Antineoplastic Cytotoxicity Research Articles

Phase I Trial of Allogeneic Donor Gamma Delta T Cell Infusion Post-Hematopoietic Cell Transplantation, an Interim Safety Report

Background Patients with 2017 European LeukemiaNet (ELN) adverse risk acute myeloid leukemia (AML) are at 40% risk of relapse leading to poor survival after allogeneic hematopoietic cell transplantation (allo-HCT) (Hansen TCT 2021, Jimenez BMT 2021). Ex vivo donor-derived gamma delta T cells (GDT) expanded with artificial antigen presented calls (aAPC) are a novel therapy with potent MHC-independent antineoplastic cytotoxicity. An ongoing phase 1/1b study is evaluating the safety and potential efficacy of ex vivo expanded donor GDT after reduced-intensity conditioning (RIC) allo-HCT in patients with ELN adverse risk AML ( NCT05015426). We report on manufacturing, safety, and correlative biology of expanded donor GDT after allo-HCT in the first 5 treated patients (Table 1). Methods The primary endpoint is the maximum-tolerated dose (MTD) of allogeneic donor GDT as a single infusion at day 60 after RIC allo-HCT. Next generation sequencing was used for measurable residual disease (MRD) assessment pre-HCT and post-GDT infusion. Donor GDT collected from the same donor of the allo-HCT were enriched from non-mobilized leukapheresis product by stimulation with zoledronic acid and IL-2 for 7 days followed by alpha beta T cell depletion using the Miltenyi CliniMACS. Enriched GDT (>90% TCRVδ2) were then co-cultured with irradiated aAPCs in Wilson Wolf G-Rex 100MCS over 10 days (Boucher J. Immunother 2023). This single center, investigator-initiated phase 1 trial is testing 3 distinct dose cohorts of infused GDT (5 x 10 6 cells/kg, 2.5 x 10 7 cells/kg, and 1 x 10 8 cells/kg) with acceptable range of 25% margin. A Bayesian optimal interval design is used to guide dose escalation/de-escalation decisions. Results First 3 patients enrolled in dose level 1 (DL1) cohort received GDT dose of 6.25 x 10 6 cells/kg, while last 2 patients enrolled in DL2 cohort received 3.13 x 10 7 cells/kg dose. Dose-limiting toxicities (DLT) were evaluated for 42 days following GDT infusion and no DLT were observed. None of the treated patients experienced cytokine release syndrome (CRS), Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS) or graft-versus-host disease (GVHD) during DLT period. Among patients treated on DL1, patient #01 had ASXL1 AML with MRD pos (VAF 43.9%) prior to RIC haploidentical (Haplo) HCT. He is in ongoing complete remission (CR) 9 months post-HCT and has had no GVHD. Patient #02 had MRD pos (VAF 61.7%) AML with TP53 mutation/complex karyotype prior to RIC matched sibling donor allo-HCT. He is in CR and has mild chronic GVHD at 7 months post-HCT. Patient #03 with pre-HCT MRD negRUNX1 AML is in ongoing CR 7 months post-RIC Haplo HCT with no GVHD. At day 28 and day 120 post-GDT, all 3 patients were MRD neg in bone marrow and had 100% bone marrow and peripheral blood leukocyte and granulocyte chimerism. In DL2 cohort, patient #04 with RUNX1 AML and patient #05 with t(9;22) AML both were MRD neg prior to RIC Haplo HCT and remain in CR with no GVHD at 5 and 4 months post-HCT, respectively. At day 28 post-GDT, both patients were MRD neg and had 100% chimerism. Patient #05 had a fall during DLT period in a setting of orthostatic hypotension requiring hospitalization for 24 hours for hydration. This was the only grade 3 adverse event observed in this trial. Serum cytokines analyzed included IL2, IL6, IL15, IFNγ, TNFα, Angiopoietin 1&2, and GM-CSF (Figure 1). We compared results prior and up to 90 days post-GDT infusion and found no significant differences, which can explain no CRS or ICANS events yet observed on this trial. Immunophenotype (TCRVδ2, TCRVδ1, PD1, CTLA4, TIGIT and CD28) of GDT was analyzed in samples of donor apheresis, pre- and post-enrichment, infused GDT product and at days 28 and 90 post-infusion. GDT (TCRVδ2) persisted at day 28 and day 90 post-infusion and expressed low levels of cell surface inhibitory receptors. Conclusion This preliminary interim report demonstrates favorable safety and tolerability of donor GDT therapy with no CRS, ICANS or GVHD in first 5 patients treated in this trial. All these patients with adverse risk AML are in ongoing MRD neg CR following RIC allo-HCT, including 2 pre-HCT MRD pos cases with TP53 AML and AXSL1 AML. Early results of this trial are promising, which provides validation for continued testing of adoptive transfer of ex vivo expanded donor GDT. Updated results of this trial will be presented at the annual conference.

New Antineoplastic Naphthohydroquinones Attached to Labdane and Rearranged Diterpene Skeletons.

Terpenylquinones are mixed biogenesis primary or secondary metabolites widespread in Nature with many biological activities, including the antineoplastic cytotoxicity, that have inspired this work. Here, we present a cytotoxic structure-activity relationship of several diterpenylhydroquinone (DTHQ) derivatives, obtained from the natural labdane diterpenoid myrceocommunic acid used as starting material. Different structural modifications, that changed the functionality and stereochemistry of the decalin, have been implemented on the bicyclic core through epoxidation, ozonolysis or decarboxylation, and through induction of biomimetic breaks and rearrangements of the diterpene skeleton. All the isomers generated were completely characterized by spectroscopic procedures. The resulting compounds have been tested in vitro on cultured cancer cells, showing their relevant antineoplastic cytotoxicity, with GI50 values in the μM and sub-μM range. The rearranged compound 8 showed the best cytotoxic results, with GI50 at the submicromolar range, retaining the cytotoxicity level of the parent compounds. In this report, the versatility of the labdane skeleton for chemical transformation and the interest to continue using structural modifications to obtain new bioactive compounds are demonstrated.

