Intellectual Property Landscape of Probe Structure

A specific and general scaffold search was performed in SciFinder on July 6, 2011 related to the ML230 probe structure. The exact structure search of ML230 did not reveal any publications or patents. A general scaffold search related to ML230 revealed patents of the following relevancies with respect to structure (no patents or literature describe this scaffold intended for transporter inhibition). There appears to be space to operate due to limitations in claims and/or use.

Blake, J.F., et al. Bioorganic & Medicinal Chemistry Letters (2010), 20(19), 5607–5612. “Discovery of pyrrolopyrimidine inhibitors of Akt.” The discovery and optimization of a series of pyrrolopyrimidine based protein kinase B (Pkb/Akt) inhibitors discovered via HTS and structure based drug design is reported. The compounds demonstrate potent inhibition of all three Akt isoforms and knockdown of phospho-PRAS40 levels in LNCaP cells and tumor xenografts. Structural modifications are focused on the head group (blue) and a modified pyrrolopyrimidine core, optimized to an indolopyrimidine moiety. Head group changes of this type were not tolerated for the ABC G2 project.

Nakai, H., et al. (2005), WO 2005026126 A1 20050324. (Japanese) “Preparation of fused pyrimidine and related compounds as CRF antagonists.” Compounds of the type indicated are claimed useful for the treatment of neuropsychiatric and digestive diseases. Structural modifications that could be discerned from a patent written in Japanese appeared to be focused on head groups of the type shown in green and with substitution of the pyrimidine core (orange) that is not tolerated in the ABC G2 project.

Paruch, K., et al. (2009) WO 2007/070567 “2-Fluoropyrazolo [1,5-A]pyrimidines as protein kinase inhibitors. Invention describes exclusively fluorinated pyrimidine cores with various functionality attached as useful compounds as CDK-2 or CHK-1 inhibitors.

Chang, E., et al. (2009), WO 2009123986 “Preparation of pyrazolo[1,5-a]pyrimidine derivatives as apoptosis signal-regulating kinase 1 inhibitors” All covered compounds have amine-linkages in place of the direct aryl substituents that appear in the ABC G2 project.

Goldfarb, D.S. US 20090163545 (2009), “Method using lifespan-altering compounds for altering the lifespan of eukaryotic organisms, and screening for such compounds.” Describes the preliminary hits from a HTS screen which show activity in a DeaD assay. Screening compounds were taken from the MLPCN and are listed by SID. A structure similar to ML230 was not identified, but it is assumed that any present would be those predating this project and which were also part of our screening deck for this project. Since compounds of interest in our project were synthesized recently, they are not covered by this document.

Bearss, D.J., et al. (2008), WO 2008058126 “Preparation of imidazo[1,2-b]pyridazine and pyrazolo[1,5-a]pyrimidine derivatives as protein kinase inhibitors.” Claims require substitutions of the core that are known to be orthogonal to those necessary for activity on the ABC G2 target.

Paruch, K., et al. (2006) US 20060094706 “Preparation of a novel class of pyrazolopyrimidines as inhibitors of protein and checkpoint kinases useful in treatment and prophylaxis of HCV infection and other diseases such as cancer.” Claims require substitutions of the core that are known to be orthogonal to those necessary for activity on the ABC G2 target.

General Information

The ABCB1 over-expressing drug-resistant cell line, CCRF-Adr 5000, and its parental CCRF-CEM cells were kindly provided by Dr. T. Efferth (Pharmaceutical Biology, German Cancer Research Center, Heidelberg, Germany). We have previously described the generation of the Jurkat-DNR ABCB1 over-expressing cell line.⁶⁰ We have also developed and previously characterized a SupT1-vincristine (Vin) drug-resistant cell line that selectively over-expresses ABCC1.⁶¹ Ovarian Ig-MXP3 (ABCG2) and its parental Igrov1-sensitive cells were kindly provided by Dr. D. Ross (Department of Medicine, University of Maryland Greenebaum Cancer Center, Baltimore, MD). Cells are grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT), 2 mM L-glutamine, 10 mM HEPES, 10 U/mL penicillin, 10 μg/mL streptomycin, and 4 μg/mL ciprofloxacin. Selective pressure for the ABCB1 over-expressing CCRF-ADR 5000 and Jurkat-DNR cells is maintained by growth in 20 nM daunorubicin (DNR). Selective pressure for the ABCG2 over-expressing Ig-MXP3 cells is maintained by treatment with 340 nM mitoxantrone (MTX) for 1 hr. prior to harvest. Selective pressure for the ABCC1 over-expressing SupT1-Vin cells is maintained by growth in 150 nM vincristine (Vin).

The fluorescent reporter dye JC-1 and cell type differentiation dye CellTrace™ Far Red DDAO-SE were obtained from Invitrogen™ (Eugene, OR). Nicardipine hydrochloride, daunorubicin hydrochloride, mitoxantrone dihydrochloride, vincristine sulfate, and Fumitremorgin C were purchased from Sigma-Aldrich (St. Louis, MO). XR9051, reversan, MK 571, and Ko 143 were purchased from Tocris Bioscience (St. Louis, MO). Compounds ordered for SAR by commerce were purchased from ChemDiv (San Diego, CA) and Ryan Scientific (Mt. Pleasant, SC). Unless otherwise indicated, all compound solutions were maintained and diluted in DMSO prior to addition to assay wells. Final DMSO concentrations were no more than 1% v/v. A Biomek® NX Multichannel (Beckman-Coulter, Fullerton, CA) was used for all cell and compound solution transfers for volumes greater than 1 μL. Low volume transfers (100 nL) were done via pintool (V&P Scientific, Inc., San Diego, CA). Compound dose response plates were generated with the Biomek® NX Span-8 (Beckman-Coulter, Fullerton, CA).

