Histamine Dihydrochloride Research Articles

Abstract CT116: Dynamics of cytotoxic T-cell subsets during immunotherapy predicts outcome in acute myeloid leukemia

Abstract Preventing relapse after the completion of chemotherapy remains a challenge in acute myeloid leukemia (AML), in particular in older subjects. For the present study, we assessed the distribution of CD8+ (cytotoxic) T cell subsets in the peripheral blood of AML patients undergoing immunotherapy with histamine dihydrochloride and low-dose interleukin-2 (HDC/IL-2) for relapse prevention. Eighty-four non-transplanted AML patients (age 18-79) in first complete remission (CR) received HDC/IL-2 in an international phase IV trial (ClinicalTrials.gov; NCT01347996). Mononuclear cells were recovered from blood samples drawn during 3-week cycles of immunotherapy and analyzed for CD8+ T cell phenotypes by flow cytometry. Patients were followed for leukemia-free survival (LFS) and overall survival (OS) for 2 years after the initiation of immunotherapy. A reduction of the frequency of CD8+ T effector memory cells (TEM cells with CD8+45RO+CCR7-phenotype) during the first cycle of HDC/IL-2 prognosticated LFS (HR 0.25, CI 0.12-0.60, P = 0.001) and OS (HR 0.24, CI 0.08-0.69, P = 0.009). Similarly, induction of T effector cells (CD8+45RO-CCR7-) during cycle 1 impacted favorably on outcome (P = 0.048 for OS). Patients with a concomitant reduction of TEM cells and induction of Teff cells (“TEM to Teff transition”) during immunotherapy showed favorable outcome in terms of LFS (HR 0.19, CI 0.07-0.50, P = 0.001) and OS (HR 0.13, CI 0.03-0.59, P = 0.008). The altered distribution of cytotoxic T cells during treatment with HDC/IL-2 significantly prognosticated LFS and OS within the group of older patients (more than 60 years old, table 1). The reported predictors of clinical outcome remained significant when taking potential confounders (age, risk group classification, number of induction courses required to achieve CR and number of consolidation courses) into account in multivariable analysis. Presence of CD8+ T cells with specificity for leukemia-associated antigens identified patients with low relapse risk (P = 0.01 for LFS). Our results point to novel aspects of T cell-mediated immunosurveillance in AML and provide conceivable biomarkers in relapse-preventive immunotherapy. Multivariable analysis of the impact of T cell distribution on outcome in older AML patientsDynamics of CD8+ T cell phenotypes during cycle 1 (n=27)Hazard ratioConfidence intervalP-valueReduction of TEM vs. LFS0.100.03-0.390.001Reduction of TEM vs. OS0.130.03-0.570.006Induction of Teff vs. LFS0.260.08-0.870.03Induction of Teff vs. OS0.100.02-0.510.005Transition of TEM to Teff vs. LFS<0.001n.a.n.a.Transition of TEM to Teff vs. OS<0.001n.a.n.a. Citation Format: Frida Ewald Sander, Anna Rydström, Elin Bernson, Roberta Kiffin, Rebecca Riise, Johan Aurelius, Mats Brune, Kristoffer Hellstrand, Fredrik Bergh Thorén, Anna Martner. Dynamics of cytotoxic T-cell subsets during immunotherapy predicts outcome in acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT116.

Evaluation of Skin Reactivity during (Immuno-) Therapy. Validation of Methods for Estimation of Changes in Skin Reactivity and Correlation to Shock Organ Sensitivity

Background: Parallel line bioassay (PLBA) has been acknowledged to be the gold standard for estimation of changes in reactivity, e.g., in RAST and ELISA inhibition tests. Objective: To study correlations between two simple methods for evaluation of changes in skin prick test (δSPT), using the slope of the allergen dose response (drra) in relation to PLBA. Methods: Skin prick test data from two published immunotherapy trials were used. In a D. farinae trial we used duplicate tests with three fixed ten-fold concentrations and in a P. judaica trial three tenfold individually chosen allergen concentrations causing wheals of similar size to that of histamine dihydrochloride 10 mg/mL, tenfold lower and tenfold higher concentration. Evaluation of the δSPT by PLBA, and two simple methods were correlated. In the D. farinae trial δSPT was compared to the change of conjunctival threshold concentration. Results: The δSPT as measured by both the simple methods gave similar results to that of PLBA (p<0.001). The δSPT was around 30-fold, i.e., about 3% of the pre-treatment reactivity. The δSPT correlated with the δCPT threshold concentration. Conclusions: Estimation of the δSPT during therapy expressed as change in concentration using simple methods based on the slope of the drra correlated well to changes by PLBA and CPT and should therefore be used both in clinical research and in practice.

Spinal histamine in attenuation of mechanical hypersensitivity in the spinal nerve ligation-induced model of experimental neuropathy

Here we studied whether and through which mechanisms spinal administration of histamine dihydrochloride (histamine) attenuates pain behavior in neuropathic animals. Experiments were performed in rats with spinal nerve ligation-induced neuropathy and a chronic intrathecal catheter for spinal drug delivery. Mechanical hypersensitivity was assessed with monofilaments while radiant heat was used for assessing nociception. Ongoing neuropathic pain and its attenuation by histamine was assessed using conditioned place-preference test. Following spinal administration, histamine at doses 0.1–10µg produced a dose-related mechanical antihypersensitivity effect. With prolonged treatment (twice daily 10µg for five days), the antihypersensitivity effect of spinal histamine was reduced. In place-preference test, neuropathic animals preferred the chamber paired with histamine (10µg). Histamine (10µg) failed to influence heat nociception in neuropathic animals or mechanically induced pain behavior in a group of healthy control rats. Histamine-induced mechanical antihypersensitivity effect was prevented by spinal pretreatment with zolantidine (histamine H2 receptor antagonist), prazosine (α1-adrenoceptor antagonist) and bicuculline (γ-aminobutyric acid subtype A, GABAA, receptor antagonist), but not by pyrilamine (histamine H1 receptor antagonist), atipamezole (α2-adrenoceptor antagonist), or raclopride (dopamine D2 receptor antagonist). A-960656, a histamine H3 receptor antagonist alone that presumably increased endogenous histamine levels reduced hypersensitivity. Additionally, histamine prevented central (presumably postsynaptically-induced) facilitation of hypersensitivity induced by N-methyl-d-aspartate. The results indicate that spinal histamine at the dose range of 0.1–10µg selectively attenuates mechanical hypersensitivity and ongoing pain in neuropathy. The spinal histamine-induced antihypersensitivity effect involves histamine H2 and GABAA receptors and (presumably neuropathy-induced) co-activation of spinal α1-adrenoceptors.

A histamine-independent itch pathway is required for allergic ocular itch

Non-histaminergic TRPA1 neural pathway is required for the development of allergic ocular itch. Pharmacological inhibition of TRPA1 channel can lead to novel therapeutic strategies to treat ocular itch in severe allergic conjunctivitis.

Pretreatment With Inactivated Bacillus Calmette-Guerin Increases CD4+CD25+ Regulatory T Cell Function and Decreases Functional and Structural Effects of Asthma Induction in a Rat Asthma Model.

Bacillus Calmette-Guerin (BCG) has been shown to have therapeutic effects on asthma through CD4+CD25+ regulatory T cells (Tregs). We sought to assess pretreatment with inactivated BCG on CD4+CD25+ Tregs and its functional and structural effects in rat asthma model. The rat asthma model was established using ovalbumin (OVA) sensitization and challenge. Ten rats were pretreated with BCG prior to OVA and received continued BCG injections during OVA challenge (BCG+OVA group), 10 rats were treated with OVA alone (OVA group), and 10 rats were treated with saline (control group). After 9 weeks, histamine dihydrochloride effect on airway resistance was measured. Number of CD4+CD25+ Tregs was measured by flow cytometry, expression of Foxp3 and CTLA-4 mRNA was measured, and serum TGF-β levels were determined. Differential cell count in bronchoalveolar lavage fluid (BALF) was determined, and lung tissue was processed and stained with hematoxylin and eosin, Masson's trichrome, and alcine blue and periodic acid Schiff's reaction to evaluate inflammatory cell infiltration, collagen deposition, and presence of goblet cells, respectively. BCG treatment led to an increase in CD4+CD25+ Tregs, as well as an increase in Foxp3 and CTLA-4 expression and serum TGF-β levels. In addition, we observed a decrease in histamine dihydrochloride-induced airway resistance, a decrease in inflammatory leukocytes in BALF, and a decrease in airway remodeling indicators in BCG+OVA-treated rats compared with OVA-treated rats. Intradermally injected inactivated BCG has the potential to improve airway inflammation, airway resistance, and airway remodeling through a mechanism that may involve CD4+CD25+ Tregs.

Abstract B78: Histamine dihydrochloride in the treatment of mesothelioma

Abstract Our recent adoptive T cell transfer trial for mesothelioma revealed a striking anti-tumor response in a patient which was accompanied by anaphylaxis, suggesting endogenous histamine release. In animal models, histamine has direct and immune-based anti-tumor effects. In fact, histamine dihydrochloride is approved in Europe in combination with interleukin-2 (IL2) for leukemia treatment. We thus explored the anti-tumor role of histamine in mesothelioma. An in vitro MTT assay was used to measure cell proliferation. Daily doses of histamine (1 mg/kg) – alone, or in combination with other immunotherapies – was used to treat four subcutaneous flank tumor models in three different mouse strains. Flow cytometric analyses were subsequently performed on tumors. Histamine had in vitro anti-tumor activity against all five cell lines tested with an IC50 of 1.8 mM. Histamine also significantly reduced tumor volume in all four in vivo models without toxic side effects. Immunodepletion studies revealed that the effect was partially due to T cells. When combined with two immunotherapy approaches (adenovirally-delivered interferon-α or a tumor antigen), anti-tumor efficacy was augmented. Histamine resulted in more interferon-γ and IL2 production by tumor-infiltrating lymphocytes (TIL), downregulation of CD206 expression on tumor-infiltrating macrophages, and reduced numbers of T regulatory cells (Tregs). Histamine has direct anti-tumor effects and immune-stimulatory effects within the tumor microenvironment (as seen with macrophage polarization, increase in cytokine production, and reduction in Tregs). This pre-clinical data suggests that histamine alone, or in combination with immunotherapy, should be explored in the treatment of mesothelioma. Citation Format: Kheng Newick, Shaun O'Brien, Veena Kapoor, Liang-chuan Wang, Edmund Moon, Andrew Haas, Evan Alley, Daniel Sterman, Steven Albelda. Histamine dihydrochloride in the treatment of mesothelioma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B78.

Pharmacokinetic Properties and Safety Profile of Histamine Dihydrochloride Injection in Chinese Healthy Volunteers: A Phase I, Single-center, Open-label, Randomized Study

PurposeHistamine dihydrochloride (HDC) injection has been approved in Europe for the treatment of adults with acute myeloid leukemia, used in combination therapy with the T-cell−derived cytokine interleukin-2. Despite years of clinical applications of HDC in Europe, no data are available on its tolerability and pharmacokinetic properties in Chinese patients. The objective of this study was to determine the safety profile and pharmacokinetic properties of HDC in Chinese healthy volunteers (HVs). MethodsIn this Phase I, single-center, open-label, randomized study, 20 Chinese HVs were randomized to receive a single dose of 0.5 or 1.0 mg HDC via a 10-minute subcutaneous injection. Whole-blood and urine samples were collected at designated time points after dosing. Plasma and urine concentrations of histamine and metabolite N-methyl histamine were measured using a validated HPLC-MS/MS method. Pharmacokinetic parameters were estimated through noncompartmental procedures based on concentration-time data. Adverse events and evaluation of clinical laboratory tests were used to assess the safety profile. The pharmacokinetic profile for a single-dose of 1.0 mg HDC in Chinese HVs was compared with that in Western HVs. FindingsNo severe adverse events occurred in this study, and the severity of all adverse events was grade I according to the Common Terminology Criteria for Adverse Events, version 4.0. For the pharmacokinetic parameters of histamine at the 0.5-mg and 1.0-mg dose levels, t½ was 0.50 and 1.02 hours; Tmax was 0.15 and 0.14 hours; mean Cmax was 26.59 and 71.01 nmol/L; AUC0−t was 8.35 and 20.43 nmol/h/L; AUC0−∞ was 9.61 and 22.69 nmol/h/L; accumulated amount excreted in urine within 24 hours was 125.93 and 145.52 nmol; and maximum urine excretion rates were 21.85 and 38.94 nmol/h, respectively. For N-methyl histamine at the 0.5-mg and 1.0-mg dose levels, t½ was 0.58 and 0.66 hours; Tmax was 0.28 and 0.26 hours; mean Cmax was 17.01 and 23.54 nmol/L; AUC0−t was 7.72 and 17.08 nmol/h/L; AUC0−∞ was 9.01 and 19.62 nmol/h/L; accumulated amount excreted in urine within 24 hours was 331.7 and 583.21 nmol; and maximum urine excretion rates were 53.29 and 133.53 nmol/h, respectively. ImplicationsBoth single-dose 0.5 mg and 1.0 mg HDC were well tolerated in Chinese HVs, and the pharmacokinetic profile of HDC in Chinese HVs was characterized in this study. A single dose of 1.0 mg HDC had a more rapid but similar extent of absorption, a wider distribution, and a little more rapid elimination in Chinese HVs compared with Western HVs. Findings from this study support additional clinical trials for HDC using in Chinese patients. Chinese Clinical Trial Registry identifier: ChiCTR-ONC-13003954.

Abstract 4293: Histamine dihydrochloride as an immune modulator for solid tumors

Abstract Our recent adoptive T cell transfer trial for mesothelioma revealed a striking anti-tumor response in a patient which was accompanied by anaphylaxis, suggesting endogenous histamine release. In animal models, histamine has direct and immune-based anti-tumor effects. In fact, histamine dihydrochloride is approved in Europe in combination with interleukin-2 (IL2) for leukemia treatment. We thus explored the anti-tumor role of histamine in mesothelioma and lung cancer. An in vitro MTT assay was used to measure cell proliferation. Daily doses of histamine (1 mg/kg) - alone, or in combination with other immunotherapies - was used to treat four subcutaneous flank tumor models in three different mouse strains. Flow cytometric analyses were subsequently performed on tumors. Histamine had in vitro anti-tumor activity against all five cell lines tested with an IC50 of 0.1 mg/ml. Histamine also significantly reduced tumor volume in all four in vivo models without toxic side effects. Immunodepletion studies revealed that the effect was partially due to T cells. When combined with two immunotherapy approaches (adenovirally-delivered interferon-α or a tumor antigen), anti-tumor efficacy was augmented. Histamine resulted in more interferon-γ and IL2 production by tumor-infiltrating lymphocytes, downregulation of CD206 expression on tumor-infiltrating macrophages, and reduced numbers of T regulatory cells (Tregs). This pre-clinical data suggests that histamine alone, or in combination with immunotherapy, should be explored in the treatment of mesothelioma, lung cancer, and perhaps other cancers. Citation Format: Kheng Newick, Shaun O'Brien, Veena Kapoor, Liang-chuan Wang, Edmund Moon, Daniel Sterman, Steven Albelda. Histamine dihydrochloride as an immune modulator for solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4293. doi:10.1158/1538-7445.AM2015-4293

Role of natural killer cell subsets and natural cytotoxicity receptors for the outcome of immunotherapy in acute myeloid leukemia

In a phase IV trial, 84 patients (age 18–79) with acute myeloid leukemia (AML) in first complete remission (CR) received cycles of immunotherapy with histamine dihydrochloride (HDC) and low-dose human recombinant interleukin 2 (IL-2) for 18 months to prevent leukemic relapse. During cycles, the treatment resulted in expansion of CD56bright (CD3−/16−/56bright) and CD16+ (CD3−/16+/56+) natural killer (NK) cells in the blood along with increased NK cell expression of the natural cytotoxicity receptors (NCRs) NKp30 and NKp46. Multivariate analyses correcting for age and risk group demonstrated that high CD56bright NK cell counts and high expression of NKp30 or NKp46 on CD16+ NK cells independently predicted leukemia-free survival (LFS) and overall survival (OS). Our results suggest that the dynamics of NK cell subsets and their NCR expression may determine the efficiency of relapse-preventive immunotherapy in AML.

Self-assembly and magnetic behavior of 2-aldehyde-8-hydroxyquinolinate-based lanthanide complex

Self-assembly reaction of Ln(NO3)3.6H2O, 2-aldehyde-8-hydroxyquinoline and histamine dihydrochloride affords two mononuclear complex [Ln(hma)(NO3)2(CH3OH)]∙0.5C4H10 (Ln=Tb (1), Dy (2); Hhma=N-(2-(8-hydroxylquinolinyl)methane(2-(4-imidazolyl)ethylamine)). The structural analysis indicates that they are isomorphous where the Ln3+ is ligated to one ligand, two nitrates and one methanol molecule. Variable temperature magnetic susceptibility studies reveal the presence of antiferromagnetic interactions in 2 and the dynamic measurement reveals that 2 displays single molecular magnet behavior below 10K under an applied field of 2000Oe.

Evaluation of Methods for the Estimation of Threshold Concentrations by the Skin Prick Test

Background: The allergen dose-response curve is flat; thus, small changes in wheal size reflect large differences in skin sensitivity. The sensitivity as measured by provocation tests is given by the threshold concentration that causes symptoms and/or objective signs. The threshold concentrations differ by several magnitudes between the most and the least sensitive individuals clinically allergic to the same allergen. Variation in technique can be minimized by relating allergen responses to that to histamine. The aim here is to present and validate simple methods for estimation of the skin sensitivity given as the concentration inducing a wheal of the same size as that with the positive reference, 10 mg/ml of histamine HCl, in the same patient. Methods: Data from previously reported trials on the biological equilibration of allergen extracts were used to document a method to calculate the concentration of allergen required to induce a wheal of the same size as that with 10 mg/ml of histamine dihydrochloride in the same patient, and to validate the methods using the parallel line bioassay as the gold standard. Results: The validated methods correlated well with the results obtained using the gold standard method and provide results of skin prick testing based on threshold concentrations of allergen. Conclusions: The validated methods reduce the error of differences in testing techniques and make it possible to report skin sensitivity at threshold concentrations. A simple method to be used in clinical practice and a method suitable to describe changes in skin reactivity over time or during treatment are proposed.

Allergen Skin Prick Test Should Be Adjusted by the Histamine Reactivity

Background: Skin prick test results are mostly reported as mean wheal diameter obtained with one concentration of allergen. Differences in technique between personnel causes variation in wheal size. The research question was whether the influence of differences in skin prick test technique among assistants and centers can be reduced by relating the allergen wheal response to that of histamine. Methods: Two methods for estimating skin reactivity, the method of Nordic Guidelines using histamine as a reference and the method of Brighton et al. [Clin Allergy 1979;9:591-596] not using histamine as a reference, were applied to data from two biological standardization trials, using the same batch of freeze-dried timothy pollen preparation. Results: The concentration defining the Nordic biological unit, defined as a concentration of allergen eliciting a wheal of the same size as that of histamine dihydrochloride 10 mg/ml, did not differ between the centers. When not using histamine as a reference, applying the method of Brighton et al., there was a 15-fold difference in the estimate of the biological activity between the trials that was eliminated by adjusting the allergen response to that of the histamine reference. Conclusions: To reduce the influence of differences in test technique among assistants and centers responses to allergen-induced skin prick tests should be compared to that of histamine.

Novel phenotype in beagle dogs characterized by skin response to compound 48/80 focusing on skin mast cell degranulation.

Beagle dogs have long been employed in toxicology studies and as skin disease models. Compared with other experimental animal species, they are known to be susceptible to skin responses, such as rashes, from exposure to various chemical compounds. Here, a unique dog phenotype was identified that showed no skin response to compound 48/80, a mast cell degranulating agent. Although the skin responses to intradermal injection of polyoxyethylene castor oil derivative (HCO-60, a nonionic detergent), histamine dihydrochloride, concanavalin A (IgE receptor-mediated stimuli), or calcium ionophore A23187 were comparable in wild-type (WT) dogs and these nonresponder (NR) dogs, only the response to compound 48/80 was entirely absent from NR dogs. The skin mast cell density and histamine content per mast cell were histologically comparable between WT and NR dogs. By checking for skin responses to compound 48/80, NR dogs were found to exist at the proportion of 17–20% among four animal breeders. From retrospective analysis of in-house breeding histories, the NR phenotype appears to conform to the Mendelian pattern of recessive inheritance. The standard skin response in WT dogs developed at 2–4 months of age. In conclusion, this unique phenotype, typified by insensitivity in the compound 48/80-induced degranulation pathway in mast cells, has been widely retained by recessive inheritance in beagle dogs among general experimental animal breeders. The knowledge concerning this phenotype could lead to better utilization of dogs in studies and aid in model development.

Maintenance with Histamine and IL-2 Induces a Marked Expansion of Activated CD56bright NK Cells in Acute Myeloid Leukemia

In acute myeloid leukemia (AML), residual leukemic (stem) cells that escape initial chemotherapy are considered a major source of relapse. Clinical trials have used IL-2 for AML patients with the aim to reduce relapse rates by eliminating residual leukemic cells through activation of NK and T cells. However, monotherapy with IL-2 has led to disappointing results. Nevertheless, recent clinical trials have shown that the co-administration of IL-2 and histamine dihydrochloride (HDC) provides maintenance of remission in AML. Histamine suppresses the formation of reactive oxygen species (ROS) thereby protecting NK and T cells from ROS-induced dysfunction and apoptosis. In addition, IL-2 is considered to maintain the anti-leukemic activity of NK cells. However, the direct effect of this treatment on NK cell numbers and anti-AML activity has not been studied in detail so far.In this study, we analyzed the immunophenotype and function of NK cells in a cohort of 7 AML patients (FAB M2, n=1; M4, n=2; M4-eo with inv 16, n=2; M5, n=2) treated with HDC plus IL-2. All patients had received induction chemotherapy with daunorubicin (45 mg/m² i.v. days 1-3), cytosine arabinoside, ARA-C (2 x 100 mg/m² i.v., days 1-7) and etoposide (100 mg/m² i.v., days 1-5) as well as at least 3 cycles of consolidation chemotherapy with high dose or intermediate dose ARA-C (Sperr et al, Clin Cancer Res 2004;10:3965-3971 and Krauth et al, J Immunol 2006;176:1759-1768). After having achieved a complete hematologic remission, patients were treated with HDC (0.5 mg) plus recombinant IL-2 (0.9 x 106 IU) twice daily s.c. for 21 days per cycle. Blood was drawn before and during treatment with HDC plus IL-2.We found that after one week of treatment with HDC plus IL-2, NK cell numbers increased in peripheral blood (from 101.8 ± 28.25 cells/µl before therapy to 208.2 ± 38.27 cells/µl after therapy, p<0.05). In the NK cell fraction, we observed an astonishing increment of CD56bright NK cells in all treated patients (from 7.2±0.97% or 17.6±5.8 cells/µl before therapy to 38.8±4.4% or 104±19.4 cells/µl after therapy, p<0.05; see Fig.1A/B). The cytotoxic activity of the CD56bright cells, as determined by NK cell degranulation and target cell lysis using the cell line K562, showed a significant increase in comparison to cells obtained before treatment (p<0.05). This was associated with an increased expression of KIR as well as the activation markers NKp44 (see Fig.1C), NKp46, and CD25 on NK cells. Furthermore, we observed a significant increase in expression of CD56 on NK cells after treatment with HDC plus IL-2 in our AML patients (2.5 ± 0.55 fold increase in the mean fluorescence intensity of CD56, p=0.02), whereas CD16 expression did not change significantly. In addition, treatment with HDC+IL-2 also induced an increased proportion of circulating CD4+CD25highCD127low/neg regulatory T cells (Treg). Finally, in vitro stimulation of NK cells with histamine plus IL-2 mimicked the effects observed in vivo. Interestingly, the in vitro treatment was also associated with an increased expression of CD56 without altered expression of CD16, suggesting that this effect could be a specific and reliable indicator of in vivo responses of NK cells to HDC plus IL-2 therapy.In conclusion, treatment with HDC plus IL-2 causes a striking increase in CD56bright NK cells. These specifically expanded NK cells exhibit an activated phenotype with enhanced potential to kill leukemic cells. We propose that the maintenance of remission in AML patients treated with HDC plus IL-2 might, at least in part, be the result of an improved anti-leukemic NK cell function. [Display omitted] DisclosuresSperr:MEDA Pharma GmbH & Co. KG: Speakers Bureau. Valent:MEDA Pharma GmbH & Co. KG: Speakers Bureau.

Reactive Oxygen Species Produced By Myeloid Cells Block CD20-Antibody Dependent NK Cell-Mediated Cytotoxicity Against Chronic Lymphocytic Leukemia Cells

Introduction:The addition of the anti-CD20 antibodies rituximab or ofatumumab to chemotherapy has improved the outcome for patients with chronic lymphocytic leukemia (CLL), but the efficacy of anti-CD20 monoclonal antibodies (mAbs) as single agents is limited. Immunosuppressive reactive oxygen species (ROS), produced by myeloid cells, including monocytes, may be a limiting factor for the efficacy of ADCC-dependent therapy. With the aim of reducing a potentially inhibitory role of ROS on mAb-dependent NK cell cytotoxicity against CLL cells, we performed in vitro cytotoxicity experiments in the presence of monocytes and the ROS formation inhibitor histamine dihydrochloride (HDC).Methods:After informed consent, blood samples were collected from Binet stage A CLL patients seen at Sahlgrenska University Hospital, Gothenburg, Sweden. NK cells, monocytes and CLL cells were isolated from PBMCs by immunomagnetic negative selection or flow cytometry. ROS production by monocytes was assessed by isoluminol-enhanced chemiluminescense upon stimulation with the chemotactic peptide fMLF in the presence or absence of CLL cells, rituximab (Roche), ofatumumab (GSK), HDC or the NADPH oxidase inhibitor DPI. NK cell death was assessed by flow cytometry after over-night culture of monocytes and lymphocytes. Autologous ADCC assays were performed with NK cells, monocytes and CFSE-labeled CLL cells in the presence or absence of rituximab, ofatumumab, HDC, catalase and interleukin-2 (IL-2). CLL cell death was determined by flow cytometry after live/dead staining. The study was approved by the local ethical review board of Gothenburg, and conducted in accordance with the declaration of Helsinki.Results:Rituximab and ofatumumab both triggered extracellular ROS production by monocytes. ROS production was blocked by the NADPH oxidase inhibitor DPI. Furthermore, monocytes induced ROS-dependent apoptosis in adjacent NK cells. In the cytotoxicity experiments, rituximab-mediated NK cell dependent cytotoxicity against CLL cells was significantly inhibited by the presence of autologous monocytes. This suppression, as well as the induction of NK cell apoptosis, were dependent on ROS, since both HDC and catalase significantly increased cytotoxicity and rescued NK cells from monocyte-induced apoptosis. Expectedly, IL-2 augmented NK cell-mediated ADCC.Conclusion:The results imply that CLL patients harbor immunosuppressive myeloid cells that reduce CD20-antibody dependent NK cell-mediated cytotoxicity against leukemic cells by producing ROS. We propose that treatment with HDC, preferably in conjunction with NK cell stimulatory cytokines, may improve anti-CD20-based immunotherapy in CLL. DisclosuresNo relevant conflicts of interest to declare.

Natural Killer Cell Expression of Natural Cytotoxicity Receptors Determines Relapse Risk in Elderly Patients with Acute Myeloid Leukemia Receiving Immunotherapy with Histamine Dihydrochloride and Interleukin-2

Background: The insufficiency of natural killer (NK) cells in acute myeloid leukemia (AML) and the purported role of NK cells for prognosis have inspired the use of NK cell-based immunotherapies for relapse prevention, including the administration of NK cell-activating cytokines. A previous phase III trial using immunotherapy with histamine dihydrochloride and low-dose interleukin-2 (HDC/IL-2) was shown to significantly prevent relapse in patients who had achieved complete remission (CR) and completed the phase of consolidation chemotherapy (Brune et al., Blood108:88). Details regarding the mechanisms of relapse prevention are only partly understood.Trial design and methods: In a single-armed multicenter phase IV study (Re:Mission Trial, NCT01347996) we aimed at assessing the immunomodulatory effects of HDC/IL-2 with a focus on NK cell biology and to correlate NK cell markers with relapse risk. Eighty-four patients (age 18-79, median 61.2) with AML in first CR received post-consolidation immunotherapy with HDC/IL-2 for 18 months in ten 3-week cycles with 3 (cycle 1-6) or 6 week (cycle 7-10) intervals between cycles. NK cell counts in blood and natural cytotoxicity receptor (NCR) expression were determined by flow cytometry before and after cycles 1 and 3. The trial protocol specified separate analyses in younger (<60 years old, n=37) and elderly patients (>60 years old, n=47).Results: During cycle 1, CD56bright (CD3–/16–/56bright) and CD16+ (CD3–/16+/56+) NK cell counts increased in the blood (P=3x10-8 and P=2x10-5, respectively, paired t-test, n=47, all evaluable patients) along with an increased expression intensity of the NCRs NKp30 and NKp46 in the cytotoxic CD16+ subset (P=2x10-7 and P=6x10-8, respectively, paired t-test n=47). NK cell counts declined between the end of treatment cycle 1 and the start of cycle 3. CD56bright NK cell counts typically returned to pre-treatment levels whereas CD16+ NK cell counts remained modestly elevated at the onset of cycle 3 (P=.003, paired t-test; n=46). A renewed accumulation of CD56bright and CD16+ NK cells in the blood was observed during treatment cycle 3 (P=2x10-6 and P=.007, respectively, paired t-test, n=40). Young and elderly patients showed similar fluctuations in counts of CD56bright and CD16+ NK cell within and between treatment cycles. Also, significant induction of the expression intensity of NKp30 and NKp46 on CD16+ NK was observed in young and elderly patients.Among elderly patients, expression of NKp46 in CD16+ NK cells after the first treatment cycle was associated with favorable outcome (RFS for above-median NKp46 expression in CD16+ NK cells; P=.002, HR=4.1, CI: 1.7-10.0; logrank test). Similar results, with increased likelihood of RFS, were obtained in elderly patients with NK cells showing above-median expression of NKp30. The expression intensity of NKp30 and NKp46 did not predict relapse in younger patients.Conclusion: These results highlight the impact of NK cells and NCRs for AML prognosis and suggest that NK cell expression of NCRs may determine the efficacy of immunotherapy for remission maintenance in elderly patients. DisclosuresMartner:Study sponsor Meda Pharma GmbH & Co, Bad Homburg, Germany: Honoraria, Research Funding. Brune:Study sponsor Meda Pharma GmbH & Co, Bad Homburg, Germany: Consultancy, Honoraria. Foa:Study sponsor Meda Pharma GmbH & Co, Bad Homburg, Germany: Consultancy. Hellstrand:Study sponsor Meda Pharma GmbH & Co, Bad Homburg, Germany: Consultancy, Honoraria, related to the use of HDC/IL-2 in AML Patents & Royalties, Research Funding. Off Label Use: HDC/IL-2 is approved for use within the EU for AML patients in CR <60 years old. In the abstract we discuss the potential usefulness of the drug for patients >60 years old. Thorén:Study sponsor Meda Pharma GmbH & Co, Bad Homburg, Germany: Honoraria, Research Funding.

Endpoints of Clinical Trials for Marketing Authorization of Drugs for Hematologic Malignancy in Japan, the US and the EU

Background: Pharmaceuticals and Medical Devices Agency (PMDA) in Japan, the US Food and Drug Administration (FDA) in the US and the European Medicines Agency (EMA) in the EU review drug applications for market approval. It was shown that most new molecular entity (NME) agents were first approved in the US because the FDA has reviewed applications more quickly (N Engl J Med. 2012; 366: 2284-93). However, differences in regulatory actions among Japan, the US and the EU in terms of relationship between the approval date and primary endpoints of pivotal trials in marketing applications including supplemental applications for additional indications have never been analyzed comprehensively. Methods: 93 indications of 55 hematologic anticancer drugs approved in Japan, the US or the EU (only central marketing authorization by the European Commission) between August 2000 and June 2014 were examined in this research. Information concerning regulatory decisions made by PMDA, the FDA and the EMA was obtained from publicly accessible data only such as PMDA’s review reports, the PMDA website, the FDA’s review reports, the Drugs@FDA website, the European public assessment reports published by the EMA’s Committee for Medicinal Products for Human Use (CHMP) and the EMA website. Results: 24, 60, and 9 indications were first approved in Japan, the US, and the EU, respectively. 2 NME agents were first approved in Japan and the EU each; mogamulizumab for relapsed CCR4-positive adult T-cell leukemia/lymphoma as well as relapsed CCR4-positive peripheral T-cell lymphoma/cutaneous T-cell lymphoma and tamibarotene for relapsed acute promyelocytic leukemia in Japan and pixantrone dimaleate for relapsed aggressive non-Hodgkin B-cell lymphoma and histamine dihydrochloride for acute myeloid leukemia in the EU. 17 indications were approved only in the US and 13 of these were approved based on response rate in single-arm trials. The other 4 indications were approved based on results of comparative trials. Of the 13 indications approved based on single-arm trials, 8 indications were approved through the accelerated approval scheme in the US. 5 indications (pixantrone dimaleate, histamine dihydrochloride, temsirolimus for relapsed mantle cell lymphoma, rituximab as maintenance therapy for relapsed follicular lymphoma and decitabine for acute myeloid leukemia) were approved only in the EU and 4 other indications (ibritumomab tiuxetan as consolidation therapy for follicular lymphoma and rituximab for diffuse large B-cell lymphoma, newly diagnosed chronic lymphocytic leukemia, and relapsed chronic lymphocytic leukemia) were approved the earliest in the EU. All these 9 indications were approved based on results of comparative trials. Of these 9 indications, only pixantrone dimaleate was approved through the conditional approval scheme and only histamine dihydrochloride was approved through the approval under exceptional circumstances scheme. Although 23 indications were approved only in Japan, most of them were additional indications of old drugs and not based on results of comparative trials. Primary endpoints were different between the US and the EU in 3 of 41 indications approved in both the US and the EU. All these 3 indications were approved earlier in the US and response rate or time to progression (TTP) in the US instead of progression-free survival (PFS) or overall survival (OS) in the EU was adopted as the primary endpoints as follows; thalidomide for newly diagnosed multiple myeloma (TTP in the US and OS in the EU), pomalidomide for relapsed multiple myeloma (overall response rate in the US and PFS in the EU), and azacitidine for myelodysplastic syndrome (complete remission rate in the US and OS in the EU). Conclusions: The conditional approval scheme or the approval under exceptional circumstances scheme in the EU and the accelerated approval scheme in the US are available, while there are no similar schemes for oncologic drugs in Japan. However, this analysis suggests that the FDA has taken a more active attitude to acceptance of surrogate endpoints. Therefore, not only shorter review time but also this attitude may lead to earlier marketing authorization of drugs for hematologic malignancy in the US. Disclosures Off Label Use: I will discuss off-label drug use in the US which has been approved in the EU or Japan..

Abstract 2545: Histamine dihydrochloride and interleukin-2 for relapse prevention in AML: Initial results of the Re:MISSION phase IV trial

Abstract BACKGROUND AND METHODS. Immunotherapy with histamine dihydrochloride and low-dose interleukin-2 (HDC/IL-2) has been approved within the EU for relapse prevention in the post-consolidation phase of acute myeloid leukemia (AML) (Blood 108:88). We have performed a phase IV trial (Re:MISSION) to assess the immunomodulatory properties of this regimen and to correlate potential biomarkers of immunity with outcome. Eighty-nine patients (age 19-80) with AML in first complete remission received HDC (0.5 mg sc bid) and human recombinant IL-2 (1MIU sc bid) in ten repeated three-week cycles for 18 months or until relapse. Mononuclear cells were analyzed for NK and T cell phenotypes by flow cytometry at onset and during cycles 1 and 3. INDUCTION OF NK CELLS AND NCR EXPRESSION DURING THERAPY. Treatment with HDC/IL-2 resulted in a 3-fold expansion of both CD56bright (CD56/CD16-) and CD16+ (CD56+/CD16+) NK cells during cycle 1 (CD56+, P= 10-6, n=47; paired samples t-test). During cycle 1, the median expression intensity of the natural cytotoxicity receptors (NCRs) NKp46 and NKp30 on CD16+ NK cells increased by 50% and 30%, respectively (P=6 x10-8 for NKp46; P=2 x10-7 for NKp30, n=56). For CD56bright cells, the results were discordant for the NCRs with pronounced induction of NKp30 (P=4 x10-13) during cycle 1 but no induction of NKp46 (P=0.51, n=56). ROLE OF NCR EXPRESSION FOR MAINTENANCE OF REMISSION. An interim analysis of immune markers vs. relapse-free survival (RFS) revealed a strong impact of NK cell NKp46 expression. Patients with NKp46 expression above the median after one 21-day cycle of HDC/IL-2 thus showed significantly higher RFS than those with lower expression (CD16+, P=0.003, HR=3.0, CI 1.4-6.4; CD56bright, P=0.002, HR=3.3, CI: 1.5-7.0; logrank test). High NK cell expression of NKp46 after a treatment cycle also identified a population of elderly patients (&amp;gt;60 years old) with favorable outcome (CD16+, P=0.002, HR=4.1, CI: 1.6-10.2; logrank test). We observed a pronounced expansion of CD4+/CD25+/Foxp3+ regulatory T cells during cycles that did not impact on relapse risk. CONCLUSION. These results highlight the role of NK cells and NCR expression for relapse-free survival in AML. Citation Format: Anna Martner, Anna Rydström, Rebecca Riise, Johan Aurelius, Mats Brune, Kristoffer Hellstrand, Fredrik Bergh Thorén. Histamine dihydrochloride and interleukin-2 for relapse prevention in AML: Initial results of the Re:MISSION phase IV trial. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2545. doi:10.1158/1538-7445.AM2014-2545

Histamine Dihydrochloride for Maintaining Remission in Acute Myeloid Leukemia

SUMMARY Maintaining complete remission in patients with acute myeloid leukemia (AML) is a great challenge. Targeting the remaining malignant clones, using different regimens of chemotherapeutic agents did not prove to be fruitful. Host residual leukemia stem cells may be eradicated by donor immune cells in case of allogeneic stem cell transplantation. However, only a minority of AML patients may benefit from this procedure. IL-2 is very well known in its ability to enhance activation and proliferation of natural killer and T cells, albeit in the clinical setting this effect is suppressed by myeloid cells derived oxygen species. Histamine dihydrochloride can inhibit myeloid cells activity, therefore reaugment antileukemic natural killer/T-cell cytotoxic activity. This review summarizes different strategies to maintain complete remission in AML patients focusing on the use of histamine dihydrochloride/IL-2 treatment.

Model of the Deposition of Aerosol Particles in the Respiratory Tract of the Rat. II. Hygroscopic Particle Deposition

Rats are frequently used to study the pharmacological and toxicological effects of inhaled aerosol particles. The deposition behavior of aerosol particles in airways is affected by their hygroscopic properties, which accordingly influence the results of such studies. A recently published nonhygroscopic aerosol particle deposition model for rat airways was extended with equations for hygroscopic particle growth in humid air and with a model to mimic the temperature and relative humidity conditions in the rat airways transformed from the upper human airways. As there are no experimental data available for hygroscopic deposition in rat lungs, several model assumptions were made for the humidity distribution in the upper rat airways. The total and regional deposition probability of salt particles in the diameter range 0.02 to 5 μm in rat lung was significantly changed by the hygroscopic properties. The maximum ratios of the total deposition of inhaled initially dry sodium chloride, cobalt chloride, and zinc sulfate particles compared with nonhygroscopic particles were 3.28, 2.44, and 2.13, respectively, and the minimum ratios 0.57, 0.63, and 0.70, respectively. The corresponding maximum (and minimum) ratios for the hygroscopic drugs histamine dihydrochloride, carbenicillin disodium, and atropine sulfate were 1.86 (0.65), 1.53 (0.70), and 1.35 (0.76), respectively. Total deposition was about 20% higher in human airways than in rat airways. The flow regime in the rat upper airways influenced total and regional deposition much less than it did in human airways. The hygroscopicity of salt and drug aerosol particles is an important factor in rat lung deposition.