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WO1997014413A9 - Derives de chelate utilises comme protecteurs contre les lesions tissulaires - Google Patents

Derives de chelate utilises comme protecteurs contre les lesions tissulaires

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Publication number
WO1997014413A9
WO1997014413A9 PCT/US1996/016722 US9616722W WO9714413A9 WO 1997014413 A9 WO1997014413 A9 WO 1997014413A9 US 9616722 W US9616722 W US 9616722W WO 9714413 A9 WO9714413 A9 WO 9714413A9
Authority
WO
WIPO (PCT)
Prior art keywords
compound
doxorubicin
mpedta
mpeg
edta
Prior art date
Application number
PCT/US1996/016722
Other languages
English (en)
Other versions
WO1997014413A1 (fr
Filing date
Publication date
Application filed filed Critical
Priority to AU74547/96A priority Critical patent/AU7454796A/en
Publication of WO1997014413A1 publication Critical patent/WO1997014413A1/fr
Publication of WO1997014413A9 publication Critical patent/WO1997014413A9/fr

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Definitions

  • the present invention is directed to substances useful in the protection of living organisms against damage due to free radical reactions.
  • Iron is involved in the pathogenesis of free radical tissue injury following inflammation. Activated leukocytes thus produce superoxide radicals which undergo dismutation to hydrogen peroxide and oxygen, and simultaneously reduce iron in ferritin. Fe . in turn, reacts with hydrogen peroxide, producing the destructive hydroxyl radical.
  • the radical reactions may be summarized:
  • Iron chelators including def roxamine (DEF) have shown some effectiveness in reducing tissue injury, such as myocardial ischemia-reperfusion injury.
  • DEF must be given parenterally, but its toxicity and rapid excretion limit its effectiveness.
  • high molecular weight substances such as hydroxyethylstarch, the circulation lifetime was increased and toxicity decreased (Hallaway, et al., Proc . Nat . Acad . Sci . USA,
  • PEG polyethylene glycol
  • MPEG polyethylene glycol
  • the low molecular weight polymers PEG 200-600 are absorbed through the gastrointestinal tract when ingested orally and excreted unchanged in the urine. PEG is absorbed along with water directly through the intestinal mucosal cell membrane.
  • PEG 200-600 being nontoxic and biologically inert, has often been used as a vehicle for administration of drugs insoluble in water. In several investigations, the PEG vehicle alone was empirically found to exhibit significant biological activity, leading to further studies of low molecular weight PEG.
  • PEG 400 when given intraperitoneally (ip) either before or shortly after x- irradiation of mice, conferred significant protection against lethality and morbidity (Shaeffer and Schellenberg, Int . J. Radiat . Oncol . Biol . Phys . , 10:2329, 1984; Shaeffer, et al., Radiat . Res . , 107:125, 1986).
  • PEG 300 ip was shown to reduce the CNS sequelae of experimental concussive brain injury (Clifton, et al., J. Neurotrama , 6:71, 1989).
  • PEG with a molecular weight around 400 is thus a uniquely nontoxic substance that exhibits a protective effect against injury to tissues.
  • PEG with a molecular weight greater than 700 is not absorbed through the GI tract.
  • the mechanism of the protective action of low molecular weight PEG has not been established, but probably involves interaction of PEG with the surface of lipid membranes or protein components. PEG aggregates near cell membranes, reduces water polarity at membrane surfaces, and increases hydrophobic interactions (Hoekstra, et al., J. Biol . Chem . , 264:6786, 1989).
  • MPEG chelates can be effective iron chelators.
  • MPEG can be linked with iminodiacetate terminus (MIDA) .
  • MIAA iminodiacetate terminus
  • MPEG 550- deferoxamine (ferrioxamine) , prepared by reacting MPEG molecular weight 550 with carbonyldiimidazole, followed by reaction of the resulting imidazolecarbonyl ester with deferoxamine base, forming a urethane linkage.
  • the material was produced as a chelate for gadolinium, to be used as a renal magnetic resonance contrast agent (Duewell, et al., Invest . Radiol . , 26:50, 1991).
  • Deferoxamine is known to be an effective chelator for ferric iron.
  • SUBSTITUTESHEET(RULE2S) MIDA can be prepared by converting MPEG 350 to the chloride by reaction with thionyl chloride, and to the iminodiacetate by reaction of the chloride with sodium iminodiacetate ( uenschell et al., J. Chromatog. 543: 345- 354, 1991) .
  • the methyl ester can be prepared with methanolic HCI.
  • the present invention is directed to methoxypolyethylene glycols (MPEG) , which are modified by chemically attaching chelating groups onto the nonmethyl end of the polymer.
  • chelating groups include ethylene-diamine tetraacetic acid (EDTA) , diethylene triamine pentaacetic acid (DTPA) , and ethylene glycol aminoethyl ether tetraacetic acid (EGTA) .
  • the present invention is directed to a derivative of methoxypolyethylene glycol having the formula:
  • n 3 to 8.
  • one embodiment of the present invention converts the terminal hydroxyl of MPEG to an amine, and then links the amine to ethylenedia inetetraacetate to produce methoxypolyethylene glycol amide of EDTA (MPEDTA) .
  • MPEDTA methoxypolyethylene glycol amide of EDTA
  • the present invention is also directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound as recited in claim 1, together with a pharmaceutically acceptable carrier.
  • the present invention is further directed to a method of preventing tissue damage during radiation treatments.
  • the present invention is directed to a method of preventing renal toxicity during treatment of cancer.
  • the present invention is directed to a method of preventing cardiac toxicity of doxorubicin during treatment of cancer.
  • Figure 1 depicts the average scores of different treatment groups for radioprotection of compounds in mice.
  • Figure 2 depicts the daily scores for different treatment groups for radioprotection of compounds in mice.
  • Figure 3 depicts doxorubicin treatment results for different treatment groups.
  • Figure 4 depicts the lack of effect of MPEDTA on tumor growth lag produced by doxorubicin.
  • Figure 5 depicts the protection by MPEDTA against heart weight loss caused by doxorubicin.
