Erik Falch

Kinetic Characterization of Inhibition of γ‐Aminobutyric Acid Uptake into Cultured Neurons and Astrocytes by 4,4‐Diphenyl‐3‐Butenyl Derivatives of Nipecotic Acid and Guvacine

The effects of N‐(4,4‐diphenyl‐3‐butenyl) derivatives of nipecotic acid (SKF‐89976‐A and SKF‐100844‐A) and guvacine (SKF‐100330‐A) on neuronal and astroglial γ‐aminobutyric acid (GABA) uptake were investigated. In addition, the uptake of SKF‐89976‐A was studied using the tritiated compound. All of the compounds were found to be competitive inhibitors of GABA uptake irrespective of the cell type, with Ki values similar to or lower than those of the parent amino acids. Moreover, none of the compounds exhibited selectivity with regard to inhibition of neuronal and glial GABA uptake. In spite of the competitive nature of SKF‐89976‐A, the compound was not transported by the GABA carriers in the two cell types, because no saturable uptake could be demonstrated.

First Demonstration of a Functional Role for Central Nervous System Betaine/γ-Aminobutyric Acid Transporter (mGAT2) Based on Synergistic Anticonvulsant Action among Inhibitors of mGAT1 and mGAT2

In a recent study, EF1502 [N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo [d]isoxazol-3-ol], which is an N-substituted analog of the GAT1-selective GABA uptake inhibitor exo-THPO (4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol), was found to inhibit GABA transport mediated by both GAT1 and GAT2 in human embryonic kidney (HEK) cells expressing the mouse GABA transporters GAT1 to 4 (mGAT1–4). In the present study, EF1502 was found to possess a broad-spectrum anticonvulsant profile in animal models of generalized and partial epilepsy. When EF1502 was tested in combination with the clinically effective GAT1-selective inhibitor tiagabine [(R)-N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid] or LU-32-176B [N-[4,4-bis(4-fluorophenyl)-butyl]-3-hydroxy-4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol], another GAT1-selective N-substituted analog of exo-THPO, a synergistic rather than additive anticonvulsant interaction was observed in the Frings audiogenic seizure-susceptible mouse and the pentylenetetrazol seizure threshold test. In contrast, combination of the two mGAT1-selective inhibitors, tiagabine and LU-32-176B, resulted in only an additive anticonvulsant effect. Importantly, the combination of EF1502 and tiagabine did not result in a greater than additive effect in the rotarod behavioral impairment test. In subsequent in vitro studies conducted in HEK-293 cells expressing the cloned mouse GAT transporters mGAT1 and mGAT2, EF1502 was found to noncompetitively inhibit both mGAT1 and the betaine/GABA transporter mGAT2 (Ki of 4 and 5 μM, respectively). Furthermore, in a GABA release study conducted in neocortical neurons, EF1502 did not act as a substrate for the GABA carrier. Collectively, these findings support a functional role for mGAT2 in the control of neuronal excitability and suggest a possible utility for mGAT2-selective inhibitors in the treatment of epilepsy.

Piperidine‐4‐sulphonic Acid, a New Specific GAB A Agonist

The use of GABA agonists may be relevant for the treatment of certain neurological and psychiatric diseases, for which malfunctions of the GABA system have been demonstrated (for references see Roberts et al., 1976; Krogsgaard-Larsen et al., 1979~) . Accordingly there is an interest in the development of specific GABA agonists with pharmacokinetic and toxicological properties acceptable for use in humans. Furthermore, specific GABA agonists are important tools for studies of the molecular mechanisms for the interactions between clinically useful drugs such as the benzodiazepines and the central GABA receptors (Tallman et al., 1978; Karobath et al., 1979). Isoguvacine and THIP are cyclic analogues of trans-4aminocrotonic acid and muscimol, respectively. This class of potent and specific GABA agonist, in which the amino groups are incorporated into six-membered rings (Krogsgaard-Larsen et al., 1977), has now been extended with piperidine-4-sulphonic acid (P4S) (Table l) , which is a cyclic analogue of 3-aminopropanesulphonic acid (3APS). The latter compound is a potent GABA agonist (Curtis and Watkins, 1965) and also a weak inhibitor of GABA uptake into rat brain slices (Beart and Johnston, 1973).

