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.

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, and wherein the extracorporeal perfusion apparatus comprises a dispenser provided separately from the filter 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.

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 3, wherein the endotoxin-binding lipopeptide adsorbed at the surface of the first carrier is present in a quantity that, when the lipopeptide is fed, gives a lipopeptide serum concentration from 0.01 g/ml to 0.8 g/ml.

5. The extracorporeal perfusion apparatus according to claim 3, wherein the first 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.

6. 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 m or smaller.

7. 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.

8. 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.

9. The extracorporeal perfusion apparatus according to claim 8, wherein the lipopeptide feed point is arranged in the extracorporeal blood circuit downstream of the filter.

10. The extracorporeal perfusion apparatus according to claim 9, 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.

11. The extracorporeal perfusion apparatus according to claim 9, 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.

12. The extracorporeal perfusion apparatus according to claim 8, 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.

13. 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.

14. The extracorporeal perfusion apparatus according to claim 1, wherein the first carrier is formed from a neutral polymer.

15. The extracorporeal perfusion apparatus according to claim 14, wherein the polymer is selected from a cross-linked polystyrene polymer or a cross-linked ethylene divinylbenzene polymer.

16. The extracorporeal perfusion apparatus according to claim 1, wherein the first carrier is porous and has a mean pore size of 100 nm or less.

17. The extracorporeal perfusion apparatus according to claim 16, wherein the first carrier has a mean pore size of 20 nm or less or a mean pore size from 80 to 100 nm.

18. The extracorporeal perfusion apparatus according to claim 1, wherein the first carrier is fibre-like or is in particle form.

19. The extracorporeal perfusion apparatus according to claim 18, wherein the first carrier has the form of microparticles having a mean particle size of 300 m or smaller.

20. The extracorporeal perfusion apparatus according to claim 19, wherein the first carrier has a mean pore size from one of either 10 nm to 20 nm or 80 nm to 100 nm and a mean particle size from 75 to 150 m.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) Numerous embodiments of the invention will be explained hereinafter in greater detail on the basis of non-limiting examples and drawings. In the drawings:

(2) 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,

(3) 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,

(4) 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.

(5) 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,

(6) 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,

(7) 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

(8) 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.

(9) FIG. 8 shows a data plot showing the desorption of PMB in accordance with the PMB coating concentration.

(10) FIG. 9 shows a data plot shows ann LPS inactivation of more than 50% was able to be achieved at the lowest coarted PMB concentration (EU=endotoxin units).

(11) 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.

(12) 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.

(13) FIG. 12 shows the distrobution of TNF-alpha cytokines 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).

(14) FIG. 13 shows the distribution of IL-1beta cytokines 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).

(15) FIG. 14 shows the distribution of IL-6 cytokines 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).

(16) FIG. 15 shows the distribution of IL-8 cytokines 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).

(17) FIG. 16 shows the PMB total clearance (Ctotal) given by addition from the individual PMB clearance rates.

(18) FIG. 17 shows the improved adsorption of TNF- cytokines by use of an Albuflow filter compared with a plasma filter.

(19) FIG. 18 shows the improved adsorption of IL-6 cytokines by use of an Albuflow filter compared with a plasma filter.

(20) FIG. 19 shows the improved adsorption of IL-10 cytokines by use of an Albuflow filter compared with a plasma filter.

(21) 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.

(22) FIGS. 21 to 24 show the desorption rate of polymyxin in plasma across various available carrier surfaces.

(23) FIG. 25 shows the equilibrium concentration of desorbed PMB in the plasma over time.

(24) FIG. 26 shows cytokine analysis performed with the aid of a Luminex apparatus.

DETAILED DESCRIPTION OF THE INVENTION

(25) 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 type (manufacturer: Fresenius Medical Care; material: polysulfone hollow fibres; sieving coefficient for albumin of 0.6 and for fibrinogen 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.

(26) 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 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-, 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.

(27) 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 type (manufacturer: Fresenius Medical Care; material: polysulfone hollow fibres; sieving coefficient for albumin of 0.6 and for fibrinogen 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.

(28) 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 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-, 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.

