CN117295508A

CN117295508A - Opioid dosage reduction compositions and methods of use thereof

Info

Publication number: CN117295508A
Application number: CN202280014124.8A
Authority: CN
Inventors: S·伊茨库; R·布朗
Original assignee: Mesoblast International SARL
Current assignee: Mesoblast International SARL
Priority date: 2021-01-22
Filing date: 2022-01-21
Publication date: 2023-12-26

Abstract

The present disclosure relates to opioid dosage reduction compositions and uses thereof for reducing opioid use. Such compositions and methods can be used to reduce pain in a subject using an opioid or reduce opioid use by the subject or increase the mobility of the subject.

Description

Opioid dosage reduction compositions and methods of use thereof

Cross Reference to Related Applications

The present application claims priority from australian application No. 2021900147 filed on month 22 of 2021, australian application No. 2021900250 filed on month 4 of 2021, and U.S. application No. 63/298,391 filed on month 11 of 2022, each of which are incorporated herein by reference in their entirety.

Technical Field

Background

Opioids, also known as opioid agonists, are a group of drugs that exhibit opioid or morphine-like properties. Opioids are mainly used as moderate to severe analgesics, but there are many other pharmacological effects including sleepiness, respiratory depression, mood changes and mental retardation without causing loss of consciousness. Prolonged use of opioids is associated with various types of pain.

Lower back pain is a chronic condition associated with inflammation and pain that requires continued use of opioids as part of conventional pain management. This condition affects approximately two-thirds of the adult population in the united states, resulting in a significant increase in doctor's office visits and a significant impact on disabilities.

Although various therapeutic agents have been used to treat pain and/or inflammation, including chronic pain and/or inflammation, treatment is often ineffective even after chronic administration of these therapeutic agents.

Clearly, there is a need in the art for improved methods of managing pain in opioid users.

Disclosure of Invention

The inventors surprisingly found that administration of mesenchymal lineage precursors or stem cells (MLPSCs) can reduce pain or reduce opioid use by a subject using an opioid or increase EQ-5D score of the subject. Thus, in one example, the disclosure relates to a method of reducing pain or reducing opioid use or increasing EQ-5D score, the method comprising administering to a subject a composition comprising mesenchymal lineage precursors or stem cells (MLPSCs), wherein the subject is using an opioid. For example, the present disclosure relates to a method of reducing pain comprising administering to a subject a composition comprising mesenchymal lineage precursors or stem cells (MLPSCs), wherein the subject is using an opioid. In another example, the disclosure relates to a method of reducing opioid use, the method comprising administering to a subject a composition comprising mesenchymal lineage precursors or stem cells (MLPSCs), wherein the subject is using an opioid. In another example, the disclosure relates to a method of increasing EQ-5D score comprising administering to a subject a composition comprising mesenchymal lineage precursors or stem cells (MLPSCs), wherein the subject is using an opioid.

In one example, the composition includes Hyaluronic Acid (HA). In one example, the opioid reduced from the composition is morphine.

In one example, the methods of the present disclosure reduce pain and reduce opioid use. In another example, the methods of the present disclosure reduce pain and improve function while also reducing opioid use.

In one example, the subject is using opioids for pain. In one example, the pain is axial pain. In one example, the axial pain is caused by MRI-defined nerve root compression.

In view of the findings of the present inventors, it has been achieved an opioid dosage reduction composition that can be administered to reduce opioid use over time. Thus, in another example, the disclosure relates to opioid dosage reduction compositions comprising mesenchymal lineage precursors or stem cells (MLPSCs). In one example, the composition further comprises Hyaluronic Acid (HA). For example, the composition may include 1% HA. In one example, the composition includes 1% HA and culture amplified MLPSCs. In one example, the opioid dosage reduction composition is administered to a subject using an opioid, preferably wherein the subject is using the opioid for pain. In one example, the opioid use-reducing composition reduces opioid use by the subject. In one example, opioid use is reduced 36 months after administration of the composition. In one example, the opioid use-reducing composition stops opioid use by the subject. In one example, opioid use is stopped 36 months after administration of the composition.

The compositions and methods of the present disclosure may be used in the context of different types of pain. In one example, the pain is chronic pain. In another example, the pain is lower back pain. In one example, the lower back pain is associated with a degenerated disc. In another example, the lower back pain is associated with an intervertebral disc. For example, the disc height of the disc may not be significantly reduced compared to the disc height of an adjacent healthy disc of the subject. In another example, the origin of the lower back pain is non-rooted. In another example, the pain is associated with one or more of: herniated disc up to 3mm herniated; nerve ingrowth into the disc; or inflammation in the intervertebral disc. In one example, the nerve ingrowth or the inflammation is in the disc space or nucleus pulposus or annulus fibrosus of the disc.

In one example, opioid use is reduced 1 month after administration of the composition. In another example, opioid use is reduced 3 months after administration of the composition. In another example, opioid use is reduced 12 months after administration of the composition. In another example, opioid use is reduced 24 months after administration of the composition. In another example, opioid use is reduced 36 months after administration of the composition. In another example, opioid use is stopped 36 months after administration of the composition.

In one example, opioid use is reduced relative to the average baseline morphine equivalent dose for the subject prior to administration of the composition.

In one example, the subject has been using an opioid for at least 1 month prior to administration of the composition. In another example, the subject has used an opioid for at least 3 months prior to administration of the composition. In another example, the subject has used an opioid for at least 6 months prior to administration of the composition.

In one example, the subject has experienced pain for 6 months to 68 months. In another example, the subject has not experienced pain for more than 68 months.

In one example, the opioid use of the subject is reduced by about 20% relative to its baseline opioid use prior to administration of the composition. In another example, the opioid use of the subject is reduced by about 30% relative to its baseline opioid use prior to administration of the composition. In another example, the opioid use of the subject is reduced by about 40% relative to its baseline opioid use prior to administration of the composition.

In one example, the subject's average Visual Analog Scale (VAS) score is reduced relative to its VAS score prior to administration of the composition. In another example, the subject has a VAS pain response of 30%. In another example, the subject has a VAS pain response of 50%.

In one example, the average EQ-5D score of the subject is increased relative to baseline. In one example, the increase is determined from an EQ-5D VAS score. In one example, the increase is observed 12 months after administration of the composition of the present disclosure. In one example, the increase is observed 36 months after administration of the composition of the present disclosure. In one example, the increase is determined from an EQ-5D index score. In one example, the increase is observed 12 months after administration of the composition of the present disclosure. In another example, the increase is observed 24 months after administration of the composition of the present disclosure. In another example, the increase is observed 36 months after administration of the composition of the present disclosure.

In one example, the subject achieves an ODI 10 score function response. In another example, the subject achieves an ODI 15 score function response. In another example, the subject achieves an ODI 10 score function response and a 30% VAS pain response. In another example, the subject achieves an ODI 15 score function response and a VAS pain response of 50%. In one example, a composition comprising MLPSC and HA is administered to these subjects. In one example, the subject achieves an ODI 10 sub-functional response 12 months after administration of the composition of the disclosure. In one example, the subject does not use an opioid prior to administration of the composition. In one example, the subject achieves an ODI 10 sub-functional response 36 months after administration of the composition of the disclosure. In one example, the subject is using an opioid prior to administration of the composition.

In another example, the subject achieved an ODI 10 score function response and a 30% VAS pain response, wherein the subject was using an opioid prior to administration of the composition of the present disclosure, and the subject had not reported lower back pain for more than 68 months. In one example, an ODI 10 score function response and a 30% VAS pain response were observed 24 months after administration of the compositions of the present disclosure. In one example, an ODI 10 score function response and a 30% VAS pain response were observed 36 months after administration of the compositions of the present disclosure.

In one example, the subject has no significant pain 12 months after administration of the composition. In another example, the subject has no significant pain 24 months after administration of the composition.

In one example, the subject has an ODI score of between 25% and 70%. In another example, the subject has an ODI score of between 30% and 60%.

In one example, the MLPSC is STRO-1+. In one example, the MLPSC is a Mesenchymal Stem Cell (MSC). In one example, the cells are allogeneic. In one example, the cells are expanded by culture. In one example, the cells are tnap+ prior to their culture expansion. In one example, the cells have been cryopreserved.

In one example, the methods of the present disclosure include administering 1x 10 ⁷ Up to 2x 10 ⁸ Individual cells. Thus, the compositions of the present disclosure may include 1x 10 ⁷ Up to 2x 10 ⁸ Individual cells.

In one example, the compositions of the present disclosure include human bone marrow derived allogeneic Mesenchymal Precursor Cells (MPCs) isolated from bone mononuclear cells using anti-STRO-3 antibodies, expanded ex vivo, and cryopreserved.

In one example, the subject stops opioid use 36 months after administration of the composition.

In another example, the disclosure relates to preparing a medicament for use in stopping opioid use by a subject. In one example, the compositions disclosed herein are used to prepare the medicament. In one example, opioid use is stopped 36 months after administration of the drug disclosed herein to a subject. For example, the medicament may be administered to a subject suffering from chronic pain. In one example, the subject has lower back pain.

Drawings

Fig. 1: changes in LS mean VAS pain from baseline for opioid users, mpc+ha achieved significantly greater mean pain relief from baseline at each time point over 24 months compared to placebo.

Fig. 2: opioid users had 50% and 30% VAS pain responses, with mpc+ha significantly increased the proportion of patients with 50% and 30% VAS pain responses at 24 months compared to placebo.

Fig. 3: the minimum pain to no pain (LBP VAS. Ltoreq.20) for opioid users significantly increased the proportion of patients with minimum pain/no pain at 6 months, 12 months, 18 months and 24 months compared to placebo.

Fig. 4: comparison of ODI and EQ5D indices as functional metrics for opioid users, average changes in EQ5D index from baseline better identify MPC therapeutic effects.

Fig. 5: EQ5D is a better identification feature than ODI in determining MIC treatment success rate for opioid users.

Fig. 6: mpc+ha significantly increased the proportion of patients who achieved a combined therapeutic response of 50% VAS/0.03EQ5D and 30% VAS/0.03EQ5D at 24 months.

