Targeted Bioavailability : Mechanisms Influencing the ...
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Targeted Bioavailability : Mechanisms Influencing the Tissue Distribution of Drugs
June 8, 2005 The New England Drug Metabolism
Discussion Group Summer Symposium University of Massachusetts Medical School
Shrewsbury, Massachusetts
William F. Elmquist, Pharm.D., Ph.D. Department of Pharmaceutics
University of Minnesota-Twin Cities
brain
Outline
Background
1) Drug transport proteins in the body and the CNS drug disposition (overview)
Experimental Studies 2) Delivery of Anti-tumor Agents (Gleevec) to CNS
Summary
3) CNS drug delivery program and efflux transporters
Variability in Drug Response in the CNS
- variability in distribution central - variability in receptors
- variability in elimination and metabolism peripheral - variability in absorption
Why study drug transporters in the CNS barriers?
Bioavailability Definitions:
FDA - “the rate and extent to which the active ingredient or active moiety is absorbed from a drug product and becomes available at the site of action. For drug products that are not intended to be absorbed into the bloodstream, bioavailability may be assessed by measurements intended to reflect the rate and extent to which the active ingredient or active moiety becomes available at the site of action.”
21CFR320.1 Code of Federal Regulations, Title 21, Volume 5, Parts 300 to 499, 1999. ; Guidance for Industry, CDER, FDA, Bioavailability and Bioequivalence Studies for Orally Administered Drug Products – General Considerations, 2003.
Commonly accepted definition: Typically, one modifies this definition to limit the delivery path from the site of administration to the bloodstream, i.e., the term bioavailability is “the fraction of the oral dose that actually reaches the systemic circulation”, and “commonly applied to both the rate and extent of drug input into the systemic circulation”.
Explain variability in drug response to PK and PD
Genetic Factors - drug targets - drug transporters - drug metabolizing enzymes
Environmental Factors - induction - inhibition
Physiological Factors - age, disease, etc.
Sources of Variability in Drug Response Variability Cycle
“Locations” of Variability in Drug Response
Presystemic bioavailability questions (“traditional” bioavailability)
Site-specific bioavailability questions (drug targeting)
Oral dosage form
Systemic circulation
Drug action
Targeted Bioavailability
Intestinal absorption
Liver metabolism
Cellular delivery
Intestinal metabolism
Tissue distribution Target site
Physicochemical Properties Ph
ysio
logy
/ Pa
thol
ogy Drug
Metabolism Receptor Affinity
Protein Binding
Membrane Permeability
Drug Transport
Gene Regulation
Targeted
Bioavailability
Dosage R
egimen
Protein Expression
Pharmacological / Toxicological Response
“Locations” of Variability in Drug Response
Presystemic bioavailability questions (“traditional” bioavailability)
Site-specific bioavailability questions (drug targeting)
Oral dosage form
Systemic circulation
Drug action
Targeted Bioavailability
Intestinal absorption
Liver metabolism
Cellular delivery
Intestinal metabolism
Tissue distribution Target site
Mechanisms influencing intestinal absorption: - solubility - permeability - transit time - carrier-mediated influx and efflux - stability - dosage form performance (disintegration, dissolution)
“Locations” of Variability in Drug Response
Presystemic bioavailability questions (“traditional” bioavailability)
Site-specific bioavailability questions (drug targeting)
Oral dosage form
Systemic circulation
Drug action
Targeted Bioavailability
Intestinal absorption
Liver metabolism
Cellular delivery
Intestinal metabolism
Tissue distribution Target site
Mechanisms influencing the intestinal first-pass metabolism : - carrier-mediated transport - phase I metabolism (e.g., CYP3A4) - phase II metabolism (e.g., UGTs) - enzyme induction - enzyme inhibition - locations of windows of absorption - intestinal motility / transit time - genetic expression patterns
“Locations” of Variability in Drug Response
Presystemic bioavailability questions (“traditional” bioavailability)
Site-specific bioavailability questions (drug targeting)
Oral dosage form
Systemic circulation
Drug action
Targeted Bioavailability
Intestinal absorption
Liver metabolism
Cellular delivery
Intestinal metabolism
Tissue distribution Target site
Mechanisms influencing the intestinal first-pass metabolism :
- carrier-mediated transport influx and efflux transporters - phase I (e.g., CYP3A4) - phase II (e.g., UGTs) - enzyme induction - enzyme inhibition - blood flow - protein binding - genetic expression patterns
