CA2009695A1 - Polymer-bound methotrexate, a process for its preparation and its use - Google Patents
Polymer-bound methotrexate, a process for its preparation and its useInfo
- Publication number
- CA2009695A1 CA2009695A1 CA002009695A CA2009695A CA2009695A1 CA 2009695 A1 CA2009695 A1 CA 2009695A1 CA 002009695 A CA002009695 A CA 002009695A CA 2009695 A CA2009695 A CA 2009695A CA 2009695 A1 CA2009695 A1 CA 2009695A1
- Authority
- CA
- Canada
- Prior art keywords
- methotrexate
- polymer
- bound
- molecular weight
- poly
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- 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
-
- 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
- A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
-
- 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
- A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
-
- 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
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
Abstract of the Disclosure:
Polymer-bound methotrexate, a process for its preparation and its use The crosslinking of the carboxyl groups of methotrexate or analogs with hydroxyl groups of water-soluble, biocom-patible polymers results in a pro-drug form of the active substance with good relevant properties for human anti-tumor therapy.
Polymer-bound methotrexate, a process for its preparation and its use The crosslinking of the carboxyl groups of methotrexate or analogs with hydroxyl groups of water-soluble, biocom-patible polymers results in a pro-drug form of the active substance with good relevant properties for human anti-tumor therapy.
Description
HOECHST AXTIENGESEL~SCHAPT HOE 89/P 050 Dr.~H/Le Description Poly er-bound ethotre ate, a proee~s for it~ preparation ~nd it~ use ,~
S There has for years been world-w$de research into the development of pXarmacologically active polymers, includ-ing, in particular, polymeric antitumor agents (US
4,460,560, US 4,551,502, ~P 0,040,506, WO 875,031, DE
3,515,178, DE 902,344 ~nd ~P 0,111,388).
An essential aim in this eonnection is the preparation of cytostatics whose side effects have been reduced or eliminated ~nd whose therapeutic range hss been improved.
The problems with the cytostatics in eurrent clinical use derive less from insuffieient eytotoxieity and more from in~dequate selectivity. This me~ns that the wide range of highly eytotoxie eompounds aet not only in tumor eells but also in healthy cells in the body and, in many eases, also more strongly in individual organs.
Hence there is a need for ~n administration form in wh$eh a level of eytostatie in the blood whieh is within the therapeutie range, i.e. with whieh tumor eell~ are greatly da~aged but healthy eells in the body are damaged only slightly or not at all, ean be reaehed ~nd main-tained for a prolonged period, in order to ~ehieve a greater tumor toxieity with, at the same time, diminished overall toxieity using doses of ehemotherapeutie whieh are eon~iderably lower in total.
Variou~ developments have been followed up to dates - Polymers as eytostaties (poly~nions sueh ~8 pyran eopolymers, polyvinyl sulfonates ete.) whieh ~re intended to have a tumor-inhibiting aetion per se.
Frequent disadvantages toxic side effeets and narrow limits in the molecular weight range (nephrotoxicity > I~W 50 I~D).
S There has for years been world-w$de research into the development of pXarmacologically active polymers, includ-ing, in particular, polymeric antitumor agents (US
4,460,560, US 4,551,502, ~P 0,040,506, WO 875,031, DE
3,515,178, DE 902,344 ~nd ~P 0,111,388).
An essential aim in this eonnection is the preparation of cytostatics whose side effects have been reduced or eliminated ~nd whose therapeutic range hss been improved.
The problems with the cytostatics in eurrent clinical use derive less from insuffieient eytotoxieity and more from in~dequate selectivity. This me~ns that the wide range of highly eytotoxie eompounds aet not only in tumor eells but also in healthy cells in the body and, in many eases, also more strongly in individual organs.
Hence there is a need for ~n administration form in wh$eh a level of eytostatie in the blood whieh is within the therapeutie range, i.e. with whieh tumor eell~ are greatly da~aged but healthy eells in the body are damaged only slightly or not at all, ean be reaehed ~nd main-tained for a prolonged period, in order to ~ehieve a greater tumor toxieity with, at the same time, diminished overall toxieity using doses of ehemotherapeutie whieh are eon~iderably lower in total.
Variou~ developments have been followed up to dates - Polymers as eytostaties (poly~nions sueh ~8 pyran eopolymers, polyvinyl sulfonates ete.) whieh ~re intended to have a tumor-inhibiting aetion per se.
Frequent disadvantages toxic side effeets and narrow limits in the molecular weight range (nephrotoxicity > I~W 50 I~D).
- 2 - ~
- Polymers which have a cytostatic action and in which constituents of the ~polymer backbone~ have a certain activity after degradation. Prequent disad-vantages: limited choice of the building block~ of the polymer, immunogenicity, side effects of the polymer~ and, ln particular, insolubillty of the products in water ~J 58/174,409, DE 3,026,448, DE
- Polymers which have a cytostatic action and in which constituents of the ~polymer backbone~ have a certain activity after degradation. Prequent disad-vantages: limited choice of the building block~ of the polymer, immunogenicity, side effects of the polymer~ and, ln particular, insolubillty of the products in water ~J 58/174,409, DE 3,026,448, DE
3,515,178, D~ 3,026,574, DE 902,344 and DE
3,026,575).
10 - Polymer-bound cytostatics on functional amide side-groups (WO 875,031, EP 0,111,388, D~ 3,539,951, ~O
8,700,056, EP 0,190,464, US 4,551,502, JP
57/143,325, EP 0,040,506 and JP 57/143,326).
Although the liberation of these cytostatics from the linkage with the polymer can in theory take place on endocytosis of the complete con~ugate in the acid pH of the lysosome, it has been shown in practice that endo-cytosis takQs place to only a small extent even in the case of polymer con~ugations with target-~pecific modifi-cations (for example antibody con~ugates). Purthermore, ma~or problems arise from the introduction of spacers, the frequently low loading density and difficult syn-theses. The in vivo results with such con~uqates to date have therefore been disappointing.
Phase-specific cytostatics, for example methotrexate, represent a special case because they act only in a particular phase of the cell cycle (synthesis phase in the case of the dihydrofolate reductase inhibitor metho-trexate). Given the relatively short biological half-life of methotrexate, a single administration frequently has no antitumor action whatever. A therapeutic effect is shown only by multiple individual administrations with very high doses. A ~rescue therapy~ with antagonists is frequently necessary in order subsequently to avoid, with a time lag, too much damage in the healthy cells.
~5 A polymer-bound form of methotrexate i8 of particular interest because of the~e problems. However, no satisfac-tory solution has to date been possible by b$nding methotrexate to poly-L-lysine, poly-lactide-glycolide (PLG) derivatives etc. (see W0 875,031).
Surprisingly, very good in vitro and in vivo properties with relevance for human therapy have now been achieved by binding methotrexate to water-soluble, biocompatible polymers carrying hydroxyl groups.
Hence the invention relates to~
1. A polymer-bound methotrexate or methotrexate deriva-tives, having the following features - an ester linkage of the ~- and/or 7-carboxyl group of the methotrexate or of the methotrexate - 15 derivatives with hydroxyl groups of water-soluble biocompatible polymers;
- bringing about a prolongation of life of more than 125~ compared with untreated controls in mice with L1210 leukemia on a ~ingle intraperi-toneal administration of 60 mg of methotrexate or methotrexate derivative, which is bound to the abovementioned polymer, per kg of body weight.
2. A proce~s for the preparation of the polymer-bound methotrexate with the features specified in 1. or of the polymer-bound methotrexate derivatives, which comprises the methotrexate which carries ~- and/or 7-carboxyl groups, or the corresponding derivatives of methotrexate, being reacted in the presence of water-abstracting coupling reagents with a water-soluble biocompatible polymer carrying hydroxyl groups.
3. The use of the polymer-bound methotrexate with the features specified in 1., or of the polymer-bound methotrexate derivatives, as tumor therapeutic.