Fig. 7: the LBP-EQ5D index overall treatment success rate and MIC overall treatment success rate for opioid users significantly increased the proportion of patients who achieved 30% VAS/0.03EQ5D integrated treatment response at both 12 months and 24 months.

Fig. 8: the LBP-EQ5D index overall treatment success rate and MIC overall treatment success rate for opioid users significantly increased the proportion of patients who achieved 30% VAS/0.03EQ5D integrated treatment response at both 12 months and 24 months.

Fig. 9: mpc+ha significantly reduced morphine milliequivalent usage of opioid for 24 months in patients who used opioid at baseline.

Fig. 10: mpc+ha reduces the morphine milliequivalents for opioid users over 24 months.

Fig. 11: mpc+ha: VAS 30% + opioid reduction ^* 。( ^* All subjects were opioid users at baseline, and had opioid values at each time point assessed).

Fig. 12: mpc+ha: VAS 50% + opioid reduction ^* 。

Fig. 13: mpc+ha: comprehensive response; ODI-10+ opioid reduction ^* 。

Fig. 14: mpc+ha: comprehensive response; ODI-15+ opioid reduction ^* 。

Fig. 15: mpc+ha: comprehensive response; VAS 30% + ODI-10+ opioid reduction ^* 。

Fig. 16: mpc+ha: comprehensive response; VAS 50% + ODI-15+ opioid reduction ^* 。

Fig. 17: mpc+ha: success rate of treatment; VAS+ODI response+opioid reduction from baseline at both 12 months and 24 months ^* And without intervention. Therapeutic success VAS/ODI: VAS evaluation by subjects at both 12 months and 24 months50% decrease from baseline and 15% increase in ODI score from baseline, and no post-treatment intervention for 24 months, and opioid decrease at 24 months. The subject's VAS score was reduced by 30% relative to baseline at both 12 months and 24 months, and the ODI score was increased by 10 points relative to baseline, with no post-treatment intervention for 24 months, and opioid reduction at 24 months.

Fig. 18: mpc+ha: success rate of treatment; VAS+EQ5D response+opioid reduction from baseline at both 12 months and 24 months ^* And without intervention. Therapeutic success VAS/EQ5D index: the subject's VAS score was reduced by 50% relative to baseline at both 12 months and 24 months, and the EQ5D index score was increased by 0.03 score relative to baseline, with no post-treatment intervention for 24 months, and opioid was reduced at 24 months. The subject's VAS score was reduced by 30% relative to baseline at both 12 months and 24 months, and the EQ5D index score was increased by 0.03 score relative to baseline, with no post-treatment intervention for 24 months, and opioid was reduced at 24 months.

Fig. 19: changes in back pain from baseline under LS mean VAS-all subjects (n=391).

Fig. 20: changes in back pain from baseline at LS mean VAS-CLBP < median (n=194).

Fig. 21: changes in LS mean ODI from baseline-CLBP < median (n=197).

Fig. 22: LHS: ODI 10 score MIC responders-CLBP is less than or equal to the median value; RHS: MIC 30% VAS/10 min ODI treatment success rate-CLBP is less than or equal to the median value.

Fig. 23: EQ5D index CFB-all subjects (LHS); CLBP duration < median (RHS).

Fig. 24: EQ5D MCIC index responders-all subjects (LHS); CLBP duration < median (RHS).

Fig. 25:30% VAS-EQ5D 0.03 score index MIC treatment success rate-all subjects (LHS); CLBP duration < median (RHS).

Fig. 26:50% VAS-EQ5D 0.03 score index MIC treatment success rate-all subjects (LHS); CLBP duration < median (RHS).

Fig. 27: LHS: VAS 30%/ODI 10% MIC treatment success rate, CLBP duration < median; RHS: AS 30%/EQ5d 0.03 min MIC treatment success rate, CLBP < median. ODI MIC treatment success-lower back pain VAS score of subjects was reduced by 30% relative to baseline, and ODI was increased by 10 score relative to baseline, with no post-treatment intervention at the evaluation time point. EQ5D index score MIC treatment success-subject's lower back pain VAS score was reduced by 30% relative to baseline, and EQ5D index score was increased by 0.03 score relative to baseline, with no post-treatment intervention at the evaluation time point.

Fig. 28: mean change in LS for mean pain over 24 hours measured by VAS-a subset of baseline opioid users pre-specified based on patient electronic diary pain drug usage entries.

Fig. 29: mean change in LS of average pain over 24 hours measured by VAS-all subjects.

Fig. 30: the proportion of subjects with reduced average pain medication relative to baseline at 24 months.

Fig. 31: ratio of baseline opioid users who did not use opioid at 6 months, 12 months, 18 months, 24 months, and 36 months.

Fig. 32: average pain least significant change (MIC; 30% reduction) respondents analysis-a subset of subjects with a pre-specified duration CLBP less than median (68.8 months).

Fig. 33: LS mean function change measured by ODI-duration CLBP was less than the median of 36 months for opioid users and opioid non-users.

Fig. 34: mean LS change for morphine equivalent dose for opioid users for 36 months.

Detailed Description

General techniques and definitions

Unless specifically stated otherwise, all technical and scientific terms used herein should be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular biology, stem cell culture, immunology, and biochemistry).

Unless otherwise indicated, cell culture techniques and assays used in the present disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained in the literature of the following sources: such as J.Perbal, molecular cloning Utility Specification (A practical Guide To Molecular Cloning), john Wili's father-son publishing company (John Wiley and Sons) (1984); sambrook et al, molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), cold spring harbor laboratory Press (Cold Spring Harbour Laboratory Press) (1989); brown (edit), "basic molecular biology: practical methods (Essential Molecular Biology: A Practical Approach), volumes 1 and 2, IRL Press (1991); glover and B.D.Hames (editions), and F.M.Ausubel et al (editions), guidelines for contemporary molecular biology experiments (Current Protocols in Molecular Biology), greene Pub.associates, and Wiley International science publication (Wiley-Interscience) (1988, including all updates to date); ed Harlow and David Lane (edit) antibody: laboratory manuals (Antibodies: A Laboratory Manual), cold spring harbor laboratory, (1988); and J.E.Coligan et al (editions), "contemporary immunology guidelines (Current Protocols in Immunology)," John Wili father-son publishing company (including all updates so far).

The term "and/or", for example, "X and/or Y" is understood to mean "X and Y" or "X or Y", and should be considered as providing explicit support for both meanings or either meaning.

As used herein, unless specified to the contrary, the term "about" refers to +/-10%, more preferably +/-5% of the specified value.

The terms "level" and "amount" are used to define the amount of a particular substance in a sample from a subject or in a cell culture medium (or sample therefrom). For example, a particular concentration, weight, percentage (e.g., v/v%) or ratio may be used to define the level of a particular substance in a sample. In one example, the level is expressed in terms of how much of a particular marker is expressed by a cell of the present disclosure under culture conditions. In one example, expression refers to cell surface expression. In another example, the level is expressed in terms of how much of a particular marker is released from the cells described herein under culture conditions. In one example, a sample (e.g., a blood sample) is obtained from a patient or subject, and the level of a substance in the sample is measured to determine the level of the substance in the sample.

In one example, the level is expressed in pg/ml. In another example, the level is at every 10 ⁶ Pg of individual cells.

In one example, the level of a particular marker in the cell culture medium is determined under culture conditions. The term "culture conditions" is used to refer to cells that are grown in culture. In one example, culture conditions refer to actively dividing cell populations. In one example, such cells may be in an exponential growth phase. For example, the level of a particular marker can be determined by taking a sample of cell culture medium and measuring the level of the marker in the sample. In another example, the level of a particular marker may be determined by taking a sample of cells and measuring the level of the marker in the cell lysate. The skilled person will measure secreted markers by sampling the medium, whereas markers expressed on the cell surface can be measured by evaluating samples of cell lysates. In one example, the sample is obtained while the cells are in an exponential growth phase. In one example, the sample is obtained after at least two days of culture.

The expansion of cells in culture from cryopreserved intermediates means that cells subjected to low temperature freezing are thawed and cultured in vitro under conditions suitable for cell growth.

In one example, the "level" or "amount" of a particular marker is determined after cells are cryopreserved and then re-inoculated into culture. For example, the level is determined after the first cryopreservation of the cells. In another example, the level is determined after a second cryopreservation of the cells. For example, cells may be culture expanded from a cryopreserved intermediate, and a second cryopreservation performed prior to re-seeding in culture, so that the level of a particular marker may be determined under culture conditions.

The term "opioid use reducer" is used in the context of the present disclosure to refer to a composition that may be administered to a subject using an opioid to facilitate reducing opioid intake by the subject. In other words, subjects administered the opioid dosage reducer of the present disclosure are able to take lower doses of opioid over time. In one example, the opioid use reducer reduces pain and/or increases the mobility of the subject.

As used herein, the terms "treating", "alleviating" and "reducing opioid use", "increasing activity" comprise administering mesenchymal lineage stem cells or precursor cells and/or their progeny and/or soluble factors derived therefrom and/or extracellular vesicles derived therefrom to thereby alleviate pain and/or reduce opioid use and/or increase activity in a subject using opioid.

As used herein, the term "subject" refers to a human subject. For example, the subject may be an adult. In another example, the subject may be a child. In another example, the subject may be an adolescent. Terms such as "subject," "patient," or "individual" are terms that may be used interchangeably in the context of the present disclosure. The subject in need of treatment comprises a subject using an opioid. For example, the subject may be using opioids for pain (e.g., back pain as follows). In one example, pain and opioid use in a subject is underway. For example, the subject may have pain for more than 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 24 months, or longer. Thus, in one example, the subject may use the opioid for more than 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 24 months, or longer. In one example, the subject is using morphine. In one example, the subject has not reported lower back pain for more than 68 months.

