WO2024042376A1 - Formulation of analgesic and anti-inflammatory ointment containing the leptucin, mcd and adolapine peptides - Google Patents

Formulation of analgesic and anti-inflammatory ointment containing the leptucin, mcd and adolapine peptides Download PDF

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WO2024042376A1
WO2024042376A1 PCT/IB2023/053414 IB2023053414W WO2024042376A1 WO 2024042376 A1 WO2024042376 A1 WO 2024042376A1 IB 2023053414 W IB2023053414 W IB 2023053414W WO 2024042376 A1 WO2024042376 A1 WO 2024042376A1
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peptides
peptide
leptucin
adolapine
venom
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PCT/IB2023/053414
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French (fr)
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Maria BEIHAGHI
Ilia TEHRANI
Vahid BAHRAMNIA GHOJEHBIGLOU
Saeed BAHRAMNIA GHOJEHBIGLOU
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Beihaghi Maria
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/63—Oleaceae (Olive family), e.g. jasmine, lilac or ash tree
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/63—Arthropods
    • A61K35/64—Insects, e.g. bees, wasps or fleas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/63—Arthropods
    • A61K35/646—Arachnids, e.g. spiders, scorpions, ticks or mites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin

Definitions

  • the formulation of the analgesic and anti-inflammatory ointment includes Adolapine and MCD peptides from bee venom as well as Leptucin peptide from Hemiscorpius lepturus scorpion venom, along with olive oil and fillers (Eucerin, lanolin, honey base), moisturizing agents (alexin), absorbing agents (aloe vera juice), penetrants (salicylic acid or alginate), as well as supplements (shikonin from Acium or levisticum).
  • This work can be generated as sprays, ointments, hydrogel pads, and other therapeutic products.
  • this ointment includes regulating the cell cycle and the many cytokines involved in this cycle, as well as activating the relevant genes. Three courses are required to complete a full recovery. Depending on whether it is used topically or through injection, the amount consumed and the percentage of use vary.
  • the present invention relates to TRPV1 selective agonist topical compositions including capsaicinoid and analgesic agent compositions and methods of manufacture and methods of providing pain relief as well as treating a variety of disorders with such compositions.
  • compositions and methods for brain delivery of analgesic peptides are provided.
  • the present invention relates to non-invasive brain delivery technology for centrally-acting analgesic peptides. Specifically, the invention is directed to compounds comprising an antibody or fragment thereof capable of transmigrating across the blood brain barrier (BBB) and an analgesic peptide. Compositions and methods of using the compounds or compositions are also provided.
  • BBB blood brain barrier
  • Bee venom may be administered in a standardized formulation with or without relatively small amounts of anesthetic.
  • results of the combination of venom and anesthetic dramatically decreased pain and discomfort for patients undergoing apitherapy.
  • the invention relates to a pre-filled bee venom syringe, in particularly to a precise syringe which is easy to operate and capable of guaranteeing safety injection dosage.
  • all bee venom injections including bee venom skin test injection and other bee venom injections in different dose, for one course of treatment are customized in advance as needed, and thus, treatment of various diseases such as rheumatic arthritis, rheumatoid arthritis, ankylosing spondylitis and neuritis by bee venom is facilitated.
  • bee venom can also be applied to treatment of gout, neurasthenia, sciatica, cervical spondylosis, lumbar intervertebral discs diseases, prosopalgia, migraine, osteoarticular diseases, chronic ulcer of legs, annexitis, pelvic inflammation, insomnia, stiff neck, contusion, cancer pain and the like.
  • gout neurasthenia, sciatica, cervical spondylosis, lumbar intervertebral discs diseases, prosopalgia, migraine, osteoarticular diseases, chronic ulcer of legs, annexitis, pelvic inflammation, insomnia, stiff neck, contusion, cancer pain and the like.
  • bee venom in good dose can be a precious good medicine.
  • Leptucin, MCD, and adolapine peptides are used in analgesic and anti-inflammatory ointments in the fields of pharmaceuticals, medicine, cellular and molecular biology, and apitherapy.
  • One of the main problems in the treatment of pain is drug dependence and addiction.
  • the current drugs cause serious problems in the liver and kidneys.
