U.S. patent application number 14/411665 was filed with the patent office on 2015-11-19 for extracorporeal perfusion apparatus.
This patent application is currently assigned to Fresenius Medical Care Deutschland GmbH. The applicant listed for this patent is Fresenius Medical Care Deutschland GmbH. Invention is credited to Dieter Falkenhagen, Stephan Harm, Jens Hartmann.
Application Number | 20150328387 14/411665 |
Document ID | / |
Family ID | 47747528 |
Filed Date | 2015-11-19 |
United States Patent
Application |
20150328387 |
Kind Code |
A2 |
Falkenhagen; Dieter ; et
al. |
November 19, 2015 |
EXTRACORPOREAL PERFUSION APPARATUS
Abstract
Embodiments of the invention relate to an extracorporeal
perfusion apparatus comprising an extracorporeal blood circuit for
conveying blood, a filtrate circuit for conveying blood plasma, and
a controller, wherein the filtrate circuit is connected to the
extracorporeal blood circuit by means of a filter, wherein the
filter has a sieving coefficient of 5% for substances having a
molar mass of 340,000 g/mol (relative molecular mass of 340 kDa),
and wherein a depletion agent comprising a first carrier having a
neutral, hydrophobic surface is arranged in the filtrate circuit,
wherein the perfusion apparatus comprises a dispensing means for
feeding an endotoxin-binding lipopeptide into the extracorporeal
blood circuit, wherein the endotoxin-binding lipopeptide is
selected from the group consisting of polymyxins, polymyxin
derivatives, prodrugs thereof, and a combination thereof.
Inventors: |
Falkenhagen; Dieter; (Krems,
AT) ; Hartmann; Jens; (Furth, AT) ; Harm;
Stephan; (Furth, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Fresenius Medical Care Deutschland GmbH |
Bad Homburg |
|
DE |
|
|
Assignee: |
Fresenius Medical Care Deutschland
GmbH
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20150157779 A1 |
June 11, 2015 |
|
|
Family ID: |
47747528 |
Appl. No.: |
14/411665 |
Filed: |
June 27, 2013 |
PCT Filed: |
June 27, 2013 |
PCT NO: |
PCT/EP2013/063498 PCKC 00 |
371 Date: |
December 29, 2014 |
Current U.S.
Class: |
210/96.2;
210/202 |
Current CPC
Class: |
A61M 1/3679 20130101;
B01J 20/28064 20130101; A61K 38/12 20130101; A61P 7/00 20180101;
B01J 20/28004 20130101; B01J 20/28066 20130101; B01J 20/28085
20130101; A61P 29/00 20180101; A61M 1/3472 20130101; A61P 31/04
20180101; A61K 31/745 20130101; B01J 20/321 20130101; B01J 20/3274
20130101; B01J 20/261 20130101; A61M 2202/0456 20130101; A61M
2205/3303 20130101; B01J 20/3214 20130101; B01J 20/28083 20130101;
B01J 20/264 20130101; B01J 20/28019 20130101; B01J 20/28061
20130101; B01J 20/3246 20130101; A61M 2205/75 20130101; A61P 1/16
20180101; A61M 1/3486 20140204; A61M 2202/0413 20130101; A61M
2202/049 20130101; B01J 20/28016 20130101 |
International
Class: |
A61M 1/34 20060101
A61M001/34; A61M 1/36 20060101 A61M001/36 |
Claims
1. An extracorporeal perfusion apparatus comprising an
extracorporeal blood circuit for conveying blood, a filtrate
circuit for conveying blood plasma, and a controller, wherein the
filtrate circuit is connected to the extracorporeal blood circuit
via a filter, wherein the filter has a sieving coefficient of 5%
for substances having a molar mass of 340,000 g/mol (relative
molecular mass of 340 kDa), and wherein a depletion agent
comprising a first carrier having a neutral, hydrophobic surface is
arranged in the filtrate circuit, wherein the extracorporeal
perfusion apparatus comprises a dispenser configured to feed an
endotoxin-binding lipopeptide into the extracorporeal blood
circuit, wherein the endotoxin-binding lipopeptide is selected from
the group consisting of polymyxins, polymyxin derivatives, prodrugs
thereof, and a combination thereof.
2. The extracorporeal perfusion apparatus according to claim 1,
wherein the endotoxin-binding lipopeptide is a polymyxin selected
from the group consisting of polymyxin B, Colistin, and prodrugs
thereof.
3. The extracorporeal perfusion apparatus according to claim 1,
wherein the depletion agent comprises the dispenser configured to
feed the endotoxin-binding lipopeptide, wherein the surface of the
first carrier has an adsorptive coating formed of the
endotoxin-binding lipopeptide.
4. The extracorporeal perfusion apparatus according to claim 1,
wherein the dispenser configured to feed the endotoxin-binding
lipopeptide is arranged in the filtrate circuit downstream of the
depletion agent, wherein the dispenser comprises a second carrier
having a neutral, hydrophobic surface, wherein the surface of the
second carrier has an adsorptive coating formed of the
endotoxin-binding lipopeptide.
5. The extracorporeal perfusion apparatus according to claim 3,
wherein the endotoxin-binding lipopeptide adsorbed at the surface
of the first or second carrier is present in a quantity that, when
the lipopeptide is fed, gives a lipopeptide serum concentration
from 0.01 .mu.g/ml to 0.8 .mu.g/ml.
6. The extracorporeal perfusion apparatus according to claim 3,
wherein the first or the second carrier has a total surface from
100 to 1500 m.sup.2/g, wherein 50 to 2000 mg of endotoxin-binding
lipopeptide in relation to the total carrier surface are bonded
adsorptively at the surface of the first or second carrier.
7. The extracorporeal perfusion apparatus according to claim 1,
wherein the dispenser configured to feed the endotoxin-binding
lipopeptide comprises a dosing device for feeding the
endotoxin-binding lipopeptide into the extracorporeal blood circuit
at a lipopeptide feed point associated with the extracorporeal
blood circuit.
8. The extracorporeal perfusion apparatus according to claim 7,
wherein the lipopeptide feed point is arranged in the
extracorporeal blood circuit downstream of the filter.
9. The extracorporeal perfusion apparatus according to claim 8,
wherein a dialyser is arranged in the extracorporeal blood circuit
downstream of the filter, wherein the lipopeptide feed point is
arranged in the extracorporeal blood circuit downstream of the
dialyser.
10. The extracorporeal perfusion apparatus according to claim 8,
wherein a sensor configured to measure the concentration of the
endotoxin-binding lipopeptide is arranged downstream of the filter
or of the dialyser and upstream of the lipopeptide feed point.
11. The extracorporeal perfusion apparatus according to claim 7,
wherein the controller of the perfusion apparatus is configured,
when the lipopeptide is dosed into the blood conveyed in the
extracorporeal blood circuit, to take into consideration the
lipopeptide clearance of the body, the lipopeptide clearance of the
depletion agent and/or the lipopeptide clearance of the
dialyser.
12. The extracorporeal perfusion apparatus according to claim 1,
wherein the dispenser configured to feed the endotoxin-binding
lipopeptide comprises a dialyser arranged in the extracorporeal
blood circuit downstream of the filter, said dialyser being
configured to supply the endotoxin-binding lipopeptide to the
extracorporeal blood circuit using a dialysis fluid conveyed
through the dialyser.
13. The extracorporeal perfusion apparatus according to claim 1,
wherein the first and the second carrier are formed from a neutral
polymer.
14. The extracorporeal perfusion apparatus according to claim 13,
wherein the polymer is selected from a cross-linked polystyrene
polymer or a cross-linked ethylene divinylbenzene polymer.
15. The extracorporeal perfusion apparatus according to claim 1,
wherein the first or the second carrier is porous and has a mean
pore size of 100 nm or less.
16. The extracorporeal perfusion apparatus according to claim 15,
wherein the first or the second carrier has a mean pore size of 20
nm or less or a mean pore size from 80 to 100 nm.
17. The extracorporeal perfusion apparatus according to claim 1,
wherein the first or the second carrier is fibre-like or is in
particle form.
18. The extracorporeal perfusion apparatus according to claim 17,
wherein the first or the second carrier has the form of
microparticles having a mean particle size of 300 .mu.m or
smaller.
19. The extracorporeal perfusion apparatus according to claim 18,
wherein the first carrier has a mean pore size from 10 to 20 nm or
80 to 100 nm and a mean particle size from 75 to 150 .mu.m.
20. The extracorporeal perfusion apparatus according to claim 3,
wherein the filtrate circuit leads into the filter, and in that the
first carrier has the form of microparticles and the filtrate
circuit comprises a suspension of these microparticles, wherein the
microparticles have a mean particle size of 20 .mu.m or smaller.
Description
[0001] The invention generally relates to an extracorporeal
perfusion apparatus comprising an extracorporeal blood circuit for
conveying blood, a filtrate circuit for conveying blood plasma, and
a controller, wherein the filtrate circuit is connected to the
extracorporeal blood circuit by means of a filter, wherein the
filter has a sieving coefficient of 5% for substances having a
molar mass of 340 000 g/mol (relative molecular mass of 340 kDa),
and wherein a depletion agent comprising a first carrier having a
neutral, hydrophobic surface is arranged in the filtrate
circuit.
[0002] Sepsis and associated complications contribute to a not
inconsiderable extent to morbidity and mortality in humans. In most
cases, sepsis can be attributed to an infection with gram-negative
bacteria when high endotoxin concentrations reach the body and have
a systemic effect.
[0003] Endotoxins are lipopolysaccharides (LPSs) in the cell wall
of gram-negative bacteria and are released by cell lysis and cell
division. In fact, lipopolysaccharides are the most common lipid
component of the outer cell membrane of gram-negative bacteria.
Endotoxins are pyrogenic substances, and the individual affected
responds with a strong inflammatory reaction and fever when
endotoxins enter the body, for example during the course of
microbial poisoning, and, as key mediators, cause an uncontrolled
activation of the mononuclear phagocyte system. An accumulation of
endotoxins in the blood circuit as a result of endotoxemia leads to
an uncontrolled activation of the immune cells and to an imbalance
of the coagulation system. This can lead to sepsis, which is
characterised inter alia by high fever, low blood pressure and, in
severe cases, by multi-organ failure. Sepsis is a condition to be
taken very seriously; the lethality of individuals with severe
sepsis or septic shock is approximately 30-60% depending on the
degree of severity of the condition. Endotoxemia as a result of an
infection with gram-negative bacteria is one of the most common
causes for the occurrence of a systemic inflammatory response
("systemic inflammatory response syndrome", SIRS), sepsis, severe
sepsis or septic shock and the resultant serious complications.
Patients with jeopardised immune defence, such as liver patients or
chemotherapy patients, are susceptible to bacterial infections and
thus display symptoms of endotoxin poisoning. Endotoxemia may also
occur in the case of acute liver failure or acute decompensation
with chronic liver failure, thus resulting in the development of
states that are very similar (from a biochemical viewpoint) to
sepsis. By way of example, acute decompensation may occur in
patients with chronic liver failure. In this state, the endotoxins
originating from the normal intestinal flora pass the intestinal
barrier and stimulate the release of inflammation mediators in the
body and therefore cause a sepsis-like state.
[0004] Furthermore, septic states can also be triggered by
gram-positive bacteria, viruses and fungi.
[0005] As mentioned, it is generally known that an uncontrolled
activation of the immune cells and an imbalance of the coagulation
system may occur in the case of sepsis and other serious
conditions. The uncontrolled activation of the mononuclear
phagocyte system stimulates an excessive release of inflammation
mediators, in particular of cytokines (also referred to as cytokine
storm or hypercytokinemia). Cytokines are key mediators in the case
of sepsis and septic shock. Tumour necrosis factor (TNF-.alpha.,
often also referred to merely as TNF) and interleukin-1.beta.
(IL-1.beta.) can be cited as the most important pro-inflammatory
examples. Further important pro-inflammatory cytokines include IL-6
and IL-8. The initially released cytokine TNF-.alpha. triggers a
biological signal amplification via a mediator cascade, thus
resulting in physiological changes, including severe disruptions to
the biological balance and subsequently to circulatory collapse and
multi-organ failure. The clinical picture of sepsis correlates with
high blood concentrations of the key mediator TNF-.alpha., but also
of other cytokines, such as IL1-.beta., IL-6 and IL-8 in the case
of the pro-inflammatory phase or IL-10 or IL-13 with the occurrence
of an anti-inflammatory phase, in which the pro-inflammatory
mediators inclusive of cytokines have very low concentrations.
Furthermore, other serious conditions, such as chronic inflammatory
intestinal diseases, psoriasis and rheumatoid arthritis are also
associated with excessive TNF-.alpha. release.
[0006] Besides the intensive medical treatment applied as standard,
antibiotics or corticosteroids, immunoglobulins and also
circulation-assisting drugs in particular are used for the
treatment of sepsis.
[0007] A disadvantage of antibiotic therapy is the increasing
spread of antibiotic-resistant bacteria. Furthermore, endotoxins
are increasingly released by the antibiotic and the accompanying
destruction of the bacteria cells, which in turn leads to an
increased distribution of inflammation mediators. In addition, an
administration of antibiotics is often associated with side
effects, such as changes to the intestinal flora or allergic
reactions. The attempt to use antibiotics against the key factor
TNF-alpha failed, since with this method the reduction of the TNF
concentration to zero or very low values appeared to trigger an
anergic situation, which was accompanied by a higher mortality
compared with the control group. The therapeutic use of specific
antibodies against LPS and TNF-.alpha. is technically very complex
and is therefore associated with high costs.
[0008] By means of extracorporeal blood or plasma purification
systems (therapeutic apheresis method), it is therefore attempted,
as will be described in greater detail hereinafter, to remove the
aforementioned cytokines, in particular the factor TNF-.alpha., in
such a way as to normalise the concentrations of these cytokines so
as to thus avoid the anergic (anti-inflammatory) phase. Endotoxins
can be eliminated by means of what are known as LPS-adsorbers (for
example the adsorber Toraymyxin.RTM.) so as to thus avoid a release
of the pro-inflammatory cytokines, which naturally also reduces the
anti-inflammatory response.
[0009] Apheresis methods are methods carried out extracorporeally,
in which pathophysiologically relevant blood and plasma components,
for example biomolecules such as (glycol) proteins, peptides,
lipids, lipoproteins and lipopolysaccharides, but also blood cells
and blood plasma, are removed. Apheresis methods can be used on the
one hand for diagnostic and therapeutic purposes, but on the other
hand also constitute a very effective possibility for obtaining
certain blood components from healthy individuals in sufficient
quantity and with sufficiently high purity. Great importance is
attributed to therapeutic apheresis, since, with certain
indications, this is often a very effective alternative, at the
same time having few side effects, compared to treatment with
drugs. In the case of plasma apheresis methods, the plasma can thus
either be completely separated or replaced by a substitute
solution, or only certain components, such as cytokines LDL,
endotoxins or immunoglobulins, are removed therefrom by means of an
adsorber, and the plasma is then returned again to the
donor/patient. Compared with the aforementioned treatment
strategies using drugs, therapeutic apheresis methods also have the
advantage that the treatment is stopped at any time with immediate
effect by switching off the apheresis apparatus.
[0010] Apheresis methods and adsorber materials for eliminating
toxic and/or harmful blood components are well known in the prior
art. Adsorber materials which specifically adsorb cytokines, in
particular TNF-.alpha., and/or endotoxins (LPSs), and remove these
from bodily fluids such as blood or plasma are also known.
[0011] Document US 2001/0070424 A1 discloses an adsorber material
based on a porous polymer, which has at least one transport pore
with a diameter from 25 to 200 nm and also effective pores with a
diameter from 10 to 25 nm. Inter alia, the polymer may also be a
non-ionic resin (neutral resin). The adsorber is used to remove
protein molecules, in particular cytokines and .beta.2
microglobulin.
[0012] Document WO 2011/123767 A1 discloses a method for treating
inflammation, wherein a therapeutically effective dose of porous
adsorber particles for adsorbing inflammation mediators is
administered to a patient, wherein the total pore volume with a
pore size from 5 to 300 nm is greater than 0.5 cc/g to 3.0
cc/g.
[0013] In WO 2003/090924 a porous separation matrix for separating
blood components is described in conjunction with inflammation
processes. The separation matrix has a pore size from 5 .mu.m to
500 .mu.m and also at least one functional group arranged on the
matrix.
[0014] DE 19515554 A1 discloses methods and apparatuses for
simultaneous extracorporeal elimination of TNF-.alpha. and
lipopolysaccharides from whole blood and/or blood plasma. Here, the
blood or blood plasma is guided in an extracorporeal perfusion
system via a porous cation exchanger material and an anion
exchanger material. The porous carrier materials described therein
have a mean pore diameter of <30 nm and/or a molecular exclusion
size for globular proteins of <10.sup.6 Dalton and in particular
<2.times.10.sup.4 Dalton.
[0015] Neutral resins for removing toxic components, including
cytokines, from a bodily fluid are also disclosed in WO 2005/082504
A2. WO 2005/082504 A2 describes a detoxification apparatus, which
comprises activated carbon and at least one non-ionic resin having
a mean pore size of 30 nm and a mean particle diameter of 35-120
.mu.m (Amberchrom CG300C) or having a means pore size of 45 nm and
mean particle diameter of 560 .mu.m (resin based on aliphatic
esters-Amberlite XAD-7HP).