The HyperCyt® high throughput flow cytometry platform (IntelliCyt™, Albuquerque, NM) was used to sequentially sample cells from 384-well microplates (2 μL/sample) for flow cytometer presentation at a rate of 40 samples per minute.^62–63 Flow cytometric analysis was performed on a CyAn™ flow cytometer (Beckman-Coulter, Fullerton, CA). The resulting time-gated data files were analyzed with HyperView® software to determine compound activity in each well. Inhibition response curves were fitted by Prism® software (GraphPad Software, Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose response model with variable slope, also known as the four-parameter logistic equation. This type of time-gated flow cytometric data analysis was described in detail for a previous ABC transporter screen from our group.⁵²

2.1. Assays

2.2. Probe Chemical Characterization

A. Probe Chemical Structure, Physical Parameters and Probe Properties

Figure 2Property summary of probe compound SID 88095709 (CID 44640177)

D. Stability

Aqueous stability was measured at room temperature (23ºC) in PBS (no antioxidants or other protectants and DMSO concentration below 0.1%). The stability of probe compound SID 88095709 (CID 44640177), determined as the percent of compound remaining after 48 hours, was 10%.⁶⁴ Stability data are depicted as a graph showing the loss of compound with time over a 48 hour period with a minimum of 6 time points and provide the percent remaining compound at end of the 48 hours (Figure 3, closed circles).

Figure 3

Aqueous stability of compound SID 88095709 (CID 44640177) in PBS and no acetonitrile (closed circles), and aqueous stability of compound SID 88095709 (CID 44640177) in PBS with the addition of acetonitrile (50% v/v final, closed triangles).

It has been reported by several MLPCN partners and our collaborators at the Sanford-Burnham Institute who perform this assay, that the conditions for the stability assay as described previously is fundamentally unreliable for compounds that exhibit moderate to poor aqueous solubility. Given the low solubility of probe SID 88095709 (0.51 μg/mL, reported in Section C), the stability experiment was repeated with the addition of acetonitrile (50% v/v final) which has been reported to routinely resolve any contributions due to insolubility. The stability data under these conditions overlain in the Figure 3 and is also depicted as a graph showing the loss of compound with time over a 48 hour period with a minimum of 6 time points and provide the percent remaining compound at end of the 48 hours (Figure 3, closed triangles). The addition of acetonitrile appears to have solubilized the compound and provided a more accurate reading of the stability, which is now 100% remaining after 48 hours.

E. Synthetic Route

The probe compound SID 88095709 (CID 44640177) and many analogues were synthesized by the method shown (sequence a–c, Figure 4). Commercial substituted aminopyrazoles 1 were treated with the appropriate dialkylmalonate or b-ketoester to give intermediate 2, followed by chlorination to afford the pyrazolo[1,5-a]pyrimidine core intermediate 3. Installation of the piperazine moiety afforded the probe compound directly. In some cases, a Suzuki or Molander type coupling was preferred to install aryl functionality at a late stage in the synthesis (sequence d–f). Specific experimental details for the probe are detailed in section 2.3, entitled: “Probe Preparation” and experimentals for the analogues are provided in Appendix B.

Figure 4

General synthetic route for probe and associated analogues.

F. Submission of Five Related Analogues to the MLSMR

Five analogues have been fully characterized and prepared in sufficient quantity for submission to the MLSMR. The five selected analogs are shown in Figure 5.

Figure 5

Five analogues submitted to support probe SID 88095709 (CID 44640177).

2.3. Probe Preparation

General experimental and analytical details

¹H and ¹³C NMR spectra were recorded on a Bruker AM 400 spectrometer (operating at 400 and 101 MHz respectively) or Bruker AM 500 spectrometer (operating at 500 and 125 MHz respectively) in CDCl₃ with 0.03% TMS as an internal standard or DMSO-d₆. The chemical shifts (d) reported are given in parts per million (ppm) and the coupling constants (J) are in Hertz (Hz). The spin multiplicities are reported as s = singlet, br. s = broad singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet and m = multiplet. The LCMS analysis was performed on an Agilent 1200 RRL chromatograph with photodiode array UV detection and an Agilent 6224 TOF mass spectrometer. The chromatographic method utilized the following parameters: a Waters Acquity BEH C-18 2.1 × 50 mm, 1.7 μM column; UV detection wavelength = 214 nm; flow rate = 0.4 mL/min; gradient = 5 – 100% acetonitrile over 3 minutes with a hold of 0.8 minutes at 100% acetonitrile; the aqueous mobile phase contained 0.15% ammonium hydroxide (v/v). The mass spectrometer utilized the following parameters: an Agilent multimode source which simultaneously acquires ESI+/APCI+; a reference mass solution consisting of purine and hexakis(1H, 1H, 3H-tetrafluoropropoxy) phosphazine; and a make-up solvent of 90:10:0.1 MeOH:Water:Formic Acid which was introduced to the LC flow prior to the source to assist ionization. The melting point was determined on a Stanford Research Systems OptiMelt apparatus.

