WO2000065030A2

WO2000065030A2 - Microencapsulated genetically engineered e. coli dh 5 cells for the removal of undesired electrolytes and/or metabolites

Info

Publication number: WO2000065030A2
Application number: PCT/CA2000/000482
Authority: WO
Inventors: Satya Prakash; Thomas M. S. Chang
Original assignee: Mcgill University
Priority date: 1999-04-28
Filing date: 2000-04-27
Publication date: 2000-11-02
Also published as: WO2000065030A3; AU4281400A

Abstract

The present invention relates to a composition for the removal of at least one undesired electrolyte and/or metabolite in a patient, which comprises a genetically engineered E. coli DH5 cells microencapsulated in artificial cells to be capable of removing said undesired electrolyte and/or metabolite, wherein said undesired electrolyte is selected from the group consisting of K, Mg, P, Na, Cl and said undesired metabolite is selected from the group consisting of uric acid, cholesterol, bilirubin, and creatinine, wherein said removal of undesired electrolyte and/or metabolite lowers the undesired chemical concentration to a therapeutically acceptable level.

Description

ARTIFICIAL CELLS MICROENCAPSULATED GENETICALLY

ENGINEERED E. COLI DH 5 CELLS FOR THE REMOVAL OF

UNDESIRED ELECTROLYTES AND/OR METABOLITES

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The invention relates to artificial cells for the removal of at least one undesired electrolyte and/or metabolite in a patient and compositions thereof.

(b) Description of Prior Art

High level of one or more systemic K, Mg, P, Na, Cl , uric acid, bilirubin, cholesterol, and creatinine occurs in a number of diseases. The most common example is in acute or terminal kidney failure resulting in elevation of many of these electrolytes and metabolites. Thus, in acute renal failure, rapid increase in systemic potassium level can cause the death of the patient. In terminal renal failure, K, Mg, P, Na, Cl , uric acid and creatinine need to be lowered. Other examples include bilirubin in liver failure, hyperbilirubinemia and other conditions. Increase in cholesterol is related to arteriosclerosis that can cause cardiovascular diseases and stroke. Uric acid is markedly increased in gout and in other conditions .

At present lowering of these metabolites is done by using dialysis, oral adsorbents and other techniques. Dialysis for kidney failure is expensive and inconvenient. Removal of bilirubin, uric acid, cholesterol etc is difficult.

Therefore, a suitable affordable method to lower these metabolites from the body fluid compartment is required. In earlier studies, Applicants have shown that using the artificial cell microencapsulated genetically engineered E. coli DH5 cells it is possible to lower the plasma urea and ammonia effectively both in vi tro and from renal failure experimental uremic rats (PCT Application published under No. WO 97/26903 on July 31, 1997) . However, removing urea and ammonia alone is not enough to treat kidney failure or liver failure respectively.

It would be highly desirable to be provided with a tool for lowering of K, Mg, P, Na, Cl , uric acid, cholesterol, bilirubin, and creatinine in patients.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided a composition for the removal of at least one undesired electrolyte and/or metabolite in a patient, which comprises a genetically engineered E. coli DH5 cells microencapsulated in artificial cells to be capable of removing said undesired electrolyte and/or metabolite, wherein said undesired electrolyte is selected from the group consisting of K, Mg, P, Na, Cl and said undesired metabolite is selected from the group consisting of uric acid, cholesterol, bilirubin, and creatinine, wherein said removal of undesired electrolyte and/or metabolite lowers the undesired chemical concentration to a therapeutically acceptable level.

The microorganism, E. coli DH5 cells, is microencapsulated using any microcapsule material which can retain the E. coli DH5 cells and allows the undesired electrolyte and/or metabolite for removal to enter the microcapsules .

The E. coli DH5 cells are entrapped within a carrier using any entrapment material which can retain the cells and allows the undesired electrolyte and/or metabolite for removal to enter in contact with the entrapped cells.

The E. coli DH5 cells are microencapsulated using any material selected from the group consisting of nylon, silicon rubber, nylon-polyethylenimine, polylactic acid, polyglycolic acid, chitosan-alginate, cellulosesulphate-poly (dimethyldiallyl) -ammonium chloride, hydroxy-ethyl methacrylate-methyl methacrylate, chitosan-carboxymethyl -cellulose and alginate-polylysine-alginate .

In accordance with the present invention, there is provided a method of treatment of a disease with elevated level of undesired electrolytes and/or metabolites in plasma of a patient, which comprises treating said patient with a composition of the present invention for the removal of at least one undesired electrolyte and/or metabolite.