. . .. . - ,: .
~ 4 ~
The invention is described in detail hereinafter, e~pe-cially in its preferred embodiments. The lnvention 18 furthermore defined by the content~ of the clalm~.
~ethotrexateorN-t4-[[(2,4-diamino-6-pteridinyl)methyl]-methylamino]benzoyll-glutamic acid is de~cribed a~ com-pound with its essential characteristics in the article by A.R. Chamberlain et al. in Analytical Profiles of Drug Substances, volume 5, edited by ~. ~lorey, Academic Pre~s, New York (1976), pages 283-306. The preparation of the compound is described by Seeger et al. tJ. Am. Soc.
11, 1753 (1949) or Rahmann et al., Medic. Res. Rev., 8, 95 (1988)].
The compounds which can be employed as methotrexate derivatives or analogs are those which have been modified on the pteridine ring or in the bridge region, a~ well as those which have been derivatized on the aromatic ring or in the glutamic acid iety. However, an e~ential condition is that at least one carboxyl group which is neces~ary for linkage to the polymer is retained. Analogs of thi~ type and processes for the prep~ration thereof are de~cribed in detail in the article by Rahoann et al.
(~ee above). Particularly preferred derivatives which can be u~ed are methopterin (~erck Index 10 (1983) 5860) and aminopterin (Merck Index 10 (1983) 477).
The said compounds can be bound via an e~ter linkage to water-soluble, biocompntible polymers carrying hydroxyl groups. By biocompatible polymers are eant compounds which are physiologically tolerated and can be degraded and/or excreted in the body. Polymers which can prefe-rably be u~ed are those in which the proportion of ionizable groups before the loading with the active substance is below 10 mol-~.
Preferably employed are water-soluble starches with a mean molecular weight of 1000 to 200,000, preferably 5000 to 50,000, which can al~o be dified. Also used are - s -cellulose acetates, especially water-soluble cellulose acetate from the company (Celanese/WSCA)~ or destrans, with a mean molecular weight between 1000 and 200,000, e~pecially with a mean molecular weight of 40,000 to 70,000, or inulin.
It is also possible to use appropriate synthetic poly-mers. One esample is poly-~,~-(hydroxyethyl)-D,L-spartamide of the fon~ula I
C - NN~ C -CH2~ ~ - CH~
r ~ C~
(CH2)2 (CIH2)2 CH2OH ~ CH2OH n in which the ratio of m to n is in the range 0-1 to 1-0, preferably 0.7s0.3 to 0.95s0.05. The molecular weight of compounds.of this type is appro~imately between 2000 and 100,000, preferably between 5000 and 50,000. Appropriate compounds and procesffe~ for the preparation thereof are described in detail in German Offenlegungsschr.ift 3,700,128.
Another example of a ~ynthetic polymer is Am~dated polylysine fumaramide/polylys~ne glutaramide of the formula II
:: : : ....... .. . ....
O O
Il 11 -(-NH-(CH2)~ - CH - NH-C-X-C-) D- II
C'O
(CH2)~OH
in which X can be -(CH2)3 or -CH-CH-, m can be the numbers 1 to 10, preferably 2, and n can be the number 5 to 2000.
~hese polymers have a molecular weight between 1000 and 300,000. Polymers of this type and processes for the preparation thereof are described in German Offenlegungs-schrift 3,707,369.
A further example is 8 copolymer of the formula IIIcomposed of poly-~,~-(2-hydroxyethyl)-D,L-aspartamide (compound of the formula I) and polysuccinimide (formul~ I) ~ ~ III
in which the ratio of y to z is in the range from about O.99sO.Ol to O.OlsO.99, preferably 2.5s7.5 to 7.Ss2.5.
Particularly preferably employed as polymers in the process according to the invention are the synthetic compounds depicted in formulae I, II and III.
, 2S For the prepsration o:E the polymer-bound methotrexate or of the corresponding derivatives, the active substance is I dissolved, for example in water, dimethyl sulfoxide ; (DMSO), form~mide, N,N-dimethylformamide or methylene chloride or a mixture of the last three solvents. The appropriate polymer is added to the same solvent which has also been u~ed to dissolve the active substance. ~he '' .
.' r' .. ,;~ , .
two mixtures are combined and can be incubated in the presence of a water-abstracting coupling reagent, where appropriate with exclusion of light, at a pH in the range from 7 to 9, preferably 8 to 8.S, and at a temperature of S 0 to 100C, preferably 20 to 30C, for a period of nbout 1 to 29 hours, preferably with stirring.
It iB poBBible to use as water-abstracting coupling reagent carbodiimides, alkylphosphonic anhydrides, carbonyldi~mines etc. Carbonyldiimidazole and dicyclo-hexylcarbodiimide are particularly preferably used.
The resulting crude product can be purified by precipita-tion with a ~olvent in which the polymer is insoluble. It is possible to use for this purpose, for example, tetra-hydrofuran, acetone, dioxane and alcohols. Further lS purification can take place using methods for molecular weight partition such as, for example, ultrafiltration, dialysis and gel permeation.
The process according to the invention results in a polymer-bound methotrexate product with a loading of methotrexate or analogs thereof from 1 to 85%, preferably lS to 7S%, based on the weight of the polymer-bound product. The product furthermore has an extinction coefficient between 0.0001 l/mg and 0.05 l/mg, preferably between 0.005 l/mg and 0.03 l/mg, in aqueous solution at pH 7 to 8.5 and a wavelength of 302 nm for methotrexate and methopterin, or an extinction coefficient of 0.0005 l/mg to 0.04 l/mg at the said pH and a wavelength of 282 nm for aminopterin.
The active substance can be slowly released from the polymer-bound methotrexates according to the invention into the body by simple hydrolysis, in contrast to the stronger binding via functional amide groups, from which the active substance can be liberated only by enzymatic cleavage.
- -5i:
: ~ . :
:i i' .~
. ; ~ :
~,~ : - , , ' ~
. ~
- 8 - ~ ~
With these polymer-methotrexate con~ugates according to the invention in vivo, for example in mice with L1210 leukemia, prolongations of life of > 125%, preferably > 150~, compared with untreated controls are achieved even with a single administration ~intraperitoneal i.p.) of 60 mg~kg of body weight methotrexate or derivatives which are linked according to the invention to the water-soluble, biocompatible polymer carrying hydroxyl groups (equivalent of methotrexate or derivative). These figures were determined as described in Example 7. Methotrexate or the derivatives are not themselves active under the said conditions. With 330 mg/kg equivalent of metho-trexate or derivative it was possible to observe complete remission without relapse up to the termination of the experiment a~ter 60 days in 2 of 5 animals. This dose is itself above the LD50 of free methotrexate.
In addition, the polymer-methotrexate con~ugates accord-ing to the invention have a higher IC50 in vitro than free methotrexate (for example on L1210, HT 29, A549 cells).
In con~unction with the in vivo results described sbove, this is to be regarded as evidence of a desired slow liberation of methotrexate.
The invention is illustrated by means of examples herein-after. Unless indicated otherwise, percentage data relate to weight.
Fsample 1: Preparation of a poly-methotrexate-poly-~,0-(2-hydroxyethyl)-D,L-aspartamide ester using carbonyldiimidazole Methotrexate was purchased from Sigma for all the examples. The polymer poly-~,0-(2-hydroxyethyl)-D,L-aspartamide is prepared by the method of P. Neri, G.
Antoni, F. aenvenuti, F. Cocola, G. Gazzei, J. Med. Chem.
16, 893 (1973).
. r~ ~
. .
. ~.
