Patent Application
20240000829
The present invention relates to a copolymer containing poly(ethylene glycol) and poly(L-amino acid derivative), in particular, a copolymer containing a poly(ornithine) segment or containing a poly(aspartic acid ester), poly(acylated arginine) or poly(ornithine)-co-poly(aspartic acid) segment, microparticles thereof, and use thereof in a pharmaceutical composition.
In patients with hepatic disorders such as hepatic cirrhosis, pathological conditions such as hyperammonemia due to abnormal urea cycle may be developed. It has been reported that L-ornithine, when administered to such patients, not only functions as a substrate for ureagenesis, but also activates urea cycle enzymes including carbamyl phosphate synthetase (CPS) and ornithine transcarbamylase (OTC), resulting in improved urea cycles and reduced ammonia levels (Non-Patent Document 1).
It has been reported that, thus, L-ornithine and a mixture of L-ornithine and L-aspartic acid (LOLA) have a beneficial effect of reducing an ammonia concentration.
On the other hand, in Non-Patent Document 2, it has been confirmed, by a double-blind, randomized, placebo-controlled study, that infusion of the amino acid mixture is extremely weak in efficacy. This is considered to be because water-soluble low-molecular-weight amino acids have a low amount of uptake into the living body and are extremely rapidly metabolized.
In order to solve such problems, the inventors have so far designed a copolymer (polycationic polymer) of poly(ethylene glycol) and poly(arginine), and proposed a polyion complex (PIC) obtained by combining the copolymer with a polyanion such as chondroitin sulfate (Patent Document 1). In addition, the inventors have demonstrated that the retention in blood and accumulation in the liver of a self-assembling drug composed of PIC in which ornithine is incorporated (abbreviated as NanoORN) are greatly improved, and further that, upon administration of NanoORN to mice with acute hepatic disorders caused by acetaminophen (APAP), not only was the blood ammonia significantly increased by APAP hepatic disorders reduced to a normal level but levels of AST and ALT, which are hepatic function markers, were significantly reduced, resulting in an improved liver function (Non-Patent Document 3). In addition, it has been confirmed that NanoORN brings about a liver function improving effect also in NASH model mice.
Thus, by incorporating L-ornithine into a self-assembling drug and subcutaneously administering the drug, the bioavailability of L-ornithine was improved and the liver function improving effect could be obtained.
On the other hand, NanoORN using PIC as a driving force is unstable against changes in ionic strength and pH, and may be disadvantageous for oral administration, for example, it may liberate a polycation and cause toxicity.
In order to solve the above problems, it is an object of the present invention to provide an assembled body of ornithine low in toxicity, effective even in oral administration and high in bioavailability. Another object of the present invention is to provide an ornithine assembled body which is effective in preventing or treating hepatic disorders.
The inventors have found that, by newly introducing an acyl group or the like into an ornithine side chain amino group of poly(ethylene glycol)-b-poly(ornithine), a copolymer capable of forming a novel assembled body which does not require formation of PIC can be provided. Also, the inventors have found that the thus-provided copolymer is made into a composition with a copolymer containing another poly(ethylene glycol) segment and a poly(side chain protected L-amino acid) segment, and thus the natures, functions or effects of the former copolymer may be enhanced.
Accordingly, the present invention provides the following aspects.
The self-assembling drug containing the copolymer of the present invention as an active ingredient is orally administered and used as a novel therapeutic drug for hepatic disorders. In particular, when orally administered, the self-assembling drug accumulates in the intestinal mucosa, is hydrolyzed by a degrading enzyme, and gradually liberates ornithine, resulting in high bioavailability of ornithine in the body and, besides, sustained effect. On the other hand, the toxicity of the self-assembling drug of the present invention is low.
The technical terms used herein have meanings commonly used in the art, unless otherwise defined.
The copolymer according to an embodiment of the present invention is as represented by formula (I) of aspect (1).
In formula (I), A
The linking group(s) L and/or L′ in formula (I) can be any organic divalent group(s) as long as it/they does/do not adversely affect the formation of the ornithine microparticles, but generally represent(s) —O—(CH2)a—NH—, —O—(CH2)a—O—, —(CH2)a—NH— or —(CH2)a—O—, preferably —O—(CH2)a—NH—, —(CH2)a—NH—, wherein a is an integer of from 1 to 6, preferably from 1 to 3. The bonding directionality of these linking groups is forward directionality of each moiety in the structural formula of each formula with respect to L. For example, taking —O—(CH2) a —NH— as an example, the linker on the —O—(CH2) moiety side is covalently bonded to the methylene of formula (I), and the NH— moiety is covalently bonded to the carbonyl group. On the other hand, the directionality of L′ is opposite to that of L.
In formula (I), Y and Y′ each independently represent a hydrogen atom, unsubstituted C1-21 alkylcarbonyl, substituted C1-4 alkylcarbonyl, unsubstituted or substituted C3-7 cycloalkylcarbonyl, unsubstituted or substituted arylcarbonyl, or unsubstituted or substituted 5- or 6-membered heteroarylcarbonyl.
