Evaluation Of CYP450 and Transporter-Mediated Drug-Drug Interaction Potential Of Pomalidomide In Healthy Men and In Vitro

Abstract Introduction Pomalidomide (POM) is an IMiD® immunomodulatory agent recently approved by the FDA for the treatment of patients with relapsed/refractory multiple myeloma. The prevalence of polypharmacy in disease treatment raises the possibilities of drug-drug interactions. Therapies that are CYP450 inhibitors, inducers, or transporter inhibitors, may alter the clinical exposure of substrates of these enzymes and transporters when they are administered concomitantly. POM is known to be predominantly metabolized via CYP450-mediated hydroxylation.1 The aim was to investigate the inhibition/induction potential of POM and its sensitivity as a CYP450 substrate in vitro, and to evaluate the impact of strong CYP450 inhibitors/inducers and a P-glycoprotein (P-gp) inhibitor on the pharmacokinetics (PK) of POM in healthy male subjects. Methods In vitro analyses were conducted using standard published methodologies.2-7 The in vitro data were followed up with a phase 1, open-label, non-randomized study in healthy men with single doses (SD) of POM. The study comprised a screening phase, 2 parallel treatment groups and a safety follow-up. Subjects in Part 1 received POM (4 mg SD) alone; ketoconazole (KETO 200 mg twice daily [BID] for 7 days) + POM (4 mg SD on day 5); and KETO (200 mg BID) + fluvoxamine (FLUV 50 mg BID) for 7 days + POM (4 mg SD on day 5) in three sequential periods to evaluate the effect of CYP3A4 and P-gp (KETO) and CYP1A2 (FLUV) inhibition on POM PK. Subjects in Part 2 received POM (4 mg SD) alone followed by carbamazepine (CARB 200 mg BID) for 11 days + POM (4 mg SD on day 10) in two sequential periods to evaluate the effect of CYP3A4 induction on POM PK. Serial blood samples were collected to determine the plasma concentrations of POM, FLUV, KETO, CARB and CARB-10, 11-epoxide by validated LC-MS/MS assay. PK and safety data were summarized using descriptive statistics. An analysis of variance model was performed on natural log transformed PK data, and used to estimate the ratio of geometric means between POM and the other drugs with treatments as fixed effects and subjects as random effect. Results In vitro, POM did not inhibit or induce CYP450 enzymes, nor the transporters P-gp, BCRP, OAT1, OAT2, OAT3, OATP1B1, or OATP1B3. The in vitro oxidative metabolism of POM was predominately mediated by CYP3A4 and CYP1A2. In the clinical trial, all 32 enrolled subjects (age 20–54 years; BMI 19.3–30.3 kg/m2) were included in the PK and safety analyses. Statistical comparison showed that the mean exposure of POM administered after multiple doses of KETO was increased by ∼19% (AUC0-inf) and 7% (Cmax) compared to exposure of POM alone (Table). Addition of FLUV to the POM + KETO regimen increased mean exposure of POM by ∼107% (AUC0-inf) and 13% (Cmax) compared to POM + KETO. Overall, co-administration of POM + KETO + FLUV increased mean exposure of POM by ∼146% (AUC0-inf) and 21% (Cmax) compared to POM alone. The mean exposure of POM when administered after multiple doses of CARB decreased by ∼20% (AUC0-inf) and 25% (Cmax) compared to exposure of POM alone. The mean estimated terminal half-life of POM was similar with or without KETO or CARB (range 5.85 to 6.77 h), but increased in the presence of KETO + FLUV (12.37 h), suggesting that the predominant interaction may be on the clearance (CL/F) rather than the absorption phase of POM. POM CL/F was decreased by ∼16% in the presence of KETO (6.96 L/h), and decreased by ∼56% in the presence of KETO + FLUV (3.64 L/h), compared to POM alone (8.27 L/h). In contrast, POM + CARB increased POM CL/F compared to POM alone (9.49 versus 7.56 L/h). POM 4 mg SD was generally well tolerated either alone or with the other drugs. The most common POM-related adverse event (AE) was nausea (6.3% of subjects). No deaths or serious AEs were reported. Conclusions POM appears to have a low potential for drug-drug interactions. POM is not a CYP450 inhibitor, inducer, or transporter inhibitor, in vitro and is therefore unlikely to affect the exposure of other drugs clinically. Co-administration of POM with the strong CYP3A4/5 and P-gp inhibitor KETO, or the strong CYP3A4/5 inducer CARB, had no clinically relevant effect on exposure to POM. Co-administration of POM with a strong CYP1A2 inhibitor (FLUV) will likely increase systemic exposure to POM and subjects receiving these concomitantly should be closely monitored for the occurrence of side-effects. POM 4 mg was generally well tolerated. Disclosures: Kasserra: Celgene: Employment, Equity Ownership. Assaf:Celgene Corporation: Employment, Equity Ownership. Hoffmann:Celgene Corporation: Employment, Equity Ownership. Li:Celgene Corporation: Employment, Equity Ownership. Liu:Celgene Corporation: Employment, Equity Ownership. Wang:Celgene Corporation: Employment, Equity Ownership. Kumar:Celgene Corporation: Employment, Equity Ownership. Palmisano:Celgene Corporation: Employment, Equity Ownership.

