RESUMO
Epilepsy remains a devastating neurological disorder associated with recurrent, unprovoked, spontaneous epileptic seizures. Current treatments involve seizure suppression using antiepileptic drugs (AEDs); however, many patients remain refractory to current treatments or suffer serious side effects. In view of this continued need for more effective and safer AEDs, we have designed a novel compound, 3-hydroxy-3-(4-methoxyphenyl)-1-methyl-1,3-dihydro-indol-2-one (YWI92), based on a lactam structural class, and evaluated its modulation of human neuronal sodium channel isoform (hNa(v))1.2 currents and hippocampal neuron action potential firing. Furthermore, we have tested its AED activity using a chronic and acute rat seizure model. In a similar manner to lamotrigine, a clinically used AED, YWI92 exhibited tonic block of hNa(v)1.2 channels and caused a hyperpolarizing shift in the steady-state inactivation curve when using a 30-s inactivating prepulse. YWI92 also delayed the time constants of channel repriming after a 30-s inactivating prepulse and exhibited use-dependent block at 20-Hz stimulation frequency. In membrane excitability experiments, YWI92 inhibited burst firing in CA1 neurons of animals with temporal lobe epilepsy at concentrations that had little effect on CA1 neurons from control animals. These actions on neuronal activity translated into AED activity in the maximal electroshock acute seizure model (ED(50) = 22.96 mg/kg), and importantly, in a chronic temporal lobe epilepsy model, in which the mean number of seizures was reduced. Notably, YWI92 exhibited no sedative/ataxic side effects at concentrations up to 500 mg/kg. In summary, greater affinity for inactivated sodium channels, particularly after long depolarizing prepulses, may be important for both anticonvulsant activity and drug tolerability.
Assuntos
Anticonvulsivantes/farmacologia , Epilepsia/tratamento farmacológico , Indóis/farmacologia , Lactamas/farmacologia , Sistema Límbico/fisiopatologia , Convulsões/tratamento farmacológico , Convulsões/prevenção & controle , Canais de Sódio/fisiologia , Animais , Anticonvulsivantes/uso terapêutico , Modelos Animais de Doenças , Eletrofisiologia , Epilepsia do Lobo Temporal/tratamento farmacológico , Epilepsia do Lobo Temporal/fisiopatologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Humanos , Rim/efeitos dos fármacos , Rim/embriologia , Rim/fisiologia , Lactamas/uso terapêutico , Sistema Límbico/efeitos dos fármacos , Masculino , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Canais de Sódio/efeitos dos fármacosRESUMO
The voltage-gated sodium channel remains a rich area for the development of novel blockers. In this study we used comparative molecular field analysis (CoMFA), a ligand-based design strategy, to generate a 3D model based upon local anesthetics, hydantoins, and alpha-hydroxyphenylamides to elucidate a SAR for their binding site in the neuronal sodium channel. Correlation by partial least squares (PLS) analysis of in vitro sodium channel binding activity (expressed as pIC(50)) and the CoMFA descriptor column generated a final non-cross-validated model with q(2)=0.926 for the training set. The CoMFA steric and electrostatic maps described a binding site predominately hydrophobic in nature. This model was then used to design and predict a series of novel sodium channel blockers that utilized overlapping structural features of phenytoin, hydroxy amides, and the local anesthetic lidocaine. Synthesis and evaluation of these compounds for their ability to inhibit [(3)H]-batrachotoxin revealed that these compounds have potent sodium channel blockade. Furthermore, the CoMFA model was able to accurately predict the binding of these compounds to the neuronal sodium channel. Synthesis and subsequent sodium channel evaluation of compound 37 (predicted IC(50)=7 microM, actual IC(50)=6 microM), established that novel compounds based on overlapping regions of phenytoin and lidocaine are better binders to the sodium channel than phenytoin itself (IC(50)=40 microM).
