WO2022244690A1 - POLY(ETHYLENE GLYCOL)-b-POLY(4-NYLON) AND NANOPARTICLES THEREOF - Google Patents

POLY(ETHYLENE GLYCOL)-b-POLY(4-NYLON) AND NANOPARTICLES THEREOF Download PDF

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WO2022244690A1
WO2022244690A1 PCT/JP2022/020180 JP2022020180W WO2022244690A1 WO 2022244690 A1 WO2022244690 A1 WO 2022244690A1 JP 2022020180 W JP2022020180 W JP 2022020180W WO 2022244690 A1 WO2022244690 A1 WO 2022244690A1
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block copolymer
substituted
nanoparticles
gaba
group
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幸夫 長崎
ドク トリ ブイ
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国立大学法人筑波大学
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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
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/40—Polyamides containing oxygen in the form of ether groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/12—Powdering or granulating
    • C08J3/14—Powdering or granulating by precipitation from solutions

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  • the present invention relates to novel drug delivery systems for ⁇ -aminobutyric acid (GABA), more specifically poly(ethylene glycol) (PEG)-b-poly(4-nylon) copolymers and their oral or parenteral administration. Nanoparticles of GABA-containing polymers suitable for and uses thereof.
  • GABA ⁇ -aminobutyric acid
  • GABA ⁇ -aminobutyric acid
  • the present inventors have found that by polymerizing low molecular weight agents, further converting the polymers into so-called pegylated copolymers, and nanoparticles with polymeric properties, the biosphere, including the blood circulation system, can be It was found that the retention of the active substance in the body can be improved, and was published (see, for example, Patent Documents 1 and 2).
  • combinations of PEG-b-poly(arginine) and polyanionic polymers can form nanoparticles of the polymer in aqueous media;
  • a hydrophilic-hydrophobic block copolymer can be formed by converting the 3,4-hydroxyl group of the 3,4-hydroxyl group into a low-molecular-weight alkyl ester to form polymer nanoparticles. It has been reported that it can improve the properties of the compound, release arginine and L-DOPA in vivo, and exhibit the desired effects based on the functions of these low-molecular-weight compounds themselves.
  • GABA ⁇ -aminobutyric acid
  • pegylated, and pegylated copolymers can be polymer nanoparticles in the same way as arginine and the like, and further, the nanoparticles can convert free GABA in vivo. Sustained release could provide novel GABA delivery systems that would be useful in the art.
  • polyGABA poly( ⁇ -aminobutyric acid (GABA)
  • GABA poly( ⁇ -aminobutyric acid
  • nylon 4 is one of the polyamides with 4 carbon atoms in the repeating unit.
  • nylon 4 is not commercially available and its uses are limited compared to other members of the polyamide family.
  • Nylon can be synthesized from biomass or by ring-opening polymerization. Kamal et al. reported the synthesis of nylon 4 and nylon 6 copolymers by bulk ring-opening polymerization of 2-pyrrolidone and ⁇ -caprolactam using low molecular weight initiators (3). However, to the best of the inventor's knowledge, block copolymers based on nylon 4 have not been reported so far.
  • nylon 4 which is a homologue of polyamides, can be considered a polymer of ⁇ -aminobutyric acid (GABA). Therefore, nylon 4 may be a strong candidate material as a source of GABA, as it releases GABA when hydrolyzed.
  • nylon 4 has strong hydrogen bonding between and within polymer molecules, making it difficult to prepare nanoparticles for application in biological environments. Moreover, so far, as far as the present inventors know, there is no known publication reporting the successful preparation of nanoparticles based on nylon 4 without using surfactants or stabilizers. not
  • the purpose of the present invention is to provide a novel drug delivery system for ⁇ -aminobutyric acid (GABA), and more specifically, to provide nanoparticles of GABA-containing polymers suitable for oral or parenteral administration.
  • GABA ⁇ -aminobutyric acid
  • the present inventors have found that the polymer form of GABA (nylon 4) and the hydrophilic polymer poly(ethylene Poly(ethylene glycol)-b-nylon 4 (hereinafter, PEG-b-Nylon4 unexpectedly found that polymer nanoparticles (sometimes abbreviated as Nano BABA ) can be efficiently formed.
  • PEG-b-Nylon 4 does not fall into the category of hydrophilic-hydrophobic block copolymers, like the block copolymers disclosed in US Pat.
  • GABA or Nylon 4
  • the presence of the PEG segment suppresses hydrogen-bonding interactions in Nylon 4, leading to the formation and aggregation of large particles in an aqueous environment. It can be understood that it was possible to prevent this and thus form stable nanoparticles suitable for administration by oral or parenteral route.
  • a 1 and A 2 independently represent unsubstituted or substituted C 1 -C 12 alkyloxy and the substituents, if substituted, represent a formyl group or a group of the formula R′R′′ CH— , wherein , R ′ and R ′′ are independently C 1 -C 4 alkoxy or R ′ and R ′′ together are —OCH 2 CH 2 O—, —O(CH 2 ) 3 O— or —O(CH 2 ) represents 4 O-
  • Aspect 4 The block copolymer of Aspect 3, wherein said block copolymer forms nanoparticles of the polymer in an aqueous medium.
  • Aspect 6 Dissolving the block copolymer of Aspect 3 in an aqueous solution containing formic acid (HCOOH); dialysis of the dissolved solution of the polymer against water through a dialysis membrane; treating the resulting polymer suspension in an ultrasonic homogenizer to break up particles in the suspension; A method of making nanoparticles of the block copolymer, comprising filtering a dispersion of divided particles through a membrane with pores.
  • a pharmaceutical formulation comprising the block copolymer of aspect 3 as an active ingredient and an additive or diluent commonly used in the art.
  • a pharmaceutical formulation of aspect 7 for use in the prevention or treatment of depression.
  • the block copolymers disclosed herein are novel and have unique physicochemical and pharmacological properties. Specifically, said block copolymers are capable of forming stable polymeric nanoparticles in certain aqueous media, and such nanoparticles gradually disintegrate in the gastrointestinal tract, especially upon oral administration to a patient. , which is capable of sustainably releasing GABA while exhibiting little or no side effects, thus effectively preventing or preventing, by oral or parenteral administration, diseases or disorders for which or involving low molecular weight GABA itself. It can be treated.
  • FIG. 1 is a schematic representation of a scheme for making nanoparticles from the block copolymers disclosed herein.
  • 1 is a 1 H NMR spectrum of the PEG-b-Nylon-4 block copolymer obtained in Production Example 1.
  • FIG. 1 is a MALDI-TOF mass spectrum of the PEG-b-Nylon-4 block copolymer obtained in Production Example 1.
  • FIG. 2 is a graphical representation of the results of measuring the hydrodynamic diameter of particles in the solution of GABA-containing polymer nanoparticles (Nano GABA ) obtained in Preparation Example 2, and a digital image of the aqueous solution of Nano GABA .
  • FIG. 2 is a graphical representation showing the correlation between the dose of Nano GABA and depressive-like behavior in Test Example 1.
  • FIG. 1 is a graphical representation of the results of observing depression-like behavior in mice treated with Nano GABA or GABA in Test Example 1.
  • FIG. 2 is a graphical representation of serum stress hormone concentrations in a depression model of each treatment example in Test Example 2.
  • FIG. 10 is a graphical representation of observation results of depressive behavior in depressive model mice in Test Example 2.
  • FIG. 2 is a graphical representation of changes in body weight of test animals as an index showing acute toxicity of Nano GABA to mice in Test Example 3.
  • FIG. It is a graphical representation showing changes in the weight of various organs of a test animal as indicators of the acute toxicity, and measurement results of organ function-related biomarkers and blood composition.
  • FIG. 4 is a graphical representation showing the properties of Nano GABA in Test Example 4 and the measurement results of the stability of drug administration thereof.
  • 10 is a graphical representation of changes in body weight of test animals due to free oral intake of Nano GABA in Test Example 5.
  • FIG. Graph showing measurement results of changes in weight of various organs of test animals and organ function-related biomarkers (A), and amounts of blood composition and related biomarkers (B) of test animals due to free oral ingestion of Nano GABA in Test Example 5 display.
  • the block copolymer referred to herein includes a PEG segment and a Nylon-4 (or polyGABA) segment as blocks constituting the copolymer.
  • an aqueous medium e.g., formic acid-containing aqueous solution
  • the ⁇ -terminal and ⁇ -terminal and the divalent linking groups of both segments can be any organic groups, moieties, etc., without limitation, as long as they are capable of forming nanoparticles of the copolymer therein.
  • the nanoparticles may be nanoparticles composed of the block copolymer itself, meaning particles whose average diameter is in the nanometer size.
  • the nanometer size can be in the range of 40 nm to 400 nm, or 40 nm to 250 nm, or 60 nm to 200 nm, as measured by dynamic light scattering, in aqueous solutions of the particles.
  • the linking groups generally refer to groups containing up to 34, or up to 18, or up to 10 carbon atoms and optionally oxygen and nitrogen atoms. More specifically, it can be the groups defined for L 1 and L 2 in the aspect 5 above.
  • the orientation of binding of these linking groups is described as being compatible with the orientation of L 1 and L 2 in Formulas IIIa and IIIb, respectively.
