NOVEL USES OF DRUG TRANSPORTERS FOR

DRUG DELIVERY:
A CASE STUDY WITH GABAPENTIN
 REFERENCED
KENNETH C. CUNDY, Ph.D.; XENOPORT,
INC., SANTA CLARA, CA; PARSIPPANY, NJ
MARCH 8, 2005
INTESTINAL ABSORPTION OF ORAL DRUGS

Passive Diffusion Active Transport

• Nutrients (small peptides, amino acids,

• Most approved oral drugs vitamins, fatty acids, etc.)
• Selected drugs: valacyclovir, ACE inhibitors
cephalosporins, pravastatin, etc.
 ENGINEERED DRUG TRANSPORT

Proactive targeting of a drug to an endogenous nutrient transport

pathway

Targeting active transport can overcome a range of problems that

limit drug development:
• Poor intestinal permeability
• Narrow absorption window
• Short half-life
• High first-pass metabolism
• Rapid drug efflux
• Poor absorption due to low solubility
• Poor CNS penetration
• Poor tissue targeting
ENGINEERING DRUG TRANSPORT - STEP 1

• Identify appropriate transporter protein

• Develop in vitro assays
• Identify chemical features required for transport
(XenoPorter Module)

Nutrient XenoPorter
Module

Transporter Transporter
protein protein
ENGINEERING DRUG TRANSPORT - STEP 2

• Combine XenoPorter Module with Drug

• Confirm in vitro transport and cleavage
• Evaluate bioavailability in animals

Drug with Poor XenoPorter

Bioavailability Module

Cleavable Permanent Transformed

Prodrug Conjugate Drug
 Gabapentin

OH Neurontin®
H2N
(Pfizer) and
O Generics

• Approved for treatment of epilepsy and post-herpetic neuralgia

• Worldwide sales $2.7 B in 2003, generic in October 2004
• Used in multiple CNS indications
Neuropathic pain (>50%) Epilepsy
Bipolar disorder Anxiety Disorders
Depression Migraine & Headache
Sleep disorders
Gabapentin: Sub-Optimal Pharmacokinetic Properties

• Blood (and brain) levels not proportional to oral dose

• Maximum limit to attainable blood levels

• High inter-patient variability in oral absorption

• Short half-life: 5-7 hours

• Administered 3-4 times a day

• SR (SUSTAINED RELEASE) formulation not possible

Gabapentin is believed to be a substrate for a low capacity amino

acid transporter localized in the small intestine
 Saturation of Gabapentin Absorption

Dose-Dependent Oral Bioavailability of Neurontin® in Humans

70

60
Bioavailability (%)

50

40

30

20
400 600 800 1000 1200 1400 1600 1800

Gabapentin Dose (mg)

Source: Gabapentin SBA
Gabapentin Has a Limited GI Absorption Window

Limited Capacity
Absorption Window

Stomach Small Intestine Colon

1 to 6 hours 2 to 4 hours 8 to 18 hours

Transit Time in Humans

• Saturable uptake – exposure not proportional to dose

• Variable capacity/transit times - inter-subject variability in PK
• No colonic absorption - SR formulation not possible
 Uptake by Transporter-Expressing Oocytes

Gabapentin Uptake (pmol/oocyte) L-Dopa Uptake (pmol/oocyte)

OH 0.6

OH
0.4

H2N CO2H
0.2

L-Dopa
0.0
LAT2 LAT1 B0+ ATB0+ y+LAT2 y+LAT1 XCT TAT1
0.6

H2N CO2H
0.4

0.2
Gabapentin
0.0

LAT2 LAT1 B0+ ATB0+ y+LAT2 y+LAT1 XCT TAT1

 PRODRUGS OF GABAPENTIN

Desired Product Profile:

• Cleavable prodrug of gabapentin suitable for b.i.d. dosing
• Novel patentable compound
• Absorbed by active transport pathways throughout intestine
• Good chemical stability, single polymorph
• Rapid conversion to gabapentin after absorption
• High oral bioavailability
• Dose proportional exposure to gabapentin
• Minimal exposure to the intact prodrug
• No new toxicity from released promoieties
MENGATASI KERTEBATASAN ABSORPTION WINDOW
Modify the drug for recognition by high capacity transporters located
throughout the intestine:

