A drug assembly and its preparation method and application

Abstract

The present invention relates to the field of pharmaceutical engineering technology, and specifically to a drug assembly and a preparation method and application thereof. The drug assembly provided by the present invention is obtained by self-assembly of a protein polypeptide calcium channel blocking drug and an amide local anesthetic; the protein polypeptide calcium channel blocking drug includes conotoxin or its derivatives. The present invention can not only quickly block nerve conduction to achieve rapid analgesia, but also prolong the analgesic time, and minimize systemic toxicity. The drug preparation can maintain long-term analgesia, providing a powerful tool for the use of biological toxin molecular sustained / controlled release preparations as non-addictive drugs to control severe pain.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical engineering, and in particular to a pharmaceutical assembly and a preparation method and application thereof. Background Art

[0002] During the development of malignant tumors, the tumor or its environment continuously stimulates peripheral nociceptors, which in turn causes pain. As the malignant tumor continues to develop, this pain sensation continues to be transmitted to the central nervous system, eventually causing severe central pathological pain sensitization. Cancer pain is one of the main causes of suffering in patients with advanced cancer. Among patients with pain, 50% to 80% of the pain is not effectively controlled due to various reasons. Cancer pain, as a global problem, affects the lives of countless patients every day.

[0003] For pain management, the World Health Organization (WHO) has proposed a "three-step" analgesic treatment guideline. The first step is non-steroidal anti-inflammatory analgesics (NSAIDs), which are mainly used to treat mild pain, and non-steroidal anti-inflammatory drugs are preferred for analgesia; the second step is weak opioids. When cancer pain changes from mild to moderate, opioid analgesics such as tramadol and codeine are gradually used; the third step is strong opioids, which are mainly used for severe cancer pain, such as morphine, oxycodone, and hydroxymorphine. The pain caused by cancer is severe, usually moderate to severe pain, and opioid analgesics are mainly selected for clinical treatment. However, opioids are addictive and tolerant, and their side effects are also very obvious. At the same time, nearly 40% of patients are not sensitive to opioids, and cancer pain cannot be relieved. Therefore, the development of non-opioid analgesics is a major demand for clinical cancer pain treatment.

[0004] Ziconotide is an attenuated derivative of ω-conotoxin that can specifically and efficiently block N-type calcium channels and hinder pain signaling by blocking the release of neurotransmitters. Studies have shown that the analgesic efficacy of ziconotide in inhibiting chronic severe pain is 1,000 times that of morphine. Ziconotide has been marketed in Europe and the United States and is clinically used for analgesia in patients with severe chronic pain who are intolerant or ineffective to systemic analgesics, intrathecal morphine, and other regimens. However, the dosage window of ziconotide is narrow, and the dosage needs to be controlled at the microgram level. During clinical use, it is accompanied by neurological side effects such as ataxia, memory deficits, abnormal gait, drowsiness, and dizziness. In addition to the risk of toxic side effects, the hysteresis of ziconotide's efficacy is also an urgent problem to be solved. Since the N-type calcium channel targeted by ziconotide is not directly involved in the conduction of neuronal electrical signals, ziconotide cannot quickly achieve pain suppression. In order to make up for this defect without affecting the analgesic effect, combined drug strategies are also being tried in clinical practice. Since sodium ions are fast-responding ions that carry action potentials, the combined blocking of sodium ion channels and calcium ion channels may not only solve the problem of delayed analgesia of ziconotide, but also enhance the analgesic efficacy. However, it may also increase the side effects of ziconotide.

[0005] In order to fully utilize the combined blockade of sodium ion channels and calcium ion channels to achieve rapid and effective suppression of pain caused by malignant tumors, it is necessary to construct a new drug formulation that can slowly / controlled release ziconotide based on the combination of drugs. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a drug assembly and a preparation method and application thereof. The assembly preparation based on conotoxin or its derivatives and amide local anesthetic drugs provided by the present invention is particularly used as an analgesic drug delivery system to achieve rapid, efficient, long-term and safe analgesia for severe chronic pain such as cancer pain.

[0007] In the present invention, conotoxin or its derivatives (ziconotide, etc.) have excellent analgesic effect on chronic severe pain, and do not have the side effects of opioids and the characteristics of easily inducing drug resistance, which provides a possibility for the development of non-opioid potent analgesics, but its own efficacy is delayed and the therapeutic window is narrow. Therefore, the purpose of the present invention is to compensate for the hysteresis of the efficacy of conotoxin or its derivatives while releasing them slowly, and the developed new preparation can achieve rapid, efficient, long-acting and safe analgesia.