[0016] EP 0787500 B1 and EP 0958839 B1 disclose a hydrophobic
carrier material having a pore size from 10 to 30 nm and particle
sizes from 20 to 350 .mu.m, preferably 10 to 100 .mu.m or 250 to
350 .mu.m, for removing toxic components, in particular cytokines,
from a bodily fluid.
[0017] EP 1 944 046 B1 discloses a carrier material based on a
polystyrene-divinylbenzene copolymer having a pore size of 30 nm
and a particle size from 75 to 120 .mu.m.
[0018] Tetta et al. (Tetta et al. 1998. Nephrol Dial Transplant
13:1458-1464) describe an adsorber of the Amberchrom CG 300md type
having a pore size of 30 nm for removing cytokines from a bodily
fluid.
[0019] The publication by Cantaluppi et al. (Cantaluppi et al.
2010. Critical Care 14:R4) describes an adsorber of the Amberchrom
CG161m type for cytokine adsorption.
[0020] It has also been found that anion exchanger resins (for
example DEAE or PEI groups bound to cellulose) are very well suited
for endotoxin binding. However, the undesirable binding of key
factors of the intracorporeal coagulation system, such as protein C
and protein S, and the associated coagulation problems are
disadvantageous. These coagulation problems can be avoided by the
use of a specific adsorber which comprises immobilised antibodies
against endotoxins. However, this possibility can only be applied
to a limited extent for economical reasons.
[0021] In DE 199 13 707 A1, an immune adsorber for use in sepsis
therapy for plasmapheresis is described, consisting of a carrier
material formed from organic or synthetic polymers and polyclonal
or monoclonal antibodies bonded thereto and directed against
complement factors, lipopolysaccharides and also against further
sepsis mediators, such as TNF-.alpha. and interleukins.
[0022] DE 10 2004 029 573 A1 discloses an apheresis material or
adsorbent and also a method for removing, depleting or inactivating
the cytokine MIF (macrophage migration inhibitory factor) from
blood, blood plasma or other bodily fluids. The adsorbent comprises
a fixed carrier material on the surface of which MIF-binding
molecules or functional groups are immobilised.
[0023] DE 10 2005 046 258 A1 discloses an immune adsorber for
treating insulin resistance and/or the metabolic syndrome, wherein
the immune adsorber comprises carrier materials with bonded ligands
which are specific for IL-6, IL-4 and C5a.
[0024] A therapy form already used for a long time in clinical
application is constituted by the parenteral administration of
polymyxins. Polymyxins are antibiotic substances which originate
initially from the bacteria Bacillus polymyxa and which have
already been used for decades in humans and animals in order to
treat infections with gram-negative bacteria. Polymyxins interfere
with the cell wall structure by increasing the permeability of the
cell membrane, thus resulting in cell lysis. Polymyxins bind not
only phospholipids, but also endotoxins (LPS) so as to form a
polymyxin-endotoxin (LPS) complex with high affinity. The
anti-bacterial mechanism of polymyxins is described in detail for
example in a publication by Tony Velkov et al. (Tony Velkov et al.
2010. Journal of Medicinal Chemistry: 53(5):1898-1916).
[0025] Due to the neurotoxic and nephrotoxic effect of polymyxins,
only polymyxin B and polymyxin E (Colistin) have gained a certain
therapeutic importance as antibiotic. Until now, these two
polymyxins were the only therapeutically admissible representatives
of their substance class. Polymyxin B and Colistin are authorised
in the USA by the FDA for parenteral infusion. Polymyxin B and
Colistin have been used for decades for oral or topical therapy
forms. However, for parenteral systemic treatment of conditions and
states caused by an infection with gram-negative bacteria, they are
only used as antibiotic in a therapeutic context as a last resort
due to their neurotoxic and nephrotoxic side effects. Colistin
appears to be less nephrotoxic than polymyxin B, however this is
offset at least in part by the necessary higher dosing, and
therefore nephrotoxic reactions are to be expected to approximately
the same extent in everyday clinical practice. However, there is
not currently sufficient data available regarding the
nephrotoxicity of the two antibiotics. Infectologists from New York
(USA) describe kidney failure in 14% of 60 patients treated with
polymyxin B. Doctors in Greece describe significant nephrotoxicity
in the majority of patients in which renal insufficiency was
already present at the start of therapy. By contrast, in patients
with normal kidney function, no significant changes were
established. A detailed overview concerning the toxicity of
polymyxins can be found in a publication by Falagas and Kasiakou
(Falagas and Kasiakou. 2006. Critical Care 10:R27). The dosing of
polymyxins consequently plays a central role in the avoidance or
minimisation of toxic side effects, in particular nephrotoxic side
effects.
[0026] Due to the occurrence, observed frequently in recent years,
of severe progressions of disease caused by infections with
multi-resistant pathogenic strains, for example in the case of
acute infections with strains of the bacterium Pseudomonas
aeruginosa, polymyxins are increasingly being administered
parenterally as antibiotic by necessity, in spite of their
toxicity. A source of supply for polymyxin B in the form of the
sulphate salt of polymyxin B1 and B2 for parenteral administration
is currently offered by Bedford Laboratories ("Polymyxin B for
Injection 500 000 Units", manufacturer: Bedford Laboratories). In
accordance with manufacturer information, the parenteral
administration is carried out intravenously, intramuscularly or, in
the case of meningitis, intrathecally, wherein the specified
maximum daily dose is generally 2.5 mg/kg body weight per day,
divided between two to three infusions. The serum concentration of
polymyxin following administration typically lies in a range from 1
to 6 .mu.g/ml. in severe cases this may also be higher in a range
from 6 to 50 .mu.g/ml. Colistin is administered predominantly in
the form of Colistin methanesulfonate, wherein the serum
concentration lies in a range from approximately 1 to 3 .mu.g/ml.
Colistin (polymyxin E) is used in a manner similar to polymyxin B,
usually in higher dosage.
[0027] A resistance to polymyxin B is rather unusual, but may
develop if the antibiotic does not reach the cytoplasma membrane
due to changes in the outer membrane. Polymyxins are effective
against many gram-negative pathogens, such as E. coli,
Enterobacter, Klebsiella spp. and also against P. aeruginosa.
Proteus types and S. marcescens, which are normally resistant; the
sensitivity of B. fragilis is variable. The minimum inhibitory
concentrations for E. coli lie in the range from 0.04-3.7 mg/l and
for P. aeruginosa between 1.2 and 33.3 mg/l (Garidel and
Brandenburg. 2009. Anti-Infective Agents in Medicinal Chemistry,
8:367-385).
[0028] Since the dosages for polymyxin B and Colistin used
previously in clinical application in the case of parental
administration induce nephrotoxic and neurotoxic side effects, new
treatment strategies and therapy approaches have been developed in
the past in conjunction with the application of endotoxin-binding
lipopeptides such as polymyxin.
[0029] The extracorporeal blood and/or blood plasma purification
methods, already mentioned previously, with use of suitable
adsorber materials have become established as frequently applied
alternatives to the administration of polymyxins in the form of a
drug.
[0030] Known adsorber materials comprise porous or fibre-like
carrier materials, on the surfaces of which polymyxin B is
immobilised. Known neurotoxic and nephrotoxic side effects have
been reported previously in conjunction with adsorber materials of
this type, which are used to a large extent in the treatment of
septic states.
[0031] In EP 0110 409 A, polymyxin B-immobilised carriers formed
from porous glass (FPG 2000) and also polymyxin B-immobilised
polysaccharide carriers based on cellulose (Cellulofine A-3) are
disclosed. Microparticles formed from cellulose or derivatised
cellulose, to which polymyxin B is covalently bonded, are also
known (Weber V., Loth F., Linsberger I., Falkenhagen D.: Int. J.
Artif. Organs 25(7), 679). EP 0 129 786 A2 describes an endotoxin
detoxification material with a fibre-like carrier, on which
polymyxin is covalently immobilised. The fibre-like carrier is
equipped with functional groups in order to bind polymyxin
covalently to the surface of the carrier. Disadvantages of the
specified endotoxin adsorbers include the low endotoxin binding
capacity and speed. The efficacy and quality of the treatment in
relation to fibre-like carriers with covalently bonded polymyxin B
have been described as sub-optimal (Cruz D N et al. 2007.
Effectiveness of polymyxin B-immobilized fiber column in sepsis: a
systematic review. Crit. Care 11(3):137).
[0032] WO 2010/083545 and WO 2011/160149 describe adsorber
materials with which polymyxin is immobilised on hydrophobic
carrier surfaces via non-covalent interactions (adsorption). WO
2007/142611 A1 and U.S. Pat. No. 5,510,242 describe hydrophobic
carrier surfaces with adsorptively bonded polymyxins. The use of
polymyxin-coated polyester fabrics for binding LPS antigens of
Salmonella typhimurium was described by Blais and Yamazaki (Blais
and Yamazaki. 1990. Use of polymyxin-coated polyester cloth in the
enzyme immunoassay of Salmonella lipopolysaccharide antigens.
International journal of Food Microbiology 11:195-204).
[0033] WO 2011/133287 A1 discloses a blood filtration apparatus,
which comprises a microfluidic separation apparatus and with which
undesirable substances such as toxins, drugs, pathogens and the
like, can be removed from the blood. The apparatus may comprise
sensors which monitor the blood in terms of the presence or
concentration of the undesirable substances. The monitoring may
also include the infusion of therapeutic active ingredients, such
as an antibiotic, into the blood of the patient.
[0034] An extracorporeal perfusion apparatus of the type mentioned
in the introduction has been described for example by Falkenhagen
et al. (Falkenhagen et al. 2006. Fluidized Bed Adsorbent System for
Extracorporeal Liver Support. Therapeutic Apheresis and Dialysis
10(2):154-159). The filter described therein is obtainable under
the trade name "Albuflow.RTM." (Fresenius Medical Care,
Germany).
[0035] Although the lethality of patients suffering from
endotoxemia-induced conditions, in particular sepsis, could be
reduced by the clinical application of the above-mentioned
polymyxin-based adsorber materials, the lethality of patients with
severe sepsis and septic shock is still very high in spite of
maximum therapy. For this reason and also due to the
ever-increasing problem of the multi-resistance of bacteria to
antibiotics and the associated rising incidence of severe
progressions of disease, there is also a high demand for improved
therapy forms and more efficient extracorporeal perfusion
apparatuses, which additionally are quite safe in clinical
application.
[0036] The object of many embodiments of the invention is therefore
to provide an extracorporeal perfusion apparatus of the type
mentioned in the introduction, with which an improved treatment of
sepsis and sepsis-like states is possible.
[0037] The object is achieved by an extracorporeal perfusion
apparatus of the type mentioned in the introduction, which is
characterised in accordance with many embodiments of the invention
in that the perfusion apparatus comprises a dispensing means for
feeding an endotoxin-binding lipopeptide into the extracorporeal
blood circuit, wherein the endotoxin-binding lipopeptide is
selected from the group consisting of polymyxins, polymyxin
derivatives, prodrugs thereof and a combination thereof.
[0038] Thanks to numerous embodiments of the invention, an improved
treatment of sepsis and sepsis-like states compared to the
previously known therapy approaches is possible.
[0039] A first major advantage of the perfusion apparatus according
to many embodiments of the invention lies in the fact that the
filter not only constitutes a barrier for endotoxins and other
high-molecular plasma components, but also for the formed
endotoxin-lipopeptide complexes, such that endotoxin-lipopeptide
complexes present in the blood of the patient, which circulate in
the extracorporeal blood circuit prior to being broken down in the
liver, cannot enter the filtrate circuit and cannot reach the
carrier. Contact with the carrier, due to competitive interaction
processes between complex and first carrier surface, would lead to
a dissolution of the endotoxin-lipopeptide complex, whereby this
may worsen the state of a patient with sepsis. The renewed supply
of endotoxins caused by the dissociation of the
lipopeptide-endotoxin complexes causes a renewed intensification of
the activation process of the complement or coagulation system and
also cellular systems (monocytes) caused by endotoxins, these
activation processes being associated with corresponding clinical
consequences such as the initiation or intensification of
multi-organ failure or the initiation of the anergic stage of
sepsis, that is to say the stage in which the immune system is
weakened. The consequence of this means that the release of
endotoxins should be prevented in any case.
[0040] Thanks to a number of embodiments of the invention,
endotoxin-binding lipopeptides can be fed to the blood by means of
the dispensing means, and endotoxins can be eliminated by complex
formation, and at the same time undesirable blood components, in
particular cytokines, can be depleted by the depletion agent,
whereby maximum therapy without additional safety risk for the
patient is possible.
[0041] A further key advantage of the perfusion apparatus according
to many embodiments of the invention also lies in the fact that
undesirable blood components, in particular cytokines, can be
removed from the blood plasma by adsorption at the surface of the
first carrier due to the depletion agent arranged in the filtrate
circuit. The inventors have surprisingly found that the adsorption
efficiency for cytokines, first and foremost TNF-.alpha., with use
of the filter used in accordance with numerous embodiments of the
invention is significantly better compared with a plasma filter
that retains only cellular blood components, although fewer
cytokines from the extracorporeal blood circuit pass through the
filter into the fractionated plasma conveyed in the filtrate
circuit. The filter used in accordance with many embodiments of the
invention practically completely prevents the permeation of
proteins or lipoproteins and glycoproteins having a relative molar
mass above 300,000. It has been found that this advantage
guarantees a much better reproducibility of the cytokine
elimination compared with the use of a plasma filter that retains
only cellular components.
[0042] The filter used in accordance with a number of embodiments
of the invention allows fractionated blood plasma to pass through,
such that high-molecular blood components, endotoxins and also
endotoxin-lipopeptide complexes are retained, whereas smaller blood
components can pass through the filter membrane. A suitable filter
is obtainable under the trade name "Albuflow.RTM." (manufacturer:
Fresenius Medical Care; material: polysulfone hollow fibres;
sieving coefficient for albumin of .gtoreq.0.6 and for fibrinogen
.ltoreq.0.1).
[0043] The term "blood plasma" used herein, in so far as this is
conveyed in the filtrate circuit of the apparatus according to many
embodiments of the invention, therefore relates to fractionated
blood plasma.
[0044] The expression "carrier having a neutral, hydrophobic
surface" within the scope of this disclosure relates to a
water-insoluble solid, which has a neutral and hydrophobic surface.
The term "neutral" is to be understood non-ionically. The carrier
can be in fibre or particle form. The carrier may also be porous
and may have outer and inner surfaces. The outer and inner surfaces
are neutral and hydrophobic. The term "inner surface" of the
carrier denotes the totality of the surfaces of the pores. The term
"outer surface" by contrast relates to the totality of the surfaces
of the carrier that are directly accessible from outside.
[0045] The terms "polymyxin" and "polymyxins" as used herein relate
to known, naturally occurring chemical compounds which originate
initially from the bacterium Bacillus polymyxa (polymyxin B) and
also Bacillus colistinus (polymyxin E). The polymyxins can either
be isolated from bacteria or can be produced synthetically.
Polymyxin B originating from the bacterium is composed of 6
derivatives referred to as polymyxin B1, polymyxin B2, polymyxin
B3, polymyxin B4, polymyxin B5 and polymyxin B6. By contrast, the
polymyxin authorised by the FDA for parenteral infusion is composed
only of polymyxin B1 to B4. As mentioned previously, only polymyxin
B and Polymyxin E (Colistin) are of clinical relevance.
[0046] The term "prodrug" as used herein relates to precursor
compounds of polymyxins as defined above, wherein the precursor
compounds are converted in vivo into the active polymyxin.
Representative examples include the prodrugs Colistin
methanesulfonate and polymyxin B methanesulfonate sodium.
[0047] The term "polymyxin derivative" relates to a compound
derived from polymyxin, which compound is obtainable by
modification of naturally occurring polymyxins, for example by
chemical modification of the Dab side chains, the cyclic peptide
ring or the fatty acid chain of the polymyxin molecule structure. A
detailed overview of polymyxin-based antibiotics, analogues and
derivatives is described in the publication by Velkov et al.
(Velkov et al. 2010. Journal of Medicinal Chemistry,
53(5):1898-1916). The suitability of a polymyxin derivative for use
in many embodiments of the present invention can be tested by a
person skilled in the art on the basis of simple routine tests.
[0048] The term "endotoxemia" is used herein for all disease
patterns in which clinically relevant quantities of endotoxins are
found in the blood of the patient and lead subsequently to disease
patterns such as sepsis and SIRS.
[0049] The term "depletion agent" relates to an agent with which
undesirable components can be removed from the blood plasma
conveyed in the filtrate circuit. Depletion agents that comprise a
carrier having a neutral, hydrophobic surface have proven
themselves in the past to be particularly favourable for the
elimination of inflammation mediators such as cytokines by
adsorption at the carrier surface thereof. These cytokines are
advantageously potentially harmful pro-inflammatory cytokines.
Representative examples for pro-inflammatory cytokines include
TNF-.alpha., IL-1.beta., IL-6 and IL-8, wherein TNF-.alpha. is of
particular importance as an initial pro-inflammatory inflammation
mediator. The depletion agent used in accordance with a number of
embodiments of the invention is therefore particularly favourable
for the treatment of conditions and states attributed to the toxic
effects of TNF-.alpha.. By way of example, in the case of sepsis,
the values for the TNF-.alpha. in the pro-inflammatory phase are at
least greater than 100-200 ng/ml. The examples specified below
provide proof that TNF-.alpha., IL-1.beta., IL-6, IL-8 and also the
anti-inflammatory IL-10 are eliminated as efficiently as possible
and that the apparatus according to many embodiments of the
invention is extraordinarily well suited for the treatment of
sepsis, septic shock and sepsis-like states.
