Post on 27-Mar-2015
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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 EfficacyEfficacy
Pharm
acokinetic
Pharm
acodynamy
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 = ABC8
ABCA2ABCA2 ABCB2 = TAP1 (RE)ABCB2 = TAP1 (RE) ABCC2 = MRP2ABCC2 = MRP2 ABCG = BCRPABCG = BCRP
ABCA3ABCA3 ABCB2 = TAP1 (RE)ABCB2 = TAP1 (RE) ABCC3 = MRP3ABCC3 = MRP3 ABCG4ABCG4
ABCA4ABCA4 ABCB4 = MDR2-3ABCB4 = MDR2-3 ABCC4 = MRP4ABCC4 = MRP4 ABCG5ABCG5
ABCA5ABCA5 ABCB5ABCB5 ABCC5 = MRP5ABCC5 = MRP5 ABCG8ABCG8
ABCA6ABCA6 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 saltsConjuguated/anionsConjuguated/anions
nucleotidesnucleotidesSterols/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 Apical
1, 3 Basolatéral1, 3 BasolatéralABCC1,2, 3ABCC1,2, 3Multidrug rresistant Multidrug rresistant
proteinprotein
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
ATPIn
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, Verapamil, amiodarone, and quinidine increase plasma levels, reduce renal and non-renalreduce renal and non-renal clearance, increase blood/brain clearance, increase blood/brain barrier transport. barrier transport. Dose adjustment may be needed in 50% of Dose adjustment may be needed in 50% of cases. 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%, -increases in digoxin AUC (+211%) and decreases in total body, renal, and non-renal clearance (-67%, -73%, -58%) after 5 days.73%, -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
O
O
O
O
OH
O
O
OO
O
R2
H
H
13
25AVERMECTINAVERMECTIN
MILBEMYCINMILBEMYCIN
O
O
OH
O
O
H
O
H
O
H
H
N
O
Plasma
Intestine
Biliairyelimination
Biotransformation
Intestinal secretion
Storage
Liver
Entero-hepatic
cycle
Adipose tissue
Efficacy
Route administrationMoleculeSpeciesPathophysiology
FecesMilk
Toxicity
Brain
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
Neurotoxicity
Murray-Grey ?
Mdr1ab -/-
Genetic disorder in Ivm sensitive Colley
Beagle of reference
tgctggtttttggaaacatgacag - - - - ctttgcaaatgcaggaatttcaagaaacaaaacttttccagttataattaatgaa Ivm Sensitive male
tgctggtttttggaaacatgacag - - - - ctttgcaaatgcaggaatttcaagaaacaaaacttttccagttataattaatgaa Ivm sensitive female
tgctggtttttggaaacatgacagatagctttgcaaatgcaggaatttcaagaaacaaaacttttccagttataattaatgaaBeagle of reference
Lack of protein in tissues
Colley sensitive
Beagle
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 V
StopA L Q M Q E F Q E T K L F Q L
N
N
M
M
T
TSensitive Colley- - - - - - - - -
- - - - - - - - -
……
Colley Deletion
Western BlotIntestine
Small Intestine
Bile
Dose (µg/kg)
0
1
2
3
4
5
100 200 400
Iver
mec
tin
(µ
g/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
I
ve
rme
cti
n (
ng
/ c
m /
kg
)
0
10
20
30
40
50
duodenum jejunum ileum
**
Control
Verapamil
Involvement of ABC transporters
Pharmacokinetics of ivermectin
0
25
50
75
100
0 6 12 18 24 30 36 42 48
Time (hr)
StandardPgp-/-
P-gp deficient mice mdr1ab-/-Iv
erm
ect
in c
once
ntr
ati
on
in p
lasm
a (
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
ecti
n c
once
ntr
atio
n
(ng/
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
O
O
O
R1O
O
OO
O
R2
H
H
H
H
H
Eimination
Dose administrée
Transporteurs ABC
Transporteurs ABC
Tissu adipeux