JP2012229179A - ANTI-CYTOKINE MEDIATED DISEASE AGENT INCLUDING α-LIPOYL VITAMIN E DERIVATIVE AS ACTIVE INGREDIENT - Google Patents

Abstract

〔式中、Ｒ_１、Ｒ_２およびＲ_３は、同一または異なって、水素原子またはメチル基を示す〕。
【選択図】なし[PROBLEMS] To provide a pharmaceutical composition effective for the prevention or treatment of diseases caused or exacerbated by inflammatory cytokines, particularly septic shock, acute organ damage or organ failure, and various organ disorders caused by ischemia reperfusion, and the like It aims at providing the novel compound which is an active ingredient.
An α-lipoyl vitamin E derivative represented by the following general formula (I) or a pharmacologically acceptable salt thereof is used as an active ingredient:

[Wherein, R ₁ , R ₂ and R ₃ are the same or different and represent a hydrogen atom or a methyl group].
[Selection figure] None

Description

  The present invention relates to an anti-cytokine-mediated disease agent that is particularly effective for the prevention and treatment of diseases (cytokine-mediated diseases) that develop or worsen mediated by inflammatory cytokines such as TNF-α and IL-6.

  In many pathologies, increased cytokines have been shown to be closely related to the worsening of the pathology. Although many types of cytokines exist, tumor necrosis factor α (hereinafter referred to as “TNF-α”) is stimulated to various cells (for example, lipopolysaccharide (LPS)) or external cellular stress (for example, It is an important cytokine secreted by various cells including monocytes and macrophages in response to osmotic shock and peroxide).

  Increased levels of TNF-α above basal levels are associated with the mediation or exacerbation of several disease states, including Paget's disease; osteoporosis; multiple myeloma; acute and chronic myeloid Leukemia; Pancreatic β-cell destruction; Inflammatory bowel disease; Adult respiratory distress syndrome (ARDS); Psoriasis; Crohn's disease; Ulcerative colitis; Anaphylaxis; Contact dermatitis; Asthma; Bone resorption; Graft-versus-host reaction; Ischemic reperfusion injury; Atherosclerosis; Brain trauma; Multiple sclerosis; Cerebral malaria; Sepsis; Septic shock; Toxin shock syndrome; , And muscle pain from infection. HIV-1, HIV-2, HIV-3, cytomegalovirus (CMV), influenza, adenovirus, herpesvirus (including HSV-1, HSV-2), and herpes zoster are also exacerbated by elevated TNF-α It is known.

  Furthermore, TNF-α has been reported to play an important role in organ damage in head trauma, stroke and ischemia. For example, in head trauma rats, TNF-α levels increased in bruised hemispheres (Non-Patent Document 1), and in ischemic rats in which the central cerebral artery was occluded, TNF-α mRNA increased. (Non-Patent Document 2), which also shows that cytokines play an important role in organ damage.

  As a function of TNF-α in specific tissues, it has been reported that administration of TNF-α to the rat cortex results in significant deposition of neutrophils in capillaries and attachment within small blood vessels. This TNF-α is an upstream factor in a complex cytokine cascade. As a result, elevated levels of TNF-α can lead to elevated levels of various other cytokines, such as IL-1, IL-6, and IL-8. Therefore, it has been reported that TNF-α promotes infiltration of other cytokines (IL-1β, IL-6) and chemokines, and this promotes neutrophil infiltration into the infarct region (Non-patent Document 3). .

  IL-6 is a lectin produced by cells such as T cells and macrophages, and is one of cytokines that control humoral immunity. IL-6 was cloned in 1986 as a complementary DNA (cDNA), and it was subsequently revealed that IL-6 is involved in various physiological phenomena and mechanisms of inflammation and immune diseases. Moreover, deterioration and / or onset of many disease states mediated by IL-6 production at sites of inflammation or injury (eg ischemia) has been associated (Non-patent Document 4). Furthermore, it has been reported that suppression of IL-6 also has an effect of improving organ damage even in ischemia reperfusion injury (Non-patent Document 5). Thus, it can be said that such disease states include, for example, asthma, inflammatory bowel disease, psoriasis, adult respiratory distress syndrome, heart and kidney reperfusion injury, thrombosis, and glomerulonephritis.

  Several approaches have been taken to block the effects of TNF-α. One approach is the use of soluble receptors for TNF-α (eg, TNFR-55 or TNFR-75), and efficacy has been observed in animal models of TNF-α mediated disease states. The second approach is a method of neutralizing TNF-α using a monoclonal antibody specific to TNF-α, cA2 (Non-patent Document 6). In addition, suppression of IL-6 has also been reported to improve pathology in ischemia reperfusion injury (Non-Patent Document 5), and these approaches involve either TNF-α and receptor antagonism by either protein segregation or receptor antagonism. This is to block the effect of IL-6.