  • the presence of pain-relieving and anti-inflammatory peptides by inhibiting different receptors, regulating cytokines, inhibiting cyclooxygenase, and also changing the process of sodium and potassium channels inhibits pain and inhibits inflammation.
  • Another advantage of this invention is the separation and use of peptides instead of the venom itself, which eliminates complications and problems caused by other problems caused by the venom.
  • the current formulation does not have any toxicity even in higher doses and is not dependent and addictive.
  • the duration of effect and the speed of effect are more than other available drugs.
  • Inflammation and pain are one of the main problems in the treatment of many diseases. Meanwhile, more than a third of the world's population suffers from constant or frequent pain. Chronic and persistent pain is one of the main reasons people go to medical and clinical care centers. On the other hand, due to heavy sports activities and improper diet, pains such as back and leg pain have increased. The existing treatments to solve this issue are mostly chemical and on the other hand, they cause many side effects such as liver damage, breathing problems, intolerance and most importantly, they are addictive. Also, some of these drugs also cause common drug resistance. On the other hand, another major problem of these drugs is toxicity in higher doses and low effect per unit of time.
  • the animal venom itself is used in total form, which in these cases, due to the lack of specificity and due to the presence of some substances, including other peptides and proteins, causes serious problems such as anaphylaxis. So that these problems can sometimes even lead to death.
  • the use of peptides is purified and in appropriate doses, in addition to having no risks, it is introduced as next generation drugs. Since peptide molecules are more targeted and also have fewer side effects and in most clinical cases are free of side effects than chemical molecules, analgesic peptides can be a new revolution in pain relief.
  • the existing ointment is formulated with the combination of Adolapine, MCD (Mast cell degranulating) and Leptucin peptides, which do not have the side effects of existing drugs. On the other hand, it is not addictive and does not give drug resistance. This compound does not have toxicity in higher doses, and on the other hand, compared to existing drugs, it has a faster effect and a longer duration of effectiveness and toxin than other existing drugs, and it has high structural stability. Due to the separation at the peptide level, this combination has also solved the problems of other substances in the venom that cause any allergies and problems.
  • bee venom was collected according to the protocol (Benton AW et al. 1963) using a glass plate and using a low electric shock.
  • Scorpion venom was also collected using a low shock according to the protocol (Ozkan and Filazi. 2004) using A low voltage was collected with the help of electric shock.
  • the venom peaks were determined by HPLC. Then the desired peptides were separated by RP-HPLC.
  • Adolapine, MCD, and leptucin peptides from bee venom and Hemiscorpius lepturus scorpion venom are utilized to make this analgesic and anti-inflammatory ointment (extracted with a cold compress according to the protocol).
  • these peptides are utilized, as well as fillers like eucerin, lanolin, honey-based substances, moisturizing agents like alexin, absorption agents like aloe vera juice, penetrants like salicylic acid or alginate, and supplements like Shikonin from Echium candicans or Levisticum.
  • honey bee venom Due to the inclusion of several peptides and apitoxins, including MCD, Melittin, Apamin, Secapin, Adolapine, etc., honey bee venom possesses anti-inflammatory characteristics. While the adolapine peptide also acts as an analgesic, the MCD peptide is a potent anti-inflammatory peptide. Additionally, leptucin peptide possesses potent analgesic properties.
  • Leptucin serves as the main peptide in this work, with adolapine and MCD peptides added as side peptides to enhance the synergistic effects of the combination.
  • adolapine and MCD peptides added as side peptides to enhance the synergistic effects of the combination.
  • mice Wistar female rats weighing about 250 grams were utilized to evaluate the mixture.
  • the mice were first placed on a hot plate set to 55 °C, and their pain reactions—including the removal of their arms and legs, licking, and/or shaking—were observed.
  • Six groups of mice with normal pain tolerance were randomly assigned (four different doses of peptides, a negative control, and a positive control).
  • negative and positive controls respectively, normal saline and morphine (10 mg/kg) were utilized.
  • Each group's response to pain was recorded after 30 minutes. The 40-second cutoff was established. At intervals of 30 to 300 minutes following delivery, time periods for recording pain responses were defined. Each time point's analgesic activity was calculated in accordance with the protocol (Moghadasi, Z et al. 2018).
