EP4308101A1 - N-acétyl cystéine pour une utilisation neuraxiale en tant qu'inhibiteur du récepteur de la tyrosine kinase trka pour le traitement de la douleur aiguë et chronique - Google Patents

N-acétyl cystéine pour une utilisation neuraxiale en tant qu'inhibiteur du récepteur de la tyrosine kinase trka pour le traitement de la douleur aiguë et chronique

Info

Publication number
EP4308101A1
EP4308101A1 EP22723443.2A EP22723443A EP4308101A1 EP 4308101 A1 EP4308101 A1 EP 4308101A1 EP 22723443 A EP22723443 A EP 22723443A EP 4308101 A1 EP4308101 A1 EP 4308101A1
Authority
EP
European Patent Office
Prior art keywords
nac
ngf
trka
treatment
pain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22723443.2A
Other languages
German (de)
English (en)
Inventor
Piercarlo Fantucci
Stefano Govoni
Alessia Angela PASCALE
Nicoletta MARCHESI
Massimo Allegri
Emilio VANOLI
Laura CALVILLO
Paolo Reggiani
Valentina PACE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Redyneuheart Srl
Original Assignee
Redyneuheart Srl
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Redyneuheart Srl filed Critical Redyneuheart Srl
Publication of EP4308101A1 publication Critical patent/EP4308101A1/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A61P29/02—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] without antiinflammatory effect

