WO2004052406A1 - Coupling of dicarboxylic compounds by convertion into anhdrids - Google Patents
Coupling of dicarboxylic compounds by convertion into anhdrids Download PDFInfo
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- WO2004052406A1 WO2004052406A1 PCT/PL2003/000138 PL0300138W WO2004052406A1 WO 2004052406 A1 WO2004052406 A1 WO 2004052406A1 PL 0300138 W PL0300138 W PL 0300138W WO 2004052406 A1 WO2004052406 A1 WO 2004052406A1
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- methotrexate
- dicarboxylic
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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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 invention is a procedure for modification of a chemical substance, which has at least one available amine and/or hydroxyl residue, by coupling to dicarboxylic molecule.
- the procedure allows obtaining conjugates of carrier with hapten, and drugs with carrier proteins in particular.
- Modified macromolecules are important tools of exploration in medicine and biotechnology, and can be utilized to generate new trends in therapy of various disorders.
- Conjugated and/or complexed drugs exhibit many beneficial properties.
- the modified drug is characterized by different half-life, and causes different side effects. Its toxicity and transport to the cell is altered. Cells that developed the resistance to the native drug can be sensitive to the modified one.
- Carrier can have the ability to selectively cumulate in target cells and/or tissues, and when coupled with a drug can augment the treatment of disease. Therefore chemical modification of both carriers and drugs, and subsequent conjugation are a perspective for therapy.
- Conjugates can be obtained on various pathways.
- a correct selection of drug and carrier as well as type of chemical modification is important.
- the conditions of modification reaction should allow retention of biological activities of both the medicine and the carrier. It can be achieved for example by carefully adjusting the conjugation level.
- amine and sulphydryl residues in protein molecule and carboxylic, amine, hydroxyl, and sugar residues in drug molecule are of interest.
- One of the means to improve therapeutic activity of dugs is their conjugation with biological or synthetic macromolecules .
- the drug molecule can be either covalently bound or complexed with them.
- the idea of the carrier is to provide selectivity to the system.
- Drug or drug-like molecule when coupled to antibody (1) or its fragments (2) can be cumulated in cells and/or tissues in sites, where target antigens are presented.
- Experimental therapies were developed using antibodies coupled with drugs or radioactive nuclides. The latter also found use in diagnostics (3) .
- Other macromolecules such as glycoproteins (4), lipoproteins (5), fibrinogen (6) and other proteins and polymers (7) can be employed as carriers for therapeutic agents, genes and diagnostic markers.
- diagnostic and therapeutic anti-tumour utility of conjugates of radioactive isotopes i.e. 131 I (8), 90 Y (9) with antibodies and their fragments with is exploited.
- Doxorubicin-monoclonal antibody conjugate is studied in the phase II clinical trial (11) and human serum albumin- methotrexate conjugate is in phase I clinical trial (10) .
- the conjugates obtained this way were characterized by reasonable uniformity and purity, lower hydrophobic and lack of cross-linked fractions.
- the method thus deprived the conjugates from the undesired toxic activity.
- the object of the invention is a method of modification of chemical substance having at least one available amine and/or hydroxyl residue, where the other component a dicarboxylic acid is first activated by transformation into anhydride.
- Acid anhydride is coupled to the substrate in aqueous solutions (pH maintained between 4.5 and 9.0) at 0 to 40 °C. At these conditions only one of the two carboxylic groups can acylate, thus cross-linking is avoided.
- dicarboxylic compound is methotrexate or tomudex
- the modified macromolecule comprises a compound selected from the group consisting of: polipeptydes, aminoacids, carbohydrates, and synthetic polymers.
- proteins In specific applications of the invention proteins, peptides, aminoacids, carbohydrates and polymers are modified.
- the modified macromolecule is a protein, especially an antibody or fibrinogen
- the modified macromolecule is a sugar.
- said sugar is dextran or mannan.
- the pure, not cross-linked conjugate of hapten and a carrier protein obtained in reaction dissolves further or dilutes in aqueous medium.
