CN115364101A

CN115364101A - Method and pharmaceutical composition for treating diabetic peripheral neuropathy

Info

Publication number: CN115364101A
Application number: CN202110560773.5A
Authority: CN
Inventors: 不公告发明人
Original assignee: Chengdu Wending Technology Development Co ltd
Current assignee: Chengdu Wending Technology Development Co ltd
Priority date: 2021-05-21
Filing date: 2021-05-21
Publication date: 2022-11-22

Abstract

The present invention relates to a method and a pharmaceutical composition for treating diabetic peripheral neuropathy. The method and the pharmaceutical composition of the invention relate to the combined use of doxazosin, pramipexole and metoprolol (or pharmaceutically acceptable salts thereof), which can obviously improve the nerve conduction velocity, particularly the sensory nerve conduction velocity of the sural nerve, thereby effectively controlling and relieving the development of diabetic peripheral neuropathy and neuropathic pain.

Description

Method and pharmaceutical composition for treating diabetic peripheral neuropathy

Technical Field

The invention relates to the field of disease treatment and medicine, in particular to treatment of diabetic peripheral neuropathy.

Background

Diabetes is an increasingly prevalent disease with a significant impact on health and quality of life. According to estimates, the population of diabetics aged between 20 and 79 years worldwide will reach 6.42 billion in 2040 years with a prevalence of about 8.8%. Up to 50% of Diabetic patients develop Diabetic Peripheral Neuropathy (DPN), a chronic microvascular complication of diabetes with a very high incidence and high disability rate, which can cause severe Neuropathy such as numbness of limbs, hyperalgesia, foot ulcer, gangrene and the like in early stage, and the amputation rate of the patients can exceed 80%, wherein at least about 20% of the patients develop neuropathic Pain, namely Diabetic Peripheral neuropathic Pain (DPNP, also called Diabetic Peripheral Neuropathy). DPN patients require higher care costs. DPN has resulted in a tremendous physiological, psychological and socio-economic burden on patients. After DPN has occurred, no drug is currently available to reverse the progression of Neuropathy and clinical management is very difficult (Zakin E, abrams R, simpson D M. Diabetic neuropathies [ J ]. Sensiars in Neurology,2019,39 (05): 560-569).

Neuroelectrophysiology (EMG) is widely used to evaluate various neurological dysfunctions in DPN patients, and the EMG can make objective and quantitative diagnoses of neurological functions. Neuroelectrophysiology studies have demonstrated a slower sensory nerve conduction rate in 5-10 year diabetic patients. Abnormal degree and abnormal rate of sensory nerve conduction velocity (SCV) in patients with DPN are usually more severe than abnormal motor nerve conduction velocity (MCV), and the patient's neurological dysfunction is that the lower limbs are more severe than the upper limbs.

Because of the non-specific treatment medicines for the peripheral nerve of diabetes, the related symptoms can be relieved only by blood sugar control, nerve repair and other neurotrophic factor auxiliary treatments at present. The polyol pathway is the most common target for the treatment of diabetic neuropathy. Epalrestat (Epalrestat) is a commonly used Clinical DPN therapeutic drug, is a novel Aldose Reductase Inhibitor of polyol pathway, can improve symptoms of Diabetic Peripheral Neuropathy and Peripheral nerve conduction velocity, but the use of Epalrestat can cause some side Effects, especially can cause allergy, liver and kidney function and digestive system adverse reaction after long-Term use, and lead to poor therapeutic effect and can not reverse the progress of Neuropathy (Hotta, nigishi, akanuma et al Long-Term Clinical Effects of Epalrestat, an Aldose Reductase Inhibitor, on Diabetes Peripheral Neuropathy [ J ]. Diabetes Care, 2006). Therefore, the existing therapeutic means and drugs are still poor in the aspects of prevention and control of DPN development and effectiveness, and new drugs and therapies are urgently needed for prevention and treatment of diabetic peripheral neuropathy.

Disclosure of Invention

It is therefore an object of the present invention to provide an alternative treatment method and medicament for the treatment of diabetic peripheral neuropathy.

A first aspect of the present invention relates to a pharmaceutical composition for treating diabetic peripheral neuropathy, which comprises doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole or a pharmaceutically acceptable salt thereof, and metoprolol (or a pharmaceutically acceptable salt thereof) as active ingredients.

A second aspect of the present invention relates to the use of a combination of doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole (or a pharmaceutically acceptable salt thereof) and metoprolol (or a pharmaceutically acceptable salt thereof) for the preparation of a pharmaceutical composition for the treatment of diabetic peripheral neuropathy in a subject in need thereof.

A third aspect of the present invention relates to the use of a combination of doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole (or a pharmaceutically acceptable salt thereof) and metoprolol (or a pharmaceutically acceptable salt thereof) for the treatment of diabetic peripheral neuropathy in a subject in need thereof.

A fourth aspect of the present invention relates to a method of treating diabetic peripheral neuropathy, the method comprising administering to a subject in need thereof a therapeutically effective amount of doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole (or a pharmaceutically acceptable salt thereof), and metoprolol (or a pharmaceutically acceptable salt thereof).

The inventors simulated human DPN patients with a rhesus monkey with idiopathic diabetic peripheral neuropathy as an animal model and surprisingly found that the use of a combination of doxazosin, pramipexole and metoprolol significantly improved nerve conduction velocity in the animal model, controlled alleviation of DPN progression, and showed good safety during administration; the curative effect intensity of the medicine composition is equivalent to that of the first-line DPN medicine epalrestat. Thus, the pharmaceutical combination of the present invention represents a novel therapy for diabetic peripheral neuropathy, including diabetic peripheral neuropathic pain.

Detailed Description

The present application relates to combinations of multiple GPCR agents and corresponding combination therapy methods. G protein (guanine nucleotide-binding protein) coupled receptors (GPCRs) are a large class of transmembrane proteins that regulate intracellular signaling and are essential for cellular homeostasis. Research in the literature indicates that GPCR signaling pathways are related to diabetes-induced peroxide production (Du, Y., et al., adrenergic and serotonin receptors extract/superoxide generation in diabetes mellitus: relationship to vascular injury and lipid metabolism.2015.29 (5): p.2194), and oxidative stress causes vascular injury and leads to endogenous hypoxia injury and reduced neurological function, which are important mechanisms in the development of DPN, and suggests that DPN development may be related to GPCR signaling pathways, so that GPCR drugs may regulate diabetes-induced peroxide production and improve microvascular injury, thereby potentially treating DPN. However, current research is limited to the pathogenesis level, and GPCR-based drugs cover hundreds of different substances, from which screening of clinically effective drugs for DPN is difficult.

In addition, most of the DPN animal models commonly used in the current research are rodents, and for example, a plurality of rat and mouse models are established by technologies such as ischemia reperfusion, a neurotomy method, a single STZ (streptozotocin) induction method, transgenosis and the like. However, the pathogenesis of the DPN is complex and the involvement of the DPN is many, so that the damage of the nerve function in the animal models hardly accurately represents the disease course of the DPN of the patients. Rhesus monkeys are very similar to humans in physiological, biochemical and systemic biology, and it has been found that the disease profile of idiopathic DPN rhesus monkeys is very similar to clinical human DPN patients. In 1989 Cornblath et al (Cornblath D R, hillman M A, striffler J S, et al. Peripheral neuropathy in Diabetes mongytes [ J ]. Diabetes,1989,38 (11): 1365-1370.; cornblath D R, dellon A L, mackinnon S E.Spontaneous Diabetes mellitus in a rhesus monkey model: neurophysiological students [ J ]. Muscle & Nerve,1989,12 (3): 233-235.) have reported spontaneous Diabetes DPN rhesus models which exhibit significant reduction in motor and sensory Nerve conduction velocities compared to non-Diabetes, and have pathogenesis and clinical features similar to human patients. Therefore, the spontaneous diabetes DPN rhesus monkey model can provide an important research means for DPN research and new drug development and provide a more accurate conclusion relative to other animal models.

