This document provides an overview of in vitro ADMET (absorption, distribution, metabolism, excretion, toxicity) assays that are used during drug discovery and development. Key points:
- In vitro assays are designed to mimic what happens to a compound in vivo and provide early data on absorption, distribution, metabolic transformations, potential toxicity, and more.
- Common assays examine solubility, permeability, protein binding, metabolic stability, metabolism pathways, toxicity, and effects of transporters and drug-drug interactions.
- The data generated from these assays are used throughout the drug development process to inform compound selection, design better candidates, and identify liabilities early. Understanding a compound's properties helps optimize the likelihood of success
Drug discovery take years to decade for discovering a new drug and very costly
Effort to cut down the research timeline and cost by reducing wet-lab experiment use computer modeling
Others have done the work. Some have used the work. I have spoken only on behalf of their behalf.
molecular docking its types and de novo drug design and application and softw...GAUTAM KHUNE
This ppt deals with all the aspects related to molecular docking ,its types(rigid ,flexible and manual) and screening based on it and also deals with de novo drug design , various softwares available for docking methodologies and applications for molecular docking in new drug design
In silico drug designing is the drug design which can be carried out in silicon chip,i.e., within computers. The slides are helpful to know a brief description about in silico drug designing.
ADMET properties prediction using AI will accelerate the process of drug discovery.
This slide mostly focuses on using graph-based deep learning techniques to predict drug properties.
Drug discovery take years to decade for discovering a new drug and very costly
Effort to cut down the research timeline and cost by reducing wet-lab experiment use computer modeling
Others have done the work. Some have used the work. I have spoken only on behalf of their behalf.
molecular docking its types and de novo drug design and application and softw...GAUTAM KHUNE
This ppt deals with all the aspects related to molecular docking ,its types(rigid ,flexible and manual) and screening based on it and also deals with de novo drug design , various softwares available for docking methodologies and applications for molecular docking in new drug design
In silico drug designing is the drug design which can be carried out in silicon chip,i.e., within computers. The slides are helpful to know a brief description about in silico drug designing.
ADMET properties prediction using AI will accelerate the process of drug discovery.
This slide mostly focuses on using graph-based deep learning techniques to predict drug properties.
Introduction to proteomics, techniques to study proteomics such as protein electrophoresis, chromatography and mass spectrometry and protein database analysis, case studies derived from scientific literature including comparisons between healthy and diseased tissues, new approaches to analyse metabolic pathways, comprehensive analysis of protein-protein interactions in different cell types.
Biomarkers – in Toxicology and Clinical Researchsuruchi71088
A small presentation on growing use of Biomarkers in the field of toxicology and Clinical Research... basically use of various types of bio-markers and its role in drug development process...
ADME – A Key To An Effective And Safe Drug – Selvita.pdflizseyi
ADME is an acronym used in pharmacology. It stands for Absorption, Distribution, Metabolism, and Excretion. In short, these are the processes that take place in our body in the context of foreign substances, including drugs. It is how drugs are absorbed, transported around our body, metabolized, and excreted that affects whether a drug is effective (reaches its destination) and safe (does not cause side effects).
Introduction to drug metabolism case studies for its impacts on drug discover...SAPA-GP
2014/10/02 SAPA-GP Webinar:
Introduction to drug metabolism case studies for its impacts on drug discovery and development
Zhoupeng Zhang
Dept of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism
Merck Research Laboratories
Sino-American Pharmaceutical Professionals Association (SAPA)
– A lecture for Medicinal Chemists
(October 2, 2014)
Metabolomics-Introduction, metabolism, intermediary metabolism, metabolic pathways, metabolites, metabolome, metabolic turnover, techniques used in metabolomics, metabolite profiling methods, data analysis, metabolomic resources, role of metabolomics in system biology.
