Module 1: Toxicokinetics (ADMMET)

what is toxiokinetic?

what the body does to a chemical

* study of mvmt of cmpd w/in the body ie ADME

concentration time curves

* determine administration route

what is toxicodynamics?

what the chemical does to the body

* study of pharm/tox actions of a cmpd

first order kinetics

* linear

• constant fraction of drug is eliminated/time (1/4 each time)

• amount eliminated is proportional to amount in body

• elimination is NOT saturated

• most drugs eliminated this way

zero order kinetics

* non-linear

• constant amount of drug is eliminated/time (8 mg)

  • rate is not proportional to concentration or dose

• w/inc dose → saturation occurs

kinetic models

• empirical models: if limited info

• compartmental models

  • groups all tissues into 1 or more compartments

  • well stirred

  • based on linear assumptions using linear differential eqns

• physiological based pharmacokinetic model: based on known anatomic/phsyio

1 compartment model

• instantaneous distribution = all tissues behave the same

• one slope

• 1 compartment of blood & highly perfused tissues → drug well represented is hydrophilic

2 compartment model

• central & peripheral compartment (poorly perfused tissues ie fat)

• 2 slopes = 2 compartments

  • drug well-represented is small, non-ionized

• plasma > peripheral tissue distribution

3 compartment model

• bone & adipose tissue

• perfusion limited

• slow to accumulate/slow to release → not clinically significant but can be more toxicity

• 3 slopes = 3 compartments

• plasma > peripheral tissue > deep compartment distribution

Cmax & Tmax

max measured concentration & time of max concentration

Area under the Curve (AUC)

measure of total systemic exposure (how much actually got into circulation from dose)

bioavailability (F)

fraction of cmpd reaching systemic circulation intact

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F = AUCext/AUCiv \* Doseiv/Doseext

poor oral bioavailability reasons

• poor drug absorption from GIT

  • cmpd is ionized at all pH of GIT

  • binds to food

  • unstable at stomach pH

  • gut microflora metabolize

  • cmpd by metabolic enzymes

  • Pgp efflux

  • cmpd doesn’t dissolve

  • poor blood flow at site of administration

• hepatic 1st pass metabolism

volume of distribution

apparent vol w/in which a drug is distributed in the body

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Vd = dose/C0 = mg drug/Cp (recall dec w/PPB inc)

C0= y-intercept

total body clearance (CL)

• vol of blood completely cleared of drug

• indicates rate at which cmpd is removed from plasma

CL = dose/AUC

elimination half life (t1/2)

time for plasma conc to decline by 1/2

• time course of drug elimination → done is ~5 half-lives

• time course of drug accumulation

t1/2 = 0.693/Kel = (0.693*Vd)/CL

multiple exposures & steady state

multiple exposures → leftovers → accumulations → input=output → depends solely on elimination t1/2

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steady-state = 4-5 elimination t1/2

factors that can alter kinetics

• age

• chemical interactions: PPB, metabolism

• disease

• pharmacogenomics

elimination

• metabolism

• excretion

removal of a cmpd from plasma → dec in conc

• cmpd is chemically transformed into metabolites

• process by which a cmpd is eliminated from body w/o a chemical change

routes of elimination

• renal

• non-renal?

• feces (bile, 2nd most common way)

• sweat

• lungs

• saliva

• milk

kidney fxn

• cleansing of blood

• regulation & maintenance of fluid/chemical balance

• production of urine to remove waste

nephron flow of filtration

blood enters from renal arteries into afferent arterioles → glomerulus capillaries → in efferent arterioles which surround tubules → bowman’s capsule (filtering) → PCT → LOH → DCT → CD → reabsorption into peritubular capillaries

total renal elimination = ?

* flow of glomerular filtration? secretion? reabsorption?

filtration rate + secretion - reabsorption = ?

• glomerular filtration = blood from afferent arteriole to lumen of tubules

• secretion = blood from peritubular capillaries to lumen

• reabsorption = tubular lumen to blood from peritubular capillaries

glomerular filtration

• substance MW that are freely filtered?

• drive by ?

• GFR?

• ~25% CO reaches glomerulus

• MW < 5,000 are freely filtered

  • 70,000 are not

• GFR = vol of plasma filtered by kidneys/min

if a molecule is bound to albumin it won’t filter through glomerulus, why?

serum albumin MW = 69,000

active tubular secretion

• mvmt from blood to tubular fluid

• active transport process

• ionized drug

• not usually affected by PPB

how does addition of Probenecid to Penicillin prolong therapeutic effect?

active tubular secretion is slowed bc Probenecid and Penicillin are competing for the same transporter → Abx is cleared slower

passive tubular reabsorption

• depends on ?