- Conjugations utilizing a coupling of only one of the two carboxylic functionalities resulted in many beneficial properties of such formed molecules. Conjugates obtained according to this procedure are not chemically cross-linked.
- Methotrexate activation 1.0 ml of methotrexate solution in dimethylformamide (45 mg/ml) is mixed with 45 dicyclohexylocarbodiimide at +4°C for 40 hrs. The supernatant is dried under vacuum or in the stream of neutral gas. Dry methotrexate anhydride is washed several times with dichloromethane
- Obtained conjugates contained 2-4 moles of methotrexate per one mole of dextran.
- Example 5 Synthesis of active esters of methotrexate. 10 mg of methotrexate anhydride (example 1) in 1 ml N,N- dimethyloformamide is mixed with 10 mg of N- hydroxysuccynimide for 10 hrs. The conjugates of proteins with N-hydroxysuccinimied active ester of methotrexate were prepared
- Example 6 Antitumor activity of native and glycated fibrinogen-methotrexate conjugates
- mice (C57B1/6 x DBA/2) FI male mice, 12-14-week-old, weighting 20-25 g were applied. Mice were supplied from Animal Breeding Centre of the Institute of Immunology and Experimental Therapy, Wroclaw, Tru, and were maintained in standard laboratory conditions. Animal experiments were performed according to the International Laboratory Animal Care Convention and after acceptance of the experimental protocol by the Local Ethical Committee for the use of Laboratory Animals.
- P388 leukemia cell line was used. Passages of leukemia cells in DBA/2 mice were carried out according to the NIH/NCI standard screening protocols in vivo (Method described in: Experimental Therapeutics Program. In vivo cancer models, 1976-1982. Bethesda, MD: NIH Publication No. 84-2635) . Design of experiment: Animals were randomly divided into 5 groups: 1) nontreated control, 2-5) treated respectively with free methotrexate, conjugate with native bovine fibrinogen (F-MTX) , conjugate with fibrinogen glycated at 65°C (F65-MTX) , and conjugate with fibrinogen glycated at 73°C (F73-MTX) .
- F-MTX native bovine fibrinogen
- F65-MTX conjugate with fibrinogen glycated at 65°C
- F73-MTX conjugate with fibrinogen glycated at 73°C
- mice were injected with 10 6 leukemia (P388) cells i.p. and 24 hours later each mouse was injected with appropriate agent (40 mg/kg of pure MTX) (i.p.).
- Antitumor effect was evaluated using the following parameters: Increase in life span of treated mice over control (ILS%), calculated from the following formula: (AST T JASTc) 100 - 100%, where AST T is the average survival time of treated animals, and AST C is the average survival time of untreated control mice. All mice were weighted every day until end of experiment. Long-term survivors (LTS) - expressed as percentage of all treated mice which survived tumor-free for at least 2 month.
- ILS Increase in life span of treated mice over control
- LTS long-term survivor
- mice (C57B1/6 x DBA/2) FI male mice, 12-14-week-old, weighting 20-25 g were applied. Mice were supplied from Animal Breeding Centre of the Institute of Immunology and Experimental Therapy, Wroclaw, Tru, and were maintained in standard laboratory conditions. Animal experiments were performed according to the International Laboratory Animal Care Convention and after acceptance of the experimental protocol by the Local Ethical Committee for the use of Laboratory Animals.
- P388 leukemia cell line was used. Passages of leukemia cells in DBA/2 mice were carried out according to the NIH/NCI standard screening protocols in vivo (Method described in: Experimental Therapeutics Program. In vivo cancer models, 1976-1982. Bethesda, MD: NIH Publication No. 84-2635) . Design of experiment: Animals were randomly divided into 3 groups: 1) nontreated control, 2) treated with free methotrexate, 2) treated with dextran-methotrexate conjugate. Mice were injected with 10 ⁇ leukemia (P388) cells i.p. and 24 hours later each mouse was injected with appropriate agent (40 mg/kg of pure MTX) (i.p.).
