Technical Field

[0001] The present invention relates to the field of medical technology, especially to applications involving binary naltrexone derivatives.

Background Techniques

[0002] Toll-like receptor 4 (TLR4), a typical innate immune receptor, recognizes inflammatory immune response induced by exogenous pathogen-associated molecular patterns (PAMPs), endogenous damage-associated molecular patterns (DAMPs), and xenobiotic-associated molecular patterns (XAMPs). The immune response protects the body, but a persistent inflammatory response can cause damage to the body. Among them, the abnormality of the innate immune TLR4 signaling pathway is closely related to the molecular causes of many diseases, such as autoimmune diseases (systemic lupus erythematosus and rheumatoid arthritis), cancer (lung cancer, breast cancer, and ovarian cancer), and cardiovascular diseases. Therefore, small molecule modulators targeting TLR4 and its accessory protein MD-2 have been extensively studied.

[0003] A series of recent studies have found that narcotics such as morphine, cocaine, and methamphetamine act as xenobiotics, activate microglia through the natural immune receptor TLR4, produce a large number of inflammatory factors, enhance neuronal excitability, and lead to drag dependence and addiction. These data suggest that abnormalities in the TLR4 signaling pathway are also associated with molecular mechanisms of dmg addiction and neuropathic pain. These data suggest that abnormalities in the TLR4 signaling pathway are also associated with molecular mechanisms of dmg addiction and neuropathic pain. Recent studies have shown that naltrexone inhibits lipopolysaccharides (LPS)-induced gene expression in HEK-TLR4 cells, and the (+) and (-) configuration enantiomers exhibit very similar inhibitory effects. In addition, naltrexone and its derivatives have good blood-brain barrier permeability. Among them, (+)-naltrexone has good dmg compliance, universality, and synergy, though (+)- naltrexone has low inhibitory activity on TLR4 and has no stereoselectivity.

[0004] Therefore, it is very necessary to develop an inhibitor with high TLR4 inhibitory activity.

Content of Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide the application of binary naltrexone derivatives. The present invention experimentally found that binary naltrexone derivatives can be used to inhibit the TLR4 signaling pathway, thereby inhibiting the expression of inflammatory factors.

[0006] The present invention provides an application of binary naltrexone derivatives in the preparation of TLR4 signaling pathway inhibitors.

[0007] The present invention provides an application of binary naltrexone derivatives in the preparation of inflammatory factor expression inhibitors.

[0008] Preferably, the inflammatory factor comprises one or more of TNF-a and IL-1B.

[0009] The present invention provides the application of binary naltrexone derivatives in the preparation of inflammatory signaling pathway activation inhibitors.

[0010] Preferably, the inhibitors are used to inhibit activation of TLR4 signaling pathways, the expression of inflammatory factors, or the release of nitric oxide by LPS.

[0011] Preferably, the binary naltrexone derivatives are as shown in formula (I), formula (II) or formula (III).

Formula (II) imnt

Formula (III)

[0013] The present invention provides an application of binary naltrexone derivatives in the preparation of drags for the prevention of autoimmune diseases, cancers, cardiovascular disease, drag addiction, or neuropathic pain.

[0014] Preferably, the autoimmune diseases include systemic lupus erythematosus, and rheumatoid arthritis, and the cancers include lung cancer, breast cancer, and ovarian cancer.

[0015] The present invention provides a drag for controlling an autoimmune disease, cancer, cardiovascular disease, drag addiction, or neuropathic pain, which comprises a binary naltrexone derivative and a pharmaceutically acceptable excipient.

[0016] Preferably, the pharmaceutical dosage form comprises one of an injection preparation, an oral preparation, and a spray preparation.

[0017] Compared to the prior art, the present invention provides the application of binary naltrexone derivatives in the preparation of TLR4 signaling pathway inhibitors. It also provides the application of binary naltrexone derivatives in the preparation of inflammatory factor expression inhibitors, binary naltrexone. The derivatives are used in the preparation of inflammatory signaling pathway activation inhibitors. The present invention confirmed in vitro that the levels of nitric oxide and downstream inflammatory factors were significantly increased in glial cells activated by lipopolysaccharides (LPS). Binary naltrexone derivatives significantly inhibited the release of nitric oxide and the expression of downstream inflammatory factors. The inhibitory activities of TLR4 were for formula

(II) (4.7 ± 0.48 mM) and for formula (III) (6.23 ± 1.82 mM), respectively. Formula (I) has no significant inhibitory effect. This means that binary naltrexone derivatives play a key role in the innate immune receptor TLR4 mediated immune response and inhibit the TLR4 signaling pathway, with selectivity indicated in formula (II) and formula

(III).

