Methods, systems, and compositions related to use of mu opioid receptor (MOR) antagonists

Abstract

This disclosure relates to using Mu Opioid Receptor (MOR) antagonists.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US Provisional Patent Application Ser. No. 63 / 603,162, filed December 21, 2023, the entire contents of which are incorporated by reference herein. GOVERNMENT INTEREST

[0002] This invention was made with government support under R01DE033473, NIHS10OD032141, and P30CA047904 awarded by the NIH. The government may have certain rights in the invention. TECHNICAL FIELD

[0003] This disclosure relates to using Mu Opioid Receptor (MOR) antagonists, in particular, axelopran. BACKGROUND

[0004] It has been recognized that PD1 / PDL1 blockade can enhance antitumor effects in different solid tumors including non-small cell lung cancer (NSCLC), melanoma, head and neck squamous cell carcinoma, renal cell carcinoma, urothelial carcinoma, microsatellite instability-high colorectal cancer, cervical cancer, Hodgkins lymphoma, hepatocellular carcinoma, gastric cancer. However, clinical data of anti-PDl / PDLl therapy continues to show limited response rates, from 20-50%. Previous investigations have revealed that a large group of patients suffer primary resistance and do not respond to PD1 / PDL1 blockade. In addition, some responders develop acquired resistance after initial responses.

[0005] Furthermore, treatment of cancer can be complicated by the use of opioids. Cancer and treatment of cancer can be painful to patients. As a result, opioids are regularly prescribed to cancer patients to ameliorate the pain. More recently, outcomes of clinical studies using anti-PDl therapies have been analysed by opioid use (and / or opioid exposure level), and the results also show an association. Numerous observational studies across multiple tumor types indicate that opioids prescribed for pain are associated with worse outcomes for patients receiving anti-PD-l / PD-Ll immunotherapy. Meta- analysis of these observational studies suggests that opioids reduce expected ORR by -50% and are associated with increased hazard ratios for adverse outcome in progression free survival (HR 1.61; 95% CI 1.37-1.89) and overall survival (HR 1.67; 95% CI 1.30-2.14).

[0006] Accordingly, there is a need for enhancing the efficacy of PD1 / PDL1 anticancer therapies. SUMMARY

[0007] This disclosure relates to using Mu Opioid Receptor (MOR) antagonists.

[0008] Embodiments include a method which involves diagnosing a patient with a cancer-associated pain, administering an opioid to the patient to ameliorate the cancer-associated pain, diagnosing a cancer in the patient, administering to the patient a peripherally active mu-opioid receptor antagonist (PAMORA), and treating the cancer in the patient with a primary treatment, wherein the primary treatment is not the PAMORA, wherein the PAMORA is maintained at a threshold level during the primary treatment.

[0009] Further embodiments include a method of treating cancer, involving: administering axelopran to establish a threshold concentration of axelopran in a cancer patient, wherein said cancer patient is concurrently receiving an opioid, treating the cancer patient with a PD-1 / PD-L1 anti-cancer therapy after establishing the threshold concentration of axelopran; and maintaining the threshold concentration of axelopran during the treating.

[0010] An additional embodiment in the disclosure includes a method of treating immunosuppression secondary to opioid administration by diagnosing a patient with pain; administering an opioid to the patient to ameliorate the pain; diagnosing deficient immune function in the patient; administering to the patient a peripherally active mu-opioid receptor antagonist (PAMORA); and treating the patient with a primary treatment, wherein the primary treatment is not the PAMORA, where the PAMORA is maintained at a threshold level during the primary treatment. BRIEF DESCRIPTION OF DRAWINGS

[0011] FIG. 1 shows a flow diagram of experimental animal treatments.

[0012] FIG. 2 shows a two-dimensional display of tumor volume change versus CD8+ T cell infiltration. N=12 for each treatment condition.

[0013] FIG. 3 shows a two-dimensional display of tumor volume change versus CD8+ T cell infiltration for each treatment shown in Fig 2 separated according to opioid status prior to immunotherapy.

[0014] FIG. 4 shows tumor size as a function of CD8+ T cell infiltration for all treatment groups.

[0015] FIG. 5 shows a summary of tumor size as a function of CD8+ T cell infiltration for the 3 identified immunoresponse categories which are dependent on opioid status.

[0016] FIG. 6 shows results from a bioinformatics study; panels (A-C) show In-silico analysis of scRNAseq of tumor-infiltrating leukocytes (TIL) from HNSCC patients, comparing those exposed to opioids with those who were not. Opioid usage decreases 0PRM1 expression across TIL, including exhausted CD8+ T cells; panels (D-E) show proportions and average expression levels of 0PRM1 across various cell clusters compared between non-users and opioid-exposed groups; panel (F) shows intra-tumoral 0PRM1+ CD8+ T cells overexpress several immune checkpoint markers (TOX, ENTPD1, and receptors PDCD1, HAVCR2, LAG3, CTLA4) compared to 0PRM1- CD8+ T cells; panel (G) shows box plots comparing bulk 0PRM1 expression (log2 of TPM) between tumor and normal tissues across various TCGA tumor types. Each pair of box plots represents a specific tumor type, with statistical significance indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). Head and neck tumors show increased OPRM1 expression when compared to normal tissue.

[0017] FIG. 7 shows 0PRM1 average expression proportions panel (A) and levels panel (B) across various cell clusters in both the vehicle and opioid-exposed groups. The data show that opioid usage by head and neck cancer patients leads to lower expression of 0PRM1 across tumor-infiltrating leukocytes, including exhausted CD8+ T-cells (identified as Lag3+CTLA4+). Panel (C) shows in silico analysis of single-cell RNA sequencing of head and neck cancer patients not prescribed opioids reveals that intra-tumoral Oprm / -positive CD8+ T-cells strongly overexpress several immune checkpoint receptors (PDCD1, TIGIT, LAG3, CTLA4) in comparison to 0PRM1-negative CD8+ T cells. Panel (D) shows in silico analysis of single-cell RNA sequencing of opioid exposed head and neck tumor reveals that intra-tumoral 0PRM1-expressing CD8+ T-cells do not strongly overexpress immune checkpoint receptors in comparison to OPRM1 -negative CD8+ T cells.