  • the compounds of the present invention are enhanced chelators in which the chelate is combined with low molecular weight PEG or other toxicity-lowering moiety.
  • the present invention is directed to methoxypolyethylene glycols with an average weight of 200 to 600, and preferably 350 (MPEG 350) , which are modified by chemically attaching chelating groups onto the nonmethyl end of the polymer.
  • the chelating groups that can be used to modify the methoxypolyethylene glycols include ethylene diamine tetraacetic acid (EDTA) , diethylene triamine pentaacetic acid (DTPA) , and ethylene glycol a inoethyl ether tetraacetic acid (EGTA) .
  • EDTA ethylene diamine tetraacetic acid
  • DTPA diethylene triamine pentaacetic acid
  • EGTA ethylene glycol a inoethyl ether tetraacetic acid
  • a particular embodiment of the present invention is the chelator obtained when methoxypolyethylene glycol amide is combined with EDTA (MPEDTA) .
  • n 3 to 8.
  • MPEDTA can be produced, for example, by first converting MPEG 350 to the chloride by reaction with thionyl chloride, then to an amine by reaction of the chloride with ammonia, and then coupling the amine with ethylenediamine tetraacetate monoanhydride (Takeshita et al., J. Am . Oil Chem . Soc , 59: 104, 1982).
  • MPEDTA can be produced by any standard method for amide formation, including the following: EDTA anhydride can be reacted with an amine in 0.3 M HEPES buffer at room temperature to form the amide (Lin et al., Biochemistry, 28:1054, 1989). EDTA in excess plus the amine in dimethylformamide at 120 degrees results in the amide (Hertzberg et al., J. Am . Chem . Soc , 104:313, 1982).
  • EDTA triethyl ester plus the amine, in dimethylformamide at 120 degrees results in the amide, and EDTA triester plus carbonyl diimidazole in dimethylformamide at room temperature, followed by the addition of amine, result in the amide (Hertzberg et al., Biochemistry, 23:3934, 1984).
  • EDTA triester, an amine, and dicycloxexyl ⁇ arbodiimide react to form the amide (Yanagisawa et al., Prostaglandins , 31:1063, 1986).
  • EDTA triester, an amine, ethyl- dimethylaminopropylcarbodiimide, and dimethylaminopyridine as a catalyst result in formation of the amide (Mazzarelli et al. , Biochemistry, 32:2979, 1993).
  • a novel method employed the cobaltic salt of EDTA which left one free carboxyl group to react with an amine in water catalyzed by ethyl-3-dimethylaminopropylcarbodiimide (Haner et al., Arch . Biochem . Biophys . , 231:477, 1984). The cobalt was later removed by extraction with dithizone in CCI .
  • MPEDTA is extremely effective in preventing damage from radiation or doxorubicin.
  • the present invention provides a pharmaceutical formulation comprising MPEDTA or a pharmaceutically acceptable salt or solvate thereof, together with one or more pharmaceutically acceptable carriers thereof and optionally one or more other therapeutic ingredients.
  • the carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • the present invention may be administered by the following routes: intravenous; intraarterial; intraperitoneal; intrathecal; intramuscular; oral; sublingual; buccal; aerosol (inhalant or topical) ; subcutaneous; nasal drops; eye drops; ear drops; topical (both direct and as patches, for use on skin and on internal organs); intracranial; intracardiac; suppository; intravaginal; extracorporeal (for dialysis, dosing blood organs, and perfusion solutions) ; and electroporetic.
  • routes may depend upon, for example, the condition and disorder of the recipient.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association MPEDTA or a pharmaceutically acceptable salt or solvate thereof ("active ingredient") with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired formulation.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units, such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion.
  • the active ingredients may also be presented as a bolus, electuary or paste.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form, such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface- active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored, and may be formulated so as to provide slow or controlled release of the active ingredient therein.
  • Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non ⁇ aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Formulations for rectal administration may be presented as a suppository with the usual carriers, such as cocoa butter or polyethylene glycol.
  • Formulations for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis, such as gelatin and glycerin or sucrose and acacia.
  • Preferred unit dosage formulations are those containing an effective dose, as hereinbelow recited, or an appropriate fraction thereof, of the active ingredient. It should be understood that, in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
  • the present invention may be administered in the following doses: internal l pg/kg to 10 g/kg; topical 0.00001% to 100%, formulated as preparations for immediate or sustained release.
  • the dosing regimens of the present invention include discrete doses of between 1 and 8 administrations per day, acute single dose, and chronic multiple dose, as drip (constant IV or other infusion) , or as bolus.
  • the invention may be formulated with solvent and other agents or compounds.
  • the precise amount of MPEDTA compound administered to a patient will be the responsibility of the attendant physician. However, the dose employed will depend on a number of factors, including the age and gender of the patient, the precise disorder being treated, and its severity. Also, the route of administration may vary depending on the condition and its severity.
  • the compounds of the present invention are useful in the treatment and prevention of tissue injury mediated by free radicals.
  • Free radicals can be produced from ionizing radiation, cardiac toxicity from certain drugs (e.g., doxorubicin, trauma, and ischemia-reperfusion of myocardial infarction and stroke.)
  • a free radical component may also be involved in auto-immune, chronic inflammatory, and viral diseases.
  • the present invention is also directed to a method of preventing tissue damage during radiation treatments.
