LOW MOLECULAR WEIGHT PECTIN COLUMN FOR APHERESIS

Abstract

An apheresis column for the treatment of mammals to reduce galectin-3 levels is set forth. The column features, as a stationary or adsorbent phase, pectin molecules, which naturally bind gal-3. Modified citrus pectin, having a molecular weight of about 40 kD or less, preferably about 25 kD or less, is a preferred adsorbent. The columns are disposable, and are used in the fashion for apheresis in general. Other targets, particularly including other galectins, as well as toxins, heavy metals and the like may also be withdrawn from the blood or plasma through this method. Gal-3 level reductions of 10%, 30% and even more may be achieved in a single treatment.

Claims

1. An apheresis column for the ex vivo treatment of a mammal, comprising a column with a pathway through which at least a portion of the mammal's blood is directed, wherein said pathway is provided with units of immobilized low molecular weight pectin to which said blood is exposed, wherein at least galectin-3 in said blood is bound by said pectin molecules in said column, after which said blood is returned to said mammal, wherein following apheresis, galectin-3 levels in the mammal are reduced by at least 10%.

2. The column of claim 1, wherein said blood is separated into plasma and non-plasma components prior to entering said pathway, and wherein only said plasma is passed through said pathway.

3. The column of claim 1, wherein the level of galectin-3 in said patient following apheresis treatment in said column is reduced by at least 30 percent.

4. The column of claim 1, wherein said immobilized pectin is comprised of modified citrus pectin having a molecular weight less than 40 kD.

5. The column of claim 4, wherein said modified citrus pectin is of a molecular weight less than 25 kD.

6. A process for the ex vivo removal of galectin-3 from the blood of a mammal, comprising: directing said blood to a column comprising a pathway provided with units of immobilized low molecular weight pectin to which said blood is exposed, wherein at least galectin-3 in said blood is bound by said pectin units in said column, thereafter returning said blood to said mammal, whereby at least 10% of the galectin-3 in the blood of said mammal is removed.

7. The process of claim 6, wherein said blood is separated into cellular components and plasma prior to being directed to said column, and wherein only said plasma is directed to said column.

8. The process of claim 7, wherein at least 80% of the galectin-3 in the blood directed to said column is removed.

9. The process of claim 6, wherein, immediately following said process, the mammal so treated exhibits a galectin-3 level at least ten percent lower than the galectin-3 level of the blood of said mammal immediately prior to that process.

10. The process of claim 9, wherein immediately following said process, the mammal so treated exhibits a galectin-3 level at least 30% lower than the galectin-3 level of said mammal immediately prior to that process.

11. The process of claim 6, wherein said process is accompanied by treatment of said mammal with one or more agents which are therapeutically more effective in the presence of reduced levels of galectin-3.

12. The method of claim 6, wherein said mammal is a human being.

Description

DESCRIPTION OF THE FIGURES

[0010] This application makes use of the ability of pectin, particularly modified citrus pectin, which has been proven safe and well tolerated in mammals, to bind gal-3. Thus, the molecular character or structure of both MCP and gal-3 are of importance to this invention.

[0011] FIG. 1 illustrates the molecular structure of MCP.

[0012] FIG. 2 illustrates the molecular structure of galectins in general, and gal-3 in particular, which may adopt a monomeric, dimeric or chimeric molecular structure.

DETAILED DESCRIPTION OF THE INVENTION

[0013] Apheresis is a well-known procedure that was developed in the early 1970s. Since that time, the use of apheresis has become widespread. This application simply applies this wide knowledge of extracorporeal practice to the use of a specific apheresis columnone using modified pectin, preferably MCP. There is an extensive collection of articles and scientific discussion of apheresis technology in de Sousa G, and Seghatchian J. Transfusion and Apheresis Science. 2006 February;34(1):107-23. Although much has been developed since that time as well, the invention here addresses a new column, rather than a specific new use. With the new column, of course, goes a new specific goalthe reduction of gal-3 in the blood by apheresisbut other than specific adaptations due to the use of modified pectin or MCP in particular, the general practice remains the same. Thus, this discussion begins with a consideration of how the column is prepared.