Water-soluble, solution-stable, and biolabile N-substituted (aminomethyl)benzoate ester prodrugs of acyclovir

Various N-substituted 3- or 4-(aminomethyl)benzoate esters of acyclovir were synthesized and evaluated as water-soluble prodrug forms with the aim of improving the delivery characteristics of acyclovir, in particular its parenteral administration. The esters showed a high solubility in weakly acidic solutions and, as demonstrated with the 3-(N,N-dipropylaminomethyl)benzoate ester, a high stability in such solutions, allowing storage for several years. The esters combine these properties with a high susceptibility to undergo enzymatic hydrolysis in plasma. The half-lives of hydrolysis in 80% human plasma ranged from 0.8 to 57 min, the rate being highly dependent on the position (3 or 4) of the aminomethyl group relative to the ester moiety. All esters were more lipophilic than acyclovir in terms of octanol–pH 7.4 buffer partition coefficients. These properties make N-substituted (aminomethyl)-benzoate esters a promising new prodrug type for acyclovir to enhance its delivery characteristics.

Prodrugs of 5-fluorouracil. IV. Hydrolysis kinetics, bioactivation and physicochemical properties of various N-acyloxymethyl derivatives of 5-fluorouracil

Abstract The kinetics and mechanism of hydrolysis of various 1-, 3- and 1,3-acyloxymethyl derivatives of 5-fluorouracil were studied to assess their potential as prodrugs with the aim of enhancing the delivery characteristics of the parent drug. All the derivatives hydrolyzed to yield 5-fluorouracil in quantitative amounts, passing through an unstable N-hydroxymethyl-5-fluorouracil intermediate. The pH-rate profiles obtained revealed the occurrence of specific acid and base catalysis as well as of a water-catalyzed reaction. The rates of hydrolysis were accelerated markedly in the presence of human plasma or rat liver homogenate, suggesting the utility of the derivatives as prodrugs. The derivatives were all more lipophilic than 5-fluoro-uracil as determined by partition experiments in octanol-aqueous buffer systems but the aqueous solubility was only slightly reduced or, for some derivatives, even greater than that of 5-fluorouracil. This behaviour was attributed to differences in the crystal lattice energy, and relationships between melting points, partition coefficients and water-solubilities for these and 11 other prodrug derivatives of 5-fluoro-uracil were established.

Action of bicyclic isoxazole GABA analogues on GABA transporters and its relation to anticonvulsant activity

The inhibitory action of bicyclic isoxazole gamma-aminobutyric acid (GABA) analogues and their 4,4-diphenyl-3-butenyl (DPB) substituted derivatives has been investigated in cortical neurones and astrocytes as well as in human embryonic kidney (HEK 293) cells transiently expressing either mouse GABA transporter-1 (GAT-1), GAT-2, -3 or -4. It was found that 4,5,6,7-tetrahydroisoxazolo(4,5-c)pyridin-3-ol (THPO) and 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-c]azepin-3-ol (THAO) displayed some inhibitory activity on GAT-1 and GAT-2, where the compounds exhibited a slightly lower potency on GAT-2 compared to GAT-1. DPB substituted THPO displayed higher inhibitory potency than the parent compound regarding the ability to inhibit GABA uptake via GAT-1 and GAT-2. Concerning the inhibitory mechanism, THPO, THAO and DPB-THPO were competitive inhibitors on GAT-1 transfected HEK 293 cells and the same mechanism was observed for THPO in GAT-3 transfected cells. Regarding GABA uptake into neurones and astroglia cells THAO and DPB-THAO both displayed competitive inhibitory action. The observations that THPO, THAO as well as their DPB derivatives act as competitive inhibitors together with earlier findings such as potent anticonvulsant activity, lack of proconvulsant activity and the ability of THPO to increase extracellular GABA concentration, indicate that these bicyclic isoxazole GABA analogues and their DPB derivatives may be useful lead structures in future search for new antiepileptic drugs.