~ ~, ., ~ . :
3 g of methotrexate are dissolved in 15 ml of a mixture of formamide s N,N-dimethylformamide : CH2Cl2 (10 : 9 s 1). To this is added a solution of 300 mg of pyrrolidinopyridine and 2.44 g of carbonyldiimidazole in 5 ml of the above solvent mixture. The reaction mixture is stirred at room temperature (RT) for 1.5 h. At the same time, 3.1 g of poly-~,~-(2-hydroxyethyl)-D,$-aspar-tamide (PHEA) are dissolved in 9 ml of the solvent mixture described and stirred at RT. The two solutions are combined and stirred at RT with exclusion of light for 20 h. The crude product is precipitated by pouring into 250 ml of acetone. The yellow precipitate is sepa-rated off, washed with acetone, dried and then taken up in 25 ml of aqueous NaHC03 solution (pH 8-9).
The amber-colored aqueous solution is poured onto a Sephadex gel chromatography column (PD10, Pharmacia) and separated into a low molecular weight and a high molecu-lar weight fraction by eluti~n with water. The high molecular weight fraction is freeze-dried. The metho-trexate content is determined by UV spectroscopy at~ - 302 nm in aqueous solution. The product is charac-terized by lH NNR, CHN analysis and thin-layer chroma-tography. The lH NMR in D20 corresponds to a sum of the spectra of methotrexate and PHEA, it being possible to establish the degree of occupancy from the integral ratios. The degree of occupancy of 22~ by weight metho-trexate measured by HPLC corre8pond8 to the result of the W determination. The coupling reagents migrate, whereas methotrexate and product remain at Rf = 0, in the TLC in diethyl ether. The presence of free methotrexate could be ruled out by ultrafiltration with m~mhranes with various exclusion limits.
I
Yield: 3 g of polymer-bound methotrexate (about 20% of theory based on methotrexate). Most of the unreacted methotrexate is recovered by reprecipitation from the low molecular weight fraction.
., .
i .
.. ~, ~. . .
' ~ .
:,...
. . .
,.. ~. :
,:x. : :
- 10 - ~09695 Xxample 2s Preparation of a poly-methotrexate-dextran (40,000) ester usinq carbonyldiimidazole Dextran 40,000 was purchased from Fluka, Buchs, Switzerland. 3 g of methotrexate are dissolved in 10 ml of a mixture of formamide : N,N-dimethylformamide s CH2Cl2 ( 10 S 9 5 1), and 2.39 g of carbonyldiimidazole and 0.29 g of pyrrolidinopyridine are mixed in 5 ml of the ~bove solvent mixture and stirred at RT for 2 h. Then a solu-tion of 3.2 g of dextran 40,000 in 20 ml of the same solvent mixture is added thereto, and the mixture is stirred at RT with exclusion of light for 20 h. The crude product i8 precipitated in 350 ml of dry acetone, washed with acetone and dried.
The solid is taken up in 25 ml of H20 and poured onto a PD10 gel chromatography column. Elution with H20 yields a high and a low molecular weight fraction. The high molecular weight fraction is freeze-dried and analyzed as described in Example 1. Unreacted methotrexate can be recovered by reprecipitation.
Yields 3 g of polymer-bound methotrexate Occupancys 17.3% by weight methotrexate F~ample 3s Preparation of apoly-methotrexate-dextran (40,000) ester using dicyclohexylcar-bodiimide (DCC) Dextran 40,000 was purchased from Fluka, Buchs, Switzerland. 3 g of methotrexate are dissolved in 10 ml of a mixture of formamide s N,N-dimethylformamide : CH2Cl2 (10 s 9 s 1), and a solution of 2.9 g of DCC with 1.8 g of N,N-dimethylaminoE~yridine (DMAP) in 5 ml of solvent mixture (as described above) is added thereto. After stirring briefly, a solution of 3.2 g of dextran 40,000 in 20 ml of the same solvent mixture is added, and the mixture is stirred at RT with exclusion of light for 20 h. After a precipitate has been filtered off, the crude product is precipitated by pouring into 350 ml of . , , ,~
.' ~ .
, . ,~
~....... . . ~
.. .
.
ll ~
- -scetone and washed with scetone and then dried.
The solid is taken up in 30 ml of H20 and poured into a PD10 gel permeation chromatography coluimn and eluted with H20. The high molecular weight fraction i8 freeze-dried and analyzed as described in Example 1.
Yield: 3 g of polymer-bound methotrexate Oeeupaney: 8.5% by weight methotrexate;
methotrexate reeovery by reprecipitation.
B~ample 4s Preparation of a poly-methotrexate-(,~-(2-hydroxyethyl)-D,L-aspartamide/polysuc-cinimide) ester 10 g (103 mmol) of polyanhydroaspartic acid (polysuc-cinimide) are eonverted only partially with 1.83 g (30 mmol) of 2-aminoethanol into poly-,~-(2-hydroxy-ethyl)-D,L-aspartamide. The polyanhydroa6partic acid-co-~,~-(2-hydroxyethyl)-D,L-aspartamide is charaeterized by NMR speetroseopy and contains about 30% hydroxyethyl groups and, in contrast to homo-poly-~,~-(2-hydroxy-ethyl)~D,L-aspartamide which can be clissolved in cold water, is now soluble only in hot water.
The xeaction is carried out in analogy to Example 1, but employing the copolymer in place of poly-~,~-(2-hydroxy-ethyl)-D,L-aspartamide. The amount of polymer employed depends on the poly-,~-(2-hydroxyethyl)-D,L-aspartamide proportion in the copolymer. Polymer eorresponding to 1 mole of OH groups is added for each 1 mole of metho-trexate. The high molecular weight final product from gel chromatography contains 20% by weight bound methotrexate (determined by HPLC). Yield about 20% of theory based on methotrexate employec!l.
. .
,.,~,,,:
.,~
~' .~ t', ; ' .' E~ample 5s Preparation of a poly-methotrexate-inulin ester The reaction iB carried out in anslogy to Example 1 but employing inulin (Fluka AG, Buchs, Switzerland) in place of poly-~,~-(2-hydroxyethyl)-D,L-aspartamide. An ~mount of inulin corresponding to 1 mole of monomer units iB
added for 1 mole of methotrexate. The high molecular weight final product from gel chromatography contains 68~
by weight bound methotrexate. Yield about 70% of theory based on methotrexate employed.
~a~ple 6: Preparation of a poly-methotrexate-poly-[(2-hydroxyethyl-~mido)-lysine fumaramide]
ester using carbonyldiimidazole The polymer is prepared in analogy to German Offen-legungsschrift 3,707,369 ~xample 2, employing (2-hydroxy-ethyl)amidolysine in place of lysine methyl ester.
3 g of methotrexate are dissolved in 15 ml of the solvent mixture described in Examples 1 to 3, and a solution of 2.39 g of carbonyldiimidazole and 0.29 g of pyrrolidino-pyridine in 5 ml of the solvent mixture are added. After stirring at RT for 2 h, a solution of 3.3 g of the polymer in 20 ml of the solvent mixture is added, and the mixture is stirred at RT with exclusion of light for 20 h. Precipitation is then carried out by pouring into 300 ml of acetone, and the residue i8 washed with acetone and dried. The solid is dissolved in 25 ml of H20 and separated into high and low molecular weight fractions on a PD-10 gel chromatography column. The dried high molecu-lar weight fraction is analyzed as described in ~xample 1.
Yield: 3.1 g of polymer-bound methotrexate occupancys 19.1~ by weight methotrexate;
methotrexate recovery by reprecipitation.
.; . , :.,~. ~ , . . ..
''~,7, '`' ' '" ' ' , ' "
Zl)09~;95 ~nmple 7s Preparation of apoly-methotrexate-(water-soluble starch) ester using carbonyl-diimidazole Various commercially available water-soluble starches were employed. 3 g of methotrexate are dissolved in 15 ml of the solvent mixture described in ~xamples 1 to 3, and a solution of 2.39 g of carbonyldiimidazole and 0.29 g of pyrrolidinopyridine in 5 ml of the same mixture is added.
