Initial Rapid Clearance Research Articles

Abstract 6313: IND-enabling studies for GrB-Fc-IT4: in vivo efficacy, pharmacokinetics, schedule optimization and maximum tolerated dose

Abstract Fn14 (fibroblast growth factor-inducible 14) is the only known receptor of TWEAK (tumor necrosis factor-related weak inducer of apoptosis). The binding of TWEAK to Fn14 activates several intracellular signal transduction cascades that lead to cell death, proliferation, migration, or survival depending on the cellular contexts. Several studies have shown that Fn14 is upregulated in many solid tumors when compared with healthy tissues. The activation of TWEAK/Fn14 signaling increases the proliferation, invasion, and migration of tumor cells. Moreover, angiogenesis, pro-inflammatory cytokine expression, and epithelial-mesenchymal transitions are promoted upon TWEAK/Fn14 activation. Therefore, Fn14 is considered a potential therapeutic target, and attempts to generate targeted therapeutics have achieved limited success in pre-clinical and clinical trials. We generated a completely human fusion protein, GrB-Fc-IT4, consisting of the IT4 scFv antibody targeting the Fn14 receptor and the serine protease granzyme B (GrB) as the cytotoxic payload. Comprehensive mechanism of action studies showed that GrB-Fc-IT4 effectively induced cell death in vitro against a broad selection of tumor types with high specificity. The intracellular pathway included rapid, irreversible activation of the caspase cascade and independent mitochondrial depolarization leading to intense apoptotic cellular damage. Pharmacokinetic studies in mice showed that GrB-Fc-IT4 cleared bi-exponentially from plasma with a rapid initial clearance (t ½α = 0.36 hours) followed by a prolonged terminal-phase plasma half-life (t 1/2β = 35 hours), similar to that of IgGs. Based on our PK study, we compared the in vivo antitumor efficacy of GrB-Fc-IT4 against A549 (NSCLC) tumors, using two different dosing schedules (QODx5 vs QWx5). Tumor efficacy showed a clear dose and schedule dependence. Mice bearing A549 tumors treated with 32 mg/kg/dose (QOWx5) exhibited complete tumor growth inhibition (7/10 tumors regressed) with 3/10 tumors showing no growth up to 50 days after the last dose. Thus, GrB-Fc-IT4 displays impressive in vitro and in vivo cytotoxic effects. Even though GrB-Fc-IT4 cross-reacts with the murine Fn14 antigen, maximum tolerated dose studies in mice have confirmed that GrB-Fc-IT4 is a safe product, as mice treated with the drug up to 500 mg/kg showed no evidence of toxicity (changes in respiration, rough hair coat, unusual behavior or hunched posture). Taking in consideration these results we estimate that the therapeutic index for this class of drug is higher than four. Our data suggest that GrB-Fc-IT4 is a novel antitumor agent with a unique mechanism of action compared to all other therapeutic agents. The exceptionally safe and effective profile of this drug makes it an ideal candidate for advancing to clinical trials. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Mihai Gagea, Michael G. Rosenblum. IND-enabling studies for GrB-Fc-IT4: in vivo efficacy, pharmacokinetics, schedule optimization and maximum tolerated dose [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6313.

Abstract 5216: Optimizing antitumor efficacy of granzyme B human fusion constructs targeting the Fn14 antigen in solid tumors. Pre IND pharmacokinetics, schedule, dose optimization and MTD studies

Abstract We previously demonstrated that a human fusion protein construct targeting the Fn14 receptor and containing the cytotoxic granzyme B (GrB) payload (GrB-Fc-IT4) displays impressive in vitro and in vivo cytotoxic effects. Comprehensive mechanism of action studies show that GrB-Fc-IT4 effectively induces cell death in vitro against a broad selection of tumor types with high specificity. The intracellular pathway includes irreversible activation of the caspase cascade and independent mitochondrial depolarization leading to an intense apoptotic cellular damage. This activation occurs quickly and efficiently. Pharmacokinetic studies in mice showed that GrB-Fc-IT4 was cleared bi-exponentially from plasma with a rapid initial clearance (t ½alpha = 0.36 hours) followed by a prolonged terminal-phase plasma half-life (t 1/2beta = 35 hours) similar to that of IgGs. Based on our pharmacokinetic study, we employed a QODX5 therapy schedule and demonstrated impressive antitumor activity against several tumor models including PDX-lung, A459 (NSCLC) and MDA-MB231(TNBC) tumor models. In the present study we compared the in vivo antitumor efficacy of GrB-Fc-IT4 against A549 (NSCLC) tumor cell line, using two different dosing schedules (QODx5 vs QWx5). There was a clear dose and schedule dependence and that treating mice bearing A549 tumors with 32mg/kg/dose using a QOWx5 schedule induced a complete tumor growth inhibition (7/10 tumors regressed) with 3/10 tumors showing no growth up to 50 days after the last dose. Preliminary results suggest that GrB-Fc-IT4 may reduce the formation of spontaneous A549 metastasis into lungs. Further studies are planned to examine the potential of the fusion construct to prevent spontaneous metastatic spread in lung tumor models. Although the fusion construct cross-reacts with the murine Fn14 antigen, maximum tolerated dose studies have confirmed that GrB-Fc-IT4 is a safe product, showing no evidence of toxicity (weight loss) in mice treated with up to 500mg/kg. This suggests that the therapeutic index (TI) for this class of agents is >4. Our data suggest that GrB-Fc-IT4 is a novel class of antitumor agents with a unique mechanism of action. This agent seems to be an exceptionally safe and effective drug and appears to be an ideal candidate for advancing to clinical trials. Research conducted, in part, by Clayton Foundation for Research. Citation Format: Ana Alvarez De Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Mihai Gagea, Michael G. Rosenblum. Optimizing antitumor efficacy of granzyme B human fusion constructs targeting the Fn14 antigen in solid tumors. Pre IND pharmacokinetics, schedule, dose optimization and MTD studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5216.

