Pharmacokinetic analysis of Carotid BPA-mannitol delivery to Human GBM indicates three compartment...

Pharmacokinetic analysis of Carotid BPA-mannitol delivery to Human GBM indicates three

compartment tumour uptake kinetics enhanced by LAT activity in the Brain Around Tumour after

resection

G. Cruickshank, D. Ngoga, S. Green, A. Detta, N.D James, C Wojnecki, J Doran, F. Lowe, Z. Ghani, G Halbert, M Elliot ,

S Ford, R Braithwaite, TMT Sheehan, J Vickerman, N Lockyer, G. Croswell, R Sugar, A. Boddy, A. King,.

Glioblastoma Before and After resection

What is the Target for Radiation ?

Invasio

Diffuse vs Focal

Visible on ScanningMRI/CT

Invasive ‘Guerilla’cells diffusely separate from main tumourNot seen on ScansNot seen at Surgery

RT issues:Dose limitationTumour targeting

Invasive vs Proliferative

Conventional Radiation

1.8 - 2.0Gy 6 x 5days30 fractions

Radiation portals set to encompass lesionplus 2cm border

Dose TCP Tox45Gy 32% 0%60Gy 43% 10%70Gy 68% 30%

Survival2/52 > 37/52

Walker 1978

Thus have to include normal brainin radiation field

Estimated Tumour Control Probability with Radiation Therapy: Glioblastoma

1.0

0.5

50 60 70Dose in Gy

nb. Effect of dose escalation

Why BNCT?Boron neutron capture therapy (BNCT) allows target cell dose escalation by virtue of its:

Biological targeting Insensitivity to hypoxia Irreparable double strand DNA

breaks Insensitivity to cell cycle status

BNCT offers the only logical radio-therapeutic approach to control of discontiguous tumours

Boron Neutron Capture Therapy

Cell

B 10

neutrons

B 11

Li7 0.84 MeV

alpha1.47 MeV

photon 0.478 MeV

*Ion combined range ~ 8-9 microns . Cell diameter ~ 10 microns.

=> radiation damage within cell

Intracellular reaction *- Double strand breaks- Cytotoxic effect not limited by hypoxia and cell cycle state - Targeting entirely dependent on biological uptake of boron by tumour cells

The Challenge: Radiation Therapy to brain Tumours

• How can we increase dose to tumour cells?• Without increasing the dose to normal cells?

What are the biological requirements for Boron ?

• Increasing bioavailability in brain (ecf)• Increasing uptake into tumour (intracellular)• Increasing retention in tumour cells

• BoronoPhenylAlanine = BPA

S

Multi-Compartment Model

BBBSystem A

LAT-1

TumourCell

GlialCell

PassiveDiffusion

Luminal(Blood)

Abluminal(Brain)

Na+

After Amo, et al Europ. J.Pharm.Sci 2008 35:161-174

Compartment 1 Compartment 2

Compartment 3

Microdialysis

Mastroberardino et al Nature 395 : 288 (1998)

Heterodimeric LAT-1 Transporter

L System AA TransportersSodium Independent

Associates with transmembrane 4F2hc for function

Found: BBB, Placenta, ~glia+++glioma, other tumours

Control of expression reversed in Glioma*

glia, liver glioma, tumour

*Cruickshank, Detta , Jones 2010

Results for counted stained cell populations in GBMs

A

0

10

20

30

40

50

60

70

80

90

100

LAT + PCNA + LAT + PCNA +

LAT + X-Bar = 72.6 ± 16.9

PCNA + X-Bar = 22.8 ± 16.9

LAT + PCNA + X-Bar = 4.8 ± 2.2n = 29

60-90 % of tumour cells express LAT-1

A much lower proportion are proliferating

Detta and Cruickshank, Cancer Res 2009

LAT-1 Transporter• 3 times more LAT-1+ cells (73%) than PCNA+ cells in

GBM, even in dispersed cells of BAT

• LAT-1 expression is not related to proliferation in malignant brain tumours and some metastases

• BPA uptake specifically inhibitable by LAT-1 substrates indicates LAT-1 critical for BPA

uptake

Detta, A., & Cruickshank, G.S. L-amino acid transporter-1 and boronophenylalanine-based boron neutron capture therapy of human brain tumors. Cancer Res. 69, 2126-2132 (2009).