Description of Attached Figures

[0018] Figure 1 depicts the inhibitory effect of binary naltrexone derivatives formula (II) on the activation of TLR4 signaling pathway in BV-2 microglia in embodiment 1 of the present invention.

[0019] Figure 2 depicts the inhibitory effect of binary naltrexone derivatives formula (III) in TLR4 signaling pathway activation in BV-2 microglia in embodiment 1 of the present invention.

[0020] Figure 3 depicts the inhibitory effect of inflammatory factor TNF-a expression in BV-2 microglia in embodiment 2 of the present invention.

[0021] Figure 4 depicts the inhibitory effect of binary naltrexone derivatives formula (II) in inflammatory factor IL-1B expression in BV-2 microglia in embodiment 2 of the present invention.

[0022] Figure 5 depicts the inhibitory effect of binary naltrexone derivatives formula (II) in TLR4 signaling pathway activation in primary microglia in embodiment 3 of the present invention. Cb

[0023] Figure 6 depicts the effect of binary naltrexone derivatives formula (II) in inflammatory factor TNF-a expression in primary microglia in embodiment 4 of the present invention.

[0024] Figure 7 depicts the inhibitory effect of binary naltrexone derivatives formula (II) in TLR4 signaling pathway activation in primary astrocytes in embodiment 5 of the present invention.

[0025] Figure 8 depicts the effect of binary naltrexone derivatives formula (II) in inflammatory factor TNF-a expression in primary astrocytes in embodiment 6 of the present invention.

Detailed Description of the Preferred Embodiments

[0026] The present invention provides an application of the binary naltrexone derivatives, and those skilled in the art can refer to the contents of this document and improve it as appropriate. It is to be noted that all similar substitutions and modifications will be apparent to those skilled in the art. They all fall within the scope of the present invention. The method and application of the present invention have been described in the preferred embodiments, and it is obvious that the method and application of the present invention can be modified or combined and modified to achieve and use the present invention without departing from the content, spirit, and scope of the technology of the present invention.

[0027] The present invention provides the application of binary naltrexone derivatives in the preparation of TLR4 signaling pathway inhibitors.

[0028] According to the present invention, the inhibitors are preferably used to inhibit TLR4 signaling pathway activation by LPS.

[0029] The TLR4 described in the present invention preferably has a primary expression in BV-2 microglia, primary microglia, or primary astrocytes.

[0030] The binary naltrexone derivatives of the present invention are shown as formula (I), formula (II) or formula (III); preferably, the structure is as shown in formula II or formula I

Formula (II)

[0031]

Formula (III)

[0032] The present invention is not limited in its source, and is commercially available or prepared according to a conventional method of the prior art. The preparation method of the compound of the present invention is preferably according to the literature (Portoghese, PS; Nagase, H.; Takemori, AE Stereochemical Studies on 31. Only One Pharmacophore Is Required for the K-Opioid Antagonist Selectivity of Norbinaltorphimine. Journal of Medicinal Chemistry, 31 (1988), 1344-1347). [0033] As a typical natural immune receptor, TLR4 can recognize inflammatory immune responses induced by exogenous pathogen-associated molecular patterns (PAMPs), endogenous damage-associated molecular patterns (DAMPs), and xenobiotic -associated molecular patterns (XAMPs). The immune response can protect the body, but the persistent inflammatory response can cause damage to the body. Among them, the natural immune TLR4 signaling pathway abnormalities are closely related to the molecular causes of many diseases, such as autoimmune diseases (systemic lupus erythematosus and rheumatoid arthritis), cancers (lung cancer, breast cancer, and ovarian cancer) and cardiovascular diseases.

[0034] The prior art discloses the activation of microglia by the natural immune receptor TLR4, which produces a large number of inflammatory factors and enhances neuronal excitability, resulting in a pharmaceutical dependence and addiction.

[0035] The inventor experimentally confirmed that binary naltrexone derivatives play an important role in the immune response mediated by the natural immune receptor TLR4 and are the major TLR4 signaling pathway inhibitors.

[0036] Through in vivo natural immune pathway activation signal nitric oxide and downstream inflammatory factor assay, the present invention found that binary naltrexone derivatives have significant inhibitory effects on the natural immune receptor TLR4-mediated signaling pathways. Moreover, different binary naltrexone derivatives showed different inhibitory activities against TLR4-mediated signaling pathways.

