PHARMACEUTICALS

EARLY PRECLINICAL RESEARCH

Early preclinical research represents an important phase of drug discovery and development, designed to identify a lead candidate from several hits.

Galileo helps customers in the design of the most suitable strategy for selecting the lead drug candidate, thus effectively accelerating the development of new medicines and reducing attrition rate.

The following tests can be proposed to evaluate “drugability”, absorption and metabolism, toxicity and efficacy.

Aqueous solubility is an important determinant of the usefulness of a drug candidate that may have a marked impact on the whole process of drug discovery and development.

The partition coefficient is a measure of differential solubility of a compound in a hydrophobic solvent (octanol) and a hydrophilic solvent (water). The logarithm of these two values enables compounds to be ranked in terms of hydrophilicity (or hydrophobicity).

One of the keys to understanding how a drug will dissolve in vivo, after oral administration, is to know how soluble it will be in the very different environments of the stomach, small intestine, and colon. Test systems consist in Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF), in fed/fasted conditions.

The binding of drug to plasma (and tissue) proteins is a major determinant of drug disposition (distribution). Binding has a very important effect on pharmacodynamics since usually only the unbound drug interacts with its molecular target. Protein binding assay can be done on plasma originating from any animal species. Usually human and rat plasma are tested in order to facilitate comparison between non-clinical and clinical data.

These parameters are very important in defining the pharmacological and toxicological profile of drugs. Since rats are one of the animal species recommended for toxicological studies, it is important to have metabolic stability data from rat microsomes, to be compared with the human data.

Useful to understand degradation to predict efficacy, right timing for PK analysis. Can be performed in plasma from different species, to allow comparison between non-clinical and clinical data.

A test on static or dynamic in vitro model of BBB to predict the passage of drugs in the brain.

Fundamental to understand bioavailability of CNS drugs. This test is done in vivo, on a small number of mice.

A test to predict the oral absorption of drugs. The assay measures drug trasport across a Caco-2 cell monolayer bidirectionally, thus predicting whether the molecule undergoes active efflux.

Important to determine the role of P-glycoprotein in the active transport of the compound across Caco-2 cell monolayers.

 

Reconstructed Human Epidermidis is used as the test system to evaluate transport through the skin.

Gives information on potential drug-drug interactions. CYP1A, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 are the most important proteins; other isoforms are available. Test systems are represented by human hepatocytes, microsomes or recombinant isoforms. The same substrates from rats can also be used.

Glutathion conjugation, Glucuronate conjugation.

In vitro/in vivo models are listed in the section of Pharmacodynamic Assays. We can propose an extensive list of preclinical disease models in the field of cancer, bone and cartilage diseases, skeletal muscle pathologies, CNS disorders and dermatological pathologies.

In order to detect any toxic effect as early as possible in the development process, Galileo Research proposes customised safety testing strategies, that include in vitro and in vivo (rodents) toxicity studies, optimizing costs and timing. The strategies can also include toxicogenomics analysis as a complementary approach to analyze the gene expression profile and identify changes associated with drug-induced toxicities. TGx is not yet a substitute for regulatory toxicity testing, but it can be informative for internal decision-making during preclinical development and can be applied to derisk compound development looking for undesirable mechanistic signatures in appropriate cellular models.

·      Single dose toxicity

·      Repeated-dose toxicity (14-day)

·      Genotoxicity (Ames’ test, Mouse Lymphoma assay)

·      In vitro cytotoxicity

REGULATORY DEVELOPMENT OF DRUGS

Verifying the efficacy of your lead molecules is of crucial importance prior to investing further on lead optimization. A number of in vitro and in vivo well-established models to study the effect of the drug in different therapeutic applications are available. In vitro systems are often proposed in the early phases of drug discovery, while the in vivo models are used to support the regulatory strategy.

All our studies are tailored on Customer’s requests and novel disease models can be developed to meet specific requirements.

A number of technologies can be offered to predict mechanism of action at the molecular level and to learn as much as possible from the experimental model about your compound: gene expression analysis, proteomics, histopathology, clinical chemistry, hematology, immunohistochemistry, blotting techniques, fluorescence microscopy.

Even if GLP standards are not required for pharmacodynamic studies, Galileo Research offers “GLP-like” standards, and works according to a global quality strategy.