  It has also been proposed to use heterocyclic compounds such as 1,4,5-trisubstituted imidazole, condensed triazole, and indazole for the treatment of diseases mediated by cytokines such as TNF-α (Patent Documents 1 and 2). reference).

JP 2004-99622 A Special Table 2008-504294 JP 2004-217669 A JP 2009-227632 A Special Table 2003-535134

Shohami et al., J. Cereb. Blood Flow Metab. 14, 615 (1994) Feurstein et al., Neurosci. Lett. 165, 125 (1993) Feurstein, Stroke 25, 1481 (1994) Frink M et al., Scand J Trauma Resusc Emerg Med. 2009 Sep 27; 17 (1): 49. Kimizuka K et al., Am J Transplant. 2004 Apr; 4 (4): 482-94. Feldmann et al. 5 Immunological Reviews, pp.195-223 (1995)

  The present invention relates to a pharmaceutical composition effective for the prevention or treatment of a disease that develops or worsens mediated by inflammatory cytokines (particularly TNF-α, IL-6). The object is to provide a product (in the present invention, this is referred to as an “anti-cytokine-mediated disease agent”). More specifically, it is useful for preventing or treating inflammation and various pathologies caused by inflammation such as septic shock, acute organ damage and organ failure, and protecting various organ damage caused by ischemia reperfusion. The object is to provide a pharmaceutical composition.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have changed the α lipoylvitamin E derivative represented by the following general formula (1) to a cell stimulating factor (lipopolysaccharide (LPS)). It has the effect of significantly suppressing the inflammation of tissues, and the induction of inflammatory cytokines (TNF-α, IL-6) and cytokine mediator (HMGB1), and the effects of inflammatory molecules (iNOS, ICAM-1) by inflammatory cytokines It has been found that expression induction is also significantly suppressed, and from this finding, α-lipoylvitamin E derivative (I) is effective as an anti-inflammatory agent, and inflammatory cytokines (particularly TNF-α and IL-6) Is effective as a preventive or therapeutic agent for diseases that develop or worsen through intervention.

  Incidentally, although α-lipoic acid and vitamin E have been known to have anti-inflammatory activity (see, for example, Patent Documents 3 to 5), α-lipoic acid has poor stability and is not used. There were problems such as limitations, and the establishment of technology to stabilize them was required. It is also known that vitamin E is also easily oxidized and susceptible to deterioration such as coloring. On the other hand, the α-lipoyl vitamin E derivative provided by the present invention is a compound that is more stable than α-lipoic acid and vitamin E, and can be widely used without being restricted by usage or handling.

The present invention has been completed based on the above findings and includes the following embodiments.
(I) α-lipoyl vitamin E derivative and production method thereof (I-1) α-lipoyl vitamin E derivative represented by the following general formula (I):

(Wherein R ₁ , R ₂ and R ₃ are the same or different and represent a hydrogen atom or a lower alkyl group having 1 to 3 carbon atoms).

(I-2) The α lipoyl vitamin E derivative according to (I-1), wherein the lower alkyl group having 1 to 3 carbon atoms is a methyl group.
(I-3) The α-lipoylvitamin E derivative according to (I-1), excluding compounds in which R ₁ , R ₂ and R ₃ in the general formula (I) are simultaneously hydrogen atoms.
(I-4) The α-lipoylvitamin E derivative according to (I-1), wherein R ₁ , R ₂ and R ₃ are all methyl groups in the general formula (I).
(I-5) α-lipoyl according to any one of (I-1) to (I-4), comprising a step of ester-linking α-lipoic acid and a vitamin E derivative represented by the following general formula (II) Method for producing vitamin E derivative (I):

(Wherein R ₁ , R ₂ and R ₃ are the same or different and represent a hydrogen atom or a lower alkyl group having 1 to 3 carbon atoms).

 (I-6) The production method according to (I-5), wherein the ester bonding step is a step of ester bonding using a mixed acid anhydride method.

(II) Pharmaceutical composition (II-1 ) A pharmaceutical composition comprising the α-lipoyl vitamin E derivative (I) described in any one of (I-1) to (I-4) as an active ingredient.
(II-2) The pharmaceutical composition according to (II-1), which is an anti-cytokine-mediated disease agent for preventing or treating a cytokine-mediated disease.
(II-3) The pharmaceutical composition according to (II-2), wherein the cytokine-mediated disease is an inflammatory disease that develops or worsens mediated by inflammatory cytokines, and the anti-cytokine-mediated disease agent is an anti-inflammatory agent object.
(II-4) Cytokine-mediated diseases are Paget's disease; osteoporosis; multiple myeloma; acute and chronic myelogenous leukemia; pancreatic beta cell destruction; inflammatory bowel disease; adult respiratory distress syndrome (ARDS); Ulcerative colitis; anaphylaxis; contact dermatitis; asthma; muscle degeneration; cachexia; Reiter's syndrome; type I and type II diabetes; bone resorption; graft-versus-host reaction; Atherosclerosis; brain trauma; multiple sclerosis; cerebral malaria; sepsis; septic shock; toxin shock syndrome; fever and muscle pain from infection (II-2) ).
(II-5) The pharmaceutical composition according to (II-2), wherein the anti-cytokine mediated disease agent is a preventive or therapeutic agent for septic shock, acute organ injury or organ failure.
(II-6) The pharmaceutical composition according to (II-2), wherein the anti-cytokine mediated disease agent is an organ or tissue protective agent against organ damage caused by septic shock or ischemia reperfusion injury.

