Application of pharmaceutical composition in treating diabetic peripheral neuropathy

Abstract

The present invention relates to a composition for preparing a drug for treating diabetic peripheral neuritis. The drug composition comprises cobra phospholipase A2, or a combination of cobra phospholipase A2 and at least one other drug for treating diabetic peripheral neuropathy. The diabetic peripheral neuropathy is manifested as a slowing of the patient's motor nerve conduction velocity and / or sensory nerve conduction velocity, and / or decreased or absent sensation in both lower limbs. The drug composition can be used to improve the above clinical symptoms of patients with diabetic peripheral neuropathy, and belongs to the field of biomedicine.

Description

Technical Field The invention relates to application of a composition in preparing a medicine for treating diabetic peripheral neuropathy, and belongs to the field of biomedicine. Background Art

[0001] Diabetes is a metabolic disorder syndrome represented by increased blood sugar. Diabetic peripheral neuropathy, also known as diabetic peripheral neuritis (DPN), is the most common chronic complication of diabetes, with an incidence rate of more than 50%. This is a group of peripheral neuropathy with sensory nerve and motor nerve symptoms as the main clinical manifestations. Current studies have shown that genetic susceptibility, insufficient blood supply to nerve vessels caused by diabetic macrovascular and microvascular diseases, and abnormal lipid and protein metabolism in peripheral nerves caused by glucose utilization disorders are the main pathogenic mechanisms. The onset of DPN is also related to the degeneration and demyelination of peripheral nerve terminals. The patient's peripheral nerves will progressively show an increase in vibration and thermal perception thresholds from the terminal to the proximal end, followed by sensory loss, numbness and other abnormal sensations.

[0002] In clinical practice, the most important objective indicator for diabetic peripheral neuritis is the nerve electromyography examination, which can be used to diagnose diabetic peripheral neuritis and detect subclinical nerve damage. In the early stages of diabetes, even before the onset of clinical symptoms, the nerve electromyography has obvious changes, mainly manifested as a slowing of nerve conduction velocity, including a slowing of sensory nerve conduction velocity (SCV) and motor nerve conduction velocity (MCV). Summary of the invention

[0003] In order to solve the above problems, the present invention provides a pharmaceutical composition capable of preventing the progression of diabetic peripheral neuritis, improving motor and / or sensory nerve conduction velocity, and / or improving sensory loss or loss of both lower limbs.

[0004] The present invention relates to an application of a composition in preparing a drug for treating diabetic peripheral neuritis in patients, improving motor and / or sensory nerve conduction velocity, and / or improving sensory loss or loss of both lower limbs, characterized in that the composition contains cobra phospholipase A2, and the patient has a history of diabetic peripheral neuritis.

[0005] The present invention relates to an application of a composition for treating diabetic peripheral neuritis in patients, improving motor and / or sensory nerve conduction velocity, and / or improving sensory loss or loss of both lower limbs, characterized in that the composition contains cobra phospholipase A2, and the patient has a history of diabetic peripheral neuritis.

[0006] The present invention relates to a composition for treating diabetic peripheral neuritis, improving motor and / or sensory nerve conduction velocity, and / or improving sensory loss or loss of both lower limbs, wherein the composition comprises 0.01%-100% of cobra phospholipase A2 and 99.9%-0% of one or more components of cobra cardiotoxin (CDX) or / and cobra neurotoxin (NTX) to prepare 100% active component of the composition,

[0007] The patient had a history of diabetic peripheral neuritis.

[0008] Streptozotocin (STZ) combined with a high-fat, high-sugar diet induced diabetic peripheral neuritis in rats can cause typical diabetic peripheral neuritis symptoms in rats, and the pathological changes in the animal model are similar to the minimal lesions of human diabetic peripheral neuritis. The present invention establishes a diabetic peripheral neuritis model induced by streptozotocin (STZ) combined with a high-fat, high-sugar diet recognized by professionals in the field to observe the therapeutic efficacy of cobra phospholipase A2 or cobra phospholipase A2 combined with one or more components of cobra cardiotoxin (CDX) or / and cobra neurotoxin (NTX) in improving the motor and / or sensory nerve conduction velocity of diabetic peripheral neuritis, and / or improving the sensory loss or loss of both lower limbs.

