Nanoparticle composition for treating neuropathic pain

Abstract

The present invention relates to a novel and innovative composition for treating neuropathic pain (NP). Specifically, the present invention relates to HfO2 nanoparticles and / or aggregates of HfO2 nanoparticles, compositions containing said nanoparticles and / or aggregates of nanoparticles, and their use in the treatment of NP.

Description

Technical Field

[0001] The present invention relates to novel and innovative compositions for treating neuropathic pain (NP). Specifically, the present invention relates to nanoparticles and / or aggregates of nanoparticles, compositions comprising said nanoparticles and / or aggregates of nanoparticles, and their use in the treatment of NP.

Background Art

[0002] Neuropathic pain (NP) is characterized by abnormal hypersensitivity to stimuli (hyperalgesia) and pain responses to non-noxious stimuli (allodynia). In these patients with hyperalgesia, the pain sensation produced by the pain-causing stimulus is increased, while in patients with allodynia, the pain sensation caused by a stimulus that normally does not cause pain is increased. NP patients may also suffer from paresthesia (abnormal sensations such as being pricked by a needle, tingling, itching), or even sensory loss. NP has a persistent and / or episodic (paroxysmal) element. The latter resembles a puncture or an electric shock.

[0003] Many different conditions can cause NP, for example, metabolic disorders such as diabetes, viral infections (e.g., postherpetic neuralgia), and autoimmune diseases that affect the central nervous system (CNS) and peripheral nervous system (PNS) such as multiple sclerosis and Guillain-Barré syndrome, respectively. Neuropathic pain is common in cancer patients as a direct result of cancer on the peripheral nerves (e.g., compression by a tumor), radiation injury, or surgery, or as a side effect of chemotherapy (chemotherapy-induced peripheral neuropathy (CIPN)). For example, oxaliplatin, a platinum-based chemotherapeutic agent, is commonly used in the treatment of various types of cancer. Oxaliplatin is currently approved in many countries as an initial treatment for colorectal cancer. However, the clinical value of oxaliplatin is reduced by acute and chronic forms of peripheral neuropathy such as mechanical hyperalgesia, which appear as side effects in both humans and rodents. This peripheral neuropathy caused by oxaliplatin is the most common treatment-related dose-limiting toxicity, and there is currently no available treatment.

[0004] Other causes of neuropathic pain include injury to the nervous system due to trauma such as spinal cord injury (SCI), peripheral nerve injury including post-traumatic NP or post-surgical NP. Complications such as post-traumatic and / or post-surgical nerve tumors may also cause NP.

[0005] Certain inflammatory diseases, genetic neuropathies, and channelopathies also exist [Colloca L, Ludman T, Bouhassira D, et al. (2017) Neuropathic pain. Nature Reviews Disease Primers 3:17002]. The prevalence of neuropathic pain in the general population is estimated to be 3% - 17% [Cavalli, E., et al., (2019) The neuropathic pain: An overview of the current treatment and future therapeutic approaches, Int. J. Immunopathology & Pharmacology, Vol. 33: 1-10].

[0006] Neuropathic pain may be associated with ectopic centripetal discharge. Ectopic centripetal discharge is caused by the high excitability and repetitive firing ability of the affected neurons / nerves, potentially due to changes in the membrane potential threshold caused by disease / disorder [Amir, R. et al., (1999) Membrane Potential Oscillations in Dorsal Root Ganglion Neurons: Role in Normal Electrogenesis and Neuropathic Pain, The Journal of Neuroscience, 19 (19):8589-8596]. Changes in the vector transport of Na + channels and / or Na + channel upregulation-induced Na +An increase in conductance is implicated as a factor in the generation of neuropathic (ectopic) afferent discharges in animals and humans.

[0007] Therefore, we understand that NP can occur due to disorders of the peripheral nervous system or the central nervous system (brain and spinal cord). Thus, NP may be classified as peripheral NP, central NP, or mixed (peripheral and central) NP.

[0008] Painful polyneuropathies such as trigeminal neuralgia, painful radiculopathy, postherpetic neuralgia, chemotherapy-induced peripheral neuropathy (CIPN, also called chemotherapy-induced neuropathic pain or CINP), and diabetic polyneuropathy, as well as pain due to peripheral nerve injury, are classified as peripheral NP. On the other hand, central NP includes pain states such as neuropathic pain after spinal cord injury (SCI), central pain in multiple sclerosis, and pain after central stroke (see Figure 4 in Finnerup, N.B, et al. (2021) Neuropathic Pain: From Mechanisms To Treatment, Physiol Rev 101: 259-301).

[0009] Current clinical solutions for treating NP can be either systemic or local therapies. For example, gabapentinoids, tricyclic antidepressants (TCA), and selective serotonin–norepinephrine reuptake inhibitors (SNRI), all of which are systemic therapies, are usually considered first-line treatments.

[0010] The gabapentinoids gabapentin and pregabalin bind to voltage-dependent channels to reduce Ca 2+ influx into cells and are used in the treatment of diabetic pain, postherpetic neuralgia, SCI, and phantom limb syndrome. However, both gabapentin and pregabalin are associated with side effects such as drowsiness, vertigo, peripheral nerve swelling, and blurred vision. 2+

[0011] TCA is used in the treatment of neuropathic pain, postherpetic neuralgia, central postpartum pain, and pain after SCI. However, TCA and SNRI are contraindicated in patients with heart disorders. They are also associated with some side effects such as nausea and drowsiness. Opioids such as tramadol and tapentadol have been proposed as secondary treatment for NP, but are associated with side effects such as nausea / vomiting, constipation, and drowsiness. Drug addiction / dependence and drug abuse also constitute a major problem with opioid treatment.

[0012] The topical therapies lidocaine and capsaicin are used for the topical treatment of neuropathic pain and are generally applied as patches. Capsaicin (the component that makes chili peppers spicy) is a vanilloid receptor 1 agonist. The application of capsaicin-containing patches is performed by a physician, may be painful, and skin burning sensations are frequently reported [https: / / www.vidal.fr / medicaments / qutenza-179-mg-patch-cutane-gel-nettoyant-93564.html]. Single application is designed to relieve pain for up to approximately three months.

[0013] The tertiary treatments for NP include systemic therapies - tramadol, strong opioids (morphine and oxycodone), and locally applied botulinum toxin A (BTX-A). Intradermal injection of BTX-A is currently used in the treatment of chronic local painful neuropathy. Botulinum toxin A can be administered by multiple intradermal injections. The painful area is divided into a checkerboard of multiple sites, and usually 5 U of BTX-A is injected into each site [Park, JH., et al. (2017) Botulinum toxin for the treatment of neuropathic pain, Toxins, 9, 260]. However, botulinum toxin A can cause muscle paralysis and thus may affect the quality of life [Mittal, SO et al. (2016) Botulinum Toxin Treatment of Neuropathic Pain, Seminars in Neurology. 36 (1): 73-83].

[0014] Overall, despite the treatments listed above, neuropathic pain is difficult to treat, and only about 40 - 60% of patients achieve partial relief [Dworkin, RH, et al. (2007) Pharmacologic management of neuropathic pain: evidence-based recommendations, Pain, 132 (3): 237-51].

[0015] Several physical approaches are being clinically evaluated for patients with refractory neuropathic pain. These approaches include non-invasive transcranial brain stimulation techniques such as repetitive transcranial magnetic stimulation (rTMS), which generates an electric current in the cortex by a transient magnetic field. In transcranial direct current stimulation (tDCS), a low-voltage current (1-2 mA) is passed transcranially. TENS and PENS are peripheral electrical stimulation methods used in the treatment of NP. Repetitive spinal magnetic stimulation (SMS) and frequency-modulated electromagnetic stimulation (FREMS) are magnetic-based techniques that have been tested in the treatment of NP. A common drawback of these latter external stimulation techniques is that their effectiveness is temporally limited (days or weeks), and re-stimulation is usually required to maintain the effect. Spinal cord posterior column stimulation (spinal cord stimulation, SCS) is an invasive technique used to suppress the hyperexcitability of central nerve cells. The spinal cord posterior column is electrically stimulated with 50 Hz pulses from an implanted generator within the spinal cord posterior column. Patients undergoing this treatment are usually those who cannot relieve pain by other non-invasive methods. Zeng, H. et al. recently reviewed the effects of non-invasive neuromodulation (NINM) on peripheral diabetic neuropathy (PDN, or diabetic neuropathic pain, DNP), and found that the effects of NINM were higher with intensive protocols and in populations with tolerance or intolerance to analgesics [Zeng, H. et al. (2020) Non-invasive neuromodulation effects on painful diabetic peripheral neuropathy: a systematic review and meta-analysis. Scientific Reports 10].