“Locations” of Variability in Drug Response
Presystemic bioavailability questions (“traditional” bioavailability)
Site-specific bioavailability questions (drug targeting)
Oral dosage form
Systemic circulation
Drug action
Targeted Bioavailability
Intestinal absorption
Liver metabolism
Cellular delivery
Intestinal metabolism
Tissue distribution Target site
Systemic clearance
Mechanisms that influence the fraction of the drug in the systemic circulation that is available for distribution to target tissue and the exposure of the tissue to the drug
- distribution of blood flow - ratio of total clearance to a distributional clearance
Distributional clearance - membrane permeability, carrier-mediated transport (influx or efflux), protein-binding, intracellular metabolism, tissue transit time, capillary structure Total clearance – will affect the availability of the drug in the blood to distribute to the tissue
“Locations” of Variability in Drug Response
Presystemic bioavailability questions (“traditional” bioavailability)
Site-specific bioavailability questions (drug targeting)
Oral dosage form
Systemic circulation
Drug action
Targeted Bioavailability
Intestinal absorption
Liver metabolism
Cellular delivery
Intestinal metabolism
Tissue distribution Target site
Mechanisms that influence cellular delivery - membrane permeability - carrier-mediated transport
(influx / efflux) - intracellular metabolism - receptor-mediated transport - binding
“Locations” of Variability in Drug Response
Presystemic bioavailability questions (“traditional” bioavailability)
Site-specific bioavailability questions (drug targeting)
Oral dosage form
Systemic circulation
Drug action
Targeted Bioavailability
Intestinal absorption
Liver metabolism
Cellular delivery
Intestinal metabolism
Tissue distribution Target site
Mechanisms influencing the drug action at the target site - intracellular signaling - intracellular transport - expression of target receptors - receptor affinity - availability of cofactors
“Locations” of Variability in Drug Response
Presystemic bioavailability questions (“traditional” bioavailability)
Site-specific bioavailability questions (drug targeting)
Oral dosage form
Systemic circulation
Drug action
Targeted Bioavailability
Intestinal absorption
Liver metabolism
Cellular delivery
Intestinal metabolism
Tissue distribution Target site
Examine a location
Physicochemical Properties Ph
ysio
logy
/ Pa
thol
ogy
Drug Metabolism
Receptor Affinity
Protein Binding
Membrane Permeability
Drug Transport
Gene Regulation
Targeted
Bioavailability
Dosage R
egimen
Protein Expression
Pharmacological / Toxicological Response
Examine a location considering the interplay between external factors and mechanism
Mechanisms
Targeting modalities
100 mg
20 mg
20 mg
80 mg 60 mg
30 mg
30 mg
F = .80 x .75 x .5 = 0.3
Fg = 0.75 Fa = 0.80 Fh = 0.50
“Traditional Bioavailability” F = Fa x Fg x Fh
Influence of fruit juices on oral bioavailability “traditional bioavailability”
At each location, several mechanisms at play:
F = fa x fg x fh
basolateral apical intracellular
Complexity of the Transporter Problem at Various Barriers
Many processes can be occurring simultaneously!
PSin
PSout
PSout
PSin
Carrier in Carrier in
Carrier out
Carrier out
Bidirectional
Bidirectional
“Targeted” Bioavailability F = Fa x Fg x Fh and Fd , Fc , Ft
Presystemic bioavailability questions (“traditional” bioavailability)
Site-specific bioavailability questions (drug targeting)
Oral dosage form
Systemic circulation
Drug action
Targeted Bioavailability
Intestinal absorption
Liver metabolism
Cellular delivery
Intestinal metabolism
Tissue distribution Target site
Fa Fg Fh Fd Fc Ft
Importance of Transporters in the Disposition of Drugs
From Lee and Gottesmann. Journal of Clinical Investigation, 1998 illustration by Naba Bora, Medical College of Georgia.
Compartmental model for solute exchange in the brain
Adapted from Quentin Smith
Pardridge, 1998
Transgenic mice
Microdialysis
Blood-Brain Barrier (BBB)
Basolateral membrane
Apical membrane
Cooray et al., 2002 NeuroReport
microvessel from normal brain stained for GLUT-1 and BCRP
GLUT-1 on both inner and outer walls of the microvessel
BCRP shows a more narrow distribution
Apical -- blood
Basolateral -- brain
MCT1
BBB endothelial cell
OATP2
OATP2
MCT1
Background Selected Drug transport systems in BBB and BCSFB
MCT1-- monocarboxylate transporter OATP2-- organic anion transport protein 2 MRP -- multidrug resistant-associated protein (ABCCX) P-gp -- p-glycoprotein (ABCB1) BCRP – breast cancer resistance protein (ABCG2)
?
?
