Workshop on imaging science development for cancer prevention and preemption

Flavonoids have been used as antioxidant, chemopreventive and chemosensitizing agents. In this study, eleven flavonoids containing a variety of hydroxy (OH) and/or methoxy (OMe) groups were evaluated for their antioxidant, cytotoxic and chemosensitizing effects to create a structure-activity relationship (SAR). 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical solution-based chemical assay and and 2',7'-dichlorofluorescin diacetate (DCFH-DA) cellular-based assay were used to compare the free radical scavenging activity on the same molar concentration basis using the AML-2/DX100 cells which are characterized by the down-regulated expression of catalase and resulting supersensitiviy to hydrogen peroxide. The chemosensitization and cytotoxicity were determined by the MTT assay in the presence or absence of an anticancer drug using the P-glycoprotein-overexpressing AML-2 subline AML-2/D100 cells. The antioxidant activity of the flavonoid (3,5,7,3',4'-OH) was higher than that of the flavonoid (5,7,3',4'-OH). Flavonoids substituted with the various number of OMe decreased antioxidant activity. Flavonoids with 7-OH or 5,7-OH groups have the highest cytotoxicity, and flavonoids with 5,7-OMe group intermediate cytotoxicity. The IC50 values of flavonoid (5,7-OMe, 3',4',5'-OMe) and flavonoid (5,7-OMe, 4'-OMe), 0.4 M and 1.4 M. The IC50 values of flavonoid (5,6,7-OMe, 3',4'-OMe) and flavonoid (5,6,7-OMe, 3',4',5'-OMe), 3.2 uM and 0.9 M, respectively, and those of flavonoid (5,6,7-OMe, 3',4',5'-OMe) and flavonoid (5,7-OMe,3',4',5'-OMe) were 0.9 M and 0.4 M, respectively. These results suggest that flavonoids with 3-OH group play a positive role in antioxidant activities, flavonoids with 5-OH and/or 7-OH groups show the higher cytotoxicity, and flavonoids with 3'-OMe and/or 5'-OMe groups plays positive but 6-OMe groups negative roles in the P-glycoprotein (Pgp) inhibition. It is believed that these SAR results can be taken into account for the development of flavonoids with high therapeutic index.

Biotherapeutics can elicit an immune response that may alter their PK, efficacy and safety. Novel biotherapeutics such as antibody– drug conjugates (ADCs) contain chemical domains and have hapten-like structures that may increase their immunogenicity risk. ADCs are composed of a cytotoxic drug linked to a monoclonal antibody (mAb) which recognizes a tumor-associated antigen. The mechanism of action involves targeted delivery of the cytotoxic drug into cancer cells. Compared with traditional chemotherapeutics, ADCs offer the promise of reduced systemic exposure, lower off-target side effects and a higher therapeutic index. Although the concept of ADCs is not new, only two examples are approved for the US market: brentuximab vedotin (Adcetris, Seattle Genetics) and ado-trastuzumab emtansine (Kadcyla, Genentech-Roche). Another ADC that gained approval but was later removed from the US market is gentuzumab ozogamicin (Mylotarg, Pfizer). These ADCs and most of those in clinical development [1] use conventional lysine or cysteine conjugation chemistries that result in a product with heterogeneous drug–antibody ratios, the complexity of which further increases in vivo [2]. Companies are working to make ADCs with reduced drug–antibody ratio heterogeneity, improved conjugate stability and the possibility of an increased therapeutic window [1]. From an immunogenicity perspective, conventional and next-generation ADCs have a hapten-like structure with potentially increased immunogenicity compared with the stand-alone corresponding mAb. The cytotoxic drug component in ADCs is the hapten while the mAb is the carrier. When administered by itself, a hapten is unlikely to induce immunogenicity, but may do so when coupled to a carrier [3]. Humoral immune responses to ADCs can include anti-therapeutic antibodies (ATAs) against all components of the ADC, including the cytotoxic drug, the mAb and neoepitopes formed in the mAb as a result of the conjugation [3]. ATAs against different domains of an ADC could have different effects on PK, efficacy and safety; this is an important point to consider when developing immunogenicity strategies. Immunogenicity strategies followed for other biotherapeutics are appropriate for ADCs, but with additional characterization to better understand the ATA specificity [4–6]. These strategies should include a risk-based assessment and assays that are consistent with industry best practices [7].