[0008] Specifically, in the first aspect, the present invention provides a drug assembly, which is obtained by self-assembly of a protein polypeptide calcium ion channel blocking drug and an amide local anesthetic; the protein polypeptide calcium ion channel blocking drug includes conotoxin or a derivative thereof.

[0009] The present invention provides an assembly method of amide local anesthetics and conotoxins and their derivatives, and a pharmaceutical preparation formed thereby. The pharmaceutical preparation is formed by supramolecular assembly of amide local anesthetic molecules and protein polypeptide calcium channel blockers (such as conotoxins, ziconotide, etc.), which can not only quickly block nerve conduction to achieve rapid analgesia, but also prolong the analgesic time and minimize systemic toxicity. In vivo experiments have shown that the pharmaceutical preparation assembled from amide local anesthetics (such as bupivacaine and ropivacaine) and ziconotide can maintain long-term analgesia, providing a powerful tool for the use of biotoxin molecular sustained / controlled release preparations as non-addictive drugs to control severe pain.

[0010] In the present invention, the conotoxin or its derivative is a protein polypeptide calcium channel blocker, which has analgesic effect. Ziconotide is preferred; amide local anesthetics are sodium channel blockers, which are commonly used in clinical practice for preoperative, intraoperative and postoperative anesthesia and analgesia, preferably bupivacaine and ropivacaine. Conotoxin or its derivative is first dispersed in a weakly alkaline environment, and then hydrochloric acid amide local anesthetics are gradually added in a certain molar ratio. During this process, the hydrogen ions of the local anesthetic are neutralized by the base and converted into hydrophobic molecules, which are co-assembled with conotoxin or its derivative. In the present invention, the final form of the amide local anesthetic in the present invention is a non-hydrochlorinated molecule.

[0011] Preferably, the amide local anesthetic and the conotoxin or its derivative realize self-assembly through intermolecular interaction to obtain a micro-nano sized drug assembly.

[0012] In the present invention, the basic units are amide local anesthetics and conotoxin or its derivatives (such as ziconotide), and self-assembly is achieved by the intermolecular interaction between the hydrophobic part of the amide local anesthetic molecules and conotoxin or its derivatives to form a nano / micron size assembly.

[0013] Preferably, the tertiary amine nitrogen atom in the amide local anesthetic molecule is not bound to a proton.

[0014] In the present invention, the tertiary amine nitrogen atom in the amide local anesthetic molecule is not bound to a proton, so as to ensure that the amide local anesthetic and the conotoxin or its derivatives are self-assembled.

[0015] Preferably, the molecular structure of the amide local anesthetic contains structural units of a benzene ring, a hydrophobic alkyl group and a tertiary amine group.

[0016] More preferably, the amide local anesthetic includes bupivacaine, lidocaine or ropivacaine.

[0017] Preferably, the molecular structure of the conotoxin or its derivative contains positively charged amino acids, and the positively charged amino acids include arginine and / or lysine.

[0018] More preferably, the conotoxin derivative is ziconotide.

[0019] Preferably, the molar ratio of the conotoxin or its derivative to the amide local anesthetic is 1:10-1:5000.

[0020] More preferably, the molar ratio of the conotoxin or its derivative to the amide local anesthetic is 1:150-1:600.

[0021] In a second aspect, the present invention also provides a method for preparing the drug assembly, comprising the following steps:

[0022] S1. Disperse conotoxin or its derivatives into a medical injection solution, add sodium hydroxide, and adjust the pH to a weak alkaline state.

[0023] S2. Under shaking conditions, mix the solution obtained in step S1 with an amide local anesthetic.

[0024] Preferably, the medical injection solution is 5% glucose injection; and / or, an equimolar amount of sodium hydroxide is added according to the dosage of the amide local anesthetic, and the pH is adjusted to 8-9.

[0025] In a third aspect, the present invention further provides a pharmaceutical preparation comprising the above-mentioned drug assembly or the drug assembly obtained by the above-mentioned method for preparing the drug assembly.

[0026] In a fourth aspect, the present invention also provides the use of the above-mentioned drug assembly or the drug assembly obtained by the method for preparing the above-mentioned drug assembly in the preparation of potent anesthetic drugs or analgesics.

[0027] Preferably, the drug assembly is used in any of the following applications:

[0028] 1) Used for preparing analgesic drug delivery or sustained release system.

[0029] 2) Used for the preparation of drugs for severe chronic pain, preferably drugs for suppressing cancer pain.

[0030] 3) Used to prepare combination medications for multimodal analgesia.