• effect of charged/uncharged?

• conc gradient

• urine flow

• lipid solubility (Kow)

• urine pH & ionization

  • drugs

  • pathophysio

  • diet

    • carnivores (pH 5.5-7)

    • herbivores (7.5-10.5)

  • uncharged → chance for reabsorption

  • charged/ionized → stays in tubules to excretion

urinary pH & excretion of drugs - ion trapping

recall if pKa < pH → more H+ in soln → molecule is ionized → more likely to be eliminated

renal clearance & lipophilicity & ionization

• inc lipophilicity → dec renal CL

  • → inc passive reabsorption

  • → inc PPB

• uncharged → dec renal CL

  • → inc passive reabsorption

• charged → trapped in urine

t/f: cmpds which are weak acids are excreted faster when the urinary pH is more alkaline as compared to acidic.

true

factors affecting renal CL

• urine pH, pKa of drug

• urine flow rate

• physiochemical properties of drug

• blood flow to kidneys

• disease states

• drug interactions

renal clearance (CLr)

rate at which plasma is cleared of cmpd by kidney

filtration only: CL = Fu\*GFRplasma

active secretion > reabsorption: CL > Fu\*GFRplasma

reabsorption>active secretion: CL > Fu\*GFRplasma

t/f: the most rapidly eliminated cmpds are those w/high GFR & that are actively secreted but aren’t passively reabsorbed.

true

fecal elimination/excretion

• direct elimination of non-absorbed cmpds

• efflux pumps

• via bile

bile

• made in liver/stored in gallbladder

• functions

  • facilitate intestinal absorption of ingested lipids

  • major route for cholesterol elimination

  • elimination route drugs

biliary elimination of drugs

• predominantly cmpds w/high polarity/some lipophilicity

• MW >325; >500 most favorable)

• glucoronide or GST conjugates

• active transporter: drug-drug interactions (DDI)

enterohepatic recycling

from liver/gallbladder → common bile duct → small intestine → hepatic portal vein → liver

• exists to recycle bile

• may prolong drug t1/2

what processes does phase 2 metabolism include? what is the purpose?

• sulfation**

• glucoronidation**

• glutathione conjugation ****

• acetylation

• amino acid conjugation

• methylation

inc hydrophilicity & tagging

t/f: phase II conjugation enzymes are ‘faster’ than phase I CYPs.

true, so Phase I is generally the rate limiting step of elimination

phase II conjugation rxns occurs mainly in which organelles? which main organ?

• cytosol, S & R ER

• liver, but also skin, intestine, brain, spleen, nasal mucosa

Glucoronidation

• enzyme

• what is being added? from what cofactor?

• purpose

• UDP-glucornosyltransferases (UGTs)

• glucuronic acid from uridine-diP (UDP) gluconic acid to lipophilic drug

• the sugar acid moiety of glucuronides makes cmpd more polar + water-soluble

liver microsome assay

for any new drug → mix w/liver microsomes & do mass spec → can ID metabolites to see which transferases involved

UGT families and substrates

* nomenclature

UGT1A1 (UGT + family + sub-family + isoform)

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UGT1A8 = androgens, morphine

UGT1A9 = ibuprofen, estradiol

UGT2B7 = NSAIDs, morphine

morphine

• skips what phase?

• what specfic enzyme?

• _ gluconoridation → 2 products, which one has analgesic potency?

• skips phase I → glucoronidated directly

• UGT2B7

• regioselective → morphine-6-glucornide has higher analgesic/pain relieving potency (made only 10% of time

  • but 90% of time: morphine-3-glucoronide → no analgesic effect

sulfotransferases (SULT)

• transfer what group? from what cofactor?

• phase I substrates?

• purpose

• sulfonate group from phosphoadenosine-phosphosulfates (PAPS)

• R-OH, -NH2, NH-OH, phenols

• makes cmpd more polar + water-soluble

SULT nomenclature

SULT + family + sub-family + isoform (same as UGT)

how does safrole (from Sassafras tree) become a carcinogenic metabolilte?

1. P450-mediated hydroxylation

2. SULT-mediated conversion to sulfate ester (unstable)

3. ester decomposes to (allyl) carbonium ion → reactive E+ → binding to G in DNA → liver carcinogen

SULT vs UGT

* how does their activity vary at high or low concentrations of drug?

• SULT are low capacity, but high affinity → work better at low doses

• UGT are high capacity, but low affinity → work better at high doses (SUGAR RUSH)

Glutathione-S-Transferase (GST)

• structure of cofactor GSH (glutathione), where is it mostly located?

