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Hydrochloride Salt of the GABAkine KRM-II-81.

ABSTRACT: Imidazodiazepine (5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazole[1,5-α][1,4]diazepin-3-yl) oxazole or KRM-II-81) is a potentiator of GABA_A receptors (a GABAkine) undergoing preparation for clinical development. KRM-II-81 is active against many seizure and pain models in rodents, where it exhibits improved pharmacological properties over standard-of-care agents. Since salts can be utilized to create opportunities for increased solubility, enhanced absorption, and distribution, as well as for efficient methods of bulk synthesis, a hydrochloride salt of KRM-II-81 was prepared. KRM-II-81·HCl was produced from the free base with anhydrous hydrochloric acid. The formation of the monohydrochloride salt was confirmed by X-ray crystallography, as well as ¹H NMR and ¹³C NMR analyses. High water solubility and a lower partition coefficient (octanol/water) were exhibited by KRM-II-81·HCl as compared to the free base. Oral administration of either KRM-II-81·HCl or the free base resulted in high concentrations in the brain and plasma of rats. Oral dosing in mice significantly increased the latency to both clonic and tonic convulsions and decreased pentylenetetrazol-induced lethality. The increased water solubility of the HCl salt enables intravenous dosing and the potential for higher concentration formulations compared with the free base without impacting anticonvulsant potency. Thus, KRM-II-81·HCl adds an important new compound to facilitate the development of these imidazodiazepines for clinical evaluation.

SUBMITTER: Mian MY

PROVIDER: S-EPMC9366947 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Hydrochloride Salt of the GABAkine KRM-II-81.

Mian Md Yeunus MY Divović Branka B Sharmin Dishary D Pandey Kamal P KP Golani Lalit K LK Tiruveedhula V V N Phani Babu VVNPB Cerne Rok R Smith Jodi L JL Ping Xingjie X Jin Xiaoming X Imler Gregory H GH Deschamps Jeffrey R JR Lippa Arnold A Cook James M JM Savić Miroslav M MM Rowlett James J Witkin Jeffrey M JM

ACS omega 20220727 31

Imidazodiazepine (5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazole[1,5-α][1,4]diazepin-3-yl) oxazole or KRM-II-81) is a potentiator of GABAA receptors (a GABAkine) undergoing preparation for clinical development. KRM-II-81 is active against many seizure and pain models in rodents, where it exhibits improved pharmacological properties over standard-of-care agents. Since salts can be utilized to create opportunities for increased solubility, enhanced absorption, and dist ...[more]

PMID: 35967038

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Project description:To provide back-up compounds to support the development of the GABAA receptor (GABAAR) potentiator KRM-II-81, three novel analogs were designed: replacing the pyridinyl with 2'-Cl-phenyl (FR-II-60), changing the positions of the N and O atoms in the oxazole ring with addition of an ethyl group (KPP-III-34 and KPP-III-51), or substituting a Br atom for the ethynyl of KRM-II-81 (KPP-III-34). The compounds bound to brain GABAARs. Intraperitoneal administration of FR-II-60 and KPP-III-34 produced anticonvulsant activity in mice [maximal electroshock (MES)-induced seizures or 6 Hz-induced seizures], whereas KPP-III-51 did not. Although all compounds were orally bioavailable, structural changes reduced the plasma and brain (FR-II-60 and KPP-III-51) exposures relative to KRM-II-81. Oral administration of each compound produced dose-dependent increases in the latency for both clonic and tonic seizures and the lethality induced by pentylenetetrazol (PTZ) in mice. Since KPP-III-34 produced the highest brain area under the curve (AUC) exposures, it was selected for further profiling. Oral administration of KPP-III-34 suppressed seizures in corneal-kindled mice, hippocampal paroxysmal discharges in mesial temporal lobe epileptic mice, and PTZ-induced convulsions in rats. Only transient sensorimotor impairment was observed in mice, and doses of KPP-III-34 up to 500 mg/kg did not produce impairment in rats. Molecular docking studies demonstrated that all compounds displayed a reduced propensity for binding to α1His102 compared with the sedating compound alprazolam; the bromine-substituted KPP-III-34 achieved the least interaction. Overall, these findings document the oral bioavailability and anticonvulsant efficacy of three novel analogs of KRM-II-81 with reduced sedative effects. SIGNIFICANCE STATEMENT: A new non-sedating compound, KRM-II-81, with reduced propensity for tolerance is moving into clinical development. Three new analogs were orally bioavailable, produced anticonvulsant effects in rodents, and displayed low sensorimotor impairment. KPP-III-34 demonstrated efficacy in models of pharmacoresistant epilepsy. Docking studies demonstrated a low propensity for compound binding to the α1His102 residue implicated in sedation. Thus, three additional structures have been added to the list of non-sedating imidazodiazepine anticonvulsants that could serve as backups in the clinical development of KRM-II-81.