HOW THE COLUMN AND MATERIALS ARE MADE

[0014] Covalent immobilization of low molecular mass pectin using EDC [1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl]/NHS (N-hydroxysuccinimide esters) primary amine linking chemistry [link through a galUA (galacturonic acid) carboxylate on MCP] to agarose beads bearing an amine group. The carboxyl-containing (COOH) galUAs to a porous, beaded agarose resin for use in affinity purification procedures using extracorporeal apheresis of plasma or whole blood. The crosslinked beaded agarose is activated with diamino-dipropylamine (DADPA) to contain long spacer arms, each with a primary amine at the end. When incubated with the resin and the carbodiimide crosslinker EDC, carboxyl-containing molecules become permanently attached to the support by stable amide bonds.

[0015] Additional details on the immobilization chemistry for carboxyl-containing molecules required to prepared the column begins with the effective crosslinking agents. EDC crosslinker reacts with carboxylates to create an amine-reactive intermediate, resulting in a covalent attachment to the DADPA-activated Resin General References (for EDC coupling chemistry), Gilles, M. A. et al., Anal Biochem 1990;184:244-8; Grabarek, Z., and Gergely, J. Anal Biochem 1990;185:131-5; Williams, A., and Ibrahim, I. A. J Am Chem Soc 1981;103:7090-5.

[0016] Prior art characterizations of MCP focus on water-soluble supplements intended for oral, or, where appropriate, IV administration. Other routes of administration, such as via rectal or vaginal suppositories, may be available. The invention of this application is, however, directed to a stable, chemically modified version of MCP or other pectin or similar which is characterized as follows.

[0017] Pectin's ability to bind a variety of molecules, and galectins, including gal-3 in particular, are key to the effective use of the column of this invention. The structure of low molecular weight pectin is illustrated in FIG. 1 of this application. Pectin consists of four different types of polysaccharides, and their structures are shown. (Abbreviations: Kdo, 3-Deoxy-d-manno-2-octulosonic acid; DHA, 3-deoxy-d-lyxo-2-heptulosaric acid.) HG and RGI are much more abundant than the other components.

[0018] Pectin is a complex plant polysaccharide consisting of homogalacturonan (HG), which is partially methyl esterified; rhamnogalacturonan I (RGI), consisting of alternating rhamnose and galacturonic acid residues with arabinan, galactan and/or arabinogalactan attached to the rhamnose residues; rhamnogalacturonan II (RGII), with a homogalacturonan backbone and complex branches containing neutral and acidic sugars including 2-keto-3-deoxyoctonic acid (KDO); and xylogalacturonan, with xylose attached to some of the galacturonic acid residues.

[0019] The ratio between HG, XGA, RGI, and RGII is variable, but typically HG is the most abundant polysaccharide, constituting about 65% of the pectin, while RGI constitutes 20% to 35%. XGA and RGII, each representing 10% or less. The different pectic polysaccharides are not separate molecules but covalently linked domains. Unbranched homopolymer chains of -1,4-linked d-GalUA are described as HG. The backbone of GalUA residues can be substituted at various positions with other sugar moieties, including Xyl (XGA) and apiofuranose (apiogalacturonan). In XGA, a single Xyl is attached to the O-3 position of some GalUA residues. Additional Xyl residues can be attached to the first Xyl with -1,4 linkage.

[0020] The size and composition of MCP are consistent with reduced molecular weight and debranched pectin consisting of a homogalacturonan backbone with little methyl esterification. The galacturonic acid content of MCP analyzed by USDA-ARS pectin scientist (Eliaz et al., Phytother Res. 2006 October;20(10):859-64) was 74.6% (3.8), the degree of esterification was 3.8%, the KDO content was 0.46% (0.0) and the total neutral sugar carbohydrate content was 10.1% (0.7). The neutral monosaccharides in MCP were consistent with the neutral sugar-rich side chains in the rhamnogalacturonan region of pectin (Ridley et al., Phytochemistry. 2001 July;57(6):929-67.).

DETAILED DESCRIPTION REGARDING USE OF THE COLUMN

[0021] The blood or plasma of a person or other mammal is passed through the affinity column to separate out lectins, such as galectins in the circulation before returning the remainder back to the circulation. It is thus an extracorporeal therapy. Galectins are carbohydrate-binding proteins that are involved in many physiological functions, such as inflammation, immune responses, cell migration, autophagy, and signaling. They are also linked to diseases such as fibrosis, cancer and heart disease. The apheresis procedure lasts several hours depending on a multiple of variables such as patient blood volume, flow rate, and binding capacity, final column size and dimensions.