Assuntos
Desenho de Fármacos , Lidocaína/química , Fenitoína/química , Bloqueadores dos Canais de Sódio/síntese química , Sistemas Inteligentes , Concentração Inibidora 50 , Lidocaína/farmacologia , Ligantes , Modelos Moleculares , Fenitoína/farmacologia , Relação Estrutura-AtividadeRESUMO
Phenytoin (DPH) is a clinically useful sodium (Na) channel blocker with efficacy against partial and generalized seizures. We have developed a novel hydantoin compound (HA) using comparative molecular field analysis (CoMFA) and evaluated its effects on hNa(v)1.2 channels. Both DPH and HA demonstrated affinity for resting (K(r)=13.9microM for HA, K(r)=464microM for DPH) and slow inactivated channels (K(I)=975nM for HA, K(I)=20.6microM for DPH). However, HA also exhibited an affinity for fast inactivated channels (K(I)=2.5microM) and shifted the V(1/2) for activation in the depolarizing direction. Furthermore, HA exhibited profound use dependent block at both 5 and 10Hz stimulation frequencies. In the 6Hz seizure model (32mA) HA had an ED(50) of 47.1mg/kg and a TD(50) of 131mg/kg (protective index (PI)=2.8). In comparison, the ED(50) for DPH was approximately 27.5mg/kg with a TD(50) of 35.6mg/kg (PI approximately 1.3). These findings provide evidence for the utility of CoMFA in the design of novel anticonvulsants and support the hypothesis that states selectivity plays an important role in achieving optimal protection with minimal side effects.
Assuntos
Anticonvulsivantes/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Fenitoína/farmacologia , Convulsões/tratamento farmacológico , Canais de Sódio/efeitos dos fármacos , Animais , Anticonvulsivantes/química , Comportamento Animal/efeitos dos fármacos , Linhagem Celular Transformada , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Avaliação de Medicamentos , Estimulação Elétrica/métodos , Humanos , Hidantoínas/química , Hidantoínas/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Modelos Moleculares , Técnicas de Patch-Clamp/métodos , Fenitoína/química , Convulsões/etiologia , Canais de Sódio/fisiologia , Transfecção/métodosRESUMO
Sodium (Na) channel blockers are known to possess antihyperalgesic properties. We have designed and synthesized a novel Na channel antagonist, alpha-hydroxyphenylamide, and determined its ability to inhibit both TTX-sensitive (TTX-s) and TTX-resistant (TTX-r) Na currents from small dorsal root ganglion (DRG) neurons. alpha-Hydroxyphenylamide tonically inhibited both TTX-s and TTX-r Na currents yielding an IC(50) of 8.2+/-2.2 microM (n=7) and 28.9+/-1.8 microM (n=8), respectively. In comparison, phenytoin was less potent inhibiting TTX-s and TTX-r currents by 26.2+/-4.0% (n=8) and 25.5+/-2.0%, respectively, at 100 microM. alpha-Hydroxyphenylamide (10 microM) also shifted equilibrium gating parameters of TTX-s Na channels to greater hyperpolarized potentials, slowed recovery from inactivation, accelerated the development of inactivation and exhibited use-dependent block. In the chronic constriction injury (CCI) rat model of neuropathic pain, intraperitoneal administration of alpha-hydroxyphenylamide attenuated the hyperalgesia by 53% at 100mg/kg, without affecting motor coordination in the Rotorod test. By contrast, the reduction in pain behavior produced by phenytoin (73%; 100mg/kg) was associated with significant motor impairment. In summary, we report that alpha-hydroxyphenylamide, a sodium channel antagonist, exhibits antihyperalgesic properties in a rat model of neuropathic pain, with favorable sedative and ataxic side effects compared with phenytoin.
Assuntos
Amidas/química , Amidas/uso terapêutico , Dor/tratamento farmacológico , Fenitoína/análogos & derivados , Fenitoína/farmacologia , Neuropatia Ciática/tratamento farmacológico , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/uso terapêutico , Canais de Sódio/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Amidas/farmacologia , Animais , Relação Dose-Resposta a Droga , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/fisiologia , Masculino , Dor/fisiopatologia , Fenitoína/química , Fenitoína/uso terapêutico , Ratos , Ratos Sprague-Dawley , Neuropatia Ciática/fisiopatologia , Bloqueadores dos Canais de Sódio/farmacologiaRESUMO
BACKGROUND: Nitroglycerin can induce relaxation of swine carotid artery without sustained reductions in [Ca2+]i or myosin regulatory light chain (MRLC) phosphorylation. This has been termed force suppression and been found to correlate with ser16-phosphorylation of heat shock protein 20 (HSP20). We tested for the existence of this mechanism in a smooth muscle that is not responsive to nitric oxide. METHODS: Isometrically mounted mucosa free rabbit bladder strips were contracted with carbachol and relaxed with 8-Br-cGMP, forskolin, or isoprenaline. RESULTS: Contraction was associated with a highly cooperative relation between MRLC phosphorylation and force such that very small increases in MRLC phosphorylation induced large increases in force. Relaxation induced by 8-Br-cGMP, forskolin, or isoprenaline did not shift the MRLC phosphorylation-force relation from that observed with carbachol alone, i.e. there was no force suppression. HSP20 content was negligible (approximately two hundred-fold less than swine carotid). CONCLUSION: The lack of force suppression in the absence of HSP20 is consistent with the hypothesized role for HSP20 in the force suppression observed in tonic smooth muscles.