  • the block copolymers disclosed herein have the OH at either terminus of PEG optionally protected with protecting groups well known in the art, and separately at either terminus of Nylon-4 (NH 2 or COOH) are protected if necessary with protecting groups well known in the art, and the other termini are optionally linked together to be compatible with the groups defined for L 1 and L 2 above.
  • a compound containing both of these segments or blocks can be prepared by linking by coupling or linking methods known in the art. However, they can conveniently be prepared according to Scheme 1 below.
  • a desired block copolymer can be obtained by preparing a PEG initiator and subjecting it to anionic ring-opening polymerization of 2-pyrrolidone to extend the nylon 4 segment.
  • Nanoparticles of the copolymers disclosed herein can conveniently be made according to Scheme 2 in FIG.
  • an aqueous medium of a block copolymer containing PEG and Nylon-4 segments can be processed in a Top-down method to provide nanoparticles composed of the block copolymer. can.
  • the block copolymer of embodiment 2 is added to a formic acid (HCOOH)-containing aqueous solution, for example, 20% to 90% by volume, or 30% to 75% by volume, or 35% to 60% by volume.
  • a formic acid (HCOOH)-containing aqueous solution for example, 20% to 90% by volume, or 30% to 75% by volume, or 35% to 60% by volume.
  • Polymer nanoparticles can be separated from their solutions or dispersions by, for example, freeze-drying, centrifugation, etc., so that they can be redissolved or suspended in physiological saline, if necessary, and administered orally or parenterally. It can be a formulation for When preparing a formulation for oral administration, if necessary, it can be provided as tablets, pills, or granules using additives, diluents, and the like commonly used in the art. Additives or diluents include, but are not limited to, chlorocarmellose sodium, microcrystalline cellulose, sodium lauryl sulfate, magnesium stearate, macrogol 4000, titanium oxide, and the like.
  • composition containing such nanoparticles as an active ingredient cannot be unambiguously specified because the optimal dose may vary depending on the severity of the disease to be treated, the age, sex, and body weight of the patient to be administered. It can be determined by specialists based on effective data obtained through small-scale clinical trials.
  • Step 2 Polymerization (1) Prior to use, commercially available 2-pyrrolidone was heated at 110° C. under reduced pressure for 2 hours to remove moisture for purification. (2) In a reaction flask, potassium tert-butoxide (t-BuOK, 1.35 g, 12 mmol) was dried by heating at 110° C. for 2 hours under reduced pressure. Purified 2-pyrrolidone (7.6 mL, 100 mmol) was then added to the reaction flask under a stream of nitrogen at 50° C. to completely dissolve the t-BuOK. A vacuum was applied to the reaction flask to distill off the by-products until no effervescence remained.
  • t-BuOK potassium tert-butoxide
  • reaction was put back under a stream of nitrogen and the PEG macroinitiator prepared above (15 mL, 2 mmol) was added.
  • the reaction was placed under vacuum again to remove THF and the vacuum was maintained until the reaction mixture was immobilized and the stir bar stopped moving. Finally, the nitrogen stream was put back on and the reaction was sealed for two days.
  • (3) Purification process The reaction mixture was dissolved in 40 mL of 80% HCOOH, precipitated with 350 mL of acetone, and centrifuged at 13000 rpm to collect the precipitate. The precipitate was then redispersed in hexane and collected again by centrifugation (repeated twice). Finally, the crude product was dried under reduced pressure until it was completely dry.
  • the product was 4.6 g of PEG-b-Nylon-4 as can be identified from the data in FIGS.
  • Molecular weight calculated from the 1 H NMR spectrum of FIG. 2: M 5,000 (containing 35 units of GABA).
  • the high mass region of the MALDI-TOF mass spectrum in Figure 3 shows repetitive signals of PEG and Nylon 4 with spacings of 44 m/z and 85 m/z, respectively.
  • Preparation Example 2 Preparation of GABA-containing polymer nanoparticles (Nano GABA ) 3.0 g of PEG-b-Nylon-4 was completely dissolved in 40 mL of 80% HCOOH and passed through a 3.5 kDa RC dialysis membrane to 3 Dialyzed against 2 L of MiliQ water daily (water changed twice daily). The polymer suspension was then physically dispersed again under the influence of an ultrasonic homogenizer (pulse: 50%, duration: 300 s, repetition: 3 times, power 4, ice cooling).
  • an ultrasonic homogenizer pulse: 50%, duration: 300 s, repetition: 3 times, power 4, ice cooling.
  • FIG. 4 is a graphical representation of hydrodynamic diameter data and a digital image of an aqueous solution of Nano GABA . These show that the nanoparticles in solution produced have a hydrodynamic diameter of 80 nm, are stable in aqueous solutions, and are suitable for administration by oral and parenteral injection routes.
  • Test Example 1 Antidepressant-Like Effect in an Acute Model Male C57BL/6 mice (6-10 weeks old) were treated with different doses of Nano GABA or GABA via free drink bottles for 3 days. After 3 days of treatment, these mice underwent the Forced Swim Tests as described by Porsolt et al. (Nature 1977, 266: 730-732) with minor modifications. In this test, each mouse was placed in a 2 L plastic beaker filled with 25° C. water to a level where the mice could not reach the bottom with their tails, and the behavior of the model animal was recorded with a video recorder for 6 minutes. The swimming (mobility) period and the floating behavior (immobility) period were measured.
  • Test Example 2 Therapeutic Effect in a Depression Model
  • male C57BL/6 mice (6-10 weeks old) were placed in confined spaces in 50 mL clear centrifuge tubes for 3 hours per day for a period of 14 days. Locked up.
  • Ad libitum Nano GABA treatment began on day 7 and continued until the last day of the study.
  • tail vein blood was collected and the concentration of stress hormone (corticosterone) was measured.
  • Tial suspension test was Another similar test to measure depressive behavior. In this test, each mouse was suspended by attaching its tail with adhesive tape to a horizontal bar at a height where the front hind leg of the mouse did not touch the bottom surface, and the behavior of the mouse was recorded with a video recorder for 6 minutes. .
  • Nano GABA treatment group decreased the concentration of stress hormone (see FIG. 7 (experimental animals: 6 animals per group)), and weakened depressive behavior in depressive model mice (FIG. 8 (experimental animals: 8 or 9 animals per group). )reference.). 7 and 8, **: p ⁇ 0.005 and ***: p ⁇ 0.0005, respectively.
  • Test Example 3 In vivo Toxicity ICR male mice (5 weeks old, 3 per group) were injected intraperitoneally with a high dose of Nano GABA , GABA or MiliQ water. After 3 days, mice were euthanized and blood and organs were collected. Blood composition and organ function-related biomarkers were also measured to assess the acute toxicity of Nano GABA . ⁇ Results> The Nano GABA -treated group had no significant effect on body weight at a dose of 72 mM GABA content (see Figure 9), and also significant effects on organ weights, organ function-related biomarkers and blood composition. did not affect This indicates that Nano GABA did not induce acute toxicity in mice in this test (see Figure 10).
  • Test Example 4 Properties of nanoparticles and their stability with respect to drug administration.
  • a digital image of the aqueous solution of is shown in FIG. 11(A). Again, it can be seen that the nanoparticles have an average particle size of about 80 nm, exhibit excellent dispersibility, and the method for producing the nanoparticles exhibits high reproducibility and productivity.
  • the rheological parameters of the nanoparticle solution were checked by dynamic light scattering measurements at 25°C.
  • FIG. 11(C) Furthermore, mimicking physiological conditions, e.g. stomach (pH 2-3), intestine (pH 7-8), tumor area (pH 5.5-7), kinetics for the stability of the nanoparticles under various pH conditions. measured the scattered light.
  • FIG. 11(D) It can also be seen from these figures that the nanoparticles exhibit high stability over the measured storage period and also exhibit high stability suitable for drug administration under various pH.
  • the sterilization potential of the nanoparticles was investigated by filtration, which is a sterilization method commonly used in the pharmaceutical industry.
  • the nanoparticle solution was filtered through a sterile cellulose acetate membrane filter unit with pores of average size 0.20 ⁇ m to remove bacteria.
  • the concentration of nanoparticles before and after filtration was then checked again by the evaporation method. The results are shown in FIG. 11(B). There was no significant change in the concentration of nanoparticles resulting from these sterilization methods, indicating that the nanoparticle solutions are suitable for preparations requiring high sterilization requirements.
  • Test Example 5 Toxicity of Nano GABA by free-drinking of nanoparticles
  • Test Example 5 Toxicity of Nano GABA by free-drinking of nanoparticles
  • FIG. 12 shows changes in body weight of the test mice on days 0, 1, 2, 3 and 4 of administration. After 4 days, the mice were also euthanized and examined by DRI-CHEM for organ weight, organ function, and blood composition by CELL-TAG system. The results are shown in FIGS. 13A and 13B.
  • Nano GABA does not affect the body weight, organ weights and blood composition of the test animals. There are also some trends in renal and liver function parameters, but they do not show statistical significance. Therefore, Nano GABA does not show significant toxicity to mice upon oral administration.
  • the PEG-b-Nylon-4 block copolymers according to the present invention have unique physico-chemical or pharmacological properties per se, and nanoparticles of the polymer itself or made therefrom can be administered, for example, orally or Since it is possible to prevent or treat depression in patients by parenteral administration, there is a possibility that it can be used in industries that provide or use various organic materials or pharmaceutical manufacturing industries.