?
P-gp MRP1, MRP4, MRP5
BCRP
Murakami, 2000
PS (in situ perf) versus logP(MW- ½)
Efflux
Barriers to Drug Delivery
Presystemic bioavailability questions (“traditional” bioavailability)
Site-specific bioavailability questions (drug targeting)
Oral dosage form
Systemic circulation
Drug action
Targeted Bioavailability
Intestinal absorption
Liver metabolism
Cellular delivery
Intestinal metabolism
Tissue distribution Target site
location of variability
location of variability
Outline
Background
1) Drug transport proteins in CNS drug disposition (overview)
Experimental Studies 2) Delivery of Anti-tumor Agents to CNS
Summary
3) CNS drug delivery program and efflux transporters
CNS Delivery of Anti-tumor Agents 1) Brain Around Tumor - BAT (growing edge) 2) Membrane permeability vs. efflux transport 3) Tumor vasculature 4) Primary and secondary tumors (metastases)
CH3SO3H
N
N
N NH
NH
O
N
N
STI-571 (GleevecTM)
Recently approved for chronic myelogenous leukemia (CML) Several clinical trials currently underway for glioma
Mechanism of Action of STI-571
“Molecularly Targeted” Therapy
Kaplan-Meier survival plots intracerebral human glioma nude mice STI-571 txt bid PO 50 mg/kg/day
Kilic, et al., Canc.Res., 2001
Ras transformed negative control
PDGF
glioma
qd dosing
8 hours
Fur
Intestinal content
1 hour
SpleenKidney Pineal glandBrown fatSkin
7 days
Eye
Fur
From Novartis Pharma
5 min.
Pineal glandLungAdrenal gland
Pituitary glandIntestinal mucosa Liver
Whole-body Autoradiography of Pigmented Rats
Methods • The directional flux of STI-571 was
studied in MDCKII epithelial cell monolayers, using Transwell inserts. The effect of P-glycoprotein inhibition on the directional flux was also determined.
• In vivo brain distribution studies were done using wild-type and mdr-1a/b (-/-) knockout mice. Mice were administered 25 mg/kg 14C-STI-571 orally, or STI-571 intravenously and brain-to-plasma concentration ratios of STI-571 were determined 30, 60 and 120 minutes post dose. RP-HPLC was used to determine the radioactivity associated with parent drug or LC-MS was used .
wild type mice mdr 1a/b (-/-) knockout mice
In Vitro Experiment
Figure 1. Directional flux of STI-571 Across MDCKII Monolayers July 31, 2001
Time (minutes)0 30 60 90 120 150 180
% F
lux
of S
TI-5
71 a
cros
s e
pith
elia
l cel
l mon
olay
er
0
10
20
30
40 A to B flux wild-type cells n=5A to B flux mdr1 cells n=5B to A flux wild-type cells n=4B to A flux mdr1 cells n=4
mean + stdev
directional flux mdckII jul 31-01.JNB
STI-571 Flux Across MDCKII Cell Monolayer(31 July 2001)
% F
lux
of S
TI-5
71 A
cros
s M
DC
KII
Mon
olay
ers
(at 1
80 m
in)
0
10
20
30
40
50
A to BWT cells
B to AWT cells
A to BMDR cells
B to AMDR cells
(mean + S.D., n = 4-5)
5.4 folddifference
24.3 folddifference
enhancement due to MDR-transfection = 4.5 fold
STI-571 Flux MDR1-transfected MDCKII epithelium
“Corrected” flux = (B-to-A / A-to-B)mdr (B-to-A / A-to-B)wt
Corrected flux ratio
4.5-fold
In Vitro Experiment
In Vitro Experiment
Figure XX. Directional flux of STI-571 Across MDCKII Monolayers September 11, 2001
(effect of 1µM LY335979)
Time (minutes)0 30 60 90 120 150 180
% F
lux
of S
TI-5
71 a
cros
s e
pith
elia
l cel
l mon
olay
er
0
5
10
15
20
25
A to B flux control mdr1 cells, n = 4B to A flux control mdr1 cells, n = 4A to B flux txt LY335979, n = 5B to A flux txt LY335979, n = 5
mean + stdev
STI directional flux mdckII sept-11-01 w-979.JNB
Effect of Specific P-gp inhibition on STI-571 Flux
Directional flux of STI-571 Across MDCKII Monolayers
Time (minutes)0 20 40 60 80 100 120 140 160 180 200
% F
lux
of S
TI-5
71 a
cros
s e
pith
elia
l cel
l mon
olay
er
0
10
20
30
40
WT a2b pgp a2b WT b2a Pgp b2a
The directional flux of 14C-STI-571 was studied in MDCKII epithelial cell monolayers.