4,5,6,7-Tetrahydroisothiazolo[5,4-c]pyridin-3-ol and related analogues of THIP. Synthesis and biological activity.

The thio analogues of the GABA (gamma-aminobutyric acid) agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), the GABA uptake inhibitor THPO (4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol), and the glycine antagonist THAZ (5,6,7,8-tetrahydro-4H-isoxazolo[4,5-d]azepin-3-ol) have been synthesized and tested biologically on single neurons in the cat spinal cord and in vitro by using synaptic membrane preparations obtained from rat brains. In contrast to THIP, thio-THIP (4,5,6,7-tetrahydroisothiazolo[5,4-c]pyridin-3-ol, 5) was only a weak GABA agonist. Thio-THPO (4,5,6,7-tetrahydroisothiazolo[4,5-c]pyridin-3-ol, 10) was slightly weaker than THPO as an inhibitor of GABA uptake in vitro, and these two compounds were approximately equipotent in enhancing the inhibition of the firing of cat spinal neurons by GABA. Like THAZ and structurally related bicyclic isoxazole zwitterions, thio-THAZ (5,6,7,8-tetrahydro-4H-isothiazolo[4,5-d]azepin-3-ol, 15) was an antagonist at glycine receptors on cat spinal neurons. The I/U ratios, which reflect the ability of neutral amino acids to penetrate the blood-brain barrier (BBB), were calculated for 5 (I/U = 16), 10 (63), and 15 (200). These low I/U ratios, compared with the findings that THIP (I/U = 500 or 1500) and THPO (I/U = 2500) enter the brain after systemic administration, suggest that the thio analogues may penetrate the BBB very easily.

GABA‐Mimetic Activity and Effects on Diazepam Binding of Aminosulphonic Acids Structurally Related to Piperidine‐4‐Sulphonic Acid

The relationship between structure, in vivo activity, and in vitro activity of some analogues of the γ‐aminobutyric acid (GABA) agonist piperidine‐4‐sulphonic acid (P4S) was studied. The syntheses of 1,2,3,6‐tetrahydropyridine‐4‐sulphonic acid (DH‐P4S) and (RS)‐pyrrolidin‐3‐yl‐methanesulphonamide (PMSA‐amide) are described. Like P4S, its unsaturated analogue DH‐P4S and the five‐ring isomer (RS)‐pyrrolidin‐3‐yl‐methanesulphonic acid (PMSA) were bicuculline methochloride (BMC)‐sensitive inhibitors of the firing of neurones in the cat spinal cord. Whereas isonipecotic acid was less potent than its unsaturated analogue isoguvacine as a GABA‐mimetic and as an inhibitor of GABA binding, the opposite relative potencies of P4S and DH‐P4S were observed, P4S being proportionally more potent than DH‐P4S. In contrast with P4S and DH‐P4S, PMSA, which is an analogue of the potent GABA uptake inhibitor and BMC‐sensitive GABA‐mimetic homo‐β‐proline, was a relatively weak inhibitor of GABA uptake in vitro. PMSA‐amide was more than two orders of magnitude weaker than PMSA as an inhibitor of GABA binding and did not significantly affect GABA uptake in vitro. The effects of 3‐aminopropanesulphonic acid (3‐APS), PMSA, P4S, and DH‐P4S on the binding of [3H]diazepam in vitro at 30°C, in the presence or absence of chloride ions, were studied and compared with those of the structurally related amino acids GABA, homo‐β‐proline, isonipecotic acid, and isoguvacine. Under these conditions the aminosulphonic acids were weaker than the respective amino acids in enhancing [3H]diazepam binding, the difference being more pronounced in the absence of chloride.

