Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Animal or plant cell
Reexamination Certificate
2001-12-14
2003-11-11
Witz, Jean C. (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Animal or plant cell
C424S489000
Reexamination Certificate
active
06645488
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the animal cell medicine, specifically to a microencapsulated medicine of ox adrenal medulla pheochromocyte (BCC) for treating pain.
BACKGROUND ART
Pain is a common symptom caused by several disease factors and causing serious soreness to patients. Although current medicines, such as morphine-like, can bring analgesic effects in short-term, its repeating administration usually result in drug-resistance and habituation. As adrenal medulla pheochromocyte can secrete certain substance in association with analgesic effects, such as methionine enkephalin(MEK), leucineenkephalin (LENK) and monamine substances, so once pheochromocyte had been implanted into subarachnoid space of spinal of human or animal, it can act as a “mini bio-pump” and release analgesic substances continuously in a long period, and wouldn't result in drug-resistance and habituation. So in the beginning of 1980's, two research groups from USA and Switzerland tried to implant homogenous (human, rat) adrenal medulla tissues and chromaffin cells into subarchnoid space of spinal cord for treating pain and had finally obtained satisfactory results. Because of the less sources of human adrenal medulla tissues and chromaffin cells, in 1990's, the research group from USA had tried to implant heterogeneous ox adrena medulla pheochromocyte (hereafter abbreviated as BCC) into subarachnoid space of spinal cord of cancer patients to cure pain. In order to overcome the immuneexclusion reaction, they used polyacrylamide hollow fiber tube of 5 cm in length and 1 mm in diameter to coat BCC. As the said hollow fiber tube only allow small molecules to pass through, the secreta of BCC can diffused slowly and uniformly from the fiber tube to perform the analgesic effect, while macromolecular immunoglobulin in host body can't pass through the hole of tube wall, so that the cells can survive in host body in a long period(about 1 year) and allows continuous delivery of analgesic substances to relief the pain of patients. But the hollow fiber tube has a large volume. On one side, the dead volume of tube affect the dispersion of nutrients and metabolites, which make the cells inside tube can't survive chronically. On the other hand, implantation of fiber tube with large volume into subarachnoid space of spinal cord would stimulate and oppress spinoneure, thereby cause many undesirable side effects. Moreover, as the volume of hollow fiber tube is large, it must to be implanted into subarachnoid space by surgical operation, which would injure patient more or less. In the meantime, the hollow fiber tube made of materials such as chitosan, polyacrylamide and carboxymethyl cellulose (used in USA) have poor tissue biocompatibility, which cause tissue reaction in host body. On the other side, the microcapsules with three layer membrane structure of sodium alginatepolylysine-sodium alginate (APA microcapsule) have little volume (200-1000 &mgr;m in diameter) and high biocompatibility, which contribute that microcapsule survive chronically and well in host cell and the cells inside microcapsule survive chronically. The experiments have indicated that microcapsule membrane can cut off macromolecule with molecular weight beyond 110,000 Kd (dalton), prevent immunoglobulin and immunological competence cell pass through the said membrane into microcapsule to destroy inside animal cell, which thus provide proper immunity protection. Experiments also indicated that APA microcapsules have proper biocompatibility, and have long term existence (about one year and half) in small or big animal body. The research on implantation of islands of Langerhans, liver cells, parathyroid and genetic recombinant growth-hormone secretory cells for treating disease model animal have provided the evidence that the said APA microcapsule protect heterogeneous implant from host immune system. However, so far, there is no report about using APA microencapsulated pheochromocyte of ox adrenal medulla as medicine for curing pain.
DESCRIPTION OF THE INVENTION
The object of the present invention is to provide APA microencapsulated medicine of ox adrenal medulla pheochromocyte for curing pain, which have advantages of proper biocompatibility, long acting time, lower side effect and can operate easily.
The present inventor has conducted deep research based on aforementioned existing technique, found that employing following embodiment can arrive at said aims, thus accomplish the present invention.
Embodiment of the present invention described as follows:
1. Microencapsulated medicine of ox adrenal medulla pheochromocyte for treating pain, which is prepared through the following steps:
(1) mixing the ox adrenal medulla pheochromocyte with 10-20 g/L solution of sodium alginate to form suspension, the said suspension contains 0.1-1×10
10
cells per liter.
(2) dispersing the suspension of step (1) into 80-120 mmol/L calcium chloride solution or calcium lactate solution, in the form of microdrop of diameter of 150-1000 &mgr;m by spray device, the proportion of the two solution is to let 1 liter mixture contains 0.1-1×10
8
cells, setting 5-20 minutes, removing the supernatant after depositing completely, obtaining the calcium alginate bead deposit which contains ox adrenal medulla pheochromocyte;
(3) adding the deposit derived from step (2) into the 0.3-0.7 g/L polylysine solution, the proportion of the two solution is to let 1 liter mixture contains 0.2-2×10
8
cells, mixing it uniformly, setting 5-20 minutes, removing the supernatant after deposit completely, getting the deposit;
(4) adding the deposit derived from step (3) into the 1.0-2.0 g/L sodium alginate solution, the proportion of the two solution is to let 1 liter mixture contains 0.2-2×10
8
cells, mixing it uniformly, setting 3-15 minutes, removing the supernatant after deposit completely, getting the deposit;
(5) adding the deposit derived from step (4) into 40-70 mmol/L sodium citrate solution, the proportion of the two solution is to let 1 liter mixture contains 0.2-2×10
8
cells, mixing it uniformly, setting 5-20 minutes, removing the supernatant after deposit completely, obtaining the microencapsulated medicine of ox adrenal medulla pheochromocyte deposit;
(6) washing the deposit derived from step (5) by adding it into the 9 g/L sodium chloride solution, finally transferring the deposit into a cell culture, and storing it as microencapsulated medicine of ox adrenal medulla pheochromocyte.
2. The microencapsulated medicine of ox adrenal medulla pheochromocyte in item 1, wherein the said ox adrenal medulla pheochromocyte has a purity of at least 80%.
3. The microencapsulated medicine of ox adrenal medulla pheochromocyte in item 1, which is characterized in that in said step (2) dispersing the suspension derived from step (1) in microdrop state with a diameter of 180-500 &mgr;m.
As for use, the APA-microencapsulated BCC (2~9×10
6
cells) was injected into subarachnoid space of spinal cord of patients (e.g. cancer patient),which would cause analgesic effect within 4-24 hours and the effect could last for over 9 months by each injection.
The detailed explanation of embodiments of the invention is described as following.
Within the forementioned several embodiments, the 1st item is essential characteristic, and that 2nd and 3rd are preferred.
The said ox adrenal medulla pheochromocyte (BCC) of present invention refers to the ox adrenal medulla cells capable of being dyed with dye containing chromium, which can secrete monamine, enkephalin (including monamine enkephalin (MEK), leucineenkephalin) and neurotrophy factors et al, these substances have analgesic effect.
The methods of acquiring BCC and its purification methods belong to conventional technology. For example, make ox adrenal react with collagenase to decompose collagen tissue, then seperate ox adrenal tissue into single cell by mechanical method, then filter by filter screen of 170 mesh (88 &mgr;m), ox adrenal medulla cell (which contains pheochromocyte, endoth
He Limin
Li Xinjian
Wang Zhengfu
Xue Yilong
Zhang Li
Ladas & Parry
Witz Jean C.
LandOfFree
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