Diphenylhydantoin: pre- and postnatal administration alters diazepam binding in developing rat cerebral cortex.

Close correlations between the development of the anticonvulsant effects of diphenylhydantoin and increases in tritiated diazepam binding were observed in rats from fetal day 16 to maturation. In contrast, significant decreases in tritiated diazepam binding were observed in 2- and 3-week-old rats that were exposed in utero to diphenylhydantoin. These changes can be correlated with reported increases in seizure susceptibility after prenatal exposure to diphenylhydantoin.

Differences in the beta-adrenergic responsiveness between high and low passage rat glioma C6 cells.

Responsiveness to catecholamines was studied in two different strains of rat glioma C6 cells. The C6 cells of low passage possessed a high capacity to accumulate cyclic AMP in response to (-)-isoproterenol. Cholera toxin was also able to stimulate cyclic AMP accumulation in these cells. High passage C6 cells were unresponsive to (-)-isoproterenol or to cholera toxin except in the presence of a high concentration of phosphodiesterase inhibitor. The affinity of beta-adrenergic receptors on both strains for (-) [3H] dihydroalprenolol was similar; however, C6 low passage possessed several times the number of beta-adrenergic receptors found in C6 high passage. This difference correlated with the difference found in (-)-isoproterenol-stimulated adenylate cyclase between C6 low passage and high passage. The sodium fluoride-stimulated adenylate cyclase was similar in both strains. Cyclic AMP phosphodiesterase activity was 2-3 times higher in homogenates of C6 high passage than in low passage. In intact cells, the rate of breakdown of cyclic AMP was 5-times faster in C6 high passage than in low passage. Thus, differences in beta-adrenergic receptor number and phosphodiesterase activity explain in part the lack of responsiveness of C6 high passage. Our studies indicate that continuous subculturing of rat glioma C6 cells led to complex alterations in the beta-adrenergic receptor-adenylate cyclase system.

Sulfonylmethanesulfonamide inhibitors of carbonic anhydrase.

A series of sulfonylmethanesulfonamide derivatives is described, which are inhibitors of carbonic anhydrase (CA). The most potent of these is the racemic fluoro sulfone 9, which inhibits carbon dioxide hydration catalyzed by human CA II (CA-II) with an IC50 of 3 nM. Binding competition studies versus dansylamide indicate that the enantiomers of 9 have different affinities for CA-II, with equilibrium dissociation constants of 3.6 and 0.6 nM. QSAR analysis suggests that the key factors involved in achieving high affinity in this series are sulfonamide acidity, hydrophobicity, and minimization of steric demands at the carbon atom adjacent to the sulfonamide group.

4-substituted thiophene- and furan-2-sulfonamides as topical carbonic anhydrase inhibitors.

A series of 4-substituted thiophene- and furan-2-sulfonamides was prepared and was found to possess nanomolar-level potency for inhibition of human carbonic anhydrase II in vitro. Selected examples from this group were further evaluated for their potential to act as topically effective ocular hypotensive agents in the ocular normotensive albino rabbit and the ocular alpha-chymotrypsinized rabbit. Solubility studies in water and pH 7.4 buffer were carried out to estimate the ability of compounds to be formulated in solution. The sensitization potential of key representative structures was determined by in vitro glutathione reactivity studies and guinea pig maximization testing.

3-substituted thieno[2,3-b][1,4]thiazine-6-sulfonamides. A novel class of topically active carbonic anhydrase inhibitors.

3-Aminoalkyl derivatives of thieno[2,3-b][1,4]thiazine-6-sulfonamide were prepared for evaluation as topically active ocular hypotensive agents. The compounds described were found to be excellent in vitro inhibitors of carbonic anhydrase II and in vivo to lower intraocular pressure in three rabbit models of ocular hypertension. Compounds 20A, 20B, and 20C met the requirement of formulation as a 1% solution at pH 5.2, but none of the compounds described exhibited greater activity in the normotensive albino rabbit, the alpha-chymotrypsin-treated albino rabbit, or the normotensive pigmented rabbit than MK-927 or MK-507, the present clinical candidates.

New isomeric classes of topically active ocular hypotensive carbonic anhydrase inhibitors: 5-substituted thieno[2,3-b]thiophene-2-sulfonamides and 5-substituted thieno[3,2-b]thiophene-2-sulfonamides.

