WR1065 dihydrochloride [2-(3-aminopropyl)aminoethanethiol dihydrochloride, empirical formula C5H14N2S·2HCl, CAS#14653-77-1, 207.16 Da; catalog#W2020] and 4-arm-PEG-SH [pentaerythritol core, formula C(CH₂O(CH₂CH₂O)nCH₂CH₂SH)₄, 10,000 Da average; catalog#JKA7008] were purchased from Sigma-Aldrich (St. Louis, MO, USA). m-PEG₆-thiol (formula C13H28O6S, CAS#441771-60-4, 312.4 Da; catalog#BP-22084) was obtained from BroadPharm (San Diego, CA, USA). Additional high purity chemicals used for synthesizing 4SP65 or 1LP65 were acquired by MedChem Partners. Amifostine was purchased from Moravek Biochemicals (Brea, CA, USA). Other pharmaceuticals or reagents including cisplatin (DDP) (Tocris Bioscience; catalog#2251/250), difluoromethylornithine (DFMO) (Selleck Chemical LLC; catalog#50-217-3144), gefitinib (LC Laboratories; catalog#G44081G), Trypan Blue, alamarBlue™ cell viability reagent (Invitrogen), and CyQUANT^®-NF cell proliferation assay kits were obtained via ThermoFisher Scientific (Waltham, MA, USA).

4SP65 was synthesized as the first BRG prodrug using a novel multi-step scheme described in brief in Supplementary Material and in detail in a Composition of Matter patent (WO2017087668), entitled Methods for improved protection and delivery of aminothiols and analogs thereof. 4SP65 (average 10,532 Da), as shown below, is the abbreviation for the trifluoroacetic acid salt of the prodrug or conjugate 7 in Figure S1 .

1LP65 was synthesized using the same multistep method in Figure S1 except for the substitution of m-PEG₆-SH shown below for compound 5,

to generate Boc-protected conjugate 6 and then the final product conjugate 7 or 1LP65 following Boc deprotection:

1LP65 is the abbreviation for the di-trifluoroacetic acid salt of this prodrug, weighing 483 Da once dissolved in solution ( Figures S3, S4 ). Figures S1–S4 and experimental findings related to the solubility, storage, and stability of 1LP65 and 4SP65 are presented in Supplementary Material.

Normal human mammary epithelial cell (NHMEC) strains used to assess potential cytotoxicity of 4SP65 and 1LP65 were established from reduction mammoplasty tissue from a healthy female donor (M99005 or ‘Strain-1’) (53) or purchased from ATCC (Manassas, VA, USA; batch#70043304 or ‘Strain-2’) or Cell Applications (San Diego, CA, USA; lot#1669 or ‘Strain-3’). Human cancer cell lines including HCC38, MDA-MB-231, A549, National Cancer Institute (NCI)-H460, NCI-H1437, NCI-H1975, DU145, LNCaP, PC3, PANC1, SKOV3, and HL60, which were free of Mycoplasma, were purchased from ATCC. HMESO1, PPMMill, and TOV21G cells, obtained from Dr. Brian Cunniff, were verified to be Mycoplasma negative using the LookOut^® Mycoplasma PCR Detection Kit (Sigma-Aldrich) and to match previously annotated DNA fingerprints by the Vermont Integrative Genomics Resource DNA Analysis Facility.

Cell culture medium components from various vendors were obtained through ThermoFisher Scientific. Normal mammary epithelial cells were cultured in Mammary Epithelium Growth Medium (Lonza, Lexington, MA, USA), containing BulletKit™ growth supplements, at 37°C in a humidified incubator with 5% CO₂. Mesothelioma and ovarian cancer cell lines were maintained in 50:50 DMEM (Corning, Manassas, VA, USA)/F12 medium (Lonza) supplemented with 10% (vol/vol) fetal bovine serum (FBS; Corning) and 100 U/mL penicillin-streptomycin (Corning). H1975 cells were cultured in an ATCC modified RPMI-1640 medium (Gibco, Waltham, MA, USA) with low L-glutamine, 10% FBS, and Pen-Strep. All other human cancer cell lines were grown in standard RPMI-1640 medium (Corning) supplemented with FBS and Pen-Strep.

To determine drug stability in tissue culture medium for short-term treatments, 4SP65 was added to medium containing alamarBlue reagent in 96-well plates and incubated in the absence of cells at 37°C for 72 h. After this incubation period, wells with medium containing alamarBlue alone or both alamarBlue and 4SP65 were assessed for fluorescence readings using a Tecan Infinite 200 PRO plate reader (San Jose, CA, USA).

To compare the utility of the alamarBlue assay and the CyQUANT^®-NF cell proliferation assay for determining changes in cell numbers following test agent exposures, H1437 non-small cell lung cancer (NSCLC) cells were exposed for 48 h to 0 - 12.5 µM 4SP65 or to 0 - 8 µM cisplatin. After treatments, fluorescence counts were determined using alamarBlue reagent or CyQUANT^® dye according to manufacturers’ instructions using a plate reader. Both logarithmic and polynomial curves were fitted to each relative cell survival curve, and R² values were determined using trendlines and formulas calculated with Excel software.