Ohio State's ASH Review 2017 - Myeloproliferative DisordersOSUCCC - James
Katherine Walsh, MD
Assistant Professor of Clinical Internal Medicine
The Ohio State University Comprehensive Cancer Center -
Arthur G. James Cancer Hospital and Richard J. Solove Research Institute
Ohio State's ASH Review 2017 - Blood and Marrow TransplantationOSUCCC - James
Basem M. William, MD, MRCP(UK), FACP
Assistant Professor of Internal Medicine
Blood and Marrow Transplant Program
The Ohio State University Comprehensive Cancer Center -
Arthur G. James Cancer Hospital and Richard J. Solove Research Institute
Ohio State's ASH Review 2017 - Benign HematologyOSUCCC - James
Spero R. Cataland, MD
Professor of Clinical Internal Medicine
The Ohio State University Comprehensive Cancer Center -
Arthur G. James Cancer Hospital and Richard J. Solove Research Institute
Ohio State's ASH Review 2017 - Update in MyelomaOSUCCC - James
Don M. Benson Jr., MD, PhD, FACP
Associate Professor of Medicine
Head of Translational Research
Division of Hematology
The Ohio State University Comprehensive Cancer Center -
Arthur G. James Cancer Hospital and Richard J. Solove Research Institute
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Cancer cell metabolism: special Reference to Lactate Pathway
In Vitro ADMET Considerations for Drug Discovery and Lead Generation
1. IN VITRO ADMET CONSIDERATIONS
FOR DRUG DISCOVERY AND
LEAD GENERATION
28 March 2017
James Drug Development Institute Symposium
Ohio State University
EVERY STEP OF THE WAY
EVERY STEP OF THE WAY1
2. Overview
• Overview of In Vitro ADMET (ADME/Tox; DMPK)
• Attractive Compound Properties
• Approaches for Obtaining In Vitro Data
• Metabolic Transformations
• Permeability (Transport, Efflux)
• Protein Binding
3. In Vitro Assays to Mimic In Vivo
In vitro assays are designed to mimic fate of compound in vivo
(for oral pill, usually preferred)
• General aqueous solubility
• Absorption from gut into bloodstream
• Binding to plasma proteins and RBCs (free drug fraction
for efficacy)
• Degradation in bloodstream or tissue
• Metabolism (liver, GI tract, etc.)
• Possible drug-drug interactions (drug as substrate or
inhibitor of enzymes, transporters)
• Possible toxicity to cells
• Excretion/elimination
4. Uses of In Vitro Metabolism (ADMET) Data
ADMET of a drug = Absorption
Distribution
Metabolism
Excretion
Toxicity
Used at many stages of drug discovery/development process:
Compound library design
Prioritizing screening “hits” and chemotypes during lead generation
Lead optimization
Compound selection for in vivo studies
IND-enabling studies (even NDA)
Development of in silico (computer) models
Key = eliminate “dud” compounds unlikely to succeed (drug
discovery/development is like very high-stakes legalized gambling)
6. How In Vitro ADMET Assay Data Help Early Drug Discovery
Assay Deliverable What question does assay answer?
Aqueous solubility Limit of solubility (µM) Is compound soluble? Soluble under assay conditions?
Octanol/water partitioning LogD How hydrophobic is the compound?
Plasma stability % remaining or T1/2 How rapidly is compound degraded in plasma/blood?
Metabolic stability % remaining or T1/2 How rapidly is compound metabolized?
Permeability Papparent, efflux ratio Is compound likely to be absorbed from gut into bloodstream?
Protein binding % free or % bound or Kd Is compound highly protein bound?
Tissue binding % free or % bound or Kd Is compound highly tissue bound?
RBC partitioning (blood/plasma ratio) Blood/plasma ratio, Kp
CYP450 inhibition % inhibition or IC50 or Ki Will compound have drug-drug interaction issues?
CYP450 induction Fold induction Will compound have drug-drug interaction issues?
Metabolite profiling/ID Masses found over time What does compound get metabolized to?
CYP450 reaction phenotyping T1/2 +/- inhibitors Which CYP450(s) metabolize compound?
Hemolysis (blood compatibility) % lysis Does the compound lyse RBCs?
Melanin binding % bound, Kd, Bmax Is the compound likely to accumulate in pigmented tissues?
Cell viability % inhibition or IC50 Does compound kill cells?
Cell proliferation % inhibition or IC50 Does compound interfere with normal cell growth?
HERG inhibition % inhibition or IC50 Is HERG-related cardiotoxicity a potential problem?
Ames/genotox Does compound cause genetic mutations?
7. Example of Compound Properties “Sweet Spots”
[Personal opinions, no single industry-accepted criterion; always exceptions]
• Overall goal is selective, potent & safe in target, cell-based, in vivo models
• Lipinsky’s rules
• High aqueous buffer solubility (e.g., >100 µM)
• High permeability (Papp>1E-06cm/sec), low efflux (<2)
• IC50 >1µM for multiple CYP450s (potent 2D6 and 3A4 inhibition could be an issue)
• Stability in plasma
• Moderate metabolic T1/2 (~1 hr LMs), hepatic clearance (CLint, CLhep)
• Moderate plasma protein binding (~70-90%), preferably not just to AGP
• No unique human metabolite
• Patentable
• Ease of chemical synthesis & kg scale up
• Other intangibles (no bad smell, color, etc.)