The alkyl as each group or a part of each group above can be straight or branched chain, and can be, but is not limited to, applicable one among methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, nonyl, undecyl, tridecyl, heptadecyl, nonadecyl and the like. Preferred aryl is selected from C1-6 alkyl. The C3-7 cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl. The aryl can be phenyl, biphenyl, or naphthyl. The 5- or 6-membered heteroaryl is an unsaturated heterocyclic radical containing one or two identical or different heteroatoms selected from oxygen, nitrogen and sulfur atoms, and may be thienyl, furyl, pyranyl, pyrrolyl, isoxazole, pyrazolyl, imidazolyl, pyridyl, pyrazinyl, or pyrimidinyl, and these heterocyclic rings may also be benzo-fused. Such fused rings include, for example, isoindolyl, indolyl, quinolyl, isoquinolyl, quinoxalinyl, quinazolinyl, and phenanthridinyl.
The substituent of the substituted C1-4 alkylcarbonyl is selected from the group consisting of halogen atoms (Cl, F, Br, and I), hydroxyl, carboxyl, unsubstituted or substituted C3-7 cycloalkyl, unsubstituted or substituted aryl and unsubstituted or substituted 5- or 6-membered heteroaryl, unsubstituted or substituted adamantyl, and unsubstituted or substituted cholesterol residues. When the substituent is substituted, a substituent may be C1-4 alkyl, C1-4 alkyloxy, hydroxyl, carboxyl, cyano, nitro, halogen atom, or mono- or di-C1-4 alkylamino. The last substituent is also applied to substituted C3-7 cycloalkylcarbonyl, substituted arylcarbonyl, or substituted 5- or 6-membered heteroarylcarbonyl.
In the above definitions, the cholesterol residue can be a residue in which any H on the 22nd to 27th carbons in the cholesterol molecule is eliminated or a hydrocarbon chain containing any of the 22nd to 27th carbons is eliminated. Examples of the substituted alkylcarbonyl of such a residue include cholic acid and chenodeoxycholic acid. Preferred as Y is C1-6 alkylcarbonyl.
R10 and R10′ in formulae (I) and (ii) are independently a hydrogen atom or R11—(C═O). Without being bound by theory, when R10 and R10′ include R11—(C═O), it is assumed that the amide bond between this and the δ-amino group of the polyornithine segment and the amide (or peptide) bond of the polyornithine main chain are each subjected to enzymatic degradation, resulting in release of free ornithine. Also, it is assumed that they are degraded, for example, by digestive enzymes in the digestive tract, resulting in gradual release of ornithine.
R11 is substituted or unsubstituted C1-C21 alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted C1-C21 alkoxy. When the R11 is substituted, a substituent is C1-4 alkyl, C1-4 alkoxy, or aryl, and, more preferably, R11 can be a methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, (optionally branched) pentyl, (optionally branched) hexyl, (optionally branched) heptyl, (optionally branched) octyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, phenyl, naphthyl, benzyloxy, or tert-butyloxy group. These groups are preferable from the perspective of efficiency in assembling the copolymer and forming the ornithine microparticles. On the other hand, from the perspective of ease of synthesis, R11 is preferably a methyl, propyl, isopropyl, butyl, isobutyl, phenyl (which may have a substituent), or benzyl (which may have a substituent) group.
For example, when R11 is methyl, it can be obtained by using, for example, acetic anhydride as a carboxylic acid ester in a condensation reaction of formula (II) which will be described later in the “production of the copolymer” section. Similarly, the desired R11 can be obtained by changing the kind of carboxylic acid, for example, using propionic anhydride when R11 is ethyl, using butyric anhydride when R11 is propyl, using isobutyric anhydride when R11 is isopropyl, using pentanoic anhydride when R11 is butyl, using isopentanoic anhydride when R11 is isobutyl, using benzoic anhydride when R11 is phenyl, and using bisphenylacetic anhydride when R11 is benzyl. By using a carboxylic acid ester having a substituent or a derivative thereof, R11 can also have a substituent.
m and m′ may independently be an integer of preferably from 2 to 300, more preferably from 15 to 150, and most preferably from 15 to 100, from the perspective of stability of particles formed by the copolymer.
Similarly, n can be an integer of preferably from 2 to 1000, more preferably from 10 to 500, and most preferably from 30 to 400.
The m and m′ (R10)'s and (R10′)'s in formula (I) can independently be hydrogen atoms in a proportion of generally up to 80%, preferably up to 60%, more preferably up to 30%, most preferably up to 10%, and, particularly most preferably, all (100%) of the m and m′ (R10)'s and (R10′)'s are (R11—(C═O))'s.
An example of a scheme for synthesizing the copolymer will be shown below. However, the following is merely an example, and the present invention is not limited by the example.
The copolymer may contain the corresponding PEG segment and polyornithine segment produced by any method as long as it is consistent with the objectives of the present invention. However, the copolymer is preferably a copolymer having a narrow molecular weight distribution and capable of providing ornithine microparticles formed by self-assembly in an aqueous medium with an appropriate average diameter.
The copolymer can be provided by preparing a copolymer represented by the following formula (II) in advance, and then adding a compound such as a carboxylic acid ester for the purpose of introducing an acyl group into the 6-amino group in the segment derived from L-ornithine in the formula.