  • the MTT assay was carried out on the HEK-293 cell line to determine the cytotoxic effect of peptides.
  • the cells (2 x 104 each) were cultured in 96-well plates with 10% FBS-supplemented DMEM medium before being incubated for 24 hours at 37 °C in a 5% CO2 and 95% air environment.
  • DMEM medium peptides were produced at concentrations ranging from 16 to 0.25 g/ml, administered to each well, and then incubated for 24 hours.
  • a 10 ⁇ l solution of freshly prepared MTT (5 mg/ml in PBS) was added to each well and incubated for another 4h. Following that, 100 ⁇ l of isopropanol was added, and the plates were gently shaken. Viability was assessed after the absorbance at 570 nm wavelength was recorded using an ELISA reader (ELx808, BioTek, Winooski, VT, USA). This was carried out in accordance with the protocol (Aghazadeh, H. 2019).
  • mice were injected intraperitoneally in doses of 0.16, 0.32, 0.48 and 0.64 mg/kg to mice. These doses were selected as drugs according to their effectiveness. And the mice were sacrificed after 48 hours of administration of peptides. Brain and liver tissues were collected and fixed in 10% buffered formalin solution. After 4 days, the organ sections were routinely processed in an automated tissue processor and embedded in paraffin wax. Tissue sections were prepared and examined with hematoxylin and eosin stain (H&E) for histopathological studies with a light microscope. In this test, the focus was more on the brain to test the effect of peptides on the necrosis of neuronal cells. This work was important for us because peptides are more related to neuronal receptors.
  • H&E hematoxylin and eosin stain
  • mice were first put inside this equipment. This machine was run for 5 minutes at a speed of 4 rpm. The mice that failed to make it through the test were taken out of the group, and the remaining mice received injections of the peptide mixture at doses of 0.32, 0.48, and 0.64 mg/kg. The second rotarod test was conducted 30 minutes after the first. Mice that could not perform the test were known as "mice with movement defects," which were not found in this test. The results of this test show that there are no movement issues or disturbances with the current mixture.
  • plasticity is used to describe the changes that take place in the nervous system. Increased functional activity of neurons in the pain pathway can eventually result from changes in brain structure, connections between neurons, and the quantity and characteristics of neurotransmitters, receptors, and ion channels. On the other hand, plasticity can weaken the body's natural pain-inhibitory mechanisms, which ultimately makes pain worse. By boosting excitatory or suppressing inhibitory pathways, injury, inflammation, and disease can all induce brain plasticity and intensify pain. Flexibility can lead to long-term changes that could be permanent or short-term alterations that span anywhere from a few minutes to several hours.
  • the primary output from the dorsal horn to the brain is formed by projection neurons in layers I and V. These neurons are the starting point of a variety of ascending pathways, such as the spinothalamic and spinoreticulothalamic pathways, which send pain signals to the thalamus and brain stem, respectively.
  • Adolapine which is present in this composition, functions as a cyclooxygenase inhibitor and a naturally occurring inhibitor of prostaglandins.
  • MCD and leptucin peptides also play a role by acting on sodium channels, calcium channels, N-Methyl-D-aspartate receptors, and neurotensin receptors via nicotinic acetylcholine receptors (- and O-conotoxins), sodium (contulakins).
  • Cysteine which is abundant in leptucin and alters sodium and potassium channels, has immediate and lasting effects.
  • peptides reduce inflammation by blocking interleukins, inflammatory pathways, tumor necrosis factors, and pro-inflammatory molecules. They also reduce pain by blocking cyclooxygenase and causing alterations in sodium and potassium channels.
  • This ointment is used to regulate the cell cycle, the different cytokines involved in this cycle, and to activate the related genes. So that three courses are needed to finish the healing process. Depending on whether it is used topically or through injection, the amount consumed and the percentage of use vary. However, the results of the animal and human phase studies indicate that 200 ppm of the total peptides may be helpful. Due to the medication's slower absorption when applied topically, this quantity may be larger in some circumstances. On the other hand, chemicals like salicylic acid increase permeability when they are present.