[0018] FIG. 8 shows in panel (A) heatmap showing the Spearman's correlation coefficients between 0PRM1 and various immune exhaustion marker genes and immune activation marker genes across different TCGA tumor types. The color gradient indicates the strength and direction of the correlation, with significant correlations (p < 0.05) highlighted by filled squares, while non-significant correlations are marked with an "X". Panel (B) shows a heatmap illustrating the hazard ratios for 0PBM1 expression in determining outcomes across various TCGA tumor types. Positive hazard ratios indicate a higher risk of adverse outcomes with increased 0PRM1 expression, while negative hazard ratios indicate a protective effect. Significant hazard ratios (p < 0.05) are denoted by filled squares, while non-significant ratios are marked with an "X". Panel (C) shows gene module analysis visualizing the correlation between 0PBM1 expression and immune infiltration levels across various TCGA cancer types. Results are shown using various computational methods to estimate the proportions of different immune cell types within tumor samples from bulk gene expression data. The heatmap represents the purity-adjusted partial Spearman's correlation coefficients (rho) between 0PBM1 expression and different immune cell types. The color gradient indicates the strength and direction of the correlation, with red representing a positive correlation and blue representing a negative correlation. Significant correlations (p < 0.05) are indicated by filled squares, while non-significant correlations are marked with an "X".

[0019] FIG. 9 shows in panel (A) relative expression (2-ACT) of Oprml and Hr4 genes using quantitative real time PCR in mouse oral cancer cell lines and mouse trigeminal ganglia (neural tissue, positive control); housekeeper = Actb. Panels (B-C) show quantitative analysis of M0C1 cell proliferation and cell migration after continued treatment with l-100pM morphine for 24 and 48 hours. Control for scratch assay was 0.5% FBS in the media. One-way ANOVA within timepoint, *p<0.05. (D) Relative expression (2-ACT) of 0PBM1 and TILR4 genes using quantitative real time PCR in human oral cancer cell lines; housekeeper = ACTB. Panel (E) shows fold change (2-AACT) in IL2 and IFNY genes in cultured CD8+ donor PBMCs after 48 hr administration of CD3 / CD28 plus 0.1-10pM morphine, lOpM morphine + lOpM MNTX, or lOpM morphine + IpM axelopran, relative to vehicle control, which was established by administration of saline and DMSO at the same volume (n=4 healthy donors). DETAILED DESCRIPTION

[0020] Before certain embodiments are described in greater detail, it is to be understood that this disclosure is not limited to certain embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing certain embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0021] Described herein are several definitions. Such definitions are meant to encompass grammatical equivalents.

[0022] The use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the terms “comprising,” “having,” “including,” as well as other forms, such as “includes” and “included,” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0023] As used herein, the term “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations. Such variations, however, are dependent on the specific component referred to and the context as understood by a person of ordinary skill in the art.

[0024] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, representative illustrative methods, and materials are now described.

[0026] Each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0027] Embodiments of the disclosure can be practiced without any component not specifically mentioned in this disclosure.

[0028] Opioids as use herein includes tramadol, hydromorphone, methadone, morphine, oxycodone, hydrocodone, codeine, oxymorphone, fentanyl, buprenorphin, and tapentadol

[0029] Peripherally active mu-opioid receptor antagonist (PAMORA) includes at least one of bevenopran, methylnaltrexone, axelopran, naloxegol, naldemedine, alvimopan or naloxone methiodide (NLXM).

[0030] Primary treatment as used herein includes pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, cemiplimab, sintilimab, dostarlimab, tislelizumab, retifanlimab, envafolimab, toripalimab, camrelizumab, spartalizumab, sunitinib, regorafenib, sorafenib, pazopanib, cabozantinib, axitinib, lenvatinib, tivozanib, temsirolimus, everolimus, aflibercept, and bevacizumab

[0031] Embodiments described herein may comprise, consist essentially of, or consist of the elements therein.

[0032] Unless otherwise stated percentages are given as percentages by total weight and all embodiments and preferred features may be combined in any combination.

[0033] As used herein, the term “axelopran” includes axelopran which has the structure shown below: and includes pharmaceutically acceptable salts of axelopran and stereoisomers of axelopran.

[0034] Many mechanisms of resistance have been proposed and investigated including, insufficient tumor immunogenicity, disfunction of MHCs, irreversible T cell exhaustion, resistance of IFN-y signaling, immunosuppressive microenvironment, classic oncogene mutation, and acquired resistance.

[0035] Emerging evidence has also shown that the diversity and composition of the gut microbiota can also influence anti-PDl / PDLl efficiency. Differences among tumor bearing mice rely on distinction of commensal microbiota. Oral administration of Bifidobacterium can promote the efficacy of anti-PDLl antibody in mice. In patients, commensal microbial composition was found associated with clinical anti-PDl / PDLl response rate, and fecal microbiota transplantation from responders into germ-free or antibiotic-treated mice leveraged the restraint of PD1 blockade.

[0036] Many strategies for overcoming resistance have been proposed, including: enhancing T cell priming, reversing T cell exhaustion, increasing T cell infiltration, and combination with other therapies. This latter strategy has proven successful in certain combinations, however, the increased immunoreactivity due to multiple agents has also led to an increase in off-target immunoreactivity, resulting in dose limiting side effects such as colitis, pneumonitis, hypothyroidism, arthralgia and vitiligo.

[0037] The presence of CD8+ T cells in the TME is a prerequisite for anti-PDl / PDLl therapy. Accordingly, the density of TILs is positively correlated with PD1 / PDL1 blockade efficacy. Thus, primary ICI resistance, is in general, accompanied by a decline in the density of intra-tumoral CD8+ T cells. Conversely, therapeutic strategies developed to overcome ICI-resistance should result in an increase in intra-tumoral CD8+ T cells.

[0038] A previously overlooked mechanism of ICI-resistance in all solid tumor studies conducted to date is the role of the mu-opioid receptor (MOR) in tumor progression and in immunosuppression. A large proportion of solid tumor patients experience cancer-associated pain which is overwhelmingly managed with prescription opioids. This ranges from 25% to as high as 90%, depending on the cancer type and stage of progression. When analyzed as a subgroup, opioid use has been consistently associated with poorer outcome and reduced survival. It is plausible that opioid use may be a marker for greater pain associated with more aggressive disease. Some studies have attempted to partially control for such confounding by stratifying patients by both baseline pain and opioid use and comparing outcomes with patients receiving other non-opioid analgesics such as NSAIDS and gabapentinoids. In each case, an independent association of opioids with poor outcome has still been observed. Association of opioids with outcomes has been seen in retrospective analyses of large randomized controlled trials, in which inclusion / exclusion criteria will have partially standardized the study population. Opioid association with impaired survival also persisted after propensity score matching among NSCLC patients receiving checkpoint inhibitors.