[0016] Based on the above, new treatments are needed for patients suffering from NP, and researchers continue to explore new treatment methods. For example, patent application US20210402004A1 describes the use of targeted cerium-containing nanoparticles for treating NP by protecting microglia from oxidative activation. The authors thereof found that Ce 3+ / Ce 4+It has been shown that by converting the oxidation state, particles can remove reactive oxygen species (ROS) and downregulate microglia with overactivated state. However, the demerit of the latter system is the well-known cytotoxicity associated with the ROS activity of cerium oxide nanoparticles [Kumari, M, et al. (2014) Toxicity study of cerium oxide nanoparticles in human neuroblastoma cells. Int J Toxicol. 33(2):86-97.].

[0017] Therefore, there is still a need for new NP therapies with fewer side effects compared to currently available treatments as described above. Furthermore, there is a need for treatments that are effective over a relatively long period so that patients can experience pain relief for as long as possible, for example, for more than 3 months (the current average period for which local therapies are effective).

[0018] In particular, there is a need for the treatment of peripheral neuropathic pain conditions. There is a need for the treatment of patients suffering from peripheral diabetic neuropathy (PDN or DNP). There is a need for the treatment of patients suffering from postherpetic neuralgia. There is a need for the treatment of patients suffering from chemotherapy-induced NP, i.e., CIPN or CINP. There is a need for the treatment of patients suffering from NP after peripheral nerve injury which is post-traumatic and / or postoperative and / or due to a nerve tumor.

[0019] There is a need for the treatment of local neuropathic pain. There is a need for the treatment of refractory and / or chronic neuropathic pain.

[0020] There is a need for a treatment for patients suffering from peripheral neuropathic pain. There is a need for the treatment of patients suffering from NP caused by autoimmune diseases affecting the peripheral nervous system (PNS) such as Guillain-Barré syndrome. There is a need for the treatment of patients suffering from ectopic centripetal discharges associated with NP. Generally, there is a need for a treatment for the above patients such that the perceived pain is reduced.

[0021] The present invention advantageously provides a solution for treating NP, particularly patients suffering from peripheral NP, using non-toxic (chemically inert) and non-biodegradable nanoparticles different from those described in the prior art. This treatment is locally administered with minimal discomfort, minimally invasive, has a long-lasting effect, and has no side effects associated with currently available systemic therapies. Therefore, the present invention provides an advantageous solution for each of the above needs.

Summary of the Invention

[0022] In one aspect, the present invention relates to HfO2 nanoparticles or aggregates thereof for treating neuropathic pain (“NP”).

[0023] In an aspect, the present invention relates to a method for treating a patient suffering from neuropathic pain.

[0024] In one aspect of the present invention, the nanoparticles may be used to treat patients suffering from trigeminal neuralgia, neuropathic pain after peripheral nerve injury (including post-traumatic neuropathic pain, postoperative neuropathic pain, or neuropathic pain due to nerve tumors), painful diabetic polyneuropathy, human immunodeficiency virus (HIV), Fabry disease, mutations in sodium channel genes, autoimmune diseases (such as Guillain-Barré syndrome, vasculitis, chronic inflammatory demyelinating polyneuropathy, amyloidosis, frostbite, tumor-associated syndromes, or leprosy), chemotherapy-induced peripheral neuropathy (CIPN), postherpetic neuralgia, or painful radiculopathy.

[0025] In a particular aspect, the nanoparticles may be used to treat patients suffering from peripheral nerve injury (including post-traumatic neuropathic pain, postoperative neuropathic pain, or neuropathic pain due to nerve tumors), postherpetic neuralgia, painful diabetic polyneuropathy, CIPN, or HIV.

[0026] In a preferred aspect of the present invention, the nanoparticles may be used to treat patients suffering from chemotherapy-induced peripheral neuropathy (CIPN).

[0027] In another preferred embodiment of the present invention, the nanoparticles may be used for treating patients suffering from diabetic polyneuropathy, also identified herein as "painful diabetic polyneuropathy" or "peripheral diabetic neuropathy".

[0028] In another embodiment of the present invention, the nanoparticles may be used for treating patients suffering from neuropathic pain after peripheral nerve injury, including post-traumatic neuropathic pain, post-surgical neuropathic pain, or neuropathic pain due to nerve tumors.

[0029] In another embodiment of the present invention, the nanoparticles may be used for treating patients suffering from ectopic centripetal discharge associated with neuropathic pain.

[0030] Treatment is needed for patients suffering from peripheral neuropathic pain.

[0031] In another embodiment of the present invention, the nanoparticles may be used for treating patients suffering from local neuropathic pain.

[0032] In another embodiment of the present invention, the nanoparticles may be used for treating patients suffering from intractable and / or chronic neuropathic pain.

[0033] In one embodiment herein, the nanoparticle or nanoparticle aggregate material may be an insulating material having a relative permittivity ε ijk of 100 or less, wherein the metal is selected from insulating materials selected from Zr, Hf and / or Re (e.g., HfO2 and / or ZrO2).

[0034] In one embodiment herein, the nanoparticle or nanoparticle aggregate material may be a metal conductor selected from Ir, Pd, Pt, Au, and any mixtures thereof.

[0035] In another aspect of the present specification, the nanoparticle or nanoparticle aggregate material has adjacent sp in the structure 2 It can be an organic material having a hybrid carbon center (i.e., an aromatic ring containing a heteroatom (usually N or S) inside or outside the carbon double bond or ring). Preferred organic materials are selected from polyaniline, polypyrrole, polyacetylene, polythiophene, polycarbazole, polystyrene, and / or polypyrene. More preferred organic materials are poly(3,4-ethylenedioxythiophene) (PEDOT), poly(styrenesulfonic acid) (PSS), or a mixture thereof.

[0036] In one aspect of the present specification, when the nanoparticle material is a metal conductor, the median core size in the population of nanoparticles or nanoparticle aggregates is at least 45 nm.

[0037] In one aspect of the present invention, the core of the nanoparticle or aggregate of nanoparticles is coated with a biocompatible coating that provides a neutral or negative surface charge when measured in an aqueous solution having an electrolyte concentration of 0.001 - 0.2 M, a concentration of the nanoparticle material or nanoparticle aggregate material of 0.01 - 10 g / L, and a pH of 6 - 8.

[0038] In another aspect, the nanoparticles or aggregates thereof may be administered by intradermal and / or intraepidermal injection. The nanoparticles or aggregates thereof may be administered using a dissolving microneedle patch.

[0039] In another aspect of the present invention, the dose of the nanoparticles or aggregates thereof to be administered is preferably at least 60 μg / cm 2 of the skin surface to be injected.

[0040] In another aspect of the present invention, when the patient suffers from painful diabetic polyneuropathy, the dose of the nanoparticles or aggregates thereof to be administered is preferably at least 60 μg / cm 2 of the skin surface to be injected.

[0041] In one aspect of the present specification, when a patient suffers from CIPN and the material of the nanoparticles or their aggregates is selected from metal oxides or mixed metal oxides (such as HfO2 and / or ZrO2), the dose of the nanoparticles or their aggregates to be administered is preferably at least 250 μg / cm 2 , more preferably at least 390 μg / cm 2 of the skin surface to be injected.

[0042] In another aspect of the present specification, when a patient suffers from CIPN and the nanoparticles or their aggregates are selected from metal conductors selected from Ir, Pd, Pt, Au, and any mixtures thereof, the dose of the nanoparticles or their aggregates to be administered is preferably at least 70 μg / cm 2 , for example 78 μg / cm 2 of the skin surface to be injected.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0044] Definition The terms "treatment" or "therapy" refer to both therapeutic or means and prophylactic or preventive treatments or means that can significantly slow disease progression or treat symptoms (e.g., relieve pain).

[0045] Such treatment or treatment is directed at a subject in need thereof, usually a human (also identified herein as a human patient or patient).

[0046] "Local neuropathic pain" means neuropathic pain with a surface area of less than 15 cm 2 Neuropathic pain less than.

[0047] "Refractory neuropathic pain" means long-term neuropathic pain that is not reduced by standard pharmacological interventions such as standard antidepressants, anticonvulsants, analgesics, etc. Experts recognize that the definition of "refractory neuropathic pain" is at least 1 year in duration and at least 4 drugs with known efficacy have been tried for 3 months or the maximum duration of durability in NP [Smith B.H., et al. (2012) Towards a definition of refractory neuropathic pain for epidemiological research. An international Delphi survey of experts, BMC Neurology, 12:29].

[0048] "Chronic neuropathic pain" means neuropathic pain that occurs regularly over several months [J. Smith et al. (2018) J. Neurol. 265, pp231-238].

