Pharmacology and pharmacokinetics of tazemetostat.

Tazemetostat, a novel oral selective inhibitor of enhancer of zeste homolog 2 (EZH2), was approved by the Food and Drug Administration (FDA) in 2020 for use in patients with advanced epithelioid sarcoma or relapsed/refractory (R/R) EZH2-mutated follicular lymphoma. These indications were approved by the FDA trough accelerated approval based on objective response rate and duration of response that resulted from phase 2 clinical trials. Tazemetostat competes with S-adenosylmethionine (SAM) cofactor to inhibit EZH2, reducing the levels of trimethylated lysine 27 of histone 3 (H3K27me3), considered as pharmacodynamic marker. Tazemetostat is orally bioavailable, characterized by rapid absorption and dose-proportional exposure, which is not influenced by coadministration with food or gastric acid reducing agents. It highly distributes in tissues, but with limited access to central nervous system. Tazemetostat is metabolized by CYP3A in the liver to 3 major inactive metabolites (M1, M3, and M5), has a short half-life and is mainly excreted in feces. Drug-drug interactions were shown with moderate CYP3A inhibitors as fluconazole, leading the FDA to recommend a 50% dose reduction, while studies investigating coadministration of tazemetostat with strong inhibitors/inducers are ongoing. No dosage modifications are recommended based on renal or hepatic dysfunctions. Overall, tazemetostat is the first-in-class EZH2 inhibitor approved by the FDA for cancer treatment. Current clinical studies are evaluating combination therapies in patients with several malignancies.

Phase II study of ceralasertib (AZD6738) in combination with durvalumab in patients with advanced gastric cancer.

BACKGROUND: Targeting the DNA damage repair (DDR) pathways is an attractive strategy for boosting cancer immunotherapy. Ceralasertib (AZD6738) is an oral kinase inhibitor of ataxia telangiectasia and Rad3 related protein, which is a master regulator of DDR. We conducted a phase II trial of ceralasertib plus durvalumab in patients with previously treated advanced gastric cancer (AGC) to demonstrate the safety, tolerability, and clinical activity of the combination. METHODS: This phase II, open-label, single-center, non-randomized study was designed to evaluate the efficacy and safety of ceralasertib in combination with durvalumab in patients with AGC. The study drug regimen was ceralasertib (240 mg two times a day) days 15-28 in a 28-day cycle in combination with durvalumab (1500 mg) at day 1 every 4 weeks. The primary end point was overall response rate (ORR) by Response Evaluation Criteria in Solid Tumors (V.1.1). Exploratory biomarker analysis was performed using fresh tumor biopsies in all enrolled patients. RESULTS: Among 31 patients, the ORR, disease control rate, median progression-free survival (PFS), and overall survival were 22.6% (95% CI 9.6% to 41.1%), 58.1% (95% CI 39.1% to 75.5%), 3.0 (95% CI 2.1 to 3.9) months, and 6.7 (95% CI 3.8 to 9.6) months, respectively. Common adverse events were manageable with dose modification. A subgroup of patients with a loss of ataxia telangiectasia mutated (ATM) expression and/or high proportion of mutational signature attributable to homologous repair deficiency (sig. HRD) demonstrated a significantly longer PFS than those with intact ATM and low sig. HRD (5.60 vs 1.65 months; HR 0.13, 95% CI 0.045 to 0.39; long-rank p<0.001). During the study treatment, upregulation of the innate immune response by cytosolic DNA, activation of intratumoral lymphocytes, and expansion of circulating tumor-reactive CD8 +T cell clones were identified in responders. Enrichment of the tumor vasculature signature was associated with treatment resistance. CONCLUSIONS: Ceralasertib plus durvalumab has promising antitumor activity, with durable responses in patients with refractory AGC. Thus, a biomarker-driven trial is required. TRIAL REGISTRATION: NCT03780608.

Phase 2 Trial of Iberdomide in Systemic Lupus Erythematosus.