[0039] As discussed above, outcomes of clinical studies using anti-PDl therapies have also been analyzed by opioid use (and / or opioid exposure level), and the results also show an association. Numerous observational studies across multiple tumor types indicate that opioids prescribed for pain are associated with worse outcomes for patients receiving anti-PD-l / PD-Ll immunotherapy. Meta- analysis of these observational studies suggests that opioids reduce expected ORR by -50% and are associated with increased hazard ratios for adverse outcome in progression free survival (HR 1.61; 95% CI 1.37-1.89) and overall survival (HR 1.67; 95% CI 1.30-2.14).

[0040] The primary mechanism of opioid-mediated, poor clinical outcome has not been identified in any of these studies, however, where ICI therapies have been studied, some limited data have been reported. A recent retrospective study reported on immune function and outcomes in a population of 66 R / M HNSCC patients who received anti-PD-1 monoclonal antibody treatment for R / M HNSCC, stratifying according to prescription opioid exposure. 63% of patients were taking opioids for pain prior to treatment with anti-PD-1; higher opioid usage in this cohort was associated with lower inter-tumoral CD8+ T cell density in tumor biopsy tissue prior to treatment, and significantly lower PFS and OS with anti-PD-1 treatment. Notably, objective response rate in the 22 patients that did not use opioids for pain control was 27.3% vs. 11.4% in the 44 patients that did use opioids, and the only complete responses seen in the study occurred in patients not taking opioids for pain control.

[0041] Mu-opioid receptor antagonists with peripheral selectivity can synergistically enhance the activity of anti-PDl therapies in the absence of exogenous opioids. These agents are referred to as PAMORAs (perpherially active mu-opioid receptor antagonists) and do not cross the blood-brain barrier, leaving opioid-induced analgesia intact.

[0042] In one embryonic model (chicken egg), the PAMORA axelopran enhanced immune cell infiltration into the grafted tumor when given with both anti-PDl and bevacizumab (an inhibitor of VEGF). In the syngeneic mouse model, the PAMORA axelopran significantly reduced colon cancer tumor growth and increased survival when given with anti-PDl, even when the anti-PDl given alone showed no significant activity. While the mechanisms responsible could be due to inhibition of endogenous opioid activity of the mu-receptor at the tumor or immune system level, it was unknown if such activity would replicate in subjects treated with exogenous opioids at levels used for control of cancer pain.

[0043] A variety of MOR antagonists can be used in the methods of the disclosure. For example, in one embodiment, Mu receptor opioid (MOR) antagonist is axelopran, naloxegol, naldemedine, alvimopan, or combinations thereof. naldemedine naloxegol alvimopan

[0044] In another embodiment, the MOR antagonist is axelopran, naloxegol, or combinations thereof. In a preferred embodiment, the MOR antagonist is axelopran. In certain embodiments, the MOR antagonist is a peripheral MOR antagonist. In one embodiment, the MOR antagonist does not comprise methylnaltrexone. In another embodiment, the MOR antagonist is a peripheral MOR antagonist excluding methylnaltrexone. In another embodiment, the MOR antagonist does not comprise methylnaltrexone, naltrexone and / or naloxone.

[0045] In certain embodiments, the methods require administering a pharmaceutical composition containing the MOR antagonist (e.g. axelopran). Examples of suitable pharmaceutical compositions are described below. In one embodiment, the pharmaceutical composition is formulated for modified release. Alternatively, the pharmaceutical composition is formulated for modified release, topical administration, or intravitreal injection. For example the pharmaceutical composition is formulated for “modified release” when it is formulated for rapid / accelerated release, site-directed release, controlled / pulsatile release, delayed release into lower gut, or sustained release.

[0046] The methods include administration of the MOR antagonist (e.g. axelopran) by a variety of routes including any of the administration routes described below. In one embodiment, the methods include orally administering the MOR antagonist. In another embodiment, the methods include subcutaneously administering the MOR antagonist. In yet another embodiment, the methods include intravitreally administering the MOR antagonist. In an alternate embodiment, the methods include topically administering the MOR antagonist.

[0047] In further embodiments, the methods including screening the patient for the presence of a cancer. These embodiments include taking a biological sample from the patient and then testing the sample for the presence of cancer cells.

[0048] Other embodiments of the disclosure are directed to using a MOR antagonist (e.g. axelopran, naloxegol, or combinations thereof) in the manufacture of medicament for treating cancer. Further embodiments are directed use of a MOR antagonist (e.g. axelopran, naloxegol, or combinations thereof) for treating cancer.

[0049] In addition, the disclosure provides for combination therapy comprising using a MOR antagonist in combination with a checkpoint inhibitor and / or a VEGF antagonist. In some embodiments, the combination therapy is used to treat angiogenesis or cancer.

[0050] Two or three drugs are administered to a subject “in combination” when the drugs are administered as part of the same course of therapy. A course of therapy refers to administration of combinations of drugs believed by the medical professional to work together additively, complementarity, synergistically, or otherwise to produce a more favorable outcome than that anticipated for administration of a single drug. A course of therapy can be for one or a few days, but more often extends for several weeks.

[0051] When two drugs are administered in combination, a variety of schedules can be used. In one case, for example and without limitation, Drug 1 (e.g. the MOR antagonist) is first administered prior to administration of Drug 2 (e.g. the checkpoint inhibitor and / or the VEGF antagonist), and treatment with Drug 1 is continued throughout the course of administration of Drug 2; alternatively Drug 1 is administered after the initiation or completion of Drug 2 therapy; alternatively, Drug 1 is first administered contemporaneously with the initiation of the other cancer therapy. As used in this context, “contemporaneously” means the two drugs are administered the same day, or on consecutive days.

[0052] Although in principle certain drugs can be co-formulated, in general they are administered in separate compositions. Similarly, although certain drugs can be administered simultaneously, more often (especially for drugs administered by infusion) drugs are administered at different times on the same day, on consecutive days, or according to another schedule.