Assuntos
Proteínas de Choque Térmico HSP20/deficiência , Contração Muscular/fisiologia , Bexiga Urinária/fisiologia , Adenilil Ciclases/metabolismo , Animais , Carbacol/farmacologia , Artérias Carótidas/metabolismo , Colforsina/farmacologia , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Proteínas de Choque Térmico HSP20/metabolismo , Técnicas Imunológicas , Técnicas In Vitro , Isoproterenol/farmacologia , Masculino , Contração Muscular/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Fosforilação , Coelhos , Coloração e Rotulagem , Suínos , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismoRESUMO
The purpose of this investigation was to evaluate the role of corticotropin releasing factor (CRF) on micturition. CRF is involved in the endocrine and central nervous system responses to stress and is also expressed in sites responsible for the control of micturition. In this investigation, cystometric experiments were performed in awake and unrestrained Wistar rats and on Spontaneous Hypertensive Rats, which are used as a rodent model of detrusor overactivity and anxiety. In vitro effects of CRF were evaluated using strips of detrusor muscle in an organ bath preparation. CRF (6.0 microg) administered via intrathecal and intraperitoneal routes, but not intracerebroventricularly, lowered the micturition threshold. CRF reduced the intercontraction interval by 28% and 26% after intrathecal or intraperitoneal administration, respectively, and reduced micturition volume by 34.7% and 30.2%, respectively. In Wistar-Kyoto rats, 6.0 microg intrathecal CRF significantly reduced intercontraction interval (423 +/- 79 vs. 669 +/- 59 s) and micturition volume (0.30 +/- 0.04 vs. 0.69 +/- 0.07 ml) compared to controls that received saline vehicle. These effects were blocked by pretreatment with 6.0 mug intrathecal astressin, a potent CRF antagonist, demonstrating that the effects are CRF receptor mediated. In Spontaneous Hypertensive Rats, 6.0 mug intrathecal CRF was found to have minimal stimulatory effects on the bladder, whereas astressin reduced baseline detrusor overactivity. CRF had no direct contractile effects on detrusor muscle strips. These results demonstrate that in the absence of detrusor overactivity, CRF stimulates micturition when administered via the intrathecal or intraperitoneal routes. Further studies are needed to explore the possibility whether CRF antagonists are effective for detrusor overactivity and the overactive bladder syndrome.
Assuntos
Hormônio Liberador da Corticotropina/antagonistas & inibidores , Hormônio Liberador da Corticotropina/fisiologia , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/farmacologia , Micção/efeitos dos fármacos , Animais , Ansiedade/fisiopatologia , Cateterismo , Hormônio Liberador da Corticotropina/farmacologia , Feminino , Técnicas In Vitro , Injeções Intraperitoneais , Injeções Intraventriculares , Injeções Espinhais , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Ratos Wistar , Medula Espinal/fisiologia , Bexiga Urinaria Neurogênica/fisiopatologia , Cateterismo UrinárioRESUMO
The development of new therapies for chronic pain is an area of unmet medical need. Central to pathways of chronic pain is the upregulation of voltage-gated sodium channels. The use of tricyclic antidepressants, which also have sodium channel activity, in chronic pain therapy prompted us to develop novel compounds from this scaffold. Herein, we show that the tricyclic moiety is not needed for effective inhibition of the [(3)H]-BTX binding site and sodium currents of hNa(v)1.2. Our lead compound 6, containing a diphenyl amine motif, demonstrated a 53% inhibitory block of Na(v)1.2 currents at 10microM, which is greater than 50% increase in current block in comparison to the amitriptyline standard. Altogether our study establishes that the tricyclic motif is unnecessary for hNa(v)1.2 activity and modification of the amine portion is detrimental to sodium channel block.
