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Reduced intensity conditioning (RIC) is fraught with risk for disease relapse. This may be overcome by donor T cell alloreactivity. Reducing the duration of intense immune suppression in the early term following transplantation may create an immunologic environment favoring rapid T cell reconstitution to influence longer term transplant outcomes. Twenty-six patients were adaptively randomized based on donor-derived T cell recovery, between 2 different dosing schedules of mycophenolate mofetil (MMF): MMF for 30 days post-transplant, with filgrastim for cytokine support (MMF30 arm, 11 patients), or MMF for 15 days post-transplant, with sargramostim (MMF15 arm, 15 patients). All patients were treated with anti-thymocyte globulin at a dose of 1.7 mg/kg/day from day - 9 through day -7 and total body irradiation, 450 cGy given in 3 fractions. Patients were well matched between the study arms and underwent HLA matched related and unrelated donor hematopoietic cell transplantation (HCT). The MMF15 arm demonstrated superior T cell recovery in the first month. This difference persisted through the first year for total T cells, T cell subsets and NK cells. T cell repertoire tended to be more diverse in the MMF15 arm. The long term superior immune recovery in the MMF15 arm is consistent with a disproportionate impact of early interventions in HCT. Clinically, shorter course MMF post-transplant was not associated with increased risk of acute or chronic graft vs. host disease (GVHD), and relapse and there was a trend toward improved overall survival in the MMF15 arm. Reducing the duration of intense immunosuppression in the early term and the use of sargramostim following allogeneic HCT is feasible and leads to superior long-term T cell recovery. This regimen should be studied to improve immune recovery in large cohorts of patients undergoing HCT with RIC.

Alloreactivity forms the basis of allogeneic hematopoietic cell transplantation (HCT), with donor derived T cell response to recipient antigens mediating clinical responses either in-part or entirely. These encompass the different manifestations of graft vs. host disease (GVHD), infection risk as well as disease response. Whilst the latter is contingent upon disease biology and thus may be less predictable, the former two are more likely to be directly proportional to the magnitude of donor derived T cell recovery. Herein we explore the quantitative aspects of immune cell recovery following allogeneic HCT and clinical outcomes in two cohorts of HLA matched allograft recipients who received rabbit anti-thymocyte globulin (ATG) on different schedules (days -9 to -7 vs. -3 to -1). Monocyte as well as donor derived T cell (ddCD3) recovery was superior in those given ATG early in their course (days -9/-7). This difference was related to a more rapid rate of ddCD3 recovery, largely driven by CD3+/8+ cells in the first month following transplantation. Early monocyte recovery was associated with later T cell recovery and improved survival. In contrast rapid and early ddCD3 expansion out of proportion to monocyte recovery was associated with a high likelihood of acute GVHD and poor survival. This analytic methodology demonstrates that modelling ‘early-term immune reconstitution' following HCT yields insights that may be useful in management of post-transplant immunosuppression and adaptive cellular therapy to optimize clinical outcomes.

Azacitidine (Aza) may promote cytotoxic effects against hematologic tumor cells when combined with donor lymphocyte infusions (DLIs). This study sought to verify Aza-DLI's efficacy and tolerability in patients with relapsed acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) following allogeneic stem cell transplantation (SCT) and identify cohorts benefitting most from therapy. Twenty-eight patients with recurrent AML or MDS following SCT received Aza-DLI. One-year overall survival (OS) after therapy initiation was 44%; two-year OS was 35%. Molecular/cytogenetic-only relapse, the development of cGVHD after therapy initiation, and a greater number of Aza and DLI were associated with remission. There was a trend toward higher absolute CD4+ cell count in those achieving remission. This study demonstrates Aza-DLI to be effective and highlights the importance of minimal residual disease testing and alloreactivity in managing post allograft relapsed hematological malignancy.