[0053] Accordingly, one embodiment of the disclosure is directed to methods of treating cancer comprising administering a MOR antagonist and a checkpoint inhibitor and / or an inhibitor of VEGF (e.g. a MOR antagonist in combination with a checkpoint inhibitor and / or an inhibitor of VEGF (e.g. bevacizumab)) to a cancer patient. In preferred embodiments of these methods, the MOR antagonist and the checkpoint inhibitor and / or an inhibitor of VEGF synergistically treat the cancer. In one embodiment, the MOR antagonist and the checkpoint inhibitor (an anti-PDl antibody) synergistically treat cancer. One specific embodiment is a method of treating cancer with a synergistic combination of axelopran and pembrolizumab (an anti-PDl antibody). Another specific embodiment is a method of treating cancer with a synergistic combination of axelopran, bevacizumab (an anti-VEGF antibody), and pembrolizumab (an anti-PDl antibody). In other embodiments, the methods include administering the MOR antagonist in combination with the checkpoint inhibitor. In alternate embodiments, the methods include administering the MOR antagonist in combination with the VEGF antagonist.

[0054] Another embodiment of the disclosure is directed methods improving the immune response to a tumor comprising administering a MOR antagonist and a checkpoint inhibitor and / or an inhibitor of VEGF to a cancer patient. In some embodiments, the methods comprise administering a MOR antagonist in combination with a checkpoint inhibitor. In other embodiments, the methods comprise administering a MOR antagonist in combination with a VEGF antagonist. In certain embodiments, the methods increase infiltration of immune cells into the tumor. In specific embodiments, the methods increase infiltration of NK cells, lymphocytes and / or monocytes / macrophages. Alternatively, the methods increase infiltration of CD3+, CD244+, and MMD+ immune cells. In certain embodiments, the MOR antagonist and the checkpoint inhibitor synergistically improve the immune response to the tumor.

[0055] In certain specific embodiments of the combination therapy, the MOR antagonist does not comprise methylnaltrexone and / or naloxone.

[0056] A variety of checkpoint inhibitors can be used in combination with the MOR antagonists such as e.g. axelopran. In certain embodiments, the checkpoint inhibitor is an inhibitor of the PD-1 / PD-L1 pathway such as e.g. an antibody. In other embodiments, the checkpoint inhibitor is an inhibitor of: CTLA-4; LAG-3; TIM-3; B7-H3; B7-H4; A2aR; NKG21; PVRIG; PVRL2; CEACAM 1; CEACAM 5; CEACAM 6; FAK; CCL2 / CCR2; LIF; CD45; CSF-1; SEMA4D; CLEVER-1; OX-40; IL-1; IL-6; or IL-8. In certain embodiments, the checkpoint inhibitor is an antibody. Other examples of suitable checkpoint inhibitors are disclosed in Marin Acevedo et al. J Hematol Oncol (2021) 14:45, the disclosure of which is incorporated as it pertains to checkpoint inhibitors. In some embodiments, the checkpoint inhibitor is an antibody that binds to PD-1 such as e.g. a humanized antibody. In one embodiment, the checkpoint inhibitor is pembrolizumab.

[0057] In some embodiments of the combination therapy, the methods include orally or subcutaneously administering the MOR antagonist. In other embodiments, the method comprises administering a pharmaceutical composition comprising the MOR antagonist and a pharmaceutically acceptable carrier as described herein. For example, the method includes administering a pharmaceutical composition formulated for modified release. For example, the pharmaceutical composition is formulated for “modified release” when it is formulated for rapid / accelerated release, site-directed release, controlled / pulsatile release, delayed release into lower gut, or sustained release. When used, the checkpoint inhibitor is administered concurrently, before, or after the MOR antagonist (e.g. axelopran).

[0058] In certain embodiments, the methods include administering an inhibitor of angiogenesis. The inhibitor of angiogenesis can be used alone or in combination with a checkpoint inhibitor. In some embodiments, the inhibitor of angiogenesis is an inhibitor of VEGF. The VEGF inhibitor can be used alone or in combination with the checkpoint inhibitor (e.g. an antibody that binds to PD-1 such as e.g. pembrolizumab). In some embodiments, the VEGF inhibitor is an anti-VEGF antibody, such as e.g. an anti-VEGF-A antibody. In some embodiments, the VEGF inhibitor is a humanized antibody, the antibody is an anti-VEGF-A antibody. In other embodiments, the inhibitor of VEGF is bevacizumab. Other suitable inhibitors of VEGF include but are not limited to Ziv-aflibercept, aflibercept, ranibizumab, pegaptanib, or faricimab. In some embodiments of the methods (combination therapy), the inhibitor of VEGF is intravenously injected. When used, the inhibitor of VEGF is administered concurrently, before, or after the MOR antagonist. In other embodiments, when used in combination with a checkpoint inhibitor, the inhibitor of VEGF is administered concurrently, before, or after the check point inhibitor such as e.g. the inhibitor of the PD-1 / PD-L1 pathway.

[0059] In some embodiments, the disclosure is directed to use of a MOR antagonist, as described above, and an inhibitor of the PD-1 / PD-L1 pathway, as described above, in the manufacture of a medicament for treating cancer. The disclosure is also directed use of a MOR antagonist, as described above, and an inhibitor of the PD-1 / PD-L1 pathway, as described above, in the manufacture of a kit for treating cancer. Additionally, the disclosure is directed to use of a MOR antagonist, as described above, and an inhibitor of the PD-1 / PD-L1 pathway, as described above, for treating cancer.

[0060] In further embodiments, the disclosure is directed to use of a MOR antagonist, as described above, and an inhibitor of VEGF, as described above, in the manufacture of a medicament for treating cancer. Alternatively, the disclosure is directed to use of a MOR antagonist, as described above, and an inhibitor of VEGF, as described above, for treating cancer. Additionally, the disclosure is directed to a MOR antagonist, as described above, and an inhibitor of VEGF, as described above, in the manufacture of a medicament for treating cancer. Furthermore, the disclosure is directed to use of a MOR antagonist, as described above, and an inhibitor of VEGF, as described above, in the manufacture of a kit for treating cancer.

[0061] Additionally, the disclosure is directed to use of a MOR antagonist, as described above, an inhibitor of the PD-1 / PD-L1 pathway, as described above, and an inhibitor of VEGF, as described above, in the manufacture of a medicament for treating cancer. Furthermore, the disclosure is directed to use of a MOR antagonist, as described above, an inhibitor of the PD-1 / PD-L1 pathway, as described above, and an inhibitor of VEGF, as described above, for treating cancer.