Since the BRG prodrugs are composed of WR1065 as one of the major constituents, assays were conducted to determine if factors reported to modulate the activity of amino acid and polyamine plasma membrane transport systems impacted the efficacy of 4SP65 or 1LP65 in selected cancer cell lines. HL60 cells were exposed to 4SP65 with up to 0.5 mM NaCl, MBA-MD-231 cells were tested in the presence and absence of medium supplemented with estradiol plus insulin (in the BulletKit™ for growth supplements for NHMECs), HCC38 cells were exposed to 4SP65 with and without 10 µM insulin added to the growth medium, and multiple cells lines were pretreated for 24 h with 1 mM DFMO prior to starting exposures to 4SP65.

To test anticancer effectiveness of individual drugs or drug combinations, the CyQUANT^®-NF proliferation assay was used with a plate reader (i) to generate standard curves for cell numbers for each cell line, (ii) to measure the starting cell numbers at initiation of drug treatment, and (iii) to obtain fluorescence readings from cells in 96-well plates for comparisons to a standard curve for the relevant cancer cell line and quantifying cell viability after drug treatments. DNA dyes like CyQUANT are a highly reliable method for determining the effects of anticancer agents on cell proliferation (54). Trypan blue exclusion was used to estimate cell numbers in a few pilot experiments and thereafter was applied to confirm very low viable cell numbers and cell death.

To assess the effects of 4SP65 or 1LP65 on the growth of normal epithelium, experiments were performed using NHMEC strains from differing donors (53). Strain-1 NHMECs were plated at 5,000 cells/well in 24-well plates and allowed to reach 50% confluence before treatment with 0-150 µM 4SP65 for 48 h. Cell viability was determined using a hemocytometer and trypan blue exclusion assay. Two other NHMEC strains, exposed to 0-150 µM 4SP65 or 0-500 µM 1LP65 for 48 h, were handled in the same fashion except that viability of cells in 96-well plates was determined using CyQUANT dye. Due to the short life span of these NHMECs, standard curves were not generated and fluorescence readings were used as a surrogate marker of cell number (54).

A series of experiments was conducted to determine the relative efficacy of 4SP65, 1LP65, and amifostine as single agents in the same human cancer cell lines. The effectiveness of 4SP65 was tested against 15 cancer cell lines derived from seven tissues while the efficacy of 1LP65 was examined against 11 cancer cell lines developed from four tissues. For short-term cell viability assays, 2000–5000 cells/well were plated in 96-well dishes and incubated for 24 h to allow cells to enter log-phase growth. Then starting cell numbers were determined and cells in the remaining wells were treated with 0 to ≤50 µM 4SP65 or 0 to ≤200 µM 1LP65 to characterize drug effectiveness following a single 48-h exposure. The efficacy of 0 to ≤500 µM amifostine was tested in a parallel manner in 14 cancer cell lines reported to have either medium, high, or no expression of plasma membrane-anchored alkaline phosphatase, the enzyme required for metabolism of amifostine to WR1065 and its subsequent efficient uptake into cells (55).

Additional series of experiments were performed in a parallel fashion to define the relative effectiveness of 4SP65 alone versus binary pairs of 4SP65 with cisplatin or gefitinib against selected human cancer cell lines. First, drug combination studies were conducted to compare dose-response metrics for 4SP65 alone, cisplatin alone, or both drugs combined against six human cancer cell lines derived from three tissues. Then, the dose-response metrics for 4SP65 alone, gefitinib alone, or both drugs combined were evaluated in A549 NSCLC cells. Starting cell numbers were determined 24 h after plating and remaining cells were treated with 0 to ≤50 µM 4SP65 alone, 0 to >15 µM cisplatin alone, 0 to 25 µM gefitinib alone, or with 4SP65 combined with cisplatin or gefitinib at selected ratios to measure dose-response metrics for drug effectiveness following single 48-h exposures.

The experimental design followed guidelines for comparisons between drugs used by the NCI (56) along with recommended modifications (54). For drug combination studies, experimental design methods recommended by Chou et al. (57) were used. Data analyses used NCI methodology (56) along with those of Brooks et al. (58) to characterize concentration parameters that measure drug effectiveness following a single antagonist cancer drug treatment (Tong 2010 https://escholarship.org/uc/item/9295t422). Five metrics were calculated and used to characterize the shape of the dose-response curve following exposure to a single test agent. Using cell numbers based on fluorescence measurements and standard curves, the percentage growth was calculated at each drug concentration level. The percentage of growth inhibition was calculated as follows:

where Tz = cell numbers or fluorescence counts at time xero, C = vehicle-exposed control cell numbers at the end of the assessment time period, and Ti = test agent-exposed cell numbers or fluorescence counts at each drug concentration. The inhibitory concentration 50% (IC50) was calculated as the concentration of the drug that reduced the growth of treated cells by 50% compared to vehicle-exposed control cells. However, IC50 values do not consider the initial cell population at time zero, leading to the development of three new special concentration parameters to improve the measurement of drug effectiveness and to enhance comparisons between drugs (see Figure 1A ). The growth inhibition of 50% (GI50) was calculated using Equation 1 = 50. The drug concentration resulting in total growth inhibition (TGI) was calculated using Equation 1 = 0. The LC50/99 was calculated using Equation 2 = -50 and Eq. 2 = -99, respectively. To define dose-response metrics in cancer cell lines, relationships between drug exposure levels and cell counts were used to calculate growth inhibition values for each exposure level, plotted on semi-log curves, and modelled using best fit regression curves and trend line formulas generated by Excel. For NHMECs, the relationships between drug exposure levels and fluorescence units were used to define dose-response metrics. The selectivity index values for individual drugs were calculated using IC50 levels in NHMECs divided by the IC50 value for each cancer cell line. Statistical comparisons of differences in individual dose-response metrics between drug treatments and cell lines were conducted using Student’s t-test, with P-values <0.05 considered significant.

Possible synergy between treatments with 4SP65-cisplatin or 4SP65-gefitinib drug pairs was explored further using SynergyFinder 2.0 (https://synergyfinder.fimm.fi) (59), a stand-alone web-application for interactive analysis that allows for inputting data from independent replicate experiments in order to calculate a 95% confidence interval for synergy scoring. Relative cell growth inhibition values for differing levels of each drug alone and in combination were input into SynergyFinder to generate dose-response curves for each drug alone and to produce a dose-response matrix. The dose-response matrix data then were used in generating a two-dimension heatmap and three-dimension volcano plot to visualize the distribution of areas of differing degrees of synergism and to calculate average and maximum synergy scores in selected human cancer cell lines. The expected drug combination responses were calculated using both the Highest Single Agent (HSA) model and the Zero interaction potency (ZIP) model. The HSA model, or Gaddum’s non-interaction model, assumes that the expected combination effect equals the higher individual drug effect at the dose in the combination, representing the idea that a synergistic drug combination should produce additional benefits on top of what its components can achieve alone (60). The ZIP model captures the drug interaction relationships by comparing the change in the potency (effect at a given dose level) of the dose–response curves between individual drugs and their combinations (60). Synergy scores < −10 indicate that the interaction between two drugs is likely to be antagonistic; scores from −10 to +10 indicate likely additive effects; and scores >10 indicate likely synergistic effects (59).

To determine 4SP65 prodrug stability in standard growth medium, reduction of alamarBlue by 4SP65 was evaluated in a cell free system for 72 h. At this timepoint, there was no significant change in the fluorescence of alamarBlue reagent in wells containing 4SP65 compared to control wells with medium containing alamarBlue without 4SP65 (data not shown). This finding indicates that 4SP65 did not induce detectable conversion of resazurin to resorufin in the absence of cells.

A series of experiments was conducted to determine the relative capacity of alamarBlue reagent or CyQUANT^® dye to measure cell viability following 48-h exposures to 4SP65 ( Supplementary Figure 5 ). The resulting data show that goodness-of-fit testing did not achieve R² values of 0.95 or greater for logarithmic or polynomial curves fitted to data for 4SP65-exposed H1437 NSCLC cells using alamarBlue reagent. However, a R² value of 0.996 was obtained for a polynomial curve fit to the relative cell survival data using CyQUANT^® dye in 4SP65-exposed H1437 cells. These findings demonstrate that conversion of alamarBlue reagent is non-linear in cells exposed to 4SP65 and the data are consistent with reported activity of WR1065 as a potent reducing agent and hydrogen donor in populations of normal and cancer cells (61–64). As a control, simultaneous studies were conducted using H1437 cells exposed to increasing levels of cisplatin. After removing the relative cell survival data for the two highest cisplatin concentrations where the curve flattened, curves fitted to either alamarBlue or CyQUANT^® fluorescence counts were linear across the change in drug levels and had R² values ≥ 0.95, showing that cisplatin had negligible effects upon the reactions involved in alamarBlue conversion in cancer cells.