8. In Vitro ADMET Assay Details/Parameters
Non-GLP is fine (not required to be GLP)
Cytochrome P450 inhibition
• Drug-drug interactions (DDIs)
• 7 Major human isozymes (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4)
• Drug substrates (typically LC/MS/MS detection vs. fluorescence)
• Time-dependent inhibition (TDI; IC50 shift +/- 30 min pre-incubation)
Metabolic stability (% remaining)
• Microsomes (liver, intestinal) and S9 (multiple species)
• Hepatocytes (multiple species, cryopreserved)
Matrix stability (plasma, serum, blood, buffer, SIF, SGF)
Cytochrome P450 and UGT reaction phenotyping
• Selective chemical inhibitors or enzymes to determine which enzymes involved
Protein binding:
• Equilibrium dialysis (e.g., Thermo RED device)
• Ultra-centrifugation (500,000 x g for 2.5 hours)
• Ultra-filtration (has non-specific binding problems)
Tissue binding
• Tissue homogenates (e.g., brain, kidney, tumor, etc.)
9. In Vitro ADMET Assay Details (cont.)
RBC partitioning (blood/plasma ratio)
• To determine if compound binds RBCs vs. stays in plasma
Melanin binding (synthetic melanin pigment)
CYP450 induction
• Drug-drug interactions (DDI)
• Measures if compound increases CYP450 enzyme activity or mRNA levels in hepatocytes (flip
side of CYP inhibition)
Permeability (Caco-2, MDCK, MDCK-MDR1)
• Tests if compound can be absorbed or transported across intestinal cell layer, or is effluxed
(e.g., by Pgp) out of cell (“escort” vs. “bouncer”)
Aqueous solubility
• Kinetic: measures turbidity using plate reader (cloudy solution)
• Thermodynamic: equilibrium (overnight), measure concentration by LC/MS/MS
Metabolite profiling/ID (LC/MS/MS using Q-Trap or high-resolution instrument)
Hemolysis (blood compatibility)
LogD (octanol/water partitioning)
• Measures how greasy a molecule is (lipophilic or hydrophilic)
Cell viability (cytotoxicity)
• Typically fluorescent or absorbance readouts (plate reader)
• Generic cell lines (e.g., L929, HepG2, HEK293, HeLa, Hepatocytes)
10. In Vitro Study Strategies, Considerations
In the early discovery stages, minimize costs & time (e.g., Excel results
should be fine; don’t need to pay for Word study reports until later stage).
Simplest assay is most cost-effective; only pay for needed options:
• Single time point vs. T1/2; single compound concentration vs. IC50
• Can be helpful to do bi-directional permeability (to assess efflux);
concurrent matrix stability control
• Testing compounds side-by-side gives more robust comparisons
• Standard curves not needed for early stage discovery
• Identify and focus on critical path experiments (avoid “interesting” or
“bells and whistles” experiments); try to prioritize/kill compounds early.
11. What to Consider re. Internal vs. Collaborator vs. CRO Lab
• Who already has assay established and is experienced?
• Are you going to run assay routinely or once-in-a-while?
• Need fast initiation and turnaround time (3-5 days) to keep lead
optimization cycles short
• Client responsiveness, attention to detail
• Direct access to scientist/Study Director for suggestions and questions
• If outsourced, need competitive price (~mid-point, not cheapest)
• “Good, fast, cheap – pick 2!”
