Pilot Clinical Trial In Patients Research Articles

OR33-04 Mechanistic Dissection Of Circadian-Specific Glucocorticoid Effects On Exercise Tolerance And Muscle Glucose Utilization

Abstract Disclosure: A. Prabakaran: None. M. Wintzinger: None. K. Piczer: None. K. Miz: None. A. Walton: None. H. Durumutla: None. M. Quattrocelli: None. Glucocorticoids (GCs) are circadian hormones regulating metabolism by activating the glucocorticoid receptor (GR) as pleiotropic transcription factor. Timing of exogenous GC drug regimens is emerging as a key determinant of their metabolic effects. This was shown by the surprising finding that once-weekly GC intermittence reverses many of the pro-obesogenic side effects of once-daily GC dosing in mice and humans. However, despite the intrinsic circadian nature of this signaling, the impact of time-of-day of exogenous GC dosing, i.e. chrono-pharmacology, remains remarkably unknown for metabolic physiology and exercise tolerance. Relevance of this question for humans is supported by the initial findings of increased lean mass and motor function with circadian-restricted prednisone intermittence in a recent pilot clinical trial in patients with genetic myopathies. However, mechanisms to support significance of these effects for metabolic diseases and aging remain unknown. We studied 12-week-long intermittent regimens of the exogenous GC prednisone in mice in conditions of diet-induced obesity or advanced aging, comparing dosing at light-phase start (ZT0) versus dark-phase start (ZT12). We found that, compared to dark-phase, light-phase dosing enhanced the regimen-driven increase in body-wide lean mass, muscle mass and muscle function in both obese and aged mice. Intriguingly, experiments with adiponectin-knockout mice showed that these effects were dependent on adiponectin, which promotes muscle insulin sensitivity. We then used tissue-specific inducible knockout models to dissect non-muscle versus muscle-autonomous effects of GC chrono-pharmacology. We found that the GC effects on total and high-molecular-weight adiponectin upregulation were dependent on regimen-specific engagement of the adipocyte-specific GR on the adiponectin gene promoter. Complementary to adipose-driven adiponectin production, we found that adiponectin receptor AdipoR1 upregulation in muscle depended on circadian-specific engagement of the myocyte-specific GR on the AdipoR1 promoter. Consistent with the adiponectin action on muscle insulin sensitivity, ablation of myocyte-specific GR blocked the regimen-driven effects on insulin-sensitive muscle glucose uptake and muscle mass. Furthermore, we found that light-phase GC dosing engaged the muscle GR for a non-canonical interaction with the clock factor BMAL1 to upregulate the mitochondrial regulator PGC1alpha. Indeed, we found that the regimen-driven increase in glucose oxidation and amino acid biogenesis from TCA cycle intermediates in muscle was dependent on the myocyte-specific PGC1alpha. In summary, we present here novel circadian and molecular mechanisms reconverting glucocorticoid drugs from deleterious to re-energizing agents for potential chrono-treatment of metabolic conditions and aging. Presentation: Sunday, June 18, 2023

Mesenchymal stromal cells ameliorate chronic GVHD by boosting thymic regeneration in a CCR9-dependent manner in mice.

Chronic graft-versus-host disease (cGVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Mature donor T cells within the graft contribute to severe damage of thymic epithelial cells (TECs), which are known as key mediators in the continuum of acute GVHD (aGVHD) and cGVHD pathology. Mesenchymal stromal cells (MSCs) are reportedly effective in the prevention and treatment of cGVHD. In our previous pilot clinical trial in patients with refractory aGVHD, the incidence and severity of cGVHD were decreased, along with an increase in levels of blood signal joint T-cell receptor excision DNA circles after MSCs treatment, which indicated an improvement in thymus function of patients with GVHD, but the mechanisms leading to these effects remain unknown. Here, we show in a murine GVHD model that MSCs promoted the quantity and maturity of TECs as well as elevated the proportion of Aire-positive medullary TECs, improving both CD4+CD8+ double-positive thymocytes and thymic regulatory T cells, balancing the CD4:CD8 ratio in the blood. In addition, CCL25-CCR9 signaling axis was found to play an important role in guiding MSC homing to the thymus. These studies reveal mechanisms through which MSCs ameliorate cGVHD by boosting thymic regeneration and offer innovative strategies for improving thymus function in patients with GVHD.