The disease may be a kidney failure-causing disease, a liver failure-causing disease or a hyperammonemia with elevated ammonia level.

In accordance with the present invention, there is provided artificial cells for the in vi tro removal of at least one undesired electrolyte and/or metabolite in plasma of a patient, which comprises genetically engineered E. coli DH5 cells microencapsulated to be capable of removing said undesired electrolyte and/or metabolite, wherein said undesired electrolyte is selected from the group consisting of K, Mg, P, Na, Cl and said undesired metabolite is selected from the group consisting of uric acid, cholesterol, bilirubin, and creatinine, wherein said removal of undesired electrolyte and/or metabolite lowers the undesired chemical concentration to a therapeutically acceptable level .

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates plasma potassium removal by free genetically engineered E. coli DH5 cells and APA- me brane artificial cell containing genetically engineered E. coli DH5 cells;

Fig. 2 illustrates plasma phosphorous removal by free genetically engineered E. coli DH5 cells and APA- membrane artificial cell containing genetically engineered E. coli DH5 cells;

Fig. 3 illustrates plasma magnesium removal by free genetically engineered E. coli DH5 cells and APA- membrane artificial cell containing genetically engineered E. coli DH5 cells;

Fig. 4 illustrates plasma sodium removal by free genetically engineered E. coli DH5 cells and APA- membrane artificial cell containing genetically engineered E. coli DH5 cells; Fig. 5 illustrates plasma chloride removal by free genetically engineered E. coli DH5 cells and APA- membrane artificial cell containing genetically engineered E. coli DH5 cells;

Fig. 6 illustrates plasma cholesterol removal by free genetically engineered E. coli DH5 cells and APA- membrane artificial cell containing genetically engineered E. coli DH5 cells;

Fig. 7 illustrates plasma bilirubin removal by free genetically engineered E. coli DH5 cells and APA- membrane artificial cell containing genetically engineered E. coli DH5 cells;

Fig. 8 illustrates plasma creatinine removal by free genetically engineered E. coli DH5 cells and APA- membrane artificial cell containing genetically engineered E. coli DH5 cells; Fig. 9 illustrates plasma uric acid removal by free genetically engineered E. coli DH5 cells and APA- membrane artificial cell containing genetically engineered E. coli DH5 cells; Fig. 10 illustrates in vivo plasma uric acid removal by oral administration of APA-membrane artificial cell containing genetically engineered E. coli DH5 cells;

Fig. 10 illustrates in vivo plasma uric acid removal by oral administration of APA-membrane artificial cell containing genetically engineered E. coli DH5 cells;

Fig. 11 illustrates in vivo plasma chloride removal by oral administration of APA-membrane artificial cell containing genetically engineered E. coli DH5 cells;

Fig. 12 illustrates in vivo plasma cholesterol removal by oral administration of APA-membrane artificial cell containing genetically engineered E. coli DH5 cells;

Fig. 13 illustrates in vivo plasma creatinine removal by oral administration of APA-membrane artificial cell containing genetically engineered E. coli DH5 cells; Fig. 14 illustrates in vivo plasma potassium removal by oral administration of APA-membrane artificial cell containing genetically engineered E. coli DH5 cells; and

Fig. 15 illustrates in vivo plasma phosphate removal by oral administration of APA-membrane artificial cell containing genetically engineered E. coli DH5 cells. DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, Applicants reports the use of artificial cells microencapsulated genetically engineered E. coli DH5 cells for lowering of K, Mg, P, Na, Cl , uric acid, cholesterol, bilirubin, and creatinine in a patient. Result shows that this novel approach has great ability to significantly lower these metabolites from the plasma and has much potential to provide a novel method to the existing system for the purpose.

MATERIALS AND METHODS

Chemicals : Alginic acid (low viscosity, Lot 611994) and poly-L-lysine (MW 16,100, Lot 11H5516) were purchased from Kelco and Sigma Chemical Co. (St. Louis, MO, USA) respectively. Unless specified, chemicals were obtained commercially and not further purified before use and they were of analytical reagent grade. Uric acid (lot 37H1291, molecular weight 168.10) used in this study were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and has the following impurities: Al<0.0005%, Ca<0.01%, Cu<0.0005%, Fe<0.0005%, Mg<0.001%, Na<0.01%, NH4+1<0.05%, P<0.005%, Pb<0.001%, Zn<0.0005%.

