Characterization of metabolic features and potential anti-osteoporosis mechanism of pinoresinol diglucoside using metabolite profiling and network pharmacology.

RATIONALE: Eucommia cortex is the core herb in traditional Chinese medicine preparations for the treatment of osteoporosis. Pinoresinol diglucoside (PDG), the quality control marker and the key pharmacodynamic component in Eucommia cortex, has attracted global attention because of its definite effects on osteoporosis. However, the in vivo metabolic characteristics of PDG and its anti-osteoporotic mechanism are still unclear, restricting its development and application. METHODS: Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used to analyze the metabolic characteristics of PDG in rats, and its anti-osteoporosis targets and mechanism were predicted using network pharmacology. RESULTS: A total of 51 metabolites were identified or tentatively characterized in rats after oral administration of PDG (10 mg/kg/day), including 9 in plasma, 28 in urine, 13 in feces, 10 in liver, 4 in heart, 3 in spleen, 11 in kidneys, and 5 in lungs. Furan-ring opening, dimethoxylation, glucuronidation, and sulfation were the main metabolic characteristics of PDG in vivo. The potential mechanism of PDG against osteoporosis was predicted using network pharmacology. PDG and its metabolites could regulate BCL2, MARK3, ALB, and IL6, involving PI3K-Akt signaling pathway, estrogen signaling pathway, and so on. CONCLUSIONS: This study was the first to demonstrate the metabolic characteristics of PDG in vivo and its potential anti-osteoporosis mechanism, providing the data for further pharmacological validation of PDG in the treatment of osteoporosis.

Clinical implications of aberrant PD-1 expression for acute leukemia prognosis.

BACKGROUND: Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are the most common types of leukemia in adults with an overall poor prognosis. PD-1 alone or combined with other immune checkpoint blockade is a promising research direction for the treatment of acute leukemia (AL) patients. However, clinical Implications of aberrant PD-1 expression in peripheral CD4+ and CD8+ T lymphocytes of AML and ALL patients in assessing the prognosis of diseases, remains inconclusive. METHODS: In the present study, we used flow cytometry to evaluate PD-1 expression on the surface of CD4+ and CD8+ T lymphocytes in the peripheral circulation of AML and ALL patients and its clinical significance. A total of 53 AML patients, 44 ALL patients and 28 healthy controls were enrolled in this study and peripheral blood specimens were detected by flow cytometry. RESULTS: Our results indicated that percentages of CD4+ PD1+ and CD8+ PD1+ T lymphocytes in newly diagnosed and non-remission groups were significantly higher than healthy control both in AML and ALL patients. The high level of CD4+ PD1+ and CD8+ PD1+ T lymphocytes were respectively poor prognostic indicators of AML patients and ALL patients but had no significant correlation with most common clinical risks. CONCLUSIONS: Our findings show that aberrant PD-1 expression correlates with the prognosis of AL patient and may thus serve as poor prognostic indicators. Immunotherapy using PD-1 inhibitors may be a promising strategy for AML and ALL patients with peripheral circulating CD4+ PD1+ and CD8+ PD1+ T lymphocytes positively expressed, respectively.

CD4+CD25+CD127low regulatory T cells associated with the effect of CD19 CAR-T therapy for relapsed/refractory B-cell acute lymphoblastic leukemia.