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Comprehensive Analysis Of The In Vitro Potency Of Ponatinib, and All Other Approved BCR-ABL Tyrosine Kinase Inhibitors (TKIs), Against a Panel Of Single and Compound BCR-ABL Mutants

Blood ◽

10.1182/blood.v122.21.3992.3992 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3992-3992 ◽

Cited By ~ 8

Author(s):

Joseph M Gozgit ◽

Alexa Schrock ◽

Tzu-Hsiu Chen ◽

Tim Clackson ◽

Victor M. Rivera

Keyword(s):

Steady State ◽

Plasma Concentration ◽

Effective Concentration ◽

The Other ◽

Single Mutation ◽

Employment Equity ◽

Clinical Resistance ◽

Equity Ownership ◽

Effective Plasma Concentration

Abstract Background Secondary mutations in BCR-ABL are the most common cause of resistance to TKIs in patients (pts) with chronic myeloid leukemia (CML). Ponatinib is a potent pan-BCR-ABL TKI that has been shown to suppress the emergence of any single mutation in vitro, including T315I, at clinically achievable concentrations (40 nM), though higher concentrations were required to suppress emergence of certain compound mutations (2 mutations in the same BCR-ABL allele). Ponatinib has demonstrated significant clinical activity in pts in the phase 2 PACE trial, 60% of whom received 3 or more prior TKIs. Responses were observed for each of the 15 mutations present in >1 chronic phase CML pt at baseline, and no single mutation conferring resistance to ponatinib has emerged to date, though in some cases development of compound mutations has been observed. To gain a more precise understanding of the effects of specific mutations on the clinical efficacy of ponatinib, IC50s for ponatinib, and all other approved TKIs, against 31 single or compound BCR-ABL mutants were determined. To explore the relationship between in vitro potency and clinical efficacy, IC50s were related to “effective” TKI levels achieved in patients. Methods TKI potency was assessed in engineered Ba/F3 cells by measuring cell viability at 72 hours. The effective plasma concentration for each TKI was calculated from published average steady-state concentration values for the recommended dose, and adjusted for the functional effects of protein binding. These effects were assessed by determining the degree to which TKI potency was reduced by the presence of physiological concentrations of human serum albumin (HSA) and alpha 1-acid glycoprotein (AAG). Results The activity of ponatinib, and 5 other TKIs, against 21 single BCR-ABL mutants is shown in Figure A. Ponatinib potently inhibited viability of native BCR-ABL and all mutants, including T315I (IC50s (nM): 3-16). The IC50s for the other TKIs, excluding T315I (>4000 for all) ranged from: 201-10,000 (imatinib), 12-784 (nilotinib), 2-104 (dasatinib), 40-1,280 (bosutinib) and 18-5,216 (radotinib). IC50 values were compared to the effective plasma concentration for each TKI (Figure A). Mutants that have previously been associated with clinical resistance to a particular TKI tended to have IC50s that approached or substantially exceeded the effective concentration for that TKI, including most mutants for imatinib, E255K/V, Y253H, L248R, T315I for nilotinib, and V299L, F317C/I/V, T315A/I for dasatinib. The most problematic mutants for bosutinib predicted by this analysis were F317V, L248R, V299L, and T315I. Notably, all mutant IC50s fell below the effective concentration for ponatinib. The activity of all TKIs against 10 clinically-observed BCR-ABL compound mutants was also assessed. Four compound mutants had IC50s near or above the effective concentration for ponatinib (T315I+M351T, E255V+F317I, T315I+E255K, T315I+E255V) (Figure B). All 4, plus others, are also predicted to be problematic for the other TKIs. Conclusions Relating in vitro TKI potency to “effective” steady-state plasma concentrations in patients identified mutations known to confer clinical resistance to imatinib, nilotinib and dasatinib. This method of analysis suggests that ponatinib may be able to inhibit all single BCR-ABL mutants, but not all compound mutants, a prediction that is thus far consistent with results observed in patients. Compound mutations that are predicted to confer resistance to ponatinib are also predicted to confer resistance to all other approved TKIs. Early introduction of ponatinib may prevent the emergence of single mutations, and thus the sequential development of compound mutations. Disclosures: Gozgit: ARIAD: Employment, Equity Ownership. Schrock:ARIAD: Employment, Equity Ownership. Chen:ARIAD: Employment, Equity Ownership. Clackson:ARIAD: employees of and own stock/stock options in ARIAD Pharmaceuticals, Inc Other, Employment. Rivera:ARIAD: Employment.

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Evaluation Of Drug Interaction Profile Of Idelalisib and Its Major Circulating Metabolite, GS-563117, In Healthy Subjects

Blood ◽

10.1182/blood.v122.21.5574.5574 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5574-5574

Author(s):

Feng Jin ◽

Michelle Robeson ◽

Huafeng Zhou ◽

Candra Moyer ◽

Sibylle Wilbert ◽

...

Keyword(s):

