Veterinary drugsSpecificity:

• Legal substances

• Large usage • Need• Treatment and prevention

• Usage in different species

Veterinary drugs: endectocides

Environment

80% of parental compound isexcreted in feces

Efficacy

Target: parasites

Meat

FatMilk

Food security

PharmacologyPharmacology

DrugDrug

AbsorptionAbsorption DistributionDistribution MetabolismeMetabolisme EliminationElimination

ToxicityToxicity EfficacyEfficacyPharm

acokinetic

Pharmacodynam

y

Many complex mecanisms

3 main famillies of actors act in synergy

PharmacokineticsFate of drugs in the host organism

Fate of drugs in the host organism

Exemple of a hepatic cell

Phase I

Cytochromes

Metabolisation

X-OH

Transferases

Transfer of gluthationGlucuronide, sulfone

Phase II X-Glu

X Xenobiotic

Transporters

Efflux

Phase III MRPMRP

X-Glu

X

X

Pgp

3 main actors3 main actors Cytochromes Phase ICytochromes Phase I

Transférase Phase IITransférase Phase II

Efflux ABC transporters Phase IIIEfflux ABC transporters Phase III

2- Mechanisms of transmembrane 2- Mechanisms of transmembrane transport of drugs – Examplestransport of drugs – Examples

Paracellular diffusionParacellular diffusion o ions, mannitol, polymersions, mannitol, polymers

Passive diffusion across lipid bilayerPassive diffusion across lipid bilayero fluoroquinolones, tetracycline (hydrophobic)fluoroquinolones, tetracycline (hydrophobic)

Diffusion through OM channels and porinsDiffusion through OM channels and porins -lactams, tetracyclins (hydrophilic, charged)-lactams, tetracyclins (hydrophilic, charged)

Facilitated diffusionFacilitated diffusiono imipenem, catechols, albomycin, albicinimipenem, catechols, albomycin, albicin

Active TransportActive Transporto aminoglycosides, cycloserine, phosphomycin, alaphosphinaminoglycosides, cycloserine, phosphomycin, alaphosphin

Vesicle Trafficking Mediated TransportVesicle Trafficking Mediated Transporto polymers, peptide hormones, targeted deliverypolymers, peptide hormones, targeted delivery

Active transportersActive transporters

Multiplicity Multiplicity Rates > passive Rates > passive Non-symmetrical Non-symmetrical (k(kinin k koutout at [S at [Sii] = [S] = [Soo])]) Saturable transport -Saturable transport - Michaelis-MentenMichaelis-Menten Inhibitable- Inhibitable- competitivecompetitive, , non-competitivenon-competitive Regulated-Regulated- inducibility & repressioninducibility & repression Tissue specific-Tissue specific- differential expression differential expression

Energy dependent-Energy dependent- active transportactive transport• primary pumps - respiration, photosyn, ATPaseprimary pumps - respiration, photosyn, ATPase• secondary transporters (coupled to Hsecondary transporters (coupled to H++, Na, Na++ etc.) etc.)

Active efflux ABC-transportersATP-Binding Cassette transporters :

ATP dependant transport

Active efflux of a large amount of substrates: ions, steroïdes, phospholipids, conjugated molecules, peptides…..drugs…

Nucleotide-binding-domains:ATPase activity

Trans-membrane domaines

ABCA (13)ABCA (13) ABCB =MDR ABCB =MDR (11)(11)

ABCC = MRP ABCC = MRP (13)(13)

ABCG (5)ABCG (5)

ABCA1ABCA1 ABCB1/MDR1ABCB1/MDR1 ABCC1 = MRP1ABCC1 = MRP1 ABCG = ABC8ABCG = ABC8ABCA2ABCA2 ABCB2 = TAP1 (RE)ABCB2 = TAP1 (RE) ABCC2 = MRP2ABCC2 = MRP2 ABCG = BCRPABCG = BCRP