Targeting cancer stem cells with a pan-BCL-2 inhibitor in preclinical and clinical settings in patients with gastroesophageal carcinoma

ObjectiveGastro-oesophageal cancers (GEC) are resistant to therapy and lead to poor prognosis. The cancer stem cells (CSCs) and antiapoptotic pathways often confer therapy resistance. We sought to elucidate the antitumour...

A pilot clinical trial of oral tetrahydrouridine/decitabine for noncytotoxic epigenetic therapy of chemoresistant lymphoid malignancies

One mechanism by which lymphoid malignancies resist standard apoptosis-intending (cytotoxic) treatments is genetic attenuation of the p53/p16-CDKN2A apoptosis axis. Depletion of the epigenetic protein DNA methyltransferase 1 (DNMT1) using the deoxycytidine analog decitabine is a validated approach to cytoreduce malignancy independent of p53/p16. In vivo decitabine activity, however, is restricted by rapid catabolism by cytidine deaminase (CDA). We, therefore, combined decitabine with the CDA-inhibitor tetrahydrouridine and conducted a pilot clinical trial in patients with relapsed lymphoid malignancies: the doses of tetrahydrouridine/decitabine used (∼10/0.2 mg/kg orally (PO) 2×/week) were selected for the molecular pharmacodynamic objective of non-cytotoxic, S-phase dependent, DNMT1-depletion, guided by previous Phase 1 studies. Patients with relapsed/refractory B- or T-cell malignancies (n = 7) were treated for up to 18 weeks. Neutropenia without concurrent thrombocytopenia is an expected toxicity of DNMT1-depletion and occurred in all patients (Grade 3/4). Subjective and objective clinical improvements occurred in 4 of 7 patients, but these responses were lost upon treatment interruptions and reductions to manage neutropenia. We thus performed parallel experiments in a preclinical in vivo model of lymphoma to identify regimen refinements that might sustain DNMT1-targeting in malignant cells but limit neutropenia. We found that timed-alternation of decitabine with the related molecule 5-azacytidine, and combination with inhibitors of CDA and de novo pyrimidine synthesis could leverage feedback responses of pyrimidine metabolism to substantially increase lymphoma cytoreduction but with less neutropenia. In sum, regimen innovations beyond incorporation of a CDA-inhibitor are needed to sustain decitabine DNMT1-targeting and efficacy against chemo-resistant lymphoid malignancy. Such potential solutions were explored in preclinical in vivo studies.

DDEL-09. HIGH DOSE MTX110 (SOLUBLE PANOBINOSTAT) SAFELY ADMINISTERED INTO THE FOURTH VENTRICLE IN A NON-HUMAN PRIMATE MODEL

OBJECTIVEThis study tested the safety and pharmacokinetics of short-term and long-term administration of MTX110 (soluble panobinostat; Midatech Pharma, UK) into the fourth ventricle of non-human primates.METHODSFour rhesus macaque monkeys underwent posterior fossa craniectomy and catheter insertion into the fourth ventricle. In Group I (n=2), catheters were externalized and lumbar drain catheters were placed simultaneously to assess cerebrospinal fluid (CSF) distribution after short-term infusions. MTX110 (0.5 ml of 300 μM panobinostat solution) was infused into the fourth ventricle daily for five consecutive days. Serial CSF and serum panobinostat levels were measured. In Group II (n=2), fourth ventricle catheters were connected to a subcutaneously-placed port for subsequent long-term infusions. Four cycles of MTX110, each consisting of 5 daily infusions (0.5 ml of 300 μM panobinostat solution), were administered over 8 weeks. Animals underwent detailed neurological evaluations, MRI scans, and post-mortem histological analysis.RESULTSNeurological assessments, MRI, and histology confirmed catheter placement and an absence of neurotoxicity. Panobinostat was undetectable in serum collected two and four hours after infusions in all samples in both groups. In Group I, mean peak panobinostat level in fourth ventricle CSF (6242 ng/ml) was significantly higher than in lumbar CSF (9 ng/ml; p < 0.0001). In Group II, mean peak CSF panobinostat level (11,042 ng/ml) was significantly higher than mean trough CSF level (33 ng/ml; p<0.0001).CONCLUSIONMTX110 can be safely delivered via 4th ventricle at supra-therapeutic doses. These results provide data for a pilot clinical trial in patients with recurrent medulloblastoma.