  • a patient in need thereof is given an effective amount of MPEDTA by any of the effective routes of administration.
  • the present invention is also directed to a method of preventing cardiac and renal toxicity of doxorubicin during treatment of cancer.
  • a patient in need thereof is injected with an effective amount of MPEDTA.
  • the MPEG-linked chelates were tested biologically for LD 50, radioprotection, and protection against doxorubicin toxicity. Attachment of MPEG to IDA, and EDTA results in derivatives that are of comparable toxicity to the parent compounds. Comparative Example 1 Methoxypolyethylene ⁇ l ⁇ col iminodiacetate fMTDA) .
  • MPEG 350 was converted to the chloride by reaction with thionyl chloride, and to the iminodiacetate by reaction of the chloride with sodium iminodiacetate (Wuenschell, et al., J. Chromatog. , 543:345-354, 1991).
  • the product was purified by charcoal decolorization, gel filtration and ion exchange chromatography. Viscous oil, yield 35-48%.
  • the methyl ester was prepared with methanolic HCI. TLC (methanol) MIDA Rf .39, MIDA methyl ester Rf .77.
  • High resolution FAB MS, n 5, C 17 H 34 0 9 N, MH + Calc. 396.2236. Found
  • Methoxypolyethylene glycol amide with EDTA (MPEDTA.. MPEG 350 was first converted to the chloride by reaction with thionyl chloride, then to the amine by reaction of the chloride with ammonia, and then the amine was coupled with ethylenediamine tetraacetate monoanhydride (Takeshita, et al., J. Am. Oil Chem . Soc , 59:104, 1082). The product was purified by decolorizing charcoal, gel filtration, and ion exchange chromatography. Fractions containing product as assayed by titration with calcium and copper were pooled and lyophilized. Viscous oil, yield 25-30%. TLC
  • EDTA and MPEDTA the calcium salts were used.
  • Preliminary determination of the approximate toxicity level of the substances was by intraperitoneal (ip) injection of increasing doses into individual Swiss-Webster female mice until toxicity was evident.
  • the LD 50 (ip) was determined by using at least three doses in 12 mice at each level, bracketing the approximate LD 50.
  • the mice were followed for at least one week and examined for weight changes, general condition, and behavior.
  • the LD 50 with lower and upper confidence limits was calculated from the data.
  • the LD 50 millimoles per kilogram
  • LL and UL lower and upper limits for the substances and for parent compounds EDTA, iminodiacetate (IDA) , and MPEG are shown in Table I.
  • RAPIPPRQTECTIQN The effect of PEG derivatives on tissue tolerance to radiation was tested. Groups of 12 female Swiss-Webster mice were given IP solutions of the derivatives at a dose of approximately one-half of the LD50. Ten minutes later, pentobarbital (PB) 0.262 mmole/kg was given IP, and twenty minutes after the first injection, the right hind leg only was irradiated with 30 Gy using the Philips RT-250 unit operated under the following conditions: 200 kVp, 20 mA, 0.2 mm Cu added filtration, HVL 0.57 mm Cu, dose rate of 1.834 Gy/min.
  • PB pentobarbital
  • mice were irradiated in groups of 12, and their legs were arranged within a 20 x 24 cm aperture cone at a 50 cm target-to-skin distance such that all their legs were within a 95% isodose.
  • the output of the X-ray unit was calibrated using a Capintec PT-06C Farmer chamber.
  • mice were examined daily by two independent observers for 24 days, and the limbs were scored for radiation damage graded on a scale of 0 to 3.5 according to criteria previously established (Fowler, et al., Int . J. Rad . Biol . , 9:241-252, 1964). Briefly, a score of 1 corresponds to definite reddening of the foot compared to normal, and a score of 2 is marked moist desquamation, loss of hair and skin, and sticking together of toes. In the control irradiated mice given saline, the irradiated limbs began to show evidence of tissue damage at day 12, reaching a maximum average score on day 16-18, and gradually healing thereafter. The average scores in the different treatment groups for days 12-24 are shown in Table II and Figure 1, and the daily scores in Figure 2.
  • mice were followed for weight change and general condition for 27 days, after which the blood urea nitrogen levels (BUN) were determined.
  • BUN blood urea nitrogen levels
  • the mean and SEM of the different treatment groups are shown in Figure 3.
  • Doxorubicin treatment resulted in markedly increased BUN compared with the untreated control group. This was completely prevented by MPEDTA, but not EDTA treatment, reflecting the enhanced effectiveness of the novel compound.
  • Tumor length is defined as the diameter in the plane parallel to the body, while width is defined as the diameter perpendicular to the body.
  • Diameter product (in mm 2) was used as the index of tumor growth. The tumor areas were averaged and plotted vs. time to show growth delay as a function of the various parameters
  • mice were killed by cervical dislocation.
  • the results are summarized in Fig. 4.
  • the solid circles indicate the groups receiving saline-saline or MPEDTA-saline, the solid squares indicate the group receiving saline-doxorubicin, and the solid triangles indicate the group receiving
  • mice were used similarly to the tumor growth lag studies, except that nontumor-bearing C57BL/6 mice were used. Groups of 12 mice were injected with either saline-saline, ⁇ aline-doxorubicin, MPEDTA-saline, or MPEDTA-doxorubicin, followed for 28 days, and tissues taken for study. A striking indication of cardiac toxicity was loss of heart weight in mice receiving doxorubicin. Results are summarized in Fig. 5. Doxorubicin alone caused a significant weight loss, which was partially prevented by the single dose of MPEDTA. MPEDTA alone was similar to the saline control. Thus, MPEDTA reduced the cardiac damage caused by doxorubicin.
  • the new chelates described here have been shown to possess similar toxicity to parent compounds, and have been found to be potentially useful in reducing damage from radiation and doxorubicin, and, by implication, other disorders due to free radicals.
  • the compounds may be useful in other clinical situations requiring chelation, such as iron-overload diseases, including thalassemia, sickle-cell disease, and hemochromatosis, and in the treatment of lead, mercury, or other heavy metal poisoning.

