A sterile dipentadol sebacate and its preparation method and use

Abstract

The present invention relates to sterile dipentadol sebacate, a preparation method thereof, and pharmaceutical uses thereof. The sterile dipentadol sebacate has a particle size D50 of 0.5 to 10 μm. The present invention also provides the use of dipentadol sebacate in the preparation of various remedies for acute and chronic pain, which exhibits significant long-lasting effects.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical chemistry, and in particular to a sterile dipentadol sebacate, a preparation method thereof, a pharmaceutical preparation thereof and medical uses thereof. Background Art

[0002] Most patients with moderate to severe pain, such as postoperative pain, post-traumatic pain, and burn pain, usually need to control pain within the first 3 days after injury. For this reason, analgesics with a long-lasting effect of about 3 days may be particularly valuable (KS. Chu, et al. (2003), Anesthesia Analgesia, Vol. 97, 806-809). Currently, opioids and non-steroidal anti-inflammatory drugs are often used in this field, but they are all short-acting drugs. Prolonging the duration of action will make (for example, long-acting opioids) more valuable in the clinical treatment of long-term pain. In recent years, extending the duration of drug analgesia and reducing the frequency of opioid use after surgery have gradually become the needs of clinical analgesics, so the application of long-acting analgesic sustained-release injections after surgery has become a research hotspot.

[0003] Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage. Pain can be categorized by etiology, duration, and severity. Etiologically, pain can be classified as somatic (i.e., organic) or psychogenic (occurring in the absence of an associated organic pathology sufficient to explain the severity and / or duration of the pain). Somatic pain can be further subdivided into nociceptive pain (caused by irritation of somatic or visceral pain-sensitive nerve fibers) or neuropathic pain (caused by dysfunction of the nervous system). Based on duration, pain is typically classified as acute or chronic. Chronic, persistent pain can lead to significant impairment in physical and mental health, as well as social responsibilities (including work and family life). Chronic pain is described as pain that lasts for at least five days and up to six months. Chronic pain is often associated with conditions such as surgery, cancer, and serious injuries. Opioids are commonly used to manage severe chronic pain conditions. Although opioids are potent analgesics, their relatively short half-life limits their benefit. Because pain from these procedures can persist for several days, these analgesics must be administered multiple times for effective pain control.

[0004] Tapentadol is the most effective of the dual opioid analgesics. The chemical name of tapentadol is

[0005] 3-((1R,2R)-3-(dimethylamino)-1-ethyl-2-methylpropyl)phenol has a molecular weight of 221.34 and is represented by the following structural formula:

[0006]

[0007] Tapentadol is an opioid analgesic with both μ-opioid receptor agonist and norepinephrine (NE) reuptake inhibitory activity, with minimal inhibition of serotonin reuptake. This dual mode of action makes tapentadol particularly useful in the treatment of nociceptive and neuropathic pain. Clinical trial evidence from acute and chronic non-cancer pain and neuropathic pain supports an opioid-sparing effect, thereby reducing some typical opioid-related adverse effects. Specifically, tapentadol treatment is associated with fewer gastrointestinal adverse effects compared to equivalent analgesic pure μ-opioid receptor agonists, thereby improving tolerability and treatment adherence.

[0008] US Patent No. 6,248,737 discloses tapentadol and its hydrochloride. Tapentadol is sold under the trade name It is marketed as an immediate-release oral tablet for the relief of moderate to severe acute pain. RETARD comes as an extended-release tablet for severe chronic pain.

[0009] When tapentadol is taken orally, it undergoes extensive first-pass metabolism, which results in a low bioavailability (32%). Approximately 97% of the parent compound is metabolized. None of the metabolites contribute to the analgesic activity. Ultimately, the desired effect can only be achieved with high doses of tapentadol. Immediate-release oral tapentadol is administered every 4-6 hours, while extended-release tablets are administered every 12 hours. As an opioid analgesic, tapentadol is used to treat severe pain, such as postoperative pain, cancer pain, etc. In such conditions, nausea and vomiting are often associated problems, resulting in poor patient compliance with oral administration. Some disadvantages of oral administration are that the patient may improperly modify the unit dose, leading to a dangerous overdose, or the patient may be unable to swallow the medication.

[0010] Tapentadol has a short duration of action, forcing patients to take it frequently. Furthermore, like other opioids, tapentadol is considered to have abuse potential. To address this issue, U.S. Patent No. 8,075,872 provides an abuse-resistant controlled-release formulation of tapentadol for oral administration twice daily.