Safety, pharmacokinetics, and antimalarial activity of the novel triaminopyrimidine ZY-19489: a first-in-human, randomised, placebo-controlled, double-blind, single ascending dose study, pilot food-effect study, and volunteer infection study

New antimalarials with novel mechanisms of action are needed to combat the emergence of drug resistance. Triaminopyrimidines comprise a novel antimalarial class identified in a high-throughput screen against asexual blood-stage Plasmodium falciparum. This first-in-human study aimed to characterise the safety, pharmacokinetics, and antimalarial activity of the triaminopyrimidine ZY-19489 in healthy volunteers. A three-part clinical trial was conducted in healthy adults (aged 18-55 years) in Brisbane, QLD, Australia. Part one was a double-blind, randomised, placebo-controlled, single ascending dose study in which participants enrolled into one of six dose groups (25, 75, 150, 450, 900, or 1500 mg) were randomly assigned (3:1) to ZY-19489 or placebo. Part two was an open-label, randomised, two-period cross-over, pilot food-effect study in which participants were randomly assigned (1:1) to a fasted-fed or a fed-fasted sequence. Part three was an open-label, randomised, volunteer infection study using the P falciparum induced blood-stage malaria model in which participants were enrolled into one of two cohorts, with participants in cohort one all receiving the same dose of ZY-19489 and participants in cohort two randomly assigned to receive one of two doses. The primary outcome for all three parts was the incidence, severity, and relationship to ZY-19489 of adverse events. Secondary outcomes were estimation of ZY-19489 pharmacokinetic parameters for all parts; how these parameters were affected by the fed state for part two only; and the parasite reduction ratio, parasite clearance half-life, recrudescent parasitaemia, and pharmacokinetic-pharmacodynamic modelling parameters for part three only. This trial is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12619000127101, ACTRN12619001466134, and ACTRN12619001215112). 48 participants were enrolled in part one (eight per cohort for 25-1500 mg cohorts), eight in part two (four in each group, all dosed with 300 mg), and 15 in part three (five dosed with 200 mg, eight with 300 mg, and two with 900 mg). In part one, the incidence of drug-related adverse events was higher in the 1500 mg dose group (occurring in all six participants) than in lower-dose groups and the placebo group (occurring in one of six in the 25 mg group, two of six in the 75 mg group, three of six in the 150 mg group, two of six in the 450 mg group, four of six in the 900 mg group, and four of 12 in the placebo group), due to the occurrence of mild gastrointestinal symptoms. Maximum plasma concentrations occurred 5-9 h post-dosing, and the elimination half-life was 50-97 h across the dose range. In part two, three of seven participants had a treatment-related adverse event in the fed state and four of eight in the fasted state. Dosing in the fed state delayed absorption (maximum plasma concentration occurred a median of 12·0 h [range 7·5-16·0] after dosing in the fed state vs 6·0 h [4·5-9·1] in the fasted state) but had no effect on overall exposure (difference in area under the concentration-time curve from time 0 [dosing] extrapolated to infinity between fed and fasted states was -0·013 [90% CI -0·11 to 0·08]). In part three, drug-related adverse events occurred in four of five participants in the 200 mg group, seven of eight in the 300 mg group, and both participants in the 900 mg group. Rapid initial parasite clearance occurred in all participants following dosing (clearance half-life 6·6 h [95% CI 6·2-6·9] for 200 mg, 6·8 h [95% CI 6·5-7·1] for 300 mg, and 7·1 h [95% CI 6·6-7·6] for 900 mg). Recrudescence occurred in four of five participants in the 200 mg group, five of eight in the 300 mg group, and neither of the two participants in the 900 mg group. Simulations done using a pharmacokinetic-pharmacodynamic model predicted that a single dose of 1100 mg would clear baseline parasitaemia by a factor of 109. The safety, pharmacokinetic profile, and antimalarial activity of ZY-19489 in humans support the further development of the compound as a novel antimalarial therapy. Cadila Healthcare and Medicines for Malaria Venture.

Pharmacokinetics and Safety of the Nucleoside Analog Antiviral Drug Galidesivir Administered to Healthy Adult Subjects.

Galidesivir (BCX4430) is an adenosine nucleoside analog broadly active in cell culture against multiple RNA virus families, and active in animal models of viral diseases associated with Ebola, Marburg, yellow fever, Zika, and Rift Valley fever. Current studies demonstrated the pharmacokinetics and safety of the first-in-human evaluations of galidesivir as intramuscular (IM) and intravenous (IV) formulations. Two double-blind, placebo-controlled, dose-ranging studies were conducted enrolling 126 healthy subjects. Study 1 evaluated the safety and tolerability of IM galidesivir over single day dosing, single day dosing ± lidocaine, and 7-day dosing with lidocaine. Study 2 evaluated the safety and tolerability of single ascending doses of IV galidesivir. Safety and tolerability were evaluated via clinical and laboratory monitoring. The plasma concentration-time profile of galidesivir at doses 0.3 to 10mg/kg IM was characterized by rapid absorption, an initial rapid distribution and clearance phase, and an extended terminal elimination phase. The initial rapid distribution and extended terminal elimination were mimicked in the profile of galidesivir at doses 5 to 20mg/kg IV. No fatal events or related serious adverse events were reported. No clinically significant dose-related trends in laboratory values, vital signs, electrocardiograms, or echocardiograms were noted. Galidesivir was safe and generally well tolerated.