In the formula,
A typical example of such production is a condensation reaction between the copolymer represented by formula (II) and a carboxylic acid ester as shown in the following scheme.
Examples of the carboxylic ester include carboxylic halides, carboxylic anhydrides, carboxylic azides and active esters, and specific examples thereof include acetic, propionic, isobutyric, butyric, oxalic, succinic, glutaric, adipic, maleic, phthalic, benzoic, and bisphenylacetic anhydrides.
When A in formula (II) in the above reaction scheme corresponds to that defined in (ii)′ above, the δ-amino group in the segments derived from ornithine corresponding to the m′ repeat units in the formula described therein undergoes a condensation reaction in the same manner as that of the m repeat unit.
Further, for the purpose of providing the copolymer according to an embodiment of the present invention having a narrow molecular weight distribution, the precursor copolymer represented by formula (II) (taking the case where A in formula (II) is represented by (I)′ as an example) is preferably produced according to the following synthesis scheme.
Here, compound 1 is commercially available or prepared by a method according to its provision method and has a molecular weight distribution as narrow as possible; compound 2 is produced by subjecting the compound to living ring-opening polymerization with N-carboxylic anhydride of ornithine in which the amino group at the δ-position is protected, and then terminating the reaction using a living terminal modifier such as acetic anhydride by a method known per se; and further an amino protecting group in the poly(L-ornithine) segment is protected.
According to such a reaction treatment using the compound 1 as a raw material, the poly(L-ornithine) segment can also be provided as a precursor copolymer having an extremely narrow molecular weight distribution.
Therefore, the finally obtained block copolymer represented by formula (I) according to an embodiment of the present invention has a molecular weight distribution of from 1.01 to 1.20, preferably from 1.01 to 1.06.
On the other hand, when A in formula (II) is defined as (ii)′, the compound 1 as the starting material in the above reaction scheme may be NH2CH2CH2—(OCH2CH2)n—NH2, hereinafter, the A moiety of each of compounds 2 to 4 may be repeat units corresponding to the m repeat units. The triblock copolymer thus obtained, in which A in formula (II) is defined as (ii)′, is represented by the following formula (III):
As another example, the copolymer can also be synthesized by a synthesis method as represented by the following scheme, starting from the compound 1 described above.
The ornithine microparticles can be produced by assembling the copolymer represented by formula (I) in water or an aqueous medium (which may contain a water-soluble organic solvent or a buffer). It is considered that, since the polyornithine segment containing R10 and R10′ exhibits hydrophobicity, the copolymer aggregates and self-assembles, and forms ornithine microparticles having the polyornithine segment as a core and the PEG segment as a shell. For example, ornithine microparticles can be obtained by dissolving the copolymer represented by formula (I) in a polar organic solvent such as dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) and dialyzing the solution in water, an aqueous solution or a buffer solution.
The ornithine microparticles (or assembled bodies) are not particularly limited, but from the perspective of ease of administration or production, the average particle diameter is preferably 1 millimeter or less, more preferably from 1 nanometer to 1 millimeter, and still more preferably from 1 nanometer to 100 micrometers. More preferably, it is from 10 to 200 nanometers, and most preferably from 20 to 70 nanometers. The particle diameter can be measured by dynamic light scattering (DLS) or the like in water or an aqueous medium.
For example, polyion complex particles (PIC) can be obtained by adding a solution of a polyanionic compound polymer such as chondroitin to compound 3 or formula (II) and thus forming a complex thereof. On the other hand, the ornithine microparticles of the present invention are characterized in that they are obtained by self-assembly of a poly(ethylene glycol)-b-poly(ornithine) copolymer or a derivative thereof alone in an aqueous medium and are not formed into a complex with another polyanionic compound polymer. Therefore, the ornithine microparticles of the present invention are distinguished from PIC.
Thus, preferably, the ornithine particles of the present invention is characterized in that they contain a poly(ethylene glycol)-b-poly(ornithine) polymer or a derivative thereof as a main component, and do not contain a polyanionic polymer or the like. Examples of the polyanionic polymer can include polyacrylic acid, polymethacrylic acid, polysulfonic acid, polyanionic polysaccharide, and anionic protein, and, more specifically, can include chondroitin sulfate, carrageenan, heparin, carboxymethyl dextran, xanthan gum, hyaluronic acid, polyaspartic acid, polyglutamic acid, polyacrylic acid, polymethacrylic acid, polyvinyl sulfate, and polystyrene sulfonic acid. However, the ornithine microparticles of the present invention may contain an aqueous medium, a solvent or the like as long as the stability of the particles is not adversely affected.
The obtained ornithine microparticles can be separated by a separation means such as centrifugation, or can be stored as a dry composition by freeze-drying, and can be reconstituted in an aqueous medium as needed. Such a dry composition can be provided as an aqueous solution of ornithine microparticles optionally containing a physiologically acceptable diluent or excipient. Such a diluent may be sterile water, physiological saline, a solution containing a physiologically acceptable buffer, or the like, and may further contain, as an additive, for example, sorbitol, dextrin, glucose, mannitol, an amino acid (for example, glycine, isoleucine, valine, methionine, glutamic acid, or the like), or the like.
