovery from sciatic nerve crush in mice result in elevated serum levels of IGF-I which are similar to those obtained following subcutaneous injections of formulated recombinant human IGF-I (Myotrophin) in normal human subjects. Based on these findings, together with evidence of safety in animals and man, clinical trials of recombinant human IGF-I have been initiated in patients with amyotrophic lateral sclerosis and are planned to begin soon in patients with chemotherapy-induced peripheral neuropathies.

CEP‐1347/KT‐7515, an inhibitor of SAPK/JNK pathway activation, promotes survival and blocks multiple events associated with Aβ‐induced cortical neuron apoptosis

Although the mechanism of neuronal death in Alzheimers disease (AD) has yet to be elucidated, a putative role for c‐jun in this process has emerged. Thus, it was of interest to delineate signal transduction pathway(s) which regulate the transcriptional activity of c‐jun, and relate these to alternate gene inductions and biochemical processes associated with beta‐amyloid (Aβ) treatment. In this regard, the survival promoting activity of CEP‐1347, an inhibitor of the stress‐activated/c‐jun N‐terminal (SAPK/JNK) kinase pathway, was evaluated against Aβ‐induced cortical neuron death in vitro. Moreover, CEP‐1347 was used as a pharmacologic probe to associate multiple biochemical events with Aβ‐induced activation of the SAPK/JNK pathway. CEP‐1347 promoted survival and blocked Aβ‐induced activation of JNK kinase (MKK4, also known as MEK‐4, JNKK and SEK1) as well as other downstream events associated with JNK pathway activation. CEP‐1347 also blocked Aβ‐induction of cyclin D1 and DP5 genes and blocked Aβ‐induced increases in cytoplasmic cytochrome c, caspase 3‐like activity and calpain activation. The critical time window for cell death blockade by CEP‐1347 resided within the peak of Aβ‐induced MKK4 activation, thus defining this point as the most upstream event correlated to its survival‐promoting activity. Together, these data link the SAPK/JNK pathway and multiple biochemical events associated with Aβ‐induced neuronal death and further delineate the point of CEP‐1347 interception within this signal transduction cascade.

Aurintricarboxylic acid protects hippocampal neurons from NMDA- and ischemia-induced toxicity in vivo

Abstract: The polymeric dye aurintricarboxylic acid (ATA) has been shown to protect various cell types from apoptotic cell death, reportedly through inhibition of a calcium‐dependent endonuclease activity. Recent studies have indicated that there may be some commonalities among apoptosis, programmed cell death, and certain other forms of neuronal death. To begin to explore the possibility of common biochemical mechanisms underlying ischemia‐or excitotoxin‐induced neuronal death and apoptosis in vivo, gerbils or rats subjected to transient global ischemia or NMDA microinjection, respectively, received a simultaneous intracerebral infusion of ATA or vehicle. As a biochemical marker of neuronal death, spectrin proteolysis, which is mediated by activation of calpain I, was measured in hippocampus after 24 h. ATA treatment resulted in a profound reduction of both NMDA‐and ischemia‐induced spectrin proteolysis, consistent with the possibility of some common mechanism in apoptosis and other forms of neuronal death in vivo.

CEP‐751 inhibits trk receptor tyrosine kinase activity in vitro and exhibits anti‐tumor activity

The present report describes the in vitro and in vivo profile of CEP‐751, a novel receptor tyrosine kinase inhibitor. CEP‐751 at 100 nM inhibits the receptor tyrosine kinase activity of the neurotrophin receptors trkA, trkB and trkC. CEP‐751 has no effect on activity of receptors for EGF, IGF‐I, insulin or on erbB2; inhibition of receptors for PDGF and bFGF was observed but occurred with lesser potency than inhibition of trk. CEP‐751 exhibited anti‐tumor efficacy against tumors derived from NIH3T3 cells transfected with trkA. Inhibition of trk phosphorylation could also be measured in these tumors, suggesting that anti‐tumor efficacy of CEP‐751 is related to inhibition of trk receptor tyrosine kinase activity. CEP‐751 was found to be without effect when administered to nude mice bearing SK‐OV‐3 tumors, which overexpress erbB2 receptors, providing further evidence that inhibition of tumor growth may be related to inhibition of trk receptor tyrosine kinase activity. Our data indicate that CEP‐751 is a potent trk inhibitor which possesses anti‐tumor activity. Int. J. Cancer 72:673–679, 1997.

Chronic 1,25-dihydroxyvitamin D3-mediated induction of nerve growth factor mRNA and protein in L929 fibroblasts and in adult rat brain

We have proposed that elevating levels of nerve growth factor (NGF) in the CNS is a rational strategy for treating certain neurodegenerative disorders. The present studies were conducted to determine: (1) if pharmacologically induced levels of NGF could be sustained for an extended time, and (2) if correlations exist between increases in NGF mRNA and NGF protein in L929 cells and in vivo. Short-term treatment of L929 cells with 1,25-dihydroxyvitamin D3 resulted in a two-fold increase in both NGF mRNA and NGF protein. These increases were sustained for up to 48 h with continuous exposure to 1,25-dihydroxyvitamin D3. In rats, 1,25-dihydroxyvitamin D3 (2.5 nmol; i.c.v.) induced NGF mRNA transiently, with peak two-fold increases observed 4 h post-injection. In contrast to L929 cells, 1,25-dihydroxyvitamin D3 did not elicit an increase in NGF protein after a single administration in vivo. However, consistent with long-term exposure in L929 cells, chronic 6 day infusion of 1,25-dihydroxyvitamin D3 resulted in induction of both NGF mRNA and NGF protein in the brain. These results indicate that 1,25-dihydroxyvitamin D3-mediated NGF induction in cultured L929 cells may predict of NGF induction in vivo, suggesting that L929 cells may have utility in studying underlying mechanisms of NGF induction by 1,25-dihydroxyvitamin D3. On the basis of NGFs ability to increase cholinergic function in animal models of cholinergic degeneration, these results are supportive of a role for NGF inducers as potential drugs for neurodegenerative disorders.

