CARBON BLOCK VS. GRANULAR COLUMNS FOR BINDING OF LIVER FAILURE

TOXINS

Stephen R. Ash MD FACP and David J. Carr MsChe

HemoCleanse, Inc. and Clarian Arnett Health Lafayette, IN

ESAO 2007, Krems, Austria

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The Problem

• Capacity for high molecular weight and protein-bound toxins has limited carbon’s efficacy in extracorporeal therapy (and other sorbents). Most of the active carbon surface in granules is in the interior, hidden from the flowing stream and protein-bound toxins.

• Small particle size allows direct interaction of sorbents with macromolecules and bound toxins. However, particles of 1-10 microns are impossible to directly fabricate into columns.

• Sorbent suspensions are difficult to retain during convection at membranes.

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Adsorption Background

Transport ProcessesBulk ConvectionAxial Dispersion

Film DiffusionPore Diffusion

Surface Diffusion

Adsorption ProcessesAggregationAdsorption

DenaturationInterference

Solid Phase Reaction

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Adsorption ProcessesAdsorption Processes Diagram

Diagram courtesy Dr.

N.-H. L. Wang

S o rb en t P ar tic le

B u lk C on vection

A xia l D isp ersion

P ore D iffu sion

F ilm D iffu sion A d sorp tion S ites

P ore F lu id

S u rface D iffu sion

B u lk F lu id

In terferen ce

A d sorp tion

D en atu ra tion

S o lid P h ase R eaction

A ggregation

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Micropores vs. Mesopores

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Using sorbent regeneration avoids need for large amounts of plasma and sterile replacement fluid, making the system

easier to implement and control...

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But if the sorbents saturate, there is decreasing clearance of hepatic toxins during the treatment. Example: decreasing clearance of bilirubin over time in the MARS system (partly due to column saturation):

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Further evidence for sorbent capacity limitations

PrometheusTM : “Blood clearances of protein-bound toxins decrease over time. The rate and the efficiency of removal of albumin-bound toxins are interrelated to both the strength of the albumin binding and the saturation of the adsorption columns” (Cl tB 29.3 ± 5.1 vs. 13.7 ± 3.7)

*P. Evenepoel, Y. Vanrenterghem et al., Detoxifying Capacity and Kinetics of Prometheus® - A New Extracorporeal System for the Treatment of Liver Failure , Blood Purification

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Our Project Goals:

• Develop method of screening sorbents for detoxification applications to predict removal of small and protein-bound toxins.

• Compare efficacy of toxin removal by mesoporous carbons in several physical forms.

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Activated Carbons TestedDescription Surface Area,

m2/gramBulk Density, g/mL

Preliminary Study

MaxsorbPellets

1.5mm diameter 2,060 0.31

MaxsorbPowder

25 to 75 μm 2,060 0.31

Norit A Powder, 1 to 25 μm

1,700 0.22

Granular Norit C Gran:840 -1,700 μm

1,400 0.20

HSGD Synthetic beads, 100 -1,000 μm

~1,600 0.10

Block Immobilized powder 1,300 0.41Nanofiber Immobilized powder 800 0.21

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HSGD

500 microns 50 microns

10.0 microns 2.0 microns 1.0 microns

Micrographs courtesy Dr. VG Nikolaev

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Maxsorb Pellets

Carbon Block

Nanofiber

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Preliminary Study

Bilirubin adsorption of two carbons with similar surface areas was compared as a function of particle size. Maxsorb carbon is commercially available in pellets. It was tested as pellets and as powder after grinding in a mortar and pestle and sieving.

Equilibrium binding of bilirubin in 5% albumin was tested for these carbons. Initial [bilirubin] was up to 12 mg/dL.

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Bilirubin Adsorption by Activated CarbonPowdered vs. Granular

Bilirubin in 5% albumin at 37C

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

Equilibrium Concentration, mg bilirubin / dL

Am

ou

nt

Bo

un

d, m

g /

g c

arb

on

Norit Powder Maxsorb Powder Maxsorb Pellets

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Results of Preliminary StudyLangmuir coefficients for bilirubin

Powdered carbons had much higher bilirubin capacity than granular carbon.

Maximum Capacity,

mg/g carbon

Relative Capacity

Binding Constant,mL/mg bilirubin

Relative Binding

Constant

Maxsorb Pellets 0.069 1 32.8 17.4

Maxsorb Powder 3.5 51 1.9 1.0

Norit A 20.7 300 4.0 2.1

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Materials & MethodsActivated carbons were tested as powders in mixed

suspension and as columns of beads or immobilized particles. Test conditions were scaled from human clinical application. Isothermal adsorption of 3 compounds from aqueous solution at low concentration (50-100 ppm) was used as a screening criterion: methylene blue (MW 320), albumin (MW 66,000) and blue dextran (MW 2,000,000).

Three carbons with the highest large-molecule adsorption were tested in columns. Adsorption at 37°C and constant pH of bilirubin (MW 585) or cytokines (IL-1β, IL-6, & IL-10) from plasma was tested in a system that recirculated treated plasma to a tank simulating a patient for 10 hours.

Removal efficiency is the final toxin concentration in the tank is expressed as a percentage of the initial tank concentration.

