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The increasing prevalence of the highly antibody-evasive Omicron sublineages increases the risk of breakthrough infections and leaves high-risk and vulnerable immunocompromised individuals with no effective options for prophylactic or therapeutic antibody treatments. Here, we report a heavily mutated anti-RBD monoclonal antibody, Acovimab, directed against a site in the receptor-binding motif (RBM) region of the CoV2 receptor-binding domain (RBD), that possesses very broad and highly potent neutralizing activity against CoV2 variants, including many Omicron variants. This antibody is derived from the IGHV1-58*01 germline sequence and possesses a relatively high level of mutation (15.5% of the VH aa sequence), which is unusual for anti-RBD antibodies. Neutralizing activity was very potent (IC50s range of 1-9 ng/ml) for early Omicron subvariants that possess an unmutated F486 residue and is retained but less potent (IC50s of 200-650 ng/ml) for more resistant Omicron subvariants which contain the F486V mutation (BA4/5, BA4.6, and BQ1.1), but is lost for the later ultra-resistant variants that contain F486S (XBB) or F486P (XBB.1.5) mutations. Based on these specificities, it is predicted that Acovimab by itself should protect against CoV2 infections other than those caused by the XBB/XBB.1.5 family. Acovimab also shows strong synergy in neutralization when combined with Sotrovimab, which neutralizes all Omicron variants, including XBB.1.5. Plasma from subjects with hybrid immunity (induced by vaccination + infection) possessed low levels of XBB.1.5 RBM-targeting plasma-neutralizing antibodies, and these also neutralized synergistically when combined with Sotrovimab. These results suggest potentially novel immunotherapeutic options for treating most of the CoV2 variants responsible for current infections.

Simple Summary Adavosertib is a first-in-class Wee1 inhibitor that has demonstrated activity against certain cancers. We evaluated the effects of adavosertib in treating differentiated thyroid cancer (DTC) using four DTC cell lines (BHP7-13, K1, FTC-133, FTC-238). Adavosertib accumulated cells in the G2/M phase and induced apoptosis in four DTC cell lines. Baseline Wee1 levels correlated with adavosertib sensitivity. Single-agent adavosertib therapy was sufficient to inhibit the growth of K1 and FTC-133 tumor models. Adavosertib potentiated the anti-tumor effect of dabrafenib and trametinib in K1 xenografts harboring the BRAFV600E mutation, with promising safety profiles. Adavosertib also improved the anti-tumor efficacy of lenvatinib in FTC-133 xenografts. Our results suggest the clinical efficacy of adavosertib for DTC patient therapy. Abstract Differentiated thyroid cancer (DTC) patients are usually known for their excellent prognoses. However, some patients with DTC develop refractory disease and require novel therapies with different therapeutic mechanisms. Targeting Wee1 with adavosertib has emerged as a novel strategy for cancer therapy. We determined the effects of adavosertib in four DTC cell lines. Adavosertib induces cell growth inhibition in a dose-dependent fashion. Cell cycle analyses revealed that cells were accumulated in the G2/M phase and apoptosis was induced by adavosertib in the four DTC tumor cell lines. The sensitivity of adavosertib correlated with baseline Wee1 expression. In vivo studies showed that adavosertib significantly inhibited the xenograft growth of papillary and follicular thyroid cancer tumor models. Adavosertib therapy, combined with dabrafenib and trametinib, had strong synergism in vitro, and revealed robust tumor growth suppression in vivo in a xenograft model of papillary thyroid cancer harboring mutant BRAFV600E, without appreciable toxicity. Furthermore, combination of adavosertib with lenvatinib was more effective than either agent alone in a xenograft model of follicular thyroid cancer. These results show that adavosertib has the potential in treating DTC.

Polo-like kinases (PLKs) are pivotal regulators of cell proliferation and cell survival; therefore, PLKs may be potential targets in the treatment of malignancy. The therapeutic effects of volasertib, a PLKs inhibitor for papillary and follicular thyroid cancer (known as well-differentiated thyroid cancer (WDTC)), were evaluated in this study. Volasertib inhibited cell proliferation in two papillary and two follicular thyroid cancer cell lines in a dose-dependent manner. Volasertib treatment reduced cells in the S phase and increased cells in the G2/M phase. Volasertib activated caspase-3 activity and induced apoptosis. Drug combinations of volasertib and sorafenib showed mostly synergism in four well-differentiated thyroid carcinoma cell lines in vitro. Volasertib treatment in vivo retarded the growth of a papillary thyroid tumor model. Furthermore, the combination of volasertib with sorafenib was more effective than a single treatment of either in a follicular thyroid cancer xenograft model. Promising safety profiles appeared in animals treated with either volasertib alone or volasertib and sorafenib combination therapy. These findings support volasertib as a potential drug for the treatment of patients with WDTC.