Considering the limitation of rodent animal models in the study of human diabetic peripheral neuropathy, the inventors have conducted extensive and intensive studies and extensive screening of numerous GPCR-based drugs in simulating human patients with idiopathic type 2 diabetes combined with peripheral neuropathy as an animal model. The results unexpectedly found that certain GPCR-based agents when combined together show significant efficacy for DPN. In particular, it has been found that the use of a combination of doxazosin, pramipexole and metoprolol enables a significant improvement of nerve conduction velocity, in particular gastrocnemius nerve sensory nerve conduction velocity, in a spontaneous DPN rhesus model, enables control and alleviation of DPN progression, and shows good safety during administration, and surprisingly has been found to be comparable in therapeutic efficacy to the first line commonly used clinical drug epalrestat. Based on this, the application discloses a pharmaceutical composition for treating diabetic peripheral neuropathy and medical use thereof.

1. Pharmaceutical composition

The first aspect of the present invention provides a pharmaceutical composition for treating diabetic peripheral neuropathy, which comprises doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole (or a pharmaceutically acceptable salt thereof), and metoprolol (or a pharmaceutically acceptable salt thereof) as active ingredients.

Certain terms are used throughout the description, examples, and claims of this application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The terms "comprising," "including," and "having" are used in an inclusive, open sense and mean that additional elements may be included in addition to the elements specified. The terms "such as", "for example" as used herein are non-limiting and are used for illustrative purposes only. "include" and "include, but are not limited to" are used interchangeably.

The term "or," as used herein, should be understood to mean "and/or" unless the context clearly indicates otherwise.

The term "treating" refers to inhibiting a disease, disorder, and/or symptom in a subject, e.g., arresting its progression; and relieving the disease, disorder and/or symptom, e.g., causing regression of the disease, disorder and/or symptom. Treating a disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected. In particular, "treatment" in this application also covers the meaning of reducing the risk of developing a disease by administering a drug, i.e. "treatment" includes both pre-morbid prevention and post-morbid remission, inhibition and cure of the disease.

The term "pharmaceutical composition" refers to a combination of substances having a particular medical or biological use, typically intended to have a therapeutic or prophylactic effect on a particular disease after being administered to a subject. The pharmaceutical compositions may comprise the specified active ingredient (biologically active substance) alone or may be provided for various uses together with conventional pharmaceutically acceptable carriers. The term "composition" in the present application should be interpreted broadly. As an implementation of the pharmaceutical composition of the invention, it can be provided, for example, by blending together the specified active ingredients (and optionally a pharmaceutically acceptable carrier) in the form of an indistinguishable mixture of the individual ingredients. As another implementation of the pharmaceutical composition of the present invention, it is also possible to individually package each of the specified active ingredients in small portions, and to hold these small individual packages together in a larger container to provide the "pharmaceutical composition" of the present invention.

The terms "active ingredient" and "biologically active substance" refer to molecules and other agents of biologically, physiologically, or pharmaceutically active substances that are effective in treating a disease or disorder (e.g., diabetic retinopathy) in a patient or subject. The term is used with respect to "pharmaceutically acceptable carrier", "excipient", "adjuvant", and like terms. "active ingredients" (or "biologically active substances") include, but are not limited to, pharmaceutically acceptable salts and prodrugs thereof. These agents may be acids, bases or salts; they may be neutral molecules, polar molecules or molecular complexes capable of hydrogen bonding; they may be prodrugs in the form of ethers, esters, amides, and the like, which are bioactivated when administered to a patient or subject.

As used herein, the term "Doxazosin" refers to the English name of Doxazosin (DOX for short) and the IUPAC name of (RS) -2- [4- (2, 3-dihydro-1, 4-benzodioxine-2-carbonyl) piperazin-1-yl]-6, 7-dimethylOxy-4-amine (formula C) ₂₃ H ₂₅ N ₅ O ₅ Molecular weight: 451.475 g/mol) of the following molecules:

. The term "doxazosin" also encompasses isotopically labeled compounds thereof, or optical isomers, geometric isomers, tautomers or isomer mixtures thereof, or prodrugs thereof (i.e., compounds which react in vivo to give the above-described molecules).

Doxazosin is a marketed drug approved by various major medical regulatory agencies (e.g., FDA), and is a selective α 1 receptor antagonist that inhibits the binding of norepinephrine (released from sympathetic nerve endings) to α -1 receptors on the membranes of vascular smooth muscle cells, and is commonly used to treat essential hypertension.

The pharmaceutical composition of the present invention may comprise doxazosin or a pharmaceutically acceptable salt thereof as an active ingredient. Most of the commercially available doxazosin is in the form of a salt, particularly a mesylate salt thereof, for example, doxazosin mesylate controlled release tablet (kuwa) available from pfeizu pharmaceutical company, doxazosin mesylate available from zenjamin pharmaceutical co.

The term "Pramipexole" as used herein refers to the English name Pramipexole (PMP for short) and the IUPAC name (S) -N6-propyl-4, 5,6, 7-tetrahydro-1, 3-benzothiazole-2, 6-diamine (formula C) ₁₀ H ₁₇ N ₃ S, molecular weight: 211.324 g/mol) of the following molecules:

. The term "pramipexole" also covers its isotopically labeled compounds, or its optical isomers, geometric isomers, tautomers or isomer mixtures, or prodrugs thereof (i.e., compounds which react in vivo to give the above-mentioned molecules).

Pramipexole, an antihistamine that is approved by various major medical regulatory agencies, such as the FDA, is used clinically primarily in the treatment of parkinson's disease, either alone (without levodopa) or in combination with levodopa, as a D2/D3 agonist of dopamine receptors. Pramipexole is also sometimes referred to in the literature as "mirapra" or "Mirapex", "Mirapexin", "Sifrol", etc.

The pharmaceutical composition of the present invention may contain pramipexole or a pharmaceutically acceptable salt thereof as an active ingredient. The commercially available pramipexole is often available in salt form, particularly as its hydrochloride salt, such as pramipexole dihydrochloride tablets (forest fortro) available from the brigrel invar pharmaceutical company.

The term "Metoprolol" as used herein refers to the English name Metoproll (MTP for short) and the IUPAC name (RS) -1- [4- (2-methoxyethyl) phenoxy]-3- [ (prop-2-yl) amino]Propan-2-ol (formula C) ₁₅ H ₂₅ NO ₃ 267.37 g/mol) of the following molecules:

. The term "metoprolol" also encompasses isotopically labeled compounds thereof, or optical isomers, geometric isomers, tautomers or isomer mixtures thereof, or prodrugs thereof (i.e., compounds that react in vivo to give the above-described molecules).