BACKGROUND: Iberdomide, a cereblon modulator promoting degradation of the transcription factors Ikaros and Aiolos, which affect leukocyte development and autoimmunity, is being evaluated for the treatment of systemic lupus erythematosus (SLE). METHODS: In this phase 2 trial, we randomly assigned patients in a 2:2:1:2 ratio to receive oral iberdomide (at a dose of 0.45, 0.30, or 0.15 mg) or placebo once daily for 24 weeks, in addition to standard medications. The primary end point at week 24 was a response on the SLE Responder Index (SRI-4), which was defined as a reduction of at least 4 points in the Systemic Lupus Erythematosus Disease Activity Index 2000 score (a 24-item weighted score of lupus activity that ranges from 0 to 105, with higher scores indicating greater disease activity), no new disease activity as measured on the British Isles Lupus Assessment Group 2004 index, and no increase of 0.3 points or more in the Physician's Global Assessment score (on a visual-analogue scale ranging from 0 [no disease activity] to 3 [maximal disease]). RESULTS: A total of 288 patients received the assigned intervention: 81 received iberdomide at a dose of 0.45 mg, 82 received iberdomide at a dose of 0.30 mg, 42 received iberdomide at a dose of 0.15 mg, and 83 received placebo. At week 24, the percentages of patients with an SRI-4 response were 54% in the iberdomide 0.45-mg group, 40% in the iberdomide 0.30-mg group, 48% in the iberdomide 0.15-mg group, and 35% in the placebo group (adjusted difference between the iberdomide 0.45-mg group and the placebo group, 19.4 percentage points; 95% confidence interval, 4.1 to 33.4; P = 0.01), with no significant differences between the groups that received the lower doses of iberdomide and the group that received placebo. Iberdomide-associated adverse events included urinary tract and upper respiratory tract infections and neutropenia. CONCLUSIONS: In this 24-week, phase 2 trial involving patients with SLE, iberdomide at a dose of 0.45 mg resulted in a higher percentage of patients with an SRI-4 response than did placebo. Data from larger, longer trials are needed to determine the efficacy and safety of iberdomide in SLE. (Funded by Bristol Myers Squibb; ClinicalTrials.gov number, NCT03161483; EudraCT number, 2016-004574-17.).

Phosphoproteomics of ATR signaling in mouse testes.

The phosphatidylinositol 3' kinase (PI3K)-related kinase ATR is crucial for mammalian meiosis. ATR promotes meiotic progression by coordinating key events in DNA repair, meiotic sex chromosome inactivation (MSCI), and checkpoint-dependent quality control during meiotic prophase I. Despite its central roles in meiosis, the ATR-dependent meiotic signaling network remains largely unknown. Here, we used phosphoproteomics to define ATR signaling events in testes from mice following chemical and genetic ablation of ATR signaling. Quantitative analysis of phosphoproteomes obtained after germ cell-specific genetic ablation of the ATR activating 9-1-1 complex or treatment with ATR inhibitor identified over 14,000 phosphorylation sites from testes samples, of which 401 phosphorylation sites were found to be dependent on both the 9-1-1 complex and ATR. Our analyses identified ATR-dependent phosphorylation events in crucial DNA damage signaling and DNA repair proteins including TOPBP1, SMC3, MDC1, RAD50, and SLX4. Importantly, we identified ATR and RAD1-dependent phosphorylation events in proteins involved in mRNA regulatory processes, including SETX and RANBP3, whose localization to the sex body was lost upon ATR inhibition. In addition to identifying the expected ATR-targeted S/T-Q motif, we identified enrichment of an S/T-P-X-K motif in the set of ATR-dependent events, suggesting that ATR promotes signaling via proline-directed kinase(s) during meiosis. Indeed, we found that ATR signaling is important for the proper localization of CDK2 in spermatocytes. Overall, our analysis establishes a map of ATR signaling in mouse testes and highlights potential meiotic-specific actions of ATR during prophase I progression.

The Solution Is a Solution.