Definitions

  • the present invention relates to the field of medical preparations containing organic active ingredients, in particular it relates to a new use and route of administration of N-acetylcysteine (NAC) based on the discovery of a new mechanism of action of the molecule and the use thereof for the treatment of acute and chronic pain.
  • NAC N-acetylcysteine
  • Nerve growth factor is a protein which regulates the growth and survival of sympathetic and sensory nerve cells (neurons). Therefore, NGF belongs to a family of growth factors called neurotrophins.
  • NGF binds with great affinity and activates the TrkA tyrosine kinase receptor.
  • TrkA receptor dimerizes and autophosphorylates, thus initiating an important signalling process for the growth and survival of sympathetic neurons.
  • NGF nerve growth factor
  • NGF has also been identified as an important pain mediator, in particular chronic pain, and as such is to be considered an important target in the search for new pharmacological strategies for treating pain.
  • An approach in this regard employs the sequestration of NGF using anti-NGF antibodies such as Tanezumab (Pfizer), Fulranumab (Johnson and Johnson), REGN475/SAR164877 (Regeneron together with Sanofi Aventis) or medi578 (AstraZeneca).
  • anti-NGF antibodies such as Tanezumab (Pfizer), Fulranumab (Johnson and Johnson), REGN475/SAR164877 (Regeneron together with Sanofi Aventis) or medi578 (AstraZeneca).
  • Acetylcysteine (or N-acetylcysteine, NAC) is an N-acetylated derivative of the sulfur amino acid cysteine.
  • the drug is used (in a dose of about 3- 9 mg/kg) orally or as an expectorant mucolytic and in case of tear deficiency or for its important systemic antioxidant action (an important patho-physiological element in the genesis of pain).
  • the drug is also considered a lifesaver in the event of an overdose of paracetamol, especially to prevent the development of fulminant hepatitis which is often the result.
  • paracetamol intoxication the intravenous administration route is used (150 mg/kg attack doses).
  • NAC is capable of inhibiting the autophosphorylation of TrkA, acting at the level of the cysteine groups thereof including in particular, but not exclusively, the 300 and 345 cysteines.
  • NAC is used as an antagonist of the TrkA tyrosine kinase receptor in the treatment and/or management of acute and chronic pain conditions.
  • NAC is used even more preferably in the treatment and/or management of pain by neuraxial infusion.
  • NAC is also used in the treatment and/or management of acute and chronic pain associated with high levels of NGF and chronicity after an episode of acute pain.
  • NAC is preferably administered at the neuraxial level, i.e., where the condition due to hyperactivity of the nerve growth factor (NGF) manifests itself.
  • NAC neuraxial (intrathecal or intraspinal).
  • NAC according to the present invention is suitable for use in the treatment and/or management in single-administration and in co-administration with local anaesthetic (ropivacaine or bupivacaine) of acute postoperative and radiculopathic pain and chronic pain such as root, spine, spasticity central pain and as an adjuvant in osteoarthritic pain, rheumatoid arthritis, interstitial cystitis, chronic pelvic pain syndrome, and cancer pain.
  • local anaesthetic ropivacaine or bupivacaine
  • the effective dose of said NAC to be administered neuraxially in adult patients is comprised between 0.5 and 15 mg/Kg, preferably 12 mg/Kg, possibly in combination with the local anaesthetic.
  • NAC according to the present invention can be used in the treatment and prophylaxis of the above conditions in animals such as primates, pigs, ruminants (cows, sheep, goats), horses, cats, dogs, poultry (for example, chickens, ducks, geese, quails, pigeons, turkeys or ornamental birds), as well as productive and ornamental fish, reptiles and amphibians.
  • animals such as primates, pigs, ruminants (cows, sheep, goats), horses, cats, dogs, poultry (for example, chickens, ducks, geese, quails, pigeons, turkeys or ornamental birds), as well as productive and ornamental fish, reptiles and amphibians.
  • pain management refers to all treatment regimens which do not completely eliminate pain from the patient, but reduce pain to improve or significantly improve the patient's quality of life.
  • compositions according to the invention are, among others, the preparations for injection and infusion in the form of sterile solutions, suspensions, emulsions, lyophilisates or powders.
  • NAC non-pharmaceutical agent
  • other pharmaceutically active compounds such as local anaesthetics (e.g., ropivacaine).
  • opioids such as morphine and fentanyl (opioids indicated for neuraxial use).
  • the minimum amount of NAC of the invention to be administered is a therapeutic amount.
  • therapeutically effective amount means an amount of compound which prevents the onset of or alleviates symptoms, manages, stops the progression and/or eliminates a disease, a disorder or a condition by virtue of the inhibition of the TrkA receptor, acting in diseases linked to high levels of NGF.
  • doses of NAC are administered as a single dose. In other embodiments of the invention, doses of NAC are administered every day or every 3, 5, 7 or 10 days.
  • Fig. 1 - shows the NGF-TrkA complex.
  • Fig. 2 - shows the characteristic disulphide bridges between the 6 beta-sheets of TrkA.
  • Fig. 3 - shows the results of in vitro assays of inhibition of the phosphorylation of TrkA in the presence of 0.4 mM DTT (dithiothreitol, a non-specific reductant).
  • Fig. 4 - shows the results of in vitro TrkA phosphorylation inhibition assays
  • Fig. 5 - shows the result of the NAC-TrkA in silico docking.
  • Fig. 6 - shows the result of the in silico covalent binding of NAC-TrkA.
  • Fig. 7 - shows the results of a formalin test on mice and in particular the effect of intrathecal NAC (5 microlitres, containing 0.3 mg of NAC) in the mouse against the pain induced by the administration of formalin in the hind paw.
  • NAC enzymatic immunosorbent assay
  • This ELISA kit is commercially available, for example from Cell Signalling Technologies under the name PathScan® Phospho-TrkA (Tyr674/675) Sandwich ELISA Kit (catalogue number 7212).
  • the test is used according to the manufacturer's instructions by exposing the PC 12 and/or SH- SY5Y cell lines, as cell types expressing TrkA receptors, to the compounds of the invention.
  • the cells grow in a medium suitable for cell culture (for example: 60-mm Falcon ⁇ ) at a certain cell density (example: 0.5-1x106 cells/ml) in a specific and complete culture medium, as for example reported in Marchesi et al. (J Cell Physiol. 2014; 229(11): 1776-86) for the SH-SY5Y cell line and Rossi et al. (Bioorg Med Chem. (2011); 19 (21):6210-24) for the PC12 cell line.
  • the procedure for the adhering cells is as follows: when the cell culture reaches a confluence of 80-90%, the culture medium is changed with a fresh medium containing a low percentage of bovine serum (Fetal Bovine Serum/FBS) and for a predetermined period of time. After washing with a phosphate buffered saline, 0.4/0.5 ml of cold IX Cell Lysis Buffer (specific buffer name) containing protease inhibitors are added to each plate, in contact with the cells, for 5 minutes in ice. The cells are removed from the plate with a scraper and transferred to an appropriate tube stored in ice where they will be sonicated.
  • FBS bovine Serum/FBS
  • the suspension is centrifuged for 10 minutes (at 13- 14000 rpm) at 4°C and the supernatant is transferred to a new tube and stored at -80°C.
  • the percentage of phosphorylated TrkA after exposure to the compounds of the invention can be compared to a calibration curve performed using NGF as a reference.
  • the compounds having antagonistic activity prevent activation by NGF.
  • NAC neurodegenerative disease 2019
  • a nociceptive and peripheral neuropathic pain model is used. Such a model involves the intra-plantar injection of formalin (or possibly other substances), which induces an increase in the thermal and mechanical sensitivity at the injection site (primary hypersensitivity). Thereafter, the animals are treated one or more times with different doses of NAC.
  • the ability of NAC to treat pain can be observed through behavioural and pain assessment tests.
  • TrkA and NGF form a single complex consisting of two NGF molecules and two TrkA molecules, forming two homo- dimers.
  • the present inventors have mapped the short contact distances between the two TrkA molecules and the two NGF molecules to find the smallest example of TrkA-NGF complex. This research has shown that the largest number of short-range interactions occurs between the first NGF molecule and the second TrkA molecule (Fig. 1).
  • DTT dithiothreitol
  • cysteines were observed at the binding interface between TrkA and NGF, and it was thus possible to verify that such cysteine residues are engaged in a disulphide bridge (Fig. 2).
  • the cysteines (cys) ascribed in this bond are in position 300 and 345.
  • the disulphide bridges have a structural role within the proteins, as mentioned above, i.e., they serve to maintain the correct three-dimensional structure of the protein.
  • the two cysteines form a well-characterized disulphide bridge between two loops of a beta-sheet; the cys in position 300 in sheet B and the cys in position 345 in sheet E. Therefore, it is possible to assume that such an interaction serves to maintain the receptor structure and its correct conformation in space intact.
  • the docking highlighted an ever-increasing affinity of DTT for TrkA as the DTT chain grows (affinity for NGF tri-DTT > bi-DTT > mono-DTT).
  • the NAC molecule was tested in vitro in the cell model and also in this case an inhibition of the phosphorylation activity of TrkA (pTrkA) occurred following response with NGF.
  • pTrkA an inhibition of the phosphorylation activity of TrkA
  • Fig. 4 it was possible to observe how the inhibition of pTrkA after NAC + NGF co-treatment with respect to NGF alone resulted in a 40% inhibition of phosphorylation at a NAC concentration of 20mM (Fig. 4).
  • This fact combined with the reducing power of NAC comparable to that of DTT, leads to the belief that the inhibitory activity of the latter molecule is due to the reduction of the disulphide bridge as observed with the reducing molecule DTT.
  • NAC is capable of binding TrkA near the disulphide bridge formed by CYS_300 and CYS_345.
  • the calculated binding AG is 12.8 kcal/mol.
  • Figure 5 shows the result of the docking, the NAC sulphur is located at a distance of 4.0 A and 5.3 A from the two sulphur atoms of the disulphide bridge.
  • Control group (8 animals): treated intrathecally with injectable H20 (vehicle), corresponding to the columns "CTRL” in figure 7;
  • Intraperitoneal administration of anaesthetic 5 m ⁇ Zoletil 20 mg/ml
  • the intrathecal administration was performed as follows:
  • the animal was anaesthetized with Isoflurane and placed on a flat operating surface in the prone position, the back was disinfected with a skin antiseptic and shaved, then a nitrile glove (cut in half) was placed over the head and on the upper part of the animal's body in order to avoid stress.
  • the animal was gripped firmly by the iliac crest using the thumb and index finger and the skin was stretched over the spine to identify on the back, by palpation, the spinal process L5 and L6 representing the cutaneous injection site.
  • the column was gently curved to open the intervertebral space between the spinous process L5 and L6 and a 10 m ⁇ syringe was inserted vertically, connected to a sterile, disposable 30Gxl/2 needle. Once contact with the bone of the spine was detected, the angle of the syringe was reduced to about 30° and the syringe was carefully inserted into the intervertebral space. The puncture of the dura mater is reliably indicated by a reflexive movement of the tail or by the formation of an "S" shape by the tail.
  • Formalin test subcutaneous administration (right paw) of 20 m ⁇ Formalin 5% in 0.9% NaCl
  • the results show an acute tolerability and an analgesic effect through the intrathecal route of administration of NAC.
  • NAC was administered through intratechal admninistration that is exactly a neuroaxial administration.
  • This route of administration allowed to deliver at spinal level amounts of NAC (300 micrograms) consenting to tribute the effect of NAC to the inhibition of the NGF activation of TrkA through the interference with the recognized cysteine bond at the indicated residues.
  • Our animal model demonstrates the clinical activity of neuraxial NAC in reducing pain.