After stirring at RT for 2 h, 3.2 g of water-~oluble starch fraction in 30 ml of the ~olvent mixture are added, and the reaction solution iB stirred at RT with exclusion of light for 20 h. The crude product is precipitated by pouring into 300 ml of acetone and, after washing with acetone, dried. ~he residue is taken up in 30 ml of H20 and sub~ected to ultrafiltration. The high molecular weight phase (retentate, membrane exclusion limit = 5000) is freeze-dried and analyzed as described in Example 1.
Yields 3 g of polymer-bound methotrexate Occupancys 8% by weight methotrexate;
methotrexate recovery by reprecipitation.
~ample 8s In vitro action of methotrexate-dextran ester on tumor cell lines.
Proliferation test (methotrexate reduction) .
L1210, A 549 or HT 29 in the exponential phase of growth are incubated in a cell density of 5 x 103 cells/ml in "Rosswell Park Memorial Institute" (RPMI) 1640 medium in a microtiter plate with 96 wells with various concentra-tions of the test substance at 37~C, 5% CO2 and 95%
relative humidity for 72 hours. Control experiments receive merely growth medium in place of test substance.
Quadruplicate determinations are set up for each test substance and for the control. After incubation for 65 hours, 5 ~1 of a methotrexate solution (2.5 mg/ml in phosphate-buffered saline solution) are added. In the .. : . , ~;, , , . , -: ' . ,..;.
presence of live cells, methotrexate is reduced to a dark red insoluble formazan dyestuff. This reaction is com-plete after 7 hours (L1210 cells) or after 24 hours (A
549, HT 29 cells), and the supernatant medium i8 careful-ly aspirated off. The insoluble dyestuff is dissolved byadding 100 ~1 of DMSO, and the extinction of the result-ing solution is subsequently measured for each well at a wavelength of 492 nm in a Multiscan Photometer 340 CC
from Flow.
The ratio of the extinctions of treated and untreated cells yield~ a dose-effect plot from which the concentra-tion which kills ~ust 50% of the cells (IC50) can be resd off. The coe~fficient of variation is less than 15% for repeat experiments.
Table 1 Substance Cell IC50 (ug/ml) .
L1210 0.01 Methotrexate (MTX) HT 29 0.008 A 549 0.01 L1219 0.13 NTX-dextran ester HT 29 0.41 A 549 0.58 , ~sample 9: In vivo activity on L1210 leukemia in mice Obtaining tumors:
Ascites fluid is removed under sterile conditions from DBA2 mice (female, 18 to 20 g) 7 days after tumor implan-tation. The ascites fluid is washed three times with PBS
(phosphate-buffered saline), counted and subsequently diluted in PBS to a final concentration of 10~ cells per 0.2 ml.
i ..,"-- .: - ": .
,., . ~ .
^ f`
~"~ :
- 15 - 2(10~
lO~ cells in 0.2 ml of PBS are administered intraperi-toneally to DBA2 mice (female, 18 to 20 g). This transfer is repeated once a wee~.
Determination of the antitumor effect:
105 cells from the ascites fluid in 0.2 ml of PBS are administered intraperitoneally to BDF1 mice ~female, 18 to 20 g). 6 animals are employed for each substance concentration and for the control.
a) The animals are weighed on day l and day 5 after the tumor cell implantation. A lo88 of weight of more than 20% on day 5 is used as indicator of a toxic effect of the substance.
b) At the end of the experiment (death of all animals or day 60 reached), the median survival time of the treated groups i8 determined as long as--the latter contained 65%
surviving animals on day 5. The median survival time iB
determined in accordance with the formulas median survival time (MST) = (X + Y) In this formula, X is the earliest day on which the number of surviving animals is N/2, and Y is the earliest day on which the number of surviving animals is (N/2)-l.
In the case where N is an odd number, the median survival time corresponds to the time X.
The median ~urvival 1ime is determined only for animals dying during the course of the experiment. Cured animals (long-time survivors, LTS) are excluded from the deter-mination of the median survival time and are listed separately.
The antitumor effect tumor/control (T/C) is determined from the median survival time of thé treated groups Z(~OX95 (MSTs~or) and control groups (~STCon~rol) in accordance with the formula MST~
T/C % x 100 MSTC
T/C values of more than 125~ are regarded as an indicator of a significant antitumor activity of the test compound.
The doses which bring about the greatest antitumor effect in each case (optimal dosage) are listed in Tab. 2.
Animals still alive on day 60 are regarded as cured (LTS).
Resultss See Table 2 Discussions It is evident from these results that the esterified methotrexate polymers are, with a single i.p. administra-tion, superior to pure methotrexate, which indicates a slow release action of the polymers.
~"" '~ ""':
. ~ ~ ,.................. ..
.
;: ~
- 17 - Z~)(~9~;95 U I N¦ Ir~
C dP N U:l It~ ~r ~ CID
_I C~ 0~ N ~0 ON 1`
~-~3~ ~ o ~ 3 ~ ~ X
N ~ ~ $ j ¦ $ " ~ ~ --~ N ~ ~3
3,026,575).
10 - Polymer-bound cytostatics on functional amide side-groups (WO 875,031, EP 0,111,388, D~ 3,539,951, ~O
8,700,056, EP 0,190,464, US 4,551,502, JP
57/143,325, EP 0,040,506 and JP 57/143,326).
Although the liberation of these cytostatics from the linkage with the polymer can in theory take place on endocytosis of the complete con~ugate in the acid pH of the lysosome, it has been shown in practice that endo-cytosis takQs place to only a small extent even in the case of polymer con~ugations with target-~pecific modifi-cations (for example antibody con~ugates). Purthermore, ma~or problems arise from the introduction of spacers, the frequently low loading density and difficult syn-theses. The in vivo results with such con~uqates to date have therefore been disappointing.
Phase-specific cytostatics, for example methotrexate, represent a special case because they act only in a particular phase of the cell cycle (synthesis phase in the case of the dihydrofolate reductase inhibitor metho-trexate). Given the relatively short biological half-life of methotrexate, a single administration frequently has no antitumor action whatever. A therapeutic effect is shown only by multiple individual administrations with very high doses. A ~rescue therapy~ with antagonists is frequently necessary in order subsequently to avoid, with a time lag, too much damage in the healthy cells.
~5 A polymer-bound form of methotrexate i8 of particular interest because of the~e problems. However, no satisfac-tory solution has to date been possible by b$nding methotrexate to poly-L-lysine, poly-lactide-glycolide (PLG) derivatives etc. (see W0 875,031).
Surprisingly, very good in vitro and in vivo properties with relevance for human therapy have now been achieved by binding methotrexate to water-soluble, biocompatible polymers carrying hydroxyl groups.
Hence the invention relates to~
1. A polymer-bound methotrexate or methotrexate deriva-tives, having the following features - an ester linkage of the ~- and/or 7-carboxyl group of the methotrexate or of the methotrexate - 15 derivatives with hydroxyl groups of water-soluble biocompatible polymers;
- bringing about a prolongation of life of more than 125~ compared with untreated controls in mice with L1210 leukemia on a ~ingle intraperi-toneal administration of 60 mg of methotrexate or methotrexate derivative, which is bound to the abovementioned polymer, per kg of body weight.
2. A proce~s for the preparation of the polymer-bound methotrexate with the features specified in 1. or of the polymer-bound methotrexate derivatives, which comprises the methotrexate which carries ~- and/or 7-carboxyl groups, or the corresponding derivatives of methotrexate, being reacted in the presence of water-abstracting coupling reagents with a water-soluble biocompatible polymer carrying hydroxyl groups.
3. The use of the polymer-bound methotrexate with the features specified in 1., or of the polymer-bound methotrexate derivatives, as tumor therapeutic.
. . .. . - ,: .
~ 4 ~
The invention is described in detail hereinafter, e~pe-cially in its preferred embodiments. The lnvention 18 furthermore defined by the content~ of the clalm~.