[0050] The expression "dispensing means for feeding an
endotoxin-binding lipopeptide into the extracorporeal blood
circuit" relates on the one hand to dispensing means for feeding
the lipopeptide directly into the extracorporeal blood circuit. On
the other hand, this expression also relates to dispensing means
for indirectly feeding the lipopeptide into the extracorporeal
blood circuit in that the lipopeptide is dispensed into the
filtrate circuit by the dispensing means and the lipopeptide then
passes from there into the extracorporeal blood circuit.
[0051] Since naturally occurring polymyxins, which originate
initially from the bacteria Bacillus polymyxa and also Bacillus
colistinus, are among the peptide antibiotics studied to the
greatest extent and have already been used for decades in the
treatment of conditions and states caused by endotoxemia, it is
preferable if the endotoxin-binding lipopeptide is a polymyxin. The
lipopeptide is particularly preferably selected from the group
consisting of the only polymyxins previously authorised for
clinical use: polymyxin B and Colistin (polymyxin E) and prodrugs
thereof. Representative examples include the prodrugs Colistin
methanesulfonate and polymyxin B methanesulfonate sodium. However,
polymyxin B and prodrugs thereof is most preferred since this has
proven to be the most successful for use in the field of human
medicine.
[0052] In accordance with a first advantageous embodiment, the
depletion agent comprises the dispensing means for feeding the
endotoxin-binding lipopeptide, wherein the surface of the first
carrier of the depletion agent has an adsorptive coating formed
from the endotoxin-binding lipopeptide. In this embodiment, the
depletion agent thus also acts as a dispensing means for the
endotoxin-binding lipopeptide, since the first carrier is coated
adsorptively with the lipopeptide. The lipopeptide is released into
the filtrate circuit continuously by desorption in small quantity
and is fed further from there to the extracorporeal blood circuit.
The lipopeptide is thus fed into the extracorporeal blood circuit
by dispensing of the lipopeptide (desorption from the first
carrier) into the filtrate circuit, where it then passes on into
the blood circuit. In a sub-variant, the filtrate circuit can be
formed as an open filtrate circuit, which leads downstream of the
filter into the extracorporeal blood circuit. In another
sub-variant, the filtrate circuit can be formed as a circuit that
is closed in the filtrate region and that leads into the filter. It
has been found in laboratory tests that the adsorptive coating of
the carrier surface with the lipopeptide has no disadvantageous
effects on the adsorption of the cytokines (see Example 11
below).
[0053] The term "adsorptive coating" used in this disclosure is to
be understood to mean that the endotoxin-binding lipopeptides bind
to the neutral, hydrophobic carrier surface via non-covalent,
adsorptive processes and interactions. It is to be assumed that in
particular the hydrophobic interaction plays an important role. The
hydrophobic interaction is of great biochemical importance and is
based on the phenomenon that hydrophobic molecules in a polar
environment tend toward association. The hydrophobic interaction
therefore is not a force per se, but is enforced by a polar
environment. Other non-covalent interactions, including, without
limitation, ionic bonds, hydrogen bridge bonds and van der Waals
interactions, may also play a role in the adsorption of
endotoxin-binding lipopeptides such as polymyxin. The binding of
endotoxin-binding lipopeptides such as polymyxin via non-covalent
interactions to hydrophobic carrier surfaces of various pore and
particle sizes has already been described in WO 2010/083545, WO
2011/160149, WO 2007/142611 A1 and U.S. Pat. No. 5,510,242. The
endotoxin-binding lipopeptide bound adsorptively on the carrier is
selected in accordance with a number of embodiments of the
invention from the group consisting of polymyxins, prodrugs
thereof, and a combination thereof.
[0054] Alternatively to the first embodiment, the dispensing means
for feeding the endotoxin-binding lipopeptide is arranged in the
filtrate circuit downstream of the depletion agent in a second
advantageous embodiment, wherein the dispensing means comprises a
second carrier having a neutral, hydrophobic surface, wherein the
surface of the second carrier has an adsorptive coating formed of
the endotoxin-binding lipopeptide. The lipopeptide is released
continuously into the filtrate circuit by desorption in small
quantity and is then fed further from there to the extracorporeal
blood circuit. The lipopeptide is thus fed into the extracorporeal
blood circuit by dispensing of the lipopeptide (desorption from the
second carrier) into the filtrate circuit, where it then passes on
into the blood circuit. In a sub-variant of the second embodiment,
the filtrate circuit can be formed as an open filtrate circuit,
which leads downstream of the filter into the extracorporeal blood
circuit. In another sub-variant of the second embodiment, the
filtrate circuit can be formed as a circuit that is closed in the
filtrate region and that leads into the filter. Due to the lower
design/equipment outlay however, the first embodiment is preferred
compared with the second embodiment.
[0055] The development of the two above-mentioned embodiments
(first and second embodiment) of the perfusion apparatus according
to the invention is based on the surprising fact that with carriers
that have a neutral, hydrophobic surface and that have an
adsorptive coating formed of polymyxin, the endotoxin elimination
is not implemented, as previously assumed, by adsorption of the
endotoxins at the polymyxin molecules immobilised on the carrier,
but via a very small quantity of desorbed polymyxin molecules that
have transferred into the blood or blood plasma. This surprising
and unforeseeable finding is based on the fact that, following
selective washing of a carrier coated adsorptively with polymyxin,
no endotoxin adsorption could be determined by the polymyxin
molecules still immobilised on the carrier surface. The inventors
could therefore determine that the excellent endotoxin elimination
efficiency of neutral, hydrophobic carrier polymers, on the
surfaces of which polymyxin is adsorptively bonded, is to be
attributed to a very small quantity of free polymyxin molecules
desorbed from the carrier material and released into the bodily
fluid, that is to say blood or blood plasma. The finding that the
small quantities of released polymyxin, which, depending on the
polymyxin quantity adsorbed at the carrier, give a polymyxin serum
concentration from approximately 0.01 .mu.g/ml to approximately 0.8
.mu.g/ml, are already sufficient to inhibit the activity of
endotoxins, wherein neurotoxic and nephrotoxic side effects are
excluded, was also surprising.
[0056] Only on the basis of this surprising finding was it possible
for the inventors to develop the first and second advantageous
embodiments of the perfusion apparatus according to the invention.
Before this, it was always assumed that the endotoxin elimination
was implemented by binding of the endotoxins to the polymyxin
molecules adsorbed at the carrier. In view of the fact that a
filter with a sieving coefficient of 5% for substances with a molar
mass of 340 000 g/mol (relative molecular mass of 340 kDa)
constitutes a barrier for endotoxins (LPS) and the endotoxins
therefore cannot reach an adsorber material for endotoxins arranged
in the filtrate circuit, there would have been an incentive for the
first time, in the knowledge of this new surprising fact, to
combine with a filter of this type a carrier that has an adsorptive
coating with an endotoxin-binding lipopeptide and that dispenses a
predefinable quantity of lipopeptide into the blood plasma. Due to
the dispensing means arranged in the filtrate circuit, a prolonged
release of very small and above all uniform quantities of
endotoxin-binding lipopeptides into the blood plasma conveyed in
the filtrate circuit over the total treatment period, preferably
from 4 to 10 hours daily over a period from 2 to 8 days, is
therefore achieved by the dispensing means arranged in the filtrate
circuit. From here, the lipopeptides pass into the extracorporeal
blood circuit, where they form a complex with the endotoxins (LPS)
located in the blood and therefore make these harmless. Thanks to
the filter, these complexes as already described above can no
longer pass into the filtrate circuit and be dissolved again,
whereby patient safety is kept high. The endotoxin-lipopeptide
complexes are then broken down predominantly in the liver of the
patient.
[0057] The endotoxin-binding lipopeptide adsorbed at the surface of
the first or second carrier is preferably present in a quantity
which, when the lipopeptide is dispensed, gives a lipopeptide serum
concentration from 0.01 .mu.g/ml to 0.8 .mu.g/ml as already
mentioned. It has surprisingly been found that the very low
desorption of the lipopeptide from the first or second carrier is
sufficient to obtain lipopeptide serum concentrations from 0.01
.mu.g/ml to 0.8 .mu.g/ml. It has been found that at these serum
concentrations the activity of endotoxins is inhibited, wherein
neurotoxic and nephrotoxic effects are to be excluded. The
lipopeptide serum concentration preferably lies in a range from 0.1
.mu.g/ml to 0.6 .mu.g/ml, preferably 0.1 .mu.g/ml to 0.4 .mu.g/ml,
most preferably between 0.1 .mu.g/ml to 0.25 .mu.g/ml, since at
these serum concentrations, even with severe progressions of
disease such as sepsis, severe sepsis or septic shock, efficient
therapy can be carried out without neurotoxic and nephrotoxic side
effects.
[0058] The first or second carrier preferably has a total surface
from 100 to 1500 m.sup.2/g, wherein 50 to 2000 mg are bound
adsorptively at the surface of the first or second carrier to
endotoxin-binding lipopeptide in relation to the total carrier
surface. In this way, a lipopeptide serum concentration from
approximately 0.01 .mu.g/ml to approximately 0.8 .mu.g/ml can be
obtained by desorption of the endotoxin-binding lipopeptide from
the carrier surface. For a person skilled in the art, the
anticipated lipopeptide serum concentration in relation to the used
carrier under consideration of the average pore size and/or average
particle size can be determined on the basis of simple routine
tests and calculations; calculation examples are specified further
below in the examples.
[0059] Alternatively to the above-mentioned embodiments, which are
based on the desorption of very low quantities of the lipopeptide
from a carrier surface, the dispensing means for feeding the
endotoxin-binding lipopeptide comprises a dosing device in
accordance with a third advantageous embodiment for feeding the
endotoxin-binding lipopeptide into the extracorporeal blood circuit
at a lipopeptide feed point associated with the extracorporeal
blood circuit. The lipopeptide feed point is preferably arranged
downstream of the filter. If a dialyser is additionally arranged in
the extracorporeal blood circuit downstream of the filter, it is
then favourable if the lipopeptide feed point in the extracorporeal
blood circuit is arranged downstream of the dialyser. In a
sub-variant of the third embodiment, the filtrate circuit can be
formed as an open filtrate circuit, which leads downstream of the
filter into the extra corporeal blood circuit. In another
sub-variant of the third embodiment, the filtrate circuit can be
formed as a circuit that is closed in the filtrate region and that
leads into the filter.
[0060] In this embodiment, the lipopeptide is infused into the
extracorporeal blood circuit by means of a dosing device. The
lipopeptide is preferably present in the form of a preparation for
parental administration, for example as an infusion solution. The
preparation may optionally comprise at least one pharmaceutically
acceptable carrier and/or excipient. The preparation may comprise
only one type of an endotoxin-binding lipopeptide or a mixture of
two or more lipopeptides, for example a mixture of polymyxin B1,
B2, B3 and B4. A "pharmaceutically acceptable carrier and/or
excipient" may be any substance that is known for production of
parenteral administration forms such as injections, infusion
solutions and the like. Formulations for infusion solutions
suitable for many embodiments of the invention are specified
further below in Examples 4 and 5.
[0061] The endotoxin-binding lipopeptide is preferably present in
the form of a lyophilised powder for production of a sterile
aqueous injection preparation or infusion preparation, wherein the
powder can be dissolved for example in sterile water, 5% dextrose
solution, a ringer solution or a physiological sodium chloride
solution. Polymyxin B is preferably used in the form of polymyxin B
sulphate. The lipopeptide is present in the preparation in
dissolved form, preferably in a concentration from 0.04 mg/1 to 13
mg/l, more preferably from 0.1 mg/1 to 7 mg/l, most preferably from
0.5 mg/1 to 4 mg/l. The dosing is preferably set such that the
lipopeptide serum concentration lies in range from 0.1 .mu.g/ml to
0.6 .mu.g/ml, preferably 0.1 .mu.g/ml to 0.4 .mu.g/ml, most
preferably between 0.1 .mu.g/ml to 0.25 .mu.g/ml, since at these
serum concentrations an efficient therapy can be carried out
without neurotoxic and nephrotoxic side effects, even with severe
progressions of disease, such as sepsis, severe sepsis or septic
shock.
[0062] The dosing device is formed, as is known per se by a person
skilled in the art, by infusion units that typically comprise a
container containing the infusion solution (for example infusion
bag or infusion bottle), a tube system and a pump means for dosing
a desired volume per unit of time.
[0063] The infusion rate is dependent on the serum half-life for
the lipopeptide in the patient. By way of example, the serum
half-life for polymyxin B in patients with normal kidney function
is typically 13 hours, and that for Colistin is 6 to 7.4 hours in
accordance with the literature. In the case of treatment by means
of the perfusion apparatus, the clearance of the filter and/or the
clearance of the depletion agent for the administered lipopeptide
is also to be taken into consideration, as described in detail
further below. Formulations for infusion solutions suitable for
this purpose and also dosing instructions are specified further
below in the examples.
[0064] In order to monitor the concentration of the
endotoxin-binding lipopeptide and in order to be able to adjust the
dosing of the infusion solution accordingly, it is advantageous if
a measuring means for measuring the concentration of the
endotoxin-binding lipopeptide is arranged downstream of the filter
or dialyser and upstream of the lipopeptide feed point. Suitable
measuring means, for example sensors, which can be used for this
purpose are described in the prior art, for example by Jiang et al.
(Jiang et al. 2004. A synthetic peptide derived from
bactericidal/permeability-increasing protein neutralizes endotoxin
in vitro and in vivo. International Immunopharmacology 4:527-537).
For the measurement, a small quantity of blood is preferably
conveyed from the extracorporeal blood circuit via a branch line to
the measuring means/sensor and is rejected once the concentration
has been determined. An insertion of the measuring means/sensor
directly into the extracorporeal blood circuit is indeed possible
in principle, but is less preferable, since in this case high
demands are placed on the state of the measuring means/sensor in
terms of sterility and biocompatibility. For these reasons, the
variant with the branch line to the measuring means/sensor is to be
preferred.
[0065] The perfusion apparatus may also be assigned a control
circuit controlled by means of the controller, wherein, by
actuating the infusion pump, the infused quantity of the
lipopeptide is controlled with respect to a predefinable target
value or target value range depending the lipopeptide current value
(lipopeptide serum concentration) measured by the measuring means.
The target value or target value range of the lipopeptide serum
concentration typically lies in a range of 0.01-0.8 .mu.g/ml.
[0066] With the third embodiment it is also advantageous if the
controller of the perfusion apparatus is designed, when dosing the
lipopeptide into the blood conveyed in the extracorporeal blood
circuit, to take into consideration the lipopeptide clearance of
the body, the lipopeptide clearance of the depletion agent and/or
the lipopeptide clearance of the dialyser. By way of example, it is
known that carriers formed of a polystyrene divinylbenzene
copolymer, besides pathophysiologically relevant components such as
cytokines, also adsorb lipopeptides such as polymyxins, such that
the consideration of the lipopeptide clearance of the first carrier
(that is to say carrier of the depletion agent) is advantageous for
the dosing of the infused lipopeptide.
[0067] Alternatively to the above-mentioned embodiments, the
dispensing means for feeding the endotoxin-binding lipopeptide
comprises, in a fourth embodiment, a dialyser, which is arranged in
the extracorporeal blood circuit downstream of the filter and which
is designed to feed the endotoxin-binding lipopeptide to the
extracorporeal blood circuit by means of a dialysis fluid conveyed
through the dialyser. In a sub-variant, the filtrate circuit can be
formed as an open filtrate circuit, which leads downstream of the
filter into the extracorporeal blood circuit. In another
sub-variant, the filtrate circuit can be formed as a circuit that
is closed in the filtrate region and that leads into the filter.
The used dialysers are preferably hydrophilic polysulfone membranes
with a surface of 1.4-2.0 m.sup.2, which have been produced by
blending with PVP (polyvinylpyrrolidone). By way of example, these
membranes are used in filters provided by the company Fresenius
Medical Care, inter alia in the models AF 1000 and FX60. These
dialysis filters have a sieving coefficient for albumin below 0.1%.
The use of what is known as a high cut-off filter, which is also
based on the use of hydrophilic polysulfone membranes having a
sieving coefficient of approximately 4% for albumin, is also
conceivable. Under dialysis conditions, that is to say a
diffusion-controlled elimination of the substances intended for
removal is primarily used, the albumin loss is less than 5-10 g per
treatment. An example of such a dialysis filter is constituted by
the EMiC.sup.2 filter produced by the company Fresenius Medical
Care. The flow conditions at which such filters are operated in
clinical use are selected appropriately for the blood flow 80-300
ml/min depending on use conditions: under the conditions of what is
known as continuous veno-venous haemodialysis, blood flows of 60-80
ml/min are used, whereas in the case of dialysis unit-assisted
intermittent haemodialysis, blood flows of 150-300 ml/min are used
in acute cases, that is to say in patients with acute kidney
failure, which also occurs very frequently in the case of sepsis.
The dialysate flow is preferably set to 500 ml/min in the case of
intermittent haemodialysis, whereas in the case of continuous
veno-venous haemodialysis, dialysate flows in a ratio of 1:1 to the
blood flow are conventional. The concentration of the
lipopeptide/polymyxin in the dialysis fluid should lie in the range
of 0.2-1.0 .mu.g/l, that is to say should be slightly higher than
the controlled serum concentration value of the patient to be
treated, since the sieving coefficient of the aforementioned
dialysis filter is between 0.8 (AF 1000) and 0.9 (EMiC.sup.2), that
is to say between 80 and 90%.