Anti-Neoplastic Cytotoxicity of Gemcitabine-(C4 -amide)-[anti-EGFR] in Dual-combination with Epirubicin-(C3 -amide)-[anti-HER2/neu] against Chemotherapeutic-Resistant Mammary Adenocarcinoma (SKBr-3) and the Complementary Effect of Mebendazole

Anti-Neoplastic Cytotoxicity of Gemcitabine-(C4 -amide)-[anti-EGFR] in Dual-combination with Epirubicin-(C3 -amide)-[anti-HER2/neu] against Chemotherapeutic-Resistant Mammary Adenocarcinoma (SKBr-3) and the Complementary Effect of Mebendazole

Carnosic Acid, Tangeretin, and Ginkgolide-B Anti-neoplastic Cytotoxicity in Dual Combination with Dexamethasone-[anti-EGFR] in Pulmonary Adenocarcinoma (A549).

Traditional chemotherapeutics of low-molecular weight diffuse passively across intact membrane structures of normal healthy cells found in tissues and organ systems in a non-specific unrestricted manner which largely accounts for the induction of most sequelae which restrict dosage, administration frequency, and duration of therapeutic intervention. Molecular strategies that offer enhanced levels of potency, greater efficacy and broader margins-of-safety include the discovery of alternative candidate therapeutics and development of methodologies capable of mediating properties of selective "targeted" delivery. The covalent immunopharmaceutical, dexamethasone-(C21-phosphoramidate)-[anti- EGFR] was synthesized utilizing organic chemistry reactions that comprised a multi-stage synthesis regimen. Multiple forms of analysis were implemented to vadliate the successful synthesis (UV spectrophotometric absorbance), purity and molar-incorporation-index (UV spectrophotometric absorbance, chemical-based protein determination), absence of fragmentation/polymerization (SDS-PAGE/chemiluminescent autoradiography), retained selective binding-avidity of IgG-immunoglobulin (cell-ELISA); and selectively "targeted" antineoplastic cytotoxicity (biochemistry-based cell vitality/viability assay). The botanicals carnosic acid, ginkgolide-B and tangeretin, each individually exerted maximum antineoplastic cytotoxicity levels of 58.1%, 5.3%, and 41.1% respectively against pulmonary adenocarcinoma (A549) populations. Dexamethasone-(C21-phosphoramidate)-[anti-EGFR] formulated at corticosteroid/ glucocorticoid equivalent concentrations produced anti-neoplastic cytotoxicity at levels of 7.7% (10-9 M), 26.9% (10-8 M), 64.9% (10-7 M), 69.9% (10-6 M) and 73.0% (10-5 M). Ccarnosic acid, ginkgolide-B and tangeretin in simultaneous dual-combination with dexamethasone-(C21-phosphoramidate)-[anti-EGFR] exerted maximum anti-neoplastic cytotoxicity levels of 70.5%, 58.6%, and 69.7% respectively. Carnosic acid, ginkgolide-B and tangeretin botanicals exerted anti-neoplastic cytotoxicity against pulmonary adenocarcinoma (A549) which additively contributed to the anti-neoplastic cytotoxic potency of the covalent immunopharmaceutical, dexamethasone-(C21-phosphoramidate)-[anti-EGFR]. Carnosic acid and tangeretin were most potent in this regard both individually and in dual-combination with dexamethasone-(C21- phosphoramidate)-[anti-EGFR]. Advantages and attributes of carnosic acid and tangeretin as potential monotherapeutics are a wider margin-of-safety of conventional chemotherapeutics which would readily complement the selective "targeted" delivery properties of dexamethasone-(C21-phosphoramidate)-[anti-EGFR] and possibly other covalent immunopharmaceuticals in addition to providing opportunities for the discovery of combination therapies that provide heightened levels of anti-neoplastic efficacy.

Chemotherapy-induced oral mucositis is associated with detrimental bacterial dysbiosis

BackgroundGastrointestinal mucosal injury (mucositis), commonly affecting the oral cavity, is a clinically significant yet incompletely understood complication of cancer chemotherapy. Although antineoplastic cytotoxicity constitutes the primary injury trigger, the interaction of oral microbial commensals with mucosal tissues could modify the response. It is not clear, however, whether chemotherapy and its associated treatments affect oral microbial communities disrupting the homeostatic balance between resident microorganisms and the adjacent mucosa and if such alterations are associated with mucositis. To gain knowledge on the pathophysiology of oral mucositis, 49 subjects receiving 5-fluorouracil (5-FU) or doxorubicin-based chemotherapy were evaluated longitudinally during one cycle, assessing clinical outcomes, bacterial and fungal oral microbiome changes, and epithelial transcriptome responses. As a control for microbiome stability, 30 non-cancer subjects were longitudinally assessed. Through complementary in vitro assays, we also evaluated the antibacterial potential of 5-FU on oral microorganisms and the interaction of commensals with oral epithelial tissues.ResultsOral mucositis severity was associated with 5-FU, increased salivary flow, and higher oral granulocyte counts. The oral bacteriome was disrupted during chemotherapy and while antibiotic and acid inhibitor intake contributed to these changes, bacteriome disruptions were also correlated with antineoplastics and independently and strongly associated with oral mucositis severity. Mucositis-associated bacteriome shifts included depletion of common health-associated commensals from the genera Streptococcus, Actinomyces, Gemella, Granulicatella, and Veillonella and enrichment of Gram-negative bacteria such as Fusobacterium nucleatum and Prevotella oris. Shifts could not be explained by a direct antibacterial effect of 5-FU, but rather resembled the inflammation-associated dysbiotic shifts seen in other oral conditions. Epithelial transcriptional responses during chemotherapy included upregulation of genes involved in innate immunity and apoptosis. Using a multilayer epithelial construct, we show mucositis-associated dysbiotic shifts may contribute to aggravate mucosal damage since the mucositis-depleted Streptococcus salivarius was tolerated as a commensal, while the mucositis-enriched F. nucleatum displayed pro-inflammatory and pro-apoptotic capacity.ConclusionsAltogether, our work reveals that chemotherapy-induced oral mucositis is associated with bacterial dysbiosis and demonstrates the potential for dysbiotic shifts to aggravate antineoplastic-induced epithelial injury. These findings suggest that control of oral bacterial dysbiosis could represent a novel preventive approach to ameliorate oral mucositis.