The probe was synthesized using the following protocols:

5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidin-7(4H)-one: A mixture of 3-phenyl-1H-pyrazol-5-amine (0.318 g, 2.0 mmol, 1.0 eq) and methyl 3-(furan-2-yl)-3-oxopropanoate (0.370 g, 2.2 mmol, 1.10 eq) was heated in acetic acid (2.0 mL) at 100 °C for 4 hr. After cooling down to rt, the precipitate was collected by filtration. The precipitate was rinsed with EtOH (15 mL) and dried under air to afford 5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidin-7(4H)-one (0.358 g, 65%) as a white solid. ¹H-NMR (400 MHz, DMSO-d₆) δ 12.70 (s, 1H), 8.06 (m, 1H), 8.00 (m, 1H), 7.98 (m, 1H), 7.51-7.47 (m, 3H), 7.44-7.42 (m, 1H), 6.81 (dd, J = 3.7, 1.8 Hz, 1H), 6.64 (s, 1H), 6.15 (s, 1H).

7-chloro-5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidine: A mixture of 5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidin-7(4H)-one (0.277 g, 1.0 mmol, 1.0 eq), POCl₃ (0.613 g, 4.0 mmol, 4.0 eq), N-benzyl-N,N,N-triethylethanaminium chloride (0.456 g, 2.0 mmol, 2.0 eq) and N,N-dimethylaniline (0.121 g, 1.0 mmol, 1.0 eq) in acetonitrile (5.0 mL) was heated at 80 °C for 4 hr. The completed reaction was diluted with CHCl₃ (20 mL), washed with H₂O (10 mL), and the separated organic layers were dried (MgSO₄) and concentrated. The residue was purified by chromatography (Biotage 25 g, EtOAc/Hexane) to afford 7-chloro-5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidine (0.247 g, 84%) as a yellow solid. ¹H-NMR (400 MHz, CDCl₃) δ 7.97-7.94 (m, 2H), 7.56 (m, 1H), 7.43-7.39 (m, 2H), 7.36-7.34 (m, 1H), 7.19 (s, 1H), 7.17 (dd, J = 3.5, 0.5 Hz, 1H), 6.97 (s, 1H), 6.54 (dd, J = 3.5, 1.7 Hz, 1H).

(4-(5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidin-7-yl)piperazin-1-yl)(furan-3-yl)methanone (SID 88095709/ CID 44640177): A mixture of 7-chloro-5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidine (0.148 g, 0.5 mmol, 1.0 eq), furan-3-yl(piperazin-1-yl)methanone (0.180 g, 1.0 mmol, 2.0 eq,) and N-ethyl-N-isopropylpropan-2-amine (0.129 g, 1.0 mmol, 2.0 eq) in acetonitrile (5.0 mL) was heated at 100 °C for 3 hr. The completed reaction was purified by chromatography (Biotage 25 g, EtOAc/Hexane) to afford (4-(5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidin-7-yl)piperazin-1-yl)(furan-3-yl)methanone (0.218 g, 99%) as a white solid. ¹H-NMR (400 MHz, CDCl₃) δ 8.03-8.00 (m, 2H), 7.83 (m, 1H), 7.62 (m, 1H), 7.52-7.47 (m, 3H), 7.45-7.41 (m, 1H), 7.23 (dd, J = 3.5, 0.7 Hz, 1H), 6.93 (s, 1H), 6.65 (m, 1H), 6.62 (dd, J = 3.5, 1.8 Hz, 1H), 6.60 (s, 1H), 4.08 (b, 4H), 3.92 (b, 4H). ¹³C NMR (125 MHz, CDCl₃) δ 164.1, 155.4, 152.4, 151.8, 150.1, 148.5, 144.3, 143.8, 143.2, 132.9, 129.0, 128.7, 126.4, 120.6, 112.6, 110.9, 110.1, 92.9, 88.9, 48.3. LCMS retention time: 3.20 min; purity at 215 nm = 100%. HRMS m/z calculated for C₂₄H₂₇N₅O₂ ([M+H]⁺): 440.1717, found 440.1715.

Submitted Probe Analogs

Experimental details and supporting information for the five submitted analogues can be found in the Appendix B.

3.1. Summary of Screening Results

The primary screening results are summarized with a flow chart showing the overall workflow for project to include the AIDs uploaded to date (Figure 6). For the primary screening protocol, a duplex assay was constructed in which ABCB1 and ABCG2 transporters were evaluated in parallel using fluorescent JC-1 as the efflux reporting substrate. ABCB1 over-expressing cells (CCRF-Adr) were color-coded to allow their distinction from ABCG2-expressing cells (Ig-MXP3). As described in detail previously, the assay was a high throughput no-wash procedure conducted in 384-well format micro plates.

Figure 6

Screening flow chart.

The primary screening results were uploaded as two AIDs; 1326 - High-throughput multiplex screening for ABC transporter inhibitors: specifically ABCB1 screen, ABCG2 counter-screen, and 1325 - High-throughput multiplex screening for ABC transporter inhibitors: specifically ABCG2 screen, ABCB1 counter-screen. A total of 194,394 compounds were tested with 200 and 130 actives noted in ABCB1 and ABCG2 respectively. Compounds were deemed active if the percent inhibition was greater than 80%. Activity was determined on the basis of the median fluorescence intensity (MFI) of JC-1. Percent inhibition was calculated as 100 × [1 - (MFI_PC - MFI_Test)/(MFI_PC - MFI_NC)] in which MFI_Test, MFI_PC and MFI_NC represent the MFI of cells in wells containing test compound, the average MFI of cells in positive control wells (maximum fluorescence intensity) and the average MFI of cells in negative control wells (minimum fluorescence intensity), respectively. The PUBCHEM_ACTIVITY_SCORE is equal to the calculated percent inhibition, except when percent inhibition is greater than 100 and less than 0.