[0037] The present invention confirmed by experiments that TLR4 inhibitory activity was for formula (II) (4.7 ± 0.48 mM) and formula (III) (6.23 ± 1.82 pM), meaning that binary naltrexone derivatives played a key role in the natural immune receptor TLR4-mediated immune response. Moreover, formula (II) and formula (III) showed selectivity during the inhibition of TLR4 signaling pathways.

[0038] The present invention provides the application of binary naltrexone derivatives in the preparation of inflammatory factor expression inhibitors.

[0039] The inflammatory factor described in the present invention preferably includes one or more of TNF-a and IL-1B.

[0040] According to the present invention, the inhibitors are preferably used to inhibit inflammatory factor expression or nitric oxide release by LPS.

[0041] The inventor experimentally confirmed that the inhibitors formula (II) and formula (III) described by the present invention significantly inhibited the release of nitric oxide, and the inhibitory activities for formula (II) and (III) were (4.7 ± 0.48 pM) and (6.23 ± 1.82 pM), respectively.

[0042] The present invention confirmed by in vitro experiments that the levels of nitric oxide and downstream inflammatory factors were significantly increased in glial cells treated with lipopolysaccharides (LPS). Binary naltrexone derivatives significantly inhibited the release of nitric oxide and the expression of downstream inflammatory factors.

[0043] The present invention also provides the application of binary naltrexone derivatives in the preparation of inhibitors of inflammatory signaling pathways.

[0044] According to the present invention, the described inhibitors are used to inhibit TLR4 pathway activation, inflammatory factor expression or nitric oxide release caused by LPS.

[0045] The present invention experimentally confirmed that the binary naltrexone derivatives can significantly inhibit the release of nitric oxide and the expression of downstream inflammatory factors, thus confirming that binary naltrexone derivatives can inhibit the activation of signaling pathways.

[0046] The binary naltrexone derivatives of the present invention are shown as formula (I), formula (II) or formula (III); preferably, the structure is as shown in formula (II) or formula (III).

ormu a ( )

[0048] The binary naltrexone derivatives of the formula (I) structure may be labeled as (-)-l, and the binary naltrexone derivatives of the formula (II) structure may be labeled as (+)-l; the binary naltrexone derivatives of the formula (III) structure may be labeled as 2.

[0049] The present invention is not limited in its source, and is commercially available or prepared according to a conventional method of the prior art. The preparation method of the compound of the present invention is preferably according to the literature (Portoghese, PS; Nagase, H.; Takemori, AE Stereochemical Studies on 31. Only One Pharmacophore Is Required for the K-Opioid Antagonist Selectivity of Norbinaltorphimine. Journal of Medicinal Chemistry, 31 (1988), 1344-1347).

[0050] The present invention provides application of binary naltrexone derivatives in the preparation of drags for the prevention of autoimmune diseases, cancers, or cardiovascular disease.

[0051] The autoimmune diseases in the present invention include systemic lupus erythematosus, and rheumatoid arthritis, and the cancers include lung cancer, breast cancer, and ovarian cancer

[0052] Since the abnormality of the TLR4 signaling pathway is also related to the molecular mechanism of drag addiction and neuropathic pain, the present invention finds that binary naltrexone derivatives can reduce drag dependence and addiction by inhibiting the TLR4 signaling pathway. This invention will provide an effective therapeutic approach for drag addiction and neuropathic pain.

[0053] The present invention provides a drag for controlling autoimmune disease, cancer, cardiovascular disease, drag addiction, or neuropathic pain, which comprises a binary naltrexone derivative and a pharmaceutically acceptable excipient.

[0054] The dosage form of the drag described in the present invention comprises one of an injection preparation, an oral preparation, and a spray preparation. The present invention is not limited to the pharmaceutically acceptable excipients, and is well known to those skilled in the art.

[0055] The present invention also provides the application of binary naltrexone derivatives in the preparation of health products for improving autoimmune diseases, cancer, or cardiovascular diseases.

