Ivosidenib and Azacitidine in IDH1-Mutated Acute Myeloid Leukemia.

BACKGROUND: The combination of ivosidenib - an inhibitor of mutant isocitrate dehydrogenase 1 (IDH1) - and azacitidine showed encouraging clinical activity in a phase 1b trial involving patients with newly diagnosed IDH1-mutated acute myeloid leukemia. METHODS: In this phase 3 trial, we randomly assigned patients with newly diagnosed IDH1-mutated acute myeloid leukemia who were ineligible for intensive induction chemotherapy to receive oral ivosidenib (500 mg once daily) and subcutaneous or intravenous azacitidine (75 mg per square meter of body-surface area for 7 days in 28-day cycles) or to receive matched placebo and azacitidine. The primary end point was event-free survival, defined as the time from randomization until treatment failure (i.e., the patient did not have complete remission by week 24), relapse from remission, or death from any cause, whichever occurred first. RESULTS: The intention-to-treat population included 146 patients: 72 in the ivosidenib-and-azacitidine group and 74 in the placebo-and-azacitidine group. At a median follow-up of 12.4 months, event-free survival was significantly longer in the ivosidenib-and-azacitidine group than in the placebo-and-azacitidine group (hazard ratio for treatment failure, relapse from remission, or death, 0.33; 95% confidence interval [CI], 0.16 to 0.69; P = 0.002). The estimated probability that a patient would remain event-free at 12 months was 37% in the ivosidenib-and-azacitidine group and 12% in the placebo-and-azacitidine group. The median overall survival was 24.0 months with ivosidenib and azacitidine and 7.9 months with placebo and azacitidine (hazard ratio for death, 0.44; 95% CI, 0.27 to 0.73; P = 0.001). Common adverse events of grade 3 or higher included febrile neutropenia (28% with ivosidenib and azacitidine and 34% with placebo and azacitidine) and neutropenia (27% and 16%, respectively); the incidence of bleeding events of any grade was 41% and 29%, respectively. The incidence of infection of any grade was 28% with ivosidenib and azacitidine and 49% with placebo and azacitidine. Differentiation syndrome of any grade occurred in 14% of the patients receiving ivosidenib and azacitidine and 8% of those receiving placebo and azacitidine. CONCLUSIONS: Ivosidenib and azacitidine showed significant clinical benefit as compared with placebo and azacitidine in this difficult-to-treat population. Febrile neutropenia and infections were less frequent in the ivosidenib-and-azacitidine group than in the placebo-and-azacitidine group, whereas neutropenia and bleeding were more frequent in the ivosidenib-and-azacitidine group. (Funded by Agios Pharmaceuticals and Servier Pharmaceuticals; AGILE ClinicalTrials.gov number, NCT03173248.).

Mutant Isocitrate Dehydrogenase 1 Inhibitor Ivosidenib in Combination With Azacitidine for Newly Diagnosed Acute Myeloid Leukemia.

