Search

Tumor-associated macrophages (TAM) are the most abundant immune cells in the tumor microenvironment. They consist of various subsets but primarily resemble the M2 macrophage phenotype. TAMs are known to promote tumor progression and are associated with poor clinical outcomes. CD47 on tumor cells and SIRPα on TAMs facilitate a “don't-eat-me” signal which prevents cancer cells from immune clearance. Therefore, blockade of the CD47–SIRPα interaction represents a promising strategy for tumor immunotherapy. Here, we present the results on ZL-1201, a differentiated and potent anti-CD47 antibody with improved hematologic safety profile compared with 5F9 benchmark. ZL-1201 enhanced phagocytosis in combination with standards of care (SoC) therapeutic antibodies in in vitro coculture systems using a panel of tumor models and differentiated macrophages, and these combinational effects are Fc dependent while potently enhancing M2 phagocytosis. In vivo xenograft studies showed that enhanced antitumor activities were seen in a variety of tumor models treated with ZL-1201 in combination with other therapeutic mAbs, and maximal antitumor activities were achieved in the presence of chemotherapy in addition to the combination of ZL-1201 with other mAbs. Moreover, tumor-infiltrating immune cells and cytokine analysis showed that ZL-1201 and chemotherapies remodel the tumor microenvironment, which increases antitumor immunity, leading to augmented antitumor efficacy when combined with mAbs. Significance: ZL-1201 is a novel anti-CD47 antibody that has improved hematologic safety profiles and combines with SoC, including mAbs and chemotherapies, to potently facilitate phagocytosis and antitumor efficacy.

CLDN18.2 (Claudin18.2)-targeting therapeutic antibodies have shown promising clinical efficacy in approximately 30% of gastric cancers expressing high levels of CLDN18.2 and less pronounced activity in low expressing malignancies. Here, we report that ZL-1211 is a mAb targeting CLDN18.2 engineered to promote enhanced antibody-dependent cellular cytotoxicity (ADCC) with the goal of achieving more potent activity in a wider spectrum of high- and low-CLDN18.2 expressing tumors. ZL-1211 demonstrated more robust in vitro ADCC activity than clinical benchmark not only in CLDN18.2-high but also CLDN18.2-low expressing gastric tumor cell lines. Greater antitumor efficacy was also observed in mouse xenograft models. Natural killer (NK) cell played critical roles in ZL-1211 efficacy and NK-cell depletion abrogated ZL-1211–mediated ADCC activity in vitro. ZL-1211 efficacy in vivo was also dependent on the presence of an NK compartment. Strikingly, NK cells strongly induced an inflammatory response in response to ZL-1211 treatment, including increased IFNγ, TNFα, and IL6 production, and were recruited into tumor microenvironment in patient-derived gastric tumors expressing CLDN18.2 upon ZL-1211 treatment to lyse the tumor cells. Taken together, our data suggest that ZL-1211 more effectively targets CLDN18.2-high gastric cancers as well as -low expressing malignancies that may not be eligible for treatment with the leading clinical benchmark by inducing enhanced ADCC response and activating NK cells with robust inflammation to enhance antitumor efficacy. Clinical activity of ZL-1211 is currently under evaluation in a phase I clinical trial (NCT05065710). Significance: ZL-1211, anti-CLDN18.2 therapeutic antibody can target CLDN18.2-high as well as -low gastric cancers that may not be eligible for treatment with clinical benchmark. ZL-1211 treatment induces NK-cell activation with robust inflammation to further activate antitumor immunity in tumor microenvironment.

Tumor-associated macrophages (TAM) are the most abundant immune cells in the tumor microenvironment. They consist of various subsets but primarily resemble the M2 macrophage phenotype. TAMs are known to promote tumor progression and are associated with poor clinical outcomes. CD47 on tumor cells and SIRPα on TAMs facilitate a “don't-eat-me” signal which prevents cancer cells from immune clearance. Therefore, blockade of the CD47–SIRPα interaction represents a promising strategy for tumor immunotherapy. Here, we present the results on ZL-1201, a differentiated and potent anti-CD47 antibody with improved hematologic safety profile compared with 5F9 benchmark. ZL-1201 enhanced phagocytosis in combination with standards of care (SoC) therapeutic antibodies in in vitro coculture systems using a panel of tumor models and differentiated macrophages, and these combinational effects are Fc dependent while potently enhancing M2 phagocytosis. In vivo xenograft studies showed that enhanced antitumor activities were seen in a variety of tumor models treated with ZL-1201 in combination with other therapeutic mAbs, and maximal antitumor activities were achieved in the presence of chemotherapy in addition to the combination of ZL-1201 with other mAbs. Moreover, tumor-infiltrating immune cells and cytokine analysis showed that ZL-1201 and chemotherapies remodel the tumor microenvironment, which increases antitumor immunity, leading to augmented antitumor efficacy when combined with mAbs.Significance:ZL-1201 is a novel anti-CD47 antibody that has improved hematologic safety profiles and combines with SoC, including mAbs and chemotherapies, to potently facilitate phagocytosis and antitumor efficacy.

Body weights of mice in HCC1954(A), SKOV3(B), ST-02-0077(C), FaDu (D), and Raji(E) xenograft efficacy studies shown in Figure 5 at days post-treatment as shown

Supplementary Figure 3 shows CLDN18.2 expression in gastric tumor cell lines and ZL-1211-induced CDC for the gastric tumor cell lines.