Metoprolol is a marketed drug approved by various major medical regulatory agencies (e.g., FDA), is a selective adrenergic beta 1 receptor blocker, and is commonly used for the treatment of hypertension and angina pectoris. Metoprolol or a pharmaceutically acceptable salt thereof may be included as an active ingredient in the pharmaceutical composition of the present invention. Most commercially available metoprolol is in the form of a salt, particularly a tartrate salt, such as metoprolol tartrate tablet (betalenk) available from astragal pharmaceuticals, inc., guangzhou, controlled release metoprolol tartrate tablet (licarin) available from pice pharmaceuticals, inc.

The active ingredients (doxazosin, pramipexole, metoprolol) contained in the compositions of the invention may be replaced by pharmaceutically acceptable salts thereof. The term "pharmaceutically acceptable salt" of a compound refers to a salt that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. As used herein, a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include, but are not limited to, inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, pyrophosphoric acid, hydrobromic acid, or nitric acid, and organic acids such as citric acid, fumaric acid, maleic acid, malic acid, ascorbic acid, succinic acid, tartaric acid, benzoic acid, acetic acid, methanesulfonic acid, ethanesulfonic acid, salicylic acid, stearic acid, benzenesulfonic acid, or p-toluenesulfonic acid. Pharmaceutically acceptable bases include alkali metal (e.g., sodium or potassium) and alkaline earth metal (e.g., calcium or magnesium) hydroxides, and organic bases such as alkyl, aryl or heterocyclic amines. For the avoidance of doubt, a "pharmaceutically acceptable salt" of an active ingredient as defined herein also includes a pharmaceutically acceptable salt formed from an isotopically-labelled compound of the active ingredient, or an optical isomer, geometric isomer, tautomer or mixture of isomers thereof, or prodrug thereof.

Pharmaceutically acceptable salts can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. In general, these salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two; generally, nonaqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. A list of salts can be found in Remington's Pharmaceutical Sciences 18 th edition (Mack Publishing Company, 1990). For example, salts can include, but are not limited to, hydrochloride, tartrate, mesylate, and the like, of the compounds of the invention.

It will be understood that all references to various active ingredients or pharmaceutically acceptable salts include solvent addition forms (solvates, e.g., hydrates, ethanol solvates, acetone solvates, etc.) of the same active ingredient or salt, or various crystalline forms (e.g., amorphous forms, polymorphs, etc.) thereof.

The content of doxazosin (or pharmaceutically acceptable salt thereof), pramipexole (or pharmaceutically acceptable salt thereof) and metoprolol (or pharmaceutically acceptable salt thereof) in the pharmaceutical composition of the invention can be adjusted according to actual needs. For example, the content or ratio of each drug in the pharmaceutical composition may be changed according to the administration mode (oral or injection, etc.) of the pharmaceutical composition.

In some embodiments, the pharmaceutical composition of the present invention generally comprises 0.5 to 45 parts by weight (preferably 1 to 10 parts by weight) of pramipexole or a pharmaceutically acceptable salt thereof, 5 to 160 parts by weight (preferably 10 to 80 parts by weight) of doxazosin or a pharmaceutically acceptable salt thereof, and 50 to 2000 parts by weight (preferably 50 to 1000 parts by weight) of metoprolol or a pharmaceutically acceptable salt thereof.

In the pharmaceutical composition of the present invention, the amount of pramipexole or a pharmaceutically acceptable salt thereof is usually 0.5 to 45 parts by weight, for example, it may be 0.5 to 42 parts by weight, 0.5 to 40 parts by weight, 0.5 to 35 parts by weight, 0.5 to 30 parts by weight, 0.5 to 25 parts by weight, 0.5 to 20 parts by weight, 0.5 to 15 parts by weight, 0.5 to 10 parts by weight, 1 to 45 parts by weight, 1 to 42 parts by weight, 1 to 40 parts by weight, 1 to 35 parts by weight, 1 to 30 parts by weight, 1 to 25 parts by weight, 1 to 20 parts by weight, 1 to 15 parts by weight, 1 to 10 parts by weight, 2 to 45 parts by weight, B2 to 40 parts by weight, 2 to 35 parts by weight, 2 to 30 parts by weight, 2 to 25 parts by weight, 2 to 20 parts by weight, 2 to 15 parts by weight, 2 to 10 parts by weight, 5 to 45 parts by weight, 5 to 40 parts by weight, 5 to 35 parts by weight, 5 to 30 parts by weight, 5 to 25 parts by weight, 5 to 20 parts by weight, 5 to 15 parts by weight, 5 to 10 parts by weight, 10 to 40 parts by weight, 10 to 30 parts by weight, 10 to 25 parts by weight, and the like. In a preferred embodiment of the present invention, the pramipexole or a pharmaceutically acceptable salt thereof is contained in an amount of 1 to 10 parts by weight.

In the pharmaceutical composition of the present invention, the amount of doxazosin or a pharmaceutically acceptable salt thereof is usually 5 to 160 parts by weight, for example, it may be 5 to 150 parts by weight, 5 to 130 parts by weight, 5 to 120 parts by weight, 5 to 100 parts by weight, 5 to 80 parts by weight, 5 to 60 parts by weight, 5 to 50 parts by weight, 5 to 40 parts by weight, 10 to 150 parts by weight, 10 to 130 parts by weight, 10 to 120 parts by weight, 10 to 100 parts by weight, 10 to 80 parts by weight, 10 to 60 parts by weight, 10 to 50 parts by weight, 10 to 40 parts by weight, 15 to 160 parts by weight, 15 to 150 parts by weight, 15 to 130 parts by weight, 15 to 120 parts by weight, 15 to 100 parts by weight, 15 to 80 parts by weight, 15 to 60 parts by weight, 15 to 50 parts by weight, 15 to 40 parts by weight, 20 to 160 parts by weight, 20 to 150 parts by weight, 20 to 130 parts by weight, 20 to 20 parts by weight, 20 to 40 parts by weight, 20 to 60 parts by weight, or the like. In a preferred embodiment of the present invention, the doxazosin or a pharmaceutically acceptable salt thereof is contained in an amount of 10 to 80 parts by weight.

In the pharmaceutical composition of the present invention, the amount of metoprolol or a pharmaceutically acceptable salt thereof is usually 25 parts by weight to 2000 parts by weight, for example, 25 to 1800 parts by weight, 25 to 25 parts by weight, 25 to 1500 parts by weight, 25 to 1300 parts by weight, 25 to 1200 parts by weight, 25 to 1000 parts by weight, 25 to 800 parts by weight, 25 to 600 parts by weight, 25 to 500 parts by weight, 25 to 400 parts by weight, 50 to 1800 parts by weight, 50 to 1600 parts by weight, 50 to 1500 parts by weight, 50 to 1300 parts by weight, 50 to 1200 parts by weight, 50 to 1000 parts by weight, 50 to 800 parts by weight, 50 to 600 parts by weight, 50 to 500 parts by weight, 50 to 400 parts by weight, 100 to 100 parts by weight, 100 to 1600 parts by weight, 100 to 1500 parts by weight, 100 to 1300 parts by weight, 1 to 1300 parts by weight, or more 100 to 1200 parts by weight, 100 to 1000 parts by weight, 100 to 800 parts by weight, 100 to 600 parts by weight, 100 to 500 parts by weight, 100 to 400 parts by weight, 150 to 2000 parts by weight, 150 to 1800 parts by weight, 150 to 1600 parts by weight, 150 to 1500 parts by weight, 150 to 1300 parts by weight, 150 to 1200 parts by weight, 150 to 1000 parts by weight, 150 to 800 parts by weight, 150 to 600 parts by weight, 200 to 2000 parts by weight, 200 to 1800 parts by weight, 200 to 1600 parts by weight, 200 to 1500 parts by weight, 200 to 1300 parts by weight, 200 to 1200 parts by weight, 200 to 1000 parts by weight, 200 to 800 parts by weight, 200 to 600 parts by weight, and the like. In a preferred embodiment of the present invention, metoprolol or a pharmaceutically acceptable salt thereof is contained in an amount of 50 parts by weight to 1000 parts by weight.