Xanthohumol, a prenylflavonoid derived from hops induces apoptosis and inhibits NF-kappaB activation in prostate epithelial cells

Research in cancer chemoprevention provides convincing evidence that increased intake of vegetables and fruits may reduce the risk of several human malignancies. Phytochemicals present therein provide beneficial anti-inflammatory and antioxidant properties that serve to improve the cellular microenvironment. Compounds known as flavonoids categorized anthocyanidins, flavonols, flavanones, flavonols, flavones, and isoflavones have shown considerable promise as chemopreventive agents. Apigenin (4', 5, 7-trihydroxyflavone), a major plant flavone, possessing antioxidant, anti-inflammatory, and anticancer properties affecting several molecular and cellular targets used to treat various human diseases. Epidemiologic and case-control studies have suggested apigenin reduces the risk of certain cancers. Studies demonstrate that apigenin retain potent therapeutic properties alone and/or increases the efficacy of several chemotherapeutic drugs in combination on a variety of human cancers. Apigenin's anticancer effects could also be due to its differential effects in causing minimal toxicity to normal cells with delayed plasma clearance and slow decomposition in liver increasing the systemic bioavailability in pharmacokinetic studies. Here we discuss the anticancer role of apigenin highlighting its potential activity as a chemopreventive and therapeutic agent. We also highlight the current caveats that preclude apigenin for its use in the human trials.

Cancer Prevention with Green Tea and Its Principal Constituent, EGCG: from Early Investigations to Current Focus on Human Cancer Stem Cells.

BACKGROUND Innate immune signaling mediated through STING (Stimulator of Interferon Genes) generates a Type I interferon (Type 1 IFN) signal that increases T cell infiltration into cold or non-inflamed tumors leading to significant regression (Woo et al., 2015). Comprehensive data from one of our STING agonists was presented at the AACR Tumor Immunology meeting in 2017. Here we describe CRD5500, a versatile next generation compound that can be dosed either by intra-tumoral route, systemically (IV or SC) or as an antibody drug conjugated to Trastuzumab (ADC). The ADC approach allows for tumor targeting through systemic delivery with low systemic exposure of the payload and should result in a higher Therapeutic Index (TI) in the clinic. METHODS We have described the discovery of small molecule activators of human STING that generate potent anti-tumor innate and adaptive responses in recently published patents. CRD 5500 was evaluated in (1) in reporter gene assays on HEK293T cells containing stably transfected hSTING polymorphs, (2) by immunoblots to confirm pSTING, pTBK1, pIRF3, (3) by real-time PCR and ELISA monitoring induction of Type I cytokines in human tumor cell lines, isolated human PBMCs and dendritic cells (hMo-DC). Direct STING binding was demonstrated by (1) Cellular Thermal Shift Assays (CETSA) and (2) in vitro kinase assay. Anti-tumor activity of CRD5500 was demonstrated by multiple routes in syngenic tumors containing human STING. Libraries of linkers were used to permit conjugation of CRD5500 with Trastuzumab. Prior to conjugation, penultimate constructs (Pre-Ab) were screened for their ability to preferentially release CRD5500 in tumor homogenate with reduced plasma exposure. Antibody drug conjugates were synthesized at Abzena, UK and were tested in HER2 expressing cells to assess release of free payload . RESULTS Treatment of an isolated cell free system containing purified recombinant STING and TBK1 leads to STING phosphorylation confirming that CRD-5500 is a direct STING binder. Evidence of direct binding also comes from CETSA. CRD-5500 activates all five common hSTING variants with agonist EC50 comparable to CDNs in the pIRF3 reporter assay. Treatment of hSTING transfected cells, human tumor cell lines or hPBMC with CRD5500 leads to phosphorylation of IRF3, TBK1 and STING. CRD-5500 causes the maturation of hDCs and the release of innate and adaptive inflammatory cytokines such as IFNβ and TNFα from hDCs and hPBMCs. In vivo administration of CRD5500 IT or systemically caused tumor regression in CT26 syngeneic tumors containing human STING. CRD5500 demonstrated the abscopal effect and when combined with check point blockade dramatically reduced tumor mass. Interestingly, when Pre-Ab constructs that were not conjugated to antibody were delivered IV, they combined with an anti-CTLA4 antibody to eliminate tumors in the hSTING.CT26 syngenic tumor model. Conjugation with Trastuzumab was demonstrated and the ADC thus formed was screened in HER2 expressing cell lines. CONCLUSIONS The ability of hSTING agonists to generate Type 1 IFN anti-tumor immune responses makes them a promising therapeutic option either as a single agent or in combination with other therapies. CRD5500 is a small molecule STING agonist that can be delivered in multiple formats making it well suited for further development as an anti-cancer agent. Citation Format: Monali Banerjee, Sourav Basu, Sandip Middya, Ritesh Shrivastava, Rajib Ghosh, David C. Pryde, Dharmendra Yadav, Gopal Bhattacharya, Thanilsana Soram, Kavita Puniya, Ritika Raina, Vijay Kadam, Sujay Garai, Priti Sharma, Anuj Singh, Vaibhav Shinde, Nidhi Rawat, Anindita Middya, Arghyotri Sinha, Sunita Chandel, Ganesh Narisipuram, Abhisek Chatterjee, Nagaswamy Mane, Arjun Surya. CRD5500: A versatile small molecule STING agonist amenable to bioconjugation as an ADC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-061.