Assuntos
Difenilamina/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Bloqueadores dos Canais de Sódio/farmacologia , Amitriptilina/farmacologia , Antidepressivos Tricíclicos/farmacologia , Células Cultivadas , Difenilamina/síntese química , Difenilamina/química , Eletrofisiologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Rim/citologia , Rim/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.2 , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/química , Canais de Sódio/metabolismoRESUMO
PURPOSE: Bladder outlet obstruction induces detrusor hypertrophy and it can eventually lead to decreased bladder smooth muscle contractility. Heat shock protein 20 is the proposed mediator of force suppression in vascular smooth muscle. We investigated whether heat shock protein 20 could also mediate the decreased contractility observed in partially obstructed rat bladders. MATERIALS AND METHODS: Female Wistar rats (Harlan Laboratories, Indianapolis, Indiana) were randomized to partial urethral ligation or sham ligation. After 3 weeks the rats were sacrificed, and the bladders were harvested, frozen, homogenized and analyzed for heat shock protein 20 content by Western blot immunoreactivity. The content of myosin regulatory light chain, a constitutively expressed protein, was determined as a control. Bladder smooth muscle strips were dissected from some rats and mounted for force generation measurement. RESULTS: At cystectomy obstructed bladders were significantly heavier and had more residual urine compared to sham operated bladders. Heat shock protein 20 immunoreactivity was significantly increased a mean +/- 1 SEM of 1.9 +/- 0.3-fold in obstructed vs sham operated bladders. Control protein myosin regulatory light chain immunoreactivity did not significantly differ in obstructed and sham operated bladders. Maximal stress, that is force per cross-sectional area, was significantly decreased in obstructed vs sham operated bladders. Human bladder was found to express immunoreactive heat shock protein 20. CONCLUSIONS: We noted that partially obstructed rat bladders 1) express higher levels of heat shock protein 20 and 2) generate less stress than sham operated bladders. These data suggest the possibility that heat shock protein 20 over expression could at least partially mediate the decreased contractile activity observed with partial bladder outlet obstruction. The mechanism for increased heat shock protein 20 expression is unknown but it may involve increased mechanical stress or hypoxia from urethral obstruction. Human bladder expressed immunoreactive heat shock protein 20, suggesting that a similar mechanism could potentially occur in humans. If confirmed in humans, patients with clinical conditions that result in detrusor hypocontractility could potentially benefit from pharmacological interventions aimed at inhibiting heat shock protein 20.
Assuntos
Proteínas de Choque Térmico HSP20/metabolismo , Contração Muscular/fisiologia , Músculo Liso/fisiopatologia , Obstrução do Colo da Bexiga Urinária/metabolismo , Obstrução do Colo da Bexiga Urinária/fisiopatologia , Bexiga Urinária/metabolismo , Animais , Feminino , Humanos , Cadeias Leves de Miosina/metabolismo , Ratos , Ratos WistarRESUMO
Serine 19 phosphorylation of the myosin regulatory light chain (MRLC) appears to be the primary determinant of smooth muscle force development. The relationship between MRLC phosphorylation and force is nonlinear, showing that phosphorylation is not a simple switch regulating the number of cycling cross bridges. We reexamined the MRLC phosphorylation-force relationship in slow, tonic swine carotid media; fast, phasic rabbit urinary bladder detrusor; and very fast, tonic rat anococcygeus. We found a sigmoidal dependence of force on MRLC phosphorylation in all three tissues with a threshold for force development of approximately 0.15 mol P(i)/mol MRLC. This behavior suggests that force is regulated in a highly cooperative manner. We then determined whether a model that employs both the latch-bridge hypothesis and cooperative activation could reproduce the relationship between Ser(19)-MRLC phosphorylation and force without the need for a second regulatory system. We based this model on skeletal muscle in which attached cross bridges cooperatively activate thin filaments to facilitate cross-bridge attachment. We found that such a model describes both the steady-state and time-course relationship between Ser(19)-MRLC phosphorylation and force. The model required both cooperative activation and latch-bridge formation to predict force. The best fit of the model occurred when binding of a cross bridge cooperatively activated seven myosin binding sites on the thin filament. This result suggests cooperative mechanisms analogous to skeletal muscle that will require testing.