WO2006058486A1 - Use of arginase in combination with 5fu and other compounds for treatment of human malignancies - Google Patents
Use of arginase in combination with 5fu and other compounds for treatment of human malignancies Download PDFInfo
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- WO2006058486A1 WO2006058486A1 PCT/CN2005/002001 CN2005002001W WO2006058486A1 WO 2006058486 A1 WO2006058486 A1 WO 2006058486A1 CN 2005002001 W CN2005002001 W CN 2005002001W WO 2006058486 A1 WO2006058486 A1 WO 2006058486A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/51—Lyases (4)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the present invention is related to pharmaceutical compositions for the treatment of human malignancies containing arginase.
- Cancer remains one of the most difficult to treat human diseases.
- certain forms of cancer such as liver cancer, there is no known effective drug.
- Arginine degrading enzymes can be developed as drugs to treat cancer.
- Arginase I (EC 3.5.3.1; L-arginine amidinohydrolase), is a key mammalian liver enzyme that catalyses the final step in urea formation in the Urea cycle, converting arginine into ornithine and urea.
- PCT publication WO 2004/000349 discloses a pharmaceutical composition containing human recombinant arginase.
- US patent application no. 10/757,843 discloses a different arginine degrading enzyme, arginine deiminase modified with polyethylene glycol, used for the treating of cancer, and the treating and/or inhibiting of metastasis.
- 09/905,201 discloses a therapeutic composition and method for treatment of cancer comprising arginine decarboxylase of E. coli and modifications thereof. [0004] It is an object of the present invention to provide improved methods of treatment and compositions for the treatment of cancer.
- one aspect of the present invention teaches a kit comprising at least one therapeutic dose of an arginine reducing compound and at least one therapeutic dose of an anti-neoplastic compound such as 5-fmorouracil (5FU), for the treatment of human malignancies.
- the arginine reducing compound is an enzyme or a compound that is capable of degrading or removing arginine from the subject, so as to achieve a physiological arginine level of below 10 ⁇ M.
- Some examples of arginine reducing compounds include, but are not limited to, arginine degrading enzymes such as arginase, human arginase I, arginine deiminase and arginine decarboxylase, modifications thereof or combinations thereof.
- An antineoplastic compound may be, but is not limited to, an alkylating agent, antimetabolite agent, antimitotic agent, DNA production inhibiting agent or DNA repair inhibiting agent, etc.
- a method for treating cancer in a human patient comprising reducing the physiological arginine levels in the patient to below 10 ⁇ M combined with a suitable anti-neoplastic or antimetabolite compound such as 5FU.
- the reduction of physiological arginine levels in a human patient may be achieved through various treatments which include, but are not limited to, embolization, dialysis or administration of an arginine reducing compound.
- a pharmaceutical composition comprising an arginine reducing compound and an anti-neoplastic compound.
- an arginine reducing compound in combination with an anti-neoplastic compound for the manufacture of a medicament for the treatment of cancer.
- the arginine reducing compound may be an arginine degrading enzyme.
- Some examples of such arginine degrading enyzmes include, but are not limited to, arginase, arginine deiminase, arginine decarboxylase, or modifications and combinations thereof.
- the arginase may also be human arginase I or pegylated human arginase.
- the anti-neoplastic compound may, for example, be an alkylating agent, antimetabolic agent, antibiotic agent, DNA production inhibiting agent or DNA repair inhibiting agent, etc.
- the anti-neoplastic compound may preferably be 5-fluorouracil.
- Figure 1 shows plasmid map of pABlOl. This plasmid carries the gene encoding Arginase (arg) and only replicates in E. coli but not in B. subtilis.
- Figures 2A, 2B and 2C show nucleotide sequence and its deduced amino acid sequence of the human Arginase I.
- Figure 2A shows the nucleotide sequence (SEQ ID NO: 1) from EcoRVMunl to Xb al sites of plasmid pABlOl.
- Nucleotide (nt) 1-6 EcoRllMunl site; nt 481-486, -35 region of promoter 1; nt 504-509, -10 region of promoter 1; nt 544-549, -35 region of promoter 2; nt 566-571, -10 region of promoter 2; nt 600-605, ribosome binding site; nt 614-616, start codon; nt 632- 637, Ndel site; nt 1601-1603, stop codon; nt 1997-2002, Xbal site.