• “You get what you pay for”
12. Drug-Drug Interactions
• One drug can interfere with metabolism of another drug via:
• Competition for metabolic pathway (inhibition)
• Up-regulation of metabolic enzymes (induction)
• Effect of transporters or efflux pumps
• The FDA and EMA offer guidance for metabolism data needed for regulatory submission (IND)
13. Types of Metabolic Transformations
Types of metabolic transformations:
• Phase 1 (oxidation, hydrolytic, reductive)
• Usually more hydrophilic
• Enzymes: Cytochrome P450s, aldehyde
oxidase (AO), xanthine oxidase (XO),
monamine oxidases (MAOs), flavin-
containing monooxygenases (FMOs)
• Phase 2 (Conjugative: glucuronidation, sulfation,
acetylation, methylation, glutathione, amino acid)
• Usually more polar and less toxic
• Enzymes: UGTs, SULTs, GSTs, NATs
• Metabolites may possibly be more potent or
toxic than parent
• CYP and UGT levels can vary dramatically
between patients, populations (polymorphisms)
Description Mass Shift (amu)
Oxidation (Phase 1)
N-dealkylation
Deethylation -28
Loss of water -18
Demethylation -14
Dehydrogenation -2
Oxidative deamination +1
Hydrogenation +2
Hydroxylation/N,S-oxidation +16
Hydration +18
Di-oxidation +32
Acetylation +42
Conjugation (Phase 2)
Glycine +57
Sulfation (sulfonation) +80
Taurine +107
Cystein conjugation +121
Glutamine +145
N-acetyl-cysteine conjugation +163
Glucuronidation +176
Oxidation with O-glucuronidation +192
Glutathione conjugation +305
14. Importance of Metabolic Transformations
• Acetaminophen (Tylenol, APAP, paracetamol; metabolite of phenacetin which was withdrawn due to
nephrotoxicity and carcinogenesis); typical dose 1-2 g/day, max 4 g/day.
• Mainly glucuronidated and sulfated before elimination in bile.
• At >7 g/day (in adult; >150 mg/kg in child), phase 2 pathways are saturated and phase 1 (CYP450
3A4 and 2E1) pathways generate NAPQI (N-acetyl-p-benzoquinone imine) via hydroxylation and
dehydration. NAPQI is a nephrotoxic and hepatotoxic metabolite.
• NAPQI can be detoxified by glutathione pathway, but pathway can be overwhelmed.
• Acetaminophen overdose is most common drug-related toxicity reported to poison centers, and is
main cause of acute liver failure in U.S. Lethal dose is 10-15 g. Chronic alcohol consumption
depletes glutathione levels.
15. Microsomal Stability: Example of Species Differences
SPECIES PROFILING OF IMIPRAMINE
HEPATIC MICROSOMAL METABOLIC STABILITY
-10
0
10
20
30
40
50
60
70
80
90
100
-15 0 15 30 45 60 75 90
INCUBATION TIME (min)
%TestCompoundRemaining
RAT
MOUSE
DOG
GUINEA PIG
CYNO. MONKEY
RHESUS MONKEY
HUMAN
SPECIES PROFILING OF
HEPATIC MICROSOMAL METABOLIC STABILITY
0
20
40
60
80
100
Verapamil Testosterone Labetalol Imipramine
%TestCompoundRemaining@15minutes
RAT
MOUSE
GUINEA PIG
DOG
CYNO. MONKEY
RHESUS MONKEY
HUMAN
• Metabolic rates can vary dramatically between species
• In vitro species comparisons facilitate selection of appropriate animal
models
16. Metabolite Mass Monitoring
• Leverages metabolic stability assays
• Can concurrently monitor level of parent + putative metabolite masses vs. time
• Detect common and abundant phase 1 (NADPH-dependent; oxidation, dealkylation) or
phase 2 (glucuronidation, sulfonation) metabolites
18. Permeability (Caco-2 and MDCK)
Caco-2
• Human colon cancer cell line
• Takes 21 days to grow before ready to use in assay
• Has full range of human transporters (including Pgp, BCRP, MRP2, etc.)
• Test unidirectional (A-B, SDL-1) or bidirectional (B-A/A-B, efflux ratio, SDL-2)
• Multiple control drugs [warfarin (high), ranitidine (low), talinolol (efflux)]
• Deliverable: default Excel summary; Papp and/or efflux ratio
MDCK & MDCK-MDR1 [MDR1 = Pgp = P-glycoprotein]
• Dog cell line (mimics human Caco-2; Madin-Darby Canine Kidney)
• Takes only 5-7 days to grow before ready to use in assay
• MDCK wild type has full range of dog transporter enzymes
• MDCK-MDR1 transfected cell has full range of dog transporter enzymes plus human MDR1 (Pgp)
To study human Pgp involvement, options are: 1) Caco-2 ± Pgp inhibitor; or 2) MDCK-MDR1 ± Pgp
inhibitor; or 3) MDCK wild type vs. MDCK-MDR1.