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Abstract

[Problem] To provide a novel drug delivery system for γ-aminobutyric acid (GABA). [Solution] A block copolymer containing a poly(ethylene glycol) (or PEG) segment and a poly(GABA) (or Nylon 4) segment, and nanoparticles of the block copolymer. The nanoparticles are useful for preventing or treating depression.

Description

ポリ（エチレングリコール）－ｂ－ポリ（４－ナイロン）及びそのナノ粒子Poly(ethylene glycol)-b-poly(4-nylon) and its nanoparticles
　本発明は、γ－アミノ酪酸（ＧＡＢＡ）の新規薬物デリバリー系に関し、より具体的には、ポリ（エチレングリコール）（ＰＥＧ）－ｂ－ポリ（４－ナイロン）のコポリマー並びにその経口又は非経口投与に適するＧＡＢＡ含有ポリマーのナノ粒子及びその使用に関する。 The present invention relates to novel drug delivery systems for γ-aminobutyric acid (GABA), more specifically poly(ethylene glycol) (PEG)-b-poly(4-nylon) copolymers and their oral or parenteral administration. Nanoparticles of GABA-containing polymers suitable for and uses thereof.
　薬理学の観点に立つと、γ－アミノ酪酸（ＧＡＢＡ）はヒトの幾つかの疾患において主としてシグナル伝達経路及び病理学に関与する脳内の重要な神経伝達物質である。そのため、ＧＡＢＡのデリバリー能を増大することはＧＡＢＡ関連疾患を処置する上で有益であることを意味する（非特許文献１、非特許文献２）。例えば、抑うつ病では、ＧＡＢＡは腸管神経系を介して気分や感情に影響を及ぼし得るとの証拠が増加しつつある。ところで、ＧＡＢＡは低分子量の親水性化合物であることから、経口摂取された後、迅速に全身の循環系に入り、次いで、体内から排泄されるので腸管神経系を標的とすることに限界がある。したがって、胃腸管で持続可能にＧＡＢＡを放出する新規薬物デリバリー系の開発は、神経疾患（又は障害）の処置にとって旧来のＧＡＢＡに比べてより有益であろう。 From a pharmacological point of view, γ-aminobutyric acid (GABA) is an important neurotransmitter in the brain that is primarily involved in signal transduction pathways and pathology in several human diseases. Therefore, it means that increasing the delivery ability of GABA is beneficial in treating GABA-related diseases (Non-Patent Document 1, Non-Patent Document 2). For example, in depressive depression there is increasing evidence that GABA can influence mood and emotions via the enteric nervous system. By the way, since GABA is a low-molecular-weight hydrophilic compound, after it is orally ingested, it rapidly enters the systemic circulatory system and is then excreted from the body, limiting its ability to target the enteric nervous system. . Therefore, the development of new drug delivery systems that release GABA sustainably in the gastrointestinal tract would be more beneficial than traditional GABA for the treatment of neurological diseases (or disorders).
　本発明者等は、低分子量の作用物質をポリマー化し、さらに当該ポリマーを、所謂、ペグ化してコポリマーとし、さらにポリマーの性質をもつナノ粒子とすることにより、血液循環系をはじめとする、生体内での前記作用物質の滞留性を改善できることを見出し、公表した（例えば、特許文献１、特許文献２、参照。）。特許文献１では、ＰＥＧ－ｂ－ポリ（アルギニン）とポリアニオン性ポリマーとの組合せ物が水性媒体中でポリマーのナノ粒子を形成でき、特許文献２では、ポリ（Ｌ－ＤＯＰＡ）のＬ－ＤＯＰＡ中の３，４－ヒドロキシル基を低分子アルキルエステル化することで、親水－疎水性ブロックコポリマーとすることによりポリマーのナノ粒子を形成することでき、こうして形成されたナノ粒子は、それぞれ、生体内滞留性を改善するとともに、生体内でアルギニン及びＬ－ＤＯＰＡを放出でき、これらの低分子化合物それら自体が有する機能に基づく所期の効果を奏する旨報告されている。 The present inventors have found that by polymerizing low molecular weight agents, further converting the polymers into so-called pegylated copolymers, and nanoparticles with polymeric properties, the biosphere, including the blood circulation system, can be It was found that the retention of the active substance in the body can be improved, and was published (see, for example, Patent Documents 1 and 2). In WO 2005/020000, combinations of PEG-b-poly(arginine) and polyanionic polymers can form nanoparticles of the polymer in aqueous media; A hydrophilic-hydrophobic block copolymer can be formed by converting the 3,4-hydroxyl group of the 3,4-hydroxyl group into a low-molecular-weight alkyl ester to form polymer nanoparticles. It has been reported that it can improve the properties of the compound, release arginine and L-DOPA in vivo, and exhibit the desired effects based on the functions of these low-molecular-weight compounds themselves.
　仮に、γ－アミノ酪酸（ＧＡＢＡ）もアルギニン等と同様にポリマー化、ペグ化、かつ、ペグ化コポリマーをポリマーのナノ粒子とすることができ、さらに、当該ナノ粒子が生体内で遊離のＧＡＢＡを持続可能に放出できるものであれば、当該技術分野で有益な新規なＧＡＢＡデリバリー系を提供できるかも知れない。 Hypothetically, γ-aminobutyric acid (GABA) can also be polymerized, pegylated, and pegylated copolymers can be polymer nanoparticles in the same way as arginine and the like, and further, the nanoparticles can convert free GABA in vivo. Sustained release could provide novel GABA delivery systems that would be useful in the art.
　ポリ（γ－アミノ酪酸（ＧＡＢＡ））について物質の観点に立つと、ポリＧＡＢＡ（又はナイロン４）は反復単位中に４個の炭素原子を有するポリアミドの一つである。現在のところ、ナイロン４は市場から入手できず、その用途も他のポリアミドファミリー群に比べ限定されている。ナイロンはバイオマスから、又は開環重合により合成できる。 Ｋａｍａｌ他は、低分子量開始剤を用いて２－ピロリドンとε－カプロラクタムのバルク開環重合によるナイロン４とナイロン６のコポリマーの合成について報告している（非特許文献３）。しかし、本発明者の知る限りナイロン４をベースとするブロックコポリマーは今までのところ報告されていない。別の観点に立てば、ナイロン４はポリアミドの同族体であるが、γ－アミノ酪酸（ＧＡＢＡ）のポリマーであると考えることができる。したがって、ナイロン４は、加水分解されるとＧＡＢＡを放出するので、ＧＡＢＡの供給源として有力な候補材料であり得る。 From the material standpoint of poly(γ-aminobutyric acid (GABA)), polyGABA (or nylon 4) is one of the polyamides with 4 carbon atoms in the repeating unit. Currently, nylon 4 is not commercially available and its uses are limited compared to other members of the polyamide family. Nylon can be synthesized from biomass or by ring-opening polymerization. Kamal et al. reported the synthesis of nylon 4 and nylon 6 copolymers by bulk ring-opening polymerization of 2-pyrrolidone and ε-caprolactam using low molecular weight initiators (3). However, to the best of the inventor's knowledge, block copolymers based on nylon 4 have not been reported so far. From another perspective, nylon 4, which is a homologue of polyamides, can be considered a polymer of γ-aminobutyric acid (GABA). Therefore, nylon 4 may be a strong candidate material as a source of GABA, as it releases GABA when hydrolyzed.
　しかし、ポリアミドの一般的な性質として、ナイロン４は、ポリマー分子間及びポリマー分子内で強い水素結合性を有し、生物学的環境に適用するためのナノ粒子を調製することが困難である。また、今までのところ、本発明者が知る限り、界面活性剤や安定化剤を用いることなく、ナイロン４をベースとするナノ粒子の作製に成功したことを報告する刊行物の存在は知られていない。 However, as a general property of polyamide, nylon 4 has strong hydrogen bonding between and within polymer molecules, making it difficult to prepare nanoparticles for application in biological environments. Moreover, so far, as far as the present inventors know, there is no known publication reporting the successful preparation of nanoparticles based on nylon 4 without using surfactants or stabilizers. not
ＷＯ　２０１６／１６７３３３パンフレットWO 2016/167333 pamphlet ＷＯ　２０２０／１６６４７３パンフレットWO 2020/166473 pamphlet
　本発明は、γ－アミノ酪酸（ＧＡＢＡ）の新規薬物デリバリー系を提供することを目的とし、具体的には、経口又は非経口投与に適するＧＡＢＡ含有ポリマーのナノ粒子を提供することを目的として鋭意研究を重ねた。本発明者は、所謂、トップ－ダウン法（ｔｏｐ－ｄｏｗｎ ｍｅｔｈｏｄ：超音波、マイクロフルイダイザーの使用)により得ることができた、ポリマー形状のＧＡＢＡ(ナイロン４)と親水性ポリマーであるポリ（エチレングリコール）（以下、また、ＰＥＧと略記する場合もある）の間を、アミド結合を介して連結した新規ブロックコポリマーである、ポリ（エチレングリコール）－ｂ－ナイロン４（以下、ＰＥＧ－ｂ－Ｎｙｌｏｎ４ともいう。）は、意外なことに、ポリマーのナノ粒子（ＮａｎｏＢＡＢＡと略記する場合もある）を効率よく形成できることを見出した。理論により、本発明の技術的範囲の解釈に影響を受けるものでないが、ＰＥＧ－ｂ－Ｎｙｌｏｎ ４は、特許文献２に開示されるブロックコポリマーのように、親水－疎水性ブロックコポリマーの範疇に入らないものの、ＧＡＢＡ（又はＮｙｌｏｎ ４）それ自体に何らの修飾を行わなくとも、ＰＥＧセグメントの存在が、ナイロン４における水素結合を介する相互作用を抑制し、水性環境下で大きな粒子の形成及び凝集を防ぐことができ、こうして、経口又非経口経路による投与に適する安定なナノ粒子を形成することができた、ものと理解できる。 The purpose of the present invention is to provide a novel drug delivery system for γ-aminobutyric acid (GABA), and more specifically, to provide nanoparticles of GABA-containing polymers suitable for oral or parenteral administration. I did a lot of research. The present inventors have found that the polymer form of GABA (nylon 4) and the hydrophilic polymer poly(ethylene Poly(ethylene glycol)-b-nylon 4 (hereinafter, PEG-b-Nylon4 unexpectedly found that polymer nanoparticles (sometimes abbreviated as Nano BABA ) can be efficiently formed. By theory, without being bound by the interpretation of the scope of the invention, PEG-b-Nylon 4 does not fall into the category of hydrophilic-hydrophobic block copolymers, like the block copolymers disclosed in US Pat. However, even without any modification to GABA (or Nylon 4) itself, the presence of the PEG segment suppresses hydrogen-bonding interactions in Nylon 4, leading to the formation and aggregation of large particles in an aqueous environment. It can be understood that it was possible to prevent this and thus form stable nanoparticles suitable for administration by oral or parenteral route.
　したがって、本発明は、主たる態様として次のものを挙げることができる
〔態様１〕式Ｉ：
 
　　　－（ＣＨ２ＣＨ２－Ｏ）ｍ－ (I)
 
式中、ｍは２～５００、又は５～３００，又は１０～２００の整数である、
 
で表される反復単位のセグメントと、
式ＩＩ:
 
　　－（ＮＨＣＨ２ＣＨ２ＣＨ２Ｃ（＝Ｏ））ｎ－ (II)
 
式中、ｎは、５～１０００、又は１０～６００、又は１５～４００の整数である、
で表される反復単位のセグメントを、含むブロックコポリマー。
〔態様２〕態様１のブロックコポリマーであって、水性媒体中でポリマーのナノ粒子を形成する、前記ブロックコポリマー。
〔態様３〕次式IIIａ又は式IIIｂで表されるブロックコポリマー。
式IIIａ：
 
　Ａ１－（ＣＨ２ＣＨ２－Ｏ）ｍ１－Ｌ1－（ＮＨＣＨ２ＣＨ２ＣＨ２Ｃ（＝Ｏ））ｎ１－Ｗ１
 
　　　　　　　　　　　　（IIIａ）
又は式IIIｂ:
 
　Ａ２－（ＣＨ２ＣＨ２－Ｏ）ｍ２－Ｌ２-（Ｃ（＝Ｏ）ＣＨ２ＣＨ２ＣＨ２ＮＨ）ｎ２－Ｗ２
 
　　　　　　　　　　　　（IIIｂ)
 
で表され、各式中、
　Ａ1及びＡ2は独立して、非置換又は置換Ｃ1－Ｃ12アルキルオキシを表し、置換されている場合の置換基は、ホルミル基又は式Ｒ'Ｒ"ＣＨ－基を表し、ここで、Ｒ'及びＲ"は独立してＣ1－Ｃ4アルコキシ又はＲ'とＲ"は一緒になって－ＯＣＨ2ＣＨ2Ｏ－、－Ｏ（ＣＨ2）3Ｏ－若しくは－Ｏ（ＣＨ2）4Ｏ－を表し、
　Ｌ１及びＬ２は独立して、二価の連結基又は存在する場合には直接結合を表し、
　Ｗ１は、ヒドロキシル基、又は非置換若しくは置換Ｏ－Ｃ1－Ｃ６アルキルを表し、置換されている場合の置換基はハロゲン原子、ヒドロキシル基又はＣ1－Ｃ３アルキル基を表し、
　Ｗ２は、水素原子、又は非置換若しくは置換Ｃ1－Ｃ６アルキルを表し、置換されている場合の置換基はハロゲン原子、ヒドロキシル基若しくはＣ1－Ｃ３アルキルを表し、又は非置換若しくは置換Ｃ（＝Ｏ）Ｃ1－Ｃ６アルキルを表し、置換されている場合の置換基はハロゲン原子、ヒドロキシル基若しくはＣ1－Ｃ３アルキル基を表し、
　ｍ１及びｍ２は独立して、２～５００、又は５～３００，又は１０～２００の整数であり、かつ、
　ｎ１及びｎ２は独立して、５～１０００、又は１０～６００、又は１５～４００の整数である。
〔態様４〕態様３のブロックコポリマーであって、水性媒体中でポリマーのナノ粒子を形成する、前記ブロックコポリマー。
〔態様５〕態様３のブロックコポリマーであって、
　Ｌ１は、（ＣＨ２）ｐＣ（＝Ｏ）、Ｃ（＝Ｏ）（ＣＨ２）ｑ又はＣ（＝Ｏ）（ＣＨ２）ｒＣ（＝Ｏ）を表し、
　Ｌ２は、直接結合、（ＣＨ２）ｓＯ又は（ＣＨ２）ｔＮＨを表し、
　ｐ、ｑ、ｒ、ｓ、及びｔは、それぞれ独立して、１～６の整数である、ブロックコポリマー。
〔態様６〕態様３のブロックコポリマーをギ酸（ＨＣＯＯＨ）含有水溶液に溶解するステップと、
　前記ポリマーの溶解した溶液を透析膜を介して水に対して透析するステップと、
　得られたポリマー懸濁液を超音波ホモジナイザー中で処理して懸濁液中の粒子を分割するステップと、
　粒子を分割した分散液を細孔を備えた膜でろ過するステップを含む、前記ブロックコポリマーのナノ粒子の作製方法。
〔態様７〕有効成分としての態様３のブロックコポリマーと当該技術分野で常用されている添加剤又は希釈剤を含む製薬学的製剤。
〔態様８〕抑うつの予防又は治療に使用するための態様７の製薬学的製剤。 Therefore, the main aspects of the present invention include the following [Aspect 1] Formula I:

—(CH 2 CH 2 —O) m — (I)

wherein m is an integer of 2 to 500, or 5 to 300, or 10 to 200;

a segment of the repeating unit represented by , and
Formula II:

- ( NHCH2CH2CH2C (=O)) n- ( II )

wherein n is an integer from 5 to 1000, or from 10 to 600, or from 15 to 400;
A block copolymer comprising a segment of repeating units represented by
Aspect 2. The block copolymer of Aspect 1, wherein said block copolymer forms nanoparticles of the polymer in an aqueous medium.
[Mode 3] A block copolymer represented by the following formula IIIa or IIIb.
Formula IIIa:

A 1 -(CH 2 CH 2 -O) m1 -L 1 -(NHCH 2 CH 2 CH 2 C(=O)) n1 -W 1

(IIIa)
or Formula IIIb:

A 2 —(CH 2 CH 2 —O) m2 —L 2 —(C(=O)CH 2 CH 2 CH 2 NH) n2 —W 2

(IIIb)

In each formula,
A 1 and A 2 independently represent unsubstituted or substituted C 1 -C 12 alkyloxy and the substituents, if substituted, represent a formyl group or a group of the formula R′R″ CH— , wherein , R ′ and R ″ are independently C 1 -C 4 alkoxy or R ′ and R ″ together are —OCH 2 CH 2 O—, —O(CH 2 ) 3 O— or —O(CH 2 ) represents 4 O-,
L 1 and L 2 independently represent a divalent linking group or a direct bond when present;
W 1 represents a hydroxyl group or an unsubstituted or substituted O—C 1 -C 6 alkyl group, where the substituent group represents a halogen atom, a hydroxyl group or a C 1 -C 3 alkyl group;
W 2 represents a hydrogen atom, or unsubstituted or substituted C 1 -C 6 alkyl, where the substituent when substituted represents a halogen atom, hydroxyl group or C 1 -C 3 alkyl, or unsubstituted or substituted C(=O)C 1 -C 6 alkyl, where the substituents, if substituted, are halogen atoms, hydroxyl groups or C 1 -C 3 alkyl groups;
m1 and m2 are independently integers of 2 to 500, or 5 to 300, or 10 to 200, and
n1 and n2 are independently integers of 5-1000, or 10-600, or 15-400.
Aspect 4. The block copolymer of Aspect 3, wherein said block copolymer forms nanoparticles of the polymer in an aqueous medium.
[Aspect 5] The block copolymer of Aspect 3,
L 1 represents (CH 2 ) p C(=O), C(=O)(CH 2 ) q or C(=O)(CH 2 ) r C(=O);
L 2 represents a direct bond, (CH 2 ) s O or (CH 2 ) t NH;
A block copolymer wherein p, q, r, s, and t are each independently an integer from 1 to 6.
[Aspect 6] Dissolving the block copolymer of Aspect 3 in an aqueous solution containing formic acid (HCOOH);
dialysis of the dissolved solution of the polymer against water through a dialysis membrane;
treating the resulting polymer suspension in an ultrasonic homogenizer to break up particles in the suspension;
A method of making nanoparticles of the block copolymer, comprising filtering a dispersion of divided particles through a membrane with pores.
[Aspect 7] A pharmaceutical formulation comprising the block copolymer of aspect 3 as an active ingredient and an additive or diluent commonly used in the art.
[Aspect 8] A pharmaceutical formulation of aspect 7 for use in the prevention or treatment of depression.
　本明細書で開示されるブロックコポリマーは新規であり、これらは理化学的にも、薬理学的にも特有の性質を有する。具体的には、前記ブロックコポリマーは特定の水性媒体中で、安定なポリマーのナノ粒子を形成でき、かようなナノ粒子は、特に、患者に経口投与すると、胃腸管においてそれらを徐々に崩壊させ、ＧＡＢＡを持続可能に放出することができる一方で副作用を殆ど又は何ら示さないので、低分子量ＧＡＢＡそれ自体が効能を有するかまたは関与する疾患又は障害を経口又は非経口投与により効果的に予防又は治療できる。 The block copolymers disclosed herein are novel and have unique physicochemical and pharmacological properties. Specifically, said block copolymers are capable of forming stable polymeric nanoparticles in certain aqueous media, and such nanoparticles gradually disintegrate in the gastrointestinal tract, especially upon oral administration to a patient. , which is capable of sustainably releasing GABA while exhibiting little or no side effects, thus effectively preventing or preventing, by oral or parenteral administration, diseases or disorders for which or involving low molecular weight GABA itself. It can be treated.
本明細書で開示されるブロックコポリマーからナノ粒子を作製するためのスキームの略図的表示である。1 is a schematic representation of a scheme for making nanoparticles from the block copolymers disclosed herein. 製造例１で得られたＰＥＧ－ｂ－Ｎｙｌｏｎ－４ブロックコポリマーの1Ｈ ＮＭＲスペクトルである。1 is a 1 H NMR spectrum of the PEG-b-Nylon-4 block copolymer obtained in Production Example 1. FIG. 製造例１で得られたＰＥＧ－ｂ－Ｎｙｌｏｎ－４ブロックコポリマーのＭＡＬＤＩ－ＴＯＦマススペクトルである。1 is a MALDI-TOF mass spectrum of the PEG-b-Nylon-4 block copolymer obtained in Production Example 1. FIG. 製造例２で得られたＧＡＢＡ含有ポリマーのナノ粒子（ＮａｎｏＧＡＢＡ）溶液における粒子の流体力学的直径を測定した結果のグラフ表示及びＮａｎｏＧＡＢＡの水溶液のデジタル映像である。FIG. 2 is a graphical representation of the results of measuring the hydrodynamic diameter of particles in the solution of GABA-containing polymer nanoparticles (Nano GABA ) obtained in Preparation Example 2, and a digital image of the aqueous solution of Nano GABA . FIG. 試験例１におけるＮａｎｏＧＡＢＡの用量と抑うつ様挙動間の相関性を示すグラフ表示である。2 is a graphical representation showing the correlation between the dose of Nano GABA and depressive-like behavior in Test Example 1. FIG. 試験例１におけるＮａｎｏＧＡＢＡ又はＧＡＢＡで処置したマウスの抑うつ様挙動を観察した結果のグラフ表示である。1 is a graphical representation of the results of observing depression-like behavior in mice treated with Nano GABA or GABA in Test Example 1. FIG. 試験例２における各処置例の抑うつモデルおける血清中ストレスホルモン濃度のグラフ表示である。2 is a graphical representation of serum stress hormone concentrations in a depression model of each treatment example in Test Example 2. FIG. 試験例２における抑うつモデルマウスにおける抑うつ挙動の観察結果のグラフ表示である。10 is a graphical representation of observation results of depressive behavior in depressive model mice in Test Example 2. FIG. 試験例３におけるＮａｎｏＧＡＢＡのマウスに対する急性毒性を示す指標としての被験動物の体重変化のグラフ表示である。2 is a graphical representation of changes in body weight of test animals as an index showing acute toxicity of Nano GABA to mice in Test Example 3. FIG. 前記急性毒性を示す指標としての被験動物の各種臓器の重量変化、並びに臓器の機能関連バイオマーカー及び血液の組成の量の測定結果を表すグラフ表示である。It is a graphical representation showing changes in the weight of various organs of a test animal as indicators of the acute toxicity, and measurement results of organ function-related biomarkers and blood composition. 試験例４におけるＮａｎｏＧＡＢＡの特性及びその薬剤投与に関する安定性の測定結果を示すグラフ表示。4 is a graphical representation showing the properties of Nano GABA in Test Example 4 and the measurement results of the stability of drug administration thereof. 試験例５におけるＮａｎｏＧＡＢＡの経口自由摂取による被験動物の体重変化のグラフ表示。10 is a graphical representation of changes in body weight of test animals due to free oral intake of Nano GABA in Test Example 5. FIG. 試験例５におけるＮａｎｏＧＡＢＡの経口自由摂取による被験動物の各種臓器の重量変化及び臓器の機能関連バイオマーカー（Ａ）、並びに血液の組成の量及び関連するバイオマーカー（Ｂ）の測定結果を表すグラフ表示である。Graph showing measurement results of changes in weight of various organs of test animals and organ function-related biomarkers (A), and amounts of blood composition and related biomarkers (B) of test animals due to free oral ingestion of Nano GABA in Test Example 5 display.
発明の具体的な記述Specific description of the invention
　本明細書に開示される態様又は内容に関して記述される用語等は、特に言及しない限り当該技術分野で常用されている意味又は内容を有するものとして使用されており、当該態様又は内容につて、さらに、以下の説明を行うことができる。 The terms and the like described with respect to the aspects or contents disclosed in this specification are used as having the meanings or contents commonly used in the technical field unless otherwise specified, and with respect to the aspects or contents, further , the following explanation can be given.
　本明細書にいうブロックコポリマーは、当該コポリマーを構成するブロックとして、ＰＥＧセグメントとＮｙｌｏｎ－４（又はポリＧＡＢＡ）セグメントを含み、具体的には後述するように、水性媒体（例えば、ギ酸含有水溶液）中で当該コポリマーのナノ粒子を形成できるものであれば、限定されることなく、そのα末端やω末端や両セグメントの二価の連結基が如何なる有機の基、部分、等であることもできる。当該ナノ粒子は、前記ブロックコポリマーそれ自体から構成されるナノ粒子であることができ、粒子の平均直径がナノメーターのサイズにある粒子を意味する。ナノメーターのサイズは、粒子の水溶液における、動的光散乱法により測定される流体力学的直径として、４０ｎｍ～４００ｎｍ、又は４０ｎｍ～２５０ｎｍ、又は６０ｎｍ～２００ｎｍの範囲内にあることができる。 The block copolymer referred to herein includes a PEG segment and a Nylon-4 (or polyGABA) segment as blocks constituting the copolymer. Specifically, as described later, an aqueous medium (e.g., formic acid-containing aqueous solution) The α-terminal and ω-terminal and the divalent linking groups of both segments can be any organic groups, moieties, etc., without limitation, as long as they are capable of forming nanoparticles of the copolymer therein. . The nanoparticles may be nanoparticles composed of the block copolymer itself, meaning particles whose average diameter is in the nanometer size. The nanometer size can be in the range of 40 nm to 400 nm, or 40 nm to 250 nm, or 60 nm to 200 nm, as measured by dynamic light scattering, in aqueous solutions of the particles.
