JP5936182B2 - Preparations for oral administration of polymerized cyclic nitroxide radical compounds - Google Patents

Description

  The present invention relates to a preparation for oral administration containing a polymerized cyclic nitroxide radical compound as an active ingredient, for delivery of the compound into the bloodstream by oral administration, or for prevention or treatment of inflammation by oral administration.

  In recent years, excessive reactive oxygen species (ROS) are produced in vivo due to irregular lifestyle, aging, social stress, etc., and various chronic diseases (arteriosclerosis, diabetes, etc.) and intractable diseases (Alzheimer, Parkinson's disease, etc.) has been revealed. In addition, ROS normally regulates the balance of oxidation-antioxidation (redox) in vivo, but if this ROS is produced excessively, the balance of oxidation-antioxidant factor is in the direction of oxidation. Tilt. This state is called “oxidative stress”, and it is also known to cause or induce various diseases including the above-mentioned diseases. Although studies have been made on the prevention of chronic diseases using various antioxidants so far, in the case of small molecule antioxidants, administration of such high concentrations is essential for living organisms such as mitochondrial electron transport systems. In addition to inhibiting the reaction, there were problems such as low effective concentration due to renal excretion and metabolism, and diffusion to the whole body, causing systemic side reactions. In addition, although treatment and prevention of oxidative stress diseases using low molecular weight antioxidants have been carried out, no significant effect has been observed.

  In fact, increasing oxidative stress in the body has been shown to cause various diseases such as intractable neurological diseases such as Alzheimer and Parkinson's disease, arteriosclerosis, cerebral infarction, myocardial infarction, renal failure, diabetes, and cancer. . For this reason, provision of the chemical | medical agent or administration system which can treat an oxidative stress disorder | damage efficiently is calculated | required.

  In consideration of such circumstances, the present inventors use a self-organized nanoparticle (RNP) encapsulating a catalytically functioning ROS scavenger nitroxide radical and erase ROS where necessary. New nanotherapeutic methods have been developed (see Patent Document 1). This RNP has a high therapeutic effect on cerebral infarction, myocardial infarction, and acute renal failure by staying in blood vessels for a long time after intravenous administration and collapsing in tissues where oxidative stress occurs. It is clear to show (patent document 1, patent document 2).

WO2009 / 133647 Japanese Patent Application No. 2010-260471

  However, in view of providing for the prevention or treatment of the above-mentioned various disorders or diseases, such a polymerized cyclic nitroxide radical compound can increase its blood concentration by oral administration, and can be used as a desired organ or organ. If it can be delivered to the surface and can exert the ROS elimination effect, it will be able to greatly contribute to the technical field.

  Accordingly, an object of the present invention is to provide a preparation for oral administration containing a polymerized cyclic nitroxide radical compound as an active ingredient.

The present inventor forms self-assembled nanoparticles (polymer micelles encapsulating cyclic nitroxide radicals) in an aqueous medium among the polymerized cyclic nitroxide radical compounds described in Patent Document 1, but under acidic pH conditions. When such a polymer micelle collapses and a compound in which radicals are released into the surrounding environment is orally administered, it can move into the bloodstream despite having a molecular weight exceeding 10,000, It was found that it can stay in the bloodstream for a long time. Incidentally, although it is a polymerized cyclic nitroxide radical compound similar in structure, the cyclic nitroxide radical as a pendant group is imino (—NH—, and in this specification, the functional group may be referred to as “imino group”). In view of the fact that it has been observed that a compound that does not exhibit pH responsiveness polymerized via an ether bond and does not exhibit pH response does not migrate into the bloodstream when administered orally, The actions and effects are very unique and interesting.

Therefore, as means for solving the above-mentioned problems, a preparation for oral administration containing a polymerized cyclic nitroxide radical compound as an active ingredient, wherein the polymerized cyclic nitroxide radical compound is represented by the general formula (I)
PEG-CNR (I)
(Wherein PEG is a segment containing poly (ethylene glycol),
A CNR is a polymer segment comprising a repeating unit that includes a cyclic nitroxide radical as part of a pendant group attached to the polymer backbone through a linking group having at least one imino group)
A formulation for oral administration represented by is now provided herein.

