Application in Preclinical and Clinical Research Stages An ... · 3 Drug Metabolism Strategic Advantages Organizational Structure Why it works: –Leadership. –Preclinical regulatory

Open Access Central Laboratory’s: Application in Preclinical and

Clinical Research Stages An Overview

Rasmy E. Talaat, PhD Wyeth/Pfizer Fellow-Retired

[email protected]

ALEX: 29 November 2017 1

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Drug MetabolismMission Statement

The Mission of the Drug Metabolism Department is to conduct early DMPK studies to guide the selection, optimization and advancement of Discovery phase

candidates, and to provide detailed metabolic disposition characterization of Development compounds

in support of clinical development and regulatory submissions

- Both Small Molecules and Proteins from all therapeutic areas

ALEX: 29 November 2017

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Drug Metabolism Strategic Advantages Organizational Structure

Why it works:

– Leadership.

– Preclinical regulatory considerations to early support clinical development strategies

– Efficient processes and workflow permits timely dissemination of data

– Maximizes efficiencies through harmonization, standardization and automation of routine methodological processes

– Innovative approaches using state-of-the-art technology to characterize potential development candidates

Consequence: – Improved productivity, elimination of redundant studies to produce better

quality compounds advancing into Development via consensus on decisions to advance or terminate a NCI.

– Proven productivity track record of supporting 8+ INDs and 2-3 NDA per year


LEADERSHIP

• Experience: UW, ABC Labs, Covance, Parke Davis, Wyeth/Pfizer

• Open and Free Training across departments as well as Clients

• UMBC Undergraduate and Graduate students Hands on Training

• Training 200+ users

• Customized levels of training

• Independent operating research with Free guidance

• Free consultation to external MS users

• Intra-, Inter-, and External Collaboration between team researchers

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Drug Metabolism - Strategic Advantages Staff Resources and Technology Platforms

• Centralized, highly integrated, full-service Drug Metabolism

laboratories

• Highly skilled, flexible, diverse and multi-disciplined workforce provides a complement of expertise that can be used to address DMPK/Safety/Toxicology in Discovery and Development

– 39% of Staff have Ph.Ds, with expertise in a range of disciplines, including Drug Metabolism, PK, Mass Spectrometry, Toxicology, Molecular Biology and Analytical Chemistry

• Laboratories equipped with sophisticated, state-of-the-art instruments capable of supporting both Discovery and Development phase studies from all therapeutic areas and platforms


Mass Spectrometry

Principle of operation

A Mass spectrometer is a “Molecule Smasher”

Measures molecular and atomic masses of whole molecules, molecular fragments by generation and detection of the corresponding gas phase of ionized molecules in vacuum, separated according to their mass-to-charge ratio (m/z) with the aim of one or more of the following:

• Molecular weight determination • Structural characterization • Gas phase reactivity study • Qualitative and Quantitative analysis of components in a mixture.


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Drug Metabolism Capital Equipment Mass Spectrometry, Automation, NMR

• Mass Spectrometers - Total of 31 in Drug Metabolism Department (35 users-40% PhD)

– Quantitative Analyses : Seventeen

Triple Quadrupole MS

– Metabolite Identification : Fourteen

High Resolution Mass Spectrometers e.g.

LTQ Orbitrap, Q-TOF-2,…….

• LC-MS/MS-NMR 600 MHz Bruker NMR Magnet (1 PhD and 1 MSc)

• IT (MSc)

• Automation (3 MSc)

– Eight Liquid Handlers e.g.

Tomtec Quadra-4, Tecan Freedom, Packard Multiprobe II,…….


Major Functions of Mass Spectrometry Discovery/Development Metabolism

• In vitro Metabolite Profiling:

• Provide metabolite profiles for drug candidates generated from in vitro systems (liver microsomes + Cytosole, Hepatocytes) to assist BETTER drug candidate design.

• In vivo Metabolite Profiling of PK/TK Plasma: Associate in vitro metabolism with the in vivo

• Examine the correlation of metabolite with adverse effects.