High Capacity Transporter

Stomach Small Intestine Colon

1 to 6 hours 2 to 4 hours 8 to 18 hours

• Increased bioavailability
• Greater dose proportionality
• Lower inter-patient variability
• Reduced dosing frequency (sustained release)
 TARGETING COLONIC TRANSPORTERS

Selection of Transporter Targets in the Colon:

• Obtain fresh human tissue biopsies (endoscopy, colonoscopy)

• Determine absolute expression level, inter-subject variability, and

regional distribution (laser capture microdissection, qPCR)
• Determine subcellular location (antibodies, confocal microscopy)

• Examine species differences in expression

• Establish in vitro assays (competition, electrophysiology, pH change,

direct uptake by LC/MS/MS)
• Determine SAR for transport (commercial compounds, NCEs)
 OBTAINING HUMAN TISSUE SAMPLES

Commercial Sources of Tissue/RNA:

• Unreliable documentation of sample history
• Samples may be from single individuals
• Contain mixture of enterocytes and other cells
• Samples often degraded after collection

Fresh Human Biopsies

• Collaboration with Stanford University
• Biopsy samples from routine endoscopies
• Many sites can be sampled
• Samples available from many individuals
• Extensive documentation available (disease, treatment history)
• RNA more representative of enterocytes
 Intestinal Mucosal Biopsy Samples Obtained
by Upper or Lower Endoscopy
80 unique samples: Frozen Sample
13 duodenum OCT-embedded Sample
for qPCR for Immunostaining
25 ileum
40 colon

Fluorescence
Upper Endoscopy

0 10 20 30 40

Colonoscopy

Stomach Small Intestine Colon

 XenoPort Transporter Expertise

• Transporter Selection and Validation

- Expression data on > 200 transporters in human, rat and mouse tissues
- 32 antibodies in hand against 28 human transporters
- 25+ antibodies in progress against human and rat transporters

• Transporter Assays
In vitro assays for >80 human transporters

• Transporter Chemistry / Prodrug Design

Chemistry and SAR on members of ~10 SLC families of greatest interest

• In vivo Pharmacokinetics
> 700 PK studies in rats, dogs, and monkeys
 XenoPort’s Transporter Primers for qPCR

Human: ~240 genes Includes nearly all organic

solute carriers

Mouse: >200 genes Sequenced mouse genome provides a

nearly complete set of homologs

Rat: >200 genes Many new rat gene sequences now

available

Monkey: ~10 genes Methodology in place to rapidly identify

primers for “un-sequenced” species

Panel of housekeeping genes (GAPDH, B-Actin, B-microglobulin,

PLSCR, FCRN) are used to normalize data for cross-tissue comparisons
 Transporter Expression (>200 genes) by qPCR
Gene clusters
Biopsy
based on
clusters based
expression
on similar
across
expression
all biopsy
patterns
samples
across 265
unique genes

• Biopsy results differ greatly from those from commercial tissue/RNA sources
• Several transporters have higher expression in human colon
Example of Transporter in Human Colon: MCT-1
40X 100X
Control
MCT
 Monocarboxylate Transporters (MCTs)
• Family of 14 H+-coupled monocarboxylate transporters
• Recognize structurally diverse monocarboxylates
• Absorbs short-chain fatty acids from carbohydrate fermentation
by bacteria in colon Natural Substrates
O
O OH O O O
HO
HO HO HO
out N

Known Drug Substrates (MCT-1)

HO O
in HO
O
O O OH
O H H H O
N S
H H
+ -
1H 1MCT N HO
O
O
HO
Carindacillin Pravastatin
 MCT Transporters in the Human Intestine
Transporter mRNA Abundance

Stomach
80000
Duodenum
Ileum
60000
Cecum
Asc Colon
40000
Trans Colon
Sigmoid Colon
20000
Rectum

0
MCT-1 MCT-2 MCT-3 MCT-4 MCT-5 MCT-6

 MCT-1 highly expressed in all segements of the colon

- also well expressed in upper GI
 Making an MCT Substrate from Gabapentin

H2N CO2H Gabapentin:

Substrate for amino acid transporter but no
interaction with monocarboxylate transporters

Promoiety Prodrug:
N CO2H Blocking positive charged amine of gabapentin
H
with enzymatically labile promoiety generates
prodrugs that are transported by MCTs
 Final Clinical Candidate

O O
OH
O O N
H
O

XP13512

XP13512 undergoes enzymatic hydrolysis in vivo to liberate

gabapentin, isobutyrate, acetaldehyde, and CO2
 XP13512 is a Substrate for MCT-1

MCT-1 Competition Assay Cells incubated with 1 mM XP13512 for 30 min.