Alloreactivity forms the basis of allogeneic hematopoietic cell transplantation (HCT), with donor derived T cell response to recipient antigens mediating clinical responses either in part or entirely. These encompass the different manifestations of graft vs. host disease (GVHD), infection risk as well as disease response. Whilst the latter is contingent upon disease biology and thus may be less predictable, the former two are more likely to be directly proportional to the magnitude of donor derived T cell recovery. Herein we explore the quantitative aspects of immune cell recovery following allogeneic HCT and clinical outcomes in two cohorts of HLA matched allograft recipients who received rabbit anti-thymocyte globulin (ATG) on different schedules (days -9 to -7 vs. -3 to -1). Monocyte as well as donor derived T cell (ddCD3) recovery was superior in those given ATG early in their course (days -9/-7). This difference was related to a more rapid rate of ddCD3 recovery, largely driven by CD3+/8+ cells in the first month following transplantation. Early monocyte recovery was associated with later T cell recovery, improved survival, and less chronic GVHD. In contrast rapid and early ddCD3 expansion out of proportion to monocyte recovery was associated with a high likelihood of acute GVHD and poor survival. This analytic methodology demonstrates that modelling ‘early-term immune reconstitution’ following HCT yields insights that may be useful in management of post-transplant immunosuppression and adaptive cellular therapy to optimize clinical outcomes.HighlightsATG given early (day -9 to -7) prior to HCT leads to improved survival and non-relapse mortality. Improved monocyte counts as well as donor-derived T cell counts are seen in recipients of early ATG. Improved clinical outcomes are observed in patients with enhanced monocyte and T cell recovery after HCT.

Introduction Thrombin is a primary target of most anticoagulants. Yet, thrombin's dual and opposing role in pro- as well as anti- coagulant processes imposes considerable challenges in discovering finely tuned regulators that maintain homeostasis, rather than disproportionately changing the equilibrium to one side. In this connection, we have been studying exosite 2-mediated allosteric modulation of thrombin activity using synthetic agents called low molecular weight lignins (LMWLs). Although the aromatic scaffold of LMWLs is completely different from the polysaccharidic scaffold of heparin, the presence of multiple negatively charged groups on both ligands induces binding to exosite 2 of thrombin. This work characterizes the nature of interactions between LMWLs and thrombin to understand the energetic cooperativity between exosite 2 and active site of thrombin. Materials and methods The thermodynamics of thrombin–LMWL complexes was studied using spectrofluorimetric titrations as a function of ionic strength and temperature of the buffer. The contributions of enthalpy and entropy to binding were evaluated using classic thermodynamic equations. Label-free surface plasmon resonance was used to assess the role of sodium ion in LMWL binding to thrombin at a fixed ionic strength. Results and conclusions Exosite 2-induced conformational change in thrombin's active site is strongly dependent on the structure of the ligand, which has consequences with respect to regulation of thrombin. The ionic and non-ionic contributions to binding affinity and the thermodynamic signature were highly ligand specific. Interestingly, LMWLs display preference for the sodium-bound form of thrombin, which supports the existence of an energetic coupling between exosite 2 and sodium-binding site of thrombin.