CLDN18.2 (Claudin18.2)-targeting therapeutic antibodies have shown promising clinical efficacy in approximately 30% of gastric cancers expressing high levels of CLDN18.2 and less pronounced activity in low expressing malignancies. Here, we report that ZL-1211 is a mAb targeting CLDN18.2 engineered to promote enhanced antibody-dependent cellular cytotoxicity (ADCC) with the goal of achieving more potent activity in a wider spectrum of high- and low-CLDN18.2 expressing tumors. ZL-1211 demonstrated more robust in vitro ADCC activity than clinical benchmark not only in CLDN18.2-high but also CLDN18.2-low expressing gastric tumor cell lines. Greater antitumor efficacy was also observed in mouse xenograft models. Natural killer (NK) cell played critical roles in ZL-1211 efficacy and NK-cell depletion abrogated ZL-1211–mediated ADCC activity in vitro. ZL-1211 efficacy in vivo was also dependent on the presence of an NK compartment. Strikingly, NK cells strongly induced an inflammatory response in response to ZL-1211 treatment, including increased IFNγ, TNFα, and IL6 production, and were recruited into tumor microenvironment in patient-derived gastric tumors expressing CLDN18.2 upon ZL-1211 treatment to lyse the tumor cells. Taken together, our data suggest that ZL-1211 more effectively targets CLDN18.2-high gastric cancers as well as -low expressing malignancies that may not be eligible for treatment with the leading clinical benchmark by inducing enhanced ADCC response and activating NK cells with robust inflammation to enhance antitumor efficacy. Clinical activity of ZL-1211 is currently under evaluation in a phase I clinical trial (NCT05065710).Significance:ZL-1211, anti-CLDN18.2 therapeutic antibody can target CLDN18.2-high as well as -low gastric cancers that may not be eligible for treatment with clinical benchmark. ZL-1211 treatment induces NK-cell activation with robust inflammation to further activate antitumor immunity in tumor microenvironment.

Supplemental Figure 2. AMG 232 treatment causes p53 stabilization, and increased p21 and MDM2 proteins in p53 wild-type cells.

Supplemental Figure 3. Body weights of mice in xenograft efficacy studies

Supplemental Figure 1. AMG 232 chemical structure, and 3-D model of AMG 232 bound to MDM2 protein.

Background: The routine perioperative use of opioids has recently been questioned due to opioid-related side effects, which can be potentially harmful in geriatric patients. This study aimed to evaluate the effects of opioid-free anesthesia in geriatric patients undergoing hip surgery. Methods: A total of 121 patients, aged 60 years or older, undergoing elective hip surgery were randomized to receive either opioid-free anesthesia with dexmedetomidine and esketamine (OFA group) or balanced anesthesia with opioids (CON group). All patients received a preoperative fascia iliaca block and postoperative patient-controlled analgesia using tramadol. The primary outcome was the incidence of a composite of anesthetic-related complications (nausea and vomiting, hypoxemia, ileus, urinary retention and delirium) within 48 hours postoperatively. The hemodynamics, postoperative pain and quality of life were also assessed. Results: The incidence of composite adverse events was significantly reduced in the OFA group compared with the CON group (35.0% vs. 62.3%, estimated difference: 27.3%, 95% confidence interval: 10.2%–44.4%, P = 0.003). Notably, patients in the OFA group experienced less postoperative nausea and vomiting (P = 0.040), and hypoxemia (P = 0.025) compared with those in the CON group. However, the incidences of postoperative ileus, urinary retention and delirium were comparable between the two groups. Also, patients in the OFA group had less pain in motion at 24 h postoperatively, as well as less risks of intraoperative hypotension and bradycardia (P Conclusions: Opioid-free anesthesia with dexmedetomidine and esketamine reduced postoperative anesthetic-related complications and provided improved hemodynamic stability in geriatric patients undergoing hip surgery. Trial registration: Chinese Clinical Trial Registry, retrospectively registered on 05/02/2022 (www.chictr.org.cn; ChiCTR2200056421)

Background The application of bedside ultrasound to evaluate gastric content and volume can assist in determining aspiration risk. Applying positive pressure ventilation via supraglottic airway devices (SAD) can result in a degree of gastric insufflation. This study assessed and compared the antral cross-sectional area (CSA) in patients undergoing laparoscopic gynecological surgery when managed with different SAD. Methods One hundred American Society of Anesthesiologists I or II female patients were assessed for inclusion in this study and divided into three groups of different ventilation devices. Patients were randomly allocated into three groups to receive LMA-Supreme (Group S), I-gel (Group I) or tracheal tube (Group T). The primary outcome was the antral cross-sectional area and secondary outcomes included haemodynamic parameters and postoperative morbidity such as sore throat, hoarseness, dry throat, nausea and vomiting. Results The antral CSA was not significantly different among three groups before induction (P = 0.451), after induction (P = 0.456) and at the end of surgery (P = 0.195). The haemodynamic variables were significantly higher in the tracheal tube group than in the LMA-Supreme and I-gel groups after insertion (P P Conclusions The SADs do not cause obvious gastric insufflation. Thus, LMA-Supreme and I-gel can be widely used as alternative to endotracheal intubation for the short laparoscopic gynecological surgery. Trial registration This trial was registered at the Chinese Clinical Trial Registry (ChiCTR1800018212, data of registration, September 2018).

