Title:  Method for removing pyrogens from plasma and blood for treatment of septic shock

United States Patent:  6,803,183

Issued:  October 12, 2004

Inventors:  Amoureux; Marie-Claude (San Diego, CA); Hegyi; Edit (Valley Center, CA); Grandics; Peter (Rancho Santa Fe, CA); Szathmary; Susan (Rancho Santa Fe, CA)

Assignee:  Clarigen, Inc. (Carlsbad, CA)

Appl. No.:  198774

Filed:  July 18, 2002

Abstract

In general, a method for removal of pyrogens from plasma or blood according to the present invention comprises: (1) passing the biological fluid through a resin comprising two components: (a) a first component comprising a polymeric support with a multiplicity of ligands comprising a C₁₀ -C₂₄ alkylamino group, the ligands specifically binding endotoxin; and (b) a second component comprising a halogenated quinolone moiety immobilized on an amino-group-containing solid support by the formation of an amide linkage, the first component and the second component being present in the resin at a ratio of from about 20:80 (v/v) to about 80:20 (v/v); and (2) collecting the biological fluid from which pyrogens have been removed. Preferably, the alkylamino group is a stearylamino group, and the ligands are cross-linked and quaternized by reaction with a dibromoalkanol. Preferably, the polymeric support of the first component is agarose in particulate form. Preferably, the halogenated quinolone moiety is (+)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H- pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid. The invention also includes resins useful in methods according to the present invention and cartridges incorporating such resins.

INVENTION SUMMARY

One aspect of the present invention is a method for removing pyrogens from a biological fluid selected from the group consisting of plasma and blood comprising the steps of:

(1) passing the biological fluid through a resin comprising two components: (a) a first component comprising a polymeric support with a multiplicity of ligands comprising a C₁₀ -C₂₄ alkylamino group, the ligands specifically binding endotoxin; and (b) a second component comprising a halogenated quinolone moiety immobilized on an amino-group-containing solid support by the formation of an amide linkage, the first component and the second component being present in the resin at a ratio of from about 20:80 (v/v) to about 80:20 (v/v); and

(2) collecting the biological fluid from which pyrogens have been removed.

Typically, the alkylamino group is a stearylamino group.

Typically, the polymeric support of the first component is agarose.

Typically, at least a proportion of the ligands of the first component are cross-linked. Preferably, the cross-linking is at the amino moiety of the alkylamino group, such as the stearylamino group. Preferably, the ligands are cross-linked by quaternization with an alkyl group, such as by reaction with a dibromoalkanol. A particularly preferred dibromoalkanol is 2,3-dibromopropanol.

Typically, the polymeric support of the first component is in particle form. Typically, the particles are from about 20 microns to about 900 microns in diameter. Preferably, the particles are from about 60 microns to about 300 microns in diameter. More preferably, the particles are from about 60 microns to about 100 microns in diameter.

Typically, the halogenated quinolone moiety of the second component is of formula (I) wherein R₁ is lower alkyl, R₂ is lower alkyl, and X is a halogen selected from the group consisting of fluoro, chloro, bromo, and iodo. Preferably, R₁ is methyl, R2 is methyl, X is fluoro, and the halogenated quinolone moiety is (+)9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-p yrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid, known as Floxin.

Typically, the amino-derivatized support of the second component is amino-derivatized agarose.

Typically, the halogenated quinolone moiety is immobilized to the amino-containing solid support by reaction with a carbodiimide. Preferred carbodiimides include, but are not limited to, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, and N,N'-dicyclohexylcarbodiimide. A particularly preferred carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

Typically, the first component and the second component are present in a ratio of from about 60:40 (v/v) to about 40:60 (v/v). Preferably, the first component and the second component are present in a ratio of about 50:50 (v/v).

Preferably, the method removes pyrogens below the level of 0.25 EU/ml. Preferably, the method removes endotoxin.

Another aspect of the present invention is a resin comprising two components: (1) a first component comprising a polymeric support with a multiplicity of ligands comprising a C₁₀ -C₂₄ alkylamino group, the ligands specifically binding endotoxin; and (2) a second component comprising a halogenated quinolone moiety immobilized on an amino-group-containing solid support by the formation of an amide linkage, the first component and the second component being present in the resin at a ratio of from about 20:80 (v/v) to about 80:20 (v/v). Particularly preferred resins are those described above in terms of the method.