- Figure 2B shows the encoding nucleotide sequence (SEQ ID NO: 2) and its corresponding encoded amino acid sequence (SEQ ID NO: 3) of a modified human Arginase.
- Nucleotide 614-1603 from Figure 2 A is an encoding region for the amino acid sequence of the modified Arginase.
- the 6xHis (SEQ ID NO: 4) tag at the N-terminus is underlined. Translation stop codon is indicated by asterisk.
- Figure 2C shows the encoding nucleotide sequence (SEQ ID NO: 8) and its corresponding encoded amino acid sequence (SEQ ID NO: 9) of the normal human Arginase I.
- Figure 3 is a schematic drawing of the construction of a B. subtilis prophage allowing expression of Arginase.
- Figure 4 shows the comparison of average tumor size for four groups of nude mice which have tumors induced by implantation with tumor cells.
- the four groups are negative control, arginase (BCT) alone, arginase (BCT) and arginase (BCT) in combination with 5FU, an anti-neoplastic and antimetabolite compound.
- Combined administration or “combined with administering” merely refers to a general period of time in which both arginase and an anti-neoplastic agent are administered to the human body for the treatment of human malignancies. It does not restrict the method of treatment to a simultaneous administration of the two types of compounds.
- combined with merely refers to a general period of time in which the two steps of treatment for cancer are performed, this includes and is not limited to the possibility of simultaneous performance of the two steps.
- the term “medicament” may refer to two different compounds applied at different times, as long as the two compounds belong to the same combination treatment.
- pegylated Arginase refers to Arginase of present invention modified by pegylation to increase the stability of the enzyme and minimize immunoreactivity.
- arginase I in both its modified and unmodified forms is the preferred arginase.
- Pegylated arginase I may also be referred to as "BCT" and is used interchangeably in this application.
- Human arginase I and other amino acid sequences as used herein include amino acid sequences that are substantially the same, meaning that they may have "slight and non-consequential sequence variations" from the actual sequences disclosed herein. Species with sequences that are substantially the same are considered to be equivalent to the disclosed sequences and as such are within the scope of the appended claims.
- “slight and non-consequential sequence variations” means that the amino acid sequences are functionally equivalent to the sequences disclosed and/or claimed herein. Functionally equivalent sequences will function in substantially the same manner to produce substantially the same compositions as the amino acid compositions disclosed and claimed herein.
- half-life refers to the time that would be required for the concentration of the Arginase in human plasma in vitro, to fall by half.
- Arginase may be obtained from Ikemoto et al. (Ikemoto et al. Biochem J. 1990. 270: 697-703) or by the method disclosed in PCT publication WO 2004/000349. The arginase may also be produced using the methods described below.
- CCAAACCATATGAGCGCCAAGTCCAGAACCATA-3' (SEQ ID NO: 5) and Arg2 (3'-CCAAACTCTAGAATCACATTTTTTGAATGACATGGACAC-S ⁇ (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 Argl contains a Ndel restriction enzyme recognition site (underlined) and primer Arg2 contains a Xbal site (underlined). These two primers (final concentration 300 nM of each) were added to 5 ⁇ l of the human liver 5'- stretch plus cDNA library (Clontech, California, USA) in a 0.2-ml micro-tube.
- DNA polymerase (2.6 units, 0.75 ⁇ l), the four deoxyribonucleotides (4 ⁇ l of each; final concentration 200 ⁇ M of each) and reaction buffer (5 ⁇ l) and dH 2 O (17.75 ⁇ l) were also added.
- PCR was performed using the following conditions: pre-PCR (94 degree C, 5 min), 25 PCR cycles (94 degree C, 1 min; 57 degree C, 1 min; 72 degree C, 1 min), post-PCR (72 degree C, 7 min).
- PCR product (5 ⁇ l) was analyzed on a 0.8% agarose gel and a single band of 1.4 kb was observed. This DNA fragment contains the gene encoding Arginase.
- Plasmid pSGl 113 which is a derivative of plasmid pSG703 (Thornewell SJ et al. Gene. 1993. 133: 47-53), was isolated from the E. coli DH5 ⁇ clone carrying pSG1113 by using the Wizard Plus Minipreps DNA Purification System (Promega, Wisconsin, USA) following the manufacturer's instruction. This plasmid, which only replicates in E. coli but not in B. subtilis, was used as the vector for the sub-cloning of the Arginase gene.