Structure-based modification of mifepristone (1) led to the discovery of novel mifepristone derivatives with improved selectivity profile. Addition of a methyl group at the C10 position of the steroid has a significant impact on progesterone receptor (PR) and androgen receptor (AR) activity. Within this series, OP-3633 (15) emerged as a glucocorticoid receptor (GR) antagonist with increased selectivity against PR and AR, improved cytochrome P450 inhibition profile, and significantly improved pharmacokinetic properties compared to 1. Furthermore, 15 demonstrated substantial inhibition of GR transcriptional activity in the GR positive HCC1806 triple negative breast cancer xenograft model. Overall, compound 15 is a promising GR antagonist candidate to clinically evaluate the impact of GR inhibition in reversal or prevention of therapy resistance.

Ventrolateral medulla(VLM) was one of the essential part of central pattern generator(CPG) in swallowing and electro-acupuncture(EA) was an important intervention in swallowing disorder. But the effect and mechanism of EA at acupoints on swallowing were unknown. The present aim to detect the effect of EA at Lianquan (CV23) on swallowing and swallowing-related(SR) interneuron in VLM. Thirty-six Sprague-Dawley rats were operated and the swallowing reflex was induced through Double distilled water (DDW) infusion. Simultaneously, the numbers of swallowing were recorded. Then EA was given at Lianquan and Neiguan (PC6) and the neuron discharges in VLM were detected. A total of 72 neurons were recorded, 60 of which were correctly recorded after histology identification. Two types of SR neurons were found and the numbers of swallowing increased after EA at CV23 and PC6 compared with no EA group. The neuron response rates were 78.3% and 50% for EA at CV23 and PC6 respectively with significant difference (P 0.05). Meanwhile, the neuron spike patterns were changed after EA at CV23 and PC6. In addition, twenty-four rats were used for immunofluorescence after EA at CV23 and PC6. The results showed that c-fos positive cells in CV23 group were 20.63±2.35, while PC6 group was 14.13±1.78 and 6.88±1.42 in control group. There were significant difference between them (P 0.05). These results indicated that EA could regulate the swallowing function via activating the SR interneurons in VLM under the physiological condition.

Despite the promise of immunotherapy for cancer treatment, nearly 80% of patients fail to respond to checkpoint inhibitor (CPI) therapy. Regulatory T cells (Tregs), which inhibit immune responses in the tumor microenvironment via multiple suppressive mechanisms, have been proposed to play a key role in those patients who are not responding to CPI therapy. Therefore, targeted depletion of Tregs should promote more effective antitumor immunity. CCR8 is a chemokine receptor that is selectively expressed on highly activated human tumor-resident Tregs, and these intratumoral CCR8+ Tregs have been shown to drive immunosuppression that leads to poor prognosis for cancer patients. Here, we demonstrate that CCR8 is highly expressed on intratumoral FoxP3+ Treg cells in multiple cancers and is absent on other major immune cell populations in tumor microenvironment including effector T cells, conventional CD4 T cells, B cells, NK cells, some FoxP3+ cells, and myeloid cells. Importantly, no CCR8 protein expression was observed on any peripheral human leukocyte subset examined. These results provide strong rationale for targeting CCR8 as a cancer immunotherapy by selectively depleting the most suppressive intratumoral Treg cells. We have developed a humanized therapeutic antibody, ZL-1218, that binds to human CCR8 with high affinity and specificity and can induce potent ADCC activity enabling strong NK cell-mediated killing of CCR8-expressing Tregs. We show that in human CCR8 knock-in mouse models bearing syngeneic tumors, ZL-1218 reduces intratumoral Treg cells and thus elicits significant tumor growth inhibition in a dose-dependent manner. We have recently explored the potential for ZL-1218 in combination immunotherapy, examining the enhanced antitumor activity when ZL-1218 is combined with anti-PD-1. Using human dissociated tumor samples, we further observed that different tumor types may induce different CCR8 expression levels on intratumoral Tregs leading to multiple, distinct CCR8+ subsets in various indications. We are currently exploring the significance of these distinct populations and the impact of ZL-1218-mediated depletion of both CCR8 high- and CCR8 low-expressing subsets in multiple indications. Together, these data support the advancement of ZL-1218 into clinical evaluation as a novel therapeutic candidate to treat human solid tumors. Citation Format: Jing Zhang, Wenhua Shi, Min Chen, Xinchuan Dai, Hongshui Liu, Shou Li, Lina Wang, Bee-Chun Sun, Monica You, Vivian Morton, Qiuping Ye, Lishan Kang, Bing Wan, Peter Brams, David I. Bellovin. ZL-1218, a novel anti-CCR8 antibody, exerts potent antitumor effect by depleting intratumoral regulatory T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3590.