Cyclin-Dependent Kinases (CDK) Signaling Blockade Potentiates NK Cell Mediated Cytotoxicity Against Acute Myelogenous Leukemia

▪Dinaciclib, a CDK 1, 2, 5, and 9 inhibitor, has been shown to have potent anti-cancer activity against several malignancies. Our group previously demonstrated a direct RALB-dependent proapoptotic activity of dinaciclib against acute myeloid leukemia (AML) (Oncogene (2017) 36, 3263-3273). Furthermore, there is emerging evidence that dinaciclib can influence the recognition and killing of cancer cells by the immune system suggesting that it could have multimodal anti-cancer activities. As natural killer (NK) cells have a critical role in antineoplastic cytotoxicity, we hypothesized that dinaciclib may influence NK cell mediated cytotoxicity against human AML cells. To address this hypothesis, we evaluated the effects of dinaciclib on the NK cytotoxicity against human AML target cells and expression of known regulators of NK cell function. Specifically, we treated THP-1 and KG-1 cells with vehicle or dinaciclib at 5-20 nM for 24 hours, then washed the AML targets and added purified NK cells from PBMCs of healthy donors at an effector: target (E:T) ratio of 2:1. After 24 hours, viable AML target cells were enumerated using Fixable Aqua Dead Cell Stain and AccuCheck Counting Beads (Thermo Fisher Scientific) by flow cytometry. Relative NK cell target killing (%) was calculated by comparing the percentage of AML targets killed with drug treatment combined with donor NK cells relative to drug treatment alone in the absence of NK cells (Relative NK killing %). We assessed NK cell activation by measuring intracellular interferon-ɣ production and CD107a expression as well as NK ligand expression on dinaciclib-treated AML targets using flow cytometry. Dinaciclib treatment of AML targets enhanced the relative NK killing % for both KG-1 and THP-1 AML targets compared to control treatment (for KG-1, 45.6% vs 14.5%, p=0.05; for THP-1, 63.7% vs 31.7%, p=0.01) (Figure 1A, 1B) and was associated with enhanced NK cell degranulation as measured by CD107a expression (DMSO group, 8.3% vs dinaciclib 20nM group, 17.7%, p=0.002) and inflammatory cytokine production as measured by intracellular interferon-ɣ expression (DMSO group, 10.4% vs dinaciclib 20nM group 17.3%, p=0.01) (Figure 1C, 1D) supporting the ability of dinaciclib to sensitize AML targets for NK cell-based immunotherapy. To investigate potential mechanisms that promote NK cell activation by dinaciclib-treated AML targets, we assessed expression of several key NK cell ligands on AML targets and found that dinaciclib treatment led to decreased expression of inhibitory ligands including HLA class I, CD112, CD155 and increased expression in activating ligands such as TRAILR1, CD48 (Figure 1E, 1F) providing potential mechanistic insights. Notably, preliminary studies using a primary, patient-derived AML sample treated with dinaciclib resulted in a similar increase in the proportion of CD107a+ NK cells compared to the control group (44.7% increase, p=0.02, Figure 1G, 1H). While the detailed mechanisms for our findings remain to be determined, this is the first report to our knowledge that inhibiting CDK signaling can sensitize AML targets to NK cell based immunotherapy, a completely novel treatment approach. Further studies are ongoing to investigate the potential for combined targeted therapy with dinaciclib and adoptive NK cell therapy including patient derived xenograft (PDX) mice to validate its translational potential. [Display omitted] DisclosuresFelices:GT Biopharma: Research Funding.

Anti-neoplastic cytotoxicity by complementary simultaneous selective “targeted” delivery for pulmonary adenocarcinoma: fludarabine-(5′-phosphoramidate)-[anti-IGF-1R] in dual-combination with dexamethasone-(C21-phosphoramidate)-[anti-EGFR

Selective “targeted” delivery of chemotherapeutic agents represents a molecular strategy for achieving enhanced margins-of-safety and greater potency compared to conventional small-molecular weight chemotherapeutics. Greater margins-of-safety are possible because extensive un-intentional diffusion of therapeutics across intact cell membranes of normal healthy cells residing within tissues and organ systems is minimized or avoided. Simultaneous, independent “targeted” delivery of one or more therapeutics at multiple sites on the external surface membrane of neoplastic cells has not been described extensively. Fludarabine-(5′-phosphoramidate)-[anti-IGF-1R] and dexamethasone-(C21-phosphoramidate)-[anti-EGFR] were synthesized by reacting fludarabine or dexamethasone-C21-monophosphate with a carbodiimide reagent and imidazole for synthesis of covalent fludarabine-(5′-phosphoramidate)-[anti-IGF-1R] and dexamethasone-(C21-phosphoramidate)-[anti-EGFR] immunopharmaceuticals. The pharmaceutical molar-incorporation-indexes for fludarabine-(C5-phosphoramidate)-[anti-IGF-1R] and dexamethasone-(C21-phosphoramidate)-[anti-EGFR] were 3.67:1 and 6.95:1 respectively. Simultaneous dual-combination selective “targeted” delivery with fludarabine-(5′-phosphoramidate)-[anti-IGF-1R] with dexamethasone-(C21-phosphoramidate)-[anti-EGFR] produced the most profound enhancement in anti-neoplastic cytotoxitity at the pharmaceutical-equivalent concentations of 10− 9 M (14.7% ± 0.80 SE) and 10− 8 M (54.0% ± 0.9 SE) respectively. Simultaneous dual “targeted” anti-neoplastic cytotoxicity can potentially be evoked ex-vivo and in-vivo with fludarabine-(5′-phosphoramidate)-[anti-IGF-1R] and dexamethasone-(C21-phosphoramidate)-[anti-EGFR] through the synergistic and additive efficacy achieved through the combined mechanisms-of-actions associated with; (1) covalently bound therapeutic moieties; (2) anti-trophic receptor IgG-immunoglobulin; and (3) activation of three host immune responses.