[0022] It is difficult to estimate the consequence of treatment in a column of the invention due to the fact that as the patient is treated, additional gal-3 is shed by the patient's tissues into the blood. For purposes of analysis, set a standard plasma volume, in a 70 KG adult, as about 3,000 ml (a close estimate). Apheresis using this column has been demonstrated to reduce nearly 100% of the gal-3 in the plasma being treated prior to treatment. In a chronic kidney disease patient with end-stage renal disease, up to 70 ng/ml gal-3, or about 210 micrograms, may be pulled from the blood being treated. It is important to understand that due to gal-3 replacement from tissues, the kinetics are such that in two plasma volumes, you remove only 80%, meaning 160 micrograms.

[0023] The principal target to be bound ex vivo through the use of the pectin-based column of this invention is galectin-3. As noted, gal-3 is the one galectin that appears not only as a monomer and dimer, but a chimeric form, sometimes considered a pentamer, as well. Gal-3 is not the only potential target for binding by MCP, however. Pectins, in general, can bind other environmental toxins in addition to the heavy metals mentioned, such as DDT (Zhan et al., Environment Intern 2019 September;130:104861). Evidence demonstrates the ability of pectin, and MCP in particular, to bind dioxin and dioxin-like toxins (Aozasa et al., Chemosphere 2001 October;45(2):195-200), radioactive isotopes (Nesterenko et al., Swiss Med Wkly, 2004 Jan. 10;134(1-2):24-7), and there is evidence that at least MCP can trap mycotoxins (Tamura et al., Carbohydr Polym. 2013 Apr. 2;93(2):747-52). Pectin binds to cholesterol in the gastrointestinal tract and slows glucose absorption by trapping carbohydrates. Thus, this invention is directed to and explains in detail the use of pectins, such as MCP, in a column to bind gal-3, and thereby reduce gal-3 levels in a patient through apheresis. The same apheresis treatment may be used to address other targeted or selective removal at the same time, where conditions warrant.

[0024] To remove specific blood components such as lectins in general (partners that bind particular carbohydrate structures), and specifically galectins [with an affinity to -galactose-containing glycoconjugates and share primary structural homology in their carbohydrate-recognition domains (CRDs)], as well as other carbohydrate bind proteins found in the circulation. In general, other components found in the blood which have previously been established as being well-bound by pectin, with MCP as an example, maybe targeted for removal by this process. Thus, in addition to gal-3 in particular, and lectins more generally, components associated with morbidity and mortality, including oxidized lipids, positively charged heavy metals, toxins and the like may be effectively removed using the column of the invention.

[0025] The column of this invention, specifically, an apheresis column comprising pectin or another polyuronide, preferably modified citrus pectin having a molecular weight of less than 40 kD, is used in a fashion not dissimilar from other apheresis columns. This invention allows apheresis to be performed on either whole blood or plasma. Whole blood may offer advantages, which can be achieved using larger columns with longer and larger pathways. Specifically, blood is withdrawn from a patient. Blood may be separated, often by centrifugation, into plasma and cell components. If treating plasma only, the components are returned to the body. The plasma is directed under low pressure through a tube or module which provides a pathway for the plasma through the adsorbent phase. A prior art adsorbent phase use in apheresis for removal of low-density lipids, or liposorption, uses dextran sulfate as the adsorbent phase. The column of the claimed invention is used in a very similar manner, but offers a different adsorbentpectinto remove a different blood/plasma componentgal-3. The apheresis treatment can be continuous or discontinuous, depending on the pump and other apparatus available.