BPA Uptake from fresh incubated Human tumour (GBM) and brain tissue (as Boron) :

NB: Saturation Kinetics: Well above Km for LAT1 thus uptake rate independent of BPA concentration

`

Viability BPA +PA +Tyr +BCH

`


`


Means and SDN = 4

NB. Fresh Heterogeneous tissue explants NOT homogeneous cell lines.....Hence more realistic

S


BBBSystem A

LAT-1

TumourCell

GlialCell

PassiveDiffusion

Luminal(Blood)

Abluminal(Brain)

Na+



Compartment 3

Microdialysis

Birmingham Data: BPA formulation

• BPA-Mannitol ~100mg/ml vs BPA-Fructose 30mg/ml • Improved formulation of BPA means higher

concentration delivery for volume• No issues with renal function eg crystallisation• Intrinsically better BBB penetration• Reasonable infusion periods

Study aims

Evaluate the toxicity of the BPA-Mannitol. Evaluate alternate routes of infusion (intra-

venous vs intra-arterial to 10B To investigate the impact of BBB disruption on

BPA availability in ECF and uptake into tumours. To investigate pharmacokinetic behaviour of BPA. To incorporate data on LAT-1 amino acid

transporter activity and expression into a final pharmacokinetic model.

Eligibility Criteria

45-75 years Presumptive diagnosis of high grade glioma Undergoing surgery or image guided biopsy

for diagnosis WHO performance status Lab tests EDTA renal clearance test

Sampling

Blood for B PK assay (-0.5h to +48h post start of Infusion)

Brain biopsies for pathology & B assays (3h, 3.5 and 4h post infusion)

CSF for B assay (at time of biopsies if accessible)

ECF (Via Brain microdialysis) for B assay (0h to +48h)

Urine for B for assay (-0.5h to +48h)

Trial Design II

• Route of delivery Using single dose BPA (175mg/kg/h for 2h) via– central venous – intra-carotid artery (close arterial infusion)

• Effect of Mannitol on BBB absorption of BPA With and without rapid (30s) Mannitol

infusion (300ml 20%)

Study Plan

BPA route Mannitol BBB

Status

Cohort 1 3 Patients

IV No Completed

Cohort 2 3 Patients

IV Yes Completed

Cohort 3 3 Patients

IA No Completed

Cohort4 1 Patients

IA Yes Completed

Suspected GBMCT/MRI

RecruitmentPlanning Optimization

•CV/IA lines•Microdialysis probes•Pre dose sampling

GA

Study Pathway

Suspected GBMCT/MRI



BPA infusion+/- rapid mannitol

2h

GA

Study Pathway

Suspected GBMCT/MRI




Biopsies:Tumour &BAT

Imprinting for SIMS analysis

2h 1h 30 min

30 min

Bx1 Bx 2

ICP-MS

GA

Study Pathway

Sampling of ECF, Blood, Urine up to 48hrs after start of infusion.

Suspected GBMCT/MRI




Biopsies:Tumour &BAT

Imprinting for SIMS analysis

2h 1h 30 min

Surgical Debulking based on clinical need

30 min

Bx1 Bx 2

ICP-MS

GA

48hrs

Study Pathway

Sampling of ECF, Blood, Urine up to 48hrs after start of infusion.

Results: Blood

Average Blood Data by Cohort

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

0 2 4 6 8 10 12

Times from infusion start (hrs)

Bo

ron

Co

nc

en

tra

ion

(mic

rog

/g)

Cohort 1 Average

Cohort 2 Average

Cohort 3 Average

Effect of Infusion Route on BBB concentration gradient

BloodConcentrationIn Cerebral Circulation

Time

IV infusion

Close arterial(carotid infusion)

Blood concentration plateaus due to rapid renal clearance

Birmingham Data: Route of Delivery

• Iv infusion- max blood level governed by renal clearance.