(29) 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 type (manufacturer: Fresenius Medical Care; material: polysulfone hollow fibres; sieving coefficient for albumin of 0.6 and for fibrinogen 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).

(30) 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 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-c, IL-6 and IL-10, by adsorbing these at the carrier and removing them from the plasma.

(31) 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.

(32) 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 g/ml.

(33) 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 type (manufacturer: Fresenius Medical Care; material: polysulfone hollow fibres; sieving coefficient for albumin of 0.6 and for fibrinogen 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).

(34) 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 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-, IL-6 and IL-10, by adsorbing these at the carrier and removing them from the plasma.

(35) 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.

(36) 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 g/ml.

(37) 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 type (manufacturer: Fresenius Medical Care; material: polysulfone hollow fibres; sieving coefficient for albumin of 0.6 and for fibrinogen 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.

(38) 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 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-, IL-6 and IL-10, by adsorbing these at the carrier and removing them from the plasma.

(39) 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 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.

(40) 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 type (manufacturer: Fresenius Medical Care; material: polysulfone hollow fibres; sieving coefficient for albumin of 0.6 and for fibrinogen 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).

(41) 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.

(42) 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 m+/3-4 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-, 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.

(43) 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 type (manufacturer: Fresenius Medical Care; material: polysulfone hollow fibres; sieving coefficient for albumin of 0.6 and for fibrinogen 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.

(44) 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 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-, IL-6 and IL-10, by adsorbing these at the carrier and removing them from the plasma.

(45) 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.

(46) 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 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%.

(47) 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 m, Mean Pore Size: 15-20 nm)

(48) 1.1 PMB Coating

(49) Carrier:

(50) Amberchrom CG161c (polystyrene-divinylbenzene copolymer, Dow Chemical Company), mean particle size 120 am, 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).

(51) Polymyxin B (PMB):

(52) polymyxin B sulphate (Sigma Aldrich)

(53) The PMB solution (10 mg/ml in dist. water) is autoclaved at 121 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

(54) TABLE-US-00001 TABLE 1.1 PMB coating PMB in mg per ml Carrier solution NaCl 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

(55) 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.

(56) 1.2 Batch Test

(57) Freshly frozen plasma (citrate plasma) was fractionated with the aid of the Albuflow filter (Fresenius Medical Care, Germany) and was frozen at 20 C. together with the whole plasma.

(58) A: whole plasma

(59) B: fractionated plasma

(60) TABLE-US-00002 TABLE 1.2 Batch approaches 0 mg/ml 1 mg/ml 2.5 mg/ml 5 mg/m1 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

(61) 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 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).

(62) 1.3 Results

(63) 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).

(64) 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

(65) 2.1 Test Structure

(66) Conditioned carrier (Amberchrom CG161c: ethylvinylbenzene-divinylbenzene copolymer (Dow Chemical Company), mean particle size 120 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.

(67) 2.2 Test Execution

(68) PMB Coating:

(69) Carrier samples with different PMB concentrations (5 mg, 10 mg, 15 mg und 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 C. for 30 min, and the carrier is coated in 15 ml Greiner tubes with PMB as follows (Table 2.2):

(70) TABLE-US-00003 TABLE 2.2 50% adsorber PMB mg PMB/g suspension 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

(71) 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.

(72) Production of Serum:

(73) 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).

(74) Endotoxin (LPS) Solution:

(75) LPS: Pseudomonas aenruginosa, L-7018 company Sigma batch: 128K4115, storage 70 C., at 100 l 10.sup.3 g/ml (1 mg/ml)

(76) An LPS solution with a concentration of 10 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 l LPS solution with a concentration of 10 g/ml are pipetted into 20 ml serum. The batch approach is performed in 2 ml blood-sampling tubes in a triplicate approach.

(77) 2.3 Results:

(78) 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

(79) 3.1. Objective

(80) 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).

(81) 3.2. Blood Donor

(82) 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:

(83) 3.3. LPS Spike, Polymyxin B Solutions and Batch Test I

(84) LPS: Pseudomonas aeruginosa (L-7018 company Sigma batch: 128K4115, 70 C., at 100 l 103 g/ml (1 mg/ml))

(85) LPS: E. coli (L-4130 company Sigma batch: 110M4086M, 70 C., at 100 l 103 g/ml (1 mg/ml))

(86) 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 C. (see Table 3.5).