Intravenous (IV) ascorbic acid (AA) improves organ function and reduces inflammation in sepsis, an inflammatory state similar to the post-hematopoietic cell transplant (HCT) milieu. This salutary effect is mediated by antioxidant activity as well transcriptional modulation by AA. HCT recipients are deficient in AA, therefore we evaluated the safety and efficacy of patients receiving parenteral AA after myeloablative conditioning for allogeneic HCT compared to similarly treated historical controls who did not receive AA. Methods: Patients with hematologic malignancies, AML (48% of patients), ALL (28%), and CML+MDS (25%) were enrolled in an IRB approved prospective phase 2 clinical trial (NCT03613727). IV AA 50 mg/kg/d divided in 3 doses was given on days 1-14 after HCT, followed by oral AA 500 mg bid from day 15 until 6 months post HCT (FDA-IND 138924). Conditioning regimens utilized included; fludarabine & melphalan (45%), cyclophosphamide with either busulfan (30%) or total body irradiation (25%). GVHD prophylaxis included calcineurin inhibitors and methotrexate or cellcept along with anti-thymocyte globulin (ATG). Primary endpoint was reduction in TRM at 1 year. Propensity score matching was used for matching study patients with similarly treated historical controls, matching for diagnosis, conditioning regimen, and CIBMTR disease risk category for comparison of clinical outcomes. Cox-proportional hazard models were used to estimate adjusted hazard ratios (AHR) between the time-to-event outcomes and study group, adjusted for patient age, donor type, stem cell source, diagnosis, conditioning regimen, and CIBMTR disease risk. Results of an interim analysis following a period of COVID 19 mandated suspension of study accrual are reported. Results: As of March 2020, 40 patients have received IV AA: these include HLA-matched related donor (MRD; n=11), and either 10/10 or 9/10 HLA- matched unrelated donor (MUD; n=22 & 7 respectively) recipients. Graft source was either peripheral blood (n=38) or bone marrow (n=2); 88% patients had CIBMTR high risk disease. Median age was 55 years; males (19). All patients enrolled were deficient in AA at day 0, median AA level 0.3 mg/dL (range: 0.1-0.5); post AA infusion level was normal at 1.6 (1.2-5.7) on day 14. Median neutrophil and platelet recovery was by 12 days (range: 9-15 & 8-21 days respectively) with sustained donor engraftment. Median absolute CD3+ cell count at day 30 was 330 cells/microL. With a median follow up of 220 days in AA recipients, no statistically significant difference was observed in transplant related mortality between propensity matched historical controls and study patients (AHR 0.6, 95% CI: 0.2-1.5; p-value = 0.27); univariate survival analysis is depicted in Figure 1. Relapse was also similar (AHR 1.2, 95% CI: 0.3-4.5; p-value = 0.82), and despite a larger number of HLA mismatched unrelated donor recipients, acute GVHD (Grade II-IV) rates were similar in the two groups for both grade II-IV (AHR 0.8, 95% CI: 0.7-1.7; p-value = 0.65) and grade III-IV disease (AHR 0.6, 95% CI: 0.2-1.6; p-value = 0.32). Chronic GVHD rates were also similar (AHR 0.4, 95% CI: 0.1-2.7; p-value = 0.74). There are no attributable grade 3 - 4 toxicities from AA; CMV and EBV reactivation rates were not different in the two groups. Conclusions: In patients undergoing myeloablative allogeneic HCT the administration of IV ascorbic acid is safe and does not negatively impact myeloid engraftment or immune reconstitution. In this interim analysis, transplant related mortality, relapse and GVHD are not increased in IV AA recipients compared to historical controls. Thus, given its safety and tolerability, and possible salutary impact on survival and relapse in these high-risk patients, we posit the feasibility of a randomized phase 3 trial with IV AA in the post-transplant setting to determine its effect on relapse and TRM. Disclosures No relevant conflicts of interest to declare.

Intravenous (IV) ascorbic acid (AA) improves organ function and reduces inflammation in sepsis, an inflammatory state similar to post-hematopoietic cell transplant (HCT) milieu. This salutary effect is mediated by antioxidant activity as well transcriptional modulation by AA. We evaluated the safety and efficacy of IV AA in ameliorating the inflammatory milieu following myeloablative conditioning and allogeneic HCT. Methods: Patients with advanced hematologic malignancies (CML, AML, ALL, MDS) were enrolled in an IRB approved prospective phase 2 clinical trial (NCT03613727). IV AA 50mg/kg/d divided in 3 doses was given on days 1-14 after HCT followed by oral AA 500mg bid from day 15 until 6 months post HCT (IND 138924). The treatment regimens included myeloablative fludarabine & melphalan, or cyclophosphamide with either busulfan or total body irradiation. GVHD prophylaxis included ATG. FDA mandated safety lead-in cohort enrollment for the trial is complete with ongoing accrual to target primary end point of reducing 1-year non-relapse mortality. Cytokines were measured at various time points following HCT. Results: As of October 2019 21 patients have received IV AA: these include HLA-MRD (n=5), and 10/10 or 9/10 HLA-MUD (12 & 4 respectively) recipients. Graft source was either peripheral blood (19) or bone marrow (2). Median age was 56 years; males (11). All patients enrolled were deficient in AA at day 0, median 0.3 mg/dL (range: 0.1-0.5), day 14, post AA infusion level was normal at 1.6 (1.2-5.7). Neutrophil recovery was by 11 days (9-15 days) and platelets by 12 (9-19) with sustained donor engraftment. Median absolute CD3+ cell count at day 30 was 390/mL (55-2288) with 100% donor chimerism. Pro-inflammatory cytokines IL-1b, IL-2, IL-6, IL-12, TNF-α, IFN-γ, as well as soluble thrombomodulin remained unchanged between day 0 and day 14 & day 30 after HCT (P=NS for all comparisons Figure 1). At a median follow up of 201 days (13-335) there is a 95% survival observed (Figure 2). There was no VOD and no attributable grade 3 and 4 toxicities to AA. Mucositis was mild and TPN was required in only 43% of patients. The cumulative incidences of grades II–IV and grades III–IV acute GVHD were 33% and 9% respectively, and moderate chronic GVHD 21%. No severe cGVHD has been observed, only one relapse has occurred. Conclusions: In patients undergoing myeloablative allogeneic HCT the administration of IV ascorbic acid is safe and does not negatively impact myeloid engraftment or immune reconstitution. Pro-inflammatory cytokines and endothelial injury markers remain around baseline levels, with no VOD and low rates of GVHD observed. IV AA requires further investigation in HCT given its safety, and low cost (∼$450 for 14 day infusion in a 80 KG recipient) and potential for worldwide use.