- AST T /AST C 100 - 100%, where AST T is the average survival time of treated animals, and AST C is the average survival time of untreated control mice. All mice were weighted every day until end of experiment. Results
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Abstract
A procedure for modification of a chemical molecule, containing at least one amine or hydroxyl group, allows a dicarboxylic molecule to be coupled with it without formation of undesired products. The procedure is to be employed in obtaining carrier-hapten conjugates, and particularly drugs with protein or carbohydrate carriers.
Description
Authors :
Janusz Boratyήski ohair-ed Sala Omar Urszula Kaήska eanata Budzyήska Monika Jagiello Dmitry Nevozhay Joanna ietrzyk Anna Nasulewicz Adam Opolski Andrzej Gόrski
The invention is a procedure for modification of a chemical substance, which has at least one available amine and/or hydroxyl residue, by coupling to dicarboxylic molecule. The procedure allows obtaining conjugates of carrier with hapten, and drugs with carrier proteins in particular.
Modified macromolecules are important tools of exploration in medicine and biotechnology, and can be utilized to generate new trends in therapy of various disorders. Conjugated and/or complexed drugs exhibit many beneficial properties. The modified drug is characterized by different half-life, and causes different side effects. Its
toxicity and transport to the cell is altered. Cells that developed the resistance to the native drug can be sensitive to the modified one. Carrier can have the ability to selectively cumulate in target cells and/or tissues, and when coupled with a drug can augment the treatment of disease. Therefore chemical modification of both carriers and drugs, and subsequent conjugation are a perspective for therapy.
Conjugates can be obtained on various pathways. A correct selection of drug and carrier as well as type of chemical modification is important. The conditions of modification reaction should allow retention of biological activities of both the medicine and the carrier. It can be achieved for example by carefully adjusting the conjugation level. It is important for the carrier-drug conjugate to selectively modulate physiology of cells and tissues. For coupling reactions amine and sulphydryl residues in protein molecule, and carboxylic, amine, hydroxyl, and sugar residues in drug molecule are of interest.
One of the means to improve therapeutic activity of dugs is their conjugation with biological or synthetic macromolecules . The drug molecule can be either covalently bound or complexed with them.
The idea of the carrier is to provide selectivity to the system. Drug or drug-like molecule when coupled to antibody (1) or its fragments (2) can be cumulated in cells and/or tissues in sites, where target antigens are presented. Experimental therapies were developed using antibodies coupled with drugs or radioactive nuclides. The latter also found use in diagnostics (3) .
Apart from antibodies other macromolecules such as glycoproteins (4), lipoproteins (5), fibrinogen (6) and other proteins and polymers (7) can be employed as carriers for therapeutic agents, genes and diagnostic markers. Currently several clinical trials, in different stages are conducted. Diagnostic and therapeutic anti-tumour utility of conjugates of radioactive isotopes i.e. 131I (8), 90Y (9) with antibodies and their fragments with is exploited. Doxorubicin-monoclonal antibody conjugate is studied in the phase II clinical trial (11) and human serum albumin- methotrexate conjugate is in phase I clinical trial (10) .
Past reports describe synthesis, chemical and anti- tumour assays of methotrexate-carrier conjugates (12). The obtained conjugates increased the life span of mice inoculated with P338 leukaemia by twofold in comparison to mice treated with free drug. High selectivity was spoiled by toxicity, which was attributed to interactions with lipids, lipoproteins and membranes. The aim of the further research was to design a pathway to synthesize conjugates of reduced hydrophobicity and high purity.
Further research on conjugates of methotrexate- fibrinogen conjugates (Polish patent PL 130458, polish patent pending P.352435 and P.357644) resulted in an invention of novel preparation method that yielded products having high anti-tumour activity.
The conjugates obtained this way were characterized by reasonable uniformity and purity, lower hydrophobic and lack of cross-linked fractions.
The method thus deprived the conjugates from the undesired toxic activity.
The object of the invention is a method of modification of chemical substance having at least one available amine and/or hydroxyl residue, where the other component a dicarboxylic acid is first activated by transformation into anhydride. Acid anhydride is coupled to the substrate in aqueous solutions (pH maintained between 4.5 and 9.0) at 0 to 40 °C. At these conditions only one of the two carboxylic groups can acylate, thus cross-linking is avoided.