[0056] The present invention provides the application of binary naltrexone derivatives in the preparation of TLR4 signaling pathway inhibitors. The present invention also provides the application of binary naltrexone derivatives in the preparation of inflammatory factor expression inhibitors and the application of binary naltrexone derivatives in the preparation of inflammatory signaling pathway activation inhibitors. The present invention confirmed by in vivo experiments that the levels of nitric oxide and downstream inflammatory factors were significantly increased in glial cells activated by lipopolysaccharides (LPS), such that binary naltrexone derivatives can significantly inhibit the release of nitric oxide and the expression of downstream inflammatory factors. The inhibitory activities of TLR4 were formula (II) (4.7 ± 0.48 mM) and formula (III) (6.23 ± 1.82 pM). Formula (I) has no significant inhibitory effect. This means that binary naltrexone derivatives play a key role in the natural immune receptor TLR4-mediated immune response, and formula (II) and formula (III) show selectivity during the inhibition of TLR4 signaling pathway. [0057] The compound binary naltrexone derivatives of the present invention have good blood-brain barrier permeability. Experimental data indicate that binary naltrexone derivatives formula (II) or formula (III) play an important role in the natural immune receptor TLR4-mediated immune response and are considered to be the major TLR4 signaling pathway inhibitors. The present invention can increase TLR4 inhibitor activity and stereostructural selectivity. This will provide an effective therapeutic approach for drag addiction and neuropathic pain.

[0058] To further illustrate the present invention, the applications of the binary naltrexone derivatives provided by the present invention are described in detail below in the following embodiments.

[0059] For the preparation of the compounds involved in the present invention, see the literature (Portoghese, P. S.; Nagase, H.; Takemori, A.E. Stereochemical Studies on Medicinal Agents. 31. Only One Pharmacophore Is Required for the K-Opioid Antagonist Selectivity of Norbinaltorphimine. Journal of Medicinal Chemistry, 31 (1988), 1344- 1347).

[0060] Embodiment 1

[0061] (1) BV-2 microglia were cultured in an incubator at 37°C (containing 5% carbon dioxide). Medium: DMEM + 10% FBS; when cell density reaches 90% full plate rate, passaging conducted. BV-2 microglia were resuspended in medium (cell density: 2 MO ⁵ cells/ml), plate in 96-well plate overnight. Medium replaced with DMEM without FBS, add 200ng/ml LPS and different concentrations of inhibitors (formula (I), formula (II), or formula (III)). After 24 hours, the supernatant was collected. The concentration of nitric oxide was determined by the 2,3-diaminonaphthalene fluorescence method.

[0062] Embodiment 1 of the present invention characterizes the inhibitory activity of binary naltrexone derivatives. The results are shown in Figure 1 and Figure 2, where Figure 1 depicts the inhibitory effect of binary naltrexone derivatives on the activation of the TLR4 signaling pathway in BV-2 microglia in embodiment 1 of the present invention; Figure 2 depicts the inhibitory effect of binary naltrexone derivatives on the activation of the TLR4 signaling pathway in BV-2 microglia in embodiment 1 of the present invention.

[0063] As shown in Figure 1 and Figure 2, the inhibitors formula (II) and formula (III) can inhibit the release of nitric oxide, and the inhibitory activities are for formula (II) (4.7 ± 0.48 mM) and formula (III) (6.23± 1.82mM). Formula (I) has no significant inhibitory activity.

[0064] Embodiment 2

[0065] (2) BV-2 microglia were cultured in an incubator at 37°C (containing 5% carbon dioxide). Medium: DMEM + 10% FBS; when cell density reaches 90% full plate rate, passaging conducted. BV-2 microglia resuspended in medium (cell density: 2 MO ⁵ cells/ml), plate in 96-well plate overnight. Replace medium with DMEM without FBS, add 200mg/ml LPS and different concentrations of inhibitors (formula (II) or formula (I)) for 24 hours. TNF-a and IL-1B levels in the supernatant due to TLR signaling pathway activation tested for using the commercial ELISA kit (BD Biosciences).

[0066] Embodiment 2 of the present invention characterizes the inhibitory activity of binary naltrexone derivatives. The results are as shown in Figure 3 and Figure 4, where Figure 3 depicts the inhibitory effect of binary naltrexone derivatives on inflammatory factor TNF-a expression in BV-2 microglia in embodiment 2 of the present invention

F binary naltrexone derivatives; Figure 4 depicts the inhibitory effect of binary naltrexone derivatives on inflammatory factor IL-1B expression in BV-2 microglia in embodiment 2 of the present invention.

[0067] As can be seen from Figure 3 and Figure 4, inhibitor formula (II) described in the present invention can significantly inhibit inflammatory factor expression. Formula (I) has no significant inhibitory effect.

