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WO2000067760A1 - Preparation de liposomes a base de medicaments antitumoraux liposolubles - Google Patents

Preparation de liposomes a base de medicaments antitumoraux liposolubles Download PDF

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Publication number
WO2000067760A1
WO2000067760A1 PCT/JP2000/002993 JP0002993W WO0067760A1 WO 2000067760 A1 WO2000067760 A1 WO 2000067760A1 JP 0002993 W JP0002993 W JP 0002993W WO 0067760 A1 WO0067760 A1 WO 0067760A1
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Prior art keywords
ribosome
composition ratio
preparation according
lipid
polyethylene glycol
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PCT/JP2000/002993
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English (en)
Japanese (ja)
Inventor
Yuji Kasuya
Junichi Okada
Kenji Hanaoka
Shinichi Kurakata
Akira Matsuda
Takuma Sasaki
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Sankyo Company, Limited
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Application filed by Sankyo Company, Limited filed Critical Sankyo Company, Limited
Priority to AU44302/00A priority Critical patent/AU4430200A/en
Publication of WO2000067760A1 publication Critical patent/WO2000067760A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to 1- (2′-cyano 2′-, j3-D-arabinopentofuranosyl) 1 N having excellent antitumor activity.
  • one of its derivatives, 1 _ (2 '—cyanone 2' —doxy) 3—D—arabino-pentofuranosyl ⁇ 4 —palmitoylcytosine (l- (2'-cyano-2, -deoxy - ⁇ -D-arabino-pentofuranosyl) -N 4 -palmitoylcytosine 0 or less, referred to as AL-CNDAC) is a fat-soluble drug in which the N 4 position of C NDAC is palmitoylated (Tokuhei 5-1). No. 94497).
  • PAL-CNDAC shows excellent antitumor activity by oral administration
  • a formulation that has even better antitumor activity and that can be administered by other than oral administration has been demanded.
  • fat-soluble drugs such as PAL-CNDAC have extremely low water solubility, and to enable intravenous administration, a water-soluble surfactant or organic solvent must be added to the injection. It is necessary to use large quantities of the substance, and there is concern about toxicity.
  • antitumor drugs have been developed to improve drug transferability to tumor tissues and retention in tumor tissues, thereby obtaining better antitumor activity and reducing side effects. Pharmaceutical innovations for ribosome preparations are generally performed.
  • a drug that can be included in a ribosome preparation is limited to about 3 mol% of the total lipid mole (Mori et al., Pliarm. Res. 10, 507-514 ( 1993).), It is considered that it is difficult to administer the therapeutically necessary amount, or the drug does not have sufficient retention properties on the ribosome, and it is difficult to enhance drug activity or reduce side effects.
  • the present inventors have conducted intensive studies on lipids constituting ribosomes and their composition ratios.
  • lipids have specific lipids, in particular, when those specific lipids constitute liposomes with specific composition ratios, they are generally PAL-CNDAC, a highly lipid-soluble drug that can only be encapsulated in a small amount, can retain a large amount of PAL-CNDAC, and the ribosome preparation has high drug transferability to tumor tissue
  • PAL-CNDAC a highly lipid-soluble drug that can only be encapsulated in a small amount, can retain a large amount of PAL-CNDAC, and the ribosome preparation has high drug transferability to tumor tissue
  • the present inventors have found that a liposomal preparation having high retentivity at high temperature and having practically usable stability can be obtained, and thus completed the present invention.
  • the present invention provides a PAL-CNDAC-containing liposomal preparation having high drug transferability to tumor tissue, high retention in tumor tissue, and practical use.
  • the present invention provides a PAL-CNDAC-containing liposomal preparation having high drug transferability to tumor tissue
  • Lipids chemically modified with polyethylene glycols which are the lipid components of liposomes, are N-monomethoxy polyethylene glycol succinyl phosphatids.
  • Dilhetano-lamines N-monomethoxypolyethylene glycol (2-chloro-1,3-, 5-triazine-1,4-diyl) succinylphosphatidyl-etano-lamines, N-monomethoxypolyethylene glycolone-carbo-norrephosphatidyl
  • lipid chemically modified with polyethylene dalicols which is a lipid component of liposomes, is converted to N-monomethoxypolyethyleneglycolsuccinylphosphatidylethanolamines or N-monomethoxypolyethylene glycol carbohydrate.
  • the ribosome preparation according to 2> or ⁇ 3> characterized in that it is a phosphatidylethanolamine.
  • phosphatidylcholine which is a lipid component constituting the ribosome, is any of dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine.
  • phosphatidylcholine that is a lipid component constituting the liposome is either dipalmitoyl phosphatidylcholine or distearoylphosphatidylcholine.