[0009] The present invention relates to a composition for use in preparing a composition for treating diabetic peripheral neuritis, improving motor and / or sensory nerve conduction velocity, and / or improving sensory loss or loss of both lower limbs. The composition comprises cobra phospholipase A2 or a combination of cobra phospholipase A2 and one or more components of cobra cardiotoxin (CDX) or / and cobra neurotoxin (NTX). For patients with early diabetic peripheral neuritis, once abnormalities in nerve electromyography examination or sensory loss of both lower limbs are found, timely treatment is given to improve motor and / or sensory nerve conduction velocity, and / or improve sensory loss or loss of both lower limbs, thereby preventing or delaying the progression of diabetic peripheral neuritis patients to the appearance of obvious clinical symptoms.

[0010] Phospholipase A2 (PLA2) is a widely distributed enzyme family, present in various animal tissues, especially in the venom of animals such as snake venom (type I) and the pancreatic secretions of mammals (type II). The main structural feature of phospholipase A2 in cobra snakes is a reverse parallel long-chain helical structure platform connected by two disulfide bonds. Their amino acid sequences maintain a high degree of homology and some functional structural similarities. They contain 7 disulfide bonds in their molecules. They all have the typical characteristic disulfide bonds at positions 11-77 of type I PLA2. Their mature proteins have a high degree of homology in amino acid sequence, with a molecular weight of about 12-14KD and 118 or 119 amino acid residues. The common functional structure makes them have common pharmacological activities, such as anticoagulant activity, and the anticoagulant activity can inhibit various complications caused by microcirculatory obstruction caused by diabetes due to increased blood viscosity. Therefore, the same functional structure of phospholipase A2 in cobra snakes makes them have common pharmacological functions.

[0011] Our animal model study found for the first time that snake phospholipase A2 from the cobra family or the combined use of cobra phospholipase A2 with one or several components of cobra cardiotoxin (CDX) or / and cobra neurotoxin (NTX) can treat and improve the symptoms of diabetic peripheral neuritis in rats. After 8 weeks of treatment of the diabetic DPN group with snake phospholipase A2 from the cobra family or the combined use of cobra phospholipase A2 with one or several components of cobra cardiotoxin (CDX) or / and cobra neurotoxin (NTX), the symptoms of slowed conduction velocity of motor nerves and sensory nerves in rats with diabetic peripheral neuritis were reversed, and the treatment significantly improved the phenomenon of decreased sensation or loss in both lower limbs of rats with diabetic peripheral neuritis. This means that the combined use of snake phospholipase A2 from the cobra family or cobra phospholipase A2 with one or several components of cobra cardiotoxin (CDX) or / and cobra neurotoxin (NTX) has a certain therapeutic effect on the symptoms of diabetic peripheral neuritis in rats.

[0012] The amino acid sequence (FASTA) of the mature protein of phospholipase A2 from the family Elapidae has the following characteristics: (SEQ ID No. 1) nly qfknmiqctv psrswwdfad ygcycgkggs gtpvddldrc cqvhdxcyxe aekisgcwpyxktysyecsq gtltckggnn acaaavcdcd rlaaicfaga pyxxxxynix lkarcq

[0013] Further, in some embodiments, the following four amino acid sequences of phospholipase A2 all show a common therapeutic effect on diabetic peripheral neuritis in rats.