BACKGROUND: Infections in neonates with herpes simplex virus 1 (HSV-1) following circumcision due to Metzitzah Be'Peh (MBP) performed by a Mohel occur each year in small numbers. One solution to this problem is the use of a mucus extractor device instead of MBP, which has been authorized by some rabbis. Yet, using a mucus extractor remains controversial among ultra-Orthodox Jews; thus, creating a need for additional solutions. OBJECTIVES: To seek to reduce HSV-1 infection of neonates due to MBP. METHODS: We tested several oral rinse solutions for their ability to destroy virus infectivity following incubation for 30 seconds and using plaque reduction assays. RESULTS: Corsodyl, Decapinol, and Listerine® all destroyed plaques formation of spiked virus, while Gengigel and Tantum Verde were found to be less effective. We focused specifically on Listerine® due to its efficacy in eliminating contagious HSV-1 from saliva after a 30-second oral rinse. Five different products of Listerine® reduced the infectivity of a spiked virus by more than 4 orders of magnitude in 30 seconds. We also showed that Listerine (up to 7% v/v) can stay in the mouth but did not harm living cells and therefore will not cause any damage to the injured tissue. CONCLUSIONS: Significant reduction in cases of infection with HSV-1 due to MBP can be achieved if Mohalim consistently adopt the practice of careful mouth washing with Listerine® just before performing MBP.

Follicular Lymphoma: a Focus on Current and Emerging Therapies

Follicular lymphoma (FL) is the most common indolent lymphoma and is characterized by a relapsing and remitting course. In addition to significant biologic heterogeneity, the clinical trajectory for patients is variable, with some being observed for many years, and others having aggressive disease requiring multiple treatment courses. Unfortunately, FL remains incurable, and continues to cause early mortality. Improved understanding of the genetic and immune biology of FL has led to several FDA-approved therapies in the relapsed and refractory setting, including PI3K inhibitors; immunomodulatory agents; the EZH2 inhibitor, tazemetostat; and anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, axicabtagene ciloleucel. This review outlines the current approach to the diagnosis and treatment of FL with a focus on emerging investigational therapies, including targeted protein inhibitors, antibody-drug conjugates, monoclonal antibodies, bispecific antibodies, and novel combination strategies.

Dose-dependent bioavailability and tissue distribution of the ATR inhibitor AZD6738 (ceralasertib) in mice.

PURPOSE: Ataxia telangiectasia and Rad3-related (ATR) initiates and regulates cellular responses to DNA damage, such as those caused by cancer treatments. Several ATR inhibitors (ATRi) are in clinical development including AZD6738. Therapeutic indices among ATRi may differ as a result of varying potencies and concentrations at both tumor and off-target sites. Additionally, AZD6738 contributes to anti-tumor immune responses necessitating evaluation of exposure at immunological sites. METHODS: Using mouse models and a highly sensitive LC-MS/MS assay, the pharmacokinetics of AZD6738 were studied, including dose linearity, bioavailability, metabolism, and tissue distribution in tumor-bearing mice. RESULTS: Initial studies identified dose-dependent bioavailability, with greater than proportional increases in exposure as dose increased resulting in a ~ twofold increase in bioavailability between the lowest and highest investigated doses. These behaviors were successfully captured with a compartmental PK model. Analysis of metabolite PK revealed decreasing metabolic ratios with increasing dose, indicative of saturable first-pass metabolism. Further analysis revealed that intestinal and gut metabolism contribute to metabolism and these saturable mechanisms. Studies of tumor and tissue distribution found rapid and extensive drug distribution to most tissues except brain and spinal cord. CONCLUSION: The complex non-linear behavior of AZD6738 PK in mice was due to pre-systemic saturation and which appears to be recapitulated clinically at low doses. PK reported here will allow future correlation of tissue related toxicities with drug exposure as well as exposure with immunological responses. These results can also be compared with those from similar studies of other ATRi to contrast drug exposure with responses.

Effects of dual inhibition of AKT and ERK1/2 pathways on endometrial pro-inflammatory, hormonal, and epigenetic microenvironment in endometriosis.