[0011] Long-lasting analgesia is particularly desirable for patients with pain, such as postoperative pain, post-traumatic pain, and burn pain, which may persist for approximately three days. Tapentadol has potent but short-acting analgesic activity. Prolonging its duration of action would make tapentadol more valuable in the clinical treatment of pain.

[0012] Therefore, there is a need for an alternative dosage form that provides prolonged action of tapentadol, thereby reducing the frequency of dosing. Additionally, there is a need for an alternative dosage form that overcomes the problems associated with oral administration and reduces the opportunity for abuse so that the release of the analgesic cannot be manipulated by the patient or other external sources.

[0013] Patent Literature:

[0014] US20130090379, Compositions comprising enzymatically cleavable phenol-modified tapentadol prodrugs

[0015] US20100227921, Amino acid and peptide carbamate prodrugs of tapentadol and their uses

[0016] JP202307524 Parenteral administration of tapentadol

[0017] US10898452 Stable formulation of tapentadol for parenteral administration

[0018] CN2014071658 Tapentadol hydrochloride injection and preparation method thereof

[0019] Several tapentadol prodrugs have been synthesized and reported. However, to the applicant's knowledge, none of the known tapentadol esters have been used for long-acting purposes.

[0020] Therefore, there is still a need in the art to develop a suitable pharmaceutical composition that can enable tapentadol to exhibit a long-lasting analgesic effect. Summary of the Invention

[0021] The present invention modifies the structure of tapentadol to produce a prodrug with long-lasting properties. This drug is formulated into a formulation that can be injected intramuscularly, subcutaneously, or intravenously. After intramuscular, subcutaneous, or intravenous injection, it forms a drug reservoir in the body. The drug is slowly, continuously, and stably released from the reservoir and converted into tapentadol, thereby exerting a long-lasting effect. The present invention utilizes the following technical solutions to achieve this:

[0022] On the one hand, the present invention aims to remedy the deficiencies in the prior art and provide a ditapentadol sebacate represented by formula (I):

[0023]

[0024] The present invention refers to a sterile raw material drug, and the particle size D of the raw material of ditapentadol sebacate is 50 0.5~10μm.

[0025] Another aspect of the present invention is to provide a crystal of bispentadol sebacate, which does not contain crystal water or solvent, and has a TGA value of about 250° C. and a DSC value of 51.90° C.

[0026] Another aspect of the present invention is to provide a method for preparing tapentadol sebacate, which comprises esterifying tapentadol hydrochloride with sebacoyl chloride in an organic solvent and an alkaline acid-binding agent, followed by quenching, washing, column chromatography, and crystallization. The reaction formula is as follows:

[0027]

[0028] The preparation of tapentadol hydrochloride was carried out according to Chinese Journal of Medicine, 2013, 44(6): 554-556. A series of crystallization methods were screened when preparing the crystals of tapentadol sebacate. The results are as follows:

[0029]

[0030]

[0031]

[0032] The solvent of the crystallization process we provide is selected from one or more of methyl tert-butyl ether, isopropyl ether, petroleum ether, n-hexane, cyclohexane, and n-pentane; preferably one or more of methyl tert-butyl ether, isopropyl ether, n-hexane, and petroleum ether; the temperature of the crystallization process of the present invention is preferably -15 to 0°C.

[0033] Another aspect of the present invention is to provide a sterile dipentadol sebacate, which is prepared by pre-filtration with a 0.45 μm microporous filter membrane before crystallization, and then undergoes two stages of 0.22 μm sterilization filtration and vacuum drying.

[0034] In yet another aspect, the present invention provides a pharmaceutical composition of dipentadol sebacate, which is administered by injection and preferably in the form of an injection solution, a lyophilized powder, or the like.

[0035] The pharmaceutical composition contains dipentadol sebacate and pharmaceutically acceptable excipients.

[0036] The injection is prepared from the active sterile raw material ditapentadol sebacate and related pharmaceutical excipients according to a general method in pharmaceutical pharmacy. The excipients include those necessary in pharmaceutical pharmacy, such as suspending agents, antioxidants, stabilizers, wetting agents, preservatives, etc.

[0037] Another aspect of the present invention is to provide a ditapentadol sebacate pharmaceutical composition for use in preparing various acute and chronic pain medications.

[0038] The main points of the present invention are:

[0039] The sterile dipentadol sebacate of the present invention has a moderate melting point, a moderate particle size, and low solubility. It can be formulated into a suspension for intramuscular or subcutaneous injection, forming a drug reservoir in the body, prolonging the drug's release rate and achieving long-lasting therapeutic effects. Furthermore, the compound is essentially non-toxic to normal human hepatocytes. Animal efficacy and safety studies have demonstrated good efficacy, a long-lasting analgesic effect, and no significant irritation to local tissues, demonstrating good safety and tolerability. Animal pharmacokinetic studies have shown that the compound has a long half-life, enabling a long-lasting release effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific 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 any creative work.