Single Dose ◽

Vital Signs ◽

Therapeutic Index ◽

Major Metabolite ◽

Employment Equity ◽

Metabolizing Enzymes ◽

Relevant Concentration ◽

Multiple Doses ◽

Equity Ownership

Abstract Abstract 5574 Background Idelalisib (IDELA) is a potent PI3K inhibitor in Phase 3 development for hematologic malignancies. IDELA is metabolized primarily by aldehyde oxidase to form GS-563117 and to a lesser extent by CYP3A and UGT1A4. In vitro, IDELA inhibits Pgp (IC50: 7.7 mM), OATP1B1 (IC50: 10.1 mM), OATP1B3 (IC50: 7.0 mM), but is not an inhibitor of common metabolizing enzymes and other uptake or secretory transporters at clinically relevant concentration. GS563117 shows time-dependent inhibition of CYP3A (IC50: 5.1 mM, KI: 0.18 mM, and kinact: 0.033 min-1), but is not an inhibitor of other common metabolizing enzymes, or of uptake or secretory transporters at clinically relevant concentration. The present study evaluated the potential for IDELA to affect Pgp and OATP1B1/OATP1B3, GS-563117 to affect CYP3A, and effect of a strong inducer, rifampin, on IDELA pharmacokinetics (PK). Methods Probe substrates of Pgp (digoxin), OATP1B1/1B3 (rosuvastatin), and CYP3A (midazolam) were each given orally as a single dose either alone or in combination with multiple doses of IDELA 150 mg BID. Additionally, a single dose of IDELA 150 mg was administered either alone or in combination with multiple doses of rifampin at 600 mg QD. Plasma exposures of digoxin, rosuvastatin, midazolam and 1'-hydroxy midazolam, rifampin, IDELA, and GS-563117 were determined using LC/MS/MS. Analysis of variance (ANOVA) using a mixed-effects model was fitted to the natural logarithmic transformation of PK parameters. The 90% confidence intervals were constructed for the ratio of geometric means of PK parameters when each of the probe drugs (or IDELA) is dosed in combination with IDELA (or rifampin) versus when dosed alone, with 70-143% defined as the lack of interaction boundaries. Safety assessments were performed throughout the study. Results A total of 24 subjects were enrolled in the study and randomized to two cohorts. The majority of subjects were male, white, and median age was 38. The most frequently reported adverse events (AE) were headache (∼25% subjects) and pyrexia (∼17%). Treatment-emergent Grade 3 increase in transaminases occurred in 5/24 subjects, and were reversible. Two subjects experienced serious AE following completion of study treatment. There were no clinically significant changes in vital signs or safety ECGs. Digoxin and rosuvastatin PK were unaffected when given in combination with IDELA 150 mg BID vs dosing alone (Table 1). Coadministration with IDELA resulted in increased plasma exposures of midazolam increased and decreased 1'-OH-midazolam consistent with the in vitro finding of CYP3A inhibition by IDELA's major metabolite, GS-563117. Coadministration of IDELA with rifampin caused a substantial decrease in IDELA and GS-563117 exposures, indicating greater contribution of CYP3A to IDELA metabolism under a strongly induced state. Conclusion GS-563117, which is the major metabolite of IDELA, is a moderate inhibitor of CYP3A; accordingly, caution is necessary when coadministering narrow therapeutic index agents that are CYP3A substrates with IDELA. Coadministration of strong inducers of CYP3A with IDELA should also be avoided to prevent decreased exposure to IDELA. Overall, IDELA and its metabolite do not affect common intestinal, hepatic or renal drug transporters. Disclosures: Jin: Gilead Sciences: Employment, Equity Ownership. Robeson:Gilead Sciences: Employment, Equity Ownership. Zhou:Gilead Sciences: Employment, Equity Ownership. Moyer:Gilead Sciences: Employment, Equity Ownership. Wilbert:Gilead Sciences: Employment, Equity Ownership. Murray:Gilead Sciences: Employment, Equity Ownership. Ramanathan:Gilead Sciences: Employment, Equity Ownership.

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Aurora Kinase Is a Therapeutic Target in Ibrutinib-Resistant Waldenstrom Macroglobulinemia: In-Silico Target Identification and in-Vitro Validation

Blood ◽

10.1182/blood.v126.23.2754.2754 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2754-2754

Author(s):

Aneel Paulus ◽

Kasyapa S. Chitta ◽

Sharoon Akhtar ◽

Hassan Yousaf ◽

Davitte Cogen ◽

...

Keyword(s):