PURPOSE: Ivosidenib is an oral inhibitor of the mutant isocitrate dehydrogenase 1 (IDH1) enzyme, approved for treatment of IDH1-mutant (mIDH1) acute myeloid leukemia (AML). Preclinical work suggested that addition of azacitidine to ivosidenib enhances mIDH1 inhibition-related differentiation and apoptosis. PATIENTS AND METHODS: This was an open-label, multicenter, phase Ib trial comprising dose-finding and expansion stages to evaluate safety and efficacy of combining oral ivosidenib 500 mg once daily continuously with subcutaneous azacitidine 75 mg/m2 on days 1-7 in 28-day cycles in patients with newly diagnosed mIDH1 AML ineligible for intensive induction chemotherapy (ClinicalTrials.gov identifier: NCT02677922). RESULTS: Twenty-three patients received ivosidenib plus azacitidine (median age, 76 years; range, 61-88 years). Treatment-related grade ≥ 3 adverse events occurring in > 10% of patients were neutropenia (22%), anemia (13%), thrombocytopenia (13%), and electrocardiogram QT prolongation (13%). Adverse events of special interest included all-grade IDH differentiation syndrome (17%), all-grade electrocardiogram QT prolongation (26%), and grade ≥ 3 leukocytosis (9%). Median treatment duration was 15.1 months (range, 0.3-32.2 months); 10 patients remained on treatment as of February 19, 2019. The overall response rate was 78.3% (18/23 patients; 95% CI, 56.3% to 92.5%), and the complete remission rate was 60.9% (14/23 patients; 95% CI, 38.5% to 80.3%). With median follow-up of 16 months, median duration of response in responders had not been reached. The 12-month survival estimate was 82.0% (95% CI, 58.8% to 92.8%). mIDH1 clearance in bone marrow mononuclear cells by BEAMing (beads, emulsion, amplification, magnetics) digital polymerase chain reaction was seen in 10/14 patients (71.4%) achieving complete remission. CONCLUSION: Ivosidenib plus azacitidine was well tolerated, with an expected safety profile consistent with monotherapy with each agent. Responses were deep and durable, with most complete responders achieving mIDH1 mutation clearance.

Taxane resistance in prostate cancer is mediated by decreased drug-target engagement.

Despite widespread use of taxanes, mechanisms of action and resistance in vivo remain to be established, and there is no way of predicting who will respond to therapy. This study examined prostate cancer (PCa) xenografts and patient samples to identify in vivo mechanisms of taxane action and resistance. Docetaxel drug-target engagement was assessed by confocal anti-tubulin immunofluorescence to quantify microtubule bundling in interphase cells and aberrant mitoses. Tumor biopsies from metastatic PCa patients obtained 2 to 5 days after their first dose of docetaxel or cabazitaxel were processed to assess microtubule bundling, which correlated with clinical response. Microtubule bundling was evident in PCa xenografts 2 to 3 days after docetaxel treatment but was decreased or lost with acquired resistance. Biopsies after treatment with leuprolide plus docetaxel showed extensive microtubule bundling as did biopsies obtained 2 to 3 days after initiation of docetaxel or cabazitaxel in 2 patients with castration-resistant PCa with clinical responses. In contrast, microtubule bundling in biopsies 2 to 3 days after the first dose of docetaxel was markedly lower in 4 nonresponding patients. These findings indicate that taxanes target both mitotic and interphase cells in vivo and that resistance is through mechanisms that impair drug-target engagement. Moreover, the findings suggest that microtubule bundling after initial taxane treatment may be a predictive biomarker for clinical response.

Convergent synthesis of hydrophilic monomethyl dolastatin 10 based drug linkers for antibody-drug conjugation.

We report a modular approach to synthesize maleimido group containing hydrophilic dolastatin 10 (Dol10) derivatives as drug-linkers for the syntheses of antibody-drug conjugates (ADCs). Discrete polyethylene glycol (PEG) moieties of different chain lengths were introduced as part of the linker to impart hydrophilicity to these drug linkers. The synthesis process involved construction of PEG maleimido derivatives of the tetrapeptide intermediate (N-methylvaline-valine-dolaisoleucine-dolaproine), which were subsequently coupled with dolaphenine to generate the desired drug linkers. The synthetic method reported in this manuscript circumvents the use of highly cytotoxic Dol10 in its native form. By using trastuzumab (Herceptin®) as the antibody we have synthesized Dol10 containing ADCs. The presence of a discrete PEG chain in the drug linkers resulted in ADCs free from aggregation. The effect of PEG chain length on the biological activities of these Dol10 containing ADCs was investigated by in vitro cytotoxicity assays. ADCs containing PEG6 and PEG8 spacers exhibited the highest level of in vitro anti-proliferative activity against HER2-positive (SK-BR-3) human tumor cells. ADCs derived from Herceptin® and PEG8-Dol10, at a dose of 10 mg kg-1, effectively delayed the tumor growth and prolonged the survival time in mice bearing human ovarian SKOV-3 xenografts.