  Further, a pharmaceutical preparation for delivering nitroxide radicals into the bloodstream or preventing or treating inflammation by oral administration containing a polymerized cyclic nitroxide radical compound represented by the above general formula (I) as an active ingredient Provided.

As the polymerized cyclic nitroxide radical compound, a cyclic nitroxide radical is o- or p-phenylene-C _1-6 alkylene-NH— (C _1-6 alkylene) _q — (where q is 0 or 1). And a cyclic nitroxide radical is linked to 2,2,6,6-tetramethylpiperidin-1-oxyl-4-yl, 2,2,5, 5-tetramethylpyrrolidin-1-oxyl-3-yl, 2,2,5,5-tetramethylpyrrolin-1-oxyl-3-yl and 2,4,4-trimethyl-1,3-oxazolidine-3- Oxyl-2-yl, 2,4,4-trimethyl-1,3-thiazolidine-3-oxyl-2-yl and 2,4,4-trimethyl-imidazolin-3-oxyl-2 -Selected from the group consisting of yl,
Here, it is preferable that the polymer main chain is derived from a polymerizable unsaturated double bond, and the unbonded end of phenylene is bonded to the main chain.

  More preferred polymerized cyclic nitroxide radical compounds for use in the present invention include those represented by the general formula (II)

Where
A represents unsubstituted or substituted C ₁ -C ₁₂ alkoxy, and when substituted, the substituent is a formyl group, the formula R ¹ R ² CH—, where R ¹ and R ² are independently C ₁ -C ₄ alkoxy or ^{R 1} and ^{R 2} are -OCH ₂ CH ₂ O together _{-, - O (CH 2)} 3 O- or a group of -O _{(CH 2)} represents a 4 O-). Represents
L ₁ is selected from the group consisting of a single bond, — (CH ₂ ) _c S—, —CO (CH ₂ ) _c S—, wherein c is an integer of 1 to 52;
L ₂ is —C _1-6 alkylene-NH— (C _1-6 alkylene) _q —, where q is an integer 0 or 1, and R is independently at least a total number n of R , 50%, preferably 70%, more preferably 90%, particularly preferably 95% 2,2,6,6-tetramethylpiperidin-1-oxyl-4-yl, 2,2,5,5-tetra Methylpyrrolidin-1-oxyl-3-yl, 2,2,5,5-tetramethylpyrrolin-1-oxyl-3-yl and 2,4,4-trimethyl-1,3-oxazolidine-3-oxyl-2 Cyclic selected from the group consisting of -yl, 2,4,4-trimethyl-1,3-thiazolidin-3-oxyl-2-yl and 2,4,4-trimethyl-imidazolindin-3-oxyl-2-yl Nitroxydrazica Represents a residue of a compound, if present, the remaining R is a hydrogen atom, a halogen atom or a hydroxy group,
m is an integer from 20 to 5,000, and n is independently an integer from 3 to 1,000,
The compound represented by these can be mentioned.

  Further, in the compound represented by the general formula (II), R is represented by the following formula:

In the formula, R ′ is a methyl group.
The group represented by the above formula occupies at least 80% of the total number n of R,
Are particularly preferably used in the present invention.

<Detailed Description of the Invention>
In order to facilitate the understanding of the present invention, a typical compound used in the present invention will be described as an example with reference to the conceptual diagram shown in FIG. When we designed nanoparticles that disintegrate in response to changes in pH and non-disintegrating nanoparticles in an aqueous medium (water, buffered water, etc.) and orally administer the two nanoparticles, As shown in FIG. 1, nanoparticles that do not disintegrate at pH (O-RNP) are not taken into the blood at all, but pH-disintegrating nanoparticles (N-RNP) are effectively taken into the blood for several tens of hours. It was observed that it stayed over. This is not limited by theory, but N-RNP once collapses in the acidic environment of the stomach and is concentrated in the vicinity of the intestinal tract, and even a high molecular weight drug with a molecular weight of about 10,000 passes through the intestinal tract. It is considered that the blood stayed in the blood for a long time by being taken in and complexed with serum proteins such as albumin in the blood. Thus, as far as the present inventors know, there is no report example describing that a polymer drug administered orally can be delivered to a diseased site through a gastrointestinal disease site or blood.