[0026] As noted, this invention is focused on the ability to remove gal-3 and lower gal-3 serum and blood levels. Gal-3 mediates a wide variety of diseases and complications. Among the most profoundly impacted are kidney patients, including those suffering from chronic kidney disease (CKD) and acute kidney disease (AM). Treatments generally adopted for such patients may be advantageously administered with or immediately after apheresis to reduce gal-3 levels. Among the more well-known and generalized therapies are the administration of diuretics, and calcium and insulin to avoid dangerous increases in blood potassium levels. Vasodilators, such as fenoldopam and minoxidil, may be used to reduce the need for renal replacement therapy and lower the mortality rate in patients with AM. ACE inhibitors and angiotensin receptor blockers (ARBs) may be used as well as anti-inflammatory and kidney protective compounds with greater effectiveness given reduced Gal-3 levels and the reduced levels of inflammation and fibrosis formation resulting. Another group of patients fundamentally impacted by gal-3 levels and the conditions mediated by gal-3 are cancer patients. A wide variety of tumor-based cancers are mediated, at least in part, by gal-3 levels.

[0027] Other more targeted therapies may be made more effective by being practiced together with Gal-3 apheresis. AM occurs in about fifty percent of patients with septic shock. Traditional treatments for septic shock, such as vasopressors, inotropic (e.g., clonidine) and the like are advantageously combined in acute patients with Gal-3 apheresis. Angiopoietin levels are closely associated with the pathogenesis of vascular permeability, and established angiopoietin disciplines may be more effective at low Gal-3 systemic levels. Ebihara et al., Ther Apher Dial. 2016;20(4):368-75. As a general proposition, most of the therapeutics used to try to slow the progress of kidney damage and disease will be more effective following and/or combined with Gal-3 apheresis, as it will reduce the tendency of fibrotic growth to block the damaged tissue from the pharmaceutical agents systemically administered. To this end, conventionally used cardiac, renal and circulatory medications may be employed to further support the patient together with the selective removal of Gal-3. In addition to the therapeutics discussed above, conventionally used therapeutics include beta-blockers, calcium channel blockers, direct renin inhibitors, alpha-blockers and alpha 2 agonists (e.g., clonidine). Erythropoietin (rhEPO) and iron replacement therapy may be effective, as well as other supplements, such as vitamins and the like. To further increase effectiveness, such agents can be administered to the plasma or blood being returned to the patient after Gal-3 removal. In this respect, the enhancement of traditional dialysis by the use of gal-3 selective removal through apheresis may greatly reduce inflammation and fibrotic kidney injury. This will allow a significant increase in the percentage of patients having a complete recovery of kidney function for both short and long term care solutions.

[0028] The reduction of gal-3 levels is the specific goal of this invention. Gal-3 is ubiquitously expressed in the heart, the kidney, blood vessels, and macrophages play a vital role in tissue fibrosis, immunity, and the inflammatory response. Furthermore, extracellular gal-3 cross-links glycoconjugates and form lattices. Formation of galectin/glycoconjugate lattices on the plasma membrane has been observed to influence the expression time, localization, and activity of several cell surface receptors, thus affecting numerous biological functions such as cell signaling, cell migration, and cell adherence. MCP, as administered, disrupts the lattice formation with positive consequences for several physiological processes related to immune responses and inflammation, as well as pathological conditions such as fibrosis, cancer, and heart disease. This invention takes advantage of the binding properties of low molecular weight pectin, generally, and MCP specifically, to reduce gal-3 levels, ex vivo, which makes possible a wide variety of applications.

[0029] MCP provides multiple carbohydrate sites for binding up the receptor CRD regions of gal-3 antagonizing its proinflammatory and tumorigenesis effects. The binding affinity of the galectin chain is proportional to its length up to 4 gal residues and mostly unchanged after that. MCP can bind to all galectin, but at different affinities. The strength of ligand binding is determined by several factors: The multivalency of both ligand and the galectin, the length of the carbohydrate, and the mode of presentation of ligand to the CRD. Different galectins have distinct binding specificities for binding oligosaccharides. The only chimeric type, gal-3, with its ability to oligomerize to a pentamer, provides a particularly apt target for binding by MCP, offering a wide array of carbohydrate ligands on its side chains and backbone makes for high-affinity selective binding. With gal-3 being overexpressed into the circulation in the medical conditions mentioned above, the most prevalent extracellular galectin found in circulation, gal-3 would be the predominant target and most effectively removed agent using the column of this invention. The molecular characteristics of gal-3 demonstrating the particular suitability of the column of this invention, are illustrated in FIG. 2 of this Application.