[0014] (SEQ ID No.2)

[0015] nly qfknmiqctv psrswwdfad ygcycgrggs gtpvddldrc cqvhdncyne aekisgcwpyfktysyecsq gtltckggnn acaaavcdcd rlaaicfaga pynnnnynid lkarcq

[0016] (SEQ ID No.3)

[0017] nly qfknmiqctv psrswwdfad ygcycgrggs gtpvddldrc cqvhdhcyne aekisgcwpysktysyecsq gtltckggnn acaaavcdcd rlaaicfaga pynnnnynid lkarcq

[0018] (SEQ ID No.4)

[0019] nly qfknmvqctv pnrswwdfad ygcycgrggs gtpvddldrc cqvhdncyge aekisrcwpyfktysyecsq gtltckggnn acaaavcdcd rlaaicfaga pyndnnynid lkarcq

[0020] (SEQ ID No.5)

[0021] nlyqfknmiq ctvpsrswwd fadygcycgr ggsgtpvddl drccqvhdnc yneaekisgcwpyfktysye csqgtltckg gnnacaaavc dcdrlaaicf agapyndndy ninlkarc

[0022] Cobra phospholipase A2 can be extracted from snake venom or obtained through recombinant or synthetic technology.

[0023] In some embodiments, the amino acid sequence of cobra phospholipase A2 in the composition is SEQ ID No2.

[0024] In some embodiments, the amino acid sequence of cobra phospholipase A2 in the composition is SEQ ID No 3.

[0025] In some embodiments, the amino acid sequence of cobra phospholipase A2 in the composition is SEQ ID No4.

[0026] In some embodiments, the amino acid sequence of cobra phospholipase A2 in the composition is SEQ ID No5.

[0027] In some embodiments, the composition comprises, in addition to cobra phospholipase A2, one or more components of cobra cardiotoxin (CDX) and / or cobra neurotoxin (NTX). The results show that the combined use of two or more of the above components will bring better therapeutic effects on the symptoms of slowed conduction velocity of motor nerves and / or sensory nerves in rats with diabetic peripheral neuritis, and / or the symptoms of decreased or absent sensation in both lower limbs of rats with diabetic peripheral neuritis.

[0028] In some embodiments, the amino acid sequence of the cobra neurotoxin in the composition is SEQ ID No6.

[0029] In some embodiments, the amino acid sequence of the cobra neurotoxin in the composition is SEQ ID No7.

[0030] In some embodiments, the amino acid sequence of the cobra neurotoxin in the composition is SEQ ID No8.

[0031] In some embodiments, the amino acid sequence of the cobra neurotoxin in the composition is SEQ ID No9.

[0032] In some embodiments, the amino acid sequence of cobra cardiotoxin in the composition is SEQ ID No10.

[0033] In some embodiments, the amino acid sequence of cobra cardiotoxin in the composition is SEQ ID No11.

[0034] In some embodiments, the amino acid sequence of cobra cardiotoxin in the composition is SEQ ID No12.

[0035] In some embodiments, the amino acid sequence of cobra cardiotoxin in the composition is SEQ ID No13.

[0036] In some embodiments, the ratio of cobra phospholipase A2 to one or more components of cobra cardiotoxin (CDX) or / and cobra neurotoxin (NTX) in the composition is any ratio of cobra phospholipase A2 0.01%-100% to one or more components of cobra cardiotoxin (CDX) or / and cobra neurotoxin (NTX) 99.9%-0%.

[0037] In some embodiments, the composition is administered once daily for 15-180 consecutive days.

[0038] In some embodiments, the composition is administered once every 12 hours, twice daily, for 15-180 consecutive days.

[0039] In some embodiments, the composition is administered once every 4 hours, 3 times a day, for 15-180 consecutive days.

[0040] In some embodiments, the dosage of cobra phospholipase A2 administered in the composition is 10-100 μg / kg once.

[0041] In some embodiments, the dosage of cobra phospholipase A2 administered in the composition is 80 μg / kg once.

[0042] In some embodiments, the dosage of cobra phospholipase A2 administered in the composition is 60 μg / kg once.

[0043] In some embodiments, the dosage of cobra phospholipase A2 administered in the composition is 40 μg / kg once.

[0044] In some embodiments, the composition is in the form of a sublingual film.

[0045] In some embodiments, the composition is in an oral dosage form.

[0046] In some embodiments, the composition is in the form of an injection.

[0047] In some embodiments, the composition is in the form of a nasal spray.

[0048] In some embodiments, the composition is in a transdermal dosage form.

[0049] In some embodiments, the pharmaceutically acceptable excipient is selected from one or more of a protein stabilizer, a membrane excipient, a mucosal permeation promoter, a cosolvent or a solvent.