2. Pharmaceutical dosage form

In some embodiments, the pharmaceutical compositions of the present invention may be provided as a bulk drug substance (e.g., a homogeneous mixture or separate packages of the ingredients). In other embodiments, the pharmaceutical composition of the present invention may be formulated into various pharmaceutical dosage forms (formulations) by adding a pharmaceutically acceptable carrier as necessary. For this purpose, various liquid or solid fillers, diluents, excipients, solvents or encapsulating materials can be used as "pharmaceutically acceptable carriers". In the present application, the phrase "pharmaceutically acceptable" refers to compounds, compositions, polymers and other materials which are, within the scope of sound medical judgment, compatible with the other ingredients of the compositions of the present application and suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication. In certain preferred embodiments, the pharmaceutically acceptable carrier is pyrogen-free. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) Cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) maltodextrin; (6) gelatin; (7) talc powder; (8) auxiliary materials such as cocoa butter and suppository waxes; (9) Oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) Polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) ringer's solution; (19) ethanol; (20) phosphate buffer solution; (21) other non-toxic compatible substances used in pharmaceutical formulations.

The pharmaceutical composition of the present invention can be formulated as required into dosage forms suitable for oral, parenteral (including subcutaneous, intramuscular, cortical and intravenous), bronchial, nasal and the like routes of administration. Among them, preferably, the pharmaceutical composition of the present invention is formulated into a dosage form (preparation) suitable for oral administration.

If a solid carrier is used, the formulation may be tableted, placed in a hard gel capsule in powder or granule form, or in the form of a lozenge or troche. Solid carriers may include conventional excipients such as binders, fillers, tableting lubricants, disintegrants, wetting agents and the like. The tablets may be film coated by conventional techniques if desired. If a liquid carrier is used, the formulation may be in the form of a syrup, emulsion, soft gel capsule, sterile vehicle for injection, aqueous or non-aqueous liquid suspension, or may be in the form of a dry product for reconstitution with water or other suitable vehicle before use. Liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, wetting agents, non-aqueous vehicles including edible oils, preservatives, and flavoring and/or coloring agents. For parenteral administration, the carrier will typically comprise, at least in large part, sterile water, although saline solutions, dextrose solutions, and the like may also be used. Injectable suspensions may also be used, in which case conventional suspending agents may be used. Conventional preservatives, buffering agents and the like may also be added to the parenteral dosage form.

Dosage forms suitable for parenteral injection may include physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate).

These pharmaceutical dosage forms may also contain various excipients, for example, preservatives, wetting agents, emulsifying agents, and dispersing agents. Inhibition of the action of microorganisms can be ensured by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, and the like). Isotonic agents, for example, sugars, sodium chloride, and the like, may also be included. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is combined with at least one inert excipient (or carrier) (e.g., sodium citrate or dicalcium phosphate) which may also include: (a) Fillers or extenders (e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid); (b) Binders (e.g., carboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and gum arabic); (c) humectants (e.g., glycerol); (d) Disintegrants (e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain synthetic silicates, sodium carbonate); (e) solution retarding agents (e.g., paraffin); (f) absorption accelerators (e.g., quaternary ammonium compounds); (g) Wetting agents (e.g., cetyl alcohol and glycerol monostearate); (h) Adsorbents (e.g., kaolin and bentonite) and (i) lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate) or mixtures thereof.

Solid compositions of a similar type may also be employed as fillers in soft-filled and hard-filled gelatin capsules using, for example, lactose and high molecular weight polyethylene glycols and the like as excipients.

Solid dosage forms (e.g., tablets, dragees, capsules, pills, and granules) can be prepared using coatings and shells (e.g., enteric coatings and others known in the art). They may contain opacifying agents, or they may be of a composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active ingredient may also be in microencapsulated form, if appropriate with one or more of the abovementioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, dispersions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art (e.g., water or other solvents), solubilizing agents and emulsifiers (e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 butylene glycol, dimethylformamidinium), oils (in particular, cottonseed oil, groundnut oil, corn oil, olive oil, castor oil, sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, or mixtures of these substances, and the like.

In addition to these inert diluents, the pharmaceutical dosage forms can also include, for example, wetting agents, emulsifying and suspending agents, perfuming agents, flavoring agents, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, sorbitan esters, microcrystalline fibres, aluminium metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.

Pharmaceutical dosage forms of the invention also include ointments, powders, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any required preservatives, buffers, or propellants.

The amount of the active ingredient in the pharmaceutical composition and the pharmaceutical dosage form may be suitably determined by the skilled person as desired, e.g. each active ingredient is typically present in a therapeutically effective amount in the pharmaceutical composition or dosage form.

For example, the pharmaceutical composition of the present invention may be formulated into an oral dosage form (preferably an oral dosage form or a long-acting oral preparation 2 to 3 times daily), an intravenous injection dosage form, or an intramuscular injection dosage form.

In a preferred embodiment of the invention, the pharmaceutical composition of the invention is in a dosage form for oral administration. In a further preferred embodiment of the invention, the pharmaceutical composition of the invention is an oral dosage form 2 times daily, an oral dosage form 2 to 3 times daily or a long-acting sustained release oral formulation.

In a preferred embodiment of the invention, the pharmaceutical composition of the invention is in a dosage form for oral administration. In a further preferred embodiment of the invention, the pharmaceutical composition of the invention is a daily oral dosage form.

3. Pharmaceutical composition and use of pharmaceutical dosage form

It has been found that the pharmaceutical compositions and pharmaceutical dosage forms of the present invention can be used for the treatment of Diabetic Peripheral Neuropathy (DPN), including diabetic peripheral neuropathic pain.

Thus, a second aspect of the present invention relates to the use of a combination of doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole (or a pharmaceutically acceptable salt thereof) and metoprolol (or a pharmaceutically acceptable salt thereof) for the preparation of a pharmaceutical composition for the treatment of diabetic peripheral neuropathy in a subject in need thereof.

A third aspect of the invention relates to the use of a combination of doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole (or a pharmaceutically acceptable salt thereof) and metoprolol (or a pharmaceutically acceptable salt thereof) for the treatment of diabetic peripheral neuropathy, including diabetic neuropathic pain, in a subject in need thereof.

A fourth aspect of the present invention relates to a method of treating diabetic peripheral neuropathy, including diabetic neuropathic pain, comprising administering to a subject in need thereof a therapeutically effective amount of doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole (or a pharmaceutically acceptable salt thereof), and metoprolol (or a pharmaceutically acceptable salt thereof).

The pharmaceutical composition and the dosage form of the invention are suitable for the treatment of diabetic peripheral neuropathy at various stages, and are also suitable for preventive treatment before onset of the disease.

The "patient" or "subject" to be treated by the method of the invention is applicable to any mammal, e.g. human, primate, rat, mouse, dog, cat, cow, horse and pig, preferably said "patient" or "subject" is a primate, especially a human.

As used herein, "diabetic peripheral neuropathy" includes its various symptoms, complications, secondary symptoms, and particularly encompasses Diabetic Peripheral Neuropathic Pain (DPNP).

