WO2024160030A1 - Composition and use thereof in preparation of drug for treating neuropathic pain - Google Patents

Abstract

A composition and the use thereof in the preparation of a drug for treating neuropathic pain. The composition comprises a pregabalin moiety and a duloxetine moiety, wherein the pregabalin moiety comprises pregabalin, the duloxetine moiety comprises duloxetine, and the mass ratio of the pregabalin bulk drug to the duloxetine bulk drug is (2.50-3.75):1. The composition can improve the bioavailability of pregabalin and enhance the hypnotic effect thereof. Moreover, the composition can be used for overcoming sleep disorders of a pain-related patient, shortening the sleep latency time thereof, prolonging sleep time and improving sleep quality, and has an adjuvant therapy effect.

Description

A composition and its application in preparing medicine for treating neuropathic pain Technical Field

The present invention belongs to the field of medicine, and in particular relates to a composition and its application in preparing a medicine for treating neuropathic pain.

Background Art

Neuropathic pain (NP; also referred to as "neuropathic pain" in this application) is a common chronic intractable pain caused by damage or disease of the somatosensory nervous system, which causes patients to experience sleep disorders and even depression, seriously affecting their quality of life. Most neuropathic pain can be divided into one of three sensory phenotypes: sensory loss, mechanical hyperalgesia, and thermal hyperalgesia. Neuropathic pain is related to changes in the central or peripheral nervous system.

Most anti-tumor drugs are prone to cause peripheral neuropathy (CIPN), which is a common and serious adverse reaction after chemotherapy. It can manifest as sensory disorders such as pain and numbness, seriously affecting the quality of life of patients. Paclitaxel and cisplatin are widely used to treat various cancers (including breast cancer, cervical cancer, ovarian cancer, pancreatic cancer and lung cancer). They are the most common chemotherapy drugs that cause CIPN. The neuropathic pain is mainly manifested as thermal allergy and mechanical hypersensitivity.

Diabetic neuropathy is a group of clinical syndromes with diverse manifestations caused by different pathophysiological mechanisms. It is the most common chronic complication of diabetes. Common types of diabetic neuropathy are distal symmetric polyneuropathy (DSPN) and autonomic neuropathy, of which DSPN is the most common type and is also commonly known as diabetic peripheral neuropathy.

Diabetic neuropathy is the most common chronic complication of type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). About 50% of diabetic patients will eventually develop DSPN. In addition, DSPN can occur in the pre-diabetes stage.

DSPN generally presents as symmetrical polysensory neuropathy, which initially affects the distal lower limbs and gradually develops upward as the disease progresses, forming typical "stocking-like" and "glove-like" sensations. The most common early symptoms are caused by diabetic small fiber neuropathy (SFN), which manifests as pain and paresthesia. 50% of diabetic patients experience pain caused by DSPN (also known as painful DSPN), which manifests as burning pain, electric shock-like pain, and sharp pain; followed by soreness, itching, cold pain, and induced pain.

Pregabalin is the most thoroughly studied drug for the treatment of DSPN. Most studies have shown that pregabalin can improve DSPN pain by at least 30% to 50%. Some studies have not obtained data on its effectiveness, especially for those painful neuropathy that is resistant to treatment. Duloxetine is considered an effective drug for the treatment of painful DSPN. It is a selective norepinephrine and serotonin reuptake inhibitor.

However, the monotherapy effects of pregabalin and duloxetine are not yet ideal. Clinical research data of original drugs show that more than half of the patients do not achieve a pain score improvement of ≥50% compared with the baseline level after monotherapy.

In Pfizer's LYRICA study DPN 1 (LYRICA FDA label), only 46% of patients who used pregabalin for 5 weeks had a pain score improvement of ≥50% compared with baseline levels; in study DPN 2 (LYRICA medical reviews), only 32.89% of patients who used pregabalin for 8 weeks had a pain score improvement of ≥50% compared with baseline levels.

In the US instructions for Eli Lilly's Cymbalta, approximately 45% and 42% of patients in the DPNP-1 and DPNP-2 studies, respectively, were treated with duloxetine for 12 weeks and achieved an improvement of ≥50 in the 24-hour average pain score.

Therefore, will the combination of these two drugs achieve a more significant therapeutic effect? What ratio and dosage can achieve a more significant therapeutic effect? These are technical issues worth exploring.

At the same time, cancer pain is also a relatively large type of pain in the real world. Almost all patients with solid tumors will experience unbearable pain when they develop to the late stage. The pain brings great physical and mental torture to patients. Most patients with a history of pain also have sleep disorders, which are characterized by long sleep induction time and short sleep time.

Summary of the invention

In order to enhance the therapeutic effect on neuropathic pain (including diabetic peripheral neuropathy pain and cancer pain), the purpose of the present invention is to provide a composition containing a specific ratio of pregabalin and duloxetine, which significantly increases (extends) the pain threshold and plays a synergistic analgesic effect; at the same time, the composition can also relieve the patient's sleep disorder and improve sleep quality; under the combined use of the composition, the bioavailability of pregabalin can also be improved. Pregabalin and duloxetine, when matched in a specific ratio of the present invention, play a synergistic effect and have good clinical application prospects.

The purpose of the present invention is achieved through the following technical solutions:

A composition comprises a pregabalin part and a duloxetine part; the pregabalin part comprises pregabalin (raw material), the duloxetine part comprises duloxetine (raw material), and the mass ratio of pregabalin to duloxetine is (2.50-3.75):1, preferably (3.00-3.75):1.

According to some embodiments, the composition contains pregabalin (75-150) mg and duloxetine (20-50) mg.

According to some embodiments, the composition contains 150 mg of pregabalin and (40-50) mg of duloxetine.

According to some other embodiments, the composition contains 75 mg of pregabalin and (20-25) mg of duloxetine.

The composition can be prepared into various dosage forms, including but not limited to granules, suspensions, tablets, capsules, etc. preparation.

According to some embodiments, in the composition, the pregabalin portion is preferably present in the form of a gastric soluble immediate release preparation.

According to some embodiments, the preparation form of the pregabalin portion may be granules, tablets, etc.

According to some embodiments, the pregabalin portion comprises pregabalin, and one or more of a filler, a binder, a wetting agent, a disintegrant, a glidant, and a lubricant.

According to some embodiments, the pregabalin partial granules are prepared according to conventional formulation techniques, such as dry granulation, wet granulation, direct mixing, etc.

According to some embodiments, the pregabalin partial particles are prepared by a direct mixing process, wherein the pregabalin API particle size range is D90: (50-500) μm, and the pregabalin API is uniformly mixed with the glidant micropowder silica gel and the lubricant magnesium stearate, or further filled with microcrystalline cellulose, silicified microcrystalline cellulose, lactose, etc., and mixed evenly.

According to some embodiments, the mass ratio of pregabalin API, glidant micropowder silica gel, and lubricant magnesium stearate in the pregabalin partial granules is 150:(0.5-10):(0-10).

According to some embodiments, the pregabalin partial granules may be prepared by a wet granulation process, and the mass ratio of pregabalin to the filler and binder is 150:(50-300):(0-10).

According to some embodiments, the pregabalin partial granules can be formed into tablets by a tableting process.

According to some embodiments, in the composition, the duloxetine portion is in the form of a gastric immediate-release preparation or an enteric-coated preparation, preferably in the form of an enteric-coated preparation.

According to some embodiments, when the duloxetine portion is in the form of a gastric-soluble immediate-release preparation, granules, tablets, etc. may be selected.

According to some embodiments, the partially gastric-soluble rapid-release preparation of duloxetine comprises duloxetine and one or more of a filler, a binder, a wetting agent, a disintegrant, a glidant and a lubricant.

According to some embodiments, the duloxetine partially gastric soluble rapid-release preparation is prepared according to conventional preparation techniques, such as dry granulation, wet granulation, direct mixing, etc.

According to some embodiments, when the duloxetine portion is in the form of an enteric-coated preparation, enteric-coated granules, enteric-coated micropellets, enteric-coated microtablets, and enteric-coated tablets may be selected.