• pKa depression

• GSH is tripeptide, most abundant antioxidant in cells

• GST conjugates Nu- GSH cofactor to E+ cmpds

• pKa depression = allows GST-bound GSH to act as a Nu- at a physiological pH

  • thiol group pKa = 9.5 → pKa 6 → Nu- activity at pH 7.4 (deprotonated S-)

GST nomenclature

GST + sub-family + isoform

3 main families

• cytosolic, mitochondrial, microsomal

  • cytosolic have 11 subfamilies

GSH conjugation of acetominophen (Tylenol)

• phase I metabolite, NAPQI → conjugated w/GSH → excreted

• if acetaminophen is OD’d → GST saturation → depletes GSH in liver → fatal liver toxicity (3-4days)

where does biotransformation occur?

• in body?

• in cell?

• liver = richest source for phase I & II

• primarily in S&R ER (microsomes)

  • also in cytoplasm, mitochondria, lysosomes, nuclei

a fat-soluble toxins → phase I via cytochrome P450 enzymes (CYPs) which include:

* how?

• oxidation

• reduction

• hydrolysis

• hydration

• dehalogenation

goal = conversion of hydrophobic drugs → more water-soluble via

• expose/introduce functional groups ie -OH, NH2, SH, COOH

• to inc hydrophilicity & polarity

CYP nomenclature

* how many human CYPs?

CYP + family + subfamily + isoform

• each CYP isoform comes from a different gene

what are the 6 CYPs that account for 86% of drug biotransformation?

• CYP1A2

• CYP2B6

• CYP2C9

• CYP2C19

• CYP2D6

• CYP3A4/5

what are CYP polymorphisms?

minor nucleotide sequence changes → affects dose & toxicity of drug

2 types of drug interactions w/CYPs

• inhibition

  • competitive binding

  • enzyme saturation

• induction

alicyclic oxidation

epoxide + H2O → diol

via epoxide hydrolase (or reverse: epoxygenase)

* present in all tissues

\
=> inc hydrophilicity

aliphatic oxidation

* ibuprofen ex

w-1 oxidation (2nd to last CH3) via CYP2C9 → alcohol metabolite

or

w-oxidation (last CH3) via CYP2C8 → carboxylic acid metabolite

\
both non-toxic & easily eliminated

aliphatic oxidation

* valproic acid

valproic acid

w oxidation (CYP2C19)

• 2-n-propylglutaric acid (non-toxic)

w-1 oxidation (CYP2C9)

• 4-Hydroxyvalproic acid (4-HPA) → inhibits mitochondrial fatty acid oxidation → hepatotoxic/mitotoxic

alkene epoxidation

* carbamazepine + valproic acid DDI

carbamazepine →

carbamazepine-10,11-epoxide (forms adducts w/DNA & proteins) via epoxide hydrolase →

trans-10,11-dihydroxy carbamazepine (diol)

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\*\*DDI: valproic acid inhibits epoxide hydrolases → inc carbamazepine-10,11-epoxide

\

polycyclic aromatic hydroxylation

1. procarcinogen & PAH benzo[a]pyrene (BaP) oxidized to epoxide

2. BaP epoxide gets hydrolyzed to diol

3. BaP diol gets further oxidized to the carcinogen BaP diol epoxide (BPDE)

BPDE is resistant to hydrolysis by epoxide hydrolase → forms DNA adducts

what are the 4 properties of chemicals that dictate its site of storage/toxic action?

• Kow

• pKa/pKb

• Vd

• PPB

Kow

* correlation w/bioconcentration factor

Kow = octanol/water partition coefficient

• at equilibrium → if Kow if high → high lipophilicity

• positive correlation of logBCF and logKow

BCF & Kow relationship

• hydrophilic cmpds show (-) logKow & low BCF

• logKow 1-7 → linear relationship w/BCF

• logKow > 7 → can precipitate

• why are there outliers? transporters!

pH and pKa

the smaller pKa, the stronger the acid

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* if pKa > pH → low pH, high \[H+\] → cmpd is protonated

which one will cross plasma membrane faster? which one will accumulate in fat over longer period of time?

* PCB by high logKow and not ionizable

volume of distribution (Vd)

* PPB effect on Vd

• Vd = vol in which current amount of drug in body must be dispersed to give current Cp; indicates if drug tends to stay in plasma or go to other tissues

  • Vd = mg drug in body/ mg/L drug in plasma

• high Vd = cmpd goes to extravascular compartments → higher dose req’d

• low Vd = cmpd tends to remain in plasma → lower dose req’d

• if PPB → low Vd bc cmpd stays in plasma

Vd: hydrophobic drug in fat vs skinny dog

fat dog has larger Vd bc has more fat for drug to sequester into and out of plasma → requires larger dose

how does warfarin have a high Cp but a low Kow?