  • This ointment is safe for people and comes from a natural source. Secondly, it can be a good idea to produce a low-dose mixture of leptucin, adolapine, and MCD peptides from scorpion and bee venom to induce an anti-inflammatory effect and penetrate inflamed tissues. This will help to lessen all types of pain and inflammation. Because these substances can block the expression of interleukins and tumor necrosis factors, lowering the inflammatory pathway. Nevertheless, they quickly decrease pain by blocking cyclooxygenase and altering sodium-potassium channel function. Using such a formulation with the current composition has never been done before. The synergistic usage of all three peptides and the use of the peptide in the ointment in a targeted and specific form are the two most crucial aspects of this innovation. Additionally, a spray version of this mixture can be created and applied.
  • This ointment has undergone all essential laboratory testing, including animal experiments, and approval has been requested for clinical trials. It was developed and produced in a laboratory setting. This compound has been used with consumer satisfaction because it can be applied topically, and the outcomes are remarkable.

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Abstract

The formulation of the analgesic and anti-inflammatory ointment includes Adolapine and MCD peptides from bee venom as well as Leptucin peptide from Hemiscorpius lepturus scorpion venom, along with olive oil and fillers (Eucerin, lanolin, honey base), moisturizing agents (alexin), absorbing agents (aloe vera juice), penetrants (salicylic acid or alginate), as well as supplements (shikonin from Acium or levisticum). This work can be generated as sprays, ointments, hydrogel pads, and other therapeutic products. The use of this ointment includes regulating the cell cycle and the many cytokines involved in this cycle, as well as activating the relevant genes. Three courses are required to complete a full recovery. Depending on whether it is used topically or through injection, the amount consumed and the percentage of use vary.

Description

Formulation of analgesic and anti-inflammatory ointment containing the leptucin, MCD and adolapine peptides
The formulation of the analgesic and anti-inflammatory ointment includes Adolapine and MCD peptides from bee venom as well as Leptucin peptide from Hemiscorpius lepturus scorpion venom, along with olive oil and fillers (Eucerin, lanolin, honey base), moisturizing agents (alexin), absorbing agents (aloe vera juice), penetrants (salicylic acid or alginate), as well as supplements (shikonin from Acium or levisticum). This work can be generated as sprays, ointments, hydrogel pads, and other therapeutic products. The use of this ointment includes regulating the cell cycle and the many cytokines involved in this cycle, as well as activating the relevant genes. Three courses are required to complete a full recovery. Depending on whether it is used topically or through injection, the amount consumed and the percentage of use vary.
A61K
PAIN RELIEF COMPOSITIONS, MANUFACTURE AND USES
United States Patent Application 20210100759
The present invention relates to TRPV1 selective agonist topical compositions including capsaicinoid and analgesic agent compositions and methods of manufacture and methods of providing pain relief as well as treating a variety of disorders with such compositions.
Compositions and methods for brain delivery of analgesic peptides
United States Patent 8986689
The present invention relates to non-invasive brain delivery technology for centrally-acting analgesic peptides. Specifically, the invention is directed to compounds comprising an antibody or fragment thereof capable of transmigrating across the blood brain barrier (BBB) and an analgesic peptide. Compositions and methods of using the compounds or compositions are also provided.
Bee venom treatment without the sting
United States Patent Application 20040081702
Bee venom may be administered in a standardized formulation with or without relatively small amounts of anesthetic. In particular, the results of the combination of venom and anesthetic dramatically decreased pain and discomfort for patients undergoing apitherapy.
Pre-filled bee venom syringe
CN106668990A
The invention relates to a pre-filled bee venom syringe, in particularly to a precise syringe which is easy to operate and capable of guaranteeing safety injection dosage. According to the course of treatment, all bee venom injections, including bee venom skin test injection and other bee venom injections in different dose, for one course of treatment are customized in advance as needed, and thus, treatment of various diseases such as rheumatic arthritis, rheumatoid arthritis, ankylosing spondylitis and neuritis by bee venom is facilitated. Besides, bee venom can also be applied to treatment of gout, neurasthenia, sciatica, cervical spondylosis, lumbar intervertebral discs diseases, prosopalgia, migraine, osteoarticular diseases, chronic ulcer of legs, annexitis, pelvic inflammation, insomnia, stiff neck, contusion, cancer pain and the like. On the basis of the theory of traditional Chinese medicine that 'moderate material always has therapeutic effect but excess material may be poison', bee venom in good dose can be a precious good medicine.