[0041] Figure 1 The diagram shows the compound structure of dipentadol sebacate.

[0042] Figure 2 This is the hydrogen spectrum of dipentadol sebacate.

[0043] Figure 3 This is the carbon spectrum of dipentadol sebacate.

[0044] Figure 4 It is ditapentadol sebacate DSC.

[0045] Figure 5 It is bisapentadol sebacate IR.

[0046] Figure 6 These are the results of analgesic pharmacodynamic experiments on dipentadol sebacate in rats. DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0048] The present invention will be further explained below with reference to specific embodiments.

[0049] Example 1:

[0050] A 1000mL three-necked flask was added with 400mL of dichloromethane and 25.70g of tapentadol hydrochloride, and 20.25g of triethylamine was added under stirring. After a few minutes, the material was completely dissolved, cooled to -5~0°C, 12.00g of sebacoyl chloride was added dropwise, the temperature rose significantly, the internal temperature was controlled at 0~10°C, and insoluble matter gradually precipitated in the system. The reaction was stirred at 0~10°C for 2 hours, and TLC monitoring (λ=254nm, PE:EA=2:1, two drops of triethylamine were added) tapentadol content ≦0.5%, 150mL of water was added to quench the reaction, and after separation, 400mL*2 water was added and washed twice. The dichloromethane layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 20.70g of a light yellow oil, which was placed in the refrigerator overnight as a white solid. The purity of HPLC was 97.68%.

[0051] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 7.339-7.300 (t, 2H), 7.047-7.028 (d, 2H), 6.938-6.883 (m, 4H), 2.585-2.549 (t, 4H), 2.445-2.394 (m, 2H), 2 .043(s, 12H), 1.955-1.914(m, 2H), 1.886-1.788(m, 4H), 1.758-1.525( m, 8H), 1.447-1.355 (m, 8H), 0.876-0.860 (d, 6H), 0.687-0.651 (t, 6H).

[0052] Example 2:

[0053] 800 mL of dichloromethane and 51.40 g of tapentadol hydrochloride were added to a 2000 mL three-necked flask, and 40.50 g of triethylamine was added under stirring. After a few minutes, the material was completely dissolved, cooled to -5 to 0 ° C, 24.00 g of sebacoyl chloride was added dropwise, the temperature rose significantly, the internal temperature was controlled at 0 to 10 ° C, and insoluble matter gradually precipitated in the system. The temperature was controlled at 0 to 10 ° C and stirred for 3 hours. TLC monitoring (λ = 254 nm, PE: EA = 2: 1, two drops of triethylamine were added) tapentadol ≤ 0.5%, 300 mL of water was added to quench the reaction, and after separation, 800 mL * 2 water was added and washed twice. The dichloromethane layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 42.40 g of a light yellow oil, which was placed in the refrigerator overnight as a white solid.

[0054] Example 3:

[0055] 1500 mL of dichloromethane and 102.80 g of tapentadol hydrochloride were added to a 3000 mL three-necked flask, and 81.00 g of triethylamine was added under stirring. After a few minutes, the material was completely dissolved, cooled to -5 to 0 ° C, 47.80 g of sebacoyl chloride was added dropwise, the temperature rose significantly, the internal temperature was controlled at 0 to 10 ° C, and insoluble matter gradually precipitated in the system. The temperature was controlled at 0 to 10 ° C and stirred for 4 hours. TLC monitoring (λ = 254 nm, PE: EA = 2: 1, two drops of triethylamine were added) tapentadol ≤ 0.5%, 600 mL of water was added to quench the reaction, and after separation, 1500 mL * 2 water was added and washed twice. The dichloromethane layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 80.00 g of a light yellow oil, which was placed in the refrigerator overnight as a white solid.

[0056] Example 4:

[0057] 400 mL of redistilled toluene and 25.70 g of tapentadol hydrochloride were added to a 1000 mL three-necked flask, and 20.25 g of triethylamine was added under stirring. The material was insoluble and the temperature was lowered to -5 to 0 ° C. 12.00 g of sebacoyl chloride was added dropwise. The temperature rose significantly. The internal temperature was controlled at 0 to 10 ° C and stirred for 2 hours. TLC monitoring (λ = 254 nm, PE: EA = 2: 1, two drops of triethylamine were added) tapentadol ≤ 0.5%, 150 mL of water was added to quench the reaction, and after separation, the organic phase was washed twice with 400 mL * 2 water. The toluene layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 21.50 g of a light yellow oil, which became a white solid after being placed in the refrigerator overnight.

