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

[0012]In another preferred embodiment, the Arginase of the present invention is modified to have sufficiently high enzymatic activity and stability to maintain “adequate arginine deprivation” (hereinafter referred to as “AAD”) in a patient for at least 3 days. One preferred method of modification is an amino-terminal tag of six histidines. Another preferred modification is pegylation to increase the stability of the enzyme and minimize immunoreactivity illicited by the patient thereto. In the example described below, the Arginase has a plasma ½-life of at least about 3 days and specific activity of at least about 250 I.U. / mg.
[0013]In another aspect of the present invention, a method is provided for producing a recombinant protein comprising the steps of (a) cloning a gene encoding the protein; (b) constructing a recombinant Bacillus subtilis strain for expression of said protein (c) fermenting said recombinant B. subtilis cells using fed-batch fermentation; (d) heat-shocking said recombinant B. subtilis cells to stimulate expression of said recombinant protein; and (e) purifying said recombinant protein from the product of said fermentation. In the preferred embodiment, a prophage is used as the recombinant strain. Using the fed-batch method of fermentation and prophage described above for the cloning and expression of human recombinant arginase, there is more than a 4-fold increase in maximum optical density at wavelength of 600 nm (OD) reached, and more than 5 times improvement in both the yield and productivity of the Arginase as shown in Example 3 in the next section. In a further embodiment, the fermenting step can be scaled up for producing the recombinant protein. In a further embodiment, the fermenting step is performed using a well-defined feeding medium of 180-320 g / L glucose, 2-4 g / L MgSO4.7H2O, 45-80 g / L tryptone, 7-12 g / L K2HPO4 and 3-6 g / L KH2PO4. The use of a well-defined medium prevents undesirable material from being purified together with the recombinant protein, making the method safe and efficient for the production of pharmaceutical grade recombinant material.
[0014]In yet another preferred embodiment, the human Arginase gene is provided with an additional coding region that encodes six additional histidines at the amino-terminal end thereof, and the purifying step comprises a chelating column chromatography step. In a further preferred embodiment, the Arginase enzyme is further modified by pegylation to improve stability.
[0015]In another aspect of the present invention, there are further provided pharmaceutical compositions comprising Arginase. In the preferred embodiment, the Arginase has sufficiently high enzymatic activity and stability to maintain AAD in a patient for at least 3 days. In the most preferred embodiment, the Arginase is further modified by pegylation to improve stability and minimize immunoreactivity.

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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Effect test

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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