PPB

effect of PPB bound to drug

ex) HSA = human serum albumin which interacts w/most drugs

cannot

• cross cell membranes

• interact w/receptor to produce effect

• be metabolized

• be excreted

absorption/administration routes

enteral

• oral, sublingual/buccal, rectal

parenteral

• IV, IM, SC, ID, IA, IP, IT

topical

• transdermal, inhalation, intranasal, intraocular, intravaginal

parenteral injection routes

directly into systemic circulation

• IM: slower absorption than IP but steady

• SC: bypasses epidermal barrier, slow

• IV: direct into circulation

intraperitoneal (IP, into peritoneal/abdominal cavity)

• GI tract, gall bladder, pancreas, spleen

• quick absorption bc high vascularization

• primarily absorbed into portal circulation (liver) & systemic circulation → cmpd needs metabolic activation in liver

t/f: if toxicant undergoes 1st pass metabolism, it will be often less toxic if administered orally than IV.

true, but does not apply for toxicants w/selective organ toxicity or bioactivated in liver

rank toxicity of absorption routes

IV > inhalation > IM/SC > dermal > IP > oral

only IP & oral are metabolized by liver/GI first

cardiovascular system of ingestion vs inhalation

ingestion

1. stomach

2. intestine

3. liver

4. R-heart

5. R-lung

inhalation

1. L-heart

   1. body tissue

   2. brain

2. R-heart

3. R-lung

t/f: toxicant cannot produce systemic injury until absorbed.

false only if toxicant doesn’t have caustic/burning properties

where are most digested fats, proteins, drugs absorbed?

small intestines

mode transport for

• weak acids/bases

• hydrophilic substances

• hydrophobic substances

weak a/b → simple diffusion (in non-ionic form)

hydrophilic → facilitated diffusion via pores/channels

• if very → active SLC transport

hydrophobic → simple diffusion

• very → absorbed into lymphatics (lacteals) via chylomicrons

lymphatics system

stomach → intestines → AVOID LIVER, lymphatic system → R-heart → lungs → L-heart → body tissue

polar vs non-polar elimination

polar

• to liver via portal vein → excreted into bile w/o entering systemic circulation

non-polar

• CM → skip liver → selective toxicity

chemosensitization to combat MDR

process where non-toxic cmpd sensitizes a cell toward uptake/effect of another cytotoxic cmpd

* ‘distracts’ drug export pump via competitive or allosteric binding → allows drug uptake into cell

competitive dye uptake assay

1. calcein-AM is non-fluorescent, cell permeable (transporter substrate)

2. intracellular esterase cleave AM → calcein

3. calcein is fluorescent, membrane-impermeable → accumulates (can’t use transporter)

• if a drug interacts w/transporter → blocks CAM export → bright cell

transport proteins abundance

solute carriers (SLCs) > channels > active transporters

types of active transporters

• ATP binding cassette type (ABC)

• secondary active - solute carrier (SLC)

• most euk ABC-transporters are efflux transporters

  • most euk SLC are uptake transporters

ABC vs SLC: which is in all kingdoms of life?

ABC-type

human ABC transporter superfamily

ABCB, ABCC, ABCG

ABC transporter nomenclature

ABC (family) + subfamily + member + isoform

types of multi-drug resistance (MDR) ABC transporters

• chemotherapy

• AbR

• insecticide resistnance

which ABC are brother in arms?

ABCB1 + ABCG2 = 1st line of defense in concert w/CYPs → work to reduce drug bioavailability

drug design to combat MDR

• inhibitors

• downregulation

• non-recognition

molecular methods to combat MDR

RNAi

• prevent transcription of transporter protein

CRISPR/Cas9

• knock out gene that encode transporter

toxic agents

• chemical

• physical

• biological

• industrial chemicals, food additives, drugs, gases

• dust, abestos, radiation, nanoparticles

• bacterial, fungal, plant, animals

toxic agent terms

• toxicants = synthetic, man-made

• toxins = biological in nature

  • venoms = injected by bite or sting

• poisons = touch, ingestion

persistent organic pollutants (POPs)

natural chemical elements or pharmaceuticals

types of assessments

• exposure

• concentration

• dose

• exposure = extent of human contact

• concentration = level or amount of substance

• dose = specified quantity of a substance administered

dose/concentration curve

potency metrics

ED50 = median effective dose in 50% of population

TD50 = median toxic dose “ “

LD50 = median lethal dose “ “

therapeutic index (TI)

range of blood levels wherein a substance is producing the desired effect w/o toxicity

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TI = TD50/ED50 (toxic dose/effective dose)

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low TI → more toxic → need to be given more frequently at lower doses (microdosing)

high TI → less toxic