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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

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.

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.

A series of 5-(9-acridinylamino)anisidines were synthesized by condensing methoxy-substituted 1,3-phenylenediamines (10 and 11) with 9-chloroacridine derivatives to form 5-(9-acridinylamino)-m-anisidines (AMAs, 14a-e) and 5-(9-acridinylamino)-o-anisidines (AOAs, 15a-e). 5-(9-Acridinylamino)-p-anisidines (APAs, 17a-e) were synthesized by reacting 2-methoxy-5-nitroaniline (12) with 9-anilinoacridines, followed by reduction. The cytotoxic inhibition of growth of various human tumor cells in culture, inhibitory effects against topoisomerase II, and DNA interaction of these agents were studied. The structure-activity relationship studies revealed the following degree of potency: AOAs > AMAs > APAs. They also revealed that the newly synthesized derivatives bearing CONH(2)NH(2)NMe(2) and Me substituents at C4 and C5 positions of the acridine chromophore (i.e., AMA 14e, AOA 15e, and APA 17e) exhibited significant cytotoxicity against human tumor cell growth in vitro. AOA (15e) was the most potent among these derivatives, which resulted in 60% suppression of tumor volume at a dose of 20 mg/kg (Q2D x 9), intravenous injection on day 26 in nude mice bearing human breast carcinoma MX-1 xenografts.

Oncolytic herpes viruses are attenuated, replication-competent viruses that selectively infect, replicate within, and lyse cancer cells and are highly efficacious in the treatment of a wide variety of experimental cancers. The current study seeks to define the pharmacologic interactions between chemotherapeutic drugs and the oncolytic herpes viral strain NV1066 in the treatment of pancreatic cancer cell lines. The human pancreatic cancer cell lines Hs 700T, PANC-1, and MIA PaCa-2 were treated in vitro with NV1066 at multiplicities of infection (MOI; ratio of the number of viral particles per tumor cell) ranging from 0.01 to 1.0 with or without 5-fluorouracil (5-FU) or gemcitabine. Synergistic efficacy was determined by the isobologram and combination-index methods of Chou and Talalay. Viral replication was measured using a standard plaque assay. Six days after combination therapy, 76% of Hs 700T cells were killed compared with 43% with NV1066 infection alone (MOI = 0.1) or 0% with 5-FU alone (2 micromol/L) (P.01). Isobologram and combination-index analyses confirmed a strongly synergistic pharmacologic interaction between the agents at all viral and drug combinations tested (LD5 to LD95) in the three cell lines. Dose reductions up to 6- and 78-fold may be achieved with combination therapy for NV1066 and 5-FU, respectively, without compromising cell kill. 5-FU increased viral replication up to 19-fold compared with cells treated with virus alone. Similar results were observed by combining gemcitabine and NV1066. We have demonstrated that 5-FU and gemcitabine potentiate oncolytic herpes viral replication and cytotoxicity across a range of clinically achievable doses in the treatment of human pancreatic cancer cell lines. The potential clinical implications of this synergistic interaction include improvements in efficacy, treatment-associated toxicity, tolerability of therapeutic regimens, and quality of life. These data provide the cellular basis for the clinical investigation of combined oncolytic herpes virus therapy and chemotherapy in the treatment of pancreatic cancer.

A series of N-mustard derivatives of 9-anilinoacridine was synthesized for antitumor and structure-activity relationship studies. The alkylating N-mustard residue was linked to the C-3' or C-4' position of the anilino ring with an O-ethylene (O-C(2)), O-butylene (O-C(4)), and methylene (C(1)) spacer. All of the new N-mustard derivatives exhibited significant cytotoxicity in inhibiting human lymphoblastic leukemic cells (CCRF-CEM) in culture. Of these agents, (3-(acridin-9-ylamino)-5-{2-[bis (2-chloroethyl)amino]ethoxy}phenyl)methanol (10) was subjected to antitumor studies, resulting in an approximately 100-fold more potent effect than its parent analogue 3-(9-acridinylamino)-5-hydroxymethylaniline (AHMA) in inhibiting the growth of human lymphoblastic leukemic cells (CCRF-CEM) in vitro. This agent did not exhibit cross-resistance against vinblastine-resistant (CCRF-CEM/VBL) or Taxol-resistant (CCRF-CEM/Taxol) cells. Remarkably, the therapeutic effect of 10 at a dose as low as one tenth of the Taxol therapeutic dose [i.e., 1-2mg/kg (Q3Dx7) or 3mg/kg (Q4Dx5); intravenous injection] on nude mice bearing human breast carcinoma MX-1 xenografts resulted in complete tumor remission in two out of three mice. Furthermore, 10 yielded xenograft tumor suppression of 81-96% using human T-cell acute lymphoblastic leukemia CCRF-CEM, colon carcinoma HCT-116, and ovarian adenocarcinoma SK-OV-3 tumor models.