We report an unusual 3-substituted pyridine polyketide, onydecalin A (1), which was obtained along with 2 as a major constituent from the fungus Aioliomyces pyridodomos (Order: Onygenales) following a two-month fermentation. Feeding studies demonstrated that the pyridine subunit originates via an unprecedented biosynthetic process in comparison to other polyketide-linked pyridines or derivatives such as pyridones. The slow growth of the fungus led us to perform a one-year fermentation, leading to production of compounds 2-4 as the major constituents. These compounds showed modest but selective inhibition against a variety of transient receptor potential channels, as well as against the human pathogenic fungus, Histoplasma capsulatum.

A racemic, prenylated polyketide dimer, oxazinin A (1), was isolated from a novel filamentous fungus in the class Eurotiomycetes, and its structure was elucidated spectroscopically. The pentacyclic structure of oxazinin A (1) is a unique combination of benzoxazine, isoquinoline, and a pyran ring. Oxazinin A (1) exhibited antimycobacterial activity and modestly antagonized transient receptor potential (TRP) channels.

We recently designed a group of novel exosite-2-directed sulfated, small, allosteric inhibitors of thrombin. To develop more potent inhibitors, monosulfated benzofuran tri- and tetrameric homologues of the parent designed dimers were synthesized in seven to eight steps and found to exhibit a wide range of potencies. Among these, trimer 9a was found to be nearly 10-fold more potent than the first generation molecules. Michaelis-Menten studies indicated an allosteric mechanism of inhibition. Competitive studies using a hirudin peptide (exosite 1 ligand) and unfractionated heparin, heparin octasaccharide, and γ'-fibrinogen peptide (exosite 2 ligands) demonstrated exosite 2 recognition in a manner different from that of the parent dimers. Alanine scanning mutagenesis of 12 Arg/Lys residues of exosite 2 revealed a defect in 9a potency for Arg233Ala thrombin only confirming the major difference in site of recognition between the two structurally related sulfated benzofurans. The results suggest that multiple avenues are available within exosite 2 for inducing thrombin inhibition.

Sulfated, low molecular weight lignins (LMWLs), designed recently as macromolecular mimetics of the low molecular weight heparins (LMWHs), were found to exhibit a novel allosteric mechanism of inhibition of human thrombin, factor Xa and plasmin, which translates into potent human blood anticoagulation potential. To identify the site of binding of sulfated LMWLs, a panel of site-directed thrombin mutants was studied. Substitution of alanine for Arg(93) or Arg(175) induced a 7-8-fold decrease in inhibition potency, while Arg(165)Ala, Lys(169)Ala, Arg(173)Ala and Arg(233)Ala thrombin mutants displayed a 2-4-fold decrease. Other exosite 2 residues including those that play an important role in heparin binding, such as Arg(101), Lys(235), Lys(236) and Lys(240), did not induce any deficiency in sulfated LMWL activity. Thrombin mutants with multiple alanine substitution of basic residues showed a progressively greater defect in inhibition potency. Comparison of thrombin, factor Xa, factor IXa and factor VIIa primary sequences reiterated Arg(93) and Arg(175) as residues likely to be targeted by sulfated LMWLs. The identification of a novel site on thrombin with capability of allosteric modulation is expected to greatly assist the design of new regulators based on the sulfated LMWL scaffold.

