Pharmaseed

• Parkinson’s disease (6-hydroxydopamine, MPTP)
  
• Alzheimer’s disease
  
• Vascular dementia
  
• Hippocampal neurodegeneration (Kainic acid)
  
• Epilepsy (Pilocarpine, Kainic acid, Pentylenetetrazol (PTZ))
  
• Spinal cord and traumatic brain injuries (Close/Open head)
  
• Ototoxicity (cisplatin)
  
• Itch model (Substance P/ capsaicin)
  
• Complementary ex-vivo models – organotypic brain slices
  
• Schizophrenia (Amphetamine, Ketamine, Apomorphine, PCP)
  
• PTSD (corticosterone)
  
• Autism (Poly IC)
  
• Depression testing (Force swimming, Tail suspension)
  
• Anxiety testing (open field, elevated maze)
  
• Neuropathic pain (spinal nerve ligation, sciatic nerve ligation, sciatic nerve inflammation)
  
• Acute pain (post-operative pain, formalin/capsaicin test)
  
• Chronic/inflammatory pain (CFA/Carrageenan)
  
• Aged mice (motor and cognition testing: Elevated Maze, Nose Poke, Object Recognition, Social Recognition)
  
• Complementary ex vivo models for neurodegenerative diseases, such as organotypic brain slices.

Our Expertise and Models for Autoimmune and Inflammatory Diseases
We offer a unique battery of pre-clinical models that allows to screen potential anti-inflammatory agents as well as identify novel therapeutic targets. Screening of compounds can be performed in existing well-characterized animal models as well as in newly developed animal models tailored to a specific tested item.

Our expanded battery of models includes studies of inflammation and inflammatory pathways, including in vivo models of inflammation and autoimmune diseases, as well as complementary in vitro and ex vivo analyses:

Acute and chronic inflammation models:
- Carrageenan-induced inflammation
- LPS-induced inflammation
- Delayed-type hypersensitivity (sRBC)
- Granuloma (cotton-pellet induced)
- Formalin-induced sub-acute inflammation
- Peritonitis model
- Bleomycin-induced lung fibrosis
- Croton oil-induced ear inflammation

Neuroinflammatory disease models:
- Experimental autoimmune encephalomyelitis (MOG/PLP/Spinal Cord Homogenate)
- Viral-induced neuroinflammation

Intestinal Bowel Disease/Colitis models in several species (mouse, rats):
- DSS-induced
- TNBS-induced
- T-cell mediated

Rheumatoid Arthritis and Osteoarthritis models:
- Collagen-induced
- CFA-induced
- MMT model in rats (transaction of the medial collateral ligaments and medial meniscus)

Dermatology models:
- Imiquimod-induced psoriasis
- Ex vivo human explants evaluation
- Itch model (Substance P/capsaicin)

• Neurodegenerative diseases (i.e. Parkinson's Disease)
  
• Stroke and HLI
  
• Erectile Dysfunction
  

Designing better translational models for neurodegenerative diseases

Not Young But Relevant Models
- Species: mouse, rat
- Young vs. old animals
- Gender differences

Environmental factors
- Inflammation: LPS-induction
- Environmental toxins: Complex I inhibitor MPTP-induction
- Oxidative stress: Western high-fat diet
- Genetics predisposition: alpha-synuclein, PAC-Tg(SNCAWT), Snca-/- mice

Behavioral tests
- Neurological score
- Adhesive removal test
- Body swing test
- Beam walking test
- Rota-Rod test
- Water maze, Radial maze
- Object recognition test, social recognition test
- Forelimb placing test
- Stepping test
- Staircase skilled reaching test
- Cylinder test

Biochemistry and Histology
- Quantification of brain DA, DOPAC, HVA-TH, alpha-synucleic immunostaining

Biodistribution studies:
- Injection of human cells or biomaterials loaded with human cells in NOD/SCID mice
- qPCR analysis for evaluation of human DNA
- histopathology analysis
- FACS

Hemi-crush in rats (moderate spinal cord injury)
- MRI Imaging
- Flexion Reflex Test
- Locomotor function: Basso, Beattie and Brensahan (BBB) test
- Grid walking

Ferric-chloride thrombosis model
- Evaluation of thrombi size and weight
- Laser Doppler analysis
- APTT and PT
- Blood analyses for coagulation factors through ELISA

• Weight bearing test
  
• Von Frey test
  
• Writhing Test
  
• Pin Prick Test
  
• Hot and Cold plate test
  
• Edema scores
  
• Protein extraversion-vasodilatation [migraine]

Isolation and in vitro assays of rat and murine pancreatic islets.