[0068] In practice, it is particularly expedient if the first or
second carrier is a neutral, preferably synthetic polymer. A good
reproducibility of the carrier material can thus be ensured. If it
is a porous polymer, a good reproducibility in particular in terms
of the porosity and particle size can then be ensured. The porosity
and particle size can additionally vary very widely. The polymer
may be both a homopolymer and a hetropolymer. These polymers are
also known under the name "non-ionic macroreticular polymer resins"
and for example are obtainable under the trade names "Amberchrom"
and "Amberlite XAD" (Rohm&Haas/Dow Chemical Company).
[0069] For practical application, cross-linked polystyrene polymers
and cross-linked ethylvinylbenzene polymers have proven to be
particularly favourable. In the case of extracorporeal blood
purification, high demands are placed on the sterility of the
apparatus parts that come into contact with the bodily fluids of
the patient. Cross-linked polystyrene polymers and cross-linked
ethylvinylbenzene polymers are characterised by a high stability to
heat and chemicals and are already established in clinical
practice. In an advantageous variant, the cross-linked polystyrene
polymer is a polystyrene divinylbenzene copolymer. In a further
advantageous variant, the cross-linked ethylvinylbenzene polymer is
an ethylvinylbenzene-divinylbenzene copolymer.
[0070] It is of course also possible, instead of the preferred
polystyrene-divinylbenzene copolymer or
ethylvinylbenzene-divinylbenzene copolymer, to use other neutral
resins of high hydrophobicity which are well known to a person
skilled in the relevant art. Representative examples for other
neutral, hydrophobic polymers suitable for many embodiments of the
invention include, for example, polymers from styrene and
ethylvinylbenzene monomers cross-linked with trivinylcyclohexane,
trivinylbenzene, divinylnaphthalene, divinyl sulfone,
trimethylolpropane triacrylate, trimethylpropane trimethacrylate or
resins based on aliphatic esters, and mixtures thereof.
[0071] The first or the second carrier is advantageously porous and
has a mean particle size of 100 nm or smaller, preferably in a
range from 1 to 100 nm. A particularly large inner surface is thus
created for the depletion of undesirable components, such as
cytokines, or for the adsorptive coating with endotoxin-binding
lipopeptide.
[0072] Although a person skilled in the art is familiar with the
meaning of the term "mean pore size" and the way in which the
porosity or the mean pore size can be adjusted purposefully, this
term will still be defined briefly at this juncture for reasons of
clarity. The mean pore size relates to the mean diameter of the
pores. With a Gaussian size distribution of the pore diameters of a
porous material, the mean pore diameter is that corresponding to
the maximum of the distribution curve. The mean pore diameter can
be determined for example by means of nitrogen adsorption (as
described in Weber et al. 2008; Neutral styrene divinylbenzene
copolymers for adsorption of toxins in liver failure.
Biomacromolecules 9(4):1322-1328)) or by means of mercury
intrusion. The pore size of a polymer is adjusted by varying the
concentration of the involved monomers or co-monomers, the solvent
or the modulator. The smaller the pores of the polymer are
selected, the greater is the inner surface of the polymer that is
available for the adsorption of molecules (in this case undesirable
blood components such as cytokines and/or endotoxin-binding
lipopeptides). The larger the pores, the better is the
accessibility of the pores for larger molecules. A production
method for a synthetic, hydrophobic polymer of defined pore size,
as can be used for many embodiments of the invention, is described
in the above-mentioned publication by Weber et al.
[0073] The production of such carriers by copolymerisation of
monovinyl and polyvinyl aromatic monomers, carried out as
suspension polymerisation, is also known from U.S. Pat. No.
4,382,124. The polyvinyl aromatic monomers are used as
cross-linkers of the polymers. For example, styrene and/or
ethylstyrene, preferably ethylstyrene, is/are used as monovinyl
aromatic compounds. Divinylbenzene is preferably used as polyvinyl
aromatic compound. The porosity is obtained by addition of porogens
to the monomers, which are removed again following the
polymerisation. The porogens may have hydrophobic or hydrophilic
properties. Examples include toluene and xylene for hydrophobic
porogens and C4-C10 alcohols for hydrophilic porogens. However,
mixtures of these two porogens classes can also be used. The pore
sizes of the polymer carriers can be varied within wide limits by
the degree of cross-linking of the polymers, the type and quantity
of the porogens and the reaction conditions during polymerisation.
A person skilled in the relevant art will know which parameters to
select in order to obtain a neutral, hydrophobic carrier of
desirable pore size.
[0074] A special embodiment of the production of porous hydrophobic
carriers is constituted by preferably post cross-linking
styrene-divinylbenzene copolymers by "hyper cross-linking"
(Davankov et al. J. Polymer Science, 47, 95-101 (1974). Examples of
such carriers include the Hypersol-Macronet sorbents by the
Purolite Company. Carriers with very large inner surface can thus
be produced, which contain practically only micropores <2
nm.
[0075] Carriers with neutral, hydrophobic surface and variable mean
pore size and particle size can be acquired for example from
Rohm&Haas/Dow Chemical Company and are obtainable under the
trade names "Amberchrom CG" and "Amberlite XAD". Fine-pore carriers
with neutral, hydrophobic surface and a mean pore size of
approximately 2.5 nm and smaller are obtainable from the company
Purolite (for example Hypersol-Macronet MN270, polystyrene
divinylbenzene polymer with a mean pore size of 2.5 nm (25
.ANG.)).
[0076] The total porosity of a carrier having a mean pore size from
1 to 100 nm is preferably 0.3-0.8 cm.sup.3/g polymer. The inner
surfaces (or total surfaces) preferably lie in a range from 100 to
1500 m.sup.2/g.
[0077] The mean pore size of the first or of the second carrier
advantageously lies in a range of 20 nm smaller, preferably 1 to 20
nm, or in a range from 80 to 100 nm, since in these specific pore
size ranges an adsorption of protein C by the first carrier or the
second carrier is much less pronounced than in a pore size range of
greater than 20 nm and less than 80 nm. With pore sizes <20 nm,
less protein C passes into the pores. The protein C adsorption by
the carrier rises with increasing pore size and decreases again
with pore sizes greater than 80 nm. Although the protein C binding
can be lowered to a minimum with larger selected pore sizes
(greater than 80 nm), it is favourable for clinical application if
the mean pore size is selected so as to be no greater than 100 nm,
since the inner surface of the carrier may otherwise become too
small. Protein C, a vitamin K-dependent protein in the blood
plasma, can pass through the filter used in accordance with many
embodiments of the invention and consequently comes into contact
with the first or second carrier arranged in the filtrate circuit.
Protein C is an important regulator of the blood clotting process
and has an anti-coagulatory effect. It can be assumed that the
adsorption of protein S is also much lower, since protein S (62 000
Da) has a similar relative molecular weight compared with the
protein C (69 000 Da). The same considerations apply to coagulation
factors of similar molecular weight, such as factor VII, factor IX
and factor X.
[0078] The total porosity of a carrier having a mean pore size of
20 nm or less is preferably 0.4 to 0.8 cm.sup.3/g polymer. The
inner surfaces (or total surfaces) preferably lie in a range from
300 to 1500 m.sup.2/g.
[0079] The total porosity of a carrier having a mean pore size from
80 to 100 nm is preferably 0.4 to 0.8 cm.sup.3/g polymer. The inner
surfaces (or total surfaces) preferably lie in a range from 100 to
500 m.sup.2/g.
[0080] In further variants, the first and the second carrier may be
fibre-like or may be in particle form. The first and the second
carrier, however, are preferably in particle form. Carriers in
particle form are easier to handle compared with fibrous carriers.
In addition, the porosity of particles can be produced and adjusted
more easily.
[0081] The mean particle size of polymer carriers can be adjusted
during polymerisation in the known manner, for example by type and
quantity of suspension stabiliser and geometry and rotational speed
of the agitator. Hydrophobic carriers with neutral, hydrophobic
surface and variable mean pore size and mean particle size can be
acquired for example by Rohm/Haas/Dow Chemical Company and are
obtainable under the trade names "Amberchrom CG" and "Amberlite
XAD". Fine-pore carriers of variable mean pore size and mean
particle size are produced for example by the company Purolite.
[0082] AC fibres from the company Mast Carbon (UK) constitute
examples for a suitable fibre-like carrier.
[0083] The first or the second carrier preferably has the form of
microparticles having a mean particle size of 300 .mu.m or smaller,
preferably 2 to 300 .mu.m. With larger particle sizes from this
range, improved blood compatibility is provided by the smaller
outer surface, whereas smaller particles are characterised by
higher dynamic efficacy.
[0084] In a particularly preferred embodiment, the first carrier
has a mean pore size from 10 to 20 nm or a mean pore size from 80
to 100 nm and an mean particle size from 75 to 150 .mu.m. it has
been found that this particle size range is advantageous in terms
of the undesirable adsorption of protein C (see example 8 below).
Coagulation complications (venous thromboses, pulmonary embolisms),
which are caused by an undesirable protein C adsorption, can thus
be minimised. With a particle size from 75 to 150 .mu.m, the outer
surface of the carrier still appears to be small enough to bind
protein C to an insignificant extent, if at all; however, it is
clearly still large enough to keep small the diffusion paths for
the substances to be adsorbed. In this preferred embodiment, the
particle size is at least 75 .mu.m, since physiologically relevant
quantities of protein C still remain here in the blood or blood
plasma, even after relatively long incubation. In an advantageous
sub-variant, the first carrier has a mean pore size from 10 to 20
nm and a mean particle size from 75 to 150 .mu.m. With a mean pore
size of less than 10 nm, the adsorption efficiency for the
inflammation mediators to be removed, such as cytokines, in
particular TNF-.alpha., decreases again. With a pore size of more
than 20 nm, the inner surface is smaller and the adsorption
capacity for cytokines decreases. This embodiment of the perfusion
apparatus according to the invention inclusive of the advantageous
sub-variants described hereinafter is therefore extraordinarily
well suited for the treatment of sepsis, in particular septic
shock, and sepsis-like states.
[0085] In a particularly advantageous and preferred sub-variant of
this embodiment, the first carrier has a mean pore size from 10 to
20 nm or a mean pore size from 80 to 100 nm, preferably a mean pore
size from 10 to 20 nm and a mean particle size from 75 to 150
.mu.m, wherein the surface of the first carrier has an adsorptive
coating formed of the endotoxin-binding lipopeptide, that is to say
in this sub-variant the depletion agent also acts, as described
above in detail with reference to the first embodiment, as a
dispensing means for the endotoxin-binding lipopeptide (see also
FIGS. 1 and 2 further below). As already mentioned, it has been
found in laboratory tests that the adsorptive coating of the
carrier surface of the lipopeptide has no disadvantageous effects
on the adsorption of the cytokines. In embodiments in which a
second carrier is provided (see above or the comments below with
regard to FIG. 5), it is advantageous in accordance with a further
sub-variant if the second carrier has a pore size of 20 nm or
smaller.
[0086] Since the protein C adsorption decreases with rising mean
particle size, the first carrier in a further advantageous
sub-variant has a mean particle size from 100 to 150 m The carrier
more preferably has a mean particle size from 110 to 130 .mu.m,
ideally a mean particle size of approximately 120 .mu.m, since at
these particle sizes on the one hand toxic substances, such as
cytokines, and on the other hand important coagulation factors,
such as protein C, can hardly continue to be adsorbed, even if the
bodily fluid (blood plasma) is brought into contact with the first
carrier over a relatively long period of time.
[0087] In a variant of the extracorporeal perfusion apparatus, the
filtrate circuit leads into the extracorporeal blood circuit at a
position downstream of the filter. In this variant the filtrate
circuit is open and the fractionated plasma is fed directly to the
extracorporeal blood circuit downstream of the filter by passing
through the filtrate circuit (see schematic illustration of this
variant in FIGS. 1, 3, 5 and 7). The basic principle of this
variant is currently used in immunoadsorption (for example
apheresis apparatuses from the company AsahiKasei, Japan). The
depletion agent or the dispensing means (first or second carrier)
is preferably arranged in this variant in a device which is
arranged in the filtrate circuit and through which blood plasma can
flow and which for example can be formed as a column or
cartridge.
[0088] In another variant of the extracorporeal blood circuit, the
filtrate circuit is closed and the fractionated plasma passes via
the membrane of the filter back into the blood flowing in the
extracorporeal blood circuit (see schematic illustration of this
variant in FIGS. 2, 4 and 6). The basic principle of the second
variant is known by the blood purification system Prometheus.RTM.
(Fresenius Medical Care GmbH, Germany) [Falkenhagen D, Strobl W,
Vogt G, Schrefl A, Linsberger I, Gerner F J, Schoenhofen M.:
Fractionated plasma separation and adsorption system: a novel
system for blood purification to remove albumin bound substances.
Artif Organs. 1999 January; 23(1):81-6].). The depletion agent or
the dispensing means (first or second carrier) can be arranged in a
device which is arranged in the filtrate circuit and through which
blood plasma can flow and which for example can be formed as a
column or cartridge.
[0089] In an advantageous embodiment the filtrate circuit leads
into the filter and thus forms a circuit that is closed in the
filtrate region, wherein the first carrier has the form of
microparticles and the filtrate circuit comprises a suspension of
these microparticles, wherein the microparticles have a mean
particle size of 20 .mu.m or smaller, preferably a mean particle
size of 8 .mu.m or smaller, ideally a mean particle size of 5 .mu.m
or smaller. This embodiment is a development of the above-mentioned
embodiments, in which only the first carrier (with or without an
adsorptive coating with endotoxin-binding lipopeptide) is arranged
in the filtrate circuit. The microparticle-shaped first carrier
(with or without lipopeptide coating) circulates here as a
suspension in the filtrate circuit. Due to the particle sizes
selected so as to be very small, the risk of a pulmonary embolism
can be avoided, should the microparticles pass for example by means
of a filter leak into the extracorporeal blood circuit and then
into the body of the patient. An extracorporeal plasma circuit, in
which a suspension of microparticles is contained, constitutes a
key component of a microspheres-based detoxification system (MDS)
and has already been described in EP 0776223 B and U.S. Pat. No.
5,855,782.
[0090] Many embodiments of the invention are advantageously used
for the treatment of an infection with gram-negative bacteria, in
particular for the prophylaxis or treatment of a systemic
inflammatory reaction (SIRS), sepsis, severe sepsis or septic
shock. Representative examples for disease patterns that can be
treated by means of the perfusion apparatus according to many
embodiments of the invention are those that occur following an
infection with gram-negative bacteria and may then lead to SIRS,
sepsis, severe sepsis with multi-organ failure, or septic
shock.
[0091] Representative examples for gram-negative bacteria include
Escherichia spp, Haemophilus influenzae, Pseudomonas aeruginosa,
Pasteurella, Enterobacter spp., Salmonella spp. and Shigella spp.
Many embodiments of the invention are particularly advantageous in
the case of gram-negative bacteria for which an increased
occurrence of multi-resistant strains has been observed, wherein
Pseudomonas aeruginosa is to be highlighted here as a particularly
relevant example.
[0092] As already mentioned above, an increased endotoxin
distribution occurs or may occur with the use of antibiotics in the
case of an infection with gram-negative bacteria and as a result of
the cell lysis induced by the administration of antibiotics. An
increased endotoxin distribution has been described for example for
antibiotics that preferably bind to PBP-3 (penicillin-binding
protein-3), for example the most commonly used antibiotics in the
group of cephalosporins, such as ceftazidime. Many embodiments of
the invention are therefore used advantageously as an additional
therapeutic or prophylactic measure in the scope of conventional
treatment of bacterial infections by means of antibiotics in order
to resist endotoxin distribution induced by the administration of
antibiotics and induction of cytokines.
[0093] In a further aspect, many embodiments are advantageously
used for the prophylaxis or treatment of an inflammatory reaction
as a result of acute liver failure or acute decompensation in the
case of chronic liver failure, in particular a systemic
inflammatory reaction (SIRS), sepsis, severe sepsis with
multi-organ failure, or septic shock. In patients with intact liver
functions, the endotoxins passing from the intestine into the
bloodstream are eliminated from the reticuloendothelial system
(RES) or Kupffer cells by endocytosis. Acute decompensation may
occur in patients with chronic liver failure. In this case,
endotoxins of the normal intestinal flora pass the intestinal
barrier and therefore pass unimpeded into the liver and lead to a
systemic inflammatory reaction (SIRS), sepsis, severe sepsis with
multi-organ failure, or septic shock.
[0094] Many embodiments also relate to a method for the prophylaxis
or treatment of conditions and states caused by endotoxemia by
means of an extracorporeal perfusion apparatus according to some
embodiments of the invention. The above-specified definitions and
developments are to be applied equally to the method.
[0095] Prior to starting the treatment of the patient by means of
the perfusion apparatus according to several embodiments of the
invention, a bolus is preferably administered once in order to
quickly break down and adjust a lipopeptide serum concentration
from preferably 0.01 .mu.g/ml to 0.8 .mu.g/ml. Within the scope of
this disclosure, the term "bolus" is thus understood to mean a
one-time parenteral administration of the endotoxin-binding
lipopeptide in the form of the preparation, preferably in the form
of an injection or infusion preparation. The bolus administration
can be used advantageously in conjunction with all above-mentioned
embodiments of the apparatus according to the invention. Examples
for injection solutions for the bolus administration are specified
below in Example 4.