In other words, as described above, one embodiment of the present invention provides the following method for producing ornithine microparticles. That is, the present invention provides a method for producing ornithine microparticles, including reacting a copolymer represented by the above formula (II) with a carboxylic acid ester compound; adding a substituent composed of R10 to a δ-amino group in a segment derived from ornithine, wherein R10 is R11—(C═O)—, R11 is substituted or unsubstituted C1-C21 alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted C1-C21 alkoxy, and when these substituents are substituted, substituents are C1-4 alkyl, C1-4 alkoxy, or aryl; and assembling the resulting copolymer in water.
<Further Copolymers which can Also be Used as Other Additives Together with or Independently of Additives Described Above or Below (which May Include Novel Compound)>
The copolymer represented by formula (I) or the ornithine microparticles from the copolymer may be provided as pharmaceutical compositions containing the following copolymer together with or independently of those described above as additives, for example, pharmaceutical compositions for preventing or treating hepatic dysfunction.
Copolymer Represented by Formula (IV):
In the formula, AASP and LASP have the same definitions as A (i) and L in formula (I), and specifically, the description for L in formula (I) can be applied. YASP has the same definition as Y in formula (I). Further, RASP is a hydrogen atom, substituted or unsubstituted C1-C21 alkyl, or substituted or unsubstituted aryl, and when these substituents are substituted, substituents are C1-4 alkyl, C1-4 alkoxy, or aryl. With respect to the C1-C21 alkyl and aryl, the description for R11 in formula (I) can be applied. Specific examples of the linking group as LASP are the same as those specifically described for L.
mA is an integer of from 2 to 300, preferably from 15 to 150, more preferably from 15 to 100, and
nA is an integer of from 2 to 1,000, preferably from 10 to 500, and more preferably from 30 to 400.
As the copolymer represented by formula (IV), a compound in which m protected ornithine units in the compound 2 are replaced with protected aspartic acid units can be provided by living ring-opening polymerization of the omega (ω) terminal amino group of compound 1 using, for example, N-carboxylic anhydride of benzylated or alkylated O-carboxyl of aspartic acid instead of N-carboxylic anhydride of ornithine having a protected δ-amino group in a reaction scheme from the compound 1 to compound 4. The compound thus provided can be obtained, if necessary, through protective group elimination, esterification, alkyl group exchange reaction, and the like which are known per se. When RASP is a hydrogen atom or benzyl, compounds known per se may be included, but other compounds represented by formula (IV-a) are considered to be copolymers which are not described in prior art documents as far as the present inventors know. Such copolymers can also be automatically assembled in an aqueous medium and form microparticles having a size similar to that of the copolymer represented by formula (I).
Since low-molecular-weight L-aspartic acid also has an action of discharging harmful ammonia similar to low-molecular-weight L-ornithine to the outside of the body, the copolymer represented by formula (IV) can also enhance the action or effect of the copolymer of formula (I), or can enhance the stability or the like of a pharmaceutical composition containing the copolymer of formula (I) or microparticles (assembled bodies) thereof.
Copolymer Represented by Formula (V):
In the formula, ACO, LCO, nCO, RASP, and mA have the same meanings as AASP, LASP, nA, RASP, and mA, respectively, defined for formula (IV) above. AORN, RORN, mO and YCO have the same definitions as A (i), R, m, n and Y, respectively, defined for formula (I). In this copolymer, the respective mASP units and the respective mORN units can be each present at random.
The copolymer represented by formula (V) can be provided as a polymer in which m protected ornithine units in the compound 2 are randomly replaced with protected aspartic acid units and protected ornithine units, by living ring-opening polymerization of N-carboxylic anhydrides to the omega (co) terminal amino group of the compound 1 and the newly generated amino group in the reaction scheme from the compound 1 to the compound 4 using, for example, a mixture of N-carboxylic anhydride of benzylated β-carboxyl of aspartic acid and N-carboxylic anhydride of protected ornithine instead of N-carboxylic anhydride of δ-amino group-protected ornithine, and can be obtained through protective group elimination, esterification and/or acylation known per se. The copolymer thus provided can also be assembled similarly to the copolymer of formula (I).
The copolymer represented by formula (V) can also have the same action and effect as those of the copolymer represented by formula (I) or (IV) and the assembled product thereof.
Copolymer Represented by Formula (VI):
Wherein AARG, LARG, nG, mG, and YARG have the same meanings as AASP, LASP, nA, mA, and YARG, respectively, defined for formula (IV) above, and RARG and RARG′ independently have the same meanings as RASP defined for formula (IV) above. The copolymer of formula (IV) can conveniently be produced by a reaction of the copolymer described in WO2016/167333 A1 wherein RARG and RARG′ are each H and YARG may have the same definition as YASP in formula (IV) with anhydrides or carboxylic acids capable of forming RARG and RARG′. The copolymers thus prepared can be assembled without the use of polyanionic polymers. These copolymers or assembled products thereof can have the same actions and effects as the copolymers of formula (I) and assembled products thereof, except that they have been confirmed to exhibit anti-tumor effects on lung cancer-metastasis model mice and subcutaneously transplanted cancer-model mice upon oral administration and to greatly reduce the adhesion of melanoma cells to the lungs (data unpublished), in addition to the actions and effects (anti-tumor effects) described in WO2016/167333 A1.
