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Bioanalytical Assays for Clinical Trials

  • LC/MS/MS assays of drug candidates, metabolites and small molecule biomarkers in various biological matrices supporting clinical Phase I to IV studies
  • Existing small molecule assays for quantiation of biomarkers in biological fluids
  • Existing drug assays supporting clinical DDI and BA/BE studies (please inquire)
  • Established protocols for validation of proprietary or commercial ELISA or cell-based assays supporting GLP and cGMP studies
  • Assay systems include LC/MS/MS, LC/MS, GC/MS, HPLC/UV, GC/FID and ELISA

PK Sample Collection Kit and Equipment Rental

  • Customize chemical stabilization kit for blood and plasma clinical PK sample collection
  • Venous / capillary blood / 24-Hr urine / saliva / nail clipping collection kits & handling protocol customized to clinical design
  • Clinical trial equipment rental of portable refrigerated centrifuges and -70°C freezers
  • PK sample storage & stability assessment
  • Cross-Border custom / TDG shipment documentation support & clinic Webinar training

Vast Experience in Bioanalytical Assays for Clinical Trials

  • Drug metabolite assay in conformance with US FDA MIST guidance
  • Personalized medicine based on translational study & therapeutic drug monitoring
  • Botanical drug candidate IND and NDA enabling development
  • Assay of uncommon PK sample matrices: neonatal dried-blood spot, CSF and others

Automated Barcode Sample Inventory & Data Handling

  • Thermo-Watson™ LIMS barcode sample and data handling of clinical PK samples
  • WinNonlin™ non-compartmental model PK parameter determination

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Clinical Trial Equipment Rental & PK Sample Collection Kit Supporting PK Sample Collection, Handling & -70°C Storage

Provide significant cost savings and logistic support for PK sample collection and storage at clinical sites prior to delivery to bioanalytical sites

Design & provide training of PK sample collection protocol, sample collection kits, and sample LIMS inventory using Thermo-Watson LIMS™ barcode system

Compact Ultra Low -70°C to -80°C Revco™ Freezer

  • L:W:H (30”:30”:44”)
  • Capacity 3 cuft
  • Power 110V/15amp (domestic US/Canada)
  • Rental (Initial 3-month term, monthly thereafter plus shipping/handling)

Refrigerated Low Speed Eppendorf™ Centrifuge

  • Variable speed 1000 to 4400 xg
  • Temperature range -9°C to 40°C
  • Power 110V/15amp (domestic US/Canada)
  • Rental (Initial 3-month term, monthly thereafter plus shipping/handling)

PK Blood / Plasma PK Sample Collection Supplies

  • Customize Chemical Stabilization Kit for Blood and Plasma PK Samples
  • Venous / Capillary Blood / 24-Hr Urine / Saliva Collection Supplies
  • PK Sample Collection & Handling Protocol Customized to Clinical Design
  • PK Sample Storage & Stability Assessment
  • Cross-Border Shipment Documentation Support & Webinar Training


Additional Clinical Trial Equipment Available Upon Request

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In Vitro DMPK & ADME

BRIVAL™ is our in vitro drug metabolism group co-founded and operated by Dr. Albert P Li and Ms. Clara Faan, featuring a full suite of in vitro metabolism studies based on human and animal hepatocytes, microsomes, S9 fractions and recombinant CYP450 and Phase II enzymes.

          In Vitro Drug Metabolism Studies
     Species
  • Human, Cyno monkey, dog, rat, mouse and others
     Materials
  • Cryopreserved human primary hepatocytes (intact system)
  • Microsomes (mainly oxidative pathways)
  • S9 (soluble oxidases, hydrolyases, transferases phase II)
  • Purified rCYPs
     Study Designs
  • Proprietary Assessment of Drug Candidate Solubility in Microsome or Hepatocyte Buffers to Ensure Dose Linearity
  • Metabolic Stability & Determination of CLint
  • Metabolite Profile, Structural Elucidation & Inter-Species 14C-Labeled Study
  • CYP450 Pathway Phenotyping
  • Metabolic Inhibition of Major CYPs (Determination of IC50, Ki, KI/kinact)
  • Metabolic Induction Screening
  • Covalent Binding Screening and Rank-Ordering

Unknown Metabolite Profile Using Microsomes or Hepatocytes

The use of 14C-labeled materials can provide time efficient identification of metabolites across species of animals

Laura ™ - Radio-chromatography Data System &
SoFie ™ - Stop-Flow Controller

The combination of SoFie™ controller and Laura™ software allows radiochemical measurement at either the threshold of detection or at any measurement level required by the user.