[0062] In addition, in one embodiment, the disclosure is directed to a combination comprising a MOR antagonist, as described above (e.g. axelopran), and an inhibitor of the PD-1 / PD-L1 pathway (e.g. pembrolizumab), as described above, for use in a method of treating cancer, the method comprising administering the combination to a subject in need thereof. In certain embodiments, the combination also includes an inhibitor of VEGF, as described above (e.g. bevacizumab).

[0063] In specific embodiments of the methods and uses described above, the MOR antagonist is axelopran and the checkpoint inhibitor is an anti-PDl antibody, such, as for example, pembrolizumab. In alternate embodiments of the methods and uses described above, axelopran and pembrolizumab act in synergy. In other embodiments of the methods and uses described above, the MOR antagonist is axelopran, the checkpoint inhibitor is an anti-PDl antibody, such, as for example, pembrolizumab, and the inhibitor of VEGF is bevacizumab. In further embodiments of the methods and uses described above, axelopran, pembrolizumab, and bevacizumab act in synergy.

[0064] One embodiment is a composition comprising a synergistic amount of a MOR antagonist and an inhibitor of the PD-1 / PD-L1 pathway. In certain embodiments of the composition, the MOR antagonist comprises axelopran, naloxegol, methylnaltrexone, or combinations thereof. In one embodiment, the MOR antagonist is axelopran. The inhibitor of the PD-1 / PD-L1 pathway is an antibody, such as an antibody that binds to PD-1 (e.g. pembrolizumab). The antibody can be humanized.

[0065] In some embodiments, the invention is directed to pharmaceutical compositions containing a MOR antagonist configured for use in the methods described herein. In one preferred embodiment, the MOR antagonist is axelopran.

[0066] In certain embodiments, the invention is directed to pharmaceutical compositions comprising a MOR antagonist and a checkpoint inhibitor and / or a VEGF antagonist. In another embodiment, the invention is directed a pharmaceutical composition comprising a MOR antagonist and a separate pharmaceutical composition comprising a checkpoint inhibitor and / or a VEGF antagonist.

[0067] Provided herein is a pharmaceutical composition for treating a cancer in a subject in need thereof. The pharmaceutical composition comprises a MOR antagonist, and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises a checkpoint inhibitor and / or a VEGF antagonist.

[0068] In one embodiment, the pharmaceutical composition comprises axelopran, pembrolizumab, and / or bevacizumab. In another embodiment, the pharmaceutical composition comprises axelopran and pembrolizumab.

[0069] Such a pharmaceutical composition is in a form suitable for administration to a subject, or the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The various components of the pharmaceutical composition may be present in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0070] Pharmaceutical compositions that are useful in the methods of the invention may be suitably developed for inhalational, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, intravenous or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations. The route(s) of administration is readily apparent to the skilled artisan and depends upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.

[0071] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multidose unit.

[0072] The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0073] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it is understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs. In one embodiment, the subject is a human or a non-human mammal such as but not limited to an equine, an ovine, a bovine, a porcine, a canine, a feline and a murine. In one embodiment, the subject is a human.

[0074] In one embodiment, the compositions are formulated using one or more pharmaceutically acceptable excipients or carriers. In one aspect a pharmaceutical composition is disclosed for treating a cancer in a subject. The pharmaceutical composition comprises a MOR antagonist. In another embodiment, the pharmaceutical compositions also contain a checkpoint inhibitor, a VEGF antagonist, or a combination thereof. Pharmaceutically acceptable carriers, which are useful, include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0075] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.

[0076] The compositions may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention included but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea, and combinations thereof. A particularly preferred preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

[0077] The compositions may include an antioxidant and a chelating agent which inhibit the degradation of the compound. Preferred antioxidants for some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid in the preferred range of about 0.01% to 0.3% and more preferably BHT in the range of 0.03% to 0.1% by weight by total weight of the composition. Preferably, the chelating agent is present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Particularly preferred chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20% and more preferably in the range of 0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition which may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are the particularly preferred antioxidant and chelating agent respectively for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.

[0078] The pharmaceutical composition disclosed herein may be used in combination with an additional therapeutic agent such as an anti-tumor agent, including but not limited to a chemotherapeutic agent, an anti-cell proliferation agent or any combination thereof. For example, any conventional chemotherapeutic agents of the following nonlimiting exemplary classes are included in the invention: alkylating agents; nitrosoureas; antimetabolites; antitumor antibiotics; plant alkyloids; taxanes; hormonal agents; and miscellaneous agents. In another aspect, the pharmaceutical composition disclosed herein may be used in combination with a radiation therapy.

[0079] In certain embodiments of the invention, the MOR antagonist, and the checkpoint inhibitor and / or the VEGF antagonist are administered at the same time. In other embodiments, the checkpoint inhibitor and / or the VEGF antagonist is administered before the MOR antagonist is administered. In another embodiment, the checkpoint inhibitor and / or the VEGF antagonist is administered after MOR antagonist administration.

[0080] The regimen of administration may affect what constitutes an effective amount. For example, the therapeutic formulations may be administered to the patient subject either prior to or after a surgical intervention related to cancer, or shortly after the patient was diagnosed with cancer. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0081] The volume of the composition can be any volume, and can be for single or multiple dosage administration, including, but not limited to, from or from about 0.01 mL to 100 mL, 0.1 mL to 100 mL, 1 mL to 100 mL, 10 mL to 100 mL, 0.01 mL to 10 mL, 0.1 mL to 10 mL, 1 mL to 10 mL, 0.02 mL to 20 mL, 0.05 mL to 5 mL, 0.5 mL to 50 mL, or 0.5 mL to 5 mL, each inclusive.

[0082] Administration of the compositions of the present invention to a patient subject, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat cancer in the subject. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound is from about 0.01 to about 50 mg / kg of body weight / per day.

[0083] The MOR antagonist and the checkpoint inhibitor and / or the VEGF receptor antagonist can be administered to a subject as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose is readily apparent to the skilled artisan and depends upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, and the type and age of the animal. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0084] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound for treating cancer or other conditions in a patient.

[0085] One skilled in the art will recognize that although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. In certain embodiments of the invention, the MOR antagonist, and the checkpoint inhibitor and / or the VEGF antagonist are administered via the same route of administration. In other embodiments, the MOR antagonist, and the checkpoint inhibitor and / or the VEGF antagonist are administered via different routes of administration.