[0049] "At least partial temperature sensation" means that the afferent fibers that cause "induced" pain, more specifically, a feeling of cold or warm allodynia / hyperalgesia in the patient, are preserved.

[0050] Temperature sensation, more specifically cold sensation and / or heat allodynia and / or hyperalgesia, may be measured according to methods known to those skilled in the art, including protocols for quantitative sensory testing such as the DFNS (German Research Network on Neuropathic Pain) protocol [Vollert, J., et al. (2015) Quantitative Sensory Testing using DFNS protocol in Europe: an evaluation of heterogeneity across multiple centers in patients with peripheral neuropathic pain and healthy subjects, Pain, 157(3)] and the NPSI (Neuropathic Pain Symptom Inventory) protocol [Bouhassira, D., et al. (2004) Development and validation of the Neuropathic Pain Symptom Inventory, Pain, 108:248-257].

[0051] "Temperature sensation" or "at least partial temperature sensation" means that the patient still (partially) retains the afferent fibers (of the epithelium).

[0052] "Level of neuropathic pain" means the level of pain that can be measured using standard questionnaires known to those skilled in the art, such as the Numerical Rating Scale (NRS) for pain, the Leeds Assessment of Neuropathic Symptoms and Signs (LANSS), the Neuropathic Pain Questionnaire, DN4, the pain DETECT questionnaire, ID pain, the Neuropathic Pain Scale (NPS), the DFNS protocol, and the Neuropathic Pain Symptom Inventory (NPSI) [Haanpaeae, M., et al. (2011) NeuPSIG guidelines on neuropathic pain assessment, Pain 152 14-27]. Preferably, the NRS for pain and / or the DFNS protocol and / or the NPSI are used.

[0053] Nanoparticles and / or aggregates of nanoparticles Size In the context of the present invention, the term "nanoparticle" refers to products of nanometer size, generally from about 1 nm to about 1000 nm, preferably from about 1 nm to about 500 nm, and even more preferably from about 1 nm to about 100 nm, particularly synthetic products.

[0054] According to one aspect of the present invention, the median of the maximum size of the core in the population of nanoparticles or aggregates of nanoparticles is from about 10 nm to about 200 nm. The term "aggregate of nanoparticles" refers to an aggregate of nanoparticles.

[0055] The size of the nanoparticles and / or aggregates of nanoparticles can generally be measured by electron microscopy (EM) techniques such as transmission electron microscopy (TEM) or cryo-TEM, as is well known to those skilled in the art. The sizes of at least 100 nanoparticles and / or aggregates of nanoparticles are generally measured, and the median size in the population of nanoparticles and / or aggregates of nanoparticles is reported as the size of the nanoparticles and / or aggregates of nanoparticles.

[0056] Shape Since the shape of the nanoparticles and / or aggregates of nanoparticles can affect their "biocompatibility", nanoparticles and / or aggregates of nanoparticles having a very uniform shape are preferred. For pharmacokinetic reasons, nanoparticles and / or aggregates of nanoparticles that are essentially spherical, circular, or oval are therefore preferred. Such shapes are also advantageous for the interaction of nanoparticles and / or aggregates of nanoparticles with cells or their uptake by cells.

[0057] Composition / structure Nanoparticles prepared from an insulating material having a low relative permittivity, i.e., 100 or less Nanoparticles prepared from (e.g., containing) or consisting of an insulating material with a low relative permittivity generally have a band gap Eg of 3.0 eV or more when measured at room temperature (about 25 °C), and generally have a relative permittivity ε 2 measured between 10 ijk Hz and infrared frequencies in the range of 20 °C to 30 °C and 100 or less, preferably 50 or less, or 20 or less (instance table 12-45 "Permittivity (dielectric constant) of inorganic solid"; Handbook of chemistry and physics; David R. Lide; 88 thEdition; Compilation of the static dielectric constant of inorganic solid. See K.F. Young and H.P.R. Frederikse, J. Phys. Chem. Ref. Data, Vol. 2, No. 2, 1973).

[0058] Such nanoparticles are generally prepared from a dielectric material selected from metal oxides, mixed metal oxides, metal elements of the 3rd, 5th, or 6th period of the Mendeleev periodic table or lanthanides, and carbon materials. The dielectric material is preferably selected from La2O3, SnO2, Ta2O5, ReO2, ZrO2, HfO2, and carbon diamond. More preferably, the dielectric material is a metal oxide selected from ZrO2, HfO2, and any mixtures thereof. Dielectric materials selected from ZrO2 and HfO2 are particularly preferred. In certain preferred embodiments, the metal oxide is not CeO2 (cerium oxide). Cerium oxide is also known as "ceria". As described above, cerium oxide is known to be cytotoxic. In fact, Pulido-Reyes et al. (2015) demonstrated that the main factor contributing to the toxicity of ceria nanoparticles is the surface content of Ce 3+ sites, and that the higher the Ce 3+ / Ce 4+ ratio, the higher the toxicity ["Untangling the biological effects of cerium oxide nanoparticles: the role of surface valence states," Scientific reports DOi: 10.1038 / srep15613]. This is also known from Soh et al. 2017 [Angew. Chem. Int. Ed. 10.1002 / anie.201704904]. Therefore, incorporating Zr 4+ into the cerium oxide lattice results in a decrease in Ce 4+ to Ce 3+It is advantageous for conversion, and the toxicity of the nanoparticles increases. Therefore, cerium oxide / zirconium nanoparticles are expected to be more toxic than cerium oxide nanoparticles and zirconia nanoparticles due to the redox reaction occurring on the surface, compared to the known surface inertness of zirconium oxide nanobiomaterials [Shtansky et al., (2015): Multifunctional bioactive nanostructured films in “Hydroxyapatite (HAp) for Biomedical Applications” Elsevier, pp 162].

[0059] In another specific preferred embodiment, the metal oxide or mixed metal oxide does not contain cerium (Ce).

[0060] In a particular embodiment, when the material is selected from ReO2, ZrO2, HfO2, preferably from ZrO2 and HfO2 (for example, when the material is HfO2), the median of the maximum size of the core in the population of nanoparticles or aggregates of nanoparticles is at least 10 nm and less than 500 nm, preferably about 10 nm to about 200 nm, even more preferably about 20 - 100 nm.

[0061] Nanoparticles prepared from a conductor material Nanoparticles prepared from a conductor material are organic nanoparticles or inorganic nanoparticles.

[0062] Inorganic nanoparticles prepared from a conductor material are generally prepared from a metal element having a standard reduction potential E° of about 0.01 or more, more preferably about 0.1, 0.2, 0.3, 0.4, or 0.5 or more when measured against a standard hydrogen electrode at 25 °C and 1 atmosphere (Table 2 “reduction reactions having E° values more positive than that of the standard hydrogen electrode”, 8 - 25, Handbook of chemistry and physics; David R. Lide; 88th (see Edition). The typical metal elements used to prepare the nanoparticles may be selected from Pd, Ir, Pt, Au, and any mixtures thereof. Preferably, the metal elements that can be used as the conductor material for preparing the nanoparticles are selected from Ir, Pd, Pt, Au, and any mixtures thereof, and even more preferably from Au, Pt, Pd, and any mixtures thereof. Particularly preferred materials are Au and Pt.

[0063] Generally, gold nanoparticles exhibit catalytic activity when their size is reduced to several nanometers (Auffan, M., et al. (2009) Towards a definition of inorganic nanoparticles from an environmental, health and safety perspective, Nature Nanotechnology, 4(10), 634 - 641). To reduce the surface area / volume ratio and minimize the contribution of the gold nanoparticle surface to catalytic activity, the median of the maximum size of the core in the population of nanoparticles or aggregates of nanoparticles is preferably at least 30 nm, generally at least 40 nm, or at least 45 nm.

[0064] In certain embodiments, when the material is a metallic material, generally a metal with a standard reduction potential E° greater than 0.2, particularly any of Ir, Au, Pt, Pd, and any mixtures thereof, the median of the maximum size of the core in the population of nanoparticles or aggregates of nanoparticles is, as described above, at least 30 nm, or at least 40 nm, preferably less than 500 nm. For example, the median of the maximum size of the core in the population of nanoparticles or aggregates of nanoparticles can be from about 40 to about 200 nm, preferably from about 45 to about 100 nm.

[0065] The organic nanoparticles prepared from the conductor material have adjacent sp in the structure 2It is generally prepared from an organic material having a hybrid carbon center (i.e., an aromatic ring containing a heteroatom (usually N or S) inside or outside the carbon double bond or ring). Preferred organic materials are selected from polyaniline, polypyrrole, polyacetylene, polythiophene, polycarbazole, polypyrene, poly(3,4-ethylenedioxythiophene), and / or poly(3,4-ethylenedioxythiophene) polystyrene sulfonic acid. More preferably, the organic material is poly(3,4-ethylenedioxythiophene) or poly(3,4-ethylenedioxythiophene) polystyrene sulfonic acid. In another preferred embodiment, the organic material is poly(3,4-ethylenedioxythiophene) (PEDOT) and / or poly(styrene sulfonic acid) (PSS).