According to some embodiments, when the duloxetine portion is in the form of enteric granules, enteric materials and duloxetine may be co-granulated, and other optional excipients such as fillers, plasticizers, glidants, lubricants, etc. may also be included.

According to some embodiments, the structure of the duloxetine partially enteric-coated pellets may include a pill core, an optional isolation coating layer, an enteric coating layer, and an optional protective coating layer.

According to some embodiments, the drug-containing pellet core can be obtained by loading the drug onto micropellets, or granulating the drug into pellets.

According to some embodiments, when the duloxetine-containing pill core is coated with micro-pellets, the pill core comprises duloxetine, a pill core, a binder, an anti-adhesive agent, and the like.

According to some embodiments, in the drug-containing pill core, the mass ratio of duloxetine, pill core, binder, and anti-adhesive agent is 40:(30-100):(10-30):(2-10).

According to some embodiments, the pill core containing the drug pill core is one or more of a sugar pill, a microcrystalline cellulose pill or a silicon dioxide pill, and the particle size of the pill core is 60 mesh to 30 mesh.

Furthermore, when the duloxetine-containing pill core is granulated into pills using drugs, the pill core contains duloxetine and one or more combinations of fillers, disintegrants, binders or anti-adherents, and can be obtained by high shear, extrusion spheronization or fluidized bed granulation, preferably by extrusion spheronization.

According to some embodiments, the duloxetine partially containing pill core is prepared by extrusion spheronization process, wherein the mass ratio of duloxetine, filler, binder, wetting agent and anti-adherent agent is 40:(20-300):(1-40):(4-60):(1-20).

According to some embodiments, in the duloxetine portion, the isolation coating layer comprises a film-forming material and an anti-adhesive agent.

According to some embodiments, the preparation of the isolation coating layer includes adding a film-forming material and an anti-adhesive agent into a solvent to prepare a solution, coating the solution on the surface of the drug-containing pill core through a fluidized bed, and drying to obtain isolation pellets.

According to some embodiments, when the isolation coating layer is prepared, the mass ratio of the film-forming material, the anti-adherent agent, and the solvent is (5-15):(1-10):(75-94).

According to some embodiments, in the duloxetine part, the enteric coating layer includes an enteric material, a plasticizer, and an anti-adherent. According to some embodiments, the preparation of the enteric coating layer includes adding the enteric material, plasticizer, and anti-adherent to a solvent to prepare a solution, coating it on the surface of the pill core or the isolation coating layer through a fluidized bed, and drying to obtain duloxetine enteric micropellets.

According to some embodiments, when the enteric coating layer is prepared, the mass ratio of the enteric material, the plasticizer, the anti-adherent agent, and the solvent is (5-15):(0.5-3):(1-15):(60-150).

According to some embodiments, in the duloxetine part, the protective coating layer includes a film-forming material and an anti-adhesive agent. According to some embodiments, the preparation of the protective coating layer includes adding the above-mentioned film-forming material and anti-adhesive agent to a solvent to prepare a solution, and further adding a pigment, a sunscreen, etc., coating the solution on the surface of the enteric coating layer through a fluidized bed, and drying to obtain duloxetine enteric micropills.

According to some embodiments, when the protective coating layer is prepared, the mass ratio of the film-forming material, the anti-adherent agent, and the solvent is (5-15):(1-10):(75-94).

According to some embodiments, when the duloxetine portion is in the form of an enteric-coated microtablet or an enteric-coated tablet, it includes a circular tablet with a long diameter of 2 to 5 mm, a capsule-shaped tablet or a special-shaped duloxetine enteric-coated microtablet, or a circular tablet with a long diameter of 5 to 12 mm, a capsule-shaped tablet or a special-shaped enteric-coated microtablet. The structure of the duloxetine enteric-coated tablets or special-shaped tablets may include a tablet core, an optional isolation coating layer, an enteric coating layer, and an optional protective coating layer.

According to some embodiments, the filler described in the present invention is selected from one or more of starch, dextrin, polyethylene glycol, lactose, microcrystalline cellulose, silicified microcrystalline cellulose, lactose-microcrystalline cellulose complex, and ethyl cellulose; the wetting agent and solvent are selected from one or more of water, ethanol, acetone, and isopropanol; the binder and film-forming material are selected from one or more of hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; the disintegrant is selected from one or more of sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, cross-linked calcium carboxymethyl cellulose, cross-linked magnesium carboxymethyl cellulose, and cross-linked polyvinylpyrrolidone; the glidant is selected from micropowder silica gel , talc, and magnesium stearate; the lubricant is selected from one or more of magnesium stearate, sodium stearyl fumarate and micro-powder silica gel; the enteric material is selected from one or more of medicinal acrylic resin, hydroxypropyl methylcellulose phthalate (HPMCP), hydroxypropyl methylcellulose acetate succinate (HPMCAS), cellulose acetate phthalate (CAP), Eudragit L30D-55, Eudragit L100-55, Eudragit L100, Eudragit NE30D, polyvinyl phthalate (PVAP) and cellulose acetate trimellitate (CAT); the plasticizer is selected from triethyl citrate, polyethylene glycol 6000, tributyl citrate and dibutyl sebacate, etc.

In the composition of the present invention, when the pregabalin part is in the form of granules and the duloxetine part is in the form of granules, enteric-coated granules or enteric-coated pellets, they can be mixed evenly and then filled into capsules or compressed into tablets; the pregabalin part and the duloxetine part can also be separately filled into the same capsule in steps; or double-layer tablets can be compressed, with the pregabalin part and the duloxetine part being in different layers.

In the composition of the present invention, when the pregabalin portion is in the form of granules and the duloxetine portion is in the form of enteric-coated microtablets or enteric-coated tablets, or when the pregabalin portion is in the form of tablets and the duloxetine portion is in the form of enteric-coated granules or enteric-coated micropellets, the pregabalin portion and the duloxetine portion can be separately filled into the same capsule according to the steps.

The composition of the present invention can effectively improve the bioavailability of pregabalin; within 24 hours of administration to rats, under combined administration (administered once a day), the AUC _0-24 of pregabalin is more than 118% of that of pregabalin administered alone, preferably (161-208)%, and particularly preferably (161-196)%.

The composition of the present invention comprises pregabalin and duloxetine in a specific ratio, and can be used to prepare a drug for treating neuropathic pain, especially for diabetic peripheral neuropathy and cancer pain. When used in combination, the pain threshold can be synergistically increased (prolonged), achieving a synergistic analgesic effect.

Therefore, the present invention also provides the use of the composition in the preparation of a drug for treating neuropathic pain and/or a drug for treating diabetic peripheral neuropathy.

Furthermore, the present invention provides a method for treating neuropathic pain and/or diabetic peripheral neuropathy, comprising: The composition of the present invention is preferably administered 1 to 3 times a day.

Cancer pain refers to the pain caused by various solid tumors compressing or infiltrating nearby parts of the body, or the pain caused by solid tumor treatment and diagnostic procedures. It may also be discomfort caused by changes in the skin, nerves and other hormones caused by imbalance or immune response.

The solid tumors include, but are not limited to, colorectal cancer, ovarian cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer (including melanoma, squamous cell carcinoma), cancer of the head or neck, breast cancer, lung cancer (including non-small cell lung cancer), uterine cancer (including endometrial cancer), cancer of the anal region, stomach cancer, testicular cancer, cancer of the fallopian tubes, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma (including Kaposi's sarcoma), urethral cancer, penile cancer, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, central nervous system (CNS) cancer, primary CNS lymphoma, spinal tumors, brain stem nerve Gliomas, pituitary adenomas, hematological malignancies (including, for example, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma/primary mediastinal B-cell lymphoma, non-Hodgkin lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma and precursor T-lymphoblastic lymphoma), and any combination of the aforementioned cancers and treatment of metastatic cancers.

The present invention also provides a combined medication method, which comprises administering pregabalin and duloxetine to a patient in combination, 1-3 times a day, preferably 2 times a day; the mass ratio of pregabalin to duloxetine is (2.50-3.75):1, preferably (3.00-3.75):1.