In another example, the subject has an ostyle disability index (Oswestry Disability Index, ODI) score of between 25% and 70%. In another example, the subject has an ODI score of between 30% and 60%. The ODI score can be used to define a patient as follows: 0% -20%: minimal disability; 21% -40%: moderate disability; 41% -60%: severe disability; 61% -80%: disabling back pain; 81% -100%: bedridden or exaggerated symptoms. In one example, the subject has experienced pain for at least one month. In another example, the subject has experienced pain for at least 3 months. In another example, the subject has experienced pain for at least 6 months. In another example, the subject has experienced pain for at least 12 months. In another example, the subject has experienced pain for at least 24 months. In another example, the subject has experienced pain for at least 36 months. In another example, the subject has experienced pain for at least 48 months. In another example, the subject has experienced pain for at least 60 months. In another example, the subject has experienced pain for 6 months to 78 months. In another example, the subject has experienced pain for 6 months to 70 months. In another example, the subject has experienced pain for 2 months to 68 months. In another example, the subject has experienced pain for 3 months to 68 months. In another example, the subject has experienced pain for 6 months to 68 months. In another example, the subject has experienced pain for 12 months to 68 months. In another example, the subject has not experienced pain for more than 68 months. In one example, pain in a subject is in need of treatment with an analgesic. In one example, the analgesic is an opioid. In one example, the opioid is morphine. For example, the subject may have experienced chronic lower back pain for 3 months to 68 months. In this example, the subject may have been treated with an opioid. In one example, the subject has experienced chronic lower back pain for at least 6 months. In one example, the subject has experienced chronic lower back pain for 6 months to 12 months.

In certain embodiments, the present disclosure relates to methods and compositions for reducing pain, such as chronic lower back pain. The reduction of pain may be determined using various methods as discussed herein (e.g., VAS, EQ-5D, ODI). The term "axial pain" is used in the context of the present disclosure to refer to localized pain (e.g., neck pain or leg pain) localized to a certain location or area.

In one example, the compositions of the present disclosure include mesenchymal precursor lineages or stem cells that have not been genetically modified. As used herein, the term "non-genetically modified" refers to cells that have not been modified by transfection with a nucleic acid. For the avoidance of doubt, in the context of the present disclosure, mesenchymal lineage precursors or stem cells transfected with a nucleic acid encoding a protein will be considered genetically modified.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step or group of elements, integers or steps, but not the exclusion of any other element, integer or step or group of elements, integers or groups of steps.

Throughout this specification, unless the context clearly indicates otherwise, reference to a single step, composition of matter, group of steps, or group of compositions of matter should be taken to encompass one or more (i.e., one or more) of those steps, compositions of matter, group of steps, or group of compositions of matter.

It will be appreciated by those skilled in the art that variations and modifications other than those specifically described may be made to the disclosure described herein. It is to be understood that the present disclosure encompasses all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

The scope of the present disclosure is not limited by the specific embodiments described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods, as described herein, are clearly within the scope of the disclosure.

Any examples disclosed herein should be considered applicable to any other examples, mutatis mutandis, unless explicitly stated otherwise.

Opioid use

The methods of the present disclosure relate to subjects using opioids. In one example, the subject is using opioids for pain. In one example, the pain is chronic pain. For example, the pain may be lower back pain. Opioids are a group of analgesic drugs that exhibit opioid or morphine-like properties. In one example, the opioid may be referred to as an opioid receptor agonist.

Exemplary opioids include alfentanil, allyl protamine, alfamodine, anilidine, benzomorphine, benzonitrile, buprofezin, clonazepine, codeine, and dihydrodeoxymorphine (dextromorphine), dextromorphine (dezocine), dezocine (diampro-mide), dimorphine (diamorphine), dihydrocodeine (dihydrocodeine), dihydromorphine (dihydroomphin), dimefloxadol (dimefadiol), dimefoheptanol (dimefeptanol), dimethylthiodine (dimethylthiamine) butyl benzene (dioxaphetyl butyrate), dipiperidone (dipiperazine), etazocine (eptazocine), ethoheptzine (ethohepsin), ethyltimothy (ethyltimibene), etomizole (etonitazene), fentanyl (Fentanyl), heroin (hermin), hydrocodone (hydrocodone), hydromorphone (hydromorphone), hydroxy pethidine (hydroypetidine), isomethadone (isomethadone), ketomide (ketobemidone), levorphanol (levorphanol), lofenadine (lofenadine), meptazin (meperidine), meptazinol (meptazinol), methadone (methadone), and methadone, morphine (morphone), meflorphine (myophine), papaverine (narcisline), nicomorphine (nicomorphine), norlevorphanol (norlevvoranol), normethadone (normethazine), nalorphine (nalorphine), nalbuphine (nalbephine), normorphine (normorphine), nopiperazine (norpiprazole), opium (opium), oxycodone (oxymorphone), oxymorphone (oxymorphone), opium (paverine), pantoprazole (pentazocine), benzoepine (phenoxazone), fenoxaprop (phenoxazone), desidine (piridine), benazepine (propitiapine), benazepine (propiverine), propiverine (propitiapine), propimorph (propimorph), and the like. In one example, the opioid is morphine or an analog thereof that interacts with morphine receptors.

In one example, the subject has been using an opioid for at least one month. In another example, the subject has been using an opioid for at least 3 months. In another example, the subject has been using an opioid for at least 6 months. In another example, the subject has been using an opioid for at least 12 months. In another example, the subject has been using an opioid for at least 24 months. In another example, the subject has been using an opioid for at least 36 months. In another example, the subject has been using an opioid for at least 48 months. In another example, the subject has been using an opioid for at least 60 months. In another example, the subject has used an opioid for 6 months to 78 months. In another example, the subject has used an opioid for 6 months to 70 months. In another example, the subject has used an opioid for 6 months to 68 months. In another example, the subject has not used opioid for more than 68 months.

In one example, the mean baseline Morphine Equivalent Dose (MED) for the subject is <75 mg/day. In one example, the MED is determined by an electronic diary entry of the subject.

In another example, the subject is using opioid for at least 6 months for chronic lower back pain. In another example, the subject fails conservative back pain care for at least 2 months, 3 months, 4 months, 5 months, or 6 months. Examples of conservative treatment regimens may include any or all of the following: initial rest, medications such as anti-inflammatory, analgesic, anesthetic/opioid, muscle relaxant, or other interventions such as massage, acupuncture, spinal correction, activity adjustment, home directed lumbar exercise program.

In another example, subjects using opioids receive supervised physical therapy such as daily walking, therapeutic exercise, and back education programs specifically for the treatment of lower back pain.

In another example, a subject using an opioid has a modified pfirmann score of 3, 4, 5, or 6 at a landmark disc (index disc) on MRI and/or a Modic class II change or less at a landmark disc on MRI. In one example, a subject using an opioid may or may not have an included disc herniation at a landmark disc on MRI.

In one example, a subject using an opioid may have a 90mm lower back pain of at least 40mm and no more than 100mm in lower back pain VAS (average pain over 24 hours).

In another example, a subject using an opioid has lower back pain associated with a degenerated disc. In another example, the lower back pain is associated with an intervertebral disc. In one example, the disc height of the disc is not significantly reduced compared to the disc height of an adjacent healthy disc of the subject. In another example, the origin of the lower back pain is non-rooted and/or the lower back pain is associated with one or more of: herniated disc up to 3mm herniated; nerve ingrowth into the disc; inflammation in the intervertebral disc. In one example, the nerve ingrowth or the inflammation is in the disc space or nucleus pulposus or annulus fibrosus of the disc.

In another example, the pain is axial pain. Thus, in one example, the present disclosure relates to a method of treating axial pain in a subject, the method comprising administering a composition of the present disclosure. In one example, the subject is using an opioid. In one example, the composition includes an MLPSC and HA. In one example, the axial pain is leg pain. In one example, the axial pain is caused by MRI-defined nerve root compression. In one example, the subject has not reported chronic pain for more than 68 months. In another example, the subject has not used opioid for more than 68 months.

Pain relief or opioid use reduction or locomotor activity enhancement

In one example, the methods of the present disclosure reduce pain in a subject using an opioid. In one example, administration of a composition of the present disclosure reduces Visual Analog Scale (VAS) pain response in a subject. VAS is a measurement tool commonly used to measure pain because it facilitates measurement of characteristics or attitudes believed to encompass continuous values that are not readily measured directly. In short, pain VAS is a one-dimensional measurement of pain intensity. The patient marks on the line the point that he perceives to be representative of his current pain state. The VAS score is determined by measuring the point from the left hand end of the line to the patient marker in millimeters. The VAS scores can be compared over time to measure changes in pain levels. Thus, it may be a useful indicator of pain relief. In one example, administration of the compositions of the present disclosure reduces the Visual Analog Scale (VAS) pain response of a subject by 10%. In another example, administration of a composition of the present disclosure reduces VAS in a subject by 20%. In another example, administration of the compositions of the present disclosure reduces VAS in a subject by 30%. In another example, administration of a composition of the present disclosure reduces VAS in a subject by 50%. In these examples, the reduction in VAS can be determined by comparison to a baseline VAS score obtained prior to the treatment disclosed herein. In one example, a decrease in VAS is observed 12 months after treatment. In another example, a decrease in VAS is observed 24 months after treatment.

In one example, the subject has no significant pain after administration of the composition of the present disclosure. In one example, the subject has no significant pain 12 months after administration of the composition of the present disclosure. In another example, the subject has no significant pain 24 months after administration of the composition of the present disclosure.

The inventors have identified that the compositions of the present disclosure can be used as opioid dosage reducers. Thus, in another example, the methods of the present disclosure reduce opioid use by a subject. In one example, opioid use is reduced 1 month after administration of the composition. In another example, opioid use is reduced 3 months after administration of the composition. In another example, opioid use is reduced 6 months after administration of the composition. In another example, opioid use is reduced 12 months after administration of the composition. In another example, opioid use is reduced 18 months after administration of the composition. In another example, opioid use is reduced 24 months after administration of the composition. In another example, opioid use is reduced 36 months after administration of the composition. In one example, the subject's opioid use is reduced relative to its baseline opioid use prior to administration of the composition of the present disclosure. For example, opioid use by the subject may be reduced relative to the average baseline morphine equivalent dose for the subject prior to administration of the composition.