IR data of α-lipoyl α-tocopherol (α-lipoic acid α-tocopherol ester) produced in Production Example 1 are shown. Among the rats (control group, LPS group, Lipoyl-VE + LPS group, VE + LPS group) divided into each group in Experimental Example 1, the control group (FIGS. AC), LPS group (FIGS. DF), and Lipoyl-VE + LPS The group (FIGS. GI) is administered lipopolysaccharide (LPS), and light microscope images of lung tissue collected 12 hours later are shown. Note that the image display magnification is increased in the order of FIG. A → B → C, FIG. D → E → F, and FIG. G → H → I. FIG. 1 shows the histological score of each group (control group, LPS group, Lipoyl-VE + LPS group) for each parameter of congestion, edema, inflammation and bleeding (black bar: control). Group, white bar: LPS group, slanted bar: Lipoyl-VE + LPS group). Results are shown as mean ± SD. * Indicates that there is a significant difference (p <0.05) from the LPS group. For LPS group (-□-), Lipoyl-VE + LPS group (-●-), and VE + LPS group (-▲-), blood was collected before LPS administration, at 3, 6, and 12 hours after LPS administration. The results of measuring the TNF-α concentration in each serum (FIG. 4A) and the IL-6 concentration in the serum (FIG. 4B) are shown (Experimental Example 1). For LPS group (-■-) and Lipoyl-VE + LPS group (-●-), blood was collected at 3 hours, 6 hours and 12 hours after LPS administration, and the results of measuring the HMGB1 concentration in each serum This is shown (Experimental Example 1). Data are shown as mean ± SD. * Indicates that there is a significant difference (P <0.05) from the LPS group. The result of having measured the expression of iNOS, ICAM-1, and HMGB1 in the lung tissue of a control group, a LPS group, and a Lipoyl-VE + LPS group is shown (Experimental example 1 (2-4)).

  The present invention relates to a novel α-lipoyl vitamin E derivative represented by the following general formula (I). The present invention also relates to a pharmaceutical composition comprising an α-lipoyl vitamin E derivative represented by the following general formula (I) or a pharmacologically acceptable salt thereof as an active ingredient.

[In formula, R < ₁ >, R < ₂ > and R < ₃ > are the same or different, and show a hydrogen atom or a C1-C3 lower alkyl group. ].

    The α-lipoyl vitamin E derivative (hereinafter, also referred to as “the present compound” in the present specification) is represented by α-lipoic acid and the following formula (II) as represented by the formula (I): It has a chemical structure formed by ester bonding with a vitamin E derivative.

[Wherein R ₁ , R ₂ and R ₃ have the same meaning as described above. ].

In the above formulas (I) and (II), examples of the lower alkyl group having 1 to 3 carbon atoms represented by R ₁ , R ₂ or R ₃ include a methyl group, an ethyl group, a propyl group, and an isopropyl group. be able to. A methyl group and an ethyl group are preferable, and a methyl group is more preferable. In the general formulas (II) and (I), the compounds in which R ₁ , R ₂ and R ₃ are all methyl groups are α-tocopherol (II-1) and α-lipoyl α-tocopherol (I-1), respectively. (See Production Example 1); Compounds in which R ₁ and R ₂ are methyl groups and R ₃ is a hydrogen atom are β-tocopherol (II-2) and α-lipoyl β-tocopherol (I-2) (Production Example), respectively. 4); compounds in which R ₁ and R ₃ are methyl groups and R ₂ is a hydrogen atom are γ-tocopherol (II-3) and α-lipoyl γ-tocopherol (I-3) (see Production Example 2), respectively. The compounds in which R ₁ is a methyl group and R ₂ and R ₃ are hydrogen atoms are δ-tocopherol (II-4) and α-lipoyl δ-tocopherol (I-4) (see Production Example 3), respectively.

As the α-lipoyl vitamin E derivative represented by the general formula (I), α-lipoyl α-tocopherol in which R ₁ , R ₂ and R ₃ are all methyl groups is preferable.