Preferably, the combined drug administration method is to administer pregabalin (75-150) mg and duloxetine (20-50) mg to the patient each time, 1-3 times a day, preferably twice a day.

Particularly preferably, the combination therapy method is for the initial treatment of patients treated with low doses and patients treated with high doses, and each time 75 mg of pregabalin and 20-25 mg of duloxetine can be administered in combination, 1-3 times a day, preferably twice a day; for the maintenance treatment of patients treated with high doses, 150 mg of pregabalin and 40-50 mg of duloxetine can be administered in combination, 1-3 times a day, preferably twice a day.

The combined drug method can effectively improve the bioavailability of pregabalin; when the total dosage is consistent, within 24 hours of administration to rats, the AUC _0-24 of pregabalin under twice-a-day administration is more than 110% of that under once-a-day administration, preferably (110-130)%.

The present application unexpectedly found that, compared with the regimen of pregabalin alone and duloxetine once a day in combination administration, duloxetine twice a day in combination administration in the present application can delay the metabolic rate of pregabalin and significantly improve the metabolism of pregabalin. Bahrain's bioavailability improves therapeutic efficacy.

In the pharmaceutical composition of the present invention, pregabalin is a structural derivative of the inhibitory neurotransmitter aminobutyric acid (GABA), which has a high affinity to the α2-δ site in the central nervous system tissue. The mechanism of action of pregabalin has not been fully elucidated, and it may be achieved by interfering with the transport of calcium channels containing α2-δ subunits and/or reducing calcium currents, or it may be through the interaction with the descending norepinephrine (NE) and 5-hydroxytryptamine (5-HT) pathways from the brainstem to regulate pain transmission in the spinal cord.

Duloxetine is a selective 5-HT and NE reuptake inhibitor. The exact mechanism of duloxetine's antidepressant and central analgesic effects has not yet been clarified, but it is believed to be related to its enhancement of the 5-HT and NE functions in the central nervous system.

Current studies have shown that the analgesic mechanisms of pregabalin and duloxetine are different, but there is a potential correlation. The inventor has conducted a large number of animal experiments and found that the specific combination of pregabalin and duloxetine is effective in treating neuropathic pain and has significant advantages over single drugs. In addition, it was unexpectedly found in the study that the combination of pregabalin and duloxetine in a specific ratio can significantly improve the bioavailability of pregabalin and potentially improve the analgesic and hypnotic effects of pregabalin.

Compared with the prior art, the present invention has the following advantages and effects:

1. The composition of the present invention comprises pregabalin and duloxetine in a specific ratio, and can be used to prepare a drug for treating neuropathic pain, especially for diabetic peripheral neuropathy pain and cancer pain. In the case of combined use, it can synergistically increase (prolong) the pain threshold and achieve a synergistic analgesic effect, which is significantly better than using the drug alone.

2. The composition of the present invention can affect the pharmacokinetic properties and metabolic rate of pregabalin, significantly improve the bioavailability of pregabalin, and enhance the analgesic effect.

3. The composition of the present invention can improve the bioavailability of pregabalin, enhance its hypnotic effect, overcome the sleep disorders of pain-related patients, shorten their sleep time, prolong sleep time, improve sleep quality, and play an auxiliary therapeutic effect.

4. The present invention further found that compared with administration once a day, administration twice a day can further improve the bioavailability and analgesic effect of the pregabalin and duloxetine combination drug.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

Example 1

Pregabalin and duloxetine compound granules, the composition and formula of which are shown in Table 1:

Table 1: Pregabalin and duloxetine compound granules formula

The preparation method of pregabalin and duloxetine compound granules provided by the present invention comprises the following steps:

1. Preparation of pregabalin: The active component A pregabalin is fully mixed with the glidant micropowder silica gel and the lubricant magnesium stearate to obtain pregabalin granules.

2. Preparation of Duloxetine:

(1) adding active ingredient B duloxetine, binder L-HPC, and anti-adhesive talc into solvent water and stirring evenly to prepare a drug solution, and then coating the drug solution on the surface of the pill core sugar pill by fluidized bed coating to obtain a pill core containing the drug;

(2) preparing a suspension of a film-forming material and an anti-adhesive talc powder, and coating the suspension on the surface of a pill core through a fluidized bed to obtain isolated pellets;

(3) The enteric material and plasticizer of the enteric layer are sequentially added to the solvent, and after being dissolved, an anti-adhesive agent is added to form a suspension, which is then coated on the surface of the isolation micro-pellets to prepare the duloxetine enteric micro-pellets.

3. Total mixing: fully and evenly mix the pregabalin granules and duloxetine enteric-coated pellets to obtain pregabalin duloxetine compound granules.

Example 2

Pregabalin and duloxetine compound tablets, the composition and formula of which are shown in Table 2:

Table 2: Pregabalin and duloxetine combination tablet formulation

The preparation method of the pregabalin-duloxetine compound tablet provided by the present invention comprises the following steps:

1. Preparation of pregabalin part: The active component A pregabalin is fully mixed with the filler microcrystalline cellulose and the filler polyethylene glycol, and a povidone aqueous solution is added. The mixture is granulated using a high shear granulator and dried to obtain pregabalin part granules.

2. Preparation of Duloxetine:

(1) The active component B duloxetine is mixed evenly with the filler microcrystalline cellulose, the binder povidone, and the anti-adhesive talc, and the wet material is added and stirred to obtain a wet material, and the wet material is prepared into a pill core by an extrusion spheronizer, and the D90 is 42 mesh;

(2) Adding enteric material hydroxypropyl methylcellulose phthalate of the enteric layer into 80% ethanol aqueous solution, and then adding anti-sticking agent talcum powder to make a suspension after dissolving, and then coating it on the surface of the pill core to make the duloxetine enteric micro-pellets.

3. Total mixing: Add part of the pregabalin granules, duloxetine enteric-coated pellets and lubricant magnesium stearate into a mixing barrel and mix thoroughly to obtain pregabalin and duloxetine compound granules.

4. Tablet compression: Add the pregabalin and duloxetine compound granules into the tablet press and compress them into tablets according to the target tablet weight. Each tablet contains 150 mg of pregabalin and 40 mg of duloxetine.

Example 3

Prescription and preparation process of pregabalin and duloxetine compound capsules:

The pregabalin and duloxetine compound granules in Example 1 are used, capsule shells of appropriate volume are selected, and filling is performed to obtain pregabalin and duloxetine compound capsules. The pregabalin and duloxetine compound capsules contain 150 mg of pregabalin and 40 mg of duloxetine; or 75 mg of pregabalin and 20 mg of duloxetine.

Example 4

Effect of the pharmaceutical composition of the present invention on diabetic peripheral neuropathic pain model rats

1. Experimental animals and modeling reagents

Healthy SD rats, 180-220 g, were kept in a room with a temperature of 20-25°C, a relative humidity of 40-70%, good ventilation, a lighting cycle of 12 h, and free access to food and water;

Streptozotocin (STZ) was dissolved in a citric acid buffer solution with a pH value of 4.0 and a concentration of 0.1 mol/L to prepare a STZ solution with a concentration of 0.5 g/100 mL;

2. Animal modeling

Six of the 80 rats were randomly selected as the normal control group, and the remaining rats were fasted for 12 hours and then intraperitoneally injected with 75 mg/kg STZ solution.

Blood glucose measurement: 7 days after injection of STZ solution, tail vein blood glucose was measured. Blood glucose ≥ 16mmol/L is one of the conditions for the diabetes model. Blood glucose concentration of rats was measured at intervals during the experiment, and rat samples with a concentration < 16mmol/L were discarded;

Mechanical pain threshold measurement: The mechanical paw withdrawal threshold (MPWT) of rats is used to reflect their mechanical pain sensitivity behavior. The rats were placed in a plexiglass box (30cm×20cm×15cm) with a hollow metal grid at the bottom. The rats were adapted to the environment for 20 minutes. When they were in a resting state, the measurement was started. The Von Frey wire vertically stimulated the skin of the rat's paw pad from bottom to top. The stimulation intensity was set to gradually increase from 0 within 10 seconds. When a rapid paw withdrawal reaction occurred, the stimulation stopped automatically and the pressure value was recorded. The measurement was performed 3 times, each with an interval of 3 minutes. The average value was taken as the MPWT at that time point, also known as the mechanical pain threshold.