[0058] Example 5:

[0059] A 500mL three-necked flask was added with dry N,N-dimethylformamide 130mL and tapentadol hydrochloride 25.70g, and triethylamine 20.25g was added under stirring. After stirring for a few minutes, the material was completely dissolved, cooled to -5~0°C, and 12.00g of sebacoyl chloride was added dropwise. The temperature rose significantly, and the internal temperature was controlled at 0~10°C and stirred for 2 hours. TLC monitoring (λ=254nm, PE:EA=2:1, two drops of triethylamine were added) tapentadol ≤0.5%, 150mL of water was added to quench the reaction, and the layers were extracted with 500mL of ethyl acetate. After separation, the organic phase was washed twice with 400mL*2 water. The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 18.60g of a light yellow oil, which became a white solid after being placed in the refrigerator overnight.

[0060] Example 6:

[0061] A 500mL three-necked flask was added with dry tetrahydrofuran 150mL and tapentadol hydrochloride 25.70g, and triethylamine 20.25g was added under stirring. After stirring for several minutes, the material was not dissolved. The temperature was lowered to -5~0°C, and 12.00g of sebacoyl chloride was added dropwise. The temperature rose significantly, and the internal temperature was controlled at 0~10°C and stirred for 2 hours. TLC monitoring (λ=254nm, PE:EA=2:1, two drops of triethylamine were added) tapentadol ≤0.5%, 150mL of water was added to quench the reaction, and the layers were extracted with 500mL of ethyl acetate. After separation, the organic phase was washed twice with 400mL*2 water. The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 19.50g of a light yellow oil, which became a white solid after being placed in the refrigerator overnight.

[0062] Example 7:

[0063] A 1000mL three-necked flask was added with 400mL of dichloromethane and 25.70g of tapentadol hydrochloride, and 20.25g of triethylamine was added under stirring. After a few minutes, the material was completely dissolved, cooled to -5~0°C, and 23.90g of sebacoyl chloride was added dropwise. The temperature rose significantly, and the internal temperature was controlled at 0~10°C. Insoluble matter gradually precipitated from the system. The reaction was stirred at 0~10°C for 2 hours. TLC monitoring (λ=254nm, PE:EA=2:1, two drops of triethylamine were added) tapentadol ≤0.5%, 500mL of water was added to quench the reaction, and after separation, 500mL*2 water was added and washed twice. The dichloromethane layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 19.30g of a light yellow oil, which was a white solid after being placed in the refrigerator overnight.

[0064] Example 8:

[0065] A 1000mL three-necked flask was added with 400mL of dichloromethane and 25.70g of tapentadol hydrochloride, and 20.25g of triethylamine was added under stirring. After a few minutes, the material was completely dissolved, cooled to -5~0°C, 18.00g of sebacoyl chloride was added dropwise, the temperature rose significantly, the internal temperature was controlled at 0~10°C, and insoluble matter gradually precipitated in the system. The temperature was controlled at 0~10°C and stirred for 2 hours. TLC monitoring (λ=254nm, PE:EA=2:1, two drops of triethylamine were added) tapentadol ≤0.5%, 400mL of water was added to quench the reaction, and after separation, 400mL*2 water was added and washed twice. The dichloromethane layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 20.30g of a light yellow oil, which was placed in the refrigerator overnight as a white solid.

[0066] Example 9:

[0067] Take 1.00 g of the white solid obtained in Example 1, add 2 mL of methyl tert-butyl ether and 5 mL of isopropyl ether, stir and heat until completely dissolved, add an appropriate amount of activated carbon for injection and keep warm for 15 min, pre-filter with a 0.45 μm microporous filter membrane, and then pass through two stages of 0.22 μm sterile filtration, then slowly cool to 15-25 ° C, continue to cool to -5-0 ° C, keep warm at -5-0 ° C for 3 days for crystallization, and filter the obtained solid under reduced pressure and vacuum dry at 40 ° C to obtain 0.89 g of a white solid. The HPLC test result was: 98.59%, and the product was sent for infrared, TGA and DSC detection.