The ornithine microparticles (or assembled products) formed from the copolymer of formula (I) according to an embodiment of the present invention are used as a composition for the prevention and treatment of hepatic disorders. That is, as one embodiment, the present invention provides a pharmaceutical composition for use in preventing or treating hepatic dysfunction, which contains the ornithine microparticles according to an embodiment of the present invention as an active ingredient.
Ornithine functions as a substrate for ureagenesis or activates urea cycle enzymes including carbamoylphosphate synthase (CPS) and ornithine transcarbamylase (OTC). Through these actions, effects of prevention and treatment of hepatic disorders are expected.
When orally administered, the ornithine microparticles according to an embodiment of the present invention accumulate in the intestinal mucosa, gradually undergo enzymatic degradation, and cause hydrolysis of the amide bond in the polymer, resulting in release of ornithine. Therefore, the ornithine microparticles effectively act in the living body, have high bioavailability, and are very effectively used in the prevention or treatment of hepatic dysfunction as shown in model animal experiments in the Examples.
The hepatic dysfunction includes viral liver disease, drug- and alcohol-related liver disease, immune-mediated liver disease, metabolic liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), hepatic failure, fulminant hepatic failure, hepatocellular carcinoma, and liver transplantation complications.
The pharmaceutical composition according to an embodiment of the present invention, ornithine microparticles or an aqueous solution thereof, can be orally administered to a mammal, particularly a human, in need thereof. Since the pharmaceutical composition according to an embodiment of the present invention has low toxicity, is easily accumulated in the liver by injection or the like, and is hydrolyzed there, resulting in release of ornithine, it can be used by direct administration to a vein, an artery, a subcutaneous site, an abdominal cavity or the like, but is more advantageously used as an orally administered agent because it more efficiently exerts its effect in the intestine.
Examples of the dosage form of the pharmaceutical composition include liquid preparations (including solutions for internal use, suspensions, emulsions, and syrups), solid preparations (including tablets, pills, sublingual tablets, capsules, drops, and troches), granules, powders, and powdered drugs. If necessary, the solid preparation can be further formulated into a dosage form coated with a coating known in the art, for example, a sugar-coated tablet, a gelatin-coated tablet, an enteric coated tablet, a film-coated tablet, a double tablet or a multilayer tablet. The shape and size of each dosage form may be within the range of dosage forms known in the art, and additives such as solubilizers, pH adjusters, carriers, excipients, diluents, binders, disintegrators, lubricants, emulsifiers, extenders, colorants, flavoring agents, sweeteners, stabilizers, and preservatives can be contained.
The dose and administration frequency are applied in an optimal concentration range depending on the age, sex, condition, degree of disease, and the like of the patient, but an amount of ornithine to be administered per day is preferably from about 10 μmg to about 1 g, more preferably from 0.1 mg to 100 mg per Kg of body weight, and this amount may be divided into about 1 to 3 times. However, since the pharmaceutical composition agent according to an embodiment of the present invention has high availability, the effect can be expected even if it is administered once a day.
With respect to the dose, administration method, and administration frequency, those skilled in the art can determine an optimum method through animal experiments or experimental administration to humans with reference to various literatures and the like.
Hereinafter, the present invention will be described in more detail with reference to specific examples, but is not limited to these examples.
To commercial CH3O—(CH2CH2O)n—H (MW=5,000, 60 g, 12 mmol), THF (200 mL) and 14.4 mL of butyllithium (23 mmol, 1.6 M-hexanes) were added. Then, methanesulfonyl chloride (CH3SO2Cl) (1.5 mL, 2.3 g, 20 mmol) was added, and they were reacted at 40° C. for 1 day. After precipitation in 2-propanol (IPA), the precipitate is dissolved in methanol for precipitation. This operation was repeated twice, and the precipitate was dried under reduced pressure to give a target product (yield: 60 g).
The CH3O—(CH2CH2O)n-CH2CH2OSO2CH3 (60 g) obtained in Production Example 1 was added with 600 mL of 28% aqueous ammonia, and they were reacted at 50° C. for 1 day. Then, the reaction product was extracted with 50 mL of chloroform, and the chloroform phase was dehydrated with NaHSO4 and filtered, followed by precipitation in 2-propanol (IPA), and the obtained precipitate was dissolved in methanol for precipitation. This operation was repeated twice, and the precipitate was dried under reduced pressure to give a target product (yield: 54 g).
Boc-ornithine (Z)—OH (Boc-Orn (Z)—OH, 15 g) was dissolved in 100 mL of THF, and ice-cooled to 0° C., and added with a solution of thionyl chloride (SOCl2, 10 mL, 16 g) in THF (50 mL), and they were reacted at 0° C. for 1 hour and at room temperature for 3 hours. The reaction solution was poured into 1 L of hexane, and the precipitate was filtered, dissolved in 100 mL of ethyl acetate, precipitated in 1 L of hexane, and filtered. This operation was repeated twice, and the resulting precipitate was dried under reduced pressure to give a target product (yield: 10 g).