In the present invention, the pendant group means a side chain having a certain functional group as generally recognized in the art. Specifically, the general formula (II) - phenylene _{-C 1-6} alkylene -NH- _{(C 1-6 alkylene)} pendant group by referring to the side chain represented by q -R more clearly You can understand. The main chain to which such a pendant group is bonded is not limited, but preferably has a polymerizable unsaturated double bond, for example, a polymerizable having an unsaturated double bond such as substituted ethylene. It means a main chain formed by radical polymerization of monomers. As specific examples of such a main chain, those described in Patent Document 1 can be referred to.

As described above, in the polymerized cyclic nitroxide radical compound that can be preferably used in the present invention, the cyclic nitroxide radical is o- or p-phenylene-C _1-6 alkylene-NH— (C _1-6 alkylene) _q — (here And q is 0 or 1), preferably via a linking group of p-phenylene-C _1-6 alkylene-NH— (C _1-6 alkylene) _q — (where q is 0). And a cyclic nitroxide radical bonded to the polymer main chain is 2,2,6,6-tetramethylpiperidin-1-oxyl-4-yl, 2,2,5,5-tetramethylpyrrolidine-1- Oxyl-3-yl, 2,2,5,5-tetramethylpyrrolin-1-oxyl-3-yl and 2,4,4-trimethyl-1,3-oxazolidine-3-oxyl Selected from the group consisting of 2-yl, 2,4,4-trimethyl-1,3-thiazolidin-3-oxyl-2-yl and 2,4,4-trimethyl-imidazolin-3-oxyl-2-yl . In the present specification, the term “bond” means a covalent bond unless otherwise specified.

  The polymerized cyclic nitroxide radical compound that can be more preferably used in the present invention has the general formula (II)

Represented by
A represents unsubstituted or substituted C ₁ -C ₁₂ alkoxy, and when substituted, the substituent is a formyl group, the formula R ¹ R ² CH—, where R ¹ and R ² are independently C ₁ -C ₄ alkoxy or ^{R 1} and ^{R 2} are -OCH ₂ CH ₂ O together _{-, - O (CH 2)} 3 O- or a group of -O _{(CH 2)} represents a 4 O-). Represents
L ₁ is selected from the group consisting of a single bond, — (CH ₂ ) _c S—, and —CO (CH ₂ ) _c S—, where c is an integer from 1 to 5, preferably 2.
L ₂ is —C _1-6 alkylene, —NH— (C _1-6 alkylene) _q —, where q is an integer 0 or 1, preferably 0, and R is independently R At least 50%, preferably 70%, more preferably 85%, particularly preferably 95% of the total number n of 2,2,6,6-tetramethylpiperidin-1-oxyl-4-yl, 2,2, 5,5-tetramethylpyrrolidin-1-oxyl-3-yl, 2,2,5,5-tetramethylpyrrolin-1-oxyl-3-yl and 2,4,4-trimethyl-1,3-oxazolidine- Consists of 3-oxyl-2-yl, 2,4,4-trimethyl-1,3-thiazolidin-3-oxyl-2-yl and 2,4,4-trimethyl-imidazolin-3-oxyl-2-yl A ring selected from the group Represents the residue of Loki Sid radical compound, if present, the remaining R is a hydrogen atom, a halogen atom or a hydroxy group,
m is an integer of 20 to 5,000, preferably 20 to 1,000, more preferably 20 to 500, and n is independently 3 to 1,000, preferably 3 to 500, More preferably, it is an integer of 3 to 100.
It is represented by

The alkyl group in C ₁ -C ₁₂ alkoxy or C ₁ -C ₄ alkoxy can be linear or branched and includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, It can be hexyl, decyl, octyl, undecyl.

