SARM1 agents for use in therapy and cosmetics

Abstract

The present invention relates to the therapeutic use of SARM1 agents (e.g. Molecules that cause neurodegeneration and / or neuronal dysfunction in a SARM1-dependent manner) such as Vacor, metabolites, analogs and derivatives thereof for the treatment and / or prevention of various diseases, the diseases include neurological, ophthalmological, dermatological, gastroenterological, colorectal, urinary system, gynecological, rheumatological, orthopedic, dental and / or ear-nose-throat diseases. Also provided are non-therapeutic uses of SARM1 agents, metabolites, analogs, and derivatives thereof, for example in methods for reducing or preventing skin changes such as wrinkles, contouring, and ameliorating oily skin.

Description

[0001] The present invention relates to the use of a SARM1 agent for use as a medicament, wherein the SARM1 agent is any molecule that causes neurodegeneration and / or neuronal dysfunction in a SARM1-dependent manner. Thus, the present invention includes the use of SARM1 activators such as Vacor, its metabolites, analogs, and derivatives as medicaments. In particular, SARM1 agents can be used to treat and / or prevent a variety of diseases, including neurological, ophthalmic, dermatological, gastroenterological, colorectal, urological, gynecological, rheumatological, orthopedic, dental, and / or otolaryngological diseases. The present invention further relates to the use of SARM1 agents, their metabolites, analogs, and derivatives in non-therapeutic methods for reducing or preventing skin changes such as wrinkles, contouring, and improving oily skin. Background Art

[0002] SARM1 (sterile alpha and Toll / interleukin-1 receptor motif protein 1) is a central regulator of programmed axonal death and is required for initiating axonal self-destruction following traumatic and toxic insults to the nervous system. SARM1 is a prodegenerative enzyme with key NAD+ (nicotinamide adenine dinucleotide)-consuming activity (Essuman et al., 2017). SARM1 also consumes NADP+ and catalyzes base exchange involving either cofactor (Angeletti et al., 2022; Zhao et al., 2019). In cells, SARM1 activity is regulated by the axon survival and NAD+ synthase NMNAT2 (nicotinamide mononucleotide adenylyltransferase 2). Loss of NMNAT2 in axons leads to increased levels of the NMNAT2 substrate NMN (nicotinamide mononucleotide) and NAD+ depletion, both of which act to increase the NMN:NAD+ ratio (Di Stefano et al., 2015, 2017; Gilley and Coleman, 2010; Figley et al., 2021). This increase drives SARM1 activation in axons, greatly increasing the rate of SARM1-mediated NAD+ consumption, leading to further, more rapid NAD+ depletion and accumulation of SARM1 products (such as cyclic ADP-ribose (cADPR)), which in turn leads to axonal degeneration.

[0003] Thus, SARM1 is a pro-neurodegenerative enzyme that plays a central role in programmed axonal death, a well-characterized and preventable / druggable mechanism of axonal degeneration (Coleman and 2020; Osterloh et al., 2012). In many neurodegenerative disorders, axonal loss occurs early. Therefore, preventing axonal loss is crucial for disease-modifying therapies. In view of this, the development of SARM1 inhibitors has been attracting much attention. The prior art discloses that SARM1 inhibitors play a role in axonal protection. For example, Sasaki et al. (Exp Neurol. [Experimental Neurology] 2021 Nov; 345: 113842) disclose that allosteric SARM1 inhibitors can be used to promote axonal protection. Similarly, US8889126 B2 discloses that SARM1 inhibitors can be used to treat axonal lesions, which occur in various neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, as well as in traumatic, toxic or ischemic damage to neurons. US11253503 B2 also discloses that compounds that can be used as inhibitors of SARM1 NAD enzyme activity can be used to treat neurodegenerative diseases or neurological diseases. Therefore, it is evident that SARM1 inhibitors have been exploited to a large extent for their neuroprotective effects.

[0004] In contrast, SARM1 agents that cause neurodegeneration and / or neurological dysfunction have not been used for reasonable medical purposes, which are cell-selective, topically applied, and have a temporary effect and are reversible. For example, Wu et al., 2021. disclose the injection of SARM1 agonists into nerves (chemical neurolysis) to treat neuropathic pain (particularly pancreatic cancer-related pain, facetogenic pain, and trigeminal neuralgia). However, the application of these agents proposed by Wu et al. for treating subjects is unreliable. As demonstrated by DeWitt, patients who took Vacor in the 1980 NEJM case series, injection into the pancreas will cause pancreatic degeneration and type 1 diabetes. The treatment of trigeminal neuralgia will require injection into the trigeminal ganglion, which will cause extensive nerve damage and several serious undesirable side effects, such as loss of saliva and tear production, sweating, permanent numbness of the entire face, and the risk of unilateral facial paralysis due to the proximity of the facial nerve to the trigeminal ganglion. In addition, injection near the spinal cord for low back pain of facet joint origin will likely cause irreversible limb paralysis through local leakage through motor neuron degeneration. For example, the SARM1 agent Vacor (a pyridine derivative and nicotinamide analog) and related compounds are well known for their bactericidal, fungicidal, herbicidal, insecticidal and rodenticidal properties. Vacor is a powerful neurotoxin associated with human peripheral and central nervous system disorders (Gallanosa et al., 1981; LeWitt, 1980) and rat axonal degeneration (Watson and Griffin, 1987). It is mainly used as a rodenticide to control the number of common rats, namely Rattus norvegicus, which poses a serious threat to human health and well-being. Vacor is now an obsolete pesticide.

[0005] US20220409648 A1 (which is based on data provided only by Wu et al., 2021) discloses Vacor and related pyridine derivatives that activate SARM1 by direct binding of the allosteric pocket for the proposed clinical use as a neuroablative agent, the mode of action of which is similar to that of botulinum toxin. US20220409648A1 (and Wu et al. 2021) assume that treatment with the "SARM1 activator" described therein will cause permanent neurodegeneration. However, this is not an ideal characteristic for the treatment and cosmetic applications of conditions (such as dystonia, spasticity, neuropathic pain) that require temporary and reversible inhibition of neurotransmission. Permanent neurodegeneration will lead to long-term paralysis, which may cause swallowing and breathing difficulties and numbness of the affected body parts, and may cause further disability. Effective neuroablation treatment for the above-mentioned conditions ideally needs to be temporary and fully reversible, that is, neurons can regenerate after neuroablation to avoid permanent disability. There is currently no prior art that proposes or indicates that “SARM1 activators” or SARM1 agents can cause temporary or reversible neurodegeneration. US20220409648 A1 also discloses that the therapeutic mechanism of “SARM1 activators” is “neurospecific” and will “not damage non-neural tissue”. US20220409648 A1 assumes that “SARM1 activators” are neurospecific, which is the basis for use as specific neuroablative agents. US20220409648A1 also discloses that SARM1 is only present in neurons, and this is also the basis for the disclosed use as specific neuroablative agents. US20220409648A1 proposes that SARM1 agents have a mode of action similar to other prior art neurolytics (such as phenol). US20220409648 A1 also discloses that “SARM1 activators” can be used in any condition where botulinum toxin is used.

[0006] US20220409648 A1 does not make any suggestion, nor does it provide any data indicating that the SARM1 activators or SARM1 agents disclosed therein have a transient, reversible and selective effect on cells expressing Sarm1 (such as neurons), but not on surrounding cells (such as skin cells, muscle cells and Schwann cells) that do not express Sarm1 or express low levels of Sarm1 at the application site.

[0007] US20220409648 A1 does not make any suggestion, nor does it provide any data to suggest that SARM1 agents should be administered and act peripherally to achieve therapeutic effects without causing disability.

[0008] SARM1 agents, such as Vacor and its metabolite VMN, act on SARM1. Specifically, Vacor is metabolized to vacor mononucleotide (VMN) and vacor adenine dinucleotide (VAD) ( Figure 2 ). VMN (an NMN analog) then binds to and activates SARM1, causing axonal degeneration. NMN (an endogenous SARM1 activator) and NAD both bind to the SARM1 allosteric pocket (activation pocket) domain in the ARM (Armadillo / β-catenin-like repeat) domain. NMN binds to the ARM domain, leading to SARM1 activation and axonal degeneration. In contrast, NAD binding in the same allosteric site keeps SARM1 inactive ( Figure 1 Therefore, NMN and NAD compete for binding, with opposing consequences for SARM1 activity. Increased NMN levels and the NMN / NAD ratio lead to SARM1 activation (Figley and DiAntonio, 2020; Figley et al., 2021). Therefore, when NMN levels are high, NMN binds to the allosteric pocket on the SARM1 ARM and activates SARM1, leading to increased SARM1-dependent NAD(P)+ depletion and axonal degeneration.

[0009] Nerve ablative drugs currently known in the art include the use of botulinum toxin (botox), capsaicin, and phenol. These agents can be injected locally to treat, for example, neuropathic pain and other conditions such as spasticity and dystonia. Botulinum toxin can also be used in non-therapeutic procedures to remove or prevent skin wrinkles when injected locally.

[0010] Both botulinum toxin and capsaicin act by causing a temporary interruption between nerves and target organs (Lim and Seet 2010; Simone et al. 1998).

[0011] In the case of botulinum toxin, the target organs are primarily skeletal muscle for conditions characterized by dystonia, dyskinesia, and spasticity, and smooth muscle for gastrointestinal and urinary dyskinesias, although injections into sphincters and muscles for gynecological conditions may involve injections into a combination of skeletal and smooth muscle. Injection of botulinum toxin into muscle prevents the release of neurotransmitters from presynaptic terminals and results in decreased muscle tone and symptom relief. Botulinum toxin is also used to target secretory glands innervated by the parasympathetic nervous system, particularly salivary and sweat glands. In this case, botulinum toxin blocks the release of acetylcholine from presynaptic terminals and activates the target secretory glands, thereby reducing saliva or sweat production (Bach and Simman 2022; Lim and Seet 2010).

[0012] With capsaicin, the target organ is the pain-sensing nerve endings in the skin. Capsaicin binds to TRPV1 receptors on pain-sensing neurons, initially activating the receptors but then causing inhibition and temporary degeneration, which results in a reduction in pain transmission (Simone et al. 1998).

[0013] In addition to inhibiting the release of neurotransmitters between nerves and muscles, botulinum toxin can also induce changes similar to denervation (Thesleff, Molgó, and 1990; Witzemann, Brenner, and Sakmann 1991) and anatomical muscle denervation followed by spontaneous regeneration (Velasco et al. 2008). Many other naturally occurring neurotoxins, such as latrotoxin from the black widow spider, cause a temporary interruption of neuromuscular synaptic transmission by causing degeneration of nerve endings followed by spontaneous regeneration of nerve endings (Duchen, Gomez, and Queiroz 1981; Harris et al. 2000; Rigoni and Montecucco 2017). Similarly, capsaicin causes activation of sensory nerve endings when applied to human skin, followed by inhibition and subsequent degeneration, and this cellular mechanism is the basis of its clinical efficacy (Simone et al. 1998).

[0014] In addition to the above-mentioned disadvantages, the effects caused by, for example, phenol are also irreversible. Phenol injections can cause permanent neurological damage and extensive fibrosis. Phenol injections cause severe pain and are therefore only used as a last resort and when the patient has no sensation in the affected body part (Kheder et al. 2012. Pract Neurol [Practical Neurology]; 12: 289). Although botulinum toxin is a very effective treatment, it has many disadvantages. First, there are primary and secondary non-responders to treatment. Many of these cases are caused by the development of immune resistance because the immune system reacts to bacterial proteins. In addition, because botulinum toxin is a very large molecule (150kDa), it cannot be applied via a simple transdermal patch, which limits its use. In addition, there is no reversal agent for botulinum toxin. If the toxin spreads away from the injection site, this can cause patients to suffer from weeks to months of unexpected muscle weakness, such as speech and swallowing difficulties caused by injections for cervical dystonia (Lim and Seet. 2010. Nat. Rev. Neurol. 6, 624; Tucker et al. 2021. Mov Dis Clin Pract; 8:541; Marion et al. 2016. Pract Neurol; 16:288). High-dose capsaicin (8%) patch treatment causes severe burning pain and extensive skin erythema when applied to approximately 50% of patients, often requiring co-administration of local anesthetics or opioid analgesics (Burness and McCormack. 2016. Drugs; 76:123-134). Therefore, there is a need to develop alternative therapies for conditions such as spasticity, dystonia, neuropathic pain, sphincter and autonomic dysfunction.

[0015] It is therefore an object of the present invention to provide new and / or improved treatments for neuropathic pain and other conditions such as spasticity, dystonia, sphincter and autonomic dysfunction.

[0016] It is another object of the present invention to provide new and / or improved methods of cosmetic treatment of skin changes. Summary of the Invention

[0017] The inventors have surprisingly discovered that SARM1 agents (molecules that cause SARM1-dependent neurodegeneration and / or neuronal dysfunction) that lead to SARM1 activation and have traditionally been used as neurotoxins, such as Vacor, have a first medical use. The inventors have determined that SARM1 agents, such as, but not limited to, activators of SARM1, can achieve selective inactivation / ablation of axons / neurons. The basis of the present invention is the discovery that differential expression of SARM1 in different cell types and the insensitivity of certain cell types to SARM1 agents establish their use as cell-selective therapies for the clinical conditions disclosed herein. Data in muscle cells, skin cells, and Schwann cells (axon-supporting cells in the peripheral nervous system (PNS)) indicate that SARM1 is expressed differently in different cell types and that Vacor and other SARM1 agents (such as, but not limited to, activators of SARM1) are non-toxic to these cells ( Figure 3 、 5 , 6, 7, 8, 9, 15, 17, 20). The present inventors determined that SARM1 protein is absent in mouse Schwann cells both in vivo and in vitro ( Figure 3 and 7 Cultured mouse Schwann cells were also completely insensitive to high doses of SARM1 activators such as vacor and 3-AP ( Figure 8 and 9 Such cells did not show a decrease in cellular NAD+ levels ( Figure 8 B). A further finding was that application of high doses of Vacor to cultures of human muscle cells (myoblasts) had no effect on these cell types ( Figure 15 Furthermore, applying high doses of Vacor to human dermal skin fibroblasts had no effect on this cell type ( Figure 17 ).

[0018] The insensitivity of Schwann cells, myoblasts, and dermal skin fibroblasts to SARM1 agents is important for applying these agents to, for example, the skin or muscle, and this discovery can be used in therapeutic and cosmetic treatments to induce selective degeneration of nerve endings in the area where these agents are applied, without affecting surrounding tissues. Vacor and other SARM1 agents are such novel neuroablative agents that specifically target neurons without affecting other cell types where SARM1 expression is absent or low. Therefore, Vacor and other SARM1 agents are ideal for treating and / or preventing conditions that require peripheral nerve ablation. Such applications include medical treatments such as pain relief and spasticity treatments, as well as cosmetic treatments to prevent wrinkles, where highly desirable results can be achieved by injecting SARM1 agents into affected areas of the body.

[0019] Furthermore, SARM1 agents, such as, but not limited to, activators of SARM1, act in a manner distinct from other drugs used in nerve ablation procedures, such as botulinum toxin, capsaicin, and phenol. Advantageously, the effects of SARM1 agents are temporary and / or reversible, thereby allowing for the regeneration of axons and terminal branches following induction of neurodegeneration, such as Figure 18 SARM1 agents are also reversible by reversal agents such as nicotinamide, making them particularly suitable for both therapeutic and non-therapeutic treatments. Nicotinamide (NAM) competes with Vacor for NAMPT, thereby reducing the conversion of Vacor to the toxic metabolite VMN and limiting SARM1 activation. It is also a precursor of NAD+ and would therefore counteract the rapid NAD+ depletion caused by Vacor-mediated SARM1 activation. The combination of these two activities may be responsible for the protective effects of NAM against Vacor neurotoxicity ( Figure 2 )(Loreto et al., 2021).

[0020] Furthermore, the insensitivity of Schwann cells, human myoblasts, and human dermal skin fibroblasts to SARM1 agents is important for applying these agents to the skin or muscle to act as a therapy that causes selective axon / terminal branch / synaptic terminal degeneration. Since Schwann cells are key to promoting nerve repair and regeneration (Jessen and Arthur-Farraj; Glia. [Glial cells] March 2019; 67(3): 421-437), the fact that they remain unaffected by high-dose SARM1 agents will allow for eventual axon regeneration and target organ reinnervation after SARM1 agents induce axon / terminal branch / synaptic terminal degeneration. Sarm1 mRNA is also present at very low levels in bone marrow-derived mouse monocytes / macrophages and was undetectable in one study, and previous observations have shown that Sarm1 deletion has no effect on macrophage function and gene expression (Kim et al., 2007; Szretter et al., 2009; Uccellini et al., 2020). This suggests that SARM1 agents are unlikely to cause any toxicity to peripheral monocyte / macrophage populations. This is important because macrophages also play an important role in supporting nerve regeneration (Cattin et al., 2015 Cell; Barrette et al., 2008. J Neurosci). In addition, it has been shown in certain cell culture situations that Schwann cells can delay the rate of axonal degeneration (Babetto et al., 2020; Mutschler et al., 2023), and therefore they may be able to modulate the effects of SARM1 agents on promoting targeted and selective axonal / terminal branching / synaptic terminal degeneration or dysfunction. Therefore, a one-time treatment with a SARM1 agent would be temporary and reversible.

[0021] In contrast to the prior art, it has surprisingly been found that "SARM1 activators" or SARM1 agents can cause temporary or reversible neurodegeneration while also allowing for neuroregeneration following administration ( Figure 18 ). This discovery makes it possible for the first time that SARM1 agents can be used to treat conditions such as spasticity, dystonia, peripheral neuropathic pain, autonomic dysfunction, and for cosmetic applications without causing harm or permanent disability. This is due to the fact that the permanent effects of SARM1 agents cause permanent and or long-lasting neurodegeneration, which is not an ideal characteristic for the treatment and cosmetic applications of conditions that require temporary and reversible inhibition of peripheral neurotransmission (such as dystonia, spasticity, peripheral neuropathic pain, autonomic dysfunction). Permanent neurodegeneration may lead to long-term paralysis, which may include loss of limb muscle function, difficulty swallowing and breathing, aspiration and risk of death, vision loss, intestinal and bladder incontinence, impotence, and numbness of the affected body parts, leading to further disability and or the need for corrective treatment and or surgery. Effective neuroablation treatments for the above-mentioned conditions ideally need to be temporary and fully reversible, that is, neurons can regenerate after neuroablation to avoid permanent disability.

[0022] Furthermore, the present inventors unexpectedly and surprisingly found that Schwann cells are insensitive to SARM1 agents ( Figure 8 and 9 ). This is in contrast to the prior art, which suggests that SARM1 agents are “neuronally specific” and “will not damage non-neural tissue”. This definition ignores that for “SARM1 activators” or SARM1 agents used as therapy in the above-mentioned conditions, they must not harm non-neuronal cells contained in the neural tissue. In particular, Schwann cells that encase axons and / or are closely associated with axons in nerve roots and nerve trunks (in the case of myelinating and non-myelinating Schwann cells), as well as Schwann cells associated with the terminal branches of neurons in the nervous tissue (in the case of terminal / perisynaptic and pain-sensing Schwann cells), are key to promoting nerve repair and regeneration by converting into injury-activated and / or repair Schwann cells (Jessen and Arthur-Farraj; Glia. [Glia] 2019 March; 67(3): 421-437). The insensitivity of Schwann cells to “SARM1 activators” or SARM1 agents is key to the temporary and reversible nature of the proposed treatment.

[0023] It is not clear in the prior art whether SARM1 is present and functional in myelinating glial cells in the CNS or PNS, as well as in non-neuronal cells of the PNS and cells of target organs (i.e., skin and muscle) innervated by the PNS. The present inventors surprisingly found that SARM1 protein is present and functional in myelinating glial cells and oligodendrocytes of the central nervous system, but is almost completely absent or present at a functionally negligible level in Schwann cells in the peripheral nervous system and Schwann cells after nerve injury ( Figure 3 、 5 , 7, 8, 9, 20). The finding of comparative SARM1 levels between central nervous system glial cells and peripheral nervous system glial cells is the basis for the use of the SARM1 agents disclosed herein as a temporary and reversible method of peripheral nerve ablation in all of the above-mentioned pathologies. Furthering this, the inventors have also surprisingly shown that other peripheral cells, human myoblasts and human dermal skin fibroblasts, are insensitive to high concentrations of SARM1 agents, strongly suggesting that they also contain very low levels of functional SARM1 ( Figure 15 and 17 ).

[0024] Furthermore, the present inventors have discovered that for the treatment of some conditions, SARM1 agents can be administered peripherally to have a therapeutic effect without causing harm. This is in contrast to the prior art, which includes treatment of conditions such as post-amputation pain, which is a central pain condition and would require administration of SARM1 agents in or near the spinal cord or brain, which would cause severe disability or even death. The present inventors have discovered that CNS myelinating glial cells contain functional SARM1 ( Figure 3 、 5 and 7), a finding that further discredits the proposed use of SARM1 agents to treat conditions that require administration of such agents in or near the CNS because such agents are not cell-selective, target multiple cell types, cause widespread damage, and cause persistent disability.

[0025] In a first aspect of the present invention, a SARM1 agent is provided for use as a medicament.

[0026] In a first aspect of the present invention, a SARM1 agent is provided for use in a method of treating and / or preventing neurological, ophthalmic, dermatological, gastroenterological, colorectal, urological, gynecological, rheumatological, orthopedic, dental and / or otolaryngological diseases.

[0027] In certain embodiments, the disease is defined by autonomic dysfunction, neuropathic pain, dystonia, spasticity, movement disorders, sphincter dysfunction, genitourinary dysfunction, and / or a skin condition.

[0028] In certain embodiments, the autonomic dysfunction comprises excessive sweating (hyperhidrosis). In certain embodiments, the autonomic dysfunction comprises excessive saliva production (sialorrhea).

[0029] In certain embodiments, the autonomic dysfunction comprises autonomic dysfunction caused by an inherited and / or genetic neurological disease. In certain embodiments, the inherited and / or genetic neurological disease comprises hereditary autonomic neuropathy and / or sensory autonomic neuropathy.

[0030] In certain embodiments, the autonomic dysfunction comprises autonomic dysfunction caused by a degenerative neurological disease. In certain embodiments, the degenerative neurological disease comprises amyotrophic lateral sclerosis, Parkinson's disease, Parkinson's syndrome, multiple system atrophy, cerebral palsy and / or a degenerative neurological disease caused by injury.

[0031] In certain embodiments, the autonomic dysfunction includes autonomic dysfunction caused by autonomic nervous system diseases caused by acquired conditions (secondary autonomic disorders). In certain embodiments, autonomic nervous system diseases caused by acquired conditions (secondary autonomic disorders) include infections (COVID and long-term COVID), autoimmune conditions, postural orthostatic tachycardia syndrome (POTS) and / or endocrine conditions. In certain embodiments, endocrine conditions include diabetes and / or thyroid dysfunction.

[0032] In certain embodiments, the autonomic dysfunction comprises autonomic dysfunction caused by small fiber neuropathy.

[0033] In certain embodiments, the autonomic dysfunction comprises autonomic dysfunction caused by a connective tissue disease.

[0034] In certain embodiments, the autonomic dysfunction comprises autonomic dysfunction caused by peripheral nerve damage.

[0035] In certain embodiments, the autonomic dysfunction comprises autonomic dysfunction caused by surgical repair of peripheral nerve injury.

[0036] In certain embodiments, the autonomic dysfunction comprises drug-induced autonomic dysfunction.

[0037] In certain embodiments, the dysautonomia comprises dysautonomia caused by idiopathic dysautonomia.

[0038] In certain embodiments, neuropathic pain comprises peripheral neuropathic pain; chronic back pain; cancer-related pain; myofascial pain; fibromyalgia; complex regional pain disorder; chronic pelvic pain; dyspareunia; vulvodynia; and / or painful bladder syndrome (interstitial cystitis). In certain embodiments, neuropathic pain comprises peripheral neuropathic pain.

[0039] In certain embodiments, neuropathic pain includes neuropathic pain caused by: diabetic neuropathy; peripheral nerve injury; infectious and post-infectious neuropathies; metabolic neuropathies; small fiber neuropathy; endocrine conditions; genetic neuropathies; paraproteinemic neuropathy; paraneoplastic neuropathy; tumor neuropathy; vasculitis; autoimmune, inflammatory and demyelinating neuropathies; idiopathic neuropathies, peripheral nerve injury and / or surgical repair of peripheral nerve injury.

[0040] In certain embodiments, dystonia includes: primary dystonia; and / or dystonia plus conditions.

[0041] In certain embodiments, dystonia includes dystonia occurring in conjunction with a neurodegenerative disease.

[0042] In certain embodiments, dystonia includes secondary dystonia occurring as a result of central nervous system (CNS) trauma, congenital malformations, genetic and chromosomal disorders, infection, tumors, ischemic; hemorrhagic stroke, inflammation, demyelination, drugs, toxins, and / or metabolic disorders;

[0043] In certain embodiments, spasticity includes upper limb spasticity, lower limb spasticity; bladder spasm / neurogenic bladder; spastic disorders of the esophagus.

