Use of heparin and carbohydrates to treat cancer

Abstract

The method is described as the removal of mediators that contribute to the pathogenesis of cancer from blood by contacting the blood with a solid, essentially non-micro-porous substrate which has been surface treated with heparin, heparan sulfate and, optionally, other molecules or chemical groups (the adsorbent media or media) having a binding affinity to the mediator, and wherein the size of the interstitial channels within said substrate are balanced with the amount of interstitial substrate surface area such that high flow rates of blood past said substrate creates a flow transport that is characterized by convection transport more than Brownian diffusion transport.

Claims

1. A method for treating cancer progression or cancer metastasis by removing circulating cancer cells from mammalian blood, the method comprising: contacting mammalian blood with a solid substrate coated with end-point attached heparin, which end-point attached heparin has a binding affinity for the circulating cancer cells, wherein circulating cancer cells are bound to end-point attached heparin coated on the solid substrate to thereby remove circulating cancer cells from mammalian blood and thereby treat cancer or cancer metastasis.

2. A method for treating cancer, the method comprising: removing circulating cancer cells from blood of a mammal that has undergone surgery, wherein the circulating cancer cells have been generated by the surgery, by contacting the mammalian blood with a solid substrate coated with end-point attached heparin, wherein the circulating cancerous cells are bound to end-point attached heparin coated on the solid substrate, and returning the blood to the mammal to treat cancer.

3. The method according to any one of claims 1-2, wherein said contacting blood from a patient is conducted during and/or after surgery to remove a tumor.

4. The method of claim 1 or 2, wherein said solid substrate comprises a packed column of non-porous rigid beads or particles, a column packed with a rigid reticulated foam, a column packed with a rigid monolithic bed of sintered beads or other sintered solid media with internal flow channels, a column packed with woven or non-woven rigid fabric, a column packed with a rigid yarn or optionally hollow monofilament fibers, a spiral wound cartridge, or a combination of at least two members selected from the group consisting of beads, rigid reticulated foam, sintered beads, fabric, yarn and monofilament.

5. The method according to claim 4, wherein the solid substrate comprises polyethylene beads.

6. The method according to claim 1 or 2, further comprising at least one additional polysaccharide coated on the solid substrate selected from the group consisting of heparan sulphate, hyaluronic acid, sialic acid, carbohydrates with mannose sequences, and chitosan.

Description

(1) 5. Examples

(2) The various aspects of the invention are further described in the following examples. These examples are not intended to be limiting.

Example 1

(3) Preparation of Heparin Column

(4) Polyethylene (PE) beads, with an average diameter of 0.3 mm (lot no. 180153), are supplied by the Polymer Technology Group (Berkeley, USA) and the columns (Mobicol, 1 mL) are obtained from MoBiTec (Germany). Heparin and polyethyleneimine (PEI) are purchased from Scientific Protein Laboratories (Waunakee, Wis., USA) and BASF (Ludwigshafen, Germany) respectively. All chemicals used are of analytical grade or better.

(5) Immobilization of heparin onto the beads was performed as described by Larm et al. (Larm O, Larsson R, Olsson P. A new non-thrombogenic surface prepared by selective covalent binding of heparin via a modified reducing terminal residue. Biomater Med Devices Artif Organs 1983; 11: 161-173).

(6) The polymeric surface was heparinized using the general procedure described below.

(7) 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. For example, the PE-beads are etched with an oxidizing agent (potassium permanganate in sulphuric acid). These hydrophilized beads, inter alia containing OH-groups and double bonds, are later used as controls.

(8) 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.

(9) 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.

(10) 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.−).

(11) In this alternate method, the aminated media is 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 media.

(12) 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.

(13) The resulting PE-beads, with covalently end-point attached heparin, are sterilized with ethylenoxide (ETO) and rinsed with 0.9% sodium chloride and ultra pure water. The amount heparin was determined to be 2.0 mg heparin/g bead with the MBTH method. (Larm O, Larsson R, Olsson P. A new non-thrombogenic surface prepared by selective covalent binding of heparin via a modified reducing terminal residue. Biomater Med Devices Artif Organs 1983; 11: 161-173 and Riesenfeld J, Roden L. Quantitative analysis of N-sulfated, N-acetylated, and unsubstituted glucosamine amino groups in heparin and related polysaccharides. Anal Biochem 1990; 188: 383-389).

(14) The polyethylene beads had a mean diameter of 0.3 mm and are heparinized with a technology that guaranteed that the heparin molecules are covalently end point attached to the surface, thereby making the carbohydrate chains more accessible for proteins with affinity for heparin/heparan sulphate. The mean molecular weight of the immobilized heparin was about 8 kDa, while 2 mg (equal to approximately 360 IU) was coupled to each gram of beads. The integrity of this surface was verified by the expected removal of 75% of antithrombin (AT) concentrations from the blood passed over heparinized, but not non-heparinized, beads.