In multiple species: mice, rats, rabbits, dogs, pigs.

In multiple species: rats, mice, dogs, pigs.

• Pilocarpine
  
• Kainic acid
  
• Pentylenetetrazol

Models for muscular dystrophy (MDX, alpha-sarcoglycan mice).
Evaluation of clinical parameters:
- Body weight
- Rotarod (coordination)
- Grip strenght test and four limbs hang test (muscle force)
- Histological evaluation: fiber diameter, number of centrally nucleated fibers, sarcolemma integrity, inflammation and fibrosis
- Biochemical: plasma CK levels

Medical cannabis studies are performed in our laboratories in Israel
• Multiple Sclerosis
• Glaucoma
• ALS
• Peripheral Neuropathic Pain
• Post Operative Pain
• Spinal Cord Injury
• Anxiety – PTSD
• Parkinson’s Disease
• Cachexia, Nausea& Vomiting – Cancer
• Rheumatoid Arthritis
• Inflammatory Bowel Diseases
• Epilepsy

Pharmacology: full analysis capabilities
• Analysis of cannabinoids composition (CBD vs THC); bioinformatic evaluation
• Binding and activity assays CB1/CB2 (cAMP)
• Full panel receptor profile binding
• Basic efficacy in vitro assays
• Animal disease efficacy studies [POC]
• Basic safety (in vivo and in vitro): GLP & non–GLP

Formulation: on-site development and optimization
• Optimization of the bioavailability and delivery standardization, based on the selected clinical indication
• Development of standard carrier (formulation) that will address stability and delivery unmet needs:sublingual, cream/patches, etc.

• MOG-induced Experimental Autoimmune Encephalomyelitis (EAE)
  

• Cuprizone-induced model

• Behavioral tests (activity box, wheel-running test, social interaction test)

• Histological evaluation

• Xenograft models
  
• Syngenic tumor models (LLC, HCC, EMT6/CTX, MBT-2, C6)
  
• Orthotopic tumor models

DSS-induced colitis in mice and rats
TNBS-induced colitis in mice and rats
T-cell-mediated colitis

Neuropathic pain models:

• STZ-induced Diabetic neuropathy (Rats)
  
• Spinal Nerve Ligation (Chung model, Mice)
  
• Chronic Constriction Injury (Bennett Model)
  
• Spared Nerve Injury (Mice)
  

• Smear evaluation –Years of experience in reading cytological smears including
  
  • Malignancy
    
  • Inflammation (types and severity)
    
  • Unique cytological features.
    
• Professional fixation , staining and IHC of cytological smears.
  
• Cytospin – A popular technique for the concerntration of cells in a low density sample, yielding high quality smears
  
• Bronco alveolar lavage (BAL) – Smears taken from the tracheal wash or lung lavage
  
• Bone marrow evaluation – Cell type analysis, including cell counts and interpretation.
  
• Cerebrospinal fluid evaluation – CSF cellular components analysis using Cytospin
  
• Synovial fluid evaluation – characterizing cell populations and interpretations.

• Sample methods: sample treatment and harvesting methods (full organ, biopsies, true cuts, fine needle aspirations, wet smears, damp smears etc.)
  
• Different fixative media (formaldehyde, alcohols, Davidson, HOPE and other fixatives for tissues and cells)
  
• Supporting animal models: Examining animal models with relevance to human diseases.
  
• Organs measurements and weight, organ evaluation: Diagnosis of normal and abnormal organ morphology
  
• Comparative anatomy & pathology
  
• Documentation: Results are formatedd in tables and images, according to the client’s demand.
  

Our Histology Unit

Our histology unit is aimed at providing expert and cost effective histology and pathology services to our clients, in a complementary or stand-alone service setting. The Histology unit is managed by an expert in-house veterinarian pathologist.

The strength of our histology unit is in our understanding of the experimental design of pre-clinical efficacy or safety studies.

We have extensive experience in the development and implementation of different investigational methodologies utilizing a large array of techniques, including “free floating sections” as a modern method.

We provide researchers with valuable input at the early planning phase of a study, or carry out histology and pathology analysis at any stage of a project, customized to your needs.