[0096] Numerous embodiments of the invention will be explained
hereinafter in greater detail on the basis of non-limiting examples
and drawings. In the drawings:
[0097] FIG. 1 shows a schematic illustration of an embodiment of an
extracorporeal perfusion apparatus according to an embodiment of
the invention with open filtrate circuit, wherein the depletion
agent arranged in the filtrate circuit also acts simultaneously as
dispensing means for polymyxin B,
[0098] FIG. 2 shows a schematic illustration of a further
embodiment of an extracorporeal perfusion apparatus according to an
embodiment of the invention with closed filtrate circuit, wherein
the depletion agent arranged in the filtrate circuit also acts
simultaneously as dispensing means for polymyxin,
[0099] FIG. 3 shows a schematic illustration of a further
embodiment of an extracorporeal perfusion apparatus according to an
embodiment of the invention with open filtrate circuit and a dosing
device for polymyxin,
[0100] FIG. 4 shows a schematic illustration of two further
embodiments of an extracorporeal perfusion apparatus according to
an embodiment of the invention with closed filtrate circuit and a
dosing device for polymyxin,
[0101] FIG. 5 shows a schematic illustration of a further
embodiment of an extracorporeal perfusion apparatus according to an
embodiment of the invention with open filtrate circuit, wherein the
dispensing means for polymyxin is arranged downstream of the
depletion agent in the filtrate circuit,
[0102] FIG. 6 shows a schematic illustration of a further
embodiment of an extracorporeal perfusion apparatus according to an
embodiment of the invention with closed filtrate circuit, wherein
the dispensing means for polymyxin is present in the filtrate
circuit as microparticle suspension, and
[0103] FIG. 7 shows a schematic illustration of a further
embodiment of an extracorporeal perfusion apparatus according to an
embodiment of the invention with open filtrate circuit and a
dialyser for dispensing polymyxin arranged downstream of the filter
in the extracorporeal blood circuit.
[0104] FIG. 1 shows a schematic illustration of an extracorporeal
perfusion apparatus 100 (extracorporeal blood purification
apparatus 100). The perfusion apparatus 100 has an extracorporeal
blood circuit 102 with an arterial inflow 102a (arterial branch)
from a patient 101 to a filter 104 and a venous outflow 102b
(venous branch) from the filter 104 to the patient 101. The
patient's blood is conveyed in the blood circuit 102 by means of a
blood pump 103 (pump rate Q.sub.Blut=60-300 ml/min depending on
treatment method). The filter 104 has a sieving coefficient of 5%
for substances with a molar mass of 340 000 g/mol (340 kDa), here a
filter of the Albuflow.RTM. type (manufacturer: Fresenius Medical
Care; material: polysulfone hollow fibres; sieving coefficient for
albumin of .gtoreq.0.6 and for fibrinogen .ltoreq.0.1). Some of the
blood plasma (=fractionated plasma) is filtered off by the filter
104 and fed to a filtrate circuit 105. A filter with a sieving
coefficient of 5% for substances with a relative molar mass of 340
kDa allows fractionated plasma to pass through, such that
high-molecular blood plasma components such as fibrinogen,
immunoglobulins, LDL, HDL, etc. are retained, whereas smaller blood
components such as albumin or protein C pass through the filter
membrane. The filtrate circuit 105 is formed as an open circuit,
which leads downstream of the filter 104 into the venous branch
102b. The fractionated blood plasma is conveyed through the
filtrate circuit 105 by means of a filtrate pump 106 (pump rate
Q.sub.frakt. Plasma=15-20% of Q.sub.Blut). The perfusion apparatus
100 is also assigned a controller 110 for the automated control of
the apparatus 100, said controller also being connected to the
pumps 103, 106 via signal connections. The controller 110 is
usefully also configured for central data acquisition and data
output.
[0105] The fractionated plasma conveyed through the filtrate
circuit 105 is guided through a column 107 arranged in the filtrate
circuit 105. The column 107 contains an adsorber bed 107a formed of
a carrier having a neutral, hydrophobic surface, wherein the
carrier surface has an adsorptive coating formed of lipopeptide
molecules, here polymyxin. In FIG. 1, the carrier is a polystyrene
divinylbenzene polymer with a mean particle size of 120 .mu.m and a
mean pore size from 15 to 20 nm, wherein the surface of the polymer
has an adsorptive coating with polymyxin (production of a polymer
coated with polymyxin, see example 1). The adsorber bed 107a
therefore functions on the one hand as a depletion agent for
cytokines such as TNF-.alpha., IL-6 and IL-10, since these are
adsorbed at the carrier and are removed from the plasma. On the
other hand, the adsorber bed 107a also acts as a dispensing means
for continuously dispensing polymyxin into the blood plasma by
continuously dispensing a very small quantity of polymyxin into the
fractionated blood plasma conveyed in the filtrate circuit 105
(desorption). The polymyxin passes on from there into the venous
outflow 102b of the extracorporeal blood circuit 102, where it
forms a complex with the endotoxins present in the blood and makes
these harmless.
[0106] FIG. 2 shows a schematic illustration of an extracorporeal
perfusion apparatus 200 (extracorporeal blood purification device
200). The perfusion apparatus 200 has an extracorporeal blood
circuit 202 with an arterial inflow 202a (arterial branch) from a
patient 201 to a filter 204 and a venous outflow 202b (venous
branch) from the filter 204 to the patient 201. The patient's blood
is conveyed in the blood circuit 202 by means of a blood pump 203
(pump rate Q.sub.Blut=30-70 ml/min). The filter 204 has a sieving
coefficient of 5% for substances with a molar mass of 340 000 g/mol
(340 kDa), here a filter of the Albuflow.RTM. type (manufacturer:
Fresenius Medical Care; material: polysulfone hollow fibres;
sieving coefficient for albumin of .gtoreq.0.6 and for fibrinogen
.ltoreq.0.1). Some of the blood plasma (=fractionated plasma) is
filtered off by the plasma filter 204 and is fed to a filtrate
circuit 205. A filter having a sieving coefficient of 5% for
substances with a relative molar mass of 340 kDa allows
fractionated plasma to pass through, such that high-molecular blood
plasma components such as fibrinogen, immunoglobulins, LDL, HDL,
etc. are retained, whereas smaller blood components such as albumin
or protein C pass through the filter membrane. The filtrate circuit
205 is formed as a circuit that is closed in the filtrate region,
wherein the fractionated blood plasma is conveyed thorough the
filtrate circuit 205 by means of a filtrate pump 206 (pump rate
Q.sub.frakt. Plasma=15 25% of Q.sub.Blut). The perfusion apparatus
200 is also assigned a controller 210 for the automated control of
the apparatus 200, said controller also being connected to the
pumps 203, 206 via signal connections. The controller 210 is
usefully also configured for central data acquisition and for data
output.
[0107] The fractionated plasma conveyed through the filtrate
circuit 205 is guided through a column 207 arranged in the filtrate
circuit 205. The column 207 contains an adsorber bed 207a formed of
a carrier having a neutral, hydrophobic surface, wherein the
carrier surface has an adsorptive coating formed of lipopeptide
molecules, here polymyxin. In FIG. 2, the carrier is a polystyrene
divinylbenzene polymer with a mean particle size of 120 .mu.m and a
mean pore size from 15 to 20 nm, wherein the surface of the polymer
has an adsorptive coating with polymyxin (production of a polymer
coated with polymyxin, see example 1). The adsorber bed 207a
therefore functions on the one hand as a depletion agent for
cytokines such as TNF-.alpha., IL-6, IL-10, since these are
adsorbed at the carrier and are removed from the plasma. On the
other hand, the adsorber bed 207a also acts as a dispensing means
for continuously dispensing polymyxin into the blood plasma by
continuously dispensing a very small quantity of polymyxin into the
fractionated blood plasma conveyed in the filtrate circuit 205
(desorption). The polymyxin passes on from there into
extracorporeal blood circuit 202. The polymyxin molecules then form
a complex with the endotoxins present in the blood.
[0108] FIG. 3 shows a schematic illustration of an extracorporeal
perfusion apparatus 300 (extracorporeal blood purification
apparatus 300). The perfusion apparatus 300 has an extracorporeal
blood circuit 302 with an arterial inflow 302a (arterial branch)
from a patient 301 to a filter 304 and a venous outflow 302b
(venous branch) from the filter 304 to the patient 301. The
patient's blood is conveyed in the blood circuit 302 by means of a
blood pump 303 (pump rate Q.sub.Blut=60-300 ml/min). The filter 304
has a sieving coefficient of 5% for substances with a molar mass of
340 000 g/mol (340 kDa), here a filter of the Albuflow.RTM. type
(manufacturer: Fresenius Medical Care; material: polysulfone hollow
fibres; sieving coefficient for albumin of .gtoreq.0.6 and for
fibrinogen .ltoreq.0.1). Some of the blood plasma (=fractionated
plasma) is filtered off by the filter 304 and fed to a filtrate
circuit 305. A filter with a sieving coefficient of 5% for
substances with a relative molar mass of 340 kDa allows
fractionated plasma to pass through, such that high-molecular blood
plasma components such as fibrinogen, immunoglobulins, LDL, HDL,
etc. are retained, whereas smaller blood components such as albumin
or protein C pass through the filter membrane. The filtrate circuit
305 is formed as an open circuit, which leads downstream of the
filter 304 into the venous branch 302b. The fractionated blood
plasma is conveyed through the filtrate circuit 305 by means of a
filtrate pump 306 (pump rate Q.sub.frakt. Plasma=15-25% of
Q.sub.Blut).
[0109] The fractionated plasma conveyed through the filtrate
circuit 305 is guided through a column 307 arranged in the filtrate
circuit 305. The column 307 contains an adsorber bed 307a formed of
a carrier with a neutral, hydrophobic surface. In FIG. 3 the
carrier is a polystyrene divinylbenzene polymer with a mean
particle size of 120 .mu.m and a mean pore size from 15 to 20 nm.
The adsorber bed 307a functions as a depletion agent for cytokines,
such as TNF-.alpha., IL-6 and IL-10, by adsorbing these at the
carrier and removing them from the plasma.
[0110] In order to dispense an endotoxin-binding lipopeptide, the
perfusion apparatus 300 is assigned an infusion device 308 known
per se comprising an infusion container 309 (for example infusion
bottle or infusion bag) containing a lipopeptide infusion solution,
here a polymyxin infusion solution, an infusion tube 311 and an
infusion pump 312. Suitable infusion solutions are described
further below in Example 5. The polymyxin is infused at a
lipopeptide feed point 313 into the venous outflow 302b of the
extracorporeal blood circuit 302. The polymyxin molecules then form
a complex with the endotoxins present in the blood.
[0111] FIG. 3 also shows an advantageous development, in which a
polymyxin sensor 314 is arranged downstream of the filter 304 and
upstream of the lipopeptide feed point 313. By way of example, a
polymyxin sensor as described previously by Jiang et al. (Jiang et
al. 2004. A synthetic peptide derived from
bactericidal/permeability-increasing protein neutralizes endotoxin
in vitro and in vivo. International Immunopharmacology 4:527-537)
can be used for this purpose. For the measurement, a small quantity
of blood is preferably conveyed from the extracorporeal blood
circuit 302 via a branch line to the sensor 314 and is rejected
once the concentration of the polymyxin has been determined. The
perfusion apparatus 300 is also assigned a controller 310 for the
automated control of the apparatus 300, said controller also being
connected to the pumps 303, 306, 311 and where applicable to the
polymyxin sensor 314 via signal connections. The controller 310 is
expediently also configured for central data acquisition and for
data output. The perfusion apparatus 300 may also be assigned a
control circuit controlled by means of the controller 310, wherein,
by actuating the infusion pump 312, the infused quantity of
polymyxin is controlled with respect to a predefined target value
or target value range depending on the polymyxin current value
(polymyxin serum concentration) measured by the sensor 314. The
target value or target value range of the polymyxin serum
concentration is typically in a range from 0.01-0.8 .mu.g/ml.
[0112] FIG. 4 shows a schematic illustration of an extracorporeal
perfusion apparatus 400 (extracorporeal blood purification device
400). The perfusion apparatus 400 has an extracorporeal blood
circuit 402 with an arterial inflow 402a (arterial branch) from a
patient 401 to a filter 404 and a venous outflow 402b (venous
branch) from the filter 404 to the patient 401. The patient's blood
is conveyed in the blood circuit 402 by means of a blood pump 403
(pump rate Q.sub.Blut=60-300 ml/min). The filter 404 has a sieving
coefficient of 5% for substances with a molar mass of 340 000 g/mol
(340 kDa), here a filter of the Albuflow.RTM. type (manufacturer:
Fresenius Medical Care; material: polysulfone hollow fibres;
sieving coefficient for albumin of .gtoreq.0.6 and for fibrinogen
.ltoreq.0.1). Some of the blood plasma (=fractionated plasma) is
filtered off by the filter 404 and is fed to a filtrate circuit
405. A filter having a sieving coefficient of 5% for substances
with a relative molar mass of 340 kDa allows fractionated plasma to
pass through, such that high-molecular blood plasma components such
as fibrinogen, immunoglobulins, LDL, HDL, etc. are retained,
whereas smaller blood components such as albumin or protein C pass
through the filter membrane. The filtrate circuit 405 is formed as
a circuit that is closed in the filtrate region, wherein the
fractionated blood plasma is conveyed thorough the filtrate circuit
405 by means of a filtrate pump 406 (pump rate Q.sub.frakt.
Plasma=15-25% of Q.sub.Blut).
[0113] The fractionated plasma conveyed through the filtrate
circuit 405 is guided through a column 407 arranged in the filtrate
circuit 405. The column 407 contains an adsorber bed 407a formed of
a carrier having a neutral, hydrophobic surface. In FIG. 4, the
carrier is a polystyrene divinylbenzene polymer with a mean
particle size of 120 .mu.m and a mean pore size from 15 to 20 nm.
The adsorber bed 407a acts as a depletion agent for cytokines such
as TNF-.alpha., IL-6 and IL-10, by adsorbing these at the carrier
and removing them from the plasma.
[0114] In order to dispense an endotoxin-binding lipopeptide, the
perfusion apparatus 400 is an infusion device 408 known per se
comprising an infusion container 409 (for example infusion bottle
or infusion bag) containing a lipopeptide infusion solution, here a
polymyxin infusion solution, an infusion tube 411 and an infusion
pump 412. Suitable infusion solutions are described further below
in Example 5. The polymyxin is infused at a lipopeptide feed point
413 into the venous outflow 402b of the extracorporeal blood
circuit 402. The polymyxin molecules then form a complex with the
endotoxins present in the blood.
[0115] Similarly to FIG. 3, FIG. 4 further shows an advantageous
development, in which a polymyxin sensor 414 is arranged downstream
of the filter 404 and upstream of the lipopeptide feed point 413.
By way of example, a polymyxin sensor as described previously by
Jiang et al. (Jiang et al. 2004. A synthetic peptide derived from
bactericidal/permeability-increasing protein neutralizes endotoxin
in vitro and in vivo. International Immunopharmacology 4:527-537)
can be used for this purpose. For the measurement, a small quantity
of blood is preferably conveyed from the extracorporeal blood
circuit 402 via a branch line to the sensor 414 and is rejected
once the concentration of the polymyxin has been determined. The
perfusion apparatus 400 is also assigned a controller 410 for the
automated control of the apparatus 400, said controller also being
connected to the pumps 403, 406, 411 and where applicable to the
polymyxin sensor 414 via signal connections. The controller 410 is
expediently also configured for central data acquisition and for
data output. The perfusion apparatus 400 may also be assigned a
control circuit controlled by means of the controller 410, wherein,
by actuating the infusion pump 412, the infused quantity of
polymyxin is controlled with respect to a predefined target value
or target value range depending on the polymyxin current value
(polymyxin serum concentration) measured by the sensor 414. The
target value or target value range of the polymyxin serum
concentration is typically in a range from 0.01-0.8 .mu.g/ml.
[0116] FIG. 5 shows a schematic illustration of an extracorporeal
perfusion apparatus 500 (extracorporeal blood purification device
500). The perfusion apparatus 500 has an extracorporeal blood
circuit 502 with an arterial inflow 502a (arterial branch) from a
patient 501 to a filter 504 and a venous outflow 502b (venous
branch) from the filter 504 to the patient 501. The patient's blood
is conveyed in the blood circuit 502 by means of a blood pump 503
(pump rate Q.sub.Blut=60-300 ml/min). The filter 504 has a sieving
coefficient of 5% for substances with a molar mass of 340 000 g/mol
(340 kDa), here a filter of the Albuflow.RTM. type (manufacturer:
Fresenius Medical Care; material: polysulfone hollow fibres;
sieving coefficient for albumin of .gtoreq.0.6 and for fibrinogen
.ltoreq.0.1). Some of the blood plasma (=fractionated plasma) is
filtered off by the filter 504 and is fed to a filtrate circuit
505. A filter having a sieving coefficient of 5% for substances
with a relative molar mass of 340 kDa allows fractionated plasma to
pass through, such that high-molecular blood plasma components such
as fibrinogen, immunoglobulins, LDL, HDL, etc. are retained,
whereas smaller blood components such as albumin or protein C pass
through the filter membrane. The filtrate circuit 505 is formed as
an open circuit, which leads downstream of the filter 504 into the
venous branch 502. The fractionated blood plasma is conveyed
thorough the filtrate circuit 505 by means of a filtrate pump 506
(pump rate Q.sub.frakt. Plasma=15-25% of Q.sub.Blut). The perfusion
apparatus 500 is also assigned a controller 510 for the automated
control of the apparatus 500, said controller also being connected
to the pumps 503, 506 via signal connections. The controller 510 is
usefully also configured for central data acquisition and for data
output. In FIG. 5 the filtrate circuit 505 is formed as an open
circuit. However, the filtrate circuit 505 can also be formed as a
closed circuit.