The treatment methods of the present invention involve the combination of multiple active ingredients, also referred to as "combination therapy" or "combination therapy". By "combination" or "combination therapy" is meant administration of a plurality of active ingredients as described herein such that they act together to provide a beneficial effect. The beneficial effects of the above combinations include, but are not limited to, the combined pharmacokinetic and pharmacodynamic effects of the above combinations of active ingredients. The combined administration of these active ingredients is usually done over a defined period of time (usually minutes, hours, days or weeks, depending on the judgment of the physician). "combination" or "combination therapy" is intended to encompass the administration of the active ingredients in a sequential manner, i.e., each of the active ingredients is administered at a different time, as well as the administration of the active ingredients in a substantially simultaneous manner, or the administration of at least two of the active ingredients. Substantially simultaneous administration may be accomplished, for example, by: administering to said host a single capsule containing a fixed ratio of each active ingredient, or administering to said host a plurality of capsules each containing one of said active ingredients. Sequential or substantially simultaneous administration of each active ingredient may be effected by any suitable route, including, but not limited to, oral, intravenous, intramuscular, intraocular, and direct absorption through mucosal tissue. The active ingredients may be administered by the same route or by different routes. For example, a first active ingredient of a selected combination may be administered by intravenous injection, while the other active ingredients of the combination may be administered orally. Alternatively, for example, all of the active ingredients may be administered orally, or all of the active ingredients may be administered by intravenous injection. The order of administration of the active ingredients is not strictly limited.

"combination" or "combination therapy" also encompasses the combined administration of the active ingredients described hereinabove, further in combination with other biologically active ingredients, as well as non-drug therapy (e.g., surgical therapy or mechanical therapy). When the combination therapy further comprises a non-drug treatment, the non-drug treatment may be carried out at any suitable time as long as the beneficial effects of the combination of the active ingredients and the non-drug treatment are achieved. For example, the beneficial effect can still be achieved, where appropriate, after temporary cessation of the non-drug treatment from administration of the active ingredient, where the cessation can be days or even weeks.

In order to achieve the desired effect, it is generally necessary to administer to a patient or subject a therapeutically effective amount of a pharmaceutical composition or dosage form of the invention or each active ingredient alone.

The phrase "therapeutically effective amount" is a term recognized in the art. In certain embodiments, the term refers to an amount necessary or sufficient to eliminate, reduce, or maintain the goal of a particular therapeutic regimen. The effective amount may vary depending on factors such as the disease or disorder being treated, the particular targeting construct being administered, the size of the subject, or the severity of the disease or disorder. One of ordinary skill in the art or a physician can empirically determine an effective amount of a particular compound without undue experimentation. In certain embodiments, a therapeutically effective amount of a therapeutic agent used in vivo may depend on a number of factors, including: the mode and method of administration; any other material in addition to the medicament that is contained in the medicament. In vitro or in vivo assays may optionally be used to help determine the optimal dosage range.

In some embodiments of the invention, the daily dose of doxazosin or a pharmaceutically acceptable salt thereof may range from 0.5mg to 16mg when orally administered to an adult human having a normal body weight of about 60 kg, for example, it may be 0.5 to 15mg, 0.5 to 13mg, 0.5 to 12mg, 0.5 to 10mg, 0.5 to 8mg, 0.5 to 6mg, 0.5 to 5mg, 0.5 to 4mg, 1 to 15mg, 1 to 13mg, 1 to 12mg, 1 to 10mg, 1 to 8mg, 1 to 6mg, 1 to 5mg, 1 to 4mg, 1.5 to 16mg, 1.5 to 15mg, 1.5 to 13mg, 1.5 to 12mg, 1.5 to 10mg, 1.5 to 8mg, 1.5 to 6mg, 1.5 to 5mg, 1.5 to 4mg, 2 to 16mg, 2 to 15mg, 2 to 13mg, 2 to 12mg, 2 to 10mg, 2 to 8mg, 2 to 6mg, 2.5 to 5mg, 2 to 4mg, 2 to 16mg, 2 to 15mg, 2 to 13mg, 2 to 12mg, 2 to 10mg, 2 to 8mg, 2 to 6mg, 2 to 4mg, and the like. In a preferred embodiment of the present invention, the daily dose of doxazosin, or a pharmaceutically acceptable salt thereof, may be about 2mg. In another preferred embodiment of the invention, the daily dose of doxazosin is about 4mg.

In some embodiments of the invention, the daily dose of pramipexole or a pharmaceutically acceptable salt thereof may range from 0.05mg to 4.5mg when orally administered to an adult human normally weighing about 60 kg, for example, it may be 0.05mg to 4.2mg, 0.05mg to 4mg, 0.05mg to 3.5mg, 0.05mg to 3mg, 0.05mg to 2.5mg, 0.05mg to 2mg, 0.05mg to 1.5mg, 0.05mg to 1mg, 0.1mg to 4.5mg, 0.1mg to 4.2mg, 0.1mg to 4mg, 0.1mg to 3.5mg, 0.1mg to 3mg, 0.1mg to 2.5mg, 0.1mg to 2mg, 0.1mg to 1.5mg, 0.1mg to 1mg, 0.2mg to 4.5mg 0.2mg to 4.2mg, 0.2mg to 4mg, 0.2mg to 3.5mg, 0.2mg to 3mg, 0.2mg to 2.5mg, 0.2mg to 2mg, 0.2mg to 1.5mg, 0.2mg to 1.0mg, 0.5mg to 4.5mg, 0.5mg to 4.2mg, 0.5mg to 4mg, 0.5mg to 3.5mg, 0.5mg to 3mg, 0.5mg to 2.5mg, 0.5mg to 2mg, 0.5mg to 1.5mg, 0.5mg to 1mg, 1mg to 4mg, 1mg to 3mg, 1mg to 2.5mg, and the like. In a preferred embodiment of the present invention, the daily dose of pramipexole or a pharmaceutically acceptable salt thereof is in the range of about 0.1mg to about 1mg. In a preferred embodiment of the invention, the daily dose of pramipexole is about 0.0625mg. In another preferred embodiment of the invention, the daily dose of pramipexole is about 0.125mg.

The daily dose of metoprolol or a pharmaceutically acceptable salt thereof may range from 2.5mg to 200mg, such as from 5mg to 200mg, preferably from 5mg to 100mg. In some embodiments of the invention, the daily dosage of metoprolol or a pharmaceutically acceptable salt thereof can range from 2.5mg to 200mg when orally administered to an adult human with a normal body weight of about 60 kg, for example, 5mg to 180mg, 5mg to 160mg, 5mg to 150mg, 5mg to 130mg, 5mg to 120mg, 5mg to 100mg, 5mg to 80mg, 5mg to 60mg, 5mg to 50mg, 5mg to 40mg, 10mg to 180mg, 10mg to 160mg, 10mg to 150mg, 10mg to 130mg, 10mg to 120mg, 10mg to 100mg, 10mg to 80mg, 10mg to 60mg, 10mg to 50mg, 10mg to 40mg, 15mg to 200mg, 15mg to 180mg, 15mg to 160mg, 15mg to 150mg, 15mg to 130mg, 15mg to 120mg, 15mg to 100mg, 15mg to 80mg, 15mg to 60mg, 20mg to 200mg, 20mg to 180mg, 20mg to 160mg, 20mg to 150mg, 20mg to 130mg, 20mg to 120mg, 20mg to 100mg, 20mg to 80mg, 20mg to 600 mg, 600 to 600 mg. In a preferred embodiment of the invention, the daily dose of metoprolol or a pharmaceutically acceptable salt thereof is in the range of 5mg to 100mg. In a preferred embodiment of the invention, the daily dose of metoprolol is about 10-20mg. In another preferred embodiment of the invention, the daily dose of metoprolol is about 50mg.

