Method for extracorporeal removal of pathogenic microbe, an inflammatory cell or an inflammatory protein from blood

Abstract

The present invention relates to a method for extracorporeal removal of a pathogenic microbe, an inflammatory cell or an inflammatory protein from mammalian blood/use of a device comprising a carbohydrate immobilized on a solid substrate, said carbohydrate having a binding affinity for a pathogenic microbe, an inflammatory cell or an inflammatory protein, for extracorporeal removal of said pathogenic microbe, inflammatory cell or inflammatory protein from mammalian blood/use of a carbohydrate having a binding affinity for a pathogenic microbe, an inflammatory cell or an inflammatory protein, wherein said carbohydrate is immobilized on a solid substrate, in the preparation of a device for treatment of a condition caused or aggravated by said pathogenic microbe, inflammatory cell or inflammatory protein and a method for treatment of a mammalian subject suffering from a condition caused or aggravated by a pathogenic microbe, an inflammatory cell or an inflammatory protein.

Claims

1. A method for treating a subject in need thereof, by extracorporeal removal of a bacterium or pro-inflammatory cytokine, said method comprising: a) contacting said subject's whole blood with heparin immobilized on a solid substrate, said heparin having a terminal residue, wherein heparin immobilization consists of a single covalent link of said terminal residue to said solid substrate by covalent end-point attachment, under conditions allowing binding of said bacterium or pro-inflammatory cytokine in said subject's whole blood sample to the heparin; b) separating the whole blood from the solid substrate; c) recovering said whole blood containing a reduced amount of said bacterium or pro-inflammatory cytokine; and d) reintroducing into said subject said whole blood containing a reduced amount of said a bacterium or pro-inflammatory cytokine.

2. The method of claim 1, wherein said solid substrate comprises microparticles.

3. The method of claim 1, wherein said solid substrate comprises fibers.

4. The method of claim 1, wherein the material of said solid substrate is at least one member selected from the group consisting of glass, cellulose, cellulose acetate, chitin, chitosan, crosslinked dextran, crosslinked agarose, polypropylene, polyethylene, polysulfone, polyacrylonitrile, silicone, Teflon and polyurethanes.

5. The method of claim 1, wherein said bacterium is selected from the group consisting of Streptococcus sanguis, Streptococcus mutans, Escherichia coli, Pseudomonas aureginosa and Mycobacterium tuberculosis.

6. The method of claim 1, wherein said pro-inflammatory cytokine is selected from the group consisting of tumor necrosis factor alpha (TNF-α), tumor necrosis factor beta (TNF-β), interleukin-1 (IL-1), and interleukin-6 (IL-6).

Description

EXAMPLES

Preparatory Example 1

(1) Amination of Sephadex G 25

(2) Sodium metaperiodate (NaIO.sub.4, 6.0 g) was dissolved in water (994 ml) and added to Sephadex G 25 (Pharmacia Biotech, Uppsala, Sweden) (50 g) in 1 l water. The mixture was kept in the dark under shaking for 24 h. After filtration and washing with water 5×11 and finally 0.1 M phosphate buffer, pH 7.0, the resulting product was suspended in phosphate buffer, pH 7.0 (350 ml) and a solution of polyethylenimine (100 ml Lupasol (BASF, Germany), 5% in water) was added. The gel was stabilized by addition of an aqueous solution of NaBH.sub.3CN, sodium cyanoborohydride (0.5 g in 100 ml, phosphate buffer, 0.1 M, pH 7.0). The gel was filtered and washed as described above and finally washed with acetate buffer (500 ml, 0.1 M, pH 4.0), yielding aminated Sephadex G 25 (85 g).

Preparatory Example 2

(3) Covalent End-Point Attachment of Heparin onto a Chromatographic Gel

(4) Aminated Sephadex G 25 (85 g) obtained as described in Preparatory example 1 was suspended in acetate buffer (800 ml, 0.1 M, pH 4.0) and 4.0 g nitrous acid degraded heparin (heparin from Pharmacia, Sweden) was added. After shaking for 0.5 h, NaBH.sub.3CN (0.4 g) was added. The reaction mixture was shaken for 24 h and then processed as above, yielding heparinized Sephadex G 25 (80 g).