Parallel Collection of HPLC/DAD, MS Full Scan and Radiochemical Data

β-RAM - RadioHPLC detector (LabLogic Systems Ltd)

The β-RAM flow-through monitor for HPLC peaks combines a proven detection system with the latest computer technology, enabling the sharpest peak-shape resolution and the lowest amounts of radioactivity quantitation


Free Drug Determination by In vitro Equilibrium Dialysis & Ultrafiltration Assays of Protein Binding in Clinical & Preclinical Samples

  • Dialysis equilibration at 37°C in a 5% CO2 Chamber
  • Assay of dialysate and dialyzed plasma for free fraction calculation
  • Qualified LC/M/MS assays of all dialysate and plasma samples
  • Verify and correct volume shift for compounds requiring long dialysis equilibration time
  • Cross-Species comparison of free fraction
  • Evaluate non-specific adsorptive loss over therapeutic concentrations and plasma species for filter suitability
  • Pre-warm centrifuge rotor to 37°C and adjust plasma pH to 7.4 with PO4 buffer 10%v/v
  • Assay ultrafiltrate (free drug) and plasma (total drug) for free fraction calculation
  • Cross-species comparison of free fractions

In Vitro vs In Vivo Studies on Reactive Metabolites

Established in vitro assays and protocols are available to determine the presence and extent of irreversible binding of 14C-radiolabeled drug candidates to human and rat liver microsomes and hepatocytes to characterize the nature of reactive metabolites and Rank-Ordering of Candidates

HLM / RLM + 14C-Drug Candidate

Reaction for 60 minutes
Exhaustive Extraction of Non-Bound Drug & Metabolites
Collect Protein for 14C Liquid Scintillation Counting
Measure Protein Concentration

Covalent Binding Rank-Ordering of Candidates

      References:

  1. Evans DC, Watt AP, Nicoll-Griffith DA, and Baillie TA (2004) Drug-protein adducts: an industry perspective on minimizing the potential for drug bioactivation in drug discovery and development. Chem Res Toxicol 17:3–16

  2. Takakusa H, Masumoto H, Yukinaga H, et.al. (2008) Covalent binding and tissue distribution/retention assessment of drugs associated with idiosyncratic drug toxicity. Drug Metab Dispos. Sep;36(9):1770-9. Epub 2008 May 28.

  3. Zhou S, Chan E, Duan W, Huang M, and Chen YZ (2005) Drug bioactivation, covalent binding to target proteins and toxicity relevance. Drug Metab Rev 37:41–213.

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In Vivo DMPK & ADME

GLP Rodent Facility for ADME, DMPK & Xenograft Efficacy Studies

  • Pharmacokinetics & oral bioavailability studies
  • Urine, bile and feces drug and metabolite excretion mass balance to determine the major routes of elimination
  • CNS cannulated rat model for collection of CSF
  • 14C-Labeled drug tissue distribution mass balance
  • Patient-Derived Xenograft oncology efficacy mouse models by Oncograph™

A Mouse Dried-Blood Spot (DBS) Rapid PK Model Based on Serial-Sampling for Evaluation of Blood PK & Tissue Drug Distribution

Click here to open the pdf file for "LC/MS/MS Assay Method for Quantitation of Drug in Dried Blood Spot (DBS)"

Traditional mouse PK studies usually require a single terminal PK blood draw from each animal to provide a large sample volume at each time point. This single terminal blood draw sparse sampling approach requires a large number of animals.

The dried blood spot (DBS) sampling technique has allowed the sampling of 20 µL whole blood at each serial time point from a mouse over the entire PK profile thereby providing improved PK data quality and reduced number of animals and cost. A parallel rider group of mouse can also be dosed to provide target organ tissue distribution data.