In Silico ◽

Copy Number ◽

Aurora Kinase ◽

Gene Copy ◽

Molecular Architecture ◽

Employment Equity ◽

In Silico Modeling ◽

Equity Ownership ◽

The Impact

Abstract Background: Current tumor profiling analytics provide some insight into the various molecular abnormalities and their individual consequences on oncogenic signaling. However, these analyses are limited by their lack of integration where the combined effect of individual mutations, gene copy number variations and chromosomal aberrations are not consolidated to create the global molecular architecture that supports neoplastic growth, particularly in the context of drug resistance. Consequentially, identities of the preferential oncogenic pathway(s) tumor cells employ to oppose the effects of targeted therapies remain cryptic and unactionable. Here we present a simulation-based method, which not only replicates the molecular architecture of ibrutinib-resistant Waldenstroms Macroglobulinemia (WM, for which ibrutinib is the only FDA-approved agent) in silico, but also predicts cell sensitivity towards existing drugs, which we validated experimentally for potential clinical translation. Materials: We used the newly established human WM cell line, RPCI-WM1/IR, as a surrogate model of ibrutinib-refractory WM. Genomic data including whole exome sequencing (WES) and copy number analysis (CNA) was utilized for the creation of an avatar of RPCI-WM1/IR, which through simulation identified the salient and prominently dysregulated cellular pathways. Importantly, illustrating these pathways highlights common convergence points on increased proliferation and viability. These convergence points were then directly and indirectly targeted by simulated testing of a library of FDA approved drugs and those impacting these dysregulated pathways were nominated. Importantly, this simulation avatar approach not only looks for agents acting on the specific gene mutation, but also predicts the convergence points to be attacked. The personalized simulation avatar technology is a comprehensive functional proteomics representation of the WM physiology network. A standardized library of equations models all the biological reactions such as enzymatic reactions, allosteric binding and protein modulation by phosphorylation, de-phosphorylation, ubiquitination, acetylation, prenylation and others. Results: Several genomic aberrations were used to create the RPCI-WM1/IR simulation avatar. Functional activity (based on mutation or copy number alteration) of several ibrutinib targets or transcription factors associated with BTK activity such as FYN, SP1, BMX and FRK were predicted to be lost. Increased expression of CAV1, which also inhibits BTK mediated signaling, was increased. An increase in CSNK2B, which activates PU.1- a transcriptional target of BTK, was also observed. Of note, no CXCR4 mutations, which have been shown to impact ibrutinib response, were observed. Next, the cytotoxic potential of over 150 FDA approved drug (and some in experimental stages) were simulated individually and in combination on the RPCI-WM1/IR avatar. In silico modeling predicted aberrant activity of aurora kinase A (AURKA) and its associated signaling partners, which could be disrupted with the (AURKA) inhibitor, tozasertib. AURKA activation was predicted as upregulated due to alterations in several genes: RASA1 loss and SOS1 increase --> increased ERK --> increased ETS1 --> increased AURKA. High beta-catenin signaling (high CTNNB1 and FZD1/4 and low AXIN1 and GSK3B) was also shown to increase AURKA. The simulation predictions were experimentally validated in vitro where AURKA inhibition with tozasertib significantly inhibited proliferation of RPCI-WM1/IR cells (IC50~14nM) as well as inducing apoptosis (48hr, 20nM treatment) and cell-cycle arrest. Conclusions: Our data demonstrates the potential of in silico modeling in predicting novel drug targets, allowing guidance in 1.) Delineating operational oncogenic circuits in an ibrutinib-resistant state by reanimation of the molecular architecture in silico, 2.) Calculating the impact of individual genomic abnormalities and their collective influence on maintaining tumor survival and 3.) Performing a rapid in-silico drug-sensitivity screen directed by the pathway analyses, which can be validated experimentally using standard assays. This novel approach holds tremendous potential in creating highly personalized therapies for ibrutinib-refractory WM patients based on unique genetic signatures. Disclosures Vali: Cellworks Group, Inc.: Employment, Equity Ownership. Kumar:Cellworks Group, Inc.: Employment. Singh:Cellworks Group, Inc.: Employment. Abbasi:Cellworks Group, Inc.: Employment, Equity Ownership.

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Phase I Study Of P-Glycoprotein (P-gp) Mediated Drug Interactions Between Lenalidomide (LEN) and Quinidine Or Temsirolimus

Blood ◽

10.1182/blood.v122.21.5395.5395 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5395-5395

Author(s):

Nianhang Chen ◽

Daniel Weiss ◽

Josephine Reyes ◽

Liangang Liu ◽

Claudia Kasserra ◽

...

Keyword(s):

Single Dose ◽

Drug Interactions ◽

Oral Absorption ◽

Renal Excretion ◽

Systemic Exposure ◽

Plasma Exposure ◽

Employment Equity ◽

Healthy Men ◽

Relevant Effect ◽

Equity Ownership

Abstract Introduction Lenalidomide (LEN) is a weak substrate of P-glycoprotein (P-gp) in vitro and renal excretion of LEN is the primary elimination route following oral administration. A P-gp inhibitor may have the potential to increase systemic exposure to LEN by reducing renal elimination at the tubular level and enhancing oral absorption at the gut level. Recently, a single uncontrolled phase 1 study (Hofmeister, 2011) and a case report (Takahashi, 2012) described that plasma exposure to LEN and temsirolimus was increased in multiple myeloma patients when lenalidomide was co-administered with a P-gp inhibitor (temsirolimus and intraconazole, respectively). This clinical study assessed LEN-drug interactions via P-gp using two probe drugs under controlled conditions. Quinidine, a P-gp inhibitor with high in vivo inhibition potential and proven effect on plasma exposure of the prototype P-gp substrate digoxin in humans, was used to maximize the likelihood of detecting drug-drug interactions via P-gp. Temsirolimus, a P-gp inhibitor/substrate, was used to evaluate P-gp mediated interactions on either drug in comparison with the results reported in literature. Methods This was a phase 1, single-center, open-label, 2-part, fixed-sequence crossover study conducted in healthy men. Part 1 comprised of 2 treatment periods with LEN alone (25 mg single dose on Day 1) in period 1, followed by LEN (on Day 4) plus quinidine (300 mg twice daily [BID] on Day 1 and 600 mg BID on Days 2–5) in period 2. Part 2 consisted of three treatment periods with LEN (25 mg single dose on Day 1) alone in period 1, temsirolimus (25 mg single dose intravenously [IV] on Day 1) alone in period 2, and LEN plus temsirolimus in period 3 (Day 1). Treatment periods were separated by a washout of 7–10 days. Serial samples were collected to determine the plasma, whole blood or urine concentrations of LEN, quinidine, and temsirolimus (and its active metabolite, sirolimus [also a P-gp inhibitor/substrate]). Safety was monitored throughout the study. Results A total of 31 healthy men, aged 22 to 62 years; were enrolled (14 in Part 1 and 17 in Part 2). Renal excretion of LEN was almost complete at 12 h post dose for all treatments (Figure 1). In the absence or presence of a P-gp inhibitor, the mean percentage LEN dose excreted in the urine (74% vs 70% in Part 1, respectively; 81% vs 80% in Part 2) and renal clearance (227 vs 245 mL/min in Part 1; 251 vs 229 mL/min in Part 2) were similar, demonstrating that the rate and capacity of LEN renal excretion were not reduced by P-gp inhibition. Both the median time (1 h) to reach the maximum concentration (Cmax) and the oral bioavailability (70–80% of the administrated dose, as indicated by the renal excretion of unchanged drug) of LEN, were comparable in the absence or presence of a P-gp inhibitor (0.5–1 h and 74–81% of the dose, respectively); therefore, the rate and extent of LEN oral absorption were also not altered by P-gp inhibition. Consequently, there was no significant change in the plasma exposure to LEN in the presence of a P-gp inhibitor (Figure 1). The 90% confidence intervals (CIs) for the ratio of geometric means between LEN alone and LEN plus a P-gp inhibitor were completely contained within the equivalence limits of 80–125% for Cmaxand area under the concentration-time curve (AUC) of LEN. In addition, LEN had no effect on blood exposure to temsirolimus and sirolimus, with the 90% CIs for the ratio of their geometric mean Cmax and AUC between temsirolimus alone and temsirolimus plus LEN between 80–125%. No significant safety findings were reported when LEN was given with quinidine or temsirolimus compared to LEN alone. Conclusions Co-administration of either the P-gp inhibitor quinidine or temsirolimus had no clinically relevant effect on the systemic exposure of LEN. Similarly, co-administration of LEN had no clinically relevant effect on the systemic exposure of the P-gp substrates temsirolimus and sirolimus. A single dose of LEN was well tolerated when co-administered with quinidine or temsirolimus in healthy men. Disclosures: Chen: Celgene Corporation: Employment, Equity Ownership. Weiss:Celegene Corporation: Employment, Equity Ownership. Reyes:Celgene Corporation: Employment, Equity Ownership. Liu:Celgene Corporation: Employment, Equity Ownership. Kasserra:Celgene: Employment, Equity Ownership. Wang:Celgene Corporation: Employment, Equity Ownership. Zhou:Celgene Corporation: Employment, Equity Ownership. Kumar:Celgene Corporation: Employment, Equity Ownership. Weiss:Celgene Corporation: Employment, Equity Ownership. Palmisano:Celgene Corporation: Employment, Equity Ownership.