[0086] Routes of administration of the disclosed compositions (containing a MOR antagonist or MOR antagonist, and the checkpoint inhibitor and / or the VEGF antagonist) include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration. Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder, or aerosolized formulations for inhalation, compositions, and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein. In one embodiment, the MOR antagonist treatment and / or treatment with the checkpoint inhibitor and / or the VEGF antagonist comprises an administration route selected from the group consisting of inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intra-hepatic arterial, intrapleural, intrathecal, intra-tumoral, intravenal, and any combination thereof.

[0087] The invention also includes kits containing the MOR antagonist, checkpoint inhibitor, and / or VEGF antagonist whereby the kits are used to treat a cancer. In one embodiment, the kit comprises a pharmaceutical a pharmaceutical composition comprising the MOR antagonist and a pharmaceutically acceptable carrier. In one embodiment, the kit includes a pharmaceutical composition comprising a Mu receptor (e.g. axelopran) antagonist and the checkpoint inhibitor (e.g. pembrolizumab). In certain embodiments, the pharmaceutical composition also contains an inhibitor of VEGF. The pharmaceutical composition can be formulated for modified release, topical administration, or intravitreal injection.

[0088] In one embodiment, the kit includes a pharmaceutical composition comprising a Mu receptor (e.g. axelopran) antagonist and the checkpoint inhibitor (e.g. pembrolizumab). The kit also includes a separate pharmaceutical composition comprising the VEGF inhibitor (e.g. pembrolizumab). Examples

[0089] A syngeneic murine model of orthotopic oral cancer, using the M0C1 cell line was used as a test model. M0C1 is an optimal preclinical model to investigate the effect of morphine on immunotherapy response given that a majority (80%) of M0C1 tumors show complete response to anti-PD-1 treatment in the absence of exogenous opioids as measured by reduction in tumor volume as well as up to a 20% increased total CD8+ TIL and granzyme B producing CD8+ TIL compared to control (Zhou et al, 2022).

[0090] In the study, 3 drugs and their respective controls were used: vehicle (saline) vs morphine (lOmg / kg i.p. 2x / day); vehicle (pH 7 PBS vs axelopran (Img / kg i.p. coinjected with morphine or with aPD-1); Isotype IgG vs anti-PD-1 (250ug / injection, 3 injections every other day). No sex differences were identified in previous studies related to morphine-induced immunosuppression or anti-PDl immunotherapy response in the M0C1 transplant model, so mice groups were allocated evenly by sex; total 48 mice).

[0091] FIG. 1 is a flow diagram of the timing of the experimental treatments. Mice were inoculated with M0C1 cancer cells (7.5xl05) in a 1:1 Matrigel dilution in cell culture media (30pl total) into the anterior lateral portion of the tongue and the tumors grew to 200mm3 or larger (about 4 weeks). Tumor size was measured by caliper and tumor volume (mm3) was calculated. Mice were then treated with morphine or morphine+axelopran for 4 days. On the fifth day, mice began anti-PDl immunotherapy alone or co-injected with axelopran for 3 treatments (5th, 7th, and 9th day). Tumor diameters were measured with calipers on day 10 and relative change in tumor volume (RCTV) was calculated between treatment groups as the change in tumor volume (TV) from the start of treatment (TV0) to the TV at the endpoint (TVn) divided by TV0 (RCTV=[TVn-TV0] / TV0). Following measurements, tongue tissue was harvested for flow cytometry analysis of tumor infiltrating lymphocytes (TIL) on day 10.

[0092] The experiment was designed to replicate a commonly observed clinical population of HNC patients where 50% of the subjects are receiving opioid treatment for pain control prior to their first cycle of anti-PDl treatment and 50% are opioid-free. The therapeutic treatment modalities were 1) no treatment, 2) axelopran, 3) anti-PDl and 4) anti-PD-1 plus axelopran. The results are shown in FIG. 2 where tumor size is presented as a function of CD8+ lymphocyte infiltration.

[0093] In this subject population, where 50% of subjects were exposed to an exogenous opioid before anti-PDl treatment, tumor growth was not significantly inhibited by anti-PDl alone (about 20% reduction). The modest effect of anti-PDl treatment in this model study is similar to the 20% clinical response seen in the previously published study of an HNC patient population comprised of 63% opioid and 37% non-opioid patients (25).

[0094] In contrast to anti-PDl treatment, tumor growth was significantly slowed by both axelopran (44% reduction, P=0.0204) and axelopran + anti-PDl (75% reduction, P=0.001). The combination of both agents together, produces a synergistic activity, i.e. the combination produces a therapeutic outcome that is greater than the additive effect of each agent alone.

[0095] Under conditions where 50% of the animals received prior morphine treatment, neither anti-PDl alone nor axelopran alone significantly altered the infiltration of CD8+ T cells into the tumor stroma (FIG. 2). However, when the two drugs were used together, CD8+ T cells increased significantly, greater than 250% over the vehicle contol (P=0.001).

[0096] A surprising finding is that the efficacy of axelopran used as a single agent to reduce tumor growth appears to occur in the absence of significantly increased CD8+ T cell infiltration into the tumor stroma. In contrast, the combination therapy of axelopran and anti-PDl, significantly increases CD8+ T cell infiltration into the tumor, coincident with a significant decrease in tumor grwoth. This suggests that axelopran may in part reduce tumor growth by a mechanism(s) distinct from that of anti-PDl.

[0097] The data displayed in FIG. 2 was further investigated by analyzing the individual sub-groups by opioid status, either endogenous or endogenous plus exogenous (morphine), during the four days prior to the immunotherapy regimen. The data are visually displayed in FIG. 3, where tumor size is shown as a function of percentage of CD8+ lymphocyte infiltration into the intra-tumoral stroma.

[0098] Opioid status is a determinant of treatment outcome when anti-PDlis used alone. Animals treated with anti-PDl alone clearly separate into two significantly different populations. Those pre-treated with morphine show the largest sized tumors which are also devoid of significant CD8+ lymphocyte infiltration. Morphine therefore produces a complete failure of the anti-PDl treatment regimen.

[0099] In contrast, animals treated with anti-PDl under endogenous opioid conditions show the highest percentage of CD8+ TILs (p=0.005), and a significant reduction in tumor size (p=0.001). Under the other three treatment conditions, there is little separation between groups treated with morphine or not for either tumor size or CD8+ T cell infiltration.