  This compound is prepared by the production of α-lipoic acid and a vitamin E derivative represented by the above formula (II) (preferably α-tocopherol, β-tocopherol, γ-tocopherol or δ-tocopherol) by polymerization of α-lipoic acid. In order to prevent this, the mixed acid anhydride method, which is an esterification method under basic conditions, is suitable. As an example, as shown in Production Examples 1 to 4, α-lipoic acid is dissolved in a nonpolar solvent such as acetonitrile or tetrahydrofuran (THF) together with triethylamine, and this is mixed with ethyl chlorocarbonate or 2,4,6. -By reacting vitamin E derivative (II) under low temperature conditions (for example, about -5 ° C to 0 ° C) using trichlorobenzoic acid chloride or the like, it is less expensive than other esterification methods. This compound (α-lipoyl vitamin E derivative) can be produced in a high yield.

  The compound thus produced may be in the form of a crude product (oil composition), but after extracting the reaction product with isopropyl ether, the extract is washed with alkaline water or water, and then a silica gel column. It can also be purified using chromatography or the like.

  As shown in Experimental Examples 1 (2-2) and (2-3), which will be described later, this compound including α-lipoyl α-tocopherol (referred to as “lipoyl-VE” in Experimental Example 1) The production or secretion of inflammatory cytokines (IL-6 and TNF-α) and cytokine mediators (HMGB1) induced by saccharides (LPS), although not complete, can be suppressed.

  These cytokines (IL-6 and TNF-α) are usually strongly induced by LPS and are produced and secreted at an early stage of the inflammatory response. In particular, TNF-α is a major factor mediating inflammation, and its release is also known to activate other cytokines such as IK-1β and IL-6, and to be involved in cell damage (Bone RC. Crit Care Med 1996; 24: 163-172). TNF-α also plays a crucial role in the pathogenesis of early shock states (ie hypotension, fever) and organ failure associated with septic shock (Russell JA. N Engl J Med2006; 355: 1699-1713 ). As shown in Experimental Example 1, the present compound remarkably suppresses the production or secretion of these inflammatory cytokines. Therefore, according to the compound, the onset or worsening of the inflammatory cytokine may occur. It is thought that the disease (cytokine mediated disease) to be prevented or improved.

  On the other hand, IL-6 is well associated with the severity and mortality of sepsis and is also a predictor of sepsis (Remick DG, et al., Shock2002; 17: 463-467). IL-6 is also thought to play an important role in the pathogenesis of septic shock and organ damage (Frink M, et al., Scand J Trauma Resusc Emerg Med. 2009 Sep 27; 17 (1): 49 .). Some data indicate that IL-6 upregulation is associated with cytotoxicity and organ damage associated with subsequent IL-6 elevation (Frink M, et al., Scand J Trauma Resusc Emerg Med. 2009 Sep 27; 17 (1): 49.). Furthermore, it is suggested that the control of IL-6 may have an action of reducing organ damage (Kimizuka K, et al., Am J Transplant. 2004 Apr; 4 (4): 482-94. ). In Experimental Example 1 described later, it was confirmed that LPS-induced inflammatory cytokines (IL-6, TNF-α) were decreased in vivo by oral administration of this compound. Inhibition of inflammatory cytokines by oral administration of this compound is a direct effect on cells, or as shown in Experimental Example 1 (2-4), suppresses cytokine production through suppression of iNOS and ICAM-1 expression. In any case, according to the present compound, the production of inflammatory cytokines (IL-6, TNF-α) is suppressed and cytotoxicity by IL-6 may be caused. It is considered that septic shock and organ damage can be improved.

  As an experiment supporting this, in Experimental Example 1 (2-1), which will be described later, acute organ damage in the lung seen in LPS-induced systemic inflammation model rats is significantly prevented or suppressed by oral administration of this compound. Which indicates that.

  In addition, it has been suggested that organ damage caused by ischemia reperfusion is related to inflammatory cytokine production, and inflammatory cytokines are increased by ischemia reperfusion (I / R) of myocardium and cerebrum. (Zhang M, Chen L. Cardiovasc Hematol Disord Drug Targets. 2008 Sep; 8 (3): 161-72.). As described above, the present compound has an action of reducing inflammatory cytokines, but as a result of significantly reducing serum cytokine concentration based on the cytokine reducing action, ischemic properties such as renal ischemia reperfusion injury and cerebral ischemia reperfusion injury It is thought that reperfusion failure is protected and prevented.

  As described above, in Experimental Example 1 (2-3), the present compound including α-lipoyl α-tocopherol can suppress the production or secretion of LPS-induced cytokine mediator (HMGB1). HMGB1 is a substance known to amplify late inflammatory response and promote cytokine secretion (van Zoelen MA, et al., Shock 2009; 31: 280-284). Recently, it is known to function as an end-stage inflammatory mediator during septic shock and as an early mediator of ischemia reperfusion injury (Tsung A, et al., J Exp Med 2005; 201: 1135 -1143).

  Based on the above, the present compounds are useful for diseases that develop or worsen mediated by inflammatory cytokines, particularly TNFα and IL-6 (cytokine-mediated diseases), particularly systemic inflammation and associated diseases (for example, septic shock, A pharmaceutical composition of the present invention that is useful as an active ingredient for preventing or treating acute organ damage, organ failure, etc.) and comprising the present compound as an active ingredient is a prophylactic or therapeutic agent for such cytokine-mediated diseases, and a cytokine. It is useful as a protective agent that protects against intervening diseases.