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ANX005, an Inhibitory Antibody Against C1q, Blocks Complement Activation Triggered By Cold Agglutinins in Human Disease

Blood ◽

10.1182/blood.v128.22.1265.1265 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1265-1265 ◽

Cited By ~ 10

Author(s):

Morie A. Gertz ◽

Haiyan Qiu ◽

Laura Kendall ◽

Mario Saltarelli ◽

Ted Yednock ◽

...

Keyword(s):

Complement Activation ◽

Research Funding ◽

Cold Agglutinin ◽

Employment Equity ◽

Cold Agglutinins ◽

Equity Ownership ◽

The Impact ◽

Human Rbcs ◽

Classical Complement

Abstract Cold agglutinin disease (CAD) is an autoimmune hemolytic anemia characterized by the presence of autoantibodies (cold agglutinins) that bind to red blood cells (RBC) at low temperatures. Cold agglutinin binding to RBCs results in antibody-mediated classical complement activation with deposition of complement C4 and C3 fragments onto the RBC surface. Complement-opsonized RBCs are removed from the circulation by macrophage-driven phagocytosis in the liver or spleen and via intravascular hemolysis following assembly of the terminal lytic complex (C5b - C9), together resulting in clinical anemia. C1q is the initiating molecule for cold agglutinin-mediated complement activation on the surface of human RBCs. We hypothesized that directly blocking C1q recruitment onto cold agglutinin-sensitized RBCs will prevent complement activation and opsonization and reduce hemolysis. To this end, we have developed a humanized monoclonal antibody (ANX005) that binds with high-affinity (~10 pM) to C1q and blocks classical complement activation & hemolysis in an in vitro sheep RBC assay. We evaluated the impact of ANX005 on hemolysis and complement deposition on human RBCs that were pre-sensitized with sera from CAD subjects. ANX005 showed a dose-dependent reduction in hemolysis using both individual and pooled CAD sera as the source of cold agglutinin. We further demonstrated that C1q blockade led to a robust reduction in C4 and C3 fragment deposition onto human RBCs. These results demonstrate that C1q inhibition is an effective way to impede C4 and C3 activation and downstream assembly of the lytic complex in sera from CAD patients, and support the clinical development of ANX005 in CAD and other antibody-mediated diseases. Disclosures Gertz: Prothena Therapeutics: Research Funding; Sandoz Inc: Honoraria; NCI Frederick: Honoraria; Celgene: Honoraria; Med Learning Group: Honoraria, Speakers Bureau; Research to Practice: Honoraria, Speakers Bureau; Alnylam Pharmaceuticals: Research Funding; Novartis: Research Funding; Ionis: Research Funding; Annexon Biosciences: Research Funding; GSK: Honoraria. Qiu:Annexon Biosciences: Employment, Equity Ownership. Kendall:Annexon Biosciences: Employment, Equity Ownership. Saltarelli:Mallinckrodt: Equity Ownership; Abbvie: Equity Ownership; Annexon Biosciences: Employment, Equity Ownership, Patents & Royalties. Yednock:Annexon, Inc: Employment, Equity Ownership. Sankaranarayanan:Annexon Biosciences: Employment, Equity Ownership.