Since 4SP65 is larger than 500 - 600 Da, and because previous studies of WR1065 implicated the involvement of an amino acid/polyamine plasma membrane transport system in drug uptake (65), it was postulated that some kind of active transport process was involved in BRG prodrug entry into cells. Thus, a series of pilot experiments was performed to obtain qualitative data for the effect of plasma membrane transport inducers upon the effects of BRG prodrugs in differing cancer cell lines. The overall goal was to determine if repeated evidence of enhanced anticancer effects was observed in the presence versus the absence of an inducer of one or more plasma membrane transport systems. The effects of insulin alone or insulin plus estradiol, known inducers of plasma membrane transport, were evaluated (66). HCC38 and MDA-MB-231 human mammary cancer cell lines were chosen so that estradiol and insulin, reagents provided in the BulletKit™ for growth of NHMECs, could be used. The first experiment was performed by allowing HCC38 cells to reach 99% confluence and then exposing cells for 48 h to 4SP65 at 10 µM with and without 10 µM insulin. Relative cell inhibition was 3.4 ± 10.9% in cells exposed to 4SP65 alone and 42 ± 6.5% in cells exposed to 4SP65 with added insulin, representing a 12.4-fold increase in drug efficacy in growth-arrested cells. In exponentially-growing MDA-MB-231 cells exposed for 48 h to 4SP65 with or without insulin plus estradiol, drug efficacy at the TGI level was increased by nearly 4-fold with hormones (TGI = 1.2 µM) versus without hormones (TGI = 4.6 µM). In all cases, induction of cell death by BRG prodrugs occurred at lower doses in the presence of a plasma membrane transport inducer compared to in its absence.

Another series of experiments was performed to assess the impact of DFMO on the anticancer efficacy of the BRG prodrugs. DFMO is an irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme involved with polyamine biosynthesis that also impacts polyamine transport (67). Results for A549 NSCLC cells treated with 1LP65 are illustrative of the influence of DFMO pretreatment in most human cancer cell lines tested. A549 cells were pretreated with DFMO at 1 mM for 24 h before being exposed to increasing concentrations of 1LP65 for 48 h. In A549 cells, the IC50 for 1LP65 decreased from 51 µM without DFMO pretreatment to 33.1 µM with DFMO; while the GI50 decreased from 38.6 to 17.1 µM without and with DFMO, respectively. Taken together, these sets of studies support the conclusion that both 4SP65 and 1LP65 are actively transported by one or more to-be-characterized membrane transport systems.

For this report, initial evaluations of the safety of 4SP65 and 1LP65 focused on their effects upon the growth of NHMECs from different donors. In a trial experiment, Strain-1 cells were exposed for 48 h to 0, 5, 15, 50, or 150 µM 4SP65 ( Figure 1B ). Treatments with 5 and 15 µM 4SP65 greatly enhanced the growth of Strain-1 cells by 336% and 286%, respectively, compared to sham-exposed control cells. In contrast, the growth of Strain-1 cells exposed to an intermediate concentration of 50 µM 4SP65 was 68% of the control cell growth. Remarkably, treatment of Strain-1 cells with a higher concentration of 150 µM 4SP65 re-established a predominant pattern of growth enhancement by the drug. In subsequent repetitive experiments, 48-h treatments of Strain-2 and Strain-3 cells with 4SP65 induced dose-response curves with growth enhancement of 162% ± 7% at 3 µM drug and concentration-dependent decreases in growth relative to control cells of 78% ± 12%, 52% ± 8%, 21% ± 4%, and 8.7% ± 2% after respective exposures to 9, 30, 90, and 150 µM drug ( Figure 1B ). Notably, the average effect per unit dose of 4SP65 (% growth ÷exposure concentration) for 9, 30, 90, and 150 µM drug exposures was 8.7, 1.70, 0.23, and 0.058, respectively, indicating that 4SP65-mediated growth inhibitory effects reached saturation, consistent with the induction of p53-mediated cell-cycle arrest in normal cells (3, 31). Last, while IC50 values could not be predicted for Strain-1 and Strain-2 NHMECs exposed to 4SP65, an IC50 of 76 ± 14 µM was calculated for Strain-3 cells.

Treatment of Strain-2 and Strain-3 cells for 48 h with 0-500 µM 1LP65 also induced concentration-dependent decreases in cell growth, reaching 19.5% and 12.5% of control cell values, respectively, at the highest drug level ( Figure 1B ). Following exposures to 90, 150, 300, or 500 µM 1LP65, an average effect per unit dose of 2.9, 0.31, 0.06, and 0.03, respectively, was observed, providing evidence that 1LP65-mediated growth inhibition approached saturation in both strains of normal cells. However, the responses to 1LP65 exposure in these two strains of NHMECs were sufficiently different that the average IC50 values were calculated to be >500 µM in Strain-2 cells and 210 µM in Strain-3 cells, averaging >355 µM for the two normal cell strains.