  • ribosome preparation according to any one of ⁇ 2> to ⁇ 6>, further comprising a sterol as a lipid component constituting the ribosome.
  • ⁇ 8> The liposome preparation according to ⁇ 7>, wherein the sterols, which are lipid components constituting ribosomes, are cholesterol.
  • the phosphatidylglycerol which is a lipid component constituting the ribosome is dimyristoyl phosphatidyl glycerol, dipalmitoyl phosphatidyl glycerol or distear yl phosphatidyl glycerol.
  • phosphatidylglycerols which are lipid components constituting ribosomes, are dipalmitoylphosphatidylglycerol or disteareinolephosphatidinoleglycerol, 9> or 10> Ribosome production described in, 1
  • ribosome preparation according to any one of ⁇ 1> to ⁇ 11>, further comprising a cationic lipid as a lipid component constituting the ribosome, ⁇ 13>
  • cationic lipid which is a lipid component constituting the liposome is N-hytrimethylammonioacetyldidodecyl-D-glutamate chloride.
  • ⁇ 14> The liposomal preparation according to any one of ⁇ 2> to ⁇ 13>, wherein the ribosome has a volume average particle diameter of 25 nm to 400 nm.
  • the lipid component constituting the liposome is
  • Liposomal preparation according to any one of 1) to 1),
  • composition ratio of monomethoxypolyethyleneglycolsuccinyldistaroylphosphatidylethanolamines or N-monomethoxypolyethyleneglycolcarbonylphosphatidylethanolamines is 0.5 mol% to 10 mol%.
  • composition ratio of sterols is 4 Omo 1% to 7 Omo 1. / o,
  • the lipid component of liposomes is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • composition ratio of phosphatidylcholine leak is 10 m0 1% to 70 m0 1%
  • composition ratio of sterols is 1 Omo1. /. ⁇ 6 Omo 1%,
  • the lipid component constituting the liposome is
  • composition ratio of phosphatidylcholines is from 1 Omo 1% to 7 Omo1. /. (3) the composition ratio of the sterols is 10 mo 1% to 60 mo 1%, (4) The composition ratio of the phosphatidylglycerols is l mo 1% to 1 Omo 1%,
  • the lipid component of liposomes is
  • composition ratio of phosphatidylcholines is 1 Omo 1% to 7 Omo 1%
  • composition ratio of monomethoxy polyethylene glycol succinyl distearoyl phosphatidyl ethanolamines or N-monomethoxy polyethylene glycol carboxy phosphatidyl ethanolamines is 0.5 mol% to l Omo.
  • composition ratio of phosphatidylglycerols is l mo 1. /. ⁇ 1 Omo 1%
  • the lipid component constituting the liposome is
  • composition ratio of the phosphatidylcholines is 10 mol 1% to 70 mol 1%
  • composition ratio of the sterols is 10 mO 1% to 60 mO 1%
  • composition ratio of monomethoxypolyethylene glycol succidyl distearoyl phosphatidyl hetanolamines or N-monomethoxy polyethylene glycol carboxy phosphatidyl hetamines is 0.5 mol% to l Omo. 1%,
  • composition ratio of phosphatidylglycerols is lmo 1. /. ⁇ 1 Omo 1%
  • ribosome preparation is a force that means a closed vesicle composed of a lipid assembled in a membrane and an internal aqueous phase (DDLasic, “uposomes: irom basic to applications”, Elsevier Science Publishers ⁇ pp.1- 171 (1993)). In the present invention, it means the whole fine particles in which lipids are aggregated, irrespective of whether or not they have an internal aqueous phase.
  • the “ribosome preparation” of the present invention preferably comprises 1- (2,1-cyano-12′-doxy-1) 3-D-arabinopentofuranosyl) 1-N 4 -palmitoylcytosine,
  • the lower limit is 3 mo 1% based on the total lipid content (including PAL-CNDAC) (Preferably 25 mo 1% as a lower limit) and 65 mo 1% as an upper limit (preferably 5 O mo 1% as an upper limit).
  • the “ribosome preparation” of the present invention is preferably a “lipid chemically modified with polyericols”, “phosphatidylcholines”, “phos ′, lysethols”, “sterols”, or “cationic”.
  • lipid chemically modified with polyethylene glycols means a lipid covalently bonded to a polyethylene dalicol having various molecular weights and a lipid, and preferably the lipid Are phosphatidylethanolamines, for example, of the general formula
  • n 10 to 100
  • 1 NH_PE represents phosphatidylethanolamine.
  • N-monomethoxypolyethyleneglycol-l-succinylphosphatidylethanolamines represented by the following formula:
  • n 10 to 100
  • 1 NH_PE represents phosphatidylethanolamine.
