Use of pharmaceutical composition in treating diabetic peripheral neuropathy

Abstract

Provided is a composition for preparing a medicament for treating diabetic peripheral neuropathy. The pharmaceutical composition comprises cobra venom phospholipase A2, or a combination of cobra venom phospholipase A2 and at least one additional drug for treating diabetic peripheral neuropathy. The diabetic peripheral neuropathy is characterized by a slowdown in motor nerve conduction velocity and / or sensory nerve conduction velocity of a patient, and / or hypoesthesia or loss of sensation in both lower limbs. The pharmaceutical composition can be used to ameliorate the clinical symptoms described above in patients with diabetic peripheral neuropathy, and belongs to the field of biopharmaceuticals.

Description

Application of pharmaceutical composition in treating diabetic peripheral neuropathy Technical Field

[0001] 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

[0002] Diabetes is a metabolic syndrome characterized by elevated blood sugar levels. Diabetic peripheral neuropathy (DPN), also known as diabetic peripheral neuropathy, is the most common chronic complication of diabetes, with an incidence exceeding 50%. This disorder primarily manifests as sensory and motor neurological symptoms. Current research suggests that the primary pathogenic mechanisms are genetic susceptibility, insufficient neurovascular blood supply due to diabetic macrovascular and microvascular disease, and abnormal lipid and protein metabolism in peripheral nerves caused by impaired glucose utilization. The onset of DPN is also associated with peripheral nerve terminal degeneration and demyelination. Patients experience a progressive increase in the threshold for vibration and thermal perception from the distal to the proximal end of the peripheral nerve, leading to sensory loss, numbness, and other abnormal sensations.

[0003] In clinical practice, the most important objective indicator for diabetic peripheral neuritis is the neuroelectromyography examination, which can be used to diagnose diabetic peripheral neuritis and detect subclinical nerve damage. In the early stages of diabetes, even before clinical symptoms appear, there are obvious changes in the neuroelectromyography, which is 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

[0004] In order to solve the above problems, the present invention provides a pharmaceutical composition that can prevent the progression of diabetic peripheral neuritis, improve motor and / or sensory nerve conduction velocity, and / or improve sensory loss or loss in both lower limbs.

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

[0007] 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 in both lower limbs. The composition is characterized in that 0.01%-100% of cobra phospholipase A2 and 99.9%-0% of one or more components of cobra cardiotoxin (CDX) and / or cobra neurotoxin (NTX) are mixed in any ratio to form 100% of the active ingredient of the composition.

[0008] The patient had a history of diabetic peripheral neuritis.

[0009] Streptozotocin (STZ) combined with a high-fat, high-sugar diet induces typical diabetic peripheral neuritis in rats, and the pathological changes in this animal model are similar to those of minimal change disease in human diabetic peripheral neuritis. This study establishes a streptozotocin (STZ) combined with a high-fat, high-sugar diet-induced diabetic peripheral neuritis model, recognized by professionals in the field, to observe the effects of cobra phospholipase A2 or the combination of cobra phospholipase A2 with one or more components of cobra cardiotoxin (CDX) and / or cobra neurotoxin (NTX) on improving motor function in diabetic peripheral neuritis.

[0010] and / or sensory nerve conduction velocity, and / or therapeutic efficacy in improving sensory loss or loss in both lower limbs.

[0011] 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 decreased sensation or loss in 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-stage diabetic peripheral neuritis, once abnormalities in nerve electromyography (EMG) examination or decreased sensation in both lower limbs are found, timely treatment is provided to improve motor and / or sensory nerve conduction velocity, and / or improve decreased sensation or loss in both lower limbs, thereby preventing or delaying the progression of diabetic peripheral neuritis to the onset of obvious clinical symptoms.

[0012] Phospholipase A2 (PLA2) is a family of enzymes widely distributed across various animal tissues, particularly in the venoms of snakes (type I) and the pancreatic secretions of mammals (type II). The primary structural characteristic of phospholipase A2 in cobra snakes is a platform of two antiparallel, long-chain helices connected by disulfide bonds. These phospholipases share a high degree of amino acid sequence homology and some functional structural similarities. They contain seven disulfide bonds, characteristic of type I PLA2 at positions 11-77. Their mature proteins share a high degree of amino acid sequence homology, with molecular weights ranging from 12 to 14 kDa and 118 or 119 amino acid residues. This shared functional structure contributes to their shared pharmacological activities, such as anticoagulant activity, which can inhibit the various complications of diabetes caused by microcirculatory obstruction due to increased blood viscosity. Therefore, the shared functional structure of phospholipase A2 in cobra snakes contributes to their shared pharmacological functions.