N819 (5 g) obtained in Production Example 2 was dissolved in 30 mL of DMF, and a solution of N831 (5 g) obtained in Production Example 3 in DMF (20 mL) was added thereto, followed by stirring at room temperature for 2 days. The solution was added to hexane:2-propanol (8:2, 500 mL), and centrifuged to give a polymer, which was dried under reduced pressure to give a target product (6.3 g).
NMR measurement results of the obtained N857 are illustrated in
N833 (4.3 g) obtained in Production Example 4 was dissolved in 45 mL of trifluoroacetate, ice-cooled to 0° C., and added with 3% HBr (acetic acid solution, 15 mL), and they were reacted for 4 hours. This was added to cold 2-propanol of 800 mL to give a precipitate, which was dried under vacuum to give a target product (yield: 3.3 g).
NMR measurement results of the obtained N967 are illustrated in
N967 (1 g) obtained in Production Example 5 was dissolved in 1 mL of DMF, and added with 5 mL of triethyl amine and 10 mL of acetic anhydride, and they were reacted at room temperature for 1 hour. This was added to hexane:2-propanol (8:2, 500 mL), and centrifuged to give a polymer, which was dried under reduced pressure to give a target product (1.0 g).
NMR measurement results of the obtained N977 are illustrated in
A target polymer (1.4 g) was obtained in exactly the same manner as in Production Example 6, except that isobutyric anhydride was used instead of acetic anhydride.
NMR measurement results of the obtained N841 are illustrated in
PEG-b-POrn (N836, 1 g) prepared in the same manner as in Production Example 5 was dissolved in 20 mL of DMF, and added with 20 mL of water, the solution was placed in a dialysis membrane (molecular weight cut off (MWCO)=3.5 KDa), and dialyzed against 0.01 M HCl (2 L) for 24 hours. Thereafter, dialysis was performed for 48 hours against 2 L of distilled and ion-exchanged (DI-water) (dialysis water was exchanged every 12 hours) to give an aqueous solution (85 mL). The total amount of cationic charges in the aqueous solutions was calculated, and a chondroitin sulfate solution (2.7 mg/mL, 115 mL) was added for attaining a ratio of the total amount of cationic charges to the total amount of anionic charges of 1:1. Then, the mixed solution was stirred for 30 minutes to give polyion complex (PIC) particles (NanoORN).
N833 (1 g) synthesized in Production Example 4 was dissolved in 20 mL of DMF, 20 mL of DI-water was added, the solution was placed in a dialysis membrane (molecular weight cut off (MWCO)=12 KDa-14 KDa), and dialyzed against 2 L of DI-water 2 L for 72 hours (dialysis water was exchanged every 12 hours) to give an aqueous solution.
This assembled body was made in exactly the same manner as in Production Example 9 except that the N977 (1 g) synthesized in Production Example 6 was used.
This assembled body was made in exactly the same manner as in Production Example 9 except that the N841 (1 g) synthesized in Production Example 7 was used. Results of dynamic light scattering measurement of the assembled body (NanoORN (iBu)) are illustrated in
Changes in size of the nano-assembled bodies prepared in Production Examples 9 to 11 depending on pH are illustrated in
Six C57BL/6N mice (male) per group were allowed to freely drink the following samples (for groups 3 to 7, the ornithine content was adjusted to about 200 mg/kg). Three days after the start of administration, an aqueous APAP solution (15 mg/mL, 0.5 mL, 300 mg/Kg-BW) was intraperitoneally administered. After 24 hours, blood and livers were collected, whole blood were used for measurement of a blood ammonia concentration, plasmas were used for measurement of hepatic disorder markers AST and ALT, and livers were subjected to hematoxylin-eosin staining for histological diagnosis.
As illustrated in
Views of hematoxylin and eosin (H & E) staining of liver tissue sections from these tests are illustrated in
Six C57BL/6N mice (male) per group were forced to be orally administered with the following samples (for groups 2 to 7, the ornithine content was adjusted to about 200 mg/kg) by use of a 0.5 mL sonde. Their body weight was measured every day, and 4 days after the start of administration, whole blood was used for measurement of blood cell count, and plasma was used for measurement of hepatic disorder markers AST and ALT.
Six C57BL/6N mice (male) per group were forced to be orally administered with the following samples (for groups 3 to 5, the ornithine content was adjusted to about 200 mg/kg) by use of a 0.5 mL sonde. Three days after the start of administration, an aqueous APAP solution (15 mg/mL, 0.5 mL, 300 mg/Kg-BW) was intraperitoneally administered. Mouse survival was then checked for 14 days.