• Use of close carotid route allows artificial elevation of intracranial BPA level – direct pass effect thereby increasing transport across BBB into brain ecf ie increased bioavailability to tumour cells

CEREBRAL CIRCULATION

The brain comprises only 2% of the body's weight but receives 20% of the blood supply.

1 Close arterial infusion2 Opening Blood Brain Barrier

Exploiting the regional circulation to the Brain

Blood Brain Barrier• Brain is a privileged site• Protected from the systemic circulation by BBB.• Comprised of endothelial cell (with tight

junctions), pericyte and astrocyte end foot.• MW /Lipid solubility/selective transporters

• BUT: Difficult to get many drugs into the brain tissue

Intra-Carotid BPA infusion

Intra-carotid and central venous catheters

S


BBBSystem A

LAT-1

TumourCell

GlialCell

PassiveDiffusion

Luminal(Blood)

Abluminal(Brain)

Na+



Compartment 3

Microdialysis

Blo

od

cap

illary

Mic

rod

ialy

sis

cath

ete

r

The Role of Microdialysis.Perfusion fluid (0.3 µl/min)

Cell

ECF

Dialysate containing analyte and drug

The Role of Microdialysis.

• The estimation of tumour to normal tissue concentrations of 10B is difficult.

• Peripheral venous levels a poor indicator of normal brain 10B levels.

• Microdialysis is an established method of continuous sampling of extracellular fluid (ECF).

• Used in the the Studsvik BNCT study.

Bergenheim, et al. Journal of Neuro-Oncology (2005) 71: 287–293

Results: ECF (Brain Microdialysis)

0 2 4 6 8 10 120.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

Average ECF Data by Cohort

Cohort 1 Average

Cohort 2 Average

Cohort 3 Average

Patient 10

Time from infusion start (hrs)

Bo

ron

co

nc

en

tra

ion

(m

ico

rg/g

)

Results: ECF [B] Integrated curves

0 1 2 3 4 5 60.0

20.0

40.0

60.0

80.0

100.0

120.0Cohort 1 average: Peripheral vein

Cohort 2 average: peripheral vein plus mannitol

Cohort 3 average: carotid artery

Patient 10: carotid artery plus mannitol

Time from start of infusion (hrs)

Tim

e-in

tegr

ated

10B

leve

l EC

F in

mg/

l. hr

s

Boron Gets Inside Tumour Cells

Secondary Ion Mass Spectrometry SIMS

Allows direct measurement of distribution and amount of Boron in tumour cells

This allows a direct estimation of energy deposition in terms of alpha particle effect

From this a precise calculation of dose comparable with Xray dosimetry can be made

BloodCompartment1

Renal Clearance

TumourCells 3

BrainECF 22

K0

K12

K23

Ka12

Ke

Nb K12<Ka12 K12 and Ka12>> K23 K32 = K23

Compartmental Analysis:Three compartment model

3

~4 Hrs from Pk [BPA] > Km

Km of LAT1 in tumour

Blood [BPA}

Brain ecf [BPA]BPA level in tumour

Time Course of BPA handling by LAT-1 Tumour Cells

*

* Uptake into tumour cell continues after plasma level drops as long as BPA concentration in Brain ecf stays above Km for LAT1- --BPA washout must necessarily have at worst same or probably slower time constant as uptake resulting in prolonged level of BPA in these cells (up to 8hours post infusion) when plasma level is near zero.

1

2

3

ICA

IV

Birmingham Data: BBB opening• Evidence shows preinfusion BBB disruption with

Mannitol leads to increased delivery across BBB.• More likely to be reliably achieved with close Carotid

infusion, as IV infusion level governed by renal clearance.

• ECF microdialysis data confirm better bioavailability* with IC and Mannitol with extension of cell uptake time due to uptake kinetics of LAT1.

• LAT1 uptake will continue as long as [BPA ecf ] stays above Km.