(87) In batch test 1, PMB concentrations with 0 (without PMB), 10, 100, 250, 500 and 1000 ng/ml are used. Sterile PMB solutions (autoclaved pyrogen-free at 121 C., 90 min) are produced for this purpose with the following concentrations (Table 3.3):

(88) TABLE-US-00004 TABLE 3.3 PMB PMB [ng/ml] [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

(89) The endotoxins are measured in the form of EU/ml with the aid of a Limulus Amebocyte Lysate test (LAL) by Charles River.

(90) 3.5. Cytokine Batch (Batch Test II)

(91) 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 C. on a roll mixer (5 revolutions/min), samples were taken, centrifuged off and 50 l plasma were frozen at 80 C. for the subsequent cytokine quantification. The test data for the cytokine batch is listed in Table 3.5.

(92) TABLE-US-00005 TABLE 3.5 PMB Plasma + PMB [ng/ml] solution 0.5 ng/ml LPS Incubation LAL EU/ml Cytokine Batch Sample 4 h Sample 12 h LPS Pseudomonas aeruginosa 0 100 l NaCl 1400 l 60 min #1 0.333 1500 l cell concentrate + 250 l .fwdarw. 250 l .fwdarw. 0 100 l NaCl 1400 l 60 min #2 0.229 1500 l LPS-PMB plasma 50 l plasma 80 C. 50 l plasma 80 C. 10 100 l sol A 1400 l 60 min #3 0.178 10 100 l sol A 1400 l 60 min #4 0.167 100 100 l sol B 1400 l 60 min #5 0.112 100 100 l sol B 1400 l 60 min #6 0.137 250 100 l sol C 1400 l 60 min #7 0.108 1500 l cell concentrate + 250 l .fwdarw. 250 l .fwdarw. 250 100 l sol C 1400 l 60 min #8 0.123 1500 l LPS-PMB plasma 50 l plasma 80 C. 50 l plasma 80 C. 500 100 l sol D 1400 l 60 min #9 0.091 1500 l cell concentrate + 250 l .fwdarw. 250 l .fwdarw. 500 100 l sol D 1400 l 60 min #10 0.081 1500 l LPS-PMB plasma 50 l plasma 80 C. 50 l plasma 80 C. 1000 100 l sol E 1400 l 60 min #11 0.062 1500 l cell concentrate + 250 l .fwdarw. 250 l .fwdarw. 1000 100 l sol E 1400 l 60 min #12 0.061 1500 l LPS-PMB plasma 50 l plasma 80 C. 50 l plasma 80 C. LPS E. coli 0 100 l NaCl 1400 l 60 min #13 1.8 1500 l cell concentrate + 250 l .fwdarw. 250 l .fwdarw. 0 100 l NaCl 1400 l 60 min #14 1.841 1500 l LPS-PMB plasma 50 l plasma 80 C. 50 l plasma 80 C. 10 100 l sol A 1400 l 60 min #15 0.77 10 100 l sol A 1400 l 60 min #16 0.871 100 100 l sol B 1400 l 60 min #17 0.379 100 100 l sol B 1400 l 60 min #18 0.382 250 100 l sol C 1400 l 60 min #19 0.281 1500 l cell concentrate + 250 l .fwdarw. 250 l .fwdarw. 250 100 l sol C 1400 l 60 min #20 0.29 1500 l LPS-PMB plasma 50 l plasma 80 C. 50 l plasma 80 C. 500 100 l sol D 1400 l 60 min #21 0.209 1500 l cell concentrate + 250 l .fwdarw. 250 l .fwdarw. 500 100 l sol D 1400 l 60 min #22 0.209 1500 l LPS-PMB plasma 50 l plasma 80 C. 50 l plasma 80 C. 1000 100 l sol E 1400 l 60 min #23 0.154 1500 l cell concentrate + 250 l .fwdarw. 250 l .fwdarw. 1000 100 l sol E 1400 l 60 min #24 0.16 1500 l LPS-PMB plasma 50 l plasma 80 C. 50 l plasma 80 C.