(5) The gel contains 2% heparin (w/w, sulfur analysis). The Sephadex G 25 beads have an average diameter of 50-150 μm. A rough calculation reveals that 1 cm.sup.3 contains 10.sup.6 beads which gives a bead surface area of 0.03 m.sup.2/cm.sup.3. Further, if heparin is attached only to the surface of the beads, a heparinized Sephadex G 25 with 2% heparin w/w has about 0.003 μg heparin/cm.sup.2.

Preparatory Example 3

(6) Covalent Attachment of Heparin onto Aminated Glass Wool

(7) A glass wool material is heparinized using the general procedure described below.

(8) Glass wool is thoroughly cleaned with acid (HCl), rinsed with absolute ethanol, and dried in an oven at 100° C. for 4 hours.

(9) Reactive amino functions are introduced on the glass wool surface by treatment with an aqueous solution of polyamine, polyethylenimine (PEI) or chitosan. For some purposes, the polyamines may be stabilized on the surface by crosslinking with bifunctional reagents, such as crotonaldehyde or glutaraldehyde.

(10) The coating is further stabilized by ionic cross linking with a sulfated polysaccharide (dextran sulfate or heparin). If necessary, these steps are repeated and a sandwich structure is built up. Careful rinsing (water, suitable buffers) should be performed between each step. After a last addition of PEI or chitosan, end-point attachment (EPA) to the aminated surface of native heparin is done by reductive amination, utilizing the aldehyde function in the reducing terminal residue in native heparin. The coupling is performed in aqueous solution, by reductive amination (cyanoborohydride, CNBH.sub.3.sup.−) essentially as described in Preparatory example 2.

(11) Surface analysis as described in Preparatory example 2 reveals that approximately 10 mg/cm.sup.2 of heparin is coupled to the glass surface.

Preparatory Example 4

(12) Covalent Attachment of Heparin onto Aminated Polymeric Surfaces

(13) A polymeric surface was heparinized using the general procedure described below.

(14) The polymeric surface is etched with a oxidizing agent (potassium permanganate, ammoniumperoxidisulfate) in order to introduce hydrophilic characteristics together with some reactive functional groups (—SO.sub.3H, —OH, —C═O, —C═C—). The surface can also be etched with plasma or corona.

(15) Reactive amino functions are introduced by treatment with a polyamine, polyethylenimine (PEI) or chitosan. For some purposes the polyamines may be stabilized on the surface by cross linking with bifunctional reagents, such as crotonaldehyde or glutaraldehyde.

(16) The coating is further stabilized by ionic cross linking with a sulfated polysaccharide (dextran sulfate or heparin). If necessary these steps are repeated and a sandwich structure is built up. Careful rinsing (water, suitable buffers) should be performed between each step. After a last addition of PEI or chitosan, end-point attachment (EPA) to the aminated surface of native heparin is done by reductive amination, utilizing the aldehyde function in the reducing terminal residue in native heparin. A more reactive aldehyde function in the reducing terminal residue can be achieved by partial, nitrous degradation of heparin. This shortens the reaction time, but the immobilized heparin will have a lower molecular weight. The coupling is performed in aqueous solution, by reductive amination (cyanoborohydride, CNBH.sub.3.sup.−) essentially as described in Preparatory example 2.

(17) 1-10 μg/cm.sup.2 of heparin can be coupled to all hydrophilic surfaces like glass, cellulose, chitin etc, and more or less all hydrophobic polymers like polyvinyl chloride, polyethylene, polycarbonate, polystyrene, PTFE etc.

Preparatory Example 5

(18) Covalent Single- or Multipoint Attachment of Heparin onto Polymeric Surfaces

(19) Performed as described in Preparatory example 2, with the exception that the aldehyde functions were introduced in the heparin chain by oxidation with sodium periodate in aqueous solution.

Preparatory Example 6

(20) Attachment of Heparin onto the Inner Lumen of Hollow Fibers

(21) In this preparatory example, a pediatric haemoflow dialyzer was used. The fibers of the dialyzer were made of polysulfone with an inner diameter of 200 microns and a wall thickness of 40 microns. The total surface area of the blood contacting material was 4000 cm.sup.2 and the priming volume was 28 ml.