A subsequent SMR cherry pick resulted in single point confirmatory testing of 273 compounds (AIDs 1451 and 1453) resulting in 18 and 16 actives in ABCB1 and ABCG2, respectively. A set of related 488/530 nm fluorescence compound profiling data was also associated with the SMR cherry pick set. These data were uploaded as two AIDs (1480 and 1483) where the 273 compounds were tested with 83 and 89 compounds noted as active (i.e. fluorescent) in ABCB1 and ABCG2, respectively. Autofluorescence was calculated on the basis of MFI detected in the green fluorescence channel (530 +/− 20 nM) and the subtraction of the autofluorescence of the cells, as shown in the following equation: Fluorescence = MFI_CMPD - MFI_CELL where MFI_CMPD is the MFI of cells in the presence of test compound and MFI_CELL is the MFI of cells in the presence of the DMSO control. The compound was noted active if the fluorescence was greater than the average plus three standard deviations of the control wells on the plate. The activity score was calculated based on normalizing the fluorescence to the maximum measured.

The aforementioned single point screen of the 273 compound SMR cherry pick set resulted in the dose dependent screening of 40 compounds. This data set was uploaded under the AIDs 1689 and 1690 and resulted in 9 actives for ABCB1 and 16 active for ABCG2. Inhibitory activity was again determined on the basis of the MFI of JC-1. Innate fluorescence of test compound was subtracted out based on MFI of unstained cells before calculation of percent inhibition of efflux pump activity. The following equations were used to calculate percent inhibition: %Inhibition = 100 × (FluorDelta_Sample - FluorDelta_NC)/(FluorDelta_PC - FluorDelta_NC) in which FluorDelta_Sample (FluorDelta = MFI_JC-1 - MFI_non-JC-1 in which MFI_JC-1 are the MFI of cells in wells in the presence of JC-1 and MFI_non-JC-1 are the MFI of cells in wells without JC-1) are the differences of JC-1 minus non-JC-1 from wells with test compound, FluorDelta_NC are differences from negative control wells (cells with DMSO, minimum fluorescence intensity) and FluorDelta_PC are differences from nicardipine positive control wells. These dose response of %Inhibition data were fitted via GraphPad Prism to a sigmoidal dose response curve with variable hillslope: %Inhibition = Bottom + (Top-Bottom)/(1+10^((LogEC50-LogCmpd)*HillSlope)) where LogCmpd is the log of compound concentration in micromolar and Top-Bottom is the FIT_PERCENT_SPAN. Prism reports estimated values and fitted statistics for the four parameters (Bottom, Top, Hillslope and EC₅₀).

Dose response fits assessed as decent were those with Residual square < 0.5, Hillslope < 5, Standard deviation of estimated LogEC₅₀ <3, and standard deviation of top/estimate for top < 0.5. Only the fits that passed this filter were reported. The activity score was calculated based on two weighted criteria; EC₅₀ < 10 micromolar and FIT_PERCENT_SPAN > 20% by the following equation: Activity Score = 75 * (EC₅₀Cutoff - EC₅₀)/EC₅₀Cutoff + 25*(Span - SpanCutoff)/SpanCutoff. Active compounds have activity scores greater than 52, inactive compounds have scores less than 52. Values above 100 or below 0 were adjusted to 100 or 0 respectively.

Limited SAR was revealed through the first round of cherry pick analysis, and about a third of the compounds were observed to be fluorescent artifacts. However, preliminary secondary screening efforts confirmed activity of several compounds including MLS000527783 (SID 17388272, CID 1434724) with which micromolar potentiation and low toxicity was observed, but little pump specificity (data not shown). Resupply of this compound (new SID 85752814, CID 1434724) confirmed the efflux inhibition, and a series of compounds similar in structure was ordered around this SMR hit. These 31 compounds were tested in dose response in two over-expressing cell lines: Jurkat-DNR (ABCB1) and Ig-MXP3 (ABCG2). Expansion of this piperazine substituted pyrazolo[1,5-a]pyrimidine substructure resulted in a selectivity profile significantly biased toward ABCG2. Of the hits that were identified through this commercial SAR endeavor, SID 85240370 (CID 1441553) had attractive efflux potency towards ABCG2 and marginal selectivity over ABCB1 (Fig 7). The KU SCC launched an SAR campaign aimed at further understanding the origin of potency and selectivity and set out to optimize the compound profile to meet the probe criteria for potency and selectivity in the efflux assay. Compounds meeting these criteria were then screened in a subsequent chemoreversal assay, a cell killing secondary assay that shows potentiation of specific chemotherapeutics for each cell line as compared to the compound’s inherent toxicity. The chemoreversal assays quantitatively show the potentiation of known killing agents for each cell line with efflux inhibitory compounds. The original 15 compounds tested in these secondary assays are reported in AIDs 2830 and 2833. The subsequent SAR related analogs to SID 17388272 (CID 1434724) and SID 85240370 (CID 1441553; Figure 7) are reported in AIDs 488973, 488974, 504476, and 504477.

Figure 7

Validated hit resulting from the preliminary commercial SAR effort.

The probe criteria were set such that micromolar potentiation is desired and the TD₅₀/CR₅₀ ratio must be > 10 with overall toxicity > 15 μM. One compound was found to meet these secondary assay criteria for ABCB1, while 5 compounds matching this profile were identified for ABCG2. Structure activity relationships remained unclear at this stage. As compared to the efflux inhibition activity for SID 85240370 (CID 1441553), selectivity in the potentiation assay maintained a slight selectivity for ABCG2 with CR₅₀ = 0.14 μM vs. 0.70 μM in ABCB1. However, there was significant toxicity noted for both cell lines (TD₅₀ = 6.0 and 3.2 μM for ABCG2 and ABCB1 respectively). With these preliminary results in hand, an extensive SAR initiative was undertaken.