C _1-6 alkylene includes, but is not limited to, a corresponding alkyl diyl group such as methylene, 1,2-propanediyl, 1,3-propanediyl, 1,4-butanediyl, and the like. .

  The residue of the cyclic nitroxide radical or cyclic nitrooxide radical compound of the R group is preferably of the formula

In the formula, R ′ is a methyl group.
The group represented by these can be mentioned.

Such a polymer cyclic nitroxide radical compound is described in Patent Document 1 or can be produced by the described method. Further, as described in Patent Document 1, such a polymer cyclic nitroxide radical compound forms a surface layer with a hydrophilic PEG segment in an aqueous medium to form a CNR segment core, so-called core-shell. Type polymer micelles are understood to form.

  In the preparation of the present invention, depending on the dosage form, the polymer cyclic nitroxide radical compound can exist in the form of self-assembled polymer micelles as described above, for example, in an aqueous medium. The compound in any state can be included, but it is preferable to include a compound that has been once formed in an aqueous medium and then dried by freeze-drying or the like.

  The preparation for oral administration can contain diluents, excipients, and additives that can be included in oral preparations well known in the art as long as the purpose of the present invention is met. There can also be. When the preparation for oral administration of the present invention is a solid, the polymer cyclic nitroxide radical compound is converted into sucrose, lactose, mannitol, cellulose, trehalose, maltitol, dextran, starch, agar, gelatin, casein, albumin, glyceride, etc. It can contain by the combination of 1 or more types chosen from. In addition, other inert diluents, lubricants such as magnesium stearate, parabens, sorbic acid, preservatives such as α-tocopherol, antioxidants such as cysteine, disintegrants, binders, buffers, Sweeteners and the like can also be included.

  Liquid preparations for oral administration include physiologically acceptable emulsions, syrups, elixirs, suspensions and pre-solution preparations. These formulations may contain a commonly used inert diluent such as water. Such an aqueous solution may contain the above-mentioned sugars and polyethylene glycol having a molecular weight of about 200 to 100,000.

Such a preparation can be rapidly transferred into the blood and retained in the bloodstream for a long time by oral administration to mammals, particularly humans. In this way, it is possible to suppress or reduce active oxygen or oxidative stress that is delivered to a cancer, an inflammatory site, or an oxidative stress disorder site and is present in a lesion or affected area. Accordingly, it is possible to prevent the onset of a disorder or disease that can be mediated by active oxygen, and to treat the onset of the disease. Such disorders or diseases include, but are not limited to:
Arteriosclerosis, diabetes, cancer, arthritis, sepsis, Alzheimer, Parkinson's disease, etc. are included.

The conceptual diagram of the nanoparticle which the polymeric nitroxide radical compound used by this invention forms. The graph which shows the ESR signal intensity | strength in blood after oral administration of N-RNP and O-RNP. The graph which shows the pharmacokinetics after oral administration using N-RNP and O-RNP which labeled the radioisotope. The graph which shows the measurement result of the thickness (inflammation marker) of the hind limb of an arthritis model mouse. The graph which shows the result of the escape reflex test with respect to the thermal stimulation of an arthritis model mouse. The graph which shows the result of activation of the neutrophil of an arthritis model mouse. The graph which shows the result of the object recognition test of Alzheimer model mouse. The graph which shows the result of the hippocampus dependence learning and memory test of an Alzheimer model mouse. The graph which shows the measurement result of the lipid peroxide in the brain of an Alzheimer model mouse. The graph which shows the result of having investigated the influence of the toxicity after N-RNP oral administration. The photograph changed into the figure which shows the arteriosclerosis therapeutic effect by N-RNP oral administration.

<Specific examples for carrying out the invention>
Hereinafter, the present invention will be specifically described, but the present invention is not intended to be limited to these embodiments. For the sake of brevity, as the polymerized nitroxide radical compound, the PEG-prepared in Production Example 4 of the above-mentioned Patent Document 1 (the disclosure of which is incorporated herein by reference). An example using nanoparticles by b-PCMS-N-TEMPO (hereinafter also referred to as N-RNP) will be mainly described.