Project description:Introduction: Dravet syndrome (DS) is an intractable epilepsy syndrome concomitant with neurodevelopmental disorder that begins in infancy. DS is dominantly caused by mutations in the SCN1A gene, which encodes the α subunit of a voltage-gated Na channel. Pre-synaptic inhibitory dysfunction is regarded as the pathophysiological mechanism, but an effective strategy for ameliorating seizures and behavioral problems is still under development. Here, we evaluated the effects of KRM-II-81, a newly developed positive allosteric modulator for α 2/3 subunit containing GABAA receptors (α2/3-GABAAR) in a mice model of DS both in vivo and at the neuronal level. Methods: We used knock-in mice carrying a heterozygous, clinically relevant SCN1A mutation (background strain: C57BL/6 J) as a model of the DS (Scn1a WT/A1783V mice), knock-in mouse strain carrying a heterozygous, clinically relevant SCN1A mutation (A1783V). Seizure threshold and locomotor activity was evaluated by using the hyperthermia-induced seizure paradigm and open filed test, respectively. Anxiety-like behavior was assessed by avoidance of the center region in locomotor activity. We estimated a sedative effect by the total distance traveled in locomotor activity and grip strength. Inhibitory post synaptic currents (IPSCs) were recorded from a hippocampal CA1 pyramidal neuron in an acutely prepared brain slice. Results: KRM-II-81 significantly increased the seizure threshold of Scn1a WT/A1783V mice in a dose-dependent manner. A low dose of KRM-II-81 specifically improved anxiety-like behavior of Scn1a WT/A1783V mice. A sedative effect was induced by relatively high dose of KRM-II-81 in Scn1a WT/A1783V mice, the dose of which was not sedative for WT mice. KRM-II-81 potentiated IPSCs by increasing its decay time kinetics. This effect was more prominent in Scn1a WT/A1783V mice. Discussion: Higher activation of α2/3-GABAAR by KRM-II-81 suggests a compensatory modification of post synaptic inhibitory function against presynaptic inhibitory dysfunction in Scn1a WT/A1783V. The increased sensitivity for KRM-II-81 may be relevant to the distinct dose-dependent effect in each paradigm of Scn1a WT/A1783V mice. Conclusion: Selective activation for α2/3-GABAAR by KRM-II-81 could be potential therapeutic strategy for treating seizures and behavioral problems in DS.

Project description:The imidizodiazepine, 5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)oxazole (KRM-II-81), is selective for α2/3-containing GABAA receptors. KRM-II-81 dampens seizure activity in rodent models with enhanced efficacy and reduced motor-impairment compared with diazepam. In the present study, KRM-II-81 was studied in assays designed to detect antiepileptics with improved chances of impacting pharmaco-resistant epilepsies. The potential for reducing neural hyperactivity weeks after traumatic brain injury was also studied. KRM-II-81 suppressed convulsions in corneal-kindled mice. Mice with kainate-induced mesial temporal lobe seizures exhibited spontaneous recurrent hippocampal paroxysmal discharges that were significantly reduced by KRM-II-81 (15 mg/kg, orally). KRM-II-81 also decreased convulsions in rats undergoing amygdala kindling in the presence of lamotrigine (lamotrigine-insensitive model) (ED50 = 19 mg/kg, i.p.). KRM-II-81 reduced focal and generalized seizures in a kainate-induced chronic epilepsy model in rats (20 mg/kg, i.p., three times per day). In mice with damage to the left cerebral cortex by controlled-cortical impact, enduring neuronal hyperactivity was dampened by KRM-II-81 (10 mg/kg, i.p.) as observed through in vivo two-photon imaging of layer II/III pyramidal neurons in GCaMP6-expressing transgenic mice. No notable side effects emerged up to doses of 300 mg/kg KRM-II-81. Molecular modeling studies were conducted: docking in the binding site of the α1β3γ2L GABAA receptor showed that replacing the C8 chlorine atom of alprazolam with the acetylene of KRM-II-81 led to loss of the key interaction with α1His102, providing a structural rationale for its low affinity for α1-containing GABAA receptors compared with benzodiazepines such as alprazolam. Overall, these findings predict that KRM-II-81 has improved therapeutic potential for epilepsy and post-traumatic epilepsy. SIGNIFICANCE STATEMENT: We describe the effects of a relatively new orally bioavailable small molecule in rodent models of pharmaco-resistant epilepsy and traumatic brain injury. KRM-II-81 is more potent and generally more efficacious than standard-of-care antiepileptics. In silico docking experiments begin to describe the structural basis for the relative lack of motor impairment induced by KRM-II-81. KRM-II-81 has unique structural and anticonvulsant effects, predicting its potential as an improved antiepileptic drug and novel therapy for post-traumatic epilepsy.

Dataset Information

Hydrochloride Salt of the GABAkine KRM-II-81.

Publications

Hydrochloride Salt of the GABAkine KRM-II-81.

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