Wee1 is a kinase that regulates the G2/M progression by the inhibition of CDK1, which is critical for ensuring DNA damage repair before initiation of mitotic entry. Targeting Wee1 may be a potential strategy in the treatment of anaplastic thyroid cancer, a rare but lethal disease. The therapeutic effects of adavosertib, a Wee1 inhibitor for anaplastic thyroid cancer was evaluated in this study. Adavosertib inhibited cell growth in three anaplastic thyroid cancer cell lines in a dose-dependent manner. Cell cycle analysis revealed cells were accumulated in the G2/M phase. Adavosertib induced caspase-3 activity and led to apoptosis. Adavosertib monotherapy showed significant retardation of the growth of two anaplastic thyroid cancer tumor models. The combination of adavosertib with dabrafenib and trametinib revealed strong synergism in vitro and demonstrated robust suppression of tumor growth in vivo in anaplastic thyroid cancer xenograft models with BRAFV600E mutation. The combination of adavosertib with either sorafenib or lenvatinib also demonstrated synergism in vitro and had strong inhibition of tumor growth in vivo in an anaplastic thyroid cancer xenograft model. No appreciable toxicity appeared in mice treated with either a single agent or combination treatment. Our findings suggest adavosertib holds the promise for the treatment of patients with anaplastic thyroid cancer.

Altered cyclin-dependent kinase activity is observed in many human malignancies. Cyclin-dependent kinases that promote cell cycle progression may be promising targets in the treatment of cancer. The therapeutic effects of roniciclib, a cyclin-dependent kinase inhibitor for medullary thyroid cancer were investigated in the present study. Roniciclib inhibited medullary thyroid cancer cell proliferation in a dose-dependent manner. Roniciclib induced caspase-3 activity and contributed to apoptosis. Cell cycle progression was arrested in the G2 phase. In vivo, roniciclib treatment retarded the growth of tumors of medullary thyroid cancer xenografts. In addition, roniciclib in combination with sorafenib was more effective than either single treatment in a xenograft model. No morbidity was observed in animals treated with single roniciclib therapy and combination treatment of roniciclib and sorafenib. These data provide a rationale for clinical assessment of using roniciclib in the treatment of patients with medullary thyroid cancer.

Preclinical animal studies or clinical protocol designs frequently limited by the dose-number or dose-range involved, especial for multiple candidate drugs and their combinations. The unified theory of the Mass-Action Law Pharmacodynamics and Bioinformatics [MAL-PD/BI] and its combination index (CI) theory provide general principle for drug evaluation in vitro, in animals and in clinical trials. Thus, all single drugs and their combinations can be compared/ranked under the same MAL-PD principle and the same protocol design-conditions for the unified ranking assessments. Using the MAL-PD/BI/CI and its computer software, anti-HIV combination clinical trials for AZT + 3TC (Mildvan D. et al, Antivir. Ther. 1: 77-88, 1996) was successfully carried out for synergism quantification/simulation (CI5 patients, using CompuSyn for instant synergy simulations. The benefit for the MAL-PD/CI is that all single drug and their combinations efficacy/synergy in vitro and in vivo (Covid genetic, antigen or neutralization assays or surrogate markers) are compared/ranked under the same MAL-PD principle and the same protocol design-conditions for the unified ranking assessments. The evaluations of other candidate compounds/agents can be designed and analyzed in the same way, just by adjusting the dose-ranges and the combination-ratios. Citation Format: Ting-Chao Chou. Pharmacodynamics algorithm integrated assessments-and-ranking of multiple candidate drugs in preclinical and clinical protocol design and quantitative computerized simulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB021.

// Shu-Fu Lin 1 , Jen-Der Lin 1 , Chuen Hsueh 2 , Ting-Chao Chou 3, 4 and Richard J. Wong 5 1 Department of Internal Medicine, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, Taiwan 2 Department of Pathology, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, Taiwan 3 Laboratory of Preclinical Pharmacology Core, Memorial Sloan Kettering Cancer Center, New York, NY, USA 4 Current address: PD Science, Inc., Paramus, NJ, USA 5 Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA Correspondence to: Shu-Fu Lin, email: mmg@cgmh.org.tw Keywords: roniciclib, cyclin-dependent kinase, anaplastic thyroid cancer Received: March 02, 2017 Accepted: May 23, 2017 Published: July 08, 2017 ABSTRACT Many human cancers have altered cyclin-dependent kinase activity. Inhibition of cyclin-dependent kinases may arrest cell cycle progression and represents an important strategy in the treatment of malignancies. We evaluated the therapeutic effects of roniciclib, a cyclin-dependent kinase inhibitor, as a treatment for anaplastic thyroid cancer. Roniciclib inhibited anaplastic thyroid cancer cell proliferation in a dose-dependent manner. Roniciclib activated caspase-3 activity and induced apoptosis. Cell cycle progression was arrested in G2/M phase. In vivo , the growth of anaplastic thyroid cancer xenograft tumors was retarded by roniciclib treatment without evidence of toxicity. These data provide a rationale for further clinical evaluation using roniciclib in the treatment of patients with anaplastic thyroid cancer.