Yet another aspect of the present invention is a cartridge for removal of pyrogens comprising:

(1) a housing for holding a resin, the housing having first and second ends;

(2) a resin according to the present invention as described above;

(3) an inlet attached to the first end of the housing for allowing a biological fluid selected from the group consisting of blood and plasma to flow into the housing; and

(4) an outlet attached to the second end of the housing for allowing the biological fluid from which pyrogens have been removed to flow out of the housing.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One aspect of the present invention is a method for removing pyrogens from a biological fluid selected from the group consisting of blood and plasma. The method comprises the steps of:

(1) passing the biological fluid through a resin comprising two components: (1) a first component comprising a polymeric support with a multiplicity of ligands comprising a C₁₀ -C₂₄ alkylamino group, the ligands specifically binding endotoxin; and (2) a second component comprising a halogenated quinolone moiety immobilized on an amino-group-containing solid support by the formation of an amide linkage, the first component and the second component being present in the resin at a ratio of from about 20:80 (v/v) to about 80:20 (v/v); and

(2) collecting the biological fluid from which pyrogens have been removed.

Typically, the alkylamino group is a stearylamino group.

Typically, the polymeric support of the first component is agarose.

Typically, at least a proportion of the ligands of the first component are cross-linked. Preferably, the cross-linking is at the amino moiety of the alkylamino group, such as the stearylamino group. Preferably, the ligands are cross-linked by quaternization with an alkyl group, such as by reaction with a dibromoalkanol. A particularly preferred dibromoalkanol is 2,3-dibromopropanol.

Typically, the polymeric support of the first component is in particle form. Typically, the particles are from about 20 microns to about 900 microns in diameter. Preferably, the particles are from about 60 microns to about 300 microns in diameter. More preferably, the particles are from about 60 microns to about 100 microns in diameter.

Typically, the halogenated quinolone moiety of the second component is of formula (I) wherein R₁ is lower alkyl, R₂ is lower alkyl, and X is a halogen selected from the group consisting of fluoro, chloro, bromo, and iodo. As used herein, the term "lower alkyl" refers to an unbranched or branched alkyl group of from 1 to 6 carbon atoms; typically, "lower alkyl" refers to an unbranched alkyl group of from 1 to 6 carbon atoms. Preferably, R₁ is methyl, R₂ is methyl, X is fluoro, and the halogenated quinolone moiety is (+)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H- pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid, known as Floxin.

Typically, the amino-derivatized support of the second component is amino-derivatized agarose.

Typically, the halogenated quinolone moiety is immobilized to the amino-containing solid support by reaction with a carbodiimide. Preferred carbodiimides include, but are not limited to, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, and N,N'-dicyclohexylcarbodiimide. A particularly preferred carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

Typically, the first component and the second component are present in a ratio of from about 60:40 (v/v) to about 40:60 (v/v). Preferably, the first component and the second component are present in a ratio of about 50:50 (v/v).

Preferably, the method removes pyrogens below the level of 0.25 EU/ml. Preferably, the method removes endotoxin.

The technology of the present invention utilizes a patented proprietary large particle based support (U.S. Pat. No. 5,466,377, incorporated herein by this reference) that has high binding capacity and allows the free passage of cells between the particles, without requiring any fluidization. The immobilization chemistry has been extensively characterized for leaching and has been found to be stable. When the support is in the form of particles, the particles are preferably in the size range of 60-160 .mu.m beads for ET adsorption from plasma and 200-300 .mu.m range beads for ET adsorption from whole blood.

One of the resins used in this new method is Acticlean Etox.TM. (Sterogene Bioseparations, Inc., Carlsbad, Calif., USA) and it is extensively used as a solid phase reagent in bioprocesses (Smith and Grandics, Am Biotech Lab 2000, 18:39-40). It effectively removes ET from several biological media including DNA, proteins and bovine serum. Affinity of the immobilized ligand for proteins is very low, delivering a mass yield of proteins that exceeds 95%. The ET binding capacity of Acticlean Etox has been measured in buffers and protein solutions (up to 500,000 EU/ml). Acticlean Etox has favorable features such as it dissociating ET-protein complexes. This ligand is stable and non-toxic. This resin is further described in U.S. Pat. No. 6,106,723 by Grandics et al., incorporated herein in its entirety by this reference. In general, the resin is prepared by a process of: derivatizing a polymeric support with an epihalohydrin; reacting the derivatized support with a (C₁₀ -C₂₄) alkylamine; and crosslinking at least a portion of the alkylamino groups and quaternizing at least a portion of the amino moieties by reacting the support with a halogenated alkyl alcohol. Preferably, the epihalohydrin is epibromohydrin.

The final resin of the invention is a mixture of Acticlean Etox and Floxin immobilized resins, preferably in equal amounts (50/50; v/v).

We have used a supernatant of Pseudomonas maltophilia, a Gram^- bacteria, as a model to spike plasma or whole blood and challenge the adsorbent.

The plasma or whole blood was pumped through the column at a flow rate of 0.6 ml/min. Samples were collected using sterile needle and syringes via a sample port.