(15) These data corresponds well with the previous observations from extracorporeal lung assistance (ECLA) on septic patients using surface heparinized oxygenators published by Bindslev et al. (Bindslev L, Eklund J, Norlander O, Swedenborg J, et al. Treatment of acute respiratory failure by extracorporeal carbon dioxide elimination performed with a surface heparinized artificial lung. Anesthesiology 1987; 67: 117-120.)

(16) Mixture of Beads with Different Surface Functionality

(17) Heparin is well known to be a biologically active carbohydrate that can bind cytokines, pathogens, and many other proteins. In addition, heparin has the advantage of being safe as it is also a well-known anticoagulant. Manufacturers have coated medical devices with heparin for years to improve their safety. Therefore, the heparinized surface in the adsorption cartridges described here provides both the safety and efficacy of the device for removing harmful substances from blood or other biological fluids.

(18) In addition to heparin and heparin sulfate, there are other biologically active carbohydrates that can remove different types of harmful substances from blood and biological fluids. Other carbohydrates of interest include sialyic acic, heparan sulfate, chondroitin sulfate, dermatan sulfate, and hyaluronic acid. However, these carbohydrate surfaces may be significantly less blood compatible than heparinized surfaces and can lead to increased thrombogenicity. A cartridge containing these additional carbohydrate surfaces as the bioactive adsorbant could be assembled to remove different cancer mediators from blood, however, due to the clotting risk of the device, the patient would need a high dose of systemic anticoagulation which could lead to a bleeding risk.

(19) By assembling an adsorption cartridge with both heparinized surfaces and additional carbohydrate chemistry, many different cancer mediators can all be removed from blood or biological fluid while maintaining the safety of the device.

(20) Use of heparinized cartridge during tumor excision surgery.

(21) When a tumor is excised, there is a high potential of release of metastatic cancer cells into the blood stream that can then spread the cancer to additional parts of the body. A heparinized cartridge is used to remove circulating cancer cells during and after the surgical procedure. The flow rate of blood through the circuit is maintained at 150 ml/min. After the surgery is complete, the cartridge continues to cleanse the blood for 2- or more hours to remove circulating tumor cells.

(22) Use of Heparinized Cartridge to Circulating Tumor Cells

(23) In combination with an assay that detects circulating tumor cells in cancer patients, a heparinized cartridge is used to selectively remove the circulating tumor. The filtration is initiated when either a biosensor or bioassay, sensitive to tumor cells, detect the presence of tumor cells in blood. An example of an FDA approved circulating tumor cell detection technology is the CellSearch® Circulating Tumor Cell (CTC) Test from Veridex.

(24) Use of Heparinized Cartridge to Remove Circulating Heparanases

(25) Heparanases are also implicated in cancer metastases by degrading HS segments on the ECM which then compromises endothelial cells for tumor cell invasion. A heparinized cartridge is used to selectively remove heparanases to protect the ECM. The filtration is initiated when either a biosensor or bioassay, sensitive to heparanase, detects the presence of heparanase.

(26) Use of Heparinized Cartridge to Remove Circulating Growth Factors

(27) Growth factors such as (VEGF) and basic fibroblast growth factor (bFGF) are implicated in cancer angiogenesis. A heparinized cartridge is used to selectively remove growth factors to prevent angiogenesis. The filtration is initiated when either a biosensor or bioassay, sensitive to growth factors, detect the presence of growth factors.

(28) Use of Heparinized Cartridge in Combination with Radiation and Chemotherapy

(29) The heparinized adsorption cartridge can bind cancer mediators circulating in blood, however it cannot treat the tumor or cancerous cells not circulating in blood. The heparinized cartridge can be used in combination with traditional cancer therapy such as radiation and chemotherapy. The radiation or chemotherapy can treat the non circulating tumors and cancer cells while the heparinized cartridge removes circulating cancer mediators.

(30) Other Examples

(31) A device according to the present invention can also take other forms, depending upon the specific environment for use of the device:

(32) 1) Wearable and portable integrated devices, such as: a. Low pressure drop optimized heparinized cartridge pumped by arterial pressure. b. Wearable for prolonged duration when risk of metastases is high. c. Simple cartridge exchange system for home use d. Sensors to close valves in case of clot formation e. On-board diagnostics

(33) The method according to the invention permits access to blood from vasculature e.g., before, during and/or after tumor surger, thereby allowing for immediate capture of mediators released from a tumor site.

(34) The invention being thus described, it will be apparent to one of ordinary skill in the art that various modifications of the materials and methods for practicing the invention can be made. Such modifications are to be considered within the scope of the invention as defined by the following claims.

(35) Each of the references from the patent and periodical literature cited herein is hereby expressly incorporated in its entirety by such citation.