// Shu-Fu Lin 1 , Jen-Der Lin 1 , Chuen Hsueh 2 , Ting-Chao Chou 3, 7 , Chun-Nan Yeh 4 , Ming-Huang Chen 5 and Richard J. Wong 6 1 Department of Internal Medicine, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, Taiwan 2 Department of Pathology, Chang Gung Memorial Hospital, Taoyuan, Taiwan 3 Laboratory of Preclinical Pharmacology Core, Memorial Sloan-Kettering Cancer Center, New York, NY, USA 4 Department of Surgery, Chang Gung Memorial Hospital, Taoyuan, Taiwan 5 Department of Internal Medicine, Taipei Veterans General Hospital, Taipei, Taiwan 6 Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY, USA 7 Current address: PD Science, Inc., Paramus, NJ, USA Correspondence to: Shu-Fu Lin, email: mmg@cgmh.org.tw Keywords: HSP90 inhibitor, ganetespib, thyroid cancer Received: October 29, 2016 Accepted: March 30, 2017 Published: April 18, 2017 ABSTRACT Heat shock protein 90 is a molecular chaperon that maintains the correct folding and function of multiple client proteins. The inhibition of heat shock protein 90, which leads to the simultaneous degradation of multiple proteins involved in oncogenic signaling pathways, has revealed an innovative strategy to treat a variety of cancer types. We evaluated the therapeutic effects of ganetespib, a heat shock protein 90 inhibitor, in treating thyroid cancer. Ganetespib effectively inhibited cell proliferation in a dose-dependent manner in eight cell lines originating from four major histologic types of thyroid cancer (papillary, follicular, anaplastic and medullary). Ganetespib decreased cyclin-dependent kinase 1 and arrested cell cycle progression in G2/M phase. The expression of proteins involved in RAS/RAF/ERK and PI3K/AKT/mTOR signaling pathways was also inhibited. The RET level was decreased in a medullary thyroid cancer cell line. Ganetespib increased Bim expression, activated caspase-3 and induced apoptosis. In vivo , ganetespib retarded the tumor growth of anaplastic and medullary thyroid cancer xenografts with acceptable safety profiles. These findings indicate that ganetespib has potential in the treatment of patients with thyroid cancer.

Biological system-analysis by the mass-action law with biophysical, biochemical and mathematical induction-and-deduction leads to the derivation of >300 reaction rate equations. These derived equations led to general biodynamics/pharmacodynamics and bioinformatics (MAL-BD/PD/BI) general paradigms for “Dose” and “Effect”. 1. Median-Effect Eq. (MEE) for single drug or entity, regardless, in vitro, in animals or in human, where Dm and m values signify the median-effect dose “potency” and “shape” (dynamic order), respectively. 2. Combination Index Eq. (CIE), where CI1 indicates synergism, additive effect and antagonism, respectively, in combinations. Since all terms in MEE and CIE are relativity ratios, they are dimensionless quantities. Therefore, MEE and CIE are fundamental MAL-BD/PD/BI/CI principle independent to entity’s physical nature (chemicals, biologicals, natural products, radiation, liquid, or gas); independent to unit (ug, nM, mg/ml. mg/kg, IU, rad or multiples of infection); and independent to mode or mechanism of actions (competitive, noncompetitive, uncompetitive, sequential, ordered, ping-pong or random). Comprehensive details are available in Chou TC, Pharmacol. Rev. 58: 621-681, 2006 (specifically on p.655 and p.660), It also discussed in Chou, Integrative Biol. 3: 548-559, 2011, and many more recent papers on chemotherapy and radiation therapy combinations, as indicated in Google scholars. The above 2006 Chou article, as of January 12, 2021, has 3,835 citations in 1,054 biomedical journals internationally, encompassing nearly all disciplines in biomedical sciences. Citation Format: Ting-Chao Chou. Why drug and radiation combination synergism or antagonism can be quantitatively determined with the mass-action law based pharmacodynamics equation, algorithm and computer simulation [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-062.