[0066] Biocompatible coating of nanoparticles or aggregates of nanoparticles In a preferred embodiment, the core of the nanoparticles or aggregates of nanoparticles used in the context of the present invention to prepare the composition of interest can be coated with a biocompatible material that provides a negative charge to the surface of the nanoparticles or aggregates of nanoparticles. Substances that form a negative charge on the surface of the nanoparticles or aggregates of nanoparticles can be, for example, phosphates (such as polyphosphates, metaphosphates, pyrophosphates, etc.), carboxylates (such as citrate or dicarboxylic acids, especially succinic acid), or sulfates.

[0067] In a preferred embodiment, the core of the nanoparticles or aggregates of nanoparticles is coated with a biocompatible material (i.e., a coating agent) selected from hydrophilic materials that exhibit a neutral surface charge or that impart a neutral surface charge to the nanoparticles.

[0068] A hydrophilic substance that gives a neutral surface charge to the core of nanoparticles or aggregates of nanoparticles can be a substance having a functional group selected from alcohols (R-OH), aldehydes (R-COH), ketones (R-CO-R), esters (R-COOR), acids (R-COOH), thiols (R-SH), saccharides (such as glucose, fructose, ribose), anhydrides (RCOOOC-R), and pyrrole. A hydrophilic substance that gives a neutral surface charge to the core of nanoparticles or aggregates of nanoparticles can be a monomer, dimer, oligomer, polymer, or copolymer. When the substance is an oligomer, it may be an oligosaccharide such as cyclodextrin. When the substance is a polymer, it can be a polyester (such as poly(lactic acid) or polyhydroxyalkanoic acid), polyether, polyethylene oxide, polyethylene glycol, polyvinyl alcohol, polycaprolactone, polyvinylpyrrolidone, a polysaccharide such as cellulose, polypyrrole, etc.

[0069] Furthermore, a hydrophilic substance that gives a neutral surface charge to the core of nanoparticles or aggregates of nanoparticles can be a substance having a specific group (X-) that can interact with the surface of the nanoparticles or aggregates of nanoparticles. X is generally selected from thiol groups, silane groups, carboxylic acid groups, and phosphate groups.

[0070] When the core of the nanoparticles or aggregates of nanoparticles is a conductor and a metal nanoparticle, X is preferably a thiol group, thioether group, thioester group, dithiolane group, or carboxylic acid group. Preferably, the hydrophilic and neutral coating agent is selected from thioglucose, 2-mercaptoethanol, 1-thioglycerol, thiodiglycol, hydroxybutyric acid, and mercaptopolyethylene glycol. Preferably, the hydrophilic substance that gives a negative surface charge at physiological pH is dimercaptosuccinic acid.

[0071] When the core of the nanoparticle or aggregate of nanoparticles is an insulator, metal oxide, or mixed metal oxide nanoparticle, X is preferably a silane group or a phosphate group. Preferably, the hydrophilic and neutral coating agent is silane-polyethylene glycol. Preferably, the hydrophilic substance that gives a negative surface charge at physiological pH is a polyphosphate molecule, more preferably hexametaphosphate.

[0072] According to a preferred embodiment, the core of the nanoparticle or aggregate of nanoparticles consists of HfO2. Preferably, the core is coated with a biocompatible coating that results in a negative surface charge, for example a coating presenting phosphate groups. In one embodiment, the core HfO2 is coated with a hexametaphosphate biocompatible coating.

[0073] According to one embodiment of the present invention, the core is coated with a biocompatible hydrophilic neutral coating agent selected from hydroxymethyltriethoxysilane, fructose 6-phosphate, or glucose 6-phosphate compounds.

[0074] The hydrophilic substance that gives a neutral surface charge to the core of the nanoparticle or aggregate of nanoparticles can be an amphoteric ionic compound such as an amino acid, peptide, polypeptide, vitamin, or phospholipid.

[0075] As is well known to those skilled in the art, the surface charge of nanoparticles or aggregates of nanoparticles is generally determined by zeta potential measurement in an aqueous (solution) where the concentration of the material of the nanoparticles or the material of the aggregates of nanoparticles is 0.01 to 10 g / L, the pH is 6 to 8, and the electrolyte concentration (in water) is generally 0.001 to 0.2 M, for example 0.01 M or 0.15 M. Under the conditions defined above, the surface charge of nanoparticles or aggregates of nanoparticles is generally -80 mV to +15 mV, -60 mV to +10 mV, or -45 mV to +10 mV. When neutral, the surface charge of nanoparticles or aggregates of nanoparticles is generally between -10 mV, -9 mV, -8 mV, -7 mV, -6 mV, -5 mV, -4 mV, -3 mV, -2 mV, or -1 mV and 1 mV, 2 mV, 3 mV, 4 mV, 5 mV, 6 mV, 7 mV, 8 mV, 9 mV or 10 mV. When negative, the surface charge of nanoparticles or aggregates of nanoparticles is generally -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, -28 mV, -29 mV, -30 mV, -31 mV, -32 mV, -33 mV, -34 mV, -35 mV, -40 mV, or less than -45 mV.

[0076] A full biocompatible coating of nanoparticles or aggregates may be advantageous in avoiding any charge on the nanoparticle surface in the context of the present invention when the nanoparticles exhibit a hydrophilic and neutral surface charge. "Full coating" means the presence of a very high density / compact biocompatible molecule capable of forming at least half a monolayer, or preferably at least a complete monolayer, on the surface of the particles.

[0077] Biocompatible coatings provide stability to nanoparticles, especially in liquids such as physiological fluids (blood, plasma, serum, etc.) or any isotonic or physiological solution required for pharmaceutical administration.

[0078] The stability is confirmed by quantification of the dried extract using a dryer and is generally measured for the nanoparticle suspension before and after filtration through a 0.45 μm filter.

[0079] Advantageously, the coating preserves the integrity of the particles in vivo, ensures or improves their biocompatibility, and promotes any functionalization thereof (e.g., by spacer molecules, biocompatible polymers, target drugs, proteins, etc.).

[0080] The biocompatible nanoparticles or aggregates of nanoparticles of the present invention should generally not dissolve (i.e., at physiological pH) after in vivo administration to release potentially toxic chemical species or exhibit redox behavior in order for the nanoparticles or aggregates of nanoparticles to be considered biocompatible, i.e., to be safely used in a subject, particularly in a mammal, preferably in a human.

[0081] Another specific object described herein relates to a composition, particularly a pharmaceutical composition, comprising nanoparticles and / or aggregates of nanoparticles (such as those described above), preferably together with a pharmaceutically acceptable carrier or vehicle.

[0082] In certain embodiments, the composition may comprise the nanoparticles or aggregates of nanoparticles of the present invention together with a therapeutic agent, such as an anti-inflammatory agent, for topical administration.

[0083] The composition can be in the form of a solid, liquid (particles in suspension), aerosol, gel, paste, and the like. Preferred compositions are liquid or gel. Particularly preferred compositions are liquid.

[0084] The pharmaceutically acceptable support or carrier utilized can be any classical support for those skilled in the art, such as saline, isotonic solutions, sterile solutions, buffer solutions, vehicle non-aqueous solutions, and the like.

[0085] The composition may also include stabilizers, surfactants, polymers, and the like.

[0086] It can be formulated, for example, as an ampoule or a liquid for injection, by using pharmaceutical formulation techniques known to those skilled in the art.

[0087] Subject to be treated The nanoparticles are administered to treat at least one peripheral neuropathic pain condition.

[0088] According to one embodiment of the present invention, the subject suffers from peripheral diabetic neuropathy (PDN).

[0089] According to one embodiment of the present invention, the subject suffers from postherpetic neuralgia.

[0090] According to one embodiment of the present invention, the subject suffers from CIPN.

[0091] According to one embodiment of the present invention, the subject suffers from painful radiculopathy.

[0092] According to one embodiment of the present invention, the subject suffers from peripheral nerve injury including post-traumatic neuropathic pain, postoperative neuropathic pain, or neuropathic pain due to a neuroma.

[0093] According to one embodiment of the present invention, the patient suffers from (peripheral) neuropathic pain caused by an autoimmune disease such as Guillain - Barré syndrome, human immunodeficiency virus (HIV), Fabry disease, mutations in sodium channel genes, vasculitis, chronic inflammatory demyelinating polyneuropathy, amyloidosis, frostbite, paraneoplastic syndrome, or Hansen's disease.