In one example, the opioid use of the subject is reduced by about 20% relative to its baseline opioid use prior to administration of the composition. In another example, the opioid use of the subject is reduced by about 30% relative to its baseline opioid use prior to administration of the composition. In another example, the opioid use of the subject is reduced by about 40% relative to its baseline opioid use prior to administration of the composition. In one example, the opioid is morphine and the reduction is relative to the average baseline morphine equivalent dose for the subject prior to administration of the composition.

In one example, the subject stops opioid use after administration of the composition of the present disclosure. In one example, the subject stops opioid use 12 months after administration of the composition of the present disclosure. In another example, the subject stops opioid use 18 months after administration of the composition of the present disclosure. In another example, the subject stops opioid use 24 months after administration of the composition of the present disclosure. In another example, the subject stops opioid use 36 months after administration of the composition of the present disclosure. In another example, the subject stops opioid use 18 months to 36 months after administration of the composition of the present disclosure.

In one example, the methods of the present disclosure increase the mobility and/or function of a subject using an opioid. In one example, activity capability and/or function is described as subject function based on well established measurements of criteria such as ODI and/or EQ-5D. In one example, administration of a composition of the present disclosure increases the EuroQol-5 dimension (EQ-5D) score of a subject relative to baseline. Thus, in one example, the methods of the present disclosure increase the EQ-5D score of subjects using opioids. EQ-5D provides a simple descriptive profile and a single index of health. The EQ-5D self-reporting questionnaire contains a Visual Analog Scale (VAS) that records the self-rated health status of respondents in a hierarchical (0-100) scale, where the higher the health-related quality of life, the higher the score. In certain examples, EQ-5D also includes an EQ-5D descriptive system that includes 5 health dimensions: mobility, self care, daily activities, pain/discomfort and anxiety/depression. This descriptive system may be used as a health profile or converted to an exponential score (EQ-5D exponent) that represents the von Neumann-morgan utility value (von Neumann-Morgenstern utility value) of current health.

In one example, the EQ-5D increase lasts at least 1 month to 6 months. In one example, the EQ-5D increase lasts at least 6 months to 12 months. In one example, EQ-5D increases by at least 0.01. In one example, EQ-5D increases by at least 0.02. In one example, EQ-5D increases by at least 0.03. In one example, EQ-5D increases by 0.01 to 0.04. In one example, the increase is determined based on the EQ-5D VAS.

In another example, administration of a composition of the present disclosure can reduce the ODI score of a subject. In one example, the ODI is reduced by at least 10 minutes or at least 15 minutes for at least 1 month to 6 months. In one example, the ODI is reduced by at least 10 minutes or at least 15 minutes for at least 6 months to 12 months. In another example, the ODI is reduced by at least 10 minutes or at least 15 minutes for at least 24 months.

In another example, the methods of the present disclosure relate to treating a subject, the methods comprising administering a composition according to the present disclosure and an opioid, wherein a plurality of doses of the opioid are administered to the subject, and the doses of the opioid are reduced throughout the course of treatment. For example, the dosage of opioid may be reduced by about 20% -50% during treatment following administration of the compositions of the present disclosure.

In another example, the present disclosure relates to a method of reducing opioid use in a subject, the method comprising administering a composition of the present disclosure. For example, the method can include administering a composition that includes MLPSC and HA. In one example, the subject has chronic lower back pain. In one example, the subject has been free of chronic lower back pain for more than 68 months. In one example, opioid use is reduced from 12 months to 36 months after administration of the compositions of the present disclosure. In one example, the reduction in opioid use persists for 36 months after administration of the compositions of the present disclosure. In one example, opioid use is stopped 18 months to 36 months after administration of the composition of the present disclosure.

Mesenchymal precursor cells

As used herein, the term "mesenchymal lineage precursor or stem cells (MLPSC)" refers to undifferentiated pluripotent cells that have the ability to self-renew while retaining the ability to differentiate into cell types of many mesenchymal sources (e.g., osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and tendons) or non-mesodermal sources (e.g., hepatocytes, neural cells, and epithelial cells). For the avoidance of doubt, "mesenchymal lineage precursor cells" refers to cells that can differentiate into mesenchymal cells such as bone, cartilage, muscle and fat cells, and fibrous connective tissue.

The term "mesenchymal lineage precursor or stem cell" encompasses the parental cell and its undifferentiated progeny. The term also encompasses mesenchymal precursor cells, pluripotent stromal cells, mesenchymal Stem Cells (MSCs), perivascular mesenchymal precursor cells, and undifferentiated progeny thereof.

The mesenchymal lineage precursor or stem cells can be autologous, allogeneic, xenogeneic, syngeneic or isogenic. Autologous cells are isolated from the same individual in which they are to be re-implanted. Allogeneic cells are isolated from a donor of the same species. The xenogeneic cells are isolated from a donor of another species. Homologous or isogenic cells are isolated from genetically identical organisms, such as twins, clones or highly inbred research animal models.

In one example, the mesenchymal lineage precursor or stem cells are allogeneic. In one example, allogeneic mesenchymal lineage precursors or stem cells are expanded in culture and cryopreserved.

Mesenchymal lineage precursors or stem cells are found predominantly in bone marrow, but are also shown to be present in a variety of host tissues including, for example, umbilical cord blood and cord, adult peripheral blood, adipose tissue, trabecular bone, and dental pulp. It is also present in skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicle, intestine, lung, lymph node, thymus, ligament, tendon, skeletal muscle, dermis and periosteum; and is capable of differentiating into a germ line, such as mesoderm and/or endoderm and/or ectoderm. Thus, mesenchymal lineage precursors or stem cells can differentiate into a wide variety of cell types including, but not limited to, fat, bone, cartilage, elastic tissue, muscle, and fibrous connective tissue. The particular lineage commitment and differentiation pathway that these cells enter depends on various effects from mechanical influences and/or endogenous bioactive factors such as growth factors, cytokines, and/or local microenvironment conditions established by the host tissues.

The term "enriched", "enriched" or variants thereof are used herein to describe a population of cells having an increased proportion of one particular cell type or of a plurality of particular cell types as compared to an untreated population of cells (e.g., cells in their natural environment). In one example, the population enriched for mesenchymal lineage precursors or stem cells includes at least about 0.1%, or 0.5%, or 1%, or 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 50%, or 75% of mesenchymal lineage precursors or stem cells. In this regard, the term "cell population enriched for mesenchymal lineage precursors or stem cells" will be employed to expressly support the term "cell population comprising X% mesenchymal lineage precursors or stem cells," where X% is a percentage as described herein. In some examples, the mesenchymal lineage precursor or stem cells can form clonogenic colonies, e.g., CFU-F (fibroblasts) or a subset thereof (e.g., 50%, or 60%, or 70%, or 90%, or 95%) can have this activity.

In an example of the present disclosure, the mesenchymal lineage precursor or stem cells are Mesenchymal Stem Cells (MSCs). MSCs may be of homogeneous composition or may be a mixed cell population enriched in MSCs. Homogeneous MSC compositions can be obtained by culturing adherent bone marrow or periosteal cells, and MSCs can be identified by specific cell surface markers identified with unique monoclonal antibodies. For example, in U.S. Pat. No. 5,486,359, a method for obtaining a population of cells enriched in MSC is described. Alternative sources of MSCs include, but are not limited to, blood, skin, cord blood, muscle, fat, bone, and perichondrium. In one example, the MSC is allogeneic. In one example, the MSC is cryopreserved. In one example, MSCs are expanded by culture and cryopreserved.

In another example, the mesenchymal lineage precursor or stem cell is cd29+, cd54+, cd73+, cd90+, cd102+, cd105+, cd106+, cd166+, MHC1+ MSC.

Isolated or enriched mesenchymal lineage precursors or stem cells can be expanded in vitro by culture. Isolated or enriched mesenchymal lineage precursors or stem cells can be cryopreserved, thawed, and then expanded in vitro by culture.

In one example, the isolated or enriched mesenchymal lineage precursor or stem cells are at 50,000 viable cells/cm ² Inoculated in a medium (serum-free or serum-supplemented), for example, an αminimum essential medium (αMEM) supplemented with 5% Fetal Bovine Serum (FBS) and glutamine, and maintained at 37℃at 20% O ₂ Adhere to the culture vessel overnight. The medium is then replaced and/or changed as required, and the cells are incubated at 37℃with 5% O ₂ Further culturing for 68-72 hours.

As will be appreciated by those skilled in the art, cultured mesenchymal lineage precursors or stem cells are phenotypically different from in vivo cells. For example, in one embodiment, it expresses one or more of the following markers: CD44, NG2, DC146, and CD140b. The cultured mesenchymal lineage precursor or stem cells are also biologically different from in vivo cells, with higher proliferation rates than most non-circulating (quiescent) cells in vivo.

In one example, the population of cells is enriched from a cell preparation comprising an alternative form of STRO-1+ cells. In this regard, the term "selectable form" will be understood to mean that the cells express a marker (e.g., a cell surface marker) that allows selection of STRO-1+ cells. The marker may be, but is not necessarily, STRO-1. For example, as described and/or exemplified herein, cells expressing STRO-2 and/or STRO-3 (TNAP) and/or STRO-4 and/or VCAM-1 and/or CD146 and/or 3G5 (e.g., mesenchymal precursor cells) also express STRO-1 (and may be STRO-1 bright). Thus, the indication that the cell is STRO-1+ does not mean that the cell is selected by STRO-1 expression alone. In one example, cells are selected based at least on STRO-3 expression, e.g., which is STRO-3+ (TNAP+).

References to the selection of cells or populations thereof do not necessarily require selection from a particular tissue source. STRO-1+ cells may be selected from or isolated or enriched from a variety of sources, as described herein. That is, in some examples, these terms provide support for selection from any tissue or vascularized tissue comprising STRO-1+ cells (e.g., mesenchymal precursor cells) or tissue comprising pericytes (e.g., STRO-1+ pericytes) or any one or more of the tissues described herein.