The median-effect equation (MEE) of the mass-action law and the combination index (CI) theorem have been used for quantitative determination of synergism (CI 1) and additive effect (CI = 1) in animals in vivo. Experimental design, the theoretical algorithm and the CompuSyn software simulation have been used to illustrate step-by-step for the combination of two anti-cancer agents, Taxotere and T607 compound, with similar mode of actions of targeting microtubule polymerization, but with distinct chemical structures. These two compounds acted synergistically against human colon carcinoma HCT-116 xenograft tumor in athymic nude mice. In all, only 78 nude mice have been used. The synergy is especially significant (p

The mass-action law (MAL) based Median-Effect Equation (MEE) for single- drug, and the Combination Index Equation (CIE) for drug-combinations, are the basis for unified Pharmacodynamics (PD) and Biodynamics (BD). To date, the MAL-PD/BD theory, in 3 articles alone, have 12,577 citations in 1,248 biomedical journals (Google Scholar Citation-Ting-Chao Chou). The applications mainly in vitro, relatively few in vivo [see, ChouTC, Pharmacol Rev. 58: 621-681, 2006 (cited 3,301 times in 914 journals) or Synergy 1: 3-21, 2014]. In recent presentations at JHU (4.9.2019) and at FDA (5.7.2019 & 11.7.2019), Chou presented, the PD-based clinical protocol design using 2-3 dose-data points for single-drug, and 10 data points for 2-drug combinations synergy quantification, e.g., A (3), B (3), and A+B (3), at constant combination ratio, plus a control. For the MAL-PD/BD based protocol design in vivo (dose-range and dose density, combination ratio), the automated CompuSyn simulation/quantitative PD-parameters (for potency and PD-shape)/diagnostic plots/digitalized conclusions/and automated print-out report, usually takes a few seconds, and print-out may take 1-2 minutes. All drugs (chemical, biological, radiation) should have a Dose-Effect curves (DEC). The MEE indicates that all PD-DEC can be linearized by the Median-Effect Plot (MEP), x= Log (dose) vs y= Log {(fraction affected (fa)/(fraction unaffected (fu)}, where fa+fu=1, and when fa=fu, gives the Median-Effect Dose (Dm). The slope gives “m” value (signifying the shape of DEC), and the antilog of x-intercept gives the “Dm” value, (signifying the potency of half-affected). Since MEP linearizes the DEC, it requires a minimum of two data pints to simulate entire DEC. Thus, the Two-Data-Point Theory is extremely cost-effective for in vivo clinical trial protocol designs, as only small number data points is required to achieve efficient computer simulations, with digital/index conclusions. In CIE, CI1, quantitatively defines Synergism, Additive Effect, and Antagonism, respectively, for 2 to n drug combs, using CompuSyn. PD requires multiple doses since “single dose” yields a data-Point, but “a point” has “no shape”, thus, it is never a PD study. Earlier examples with PD-CI method in vivo are: 1. Anti-HIV Combos (AZT+IFN, using only 36 AIDS patients), and 2. Anti-cancer Combos (Taxotere+T607, against HCT-116 colon carcinoma xenograft in nude mice), both used only 10 data points and both achieved quantitative determination of Synergism. This report reaffirm Example 2 with the same drug combos against MX-1 mammary carcinoma xenograft in nude mice, using only 10 data points, which showed similar synergy at ED75 to ED97, CI= 0.82-0.79 for MX-1, and CI= 0.98-0.64 for HCT-116. In conclusion, the MAL-PD/BD and its equations, algorithms and computer simulation provide a rigorous, cost-effective, automated, quantitative biomedical R and D and general drug evaluation guidance in clinical trial design and data analysis, that save resources, time and efforts. Citation Format: Ting-Chao Chou. General pharmacodynamics algorithm-based clinical protocol design with two to three dose-data points for single-drug, and ten data points for two-drug-combination synergy quantification, using computer simulation for digitalized data analysis and conclusions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT155.

The Median-Effect Equation (MEE) for single drug, and the Combination Index Equation (CIE) for multiple drug combinations, are basis for general Pharmacodynamics (PD), and Biodynamics (BD). Both, MEE and CIE are derived from system analysis of the Mass-Action Law (MAL). The MAL-PD/BD theory, equations and algorithms allow automated computer simulations for diagnostic plots and quantitative, digital/indexed conclusions. To date, the MAL dynamic theory is cited over 15,000 times, in over 1,248 biomedical journals. The publications include studies in vitro, in cell, in animal and in clinical trial levels, as indicated in Google Scholar Citations -Ting-Chao Chou, or in Pharmacol. Rev. 58: 621-681, 2006 (cited 3,119 times in 914 journals). MEE indicates dose and effect are interchangeable, and all PD Dose-Effect curves (DEC) can be linearized by the Median-Effect Plot (MEP), x= Log (dose) vs y= Log {(fraction affected (fa)/(fraction unaffected (fu)}, where fa+fu=1, and when fa=fu, gives the Median-Effect Dose (Dm). The slope gives “m” value (dynamic order, signifying the shape of DEC), and the Antilog of x-intercept gives the “Dm” value, (signifying the potency of half-affected, such as IC50, ED50, or LD50). The MEE, is the unified theory of the Michaelis-Menten Eq. for enzyme half-saturation, Handerson-Hasselbalch Eq. for pH half-ionization, Hill Eq. for ligand half-occupancy, and the Scatchard Eq, for receptor half-bond and half-free. Since MEP linearizes the DEC, it requires only a minimum of two data pints to simulate entire DEC. Thus, the Two-Data-Point Theory is extremely cost-effective for animal studies and clinical trial protocol designs, as only small number data points is required to achieve efficient computer simulations, with digital/index conclusions. In CIE, CI1, quantitatively defines Synergism, Additive Effect, and Antagonism, respectively, for 2 to n drug combs, using CompuSyn. PD requires multiple doses (3-4 doses in animals; 2-3 doses in clinical trials) since single dose yields a data Point, but “a point” which has “no shape”, thus, it is Not a PD in Vivo. For 2-drug-combos using the CI method in vivo for drug A, B, and A+B, require only 10 data points, (3+3+3 plus one control). Earlier examples with CI method in vivo are: 1. Anti-HIV Combos (AZT+IFN, using only 36 AIDS patients), and 2. Anti-cancer Combos (Taxotere+T607, against HCT-116 colon carcinoma xenograft in nude mice), both used only 10 data points and both achieved quantitative determination of Synergism. This report reaffirm Example 2 with the same drug combos against MX-1 mammary carcinoma xenograft in nude mice, using only 10 data points, showed similar synergy at ED75 to ED97, CI= 0.82-0.79 for MX-1, and CI= 0.98-0.64 for HCT-116. Citation Format: Ting-Chao Chou. Mass-action law based general pharmacodynamics theory and algorithm for quantitative computer simulation of drug-evaluations in cells, in animals and in clinical trials [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5510.

