Synthetic resins or natural rubbers -- part of the class 520 ser – Synthetic resins – Mixing of two or more solid polymers; mixing of solid...
Reexamination Certificate
2000-02-07
2001-07-17
Riley, Jezia (Department: 1656)
Synthetic resins or natural rubbers -- part of the class 520 ser
Synthetic resins
Mixing of two or more solid polymers; mixing of solid...
C521S134000, C585S500000, C585S507000, C585S832000, C525S054200, C525S050000
Reexamination Certificate
active
06262172
ABSTRACT:
This application claims priority under 35 U.S.C. §§119 to 98102355.X filed in People's Republic of China on Jun. 8, 1998, the entire contents of which are hereby incorporated by reference.
FIELD OF THE INVENTION
This invention relates to clinically useful material, and in particular to an immunoadsorbent resin prepared by immobilizing biologically active DNA to a carbonized resin as a carrier, adapted for hemoperfusion for the treatment of systemic lupus erythematosus (SLE).
BACKGROUND OF THE INVENTION
Systemic lupus erythematosus is an autoimmune disease. Its etiology is still under investigation and currently there is no effective treatment. Mortality is usually caused by multiple organ failure in SLE patients, and the death rate is fairly high.
Utilization of extracorporeal immunoadsorbent to treat SLE was first introduced in 1987. This was described in the patent of Jnes, Frank R., EP 0 272 792 A1 (1987). The hemoperfusion in STE therapy with DNA immunoadsorbent utilizes the specificity of the immobilized DNA on the stationary resin to remove the disease-causing anti-DNA antibody in the circulating blood of SLE patients, thereby reversing the clinical symptoms, and improving the immune function of the SLE patients, allowing them to recover gradually. This treatment method is attracting increasing attention in clinical medicine.
Terman D. S. et al reported the first usage in extracorporeal immunoadsorption by DNA immobilized by enclosure with collodion membrane adsorbed to active charcoal to treat a 29-year-old woman with severe lupus nephritis (Lancet, 1979, 2(8147):824-827). Her life span was extended by 31 days by the hemoperfusion. This opened up a new research era of SLE therapy. However, because Terman employed active charcoal as carrier, the mechanical strength and resilience of the adsorbent were poor, with shedding of fine charcoal particles that would clog the blood capillaries.
Yang Y. et al employed carbonized resin from Japan as carrier for the collodion membrane-enclosed DNA adsorbent, where the DNA was pretreated by blue tetrazolium (Chinese Journal of Biomedical Engineering, 1985, 4(2):88-95, Chemical Journal of Chinese Universities, 1985, 6(9):843-847). This immunoadsorbent showed greatly enhanced mechanical strength, and lack of shedding of the immobilized DNA. It was used successfully for the clinical treatment of SLE patients in China with evident therapeutic effects. However, the presence of endotoxin was indicated by patients' shivering. In addition, improvement in the enclosure method is required in order to increase the adsorbent function. Furthermore, there is need to improve the method of preparation of the carbonized resin, and lowering its cost, in order to advance the clinical application of the adsorbent.
SUMMARY OF INVENTION
Because the structure and function of the resin carrier and its compatibility with blood directly affect the clinical utilization of the DNA immunoadsorbent resin, the present invention describes an improved method of preparing a carbonized resin DNA immunoadsorbent. The present invention improves the DNA enclosure procedure, enhances the immunoadsorption function, and makes possible the ready avoidance the introduction of pyrogens, in order to increase clinical effectiveness. The production cost of the material is also substantially reduced.
In this invention, the DNA immunoadsorbent uses carbonized resin as carrier for immobilizing an effective amount of DNA. The DNA content attains the level of 0.3-0.5 mg/ml resin. Briefly, the two-staged preparation steps are:
(a) Preparation of carbonized resin
Styrene and acrylonitrile were used as monomers, divinylbenzene as crosslinker, toluene and liquid paraffin (equivalent to three times by weight the total monomers and crosslinker) as porogens, benzoyl peroxide as initiator, and 1-2% of polyvinyl alcohol as dispersing agent. Polymerization was carried out at 80° C. for 5 hours. Toluene was distilled off at 96° C. The globular polymer was heated at 100° C. for 2-3 hours for further polymerization and hardening, washed with hot water, and heat dried at 50° C. Petroleum ether was applied to remove the paraffin. The result was a tri-component macroporous resin.