[0044] In certain embodiments, spasticity includes spasticity caused by: demyelinating conditions; congenital malformations; chromosomal disorders; genetic conditions; CNS infections; tumors; drugs; toxins; metabolic disorders; paraneoplastic conditions; post-infectious conditions; autoimmune conditions; endocrine conditions, peripheral nerve injury, and / or surgical repair of peripheral nerve injury;

[0045] In certain embodiments, the movement disorder comprises: hemifacial spasm with facial nerve synkinesis; and / or palatal myoclonus / tremor.

[0046] In certain embodiments, movement disorders include movement disorders caused by: tics; tremors; myokymia, trauma, infection, autoimmune conditions; neuromyotonia caused by autoimmune and / or genetic conditions;

[0047] In certain embodiments, the sphincter dysfunction is caused by: body sphincter dysfunction; pyloric sphincter; sphincter of Oddi dysfunction leading to gastrointestinal dysmotility; pancreatic sphincter; urethral sphincter; anal fissure; and / or Hirshsprungs disease.

[0048] In certain embodiments, the urogenital dysfunction comprises: vaginismus; and / or urinary dysfunction.

[0049] In certain embodiments, the skin condition comprises: an ulcer; acne; and / or Raynaud's phenomenon / disease.

[0050] In certain embodiments, the orthopedic condition comprises: sports injury; post-traumatic elbow stiffness; clubfoot; and / or piriformis syndrome.

[0051] In certain embodiments, the dental condition comprises: toothache; and / or root canal treatment.

[0052] In a third aspect of the invention there is provided a non-therapeutic method for: (i) Removal and / or prevention of skin changes; (ii) contouring; and / or (iii) Improve oily skin; The method comprises administering a SARM1 agent.

[0053] In certain embodiments, the skin changes include one or more of skin wrinkles, frown lines / glabellar lines, forehead lines, lateral canthal lines / crow's feet, transverse nasal creases, neck bands, platysmal neck lines, smoker's lines, bunny lines, upper lip wrinkles, lateral commissure ptosis, marionette lines, gummy smile, chin dimples, cellulite chin, sunken skin, drooping eyebrows, drooping eyelids, drooping lips, square jaw, and downturned nose tip.

[0054] In certain embodiments, contouring includes (i) lip, cheek, jaw, and / or temple shaping; and / or (ii) eyebrow contouring.

[0055] In certain embodiments of any aspect of the invention, the SARM1 agent causes SARM1-dependent neurodegeneration and / or neuronal dysfunction. In certain embodiments of any aspect of the invention, the SARM1 agent activates SARM1 and neurodegeneration and / or neuronal dysfunction.

[0056] In certain embodiments of any aspect of the present invention, the SARM1 agent activates SARM1 by binding to SARM1. In certain embodiments of any aspect of the present invention, the SARM1 agent increases intracellular levels of NMN. In certain embodiments, the SARM1 agent decreases intracellular levels of NAD. In certain embodiments, the SARM1 agent increases the intracellular NMN / NAD ratio.

[0057] In certain embodiments, the neurodegeneration comprises degeneration of axons, terminal branches or synaptic terminals and / or neurolytic death, optionally wherein the axonal degeneration, terminal branches and / or synaptic terminals and / or neurolytic death is reversible. In certain embodiments, the neurodegeneration is temporary. In certain embodiments, the neurodegeneration allows for nerve regeneration to occur after the neurodegeneration. That is, after the SARM1 agent induces neurodegeneration, neuronal cells may be able to regenerate. For example, neurons may regenerate their axons after degeneration caused by administration of a SARM1 agent as described herein. For example, topically.

[0058] In certain embodiments, the neurodegeneration and / or neuronal dysfunction is reversible by a reversal agent.

[0059] In certain embodiments, neurodegeneration and / or neuronal dysfunction: (ii) occurs in a cell expressing SARM1, optionally wherein the cell expressing SARM1 comprises a neuron; and (ii) is not present in cells that lack SARM1 or express a functionally insignificant amount of SARM1 protein, optionally wherein the cells that lack SARM1 or express a functionally insignificant amount of SARM1 include Schwann cells (myelinating, non-myelinating / Remak, perisynaptic / peripheral, pain-sensing or injury-activated / repair Schwann cells); muscle cells, skin cells (such as dermal fibroblasts), HEK293T cells, HeLa cells, SK mel 2 cells, SK mel 5 cells, SK mel 28 cells, CAPAN cells, JURKAT cells, C6 cells, HL-60 cells, LP-1 cells, U937 cells and / or MEWO cells, monocytes, macrophages and / or RAW264.7 cells (Uccellini et al., 2020).

[0060] In certain embodiments, the SARM1 agent is selected from vacor; vacor mononucleotide (VMN); 3-acetylpyridine (3-AP); 3-acetylpyridine mononucleotide (3-APMN); 2-aminopyridine (2-AnP); 2-AnP mononucleotide (2-AnPMN); nicotinamide mononucleotide (NMN), sulfo-ara-F-NMN (CZ-48), sulfo-ara-F-VMN; sulfo-ara-F-3-APMN; sulfo-ara-F-2-AnPMN, S-NMN, ara-F-NMN (CZ-17) ; pyridine and molecules containing a pyridine ring; vacor nucleoside (VR), vacor nucleoside (VR) analogs, nicotinamide (NAM) analogs; nicotinamide riboside (NR), nicotinamide riboside (NR) analogs; nicotinic acid (NA) analogs; nicotinic acid riboside (NaR) analogs, nicotinic acid mononucleotide (NaMN) analogs, molecules that increase intracellular NMN levels, decrease NAD levels and / or increase the NMN / NAD ratio, thereby leading to SARM1 activation; NMNAT1-2-3 inhibitors or molecules that decrease NMNAT1-2-3 levels; and / or metabolites, analogs and derivatives thereof.

[0061] In certain embodiments, the analogs and derivatives increase the permeability and / or solubility of the SARM1 agent.

[0062] In certain embodiments, the SARM1 agent is administered topically, optionally by: (i) transdermal patch or (ii) injection into tissues such as (a) skin or muscle, optionally intravesically into the detrusor / urethral sphincter with or without ultrasound, radiographic and / or electromyographic guidance, (b) neuromas, optionally for peripheral nerve regeneration and surgical nerve repair, and / or (c) root canals, optionally for use in dental surgery; (iii) endoscopic injection, such as injection into the esophageal sphincter, pyloric sphincter, sphincter of Oddi; cystoscopic injection into the urethral sphincter; (iv) injection into the salivary glands, such as the sublingual and submandibular glands; or (v) Topical administration.

[0063] In certain embodiments, two or more SARM1 agents as described herein are co-administered, optionally with the administration of an additional agent, such as an antidote and / or analgesic / anesthetic agent.

[0064] In a further aspect, there is provided a composition comprising a SARM1 agent as described herein; optionally wherein the composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable excipient, diluent and / or carrier.

[0065] In certain embodiments, the compositions described herein are for use in any of the methods described herein.

[0066] In certain embodiments, the composition is a topical composition; optionally wherein the composition is a cream or ointment.

[0067] In certain embodiments of the third aspect, the SARM1 agent is contained in a composition as described herein.

[0068] In a further aspect, a syringe is provided comprising a SARM1 agent or composition as described herein.

[0069] In a further aspect, a transdermal patch is provided, comprising a SARM1 agent or composition as described herein.

[0070] In a further aspect, a kit is provided comprising: a. a SARM1 agent or composition as described herein; and b. an applicator configured to administer the SARM1 agent.

[0071] In certain embodiments, the applicator comprises a transdermal patch as described herein.

[0072] Throughout the description and claims of this specification, the words “comprise” and “comprising” and variations thereof mean “including but not limited to,” and they are not intended to (and do not) exclude other parts, additives, components, integers or steps.

[0073] Throughout the description and claims of this specification, the singular encompasses the plural, unless the context requires otherwise. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0074] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Embodiments of the present invention are further described below with reference to the accompanying drawings, in which:

[0076] Figure 1Schematic diagram of (A) the programmed axonal death pathway and (B) the SARM1 activation mechanism are shown.

[0077] Figure 2 Schematic diagram showing (A) how SARM1 activation leads to axonal degeneration and (B) how reversal of SARM1 activation by nicotinamide is achieved.

[0078] Figure 3 : Detection of SARM1 protein associated with CNS myelinating glial cells, but not PNS myelinating glial cells, is shown. A) Representative immunofluorescence image of a Wt mouse tibial nerve cross section, showing colocalization of SARM1 with NF-200 (neurofilament light chain) in axons. The magnified area is indicated by a white bounding box. The scale bar is 10 μm. B) Representative immunofluorescence image of a Wt mouse optic nerve cross section, showing SARM1 expression in TUJ1 (anti-βIII tubulin)-positive axons. The magnified area is indicated by a white bounding box. The scale bar is 10 μm. C) Representative immunofluorescence image of a Wt mouse DRG cross section, showing colocalization of SARM1 with NF-200-positive neurons. The scale bar is 25 μm. D) Representative immunofluorescence image of a Wt and Sarm1 KO mouse tibial nerve cross section, showing SARM1 and SOX10 expression. The white arrow shows colocalization of DAPI with SOX10, but no SARM1 expression. The magnified area is indicated by a white bounding box. The scale bar is 10 μm. E) Representative immunofluorescence images of cross-sections of DRG from Wt and Sarm1 KO mice, showing SARM1 and SOX10 expression. White arrows point to SOX10-positive satellite glial cells and DAPI, with no colocalization of SARM1. The scale bar is 10 μm. F) Representative immunofluorescence images of cross-sections of optic nerves from Wt and Sarm1 KO mice, showing SARM1 and SOX10 expression. White arrows point to SOX10-positive oligodendrocytes and DAPI, with perinuclear SARM1 staining. The scale bar is 10 μm. n = 3 for all experiments (3 animals, 2 nerves per animal, 5 sections per nerve).

[0079] Figure 4: First layer SARM1 antibody control not shown. A) Representative immunofluorescence images of transverse sections of dorsal root ganglia (DRG) from Wt and Sarm1 KO mice, showing the lack of SARM1 signal in the absence of added primary antibody. NF-200 (neurofilament light chain) positive neurons are shown. Scale bar, 25 μm. B) Representative immunofluorescence images of transverse sections of dorsal root ganglia (DRG) from Wt and Sarm1 KO mice, showing the lack of SARM1 signal in the KO sample in the presence of primary antibody. The Wt image shows colocalization of NF-200-positive neurons with SARM1. Scale bar, 25 μm.

[0080] Figure 5 Figure 1: Shows sarm1 expression in the posterior lateral line nerve (PLLn) and spinal cord of zebrafish larvae. A) Maximum projection, lateral view of the PLLn of a 5 dpf larva, demonstrating in situ HCR labeling for sox10 and sarm1 mRNAs and DAPI staining for nuclei. B) Maximum projection, lateral view of the spinal cord of a 5 dpf larva, demonstrating in situ HCR labeling for sox10 and sarm1 mRNAs and DAPI staining for nuclei. C) Single confocal z-plane, 616 nm optical depth, lateral view of the PLLn of a 5 dpf larva. Arrows mark nuclei labeled with DAPI that are positive for sox10 and sarm1 mRNAs. Scale bar, 25 μm, for all images. n = 7 biological replicates for all experiments.

[0081] Figure 6 Shown are sarm1 expression in the hypothalamus and retina of zebrafish larvae. A) Maximum projection, lateral view of the PLLn of a 5 dpf larva, without application of either a probe targeting sox10 or a probe targeting sarm1 (no first layer control). Images show DAPI and hairpins labeled with Alexa647 and Alexa546. Scale bar, 25 μm. B) Maximum projection, lateral view of the spinal cord of a 5 dpf larva, without application of either an antisense probe targeting sox10 or an antisense probe targeting sarm1. Images show DAPI and hairpins labeled with Alexa647 and Alexa546. Scale bar, 25 μm. C) Maximum projection, lateral view of the hypothalamus of a 5 dpf larva, showing sox10 (magenta) and sarm1 (green) mRNA expression. Nuclei are labeled with DAPI (grey). Scale bar, 25 μm. D) Maximum projection, lateral view of the retina of a 5 dpf larva, showing sox10 and sarm1 mRNA expression. Nuclei were labeled with DAPI. Scale bar, 25 μm.

[0082] Figure 7Sarm1 mRNA, but not SARM1 protein, is detected in mouse Schwann cells. A) RT PCR of cDNA from cultured mouse Schwann cells from Wt, Sarm1 knockout mouse Schwann cells, and mouse dorsal root ganglion neurons cultured from Wt and Sarm1 knockout embryos (E14.5) (n=4: four separate cultures prepared from four separate timed matings). B) qPCR analysis of Sarm1 expression in cultured mouse Wt and Sarm1 knockout DRGs and Wt and Sarm1 knockout Schwann cells (SC, n=4 (four biological replicates: four sciatic nerves from two animals combined per biological replicate); **p<0.01). C) Freshly plated Wt and Sarm1 knockout, P2-dissociated mouse Schwann cells all have undetectable levels of SARM1 protein. Cultures were labeled for DAPI and SOX10 (n=3: two to four mice, each from three litters, resulting in three separate cultures). Scale bar, 50 μm. D) Cultured rat oligodendrocytes (SOX10-positive) expressing SARM1 protein using a commercially available polyclonal anti-SARM1 antibody. Magnified region (indicated by the dotted box). Arrows indicate SOX10-positive and SARM1-positive cells. Scale bar, 50 μm.

[0083] Figure 8 Cultured oligodendrocytes, but not Schwann cells, are sensitive to vacor-induced cell death. A) Dissociated and freshly plated P2 mouse Schwann cells cultured for 72 hours in DMSO or 100 μM vacor showed no cell death as judged by propidium iodide (PI) and DAPI nuclear staining and SOX10 immunocytochemistry (n=3: two to four mice, each from three litters, resulting in three separate cultures). Scale bar, 25 μm. B) Intracellular NAD in Schwann cells treated with 250 mM 3AP or 100 μM vacor for 72 hours compared to DMSO control conditions. +Levels did not change. C) Rat oligodendrocytes cultured in DMSO or 100 μM vacor for 72 hours showed massive cell death (n=3: one rat from three litters, resulting in three separate cultures) as judged by DAPI nuclear staining and SOX10 immunocytochemistry. Scale bar is 50 μm. D) Quantification of SOX10-positive oligodendrocyte survival in response to 100 μM vaco treatment compared to DMSO control cultures. E) Cultured rat oligodendrocytes (SOX10 positive) expressed SARM1 protein using a polyclonal anti-SARM1 antibody generated by Yi-Ping Hsueh. Amplified area (indicated by a dotted box). Arrows indicate SOX10-positive SARM1-positive cells. Scale bar is 50 μm.

[0084] Figure 9 Cultured Schwann cells are shown to be insensitive to 3-AP-induced cell death. A) Dissociated and freshly plated P2 mouse Schwann cells cultured in water or 250 μM 3-AP for 72 hours showed no cell death as judged by propidium iodide (PI) and DAPI nuclear staining and SOX10 immunocytochemistry (n=3: two to four mice, each from three litters, resulting in three separate cultures). Scale bar, 25 μm. B) Intracellular NAD in HEK293 cells treated with 100 μM vacor for 72 hours. + The level is reduced.

[0085] Figure 10 Figure 3: Axonal degeneration is significantly delayed after two-photon laser axotomy of PLLn in zebrafish larvae in the absence of functional Sarm1. A) Schematic overview of two-photon laser axotomy of PLLn (green) in larval zebrafish. B) Injection of wt and sarm1 cells at the one-cell zygote stage with the neuroD:tdtomato DNA construct. SA11193 / SA11193 (sarm1 mutant) fish were imaged and two-photon laser axotomy was performed in both genotypes at 4 dpf. While wt PLLn axons began to degenerate around three hours after axotomy and were completely degenerated by six hours, PLLn axons in sarm1 mutant larvae remained intact until at least 26 hours after imaging (imaging was discontinued due to UK Home Office regulations) (n=5). Scale bar, 100 μm.

[0086] Figure 11 Figure 3: PLLn and spinal cord myelination are not affected by the loss of functional Sarm1 in zebrafish. A) Wild-type (wt) Tg[mbp:EGFP-CAAX] and sarm1 SA11193 / SA11193Maximum projection of a lateral view of a 5dpf (sarm1 mutant, mut) Tg[mbp:EGFP-CAAX] larva. Arrowhead (white) delineates the dorsal spinal cord; arrowhead (white) marks the ventral spinal cord; arrowhead (gray) pinpoints the PLLn. Scale bar, 100 μm. B) Magnified lateral view of the spinal cord and PLLn (area indicated by the box in A). Arrowhead (white) delineates the dorsal spinal cord; arrowhead (white) marks the ventral spinal cord; arrowhead (gray). Scale bar, 25 μm. C) Relative GFP intensity in the PLLn of Tg[mbp:EGFP-CAAX]wt and sarm1 mutant (mut) (n = 9; p = 08633). D) Relative GFP intensity in the dorsal and ventral spinal cords of a combination of Tg[mbp:EGFP-CAAX]wt and sarm1 mutant (mut) (n = 9; p = 08633). E) Relative GFP intensity in the dorsal spinal cord of Tg[mbp:EGFP-CAAX]wt and sarm1 mutants (mut) (n=9; p=09314). F) Relative GFP intensity in the ventral spinal cord of Tg[mbp:EGFP-CAAX]wt and sarm1 mutants (mut) (n=9; p=08633). G) Maximum projection, lateral view of the PLLn of 5dpf wt and sarm1 mutants (mut), showing mbp mRNA expression. Nuclei are labeled with DAPI (grey). Scale bar, 25 μm. Heatmap of nuclear mbp mRNA expression intensity (mbp*) in the PLLn of 5dpf wt and sarm1 mutants (mut). Dark nuclei represent cells with high nuclear mbp expression. Scale bar, 20 μm. H) Quantification of mbp mRNA signal intensity / μm in the PLLn of wt and sarm1 mutant (mut) 5 dpf juveniles (WT n=6; MUT n=7; p=0.8357). J) Quantification of DAPI signal intensity / μm in the PLLn of wt and sarm1 mutant (mut) 5 dpf juveniles (WT n=6; MUT n=7; p=0.7308). K) Maximum projection, lateral view of the spinal cord of wt and sarm1 mutant (mut) 5 dpf juveniles, showing mbp mRNA expression. Nuclei are labeled with DAPI. Scale bar, 25 μm. L) Quantification of mbp mRNA signal intensity / μm in the combined dorsal and ventral spinal cords of wt and sarm1 mutant (mut) 5 dpf juveniles (WT n=6; MUT n=7; p=0.4452). M) Quantification of DAPI signal intensity / μm in the spinal cords of wt and sarm1 mutant (mut) 5 dpf larvae (wt n=6; mut n=7; p=0.7308).

[0087] Figure 12Figure 3. PNS myelination and myelin maintenance are normal in Sarm1-null mice. A) Representative electron micrographs taken at 3000x magnification of postnatal day 2 (P2) tibial nerves from Wt and Sarm1 knockout (KO) mice. Scale bar, 5 μm. B) The total number of axons >1.5 mm, quantified by neural profiling, was not significantly different between Wt and Sarm1 KO nerves (n=5; p=0.3095). C) The number of myelinated axons, quantified by neural profiling, was similar in both Wt and Sarm1 KO nerves (n=5; p=0.3095). D) The number of unmyelinated axons >1.5 μm was not significantly different between Wt and Sarm1 KO nerves (n=5; p=0.1508). E) Based on neural profiling, the number of unmyelinated axons >1.5 μm at a 1:1 ratio was slightly higher in Sarm1 KO nerves compared to Wt; however, this did not reach significance (n=5; p=0.0794). F) The percentage of myelinated to unmyelinated axons present in Wt and Sarm1 KO nerves was not significantly different (n=5; p=0.1508). G) The number of Schwann cell nuclei quantified by neural profiling was not significantly different between Wt and Sarm1 KO nerves (n=5; p=0.5). H) Wt (29131 mm 2 ) and Sarm1 KO (35291mm 2 ) There was no significant difference in the mean total nerve area between the nerves (n = 5; p = 0.1508). J) Representative electron micrographs taken at 3000x magnification from adult Wt and Sarm1 KO tibial nerves at P60. There were no ultrastructural differences in the adult Sarm1 KO tibial nerve compared to the WT tibial nerve. Scale bar, 5 μm. K) Based on nerve profiling, the total number of axons >1.5 μm quantified was similar in Wt and Sarm1 KO nerves (n = 4; p = 0.3429). L) There was no significant difference in the number of myelinated axons present in Wt and Sarm1 KO nerves (n = 4; p = 0.3429). M) Based on nerve profiling, there was no significant difference in the number of unmyelinated axons >1.5 μm in Wt and Sarm1 KO nerves (n = 4; p = 0.3429). N) The number of Schwann cell nuclei was similar in both Wt and Sarm1 KO neurons (n=4; p=0.4857). P) The thickness of myelin sheaths, as depicted by g-ratio, was similar in both Wt and Sarm1 KO neurons (n=4; p=0.4857). Q) The percentage of myelinated versus unmyelinated axons present in both Wt and Sarm1 KO neurons did not differ (n=4; p=0.3429). R) Based on the neuronal profile, Wt (156715 μm 2 ) and Sarm1 KO (154927 μm2 ) nerves. The total nerve area of ​​the two nerves was very similar (n=4; p=0.8857).

[0088] Figure 13 Figure 3: Myelin gene expression is normal in Sarm1-null peripheral nerves. A) Relative mRNA expression of chemokines, Schwann cell damage, and myelin genes in P60 intact tibial nerves from Wt and Sarm1 KO mice. All fold change values ​​are normalized to intact Wt tibial nerves (n=4; *p<0.05, **p<0.01). B) Representative Western blot images of tibial nerve protein extracts from P60 Wt and Sarm1 KO mice. Images show that EGR2 and JUN levels do not differ between Wt and Sarm1 KO nerves. C) EGR2 levels are not significantly different between Wt and Sarm1 KO nerves (n=7; p=0.9118). EGR2 protein levels are normalized to those in Wt nerves, which are set to 1. D) JUN levels are not significantly different between Wt and Sarm1 KO nerves (n=7; p=0.5787). Quantification results were normalized to the levels in Wt nerves, which were set to 1. E) Representative Western blot images showing that the levels of myelin zero protein (MPZ) and myelin basic protein (MBP) were not different between P60 Wt and Sarm1 KO nerves. F) There was no significant difference in MPZ levels between Wt and Sarm1 KO nerves (WT n=6; KO n=5; p=0.3176). Quantification results were normalized to the levels in Wt nerves, which were set to 1. G) There was no significant difference in MBP levels between Wt and Sarm1 KO nerves (n=7; p>0.9999). Quantification results were normalized to the levels in Wt nerves, which were set to 1.

[0089] Figure 14Figure 3: Sarm1 null mice show normal optic nerve myelination. A) Representative electron micrographs taken at 3000x (left panel) and 12,000x (right panel) magnification from adult Wt and Sarm1 KO optic nerves at postnatal day 60 (P60). There are no ultrastructural differences in the optic nerves of Sarm1 KO compared to Wt animals. Scale bars are 5 μm (3000x) and 2 μm (12,000x). B) The total number of axons quantified by optic nerve spectra was not significantly different between WT and Sarm1 KO (n=6 WT; n=5 KO; p=0.9307). C) The number of myelinated axons quantified in Wt and Sarm1 KO optic nerves was similar (n=6 WT; n=5 KO; p=0.4286). D) There was no significant difference in the percentage of myelinated axons quantified in Wt and Sarm1 KO optic nerves (n=6 WT; n=5 KO; p=0.0736). E) Based on the neuronal profile, Wt (53686 μm 2 ) and Sarm1 KO (56979μm 2 ) There was no significant difference in the total nerve area of ​​the optic nerve (n=6 WT; n=5 KO; p=0.1255). F) Relative mRNA expression of chemokine, oligodendrocyte, and myelin genes in the intact optic nerve of Wt and Sarm1 KO mice at P60. All fold change values ​​were normalized to the intact WT optic nerve (n=4; *p<0.05, **p<0.01). G) Representative Western blot images of optic nerve protein extracts show that MBP levels are not different between Wt and Sarm1 KO nerves. H) There was no significant difference in MBP expression between Wt and Sarm1 KO optic nerves (n=6; p=0.3939). Quantification results were normalized to the levels in the intact Wt nerve, which was set to 1.

[0090] Figure 15 Cultured human myoblasts are shown to be resistant to vacor-induced cell death. Representative images of cultured human myoblasts treated with DMSO or vacor for 24 hours. Propidium iodide (PI) staining shows that cell death does not occur even in the presence of vacor.

[0091] Figure 16Shown are A) NMN-interacting residues in Drosophila and human SARM1, based on the dSARM1ARM crystal structure (from Figley et al. Neuron 2021). B) VMN-interacting residues (in parentheses) in Drosophila and human SARM1, based on the dSARM1ARM crystal structure (from Loreto et al. eLife 2021). C) ARMSAM:NMN interaction between the ARM domain of human SARM1 and NMN (stick representation). Polar contacts are shown as yellow dashed lines (from Shi et al. Mol Cell 2022).

[0092] Figure 17 Figure 1 shows that cultured human dermal skin fibroblasts are resistant to vacor-induced cell death. Representative images of cultured human dermal skin fibroblasts treated with DMSO or vacor for 72 hours. Propidium iodide (PI) staining shows that no cell death occurs in the presence of high concentrations of vacor.

[0093] Figure 18 Figure 2 shows human fetal DRG axon regeneration after local Vacor treatment. (A) Human fetal DRG was cultured in a microfluidic chamber to allow local treatment of axons (green compartment). (B) 24 hours after adding 25 μM Vacor to the axon compartment, DRG axons degenerated, while untreated neuronal cell bodies remained healthy. 24 hours after Vacor treatment, when the axons had degenerated, the Vacor was washed off and fresh culture medium was added to the axon compartment. DRG axons began to grow again and completely refilled the axon compartment 120 hours after Vacor removal. This shows that local Vacor treatment is temporary and / or reversible and / or allows neuronal regeneration.

[0094] Figure 19Showing that highly expanded / passaged mouse Schwann cells in culture are sensitive to SARM1 agents, vacor, and 3-AP. A) Phase contrast (phase contrast) microscopy and low magnification (10x) image montage of individual cultures of mouse Schwann cells passaged three times in proliferation medium before being treated with 100 μM vacor or DMSO as a control for 72 hours, and propidium iodide (PI) staining. Scale bar is 200 μm. B) Phase contrast (phase contrast) microscopy and high magnification (40x) image of individual cultures of mouse Schwann cells passaged three times in proliferation medium before being treated with 100 μM vacor or DMSO as a control for 72 hours. Arrows show colocalization (merge) of PI staining with dying or dead cells identified by phase contrast microscopy. Scale bar is 100 μm. In both A) and B), the significant increase in PI staining in vacor-treated cultures indicates increased cell death. C) Phase contrast (phase contrast) microscopy and low-magnification (10x) image montage of individual cultures of mouse Schwann cells passaged three times in proliferation medium before treatment with 250 μM 3-AP or water as a control for 72 hours, and propidium iodide (PI) staining. Scale bar, 200 μm. D) Phase contrast (phase contrast) microscopy and high-magnification (40x) image of individual cultures of mouse Schwann cells passaged three times in proliferation medium before treatment with 250 μM 3-AP or water as a control for 72 hours, and propidium iodide (PI) staining. Arrows show colocalization of PI staining with dying or dead cells identified by phase contrast microscopy (merge). Scale bar, 100 μm. In both C) and D), the significant increase in PI staining in 3-AP-treated cultures indicates increased cell death.

[0095] Figure 20 The results show that Schwann cells do not express detectable SARM1 after nerve injury. Immunohistochemistry for SARM1, SOX10 (a marker of Schwann cells), neurofilament light chain (NF), and DAPI nuclear stain were performed in intact and injured tibial nerves from 6–8-week-old C57BL / 6J mice. The sciatic nerve was injured (transected) at the sciatic notch, and the distal tibial nerve was harvested at least 1–1.5 cm from the lesion site, fixed, and processed for immunocytochemistry. The distal tibial nerve was removed and analyzed 24 and 48 hours and 7 days after injury. SARM1 staining did not colocalize with SOX10 at any time point after nerve injury, indicating that Schwann cells do not express detectable SARM1 protein in vivo after nerve injury.

[0096] The patents, scientific and technical literature referred to herein establish the knowledge available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications cited herein are hereby incorporated by reference to the same extent as if each were specifically and individually indicated to be incorporated by reference. In the event of any inconsistency, the present disclosure controls.

[0097] Various aspects of the invention are described in further detail below. DETAILED DESCRIPTION

[0098] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2nd edition, John Wiley and Sons, New York (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, New York (1991) provide a general dictionary of many terms used in the present invention for those skilled in the art. Although any methods and materials similar or equivalent to the methods and materials described herein can be used in the practice of the present invention, preferred methods and materials are described herein. Therefore, the terms defined immediately below are more fully described by reference to the entire specification. In addition, as used herein, unless the context clearly indicates otherwise, the singular terms "a / an" and "the" include plural referents. Unless otherwise indicated, nucleic acids are written from left to right in 5' to 3' orientation; amino acid sequences are written from left to right in amino to carboxyl orientation, respectively. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary depending on the context in which they are used by one skilled in the art.

[0099] SARM 1 potion

[0100] A SARM1 agent may be a molecule that causes SARM1-dependent neurodegeneration and / or neuronal dysfunction.

[0101] SARM1 is a multifunctional, pro-mutagenic enzyme with nicotinamide adenine dinucleotide phosphate (NAD(P))-consuming activity. SARM1 possesses NAD(P) hydrolysis, cyclization, and base exchange activities. Under physiological conditions, SARM1 has basal NAD(P) enzyme activity, which slowly consumes NAD and NADP while producing nicotinamide, cyclic ADP-ribose (cADPR), ADP-ribose (ADPR), cyclic ADP-ribose phosphate (cADPRP), and ADP-ribose phosphate (ADPRP). Under physiological conditions, basal SARM1 activity does not lead to neurodegeneration and / or neuronal dysfunction. In the presence of toxic stimuli, or following administration of SARM1 agents or mutations in the SARM1 coding sequence, SARM1 enzymatic activity increases, implying an increased rate of NAD(P) consumption and increased levels of SARM1 products (e.g., nicotinamide, cADPR, ADPR, cADPRP, ADPRP). SARM1 agents may work by activating SARM1 and increasing NAD(P) depletion (Angeletti et al. 2022, iScience). Methods are described herein to determine when SARM1 is activated and when SARM1 neurodegeneration occurs.

[0102] In general, direct SARM1 agents can bind directly to SARM1, thereby activating SARM1 and causing SARM1-dependent neurodegeneration and / or neuronal dysfunction. Indirect SARM1 agents can activate SARM1 via alternative mechanisms (not directly binding to SARM1) and cause SARM1-dependent neurodegeneration and / or neuronal dysfunction. For example, via one or more binding partners or via other molecules that modulate the activity of SARM1. SARM1 agents can also be molecules that do not activate SARM1, do not bind to SARM1, but still cause SARM1-dependent neurodegeneration and / or neuronal dysfunction via alternative mechanisms.

[0103] Examples of each category are provided below.

[0104] The SARM1 agent may be a direct SARM1 activator, for example, an activator or agonist that binds directly to SARM1 to activate SARM1, such as a molecule that binds to the allosteric pocket of SARM1 (the activation pocket in the ARM domain of SARM1). Examples of direct SARM1 activators are the mononucleotides NMN, VMN, and 3-APMN, which bind to the activation pocket in the N-terminal autoregulatory ARM domain of SARM1. The SARM1 agent may also be a molecule that binds to the allosteric pocket in the N-terminal autoregulatory ARM domain of SARM1 in a manner similar to NMN and / or VMN, thereby interacting with some or all residues involved in the binding of VMN or NMN to SARM1. A list of NMN and / or VMN residues that interact with SARM1 can be found in Figure 16 and Figley et al. (Neuron. 2021 Apr 7;109(7):1118-1136.e11), Loreto et al. (Elife. 2021 Dec 6;10:e72823), and Shi et al. (Mol Cell. 2022 May 5;82(9):1643-1659.e10).

[0105] SARM1 agents can also be molecules that do not bind to the allosteric pocket of SARM1, such as Vacor, 3-AP, and their analogs or derivatives, as well as other molecules disclosed herein. For example, Vacor is converted to the metabolite VMN (Vacor mononucleotide), and 3-AP is converted to 3-APMN. These metabolites (e.g., NMN, VMN, 3-APMN, and other metabolites listed below) act as activators that bind to the activation pocket in the N-terminal autoregulatory ARM domain of SARM1, thereby causing SARM1-dependent neurodegeneration and / or neuronal dysfunction. SARM1 agents can also be prodrugs, which require intracellular conversion to cause SARM1-dependent neurodegeneration and / or neuronal dysfunction. Vacor, 3-AP, 2-AnP, and NMN precursors (e.g., nicotinamide riboside and other substances listed below) are examples of prodrugs because they do not directly activate SARM1, but are converted into direct SARM1 activators such as VMN, 3-APMN, and NMN in the cell.

[0106] The SARM1 agent may be a pyridine derivative, such as Vacor. It may be a nicotinamide analog, such as Vacor. Any pyridine derivative, Vacor analog, 3-AP, nicotinamide, nicotinamide riboside, Vacor nucleoside, Vacor nucleoside analog, nicotinamide riboside analog; nicotinic acid analog; nicotinic acid riboside analog, nicotinic acid mononucleotide analog has the potential to interact with SARM1 or generate metabolites that act as SARM1 activators. The SARM1 agent may also be pyridoxine or 6-aminonicotinamide. Generally speaking, the SARM1 agent may have a common pyridine ring. The SARM1 agent may also be an analog of a mononucleotide known to directly activate SARM1, such as NMN, VMN, and 3-APMN. Generally speaking, examples of analogs of the mononucleotides NMN, VMN, and 3-APMN may be composed of a sugar (typically ribose), a phosphate group, and a nitrogenous base (e.g., pyridine). These analogs can be SARM1 agents and can activate SARM1 by directly binding or in an indirect manner and cause SARM1-dependent neurodegeneration and / or neuronal dysfunction.

[0107] SARM1 agents can be prodrugs (such as Vacor, 3-AP, 2-AnP) and analogs of NMN precursors (e.g., analogs of nicotinamide, nicotinamide riboside, and other substances listed below), and are generally pyridine and molecules containing pyridine rings. These analogs can activate SARM1 by direct binding or indirect means and cause SARM1-dependent neurodegeneration and / or neuronal dysfunction.

[0108] SARM1 is a protein that has NAD(P) + Consumes active catalytic enzymes. This activity is controlled by axonal survival and NAD + Regulation of the synthase NMNAT2. NMNAT2 depletion or inhibition leads to the accumulation of the NMNAT2 substrate NMN and an increase in the NMN / NAD ratio. When NMN levels are high, NMN binds to the allosteric pocket (activation pocket) on the SARM1 ARM domain and activates SARM1, resulting in SARM1-dependent NAD(P) +Increased depletion and neurodegeneration. Therefore, SARM1 agents (exogenous molecules such as vacor and 3-AP) can be converted into metabolites in cells, namely NMN analogs (in this case, VMN and 3-APMN), which also bind to and activate SARM1, thereby causing neurodegeneration. Therefore, SARM1 agents may cause neurodegeneration and / or neuronal dysfunction in the manner described. SARM1 agents can be indirect SARM1 activators, which act by reducing the level and / or enzymatic activity of NMNAT1-2-3 (particularly the axonal isoform and the endogenous SARM1 regulator NMNAT2), thereby leading to SARM1 activation and SARM1-dependent neurodegeneration and / or neuronal dysfunction. These agents can be direct inhibitors of NMNAT1-2-3, which lead to loss of enzymatic activity, or can be molecules that deplete NMNAT1-2-3 levels. This may occur in a variety of ways, including mitochondrial dysfunction, protein synthesis blockade, increased protein degradation, impaired axonal transport, NMNAT1-2-3 mutations, molecules that affect NMNAT1-2-3 stability (e.g., effects on turnover, degradation, half-life, stability), and selective inhibition of NMNAT1-2-3 activity (Merlini et al., 2022). SARM1 agents can encompass any agent that causes SARM1 activation via any one or more of these mechanisms. SARM1 activation can be determined by any means known in the art, as described below.

[0109] SARM1 agents may be indirect SARM1 activators that activate SARM1 and cause SARM1-dependent neurodegeneration and / or neuronal dysfunction via alternative mechanisms (not directly binding to SARM1), via one or more binding partners, or by modulating the activity of SARM1 via other molecules. Examples of indirect SARM1 agents include chemotherapeutic drugs such as vincristine, which impair axonal transport and lead to decreased levels of the endogenous SARM1 regulator NMNAT2. Other examples are mitochondrial toxins such as CCCP, rotenone, etc., which also lead to decreased levels of NMNAT2 (Merlini et al. 2022).

[0110] Importantly, changes in NMN and NAD levels can trigger SARM1 activation and SARM1-dependent neurodegeneration and / or neuronal dysfunction (Gilley and Coleman, 2010; Figley et al. 2021; Coleman and Hoke, 2020). Therefore, SARM1 agents can be molecules that cause changes in NMN, NAD, and the NMN / NAD ratio, thereby leading to SARM1 activation and SARM1-dependent neurodegeneration and / or neuronal dysfunction. Specifically, molecules that increase NMN levels, decrease NAD levels, and / or increase the NMN / NAD ratio have the potential to activate SARM1. These molecules can act on NMNAT1-2-3 levels and / or activity as described above, but can also act via other mechanisms, such as through the activation of NMN synthases such as NAMPT (e.g., SBI-797812, PC73 Gardell, SJ, Hopf, M., Khan, A. et al. Boosting NAD+ with a small molecule that activates NAMPT. [Boosting NAD+ with a small molecule that activates NAMPT.] Nat Commun [Nature Communications] 10, 3241 (2019) https: / / doi.org / 10.1038 / s41467-019-11078-z, and Wang G, Han T, Nijhawan D et al. P7C3 neuroprotective chemicals function by activating the rate-limiting enzyme in NAD salvage. [P7C3 neuroprotective chemicals act by activating the rate-limiting enzyme in NAD salvage] Cell. [Cell] 2014; 158(6): 1324-1334. doi: 10.1016 / j.cell.2014.07.040) and / or NRK1,2 activation and / or increased activation and / or expression of NAD+ consuming enzymes such as poly (ADP-ribose) polymerase (PARP), sirtuins and CD38 (Xie et al. 2020). SARM1 agents can encompass any agent that causes SARM1 activation via any one or more of these mechanisms.

[0111] A SARM1 agent can be a molecule that does not cause SARM1 activation (see definition of SARM1 activation) when used alone or in combination with any other SARM1 agent and / or SARM1-dependent neurodegenerative mechanism described herein, but still causes SARM1-dependent neurodegeneration and / or neuronal dysfunction. For example, SARM1 has base exchange activity and can convert molecules into toxic metabolites even through basal activity. These products may contribute to SARM1-dependent neurodegeneration and / or neuronal dysfunction. For example, Vacor is used for base exchange to produce VAD. VAD is known to inhibit enzymes in the NAD biosynthesis pathway, such as NMNAT1-2-3 (Loreto et al. 2021elife). This may reduce compensatory mechanisms downstream of SARM1 activation and contribute to the toxicity and SARM1-dependent neurodegeneration and / or neuronal dysfunction caused by vacor and its analogs. A list of other molecules used for base exchange by SARM1 and that could potentially be SARM1 agents can be found in (Angeletti et al. 2022, iScience and https: / / elifesciences.org / articles / 67381.pdf) and Table S2 (Shi et al. 2022 Mol Cell).

[0112] The SARM1 agent can be any molecule that causes a change in SARM1 enzymatic activity, thereby causing SARM1-dependent neurodegeneration and / or neuronal dysfunction. The effect can act on any enzymatic activity described for SARM1 (NAD(P) consumption activity, NAD(P) hydrolysis, cyclization, and base exchange activity) (for more information, see Angeletti et al. 2022, iScience). SARM1-dependent neurodegeneration and / or neuronal dysfunction and activation can be determined by any means known in the art, as described below.

[0113] The SARM1 agent may be any molecule that causes SARM1-dependent neurodegeneration and / or neuronal dysfunction. SARM-1-dependent neurodegeneration and / or neuronal dysfunction may be determined by any means known in the art, as described below.

[0114] A SARM1 agent can bind directly or indirectly to a molecule comprising an amino acid sequence identified by UniProtKb numbering Q6SZW1. A SARM1 agent can bind directly or indirectly to a molecule encoded by a nucleotide sequence encoding the SARM1 protein sequence identified by UniProtKb numbering Q6SZW1. SARM1 can comprise a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence identified by UniProtKb numbering Q6SZW1. SARM1 can be encoded by a nucleic acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence encoding the SARM1 protein identified by UniProtKb numbering Q6SZW1. In some examples, the SARM1 protein can be a homolog or variant of the protein identified by UniProtKb numbering Q6SZW1.

[0115] As disclosed herein, SARM1 agents can be mononucleotides, such as NMN, CZ-48, VMN, and 3-APMN. VMN and 3-APMN are mononucleotides containing a negative charge. The charge of the mononucleotide can be adjusted to increase permeability and / or solubility using any means known in the art. Therefore, analogs and derivatives of SARM1 agents that increase the permeability and / or solubility of the SARM1 agent can be used in the present invention. For example, CZ-48 is a modified version of NMN in which the ribose sugar is replaced with 2-deoxy-2-fluoro-D-arabinose and one of the oxygens of the phosphate group is replaced with sulfur to increase cell permeability (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC6531917). These (or similar) modifications can be applied to any SARM1 agent, such as SRAM1 activators, to increase permeability, including VMN, 3-APMN, and their analogs. Permeability can also be enhanced by commonly known methods, such as by using permeation enhancers, ion pairing, encapsulation (such as nanoencapsulation), or nanosizing.

[0116] Thus, the SARM1 agent used in any of the methods disclosed herein can be selected from vacor; vacor mononucleotide (VMN); 3-acetylpyridine (3-AP); 3-acetylpyridine mononucleotide (3-APMN); 2-aminopyridine (2-AnP); 2-AnP mononucleotide (2-AnPMN); nicotinamide mononucleotide (NMN), sulfo-ara-F-NMN (CZ-48); sulfo-ara-F-VMN; sulfo-ara-F-3-APMN; sulfo-ara-F-2-AnPMN, S-NMN, ara-F-NMN N(CZ-17); pyridine and molecules containing a pyridine ring; vacor nucleoside (VR), vacor nucleoside analogs; nicotinamide (NAM) analogs; nicotinamide riboside (NR), nicotinamide riboside analogs; nicotinic acid (NA) analogs; nicotinic acid riboside (NaR) analogs, nicotinic acid mononucleotide (NaMN) analogs, molecules that increase intracellular NMN levels, decrease NAD levels and / or increase the NMN / NAD ratio, thereby leading to SARM1 activation; NMNAT1-2-3 inhibitors or molecules that decrease NMNAT1-2-3 levels; and / or their metabolites, analogs and derivatives.

[0117] Vacor analogs can act in a similar manner to each other. That is, Vacor analogs can be converted into metabolites by NAMPT, NRK (nicotinamide riboside kinase) or NaPRT or any other similar enzyme. The resulting metabolite / mononucleotide can then activate SARM1 by binding to the ARM domain, thereby causing neurodegeneration and / or neuronal dysfunction. For example, in the case of Vacor nucleoside, Vacor nucleoside can be converted to VMN in the cell by NRK. Other SARM1 agents can interact with SARM1 in different ways, for example, by binding to another region of SARM1. For example, Vacor can also be used directly by SARM1 for base exchange (where conversion to VMN is not required). This reaction is mediated by interaction with another SARM1 domain (TIR domain) ( Figure 1(Angeletti et al. 2022 iScience). Vacor's base exchange activity by SARM1 may not be sufficient to cause neurodegeneration and / or neuronal dysfunction alone, but may lead to neurodegeneration and / or neuronal dysfunction when combined with any or all of the above mechanisms. Vacor may require conversion to VMN for toxicity. However, a SARM1 agent may cause neurodegeneration and / or neuronal dysfunction without conversion to a metabolite, for example, by using it for base exchange by SARM1. Thus, a SARM1 agent can be a molecule that interacts with SARM1 in any domain of the enzyme, not limited to the activation pocket in the N-terminal ARM domain.

[0118] SARM1 has basal enzymatic activity (see definition above). SARM1 agents can be molecules that are converted to toxic metabolites by SARM1 via base exchange (activated SARM1 and / or via basal SARM1 activity). The products of this activity can cause SARM1-dependent neurodegeneration and / or neuronal dysfunction, for example by blocking enzymes in the NAD biosynthesis pathway, thereby reducing compensatory mechanisms downstream of SARM1 and NAD(P) depletion.

[0119] SARM1 agents can be molecules that block compensatory mechanisms, such as impairing the levels and or activity of enzymes involved in NAD biosynthesis. In this case, basal SARM1 activity may lead to NAD depletion, thereby causing SARM1-dependent neurodegeneration and / or neuronal dysfunction.

[0120] SARM1 agents may be used in combination with some, all, or any of the methods described to achieve the desired therapeutic outcome. This includes combining more than one SARM1 agent, as well as co-administering a reversal agent.

[0121] For all of the above applications, SARM1 activation and / or SARM1-dependent neurodegeneration and / or neuronal dysfunction can be determined by any means known in the art. As described above, changes in the levels of NMN, NAD, NMN / NAD ratio, SARM1 products such as nicotinamide, cADPR, ADPR, cADPRP, ADPRP are indicators of SARM1 activation. Specifically, an increase in NMN, NMN / NAD ratio, nicotinamide ratio, cADPR, ADPR, cADPRP, ADPRP indicates SARM1 activation. A decrease in NAD, NADP, and NMNAT1-2-3 levels also indicates SARM1 activation (Figley et al. 2021 Neuron, Angeletti et al. 2021 iScience). The amount of NMN and NAD can be measured using any method known in the art. The ratio of NMN / NAD and the increase in the ratio can then be determined by measuring the amount of NMN and NAD. The increase or decrease in the level of molecules (such as NMN, NAD, nicotinamide, cADPR, ADPR, cADPRP, ADPRP or NMNAT2) can be determined by methods known in the art. Examples of assays for determining SARM1 activation and / or SARM1-dependent neurodegeneration and neuronal dysfunction are metabolomics analysis using an NAD glo assay kit, HPLC and LC-MS (Angeletti et al., 2022iSciece [cross science]; Figley et al. 2021Neuron [neuron], Loreto et al. 2021elife [electronic life] and https: / / pubmed.ncbi.nlm.nih.gov / 32087251 / ) for measuring changes in the levels of NMN, NAD, NMN / NAD ratios, and changes in the levels of SARM1 active products, nicotinamide, cADPR, ADPR, cADPRP, ADPRP. Other examples are the use of in vivo fluorescent probes, such as PC6, as described in (https: / / elifesciences.org / articles / 67381.pdf). Other examples are the use of antibodies that bind to the active conformation of SARM1 (e.g., Nb-C60), as described in (https: / / www.biorxiv.org / content / 10.1101 / 2022.03.25.485784v2.full). Other examples are the use of cryo-EM and X-ray crystallography to study structural changes associated with SARM1 activation (Loreto et al. 2021 elife, Figley et al. 2021 Neuron, Shi et al. 2022 Mol Cell).Other examples include performing enzymatic assays on recombinant SARM1 to study molecules that affect SARM1 activity, as described in (Angeletti et al. 2022 iScience, Loreto et al. 2021 eLife; Gilley et al. 2021 eLife). Other examples include methods for detecting the level and / or enzymatic activity of NMNAT1-2-3, such as Western blots and enzymatic assays. An increase or decrease in the above molecules can be determined by reference to baseline levels or amounts before administration of the SARM1 agent. The control sample can be the level of NMN, NAD, NMN / NAD ratio, NMNAT1-2-3, SARM1 products such as nicotinamide, cADPR, ADPR, cADPRP, ADPRP NMN, NAD, cADRP, ADPR, and other SARM1 activation markers disclosed herein before SARM1 activation / addition of the SARM1 agent to the sample. For example, compared to a control sample, an increase in the level of the above-mentioned molecule in a sample can be an amount that is at least about 3% greater, such as at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% greater than the sample population. Compared to a control sample, a decrease in a sample can be an amount that is at least about 3% lower, such as at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% lower than the sample population. For example, an increase in level can be at least about 1.1 times greater, up to 10 times greater. For example, the reduction in levels can be at least about a 1.1-fold reduction, up to a 10-fold reduction. For example, an increase of about 3% or more in NMN, the NMN / NAD ratio, nicotinamide, cADPR, ADPR, cADPRP, or ADPRP can identify a molecule, metabolite, analog, or derivative thereof as a SARM1 agent. A reduction of about 3% or more in NAD, NADP, and NMNAT1-2-3 levels can identify a molecule, metabolite, analog, or derivative thereof as a SARM1 agent. A reduction of about 3% or more in NMNAT2 levels can identify a molecule, metabolite, analog, or derivative thereof as a SARM1 agent. An increase of about 3% or more in the NMN / NAD ratio can identify a molecule, metabolite, analog, or derivative thereof as a SARM1 agent. Furthermore, SARM1-dependent neurodegeneration and / or neuronal dysfunction can be determined using any means known in the art.Examples are studies of neuronal and / or neurite morphology using microscopy (Loreto et al. 2021 eLife), measurement of neurofilament light chain (NfL) levels in in vitro assays (e.g. culture supernatants), serum and cerebrospinal fluid (CSF) (https: / / www.sciencedirect.com / science / article / pii / S2211124720315771?via%3Dihub), metabolomic assays as described above, electrophysiology – nerve conduction studies / electromyography, mitochondrial dynamics and function, axonal transport, metabolomic changes, serum / CSF biomarkers such as neurofilaments. Motor / sensory neurological examination (including MRC scale and total score), cognitive tests (such as Addenbrookes Cognitive Examination III), methods for measuring peak volitional muscle contraction force (such as dynamometer and muscle fatigue, such as neurological examination and neurophysiology), timed walking test, objective sensory examination (such as Von frey hair), thermal threshold, proprioception test (such as 9-hole peg test). Intraepidermal nerve fiber analysis.

[0122] The SARM1 agent can be any of a variety of the following compounds:

[0123] Vacor

[0124] Vacor can be converted to VMN for neuronal toxicity. Vacor interacts with the SARM1 active site (TIR domain) to undergo a base exchange reaction that modestly increases SARM1 activity (Loreto et al., 2021; Angeletti et al., 2022).

[0125] VMN (Vacor Mononucleotide)

[0126] VMN is a Vacor metabolite and binds to SARM1 in an allosteric site (ARM domain) and potently activates SARM1. It is likely not cell permeable (Loreto et al., 2021, ELife [Electronic Life] December 6), but cell permeability can be increased by common known methods (such as by using penetration enhancers, ion pairing, encapsulation (such as nanoencapsulation) or nanosizing). For example, CZ-48 is a modified version of NMN in which ribose is replaced by 2-deoxy-2-fluoro-D-arabinose and one of the oxygens of the phosphate is replaced by sulfur to increase cell permeability. The same modifications can be used for VMN and other mononucleotides listed herein.

[0127] 3-Acetylpyridine (3-AP)

[0128] 3-AP needs to be converted to 3-APMN for toxicity in neurons. 3-AP can interact with the SARM1 active site (TIR domain) to undergo a base exchange reaction that leads to increased SARM1 activity. (Angeletti et al., 2022, iScience Jan 25;25(2):103812; Wu et al., 2021, Cell Rep Oct 19:37(3):109872).

[0129] 3-AP mononucleotide (3-APMN)

[0130] 3-APMN is a 3-AP metabolite that is predicted to bind to SARM1 in an allosteric site (ARM domain) and activate SARM1. It is most likely not cell permeable (Wu et al., 2021), but cell permeability can be increased by common known methods, such as by using penetration enhancers, ion pairing, encapsulation (such as nanoencapsulation), or nanosizing. For example, CZ-48 is a modified version of NMN in which the ribose sugar is replaced by 2-deoxy-2-fluoro-D-arabinose and one of the oxygens of the phosphate group is replaced by sulfur to increase cell permeability. The same modifications can be used for 3-APMN and the other mononucleotides listed herein.

[0131] 2-Aminopyridine (2-AnP): 2-AnP can bind to the allosteric site of SARM1 and induce SARM1-dependent neurodegeneration, such as axonal degeneration.

[0132] Nicotinamide mononucleotide (NMN)

[0133] NMN is an endogenous SARM1 agent / activator. It binds directly to SARM1 in the allosteric site (ARM domain) and potently activates the SARM1. The precursors of NMN are nicotinamide and nicotinamide riboside (both part of the vitamin B3 family) (Figley et al., 2021, Neuron [Neuron], April 7: 109 (7): 1118-1136). It is most likely not cell permeable (Wu et al., 2021), but cell permeability can be increased by common known methods (such as by using permeation enhancers, ion pairing, encapsulation (such as nanoencapsulation) or nanosizing). For example, CZ-48 is a modified version of NMN in which ribose is replaced by 2-deoxy-2-fluoro-D-arabinose and one of the oxygens of the phosphate is replaced by sulfur to increase cell permeability.

[0134] CZ-48(sulfo-ara-F-NMN)

[0135] CZ-48 is an NMN analog. It can activate SARM1 in both in vitro and cellular assays. It is modified (F and S) to improve cell permeability (Zhao et al., 2019, iScience May 31;15:452-466).

[0136] CZ-17(ara-F-NMN)

[0137] C-17 is an NMN analog. C-17 can activate SARM1 in an in vitro assay (Zhao et al., 2019).

[0138] S-NMN

[0139] S-NMN is an NMN analog that can activate SARM1 in in vitro assays (Zhao et al., 2019).

[0140] SARM1 agents may include those with chemical modifications of the compounds disclosed herein. SARM1 agents may also be derivatives, prodrugs, or analogs of any of the compounds disclosed herein. For example, modification of a compound with F and / or S groups may improve cell permeability, as demonstrated for CZ-48. Such chemical modifications may be applied to other SARM1 agents, such as VMN and 3-APMN.

[0141] SARM1 agents for use as pharmaceuticals

[0142] SARM1 agents may be associated with a first medical use. For example, SARM1 agents that result in SARM1 activation, such as Vacor and related compounds, have been used for non-medical purposes, such as as rodenticides for pest control. The present inventors have for the first time identified such SARM1 agents as being useful for treating and / or preventing the diseases / conditions disclosed herein. Thus, disclosed herein are SARM1 agents for use as pharmaceuticals.

[0143] SARM1 agents for use in methods of treating and / or preventing disease

[0144] The SARM1 agents disclosed herein can be used in methods for treating and / or preventing diseases. The diseases can be neurological, ophthalmic, dermatological, gastroenterological, colorectal, urological, gynecological, rheumatological, orthopedic, dental and / or otolaryngological diseases.

[0145] In some instances, the disease is a SARM1-dependent neurological disorder. A SARM1-dependent neurological disorder refers to any disease or condition that is regulated by, caused by, or has symptoms associated with SARM1 dysregulation. For example, the disease or condition and any symptoms thereof may be caused by or associated with decreased SARM1 activity. Decreased activity refers to decreased enzymatic activity of the SARM1 protein in a subject and / or decreased expression of SARM1 mRNA. The decrease can be, for example, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to SARM1 activity in a healthy subject.

[0146] The disease or condition to be treated or prevented can also be defined as autonomic dysfunction, neuropathic pain, dystonia, spasm, movement disorder, sphincter dysfunction, urogenital dysfunction and / or skin condition. The disease or condition can be prevented or treated in whole or in part. That is, if "whole" is prevented or treated, any of these diseases or conditions or conditions may not exist at all after treatment or prevention. Alternatively, if these diseases or conditions can be partially prevented or treated, if "partially" is prevented or treated, any of these diseases or conditions or conditions may exist partially after treatment or prevention. For example, if neuropathic pain does not exist at all after treatment or prevention with a SARM1 agent, then neuropathic pain can be prevented or treated in whole. As another example, if neuropathic pain is alleviated after treatment or prevention with a SARM1 agent, then neuropathic pain can be prevented or treated in part.

[0147] As used herein, the terms "treat," "treating," and "treatment" are intended to include interventions performed to prevent the development of a condition, disorder, or symptom or to alter the pathology of a condition, disorder, or symptom. Thus, "treatment" refers to both therapeutic treatment and preventative or prophylactic measures, wherein the object is to prevent or slow down (lessen) the condition, disorder, or symptom of interest. In other words, the terms "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progression of a condition, disorder, or symptom.

[0148] In some instances, treatment can be administered after one or more signs or symptoms of disease have developed or have been observed. In other instances, treatment can be administered when there is no sign or symptom of disease. For example, treatment can be administered to susceptible subjects before the onset of symptoms (for example, according to symptom history). After symptom subsidence, treatment can also be continued, for example, to delay and / or prevent recurrence. Compared with the expected life span in the case of not receiving treatment, treatment can bring about the life span of extension. Alternatively or additionally, compared with the expected standard of living in the case of not receiving treatment, treatment can provide the standard of living of improvement for the patient.

[0149] "Autonomic dysfunction," also known as "dysautonomia," "autonomic neuropathy or ganglionopathy," or "primary or secondary autonomic failure," refers to a condition in which the autonomic nervous system (ANS) does not function properly. This may affect the function of the heart, bladder, intestines, sweat glands, pupils, smooth muscle, and / or blood vessels. Autonomic dysfunction has many causes, not all of which can be classified as neurological. The main symptoms of autonomic dysfunction, which may vary from person to person, include: orthostatic hypotension; syncope and presyncope; hypothermia; hyperthermia; dry mouth; drooling; fast / slow / irregular heart rate; pupillary dysfunction; labile blood pressure; dysphagia; bowel incontinence; blurred vision; urinary incontinence, urinary retention, and bladder emptying dysfunction; gastroparesis; constipation; postprandial symptoms; anhidrosis; hyperhidrosis; and sexual dysfunction, such as erectile / clitoral dysfunction and loss of libido, fatigue, exercise intolerance, anxiety, headache, brain fog, and shoulder, back, and neck pain (coat hanger pain). Autonomic dysfunction may be due to inherited, genetic, or degenerative neurological diseases (primary autonomic dysfunction), or it may occur due to damage / disease of the autonomic nervous system caused by acquired conditions (secondary autonomic dysfunction), such as infections (such as COVID and long COVID), postural orthostatic tachycardia syndrome (POTS), autoimmune conditions, blood conditions, amyloidosis, endocrine conditions (such as diabetes and thyroid dysfunction), neuropathy secondary to paraneoplastic, inflammatory, metabolic, and vasculitic causes, small fiber neuropathies, connective tissue diseases (such as Ehlers-Danlos spectrum disorders), menopause, or medications. The cause may also be unknown (idiopathic).

[0150] Dysautonomia can include any one or more of excessive sweating and / or excessive saliva production (also known as sialorrhea). Dysautonomia can be primary or secondary to neurodegenerative diseases such as amyotrophic lateral sclerosis, Parkinson's disease, Parkinson's syndrome, multiple system atrophy, cerebral palsy, and medications.

[0151] Known treatments for autonomic dysfunction include glycopyrronium, submandibular gland / parotid gland injection of botulinum toxin A / B for salivation or topical NH3CL antiperspirant / iontophoresis or axillary sweat gland and other parts of the sweat gland injection of botulinum toxin A / B for hyperhidrosis (excessive sweating). The SARM1 agents described herein can be used as a supplement to or in combination with any of the above-mentioned known autonomic dysfunction treatments.

[0152] Thus, in some examples, provided are SARM1 agents as described herein for use in treating or preventing autonomic dysfunction, or for use in a method of treating or preventing autonomic dysfunction.

[0153] Neuropathic pain refers to the pain caused by nerve damage or neurological dysfunction, and can also include spontaneous pain that also occurs when there is no pain stimulation, the abnormal pain caused by the stimulation that usually does not cause pain, the hyperalgesia and other abnormal responses worsened by noxious stimulation, such as paresthesia or dysesthesia. When " neuropathic pain " is divided into central type and peripheral type, central neuropathic pain may be caused by the central nervous system damage (such as brain tumor, cerebral hemorrhage, syringomyelia and acquired immunodeficiency syndrome) that stimulates the spinal cord, brainstem, thalamus and cortex. Peripheral neuropathic pain can refer to the pain caused by peripheral nervous system abnormality, such as postherpetic neuralgia, diabetic neuropathy and type II complex regional pain syndrome (Duck Mi Yoon, 2011).

[0154] Neuropathic pain includes peripheral neuropathic pain; chronic back pain; cancer-related pain; myofascial pain secondary to bruxism or temporomandibular joint dysfunction; fibromyalgia; complex regional pain disorders; chronic pelvic pain; dyspareunia; vulvodynia; painful bladder syndrome (interstitial cystitis) or neuropathic pain caused by diabetic neuropathy, peripheral nerve injury and neuralgia following surgical repair of nerve injury, infectious and post-infectious neuralgia such as postherpetic neuralgia and long-term COVID, HIV neuropathy and Lyme disease; chemotherapy, toxic and drug-induced neuropathies; metabolic neuropathies, such as those secondary to renal or liver dysfunction / failure; small fiber neuropathies; endocrine conditions, such as thyroid, parathyroid and pituitary dysfunction; genetic neuropathies, such as Charcot-Marie-Tooth disease. Disease), hereditary sensory neuropathy (HSN) and hereditary sensory and autonomic neuropathy (HSAN); paraproteinemic, paraneoplastic and tumorous neuropathy; vasculitis; autoimmune, inflammatory and demyelinating neuropathy, such as Guillain-Barre syndrome and chronic inflammatory demyelinating polyradiculoneuropathy; and idiopathic neuropathy. In some instances, the neuropathic pain is peripheral neuropathic pain.

[0155] Cancer pain refers to cancer-induced pain. According to the three-step analgesic ladder created as a guideline for cancer pain management and advocated by the World Health Organization (WHO), the following are recommended: Step 1: non-opioid analgesics for mild pain, Step 2: opioid analgesics for moderate pain, and Step 3: strong opioid analgesics for severe pain. The degree of pain varies, and for example, when the pain is severe, it is considered an emergency requiring prompt treatment, and may be associated with pain that is severe enough to cause general weakness, mental illness, etc. Therefore, such pain may refer to pain that requires active analgesic treatment, and its classification is different from other causes of pain.

[0156] Known treatments for neuropathic pain, such as peripheral neuropathic pain, include gabapentin, pregabalin, serotonin norepinephrine reuptake inhibitors (such as duloxetine or venlafaxine), tricyclic antidepressants (which may be first-line treatment), capsaicin 8% patch, lidocaine patch, tramadol (which may be second-line treatment), or botulinum toxin A (which may be third-line treatment) (Finnerup et al. 2015 Lancet Neurol. 2015 Feb;14(2):162-73., NICE guidelines on neuropathic pain cg173–2013; Attal et al. 2010. Eur J Neurol. 2010 Sep;17(9):1113-e88). The SARM1 agents described herein may be used in addition to or in combination with any of the known neuropathic pain treatments described above.

[0157] In some instances, a SARM1 agent as described herein is provided for use in treating or preventing neuropathic pain, or for use in a method of treating or preventing neuropathic pain. In some instances, the SARM1 agents and compositions described herein can be administered via a transdermal patch for use in treating neuropathic pain.

[0158] Dystonia refers to sustained muscle contractions that cause repetitive movements or twisting and other abnormal postures. In some embodiments, dystonia can occur in a limb (e.g., a hand or foot).

[0159] Dystonias include primary dystonias, such as focal, segmental, genetic, and idiopathic types, such as blepharospasm, strabismus, oromandibular dystonia, laryngeal dystonia, writer's cramp, and cervical dystonia; superimposed dystonia conditions, such as dopa-responsive dystonia (DRD) or Segawa syndrome, rapid-onset dystonia-parkinsonism (RDP), and myoclonus-dystonia, as well as dystonias that occur with neurodegenerative diseases, such as Parkinson's disease, multiple system atrophy, Huntington's disease, Wilson's disease, and neuroferritinopathy; secondary dystonias that occur due to central nervous system (CNS) trauma, congenital malformations, genetic and chromosomal disorders, infections, tumors, ischemic or hemorrhagic stroke, inflammation, demyelination, drugs, toxins, metabolic disorders (such as secondary to renal or liver dysfunction / failure), paraneoplastic, post-infectious autoimmune, and endocrine conditions, such as thyroid, parathyroid, and pituitary dysfunction.

[0160] Known treatments for dystonia include botulinum toxin A (for cranial / cervical dystonia); botulinum toxin B (for cervical dystonia), which are sometimes combined with adjunctive oral medications such as trihexyphenidyl, dopamine replacement or agonist drugs, benzodiazepines, and steroids. Drugs such as diazepam, and surgical interventions such as deep brain stimulation (DBS), for example, Paladin DBS for cervical dystonia (Phukan et al. 2011. Lancet Neurol. [Lancet Neurology] Dec; 10(12): 1074-85., Marion et al. 2016. Pract Neurol [Practical Neurology] 2016; 16: 288-295). The SARM1 agents described herein can be used in addition to or in combination with any of the above-mentioned known dystonia treatments.

[0161] In some examples, provided are SARM1 agents as described herein for use in treating or preventing dystonia, or for use in a method of treating or preventing dystonia.

[0162] Spasticity refers to a condition in which certain muscles contract continuously or abnormally. This contraction causes muscle stiffness or tightness and may interfere with the normal movement of the face, limbs, trunk and / or sphincter, resulting in defects such as speech, gait and / or bladder and bowel function. Spasticity is a condition that occurs in many CNS disorders that affect brain and / or spinal cord function, including, for example, traumatic injury to the brain or spinal cord, multiple sclerosis, cerebral palsy, stroke or other conditions. Although the underlying conditions are different, when the characteristics of motor neurons change in response to the condition and excessively generate electrical impulses, resulting in excessive muscle contraction, spasticity develops. Damage causes changes in the signal balance between the nervous system and the muscles, resulting in increased muscle excitability. Spasticity is common in conditions in which the brain and / or spinal cord are damaged or cannot develop normally; these include cerebral palsy, multiple sclerosis, spinal cord injury and acquired brain injury (including stroke).

[0163] Spasticity may include one or more of the following: upper limb spasticity, lower limb spasticity, or bladder spasticity / neurogenic bladder; spastic disorders of the esophagus, such as esophageal spasm and nutcracker esophagus; or spasticity due to ischemic or hemorrhagic stroke; demyelinating conditions, such as multiple sclerosis; traumatic spinal cord or brain injury; congenital malformations, such as cerebral palsy; chromosomal disorders; genetic conditions, such as hereditary spastic paraplegia; CNS infections; tumors; drugs and toxins; metabolic disorders, such as secondary to renal or liver dysfunction / failure; paraneoplastic disorders; post-infectious conditions; autoimmune conditions; endocrine conditions, such as thyroid, parathyroid, and pituitary dysfunction.

[0164] Known treatments for spasticity include oral and intrathecal baclofen, benzodiazepines, The SARM1 agents described herein can be used in addition to or in combination with any of the above-mentioned known spasticity treatments, such as tizanidine, cannabinoids (such as nabiximol), dantrolene or botulinum toxin A / B, intraneural, intramuscular and intrathecal phenol injections, and surgical interventions such as rhizotomy (Kheder et al., Pract Neurol. 2012 Oct; 12(5): 289-98, Chang et al. 2013. Crit Rev Phys Rehabil Med. 2013; 25(1-2): 11-22).

[0165] In some examples, provided are SARM1 agents as described herein for use in treating or preventing spasticity, or for use in a method of treating or preventing spasticity.

[0166] Movement disorders are abnormal or uncontrollable movements and include, but are not limited to, chorea, tremors, tics, dystonia, athetosis, myoclonus, and tics.

[0167] Movement disorders may include one or more of the following: hemifacial spasm with facial nerve synkinesis; palatal myoclonus / tremor; tics, such as motor and vocal tics in Tourette's syndrome; tremors, such as essential tremor, dystonic tremor, Parkinsonian tremor, and rubrostral tremor; myokymia, such as idiopathic eyelid, facial, limb myokymia, or secondary to tumors, trauma, infection, autoimmune conditions (including autoimmune encephalitis), demyelination (such as multiple sclerosis), and neuropathies (such as Guillain-Barré syndrome); neuromyotonia caused by autoimmune, paraneoplastic, or genetic conditions (Fuller. 2010. Practical Neurology 10: 114-123).

[0168] Known treatments for movement disorders include beta-blockers, gabapentin, opioids, benzodiazepines, Drugs that have been used for treatment include narcotics and barbiturates, anticholinergics (such as procyclidine and trihexyphenidyl), dopamine antagonist drugs, tetrabenazine, botulinum toxin A / B, and deep brain stimulation (Fuller. 2010. Practical Neurology 10: 114-123.).

[0169] Neuromyotonia is a rare condition of spontaneous, sustained muscle activity of peripheral nerve origin. Its clinical features are muscle twitching (visible tremors) at rest, spasms that can be triggered by voluntary or induced muscle contractions, and impaired muscle relaxation (pseudomyotysis) (Maddison Practical Neurology, 2002, 2, 225–229).

[0170] Known treatments for neuromyotonia include anticonvulsant drugs and immunosuppressive therapies such as steroids, immunoglobulins, azathioprine, and plasma exchange (Maddison Practical Neurology, 2002, 2, 225–229).

[0171] In some examples, provided are SARM1 agents as described herein for use in treating or preventing a movement disorder, or for use in a method of treating or preventing a movement disorder.

[0172] Sphincter dysfunction may be caused by sphincter dysfunction in the body, including the esophageal sphincter, such as achalasia caused by genetic / inherited infections, such as Chagas disease, and autoimmune conditions in addition to idiopathic achalasia; the pyloric sphincter, such as gastroparesis caused by conditions such as diabetes; sphincter of Oddi dysfunction leading to gastrointestinal dysmotility; the pancreatic sphincter, such as recurrent pancreatitis; the urethral sphincter, such as detrusor-sphincter dyssynergia, primary or secondary to brain or spinal cord injury, multiple sclerosis, hemorrhagic or ischemic stroke, CNS tumors, Fowler syndrome; anal sphincter dysfunction, such as anal spasm; anal fissures, primary or secondary to inflammatory bowel disease, HIV infection, anal abscesses, and perianal tumors / cancers; and Hirschsprung disease (Bach and Simman, Plast Reconstr Surg Glob). Open. [Plastic and Reconstructive Surgery - Global Open] 2022 Apr 6;10(4):e4228, Apostolidis A., Eur Urol. [European Journal of Urology] 2009 Jan;55(1):100-19, Lacy et al. Gastroenterol Hepatol [Gastroenterology and Hepatology] (New York). 2008 Apr;4(4):283–295).

[0173] Known treatments for sphincter dysfunction include botulinum toxin A / B injections and surgical options (Bach and Simman, Plast Reconstr Surg Glob Open. 2022 Apr 6;10(4):e4228, Apostolidis A., Eur Urol. 2009 Jan;55(1):100-19, Lacy et al. Gastroenterol Hepatol. (New York). 2008 Apr;4(4):283–295).

[0174] In some examples, provided are SARM1 agents as described herein for use in treating or preventing sphincter dysfunction, or for use in a method of treating or preventing sphincter dysfunction.

[0175] Genitourinary dysfunction refers to conditions affecting the urinary and reproductive organs, for example, vaginismus, primary or secondary to childbirth, infection, trauma, iatrogenic or psychological conditions; urinary dysfunction, such as after pelvic surgery; pelvic floor muscle dysfunction (Moga et al., 2018, Toxins 2018, 10, 169).

[0176] Known treatments for genitourinary dysfunction include lubricants, anxiolytics, and botulinum toxin A / B injections (Moga et al., 2018, Toxins 2018, 10, 169).

[0177] In some examples, provided are SARM1 agents as described herein for use in treating or preventing urogenital dysfunction, or for use in a method of treating or preventing urogenital dysfunction.

[0178] Skin conditions may include one or more of the following: ulcers, primary or secondary to diabetes, vascular disease (including arterial and venous or disorders), autoimmune conditions, infection, neuropathy, and trauma; acne; hypertrophic scars, such as keloids; rosacea and facial flushing; pruritus; Hailey-Hailey disease; hidradenitis suppurativa; alopecia; back pain with paresthesia; psoriasis; eczema; Raynaud's phenomenon / disease, primary or secondary to autoimmune diseases such as systemic lupus erythematosus and limited and systemic sclerosis (see Kim et al., 2017. doi: 10.3390 / toxins9120403; Bach and Simman, Plast Reconstr Surg Glob ... Open. [Plastic and Reconstructive Surgery - Global Open] 2022 Apr 6;10(4):e4228; Campanati et al., 2017. doi:10.1159 / 000452341; and Winayanuwattikun and Vachiramon 2022. doi:10.3390 / toxins14060406).

[0179] In some examples, provided are SARM1 agents as described herein for use in treating or preventing a skin condition, or for use in a method of treating or preventing a skin condition.

[0180] Orthopedic diseases or conditions may refer to sports injuries; post-traumatic elbow stiffness; clubfoot; and / or piriformis syndrome (REFBach and Simman, Plast Reconstr Surg Glob Open. 2022 Apr 6;10(4):e4228).

[0181] In some examples, provided are SARM1 agents as described herein for use in treating or preventing an orthopedic disease or condition, or for use in a method of treating or preventing an orthopedic disease or condition.

[0182] Dental diseases or conditions may refer to toothache, root canal surgery, and / or dental surgery (Pergolozzi et al., Expert Opin Pharmacother. 2020 Apr; 21(5): 591-601). In some instances, a SARM1 agent as described herein is provided for use in treating or preventing a dental disease or condition, or for use in a method of treating or preventing a dental disease or condition. In some instances, a SARM1 agent is provided for use in a method of performing a dental surgery or other surgical procedure. In some instances, the use of a SARM1 agent in a dental surgery or other surgical procedure is provided.

[0183] Therapeutic treatment can result in a decrease in the severity of disease symptoms, or an increase in the frequency or duration of symptom-free periods. An amount sufficient to achieve this is defined as a "therapeutically effective amount". In preventive applications, SARM1 agents and any products containing SARM1 agents are administered to subjects who have not yet shown symptoms of a disease or condition in an amount sufficient to prevent or delay the development of symptoms. Such an amount is defined as a "prophylactically effective amount". The subject may have been identified as being at risk of developing a disease or condition by any appropriate means. Therefore, the present invention also provides the SARM1 agents disclosed herein for use in the treatment of the human or animal body.

[0184] In some examples, the SARM1 agents described herein and the compositions described herein can be used in a method of treating one or more of strabismus, blepharospasm, cervical dystonia, upper limb spasticity, lower limb spasticity, overactive bladder, neurogenic bladder, hyperhidrosis, sialorrhea, neuropathic pain (e.g., peripheral neuropathic pain).

[0185] Also provided herein is a method for preventing or treating a disease or condition as disclosed herein in a subject, the method comprising administering a SARM1 agent to the subject in a preventive or therapeutically effective amount. The SARM1 agent can be co-administered with another agent. The method of administering a SARM1 agent can be carried out by any suitable route of administration or as disclosed herein.

[0186] The appropriate dosage of the SARM1 agent of the present invention can be determined by a skilled practitioner. The actual dosage level of the SARM1 agent can be varied to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors, including: the activity of the specific antibody employed; the route of administration; the time of administration; the excretion rate of the antibody; the duration of treatment; other drugs, compounds, and / or materials used in combination with the specific compound employed; the age, sex, weight, condition, general health, and previous medical history of the patient being treated; and similar factors well known in the medical field.

[0187] The suitable dosage of the SARM1 agent can be, for example, in the range of about 0.1 μg / kg to about 100 mg / kg of the patient's body weight to be treated. For example, a suitable dosage can be about 1 μg / kg to about 10 mg / kg body weight per day or about 10 μg / kg to about 5 mg / kg body weight per day. The amount of the SARM1 agent administered can be any appropriate amount, such as between 0.01 mg / kg body weight and 2 mg / kg body weight, between 0.04 and 2 mg / kg body weight, between 0.12 mg / kg body weight and 2 mg / kg body weight, preferably between 0.24 mg / kg and 2 mg / kg body weight, and most preferably between 1 mg / kg and 2 mg / kg body weight. The SARM1 agent can be administered to the same subject multiple times.

[0188] The dosage regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus can be administered, or the method can include several divided doses administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation, provided that the desired interval is ensured. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and dosage uniformity. As used herein, dosage unit form refers to physically discrete units suitable as unit doses for a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect and the required pharmaceutical carrier.

[0189] Any of the SARM1 agents described herein can be formulated into vaccines for prophylaxis or for therapeutic administration. Any such vaccine formulation can be administered directly to an animal for prophylaxis or for therapeutic administration. Any such vaccine formulation can be administered to the muscles of a subject. For example, the vaccine formulation can be administered (e.g., injected) into the facial muscles. Any such vaccine formulation can be in the form of a composition comprising a SARM1 agent of the present invention.

[0190] The present invention also provides a SARM1 agent for use in the manufacture of a medicament for treating or preventing a disease or condition as disclosed herein. The present invention also provides a method of treating and / or preventing a disease or condition as disclosed herein by administering a SARM1 agent to a subject in need thereof. Therefore, any reference to or examples involving the use of a SARM1 agent for use in treating and / or preventing a disease or condition as disclosed herein may apply to a SARM1 agent for use in the manufacture of a medicament for treating or preventing a disease or condition and in a method of treating and / or preventing a disease or condition.

[0191] The SARM1 agent described herein can be part of a pharmaceutical composition. As used herein, a "pharmaceutical composition" refers to a composition comprising one or more compounds that is formulated for administration to a subject. A pharmaceutical composition typically comprises one or more active ingredients (e.g., a SARM1 agent in this example) and one or more pharmaceutically acceptable materials. Therefore, the pharmaceutical composition described herein may comprise a SARM1 agent and one or more other components. For example, a pharmaceutical composition may comprise a SARM1 agent and a pharmaceutically acceptable excipient, diluent, and / or carrier. Pharmaceutical compositions may conventionally contain pharmaceutically acceptable concentrations of salt, buffers, preservatives, compatible carriers, supplemental immune enhancers (such as adjuvants and cytokines), and optional other therapeutic agents or compounds.

[0192] As used herein, "pharmaceutically acceptable" refers to a material that does not have biological or other adverse effects, i.e., the material can be administered to a subject together with the selected compound (e.g., SARM1 agent) without causing any undesirable biological effects or interacting in a deleterious manner with any other components of the pharmaceutical composition in which it is contained.

[0193] In some instances, the pharmaceutical composition comprises a pharmaceutically acceptable diluent. A diluent is a diluent. Pharmaceutically acceptable diluents are well known in the art. Thus, one of ordinary skill in the art can readily identify a suitable diluent.

[0194] In some instances, the pharmaceutical composition comprises a pharmaceutically acceptable excipient. An excipient is a natural or synthetic substance formulated with an active ingredient for the purpose of expanding the formulation or imparting therapeutic enhancement to the active ingredient in the final dosage form, such as promoting drug absorption or dissolution. Excipients can also be used in the manufacturing process, and in addition to contributing to in vitro stability (such as preventing denaturation within the expected shelf life), they also help to handle the relevant active substance, such as by promoting powder flowability or non-stick properties. Pharmaceutically acceptable excipients are well known in the art. Therefore, one of ordinary skill in the art can easily identify suitable excipients. For example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose solution, glycerol, ethanol, etc.

[0195] In some examples, the pharmaceutical composition may include a SARM1 agent and a pharmaceutically acceptable carrier. The carrier is non-toxic to the recipient at the dose and concentration employed and is compatible with the other ingredients of the formulation. The term "carrier" refers to a natural or synthetic organic or inorganic ingredient with which the active ingredient is combined to facilitate application. Pharmaceutically acceptable carriers are well known in the art. Therefore, one of ordinary skill in the art can readily identify suitable carriers.

[0196] Non-therapeutic methods

[0197] The non-therapeutic methods disclosed herein can be used to (i) remove and / or prevent skin changes, (ii) contour skin, and / or (iii) improve oily skin comprising administering a SARM1 agent.

[0198] Skin changes may include one or more of skin wrinkles, frown lines / glabellar lines, forehead lines, lateral canthal lines / crow's feet, transverse nasal creases, neck bands, platysmal neck lines, smoker's lines, bunny lines, upper lip wrinkles, lateral commissure ptosis, marionette lines, gummy smile, chin dimples, cellulite chin, sunken skin, drooping eyebrows, drooping eyelids, lip drooping, square jaw, and downturned nasal tip.

[0199] Thus, administration of one or more SARM1 agents can prevent any one or more of the skin changes described herein. Thus, removing skin changes can be removing any one of a variety of these skin changes over a period of time. Thus, preventing skin changes can be preventing any one or more of these changes over a period of time. That is, as disclosed herein, the effects of the methods may be temporary.

[0200] Contouring may include (i) lip, cheek, jaw, and / or temple shaping; and / or (ii) eyebrow contouring. One or more SARM1 agents may be used to contour / sculpt any one or more of these facial features. Contouring / sculpting may also be performed on other body features, such as the gums, neck, chest, abdomen, back, legs, calves, arms, and shoulders. As disclosed herein, contouring may be a temporary effect (i.e., lasting a limited period of time).

[0201] Improvement in oily skin can be a decrease in oil from the skin after application of a non-therapeutic method. As disclosed herein, this effect can be temporary (ie, lasting for a limited period of time).

[0202] As disclosed herein, the effects of non-therapeutic methods may be temporary. Temporary may mean that the effect (e.g., removal / prevention of skin wrinkles) lasts for up to 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the method is applied.

[0203] The non-therapeutic methods disclosed herein may be used for cosmetic purposes only. They may not encompass medical uses.

[0204] Neurodegeneration

[0205] The SARM1 agents described herein can cause SARM1-dependent neurodegeneration and / or neuronal dysfunction and / or lead to SARM1 activation. SARM1 is composed of an autoinhibitory N-terminal armadillo repeat (ARM) domain, a tandem sterile alpha motif (SAM) domain that mediates octamerization, and an enzymatically active C-terminal Toll / interleukin-1 receptor (TIR) ​​domain. In healthy neurons in vivo, SARM1 is maintained in an autoinhibitory state via multiple intramolecular and intermolecular interactions, including binding of the N-terminal ARM domain to the C-terminal TIR domain. SARM1 autoinhibition is regulated by an allosteric binding site within the autoinhibitory ARM domain that can bind NAD+ or its precursor, nicotinamide mononucleotide (NMN). Axonal damage leads to the loss of the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), resulting in an increase in the NMN / NAD+ ratio, which favors NMN binding to the allosteric site. The switch from NAD+ to NMN binding induces compaction of the autoinhibitory ARM domain and allows the formation of TIR-TIR interactions that activate SARM1. Activation of SARM1 may lead to neurodegeneration or neuronal dysfunction. Neurodegeneration refers to the loss of functional activity and trophic degeneration of neuronal cell bodies. Neuronal dysfunction can refer to the loss of functional activity and / or disruption of neural signaling in the absence of gross morphological degeneration of axons / terminals or cell body death.

[0206] Neurodegeneration can include axonal degeneration and / or neurolytic death. Axonal degeneration refers to the loss of functional activity and nutritional degeneration of axons and their terminal branches. Neurolytic death or neurolysis refers to the degeneration of parts of neurons or nerve cells or any combination thereof, such as cell bodies, axons, axon hillocks, dendrites, terminal branches (terminal dendrites), regenerated axons, regenerated terminal branches, synaptic endings, including neuromuscular junctions and sensory receptors. When any part of a nerve cell degenerates, it causes an interruption in the transmission of neural signals.

[0207] With regard to the SARM1 agents disclosed herein, whether for use in a medical method or a non-therapeutic method, neurodegeneration (which may include axonal degeneration and / or neurolytic death) may be reversible by a reversal agent, such as nicotinamide, NAD+ precursors such as nicotinic acid, NAMPT inhibitors such as FK866 and CHS-828 (also known as GMX1778), SARM1 inhibitors and NMNAT (including NMNAT1-2-3) activators, and NMNAT stabilizers that reduce NMNAT degradation / turnover. The reversal agent can be any agent that can reverse the effect of the SARM1 agent.

[0208] Neurodegeneration / degeneration may occur in (i) cells expressing SARM1. For example, in neurons. Neurons refer to nerve cells, the main functional unit of the nervous system. Neurons are composed of a cell body and its processes - an axon, one or more dendrites, its terminal branches or terminal dendrites, and synaptic terminals. Neurons transmit information to other neurons or cells by releasing neurotransmitters at synaptic terminals. Examples of neurons include dopaminergic neurons, GABAergic neurons, glutamatergic neurons, spinal neurons, such as sensory neurons, pain neurons, dorsal root ganglion neurons, sympathetic and parasympathetic preganglionic and postganglionic neurons, enteric neurons such as dogiel neurons, motor neurons, interneurons, monopolar cells, bipolar cells, multipolar cells, pseudomonopolar cells, pyramidal cells, basket cells, astrocytes, Purkinje cells, Bates cells, amacrine cells, granule cells, oval cells, medium spiny neurons and large spiny neurons, as well as all types of neurons.

[0209] Neurodegeneration / degeneration may not occur in cells that lack SARM1 or express low levels of SARM1, such as Schwann cells (including myelinating, non-myelinating / Remak, perisynaptic / terminal, pain-sensing, injury-activated / repair Schwann cells), muscle cells, skin cells (such as dermal fibroblasts), HEK293 cells, HEK293T cells, HeLa cells, SKmel 2 cells, SKmel 5 cells, SKmel 28 cells, CAPAN cells, JURKAT cells, C6 cells, HL-60 cells, LP-1 cells, U937 cells, MEWO cells, monocytes / macrophages, and / or RAW264.7 cells (Uccellini et al., 2020; Buonvicino et al., 2018; Zhao et al., 2019) ( Figure 3 、 5, 7, 15, 17 and 20). Neurodegeneration induced by SARM1 activation (e.g., by the SARM1 agents described herein) is selective for some cells, making it suitable for medical and non-therapeutic methods disclosed herein. In some examples, low levels of expression can be functionally insignificant levels. In some examples, functionally insignificant refers to a level of SARM1 that does not provide a level of SARM1 that can cause an effect on the cell (such as neurodegeneration and / or neuronal dysfunction). In some examples, functionally insignificant refers to an expression level below a detectable threshold by methods known in the art and described herein. In some examples, functionally insignificant can refer to an expression level below a baseline level, for example, compared to one or more housekeeping or control genes. In some examples, techniques for detecting RNA (i.e., RT-PCR, PCR, hybridization) or protein (i.e., ELISA, immunohistochemistry) can be used to determine the expression level of SARM1. In some examples, the Schwann cells can be injury-activated Schwann cells (e.g., repair Schwann cells).

[0210] In some instances, after administration and neurodegeneration, neuronal cells may be able to regenerate. For example, neurodegeneration may allow nerve regeneration. Without being bound by theory, regeneration may occur due to the temporary and / or reversible nature of the mode of action of the SARM1 agent. In addition, the insensitivity of Schwann cells (including the terminal SC responsible for the reinnervation of the neuromuscular junction and the pain Schwann cells associated with the pain fiber nerve endings) to the SARM1 agent may also allow neuronal regeneration to occur. The occurrence of neurodegeneration can provide advantages in the treatment of certain conditions described herein and in cosmetic uses, because the SARM1 agent may not cause permanent neurodegeneration and possible paralysis (such as paralysis of facial muscles in cosmetic uses), which may cause adverse side effects or results.

[0211] Neurodegeneration / degeneration can be measured using any method known in the art. For example, neurodegeneration can be quantitatively or qualitatively measured by one or more techniques selected from the group consisting of electroencephalography (EEG), electromyography (EMG), evoked potentials, neuroimaging, functional MM, structural MI, diffusion tensor imaging (DTI), [18F] fluorodeoxyglucose (FDG) PET, agents that label amyloid, [18F] F-dopa PET, radiotracer imaging, volumetric analysis of regional tissue loss, specific imaging markers of abnormal protein deposition, multimodal imaging, microscopy of pathological specimens, and biomarker analysis (including clinical neurological motor sensory examination, olfactory examination, cognitive testing, sleep studies, circadian rhythm analysis, serum / cerebrospinal fluid biomarkers).

[0212] Neuronal dysfunction can be determined using any means known in the art. Examples are studies of neuronal and / or neurite morphology using microscopy (Loreto et al. 2021 eLife), measurement of neurofilament light chain (NfL) levels in in vitro assays, (e.g., culture supernatants) serum, and cerebrospinal fluid (CSF) (see https: / / www.sciencedirect.com / science / article / pii / S2211124720315771?via%3Dihub), metabolomics assays as described above, electrophysiology - nerve conduction studies / electromyography, mitochondrial dynamics and function, axonal transport, metabolomic changes, serum / CSF biomarkers such as neurofilaments. Motor / sensory neurology examination (including MRC scale and total score), cognitive tests (such as Addenbrookes Cognitive Examination III), olfactory examination, sleep study, circadian rhythm analysis, methods for measuring peak volitional muscle contraction force (such as myometry and muscle fatigue, such as neurological examination and neurophysiology), timed walking test, objective sensory examination (such as ciliary mechanical stimulation needle), thermal threshold, proprioception test (such as 9-hole plug test). Intraepidermal nerve fiber analysis.

[0213] Application

[0214] SARM1 agents can be administered by any suitable route, including, for example, injection, intravenous infusion, intradermal, subcutaneous, transdermal, intramuscular, intraarterial, intraperitoneal, intraarticular, intraosseous, intracapsular, transdermal, oral, topical or other appropriate routes of administration. Administration can be "parenteral administration", meaning a mode of administration other than enteral and topical administration, usually by injection. Alternatively, SARM1 agents can be administered via non-parenteral routes, such as topical, epidermal or mucosal administration routes. Topical administration can also be performed, including around the tumor, beside the tumor, within the tumor, within the lesion, around the lesion, intracavitary infusion, intracapsular administration and inhalation. In some instances, SARM1 agents can be administered peripherally. For example, peripheral administration includes intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, transdermal, by inhalation, buccal, intranasal, rectal, oral, parenteral, sublingual or nasal. In preferred embodiments, the SARM1 agents disclosed herein are administered via a transdermal patch, composition (such as an ointment or cream) or via a syringe (such as a pre-filled syringe). Other useful routes are routes that can be used to deliver the SARM1 agent to the skin or muscle.

[0215] The compositions can be administered using any suitable route of administration, including, but not limited to, oral, aerosol or other device for delivery to the lungs, nasal spray, intravenous, intramuscular, intraperitoneal, intrathecal, vaginal, rectal, topically, by lumbar puncture, intrathecal, and direct application to the brain and / or meninges.

[0216] In particular, the SARM1 agents disclosed herein can be administered topically. For example, via a transdermal patch. In some instances, by injection into the skin or muscle, including the anal sphincter. In some instances, by intravesical injection into the detrusor / urethral sphincter with or without ultrasound, radiography, and / or electromyography guidance. In some instances, by endoscopic injection, such as injection into the esophageal sphincter, pyloric sphincter, sphincter of Oddi; cystoscopic injection into the urethral sphincter. In some instances, by injection into the salivary glands (such as the sublingual and submandibular glands) or into the axilla or groin.

[0217] Topical application can be achieved by using a topical composition of a SARM1 agent as described herein. A topical composition refers to a composition suitable for application to mammalian (e.g., human) skin. Non-limiting examples of topical compositions include skin care formulations such as cleansers, toners, serums, sticks, wipes, masks, lotions, creams, ointments, balms, oils, scrubs, liquids, strips, gels, oils, foams, or treatments; and beauty products, including but not limited to foundations, eyeliners, eyeshadows, blushes, contouring, highlights, lip liners, eyebrow pencils, blemish / beauty creams, color correction / control creams, lipsticks, mascara, lip gloss, lip balms, concealers, and pressed powders.

[0218] In some instances, the topical composition is a cream or ointment. Cream refers to a soft, semisolid, pharmaceutically and cosmetically acceptable formulation that is intended for external application to the skin or other tissues. Creams typically include an aqueous base formulated as a water-in-oil emulsion or an oil-in-water emulsion. Ointment refers to a uniform, viscous formulation that can be applied to a subject. Ointment can be considered synonymous with lotion, cream, emulsion, gel, or emollient.

[0219] The SARM1 agents disclosed herein can also be applied to the scalp, gums, legs, calves, arms, armpits, palms, soles of feet, groins and shoulders and any other body parts. Administration can be carried out via any means known in the art, such as by injection. The SARM1 agents can be transdermally administered to the subject for the treatment of the conditions disclosed herein, such as undesirable facial muscles or other muscle spasms, hyperhidrosis, acne or the conditions of other parts of the body that need to relieve muscle pain or spasms. The SARM1 agents can be administered by injection or topically for transdermal delivery to muscles or other skin-related structures. The administration can, for example, be applied to legs, shoulders, back (including lower back), armpits, palms, feet, neck, groin, back of hands or back of feet, elbows, upper arms, knees, thighs, buttocks, trunk, pelvis or any other part of the body that needs to be administered SARM1 agents.

[0220] SARM1 agents, compositions and formulations comprising SARM1 agents can be applied so as to administer an effective amount of the SARM1 agent. As used herein, the term "effective amount" means an amount of the SARM1 agent as defined above, which is sufficient to produce the desired effect (e.g., removing / preventing wrinkles), induce muscle paralysis, or other biological or aesthetic effects as disclosed herein, but implicitly a safe amount, i.e., an amount low enough to avoid serious side effects. The desired effect may include relaxing certain muscles (for example, to reduce the appearance of fine lines and / or wrinkles, particularly on the face, or to otherwise adjust the facial appearance, such as widening the eyes, lifting the corners of the mouth, or smoothing wrinkles radiating from the upper lip), or generally relieving muscle tension. The last-mentioned effect, i.e., generally relieving muscle tension, can be achieved on the face or other parts of the body. A suitable effective amount of the SARM1 agent for application may be a single-dose treatment, or may be more concentrated for dilution at the site of application or for use in multiple applications.

[0221] The SARM1 agents disclosed herein can be co-administered. For example, more than one SARM1 agent can be administered together as a pharmaceutical, whether simultaneously or sequentially, for example, in a method of treating any disease or condition as disclosed herein. For example, one SARM1 agent can be administered first, and the second SARM1 agent can be administered up to 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the administration of the first SARM1 agent. The same SARM1 agent can also be administered repeatedly.

[0222] The amount of a SARM1 agent as disclosed herein (e.g., Vacor, its metabolites, analogs and derivatives) required for use in therapy will of course vary not only with the specific compound, but also with the route and form of administration, the nature and severity of the condition being treated, and the type, age and condition of the organism. Accordingly, appropriate concentrations of the SARM1 agent to be incorporated into the compositions and formulations can be routinely determined by one skilled in the art according to standard practice.

[0223] Any of the above-mentioned SARM1 agents and products containing SARM1 agents may further comprise one or more additional agents. One or more additional agents may be selected from the group consisting of: botulinum toxin and capsaicin, local anesthetics, sympathomimetics, steroids, reversal agents, and molecules that interfere with enzymes in the NAD biosynthesis pathway. Local anesthetics may include lidocaine, bupivacaine; opioid analgesics such as morphine, fentanyl, and oxycodone. Sympathomimetics may include epinephrine and norepinephrine. Steroids may include prednisolone, betamethasone, dexamethasone, and hydrocortisone. Reversal agents may include direct SARM1 competitive / non-competitive inhibitors and indirect inhibitors such as nicotinamide. Molecules that interfere with enzymes in the NAD biosynthetic pathway may include NAMPT inhibitors (such as FK866), inhibitors of NMNAT (1-2-3), and products of SARM1 base exchange activity (such as VAD), among others (Loreto et al., 2021, eLife; Shi et al., 2022 Mol Cell).

[0224] Any of the above-mentioned SARM1 agents and additional agents can be co-administered or administered sequentially. For example, an analgesic can be co-administered with one or more SARM1 agents. As another example, two or more SARM1 agents can be co-administered. As another example, a reversal agent can be administered after administration of any of the various SARM-1 agents disclosed herein.

[0225] Transdermal patch, syringe, composition

[0226] Also disclosed herein are products such as transdermal patches comprising a SARM1 agent as disclosed herein, syringes comprising a SARM1 agent as disclosed herein, and compositions comprising a SARM1 agent as disclosed herein. Such products can be used to deliver a SARM1 agent to a subject.

[0227] Transdermal patches can allow one or more SARM1 medicaments to be delivered to the subject's system at a controlled rate through the skin. SARM1 medicaments can be co-administered with any other medicament (such as a drug, etc.). Patch or pump equipment or other systems can be placed in contact with the skin area to transport molecules (such as SARM1 medicaments, other medicaments or drugs, etc.) transdermally via many different mechanisms (such as maintaining simple contact of molecules on the skin surface for absorption (with or without chemical penetration enhancers), iontophoresis, needles, indwelling catheters, etc.). Patch or pump can be removably adhered to or directly placed on the user's skin surface via a variety of methods, such as via a temporary adhesive layer or optionally directly adhered to the skin by utilizing the direct pressure of a band or belt.

[0228] Transdermal patches can allow the SARM1 agent and any other selected agent to penetrate the skin surface by passive or active mechanisms. Examples of passive mechanisms can include simple diffusion or absorption through the skin, osmosis, and the like, and some examples of active mechanisms can include introduction through the skin via mechanical insertion through a needle, abrasion, and the like, or by electrical methods such as iontophoresis, in which a suspension of drug molecules is subjected to an electric field to enter or pass through the adjacent skin surface and into the patient's bloodstream.

[0229] In one embodiment, the patch of iontophoresis is used to deliver a drug formulation to the skin using an electric current or voltage applied. Electric current can be programmed to regulate and control the speed of drug delivery to the patient's skin. Other methods, such as using chemical penetration enhancers, heat or ultrasound, can also be used to increase the delivery rate by the skin barrier. Other patches or pumps include infusion pumps, which deliver molecules via a needle or a catheter inserted into the skin for symptom control, such as the infusion pump used in the insulin therapy for the treatment of diabetes. Such patches or pumps deliver molecules via a fluid vehicle, wherein fluid is retained in a reservoir conventionally, and the reservoir is coupled to delivery mechanisms such as a film, a needle or a catheter according to a delivery mode.

[0230] The patch, pump or other system for delivery can include many features that promote the delivery of molecules (SARM1 agents, other agents, drugs, etc.) to the subject. For example, the patch or pump assembly can seal or contain a fluid reservoir in a housing to contain molecules or be suspended in a fluid medium. The reservoir can be connected via a microchannel lumen fluid, and fluid or medicine can be transported to the transdermal drug delivery mechanism that contacts or approaches the skin surface of the lower layer by the microchannel lumen. The pump can be used to drive or promote molecules from the reservoir and through the skin via one of the drug delivery mechanisms (such as by a microneedle array). The pump can also be used to deliver drugs from the reservoir to be placed directly on the skin surface, where the drug can remain in place or maintain contact with the skin surface for absorption, such as by maintaining contact with the skin with a microporous membrane. When molecules are placed on the skin surface (rather than introducing or promoting molecules by the skin layer), many chemical enhancers can be utilized to promote drug absorption into the skin surface. For example, agents such as propylene glycol, ethanol, dimethyl sulfoxide, and the like can be placed on the skin surface before, during, or after the drug is placed on the skin, or such agents can be combined with the drug in a fluid vehicle so that they are delivered directly to the skin surface along with the drug.

[0231] To control the pumping of molecules and different treatment plans, an electronic control circuit system can also be positioned on the housing and electrically connected to the pump. A battery (which can be rechargeable or replaceable) can also be positioned along the housing to provide power to the pump, control circuit system, and any other features as needed. For example, the electronic control circuit system can provide various functions and determine when the pump should be active. By controlling when the pump is active or inactive, the electronic circuit system can be used to control when the fluid from the reservoir is pumped to the skin. The control circuit system can also include diagnostic algorithms and indicators, such as monitoring battery charge, pump operation, circuit integrity, etc. Another element that can be included in the control circuit system is an on-chip clock that tracks the time and date to facilitate adjustment of the fluid delivery schedule controlled by the microprocessor, particularly for chronotherapeutic drug formulations, where the delivery of the drug is carried out according to a timed schedule corresponding to the date and / or time of day. Additionally or optionally, a flow rate monitor can also be included in the control circuit system to monitor the amount of fluid that has been delivered to or through the skin. Further, any microchannels that fluidically connect various features (such as reservoirs, pumps, and microporous membranes) can be formed directly within or along the housing and sized to have cross-sectional dimensions ranging from 1 micron to 5000 microns and lengths from 1 millimeter to 1 meter or longer. Due to their size, microchannels can facilitate the passage of fluids, such as by capillary action, so that fluid delivery through the channels is consistent regardless of the orientation or angle of the assembly. In addition, microchannels can also help suppress or prevent the formation of bubbles within the channels so that molecular delivery can be consistently metered to the patient.

[0232] Using microchannels as molecular reservoirs can also allow for different types of pump configurations. Instead of using a pump to extract liquid from a microchannel reservoir and pump it out to ultimately reach the user's skin, an air or gas pump can be used to push the liquid down the length of the microchannel reservoir, ultimately depositing it on the user's skin.

[0233] The reservoir can also be configured into various other patterns, such as spiral or any other configuration that allows fluid to be stored in or along the housing. For example, a microchannel reservoir can be formed to have one or more separate channels aligned parallel to each other. Each channel in these separate parallel channels can converge into a single microchannel, and the single microchannel fluid is connected to a pump or other mechanism. The number of channels and the length of each channel can be uniform or individually varied. In addition, one or more microchannels in the microchannel may contain different formulations or different dosages, depending on the desired dosage obtained and the drug combination to be infused into the patient's body. Another modification of the microchannel reservoir can include, for example, a first microchannel reservoir and a second microchannel reservoir that is separate and different from the first microchannel reservoir. In addition, other modifications can include one or more microchannel reservoirs aligned along multiple geometric planes in the housing. For example, the first reservoir can be located in a first plane along the housing, and the second reservoir can be located in a second plane below or above the first reservoir. In this way, multiple reservoirs can be "stacked" on top of or below each other in several adjacent planes that can be separated from each other, or fluid interconnections can be formed between two or more reservoirs between their respective planes. The microchannel reservoirs can each contain the same or different formulations or dosages and can each be coupled to one or more valves that can be electronically controlled to meter or control the volume of one or two reservoirs to be pumped. The microchannel reservoir can also be removed completely from the patch or pump. The microchannel reservoir can reside in a removable package or cartridge that can be securely inserted into an interface or receiving channel defined in the housing. Once the reservoir is exhausted, the reservoir can be refilled, or the cartridge can be removed completely from the housing and replaced with another cartridge without removing the housing from the patient's skin.

[0234] The programmable electronic circuit system of patch or pump assembly can be included and be equipped with a transmitter and / or receiver, and described transmitter and / or receiver allows it to communicate wirelessly or otherwise with external controllers such as computers or handheld devices.Doctors or patients can use external controllers to program parameters, such as the drug delivery time curve of specific patients, thereby customizing the delivery rate curve according to the needs of specific patients for specific medicines.Another aspect of transdermal patch or pump assembly can provide the ability of "on demand" controlled drug delivery determined by patients.The response initiated by the user can be used as the signal of programmable electronic circuit system, to indicate the appropriate dosage curve to be used from this point on or until the signal initiated by a new user is received.When the user of patch or pump needs, these signals can also be used for example to start "on demand" injection to carry out drug delivery.When pressing the "on demand" button, the amount and rate of molecule delivery can be predetermined by circuit system. When the patient wishes to administer a small dose of a medication (such as an analgesic to relieve pain or a stimulant to help maintain consciousness and alertness), the patient can actuate a control device (such as pressing a button on a transdermal patch or pump) to release a preset bolus of the medication / formulation. The control device can be part of the electronic control device itself.

[0235] Transdermal patch can be constructed according to any transdermal patch known in the art.For example, for the transdermal patch for applying the molecule disclosed herein (for example, SARM1 medicament, other medicaments or medicine) to the patient's skin, a backing layer, a liner and an adhesive layer containing molecules arranged between the backing layer and the liner. The adhesive layer containing molecules can include a plasticizer (wherein the ratio of plasticizer to polyisobutylene) of polyisobutylene, at least 5 % by weight and a molecular component comprising molecules to be delivered, the molecule can be appropriately dissolved in the plasticizer. Preferably, the ratio of plasticizer to polyisobutylene is between about 0.05 and 0.8.

[0236] Regarding transdermal patches, the adhesive layer containing molecules can be in direct contact with the backing layer, so that when the backing layer is removed and the transdermal patch is applied to the skin, the adhesive layer is in direct contact with the skin. The filler of the transdermal patch can be a metal oxide, an inorganic salt, a polymer filler, a clay component and / or its mixture. Preferably, the metal oxide can be an oxide of zinc, magnesium, calcium or titanium. The inorganic salt can include calcium carbonate, magnesium carbonate and sodium carbonate, calcium sulfate and magnesium sulfate, and calcium phosphate. The clay component can include talc, kaolin and bentonite. The polyisobutylene of the transdermal patch can include a mixture of high molecular weight polyisobutylene and low molecular weight polyisobutylene. Preferably, the viscosity-average molecular weight of the high molecular weight polyisobutylene is between about 450,000 and 2,100,000. Preferably, the average molecular weight of the low molecular weight polyisobutylene is between about 1,000 and 450,000. Preferably, the ratio of high molecular weight polyisobutylene:low molecular weight polyisobutylene is between about 20:80 and 70:30.

[0237] Transdermal patches can be used to deliver one or more molecules, such as one or more SARM1 agents, other agents such as drugs, etc., including additional agents disclosed herein. Any drug / agent component of the transdermal patch can include additional agents disclosed herein, such as local anesthetics, sympathomimetic drugs, steroid drugs, reversal agents, and molecules that interfere with enzymes in the NAD biosynthetic pathway. Local anesthetics can include lidocaine, bupivacaine; opioid analgesics, such as morphine, fentanyl, and oxycodone. Sympathomimetic drugs can include epinephrine and norepinephrine. Steroid drugs can include prednisolone, betamethasone, dexamethasone, and hydrocortisone. Reversal agents can include direct SARM1 competitive / non-competitive inhibitors and indirect inhibitors, such as nicotinamide. Molecules that interfere with enzymes in the NAD biosynthetic pathway may include NAMPT inhibitors (such as FK866), inhibitors of NMNAT (1-2-3), and products of SARM1 base exchange activity (such as VAD), among others (Loreto et al., 2021, eLife; Shi et al., 2022 Mol Cell).

[0238] The plasticizer of transdermal patch can comprise hydrophobic liquid, for example, and described hydrophobic liquid has between about 12 and 18 (J / cm )1 / 2 solubility parameter between.Plasticizer can comprise mineral oil, linseed oil, octyl palmitate, squalene, squalane, silicone oil, isobutyl myristate, isostearyl alcohol and oleyl alcohol etc.Mineral oil can exist with the amount between about 10 % by weight and 40 % by weight.

[0239] In a specific embodiment, a transdermal patch can comprise a backing layer, a backing layer, and an adhesive layer containing a SARM-1 agent disposed between the backing layer and the backing layer, wherein the SARM-1-containing adhesive layer comprises polyisobutylene, a plasticizer for the polyisobutylene comprising mineral oil, and at least 5% by weight of a filler, wherein the ratio of the mineral oil to the polyisobutylene is between about 0.5 and 0.8.

[0240] The syringes disclosed herein may contain one or more molecules, such as one or more SARM1 agents, other agents such as drugs, and the like, including additional agents disclosed herein. The syringe may be any syringe known in the art. For example, any drug / agent component of the syringe may include additional agents, such as local anesthetics, sympathomimetics, steroids, reversal agents, and molecules that interfere with enzymes in the NAD biosynthesis pathway. Local anesthetics may include lidocaine and bupivacaine; opioid analgesics may include morphine, fentanyl, and oxycodone. Sympathomimetics may include epinephrine and norepinephrine. Steroids may include prednisolone, betamethasone, dexamethasone, and hydrocortisone. Reversal agents may include direct SARM1 competitive / noncompetitive inhibitors and indirect inhibitors, such as nicotinamide. Molecules that interfere with enzymes in the NAD biosynthetic pathway may include NAMPT inhibitors (such as FK866), inhibitors of NMNAT (1-2-3), and products of SARM1 base exchange activity (such as VAD), among others (Loreto et al., 2021, eLife; Shi et al., 2022 Mol Cell).

[0241] Syringes can be prefilled for ease of administration. Such syringes are known in the art, in which the syringe barrel is prefilled with medication. Such prefilled syringes are equipped with a nozzle for attaching an injection needle at one end of the syringe barrel, which forms the syringe body. At the other end, an opening is provided through which a plunger, with a gasket at the tip of the plunger rod, can move freely backward and forward. After filling with medication, the nozzle is typically covered with a rubber cap to prevent leakage. The entire package is then packaged in blister packs or pillow packs with the plunger in place. Prefilled syringes are also known, in which the capped nozzle is then covered with a heat-shrinkable resin film (shrink film) to prevent the cap from falling off and to protect the filled medication from tampering. If the syringe is made of plastic, to prevent degradation of the medication filled in the syringe barrel, it is sometimes necessary to store the syringe in barrier packaging with gas barrier properties. If the syringe is stored in barrier packaging, the gas barrier properties prevent the medication filled in the syringe barrel from degrading.

[0242] The compositions disclosed herein can be any formulation known in the art. The compositions can be applied topically to deliver one or more molecules, such as one or more SARM1 agents, other agents such as drugs, etc. For example, the compositions disclosed herein can comprise solutions, emulsions (including microemulsions), suspensions, creams, lotions, gels, powders, or typical solid or liquid compositions for application to skin and other tissues, in which molecules (e.g., SARM1 agents and / or other agents) can be used. For example, the compositions can be formulations designed for efficient topical application to the skin. In addition to the SARM1 agent and any carrier, such a composition may also contain other ingredients commonly used in such products, such as antimicrobial agents, moisturizers and hydrating agents, penetrants, preservatives, emulsifiers, natural or synthetic oils, solvents, surfactants, detergents, emollients, antioxidants, fragrances, fillers, thickeners, waxes, odor absorbers, dyes, colorants, powders, and optionally include anesthetics, antipruritic additives, plant extracts, conditioners, darkening agents or brightening agents, glitter, moisturizers, mica, minerals, polyphenols, silicones or their derivatives, sunscreens, vitamins and botanicals. The composition may also contain a gelling agent and / or viscosity modifier. These agents are typically added to increase the viscosity of the composition, thereby making the application of the composition easier and more accurate. In addition, these agents help prevent the aqueous SARM1 agent / carrier solution from drying out, which often causes the activity of the SARM1 agent to decrease. Particularly preferred agents are those with an electric charge that does not interfere with the activity of the SARM1 agent or the efficiency of the neurotoxin-carrier complex to pass through the skin. For example, the gelling agent can be certain cellulose-based gelling agents, such as hydroxypropyl cellulose (HPC). In some embodiments, the SARM1 agent / carrier complex is formulated in a composition having 2-4% HPC. Alternatively, the viscosity of the solution containing the SARM1 agent / carrier complex can be altered by adding polyethylene glycol (PEG). In other embodiments, the SARM1 agent / carrier solution is mixed with a premixed viscous agent (such as Moisturizing cream) combination.

[0243] The composition may optionally include a spacer. As used herein, a "spacer" is any substance or additive that has the property of preventing or minimizing unwanted or uncontrolled aggregation of the SARM1 agent and any molecule to be delivered using the vehicle of the present invention. For example, a spacer may be useful when a concentrated solution containing the SARM1 agent must be used due to volume limitations. In these cases, the spacer keeps the SARM1 agent dispersed, thereby preventing aggregation that would occur in the absence of the spacer. Typically, the spacer (1) is non-irritating, (2) does not destroy the active agent, such as SARM1, (3) does not impart any increase in permeability, (4) provides a reliable and stable particle size, (5) has a relevant charge, and (6) does not interfere with the complex of the neurotoxin and the transdermal vehicle. An example of a suitable spacer is ethanol (EtOH). In a preferred embodiment, EtOH is less than 20% of the composition, and most preferably, less than 5% of the composition.

[0244] Oligo- or polyanionic bridges may be added to the SARM1 formulation to increase the efficiency and efficacy of topical administration. Examples of such oligo / polyanionic bridges include sodium phosphate (5%), PBS, or 5% poly-L-aspartic acid (e.g., MW 3000).

[0245] The compositions disclosed herein may be in the form of controlled-release or sustained-release compositions, wherein the SARM1 agent and any carrier are encapsulated or otherwise contained within a material such that they are released to the skin in a controlled manner over time. The SARM1 agent and any carrier may be contained within a matrix, liposomes, vesicles, microcapsules, microspheres, etc., or within a solid particulate material, all of which are selected and / or constructed to provide for the release of the SARM1 agent over time. The SARM1 agent and any carrier may be encapsulated together (e.g., in the same capsule) or separately (in separate capsules).

[0246] Thus, the compositions described herein can be used to deliver molecules (e.g., SARM1 and any carrier, agent, etc.) in effective amounts to the muscles beneath the skin, or to glandular structures within the skin to produce paralysis, produce relaxation, relieve contraction, prevent or relieve spasms, reduce glandular output, or other desired effects. Local delivery of SARM1 agents in this manner can reduce dosage, reduce toxicity, and allow for more precise dosage optimization to achieve the desired effect relative to injectable or implantable materials.

[0247] Kit

[0248] The SARM1 agent or composition thereof can be provided in the form of a kit. For example, the kit can include a SARM1 agent or composition thereof as described herein and an applicator for administering the SARM1 agent to a subject. For example, the applicator can be a syringe or a transdermal patch as described herein. In some instances, the SARM1 agent or composition thereof can be in unit dosage form.

[0249] The kit may also include instructions for use.

[0250] Other definitions

[0251] The subject may be referred to as a patient in this article. The terms "subject," "individual," and "patient" are used interchangeably herein. As used herein, the term "subject" is intended to include humans and animals. Generally, a subject refers to a human or animal having homologous recombination-deficient cells in its body. Examples of subjects include mammals, such as humans, dogs, cattle, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In some instances, the subject includes companion animals, such as dogs, cats, rabbits, and rats. In some instances, the subject includes livestock, such as cattle, pigs, sheep, goats, and rabbits. In some instances, the subject includes purebred animals or ornamental animals, such as horses, pigs, cattle, and rabbits.

[0252] Any suitable algorithm can be used to calculate amino acid identity. For example, PILEUP and BLAST algorithms can be used to calculate identity or to sort sequences (such as identifying equivalent or corresponding sequences (usually according to their default settings), for example, as described in Altschul SF, 1993 J Mol Evol [Journal of Molecular Evolution] 36: 290-300; Altschul, S, F et al., 1990 J Mol Biol [Journal of Molecular Biology] 215: 403-10. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with a word of the same length in a database sequence, match or satisfy a positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing the initial neighborhood word hits. Word hits are extended in both directions along each sequence until the cumulative alignment score can be increased. Extension of word hits in each direction is halted if: the cumulative alignment score decreases by an amount X from its maximum achieved value; the cumulative score becomes zero or lower due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses the following defaults: word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1992 Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 89: 10915-10919), alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both chains.

[0253] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, for example, Karlin and Altschul, 1993 Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 90: 5873-5787. A similarity measure provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability of a match occurring by chance between two nucleotide or amino acid sequences. For example, if the minimum sum probability of comparing a first sequence to a second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, a sequence is considered to be similar to another sequence. Alternatively, the UWGCG package provides the BESTFIT program (Devereux et al., 1984 Nucleic Acids Research [nucleic acid research] 12, 387-395) that can be used to calculate identity (e.g., used according to its default settings).

[0254] The amino acid sequence of any biomolecule (such as SARM1) mentioned herein may comprise the amino acid sequence of all or part of the reference sequence defined by the SEQ ID NO disclosed herein, wherein modifications such as amino acid additions, deletions or substitutions have been made relative to the reference sequence or a portion thereof. Modifications may be conservative or non-conservative amino acid substitutions. In the case where multiple modifications are present in a single polypeptide, the modifications in the polypeptide sequence may be a combination of conservative and non-conservative amino acid substitutions. Conservative substitutions replace amino acids with other amino acids having similar chemical structures, similar chemical properties or similar side chain volumes. The introduced amino acid may have similar polarity, hydrophilicity, hydrophobicity, alkalinity, acidity, neutrality or charge to the amino acid it replaces. Alternatively, conservative substitutions may introduce another aromatic or aliphatic amino acid to replace a pre-existing aromatic or aliphatic amino acid. Examples

[0255] The following examples are provided to illustrate the present invention but are not intended to limit the present invention.

[0256] Example 1 - Sarm1 / SARM1 detection in Schwann cells and oligodendrocytes, although deletion does not interfere with myelination in zebrafish and mice.

[0257] SARM1 is a central regulator of programmed axonal death and is required for initiating axonal autodestruction following traumatic and toxic insults to the nervous system. Aberrant activation of this axonal degeneration pathway is increasingly recognized as a contributing factor to human neurological diseases, and SARM1 knockdown or inhibition has emerged as an attractive therapeutic strategy to preserve axonal loss in various conditions of the peripheral and central nervous systems. Despite this, it remains unclear whether Sarm1 / SARM1 is present in myelinating glial cells and whether it plays a role in myelination in the PNS or CNS. Answering these questions will be crucial to understand whether future therapies that inhibit SARM1 function might have unintended deleterious effects on myelination. This study demonstrates that Sarm1 mRNA is identifiable in PNS and CNS glial cells in zebrafish and mice. SARM1 protein is present in oligodendrocytes, but not in myelinating cells, Remak Schwann cells, and satellite glial cells in the adult murine nervous system. Using specific SARM1 activators in cultured cells, we confirmed that Schwann cells contain negligible functional SARM1, whereas oligodendrocytes are sensitive to endogenous SARM1 activation in vitro. Furthermore, using zebrafish and mouse Sarm1 mutants, we demonstrate that SARM1 is not required for the proper initiation and maintenance of myelination in both oligodendrocytes and Schwann cells. Therefore, strategies to inhibit SARM1 function in the nervous system to treat neurological disorders are unlikely to interfere with myelination in humans.

[0258] The programmed axonal death (also known as Wallerian degeneration) pathway is becoming increasingly relevant to neurological diseases. Two of the most important regulators of this pathway are nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) and sterile alpha and Toll / interleukin-1 receptor (TIR) ​​motif protein 1 (SARM1), which are members of the MyoD88 family (Gilley and Coleman, 2010; Osterloh et al., 2012; Gerdts et al., 2013; Coleman and 2020). Complete loss-of-function mutations in Nmnat2 are associated with cases of fetal akinesia dysplasia sequence (FADS), in which the fetus is stillborn with severe skeletal dysplasia and hydrops fetalis, while partial loss-of-function mutations are associated with the development of peripheral neuropathy with erythromelalgia (Huppke et al., 2019; Lukacs et al., 2019). SARM1 function is conserved in humans, and the SARM1 locus has been identified in two genome-wide association studies (GWAS) of amyotrophic lateral sclerosis (ALS) (Fogh et al., 2014; van Rheenen et al., 2016; Chen et al., 2021). Since then, gain-of-function SARM1 mutations have been identified in sporadic ALS and other motor neurological disorders (Gilley et al., 2021; Bloom et al., 2022). In addition, the abandoned rat poison vacor, which causes highly specific activation of SARM1, causes severe neurotoxic effects in humans. (LeWitt, 1980; Loreto et al., 2021).

[0259] Axonal dysfunction and loss are hallmarks of many neurological diseases of the central nervous system (CNS) and peripheral nervous system (PNS), including Parkinson's disease, traumatic brain injury, progressive multiple sclerosis, ALS, and many inherited and acquired peripheral neuropathies (Coleman and 2020). In animal models of traumatic brain injury, metabolic neuropathy, and several models of chemotherapy-induced neuropathy, Sarm1 deficiency has been shown to have a significant protective effect against axonal loss (Geisler et al., 2016; Henninger et al., 2016; Turkiew et al., 2017; Cheng et al., 2019; Geisler et al., 2019a; Marion et al., 2019; Bosanac et al., 2021; Gould et al., 2021b). Therefore, inhibition or knockdown of SARM1 through the use of pharmacology, gene therapy, and antisense oligonucleotide approaches has become a very attractive therapeutic strategy to be tested in various neurological diseases (Geisler et al., 2019b; Coleman and 2020; Krauss et al., 2020; Arthur-Farraj and Coleman, 2021; Bosanac et al., 2021; Gould et al., 2021a; Merlini et al., 2022).

[0260] SARM1 is highly abundant in the nervous system but is not found in most other tissues, including the heart, kidney, liver, lung, skeletal muscle, spleen, or thymus (Kim et al., 2007; Chen et al., 2011). While neurons have been shown to have high levels of SARM1, it is unclear whether SARM1 is also present in myelinating glial cells in the PNS or CNS. Furthermore, due to the lack of in-depth quantitative studies of myelination, it is unclear whether SARM1 plays any role in regulating oligodendrocyte or Schwann cell myelination or myelin maintenance in a cell-autonomous or non-cell-autonomous manner. This understanding is important because it may have adverse effects on therapies designed to treat various neurological conditions by inhibiting SARM1 function.

[0261] In this example, mice and zebrafish larvae were used to demonstrate that SARM1 / sarm1 mRNA is present at low levels in developing Schwann cells and oligodendrocytes, while SARM1 protein is only detectable in oligodendrocytes and not in Schwann cells in cell culture and in the adult mouse nervous system. In cultured cells, the use of the specific SARM1 activators vacor and 3-acetylpyridine (3-AP) showed that Schwann cells contain insignificant amounts of SARM1, while cultured oligodendrocytes contain functionally relevant levels of SARM1 protein. Furthermore, the study showed that in the absence of SARM1 / Sarm1, myelination in the PNS and CNS is initiated normally in both mice and zebrafish larvae, and that PNS and CNS myelin maintenance in adult mice is unaffected.

[0262] Materials and Methods

[0263] animal

[0264] All zebrafish studies complied with the Animals (Scientific Procedures) Act 1986 and the University of Cambridge Animal Welfare and Ethical Review Body (AWERB) under project license code P98A03BF9. Zebrafish (Danio rerio) were maintained at 28°C. Tg[mbp:eGFPCAAX] fish were a gift from Dave Lyons (Almeida et al., 2011), and Sarm111193 mutant fish were obtained from ZIRC (Anon) (Kettleborough et al., 2013). Tg[mbp:eGFPCAAX] and sarm1SA11193 embryos were obtained by natural spawning and cultured in Petri dishes in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.3 mM MgSO4, and 0.1% methylene blue) until 24 hours postfertilization. Embryos were then incubated in E3 medium supplemented with 0.003% phenylthiourea (PTU) to prevent pigmentation. F2 generation fish were used for analysis. For all experiments, sarm1+ / + (wt) mice were compared with sarm1SA11193 / SA11193 (sarm1 mutant). Mouse studies complied with EU guidelines, and the experimental protocol was approved by the Bioethics and Biosafety Committee of the Alicante Neuroscience Institute (Comité de Bioética y Bioseguridad del Instituto de Neurociencias de Alicante), the Universidad Miguel Hernández de Elche, and the Spanish National Research Council (http: / / in.umh-csic.es / ), with reference number 2017 / VSC / PEA / 00022tipo 2. Sarm1 knockout mice on a C57BL / 6J background (Kim et al., 2007) were bred from heterozygotes and F1 littermates were used for analysis. Mice were genotyped as previously described (Kim et al., 2007).

[0265] Zebrafish genotyping

[0266] Zebrafish genomic DNA was isolated from adult zebrafish fins and incubated overnight at 55°C with 0.5mg / ml proteinase K in 10mM Tris-HCl buffer containing 50mM KCL, 10% Tween 20, and 10% NP40. PCR was performed using primers: forward: 5'-TCTGGAGCTGGTGGAGCCCT-3' (SEQ ID NO: 1); reverse: 5'-AGTCTAGTTTCTGCCTGACCTTGG-3' (SEQ ID NO: 2). The PCR product was then digested with the MseI (New England Biolabs) restriction enzyme at 37°C for 1 hour and then run on a 1.5% agarose gel. The WT PCR product produced a band of 233 base pairs. The mutant PCR product produced two bands of 165 and 68 base pairs.

[0267] Plasmids and microinjection

[0268] To generate neuroD:tdTomato constructs, use Gateway TM The p-5E neuroD5kb promoter entry vector (Mo and Nicolson, 2011), a gift from Dr. A. Nechiporuk, was inserted into pDestTol2pA2 along with pMEtdTomato (Oehlers et al., 2015) (Addgene plasmid #135202) and p3EpolyA, a gift from Dr. D. Tobin, as previously described (Kwan et al., 2007). To visualize posterior lateral line neurons, 20 pg of the neuroD:tdTomato construct was microinjected into one-cell embryos. Larvae were then fluorescently sorted at 4 dpf.

[0269] Two-photon axotomy of PLL axons in zebrafish larvae

[0270] Axotomy was performed at the Cambridge Advanced Imaging Centre on a TriM Scope II two-photon scanning fluorescence microscope using Imspector Pro software (LaVision Biotec) and a near-infrared laser source (Insight DeepSee, Spectra-Physics). The laser was focused through a 25x, 1.05 numerical aperture water immersion objective (XLPLN25XWMP2, Olympus). Axotomy was performed by focusing the laser on a 6×6 μm slice of the posterior lateral line using 100% laser power.

[0271] Schwann cell culture

[0272] Freshly plated mouse Schwann cells were obtained from P2 sciatic and brachial nerves. Nerves were trypsinized and collagenase digested, centrifuged and plated in defined medium on laminin / poly-L-lysine coated glass coverslips as previously described (Arthur-Farraj et al., 2011).

[0273] Intracellular NAD + Measurement

[0274] Schwann cells and HEK 293T cells were treated with 100 μM vacor, 250 μM 3AP or vehicle control and collected in culture medium at 0 and 72 hours after treatment. TM Proteins were extracted using PierceIP lysis buffer with Mini EDTA-free protease inhibitor cocktail and protein concentrations were determined by BCA assay. All samples were diluted to 0.5 μg / μL. + / NADH-Glo TM The NAD-glo assay was performed according to the manufacturer's instructions. Briefly, 25 μL of sample was incubated with 12.5 μL of 0.4 M HCl at 60°C for 15 minutes, followed by the addition of 12.5 μL of 0.5 M Tris base. 10 μL of NAD + The standard or sample was mixed with 10 μL NAD-glo master mix (1 ml fluorescein detection reagent, 5 μl reductase, 5 μl reductase substrate, 5 μl NAD + Cycling enzyme, 25 μl NAD + Mix with circulating substrate and incubate at RT for 40 minutes. Luminescence is read on an Explorer plate reader and is measured relative to NAD + Determination of NAD by standard curve + concentration.

[0275] Oligodendrocyte culture

[0276] After a lethal overdose of pentobarbital, Sprague-Dawley newborn (≤p7) rat pups were decapitated. The brains were dissected and immersed in Hibernate-A low fluorescence medium (Transnetyx Tissue, #HALF). The tissue was cut into 1-mm 3 slices in HBSS - / -The cells were washed with 4% paraformaldehyde (Gibco, #11039047) and then centrifuged at room temperature for 1 minute at 100 g. To digest the tissue, the tissue was incubated in HALF containing 34 U / mL papain (Worthington, #LS003127) and 40 μg / mL DNase I (Sigma, #D5025) and incubated on an orbital shaker (55 rpm) for 40 minutes at 37°C. To obtain a single cell suspension, the tissue was ground in HALF supplemented with 2% B27 and 2 mM sodium pyruvate, first using a 5-ml serological pipette and then using three flame-polished glass pipettes of decreasing diameter. The supernatant containing the cells was filtered through a 70-μm filter into a tube containing 90% isotonic Percoll (GE Healthcare, #17-0891-01 in 10×PBS pH 7.2 (Gibco, #70013032). The final volume was made up with DMEM / F12 (Gibco, #31331028) and then inverted several times to produce a homogenous suspension with a final Percoll concentration of 22.5%. The single cell suspension was then separated from tissue and myelin debris by gradient density centrifugation at 800 g (no brake) for 20 minutes at room temperature. The myelin debris and supernatant were aspirated, leaving only the cell pellet, which was then resuspended in HBSS. - / - The cells were then washed with 5% paraformaldehyde (PNA) to wash out the Percoll. Subsequently, erythrocytes were removed using erythrocyte lysis buffer (BD Biosciences, #555899). OPCs were isolated by positive selection using the MACS protocol according to the manufacturer's instructions using 2.5 μg of A2B5 (Merck Millipore, #MAB312) primary antibody followed by 20 μl of rat anti-mouse IgM antibody (Miltenyi, #130-047-302) per brain. To collect A2B5 +The MACS MS column (Miltenyi Biotec, #130-042-201) was removed from the magnetic stand (Miltenyi Biotec, #130-042-102) and washed with 1 ml of pre-warmed OPC medium (DMEM F / 12 containing N-acetylcysteine ​​(60 μg / ml, Sigma, #A9165), human recombinant insulin (10 μg / ml), sodium pyruvate (1 mM, Thermo Fisher Scientific)). The cells were washed from the column with 1% paraformaldehyde (Sigma, #11360-070), apo-transferrin (50 μg / ml, Sigma, #T2036), putrescine (16.1 μg / ml, Sigma, #P7505), sodium selenite (40 ng / ml, Sigma, #S5261), progesterone (60 ng / ml, Sigma, #P0130), and bovine serum albumin (330 μg / ml, Sigma, #A4919). The cells were counted and then centrifuged at 20,000 cells / cm 2 The cells were seeded at a density of 100 μg / ml on poly-D-lysine (5 μg / ml PDL, Sigma #P6407) coated plates.

[0277] Freshly isolated OPCs were cultured in OPC medium containing proliferation factors, b-FGF (30 ng / ml, Peprotech, #100-18B) and PDGF (30 ng / ml, Peprotech, #100-13a), and then replaced with OPC medium containing T3 (triiodothyronine, Sigma, #T2877) for 5-7 days to induce OPC differentiation into oligodendrocytes. During culture, cells were maintained in a humidified incubator at 37°C, 5% CO2, and 5% O2, and the medium was changed every 48 hours.

[0278] Quantification of Vacor-treated oligodendrocyte cultures

[0279] Rat oligodendrocyte cultures were fixed at different time points after DMSO and Vacor treatment. These cells were immunofluorescently labeled with DAPI and an antibody against SOX10. Three representative images taken at 20x across the cultures at different time points were quantified using Fiji for the presence of SOX10 expression. This experiment was repeated three times, and the average number of SOX10-positive oligodendrocytes counted per frame is shown.

[0280] In situ hybridization chain reaction (HCR)

[0281] Embryos were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) without calcium and magnesium and stored at -20°C in 100% MeOH. HCR 3.0 was performed as described in (Choi et al., 2018). Briefly, 2 pmol of HCR probe was hybridized overnight at 37°C in hybridization buffer and then washed repeatedly in wash buffer. The probe was detected using 30 pmol of fluorescent hairpin in amplification buffer at room temperature overnight and then washed in 5X SSC 0.001% Tween-20. The samples were finally counterstained with 1 ug / ml DAPI and mounted in 80% glycerol in MatTek glass-bottom culture dishes. DNA probes for zebrafish mbp, sarm1, sox10, fluorescent hairpins, and buffer were purchased from Merck Molecular Instruments, Inc.

[0282] Imaging and image analysis

[0283] All fish were imaged on a Zeiss LSM 700 confocal microscope with a 40x oil immersion objective. For imaging of live larvae, animals 4 or 5 days post fertilization were anesthetized in 0.01% tricaine (w / v) and embedded in 1% low melting point agarose (w / v, in E3 culture medium supplemented with 0.003% PTU). For imaging of Tg[mbp:eGFPCAAX] zebrafish, images of the spinal cord and PLLn were obtained at the level of the first, third, and seventh motor nerves distal to the urogenital opening. For live imaging after PLLn injury, images were taken every 10 minutes for up to 26 hours, and the embryos were maintained at 28°C. The survival of the fish was monitored by observing heartbeat and blood circulation. Confocal images are shown as maximum intensity projections of grids spliced ​​using Fiji (Preibisch et al., 2009; Schindelin et al., 2012). For fluorescence intensity measurements of Tg (mbp: eGFPCAAX) fish, integrated density was obtained from the region of interest. For mbp HCR quantification, images of the PLLn and spinal cord were obtained at the level of the first, third, and seventh motor nerves distal to the urogenital opening. In order to statistically encode the colors by the intensity of the mbp signal, nuclear segmentation was performed using a surface mask around the DAPI dye in Imaris (Bitplane), with a surface detail of 0.22 μm and a seed size of 4 μm for the contact surface. For signal intensity analysis, the surface of the spinal cord and posterior lateral line was drawn in Imaris (Bitplane), and the sum of the intensities of both mbp and DAPI signals was determined. The intensity was then normalized by surface area. For all experiments, seven to nine fish were imaged for each genotype (spinal cord and one PLLn).

[0284] Antibody

[0285] Immunofluorescence: SOX10 (R&D Systems, 1:100, AF2864, RRID: AB_442208), donkey anti-goat IgG (H+L) Alexa Fluor 488 (Invitrogen, 1:1000, A11057), anti-βIII tubulin (Sigma Aldrich, 1:1000, AB9354, RRID: AB_570918), NF200 (Abcam, 1:1000, ab72997, RRID: AB_1267598), SARM1 rabbit polyclonal antibody (kindly provided by Professor Hsueh, 1:500) purchased from Abcam (Ab226930, 1:2000, RRID: AB_2893433), donkey anti-goat IgG (H+L) Alexa Fluor 488 (Invitrogen, 1:1000, A11057), anti-βIII tubulin (Sigma Aldrich, 1:1000, AB9354, RRID: AB_570918), NF200 (Abcam, 1:1000, ab72997, RRID: AB_1267598), SARM1 rabbit polyclonal antibody (kindly provided by Professor Hsueh, 1:500) purchased from Abcam (Ab226930, 1:2000, RRID: AB_2893433), Fluor488 (Invitrogen, 1:1000, A11057), Cy3 donkey anti-rabbit IgG (H+L) (Jackson Immunoresearch, 1:500, 711-165-152), donkey anti-mouse IgG (H+L) highly cross-adsorbed Seco Alexa Fluor 488 (Invitrogen, 1:1000, A-21202), donkey anti-chicken IgY, Alexa Fluor 647 (Merck, 1:1000, 15389818), DAPI (Thermo scientific, 1:2000, 62248).

[0286] Western blot: Calnexin (Enzo Life Sciences, 1:1000, ADI-SPA-860-D, RRID: AB_312058), JUN (Cell Signaling Technology, 1:1000, 9165, RRID: AB_2130165), EGR2 (EMD Millipore, 1:500, ABE1374, RRID: AB_2715555), MPZ (Aves Laboratories, 1:500, ABE1374, RRID: AB_2715555), Labs), 1:2000, PZO, RRID: AB_2313561), MBP (EMD Millipore, 1:1000, AB9348, RRID: AB_2140366), SARM1 rabbit polyclonal and mouse monoclonal (kindly provided by Professor Hsueh, 1:500), β-actin (Santa Cruz, 1:3000, sc-47778HRP, RRID: AB_2714189), anti-mouse IgG HRP-linked antibody (Cell Signaling Technology, 1:2000, 7076S), anti-rabbit HRP-linked antibody (Cell Signaling Technology, 1:2000, 7074S), goat pAb anti-chicken IgYH+L (HRP) (Abcam, 1:2000, ab97135).

[0287] Immunofluorescence

[0288] Sciatic and optic nerves were dissected from Wt and Sarm1 KO mice and directly embedded in OCT compound without fixation and stored at −80°C until ready for cryosectioning. 5-μm-thick cryosections were collected onto SuperFrost plus slides. These slides were postfixed in 4% paraformaldehyde (PFA) for 10 minutes at room temperature and then washed three times with 1xPBS for 5 minutes each. The slides were then immersed in 100% methanol at −20°C for 10 minutes and then washed three times with 1xPBS for 5 minutes each. The slides were then blocked in 5% horse serum (HS) / 0.2% Triton X-100 / PBS for 30 minutes at room temperature. Primary antibodies were diluted in blocking solution, and the slides were incubated overnight at 4°C. The next day, the slides were washed once with 1XPBS for 5 minutes, washed twice with 0.1% tween / PBS for 5 minutes each time, and finally washed again with 1XPBS for 5 minutes before adding the secondary antibody. The corresponding secondary antibody was diluted at 1:500 and DAPI was diluted at 1:2000 at room temperature for 2 hours and stored in the dark. The slides were then washed once with 1XPBS for 5 minutes, washed twice with 0.1% tween / PBS for 5 minutes each time, and finally washed again with 1XPBS for 5 minutes, then sealed with Fluorsave (Calbiochem, 345789) and representative images were taken using a Leica DMI6000B at 40 times, 63 times, and 100 times. Immunofluorescence staining in rat oligodendrocyte cultures followed the same protocol and representative images were taken at 20 times using a Leica DMI8.

[0289] Western blotting

[0290] Homogenates were obtained from the sciatic or optic nerves. These lysates were prepared as previously described (Gomez-Sanchez et al., 2015). The samples were lysed in ice-cold RIPA buffer with Halt TMThe cells were homogenized with a protease inhibitor cocktail (100X) (ThermoFisher Scientific 78429). After homogenization, the samples were incubated on ice for 30 minutes for further lysis. The resulting lysate was centrifuged at 12,000 rpm at 4°C, and the supernatant was used for bicinchoninic acid (BCA) assay (ThermoFisher, 23227). The supernatant was diluted in loading buffer and boiled at 95°C for 5 minutes. For Western blot analysis of SARM1, 20 ug of protein was loaded, for JUN and EGR2, 15 μg of protein was loaded, and for analysis of myelin proteins MPZ and MBP, 7.5 μg of protein was loaded. Next, the samples were run in SDS-PAGE and transferred to a PVDF membrane. After the membrane was blocked for 1 hour in 5% skim milk (diluted in TBS / 0.1% Tween), the membrane was incubated overnight at 4°C with the listed primary antibodies. The experiment was repeated at least three times with fresh samples, and representative photographs are shown. Densitometric quantification was performed using ImageJ. Measurements were normalized to the loading control calnexin or β-actin.

[0291] RNA extraction and qPCR

[0292] RNA was extracted from P60 tibial nerves or optic nerves using Trizol (Invitrogen, 15596026) as previously described (Arthur-Farraj et al., 2017). For each biological replicate, nerves from two mice were pooled. The integrity and quantity of RNA were determined using Nanodrop (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer (Agilent Technologies). 500 ng of RNA was used for cDNA conversion per biological replicate using the QuantiTect Reverse Transcription Kit (Qiagen, 205311). qPCR was run on a BioRad CFX96 using iTaq and SYBR Green (BioRad, 1725124). Ankrd27 and Canx were used as housekeeping genes. Two technical replicates and five biological replicates were run for each experiment. The fold change was calculated using the ΔCT method. All primers were designed using Primer blast (NCBI) ( Ye et al., 2012 ). Table 1 - Primer sequences

[0293] Electron microscopy

[0294] Sciatic and optic nerves were processed as previously described (Gomez-Sanchez et al., 2015) (Gomez-Sanchez et al., 2015). Briefly, samples were fixed overnight at 4°C in 2.5% glutaraldehyde / 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4). Samples were postfixed with 1% OsO4 and embedded in Agar 100 epoxy resin. Transverse ultrathin sections were taken from neonatal (P2) and adult (P60) sciatic nerves (5 mm from the notch) or from adult (P60) optic nerves (2 mm from the chiasm) and mounted on membrane.

[0295] Photos were taken using a Jeol 1010 electron microscope with a Gatan camera and software. Images were analyzed using ImageJ. Photos of the sciatic nerve were taken at 3000x magnification to measure the number of myelinated axons, unmyelinated axons greater than 1.5 μm, and Schwann cell nuclei. Nerve area was measured based on photos taken at 200x magnification. Photos of the optic nerve were taken at 12000x magnification to measure the number of myelinated and unmyelinated axons. Nerve area was measured based on photos taken at 200x magnification. For all experiments, N=5 (5 biological replicates: 1 nerve per animal, 5 animals, 15-20 sections per nerve).

[0296] Statistical analysis:

[0297] Statistical analysis was performed using Graph-Pad Prism software (version 9.1.2). Results are expressed as mean ± SEM. Statistical significance was estimated by Mann-Whitney U test or unpaired two-tailed Student's t-test, with Bonferroni correction for multiple testing when necessary. P < 0.05 was considered statistically significant. The N numbers for all experiments are listed in the figure legends. The assessors were blinded to all quantifications.

[0298] result

[0299] Sarm1 / sarm1 mRNA expression in vertebrate Schwann cells, satellite glia, and oligodendrocytes Protein detection.

[0300] To investigate the presence of SARM1 protein in various cell types of the murine nervous system, a validated polyclonal antibody generated by C.-Y. Chen et al., 2011 was used in transverse cryosections of adult (P60) tibial and optic nerves. As expected, SARM1 expression was found to colocalize with the axonal markers neurofilaments and β-III tubulin in the sciatic and optic nerves, respectively ( Figure 3A and B). In addition, the study found that SARM1 protein is prevalent at high levels in the cell bodies of large and small dorsal root ganglia (DRG) neurons in vivo, but at lower levels in their axons ( Figure 3 C). Importantly, we observed no staining in control sections without primary antibody and in Sarm1 knockout (KO) DRG neurons ( Figure 4 A and B). To test whether detectable levels of SARM1 protein were present in myelinating and non-myelinating Schwann cells, satellite glia, and oligodendrocytes, immunohistochemistry for SARM1 and SOX10 was performed in frozen sections from tibial nerves, DRGs, and optic nerves, as well as in PNS tissues. No colocalization was found in multiple sections from three separate non-littermates ( Figure 3 However, in optic nerve sections, SARM1 protein appeared to be present in a perinuclear staining pattern around the nuclei of SOX10-positive cells ( Figure 3 F) Thus, SARM1 protein appears to be restricted to neuronal populations in the PNS and is undetectable in adult rat Schwann cells and satellite glia. However, in the CNS, SARM1 protein may be present in oligodendrocytes.

[0301] To identify whether sarm1 mRNA is expressed in myelinating glial cells, a third-generation in situ hybridization chain reaction (HCR) was used in five-day postfertilization (dpf) zebrafish larvae, where oligodendrocytes and Schwann cells in the CNS and PNS, respectively, initiate myelination (D'Rozario et al., 2017; Choi et al., 2018). HCR probes targeting sarm1 and sox10 were used to label cells of the oligodendrocyte and Schwann cell lineages. In the posterior lateral line nerve (PLLn), strong expression of sox10 was observed, but sarm1 expression was minimal, but importantly, no sarm1 signal was observed when the antisense probe was omitted ( Figure 5 A; Figure 6 In the spinal cord, while sox10 expression was observed to be primarily concentrated in the ventral and dorsal spinal tracts, sarm1 expression was visualized more diffusely throughout the spinal cord, although without much overlap with the sox10 signal ( Figure 5 B). Some overlap between sox10 and sarm1 is expected, as sox10 is also expressed in a subset of spinal cord interneurons (Almeida and Lyons, 2015). Interestingly, no substantial overlap in sox10 and sarm1 expression was found in two other regions of the nervous system: the hypothalamus and retina ( Figure 6C and D). To investigate whether sarm1 and sox10 are co-expressed in the same cells in the PNS and CNS, single confocal sections (616 nm optical depth) were examined and found that co-expression of both markers was rare only in nuclei along the PLLn. Specifically, in the PLLn, we found that 22.4% + / - 3.161 (n = 5 larvae, 5 sections per larvae) of Schwann cells expressed low levels of sarm1 ( Figure 5 C).

[0302] Taken together, these results are consistent with the view that Schwann cells and oligodendrocytes do express low levels of Sarm1 mRNA, but only oligodendrocytes may have detectable levels of SARM1 protein.

[0303] Cultured oligodendrocytes, but not Schwann cells, have detectable SARM1 protein and are prone to Specific, SARM1 activator-induced cell death. Given the observed expression of sarm1 mRNA in Schwann cells and oligodendrocytes in zebrafish, it was decided to test whether mouse Schwann cells also express Sarm1 mRNA. Although Sarm1 mRNA was detected in cultured mouse Schwann cells, it was not detected in Sarm1 KO Schwann cells, but its levels were significantly lower than those in cultured mouse DRG neurons ( Figure 7 A and B).

[0304] Since SARM1 protein could not be identified in Schwann cells, but SARM1 could potentially be detected in cells of the oligodendrocyte lineage in vivo, we decided to further explore whether these cells might contain small amounts of functional SARM1 protein. To test this hypothesis, cultures of freshly isolated mouse Schwann cells and rat oligodendrocytes were treated with two separate SARM1 activators, vacor and 3-AP. Addition of high doses of vacor or 3-AP to neuronal cultures induced rapid SARM1 activation within hours, severe nicotinamide adenine dinucleotide (NAD + ) depletion and axonal degeneration and cell death (Loreto et al., 2021; Wu et al., 2021). Studies have found that mouse Schwann cells are completely insensitive to high-dose vacor and 3-AP treatment for a long time (up to 72 hours) ( Figure 8 A and Figure 9 A) To further support this, Schwann cells treated with vacor or 3-AP for 72 hours showed that intracellular NAD + The levels did not decrease, which is different from human embryonic kidney cells, which express low levels of SARM1 protein ( Figure 8 B and Figure 9B) In contrast, the study found that oligodendrocytes were sensitive to vacor addition and died within hours, as determined by propidium iodide staining and nuclear labeling with SOX10 and DAPI ( Figure 8 C and D). Oligodendrocyte cultures were then stained and, compared to mouse Schwann cells, detectable levels of SARM1 were found using two different antibodies ( Figure 8 E and Figure 7 C and D). Thus, in contrast to Schwann cells, cultured oligodendrocytes appear to contain sufficient endogenous SARM1 protein to induce cell death.

[0305] Initiation of CNS and PNS myelination proceeds normally in Sarm1 mutant zebrafish larvae

[0306] Since inhibition of SARM1 function has been proposed to treat neurological disorders, it is crucial to determine whether loss of SARM1 (cell or non-cell autonomous) leads to any disruption of myelination in the vertebrate CNS or PNS. This question is particularly relevant given that functional SARM1 protein has been identified in oligodendrocytes. To date, no studies have performed in-depth quantitative analyses of CNS and PNS myelination in the absence of SARM1 function. To test whether SARM1 is required for the proper initiation of myelination in zebrafish, we assessed the expression of SARM1 in wild-type (wt) and sarm1 zebrafish at 5 dpf. SA11193 / SA11193 Myelination and myelin gene expression in the spinal cord and PLLn of juvenile (mutant) sarm1 SA11193 The mutant fish has a point mutation (C>A) in exon 2 that introduces a premature stop codon (Busch-Nentwich et al., 2013). SA11193 The mutants were viable, fertile, and morphologically indistinguishable from wt fish (data not shown). To confirm that sarm1 mutants functionally behaved like sarm1 null animals (which had significantly delayed axonal degeneration after traumatic injury), wt and homozygous mutant fish were injected with neuroD:tdTomato DNA constructs at the one-cell zygote stage. Two-photon laser axotomy was performed on tdTomato-labeled PLLn neurons at 4 dpf, and axons distal to the injury site were imaged live ( Figure 10 A). We found that while wt axons began to degenerate between 2 hours 40 minutes and 3 hours 20 minutes (n=7), axons in sarm1 mutant fish remained intact even 26 hours after axotomy, a time limit imposed by UK Home Office regulations for our imaging capabilities ( Figure 10 B). This confirms that sarm1 SA11193The mutant zebrafish phenotypically resemble Sarm1-null mice and are similar to sarm1-null mutant zebrafish generated by Crispr-Cas9 (Tian et al., 2020).

[0307] To assess PNS and CNS myelination, sarm1 SA11193 The mutants were crossed with Tg(mbp:EGFP-CAAX) zebrafish, which express membrane-bound GFP under the myelin basic protein (mbp) promoter (Almeida et al., 2011). SA11193 Normal formation of long GFP-labeled myelin segments was observed in the dorsal and ventral spinal cords and PLLn in both mutant and wt fish, and quantitative results showed no difference in myelination between the PLLn and spinal cord ( Figure 11 AF).

[0308] To quantify myelin gene expression between wt and sarm1 mutant zebrafish, mbp HCR was performed. SA11193 Equivalent levels of mbp expression were present in the spinal cord and PLLn of mutant fish, and importantly, the number of cells in either structure did not differ between genotypes ( Figure 11 GM).

[0309] Collectively, these experiments demonstrate that myelination proceeds normally in both the PLLn and spinal cord of zebrafish larvae in the absence of Sarm1 function.

[0310] PNS myelination and myelin maintenance are normal in Sarm1KO mice

[0311] Given that zebrafish embryos appear to myelinate normally in the absence of functional Sarm1, we next tested whether PNS myelination is properly initiated in Sarm1KO mice. PNS myelination begins in murine peripheral nerves shortly after birth (Jessen and Mirsky, 2005). Postnatal (P) day 2 transverse sections through tibial nerves were examined by transmission electron microscopy (EM) and noted that, while there was a nonsignificant trend toward slightly more axons and Schwann cells in Sarm1KO nerves compared to Wt (n=5), there was no difference in the proportion of myelinated large-caliber axons ( Figure 12 AH).

[0312] Adult (P60) tibial nerves were then examined by EM to investigate whether myelin maintenance is affected by the loss of Sarm1 in the PNS. Interestingly, at P60, the trend toward increased axon number was no longer seen in Sarm1 KO ( Figure 12JM). In addition, the proportions of unmyelinated and myelinated axons, Schwann cell nuclei, and myelin thickness (measured by g-ratio and nerve area) were similar between Wt and Sarm1 KO samples ( Figure 12 NR).

[0313] To test whether there were any gene expression differences between Wt and Sarm1 KO tibial nerves, gene and protein expression were visualized by quantitative reverse transcriptase polymerase chain reaction (qPCR) and Western blotting, respectively. Sarm1 KO nerves showed no differences in the expression of myelin genes or other Schwann cell-specific genes (Cdh1, Egr2, Mbp, Mpz, Sox10), immature or Schwann cell injury gene expression (Fos, Jun, Sox2), or cytokine or chemokine expression (Ccl2, Ccl3, Ccl4, Ccl5, Il1b, IL6, IL10). However, Sarm1 KO tibial nerves were detected to express twofold higher levels of the pro-apoptotic gene Xaf1, which has also been reported to be upregulated in mouse brains and macrophages from this particular Sarm1 KO mouse line ( Figure 13 A; Uccellini et al., 2020; Zhu et al., 2019). In addition, the study found no difference in the protein levels of EGR2 / KROX-20, JUN, and myelin proteins MPZ and MBP ( Figure 13 BG).