Immunohistochemistry and special stains:

• Stainings for fibrous tissue, stromal components, epithelial glycoproteins, calcification and more
  
• From commonly used stains (Silver stain for bone tissue, Toluidine blue, Masson Trichrome and H&E) to more specialized staining methods (Paragon and more)
  
• Special immunofluorescence for brain research, stem cells and other subjects
  
• Morphometric service: Manual (semi-quantitative) and digital histological result quantification
  
• Technical and scientific QA: We are a GLP-certified CRO. The histology unit is non-GLP, however uses a GLP compliant QA.
  

Gross pathology:

• Sample methods: sample treatment and harvesting methods (full organ, biopsies, true cuts, fine needle aspirations, wet smears, damp smears etc.)
  
• Different fixative media (formaldehyde, alcohols, Davidson, HOPE and other fixatives for tissues and cells)
  
• Supporting animal models: Examining animal models with relevance to human diseases.
  
• Organs measurements and weight, organ evaluation: Diagnosis of normal and abnormal organ morphology
  
• Comparative anatomy & pathology
  
• Documentation: Results are formatedd in tables and images, according to the client’s demand.
  

Cytology

• Smear evaluation (malignancy, inflammation, unique cytological features)
  
• Professional fixation, staining and immunohistochemistry of cytological smears
  
• Cytospin
  
• Broncho-alveolar lavage (BAL)
  
• Bone marrow evaluation
  
• Cerebrospinal fluid evaluation
  
• Synovial fluid evaluation
  

Medical Device Analysis:

• Sectioning of metal and other hard devices within the tissue, without creating artifacts through use of a diamond saw
  
• Standard device sectioning in paraffin
  
• High quality: unique plastic slides ensure smooth tissue sectioning (15-30 micron) and allow full histological evaluation in light microscopy
  
• Perfect morphometric method: measurements performed directly on the slide (tissue density, % contact tissue/device), cellular involvement
  

Free floating sections: a sectioning method that can be used for immunofluorescence staining. It allows optimal antibody penetration and staining of thick sections that are then imaged through a confocal microscope.

Other services and capabilities:

• In-situ hybridization on paraffin sections
  
• Extraction of DNA and RNA from paraffin blocks
  
• Specialized tissue fixatives (HOPE)
  
• Development of new scientific methods in the fields of imaging, histology and tissue processing.

• 6-OHDA model
  
• Chronic, subchronic and acute MPTP model
  
• MPTP model in aged animals
  

Behavioural analysis:
- Contra-lateral Rotation
- Stair Case Test
- Stepping Test
- Rotarod
- Cylinder Test

Histological (single staining & co-stainings) and bioanalytical evaluation:
- Tyrosine Hydroxylase (TH)
- BrdU
- Alpha-synuclein
- Levels of striatal dopamine & metabolites

Pharmaseed’s regulatory capabilities comprise a vast array of safety and toxicology designs, ranging from acute single-dose studies to chronic and local tolerance studies, including specialty routes such as intra-cerebroventricular administration and infusion. All studies are fully GLP-certified and are in accordance with current guidelines, or custom-designed for particular needs.

Cytokines and cellular markers measurements are done in induced/treated cells, plasma or organs

ELISA and other antibody–mediated assays are available

Safety: Cytotoxicity of Medical device

Cytotoxicity assays are also available for the evaluation of medical devices’ safety.

The medical device is immersed in both polar and non-polar solvents. A live cell culture is exposed to the extract, and the possible toxicity of the medical device is then evaluated using a cytotoxicity assay.

All assays are ISO-10993 compliant.

Pharmaseed offers cytotoxicity / cell viability assays, such as XTT, MTT and LDH kit.

Evaluation of the test item’s effect on proliferation or cell death.

An array of cell lines and/or primary cells is available for the evaluation of the test item.

Determination of EC50/IC50 values – enables the comparison to other cell culture models or materials

Pharmaseed offers cytotoxicity / cell viability assays, such as XTT, MTT and LDH kit.

Evaluation of the test item’s effect on proliferation or cell death.

An array of cell lines and/or primary cells is available for the evaluation of the test item.

Determination of EC50/IC50 values – enables the comparison to other cell culture models or materials

We specialize in Isolation of murine and human stem cells

Differentiation and maturation protocols are available upon request

We provide viability and purity testing, as well as flow cytometry (FACS) analysis for requested markers.

Permanent and transient models of stroke in rats, mice & rabbits

Neuroblastoma, breast cancer, melanoma, pancreatic cancer, colon cancer, multiple myeloma, prostate cancer.