In one example, the cells used in the present disclosure express one or more markers, either alone or in combination, selected from the group consisting of: TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90 beta), CD45+, CD146+, 3G5+, or any combination thereof.

By "individually" is meant that the present disclosure individually encompasses the markers or sets of markers, and although individual markers or sets of markers may not be individually listed herein, the appended claims may define such markers or sets of markers individually and separately from each other.

"collectively" means that the present disclosure encompasses any number or combination of the markers or sets of markers, and that although such number or combination of markers or sets of markers may not be specifically listed herein, the appended claims may define such combination or sub-combination separately and separately from any other marker combination or set of markers.

As used herein, the term "TNAP" is intended to encompass all isoforms of tissue-non-specific alkaline phosphatase. For example, the term encompasses liver isotype (LAP), bone isotype (BAP) and kidney isotype (KAP). In one example, the TNAP is BAP. In one example, TNAP as used herein refers to a molecule that can bind to STRO-3 antibodies produced by a hybridoma cell line deposited with ATCC under the provisions of the Budapest Treaty at 12/19/2005 under deposit accession number PTA-7282.

Furthermore, in one example, STRO-1+ cells are capable of producing clonogenic CFU-F.

In one example, a significant proportion of STRO-1+ cells are capable of differentiating into at least two different lineages. Non-limiting examples of lineages into which STRO-1+ cells may committed include: bone precursor cells; a hepatocyte progenitor cell having multipotency for biliary epithelial cells and hepatocytes; a neural restricted cell that can generate glial cell precursors that progress into oligodendrocytes and astrocytes; a neuron precursor that progresses to a neuron; myocardium and precursors of cardiomyocytes, glucose-responsive insulin secreting pancreatic beta cell lines. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells and chondrocytes, as well as precursor cells of: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal catheter epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelial cells, glial cells, neurons, astrocytes and oligodendrocytes.

In one example, the mesenchymal lineage precursors or stem cells are obtained from a single donor or multiple donors, where the donor samples or mesenchymal lineage precursors or stem cells are then pooled and then culture expanded.

Mesenchymal lineage precursors or stem cells encompassed by the present disclosure can also be cryopreserved prior to administration to a subject. In one example, the mesenchymal lineage precursor or stem cells are culture expanded and cryopreserved prior to administration to a subject.

In one example, the present disclosure encompasses mesenchymal lineage precursors or stem cells and their progeny, soluble factors derived therefrom, and/or extracellular vesicles isolated therefrom. In another example, the disclosure encompasses mesenchymal lineage precursors or stem cells and extracellular vesicles isolated therefrom. For example, mesenchymal precursor lineages or stem cells of the present disclosure may be expanded in culture for a period of time under conditions suitable for secretion of extracellular vesicles into the cell culture medium. Secreted extracellular vesicles can then be obtained from the culture medium for use in therapy.

As used herein, the term "extracellular vesicles" refers to lipid particles that are naturally released from cells and range in size from about 30nm to as large as 10 microns, although their size is typically less than 200nm. It may contain cells derived from the release (e.g., mesenchymal stem cells; STRO-1) ⁺ Cells), protein, nucleic acid, lipid, metabolite or organelle。

As used herein, the term "exosome" refers to a type of extracellular vesicle, typically in the range of about 30nm to about 150nm in size, and derived from a mammalian cell's endosomal compartment from which it is transported to the cell membrane and released. It may contain nucleic acids (e.g., RNA; microRNA), proteins, lipids, and metabolites, and may play a role in intercellular communication by being secreted from one cell and taken up by other cells to deliver its cargo.

In one example, the compositions of the present disclosure include cells that induce neovascularization in a target tissue.

Culture expansion of cells

In one example, the mesenchymal lineage precursor or stem cells are expanded by culture. The "culture expanded" mesenchymal lineage precursor or stem cell culture medium differs from freshly isolated cells in that it has been cultured and passaged (i.e., subcultured) in the cell culture medium. In one example, culture-expanded mesenchymal lineage precursors or stem cells are culture-expanded from about 4 passages to 10 passages. In one example, the mesenchymal lineage precursor or stem cells are expanded by culture at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, at least 10 passages. For example, mesenchymal lineage precursors or stem cells can be expanded by culture for at least 5 passages. In one example, the mesenchymal lineage precursor or stem cells can be culture expanded at least 5 passages to 10 passages. In one example, the mesenchymal lineage precursor or stem cells can be culture expanded at least 5 passages to 8 passages. In one example, the mesenchymal lineage precursor or stem cells can be culture expanded at least 5 passages to 7 passages. In one example, the mesenchymal lineage precursor or stem cells can be culture expanded for more than 10 passages. In another example, the mesenchymal lineage precursor or stem cells can be culture expanded for more than 7 passages. In these examples, stem cells can be culture expanded prior to being cryopreserved to provide an intermediate cryopreserved MLPSC population. In one example, the compositions of the present disclosure are produced by culturing cells from an intermediate cryopreserved MLPSC population or in other words a cryopreserved intermediate.

In one example, the compositions of the present disclosure include mesenchymal lineage precursors or stem cells expanded from cryopreserved intermediate cultures. In one example, a cell culture expanded from a cryopreserved intermediate culture is expanded for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 passages. For example, mesenchymal lineage precursors or stem cells can be expanded by culture for at least 5 passages. In one example, the mesenchymal lineage precursor or stem cells can be culture expanded at least 5 passages to 10 passages. In one example, the mesenchymal lineage precursor or stem cells can be culture expanded at least 5 passages to 8 passages. In one example, the mesenchymal lineage precursor or stem cells can be culture expanded at least 5 passages to 7 passages. In one example, the mesenchymal lineage precursor or stem cells can be culture expanded for more than 10 passages. In another example, the mesenchymal lineage precursor or stem cells can be culture expanded for more than 7 passages.

In one example, mesenchymal lineage precursors or stem cells expanded from cryopreserved intermediate cultures can be expanded in culture in a medium that does not contain animal proteins. In one example, mesenchymal lineage precursors or stem cells expanded from cryopreserved intermediate cultures can be expanded in culture in a xeno-free medium. In one example, mesenchymal lineage precursors or stem cells expanded from cryopreserved intermediate cultures can be expanded in culture in medium free of fetal bovine serum.

In one embodiment, the mesenchymal lineage precursors or stem cells can be obtained from a single donor or multiple donors, where the donor samples or mesenchymal lineage precursors or stem cells are then pooled and then culture expanded. In one example, the culture amplification process comprises:

i. expanding a plurality of living cells by passaging to provide a preparation of at least about 10 hundred million living cells, wherein passaging comprises establishing a primary culture of isolated mesenchymal lineage precursors or stem cells, and then continuously establishing a first non-primary (P1) culture of mesenchymal lineage precursors or stem cells isolated from a previous culture;

expanding the P1 culture of isolated mesenchymal lineage precursors or stem cells to a second non-primary (P2) culture of mesenchymal lineage precursors or stem cells by passaging expansion; and

preparing and cryopreserving an intermediate mesenchymal lineage precursor or stem cell preparation in a process obtained from P2 culture of mesenchymal lineage precursors or stem cells; and

thawing the intermediate mesenchymal lineage precursor or stem cell preparation in the cryopreserved treatment and amplifying the intermediate mesenchymal lineage precursor or stem cell preparation in the treatment by passaging expansion.

In one example, the expanded mesenchymal lineage precursor or stem cell preparation has an antigen profile and an activity profile, the antigen profile and activity profile comprising:

i. Less than about 0.75% cd45+ cells;

at least about 95% cd105+ cells;

at least about 95% cd166+ cells.

In one example, the expanded mesenchymal lineage precursor or stem cell preparation is capable of inhibiting IL2-rα expression of CD3/CD28 activated PBMCs by at least about 30% relative to a control.

In one example, the culture-expanded mesenchymal lineage precursor or stem cells are culture-expanded about 4 passages to 10 passages, with the mesenchymal lineage precursor or stem cells being cryopreserved after at least 2 passages or 3 passages before being further culture-expanded. In one example, the mesenchymal lineage precursor or stem cell is culture expanded at least 1 passage, at least 2 passages, at least 3 passages, at least 4 passages, at least 5 passages, cryopreserved, and then further culture expanded at least 1 passage, at least 2 passages, at least 3 passages, at least 4 passages, at least 5 passages prior to culturing according to the methods of the present disclosure.

The process of mesenchymal lineage precursor or stem cell isolation and ex vivo expansion can be performed using any apparatus and cell handling methods known in the art. Various culture expansion embodiments of the present disclosure employ steps that require manipulation of the cells, e.g., seeding, feeding, dissociating adherent culture, or washing. Any step of manipulating the cells may damage the cells. Although mesenchymal lineage precursors or stem cells can generally sustain a certain amount of damage during preparation, the cells are preferably manipulated by a treatment procedure and/or apparatus that adequately performs the given steps while minimizing damage to the cells.

In an example, mesenchymal lineage precursors or stem cells are washed in an apparatus comprising a cell source bag, a wash solution bag, a recycle wash bag, a rotating membrane filter with inlet and outlet ports, a filtrate bag, a mixing zone, a final product bag for washed cells, and appropriate tubing, for example, as described in US 6,251,295, which is hereby incorporated by reference.

In one example, a mesenchymal lineage precursor or stem cell composition cultured according to the present disclosure is 95% homogeneous in CD105 positive and CD166 positive and CD45 negative. In one example, this homogeneity persists through ex vivo amplification; i.e. although multiple population doublings.

In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are culture expanded in 3D culture. For example, the mesenchymal lineage precursors or stem cells of the present disclosure can be culture expanded in a bioreactor. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are first culture expanded in 2D culture, then further expanded in 3D culture. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are culture expanded from a master cell bank. In one example, mesenchymal lineage precursor or stem cells of the present disclosure are culture expanded from a master cell bank in 2D culture prior to seeding in 3D culture. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are culture expanded in a bioreactor from a master cell bank in 2D culture for at least 3 days prior to seeding in 3D culture. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are culture expanded in a bioreactor from a master cell bank in 2D culture for at least 4 days prior to seeding in 3D culture. In one example, the mesenchymal lineage precursor or stem cells of the present disclosure are culture expanded in a bioreactor in 2D culture from a master cell bank for 3 days to 5 days prior to seeding in 3D culture. In these examples, the 2D culture may be performed in a cell factory. Various cell factory products are commercially available (e.g., siemens, sigma, thermofiser).