Endometriosis is an estrogen-dependent and progesterone-resistant gynecological inflammatory disease of reproductive-age women. The prevalence of endometriosis is ~5-10% in reproductive-age women, increasing to 20-30% in women with subfertility. The current anti-estrogen therapies can be prescribed only for a short time because of the undesirable side effects on menstruation, pregnancy, bone health, and failure to prevent a recurrence. The causes of endometriosis-associated infertility are multifactorial and poorly understood. The objective of the present study was to determine the inhibitory effects of AKT and/or ERK1/2 pathways on the microenvironment of the endometrium in a xenograft mouse model of endometriosis of human origin. Results indicate that dual inhibition of AKT and ERK1/2 pathways, but not inhibition of either AKT or ERK1/2 pathway, suppresses the growth of the endometriotic lesions in vivo. Dual inhibition of AKT and ERK1/2 pathways suppresses the production of proinflammatory cytokines, decreases E2 biosynthesis and signaling, and restores progesterone receptor-B signaling components in the epithelial and stromal cells of the endometrium in a cell-specific manner. These results together suggest that dual inhibition of AKT and ERK1/2 pathways suppresses the estrogen-dominant state and concomitantly increases the progesterone-responsive state of the endometrium. Therefore, dual inhibition of AKT and ERK1/2 pathways could emerge as long-term nonsteroidal therapy for endometriosis.

Drug connectivity mapping and functional analysis reveal therapeutic small molecules that differentially modulate myelination.

Disruption or loss of oligodendrocytes (OLs) and myelin has devastating effects on CNS function and integrity, which occur in diverse neurological disorders, including Multiple Sclerosis (MS), Alzheimer's disease and neuropsychiatric disorders. Hence, there is a need to develop new therapies that promote oligodendrocyte regeneration and myelin repair. A promising approach is drug repurposing, but most agents have potentially contrasting biological actions depending on the cellular context and their dose-dependent effects on intracellular pathways. Here, we have used a combined systems biology and neurobiological approach to identify compounds that exert positive and negative effects on oligodendroglia, depending on concentration. Notably, next generation pharmacogenomic analysis identified the PI3K/Akt modulator LY294002 as the most highly ranked small molecule with both pro- and anti-oligodendroglial concentration-dependent effects. We validated these in silico findings using multidisciplinary approaches to reveal a profoundly bipartite effect of LY294002 on the generation of OPCs and their differentiation into myelinating oligodendrocytes in both postnatal and adult contexts. Finally, we employed transcriptional profiling and signalling pathway activity assays to determine cell-specific mechanisms of action of LY294002 on oligodendrocytes and resolve optimal in vivo conditions required to promote myelin repair. These results demonstrate the power of multidisciplinary strategies in determining the therapeutic potential of small molecules in neurodegenerative disorders.

Evaluation of the clinical cardiac safety of pemigatinib, a fibroblast growth factor receptor inhibitor, in participants with advanced malignancies.

Pemigatinib is a potent inhibitor of the fibroblast growth factor receptor (FGFR) family of receptors that is approved for the treatment of cholangiocarcinoma with FGFR2 fusion or other rearrangements. Data from a first-in-human clinical study were used to assess the potential for pemigatinib to produce clinically significant effects on heart rate (HR) and cardiac repolarization (QTc). A central tendency analysis for electrocardiogram (ECG) outliers and a plasma concentration-QTc analysis were conducted to assess cardiac safety in the first-in-human pemigatinib study (FIGHT-101; NCT02393248). The study included 113 participants who received at least one dose of pemigatinib as monotherapy and had at least one pair of plasma pharmacokinetic (PK) and ECG data points collected. Timed 12-lead ECGs were performed within 15 min of PK blood draws. The ECG parameters for each dose group in the study varied within expectations for patients with advanced malignancies. Categorical analysis of QT interval corrected for HR by Fridericia's method did not reveal dose dependence in the incidence of outliers, and the results of the central tendency and concentration-QTc analyses did not suggest a dose- or concentration-dependent drug effect. Least squares mean change from baseline in HR was small and did not indicate a clinically relevant effect on HR, and no effect was observed on cardiac conduction as assessed by PR and QRS intervals. In conclusion, pemigatinib does not exhibit any clinically significant prolongation of QTc or dose-dependent changes in HR. Clinical trial registration: ClinicalTrials.gov NCT02393248.