Leptucin, MCD, and adolapine peptides are used in analgesic and anti-inflammatory ointments in the fields of pharmaceuticals, medicine, cellular and molecular biology, and apitherapy. One of the main problems in the treatment of pain is drug dependence and addiction. On the other hand, the current drugs cause serious problems in the liver and kidneys. The presence of pain-relieving and anti-inflammatory peptides by inhibiting different receptors, regulating cytokines, inhibiting cyclooxygenase, and also changing the process of sodium and potassium channels inhibits pain and inhibits inflammation. Another advantage of this invention is the separation and use of peptides instead of the venom itself, which eliminates complications and problems caused by other problems caused by the venom. On the other hand, the current formulation does not have any toxicity even in higher doses and is not dependent and addictive. On the other hand, the duration of effect and the speed of effect are more than other available drugs. Among the other innovations of this formulation, we can mention its scope and usage and packaging and production in various pharmaceutical forms.
Inflammation and pain are one of the main problems in the treatment of many diseases. Meanwhile, more than a third of the world's population suffers from constant or frequent pain. Chronic and persistent pain is one of the main reasons people go to medical and clinical care centers. On the other hand, due to heavy sports activities and improper diet, pains such as back and leg pain have increased. The existing treatments to solve this issue are mostly chemical and on the other hand, they cause many side effects such as liver damage, breathing problems, intolerance and most importantly, they are addictive. Also, some of these drugs also cause common drug resistance. On the other hand, another major problem of these drugs is toxicity in higher doses and low effect per unit of time.
On the other hand, in some cases, the animal venom itself is used in total form, which in these cases, due to the lack of specificity and due to the presence of some substances, including other peptides and proteins, causes serious problems such as anaphylaxis. So that these problems can sometimes even lead to death. But the use of peptides is purified and in appropriate doses, in addition to having no risks, it is introduced as next generation drugs. Since peptide molecules are more targeted and also have fewer side effects and in most clinical cases are free of side effects than chemical molecules, analgesic peptides can be a new revolution in pain relief. The existing ointment is formulated with the combination of Adolapine, MCD (Mast cell degranulating) and Leptucin peptides, which do not have the side effects of existing drugs. On the other hand, it is not addictive and does not give drug resistance. This compound does not have toxicity in higher doses, and on the other hand, compared to existing drugs, it has a faster effect and a longer duration of effectiveness and toxin than other existing drugs, and it has high structural stability. Due to the separation at the peptide level, this combination has also solved the problems of other substances in the venom that cause any allergies and problems. 
Solution of problem
Method:
First, bee venom was collected according to the protocol (Benton AW et al. 1963) using a glass plate and using a low electric shock. Scorpion venom was also collected using a low shock according to the protocol (Ozkan and Filazi. 2004) using A low voltage was collected with the help of electric shock. The venom peaks were determined by HPLC. Then the desired peptides were separated by RP-HPLC. For separation by reverse phase high performance liquid chromatography (RP-HPLC ) using the protocol with the specifications of PMB k-100, UV detector model 2550, 20 microliter loop, 100 c18 column and chrome gate version 3.3.2 software, a linear gradient of acetonitrile from 0 to 90% (absolute acetonitrile containing 0.1 percentage of TFA) was applied with a flow rate of 1 ml/min for 90 minutes, and they were purified up to 95%. The findings were carried out and confirmed in Pasteur Institute of Iran, Tehran. Finally, the peptides were lyophilized. To compare the efficiency of the peptides, in Supernatant and pellet at four pH protocols, SDS-PAGE was performed using 15% polyacrylamide gel. The best deprotection method protocol to evaluate It was further considered in RP-HPLC. Then the peptide concentration was measured by spectrophotometer with UV estimation at a wavelength of 280 nm. The yield percentage was calculated using the following formula: 
Ointment production:
Adolapine, MCD, and leptucin peptides from bee venom and Hemiscorpius lepturus scorpion venom are utilized to make this analgesic and anti-inflammatory ointment (extracted with a cold compress according to the protocol). Along with 1000 ppm of olive oil, these peptides are utilized, as well as fillers like eucerin, lanolin, honey-based substances, moisturizing agents like alexin, absorption agents like aloe vera juice, penetrants like salicylic acid or alginate, and supplements like Shikonin from Echium candicans or Levisticum. Due to the inclusion of several peptides and apitoxins, including MCD, Melittin, Apamin, Secapin, Adolapine, etc., honey bee venom possesses anti-inflammatory characteristics. While the adolapine peptide also acts as an analgesic, the MCD peptide is a potent anti-inflammatory peptide. Additionally, leptucin peptide possesses potent analgesic properties.