A series of 5-substituted thieno[2,3-b]- and thieno[3,2-b)- and thieno[3,2-b)thiophene-2-sulfonamides was prepared and evaluated for topical ocular hypotensive activity in glaucoma models. The 5-substituents were varied to maximize both inhibitory potency against carbonic anhydrase and water solubility. At the same time, these substituents were varied in order to obtain compounds with the appropriate pKa to minimize pigment binding in the iris. All of these variables were optimized in the best compound, 5-[[(methoxyethyl)[(methoxyethyl)ethyl] amino]methyl]thieno[2,3-b]thiophene-2-sulfonamide hydrochloride (55).

Thieno[2,3-b]furan-2-sulfonamides as topical carbonic anhydrase inhibitors.

Novel 5-[(alkylamino)methyl]thieno[2,3-b]furan-2-sulfonamides were prepared and evaluated in vitro for inhibition of human carbonic anhydrase II (CA II) and ex vivo for their ability to inhibit Ca II in the albino rabbit eye after topical administration. Compound 11a was found to lower intraocular pressure (IOP) in both the alpha-CT ocular hypertensive albino rabbit and the normal albino rabbit, but was ineffective at lowering IOP in a hypertensive, pigmented monkey model. Since 11a was highly bound to ocular pigment, a series of less basic analogs was prepared. Examples in this series were both less extensively bound to ocular pigment and more active at reducing IOP in pigmented rabbits after topical dosing. Key examples displayed moderate reactivity toward glutathione.

L-653,328: an ocular hypotensive agent with modest beta receptor blocking activity.

L-653,328 is the acetate ester of L-652,698 ((S)-3-tert-butylamino-1-[4-[2(hydroxy)ethyl]phenoxy]2-propanol). The penetration of L-652,698 into the albino rabbit eye was enhanced when the compound was instilled as its prodrug acetate ester. The instillation (one drop of 50 microliter) of 0.01, 0.05 and 0.1% solutions of L-653,328 significantly decreased in a dose-dependent manner the elevated intraocular pressure (IOP) of alpha-chymotrypsinized rabbits by 3.2, 4.7 and 6.1 mm Hg, respectively. A 0.01% solution of L-652,698 failed to significantly lower IOP, whereas this dose of timolol (3.8 mm Hg) and betaxolol (3.3 mm Hg) was effective. L-652,698 was active at 0.05% and 0.1%. Extraocular beta-adrenoceptor blockade was quantified in ganglion-blocked, conscious rabbits by determining effects on heart rate and blood pressure changes to i.v. isoproterenol (0.5 microgram/kg). Doses of timolol blocking isoproterenol-induced hypotension and tachycardia by 50% were 0.0065% and 0.03%, respectively. The corresponding doses for betaxolol were greater than 3% (43% inhibition) and 0.3%. Heart rate and blood pressure changes to isoproterenol were blocked by 18 and 36%, respectively, after the instillation of a 3% solution of L-653,328. The reduced propensity of L-653,328 for extraocular beta-adrenoceptor blockade stems from the modest affinity of L-652,698, its active moiety, for beta-adrenoceptors. The Ki values of L-652,698 for displacement of 125I-iodocyanopindolol binding to beta 1-(left ventricle) and beta 2-binding sites (iris + ciliary body) in the rabbit were 5.7 microM and 7.3 microM, respectively. In marked contrast, the corresponding values for timolol were 12 nM and 1.8 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

L-662,583 is a topically effective ocular hypotensive carbonic anhydrase inhibitor in experimental animals.