  • n 10 to 100
  • 1 NH—PE represents phosphatidylethanolamine.
  • N-monomethoxypolyethylene glycol carbonyl phosphatidylethanolamine represented by the formula:
  • N-monomethoxypolyethyleneglycolethylenephosphatidylethanolamines represented by the following formula: Preferably, it is N-monomethoxypolyethylene glycol ethylene phosphatidinoleethanolamines or N-monomethoxy polyethylene glycol carbonyl phosphatidyl ethanol / reamines.
  • the molecular weight of the polyethylene glycol moiety is About 1000 to about 100000 N-monomethoxypolyethylene glycol ethylene phosphatidyl / reethanolamines or N-monomethoxypolyethylene glycol carbuel phosphatidylethanolamines (DD Lasic, " Liposomes: from basic to appl icationsj, El sevier Science Publ ishers ⁇ pp. 294-296 (1993)) 0
  • N-monomethoxypolyethylene glycol ethylene phosphatidylethanolamine or N-monomethoxypolyethylene glycol carbonyl phosphatidylethanolamine can reduce the total amount of liposomal lipids (including PAL-CNDAC). , Preferably 0.5 to 10 mol% in the liposome.
  • phosphatidylcholines include, for example, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidyl Choline or distearoyl phosphatidylcholine and the like are preferable, and preferred are dimyristyl phosphatidylcholine, dipanolemityl phosphatidylcholine, and distearoylphosphatidylcholine.
  • the phosphatidylcholine is preferably 10 mol 1 as a lower limit with respect to the total lipid amount (including PAL-CNDAC) constituting the ribosome. / o, (preferably 30 mol 1% as the lower limit), 95 mol 1% (preferably as 70 mol% as the upper limit) as the upper limit, and contained in the ribosome.
  • phosphatidylglycerols include, for example, dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dinonoremitolylphosphatidylglycerol, and distearylphosphatidylglycerol. Are dimyristoyl phosphatidyl glycerol, dipalmitoyl phosphatidyl glycerol or distearoyl phosphatidyl glycerol.
  • the phosphatidylglycerol is preferably 1 to 1 Omo 1 with respect to the total lipid amount (including PAL-CNDAC) constituting the ribosome. / o, contained in ribosome.
  • sterols include cholesterol, cholesterol hemi-succinate, 3 / 3— [N- (N ′, N′-dimethinoleaminoethane) rubamoyl] cholesterol, ergosterol, lanosterol And cholesterol is preferred.
  • the sterols preferably have a lower limit of 10 mol%, preferably a lower limit of 40 mol% based on the total lipid amount (including PAL-CNDAC) constituting the liposome. ), The upper limit being 70 mo 1% (preferably the upper limit is 6 Omo 1%), which is contained in the liposome.
  • cationic lipid for example, N— [1— (2,3—Gioreiro) Xy) propyl] -N, N, N-trimethylammonium mouth ride, ⁇ - ⁇ -trimethylammonioacetyldidodecyl-D-glutamate mouth ride, and the like.
  • the ribosome preparation of the present invention usually comprises Liposomes may contain lipids which can be used, for example, phosphatidylinositols such as dilauroylphosphatidylinositol, dimyristoylphosphatidylinositol, dipalmitoylphosphatidylinositol or distearoylphosphatidylinositol; dilauroyl Phosphatidylserines such as phosphatidylserine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine or diste
  • phosphatidylcholines phosphatidylglycerols, phosphatidylinositols, phosphatidylserines, phosphatidylethanolamines, digalactosyldiglycerides, galactosyldiglycerides, sphingomyelins, gangliosides, N-gangliosides Monomethoxypolyethylene glycol succininolephosphatidyl ethanolamines, N-monomethoxypolyethylene glycol (2-1,3,5-triazine-1,4,6-diyl) saxchelphosphatidylethanola Amines, N-monomethoxypolyethylene glycol carbonyl phosphatidylethanolamines, and N-monomethoxypolyethyleneglycol-l-ethylene phosphatidylethanolamines.