BACKGROUND: CD19-specific chimeric antigen receptor T-cell (CAR-T) therapy has shown promising clinical outcomes in relapsed/refractory acute B-cell lymphoblastic leukemia (R/R B-ALL) patients. However, some patients did not respond to this therapy or relapsed after remission. Regulatory T cells (Tregs) have shown great importance in promoting tumor escape, but little is known about their role in R/R B-ALL patients with CAR-T therapy. Our previous study has proved that higher Tregs before infusion was an independent high-risk factor for relapse-free survival (RFS). To further clarify the relationship between Tregs and the efficacy of CAR-T therapy, the present study tested the levels of CD4+CD25+CD127low Tregs in peripheral blood (PB) of R/R B-ALL patients at different stages of CD19 CAR-T therapy, and evaluate their impact on the efficacy and prognosis of CAR-T therapy. METHODS: From November 2015 to May 2019, 47 R/R B-ALL patients successfully received CD19 CAR-T therapy at our institution and followed up for at least 1 month. Among them, one patient did not tested for Tregs, so 46 patients enrolled in this study. We collected clinical information of them and dynamically detected the frequency of CD4+CD25+CD127low Tregs within CD4 + T cells at different time points (before infusion and at 1 week after infusion) by flow cytometry, and validated the relationship of circulating Tregs with clinical efficacy, OS, and recurrence of CAR-T therapy. RESULTS: Circulating Tregs of R/R B-ALL patients in pre-infusion group (median 6.67%) and in 1 week after infusion group (median 6.80%) were all higher than that of the healthy control group (median 5.04%), with statistical significance (P < 0.05). The frequencies of Tregs in not remission (NR) group at baseline (pre-infusion) and at 1 week after infusion were all significantly higher than those in remission group. With cut-off values of 11.54% (before infusion) and 13.56% (1 week after infusion), the specificity for Tregs were 94.6% and 100% , respectively. In remission group, 11 patients underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) after achieving remission by Sino 19 cell therapy. No significant differences of Tregs expression were found between transplantation and non-transplantation groups. Time-dependent Cox model showed that transplantation group had lower risk of relapse and death when compared with non-transplantation group (HR = 0.664 for RFS and HR = 0.364 for OS), however, no statistical significances were found (P = 0.403 and 0.106, respectively). Higher Tregs before infusion and at 1 week after infusion were significant associated with shorter RFS and OS by Kaplan-Meier analysis. Multivariate analysis showed that higher Tregs at 1 week after infusion was the independently factor for poor RFS (P = 0.032) and shorter OS (P = 0.025) in R/R B-ALL patients with CD19 CAR-T therapy. Besides, Tregs levels before and at 1 week after infusion were negatively correlated with the persistence time of Sino 19 cell. CONCLUSION: Higher circulating Tregs, especially 1 week after CD19 CAR-T cell infusion, was a poor predict indicator for CD19 CAR-T therapy in R/R B-ALL patients.

Influence of patient characteristics on chimeric antigen receptor T cell therapy in B-cell acute lymphoblastic leukemia.

CD19-specific chimeric antigen receptor T cell (CD19 CAR T) therapy has shown high remission rates in patients with refractory/relapsed B-cell acute lymphoblastic leukemia (r/r B-ALL). However, the long-term outcome and the factors that influence the efficacy need further exploration. Here we report the outcome of 51 r/r B-ALL patients from a non-randomized, Phase II clinical trial (ClinicalTrials.gov number: NCT02735291). The primary outcome shows that the overall remission rate (complete remission with or without incomplete hematologic recovery) is 80.9%. The secondary outcome reveals that the overall survival (OS) and relapse-free survival (RFS) rates at 1 year are 53.0 and 45.0%, respectively. The incidence of grade 4 adverse reactions is 6.4%. The trial meets pre-specified endpoints. Further analysis shows that patients with extramedullary diseases (EMDs) other than central nervous system (CNS) involvement have the lowest remission rate (28.6%). The OS and RFS in patients with any subtype of EMDs, higher Tregs, or high-risk genetic factors are all significantly lower than that in their corresponding control cohorts. EMDs and higher Tregs are independent high-risk factors respectively for poor OS and RFS. Thus, these patient characteristics may hinder the efficacy of CAR T therapy.

Immune dysregulation in primary immune thrombocytopenia patients.

OBJECTIVES: To explore the immunological abnormalities in patients with primary immune thrombocytopenia (ITP), and analyze its relationship with treatment. METHODS: Proportion of different immune cell subsets were detected in the peripheral blood of 124 ITP patients at different time points and 45 normal controls by flow cytometry. The treatments included glucocorticoids, intravenous IgG as first-line treatment and second-line drugs. RESULTS: Elevated CD4/CD8 ratio and decreased the proportion of NK and CD4 + CD25 + CD127low regulatory T cells (Tregs) were found in pre-treated ITP patients than healthy controls. The newly diagnosed group had a significantly higher CD4/CD8 ratio than the relapsed group, but no differences in the proportion of B cells, NK cells and Tregs. No relationships were found between the curative effect and the pre-treated cell subsets within both the effective and ineffective groups. Furthermore, compared with the ineffective group, the effective group had higher Tregs and lower CD4/CD8 ratio post-treatment, but no significant differences in NK and B cells. CONCLUSION: ITP patients presented with a high CD4/CD8 ratio and low levels of Tregs and NK cells, suggesting that immune deregulation was involved in the pathogenesis of ITP. The pre-treated immune status of ITP patients may not be related to the curative effect. Tregs significantly increased in the effective group post-treatment, highlighting that the mechanism of restoring Tregs may be involved in the treatment of ITP. However, whether or not the targeted regulation of Tregs is an effective treatment for ITP still requires further studies.