Population pharmacokinetic analysis of nanoparticle-bound and free camptothecin after administration of NLG207 in adults with advanced solid tumors

PurposeNLG207 (formerly CRLX101) is a nanoparticle–drug conjugate (NDC) of the potent topoisomerase I inhibitor, camptothecin (CPT). The present study sought to characterize the complex pharmacokinetics (PK) of NLG207 and better describe CPT release from nanoparticles using a population PK (popPK) model.MethodsFrom 27 patients enrolled on two phase II clinical trials (NCT02769962 and NCT03531827), dense sampling was performed up to 48 h post-administration of NLG207 during cycle one and six of treatment; samples were also collected at ~ 360 h post-dose. Conjugated and free CPT concentrations were quantified from each sample, resulting in 477 observations to build a popPK model using non-linear mixed-effects modeling.ResultsThe PK of NLG207 was characterized by combining two linear two-compartment models with first-order kinetics each to describe nanoparticle-bound (conjugated) and free CPT. Allometric scaling based on body weight provided the best body-size descriptor for all PK parameters. The typical volumes of distribution of the conjugated CPT central and free CPT central compartments were 3.16 L (BSV CV%; 18.1%) and 21.1 L (CV%; 79.8%), respectively. CPT release from the nanoparticle formulation was characterized via an initial rapid clearance of 5.71 L/h (CV%; 62.6%), which decreased via first-order decay (estimated half-life of 0.307 h) to the steady-state value of 0.0988 L/h (CV%; 33.5%) by ~ 4 h after end of infusion. Renal clearance of free CPT was 0.874 L/h (CV%; 42.2%).ConclusionThe popPK model confirmed nanoparticle behavior of conjugated CPT and mechanistically characterized CPT release from NLG207. The current analysis provides a strong foundation for future study as a potential predictive tool in ongoing NLG207 clinical trials.

Ingenol mebutate for the management of cryotherapy-resistant anogenital warts.

Ingenol mebutate (IM), as an active compound, is derived from the sap of the Euphorbia peplus, which is an FDA-approved plant for the treatment of actinic keratosis. Some reports have demonstrated that the IM gel 0.05% is safe and effective in the treatment of external anogenital warts (AGWs) but the efficacy of the drug on the recalcitrant AGWs is not clear. This article assesses the efficacy and safety of the IM gel 0.05% for cryotherapy -resistant AGWs. Totally, 15 cryotherapy-resistant patients with AGWs (including 8 men and 7 women) and a mean age of 34 years old (age range of 23-50 years old) were enrolled in this study. IM gel 0.05% was applied carefully on the AGWs every two weeks for a maximum of three cycles. The complete clearance rate and recurrence rate were assessed 1 week and 3 months after the last treatment, respectively. Safety was assessed by the occurrence of local skin reaction and the severity of pain was evaluated using the 10-point Visual Analogue Scale. Initially, the AGWs were cleared completely in 10 (66%) patients while 4 (40%) and all of (100%) the patients experienced a recurrence in the 3- and 12-months follow-ups, respectively. All the 15 patients experienced some degrees of pain and local adverse reactions. The mean score of the reported pain was equal to 5.87 ± 2.39. The use of IM gel 0.05% in the treatment of the difficult-to-treat cases of AGWs is associated with a high recurrence rate despite the initial rapid and effective clearance of the lesions. Also, the high level of local adverse reactions and severe pain are other prohibitive factors in the treatment of recalcitrant AGWs with the IM.

P1113TREATMENT OF ACUTE LITHIUM INTOXICATION WITH SUSTAINED LOW-EFFICIENCY DIALYSIS (SLED)

Abstract Background and Aims Lithium salts are widely used in psychiatry for prophylaxis and treatment of bipolar affective disorders. Given its narrow therapeutic range, drug overdosage is not infrequent, and lithium intoxication has potentially fatal neurologic and cardiac side effects. Owing to its low molecular weight (7 Da), relatively small volume of distribution, and its negligible protein binding, lithium can be efficiently removed by any extracorporeal modality of renal replacement therapy (RRT). Although conventional intermittent haemodialysis (IHD) is effective in promptly cleating lithium, postdialysis rebound is common. The use of sustained low-efficiency dialysis (SLED) in lithium intoxication seems to be a valid therapeutic strategy, but strong evidence is still lacking in the literature. We present here two cases of lithium intoxication, in one case acute (naïve patient) and in the other case acute-on-chronic (a deliberate overdose in a patient previously on lithium therapy), successfully treated with SLED. Method: Patient A - A 47 years-old woman without history of lithium consumption was admitted to the ER. after a voluntary acute lithium ingestion of 40 tablets of lithium carbonate (8.12 mEq lithium each). Notwithstanding treatment with intravenous crystalloids and gastric lavage, lithium concentration increased up to 4.18 mEq/l at about 24 h after admission. She developed mental status changes, oliguria, hypotension and bradycardia. Patient B – A 58 years-old man on lithium therapy for bipolar syndrome was admitted to ER after a voluntary acute lithium ingestion of an unknown quantity of lithium carbonate tablets. Lithium concentration, after an initial level of 0.4 mEq/L, increased up to 4.35 mEq/l about 15 h from admission, despite irst-line therapies with intravenous crystalloids and gastric lavage were promptly started. His mental status worsened, together with oliguria, bradycardia and hypotension. Results: Patient A - A 8-hour SLED session with regional citrate anticoagulation (RCA) was planned [blood flow rate (Qb) 200 mL/min; dialysis fluid rate (Qd) 300 mL/min; countercurrent flow direction]. Lithium serum levels decreased by 87% during treatment (0.55 mEq/L), and the patient fully awoke recovering a normal mental status within the first 4 h of treatment. SLED was completed safely within the prescribed time. After the end of treatment, lithium rebound was unremarkable and renal function completely recovered and the patients was discharged after 3 days. Patient B - A 12-hour SLED session with RCA was planned (Qb 200 mL/min; Qd 500 mL/min; countercurrent flow direction). Serum lithium levels decreased by 55% (2.10 mEq/L) during treatment, and the patient awoke, recovering a normal mental status. At the end of treatment, rebound of lithium concentration was observed at 4 hours from SLED end detected (from 2.1 to 2.5 mEq/L), with initial signs of mental deterioration. A second SLED (12 h) was performed, with the same operative parameters and, by the end of the second treatment, lithium concentration was 0.49 mEq/L. Afterward, mental status and renal function fully recovered and no rebound rebound was observed. The patient was discharged after 6 days. Conclusion SLED could represent a good therapeutic strategy to treat acute lithium intoxication, providing an initial rapid clearance with resolution of symptoms, and limiting major rebound.