• Reactive Metabolite Identification:

• Assess potential bio-liabilities by detection of reactive metabolites using trapping techniques (e.g. GSH, KCN, Dipeptide) along with the recommendations to minimize/avoid the formation of such reactive metabolite in drug design.


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DPK 2008 Productivity

• Small Molecules – Total Studies 1969 – Neurosciences 583

– CVMD 470

– Inflammation 353

– Oncology 419

– WH&B 144

• Proteins – Total Studies 50


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Pre-clinical PK Productivity

488

0

741

98

966

170

1284

59

1665

47

1787

122

1969

77

0

250

500

750

1000

1250

1500

1750

2000

2250

2002 2003 2004 2005 2006 2007 2008

# of Studies

# of FTEs has been reasonably constant over the years ~23

Total Studies

Contracted Studies


DISCOVERY METABOLISM

• CYP inhibition reversible IC50 cocktail

assay

•CYP inhibition mechanism-based screen

•CYP inhibition kinetic studies: Ki or KI,

kinact

•Reaction phenotype (cold studies)

•Km substrate depletion or Km, Vmax

determinations

•Intrinsic clearance and metabolite ID

•Trapping reactive intermediate metabolites

•Biomarker assays

DISCOVERY PHARMACOKINETICS

• Protein precipitation

(conventional and filter plate)

•Liquid-liquid extraction

(conventional and solid-

supported)

•Solid phase extraction

(microplate based and on-line)

•Method Development &

Optimization

DEVELOPMENT METABOLISM

• Reaction phenotype (hot studies)

•Plasma and whole blood stability

•384 well UGT assay

•Clinical BA Studies

APPLICATIONS OF THE MASS SPECTROMETRY/AUTOMATION GROUP IN

ADME-SAFETY of DRUG DISCOVERY-DEVELOPMENT STAGES

o Variable channel liquid handlers

96-channel pipettors

Robotic arms and rails

PROTEIN BINDING

• Equilibrium dialysis (hot and cold studies)

•Ultracentrifugation (hot and cold studies)

•Ultrafiltration (hot and cold studies)

Plasma, Microsomes and Brain Tissue for better

First in Human dose projections and improved PK-

PD in CNS

On-line LC/MS automated systems HIGH THROUGHPUT INCUBATION

AND EXTRACTION TOOLS

TYPICAL FACT SHEET

PHARMACOLOGY In vitroIC50 Enzymatic: 0.9 nM WBC Stat PO4: 59 nM Whole blood: 580 nM

In vivo Mouse IL-2/IFNg: ED50: 1 mg/kg DTH mode ED50: 10 mg/kg QD Mouse CIA model: MED 3 mg/kg BID 3 mg/kg single dose Cmax: 217 ng/mL AUClast =2089 ng.hr/mL Extrapolated AUC at MED = 4178 ng.hr/mL

PHYSICOCHEMICAL PROPERTIES m.w. 437 logP: 3.13 Permeability (x 10-6 cm/sec) Caco2: A-B 14.9 (DSM) ER ~2 P-gp inhibition IC50: 10 – 100 M Solubility <LOQ (pH 7.4), 0.002 mg/mL (pH 1), 0.019 mg/mL in MC/TW 0.32 mg/mL in 10% TPGS BCS: 2 Protein Binding >97% human; 92% rat and 91% mice (PCOP) Unbound 3.8% human, 4.1% DBA mouse, 6.3% rat, 4.6% dog and 3.9% monkey (DSM) Blood/Plasma: 1 (r), 0.7 (h)

PHARMACOKINETICS Balb/C Mouse iv 2 mpk CL = 12 mL/min/kg Vss = 2.1 L/kg; T1/2= 2.3 h po 10 mpk (MC/TW) AUClast = 5200 h*ng/mL Cmax = 750 ng/mL; F = 36%