[0117] The fractionated plasma conveyed through the filtrate
circuit 505 is guided through a column 507 arranged in the filtrate
circuit 505. The column 507 contains an adsorber bed 507a formed of
a carrier with a neutral, hydrophobic surface. In FIG. 5 the
carrier is a polystyrene divinylbenzene polymer with a mean
particle size of 120 .mu.m and a mean pore size from 15 to 20 nm.
The adsorber bed 507a functions as a depletion agent for cytokines,
such as TNF-.alpha., IL-6 and IL-10, by adsorbing these at the
carrier and removing them from the plasma.
[0118] In order to dispense a lipopeptide, a further column 508 is
arranged in the filtrated circuit 505, downstream of the column
507. The column 508 contains a carrier bed 508a formed of a carrier
having a neutral, hydrophobic surface, wherein the carrier surface
has an adsorptive coating formed of lipopeptide molecules, here
polymyxin. In FIG. 5 the carrier is a polystyrene divinylbenzene
polymer with a mean particle size of 120 .mu.m and a mean pore size
from 15 to 20 nm, wherein the surface of the polymer has an
adsorptive coating with polymyxin (production of a polymer coated
with polymyxin, see Example 1). The carrier bed 508a acts a
dispensing means for continuously dispensing polymyxin into the
blood plasma by continuously dispensing a very small quantity of
polymyxin into the fractionated blood plasma conveyed in the
filtrate circuit 505 (desorption). From there, the polymyxin passes
on into the extracorporeal blood circuit 502. The polymyxin
molecules then form a complex with the endotoxins present in the
blood.
[0119] FIG. 6 shows a schematic illustration of an extracorporeal
perfusion apparatus 600 (extracorporeal blood purification device
600). The perfusion apparatus 600 has an extracorporeal blood
circuit 602 with an arterial inflow 602a (arterial branch) from a
patient 601 to a filter 604 and a venous outflow 602b (venous
branch) from the filter 604 to the patient 601. The patient's blood
is conveyed in the blood circuit 602 by means of a blood pump 603
(pump rate Q.sub.Blut=60-300 ml/min). The filter 604 has a sieving
coefficient of 5% for substances with a molar mass of 340 000 g/mol
(340 kDa), here a filter of the Albuflow.RTM. type (manufacturer:
Fresenius Medical Care; material: polysulfone hollow fibres;
sieving coefficient for albumin of .gtoreq.0.6 and for fibrinogen
.ltoreq.0.1). Some of the blood plasma (=fractionated plasma) is
filtered off by the filter 604 and is fed to a filtrate circuit
605. A filter having a sieving coefficient of 5% for substances
with a relative molar mass of 340 kDa allows fractionated plasma to
pass through, such that high-molecular blood plasma components such
as fibrinogen, immunoglobulins, LDL, HDL, etc. are retained,
whereas smaller blood components such as albumin or protein C pass
through the filter membrane. The filtrate circuit 605 is formed as
a circuit that is closed in the filtrate region, wherein the
fractionated blood plasma is conveyed thorough the filtrate circuit
605 by means of a filtrate pump 606 (pump rate Q.sub.frakt.
Plasma=15-25% of Q.sub.Blut).
[0120] The perfusion apparatus 600 is also assigned a controller
610 for the automated control of the apparatus 600, said controller
also being connected to the pumps 603, 606 via signal connections.
The controller 610 is expediently also configured for central data
acquisition and data output.
[0121] Here, the filtrate circuit 605, as depletion
agent/dispensing means 607, comprises a suspension (not illustrated
in detail) of the carrier 607a, that is to say the depletion
agent/dispensing means 607 or the carrier 607a is in microparticle
form and is present as suspension distributed in the fractionated
plasma and circulates as suspension in the filtrate circuit 605.
The carrier 607a in microparticle form has a neutral, hydrophobic
surface, wherein the carrier surface has an adsorptive coating
formed of lipopeptide molecules, here polymyxin. In FIG. 6, the
carrier 607a is a polystyrene divinylbenzene polymer with a mean
particle size of 5 .mu.m+/-3-4 .mu.m and a mean pore size of 15 to
20 nm (source of polymer acquisition: Rohm&Haas), wherein the
surface of the polymer has an adsorptive coating with polymyxin
(production of a polymer coated with polymyxin, see example 1). The
carrier 607a in microparticle form thus functions on the one hand
as a depletion agent for cytokines, such as TNF-.alpha., IL-6 and
IL-10, by adsorbing these at the carrier and removing them from the
plasma. On the other hand, the carrier 607a in microparticle form
also acts as a dispensing means for continuously dispensing
polymyxin into the blood plasma by continuously dispensing a very
small quantity of polymyxin into the fractionated blood plasma
conveyed in the filtrate circuit 605 (desorption). From there, the
polymyxin passes on into the extracorporeal blood circuit 602. The
polymyxin molecules then form a complex with the endotoxins present
in the blood.
[0122] FIG. 7 shows a schematic illustration of an extracorporeal
perfusion apparatus 700 (extracorporeal blood purification device
700). The perfusion apparatus 700 has an extracorporeal blood
circuit 702 with an arterial inflow 702a (arterial branch) from a
patient 701 to a filter 704 and a venous outflow 702b (venous
branch) from the filter 704 to the patient 701. The patient's blood
is conveyed in the blood circuit 702 by means of a blood pump 703
(pump rate Q.sub.Blut=60-300 ml/min). The filter 704 has a sieving
coefficient of 5% for substances with a molar mass of 340 000 g/mol
(340 kDa), here a filter of the Albuflow.RTM. type (manufacturer:
Fresenius Medical Care; material: polysulfone hollow fibres;
sieving coefficient for albumin of .gtoreq.0.6 and for fibrinogen
.ltoreq.0.1). Some of the blood plasma (=fractionated plasma) is
filtered off by the filter 704 and is fed to a filtrate circuit
705. A filter having a sieving coefficient of 5% for substances
with a relative molar mass of 340 kDa allows fractionated plasma to
pass through, such that high-molecular blood plasma components such
as fibrinogen, immunoglobulins, LDL, HDL, etc. are retained,
whereas smaller blood components such as albumin or protein C pass
through the filter membrane. The filtrate circuit 705 is formed as
an open circuit, which leads downstream of the filter 704 into the
venous branch 702b. The fractionated blood plasma is conveyed
thorough the filtrate circuit 705 by means of a filtrate pump 706
(pump rate Q.sub.frakt. Plasma=15-25% of Q.sub.Blut). In FIG. 7 the
filtrate circuit 705 is formed as an open circuit. However, the
filtrate circuit 705 can also be formed as a closed circuit.
[0123] The fractionated plasma conveyed through the filtrate
circuit 705 is guided through a column 707 arranged in the filtrate
circuit 705. The column 707 contains an adsorber bed 707a formed of
a carrier with a neutral, hydrophobic surface. In FIG. 7 the
carrier is a polystyrene divinylbenzene polymer with a mean
particle size of 120 .mu.m and a mean pore size from 15 to 20 nm.
The adsorber bed 707a functions as a depletion agent for cytokines,
such as TNF-.alpha., IL-6 and IL-10, by adsorbing these at the
carrier and removing them from the plasma.
[0124] In order to dispense a lipopeptide, a dialyser 708 (dialysis
filter 708) is arranged in the venous branch 702b of the
extracorporeal blood circuit 702. In the dialyser, the blood is
brought into contact with the dialysis solution via a
semi-permeable membrane. The dialysis solution is pumped by means
of a dialysis solution pump 709 into the dialyser 708 via a
dialysis solution inflow 708a. After having passed through the
dialyser 708, the dialysate is removed and disposed of via a
dialysate outflow 708b. The lipopeptide, here polymyxin, is fed to
the blood by means of the dialysis solution. In the embodiment
illustrated in FIG. 7, the dialyser 708 thus acts as dispensing
means for dispensing the lipopeptide (polymyxin) into the
extracorporeal blood circuit 702. The polymyxin molecules then form
a complex with the endotoxins present in the blood.
[0125] The dialyser 708 preferably comprises a hydrophilic
polysulfone membrane with a surface of 1.4-2.0 m.sup.2, which has
been produced by blending with PVP (polyvinylpyrrolidone). By way
of example, these membranes are used in the filters from the
company Fresenius Medical Care in models AF 1000 and FX60, inter
alia. These dialysis filters have a sieving coefficient for albumin
less than 0.1%. The use of what is known as a high cut-off filter,
which is also based on the use of hydrophilic polysulfone membranes
having a sieving coefficient of approximately 4% for albumin, is
also conceivable. Under dialysis conditions, that is to say a
diffusion-controlled elimination of the substances intended for
removal is primarily used, the albumin loss is less than 5-10 g per
treatment. The EMiC.sup.2 filter produced by the company Fresenius
Medical Care can be cited as an example of a dialysis filter of
this type. The flow conditions under which filters of this type are
operated in clinical use are selected accordingly for a blood flow
of 60-300 ml/min depending on use conditions: blood flows of 60-80
ml/min are used under the conditions of what is known as continuous
veno-venous haemodialysis, whereas blood flows of 150-300 ml/min
are used with dialysis device-assisted intermittent haemodialysis
in acute cases, that is to say in patients with acute kidney
failure, which also occurs very frequently in the case of sepsis.
The dialysate flow in the case of intermittent haemodialysis is
preferably set to 500 ml/min, whereas dialysate flows in a ratio of
1:1 to the blood flow are usual in the case of continuous
veno-venous haemodialysis. The concentration of the
lipopeptide/polymyxin in the dialysis fluid should lie in the range
of 0.2-1.0 .mu.g/l, that is to say should be slightly higher than
the controlled serum concentration value of the patient to be
treated, since the sieving coefficient of the aforementioned
dialysis filter is between 0.8 (AF 1000) and 0.9 (EMiC.sup.2) that
is to say between 80 and 90%.
[0126] The perfusion apparatus 700, for the automated control of
the apparatus 700, is also assigned a controller 710, which is also
connected to the pumps 703, 706, 709 via signal connections. The
controller 710 is expediently also configured for central data
acquisition and for data output.
1. EXAMPLE 1
Polymyxin B (PMB) Desorption in Plasma and Fractionated Plasma (Use
of an Albuflow Filter) with Differently PMB-Coated Carrier (Mean
Particle Size: 120 .mu.m, Mean Pore Size: 15-20 nm)
1.1 PMB Coating
[0127] Carrier: Amberchrom CG161c (polystyrene-divinylbenzene
copolymer, Dow Chemical Company), mean particle size 120 .mu.m,
mean pore size 15 nm; accessible surface 900 m.sup.2/g polymer
(dry). The dry weight per ml moist carrier is 18% (w/v).
[0128] Polymyxin B (PMB): polymyxin B sulphate (Sigma Aldrich)
[0129] The PMB solution (10 mg/ml in dist. water) is autoclaved at
121.degree. C., for 30 min, and the carrier is then coated in 15 ml
Greiner tubes with PMB as follows (Table 1.1): 3 ml carrier with
7.5 ml PMB solution
TABLE-US-00001 TABLE 1.1 PMB coating in Carrier PMB solution NaCl
mg per ml carrier [ml] [ml] [ml] 0 3 0 7.5 1 3 0.3 7.2 2.5 3 0.75
6.75 5 3 1.5 6 7.5 3 2.25 5.25 10 3 3 4.5
[0130] The coating is carried overnight on a roll mixer at room
temperature. The carrier is then washed twice with 10 ml NaCl
solution (sterile), and a 50% suspension is produced.
1.2 Batch Test
[0131] Freshly frozen plasma (citrate plasma) was fractionated with
the aid of the Albuflow.RTM. filter (Fresenius Medical Care,
Germany) and was frozen at -20.degree. C. together with the whole
plasma. [0132] A: whole plasma [0133] B: fractionated plasma
TABLE-US-00002 [0133] TABLE 1.2 Batch approaches 0 mg/ml 1 mg/ml
2.5 mg/ml 5 mg/ml 7.5 mg/ml 10 mg/ml A whole 0A 1 + 2 1A 1 + 2 2.5A
1 + 2 5A 1 + 2 7.5A 1 + 2 10A 1 + 2 plasma B fractionated 0B 1 + 2
1B 1 + 2 2.5B 1 + 2 5B 1 + 2 7.5B 1 + 2 10B 1 + 2 plasma
[0134] In the duplicate approach (see Table 1.2), every 0.5 ml of
carrier are incubated with 4.5 ml of plasma=10% (v/v) approach at
37.degree. C. for 60 min on an Enviro-genie. The carrier is then
centrifuged off and the supernatant is used for PMB quantification
by means of ELISA (polymyxin ELISA from Beijing Kwinbon
Biotechnology Co., Ltd., China).
1.3 Results
[0135] With rising PMB concentration, the PMB solution used to coat
the carrier desorbs a higher quantity of PMB in the plasma. The
result is shown in FIG. 8 (desorption of PMB in accordance with the
PMB coating concentration).
[0136] In order to achieve in the fractionated plasma a PMB plasma
level of approximately 150 ng/ml by means of the desorption, the
carrier used in this test has to be coated with 10 mg PMB per ml of
adsorber.
2. EXAMPLE 2
Endotoxin Batch with Differently PMB-Coated Carrier in Serum
2.1 Test Structure
[0137] Conditioned carrier (Amberchrom CG161c:
ethylvinylbenzene-divinylbenzene copolymer (Dow Chemical Company),
mean particle size 120 .mu.m, mean pore size 15 nm) is coated with
different quantity of polymyxin B (PMB): 0, 5, 10, 15 and 25 mg/g
moist carrier. These are tested in a triplicate approach in an
endotoxin batch test for LPS inactivation thereof in serum.
2.2 Test Execution
[0138] PMB Coating: Carrier samples with different PMB
concentrations (5 mg, 10 mg, 15 mg and 25 mg per g moist carrier)
are produced (see protocol above in Example 1). The PMB solution
(10 mg/ml in dist. water) and the carrier in 50% suspension are
autoclaved at 121.degree. C. for 30 min, and the carrier is coated
in 15 ml Greiner tubes with PMB as follows (Table 2.2):
TABLE-US-00003 TABLE 2.2 mg PMB/g 50% adsorber suspension PMB
solution NaCl adsorber [ml] [ml] [ml] 0 2 0 3 5 2 0.5 2.5 10 2 1 2
15 2 1.5 1.5 25 2 2.5 0.5
[0139] The coating is performed for 4 hours on an overhead shaker
(Enviro-Genie, frequency: 25:50) at room temperature. The carrier
is then washed twice with 10 ml NaCl solution (sterile), and a 50%
suspension is produced.
Production of Serum:
[0140] 7 blood tubes (vacuette with serum beads for coagulation
activation) measuring 8 ml are removed from the donor. The tubes
filled with blood are left to stand for 30 min. The coagulated
blood is then centrifuged and the serum obtained (cooled in a
sterile Erlenmeyer flask).
Endotoxin (LPS) solution:
[0141] LPS: Pseudomonas aeruginosa, L-7018 company Sigma batch:
128K4115, storage -70.degree. C., at 100 .mu.l 10.sup.-3 g/ml (1
mg/ml)
[0142] An LPS solution with a concentration of 10 .mu.g/ml is
produced from this LPS stock solution with sterile NaCl solution.
The LPS is used in the batch with a final concentration of 5 ng/ml.
10 .mu.l LPS solution with a concentration of 10 .mu.g/ml are
pipetted into 20 ml serum. The batch approach is performed in 2 ml
blood-sampling tubes in a triplicate approach.
2.3 Results
[0143] The LPS inactivation of more than 50% was able to be
achieved already at the lowest coated PMB concentration. The result
is illustrated in FIG. 9 (EU=endotoxin units).
3. EXAMPLE 3
Endotoxin (LPS) Inactivation in Accordance with the
Polymyxin-concentration on the Basis of Endotoxins from E. coli and
Pseudomonas aeruginosa
3.1. Objective
[0144] The objective of this test is to determine the polymyxin B
(PMB) concentration-dependent endotoxin activation in plasma (batch
test I). Furthermore, the extent to which this endotoxin activation
results in an inhibition of cytokine distribution is to be examined
(batch test II).
3.2. Blood donor
[0145] 9 blood-sampling tubes (each measuring 9 ml) spiked with 5
IU heparin are removed from a donor. The plasma is centrifuged off
and the cell pellet incubated on a roll mixer. The plasma is spiked
with endotoxin (LPS) and used for batch test I:
3.3. LPS Spike, Polymyxin B Solutions and Batch Test I
[0146] LPS: Pseudomonas aeruginosa (L-7018 company Sigma batch:
128K4115, -70.degree. C., at 100 .mu.l 10-3 g/ml (1 mg/ml))
[0147] LPS: E. coli (L-4130 company Sigma batch: 110M4086M,
-70.degree. C., at 100 .mu.l 10-3 g/ml (1 mg/ml))
[0148] The LPS is used in the batch with a final concentration 0.5
ng/ml. The tests are carried out in 3 ml pyrogen-free glass vials.
In batch test I, different PMB concentrations (company. Sigma,
P-1004) are added in the duplicate approach and are incubated for
60 min on an overhead shaker at 37.degree. C. (see Table 3.5).