(93) 3.6. Results

(94) Endoloxin Batch (Batch Test I):

(95) 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.

(96) FIG. 11 shows the inhibition of LPS from Pseudoonas 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.

(97) 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 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.

(98) Cytokine Batch (Batch Test II):

(99) The distribution of the cytokines TNF-alpha (FIG. 12), IL-1beta (FIG. 13), IL-6 (FIG. 14) and IL-8 (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)

(100) 4.1. Bolus Administration for a PMB Serum Concentration of 100 ng/ml Plasma

(101) Assumption: patient with 70 kg body weight and 60% of the body weight are distribution volumes for PMB.fwdarw.42000 ml distribution volumes.

(102) A PMB serum concentration of 100 ng PMB/ml plasma is sought.fwdarw.a total of 4.2 mg PMB are required.

(103) 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.

(104) 4.2. Bolus Administration for a PMB Serum Concentration of 250 ng/ml Plasma

(105) Assumption: patient with 70 kg body weight and 60% of the body weight are distribution volumes for PMB.fwdarw.42000 ml distribution volumes.

(106) A PMB serum concentration of 250 ng PMB/ml plasma is sought.fwdarw.a total of 10.5 mg PMB are required.

(107) 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.

(108) 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

(109) 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.).

(110) 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.

(111) 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

(112) 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.

(113) 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: 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. 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. The PMB patient clearance was determined in the specified example from the half-life for PMB of 13.6 and is 36 ml/min.

(114) 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.

(115) The following infusion rates are given for the specified example: =>0.84 mg PMB/h during the treatment with dialysis and adsorption

(116) With 6-hour extracorporeal treatment, this gives the following PMB quantity to be infused:

(117) 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)

(118) 7.1 Batch Test Cytokine Adsorption

(119) Test Description:

(120) Adsorbers with different pore sizes (30 nm and 15-20 nm) are to be tested in terms of adsorption of TNFa, IL-6 and IL-10 in whole plasma and in fractionated plasma.

(121) Test Structure:

(122) Carrier: polystyrene-divinylbenzene copolymer, CG300c (carrier A), CG161c (carrier B), Dow Chemical Group carrier A: particle size: 120 m, pore size: 30 nm carrier B: particle size: 120 m, pore size: 15-20 nm

(123) plasma: deep-frozen unfractionated citrate plasma (fresh frozen plasma, obtained by means of blood centrifugation)

(124) fractionated citrate plasma: obtained by the use of the Albuflow-Filters (Fresenius Medical Care, Germany).

(125) Cytokine spike (TNF-, IL-6, IL-10) according to Table 7.1 below.

(126) 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 with 0.9% NaCl solution

(127) The batch test is carried out in triplicate approach both in whole plasma and in fractionated plasma (pre-treatment by the Albuflow filter).

(128) Before the test is started, the carriers are incubated with unspiked whole plasma or with fractionated plasma for 15 min, washed, lx with NaCl and then used for the batch test.

(129) Batch test: In each case 1 ml adsorber (moist)+9 ml citrate plasma on a roll mixer at 37 C. for 60 min.

(130) TABLE-US-00006 TABLE 7.1 cytokine spike Stock Expected end Stock Stock dilution concentration Measured and Batch concentration dilution to 40 ml in the plasma concentration number [g/ml] 1:10 Plasma [pg/ml] in the plasma TNF- AA27/1082 10 10 stock + 8 l 500 625 90NaCl IL-6 OJZ0411121 10 10 stock + 35 l 200 355 90NaCl IL-10 EYB0211041 10 10 stock + 80 l 300 517 90NaCl

(131) Analyses:

(132) TNFa, IL-6 and IL-10 were quantified by means of commercial ELISA from the company R&D Systems.

(133) 7.2 Results

(134) It was possible to determine improved cytokine adsorption in the fractionated plasma.

(135) FIG. 17-19 show the improved adsorption of the cytokines TNF-, 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

(136) Ethylene vinylbenzene-divinylbenzene copolymers (carrier) with identical mean pore sizes (15-20 nm), but with different mean particle sizes of 3-5 m, 35 m, 75 m and 120 m are compared in terms of the adsorption property for protein C.