Trash Your Agar Plates! Blood Stream Bacteria Are Now Quantified by in vivo Flow Cytometry.

© 2020 International Society for Advancement of Cytometry.

Resistance to Inotuzumab Ozogamicin in a B-ALL Patient with TET2 and DNMT3A Mutations and Myeloid Lineage Switch

Background: Immunologic therapies targeting lineage-specific antigens such as CD22 and CD19 can improve outcome in patients with B cell acute lymphoblastic leukemia (B-ALL). The presence of mixed lineage leukemia (MLL) gene rearrangements highlights a subset of B-ALL patients with increased risk of treatment failure after inotuzumab ozogamicin (INO), an anti-CD22 antibody-drug conjugate. In some MLL-rearranged cases, a switch of hematopoietic lineage from B-ALL to acute myeloid leukemia (AML), has been associated with resistance to lymphoid-targeted therapies. In AML, founder clones harboring mutations associated with clonal hematopoiesis of indeterminate potential (CHIP) can persist during remission as reservoirs of treatment resistance. We therefore hypothesized that B-ALL can arise in the setting of a founding CHIP clone and undergo lineage switch under the selection pressure of B cell restricted therapy.Case Description: A 65-year-old female presented with two weeks of easy bruising, fatigue, and 54% circulating blasts. Flow cytometry was consistent with B-ALL that was positive for CD19, CD22, and aberrant CD15. Cytogenetics confirmed the presence of a t(4;11) (q21;q23) MLL rearrangement. The patient received intensive induction with dose-adjusted Larson regimen and morphologic complete remission was confirmed on Day 36. No residual disease was detectable by multiparametric flow cytometry. During consolidation (Day 136), a relapse of CD19+ CD22+ B-ALL was observed in the blood and marrow and treatment with INO was initiated. Despite rapid initial clearance of circulating disease, peripheral blasts were again observed on Day 167. At this time, flow cytometry demonstrated myeloblasts (positive for CD13, CD33, and CD117, but negative for CD19 and CD22), with no evidence of the previous lymphoblast population. The AML was refractory to multiagent salvage chemotherapy.Results: We evaluated the lineage-specificity and clonal dynamics of somatic genetic alterations using targeted gene panel sequencing at 5 timepoints during the course of treatment, compared to results from cytogenetic analysis and flow cytometry. At the time of initial diagnostic evaluation, we identified somatic mutations in DNMT3A (p.Q696*) and TET2 (p.L1209P). Variant allele fractions (VAFs) were 44% and 48%, respectively, suggesting they were both present at high abundance in the leukemic clone.TET2 and DNMT3A mutations are most commonly found in individuals with myeloid neoplasms and CHIP and are not frequently observed in those with B-ALL. We therefore asked whether these mutations preceded the MLL rearrangement in the leukemia clonal hierarchy by comparing their abundance at diagnosis to their abundance during MRD-negative clinical remission. We observed persistence of the DNMT3A (VAF = 13% and 10%) and TET2 (VAF = 8% and 6%) mutations during remission (Day 39, Day 82), while the MLL rearrangement was undetectable at both remission timepoints.We next evaluated the clonal relationship between the myeloid relapse after INO treatment and the initial B-ALL. In the AML phase, we again observed DNMT3A and TET2 mutations at high VAFs (>40%), as well as a PTPN11 p.A72T mutation (VAF 15%) and tetrasomy 8 that were undetectable at prior timepoints. A new TP53 p.R249T mutation was detected at the time of clinical progression after salvage chemotherapy, despite clearance of the PTPN11 mutated subclone. Cytogenetic analysis at diagnosis, first relapse, and second relapse confirmed MLL rearrangement t(4;11)(q21;q23).Conclusion: These results provide molecular evidence that primordial founder clones may persist during clinical remissions in MLL-rearranged leukemia and enable lineage switching upon relapse. More broadly, this case suggests that somatic mutations found in CHIP and other potentially pre-neoplastic states could confer increased risk of clonal escape from lineage-restricted therapies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 4587: A human hybrid immuno-oncology construct targeting the TWEAK receptor Fn14 and containing the serine protease granzyme B