Cell culture medium

The mesenchymal lineage precursors or stem cells disclosed herein can be culture expanded in a variety of suitable growth media.

The term "medium" as used in the context of the present disclosure comprises a component of the surrounding environment of a cell. The culture medium facilitates and/or provides conditions suitable to allow cell growth. The medium may be solid, liquid, gaseous or a mixture of phases and materials. The medium may comprise a liquid growth medium and a liquid medium that does not sustain cell growth. The medium also comprises a gelatinous medium such as agar, agarose, gelatin and collagen matrix. Exemplary gaseous media comprise a gaseous phase to which cells grown on a petri dish or other solid or semi-solid support are exposed.

The cell culture medium used for culture expansion contains all essential amino acids and may also contain nonessential amino acids. Generally, amino acids are classified as essential amino acids (Thr, met, val, leu, ile, phe, trp, lys, his) and non-essential amino acids (Gly, ala, ser, cys, gln, asn, asp, tyr, arg, pro).

Those skilled in the art will appreciate that in order to obtain optimal results, the basal medium must be suitable for the cell line of interest. For example, if glucose (or other energy source) in the basal medium is found to be depleted and thus growth is restricted, it may be desirable to increase the level of this energy source, or to add glucose (or other energy source) during the culture process. In one example, the Dissolved Oxygen (DO) level may also be controlled.

In one example, the cell culture medium contains additives of human origin. For example, human serum and human platelet cell lysate may be added to the cell culture medium.

In one example, the cell culture medium contains only additives of human origin. Thus, in one example, the cell culture medium is xeno-free. For the avoidance of doubt, in these examples the medium is animal protein free. In one example, the cell culture medium used in the methods of the present disclosure is free of animal components.

In one example, the medium includes serum. In other examples, the medium is a fetal bovine serum free medium comprising growth factors that promote proliferation of mesenchymal lineage precursors or stem cells. In one embodiment, the medium is a serum-free stem cell medium. In one example, the cell culture medium comprises:

a basal medium;

platelet Derived Growth Factor (PDGF);

fibroblast growth factor 2 (FGF 2).

In one example, the medium includes Platelet Derived Growth Factor (PDGF) and fibroblast growth factor 2 (FGF 2), wherein the level of FGF2 is less than about 6ng/ml. For example, FGF2 levels may be less than about 5ng/ml, less than about 4ng/ml, less than about 3ng/ml, less than about 2ng/ml, less than about 1ng/ml. In other examples, the FGF2 level is less than about 0.9ng/ml, less than about 0.8ng/ml, less than about 0.7ng/ml, less than about 0.6ng/ml, less than about 0.5ng/ml, less than about 0.4ng/ml, less than about 0.3ng/ml, less than about 0.2ng/ml.

In another example, the level of FGF2 is between about 1pg/ml and 100 pg/ml. In another example, the FGF2 level is between about 5pg/ml and 80 pg/ml.

In one example, the PDGF is PDGF-BB. In one example, PDGF-BB levels are between about 1ng/ml and 150 ng/ml. In another example, the PDGF-BB level is between about 7.5ng/ml and 120 ng/ml. In another example, the PDGF-BB level is between about 15ng/ml and 60 ng/ml. In another example, the PDGF-BB level is at least about 10ng/ml. In another example, the PDGF-BB level is at least about 15ng/ml. In another example, the PDGF-BB level is at least about 20ng/ml. In another example, the PDGF-BB level is at least about 21ng/ml. In another example, the PDGF-BB level is at least about 22ng/ml. In another example, the PDGF-BB level is at least about 23ng/ml. In another example, the PDGF-BB level is at least about 24ng/ml. In another example, the PDGF-BB level is at least about 25ng/ml.

In another example, the PDGF is PDGF-AB. In one example, PDGF-AB levels are between about 1ng/ml and 150 ng/ml. In another example, the PDGF-AB level is between about 7.5ng/ml and 120 ng/ml. In another example, the PDGF-AB level is between about 15ng/ml and 60 ng/ml. In another example, the PDGF-AB level is at least about 10ng/ml. In another example, the PDGF-AB level is at least about 15ng/ml. In another example, the PDGF-AB level is at least about 20ng/ml. In another example, the PDGF-AB level is at least about 21ng/ml. In another example, the PDGF-AB level is at least about 22ng/ml. In another example, the PDGF-AB level is at least about 23ng/ml. In another example, the PDGF-AB level is at least about 24ng/ml. In another example, the PDGF-AB level is at least about 25ng/ml.

In other examples, additional factors may be added to the cell culture medium. In one example, the medium further comprises EGF. EGF is a growth factor that stimulates cell proliferation by binding to EGFR, its receptor. In one example, the methods of the present disclosure comprise culturing a population of stem cells in a cell culture medium that further comprises EGF that does not contain fetal bovine serum. In one example, the EGF level is between about 0.1ng/ml and 7ng/ml. For example, the EGF level may be at least about 5ng/ml.

In another example, the EGF level is between about 0.2ng/ml and 3.2 ng/ml. In another example, the EGF level is between about 0.4ng/ml and 1.6 ng/ml. In another example, the EGF level is between about 0.2ng/ml. In another example, the EGF level is at least about 0.3ng/ml. In another example, the EGF level is at least about 0.4ng/ml. In another example, the EGF level is at least about 0.5ng/ml. In another example, the EGF level is at least about 0.6ng/ml. In another example, the EGF level is at least about 0.7ng/ml. In another example, the EGF level is at least about 0.8ng/ml. In another example, the EGF level is at least about 0.9ng/ml. In another example, the EGF level is at least about 1.0ng/ml.

In the above examples, such as αMEM or StemSpan ^TM The basic culture medium can be supplementedIs filled with a reference amount of growth factors. In one example, the medium comprises alpha MEM or StemSpan supplemented with 32ng/ml PDGF-BB, 0.8ng/ml EGF and 0.02ng/ml FGF ^TM 。

In other examples, additional factors may be added to the cell culture medium. For example, the cell culture medium may be supplemented with one or more stimulating factors selected from the group consisting of: epidermal Growth Factor (EGF), 1 alpha, 25-dihydroxyvitamin D3 (1,25D), tumor necrosis factor alpha (TNF-alpha), interleukin-lbeta (IL-lbeta), and stroma-derived factor lalpha (SDF-lalpha). In another embodiment, the cells may also be cultured in the presence of at least one cytokine in an amount sufficient to support the growth of the cells. In another embodiment, the cells may be cultured in the presence of heparin or a derivative thereof. For example, the cell culture medium may contain about 50ng/ml heparin. In other examples, the cell culture medium contains about 60ng/ml heparin, about 70ng/ml heparin, about 80ng/ml heparin, about 90ng/ml heparin, about 100ng/ml heparin, about 110ng/ml heparin, about 120ng/ml heparin, about 130ng/ml heparin, about 140ng/ml heparin, about 150ng/ml heparin, or derivatives thereof. In one example, the heparin derivative is sulfate. Various forms of heparin sulfate are known in the art and comprise heparin sulfate 2 (HS 2). HS2 may be from a variety of sources including, for example, the liver of male and/or female mammals. Exemplary heparan sulfates therefore include male heparin sulfate (MML HS) and female heparin sulfate (FML HS).

In another example, the cell culture media of the present disclosure promote stem cell proliferation while maintaining stem cells in an undifferentiated state. Stem cells are considered undifferentiated when they have not yet become a specific differentiation lineage. As discussed above, stem cells exhibit morphological features that are distinct from differentiated cells. In addition, undifferentiated stem cells express genes that can be used as markers for detecting the differentiation status. The polypeptide product may also be used as a marker for detecting the differentiation status. Thus, one of skill in the art can readily determine whether the methods of the present disclosure maintain stem cells in an undifferentiated state using conventional morphological, genetic, and/or proteomic analysis.

Modification of cells

The mesenchymal lineage precursors or stem cells disclosed herein can be altered in such a way that upon administration, lysis of the cells is inhibited. The alteration of the antigen may induce immune non-responses or tolerance, thereby preventing effector phases (e.g., cytotoxic T cell production, antibody production, etc.) that induce immune responses that ultimately lead to rejection of the foreign cells in a normal immune response. Antigens that can be altered to achieve this goal include, for example, MHC class I antigens, MHC class II antigens, LFA-3, and ICAM-1.

Mesenchymal lineage precursors or stem cells can also be genetically modified to express proteins of importance for differentiation and/or maintenance of striated skeletal muscle cells. Exemplary proteins include growth factors (TGF-beta, insulin-like growth factor 1 (IGF-1), FGF), myogenic factors (e.g., myoD, myogenic factor 5 (Myf 5), myogenic Regulatory Factor (MRF)), transcription factors (e.g., GATA-4), cytokines (e.g., cardiophilin-1), neuregulin family members (e.g., neuregulin 1, 2, and 3), and homeobox genes (e.g., csx, tinman, and NKx families).

Composition and method for producing the same

In one example, the present disclosure encompasses opioid dosage reduction compositions comprising mesenchymal lineage precursors or stem cells (MLPSCs) and Hyaluronic Acid (HA). In one example, the composition is administered and then the opioid achieves a dose reduction after administration. For example, opioid may achieve dose reduction after 3 months. In another example, opioid may achieve a dose reduction after 6 months. In another example, opioid may achieve a dose reduction after 12 months. In another example, opioid may achieve a dose reduction after 18 months. In another example, opioid may achieve a dose reduction after 24 months. In another example, opioid may achieve a dose reduction at 36 months. In another example, the opioid may be deactivated after 18 months. In another example, the opioid may be deactivated at 36 months.

In one example, the opioid dosage reduction composition includes 1% HA. In another example, the opioid dosage reducing composition is administered to a subject who is using an opioid. For example, the subject may be using opioids for the treatment of pain.