[0094] According to one embodiment of the present invention, the subject suffers from local (peripheral) neuropathic pain.

[0095] According to one embodiment of the present invention, the subject suffers from intractable and / or chronic (peripheral) neuropathic pain.

[0096] According to a preferred embodiment of the present invention, the subject suffers from peripheral NP that manifests as ectopic centripetal discharge.

[0097] Patients suffering from NP may preferably still have at least partial temperature sensation (tested by cold or warm allodynia / hyperalgesia tests known to those skilled in the art).

[0098] Without being bound by any theory, the inventors believe that the preservation of at least some temperature sensation, i.e., the preservation of at least a portion of the centripetal fibers (of the epithelium), may be necessary in the subject for the subject to feel a sense of pain relief.

[0099] Administration method The affected area with pain may be outlined by a person skilled in the art (i.e., a physician or nurse), drawn on the skin with a pen, and measured. This outlining may help determine the total dosage of nanoparticles to be administered in the area with pain. The outlined area with pain should preferably be less than 15 cm 2 and should be less.

[0100] The nanoparticles or aggregates of nanoparticles of the present invention can be administered to a subject using intradermal and / or intradermal injection. Intradermal injection can be performed at a normal intradermal injection angle (e.g., an angle of 5 to 15 degrees from the site) using a 25- to 30-gauge needle and a syringe commonly used for intradermal injection (e.g., an insulin or tuberculin syringe). Intradermal injection may be performed according to the Mantoux method. In another aspect, intradermal and / or intradermal injection can be performed using hollow, solid, or dissolving microneedles. Such microneedles can be in the form of a patch [Jung, J.H., et al. (2021) Microneedle for transdermal drug delivery: current trends and fabrication, Journal of Pharmaceutical Investigation, 51, 503-517]. The nanoparticles or aggregates of nanoparticles of the present invention can be administered by intradermal and / or intradermal injection at a dose per unit site multiple times by dividing the affected area with pain into a checkerboard of multiple sites, as is usually done using botulinum toxin A to treat local neuropathic pain.

[0101] The nanoparticles or aggregates of nanoparticles may be administered at a frequency required according to the patient's pain. Administration of the nanoparticles may be required once a month, once every two months, or once every three months.

[0102] Preferably, administration of the nanoparticles is only required once every three months. More preferably, administration of the nanoparticles is only required once every four months. Even more preferably, administration of the nanoparticles is only required once every five or six months. Even more preferably, administration of the nanoparticles is only required once every seven or eight months. Most preferably, the nanoparticles are administered only once, providing pain relief for more than one year. Even most preferably, the nanoparticles are administered only once, providing pain relief for the remainder of the patient's life.

[0103] Once administered, the nanoparticles and / or aggregates of nanoparticles typically interact with the neurons / nerves of the subject. In a preferred embodiment, this interaction is a long-term interaction, i.e., an interaction lasting for hours, days, weeks, or months. In certain embodiments, the nanoparticles or aggregates of nanoparticles remain in the subject.

[0104] The nanoparticles or aggregates of nanoparticles described herein and compositions containing such nanoparticles or aggregates of nanoparticles are generally for use in a subject, typically a human patient.

[0105] Typical numbers of nanoparticles or aggregates of nanoparticles administered in the dermis and / or epidermis of the subject are 10 2 to 10 5 ~ 10 17 , 10 5 ~ 10 16 or 10 5 ~ 10 15 , preferably 10 7 ~ 10 14 , more preferably 10 9 ~ 10 13 per cm 2 of the subject, and most preferably, typical numbers of nanoparticles or aggregates of nanoparticles administered in the dermis and / or epidermis of the subject are 10 10 ~ 10 12 per cm

[0106] According to one embodiment of the invention, when a patient suffers from painful diabetic polyneuropathy, the dose of the nanoparticles or aggregates thereof administered is preferably at least 60 μg / cm 2 of the skin surface to be injected.

[0107] In one aspect, when a patient suffers from diabetic polyneuropathy and the material of the nanoparticles or aggregates thereof is a metal oxide or a mixed metal oxide (e.g., HfO2 and / or ZrO2), the dose of the nanoparticles or aggregates thereof administered is preferably at least at least 60 μg / cm 2 of the skin surface to be injected.

[0108] In one embodiment, when a patient suffers from CIPN and the material of the nanoparticles or their aggregates is a metal oxide or a mixed metal oxide (e.g., HfO2 and / or ZrO2), the dose of the administered nanoparticles or their aggregates is preferably at least 250 μg / cm 2 , more preferably at least 390 μg / cm 2 of the skin surface to be injected.

[0109] According to one embodiment, when a patient suffers from CIPN and the nanoparticles or their aggregates are a metal conductor selected from Ir, Pd, Pt, Au, and any mixtures thereof, the dose of the administered nanoparticles or their aggregates is preferably at least 70 μg / cm 2 , for example 78 μg / cm 2 of the skin surface to be injected.

[0110] Targeting The specific nanoparticles and / or aggregates of nanoparticles described herein further comprise a targeting agent that enables interaction with recognition elements present on target cells, generally on neurons / nerve cells. Such targeting agents generally act when the nanoparticles and / or aggregates of nanoparticles accumulate at the target site, generally in the skin, and even more generally in neurons / nerve. The targeting agent can be any biological or chemical structure that exhibits an affinity for molecules present in the human or animal body. For example, it can be a peptide, oligopeptide or polypeptide, protein, nucleic acid (DNA, RNA, SiRNA, tRNA, miRNA, etc.), antibody, hormone, vitamin, enzyme, ligand of a molecule / receptor expressed by neurons / nerve cells or diseased cells, particularly a ligand of an intraepidermal nerve fiber receptor, a ligand of an intradermal nerve fiber receptor, a ligand of an axonal receptor, a ligand of a cell body receptor, a ligand of a Schwann cell receptor, a ligand of myelin, a ligand of an axonal transport vesicle / endosome receptor, a ligand of a signaling endosome receptor, a ligand of a neurotrophin, a ligand of a semaphorin, a ligand of a nerve growth factor, a ligand of a growth cone receptor, or a ligand of a netrin receptor complex, a ligand of a receptor-type tyrosine phosphatase, a ligand of dynein or kinesin, or a ligand of a cytokine receptor. The targeting agent can be selected, for example, from the group of biomarkers of PGP9.5, GTPase-activating protein (including GAP-43), betaIII tubulin, vesicular glutamate transporter (VGLUT1), tyrosine receptor kinases B and C (Trk B, C), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), calretinin, and the targeting agent can be retinoic acid.

[0111] Composition Also described herein are pharmaceutical compositions comprising nanoparticles and / or aggregates of nanoparticles and a pharmaceutically acceptable carrier, vehicle, or support, such as those described above herein.

[0112] The pharmaceutical composition is suitable for use in the treatment of neuropathic pain as described above herein.

[0113] Technical effect The therapeutic efficacy of the claimed nanoparticles is illustrated by the results of experiments (detailed in the Examples) using animal models of neuropathic pain, namely the CIPN model (Examples 5 and 6) and the diabetic neuropathic pain model (Example 7).

[0114] In Example 5, several embodiments of the claimed nanoparticles were demonstrated in rats, including long-lasting efficacy (at least 11 days) in relieving pain from CIPN after a single intraplantar injection. In the experiment, animals could not be evaluated beyond 11 days.

[0115] Therefore, according to one embodiment of the present invention, the claimed nanoparticles can be used to treat CIPN.

[0116] In Examples 6 and 7, a single intraplantar injection of hafnium oxide nanoparticles with a negatively charged biocompatible coating (Example 3) resulted in long-lasting pain relief (at least 7 days for the diabetic NP model and at least 11 days for the CIPN model). In contrast, the positive control, pregabalin, required injection every two days, two hours before von Frey assessment, to achieve consistent pain relief throughout the study period. Furthermore, a dose effect (i.e., increasing the dose of nanoparticles administered increased the pain-reducing effect) was clearly observed in the experiment of Example 6.

[0117] Therefore, according to one embodiment of the present invention, the claimed nanoparticles can be used to treat diabetic neuropathic pain.

[0118] According to one embodiment of the present invention, an increase in the concentration of nanoparticles results in an increase in the therapeutic effect.

[0119] The two animal models used to demonstrate the efficacy of the present nanoparticles are considered clinically relevant.

[0120] Based on in vivo data from two different animal models presented in this specification, the inventors believe that the inventive nanoparticle composition can be used to treat all neuropathic pain states, preferably peripheral neuropathic pain states.