In Vitro / In Vivo SEDDs Formulation Design prior to ADME & DM/PK Studies

  • SEDDs are isotropic mixtures of oils, surfactants, co-surfactants forming a finely and spontaneously dispersed micro-emulsion in an aqueous medium over the physiological pH range
  • Often enhance reproducibility in the rate and the extent of oral absorption
  • Some SEDDs can increase oral bioavailability by inhibiting pre-systemic metabolism and intestinal P-gp efflux [1]

[1] Chervinsky D.S., Brecher B.L., Hoelcle M.J. Anticancer Res., 13:93-96 (1993)

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Drug Candidate Early In Vitro & In Vivo Screening

  • LCMSMS Assay Development and Performance Qualification

  • In Vivo Pharmacokinetic Screening

Rapid PK & Drug Tissue Distribution based on Dried-Blood Spot LCMSMS Assay
Rodent Mass Balance Excretion Model

  • In Vitro Drug Metabolism Screening

In Vitro CYP Inhibition / Pathway ID
In Vitro Metabolic Stability / Metabolite ID
Protein Binding Cross-Species Comparison

  • In Vivo Pharmacology / Toxicology Screening

Patient-Derived Primary Tumor Xenograft Mouse Model
Acute Tox Screening in Rodents

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Patient Tumor-Derived Xenograft Models for Drug Development & Personalized Drug Sensitivity Evaluation at Oncograph™

Oncograph™ is founded by a group of world experts in the area of cancer modeling, anti-cancer drug evaluation and personalized cancer therapy. The mission is to develop new generation of preclinical platforms for 1) better predictive of personalized anti-cancer drug sensitivity; 2) accelerating the development of oncology drugs.

At present, the most commonly used preclinical cancer xenograft models are based on use of cultured human cell lines. Such cell line xenograft models, while valuable for basic research, in general do not adequately predict the efficacy of anticancer agents in the clinic and have been highlighted as a key obstacle in the translation of major advances in basic cancer research into meaningful clinical benefits. In addition to tradition cell line models, Oncograft™ utilizes new generation of pre-clinical platform, a unique panel of patient-derived xenograft models, established by the implantation of primary human tumors under the renal capsule of immune-deficient mice. The high vascularity of this subrenal capsule graft site, compared to the subcutaneous and orthotopic sites, allows a more adequate supply of nutrients to the tumor tissue. This grafting methodology permits a high tumor take rates (>95%) for a variety of low and higher grade malignancies, including cancers of the prostate (Lin et al, 2008; Wang et al, 2005a; Wang et al, 2005b), ovary (Lee et al, 2005; Press et al, 2008), kidney (Liou et al, 2004), lung (Cutz et al, 2006; Guan et al, 2009; Dong et al.,2010), pancreas (Watahiki et al, 2006) and others (unpublished data).

Significantly, the cancer tissue xenografts and transplantable tumor lines derived from such xenografts are histologically highly similar to the donor tissues and retain important genetic and epigenetic features as well as responses to treatment with drugs (Cutz et al, 2006; Lee et al, 2005; Press et al, 2008). It is also proved that patient-derived xenografts can be used for personalized cancer therapy (Dong, et al., 2010). The advanced xenografting technology allows us develop next generation of patient-derived cancer models to support new drug candidate development and personalized oncology drug therapy evaluation for patients.