Microorganism and Culture Conditions:

Genetically engineered bacteria Escheretia coli DH5, containing the urease gene from Klebsiella aerogens, was a generous gift from Prof. R. P. Haussinger (Mobley, H. L. and Haussinger, R. P. (1989) Microbiol . Rev. 53: pp. 85-108). Luria-Bertani (LB) growth medium was used for primary cell cultivation. The composition of LB medium was of 10.00 g/L bactotryptone (Difco) , 5.00 g/L bacto yeast extract (Difco) , and 10.00 g/L sodium chloride (Sigma). The pH was adjusted to 7.5 by adding about 1.00 ml of 1.00 N NaOH. Media were then sterilized in Castle Labclaves for 30 minutes at 250°C. Incubation was carried out in 5.00 ml LB in 16.00 ml culture tubes at 37°C in an orbital shaker at 120 rpm. For the large-scale production of biomass, for microencapsulation purpose, 250 ml Erlenmeyer flask containing 100 ml LB medium was used.

Micro-organism Induction Procedure:

To increase the efficiency of the genetically engineered cells, metabolic fermentation induction was performed. For this genetically engineered E. coli DH5 cells were induced by fermentation incubation in a specially designed media called, modified media, which contains a defined chemical compositions for forty six consecutive generations. The media composition was as follows: Potassium mono hydro phosphate lg/1, Potassium di hydro phosphate 4.0 mg/1, Ammonium sulphate 20 mg/1, Magnesium sulphate septa hydrate 3.4 g/1, Vitamin Bl 0.07 g/1, and Trace metal, 5.0 ml. All the media was supplemented with glucose (lg/1) and urea (4 ml form 250 mg/ml stock / 1) filtered) autoclaved in a separate container. This was done in a 250 ml Erlenmeyer flask containing 100 ml of the medium at 37°C in an orbital shaker at 120 rpm.

Microencapsulation Procedure:

The details of microencapsulation procedures are as follows. Microcapsule containing bacterium E. coli DH5 cells were prepared as follow: Bacterial cells were suspended in an autoclaved sodium alginate in 0.9 % sodium chloride solution. The viscous alginate- bacterial suspension was pressed through a 23 gauge needle using a syringe pump (Compact Infusion Pump Model 975, Harvard App . Co. MA) . Compressed air was passed through a 16 gauge needle to shear the droplets coming out of the tip of the 23 gauge in a droplet needle. The droplets were allowed to gel for 15 minutes in a gently stirred ice-cold solution of calcium chloride (1.4 %) . After gelation in the calcium chloride, alginate gel beads were coated with polylysine (0.05 % in HEPES buffer saline, pH 7.20) for 10 minutes. The beads were then washed with HEPES and coated with an alginate solution (0.1 %) for 4.00 minutes. The alginate-poly-L-lysine-alginate capsules were then washed in a 3.00 % citrate bath (3.00 % in 1:1 HEPES-buffer saline, pH 7.20) to liquefy the gel in the microcapsules. The microcapsules formed were stored at 4°C and used for the experiments.

Microcapsule Storage Condition:

After the microencapsulation microcapsules were washed properly several times (two to three times) with sterile water. The microcapsules were resuspended in the Aggrobacteriium minimum broth (AG minimal media) at 4-10°C. This media, unlike L. B. media, does not support the growth of E. coli , it has however all the components which is necessary to maintain biochemical activity (Chang, T.M.S. (1964) Science 146:524-525). Before the use microcapsules were washed in normal saline to remove the media component from the surface and used for the experiment .

Plasma Used:

For all the studies freshly isolated non heparinized plasma from male Whister rats of 170-370 g weight range were used otherwise mentioned. In vitro Experimental Procedure:

The bacteria were grown in L B medium. Log phase bacterial cells were harvested by centrifuging at 10,000 g for 20 min. at 4°C. The cell mass was then washed five times with sterile cold water to remove media components. Cells were then weighed and used for the plasma K, Mg, P, bilirubin, uric acid and Creatinine removal studies by free genetically engineered E. coli DH5 cells. For the microencapsulated E. coli DH5 in vi tro removal of plasma K, Mg, P, and bilirubin, uric acid and Cretinine studies, equivalent masses of the cells were microencapsulated in APA membrane and used otherwise mentioned. Uremic rat plasma from different uremic rats were isolated and mixed together to make plasma pool before using them for plasma K, Mg, P, and bilirubin removal studies by free and microencapsuletd bacteria removal studies .