[0068] Embodiment 3

[0069] (3) Primary microglia were cultured in an incubator at 37°C (containing 5% carbon dioxide). Medium: DMEM + 10% FBS; when cell density reaches 90% full plate rate, passaging conducted. Primary microglia were resuspended in medium (cell density: (cell density: 2M0 cells/ml), plate in 96-well plate overnight. Medium replaced with DMEM without FBS, add 200ng/ml LPS and different concentrations of inhibitors (formula (II) or formula (I)). After 24 hours, the supernatant was collected. The concentration of nitric oxide was determined by the 2,3-diaminonaphthalene fluorescence method.

[0070] Embodiment 3 of the present invention characterizes the inhibitory activity of binary naltrexone derivatives. The results are as shown in Figure 5, where Figure 5 depicts the inhibitory effect of binary naltrexone derivatives on TLR4 signaling pathway activation in primary microglia in embodiment 3 of the present invention;

[0071] As can be seen in Figure 5, inhibitor (II) described in the present invention can significantly inhibit nitric oxide release. Formula (I) has no significant inhibitory activity.

[0072] Embodiment 4

[0073] (4) Primary microglia were cultured in an incubator at 37°C (containing 5% carbon dioxide). Medium: DMEM + 10% FBS; when cell density reaches 90% full plate rate, passaging conducted. BV-2 microglia resuspended in medium (cell density: 2 MO ⁵ cells/ml), plate in 96-well plate overnight. Replace medium with DMEM without FBS, add 200mg/ml LPS and different concentrations of inhibitors (formula (II) or formula (I)) for 24 hours. TNF-a levels in the supernatant due to TLR signaling pathway activation tested for using the commercial ELISA kit (BD Biosciences).

[0074] Embodiment 4 of the present invention characterizes the inhibitory activity of binary naltrexone derivatives. Figure 6 depicts the effect of binary naltrexone derivatives on inflammatory factor TNF-a expression in primary microglia in embodiment 4 of the present invention.

[0075] As can be seen in Figure 6, the inhibitor formula (II) described in the present invention can significantly inhibit inflammatory factor expression. Formula (I) has no significant inhibitory effect.

[0076] Embodiment 5

[0077] (5) Primary astrocytes were cultured in an incubator at 37°C (containing 5% carbon dioxide). Medium: DMEM + 10% FBS; when cell density reaches 90% full plate rate, passaging conducted. Primary microglia were resuspended in medium (cell density: (cell density: 2M0 cells/ml), plate in 96-well plate overnight. Medium replaced with DMEM without FBS, add 200ng/ml LPS and different concentrations of inhibitors (formula (II) or formula (I)). After 24 hours, the supernatant was collected. The concentration of nitric oxide was determined by the 2,3-diaminonaphthalene fluorescence method.

[0078] Embodiment 5 of the present invention characterizes the inhibitory activity of binary naltrexone derivatives. The results are as shown in Figure 7. Figure 7 depicts the inhibitory effect of binary naltrexone derivatives formula (II) on TLR4 signaling pathway activation in primary astrocytes in embodiment 5 of the present invention.

[0079] As can be seen in Figure 7, the inhibitor formula (II) described in the present invention can significantly inhibit the release of nitric oxide. Formula (I) has no significant inhibitory activity.

[0080] Embodiment 6

[0081] (6) Primary astrocytes were cultured in an incubator at 37°C (containing 5% carbon dioxide). Medium: DMEM + 10% FBS. when cell density reaches 90% full plate rate, passaging conducted. BV-2 microglia resuspended in medium (cell density: 2 MO ⁵ cells/ml), plate in 96-well plate overnight. Replace medium with DMEM without FBS, add 200mg/ml LPS and different concentrations of inhibitors (formula (II) or formula (I)) for 24 hours. TNF-a levels in the supernatant due to TLR signaling pathway activation tested for using the commercial ELISA kit (BD Biosciences).

[0082] Embodiment 6 of the present invention characterizes the inhibitory activity of binary naltrexone derivatives. The results are as shown in Figure 8. Figure 8 depicts the effect of binary naltrexone derivatives formula (II) on inflammatory factor TNF-a expression in primary astrocytes in embodiment 6 of the present invention.

[0083] As can be seen in Figure 8, inhibitor formula (II) described in the present invention can significantly inhibit inflammatory factor expression. Formula (I) has no significant inhibitory effect. [0084] The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present invention. These improvements and refinements should also be considered as the scope of protection of the present invention.