A Pilot Trial on the Effect of Levothyroxine on Proteinuria in Patients With Advanced CKD

IntroductionThyroid hormones can directly affect kidney function; elevated levels of thyroid-stimulating hormone (TSH) and chronic kidney disease (CKD) are associated with proteinuria, decreased estimated glomerular filtration rate (eGFR), and progression to end-stage renal disease. Our hypothesis is that in patients with CKD and TSH at levels considered to be in the low subclinical hypothyroidism (SCH) range, lowering TSH with levothyroxine (LVX) improves the clinical parameters of renal function.MethodsThis was a double-blind, randomized, pilot clinical trial in patients with proteinuric CKD (eGFR <60 ml/min per 1.73 m2 and proteinuria >150 mg/d) performed at the Hospital Civil de Guadalajara, with the intention of lowering TSH (levels of 1.25−2.5 μIU/l) in patients with TSH (levels of 2.6−9.9 μIU/ml with FT4 in the range of 0.7−1.8 ng/dl). Patients were randomized 1:1 to receive LVX or placebo for 12 weeks. The primary objective was to evaluate absolute levels of proteinuria at the beginning compared to the end of the study and, as a secondary objective, the changes in serum creatinine (sCr), eGFR, cholesterol, triglycerides, low-density lipoprotein (LDL), and blood pressure, and to assess the tolerability and safety of LVX.ResultsBetween March and November 2018, a total of 163 patients were assessed for eligibility; 119 patients did not meet the inclusion criteria or were excluded, and 32 patients were randomized. The demographic and clinical characteristics of the 2 study groups were essentially not different. Subjects were 66.87 (SD 12.19) years of age, 62.5% were female, 75% were diabetes mellitus, eGFR was 23.55 (±12.91) ml/min per 1.73 m2, TSH was 5.37 ± 2.13 μIU/ml, proteinuria in 24-hour urine collection was 1.52 ± 1.12, and all of them were taking angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs). Proteinuria at 12 weeks in the LVX group was 0.89 SD ± 1.28 g/d, and in the placebo group it was 1.35 SD ± 0.85 g/d; when compared to placebo, LVX showed a significant decrease in proteinuria of 1.1 g/d (P = 0.0011). The eGFR in the LVX group showed an improvement of 4 ml/min/1.73 m2 (P = 0.049); in the placebo group, there was a decrease of 1.98 ml/min per 1.73 m2. The sCr, cholesterol, triglycerides, low-density lipoprotein, systolic blood pressure, and diastolic blood pressure were not different between groups. Adverse events were reported in the LVX group in 7.14% of patients and in 11.11% of patients in the placebo group; none left the study because of adverse effects, and there were no serious adverse events.ConclusionThis single-center, randomized, double-blind, placebo-controlled pilot clinical trial in patients with advanced proteinuric CKD who already used ACEIs or ARBs demonstrated that administering LVX to obtain a TSH range close to 2.5 μIU/ml decreased proteinuria and improved eGFR. Future research is needed to confirm our results and to determine whether our findings generalize to patient groups not explicitly enrolled in this small pilot trial.

High-dose MTX110 (soluble panobinostat) safely administered into the fourth ventricle in a nonhuman primate model.