3.2. Dose Response Curves for Probe

Figure 8Efflux inhibition and chemotherapeutic potentiation of SID 88095709 (CID 44640177)

A) A representative curve showing efflux inhibition of ABCB1 in Jurkat-DNR cells (closed circles). The average IC₅₀ (n = 3) is 4.7 ± 0.5 μM. B) A representative curve showing efflux inhibition of ABCG2 in Ig-MXP3 cells (open circles). The average IC₅₀ (n = 2) is 0.13 ± 0.30 μM C) Potentiation of DNR mediated killing in Jurkat-DNR cells with SID 88095709 (n = 2 per data point). The CR₅₀ (closed triangles) is 0.55 μM while the TD₅₀ (closed squares) is 5.5 μM. Minimum allowable toxicity is set at 15 μM, thus the toxicity here is below the cut-off. D) Potentiation of MTX mediated killing in Ig-MXP3 cells (n = 2 per data point). The CR₅₀ (open triangles) is 0.31 μM while the TD₅₀ (open squares) is 18.3 μM. The minimum toxicity and the CR₅₀/TD₅₀ ratio (equal to 59) meet the cut-off criteria for a desirable compound in the chemoreversal secondary ABCG2 assay.

3.3. Scaffold/Moiety Chemical Liabilities

The pyrazolo[1,5-a]pyrimidine scaffold and its derivatives have been easily handled in terms of stability to reaction conditions, exposure to acid or base, heating, and general manipulation. Most are isolated as stable solid materials. We have not observed decomposition nor have we experienced any chemical liability with these compounds. The structure does not contain moieties that are known generally to be reactive. Stability assessment was performed in 1x PBS buffer at pH 7.4 and room temperature. After 48 hours, it was determined that only 10 percent of the parent probe compound remained when the experiment was performed in PBS buffer alone, thus possibly indicating some structural liability that at this point in time is unknown. However, as previously mentioned, the stability assay used with PBS alone is likely not suitable for compounds with lower solubility. The experiment was repeated with acetonitrile to help solubilize the compound, leading to a favorable profile in which 100% of the sample was remaining after 48 hours and indicating no loss of integrity. Comparative assessment of the solubility in multiple assay conditions indicates moderate solubility; however this does not appear to be an issue for either primary or secondary screening protocols based both on direct observation of activity as well as the flow based experimentation briefly described in Section 2.2.

In a follow up activity for which data will not be available in time for this submission, the team is submitting one of the supporting analogs, SID 97301789 (CID 46245505) for stability assessment, noting that this analogue differs structurally in three regions and possesses an improved activity profile as compared to the probe. This compound will be proposed for further enhancement going forward.

3.4. SAR Tables

A total of 165 compounds were assessed in the primary efflux assay (AIDs 489002, 489003, 504566, and 504569) and of these, 126 were synthesized by the KU SCC. A subset of compounds relating to the parent scaffold were purchased and assessed as part of the HTS effort at UNMCMD, prior to the KU SCC involvement. Of the ~ 30 commercial compounds assessed by the UNMCMD, a few showed modest ABCG2 selectivity (Figure 7, SID 85240370/CID 1441553), but gaps in the collection did not resolve the structural functionality responsible for any significant efflux potency or selectivity towards ABCG2 or ABCB1. Due to the diversity of structural changes present in the commercial set, additional compounds were needed to construct meaningful SAR, and this was done using the primary efflux data as it was more readily obtainable. The commercial set contained several members with conserved functionality that provided the basis for establishing a methodical SAR assessment. The pyrazolo[1,5-a]pyrimidine core was preserved, and exchange of the peripheral substituents were surveyed as depicted by the shaded regions in Figure 9. Several compounds of the purchased collection contained a 3-chlorophenyl substituent at R1, which also reflected the R1 moiety present in hit SID 85240370 (CID 1441553). As such, initially a series of compounds were prepared with these features maintained while adjusting R2-R4 (Tables 1 and 2).

Figure 9

Focus of SAR expansion efforts based on primary efflux data.

Table 1

SAR expansion on initial hit SID 85240370.

Table 2

Continuation of SAR expansion on initial hit SID 85240370.

One compound cluster was constructed with R1-R3 groups identical to that of the parent hit SID 85240370 (entries 3–18) while modulating R4. Notably, the substitution of the acyl-2 furan for acyl-3-furan (entry 4) produced an enhancement in selectivity for ABCG2 (8.6-fold), predominately due to erosion of ABCB1 potency, while only modestly attenuating ABCG2 potency as compared to the parent hit. Improved ABCG2 potency was achieved with installation of an acyl-3-pyridine; however, the selectivity deteriorated essentially to pan inhibition (entry 13). Following incorporation of the acyl-3-furan as the more optimal R4 substituent, a survey was then done on the R2 group while holding constant R1 and R3 (entries 19–25). Substantial potency for ABCG2 was gained when R4 was 3-pyridine (entry 23); however, once again, selectivity was negatively impacted.

Alterations in the 3-chlorophenyl R1 substituent were then made while assessing several R4 head groups, specifically toggling between acyl-2-furan, acyl-3-furan, or benzoyl functionalities (entries 26–36, Table 2). No substantial improvements were noted with these changes; however, when R1 was changed from 3-chlorophenyl to phenyl, and R2 was varied (entries 37–43), it was discovered that a 2-furan at R2 in concert with the optimized acyl-3-furan afforded a significant boost in both ABCG2 potency as well as overall ABCG2 selectivity (entry 37, SID 88095709, ABCB1 EC₅₀ = 4.65 μM; ABCG2 EC₅₀ = 0.13 μM, selectivity = 36 fold).