This report presents quantitative analysis of the synergistic interaction of azidothymidine (AZT) and cladribine (CdA) in human H9-lymphoid cell lines sensitive and resistant to AZT (H9-araC cells). H9-araC cells obtained by cultivation of H9 cells in the presence of 0.5 microM arabinosyl-cytosine (araC) had lower deoxycytidine kinase and thymidine kinase (TK) activities and expressed cross-resistance to araC and AZT. The IC(50) values of AZT and CdA were calculated by using median-effect analysis and CalcuSyn software. The IC(50) values were 0.44 and 0.82 microM for CdA and 67.8 and 30,310 microM for AZT in H9 and H9-araC cells, respectively. However, when the drugs were used in combination the IC(50) values of CdA and AZT were reduced to 0.12 and 15.5 microM in H9 cells and to 0.19 and 24.9 microM in H9-araC cells, respectively. Calculation of dose reduction index (DRI) indicated that at 50-90% growth inhibition level, the combination of the drugs caused 3.6-5.8- and 4.1-11.5-fold reduction in the dose of CdA and 4.4-37.6- and > 1000-fold reduction in the dose of AZT in H9 and H9-araC cells, respectively. The combination index (CI) values simulated from these data suggested synergistic to very strong synergistic lymphocytotoxic effects of AZT combined with CdA. These findings suggest the potential usefulness of a double-targeted approach for designing efficacious therapeutics for the kinase deficient drug resistant tumors.

A series of new analogues of 3-(9-acridinylamino)-5-hydroxymethylaniline (AHMA, 1) and AHMA-ethylcarbamate (2) were synthesized by introducing an O-alkylcarboxylic acid esters to the CH2OH function, displacing the CH2OH function with a dimethylaminocarboxamido group or with a methyl function introduced at the meta-, para -o rortho-position to the NH2 group to form 5-(9-acridinylamino)-m-toluidines (AMTs), 5-(9-acridinylamino)-p-toluidines (APTs) or 5-(9-acridinylamino)-o-toluidines (AOTs), respectively. The inhibitions of a variety of human tumor cell growth, interactions with DNA as well as inhibitory effect against topoisomerase II (Topo II) of these new agents were studied. Among AMT, APT and AOT derivatives with dimethylaminoethylcarboxamido and Me at C4 and C5 of acridine moiety (i.e., 21c, 23c and 26c) were more cytotoxic than AHMA (1) and AHMA-ethylcarbamate (2), depending upon the tumor cell line tested. Detailed structure-activity relationships of the new analogues were studied. # 2003 Elsevier Ltd. All rights reserved.

The title compound retains some of the affinity for microtubule assemblies as does 12,13-desoxyepothilone B.

The synthesis and biological evaluation of about one dozen 7-silylcamptothecin derivatives are described. Most new compounds show potencies comparable to or better than camptothecin itself. The best compound, 11-fluoro-10-amino-7-trimethylsilylcamptothecin, is more than 20 times more potent than camptothecin in cell assays.

The title compounds have been synthesized in a convergent way by recourse to a Weiler type dianion construction.

Several promising antiviral nucleosides have been tested in paired combinations against guinea pig cytomegalovirus (GPCMV) replication in guinea pig embryo (GPE) cells by plaque reduction assay; these are [9-(2-hydroxy-1-3-2-dioxaphosphorinan-5-yl)oxymethyl]-guanine P-oxide (2′nor-cGMP, compound 164), [4-amino-5-bromo-7-(2-hydroxyethoxymethyl)-pyrrolo(2,3-d)pyrimidine] (compound 102), (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC), 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), 9-(2-hydroxyethoxymethyl)guanine (acyclovir, ACV) and 3′-azido-3′-deoxythymidine (zidovudine, AZT). Various degrees of interactions were observed; i.e. synergistic reactions were noted in the presence of compound 164/compound 102 and compound 164/DHPG combinations at all concentrations tested. HPMPC/DHPG combinations were synergistic at relatively lower concentrations of DHPG, but became antagonistic as the concentration of DHPG increased. Combinations of compound 164/ACV and DHPG/AZT were antagonistic.