Advantageous for the modification of the chemical macromolecule useful in preparation of important tools in medicine and biotechnology is the evidence that dicarboxylic compound is methotrexate or tomudex, and the modified macromolecule comprises a compound selected from the group consisting of: polipeptydes, aminoacids, carbohydrates, and synthetic polymers.
In specific applications of the invention proteins, peptides, aminoacids, carbohydrates and polymers are modified.
Favourably in particular, the modified macromolecule is a protein, especially an antibody or fibrinogen
In another advantageous realisation of the method in accordance with the invention, the modified macromolecule is a sugar. Favourably, said sugar is dextran or mannan. In advantageous realisation of the method in accordance with the invention, the pure, not cross-linked conjugate of hapten and a carrier protein obtained in reaction, dissolves further or dilutes in aqueous medium. In the method according to the
invention is shown unexpectedly many advantages. Conjugations utilizing a coupling of only one of the two carboxylic functionalities resulted in many beneficial properties of such formed molecules. Conjugates obtained according to this procedure are not chemically cross-linked. They are characterized by lower hydrophobicity as compared to those obtained without control of the reactivity of dicarboxylic functionality of tomudex and/or methotrexate. These effects together reduce the non-specific interactions with hydrophobic components of tissues (lipids, lipoproteins, cellular membranes, fats) and thus emphasize the selectivity of the carrier. Furthermore the conjugates are better soluble in aqueous media. A great advantage of the invention is that the products are obtained in pure form.
Example 1
Methotrexate activation 1.0 ml of methotrexate solution in dimethylformamide (45 mg/ml) is mixed with 45 dicyclohexylocarbodiimide at +4°C for 40 hrs. The supernatant is dried under vacuum or in the stream of neutral gas. Dry methotrexate anhydride is washed several times with dichloromethane
Example 2 Synthesis of methotrexate-protein conjugates
Solution containing proteins (3-20 mg/ml) in 0.1 M sodium bicarbonate is mixed with different amounts of methotrexate anhydride solution in N,N-dimethylformamide . The level of drug substitution depends on proportion of reagents, pH and
nature of protein. The conjugation is completed during 10-60 minutes. The pure conjugates are isolated by common procedures as dialysis or gel filtration chromatography. Protein- ethotrexate conjugates are stabile at frizzed state and at +4°C at pH 4 and 10. Storage at 37°C for 4 days leads to liberation of 25% of bound drugs.
Example 3
Synthesis of tomudex-protein conjugates. 2 mg of pure tomudex (Raltitrexed) in 0.1 ml of N,N- dimethylformamide is mixed with 2 mg of N,N'- dicyclohexylocarbodiimide. The reaction was carried for 12 hr at +4°C. Precipitate of urea derivative was centrifuged and solution was dried under high vacuum. Dry tomudex anhydride was washed several times by dichloromethane. The residue was dissolved in N,N-dimethylformamide and mixed with bovine serum albumin dissolved in 0.1 M sodium bicarbonate. Obtained conjugates contained 1-3 moles of tomudex per ol of protein.
Example 4
Synthesis of conjugates of methotrexate with carbohydrates
100 mg of dextran T 40 in 4 ml of 0.1 M sodium bicarbonate was mixed with 4 mg of methotrexate anhydride in 0.2 ml N,N- dimethyloformamide. After 3 hr the excess of unreacted and decomposed anhydride was removed by dialysis against water.
Obtained conjugates contained 2-4 moles of methotrexate per one mole of dextran.
Example 5. Synthesis of active esters of methotrexate.
10 mg of methotrexate anhydride (example 1) in 1 ml N,N- dimethyloformamide is mixed with 10 mg of N- hydroxysuccynimide for 10 hrs. The conjugates of proteins with N-hydroxysuccinimied active ester of methotrexate were prepared
Example 6:Antitumor activity of native and glycated fibrinogen-methotrexate conjugates
Materials and methods Mice: (C57B1/6 x DBA/2) FI male mice, 12-14-week-old, weighting 20-25 g were applied. Mice were supplied from Animal Breeding Centre of the Institute of Immunology and Experimental Therapy, Wroclaw, Poland, and were maintained in standard laboratory conditions. Animal experiments were performed according to the International Laboratory Animal Care Convention and after acceptance of the experimental protocol by the Local Ethical Committee for the use of Laboratory Animals.