[0100] Shown in Tables 1 and 2 are the tumor growth and T cell infiltration data for each of the 8 treatment conditions displayed in Figure 3. Tumor growth in animals not treated previously with morphine is significantly inhibited by anti-PDl (p=0.0238) with a concomitant influx of CD8+ T cells into the tumors (P=0.021). Even though axelopran when given alone has no significant effect on tumor growth (P=0.4398) or CD8+ T cell infiltration (0.9699), when combined with anti-PDl, a greater than additive decrease in tumor volume is observed (P=0.008) without a concomitant increase in CD8+ T cell infiltration (P=0.9184). This suggests a permissive condition for the PAMORA.

[0101] Table 1 Vehicle No Morphine Anti-PDl Axelopran Axelopran +Anti-PDl Vehicle Anti-PDl Morphine Axelopran Axelopran +Anti-PDl N=6 N=6 N=6 N=6 N=6 N=6 N=6 N=6 Mean SEM 85.96 13.57 23.25 4.79 56.88 8.58 13.61 8.92 101.34 28.22 126.20 22.43 49.01 9.50 33.64 18.07 P-value vs Vehicle - 0.0238 0.4398 0.008 - 0.5663 0.0691 0.0137

[0102] Tumor growth in the presence of morphine increases marginally, but CD8+ T cell infiltration is significantly inhibited (p=0.001) compared to the no morphine vehicle control. Axelopran partially restores the blocked infiltration of CD8+ T cells, but not back to levels seen in the absence of morphine. In the presence of morphine, anti-PDl fails to induce infiltration of CD8+ T cells into the tumor stroma and this is accompanied by a further increase in tumor growth.

[0103] The morphine-induced failure of anti-PDl to induce CD8+ T cell infiltration can be completely prevented if axelopran is started during morphine treatment and continued together with anti-PDl treatment. The combination of axelopran and anti-PDl when given together with morphine shows infiltration levels similar to that seen with anti-PDl alone in the absence of morphine.

[0104] Table 2 No Morphine Morphine Vehicle Anti-PDl Axelopran Axelopran +Anti-PDl Vehicle Anti-PDl Axelopran Axelopran +Anti-PDl Mean 3.34 6.76 2.90 3.97 0.71 1.10 2.19 6.90 SEM 0.57 1.55 0.48 0.88 0.12 0.29 1.06 0.97 P-value vs Vehicle - 0.0201 0.9699 0.9184 *0.001 0.9771 0.4767 <0.0001 *vs no morphine vehicle

[0105] The presence of CD8+ T cells in the tumor microenvironment is not the only requirement for a successful immunotherapy outcome. The expression of exhaustion markers such and PD1 and TIM3 are also determinants of the outcome. The levels of exhaustion markers were therefore also determined.

[0106] Under endogenous opioid conditions, T cells expressing PD1 are decreased by about one-third by anti-PDl treatment and are increased by axelopran alone. However, when axelopran and anti-PDl are given together, T cells expressing PD1 are reduced by 5-fold. In the presence of morphine, cells positive for the exhaustion marker PD1 are increased relative to that seen under endogenous opioid conditions. Both axelopran and anti-PDl decrease exhaustion markers by about 50%. However, when given together there is a 10-fold reduction in the exhaustion marker PD1, as shown in Table 3.

[0107] Table 3 Vehicle No Morphine Axelopran +Anti-PDl Vehicle Morphine Axelopran + Anti-PDl Anti-PDl Axelopran Anti-PDl Axelopran Mean 32.68 10.70 45.87 6.34 40.47 19.20 20.88 4.63 SEM 7.80 1.43 6.88 0.69 10.95 4.41 6.84 1.24 P-value vs 0.0393 0.3049 0.0111 0.0476 0.0736 0.0005 Vehicle [OK 38] Under endogenous opioid conditions , none of the treatments significantly altered the expression of TIM3. However, in the presence of morphine, cells positive for the exhaustion marker TIM3 were increased relative to that seen under endogenous opioid conditions and both axelopran and anti-PDl decreased this exhaustion marker by about 40%. However, when given together there is a 10-fold reduction in the exhaustion marker TIM3, as shown in Table 4.

[0109] Table 4 Vehicle No Morphine Axelopran +Anti-PDl Vehicle Anti-PDl Morphine Axelopran Axelopran +Anti-PDl Anti-PDl Axelopran Mean 13.47 7.90 12.32 6.99 21.83 12.12 13.47 2.71 SEM 1.95 1.61 2.28 0.91 6.94 5.43 6.00 0.57 - 0.6349 0.9940 0.5254 - 0.2172 0.3256 0.0043

[0110] The results disclosed herein describe three distinct conditions that define the anti-PDl response. These are summarized in FIG. 4, FIG. 5, and Table 5.

[0111] Table5 Opioid-Induced Immune Failure Immune Permissive Immune Responsive N=12 N=18 N=18 Tumor Size Mean SEM 113.8 17.6 63.9 7.0 23.5 6.8 Tumor Size P-value 0.006 0.000007 CD8+ % Mean 0.90 2.82 5.88 SEM 0.16 0.42 0.71 CD8+ % P-value 0.001 0.000005 PD1+ Mean 29.84 33.15 7.22 SEM 6.48 4.62 0.88 PD1+ P-value n.s 0.0002 Tim3+ Mean 16.97 13.09 5.87 SEM 4.45 2.11 0.82 Tim3+ P-value n.s 0.006

[0112] In the presence of elevated opioids (morphine), tumors show the greatest growth rate of all conditions tested, CD8+ T cells fail to enter the tumor microenvironment, and those that do so, display the highest percentage of T cells positive for the exhaustion markers PD1 and TIM3.

[0113] Under endogenous opioid conditions, or in the presence of axelopran with or without morphine, tumors are of an intermediate size and CD8+ T cell infiltration increases over that seen for animals treated with morphine. This intermediate category is considered immune permissive because when anti-PDl is added to the treatment regimen, there is a significant anti-tumor response.

[0114] When anti-PDl is given under the immune permissive conditions the tumor size is lowest, CD8+ T cell infiltration is the highest and the expression of exhaustion markers is significantly reduced.

[0115] In silico bioinformatics were used to assess 0PRM1 receptor expression on tumor infiltrating immune cells in head and neck squamous cell carcinoma (HNSCC) patients.