  1. Lee CH, Xue H, Sutcliffe M, Gout PW, Huntsman DG, Miller DM, Gilks CB, Wang YZ. Establishment of subrenal capsule xenografts of primary human ovarian tumors in SCID mice: potential models. Gyn. Oncol. 96 (1): 48-55, 2005
  2. Press JZ, Kenyon JA, Xue H, Miller MA, de Luca A, Miller DM, Huntsman DG, Gilks CB, McAlpine JN, Wang YZ. Xenografts of primary human gynecological tumors grown under the renal capsule of NOD/SCID mice show genetic stability during serial transplantation and respond to cytotoxic chemotherapy. Gyn. Oncol. 110: 256-264, 2008.
  3. Wang YZ, Revelo MP, Sudilovsky D, Chen WG, Goetz L, Xue H, Sadar M, Shappell SB, Cunha GR, Hayward SW. Development and characterization of efficient xenograft models for benign and malignant human prostate tissue. Prostate 64, 149-159, 2005
  4. Wang Y, Xue H, Mawji R, Chen WG, Hayward SW, Gilks CB, Sadar MD, Gout PW, Cunha GR, Wang YZ. An orthotopic metastatic prostate cancer model in SCID mice via grafting of a transplantable human prostate tumor line. Lab. Invest. 85: 1392-1404, 2005 (Cover art and features inside Lab. Invest.)
  5. Lin D, Watahiki A, Bayani J, Zhang F, Liu L, Ling V, Sadar M, English J, Fazli L, So A, Gout P, Gleave M, Squire JA, Wang YZ. ASAP1, a Gene at 8q24, is Associated with Prostate Cancer Metastasis. Cancer Research. 68: 4352-4359. 2008.
  6. McPherson S, Hussain S, Balanathan P, Hedwards S, Niranjan B, Grant M, Chandrasiri U, Toivanen U, Wang YZ, Taylor R, Risbridger G. Estrogen receptor beta activated apoptosis in benign hyperplasia and cancer of the prostate is androgen-independent and TNFa-mediated. PNAS 107(7): 3123-3128, 2010.
  7. Cutz J-C, Guan J, Bayani J, Xue H, Sutcliffe M, English J, Flint J, LeRiche J, Yee J, Squire J, Gout PW, Lam S, Wang YZ. Establishment in SCID Mice of Subrenal Capsule Xenografts and Transplantable Tumor Lines from a Variety of Primary Human Lung Cancers: Potential Models for Studying Tumor Progression-Related Changes. Clin. Cancer Research. 12 (13): 4043-4054, 2006.
  8. Guan J, Lo M, Dockery P, Mahon S, Karp C, Buckley A, Lam S, Gout PW, Wang YZ. The xc- Cystine Transporter as a Therapeutic Target for Small Cell Lung Cancer: Use of Sulfasalazine. Cancer Chemotherapy and Pharmacology, 64(3): 463-72, 2009.
  9. Dong X, Guan J, English J, Flint J, Yee J, Evans K, Murray N, MacAulay C, Ng R, Gout PW, Lam W, Laskin J, Ling V, Lam S, Wang YZ. Patient-derived first generation xenografts of non-small cell lung cancers: Promising tools for predicting drug responses for personalized chemotherapy. Clin. Cancer Research 16:1442-1451, 2010.

Clinical Benefits of Patient-Derived Primary Tumor Xenograft Services at Oncograph™

  • Offer personalized chemotherapy for each patient based on customized xenografts developed from each patient’s biopsy tumor tissue to develop xenografts for chemotherapy evaluation within a 4-6 week period.
  • Offer cancer patients the ability to evaluate the effectiveness of 1st-line and 2nd-line chemotherapies prior to drug treatment
  • Predict the onset of chemotherapy drug resistance
  • Evaluate potential outcome of new investigational therapies for cancer patients prior to clinical trials

Patient-Derived Primary Tumor Xenograft Drug Candidate Development at Oncograph™

  • Patient-derived orthotopic and sub-renal capsule Xenograft models offer an established valuable alternative to commercial cell-lines derived from subcutaneous xenografts which are less predictive of clinical efficacy.
  • Having been developing a panel of transplantable patient-derived tumor xenografts of various cancers including prostate, ovary, lung, pancreas, skin and kidney developed for oncology drug candidate evaluation.
  • Screening and selection of clinical new drug candidates with simultaneous efficacy and pharmacokinetic correlation based on serial blood and tissue sampling from each Xenograft animal followed by LC/MS/MS assay and PK/PD assessment.

Study Designs

  • Acute toxicity and efficacy study
  • MTD
  • Short and long-term multiple dose studies
  • Identification of target organ

Common Study Safety and Efficacy Endpoints

  • Inhibition of tumor growth
  • Incidence of metastases and metastatic growth rate
  • Survival rate
  • Histopathological endpoints
  • Immunohistochemistry endpoints
  • Offering discovery and validation of small molecule tumor biomarkers based on LC/MS/MS approach in support of early clinical diagnosis of cancers and cancer treatment prognosis.
  • Offering an array of biomarker assay available based on commercially developed multiplex ELISA assays available.