1. L-662,583 was a potent inhibitor in vitro of purified, human erythrocyte carbonic anhydrase II, possessing an IC50 of 0.7 nM. The IC50 values for MK-927, acetazolamide and methazolamide were 13.0 nM, 10.8 nM and 21.2 nM, respectively. 2. A 1 h pretreatment with one 50 microliters drop of a 0.1% solution of L-662,583 blocked carbonic anhydrase activity in a homogenate of the iris + ciliary body of albino rabbits by 63%. Similar treatment with 0.1% suspensions of acetazolamide and methazolamide elicited inhibitions of 30% and 20%, respectively. This ex vivo model indirectly assesses the ability of an agent to enter the rabbit eye. 3. Concentrations of L-662,583 in the cornea, aqueous humour and iris + ciliary body of albino rabbits were determined by h.p.l.c. at predetermined times after the instillation (one drop of 50 microliters) of a 2% solution of L-662,583. Peak levels for cornea (47.4 micrograms g-1), aqueous humour (4.51 micrograms ml-1) and iris + ciliary body (9.61 micrograms g-1) occurred at 0.5, 2 and 1 h after instillation, respectively. 4. The experimentally elevated intraocular pressure of the right eye of rabbits, induced by prior intraocular injection of alpha-chymotrypsin, was maximally decreased by 4.5 mmHg, 6.2 mmHg and 9.8 mmHg after the instillation (one drop of 50 microliters) of 0.01%, 0.1% and 0.5% solutions of L-662,583, respectively. All three concentrations lowered intraocular pressure at all time points from 1 h up to and including 5 h, the last recorded time point. The unilateral instillation of L-662,583 (0.5%) into the contralateral, left eye failed to lower the elevated intraocular pressure of the untreated, right eye. This finding indicates that the site of action of topically applied L-662,583 in this paradigm is local. The ocular normotensive, albino rabbit was much less susceptible than the ocular hypertensive rabbit to the intraocular pressure lowering effect of topically applied L-662,583, with a 2% solution maximally decreasing intraocular pressure by 2.3 mmHg. 5. Unilateral ocular hypertension was elicited in the right eye of sedated, cynomolgus monkeys by argon laser-induced photocoagulation of the trabecular meshwork. The instillation (one drop of 50 microL) of L-662, 583 (2%) significantly lowered the elevated intraocular pressure of the right eye at all time points from 1 h up to and including 5 h. The maximum decline was 8.3 mmHg at 3 h and this represented a reduction of 23% from the corresponding baseline value of 36.8 mmHg. The intraocular pressure of the hypertensive, right eye was maximally decreased by 4.1 mmHg and 4.8 mmHg after the instillation of 0.5% and 1% solutions of L-662,583, respectively. Like the rabbit, the normotensive eye of cynomolgus monkeys was more resistant than the hypertensive eye to the ocular hypotensive action of L-662, 583, as indicated by the inability of 0.5% and 1% solutions of the agent to lower intraocular pressure. L-662,583 (2%) maximally reduced the intraocular pressure of normotensive monkey eyes by 2.4 mmHg at 2 h. 6. L-662,583 is structurally different from MK-927, a carbonic anhydrase inhibitor that lowers the intraocular pressure of glaucoma patients following the instillation of a 2% solution. These preclinical observations indicate that L-662,583, like MK-927, is a water-soluble carbonic anhydrase inhibitor which, on topical administration, lowers intraocular pressure by virtue of an action confined to within the eye.

Chronic elevation of brain GABA by gamma-vinyl GABA treatment does not alter the sensitivity of GABAergic or dopaminergic receptors in rat CNS.

Rat brain GABA levels were elevated chronically by daily administration of gamma-vinyl GABA, an enzyme-activated, irreversible inhibitor of GABA:2-oxo-glutarate aminotransferase (GABA-T; EC2.6.1.19). Following various periods of drug treatment and withdrawal, the sensitivity of dopamine and GABA receptors in the CNS was determined by biochemical and behavioral evaluations. In contrast to chronic haloperidol treatment, none of the treatment schedules with gamma-vinyl GABA had any significant effect on parameters such as apomorphine induced locomotor activity, [3H] spiperone binding or dopamine-stimulated adenylate cyclase in the corpus striatum; nor did gamma-vinyl GABA treatment affect [3H] GABA binding or GABA-activated [3H] diazepam binding in the cerebral cortex. Moreover, co-administration of gamma-vinyl GABA and haloperidol did not alter the ability of the neuroleptic to induce supersensitivity in the striatal dopaminergic system. Thus, it appears that, in contrast to reported studies using chronic administration of other less specific GABA-T inhibitors such as gamma-acetylenic GABA, amino-oxyacetic acid and isonicotinic acid hydrazide or direct GABA agonists such as THIP (4,5,6,7-tetrahydroisoxazolo (5,4-c-)-pyridin-3-ol) or kojic amine, gamma-vinyl GABA does not alter the sensitivity of the striatal dopaminergic system.