The mesenchymal lineage or stem cells disclosed herein can be expanded from cryopreserved intermediate cultures to produce a formulation containing at least one therapeutic dose. In one example, the methods of the present disclosure encompass the administration of one dose. In another example, more than one dose is administered.

In one example, the composition of the present disclosure includes 10x 10 ⁶ Individual cells to 35x 10 ⁶ Individual cells. In another example, the composition includes 20x 10 ⁶ Individual cells to 30x 10 ⁶ Individual cells. In other examples, the composition comprises at least 5 million cells. In another example, the composition comprises 6 million cells.

In one example, the compositions of the present disclosure include a pharmaceutically acceptable carrier and/or excipient. The terms "carrier" and "excipient" refer to compositions of matter conventionally used in the art to facilitate storage, administration and/or bioactivity of an active compound (see, e.g., remington's Pharmaceutical Sciences, 16 th edition, mark publication (Mac Publishing Company) (1980)). The carrier may also reduce any undesirable side effects of the active compound. Suitable carriers are, for example, stable, e.g., not reactive with other components of the carrier. In one example, the carrier does not produce significant local or systemic side effects in the recipient at the dosage and concentration used for treatment.

Suitable carriers of the present disclosure include those conventionally used, such as water, saline, aqueous dextrose, lactose, ringer's solution, buffer solutions, hyaluronic acid, and glycols are exemplary liquid carriers, particularly (when isotonic) for solutions. Suitable pharmaceutical carriers and excipients include starch, cellulose, dextrose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol and the like.

In another example, the carrier is a culture medium composition, e.g., in which cells are grown or suspended. Such a culture medium composition does not cause any side effects in the subject to which it is administered. Exemplary carriers and excipients do not adversely affect cell viability and/or the ability of the cells to treat or prevent disease.

In one example, the carrier or excipient provides buffer activity to maintain the cells and/or soluble factors at a suitable pH to exert biological activity, e.g., the carrier or excipient is Phosphate Buffered Saline (PBS). PBS represents an attractive carrier or excipient because of minimal interaction with and allows for rapid release of cells and factors, in which case the compositions of the present disclosure may be produced as a liquid for direct application into the blood stream or into tissue or areas surrounding or adjacent to tissue, for example by injection.

The compositions of the present disclosure may be cryopreserved. Cryopreservation of mesenchymal lineage precursors or stem cells can be performed using slow cooling methods or 'fast' freezing protocols known in the art. Preferably, the cryopreservation method maintains similar phenotypes, cell surface markers and growth rates of cryopreserved cells compared to unfrozen cells.

The cryopreserved composition may comprise a cryopreservation solution. The pH of the cryopreservation solution is typically 6.5 to 8, preferably 7.4.

The cryopreservation solution may comprise a sterile, pyrogen-free isotonic solution, such as PlasmaLyte ATM.100mL of PlasmaLyte ATM contained 526mg sodium chloride, USP (NaCl); 502mg sodium gluconate (C6H 11NaO 7); 368mg sodium acetate trihydrate, USP (C2H 3 NaO2.3H2O); 37mg potassium chloride, USP (KCl); and 30mg magnesium chloride, USP (MgCl2.6H2O). It is free of antimicrobial agents. The pH was adjusted with sodium hydroxide. The pH was 7.4 (6.5 to 8.0).

The cryopreservation solution may include Profreize ^TM . The cryopreservation solution may additionally or alternatively comprise a culture medium, such as αmem.

To facilitate freezing, cryoprotectants, such as Dimethylsulfoxide (DMSO), are typically added to the cryopreservation solution. Ideally, cryoprotectants should be non-toxic, non-antigenic, chemically inert to cells and patients, provide high survival rates after thawing and allow transplantation without washing. However, the most commonly used cryoprotectant DMSO shows some cytotoxicity. Hydroxyethyl starch (HES) may be used as a surrogate or in combination with DMSO to reduce cytotoxicity of the cryopreservation solution.

The cryopreservation solution may include one or more of DMSO, hydroxyethyl starch, human serum components, and other protein fillers. In one example, the cryopreserved solution comprises a Plasma-Lyte a (70%), DMSO (10%), HSA (25%) solution comprising 5% HSA and 15% buffer.

In one example, the cryopreservation solution may further include one or more of methylcellulose, polyvinylpyrrolidone (PVP), and trehalose.

The cryopreserved composition may be thawed and administered directly to a subject or added to another solution comprising, for example, hyaluronic acid. Alternatively, the cryopreserved composition may be thawed and the mesenchymal lineage precursor or stem cells resuspended in an alternative carrier prior to administration.

The compositions described herein may be administered alone or as a mixture with other cells. The different types of cells may be mixed with the compositions of the present disclosure immediately or shortly before administration, or they may be co-cultured together for a period of time prior to administration.

The exact amount of cells to be administered depends on a variety of factors, including the age, weight and sex of the subject, as well as the extent and severity of the condition to be treated.

Although the number of cells is provided in the composition, in one example, 50x 10 is administered ⁶ From 200x 10 ⁷ Individual cells. In other examples, 60x 10 is applied ⁶ From 200x 10 ⁶ Individual cells or 75x 10 ⁶ From 150x 10 ⁶ Individual cells. In one example, 75x 10 is applied ⁶ Individual cells. In another example, 150x 10 is applied ⁶ Individual cells. In these examples, the cells may be administered as a single dose or via multiple doses.

In one example, the composition comprises greater than 5.00x 10 ⁶ Each living cell/mL. In another example, the composition comprises greater than 5.50x10 ⁶ Each living cell/mL. In another example, the composition comprises greater than 6.00x 10 ⁶ Each living cell/mL. In another example, the composition comprises greater than 6.50x10 ⁶ Each living cell/mL. In another example, the composition comprises greater than 6.68x10 ⁶ Each living cell/mL.

In one example, the mesenchymal lineage precursor or stem cells comprise at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of the cell population of the composition.

In one example, the composition may optionally be packaged in a suitable container with written instructions for the desired purpose.

The compositions of the present disclosure may be administered systemically, e.g., by intravenous administration. In one example, the composition is applied to a painful intervertebral disc. In one example, the compositions of the present disclosure are applied to the nucleus pulposus or annulus fibrosus of an intervertebral disc.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

All publications discussed and/or mentioned herein are incorporated herein in their entirety.

The present application claims priority from AU2021900147 submitted at 22 of 2021, AU2021900250 submitted at 4 of 2021, 2 and 11 of 2022, the disclosure of which is incorporated herein in its entirety.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. This is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Examples

Composition and method for producing the same

The therapeutic composition comprises human bone marrow derived allogeneic MPCs amplified in vitro and cryopreserved. Cells were present in the composition alone (MPC) or with 1% Hyaluronic Acid (HA) (mpc+ha).

Patient(s)

Subjects with chronic lower back pain (> 6 months) and associated with moderate radiological degenerative changes of the intervertebral disc.

Baseline data

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Treatment with both MPC and mpc+ha reduced the average VAS lower back pain for all opioid-treated patients relative to baseline. The most significant reduction was observed in subjects administered mpc+ha. Indeed, mpc+ha had significantly greater average pain relief from baseline at each time point within 36 months compared to placebo (figures 1 and 28). From a broader perspective (fig. 29), treatment with mpc+ha reduced average VAS lumbago in all patients relative to baseline at 12 months (p=0.0162) and 24 months (p= 0.0426).

Despite the indication that the treatment regimen was not to be changed, opioid use was reduced by approximately 40% in patients receiving mpc+ha treatment for 24 months. In these patients, mpc+ha:

-significantly reduced average VAS score (fig. 1);

-a significant increase in the proportion of 50% and 30% VAS responders (fig. 2);

significantly increased the proportion of patients with minimal/no pain at 12 months and 24 months (figure 3). Pain responders were defined as subjects with a 50% decrease in lower back pain VAS score relative to baseline and no post-treatment intervention at the time point of evaluation;

significantly increasing the average EQ-5D score (fig. 4 and 5); and is also provided with

Significantly increasing the proportion of patients who achieved a combined pain and function treatment within 24 months (figures 6 to 8). With respect to LBP-ODI treatment success rate, overall success is defined as a 50% decrease in LBP VAS score relative to baseline and a 15 score increase in ODI score relative to baseline for subjects at both 12 months and 24 months with no post-treatment intervention for 24 months. For MIC, success was defined as a 30% decrease in LBP VAS score relative to baseline and a 10 score increase in ODI score relative to baseline for subjects at 12 months and 24 months with no post-treatment intervention for 24 months. For LBP-EQ5D, treatment success was defined as 0.03 increase in EQ5D at both 12 months and 24 months, and 50% decrease in VAS (overall) or 30% decrease in VAS (MIC) in the subject, with no intervention.

Notably, both MPC and mpc+ha reduced morphine milliequivalents for opioid users over 24 months (fig. 9 and 10), with a significant reduction in mpc+ha observed (p=0.01).

Further analysis of the therapeutic response of patients administered mpc+ha revealed:

-an increase in the proportion of opioid users who achieved both VAS 30% and VAS 50% pain responses within 24 months, while reducing daily opioid use (figures 11 and 12);

-an increase in the proportion of opioid users who achieved both an ODI 10 split functional response (fig. 13), a 15 split functional response (fig. 14), a VAS 30% + ODI 10 split functional response (fig. 15) and a VAS 50% + ODI 15 split functional response (fig. 16) within 24 months, while reducing daily opioid use; and is also provided with

The proportion of opioid users who achieved a pain/function integrated response at both 12 months and 24 months was increased, while also reducing opioid use (figures 17 and 18).

In conjunction with the findings above, these data indicate that compositions comprising MLPSCs can be used as opioid dose reducers, and especially in the case of subjects with chronic pain (such as back pain). This is an important finding in view of the sustained opioid use and its sustained side effects in this patient population. Analysis of opioid usage 24 months after cell therapy administration provides further evidence that compositions including MLPSCs can be used as opioid usage reducers. 73.7% of patients receiving mpc+ha and 63.4% of patients receiving MPC, respectively, reduced their analgesic use. In contrast, only 50% of placebo patients reduced analgesic use (fig. 30). Notably, significant increases in patients achieving opioid-free use were achieved at 18 months (p=0.0121) and 36 months (p=0.0075) after mpc+ha administration (fig. 31). The durable elimination of opioid use is significant and this represents a particularly surprising result in view of the chronic pathological nature of these patients.