- the reaction mixture was subjected to agarose gel (0.8%) electrophoresis, and the 1.4 kb DNA fragment was recovered from the gel by using the Qiaex II Gel Extraction Kit (Qiagen, California, USA). Separately, the plasmid pSG1113 was treated with the same restriction endonucleases in the same way.
- the reaction mixture was subjected to agarose gel (0.8%) electrophoresis, and a DNA fragment having a size of about 3.5 kb was recovered from the gel. This DNA fragment was joined by using T4 DNA ligase to the above 1.4 kb DNA fragment. The ligation mixture was used to transform E.
- coli XLI-Blue using the conventional calcium method (Sambrook J et al. Molecular Cloning, A Laboratory Manual, second edition. Cold Spring Harbor Laboratory Press, New York. 1989) and plated on nutrient agar plate containing 100 ⁇ g/ml ampicillin. Colonies were screened for a plasmid with the appropriate insert by restriction analysis. The plasmid constructed was designated pABlOl (Fig. 1). ORI is the E. coli origin of replication and bla is the ampicillin resistant marker gene.
- DNA sequencing was performed with primers Argl (SEQ ID NO: 5), Arg2 (SEQ ID NO: 6) and Arg6 (5'-CTCTGGCCATGCCAGGGTCCACCC-S') (SEQ ID NO: 7) to confirm the identity of the gene encoding Arginase (Fig. 2A, B, C).
- subtilis lA304( ⁇ l05MU331) according to the known method (Anagnostopoulos C and Spizizen J. J Bacteriol. 1961. 81: 741-746).
- the B. subtilis strain 1A304 ( ⁇ l05MU331) was obtained from J. Errington (Thornewell S et al. 1993. Gene. 133: 47-53). The strain was produced according to the publications by Thornewell, S. et al., 1993, Gene 133, 47-53 and by Baillie, L. W. J. et al., 1998, FEMS Microbiol. Letters 163, 43-47, which are incorporated herein in their entirety. Plasmid pABlOl (shown linearized in Fig.
- the erythromycin resistance gene (ermC) was replaced by the Arginase gene (arg) by a double crossover event in a process of homologous recombination.
- the 0.6 kb Munl-Ndel ⁇ lO5 phage DNA fragment (labeled as " ⁇ l05") and the cat gene provided the homologous sequences for the recombination.
- the Arginase gene was targeted to the expression site in the prophage DNA of B. subtilis 1A304 ( ⁇ j>105MU331) and the Arginase gene was put under the control of the strong thermoinducible promoter (Leung YC and Errington J. Gene. 1995. 154: 1-6).
- the fed-batch fermentation was carried out in a 2-liter fermentor at 37 degree C, pH 7.0 and dissolved oxygen 20% air saturation.
- the feeding medium contained 200 g/L glucose, 2.5 g/L MgSO 4 «7H 2 O, 50 g/L tryptone, 7.5 g/L K 2 HPO 4 and 3.75 g/L KH 2 PO 4 .
- 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.
- a 5-ml HiTrap Chelating column (Pharmacia, New Jersey, USA) was equilibrated with 0.1 M NiCl 2 in dH 2 O, for 5 column volumes.
- Fractions with arginase activity and high arginase purity were pooled (24 ml) and diluted ten times with start buffer [0.02 M sodium phosphate buffer (pH 7.4), 0.5 M NaCl]. This was loaded onto a second 5-ml HiTrap Chelating column (Pharmacia, New Jersey, USA), repeating the same procedure as above.
- mPEG-SPA cat. no. 2M4MOH01, Nektar, USA
- MW 5,000 5.82 g
- the mixture was then dialyzed extensively by ultra-dialysis against 15 L of PBS buffer using the F50(S) capillary dialyser (Fresenius Medical Care, Bad Homburg, Germany) to remove all the unincorporated PEG.
- the mPEG-SPA uses amino groups of lysines and the N- terminus of the protein as the site of modification.
- the measured specific activity of the pegylated Arginase was as high as 592 1.U./mg.