　前記の連結基は、一般的には、最大３４個、又は最大１８個、又は最大１０個の炭素原子、並びに任意に酸素及び窒素原子を含有する基を意味する。より具体的には、前記態様５において、Ｌ１及びＬ２について定義した基であることができる。これらの連結基の結合する方向性は、それぞれ、式IIIａ及び式IIIｂにおけるＬ１及びＬ２の記載方向に適合するものとして記載している。例えば、式IIIａにあっては、Ｌ１の（ＣＨ２）ｐＣ（＝Ｏ）は、（ＣＨ２）ｐのα末端のメチレン基の炭素原子がＣＨ２ＣＨ２－Ｏ単位の酸素原子に結合し、カルボニル基Ｃ（＝Ｏ）の炭素原子がＮＨＣＨ２ＣＨ２ＣＨ２Ｃ（＝Ｏ）単位の窒素原子に結合する方向性を有する。 The linking groups generally refer to groups containing up to 34, or up to 18, or up to 10 carbon atoms and optionally oxygen and nitrogen atoms. More specifically, it can be the groups defined for L 1 and L 2 in the aspect 5 above. The orientation of binding of these linking groups is described as being compatible with the orientation of L 1 and L 2 in Formulas IIIa and IIIb, respectively. For example, in Formula IIIa, (CH 2 ) p C(=O) of L 1 is such that the carbon atom of the α-terminal methylene group of (CH 2 ) p is replaced by the oxygen atom of the CH 2 CH 2 —O unit. oriented such that the carbon atom of the carbonyl group C (=O) is attached to the nitrogen atom of the NHCH2CH2CH2C ( =O) unit.
　本明細書で開示されるブロックコポリマーは、ＰＥＧのいずれかの末端のＯＨを当該技術分野で周知の保護基で必要があれば保護しておき、別に、Ｎｙｌｏｎ－４のいずれかの末端（ＮＨ２又はＣＯＯＨ）を当該技術分野で周知の保護基で必要があれば保護しておき、上記のＬ１及びＬ２について定義した基に適合するように他の末端を必要があれば相互に連結し得る官能基を導入しておき、これらの両セグメント又はブロックを含む化合物、それ自体当該技術分野で周知のカップリング又は連結方法により連結することにより、製造できる。しかし、都合よくは、次のスキーム1に従って製造できる。 The block copolymers disclosed herein have the OH at either terminus of PEG optionally protected with protecting groups well known in the art, and separately at either terminus of Nylon-4 (NH 2 or COOH) are protected if necessary with protecting groups well known in the art, and the other termini are optionally linked together to be compatible with the groups defined for L 1 and L 2 above. A compound containing both of these segments or blocks can be prepared by linking by coupling or linking methods known in the art. However, they can conveniently be prepared according to Scheme 1 below.
Figure JPOXMLDOC01-appb-I000001
Figure JPOXMLDOC01-appb-I000001
　スキーム１に従えば、簡潔には、ＰＥＧイニシエーターを用意し、これに２－ピロリドンをアニオン開環重合することにより、ナイロン４セグメントを伸長させることにより、目的のブロックコポリマーを得ることができる。 Briefly, according to Scheme 1, a desired block copolymer can be obtained by preparing a PEG initiator and subjecting it to anionic ring-opening polymerization of 2-pyrrolidone to extend the nylon 4 segment.
　本明細書で開示されるコポリマーのナノ粒子（ＮａｎｏＧＡＢＡ）は、都合よくは、図１のスキーム２に従って作製できる。 Nanoparticles of the copolymers disclosed herein (Nano GABA ) can conveniently be made according to Scheme 2 in FIG.
　簡潔には、ＰＥＧセグメントとＮｙｌｏｎ－４セグメントを含むブロックコポリマーの水性媒体を、トップ－ダウン法（Top-down method）で処理することにより、当該ブロックコポリマーから構成されたナノ粒子を提供することができる。 Briefly, an aqueous medium of a block copolymer containing PEG and Nylon-4 segments can be processed in a Top-down method to provide nanoparticles composed of the block copolymer. can.
　スキーム２に従えば、例えば、態様２のブロックコポリマーをギ酸（ＨＣＯＯＨ）含有水溶液、例えば、２０容量％～９０容量％、又は、３０容量％～７５容量％、又は、３５容量％～６０容量％のギ酸含有液に溶解するステップと、
　前記ポリマーの溶解した溶液を透析膜、例えば、１ｋＤａ～３．５ｋＤａのカットオフ細孔を備えたダイアライシスメンブラン、を介して水に対してギ酸が実質的に存在しなくなるまで透析するステップと、
　得られたポリマー懸濁液を超音波ホモジナイザー中で処理して、懸濁液を再度懸濁させるステップと、
　再懸濁液をマイクロフルイダイザー中で懸濁液中の微粒子を分割するステップと、次いで、必要により、
　粒子を分割した分散液を、例えば、細孔径０．１μｍ～１．０μｍを備えた膜でろ過するステップを利用することができる。 According to Scheme 2, for example, the block copolymer of embodiment 2 is added to a formic acid (HCOOH)-containing aqueous solution, for example, 20% to 90% by volume, or 30% to 75% by volume, or 35% to 60% by volume. dissolving in a formic acid-containing liquid of
dialysis of the dissolved solution of the polymer against water through a dialysis membrane, such as a dialysis membrane with a pore cut-off of 1 kDa to 3.5 kDa, until the formic acid is substantially absent;
treating the resulting polymer suspension in an ultrasonic homogenizer to resuspend the suspension;
resuspending in a microfluidizer to split the microparticles in suspension;
Filtration of the particle-divided dispersion through a membrane with, for example, a pore size of 0.1 μm to 1.0 μm can be used.
　ポリマーのナノ粒子（ＮａｎｏＧＡＢＡ）は、例えば、凍結乾燥、遠心分離、等によりそれらの溶液又は分散液から分離できるので、必要により、生理食塩水中に再溶解又は懸濁させ、経口又は非経口投与用の製剤とすることができる。経口投与用の製剤とするときは、必要があれば、当該技術分野で常用されている、添加剤、希釈剤、等を利用して、錠剤、丸薬、顆粒剤として提供できる。添加剤又は希釈剤としては、限定されるものでないが、クロロカルメロースナトリウム、結晶セルロース、ラウリル硫酸ナトリウム、ステアリン酸マグネシウム、マクロゴール４０００、酸化チタン、等を挙げることができる。 Polymer nanoparticles (Nano GABA ) can be separated from their solutions or dispersions by, for example, freeze-drying, centrifugation, etc., so that they can be redissolved or suspended in physiological saline, if necessary, and administered orally or parenterally. It can be a formulation for When preparing a formulation for oral administration, if necessary, it can be provided as tablets, pills, or granules using additives, diluents, and the like commonly used in the art. Additives or diluents include, but are not limited to, chlorocarmellose sodium, microcrystalline cellulose, sodium lauryl sulfate, magnesium stearate, macrogol 4000, titanium oxide, and the like.
　このようなナノ粒子を有効成分として含む組成物は、処置対処たる疾患の重篤度、投与対象たる患者の年齢、性別、体重により、最適の用量が変動し得るので、一義的に特定できないが、小規模の臨床試験等を通して得られる有効のデータ等に基づいて専門医が決定することができる。 The composition containing such nanoparticles as an active ingredient cannot be unambiguously specified because the optimal dose may vary depending on the severity of the disease to be treated, the age, sex, and body weight of the patient to be administered. It can be determined by specialists based on effective data obtained through small-scale clinical trials.
　以下、説明が煩雑になることを避けるため、本発明の典型例に基づいて、本発明をさらに具体的に説明するが、本発明はこれらの例に限定されるものではない。 In order to avoid complicating the description, the present invention will be described more specifically based on typical examples of the present invention, but the present invention is not limited to these examples.
製造例１：ＰＥＧ－ｂ－Ｎｙｌｏｎ－4ブロックコポリマー：
　　　　　　CH3O-(CH2CH2O)n-CH2-CO-(NHCH2CH2CH2CO)m-OH
　　　　の合成
ステップ１：ＰＥＧマクロイニシエーター（ＣＨ３Ｏ－（ＣＨ２ＣＨ２Ｏ）ｎ－ＣＨ２－ＣＯＣｌ）の合成
（１）市販のＣＨ３Ｏ－（ＣＨ２ＣＨ２Ｏ）ｎ－Ｈ (ｍＰＥＧ－ＯＨ、 ＭＷ＝２，０００、２０ ｇ, １０　ｍｍｏｌ)を、減圧下で２時間１１０ ℃で加熱し、予め水分を除去した。反応系を５５℃に冷却した。窒素流下でテトラヒドロフラン（ＴＨＦ）の５０ｍＬを反応系に加え、ｍＰＥＧ－ＯＨを完全に溶解した。室温で、ブチルリチウムの１．６ Ｍ－ヘキサン（ＢｕＬｉ, １０ ｍＬ, １６ ｍｍｏｌ）とブロモ化酢酸エチル（３．６ ｍＬ, ３０ ｍｍｏｌ）を窒素流下で前記反応系に加えた。反応混合物を室温下で１日撹拌した。その後、反応混合物を３℃で貯蔵した２－プロパノール（冷ＩＰＡ）に滴下することにより沈殿を形成させた。沈殿物を遠心 （９０００ ｒｐｍ, ５ 分） により集め、次いで、メタノール（１００ ｍＬ）に溶解させた。ＩＰＡ中での沈殿、遠心による収集、メタノールへの溶解を３回繰り返した。その後、ＩＰＡ中の沈殿物を遠心により集め、次いで、ｎ－ヘキサン中に再分散させ、遠心し、さらに減圧下で完全に乾燥するまで乾燥した。生成物は、 ＣＨ３Ｏ－（ＣＨ２ＣＨ２Ｏ）ｎ－ＣＨ２－ＣＯＯＣ２Ｈ５, １８．５ ｇであった。
（２）前記 ＣＨ３Ｏ－（ＣＨ２ＣＨ２Ｏ）ｎ－ＣＨ２－ＣＯＯＣ２Ｈ５（１８ ｇ, ９ ｍｍｏｌ）を、５０ｍＬのミリＱ水（ＭｉｌｉＱ Ｗａｔｅｒ）で溶解し、１．５Ｍの水酸化ナトリウム溶液（１０ ｍＬ、１５ ｍｍｏｌ）を用いて加水分解した。１日撹拌した後、塩酸を用いて反応系のｐＨを１～２に調節し、ジクロロメタン（ＤＣＭ, ３ ℃, 各６０ ｍＬ ）により有機相に生成物を抽出し、ＤＣＭ部を集め、次いで、ロータリーエバポラーターによりＤＣＭの量を減少させた。このＤＣＭ溶液を冷ＩＰＡにより沈殿させ、遠心（９０００ ｒｐｍ, ５ 分）により集め、メタノール中に再溶解し、冷ＩＰＡで再沈殿させた。これらの精製ステップは３回繰り返した。最後に、沈殿物をヘキサン中に分散し、遠心分離し、完全に乾燥するまで減圧下で乾燥した。生成物は、　ＣＨ３Ｏ－（ＣＨ２ＣＨ２Ｏ）ｎ－ＣＨ２－ＣＯＯＨ，１６ｇであった。
（３）前記 ＣＨ３Ｏ－（ＣＨ２ＣＨ２Ｏ）ｎ－ＣＨ２－ＣＯＯＨ （６．６ｇ, ２　ｍｍｏｌ）を、３０分間７５℃で予備加熱して水分を除去し、室温の窒素流下でＴＨＦ（１５ｍＬ）に溶解した。得られた溶液に塩化チオニル（ＳＯＣｌ２, ０．５ ｍＬ, ６．９ ｍｍｏｌ） を窒素流下で加え一夜撹拌した。反応フラスコ中のＴＨＦと過剰のＳＯＣｌ２を６０℃の減圧下で３０分間留去した。その後、この反応混合物を１５ｍＬのＴＨＦに再溶解して、生成物：マクロイニシエーターＣＨ３Ｏ－（ＣＨ２ＣＨ２Ｏ）ｎ－ＣＨ２－ＣＯＣｌのＴＨＦ溶液を得た。 Production Example 1: PEG-b-Nylon-4 block copolymer:
CH3O- ( CH2CH2O ) n - CH2 - CO-( NHCH2CH2CH2CO ) m -OH