[0068] Example 10:

[0069] Take 1.00 g of the white solid obtained in Example 1, add 2 mL of methyl tert-butyl ether, stir and heat until completely dissolved, add 5 mL of n-hexane during reflux, add an appropriate amount of needle-washed activated carbon and keep warm for 15 min, pre-filter with a 0.45 μm microporous filter membrane, and then pass through two stages of 0.22 μm sterile filtration, then slowly cool to 15-25 ° C, continue to cool to -5-0 ° C, keep at -5-0 ° C for 3 days for crystallization, and filter the obtained solid under reduced pressure and vacuum dry at 40 ° C to obtain 0.91 g of a white solid. The HPLC test result is: 98.07%.

[0070] Example 11:

[0071] Take 1.00 g of the white solid obtained in Example 1, add 7 mL of methyl tert-butyl ether, stir and heat until completely dissolved, add an appropriate amount of needle-washed activated carbon and keep warm for 15 min, pre-filter with a 0.45 μm microporous filter membrane, and then pass through two stages of 0.22 μm sterile filtration, then slowly cool to 15-25 ° C, continue to cool to -5-0 ° C, keep warm at -5-0 ° C for 3 days to crystallize, and the obtained solid is filtered under reduced pressure and dried in vacuo at 40 ° C to obtain 0.86 g of a white solid. The HPLC test result is: 96.13%.

[0072] Spectrum analysis of the dipentadol sebacate of the present invention:

[0073] Table 1 Nuclear magnetic hydrogen spectrum detection results of the compounds of the present invention

[0074]

[0075] Table 2 NMR carbon spectrum test results of the compounds of the present invention

[0076]

[0077]

[0078] Table 3 Mass spectrometry results of the compounds of the present invention

[0079] Mass-to-charge ratio (m / z) Remark 609.7 <![CDATA[[M+H] + ]]> 305.7 <![CDATA[[M / 2+H] + ]]>

[0080] Table 4 Infrared detection results of the compounds of the present invention

[0081]

[0082] Product TGA test results: From the TGA graph, it can be seen that this product does not contain crystalline water or solvent and begins to decompose at about 250°C.

[0083] Product DSC test results: The DSC chart shows that this product does not contain crystal water or crystallization solvent, and the melting point is 51.90℃.

[0084] Example 12: Preparation of Ditapentadol Nonacid and Ditapentadol Undecanoate

[0085] Referring to the synthesis method of Example 1, sebacoyl chloride was replaced with azelayl chloride or undecyl dichloride to synthesize bispentadol nonacid ester and bispentadol undecanoate.

[0086] Table 5 Mass spectra and appearance properties of dipentadol nonacid and dipentadol undecanoate

[0087]

[0088] Example 12: Preparation of Tapentadol Decanoate

[0089] A 1000mL three-necked flask was charged with 400mL of dichloromethane and 25.70g of tapentadol hydrochloride. 20.25g of triethylamine, 4.5g of DCC and 0.4g of DMAP were added under stirring. After a few minutes, the material was completely dissolved and cooled to -5 to 0°C. 19.3g of sebacic acid was added dropwise. The temperature rose significantly. The internal temperature was controlled at 0 to 10°C. Insoluble matter gradually precipitated from the system. The reaction was stirred at 0 to 10°C for 2 hours. TLC monitoring (λ = 254nm, PE: EA = 2:1, two drops of triethylamine were added) The tapentadol content was ≤0.5%. 100mL of water was added to quench the reaction. After separation, 400mL*2 water was added and washed twice. The dichloromethane layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 13.01g of a light yellow oily substance. After standing in the refrigerator overnight, it was an amorphous powder solid.

[0090] Table 6 Mass spectrum and appearance properties of tapentadol decanoate

[0091]

[0092]

[0093] Example 13: Particle size distribution detection

[0094] According to the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Chapter 0982, Method 3, the particle size of the raw material was tested. The products of Examples 9, 10, and 11 were subjected to particle size distribution testing. Specific operation: Take an appropriate amount of the crystalline powder of the products of Examples 9, 10, and 11 in a 5mL centrifuge tube, add distilled water, and shake well. Perform a background scan with distilled water as the background to determine the background value, then take an appropriate amount of the above-mentioned test solution in the sample pool and perform a particle size distribution test on it. The results are as follows:

[0095] Table 7 Particle size distribution test results of the compounds of the present invention

[0096] name D10 / μm D50 / μm D90 / μm Example 9 2.6 5.2 8.1 Example 10 2.9 5.0 7.8 Example 11 2.3 5.1 9.0

[0097] Example 14: Stability Test

[0098] The compound of the present invention will be used as a bulk drug for the preparation of suspension injections in the future, so storage is a critical issue. Other impurities may be introduced or generated during storage, affecting the use of the bulk drug. Therefore, the stability of the bulk drug of the present invention was investigated. Referring to the ICH guideline "Stability Testing of New Drug Substances and Preparations", an accelerated test was used to investigate the effects of temperature, humidity, and light on the bulk drug. The conditions were set as follows: temperature 40±2°C, humidity 75%±5%, light 4500lx, and sampling every 10 days. The results are shown in the following table:

[0099] Table 8 Stability test results of the compounds of the present invention

[0100]

[0101]

[0102] Conclusion: The table shows that the API of the present invention is relatively stable under the relevant setting conditions and does not produce obvious impurities. Therefore, after the API is prepared, it can be stored at room temperature.