- EXAMPLE 5 1 A-LIFE DETERMINATION OF PEGYLATED ARGINASE IN VITRO USING THE METHOD IN HUMAN BLOOD PLASMA
- Pegylated Arginase (305.6 ⁇ l) at a concentration of 1 mg/ml was added into human plasma (1 ml) and the final concentration of pegylated Arginase was 0.24 mg/ml. The mixture was divided into 20 aliquots in eppendorf tubes (65 ⁇ l mixture in each eppendorf tube) and then incubated at 37 0 C. A 1-2 ⁇ l portion of the mixture from each eppendorf tube was used to test the Arginase activity. In this example, the 1 A-MQ was determined to be approximately 3 days. It took about 3 days to reduce the relative activity from 100% to 50%.
- arginase BCT was used in combination with 5FU.
- Hep 3B cells obtained from the American Type Culture Collection (ATCC) were propagated through four passages according to the supplier's recommendations before 40 nude mice were implanted with a tumor of this cell line of at least 3mm 3 .
- mice were divided randomly into four groups of 10 animals each. These are: Group 1, negative control (0.2 ml of 0.9% normal saline as negative control); Group 2, 250 IU of BCT; Group 3, combination of 250 IU arginase (BCT) and 10 mg/kg 5FU (5- fluorouracil) (Ebewe Arzneistoff Ges.m.b.H., Austria, Europe) ; and Group 4, 10 mg/kg 5FU.
- the animals were treated by intraperitoneal injection of the compound(s) or normal saline once a week..
- the implanted animals were observed once every two days for growth of the solid tumor in situ by digital caliper measurements to tumor size and weight.
- the tumor size was the average of two perpendicular diameters and one diagonal diameter.
- the tumor weight was taken to be the (length x width 2 )/2; assuming a specific gravity of 1.0 g/cm 3 .
- tumour growth was measured for 71 days and is shown in Figure 4.
- Statistical analysis was performed by SPSS 11.0 software (SPSS 5 Chicago, USA), based on Kaplan-Meier estimation and groups were compared by the log-rank test.
- the blood samples of the patients may be taken daily throughout treatment for arginine levels, Arginase (BCT) activities, complete blood picture and full clotting profile. Renal and liver functions are taken at least every other day, or sooner if deemed necessary.
- BCT Arginase
- BCT On day 1 BCT is infused over 30 minutes at 2,000 IU per kg. Thereafter, BCT is infused weekly for 8-12 weeks. This may be continued if anti-tumour activity is observed. Twenty minutes before each BCT infusion, pre-medication with dipheneramine 10 mg iv. and hydrocortisone 100 mg iv. is given.
- 5FU As for 5FU, it may administered at 125mg per meter square by short infusion every day from day 1 to day 5. Each 5FU infusion from day 1 to day 5 is preceded by administration of Folinic acid of 50mg per meter square. This same 5FU and Folinic acid treatment may be repeated every 4 weeks.
- EXAMPLE 8 USING EMBOLIZATION AS A METHOD TO REDUCE PHYSIOLOGICAL ARGININE LEVELS IN COMBINATION WITH AN ANTI-NEOPLASTIC COMPOUND
- Tumor embolization is a procedure used to reduce the vascularity of a tumor.
- a particulate form such as microspheres, is administered via catheter, which is positioned in the tumor's arterial blood supply.
- the particles are released from the catheter and carried by blood flow to the arterioles and capillary bed where they embolize and thus retards blood flow to the tumor (Microspheres and Regional Cancer Therapy, CRC Press, October 20, 1993).
- embolization induces a leakage of hepatic arginase from the liver into the circulation and the hepatic arginase released into the systemic circulation, in such a way, rapidly depletes plasma arginine (Cheng PN, Leung YC, Lo WH, Tsui SM, Lam KC. Cancer Lett. 2005 Jun 16;224(l):67-80. Epub 2004 Dec 25.).
- embolization effectively acts as a natural source of arginase that becomes released into the patient's blood stream after embolization i.e. it essentially acts as the administration of arginase to a patient, and is therefore expected to produce the same synergistic effect when administered in combination with an anti-neoplastic drug.