Test Example 1: Translocation to blood after oral administration of radical-containing nanoparticles After oral administration of pH-responsive nanoparticles (N-RNP) and non-pH-responsive nanoparticles (O-RNP) at a concentration of 1200 mg / kg Blood was collected every time and the electron spin resonance (ESR) spectrum was measured. When these two nanoparticles are administered orally, nanoparticles that do not disintegrate at pH as shown in FIG. 2 are not taken into the blood at all, but the pH-disintegrating nanoparticles are effectively taken into the blood over several hours. Found to stay.

Test Example 2: Evaluation of pharmacokinetics of radioisotope-labeled radical-containing nanoparticles After radiolabeling the nanoparticles with radioisotope ¹²⁵ I, oral administration was performed. FIG. 3 shows the results of extracting the stomach, small intestine, cecum, large intestine, liver and blood at regular intervals and measuring the radiation dose with a gamma counter.

  As in FIG. 2, nanoparticles that do not disintegrate at pH (O-RNP) are not taken into the blood at all, but it is clear that pH-disintegrating nanoparticles (N-RNP) are effectively taken into the blood. It became.

Test Example 3: Arthritis Treatment N-RNP (100 mg / kg) was orally administered. One day later, a 1% carrageenan suspension was administered to the hind limbs to create an arthritis model and measure its impairment markers.

  As a measure of inflammation, the thickness of the hind limb after administration of carrageenan was measured. As a result, oral administration of N-RNP showed a higher protective effect than the low molecular compound TEMPOL (FIG. 4).

  When thermal stimulation was applied to the hind limb of the mouse and the latency of the escape reflex was measured, it was revealed that N-RNP exhibited an excellent protective effect (FIG. 5).

  Measurement of myeloperoxidase (MPO) to examine the activation of mouse neutrophils (an index of inflammation) revealed that N-RNP suppressed inflammation (FIG. 6). .

Test Example 4: Alzheimer treatment Alzheimer model mice SAMP8 10 weeks old (aging promotion model mice; model mice that spontaneously develop learning / memory impairment) were examined for Alzheimer prevention. For this model mouse, N-RNP was orally administered (300 mg / kg / day) with a sonde for 28 days, and the degree of Alzheimer was measured after administration.

When an object recognition test was performed, both N-RNP and the low molecular compound TEMPOL were improved, but N-RNP showed a very high object recognition ability compared to the low molecular compound TEMPOL. On the other hand, no improvement was observed for nanoparticles (micelle) to which TEMPO was not bound (FIG. 7).

  When Morris water maze, a behavioral evaluation, was performed to test hippocampal-dependent learning and memory, a trend of improvement was observed only in N-RNP, and the low molecular weight compounds TEMPOL and TEMPO were not introduced. (Micelle) showed no improvement trend (FIG. 8).

  As an index of oxidative stress in the brain, when the amount of lipid peroxide was evaluated using the TBARS method, suppression of the amount of lipid peroxide was observed only in N-RNP, and nanomolecules into which the low molecular compounds TEMPOL and TEMPO were not introduced Micelle was not significantly suppressed (FIG. 9).

Test Example 5: Arteriosclerosis treatment Hyperlipidemic / arteriosclerosis model LDLR-deficient male male 8 weeks old was administered 12.125 mg / mL N-RNP together with Western diet (WTD) load in a free drinking water bottle. . Twelve weeks after administration, blood parameters, blood pressure, and formation of arteriosclerotic lesions were evaluated.

  Since there was no effect on most parameters depending on the presence or absence of administration of N-RNP at the time of Western diet loading, it indicates that there is no toxicity due to oral administration of N-RNP (FIG. 10).

  Although there was no effect on most parameters depending on the presence or absence of N-RNP administration at the time of Western diet loading, atherosclerotic lesions were observed with N-RNP administration in both the full length of the aorta and the section of the origin of the aorta. A tendency to suppress the formation of was obtained (FIG. 11).