  • the volume average particle diameter of the ribosome is preferably from 25 to 400 nm, more preferably from 50 to 400 nm, which has high retentivity in blood and high accumulation in Z or tumor tissue after intravenous administration. 200 nm (see A ⁇ . Klibanov et al., Biochim. Biophys. Acta 1062, 142-148 (1991J / DDLasic, ⁇ Liposomes: from basic to applications '', Elsevier Science Publishers, pp.261-471 (1993)). ), And the volume average particle size of the ribosome can be determined based on principles such as dynamic light scattering (DD ⁇ asic,
  • the PAL-CNDAC-containing ribosome preparation of the present invention can be produced according to a method well known to those skilled in the art, and the volume average particle diameter can also be adjusted. That is, ribosomes can be produced using the above lipids and aqueous phase by thin film method, reverse phase evaporation method, ethanol injection method, ether injection method, dehydration monorehydration method, etc., ultrasonic irradiation method, freezing The volume average particle diameter can be adjusted by methods such as ultrasonic irradiation after melting, extrusion, French press, and homogenization. (See DD @ asic, "Liposomes: from basic to applications," Elsevier Science Publishers, pp.
  • the “aqueous phase” means an aqueous solution constituting the inside of the ribosome, and is not particularly limited as long as it is commonly used in this technical field.
  • sodium chloride aqueous solution, phosphate buffer, A buffer such as an acetate buffer, a glucose aqueous solution, a saccharide aqueous solution such as trehalose, and a mixed aqueous solution thereof are preferable.
  • the aqueous phase constituting the internal aqueous phase used for the production of ribosomes is extra-ribosomal, that is, it is nearly isotonic with body fluids, It is desirable that the osmotic pressure applied to the substrate be low.
  • lipid used for producing the ribosome preparation of the present invention, a commercially available product can be used, and usually, various kinds of carbon atoms and Z or non-Z, such as egg yolk lecithin and soybean lecithin, are used.
  • lipids having a degree of saturation can also be used without separating and purifying them into a single component.
  • an antioxidant such as sodium tocofunniol may be added for the purpose of antioxidant action and the like.
  • the ribosome preparation of the present invention is administered to humans, it is diluted with various aqueous solutions so that the concentration of the lipid constituting the liposome calculated from the composition at the time of formulation is 1 to 30 OmM. Or, use by concentrating by centrifugation.
  • the aqueous solution used for the dilution is not particularly limited as long as it is usually used.
  • a phosphate buffer solution for example, a sugar aqueous solution such as glucose and trehalose, a salt aqueous solution such as sodium chloride, and a physiological saline solution
  • a physiological saline solution for example, a physiological saline solution
  • intravenous injection is used, and when the final liquid volume is 100 to 100 ml, intravenous infusion is used.
  • intravenous infusion is used.
  • ribosome preparation of the present invention that is, physical stability of the liposome itself Since the chemical properties and chemical stability of the lipids forming the ribosome and the drug included therein may vary depending on the lipid used, etc., they should generally be stored in a cold place such as a refrigerator or used at normal use. Similar to the dissolvable injection preparations, etc., freeze-dried according to the method described in the literature (see DD Lasi "Shi iposomes: from basic to applications", Elsevier Science Publishers pp. 529-532 (1993)). It is better to save it in a state.
  • a desired injection preparation can be reconstituted by adding distilled water or the like at the time of use.
  • BEST MODE FOR CARRYING OUT THE INVENTION will be described in more detail with reference to Examples and Test Examples. However, the present invention is not limited to these examples.
  • lipids used in the following Examples and Test Examples other than cholesterol, were purchased and used from Nippon Oil & Fats Co., Ltd. Cholesterol was purchased from Tokyo Chemical Industry Co., Ltd. And recrystallized from hot ethanol. Furthermore, the molecular weight of PAL-CNDAC was 490.65, and the production method was according to the method described in JP-A-5-194497.
  • DSPC Distearoylphosphatidylcholine
  • DPPC dipalmitoylphosphatidylcholine c hereinafter referred to as DPPC.
  • DM PC Dilauroyl phosphatidylcholine
  • DLPC dilauroylphosphatidylcholine.
  • EPC egg yolk phosphatidylcholine
  • DSPG dipalmitoylphosphatidylglycerol
  • DPPG dipalmitoylphosphatidylglycerol
  • DLPG N-monomethylethoxypolyethylene glycol succinyl-distearoylphosphatidylethanolamine having a molecular weight of about 2,000, and N-monomethoxyethoxyethylene glycol succinyl distearoyl phosphatidyl ethanoylamine.
  • N-monomethoxypoly (ethyleneglvcol) carbonyldistearoyiphosphatidvlethanolamine N-monomethoxypoly (ethyleneglvcol) carbonyldistearoyiphosphatidvlethanolamine 0 or less) with a molecular weight of polyethylene glycol of about 5,000, and PEG5000-SC-DSPE.
  • N-H-trimethylammonioacetyldodecyl-D-glutamate chloride N-H-trimethylammonioacetyldodecyl-D-glutamate chloride; hereinafter referred to as TMAG.