In this example, the mechanical paw withdrawal threshold of rats was measured using a Von Frey dynamic plantar tactile meter before STZ injection, 7 days, 3 weeks, and 5 weeks after injection.

Thermal pain threshold measurement: The thermal paw withdrawal latency (TPWL) of rats to thermal radiation stimulation is used as an indicator to reflect thermal pain sensitivity. The measurement time point is the same as the mechanical pain threshold. A radiation thermal analgesia instrument is used to measure the pain threshold time of the plantar photothermal stimulation of rats in a free state. The rats are placed in a 22cm×12cm×12cm box with a smooth transparent glass plate at the bottom. After 20 minutes of adaptation, the thermal radiation stimulator is used to irradiate the hind limbs of the rats (either the left or right foot, but the right foot is habitually selected). When the rat lifts its foot to avoid, the irradiation is stopped and the irradiation time is recorded. This time is the thermal paw withdrawal latency of the rat. In order to prevent the sole of the rat from being scalded, the upper limit of TPWL is set to 30s. If the rat still does not lift its foot after more than 30s, the thermal stimulation will automatically terminate and be recorded as 30s. Irradiate 3 times, each time with an interval of 5 to 6 minutes, and take the average value as the TPWL at that time point, which is also the thermal pain threshold. The intensity of thermal radiation was set to an intensity that could cause normal rats to withdraw their paws within 20 seconds. The same intensity of thermal stimulation was maintained throughout the experiment.

Modeling results: 7 days after injection of STZ solution, rats with blood glucose ≥16mmol/L, significantly reduced mechanical pain threshold, and significantly shortened thermal pain threshold were considered to have successfully established the model. A total of 65 rats were successfully modeled with diabetic peripheral neuropathic pain.

3. Trial Grouping and Dosing

60 rats with successful modeling were selected, 6 of which were randomly selected as the model control group, and the remaining rats were divided into 9 groups: pregabalin treatment group, duloxetine treatment group A/group B/group C/group D, combined medication group A (P/D-A group), combined medication group B (P/D-B group), combined medication group C (P/D-C group), and combined medication group D (P/D-D group); the dosage of each group is as follows:

Normal control group: an equal volume of distilled water was given by gavage;

Model control group: an equal volume of distilled water was given by gavage;

Pregabalin treatment group (P group): 60 mg/kg of pregabalin was administered by gavage;

Duloxetine treatment group A (group D-A): 12 mg/kg duloxetine was administered by gavage;

Duloxetine-treated group B (group D-B): 16 mg/kg duloxetine was administered by gavage;

Duloxetine-treated group C (group D-C): 20 mg/kg duloxetine was administered by gavage;

Duloxetine-treated group D (group D-D): 24 mg/kg duloxetine was administered by gavage;

Combined medication group A (P/D-A group): 60 mg/kg of pregabalin and 12 mg/kg of duloxetine were administered by gavage;

Combined medication group B (P/D-B group): 60 mg/kg of pregabalin and 16 mg/kg of duloxetine were administered by gavage;

Combined medication group C (P/D-C group): 60 mg/kg of pregabalin and 20 mg/kg of duloxetine were administered by gavage;

Combined medication group D (P/D-D group): 60 mg/kg of pregabalin and 24 mg/kg of duloxetine were administered by oral gavage.

Each administration group used the corresponding part of the drug granules (pregabalin granules, duloxetine enteric-coated pellets, pregabalin duloxetine compound granules) in Example 1 and mixed them into a paste with an appropriate amount of distilled water, and administered by gavage once a day. Administration began 7 days after the injection of STZ solution, and continued for 2 weeks and 4 weeks. After 3 hours of administration, the mechanical pain threshold and thermal pain threshold of rats in each administration group were measured.

4. Data processing

SPSS 22.0 statistical software was used, and the calculated data were expressed as mean ± standard deviation. Variance analysis was used to compare multiple groups of data, and t-test was used to compare between groups. P < 0.05 was considered statistically significant.

5. Test results

The measurement results of each group are shown in Table 3.

Table 3: Effects of the pharmaceutical composition of the present invention on diabetic peripheral neuropathic pain model rats

Note: ☆Compared with normal control group, P < 0.05; #Compared with model control group, P < 0.05; *Compared with P group, P value < 0.05;

▲P/D-B group vs. D-B group, P<0.05; ◆P/D-C group vs. D-C group, P<0.05; ★P/D-D group vs. D-D group, P<0.05;

§Compared with P/D-A group, P＜0.05; ○Compared with P/D-B group, P＜0.05.

The experimental results show that:

(1) Compared with the normal group, the mechanical pain threshold and thermal pain threshold of the model group decreased significantly (p < 0.05), indicating that the diabetic neuropathic pain rat model obtained by injection of streptozotocin is ideal and meets the experimental needs.

(2) In the single-drug group: Compared with the model group, the pregabalin treatment group (P group) had significantly increased thermal pain thresholds at the second week of treatment and mechanical pain thresholds and thermal pain thresholds at the fourth week of treatment, P < 0.05. Compared with the model group, the mechanical pain thresholds and thermal pain thresholds of group A (D-A group) and group B (D-B group) treated with a slightly lower dose of duloxetine were increased at each treatment point, but the difference was not significant; compared with the model group, the mechanical pain thresholds and thermal pain thresholds of group C (D-C group) and group D (D-D group) treated with a slightly higher dose of duloxetine were significantly increased, P < 0.05.

(3) In the combination drug group: compared with the model group, the mechanical pain threshold and thermal pain threshold of pregabalin combined with different doses of duloxetine at each treatment point were significantly increased, P < 0.05.

Compared with the pregabalin treatment group (P group), the mechanical pain threshold and thermal pain threshold of the combination medication group A (P/DA group) were increased at each treatment point, but the difference was not significant; the mechanical pain threshold and thermal pain threshold of the combination medication group B (P/DB group) and the combination medication group C were increased. The mechanical pain threshold and thermal pain threshold of the two groups were significantly increased at each treatment point (P/DC group) and the combined medication D group (P/DD group), P＜0.05.

Compared with the duloxetine treatment group with the same duloxetine dosage, the mechanical pain threshold and thermal pain threshold of the combination medication group A (P/D-A group) were increased at each treatment observation point, but the difference was not significant; the mechanical pain threshold and thermal pain threshold of the combination medication group B (P/D-B group), the combination medication group C (P/D-C group), and the combination medication group D (P/D-D group) were significantly increased at each treatment observation point, P < 0.05.

The mechanical pain threshold and thermal pain threshold increased significantly from the P/D-B group to the P/D-C group, P < 0.05; however, the increase in the mechanical pain threshold and thermal pain threshold in the P/D-D group was significantly reduced when the duloxetine dosage increased from 20 mg/kg to 24 mg/kg compared with the P/D-C group, and the difference was not significant.

Example 5

The study on the effect of the pharmaceutical composition of the present invention on sleep disorders in diabetic peripheral neuropathic pain model rats was conducted on the animal group that completed the experiment in Example 4.

On the first day after 4 weeks of drug administration, the animal group of the experiment in Example 4 continued to be administered according to the original drug administration grouping scheme. Three hours later, the rats were intraperitoneally injected with a subthreshold 2.5% concentration of chloral hydrate aqueous solution (250 mg/kg). The administration time of the rats (T0) was recorded; after administration, the rats were placed alone, and the disappearance time of the righting reflex (T1) was recorded. The time from drug administration to the disappearance of the righting reflex was the sleep induction time; after sleep induction, the disappearance time of the toe pinch reflex (the time when the animal began to enter deep sleep, T2) and the recovery time (T3) were recorded, and the duration of deep sleep was calculated; finally, the righting reflex recovery time (T4) was recorded. The time interval was calculated, T1-T0 was the sleep induction time, and T4-T1 was the sleep time.