Cabazitaxel is more active than first-generation taxanes in ABCB1(+) cell lines due to its reduced affinity for P-glycoprotein.

PURPOSE: The primary aim of this study was to determine cabazitaxel's affinity for the ABCB1/P-glycoprotein (P-gp) transporter compared to first-generation taxanes. METHODS: We determined the kinetics of drug accumulation and retention using [14C]-labeled taxanes in multidrug-resistant (MDR) cells. In addition, membrane-enriched fractions isolated from doxorubicin-selected MES-SA/Dx5 cells were used to determine sodium orthovanadate-sensitive ATPase stimulation after exposure to taxanes. Custom [3H]-azido-taxane analogues were synthesized for the photoaffinity labeling of P-gp. RESULTS: The maximum intracellular drug concentration was achieved faster with [14C]-cabazitaxel (5 min) than [14C]-docetaxel (15-30 min). MDR cells accumulated twice as much cabazitaxel than docetaxel, and these levels could be restored to parental levels in the presence of the P-gp inhibitor PSC-833 (valspodar). Efflux in drug-free medium confirmed that MDR cells retained twice as much cabazitaxel than docetaxel. There was a strong association (r2 = 0.91) between the degree of taxane resistance conferred by P-gp expression and the accumulation differences observed with the two taxanes. One cell model expressing low levels of P-gp was not cross-resistant to cabazitaxel while demonstrating modest resistance to docetaxel. Furthermore, there was a 1.9 × reduction in sodium orthovanadate-sensitive ATPase stimulation resulting from treatment with cabazitaxel compared to docetaxel. We calculated a dissociation constant (Kd) value of 1.7 µM for [3H]-azido-docetaxel and ~ 7.5 µM for [3H]-azido-cabazitaxel resulting in a 4.4 × difference in P-gp labeling, and cold docetaxel was a more effective competitor than cabazitaxel. CONCLUSION: Our studies confirm that cabazitaxel is more active in ABCB1(+) cell models due to its reduced affinity for P-gp compared to docetaxel.

Design, Synthesis, and in†vitro Evaluation of Multivalent Drug Linkers for High-Drug-Load Antibody-Drug Conjugates.

A series of novel multivalent drug linkers (MDLs) containing cytotoxic agents were synthesized and conjugated to antibodies to yield highly potent antibody-drug conjugates (ADCs) with drug/antibody ratios (DARs) higher than those typically reported in the literature (10 vs. ≈4). These MDLs contain two copies of a cytotoxic agent attached to biocompatible scaffolds composed of a branched peptide core and discrete polyethylene glycol (PEG) chains to enhance solubility and decrease aggregation. These drug linkers produced well-defined ADCs, whose DARs could be accurately determined by LC-MS. Using this approach, ADCs with significantly lower aggregation and higher DAR than those of conventional drug linker design were obtained with highly hydrophobic cytotoxic agents such as monomethyldolastatin 10 (MMAD). The in vitro potencies of the MDL-derived conjugates matched that of ADCs of similar DAR with conventional linkers, and the potency increased proportionally with drug loading. This approach may provide a means to prepare highly potent ADCs from a broader range of drugs, including those with lower cytotoxicity or poor solubility, which otherwise limits their use for antibody-drug conjugates. This may also provide a means to further improve the potency achievable with cytotoxins currently used in ADCs.

Anti-Endosialin Antibody-Drug Conjugate: Potential in Sarcoma and Other Malignancies.