A375 cell line and others

HT29, HCT-116 and other cell lines

MCF7, BT474, MDA-MB-231, T47D and other human cell lines

Hindlimb ischemia models in rats & mice

Lewis Lung Carcinoma Model

LLC cells injected through the tail vein and assessment of lung metastases.

Endothelial Tube Formation
Endothelial Cell Migration & Invasion assays
Murine aortic ring assay

Modified Corneal Pocket Assay in rabbits

In vitro models (determination of NO and hormone levels);
Relaxation test in isolated corpus cavernosum in rabbits
Screening of erectile response in diabetic or hypertension animal models (STZ-induced diabetic rats or SHR)

Wound healing models in normal and diabetic rats and mice

• Osteoregeneration in Calvarias model
  
• Osteoporosis in rats (OVX)
  
• Implantation of biomaterials in the rat knee (efficacy and safety)
  
• Implantation of bone materials in the maxillae

• CFA-induced monoarthritis model in rats
  
• Collagenase-II induced OA in rats
  
• MMT model in rats
  
• ACLT-MMx model in rats
  
• Collagen-induced arthritis model in rats and mice
  
• Histo-pathological evaluation and imaging (MRI)
  
• In vitro screening in murine primary articular chondrocytes
  
• Multiplex assays
  
• RAW 264.7 test following LPS stimulation and cytokines assessment

• Imiquimod (IMQ)-induced psoriasis
  
• Humanized psoriasis model in NOD-SCID mice

Our Expertise in Oncology and Cancer Models
We offer a wide range of preclinical oncology models and tumor modeling in vivo for the study of cancer growth and metastasis in mice and rats.

The two most used models in oncology R&D for the assessment of potential efficacy of tested technology are the xenograft and the orthotopic model. We combine these in vivo models with in vitro and ex vivo analyses, such as FACS, angiogenesis and cell invasion assays (i.e. HET-CAM assay), and with high-level histological evaluation and imaging tools (Laser Doppler, MRI, X-ray).

Subcutaneous Xenograft Models for a variety of human cancers:
- Pancreatic adenocarcinoma
- Breast cancer
- Prostate cancer
- Melanoma
- Glioblastoma
- Neuroblastoma
- Colorectal carcinoma
- Liver cancer
- Lung cancer
- Lymphoid cancer

Orthotopic implantation:
- Orthotopic pancreatic cancer model
- Intra-parenchimal glioblastoma model

Metastatic models (i.e. Lewis Lung Adenocarcinoma systemic administration)

Hollow fiber assay

Complementary models:
- In vitro screening for cytotoxicity activity
- In vitro screening for anti-angiogenic activity (Endothelial Tube Formation assay in HUVEC, Aortic Ring Assay)
- Ex ovo HET-CAM assay

Histological evaluation and imaging capabilities

• Imiquimod-induced psoriasis
  
• Ex vivo human explants evaluation
  
• Itch model (Substance P/capsaicin)

Hindlimb ischemia models in different species (mouse, rat, rabbit):
- Evaluation of blood flow via Laser Doppler analysis, bubble contrast ultrasound
- X-ray analysis: angiogram
- Analysis of blood vessel functionality (immunohistochemistry CD34/Dextran)
- Supportive histological and biomolecular parameters

Stroke models in different species (mouse, rat, rabbit) – embolic and mechanical induction; permanent and transient occlusions; local or distal embolizations:
- Analysis of cortical blood flow by Transcranial Laser Doppler
- Morphological assessment
- Imaging
- Immunohistochemistry
- Behavioural tests:
- Adhesive removal
- Staircase skilled reaching
- Neuroscore
- Forelimb placing test
- Cylinder test

Ischemic Heart Disease model in different species (mouse, rat) – Transient Left Coronary Artery Occlusion:
- Computerized analysis of infarct size (2,3,5-TTC staining)
- ECG recording
- Supportive histological analysis (H&E, troponin staining)

Wound Healing model in healthy, diabetic or aged mice

Complementary Angiogenesis models:
- Ex ovo computerized HET-CAM assay
- Endothelial Tube Formation assay
- Aortic ring assay
- Modified Corneal Pocket assay in rabbit

Erectile dysfunction models in diabetic, hypertensive or naive rats:
- Relaxation test in isolated corpus cavernosum (from rabbits)
- Vasoactivity evaluation by Laser Doppler analysis
- Supportive biochemical parameters (determination of hormones and nitric oxide levels)
- Supportive behavioural parameters (mounting behaviour test, mating performances, orientation activities)