[0121] Therefore, the inventors have shown that the nanoparticles described in the claims can be used as a topical therapy for peripheral neuropathic pain for long-lasting pain relief. Topical administration is advantageous as it avoids the negative side effects of the systemic therapy detailed above. Additionally, since the nanoparticles are administered at a lower frequency compared to other topically administered neuropathic pain treatments, patient compliance is improved.

[0122] Other aspects and advantages of the present invention will become apparent from the following examples provided for purposes of illustration and not limitation.

Example

[0123] Example 1. Nanoparticles prepared from a conductor material: Synthesis of gold nanoparticles coated with a biocompatible coating exhibiting a negative surface charge Gold nanoparticles were synthesized by reducing chloroauric acid (HAuCl4) with a capping agent (sodium citrate) (see protocol G. Frens, (1973) Nature Physical Science 241, 21). In a typical experiment, the HAuCl4 solution was heated to boiling. Then, the sodium citrate solution was added. The resulting solution was boiled for an additional 5 minutes and then filtered. The gold concentration in the suspension was determined by ultraviolet-visible spectroscopy at 530 nm.

[0124] The resulting nanoparticles were coated with meso-2,3-dimercaptosuccinic acid (DMSA), a biocompatible surface coating. An amount of DMSA sufficient to achieve a monolayer coverage of at least half (2.5 molecules / nm 2 ) on the surface was added to the nanoparticle suspension.

[0125] The nanoparticle suspension was diluted with water (final concentration 0.1 g / L), and the hydrodynamic diameter (measured by intensity) was determined by dynamic light scattering (DLS) at a scattering angle of 173° and a laser wavelength of 633 nm using a Nano-Zetasizer (Malvern). The hydrodynamic diameter of the nanoparticles in the suspension thus obtained was equal to 81 nm, and the polydispersity index (dispersion of the number of nanoparticles in size) was 0.49.

[0126] The nanoparticle suspension was diluted with 1 mM NaCl solution at pH 7 (final concentration 0.1 g / L), and the zeta potential was determined by measuring the electrophoretic mobility of the nanoparticles (Nano-Zetasizer, Malvern). The zeta potential at pH 7 was equal to -32 mV.

[0127] The gold concentration was determined using inductively coupled plasma optical emission spectrometry (ICP-OES).

[0128] Example 2. Nanoparticles prepared from an insulating material having a low relative permittivity of 100 or less: Synthesis of zirconium oxide nanoparticles coated with a biocompatible coating exhibiting a negative surface charge Zirconium oxide (ZrO2) nanoparticles were synthesized by precipitating zirconium chloride (ZrCl4) with tetramethylammonium hydroxide (TMAOH) at basic pH. The resulting suspension was autoclaved at a temperature above 110 °C, washed with deionized water after cooling, acidified, and filtered (0.22 μm PES membrane filter). The nanoparticle (ZrO2) concentration was determined as the dry mass concentration.

[0129] Coating was performed as follows. Sodium hexametaphosphate in an amount sufficient to reach a monolayer coverage of at least half (2.5 molecules / nm 2 ) on the surface was added to the nanoparticle suspension.

[0130] The hydrodynamic diameter of the nanoparticles measured by DLS was 66 nm, and the polydispersity index (dispersion of the number of nanoparticles in size) was 0.10.

[0131] The zeta potential at pH 7 was -40 mV.

[0132] The zirconium concentration was determined using inductively coupled plasma optical emission spectrometry (ICP-OES).

[0133] Example 3. Nanoparticles prepared from an insulating material having a low relative permittivity of 100 or less: Synthesis of hafnium oxide nanoparticles coated with a biocompatible coating exhibiting a negative surface charge Hafnium oxide (HfO2) nanoparticles were synthesized by precipitating hafnium chloride (HfCl4) with tetramethylammonium hydroxide (TMAOH) at basic pH. The resulting suspension was autoclaved above 110 °C, cooled, washed with water, and acidified.

[0134] Surface functionalization was performed using sodium hexametaphosphate in the same manner as in Example 2.

[0135] The hydrodynamic diameter measured by DLS was 73 nm, and the polydispersity index (dispersion of the number of nanoparticles in size) was 0.09. The zeta potential at pH 7 was -39 mV.

[0136] The hafnium concentration was determined using inductively coupled plasma optical emission spectrometry (ICP-OES).

[0137] Example 4. Nanoparticles prepared from an insulating material having a low relative permittivity of 100 or less: Synthesis of hafnium oxide nanoparticles coated with a biocompatible coating exhibiting a neutral surface charge Hafnium oxide (HfO2) nanoparticles were synthesized by precipitating hafnium chloride (HfCl4) with tetramethylammonium hydroxide (TMAOH) at basic pH. The resulting suspension was autoclaved above 110 °C, cooled, washed with deionized water, and acidified.

[0138] A biocompatible coating was performed using silane-poly(ethylene) glycol 2 kDa (“Si-PEG 2 kDa”). A sufficient mass of Si-PEG 2 kDa was added to the nanoparticle suspension to reach at least half (2.5 molecules / nm 2 ) of the monolayer coverage on the surface.

[0139] The hydrodynamic diameter of the nanoparticles was equal to 81 nm, and the polydispersity index (dispersion of the number of nanoparticles in size) was 0.15.

[0140] The zeta potential at pH 7 was equal to -5 mV.

[0141] The hafnium concentration was determined using inductively coupled plasma optical emission spectrometry (ICP-OES).

[0142] Example 5: Efficacy of single-dose nanoparticles for symptomatic treatment of mechanical allodynia in rats with a chemotherapy-induced peripheral neuropathy model (oxaliplatin) The purpose of this study was to investigate the effect of a single intradermal treatment (injection volume of 20 μL each) with nanoparticles of Example 1 (NP1B) at a dose of 11 g / L, nanoparticles of Example 2 (NP2B) at a dose of 53 g / L, nanoparticles of Example 3 (NP20B) at a dose of 57 g / L, and nanoparticles of Example 4 (NP20A) at a dose of 59 g / L on mechanical allodynia (measured using von Frey) that occurs in rats after systemic administration of oxaliplatin.

[0143] Study design The experimental design of the CIPN model is shown in Table 1.

Table 1

[0144] Materials and methods All experiments were conducted in accordance with the ethical guidelines of the International Association for the Study of Pain (Zimmerman, 1983).

[0145] Experimental subjects: Seventy-two adult male Sprague-Dawley rats were used for this study. The animals were housed in groups of four in ventilated cages in a controlled environment of constant temperature and humidity (temperature: 21 ± 1 °C, 12:12-hour light-dark cycle), with free access to food and water. The animals were allowed to recover from transportation for at least one week before the experiment began.

[0146] Static mechanical (contact) allodynia evaluation:A von-Frey monofilament with graduations (force; g) (Touch-Test Sensory Evaluator; Scientific Marketing Associates) was applied to the plantar surface of the hindlimb to measure the paw withdrawal threshold (PWT). The withdrawal threshold was measured by increasing and decreasing the stimulus intensity and estimated using the Dixon up-and-down method.

[0147] 0.5 cm 2 Animals were placed on an elevated mesh-bottom platform with a grid of 2 of 0.5 cm, allowing access to the ventral side of the hindlimbs. An inverted plexiglass container was placed over each rat, and the test was conducted after an initial 15 - 20 minute acclimation / training period. From beneath the mesh floor, a von Frey filament was positioned perpendicular to the plantar surface of the hindlimb. The monofilament was held in that position for approximately 5 seconds with sufficient force to cause it to bend slightly. Only a sharp withdrawal response (or flinching) within 5 seconds of the stimulus was considered a positive response.

[0148] Test schedule: Baseline measurements of withdrawal latency (seconds) were evaluated for 3 consecutive days (-3 days, -2 days, and -1 days). The average of -2 days and -1 days was considered the baseline before oxaliplatin administration, and day 0 (D0) was defined as the day of oxaliplatin injection. Neuropathy baseline recordings were taken on day 5 (D5) and day 6 (D6) after oxaliplatin.

[0149] Animals treated with nanoparticles were given an intradermal injection into the left paw with a micro-needle device on day 7 after oxaliplatin. The injection of nanoparticles was performed only once throughout the experiment. Animals treated with pregabalin (positive control) were administered orally (p.o.) and tested 2 hours after administration. Animals treated with nanoparticles were also tested 2 hours after administration on day 7. Pregabalin was administered on each day of the von Frey test.

[0150] Mechanical allodynia was re-evaluated 2 - 3 times per week after oxaliplatin until day 26. The test days (after oxaliplatin) were D7 (the administration day of the test compound), D10, D14, D18, D22, and D26.

[0151] Data analysis Two-way repeated measures ANOVA was performed using "treatment" as the between-subjects effect and "day" as the within-subject effect (GraphPad Software, GraphPad Prism version 9.0 (Windows version), San Diego, California, USA), followed by Dunnett's multiple comparison test. Data are shown as mean ± SEM.