The resin was placed in a resistance oven, and under the protection with inert gas, heated to 100-300° C. in an oven for 8-10 hours. The temperature was then further increased to 500-800° C. Steam was introduced to activate the resin for 0.5-1.0 hour. The desired carbonized resin was obtained after cooling.
(b) Preparation of carbonized resin DNA immunoadsorbent
Calf thymus DNA was uniformly mixed with collodion. Under vigorous stirring, the carbonized resin was added and the liquid was absorbed quickly. The reaction mixture was stirred intermittently and washed for 2 hours at 50-60° C. in a water bath until the solvent was totally evaporated. The resin was vacuum dried at 50° C. for 2 hours, and washed several times with water. After packing into a column, the resin was washed further until the eluent was devoid of absorbance at 260 nm. The resin was further washed again and dried with suction at 50° C. to yield the immunoadsorbent.
The carbonized resin DNA immunoadsorbent prepared by this method contained 0.3-0.5 mg DNA/ml resin.
The present invention provides an approach for the therapeutic treatment of SLE, based on hemoperfusion of the SLE patient through a column of the carbonized resin DNA immunoadsorbent. Blood is bled from an artery of a patient, pumped through the resin column to remove disease-causing substances, and returned to the patient's body through a vein usually for 2-3 hours. The hemoperfusion has been found to be safe, simple, and inexpensive.
Below is a detailed description of the methodologies of the present invention:
1. Preparation of Carbonized Resin
The organic phase was prepared by mixing 5.0-7.5% w/w of styrene, 10.0-12.5% w/w of divinylbenzene, 7.5-10.0% w/w of acrylonitrile, and 50% w/w of toluene, with addition of 0.15-0.3% w/w of the benzoyl peroxide initiator. After complete dissolution, 25.0% w/w liquid paraffin was added and mixed in thoroughly. The total amount of toluene and liquid paraffin was 3 times the total weight of the three monomers. A volume of 1-2% aqueous solution of polyvinyl alcohol twice the volume of the organic phase was placed in a 3L three-necked flask, and was warmed to 50° C. The organic phase was added with stirring and the temperature was increased to 80° C. for 3-5 hours. The temperature was then increased to 90-100° C. to distill the toluene. The globular polymer was boiled at 100° C. for 2 hours, filtered, and washed several times with hot distilled water. After drying in a 50° C. oven, the resin was washed or warm-extracted with petroleum ether to remove liquid paraffin. The resultant resin was a tri-component macroporous resin. Standard sieves were used to select the 0.45-1.0 mm sized resin beads.
The resin was pre-heated to 100-300° C. in a tubular resistance oven for 8-10 hours. Under N
2
, the temperature was further increased at 100° C./hour to 500-800° C. Stearn was passed into the resin to activate the resin for 0.5-1.0 hour, and then allowed to cool to room temperature. About 350 ml of carbonized 0.45-0.9 mm resin was obtained.
The resin obtained was boiled in 2 volumes of 5% HCl for 30 minutes, washed with double distilled water until neutral pH, then boiled in 2 volumes of 5% NaOH, and again washed with double distilled water until neutral pH, and dried at 50° C. Finally it was extracted with hot absolute ethanol for 24 hours, and dried.
2. Preparation of Carbonized Resin DNA Immunoadsorbent
(a) Preparation of DNA solution: High purity calf thymus DNA was dissolved in 0.2 M Tris-HCl, pH 7.4 buffer solution at a concentration of 4 mg/ml. An equal volume of saturated blue tetrazolium aqueous solution was added, followed by an equal volume of absolute ethanol. The mixture was stirred or shakened until complete dissolution.
(b) Preparation of collodion: 5% collodion ether solution was diluted 7.4 fold with absolute ethanol.
(c) DNA immobili
Chen Changzhi
Kong Deling
Yu Yaoting
Burns Doane Swecker & Mathis L.L.P.
NanKai University
Riley Jezia
LandOfFree
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