ABCA3ABCA3 ABCB2 = TAP1 (RE)ABCB2 = TAP1 (RE) ABCC3 = MRP3ABCC3 = MRP3 ABCG4ABCG4ABCA4ABCA4 ABCB4 = MDR2-3ABCB4 = MDR2-3 ABCC4 = MRP4ABCC4 = MRP4 ABCG5ABCG5

ABCA5ABCA5 ABCB5ABCB5 ABCC5 = MRP5ABCC5 = MRP5 ABCG8ABCG8ABCA6ABCA6 ABCB6ABCB6 ABCC6 = MRP6ABCC6 = MRP6

ABCA7ABCA7 ABCB7 (lysosomes)ABCB7 (lysosomes) ABCC7 = CFTRABCC7 = CFTR

ABCA8ABCA8 ABCB8ABCB8 ABCC8 = SUR1ABCC8 = SUR1

ABCA9ABCA9 ABCB9ABCB9 ABCC9 = SUR2ABCC9 = SUR2

ABCA10ABCA10 ABCB11 = BSEPABCB11 = BSEP ABCC10ABCC10

ABCA12ABCA12 ABCC11ABCC11

ABCA13ABCA13 ABCC12ABCC12

lipids/cholesterollipids/cholesterol Drugs/steroïdes/Drugs/steroïdes/biliary saltsbiliary salts

Conjuguated/anionsConjuguated/anionsnucleotidesnucleotides

Sterols/lipids/Sterols/lipids/drugs drugs

Human ATP-Binding Cassette Transporters ABCMDR : multidrug resistant

Multidrog resistance transporters Multidrog resistance transporters P-gp, P-gp, MRPs, BCRPMRPs, BCRP

ATP-Binding cassette familly: ABC transporters Mediate the active efflux of xenobiotics Large specificity of substrates Involved in multidrug resistance Ubiquitus localisation

ApicalApicalABCG2ABCG2

4 Apical4 Apical5 Basolatéral5 Basolatéral

ABCC4, 5ABCC4, 5MRP 4, 5MRP 4, 5

2 Apical2 Apical1, 3 Basolatéral1, 3 Basolatéral

ABCC1,2, 3ABCC1,2, 3Multidrug rresistant proteinMultidrug rresistant protein

MRP1,2,3MRP1,2,3

ApicalApicalABCB1ABCB1P-glycoprotéineP-glycoprotéine

P-gpP-gp

Localisation Localisation cellulairecellulaire

StructureStructure secondairesecondaire

GèneGèneProtéineProtéine

Breast cancer resistant Breast cancer resistant proteinprotein

BCRP BCRP ou MXRou MXR

Substrates of P-glycoproteine

DoxorubicineAnthracyclines Daunorubicine

Epirubicine

Alcaloïdes de la vinca VincristineVinblastine

Epipodophyllotoxines EtoposideTeniposide

Taxanes PaclitaxelDocetaxel

Colchicine Actinomycine D

Agents cytotoxiques EmétineTopotecan

MithramycineMitomycine

RitonavirInhibiteurs des protéases Indinavir

Saquinavir

Rhodamine 123Hoechst 33342

Colorants Fura-2 AMAcridine

99mTc-SESTAMIBI Calcéine-AM

AldosteroneStéroides Dexamethasone

Progesterone Corticosterone

Gramicidine DValinomycine

N-Acetyl-leucyl-leucyl-norleucineNAc-Leu-Leu-norLeu-al

Peptides cyclique et linéaire NAc-Leu-Leu-Met-alLeupeptin

Pepstatine AFacteur A

Cyclosporine AValspodar (PSC 833)