For the microencapsulated E. coli DH5 uric acid and Creatinine removal studies, the equivalent masses of the cells were microencapsulated in APA membrane and used.

For the plasma uric acid and Creatinine removal studies, we used heparin free normal rat plasma with added uric acid and Creatinine from out side. In all the experiment, the ratio of the volume of the plasma used to the amount of microencapsulated bacteria used was held constant .

In all in vi tro studies, reactions were performed in 50 ml Erlenmeyer flasks at 30°C and

100 rpm, unless otherwise mentioned. The Lab-Line orbital Environ-Shaker equipped with thermal control and air quality was used for this purpose. Sampling was carried out aseptically at designated times. Bacterial cells, in the free bacteria removal studies, were removed from the sample by centrifugation at 15,000 rpm O 00/65030

for 10 minutes at 4°C and supernatant analyzed. The samples were stored at 4°C for suitable amount of time prior to analysis .

Surgical Experimental Rat Model

The surgical procedure for making the uremic rat model involved two steps, one to perform right nephrectomy and the other to ligate the left artery, vein, and ureter, was designed. Male Wister rats of 300-340 g weight range were used. The details of these two steps are as follows:

Step 1: Unilateral (Right) Nephrectomy

The anesthetized animal was placed in ventral recombency with its tail towards the surgeon. The hair in the right dorsal lumbar area was clipped and the skin was swabbed thoroughly with a surgical scrub. A 2-

3 cm incision was made into the skin caudal to the rib cage on the right side of the animal. A 2-3 cm incision was then made into the underlying muscle wall. The kidney was pulled through the muscle wall; the renal artery, vein and ureter were then ligated and the kidney was removed by incising the vessels and ureter between the kidney. The ligature remaining tissue was returned to the peritoneal cavity and the muscle wall was sutured. The remaining tissue was returned to the peritoneal cavity and the muscle wall was sutured. The skin incision was closed using 2-3 wound clips.

Step 2: Left Renal Artery / Vein / Ureter / Ligation

The left side of the rat was prepared as if to perform a left nephrectomy. After an incision (2-3 cm) was made in the muscle wall, the left renal artery, vein, and ureter were located. Using a blunt forceps, the left renal vessels and ureter were isolated and separated from the peritoneal connective tissue. The renal vessels and ureter were ligated using sterile silk suture. The muscle wall was sutured. The skin incision was closed with 2-3 metal wound clips.

In vivo Experimental Procedure

The bacteria were grown in L. B. medium to their log phase and harvested by centrifugation at 10,000 g for 20 min. at 4°C. The cell mass was then washed 5 times with sterile cold water to remove media components. Cells were then weighed and used for removal studies. For the microencapsulated uric acid removal studies an equivalent mass of the cells were microencapsulated and used. For the microencapsulated in vivo animal studies, microcapsules containing log phase bacteria were first suspended in 0.8-1.0 ml sterile normal saline (0.9%) in a 5 ml syringe. The floating microcapsules were then administered orally to the experimental rats using a curved 12G-3 1/2 stainless steel gastric lavage tube. Blood sampling was done from the rat after sedating the animals using appropriate amounts of drugs that have been reported not to have any side effects on renal or hepatic functions. The drugs used were atravet (acepromazine) and ketaset (ketamine) in concentrations of 75 mg/kg and 5-10 mg/9 kg intramuscularly, respectively. Blood was withdrawn using a small 23 Gl precision Glide needle from leg artery. Blood samples were then centrifuged immediately in an Eppendroff micro- centrifuge at 4°C and plasma was collected and analyzed for plasma uric acid concentrations.

Plasma K, P, Mg , Na, Cl, Bilirubin, and Cholesterol Determination :

For the determination of plasma K, Mg, P, Na, Cl , Bilirubin and Cholesterol suitable amount of the sample were withdrawn keeping the reaction condition sterile using a U.V. sterile chamber. The bacterial cells and microcapsule were removed from the sample immediately by centrifugation at 15,000 rpm for 10 minutes at 4°C and the sample were then stored at stored at 4°C for the analysis. The analysis of plasma K, Mg, P, Cl , Na, bilirubin, and cholesterol was carried out at McGill university animal center biochemical, toxicology and immunology analysis lab. The analysis was done using Reflotron from Manheim Boehringer. This Reflotron system is based on dry chemistry and uses fiber optics in its operation.