[0116] In silico analyses of scRNA-Seq of head and neck cancer patients' tumorinfiltrating leukocytes revealed that 0PRM1 is primarily expressed by CD8+ cells. Patients were then stratified by opioid use, delimited by an active opioid prescription at the time of resection, demonstrating that opioid use diminished immune infiltration and overall expression of 0PRM1 (FIG 6 A-E, FIG. 7). We found that OPRM1+CD8+ T cells, particularly in patients who had not been prescribed opioids prior to resection, exhibited increased expression of exhaustion markers (e.g., PDCD1, CHAP LACH TIGIT), and decreased cytokine expression (e.g., TGFB1, IFNG, TNF) (FIG 6F, FIG. 7). Using The Cancer Genome Atlas (TCGA) database, 0PRM1 expression across cancer types was compared to site-matched normal tissues; findings indicated statistically significant expression in various tumors, including head and neck squamous cells (Fig 6G; FIG. 8). 0PRM1 expression was correlated with worsened survival across tumor types, along with immune exhaustion and impaired immune infiltration (e.g., TIMER, xCell, EPIC; FIG. 8). We could not detect 0PRM1 mRNA using quantitative PCR in nine human oral squamous cell carcinoma (oSCC) cell lines and five mouse oSCC cell lines and found no proliferative or migratory changes in response to morphine treatment (FIG. 9). A further example of CD8+ T cells as targets for PAMORA-reversal of opioid-induced immunosuppression is the data shown in Figure 9E. It is clear that morphine treatment significantly reduces cytokine expression by CD8+ T cells (a marker of T-cell activation) and this inhibition can be overcome by treatment with the PAMORA agents, methylnaltrexone and axelopran. Taken together, these data indicate that OPAAfY-expressing CD8+ T cells may be the target cell for opioid-induced immunosuppression and may exhibit decreased anti-tumor capabilities.

[0117] In the bioinformatics analysis, single-cell RNA sequencing data from tumor infiltrating lymphocytes (TILs) of head and neck squamous cell carcinoma (HNSCC) patients were sourced from GSE139324, comprising 16 opioid-naive and 10 opioid-exposed patients’ scRNA-Seq data which were processed using the 10X Genomics Cell Ranger pipeline. Seurat objects were created from available raw gene expression matrices for each patient, normalized, and integrated using R with the Seurat package. Principal component analysis (PCA) was performed, followed by clustering using 20 principal components and a resolution of 0.5. The immune cells clusters were visualized using UMAP and annotated based on gene markers from PanglaoDB. Feature and violin plots, visualized with the Plotly package, were used to assess 0PRM1 expression across immune cell clusters.

[0118] The differential expression of 0PRM1 between tumor and adjacent normal tissues was evaluated using the Gene DE module of the Tumor Immune Estimation Resource version 2 (TIMER2.0) (timer.cistrome.org / ), which enables the analysis of gene expression levels across all cancer types included in The Cancer Genome Atlas (TCGA). Expression distributions for 0PPM1 in tumor and normal tissues were visualized using box plots. The statistical significance of the differential expression was calculated using the Wilcoxon test.

[0119] The many features and advantages of the disclosure are apparent from the detailed specification, and thus, it is intended by the appended claims to cover all such features and advantages of the disclosure which fall within the true spirit and scope of the disclosure. Further, since numerous modifications and variations will readily occur to those skilled in the art, it is not desired to limit the disclosure to the exact construction and operation illustrated and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the disclosure.

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Claims

1. A method, comprising:diagnosing a patient with a cancer-associated pain;administering an opioid to the patient to ameliorate the cancer-associated pain;diagnosing a cancer in the patient;administering to the patient a peripherally active mu-opioid receptor antagonist(PAMORA); andtreating the cancer in the patient with a primary treatment, wherein the primary treatment is not the PAMORA;wherein the PAMORA is maintained at a threshold level during the primary treatment.

2. The method of claim 1, wherein the opioid is selected from at least one of: tramadol, hydromorphone, methadone, morphine, oxycodone, hydrocodone, codeine, oxymorphone, fentanyl, buprenorphine, and tapentadol.

3. The method of claim 2, wherein the opioid is selected from at least morphine.

4. The method of any one of the proceeding claims, wherein the PAMORA is selectedfrom at least one of bevenopran, methylnaltrexone, axelopran, naloxegol, naldemedine, alvimopan or naloxone methiodide (NLXM).

5. The method of claim 1, wherein the PAMORA is axelopran.

6. The method of claim 1, wherein the PAMORA is methylnaltrexone.

7. The method of any one of claims 1-2 or 5-6, wherein the primary treatment includes acheckpoint inhibitor and / or an inhibitor of VEGF.

8. The method of claim 7, wherein the checkpoint inhibitor is an inhibitor of the PD-1 / PD-L1 pathway.

9. The method of claim 8, wherein the inhibitor of the PD-1 / PD-L1 pathway is an antibody.

10. The method of claim 9, wherein the antibody is an antibody that binds to PD-1.

11. The method of claim 10, wherein the antibody is a humanized antibody.

12. The method of claim 11, wherein the primary treatment includes at least one ofpembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, cemiplimab, sintilimab, dostarlimab, tislelizumab, retifanlimab, envafolimab, toripalimab, camrelizumab, spartalizumab, sunitinib, regorafenib, sorafenib, pazopanib, cabozantinib, axitinib, lenvatinib, tivozanib, temsirolimus, everolimus, aflibercept, and bevacizumab.

13. A method of treating cancer, comprising:administering axelopran to establish a threshold concentration of axelopran in a cancer patient, wherein said cancer patient is concurrently receiving an opioid;treating the cancer patient with a PD-1 / PD-L1 anti-cancer therapy after establishing the threshold concentration of axelopran; andmaintaining the threshold concentration of axelopran during the treating.

14. The method of claim 13, wherein the threshold concentration of axelopran synergizes the PD-1 / PD-L1 anti-cancer therapy.

15. The method of either claim 1 or 13, further comprising maintaining the administering of axelopran while stopping the opioid in the patient.

16. A method of treating immunosuppression secondary to opioid administration, comprising:diagnosing a patient with pain;administering an opioid to the patient to ameliorate the pain;diagnosing deficient immune function in the patient;administering to the patient a peripherally active mu-opioid receptor antagonist (PAMORA); andtreating the patient with a primary treatment, wherein the primary treatment is not the PAMORA;wherein the PAMORA is maintained at a threshold level during the primary treatment.

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