Interaction of diphenylhydantoin and benzodiazepines in the CNS.

Using extracellular unit recording and microiontophoretic techniques, the anticonvulsant diphenylhydantoin (DPH) was found to increase the physiological efficacy of the benzodiazepines. This increased biological effect could be correlated with an enhanced specific binding of benzodiazepines measured in vivo following pretreatment of rats with DPH. The increased binding of benzodiazepines is due to an increase in the total number of benzodiazepine binding sites without an alteration in the affinity of these sites for [3H]diazepam. The data show that the effects of DPH on benzodiazepine binding are qualitatively different and independent from the effects of gamma-amino-butyric acid. Based on the dose-responsive relationship between benzodiazepine binding effects and the anticonvulsant activity of DPH and reports of other convulsant, anticonvulsant compounds which alter benzodiazepine binding, it is suggested that the benzodiazepine binding site may be relevant to convulsant-anticonvulsant activity.

Effects of phenytoin on [3H]diazepam binding in dissociated primary cortical cell culture.

The effects of chronic exposure of primary dissociated cerebral cortical cells in culture to the anticonvulsant drug phenytoin have been investigated using benzodiazepine binding techniques. By separating benzodiazepine binding into pharmacologically distinct subtypes, the data indicate that clonazepam-displaceable benzodiazepine binding (associated primarily with neuronal membranes) is significantly decreased by exposure to therapeutic and toxic doses of phenytoin while R05-4864-displaceable benzodiazepine binding (associated principally with non-neuronal elements) is enhanced. The ratio of clonazepam-displaceable to R05-4864-displaceable benzodiazepine binding appears to be the most sensitive indicator for these changes.

[3H]diazepam binding sites on chick neurons in primary culture.

High affinity [3H]diazepam binding sites were identified on neurons prepared from the hemispheres of 8-day-old chick embryos and grown in serum-containing or serum-free medium. Clonazepam (IC50 = 3 nM) was more potent than Ro 5-4864 (IC50 greater than 1000 nM) in displacing [3H]diazepam binding. GABA and pentobarbital, in the presence of chloride ions were able to stimulate [3H]diazepam binding synergistically. These interactions were found to be comparable to those observed in mammalian brain.

Characterization of [3H]forskolin binding sites in the iris-ciliary body of the albino rabbit.

[3H]Forskolin binding sites were identified using membranes prepared from the iris-ciliary body of adult, albino rabbits. Scatchard analysis of saturation binding experiments demonstrated that [3H]forskolin bound to a single population of high affinity sites. The Kd and Bmax values were 8.7 +/- 0.9 nM and 119.0 +/- 30.9 fmol/mg prot. using membranes prepared from frozen tissue and 17.0 +/- 6.2 nM and 184.4 +/- 47.2 fmol/mg prot. using fresh tissue. The binding of [3H]forskolin was magnesium-dependent. The Bmax was enhanced by sodium fluoride and Gpp(NH)p, a nonhydrolyzable guanine nucleotide analog. Forskolin was the most potent inhibitor of [3H]forskolin binding; two commercially-available analogs were weaker inhibitors. In an adenylate cyclase assay, there was the same rank order of potency to enhance enzyme activity. Based upon binding affinities, magnesium-dependence, sensitivity to sodium fluoride and Gpp(NH)p, rank order of potencies of analogs and correlation of binding with adenylate cyclase activity, these studies suggest that the [3H]forskolin binding site in the iris-ciliary body is similar to the binding site in other tissues.

Characterization of serotonin receptors in the iris + ciliary body of the albino rabbit.