Activation of cyclin-dependent kinase activity is frequently observed in many human cancers; therefore, cyclin-dependent kinases that promote cell cycle transition and cell proliferation may be potential targets in the treatment of malignancy. The therapeutic effects of roniciclib, a cyclin-dependent kinase inhibitor for papillary and follicular thyroid cancer (designated as well-differentiated thyroid cancer), were investigated in this study. Roniciclib inhibited cell proliferation in two papillary and two follicular thyroid cancer cell lines in a dose-dependent manner. Roniciclib activated caspase-3 activity and induced apoptosis. Cell cycle progression was arrested in the G2/M phase. Roniciclib treatmentin vivoretarded the growth of two well-differentiated thyroid tumors in xenograft models in a dose-dependent fashion. Furthermore, the combination of roniciclib with sorafenib was more effective than either single treatment in a follicular thyroid cancer xenograft model. Acceptable safety profiles appeared in animals treated with either roniciclib alone or roniciclib and sorafenib combination therapy. These findings support roniciclib as a potential drug for the treatment of patients with well-differentiated thyroid cancer.

International audience; The global incidence of Fusarium head blight and attendant cereal grains multi-contamination by the trichothecene mycotoxins deoxynivalenol (DON) and nivalenol (NIV) are increasing as a possible result of climate change and inadequate agricultural practices. At the molecular level, these mycotoxins bind to the ribosome, activate the mitogen-activated protein kinase and induce a local and systemic inflammation. DON is of public health concern owing to the narrow margin between exposure and tolerable daily intake. The intestinal inflammatory response to DON, NIV and their mixture was analyzed to determine thresholds for their intestinal pro-inflammatory effects and characterize the type and magnitude of their interaction. Fully differentiated-three dimensional porcine jejunal explants were exposed to increasing doses of DON and NIV alone or in combination; the expression level of IL-1α, IL-1β, IL-8, IL-17A, and IL-22 was measured by RT-PCR. Doses as low as 0.16µM DON or 0.73µM NIV significantly increase the intestinal expression levels of the tested inflammation-related genes. These doses are lower than those previously reported for other intestinal toxicity endpoints. The combined pro-inflammatory activity of DON and NIV was synergistic for all the tested genes with combination-index value range of 0.23-0.8. Our results indicate that (i) inflammation is a very sensitive endpoint for the intestinal toxicity of the trichothecenes, and (ii) co-exposure to DON and NIV has a greater inflammatory effect than induced by mycotoxins alone. This synergy should be taken into account considering the frequent co-occurrence of DON and NIV in the diet.

Summary Drug combinations have been widely used in the treatment of the most dreadful diseases, such as cancer and AIDS. In the search for synergistic combinations for therapy, numerous articles have been published during the past century. However, the term “synergy” has at least 20 different definitions in literature but none supports others. The confusion on synergy claims has far reaching consequences in biomedical research, drug discovery and development, regulation, and medical care of patients. This article reviews the current status and enlists the frequently occurred pit-falls, misconceptions and common errors in drug combination studies. The questions and issues are contemplated to be answered and clarified with the physico-chemical algorithms of the mass-action law, specifically with the unified theory of the median-effect equation and its combination index theorem for drug combinations. The derived theory, algorithm and its computer simulation lead to a quantitative indexed bioinformatics, and econo-green bio-research using small number of data points.