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An in vitro Evaluation of Artificial Caries-like Lesions on Restored Overdenture Abutments

Journal of Dental Research ◽

10.1177/00220345880670031101 ◽

1988 ◽

Vol 67 (3) ◽

pp. 582-584 ◽

Cited By ~ 8

Author(s):

P.P. Kambhu ◽

R.L. Ettinger ◽

J.S. Wefel

Keyword(s):

Light Microscopy ◽

Polarized Light ◽

Polarized Light Microscopy ◽

The Other ◽

Type Ii ◽

Restorative Materials ◽

Abutment Teeth ◽

The Mean ◽

Materials Used

An acidified dialyzed gelatin gel system was used to determine the caries resistance of a variety of restorative materials used to obturate the canal orifice of overdenture abutment teeth. The restorative materials used were Tytin, Tytin + Copalite, P30 + Scotchbond, Fuji Ionomer-Type II, and Miracle Mix. Polarized light microscopy and microradiography were used to examine the caries-like lesions adjacent to the restorations. The lesions formed in the Fuji Ionomer-Type II and Miracle Mix groups appeared arrested at the wall adjacent to the restoration, and did not penetrate apically down the wall as did those associated with the other restorative materials. The mean depths of lesions adjacent to Fuji Ionomer-Type II and Miracle Mix restorations were significantly less than those of Tytin, Tytin + Copalite, or P30 + Scotchbond.

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Phenotype Does Not Always Equal Function: HDAC Inhibitors and UM171, but Not SR1, Lead to Rapid Upregulation of CD90 on Non-Engrafting CD34+CD90-Negative Human Cells

Blood ◽

10.1182/blood.v130.suppl_1.659.659 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 659-659

Author(s):

Kevin A. Goncalves ◽

Megan D. Hoban ◽

Jennifer L. Proctor ◽

Hillary L. Adams ◽

Sharon L. Hyzy ◽

...

Keyword(s):

In Vitro Culture ◽

Small Molecule ◽

Hdac Inhibitors ◽

Employment Equity ◽

Nsg Mice ◽

Cd34 Cells ◽

Equity Ownership ◽

Human Hscs

Abstract Background. The ability to expand human hematopoietic stem cells (HSCs) has the potential to improve outcomes in HSC transplantation and increase the dose of gene-modified HSCs. While many approaches have been reported to expand HSCs, a direct comparison of the various methods to expand transplantable HSCs has not been published and clinical outcome data for the various methods is incomplete. In the present study, we compared several small molecule approaches reported to expand human HSCs including HDAC inhibitors, the aryl hydrocarbon antagonist, SR1, and UM171, a small molecule with unknown mechanism, for the ability to expand phenotypic HSC during in vitro culture and to expand cells that engraft NSG mice. Although all strategies increased the number of phenotypic HSC (CD34+CD90+CD45RA-) in vitro, SR1 was the most effective method to increase the number of NOD-SCID engrafting cells. Importantly, we found that HDAC inhibitors and UM171 upregulated phenotypic stem cell markers on downstream progenitors, suggesting that these compounds do not expand true HSCs. Methods. Small-molecules, SR1, HDAC inhibitors (BG45, CAY10398, CAY10433, CAY10603, Entinostat, HC Toxin, LMK235, PCI-34051, Pyroxamide, Romidepsin, SAHA, Scriptaid, TMP269, Trichostatin A, or Valproic Acid) and UM171 were titrated and then evaluated at their optimal concentrations in the presence of cytokines (TPO, SCF, FLT3L, and IL6) for the ability to expand human mobilized peripheral blood (mPB)-derived CD34+ cells ex vivo . Immunophenotype and cell numbers were assessed by flow cytometry following a 7-day expansion assay in 10-point dose-response (10 µM to 0.5 nM). HSC function was evaluated by enumeration of colony forming units in methylcellulose and a subset of the compounds were evaluated by transplanting expanded cells into sub-lethally irradiated NSG mice to assess engraftment potential in vivo . All cells expanded with compounds were compared to uncultured or vehicle-cultured cells. Results. Following 7 days of expansion, SR1 (5-fold), UM171 (4-fold), or HDAC inhibitors (>3-35-fold) resulted in an increase in CD34+CD90+CD45RA- number relative to cells cultured with cytokines alone; however, only SR1 (18-fold) and UM171 (8-fold) demonstrated enhanced engraftment in NSG mice. Interestingly, while HDAC inhibitors and UM171 gave the most robust increase in the number and frequency of CD34+CD90+CD45RA- cells during in vitro culture, these methods were inferior to SR1 at increasing NSG engrafting cells. The increase in CD34+CD90+CD45RA- cells observed during in vitro culture suggested that these compounds may be generating a false phenotype by upregulating CD90 and down-regulating CD45RA on progenitors that were originally CD34+CD90-CD45RA+. We tested this hypothesis by sorting CD34+CD90-CD45RA+ cells and culturing these with the various compounds. These experiments confirmed that both HDAC inhibitors (33-100 fold) and UM171 (28-fold) led to upregulation of CD90 on CD34+CD90-CD45RA+ cells after 4 days in culture. Since approximately 90% of the starting CD34+ cells were CD90-, these data suggest that most of the CD34+CD90+CD45RA- cells in cultures with HDAC inhibitors and UM171 arise from upregulation of CD90 rather than expansion of true CD34+CD90+CD45RA- cells and may explain the disconnect between in vitro HSC phenotype and NSG engraftment in vivo . This was further confirmed by evaluation of colony forming unit frequency of CD34+CD90-CD45RA+ cells after culture with compounds. Conclusions. We have showed that AHR antagonism is optimal for expanding functional human HSCs using the NSG engraftment model. We also demonstrated that UM171 and HDAC inhibitors upregulate phenotypic HSC markers on downstream progenitors. This could explain the discrepancy between impressive in vitro phenotypic expansion and insufficient functional activity in the NSG mouse model. Therefore, these data suggest caution when interpreting in vitro expansion phenotypes without confirmatory functional transplantation data, especially as these approaches move into clinical trials in patients. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership. Hoban: Magenta Therapeutics: Employment, Equity Ownership. Proctor: Magenta Therapeutics: Employment, Equity Ownership. Adams: Magenta Therapeutics: Employment, Equity Ownership. Hyzy: Magenta Therapeutics: Employment, Equity Ownership. Boitano: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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Impact of Amotosalen/UVA Pathogen Inactivated Platelet Components on Outcomes after Allogeneic Hematopoetic Stem Cell Transplantation: A Single Center Analysis

Blood ◽

10.1182/blood-2019-122330 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1167-1167

Author(s):

Andreas S. Buser ◽

Laura Infanti ◽

Andreas Holbro ◽

Joerg Halter ◽

Sabine Gerull ◽

...