Abstract Targeting immune effector cells to tumors has recently emerged as a clinically-relevant approach for therapy. The underlying mechanism for immune effector killing involves T or B-cell-mediated delivery of the serine protease granzyme B to the target cells. The cell-surface receptor Fn14 for the TNF-related cytokine TWEAK has emerged as a potentially valuable target for cancer therapy because its expression is low in most normal tissues but significantly elevated in a variety of solid tumor types. The serine protease Granzyme B (GrB) is a highly cytotoxic component of human immune effector cells and induces multiple, intense pro-apoptotic signals when delivered to the cytoplasm of target cells. This protein has been well-studied, operating through multimodal pathways which are both caspase-dependent and caspase-independent. Our laboratory has designed series of new Fn14-targeted fusion constructs containing an engineered GrB payload. The GrB-Fc-IT4 construct is a completely human homodimer (~200kDa) containing an IgG Fc domain for prolonged serum half-life. This construct displays high-level protein production in an HEK293E expression system. It exhibits high affinity and selective cytotoxicity within the nanomolar range (IC50 ranged from 4 to 284 nM) when tested against a panel of 25 Fn14-positive human cancer cell lines. We are currently assessing a panel of 100 annotated NSCLC cell lines to try to define a molecular fingerprint related to cellular sensitivity or resistance to the GrB payload. Pharmacokinetic studies in mice revealed that GrB-Fc-IT4 exhibited a bi-exponential clearance from plasma with a rapid initial clearance (t ½α = 0.36 hours) followed by a prolonged terminal-phase plasma half-life (t 1/2β = 35 hours). Mice bearing MDA-MB-231 orthotopic tumor xenografts were intravenously treated with saline, or GrB-Fc-IT4 construct (QODX5). On average, tumors from saline-treated mice grew about 10-fold over 40 days. In contrast, tumors from mice treated with GrB-FC-IT4 did not grow during this period and remained the same size as at the onset of treatment. Moreover, 3/5 mice treated with GrB-Fc-IT4 showed complete tumor regression lasting beyond day 80 (end of the study). Overall, treatment with GrB-FC-IT4 was well-tolerated by mice (no loss of body weight) and resulted in significant anti-tumor efficacy. Toxicology and bio-distribution studies are ongoing and will be presented. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Ana Alvarez de Cienfuegos, Lawrence H. Cheung, Khalid A. A. Mohamedali, Jeffrey A. Winkles, Michael G. Rosenblum. A human hybrid immuno-oncology construct targeting the TWEAK receptor Fn14 and containing the serine protease granzyme B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4587. doi:10.1158/1538-7445.AM2017-4587

Predicting lung dosimetry of inhaled particleborne benzo[a]pyrene using physiologically based pharmacokinetic modeling

Benzo[a]pyrene (BaP) is a by-product of incomplete combustion of fossil fuels and plant/wood products, including tobacco. A physiologically based pharmacokinetic (PBPK) model for BaP for the rat was extended to simulate inhalation exposures to BaP in rats and humans including particle deposition and dissolution of absorbed BaP and renal elimination of 3-hydroxy benzo[a]pyrene (3-OH BaP) in humans. The clearance of particle-associated BaP from lung based on existing data in rats and dogs suggest that the process is bi-phasic. An initial rapid clearance was represented by BaP released from particles followed by a slower first-order clearance that follows particle kinetics. Parameter values for BaP-particle dissociation were estimated using inhalation data from isolated/ventilated/perfused rat lungs and optimized in the extended inhalation model using available rat data. Simulations of acute inhalation exposures in rats identified specific data needs including systemic elimination of BaP metabolites, diffusion-limited transfer rates of BaP from lung tissue to blood and the quantitative role of macrophage-mediated and ciliated clearance mechanisms. The updated BaP model provides very good prediction of the urinary 3-OH BaP concentrations and the relative difference between measured 3-OH BaP in nonsmokers versus smokers. This PBPK model for inhaled BaP is a preliminary tool for quantifying lung BaP dosimetry in rat and humans and was used to prioritize data needs that would provide significant model refinement and robust internal dosimetry capabilities.

Abstract 575: Human serine protease therapeutics- potent constructs targeting Fn14 in multiple tumor types

Abstract The cytokine TWEAK, a TNF superfamily member, and its cognate receptor, Fn14, have emerged as potentially valuable targets for cancer therapy since their expression has been frequently identified as independent negative prognostic indicators in a variety of tumor types. The serine protease Granzyme B (GrB) is a highly cytotoxic component of human immune effector cells and induces multiple, intense pro-apoptotic signals when delivered to the cytoplasm of target cells. This human protein has been well-studied, operating through multimodal pathways which are both caspase-dependent and caspase-independent. Our laboratory has designed series of new Fn14 targeted fusion constructs containing an engineered GrB payload. The GrB-Fc-IT4 containing either WTCYS or EACYS mutants of GrB, and a human scFv targeting Fn14 were expressed in HEK293E and suspension CHO cells, harvested under serum-free conditions and purified to homogeneity. Both constructs display high-level protein production (>50 mg/L), enhanced serum stability and impressive cytotoxic activity (IC50 < 20 nM) against Fn14 positive cells. The specific activity of the GrB moiety, as assessed by cleavage of a synthetic chromogenic GrB substrate, was comparable to that of human GrB for the WTCYS while the EACYS variant showed a 50% reduction in specific activity. On the other hand, in vitro stability studies showed that EACYS was more stable after 48h incubation in human serum, retaining over 60% of its initial specific activity. Against a panel of 20 human cell lines expressing Fn14, both constructs showed high affinity and selective cytotoxicity within the nanomolar range (IC50 ranged from 4 to 284 nM) and were two to over a hundred times more potent than free GrB. Mechanistic studies demonstrated that GrB-Fc-IT4 constructs activated caspase cascades and cytochrome C related pro-apoptotic mechanisms in keeping with the known intracellular functions of GrB in target cells. Pharmacokinetic studies in mice revealed that GrB-Fc-IT4 fusion proteins exhibited a bi-exponential clearance from plasma with a rapid initial clearance (t ½ α = 0.36 hours) followed by a prolonged terminal-phase plasma half-life (t 1/2 β = 35 hours). Toxicity studies in mice demonstrated that the MTD is above 100 mg/kg total dose. Murine orthotropic tumor model efficacy and bio-distribution studies are ongoing and will be presented. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Yunli Zhao, Kathryn E. Ruisaard, Jeffrey A. Winkles, Michael G. Rosenblum. Human serine protease therapeutics- potent constructs targeting Fn14 in multiple tumor types. [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 575.