(137) 8.1 Provision of Neutral, Hydrophobic Polymers

(138) 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.

(139) TABLE-US-00007 TABLE 8.1 Ethylvinylbenzene-divinylbenzene copolymers Naming of the Mean Mean ethylvinylbenzene-divinylbenzene pore size particle size copolymer [nm] [m] #2000 15-20 3-5 #1785 15-20 35 #1760 15-20 75 #2004 15-20 120

(140) 8.2. Carrier Preparation and Batch Test

(141) 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.

(142) 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.

(143) 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.

(144) For the batch approach (triplicate approach, n=3) 150 l carriers (moist) were each coated with 1350 l citrate plasma in 15 ml Greiner tubes. The tubes were shaken in the Enviro-Genie at 25/50 rpm at 37 C. for 60 min. As control (120 l NaCl+1350 l citrate plasma), a tube without carrier was included.

(145) Samples each measuring 500 l were taken after 15 min and after 60 min for the Protein C analysis. Protein C was analysed on the Sysmex (Siemens, CA560) with the associated reagents (Siemens, OUVV17).

(146) 8.3. Analyses and Results

(147) 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.

(148) 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

(149) 9.1 Carriers

(150) CG161c:

(151) 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 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).

(152) HPR10:

(153) 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 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).

(154) 9.2 Coating of the Carrier with Polymyxin B (PMB)

(155) The PMB solution (Sigma Aldrich, 10 mg/ml in dist. water) is autoclaved at 121 C. for 30 min, and the respective carrier (CG161c or HPR10) is then coated in 15 ml Greiner tubes with PMB as follows (Table 92): 3 ml carrier with 7.5 ml PMB solution

(156) TABLE-US-00009 TABLE 9.2 PMB coating PMB in mg per ml Carrier solution NaCl 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

(157) 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.

(158) 9.3 Batch Test

(159) 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 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).

(160) 9.4 Result

(161) 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).

(162) TABLE-US-00010 TABLE 9.4.1 Example CG161c Surface Dry Surface PMB mg PMB [m.sup.2/g compo- [m.sup.2/ml [g/m.sup.2 PMB/ml desorption adsorber nent % adsorber adsorber adsorber [ng/ml] (dry)] [w/v] (moist)] 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

(163) TABLE-US-00011 TABLE 9.4.2 Example HPR10 Surface Dry Surface PMB mg PMB [m.sup.2/g compo- [m.sup.2/ml [g/m.sup.2 PMB/ml desorption adsorber nent % adsorber adsorber adsorber [ng/ml] (dry)] [w/v] (moist)] 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

(164) 9.5 Calculation Examples

(165) 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 FIG. 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

(166) A PMB concentration in the plasma of 0.8 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:

(167) $\mathrm{PMB}\left[\frac{\mathrm{mg}}{g\mathrm{adsorber}}\right]=0.00000001x^2+0.0012x+1.258$

(168) x=desired PMB concentration in plasma=0.8 g/ml=800 ng/ml

(169) 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

(170) A PMB concentration in the plasma of 0.8 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:

(171) $\mathrm{PMB}\left[\frac{\mathrm{mg}}{g\mathrm{adsorber}}\right]=0.00000003x^2+0.0048x+3.0442$

(172) x=desired PMB concentration in plasma=0.8 g/ml=800 ng/ml

(173) 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

(174) A PMB concentration in the plasma of 0.1 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:

(175) $\mathrm{PMB}\left[\frac{\mathrm{mg}}{g\mathrm{adsorber}}\right]=0.00000001x^2+0.0012x+1.258$

(176) x=desired PMB concentration in plasma=0.1 g/ml=100 ng/ml

(177) 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

(178) A PMB concentration in the plasma of 0.1 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:

(179) $\mathrm{PMB}\left[\frac{\mathrm{mg}}{g\mathrm{adsorber}}\right]=0.00000003x^2+0.0048x+3.0442$

(180) x=desired PMB concentration in plasma=0.1 g/ml=100 ng/ml

(181) 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)

(182) A PMB concentration in the plasma of 0.15 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:

(183) $\mathrm{PMB}\left[\frac{\mathrm{mg}}{g\mathrm{adsorber}}\right]=2.6718\ln \left(x\right)-3.3628$

(184) x=desired PMB concentration in plasma=0.15 g/ml=150 ng/ml

(185) 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)

(186) A PMB concentration in the plasma of 0.8 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:

(187) $\mathrm{PMB}\left[\frac{\mathrm{mg}}{g\mathrm{adsorber}}\right]=2.6718\ln \left(x\right)-3.3628$

(188) x=desired PMB concentration in plasma=0.8 g/ml=800 ng/ml

(189) 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

(190) This test is intended to demonstrate that the equilibrium reaction (adsorption and desorption of polymyxin (B)) is quick and stable in plasma.

(191) 10.1 Carrier

(192) 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 j m and the accessible surface is 500 m.sup.2/g carrier (dry). The dry weight per ml moist carrier is 30% (w/v).

(193) 10.2 Coating of the Carrier with Polymyxin B (PMB)

(194) The PMB solution (Sigma, 10 mg/ml in dist. water) is autoclaved at 121 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

(195) TABLE-US-00012 TABLE 10.2 The carrier/adsorber is coated with different quantities of PMB PMB coating PMB in mg per ml Adsorber solution NaCl adsorber [ml] [ml] [ml] 5 3 1.5 6 10 3 3 4.5 25 3 0 7.5

(196) 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.

(197) 10.3 Batch Test

(198) 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 l carrier+2970 l LPS spiked plasma) at 37 C. in an overhead shaker and samples were taken at intervals (5, 15 and 60 min) for LAL analysis.

(199) 10.4 Analysis

(200) The analysis was performed using an LAL test.

(201) Used Materials for Batch Test and LAL Tests:

(202) TABLE-US-00013 Batch: 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

(203) 10.5 Result

(204) 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

(205) 11.1 Test Description

(206) 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.

(207) 11.2 Test Structure

(208) Carrier:

(209) Amberchrom CG161 (mean particle size 120 am, mean pore size 15 nm)

(210) Coating with PMB:

(211) 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):

(212) TABLE-US-00014 TABLE 11.2.1 50% adsorber PMB suspension solution NaCl [ml] [ml] [ml] 3xapproach 2 1 2

(213) 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.

(214) Batch Approach:

(215) Triplicate approach: in each case 1 ml adsorber (moist)+9 ml spike

(216) 15 ml Greiner tubes are shaken in the Enviro-Genie for 60 min at 25/50 rpm at 37 C.

(217) Cytokines:

(218) The stock solution is diluted 1:10 in plasma (freshly frozen plasma, plasma donor centre Retz) (1:10; 5 L stock+45 L plasma). The end concentration of the plasma spike (100 mL) for the used cytokines is presented in Table 11.2.2 below:

(219) TABLE-US-00015 TABLE 11.2.2 End Stock Stock concen- concen- Stock dilution tration in tration dilution to plasma the plasma Batch Nr [g/mL] 1:10 [100 mL] [pg/mL] TNF- 10 5 L + 45 l 15 L 500 IL-1 5 5 L + 45 l 65 L 250 IL-6 10 5 L + 45 l 35 L 200 IL-8 10 5 L + 45 l 35 L 200 IL-10 10 5 L + 45 l 40 L 300

(220) Endotoxins (LPS):

(221) Pseudomonas aeruginosa: L-7018 company Sigma batch: 128K4115, 70 C., at 100 l 103 g/ml (1 mg/ml).

(222) LPS is used in the batch with a final concentration of 5 ng/ml

(223) .fwdarw.50 l 10.sup.5 solution in 100 ml plasma (Tables 11.2.3 and 11.2.4)

(224) TABLE-US-00016 TABLE 11.2.3 10.sup.4 10.sup.5 0.9 0.900 NaCl 0.1 0.100 LPS 100 g/ml 10 g/ml LPS concentration

(225) 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

(226) 11.3 Analysis

(227) The cytokine analysis is performed with the aid of a Luminex apparatus (based on antibodies) from the company Biorad.

(228) 11.4 Results

(229) 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.