[0031] The beneficial effects of the present invention are at least:

[0032] (I) The present invention has successfully established an assembly of conotoxin or its derivatives and amide local anesthetics, which can achieve joint blocking of sodium and calcium ion channels. This preparation not only effectively compensates for the delayed analgesic effect of conotoxin and its derivatives, but also achieves a slow / controlled release of conotoxin and its derivatives, thereby improving the safety of conotoxin and its derivatives.

[0033] (ii) The method for preparing the assembly of the conotoxin or its derivatives and the amide local anesthetic of the present invention is simple and does not require harsh and complicated synthesis or preparation steps.

[0034] (III) The present invention realizes the construction of sustained-release preparation by assembling two drug molecules, without the need for other carrier materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 It is the structural formula of the amide local anesthetic in the embodiment of the present invention.

[0037] Figure 2 This is a schematic diagram of the assembly of a composition formed by assembling conotoxin or its derivatives with amide local anesthetics in an embodiment of the present invention.

[0038] Figure 3 This is a diagram showing the analgesic principle of a composition formed by conotoxin or its derivatives and amide local anesthetics in an embodiment of the present invention.

[0039] Figure 4 The morphology of the self-assembled ω-conotoxin derivative (ziconotide, ZP) and bupivacaine (BUP) according to the embodiment of the present invention is shown in the transmission electron microscope (TEM) image. The molar ratio of ZP to BUP is 1:50 to 1:600, ZP is the abbreviation of ziconotide, and BUP is the abbreviation of bupivacaine.

[0040] Figure 5 is the in vitro cumulative release of the ZP and bupivacaine assembly after continuous release for 48 hours in the embodiment of the present invention, wherein the molar ratio of ZP:BUP is 1:300.

[0041] Figure 6 The figure shows the comparison of the effects of the mixed solution of ZP and water-soluble bupivacaine and the assembly of ZP and hydrophobic bupivacaine on inhibiting cancer pain in the embodiments of the present invention.

[0042] Figure 7 The figure shows the comparison of the effects of the mixed solution of ZP and water-soluble ropivacaine and the assembly of ZP and hydrophobic ropivacaine on inhibiting cancer pain in the embodiments of the present invention. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] The drug assembly (polypeptide-hydrophobic small molecule composition) provided in the embodiment of the present invention is formed by the association of conotoxin or its derivatives with amide local anesthetics through supramolecular assembly, wherein the amide local anesthetic contains three structure-effect units, the lipophilic benzene ring participates in the formation of the core of the assembly, the hydrophilic tertiary amine and piperidine structure produce cation-π interaction with the conotoxin or its derivatives, and the amide between the lipophilic part and the hydrophilic part produces hydrogen bonding with the conotoxin or its derivatives. It should be noted that the assembly of conotoxin or its derivatives with amide local anesthetics depends on the hydrophilic unit tertiary amine and piperidine structure, which need to be in an alkaline environment, and the intermolecular force will be destroyed in an acidic environment. The present invention can be widely used in drug delivery systems for cancer pain inhibition. The delivery system is composed of conotoxin or its derivatives and amide local anesthetics to form a composition, and the rapid blocking effect of amide local anesthetics on sodium ion channels is used to make up for the defect of ziconotide hysteresis, so as to achieve rapid inhibition of cancer pain; the interaction between conotoxin or its derivatives and amide drug molecules is used to achieve drug controlled release, stabilize the release of conotoxin or its derivatives, and reduce its toxic and side effects.

[0045] The assembly (polypeptide-hydrophobic small molecule composition) provided in the embodiment of the present invention is formed by intermolecular interaction between spirotoxin or its derivative and amide local anesthetic, that is, the hydrophobic part of the amide drug is responsible for the core construction of the assembly, the intermediate amide bond and the hydrophilic piperidine ring are responsible for the specific binding of ziconotide, and finally the spirotoxin or its derivative is compounded with the amide local anesthetic to form a spherical assembly. It can be prepared according to the following method, including: 1) dissolving the spirotoxin or its derivative in a medical injection solution, and adding an equimolar amount of sodium hydroxide according to the amount of bupivacaine; 2) adding a specific molar ratio of amide local anesthetic hydrochloric acid solution dropwise to the obtained solution, reacting at room temperature for 30 minutes, and obtaining the polypeptide-hydrophobic small molecule composition, wherein the molar ratio of spirotoxin or its derivative to amide local anesthetic is 1:50, 1:150, 1:300, 1:600. Preferably, step S2 further comprises characterizing by negative staining of uranyl acetate (1.0% w / w) using TEM (Tecnai G2 T20; FEI Company). The average particle size of the assembly is calculated after measuring the particle sizes of ten assemblies in the TEM image using Image J. DLS (Delsa Nano; Beckman Coulter) is used to determine whether the assembly contains nanoscale materials and to detect dispersibility and solution stability.