Combining oncolytic viruses with conventional therapy such as radiation is an innovative option for pancreatic cancer. We demonstrated that combination of GLV-1h151 and radiation yielded a synergistic cytotoxic effect, with the greatest effect achieved in the AsPC-1cell line. Combination treatment significantly increased apoptosis compared with either single treatment or the control group. In mice bearing human pancreatic tumor xenografts, combination treatment resulted in significantly enhanced inhibition of tumor growth. No evidence of toxicity was observed in mice. These results indicate that the combination of GLV-1h151 and radiation has great potential for translation into clinic practice.

The unified theory of the median-effect equation (MEE) of the mass-action law (MAL) indicates that dose and effect are interchangeable, and all dose-effect curves can be transformed into straight-lines by the median-effect plot. Therefore, it allows pharmacodynamics (PD) analysis using small size experimentation for in vitro and in vivo studies. Further, extension of MEE has proven that the Combination Index Equation (CIE) which defines Synergism (CI 1), can be automatically simulated by CompuSyn software within one second after data entries. With adequate experimental accuracy of measurements, the minimum number of only 10 data points are required for quantitative synergy determination in two-drug combinations, even in animals or in clinical trials. Three articles introducing the CI method (in 1984), the review (2006), and the perspectives (2011) have been well recognized and cited 5516, 2382, and 1701 times in 1117, 768 and 530 biomedical journals, respectively (as of 2.10.2018) [Google Scholar Citations- Ting-Chao Chou]. However, the academic societies and governmental regulatory agencies are still lack of guideline on synergy definition, despite over 20 arbitrary, non-quantitative synergy definitions are still applied and reported. As drug combination is the most widely used therapies for the most dreadful diseases such as cancer and AIDS, a call for open forum on scientific credibility and accountability in these matters is warranted.

The median-effect equation (MEE) derived from the mass-action law (MAL) is the unified theory of dose-effect pharmacodynamics (PD) and biodynamics (BD). MEE enables the linearization of a dose-effect curve into a straight linedefined by two data points. Thus any two data points can represent an entire dose-effect curve. For dose-effect curves using MAL, dose-zero can serve as 3rd point, and the universal reference point, the median-effect dose (Dm) serves as 4th point. This functionality has tremendous significance for in vivo studies. Fewer data points are required for a dose-effect curve, facilitating economical and ethically sustainable PD analyses. The extension of MEE from a single drug to multiple drugs establishes the general combination index equation (CIE), which quantitatively defines synergism (CI 1]. Although the CI method is often (>6000 citations) applied in in vitro studies, it is rarely used in animal studies or clinical trials. In vivo drug combination studies that use only single dose or statistical p value analyses do not allow quantitative synergy claims. This article presents two examples for drug combinations in vivo: (i) in animals (anticancer drug combination against human HCT-116 colon carcinoma xenografts in nude mice, Taxotere + T607) and (ii) in a clinical trial (anti-retroviral drug combinations against HIV/AIDS, AZT + INF). Only 36 patients respectively only 66 nude mice were required. Both examples require only ten data points (D1, D2 and [D1+D2], each with 3 doses plus one control) to quantitatively determine synergism or antagonism with the CompuSyn software.

A series of N-mustard-quinazoline conjugates was synthesized and subjected to antitumor studies. The N-mustard pharmacophore was attached at the C-6 of the 4-anilinoquinazolines via a urea linker. To study the structure-activity relationships of these conjugates, various substituents were introduced to the C-4 anilino moiety. The preliminary antitumor studies revealed that these agents exhibited significant antitumor activity in inhibiting various human tumor cell growths in vitro. Compounds 21b, 21g, and 21h were selected for further antitumor activity evaluation against human breast carcinoma MX-1 and prostate PC-3 xenograft in animal model. These agents showed 54-75% tumor suppression with low toxicity (5-7% body-weight changes). We also demonstrate that the newly synthesized compounds are able to induce DNA cross-linking through alkaline agarose gel shift assay and inhibited cell cycle arrest at G2/M phase.

A series of linear pyrrolo[1,2-b]isoquinoline derivatives was synthesized for antitumor evaluation. The preliminary antitumor studies reveal that both bis(hydroxymethyl) and their bis(alkylcarbamate) derivatives show significant antitumor activity in inhibiting various human tumor cell growth in vitro. 1,2-Bis(hydroxymethyl)-3-methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline (20a) was selected for antitumor studies in animal models. The results show that this agent can induce complete tumor remission or significant suppression in nude mice bearing human breast (MX-1) xenograft and ovarian (SK-OV-3) xenografts, respectively. Alkaline agarose gel shifting assay showed that 20a is able to cross-link with DNA. Studies on the cell cycle inhibition revealed that this agent induces cell arrest at G2/M phase. The results warrant further antitumor investigation against other human tumor growth in animal models.