They will be blended in accordance with each peptide's ED and LD50. On the other hand, depending on the sort of production, these percentages may vary. Leptucin serves as the main peptide in this work, with adolapine and MCD peptides added as side peptides to enhance the synergistic effects of the combination. In accordance with the findings and studies based on the estimation of LD50 and ED dosages, it is possible to mix roughly 2% of the total product of leptucin peptide, 1% of adolapine peptide, and 1% of MCD peptide. However, these quantities may alter depending on the therapeutic necessity and the usage circumstances.
Medication evaluation test:
Hot plate test:
Wistar female rats weighing about 250 grams were utilized to evaluate the mixture. In this experiment, the mice were first placed on a hot plate set to 55 °C, and their pain reactions—including the removal of their arms and legs, licking, and/or shaking—were observed. Six groups of mice with normal pain tolerance were randomly assigned (four different doses of peptides, a negative control, and a positive control). The intraperitoneal administration of dosages of 0.16, 0.32, 0.48, and 0.64 mg/kg (4, 8, 12, and 16 g/rat; each rat was regarded as an average of 200–250 g) to six mice in each group. As negative and positive controls, respectively, normal saline and morphine (10 mg/kg) were utilized. Each group's response to pain was recorded after 30 minutes. The 40-second cutoff was established. At intervals of 30 to 300 minutes following delivery, time periods for recording pain responses were defined. Each time point's analgesic activity was calculated in accordance with the protocol (Moghadasi, Z et al. 2018).
MTT assay:
The MTT assay was carried out on the HEK-293 cell line to determine the cytotoxic effect of peptides. The cells (2 x 104 each) were cultured in 96-well plates with 10% FBS-supplemented DMEM medium before being incubated for 24 hours at 37 °C in a 5% CO2 and 95% air environment. In DMEM medium, peptides were produced at concentrations ranging from 16 to 0.25 g/ml, administered to each well, and then incubated for 24 hours. A 10 μl solution of freshly prepared MTT (5 mg/ml in PBS) was added to each well and incubated for another 4h. Following that, 100 μl of isopropanol was added, and the plates were gently shaken. Viability was assessed after the absorbance at 570 nm wavelength was recorded using an ELISA reader (ELx808, BioTek, Winooski, VT, USA). This was carried out in accordance with the protocol (Aghazadeh, H. 2019).
Histopathological study:
In this test, the compounds were injected intraperitoneally in doses of 0.16, 0.32, 0.48 and 0.64 mg/kg to mice. These doses were selected as drugs according to their effectiveness. And the mice were sacrificed after 48 hours of administration of peptides. Brain and liver tissues were collected and fixed in 10% buffered formalin solution. After 4 days, the organ sections were routinely processed in an automated tissue processor and embedded in paraffin wax. Tissue sections were prepared and examined with hematoxylin and eosin stain (H&E) for histopathological studies with a light microscope. In this test, the focus was more on the brain to test the effect of peptides on the necrosis of neuronal cells. This work was important for us because peptides are more related to neuronal receptors.
rotarod test:
A 1.5-inch rotarod device was employed in the test. The mice were first put inside this equipment. This machine was run for 5 minutes at a speed of 4 rpm. The mice that failed to make it through the test were taken out of the group, and the remaining mice received injections of the peptide mixture at doses of 0.32, 0.48, and 0.64 mg/kg. The second rotarod test was conducted 30 minutes after the first. Mice that could not perform the test were known as "mice with movement defects," which were not found in this test. The results of this test show that there are no movement issues or disturbances with the current mixture.
