Pharmacology

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Pharmacology

Expertise within IOS includes obtaining proof of concept for novel drug targets, and therapeutic drug target discovery, screening of biological activity and preclinical evaluation of drug candidates. Pharmacological testing of novel compounds includes investigation of biochemical and physiological effects, mechanisms of action, absorption, distribution, biotransformation and excretion. The activities of synthesized compounds are tested in models that comprise techniques of pharmacology, physiology, biochemistry, and molecular biology. The approaches range from the molecular and cellular through electrophysiology and organ systems to in vivo models and behavior:

  • Screening of compounds in the test systems selected by the customer
  • Development of screening systems for new indications
  • Investigations on the mechanisms of active compounds
  • Experiments on cells and isolated organs
  • Research on metabolism and pharmacokinetics
  • Toxicology tests

The typical biological activity testing services include:

  1. screening of compounds in the test systems selected by the customer and development of screening systems for new indications
  2. investigations on the mechanisms of action of active ingredients
  3. molecular pharmacology and biochemistry
    • receptor binding, enzyme activity assays, protein and gene expression, immunohistochemistry, ELISA, mitochondrial respiration.
  4. in vitro experiments in cell culture and isolated organs
  5. cardiovascular pharmacology in small experimental laboratory animals
    • in vivo/ex vivo heart infarction, isolated heart, aorta, ileum, vas-deference set-ups, echocardiography, blood pressure, atherosclerosis, diabetes and obesity models, heart failure and arrhythmia models
  6. CNS activity testing in vivo
    • cognition and memory processes, Middle cerebral artery occlusion model (stroke), Blood flow and oxygen saturation in the brain, inflammatory pain perception, antidepressant, behavioural tests
  7. anticancer activity screening in vitro and in vivo
    • in vitro cytotoxicity assays, in vivo mouse tumour models, 62 tumour cell lines are available for screening; multidrug resistance modulator screening, ex ovo screening of anti-angiogenic and anti-metastatic activity .
  8. drug metabolism and pharmacokinetics
  9. In vivo and in vitro ADME/toxicology tests: acute, repeated dose

Laboratory of Pharmaceutical Pharmacology uses in vivo, ex vivo and in vitro models to test cardioprotective, CNS, anti-inflammatory, metabolic activities, and to evaluate ADME and toxicological profile of novel compounds.

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Cardiovascular methods: Blood pressure measurements, Heart infarction in vivo and in vitro, Heart failure and Arrhythmia models, Atherosclerosis, Isolated aorta, ileum, vas deference preparations.

CNS methods: Cognition and memory processes, Middle cerebral artery occlusion model (stroke), Blood flow and oxygen saturation in the brain, inflammatory pain perception, antidepressant, behavioral tests.

Molecular pharmacology and biochemistry: Receptor binding, Enzyme activity assays, Protein and gene expression, Immunohistochemistry, ELISA, Mitochondrial respiration.

In vivo 3D imaging: InSyTe FLECT/CT 360º system – fluorescence tomography/x-ray computed tomography system to study true anatomical displacement of small animals and accurately measure near infrared fluorophore concentrations throughout the region of interest in vivo.

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Methods in the Group of Experimental Chemical therapy:

  • Genetically modified cell lines assay;
  • Cancer multidrug resistance;
  • In vitro cytotoxic assays, cell viability assays:
    • Crystal violet;
    • MTT cell viability assay;
    • Coomassie blue protein assay;
    • Basal cytotoxicity test (Neutral red assay, determination of LD50);
    • Lactate dehydrogenase assay
  • Apoptotic and necrotic cell detection
  • Calcium Assay, measurement of intracellular Ca2+ with Fluo-4 NW
  • Adipogenesis,
  • Matrix metalloproteinases inhibitors screening assay
  • Angiogenesis in vitro and in vivo
  • Antitumour activity detection in vivo of compounds on mice with tumour; influence on tumour growth, life length and metastasis

Equipment

The unit of pharmacological studies possesses about 300m2 of laboratory rooms with necessary equipment for both in-vitro and in-vivo routine testing of novel compounds. The animal facility fully corresponds to EU regulations of animal care.

Details

Laboratories are equipped for biochemical measurements, gene expression by qRT-PCR method and conventional WB equipment. There are incubators (Binder) and 96-well Spectrometer Zenyth 200ST plates (Anthos). Gene expression by qRT-PCR method can be performed by two Real-Time PCR Systems AB Prism 7500 (Applied Biosystems). Conventional WB equipment (Owl Electrophoresis System) as well as a SNAP id system (Millipore) and dark room can be used for protein expression experiments. In addition, a CM 1850 cryostat (Leica), FreeZone 2.5 Liter Benchtop Freeze Dry System (Labconco), and Optima™ L-80 XP ultracentrifuge (Beckman Coulter) are available. The radio-labelled laboratory materials and multi-detector instruments (MicroBeta TriLux, Perkin Elmer) for radiolabelled substances are available for liquid scintillation or luminescence detection of samples in microtitration plates, tubes or on filters.

Physiological equipment includes units for measuring blood pressure, heart rate, EKG measurements in vivo (surgery tables and inhalation anaesthesia system from Scanbur) as well as Langendorff and Working isolated heart and tissue preparations in vitro. In addition, there is a Philips iE33 Echocardiography System for detection of non-invasive cardiovascular parameters in animals. Data registration are based on ADInstruments PowerLab 8/30 systems which combine software-controlled setting of parameters, extensive signal conditioning options, variable sampling speeds and powerful real-time computations with the advantages of computer-based data displays and analysis. It fully ensures our ability to manage animals with cardiac diseases, such as heart failure and congenital heart disease. ADInstruments PowerLab system also is connected to Microelectrodes oxygen sensors for mitochondria respiration measurements.