Effe
ctiv
e P
erm
eabi
lity
of S
TI-
571
(Pef
f)(c
m/m
in)
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
A to BWT cells
B to AWT cells
A to BMDR cells
B to AMDR cells
B
***
***
RESULTS
WT MDR
Bra
in/P
lasm
a R
atio
(% ra
dioe
quiv
alen
ts)
0
1
2
3
4
5
3H-Inulin14C-STI-571
Figure 6. Brain to plasma ratio of tritiated inulin (10 min post tail vein injection) and carbon-14 STI-571 (90 min and 120 min post oral dose, 25 mg/kg) in
wild-type mice. (dual label liquid scintillation counting, total levels, not corrected for residual volume (inulin space))
14C-STI-571(90 min post PO)
3H-Inulin 3H-Inulin 14C-STI-571(120 min post PO)
14,15 nov mice inulin sti.jnb
mean + stdev
Bra
in/P
lasm
a R
atio
(%
radi
oact
ivity
)
0
1
2
3
4
P-gp KnockoutWild-type Wild-typeP-gp Modulator
Figure XX. Brain to Plasma Ratio of STI-571 Radioequivalents 60 minutes post oral dose (25 mg/kg) in wild-type mice, p-gp knockout mice, and wild-type mice treated with potent p-gp modulator.
Bra
in/P
lasm
a R
atio
(% ra
dioa
ctiv
ity)
0
1
2
3
4
5
Wild-type miceP-gp knockout mice
30 minutespostdose
60 minutespostdose
120 minutespostdose
Figure 4. Brain to plasma ratio of STI-571 radioequivalents post oral dose(25 mg/kg) at various times postdose. (mean + SD, n = 4 at each time point)
corrected for residual vascular volume (inulin space)18 sept mice wt ko 30 60 120.jnb mean + stdev
Radiopurity of 14C-STI-571 (GleevecTM)
Time (minutes)0 5 10 15 20 25
dpm
in e
fflue
nt
0
2000
4000
6000
8000
10000
12000
14000retention time of parentSTI-571 by UV detection
Tracer STI-571 injected on column
Plasma Radioactivity Profile Wild-type Mice 1-4Sept. 18, 2001 plasma collected 60 min post 14C-STI-571 dose
retention time (min)0 5 10 15 20 25
Pla
sma
Rad
ioac
tivity
(dp
m)
0
1000
2000
3000
4000
wild-type #1wild-type #2wild-type #4wild-type #3
Polar metabolites
Parent STI-571
Brai
n to
Pla
sma
Rat
io %
dpm
ass
ocia
ted
with
STI
-571
0
2
4
6
8
10
12
Wild-type Mice mdr 1 a/b (-/-) Mice
Brain-to-Plasma Ratio at 60 minutes postdose
Brain/Plasma ratio KO = 11.2 fold opportunity for Brain/Plasma ratio WT targeted bioavailability
Gleevec Brain Distribution
Time (minutes)20 40 60 80 100 120 140
Pla
sma
STI
-571
Con
cent
ratio
n(n
g/m
l)
0
2000
4000
6000
8000
Mdr 1 a/b (-/-) mice Wild type mice
A
Time (minutes)20 40 60 80 100 120 140
Bra
in S
TI-5
71 C
once
ntra
tion
(ng/
gram
bra
in ti
ssue
)
0
2000
4000
6000
8000
Mdr 1 a/b (-/-) mice Wild type mice
B
Rat
io o
f STI
-571
bra
in p
enet
ratio
n (m
dr 1
a/b
(-/-)
mic
e / w
ild ty
pe m
ice)
0
2
4
6
8
10
12
30 60 120
C
Time postdose (minutes)
A – Plasma STI-571 conc vs time B – Brain STI-571 conc vs. time
C – increase in STI-571 brain penetration in the P-gp knockout
enhanced: 6-8 fold !