DNA-damaging poisons and replicative stress commonly result in increased double-strand breaks (DSBs) in tumor cells. Non-homologous end joining (NHEJ), driven by the enzymatic function of DNA-PK, is one of the critical pathways employed to repair such DSBs. Inhibition of DNA-PK kinase activity impairs NHEJ, potentiates the activity of DNA-damaging agents, and inhibits survival of tumor cells incapable of repairing DSBs via other means, including homologous recombination (HR). We have developed an orally bioavailable, highly potent, and selective pharmacological inhibitor of DNA-PK activity, ZL-2201, for the treatment of cancer. ZL-2201 demonstrates enzymatic inhibition of DNA-PK in the single-digit nM range, with nearly 500-fold or greater selectivity against other phosphatidylinositol 3-kinase-related kinases (PIKKs). In addition, ZL-2201 showed high selectivity for DNA-PK kinase activity in a kinase panel screening study and a clean off-target profile with no appreciable inhibition against a panel of diverse, safety-relevant targets. We demonstrate that ZL-2201 effectively inhibits DNA-PK autophosphorylation and its downstream target phospho-RPA in multiple cell lines in a concentration- and time-dependent manner. DNA-PK has been shown previously to have synthetically lethal interactions with ATM. Therefore, we profiled the impact of ATM expression on the antiproliferative effects of ZL-2201. Cancer cell lines possessing defects in ATM displayed greater sensitivity to ZL-2201 compared to ATM-WT cancer cell lines. These observations were recapitulated in two ATM knockout isogenic cell lines. Moreover, ZL-2201 displayed minimal effects in a DNA-PK deficient model or in normal human and canine colon and kidney cell lines. Independent of ATM status, ZL-2201 showed strong synergy in vitro with topoisomerase II inhibitors. Following oral administration of ZL-2201 in vivo, ZL-2201 demonstrated dose-dependent and exposure-dependent antitumor activity against multiple models, with the greatest activity against ATM deficient human cancer xenografts. Antitumor activity correlated with duration of ZL-2201 exposure and level of target engagement. In addition, ZL-2201 significantly enhanced the activity of DNA-damaging agents in vivo, even in human cancer xenograft models with limited monotherapy antitumor activity for ZL-2201. The projected human PK properties, based on the ADME properties across species and PK properties in preclinical species of ZL-2201, suggest that ZL-2201 is suitable for oral administration. In conclusion, ZL-2201 demonstrates potent and selective inhibition of DNA-PK enzymatic activity, strong synergy with DNA damaging agents in vitro and in vivo, antitumor activity against multiple human xenograft models, and a favorable projected human PK profile. ZL-2201 is expected to enter clinical development in 2022. Citation Format: Shruti Lal, Bee-Chun Sun, Yuping Chen, Tom Huang, Neil Bhola, Vivian Morton, Kevin Chen, Shanghua Xia, Haoyu Zhang, Qiuping Ye, Petter Veiby, David I. Bellovin, Yuhua Ji. Discovery and characterization of ZL-2201, a potent, highly-selective, and orally bioavailable small-molecule DNA-PK inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2594.

The adenosinergic pathway represents an attractive new therapeutic approach in cancer immunotherapy. In this pathway, ecto-5-nucleotidase CD73 has the unique function of regulating production of immunosuppressive adenosine (ADO) through the hydrolysis of AMP. CD73 is overexpressed in many cancers, resulting in elevated levels of ADO that correspond to poor patient prognosis. Therefore, reducing the level of ADO via inhibition of CD73 is a potential strategy for treating cancers. Based on the binding mode of adenosine 5'-(α,β-methylene)diphosphate (AOPCP) with human CD73, we designed a series of novel monophosphonate small-molecule CD73 inhibitors. Among them, OP-5244 (35) proved to be a highly potent and orally bioavailable CD73 inhibitor. In preclinical studies, 35 completely inhibited ADO production in both human cancer cells and CD8+ T cells. Furthermore, 35 lowered the ratio of ADO/AMP significantly and reversed immunosuppression in mouse models, indicating its potential as an in vivo tool compound for further development.

The glucocorticoid receptor (GR) has been linked to therapy resistance across a wide range of cancer types. Preclinical data suggest that antagonists of this nuclear receptor may enhance the activity of anticancer therapy. The first-generation GR antagonist mifepristone is currently undergoing clinical evaluation in various oncology settings. Structure-based modification of mifepristone led to the discovery of ORIC-101 (28), a highly potent steroidal GR antagonist with reduced androgen receptor (AR) agonistic activity amenable for dosing in androgen receptor positive tumors and with improved CYP2C8 and CYP2C9 inhibition profile to minimize drug-drug interaction potential. Unlike mifepristone, 28 could be codosed with chemotherapeutic agents readily metabolized by CYP2C8 such as paclitaxel. Furthermore, 28 demonstrated in vivo antitumor activity by enhancing response to chemotherapy in the GR+ OVCAR5 ovarian cancer xenograft model. Clinical evaluation of safety and therapeutic potential of 28 is underway.

The aim of this study was to determine the optimum frequency of electroacupuncture (EA) for the treatment of dysphagia after stroke. Male C57BL/6 J mice were randomly divided into five groups: normal, model, 2 Hz, 50 Hz, and 100 Hz groups. All mice received a photochemical ischemia, except the normal group. The EA parameters were 1 mA for 15 min, with different frequencies (2, 50, and 100 Hz) applied. After a three day treatment, neuronal activation was detected by the expression of c-Fos. A multi-channel electrophysiological technique was used to assess the discharge of contralateral neurons and the neuron types in each group. The concentration of brain-derived neurotrophic factor (BDNF) in the contralateral neurons was also examined. In addition, the dysfunction of swallowing in mice was calculated according to the lick counts and the lick–lick interval within a certain period of time. The number of c-Fos neurons (P