[0050] In some embodiments, the protein stabilizer is mannitol.

[0051] In some embodiments, the film excipient is propylene glycol, polyethylene glycol, hydroxypropyl beta-cyclodextrin, Tween 80, hydroxypropyl methylcellulose (HPMC), methylcellulose, xanthan gum.

[0052] In some embodiments, the mucosal permeation promoting agent is laurocapram, poloxamer, borneol, or dextroborneol.

[0053] In some embodiments, the co-solvent is propylene glycol.

[0054] In some embodiments, the diabetic peripheral neuritis is a symptom of slowed conduction velocity of motor nerves and / or sensory nerves in diabetic patients, and / or decreased or absent sensation in both lower limbs.

[0055] Another advantage of the present invention is in production. Because the cobra phospholipase A2 disclosed in the present invention has a clear amino acid sequence, it can be produced through genetic engineering, solving the practical problem of scarce snake venom resources. Even if phospholipase A2 is obtained by separation and purification of natural snake venom, it is easier to achieve quality and purity control because of the clear amino acid sequence in the process, which lays a necessary foundation for the development of drugs for monomer components in snake venom.

[0056] The present invention is further described below in conjunction with specific embodiments, but the following embodiments are not intended to limit the present invention; at the same time, any equivalent replacements in the art made according to the disclosure of the present invention belong to the protection scope of the present invention. DETAILED DESCRIPTION

[0057] Example

[0058] Example 1: Obtaining cobra phospholipase A2 protein (SEQ ID No. 2)

[0059] Dissolve 1g of crude cobra venom in 25ml of 0.025mol pH6.0 ammonium acetate buffer, centrifuge at low temperature, and take the supernatant; balance the TSK CM-650 (M) column with 0.025mol pH6.0 ammonium acetate solution; after loading, use 0.1-0.5mol and 0.7-1.0mol, pH5.9 ammonium acetate buffer for two-compartment step gradient elution (0.1~0.5mol and 0.6~1mol), UV detection parameters: 280nm; elution flow rate: 48ml / h; collect various toxin components according to the recorded spectrum, and 12 protein peaks are eluted in the collected solution. N-terminal sequencing is performed on each protein peak, and the protein with N-terminal nlyqfknm is further sequenced, and finally various sequences of cobra phospholipase A2 are obtained. The amino acid sequence Fasta format of the primary structure of cobra phospholipase A2 (SEQ ID No.2) obtained by sequencing is:

[0060] nly qfknmiqctv psrswwdfad ygcycgrggs gtpvddldrc cqvhdncyne aekisgcwpyfktysyecsq gtltckggnn acaaavcdcd rlaaicfaga pynnnnynid lkarcq

[0061] Example 2: Effects of cobra phospholipase A2 (PLA2) treatment group and cobra phospholipase A2 + cobra neurotoxin + cobra cardiotoxin (PLA2 + NTX + CDX) treatment group on nerve conduction velocity in rats with diabetic peripheral neuritis induced by streptozotocin (STZ) combined with high-fat and high-sugar diet

[0062] 1. Experimental animals and modeling groups

[0063] Fifty male Wistar rats, weighing 180-200 g, were randomly divided into a normal control group of 10 and a modeling group of 40. Thirty rats that survived the successful modeling were randomly selected and divided into a cobra phospholipase A2 (PLA2) treatment group of 10, a cobra phospholipase A2 + cobra neurotoxin + cobra cardiotoxin (PLA2+NTX+CDX) treatment group of 10, and a rat diabetic peripheral neuritis (DPN) group of 10, and the rest were excluded.