The development of new anticancer agents with lower toxicity, higher therapeutic index, and weaker tendency to induce resistant phenotypes in tumor cells is a continuous challenge for the scientific community. Toward that end, we showed previously that a new class of soft alkylating agents designed as phenyl-3-(2-chloroethyl)ureas (CEUs) inhibits tumor cell growth in vitro and that their efficiency is not altered by clinically relevant mechanisms of resistance such as overexpression of multidrug resistance proteins, increase in intracellular concentration of glutathione and/or glutathione S-transferase activity, alteration of topoisomerase II, and increased DNA repair. Mechanistic studies have showed recently that the cytotoxic activity of several CEUs was mainly related to the disruption of microtubules. Here, we present results supporting our assumption that 4-tert-butyl-[3-(2-chloroethyl)ureido]phenyl (tBCEU) (and its bioisosteric derivative 4-iodo-[3-(2-chloroethyl)ureido]phenyl (ICEU) are potent antimicrotubule agents both in vitro and in vivo. They covalently bind to beta-tubulin, leading to a microtubule depolymerization phenotype, consequently disrupting the actin cytoskeleton and altering the nuclear morphology. Accordingly, tBCEU and ICEU also inhibited the migration and proliferation of endothelial and tumor cells in vitro in a dose-dependent manner. It is noteworthy that ICEU efficiently blocked angiogenesis and tumor growth in three distinct animal models: (a) the Matrigel plug angiogenesis assay; (b) the CT-26 tumor growth assay in mice; and (c) the chick chorioallantoic membrane tumor assay. In addition, we present evidence that CEU cytotoxicity is unaffected by additional resistance mechanisms impeding tumor response to DNA alkylating agents such as cisplatin, namely the cell adhesion mediated-drug resistance mechanism, which failed to influence the cytocidal activity of CEUs. On the basis of the apparent innocuousness of CEUs, on their ability to circumvent many classical and recently described tumor cell resistance mechanisms, and on their specific biodistribution to organs of the gastrointestinal tract, our results suggest that CEUs represent a promising new class of anticancer agents.