The above-mentioned daily dose may be administered continuously on a periodic basis, for example every 2 hours, every 6 hours, every 8 hours, every 12 hours, about once every 24 hours. Preferably, the daily dose may be administered to the patient from 2 to 3 times daily, or in sustained release tablets. The difference between the oral daily doses of the three active ingredients is large, which is determined by the pharmacokinetics of the respective active ingredients in vivo.

It will be appreciated by those skilled in the art that when the pharmaceutical compositions of the invention are formulated in other dosage forms suitable for intravenous drip or intramuscular injection, the dosage ranges for the individual active ingredients may differ from those given above for oral administration, and may be reasonably determined by one skilled in the art or by a physician in conjunction with in vivo and in vitro experimentation and taking into account the different pharmacokinetic characteristics of the various routes of administration.

In a preferred embodiment of the invention, the pharmaceutical composition of the invention is for use in a mammalian, primate subject, particularly a human subject.

It will be appreciated by those skilled in the art that the various aspects of the invention described herein can be combined separately in various ways that will be apparent to those skilled in the art without departing from the subject matter and spirit of the application. Such combinations are also included within the scope of the present application. For example, ranges for amounts of certain components recited herein include any combination of any lower limit and any upper limit set forth in the specification, as well as any range where a particular amount of that component in each particular embodiment constitutes either an upper limit or a combination of lower limits; all such ranges are encompassed within the scope of the present invention. In addition, each feature of the present invention recited in the specification may be combined with any other feature of the present invention, and such a combination is also within the scope of the present invention. Specific embodiments of the present application are described in detail below with reference to the accompanying drawings.

Drawings

Figure 1 shows sensory transmission rate effect change value individual data on diabetic peripheral neuropathy rhesus monkeys for 58 days of administration for each group, wherein placebo (n =4,7 aberrant nerves), DOX + PMP + MTP treated (n =4, 17 aberrant nerves), PMP treated (n =3,8 aberrant nerves), DOX + MTP treated (n =3,9 aberrant nerves), DOX + PMP treated (n =2,6 aberrant nerves) and epalrestat (n =4, 13 aberrant nerves).

Fig. 2 shows the sensory nerve conduction velocity effect on diabetic peripheral neuropathy rhesus monkeys on day 58 of administration in each group, in which placebo (n =4,7 abnormal nerves), DOX + PMP + MTP treated group (n =4, 17 abnormal nerves), PMP treated group (n =3,8 abnormal nerves), DOX + MTP treated group (n =3,9 abnormal nerves), DOX + PMP treated group (n =2,6 abnormal nerves) and epalrestat group (n =4, 13 abnormal nerves).

Fig. 3 shows sural sensory nerve conduction velocity change value individual data for diabetic peripheral neuropathy rhesus monkeys at 58 days of administration for each group, in which placebo (n =4,7 abnormal nerves), DOX + PMP + MTP treated (n =4,8 abnormal nerves), PMP treated (n =3,5 abnormal nerves), DOX + MTP treated (n =3,6 abnormal nerves), DOX + PMP treated (n =2,2 abnormal nerves), and epalrestat (n =4,7 abnormal nerves).

Figure 4 shows data on change in the effect of 58 days on the conduction velocity of the sural nerve sensory nerves in diabetic peripheral neuropathy rhesus monkeys for each group, in which placebo (n =4,7 abnormal nerves), DOX + PMP + MTP treated group (n =4,8 abnormal nerves), PMP treated group (n =3,5 abnormal nerves), DOX + MTP treated group (n =3,6 abnormal nerves), DOX + PMP treated group (n =2,2 abnormal nerves), and epalrestat group (n =4,7 abnormal nerves).

Examples

The inventor takes a spontaneous diabetic peripheral neuropathy rhesus monkey as an experimental animal, verifies the effectiveness and the safety tolerance of three marketed GPCR (GPCR) signal path medicines of Doxazosin (DOX), pramipexole (PMP) and Metoprolol (MTP) on diabetic peripheral neuropathy through experiments, and evaluates whether the sensory conduction and the motor conduction speed and the safety of the DPN rhesus monkey can be improved or not by 58 days of treatment and adopting neuroelectrophysiology examination (electromyography) -nerve conduction function examination. The currently common first-line DPN medicament epalrestat is used as a positive control in the experiment.

1. Experimental Material

Specifically, the following three marketed drugs were used as test products in the experiment:

note: the dose of monkey drug and the dose of human drug can be converted by a body surface area method or in vivo drug concentration exposure.

Test article 1:

name or abbreviation (english name): doxazosin mesylate (DOX)

Purity: 98% by weight HPLC

Production unit: shanghai Yangzi Biotech Co Ltd

Sample 2:

name or abbreviation (english name): pramipexole dihydrochloride (Pramipexole 2HCL monohydrate, PMP)

Purity: 99.55% HPLC

Production unit: shanghai Yangzi Biotech Co Ltd

Test article 3

Name or abbreviated name (english name): metoprolol tartrate (Metoprotrol tartratestat, MTP)

Purity: 98% by weight HPLC

Production unit: aladdin reagent, inc

Positive control drug

Name or abbreviated name: epalrestat

Batch number: RS20200323

Physical and chemical properties: white powder

Purity: 98 percent of

Production unit: TOKYO CHEMICAL INDUSTRY co.

2. Specific experimental scheme

Laboratory animal

Animal species Rhesus monkey, macaca mulatta (Rhesuus Macaque)

Grade: a normal stage. The quarantine is qualified before the test, and the contents comprise physical examination, 2 times of tubercle bacillus test, parasite, salmonella, shigella and B virus examination.

Animal identification: the neck ring is worn with stainless steel number plate engraved with Arabic numerals, and the chest is tattooed

Supply unit: yaan Promei Biotechnology GmbH (Primei Primate research center)

Producing a license number: SCXK 2019-027

The test system complies with the AAALAC requirements

A total of 20 spontaneous DPN rhesus monkeys were enrolled for the experiments, with the following inclusion criteria:

1) 20 males or females, age 13 to 25 years (equivalent to 40 to 75 years old); 13 males; female 7 were used.

2) The course of diabetes is 3-7 years: fasting blood glucose (FPG) is more than 5mmol/L, vs age-matched controls (age-matched controls) is 4.1 + -0.3 mmol/L;

3) Chronic sensorimotor distal, symmetric neuropathy (DSPN):

performing neuroelectrophysiological examination by using Electromyography (EMG), evaluating the ability of peripheral myelinated crude fiber nerve to conduct electric signals, and selecting and diagnosing to be distal and symmetric diabetic peripheral polyneuropathy (common DNP type), wherein nerve damage persists for at least 6 months. Detecting nerves includes: 1) Sensory nerve conduction function: the median nerve (left and right), ulnar nerve (left and right), sural nerve (left and right), and superficial peroneal nerve (left and right); 2) Motor nerve conduction function: the median nerve (left and right), the ulnar nerve (left and right), and the common peroneal nerve (left and right). The inclusion criteria for abnormalities in nerve conduction velocity (SNCV) in this trial met the DPN criteria of the international cooperation study (WHOPNTF).