Co-treatment with miR-21-5p inhibitor and Aurora kinase inhibitor reversine suppresses breast cancer progression by targeting sprouty RTK signaling antagonist 2.

Numerous studies have reported the regulatory effects of miR-21-5p and reversine in human breast cancer (HBC). However, the mechanism of reversine and miR-21-5p has not been fully investigated in HBC. The aim of the current study was to assess the mechanism of action of reversine, with or without miR-21-5p, in HBC progression. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot results confirmed the upregulation of miR-21-5p and downregulation of sprouty RTK signaling antagonist 2 (SPRY2) in HBC. Bioinformatics analysis and luciferase assay identified the correlation between miR-21-5p and SPRY2. Cell function experiment results indicated a decrease in migration, proliferation, and invasion of HBC cells treated with miR-21-5p inhibitor and reversine; however, an increase in apoptosis was observed in these cells. Apoptotic ability was more enhanced and migration, proliferation, and invasion were more impaired in HBC cells treated with both miR-21-5p inhibitor and reversine than in those treated individually with either inhibitors. SPRY2, downstream of miR-21-5p, participated in HBC progression with reversine. Overall, our study proved that combining the miR-21-5p inhibitor with reversine produced a synergistic effect by regulating SPRY2, thereby limiting HBC progression. This knowledge might offer insights into the clinical therapy of HBC.

Evaluation of an EZH2 inhibitor in patient-derived orthotopic xenograft models of pediatric brain tumors alone and in combination with chemo- and radiation therapies.

Brain tumors are the leading cause of cancer-related death in children. Tazemetostat is an FDA-approved enhancer of zeste homolog (EZH2) inhibitor. To determine its role in difficult-to-treat pediatric brain tumors, we examined EZH2 levels in a panel of 22 PDOX models and confirmed EZH2 mRNA over-expression in 9 GBM (34.6 ± 12.7-fold) and 11 medulloblastoma models (6.2 ± 1.7 in group 3, 6.0 ± 2.4 in group 4) accompanied by elevated H3K27me3 expression. Therapeutic efficacy was evaluated in 4 models (1 GBM, 2 medulloblastomas and 1 ATRT) via systematically administered tazemetostat (250 and 400 mg/kg, gavaged, twice daily) alone and in combination with cisplatin (5 mg/kg, i.p., twice) and/or radiation (2 Gy/day × 5 days). Compared with the untreated controls, tazemetostat significantly (Pcorrected < 0.05) prolonged survival times in IC-L1115ATRT (101% at 400 mg/kg) and IC-2305GBM (32% at 250 mg/kg, 45% at 400 mg/kg) in a dose-dependent manner. The addition of tazemetostat with radiation was evaluated in 3 models, with only one [IC-1078MB (group 4)] showing a substantial, though not statistically significant, prolongation in survival compared to radiation treatment alone. Combining tazemetostat (250 mg/kg) with cisplatin was not superior to cisplatin alone in any model. Analysis of in vivo drug resistance detected predominance of EZH2-negative cells in the remnant PDOX tumors accompanied by decreased H3K27me2 and H3K27me3 expressions. These data supported the use of tazemetostat in a subset of pediatric brain tumors and suggests that EZH2-negative tumor cells may have caused therapy resistance and should be prioritized for the search of new therapeutic targets.

Central Nervous System Distribution of an Opioid Agonist Combination with Synergistic Activity.