  • TMAG N-H-trimethylammonioacetyldodecyl-D-glutamate chloride
  • 5% by weight aqueous glucose solution or 10% by weight aqueous solution of Treha ore and 0.1 M acetate buffer at pH 4 0.1 M aqueous acetic acid solution and 0.1 M aqueous sodium acetate solution are mixed in a volume ratio of 5: 1) (Mixed at a volume ratio of 9: 1) as an aqueous phase, using Bangham et al. (See J. Mol. Biol., 8, 660-668 (1964)). According to the method, to obtain a crude dispersion of the ribosome.
  • predetermined amounts of various lipids shown in Table 1 were measured in a 25 mL eggplant-shaped flask, and 5 mL of cloper form was added per 1 ⁇ m of total lipid, and the mixture was heated and dissolved at 40 ° C.
  • the mouth form was distilled off to form a thin layer of lipid on the inner wall of the eggplant-shaped flask.
  • An aqueous phase was added to the lipid thin layer so that the ribosome dispersion had a predetermined volume, and the mixture was shaken using a vortex mixer to obtain a crude ribosome dispersion.
  • PAL-CNDAC ribosome The liposome thus obtained is hereinafter referred to as PAL-CNDAC ribosome.
  • Table 1 The letters “0” and “0” mean phosphatidylcholines and phosphatidylglycerols, respectively.
  • these acyl chains are represented by S (steroyl), p (palmitoyl) and M (myristoyl). The abbreviation of is shown. However, When egg yolk lecithin was used as PC, the abbreviation of E was shown in parentheses.
  • Formulation Examples 1 to 16, 40, and 41 a 5% by weight aqueous glucose solution was used as the aqueous phase, and the total lipid concentration was 55 to 11 O mM.
  • a 5% by weight aqueous glucose solution was used as the aqueous phase, and the total lipid concentration was 55 to 11 O mM.
  • the Formulation Examples 1 7 to 3 as an aqueous phase 1 0 wt 0/0 ⁇ ⁇ 1 M acetic acid buffer mixture of trehalose and p H 4 (volume ratio 9: 1) was used, the total lipid The concentration was between 100 and 110 mM.
  • Prescription amount of lipid component per 1 mL of liposome dispersion liquid (m o 1)
  • Prescription example PC Cholesterol PG PEG2000-SS-DSPE PAL-CNDAC
  • the average volume particle diameter of the liposome was adjusted by the extrusion method or the ultrasonic irradiation method.
  • an extruder Liposofast-Basic, manufactured by AVESTIN
  • a polycarbonate membrane filter Nomura Microscience
  • Pass the coarse dispersion of CNDAC ribosome through the membrane hereinafter, passing the liposome through the membrane using an extruder equipped with a pore size membrane filter of a predetermined size).
  • Table 2 shows the volume average particle diameter of the liposome after particle diameter adjustment. No precipitate was found in any of the production examples.
  • each of these ribosome dispersions was a homogeneous semi-clear emulsion, and at 25 ° C, no change in appearance was observed for at least one day, and was a stable dispersion.
  • the particle size of the ribosome was measured as follows. That is, by diluting the PAL-CNDAC ribosome dispersion after particle size adjustment with 150 mM aqueous sodium chloride solution, the lipid concentration was reduced to approximately 0.1 mM, and the particle size measurement device (Nicomp Particle Sizer) was used. Model 370, Nicomp particle Sizing body volume average particle diameter using Systems Corp.,) (volume-weighted diameter. below were measured to.) and D v. Table 2 shows the values of D v obtained in this way. Both ribosomes had a sufficiently small average particle size, and were of a particle size that could be administered intravenously.
  • the numbers in the prescription column for lipid components indicate the prescription example numbers in Table 1
  • the numbers in the size adjustment column indicate the respective size adjustment methods (1 is the ultrasonic irradiation method, 2 is the extinction zone). (200 nm), 3 is the extension (100 nm), and 4 is the extension (50 nm).
  • these lysates gelled after at least about 10 and 30 minutes, respectively, and formed precipitates after 30 and 60 minutes. Since no such precipitate was formed in the solution containing no PAL-CNDAC (Comparative Production Example 3 in Table 3), the precipitate formed from the lysate of PAL-CNDAC was considered Conceivable.
  • PAL-CNDAC intravenous administration of PAL-CNDAC can be made possible by a general solubilization method using a surfactant or the like. Maintaining a stable solubilized state of PAL-CNDAC proved to be extremely difficult (Table 3).
  • the ribosome dispersion was diluted with physiological saline, and the total lipid concentration in the dispersion calculated from the formulation at the time of production was 0.5 mM.