VerapamilBloqueur des canaux calciques Nifedipine

AzidopineDexniguldipine

QuinidineBépridil

RéserpineAutres Morphine

BromocriptineForskoline

Ivermectine

Substrates of MRPsSubstrates of MRPs ou cMOAT ou cMOAT (canalicular multispecific organic anion transporter)(canalicular multispecific organic anion transporter)

glutathione disulfideglutathione disulfide leukotrienes (C4, D4, E4, N-acetyl-E4)leukotrienes (C4, D4, E4, N-acetyl-E4) glutathione conjugates (e.g., DNP, bromosulfophthalein, glutathione conjugates (e.g., DNP, bromosulfophthalein,

metals Sb, As, Bi, Cd, Cu, Ag, Zn)metals Sb, As, Bi, Cd, Cu, Ag, Zn) glucuronide conjugates (bilirubin, T3, p-nitrophenol, glucuronide conjugates (bilirubin, T3, p-nitrophenol,

grepafloxacin)grepafloxacin) bile acid conjugates (glucuronides and sulfates)bile acid conjugates (glucuronides and sulfates) organic anions (folates, methotrexate, ampicillin, ceftiaxone, organic anions (folates, methotrexate, ampicillin, ceftiaxone,

cefadozime, grepafloxacin, prevastatin, temocaprilate)cefadozime, grepafloxacin, prevastatin, temocaprilate)

P-glycoprotein and “multidrug P-glycoprotein and “multidrug resistance” (MDR)resistance” (MDR)

Gene of Pgp: MDR1 in humans and Gene of Pgp: MDR1 in humans and mdr1a/1b in rodents mdr1a/1b in rodents

Phenotype of multidrug resistance (MDR)Phenotype of multidrug resistance (MDR) in tumor cellsin tumor cells

Protection against xenobioticsOverexpression in cancer cells

Localisation on main epithelial barriers

Brain liver IntestineTestisKidneyPlacenta…….

Cellular localisation

Distribution between apical and basolateral pole

At cellular level Lower intracellular bioavailability of xenobiotic (drugs) Lower toxicityLower drug efficacy

In the whole animalPhysiological compounds:

•excretion of metabolites or toxins

Xenobiotics:•Lower intestinal absorption •Increase intestinal and biliary elimination •Reduces disposable fraction of the drugs•Protects the central nervous system

Summary: role of P-gp

Models

• Cells, vesicles

• Whole animal

• Parasites

ATP ADP

Methodology

ATP

NH2

COOH

1 12

ATP In

Out

SubstrateInhibitor ATPase

Tools to study MDR transporters

Proteinexpression

Drug Interactions & Drug TransportDrug Interactions & Drug TransportCinical assays in humansCinical assays in humans

Digoxin - non-metabolized substrate for PgPDigoxin - non-metabolized substrate for PgP

• Verapamil, amiodarone, and quinidine increase plasma levels, reduce Verapamil, amiodarone, and quinidine increase plasma levels, reduce renal and non-renalrenal and non-renal clearance, increase blood/brain barrier clearance, increase blood/brain barrier transport. transport. Dose adjustment may be needed in 50% of cases. Dose adjustment may be needed in 50% of cases.

• St. John's wort (Hypericum perforatum) St. John's wort (Hypericum perforatum) decreased digoxin AUC decreased digoxin AUC by 25% after 10 days treatment through induction of PgP.by 25% after 10 days treatment through induction of PgP.

HIV Protease InhibitorsHIV Protease Inhibitors

• Amprenavir clearance reduced by nelfinavir (-41%) and by indinavir (-Amprenavir clearance reduced by nelfinavir (-41%) and by indinavir (-54%), but not saquinavir.54%), but not saquinavir.

• FDA warning against Hypericum supplementsFDA warning against Hypericum supplements

Drug Resistance & ReversalDrug Resistance & Reversal MDR1 (P-glycoprotein) – drug efflux pumpMDR1 (P-glycoprotein) – drug efflux pump

• Multiple trials of multiple agents – recent efforts at inhibiting transcription Multiple trials of multiple agents – recent efforts at inhibiting transcription • Steady state digoxin therapy was established in normal healthy volunteers (1 mg then 0.125 mg/day). Steady state digoxin therapy was established in normal healthy volunteers (1 mg then 0.125 mg/day).