Early intervention

Single dose MPC treatment reduced pain in all patients over 36 months (fig. 19 and 32). The median Chronic Lower Back Pain (CLBP) duration of these patients was 68 months. Further analysis of the data for patients with CLBP less than 68 months revealed that the therapeutic effect of MPC on pain (both MPC and mpc+ha) was most pronounced in these patients (fig. 20; fig. 21). Notably, the therapeutic effect of scoring ODI function was most pronounced in patients with CLBP duration less than 68 months (fig. 21), especially in those patients who were also opioid users (fig. 33). Further, MPC treatment (both MPC and mpc+ha) reduced morphine equivalent dose to opioid users within 36 months (fig. 34). Also notable was that mpc+ha administration significantly increased the number of integrated responders at 12 months (30% VAS/0.03EQ5D) (fig. 22; a trend of increasing numbers of integrated responders at 12 months was also observed in MPC treatment group). Patients with CLBP duration less than 68 months after both MPC and mpc+ha treatment had improved EQ5D (figures 23-26; rhs showed CLBP <68 months). These data are particularly promising because EQ5D can more effectively evaluate the therapeutic response of CLBP at the early stage. Furthermore, mpc+ha administration increased the combined therapeutic success rate of pain/function (VAS/ODI) and pain/quality of life (VAS/EQ 5D index) at both 12 months and 24 months (fig. 27).

The most significant improvement was noted in patients with CLBP less than 68 months after administration of mpc+ha:

-a significant reduction of the average VAS score;

-a significant increase in the proportion of 50% and 30% VAS responders;

significantly increasing the proportion of patients with minimal/no pain at 12 months and 24 months;

-a significant increase in average EQ-5D score; and is also provided with

Significantly increasing the proportion of patients who achieved a combined pain and function treatment within 24 months.

In summary, the above reference data indicate that early intervention in cell therapy in CLBP patients, especially intervention 68 months before CLBP, improved treatment outcome.

Relief of axial pain

Treatment with both MPC (n=15) and mpc+ha (n=14) reduced axial pain in patients with MRI-defined nerve root compression (tables 1 and 2). These data support a method of alleviating axial pain in a subject, particularly a subject with MRI-defined nerve root compression, by administering an MLPSC.

Table 1: mean change in VAS pain scores for patients with nerve root compression at baseline.

Treatment group	Numbering device	For 3 months	6 months of	For 12 months	24 months of
						Physiological saline	13	-2.1	-2.5	-2.7	1.5
6M MPC+HA	14	-25.2	-38.6	-38.9	-24.1
						6M MPC	15	-27.1	-27.1	-25	-21.7

Table 2: responders to treatment were scored for 50% of VAS pain in patients with nerve root compression at baseline.

Treatment group	Numbering device	For 3 months	6 months of	For 12 months	24 months of
						Physiological saline	13	1(8％)	1(8％)	2(15％)	0(0％)
6M MPC+HA	14	9(64％)	11(79％)	10(71％)	7(50％)
						6M MPC	15	4(27％)	10(67％)	7(47％)	7(47％)

Claims

1. A method of reducing pain or reducing opioid use or increasing EQ-5D score, the method comprising administering to a subject a composition comprising mesenchymal lineage precursors or stem cells (MLPSCs), wherein the subject is using an opioid.

2. The method of claim 1, wherein the composition comprises Hyaluronic Acid (HA).

3. The method of claim 1 or claim 2, wherein the subject is using opioids for pain.

4. An opioid dosage reduction composition comprising mesenchymal lineage precursors or stem cells (MLPSCs).

5. The composition of claim 4, further comprising Hyaluronic Acid (HA), preferably 1% HA.

6. The composition of claim 4 or claim 5, wherein the opioid dosage reduction composition is administered to a subject using an opioid, preferably wherein the subject is using the opioid for pain.

7. A method according to any one of claims 1 to 3 or a composition according to claim 6, wherein the pain is chronic pain.

8. A method according to any one of claims 1 to 3 or a composition according to any one of claims 4 to 7, wherein the pain is lower back pain, preferably wherein the lower back pain is associated with a degenerated disc.

9. The method or composition of claim 7, wherein the lower back pain is associated with an intervertebral disc, preferably wherein the intervertebral disc height of the intervertebral disc is not significantly reduced compared to the intervertebral disc height of an adjacent healthy intervertebral disc of the subject.

10. The method or composition of claim 7, wherein the origin of the lower back pain is non-rooted and/or associated with one or more of:

-a herniated disc of at most 3mm herniation;

nerve ingrowth into the disc;

-inflammation in the intervertebral disc.

11. The method or composition of claim 10, wherein the nerve ingrowth or the inflammation is in an intervertebral disc space or nucleus pulposus or annulus fibrosus of the intervertebral disc.

12. The method of any one of claims 1 to 3 or 6 to 11 or the composition of any one of claims 5 to 11, wherein opioid use is reduced 1 month, preferably 3 months, after administration of the composition.

13. The method of any one of claims 1 to 3 or 6 to 11 or the composition of any one of claims 5 to 11, wherein opioid use is reduced 6 months, preferably 12 months, more preferably 18 months, even more preferably 24 months after administration of the composition.

14. The method of any one of claims 1 to 3 or 7 to 11 or the composition of any one of claims 5 to 11, wherein opioid use is reduced for at least 12 months, preferably at least 24 months, after administration of the composition.

15. The method of any one of claims 1-3 or 6-14 or the composition of any one of claims 5-14, wherein opioid use is reduced relative to the average baseline morphine equivalent dose of the subject prior to administration of the composition.

16. The method of any one of claims 1 to 3 or 6 to 15, wherein the subject has been using an opioid for at least 1 month, preferably at least 3 months, more preferably at least 6 months, prior to administration of the composition.

17. The method of any one of claims 1-3 or 6-16, wherein the subject's opioid use is reduced by about 20%, about 30%, about 40% relative to its baseline opioid use prior to administration of the composition.

18. The method of any one of claims 1 to 3 or 6 to 17, wherein the average VAS score of the subject is reduced relative to its VAS score prior to administration of the composition.

19. The method according to any one of claims 1 to 3 or 6 to 18, wherein the subject has a Visual Analog Scale (VAS) pain response of 30%, preferably 50%.

20. The method of any one of claims 1 to 3 or 6 to 19 or the composition of any one of claims 5 to 19, wherein the opioid is morphine.

21. The method of any one of claims 1-3 or 6-20, wherein the average EQ-5D score of the subject is increased relative to baseline.

22. The method according to any one of claims 2 to 3 or 6 to 20, wherein the subject achieves an ODI 10 score function response, preferably a 15 score ODI function response.

23. The method of any one of claims 1 to 3 or 6 to 22, wherein the subject has no significant pain 12 months, preferably 24 months, after administration of the composition.

24. The method of any one of claims 1-3 or 6-23, wherein the subject stops opioid use 12 months to 24 months after administration of the composition.

25. The method of any one of claims 1-3 or 6-24, wherein the subject has an ODI score of between 25% and 70%, between 30% and 60%.

26. The method of any one of claims 1 to 3 or 6 to 25 or the composition of any one of claims 5 to 15, wherein the MLPSC is STRO-1+.

27. The method of any one of claims 1 to 3 or 6 to 26 or the composition of any one of claims 5 to 15 or 26, wherein the MLPSC is a Mesenchymal Stem Cell (MSC).

28. The method of any one of claims 1 to 3 or 6 to 27 or the composition of any one of claims 5 to 15 or 26 or 27, wherein the cells are allogeneic and/or culture expanded, preferably wherein the cells are tnap+ prior to their culture expansion.

29. The method of any one of claims 1 to 3 or 6 to 28 or the composition of any one of claims 5 to 15 or 25 to 28, wherein the cells have been cryopreserved.

30. The method of any one of claims 1 to 3 or 6 to 29, comprising administering 1x 10 ⁷ Up to 2x 10 ⁸ Individual cells.

31. The composition of any one of claims 5 to 15 or 25 to 29, comprising 1x 10 ⁷ Up to 2x 10 ⁸ Individual cells.

32. The method of any one of claims 1 to 3 or 6 to 30 or the composition of any one of claims 5 to 15 or 25 to 29 or 31, wherein the composition comprises human bone marrow-derived allogeneic Mesenchymal Precursor Cells (MPCs) isolated, ex vivo expanded and cryopreserved from bone mononuclear cells using an anti-STRO-3 antibody.

33. The method of any one of claims 1 to 3 or 6 to 32 or the composition of any one of claims 5 to 15 or 25 to 29 or 31 or 32, wherein the subject has experienced pain for 6 months to 68 months.

34. The method of any one of claims 1 to 3 or 6 to 33 or the composition of any one of claims 5 to 15 or 25 to 29 or 31 to 33, wherein the subject has not experienced pain for more than 68 months.

35. The method of any one of claims 1 to 3 or 6 to 33 or the composition of any one of claims 5 to 15 or 25 to 29 or 31 to 34, wherein the pain is axial pain.

36. The method or composition of claim 35, wherein the axial pain is caused by MRI-defined nerve root compression.

37. The method of any one of claims 1-3 or 6-36, wherein the subject stops opioid use 36 months after administration of the composition.

38. The composition of any one of claims 5-15 or 25-29 or 31-34, wherein the composition stops opioid use by a subject 36 months after administration.

39. Use of a composition according to any one of claims 5 to 15 or 25 to 29 or 31 to 34 in the manufacture of a medicament for use in stopping opioid use in a subject, wherein the opioid use is stopped 36 months after administration of the composition.

40. The use of claim 39, wherein the subject has chronic pain.

41. The use of claim 40, wherein the subject has chronic lower back pain.