[0013] 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 more 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 with snake phospholipase A2 from the cobra family, or the combined use of cobra phospholipase A2 with one or more components of cobra cardiotoxin (CDX) or / and cobra neurotoxin (NTX) in the diabetic DPN group, the symptoms of slowed motor and sensory nerve conduction velocity in rats with diabetic peripheral neuritis were reversed. In addition, treatment significantly improved the sensory loss or loss in both lower limbs of rats with diabetic peripheral neuritis. This suggests that the combined use of snake phospholipase A2 from the cobra family, or cobra phospholipase A2 with one or more components of cobra cardiotoxin (CDX) or / and cobra neurotoxin (NTX), has some therapeutic effect on the symptoms of diabetic peripheral neuritis in rats.

[0014] 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 aekisgcwpy xktysyecsq gtltckggnn acaaavcdcd rlaaicfaga pyxxxxynix lkarcq

[0015] Furthermore, in some embodiments, the following four amino acid sequences of phospholipase A2 all show a common therapeutic effect on diabetic peripheral neuritis in rats.

[0016] (SEQ ID No. 2)

[0017] nly qfknmiqctv psrswwdfad ygcycgrggs gtpvddldrc cqvhdncyne aekisgcwpy fktysyecsq gtltckggnn acaaavcdcd rlaaicfaga pynnnnynid lkarcq

[0018] (SEQ ID No. 3)

[0019] nly qfknmiqctv psrswwdfad ygcycgrggs gtpvddldrc cqvhdhcyne aekisgcwpy sktysyecsq gtltckggnn acaaavcdcd rlaaicfaga pynnnnynid lkarcq

[0020] (SEQ ID No. 4)

[0021] nly qfknmvqctv pnrswwdfad ygcycgrggs gtpvddldrc cqvhdncyge aekisrcwpy fktysyecsq gtltckggnn acaaavcdcd rlaaicfaga pyndnnynid lkarcq

[0022] (SEQ ID No. 5)

[0023] n lyqfknmiq ctvpsrswwd fadygcycgr ggsgtpvddl drccqvhdnc yneaekisgc wpyfktysye csqgtltckg gnnacaaavc dcdrlaaicf agapyndndy ninlkarc

[0024] Cobra phospholipase A2 can be extracted from snake venom or obtained through recombinant or synthetic technology.

[0025] In some embodiments, the amino acid sequence of the cobra phospholipase A2 in the composition is SEQ ID No. 2.

[0026] In some embodiments, the amino acid sequence of the cobra phospholipase A2 in the composition is SEQ ID No. 3.

[0027] In some embodiments, the amino acid sequence of the cobra phospholipase A2 in the composition is SEQ ID No. 4.

[0028] In some embodiments, the amino acid sequence of cobra phospholipase A2 in the composition is SEQ ID No. 5.

[0029] 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 can 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 the lower limbs of rats with diabetic peripheral neuritis.

[0030] In some embodiments, the amino acid sequence of the cobra neurotoxin in the composition is SEQ ID No. 6.

[0031] In some embodiments, the amino acid sequence of the cobra neurotoxin in the composition is SEQ ID No. 7.

[0032] In some embodiments, the amino acid sequence of the cobra neurotoxin in the composition is SEQ ID No. 8.

[0033] In some embodiments, the amino acid sequence of the cobra neurotoxin in the composition is SEQ ID No. 9.

[0034] In some embodiments, the amino acid sequence of cobra cardiotoxin in the composition is SEQ ID No. 10.

[0035] In some embodiments, the amino acid sequence of cobra cardiotoxin in the composition is SEQ ID No. 11.

[0036] In some embodiments, the amino acid sequence of cobra cardiotoxin in the composition is SEQ ID No. 12.

[0037] In some embodiments, the amino acid sequence of cobra cardiotoxin in the composition is SEQ ID No. 13.

[0038] 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%.

[0039] In some embodiments, the composition is administered once daily for 15-180 consecutive days.

[0040] In some embodiments, the composition is administered every 12 hours, twice daily, for 15-180 days.

[0041] In some embodiments, the composition is administered every 4 hours, 3 times daily, for 15-180 days.

[0042] In some embodiments, the dosage of Naja phospholipase A2 in the composition is 10-100 μg / kg once.

[0043] In some embodiments, the dosage of cobra phospholipase A2 administered in the composition is 80 μg / kg once.

[0044] In some embodiments, the dosage of cobra phospholipase A2 administered in the composition is 60 μg / kg once.

[0045] In some embodiments, the dosage of cobra phospholipase A2 administered in the composition is 40 μg / kg once.

[0046] In some embodiments, the composition is in the form of a sublingual film.

[0047] In some embodiments, the composition is in an oral dosage form.

[0048] In some embodiments, the composition is in the form of an injection.