With this information in hand, the team followed up with an SAR effort aimed at demonstrating supportive SAR for compounds bearing an R2 = 2-furyl group while also attempting to improve upon the profile of the new lead, SID 88095709 (Fig 10).

Figure 10

Parent hit SID 85240370 modified to a new lead, SID 88095709.

The new lead scaffold, represented by SID 88095709, was further studied by adjusting physiochemical and spatial elements in R1 (Table 3).

Table 3

Summary of modifications for R1.

Switching out the phenyl ring of the lead with a t-butyl group erased much of the gains towards ABCG2 selectivity (entry 2, Table 3). Traditional phenyl replacements such as thiophene or furan were tolerated, but only led to modest selectivity and potencies. The installation of a 4-chlorophenyl substituent led to a reduced impact on ABCB1, resulting in selectivity in the efflux assay of 22-fold (entry 6); however, the change also marginalized the potency on ABCG2. Renovating the phenyl substituent with electron donating groups did not appear to be beneficial.

An examination of 2-furan replacements at R2 was also undertaken (Table 4). Simple alkyl units such as methyl or t-butyl degraded potency and fold-selectivity for both transporters. Notably, use of t-butyl actually reversed selectivity for ABCB1, albeit at the expense of potency (entry 5).

Table 4

Summary of modifications for R2.

Some R2 revisions resulted in impressive ABCG2 selectivities and potencies. The choice of 2-F-phenyl (entry 10, Table 4) slightly degraded potency for ABCB1 as compared to the parent (entry 1), leading to a 10-fold selectivity in favor of ABCG2. For the fluorinated series (entries 10–12), the potency for both transporters decreased as the fluorine atom was migrated from the 2- to 3- to 4-position of the aromatic R2 ring. Interestingly, the use of the use of a 3-MeO-phenyl group impeded potency for ABCB1 activity while retaining sub micromolar ABCG2 potency on par with the parent, leading to an improved 83-fold selectivity between the transporters (entry 8). In this series (entries 7–9), however, a trend was not observed as the substituent was shifted from each position. Additional compounds prepared with the 3-MeO-phenyl group at R2 did not show a consistent SAR (data not shown).

The commercial set of compounds contained a few scaffolds bearing a methyl group at R3. SAR generated in the early phases demonstrated some benefit to the presence of small alkyl groups at R3; however, this was highly dependent on the identity of groups at R1, R2 and R4. For the purposes of expanding the SAR around SID 88095709, one analogue was prepared to quickly evaluate the effect of this substitution pattern in concert with our chosen functionalities at R1, R2 and R4 (Fig 11). ABCB1 potency was encouragingly impaired, but not without also effecting G2, resulting in a marginal selectivity profile.

Figure 11

Effect of R3 alkyl substitution.

Attention was then turned to investigating the effect of different R4 functionality appended to the piperazine (Table 5).

Table 5

Summary of modifications for R4.

In earlier SAR sets, activity was found to be sensitive to the identity of R4 and the pairing of groups at R2 and R3. In the context of our new lead, SID 88095709, we wanted to better understand the effect of R4 with the chosen substituents. It was confirmed that an acyl-3-furan was preferred to an acyl-2-furan (entry 2), and simple alkyl substitution of the 3-furan (entries 5–7) or a larger benzofuran (entry 8), while tolerated, did not reveal any benefits. However, the most influential effects on ABCB1 were observed when the acyl furan was exchanged for a benzyl ester (entry 11). While ABCG2 potency was compromised compared to the lead, ABCB1 potency was completely lost, yielding a selectivity of ~ 19 fold.

In a more aggressive effort, the entire “top piece” of the scaffold, consisting of the piperazine and the R4 group, was modified (Table 6). Ring-opened piperazine equivalents, truncated amino groups, piperidine amides, ring-expanded amines (not shown) and various structural variations on a theme did not produce a profile superior to that which had already been observed.

Table 6

Summary of piperazine and combined R4 modifications (X).

In the process of evaluating these structural modifications, several compounds were prepared singly to target possible oversights in SAR, as every possible R1-R4 combination cannot be prepared and assessed in a timely way. Others were targeted as a means of inserting the best combinations as gleaned from the preceding generations of SAR. These compounds were more recently pursued to probe-specific structural combinations and are summarized in Table 7. Data obtained early on had indicated that the acetyl group at R4 was more advantageous than other changes that had been surveyed (including the benzyl ester modification), though later refinement of these data does not now stand out as particular SAR of interest. Based on the information in hand at the time, substituted phenyl derivatives possessing a disparate electronic nature at R1 were incorporated with the acetyl R4 group in place (entries 2 and 3). No advantages were found.

Table 7

Analogues with R1 – R4 substitution patterns in combination.

We were interested in also surveying the effect of reducing the carbonyl of the R4 head group to give an amine in place of the amide when some of the preferred R1-R3 substituents were incorporated (entry 4) and, as an experimental one-off compound, in other pockets of SAR that seemed to be unrelated but were still interesting (entry 6). While the effect did not enhance the profile in the case of the most closely related analogue to the lead SID 88095709 (entry 4, SID 99376134), a profound effect was observed for the alternatively substituted compound with SID 97301789 (entry 6). As previously mentioned, the combination of R1-R4 has been found to radically influence the profile of the compounds towards ABCB1 or ABCG2. For example, methylation at R2 and R3 in concert with previously surveyed R1 and R4 moieties delivered an analogue which amounts to pan inhibition (entry 5). The combination effect cannot be underestimated, as shown with entry 6, in which methylation at R2 and R3, in concert with the newly discovered R4 head group of CH₂-3-furan, led to a 233-fold selectivity for G2 with comparable potency to the lead compound and abolished activity for ABCB1 in the efflux assay! The improved selectivity observed with the CH₂-3-furan in the R4 position could be due to a number of effects. Removal of the carbonyl of the parental acyl-3-furan R4 group confers basicity to the head group that was previously missing. Removal of the carbonyl also changes the conformation of the R4 group in relation of the piperidine ring (sp³ hybridization) versus when the amide of the parent is intact (sp² hybridization). Although these aspects are likely not the only contributors to the observed benefits in selectivity, as a previously screened benzyl R4 group did not result in analogous improvements. Still, these effects are advantageous only when put in play with a select group of R1-R3 substitutions.