[0152] Results The values of the paw withdrawal threshold (PWT) in rats after induction of mechanical allodynia by oxaliplatin are shown in Figure 1. Figure 2 shows photographs of the paws of rats immediately after intradermal administration of the nanoparticles of Example 1 (D0) and immediately before sacrificing the rats (D26).

[0153] Conclusion Administration of oxaliplatin induced a consistent mechanical allodynia response as indicated by the paw withdrawal threshold (PWT) level (Figure 1). Pregabalin systemic therapy as a positive control abolished the allodynia response and restored the PWT to a healthy baseline level.

[0154] The nanoparticles of Example 1 (NP1B) showed a significant increase in PWT, especially on and after the 14th day, compared to its neuropathy baseline (Figure 1). The nanoparticles of Example 2 (NP2B) showed some increase compared to its neuropathy baseline values (D7, D14, and D18). The nanoparticles of Example 3 (NP20B) were particularly effective on and after the 18th day, showing a significant difference from its neuropathy baseline. The nanoparticles of Example 4 (NP20A) were the most effective nanoparticle compound in reversing mechanical allodynia consistently on and after the 14th day, with a significant increase in PWT values from its neuropathy baseline. Figure 2 shows that the nanoparticles of Example 1 remained at the injection site with minimal removal for at least 26 days.

[0155] Example 6: Efficacy of single-dose nanoparticles of Example 3 for symptomatic treatment of mechanical allodynia in rats with a chemotherapy-induced peripheral neuropathy model (oxaliplatin): Dose-effect The purpose of this study was to investigate the effect of the nanoparticles of Example 3 on mechanical allodynia when administered as a single intraplantar dose at different dosages in a rat oxaliplatin model of chemotherapy-induced neuropathic pain.

[0156] Study design The experimental design of the CIPN model test is shown in Table 2.

Table 2

[0157] Materials and methods All experiments were conducted in accordance with the ethical guidelines of the International Association for the Study of Pain (Zimmerman, 1983).

[0158] Experimental subjects: Sixty adult male Sprague-Dawley rats (180 - 210 g) were used for this study. The animals were housed in groups of four in ventilated cages in a controlled environment of constant temperature and humidity (temperature: 21 ± 1°C, 12:12-hour light-dark cycle), with free access to food and water. The animals were allowed to recover from transportation for at least one week before the start of the experiment.

[0159] Static mechanical (contact) allodynia evaluation: A von-Frey monofilament with graduations (force; g) (Touch-Test Sensory Evaluator; Scientific Marketing Associates) was applied to the plantar surface of the hindlimb to measure the withdrawal threshold. The withdrawal threshold was determined by increasing and decreasing the stimulus intensity and estimated using the Dixon up-and-down method.

[0160] 0.5 cm 2 The animals were placed on an elevated mesh-bottom platform with a grid of 0.5 cm, and were allowed to be touched on the ventral side of the hindlimbs. An inverted plexiglass container was placed above each rat, and the test was performed after an initial acclimation / training period of 15 - 20 minutes. From beneath the mesh floor, the von Frey filament was positioned perpendicular to the plantar surface of the hindlimb. The monofilament was held in that position for approximately 4 seconds with sufficient force to cause it to bend slightly. Only a sharp withdrawal response (or flinching) within 5 seconds of the stimulus was considered a positive response.

[0161] Test schedule: Baseline measurements of withdrawal latency (seconds) were evaluated for 3 consecutive days (-3 days, -2 days, and -1 days). The average of -2 days and -1 days was considered the baseline before oxaliplatin administration, and day 0 (D0) was set as the injection day of oxaliplatin. The recording of the neuropathy baseline was taken on day 5 (D5) from oxaliplatin.

[0162] Animals treated with nanoparticles were injected intradermally into the plantar surface of one foot with a micro-needle device on day 6 (D6) from oxaliplatin (the opposite foot was used as a control and injected with the same volume of vehicle - saline with a micro-needle device). The injection of nanoparticles was performed only once throughout the experiment. Animals treated with pregabalin (positive control) were administered orally (p.o.) and tested for mechanical allodynia 2 hours after administration. Animals treated with nanoparticles were also tested 2 hours after administration on day 6. Pregabalin was administered on each day of the von Frey test.

[0163] Mechanical allodynia was then re-evaluated 2 - 3 times per week after injection from oxaliplatin until day 24 (D24). The test days (after oxaliplatin) were D6 (the administration day of the test compound), D9, D13, D16, D20, and D24.

[0164] Data analysis Two-way repeated measures ANOVA using "treatment" as the between-subjects effect and "day" as the within-subject effect was performed (GraphPad Software, GraphPad Prism version 9.0 (Windows version), San Diego, California, USA), followed by Dunnett's multiple comparison test. The data are shown as mean ± SEM.

[0165] Results The values of the paw withdrawal threshold (PWT) in rats after induction of mechanical allodynia by oxaliplatin are shown in Figures 3, 4, 5, 6, and 7, and the significance of the results is evaluated in Tables 3, 4, 5, 6, and 7 below.

Table 3

Table 4

Table 5

Table 6

Table 7

[0166] Conclusion Administration of oxaliplatin induced a consistent mechanical allodynia response, indicated by the paw withdrawal threshold (PWT) level of the right hind paw, on the evaluation days up to day 20 after administration (Figure 1). As known from the literature (W. H. Xiao, H. Zheng, and G. J. Bennett, Neuroscience. 2012, 203: 194-206: Characterization of oxaliplatin-induced chronic painful peripheral neuropathy in the rat and comparison to the neuropathy induced by paclitaxel), and as observed from the PWT of the untreated (right) hind paw of all groups at the baseline (before neuropathy) level on day 24, the mechanical allodynic nociceptive effect of oxaliplatin began to decrease 4 weeks later due to oxaliplatin washout. The pregabalin systemic therapy functioned as a positive control because the PWT levels were not significantly different between the left and right hind paws and the "vehicle / vehicle" group (no neuropathy) on any of the test days.

[0167] The nanoparticles of Example 3 with a concentration of 61 g / L and a dosage of 20 μL showed a higher paw withdrawal threshold (PWT) in the left paw compared to the right paw in the data, and the statistical analysis showed a significant effect between the left and right (left paw vs right paw) with a p-value < 0.05 (p-value = 0.0326; cf. Table 4). In contrast, for the nanoparticles of Example 3 with 18 g / L, a volume of 20 μL (p-value = 0.0678) and 10 μL (p-value = 0.4304), no significant effect between the left and right was shown (cf. Tables 5 and 6), thus being involved in the reduction of the pain response in the treated (left) paw. This result indicates the threshold of the minimum dosage for the nanoparticles of Example 3 to significantly and efficiently reduce mechanical allodynia induced by oxaliplatin-induced peripheral neuropathy in rats. A single injection of the nanoparticles of Example 3 with a concentration of 61 g / L (20 μL) induced pain relief from day 9 to day 20, which means that the pain relief lasted for at least 11 days. On the other hand, the positive control pregabalin needed to be injected every 2 days (2 hours before von Frey assessment).

[0168] Example 7: Efficacy of single-dose nanoparticles for symptomatic treatment of mechanical allodynia in rats with a diabetic peripheral neuropathy model (streptozotocin) The main cause of clinically observed neuropathic pain is diabetes. In rodents, this is reproduced by injecting streptozotocin (STZ) to induce diabetes. STZ is an antibiotic that inhibits insulin production by destroying pancreatic islet cells. Thus, rats develop diabetes and diabetic neuropathic pain. The rat STZ diabetes model has been successfully used to model type 1 diabetic neuropathic pain and has provided important decision-making allodynia efficacy data for the development of pregabalin, one of the very few treatments that have succeeded in translating preclinical results of neuropathic pain into clinical success.

[0169] Diabetes is confirmed by checking blood glucose levels. Symptoms of neuropathic pain, such as mechanical allodynia (measured using von-Frey filaments), usually develop 2 - 6 weeks after the injection of STZ.

[0170] The purpose of this study was to investigate the effect of a single intraplantar treatment with the nanoparticles of Example 3 at doses of 117 g / L, injection volume of 20 μL, 58 g / L, injection volume of 20 μL, and 18 g / L, injection volume of 10 μL on mechanical allodynia (evaluated using von Frey) that occurs in rats after systemic administration of streptozocin.

[0171] Study design The experimental plan for the STZ-induced diabetic neuropathy model is shown in Table 8.

Table 8

[0172] Materials and methods All experiments were conducted in accordance with the ethical guidelines of the International Association for the Study of Pain (Zimmerman, 1983). All behavioral tests were approved by the United Kingdom Home Office Animals (Scientific Procedures) Act 1986.