Thrombin is a key enzyme targeted by the majority of current anticoagulants that are direct inhibitors. Allosteric inhibition of thrombin may offer a major advantage of finely tuned regulation. We present here sulfated benzofurans as the first examples of potent, small allosteric inhibitors of thrombin. A sulfated benzofuran library of 15 sulfated monomers and 13 sulfated dimers with different charged, polar, and hydrophobic substituents was studied in this work. Synthesis of the sulfated benzofurans was achieved through a multiple step, highly branched strategy, which culminated with microwave-assisted chemical sulfation. Of the 28 potential inhibitors, 11 exhibited reasonable inhibition of human α-thrombin at pH 7.4. Structure-activity relationship analysis indicated that sulfation at the 5-position of the benzofuran scaffold was essential for targeting thrombin. A tert-butyl 5-sulfated benzofuran derivative was found to be the most potent thrombin inhibitor with an IC(50) of 7.3 μM under physiologically relevant conditions. Michaelis-Menten studies showed an allosteric inhibition phenomenon. Plasma clotting assays indicate that the sulfated benzofurans prolong both the activated partial thromboplastin time and prothrombin time. Overall, this work puts forward sulfated benzofurans as the first small, synthetic molecules as powerful lead compounds for the design of a new class of allosteric inhibitors of thrombin.

Clinical outcomes following hematopoietic cell transplant (HCT) are considered stochastic with different outcomes despite uniformity in characteristics between patients. However, immune reconstitution following SCT has features of a dynamical system, making it likely that clinical outcomes are partially deterministic. We hypothesized that early cessation of mycophenolate mofetil (MMF) in reduced intensity conditioning (RIC) transplants will hasten T cell reconstitution without adversely impacting clinical outcomes. This idea is being tested in a randomized phase 2 clinical trial comparing GVHD prophylaxis using a regimen of tacrolimus & MMF 15 mg/kg q 12 h for 30 days in the control (MMF30) vs. 15 days in the study cohort (MMF15). The MMF15 patients received GM-CSF to promote early monocyte recovery; MMF 30 got G-CSF. All patients underwent reduced intensity conditioning with ATG 1.7 mg/kg/d given on days -9 thru -7 & 450 cGy total body irradiation on d-1 & 0. Patients were stratified based on diagnosis and donor type (8/8 or 7/8 HLA MRD or MUD). A response-adaptive randomization scheme was used to increase the probability that patients were allocated to the treatment regimen which yields superior donor-derived CD3+ (ddCD3) cell count by 8 weeks; primary endpoint is relapse free, DLI free survival. Preliminary results from the first 21 evaluable patients are presented. Patients baseline characteristics are summarized in table 1. Mean ddCD3 count at 8 weeks post HCT favored the MMF group (461 vs. 329), which led to more patients allocated to MMF15 cohort. Median myeloid engraftment at day 60 was 100% in both cohorts. Mean absolute lymphocyte count, CD3+ and CD4+ T-cell counts were higher in the MMF15 cohort on day 30 (500 vs 200/mL−1, P=0.005; 254 vs. 71/mL−1, P=0.0188; 107 vs. 25/mL−1, P=0.0036 respectively). There was a trend for higher ddCD3 cell count in the MMF15 cohort (Fig. 1). Despite this there were no significant differences noted on in lymphoid recovery at later time points. Despite a shorter course of MMF there was no significant difference in the rates of acute and chronic GVHD, relapse and patient survival between the two cohorts (Fig 2). In conclusion these early data demonstrate that a shorter 15-day course of MMF plus GM-CSF led to rapid, early T cell reconstitution post HCT without a significant increase in the risk of acute or chronic GVHD, or disease relapse. This effect is consistent with immune reconstitution post HCT being a dynamical rather than a stochastic process, which may be modified in real-time by adjustments in the intensity of GVHD prophylaxis.

