KM-408, a novel phenoxyalkyl derivative as a potential anticonvulsant and analgesic compound for the treatment of neuropathic pain.

BACKGROUND: Epilepsy frequently coexists with neuropathic pain. Our approach is based on the search for active compounds with multitarget profiles beneficial in terms of potential side effects and on the implementation of screening for potential multidirectional central activity. METHODS: Compounds were synthesized by means of chemical synthesis. After antiseizure and neurotoxicity screening in vivo, KM-408 and its enantiomers were chosen for analgesic activity evaluations. Further safety studies included acute toxicity in mice, the effect on normal electrocardiogram and on blood pressure in rats, whole body plethysmography in rats, and in vitro and biochemical assays. Pharmacokinetics has been studied in rats after iv and po administration. Metabolism has been studied in vivo in rat serum and urine. Radioligand binding studies were performed as part of the mechanism of action investigation. RESULTS: Selected results for KM-408: Ki sigma = 7.2*10-8; Ki 5-HT1A = 8.0*10-7; ED50 MES (mice, ip) = 13.3 mg/kg; formalin test (I phase, mice, ip)-active at 30 mg/kg; SNL (rats, ip)-active at 6 mg/kg; STZ-induced pain (mice, ip)-active at 1 mg/kg (von Frey) and 10 mg/kg (hot plate); hot plate test (mice, ip)-active at 30 mg/kg; ED50 capsaicin test (mice, ip) = 18.99 mg/kg; tail immersion test (mice)-active at 0.5%; corneal anesthesia (guinea pigs)-active at 0.125%; infiltration anesthesia (guinea pigs)-active at 0.125%. CONCLUSIONS: Within the presented study a novel compound, R,S-2-((2-(2-chloro-6-methylphenoxy)ethyl)amino)butan-1-ol hydrochloride (KM-408) with dual antiseizure and analgesic activity has been developed for potential use in neuropathic pain treatment.

Comparative Quantitative Study of Ardisiacrispin A in Extracts from Ardisia crenata Sims Varieties and Their Cytotoxic Activities.

Ardisia crenata Sims (Primulaceae) occurs in natural habitats in two varieties, bearing red or white fruits. While roots of the red-berried ardisia are valued as a medicinal product, the pharmacological activity of which is attributed to triterpene saponins, including ardisiacrispin A, data on the white-berried variety are scarce. A TLC-densitometric method was developed and validated to estimate the levels of saponins, calculated as ardisiacrispin A, in different plant parts in both varieties. Their content amounted to 22.17±4.75 and 25.72±1.46 mg/g d.w. in roots, and 2.64±0.74 and 3.43±0.70 mg/g d.w. in fruits of red-berried and white-berried ardisia, respectively. Assessment of cytotoxicity of ardisiacrispin A and A. crenata extracts on a panel of human cancer cell lines revealed a similar effect of root extracts from both varieties, with the highest potency against melanoma WM793 and colon cancer Caco2. Thus, roots of the white-berried variety may be treated as a substitute for red-berried ardisia and serve as an alternative source for the acquisition of plant material rich in bioactive saponins.

Development and Validation of Stability-Indicating HPLC Methods for the Estimation of Lomefloxacin and Balofloxacin Oxidation Process under ACVA, H2O2, or KMnO4 Treatment. Kinetic Evaluation and Identification of Degradation Products by Mass Spectrometry.

The oxidation of lomefloxacin (LOM) and balofloxacin (BAL) under the influence of azo initiator of radical reactions of 4,4'-azobis(4-cyanopentanoic acid) (ACVA) and H2O2 was examined. Oxidation using H2O2 was performed at room temperature while using ACVA at temperatures: 40, 50, 60 °C. Additionally, the oxidation process of BAL under the influence of KMnO4 in an acidic medium was investigated. New stability-indicating HPLC methods were developed in order to evaluate the oxidation process. Chromatographic analysis was carried out using the Kinetex 5u XB-C18 100A column, Phenomenex (Torrance, CA, USA) (250 × 4.6 mm, 5 µm particle size, core shell type). The chromatographic separation was achieved while using isocratic elution and a mobile phase with the composition of 0.05 M phosphate buffer (pH = 3.20 adjusted with o-phosphoric acid) and acetonitrile (87:13 v/v for LOM; 80:20 v/v for BAL). The column was maintained at 30 °C. The methods were validated according to the ICH guidelines, and it was found that they met the acceptance criteria. An oxidation process followed kinetics of the second order reaction. The most probable structures of LOM and BAL degradation products formed were assigned by the UHPLC/MS/MS method.