19. Pgp (MDR1): Caco-2 vs. MDCK-MDR1
Assay:
Apical side (A)
Basolateral side (B) Caco-2
(human)
hPgp
Drug 1 Drug 2 Drug 1 Drug 2
hPgp dPgp
MDCK-MDR1
(dog with human)
20. Protein Binding: Equilibrium Dialysis Approach
• Protein binding can help interpret differences in potency between target and cell-
based assays (e.g., presence of 10% FBS)
• A white paper will be released for protein binding, but may not have many specifics
• Pierce Teflon RED (Rapid Equilibrium Device) unit or HTDialysis Teflon plate
• Plasma, serum, whole blood, pure proteins (HSA, AGP)
• Multiple species, anticoagulants
• Steps: Spike test article into inner dialysis tube; dialyze for 6 hours at 37°C with
shaking in CO2; matrix-match aliquots from inner and outer chambers; determine
levels by LC/MS/MS
• Calculate % Free, % Bound
Plasma to Plasma Equilbrium Ratio
versus
Percent Plasma Protein Bound
Percent Plasma Protein Bound
0 20 40 60 80 100 120
PlasmatoPlasmaEqulibriumRatio
0
10
20
30
40
50 20 hours of dialysis
Equilibrium Ratio Issues?
21. Protein Binding: Ultracentrifugation Approach
• Advantages:
• Doesn’t rely on equilibrium
• Shorter exposure time to plasma (2.5 hours)
• Smaller sample volume
• Cons:
• % bound lower than expected (but more accurate)
• Not automatable
• Ignores binding to “floating” chylomicrons and
lipoproteins
• Steps: Spike test article into matrix (plasma, etc.);
separate protein-bound compound from free by
sedimenting plasma proteins via approx. 500,000 x g for
2.5 hours, 37°C; aliquot supernatant (free drug) below
lipid layer; matrix-match samples; determine levels by
LC/MS/MS
• Calculate % Free, % Bound
Beckman table-top ultracentrifuges
Centrifuge
BoundDrugFreeDrug
Total:Drug:PlasmaMixture
Plasma
Water
Centrifuge
BoundDrugFreeDrug
Total:Drug:PlasmaMixture
Plasma
Water
23. Cytochrome P450 Reaction Phenotyping
DEXTROMETHORPHAN
Reaction Phenotyping
Incubation Time (min)
0 15 30 45 60 75 90 105 120 135 150 165 180
%ParentRemaining(±NSD)
0
20
40
60
80
100
Control A
Furafylline/CYP1A2
Omeprazole/CYP2C19
Ketoconazole/CYP3A4
Quinidine/CYP2D6
Sulfaphenazole/CYP2C9
Tranylcypromine/CYP2A6
Control B
MIDAZOLAM
Reaction Phenotyping
Incubation Time (min)
0 15 30 45 60 75 90 105 120 135 150 165 180
%ParentRemaining(±NSD)
0
20
40
60
80
100
Control A
Furafylline/CYP1A2
Omeprazole/CYP2C19
Ketoconazole/CYP3A4
Quinidine/CYP2D6
Sulfaphenazole/CYP2C9
Tranylcypromine/CYP2A6
Control B
HYDROXY-MIDAZOLAM
Formation
C
ontrolAFU
R
A
/C
Y
P
1A
2
O
M
E
P
/C
Y
P
2C
19K
E
TO
/C
Y
P
3A
4Q
U
IN
/C
Y
P
2D
6S
U
P
H
/C
Y
P
2C
9TR
C
Y
/C
Y
P
2A
6
C
ontrolB
MetaboliteFormed@15min
(%ofControl±NSD)
0
20
40
60
80
100
.
DEXTRORPHAN
Formation
C
ontrolAFU
R
A
/C
Y
P
1A
2
O
M
E
P
/C
Y
P
2C
19K
E
TO
/C
Y
P
3A
4Q
U
IN
/C
Y
P
2D
6S
U
P
H
/C
Y
P
2C
9TR
C
Y
/C
Y
P
2A
6
C
ontrolB
MetaboliteFormed@120min
(%ofControl±NSD)
0
20
40
60
80
100
.
• Determines which CYP450 isozyme(s) involved with test article metabolism and/or metabolite formation.
• Perform metabolic stability assay ± specific CYP450 inhibitors
24. Summary of Protein Binding Approaches
• Equilibrium Dialysis (RED):
• Best known method and widely accepted method
• But may over-estimate % bound for highly bound compounds due to lack of equilibrium.