[0103] Example 15: Experimental study on cytotoxicity of normal human hepatocytes in vitro

[0104] 1 Experimental Materials

[0105] 1.1 Cells: LO2 cells, a human liver cell line

[0106] 1.2 Drug: Compound of the present invention, HPLC purity 98.07%

[0107] 1.3 Reagents and instruments Modified RPMI-1640 culture medium, penicillin-streptomycin double antibody solution, 0.25% trypsin-EDTA, fetal bovine serum, MTT, dimethyl sulfoxide, CO-150 carbon monoxide incubator, SW-CJ-2F medical clean bench, CKX-41-32 inverted microscope, CU600 electric constant temperature water bath, RT-2100C enzyme label analyzer

[0108] 2 Experimental methods

[0109] 2.1 Reagent preparation

[0110] 2.1.1 Preparation of MTT: Weigh 0.25 g of MTT using a precision balance and place it in a 50 mL volumetric flask. Add an appropriate amount of PBS and place in a 50-60°C water bath. Shake well to dissolve thoroughly. Add PBS to the mark to prepare a 5 mg / kg solution. Sterilize by filtering through a 0.22 μm microporous filter membrane. Aliquot and store in a refrigerator at 4°C away from light.

[0111] 2.1.2 Preparation of cell freezing solution Cell freezing solution was prepared by mixing 20% serum, 10% DMSO and 70% 1640 culture medium and stored at -20℃.

[0112] 2.1.3 Preparation of the Compounds of the Invention The stock solution of the drug was prepared with DMSO, and then diluted with culture medium to the concentration of the drug to be used. The final concentration of DMSO was controlled to be ≤0.1%.

[0113] 2.2 LO2 cell culture Normal human LO2 cells were placed in a 25 cm 2 Add approximately 4-5 mL of RPMI-1640 medium containing 10% FBS to the cell culture flask and incubate in a 37°C cell culture incubator with 5% CO2 saturated humidity. Change the medium every two days and observe cell growth daily. Passage or cryopreserve cells when they reach 80% confluency. Select cells from passages 5-7 for formal experiments.

[0114] 2.3 Grouping and Dosing The experiment was divided into a normal cell control group, and different concentrations of the drug-dosing groups of the present invention were set according to the results of the preliminary experiment, with concentrations of 5.0, 10.0, 20.0, 40.0, 80.0, 160, 320, 640, 1280, and 1500 μmol / L, respectively.

[0115] 2.4 Hepatocyte MTT Colorimetry: LO2 cells in the logarithmic growth phase were prepared and plated into a cell suspension of 5.0 × 10 cells / mL in a 96-well plate. A normal control group and groups treated with the compound of the present invention at various concentrations of 4.0, 8.0, 16.0, 32.0, 64.0, 128, 256, 512, 800, and 1000 μmol / L were set up. After 24 hours of incubation, the culture medium was aspirated and the cells were washed two to three times with PBS. The compounds of the present invention were then added at various concentrations, with eight replicate wells per well for each concentration. The cells were incubated in a 37°C, 5% CO2 incubator. After 24, 48, and 72 hours of incubation, MTT solution was added at each time point and incubated for 4 hours in the dark. After 4 hours, the supernatant was aspirated and 150 μL of DMSO solvent was added to each well to dissolve the thiazolyl blue crystals. The plates were gently shaken to ensure uniform dissolution. The absorbance of each well was measured at 490 nm using a microplate reader. The absorbance value of each well reflects the number of cells, and the two are directly proportional. The experiment was repeated 3 times. The cell viability rate was calculated as follows: Cell viability rate (%) = absorbance of each group (OD490) × 100 / absorbance of control group (OD490)

[0116] 2.5 Statistical Analysis All data are expressed as mean ± standard deviation and processed using SPSS 17.0 statistical software package. Statistical analysis was performed using the t-test. Paired t-tests were used for intragroup comparisons, and unpaired t-tests were used for intergroup comparisons. P < 0.05 was the standard.