The term "plasticity" is used to describe the changes that take place in the nervous system. Increased functional activity of neurons in the pain pathway can eventually result from changes in brain structure, connections between neurons, and the quantity and characteristics of neurotransmitters, receptors, and ion channels. On the other hand, plasticity can weaken the body's natural pain-inhibitory mechanisms, which ultimately makes pain worse. By boosting excitatory or suppressing inhibitory pathways, injury, inflammation, and disease can all induce brain plasticity and intensify pain. Flexibility can lead to long-term changes that could be permanent or short-term alterations that span anywhere from a few minutes to several hours. The primary output from the dorsal horn to the brain is formed by projection neurons in layers I and V. These neurons are the starting point of a variety of ascending pathways, such as the spinothalamic and spinoreticulothalamic pathways, which send pain signals to the thalamus and brain stem, respectively.
Adolapine, which is present in this composition, functions as a cyclooxygenase inhibitor and a naturally occurring inhibitor of prostaglandins. MCD and leptucin peptides also play a role by acting on sodium channels, calcium channels, N-Methyl-D-aspartate receptors, and neurotensin receptors via nicotinic acetylcholine receptors (- and O-conotoxins), sodium (contulakins). Cysteine, which is abundant in leptucin and alters sodium and potassium channels, has immediate and lasting effects.
These peptides reduce inflammation by blocking interleukins, inflammatory pathways, tumor necrosis factors, and pro-inflammatory molecules. They also reduce pain by blocking cyclooxygenase and causing alterations in sodium and potassium channels.
This ointment is used to regulate the cell cycle, the different cytokines involved in this cycle, and to activate the related genes. So that three courses are needed to finish the healing process. Depending on whether it is used topically or through injection, the amount consumed and the percentage of use vary. However, the results of the animal and human phase studies indicate that 200 ppm of the total peptides may be helpful. Due to the medication's slower absorption when applied topically, this quantity may be larger in some circumstances. On the other hand, chemicals like salicylic acid increase permeability when they are present.
Advantage effects of invention
Quick healing of joint pain and reducing inflammation.
The lack of dependence and addiction of this drug compared to existing drugs that have similar problems.
Not causing problems such as MDR and drug resistance.
No toxicity in high doses.
Easy and cost-effective preparation of the product.
Production and processing based on local knowledge of the product.
Easier to use in desired places.
The complete naturalness of the product.
Reducing treatment costs.
Quick effect and help in quick and long-term recovery compared to other drugs.
Use in cases of various types of pains, including sciatica pains, as well as in other types of joint diseases.
The possibility of formulation in other forms such as ointment, spray pad, hydrogel, poultice, etc.
No need for high technology and facilities in production.
High ability to penetrate the lower layers of the skin and reach the muscles and joints and its quick effect.
Examples
This ointment is safe for people and comes from a natural source. Secondly, it can be a good idea to produce a low-dose mixture of leptucin, adolapine, and MCD peptides from scorpion and bee venom to induce an anti-inflammatory effect and penetrate inflamed tissues. This will help to lessen all types of pain and inflammation. Because these substances can block the expression of interleukins and tumor necrosis factors, lowering the inflammatory pathway. Nevertheless, they quickly decrease pain by blocking cyclooxygenase and altering sodium-potassium channel function. Using such a formulation with the current composition has never been done before. The synergistic usage of all three peptides and the use of the peptide in the ointment in a targeted and specific form are the two most crucial aspects of this innovation. Additionally, a spray version of this mixture can be created and applied.
[Chart. 1]
Chart. 1. HPLC test results for hemiscorpius lepturus venom.
Chem. 2. Three-dimensional structure of leptosin.
[Chart. 2]
Chart. 2. CD spectrum of leptosin.
[Chart. 3]
Chart. 3. Hot plate measurement
[Chart. 4]
Chart. 4. MTT test results
B
 A
D
 C
F
 E
H
 G
Chem. 2. Histopathology test results
This ointment has undergone all essential laboratory testing, including animal experiments, and approval has been requested for clinical trials. It was developed and produced in a laboratory setting. This compound has been used with consumer satisfaction because it can be applied topically, and the outcomes are remarkable.