Metal-free graphitic carbon nitride (g-C3N4) should be an ideal candidate for photocatalytic degradation of pollutants. However, its performance is limited by low adsorption and high recombination rate of electron-hole pairs. Herein, g-C3N4 nanosheets were highly dispersed on rice husk carbon (CN@RHC) by one-step sealed calcination. The specific surface area was greatly increased from 7.7 to 137 m2/g, which improves the adsorption performance. Abundant dangling CC bond defects of rice husk carbon were confirmed by electron paramagnetic resonance, which played an important role of electron acceptor and effectively inhibit the recombination of photogenerated carriers. As a result, 98% of Methylene blue was removed in 4.5 h using a 5 W light emitting diode source with visible light while only 80% of that was removed by bulk g-C3N4. The degradation performance was enhanced by the synergism of excellent adsorption performance and better photogenerated carriers transport performance. Based on the simple synthesis method, low cost and excellent visible photocatalytic performance, dangling CC bonds should be the focus for promoting photocatalysis as well as improve the added value of agricultural waste.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer and one of the most lethal cancers, with a 5-year survival rate of 8%. Chemotherapy remains the main treatment option for patients with advanced and metastatic tumors. However, intrinsic resistance to chemotherapeutics and lack of effective targeted therapies are major factors contributing to the dismal prognosis. Activation of glucocorticoid receptor (GR) signaling confers resistance to chemotherapy in solid tumors, making GR antagonism an attractive combination treatment strategy to overcome chemotherapeutic resistance. ORIC-101 is a novel selective GR antagonist under clinical evaluation in combination with anticancer therapies. We previously reported that ORIC-101 can sensitize triple-negative breast cancer cells to chemotherapy by reversing multiple GR-modulated pathways. Here, we investigate the effects of ORIC-101 in preclinical models of PDAC. Employing in vitro functional assays and gene expression analysis, ORIC-101 exhibited chemo-potentiation in a panel of PDAC cell lines. Biomarker analysis of transcription activity following ORIC-101 treatment in vitro confirmed pharmacodynamic (PD) modulation of core GR target genes, indicative of suppressed GR signaling. Extending these findings in vivo across several PDAC xenograft models (SW1990, BxPC3, and HPAC) revealed that ORIC-101 blocks GR transcriptional activity induced by dexamethasone in a dose-dependent manner. The PD modulation observed in vivo correlated with ORIC-101 plasma exposure. Consistent with ORIC-101's ability to suppress GR activity, in vivo efficacy studies in combination with paclitaxel significantly inhibited PDAC tumor growth compared to the paclitaxel control group. These results in PDAC preclinical models support the clinical development of ORIC-101 in overcoming GR-driven chemoresistance. A phase 1b study of ORIC-101 in combination with nab-paclitaxel in patients with advanced or metastatic solid tumors is ongoing (NCT03928314). Citation Format: Jessica D. Sun, Haiying Zhou, Wayne Kong, Natalie Yuen, Frank L. Duong, Shravani Barkund, Aleksandr Pankov, Dan McWeeney, Qiuping Ye, Omar Kabbarah, Lori S. Friedman, Anneleen Daemen, Melissa Junttila. ORIC-101 overcomes glucocorticoid receptor-mediated chemoresistance in pancreatic cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4123.

ORIC-101 is a novel and selective glucocorticoid receptor (GR) antagonist currently in Ph1 clinical evaluation in solid tumors. In preclinical models, ORIC-101 potently sensitized triple-negative breast cancer (TNBC) cells to chemotherapy by inhibiting GR-mediated resistance through the epithelial-to-mesenchymal transition and anti-apoptosis pathways, both in vitro and in vivo. To inform the clinical development of ORIC-101, we identified pharmacodynamic (PD) biomarkers and established a relationship between those biomarkers, pharmacokinetics (PK), and efficacy in preclinical models. Using genome-wide profiling, we developed a GR activation signature that includes the direct GR transcriptional targets FKBP5 and GILZ, and subsequently validated both genes as ORIC-101 PD biomarkers in a panel of cell lines. We demonstrated that FKBP5 and GILZ are transcriptionally induced by glucocorticoids in a dose-dependent manner, and that the degree of their transcriptional activation is correlated with basal levels of GR in these models. We showed that ORIC-101 robustly inhibits the transcriptional induction of FKBP5 and GILZ and fully reverses GR-mediated chemoresistance in vitro and in vivo. Furthermore, FKBP5 and GILZ are reliable PD biomarkers for ORIC-101 in human-derived peripheral blood mononuclear cells (PBMCs). Importantly, by integrating PK, PD, and efficacy results from numerous preclinical studies, we captured the relationship between ORIC-101 PK, PD biomarker modulation, and chemopotentiation. Altogether, FKBP5 and GILZ mRNA levels are robust PD biomarkers to measure GR pathway engagement and modulation which corresponds with efficacy in response to GR antagonist treatment in preclinical models. These findings will guide the clinical development of ORIC-101 in TNBC. Citation Format: Haiying Zhou, Qiuping Ye, Aleksandr Pankov, Wayne Kong, Shravani Barkund, Lori S. Friedman, Jessica D. Sun, Omar Kabbarah. ORIC-101 robustly inhibits the glucocorticoid pathway and overcomes chemoresistance in triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-24.