Selectively Targeted Anti-Neoplastic Cytotoxicity of Three Immunopharmaceuticals with Covalently Bound Fludarabine, Gemcitabine and Dexamethasone Moieties Synthesized Utilizing Organic Chemistry Reactions in a Multi-Stage Regimen.

Unintentional passive diffusion of conventional small molecular weight pharmaceuticals across intact membranes of normal healthy cells in tissues and organ systems induces sequelae that limit therapeutic dosage and duration of administration. Selective "targeted" delivery of pharmaceuticals is a molecular strategy that can potentially provide heightened margins-of-safety with greater potency and improved efficacy. Monophosphate analogs of fludarabine, gemcitabine, and dexamethasone were combined with a carbodiimide reagent in the presence of imidazole to produce reactive intermediates that were subsequently covalently bound to monoclonal anti-IGF-1R or anti-EGFR IgG-immunoglobulin. The resulting covalent immunopharmaceutical end-products, fludarabine-(5'-phosphoramidate)-[anti-IGF-1R], gemcitabine-(5'- phosphoramidate)-[anti-IGF-1R], and dexamethasone-(C21-phosphoramidate)-[anti-EGFR] were evaluated by SDS-PAGE/chemiluminescent autoradiography (fragmentation/polymerization detection), UV spectrophotometric absorbance (purity; molar-incorporation-index), cell-ELISA (retained selective binding-avidity), and cell vitality-viability (selectively "targeted" anti-neoplastic cytotoxicity). Maximum selectively "targeted" anti-neoplastic cytotoxicity of fludarabine-(5'-phosphoramidate)-[anti- IGF-1R], gemcitabine-(5'-phosphoramidate)-[anti-IGF-1R], and dexamethasone-(C21-phosphoramidate)-[anti- EGFR] was detected at the pharmaceutical-equivalent concentrations of 10-5 M (94.7%), 10-7 M (93.1%), and 10-7 M (64.9%) respectively. Organic chemistry reactions were optimized in a template multi-stage synthesis regimen for fludarabine-( 5'-phosphoramidate)-[anti-IGF-1R], gemcitabine-(5'-phosphoramidate)-[anti-IGF-1R], and dexamethasone-( C21-phosphoramidate)-[anti-EGFR]. Attributes of the synthesis regimen include; [-i-] covalent bonding of pharmaceutical moeities at high molar incorporation indexes, [-ii-] implementation of organic chemistry reactions in a non-dedicated synthesis regimen allowing component substitution and [-iii-] optional preservation of presynthesized amine-reactive pharmaceutical intermediates for on-demand immunopharmaceutical synthesis. Attributes of the covalent immunopharmaceuticals are; absence of any synthetically introduced chemical groups, retained IgG-immunoglobulin binding-avidity and potent selective "targeted" anti-neoplastic cytotoxic potency. Under in-vivo conditions, supplemental anti-neoplastic cytotoxicity is realized through trophic receptor inhibition and activation of multiple cytotoxic host immune responses.

Gemcitabine-(5'-phosphoramidate)-[anti-IGF-1R]: molecular design, synthetic organic chemistry reactions, and antineoplastic cytotoxic potency in populations of pulmonary adenocarcinoma (A549).

One molecular‐based approach that increases potency and reduces dose‐limited sequela is the implementation of selective ‘targeted’ delivery strategies for conventional small molecular weight chemotherapeutic agents. Descriptions of the molecular design and organic chemistry reactions that are applicable for synthesis of covalent gemcitabine‐monophosphate immunochemotherapeutics have to date not been reported. The covalent immunopharmaceutical, gemcitabine‐(5′‐phosphoramidate)‐[anti‐IGF‐1R] was synthesized by reacting gemcitabine with a carbodiimide reagent to form a gemcitabine carbodiimide phosphate ester intermediate which was subsequently reacted with imidazole to create amine‐reactive gemcitabine‐(5′‐phosphorylimidazolide) intermediate. Monoclonal anti‐IGF‐1R immunoglobulin was combined with gemcitabine‐(5′‐phosphorylimidazolide) resulting in the synthetic formation of gemcitabine‐(5′‐phosphoramidate)‐[anti‐IGF‐1R]. The gemcitabine molar incorporation index for gemcitabine‐(5′‐phosphoramidate)‐[anti‐IGF‐R1] was 2.67:1. Cytotoxicity Analysis – dramatic increases in antineoplastic cytotoxicity were observed at and between the gemcitabine‐equivalent concentrations of 10−9 M and 10−7 M where lethal cancer cell death increased from 0.0% to a 93.1% maximum (100.% to 6.93% residual survival), respectively. Advantages of the organic chemistry reactions in the multistage synthesis scheme for gemcitabine‐(5′‐phosphoramidate)‐[anti‐IGF‐1R] include their capacity to achieve high chemotherapeutic molar incorporation ratios; option of producing an amine‐reactive chemotherapeutic intermediate that can be preserved for future synthesis applications; and non‐dedicated organic chemistry reaction scheme that allows substitutions of either or both therapeutic moieties, and molecular delivery platforms.