(22) The amination procedure was performed as generally described in Preparatory example 4 with the exception that the etching step was omitted. Polysulfone is hydrophilic and does not need etching. Immobilization of heparin was performed by pumping a solution containing nitrous acid degraded heparin (heparin from Pharmacia) together with NaBH.sub.3CN as described in Preparatory example 2. As measurement of the amount of heparin is a destructive procedure, a reference dialyzer that was heparinized under identical conditions was sacrificed and its fibers are subjected to sulfur analysis. The results revealed a heparin content of about 5 μg heparin/cm.sup.2, which corresponds to a content of 20 mg heparin in the device.

Preparatory Example 7

(23) Covalent Attachment of Oligomers with Terminal Sialic Acid Residues onto the Inner Lumen of Hollow Fibers

(24) In this preparatory example, the aldehyde group at the reducing terminal residue was used for coupling. Amination of the fibers was performed as described in Preparatory example 6 and coupling of the oligosaccharide of formula I, which contains terminal sialic acid residues, was performed by circulating the compound of formula I, dissolved in acetate buffer (800 ml, 0.1 M, pH 4.0) together with NaBH.sub.3CN (0.4 g), at room temperature for 24 h. The results revealed a sialic acid content of ca. 2 μg/cm.sup.2.

(25) ##STR00001##

Comparative Example 1

(26) Binding of HSV-1 to Heparin in Solution

(27) A solution (10 μl) containing 10.sup.7 plaque forming units of virus (Herpes simplex virus type 1 strain KOS321) was incubated with 20 μl of .sup.3H-labelled heparan sulfate (HS) in a total volume of 400 μl of buffered NaCl for 30 min at 37° C. Thereafter, the solution was centrifuged through a Microsep 1 M filter, retaining virus and bound HS. 2.3% of HS was bound (479 CPM) to the virus, while 97.7% of the HS was unbound and passed through the filter.

Example 1

(28) Removal of HSV-1 and HSV-2 Virus Particles from Buffered Saline by Binding to Heparin Immobilized on Sephadex Beads

(29) Sephadex beads coated with heparin, as in Preparatory example 2, were soaked in buffered NaCl (PBS) and 0.8 ml was transferred to each of two small disposable columns, forming a gel layer of approximately 1 cm. After washing three times, 50 μl of .sup.3H-thymidine radiolabelled viruses were suspended in 150 μl of PBS. 10.sup.9 plaque forming units of HSV-1, corresponding to 10.sup.11 virus particles, were added to column 1, and 10.sup.8 plaque forming units of HSV-2, corresponding to 10.sup.10 virus particles, were added to column 2. Virus was allowed to adsorb to the respective columns. Thereafter, 0.8 ml of PBS was added to each column and the pass-through fluid was collected for estimation of unabsorbed virus.

(30) Subsequently, both columns were washed 4 times with 1 ml of PBS, and the washings were collected as fractions for quantification of washed out virus. These, and the following fractions, were transferred to scintillation vials and quantified with regard to amount of virus through determination of cpm in a beta counter. In the next step, the columns were subjected to elution of the respective heparin-bound viruses three times by 1 ml of 2 M NaCl, and the three fractions were collected from each column. Following that, elution was performed by twice adding 1 ml of 5% SDS in PBS (PBS-SDS), and the two fractions from each column were collected. Finally, the heparin-coated beads from the two columns were each suspended in 1 ml of PBS-SDS, and 200 μl aliquots were subjected to quantification of remaining bound virus particles by determination of radioactivity.

(31) The results are shown in Table 1 below. As shown, only 5.5% of HSV-1 particles and 11.7% of HSV-2 particles did not adsorb to the column. Moreover, since the viral DNA and not their heparin-binding proteins are labeled with radioactivity, these non-adsorbed particles might represent non-infectious viruses with disrupted envelopes (i.e. the outer, fragile, parts of the virus that bind to heparin). The rest of the viruses (94.5% for HSV-1 and 88.3% of HSV-2) bind to the heparin-coated beads in the column. The binding appears to be strong, judging from the fact that only 0.5% of HSV-1 and 1.1% of HSV-2 was removed by 4 successive washings. The limited ability of 2 M NaCl at 3 successive attempts to elute the viruses underscores the high-affinity characteristic of the binding of both viruses to the heparin-coated beads. In contrast, substantial quantities of HSV-1 and HSV-2 were eluted by PBS-SDS.

(32) A total of 48% of HSV-1 and 68.8% of HSV-2 were recovered from the columns. This can be attributed to the fact that 2 M NaCl spontaneously decreased the radioactivity of the samples by approximately 30% according to our past observations, and that SDS-PBS probably also has this effect.