Introduction: CD73 mediates the final dephosphorylation in the conversion of extracellular ATP to adenosine, a metabolite which signals through the A2 family of receptors. Like PD-1, adenosine appears to be part of a negative feedback loop to limit immune activation and prevent excessive inflammation in the context of tissue damage. However, intratumoral hypoxia likely drives excessive CD73-mediated adenosine generation and prevents optimal anti-tumor immune responses. We show that intratumoral adenosine levels are sufficient to drive pervasive suppression of multiple immune subsets in vitro and an orally bioavailable small molecule can prevent adenosine generation in vitro and within the tumor in vivo. Methods We examined the effects of adenosine signaling on multiple immune cell subsets through in vitro functional assays. We interrogated T cell activation, proliferation, and cytolytic activity, as well as NK cell function in vitro. Furthermore, we investigated and identified functional targets of adenosine signaling within T cells using transcriptomic and protein expression analyses. In addition, we performed in vitro myeloid differentiation assays to determine the effect of adenosine signaling on myeloid lineage maturation. Finally, we explored the activity of a novel, orally bioavailable inhibitor of CD73 in vitro and assessed its ability to rescue T cell activation and suppress AMP conversion to adenosine within the tumor microenvironment. Results and Conclusions We found that T cell-expressed CD73 was sufficient to drive adenosine generation from AMP in vitro, resulting in suppression of anti-CD3/CD28-induced T cell activation and proliferation and a reduced capacity to kill cognate antigen-expressing tumor cells. Adenosine-exposed T cells displayed decreased induction of markers associated with activation at both the mRNA and protein level, including Ki67, ICOS, and PD-1, while showing higher expression of naïve-associated CD73. Interestingly, we also observed a third subset of markers which were uniquely induced by adenosine. In addition to its activity on T cells, adenosine signaling caused functional suppression of NK cells in the presence of target Yac-1 cells. In the myeloid compartment, NECA, a stable analog of adenosine, prevented in vitro differentiation of CD103+ cross-presenting dendritic cells, a population which provides a critical stimulus to tumor-infiltrating T cells. Additionally, NECA impaired macrophage expression of CD80, a canonical M1 marker and mediator of T cell costimulation, in both the presence and absence of M1-polarizing IFNγ and LPS. Finally, we showed that a novel, orally bioavailable CD73 inhibitor was able to effectively inhibit AMP to adenosine conversion both in vitro and in vivo, while an anti-CD73 antibody had incomplete effects. Taken together, an orally bioavailable small molecule inhibitor of CD73 represents a potential therapeutic approach to reverse immunosuppression within the tumor microenvironment. Citation Format: Todd C Metzger, Brian R Blank, Brenda Chan, Chelsea Chen, Yuping Chen, Xiaohui Du, Frank L Duong, Valeria R. Fantin, Lori S Friedman, Jared T Moore, Daqing Sun, Jessica Sun, Dena Sutimantanapi, Qiuping Ye, Natalie Yuen, Tatiana Zavorotinskaya. Intratumoral immunosuppression is reversed by blocking adenosine production with an oral inhibitor of CD73 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A19. doi:10.1158/1535-7163.TARG-19-LB-A19

To observe the effect of electroacupuncture (EA) on swallowing and its underlying mechanism, 32 Sprague-Dawley (SD) rats were chose and the electrophysiology was used to detect the discharge of nucleus ambiguus (NA) after EA at CV23 (Lianquan), GV16 (Fengfu), and other acupoints. The swallowing-related motor neuron was identified by antidromic stimulation through recurrent laryngeal nerve. Meanwhile, the swallowing numbers were induced by Double-distilled water (DDW) and the neuron discharges were recorded before and after EA. Beside, 50 SD rats were used for testing the c-fos expressions in NA after EA at different acupoints and the other 80 SD rats were used for chemical damage through the microinjection to bilateral NA. 58 neurons provided complete data after histological identification. And two types of swallowing-related (SR) motor neurons were identified, named spontaneous and silent neurons. We found that the onset latency of the first swallow was shorter and the swallowing numbers were increased after EA at CV23 than the other acupoints (P 0.01). The excitatory neuron response rates were 66.67%, 71.11%, 42.22% and 35.56% for CV23, GV16, PC6 (Neiguan), and ST36 (Zusanli), respectively. The c-fos expressions on CV23 and GV16 groups were significantly higher than the other groups (P 0.05). After chemical damage, the swallowing numbers could not be regulated by EA, but could be regulated by EA after fake damage. The results of the present study demonstrate that EA at CV23 and GV16 could regulate swallowing function via activating swallowing-related motor neurons in NA.

1. AMG 232 is a novel inhibitor of the p53-MDM2 protein-protein interaction currently in Phase I clinical trials for multiple tumor indications. The objectives of the investigations reported in this article were to characterize the pharmacokinetic and drug metabolism properties of AMG 232 in pre-clinical species in vivo and in vitro, and in humans in vitro, and to predict its pharmacokinetics in humans through integrating PKDM data. 2. AMG 232 exhibited low clearance (0.25 × Qh) and moderate to high oral bioavailability in mice, rats and monkeys (42%), but high clearance (0.74 × Qh) and low oral exposure in dogs (18%). 3. Biotransformation was the major route of elimination of AMG 232 in rats, with only 7% of intravenously administered (14)C-labeled AMG 232 recovered as parent molecule in bile. The major metabolite was an acyl glucuronide as measured by in vivo rat studies and in vitro hepatocyte incubations in multiple species. 4. The in vitro-in vivo correlation of AMG 232 clearance was within 2-fold in pre-clinical species using hepatocytes. AMG 232 was predicted to exhibit low clearance, high volume distribution and long half-life in humans. The predictions are consistent with the preliminary human pharmacokinetic parameters of AMG 232 in clinical trials.