AREAS OF EXPERTISE

In vitro test: Cultures of osteoblasts, osteoclasts, synoviocytes and chondrocytes can be used to mimic the disease and assess drug functionality by measuring:

  • cytokine release
  • collagen synthesis
  • hyaluronic acid synthesis
  • GAG synthesis
  • Enzyme activities
  • Bone mineralization
  • Bone resorption/formation
  • Phenotype
  • Gene expression (PCR, qPCR, microarrays)
  • Proteome analysis

In vivo models:

  • osteoporosis (by ovariectomy, immobilization, GC-induced)
  • osteoarthritis (by ACL transaction, MIA injection)
  • rheumatoid arthritis (by collagen injection)
  • osteointegration of titanium implants
  • fracture healing

Observation: histology, histomorphometry and immunohistochemistry

Blood and urine analyses for the detection of specific markers.

Gene expression and proteomic analysis on biological samples.

Behavioural testing (rotarod, 4-hindlimbs, Von Frey filaments, incapacitance test).

In vitro test: Neurons can be cultured and submitted to different stress conditions: cumene hydroperoxyde-induced oxydative stress, oxygen/glucose deprivation.

The efficacy of the drug can be evaluated by cell viability, LDH assay, ROS inhibition, enzymatic activities.

In vivo models:

Cerebral ischemia (MCAO): we have settled a model of focal cerebral ischemia induced by transient middle cerebral artery occlusion in obese, diabetic and hypertensive rats. This model is much more robust and valid, in comparison with other methods, and is a good predictor of therapeutic efficacy since it has more similarity to the human conditions. A number of analysis can be performed on this model to monitor the evolution of the disease and to evaluate the impact of drug treatment:

  • blood pressure
  • brain circulation
  • animal behaviour
  • brain histology
  • enzyme activity
  • proteomic analysis
  • miRNA
  • gene expression

Mood disorders:

  • Elevated plus-maze
  • Black/white box
  • Chronic mild stress
  • Forced swim
  • Open field
  • Appetite behaviour

In vitro test: In vitro cell growth/viability/cytotoxicity

In vivo models: Human tumor xenografts in SCID and athymic nude mice (subcutaneous, intraperitoneal, orthotopic).

  • Psoriasis (mouse tail para/orthokeratosis)
  • Acnes vulgaris (P. acnes)
  • In vitro simulation of celiac disease in Caco-2 cells
  • In vivo induced celiac disease in NOD mice

In vitro test

C2C12 myoblasts in culture for the study of proliferation, differentiation in myotubes.

In vivo test

  • Mdx transgenic mice, for muscular dystrophy
  • Mouse model of polymyositis

NON-CLINICAL SAFETY

Toxicity studies are performed in full compliance with Good Laboratory Practice (GLP) and have the purpose to determine the toxicological profile of new compounds or to improve the knowledge on existing ones, with the aim of supporting different formulations, new therapeutic indications or administration routes.

Data supports human clinical trials and marketing authorization approval in Europe.

ICH and OECD guidelines are used as main reference guidelines.

The development of anticancer drugs is further regulated by dedicated guidelines. Galileo Research can assist the customers in the design of an appropriate program of non-clinical studies to support the development of anticancer pharmaceuticals (EMEA/CHMP/ICH/646107/2008 Note for the guidance on nonclinical evaluation for anticancer pharmaceuticals).

ICH Harmonised tripartite guideline: Guidance on Genotoxicity testing and Data Interpretation for Pharmaceuticals Intended for Human Use. S2(R1) 9 November 2011.

Test

OECD Guideline

Bacterial Reverse Mutation Test (Ames’ test)

OECD TG 471

In vitro mouse Lymphoma assay in L5178Y

OECD TG 490

In Vitro Mammalian Cell Micronucleus Test

OECD TG 487

Unscheduled DNA synthesis in vitro

OECD TG 482

In vitro gene mutations in V79 cells (HPRT)

OECD TG 476

In Vitro Mammalian Chromosomal Aberration Test

OECD TG 473

In vivo micronucleous test in mice*

OECD TG 474

*Demonstration of in vivo exposure: exposure information is generally available as part of the toxicology assessment. Demonstration of adequate exposure of the target tissue to the test compound should be obtained also during genotoxicity in vivo testing by toxicokinetic analysis.