Cells: P388 leukemia cell line was used. Passages of leukemia cells in DBA/2 mice were carried out according to the NIH/NCI standard screening protocols in vivo (Method described in: Experimental Therapeutics Program. In vivo cancer models, 1976-1982. Bethesda, MD: NIH Publication No. 84-2635) . Design of experiment: Animals were randomly divided into 5 groups: 1) nontreated control, 2-5) treated respectively with free methotrexate, conjugate with native bovine fibrinogen (F-MTX) , conjugate with fibrinogen glycated at 65°C (F65-MTX) , and conjugate with fibrinogen glycated at 73°C (F73-MTX) . Mice were injected with 106 leukemia (P388) cells i.p. and 24 hours later each mouse was injected with appropriate agent (40 mg/kg of pure MTX) (i.p.).
Antitumor effect was evaluated using the following parameters: Increase in life span of treated mice over control (ILS%), calculated from the following formula: (ASTTJASTc) 100 - 100%, where ASTT is the average survival time of treated animals, and ASTC is the average survival time of untreated control mice. All mice were weighted every day until end of experiment. Long-term survivors (LTS) - expressed as percentage of all treated mice which survived tumor-free for at least 2 month.
Results
In our preliminary investigation all newly synthesized conjugates (F-MTX, F65-MTX, F73-MTX) revealed significantly higher antitumor activity in vivo in comparison with free methotrexate (p<0.05, p<0.001 and p<0.05 respectively). There was one out of 6 (16,66%) long-term survivor (LTS) in group treated with F65-MTX conjugate, which survived more than 2 month after tumor transplantation without any manifestation of leukemia. The results of our experiment are summarized in Table 1.
Table 1. Mean survival time of tumor (P388 ) -bearing mice treated with free methotrexate or with different conjugates of methotrexate, expressed as an ILS (%) - increase of life span.
Group ΪV5 ILS % Mean + S∑f
Control 6 '. 11,17 ± 1,33
Methotrexate 6 34 15 ± 0,63
F-MTX conjugate 6 137 26,5 ± 14,8
F65-MTX conjugate c 6 151 28 ± 5,96
F73-MTX conjugate 6 91 21,33 ± 10,52 a N, number of mice b Mean survival time (days)
c In this group there was one out of 6 (16,66%) long-term survivor (LTS) which survived more than 2 month after tumor transplantation without any manifestation of leukemia.
Example 7 : Antitumor activity of dextran-methotrexate conjugate
Materials and methods Mice: (C57B1/6 x DBA/2) FI male mice, 12-14-week-old, weighting 20-25 g were applied. Mice were supplied from Animal Breeding Centre of the Institute of Immunology and Experimental Therapy, Wroclaw, Poland, and were maintained in standard laboratory conditions. Animal experiments were performed according to the International Laboratory Animal Care Convention and after acceptance of the experimental protocol by the Local Ethical Committee for the use of Laboratory Animals.
Cells: P388 leukemia cell line was used. Passages of leukemia cells in DBA/2 mice were carried out according to the NIH/NCI standard screening protocols in vivo (Method described in: Experimental Therapeutics Program. In vivo cancer models, 1976-1982. Bethesda, MD: NIH Publication No. 84-2635) . Design of experiment: Animals were randomly divided into 3 groups: 1) nontreated control, 2) treated with free methotrexate, 2) treated with dextran-methotrexate conjugate. Mice were injected with 10δ leukemia (P388) cells i.p. and 24 hours later each mouse was injected with appropriate agent (40 mg/kg of pure MTX) (i.p.).
Antitumor effect was evaluated using the following parameters: Increase in life span of treated mice over control (ILS%), calculated from the following formula:
(ASTT/ASTC) 100 - 100%, where ASTT is the average survival time of treated animals, and ASTC is the average survival time of untreated control mice. All mice were weighted every day until end of experiment.