Initiation of valspodar (400 mg followed by 200 mg twice per day) caused immediate and progressive Initiation of valspodar (400 mg followed by 200 mg twice per day) caused immediate and progressive increases in digoxin AUC (+211%) and decreases in total body, renal, and non-renal clearance (-67%, -73%, -increases in digoxin AUC (+211%) and decreases in total body, renal, and non-renal clearance (-67%, -73%, -58%) after 5 days.58%) after 5 days.

BCRPBCRP (breast cancer resistance protein or ABCG2)(breast cancer resistance protein or ABCG2)• Inhibited by fungal toxin fumitremorgin C, but neurotoxic side effectsInhibited by fungal toxin fumitremorgin C, but neurotoxic side effects• Kol143 and other derived analogs developed inhibit BCRP, but not PgP or MRPKol143 and other derived analogs developed inhibit BCRP, but not PgP or MRP• Non-toxic in mice, increased oral availability of topotecan in miceNon-toxic in mice, increased oral availability of topotecan in mice

RFC (reduced folate carrier) - antifolate drugs (methotrexate) RFC (reduced folate carrier) - antifolate drugs (methotrexate) • Resistant leukemia cell lines were selected by stepwise dosesResistant leukemia cell lines were selected by stepwise doses• Cross resistance (>2000x) to five novel hydrophilic antifolates shownCross resistance (>2000x) to five novel hydrophilic antifolates shown• Intracellular folate levels reduced, increased requirement 42xIntracellular folate levels reduced, increased requirement 42x• Hypersensitive to hydrophobic antifolatesHypersensitive to hydrophobic antifolates• Mutations clustered in exons 2 and 3, TMD1Mutations clustered in exons 2 and 3, TMD1

Exemple of veterinary drugsExemple of veterinary drugs

Macrocyclic lactones: potent Macrocyclic lactones: potent parasiticidesparasiticides

 

Lactones macrocycliques:Lactones macrocycliques:

 

• Massive utilisation versus optimized utilisation

•1/3 of veterinary drugs are parasiticides anti-parasitaires (among them 60% are macrocyclic lactones)

• High efficacy with large spectrum: endectoparasiticide

Endectocidal Macrocyclic LactonesEndectocidal Macrocyclic Lactones

Large use of MLsLarge use of MLs

Prevention and therapy = Necessity

Emergence of resistant parasites

We must give existing active compounds the best chance to work

2 2 1 3 2 Ivermectin X = -CH2 - -CH - R = CH(CH )CH CH 3 1a 3 2 3 Abamectin B

X = -CH=CH- R 1 = CH(CH )CH CH

Doramectin X = -CH=CH- R 1 = Cyclohexyl Eprinomectin B 1a X = -CH=CH- R 1 = CH(CH 3)CH 2 CH 3 R 2 = NHCOCH 3

Moxidectin

a. Généralités et Structurea. Généralités et Structure

I. I. Macrocyclic lactones (MLs) Macrocyclic lactones (MLs)

232213

4

OO

O

R1

OO

O

O

OH

O

OO

O

O

R2H

H

13

25AVERMECTINAVERMECTIN

MILBEMYCINMILBEMYCIN

O

O

OH

O

O

H

O

H

O

H

H

NO

Plasma

Intestine

Biliairyelimination

Biotransformation

Intestinal secretion

Storage

Liver

Entero-hepatic

cycle

Adipose tissue

Efficacy

Route administrationMoleculeSpeciesPathophysiology

FecesMilk

ToxicityBrain

Pharmacokinetics of MLs

O O

O MeOH

MeO

O

O

OO

O

OO

OM e

M eO

O H

OH

Low liver biotransformationHigh P-glycoprotein interaction

P-gp and Ivermectin

Brain Intestine Overall bioavailability Parasite

Ivermectin

ATPNH2

COOH

1 12

ATP In

Ivermectin

Out

O O

OMeOH

MeO

OO

OO

O

OO

OMe

MeO

OH

OH

Pgp and ivermectin neurotoxicity

Natural model Colley

CF-1

Artificial model

Brain

Ivermectin0.2 mg/kg

Alteration of Pgp functionAlteration of Pgp function

Ivm sensitivity of colley dogs

SYMPTOMS:

•• Ataxia•• Tremors•• Mydriasis•• Coma

NeurotoxicityMurray-Grey ?