Chronic Myeloid Leukemia (CML): BCR-ABL Transcript Levels Halving Time and Relative Reduction Ratio at Months 3 As New Predictors of Outcome. an Argentine Pilot Study

▪BCR-ABL transcript levels (IS%BCR-ABL) in CML patients (pts) allow to define molecular response (MR) to TKIs. MR at 3 months (M3>10%) is the strongest predictor of bad outcome and a point of divergence for changing therapy. Emerging biomarkers are being proposed in order to optimize prognosis in addition to initial MR and other clinical markers. The rate of BCR-ABL decline measured as halving time (HT) for transcript level reduction (RT0.5) at month 3 in 1GTKI (imatinib) treated pts, and the fractional clearance as a relative BCR-ABL ratio (RR3) during the first 3 months in 2GTKI (dasatinib) treated pts were recently proposed as predictive markers of outcome. Optimal responses (OR) were defined upon %ISBCR-ABL values at 3, 6 and 12 months: M3 ≤10%, M6 ≤1% y M12 ≤0,1%. Early time point prognostication could allow better outcome predictions and rapid therapeutic interventions.Aim: to determine whether the rate of BCR-ABL decline as HT and the BCR-ABL transcripts reduction as relative ratio at M3 from baseline, have predictive value in CML pts treated with 2GTKIs.Method: BCR-ABL transcript level was measured by Q-PCR according international recommendations and results were reported as BCR-ABL/ABL on the international scale (IS BCR-ABL %). Molecular monitoring was performed prior treatment (M0) and at months 3 (M3), 6 (M6), 12 (M12) and every 6 months thereafter. RT0.5 from baseline to M3 was calculated as the time to halve by considering an exponential model of reduction during the period. The median RT0.5 and quartiles, were calculated to define rapid declines as those HT in the lowest quartile. RR3 was determined as a relative ratio of transcript clearance at M3 from baseline. The median RR3and quartiles, were considered to define high BCR-ABL clearance as those in the highest quartile. Fisher statistical test was used to determine whether the proportions of good responses were statistically different depending on the decline rate.Results: A total of 77 CML pts were included, first line treatment: Dasatinib (D) 28%, Nilotinib (N) 32% and Imatinib (I) 40%. Sokal risk score was available in 70% pts: low (L) 67%, intermediate (In) 22% and high (H) 11%. Molecular assessment: prior therapy M0 (90%), M3 (60%), M6 (79%), M12 (78%) and M0+M3+M12(39%). Deep responses (MR4.0) were considered as IS%BCR-ABL<0,01 or no detectable transcripts in ≥10000 ABL copies. OR according to first line TKI (I vs D+N), were: M3≤10%: 75% vs 90% and M12 ≤0,1%: 50% vs 65%. RT0.5 median values for I vs D+N groups were 32d vs 16d and RR3 median values were 79% vs 98%. RT0.5 ≤13d and RR3 ≥99% were cut off values for fast responses and high clearance in pts treated with 2GTKI(83%). Milestone molecular result M3≤10% showed 20% pts with M12>0,1% and without MR4.0. All of these pts showed RT0.5 >13d and RR3 <99%. From total pts with OR, 80% pts achieved MR4.0 and all showed RR3≥99% meanwhile the remaining 20% pts who never achieved MR4.0 had RR3<99%. Higher frequencies of OR were observed in low Sokal risk pts, in RT0.5 ≤13d pts and in pts with RR3 ≥99%. High Sokal risk pts had poorer results at milestones molecular responses but those with rapid initial decline (RT0.5 ≤13d) and high BCR-ABL clearance (RR3 ≥99%) achieved molecular responses similar to those observed in the low risk sokal group.Conclusions: Faster BCR-ABL decline and high clearance were related to OR and MR4.0 achievement in 2GTKIs patients. No direct relation between Sokal Index and M12≤ 0,1% was observed. Patients with RT0.5 ≤13d showed higher frequencies of good responses. Statistical differences were not found probably due to low sample numbers. Our data suggests that RT0.5 in 2GTKIs (N+D) patients could have a similar predictive value as reported by Branford in 2014, even though it is known that BCR-ABL measurements tend to be less satisfactory when control gene is ABL, owing to possible non-linear ratios. Whilst there were no statistical correlations probably due to the low sample number, pts with OR but no MR4.0 had low reduction rate RR3<99%. RR3>99% could provide a stronger predictive value for milestone optimal responses achievement in high risk pts. It will be necessary to extend this study including a higher number of patients treated with 2G TKIs. [Display omitted] DisclosuresVarela:Bristol Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau. Moiraghi:Novartis: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau. Pavlovsky:Novartis: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau. Pavlovsky:Bristol Myers Squibb: Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau.