The BRG prodrug 4SP65, delivering four WR1065s/molecule intracellularly, yielded broad-spectrum in vitro anticancer effects against 15 human cancer cell lines representing four NSCLCs (A549, H460, H1437, and H1975), an acute promyelocytic leukemia (HL-60), two triple negative breast cancers (HCC38 and MDA-MB-231), three prostate cancers (DU145, LNCaP, and PC3), a serous and a clear cell ovarian carcinoma (SKOV3 and TOV21G, respectively), one pancreatic cancer (PANC1), and two malignant pleural mesotheliomas (HMESO1 and PPMMill). The results of cell proliferation assays demonstrated that, based on individual dose-response metrics listed in Tables 1 – 3 , single 48-h exposures to 4SP65 had dose-dependent activities against all cancer cell lines tested, yielding a consistent pattern where cytostatic effects transitioned to cytocidal effects as drug levels were increased ( Figure 2 ). The GI50 values show that cell growth above starting cell numbers was blocked by 50% at an average concentration of 4.1 ± 0.8 µM 4SP65, ranging from 0.7 to 10.6 µM for individual cell lines. TGI values indicate that cell growth above starting cell numbers was completely blocked at an average concentration of 6.8 ± 1.1 µM 4SP65 (range, 2.0-16.3 µM). LC50 values demonstrate that starting cell numbers were reduced by 50% at an average concentration of 9.1 ± 1.2 µM 4SP65 (range, 2.7 to 19.6 µM). LC99 values reveal that an average concentration of 11.2 ± 1.2 µM 4SP65 (range, 3.0-21.1 µM) induced near-complete cell death in all cancer cell lines, displaying a high degree of disease resolution. Moreover, if the IC50 values of >150 µM 4SP65 are used for strains 1 and 2 NHMECs and an IC50 of 76 µM 4SP65 is used for strain 3 NHMECs to calculate a selectivity index, then the average values for HCC38 and MDA-MB-231 breast cancer cells were >31 and >26, respectively, indicating that 4SP65 is a safe anticancer agent.

Tables 1 – 3 include a list of the genes with pathogenic mutations associated with each human cancer cell line treated with 4SP65. Among eight cancer cell lines with TP53 mutations (not including TP53-null cell lines), H1437 cells were much less responsive to the cytostatic/cytocidal effects of 4SP65 with significantly higher values observed for each dose-response metric compared to other TP53 mutant cancer cell lines (P-values of 2 × 10^-7 for GI50s to 6 × 10^-6 for LC99s). A pilot study suggested that the low effectiveness of 4SP65 in TP53-mutant H1437 cells is a consequence of restricted active transport of the drug. This phenomenon also was observed in H460 cells and was overcome when 4SP65 was combined with cisplatin, resulting in a dramatic increase in the efficacy of 4SP65 (36-fold at the GI50 level), postulated to be due to cisplatin-induced polyamine depletion and compensatory changes in plasma membrane transport systems (68). Among the five cell lines with wild-type TP53, TOV21G cells were much more responsive to the cytostatic/cytocidal effects of 4SP65 with significantly lower dose-response metrics compared to other TP53 wild-type cancer cell lines (P-values of 0.035 for GI50s to 0.001 for LC99s). TOV21G cells have wild-type TP53 but exhibit a ‘hypermutator’ genotype that clearly sets them apart from other ovarian cancer cell lines including SKOV3 and high-grade serous ovarian cancer cell lines (69). If H1437 and TOV21G cells are excluded from analyses, comparisons of individual dose-response metrics for the remaining seven TP53 mutant cancer cell lines to corresponding values for the other four TP53 wild-type cancer cells lines show that the TP53-mutant cell lines are significantly more responsive than TP53 wild-type cell lines to a 48-h 4SP65 treatment, with the P-values progressing from 0.03 at GI50 level cytostatic effects to 0.005 at LC99 level cytolytic effects ( Table 4 ). In other words, roughly half as much 4SP65 (2.23- to 1.58-fold lower concentrations) was required to achieve equivalent responses at each dose-response metric in TP53-mutant cancer cell lines compared to TP53 wild-type cell lines.

4SP65 also was found to be effective against all tested human cancer cell lines having a pathogenic KRAS mutation in the presence or absence of a TP53 mutation. The specific cell lines and their respective KRAS mutations responsive to the cytolytic effects of 4SP65 included A549 and H460 NSCLC cell lines with KRAS G12S and Q61H mutations, respectively, MDA-MB-231 breast cancer cells with a KRAS G13D mutation plus a highly expressed TP53 R280K mutation, TOV21G ovarian cancer cells with a KRAS G13C mutation, and PANC1 pancreatic cancer cells with a KRAS G12D mutation plus a TP53 818G>A mutation (70–73). Three of these KRAS mutations (G12D, G13D, Q61H) are reported to confer either primary resistance, or to occur as “on-target” secondary mutations causing acquired resistance, to inhibitors of the most common KRAS mutation in NSCLC, which is KRAS G12C (70–72). These findings suggest the possibility that treatments combining 4SP65 with a KRAS ^G12C inhibitor can improve therapeutic outcomes given that acquired KRAS alterations occur in ~50% of patients developing resistance to KRAS G12C inhibitors (72).