  • 100 ⁇ L of this ribosome dispersion add 100 ⁇ L of ⁇ 4 acetate buffer (consisting of 0.83 ⁇ acetic acid and 0.17 ⁇ sodium acetate) and 800 ⁇ L of methanol, and mix with a vortex mixer. The mixture was centrifuged (1 000 g x 5 mi ⁇ .), And the supernatant was analyzed by reversed-phase high-performance liquid chromatography under the conditions shown in Table 4. The concentration of PAL-CNDAC eluted at 0 minutes was measured.
  • the recovery of PAL-CNDAC was calculated as the ratio of the actual concentration of PAL-CNDAC in the dispersion to the concentration of PAL-CNDAC in the dispersion, calculated from the formulation at the time of ribosome production.
  • the drug concentration in the ribosome dispersion can be further increased, for example, by concentrating by ultracentrifugation etc. after adjusting the size, and in general, it can be concentrated to a total lipid concentration of about 30 OmM (DDLasic For example, see Liposomes: irom basic to applications, Elsevier Science Publishers ⁇ p.68 (1993).) It can be concentrated about 1-fold, and a product with a PAL-CNDAC concentration of 84 mg / mL can be obtained. On the other hand, the drug concentration can be reduced by diluting with an aqueous solution of an electrolyte or a saccharide.
  • a single intravenous administration of various formulations of PAL-CNDAC was performed in mice, and the adequacy of intravenous administration was examined.
  • the various liposomal preparations of PAL-CNDAC prepared in Example 1 and the solubilized preparation of PAL-CNDAC prepared in Comparative Example 1 were appropriately diluted at 1.5-fold intervals (the liposomal preparation was The aqueous phase used in the production of ribosomes was used as the diluting solution, and a 150 mM aqueous sodium chloride solution was used as the diluting solution for the solubilized preparation.) A female CDF 1 (body weight: 20 to 30 g) was used. Was administered from Charles River Co., Ltd.) via the tail vein at 2 OmL per kg of body weight.
  • mice By observing the survival of mice for 1 hour after administration, The maximum dose that could be administered for PAL-CNDAC in the seed formulation, ie, the highest drug concentration at which the mice survived, was determined. In determining the maximum dose that could be administered, at least 6 mice were administered and the reproducibility was examined, confirming that no single animal died.
  • MTD Maximum tolerated dose
  • the ribosome preparation of the present invention can safely administer a larger amount of PAL-CNDAC than a solubilized preparation using a surfactant or the like.
  • Formulations of PAL-CNDAC shown in Example 1 or formulations of PAL-CNDAC newly produced in the same manner as above are shown in Tables 7 and 9. )
  • the dosing schedule was four intermittent doses every three days.
  • the antitumor activity was evaluated using the tumor growth inhibitory effect and the survival rate as scales.
  • Test examples 3 (1) and 3 (2) were obtained from experiments performed on different days under the same conditions.
  • Test Example 3 Preparation of PAL-CNDAC Preparation Example Drug recovery rate after production (%) Volume average particle size (nm)
  • Comparison 6 50 Approx. 100 2 2 to 7.5 mg of PAL-CNDAC / mL Dissolve in 50 ⁇ L of toamide, 10 w / w. /.
  • a 150 mM aqueous sodium chloride solution containing a neutral surfactant (Emulphor EL-620; manufactured by Rhone Poulin 'Japan Co., Ltd.) was added to a predetermined volume.
  • Mouse colon cancer co1on26 was implanted subcutaneously into 5- to 6-week-old female CDF1 mice to engraft and grow tumor tissue. On the 7th day after transplantation, 6 animals per group were randomly divided into groups and administered the first dose. As for various preparations for intravenous administration, a volume of 2 OmLZkg was intravenously administered in the same manner as in Test Example 2.
  • the oral suspension preparation examined as a comparison was administered in a volume of 2 OmLZkg.
  • the same preparation was administered in the same volume, for a total of four administrations.
  • the MTD in this dosing schedule was also determined in the same manner as in Test Example 2.c
  • the maximum dose that could be administered was as follows: on Day 20 of the transplant, there were no deaths due to adverse drug reactions, and on the seventh day of the transplant. The maximum drug concentration in the drug product with an average weight loss of 20% or less for the eyes was determined.
  • tumor volume (mm 3 ) major axis (mm) X minor C2
  • the relative tumor volume calculated from the diameter 2 (mm 2 ) Z2 is the tumor volume on the day of the first administration. It was calculated as a relative value of the tumor volume set to 1. The smaller this value is, the stronger the tumor growth inhibitory effect is.
  • the relative tumor volume on the 20th day of transplantation was calculated as a measure of the tumor growth inhibitory effect.
  • mice After the administration of the various preparations, the mice were bred and the survival days of each mouse were determined.