The equipment for animal behavioural studies includes Gemini Avoidance System apparatus (San Diego Instruments water maze pool (150 cm Ø) with automatically lifted and lowered hydraulic platforms (Ugo Basile, Italy), EthoVision video tracking system (version 3.1., Noldus, Netherland), Painless (Stoelting), Von Frey, Randall Selitto, Plethysmometer, Grip Strength Meter and Pincher equipment (TC Penta 5-in-1, Stoelting), Hot/cold plate 35100 (Ugo Basil).

Selected references:

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Cardiovascular pharmacology

  • Liepinsh E, Makrecka-Kuka M, Kuka J, Vilskersts R, Makarova E, Cirule H, Loza E, Lola D, Grinberga S, Pugovics O, Kalvinsh I, Dambrova M. Inhibition of L-carnitine biosynthesis and transport by methyl-γ-butyrobetaine decreases fatty acid oxidation and protects against myocardial infarction. British Journal of Pharmacology, 2015,172(5):1319-1332.
  • Vilskersts R, Kuka J, Liepinsh E, Makrecka-Kuka M, Volska K, Makarova E, Sevostjanovs E, Cirule H, Grinberga S, Dambrova M. Methyl-γ-butyrobetaine decreases levels of acylcarnitines and attenuates the development of atherosclerosis. Vascul Pharmacol. 2015 doi: 10.1016/j.vph.2015.05.005.
  • Liepinsh E, Makrecka M, Kuka J, Cirule H, Makarova E, Sevostjanovs E, Grinberga S, Vilskersts R, Lola D, Loza E, Stonans I, Pugovics O, Dambrova M. Selective inhibition of OCTN2 is more effective than inhibition of Gamma-butyrobetaine dioxygenase to decrease the availability of L-carnitine and to reduce myocardial infarct size. Pharmacological Research, 2014; 85:33-38. doi: 10.1016/j.phrs.2014.05.002.
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CNS pharmacology

  • Zvejniece L, Vavers E, Svalbe B, Vilskersts R, Domracheva I, Vorona M, Veinberg G, Misane I, Stonans I, Kalvinsh I, Dambrova M. The cognition-enhancing activity of E1R, a novel positive allosteric modulator of sigma-1 receptors. Br. J. Pharmacol. 2014, 171(3): 761-771. DOI: 10.1111/bph.12506.
  • Zvejniece L, Svalbe B, Liepinsh E, Pulks E, Dambrova M. The sensorimotor and cognitive deficits in rats following 90- and 120-min transient occlusion of the middle cerebral artery. J Neurosci Methods. 2012; 208(2):197-204. doi: 10.1016/j.jneumeth.2012.05.018.
  • Zvejniece L, Svalbe B, Veinberg G, Grinberga S, Vorona M, Kalvinsh I, Dambrova M, Investigation of stereoselective pharmacological activity of phenotropil, Basic & Clinical Pharmacology & Toxicology, 2011;109(5):407-12.
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Molecular pharmacology and biochemistry

  • Liepinsh E, Makrecka M, Kuka J, Makarova E, Vilskersts R, Cirule H, Sevostjanovs E, Grinberga S, Pugovics O, Dambrova M, The heart is better protected against myocardial infarction in the fed state compared to the fasted state, Metabolism Clinical and Experimental, 2014, 63:127-136. doi: 10.1016/j.metabol.2013.09.014
  • Makrecka M, Kuka J, Volska K, Antone U, Sevostjanovs E, Cirule H, Grinberga S, Pugovics O, Dambrova M, Liepinsh E. Long chain acylcarnitine content determines the pattern of energy metabolism in cardiac mitochondria. Molecular and Cellular Biochemistry, 2014, 395(1-2):1-10. doi:10.1007/s11010-014-2106-3.
  • Liepinsh E, Skapare E, Kuka J, Makrecka M, Cirule H, Vavers E, Sevostjanovs E, Grinberga S, Pugovics O, Dambrova M. Activated peroxisomal fatty acid metabolism improves cardiac recovery in ischemia-reperfusion. Naunyn Schmiedebergs Arch Pharmacol. 2013; 386(6):541-50. doi: 10.1007/s00210-013-0849-0.
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Experimental chemotherapy


  • Jaudzems K, Tars K, Maurops G, Ivdra N, Otikovs M, Leitans J, Kanepe-Lapsa I, Domraceva I, Mutule I, Trapencieris P, Blackman M.J, Jirgensons A. Plasmepsin inhibitory activity and structure-guided optimization of a potent hydroxyethylamine-based antimalarial hit. ACS Med. Chem. Lett.  2014vol.5 (No.4), 373-377.
  • Arsenyan P, Paegle E, Domracheva I, Gulbe A, Kanepe-Lapsa I, Shestakova I. Selenium analogues of raloxifene as promising antiproliferative agents in treatment of breast cancer. Eur. J. Med. Chem. 2014(87), 471-483.
  • Ignatovich L, Romanovs V, Muravenko V, Sleiksha I, Popelis Y, Shestakova I. Synthesis and Cytotoxic Activity of 1-{3-[1-(5-Organylsilylfuran-2-yl)silinan-1-yl]propyl}amines and Some Trimethylgermyl Analogues. Chem.-Eur. J. 2014(20), 12786-12788.