  Cytokine-mediated diseases targeted by the present invention include inflammatory diseases; Paget's disease; osteoporosis; multiple myeloma; acute and chronic myelogenous leukemia; pancreatic β-cell destruction; inflammatory bowel disease; Psoriasis; Crohn's disease; ulcerative colitis; anaphylaxis; contact dermatitis; asthma; muscle degeneration; cachexia; Reiter's syndrome; type I and type II diabetes; Reperfusion injury; atherosclerosis; brain trauma; multiple sclerosis; cerebral malaria; sepsis; septic shock; toxin shock syndrome;

  In particular, the cytokine-mediated disease agent of the present invention is an agent for preventing or treating septic shock, acute organ injury or organ failure mediated by inflammatory cytokines, and further protection of organs or tissues against ischemia reperfusion injury It can be effectively used as an agent. It should be noted that ischemia-reperfusion injury (also referred to as “ischemia-reperfusion injury”) refers to various changes in the microcirculation within an organ or tissue when blood reperfusion occurs in the ischemic organ or tissue. It is a disorder caused by the production of toxic substances. Such ischemia reperfusion injury is often seen after reperfusion injury for cerebral infarction, myocardial infarction, mesenteric vascular occlusion, or after organ transplantation. The mechanism of its occurrence includes disorders due to the production of active radicals such as superoxide and hydroxy radicals and free radicals such as nitric oxide, and disorders based on the interaction between activated neutrophils and vascular endothelial cells, as well as various cytokines. Problems caused by the production of chemical mediators are considered. It is known that ischemia / reperfusion injury causes damage (remote organ injury) not only to the local area where ischemia occurs but also to the main organs of the whole body. In particular, the brain, lungs, liver, kidneys, etc. may become target organs, resulting in multiple organ failure.

When the present compound is used as an active ingredient of an anti-cytokine mediated disease agent, one or more of the present compounds can be contained in appropriate combination depending on the purpose and necessity. Among these compounds, preferably, in the above formula (I), the lower alkyl group represented by R ₁ , R ₂ or R ₃ is a methyl group, and more preferably R ₁ , R ₂ and R _3. Are α-lipoyl α-tocopherol, each of which is a methyl group.

  This compound is administered as an anti-cytokine-mediated disease agent orally or parenterally (intravenous, subcutaneous, transdermal, transpulmonary, transmucosal (eg, nasal), rectal administration, etc.) . Oral administration is preferred. The anti-cytokine-mediated disease agent is a pharmaceutically acceptable carrier (excipient, binder, disintegrant, disintegration aid, lubricant, wetting agent, etc.) that is usually used for oral or parenteral administration. And additives, granules, powders, tablets (including sugar-coated tablets), pills, buccals, capsules (including soft capsules and hard capsules), syrups, solutions, emulsions, suspensions, It can be prepared by formulating into a desired form such as a cream, ointment, eye drop, injection, drop, nasal drop and the like.

  In particular, since the present compound is oil-soluble and insoluble in water, it can also be prepared in the form of an emulsion.

  For these formulations, commonly used pharmaceutically acceptable carriers such as binders (eg syrup, gum arabic, gelatin, sorbit, tragacanth, polyvinylpyrrolidone), excipients (eg lactose, sugar, corn starch, Potassium phosphate, sorbit, glycine), lubricant (eg, magnesium stearate, talc, polyethylene glycol, silica), disintegrant (eg, potato starch), wetting agent (eg, sodium lauryl sulfate), thickener, dispersion A reabsorption accelerator, a pH adjuster, a surfactant, a solubilizer, a preservative, an emulsifier, an isotonic agent, a stabilizer and the like may be appropriately used as an agent and other additives.

  The dosage when using this compound as an anti-cytokine-mediated disease agent varies depending on the type of the compound used, the weight and age of the patient, the type and condition of the target disease, and the administration method. In terms of the amount of this compound, about 1 mg to about 30 mg once a day for adults in the case of injections, about 1 mg to about 100 mg once a day for adults such as tablets. It is good to do. In the case of a mucosal administration agent such as eye drops, it is preferable to administer a preparation having a concentration of about 0.01 to 5 (w / v)% several times a day for adults.

  The pharmaceutical composition (anti-cytokine mediated disease agent) containing the present compound may appropriately contain other anti-inflammatory agent, anti-cytokine mediated disease agent or another kind of medicinal component as long as it does not contradict the purpose of the present invention. .

  Hereinafter, the configuration and effects of the present invention will be described in more detail based on production examples and experimental examples. However, these experimental examples are merely examples, and the present invention is not limited by these experimental examples.