The sweet smell of aroma of Jasminum sambac (L.) Ait. is releasing while the flowers are blooming. Although components of volatile oil have been extensively studied, there are problematic issues, such as low efficiency of yield, flavour distortion. Here, the subcritical fluid extraction (SFE) was performed to extract fragrant volatiles from activated carbon that had absorbed the aroma of jasmine flowers. This novel method could effectively obtain main aromatic compounds with quality significantly better than solvent extraction (SE). Based on the analysis data with response surface methodology (RSM), we optimized the extraction conditions which consisted of a temperature of 44°C, a solvent-to-material ratio of 3.5:1, and an extraction time of 53 min. Under these conditions, the extraction yield was 4.91%. Furthermore, the key jasmine essence oil components, benzyl acetate and linalool, increase 7 fold and 2 fold respectively which lead to strong typical smell of the jasmine oil. The new method can reduce spicy components which lead to the essential oils smelling sweeter. Thus, the quality of the jasmine essence oil was dramatically improved and yields based on the key component increased dramatically. Our results provide a new effective technique for extracting fragrant volatiles from jasmine flowers.

Structure-based rational design and extensive structure-activity relationship studies led to the discovery of AMG 232 (1), a potent piperidinone inhibitor of the MDM2-p53 association, which is currently being evaluated in human clinical trials for the treatment of cancer. Further modifications of 1, including replacing the carboxylic acid with a 4-amidobenzoic acid, afforded AM-7209 (25), featuring improved potency (KD from ITC competition was 38 pM, SJSA-1 EdU IC50 = 1.6 nM), remarkable pharmacokinetic properties, and in vivo antitumor activity in both the SJSA-1 osteosarcoma xenograft model (ED50 = 2.6 mg/kg QD) and the HCT-116 colorectal carcinoma xenograft model (ED50 = 10 mg/kg QD). In addition, 25 possesses distinct mechanisms of elimination compared to 1.

Objective. To assess the therapeutic efficacy of acupuncture for dysphagia after stroke. Methods. Seven electronic databases were searched from their inception until 31 September 2016. All randomized controlled trials (RCTs) incorporating acupuncture or acupuncture combined with other interventions for treatment of dysphagia after stroke were enrolled. Then they were extracted and assessed by two independent evaluators. Direct comparisons were conducted in RevMan 5.3.0 software. Results. 6010 patients of 71 papers were included. The pooled analysis of efficacy rate of 58 studies indicated that acupuncture group was superior to the control group with moderate heterogeneity (RR = 1.17, 95% CI: 1.13 1.21, Z=9.08, and P<0.00001); meta-analysis of the studies using blind method showed that the efficacy rate of acupuncture group was 3.01 times that of control group with no heterogeneity (RR = 3.01, 95% CI: 1.95 4.65, Z=4.97, and P<0.00001). Only 13 studies mentioned the safety evaluation. Conclusion. The result showed that the acupuncture group was better than control group in terms of efficacy rate of dysphagia after stroke. And the combining result of those researches using blind method was more strong in proof. Strict evaluation standard and high-quality RCT design are necessary for further exploration.

We recently reported on the discovery of AMG 232, a potent and selective piperidinone inhibitor of the MDM2-p53 interaction. AMG 232 is being evaluated in human clinical trials for cancer. Continued exploration of the N-alkyl substituent of this series, in an effort to optimize interactions with the MDM2 glycine-58 shelf region, led to the discovery of sulfonamides such as compounds 31 and 38 that have similar potency, hepatocyte stability and rat pharmacokinetic properties to AMG 232.

Continued optimization of the N-substituent in the piperidinone series provided potent piperidinone-pyridine inhibitors 6, 7, 14, and 15 with improved pharmacokinetic properties in rats. Reducing structure complexity of the N-alkyl substituent led to the discovery of 23, a potent and simplified inhibitor of MDM2. Compound 23 exhibits excellent pharmacokinetic properties and substantial in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft mouse model.

We previously reported the discovery of AMG 232, a highly potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Our continued search for potent and diverse analogues led to the discovery of novel morpholinone MDM2 inhibitors. This change to a morpholinone core has a significant impact on both potency and metabolic stability compared to the piperidinone series. Within this morpholinone series, AM-8735 emerged as an inhibitor with remarkable biochemical potency (HTRF IC50 = 0.4 nM) and cellular potency (SJSA-1 EdU IC50 = 25 nM), as well as pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 41 mg/kg. Lead optimization toward the discovery of this inhibitor as well as key differences between the morpholinone and the piperidinone series will be described herein.