Dose-response metric values for cisplatin and paclitaxel from the NCI COMPARE database allowed comparisons to the corresponding values for seven human cancer cell lines treated with 4SP65 ( Table 5 ). In these cell lines, the cytostatic effects of 4SP65 largely overlapped with those of cisplatin and paclitaxel at the GI50 level. For example, the GI50 values ranged from 0.7 to 9.4 µM for 4SP65 compared to 0.1 to 5.3 µM for cisplatin in all cell lines excluding the GI50 of 22.4 µM cisplatin in MDA-MB-231 cells. In contrast, 4SP65 was 6- to >22-fold more potent than cisplatin or paclitaxel at the LC50 level, but notably the reported LC50 concentrations for cisplatin and paclitaxel are neither achievable nor safe in cancer patients (78, 79).

Table 5 also shows further comparisons of GI50 values for 4SP65 with those for PRIMA-1 or APR-246 (PRIMA-1^MET/Eprenetapopt), which are anticancer drugs that reactivate mutant p53 protein (75). Among the seven cancer cell lines available for comparisons, 4SP65 was more effective as a cytostatic agent than PRIMA-1 or APR-246 by 4.3- to 21-fold in TP53 wild-type cells, 2.3- to 30-fold in TP53 mutant cells, and 3.3- to 16-fold in TP53-null SKOV3 cells.

In vitro treatment with 1LP65, delivering only one WR1065/molecule intracellularly, also induced broad-spectrum anticancer effects ( Figure 2 and Tables 1 – 3 ). Across 11 human cancer cell lines treated for 48 h with 1LP65, the dose-response metric averages were 41.2 ± 5.4 µM (range, 21.3-77.7) for GI50s, 69.3 ± 10.3 µM (range, 32.7-126) for TGIs, 94.6 ± 12.8 µM (range, 51.3-157) for LC50s, and 126 ± 15.8 µM (range, 83.5-234) for LC99s. As was the case with 4SP65, if H1437 and TOV21G cells were omitted from the analyses, comparisons of the dose-response metrics for the remaining six TP53 mutant cancer cell lines to the corresponding values for the other three TP53 wild-type cancer cell lines showed that the TP53-mutated cell lines were significantly more responsive (by ~1.8-fold lower concentrations of drug) than TP53 wild-type cell lines to 1LP65 treatment ( Table 4 ).

Table 5 shows comparisons between dose-response metrics for 1LP65, cisplatin, and paclitaxel in six human cancer cell lines treated for 48 h. At the GI50 level, the doses of cisplatin that achieved cytostatic effects were ~5- to 14-fold lower than those of 1LP65 in four cell lines and 130-fold lower in H460 cells. MDA-MB-231 cells were the exception, where a GI50 of 22.4 µM cisplatin is toxic in cancer patients (78) while a GI50 of 41.8 µM 1LP65 is safe based upon the NHMEC studies. Similarly, at the GI50 level, the cytostatic effects of paclitaxel were achieved at approximately 6- to 14-fold lower doses than 1LP65 in four cell lines and 77-fold lower in SKOV3 cells. PC3 cells were the exception, where a GI50 of 36.4 µM paclitaxel is highly toxic in vivo (79), while a GI50 of 22.9 µM 1LP65 likely is safe and clinically achievable. Notably, the levels of cisplatin and paclitaxel required for LC50 level cytolytic effects in these cancer cell lines occur at drug concentrations that are neither safe nor achievable in cancer patients (78, 79). While in vitro studies indicate that 500 µM 1LP65 is not cytotoxic in NHMECs, pharmacokinetic studies need to be conducted to determine if concentrations of 60-155 µM 1LP65 that induce LC50 level cytolytic effects across the 11 cancer cell lines tested can be achieved safely in vivo.

The potential cytostatic and cytolytic effects of amifostine, delivering one WR1065/molecule extracellularly, were assessed in the same human cancer cell lines to enable direct comparisons to the anticancer effectiveness of 4SP65 and 1LP65 ( Figure 2 and Tables 1 – 4 ). Although plasma concentrations of 100 µM amifostine and exposures of over 3 h are not readily achievable in humans or animals due to inherent drug delivery restrictions and dose-limiting side-effects (1), for comparison purposes, cell lines were exposed for 48 h to 0-500 µM amifostine. Among 14 cancer cell lines, GI50 values of 47-81 µM amifostine were attained in four cell lines (HCC38, PC3, SKOV3, and TOV21G) reported to exhibit moderate/high expression of alkaline phosphatase (80, 81), GI50 values of 113-378 µM were achieved in seven cell lines, and exposures up to 500 µM failed to induce cytostatic effects in three cell lines. Amifostine levels >100 µM induced TGI level effects in only two cell lines, HCC38 and PC3. Cytolytic effects were not found in any cell line even at exposure levels of 500 µM amifostine. At high concentrations of 200-500 µM amifostine, the dose-response curves were U-shaped for 6/14 cell lines, indicating that exposure to higher drug levels did not induce a greater effect. In the other cell lines, the dose-response curves flattened between 200-500 µM amifostine, without achieving greater growth inhibition. In contrast, dose-response curves for 4SP65 and 1LP65 did not display a U-shape at any concentration range in cancer cell lines, and did not flatten until >97% cytolytic effects were achieved.