The following criteria were met for nerve conduction abnormalities as shown in the following Table 2-1:

TABLE 2-1 criteria for nerve conduction abnormalities

For comparison, the following table 2-2 shows the nerve conduction velocity reference values of healthy rhesus monkeys (non-diabetic) (data from the mean of measurements of 20 age-matched healthy rhesus monkeys).

TABLE 2-2 age-matched control (age-matched controls) non-diabetic monkey neural function parameters

Grouping condition:

the 20 spontaneous DPN rhesus monkeys were divided into six groups, and the detailed grouping was shown in tables 2-3 below. Tables 2-4 show the abnormal detection of the nerve conduction parameter of the spontaneous DPN rhesus monkey nerve electromyography tested in each group (determined according to the standard in table 2-1).

Table 2-3 test DPN rhesus monkey cohort cases and basic data

Dosing regimens

The experiment was performed on DOX + PMP + MTP group (n = 4), epalrestat group (n = 4), DOX + MTP group (n = 3), PMP group (n = 3), DOX + PMP group (n = 2) and placebo group (n = 4). The specific dosing regimen for each group is described below, wherein the route of administration is oral. The baseline period was 1 month, followed by 58 consecutive oral administrations to assess the effect of the 58 days of treatment on nerve conduction velocity and the ability to reduce the sustained deterioration of nerve function. The following is grouping and dosing information:

DOX + PMP + MTP group: number of animals 4

D0 to D14 DOX + PMP + MTP 0.133+0.002+0.833mg/kg 2 times a day

D15 to D28: DOX + PMP + MTP 0.133+0.002+1.667mg/kg 2 times a day

D29 to D58: DOX + PMP + MTP 0.266+0.004+1.667mg/kg for 2 times a day

Epalrestat group: the number of animals was 4; the dose is 2.5mg/kg (equivalent to a clinically equivalent dose of 40-80 mg) administered daily on D0-D58, 1 time daily on weeks 1-2 and 2 times daily on weeks 3-8.

DOX + MTP group: number of animals 3

D0-D28 DOX + MTP 0.266mg/kg +1.667mg/kg 2 times daily

D29 to D58: DOX + MTP 0.53mg/kg +3.3mg/kg 2 times daily

PMP group: number of animals 3

D0 to D58:0.004mg/kg, 2 times daily

DOX + PMP group: number of animals 2

D0 to D28 of 0.133mg/kg +0.002mg/kg 2 times a day

D29 to D58, 0.266mg/kg +0.004mg/kg, 2 times daily

Placebo group: animals were counted 4, given drinking water or fruit, and observed for 58 consecutive days.

The administration route is as follows: food-induced dosing.

Calculating the dosage: the dose for the next week was calculated as the body weight weighed each time.

3. Observation indicator and monitoring method

3.1 Main drug efficacy index

1. Neuromuscular electrical map

The detection method comprises the following steps: the compound ketamine is injected into muscles, skin preparation of four limbs is respectively pasted with a receiving electrode, a reference electrode and a ground wire after anesthesia, a stimulation electrode is used for stimulating a designated part, the current is increased until an image is stable and then stored, and the distance between the stimulation electrode and the receiving electrode (sensory conduction) or the distance between two stimulation points (motor conduction) is measured. During detection, the environment needs to be kept quiet, and the temperature of the detection environment is controlled to be 20-25 ℃. The examiner needs double blindness and dose information of the animal is unclear.

And (4) detecting items: see Table 3-1.

Detecting frequency: 1 time before administration and 1 time after administration (58 days).

And (3) detecting an instrument: haishen NDI-092 electromyography/evoked potential apparatus.

TABLE 3-1 EMG potential detection index

3.2 other indices

1. Glycolipid metabolism index and blood biochemical index

The blood sample collection method comprises the following steps: see tables 3-2, specifically: animals were fasted overnight one day prior to the blood collection procedure, and on the following day 08.

3-2 sample Collection processing information

The detection indexes and the method are as follows: see tables 3-3

Detecting frequency: 1 time before and 58 days after administration

And (3) detecting an instrument: the detection is carried out by adopting a Roche cobas6000 analyzer series C501 module

TABLE 3-3 Biochemical examination of blood

2. Observation of clinical symptoms

The observation times are as follows: the observation was done 1 time per day.

And (4) observing the content: skin, quilt hair, eyes, ears, nose, oral cavity, chest, abdomen, urogenital area, limbs, etc., and respiratory, athletic, urinary, bowel movements, behavioral changes, etc.

3.3 data processing

The results are presented in the form of individual data. Statistical data processing analysis was performed using Microsoft Office Excel 2016 software. Each measurement is expressed by means of "Mean + -SD". Taking the latest data before the administration as a baseline value, and performing statistical analysis on each index before and after the administration by adopting repeated measurement, wherein P <0.05 is the difference with statistical significance.

4. Experimental results and discussion

4.1 effects on improvement of neurological function

4.1.1 Effect on sensory nerve conduction velocity

Neuromuscular electrograms were measured on "abnormal" sensory nerves labeled in tables 2-4 before (baseline) and 58 days after (D58) administration, respectively, to evaluate the effect of the test drug on sensory nerve conduction velocity in diabetic peripheral neuropathy rhesus monkeys, as shown in table 4-1, and fig. 1 and 2.

Placebo group (n = 4): after the administration, the conduction velocity of each nerve is not obviously changed, and the model is proved to be more stable. The D58 day change in conduction velocity measurements from baseline was-0.4. + -. 1.0m/s for 7 abnormal nerves.

Epalrestat group (n = 4): compared to baseline values, a significant increase in SCV was obtained for 58 days with 13 abnormal conduction velocity nerves, with a mean increase of 4.2 ± 4.0m/s and a very significant increase compared to placebo (P <0.01vs placebo).

DOX + PMP + MTP group (n = 4): significant increases in SCV were obtained for 58 days with 17 abnormal conduction velocity nerves compared to baseline, with mean increases of 4.3 ± 3.4m/s (P <0.05vs baseline), and very significant increases compared to placebo (P <0.01vs placebo).

In addition, no significant improvement in abnormal sensory nerve conduction velocity was seen in the DOX + MTP, PMP, and DOX + PMP groups compared to the placebo group, and there was no statistical difference from the placebo group (P >0.05vs placebo group).

TABLE 4-1 Effect of 58 days of administration on sensory nerve conduction velocity in diabetic peripheral neuropathy rhesus monkeys

Note: * P is less than 0.05vs baseline value. Change = end of dose-baseline value.

TABLE 4-1 Effect of (continuous) administration on sensory nerve conduction velocity in diabetic peripheral neuropathy rhesus monkeys for 58 days

4.1.2 Effect on the sensory conduction velocity of the sural nerve

Abnormal degree and abnormal rate of sensory nerve conduction velocity (SCV) in DPN patients are more severe than abnormal motor nerve conduction velocity (MCV), and patient neurological dysfunction is usually with lower limbs (sural nerve) heavier than upper limbs, while the disease phenotype characteristics of DPN rhesus monkeys are similar to those of DPN patients. Therefore, it was evaluated whether the improvement in the gastrocnemius sensory conduction velocity of DPN rhesus monkeys 58 days after the administration was the primary efficacy index. The results of the effect of the test drugs on the sensory transduction rate of the "abnormal" gastrocnemius nerve of DPN rhesus monkeys are shown in table 4-2, fig. 3, and fig. 4. Compared with the baseline, the nerve conduction speed returns to normal after 58 days of administration, or the nerve conduction speed is increased by 2-5 m/s to be diagnosed as effective, or the nerve conduction speed is increased by more than 5m/s to be judged as effective.