[0173] Experimental subjects: Sixty-six adult male Sprague-Dawley rats (180 - 210 g) were used for this study. The animals were housed in pairs in ventilated cages in a controlled environment of constant temperature and humidity (temperature: 21 ± 1 °C, 12:12 h light-dark cycle) with free access to food and water. The animals were allowed to recover from transportation for at least one week before the experiment began.

[0174] Streptozotocin-induced diabetes model:Animals were injected with STZ (55 mg / kg, i.p., 10 mL / kg, single injection) or its vehicle solution (20 mM citrate buffer, i.p., 10 mL / kg, single injection) on day 0. For 48 hours after STZ injection, animals were allowed to consume 2% sucrose in their drinking water (choice of one bottle of water and one bottle of 2% sucrose was provided) to manage the transient hypoglycemic period that generally occurs 4 - 8 hours after STZ injection. After STZ injection, all animals were switched to a slightly high - protein diet (Labdiet 5LF5; 22% protein).

[0175] Body weight was monitored daily throughout the study period. STZ rats typically lost 5 - 10% of their baseline body weight and usually began to regain weight to baseline levels over a 4 - week period. Observable STZ - induced toxicity (less than 5% of the total n) occurred 3 - 4 days after injection. Typical symptoms in diabetic animals include polydipsia, polyuria, glycosuria, and hyperglycemia. Since diabetic animals were polydipsic, additional drinking water was provided to minimize discomfort, and the home cages were changed more frequently (daily) due to polyuria. Rats were cleaned daily after i.p. injection of STZ and after behavioral testing.

[0176] Confirmation of hyperglycemia: On day 7 (D7), a drop of tail vein blood was obtained from awake rats by puncturing the tail vein with a needle. Blood glucose levels in a drop of blood were measured using an Exactive Vital (MictoTech Medical) blood glucose monitor. Only STZ - injected rats with blood glucose concentrations above 16 mmol / L were considered diabetic and included in this study (expected value > 90%).

[0177] Static mechanical (contact) allodynia: The withdrawal threshold was measured by applying a calibrated (force; g) von - Frey monofilament (Touch - Test Sensory Evaluator; Scientific Marketing Associates) to the plantar surface of the hind paw and retracting it. The withdrawal threshold was determined by increasing and decreasing the stimulus intensity and estimated using the Dixon up - and - down method (Dixon, 1980; Chaplan et al., 1994).

[0178] 0.5 cm 2 Animals were placed on an elevated mesh-bottom platform with a grid of 2 such that their ventral side of the hindlimbs could be touched. An inverted plexiglass container was placed on top of each rat, and the test was conducted after an initial 15 - 20 minute acclimation / taming period. From beneath the mesh floor, von Frey filaments were placed perpendicular to the plantar surface of the hindlimb foot. The monofilament was held in that position for approximately 4 seconds with a force sufficient to cause the filament to bend slightly. Only a sharp withdrawal response (or flinching) within 5 seconds of the stimulus was considered a positive response.

[0179] Test schedule: Animals to be treated with nanoparticles were locally administered by intradermal injection into one foot using a micro-needle device. Pregabalin was orally administered 2 hours before each Von Frey test. Nanoparticles were administered only once on day 14 (D14). Control groups (「vehicle / vehicle」 and 「STZ / vehicle」) were injected once with saline into one foot using a micro-needle (D14).

[0180] Von Frey measurements were taken at baseline (before STZ), on D9 and D11 after STZ (neuropathy / allodynia baseline), and 2 hours after administration on D14. Von-Frey measurements were repeated twice a week until 3 weeks after STZ (1 week after nanoparticle administration).

[0181] Data analysis Two-way repeated measures ANOVA was performed using 「treatment」 as the between-subjects effect and 「day」 as the within-subjects effect (GraphPad Software, GraphPad Prism version 9.0 (Windows), San Diego, California, USA), followed by Dunnett's multiple comparison test. Data are presented as mean ± SEM.

[0182] Results The values of the paw withdrawal threshold (PWT) in rats after mechanical allodynia induction by STZ are shown in Figure 8, and the significance of the results is evaluated in Table 9 below.

Table 9

[0183] Conclusion Blood glucose level: All STZ-treated animals showed an increase in blood glucose on day 7 (D7), which was higher than 16 mmol / L (data not shown). This increase was statistically significant compared to the mean of the different groups on day 0 (D0) (STZ administration day and day 7).

[0184] The model was run until D38. However, due to abnormal behaviors in animals caused by the side effects of STZ (diarrhea, increased behavioral signs of stress response during handling), naive group animals also did not show the expected clinical symptoms, but showed some behavioral signs of stress such as restlessness during handling. Therefore, it should be noted that the results from D24 to D38 are not interpretable.

[0185] Mechanical allodynia: STZ-treated animals were scored for neuropathic mechanical allodynia on day 9 (D9) and day 11 (D11). The level of mechanical allodynia increased in all animals.

[0186] Pregabalin systemic therapy functioned as a positive control. There were significant differences in PWT levels compared to the "STZ / vehicle" group on all test days.

[0187] Treatment with different doses of nanoparticles was involved in a statistically significant increase in PWT levels at D14, D17, and D21 when compared between the group treated at a concentration of 58 g / L and the "STZ / vehicle" group (Figure 7). The lowest dose of the nanoparticles of Example 3, 18 g / L - 10 μL, resulted in a statistically significant increase in PWT levels with a p-value < 0.001 compared to the "STZ / vehicle" group at D21, which is noted to mean that this dose is sufficient for the nanoparticles of Example 3 to be effective in the symptomatic treatment of mechanical allodynia in a rat model of painful diabetic neuropathy.

Claims

1. Nanoparticles or aggregates thereof for treating peripheral neuropathic pain in a subject without being exposed to an electric field or any other external activation source, wherein (i) the nanoparticle material comprises HfO ２ or consists of HfO ２ , and (ii) the core of the nanoparticle or aggregate of nanoparticles is coated with a biocompatible coating that provides a neutral or negative surface charge when measured in an aqueous solution having an electrolyte concentration of 0.001 to 0.2 M, a concentration of the nanoparticle material or the aggregate material of the nanoparticles of 0.01 to 10 g / L, and a pH of 6 to 8.

2. The nanoparticles or aggregates thereof according to claim 1, wherein the subject to be treated has neuropathic pain after peripheral nerve injury including trigeminal neuralgia, post-traumatic neuropathic pain, postoperative neuropathic pain, or neuropathic pain due to nerve tumors, painful diabetic polyneuropathy, human immunodeficiency virus (HIV), Fabry disease, mutation of sodium channel gene, Guillain-Barré syndrome, vasculitis, chronic inflammatory demyelinating polyneuropathy, amyloidosis, non-freezing cold injury, tumor-associated syndrome, or autoimmune diseases such as Hansen's disease, chemotherapy-induced peripheral neuropathy (CIPN), postherpetic neuralgia, or painful radiculopathy.

3. The nanoparticles or aggregates thereof according to claim 2, wherein the subject to be treated has peripheral nerve injury including post-traumatic neuropathic pain, postoperative neuropathic pain, or neuropathic pain due to nerve tumors, postherpetic neuralgia, painful diabetic polyneuropathy, CIPN, or HIV.

4. The nanoparticles or aggregates thereof according to claim 3, wherein the subject to be treated has neuropathic pain after peripheral nerve injury including post-traumatic neuropathic pain, postoperative neuropathic pain, or neuropathic pain due to nerve tumors.

5. The nanoparticles or aggregates thereof according to claim 3, wherein the subject to be treated has painful diabetic polyneuropathy or CIPN.

6. The nanoparticles or aggregates thereof according to any one of claims 1 to 5, wherein the subject has local neuropathic pain.

7. The nanoparticles or aggregates thereof according to any one of claims 1 to 5, wherein the subject has intractable and / or chronic neuropathic pain.

8. The nanoparticles or aggregates thereof according to any one of claims 1 to 7, which are administered by intradermal and / or intraepidermal injection.

9. The dosage to be administered is at least 60 μg / cm ２ The nanoparticle or an aggregate thereof according to any one of claims 1 to 8, which is a skin surface to be injected.

10. wherein the patient suffers from diabetic neuropathy and the administered dose is at least 60 μg / cm ２ The nanoparticle or an aggregate thereof according to any one of claims 1 to 9, which is a skin surface to be injected.

11. wherein the patient suffers from CIPN and the administered dose is at least 390 μg / cm ２ The nanoparticle or aggregate thereof according to any one of claims 1 to 9, which is a skin surface to be injected.

12. The nanoparticles or aggregates thereof according to any one of claims 1 to 11, which are administered using a dissolving microneedle patch.

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