• Not suitable for compounds unstable in matrix (RED assay takes 4-24 hours)
• Ultracentrifugation (UC):
• Well known and accepted method
• Most suitable for highly bound compounds and/or compounds with limited stability in matrix
(UC assay only takes 2.5 hours)
• Ultrafiltration (UF):
• Easy method to perform
• Very significant non-specific binding (NSB) problems and other limitations
25. 1. Equilibrium dialysis approach
• Free drug is separated from protein-bound drug by dialysis membrane (e.g., 6-8K
MWCO)
• Plasma, serum, whole blood, purified proteins
• Multiple species, anticoagulants
• Method:
• Test article is spiked into plasma in “donor” side
• Dialyzed against buffer (“receiver” side) for 6+ hours at 37°C with agitation
• Aliquots from each side are sampled and matrix-matched
• Concentrations determined by LC/MS/MS
• Deliverable = % protein bound [= 100% - (Free/Total)]; also % recovery, equilibrium
assessment can be checked
27. Pros/cons of equilibrium dialysis approach
• Pros
• “Gold standard” and well-known, accepted
• Newer, easy-use apparatus commercially available
• Lower NSB (Teflon)
• Lower volume required
• Higher throughput (automatable)
• Cons
• Long equilibration times (even >24 hours?)
• Potential matrix stability issues
• Potential volume shifts
• May need dialyzed matrix for matrix matching
28. Is equilibrium dialysis really at equilibrium?
• If % plasma protein binding is
high, need to check for
equilibration
• Very high affinity
• Slow off-rate
• Irreversible binding
• Small differences in % bound
values can be large differences in
% free:
• 99.5% vs. 99.9% bound
equates to a 5-fold difference
in free concentration
Plasma to Plasma Equilbrium Ratio
versus
Percent Plasma Protein Bound
Percent Plasma Protein Bound
0 20 40 60 80 100 120
PlasmatoPlasmaEqulibriumRatio
0
10
20
30
40
50 20 hours of dialysis
Equilibrium Ratio Issues?
29. 2. Ultracentrifugation approach
• Advantages:
• Doesn’t rely on equilibrium
• Shorter exposure time to plasma (2.5 hours)
• Steps:
•Spike test article into plasma (serum, purified proteins)
• Separate protein-bound compound from free by sedimenting plasma proteins
by ultracentrifugation
• Approx. 500,000 x g for 2.5 hours, 37°C
• Free drug is in supernatant below lipid layer
• Matrix-match samples +/- centrifugation; determine concentrations by
LC/MS/MS
• Deliverable = % protein bound, % recovery
Beckman TL100
table-top
ultracentrifuge
30. Ultracentrifugation approach
• Pros
• Short plasma exposure, “equilibration” time
• Simple approach
• Minimizes recovery/stability issues
• Limits aqueous solubility, NSB issues
• Moderate sample volume (2 ml)
• Minimal sample handling
• Cons
• % Bound values may be lower than expected (but more accurate!)
• Very slight residual (small) plasma proteins/peptides in supernatant
• Ignores binding to “floating” chylomicrons and lipoproteins
• Dynamic protein concentration during sedimentation
• Needs expensive ultracentrifuge equipment
Centrifuge
BoundDrugFreeDrug
Total:Drug:PlasmaMixture
Plasma
Water
Centrifuge
BoundDrugFreeDrug
Total:Drug:PlasmaMixture
Plasma
Water
31. Ultracentrifugation method an accepted approach
• “Indeed, our technique accurately determined the plasma protein binding ratios of a wide range of
compounds and could be used to evaluate protein binding kinetics.”
“…our results indicate the reliability of this micro-scale ultracentrifugation technique for the
evaluation of the protein binding of drugs….” (Nakai 2003 J Pharm Sci 93, 847)
• “The UC method was confirmed to be comparable with the ED method in terms of reliability and
rather superior in terms of reproducibility, especially at low drug concentrations.” (Yasuo 2008
Chem Pharm Bull, 2948)
• Extensively used at several large pharmas
32. 3. Ultrafiltration approach
• Separation of bound drug from free drug using UF membrane
• Pros
• Rapid equilibration time (minimal recovery/stability issues)
• New methods/apparatus (improved NSB)
• Lower volume required
• Higher throughput
• Cons
• Significant NSB (particularly for hydrophobic compounds)
• Oncotic issues during concentration of protein