Endosialin/TEM1/CD248 is a cell surface protein expressed at high levels by the malignant cells of about 50% of sarcomas and neuroblastomas. The antibody-drug conjugate (ADC) anti-endosialin-MC-VC-PABC-MMAE was selectively cytotoxic to endosialin-positive cells in vitro and achieved profound and durable antitumor efficacy in preclinical human tumor xenograft models of endosialin-positive disease. MC-VC-PABC-MMAE was conjugated with anti-endosialin with 3-4 MMAE molecules per ADC. The anti-endosialin-MC-VC-PABC-MMAE conjugate was tested for activity in four human cell lines with varied endosialin levels. The HT-1080 fibrosarcoma cells do not express endosialin, A-673 Ewing sarcoma cells and SK-N-AS neuroblastoma cells are moderate expressers of endosialin, and SJSA-1 osteosarcoma cells express very high levels of endosialin. To determine whether endosialin expression was maintained in vivo, A-673 Ewing sarcoma, SK-N-AS neuroblastoma, and SJSA-1 osteosarcoma cells were grown as xenograft tumors in nude mice. The SK-N-AS neuroblastoma and the A-673 Ewing sarcoma lines were selected for in vivo efficacy testing of the anti-endosialin-MC-VC-PABC-MMAE conjugate. The treatment groups included a vehicle control, unconjugated anti-endosialin, an admix control consisting of anti-endosialin and a dose of free MMAE equivalent to the dose administered as the ADC, and the anti-endosialin-MC-VC-PABC-MMAE conjugate. The unconjugated anti-endosialin had no antitumor activity and resulted in similar tumor growth as the vehicle control. The admix control produced a modest tumor growth delay. Administration of the anti-endosialin-MC-VC-PABC-MMAE conjugate resulted in a marked prolonged tumor response of both xenograts. These proof-of-concept results break new ground and open a promising drug discovery approach to these rare and neglected tumors.

Site-specific antibody-drug conjugation through glycoengineering.

Antibody-drug conjugates (ADCs) have been proven clinically to be more effective anti-cancer agents than native antibodies. However, the classical conjugation chemistries to prepare ADCs by targeting primary amines or hinge disulfides have a number of shortcomings including heterogeneous product profiles and linkage instability. We have developed a novel site-specific conjugation method by targeting the native glycosylation site on antibodies as an approach to address these limitations. The native glycans on Asn-297 of antibodies were enzymatically remodeled in vitro using galactosyl and sialyltransferases to introduce terminal sialic acids. Periodate oxidation of these sialic acids yielded aldehyde groups which were subsequently used to conjugate aminooxy functionalized cytotoxic agents via oxime ligation. The process has been successfully demonstrated with three antibodies including trastuzumab and two cytotoxic agents. Hydrophobic interaction chromatography and LC-MS analyses revealed the incorporation of ~1.6 cytotoxic agents per antibody molecule, approximating the number of sialic acid residues. These glyco-conjugated ADCs exhibited target-dependent antiproliferative activity toward antigen-positive tumor cells and significantly greater antitumor efficacy than naked antibody in a Her2-positive tumor xenograft model. These findings suggest that enzymatic remodeling combined with oxime ligation of the native glycans of antibodies offers an attractive approach to generate ADCs with well-defined product profiles. The site-specific conjugation approach presented here provides a viable alternative to other methods, which involve a need to either re-engineer the antibody sequence or develop a highly controlled chemical process to ensure reproducible drug loading.

Micro- and mid-scale maleimide-based conjugation of cytotoxic drugs to antibody hinge region thiols for tumor targeting.

Currently, the principal chemistries for the preparation of antibody-drug conjugates (ADC) target either lysines or cysteines for coupling cytotoxic drugs for delivery to target cells expressing tumor-specific antigens. All of these chemistries generate populations of molecules which differ in critical properties which are known to affect efficacy, pharmacokinetics, and the therapeutic window. Of key interest are methods to minimize this heterogeneity to achieve reproducible product profiles and efficacy. A current trend in the development of ADC is the evaluation of suitable targets, antibodies, and payloads, occurring well before process development to produce conjugates of clinical quality. This creates a need for an ability to generate comparably high-quality products early in development and at sufficient scale for evaluating in vitro potency and in vivo efficacy, as well as the early identification of any deficiencies in critical quality attributes including solubility and stability. Here we elaborate detailed protocols using maleimide-based chemistry for the conjugation to reduce hinge disulfides in antibodies by several cytotoxic drugs. We present a method for the initial characterization of the reduction/alkylation reaction using polyethylene-glycol (PEG) as a drug surrogate, a 5 mg scale drug conjugation to provide material for initial characterization including cell proliferation assays and a 150 mg scale process for performing efficacy studies in small animals. These methods yield well-defined predictable product profiles at high yield and with low impurities. These procedures include details relevant to the execution of these methods in a safe and contained manner within a typical laboratory environment.