Surface modification of zinc oxide nanoparticles with amorphous silica alters their fate in the circulation

Nanoparticle (NP) pharmacokinetics and biological effects are influenced by many factors, especially surface physicochemical properties. We assessed the effects of an amorphous silica coating on the fate of zinc after intravenous (IV) injection of neutron activated uncoated 65ZnO or silica-coated 65ZnO NPs in male Wistar Han rats. Groups of IV-injected rats were sequentially euthanized, and 18 tissues were collected and analyzed for 65Zn radioactivity. The protein coronas on each ZnO NP after incubation in rat plasma were analyzed by SDS-PAGE gel electrophoresis and mass spectrometry of selected gel bands. Plasma clearance for both NPs was biphasic with rapid initial and slower terminal clearance rates. Half-lives of plasma clearance of silica-coated 65ZnO were shorter (initial – <1 min; terminal – 2.5 min) than uncoated 65ZnO (initial – 1.9 min; terminal – 38 min). Interestingly, the silica-coated 65ZnO group had higher 65Zn associated with red blood cells and higher initial uptake in the liver. The 65Zn concentrations in all the other tissues were significantly lower in the silica-coated than uncoated groups. We also found that the protein corona formed on silica-coated ZnO NPs had higher amounts of plasma proteins, particularly albumin, transferrin, A1 inhibitor 3, α-2-hs-glycoprotein, apoprotein E and α-1 antitrypsin. Surface modification with amorphous silica alters the protein corona, agglomerate size, and zeta potential of ZnO NPs, which in turn influences ZnO biokinetic behavior in the circulation. This emphasizes the critical role of the protein corona in the biokinetics, toxicology and nanomedical applications of NPs.

A platform technique for growth factor delivery with novel mode of action

Though growth factors allow tissue regeneration, the trade-off between their effectiveness and adverse effects limits clinical application. The key issues in current growth factor therapy largely derive from initial burst pharmacokinetics, rapid clearance, and proteolytic cleavage resulting in clinical ineffectiveness and diverse complications. While a number of studies have focused on the development of carriers, issues arising from soluble growth factor remain. In this study, we report a prodrug of growth factors constituting a novel mode of action (MoA). To mimic endogenous protein processing in cells, we developed a recombinant BMP-2 polypeptide based on a protein transduction domain (PTD) to transduce the protein into cells followed by furin-mediated protein cleavage and secretion of active growth factor. As proof of concept, a few micrograms scale of PTD-BMP-2 polypeptide sufficed to induce bone regeneration in vivo. As a simple platform, our technique can easily be extended to delivery of BMP-7 and DKK-1 as therapeutics for TGF-β and canonical Wnt signaling, respectively, to suppress the epithelial–mesenchymal transition (EMT), which constitutes a fundamental biological mechanism of many diseases. This technique largely overcomes the limitations of current soluble growth factors and opens the door to next generation growth factor therapeutics.

Homoisoflavonoids as potential imaging agents for β-amyloid plaques in Alzheimer's disease

A series of homoisoflavonoids [(E)-3-benzylidenechroman-4-ones, 3a–l] as novel potential diagnostic imaging agents targeting β-amyloid (Aβ) plaques in Alzheimer's disease (AD) were synthesized and evaluated. In vitro binding studies using Aβ1–40 aggregates with [125I]IMPY as the reference ligand showed that these compounds demonstrated high to low binding affinities at the Ki values ranged from 9.10 to 432.03 nM, depending on the substitution of the phenyl ring. Fluorescent staining in vitro indicated that one compound with a N,N-dimethylamino group intensely stained Aβ plaques within brain sections of postmortem AD patients. Biodistribution studies in normal mice after i.v. injection of the radioiodinated homoisoflavonoid displayed good initial brain uptake (2.61% ID/g at 2 min postinjection) and rapid clearance from the brain (0.18% ID/g at 60 min), which is desirable for amyloid imaging agents. The results strongly suggest that these derivatives are worthy of further study and may be useful amyloid imaging agents for early detection of amyloid plaques in the brain of AD.

Antitumor Activity of a Humanized, Bivalent Immunotoxin Targeting Fn14-Positive Solid Tumors

The TNF-like weak inducer of apoptosis (TWEAK; TNFSF12) receptor Fn14 (TNFRSF12A) is expressed at low levels in normal tissues but frequently highly expressed in a wide range of tumor types such as lung, melanoma, and breast, and therefore it is a potentially unique therapeutic target for these diverse tumor types. We have generated a recombinant protein containing a humanized, dimeric single-chain anti-fibroblast growth factor-inducible 14-kDa protein (Fn14) antibody fused to recombinant gelonin toxin as a potential therapeutic agent (designated hSGZ). The hSGZ immunotoxin is a highly potent and selective agent that kills Fn14-positive (Fn14(+)) tumor cells in vitro. Treatment of cells expressing the MDR protein MDR1 (ABCB1B) showed no cross-resistance to hSGZ. Induced overexpression of Fn14 levels in MCF7 cells through HER2 (ERBB2) signaling translated to an improved therapeutic index of hSGZ treatment. In combination with trastuzumab, hSGZ showed an additive or synergistic cytotoxic effect on HER2(+)/Fn14(+) breast cancer cell lines. Also, hSGZ treatment inhibited Erb3/Akt signaling in HER2-overexpressing breast cancer cells. Pharmacokinetic studies in mice revealed that hSGZ exhibited a biexponential clearance from plasma with a rapid initial clearance (t1/2α = 1.26 hours) followed by a seven-fold longer plasma half-life (t1/2β = 7.29 hours). At 24, 48, and 72 hours after injection, uptake of the hSGZ into tumors was 5.1, 4.8, and 4.7%ID/g, with a tumor-to-muscle ratio of 5.6, 6.2, and 9.0, respectively. Therapeutic efficacy studies showed significant tumor inhibition effects using an MDA-MB-231/Luc breast cancer xenograft model. Our findings show that hSGZ is an effective anticancer agent and a potential candidate for clinical studies.