~ethotrexateorN-t4-[[(2,4-diamino-6-pteridinyl)methyl]-methylamino]benzoyll-glutamic acid is de~cribed a~ com-pound with its essential characteristics in the article by A.R. Chamberlain et al. in Analytical Profiles of Drug Substances, volume 5, edited by ~. ~lorey, Academic Pre~s, New York (1976), pages 283-306. The preparation of the compound is described by Seeger et al. tJ. Am. Soc.
11, 1753 (1949) or Rahmann et al., Medic. Res. Rev., 8, 95 (1988)].
The compounds which can be employed as methotrexate derivatives or analogs are those which have been modified on the pteridine ring or in the bridge region, a~ well as those which have been derivatized on the aromatic ring or in the glutamic acid iety. However, an e~ential condition is that at least one carboxyl group which is neces~ary for linkage to the polymer is retained. Analogs of thi~ type and processes for the prep~ration thereof are de~cribed in detail in the article by Rahoann et al.
(~ee above). Particularly preferred derivatives which can be u~ed are methopterin (~erck Index 10 (1983) 5860) and aminopterin (Merck Index 10 (1983) 477).
The said compounds can be bound via an e~ter linkage to water-soluble, biocompntible polymers carrying hydroxyl groups. By biocompatible polymers are eant compounds which are physiologically tolerated and can be degraded and/or excreted in the body. Polymers which can prefe-rably be u~ed are those in which the proportion of ionizable groups before the loading with the active substance is below 10 mol-~.
Preferably employed are water-soluble starches with a mean molecular weight of 1000 to 200,000, preferably 5000 to 50,000, which can al~o be dified. Also used are - s -cellulose acetates, especially water-soluble cellulose acetate from the company (Celanese/WSCA)~ or destrans, with a mean molecular weight between 1000 and 200,000, e~pecially with a mean molecular weight of 40,000 to 70,000, or inulin.
It is also possible to use appropriate synthetic poly-mers. One esample is poly-~,~-(hydroxyethyl)-D,L-spartamide of the fon~ula I
C - NN~ C -CH2~ ~ - CH~
r ~ C~
(CH2)2 (CIH2)2 CH2OH ~ CH2OH n in which the ratio of m to n is in the range 0-1 to 1-0, preferably 0.7s0.3 to 0.95s0.05. The molecular weight of compounds.of this type is appro~imately between 2000 and 100,000, preferably between 5000 and 50,000. Appropriate compounds and procesffe~ for the preparation thereof are described in detail in German Offenlegungsschr.ift 3,700,128.
Another example of a ~ynthetic polymer is Am~dated polylysine fumaramide/polylys~ne glutaramide of the formula II
:: : : ....... .. . ....
O O
Il 11 -(-NH-(CH2)~ - CH - NH-C-X-C-) D- II
C'O
(CH2)~OH
in which X can be -(CH2)3 or -CH-CH-, m can be the numbers 1 to 10, preferably 2, and n can be the number 5 to 2000.
~hese polymers have a molecular weight between 1000 and 300,000. Polymers of this type and processes for the preparation thereof are described in German Offenlegungs-schrift 3,707,369.
A further example is 8 copolymer of the formula IIIcomposed of poly-~,~-(2-hydroxyethyl)-D,L-aspartamide (compound of the formula I) and polysuccinimide (formul~ I) ~ ~ III
in which the ratio of y to z is in the range from about O.99sO.Ol to O.OlsO.99, preferably 2.5s7.5 to 7.Ss2.5.
Particularly preferably employed as polymers in the process according to the invention are the synthetic compounds depicted in formulae I, II and III.
, 2S For the prepsration o:E the polymer-bound methotrexate or of the corresponding derivatives, the active substance is I dissolved, for example in water, dimethyl sulfoxide ; (DMSO), form~mide, N,N-dimethylformamide or methylene chloride or a mixture of the last three solvents. The appropriate polymer is added to the same solvent which has also been u~ed to dissolve the active substance. ~he '' .
.' r' .. ,;~ , .
two mixtures are combined and can be incubated in the presence of a water-abstracting coupling reagent, where appropriate with exclusion of light, at a pH in the range from 7 to 9, preferably 8 to 8.S, and at a temperature of S 0 to 100C, preferably 20 to 30C, for a period of nbout 1 to 29 hours, preferably with stirring.
It iB poBBible to use as water-abstracting coupling reagent carbodiimides, alkylphosphonic anhydrides, carbonyldi~mines etc. Carbonyldiimidazole and dicyclo-hexylcarbodiimide are particularly preferably used.
The resulting crude product can be purified by precipita-tion with a ~olvent in which the polymer is insoluble. It is possible to use for this purpose, for example, tetra-hydrofuran, acetone, dioxane and alcohols. Further lS purification can take place using methods for molecular weight partition such as, for example, ultrafiltration, dialysis and gel permeation.
The process according to the invention results in a polymer-bound methotrexate product with a loading of methotrexate or analogs thereof from 1 to 85%, preferably lS to 7S%, based on the weight of the polymer-bound product. The product furthermore has an extinction coefficient between 0.0001 l/mg and 0.05 l/mg, preferably between 0.005 l/mg and 0.03 l/mg, in aqueous solution at pH 7 to 8.5 and a wavelength of 302 nm for methotrexate and methopterin, or an extinction coefficient of 0.0005 l/mg to 0.04 l/mg at the said pH and a wavelength of 282 nm for aminopterin.
The active substance can be slowly released from the polymer-bound methotrexates according to the invention into the body by simple hydrolysis, in contrast to the stronger binding via functional amide groups, from which the active substance can be liberated only by enzymatic cleavage.
- -5i:
: ~ . :
:i i' .~
. ; ~ :
~,~ : - , , ' ~
. ~
- 8 - ~ ~
With these polymer-methotrexate con~ugates according to the invention in vivo, for example in mice with L1210 leukemia, prolongations of life of > 125%, preferably > 150~, compared with untreated controls are achieved even with a single administration ~intraperitoneal i.p.) of 60 mg~kg of body weight methotrexate or derivatives which are linked according to the invention to the water-soluble, biocompatible polymer carrying hydroxyl groups (equivalent of methotrexate or derivative). These figures were determined as described in Example 7. Methotrexate or the derivatives are not themselves active under the said conditions. With 330 mg/kg equivalent of metho-trexate or derivative it was possible to observe complete remission without relapse up to the termination of the experiment a~ter 60 days in 2 of 5 animals. This dose is itself above the LD50 of free methotrexate.
In addition, the polymer-methotrexate con~ugates accord-ing to the invention have a higher IC50 in vitro than free methotrexate (for example on L1210, HT 29, A549 cells).
In con~unction with the in vivo results described sbove, this is to be regarded as evidence of a desired slow liberation of methotrexate.
The invention is illustrated by means of examples herein-after. Unless indicated otherwise, percentage data relate to weight.
Fsample 1: Preparation of a poly-methotrexate-poly-~,0-(2-hydroxyethyl)-D,L-aspartamide ester using carbonyldiimidazole Methotrexate was purchased from Sigma for all the examples. The polymer poly-~,0-(2-hydroxyethyl)-D,L-aspartamide is prepared by the method of P. Neri, G.
Antoni, F. aenvenuti, F. Cocola, G. Gazzei, J. Med. Chem.
16, 893 (1973).
. r~ ~
. .
. ~.