Low dose IL-2 increase regulatory T cells and elevate platelets in a patient with immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder in which its immune system destroys platelets and leads to haemorrhage symptom. Recent studies have found that regulatory T cells (Tregs) in peripheral blood, bone marrow, and spleen were reduced in ITP patients and recovered after effective ITP therapy. Low-dose Interleukin-2 (IL-2) has been reported recently to increase Tregs and used to treat autoimmune disease including graft-versus-host disease (GVHD) after organ transplantation and HCV-related autoimmune vasculitis. However, it is unknown whether IL-2 is able to treat ITP. We have used low-dose IL-2 (1.0 million IU/day) on 5 consecutive days per week for 4 weeks in a 36-year-old patient with ITP. The result has shown that low-dose IL-2 induces expansion of Tregs significantly and increase platelet count was gradually from 36 × 109 /L to maximum 85 × 109 /L. No side effects of IL-2 have been found. This result suggested that low-dose of IL-2 may have therapeutic potential for ITP. © 2016 International Clinical Cytometry Society.

Therapeutic potential of low-dose IL-2 in immune thrombocytopenia: An analysis of 3 cases.

Immune thrombocytopenia (ITP) is an acquired immune-mediated disorder with regulatory T cells (Tregs) reduction. Recent studies have shown that low-dose interleukin-2 can preferentially induce Treg expansion in vivo, and therefore offers a therapeutic strategy against immune thrombocytopenia. We have demonstrated in a previous study that Tregs and platelet counts significantly improve in an adult with ITP following low-dose IL-2 treatment. Here we report the efficacy of low-dose IL-2 in another three adults with immune thrombocytopenia who failed the first-line treatment. All patients received a dose of 1.0 million IU IL-2/day for 5 consecutive days per week as a cycle for 2 or 4 weeks. In addition to IL-2, vincristine (2 mg IV weekly × 3 weeks) was added to one patient as a combination therapy. No specific treatment was added in the other two patients. Two cases exhibited significantly increased platelet counts with improved levels of Tregs, while no changes were observed for the remaining patient. In summary, administration of daily subcutaneous low-dose IL-2 was safe, and it may be a new therapeutic option for treatment of ITP, especially refractory ITP. © 2017 International Clinical Cytometry Society.

Discovery and characterization of a novel potent type II native and mutant BCR-ABL inhibitor (CHMFL-074) for Chronic Myeloid Leukemia (CML).

BCR gene fused ABL kinase is the critical driving force for the Philadelphia Chromosome positive (Ph+) Chronic Myeloid Leukemia (CML) and has been extensively explored as a drug target. With a structure-based drug design approach we have discovered a novel inhibitor CHMFL-074, that potently inhibits both the native and a variety of clinically emerged mutants of BCR-ABL kinase. The X-ray crystal structure of CHMFL-074 in complex with ABL1 kinase (PDB ID: 5HU9) revealed a typical type II binding mode (DFG-out) but relatively rare hinge binding. Kinome wide selectivity profiling demonstrated that CHMFL-074 bore a high selectivity (S score(1) = 0.03) and potently inhibited ABL1 kinase (IC50: 24 nM) and PDGFR α/ß (IC50: 71 nM and 88 nM). CHMFL-074 displayed strong anti-proliferative efficacy against BCR-ABL-driven CML cell lines such as K562 (GI50: 56 nM), MEG-01 (GI50: 18 nM) and KU812 (GI50: 57 nM). CHMFL-074 arrested cell cycle into the G0/G1 phase and induced apoptosis in the Ph+ CML cell lines. In addition, it potently inhibited the CML patient primary cell's proliferation but did not affect the normal bone marrow cells. In the CML cell K562 inoculated xenograft mouse model, oral administration of 100 mg/kg/d of CHMFL-074 achieved a tumor growth inhibition (TGI) of 65% without exhibiting apparent toxicity. As a potential drug candidate for fighting CML, CHMFL-074 is under extensive preclinical safety evaluation now.