Keyword(s):

Stem Cell ◽

Cox Regression ◽

Univariate Analysis ◽

Conditioning Regimen ◽

Disease Status ◽

Risk Scores ◽

Employment Equity ◽

Stem Cell Source ◽

Equity Ownership ◽

The Impact

Background: Platelet component (PC) transfusion is required for allogeneic hematopoietic stem cell transplantation (HCT) recipients. Contamination with infectious pathogens (bacteria, viruses, or protozoa) and T-cells is a risk factor for transfusion-transmitted infection (TTI) and transfusion associated graft-versus-host disease (TA-GVHD). Pathogen inactivation (PI) treatment of PC with amotosalen-UVA (PI-PC, INTERCEPT Blood System, Cerus Corp) in platelet additive solution (PAS) without bacterial screening, gamma irradiation, CMV serology, and with 7-day storage has been the standard of care in Switzerland since 2011 to manage risk of TTI and TA-GVHD. PI-PC have replaced conventional PC (C-PC) prepared in PAS with gamma irradiation and 5 day storage. We previously reported platelet usage in two consecutive five year periods at the University Hospital of Basel. Mean PI-PC dose was higher (3.0 vs. 2.8 x 1011, p=0.001) and mean storage duration longer (4.2 vs. 3.4 days: p=0.001) than with C-PC. PC expiration wastage was reduced with 7-day PI-PC storage vs. 5-day storage (1.5% vs. 8.7%). For HCT recipients, days of PC support; PC use per patient; and RBC use per patient were similar, despite 24.3% lower corrected count increments (CCI) with PI-PC. Now, we report the impact of these observations on treatment related mortality (TRM) and overall survival (OS) 100 days after HCT. Patients and Methods: A two-period retrospective cohort study was conducted to evaluate PI-PC impact on outcomes of consecutive first allogeneic HCT recipients from January 2006 to December 2010 (Period 1, P1), when gamma-irradiated apheresis C-PC were used, and Period 2 (P2) from January 2011 to December 2017, when apheresis and whole blood-derived PI-PC were used. The review utilized 100-day OS and 100-day TRM to determine the impact of PI-PC on HCT outcomes. Descriptive statistics were used for continuous variables and log-rank analysis for survival outcomes. Univariate analysis was performed using Pearson χ2 statistics. Multivariate Cox regression modelling analyses included: PC period (P1, P2), donor match (HLA identical/twin, matched related, matched unrelated), disease state (early, intermediate, late), and conditioning regimen (reduced intensity, myeloablative) with TRM as the outcome. This was an IRB approved single-center analysis. Results: In P1 and P2, 256 and 557 consecutive first-time allogeneic HCT recipients were included, respectively. By univariate analysis, the distribution of European Group for Bone Marrow Transplantation (EBMT) risk scores (grouped 0-2, 3-4, 5-7) and mean patient age were higher during P2 (p = 0.001 and p <0.001, respectively). Primary disease status (p = 0.039); stem cell source (p <0.001); GVHD prophylaxis with ATG (p <0.001); total body irradiation (p <0.001); and conditioning regimen (p <0.001) were different between P1 and P2. Donor match (p=0.084) and disease status (p = 0.628) were similar in P1 and P2. TRM at day 100 post HCT was significantly less (31/557, 5.5%) for PI-PC recipients in P2 vs. C-PC recipients in P1 (37/256, 14.5%, p<0.001). Overall proportion of survivors at day 100 post HCT was significantly greater for PI-PC recipients (507/557, 91.0 %) compared to C-PC recipients (209/256, 81.6%, p <0.001). By multivariate Cox regression analysis, P2 with PI-PC component support was associated with improved TRM (p = 0.001; adjusted hazard ratio 0.433; 95% confidence interval: 0.262, 0.716). Donor match (p = 0.019), disease state (p = 0.022), and myeloablative conditioning (p = 0.034) were associated with significantly poorer TRM (Table). Stem cell source was not significant (p=0.157) in the model. Hemorrhage was reported as cause of death in 1/50 (2.0%) patients during P2 with PI-PC and 4/47 (8.5%) patients during P1 with C-PCs. Conclusions: Universal implementation of PI-PC in routine with extended storage to 7 days in P2 was associated with reduced TRM and better overall survival 100 days post HCT, despite transplantation of older patients with higher EBMT risk scores. Multivariate analysis revealed an adjusted hazard ratio of 0.433 (95% C.I. 0.262, 0.716) for TRM by 100 days, suggesting better outcomes in P2. This retrospective analysis at a single site indicated that PI-PC treated with amotosalen /UVA stored up to 7 days did not have a negative impact on TRM and OS in HCT recipients, and was an integral part of improving clinical outcomes at our institution. . Table. Disclosures Heim: Novartis: Research Funding. Irsch:Cerus Corporation: Employment, Equity Ownership. Lin:Cerus Corporation: Employment, Equity Ownership. Benjamin:Cerus Corporation: Employment, Equity Ownership. Corash:Cerus Corporation: Employment, Equity Ownership.