* AUC µg/ml vs time

Retention of BPA: Plasma vs Tumourhours post plasma peak level

0 2 4 6 8 10 12 14 16 180

20

40

60

80

100

120

Arrows indicate Maximum difference between Plasma and Tumour levels As % of peak in eachTumour

Plasma

Retention of BPA: Plasma vs Tumourhours post plasma peak level

0 5 10 15 20 250

20

40

60

80

100

120

Tumour level offset for known delay in tumour Peak level Likely to increase availableTreatment window

Plasma

Tumour

Figure 6: All available data for measured boron levels in tumour biopsy samples for a wide range of boron infusion schedules including 300 mg/kg over 2 hrs (Brookhaven), 900 mg/kg over 6 hrs (Studsvik) and 350

mg/kg over 2 hrs (Birmingham). There is no clear indication of rapid clearance of boron from tumour in the 3-4 hours after the end of the infusion, while from blood (figure 5) the level reduces to less than 15 mg/kg

Tumour cellularity

Patient 5 tumour biopsy

Patient 2 tumour biopsy

0 100 200 300 400 500 600 7000

10

20

30

40

50

60

p1

p2

p3p4

p5

p6

p7

Correlation between boron uptake and Tumour cell number density

Cell number density

Boro

n u

pta

ke i

n t

um

ou

r m

easu

red

by

ICP

-MS

[u

g/g

]

Adjustment for cellularity

Results for counted stained cell populations in GBMs

A

0

10

20

30

40

50

60

70

80

90

100

LAT + PCNA + LAT + PCNA +

LAT + X-Bar = 72.6 ± 16.9

PCNA + X-Bar = 22.8 ± 16.9

LAT + PCNA + X-Bar = 4.8 ± 2.2n = 29

60-90 % of tumour cells express LAT-1

A much lower proportion are proliferating

Detta and Cruickshank, Cancer Res 2009

Major factors in BPA uptake

• BPA is taken by a specific exchange transporter protein that is upregulated in Glioma

• Under saturated conditions BPA uptake to plateau has a time course of four hours in fresh tumour tissue due to fixed LAT1 exchange.

• Hence increase in ambient concentration (bioavailability) of BPA will not increase uptake further, as efflux of BPA occurs.

• The level of tumour BPA under these conditions thus governed by available LAT1 - Biomarker

Figure 6: All available data for measured boron levels in tumour biopsy samples for a wide range of boron infusion schedules including 300 mg/kg over 2 hrs (Brookhaven), 900 mg/kg over 6 hrs (Studsvik) and 350

mg/kg over 2 hrs (Birmingham). There is no clear indication of rapid clearance of boron from tumour in the 3-4 hours after the end of the infusion, while from blood (figure 5) the level reduces to less than 15 mg/kg

Comparable evidence

?Different Populations of LAT1 Cells

Map of 18FET-PET kinetic analysis Kunz et al Neuro-Oncology 13 307-316 2011

Normalised Changes in BPA uptake

Tumour n=100

0.5

1

1.5

2

2.5

HGGHGG/BAT

Normalised peak BPA uptake in Human GBM explants under optimised conditionsDetta A, Cruickshank G 2014

Increasing or Retaining BPA Uptake: LAT1

• Saturable transporter – no ability to increase activity

• Alter kinetic behaviour-?dopamine – beware concentration effects.

• Increase available transporter Upregulation-evidence from mRNA studies that LAT1 can be

upregulated by pretreatment with PA Dimerisation – no evidence as yet

• What is the Target cell LAT 1 behaviour?

Target Cells: Proliferating vs Invading

Proliferative Invasive

LAT1 +/- +++

PCNA +++ +/-

Evidence1. MRI Characteristics ADC mapping2. Selective Biopsy

Acknowledgements• School of Physics and Astronomy, University of

Birmingham• Department of Neurosurgery, University Hospital

Birmingham• Department of Medical Physics, University

Hospital Birmingham• Regional Laboratory for Toxicology, Sandwell &

West Birmingham Hospitals Trust• Northern Institute for Cancer Research,

University of Newcastle, Newcastle-Upon-Tyne• CR-UK Formulation Unit, University of

Strathclyde, Glasgow• Surface Analysis Research Centre, The

University of Manchester, Manchester• CR-UK Drug Development Office, London

Many thanks!

Pharmacokinetic analysis of Carotid BPA-mannitol delivery to Human GBM indicates three compartment...

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