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API / Clinical Product Quality Control, Dosing Solutions and Materials, Dose Formulation and Stability

To support CMC development, BRI offers a variety of analytical chemistry assays supporting API / clinical product quality control, toxicology and safety study dosing samples / materials assays, dose formulation and IND-enabling stability studies conducted in conformance to cGMP and ICH requirements.

IND-Enabling API / Clinical Product Quality Control Assays

  • Manufacturing specification release assays for API and clinical products
  • Complementary LC/MS, HPLC and GC assays in support of clinical product specification release and QC testing
  • Established USP drug product and drug substance monograph assays
  • Canadian cGMP licensed testing facility

Toxicology and Safety Study Dose Solutions / Materials Assays

  • Evaluation of drug substance True Solution Solubility™ in aqueous buffers used for safety pharmacology and toxicology experimental system
  • HPLC or LC/MS assays for Verification of dose material label claim concentrations
  • Dose materials stability and homogeneity supporting toxicology and safety studies

Drug Substance and Drug Product Stability Studies

  • Characterization and identification of unknown impurity and degradation product
  • API & clinical product impurities and degradation products assays by HPLC/UV/MS assay
  • IND-Enabling API & clinical product stress and stability studies under ICH stability protocols

IND-Enabling Clinical Product Development and Manufacturing

  • Oral capsules (hard and soft gelatin), tablets, topical and parenteral product formulation development
  • Clinical product cGMP manufacturing outsourcing and project management

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Strategic Development of Botanical Drugs

BRI offers years of experience in the development of Botantical Drugs (US FDA Guidance, 2004), Dietary Supplement (DSHEA, US) and Natural Health Products (Canadian), engaging in the laboratory-scale, pilot-scale, and commercial-scale development of botanical extracts (API), formulation and oral absorption enhancement, and out-source manufacturing of clinical and commercial products.

Naturally Exact™ is a patent-pending mathematical QC control system developed and validated at BRI to ensure lot-by-lot consistency of natural botanical extracts against a pre-defined botanical extract (API) composition specifications. This proprietary process utilize post-extraction treatment of extracts from multiple botanical ingredients to arrive at an exact specification regardless of the raw material content.

BRI also regularly provides consultation and preparation of IND, CTA, Dietary Ingredient Notification, GRAS and NHP submissions in collaboration with regulatory consultants.

  • Multiple-target & system biology approach guided by in vitro and in vivo data
  • Strategic approach in defining the drug substance mixture from whole extract to partial isolate
  • Strategic approach in selecting chemical markers to guide product development
  • Achieve desirable oral bioavailability
  • Proprietary mathematical QC control approach to achieve lot-to-lot consistency of final extract
  • Botanical raw materials supply-chain planning and production strategies

Guidance for Industry

Attachment B

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Cynomologus and Rhesus Monkey Hepatocytes and Blood Products

ALIVE Laboratories™   Alive Laboratories

BRI is a Canadian commercial distributor of various cynomology and rhesus monkey and other non-human primate hepatocytes, microsome, S9 and blood products (whole blood, plasma, serum) collected using a variety of anticoagulant. Products are usually shipped within a week of receiving order from within Canada.

Please visit www.alivelaboratories.com for a list of products

Species:
  • Cynomolgous Monkey
  • Rhesus Monkey
  • African Green Monkey
  • Marmoset
  • Others (Please Inquire)
Products:
  • Blood
  • Plasma
  • Serum
  • CSF (Spinal Tap)
  • Other Blood Products (Please Inquire)
Viral Quality Control:
  • HBsAg
  • HIV 1/2 Ab,
  • HIV-1 RNA
  • HCV Ab,
  • HCV RNA
  • Herpes B
  • Additional viruses, diseases or pathogens available upon request
Chemical Quality Control Available Upon Request:
  • Extractable Chemical Profile by LC/MS
  • Total Protein
  • Total Cholesterol (Esterified and Non-Esterified)
  • Others (Please Inquire)
Options:
  • Individual Male & Female
  • Pooled Males & Females
Choices of Anticoagulants:
  • K2EDTA
  • K3EDTA
  • Lithium heparin
  • Sodium heparin
  • Others (Please Inquire)

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