[0046] The embodiment of the present invention uses an amide local anesthetic and a spirotoxin or its derivative molecules to assemble into a specific delivery system of the composition, which can not only achieve rapid nerve blockade to inhibit cancer pain, but also control the release of spirotoxin or its derivatives to reduce its toxicity. Animal experiments have shown that the nanostructured delivery system formed by spirotoxin or its derivatives and amide local anesthetics can quickly inhibit cancer pain, successfully making up for the drawbacks of the delayed efficacy of spirotoxin or its derivatives, and providing a powerful tool for non-addictive pain control.

[0047] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0048] Example 1 Construction of Ziconotide and Bupivacaine Assembly

[0049] 1. Preparation of alkaline ziconotide solution

[0050] Prepare 1 mg / mL ziconotide stock solution, the solvent is 5% glucose injection, take 6.7 μL stock solution and dissolve it in 100 μL 5% glucose injection, and add 5 μL, 15 μL, 30 μL, and 60 μL 1 mg / mL sodium hydroxide solution. Then, ultrasonicate at 100 W for 3 min at 25 °C to obtain a homogenous solution.

[0051] 2. Assembly of Bupivacaine and Ziconotide

[0052] A 10 mg / mL stock solution of bupivacaine hydrochloride was prepared using 5% glucose injection as solvent. 4.2 μL, 12.5 μL, 25 μL, and 50 μL of the stock solution (i.e., molar ratios of 1:50, 1:150, 1:300, and 1:600, respectively) were taken and diluted to 100 μL. The bupivacaine hydrochloride dilution was added to the alkaline ziconotide solution at a rate of 20 μL / min and incubated at room temperature for 30 min to generate a ziconotide-bupivacaine composition. The solution was diluted 10 times and characterized by TEM using negative staining with uranyl acetate (1.0% w / w). The average particle size of the assembly was calculated by measuring the particle size of ten assemblies in the TEM image using Image J. DLS (Delsa Nano; Beckman Coulter) was used to determine whether the assembly contained nanoscale materials.

[0053] Amide local anesthetics, assembly diagram, and analgesia principle diagram are shown in Figure 1-3 , the experimental results are shown in Figure 4 As can be seen from the figure, the assembly is spherical and gradually increases in size with the increase in the amount of bupivacaine.

[0054] Example 2 Quantitative drug release assay of ziconotide-bupivacaine assembly in vitro

[0055] 1. 1 mL of the assembly containing 0.2 μM ZP and 60 μM BUP was placed in a Slide-A-Lyzermin I dialysis device (Thermo Scientific) with a pore size of 10,000 MW and dialyzed against 4 mL of 5% glucose solution at 37 °C on a platform shaker at 100 rpm for drug release studies. The dialyzed solution was exchanged with fresh, preheated 37 °C 5% glucose solution at predetermined time intervals. The concentrations of ZP and BUP were quantified by high performance liquid chromatography (HPLC).

[0056] 2. Quantification of Ziconotide by HPLC. Prepare ZP solutions with concentrations of 1 mg / mL, 0.25 mg / mL, 0.0625 mg / mL, 0.015625 mg / mL, and 0.00390625 mg / mL, respectively. Detect the corresponding peak areas by HPLC, and calculate and draw the ZP standard curve. Transfer the combined samples collected by dialysis for a predetermined time to a liquid phase vial and detect them using the ZP method. The specific detection method for ZP is: C18 column, 85% mobile phase A (water / 0.1% TFA), 15% mobile phase B (acetonitrile / 0.1% TFA), detection wavelength 200 nm, single injection volume 30 μL, peak position 8-10 min.

[0057] 3. Quantification of Ziconotide by HPLC. Prepare BUP solutions with concentrations of 1 mg / mL, 0.25 mg / mL, 0.0625 mg / mL, 0.015625 mg / mL, and 0.00390625 mg / mL, respectively. Use HPLC to detect the corresponding peak areas, and count and draw the BUP standard curve. Transfer the combined samples collected by dialysis at the predetermined time to a liquid phase vial and detect them using the BUP method. The specific detection method for BUP is: C18 chromatographic column, 35% mobile phase A (0.02 mol / l phosphate buffer, pH 8.0), 65% mobile phase B (acetonitrile), detection wavelength 210 nm, single injection volume 10 μL, peak position 12 min. The experimental results are shown in Figure 5 .