[0149] In batch test I, PMB concentrations with 0 (without PMB),
10, 100, 250, 500 and 1000 ng/ml are used. Sterile PMB solutions
(autoclaved pyrogen-free at 121.degree. C., 90 min) are produced
for this purpose with the following concentrations (Table 3.3):
TABLE-US-00004 TABLE 3.3 PMB [ng/ml] PMB [ng/ml] in Batch (1:15)
PMB solution A 150 10 PMB solution B 1500 100 PMB solution C 3750
250 PMB solution D 7500 500 PMB solution E 15000 1000 NaCl-solution
0 0
3.4. Endotoxin Analysis
[0150] The endotoxins are measured in the form of EU/ml with the
aid of a Limulus Amebocyte Lysate test (LAL) by Charles River.
3.5. Cytokine Batch (Batch Test II)
[0151] The plasma spiked with LPS and PMB is fed back following
batch test 1 to the cell concentrate obtained from the blood donor
in the ratio 1:1 (see Table 3.5). For the cytokine batch, the
samples from batch test I were used with a PMB concentration of 0
(without PMB), 250, 500 and 1000 ng/ml. As control, a sample
without LPS and with 1000 ng/ml PMB was included. Following the
incubation times of 4 h and 12 h at 37.degree. C. on a roll mixer
(5 revolutions/min), samples were taken, centrifuged off and 50
.mu.l plasma were frozen at -80.degree. C. for the subsequent
cytokine quantification. The test data for the cytokine batch is
listed in Table 3.5.
TABLE-US-00005 TABLE 3.5 PMB Plasma + [ng/ml] PMB solution 0.5
ng/ml LPS Incubation LAL EU/ml Cytokine Batch Sample 4 h Sample 12
h LPS Pseudomonos aureginosa 0 100 .mu.l NaCl 1400 .mu.l 60 min #1
0.333 1500 .mu.l cell concentrate + 250 .mu.l.fwdarw. 50 .mu.l
plasma -80.degree. C. 250 .mu.l.fwdarw. 50 .mu.l 0 100 .mu.l NaCl
1400 .mu.l 60 min #2 0.229 1500 .mu.l LPS-PMB plasma plasma
-80.degree. C. 10 100 .mu.l sol A 1400 .mu.l 60 min #3 0.178 10 100
.mu.l sol A 1400 .mu.l 60 min #4 0.167 100 100 .mu.l sol B 1400
.mu.l 60 min #5 0.112 100 100 .mu.l sol B 1400 .mu.l 60 min #6
0.137 250 100 .mu.l sol C 1400 .mu.l 60 min #7 0.108 1500 .mu.l
cell concentrate + 250 .mu.l.fwdarw. 50 .mu.l plasma -80.degree. C.
250 .mu.l.fwdarw. 50 .mu.l 250 100 .mu.l sol C 1400 .mu.l 60 min #8
0.123 1500 .mu.l LPS-PMB plasma plasma -80.degree. C. 500 100 .mu.l
sol D 1400 .mu.l 60 min #9 0.091 1500 .mu.l cell concentrate + 250
.mu.l.fwdarw. 50 .mu.l plasma -80.degree. C. 250 .mu.l.fwdarw. 50
.mu.l 500 100 .mu.l sol D 1400 .mu.l 60 min #10 0.081 1500 .mu.l
LPS-PMB plasma plasma -80.degree. C. 1000 100 .mu.l sol E 1400
.mu.l 60 min #11 0.062 1500 .mu.l cell concentrate + 250
.mu.l.fwdarw. 50 .mu.l plasma -80.degree. C. 250 .mu.l.fwdarw. 50
.mu.l 1000 100 .mu.l sol E 1400 .mu.l 60 min #12 0.061 1500 .mu.l
LPS-PMB plasma plasma -80.degree. C. LPS E. coli 0 100 .mu.l NaCl
1400 .mu.l 60 min #13 1.8 1500 .mu.l cell concentrate + 250
.mu.l.fwdarw. 50 .mu.l plasma -80.degree. C. 250 .mu.l.fwdarw. 50
.mu.l 0 100 .mu.l NaCl 1400 .mu.l 60 min #14 1.841 1500 .mu.l
LPS-PMB plasma plasma -80.degree. C. 10 100 .mu.l sol A 1400 .mu.l
60 min #15 0.77 10 100 .mu.l sol A 1400 .mu.l 60 min #16 0.871 100
100 .mu.l sol B 1400 .mu.l 60 min #17 0.379 100 100 .mu.l sol B
1400 .mu.l 60 min #18 0.382 250 100 .mu.l sol C 1400 .mu.l 60 min
#19 0.281 1500 .mu.l cell concentrate + 250 .mu.l.fwdarw. 50 .mu.l
plasma -80.degree. C. 250 .mu.l.fwdarw. 50 .mu.l 250 100 .mu.l sol
C 1400 .mu.l 60 min #20 0.29 1500 .mu.l LPS-PMB plasma plasma
-80.degree. C. 500 100 .mu.l sol D 1400 .mu.l 60 min #21 0.209 1500
.mu.l cell concentrate + 250 .mu.l.fwdarw. 50 .mu.l plasma
-80.degree. C. 250 .mu.l.fwdarw. 50 .mu.l 500 100 .mu.l sol D 1400
.mu.l 60 min #22 0.209 1500 .mu.l LPS-PMB plasma plasma -80.degree.
C. 1000 100 .mu.l sol E 1400 .mu.l 60 min #23 0.154 1500 .mu.l cell
concentrate + 250 .mu.l.fwdarw. 50 .mu.l plasma -80.degree. C. 250
.mu.l.fwdarw. 50 .mu.l 1000 100 .mu.l sol E 1400 .mu.l 60 min #24
0.16 1500 .mu.l LPS-PMB plasma plasma -80.degree. C.
3.6. Results
Endotoxin Batch (Batch Test I):
[0152] FIG. 10 shows the inhibition of LPS from E. coli in plasma
(original LPS concentration: 0.5 ng/ml) in accordance with the PMB
concentration (n=2) following an incubation time of 60 min.
[0153] FIG. 11 shows the inhibition of LPS from Pseudomonas
aeruginosa in plasma (original LPS concentration: 0.5 ng/ml) in
accordance with the PMB concentration (n=2) following an incubation
time of 60 min.
[0154] The results clearly show that, even with a very low PMB
concentration in the plasma, that is to say in a range from 50 to
300 ng/ml (0.05 to 0.3 .mu.g/ml), a strong inhibition of LPS from
E. coli and Pseudomonas aeruginosa takes place, wherein the LPS
inhibition no longer increases significantly with rising PMB
concentration. As a result, even very low concentrations of PMB are
sufficient in order to inhibit the activity of LPS (endotoxins).
Neurotoxic and nephrotoxic side effects are to be ruled out at
these low concentrations.
Cytokine Batch (Batch Test II):
[0155] The distribution of the cytokines TNF-alpha (FIG. 12),
IL-1beta (FIG. 13), IL-6 (FIG. 14) and IL8 (FIG. 15) by the blood
cells in accordance with the PMB concentration (without PMB, 250
ng/ml, 500 ng/ml and 1000 ng/ml; control with 1,000 ng/ml without
LPS) in LPS (E. coli)-spiked plasma after 4 hours incubation is
illustrated in FIGS. 3 to 4. The results from batch test II clearly
show that, even at very low PMB concentrations, not only a strong
inhibition of LPS (see batch test I), but subsequently also a
strong inhibition of the cytokine distribution takes place. This is
particularly pronounced in the case of the inhibition of the key
mediator TNF-alpha (FIG. 12).
4. EXAMPLE 4
Examples for Formulations for Preparations for Parenteral
Administration of Polymyxin B (PMB)-Injection Solutions (for Bolus
Administration)
4.1. Bolus Administration for a PMB Serum Concentration of 100
ng/ml Plasma
[0156] Assumption: patient with 70 kg body weight and 60% of the
body weight are distribution volumes for PMB.fwdarw.42000 ml
distribution volumes.
[0157] A PMB serum concentration of 100 ng PMB/ml plasma is
sought.fwdarw.a total of 4.2 mg PMB are required.
[0158] Injection solution for bolus administration over a period of
60 min: 4.2 mg PMB in 100 ml physiological saline solution=finished
injection solution for bolus administration over a period of 60
min.
4.2. Bolus Administration for a PMB Serum Concentration of 250
ng/ml Plasma
[0159] Assumption: patient with 70 kg body weight and 60% of the
body weight are distribution volumes for PMB.fwdarw.42000 ml
distribution volumes.
[0160] A PMB serum concentration of 250 ng PMB/ml plasma is
sought.fwdarw.a total of 10.5 mg PMB are required.
[0161] Injection solution for bolus administration over a period of
120 min: 10.5 mg PMB in 100 ml physiological saline
solution=finished injection solution for bolus administration over
a period of 120 min.
[0162] As soon as the desired PMB serum concentration is set by the
bolus administration, this is maintained by PMB release by means of
the dispensing means associated with the perfusion apparatus
according to several embodiments of the invention, as described
above.
5. EXAMPLE 5
Examples for Infusion Solutions for Infusion of Polymyxin B (PMB)
in the Extracorporeal Blood Circuit at a Lipid Feed Point and Also
Dosing Instructions
[0163] Assumption: patient with 70 kg.fwdarw.distribution volume
for PMB (60% of the body mass) 42000 ml body fluid with 100 ng
PMB/ml .fwdarw.4.2 mg PMB in the distribution volume (see under
2.1.1.).
[0164] Infusion solution for a 24 h infusion with a serum half-life
of 6 h: assumed half-life for PMB in serum 6 h: 2.1 mg PMB per 6 h
or 8.4 mg PMB/day are broken down .fwdarw.8.4 mg PMB in 1 L
physiological saline solution=infusion solution for 24 h
infusion.
[0165] Infusion solution for a 24 h infusion with a serum half-life
of 14 h: half-life for PMB in serum 14 hours: 4.2 mg PMB/14 h or
7.2 mg PMB/day are broken down .fwdarw.7.2 mg PMB in 1 L
physiological saline solution=infusion solution for 24 h
infusion.
6. EXAMPLE 6
Dosing Instructions for Infusion of Polymyxin B (PMB) in the
Extracorporeal Blood Circuit at a Lipid Feed Point Under
Consideration of the PMB Total Clearance of the Perfusion
Apparatus
[0166] The following calculation example, besides the PMB patient
clearance, also takes into consideration the clearance of a
dialyser (dialysis filter) arranged in the extracorporeal blood
circuit and the clearance of the carrier of the depletion agent.
The calculation example presupposes an existing PMB serum
concentration. This is provided by administration of a bolus prior
to the start of the treatment, wherein the injection solutions
described under Example 4 can be used for this purpose.
[0167] For the calculation of the dosing of polymyxin B via
infusion in the extracorporeal blood circuit at a lipid feed point,
the PMB clearance of the patient body, of the dialyser and of the
depletion agent are taken into consideration: [0168] The PMB
dialysis clearance (CDial) can be determined experimentally and is
dependent on the plasma flow and also on the dialysis filter type
used. In the specified example, this is 60 ml/min. [0169] The PMB
clearance of the depletion agent (Cads) is dependent on the carrier
material used and also on the filtrate flow. In the specified
example, this is 45 ml/min. [0170] The PMB patient clearance was
determined in the specified example from the half-life for PMB of
13.6 and is 36 ml/min.
[0171] The PMB total clearance (Ctotal) is given by addition from
the individual PMB clearance rates. The resultant decrease of PMB
is illustrated in FIG. 16. The evident negative rise of the PMB
decrease (Ctotal) at a certain moment in time clear in FIG. 16
corresponds to the necessary PMB infusion in order to maintain the
PMB serum concentration of the associated moment in time.
[0172] The following infusion rates are given for the specified
example: [0173] =>0.84 mg PMB/h during the treatment with
dialysis and adsorption With 6-hour extracorporeal treatment, this
gives the following PMB quantity to be infused: 6 hours treatment
with dialysis and adsorption: 5.1 mg
7. EXAMPLE 7
Improved Adsorption of Cytokines by the Use of an Albuflow Filter
(Comparison of Plasma and Fractionated Plasma)
7.1 Batch Test Cytokine Adsorption
Test Description:
[0174] Adsorbers with different pore sizes (30 nm and 15-20 nm) are
to be tested in terms of adsorption of TNF-.alpha., IL-6 and IL-10
in whole plasma and in fractionated plasma.
Test Structure:
[0175] Carrier: polystyrene-divinylbenzene copolymer, CG300c
(carrier A), CG161c (carrier B), Dow Chemical Group [0176] carrier
A: particle size: 120 .mu.m, pore size: 30 nm [0177] carrier B:
particle size: 120 .mu.m, pore size: 15-20 nm plasma: deep-frozen
unfractionated citrate plasma (fresh frozen plasma, obtained by
means of blood centrifugation) fractionated citrate plasma:
obtained by the use of the Albuflow-Filters (Fresenius Medical
Care, Germany). Cytokine spike (TNF-.alpha., IL-6, IL-10) according
to Table 7.1 below.
[0178] Carriers A and B are conditioned: the carrier is washed with
2.5 volume of ethanol absolute and the carrier is centrifuged off.
The supernatant is rejected and the carrier is incubated for 1 hour
at room temperature with 2.5 times volume ethanol absolute, then
centrifuged off, and the supernatant is rejected once again. The
same procedure is then carried out with twice-distilled water and
lastly with physiological saline solution. Following the
conditioning, the carrier is additionally washed again 3.times.
with 0.9% NaCl solution
[0179] The batch test is carried out in triplicate approach both in
whole plasma and in fractionated plasma (pre-treatment by the
Albuflow filter).
[0180] Before the test is started, the carriers are incubated with
unspiked whole plasma or with fractionated plasma for 15 min,
washed, 1.times. with NaCl and then used for the batch test.
[0181] Batch test: In each case 1 ml adsorber (moist)+9 ml citrate
plasma on a roll mixer at 37.degree. C. for 60 min.
TABLE-US-00006 TABLE 7.1 cytokine spike Stock Expected end Stock
dilution concentration Measured end Batch Stock concentration
dilution to 40 ml in the plasma concentration number [.mu.g/mL]
1:10 Plasma [pg/ml] in the plasma TNF-.alpha. AA27/1082 10 10 stock
+ 8 .mu.L 500 625 90 NaCl IL-6 OJZ0411121 10 10 stock + 35 .mu.L
200 355 90 NaCl IL-10 EYB0211041 10 10 stock + 80 .mu.L 300 517 90
NaCl
Analyses:
[0182] TNF-.alpha., IL-6 and IL-10 were quantified by means of
commercial ELISA from the company R&D Systems.
7.2 Results
[0183] It was possible to determine improved cytokine adsorption in
the fractionated plasma.
[0184] FIG. 17-19 show the improved adsorption of the cytokines
TNF-.alpha., IL-6 and IL-10 by use of an Albuflow filter compared
with a plasma filter.
8. EXAMPLE 8
Testing of Ethylvinylbenzene-Divinylbenzene Copolymers of Identical
Pore Size and Different Particle Size in Terms of the Adsorption
Properties Thereof
[0185] Ethylene vinylbenzene-divinylbenzene copolymers (carrier)
with identical mean pore sizes (15-20 nm), but with different mean
particle sizes of 3-5 .mu.m, 35 .mu.m, 75 .mu.m and 120 .mu.m are
compared in terms of the adsorption property for protein C.
8.1 Provision of Neutral, Hydrophobic Polymers
[0186] The ethylene vinylbenzene-divinylbenzene copolymers used in
this example (Amberchrom CG 161, Rohm&Haas/Dow Chemical
Company) with identical mean pore size and different mean particle
size are listed in Table 8.1.
TABLE-US-00007 TABLE 8.1 Ethylvinylbenzene-divinylbenzene
copolymers Naming of the ethylvinylbenzene- Mean pore size Mean
particle size divinylbenzene copolymer [nm] [.mu.m] #2000 15-20 3-5
#1785 15-20 35 #1760 15-20 75 #2004 15-20 120
8.2. Carrier Preparation and Batch Test
[0187] The carriers #2000, #1785, #1760 and #2004 specified in
Table 8.1 were tested in the batch test in terms of the adsorption
properties thereof for protein C and were compared with one
another.
[0188] The carriers were conditioned and incubated for 15 min in
plasma directly prior to the batch test, centrifuged off and then
used in the batch test.
[0189] Conditioning of carriers: dry carriers should be conditioned
prior to use in order to enable good wetting with aqueous solutions
or with plasma. Dry, hydrophobic carriers are pretreated as
follows: the required quantity of dry carrier is placed in a 50 ml
Greiner tube and washed with 5 times volume of undenatured ethanol
(suspended and centrifuged for 5 min at 4000 rpm). The supernatant
is removed and discarded and suspended again with fresh,
undenatured ethanol and incubated for 1 h (Enviro Genie, frequency
25:50). Following incubation, the carrier suspension is centrifuged
off (centrifuged for 5 min at 4000 rpm) and the supernatant is
discarded. The carrier is then washed with 5 times volume of
distilled water (suspended and centrifuged for 5 min at 4000 rpm).
The supernatant is removed and discarded and suspended again with
fresh distilled water and incubated for 1 h (Enviro Genie,
frequency 25:50). Following incubation the carrier suspension is
centrifuged off (centrifuged for 5 min at 4000 rpm) and the
supernatant is discarded. The carrier is then washed with 5 times
volume of physiological saline solution (suspended and centrifuged
for 5 min at 4000 rpm). The supernatant is removed and discarded
and suspended again with fresh physiological saline solution and is
incubated for 1 h (Enviro Genie, frequency 25:50). Following
incubation, the carrier suspension is centrifuged off (centrifuged
for 5 min at 4000 rpm) and the supernatant is discarded. A 50%
carrier suspension is ultimately produced with physiological saline
solution and is stored in a refrigerator until use.