Experimental results:

The animals in Example 4 survived and were in good health after 4 weeks of administration, and all were studied in this example. After this study, none of the animals died or had abnormal conditions. The results are shown in Table 4.

Table 4: Effects of the pharmaceutical composition of the present invention on sleep disorders in diabetic peripheral neuropathic pain model rats

Note: ☆Compared with normal control group, P < 0.05; #Compared with model control group, P < 0.05; *Compared with P group, P value < 0.05;

△P/D-A group compared with D-A group, P＜0.05;▲P/D-B group compared with D-B group, P＜0.05;◆P/D-C group compared with D-C group, P＜0.05;★P/D-D group compared with D-D group, P＜0.05;

§Compared with P/D-A group, P＜0.05; ○Compared with P/D-B group, P＜0.05; ※Compared with P/D-C group, P＜0.05.

The experimental results show that:

(1) Compared with the normal group, the sleep induction time of the model group was significantly prolonged (p < 0.05), and the sleep time was significantly decreased (p < 0.05), indicating that the diabetic neuropathic pain rat model obtained by injection of streptozotocin would significantly affect the hypnotic effect of chloral hydrate, which met the experimental needs.

(2) In the single-drug groups: compared with the model group, the sleep induction time of the pregabalin-treated group (P group), duloxetine-treated group A (D-A group), group B (D-B group), group C (D-C group), and group D (D-D group) was slightly reduced, and the sleep time was slightly prolonged, indicating that there was some improvement in sleep, but it was not significant.

(3) In the combination drug group: Compared with the model group, the combination of pregabalin and different doses of duloxetine improved the sleep induction time and sleep time, and the combination drug group B (P/D-B group), combination drug group C (P/D-C group) and combination drug group D (P/D-D group) all showed significant improvement.

Among them, compared with the pregabalin treatment group (P group) or the same dose of duloxetine monotherapy group, the combined medication group A (P/D-A group) showed some improvement in sleep induction time and sleep time, but the difference was not significant. The combined medication group B (P/D-B group), combined medication group C (P/D-C group), and combined medication group D (P/D-D group) showed significant improvement in sleep induction time and sleep time. Among them, the sleep induction time and sleep time of the P/D-C group were significantly improved compared with the P/D-B group; however, the duloxetine dosage in the P/D-D group increased from 20mg/kg to 24mg/kg compared with the P/D-C group, and the improvement in sleep induction time and sleep time was significantly reduced.

Example 6

Effect of the pharmaceutical composition of the present invention on skin cancer pain

60 SD rats weighing 180-220 g were selected, 6 of which were kept as the normal control group, and the rest were used for modeling;

For the rats in the model group, the foot pad skin of the rats was disinfected with 75% ethanol; 200 μL of Walker256 breast cancer cell suspension with a density of 4×10 ⁷ /mL was slowly injected into the subcutaneous part of the foot pad of the rats in the model group using a 1mL syringe; the needle was slowly withdrawn after being stopped for 30s-1min; local pressure was applied for 1min. After the experimental operation was completed, the rats were returned to their original cages for continued feeding after they woke up.

On the 8th day after surgery (after modeling), the mechanical pain threshold and thermal pain threshold of the rats in the modeling group were measured (same as in Experimental Example 1), and the rats that failed the modeling were eliminated (see Table 5).

Table 5: Effects of Walker256 cell inoculation on pain threshold in rats

The results showed that after the Walker256 cells were used to establish the model, the mechanical pain threshold of the rats was reduced and the thermal pain threshold was shortened, indicating that the skin cancer pain model of the rats was established. A total of 48 rats were successfully established.

42 rats were selected from the modeling group and divided into 7 groups, 6 rats in each group, including model control group, pregabalin treatment group (P group), duloxetine treatment group (D group), combination medication group A (P/D-A group), combination medication group B (P/D-B group), combination medication group C (P/D-C group), and combination medication group D (P/D-D group); the dosage of each group is as follows:

Normal control group: an equal volume of distilled water was given by gavage;

Model control group: an equal volume of distilled water was given by gavage;

Pregabalin treatment group (P group): 60 mg/kg of pregabalin was administered by gavage;

Duloxetine treatment group (Group D): 24 mg/kg duloxetine was administered by gavage;

Combined medication group A (P/D-A group): 60 mg/kg of pregabalin and 12 mg/kg of duloxetine were administered by gavage;

Combined medication group B (P/D-B group): 60 mg/kg of pregabalin and 16 mg/kg of duloxetine were administered by gavage;

Combined medication group C (P/D-C group): 60 mg/kg of pregabalin and 20 mg/kg of duloxetine were administered by gavage;

Combined medication group D (P/D-D group): 60 mg/kg of pregabalin and 24 mg/kg of duloxetine were administered by gavage.

Each medication group used the corresponding part of the drug granules in Example 1 (pregabalin granules, duloxetine enteric-coated pellets, pregabalin and duloxetine compound granules) and mixed them into a paste with an appropriate amount of distilled water, and administered the drug by gavage once a day. After that (i.e. starting from the 8th day after modeling), the drug was administered continuously for 2 weeks and 4 weeks. The mechanical pain threshold and thermal pain threshold of the rats in each drug group were measured 3 hours after each administration. The results are shown in Table 6.

Table 6: Effect of the pharmaceutical composition of the present invention on pain in cancer rats

Note: ☆Compared with normal control group, P＜0.05; #Compared with model control group, P＜0.05;

*P value compared with group P < 0.05; △P value compared with group D < 0.05;

§Compared with P/D-A group, P＜0.05; ○Compared with P/D-B group, P＜0.05; ※Compared with P/D-C group, P＜0.05.

The experimental results show that:

(1) Compared with the normal group, the results of the model group showed that the mechanical pain threshold of rats was reduced and the thermal pain threshold was shortened after the Walker256 cell model was established, indicating that the rat skin cancer pain model was established.

(2) In the single-drug group: compared with the model group, the mechanical pain threshold and thermal pain threshold of the pregabalin treatment group (group P) and the duloxetine treatment group (group D) were significantly increased at each treatment point.

(3) In the combination drug group: compared with the model group, the mechanical pain threshold and thermal pain threshold of pregabalin combined with different doses of duloxetine at each treatment point were significantly increased.

Compared with the pregabalin treatment group (P group), the mechanical pain threshold and thermal pain threshold of the combination medication group A (P/DA group) were increased at each treatment point, but the difference was not significant; the mechanical pain threshold and thermal pain threshold of the combination medication group B (P/DB group) and the combination medication group C were increased. The mechanical pain threshold and thermal pain threshold of the two groups were significantly increased at each treatment point.

Among them, compared with the duloxetine treatment group (Group D), the mechanical pain threshold of the combination medication group A (P/D-A group) was significantly increased at each treatment observation point, but the difference in the thermal pain threshold was not significant; the mechanical pain threshold and thermal pain threshold of the combination medication group B (P/D-B group), the combination medication group C (P/D-C group), and the combination medication group D (P/D-D group) were significantly increased at each treatment observation point.

Among them, the mechanical pain threshold and thermal pain threshold of the P/D-C group were improved compared with the P/D-B group, but the improvement was not significant, P>0.05; however, the dosage of duloxetine in the P/D-D group increased from 20 mg/kg to 24 mg/kg compared with the P/D-C group, which had a significant effect on improving the mechanical pain threshold and thermal pain threshold, P<0.05.

Example 7

Effect of the pharmaceutical composition of the present invention on bone cancer pain

Eighty female SD rats were randomly divided into three groups: rats in group X served as normal blank controls (n=6), rats in group Y were injected with inactivated walker256 rat breast cancer cells into the left hind leg tibia (n=6), and rats in group Z were injected with walker256 rat breast cancer cells into the left hind leg tibia to establish a bone cancer pain model (n=68).