  • the survival rate of each treatment group was calculated by the following equation: (a ⁇ b) / b ⁇ 100 (%).
  • a and b mean the median value of the number of days alive in the treated group and the untreated group, respectively.
  • Tables 10 to 11 show the evaluation results of the antitumor activity near the MTD of each preparation.
  • Test example 3 Antitumor activity test results of (1) Production example Survival rate on day 20 of tumor implantation (%)
  • Test result of antitumor activity of test example 3 (2) Production example Survival rate on the 20th day of tumor implantation (%)
  • Test Example 3 (1) in Table 10 when a formulation prepared by solubilizing PAL-CNDAC using polyoxyethylene hydrogenated castor oil 60 and dimethyl sulfoxide (Comparative Production Example 1) was administered intravenously. In comparison, the relative tumor volume was increased and the survival rate was reduced as compared with the case where the suspension of the drug (Comparative Production Example 4) was orally administered. Therefore, an antitumor activity comparable to the excellent antitumor activity at the time of oral administration cannot be obtained by intravenous administration of the solubilized preparation, and intravenous administration of PAL-CNDAC is disadvantageous compared to oral administration. This was found to be the administration route.
  • Test Example 4 Antitumor activity Various formulations of PAL-CNDAC shown in Example 1 or a formulation of PAL-CNDAC newly produced in the same manner as the formulation (The drug recovery and volume average particle size of the tested formulations are shown in Tables 12 and 14). The antitumor activity of a different cancer type from that used in Test Example 3 was examined. In Test Example 4 (1), the intravenous administration of ribosomes and oral administration of the suspension were single doses, and in Test Example 4 (2), the suspension was administered for 5 consecutive days and then suspended. The ribosome preparation was administered for 6 weeks on an administration schedule considered to be suitable for various preparations, such as every 7 days.
  • Mouse fibrosarcoma Meth A was implanted subcutaneously into 5- to 6-week-old female CDF1 mice to engraft and grow tumor tissue. On the 7th day of transplantation, the animals were randomly divided into groups, each group consisting of 6 animals. In Test Example 4 (1), a single dose was administered, and in Test Example 4 (2), the first dose was administered. As for the preparation for intravenous administration, a volume of 20 mLZZ kg was intravenously administered in the same manner as in Test Examples 2 and 3. In addition, the oral suspension preparations tested for comparison received 20 mLZkg in Test Example 4 (1) and 1 OmLZkg in Test Example 4 (2). The MTD in Test Example 4 was determined in the same manner as in Test Examples 2 and 3.
  • the maximum concentration that can be administered is that no death occurred due to the side effects of the drug 6 days after administration (13 days after transplantation), when the single administration in Test Example 4 (1) maximized the side effects of the drug, and The maximum drug concentration in the drug product with an average weight loss of 20% or less on the 7th day of transplantation was determined.
  • the administration schedule suitable for each of the preparations in Test Example 4 (2) there were no deaths due to adverse drug reactions until 3 days after the last administration (day 45 of transplantation) and weight loss relative to day 7 of transplantation The maximum drug concentration with an average rate of 20% or less was determined.
  • Test Example 4 (1) on the 7th day of transplantation, that is, on the day of the first administration and on the 20th day, in Test Example 4 (2), on the 73rd transplantation, that is, on the day of the first administration and on the 21st, 3 On day 1, the major axis and minor axis of the tumor tissue were measured from above the skin, and the relative tumor was measured in the same manner as in Test Example 3. The volume was calculated.
  • Test Example 4 (1) the relative tumor volume on the 20th day of transplantation and in Test Example 4 (2) on the 2nd and 31st days of transplantation were calculated as a measure of the tumor growth inhibitory effect. After administration of the various preparations, the mice were bred, and the survival rate was calculated as in Test Example 3. Tables 15 and 16 show the evaluation results of the antitumor activity near the MTD of each preparation. (Table 15)
  • Test result of antitumor activity of test example 4 (1) Production example Survival rate on the 20th day of tumor implantation (%)
  • Test Example 4 Test result of antitumor activity of (2) Production example Relative tumor volume Prolonged survival rate (%)
  • PAL-CNDAC concentrations in tumors after administration of various preparations of PAL-CNDAC were measured. As shown in Tables 17 and 18, as for the administration route, the liposomal preparation and the solubilized preparation were intravenously administered, and the suspension was oral administration, as in the evaluation of antitumor activity.
  • a 6-week-old CDF1 mouse was transplanted subcutaneously with mouse colon cancer co1on26 or mouse fibrosarcoma MethA, and a single dose of the formulation shown in Tables 17 and 18 was given 10 to 14 days later did.