Results
In our preliminary investigation dextran-methotrexate conjugate revealed significantly higher antitumor activity in vivo in comparison with free methotrexate (p<0.001). The results of our experiment are summarized in Table 2.
Table 2. Mean survival time of tumor (P388) -bearing mice treated with free methotrexate or with dextran-methotrexate conjugate, expressed as an ILS (%) - increase of life span. Group _¥ ILS % Mean ± S∑f
Control 12 11,6 ± 1,24
Methotrexate 8 26 14,63 ± 0,74
D-MTX conjugate 8 49 17,25 ± 0,89 a N, number of mice b Mean survival time (days)
References:
1. Hebert C, Norris K, Sauk JJ. Targeting of human squamous carcinomas by SPA470-doxorubicin immunoconjugates . J Drug Target. 2003 Feb; 11 (2) : 101-107.
2. Donda A, Cesson V, Mach JP, Corradin G, Primus FJ, Robert B. In vivo targeting of an anti-tumor antibody coupled to antigenic MHC class I complexes induces specific growth inhibition and regression of established syngeneic tumor grafts. Cancer Immun. 2003 Aug 14;3:11
3. Lewis MR, Wang M, Axworthy DB, Theodore LJ, Mallet RW, Fritzberg AR, Welch MJ, Anderson CJ. In vivo evaluation of pretargeted 64Cu for tumor imaging and therapy. J Nucl Med. 2003 Aug; 44 (8 ): 1284-92
4. Shimura N, Sogawa Y, Kawakita Y, Ikekita M, Yamazaki N, Kojima S. Radioiodination of glycoprotein-conjugated liposomes by using the Bolton-Hunter reagent and biodistribution in tumor-bearing mice. Nucl Med. Biol. 2002 May;29 (4) :491-6
5. Xiao W, Wang L, Ryan JM, Pater A, Liu H. Incorporation of an (125) I-labeled hexa-iodinated diglyceride analog into low-density lipoprotein and high specific uptake by cells of cervical carcinoma cell lines. Radiat Res. 1999 Sep;152(3) :250-6
6. Boratyήski J. , Opolski A., Wietrzyk J. , Gόrski A., Radzikowski C: „Cytotoxic and antitumor effect of fibrinogen-methotrexate conjugate." Cancer Letters 2000, 148: 189-195. 7. Rihova B, Strohalm J, Prausova J, Kubackova K, Jelinkova M, Rozprimova L, Sirova M, Plocova D, Etrych T, Subr V, Mrkvan T, Kovar M, Ulbrich K. Cytostatic and immunomobilizing activities of polymer-bound drugs: experimental and first clinical data. J Control Release. 2003 Aug 28,-91 (1-2) :1-16
8. Matthay K. K. , Panina C, Huberty J. , Price D., Glidden D. V., Tang H. R. , Hawkins R. A., Veatch J. , Hasegawa B. : Correlation of tumor and whole-body dosimetry with tumor response and toxicity in refractory neuroblastoma treated with (131)I-MIBG. J. Nucl. Med. 2001, 42(11): 1713-21.
9. Weiden P. L., i Breitz H. B. : Pretargeted radioimmunotherapy (PRIT) for treatment of non-Hodgkin" s lymphoma (NHL). Crit. Rev. Oncol. Hematol. 2001, 40(1): 37- 51.
10. Hartung G., Stehle G., Sinn H., Wunder A., Schrenk H. H., Heeger S., Kranzle M. , Edler L., Frei E., Fiebig H. H., Heene D. L., Maier-Borst W. , Quiesser W. : Phase I trial of ethotrexate-albumin in a weekly intravenous bolus regimen in cancer patients. Phase I Study Group of the Association for Medical Oncology of the German Cancer Society. Clin Cancer Res. 1999, 5 (4) : 753-759.