Introduction: Prevention of cancer remains a primary need and new chemopreventive agents must be developed for this purpose. It is well known that the radiotherapy is associated with severe side effects and offers no firm expectation for a cure. Thus, there is a constant need for the investigation of other potentially useful options. One of the widely sought approaches is cancer chemoprevention that uses natural agents. Curcumin, a natural plant product, has been shown to have anti-inflammatory and antioxidant properties and which, thus, may be a potential anticancer drug. Material and Methods: The inhibitory effects of dietary curcumin (0.6 % in diet) on colonic precancerous lesions in rats treated with 1,2-dimethylhydrazine, (DMH, 20 mg/kg, s.c. once weekly for 6 weeks) or exposed to γ-radiation (6.5 Gy) alone or in combination were evaluated histologically and histochemically. Results: The results showed clear features of colonic dysplasia with DMH treatment. The dysplastic crypts exhibited marked hyperchromatism, loss of polarity besides nuclear and cellular stratification. Whereas, γ-irradiation revealed marked crypt dilatation accompanied by disorganized crypt structure and alteration of surface epithelium. The results also demonstrate an apparent synergy between the radiation and the chemical carcinogen in the induction of augmentation of mucosal abnormalities accompanied with increased total DNA content and cell proliferation. Consistent with these observations, changes in the type of mucous secretion with predominance of sialomucins were observed in the areas of mild to moderate dysplasia whilst the surrounding normal epithelium produced sulphated material. Mucus depletion was a common feature in areas of severe dysplasia. Addition of curcumin to the diet remarkably reduced the amount and severity of dysplastic lesions with DMH-induced colon preneoplasia without appreciable toxicity. Moreover, curcumin could prevent or improve the disruption of colonic architecture caused by γ- irradiation. Such improvement was also investigated but to a lesser extent with the combined radiation and DMH. Conclusion: These results indicated that curcumin is potent in inhibiting the preneoplastic lesions and other cellular events relevant to colon and was, therefore, of value as a safe chemotherapeutic agent.

In this review, we summarize studies that initiated further exploration of the relationship of cyclooxygenase-2 (COX-2) to cancer, and evidence for COX-2 involvement in transformation, maintenance of tumor growth, viability, and metastasis. Included are descriptions of the chronic upregulation of COX-2 in multiple human tumors, as well as the pharmacological evidence of the anti-angiogenic activity of COX-2 inhibitors in animal models. Collectively, these data have generated interest in the functional role of COX-2 during tumorigenesis, and imply COX-2 inhibitors may be effective in the prevention and/or treatment of cancer. Human trials are ongoing to assess the chemopreventive activity of COX-2 inhibitors on progression of premalignant to malignant conditions and/or recurrence. Additionally, COX-2 inhibitors are being tested in combination with standard cytotoxics, radiation, and molecular targeted agents to explore whether combination modalities enhance efficacy of anticancer agents.

In Iraq like most third world countries, attempts to extract, identify and study the anticancer activity of the active components of plants and use it as drugs. Curcumin[(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5dione] is the major yellow pigment extracted from turmeric, a commonly used spice, derived from the rhizome of the plant Curcuma longa. In India and Southeast Asia, turmeric has long been used as a treatment for inflammation, skin wounds and tumors. Curcumin has broad spectrum cancer chemo preventive activity in preclinical animal models. The extract of the herb curcumin, from Iraqi curcumin, was done by using of 95% ethanol as a solvent, then isolation of curcumin from the ethanolic extract by column chromatography, curcumin was characterized by Uv-visible, FT-IR and proton NMR spectroscopy. The study of anticancer activity of the curcumin and ethanolic extract were done in vivo on mice and in vitro on cell line. The extract showed a considerable anticancer activity against the cell line of human hepato cellular liver carcinoma. Key words: Anticancer, curcumin, ethanol, extraction.

Combining targeted alpha and beta therapy may address challenges such as toxicity, treatment resistance, and tumor heterogeneity. We evaluated the feasibility and therapeutic effectiveness of a DOTA-PRIT approach using a 177Lu/225Ac radioisotope cocktail, directly compared with monotherapies targeting GPA33 in human colorectal cancer (CRC) xenografts in mice. A three-step pretargeting regimen was employed: an anti-GPA33/anti-DOTA bispecific antibody (BsAb), a dendrimeric clearing agent, and radioligands labeled with 177Lu, 225Ac, alone or in combination. Serial biodistribution studies in GPA33(+) SW1222 xenografts evaluated how co-injection of 177Lu and 225Ac radioligands affected tumor uptake and biodistribution. iQID digital autoradiography was used to visualize isotope distribution in tumor and kidney samples. Mice bearing SW1222 and LS174T xenografts received mono- or combination-therapy regimens delivering 37-38 Gy to tumors. Dose-escalation studies, histopathology, and qPCR analysis of DNA-damage and apoptosis-related genes were also performed. Biodistribution and autoradiography confirmed that the 177Lu- and 225Ac-labeled ligand effectively bound to pretargeted GPA33(+) xenografts when co-administered. High therapeutic indices were maintained across treatment groups, with autoradiography showing general overlap of co-injected probes. Combination therapy demonstrated comparable efficacy to monotherapies. At 150 d post-treatment, no treatment group had reached median survival; 5/9 mice receiving the cocktail (62.9 MBq 177Lu + 18.5 kBq 225Ac) were alive, including two tumor-free. In comparison, 4/8 mice in the 177Lu group and 8/10 in the 225Ac group survived, with 3 and 5 tumor-free animals, respectively. Combination therapy was well tolerated, showing no significant adverse effects on body weight or blood cell counts compared to healthy controls. Combined administration was safe up to 62.9 MBq 177Lu + 37 kBq 225Ac, resulting in 10/10 histological cures. Our findings confirm the feasibility of a combined 177Lu and 225Ac DOTA-PRIT in mice with established SW1222 xenografts, demonstrating tolerability and effectiveness comparable to monotherapy at equivalent average absorbed tumor doses.