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  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Epidemiology (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

La N-Acétyl cystéine (NAC) destinée à être utilisée en tant qu'antagoniste du récepteur de la tyrosine kinase TrkA dans le traitement et/ou la gestion des états douloureux aigus et chroniques dans lesquels il y a une hyperactivité du facteur de croissance des nerfs (NGF), ladite NAC étant administrée localement où se produit l'état due à l'hyperactivité du facteur de croissance des nerfs (NGF).
EP22723443.2A 2021-04-21 2022-04-20 N-acétyl cystéine pour une utilisation neuraxiale en tant qu'inhibiteur du récepteur de la tyrosine kinase trka pour le traitement de la douleur aiguë et chronique Pending EP4308101A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT202100010181 2021-04-21
PCT/EP2022/060375 WO2022223590A1 (fr) 2021-04-21 2022-04-20 N-acétyl cystéine pour une utilisation neuraxiale en tant qu'inhibiteur du récepteur de la tyrosine kinase trka pour le traitement de la douleur aiguë et chronique

Publications (1)

Publication Number Publication Date
EP4308101A1 true EP4308101A1 (fr) 2024-01-24

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Application Number Title Priority Date Filing Date
EP22723443.2A Pending EP4308101A1 (fr) 2021-04-21 2022-04-20 N-acétyl cystéine pour une utilisation neuraxiale en tant qu'inhibiteur du récepteur de la tyrosine kinase trka pour le traitement de la douleur aiguë et chronique

Country Status (2)

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EP (1) EP4308101A1 (fr)
WO (1) WO2022223590A1 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ITRM20050290A1 (it) * 2005-06-07 2006-12-08 Lay Line Genomics Spa Uso di molecole in grado di inibire il legame tra ngf e il suo recettore trka come analgesici ad effetto prolungato.
US20120157405A1 (en) * 2010-12-19 2012-06-21 White Iii John B Methods and Compositions for the Treatment of "Burning Feet Syndrome"

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