[0049] In some embodiments, the composition is in the form of a nasal spray.

[0050] In some embodiments, the composition is in a transdermal dosage form.

[0051] In some embodiments, the pharmaceutically acceptable excipient is selected from one or more of a protein stabilizer, a membrane excipient, a mucosal permeation promoting agent, a cosolvent, or a solvent.

[0052] In some embodiments, the protein stabilizer is mannitol.

[0053] In some embodiments, the film excipient is propylene glycol, polyethylene glycol, hydroxypropyl beta-cyclodextrin, Tween 80, hydroxypropyl methylcellulose (HPMC), methylcellulose, or xanthan gum.

[0054] In some embodiments, the mucosal permeation promoting agent is laurocapram, poloxamer, borneol, or dextroborneol.

[0055] In some embodiments, the co-solvent is propylene glycol.

[0056] 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.

[0057] Another advantage of the present invention is its 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, the clear amino acid sequence in the process makes it easier to control quality and purity, which lays the necessary foundation for the development of pharmaceuticals based on monomeric components in snake venom.

[0058] The present invention will be further described below with reference to specific examples, but the following examples 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 shall fall within the scope of protection of the present invention. DETAILED DESCRIPTION

[0059] Example

[0060] Example 1: Obtaining cobra phospholipase A2 protein (SEQ ID No. 2)

[0061] Dissolve 1 g of crude cobra venom in 25 ml of 0.025 M ammonium acetate buffer, pH 6.0, centrifuge at low temperature, and collect the supernatant. Equilibrate the supernatant with 0.025 M ammonium acetate, pH 6.0, and load the column onto a TSK CM-650 (M) column. Elute the column using a two-compartment step gradient (0.1-0.5 M and 0.7-1.0 M ammonium acetate buffer, pH 5.9) with UV detection at 280 nm and a flow rate of 48 ml / h. Collect the various toxin components according to the recorded spectra. Twelve protein peaks were eluted from the collected solution. N-terminal sequencing was performed on each peak, and proteins with an N-terminal residue of nlyqfknm were further sequenced to obtain various sequences of cobra phospholipase A2. The amino acid sequence of the primary structure of cobra phospholipase A2 (SEQ ID No. 2) was obtained in Fasta format:

[0062] nly qfknmiqctv psrswwdfad ygcycgrggs gtpvddldrc cqvhdncyne aekisgcwpy fktysyecsq gtltckggnn acaaavcdcd rlaaicfaga pynnnnynid lkarcq

[0063] Example 2: Effects of the cobra phospholipase A2 (PLA2) treatment group and the 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 a high-fat and high-sugar diet

[0064] 1. Experimental Animals and Model Grouping

[0065] Fifty male Wistar rats, weighing 180-200 g, were randomly divided into a normal control group (n=10) and a modeling group (n=40). Thirty surviving rats were randomly selected after successful modeling and divided into a cobra phospholipase A2 (PLA2) treatment group (n=10), a cobra phospholipase A2 + cobra neurotoxin + cobra cardiotoxin (PLA2 + NTX + CDX) treatment group (n=10), and a rat diabetic peripheral neuritis (DPN) group (n=10). The remaining rats were excluded.

[0066] 2. Modeling method

[0067] The rats in the model 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.

[0068] (CFA). The next day, streptozotocin (STZ) solution was intraperitoneally injected at a concentration of 1% in 0.1 mmol / L citrate buffer (pH 4.5) and injected intraperitoneally at 35 mg / kg. Eight weeks after modeling, tail vein blood glucose was collected for testing. A successful diabetic peripheral neuritis model was considered established if random blood glucose remained above 16.7 mmol / L, urine glucose was 3+ to 4+, sciatic nerve sensory and motor nerve conduction velocities were significantly slowed to below 28 m / s, and the tail-flick temperature threshold increased by more than 1°C. Thirty rats with successful modeling were randomly selected and then divided into a cobra phospholipase A2 (PLA2) treatment group (10 rats), a cobra phospholipase A2 + cobra neurotoxin + cobra cardiotoxin (PLA2 + NTX + CDX) treatment group (10 rats), and a rat diabetic peripheral neuritis (DPN) group (10 rats). The remaining rats were excluded from the group. Ten normal control rats were injected with saline alone and fed a normal diet. The cobra phospholipase A2 treatment group received cobra phospholipase A2 20 μg / kg by gavage once daily, while the cobra phospholipase A2 + cobra neurotoxin + cobra cardiotoxin treatment group (PLA2 15 μg / kg + NTX 10 μg / kg + CDX 15 μg / kg) received gavage once daily for 8 weeks. The DPN group and the normal control group received normal saline by gavage once daily for 8 weeks.