Chemotherapy infusions directly into the fourth ventricle may play a role in treating malignant fourth-ventricular tumors. This study tested the safety and pharmacokinetics of short-term and long-term administration of MTX110 (soluble panobinostat; Midatech Pharma) into the fourth ventricle of nonhuman primates. Four rhesus macaque monkeys underwent posterior fossa craniectomy and catheter insertion into the fourth ventricle. In group I (n = 2), catheters were externalized and lumbar drain catheters were placed simultaneously to assess CSF distribution after short-term infusions. MTX110 (0.5 ml of 300 μM panobinostat solution) was infused into the fourth ventricle daily for 5 consecutive days. Serial CSF and serum panobinostat levels were measured. In group II (n = 2), fourth-ventricle catheters were connected to a subcutaneously placed port for subsequent long-term infusions. Four cycles of MTX110, each consisting of 5 daily infusions (0.5 ml of 300 μM panobinostat solution), were administered over 8 weeks. Animals underwent detailed neurological evaluations, MRI scans, and postmortem histological analyses. No neurological deficits occurred after intraventricular MTX110 infusions. MRI scans showed catheter placement within the fourth ventricle in all 4 animals, with extension to the cerebral aqueduct in 1 animal and into the third ventricle in 1 animal. There were no MRI signal changes in the brainstem, cerebellum, or elsewhere in the brains of any of the animals. Histologically, normal brain cytoarchitecture was preserved with only focal mild postsurgical changes in all animals. Panobinostat was undetectable in serum samples collected 2 and 4 hours after infusions in all samples in both groups. In group I, the mean peak panobinostat level in the fourth-ventricle CSF (6242 ng/ml) was significantly higher than that in the lumbar CSF (9 ng/ml; p < 0.0001). In group II, the mean peak CSF panobinostat level (11,042 ng/ml) was significantly higher than the mean trough CSF panobinostat level (33 ng/ml; p < 0.0001). MTX110 can be safely infused into the fourth ventricle in nonhuman primates at supratherapeutic doses. Postinfusion CSF panobinostat levels peak immediately in the fourth ventricle and then rapidly decrease over 24 hours. Panobinostat is detectable at low levels in CSF measured from the lumbar cistern up to 4 hours after infusions. These results will provide background data for a pilot clinical trial in patients with recurrent medulloblastoma.

Photobiomodulation in Neuroscience: A Summary of Personal Experience

This review summarizes personal experience with laser photobiomodulation and its potentials for the treatment of peripheral and central nerve system injuries. Laser photobiomodulation was shown to induce nerve cell activation, have a positive effect on metabolism of the nerve cells, and to stimulate nerve sprouting processes. Studies investigating the effects of laser photobiomodulation on injured peripheral nerves in rats reported immediate protective effects which increase the functional activity of the nerve, decrease or prevent scar tissue formation at the injured site, prevent or decrease degeneration in corresponding motor neurons of the spinal cord, and significantly increase axonal growth and myelinization. A direct application of laser on the spinal cord had a positive impact on the corresponding injured peripheral nerve and promoted recovery. A 780-nm laser phototherapy was applied following peripheral nerve reconstruction using a guiding nerve tube. Results showed myelinated axons crossing through the nerve tube and the continuation of axonal sprouting through the tube toward the distal part of the nerve. In a double-blind, placebo-controlled randomized pilot clinical trial in patients with incomplete stable long-term peripheral nerve injury (PNI), 780-nm laser irradiation progressively improved peripheral nerve function and led to substantial functional recovery. Muscle atrophy represents a major challenge in restorative medicine. Laser phototherapy was shown to increase biochemical activity and improve morphological recovery in muscle and, thus, could have a direct therapeutic application, especially during progressive muscle atrophy resulting from PNI. The effectiveness of composite implants of cultured embryonal nerve cells and the role of laser irradiation on regeneration and repair of the completely transected rat spinal cord were examined. Results suggested that laser photobiomodulation treatment accelerates the axonal growth. The significance of the performed experimental and clinical studies is in the provision of new laser technology in field of cell therapy and its therapeutic value for peripheral nerve and spinal cord injuries. Additional well-designed clinical studies are needed to evaluate the effectiveness and role of laser photobiomodulation treatment in a clinical setting.

