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Pharmaceutical Preparation and Method of Treatment of Human Malignancies with Arginine Deprivation

a technology of arginine deprivation and pharmaceutical preparation, which is applied in the direction of drug compositions, enzyme stabilisation, peptide/protein ingredients, etc., can solve the problems of inability to treat cancer in vivo with arginine depletion, failure of previous attempts to deplete arginine with various physical methods or arginine degrading enzymes, etc., to achieve “adequate arginine deprivation” and increase the stability of enzym

Inactive Publication Date: 2008-09-18
BIO CANCER TREATMENT INT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The recombinant arginase effectively maintains arginine levels below 10 μM for an extended period, achieving adequate arginine deprivation without the need for insulin, thereby providing a safe and effective treatment for malignancies while minimizing side effects.

Problems solved by technology

Despite the promising in vitro data, attempts with arginine depletion to treat cancer in vivo were unsuccessful.
Thus, previous attempts to deplete arginine with various physical methods or arginine degrading enzymes have failed because of the body's amino acid homeostatic mechanism.
Although insulin can act as a protein breakdown inhibitor, it also has far-reaching physiological effects on the human body that may cause fatal problems if blood glucose levels of the patient are not strictly maintained within the narrow normal range.

Method used

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  • Pharmaceutical Preparation and Method of Treatment of Human Malignancies with Arginine Deprivation
  • Pharmaceutical Preparation and Method of Treatment of Human Malignancies with Arginine Deprivation
  • Pharmaceutical Preparation and Method of Treatment of Human Malignancies with Arginine Deprivation

Examples

Experimental program
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example 1

Construction of the Recombinant Strain LLC101

[0066](a) Isolation of the Gene Encoding Human Arginase I

[0067]The gene sequence of human Arginase I was published in 1987 (Haraguchi, Y. et al., 1987, Proc. Natl. Acad. Sci. 84, 412-415) and primers designed therefrom. Polymerase chain reaction (PCR) was performed to isolate the gene encoding a human Arginase using the Expand High Fidelity PCR System Kit (Roche). Primers Arg1 (5′-CCAAACCATATGAGCGCCAAGTCCAGAACCATA-3′) (SEQ ID NO: 5) and Arg2 (5′-CCAAACTCTAGAATCACATTTTTTGAATGACATGGACAC-3′) (SEQ ID NO: 6), respectively, were purchased from Genset Singapore Biotechnology Pte Ltd. Both primers have the same melting temperature (Tm) of 72 degree C. Primer Arg1 contains a NdeI restriction enzyme recognition site (underlined) and primer Arg2 contains a XbaI site (underlined). These two primers (final concentration 300 nM of each) were added to 5 μl of the human liver 5′-stretch plus cDNA library (Clontech) in a 0.2-ml micro-tube. DNA polymerase...

example 2a

Batch Fermentation in a 2-Liter Fermentor

[0076]The B. subtilis LLC101 strain is maintained on a Nutrient Agar (beef extract 1 g / L, peptone 10 g / L, NaCl 5 g / L and agar 20 g / L) plate, supplemented with 5 mg / L of chloramphenicol. To prepare the innoculum for batch and fed-batch fermentation, a few colonies of the aforementioned strain were transferred from a freshly prepared Nutrient Agar plate into two 1-L flasks, each containing 80 mL of fermentation medium containing glucose 5 g / L, tryptone 10 g / L, yeast extract 3 g / L, sodium citrate 1 g / L, KH2PO4 1.5 g / L, K2HPO4 1.5 g / L, and (NH4)2SO4 3 g / L. The bacterial cell culture was cultivated at 37° C. and pH 7.0 on an orbital shaker rotating at 250 r.p.m. The cultivation was terminated when OD600 nm reached 5.5-6.0 at about 9-11 h growth time. Then the 160-mL culture broth was introduced into the 2-L fermentor containing 1440-mL fermentation medium (glucose 5 g / L, tryptone 10 g / L, yeast extract 3 g / L, sodium citrate 1 g / L, KH2PO4 1.5 g / L, K...

example 2b

Fed-Batch Fermentation in a 2-Liter Fermentor

[0077]The Fed-batch fermentation was carried out at 37 degree C., pH 7.0 and dissolved oxygen 20% air saturation. The inoculation procedure was similar to that of the batch fermentation described in Example 2A. Initially, the growth medium was identical to that used in the batch fermentation described in Example 2A. The feeding medium contained 200 g / L glucose, 2.5 g / L MgSO4.7H2O, 50 g / L tryptone, 7.5 g / L K2HPO4 and 3.75 g / L KH2PO4. The medium feeding rate was controlled with the pH-stat control strategy. In this strategy, the feeding rate was adjusted to compensate the pH increase caused by glucose depletion. This control strategy was first implemented when the glucose concentration decreased to a very low level at about 4.5-h fermentation time. If pH>7.1, 4 mL of feeding medium was introduced into the fermentor. Immediately after the addition of glucose, the pH value would decrease below 7.1 rapidly. After approximate 10 min, when the g...

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Abstract

The present invention provides an isolated and substantially purified recombinant human arginase having sufficiently high enzymatic activity and stability to maintain Adequate Arginine Depletion in a patient. The present invention also provides a pharmaceutical composition comprising the modified invention enzyme and method for treatment of diseases using the pharmaceutical composition.

Description

RELATED APPLICATION[0001]This application is a divisional of co-pending U.S. patent application Ser. No. 10 / 518,223, filed Dec. 15, 2004.FIELD OF INVENTION[0002]The present invention is related to pharmaceutical compositions containing arginase and use therefor. In particular, the present invention is related to pharmaceutical compositions that have the capability of reducing the arginine level in patients with tumours and its use for treatment of human malignancies. The present invention also relates to a method of producing a recombinant protein.BACKGROUND OF INVENTION[0003]Arginase I (EC 3.5.3.1; L-arginine amidinohydrolase), is a key mammalian liver enzyme that catalyzes the final step in the urea formation in the Urea cycle, converting arginine into ornithine and urea. Rat liver extract, which has a high content of arginase, was found to have anti-tumour properties in vitro when it was accidentally added to tumour cell culture medium (Burton et al., 1967, Cytolytic action of co...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/46A61P35/04A61K9/08C12N15/09A61K38/00A61K38/44A61P1/00A61P35/00C07H21/04C12N1/21C12N9/06C12N9/14C12N9/78C12N9/80C12N9/96C12N15/55C12N15/74C12P21/06C12R1/125
CPCA61K38/00C12N9/78C12N9/14A61P1/00A61P1/16A61P15/14A61P35/00A61P35/04
Inventor CHENG, NING MANLEUNG, YUN CHUNGLO, WAI HUNG
Owner BIO CANCER TREATMENT INT
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