The Glucocorticoid Receptor (GR) is a member of the superfamily of nuclear hormone receptors that is activated by human cortisol and synthetic glucocorticoids such as dexamethasone (Dex). Upon ligand binding, GR translocates to the nucleus and regulates the expression of a wide spectrum of genes involved in diverse biological processes, including inflammation, immunity, metabolism, cell cycle, and differentiation. Dysregulated cortisol levels are associated with poor prognosis, drug resistance, and increased cancer recurrence. Multiple studies have shown that GR inhibition reverses resistance to chemotherapy in cancers of epithelial origin including prostate, bladder, renal, ovarian, pancreatic, and triple negative breast cancer (TNBC). We have recently reported the discovery of ORIC-101, a potent GR antagonist with a unique cytochrome P450 inhibition profile that makes this compound particularly suitable for combination with taxanes such as paclitaxel (Rew Y et al, 2018). Consistent with previous reports, our data showed that activation of GR promoted growth of TNBC cells in 3D culture conditions and protected TNBC cells from paclitaxel. Treatment with ORIC-101 fully reversed these effects. To understand the molecular basis of the observed GR-mediated chemotherapy resistance, we set out to isolate the pool of TNBC cells that escaped from paclitaxel treatment in the presence of Dex. Molecular profiling of these “chemotherapy escapees” pointed to a number of glucocorticoid-regulated biological pathways, including basal stem cell lineage genes and mesenchymal markers, suggesting the acquisition of an EMT-like phenotype in chemo-resistant cells. In support of this finding, we found using ChIP-seq analysis that GR directly bound within the promoter/enhancer regions of well-established EMT genes such as SNAI2 and FN1, and regulated their expression in response to Dex treatment. Functionally, RNAi-mediated knockdown of SNAI2 partially restored sensitivity to paclitaxel, suggesting that the GR-driven EMT phenotype contributes to paclitaxel resistance in TNBC cells. Consistent with the in vitro observations, immunohistochemical analysis showed that GR activation upregulated the levels of both basal stem cell and mesenchymal markers in “chemotherapy escapees” from paclitaxel-treated TNBC xenografts. Importantly oral administration of ORIC-101 fully blocked these effects. Altogether, we found that activation of GR drove an EMT-like phenotype in TNBC cells in vitro and in vivo. ORIC-101 reversed these effects and sensitized TNBC cells to chemotherapy. Our findings thus provide mechanistic insights into the role of GR as a mediator of therapy resistance in TNBC. Clinical evaluation is being planned to assess the therapeutic potential of ORIC-101 in combination with standard-of-care chemotherapeutic agents. Citation Format: Haiying Zhou, Jessica Sun, Wayne Kong, Yosup Rew, Xiaohui Du, John Eksterowicz, Daqing Sun, Qiuping Ye, Omar Kabbarah, Valeria R. Fantin. ORIC-101 reverses a GR-driven EMT-like phenotype and sensitizes TNBC cells to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3822.

Subcritical fluid extraction (SFE), as a novel method, was applied to investigate the yield, quality, and sensory evaluation of headspace oil from Jasminum sambac (L.) Aiton in comparison with petroleum ether extraction (PEE). The results indicated that the yield of the headspace oil using SFE was significantly higher (P < 0.05) than when using PEE. SFE contributed to obtaining alcohols and ethers, prevented the thermal reaction of terpenes, and reduced α-caryophyllene and β-caryophyllene in the headspace oil. The contents of linalool (21.90%) and benzyl acetate (16.31%) were higher via SFE than PEE. In addition, the sensory evaluation of SFE was superior to PEE, indicating a fresh, jasmine-like odor and green-yellow color. Thus, SFE is an improved method for obtaining natural headspace oil from jasmine flowers.

Ischemia/reperfusion may induce inflammation and cell death through the nuclear factor (NF)‑κB signaling pathway. As a negative regulator of NF‑κB, zinc finger A20 exhibits anti-apoptotic and anti‑inflammatory effects in vitro. The present study was designed to upregulate A20 expression using an A20 transfection approach to investigate the in vivo protective effects of the A20 gene on renal ischemia reperfusion injury. The A20 gene was cloned into a pcDNA3.1 vector to construct the expression plasmid pcDNA3.1‑A20. The plasmid was wrapped with a liposome and injected intravenously into rats 48 h prior to establishing the models of renal ischemia reperfusion injury. Saline and the empty plasmid pcDNA3.1 were used as controls. Following 24 h post‑operation, A20 expression was determined using reverse transcription‑quantitative polymerase chain reaction and western blotting. The renal function and structure were assessed by analyzing the concentrations of serum creatinine (Scr), blood urea nitrogen (BUN) and histological features. Renal tissues were additionally examined for renal tubular cell apoptosis and NF‑κB activity. The results demonstrated that in vivo transfection of pcDNA3.1‑A20 induced renal A20 expression in rats. A20 overexpression in vivo significantly reduced renal injury as demonstrated by the improved levels of Scr and BUN and the reduction in histological damage. These improvements were accompanied by a suppression of renal proximal tubular epithelial cell apoptosis and an inhibition of NF‑κB activity. These results indicated that transfection of the A20 gene upregulates the expression of A20 in vivo and protects the kidneys from ischemia reperfusion injury via inhibition of the NF‑κB signal transduction pathway.

A series of spiropiperidine carbazoles were synthesized and evaluated as MCHR2 antagonists using a FLIPR assay. The pharmacokinetic properties of selected compounds have also been studied. This effort led to the discovery of potent and specific MCHR2 antagonists. Compound 38 demonstrated good pharmacokinetic properties across rat, beagle dog and rhesus monkey and had a favorable selectivity profile against a number of other receptors. These MCHR2 antagonists are considered appropriate tool compounds for study of the function of MCHR2 in vivo.

A bis-amide antagonist of Smoothened, a seven-transmembrane receptor in the Hedgehog signaling pathway, was discovered via high throughput screening. In vitro and in vivo experiments demonstrated that the bis-amide was susceptible to N-acyl transferase mediated amide scission. Several bioisosteric replacements of the labile amide that maintained in vitro potency were identified and shown to be metabolically stable in vitro and in vivo.