[0058] Example 3 Detection of the analgesic ability of ZP-BUP assembly on cancer pain

[0059] 1. Establishment of rat bone cancer pain model

[0060] Six-week-old Wistar female rats weighing 250-300 g were used in the experiment. They were housed under a 12h / 12h light / dark cycle for 7 days, with 6 rats in each group. After anesthesia with isoflurane-oxygen, Walker256 cell line was inoculated into the tibia of the left leg of the rat using a 23-G needle. After successful tumor inoculation, the rats were subjected to imaging, behavioral and molecular biological tests to evaluate the construction of the bone cancer pain model and the central sensitization caused by cancer pain.

[0061] 2. Rapid suppression of cancer pain by injection of ZP-BUP assembly

[0062] The ZP-BUP assembly was intrathecally injected into the established rat bone cancer pain model, the molar ratio of ZP to BUP in the assembly was controlled in the range of 1:50-1:600, preferably 1:150-1:300, 1:300 in the embodiment, 30 μL per rat, and the rats were subjected to pain behavioral testing (Von Frey mechanical sting test) at a predetermined time after administration, i.e. mechanical stimulation of the plantar surface of the rat's hind paw was performed, and the magnitude of the mechanical force was recorded until the animal lifted the paw. The measurement was repeated 3 times at each time interval.

[0063] The experimental results are shown in Figure 6 ZP (0.033 mg / mL, 30 μL / mouse) and ZP-BUP assembly (0.033 mg / mL ZP + 1.25 mg / mL BUP, 30 μL / mouse, i.e. 1 μg ZP + 37.5 μg BUP per mouse). As can be seen from the figure, the group containing BUP can produce mechanical analgesia within 1 hour; and the BUP-ZP assembly after forming the assembly has a longer analgesic time than the unassembled form.

[0064] 3. Rapid and long-term suppression of cancer pain by injection of ZP-ROP (ropivacaine) assembly

[0065] The ZP-ROP assembly was injected intrathecally into the rat bone cancer pain model constructed above. The molar ratio of ZP to ROP in the assembly was controlled in the range of 1:50-1:600, preferably 1:150-1:300, 1:300 in the embodiment, 30 μL per rat. After administration, the rats were subjected to pain behavioral testing (Von Frey mechanical sting test) at a predetermined time, that is, mechanical stimulation of the plantar surface of the rat's hind paw was performed, and the magnitude of the mechanical force was recorded until the animal lifted the paw. The measurement was repeated 3 times at each time interval. The experimental results are shown in Figure 7 , ZP (0.033 mg / mL, 30 μL / mouse) and ZP-ROP assembly (0.033 mg / mL ZP + 1.25 mg / mL ROP, 30 μL / mouse, i.e. 1 μg ZP + 37.5 μg ROP per mouse). As can be seen from the figure, the group containing ROP can produce mechanical analgesia within 1 hour; and the ROP-ZP assembly after forming the assembly has a longer analgesia time than the unassembled form.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A drug assembly, characterized in that: The drug assembly is obtained by self-assembly of a protein polypeptide calcium channel blocker and an amide local anesthetic; the protein polypeptide calcium channel blocker is a derivative of ω-conotoxin; The amide local anesthetic and the ω-conotoxin derivative achieve self-assembly through intermolecular interaction to obtain a drug assembly; the ω-conotoxin derivative is ziconotide, and the molar ratio of ziconotide to the amide local anesthetic is 1:150-1:600; the molecular structure of the amide local anesthetic contains structural units of a benzene ring, a hydrophobic alkyl group and a tertiary amine group, and the tertiary amine nitrogen atom in the molecule of the amide local anesthetic is not bound to a proton; The drug assembly is prepared by a method comprising the following steps: S1. Dispersing the ω-conotoxin derivative into a medical injection solution and adjusting the pH to 8-9; S2. Under shaking conditions, mix the solution obtained in step S1 with an amide local anesthetic.

2. The drug assembly according to claim 1, characterized in that: The amide local anesthetic is bupivacaine, lidocaine or ropivacaine.

3. The method for preparing the drug assembly according to claim 1 or 2, characterized in that: The steps include: S1. Dispersing the ω-conotoxin derivative into a medical injection solution and adjusting the pH to 8-9; S2, mixing the solution obtained in step S1 with an amide local anesthetic under shaking conditions; The medical injection solution is 5% glucose injection.

4. A pharmaceutical preparation, characterized in that A drug assembly comprising the drug assembly according to claim 1 or 2 or a drug assembly obtained by the preparation method of the drug assembly according to claim 3.

5. Use of the drug assembly obtained by the preparation method of the drug assembly according to claim 1 or 2 or the drug assembly according to claim 3 in the preparation of potent anesthetic drugs or analgesics.

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