(33) Taken together, the results prove the principle that HSV-1 and HSV-2 virus particles can be removed from a fluid phase by passage through a short column containing heparin-coated Sephadex beads, and that extracted viruses bind with high affinity to the columns.

(34) TABLE-US-00001 TABLE 1 Binding of radiolabelled HSV virus particles, suspended in buffered NaCl, to heparin-Sephadex beads. Binding of HSV to heparin column (% of input virus = control = 100%) HSV-1 HSV-2 Input virus (control) 100.0 100.0 Unadsorbed virus 5.5 11.7 Washed from column 1.sup.st washing 0.2 0.4 2.sup.nd washing 0.1 0.3 3.sup.rd washing 0.1 0.2 4.sup.th washing 0.1 0.2 total unadsorbed + washed 6.0 12.8 Eluted with 2M NaCl 1.sup.st elution 7.8 14.7 2.sup.nd elution 1.6 1.9 3.sup.rd elution 0.1 1.3 Eluted with 5% SDS 1.sup.st elution 10.6 11.9 2.sup.nd elution 18.5 18.3 total eluted NaCl + SDS 38.6 48.1 Uneluted from beads 4.1 7.9 total uneluted 4.1 7.9 Total recovered 48.7 68.8 (unadsorbed + washed + eluted + uneluted)

Example 2

(35) Removal of HSV-1 Virus Particles from Human Serum by Binding to Heparin Immobilized on Sephadex Beads

(36) The experimental procedure as described in Example 1 was utilized with the difference that the radiolabelled HSV-1 virus particles at a quantity of 10.sup.9 PFU equivalent to 10.sup.11 virus particles were mixed with 0.5 ml of human serum and then applied on heparin-coated beads in a disposable column. Thereafter, the procedure including elution and washing was followed as in Example 1. The results are shown in Table 2 below.

(37) TABLE-US-00002 TABLE 2 Binding of radiolabelled HSV-1 virus particles (10.sup.11), suspended in human serum, to heparin-Sephadex beads (1 cm.sup.3). Binding of HSV-1 to heparin column (% of input virus = control = 100%) Input virus (control) 100.0 Unabsorbed virus 2.4 Washed from column 1.sup.st washing 0.9 2.sup.nd washing 0.2 3.sup.rd washing 0.1 4.sup.th washing 0.2 total unabsorbed + washed 3.8 Eluted with 2M NaCl 1.sup.st elution 2.1 2.sup.nd elution 0.4 3.sup.rd elution 0.2 Eluted with 5% SDS 1.sup.st elution 3.5 total eluted NaCl + SDS 6.2 Total recovered 10.0 (unabsorbed + washed + eluted + uneluted) Remained on beads 90.0

(38) As shown, 97.6% of the HSV-1 particles suspended in human serum were bound to the column. By washing 4 times, only 3.8% of the virus particles were removed. Using 2 M NaCl, only 2.7% of the virus was eluted, and an additional 3.5% were eluted by SDS. The conclusion of these results is that suspending virus in serum, which is the real life situation during severe, disseminated infection, improved the performance of the virus-removing column as regards binding of radiolabelled virus, and that only 2.4% of the HSV-1 particles were unadsorbed. As a probable explanation, serum proteins helped to stabilize the virus particles and thereby improved the removal of HSV-1 by the heparinized Sephadex beads.

Example 3

(39) Removal of HSV-1 Virus Particles from Human Serum by Binding to Heparin Immobilized on a Hollow Fiber Haemoflow Dialyzer

(40) The experimental procedure as described in Example 1 was utilized with the difference that the radiolabelled HSV-1 virus particles at a quantity of 10.sup.9 PFU equivalent to 10.sup.11 virus particles were mixed with 0.5 ml of human serum and then applied on the heparin-coated hollow fiber dialyzer of Preparatory example 6. Thereafter, the procedure including elution and washing was followed as in Example 1. The results are shown in Table 3 below.

(41) As shown, 92.3% of the HSV-1 particles suspended in human serum were bound to the column. By washing 4 times, only 4.2% of the virus particles were removed. By 2 M NaCl, only 4.0% of the virus was eluted, and an additional 4.5% were eluted by SDS. The conclusion of these results is that the binding of virus particles suspended in human serum to heparinized fibers is comparable to that of similarly suspended virus binding to heparin-coated Sephadex beads, and that only 7.7% of the HSV-1 particles were unabsorbed.