Phase 1–2 pilot clinical trial in patients with decompensated liver cirrhosis treated with bone marrow–derived endothelial progenitor cells

The aim of this nonrandomized, open label, phase 1 clinical trial was to evaluate the safety and the feasibility of the treatment with autologous bone marrow-derived endothelial progenitor cells (EPC) in decompensated liver cirrhosis. In addition, the changes in liver function and hepatic venous pressure gradient (HVPG) and their relation with the characteristics of the cellular product were analyzed. Twelve patients with Child-Pugh ≥8 liver cirrhosis underwent bone marrow harvest for exvivo differentiation of EPC. The final product was administered through the hepatic artery in a single administration. Patients underwent clinical and radiologic follow-up for 12months. The phenotype and the ability to produce cytokines and growth factors of the final cellular suspension were analyzed. Eleven patients were treated (feasibility 91%). No treatment-related severe adverse events were observed as consequence of any study procedure or treatment. Model for end-stage liver disease score improved significantly (P0.042) in the first 90days after cells administration and 5 of the 9 patients alive at 90days showed a decreased of HVPG. There was a direct correlation between the expression of acetylated-low density lipoprotein and von Willebrand factor in the cellular product and the improvement in liver function and HVPG. The treatment with EPCs in patients with decompensated liver cirrhosis is safe and feasible and might have therapeutic potential. Patients receiving a higher amount of functionally active EPC showed an improvement of liver function and portal hypertension suggesting that the potential usefulness of these cells for the treatment of liver cirrhosis deserves further evaluation.

Circulation: Cardiovascular Interventions Editors’ Picks

<i>Circulation: Cardiovascular Interventions</i> Editors’ Picks

Interferon-γ–Dependent Infiltration of Human T Cells into Neuroblastoma TumorsIn vivo

To investigate the impact of interferon-gamma-mediated upregulation of major histocompatibility complex class I expression on tumor-specific T-cell cytotoxicity and T-cell trafficking into neuroblastoma tumors in vivo. Restoration of major histocompatibility complex class I expression by interferon-gamma treatment enhances killing of neuroblastoma cells. To understand the potential of this approach in vivo, we developed a novel model of neuroblastoma in which NOD/scid/IL2R gamma(null) immunodeficient mice are engrafted with both human T cells and tumor cells. Here, we show enhanced killing of neuroblastoma cells by patient-derived, tumor-specific T cells in vitro. In addition, interferon-gamma treatment in vivo induces efficient upregulation of major histocompatibility complex class I expression on neuroblastoma tumor cells, and this is accompanied by significantly enhanced infiltration of T cells into the tumor. In a pilot clinical trial in patients with high-risk neuroblastoma, we similarly observed augmented T-cell trafficking into neuroblastoma nests in tumor biopsy specimens obtained from patients after 5 days of systemic interferon-gamma therapy. Interferon-gamma overcomes critical obstacles to the killing of human neuroblastoma cells by specific T cells. Together, these findings provide a rationale for the further testing of interferon-gamma as an approach for improving the efficacy of T cell-based therapies for neuroblastoma and other major histocompatibility complex class I-deficient malignancies. In addition, we describe a model that may expedite the preclinical screening of approaches aimed at augmenting T-cell trafficking into human tumors.

Production of a dendritic cell-based vaccine containing inactivated autologous virus for therapy of patients with chronic human immunodeficiency virus type 1 infection.

In preparation for a pilot clinical trial in patients with chronic human immunodeficiency virus type 1 (HIV-1) infection, a novel dendritic cell (DC)-based vaccine is being manufactured. The trial will test the hypothesis that isolated endogenous virus presented by DCs serves as a potent immunogen for activation of CD8(+) and CD4(+) T cells specific for a broad range of autologous HIV-1 antigens. Production of the vaccine under good manufacture practice conditions involves (i) autologous virus isolation; (ii) superinfection of CD4(+) T cells with the virus; (iii) inactivation of the virus in CD4(+) T cells, T-cell apoptosis, and coincubation of T cells with autologous DCs; and (iv) product testing and release. Endogenous virus was isolated from peripheral blood-derived CD4(+) T cells of three HIV-1-positive subjects by coincubation with autologous OKT-3-stimulated CD4(+) T cells. CD4(+) T-cell supernatants were tested for p24 levels by enzyme-linked immunosorbent assay (>25 ng/ml) and for the 50% tissue culture infective doses (TCID(50); which ranged from 4,642 to 46,416/ml on day 19 of culture). Autologous CD4(+) T cells that were separated on immunobeads (>95% purity) and superinfected with virus-expressed p24 (28 to 54%) had TCID(50) of >400/ml on days 5 to 10. Virus inactivation with psoralen (20 microg/ml) and UVB irradiation (312 nm) reduced the TCID(50) of the supernatants from 199,986 to 11/ml (>99%). 7-Amino-actinomycin D-positive, annexin V-positive CD4(+) T cells were fed to autologous DCs generated by using the Elutra cell separation system and the Aastrom system. Flow analysis showed that DC loading was complete in 24 h. On the basis of these translational results and experience with the generation of DCs from HIV-1-infected patients in a previous clinical trial, the Investigational New Drug application for clinical vaccination was submitted and approved by the FDA (application no. BB-IND-13137).