Novel combinations of specific opioid agonists like loperamide and oxymorphindole targeting the µ- and δ-opioid receptors, respectively, have shown increased potency with minimized opioid-associated risks. However, whether their interaction is pharmacokinetic or pharmacodynamic in nature has not been determined. This study quantitatively determined whether these drugs have a pharmacokinetic interaction that alters systemic disposition or central nervous system (CNS) distribution. We performed intravenous and oral in vivo pharmacokinetic assessments of both drugs after discrete dosing and administration in combination to determine whether the combination had any effect on systemic pharmacokinetic parameters or CNS exposure. Drugs were administered at 5 or 10 mg/kg i.v. or 30 mg/kg orally to institute for cancer research (ICR) mice and 5 mg/kg i.v. to Friend leukemia virus strain B mice of the following genotypes: wild-type, breast cancer resistance protein (Bcrp-/- ) (Bcrp knockout), Mdr1a/b-/- [P-glycoprotein (P-gp) knockout], and Bcrp-/- Mdr1a/b-/- (triple knockout). In the combination, clearance of oxymorphindole (OMI) was reduced by approximately half, and the plasma area under the concentration-time curve (AUC) increased. Consequently, brain and spinal cord AUCs for OMI in the combination also increased proportionately. Both loperamide and OMI are P-gp substrates, but administration of the two drugs in combination does not alter efflux transport at the CNS barriers. Because OMI alone shows appreciable brain penetration but little therapeutic efficacy on its own, and because loperamide's CNS distribution is unchanged in the combination, the mechanism of action for the increased potency of the combination is most likely pharmacodynamic and most likely occurs at receptors in the peripheral nervous system. This combination has favorable characteristics for future development. SIGNIFICANCE STATEMENT: Opioids have yet to be replaced as the most effective treatments for moderate-to-severe pain and chronic pain, but their side effects are dangerous. Combinations of opioids with peripheral activity, such as loperamide and oxymorphindole, would be valuable in that they are effective at much lower doses and have reduced risks for dangerous side effects because the µ-opioid receptor agonist is largely excluded from the CNS.

H2S Donors and Their Use in Medicinal Chemistry.

Hydrogen sulfide (H2S) is a ubiquitous gaseous signaling molecule that has an important role in many physiological and pathological processes in mammalian tissues, with the same importance as two others endogenous gasotransmitters such as NO (nitric oxide) and CO (carbon monoxide). Endogenous H2S is involved in a broad gamut of processes in mammalian tissues including inflammation, vascular tone, hypertension, gastric mucosal integrity, neuromodulation, and defense mechanisms against viral infections as well as SARS-CoV-2 infection. These results suggest that the modulation of H2S levels has a potential therapeutic value. Consequently, synthetic H2S-releasing agents represent not only important research tools, but also potent therapeutic agents. This review has been designed in order to summarize the currently available H2S donors; furthermore, herein we discuss their preparation, the H2S-releasing mechanisms, and their -biological applications.

Dual Targeting by Inhibition of Phosphoinositide-3-Kinase and Mammalian Target of Rapamycin Attenuates the Neuroinflammatory Responses in Murine Hippocampal Cells and Seizures in C57BL/6 Mice.

Emerging evidence suggests the association of seizures and inflammation; however, underlying cell signaling mechanisms are still not fully understood. Overactivation of phosphoinositide-3-kinases is associated with both neuroinflammation and seizures. Herein, we speculate the PI3K/Akt/mTOR pathway as a promising therapeutic target for neuroinflammation-mediated seizures and associated neurodegeneration. Firstly, we cultured HT22 cells for detection of the downstream cell signaling events activated in a lipopolysaccharide (LPS)-primed pilocarpine (PILO) model. We then evaluated the effects of 7-day treatment of buparlisib (PI3K inhibitor, 25 mg/kg p.o.), dactolisib (PI3K/mTOR inhibitor, 25 mg/kg p.o.), and rapamycin (mTORC1 inhibitor, 10 mg/kg p.o.) in an LPS-primed PILO model of seizures in C57BL/6 mice. LPS priming resulted in enhanced seizure severity and reduced latency. Buparlisib and dactolisib, but not rapamycin, prolonged latency to seizures and reduced neuronal loss, while all drugs attenuated seizure severity. Buparlisib and dactolisib further reduced cellular redox, mitochondrial membrane potential, cleaved caspase-3 and p53, nuclear integrity, and attenuated NF-κB, IL-1ß, IL-6, TNF-α, and TGF-ß1 and TGF-ß2 signaling both in vitro and in vivo post-PILO and LPS+PILO inductions; however, rapamycin mitigated the same only in the PILO model. Both drugs protected against neuronal cell death demonstrating the contribution of this pathway in the seizure-induced neuronal pyknosis; however, rapamycin showed resistance in a combination model. Furthermore, LPS and PILO exposure enhanced pAkt/Akt and phospho-p70S6/total-p70S6 kinase activity, while buparlisib and dactolisib, but not rapamycin, could reduce it in a combination model. Partial rapamycin resistance was observed possibly due to the reactivation of the pathway by a functionally different complex of mTOR, i.e., mTORC2. Our study substantiated the plausible involvement of PI3K-mediated apoptotic and inflammatory pathways in LPS-primed PILO-induced seizures and provides evidence that its modulation constitutes an anti-inflammatory mechanism by which seizure inhibitory effects are observed. We showed dual inhibition by dactolisib as a promising approach. Targeting this pathway at two nodes at a time may provide new avenues for antiseizure therapies.

