DMPK

in vitro potassium release assay for liposomal formulations of Amphotericin B

• Assay performed with blood from preclinical species or with human blood
• Achieved dose- and time-dependent potassium release from red blood cells
• Assay validated for determination of potassium concentration using ICP-MS platform
• Assay conducted in compliance with GLP and in non-GLP format

Physicochemical Properties

• Aqueous solubility and stability at different pH condition
• Stability in simulated gastric and intestinal fluid
• Octanol water partitioning (Log P and Log D)
• pKa determination

Permeability 

• PAMPA
• Caco-2 cell line
• MDCKII cell line

P-gp Substrate and Inhibition Potential

• Caco-2 cell line
• MDCKII-P-gp transfected cell line

Metabolic Stability

• Stability in S9, microsomes and hepatocytes to assess potential for intestinal first pass, intrinsic clearance to evaluate in vitro clearance

Plasma Protein Binding

• Equilibrium dialysis and ultrafiltration techniques
• Blood/Plasma Partitioning and Stability

Tissue Homogenate Binding

CYP Reaction Phenotyping

• Using recombinant human CYPs as well as human liver microsomes
• Aldehyde oxidase reaction phenotyping

Metabolite Identification

• Using unlabeled and radiolabeled compound coupled with mass spectrometric detection
• Preliminary metabolite identification
  • In in vitro and in vivo study samples
  • Reactive metabolite trapping and characterization

Drug-Drug Interaction Assays

• CYP Inhibition using human liver microsomes (IC₅₀, Ki determination)
• Time dependent CYP inhibition (IC₅₀shift)
• CYP induction in human hepatocytes
• P-gp substrate and inhibition assays using Caco2 and MDCKII-P-gp cell lines

Cytotoxicity

• MTT Assay

• Species (rodent and non-rodent)
• Absolute and relative bioavailability assessment
• Cannulation techniques used: Jugular vein, femoral vein, carotid artery, portal vein and bile duct
• Dosing techniques: IV, Oral, IM, SC, intranasal instillation, endotracheal intubation, dermal application, intradermal etc.
• Brain penetration in vivo, supplemented with P-gp assessment in vitro
• Validated WinNonlin Phoenix software is used for PK data analysis

• Mass Balance / Excretion balance
• Tissue distribution
• Ocular PK study in rabbits
• Biliary excretion study
• Metabolite profiling

• Toxicokinetic data analysis and report preparation to support IND enabling studies
• Allometric scaling: FIH dose projections and human PK predictions
• Validated Phoenix WinNonlin® software

Ideally a lead compound should have > 30% oral bioavailability and < 30% liver blood flow clearance and an adequate half-life for once daily dosing. In the absence of this, to improve bioavailability, a suitable formulation needs to be developed. Eurofins Advinus has the capability to execute the following:

• Development of solution formulation for IV and PO studies of low solubility lipophilic compounds for conduct of preclinical PK studies using
  • pH adjustment
  • Co-solvents
  • Inclusion complexes (cyclodextrins)
• Attempts are made to achieve formulation using excipients acceptable for use in repeated dose studies
• The prepared formulations are assessed for accuracy using HPLC or LC/MS/MS method
• If necessary, salt forms or pro-drugs can be considered for improving the bioavailability of lead compound along with micronization of compound

• Formulation method development and validation for preclinical dose formulation sample analysis