Immunotherapy with Cytokine Induced Killer Cells in Solid and Hematopoietic Tumors: Preliminary Data.

CIK cells are a unique population of CD3+CD56+ immune effector cells capable of lysing a broad variety of tumor cells targeted through a perforin based mechanism mediated by NKG2D.We started a pilot clinical trial in patients with refractory lymphoma and solid tumors according to GMP guidelines that is currently ongoing.Methods: CIK cells were generated from PBMNC and incubated in LifeCell culture bags in the presence of IFN-γ followed by IL-1β, OKT3 and IL-2. Expansion was assessed between day 21 and 28 and flow cytometric analysis was performed every week. Patients were monitored before and after treatment. Unpaired t-test was used to analyze for statistical significance. A p-value <0.05 was considered statistically significant.Results: At present 10 patients are enrolled: 6 advanced lymphomas, 3 metastatic kidney carcinoma and 1 hepatocellular carcinoma. The median number of transferred cells per patient was 19 x109 (6–37 x109) and the absolute number of CD3+CD56+ cells infused ranged from 1 to 16 x109 (median value 5 x107/Kg). Patients affected by solid tumors received in association low doses of rhIL-2 or α-interferon. Protocol adherence was excellent and the toxicity profile was favourable. Only 2 patients developed low-grade fever during the first cycle of infusion (5%) but promptly resolved without antibiotic treatment. After CIK cell infusion, in patient's peripheral blood the absolute median count of PBLs, CD3+, CD8+ and CD3+CD56+ cells significantly increased with a p-value of 0.034, 0.025, 0.034 and 0.038, respectively. Clinical outcome appeared promising: 2 patients had complete response (1 metastatic kidney carcinoma and 1 hepatocellular carcinoma) and 2 patients had stabilization of disease (1 metastatic kidney carcinoma and 1 NHL) with a median follow-up of 12 months (range 4–14).Conclusions: These preliminary data showed that adoptive immunotherapy with CIK cells is a safe therapy with some suggestion of efficacy that significantly enhances immune functions increasing absolute numbers of effector cells without side effects. If confirmed in larger scale studies, these promising results may have a favourable impact on conventional treatment strategy of malignancies.

Lnterferon‐β gene therapy for cancer: Basic research to clinical application

Interferon-beta gene therapy for cancer is the first such protocol developed in Japan. Here we describe the development process of our interferon-beta gene therapy from basic research to clinical application. Interestingly, the biological and biochemical characteristics of interferon-beta gene therapy through transfer of the interferon-beta gene into tumor cells by means of cationic liposomes differed from those of conventional interferon-beta protein therapy. Interferon-beta gene transfer could induce apoptosis in interferon-beta protein-resistant tumor cells, such as glioma, melanoma, and renal cell carcinoma. Induction of apoptosis was related to the level of intracellular mRNA of interferon-beta, prolongation of the phosphorylation time of molecules in the interferon-beta signal transduction pathway, such as JAK1, Trk2, and STAT1, and activation of DNase gamma. In our preclinical study we developed lyophilized cationic liposomes containing interferon-beta gene (gene drug) for clinical use and confirmed their safety. Thereafter, we performed a pilot clinical trial in patients with malignant glioma and confirmed the safety and effectiveness of this interferon-beta gene therapy. In this review we also comment on the status of gene therapy regulation in Japan. Interferon-beta gene therapy is expected to become widely available for clinical use in cancer patients, and this new strategy might be extended to molecular targeting therapy, or used in combination with cell therapy or other therapies.