Well-defined aminooxy terminated N-(2-hydroxypropyl) methacrylamide macromers for site specific bioconjugation of glycoproteins.

Syntheses and characterization of aminooxy terminated polymers of N-(2-hydroxyproyl) methacrylamide (HPMA) of controlled molecular weight and narrow molecular weight distribution are presented here. Design of a chain transfer agent (CTA) containing N-tert-butoxycarbonyl (t-Boc) protected aminooxy group enabled us to use reversible addition-fragmentation (RAFT) polymerization technique to polymerize the HPMA monomer. An amide bond was utilized to link the aminooxy group and the CTA through a triethylene glycol spacer. As a result, the aminooxy group is linked to the poly(HPMA) backbone through a hydrolytically stable amide bond. By varying the monomer to initiator ratios, polymers with targeted molecular weights were obtained. The molecular weights of the polymers were determined by gel permeation chromatography (GPC) and mass spectrometry (ESI and MALDI-TOF). The t-Boc protecting group was quantitatively removed to generate aminooxy terminated poly(HPMA) macromers. These macromers were converted to rhodamine B terminated poly(HPMA) by reacting N-hydroxysuccinimide (NHS) ester of the dye with the terminal aminooxy group to form a stable alkoxyamide bond. Utility of these dye-labeled polymers as molecular probes was evaluated by fluorescence microscopy by studying their intracellular uptake by renal epithelial cells. These aminooxy terminated poly(HPMA) were also tested as biocompatible carriers to prepare chemoselective bioconjugates of proteins using transferrin (Tf) as the protein. Oxidation of the sialic acid side chains of Tf generated aldehyde functionalized protein that was reacted with aminooxy terminated poly(HPMA), which resulted in protein-polymer bioconjugates carrying oxime linkages. These bioconjugates were characterized by gel electrophoresis and MALDI-TOF mass spectrometry.

Targeting HER2-positive cancer with dolastatin 15 derivatives conjugated to trastuzumab, novel antibody-drug conjugates.

PURPOSE: Targeting tubulin binders to cancer cells using antibody-drug conjugates (ADCs) has great potential to become an effective cancer treatment with low normal tissue toxicity. The nature of the linker used to tether the tubulin binder to the antibody and the conjugation sites on the antibody and the small molecule are important factors in the ADC stability and effectiveness. METHODS: We explored the use of tubulin-targeting dolastatin 15 derivatives (Dol15) tethered covalently to a representative antibody, trastuzumab, via cleavable and non-cleavable linkers at varying antibody reactive sites (i.e., lysine residues, partially reduced hinge region disulfide bonds) and drug coupling sites (i.e., C-terminus, N-terminus), to investigate which constructs were more effective in killing HER2-positive cells in vitro and in vivo. RESULTS: We found that Dol15 conjugated to trastuzumab via lysine residues at the drug C-terminus using a non-cleavable linker (trastuzumab-amide-C-term-Dol15) produced target-dependent growth inhibition of cells endogenously expressing high HER2 levels (i.e., SK-BR-3, SK-OV-3) in vitro. This ADC was effective at varying doses (i.e., 10 and 20 mg/kg) in the SK-OV-3 human ovarian cancer xenograft. CONCLUSIONS: Tethering Dol15 via partially reduced disulfide bonds at the drug C-terminus via a non-cleavable linker (trastuzumab-MC-C-term-Dol15) resulted in an equally effective ADC in vitro, showing that site of antibody conjugation did not influence ADC activity. However, tethering Dol15 at the drug N-terminus using non-cleavable and cleavable linkers (trastuzumab-MC-N-term-Dol15 and trastuzumab-MC-VC-PABC-N-term-Dol15, respectively) resulted in ineffective ADCs. Thus, Dol15 tethered at the C-terminus may be a useful tubulin-targeting agent for conjugation at various antibody reactive sites.

