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
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.
2
Adsorption Background
Transport Processes Adsorption Processes
Bulk Convection
Aggregation
Axial Dispersion
Adsorption
Film Diffusion
Denaturation
Pore Diffusion
Interference
Surface Diffusion
Solid Phase Reaction
3
Adsorption Processes Diagram
Bulk
Fluid
Bulk
Convection
Aggregation
Axial Dispersion
Film Diffusion
Adsorption
Sites
Adsorption
Pore
Diffusion
Denaturation
Interference
Surface
Diffusion
Pore
Fluid
Sorbent
Particle

Solid Phase
Reaction
Diagram
courtesy Dr.
N.-H. L. Wang
4
Micropores vs. Mesopores
5
Using sorbent regeneration avoids need for large amounts
of plasma and sterile replacement fluid, making the system
easier to implement and control...
6
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):
7
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
8
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.
9
Activated Carbons Tested
Description
Surface Area,
m2/gram
Bulk Density, g/mL
Maxsorb
Pellets
1.5mm diameter
2,060
0.31
Maxsorb
Powder
25 to 75 μm
2,060
0.31
Powder,
1 to 25 μm
1,700
0.22
Norit C Gran:
840 -1,700 μm
1,400
0.20
Preliminary
Study
Norit A
Granular
HSGD
Synthetic beads,
100 -1,000 μm
~1,600
0.10
Block
Immobilized powder
1,300
0.41
Nanofiber
Immobilized powder
800
0.21
10
HSGD
500 microns
10.0 microns
50 microns
2.0 microns
1.0 microns
Micrographs courtesy Dr. VG Nikolaev
11
Maxsorb
Pellets
Carbon
Block
Nanofiber
12
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.
13
Bilirubin Adsorption by Activated Carbon
Powdered vs. Granular
Bilirubin in 5% albumin at 37C
Amount Bound, mg / g carbon
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
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
Norit Powder
Maxsorb Powder
Maxsorb Pellets
14
Results of Preliminary Study
Langmuir coefficients for bilirubin
Maximum
Capacity,
mg/g carbon
Maxsorb
Pellets
Maxsorb
Powder
Norit A
Relative
Capacity
Binding Constant,
mL/mg bilirubin
Relative
Binding
Constant
0.069
1
32.8
17.4
3.5
51
1.9
1.0
20.7
300
4.0
2.1
Powdered carbons had much higher bilirubin
capacity than granular carbon.
15
Materials & Methods
Activated 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
16
concentration.
Results: Binding of Marker Molecules
Methylene Blue Removal Efficiency
Initial [Methylene Blue] = 50 ppm
100%
Methylene Removal %
90%
80%
70%
60%
50%
94.04%
95.33%
99.22%
Granular
HSGD
Block
40%
30%
20%
10%
0%
Activated Carbon
17
Results: Binding of Marker Molecules
Albumin Removal Efficiency
Initial [Albumin] = 50 ppm
100%
Albumin Removal %
90%
80%
70%
60%
50%
94.13%
99.85%
40%
30%
20%
10%
19.10%
0%
Granular
HSGD
Block
Activated Carbon
18
Results: Binding of Marker Molecules
Blue Dextran Removal Efficiency
Initial [Blue Dextran] = 100 ppm
100%
Blue Dextran Removal %
90%
80%
70%
60%
55%
46%
50%
40%
30%
20%
16%
10%
0%
Granular
HSGD
Block
Activated Carbon
19
Results: Binding of Bilirubin
Bilirubin Removal Efficiency
Initial [Bilirubin] = 4 mg%
100%
90%
Bilirubin Removal %
80%
70%
67%
66%
HSGD
Block
60%
50%
40%
30%
30%
20%
10%
0%
Nanofiber
Activated Carbon
Granular
carbon=near zero
20
Results: Binding of Cytokines
Cytokine Removal Efficiency
Single Cytokine in 5% Albumin
100%
Cytokine Revmoval %
90%
80%
80%
73%
70%
59%
60%
50%
59%
49%
40%
33%
35%
29%
30%
20%
11%
10%
0%
Nanofiber
HSGD
Block
Activated Carbon
IL-1β
Granular carbon=near zero
IL-6
IL-10
21
Results 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.
22
Carbon Comparison
Carbon Block
HSGD Nanofiber
Carbon Density
+++
++
+
++
Lack of Fines
+++
--
++
--
Small Toxin Capacity
+++
+++
+++
+++
Bilirubin Capacity
+++
+++
+
+
Cytokine Capacity
++
+++
++
+
-
With
coating
Hemoperfusion
capable
Additional sorbent
capable
+
+++
-
++
Granular
23
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.
24
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.
25
Alternative Carbon to Consider: carbide-derived carbon
26
Once the artificial liver is built, how to test it? Rats!
Peritoneal implants
107 Cells in
membranes
27
Sorbent-Based Pheresis in the
Rat
Plasmafilter and Sorbent
Reactors
Animal Interface
Hydraulic Performance
Rat Blood and Plasma Treatment Volumes
700
600
Volume Treate d (mL)
500
400
300
200
100
0
5B
7B
40B
50B
52N
56N
60N
58N
52V
43V
44V
49V
Rat Number
Blood Volume Treated mL)
Plasma Volume Treated (mL)
51V
55V
61V
63V
Blood cellular and chemical component tests
Figure 14
Average IL-1b Treated vs Control
900
800
Figure 12
700
Average WBC Treated vs Control
600
pg/ml
30
400
300
200
20
100
0
4
ay
3
D
ay
D
tr
ti
Ea
n
rl
y
tr
to
ut
La
te
tr
ti
La
n
te
tr
to
ut
Pr
etr
t
Ps
t-
10
Ea
rl
y
Pr
edo
se
do
se
2h
15
tim e/day
5
P= 0.89
Treated
Control
0
day 4
Figure 15
Average IL-10 for Treated vs Control Anim als
1400
1 rat
1200
P= 0.2
1000
800
600
P= 0.1
400
200
0
P= 0.0
-200
Day 3
Control
Late trt b
Treat
Late trt a
day 3
Early trt b
day 2
Early trt a
post-trt
time/day
Pre-trt
pre-trt
Pst-dose2h
post-dose 2h
Pre-dose
pre-dose
IL-10 concentration, pg/mL
WBC Count, 1000 / uL
25
500
Tim e
Treated
Control
32
Treatment Results include survival to death or
euthanasia by defined criteria
Average survival in hours
140.00
120.00
Hours
100.00
80.00
60.00
40.00
20.00
0.00
Treated
Control
Pheresis with sorbents and/or cells is possible for a rat liver failure model
34
Artificial liver support therapy for patients with fulminant hepatic failure
currently used in Japan- TAD, Yoshiba et al.
35
36
37
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 System
Contribution of the Different Inbuilt Filters, Blood Purification
38
39
40