Figure 3 shows the results of the estimates of the relative anticancer potency of 4SP65, 1LP65, and amifostine based on the averages of individual dose-response metrics for each compound across a panel of cancer cell lines. First, 4SP65 was 45- to 63-fold (averaging 55-fold) more potent than amifostine as a cytostatic agent, far exceeding the difference predicted solely by the ratio of four versus one WR1065/molecule in 4SP65 compared to amifostine. Second, 1LP65 was 5.0- to 6.4-fold (averaging 5.9-fold) more potent than amifostine as a cytostatic agent even though both drugs delivered a single WR1065 molecule. If the analysis of the relative cytostatic potency between 4SP65 or 1LP65 versus amifostine is restricted to comparisons where amifostine achieved a GI50 value at <100 µM drug in four cell lines, then the average differences in potency remain significant (P <0.007) with a 38-fold greater effect for 4SP65 (average GI50 = 1.7 µM) than amifostine (average GI50 = 64.8 µM) and a 2.1-fold greater effect for 1LP65 (average GI50 = 30.4 µM) than amifostine. Third, 4SP65 was 9.7- to 11.3-fold (averaging 10.4-fold) more potent than 1LP65 depending on the dose-response metric, with the difference increasing as drug levels were raised to achieve LC50 or LC99 effects. Thus, the potency of 4SP65 was ~2.5-fold greater than that predicted by the ratio of four WR1065s/molecule in 4SP65 versus one in 1LP65. Last, the relative potency of 4SP65 and 1LP65 as cytocidal agents compared to amifostine is immeasurable because the latter drug did not induce cytolytic effects in any cancer cell line.

A series of experiments was conducted to assess the relative drug efficacy of 4SP65 and cisplatin as single agents or in combination against two NSCLC, two pleural mesothelioma, and two ovarian cancer cell lines. For these experiments, a human cancer cell line was considered resistant to cisplatin when the amount of drug required to achieve cytolytic effects in cell culture was ≥15 µM, which exceeds reported peak plasma levels (C_max) of free drug averaging from 3.8 ± 1.7 µM to 7.5 ± 6.8 µM cisplatin during routine 1 h IV infusions to differing sets of cancer patients (78, 82). 4SP65 was deemed to overcome resistance to cisplatin when the two drugs combined resulted in a significant fold-increase in the level of cytocidal effects for cisplatin at concentrations below 15 µM and the efficacy of 4SP65 was increased as well.

For each cancer cell line, Figures 4A–F shows the dose-response curves for cell growth after 48-h treatments with 4SP65, cisplatin, or both drugs, while Table 6 lists the corresponding dose-response metrics, the fold-increase in efficacy for each drug in the combination (with P-values indicated where increases are significant), and the synergy scores for the combination. For H460, HMESO1, PPMMill, and SKOV3 cells, cisplatin alone induced only cytostatic effects, consistent with slowing of progressive disease. In contrast, 4SP65-cisplatin combined resulted in cytolytic effects at plasma-equivalent levels of cisplatin with significant dose-dependent gains of both cytostatic and cytolytic activities by 1.7- to 6.9-fold for cisplatin in all four cancer cell lines. Co-treatment with 4SP65-cisplatin also resulted in significant fold increases in the cytostatic and cytolytic activities by 1.3- to 36-fold in H460 and SKOV3 cells, but the modest gains of effect for 4SP65 (1.1- to 1.3-fold) in HMESO1 and PPMMill cells were not significant. In A549 cells and TOV21G cells, treatment with cisplatin alone achieved a LC50 but not a LC99 at <15 µM drug. In A549 cells, combined treatment with 4SP65-cisplatin resulted in significant gains of both cytostatic and cytolytic activities by 1.9- to 2.4-fold for 4SP65 and 1.7- to 3.5-fold for cisplatin to achieve a LC99 at 8 µM 4SP65 plus 7 µM cisplatin. In TOV21G cells, co-treatment with 4SP65-cisplatin also yielded significant gains in total growth inhibition and cytolytic effects by 2.4- to 3.2-fold for 4SP65 and 1.3- to 2.9-fold for cisplatin, reducing the LC99 values of 3.0 µM for 4SP65 alone and 17.2 µM for cisplatin alone to 0.95 µM 4SP65 plus 5.9 µM cisplatin when combined.