Ocular bioavailability of pilocarpic acid mono- and diester prodrugs as assessed by miotic activity in the rabbit

Abstract Following topical ophthalmic dosing of rabbits with pilocarpic acid diester and monoester prodrug solutions, significant biological activity was observed. The response, measured as pupillary diameter, vs time profiles, showed a slightly longer time requirement for attainment of maximal activity, a plateauing region of sustained response, and a longer duration of action as compared to pilocarpine. Several monoesters were capable of maintaining durations of action 1.5 times that of pilocarpine while the diesters were active for up to 2.25 times as long, and from half the dosing concentration. The profile shapes eliminate the early spiking response seen with higher doses of pilocarpine. The bioavailability, as assessed by response, of the prodrugs relative to pilocarpine is a balance between 3 factors: prodrug lipophilicity, the kinetics of conversion from diester to monoester to pilocarpine, and ocular clearance or elimination rates. The increased bioavailability (response vs time) of the diesters is primarily a result of their lipophilicity, with an optimum being seen. For the monoesters, the increase is dependent on the rate of the monoester to pilocarpine conversion. A linear correlation has been established between the monoester structures and the activities observed following their dosing, through the use of the Taft σ ∗ values for the alcohol alkyl moieties. For the diesters, an inverted V-shaped correlation exists between the partition coefficients of the prodrugs and their relative bioavailabilities, as calculated from response data. In both cases, considerable predictability of response from prodrug structure should be possible.

AMPA receptor agonists: resolution, configurational assignment, and pharmacology of (+)-(S)- and (-)-(R)-2-amino-3-[3-hydroxy-5-(2-pyridyl)-isoxazol-4-yl]-propionic acid (2-Py-AMPA).

We have previously shown that whereas (RS)-2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid (APPA) shows the characteristics of a partial agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, (S)-APPA is a full AMPA receptor agonist and (R)-APPA a weak competitive AMPA receptor antagonist. This observation led us to introduce the new pharmacological concept, functional partial agonism. Recently we have shown that the 2-pyridyl analogue of APPA, (RS)-2-amino-3-[3-hydroxy-5-(2-pyridyl)isoxazol-4-yl]propionic acid (2-Py-AMPA), is a potent and apparently full AMPA receptor agonist, and this compound has now been resolved into (+)- and (-)-2-Py-AMPA (ee > or = 99.0%) by chiral HPLC using a Chirobiotic T column. The absolute stereochemistry of the enantiomers of APPA has previously been established by X-ray analysis, and on the basis of comparative studies of the circular dichroism spectra of the enantiomers of APPA and 2-Py-AMPA, (+)- and (-)-2-Py-AMPA were assigned the (S)- and (R)-configuration, respectively. In a series of receptor binding studies, neither enantiomer of 2-Py-AMPA showed detectable affinity for kainic acid receptor sites or different sites at the N-methyl-D-aspartic acid (NMDA) receptor complex. (+)-(S)-2-Py-AMPA was an effective inhibitor of [3H]AMPA binding (IC50 = 0.19 +/- 0.06 microM) and a potent AMPA receptor agonist in the rat cortical wedge preparation (EC50 = 4.5 +/- 0.3 microM) comparable with AMPA (IC50 = 0.040 +/- 0.01 microM; EC50 = 3.5 +/- 0.2 microM), but much more potent than (+)-(S)-APPA (IC50 = 5.5 +/- 2.2 microM; EC50 = 230 +/- 12 microM). Like (-)-(R)-APPA (IC50 > 100 microM), (-)-(R)-2-Py-AMPA (IC50 > 100 microM) did not significantly affect [3H]AMPA binding, and both compounds were weak AMPA receptor antagonists (Ki = 270 +/- 50 and 290 +/- 20 microM, respectively).