Uptake, retention and internalization of quantum dots in Daphnia is influenced by particle surface functionalization

Nanomaterials are a diverse group of compounds whose inevitable release into the environment warrants study of the fundamental processes that govern the ingestion, uptake and accumulation in aquatic organisms. Nanomaterials have the ability to transfer to higher trophic levels in aquatic ecosystems, and recent evidence suggests that the surface chemistry of both the nanoparticle and biological membrane can influence uptake kinetics. Therefore, our study investigates the effect of surface functionalization on uptake, internalization and depuration in Daphnia spp. Uncharged (polyethylene glycol; PEG), positively charged (amino-terminated: NH2) and negatively charged (carboxyl-modified; COOH) cadmium selenide/zinc sulfide quantum dots were used to monitor ingestion, uptake and depuration of nanometals in Daphnia magna and Ceriodaphnia dubia over 24h of exposure. These studies demonstrated that particles with higher negative charge (COOH quantum dots) were taken up to a greater extent by Daphnia (259.17±17.70 RFU/20 Daphnia) than either the NH2 (150.01±18.91) or PEG quantum dots (95.17±9.78), however this is likely related to the functional groups attached to the nanoparticles as there were no real differences in zeta potential. Whole body fluorescence associates well with fluorescent microscopic images obtained at the 24h timepoint. Confocal and electron microscopic analysis clearly demonstrated that all three types of quantum dots could cross the intestinal epithelial barrier and be translocated to other cells. Upon cessation of exposure, elimination of all three materials was biphasic with rapid initial clearance that likely represents elimination of material remaining in the GI tract followed by a much slower elimination phase that likely represents elimination of internalized material. These studies demonstrate that daphnids can take up intact nanomaterial from the water column and that this uptake is strongly influenced by particle surface functionalization. In addition, the usefulness of using quantum dots as a proxy for other nanometals (no acute toxicity, clear visualization in electron microscopy), in conjunction with several different imaging techniques in assessing uptake and accumulation of nanoparticles in daphnids was demonstrated.

Washout allometric reference method (WARM) for parametric analysis of [11C]PIB in human brains

Rapid clearance and disappearance of a tracer from the circulation challenges the determination of the tracer's binding potentials in brain (BPND) by positron emission tomography (PET). This is the case for the analysis of the binding of radiolabeled [11C]Pittsburgh Compound B ([11C]PIB) to amyloid-β (Aβ) plaques in brain of patients with Alzheimer's disease (AD). To resolve the issue of rapid clearance from the circulation, we here introduce the flow-independent Washout Allometric Reference Method (WARM) for the analysis of washout and binding of [11C]PIB in two groups of human subjects, healthy aged control subjects (HC), and patients suffering from AD, and we compare the results to the outcome of two conventional analysis methods. We also use the rapid initial clearance to obtain a surrogate measure of the rate of cerebral blood flow (CBF), as well as a method of identifying a suitable reference region directly from the [11C]PIB signal. The difference of average absolute CBF values between the AD and HC groups was highly significant (P < 0.003). The CBF measures were not significantly different between the groups when normalized to cerebellar gray matter flow. Thus, when flow differences confound conventional measures of [11C]PIB binding, the separate estimates of CBF and BPND provide additional information about possible AD. The results demonstrate the importance of data-driven estimation of CBF and BPND, as well as reference region detection from the [11C]PIB signal. We conclude that the WARM method yields stable measures of BPND with relative ease, using only integration for noise reduction and no model regression. The method accounts for relative flow differences in the brain tissue and yields a calibrated measure of absolute CBF directly from the [11C]PIB signal. Compared to conventional methods, WARM optimizes the Aβ plaque load discrimination between patients with AD and healthy controls (P = 0.009).

Novel Indanone Derivatives as Potential Imaging Probes for β‐Amyloid Plaques in the Brain

Molecular imaging probes to detect senile plaques (SPs) might help the early diagnosis of Alzheimer's disease (AD). In this study, a novel series of indanone derivatives were synthesized and characterized. In in vitro binding studies, compound 2e exhibited a K(i) value of 16 nM with a human AD brain homogenate. Although they displayed relatively low affinities for 2i and 2j--with K(i) values of 99 and 237 nM, respectively--the SPs in AD brain sections were positively stained by 2j. A method for in situ micro-autoradiography of AD brain was developed in this study and showed clear labeling of SPs by [(125)I]2i and [(125)I]2j. Both [(125)I]2i and [(125)I]2j had suitable lipophilicities and displayed high initial uptake and rapid clearance from the mouse brains. Furthermore, [(125)I]2i and [(125)I]2j were more stable in human brain homogenates than in mouse brain homogenates. These data suggest that such indanone derivatives might represent potential amyloid imaging agents for the detection of SPs in AD.