The skin of the left knee joint of rats in group Y and group Z was prepared, and a small incision of 1 cm was made in the upper tibia skin. After drilling holes in the left tibia, 15-20 μL of inactivated walker256 rat breast cancer cell solution and 1×10 ⁶ walker256 rat breast cancer cells (15-20 μL) were injected. Finally, the holes were sealed with medical bone wax and the wounds were sutured layer by layer.

The mechanical pain threshold was determined as in Example 4.

The mechanical pain threshold of SD rats in group X, group Y and group Z was measured 1 day before surgery, 7 days after surgery, 11 days after surgery and 14 days after surgery.

After modeling, it was found that there was no significant statistical difference in the mechanical pain thresholds of rats in group A and group B at each time point 11 days and 14 days after surgery (P>0.05); in group Z, rats with a mechanical pain threshold greater than 5g at 14 days were eliminated, and the mean mechanical pain threshold at each time point was calculated. The data showed that compared with rats in group A and group B, their mechanical pain thresholds were significantly lower, and there was no upward trend at 14 days. The results showed that the modeling was successful.

Experimental grouping and drug administration: 60 rats with successful modeling in group Z were selected, 6 of which were randomly selected as the model control group, and the remaining rats were divided into 9 groups: pregabalin treatment group, duloxetine treatment group A/group B/group C/group D, combined medication group A (P/DA group), combined medication group B (P/DB group), combined medication group C (P/DC group), and combined medication group D (P/DD group); the dosage of each group is as follows:

Model control group: an equal volume of distilled water was given by gavage;

Pregabalin treatment group (P group): 60 mg/kg of pregabalin was administered by gavage;

Duloxetine treatment group A (group D-A): 12 mg/kg duloxetine was administered by gavage;

Duloxetine-treated group B (group D-B): 16 mg/kg duloxetine was administered by gavage;

Duloxetine-treated group C (group D-C): 20 mg/kg duloxetine was administered by gavage;

Duloxetine-treated group D (group D-D): 24 mg/kg duloxetine was administered by gavage;

Combined medication group A (P/D-A group): 60 mg/kg of pregabalin and 12 mg/kg of duloxetine were administered by gavage;

Combined medication group B (P/D-B group): 60 mg/kg of pregabalin and 16 mg/kg of duloxetine were administered by gavage;

Combined medication group C (P/D-C group): 60 mg/kg of pregabalin and 20 mg/kg of duloxetine were administered by gavage;

Combined medication group D (P/D-D group): 60 mg/kg of pregabalin and 24 mg/kg of duloxetine were administered by oral gavage.

Each administration group used the corresponding part of the drug granules (pregabalin granules, duloxetine enteric-coated pellets, pregabalin duloxetine compound granules) in Example 1 and mixed them into a paste with an appropriate amount of distilled water, and administered by gavage once a day. Administration began 14 days after the injection of the breast cancer cell solution, and continued for 2 and 3 weeks. After 3 hours of administration, the mechanical pain threshold of the rats in each administration group was measured.

4. Data processing

SPSS 22.0 statistical software was used, and the calculated data were expressed as mean ± standard deviation. Variance analysis was used to compare multiple groups of data, and t-test was used to compare between groups. P < 0.05 was considered statistically significant.

5. Test results

The measurement results of each group are shown in Table 7.

Table 7: Effects of the pharmaceutical composition of the present invention on bone cancer pain model rats

Note: #Compared with the model control group, P < 0.05; *Compared with the P group, P value < 0.05;

△P/D-A group compared with D-A group, P＜0.05;▲P/D-B group compared with D-B group, P＜0.05;◆P/D-C group compared with D-C group, P＜0.05;★P/D-D group compared with D-D group, P＜0.05;

§Compared with P/D-A group, P＜0.05; ○Compared with P/D-B group, P＜0.05; ※Compared with P/D-C group, P＜0.05.

The experimental results show that:

(1) In the single-drug group: Compared with the model group, the thermal pain threshold of the pregabalin-treated group (P group) in the first week of treatment and the mechanical pain threshold in the second week of treatment were significantly increased, P < 0.05. Compared with the model group, the mechanical pain threshold of the slightly lower dose of duloxetine-treated group A (D-A group), group B (D-B group) and group C (D-C group) at each treatment point was increased, but the difference was not significant; compared with the model group, the mechanical pain threshold of the slightly higher dose of duloxetine-treated group D (D-D group) was significantly increased, P < 0.05.

(2) In the combination drug group: compared with the model group, the mechanical pain threshold of pregabalin combined with different doses of duloxetine at each treatment point was significantly increased, P < 0.05.

Among them, compared with the pregabalin treatment group (P group), the mechanical pain threshold of the combination medication group A (P/D-A group) at each treatment observation point was increased, but the difference was not significant; the mechanical pain threshold of the combination medication group B (P/D-B group), the combination medication group C (P/D-C group), and the combination medication group D (P/D-D group) at each treatment observation point was significantly increased, P < 0.05.

Compared with the duloxetine monotherapy group at the same dosage, the mechanical pain threshold of the combination therapy was significantly increased at each treatment point, P < 0.05.

The mechanical pain threshold increased significantly from the P/D-B group to the P/D-C group, P < 0.05; however, the increase in the mechanical pain threshold in the P/D-D group was significantly reduced when the duloxetine dosage increased from 20 mg/kg to 24 mg/kg compared with the P/D-C group, and the difference was not significant.

Example 8

Effect of the composition of the present invention on cancer pain induced by paclitaxel, a chemotherapy drug

70 male SD rats weighing 180-200 g were randomly divided into 2 groups: the rats in group X were intraperitoneally injected with blank solution as a blank control group (n=6), and the rats in group Y were injected with paclitaxel to establish a chemotherapy drug-induced cancer pain model group (n=64).

In group Y, paclitaxel was dissolved in DMSO and Tween 80 and sterile water were added to adjust the concentration to 2 mg/ml of paclitaxel solution, and 2 mg/kg of paclitaxel was intraperitoneally injected on days 0, 2, 4, and 6. In group X, DMSO, Tween 80, and sterile water were used in the paclitaxel solution of group Y to prepare blank solution, and 0.2 ml of blank solution was intraperitoneally injected on days 0, 2, 4, and 6.

The mechanical pain threshold of rats was measured before modeling (D0), on the 7th day (D7), on the 14th day (D14), and on the 21st day (D21). The determination method of the mechanical pain threshold was the same as in Example 4. The determination results of each group of rats showed that at D7, D14, and D21, the mechanical pain threshold of group Y was significantly lower than that of group X, and there was no upward trend until D21. The results showed that the modeling was successful.

Experimental grouping and dosing: 60 rats with successful modeling in group Y were randomly selected as the model control group, and the remaining rats were divided into 9 groups. The grouping and dosing scheme were referred to Example 7. Dosing began 21 days after the injection of paclitaxel solution, and continued for 2 and 3 weeks. The mechanical pain threshold of the rats in each dosing group was measured 3 hours after dosing.

4. Data processing

SPSS 22.0 statistical software was used, and the calculated data were expressed as mean ± standard deviation. Variance analysis was used to compare multiple groups of data, and t-test was used to compare between groups. P < 0.05 was considered statistically significant.

5. Test results

The measurement results of each group are shown in Table 8.

Table 8: Effects of the pharmaceutical composition of the present invention on rats with cancer pain model induced by paclitaxel, a chemotherapy drug

Note: #Compared with the model control group, P < 0.05; *Compared with the P group, P value < 0.05;

△P/DA group compared with DA group, P＜0.05;▲P/DB group compared with DB group, P＜0.05;◆P/DC group compared with DC group, P＜0.05;★P/DD group compared with DD group, P＜0.05;

§Compared with P/D-A group, P＜0.05; ○Compared with P/D-B group, P＜0.05; ※Compared with P/D-C group, P＜0.05.