As illustrated in
Potassium naphthalene (1 mml) and ethyleneoxide (115 mmol) were added to commercially available CH3OCH2CH2OH (1 mmol) in 50 mL of ultra-dehydrated THF in a 100 mL flask, and reacted for 1 day under water cooling, and then methanesulfonylchloride (5 mmol) was added thereto. After 20 minutes, the mixture was poured into 500 mL of 2-propanol for precipitation, and then the precipitate was dissolved in methanol for precipitation. This operation was repeated twice, and the precipitate was dried under reduced pressure to give a target product
The CH3O—C6H5—CH2O—(CH2CH2O)n-CH2CH2OSO2CH3 obtained in Experimental Example 1 was added with 600 mL of 28% aqueous ammonia, and they were reacted at 50° C. for 1 day. Then, the reaction product was extracted with 50 mL of chloroform, and the chloroform phase was dehydrated with NaHSO4 and filtered. Then, the product was precipitated in 2-propanol (IPA), and the obtained precipitate was dissolved in methanol for precipitation. This operation was repeated twice, and the precipitate was dried under reduced pressure to give a target product
N956 obtained in Reference Production Example 2 was dissolved in DMF, and a solution of commercially available N-benzyl-aspartic acid NCA in DMF was added thereto, followed by stirring at room temperature for 2 days. The solution was added to hexane:2-propanol (8:2, 500 mL), centrifuged to give a polymer, which was dried under reduced pressure to give a target product.
N962 obtained in Reference Production Example 3 was dissolved in trifluoroacetic acid, ice-cooled to 0° C., and added with 3% HBr (acetic acid), and they were reacted for 4 hours. This was added to cooled 2-propanol of 800 mL to give a precipitate, which was dried under vacuum to give a target product.
A target polymer (1.4 g) was obtained in exactly the same manner as in Production Example 7, except that N973 synthesized in Reference Production Example 4 was used instead of N841.
N978 synthesized in Production Example 125 was dissolved in DMSO, and the solution was dialyzed against 2 L of DI-water 2 L for 72 hours (dialysis water was exchanged every 12 hours) to give an aqueous solution of an assembled body. Thereafter, chloramine T and a radioisotope 125I of iodine were mixed, and the phenyl group introduced into a terminal of the PEG chain was labeled with 125I.
For examination of pharmacokinetics, three ICR mice were prepared for each of endpoints at 0.5 h, 1 h, 2 h, 4 h, and 24 h, i.e., 15 mice in total. After the labeled structures were forced to be orally administered by use of a sonde, the mice were sacrificed for each endpoint. Hearts, lungs, digestive tracts, livers, spleens, kidneys and bloods were extracted, and radioactivity of each organ was measured with a gamma counter. As illustrated in
The former as a target product was synthesized by exactly the same method as in Production Example 7, except that 1 mg of commercially available TAMRA-N═C═S was added before isobutyric anhydride was added and that the mixture was reacted for 10 minutes.
The latter as a target product was synthesized by exactly the same method as in Production Example 7, except that 1 mg of commercially available Cy5-NHS was added before isobutyric anhydride was added and that the mixture was reacted for 10 minutes.
BAEC (bovine aortic endothelial cells), L-929 (mouse fibroblasts) and RAW264.7 (mouse macrophages) were prepared, and seeded, at from 5 to 10×103 cells, in 96-well plates. Various concentrations of NanoORN (Z), NanoORN (Me), NanoORN (iBu), and NanoORN were added to each well. After 24 or 48 hours of culture, MTT reagents were added. After additional 4 hours, DMSO was added, and formazan crystals were solubilized. The absorption at a wavelength of 575 nm was measured.
The results are presented in
N819 (10 g) synthesized according to Production Example 2 was dissolved in 60 mL of DMF. Commercially available L-aspartic acid (CH2Ph)-N-carboxylic anhydride (L-ASP (Z)-NCA) (10 g) was dissolved in 40 mL of DMF. They were mixed and reacted at room temperature for 2 days. The resulting polymer was precipitated in 2-propanol (IPA), then the precipitate was dissolved in acetone and precipitated in hexane. This operation was repeated twice, and the precipitate was dried under reduced pressure to give a target product (yield: 16 g; n=114; m=15.5)
Commercially available methyl L-aspartate hydrochloride (15 g; 81.6 mmol) was dissolved in 90 mL of THF, and α-pinene (33 g; 252 mmol) and triphosgene 12.2 g (40.8 mmol) were added, and they were reacted at 50° C. for 2 hours. The reaction solution is poured into 1 L of hexane, and the precipitate is filtered, dissolved in 100 mL of 2-propanol, precipitated in 1 L of hexane, and filtered. This operation was repeated twice, and the resulting precipitate was dried under reduced pressure to give a target product (yield: 10.2 g).
N819 (5 g) synthesized according to Production Example 2 was dissolved in 30 mL of DMF. L-aspartic acid (CH3)—N-carboxylic anhydride (L-ASP (Me)-NCA) (5 g) synthesized in Production Example 15 was dissolved in 20 mL of DMF. They were mixed and reacted at room temperature for 2 days. The resulting polymer was precipitated in 2-propanol (IPA), then dissolved in methanol, and precipitated in ether. This operation was repeated twice, and the precipitate was dried under reduced pressure to give a target product (yield: 6.4 g; n=114; m=10)
PEG-NH2 (N866) (5 g; 1 mmol) synthesized according to Production Example 2 was dissolved in 30 mL of DMF.