- EXAMPLE 9 USING DIALYSIS AS A METHOD TO REDUCE PHYSIOLOGICAL ARGININE LEVELS IN COMBINATION WITH AN ANTI-NEOPLASTIC COMPOUND
- Dialysis is a method used to remove waste materials and extra fluids from the blood. This method can also be used to remove certain amino acids from the blood (Gouyon JB, Desgres J, Mousson C. Pediatr Res. 1994 Mar; 35(3):357- 61). Therefore, dialysis can also be used to reduce the physiological levels of arginine in a human patient to below 10 ⁇ M, and is thus another implementation which may be used in combination with the administration of an anti-neoplastic drug.
- arginine depletion is a new form of anti-neoplastic therapy that differs from the mode of operation of traditional anti-neoplastic drugs, such as, alkylating agents and mitotic inhibitors, etc.
- Arginine deprivation results in the rapid and selective death of culture transformed and malignant cells (Scott L, Lamb J, Smith S, Wheatley DN. Br J Cancer. 2000 Sep;83(6):800-10). It has been suggested that loss of viability in the malignant phenotypes may be the result of the loss of control primarily at the key Gl checkpoint, which normally prevents cells from reinitiating DNA synthesis under adverse conditions.
- nutrient depletion refers to the reduction of physiological arginine levels to below lO ⁇ M.
- the reduction of physiological arginine levels may be achieved by different implementations.
- one implementation of the present invention is the administration of arginine reducing compounds to the human patient.
- arginine reducing compounds may be, but are not limited to, arginine degrading enzymes (such as arginase I, arginine decarboxylase, arginine deiminase, human, bovine and other animal arginase etc.).
- Another implementation of the present invention for this purpose is the use of embolization or dialysis, or other methods of treatment to reduce arginine levels.
- anti-neoplastic drugs are directed to damaging different parts of the cell e.g. the metabolic pathways or DNA synthesis/repair/transcription.
- Families of anti-neoplastic drugs include, but are not limited to, alkylating agents such as chlorambucil, cyclophosphamide, thiotepa, and busulfan, mitotic inhibitors such as plant alkaloids (e.g. actinomycin D 5 mitomycin) and podophyllotoxins, and antibiotics (e.g. mitoxantrone, bleomycin), such as anthracyclines (e.g. doxorubicin). It is there part of the present invention that any one or combination of these drugs may be used as the cell-damaging agent referred to above.
- 5FU is an example of an antimetabolite drug, and it is anticipated that other antimetabolite drugs, such as purine antagonists (e.g. 2- chlorodeoxyadenosine) and folate antagonists (e.g. methotrexate) would also provide synergistic effects.
- 5FU is used in combination with administering arginase (BCT).
- BCT arginase
- 5FU is an analogue of uracil with a fluorine atom at the C-5 position in place of hydrogen. It rapidly enters the cell using the same facilitated transport mechanism as uracil.
- 5FU is converted intracellularly to several active metabolites, which disrupt RNA synthesis and the action of the nucleotide synthetic enzyme thymidylate synthase (TS) (Leffingwell R, Rustum Y. Fluoropyrimidines in Cancer Therapy. Humana Press. January 1, 2003. p61-62).
- TS thymidylate synthase
- Example 6 the two compounds were administered at short time intervals, one immediately after another to facilitate experimental work.
- the term "combinatorial” or “used in combination” refer to the administration of the two compounds, such as an arginine degrading enzyme (e.g. arginase or arginine deiminase) and an anti-neoplastic compound such as 5FU, either simultaneously or consecutively within a temporal interval wherein the compound first administered is still at a concentration to exert an effect with regards to the treatment target cells, tissues or organs.
- the overlapping period of time in which the two compounds may be administered can be over a period of days or weeks, such as described in Example 7 above.
- a compound such as 5FU may be administered topically.
- the dosage of 5FU When used in human patients, the dosage of 5FU may be determined by a qualified health professional depending on the condition being treated, the size and overall health of the patient, as well as the particular regimen used. It is envisaged that when used in combination with an arginine degrading enzyme such as arginase (BCT) as taught by the present invention, the dosage of the anti-neoplastic compound such as 5FU can be significantly lower, thus also eliciting fewer and less severe side effects.