Cell lysate analyzed by LC/MS/MS
3000 9
H-substrate uptake

8 Cmpd alone
7 + xs Lactate
Gabapentin
(cpm/well)

2000 6

uptake (M)
5

XP13512 4

1000
pIC50 = 3.3 3
3

1
0
0
-5 -4 -3 -2
Cmpd alone + xs Lactate
Conc. (log M) (Control Substrate)

The parent drug gabapentin is not a substrate for MCT1

 XP13512 is a Substrate for SMVT
Control substrate XP13512 (uM)
Competition Assay in 500uM 0.22 0.67 2 6 18 54
SMVT-Expressing Cells

3000
Gabapentin
H-biotin uptake
(cpm/well)

2000
10nA Oocyte
XP13512
elecrophysiology
pIC50 = 4.9 10s
1000
3

0
-9 -8 -7 -6 -5 -4 -3 -2 Biotin XP13512 (uM)
Conc. (log M) 500uM 0.22 0.67 2 6 18 54
 XP13512: Summary of In Vitro Data
• XP13512 is cleaved to gabapentin by non-specific
esterases in intestine, liver, and blood – released
gabapentin is metabolically stable

• XP13512 does not interact with CYP450 or Pgp

• In vitro transport shows that XP13512 can be transported

by:
MCT-1
SMVT
pH-dependent passive diffusion (pKa = 5.0)

• Intestinal absorption in vivo is likely to occur by a

combination of mechanisms
PK of XP13512 Sodium Salt in Monkeys
Concentrations of Gabapentin in Plasma After
Concentrations of of
Oral Administration Gabapentin
Gabapantin or in Plasma
XP13497 After Oral
to Monkeys
Administration of Gabapentin or XP13497 to Monkeys

Gabapentin
60 XP13497

Equimolar Dose = 75 mg GP eq./ kg

40 N=4

~ 5-fold increase in GP Cmax

20 ~ 3-fold increase in GP AUC
Concentration (u

0
0 6 12 18 24

Time (hr)
Low Exposure to Prodrug in Monkeys
Concentrations
Concentrations of of Gabapentin or Prodrug
Gabapentin and in Plasmain
Prodrug After
Plasma
Oral Administration of XP13497 to Monkeys
After Oral Administration of XP13497 to Monkeys

Gabapentin
60 Prodrug

40
Low Prodrug Exposure

20
Concentration (u

0
0 6 12 18 24

Time (hr)
 Effect of Dose on Gabapentin AUC in Monkeys
AUC of Gabapentin in Plasma After Oral Administration
of XP13512 or Gabapentin to Monkeys
3500
Gabapentin AUC (ug*hr / mL)

3000
XP13512 provides
Gabapentin-XP
2500
dose-proportional
exposure
2000

1500

Gabapentin*
Gabapentin*
1000
saturates
500

0
0 200 400 600 800 1000 1200

*Source: Gabapentin SBA Dose (mg-eq GP/ kg)

 Colonic Absorption in Preclinical Species
Concentrations of Gabapentin in Plasma After Intracolonic
Administration
Concentrations of Gabapentin of Gabapentin
in Plasma After Intracolonic Concentrationsor XP13512
of Gabapentin in Plasma After Intracolonic
Dosing of Gabapentin or XP13512 Suspension in Monkeys Dosing of Gabapentin or XP13512 Suspension in Rats
Monkeys Rats
12 12
Gabapentin Gabapentin
XP13512 XP13512
10 10

8 Equimolar Dose: 8
Equimolar Dose:
10 mg GP eq/kg 25 mg GP eq/kg
6 6
N=4 N=4
4 4
Concentration (ug/mL)

Concentration (ug/mL)
2 2

0 0
0 6 12 18 24 0 2 4 6 8
Time (hr) Time (hr)

>15 to 20-fold increase in gabapentin AUC

 Single Dose PK of XP13512 – Study XP006
STUDY XP006

Design:
• Five groups of 10 healthy adult subjects (male and female)
• Single oral doses of XP13512 (n = 8) or placebo (n = 2) followed
one week later by a single near-equimolar dose of Neurontin®
• All subjects fasted prior to each dose
• Blood and urine PK determined over 36 hours after each dose:

XP13512 Doses: 350, 700, 1400, 2100, 2800 mg

(Equivalent to: 182, 364, 729, 1090, 1460 mg of gabapentin)
Neurontin Doses: 200, 400, 800, 1200, 1400 mg
 XP13512 (IR) vs. Neurontin:
Concentrations of Gabapentin in Blood
Effect of Dose on Concentrations of Gabapentin in Blood
Concentrations of Gabapentin in Blood After Oral Concentrations of Gabapentin in Blood After Oral
After Oral Administration
Administration of Neurontin or XP13512
of Neurontin in Study XP006 Administration ofto Healthy
XP13512 Adults
in Study XP006

18 18
Neurontin 200 mg XP13512 350 mg
16 16
Neurontin 400 mg XP13512 700 mg
14
Neurontin 800 mg 14
XP13512 1400 mg
Neurontin 1200 mg XP13512 2100 mg
12 Neurontin 1400 mg 12 XP13512 2800 mg
10 10

8 8

6 6

4 4
Concentration (ug/mL)

Concentration (ug/mL)
2 2

0 0
0 6 12 18 24 30 36 0 6 12 18 24 30 36

Time (hr) Time (hr)

All AEs reported for XP13512 were mild and similar to those reported for Neurontin
 XP13512: Dose Proportionality of AUC
Dose Proportionality of Gabapentin in Blood After
Effect of Dose on
Oral Administration AUC oforGabapentin
of XP13512 in Blood
After
Neurontin in Study XP006
Oral Administration of XP13512 or Neurontin
180

XP13512 XP13512
160
R = 0.994
2
produced
140 Neurontin dose-
120 proportional
exposure
100

80

60 Neurontin
40
exposure
showed
Gabapentin AUC

20 saturation
0
0 200 400 600 800 1000 1200 1400 1600

Dose (mg-equivalents of Gabapentin/kg)

XP13512: High Bioavailability as Gabapentin
Effect of Doseofon
Bioavailability Bioavailability
Gabapentin as Gabapentin
After Oral Administration
After Oral Administration of XP13512
of XP13512 or Neurontin or Neurontin®
in Study XP006

to Healthy Volunteers
100

90
No saturation
80
of XP13512
70
absorption or
60 conversion to
50 gabapentin
40

30
Bioavailability (%)

20
XP13512 Neurontin absorption
10 Neurontin was saturated
0
0 200 400 600 800 1000 1200 1400 1600

Dose (mg-equivalents of Gabapentin)

 XP13512: Very Low Exposure to Prodrug
Concentrations of Gabapentin and Prodrug in Blood After
Oral
Concentrations ofAdministration
Gabapentin of 2800
andmg XP13512
Intact in Study in
XP13512 XP006
AfterBlood
Oral Administration of XP13512 to Healthy Volunteers at 2800 mg
18
Gabapentin
16 Prodrug
14

12 Gabapentin Cmax was 50-fold

higher than prodrug Cmax
10

8 Gabapentin AUC was 280-fold

higher than prodrug AUC
6

4
Concentration (ug/

0
0 6 12 18 24 30 36

Time (hr)
 Multiple Dose PK of XP13512 – Study XP018
STUDY XP018

Design:
• Four groups of 10 healthy adult subjects (male and female)
• Twice daily doses of XP13512 (n = 8) or placebo (n = 2) titrated
upward with one week at the target dose level
• All subjects fasted prior to each dose
• Steady state blood and urine PK determined over 24 hours after
the AM dose on Day 7 at the target dose level

XP13512 Doses: 350, 700, 1400, 2100 mg b.i.d.

(Equivalent to: 182, 364, 729, 1090 mg of gabapentin b.i.d.)
XP13512: Concentrations of Gabapentin in Blood
Steady State Concentrations of Gabapentin in Blood After
Steady State Concentrations
Repeated ofDosing
Twice Daily Oral Gabapentin
of XP13512inin Blood
Twice After
Study XP018
Daily Oral Administration of XP13512 to Healthy Volunteers
20
350 mg
700 mg
1400 mg
15 2100 mg