Topical neurokinin-1 receptor antagonist Fosaprepitant ameliorates ocular graft-versus-host disease in a preclinical mouse model.

PURPOSE: to assess the effect of topical administration of the Neurokin-1 receptor (NK1R) antagonist Fosaprepitant in a pre-clinical model of ocular Graft-versus-Host disease (GVHD). METHODS: BALB/c mice were pre-conditioned by myeloablative total body irradiation and subjected to allogeneic bone marrow transplantation and mature T cell infusion (BM + T). BM-transplanted mice (BM) were used as controls. Ocular GVHD was specifically assessed by quantifying corneal epithelial damage, tear secretion, blepharitis and phimosis, 3 times/week for 28 days post-transplantation. A group of BM + T mice received Fosaprepitant 10 mg/mL, 6 times/day, topically, from day 7-29 after transplantation. After sacrifice, the expression of NK1R, CD45, CD3, and CXCL10 was quantified in the cornea, conjunctiva, and lacrimal gland by immunohistochemistry. RESULTS: BM + T mice developed corneal epithelial damage (day 0-29, p < 0.001), blepharitis (day 0-29, p < 0.001), and phimosis (day 0-29, p < 0.01), and experienced decreased tear secretion (day 21, p < 0.01) compared to controls. NK1R was found upregulated in corneal epithelium (p < 0.01) and lacrimal gland (p < 0.01) of BM + T mice. Fosaprepitant administration significantly reduced corneal epithelial damage (p < 0.05), CD45+ (p < 0.05) and CD3+ (p < 0.01) immune cell infiltration in the cornea and conjunctiva (p < 0.001 and p < 0.001, respectively). In addition, Fosaprepitant reduced the expression of CXCL10 in the cornea (p < 0.05) and in the lacrimal gland (p < 0.05). CONCLUSIONS: Our results suggest that NK1R represents a novel druggable pathway for the therapy of ocular GVHD.

PKI-587 enhances radiosensitization of hepatocellular carcinoma by inhibiting the PI3K/AKT/mTOR pathways and DNA damage repair.

Radiation is an important therapeutic strategy for hepatocellular (HCC). In this study, we evaluated the role of the dual PI3K/mTOR inhibitor, PKI-587, on radiosensitization of HCC and its possible mechanism. MTT, colony formation, flow cytometry, and immunofluorescence were used to analyze the proliferation, cell cycle, formation of residual γ-H2AX foci, and apoptosis of HCC cells. A SK-Hep1 xenograft HCC model was used to assess the effects of PKI-587 in combination with ionizing radiation in vivo. The activation levels of PI3K/AKT/mTOR and DNA damage repair pathways and their downstream effector molecules were detected with Western blot. It was found that PKI-587 sensitized HCC cells to radiation by increasing DNA damage, enhancing G0/G1 cell-cycle arrest, and inducing apoptosis. In vivo, the combination of radiation with PKI-587 significantly inhibited tumor growth. These findings suggest the usefulness of PKI-587 on radiosensitization of HCC cells by inhibiting the PI3K/AKT/mTOR and DNA damage repair pathways. The combination of ionizing radiation and PKI-587 may be a strategy to improve the efficacy of treating HCC.