Placebo group (n = 4): after administration, the conduction velocity of each nerve was not significantly changed, and the mean change of the conduction velocity (SCV) measured values of 7 abnormal nerves at 58 days relative to the baseline value was-0.4. + -. 1.0m/s, which proved that the model was relatively stable. Compared to baseline, 0/7 of the nerves were effective.

Epalrestat group (n = 4): compared with the baseline value, the sensory nerve conduction velocity (SCV) of the sural nerve increased by 3.2 +/-3.9 m/s on average in 58 days after 7 abnormal nerve administrations, and was significantly increased compared with the placebo group (P <0.05vs placebo group). Compared to baseline, 4/7 neural determination of conduction velocity effectively improved.

DOX + PMP + MTP group (n = 4): sensory nerve conduction velocity (SCV) was significantly increased after 58 days of 8 abnormal sural nerve administration compared to baseline, with a mean increase of 4.6 ± 3.9m/s (P <0.05vs baseline), and very significantly increased compared to placebo (P <0.01vs placebo). Compared to baseline, 6/8 nerves judged a significant improvement in conduction velocity.

In addition, no significant improvement in abnormal gastrocnemius sensory nerve conduction velocity was seen in either the PMP group or the DOX + PMP group compared to the placebo group, and there was no statistical difference from the placebo group (P >0.05vs placebo group).

TABLE 4-2 Effect of 58 days on DNP rhesus monkey gastrocnemius sensory nerve conduction velocity

4.1.3 Effect on Motor nerve conduction velocity

In the test, the abnormal number of the motor nerve conduction velocity (MCV) of each group of the selected DPN rhesus monkeys is small and does not reach the statistical standard, so the test does not research the influence of the motor nerve conduction velocity. In addition, since abnormal degree and abnormal rate of sensory nerve conduction velocity (SCV) are generally more important than abnormal motor nerve conduction velocity (MCV) in DPN patients, the effect of SCV is more important in pharmacodynamic studies.

4.2 safety of the composition.

As marketed drugs, safe dose ranges and toxic side effects of DOX, PMP, MTP are known.

A common tablet dosage of DOX is 4-8 mg/time, 2 times daily. Adverse effects that may result when overdosing (e.g. more than 4 mg) include mainly orthostatic hypotension.

PMP recommends that the individual dose should be 0.375mg to 4.5mg per day, with an initial dose of 0.375mg per day for the treatment of idiopathic parkinson's disease, followed by an increase of the dose once every 5-7 days, with a maximum dose of 1.5mg. The incidence of lethargy increases at daily doses above 1.5mg.

The recommended dose of MTP is 100-200 mg/day, and can reach 300 mg/day or 400 mg/day if necessary, and the known adverse reaction is hypotension.

In the test, the human dose calculated based on the dosage of the rhesus monkey is far lower than the maximum value of the recommended dose, so that the DOX + PMP + MTP three-drug combination is very good in safety theoretically.

In addition, in the above experimental process, no adverse events (including blood biochemical indicators and clinical observation results) related to the administration were observed in the animals of the DOX + PMP + MTP group during the whole administration period, which further confirms that the triple drug combination of DOX + PMP + MTP of the present invention indeed has good biological safety indicators.

5. Conclusion

The above experimental results show that: three GPCR agonists (DOX + PMP + MTP) are orally taken for 58 days, can obviously improve nerve conduction speed, particularly calf nerve sensory nerve conduction speed, control and relieve DPN progress in a spontaneous DPN rhesus monkey model, and show good safety during administration, and the curative effect intensity of the GPCR agonists is equivalent to that of epalrestat which is a common clinical first-line medicament.

6. Pharmaceutical combinationEXAMPLES example 1

For 10kg of monkeys, 1.3mg of Doxazosin mesylate (DOX) raw material powder, 0.02mg of Pramipexole Dihydrochloride (PMP) raw material powder, and 8.3mg of Metoprolol Tartrate (MTP) raw material powder may be respectively and uniformly mixed to obtain the pharmaceutical composition of example 1. The pharmaceutical composition can be directly mixed with food to feed animals, or mixed with proper excipient or additive to make into oral tablet.

Although specific embodiments have been described above, those skilled in the art will appreciate that, based on the disclosure and guidance in the above description, appropriate variations and modifications can be made to the above embodiments by those skilled in the art. Therefore, it is intended that the present invention not be limited to the particular embodiments disclosed and described, but that modifications and variations are possible within the scope of the appended claims.

Claims

1. A pharmaceutical composition for treating diabetic peripheral neuropathy comprising doxazosin or a pharmaceutically acceptable salt thereof, pramipexole or a pharmaceutically acceptable salt thereof, and metoprolol or a pharmaceutically acceptable salt thereof as active ingredients.

2. The pharmaceutical composition according to claim 1, comprising 0.5 to 45 parts by weight (preferably 1 to 10 parts by weight) of pramipexole or a pharmaceutically acceptable salt thereof, 5 to 160 parts by weight (preferably 10 to 80 parts by weight) of doxazosin or a pharmaceutically acceptable salt thereof, and 25 to 2000 parts by weight (preferably 50 to 1000 parts by weight) of metoprolol or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition according to claim 1 or 2, which is an oral, intravenous or intramuscular dosage form, preferably an oral dosage form.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, sulfate, phosphate, pyrophosphate, hydrobromide or nitrate, citrate, fumarate, maleate, malate, ascorbate, succinate, tartrate, benzoate, acetate, mesylate, ethanesulfonate, salicylate, stearate, benzenesulfonate or p-toluenesulfonate.

5. Use of a combination of doxazosin or a pharmaceutically acceptable salt thereof, pramipexole or a pharmaceutically acceptable salt thereof and metoprolol or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition for the treatment of diabetic peripheral neuropathy in a subject in need thereof.

6. Use according to claim 5, wherein the pharmaceutical composition is an oral dosage form (preferably an oral dosage form 2-3 times daily or a long acting oral formulation), an intravenous dosage form, or an intramuscular dosage form, preferably an oral dosage form.

7. Use according to any one of claims 5 to 6, wherein the pharmaceutical composition is suitable for mammalian, e.g. human, primate, rat, mouse, dog, cat, cow, horse and pig, in particular human and primate subjects.

8. Use according to claim 7, wherein the daily dose of doxazosin or a pharmaceutically acceptable salt thereof is in the range of 0.5mg to 16mg, preferably 1mg to 8mg, the daily dose of pramipexole or a pharmaceutically acceptable salt thereof is in the range of 0.05mg to 4.5mg, preferably 0.1mg to 1mg, and the daily dose of metoprolol or a pharmaceutically acceptable salt thereof is in the range of 2.5mg to 200mg, preferably 5mg to 100mg.

9. The use according to any one of claims 5 to 8, wherein the pharmaceutically acceptable salt is selected from the hydrochloride, sulfate, phosphate, pyrophosphate, hydrobromide or nitrate, citrate, fumarate, maleate, malate, ascorbate, succinate, tartrate, benzoate, acetate, mesylate, ethanesulfonate, salicylate, stearate, benzenesulfonate or p-toluenesulfonate salt.

10. The use according to any one of claims 5 to 9, wherein the diabetic peripheral neuropathy is diabetic peripheral neuropathic pain.