In parallel to the described efforts, compounds were also assessed in potentiation secondary assays and associated data are presented in the preceding tables; however, the chemoreversal assay is a very low throughput assay and compound data from this assay could not be used to drive the SAR program. Key data have been collected for some of the most promising compounds (Fig 12). Evaluation of probe compound SID 88095709 shows submicromolar ABCG2 efflux inhibition activity with ~ 36-fold selectivity toward ABCG2 over ABCB1. Potent submicromolar activity has also been demonstrated in the potentiated killing of both over-expressing cell lines with preference for ABCG2 from a toxicity perspective, though the degree of selectivity observed in the cell killing assay is removed (1.8 fold in chemoreversal vs. 36-fold in efflux assay). The most recently advanced analogue, SID 97301789, presents as an exceedingly potent compound in the potentiation assays with a 4.5-fold window between the observed potencies for the two transporters, but in favor of ABCB1. Interestingly, the analogue appears to be devoid of toxicity (> 100 μM) and represents a promising tool for further refinement.

Figure 12

Refinement of structure based on efflux and associated potentiation and toxicity data.

3.5. Cellular Activity

The efflux inhibition assay and the chemoreversal secondaries are both direct indication of cellular activity in vitro. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 inhibitors is unprecedented.

3.6. Profiling Assays

Since ABC transporters are ATP dependant efflux pumps and several kinase targets have been implicated in the patent literature with structurally similar scaffolds, the probe was profiled against 50 kinases at a single concentration of 10 μM to assess promiscuity of the chemotype.⁶⁶ Probe SID 88095709 was dissolved in DMSO and tested at a final concentration of 10 μM. Prior to initiating a profiling campaign, the compound was evaluated for false positive against split-luciferase. Profiling was done in duplicate for SID 88095709 against each kinase. The Percent Inhibition and Percent Activity Remaining (shown in Table 8) are calculated using the following equation:

Table 8

Percent of activity remaining for various kinases when inhibited by SID 88095709.

The team has also submitted the probe and the analog SID 97301789 (CID 46245505) to the NIH National Cancer Institute to elucidate the effect of the probe on the cancer cell line panel.

4.1. Comparison to Existing Art and How the New Probe is an Improvement

ABCB1, ABCC1, and ABCG2 transporters are known to significantly influence the ADME-Tox properties of drugs,² and although a large number of compounds have been identified possessing ABC transporter inhibitory properties, only a few of these agents are appropriate candidates for clinical use as MDR reversing agents.⁶⁷ Clinical trials with late generation modulators (e.g. biricodar, zosuquidar, dofequidar, and laniquidar) specifically developed for MDR reversal are ongoing.^33,36,68 Efflux pumps are by design highly adaptive and potentially able to adjust to a wide array of chemotypes owing to a relatively large cavity (~6000 ų) and at least three non-overlapping binding site configurations.⁸ For example, rhodamine 123 has been used in combination with Hoechst 33342 to describe two functional transport sites in ABCB1 with complex allosteric interactions.⁶⁹ Concurrently, rhodamine 123 may bind to a different overlapping region, or potentially within the same large flexible binding site, as LDS 751.⁷⁰ It has also been shown that ABCB1 possesses two allosterically coupled drug acceptor sites where one binds vinblastine, doxorubucin, etoposide and cyclosporin A, and the other binds dexniguldipine and other 1,4-dihydropyridines.⁷¹ It is thus possible that within a single chemical sub-structure class there could be multiple binding patterns leading to both the difficulty of direct structure activity relationship comparisons but also to the possibility of a tuneable synthetic system allowing selectivity and/or cross-transporter inhibition. It should be noted, however, that acquired mutations in transporter genes introduce even more complexity, altering the pattern of resistance and improving the ability of the mutants to efflux new drugs.⁷² It was reported that various drug-selected human tumor cell lines expressed different ABCG2 variants, which were suggested to be gain-of-function mutations acquired during the course of drug exposure.⁷³ Single amino-acid changes cause an altered drug resistance profile and substrate specificity compared to the wild type ABCG2 transporter.⁷⁴ The lesson to be learned is that high efficacy and good selectivity need to be carefully compared in analogous systems and cell lines. This is readily apparent in the discrepant activity seen with FTC and Ko 143.

Figure 13 summarizes the prior art comparison to SID 88095709 (CID 44640177). XR9051 and MK 571 were chosen to verify ABCB1 activity and counterscreen ABCC1 activity, respectively in our system. XR9051 does inhibit the efflux of JC-1 in both ABCB1 and ABCG2 over-expressing cell lines (0.6 and 2.3 μM respectivly). Potentiation data indicates submicromolar chemoreversal in both Jurkat-DNR and Ig-MXP3 cells with a bias toward ABCB1 at 10 nM as compared to 700 nM for ABCG2. Not surprisingly, we didn’t observe any inhibition in ABCB1 or ABCG2 with MK 571 but we remain interested in the MK 571 response in ABCC1 over-expressing SupT1-Vin cells. It is likely that the “potentiation” seen with MK 571 is simply due to general toxicity since in both cell lines the CR₅₀ is equivalent to the TD₅₀ (curves not shown).