Introduction Melphalan 200 mg/m2 on day -2 (Mel 200) is the standard conditioning regimen in autologous stem cell transplant (ASCT) for multiple myeloma (MM). This regimen is associated with significant gastrointestinal (GI) toxicity, including diarrhea, vomiting, and mucositis; therefore the dose may be reduced to 100-140 mg/m2 based on performance status or age. As the true difference in GI toxicity in older vs. younger patients is unknown, dose reductions may unnecessarily compromise efficacy outcomes. The objective of this study was to define the incidence of GI toxicity in patients age ≥ 60 vs. patients age Objectives Review dose adjustments for melphalan conditioning prior to ASCT in MM. Identify toxicities associated with melphalan conditioning for ASCT in MM. Compare melphalan toxicity between patients age ≥ 60 vs. patients age Methods A retrospective cohort study was conducted via medical record review of adult patients treated with Mel 200 followed by first ASCT for MM from September 2012 to September 2017. The primary endpoint was incidence of GI toxicity, defined by Common Terminology Criteria for Adverse Events (CTCAE) grade 2 or higher diarrhea or grade 3 or higher mucositis during transplant admission. Secondary endpoints included subdivided primary endpoints, complete response rates, and measurements of recovery. Subgroup analyses were performed for patients age ≥ 65, and patients with renal impairment. Results A total of 167 patients were included, with 116 patients in the age Conclusion This single-center, retrospective medical record review demonstrated that patients older than age 60, particularly those 60 to 65 years of age, may receive Mel 200 with no difference in the incidence of GI toxicity compared to younger patients. Further study in a larger population of patients age 65 or older is needed to establish conclusions in this patient population.

Transient receptor potential vanilloid-3 (TRPV3) is a member of the TRPV subfamily of TRP ion channels. The physiological functions of TRPV3 are not fully understood, in part, due to a lack of selective agonists and antagonists that could both facilitate the elucidation of roles for TRPV3 in mammalian physiology as well as potentially serve as therapeutic agents to modulate conditions for which altered TRPV3 function has been implicated. In this study, the Microsource Spectrum Collection was screened for TRPV3 agonists and antagonists using alterations in calcium flux in TRPV3 overexpressing human embryonic kidney-293 (HEK-293) cells. The antispasmodic agent drofenine was identified as a new TRPV3 agonist. Drofenine exhibited similar potency to the known TRPV3 agonists 2-aminoethoxydiphenylboronate (2-APB) and carvacrol in HEK-293 cells, but greater selectivity for TRPV3 based on a lack of activation of TRPA1, V1, V2, V4, or M8. Multiple inhibitors were also identified, but all of the compounds were either inactive or not specific. Drofenine activated TRPV3 via interactions with the residue, H426, which is required for TRPV3 activation by 2-APB. Drofenine was a more potent agonist of TRPV3 and more cytotoxic than either carvacrol or 2-APB in human keratinocytes and its effect on TRPV3 in HaCaT cells was further demonstrated using the antagonist icilin. Due to the lack of specificity of existing TRPV3 modulators and the expression of multiple TRP channels in cells/tissue, drofenine may be a valuable probe for elucidating TRPV3 functions in complex biological systems. Identification of TRPV3 as a target for drofenine may also suggest a mechanism by which drofenine acts as a therapeutic agent.

Earlier, we reported on the design of sulfated benzofuran dimers (SBDs) as allosteric inhibitors of thrombin (Sidhu et al. J. Med. Chem.201154 5522-5531). To identify the site of binding of SBDs, we studied thrombin inhibition in the presence of exosite 1 and 2 ligands. Whereas hirudin peptide and heparin octasaccharide did not affect the IC(50) of thrombin inhibition by a high affinity SBD, the presence of full-length heparin reduced inhibition potency by 4-fold. The presence of γ' fibrinogen peptide, which recognizes Arg93, Arg97, Arg173, Arg175, and other residues, resulted in a loss of affinity that correlated with the ideal Dixon-Webb competitive profile. Replacement of several arginines and lysines of exosite 2 with alanine did not affect thrombin inhibition potency, except for Arg173, which displayed a 22-fold reduction in IC(50). Docking studies suggested a hydrophobic patch around Arg173 as a plausible site of SBD binding to thrombin. The absence of the Arg173-like residue in factor Xa supported the observed selectivity of inhibition by SBDs. Cellular toxicity studies indicated that SBDs are essentially nontoxic to cells at concentrations as high as 250 mg/kg. Overall, the work presents the localization of the SBD binding site, which could lead to allosteric modulators of thrombin that are completely different from all clinically used anticoagulants.