KM-416, a novel phenoxyalkylaminoalkanol derivative with anticonvulsant properties exerts analgesic, local anesthetic, and antidepressant-like activities. Pharmacodynamic, pharmacokinetic, and forced degradation studies.

Anticonvulsant drugs are used to treat a wide range of non-epileptic conditions, including chronic, neuropathic pain. We obtained a phenoxyalkylaminoalkanol derivative, KM-416 which had previously demonstrated a significant anticonvulsant activity and had also been shown to bind to 5-HT1A, α2-receptors and SERT and not to exhibit mutagenic properties. As KM-416 is a promising compound in our search for drug candidates, in the present study we further assessed its pharmacological profile (analgesic, local anesthetic, and antidepressant-like activities) accompanied with patch-clamp studies. Considering the importance of drug safety, its influence on the cardiovascular system was also evaluated. Moreover, KM-416 was subjected to forced degradation and pharmacokinetic studies to examine its stability and pharmacokinetic parameters. KM-416 revealed a significant antinociceptive activity in the tonic - the formalin test, neurogenic - the capsaicin test, and neuropathic pain model - streptozotocin-induced peripheral neuropathy. Moreover, it exerted a local anesthetic effect. In addition, KM-416 exhibited anti-depressant like activity. The results from the patch-clamp studies indicated that KM-416 can inhibit currents elicited by activation of NMDA receptors, while it also exhibited a voltage-dependent inhibition of Na+ currents. KM-416 did not influence ventricular depolarization and repolarization. Following oral administration, pharmacokinetics of KM-416 was characterized by a rapid absorption in the rat. The brain-to-plasma AUC ratio was 6.7, indicating that KM-416 was well distributed to brain. The forced degradation studies showed that KM-416 was very stable under stress conditions. All these features made KM-416 a promising drug candidate for further development against neuropathic pain and epilepsy.

Simultaneous Determination of Eight Hypotensive Drugs of Various Chemical Groups in Pharmaceutical Preparations by HPLC-DAD.

A new sensitive, simple, rapid, and precise HPLC method with diode array detection has been developed for separation and simultaneous determination of hydrochlorothiazide, furosemide, torasemide, losartane, quinapril, valsartan, spironolactone, and canrenone in combined pharmaceutical dosage forms. The chromatographic analysis of the tested drugs was performed on an ACE C18, 100 Å, 250×4.6 mm, 5 µm particle size column with 0.0.05 M phosphate buffer (pH=3.00)-acetonitrile-methanol (30+20+50 v/v/v) mobile phase at a flow rate of 1.0 mL/min. The column was thermostatted at 25°C. UV detection was performed at 230 nm. Analysis time was 10 min. The elaborated method meets the acceptance criteria for specificity, linearity, sensitivity, accuracy, and precision. The proposed method was successfully applied for the determination of the studied drugs in the selected combined dosage forms.

Determination of neomycin in the form of neomycin derivative with dabsyl chloride by thin layer chromatography and densitometry.

A thin layer chromatographic-densitometric method has been developed for identification and quantitative determination of neomycin derivative with dabsyl chloride. The analysis of antibiotic was achieved on the silica gel TLC plates with fluorescent indicator with n-butanol--2-butanone--25% ammonia--water (10 : 6 : 2 : 2, v/v/v/v) as the mobile phase. The densitometric measurements were made at 460 nm. Under these conditions good separation of chosen aminoglycoside antibiotic from reagent used to make a complex was obtained. The method is characterized by high sensitivity, LOD from 0.1953 µg per band and LOQ from 0.5918 µg per band, wide linearity range from 0.5918 to 2.1960 µg per band for neomycin. The precision of the method was good; RSD varied from 1.17 to 2.05%. Satisfactory results of validation of the method were also confirmed by determination of selected antibiotic in pharmaceutical commercial preparation. The results obtained by TLC-densitometric method were compared with those obtained by spectrophotometric method.

Photodegradation assessment of ciprofloxacin, moxifloxacin, norfloxacin and ofloxacin in the presence of excipients from tablets by UPLC-MS/MS and DSC.