This is a preclinical study of BO-0742, a derivative of 3-(9-acridinylamino)-5-hydroxymethyl-aniline (AHMA) and N-mustard, as an anti-cancer agent. MTS assays revealed a broad spectrum of anti-cancer activities in vitro , with the greatest cytotoxicity against leukemia and neuroblastoma including those with drug resistant characteristics, and a good therapeutic index with leukemia being 10–40 times more sensitive to BO-0742 than hematopoietic progenitors. Administration of BO-0742 at an optimal dose schedule based on its pharmacokinetics significantly suppressed the growth of xenografts of human breast and ovarian cancers in mice. Thus, BO-0742 is a potent anti-cancer agent worthy of further clinical development.

Imatinib mesylate (STI571, Gleevec) is a signal transduction inhibitor and novel anti-cancer agent. It selectively inhibits aberrantly activated tyrosine kinases in malignant cells, for example, bcr-abl in leukaemia, platelet-derived growth factor receptor and stem cell factor receptor (c-Kit) in solid cancers including malignant glioma. However, recently published clinical studies with imatinib monotherapy in patients with malignant glioma demonstrated only very modest anti-tumour activity. The aim of this study was to investigate the biological activity of imatinib, its cellular mechanisms of action and its synergism with other chemotherapeutic agents in human malignant glioma cells in culture. Expression of PDGF/R and c-Kit was analyzed by RT-PCR. Proliferation was measured by MTT assays and drug synergy was assessed by the Chou-Talalay method. Cell cycle and apoptosis were analyzed by flow cytometry and migration by monolayer migration assays. Multi-immunoblot was performed on imatinib-treated and control malignant glioma cells. Results indicate that imatinib is more effective in inhibiting cell colony formation and migration rather than proliferation. Imatinib treatment caused cell cycle arrest of glioma cells in G0-G1 or G2/M, with significant elevation of a few cyclin-dependent kinases. Furthermore, imatinib acted synergistically with chemotherapy agents, such as the DNA alkylating agent, temozolomide, and riboneucleotide reductase inhibitors, for example, hydroxyurea at varied effective dose levels. In conclusion, imatinib exerts varied biological effects on malignant glioma cells in culture. Synergistic interaction of imatinib with chemotherapy agents may be related to cell cycle control mechanisms and could be potentially important in a clinical setting.

The "aglycone" of pluraflavin A (2) has been synthesized. The key features of this synthesis include a 1,3-dipolar cycloaddition between a nitrile oxide (cf. 14) and an olefin (22) to yield an isoxazoline followed by subsequent conversion into the gamma-pyrone of pluraflavin A. The epoxide moiety linked to the pyrone is installed prior to Diels-Alder installation of the D ring, which allows access to a number of potentially active cytotoxic intermediates en route to the final compound. The preliminary in vitro results of two such compounds are also included with the racemic title compound exhibiting cytotoxicity in the nanomolar range.

The G2/M cell cycle checkpoint is regulated by a multitude of signaling pathways after genotoxic stress. Herein, we report that treatment with the 90-kDa heat shock protein (Hsp90) molecular chaperone inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG) selectively abrogates the G2/M checkpoint induced by 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of irinotecan, in p53-null compared with p53-intact HCT116 colon cancer cells. The basis for this selectivity can be explained in part by the lack of p21 induction in p53-null cells. In accord with published results, we could show that treatment with 17AAG resulted in depletion of Chk1, a known Hsp90 client protein. In addition, we observed a time- and dose-dependent decrease in Wee1 kinase level, a negative regulator of mitosis, after 17AAG treatment in gastrointestinal cancer cells. Depletion of Wee1 protein preceded mitotic entry induced by 17AAG, and this decrease could be partially rescued by cotreatment with a proteasome inhibitor. Coimmunoprecipitation experiments showed that Hsp90 and Wee1 interacted in whole cells, and 17AAG treatment decreased the degradative half-life of Wee1, indicating that Wee1 is another Hsp90 client in mammalian cells. Knockdown of Chk1 and Wee1 by short interfering RNA each resulted in abrogation of the G2/M checkpoint induced by SN-38. The combination of SN-38 and 17AAG was shown to be synergistic in p53-null but not in parental HCT116 cells by median effect/combination index analysis. Taken together, 17AAG specifically inhibits the G2/M checkpoint in p53-defective cells by down-regulation of two critical checkpoint kinases, Chk1 and Wee1.

The epothilones represent a promising class of natural product-based antitumor drug candidates. Although these compounds operate through a microtubule stabilization mechanism similar to that of taxol, the epothilones offer a major potential therapeutic advantage in that they retain their activity against multidrug-resistant cell lines. We have been systematically synthesizing and evaluating synthetic epothilone congeners that are not accessible through modification of the natural product itself. We report herein the results of biological investigations directed at two epothilone congeners: iso-fludelone and iso-dehydelone. Iso-fludelone, in particular, exhibits a number of properties that render it an excellent candidate for preclinical development, including biological stability, excellent solubility in water, and remarkable potency relative to other epothilones. In nude mouse xenograft settings, iso-fludelone was able to achieve therapeutic cures against a number of human cancer cell lines, including mammarian-MX-1, ovarian-SK-OV-3, and the fast-growing, refractory, subcutaneous neuroblastoma-SK-NAS. Strong therapeutic effect was observed against drug-resistant lung-A549/taxol and mammary-MCF-7/Adr xenografts. In addition, iso-fludelone was shown to exhibit a significant therapeutic effect against an intracranially implanted SK-NAS tumor.