Pharmacological Induction of Nerve Growth Factor mRNA in Adult Rat Brain

Three structurally unrelated compounds, all of which induce nerve growth factor (NGF) in cell culture systems, were assessed for their ability to induce NGF mRNA in adult rat brain using a highly sensitive RNAse protection assay. Interleukin-1 beta (0.5-1 pmol) and 1,25-dihydroxyvitamin D3 (25-25,000 pmol) were extremely potent inducers of NGF mRNA, being respectively at least 50,000 and 4000 times more potent than 4-methylcatechol. These compounds elicited an approximate twofold increase in NGF mRNA in both the hippocampus and cortex, without altering beta-actin mRNA levels after a single intracerebroventricular injection. The duration of NGF induction was dependent on the compound administered. For example, the elevation of NGF mRNA elicited by interleukin-1 beta peaked at 8 h and lasted for at least 24 h. In contrast, the induction of NGF after 1,25-dihydroxyvitamin D3 and 4-methylcatechol administration peaked between 4 and 8 h and was not apparent 24 h after injection. These results demonstrate induction of NGF mRNA in vivo by administration of physiological or pharmacological agents and differentiate these agents by potency and duration of action. Further, these findings indicate that pharmacological induction of NGF may be a viable strategy for the treatment of neurodegenerative disorders such as Alzheimers disease.

Insulin-like growth factor-I prevents development of a vincristine neuropathy in mice

Vincristine is a commonly used antitumor agent whose major dose-limiting side-effect is a mixed sensorimotor neuropathy. To assess whether insulin-like growth factor-I (IGF-I), a neurotrophic agent that supports the survival of motoneurons and enhances regeneration of motor and sensory neurons, could prevent the peripheral neuropathy produced by vincristine, mice were treated with both vincristine (1.7 mg/kg, i.p., 2 x /week) and/or IGF-I (0.3 or 1 mg/kg, s.c. daily) for 10 weeks. In mice treated with vincristine alone, there was evidence of a mixed sensorimotor neuropathy as indicated by changes in behavior, nerve conduction and histology. Caudal nerve conduction velocity was significantly slower in mice treated with vincristine alone as compared with vehicle-treated mice. Vincristine treatment alone also significantly increased hot-plate latencies and reduced gait support and stride length, but not toe spread distances. The effects of vincristine were accompanied by degeneration of sciatic nerve fibers and demyelination, indicating a peripheral neuropathy. IGF-I (1 mg/kg, s.c.) administered to vincristine-treated mice prevented the neurotoxic effects of vincristine as measured by nerve conduction, gait, response to noxious stimuli and nerve histology. At a lower dose of 0.3 mg/kg administered s.c., IGF-I partially ameliorated the neuropathy induced by vincristine as this dose only prevented the change in nerve conduction and hot-plate latencies. IGF-I administered alone had no effect on any of these parameters. These results suggest that IGF-I prevents both motor and sensory components of vincristine neuropathy and may be useful clinically in preventing the neuropathy induced by vincristine treatment.

Preservation of cholinergic activity and prevention of neuron death by CEP-1347/KT-7515 following excitotoxic injury of the nucleus basalis magnocellularis

We have identified a class of small organic molecules, derived from the indolocarbazole K-252a, that promote the survival of cultured neurons. However, many of these indolocarbazoles inhibit protein kinase C and neurotrophin-activated tyrosine kinase receptors. These kinase inhibitory activities may limit the utility of these compounds for neurological disorders. A bis-ethyl-thiomethyl analogue of K-252a, CEP-1347/KT-7515, has been identified that lacks protein kinase C and tyrosine kinase receptor inhibitory activities, yet retains the ability to promote survival of cultured neurons, including cholinergic neurons derived from the basal forebrain. In the present studies, CEP-1347/KT-7515 was assessed for neurotrophic activity on basal forebrain neurons of in vivo rats following excitotoxic insult. Ibotenate infusion into the nucleus basalis magnocellularis reduced levels of choline acetyltransferase activity in the cortex, as well as reduced numbers of choline acetyltransferase-immunoreactive and retrogradely (FluoroGold)-labelled cortically-projecting neurons in the nucleus basalis. Systemically administered CEP-1347/KT-7515 attenuated the loss of cortical choline acetyltransferase activity and the loss of the number of choline acetyltransferase-immunoreactive and retrogradely-labelled FluoroGold neurons in the nucleus basalis. Moreover, CEP-1347/KT-7515 ameliorated the loss of cortical choline acetyltransferase if administration was initiated one day, but not seven days post-lesion. Together, these results demonstrate that CEP-1347/KT-7515 protects damaged cortically-projecting basal forebrain neurons from degeneration. Thus, CEP-1347/KT-7515 may have therapeutic potential in neurodegenerative diseases, such as Alzheimers disease, in which basal forebrain cholinergic neurons degenerate.