Dexamethasone-(C21-phosphoramide)-[anti-EGFR]: molecular design, synthetic organic chemistry reactions, and antineoplastic cytotoxic potency against pulmonary adenocarcinoma (A549).

PurposeCorticosteroids are effective in the management of a variety of disease states, such as several forms of neoplasia (leukemia and lymphoma), autoimmune conditions, and severe inflammatory responses. Molecular strategies that selectively “target” delivery of corticosteroids minimize or prevents large amounts of the pharmaceutical moiety from passively diffusing into normal healthy cell populations residing within tissues and organ systems.Materials and methodsThe covalent immunopharmaceutical, dexamethasone-(C21-phosphoramide)-[anti-EGFR] was synthesized by reacting dexamethasone-21-monophosphate with a carbodiimide reagent to form a dexamethasone phosphate carbodiimide ester that was subsequently reacted with imidazole to create an amine-reactive dexamethasone-(C21-phosphorylimidazolide) intermediate. Monoclonal anti-EGFR immunoglobulin was combined with the amine-reactive dexamethasone-(C21-phosphorylimidazolide) intermediate, resulting in the synthesis of the covalent immunopharmaceutical, dexamethasone-(C21-phosphoramide)-[anti-EGFR]. Following spectrophotometric analysis and validation of retained epidermal growth factor receptor type 1 (EGFR)-binding avidity by cell-ELISA, the selective anti-neoplasic cytotoxic potency of dexamethasone-(C21-phosphoramide)-[anti-EGFR] was established by MTT-based vitality stain methodology using adherent monolayer populations of human pulmonary adenocarcinoma (A549) known to overexpress the tropic membrane receptors EGFR and insulin-like growth factor receptor type 1.ResultsThe dexamethasone:IgG molar-incorporation-index for dexamethasone-(C21-phosphoramide)-[anti-EGFR] was 6.95:1 following exhaustive serial microfiltration. Cytotoxicity analysis: covalent bonding of dexamethasone to monoclonal anti-EGFR immunoglobulin did not significantly modify the ex vivo antineoplastic cytotoxicity of dexamethasone against pulmonary adenocarcinoma at and between the standardized dexamethasone equivalent concentrations of 10−9 M and 10−5 M. Rapid increases in antineoplastic cytotoxicity were observed at and between the dexamethasone equivalent concentrations of 10−9 M and 10−7 M where cancer cell death increased from 7.7% to a maximum of 64.9% (92.3%–35.1% residual survival), respectively, which closely paralleled values for “free” noncovalently bound dexamethasone.DiscussionOrganic chemistry reaction regimens were optimized to develop a multiphase synthesis regimen for dexamethasone-(C21-phosphoramide)-[anti-EGFR]. Attributes of dexamethasone-(C21-phosphoramide)-[anti-EGFR] include a high dexamethasone molar incorporation-index, lack of extraneous chemical group introduction, retained EGFR-binding avidity (“targeted” delivery properties), and potential to enhance long-term pharmaceutical moiety effectiveness.

DHA is a newly defined STAT3 inhibitor that may be of multiple potential uses in cancer treatment

Accumulating evidence from epidemiological, pharmacological and case control studies has shown that dihydroartemisinin (DHA), one of first-line antimalarial drugs recommended by World Health Organization (WHO), possesses antineoplastic activity and selective cytotoxicity to a variety of human cancer cell model systems. Although some antitumor mechanisms of DHA have been confirmed, the findings from the previous studies are insufficient to fully reveal the whole picture of tumor suppression by DHA. In a recent investigation, we demonstrated that DHA exhibits antitumor properties against a variety of human HNSCC cells both in vitro and in vivo. The underlying mechanism involves selective inhibition of Jak2/STAT3 signaling. By specific inhibition of STAT3 activation, DHA exerted the chemotherapeutic efficacy, including reduction in cell viability and migratory capability, along with induction of G1 phase cell cycle arrest and apoptosis in HNSCC cells. All of data suggested that DHA may be a potent inhibitor of STAT3 that can be potentially used to treat patients with HNSCC, as well as other human cancers harboring STAT3 activation.

Anti-Neoplastic Cytotoxicity of SN-38-Loaded PCL/Gelatin Electrospun Composite Nanofiber Scaffolds against Human Glioblastoma Cells In Vitro

Electrospun poly(ε-caprolactone) (PCL)/gelatin (GT) scaffolds were developed to provide controlled release of 7-ethyl-10-hydroxy camptothecin (SN-38). Acetic acid was introduced to improve the miscibility of PCL and GT to produce a homogeneous nanofiber membrane mixture. The effect of SN-38 content in binary mixtures on processability, fiber morphology, water sorption, swelling, and drug release was investigated. Electrospun PCL/GT blend nonwoven fibers showed fiber surface roughness, decreased PCL crystallinity, and increased swelling with increasing drug content of 1, 2, and 4 wt %. Additionally, increasing the SN-38 concentration reduced the degradation rate of the GT. Furthermore, we hypothesize the existence of a drug content saturation point in the monoaxial fiber to explain the different drug release patterns of PG2 compared with those of PG1 and PG4. The matrix also showed good biodegradation and anti-tumor function. Our results demonstrate that SN-38-loaded PCL/GT fibers can be obtained by electrospinning. The SN-38-loaded fibers merit further evaluation as a means to potentially prevent locoregional recurrence following surgical tumor resection.

Simultaneous Dual Selective Targeted Delivery of Two Covalent Gemcitabine Immunochemotherapeutics and Complementary Anti-Neoplastic Potency of [Se]-Methylselenocysteine.