Experiments were undertaken to determine if serotonin (5-HT) radioligand binding sites were present in a membrane fraction of iris + ciliary body from adult, albino rabbits. The total binding of 3H-5-HT, 3H-spiroperidol (SPI) and 3H-ketanserin (KET), all at 2 nM, was determined in the absence and in the presence of ketanserin, mianserin, methysergide or 5-methoxytryptamine (5-MT), all at 1 microM. Except for ketanserin, which displaced 15% of 3H-KET binding, none of the agents altered 3H-SPI or 3H-KET binding. Reductions of 12% and 14% in 3H-5-HT binding were achieved by ketanserin and mianserin, respectively. In contrast, methysergide and 5-MT displaced 3H-5-HT binding by 57% and 56%, respectively. 5-HT (1 microM) also displaced 3H-5-HT binding by approximately 60% and this was used to measure nonspecific binding. Specific binding of 3H-5-HT was saturable and of high affinity with one population of binding sites being labelled. Kd and Bmax values of 1.1 nM and 57.8 fmoles/mg protein were obtained. Seven 5-HT antagonists possessing various affinities for 5-HT1 and 5-HT2 binding sites were assessed for their ability to displace specific 3H-5-HT binding. Ketanserin was the least potent (Ki greater than 3 microM). In contrast, the respective Ki values for metergoline and methysergide were 13 nM and 20 nM. These observations indicate the presence of 5-HT1 binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II receptors labelled with 125I-[Sar1, Ile8]-AII in albino rabbit ocular tissues.

High affinity binding sites for the angiotensin II antagonist 125I-[Sar1,Ile8]-AII have been identified and characterized in membrane suspensions of ocular tissues of albino rabbits. Scatchard analysis of the binding indicated a single class of sites with Kd values of 186, 92, 152, 50, 102 pM for the iris + ciliary body, choroid, ciliary process, retina and cornea, respectively. The corresponding concentrations of binding sites were 22, 68, 35, 22 and 4 fmole/mg of protein. The order of potency for several AII analogs to compete with 125I-[Sar1,Ile8]-AII at its binding sites in iris + ciliary body membranes ([Sar1,Leu8]-AII = [Sar1,Ile8]-AII greater than AII = [Sar1, Ala8]-AII greater than AIII greater than AI) resembled the order of potency found for AII receptors in other tissues. The competition curves for this tissue using AII and AIII were best explained by the existence of two populations of binding sites. The addition of the guanine nucleotide, GppNHp, to the assay resulted in a 6.7-fold and 2.3-fold decrease in the respective affinities of AII and AIII for 125I-[Sar1,Ile8]-AII binding sites without a change in the slope of the competition curves. The GppNHp-induced effect was also observed in ciliary process membranes but not in retinal or choroidal membranes. These results indicate the presence of AII receptors regulated by a GTP-binding protein in both the ciliary process and the iris + ciliary body of the rabbit. They also suggest a difference in the guanine nucleotide regulation of AII receptors in different ocular tissues.

A comparison of L-671,152 and MK-927, two topically effective ocular hypotensive carbonic anhydrase inhibitors, in experimental animals.

L-671,152 is a water-soluble, carbonic anhydrase inhibitor structurally similar to MK-927, a carbonic anhydrase inhibitor that, on topical administration, lowers the intraocular pressure (IOP) of experimental animals and humans. L-671,152 was more potent than MK-927 at inhibiting purified, human erythrocyte carbonic anhydrase II in vitro, as reflected in their respective IC50 values of 0.16 nM and 1.19 nM. Both compounds were compared for topical, ocular hypotensive activity in pigmented rabbits and cynomolgus monkeys. Ocular hypertension was induced in the latter by argon laser photocoagulation of the trabecular meshwork. A 2% solution of L-671,152 was more potent than 2% MK-927 in lowering the IOP of ocular hypertensive monkeys, the maximum reductions being 13.8 mm Hg (37%) and 9.6 mm Hg (27%) at 5 hr and 4 hr, respectively. Moreover, the duration of action of L-671,152 was superior to that of MK-927. The ocular hypotensive effect of L-671,152 was greater than that of MK-927 over a range of concentrations (0.5%-2%) in pigmented rabbits whose IOP was inherently elevated. The peak declines in the IOP of these rabbits after the instillation of 2% solutions of L-671,152 and MK-927 were 6.1 mm Hg and 4.8 mm Hg, respectively. L-671,152 was very effective in lowering the elevated IOP of alpha-chymotrypsinized rabbits and the unilateral instillation of 0.5% L-671,152 into the contralateral eye failed to decrease the elevated IOP of the alpha-chymotrypsinized eye. This finding indicates that the site of action of topically applied L-671,152 is local. The enhancement in the potency of L-671,152 over MK-927 is attributed to a greater inhibition of carbonic anhydrase activity.