  • the dose of PAL-CNDAC was close to the MTD determined in Test Example 3 for co 1 on 26 tumor-bearing mice and Test 4 (1) for Meth A tumor-bearing mice. did.
  • the PAL-CNDAC concentration in MethA tumors was investigated.
  • the PAL-CNDAC concentration in each tumor homogenate and the PAL-CNDAC concentration in the tumor tissue were determined from the dilution calculated from the weight ratio of the tumor tissue to the added aqueous sodium chloride solution.
  • PAL-CNDAC was detected for 48 to 72 hours after administration of the ribosome preparation in mouse colon cancer co 1 on 26 tumor-bearing mice.
  • PAL-CNDAC could not be detected after intravenous administration of the solubilized preparation and oral administration of the suspension.
  • Table 20 in the mouse fibrosarcoma MethA tumor-bearing mouse, PAL-CNDAC was detected in the tumor after administration of the liposomal preparation 24 hours after administration of the various preparations. After oral administration of the suspension, no detection was possible.
  • a ribosome having a high composition ratio of PAL-CNDAC to the total lipid amount can be produced, and intravenous administration of PAL-CNDAC can be performed without using a highly toxic neutral water-soluble surfactant.
  • a highly toxic neutral water-soluble surfactant could be possible.
  • FIG. 1 shows the time course of PAL-CNDAC concentration in tumor (Co 1 on 26) after administration of liposomal formulation of PAL-CNDAC.

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Abstract

L'invention concerne une préparation de liposomes contenant de la 1-(2-C-cyano-2-déoxy-β-D-arabinopentofuranosyl)-N4-palmitoylcytosine, qui agit comme agent antitumoral. Cette préparation présente un bon transfert du médicament aux tissus tumoraux ainsi qu'un fort pouvoir d'accumulation dans lesdits tissus, d'où son utilisation pratique.
PCT/JP2000/002993 1999-05-11 2000-05-10 Preparation de liposomes a base de medicaments antitumoraux liposolubles WO2000067760A1 (fr)

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AU44302/00A AU4430200A (en) 1999-05-11 2000-05-10 Liposome preparation of fat-soluble antitumor drug

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007054731A1 (fr) * 2005-11-11 2007-05-18 Cyclacel Limited Produit compose antiproliferatif comprenant cyc-682 et un agent cytotoxique
US8349792B2 (en) 2006-12-19 2013-01-08 Cyclacel Limited Combination comprising CNDAC (2′-cyano-2′-deoxy-N4-palmitoyl-1-beta-D-arabinofuranosyl-cytosine) and a cytotoxic agent

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60185716A (ja) * 1984-03-05 1985-09-21 Mitsui Pharmaceut Inc 脂質分散系制癌剤
WO1991005546A1 (fr) * 1989-10-20 1991-05-02 Liposome Technology, Inc. Procede et composition de traitement de tumeurs solides
EP0536936A1 (fr) * 1991-09-30 1993-04-14 Sankyo Company Limited Dérivés de nucléosides pyrimidiniques ayant une activité anti-tumorale, leur préparation et utilisation
WO1995013053A1 (fr) * 1993-11-12 1995-05-18 The Research Foundation Of State University Of New York Formulation de taxol
JPH08151333A (ja) * 1994-09-29 1996-06-11 Ryuichi Kamioka 脳腫瘍治療用ハイブリッド型リポソーム製剤

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60185716A (ja) * 1984-03-05 1985-09-21 Mitsui Pharmaceut Inc 脂質分散系制癌剤
WO1991005546A1 (fr) * 1989-10-20 1991-05-02 Liposome Technology, Inc. Procede et composition de traitement de tumeurs solides
EP0536936A1 (fr) * 1991-09-30 1993-04-14 Sankyo Company Limited Dérivés de nucléosides pyrimidiniques ayant une activité anti-tumorale, leur préparation et utilisation
WO1995013053A1 (fr) * 1993-11-12 1995-05-18 The Research Foundation Of State University Of New York Formulation de taxol
JPH08151333A (ja) * 1994-09-29 1996-06-11 Ryuichi Kamioka 脳腫瘍治療用ハイブリッド型リポソーム製剤

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007054731A1 (fr) * 2005-11-11 2007-05-18 Cyclacel Limited Produit compose antiproliferatif comprenant cyc-682 et un agent cytotoxique
US8349792B2 (en) 2006-12-19 2013-01-08 Cyclacel Limited Combination comprising CNDAC (2′-cyano-2′-deoxy-N4-palmitoyl-1-beta-D-arabinofuranosyl-cytosine) and a cytotoxic agent

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