~ ~, ., ~ . :
3 g of methotrexate are dissolved in 15 ml of a mixture of formamide s N,N-dimethylformamide : CH2Cl2 (10 : 9 s 1). To this is added a solution of 300 mg of pyrrolidinopyridine and 2.44 g of carbonyldiimidazole in 5 ml of the above solvent mixture. The reaction mixture is stirred at room temperature (RT) for 1.5 h. At the same time, 3.1 g of poly-~,~-(2-hydroxyethyl)-D,$-aspar-tamide (PHEA) are dissolved in 9 ml of the solvent mixture described and stirred at RT. The two solutions are combined and stirred at RT with exclusion of light for 20 h. The crude product is precipitated by pouring into 250 ml of acetone. The yellow precipitate is sepa-rated off, washed with acetone, dried and then taken up in 25 ml of aqueous NaHC03 solution (pH 8-9).
The amber-colored aqueous solution is poured onto a Sephadex gel chromatography column (PD10, Pharmacia) and separated into a low molecular weight and a high molecu-lar weight fraction by eluti~n with water. The high molecular weight fraction is freeze-dried. The metho-trexate content is determined by UV spectroscopy at~ - 302 nm in aqueous solution. The product is charac-terized by lH NNR, CHN analysis and thin-layer chroma-tography. The lH NMR in D20 corresponds to a sum of the spectra of methotrexate and PHEA, it being possible to establish the degree of occupancy from the integral ratios. The degree of occupancy of 22~ by weight metho-trexate measured by HPLC corre8pond8 to the result of the W determination. The coupling reagents migrate, whereas methotrexate and product remain at Rf = 0, in the TLC in diethyl ether. The presence of free methotrexate could be ruled out by ultrafiltration with m~mhranes with various exclusion limits.
I
Yield: 3 g of polymer-bound methotrexate (about 20% of theory based on methotrexate). Most of the unreacted methotrexate is recovered by reprecipitation from the low molecular weight fraction.
., .
i .
.. ~, ~. . .
' ~ .
:,...
. . .
,.. ~. :
,:x. : :
- 10 - ~09695 Xxample 2s Preparation of a poly-methotrexate-dextran (40,000) ester usinq carbonyldiimidazole Dextran 40,000 was purchased from Fluka, Buchs, Switzerland. 3 g of methotrexate are dissolved in 10 ml of a mixture of formamide : N,N-dimethylformamide s CH2Cl2 ( 10 S 9 5 1), and 2.39 g of carbonyldiimidazole and 0.29 g of pyrrolidinopyridine are mixed in 5 ml of the ~bove solvent mixture and stirred at RT for 2 h. Then a solu-tion of 3.2 g of dextran 40,000 in 20 ml of the same solvent mixture is added thereto, and the mixture is stirred at RT with exclusion of light for 20 h. The crude product i8 precipitated in 350 ml of dry acetone, washed with acetone and dried.
The solid is taken up in 25 ml of H20 and poured onto a PD10 gel chromatography column. Elution with H20 yields a high and a low molecular weight fraction. The high molecular weight fraction is freeze-dried and analyzed as described in Example 1. Unreacted methotrexate can be recovered by reprecipitation.
Yields 3 g of polymer-bound methotrexate Occupancys 17.3% by weight methotrexate F~ample 3s Preparation of apoly-methotrexate-dextran (40,000) ester using dicyclohexylcar-bodiimide (DCC) Dextran 40,000 was purchased from Fluka, Buchs, Switzerland. 3 g of methotrexate are dissolved in 10 ml of a mixture of formamide s N,N-dimethylformamide : CH2Cl2 (10 s 9 s 1), and a solution of 2.9 g of DCC with 1.8 g of N,N-dimethylaminoE~yridine (DMAP) in 5 ml of solvent mixture (as described above) is added thereto. After stirring briefly, a solution of 3.2 g of dextran 40,000 in 20 ml of the same solvent mixture is added, and the mixture is stirred at RT with exclusion of light for 20 h. After a precipitate has been filtered off, the crude product is precipitated by pouring into 350 ml of . , , ,~
.' ~ .
, . ,~
~....... . . ~
.. .
.
ll ~
- -scetone and washed with scetone and then dried.
The solid is taken up in 30 ml of H20 and poured into a PD10 gel permeation chromatography coluimn and eluted with H20. The high molecular weight fraction i8 freeze-dried and analyzed as described in Example 1.
Yield: 3 g of polymer-bound methotrexate Oeeupaney: 8.5% by weight methotrexate;
methotrexate reeovery by reprecipitation.
B~ample 4s Preparation of a poly-methotrexate-(,~-(2-hydroxyethyl)-D,L-aspartamide/polysuc-cinimide) ester 10 g (103 mmol) of polyanhydroaspartic acid (polysuc-cinimide) are eonverted only partially with 1.83 g (30 mmol) of 2-aminoethanol into poly-,~-(2-hydroxy-ethyl)-D,L-aspartamide. The polyanhydroa6partic acid-co-~,~-(2-hydroxyethyl)-D,L-aspartamide is charaeterized by NMR speetroseopy and contains about 30% hydroxyethyl groups and, in contrast to homo-poly-~,~-(2-hydroxy-ethyl)~D,L-aspartamide which can be clissolved in cold water, is now soluble only in hot water.
The xeaction is carried out in analogy to Example 1, but employing the copolymer in place of poly-~,~-(2-hydroxy-ethyl)-D,L-aspartamide. The amount of polymer employed depends on the poly-,~-(2-hydroxyethyl)-D,L-aspartamide proportion in the copolymer. Polymer eorresponding to 1 mole of OH groups is added for each 1 mole of metho-trexate. The high molecular weight final product from gel chromatography contains 20% by weight bound methotrexate (determined by HPLC). Yield about 20% of theory based on methotrexate employec!l.
. .
,.,~,,,:
.,~
~' .~ t', ; ' .' E~ample 5s Preparation of a poly-methotrexate-inulin ester The reaction iB carried out in anslogy to Example 1 but employing inulin (Fluka AG, Buchs, Switzerland) in place of poly-~,~-(2-hydroxyethyl)-D,L-aspartamide. An ~mount of inulin corresponding to 1 mole of monomer units iB
added for 1 mole of methotrexate. The high molecular weight final product from gel chromatography contains 68~
by weight bound methotrexate. Yield about 70% of theory based on methotrexate employed.
~a~ple 6: Preparation of a poly-methotrexate-poly-[(2-hydroxyethyl-~mido)-lysine fumaramide]
ester using carbonyldiimidazole The polymer is prepared in analogy to German Offen-legungsschrift 3,707,369 ~xample 2, employing (2-hydroxy-ethyl)amidolysine in place of lysine methyl ester.
3 g of methotrexate are dissolved in 15 ml of the solvent mixture described in Examples 1 to 3, and a solution of 2.39 g of carbonyldiimidazole and 0.29 g of pyrrolidino-pyridine in 5 ml of the solvent mixture are added. After stirring at RT for 2 h, a solution of 3.3 g of the polymer in 20 ml of the solvent mixture is added, and the mixture is stirred at RT with exclusion of light for 20 h. Precipitation is then carried out by pouring into 300 ml of acetone, and the residue i8 washed with acetone and dried. The solid is dissolved in 25 ml of H20 and separated into high and low molecular weight fractions on a PD-10 gel chromatography column. The dried high molecu-lar weight fraction is analyzed as described in ~xample 1.
Yield: 3.1 g of polymer-bound methotrexate occupancys 19.1~ by weight methotrexate;
methotrexate recovery by reprecipitation.
.; . , :.,~. ~ , . . ..
''~,7, '`' ' '" ' ' , ' "
Zl)09~;95 ~nmple 7s Preparation of apoly-methotrexate-(water-soluble starch) ester using carbonyl-diimidazole Various commercially available water-soluble starches were employed. 3 g of methotrexate are dissolved in 15 ml of the solvent mixture described in ~xamples 1 to 3, and a solution of 2.39 g of carbonyldiimidazole and 0.29 g of pyrrolidinopyridine in 5 ml of the same mixture is added.