[0117] 3 Experimental results

[0118] Table 9 Experimental results of in vitro cytotoxicity of the compounds of the present invention on normal human liver cells

[0119]

[0120]

[0121] Conclusion: After 72 hours of administration of different concentrations of the present invention, the OD values of the cells in each group showed an increasing trend, but the growth rate decreased, indicating that the cells were still in the growth stage and the growth rate was inhibited. The groups treated with different concentrations of the present invention had no inhibitory effect on the cells. Compared with the normal group, the cell survival rate was greater than 90%. In the range of 5.0-1500μmol / L, there was almost no inhibition on cell growth and no toxicity to the cells.

[0122] Example 16: Analgesic Pharmacodynamics Evaluation

[0123] 1 Experimental animals

[0124] Male Sprague-Dawley (SD) rats, weighing (200±20) g.

[0125] 2 Experimental methods

[0126] The efficacy of the present invention was studied by subcutaneous administration in a rat back incision model. The experimental animals were male SD rats (n=3). All animals were adapted to the environment for 2 to 3 days before surgery, and animals that were particularly sensitive or particularly dull were eliminated. The screened rats were anesthetized with Zotai-50. Under sterile conditions, a 2 cm longitudinal incision was made on the left side of the midline of the rat's back, directly reaching the fascia layer without damaging the muscle. The fascia and muscle were separated, and the skin was sutured discontinuously at the incision site with sutures. At 1.5 mL·kg -1 Subcutaneous injection of hydrogenated castor oil solution of the present invention (prepared by dissolving the compound of the present invention in hydrogenated castor oil), (2.5 mg·kg -1 , 5mg·kg -1 , 10mg·kg -1 The rats were randomly assigned to the incision site (low, medium, and high dose groups). A non-dose incision group served as a blank control. Von Frey fibers (providing a tactile stimulus of 0.008 to 300 g) were used to stimulate the area near the incision site before dosing and 1, 2, 3, 4, 8, 12, 24, 36, 48, 60, 72, and 84 hours after dosing. The pain threshold was defined as the number of g represented by the Von Frey fiber that elicited a withdrawal response, such as subcutaneous muscle contraction, on at least three occasions, during five consecutive tests.

[0127] 3 Experimental results

[0128] The pain threshold of each dose group of the present invention was higher than the initial value at 3 days, and the analgesic effect was long-lasting. -1 , 5mg·kg -1 , 10mg·kg -1 The pain threshold of the group at 72 hours was significantly different from that of the blank incision (****P<0.0001). Although there was no significant difference in the pain threshold at 72 hours between the different dose groups of the present invention (ns), the high-dose group had a lower pain threshold, while the medium-dose group had a higher tolerable pain threshold. Moreover, the pain threshold of the high-dose group was significantly higher than that of the low- and medium-dose groups at 84 hours, indicating a certain dose-dependency. Therefore, the low-, medium-, and high-dose groups of the present invention can all maintain analgesia for 3 days, showing a long-lasting analgesic effect.

[0129] Example 17: Safety Evaluation

[0130] The administration method was the same as above. The healing of the incision at the administration site was observed with the naked eye 3, 10, and 14 days after administration. Skin samples around the injection site were collected on the 14th day, fixed with 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin for histopathological analysis.

[0131] Conclusion: After subcutaneous administration of the present invention to rats, scabs formed on the incision 3 days later, the scabs fell off on the 10th day, and the back incision was basically healed on the 14th day, with no significant difference from the non-administered group, indicating that the present invention has no effect on postoperative incision healing. Within 3, 10, and 14 days after administration, the motor behavior of rats in each group was normal. The injection site and surrounding tissues of all animals were observed to be normal by naked eye. After 14 days, the tissue pathological changes (bleeding, foreign matter, ulcers) were no significantly different from those in the non-administered group. The present invention had no obvious irritation to local tissues and showed good safety and tolerability.