Step 1: Synthesis of PEG macroinitiator (CH 3 O—(CH 2 CH 2 O) n —CH 2 —COCl) (1) Commercially available CH 3 O—(CH 2 CH 2 O) nH ( mPEG-OH, MW=2,000, 20 g, 10 mmol) was heated at 110° C. for 2 hours under reduced pressure to remove water beforehand. The reaction system was cooled to 55°C. Under a stream of nitrogen, 50 mL of tetrahydrofuran (THF) was added to the reaction to completely dissolve the mPEG-OH. At room temperature, butyllithium in 1.6 M-hexane (BuLi, 10 mL, 16 mmol) and ethyl bromide acetate (3.6 mL, 30 mmol) were added to the reaction under a stream of nitrogen. The reaction mixture was stirred at room temperature for 1 day. A precipitate was then formed by adding the reaction mixture dropwise to 2-propanol (cold IPA) stored at 3°C. The precipitate was collected by centrifugation (9000 rpm, 5 minutes) and then dissolved in methanol (100 mL). Precipitation in IPA, collection by centrifugation, and dissolution in methanol were repeated three times. The precipitate in IPA was then collected by centrifugation, then redispersed in n-hexane, centrifuged, and dried under reduced pressure until completely dry. The product was CH 3 O—(CH 2 CH 2 O) n —CH 2 —COOC 2 H 5 , 18.5 g.
(2) The above CH 3 O—(CH 2 CH 2 O) n —CH 2 —COOC 2 H 5 (18 g, 9 mmol) was dissolved in 50 mL of MiliQ Water, and 1.5 M Hydrolyzed with sodium hydroxide solution (10 mL, 15 mmol). After stirring for 1 day, the pH of the reaction was adjusted to 1-2 with hydrochloric acid, the product was extracted into the organic phase with dichloromethane (DCM, 3° C., 60 mL each), the DCM portion was collected, and then The amount of DCM was reduced by rotary evaporator. The DCM solution was precipitated with cold IPA, collected by centrifugation (9000 rpm, 5 min), redissolved in methanol and reprecipitated with cold IPA. These purification steps were repeated three times. Finally, the precipitate was dispersed in hexane, centrifuged and dried under vacuum until completely dry. The product was CH 3 O—(CH 2 CH 2 O) n —CH 2 —COOH, 16 g.
(3) The above CH 3 O—(CH 2 CH 2 O) n —CH 2 —COOH (6.6 g, 2 mmol) was preheated at 75° C. for 30 minutes to remove water and then cooled at room temperature under a stream of nitrogen. Dissolve in THF (15 mL). Thionyl chloride (SOCl 2 , 0.5 mL, 6.9 mmol) was added to the resulting solution under a stream of nitrogen and stirred overnight. THF and excess SOCl 2 in the reaction flask were distilled off under reduced pressure at 60° C. for 30 minutes. The reaction mixture was then redissolved in 15 mL of THF to give a THF solution of the product: macroinitiator CH 3 O—(CH 2 CH 2 O) n —CH 2 —COCl.
ステップ２：重合
（１）使用する前に市販の２－ピロリドンを減圧下、２時間１１０℃で加熱し、水分を除去して精製した。
（２）反応フラスコ中で、カリウムｔｅｒｔ－ブトキシド（ｔ－ＢｕＯＫ, １．３５ ｇ,１２ ｍｍｏｌ）を減圧下、２時間１１０℃で加熱乾燥した。次いで、精製２－ピロリドン （７．６ ｍＬ, １００ ｍｍｏｌ）を、５０℃の窒素流下で反応フラスコに加えてｔ－ＢｕＯＫを完全に溶解した。この反応フラスコを減圧にして泡立ちが何ら残存しなくなるまで副産物を留去した。この反応系を窒素流下に戻し、前記で生成したＰＥＧマクロイニシエーター（１５ ｍＬ, ２ ｍｍｏｌ）を加えた。当該反応系を再度減圧下に戻してＴＨＦを除去し、反応混合物が不動化し、撹拌棒が動かなくなるまで減圧を維持した。最後に、窒素流下に戻し、反応系を２日間密封した。
（３）精製プロセス：４０ｍＬの８０％ＨＣＯＯＨに前記反応混合物を溶解し、３５０ｍＬのアセトンで沈殿させ、沈殿物を１３０００ｒｐｍで遠心して沈殿物を集めた。次いで、沈殿物をヘキサンに再分散させ、再度、遠心分離により集めた（２回繰り返す）。最後に、粗生成物が完全に乾燥するまで減圧下で乾燥した。生成物は、図２及び図３のデータから同定できるとおり、４．６ｇのＰＥＧ－ｂ－Ｎｙｌｏｎ－４であった。
図２の1Ｈ ＮＭＲスペクトルから換算された分子量：Ｍ＝５，０００（３５単位のＧＡＢＡを含む)であった。図３のＭＡＬＤＩ－ＴＯＦマススペクトルの高マス領域は、それぞれ、４４ｍ／ｚ及び８５ｍ/ｚの間隔を伴うＰＥＧ及びＮｙｌｏｎ ４の反復シグナルを示す。 Step 2: Polymerization (1) Prior to use, commercially available 2-pyrrolidone was heated at 110° C. under reduced pressure for 2 hours to remove moisture for purification.
(2) In a reaction flask, potassium tert-butoxide (t-BuOK, 1.35 g, 12 mmol) was dried by heating at 110° C. for 2 hours under reduced pressure. Purified 2-pyrrolidone (7.6 mL, 100 mmol) was then added to the reaction flask under a stream of nitrogen at 50° C. to completely dissolve the t-BuOK. A vacuum was applied to the reaction flask to distill off the by-products until no effervescence remained. The reaction was put back under a stream of nitrogen and the PEG macroinitiator prepared above (15 mL, 2 mmol) was added. The reaction was placed under vacuum again to remove THF and the vacuum was maintained until the reaction mixture was immobilized and the stir bar stopped moving. Finally, the nitrogen stream was put back on and the reaction was sealed for two days.
(3) Purification process: The reaction mixture was dissolved in 40 mL of 80% HCOOH, precipitated with 350 mL of acetone, and centrifuged at 13000 rpm to collect the precipitate. The precipitate was then redispersed in hexane and collected again by centrifugation (repeated twice). Finally, the crude product was dried under reduced pressure until it was completely dry. The product was 4.6 g of PEG-b-Nylon-4 as can be identified from the data in FIGS.
Molecular weight calculated from the 1 H NMR spectrum of FIG. 2: M=5,000 (containing 35 units of GABA). The high mass region of the MALDI-TOF mass spectrum in Figure 3 shows repetitive signals of PEG and Nylon 4 with spacings of 44 m/z and 85 m/z, respectively.
製造例２：ＧＡＢＡ含有ポリマーのナノ粒子（ＮａｎｏＧＡＢＡ）の作製
　３．０ｇのＰＥＧ－ｂ－Ｎｙｌｏｎ－４を４０ｍＬの８０％ＨＣＯＯＨに完全に溶解し、３．５ｋＤａ ＲＣ ダイアライシスメンブランを介して３日間２Ｌの ＭｉｌｉＱ 水に対して透析した（水は1日に２回交換した）。その後、ポリマー懸濁液を超音波ホモジナイザーの影響下で再度物理的に分散させた（パルス：５０％、持続時間：３００秒、反復：３回、出力４，氷冷却）。綿により大きな粒子を除去した後、ポリマー懸濁液をマイクロフルイダイザー中に移し、高圧力下でより小さな粒子に粒子を分割した（１５０００ｐｓｉ, ２００００ｐｓｉ, ２００００ｐｓｉのシステム圧で連続して３回）。最終ステップでは、ナノ粒子の溶液（分散液）を０．２μｍの膜で濾過した。濾過された粒子を集め、さらなる使用に備えて４℃で保存した。生成物：ＮａｎｏＧＡＢＡの溶液、２００ｍＬ、１１．２ｍｇ/ｍＬ、図４参照。）
図４は、流体力学的直径のデータのグラフ表示とＮａｎｏＧＡＢＡの水溶液のデジタル映像である。これらから、作製された溶液中のナノ粒子は８０ｎｍの流体力学的直径を有し、水溶液中で安定であり、かつ、経口及び非経口注入経路による投与に適することが判る。 Preparation Example 2: Preparation of GABA-containing polymer nanoparticles (Nano GABA ) 3.0 g of PEG-b-Nylon-4 was completely dissolved in 40 mL of 80% HCOOH and passed through a 3.5 kDa RC dialysis membrane to 3 Dialyzed against 2 L of MiliQ water daily (water changed twice daily). The polymer suspension was then physically dispersed again under the influence of an ultrasonic homogenizer (pulse: 50%, duration: 300 s, repetition: 3 times, power 4, ice cooling). After removal of large particles with cotton, the polymer suspension was transferred into a microfluidizer and the particles were split into smaller particles under high pressure (3 times in succession at system pressures of 15000 psi, 20000 psi, 20000 psi). In the final step, the nanoparticle solution (dispersion) was filtered through a 0.2 μm membrane. The filtered particles were collected and stored at 4°C for further use. Product: Solution of Nano GABA , 200 mL, 11.2 mg/mL, see FIG. )
FIG. 4 is a graphical representation of hydrodynamic diameter data and a digital image of an aqueous solution of Nano GABA . These show that the nanoparticles in solution produced have a hydrodynamic diameter of 80 nm, are stable in aqueous solutions, and are suitable for administration by oral and parenteral injection routes.
試験例１：急性モデルにおける抗抑うつ様効果
　雄Ｃ５７ＢＬ／６マウス（６～１０週令）を異なる用量のＮａｎｏＧＡＢＡ又はＧＡＢＡでフリードリンク瓶を介して３日間処置した。３日処置後、これらのマウスに若干改変したＰｏｒｓｏｌｔ 他 （Ｎａｔｕｒｅ １９７７, ２６６： ７３０－７３２）に記載されているフォースド・スイム・テスト（Ｆｏｒｃｅｄ Ｓｗｉｍ Ｔｅｓｔｓ ）を受けさせた。この試験では、マウスが彼らの尾を底に届くことができない水のレベルまでに２５℃の水を充填した２Ｌのプラスチックビーカー内に各マウスを置き、モデル動物の挙動を６分間ビデオレコーダーで録画し、泳いでいる（可動性）期間及び浮遊挙動（不動性）期間を測定した。
＜結果＞
　ＮａｎｏＧＡＢＡの自由摂取群は、フォースド・スイム・テストを通して抑うつ様挙動が用量依存的に減少した。この結果はＮａｎｏＧＡＢＡの抗抑うつ様効果の証左である（図５（実験動物：１群７又は８匹）参照。）。３日間自由摂取の別の同様な試験群と比べてみると、ＮａｎｏＧＡＢＡ群は、同様なＧＡＢＡ含有濃度（それぞれ、６ｍＭ）を用いる従来のＧＡＢＡ処置よりも統計的に有意に優れた抗抑うつ効果を示した（図６（実験動物：１群８匹）参照。）。なお、図６において、＊：ｐ ＜ ０．００５、＊＊： ｐ ＜ ０．００５ である。 Test Example 1: Antidepressant-Like Effect in an Acute Model Male C57BL/6 mice (6-10 weeks old) were treated with different doses of Nano GABA or GABA via free drink bottles for 3 days. After 3 days of treatment, these mice underwent the Forced Swim Tests as described by Porsolt et al. (Nature 1977, 266: 730-732) with minor modifications. In this test, each mouse was placed in a 2 L plastic beaker filled with 25° C. water to a level where the mice could not reach the bottom with their tails, and the behavior of the model animal was recorded with a video recorder for 6 minutes. The swimming (mobility) period and the floating behavior (immobility) period were measured.
<Results>