Abstract

La présente invention se rapporte à des substances utiles dans la protection d'organismes vivants contre des lésions dues aux réactions des radicaux libres. Cette invention se rapporte également à des méthoxypolyéthylène glycols (MPEG) qui sont modifiés par des groupes de chélation liés chimiquement se trouvant sur l'extrémité non méthylée du polymère. Ces groupes de chélation comprennent l'acide éthylène-diamine tétracétique (EDTA), l'acide diéthylène triamine pentacétique (DTPA) et l'acide tétracétique d'éthylène glycol aminoéthyle éther (EGTA).
PCT/US1996/016722 1995-10-20 1996-10-18 Derives de chelate utilises comme protecteurs contre les lesions tissulaires WO1997014413A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU74547/96A AU7454796A (en) 1995-10-20 1996-10-18 Chelate derivatives as protectors against tissue injury

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US54656295A 1995-10-20 1995-10-20
US08/546,562 1995-10-20

Publications (2)

Publication Number Publication Date
WO1997014413A1 WO1997014413A1 (fr) 1997-04-24
WO1997014413A9 true WO1997014413A9 (fr) 1997-07-24

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* Cited by examiner, † Cited by third party
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US6020373A (en) * 1995-10-20 2000-02-01 Eastern Virginia Medical School Chelate derivatives as protectors against tissue injury
EP2428234A1 (fr) * 2006-02-14 2012-03-14 Eastern Virginia Medical School Composition à utiliser dans le traitement de la mort cellulaire rétinienne et la réduction de la pression intraoculaire
US8999157B2 (en) 2007-07-09 2015-04-07 Ge Healthcare Bio-Sciences Ab Method for preparation of a biomolecule adsorbent

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