- BCT arginase
- Formulations of the pharmaceutical composition of the present invention can be used in the form of a solid, a solution, an emulsion, a dispersion, a micelle, a liposome, and the like, wherein the resulting formulation contains one or more of the modified arginine degrading enzyme such as human arginase in the practice of the present invention, as active ingredients, in a mixture with an organic or inorganic carrier or excipient suitable for enteral or parenteral applications.
- the active ingredients may be the arginase, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use in
- I l manufacturing preparations in solid, semisolid, or liquid form.
- auxiliary, stabilizing, thickening and coloring agents and perfumes may be used.
- the active ingredients of one or more arginase are included in the pharmaceutical formulation in an amount sufficient to produce the desired effect upon the target process, condition or disease.
- compositions containing the active ingredients contemplated herein may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
- Formulations intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical formulations.
- the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing sustained action over a longer period. They may also be coated to form osmotic therapeutic tablets for controlled release.
- formulations for oral use may be in the form of hard gelatin capsules wherein the active ingredients are mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, kaolin, or the like. They may also be in the form of soft gelatin capsules wherein the active ingredients are mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.
- the pharmaceutical formulations may also be in the form of a sterile injectable solution or suspension. This suspension may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
- the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,4-butanediol.
- Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
- any bland fixed oil may be employed including synthetic mono- or diglycerides, fatty acids (including oleic acid), naturally occurring vegetable oils like sesame oil, coconut oil, peanut oil, cottonseed oil, or synthetic fatty vehicles, like ethyl oleate, or the like. Buffers, dextrose solutions preservatives, antioxidants, and the like, can be incorporated or used as solute to dissolve the soluble enzyme as required.
- the pharmaceutical formulations may also be an adjunct treatment together with other chemotherapeutic agents.
- an arginase that has an amino acid sequence substantially the same as the sequence shown in SEQ ID No. 9 means that the sequence is at least 30% identical to that shown in SEQ ID No. 9 or that using the Arginase activity assay as described herein, there is no significant difference in the enzymatic activity between the enzyme of SEQ ID No. 9 and the one that is substantially similar.
- the six histidines are provided for ease of purification, and the additional methionine group provided at the amino terminus thereof is to allow translation to be initiated.
- SEQ ID NO. 1 (Fig. 2A) SEQ ID NO. 3: (Fig. 2B: amino acid sequence (SEQ ID NO:3)
- SEQ ID NO. 5 5'-CCAAACCATATGAGCGCCAAGTCCAGAACCATA-S'
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Cited By (4)
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EP2102231A2 (en) * | 2006-12-12 | 2009-09-23 | Bio-Cancer Treatment International Limited | Improved expression system for recombinant human arginase i |
WO2010124547A1 (en) * | 2009-03-26 | 2010-11-04 | The Hong Kong Polytechnic University | Site-directed pegylation of arginases and the use thereof as anti-cancer and anti-viral agents |
WO2014001956A3 (en) * | 2012-06-25 | 2014-02-20 | Ning Man Cheng | Combinational use of pegylated recombinant human arginase with chemotherapeutic/target therapeutic drug in cancer treatment |
EP2654776A4 (en) * | 2010-12-21 | 2016-05-11 | Bio Cancer Treatment Int Ltd | Use of pegylated recombinant human arginase for treatment of leukemia |
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WO2003063780A2 (en) * | 2002-01-25 | 2003-08-07 | Cancer Treatments International | Therapeutic composition for treatment of cancer by arginine depletion |
WO2003078578A2 (en) * | 2002-03-12 | 2003-09-25 | Lsu Medical Center | Modulation of the immune response through the manipulation of arginine levels |
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EP2102231A4 (en) * | 2006-12-12 | 2010-03-31 | Bio Cancer Treatment Int Ltd | Improved expression system for recombinant human arginase i |
WO2010124547A1 (en) * | 2009-03-26 | 2010-11-04 | The Hong Kong Polytechnic University | Site-directed pegylation of arginases and the use thereof as anti-cancer and anti-viral agents |
US8507245B2 (en) | 2009-03-26 | 2013-08-13 | The Hong Kong Polytechnic University | Site-directed pegylation of arginases and the use thereof as anti-cancer and anti-viral agents |
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WO2014001956A3 (en) * | 2012-06-25 | 2014-02-20 | Ning Man Cheng | Combinational use of pegylated recombinant human arginase with chemotherapeutic/target therapeutic drug in cancer treatment |
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