Plasma Uric Acid Determination:

The concentration of uric acid were determined based on quantitative measurements using the Sigma diagnostics kits product number 686 purchased from

Sigma Chemical Co. USA. This kit is for quantitative enzymatic determination of uric acid in serum or plasma at 520 nm. Two enzymes, uricase and peroxidase, re involved in the reaction of this test procedure. Enzyme uricase catalyses the oxidation of uric acid to allantoin, carbon dioxide, and hydrogen peroxide. In the presence of enzyme peroxidase, the hydrogen peroxide formed reacts with 4-aminoantipyrine dye (4- APP) and 3 , 5-dichloro-2-hydroxybenene at sulfonate

(DHBS) to form a quinoeimine dye with an absorbency maximum at 540 nm. The intensity of the colour produced is directly proportional to the uric acid concentration in the sample.

Plasma Creatinine Determination:

The concentration of Creatinine were determined using the Sigma diagnostics kits product number 555 purchased from Sigma Chemical Co. USA. This method is for a quantitative colorimetric determination of O 00/65030

Creatinine in serum, plasma, and urine at 500 nm optical density.

RESULTS : Experiments were designed to evaluate the use of microencapsulated genetically engineered cell for the removal of uric acid. For the experiment plasma from six different rat weight range from 170g to 370g were isolated, without using any heparin, and mixed. The isolated plasma then divided into two groups as pool of the plasma source for entire plasma in vi tro studies. To one group uric acid were added from outside and the other group was used as control plasma, with no added uric acid. The concentration of uric acid in the control pool was found to be 5.99 + 0.62 mg/dl. The addition of uric acid to the plasma resulted in increased plasma uric acid level, the plasma uric acid concentration went up to 88.88 + 4.63 mg/dl from 5.99 +

0.62 mg/dl .

Lowering of Plasma Potassium:

Experiments were designed to evaluate the use of microencapsulated genetically engineered cell for the removal of plasma potassium in vi tro . Results (Fig. 1) shows that both free E. coli DH5 cells and artificial cell microencapsulated E. coli DH5 cells were able to lower plasma potassium. Free bacteria were able to lower plasma potassium from 4.37 + 0.76 mEq/1 to 3.63 + 0.90 mEq/1 and APA encapsulated from 5.80+ 0.40 mEq/1 to 3.50 + 0.03 mEq/1 in 24 hours. Result also shows that the removal of plasma K by free bacteria and encapsulated bacteria is similar (Fig. 1) .

Lowering of Plasma Phosphorous To evaluate the use of microencapsulated genetically engineered cell for the removal of plasma phosphorous in vi tro . Results (Fig. 2) shows that both free E. coli DH5 cells and artificial cell microencapsulated E. coli DH5 cells were able to lower plasma potassium, experiment were designed. Free E. coli DH5 cells were able to lower plasma phosphate from 3.31 ± 0.016 mg/dl to 1.20 + 0.02 mg/dl and APA encapsulated from 2.20+ 0.9 mg/dl to 1.49 + 0.03 mg/dl in 24 hours. Result (Fig. 2) also shows that free bacteria have higher capacity in terms of overall P lowering than encapsulated bacteria. Also it is found that empty microcapsule were also able to lower plasma

P however there was statistically significant different was observed when the result was compared with that of microcapsules having genetically engineered E. coli DH

5 cells (Fig. 2) .

Lowering of Plasma Magnesium:

Results (Fig. 3) shows that both free E. coli

DH5 cells and artificial cell microencapsulated E. coli

DH5 cells were able to lower plasma magnesium in vi tro . Free E. coli DH5 cells were able to lower plasma magnesium from 0.84 + mg/dl to 0.74 + mg/dl and APA encapsulated E. coli DH5 cells from 0.90+ mg/dl to 0.66

+ mg/dl in 24 hours. Lowering of Plasma Sodium:

Experiment was design to evaluate the plasma sodium removal efficiency of encapsulated and free E. coli DH5 cells. Result shows that (Fig. 4) both free bacteria and encapsulated bacteria were able to lower the plasma sodium. Free bacteria were able to lower plasma Na from 175 + 10.24 mEq/1 to 132 + 5.80 mEq/1 and encapsulated bacteria was able to lower plasma Na from 172 + 11.00 mEq/1 to 129 + 6.12 mEq/1 in 24 hours

(Fig. 4) . Lowering of Plasma Chloride:

Plasma chloride concentration was determined after challenging the plasma with free E. coli DH 5 cells and encapsulated E. coli DH 5 cells. Result (Fig. 5) shows that free bacteria were able to plasma chloride concentration from 137 + 10.10 mEq/1 to 107 + 5.08 mEq/1 and encapsulated bacteria were able to lower plasma chloride from 137 + 6.60 mEq/1 to 107 + 2.00 mEq/1 in 24 hours (Fig. 5) . Result also shows (Fig. 5) that both free and encapsulated have identical efficiency for plasma chloride removal .

Lowering of Plasma Cholesterol:

Experiments were design to evaluate the plasma cholesterol lowering capacity of free and encapsulated genetically engineered E. Coli DH5 cell. Result (Fig.

6) shows that both free and encapsulated bacteria were able to lower plasma cholesterol . Free bacteria were able to lower plasma cholesterol from 1.82 + 0.13 mmol/1 to 1.13 + 0.04 mmol/1 and encapsulated bacteria were able to lower plasma cholesterol from 1.86 + 0.10 mmol/1 to 1.37 + 0.06 mmol/1 in 24 hours. The plasma cholesterol removal capacity of encapsulated bacteria, however, found smaller when compared with free bacteria (Fig. 6) .

Lowering of Plasma Bilirubin:

Results (Fig. 7) shows that both free E. coli

DH5 cells and artificial cell microencapsulated E. coli DH5 cells were able to lower plasma magnesium in vi tro .

Free E. coli DH5 cells were able to lower plasma bilirubin from 6.0 + 0.20 mg/dl to 3.0 + 0.21 mg/dl and

APA encapsulated E. coli DH5 cells from 6.00 + 0.80 mg/dl to 4.00 + 0.20 mg/dl in 24 hours (Fig. 7) . Lowering of Plasma Creatinine:

Experiments were design to evaluate the plasma

Creatinine removal efficiency of the free and encapsulated E. coli DH 5 cells. Result (Fig. 8) shows that when challenged, 80.21 + 1.00% of plasma

Creatinine was remaining in the case of free bacteria after 24 hours of incubation and 83.31 + 2.40% plasma

Creatinine was remaining after 24 hours of incubation in the case of encapsulated bacteria (Fig. 8) .

Lowering of Plasma Uric Acid:

Experiments were designed to evaluate the use of microencapsulated genetically engineered cell for the removal of uric acid. For the experiment plasma from six different rat weight range from 170 g to 370 g were isolated, without using any heparin, and mixed. The isolated plasma then divided into two groups as pool of the plasma source for entire plasma in vi tro studies. To one group uric acid were added from outside and the other group was used as control plasma, with no added uric acid. The concentration of uric acid in the control pool was found to be 5.99 + 0.62 mg/dl. The addition of uric acid to the plasma resulted in increased plasma uric acid level, the plasma uric acid concentration went up to 88.88 + 4.63 mg/ dl from 5.99 + 0.62 mg/dl .

The experiment were designed to evaluate the plasma uric acid removal capacity of the free genetically engineered E. coli DH5 cell by adding the log phase L B grown bacterial cells. Also a control was kept using the uric acid pool plasma. The obtained results shows (Fig. 9) that free bacteria were able to plasma in vi tro . The plasma uric acid level decreased to 3.44 + 0.16 from 84.80+2.80 mg/dl in 24 hours. In the control experimental group, the plasma uric acid concentration was fairly steady throughout the experiment .

Experiment were design to evaluate if the artificial cell encapsulated genetically engineered bacteria E. coli DH5 is capable of lowering the plasma uric acid in vi tro . Results are shown in Figure 9 shows that that APA encapsulated genetically engineered E. coli DH5 cells were able to lower plasma uric acid from 84.80 + 3.40 mg/ dl to 8.80 + 3.12 mg/ dl in 24 hours.

CONCLUSIONS AND SUMMARY:

High level of one or more systemic K, Mg, P, Na, Cl , uric acid, bilirubin, cholesterol, and creatinine occurs in a number of diseases. The most common example is in acute or terminal kidney failure resulting in elevation of many of these electrolytes and metabolites. Thus, in acute renal failure, rapid increase in systemic potassium level can cause the death of the patient. In terminal renal failure, K, Mg, P, Na, Cl , uric acid and creatinine need to be lowered. In the present novel approach, all these electrolytes and metabolites can be removed effectively by encapsulated E.coli DH5 cells. Based on the result obtained the levels of the electrolytes are lowered to a save level. This novel approach can also remove bilirubin and has potential for use in liver failure, hyperbilirubinemia and other conditions. The ability to remove cholesterol has potentials for use in lowering cholesterol is related to arteriosclerosis that can cause cardiovascular diseases and stroke. This approach can very effectively lower uric acid and it may have much potential in lowering uric acid in gout and in other conditions. These approaches may supplement or replace the expensive and inconvenient treatment using dialysis, plasmapheresis, oral adsorbents and medications. 00/65030 - l i

The present invention will be more readily un^¬ derstood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE I

In vi tro plasma unwanted metabolite removal efficiency of the artificial cells containing genetically engineered E. coli DH 5 cells

Example II

Lowering of high plasma uric acid levels in experimental rats by oral administration of artificial cell microencapsulated genetically engineered E. coli

DH5 cells

Microcapsules containing genetically engineered bacteria E. coli DH5 cells were prepared as described before. Male Wister rats of 300-325 g weight range were used. The experimental surgical model has a high level of plasma uric acid when compared to normal rats (Fig. 10) . A suitable quantity of encapsulated bacteria was given daily to each rat. For this purpose microcapsules were first suspended in 0.8-1.0 ml sterile saline in a 5.0 ml syringe and then administered orally using a 12 G gastric lavage tube. Besides monitoring pretreatment uric acid levels in experimental rat as internal control, we also used a control group. The control group receives empty microcapsule containing no bacteria.

Experiments were designed to evaluate the efficiency of encapsulated genetically engineered E. coli DH5 cells for lowering plasma uric acid by its oral administration. For this two groups of uremic experimental rat on normal rat chaw were selected. One group that receive empty microcapsule and the other group that receives microcapsule containing 1.00 + 0.15 mg/g bodyweight of genetically engineered E. coli DH5 cells. We followed the plasma uric acid concentration of both the groups for 7 days before giving any type of microcapsules. On day 7 we started oral administration of empty microcapsules and the microcapsule containing genetically engineered E. coli DH5 cells to the respective group and followed their plasma uric acid concentration. Results (Fig. 10) show that the encapsulated bacteria were able to lower the plasma uric acid concentration very efficiently. Encapsulated bacteria were able to lower plasma uric acid concentration from 88.66 + 23.67 to 20.33+ 17.43 mm/L 2 days later (Fig. 10) . The control uremic rat group the plasma uric acid concentration remained high at 72.00 + 12.01 mm/L on day 1, 79.00+ 27.83 mm/L on day 4. By continued daily oral administration of the encapsulated E. coli DH5 cells, the plasma uric acid concentration of nephrectomy induced uremic rats to this normal level for the entire test period. With discontinuation of oral treatment, the plasma uric acid level quickly returned to the high level. Plasma uric acid level went back to 64.67 +26.27 mm/L, on the very next day followed by 48.00 + 25.23 mm/L, 45.33 + 6.35 mm/L, 41.33 + 12.43 mm/L, 59.00 +19.00 mm/L, 43.34 + 5.68 mm/L on days 2,3,4,5,6, and day 7, respectively (Fig. 10) .

The obtained result shows that this biotechnological approach of using artificial cells microcapsules containing genetically engineered E. coli DH5 cells in vi tro has shown very strong potential to be useful for plasma uric acid lowering in various situations. When given orally, the microorganisms will remain immobilized inside the microcapsules. The microcapsules remain intact as they pass down the gastrointestinal tract. Finally, they are excreted intact with the stool in about 24 hours. The membranes of the intact microcapsules are permeable to smaller molecules like uric acid, urea, ammonia, phosphate, etc. Thus, during the passage of the intact microcapsules through the intestine smaller molecules can diffuse into the microcapsules.

We have also evaluated the other unwanted plasma metabolite removal capacity of artificial cell microencapsulated genetically engineered E. coli DH5 cells in vivo . The plasma chloride from 170 ₊ 17.03 mmol/L to 150.66 + 31.97 mmol/L on day 2, plasma cholesterol from 2.24 + 0.2816 mmol/L to 2.30 + 0.3464 mmol/L on day 2, alkaline phosphatase from 198.33 ₊ 23.50 to 149.00 + 21.93 U/L, creatinine from 34.52 + 5.29 to mm/L 33.00 + 2.0 mm/L on day 2, potassium from 5.70 + 0.96 mm/L to 5.62 + 0.450 mm/L, and the plasma phosphate from 2.57 + 0.26 mmol/L to 2.41 + 0.37 mmol/L on the day 2 of the oral administration. Example III

Lowering of plasma electrolytes and metabolites in experimental rats by oral administration of artificial cell microencapsulated genetically engineered E. coli

DH5 cells

Microcapsules containing genetically engineered bacteria E . coli DH5 cells were prepared as described before. Male Wister rats of 300-325g weight range were used. Throughout the control and treatment periods the experimental rats received normal rat chow. During the treatment, a suitable quantity of encapsulated bacteria was given daily to each rat. For this purpose microcapsules were first suspended in 0.8-1.0 ml sterile saline in a 5.0 ml syringe and then administered orally using a 12 G gastric lavage tube. The animal group receiving empty microcapsule containing no bacteria was treated as other control . A quantity of 1.0 + 0.15 mg/g body weight of log phase genetically engineered bacteria E. coli DH5 cells in microcapsules was administered daily to a group of 43 day old experimental rats. We followed the plasma electrolytes (Sodium, Potassium, Phosphate, Chloride) and metabolites (creatinine, cholesterol, bilirubin, uric acid) concentration of normal and experimental uremic rats for 27 days.

Experiments were designed to evaluate the efficiency of encapsulated genetically engineered E. coli DH5 cells for lowering plasma electrolytes and metabolites. For this two groups of uremic experimental rat were selected. One group that receive empty microcapsule and the other group that receives microcapsule containing genetically engineered E. coli DH5 cells. We followed the plasma concentration of both the groups for 7 days before giving any type of the microcapsule. On the day 7 we started oral administration of empty microcapsules and the microcapsule containing genetically engineered E. coli DH5 cells to the respective group of the experimental animals and followed their plasma concentration. Results in the Figs. 10-15 show that the encapsulated bacteria were able to lower the plasma concentration of Sodium, Potassium, Phosphate, Chloride, creatinine, cholesterol, bilirubin, and uric acid. By continued daily oral administration maintained the plasma concentration of nephrectomy induced uremic rats to this lowered level for the entire test period. With discontinuation of oral treatment, the plasma level of these electrolytes and metabolites increased to its pretreated high levels. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims .

Claims

WHAT IS CLAIMED IS:

1. A composition for the removal of at least one undesired electrolyte and/or metabolite in a patient, which comprises a genetically engineered E. coli DH5 cells microencapsulated in artificial cells to be capable of removing said undesired electrolyte and/or metabolite, wherein said undesired electrolyte is selected from the group consisting of K, Mg, P, Na, Cl and said undesired metabolite is selected from the group consisting of uric acid, cholesterol, bilirubin, and creatinine, wherein said removal of undesired electrolyte and/or metabolite lowers the undesired chemical concentration to a therapeutically acceptable level .

2. The composition of claim 1, wherein said E. coli DH5 cell is microencapsulated using any microcapsule material which can retain the E. coli DH5 cells and allows the undesired electrolyte and/or metabolite for removal to enter the microcapsules.

3. The composition of claim 1, wherein said E. coli DH5 cells are entrapped within a carrier using any entrapment material which can retain the cells and allows the undesired electrolyte and/or metabolite for removal to enter in contact with the entrapped cells.

4. The composition of claim 2, wherein said E. coli DH5 cells are microencapsulated using any material selected from the group consisting of nylon, silicon rubber, nylon-polyethylenimine, polylactic acid, polyglycolic acid, chitosan-alginate, cellulosesulph- ate-poly (dimethyldiallyl) -ammonium chloride, hydroxy- ethyl methacrylate-methyl methacrylate, chitosan- carboxymethyl -cellulose and alginate-polylysine- alginate .

5. A method of the treatment of a disease with elevated level of undesired electrolytes and/or metabolites in plasma of a patient, which comprises treating said patient with a composition according to claim 1 for the removal of at least one undesired electrolyte and/or metabolite.

6. The method of treatment of claim 5, wherein said disease is a kidney failure-causing disease.

7. The method of treatment of claim 5, wherein said disease is a liver failure-causing disease.

8. The method of treatment of claim 5, wherein said disease is a hyperammonemia with elevated ammonia level .

9. Artificial cells for the in vi tro removal of at least one undesired electrolyte and/or metabolite in plasma of a patient, which comprises genetically engineered E. coli DH5 cells microencapsulated to be capable of removing said undesired electrolyte and/or metabolite, wherein said undesired electrolyte is selected from the group consisting of K, Mg, P, Na, Cl and said undesired metabolite is selected from the group consisting of uric acid, cholesterol, bilirubin, and creatinine, wherein said removal of undesired electrolyte and/or metabolite lowers the undesired chemical concentration to a therapeutically acceptable level .