[0064] 2. Modeling method

[0065] The rats in the modeling group were fed a high-sugar and high-fat diet and had free access to food and water. Each rat was first intraperitoneally injected with 0.5 ml of Freund's complete adjuvant (CFA). The next day, streptozotocin (STZ) solution was intraperitoneally injected. Before use, 0.1 mmol / L pH 4.5 citrate buffer was prepared into a 1% concentration and intraperitoneally injected at 35 mg / kg. After 8 weeks, tail vein blood was collected from the modeling group to test blood sugar. The diabetic peripheral neuritis model was successful when random blood sugar was maintained above 16.7 mmol / L, urine sugar was 3+ to 4+, the sciatic sensory nerve conduction velocity and motor nerve conduction velocity were significantly slowed down to below 28 (m / s), and the tail swing temperature threshold increased by more than 1°C. Thirty rats with successful modeling were randomly selected and then randomly divided into 10 rats in the cobra phospholipase A2 (PLA2) treatment group, 10 rats in the cobra phospholipase A2 + cobra neurotoxin + cobra cardiotoxin (PLA2 + NTX + CDX) treatment group, and 10 rats in the rat diabetic peripheral neuritis (DPN) group. The rest were excluded. Ten normal control rats were injected with normal saline only and fed with normal feed. The cobra phospholipase A2 treatment group was gavaged with cobra phospholipase A2 20ug / kg once a day, and the cobra phospholipase A2 + cobra neurotoxin + cobra cardiotoxin treatment group (PLA2 15ug / kg +NTX10ug / kg +CDX15ug / kg) was gavaged once a day for 8 consecutive weeks. The DPN group and the normal control group were gavaged with normal saline once a day for 8 consecutive weeks.

[0066] 3. Observation indicators and detection methods

[0067] Observation and measurement of sciatic motor and sensory nerve conduction velocity in rats in the normal group, diabetic DPN control group, and two treatment groups: The conduction velocity of the right sciatic nerve of rats was detected by neuroelectrophysiology under ether inhalation anesthesia.

[0068] a) Motor nerve conduction velocity: The first stimulation site is at the sciatic fossa, and the second stimulation site is at the ankle. Two needle electrodes are inserted percutaneously, with an electrode spacing of 2 mm, grounded to the tail, and the skin temperature is maintained at 30°C. The nerve is stimulated with a square wave (10~20mA, 40 μs pulse width), and the compound muscle action potential is recorded in the interosseous muscle of the foot on the same side with two needle electrodes. The latency of 3 pairs of different M waves is recorded, and the average value is taken. The difference between the proximal and distal latencies is used as the conduction time of the motor nerve between the two stimulation sites. The distance between the two stimulation sites is measured with a foot bend gauge. Motor nerve conduction velocity (m / s) = distance / latency difference.

[0069] b) Sensory nerve conduction velocity: The first stimulation site was at the sciatic fossa, and the second stimulation site was at the ankle. Two needle electrodes were inserted percutaneously, with an electrode spacing of 2 mm, grounded to the tail, and the skin temperature was maintained at 30°C. Six pairs of H reflex latencies stimulated at the sciatic fossa and ankle were recorded using a square wave (2 mA, 40 μs pulse width). The shortest latency difference was taken, and the distance between the two stimulation sites was measured using a foot bend gauge. Sensory nerve conduction velocity (m / s) = distance / latency difference.

[0070] 4. Experimental Results

[0071] Results of nerve conduction velocity experiment in rats in normal group, diabetic DPN control group, and two treatment groups

[0072] Cobra phospholipase A2 (PLA2) SEQ ID No. 2, cobra neurotoxin (NTX) SEQ ID No. 6, cobra cardiotoxin (CDX) SEQ ID No. 10 were used for the above animal tests

[0073] Table 1

[0074] (`x±SD,n=10)

[0075]

[0076] Effects of cobra phospholipase A2 (PLA2) treatment group and cobra phospholipase A2 (PLA2) + cobra neurotoxin (NTX) + cobra cardiotoxin (CDX) treatment group on nerve conduction velocity of diabetic peripheral neuritis in rats. Compared with the diabetic DPN group, the nerve conduction velocity of the above two treatment groups was significantly increased. * indicates that the nerve conduction velocity of the above two treatment groups is compared with the diabetic DNP group P < 0.05.