The experimental results show that:

(1) In the single-drug group: Compared with the model group, the thermal pain threshold of the pregabalin-treated group (P group) at the second week of treatment and the mechanical pain threshold at the third week of treatment were significantly increased, P < 0.05. Compared with the model group, the mechanical pain threshold of the slightly lower dose of duloxetine-treated group A (D-A group) and group B (D-B group) at each treatment point was increased, but the difference was not significant; compared with the model group, the mechanical pain threshold of the slightly higher dose of duloxetine-treated group C (D-C group) and group D (D-D group) was significantly increased, P < 0.05.

(2) In the combination drug groups: compared with the model group, except for the P/D-A group, the mechanical pain thresholds of the other pregabalin and duloxetine dosage combination groups at each treatment observation point were significantly increased, P < 0.05.

Among them, compared with the pregabalin treatment group (P group), the mechanical pain threshold of the combination medication group A (P/D-A group) at each treatment observation point was slightly increased, but the difference was not significant; the mechanical pain threshold of the combination medication group B (P/D-B group), the combination medication group C (P/D-C group), and the combination medication group D (P/D-D group) at each treatment observation point was significantly increased, P < 0.05.

Compared with the duloxetine monotherapy group at the same dose, the mechanical pain threshold of the combination therapy was significantly improved at each treatment point except the P/D-A group (P < 0.05).

The mechanical pain threshold increased significantly from the P/D-B group to the P/D-C group, P < 0.05; however, the increase in the mechanical pain threshold in the P/D-D group was significantly reduced when the duloxetine dosage increased from 20 mg/kg to 24 mg/kg compared with the P/D-C group, and the difference was not significant.

Embodiment 9

The single component of pregabalin in Example 1 (Group A) and the pregabalin-duloxetine compound granules (Group B1, Group B2, Group B3 and Group B4) in Example 1 were used for pharmacokinetic study in animals. At the same time, the effect of twice-daily administration (Group A-BID, Group B1-BID, Group B2-BID, Group B3-BID and Group B4-BID) on the pharmacokinetic study of pregabalin-duloxetine compound granules in animals was further investigated.

SD rats with standard body weight (about 220g-250g) were selected as test subjects, with 6 rats in each group, and the drug was administered by gavage.

The following experimental groups were given the drug once a day. The dose of group A was 60 mg/kg of pregabalin, the doses of group B1 were 60 mg/kg and 12 mg/kg of pregabalin and duloxetine, respectively, the doses of group B2 were 60 mg/kg and 16 mg/kg of pregabalin and duloxetine, respectively, the doses of group B3 were 60 mg/kg and 20 mg/kg of pregabalin and duloxetine, respectively, and the doses of group B4 were 60 mg/kg and 24 mg/kg of pregabalin and duloxetine, respectively.

The following experimental groups were given medication once every 12 hours. The dosage of each medication was as follows: the dosage of pregabalin in the A-BID group was 30 mg/kg; the dosage of pregabalin and duloxetine in the B1-BID group was 30 mg/kg and 6 mg/kg, respectively; the dosage of pregabalin and duloxetine in the B2-BID group was In the B3-BID group, pregabalin and duloxetine were 30 mg/kg and 8 mg/kg, respectively; in the B3-BID group, pregabalin and duloxetine were 30 mg/kg and 10 mg/kg, respectively;

In the B4-BID group, pregabalin and duloxetine were 30 mg/kg and 12 mg/kg, respectively.

The rat tail venous blood was collected at different time points within 24 hours after administration to detect the content of pregabalin, and AUC _0-24 was calculated based on the drug-time curve. The obtained pharmacokinetic parameters are shown in Table 9.

Table 9: Pharmacokinetic parameters of pregabalin in different experimental groups

The results showed that when the dosage of pregabalin was consistent in each experimental group and the administration was once a day, the area under the concentration-time curve (AUC _0-24 ) of pregabalin in group A, group B1, group B2, group B3 and group B4 were 0.946×10 ⁴ h.ng/mL, 1.112× ^{10 4} h.ng/mL, 1.521 ^{×10 4} h.ng/mL, 1.853×10 4 h.ng/mL, 1.963× ^{10 4} h.ng/mL, among which the exposure of pregabalin in group B1, group B2, group B3 and group B4 was 118%, 161%, 196% and 208% of that in group A, respectively, indicating that the pharmacokinetics of pregabalin in group B2, group B3 and group B4 were significantly affected by the combination with duloxetine, the metabolism rate was slowed down and the bioavailability was improved, while the effect on group B1 was smaller, further indicating that the combination of pregabalin and duloxetine in a certain proportion affects the metabolism of pregabalin. It is proved that when the ratio of pregabalin to duloxetine per kilogram of body weight of rats is 60mg:16mg~24mg, the bioavailability of pregabalin can be greatly improved.

It can also be seen from Table 9 that, in the combination of pregabalin and different doses of duloxetine in each test group, when the total daily dosage is the same, the AUC _0-24 of pregabalin administered twice a day is greater than that administered once a day. This indicates that the bioavailability of pregabalin in the combination preparation of pregabalin and duloxetine is greater when administered twice a day than when administered once a day.

Example 10

Based on Example 4, the effects of the pharmaceutical composition of the present invention, pregabalin and duloxetine, administered once a day (QD) and twice a day (BID), on diabetic peripheral neuropathy pain model rats were studied.

1. Experimental animals and modeling reagents

Same as Example 4.

2. Animal modeling

The modeling was carried out in the same manner as in Example 4.

3. Trial Grouping and Dosing

Thirty rats with successful modeling were selected, and 6 were randomly selected as the model control group. The remaining rats were divided into 4 groups: group A administered once a day (QD-A group), group B administered once a day (QD-B group), group A administered twice a day (BID-A group), and group B administered twice a day (BID-B group); the dosage of each group is as follows:

Model control group: an equal volume of distilled water was given by gavage;

Group A, which was given once a day (QD-A group): 60 mg/kg of pregabalin and 16 mg/kg of duloxetine were given by gavage each time;

Group B was given medication once a day (QD-B group): 60 mg/kg of pregabalin and 24 mg/kg of duloxetine were given by oral gavage each time.

Group A was given twice a day (BID-A): 30 mg/kg of pregabalin and 8 mg/kg of duloxetine were given by gavage each time;

Group B was given medication twice a day (BID-B group): 30 mg/kg of pregabalin and 12 mg/kg of duloxetine were administered by oral gavage.

Each administration group used the corresponding part of the drug granules (pregabalin granules, duloxetine enteric-coated pellets, pregabalin duloxetine compound granules) in Example 1 and mixed them into a paste with an appropriate amount of distilled water, and administered by gavage once a day or twice a day (12 hours apart). Administration began 7 days after the injection of STZ solution, and continued for 2 weeks and 4 weeks. The mechanical pain threshold and thermal pain threshold of rats in each administration group were measured 3 hours and 15 hours after the first administration on the same day.

4. Data processing

SPSS 22.0 statistical software was used, and the calculated data were expressed as mean ± standard deviation. Variance analysis was used to compare multiple groups of data, and t-test was used to compare between groups. P < 0.05 was considered statistically significant.

5. Test results

The measurement results of each group are shown in Table 10.

Table 10: Effects of the pharmaceutical composition of the present invention on diabetic peripheral neuropathic pain model rats

Note: #P＜0.05 compared with the model control group; *P＜0.05 compared with the BID-A group and the QD-A group;

▲Comparison between BID-B group and QD-B group, P＜0.05.

The experimental results show that:

(1) The mechanical pain threshold and thermal pain threshold of each dosing group 3 hours after the first administration at 2 weeks and 4 weeks of administration were significantly decreased compared with the model control group (p < 0.05), but the data between the QD-A group and the BID-A group, and between the QD-B group and the BID-A group were basically similar, indicating that within the total dosage within the experimental range, the analgesic efficacy of different dosing methods of once a day and twice a day was not much different 3 hours after administration.

(2) Compared with the QD-A group, the BID-A group had higher mean mechanical pain threshold and thermal pain threshold 15 hours after administration at 2 weeks and 4 weeks, and most of the data were significantly different (p < 0.05); the same situation was observed between the QD-B group and the BID-A group, indicating that within the total dosage within the experimental range, the analgesic efficacy of different administration methods at 15 hours after administration was better when the drug was administered twice a day than once a day. This may be related to the fact that the twice-a-day administration method can maintain a higher blood drug concentration.