After stirring at RT for 2 h, 3.2 g of water-~oluble starch fraction in 30 ml of the ~olvent mixture are added, and the reaction solution iB stirred at RT with exclusion of light for 20 h. The crude product is precipitated by pouring into 300 ml of acetone and, after washing with acetone, dried. ~he residue is taken up in 30 ml of H20 and sub~ected to ultrafiltration. The high molecular weight phase (retentate, membrane exclusion limit = 5000) is freeze-dried and analyzed as described in Example 1.
Yields 3 g of polymer-bound methotrexate Occupancys 8% by weight methotrexate;
methotrexate recovery by reprecipitation.
~ample 8s In vitro action of methotrexate-dextran ester on tumor cell lines.
Proliferation test (methotrexate reduction) .
L1210, A 549 or HT 29 in the exponential phase of growth are incubated in a cell density of 5 x 103 cells/ml in "Rosswell Park Memorial Institute" (RPMI) 1640 medium in a microtiter plate with 96 wells with various concentra-tions of the test substance at 37~C, 5% CO2 and 95%
relative humidity for 72 hours. Control experiments receive merely growth medium in place of test substance.
Quadruplicate determinations are set up for each test substance and for the control. After incubation for 65 hours, 5 ~1 of a methotrexate solution (2.5 mg/ml in phosphate-buffered saline solution) are added. In the .. : . , ~;, , , . , -: ' . ,..;.
presence of live cells, methotrexate is reduced to a dark red insoluble formazan dyestuff. This reaction is com-plete after 7 hours (L1210 cells) or after 24 hours (A
549, HT 29 cells), and the supernatant medium i8 careful-ly aspirated off. The insoluble dyestuff is dissolved byadding 100 ~1 of DMSO, and the extinction of the result-ing solution is subsequently measured for each well at a wavelength of 492 nm in a Multiscan Photometer 340 CC
from Flow.
The ratio of the extinctions of treated and untreated cells yield~ a dose-effect plot from which the concentra-tion which kills ~ust 50% of the cells (IC50) can be resd off. The coe~fficient of variation is less than 15% for repeat experiments.
Table 1 Substance Cell IC50 (ug/ml) .
L1210 0.01 Methotrexate (MTX) HT 29 0.008 A 549 0.01 L1219 0.13 NTX-dextran ester HT 29 0.41 A 549 0.58 , ~sample 9: In vivo activity on L1210 leukemia in mice Obtaining tumors:
Ascites fluid is removed under sterile conditions from DBA2 mice (female, 18 to 20 g) 7 days after tumor implan-tation. The ascites fluid is washed three times with PBS
(phosphate-buffered saline), counted and subsequently diluted in PBS to a final concentration of 10~ cells per 0.2 ml.
i ..,"-- .: - ": .
,., . ~ .
^ f`
~"~ :
- 15 - 2(10~
lO~ cells in 0.2 ml of PBS are administered intraperi-toneally to DBA2 mice (female, 18 to 20 g). This transfer is repeated once a wee~.
Determination of the antitumor effect:
105 cells from the ascites fluid in 0.2 ml of PBS are administered intraperitoneally to BDF1 mice ~female, 18 to 20 g). 6 animals are employed for each substance concentration and for the control.
a) The animals are weighed on day l and day 5 after the tumor cell implantation. A lo88 of weight of more than 20% on day 5 is used as indicator of a toxic effect of the substance.
b) At the end of the experiment (death of all animals or day 60 reached), the median survival time of the treated groups i8 determined as long as--the latter contained 65%
surviving animals on day 5. The median survival time iB
determined in accordance with the formulas median survival time (MST) = (X + Y) In this formula, X is the earliest day on which the number of surviving animals is N/2, and Y is the earliest day on which the number of surviving animals is (N/2)-l.
In the case where N is an odd number, the median survival time corresponds to the time X.
The median ~urvival 1ime is determined only for animals dying during the course of the experiment. Cured animals (long-time survivors, LTS) are excluded from the deter-mination of the median survival time and are listed separately.
The antitumor effect tumor/control (T/C) is determined from the median survival time of thé treated groups Z(~OX95 (MSTs~or) and control groups (~STCon~rol) in accordance with the formula MST~
T/C % x 100 MSTC
T/C values of more than 125~ are regarded as an indicator of a significant antitumor activity of the test compound.
The doses which bring about the greatest antitumor effect in each case (optimal dosage) are listed in Tab. 2.
Animals still alive on day 60 are regarded as cured (LTS).
Resultss See Table 2 Discussions It is evident from these results that the esterified methotrexate polymers are, with a single i.p. administra-tion, superior to pure methotrexate, which indicates a slow release action of the polymers.
~"" '~ ""':
. ~ ~ ,.................. ..
.
;: ~
- 17 - Z~)(~9~;95 U I N¦ Ir~
C dP N U:l It~ ~r ~ CID
_I C~ 0~ N ~0 ON 1`
~-~3~ ~ o ~ 3 ~ ~ X
N ~ ~ $ j ¦ $ " ~ ~ --~ N ~ ~3
Claims (9)
1. A polymer-bound methotrexate or methotrexate derivatives having the following features - an ester linkage of the .alpha.- and/or .gamma.-carboxyl group of the methotrexate or of the methotrexate deriva-tives with hydroxyl groups of water-soluble biocompatible polymers - occupancy of 1 to 85% methotrexate or methotrexate derivatives - bringing about a prolongation of life of more than 125% compared with untreated controls in mice with L1210 leukemia on a single intraperitoneal adminis-tration of 60 mg of methotrexate or methotrexate derivative, which is bound to the abovementioned polymer, per kg of body weight.
2. A compound as claimed in claim 1, wherein the polymer has a proportion of ionizable groups below 10 mol-% before the loading.
3. A compound as claimed in claim 1 or 2, wherein the polymer is a) a poly-.alpha.,.beta.-(2-hydroxyethyl)-D,L-aspartamide of the formula I
I
in which the ratio of m to n is 0:1 to 1:0, with a molecular weight of 2000 to 100,000, b) a polylysine fumaramide or glutaramide of the formula II
II
in which X is -(CH2)3- or -CH=CH-, m is a number from 1 to 10 and n is a number from 5 to 2000, with a molecular weight between 1000 and 300,000 c) a poly-.alpha.,.beta.-(2-hydroxyethyl)-D,L-aspartamide/
polysuccinimide of the formula III
III
in which the ratio of y to z is in the range from about 0.99:0.01 to 0.01:0.99.
d) a dextran with a mean molecular weight between 1000 and 200,000 e) a starch fraction with a mean molecular weight between 1000 and 200,000 or f) inulin.
I
in which the ratio of m to n is 0:1 to 1:0, with a molecular weight of 2000 to 100,000, b) a polylysine fumaramide or glutaramide of the formula II
II
in which X is -(CH2)3- or -CH=CH-, m is a number from 1 to 10 and n is a number from 5 to 2000, with a molecular weight between 1000 and 300,000 c) a poly-.alpha.,.beta.-(2-hydroxyethyl)-D,L-aspartamide/
polysuccinimide of the formula III
III
in which the ratio of y to z is in the range from about 0.99:0.01 to 0.01:0.99.
d) a dextran with a mean molecular weight between 1000 and 200,000 e) a starch fraction with a mean molecular weight between 1000 and 200,000 or f) inulin.
4. A process for the preparation of the polymer-bound methotrexate or of the polymer-bound methotrexate deriva-tives as claimed in claim 1, which comprises the metho-trexate which carries .alpha.- and/or .gamma.-carboxyl groups, or the corresponding derivatives of methotrexate, being reacted in the presence of water-abstracting coupling reagents with a water-soluble biocompatible polymer carrying hydroxyl groups.