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Highly Differentiated Anti-CD47 Antibody, AO-176, Potently Inhibits Hematologic Malignancies Alone and in Combination

Blood ◽

10.1182/blood-2019-126298 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1844-1844

Author(s):

John Richards ◽

Myriam N Bouchlaka ◽

Robyn J Puro ◽

Ben J Capoccia ◽

Ronald R Hiebsch ◽

...

Keyword(s):

Multiple Myeloma ◽

Combination Therapy ◽

Tumor Growth ◽

Growth Inhibition ◽

Tumor Cells ◽

Tumor Growth Inhibition ◽

Employment Equity ◽

Equity Ownership

AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.

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Clinical Pharmacodynamic Markers and Combinations with SY1425 (tamibarotene) in a Genomically-Defined Subset of Non-APL AML

Blood ◽

10.1182/blood.v128.22.2898.2898 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2898-2898

Author(s):

Michael R McKeown ◽

Christopher Fiore ◽

Emily Lee ◽

Matthew L Eaton ◽

Christian C. Fritz

Keyword(s):

Transcription Factor ◽

Cell Lines ◽

Binding Sites ◽

Clinical Studies ◽

High Cell ◽

Preclinical Models ◽

Employment Equity ◽

Equity Ownership ◽

Maximal Induction

Abstract SY-1425, a potent and selective agonist of the retinoic acid receptor RARα, is being investigated in a Ph2 trial in a novel genomically-defined subset of non-APL AML and MDS patients (clinicaltrials.gov NCT02807558). RARa is a nuclear hormone receptor and transcription factor that regulates genes involved in cell differentiation and proliferation. We identified a super-enhancer (SE) at the RARA locus, the gene encoding RARa, in a subset of primary non-APL AML blasts. Preclinical models demonstrated a correlation between the presence of a RARA SE and sensitivity to SY-1425, providing the rationale for clinical investigation. Further research has investigated pharmacodynamics (PD) markers and combinations of drugs to support clinical development of SY-1425. In this study we identified DHRS3mRNA induction as a measure of RARα target engagement with SY-1425. We also demonstrated synergy in preclinical models with SY-1425 and hypomethylating agents. Since RARα is a transcription factor that regulates target genes when bound by a retinoid, we characterized the dynamic expression changes of a panel of RARA enhancer- high and - low non-APL AML cell lines (hereafter referred to as RARA-high and -low) in response to SY-1425 treatment. DHRS3 showed the largest expression increase following treatment in 3 RARA-high cell lines, with a range of 29 to 115 fold. In contrast, there was a much lower DHRS3 induction in 3 RARA-low cell lines (range of 1.6 to 6.1 fold). Induction was found to be both time- and dose-dependent with maximal induction at approximately 6 hours and half maximal induction near the EC50 for the anti-proliferative effect in RARA-high cell lines. DHRS3 encodes dehydrogenase/reductase (SDR family) member 3, a metabolic enzyme involved in maintaining cellular retinol homeostasis and had previously been shown to be induced by retinoids. Thus, DHRS3induction in tumor cells represents a potentially useful PD marker for clinical studies of SY-1425. To better understand the mechanism of induction of DHRS3 by SY-1425 we examined the chromosomal localization of RARα as well as the epigenomic state of the DHRS3 locus by ChIP-seq for RARα and H3K27 acetylation, the latter being an indicator of active enhancers and promoters. In the untreated state, OCI-AML3 (a typical RARA-high AML cell line) was found to have multiple RARα binding sites both within and distal to the DHRS3 gene but minimal H3K27 acetylation. Following treatment with SY-1425, the level of H3K27 acetylation at DHRS3 increased, resulting in the formation of a SE. Moreover, the SE encompassed the RARα binding sites, consistent with the model in which SY-1425 converts RARα into an activator of DHRS3expression. Similar results were seen for the CD38 locus in which SY-1425 treatment increased expression, H3K27 acetylation, and RARα binding. CD38 is a cell surface antigen and marker of myeloid maturation readily analyzed by FACS analysis, suggesting it could be an additional PD marker to be used in clinical studies. Indeed, it was found that SY-1425 induced CD38 cell surface expression at similar levels in RARA-high AML cell lines and the NB-4 APL cell line, but not in RARA-low cell lines. We also investigated combinations of SY-1425 with approved or investigational AML and MDS agents in in vitro and in vivo models to inform future clinical studies and to further explore potential PD markers unique to the combined action of the drugs. Several standard of care agents and drugs in current development were found to have synergistic interactions with SY-1425 in RARA-high but not RARA-low cell lines. In particular, azacitidine and decitabine each showed strong in vitro synergy with SY-1425. Evaluation of SY-1425 plus azacitidine in a RARA-high PDX model of non-APL AML demonstrated a better response compared to either agent alone. Additional genome-wide ChIP-seq and expression studies of RARA-high cells treated with various combinations are being investigated to identify optimal PD markers for these combinations. These studies support the use of DHRS3 mRNA induction in tumor cells as a PD marker in the recently initiated Ph2 study of SY-1425 in genomically-defined non-APL AML and MDS patients (clinicaltrials.gov NCT02807558) and further exploration as a PD marker for future combination studies. Disclosures McKeown: Syros Pharmaceuticals: Employment, Equity Ownership. Fiore:Syros Pharmaceuticals: Employment, Equity Ownership. Lee:Syros Pharmaceuticals: Employment, Equity Ownership. Eaton:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership.

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