The nature of the combined cytostatic/cytocidal effects of 4SP65-cisplatin was surveyed further via synergy reports where the upper limits for cisplatin were set at 15 µM to focus on responses at in vitro treatment levels corresponding to reported peak plasma levels of free drug. The highest average HSA synergy score of 36.4 on a scale of 60 was observed in H460 cells, where the potentiation of 4SP65-cisplatin combined was most prominent in the low-dose region of both drugs as shown in the heat map and in a ridge of high synergy score in the volcano plot of 45 to 57 at concentrations of <1 µM cisplatin combined with 1.5 to 4.6 µM 4SP65 ( Figure 5 ). A similar pattern with an average HSA synergy score of 23.6 was observed in A549 cells with, for example, treatment with 3-6 µM cisplatin and 4-8 µM 4SP65 yielding ~80-90% reduction in starting cell numbers ( Supplementary Figure 6A ) while 13.6 µM cisplatin alone reduced starting cell numbers by only 50% ( Table 6 ). The average HSA synergy score in SKOV3 cells was 18.2 with a ridge of high energy score approaching 42 in the volcano plot at concentrations of 4.5 to 6.0 µM cisplatin and 6 to 9 µM 4SP65 associated with 95-100% reduction in starting cell numbers ( Supplementary Figure 6E ). In contrast, corresponding levels of cisplatin alone were not cytolytic in SKOV3 cells. TOV21G cells were highly sensitive to cytolytic effects of 4SP65 so that, while the average synergy score for the combination treatment was 18.2, a 48-h exposure to ~6.0 µM cisplatin and 1 µM 4SP65 resulted in near-complete cell killing in the region of the most synergistic area score of 26.4 ( Supplementary Figure 6F ). On the other hand, cisplatin alone reduced starting numbers by only 50% (LC50) at 13.2 µM cisplatin in TOV21G cells. 4SP65-cisplatin combined was least effective against HMESO1 and PPMMill cells, with weak potentiation or additive effects driven primarily by significant increases in the cytolytic effects of cisplatin in the combination, effects that were not achieved by plasma-equivalent levels of cisplatin alone ( Table 6 and Supplementary Figures 6C, D ).

A limited set of experiments was conducted to determine if 4SP65 enhanced the activity of and overcame resistance to the first-generation TKI gefitinib in A549 cells, which are TP53 wild-type, KRAS mutant, and overexpress wild-type EGFR and are considered a representative cell line for innate EGFR-TKI resistance (84). For these experiments, a human cancer cell line was considered resistant to gefitinib when the amount of drug required to achieve cytolytic effects in cell culture was >4.5 µM. This upper limit was based upon reports that, in cancer patients receiving 250 mg gefitinib daily, C_max plasma concentrations were 0.5-1 µM or more (85) but trough levels ranged from 0.257−4.50 µM and 0.282−6.55 µM after 3 and 8 days of treatment, respectively, due to the long elimination half-life of 48 h for gefitinib (86). For each cancer cell line, Figure 6 shows the dose-response curves for cell growth after 48-h treatments with each drug alone or 4SP65-gefitinib combined, while Table 7 lists the corresponding dose-response metrics, the fold-increase in efficacy for each drug in the combination (with P-values indicated where increases are significant), and the synergy scores for the combination.

Treatment of A549 cells for 48 h with 4SP65-gefitinib resulted in gefitinib having a significant therapeutic effect at the GI50 and LC50 levels, respectively, when cells were exposed at clinically relevant levels ranging from 2.0 to 5.4 µM gefitinib in the drug combination ( Table 7 and Figure 6 ). By comparison, gefitinib alone exhibited a GI50 of 17.5 ± 4.4 µM in A549 cells, which was nearly four-fold greater than the highest trough concentrations of the drug observed in cancer patients. However, total growth inhibition or cytolytic effects were not induced in A549 cells at a treatment level of 25 µM gefitinib alone. In the combined treatment of A549 cells, the amplification in the effects of gefitinib in combination with 4SP65 was significant for all dose-response metrics, as shown by ~9- and ~4-fold increases in efficacy of gefitinib at the GI50 and LC99 levels, respectively. Co-exposure of A549 cells to 4SP65-gefitinib also yielded significant increases in the growth inhibitory effects of 4SP65 but not in the cytolytic effects of 4SP65 in the combination. The nature of the combined cytostatic/cytocidal effects of 4SP65 plus gefitinib was surveyed further via synergy reports where the upper limits for gefitinib were set at 6 µM to focus on responses at in vitro treatment levels corresponding to reported peak plasma trough levels of free drug at 4.50 µM and 6.55 µM after 3 and 8 days of treatment in patients with NSCLC (86). The synergy report for A549 cells shows that despite a nearly flat dose-response curve for gefitinib alone, co-treatment with 4SP65-gefitinib triggered both cytostatic and cytolytic effects at biologically-relevant concentrations of both drugs, achieving ~91% reduction in starting cell numbers at 6 µM gefitinib plus 12 µM 4SP65 ( Figure 7 ). The dose-response matrix in the synergy report shows that 48-h co-treatment with 4SP65-gefitinib resulted in ~62% to 80% reduction in starting cell numbers as the concentration of gefitinib was increased from 2 to 6 µM in combination with 6 µM 4SP65. The heat map and volcano plot further demonstrate that the average HSA synergy score of 14.7 was driven by the most synergistic area score of 20.5 occurring between 2 and 6 µM gefitinib and 3 and 6 µM 4SP65.