Mdr1ab -/-

Genetic disorder in Ivm sensitive Colley

Beagle of reference

tgctggtttttggaaacatgacag - - - - ctttgcaaatgcaggaatttcaagaaacaaaacttttccagttataattaatgaa Ivm Sensitive maletgctggtttttggaaacatgacag - - - - ctttgcaaatgcaggaatttcaagaaacaaaacttttccagttataattaatgaa Ivm sensitive female

tgctggtttttggaaacatgacagatagctttgcaaatgcaggaatttcaagaaacaaaacttttccagttataattaatgaaBeagle of reference

Lack of protein in tissues

Colley sensitiveBeagle

Premature Stop Codon

(Roulet et al 2003)

P-gp

Synthesis of a truncated protein of 78 aa

Deletion of 4 base pairs on P-gp gene (exon 2)

D I I N ES F A N A G I S R N K T F P VStopA L Q M Q E F Q E T K L F Q L

NN

MM

TTSensitive Colley

- - - - - - - - -- - - - - - - - -

……

Colley Deletion

Western BlotIntestine

Small Intestine

Bile

Dose (µg/kg)0

1

2

3

4

5

100 200 400

Iver

mec

tin (

µg/k

g B

W)

29

Laffont et al. 2002

Ratio intestine / bile = 5

Intestinal excretion of Intestinal excretion of ivermectinivermectin

In situ model Intestinal closed loop

Iv

erm

ectin

(ng

/ cm

/ kg

)

0

10

20

30

40

50

duodenum jejunum ileum

**

ControlVerapamil

Involvement of ABC transporters

Pharmacokinetics of ivermectin

0255075

100

0 6 12 18 24 30 36 42 48

Time (hr)

StandardPgp-/-

P-gp deficient mice mdr1ab-/-Iv

erm

ectin

con

cent

ratio

n in

pla

sma

(ng/

ml)

Verapamil in rat Verapamil in rat (Alvinerie et al, 1999)(Alvinerie et al, 1999) Quercetin in sheep Quercetin in sheep (Dupuy et al. 2003) (Dupuy et al. 2003)

Loperamide in sheep Loperamide in sheep (Lifschitz et al. 2004)(Lifschitz et al. 2004)

Verapamil in sheep Verapamil in sheep ((Molento et al, 2004)Molento et al, 2004) Itraconazole and valspodar in rat Itraconazole and valspodar in rat (Ballent et al, 2006)(Ballent et al, 2006)

Ketoconazole in dog Ketoconazole in dog (Alvinerie, unpublished data)(Alvinerie, unpublished data)

In vivo P-gp reversing agents

Impact on bioavailability of MLs

0

5

10

15

20

25

0 2 4 6 8 10 12

Time (days)

Ivomec

Ivomec + Ketoconazole

Iv

erm

ectin

con

cent

ratio

n (n

g/g)

Ketoconazole and ivermectin

Ivermectin concentration in dog plasma

La P-gp module l’exposition et Efficacité thérapeutique des LMs

Médicament Hôte Parasite

Parasite

Résistance

Absorption

Parasite

SNC toxicité

EfficacitéDistribution

Transporteurs ABC

BHM

Transport lipoprotéines

X

OH

O

OH

OO

OO

OR1

O

O

OO

OR2

H

H

H

H

H

Eimination

Dose administrée

Transporteurs ABC

Transporteurs ABC

Tissu adipeux