[0132] Example 18: Pharmacokinetic study in rats

[0133] 1 In vivo HPLC analysis method

[0134] Chromatographic column: 150mm×4.6mm end-capped octadecylsilane bonded silica gel column (5μm)

[0135] Column temperature: 35°C

[0136] Mobile phase: Mobile phase A: phosphoric acid-methanol-water = 0.1:10:90

[0137] Mobile phase B: phosphoric acid-water-methanol = 0.1:10:90

[0138] Table 10 Chromatographic conditions

[0139] time Mobile phase A Mobile phase B 0-2 100 0 2-44 100→61 0→39 44-44.5 61→0 39→100

[0140] Flow rate: 1.5ml / min

[0141] Detection wavelength: 215nm

[0142] Injection volume: 20 μl2 Preparation of plasma samples

[0143] Blank plasma sample: Thaw rat blank plasma, accurately measure 100 μL into a 1.5 mL centrifuge tube, add 700 μL of 4% glacial acetic acid in methanol, vortex mix for 5 minutes, centrifuge at 9000 rpm for 10 minutes to precipitate the protein, aspirate the supernatant into a 1.5 mL centrifuge tube, and evaporate to dryness at 40°C. Reconstitute the residue with 50 μL of a 0.1:10:90 ratio of phosphoric acid-methanol-water solution, vortex for 6 minutes, sonicate for 15 minutes, and centrifuge at 13000 rpm for 20 minutes. The supernatant is the blank plasma sample solution.

[0144] Plasma sample after administration: Take the plasma of the rat after administration, thaw it, accurately measure 100 μL and place it in a 1.5 mL centrifuge tube. Add 10 μL of internal standard (tramadol 2 μg / mL, dissolved in phosphoric acid-methanol-water = 0.1:10:90), and vortex for 1 minute to mix evenly. Add 700 μL of 4% glacial acetic acid in methanol, vortex for 5 minutes, centrifuge at 9000 rpm for 1 minute to precipitate the protein, aspirate the supernatant into a 1.5 mL centrifuge tube, and evaporate to dryness at 40°C. Add 50 μL of phosphoric acid-methanol-water = 0.1:10:90 to the residue for reconstitution, vortex for 6 minutes, sonicate for 15 minutes to fully dissolve the drug, and centrifuge at 13000 rpm for 20 minutes. The supernatant is the plasma sample solution after administration.

[0145] 3. Dosage regimen and sample collection

[0146] Twelve rats were randomly divided into two groups, each with six rats, and each group was injected intramuscularly with a hydrogenated castor oil suspension of the present invention (prepared by dissolving the compound of the present invention in hydrogenated castor oil) at a dose of 10 mg / kg. Blood was collected from the rats' orbital vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 96, 144, 192, and 240 hours after administration. Approximately 0.5 mL of blood was collected each time and added to a sodium heparin-treated anticoagulant tube. The blood was centrifuged at 4000 rpm for 10 minutes, and the upper plasma layer was separated and stored at -20°C for analysis.

[0147] 4 Plasma sample testing and data processing

[0148] Take the plasma sample after administration and process it according to the method under 2. Preparation of plasma sample. Inject it according to the chromatographic conditions under 1. In vivo HPLC analysis method. Substitute the peak area ratio of the active drug and the internal standard peak into the standard curve to calculate the hemorrhagic drug concentration. Process the results using Phoenix software to calculate the relevant pharmacokinetic parameters.

[0149] 5 Results

[0150] Table 11 Pharmacokinetic test results in rats

[0151]

[0152]

[0153] The results show that the compounds of the present invention can be slowly, continuously and stably released in the body and converted into tapentadol, thereby exerting a long-lasting effect. At the same time, they are released steadily to achieve a long-lasting release effect.

[0154] 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 above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Ditapentadol sebacate represented by formula (I): 。 2. The ditapentadol sebacate according to claim 1, characterized in that: Ditapentadol sebacate is a sterile drug substance.

3. The bisapentadol sebacate according to claim 2, characterized in that: The particle size D50 of the sterile bulk drug substance of dipentadol sebacate is 0.5~10μm.

4. The method for preparing dipentadol sebacate according to claim 1, characterized in that: The following steps are involved: Tapentadol hydrochloride and sebacyl chloride are acylated in an organic solvent and an acid-binding agent, followed by quenching, washing, column chromatography, and crystallization. The reaction formula is as follows: 。 5. The method for preparing dipentadol sebacate according to claim 4, characterized in that: The solvent of the crystallization process is selected from one or more of methyl tert-butyl ether, isopropyl ether, n-hexane and petroleum ether.

6. The method for preparing dipentadol sebacate according to claim 4, characterized in that: The temperature of the crystallization process is -15 to 0°C.

7. The method for preparing dipentadol sebacate according to claim 4, characterized in that: Before the crystallization process, the product is pre-filtered with a 0.45 μm microporous filter membrane, and then subjected to two stages of 0.22 μm sterilization filtration and vacuum drying.

8. A pharmaceutical composition comprising the dipentadol sebacate according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that: The pharmaceutical composition is administered by injection.

10. Use of the dipentadol sebacate according to claim 1 in preparing medicaments for treating various acute and chronic pains.

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