5. The process as claimed in claim 4, wherein the polymer employed is a) a poly-.alpha.,.beta.-(2-hydroxyl)-D,L-aspartamide of the formula I
I
in which the ratio of m to n is 0:1 to 1:0, with a molecular weight of 2000 to 100,000, b) a polylysine fumaramide or glutaramide of the formula II
II
in which X is -(CH2)3- or -CH=CH-, m is a number from 1 to 10 and n is a number from 5 to 1000, with a molecular weight between 1000 and 300,000 c) a poly-.alpha.,.beta.-(2-hydroxyethyl)-D,L-aspartamide/
polysuccinimide of the formula III
III
in which the ratio of y to z is in the range from about 0.99:0.01 to 0.01:0.99.
d) a dextran with a mean molecular weight between 1000 and 200,000 e) a starch fraction with a mean molecular weight between 1000 and 200,000 or f) inulin.
I
in which the ratio of m to n is 0:1 to 1:0, with a molecular weight of 2000 to 100,000, b) a polylysine fumaramide or glutaramide of the formula II
II
in which X is -(CH2)3- or -CH=CH-, m is a number from 1 to 10 and n is a number from 5 to 1000, with a molecular weight between 1000 and 300,000 c) a poly-.alpha.,.beta.-(2-hydroxyethyl)-D,L-aspartamide/
polysuccinimide of the formula III
III
in which the ratio of y to z is in the range from about 0.99:0.01 to 0.01:0.99.
d) a dextran with a mean molecular weight between 1000 and 200,000 e) a starch fraction with a mean molecular weight between 1000 and 200,000 or f) inulin.
6. The process as claimed in claim 4 or 5, wherein carbonyl-diimidazole or dicyclohexylcar,bDdiimide is employed as coupling reagent.
7. The process as claimed in one or more of claims 4 to 6, wherein the reaction takes place at a pH of 7 to 9 and a temperature of 0 to 100°C.
8. The use of the polymer-bound methotrexate or of the polymer-bound methotrexate derivatives as claimed in claim 1 as tumor therapeutic.
9. The polymer-bound methotrexate or methotrexate derivatives as claimed in claim 1, and substantially as described herein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3904119.0 | 1989-02-11 | ||
| DE3904119A DE3904119A1 (en) | 1989-02-11 | 1989-02-11 | POLYMER FIXED METHOTREXATE, METHOD FOR PRODUCTION AND USE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2009695A1 true CA2009695A1 (en) | 1990-08-11 |
Family
ID=6373882
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002009695A Abandoned CA2009695A1 (en) | 1989-02-11 | 1990-02-09 | Polymer-bound methotrexate, a process for its preparation and its use |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0383170A3 (en) |
| JP (1) | JPH02245028A (en) |
| CA (1) | CA2009695A1 (en) |
| DE (1) | DE3904119A1 (en) |
| HU (1) | HU900748D0 (en) |
| PT (1) | PT93104A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6844328B2 (en) | 2000-03-17 | 2005-01-18 | Eurand Pharmaceuticals Ltd. | Polysaccharidic esters of N-derivatives of glutamic acid |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09501655A (en) | 1993-08-02 | 1997-02-18 | コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガニゼーション | Therapeutic compound-fatty acid conjugate |
| NZ298712A (en) * | 1995-01-16 | 1998-12-23 | Commw Scient Ind Res Org | Fatty acid acyl group conjugates as therapeutic compounds |
| WO1998009955A1 (en) * | 1996-09-06 | 1998-03-12 | Eli Lilly And Company | Process and novel intermediates |
| CN1095472C (en) | 2000-04-17 | 2002-12-04 | 上海复康医药科技发展有限公司 | Folica acid-polysaccharide composite and its preparation and medical composition with the composite as active component |
| CN112094320B (en) * | 2019-06-18 | 2022-08-02 | 首都医科大学 | His-Gly-Glu modified methotrexate, synthesis, antitumor activity and application thereof |
| CN112094322B (en) * | 2019-06-18 | 2022-04-22 | 首都医科大学 | His-Gly-Lys modified methotrexate, synthesis, antitumor activity and application thereof |
| CN112094319B (en) * | 2019-06-18 | 2022-08-02 | 首都医科大学 | Glu-Asp-Gly modified methotrexate, synthesis, anti-transfer activity and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1541436A (en) * | 1976-02-02 | 1979-02-28 | Searle & Co | Immunological materials |
| US4182800A (en) * | 1977-07-16 | 1980-01-08 | Hercules Incorporated | Methotrexate-divinyl ether--maleic anhydride copolymer reaction product |
-
1989
- 1989-02-11 DE DE3904119A patent/DE3904119A1/en not_active Withdrawn
-
1990
- 1990-02-07 EP EP19900102364 patent/EP0383170A3/en not_active Withdrawn
- 1990-02-09 HU HU90748A patent/HU900748D0/en unknown
- 1990-02-09 JP JP2028624A patent/JPH02245028A/en active Pending
- 1990-02-09 CA CA002009695A patent/CA2009695A1/en not_active Abandoned
- 1990-02-09 PT PT93104A patent/PT93104A/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6844328B2 (en) | 2000-03-17 | 2005-01-18 | Eurand Pharmaceuticals Ltd. | Polysaccharidic esters of N-derivatives of glutamic acid |
Also Published As
| Publication number | Publication date |
|---|---|
| PT93104A (en) | 1990-08-31 |
| EP0383170A2 (en) | 1990-08-22 |
| JPH02245028A (en) | 1990-09-28 |
| DE3904119A1 (en) | 1990-08-16 |
| HU900748D0 (en) | 1990-04-28 |
| EP0383170A3 (en) | 1990-12-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0477931B1 (en) | Novel adriamycin derivatives | |
| AU679788B2 (en) | Polymer-bound camptothecin derivatives | |
| HU211291A9 (en) | Polymer-bound paclitaxel derivatives | |
| EP2258397A1 (en) | Polymer conjugate of physiologically active substance | |
| Przybylski et al. | Pharmacologically active polymers, 17. Syntheses and characterization of polymeric derivatives of the antitumor agent methotrexate | |
| US20040192644A1 (en) | DDS compound and method for measurement thereof | |
| WO2011075953A1 (en) | Multi-arm polyethylene glycol derivatives, conjugates and gels of drugs and the same | |
| NZ250375A (en) | Peg hydrazone and peg oxime linkage forming reagents and protein derivatives | |
| US9867884B2 (en) | Vascular delivery systems | |
| CA2009695A1 (en) | Polymer-bound methotrexate, a process for its preparation and its use | |
| US6646120B1 (en) | Dextran derivatives, preparation and medicinal applications | |
| Hirano et al. | Synthesis of antitumor‐active conjugates of Adriamycin or daunomycin with the copolymer of divinyl ether and maleic anhydride | |
| EP0526649B1 (en) | Carboxymethylmannoglucan and derivative thereof | |
| EA007353B1 (en) | Ph-sensitive polymeric conjugates of anthracycline cancerostatic active agents for targeted therapy | |
| JP2002508758A (en) | Biologically active material | |
| US8609860B2 (en) | Acid-labile trigger units | |
| CA2136438C (en) | Anthracycline conjugates, their preparation and their use as antitumor agents | |
| CN114404417B (en) | Dendritic polypeptide-camptothecin drug conjugate and preparation method and application thereof | |
| Tevyashova et al. | New conjugates of antitumor antibiotic doxorubicin with water-soluble galactomannan: Synthesis and biological activity | |
| US4182800A (en) | Methotrexate-divinyl ether--maleic anhydride copolymer reaction product | |
| US5401742A (en) | Polymeric conjugate of methotrexate compound and pyran copolymer and method for the preparation thereof | |
| Munck et al. | Relationship between the intracellular level and growth inhibition of a new anthracycline 4′ O-tetrahydropyranyl-Adriamycin in Friend leukemia cell variants | |
| JPH04288304A (en) | High-molecular mitomycin c derivative and production thereof | |
| Song et al. | Synthesis and antitumor activity of poly (organophosphazene)/doxorubicin conjugates | |
| CN116396365A (en) | Anti-tumor polypeptide THP-24 and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |