mRNA pain therapy

Abstract

The present invention discloses the use of a mRNA pain therapy to produce endogenous opioids for the treatment of temporary and chronic pain. In the preferred embodiment mRNA that encodes the production of B-endorphin, dynorphins A and B, and Nociceptin / Orphanin FQ, along with preferred packaging and administration options is disclosed.

Description

PCT Patent Application Docket No.: PETC 007 PCT mRNA PAIN THERAPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This international PCT application claims the benefit of and priority to U. S. Provisional Patent Application No. 63 / 741,836 filed January 4, 2025, the entire contents of which are incorporated herein.INCORPORATION BY REFERENCE STATEMENT: COMPUTER PROGRAM, SEQUENCE LISTING, OR LARGE TABLES

[0002] In compliance with 37 C. F. R. 1.52(e)(5), the sequence listing information contained in electronic file name: "mRNA Pain Therapy SequenceListing.xml"; size 7.0 KB; created on: 5 January 2025, is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION

[0003] Since researchers have discovered the endogenous opioid system and its significance in day-to-day function and survival, endogenous opioid systems should be taken into account when considering opioid drug regimens.

[0004] The system comprises three families of opioid peptides - neurotransmitters that are derived from proteolytic cleavage of more abundant precursor proteins - and three families of receptors. The three families of peptides are P-endorphin (derived from the precursor proopiomelanocortin); leucine (Leu)- and methionine (Met)-enkephalins (derived from preproenkephalin); and dynorphins, including dynorphins A and B and neoendorphins (all derived from preprodynorphin).

[0005] These opioid peptides bind to their G-protein-coupled receptors - the p-opioid receptors, the 8-opioid receptors, the K-opioid receptors, and the non-opioid receptor Nociceptin (NOC -previously called opioid receptor-like 1 receptors).

[0006] P-endorphin mainly binds to p-opioid receptors; the Met- and Leu-enkephalins primarily bind to 8-opioid receptors and p-opioid receptors, while dynorphin and related peptides mainly bind to K-opioid receptors. Nociceptin / Orphanin FQ is the peptide that binds to NOC.PCT Patent Application Docket No.: PETC 007 PCT

[0007] Orphanin FQ, also known as Nociceptin, primarily interacts with the Nociceptin opioid receptor (NOP). This receptor is part of the broader family of opioid receptors, including the p-opioid receptor (MOP), 8-opioid receptor (DOP), and K-opioid receptor (KOP), but Orphanin FQ's primary binding affinity lies with the NOP receptor.

[0008] The NOP receptor, also referred to as ORL1, or opioid receptor-like 1, is a G protein-coupled receptor (GPCR). Orphanin FQ / Nociceptin is the endogenous ligand for this receptor.

[0009] While Orphanin FQ interacts with the NOP receptor, Orphanin FQ does not have high affinity for the other classical opioid receptors (MOP, DOP, KOP). Similarly, the classical opioid peptides (like enkephalins, dynorphin, and endorphins) do not readily bind to the NOP receptor.

[0010] The NOP receptor and the Orphanin FQ system play roles in various physiological processes, including pain modulation, stress responses, and feeding behavior.

[0011] While the primary interaction is with the NOP receptor, some evidence suggests that Orphanin FQ may also have interactions with other G protein-coupled receptors, though this is not as well-established.

[0012] Current treatment therapies use synthetic opiates, such as, for example, morphine, Oxycontin, Fentanyl, and other addictive small molecule solutions. The current solutions currently are a major cause of a health crisis with tens of thousands of deaths in the past several decades. As these current treatment therapies are very addictive, a solution is needed to treat pain using a non-addictive treatment therapy to solve the current health crisis caused by the current treatments.

[0013] The present invention solves the current health crisis based on the use of highly addictive small molecules by providing pain relief via mRNA pain therapy which produces endogenous opioids for the treatment of temporary and chronic pain. In particular, the present invention uses mRNA which encodes the production of 13-endorphin, dynorphins A and B, andNoci cep tion / Orphanin FQ, along with preferred packaging and administration options.BRIEF SUMMARY OF THE INVENTIONPCT Patent Application Docket No.: PETC 007 PCT

[0014] The present invention relates generally to the use of mRNA pain therapy to produce endogenous opioids for the treatment of temporary and chronic pain.

[0015] In particular, the present invention is a method of treating chronic and temporary pain using mRNA pain therapy that produces endogenous opioids comprising the steps of providing for a patient in need of chronic or transient pain treatment thereof; synthesizing the mRNA for proteins of interest which encode endogenous opioid peptides; encapsulating the mRNA in a delivery mechanism; storing and transporting the encapsulated mRNA to prevent degradation; and delivering the encapsulated mRNA to a patient in need thereof to treat chronic and transient pain.

[0016] In some embodiments, the endogenous opioid peptide is Nociceptin.

[0017] In some embodiments, the endogenous opioid peptide is Orphanin FQ.

[0018] In some embodiments, the endogenous opioid peptide is an endorphin derived from proopiomelanocortin. In some aspects of this embodiment, the endogenous opioid peptide is 13-endorphin. In some aspects of this embodiment, the endogenous opioid peptide is a-endorphin.

[0019] In some embodiments, wherein the endogenous opioid peptide is an enkephalin derived from proenkephalin. In some aspects of this embodiment, the endogenous opioid peptide is leucine-enkephalin. In some aspects of this embodiment, the endogenous opioid peptide is methionine-enkephalin.

[0020] In some embodiments, the endogenous opioid peptide is a dynorphin derived from prodynorphin. In some aspects of this embodiment, the endogenous opioid peptide is dynorphin A. In some aspects of this embodiment, the endogenous opioid peptide is Dyn A 1-17. In some aspects of this embodiment, the endogenous opioid peptide is Dyn A 1-8. In some aspects of this embodiment, the endogenous opioid peptide is dynorphin B.

[0021] In some embodiments, the mRNA pain therapy is packaged in a lipid nanoparticle delivery mechanism. In some aspects of this embodiment, the delivery mechanism is ionizable lipids. In some aspects of this embodiment, the delivery mechanism is phospholipids. In somePCT Patent Application Docket No.: PETC 007 PCT aspects of this embodiment, the delivery mechanism is cholesterol. In some aspects of this embodiment, the delivery mechanism is PEGylated lipids.

[0022] In some embodiments, the mRNApain therapy is packaged in an exosome delivery mechanism.

[0023] In some embodiments, the encapsulated mRNAis stored and transported at approximately -70° C.

[0024] In some embodiments, the encapsulated mRNA further comprises a stabilizer.

[0025] In some embodiments, the method of delivering the packaged mRNAis via injection. In some aspects of this embodiment, the method of delivering the packaged mRNA via injection is intramuscular. In some aspects of this embodiment, the method of delivering the packaged mRNA via injection is subcutaneous. In some aspects of this embodiment, the method of delivering the packaged mRNAvza injection is intradermal.

[0026] In some embodiments, the method of delivering the packaged mRNAis via oral intake.

[0027] In some embodiments, the method of delivering the packaged mRNAis via nasal spray.

[0028] In some embodiments, the method of delivering the packaged mRNA is via transdermal methods.

[0029] In some embodiments, the method of delivering the packaged mRNA is via intravenous (IV) methods.

[0030] In some embodiments, the invention is directed to the use of a mRNA to manufacture a medicament designed to treat chronic and transient pain.

[0031] In some embodiments, the invention is directed to the use of a mRNA to manufacture a medicament designed to treat chronic and transient pain, wherein the mRNA produces particular endogenous opioids.PCT Patent Application Docket No.: PETC 007 PCT

[0032] In some embodiments, the invention is directed to the use of a mRNAto manufacture a medicament designed to treat chronic and transient pain, wherein the mRNA is synthesized for proteins that encode endogenous opioid peptides.

[0033] In some embodiments, the invention is directed to a pharmaceutical comprising synthesized mRNA designed to produce endogenous opioid peptides, wherein the pharmaceutical is encapsulated in a delivery mechanism.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The novel features that are characteristic of the present invention are set forth in the appended claims, however, the preferred embodiments of the invention, together with further objects and advantages, are better understood by reference to the following detailed description read concurrently with the accompanying drawings in which:

[0035] FIGs. 1A-1B are examples of the data generated by the optical electrophysiology platform developed by Cellectricon (Mblndal, Sweden). FIG. 1A is the imaging of neuronal excitability caused by electric field stimulation- (EFS)-induced calcium transients. The noted area is a combination of EFS and fluorescent probes used to detect action potential firing. FIG. 1B is a graphical example of electric field stimulation- (EFS)-evoked changes in intracellular calcium probe fluorescence. FIG. 1C is a chart detailing the protocol used to conduct the optical electrophysiology platform.

[0037] FIGs. 2A-2C are bar graphs depicting the results of the effect of the “inflammatory soup” on the excitability of the tested compounds for Experiment 1, as described below. The Y-axis of each graph plots the response ratio, whereas the X-axis plots the electric field stimulation (EFS) protocol. FIG. 2A, in particular, used a dimethyl sulfoxide (DMSO) control (the left bar of each graph) and DMSO as a control together with the “inflammatory soup” (the right bar of each graph) and was was performed in Plate 1. FIG. 2B, in particular, used water (H2O) as a control (the left bar of each graph) and H2O as a control together with the “inflammatory soup” (the right bar of each graph) and was performed in Plate 1. FIG. 2C, in particular, used a dimethyl sulfoxide (DMSO) control (the left bar of each graph) and DMSO as a control together with the “inflammatory soup” (the right bar of each graph) and was performed in Plate 2. P-values arePCT Patent Application Docket No.: PETC 007 PCT indicated as *p<0.5, **p<0.01, ***p<0.001 and ****p<0.0001. The “inflammatory soup” increased the excitability of the rat dorsal root ganglion (DRG) cultures in both test occasions and across all plates. The effect of the “inflammatory soup” on excitability was more prominent at higher voltage EFS protocols. Individual experiments presented as averages and individual technical replicates. A two-way analysis of variance (ANOVA) with a Sidak’s post hoc test was performed.

[0037] FIGs. 3A-3C are bar graphs depicting the results of the effect of the “inflammatory soup” on the excitability of the tested compounds for Experiment 2, as described below. The Y-axis of each graph plots the response ratio, whereas the X-axis plots the electric field stimulation (EFS) protocol. FIG.3A, in particular, used a dimethyl sulfoxide (DMSO) control (the left bar of each graph) and DMSO as a control together with the “inflammatory soup” (the right bar of each graph) and was performed in Plate 1. FIG. 3B, in particular, used water (H2O) as a control (the left bar of each graph) and H2O as a control together with the “inflammatory soup” (the right bar of each graph) and was performed in Plate 1. FIG. 3C, in particular, used a dimethyl sulfoxide (DMSO) control (the left bar of each graph) and DMSO as a control together with the “inflammatory soup” (the right bar of each graph) and was performed in Plate 2. P-values are indicated as *p<0.5, **p<0.01, ***p<0.001 and ****p<0.0001. The “inflammatory soup” increased the excitability of the rat dorsal root ganglion (DRG) cultures in both test occasions and across all plates. The effect of the “inflammatory soup” on excitability was more prominent at higher voltage EFS protocols. Individual experiments presented as averages and individual technical replicates. A two-way analysis of variance (ANOVA) with a Sidak’s post hoc test was performed.

[0037] FIGs. 4A-4C are graphical representations of the data collected from the experiments depicted in FIGs. 2A-2C and 3A-3C and described below. FIGs. 4A and 4B are bar graphs of the average percent effect of Suzetrigine normalized to tetracaine (=0% as a control for no signal) and vehicle control (=100%) at 18V 25p; mean ±SD. N= 2-3 wells per concentration. The Y-axis represents the %effect, whereas the X-axis is the concentration in pM. FIG. 4A reports the results of Experiment 1, whereas FIG. 4B reports the results of Experiment 2. FIG. 4C reports on the pooled average percent effect of Suzetrigine normalized to tetracaine (=0% as aPCT Patent Application Docket No.: PETC 007 PCT control for no signal) and vehicle control (=100%) at 18V 25p; mean ±SD. N= 2-3. The Y-axis represents the %effect, whereas the X-axis is the concentration in pM.

[0037] FIGs. 5A-5C are graphical representations of the data collected from the experiments depicted in FIGs. 2A-2C and 3A-3C and described below. FIGs. 5A and 5B are bar graphs of the average percent effect of PF-04885614 normalized to tetracaine (=0% as a control for no signal) and vehicle control (=100%) at 18V 25p; mean ±SD. N= 2-3 wells per concentration. The Y-axis represents the %effect, whereas the X-axis is the concentration in pM. FIG. 5A reports the results of Experiment 1, whereas FIG. 5B reports the results of Experiment 2. FIG. 5C reports on the pooled average percent effect of PF-04885614 normalized to tetracaine (=0% as a control for no signal) and vehicle control (=100%) at 18V 25p; mean ±SD. N= 2-3. The Y-axis represents the %effect, whereas the X-axis is the concentration in pM.

[0037] FIGs. 6A-6C are graphical representations of the data collected from the experiments depicted in FIGs. 2A-2C and 3A-3C and described below. FIGs. 6A and 6B are bar graphs of the average percent effect of Ro 64-6198 normalized to tetracaine (=0% as a control for no signal) and vehicle control (=100%) at 18V 25p; mean ±SD. N= 2-3 wells per concentration. The Y-axis represents the %effect, whereas the X-axis is the concentration in pM. FIG. 6A reports the results of Experiment 1, whereas FIG. 6B reports the results of Experiment 2. FIG. 6C reports on the pooled average percent effect of Ro 64-6198 normalized to tetracaine (=0% as a control for no signal) and vehicle control (=100%) at 18V 25p; mean ±SD. N= 2-3. The Y-axis represents the %effect, whereas the X-axis is the concentration in pM.

[0037] FIGs. 7A-7C are graphical representations of the data collected from the experiments depicted in FIGs. 2A-2C and 3A-3C and described below. FIGs. 7A and 7B are bar graphs of the average percent effect of nociceptin normalized to tetracaine (=0% as a control for no signal) and vehicle control (=100%) at 18V 25p; mean ±SD. N= 2-3 wells per concentration. The Y-axis represents the %effect, whereas the X-axis is the concentration in pM. FIG. 7A reports the results of Experiment 1, whereas FIG. 7B reports the results of Experiment 2. FIG. 7C reports on the pooled average percent effect of nociceptin normalized to tetracaine (=0% as a control for no signal) and vehicle control (=100%) at 18V 25p; mean ±SD. N= 2-3. The Y-axis represents the %effect, whereas the X-axis is the concentration in pM.PCT Patent Application Docket No.: PETC 007 PCT DETAILED DESCRIPTION OF THE INVENTION

[0035] Endogenous opioids are naturally occurring peptides in the body that bind to opioid receptors and produce effects similar to opiate drugs, such as, for example, pain relief, euphoria, and sedation. These peptides are encoded by specific genes, specific genes of which are three main classes of endogenous opioids: endorphins, enkephalins, and dynorphins. These peptides act on various opioid receptors, such as, for example, p, 8, and K receptors, to modulate pain, mood, and other physiological processes in the body.

[0036] Endogenous opioids are a group of naturally occurring peptides that bind to opioid receptors in the brain and nervous system. These peptides include endorphins, enkephalins, and dynorphins, and are derived from larger precursor proteins through enzymatic cleavage. The mRNA sequences that code for these peptides is part of the gene sequences for each respective precursor protein.ENDOGENOUS OPIOIDSA. Endorphins

[0037] Endorphins are primarily derived from a precursor protein called pro-opiomelanocortin (POMC). The endorphins include beta-endorphin and alpha-endorphin. The POMC gene is located on chromosome 2 in humans and encodes the precursor protein, which is cleaved to form multiple peptides, such as, for example, beta-endorphin. Beta-endorphins are produced from the precursor POMC, which also give rise to other hormones, such as adrenocorticotropic hormone (ACTH).

[0038] The DNA sequence for beta-endorphin is encoded by the C-terminal portion of the POMC protein and is typically produced after cleavage of the precursor.Tyr-Gly-Gly-Phe-Met-Arg-Phe-Thr-Gly-Ser-Tyr-Leu-Leu-Pro-Ser- Ala-Ser (SEQ ID NO.: 1)B. EnkephalinsEnkephalins are smaller peptides derived from the proenkephalin precursor. There are two major types of enkephalins: leucine-enkephalin (Leu-enkephalin) and methionine-enkephalin (Met-PCT Patent Application Docket No.: PETC 007 PCT enkephalin). The proenkephalin (PENK) gene is located on chromosome 8 and encodes the precursor protein that is cleaved into smaller enkephalin peptides, such as, for example, Leu-enkephalin Tyr-Gly-Gly-Phe-Leu (SEQ ID NO.: 2) and Met-enkephalin Tyr-Gly-Gly-Phe-Met (SEQ ID NO.: 3). Enkephalins are derived from proenkephalin which play a role in pain modulation and reward.C. DynorphinsDynorphins are peptides derived from the prodynorphin precursor. This group of peptides includes dynorphin A, further including Dyn A 1-17 and Dyn A 1-8, and dynorphin B. The prodynorphin (PDYN) gene is located on chromosome 20 and encodes the precursor protein Tyr-Gly-Gly-Phe-Leu- Asp- Vai -Ala-Ser- Thr-Ser-Pro-Gly-Pro-Tyr-Pro-Gly (SEQ ID NO.: 4) that is cleaved into dynorphins Tyr-Gly-Gly-Phe-Leu-Asp-Val (SEQ ID NO.: 5), such as, for example, dynorphin A, further including Dyn A 1-17 and Dyn A 1-8, and dynorphin B. Dynorphins are derived from prodynorphin, which is cleaved into various dynorphin peptides and are involved in the regulation of pain, stress, and mood.D. mRNA Sequence Coding

[0039] POMC encodes P-endorphins, ACTH, and other hormones. The mRNA sequence can be found within the coding region of the POMC gene on chromosome 2.

[0040] PENK encodes proenkephalin, which gives rise to enkephalins. The mRNA for proenkephalin comes from the PENK gene on chromosome 8.

[0041] PDYN encodes prodynorphin, from which dynorphins are cleaved. The mRNA sequence is found within the PDYN gene on chromosome 13.

[0042] POMC mRNA (P-endorphin): The POMC gene has several isoforms and the mRNA sequence specific to a species of interest (e.g., Homo sapiens) may be found in NCBI GenBank under the accession number NM 005080.

[0043] PENK mRNA (enkephalins): For human PENK, the sequence is listed under NM_001301 in GenBank.PCT Patent Application Docket No.: PETC 007 PCT

[0044] PDYN mRNA (dynorphins): For human PDYN, refer to NM_002611 in GenBank.PROCESSA. Synthesis

[0045] mRNA treatment therapies, such as the therapies developed for COVID- 19 and, for example, Pfizer®-BioNTech® and Moderna®, require careful packaging to ensure the mRNA can be safely delivered into cells where it can instruct the body to produce a protein, such as a spike protein of a virus, to trigger an immune response.

[0046] Generation: mRNA is synthesized in a laboratory using a DNA template that encodes the protein of interest (e g., the spike protein of the SARS-CoV-2 virus). This mRNA is the messenger that tells the body’s cells how to make the protein.B. Generation

[0047] Lipid Nanoparticles (LNPs): The primary challenge in delivering mRNA effectively is protecting the fragile mRNA from degradation and ensuring it can enter cells. mRNA is unstable in the body, and without protection, it could be destroyed before reaching its target cells.

[0048] Lipid nanoparticles are small particles made of lipids (fats) that encapsulate the mRNA. The lipid shell helps protect the mRNA from degradation and allows it to fuse with the cell membrane, delivering the mRNA into cells.

[0049] LNPs typically consist of ionizable lipids, phospholipids, cholesterol and PEGylated lipids. Ionizable lipids help to encapsulate the mRNA and facilitate endocytosis (the process of a mammalian cell engulfing the nanoparticle). Phospholipids help stabilize the LNPs and mimic natural cell membranes. Cholesterol helps with the overall stability of the LNPs. PEGylated lipids can help the LNPs avoid immune detection and prolong their circulation in the bloodstream.C. Delivery to CellsPCT Patent Application Docket No.: PETC 007 PCT

[0050] Once the mRNA is encapsulated in LNPs, the pain therapy is injected into the body. LNPs are designed to be taken up by cells through endocytosis, a process where the cell engulfs the nanoparticle.

[0051] Once inside the cell, the LNPs help release the mRNA into the cytoplasm. The mRNA then gets translated by the cell’s ribosomes, which use the genetic code to produce the protein specified by the mRNA (e.g., the viral spike protein in the case of COVID- 19) and pain therapies.D. Protein Production and Immune Response

[0052] The produced protein is recognized by the immune system as foreign, prompting the body to mount an immune response. This includes producing antibodies and activating T-cells that can recognize and destroy infected cells in the future.E. Stabilization and Storage and Transport

[0053] mRNA is inherently unstable so it needs to be stored and transported under specific conditions to prevent degradation. For many mRNA pain therapies this means ultra-cold storage at temperatures as low as -70°C (for Pfizer®'s pain therapy, for example) until it is ready to be used.

[0054] During production, mRNA and LNPs are often formulated with stabilizers or other excipients that help maintain the integrity of the pain therapy during storage and transport.E Administration

[0055] Once the pain therapy is ready, the therapy is injected intramuscularly (typically in the upper arm) into the patient suffering from chronic or transient pain, where the pharmaceutical can be absorbed into the bloodstream and reach the target cells, usually muscle cells and some immune cells (like dendritic cells).ENCAPSULATIONPCT Patent Application Docket No.: PETC 007 PCT

[0056] Exosome mRNA encapsulation refers to the process of loading messenger RNA (mRNA) into exosomes, which are nanosized vesicles naturally secreted by cells. Exosomes are used in various biomedical applications, especially in gene therapy and RNA-based therapies. The encapsulation of mRNA into exosomes provides a potential delivery system for mRNA therapeutics due to their unique properties, including their ability to cross biological barriers, such as the blood-brain barrier, and their relatively low immunogenicity.A. Exosome Biogenesis

[0057] Exosomes are naturally produced within cells via the inward budding of endosomes that form multivesicular bodies (MVBs). These MVBs fuse with the plasma membrane, releasing the exosomes into the extracellular space. Exosomes contain a variety of cargo, including lipids, proteins, and RNAs, including, but not limited to, mRNA (messenger), miRNA (micro), and IncRNA (long non-coding RNA).

[0058] Several methods can be employed to encapsulate mRNA into exosomes: natural loading, electroporation, sonication, and chemical fusion.

[0059] Natural Loading: Some studies suggest that mRNA can be incorporated into exosomes during their biogenesis in the cell, especially if the mRNA is upregulated or interacts with specific RNA-binding proteins.

[0060] Electroporation: This method involves applying an electric field to cells or exosomes, which creates temporary membrane pores allowing mRNA or other cargo to be introduced.

[0061] Sonication: This technique uses sound waves to facilitate the loading of mRNA into exosomes, either before or after exosome isolation.

[0062] Chemical Fusion: Certain chemicals can promote the fusion of synthetic lipid vesicles with exosomes, enhancing the encapsulation efficiency of mRNAB. Advantages

[0063] Several advantages of mRNA encapsulation exist, such as biocompatibility and low immunogenicity, targeting capability, protection of mRNA, and crossing biological barriers.PCT Patent Application Docket No.: PETC 007 PCT

[0064] Biocompatibility and Low Immunogenicity: Exosomes are derived from cells and thus tend to be biocompatible and have minimal immune responses compared to synthetic nanoparticles.

[0065] Targeting Capability: Exosomes naturally can target specific cells or tissues based on their surface proteins and lipid composition. This makes them a promising vehicle for targeted drug delivery, including mRNA therapies.

[0066] Protection of mRNA: Exosomes provide a protective environment for the mRNA, preventing degradation by RNases and improving its stability in circulation.

[0067] Crossing Biological Barriers: Exosomes have been shown to cross various biological barriers, including the blood-brain barrier, which could be beneficial for treating diseases that affect the central nervous system.C. Applications

[0068] There are several applications for exosome delivery, such as gene therapy, cancer immunotherapy, and pain therapy development.

[0069] Gene Therapy: Exosomes can deliver therapeutic mRNA that encodes proteins to replace or repair defective genes in various diseases.

[0070] Cancer Immunotherapy: Exosomes can deliver mRNA encoding antigens or immune modulatory proteins to stimulate anti-tumor immune responses.

[0071] Pain therapy Development: Exosome-based delivery systems could potentially be used for mRNA pain therapy, especially for diseases where traditional pain therapy strategies face challenges (e.g., cancer, infectious diseases).D. Challenges and Considerations

[0072] Challenges and considerations in the use of exosome encapsulation include exosome isolation and purification, encapsulation efficiency, stability and storage, and regulatory hurdles.PCT Patent Application Docket No.: PETC 007 PCT

[0073] Exosome Isolation and Purification: Obtaining high-quality exosomes with minimal contamination from other cellular components can be challenging and may impact the reproducibility and scalability of exosome-based therapies.

[0074] Encapsulation Efficiency: Achieving high levels of mRNA encapsulation without damaging exosomes is a critical issue that needs optimization.

[0075] Stability and Storage: Exosome-based mRNA formulations must be stable under storage conditions, which requires precise formulation and stabilizing agents.

[0076] Regulatory Hurdles: As with any novel therapeutic approach, there are regulatory challenges related to the clinical development of exosome-based mRNA therapies, including ensuring the safety and efficacy of these therapies.

[0077] Many of the aforementioned challenges have been solved by Rion Inc based in Rochester Minnesota.DELIVERY

[0078] mRNA pain therapies may be delivered to the body through several methods depending on the type of pain therapy and the desired immune response. The most common methods of pain therapy delivery include injection, oral intake, nasal spray, transdermal, needles, intravenous (IV), and other emerging methods.A. Injection (Intramuscular, Subcutaneous, Intradermal)

[0079] Intramuscular (IM): This is the most common method. The pain therapy is injected directly into the muscle, typically the upper arm (deltoid muscle). Pain therapies like the flu pain therapy, COVID-19 pain therapies (e.g., Pfizer® and Moderna®), and many others are delivered this way.

[0080] Subcutaneous (SC): The pain therapy is injected into the layer of fat just beneath the skin. Some pain therapies, like the MMR (measles, mumps, rubella) pain therapy, are administered this way.PCT Patent Application Docket No.: PETC 007 PCT

[0081] Intradermal (ID): The pain therapy is injected into the skin's layer, just below the surface. This method is used less frequently but has been employed for pain therapies like the BCG (tuberculosis) pain therapy.B. Oral (Ingestion)

[0082] Some pain therapies are delivered orally, meaning they are taken as a pill, liquid, or other form that is swallowed. These pain therapies are often designed to stimulate immunity in the digestive tract. A well-known example is the oral polio pain therapy (OPV), though in many countries the injectable form (IPV) is now preferred.C. Nasal Spray (Intranasal)

[0083] Certain pain therapies are delivered through the nose as a spray, where the pain therapy is absorbed by the mucous membranes of the nasal passages. The flu pain therapy (FluMist®) is one example of an intranasal pain therapy, which is a live attenuated pain therapy designed to trigger an immune response in the respiratory tract.D. Transdermal (Skin Patch)

[0084] In research and development stages, some pain therapies are being designed to be delivered via a skin patch that can deliver the pain therapy through the skin without the need for needles. These patches often contain microneedles that painlessly penetrate the skin's outer layer to deliver the pain therapy.E. Microneedles and Other Innovative Methods

[0085] Microneedle patches or arrays are small, painless devices that can deliver a pain therapy by creating tiny, microscopic punctures in the skin. Microneedle patches or arrays systems may be more comfortable for patients and potentially easier to distribute. Microneedles are being researched for a variety of pain therapy’s, including those for flu and COVID-19.E Intravenous (IV)PCT Patent Application Docket No.: PETC 007 PCT

[0086] Although not common, some pain therapy’s, especially those in clinical trials, may be administered intravenously. This method would allow pain therapy to directly enter the bloodstream, but it is not typically used for routine immunization.G. Other Emerging Methods

[0087] There are ongoing efforts to develop alternative pain therapy delivery systems, such as aerosolized pain therapies (delivered as a mist or spray that is inhaled) or nanoparticle-based pain therapies, which may offer more targeted and efficient delivery.PERIPHERAL ANALGESIC EFFECTS

[0088] Orphanin FQ, also known as Nociceptin, acts on the NOP (Nociceptin / Orphanin FQ peptide) receptor that is present both centrally and peripherally in the nervous system in mammals. The effects of Orphanin FQ on pain are complex and depend on the route of administration, pain model, and species studied.

[0089] Peripheral administration of Orphanin FQ (N / OFQ) in animal models has been shown to reduce visceral hypersensitivity and pain triggered by inflammation or stress, without affecting normal (basal) pain sensitivity.

[0090] Specifically, in rat models, peripherally injected N / OFQ significantly decreased pain responses in conditions of inflammation or stress-induced hyperalgesia, and this effect was blocked by a NOP receptor antagonist.

[0091] These findings suggest that activation of peripheral NOP receptors by Orphanin FQ can produce anti-nociceptive (pain-reducing) effects in the periphery, particularly under conditions of heightened pain sensitivity (hyperalgesia) due to inflammation or stress.

[0092] Orphanin FQ does not appear to affect normal pain thresholds when administered peripherally; its analgesic effects are most pronounced in sensitized (inflamed or stressed) states.

[0093] There is ongoing clinical development of peripherally restricted NOP agonists for chronic peripheral neuropathic pain, indicating potential for future therapeutic use in humans.PCT Patent Application Docket No.: PETC 007 PCT

[0094] The effects of Orphanin FQ can differ based on administration route: central administration (into the brain) may actually increase pain (hyperalgesia), while spinal or peripheral administration can reduce pain.BIOLOGICAL TESTING

[0095] Cellectricon (Mblndal, Sweden) was contracted by the inventor to investigate whether the peptide Nociceptin / Orphanin FQ modulates pain signal transmission in an in vitro model of the peripheral nervous system (Study No.: 501-15A, Study Title: Effects of compounds on neuronal excitability in rat dorsal root ganglia in vitro cultures). Cellectricon studied the effects of the compounds of the instant application on the neuronal excitability in rat dorsal root ganglia in vitro cultures. In particular, Cellectricon utilized its proprietary optical electrophysiology platform to assess the compounds of interest effect on neuronal excitability, as illustrated in FIGs 1-3. Cellectricon recommended using its assays under inflammatory conditions, i.e. with preincubation with an “inflammatory soup” in vitro.A. Materials and Methods

[0096] In addition to the compounds of interest, particularly the Nociceptin / Orphanin FQ, a peptide targeting the nociception (ORL.l) receptor, and having the structure:PCT Patent Application Docket No.: PETC 007 PCTNH,(Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln (SEQ ID NO:6)(Phoenix Pharmaceuticals, Inc., Buden-Wiirttemberg, Germany) three reference compounds were included in the study, including• Suzetrigene(sold under the brand name Journavx® (Vertex Pharmaceuticals, Inc., Boston, MA)), a clinically approved small molecule Navi.8 inhibitor;PCT Patent Application Docket No.: PETC 007 PCTa small molecule targeting the nociception receptor (MedChemExpress, Monmouth Junction, NJ, USA); andPF-04885614,a pre-clinical small molecule Navi.8 (Pfizer, New York, NY), using the following concentrations:Cornpourid ( ’OIK c tit r At ion Suppl itr <\it Nr. Solvent \ '. K k;-?: I l l i 'i. M -j. M:. M. I. -.-. <?< t v d t. I1L; USX J 1 Oi o l '. S: -. M ii.nl.' 1: i JL:,. Mr.h: iu: •J I tM Si ' 1 ’1 ■ i r% s:t. -. ■ ■ • '■ I i i f J:::, 1 h >. ■ • I M 1,. ■ D ' « u i 1 OdM 1S u / i ll Lj l K ' ’ ■ \: M ’ u.t' l -.. ii j d ku-.’ M’CdU he D M sriMedChemExpress is headquartered at Monmouth Junction, NJ, USA

[0097] Rat dorsal root ganglion (DRG) cultures (Cellectricon (Mblndal, Sweden)) were generated from 5-9 weeks old male, Sprague-Dawley rats (Javier Labs, Le Genest-Saint-Isle, France) according to standard practice. The DRGs were plated in 384 well format plates using 48 wells / plate and cultured for three (3) days in vitro. Inflammatory mediators were added after 24 hours in culture to establish disease-like conditions of hyperexcitabilty. Compounds werePCT Patent Application Docket No.: PETC 007 PCT investigated in triplicate at five concentrations plus vehicle in inflammatory conditions.Nociceptin / Orphanin FQ (1 pM- 0.012 pM; 1:3 dilution); Ro 64-6198 (1 pM- 0.012 pM; 1:3 dilution); Suzetrigine (1 pM - 0.012 pM; 1:3 dilution); and PF-04885614 (10 pM - 0.12 pM; 1:3 dilution).

[0098] After 72 hours in culture, DRG neurons underwent electric field stimulation (EFS) to assess changes in excitability, namely, changes of fluorescence of a calcium indicator.Excitability was measured using optical electrophysiology (Cellectricon (Mblndal, Sweden)) via calcium imaging. The compounds were added acutely (1 hour) prior to the EFS experiment together with the calcium indicator (Ca5)(Thermo Fisher Scientific, Waltham, MA). For each test occasion, two plates were prepared. Nociceptin and Suzetrigine were added to one plate, while Ro 64-6198 and PF-04885614 were added to the other plate. Nociceptin was diluted in water, and the rest of the compounds were diluted in dimethyl sulfoxide (DMSO)(Millipore Sigma, Burlington, MA). Tetracaine, an ester local anesthetic used to numb the eyes, nose, or throat (Quimdis, Levallois-Perret, France), at a concentration aimed to generate full block was included for normalization and quality control (QC) purposes. Data is delivered from two test occasions that passed QC. Some data points were excluded due to technical errors.

[0099] FIG. 2 and FIG. 3 report the results and are presented as averages and individual data points from two individual test occasions (Experiment 1 and Experiment 2), as well as pooled data of the two presented test occasions. Excitability was tested using four different EFS protocols: 10V 5Hz 25p, 18V 5Hz 25p, 30V 5Hz 25p and at a high frequency stimulation (HFS; 30V 5Hz 150p). Data from the 18V, 5Hz EFS protocol provided the best balance between sensitivity and robustness based on the QC (Z’ values between the positive control wells containing tetracaine and the vehicle control wells) for the plates. The response ratio was normalized to average of control wells of their respective vehicle with inflammatory soup and tetracaine wells. Data assessing excitability in the inflammatory condition was analyzed using a two-way Analysis of Variance (ANOVA) with Sidak’s post hoc test for multiple comparisons. Curve fitting was performed and approximation of an IC50 value was determined.B. ResultsPCT Patent Application Docket No.: PETC 007 PCT

[0100] Z’ was determined for each plate and EFS protocol. Z’ serves as a comparison between the means and standard deviations of tetracaine wells vs. vehicle control wells.( ] li.1 it.iOE ) 1 O! 1 ) O C DUol1 X pc!'i 111C3111 peri] i unit 2 / '10V ’ -0.27 0. S2isv o.s-t o.o"1Plate 130V 0.46 1T65i ll’s CS” 0. S’10V 0.41 0.10ISV 0.65 O.-tSPlate 230V 0,"0 0.60HI S 0.67 0.5^Conditions tor H2O controlExp I Exp2Z'10 V 0.18 0.21I SV 0.60 0.3S30 V 0.62 0.49HE'S 0.65 0.35PCT Patent Application Docket No.: PETC 007 PCT Z’ was determined for both vehicle controls, DMSO and H2O separately. AZ’>0.3 was used as a criteria for QC. Lowest Z’s were observed after application of the lowest EFS protocol (10V 25p) for which data was not analyzed further. At higher voltages than 10V, all EFS protocols and plates passed QC. The results for the two experiments for both plates at 10V, 18V are shown in FIGs. 2 and 3.i. _ Suzetrigine

[0101] The inventor of the claimed technology discovered that Suzetrigine decreased the excitability in both test occasions. The decrease was concentration-dependent and partial within the tested concentration range (approximate 35% change). The IC50 value was 0.29 pM. The results of the proof of concept studies are reported in FIGs. 4A-4C.ii, PF-04885614

[0102] PF-04885614 decreased the excitability in both test occasions. The decrease was concentration-dependent and partial within the tested concentration range (approximate 50% change). The IC50 value was 4.63 pM.iii, Ro 64-6198

[0103] Ro 64-6198 decreased the excitability in both test occasions, as reported in FIGs 6A-6C.The decrease was concentration-dependent and partial within the tested concentration range (approximate 25% change). The IC50 value was 0.25 pM.iv. Nociceptin

[0104] As reported in FIGs 7A-7C, nociceptin decreased the excitability in both test occasions. The decrease was concentration-dependent and partial within the tested concentration range (approximate 25% change). The IC50 value was 0.32 pM.C. Summary and Conclusion

[0105] All tested compounds, including Nociceptin, consistently decreased the EFS-evoked excitability response in a concentration-dependent manner across two independent testPCT Patent Application Docket No.: PETC 007 PCT occasions. For all tested compounds, the inhibition was partial within the tested concentration range and the IC50 values ranged from 0.25 to 4.6 pM. All plates passed QC. All tested compounds decreased the excitability of DRG cultures in a concentration-dependent manner.

[0106] Table 1: Summarized effects of compounds on neuronal excitability and the corresponding IC50 values for each tested compound.Compound Maximum % Decrease in Estimated IC50 (pM) ExcitabilitySuzetrigine 35 0.29PF-04885614 51 4.63Ro 64-6198 26 0.25Nociceptin 24 0.32EQUIVALENTS

[0107] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system,PCT Patent Application Docket No.: PETC 007 PCT article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0108] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0109] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0110] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”[oni] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, e.g., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, e.g., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to “A” only (optionally including elements other than B); in another embodiment, to “B” only (optionally including elements other than A); in yet another embodiment, to both “A and B” (optionally including other elements); etc.

[0112] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or,PCT Patent Application Docket No.: PETC 007 PCT when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (e.g., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0113] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0114] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0115] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, e.g., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g.,PCT Patent Application Docket No.: PETC 007 PCT “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

Claims

1. PCT Patent Application Docket No.: PETC 007 PCT CLAIMSI claim:

1. A method of treating chronic and transient pain using a mRNA pain therapy which produces endogenous opioids comprising:providing for a patient in need of chronic or transient pain treatment thereof; synthesizing the mRNA for proteins of interest which encode endogenous opioid peptides;encapsulating the mRNA in a delivery mechanism;storing and transporting the encapsulated mRNA to prevent degradation; and delivering the encapsulated mRNA to a patient in need thereof to treat chronic and transient pain.

2. The endogenous opioid peptides in accordance with claim 1, wherein the endogenous opioid peptide is Nociceptin.

3. The endogenous opioid peptides in accordance with claim 1, wherein the endogenous opioid peptide is Orphanin FQ.

4. The endogenous opioid peptides in accordance with claim 1, wherein the endogenous opioid peptide is an endorphin derived from pro-opiomelanocortin.

5. The endogenous opioid peptides in accordance with claim 1, wherein the endogenous opioid peptide is beta-endorphin.

6. The endogenous opioid peptides in accordance with claim 1, wherein the endogenous opioid peptide is an alpha-endorphin.

7. The endogenous opioid peptides in accordance with claim 1, wherein the endogenous opioid peptide is an enkephalin derived from proenkephalin.

8. The endogenous opioid peptides in accordance with claim 1, wherein the endogenous opioid peptide is leucine-enkephalin.PCT Patent Application Docket No.: PETC 007 PCT 9. The endogenous opioid peptides in accordance with claim 1, wherein the endogenous opioid peptide is methionine-enkephalin.

10. The endogenous opioid peptides in accordance with claim 1, wherein the endogenous opioid peptide is a dynorphin derived from prodynorphin.

11. The endogenous opioid peptides in accordance with claim 1, wherein the endogenous opioid peptide is dynorphin A.

12. The endogenous opioid peptides in accordance with claim 1, wherein the endogenous opioid peptide is Dyn A 1-17.

13. The endogenous opioid peptides in accordance with claim 1, wherein the endogenous opioid peptide is Dyn A 1-8.

14. The endogenous opioid peptides in accordance with claim 1, wherein the endogenous opioid peptide is dynorphin B.

15. The delivery mechanism in accordance with claim 1, wherein the delivery mechanism is lipid nanoparticles.

16. The delivery mechanism in accordance with claim 1, wherein the delivery mechanism is an exosome.

17. The delivery mechanism in accordance with claim 1, wherein the delivery mechanism is ionizable lipids.

18. The delivery mechanism in accordance with claim 1, wherein the delivery mechanism is phospholipids.

19. The delivery mechanism in accordance with claim 1, wherein the delivery mechanism is cholesterol.

20. The delivery mechanism in accordance with claim 1, wherein the delivery mechanism is PEGylated lipids.PCT Patent Application Docket No.: PETC 007 PCT 21. The method of storing and transporting the encapsulated mRNA according to claim 1, wherein the encapsulated mRNA is stored and transported at approximately -70° C.

22. The method of storing and transporting the encapsulated mRNA according to claim 1, wherein the encapsulated mRNA further comprises a stabilizer.

23. The method of delivering the encapsulated mRNA to a patient in need thereof to treat chronic and transient pain in accordance with claim 1, wherein the delivery method is injection, and wherein the injection is selected from one of the following: intramuscular, subcutaneous, and intradermal.

24. The method of delivering the encapsulated mRNA to a patient in need thereof to treat chronic and transient pain in accordance with claim 1, wherein the delivery method is an oral intake.

25. The method of delivering the encapsulated mRNA to a patient in need thereof to treat chronic and transient pain in accordance with claim 1, wherein the delivery method is a nasal spray.

26. The method of delivering the encapsulated mRNA to a patient in need thereof to treat chronic and transient pain in accordance with claim 1, wherein the delivery method is transdermal.

27. The method of delivering the encapsulated mRNA to a patient in need thereof to treat chronic and transient pain in accordance with claim 1, wherein the delivery method is microneedles.

28. The method of delivering the encapsulated mRNA to a patient in need thereof to treat chronic and transient pain in accordance with claim 1, wherein the delivery method is intravenous.

29. Use of one or more mRNAs to manufacture a medicament designed to treat chronic and transient pain.PCT Patent Application Docket No.: PETC 007 PCT 30. The use of one or more mRNAs to manufacture a medicament designed to treat chronic and transient pain according to claim 29, wherein the one or more mRNAs is specifically synthesized to manufacture proteins that encode desired endogenous opioid peptides.

31. The use of one or more mRNAs to manufacture a medicament designed to treat chronic and transient pain according to claim 30, wherein the medicament is encapsulated.

32. The use of one or more mRNAs to manufacture a medicament designed to treat chronic and transient pain according to claim 32, wherein the encapsulation assists with the delivery of the medicament to a patient in need of treatment for chronic and transient pain.

33. A pharmaceutical comprising one or more mRNAs, wherein said one or more mRNAs are specifically synthesized to manufacture proteins that encode desired endogenous opioid peptides.

34. The pharmaceutical comprising one or more mRNAs according to claim 33, wherein said pharmaceutical is designed to treat chronic and transient pain in a patient in need of treatment.

35. A pharmaceutical comprising one or more mRNAs according to claim 33, wherein the pharmaceutical further comprises an encapsulation element.

36. The use of one or more mRNAs to manufacture a medicament designed to treat chronic and transient pain according to claim 32, wherein the encapsulation assists with the delivery of the medicament to a patient in need of treatment for chronic and transient pain.

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