[0069] 3. Observation indicators and detection methods

[0070] 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 right sciatic nerve conduction velocity of rats was detected by neuroelectrophysiology under ether inhalation anesthesia.

[0071] a) Motor nerve conduction velocity: The first stimulation site was the sciatic fossa, and the second stimulation site was the ankle. Two needle electrodes were inserted percutaneously with a 2 mm spacing between the electrodes and grounded to the tail. The skin temperature was maintained at 30°C. The nerve was stimulated with a square wave (10-20 mA, 40 μs pulse width). Compound muscle action potentials were recorded in the ipsilateral interosseous muscles of the foot using two needle electrodes. The latency of three pairs of different M waves was recorded and averaged. The difference between the proximal and distal latencies was used as the conduction time of the motor nerve between the two stimulation sites. The distance between the two stimulation sites was measured with a foot bend gauge. Motor nerve conduction velocity (m / min)

[0072] s) = distance / latency difference.

[0073] b) Sensory nerve conduction velocity: The first stimulation site was the ischial fossa, and the second stimulation site was the ankle. Two needle electrodes were inserted percutaneously, with a spacing of 2 mm between them. The electrodes were grounded to the tail, and the skin temperature was maintained at 30°C. Six pairs of H-reflex latencies were recorded using a square wave (2 mA, 40 μs pulse width) at the ischial fossa and ankle. The shortest latency difference was used, and the distance between the two stimulation sites was measured using a foot bend gauge. Sensory nerve conduction velocity (m / s) = distance / latency difference.

[0074] 4. Experimental Results

[0075] Results of nerve conduction velocity experiments in rats in the normal group, diabetic DPN control group, and two treatment groups

[0076] 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 experiments

[0077] Table 1

[0078] (`x±SD,n=10)

[0079]

[0080]

[0081] 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 in rats with diabetic peripheral neuritis. Compared with the diabetic DPN group, the nerve conduction velocity of the above two treatment groups was significantly increased. * indicates the nerve conduction velocity of the above two treatment groups

[0082] The speed was compared with the diabetic DNP group, P<0.05.

[0083] Example 3: Effects of the cobra phospholipase A2 (PLA2) treatment group and the cobra phospholipase A2 + cobra neurotoxin + cobra cardiotoxin (PLA2 + NTX + CDX) treatment group on the increased peripheral to proximal thermal perception threshold and sensory loss in rats with diabetic peripheral neuritis induced by streptozotocin (STZ) combined with a high-fat diet

[0084] 1. Experimental Animals and Model Grouping

[0085] Example 2 The same method was used to group the experimental animals into models.

[0086] 2. Modeling method

[0087] Example 2 Same method

[0088] 3. Observation indicators and detection methods

[0089] Observation and detection of the average latency time of paw licking in rats in the normal group, diabetic DPN control group, and two treatment groups

[0090] Mice were placed individually on a (55.0±0.5)°C hot plate apparatus, and the average latency of the rats' paw licking was measured and recorded. This was used to measure abnormal sensations such as an increase in the peripheral to proximal thermal perception threshold and sensory loss. The average latency of the hind paw licking was used as an indicator of heat sensation and sensory loss.

[0091] 4. Experimental Results

[0092] Results of the experiment on the average latency time of licking the hind paw of rats in the normal group, diabetic DPN control group, and two treatment groups

[0093] 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 experiments

[0094] Table 2

[0095] (``x±SD, n=10)

[0096]

[0097]

[0098] Effects of the cobra phospholipase A2 (PLA2) treatment group and the 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. A composition for preparing a drug for treating diabetic peripheral neuritis, wherein the composition comprises a therapeutically effective dose of cobra phospholipase A2 and a pharmaceutically acceptable excipient; the amino acid sequence of the cobra phospholipase A2 is as shown in SEQ As shown in 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 the lower limbs.

3. The application according to any one of claims 1-2, wherein: 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 drugs for treating diabetic peripheral neuritis include 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 composition comprises a combination of cobra phospholipase A2 and one or more components of cobra cardiotoxin (CDX) or / and cobra neurotoxin (NTX) in a ratio of 0.01%-100% cobra phospholipase A2 and 99.9%-0% of one or more components of cobra cardiotoxin (CDX) or / and cobra neurotoxin (NTX) to form 100% of the active ingredient 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 promoting agents, cosolvents or solvents.

6. application according to claim 1, it is further characterized in that, the protein stabilizer in the acceptable excipient of the drug is mannitol, the film excipient is propylene glycol, polyethylene glycol, hydroxypropyl beta-cyclodextrin, Tween-80, hydroxypropyl methylcellulose (HPMC), methylcellulose, xanthan gum, the mucosal permeation promoting agent 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 oral dosage form, sublingual

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