L-Orn (Z)-NCA (N854) (6 g; 20 mmol) synthesized according to the Production Example and commercially available L-aspartic acid (CH2Ph)-N-carboxylic anhydride (L-ASP (Z)-NCA) (2.5 g; 10 mmol) were dissolved in 20 mL of DMF. They were mixed and reacted at room temperature for 2 days. The resulting polymer was precipitated in 2-propanol (IPA)/hexane (1:9 v), then the precipitate was dissolved in acetone and precipitated in hexane. This operation was repeated twice, and the precipitate was dried under reduced pressure to give a target product (yield: 8.8 g; n=114; number of asparagine units=15; number of ornithine units=20)
N865 (1 g) synthesized according to Production Example 14 was dissolved in 20 mL of DMF, 20 mL of DI-water was added, the solution was placed in a dialysis membrane (molecular weight cut off (MWCO)=12 KDa-14 KDa), and dialyzed for 72 hours against 2 L of DI-water 2 L (dialysis water was exchanged every 12 hours) to give an aqueous solution containing a target assembled body.
The same procedures as in Production Example 18 were repeated, except that N869 (1 g) synthesized according to Production Example 16 was used, to give an aqueous solution containing a target assembled body.
The same procedures as in Production Example 18 were repeated, except that N877 (1 g) synthesized according to Production Example 17 was used, to give an aqueous solution containing a target assembled body.
The particle diameters of the particles prepared in Production Examples 16 to 18 were measured by a dynamic light scattering method.
NanoASP (Bz) formed stable nanoparticles of about from 120 to 140 nm at a pH of up to 12 over 24 hours.
NanoASP (Me) forms an assembled body of about 10 nm for up to 6 hours at all measurement pHs. After 24 hours, aggregates of about 100 nm were observed at pH 1 and 5.
NanoASP/ORN formed an assembled body of about 100 nm for up to 24 hours at all measurement pHs.
In addition, NanoASP (Bz), NanoASP (Me), and NanoASP/ORN did not show substantial toxicity to mice similarly to the PEG-b-POrn (Z) assembled body (NanoORN (Z)) and the PEG-b-POrn (Me) assembled body (NanoORN (Me)) according to the results of changes in body weight of the orally administered (sonde) acute hepatic disorder mice.
The method of Test Example 2 was repeated except that samples for the following groups were used.
As a result of the above test, these assembled bodies provide a weight change equivalent to that of the healthy group, were hardly toxic, and provide blood ammonia levels, AST and ALT levels of the acute hepatic disorder mice as illustrated in
PEG-b-PArg (N1046; 2.4 g) synthesized in the same manner as in Example 5 of PCT/JP2016/62062 A1 was dissolved in 50 mL of DMF. Acetic anhydride (30 mL) was added to this, and there were reacted at 50° C. for 5 hours and then at room temperature for 1 day. The resulting polymer is precipitated in t-butyl methyl ether and then washed with hexane. This operation was repeated twice, and the precipitate was dried under reduced pressure to give a target product (yield: 2.4 g; n=114; m=28). A 1H-NMR chart of the target copolymer is illustrated in
A target product was obtained in exactly the same manner as in Production Example 21 except that acetic anhydride was replaced with isobutyric anhydride (yield: 2.4 g; n=114; m=28). A 1H-NMR chart of the target copolymer is illustrated in
N1047 (1 g) synthesized according to Production Example 21 was dissolved in 20 mL of DMF, 20 mL of DI-water was added, the solution was placed in a dialysis membrane (molecular weight cut off (MWCO)=12 KDa-14 KDa), and dialyzed for 72 hours against 2 L of DI-water 2 L (dialysis water was exchanged every 12 hours) to give an aqueous solution containing NanoArg (Ac). (Average particle diameter: 132 nm, ζ potential: +4.9 mV)
An assembled body was prepared in exactly the same manner as in Production Example 23 except that the N1063 (1 g) synthesized in Production Example 22 was used. Average particle diameter: 44 nm, ζ potential: −2.2 mV)
The procedure of Test Example 2 was repeated except that the acetylated arginine assembled body was used in place of the ornithine assembled body of Test Example 2. The results are shown in
Six C57BL/6J mice (male) per group were allowed to be freely orally administered with an aqueous solution of acetylated polyarginine particles (NanoArg (Ac) ([NanoArg (Ac)]=5 mg/mL) (Days 0 to 8). One day after the start of oral administration, a choline-deficient methionine-reduced high-fat diet (CADHFD) was given (Days 1 to 8). Bloods and livers were collected on Day 8 after the start of CADHFD feeding. Plasma was used for measurement of triglyceride (TG).
The results are illustrated in
The copolymer and the ornithine microparticles according to the present invention can be used as, for example, medicaments for the prevention and treatment of hepatic dysfunction, though not limited thereto, and can be utilized at least in the pharmaceutical industry.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-187688 | Nov 2020 | JP | national |
This application is a U.S. National Phase application under 35 U.S.C. § 371 of International Patent Application No. PCT/JP2021/041160 filed on Nov. 9, 2021 and claims the benefit of priority to Japanese Patent Application No. 2020-187688 filed on Nov. 11, 2020, the contents of all of which are incorporated herein by reference in their entireties. The International Application was published in Japanese on May 19, 2022 as International Publication No. WO/2022/102608 under PCT Article 21(2).
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/041160 | 11/9/2021 | WO |