10

5
Concentration (ug/m

0
0 6 12 18 24

Time (hr)
 XP13512: Dose Proportionality
Effect of Dose on Steady State AUC and Cmax of Gabapentin in
Effect ofBlood After
Dose on Steady State Twice Daily Oral Administration
AUC of Gabapentin of XP13512
Effect of Dose on Steady State Maximum Concentration of
in Blood After Twice Daily Oral Dosing of XP13512 Gabapentin in Blood After Twice Daily Oral Dosing of XP13512
AUCss Cmaxss
140 25

2
RR2 ==0.994
2
120
0.994 20
R2R==0.996
0.996

100

15

(ug/mL)
80

ss
(ug*hr/mL)
ss

60
10

40

5
20

Gabapentn Cmax
Gabapentn AUC

0 0
0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500

XP13512 Dose (mg) XP13512 Dose (mg)

XP13512 produced dose-proportional gabapentin exposure in blood

XP13512: High Bioavailability as Gabapentin
Bioavailability of Gabapentin from XP13412 at Steady
Effect State
of Dose on Steady
After Twice State
Daily Dosing Bioavailability
of XP13512 of Gabapentin
in Study XP018
From XP13512 After Twice Daily Dosing in Healthy Volunteers
100

90

80
No saturation
70 of XP13512
60
absorption or
conversion to
50
gabapentin
40

30
Bioavailability (%)

20

10

0
0 500 1000 1500 2000 2500

XP13512 Dose (mg)

 XP13512: Very Low Exposure to Prodrug
Steady State Concentrations of Gabapentin and Prodrug in Blood After
SteadyRepeated
State Concentrations of ofGabapentin
Twice Daily Oral Dosing andin XP13512
2100 mg XP13512 Study XP018 in
Blood
After Daily Oral Administration of XP13512 at 2100 mg b.i.d
20
Gabapentin
XP13512

15

Gabapentin Cmax was >50-fold

higher than prodrug Cmax
10

5
Concentration (ug/m

0
0 6 12 18 24

Time (hr)
 XP13512 – Multi-Dose PK Conclusions

• XP13512 rapidly absorbed and converted to gabapentin

• Dose-proportional exposure to gabapentin in blood at steady
state (based on AUC, Cmax, or Cmin)
• High bioavailability (≥70%) as gabapentin over the entire dose
range
• Predictable accumulation in blood compared to single doses
• Low and transient exposure to prodrug at all doses

XP13512 is now in Phase IIa studies for Post-Herpetic

Neuralgia and Restless Legs Syndrome
 MDS XP13512 SR Tablets vs Neurontin®
Concentrations of Gabapentin in Blood After Oral
Mean Concentrations ofXP13512
Administration of Gabapentin inNeurontin
Dosing
Tablets or Blood After Oral
Capsules
of XP13512 or Neurontin® in Healthy Adults (Study XP022)
6
XP13512 1200 mg (n = 10)
Neurontin 600 mg (n = 11)
5

4
2-fold increase in AUC
3-fold increase in Tmax
3 Bioavailability 75%

2
Concentration (ug/m

0
0 6 12 18 24 30 36

Time (hr)
 XP13512 –Conclusions
• XP13512 is a substrate for multiple transporters in the GI tract
• The prodrug is rapidly absorbed and converted to gabapentin
• XP13512 provides dose-proportional exposure to gabapentin
Bioavailability as gabapentin was ≥70% over wide dose range
• A sustained release tablet gave extended exposure to
gabapentin with high bioavailability
• Low and transient exposure to prodrug was seen at all doses

• XP13512 has completed a successful Phase IIa study in RLS

• A Phase IIb study X RLS started last month
• A Phase IIa study of XP13512 in Post-Herpetic Neuralgia is
ongoing
 Acknowledgements
Chemistry: Preclinical Development: Biology:
Stephen Raillard Thamil Annamalai Noa Zerangue
Adam Mann Wendy Luo Katy Woodford
Tono Estrada Shirley Liu Mark Warren
Cindy Zhou Shasha Jumbe Meera Kumar
Fenmei Yao Shubhra Upadhyay Natali Minassian
Maria Ludwikow Jeanelle Zamora Tracy Dias
Ge Peng Payal Shirsat Matt Panuwat
Thu Phan Josephine DeVera Kerry Koller
Hui Yan Allan Torneros Tania Chernov-Rogan
Quincey Wu Lin Bu
Randy Sheuerman Joan Zhou
Xuedong Dai Russell Branch Others:
Dan Canafax
Mark Gallop
Ron Barrett
Tristen Moors
The Leader in Engineered Drug Transport™