SD Rats iv 2 mpk CL = 16 mL/min/kg Vss = 1.8 L/kg; T1/2= 1.4 h po 10 mpk (10% TPGS) AUClast = 1056 h*ng/mL Cmax = 240 ng/mL; F = 9.4% Ascending po dose (10% TPGS) AUClast (h*ng/mL) = 9276 (100 mg/kg), 14231 (300 mg/kg), 24517 (1000 mg/kg)

Dogs iv 2 mpk CL =25 mL/min/kg Vss = 1.2 L/kg; T1/2= 0.8 h po 10 mpk (10% TPGS) AUClast = 2426 h*ng/mL F = 35% Cmax = 1556 ng/mL Ascending po dose (10% TPGS) AUClast (h*ng/mL) = 1213 (30 mg/kg), 6754 (100 mg/kg), 8988 (300 mg/kg)

SAFETY Moderate cytotoxicity (IC50 = 6 M) No major findings in 7-day EDS assessemnt in CD-1 mice at 10 and 100 mg/kg/day hERG (IC50) Weak activity (27% inhibition at 10 M) Ames Negative

PHARMACOKINETICS Monkeys iv 2 mpk CL =25 mL/min/kg Vss = 0.95 L/kg; T1/2= 0.6 h po 10 mpk (10% TPGS) AUClast = 35 h*ng/mL F = 0.4% Cmax = 6 ng/mL

METABOLISM Stability (DSM) Hepatocytes CLint (µL/min/10^6 cells): CD-1 Mouse(LM)/Rat/ dog/ monkey/ human 8/4.0/4.6 / 63 / 9.4

CYP450 Inhibition DSM IC50 (µM) 1A2: NI 2A6: NI 2C9: 4 2C19: 24 2D6: 47 3A (MDZ): NI (TST): 56 Not a MBI DDI: possible for CYP2C9 Induction: Reporter assay at 2 uM: 2.4 fold (CYP3A4), 3 fold (CYP1A2)

L. Leung May 13, 2009


The Bioanalytical Center • To establish an International Bioanalytical Center. • To provide superior quality analytical data with

unsurpassed turn-around time to support Preclinical and

Clinical Research and to enhance Life Sciences Research

.

• Create highly versatile scientific community

• Support and Advance e.g. Medical and Clinical studies,

Academic research, Analytical research, and

• Drug Discovery and Development

• Economically highly rewarding

• Generate Steady Income to self support


Requirements List and Inventory

The appropriate physical and material infrastructures are

required to support the Laboratory.

Construction must include:

i-Electric outlets, electric circuit distributors and breakers as specified by the Vendors

ii- Appropriate air condition capabilities specified by the ms

vendor

iii-Independent IT closed circuit iv- Un-interrupted water supply, and distilled water supply

v- Assemble the proper exhaust system for personnel safety

and efficient function of instruments

vi- Acquisition of the most sophisticated mass spectrometers vii- Furnishing the wet labs with the efficient hoods, freezers

(-20C and -70C) and refrigerators

viii- Liquid handle instruments for speed and efficient automated sample preparation, the 96-well sample preparation instrument


Personnel • Seek and hire the highest qualified Egyptian scientists.

Obtaining competent and experienced staff is crucial to the

initial success and maintenance of this effort.

• Hands-on Training of the Director, Operators and Support

Engineer on the basic instrument operation,

• The need to build upon a foundation of efficient and

committed staff is two fold:

• For this organization to be viable it must have a successful launch. Establish itself in the shortest time possible and

committed to the highest international standards.

• Hiring staff that is unqualified and inexperienced will

necessitate the training the scientists (after installation and

sign-off the specification of the instruments, it is costly

($2000/day) to call the vendor technician, time consuming,

lead to operational interruption and instability of the operation.

• Training the Quality Assurance Officer and start preparing the

required SOPs. ALEX: 29 November 2017 15

THANK YOU


Documents

Application in Preclinical and Clinical Research Stages An ... · 3 Drug Metabolism Strategic Advantages Organizational Structure Why it works: –Leadership. –Preclinical regulatory