The anti-metabolite chemotherapeutic, gemcitabine is relatively effective for a spectrum of neoplastic conditions that include various forms of leukemia and adenocarcinoma/carcinoma. Rapid systemic deamination of gemcitabine accounts for a brief plasma half-life but its sustained administration is often curtailed by sequelae and chemotherapeutic-resistance. A molecular strategy that diminishes these limitations is the molecular design and synthetic production of covalent gemcitabine immunochemotherapeutics that possess properties of selective “targeted” delivery. The simultaneous dual selective “targeted” delivery of gemcitabine at two separate sites on the external surface membrane of a single cancer cell types represents a therapeutic approach that can increase cytosol chemotherapeutic deposition; prolong chemotherapeutic plasma half-life (reduces administration frequency); minimize innocent exposure of normal tissues and healthy organ systems; and ultimately enhance more rapid and thorough resolution of neoplastic cell populations. Materials and Methods: A light-reactive gemcitabine intermediate synthesized utilizing succinimidyl 4,4-azipentanoate was covalently bound to anti-EGFR or anti-HER2/neu IgG by exposure to UV light (354-nm) resulting in the synthesis of covalent immunochemotherapeutics, gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu]. Cytotoxic anti-neoplastic potency of gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] between gemcitabine-equivalent concentrations of 10−12 M and 10−6 M was determined utilizing chemotherapeutic-resistant mammary adenocarcinoma (SKRr-3). The organoselenium compound, [Se]-methylselenocysteine was evaluated to determine if it complemented the anti-neoplastic potency of the covalent gemcitabine immunochemotherapeutics. Results: Gemcitabine-(C4-amide)-[anti-EGFR], gemcitabine-(C4-amide)-[anti-HER2/neu] and the dual simultaneous combination of gemcitabine-(C4-amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] all had anti-neoplastic cytotoxic potency against mammary adenocarcinoma. Gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] produced progressive increases in anti-neoplastic cytotoxicity that were greatest between gemcitabine-equivalent concentrations of 10−9 M and 10−6 M. Dual simultaneous combinations of gemcitabine-(C4-amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] produced levels of anti-neoplastic cytotoxicity intermediate between each of the individual covalent gemcitabine immunochemotherapeutics. Total anti-neoplastic cytotoxicity of the dual simultaneous combination of gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) was substantially higher when formulated with [Se]-methylsele-nocysteine.

Fludarabine- (C2-methylhydroxyphosphoramide)- [anti-IGF-1R]: Synthesis and Selectively "Targeted"Anti-Neoplastic Cytotoxicity against Pulmonary Adenocarcinoma (A549).

Many if not most conventional small molecular weight chemotherapeutics are highly potent against many forms of neoplastic disease. Unfortunately, majority of an administered dose unintentionally diffuses passively into normal tissues and healthy organ systems following intravenous administration. One strategy for both increasing potency and reducing dose-limited sequela is the selective "targeted" delivery of conventional chemotherapeutic agents. The fludarabine-(C2- methylhydroxyphosphoramide)-[anti-IGF-1R] was synthesized by initially reacting fludarabine with a carbodiimide to form a fludarabine carbodiimide phosphate ester intermediate that was subsequently reacted with imidazole to create an amine-reactive fludarabine- (C2-phosphorylimidazolide) intermediate. Monoclonal anti-IGF-1R immunoglobulin was combined with the amine-reactive fludarabine- (C2-phosphorylimidazolide) intermediate resulting in the synthesis of covalent fludarabine-(C2-methylhydroxyphosphoramide)- [anti-IGF-1R] immunochemotherapeutic. Residual fludarabine and un-reacted reagents were removed by serial microfiltration (MWCO 10,000) and monitored by analytical-scale HP-TLC. Retained IGF-1R binding-avidity of fludarabine-(C2- methylhydroxyphosphoramide)-[anti-IGF-1R] was established by cell-ELISA using pulmonary adenocarcinoma cell (A549) which over-expresses IGF-1R and EGFR. Anti-neoplastic cytotoxic potency of fludarabine-(C2-methylhydroxyphosphoramide)-[anti- IGF-1R] was determined against pulmonary adenocarcinoma (A549) using an MTT-based vitality stain methodology. The fludarabine molar-incorporation-index for fludarabine- (C2-methylhydroxyphosphoramide)-[anti-IGF-R1] was 3.67:1 while non-covalently bound fludarabine was not detected by analytical scale HP-TLC following serial micro-filtration. Size-separation fludarabine-(C2-methylhydroxyphosphoramide)-[anti- IGF-1R] by SDS-PAGE with chemo luminescent autoradiography detected only a single 150-kDa band. Cell-ELISA of fludarabine- (C2-methylhydroxyphosphoramide)-[anti-IGF-1R] measuring total immunoglobulin bound to exterior surface membranes of pulmonary adenocarcinoma (A549) increased with elevations in immunoglobulin-equivalent concentrations of the covalent fludarabine immunochemotherapeutic. Between the fludarabine-equivalent concentrations of 10-10 M and 10-5 M both fludarabine-(C2- methylhydroxyphosphoramide)-[anti-IGF-1R] and fludarabine had ex-vivo anti-neoplastic cytotoxic potency levels that increased rapidly between the fludarabine-equivalent concentrations of 10-6 M and 10-5 M where cancer cell death percentages increased from 24.4% to a maximum of 94.7% respectively. The molecular design and organic chemistry reaction schemes were developed for synthesizing fludarabine-(C2- methylhydroxyphosphoramide)-[anti-IGF-1R] which possessed both properties of selective "targeted" delivery and anti-neoplastic cytotoxic potency equivalent to fludarabine chemotherapeutic.

Anti-Neoplastic Cytotoxicity of Gemcitabine-(C4-amide)-[anti-EGFR] in Dual-combination with Epirubicin-(C3-amide)-[anti-HER2/neu] against Chemotherapeutic-Resistant Mammary Adenocarcinoma (SKBr-3) and the Complementary Effect of Mebendazole.