2-Methoxyestradiol (2ME) is an estradiol metabolite with anti-tumor and anti-angiogenic properties. We studied the effect of 2ME on apoptosis of MCF-7 breast cancer cells and explored a combination therapy using 2ME and a polyamine analogue, bis(ethyl)norspermine (BE-3-3-3). Determination of viable cells on day 4 of treatment with 2ME/BE-3-3-3 combinations showed synergistic effects by Chou-Talalay analysis. APO-BRDU analysis showed that there was only 1.5+/-0.5% apoptosis at 200 nM 2ME and 3.7+/-1.7% in the presence of 2.5 microM BE-3-3-3. Combination of 200 nM 2ME and 2.5 microM BE-3-3-3 resulted in 52.2+/-2.6% apoptosis. Up to 90% of the cells underwent apoptosis in the presence of 1000 nM 2ME and 2.5 microM BE-3-3-3. Combination treatments resulted in total disruption of microtubules and depletion of putrescine, spermidine and spermine. In addition, phosphorylation of Akt and nuclear localization of cyclin D1 were altered by 2ME/BE-3-3-3 combination. Our results suggest an important strategy to induce apoptosis of breast cancer cells, with potential applications in therapy.

This brief article focuses on two aims: i) To investigate the in vitro pharmaco-dynamic interactions of combining synthetic potent microtubule targeting anticancer agent, Fludelone (FD) with cyto-protective agent, Panaxytriol (PXT) derived from Panax ginseng, and ii) To illustrate step-by-step operation for conducting two-drug combination in vitro using the combination index method, in terms of experimental design, data acquisition, computerized simulation and data interpretation. The Chou-Talalay method for drug combination is based on the median-effect equation, which provides the theoretical basis for the combination index (CI)-isobologram equation that allows quantitative determination of drug interactions, where CI1 indicates synergism, additive effect and antagonism, respectively. Based on these algorithms, computer software, CompySyn, is used for determining synergism and antagonism at all doses or effect levels simulated automatically. The use of Chou-Talalay’s CI method in quantifying synergism or antagonism is increasing steadily during the past two decades, however, confusing questions and pitfalls were still frequently raised by insufficient understanding of the theory, especially reflected when researchers trying to use the computerized software to design and conduct experiments. In order to specifically address the confusions and to illustrate the practical features of this method, in this paper, a selected example is given based on our unpublished data regarding the combinational pharmacologic interactions of FD and PXT against the growth of breast cancer cell line MX-1. The step-by-step operation from experimental design to the real data analysis is illustrated. The results indicated that FD and PXT combination in vitro exerted synergistic effect when cell growth inhibition was greater than 45%, with CI ranged 0.836-0.609 for the fractional inhibition of Fa=0.50~0.90, as shown by the Fa-CI plot and by the isobologram. Thus, quantitative conclusion of synergism is obtained using the Chou-Talalay CI method, under the well-defined simple conditions for the FD and PXT combinations in vitro.

Malignant pleural mesothelioma (MPM) treated with radiotherapy (RT) has incomplete responses as a result of radiation-induced tumoral stress response that repairs DNA damage. Such stress response is beneficial for oncolytic viral therapy. We hypothesized that a combination of RT and NV1066, an oncolytic herpes virus, might exert an additive or synergistic effect in the treatment of MPM. JMN, a MPM cell line, was infected with NV1066 at multiplicities of infection of .05 to .25 in vitro with and without radiation (1 to 5 Gy). Virus replication was determined by plaque assay, cell kill by lactate dehydrogenase assay, and GADD34 (growth arrest and DNA damage repair 34, a DNA damage-repair protein) by real-time reverse transcriptase–polymerase chain reaction and Western blot test. Synergistic cytotoxicity dependence on GADD34 upregulation was confirmed by GADD34 small inhibitory RNA (siRNA). Synergism was demonstrated between RT and NV1066 across a wide range of doses. As a result of such synergism, a dose-reduction for each agent (up to 5500-fold) can be accomplished over a wide range of therapeutic-effect levels without sacrificing tumor cell kill. This effect is correlated with increased GADD34 expression and inhibited by transfection of siRNA directed against GADD34. RT can be combined with oncolytic herpes simplex virus therapy in the treatment of malignant pleural mesothelioma to achieve synergistic efficacy while minimizing dosage and toxicity.