Background: NV1066, a replication-competent oncolytic herpes simplex virus attenuated by deletion of the viral replicative gene |[gamma]|134.5, preferentially replicates in and kills malignant cells. |[gamma]|134.5 codes for the protein ICP 34.5, which is homologous to the cisplatin-induced stress response protein GADD34 (Growth Arrest and DNA Damage 34). We hypothesize that cisplatin-induced GADD34 upregulation enhances viral replication and potentiates NV1066 efficacy.

Treatment of H35 hepatoma cells with the lipid soluble dihydrofolate reductase inhibitors metoprine and trimetrexate cause a nearly 10-fold increase in the toxicity of the antipyrimidine folate analogue PDDF and the antipurine folate analogue DDATHF. Evaluation of these interactions by the combination index developed by Chou (17-20) yields results conforming to synergistic interactions. The capacity of PDDF to inhibit thymidylate synthase in intact cells as measured by tritium release from [5-3H]deoxyuridine was increased by approximately the same amount by preincubation with the dihydrofolate reductase inhibitors. The primary effect of the reductase inhibitors in causing greater activity may be a reduction in cellular folates which can cause 5,10-CH2H4PteGlun to decrease and cellular PDDF (polyglutamates) to increase. These conditions would favor inhibition of thymidylate synthase by PDDF by promoting formation of the stable, inhibited PDDF (polyglutamates)-thymidylate synthase-dUMP complex (12).

The incorporation and metabolism of 2′-fluoro-5-substituted arabinosyl pyrimidine analogs, and their selective inhibition of viral DNA synthesis in herpes simplex virus type 1 (HSV-1)-infected and mock-infected Vero cells were studied by HPLC and CsCl isopycnic density gradient analysis of isolated DNAs. The amounts of radiolabeled analogs incorporated as parent compound following 10 μM exposure for 4 h were 10-fold higher in HSV-1-infected vs mock-infected cells for 2′-fluoro-5-difluoromethyl-Ara-U (F 2 FMAU); 4.3-fold higher for 5-ethyl deoxyuridine (EdU); 2.6-fold higher for 2′-fluoro-5-methyl-Ara-U (FMAU) and 1.7-fold higher for dThd. For 2′-fluoro-5-ethyl-Ara-U (FEAU), 3.0 pmole of unchanged moiety was incorporated per 10 6 HSV-1-infected cells but no incorporation was detected in mock-infected cells. HPLC profiles showed that the percentages of radiolabeled analogs incorporated as parent compound in the DNA extracted from HSV-1-infected cells were 31.0% for F 2 FMAU, 99.6% for EdU, 83.5% for FEAU and 98.3% for FMAU; from mock-infected cells, they were 63.6% for F 2 FMAU, 96.7% for EdU, 97.3% for FMAU and no incorporation into DNA for FEAU was detected. CsCl density gradient analyses of isolated DNA showed that viral DNA synthesis was inhibited 98% by 10 μM FEAU, 92% by 10 μM F 2 FMAU, 90% by 2 μM FMAU and 80% by 50 μM EdU, whereas cellular DNA synthesis was inhibited by 53, 44, 61, 66 and 54%, respectively. We conclude that: (a) FEAU incorporation into host-cell DNA was not detectable but FEAU was selectively incorporated into HSV-infected cells; (b) FMAU and FEAU were metabolically stable; however, F 2 FMAU was extensively metabolized; (c) FEAU and F 2 FMAU were among the most selective inhibitors of HSV-1 DNA synthesis while allowing cellular DNA synthesis to continue.

