The ultimate goals of preclinical studies are to accurately model, in animals, the desired biological effect of a drug in order to predict treatment outcome in patients (efficacy), dosing and administration route (pharmacokinetic, bioavailability), and to identify and characterize all toxicities associated with a drug in order to predict adverse events (safety) for informed risk assessment.
In vivo assays are developed in accordance with the guidelines of European Union Directive 2010/63/EU on the protection of animals used for scientific purposes. Studies are performed in the Animal Facilities of the Center of Biomedical Investigation of the University of Granada (Granada, Spain).
Preclinical Development
[accordions] [accordion title=”Bioanalytical Assays”]Standard bioanalytical method development
- Sample preparation method development, chromatographic method development, detection method development and optimization
Advanced bioanalytical method development
- Linearity and sensitivity
- Accuracy and precision
- Selectivity
- Stability
- Recovery
Analytical Technologies
- High Throughput Injector CTC Pal Xt
- 5600 QTOF Mass Spectrometer (SCIEX)
- UPLC Cromatograph Agilent 1290 Infinity
- Triple Quad mass Spectrometer API4000 (SCIEX)
Analytical tools
- LC-MS/MS Agilent 1290-API4000 (SCIEX)
- LC-HRMS Agilent1290-Triple TOF5600 SCIEX
- Liquid handlers equipped with on-deck accessories; Freedom EVO, Tecan; Biomek FX and i7, Beckman, Echo® Acoustic Liquid Handling System, Beckman; EvolutionP3, PerkinElmer; and software specifically designed to automate sample preparation procedures
Drug absorption
- PAMPA (Parallel artificial membrane permeability assay). Detection method: LC-MS/MS and LC-HRMS
- Caco-2 cell permeability assay. Detection method: LC-MS/MS and LC-HRMS
Drug metabolism
- CYP Inhibition: CYP1A2 / CYP2C8 / CYP2C9 / CYP2C19 / CYP2D6 / CYP3A4. Detection method: LC-MS/MS
- CYP Induction (cryopreserved hepatocytes). Detection method: LC-MS/MS
- CYP P450 Reaction phenotyping. Detection method: LC-MS/MS
- Reactive metabolite assessment. Detection method: LC-MS/MS & HRMS
- Metabolite stability (Criopreserved hepatocyte & liver microsomes) in different species (human, mouse, rat, dog, monkey, pig). Detection method: LC-HRMS
- In vivo and in vitro metabolic profiling (Phase I and II). Comparison between species. Metabolite identification by LC-HRMS.
- Plasma stability. Detection method: LC-MS/MS
Drug distribution
- Plasma protein binding (dialysis equilibrium). Detection method: LC-MS/MS.
Physicochemical profiling – solutions properties
- Kinetic solubility. Detection method: absorbance at 620 nm
- Thermodynamic solubility. Detection method: LC-MS/MS
- Chemical stability. Detection method: LC-MS/MS
Toxicity
- Cytotoxicity: cell viability in tumor and non-tumor cells. Detection method: absorbance and HCS
- In vitro cytotoxicity of Medical Devices (non-GLP, ISO_10993_5)
- Genotoxicity:
- AMES test. Detection method: visual read-out
- Mammalian Cell Micronucleus Test (non-GLP, OECD Test Guideline 487). Detection method: HCS and in-house image analyzing App “Nucleus Finder”
- Cardiotoxicity: ion channel activity (hERG, Nav1.5, Cav 1.2). Detection method: fluorescence (FLIPR Tetra® High-Throughput Cellular Screening System (Molecular Devices)
- Neurotoxicity: neurotransmitter receptors: 5HT-1A. Detection method: Alphascreen®
[/accordion] [accordion title=”Authorization for animal studies”]
- We prepare the project and handle the documentation for approval by the Animal Experimentation Ethics Committee (CEEA), by the Animal Experimentation Authorization Committee and by the Regional Government.
- Custom in vivo studies, different administration routes (intravenous – tail vein and retro-orbital sinus administration – oral, intra-peritoneal, subcutaneous and intramuscular), dosing regimen and readouts on demand
- Various administration routes: intravenous (tail vein and retro-orbital sinus administration) oral, intra-peritoneal, subcutaneous and intramuscular
- The dosing regimen and sampling are designed according to the project needs and prior information available on the study compounds
- Metabolite identification and profiling in vivo
- Analysis of different matrices: blood, plasma, cerebrospinal fluid, tissues, bile, urine and feces.
- Solid state evaluation and enhanced formulation for problematic compounds
- Biomarker development and application within projects
- Support of pharmacodynamic and efficacy study design
- The relationship between concentration-time and response-time profiles varies with different targets and the position of a target in a biological pathway. The team provides analysis and interpretation of pharmacokinetic/pharmacodynamic (PKPD) relationships in support of PD.
- Analysis of biomarkers can also be conducted at MEDINA, as it can greatly enhance the process; integration of such information in static or ideally dynamic models form the foundation for drug discovery and safety risk assessment.
- Calculation of Pharmacokinetics parameters:
- Half-life
- Cmax
- Area under curve, AUC
- Bioavailability
- Compartment
- Volume of distribution, Vd
- Clearance
- Steady state concentration, CSS
- Various administration routes: intravenous (tail vein and retro-orbital sinus administration) oral, intra-peritoneal, subcutaneous and intramuscular
- Evaluation of body weights, clinical signs of toxicity and macroscopic organ toxicity
- Acute toxicity (non-GLP, OECD Test Guideline 425) for limiting the maximal tolerated dose (MTD)
- Short-term single dose toxicity for LD50 calculation (non-GLP, OECD Test Guideline 420)
- Short-term repeated dose toxicity (non-GLP, OECD Test Guideline 407)