(42) TABLE-US-00003 TABLE 3 Binding of radiolabelled HSV-1 virus particles, suspended in human serum, to heparinized hollow fibers. Binding of HSV-1 to heparin column (% of input virus = control = 100%) Input virus (control) 100.0 Unabsorbed virus 7.7 Washed from column 1.sup.st washing 3.1 2.sup.nd washing 0.8 3.sup.rd washing 0.1 4.sup.th washing 0.2 total unadsorbed + washed 4.2 Eluted with 2M NaCl 1.sup.st elution 3.3 2.sup.nd elution 0.5 3.sup.rd elution 0.2 Eluted with 5% SDS 1.sup.st elution 4.5 total eluted NaCl + SDS 8.5 Total recovered 12.7 (unabsorbed + washed + eluted + uneluted) Remained on beads 87.3

Example 4

(43) Removal of HSV-1 and HSV-2 Virus Particles from Human Whole Blood by Binding to Heparin Immobilized on Sephadex Beads

(44) The experimental procedure as described in Example 1 was utilized with the difference that the radiolabelled HSV-1 and HSV-2 virus particles at a quantity of equivalent to 10.sup.11 virus particles per ml were mixed with 1 ml of human blood and then applied to 1 ml heparin-coated beads in a disposable column. Thereafter, the procedure including elution and washing was followed as in Example 1.

(45) The results are shown in Table 4 below. As shown, 99.1% of the HSV-1 particles and 99.8% of the HSV-2 particles suspended in human blood were bound to the column.

(46) TABLE-US-00004 TABLE 4 Binding of radiolabelled HSV virus particles, suspended in human blood, to heparin-Sephadex beads (1 cm.sup.3). Binding of whole blood HSV to heparin column (% of input virus = control = 100%) HSV-1 HSV- 2 % % Input virus (control) 100.0 100.0 Unadsorbed virus 0.9 0.2

Example 5

(47) Removal of Influenza A virus from Human Serum by Binding to Immobilized Oligosaccharides Containing Sialic Acid

(48) Virus stocks of Influenza A H1N1 were replicated in MDCK cells grown at 35° C. under standard conditions for 3 days, after which the cells were homogenized and titrated to assess the number of focus-forming units (FFU)/ml. Virus particles were suspended in human serum to a final concentration of 10.sup.6 FFU/ml. A 10 ml suspension was applied on a sialic acid-coated hollow fiber dialyzer, prepared using the method described in Preparatory example 7. After titrating the infectivity of the Influenza A-containing serum after passage through the dialyzer and comparing it with titers of an aliquot of the same virus-containing serum not passed through the device it was concluded that 87% of the Influenza A virus FFU remained bound to the fibers.

Example 6

(49) Removal of Helicobacter pylori and Staphylococcus aureus by Binding to Immobilized Heparin

(50) Four sterile pipettes were packed with glass wool (0.5 ml) that was heparinized as described in Preparatory example 3. The “columns” thus formed were washed with 3 ml of sterile phosphate saline buffer (PBS), pH 7.2. Two different strains of H. pylori and two different strains of S. aureus were tested. Each of the four different bacteria samples, suspended in PBS buffer, were applied to a separate “column”. The amounts of bacteria in the samples were measured before application to the column and after elution from the column by optical density (OD) at 560 nm and viable counts (CFU/ml). As is evident from the table below, roughly 90% of H. pylori and roughly 50% of S. aureus bacteria were immobilized on the columns.

(51) TABLE-US-00005 TABLE 6 Binding of H. pylori and S. aureus to heparinized glass wool. OD.sub.560 CFU/ml Binding Bacteria In Out In Out % H. Pylori ATCC 8.33 0.81   2 × 10.sup.8 1.5 × 10.sup.7 ~90 43504 H. Pylori ATCC 8.33 1.86   2 × 10.sup.8   1 × 10.sup.7 ~90 43504 S. aureus CCUG 9.30 5.5 7.7 × 10.sup.9 3.6 × 10.sup.9 ~50 12600 S. aureus CCUG 9.30 6.9 7.7 × 10.sup.9 3.6 × 10.sup.9 ~50 12600