In the Nano GABA ad libitum group, depressive-like behavior decreased dose-dependently through the forced swim test. This result is proof of the antidepressant-like effect of Nano GABA (see FIG. 5 (experimental animals: 7 or 8 animals per group)). When compared to another similar test group fed ad libitum for 3 days, the Nano GABA group showed statistically significantly superior antidepressant effects over conventional GABA treatment with similar GABA-containing concentrations (6 mM each). was shown (see FIG. 6 (experimental animals: 8 animals per group)). In addition, in FIG. 6, *: p<0.005, **: p<0.005.
試験例２：抑うつモデルにおける治療効果
　抑うつモデルを誘発するために、雄Ｃ５７ＢＬ／６マウス（６～１０週令）を５０ｍＬの透明な遠沈管の狭い場所に１４日の期間中、１日当たり３時間閉じ込めた。ＮａｎｏＧＡＢＡの自由摂取処置は第７日から開始しこの試験の最終日まで続けた。第１５日目に、尾の静脈中の血液を集め、ストレスホルモン（コルチコステロン）の濃度を測定した。別に同様な試験をテイル・サスペンション・テスト（Ｔｉａｌ ｓｕｓｐｅｎｓｉｏｎ ｔｅｓｔ）により抑うつ挙動を測定した。このテストでは、マウスの正面の後ろ脚が底面に触れない高さで水平棒にマウスの尾を接着テープで差し込み、付着することにより各マウスを吊るし、６分間マウスの挙動をビデオレコーダーで録画した。
＜結果＞
　ＮａｎｏＧＡＢＡ処置群はストレスホルモンの濃度が低下し（図７参照（実験動物：１群６匹）。）、抑うつモデルマウスにおける抑うつ挙動を弱めた（図８（実験動物：１群８又は９匹）参照。）。なお、図７及び図８において、それぞれ、＊＊：ｐ ＜ ０．００５、＊＊＊：ｐ ＜ ０．０００５である。 Test Example 2: Therapeutic Effect in a Depression Model To induce a depression model, male C57BL/6 mice (6-10 weeks old) were placed in confined spaces in 50 mL clear centrifuge tubes for 3 hours per day for a period of 14 days. Locked up. Ad libitum Nano GABA treatment began on day 7 and continued until the last day of the study. On day 15, tail vein blood was collected and the concentration of stress hormone (corticosterone) was measured. Another similar test was the Tial suspension test to measure depressive behavior. In this test, each mouse was suspended by attaching its tail with adhesive tape to a horizontal bar at a height where the front hind leg of the mouse did not touch the bottom surface, and the behavior of the mouse was recorded with a video recorder for 6 minutes. .
<Results>
The Nano GABA treatment group decreased the concentration of stress hormone (see FIG. 7 (experimental animals: 6 animals per group)), and weakened depressive behavior in depressive model mice (FIG. 8 (experimental animals: 8 or 9 animals per group). )reference.). 7 and 8, **: p < 0.005 and ***: p < 0.0005, respectively.
試験例３：イン・ビボ（ｉｎ ｖｉｖｏ）毒性
　ＩＣＲ雄マウス（５週令、一群３匹）に高用量のＮａｎｏＧＡＢＡ、ＧＡＢＡ又はＭｉｌｉＱ 水を腹腔内に注入した。３日後、マウスを安楽死させ、血液と臓器を集めた。血液の組成と臓器の機能関連バイオマーカーをも測定し、ＮａｎｏＧＡＢＡの急性毒性を評価した。
＜結果＞
　ＮａｎｏＧＡＢＡ処置群は、７２ｍＭ ＧＡＢＡ含有量の用量において、体重に顕著な影響を及ぼさず（図９参照。）、さらに、臓器の重量、臓器の機能関連バイオマーカー及び血液の組成にも顕著な影響を及ぼさなかった。このことは、この試験では、ＮａｎｏＧＡＢＡがマウスに対して急性毒性を誘発しなかったことを示す（図１０参照。）。 Test Example 3: In vivo Toxicity ICR male mice (5 weeks old, 3 per group) were injected intraperitoneally with a high dose of Nano GABA , GABA or MiliQ water. After 3 days, mice were euthanized and blood and organs were collected. Blood composition and organ function-related biomarkers were also measured to assess the acute toxicity of Nano GABA .
<Results>
The Nano GABA -treated group had no significant effect on body weight at a dose of 72 mM GABA content (see Figure 9), and also significant effects on organ weights, organ function-related biomarkers and blood composition. did not affect This indicates that Nano GABA did not induce acute toxicity in mice in this test (see Figure 10).
試験例４：ナノ粒子の特性及びその薬剤投与に関する安定性
　製造例２と同様の方法で作製された別のバッチによるナノ粒子（ＮａｎｏＧＡＢＡ）の動力学的光散乱の測定結果のデータのグラフ表示との水溶液のデジタル映像を図１１の（Ａ）に示す。ここでもまた、当該ナノ粒子は平均粒径約８０ｎｍを有し、優れた分散性を示し、当該ナノ粒子の作製方法は再現性の高い生産性を示すことが判る。 Test Example 4: Properties of nanoparticles and their stability with respect to drug administration. A digital image of the aqueous solution of is shown in FIG. 11(A). Again, it can be seen that the nanoparticles have an average particle size of about 80 nm, exhibit excellent dispersibility, and the method for producing the nanoparticles exhibits high reproducibility and productivity.
　また、長期間貯蔵条件下での当該ナノ粒子の安定性について次のとおり調査した。ナノ粒子溶液（[ＮａｎｏＧＡＢＡ]＝８．０ｍｇ/ｍＬ）を冷蔵庫において４℃で貯蔵した。所定の期間後、ナノ粒子の溶液の流体力学的パラメーターを２５℃における動力学的散乱光の測定によりチェックした。結果を図１１の（Ｃ）に示す。さらに、生理学的条件を模倣する、例えば、胃（ｐＨ２‐３）、腸（ｐＨ７‐８）、腫瘍領域（ｐＨ５．５‐７）、種々のｐＨ条件下で前記ナノ粒子の 安定性について動力学的散乱光の測定を行った。結果を図１１の（Ｄ）に示す。これらの図から、当該ナノ粒子は測定された貯蔵期間を通して高い安定性を示し、また、各種ｐＨ下での薬剤投与に適する高い安定性を示すことも判る。 In addition, the stability of the nanoparticles under long-term storage conditions was investigated as follows. Nanoparticle solutions ([Nano GABA ]=8.0 mg/mL) were stored in a refrigerator at 4°C. After a given period of time, the rheological parameters of the nanoparticle solution were checked by dynamic light scattering measurements at 25°C. The results are shown in FIG. 11(C). Furthermore, mimicking physiological conditions, e.g. stomach (pH 2-3), intestine (pH 7-8), tumor area (pH 5.5-7), kinetics for the stability of the nanoparticles under various pH conditions. measured the scattered light. The results are shown in FIG. 11(D). It can also be seen from these figures that the nanoparticles exhibit high stability over the measured storage period and also exhibit high stability suitable for drug administration under various pH.
　加えて、ヒトでの薬剤投与、又はイン・ビトロ若しくはイン・ビボでの用途に関して、滅菌は重要であり、特に注射製剤では、必須である。そこで、当該ナノ粒子の滅菌可能性を製薬業界で汎用されている滅菌法である、濾過により調査した。ここでは、ナノ粒子溶液を、細菌を除去するための平均サイズ０．２０μｍの細孔を備えた滅菌酢酸セルロース膜のフィルターユニットを介して濾過した。次いで、濾過の前後のナノ粒子の濃度を蒸発法により再度確認した。結果を図１１の（Ｂ）に示す。これらの滅菌法により生ずるナノ粒子の濃度に著しい変化はみられず、前記ナノ粒子溶液は高度な滅菌要件を必要とする調製に適することを示す。 In addition, for drug administration in humans, or for in vitro or in vivo use, sterilization is important, especially for injectable formulations. Therefore, the sterilization potential of the nanoparticles was investigated by filtration, which is a sterilization method commonly used in the pharmaceutical industry. Here, the nanoparticle solution was filtered through a sterile cellulose acetate membrane filter unit with pores of average size 0.20 μm to remove bacteria. The concentration of nanoparticles before and after filtration was then checked again by the evaporation method. The results are shown in FIG. 11(B). There was no significant change in the concentration of nanoparticles resulting from these sterilization methods, indicating that the nanoparticle solutions are suitable for preparations requiring high sterilization requirements.
試験例５：ナノ粒子の自由飲用（ｆｒｅｅ－ｄｒｉｎｋｉｎｇ）によるＮａｎｏＧＡＢＡの毒性
　経口投与によるＮａｎｏＧＡＢＡの毒性の調査。１０週令Ｃ５ＢＬ/６Ｊ雄マウス（１群６匹）をＮａｎｏＧＡＢＡの１．０ｍｇ/ｍＬ溶液を含む飲用ボトルに自由に接近させた。投与０日、１日、２日、３日、４日目の、被検マウスの体重変化を図１２に示す。また、４日後、マウスを安楽死させ、ＤＲＩ－ＣＨＥＭによる臓器の重量、臓器の機能、並びに血液組成のＣＥＬＬ－ＴＡＧシステムによる、調査を行った。結果を図１３の（Ａ）及び（Ｂ）に示す。 Test Example 5: Toxicity of Nano GABA by free-drinking of nanoparticles To investigate the toxicity of Nano GABA by oral administration. Ten-week-old C5BL/6J male mice (6 per group) were given free access to drinking bottles containing a 1.0 mg/mL solution of Nano GABA . FIG. 12 shows changes in body weight of the test mice on days 0, 1, 2, 3 and 4 of administration. After 4 days, the mice were also euthanized and examined by DRI-CHEM for organ weight, organ function, and blood composition by CELL-TAG system. The results are shown in FIGS. 13A and 13B.
　これらの図から、ＮａｎｏＧＡＢＡは、被検動物の体重、臓器の重量及び血液組成に影響を及ぼさないことが判る。また、腎臓機能及び肝臓機能のパラメーターにおいて、何らかの傾向が存在するが、統計的な有意差を示していない。したがって、ＮａｎｏＧＡＢＡは経口登用によりマウスに著しい毒性を示さない。 These figures show that Nano GABA does not affect the body weight, organ weights and blood composition of the test animals. There are also some trends in renal and liver function parameters, but they do not show statistical significance. Therefore, Nano GABA does not show significant toxicity to mice upon oral administration.
　本願発明従うＰＥＧ－ｂ－Ｎｙｌｏｎ－４ブロックコポリマーは、それ自体特有の物理化学的性質又は薬理学的性質を有し、また、それ自体若しくはそれから作製されたポリマーのナノ粒子は、例えば、経口又は非経口投与することにより患者の抑うつを予防又は治療できるので、各所有機材料を提供若しくは利用する産業又は医薬製造業で利用できる可能性をもつ。 The PEG-b-Nylon-4 block copolymers according to the present invention have unique physico-chemical or pharmacological properties per se, and nanoparticles of the polymer itself or made therefrom can be administered, for example, orally or Since it is possible to prevent or treat depression in patients by parenteral administration, there is a possibility that it can be used in industries that provide or use various organic materials or pharmaceutical manufacturing industries.