[Production example]
Production Example 1
Preparation of α-lipoyl α-tocopherol (α-lipoic acid α-tocopherol ester) α-lipoic acid 2.06 g (0.01 mol) and triethylamine 1.1 g were dissolved in acetonitrile 20 ml, cooled to −5 ° C., and stirred. To this, 1.1 g of ethyl chlorocarbonate was gradually added dropwise. 5-10 minutes after the completion of the dropwise addition, a solution (10-20 ° C) of 4.3 g (0.01 mol) of α-tocopherol in 30 ml of THF was added at once and stirred for 15 minutes. Subsequently, this was returned to room temperature (25 degreeC), and also after stirring for about 1 hour, the solvent was distilled off. The residue was extracted with isopropyl ether, the extract was washed with 1% sodium hydroxide and water, and isopropyl ether was distilled off to obtain about 6 g of a residual oil.

The residual oil was subjected to column chromatography using normal phase silica gel (Wako Pure Chemical Industries, Ltd.) as a carrier, and a solvent containing n-hexane and isopropyl ether in a volume ratio of 5: 3 was used. Development and elution fractionated (temperature condition: room temperature (25 ° C.)). A portion of each fraction was subjected to thin layer chromatography using normal phase silica gel as a carrier, and developed using a solvent containing n-hexane and isopropyl ether in a volume ratio of 5: 3, and α-tocopherol. The appearance of a new spot located between the spot and the spot of α-lipoic acid was confirmed. Fractions having such spots were collected and concentrated to obtain 3.5 g (yield 56.6%) of the title target compound (α-lipoyl α-tocopherol) as a pale yellow oil. The results of subjecting the α-lipoyl α-tocopherol thus produced to IR are shown in FIG. As shown in FIG. 1, a peak corresponding to methylene groups resulting from α- tocopherol 2850～2980Cm ^-1, since the peak corresponding to a carboxyl group resulting from α- lipoyl to 1730 cm ^-1 was confirmed The compound was determined to have the structure of α-lipoyl α-tocopherol.

  Further, the obtained α-lipoyl α-tocopherol was subjected to thin layer chromatography using normal phase silica gel as a carrier and developed using a solvent containing n-hexane and isopropyl ether in a volume ratio of 5: 3. Which showed a single spot, and its Rf was 0.43.

Production Example 2
Production of α-lipoyl γ-tocopherol (α-lipoic acid γ-tocopherol ester) Using α-lipoic acid 0.5 g (0.0024 mol) and γ-tocopherol 1.0 g (0.0024 mol) as tocopherol, the same as Production Example 1 To give 530 mg (yield 35.0%) of the title compound (α-lipoyl γ-tocopherol) as a pale yellow oil.

  The obtained α-lipoylγ-tocopherol was subjected to thin layer chromatography using normal phase silica gel as a carrier, and developed using a solvent containing n-hexane and isopropyl ether in a volume ratio of 5: 3. , Showing a single spot, Rf was 0.42.

Production Example 3
Production of α-lipoyl δ-tocopherol (α-lipoic acid δ-tocopherol ester) Using 2.06 g (0.01 mol) of α-lipoic acid and 4.0 g (0.01 mol) of δ-tocopherol as tocopherol, the same as Production Example 1 To give 3.0 g (51.0%) of the title compound (α-lipoyl δ-tocopherol) as a pale yellow oil.

  The obtained α-lipoyl δ-tocopherol was subjected to thin-layer chromatography using normal phase silica gel as a carrier, and developed using a solvent containing n-hexane and isopropyl ether in a volume ratio of 5: 3. , Showing a single spot, Rf was 0.41.

Production Example 4
Production of α-lipoyl β-tocopherol (α-lipoic acid β-tocopherol ester) Using 0.5 g (0.0024 mol) of α-lipoic acid and 1.0 g (0.0024 mol) of β-tocopherol as tocopherol, the same as in Production Example 1 To give 550 mg (yield 36.3%) of the title compound (α-lipoyl β-tocopherol) as a pale yellow oil. The obtained α-lipoyl β-tocopherol was subjected to thin-layer chromatography using normal phase silica gel as a carrier, and developed using a solvent containing n-hexane and isopropyl ether in a volume ratio of 5: 3. , Showing a single spot, Rf was 0.42.

  The tocopherol esters of α-lipoic acid produced in Production Examples 1 to 4 are all superior to the existing tocopherols in that the color tone does not change and oxidation does not proceed even when left in the air. It was confirmed that Moreover, it is much more stable than α-lipoic acid which is easily polymerized and unstable.

[Experimental example]
In the following experiment, α-lipoyl α-tocopherol (this compound) represented by the following formula was used as an example of an α-lipoyl vitamin E derivative. This compound is lipophilic (oil soluble) and is characterized by stability as described above.

Experimental cases Overexpression of pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) has been implicated in shock and organ damage (Bhatia M, et al., J Pathol2004; 202: 145-156).