BACKGROUND: Ciprofloxacin (CIP), moxifloxacin (MOX), norfloxacin (NOR) and ofloxacin (OFL), are the antibacterial synthetic drugs, belonging to the fluoroquinolones group. Fluoroquinolones are compounds susceptible to photodegradation process, which may lead to reduction of their antibacterial activity and to induce phototoxicity as a side effect. This paper describes a simple, sensitive UPLC-MS/MS method for the determination of CIP, MOX, NOR and OFL in the presence of photodegradation products. RESULTS: Chromatographic separations were carried out using the Acquity UPLC BEH C18 column; (2.1 × 100 mm, 1.7 µm particle size). The column was maintained at 40°C, and the following gradient was used: 0 min, 95% of eluent A and 5% of eluent B; 10 min, 0% of eluent A and 100% of eluent B, at a flow rate of 0.3 mL min(-1). Eluent A: 0.1% (v/v) formic acid in water; eluent B: 0.1% (v/v) formic acid in acetonitrile. The method was validated and all the validation parameters were in the ranges acceptable by the guidelines for analytical method validation. The photodegradation of examined fluoroquinolones in solid phase in the presence of excipients followed kinetic of the first order reaction and depended upon the type of analyzed drugs and coexisting substances. Photodegradation process of analyzed drugs was confirmed by differential scanning calorimetry. In addition, the identification of degradation products was carried out by mass spectrometry. CONCLUSION: The developed UPLC-MS/MS method enables the determination of CIP, MOX, NOR and OFL in the presence of photodegradation products and identification of photodegradation products.

Separation and characterization of ciprofloxacin, difloxacin, lomefloxacin, norfloxacin, and ofloxacin oxidation products under potassium permanganate treatment in acidic medium by UPLC-MS/MS.

A simple, sensitive and reproducible ultra-performance liquid chromatography method for determination of ciprofloxacin, difloxacin, lomefloxacin, norfloxacin and ofloxacin oxidation stability under permanganate treatment in acidic conditions at pH from 3.0 to 6.0, was developed. Chromatographic separations were carried out using the Acquity UPLC BEH C18 column; (2.1×100 mm, 1.7 µm particle size). The column was maintained at 40°C, and eluted under isocratic conditions using 83% of eluent A and 17% of eluent B over 6.5 min, at a flow rate of 0.3 mL min(-1). Eluent A: water/formic acid (0.1 v/v%); eluent B: acetonitrile/formic acid (0.1 v/v%). An oxidation process followed kinetic of the second order reaction and depended upon solution acidity. Oxidation of fluoroquinolones proceeded at piperazine moiety yielding respective hydroxy and oxo analogs, and remaining the quinolone fragment intact. Structures of products formed were assigned on a basis of UPLC/MS/MS fragmentation pathways.

Kinetic and thermodynamic studies of moxifloxacin hydrolysis in the presence and absence of metal ions in acidic solutions.

Chromatographic and densitometric method for determination of moxifloxacin in the presence of products of acidic hydrolysis was developed. The established method had suitable specificity, precision, good accuracy, and high sensitivity. In addition, stability of moxifloxacin in acidic solutions at temperature 90 degrees C and 110 degrees C in the presence and absence of metal ions, such as Cu(II), Fe(III), Zn(II), and Al(III) was studied. It was proved that decomposition of moxifloxacin proceeds according to kinetics of the first-order reaction and is dependent on temperature, incubation time and the type of the metal ion. Based on the calculated kinetic (k, t0, and t0.5) and thermodynamic (Ea) parameters, it was observed that among studied ions the highest effect on decomposition process of moxifloxacin had Cu(II) ions. The liquid chromatography coupled with mass spectrometry detection (LC-MS) and proton nuclear magnetic resonance (1H NMR) techniques have been used to identify degradation products for the compound.

Kinetic and thermodynamic studies of moxifloxacin hydrolysis in the presence and absence of metal ions in acidic solutions.

Chromatographic and densitometric method for determination of moxifloxacin in the presence of products of acidic hydrolysis was developed. The established method had suitable specificity, precision, good accuracy and high sensitivity. In addition, stability of moxifloxacin in acidic solutions at temperature 90 degrees C and 110 degrees C in the presence and absence of metal ions, such as Cu(II), Fe(III), Zn(II), and Al(III) was studied. It was proved that decomposition of moxifloxacin proceeds according to kinetics of the first-order reaction and is dependent on temperature, incubation time and the type of the metal ion. Based on the calculated kinetic (k, t0.1, and t0.5) and thermodynamic (E(a)) parameters, it was observed that among studied ions the highest effect on decomposition process of moxifloxacin had Cu(II) ions. The liquid chromatography coupled with mass spectrometry detection (LC-MS) and proton nuclear magnetic resonance (1H NMR) techniques have been used to identify degradation products of moxifloxacin.