Phosphodiesterase 4 (PDE4) has four isoforms (PDE4A-D) with at least 25 splice variants. PDE4 subtype nonselective inhibitors produce potent antidepressant-like and cognition-enhancing effects via increased intracellular cyclic AMP (cAMP) signaling in the brain. Our previous data have demonstrated that long-form PDE4Ds appear to be involved in these pharmacological properties of PDE4 inhibitors in the normal animals. However, it is not clear whether long-form PDE4Ds are critical for the behaviors and related cellular signaling/neuronal plasticity/neuroendocrine alterations in the depressed animals. In the present study, animals exposed to the chronic unpredictable stress (CUS), a rodent model of depression, exhibited elevated corticosterone, depressive-like behavior, memory deficits, accompanied with decreased cAMP-PKA-CREB and cAMP-ERK1/2-CREB signaling and neuroplasticity. These alterations induced by CUS were reversed by RNA interference (RNAi)-mediated prefrontal cortex long-form PDE4Ds (especially PDE4D4 and PDE4D5) knock-down, similar to the effects of the PDE4 subtype nonselective inhibitor rolipram. Furthermore, these effects of RNAi were not enhanced by rolipram. These data indicate a predominant role of long-form PDE4Ds in the pharmacotherapies of PDE4 inhibitors for depression and concomitant memory deficits. Long-form PDE4Ds, especially PDE4D4 and PDE4D5, appear to be the promising targets for the development of antidepressants with high therapeutic indices.

Gene therapies and gene product-based drug candidates for normalizing and preserving tissue functions in animal models of ocular hypertension and glaucoma

Phylogenetic analysis has demonstrated that some positive-sense RNA viruses can be classified into the picornavirus-like supercluster, which includes picornaviruses, caliciviruses, and coronaviruses. These viruses possess 3C or 3C-like proteases (3Cpro or 3CLpro, respectively), which contain a typical chymotrypsin-like fold and a catalytic triad (or dyad) with a Cys residue as a nucleophile. The conserved key sites of 3Cpro or 3CLpro may serve as attractive targets for the design of broad-spectrum antivirals for multiple viruses in the supercluster. We previously reported the structure-based design and synthesis of potent protease inhibitors of Norwalk virus (NV), a member of the Caliciviridae family. We report herein the broad-spectrum antiviral activities of three compounds possessing a common dipeptidyl residue with different warheads, i.e., an aldehyde (GC373), a bisulfite adduct (GC376), and an α-ketoamide (GC375), against viruses that belong to the supercluster. All compounds were highly effective against the majority of tested viruses, with half-maximal inhibitory concentrations in the high nanomolar or low micromolar range in enzyme- and/or cell-based assays and with high therapeutic indices. We also report the high-resolution X-ray cocrystal structures of NV 3CLpro-, poliovirus 3Cpro-, and transmissible gastroenteritis virus 3CLpro- GC376 inhibitor complexes, which show the compound covalently bound to a nucleophilic Cys residue in the catalytic site of the corresponding protease. We conclude that these compounds have the potential to be developed as antiviral therapeutics aimed at a single virus or multiple viruses in the picornavirus-like supercluster by targeting 3Cpro or 3CLpro.

New 4 H-chromen-4-one and 2 H-chromene derivatives as anti-picornavirus capsid-binders