[0190] For the batch approach (triplicate approach, n=3) 150 .mu.l
carriers (moist) were each coated with 1350 .mu.l citrate plasma in
15 ml Greiner tubes. The tubes were shaken in the EnviroGenie at
25/50 rpm at 37.degree. C. for 60 min. As control (120 .mu.l
NaCl+1350 .mu.l citrate plasma), a tube without carrier was
included.
[0191] Samples each measuring 500 .mu.l were taken after 15 min and
after 60 min for the Protein C analysis. Protein C was analysed on
the Sysmex (Siemens, Calif.560) with the associated reagents
(Siemens, OUVV17).
8.3. Analyses and Results
[0192] FIG. 20 shows the protein C concentration (specification in
[%] in relation to the physiological protein C concentration in
human plasma) over time for the individual carriers. On the basis
of the curves, the dependency of the protein C adsorption on the
mean particle size is clearly evident. A pronounced protein C
adsorption was determined with carriers #2000 and #1785. In the
case of carrier #2000, protein C was removed almost completely from
the plasma after just 15 minutes. By contrast, protein C was
adsorbed from the plasma to a much smaller extent by carriers #1760
and #2004. The protein C reduction of -25% observed for carrier
#1760 (after 60 min incubation) is still in a range in which
physiologically relevant quantities of protein C remain in the
plasma. The lowest protein C adsorption, which was just 8% in
relation to the protein C starting concentration after 60 min
incubation, was determined for carrier #2004. The protein C
adsorption (in % in relation to the protein C starting
concentration) by the individual carriers is listed in Table
8.3.
TABLE-US-00008 TABLE 8.3 Protein C adsorption after Protein C
adsorption after Carrier: 15 min incubation 60 min incubation #2000
94% 99% #1785 14% 52% #1760 5% 25% #2004 1% 8%
9. EXAMPLE 9
Comparison of the PMB Desorption in Plasma Between Adsorbers with
Different Pores and Particle Size and Thus Different Available
Adsorption Surface
9.1 Carriers
CG161c:
[0193] The carrier (Rohm & Haas/Dow Chemical Company; also
referred to hereinafter as adsorber) consists of a porous
polystyrene-divinylbenzene matrix. The average pore size is 15 nm,
the average particle size is 120 .mu.m and the accessible surface
is 900 m.sup.2/g adsorber (dry). The dry weight per ml of moist
adsorber is 18% (w/v).
HPR10:
[0194] The carrier (Rohm & Haas/Dow Chemical Company; also
referred to hereinafter as adsorber) consists of a porous
polystyrene-divinylbenzene matrix. The average pore size is 30-40
nm, the average particle size is 10 .mu.m and the accessible
surface is 500 m.sup.2/g adsorber (dry). The dry weight per ml of
moist carrier is 30% (w/v).
9.2 Coating of the Carrier with Polymyxin B (PMB)
[0195] The PMB solution (Sigma Aldrich, 10 mg/ml in dist. water) is
autoclaved at 121.degree. C. for 30 min, and the respective carrier
(CG161c or HPR10) is then coated in 15 ml Greiner tubes with PMB as
follows (Table 9.2): 3 ml carrier with 7.5 ml PMB solution
TABLE-US-00009 TABLE 9.2 PMB coating in Carrier PMB solution NaCl
mg per ml carrier [ml] [ml] [ml] 0 3 0 7.5 2.5 3 0.75 6.75 5 3 1.5
6 10 3 3 4.5 15 3 4.5 3 20 3 1.5 6 25 3 0 7.5
[0196] The coating is performed overnight on a roll mixer at room
temperature. The adsorber is then washed twice with 10 ml NaCl
solution (sterile), and a 50% suspension is produced.
9.3 Batch Test
[0197] In the duplicate approach, 0.5 ml carrier suspension is
incubated in each case with 4.5 ml citrate plasma=10% (v/v) at
37.degree. C. for 60 min in an Enviro-genie. The carrier is then
centrifuged off and the supernatant is used for the PMB
quantification by means of ELISA (polymyxin-ELISA from Beijing
Kwinbon Biotechnology Co., Ltd., China).
9.4 Result
[0198] It is clear from the result (see FIG. 21, FIG. 22, FIG. 23
and FIG. 24) that the desorption rate of polymyxin in plasma is
very heavily dependent on the available carrier surface. This means
that the desorption of polymyxin is dependent on the quantity of
hydrophobically bonded polymyxin per m.sup.2. This is also clear
from the following calculation tables (Table 9.4.1 and Table
9.4.2).
TABLE-US-00010 TABLE 9.4.1 Example CG161c mg PMB/ml PMB desorption
Surface [m.sup.2/g Dry component % Surface [m.sup.2/ml PMB
[.mu.g/m.sup.2 adsorber [ng/ml] adsorber (dry)] [w/v] adsorber
(moist)] adsorber surface] 2.5 152 900 18 162 15 5 409 900 18 162
31 10 1215 900 18 162 62 15 2747 900 18 162 93 20 5732 900 18 162
123 25 7514 900 18 162 154
TABLE-US-00011 TABLE 9.4.2 Example HPR10 mg PMB/ml PMB desorption
Surface [m.sup.2/g Dry component % Surface [m.sup.2/ml PMB
[.mu.g/m.sup.2 adsorber [ng/ml] adsorber (dry)] [w/v] adsorber
(moist)] adsorber surface] 2.5 488 300 30 90 28 5 4776 300 30 90 56
10 7082 300 30 90 111 15 14100 300 30 90 167 20 19752 300 30 90 222
25 31359 300 30 90 278
9.5 Calculation examples
[0199] In order to precisely define the PMB concentration in the
plasma during a treatment by the PMB desorption from the carrier
(also referred to hereinafter as adsorber), in vitro desorption
experiments (as carried out in Example 9) are necessary for the
respective adsorber. It is possible to very accurately adjust the
desorption in the plasma and therefore the PMB concentration in the
plasma by the degree of coating of the carrier (quantity of PMB per
g adsorber) on the basis of the data obtained in the experiments
(See FIGS. 21 to 24). As shown in Example 1, the desorption rate in
the fractionated plasma could be lower and could therefore be
determined separately.
Calculation Example 1
[0200] A PMB concentration in the plasma of 0.8 .mu.g/ml is to be
obtained by the use of the PMB-coated adsorber HPR10 in the
extracorporeal blood circuit. Due to preliminary tests (See FIG.
23), the function describing the correlation between coated PMB
quantity per g adsorber and desorbed PMB quantity in the plasma was
able to be determined by way of experiment. In this case, it is as
follows:
P M B [ mg g adsorber ] = 0.00000001 x 2 + 0.0012 x + 1.258
##EQU00001##
x=desired PMB concentration in plasma=0.8 .mu.g/ml=800 ng/ml
[0201] If x=800 ng/ml is used, the PMB quantity that has to be
bonded hydrophobically per g carrier is: 2.224 mg per g carrier
(HPR10)
Calculation Example 2
[0202] A PMB concentration in the plasma of 0.8 .mu.g/ml is to be
obtained by the use of the PMB-coated adsorber CG161c in the
extracorporeal blood circuit. Due to preliminary tests (See FIG.
23), the function describing the correlation between coated PMB
quantity per g adsorber and desorbed PMB quantity in the plasma was
able to be determined by way of experiment. In this case, it is as
follows:
P M B [ mg g adsorber ] = 0.00000003 x 2 + 0.0048 x + 3.0442
##EQU00002##
x=desired PMB concentration in plasma=0.8 .mu.g/ml=800 ng/ml
[0203] If x=800 ng/ml used, the PMB quantity that must be bonded
hydrophobically per g adsorber is: 7.076 mg pro g adsorber
(CG161c)
Calculation Example 3
[0204] A PMB concentration in the plasma of 0.1 .mu.g/ml is to be
obtained by the use of the PMB-coated adsorber HPR10 in the
extracorporeal blood circuit. Due to preliminary tests (See FIG.
23), the function describing the correlation between coated PMB
quantity per g adsorber and desorbed PMB quantity in the plasma was
able to be determined by way of experiment. In this case, it is as
follows:
P M B [ mg g adsorber ] = 0.00000001 x 2 + 0.0012 x + 1.258
##EQU00003##
x=desired PMB concentration in plasma=0.1 .mu.g/ml=100 ng/ml if
x=100 ng/ml used, the PMB quantity that must be bonded
hydrophobically per g adsorber is: 1.378 mg pro g adsorber
(HPR10)
Calculation Example 4
[0205] A PMB concentration in the plasma of 0.1 .mu.g/ml is to be
obtained by the use of the PMB-coated adsorber CG161c in the
extracorporeal blood circuit. Due to preliminary tests (See FIG.
23), the function describing the correlation between coated PMB
quantity per g adsorber and desorbed PMB quantity in the plasma was
able to be determined by way of experiment. In this case, it is as
follows:
P M B [ mg g adsorber ] = 0.00000003 x 2 + 0.0048 x + 3.0442
##EQU00004##
x=desired PMB concentration in plasma=0.1 .mu.g/ml=100 ng/ml
[0206] If x=100 ng/ml used, the PMB quantity that must be bonded
hydrophobically per g adsorber is: 3.527 mg pro g adsorber
(CG161c)
Calculation Example 5
Fractionated Plasma
[0207] A PMB concentration in the plasma of 0.15 .mu.g/ml is to be
obtained by the use of the PMB-coated adsorber CG161c in the
extracorporeal blood circuit. Due to preliminary tests (See FIG.
24), the function describing the correlation between coated PMB
quantity per g adsorber and desorbed PMB quantity in the
fractionated plasma was able to be determined by way of experiment.
In this case, this is as follows:
P M B [ mg g adsorber ] = 2.6718 ln ( x ) - 3.3628 ##EQU00005##
x=desired PMB concentration in plasma=0.15 .mu.g/ml=150 ng/ml
[0208] If x=150 ng/ml used, the PMB quantity that must be bonded
hydrophobically per g adsorber is: 10.025 mg pro g adsorber
(CG161c)
Calculation Example 6
Fractionated Plasma
[0209] A PMB concentration in the plasma of 0.8 .mu.g/ml is to be
obtained by the use of the PMB-coated adsorber CG161c in the
extracorporeal blood circuit. Due to preliminary tests (See FIG.
24), the function describing the correlation between coated PMB
quantity per g adsorber and desorbed PMB quantity in the
fractionated plasma was able to be determined by way of experiment.
In this case, this is as follows:
P M B [ mg g adsorber ] = 2.6718 ln ( x ) - 3.3628 ##EQU00006##
x=desired PMB concentration in plasma=0.8 .mu.g/ml=800 ng/ml
[0210] If x=800 ng/ml used, the PMB quantity that must be bonded
hydrophobically per g adsorber is: 14.497 mg pro g adsorber
(CG161c)
10. EXAMPLE 10
Polymyxin B (PMB) Desorption Over Time
[0211] This test is intended to demonstrate that the equilibrium
reaction (adsorption and desorption of polymyxin (B)) is quick and
stable in plasma.
10.1 Carrier
[0212] HPR10: the carrier HPR10 (Rohm & Haas/Dow Chemical
Company; also referred to hereinafter as adsorber) consists of a
porous polystyrene-divinylbenzene matrix. The average pore size is
30-40 nm, the average particle size is 10 .mu.m and the accessible
surface is 500 m.sup.2/g carrier (dry). The dry weight per ml moist
carrier is 30% (w/v).
10.2 Coating of the Carrier with Polymyxin B (PMB)
[0213] The PMB solution (Sigma, 10 mg/ml in dist. water) is
autoclaved at 121.degree. C. for 30 min, and the carrier (HPR10) is
then coated in 15 ml Greiner tubes with PMB as follows (see Table
10.2): 3 ml carrier with 7.5 ml PMB solution
TABLE-US-00012 TABLE 10.2 The carrier/adsorber is coated with
different quantities of PMB PMB coating in Adsorber PMB solution
NaCl mg per ml adsorber [ml] [ml] [ml] 5 3 1.5 6 10 3 3 4.5 25 3 0
7.5
[0214] The coating is carried out overnight on a roll mixer at room
temperature. The coated carrier is then washed twice with 10 ml
NaCl solution (sterile), and a 50% suspension is produced. A tube
without adsorber was included as control.
10.3 Batch Test
[0215] The plasma with 5 IU heparin is spiked with 5 ng/ml LPS
(L-7018 Pseud. aerug. company Sigma batch: 128K4115). In the
triplicate approach, 1% PMB-coated carrier is incubated with the
LPS-spiked plasma (30 .mu.l carrier+2970 .mu.l LPS spiked plasma)
at 37.degree. C. in an overhead shaker and samples were taken at
intervals (5, 15 and 60 min) for LAL analysis.
10.4 Analysis
[0216] The analysis was performed using an LAL test.
Used Materials for Batch Test and LAL Tests:
Batch
TABLE-US-00013 [0217] Microtiter plates MT 1007 company Charles
River 1721599k.A. Lal test tubes T 200 Ch. River Endosafe 53351
Dk.A. Combitips plus 5 ml Biopur Eppendorf X131667I Pipette tips
Eppendorf V125542M Pipette tips Eppendorf W130324Q NaCl 0.9%
Mayerhofer 8G5523 2011-07 Microcentrifuge tubes Greiner 05200108
Charles RiverEndosafe Endochrome Kit. batch: A2112EK1
10.5 Result
[0218] The equilibrium concentration of desorbed PMB in the plasma
is attained very quickly. The LPS inactivation after 5 minutes is
almost the same as after 60 minutes of incubation (see FIG.
25).
11. EXAMPLE 11
Influence of the Coating of a Carrier Coated with Polymyxin B(PMB)
on Cytokine Adsorption
11.1 Test Description
[0219] The extent to which the PMB-coated adsorber CG161c is
suitable for the adsorption of cytokines compared with the uncoated
adsorber CG161c was tested in a 10% (v/v) batch test. 5 ng/ml
endotoxin (LPS) from Pseudomonas aeruginosa were also added.
11.2 Test Structure
[0220] Carrier: Amberchrom CG161(mean particle size 120 .mu.m, mean
pore size 15 nm)
Coating with PMB:
[0221] The PMB solution (Sigma Aldrich, 10 mg/ml in dist. water)
and the carrier in 50% suspension are coated in 15 ml Greiner tubes
with PMB as follows (Table 11.2.1):
TABLE-US-00014 TABLE 11.2.1 50% adsorber suspension PMB solution
NaCl [ml] [ml] [ml] 3xapproach 2 1 2
[0222] The coating was performed overnight on an Enviro-Genie
(25:50) at room temperature. The carrier was then washed twice with
10 ml NaCl solution (sterile), and a 50% suspension was
produced.
Batch Approach:
[0223] Triplicate approach: in each case 1 ml adsorber (moist)+9 ml
spike 15 ml Greiner tubes are shaken in the Enviro-Genie for 60 min
at 25/50 rpm at 37.degree. C.
Cytokines:
[0224] The stock solution is diluted 1:10 in plasma (freshly frozen
plasma, plasma donor centre Retz) (1:10; 5 .mu.l, stock+45 .mu.l,
plasma). The end concentration of the plasma spike (100 mL) for the
used cytokines is presented in Table 11.2.2 below:
TABLE-US-00015 TABLE 11.2.2 Stock Stock concen- Stock dilution End
concentration Batch tration dilution to plasma in the plasma Nr
[.mu.g/mL] 1:10 [100 mL] [pg/mL] TNF-.alpha. 10 5 .mu.L + 45 .mu.l
15 .mu.L 500 IL-1.alpha. 5 5 .mu.L + 45 .mu.l 65 .mu.L 250 IL-6 10
5 .mu.L + 45 .mu.l 35 .mu.L 200 IL-8 10 5 .mu.L + 45 .mu.l 35 .mu.L
200 IL-10 10 5 .mu.L + 45 .mu.l 40 .mu.L 300
Endotoxins (LPS):
[0225] Pseudomonas aeruginosa: L-7018 company Sigma batch:
128K4115, -70.degree. C., at 100 .mu.l 10-3 g/ml (1 mg/ml).
[0226] LPS is used in the batch with a final concentration of 5
ng/ml
.fwdarw.50 .mu.l 10.sup.-5 solution in 100 ml plasma (Tables 11.2.3
and 11.2.4)
TABLE-US-00016 TABLE 11.2.3 10.sup.-4 10.sup.-5 0.9 0.900 NaCl 0.1
0.100 LPS 100 .mu.g/ml 10 .mu.g/ml LPS concentration
TABLE-US-00017 TABLE 11.2.4 0 min 60 min Spike without adsorber 1 2
CG161c- 1 without PMB 3 CG161c - 2 without PMB 4 CG161c - 3 without
PMB 5 CG161c - 4 with PMB 6 CG161c - 5 with PMB 7 CG161c - 6 with
PMB 8 =8 samples, that is to say 100 ml citrate plasma spikes
11.3 Analysis
[0227] The cytokine analysis is performed with the aid of a Luminex
apparatus (based on antibodies) from the company Biorad.
11.4 Results
[0228] The results are shown in FIG. 26, from which it can be
clearly seen that an adsorptive coating of the carrier surface with
polymyxin B has no effects on the adsorption of the cytokines.
* * * * *