  Therefore, in the following experimental examples, the present compound inhibits the production or secretion of cytokines (TNF-α, IL-6), and thereby induces systemic inflammation model rats (sepsis model) induced by lipopolysaccharide (LPS). We investigated whether to prevent or improve organ damage in rats).

  All data are shown as mean ± SD and evaluated using one-way analysis of variance (ANOVA). A p-value <0.05 was considered statistically significant.

(1) Male rats (Wister rats ) weighing 250-300 g were used. All rats were allowed unlimited access to food and water before and after the experiment. The study was approved by the Oita University Medical School Ethics Committee for Animal Research. All protocols were in accordance with National Institutes of Health (NIH) guidelines. Anesthesia was performed using 4% sevoflurane (manufactured by Maruishi Pharmaceutical Co., Ltd.).

Experimental Example 1 Effect of α-lipoyl vitamin E on LPS-treated rats (1) Test method Randomize the above male rats, 1) control group, 2) LPS group, 3) lipoyl vitamin E group (lipoyl-VE + LPS group) And 4) Vitamin E group (VE + LPS group) divided into 4 groups, and each of these groups was divided into 1) physiological saline (0.9% NaCl aqueous solution, the same shall apply hereinafter), 2) physiological saline, 3) Lipoyl vitamin E (1 mmol / kg) and 4) Vitamin E (1 mmol / kg) were orally administered. 1 hour after administration, LPS (10 mg / kg) was intravenously injected into the tail vein of the 3 groups of rats 2) to 4), and physiological saline was intravenously injected into the tail vein of the control group 1). Injected. Blood was collected 3, 6 and 12 hours after the intravenous injection of LPS or physiological saline, and the serum cytokine (TNF-α, IL-6) concentration was measured to evaluate the change over time. After a period of time, lungs were removed from each group of rats, and histological evaluation was performed on the tissues of the removed lungs.

(2) Test results (2-1) Optical microscopic analysis Rats of each group (control group, LPS group [saline + LPS], Lipoyl-VE + LPS group) collected 12 hours after intravenous injection of LPS or physiological saline. Lung tissue specimens were fixed in formalin according to a standard method, embedded in paraffin, and sectioned on a microtome. The prepared sections were stained with hematoxylone and eosin.

  The samples were analyzed and the extent of lung injury was determined based on Murakami technology (Murakami K, et al., Shock2002; 18: 236-241). Twenty-four areas in the lung parenchyma were categorized into five levels from 0 to 4 with respect to four parameters (congestion, edema, inflammation and bleeding) (0: none or appears normal, 1: mild, 2: moderate, 3 : Severe, 4: very severe).

  The result of observation of the stained section of lung tissue with an optical microscope is shown in FIG. Although lung tissue changes were not observed in the control group (FIGS. 1A to 1C), significant interstitial edema and infiltration of inflammatory cells were observed in the lung tissue of the LPS group (saline + LPS group) ( 1D-F). In the “Lipoyl-VE + LPS group” treated with this compound, interstitial edema and inflammatory cell infiltration were significantly reduced compared to the LPS group (FIGS. 1G to I). The histological score results for lung tissue are shown in FIG. As can be seen, the histological score of the lung tissue of the LPS group (white bar) was all significantly higher than that of the control group (black bar), and the “Lipoyl-VE + LPS group” treated with this compound ( The score of (hatched bar) was in the middle.

  From this result, it was found that acute injury of lung tissue in a systemic inflammation model animal (sepsis model animal) induced by LPS administration can be significantly prevented by administering this compound in advance.

(2-2) Effect of α-lipoyl vitamin E on serum TNF-α and IL-6 levels 3, 6 and 12 hours after intravenous injection of LPS or physiological saline (control group, LPS group [physiology Serum cytokine concentrations (TNF-α, IL-6) were measured using blood collected from rats of saline + LPS], Lipoyl-VE + LPS group, and VE + LPS group, and the effect of this compound on serum cytokines was examined. The concentrations of TNF-α and IL-6 in the serum were analyzed using a commercially available ELISA kit and a rat-specific monoclonal antibody (Invitrogen) against each of TNF-α and IL-6. Absorbance was measured at 540 nm (Bio-Rad Laboratories).

  Changes over time in serum TNF-α and IL-6 concentrations are shown in FIGS. 4A and B, respectively.

  Serum TNF-α levels did not increase in the control group (results not shown), but LPS group (--- □ ---), Lipoyl-VE + LPS group (-●-), and VE + LPS group An increase was observed in (-▲-), and all reached a peak 3 hours after LPS administration. However, the degree of increase was significantly lower in the Lipoyl-VE + LPS group (-●-) than in the LPS group (--- □ ---), and even compared to the VE + LPS group (-▲-). Significantly lower (Figure 4A). Similarly, the serum IL-6 concentration did not increase in the control group (results not shown), but the LPS group (--- □ ---), the Lipoyl-VE + LPS group (-●-), Both the VE + LPS group (-▲-) reached a peak 3 hours after LPS administration (Fig. 4B). However, the extent of the increase is similar to the TNF-α concentration in the Lipoyl-VE + LPS group (-●-) compared to the LPS group (--- □ ---) and the VE + LPS group (-▲-). It was significantly lower (Figure 4B).