AimsDelineate the feasibility of simultaneous, dual selective “targeted” chemotherapeutic delivery and determine if this molecular strategy can promote higher levels anti-neoplastic cytotoxicity than if only one covalent immunochemotherapeutic is selectively “targeted” for delivery at a single membrane associated receptor over-expressed by chemotherapeutic-resistant mammary adenocarcinoma.MethodologyGemcitabine and epirubicin were covalently bond to anti-EGFR and anti-HER2/neu utilizing a rapid multi-phase synthetic organic chemistry reaction scheme. Determination that 96% or greater gemcitabine or epirubicin content was covalently bond to immunoglobulin fractions following size separation by micro-scale column chromatography was established by methanol precipitation analysis. Residual binding-avidity of gemcitabine-(C4-amide)-[anti-EG-FR] applied in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu] was determined by cell-ELIZA utilizing chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) populations. Lack of fragmentation or polymerization was validated by SDS-PAGE/immunodetection/chemiluminescent autoradiography. Anti-neoplastic cytotoxic potency was determined by vitality stain analysis of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) monolayers known to uniquely over-express EGFR (2 × 105/cell) and HER2/neu (1 × 106/cell) receptor complexes. The covalent immunochemotherapeutics gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] were applied simultaneously in dual-combination to determine their capacity to collectively evoke elevated levels of anti-neoplastic cytotoxicity. Lastly, the tubulin/microtubule inhibitor mebendazole evaluated to determine if it’s potential to complemented the anti-neoplastic cytotoxic properties of gemcitabine-(C4-amide)-[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu].ResultsDual-combination of gemcitabine-(C4-amide)-[anti-EGFR] with epirubicin-(C3-amide)-[anti-HER2/neu] produced greater levels of anti-neoplastic cytotoxicity than either of the covalent immunochemotherapeutics alone. The benzimidazole microtubule/tubulin inhibitor, mebendazole complemented the anti-neoplastic cytotoxicity of gemcitabine-(C4-amide)-[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu].ConclusionsThe dual-combination of gemcitabine-(C4-amide)-[anti-EGFR] with epirubicin-(C3-amide)-[anti-HER2/neu] produced higher levels of selectively “targeted” anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) than either covalent immunochemotherapeutic alone. The benzimidazole tubulin/microtubule inhibitor, mebendazole also possessed anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) and complemented the potency and efficacy of gemcitabine-(C4-amide)-[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu].

Anti-Neoplastic Cytotoxicity of Gemcitabine-(C4-amide)-[anti-HER2/neu] in Combination with Griseofulvin against Chemotherapeutic-Resistant Mammary Adenocarcinoma (SKBr-3).

IntroductionGemcitabine is a pyrimidine nucleoside analog that becomes triphosphorylated and in this form it competitively inhibits cytidine incorporation into DNA strands. Diphosphorylated gemcitabine irreversibly inhibits ribonucleotide reductase thereby preventing deoxyribonucleotide synthesis. Functioning as a potent chemotherapeutic, gemcitabine decreases neoplastic cell proliferation and induces apoptosis which accounts for its effectiveness in the clinical treatment of several leukemia and carcinoma cell types. A brief plasma half-life due to rapid deamination, chemotherapeuticresistance and sequelae restricts gemcitabine utility in clinical oncology. Selective “targeted” gemcitabine delivery represents a molecular strategy for prolonging its plasma half-life and minimizing innocent tissue/organ exposure.MethodsA previously described organic chemistry scheme was applied to synthesize a UV-photoactivated gemcitabine intermediate for production of gemcitabine-(C4-amide)-[anti-HER2/neu]. Immunodetection analysis (Western-blot) was applied to detect the presence of any degradative fragmentation or polymerization. Detection of retained binding-avidity for gemcitabine-(C4-amide)-[anti-HER2/neu] was determined by cell-ELISA using populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) that highly over-express the HER2/neu trophic membrane receptor. Anti-neoplastic cytotoxicity of gemcitabine-(C4-amide)-[anti-HER2/neu] and the tubulin/microtubule inhibitor, griseofulvin was established against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3). Related investigations evaluated the potential for gemcitabine-(C4-amide)-[anti-HER2/neu] in dual combination with griseofulvin to evoke increased levels of anti-neoplastic cytotoxicity compared to gemcitabine-(C4-amide)-[anti-HER2/neu].ResultsCovalent gemcitabine-(C4-amide)-[anti-HER2/neu] immunochemotherapeutic and griseofulvin exerted anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3). Covalent gemcitabine-(C4-amide)-[anti-HER2/neu] immunochemotherapeutic or gemcitabine in dual combination with griseofulvin created increased levels of anti-neoplastic cytotoxicity that were greater than was attainable with gemcitabine-(C4-amide)-[anti-HER2/neu] or gemcitabine alone.ConclusionGemcitabine-(C4-amide)-[anti-HER2/neu] in dual combination with griseofulvin can produce enhanced levels of anti-neoplastic cytotoxicity and potentially provide a basis for treatment regimens with a wider margin-of-safety. Such benefits would be possible through the collective properties of; [i] selective “targeted” gemcitabine delivery; [ii] relatively lower toxicity of griseofulvin compared to many if not most conventional chemotherapeutics; [iii] reduced total dosage requirements faciliated by additive or synergistic anti-cancer properties; and [iv] differences in sequelae for gemcitabine-(C4-amide)-[anti-HER2/neu] compared to griseofulvin functioning as a tubulin/microtubule inhibitor.