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Results: Binding of Marker Results: Binding of Marker Molecules Molecules

Methylene Blue Removal EfficiencyInitial [Methylene Blue] = 50 ppm

94.04% 95.33% 99.22%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Granular HSGD Block

Activated Carbon

Met

hyl

ene

Rem

ova

l %

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Results: Binding of Marker Results: Binding of Marker Molecules Molecules

Albumin Removal EfficiencyInitial [Albumin] = 50 ppm

19.10%

94.13% 99.85%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Granular HSGD Block

Activated Carbon

Alb

um

in R

emo

val %

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Results: Binding of Marker Results: Binding of Marker Molecules Molecules

Blue Dextran Removal EfficiencyInitial [Blue Dextran] = 100 ppm

16%

46%

55%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Granular HSGD Block

Activated Carbon

Blu

e D

extr

an R

emo

val

%

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Results: Binding of BilirubinResults: Binding of Bilirubin

Bilirubin Removal EfficiencyInitial [Bilirubin] = 4 mg%

30%

67% 66%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Nanofiber HSGD BlockActivated Carbon

Bili

rub

in R

emo

val %

Granular carbon=near zero

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Results: Binding of CytokinesResults: Binding of Cytokines

Cytokine Removal EfficiencySingle Cytokine in 5% Albumin

49%

73%

59%

11%

80%

33%29%

59%

35%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Nanofiber HSGD Block

Activated Carbon

Cy

tok

ine

Re

vm

ov

al %

IL-1β IL-6 IL-10

Granular carbon=near zero

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Results SummaryResults Summary•Methylene blue performance is similar for all the carbons tested. •Carbon adsorbs blue dextran in proportion to its mesoporous character AND to the surface area exposed to flowing fluid. •Carbons with significant blue dextran interaction also remove bilirubin and cytokines from plasma.•Carbon block (powdered) removes bilirubin and cytokines about as well as HSGD, the best clinically tested carbon.

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Carbon Comparison   Carbon Block HSGD Nanofiber

Granular

Carbon Density +++ ++ + ++Lack of Fines +++ -- ++ --

Small Toxin Capacity ++++++

++++++

Bilirubin Capacity ++++++

++

Cytokine Capacity +++++

+++

Hemoperfusioncapable -

+++

-With coating

Additional sorbent capable + - ++ -

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Conclusions

• Blue dextran adsorption from aqueous solution is indicative of in-vitro bilirubin and cytokine binding capacities.

• Mesoporous carbons with high surface area in contact with flowing fluid are the best candidates for clinically effective sorption of protein-bound toxins.

• Examples are HSGD and carbon block (pore size range = 2 to 50 nanometers)

• For reasons of density, lack of fines, flexibility, carbon block is a practical and effective choice.

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Progress towards Carbon Block for Biological Fluid Regeneration

• Carbon type, particle size, and size of block• Purity of perfusate-AANSI standards for metals,

endotoxin, bacteria• Free of organics-GCMS assay• Case design• Sterilization of product• Priming with sterile fluid• Platform definition-regenerate dialysate, then

albumin-dialysate and plasma.

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Alternative Carbon to Consider: carbide-derived carbon

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Once the artificial liver is built, how to test it? Rats!

Peritoneal implants 107 Cells in membranes

Sorbent-Based Pheresis in the Rat

Plasmafilter and Sorbent Reactors

Animal Interface

Hydraulic Performance

R at B lo o d an d Plasm a Treatm en t Volum es

0

100

200

300

400

500

600

700

5B 7B 40B 50B 52N 56N 60N 58N 52V 43V 44V 49V 51V 55V 61V 63V Rat Number

Vo

lum

e T

reated

(m

L)

Blood Volume Treated mL) Plasma Volume Treated (mL)

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Blood cellular and chemical component testsFigure 14

Average IL-1b Treated vs Control

0

100

200

300

400

500

600

700

800

900

Pre-d

ose

Pst-d

ose2h

Pre-tr

t

Early

trt i

n

Early

trt o

ut

Late

trt in

Late

trt o

utDay

3

Day 4

time/day

pg

/ml

Treated Control

Figure 12Average WBC Treated vs Control

0

5

10

15

20

25

30

pre-dose post-dose 2h pre-trt post-trt day 2 day 3 day 4

time/day

WB

C C

ou

nt,

10

00 /

uL

Treat Control

P= 0.89

Figure 15Average IL-10 for Treated vs Control Animals

-200

0

200

400

600

800

1000

1200

1400

Pre

-do

se

Pst

-do

se2h

Pre

-trt

Ear

ly t

rt a

Ear

ly t

rt b

Lat

e tr

t a

Lat

e tr

t b

Day

3

Time

IL-1

0 co

nce

ntr

atio

n, p

g/m

L

Treated Control

1 rat

P= 0.2

P= 0.0

P= 0.1

Treatment Results include survival to death or euthanasia by defined criteria

Average survival in hours

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

T re a te d C o n tro l

Ho

urs

Pheresis with sorbents and/or cells is possible for a rat liver failure model

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Artificial liver support therapy for patients with fulminant hepatic failurecurrently used in Japan- TAD, Yoshiba et al.

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Evidence for sorbent capacity limitations

MARS : The removal efficiency of albumin-bound toxins drops after the initiation of treatment to become insignificant after 6 hours due to both the strength of the albumin binding and the saturation of the adsorption columns*

*P. Evenepoel, Y. Vanrenterghem et al., Detoxifying Capacity and Kinetics of the Molecular Adsorbent Recycling SystemContribution of the Different Inbuilt Filters, Blood Purification

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