Claims (9)

  1. The formulation of the analgesic and anti-inflammatory ointment includes Adolapine and MCD peptides from bee venom as well as Leptucin peptide from Hemiscorpius lepturus scorpion venom, along with olive oil and fillers (Eucerin, lanolin, honey base), moisturizing agents (alexin), absorbing agents (aloe vera juice), penetrants (salicylic acid or alginate), as well as supplements (shikonin from Acium or levisticum).
  2. According to claim 1, the peptides from scorpion and bee venom have been produced, purified, and added in dry form.
  3. According to claim 2, initially, bee venom was collected according to protocol (Benton AW et al., 1963) using a glass plate and with the help of a low electric shock.
  4. According to claim 2, scorpion venom was also collected using low-voltage electric shocks according to the protocol (Ozkan and Filazi, 2004) with the help of a low-voltage electric shock.
  5. According to claims 3 and 4, using HPLC, the peaks of venom were determined. Peptides were then separated using RP-HPLC.
  6. According to claim 5, for separation by reverse-phase high-performance liquid chromatography (RP-HPLC) using the protocol with the specifications of a k-100 pump, UV detector model 2550, 20-microliter loop, C18 column, and chrom gate software version 3.3.2. Acetonitrile that was absolute and included 0.1% TFA was applied in a linear gradient from 0 to 90% for 90 minutes while also being purified to 95%.
  7. They will be blended in accordance with each peptide's ED and LD50. On the other hand, depending on the sort of production, these percentages may vary. In this work, the main peptide is leptucin, and adolapine and MCD peptides are added as side peptides to synergize the effects of the composition.
  8. According to claim 7, LD50, and ED dosages, roughly 2% of the total product of leptucin peptide, 1% of adolapine peptide, and 1% of MCD peptide can be combined. However, these quantities may alter depending on the therapeutic necessity and the usage circumstances.
  9. This work can be generated as sprays, ointments, hydrogel pads, and other therapeutic products. The use of this ointment includes regulating the cell cycle and the many cytokines involved in this cycle, as well as activating the relevant genes. Three courses are required to complete a full recovery. Depending on whether it is used topically or through injection, the amount consumed and the percentage of use vary.
PCT/IB2023/053414 2023-04-04 2023-04-04 Formulation of analgesic and anti-inflammatory ointment containing the leptucin, mcd and adolapine peptides WO2024042376A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017052706A (en) * 2015-09-07 2017-03-16 一丸ファルコス株式会社 Cosmetic composition containing granules

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017052706A (en) * 2015-09-07 2017-03-16 一丸ファルコス株式会社 Cosmetic composition containing granules

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BAGHERI-ZIARI SEDIGHEH, SHAHBAZZADEH DELAVAR, SARDARI SOROUSH, SABATIER JEAN-MARC, POOSHANG BAGHERI KAMRAN: "Discovery of a New Analgesic Peptide, Leptucin, from the Iranian Scorpion, Hemiscorpius lepturus", MOLECULES, vol. 26, no. 9, 28 April 2021 (2021-04-28), CH , pages 1 - 17, XP093145332, ISSN: 1420-3049, DOI: 10.3390/molecules26092580 *
HANSON JENNIFER M., MORLEY J., SORIA‐HERRERA C.: "Anti-inflammatory property of 401 (MCD-peptide), a peptide from the venom of the bee Apis mellifera (L.)", BRITISH JOURNAL OF PHARMACOLOGY, vol. 50, no. 3, 1 March 1974 (1974-03-01), UK , pages 383 - 392, XP093145337, ISSN: 0007-1188, DOI: 10.1111/j.1476-5381.1974.tb09613.x *
SHKENDEROV, S. ; KOBUROVA, K.: "Adolapin - A newly isolated analgetic and anti-inflammatory polypeptide from bee venom", TOXICON, vol. 20, no. 1, 1 January 1982 (1982-01-01), US , pages 317 - 321, XP025539817, ISSN: 0041-0101, DOI: 10.1016/0041-0101(82)90234-3 *

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