“targeted bioavailability”
Brain and Plasma Concentrations (LC-MS)
A) mice developed neurological symptoms while on imatinib
B) pathological evidence of macrophage brain and meningeal masses
(50 mg/kg am, 100 mg/kg pm orally) C) brain and spinal cord B-lymphoid cell staining
D) laser dissected regions compared using PCR primers specific for P210 Bcr/Abl normal brain brain lesion
PCR products
Wolff et al., Blood 2003 The CNS is a sanctuary for leukemic cells in mice receiving imatinib mesylate for BCR/Abl induced leukemia
Drug Metabolite
ng/ml µM ng/ml µM
Concentration
Plasma (average)
6958 (± 2082)
11.8 (± 3.5)
611 (± 179)
1.0 (± 0.3)
CSF (pooled)
45 0.08 0 0
Plasma/CSF ratio
155 NA
(n = 9)
from Wolff et al., CSF/ Plasma STI571 concentrations in the mouse model
STI
-571
con
cent
ratio
n(n
g/m
l)
0
1000
2000
3000
4000
5000
CSF Plasma
IC50
Simultaneous STI-571 Concentrations in Four Patientswith CML Lymphoid Blast Crisis
Leis et al., Low penetration of imatinib (STI571) into the CSF indicates need for standard CNS prophylaxis in patients with CML lymphoid blast crisis and Philadelphia Chromosome Positive ALL. Abstract, American Society of Hematology, December 8, 2001
improved cellular activity
provided tyrosine kinase activity
abolished protein kinase-c inhibitory activity
increased solubility and oral bioavailability
Capdeville et al., Glivec (STI571, Imatinib), a rationally developed, targeted anticancer drug.
Nature Reviews Drug Discovery, July 2002.
P-gp substrate pharmacophore model and Gleevec aligned. Blue features (hydrophobes) and green features (hydrogen bond acceptors) with vector in the direction of the putative hydrogen bond donor. (Ekins et al, Molecular Pharmacology, vol. 61, 2002)
basolateral apical intracellular
Complexity of the Transporter Problem at Various Barriers
Many processes can be occurring simultaneously !
PSin
PSout
PSout
PSin
Carrier in Carrier in
Carrier out
Carrier out Pgp
Drug delivery across barrier
Bidirectional
Bidirectional Carrier out BCRP
Cancer Res 2005; 65:(7). April 1, 2005
Breedveld et al. (BCRP and Imatinib CNS Penetration)
Cancer Res 2005; 65:(7). April 1, 2005
[A] Imatinib Brain penetration - BCRP, P-gp [B] Imatinib Brain penetration - control mice w/inhibitor
From Tsuji, 2000.
Transport Mechanisms at the Blood- Brain Barrier
X
Barriers to Drug Delivery
Presystemic bioavailability questions (“traditional” bioavailability)
Site-specific bioavailability questions (drug targeting)
Oral dosage form
Systemic circulation
Drug action
Targeted Bioavailability
Intestinal absorption
Liver metabolism
Cellular delivery
Intestinal metabolism
Tissue distribution Target site
X
Biochemistry and molecular biology
Program for CNS drug delivery evaluation
In Vitro cellular barriers (transfected cell lines and isolated brain cellular barriers)
In Vivo techniques (BUI, intravenous injection, in situ perfusion)
Specialized in vivo techniques (microdialysis, transgenic animals)
Strategies to Improve the CNS Delivery of Drugs
1) improve physiochemical properties 2) use existing influx transport systems 3) reduce affinity for efflux transporters
problem: multidrug resistance 4) temporary breakdown of the BBB
hyperosmotic (mannitol) inflammatory mediators (RMP-7)
5) drug delivery systems implants/direct delivery polymeric carriers nanoparticles peptide vectors immunodirected vectors efflux inhibitors
CNS Drug Distribution in the Future The role of various drug efflux proteins in the blood-brain and blood-CSF barriers in CNS targeting can be examined using a variety of complimentary experimental approaches. These include:
- biochemical and molecular biological methods (expression of functional protein and RNA message)
- in vitro models using transfected cell cultures
- in vitro models using cell cultures of CNS barriers
- in vivo models using novel sampling techniques
- in vivo models that have been genetically engineered
These approaches will allow a specific quantitative analysis of the biological determinants of drug action in the CNS that are related to drug delivery across the cellular barriers in the CNS.
Acknowledgements Haiying Sun Hua Yang Qin Wang Michele Fontaine Tim Spitzenberger Christine Brandquist Haiqing Dai Ying Chen Dr. Mini Kurumboor Naveed Shaik Dr. Paul Stemmer Dr. Donald Miller
Dr. Alexander Kabanov Dr. Elena Batrakova Tanya Bronich
Lilly Cancer Research Dr. Anne Dantzig Dr. James Starling
Funding National Institutes of Health Lilly Cancer Research Laboratories Novartis Pharma UNMC Graduate Fellowship
Yale University Dr. Alan Sartorelli Dr. Rick Finch
Novartis Pharma Dr. Michel Lemaire Dr. Peter Marbach
Center for Neurovirology Dr. Howard Gendelman Dr. Yuri Persidsky
Alfred Schinkel