Claims (8)

  1. 　式Ｉ：
     
    　　　－（ＣＨ２ＣＨ２－Ｏ）ｍ－ (I)
     
    式中、ｍは２～５００の整数である、
     
    で表される反復単位のセグメントと、
    式ＩＩ:
     
    　　－（ＮＨＣＨ２ＣＨ２ＣＨ２Ｃ（＝Ｏ））ｎ－ (II)
     
    式中、ｎは、５～１０００の整数である、
     
    で表される反復単位のセグメントを、含むブロックコポリマー。     Formula I:

    —(CH 2 CH 2 —O) m — (I)

    wherein m is an integer from 2 to 500,

    a segment of the repeating unit represented by , and
    Formula II:

    - ( NHCH2CH2CH2C (=O)) n- ( II )

    wherein n is an integer from 5 to 1000;

    A block copolymer comprising a segment of repeating units represented by
  2. 　請求項１のブロックコポリマーであって、水性媒体中でポリマーのナノ粒子を形成する、前記ブロックコポリマー。 The block copolymer of claim 1, which forms nanoparticles of the polymer in an aqueous medium.
  3. 　次式IIIａ又は式IIIｂで表されるブロックコポリマー。
    式IIIａ：
     
    　Ａ１－（ＣＨ２ＣＨ２－Ｏ）ｍ１－Ｌ1－（ＮＨＣＨ２ＣＨ２ＣＨ２Ｃ（＝Ｏ））ｎ１－Ｗ１
     
    　　　　　　　　　　　　（IIIａ）
    又は式IIIｂ:
     
    　Ａ２－（ＣＨ２ＣＨ２－Ｏ）ｍ２－Ｌ２-（Ｃ（＝Ｏ）ＣＨ２ＣＨ２ＣＨ２ＮＨ）ｎ２－Ｗ２
     
    　　　　　　　　　　　　（IIIｂ)
     
    で表され、各式中、
    　Ａ1及びＡ2は独立して、非置換又は置換Ｃ1－Ｃ12アルキルオキシを表し、置換されている場合の置換基は、ホルミル基又は式Ｒ'Ｒ"ＣＨ－基を表し、ここで、Ｒ'及びＲ"は独立してＣ1－Ｃ4アルコキシ又はＲ'とＲ"は一緒になって－ＯＣＨ2ＣＨ2Ｏ－、－Ｏ（ＣＨ2）3Ｏ－若しくは－Ｏ（ＣＨ2）4Ｏ－を表し、
    　Ｌ１及びＬ２は独立して、二価の連結基又は存在する場合には直接結合を表し、
    　Ｗ１は、ヒドロキシル基又は非置換若しくは置換Ｏ－Ｃ1－Ｃ６アルキルを表し、置換されている場合の置換基はハロゲン原子、ヒドロキシル基又はＣ1－Ｃ３アルキル基を表し、
    　Ｗ２は、水素原子又は非置換若しくは置換Ｃ1－Ｃ６アルキルを表し、置換されている場合の置換基はハロゲン原子、ヒドロキシル基又はＣ1－Ｃ３アルキルを表し、又は非置換若しくは置換Ｃ（＝Ｏ）Ｃ1－Ｃ６アルキルを表し、置換されている場合の置換基はハロゲン原子、ヒドロキシル基若しくはＣ1－Ｃ３アルキル基を表し、
    　ｍ１及びｍ２は独立して、２～５００の整数であり、かつ、
    　ｎ１及びｎ２は独立して、５～１０００の整数である。     A block copolymer represented by the following Formula IIIa or Formula IIIb.
    Formula IIIa:

    A 1 -(CH 2 CH 2 -O) m1 -L 1 -(NHCH 2 CH 2 CH 2 C(=O)) n1 -W 1

    (IIIa)
    or Formula IIIb:

    A 2 —(CH 2 CH 2 —O) m2 —L 2 —(C(=O)CH 2 CH 2 CH 2 NH) n2 —W 2

    (IIIb)

    In each formula,
    A 1 and A 2 independently represent unsubstituted or substituted C 1 -C 12 alkyloxy and the substituents, if substituted, represent a formyl group or a group of the formula R′R″ CH— , wherein , R ′ and R ″ are independently C 1 -C 4 alkoxy or R ′ and R ″ together are —OCH 2 CH 2 O—, —O(CH 2 ) 3 O— or —O(CH 2 ) represents 4 O-,
    L 1 and L 2 independently represent a divalent linking group or a direct bond when present;
    W 1 represents a hydroxyl group or an unsubstituted or substituted O—C 1 -C 6 alkyl group, the substituent group if substituted represents a halogen atom, a hydroxyl group or a C 1 -C 3 alkyl group;
    W 2 represents a hydrogen atom or unsubstituted or substituted C 1 -C 6 alkyl, the substituents if substituted represent a halogen atom, hydroxyl group or C 1 -C 3 alkyl, or unsubstituted or substituted C (=O) represents C 1 -C 6 alkyl, where the substituents, if substituted, represent a halogen atom, a hydroxyl group or a C 1 -C 3 alkyl group;
    m1 and m2 are independently integers from 2 to 500, and
    n1 and n2 are independently integers from 5 to 1,000.
  4. 　請求項３のブロックコポリマーであって、水性媒体中でポリマーのナノ粒子を形成する、前記ブロックコポリマー。 The block copolymer of claim 3, said block copolymer forming nanoparticles of the polymer in an aqueous medium.
  5. 　請求項３のブロックコポリマーであって、
    　Ｌ１は、（ＣＨ２）ｐＣ（＝Ｏ）、Ｃ（＝Ｏ）（ＣＨ２）ｑ又はＣ（＝Ｏ）（ＣＨ２）ｒＣ（＝Ｏ）を表し、
    　Ｌ２は、直接結合、（ＣＨ２）ｓＯ又は（ＣＨ２）ｔＮＨを表し、
    　ｐ、ｑ、ｒ、ｓ、及びｔは、それぞれ独立して、１～６の整数である、ブロックコポリマー。     4. The block copolymer of claim 3,
    L 1 represents (CH 2 ) p C(=O), C(=O)(CH 2 ) q or C(=O)(CH 2 ) r C(=O);
    L 2 represents a direct bond, (CH 2 ) s O or (CH 2 ) t NH;
    A block copolymer wherein p, q, r, s, and t are each independently an integer from 1 to 6.
  6. 　請求項３のブロックコポリマーをギ酸（ＨＣＯＯＨ）含有水溶液に溶解するステップと、
    　前記ポリマーの溶解した溶液を透析膜を介して水に対して透析するステップと、
    　得られたポリマー懸濁液を超音波ホモジナイザー中で処理して懸濁液中の粒子を分割するステップと、
    　粒子を分割した分散液を細孔を備えた膜でろ過するステップ
    を含む、ポリマーのナノ粒子の作製方法。     dissolving the block copolymer of claim 3 in an aqueous solution containing formic acid (HCOOH);
    dialysis of the dissolved solution of the polymer against water through a dialysis membrane;
    treating the resulting polymer suspension in an ultrasonic homogenizer to break up particles in the suspension;
    A method of making polymeric nanoparticles comprising filtering a dispersion of divided particles through a membrane with pores.
  7. 　有効成分としての請求項３のブロックコポリマーと当該技術分野で常用されている添加剤又は希釈剤を含む製薬学的製剤。 A pharmaceutical formulation containing the block copolymer of claim 3 as an active ingredient and an additive or diluent commonly used in the art.
  8. 　抑うつの予防又は治療に使用するための請求項７の製薬学的製剤。 The pharmaceutical preparation of claim 7 for use in preventing or treating depression.
PCT/JP2022/020180 2021-05-19 2022-05-13 POLY(ETHYLENE GLYCOL)-b-POLY(4-NYLON) AND NANOPARTICLES THEREOF WO2022244690A1 (en)

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Publication number Priority date Publication date Assignee Title
JP2002265596A (en) * 2001-03-15 2002-09-18 National Institute Of Advanced Industrial & Technology 2-pyrrolidone polymer having special structure and method of manufacturing the same
JP2003528939A (en) * 1999-09-03 2003-09-30 スクール オブ ファーマシー， ユニヴァーシティ オブ ロンドン Degradable polymer
CN105622932A (en) * 2014-11-07 2016-06-01 合肥杰事杰新材料股份有限公司 Nanometer nylon microsphere and preparation method thereof

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JP2003528939A (en) * 1999-09-03 2003-09-30 スクール オブ ファーマシー， ユニヴァーシティ オブ ロンドン Degradable polymer
JP2002265596A (en) * 2001-03-15 2002-09-18 National Institute Of Advanced Industrial & Technology 2-pyrrolidone polymer having special structure and method of manufacturing the same
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