Claims (4)

A pharmaceutical preparation for delivering a nitroxide radical into the bloodstream by oral administration comprising a polymerized cyclic nitroxide radical compound as an active ingredient,
The polymerized cyclic nitroxide radical compound has the general formula (II)
Where
A represents unsubstituted or substituted C ₁ -C ₁₂ alkoxy, and when substituted, the substituent is a formyl group, formula R ¹ R ² CH— (wherein R ¹ and R ² are independently C ₁ -C ₄ alkoxy or R ¹ and R ² are -OCH ₂ CH ₂ O together _{-, - O (CH 2)} 3 O- or a group of -O (CH ₂₎ represents a ₄ O-). Represents
L ₁ is selected from the group consisting of a single bond, — (CH ₂ ) _c S—, —CO (CH ₂ ) _c S—, wherein c is an integer of 1 to 5,
L ₂ is —C _1-6 alkylene-NH— (C _1-6 alkylene) _q —, where q is 0 or 1, and
R is independently at least 50% of the total number n of R is 2,2,6,6-tetramethylpiperidin-1-oxyl-4-yl, 2,2,5,5-tetramethylpyrrolidine-1- Oxyl-3-yl, 2,2,5,5-tetramethylpyrrolin-1-oxyl-3-yl and 2,4,4-trimethyl-1,3-oxazolidine-3-oxyl-2-yl, 2, Residue of cyclic nitroxide radical compound selected from the group consisting of 4,4-trimethyl-1,3-thiazolidin-3-oxyl-2-yl and 2,4,4-trimethyl-imidazolindin-3-oxyl-2-yl Represents a group, and when present, the remaining R is a hydrogen atom, a halogen atom or a hydroxy group,
m is an integer from 20 to 5,000, and
n is independently an integer from 3 to 1,000,
A pharmaceutical preparation represented by: A pharmaceutical preparation for preventing or treating inflammation by oral administration comprising a polymerized cyclic nitroxide radical compound as an active ingredient,
The polymerized cyclic nitroxide radical compound has the general formula (II)
Where
A represents unsubstituted or substituted C ₁ -C ₁₂ alkoxy, and when substituted, the substituent is a formyl group, formula R ¹ R ² CH— (wherein R ¹ and R ² are independently C ₁ -C ₄ alkoxy or R ¹ and R ² are -OCH ₂ CH ₂ O together _{-, - O (CH 2)} 3 O- or a group of -O (CH ₂₎ represents a ₄ O-). Represents
L ₁ is selected from the group consisting of a single bond, — (CH ₂ ) _c S—, —CO (CH ₂ ) _c S—, wherein c is an integer of 1 to 5,
L ₂ is —C _1-6 alkylene-NH— (C _1-6 alkylene) _q —, where q is 0 or 1, and
R is independently at least 50% of the total number n of R is 2,2,6,6-tetramethylpiperidin-1-oxyl-4-yl, 2,2,5,5-tetramethylpyrrolidine-1- Oxyl-3-yl, 2,2,5,5-tetramethylpyrrolin-1-oxyl-3-yl and 2,4,4-trimethyl-1,3-oxazolidine-3-oxyl-2-yl, 2, Residue of cyclic nitroxide radical compound selected from the group consisting of 4,4-trimethyl-1,3-thiazolidin-3-oxyl-2-yl and 2,4,4-trimethyl-imidazolindin-3-oxyl-2-yl Represents a group, and when present, the remaining R is a hydrogen atom, a halogen atom or a hydroxy group,
m is an integer from 20 to 5,000, and
n is independently an integer from 3 to 1,000,
Represented by
A pharmaceutical formulation , wherein the inflammation is arteriosclerosis or arthritis or Alzheimer . R is the following formula
Where R ′ is methyl.
The group represented by the above formula occupies at least 80% of the total number n of R,
The formulation of claim 1 . R is the following formula
Where R ′ is methyl.
The group represented by the above formula occupies at least 80% of the total number n of R,
The formulation according to claim 2 .