11. Smith S.: Technology evaluation: SGN-15, Seattle Genetics Inc. Curr. Opin. Mol. Ther. 2001, 3 (3) : 295-302. 12 Boratynski J, Opolski A, Wietrzyk J, Gorski A, Radzikowski C. Cytotoxic and antitumor effect of fibrinogen- methotrexate conjugate. Cancer Lett. 2000 Feb 1; 148 (2) : 189-95 Zglaszaja-cy: Instytut Immunologii i Terapii Doswiadczalnej Polskiej Akademii Nauk we Wroclawiu
Claims
1. A method of modification of a chemical molecule consisting of at least one available amine or hydroxyl residue characterised in that the coupling with amine or hydroxyl residues is carried by dicarboxylic compound that is prior converted into an anhydride form, than mixed with the substrate for modification at 0 to 40 °C and pH between 4.5 and 9.0 then only one carboxyl moiety of the coupled dicarboxylic molecule is able to react effectively.
... w forme bezwodnika, po czym zapewniaja-c pH od 4,5 do 9 i temperature od 0 do 40°C miesza sie z roztworem poddawanych modyfikacji makrocza,steczek, przy czym tylko jedna grupa karboksylowa sprzeganego zwia_zku dikarboksylowego jest zdolna do efektywnej reakcji.
2. The method according to claim 1, wherein the dicarboxylic compound is a methotrexate or tomudex, and modified macromolecule comprises a compound selected from the group consisting of: polipeptydes, aminoacids, carbohydrates, and synthetic polymers.
3. The method according to claim 2, wherein said modified macromolecule is a protein.
4. The method according to claim 3, wherein said protein is an antibody or fibrinogen.
5. The method according to claim 2, wherein the modified compound is a sugar.
6. The method according to claim 5, wherein said sugar is dextran or mannan.
7. The method according to claim 2, wherein the modified macromolecule is alcohol.
8. The method according to claim 7, wherein said alcohol is N-hydroxysuccynimide or sulpho N-hydroxysuccynimide .
9. The method according to claim 3, wherein the pure, not cross-linked conjugate of hapten and carrier proteins obtained in reaction, is further dissolved or suspended in aqueous medium.
In the method according to the invention is shown unexpectedly many advantages.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2003287110A AU2003287110A1 (en) | 2002-12-10 | 2003-12-10 | Coupling of dicarboxylic compounds by convertion into anhdrids |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PL357664A PL195813B1 (en) | 2002-12-10 | 2002-12-10 | Method of modification of the molecule |
| PLP.357664 | 2002-12-10 | ||
| PL03363422A PL363422A1 (en) | 2003-11-12 | 2003-11-12 | Method for modification of the molecule |
| PLP.363422 | 2003-11-12 |
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| WO2004052406A1 true WO2004052406A1 (en) | 2004-06-24 |
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| WO (1) | WO2004052406A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010047607A2 (en) | 2008-10-26 | 2010-04-29 | Instytut Immunologii i Terapii Doświadczalnej PAN | Method of production of polyanionic drug-carrier conjugates |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4699784A (en) * | 1986-02-25 | 1987-10-13 | Center For Molecular Medicine & Immunology | Tumoricidal methotrexate-antibody conjugate |
-
2003
- 2003-12-10 AU AU2003287110A patent/AU2003287110A1/en not_active Abandoned
- 2003-12-10 WO PCT/PL2003/000138 patent/WO2004052406A1/en not_active Application Discontinuation
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4699784A (en) * | 1986-02-25 | 1987-10-13 | Center For Molecular Medicine & Immunology | Tumoricidal methotrexate-antibody conjugate |
Non-Patent Citations (7)
| Title |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2010047607A2 (en) | 2008-10-26 | 2010-04-29 | Instytut Immunologii i Terapii Doświadczalnej PAN | Method of production of polyanionic drug-carrier conjugates |
| WO2010047607A3 (en) * | 2008-10-26 | 2010-07-22 | Instytut Immunologii i Terapii Doświadczalnej PAN | Method of production of polyanionic drug-carrier conjugates |
| US8623998B2 (en) | 2008-10-26 | 2014-01-07 | Instytut Immunologii I Terapii Doswiadczalnej Pan | Method of production of polyanionic drug-carrier conjugates |
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| AU2003287110A1 (en) | 2004-06-30 |
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