Systematic analyses of dose-effect relationships of inhibitors in the presence of substrates in enzyme catalyzed reactions with different reaction mechanisms and different types and mechanisms of inhibition have yielded equation (A), f t = 1 [1+( I 50 I )] , where ft is fractional inhibition and I50 is the concentration of inhibitor, I, that is required to produce a median-effect. Equation (A) has the same form as the Michaelis-Menten equation, v V max = 1 [1+( K m S )] , and thus share the common geometric properties. However, equation (A) is mechanism independent, contains no explicit kinetic constant, and the effect is expressed with respect to the control velocity rather than the maximal velocity. The relationship depicted by equation (A) has the following applications in analyzing experimental data, (i) The median-effect concentration and the apparent kinetic order of interaction can be conveniently determined by graphical methods which transform the dose-effect relationships into linear forms, (ii) The method described by Dixon (1972) for determining the distribution of enzyme species in single-substrate reactions in the presence of a tightly binding inhibitor can be extended to multiplesubstrate reactions with different mechanisms, (iii) The median-effect principle provides an alternative simple way to derive a variety of equations of the mass-action law including the equilibrium binding equation of Scatchard (1949) . It is also shown that by determining the ratios of Kt and I50 of different inhibitors under the same experimental conditions, it is possible to determine whether the inhibitors bind to the same fraction of the total enzyme.

The summation of the effects of two or more reversible inhibitors of various types on the initial velocity of enzyme systems obeying Michaelis-Menten kinetics is described by the the general relation: (formula: see text) wherein v1,2,3...n is the velocity of reaction in the simultaneous presence of n inhibitors, vi is the velocity observed in the presence of each individual inhibitor, and v0 is the velocity in the absence of inhibition. The derivation is based on the assumption that each enzyme species can combine with no more than one of the inhibitors (i.e. the inhibitors are mutually exclusive). The above relationship holds irrespective of the number of inhibitors, the type of inhibition (competitive, noncompetitive, or uncompetitive), or the kinetic mechanism (sequential or ping-pong) of the enzyme reaction under consideration. Deviations from this equality define synergism or antagonism of inhibitors depending on whether the value of the left side of the above equation is greater or smaller than the right, respectively. Knowledge of the kinetic constants for substrates and inhibitors is not required. If two or more inhibitors act independently (i.e. are not mutually exclusive), their combined effects are necessarily synergistic. Under certain circumstances, described in the text, mutually nonexclusive inhibitors obey the fractional velocity product relationship: v1,2,3...n/v0 = (v1/v0) x (v2/v0) x (v3/v0)...(vn/v0).

The dose-effect relationships of the reference ligands (e.g. inhibitors) in the presence of the primary ligands (e.g. substrates) in enzyme kinetic models have been analyzed systematically. Enzyme reactions with different number of substrates, different reaction mechanisms, and different types and mechanisms of inhibition yield, equation (A), f a f u = D D m , for the first-order interactions; and equation (B), f a f u = ( D D m ) m , for the m-order interactions; where D is dose, Dm is the D required for the median-effect, and fa and fu are the fractions of the system that are affected and unaffected by D, respectively. Conversion of equation (A) gives f a = 1 [1 + ( D m D )] which has the same form as the Michaelis-Menten equation, v V max = 1 [1 + ( K m S )] . The logarithmic form of equation (B) gives the Hill type equation. In equations (A) and (B), the fractional velocity is expressed with respect to the control velocity rather than the maximal velocity; and D can be the reference ligands or environmental factors rather than the restriction to the primary ligands. It is concluded that a plot of log (D) v. log [(fu)−1 − 1] or log [(fa)−1 − 1]−1 will be a useful procedure for transforming dose-effect relationships and for analysing mass action characteristics in various systems. The plot will give the slope m, and the intercepts of the dose-effect lines with the median-effect axis (i.e. log [(fu)−1 − 1] = 0) will give Dm values. Any cause-consequence relationships that give straight lines in the plot will give m and Dm values, and thus give empirical equation for the system. Both equations (A) and (B) have been compared with relevant existing equations in terms of limitations and applicabilities. It is concluded that mechanism-dependent equations involving kinetic or equilibrium constants can be transformed to a mechanism-independent general equation involving the median-effect value.

A new assay technique for ATP:l-methionine S-adenosyltransferase activity (EC 2.5.1.6) is described. The procedure eliminates the use of ion exchange resin columns which have been routinely used in the past. The principle of the assay depends upon the separation of S-adenosyl-l-[Me-14C]methionine from l-[Me-14C]-methionine by differential adsorption on cellulose phosphate ion exchange discs. The cellulose phosphate disc method is considerably simpler, more rapid, and less expensive than the column procedure while retaining high sensitivity and precision.