Synthesis, cytotoxicity and antiplasmodial activity of novel ent-kaurane derivatives

This paper reports on the syntheses and spectrometric characterisation of eleven novel ent-kaurane diterpenoids, including a complete set of 1H, 13C NMR and crystallographic data for two novel ent-kaurane diepoxides. Moreover, the antineoplastic cytotoxicity for kaurenoic acid and the majority of ent-kaurane derivatives were assessed in vitro against a panel of fourteen cancer cell lines, of which allylic alcohols were shown to be the most active compounds. The good in vitro antimalarial activity and the higher selectivity index values observed for some ent-kaurane epoxides against the chloroquine-resistant W2 clone of Plasmodium falciparum indicate that this class of natural products may provide new hits for the development of antimalarial drugs.

Synthesis, characterisation, and antineoplastic cytotoxicity of hybrid naphthohydroquinone–nucleic base mimic derivatives

From a partially degraded Diels–Alder adduct of α-myrcene and 1,4-benzoquinone, several model compounds belonging to a new series of 1,4-naphthohydroquinone derivatives have been prepared. Phenyl, pyridyl, imidazolyl and some nucleic base mimic heterocycles have been attached to the naphthohydroquinone system through linkers of different size and type, leading to potentially antineoplastic hybrid structures. The new compounds have been evaluated in vitro for their cytotoxicity against cultured human cancer cells of A-549 lung carcinoma, HT-29 colon adenocarcinoma and MDA-MB-231 breast carcinoma. GI50 values ranged in the μM level.

Lipid Nucleoside Conjugates for the Treatment of Cancer

Phospholipid nucleoside conjugates and nucleosides with chemical additions to the hydroxyl and amino moieties have been used since the 1970s in order to increase the biological activity of the parent compound. Previous investigators have found that adding lipid moieties to ara-C or chemically linking ara-C to a phospholipid creates a prodrug that exhibits superior cytotoxic activity compared to ara-C alone when used in animal tumor models. The novel ara-C molecules reveal different pharmacological profiles from the parent compound such as decreased catabolism by cytidine deaminase, increased plasma half-life, and release of nucleoside monophosphate, a reaction that bypasses the rate limiting initial nucleoside phosphorylation. Additionally, these compounds were able to penetrate the blood-brain barrier and were active against tumor cells implanted i.c. The purpose of this review is to briefly cover the history and successes of previous investigators who have synthesized and tested these phospholipid ara-C conjugates, to discuss recent phospholipid ara-C and fludarabine conjugates, and to discuss the synthetic design and synthesis of a novel phospholipid gemcitabine conjugate. These phospholipid nucleoside conjugates possess the potential to have superior anti-neoplastic cytotoxicity profiles with fewer side effects than the parent nucleoside and merit further investigation.

Amifostine: a tonic or toxin to myeloid progenitors

It should come as no surprise, that the aminothiol prodrug, Amifostine (EthyolTM), whose actions as a hematopoietic promoter were recently realized, should now be considered to exert opposing biologic effects in leukemic cells [1]. A product of the United States Defense Department research emanating from the cold war, the relative selective cytoprotection afforded by amifostine to normal cells compared to tumor cells seemed at first glance somewhat difficult to accept. Indeed, relative differences in alkaline phosphatase distribution and pH within the tumor milieu appeared plausible [2–4], but insufficient to explain the agent’s ability to potentiate antineoplastic cytotoxicity in selected malignancies while conferring no protection to leukemic cells and protecting normal tissues [5–8]. The answer to this apparent dichotomy lies in amifostine’s chemical derivation and structural resemblance to endogenous polyamines, abundant but highly regulated organic cations involved in the homeostatic balance of cell death and survival. The dephosphorylated form of amifostine, WR-1065, and its symmetrical disulfide oxidation product, WR33278, display striking homology to spermidine and spermine, respectively [9,10]. In the oxidized extracellular environment, WR-33278 is believed to predominate, gaining entry into cells as a substrate for the polyamine facilitative transporter [11,12]. The activity of these transporters are strictly regulated by the feedback inhibitor, antizyme, which serves as a gate-keeper to limit intracellular polyamine content [13,14]. Within the cell, WR-33278 can be reduced to WR-1065. These novel aminothiols can then modulate cell behavior in two ways. They can compete with the naturally occurring polyamines for binding to DNA [15] and as substrates for amine oxidases [16]. Like endogenous polyamines, they induce the activity of spermine/spermidine acetyl transferase (SSAT), the first enzyme in polyamine catabolism, which is transcriptionally activated by the erythroid-specific transcription factor GATA-1 as an early event in erythroid differentiation [16,17]. In addition, these molecules can activate unique patterns of gene expression via polyamine-independent mechanisms, such as the redox sensitive tumor suppressor gene, p53 [18]. Like the polyamines, the cationic charge of the aminothiols facilitates their avid DNA binding, where they exert their many biologic activities including activation of early response genes, thymidine kinase, NFk-B, and other genes involved in the maintenance of genomic integrity [19–21]. Polyamines and polyamine analogues, like classical thiol antioxidants, afford protection against nuclear oxidation by serving as scavengers of reactive oxygen intermediates [22–24]. How then can we expect divergent effects on cell survival in normal, myelodysplastic and leukemic progenitors? Proliferating tumor cells maintain a high cellular polyamine content via ornithine decarboxylase-controlled biosynthesis; thereby blocking cell import by the induction of antizyme. Significant changes in polyamine homeostasis in tumor cells can be catastrophic, resulting in growth arrest and/or programmed cell death. Although active import of WR33278 is inhibited [25], passive inward diffusion of WR-1065 accelerates polyamine degradation by induction of SSAT, resulting in lowered apoptotic threshold. Sensing exceedingly high intracellular polyamine content, antizyme binds to and destabilizes ornithine decarboxylase, triggering its rapid proteosome degradation [26,27]. In normal cells where polyamine generation is not upregulated, the disulfide is actively imported into the cell, affording added defense against oxidative stress and resistance to apoptosis induction. The latter is indeed the case for normal hematopoietic progenitors,