Embodiment 11

Based on Example 6, the effects of the pharmaceutical composition of the present invention, pregabalin and duloxetine, administered once a day (QD) and twice a day (BID), on skin cancer pain model rats were studied.

1. The selection of experimental animals, modeling reagents and modeling operations are the same as those in Example 6.

2. Trial Grouping and Dosing

Thirty rats with successful modeling were selected, and 6 were randomly selected as the model control group. The remaining rats were divided into 4 groups: group A administered once a day (QD-A group), group B administered once a day (QD-B group), group A administered twice a day (BID-A group), and group B administered twice a day (BID-B group); the dosage of each group is as follows:

Model control group: an equal volume of distilled water was given by gavage;

Group A, which was given once a day (QD-A group): 60 mg/kg of pregabalin and 16 mg/kg of duloxetine were given by gavage each time;

Group B was given medication once a day (QD-B group): 60 mg/kg of pregabalin and 24 mg/kg of duloxetine were given by oral gavage each time.

Group A was given twice a day (BID-A): 30 mg/kg of pregabalin and 8 mg/kg of duloxetine were given by gavage each time;

Group B was given medication twice a day (BID-B group): 30 mg/kg of pregabalin and 12 mg/kg of duloxetine were administered by oral gavage.

Each administration group used the corresponding part of the drug granules (pregabalin granules, duloxetine enteric-coated pellets, pregabalin duloxetine compound granules) in Example 1 and mixed them into a paste with an appropriate amount of distilled water, and administered by gavage once a day or twice a day (12 hours apart). After the model was successfully established (i.e., starting from the 8th day after model establishment), the administration was continued for 2 weeks and 4 weeks, and the mechanical pain threshold and thermal pain threshold of the rats in each administration group were measured 3 hours and 15 hours after the first administration on the same day.

4. Data processing

SPSS 22.0 statistical software was used, and the calculated data were expressed as mean ± standard deviation. Variance analysis was used to compare multiple groups of data, and t-test was used to compare between groups. P < 0.05 was considered statistically significant.

5. Test results

The measurement results of each group are shown in Table 11.

Table 11: Effect of the pharmaceutical composition of the present invention on pain in cancer rats

Note: #P＜0.05 compared with the model control group; *P＜0.05 compared with the BID-A group and the QD-A group;

▲Comparison between BID-B group and QD-B group, P＜0.05.

The experimental results show that:

(1) The mechanical pain threshold and thermal pain threshold of each dosing group 3 hours after the first administration on the day of 2 weeks and 4 weeks of administration were significantly decreased compared with the model control group (p < 0.05), but the data between the QD-A group and the BID-A group, and between the QD-B group and the BID-A group were basically similar, indicating that within the total dosage within the experimental range, the analgesic efficacy of different dosing methods of once a day and twice a day was not much different 3 hours after administration.

(2) Compared with the QD-A group, the BID-A group had higher mean mechanical pain threshold and thermal pain threshold 15 hours after administration at 2 weeks and 4 weeks, and most of the data were significantly different (p < 0.05); the same situation was observed between the QD-B group and the BID-A group, indicating that within the total dosage within the experimental range, the analgesic efficacy of different administration methods at 15 hours after administration was better when the drug was administered twice a day than once a day. This may be related to the fact that the administration method of twice a day can maintain a higher blood drug concentration.

The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims (22)

A composition comprises a pregabalin part and a duloxetine part; the pregabalin part comprises pregabalin, the duloxetine part comprises duloxetine, and the mass ratio of the pregabalin to the duloxetine is (2.50-3.75):1. The composition according to claim 1, characterized in that the mass ratio of pregabalin to duloxetine is (3.00-3.75):1. The composition according to claim 1, characterized in that: the composition contains pregabalin (75-150) mg and duloxetine (20-50) mg. The composition according to claim 1, characterized in that: the composition contains 150 mg of pregabalin and (40-50) mg of duloxetine. The composition according to claim 1, characterized in that: the composition contains 75 mg of pregabalin and (20-25) mg of duloxetine. The composition according to claim 1 is characterized in that the composition is in the form of granules, suspensions, tablets or capsules. The composition according to claim 1, characterized in that: in the composition, the pregabalin portion exists in the form of a gastric soluble rapid-release preparation. The composition according to claim 7, characterized in that the preparation form of the pregabalin portion is granules or tablets. The composition according to claim 1 is characterized in that: in the composition, the duloxetine portion exists in the form of a gastric-soluble immediate-release preparation or an enteric-coated preparation. The composition according to claim 9, characterized in that: When the duloxetine portion is in the form of a gastric-soluble immediate-release preparation, the preparation form of the duloxetine portion is granules or tablets; When the duloxetine portion is in the form of an enteric-coated preparation, the preparation form of the duloxetine is enteric-coated granules, enteric-coated micropellets, enteric-coated microtablets or enteric-coated tablets. The composition according to any one of claims 6 to 10, characterized in that: when the pregabalin portion is in the form of granules and the duloxetine portion is in the form of granules, enteric-coated granules, or enteric-coated pellets, the pregabalin portion and the duloxetine portion are mixed evenly and then filled into capsules or pressed into tablets; or the pregabalin portion and the duloxetine portion are separately filled into the same capsule; or a double-layer tablet is pressed, with the pregabalin portion and the duloxetine portion being in different layers. The composition according to any one of claims 6 to 10, characterized in that: when the pregabalin portion is in the form of granules and the duloxetine portion is in the form of enteric-coated microtablets or enteric-coated tablets, or when the pregabalin portion is in the form of tablets and the duloxetine portion is in the form of When the duloxetine portion is enteric-coated granules or enteric-coated micropellets, the pregabalin portion and the duloxetine portion are separately filled in the same capsule. Use of the composition according to any one of claims 1 to 12 in the preparation of a drug for treating neuropathic pain. Use of the composition according to any one of claims 1 to 12 in the preparation of a medicament for treating diabetic peripheral neuropathy. The use of the composition according to any one of claims 1 to 12 in the preparation of a drug for treating cancer pain, characterized in that the cancer pain refers to the pain caused by various solid tumors compressing or infiltrating nearby body parts, or the pain caused by solid tumor treatment and diagnostic procedures, and also includes discomfort caused by changes in the skin, nerves and other hormone imbalances or immune responses. The use according to claim 15 is characterized in that: the solid tumors include colorectal cancer, ovarian cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, breast cancer, lung cancer, uterine cancer, cancer of the anal region, stomach cancer, testicular cancer, cancer of the fallopian tube, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, central nervous system cancer, primary CNS lymphoma, spinal tumor, brain stem glioma, pituitary adenoma, hematological malignancies, and any combination of the above cancers and treatment of metastatic cancer. A combined medication method comprises administering pregabalin and duloxetine to a patient, the administration being 1-3 times a day, preferably 2 times a day; the mass ratio of pregabalin to duloxetine is (2.50-3.75):1, preferably (3.00-3.75):1. The combined drug method according to claim 17, characterized in that: when the total dosage is the same, within 24 hours of administration to rats, the AUC _0-24 of pregabalin under twice-a-day administration is more than 110% of that under once-a-day administration, preferably (110-130)%. The combined drug method according to claim 17, characterized in that: pregabalin (75-150) mg and duloxetine (20-50) mg are administered to the patient in combination, 1-3 times a day, preferably twice a day. The combined drug method according to claim 19, characterized in that: 75 mg of pregabalin and 20-25 mg of duloxetine are administered to the patient in combination, 1-3 times a day, preferably twice a day. The combined drug method according to claim 19, characterized in that: 150 mg of pregabalin and 40-50 mg of duloxetine are administered to the patient in combination, 1-3 times a day, preferably twice a day. A method for treating neuropathic pain and/or diabetic peripheral neuropathy, comprising administering the composition of any one of claims 1 to 12 to a patient, preferably 1 to 3 times a day.