Hyaluronan-tethered opioid depots: synthetic strategies and release kinetics in vitro and in vivo.

We proposed the use of opioid drug bound covalently to hyaluronan (HA) via ester linkages as a method to prolong drug delivery and to possibly increase the quality of perioperative pain management. The in vitro release profile of morphine conjugated to HA (1.3 million MW) was studied. The influence of parameters such as conjugation site and steric protection of the labile ester bonds was investigated in phosphate buffered saline (PBS) medium. HA--codeine and HA--naloxone conjugates were used as structural controls. Codeine and morphine conjugated via the allylic hydroxyl group had a release half-life of 14.0 days in PBS. Naloxone conjugated via the phenolic hydroxyl group showed a half-life of 0.3 days, and all drugs admixed in HA showed half-lives of 0.1 days. Methyl, ethyl, or n-propyl introduced in vicinal position to the ester bond prolonged release of naloxone with half-lives of 0.5, 4.0, and 4.0 days in PBS, respectively. Incorporation of a methyl group prolonged codeine release with a half-life of 55.0 days in PBS. Drugs were released chemically unaltered from the conjugates as confirmed by LC-MS/MS. Further, morphine was conjugated to divinylsulfone cross-linked HA (Hylan B) particles and the release profiles in rat plasma were studied in vitro and in vivo. Release in rat plasma was faster than in PBS with a half-life of 2.5 days, but the release was similar (ca. 12 days) when a cocktail of protease inhibitors was added to the plasma. Sustained release of morphine was observed in a rat surgical model over 30 h. Morphine was released chemically unaltered from the conjugate and morphine intermediates were not detected in significant amounts as confirmed by LC-MS/MS. These results suggest that the morphine release profile from the HA conjugates depends on the alkyl groups vicinal to the ester and the nature of the leaving group. In rat plasma, hydrolysis seems to be controlled by esterase activity.

Synthesis and evaluation of hydrolyzable hyaluronan-tethered bupivacaine delivery systems.

Local anesthetics are useful for reducing acute pain, but their short duration precludes them from use in solely managing postoperative pain. To prolong the duration of local anesthesia, we conjugated bupivacaine to native hyaluronan (HA) and divinyl sulfone cross-linked Hylan A (Hylan B particles) using a hydrolyzable linker incorporating an imide. Bupivacaine was prepared for conjugation to HA by forming the acryl imide derivative. Separately, the carboxyl group of HA was reacted with nipsylethylamine (NEA) using carbodiimide-mediated coupling to provide HA-NEA that was subsequently reduced with tris(2-carboxyethylphosphine) hydrochloride to yield HA carrying a free sulfhydryl (HA-SH). The HA-bupivacaine conjugate was assembled by reacting HA-SH with acrylbupivacaine. Characterization of the conjugates showed 22% degree of modification by 1 mol of carboxyl. In vitro release studies comparing bupivacaine admixed in HA with bupivacaine conjugated to HA showed half-lives of 0.4 +/- 0.1 h, and 16.9 +/- 0.2 h, respectively, and the bupivacaine was released chemically unaltered as confirmed by LC-MS. In vivo studies to assess the duration of anesthetic activity were performed in a rat sciatic nerve blockade model. For these studies, bupivacaine was conjugated to Hylan B following a similar procedure, and the degree of modification obtained was 14%. Free bupivacaine (3 and 16 mg/kg) and free bupivacaine (3 mg/kg) admixed with Hylan B particles showed nerve block over 4, 9, and 6 h, respectively. Free bupivacaine (3 mg/kg) admixed with bupivacaine (13 mg/kg) conjugated to Hylan B particles showed a four to 5-fold longer impairment of motor function over the free bupivacaine formulations with a total block time of 19 h. Bupivacaine conjugated to Hylan B particles has the potential to prolong the duration of local anesthesia.