[0077] Example 3: Effects of cobra phospholipase A2 (PLA2) treatment group and cobra phospholipase A2 + cobra neurotoxin + cobra cardiotoxin (PLA2 + NTX + CDX) treatment group on the upregulation of peripheral to proximal thermal perception threshold and sensory loss in rats with diabetic peripheral neuritis induced by streptozotocin (STZ) combined with high-fat diet

[0078] 1. Experimental animals and modeling groups

[0079] Example 2 The experimental animals were grouped in the same manner

[0080] 2. Modeling method

[0081] Example 2 Same method

[0082] 3. Observation indicators and detection methods

[0083] Observation and detection of the average latency time of paw licking in the normal group, diabetic DPN control group, and two treatment groups

[0084] Mice were placed one by one on a (55.0±0.5)℃ hot plate apparatus, and the average latency of the rats' paw licking was measured and recorded to measure abnormal sensations such as the increase in the peripheral to proximal thermal perception threshold and sensory loss. The average latency of licking the hind paw was used as an indicator of heat sensation and sensory loss.

[0085] 4. Experimental Results

[0086] Results of the experiment on the average latency of licking the hind paw of rats in the normal group, the diabetic DPN control group, and the two treatment groups

[0087] Cobra phospholipase A2 (PLA2) SEQ ID No. 2, cobra neurotoxin (NTX) SEQ ID No. 6, cobra cardiotoxin (CDX) SEQ ID No. 10 were used for the above animal tests

[0088] Table 2

[0089] (``x±SD, n=10)

[0090]

[0091] Effects of cobra phospholipase A2 (PLA2) treatment group and cobra phospholipase A2 + cobra neurotoxin + cobra cardiotoxin (PLA2+NTX+CDX) treatment group on thermal perception and sensory loss in rats with diabetic peripheral neuritis. Compared with the diabetic DPN group, the thermal perception and sensory loss of the above two treatment groups were significantly improved. * indicates P < 0.05 compared with the diabetic DNP group.

Claims

1. Use of a composition for preparing a drug for treating diabetic peripheral neuritis, the composition comprising a therapeutically effective dose of cobra phospholipase A2 and a pharmaceutically acceptable excipient; the amino acid sequence of the cobra phospholipase A2 is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The drug has at least one of the following applications (1)-(3): (1) The drug is used to improve motor nerve conduction velocity; (2) The drug is used to improve sensory nerve conduction velocity; (3) The drug is used to improve decreased or absent sensation in both lower limbs.

3. The use according to any one of claims 1-2, characterized in that: The composition also includes the combined use of cobra phospholipase A2 and at least one other drug for treating diabetic peripheral neuritis to improve motor nerve conduction velocity, and / or improve sensory nerve conduction velocity, and / or improve hypoesthesia or loss of lower limbs. The other drug for treating diabetic peripheral neuritis includes cobra cardiotoxin and cobra neurotoxin; the amino acid sequences of the cobra neurotoxin and cobra cardiotoxin are shown in SEQ ID NO.6 and 10, respectively.

4. The use according to claim 3, characterized in that: The ratio of cobra phospholipase A2 and one or more components of cobra cardiotoxin (CDX) or / and cobra neurotoxin (NTX) in the composition is 0.01%-100% of cobra phospholipase A2 and 99.9%-0% of one or more components of cobra cardiotoxin (CDX) or / and cobra neurotoxin (NTX) to prepare 100% active ingredients of the composition.

5. The use according to claim 1, characterized in that: The pharmaceutically acceptable excipients in the pharmaceutical composition are selected from one or more of protein stabilizers, membrane excipients, mucosal permeation promoters, cosolvents or solvents.

6. The use according to claim 1, further characterized in that the protein stabilizer in the drug acceptable excipients is mannitol, the film excipients are propylene glycol, polyethylene glycol, hydroxypropyl β-cyclodextrin, Tween-80, hydroxypropyl methylcellulose (HPMC), methylcellulose, xanthan gum, the mucosal permeation promoter is laurocapram, poloxamer, borneol, dextroborneol, and the cosolvent is propylene glycol.

7. The use according to claim 1, characterized in that: The dosage form of the composition is an oral dosage form, a sublingual dosage form, a nasal dosage form, a transdermal dosage form or an injection dosage form.

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