To determine ET content in plasma samples and blood samples, the Limulus amoebocyte lysate (LAL) assay was used directly on heparinized plasma, or serum prepared from heparinized whole blood that clotted in the glass ET-free tubes used for collection. The use of citrated plasma or blood was prevented by the interference of citrate with the LAL assay.

The new resin of the present invention is able to remove at least 50% of the loaded ET from plasma. Three cartridges of 1.5 ml placed in series result in ET depletion from 2 EU/ml (200 pg/ml) level found in plasma of sepsis patients, to <0.25 EU/ml. A volume of plasma of up to 34 column volumes can be treated and 50% of the loaded ET can consistently be removed by one cartridge.

We have found that the Floxin/Acticlean Etox resin can also remove ET from whole blood. When blood was spiked with 2.6 EU/ml ET, after passing through 1.5 ml of adsorbent, post-column levels were between 0.4 and 1.6 EU/ml. For a spike of 0.75 EU/ml, the levels were brought down to <0.33 EU/ml.

Since non-LPS (LAL-negative) pyrogens also contribute to sample pyrogenicity, an assay other than the LAL test was used to evaluate pyrogens in samples. Such a test can be the USP rabbit pyrogenicity test. This is however rather tedious and impractical for this purpose for a variety of reasons. Moreover, the rabbit test does not provide information on sub-toxic, low level inflammatory reactions induced by the sample. A more suitable test is based upon the monocyte activation/cytokine assay that is the equivalent to the rabbit test in assessing total sample pyrogenicity. Monocytes, when exposed to pyrogenic substances, secrete inflammatory cytokines (IL-1, IL-6, IL-8, TNF-.alpha., etc.) that can be detected at very low levels. Induction of TNF-.alpha. secretion by monocytes was used as an in vitro assay system for pyrogen-induced inflammatory reactions. TNF-.alpha. is an important early mediator of host responses to ET contamination, and is induced in vitro by ET challenge. TNF-.alpha. can also induce the production of IL-1 in various cell types, as well as IL-6 (Shalaby, Clin Immunol Immunopathol 1989, 53(3): 488-498). TNF-.alpha. is the only endogenous mediator capable of triggering the entire spectrum of metabolic, hemodynamic, tissue and cytokine cascade responses of septic shock (Tracey, Crit Care Med 1993, 21 (10 Suppl): S415-422). We have found that the Floxin/Acticlean Etox resin greatly reduced TNF-.alpha. production.

In order to assess the efficiency of removal of infectious agents, Pseudomonas maltophilia was used as a model of Gram^- bacteria. Floxin/Acticlean Etox (1.5 ml) was able to remove between 20 and 60% of bacteria from plasma.

Another aspect of the present invention is a resin comprising two components: (1) a first component comprising a polymeric support with a multiplicity of ligands comprising a C₁₀ -C₂₄ alkylamino group, the ligands specifically binding endotoxin; and (2) a second component comprising a halogenated quinolone moiety immobilized on an amino-group-containing solid support by the formation of an amide linkage, the first component and the second component being present in the resin at a ratio of from about 20:80 (v/v) to about 80:20 (v/v). Particularly preferred resins are those described above in terms of the method.

Yet another aspect of the present invention is a cartridge for removal of pyrogens comprising:

(1) a housing for holding a resin, the housing having first and second ends;

(2) a resin according to the present invention as described above;

(3) an inlet attached to the first end of the housing for allowing a biological fluid selected from the group consisting of blood and plasma to flow into the housing; and

(4) an outlet attached to the second end of the housing for allowing the biological fluid from which pyrogens have been removed to flow out of the housing.

Preferred resins are those described above.

The cartridges can be coupled in series, so that two or more cartridges can be coupled together, the outlet of one cartridge being attached to the inlet of the next. Accordingly, another aspect of the present invention is an assembly comprising a plurality of cartridges according to the present invention connected in series so that the outlet of each cartridge is connected to the inlet of the next cartridge.

Claim 1 of 62 Claims

What is claimed is:

1. A method for removing pyrogens from a biological fluid selected from the group consisting of plasma and blood comprising the steps of:

(a) passing the biological fluid through a resin comprising two components: (1) a first component comprising a polymeric support with a multiplicity of ligands comprising a C₁₀ -C₂₄ alkylamino group, the ligands specifically binding endotoxin; and (2) a second component comprising a halogenated quinolone moiety immobilized on an amino-group-containing solid support by the formation of an amide linkage, the first component and the second component being present in the resin at a ratio of from about 20:80 (v/v) to about 80:20 (v/v); and

(b) collecting the biological fluid from which pyrogens have been removed.

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