[0314] Thus, in the absence of Sarm1 in mice, myelination and myelin maintenance are unperturbed, and myelin gene and protein expression is normal in the PNS.

[0315] CNS myelination and myelin gene expression are normal in Sarm1KO mice

[0316] The results showed that oligodendrocytes in the dorsal and ventral spinal cords of Sarm1 mutant zebrafish larvae myelinated normally. To confirm whether CNS myelination was also unaffected in Sarm1 KO mice, adult P60 optic nerves were evaluated by EM. The study found that Sarm1 KO optic nerves were morphologically indistinguishable from Wt samples, with similar total axon numbers, ratios of myelinated to unmyelinated axons, and similar cross-sectional nerve areas ( Figure 14 AF). Furthermore, by qPCR, Sarm1 KO optic nerves showed no differences in gene expression of oligodendrocyte / myelin markers (Mbp, Olig2, Plp1, Sox10), cytokine or chemokine expression (Ccl2, Ccl3, Ccl4, Ccl5, IL1b, IL6), except for upregulation of IL10 ( Figure 14G). Interestingly, despite the significant difference in Sarm1 mRNA expression between WT and KO samples, as expected, no upregulation of Xaf1 was detected in the optic nerve ( Figure 14 G). MBP protein levels were also tested in Sarm1 KO optic nerves by western blotting and found no difference compared to WT nerves ( Figure 14 H).

[0317] In conclusion, there are no observable myelin defects in the optic nerves of adult Sarm1 KO mice.

[0318] discuss

[0319] Numerous studies investigating the role of SARM1 in the nervous system have demonstrated that PNS and CNS myelin appear normal in adult Sarm1 knockout mice. However, no in-depth quantitative studies of PNS and CNS myelination have been conducted to confirm or refute this finding (Osterloh et al., 2012; Geisler et al., 2016; Marion et al., 2019; Ko et al., 2020). Furthermore, one study generated Crispr-Cas9 sarm1 mutant zebrafish, but they did not address whether PNS or CNS myelination was normal (Tian et al., 2020). Furthermore, no studies have investigated whether SARM1 is present in myelinating glial cells in the PNS and CNS.

[0320] This study demonstrates that sarm1 mRNA is expressed at low levels in Schwann cells and oligodendrocytes of developing zebrafish larvae, and that cultured mouse Schwann cells also express Sarm1 mRNA. However, SARM1 protein was detected in oligodendrocytes, but not in Schwann cells, of the mature mouse nervous system. It also demonstrates that in the absence of Sarm1 / SARM1, PNS and CNS myelination proceeds normally in both zebrafish and mice, and that SARM1 plays no role in myelin maintenance in the murine PNS or CNS. This complements previous findings that conduction velocities measured by neurophysiological studies of the sciatic nerves of adult Sarm1 KO mice are similar to those of control mice, and that adult Sarm1 KO mice have normal numbers of myelinated axons in the corpus callosum (Geisler et al., 2016; Marion et al., 2019).

[0321] Unlike other members of the MyD88 family, SARM1 is highly abundant in the murine nervous system, particularly in neurons (Kim et al., 2007; Chen et al., 2011). A previous study demonstrated that SARM1 protein was not found in mouse microglia (Lin et al., 2014), and this paper shows that SARM1 protein is similarly present in oligodendrocytes in vivo, but not in myelinating and non-myelinating Schwann cells. Interestingly, a recent study showed that SARM1 protein is detectable in mouse astrocytes and appears to play a role in neuroinflammation (Liu et al., 2021). In the PNS, in addition to Schwann cells, this paper also shows that satellite glia do not appear to have identifiable levels of SARM1 protein. SARM1 may also not be present at high levels in PNS-resident macrophages, as studies of other peripheral macrophage populations have found relatively low levels of SARM1 in these cells, and Sarm1 loss has no effect on macrophage function or gene expression (Kim et al., 2007; Uccellini et al., 2020).

[0322] It has been hypothesized that one of the purposes of SARM1 (a toll-like adaptor protein and remnant of the innate immune system) and the broader axonal degeneration machinery is to generate compartmentalized neurodegeneration to prevent the spread of viral pathogens throughout the nervous system (Tsunoda, 2008). In mouse CNS myelinating co-cultures, loss of Sarm1 protects neuronal cell bodies from infection and cell death (Crawford et al., 2022). Zika virus preferentially infects oligodendrocytes and astrocytes in neuron / glial co-cultures and causes glial cell death (Cumberworth et al., 2017). Activation of SARM1 is known to cause neuronal cell body death independent of axonal degeneration (Sasaki et al., 2020; Loreto et al., 2021). It is currently unknown whether CNS glial cells upregulate Sarm1 expression in response to Zika infection and whether glial cell death is SARM1-dependent. Interestingly, astrocytes do appear to upregulate SARM1 expression in response to spinal cord injury (Liu et al., 2021).

[0323] In summary, our findings suggest that the use of Sarm1 mutant mice and sarm1 mutant zebrafish to model axonopathy in various disease models is a feasible approach, as myelination is unlikely to be perturbed by Sarm1 loss. Example 2

[0324] Materials and Methods

[0325] Human primary myoblasts and human dermal skin fibroblast culture, treatment, and cell death assay

[0326] Human primary myoblasts and human dermal fibroblasts were cultured according to previously described protocols (Kisiel and Klar 2019; Rozwadowska et al. 2022). Drug treatment was initiated when cells reached the desired confluence. To visualize cell death, 1 μg / ml propidium iodide (PI) (Thermo Fisher Scientific) was added to the culture medium at the same time as the drug addition (Loreto et al. 2021). Phase contrast and fluorescence microscopy images were acquired on a DMi8 upright fluorescence microscope (Leica microsystems) connected to a monochrome digital camera (Hamamatsu C4742-95). The objective lens used was HCXPL 20X / 0.40CORR.

[0327] Axonal degeneration and regeneration assays in human DRG

[0328] Human fetal DRG ganglia were dissected, dissociated, and plated in microfluidic chambers (150 μm barrier, XONA Microfluidics) in 35 mm tissue culture dishes pre-coated with poly-L-lysine (100 μg / ml for 1 hour; Merck) and laminin (20 μg / ml for 1 hour; Merck) in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 1% penicillin / streptomycin, 33 ng / ml 2.5S NGF (both from Invitrogen), and 2% B27 (Gibco). 4 μM aphidicolin (Merck) was used to reduce the proliferation and viability of a small number of non-neuronal cells. At DIV7, a 100 μl difference in culture medium was introduced between the chambers, and drugs were added to the compartment with lower hydrostatic pressure. Phase contrast microscopy images were acquired on a DMi8 upright fluorescence microscope (Leica Microsystems) coupled to a monochrome digital camera (Hamamatsu C4742-95). The objective lens used was HCXPL20X / 0.40CORR.

[0329] result

[0330] To test whether human muscle and skin cells express any functional SARM1 protein, cultures of human primary myoblasts (muscle cells) and human dermal skin fibroblasts (skin cells) were treated with the SARM1 agent vacor. The study found that human myoblasts and human dermal skin fibroblasts were completely insensitive to cell death induced by high doses of vacor (100 μM), as demonstrated by propidium iodide staining ( Figure 15 、 17These findings indicate that SARM1 protein is absent or present at functionally negligible levels in human myoblasts (muscle cells) and human dermal skin fibroblasts (skin cells). Next, we tested whether human axons could regenerate after local axonal degeneration caused by the SARM1 agent vacor. To this end, we cultured human fetal DRG axons in microfluidic chambers to allow local manipulation of axons ( Figure 18 24 hours after adding 25 μM Vacor to the axonal compartment, DRG axons degenerated, while untreated neuronal cell bodies remained healthy. Then, 24 hours after treatment, when axons had already degenerated, Vacor was removed from the axonal compartment and fresh culture medium was added to the axonal compartment. Following the addition of fresh culture medium, DRG axons began to regrow and completely repopulated the axonal compartment within 120 hours after Vacor removal. This demonstrates that human neurons can regenerate their axons after degeneration caused by topically applied SARM1 agents.

[0331] discuss

[0332] In order for SARM1 agents to have a temporary, reversible, and selective effect, it is important that these agents cause selective nerve terminal degeneration in SARM1-expressing cells (such as neurons) in the area where they are administered, rather than in surrounding cells in the application site (which do not express SARM1 or express functionally negligible levels of SARM1), such as skin cells, muscle cells, and Schwann cells. It is also important that neurons can regenerate their axons to avoid permanent disability after treatment with SARM1 agents. First, it was previously unclear whether SARM1 is present at functional levels in non-neuronal cells of the PNS and cells of target organs innervated by the PNS (i.e., skin and muscle cells). As discussed in Example 3 and the data presented herein, it is important to note that one cannot simply assume from previous studies and publications that SARM1 is present selectively in neurons. Here, it is shown for the first time that human myoblasts (muscle cells) and human dermal skin fibroblasts (skin cells) are insensitive to the SARM1 agent vacor. These findings further support the proposal to use SARM1 agents as a cell-selective approach for peripheral nerve ablation to treat conditions such as dystonia, spasticity, peripheral neuropathic pain, autonomic dysfunction, and cosmetic applications.

[0333] Furthermore, it was shown that after topical application of the SARM1 agent vacor, human DRG axons degenerated, even at doses lower than those used for other cell types (such as muscle cells, skin cells, and Schwann cells). This is the first time that axons have been shown to regenerate after degeneration caused by topical application of SARM1 agents (such as vacor). In addition to the finding that Schwann cells are insensitive to SARM1 agents (such as vacor), these findings further support the use of SARM1 agents as a temporary, reversible, and cell-selective method for peripheral nerve ablation, thereby allowing the regeneration of axons and terminal branches to treat conditions such as dystonia, spasticity, peripheral neuropathic pain, autonomic dysfunction, and for cosmetic conditions. It is important to note that these experiments were performed on primary human cells, which further supports the feasibility of the proposed use of SARM1 agents. Example 3

[0334] Materials and Methods

[0335] Sciatic nerve transection surgery

[0336] All studies were conducted in accordance with the Animals (Scientific Procedures) Act 1986, the Animal Welfare and Ethical Review Body (AWERB) of the University of Cambridge, and European Union guidelines, and the protocol was approved by the Bioethics and Biosafety Committee of the Institute of Neuroscience of Alicante, the Miguel Hernández University of Elche, and the Spanish National Research Council (http: / / in.umh-csic.es / ). Wild-type (Wt) C57BL / 6J mice were obtained from Charles River Laboratories or maintained as wild-type colonies. Surgery was performed on 6-8 week old wild-type or Sarm1 knockout mice. Mice were anesthetized with 2% isoflurane, the sciatic nerve was exposed, and an incision was made at the sciatic notch (Arthur-Farraj et al., 2012). The wound was closed using a veterinary automated clamp (AutoClip System). Nerves were harvested after 3, 6, 9, 12, 18, or 24 hours, or after 2, 5, 7, or 14 days, and the contralateral uninjured nerve was used as a control.

[0337] Mouse Schwann cell expansion

[0338] Mouse SCs were passaged on PLL / laminin-coated dishes in the presence of low serum (0.5% horse serum), 10 ng / ml β-neuregulin 1 (βNRG1), and a low concentration of cyclic adenosine monophosphate (cAMP)-elevating signal (such as 2 μM forskolin or 100 μM dibutyl cAMP) (Arthur-Farraj et al., 2011). The full protocol is described in (Mutschler et al., 2023).

[0339] All other relevant methods are described in Example 1.

[0340] result

[0341] In Example 1, freshly isolated Schwann cells from the sciatic nerves of newborn mice were used to test the sensitivity of Schwann cells to the SARM1 agents vacor and 3-AP. The study found that Schwann cells were completely insensitive to the SARM1 agonists ( Figure 7 、 8 and 9). These findings are consistent with the finding that SARM1 protein is not present in mouse Schwann cells in uninjured sciatic nerves and that sarm1 mRNA is expressed at very low levels only in a subset of Schwann cells in the zebrafish PLLn ( Figure 3 and 5 Early in the study, experiments were also conducted using expanded mouse Schwann cells that had been passaged three times in cell culture over a 2-week period after extraction from mouse nerves. When the expanded mouse Schwann cells were treated with the same concentrations of vacor or 3-AP as used in Example 1, the expanded mouse Schwann cells were found to be susceptible to cell dysfunction and death, as demonstrated by propidium iodide staining ( Figure 19 Given that cultured Schwann cells can replicate damaged Schwann cells in vivo or repair Schwann cell markers, it was desirable to test whether Schwann cells contain functional SARM1 protein after nerve injury (Arthur-Farraj et al., 2017). At different time points after nerve injury (24 and 48 hours and 7 days), sections of injured mouse tibial nerves were immunostained. Colocalization of SARM1 protein with the Schwann cell marker SOX10 was not observed, but colocalization of SARM1 with the axonal protein neurofilament light chain was observed ( Figure 20 These findings indicate that Schwann cells do not contain detectable SARM1 protein at various time points after nerve injury. However, after prolonged passage, cultured Schwann cells do become sensitive to SARM1 agents.

[0342] discuss

[0343] When axons degenerate, Schwann cells are activated by injury and transform into repair Schwann cells (Arthur-Farraj and Coleman, 2021). This injury-induced Schwann cell transformation occurs when SARM1 agents are applied to peripheral neural structures and target organs such as skin and muscle, so it was important to test whether Schwann cells alter their SARM1 expression profile in response to injury. The finding that Schwann cells in vivo do not contain detectable levels of SARM1 protein at different time points after nerve injury is consistent with the absence of SARM1 in Schwann cells from intact nerves and freshly isolated Schwann cells in culture. This finding further supports the use of SARM1 agents as a cell-selective approach for peripheral nerve ablation to treat conditions such as dystonia, spasticity, peripheral neuropathic pain, and autonomic dysfunction, as well as for cosmetic applications. The finding that prolonged passage of cultured mouse Schwann cells sensitizes them to vacor- and 3-AP-induced cell death is surprising and inconsistent with all other observations. However, long-term passage in cell culture is largely artificial, and studies have shown that Schwann cells may lose their phenotypic hallmarks and alter the expression of key markers, such as downregulation of JUN (Wagstaff et al., 2021). In addition, long-term passage of cells leads to the development of multiple somatic mutations (Rouhani et al., 2022). This finding may represent an artifact of long-term cell culture, and this is exemplified by the observation that freshly isolated Schwann cells cultured for only 24 hours were insensitive to SARM1 agents. It is important to note that this result does highlight that it is not possible to simply assume from previous studies that SARM1 is selectively present in neurons and not in other cell types, because this article surprisingly shows that, first, CNS myelinating glial cells contain functional SARM1, and second, Schwann cells do not contain functional SARM1, but may become sensitive to SARM1 agents in artificial situations (such as long-term cell culture). Importantly, the vast majority of in vitro Schwann cell experiments in the field are performed on Schwann cells that have been passaged several times. Therefore, the insensitivity of Schwann cells to SARM1 agents was only discovered when an unconventional approach was taken, using short-term Schwann cell cultures to maintain them in a physiological state closer to that in vivo. References

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Claims

1. A SARM1 agent for use as a medicine.

2. A SARM1 agent for use in a method of treating and / or preventing neurological, ophthalmic, dermatological, gastroenterological, colorectal, urological, gynecological, rheumatological, orthopedic, dental and / or otolaryngological diseases.

3. The SARM1 agent for use according to claim 2, wherein the disease is defined by autonomic dysfunction, neuropathic pain, dystonia, spasticity, movement disorders, sphincter dysfunction, urogenital dysfunction and / or skin conditions.

4. The SARM1 agent for use according to claim 2 or 3, wherein: (i) Autonomic dysfunction includes: Excessive sweating (hyperhidrosis) and / or excessive saliva production (sialorrhea), Autonomic dysfunction caused by: Hereditary and / or genetic neurological diseases, including: Hereditary autonomic neuropathy, sensory autonomic neuropathy; and / or Degenerative neurological diseases, including: Amyotrophic lateral sclerosis, Parkinson's disease, Parkinson's syndrome, multiple system atrophy, cerebral palsy and / or degenerative neurological disorders resulting from injury; and / or Autonomic nervous system disorders caused by acquired conditions (secondary autonomic disorders), including: Infections (COVID and long COVID), autoimmune conditions, postural orthostatic tachycardia syndrome (POTS), endocrine conditions including Diabetes and / or thyroid dysfunction; and / or Small fiber neuropathy; and / or Connective tissue disease; and / or Amyloidosis; and / or Peripheral nerve damage; and / or Surgical repair of peripheral nerve injuries and / or pharmaceuticals; and / or Idiopathic dysautonomia; and / or (ii) Neuropathic pain includes: Peripheral neuropathic pain; chronic back pain; cancer-related pain; myofascial pain; fibromyalgia; complex regional pain disorder; chronic pelvic pain; dyspareunia; vulvodynia; and / or painful bladder syndrome (interstitial cystitis); and / or Neuropathic pain caused by: diabetic neuropathy; neuralgia following peripheral nerve injury; infectious and post-infectious neuralgia; metabolic neuropathy; small fiber neuropathy; endocrine conditions; genetic neuropathy; paraproteinemic neuropathy; paraneoplastic neuropathy; tumor neuropathy; vasculitis; autoimmune, inflammatory, and demyelinating neuropathies; idiopathic neuropathies, peripheral nerve injury, and / or surgical repair of peripheral nerve injury; and / or (iii) Dystonia includes: Primary dystonia; Dystonia superimposed symptoms; Dystonia occurring in conjunction with a neurodegenerative disorder; and / or Secondary dystonia occurs due to: Central nervous system (CNS) trauma, congenital malformations, genetic and chromosomal disorders, infection, tumors, ischemic; hemorrhagic stroke, inflammation, demyelination, drugs, toxins and / or metabolic disorders; and / or (iv) Spasticity includes Upper limb spasticity, lower limb spasticity; bladder spasm / neurogenic bladder; spastic esophageal disorders; and / or Cramps caused by: Demyelinating conditions; congenital malformations; chromosomal disorders; genetic conditions; CNS infections; tumors; drugs; toxins; metabolic disorders; paraneoplastic conditions; post-infectious conditions; autoimmune conditions; endocrine conditions, peripheral nerve injury and / or surgical repair of peripheral nerve injury; and / or (v) Movement disorders include: Hemifacial spasm with facial nerve synkinesis; palatal myoclonus / tremor; and / or Movement disorders caused by: Tics; tremors; myokymia, trauma, infection, autoimmune conditions; neuromyotonia caused by autoimmune and / or genetic conditions; and / or (vi) Sphincter dysfunction caused by: Dysfunction of the body's sphincters; pyloric sphincter; sphincter of Oddi dysfunction leading to gastrointestinal dysmotility; pancreatic sphincter; urethral sphincter; anal fissure; and / or Hirschsprung disease; (vii) Urogenital dysfunction includes: Vaginismus; and / or urinary dysfunction; and / or (viii) Skin conditions include: ulcers; acne; and / or Raynaud's phenomenon / disease; (ix) Orthopedic diseases include: Sports injuries; post-traumatic elbow stiffness; clubfoot; and / or piriformis syndrome; and / or (x) Dental diseases include: Toothache; and / or root canal treatment.

5. A non-therapeutic method for: (i) Removal and / or prevention of skin changes; (ii) contouring; and / or (iii) Improve oily skin; The method comprises administering a SARM1 agent.

6. The non-therapeutic method of claim 5, wherein the skin changes comprise one or more of skin wrinkles, frown lines / glabellar lines, forehead lines, lateral canthal lines / crow's feet, transverse nasal bridge lines, neck bands, platysmal neck lines, smoker's lines, bunny lines, upper lip lines, lateral commissure ptosis, marionette lines, gummy smile, chin dimples, cellulite chin, sunken skin, drooping eyebrows, drooping eyelids, lip drooping, square jaw, and downturned nasal tip.

7. A non-therapeutic method according to claim 5 or 6, wherein contouring comprises (i) lip, cheek, jaw and / or temple shaping; and / or (ii) eyebrow contouring.

8. The SARM1 agent for use according to any one of claims 1-4 or the method according to claims 6-7, wherein the SARM1 agent: The SARM1 agent activates SARM1 and causes neurodegeneration and / or neuronal dysfunction; and / or Causes SARM1-dependent neurodegeneration and / or neuronal dysfunction.

9. The SARM1 agent for use according to any one of claims 1 to 5 or the method according to claims 6 to 8, wherein the SARM1 agent activates SARM1 by: Binds to SARM1; Increase the intracellular level of NMN; Reduction of intracellular NAD levels; and / or Increases the intracellular NMN / NAD ratio.

10. The SARM1 agent for use according to any one of claims 8-9 or the method according to any one of claims 8-9, wherein the neurodegeneration comprises degeneration of axons, terminal branches or synaptic terminals and / or neurolytic death, optionally wherein the axon degeneration, terminal branches and / or synaptic terminals and / or neurolytic death is reversible and / or transient, further optionally wherein neuronal regeneration occurs after the neurodegeneration.

11. The SARM1 agent for use according to any one of claims 8 to 10 or the method according to any one of claims 8 to 10, wherein the neurodegeneration and / or neuronal dysfunction is reversible by a reversal agent.

12. The SARM1 medicament for use according to any one of claims 8 to 11 or the method according to any one of claims 8 to 11, wherein the neurodegeneration and / or neuronal dysfunction: (i) occurs in a cell expressing SARM1, optionally wherein the cell expressing SARM1 comprises a neuron; and (ii) is not present in cells that lack SARM1 or express a functionally insignificant amount of SARM1 protein, optionally wherein the cells that lack SARM1 or express a low and / or functionally insignificant amount of SARM1 include Schwann cells; muscle cells, skin cells (such as dermal fibroblasts), HEK293 cells, HEK293T cells, HeLa cells, SK mel 2 cells, SKmel 5 cells, SK mel 28 cells, CAPAN cells, JURKAT cells, C6 cells, HL-60 cells, LP-1 cells, U937 cells, MEWO cells, monocytes, macrophages and / or RAW264.7 cells.

13. The SARM1 agent for use according to any one of claims 1-4 and 6-12 or the method according to any one of claims 5 to 12, wherein the SARM1 agent is selected from vacor; vacor mononucleotide (VMN); 3-acetylpyridine (3-AP); 3-acetylpyridine mononucleotide (3-APMN); 2-aminopyridine (2-AnP); 2-AnP mononucleotide (2-AnPMN); nicotinamide mononucleotide (NMN), sulfo-ara-F-NMN (CZ-48), sulfo-ara-F-VMN; sulfo-ara-F-3-APMN; Sulfo-ara-F-2-AnPMN, S-NMN, ara-F-NMN (CZ-17); pyridine and molecules containing a pyridine ring; vacor nucleoside (VR), vacor nucleoside (VR) analogs, nicotinamide (NAM) analogs; nicotinamide riboside (NR), nicotinamide riboside (NR) analogs; nicotinic acid (NA) analogs; nicotinic acid riboside (NaR) analogs, nicotinic acid mononucleotide (NaMN) analogs, molecules that increase intracellular NMN levels, decrease NAD levels and / or increase the NMN / NAD ratio, thereby leading to SARM1 activation; NMNAT1-2-3 inhibitors or molecules that decrease NMNAT1-2-3 levels; and / or their metabolites, analogs and derivatives. 13A. The SARM1 agent for use according to claim 13 or the method according to claim 13, wherein said analogs and derivatives increase the permeability and / or solubility of said SARM1 agent.

14. The SARM1 agent for use according to any one of claims 1-4 and 6-13A or the method according to any one of claims 6-13A, wherein the SARM1 agent is administered topically, optionally by: (i) transdermal patch or (ii) injection into tissues such as (a) skin or muscle, optionally intravesically into the detrusor / urethral sphincter with or without ultrasound, radiographic and / or electromyographic guidance, (b) neuromas, optionally for peripheral nerve regeneration and surgical nerve repair, and / or (c) root canals, optionally for use in dental surgery; (iii) endoscopic injection, such as injection into the esophageal sphincter, pyloric sphincter, sphincter of Oddi; cystoscopic injection into the urethral sphincter; (iv) injection into the salivary glands, such as the sublingual and submandibular glands; or (v) Topical administration.

15. The SARM1 agent for use according to any one of claims 1-4 and 6-14, or the method according to any one of claims 5 to 14, wherein two or more SARM1 agents according to any one of claims 8 to 14 are co-administered, optionally wherein an additional agent such as an anti-inflammatory, analgesic and / or anesthetic agent is administered.

16. A composition comprising a SARM1 agent according to any one of claims 8 to 13; optionally wherein the composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable excipient, diluent and / or carrier.

17. The composition according to claim 16, for use according to any one of claims 1 to 4 and 8 to 14.

18. A composition according to claim 16 or a composition for use according to claim 17, wherein the composition is a topical composition; optionally wherein the composition is a cream or ointment.

19. The method of any one of claims 5 to 15, wherein the SARM1 agent is contained in a composition according to claim 16 or 18.

20. A syringe comprising the SARM1 agent according to any one of claims 8 to 13 or the composition according to claim 16 or 18.

21. A transdermal patch comprising a SARM1 agent according to any one of claims 8 to 13 or a composition according to claim 16 or 18.

22. A kit comprising: The SARM1 agent according to any one of claims 8 to 13 or the composition according to claim 16 or 18; and An applicator configured to administer the SARM1 agent.

23. The kit of claim 22, wherein the applicator comprises the transdermal patch of claim 21 or the syringe of claim 20.

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