  • ICH Guideline: Duration of Chronic Toxicity Testing in Animals (rodent and non rodent toxicity testing) S4
  • ICH Guideline: Note for Guidance on Toxicokinetics: the Assessment of Systemic Exposure in Toxicity Studies
  • ICH Guideline: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility S5(R2)
  • ICH Topic S8 Immunotoxicity Studies for Human Pharmaceuticals (May 2006 CHMP/167235/2004)
  • ICH Topic S9 Non-clinical Evaluation for Anticancer Pharmaceuticals

Single dose toxicities in rodents, by different administration routes: oral, dermal, intravenous, intramuscular, intra-articular, subcutaneous, intraperitoneal.

Acute oral toxicity by fixed dose method

OECD TG 420

Acute oral toxicity by up-and-down procedure

OECD TG 425

Acute oral toxicity by toxic class method

OECD TG 423

Acute dermal toxicity in rodents

OECD TG 402

Repeated dose toxicity in rodents by different administration routes: oral, dermal, intravenous, intramuscular, intra-articular, subcutaneous, intraperitoneal.

  • 28-day Oral Toxicity in Rodents (OECD TG 407), including main study, recovery, toxicokinetic

5 rats/sex/group, 4 Groups, 7-Day Dosing

  • 28-day Dermal Toxicity in Rodents (OECD TG 410), including main study, recovery, toxicokinetic

5 rats/sex/group, 4 Groups, 7-Day Dosing

  • 90-day Oral Toxicity in Rodents (OECD TG 408), including main study, recovery, toxicokinetic

10 rats/sex/group, 4 Groups, 7-Day Dosing

  • 90-day Dermal Toxicity in Rodents (OECD TG 411), including main study, recovery, toxicokinetic

10 rats/sex/group, 4 Groups, 7-Day Dosing

  • 6-month Oral Toxicity in Rodents (OECD TG 452), including main study, recovery, toxicokinetic
  • Reproductive Toxicity in Rodents
    • Embryo-Foetal Development in Rats (OECD TG 414)
    • Reproduction Toxicity in Rats (OECD TG 443, 415, 416)

 

Immunotoxicity (unintended immunosuppression or enhancement). The ICH guideline recommends non-clinical testing approaches to identify compounds which have the potential to be immunotoxic, and provides a guidance on a weight-of-evidence decision making approach for immunotoxicity testing:

  • Hematology
  • Histopathology of lymphoid organs
  • NK cell activity
  • T-cell Dependent Antibody Response (Plaque assay)
  • Mixed lymphocyte reaction
  • Antigenicity (induction of humoral response)

EMA/CHMP/SWP/2145/2000 Rev. 1, Corr. 1*. 22 October 2015. Guideline on non-clinical local tolerance testing of medicinal products.

“Stand-alone” studies on local tolerance are generally not recommended by the guideline, in order to reduce the number of animals. Thus local tolerance endpoints should be included in other toxicity studies. Moreover, in vitro methods, internationally validated and accepted by regulatory bodies, and included in OECD test guidelines, may be used as a partial replacement within a tiered testing strategy or as a stand-alone replacement depending on the outcome of the study.

Single dose local tolerance

Repeated dose local tolerance (up to 4 weeks)

  • Dermal (mouse, rat)
  • Intra-articular (rat)
  • Intramuscular (all species)
  • Ocular (rat)
  • Subcutaneous (all species)
  • Intravaginal (rat)

Skin sensitization

  • Mouse Local Lymph Node Assay (OECD TG 442A)

In vitro skin irritation/corrosion

  • In vitro skin corrosion in reconstructed human epidermis (RHE) test method (OECD TG 431)
  • In vitro skin irritation in reconstructed human epidermis (RHE) test method (OECD TG 439)

ICH Guideline: Pharmacokinetics: Guidance for Repeated Dose Tissue Distribution Studies S3B

  • In vitro metabolism:
    • Metabolic stability
    • Metabolite profiling
    • CYP450 inhibition
    • CYP450 induction
    • Phase II metabolism: Glutathion conjugation, Glucuronate conjugation
    • Drug-drug interaction
    • Test systems: human and rodent microsomes, human and rodent hepatocytes, S9, human recombinant CYP450
  • In vivo pharmacokinetic in rodents, with analytical method set up and validation
  • In vitro absorption through Reconstructed Human Epidermis (RHE), Reconstructed Human Corneal Epithelium (RHCE), Reconstructed Human Vaginal Epithelium (RHVE)

Analytical methods for the determination of the active principles in biological matrixes can be developed and validated, according to ICH.

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