  From these results, it was found that all cytokines (TNF-α, IL-6) induced by LPS administration (LPS stimulation) were significantly suppressed by this compound.

(2-3) Effect of α-lipoyl vitamin E on serum HMGB1 level HMGB1 (high-mobility group box1 protein) is a 30 kD non-histone chromosome-related protein (Bianchi ME, et al., Science, 1898; 243 : 1056-1059), and its serum concentration has been reported to increase due to severe sepsis and septic shock (Wang H, et al., Science 1999; 285: 248-251). HMGB1 is known to amplify late inflammatory response and promote cytokine secretion (cytokine mediator) (van Zoelen MA, et al., Shock 2009; 31: 280-284), It has been shown to function as an end-stage inflammatory mediator during septic shock and as an early mediator of ischemia-reperfusion injury (Tsung A, et al., J Exp Med 2005; 201: 1135-1143). As for local diseases, it has been reported that HMGB1 concentration also increases in rheumatoid arthritis and inflammatory bowel disease.

  Serum HMGB1 concentration using blood collected from rats of each group (control group, LPS group [saline solution + LPS], Lipoyl-VE + LPS group) 3, 6 and 12 hours after intravenous injection of LPS or saline The effect of this compound on serum HMGB1 was examined. The concentration of HMGB1 in the serum was analyzed using a commercially available ELISA kit and a rat-specific monoclonal antibody against HMGB1 (Invitrogen). Absorbance was measured at 540 nm (Bio-Rad Laboratories).

  The time-dependent change of the HMGB1 concentration in serum is shown in FIG.

  The serum HMGB1 concentration did not increase in the control group (results not shown), but increased over time in the LPS group (-■-) and the Lipoyl-VE + LPS group (-●-). All reached a peak 12 hours after LPS administration. However, the increase was significantly lower in the Lipoyl-VE + LPS group (-●-) than in the LPS group (-■-) (Fig. 5).

  From this result, it was found that HMGB1 induced by LPS administration (LPS stimulation) is significantly suppressed by administration of this compound.

(2-4) Effect of lipoylvitamin E on the expression of iNOS, ICAM-1 and HMGB1 iNOS (inducible nitric oxide synthase) in the lung tissue of rats in each group (control group, LPS group, Lipoyl-VE + LPS group) The expression status of ICAM-1 (Intercellular adhesion molecule-1) and HMGB1 was examined using Western blotting. The results are shown in FIG. As shown in FIG. 6, the expression of iNOS, ICAM-1 and HMGB1 were all increased in the LPS group, whereas those increases were significantly suppressed in the lipoyl-VE + LPS group.

  This result suggests that the inhibitory action on the induction of inflammatory cytokines by this compound is that this compound has one mechanism that suppresses the expression of inflammatory molecules such as iNOS, ICAM-1 and HMGB1. That is, this compound is considered to suppress the expression of a protein that is the key to inflammation and exert an anti-inflammatory action based on it.

  In conclusion, from the above experimental examples, the present compound suppresses the secretion of inflammatory cytokines, thereby causing various diseases mediated by inflammatory cytokines, for example, various diseases related to inflammation and inflammation, and caused thereby It is thought to have an action of preventing the occurrence of organ and tissue damage. That is, it is considered that treatment with the present compound can protect and prevent not only inflammation but also various organ disorders (acute organ disorder, organ failure) caused by septic shock and ischemia reperfusion.

Claims (6)

Α-Lipoyl vitamin E derivative represented by the following general formula (I):

(Wherein R ₁ , R ₂ and R ₃ are the same or different and represent a hydrogen atom or a lower alkyl group having 1 to 3 carbon atoms). The α lipoyl vitamin E derivative according to (I-1), wherein the lower alkyl group having 1 to 3 carbon atoms is a methyl group. The α-lipoyl vitamin E derivative according to (I-1), wherein R ₁ , R ₂ and R ₃ are all methyl groups in the general formula (I). The α-lipoyl vitamin E derivative (I) described in any one of (I-1) to (I-3), which comprises esterifying a lipoic acid with a vitamin E derivative represented by the following general formula (II) Production method:

(Wherein R ₁ , R ₂ and R ₃ are the same or different and represent a hydrogen atom or a lower alkyl group having 1 to 3 carbon atoms). A pharmaceutical composition comprising the α-lipoyl vitamin E derivative (I) according to any one of claims 1 to 3 as an active ingredient. The pharmaceutical composition according to claim 5, which is an anti-cytokine mediated disease agent for preventing or treating a cytokine mediated disease.

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