Direct comparison of reversan in our efflux inhibition system shows low micromolar inhibition of both ABCB1 and ABCG2 (4.4 and 0.8 μM respectively) with moderate selectivity for ABCG2. In our chemoreversal potentiation assay, reversan showed micromolar activity and no apparent selectivity (2.2 and 3.6 μM in ABCB1 and ABCG2, respectively). This was coupled with significant toxicity in the Jurkat-DNR cell line. FTC showed no activity in either cell line in the efflux inhibition assay and was not tested in the potentiation assay. Ko 143 has been shown to potentiate mitoxantrone (MTX) at nanomolar levels in ABCG2 over-expressing cells.⁴⁸ In our potentiation assay there actually seemed to be a selectivity for ABCB1 over ABCG2 (CR₅₀ = 1.0 and 5.9 M respectively) with considerable toxicity in both cell lines. The efflux inhibition activity did not mirror this, showing no activity in ABCB1 and only 13.6 μM inhibition in ABCG2, potentially indicating a binding site difference versus JC-1.

The entire probe SAR series was also structurally compared via PLS analysis to the inhibitors described in Figure 13 as well as the clinically relevant potentiators; Cyclosporin A, Biricodar, Tariquidar, Zosuquidar, Elacridar, Laniquidar, Dofequidar, and ONT093. The eight biological parameters listed in Table 1 have been used as dependent variables in the Y block of the PLS analysis which was performed. They included the normalized percent response and IC₅₀ values in the primary assay, and the CR₅₀ values in the secondary assay for both ABCB1 and ABCG2 transporters, and also the associated toxicity TD₅₀ data for these two targets. The goal of this analysis was not only to cluster the compounds in the physicochemical molecular descriptors space, but also to map these biological parameters and selected physicochemical parameters (computed solubility, logD 7.4, MW, hydrogen bond donors and acceptors, etc) in the principal components space. Figure 14 illustrates the clustering. This resulted in effectively three groupings based on the similarity comparisons. The probe (SID 88095709, CID 44640177) and analogs with aryl and heteroaryl R1 and R2 groups (excluding those with modifications to the piperizine) obviously clustered together and those like SID 97301789 (CID 46245505) with alkyl R2 substituents were grouped together. The third set is included in the remainder of compared structures that did not show significant similarity to one another or the probe class scaffolds. In conjunction with the unique chemical space covered, the calculated solubility for the SAR structures appear to be potentially better than those previously described in clinical trials. Interestingly, the calculated solubility of compounds with greater selectivity toward ABCG2 efflux inhibition of JC-1 seem to generally be poorer (more lipophilic) than those for ABCB1 inhibition where greater solubility/hydrophilicity appears to be required. Although the experimental solubility of SID 88095709 is less than ultimately desirable, we are confident that the availability is sufficient for all biological conditions outlined in this report. The probe compound also shows good solution stability and appears to not be a kinase inhibitor based on the profile outlined Section 3.6.

Figure 14

Map of the computed solubility of selected compounds in the PLS principal component space. Only prior art compounds and compounds tested in the secondary assays have been included. Most of the newly synthesized compounds show higher computed solubility (more...)

The primary screening conditions outlined here are a model system where JC-1 is an efflux inhibition surrogate for chemotherapeutics and it must be noted that there is not necessarily a one to one comparison of substrate recognition by either efflux pump. Previous experimentation from our group has validated the utility of such a model.^52,75 However, this does not exclude the possibility that JC-1 efflux inhibition will not match more phenotypic cell killing assay conditions such as the potentiation assays outlined in this report. Our group has extensively looked at profiling dozens of fluorescent substrates against a panel of known efflux inhibitors in several ABC transporters and observed distinctly different activities dependent on the substrate inhibitor pairing in each over-expression system (unpublished results, manuscript in preparation). This, however, does not diminish the utility of small molecules with specific efflux inhibition profiles. Such inhibitors can be useful tools to look more closely at the nature of such pump poly-specific substrate recognition, ultimately allowing for generation of better model systems.

Probe SID 88095709 demonstrates a 36-fold better efflux inhibition of JC-1 efflux in ABCG2 over ABCB1, thus establishing its usefulness in exploration of the system from a biochemical perspective. This result, coupled with the noted cellular activity in the potentiation assay justifies the overall utility of SID 88095709 as a probe for ABCG2. SID 88095709 showed greater potency and ABCG2 selectivity than any of the aforementioned literature precedent compounds in the efflux inhibition screening conditions. Only XR9051 seems to have better activity in the potentiation assay although with reversed selectivity toward ABCB1. More work needs to be done in this subclass to understand the difference in efflux inhibitions, as compared to potentiation.

Interestingly, the structurally-related, but SAR-distinct analogue of the probe that has an improved selectivity toward ABCG2 in the efflux assessment, compound SID 97301789, was shown to have significantly more potent cell killing activity in the chemoreversal assay (with no observed toxicity) than the probe compound, however, the efflux inhibition selectivity appears to be lost. The emergence of this compound occurred after extensive supporting SAR had been established for SID 88095709, thus limiting the expansion of another arm of SAR in support of SID 97301789 as a probe in this report; however, this development provides an exciting opportunity for refinement as an extended project, particularly in the context of the observed ABCG2 tumor reduction preliminary results that were mentioned in the “Recommendations for scientific use of the probe” section.

4.2. Mechanism of Action Studies

In relation to the phenotypic readout of the potentiation assay, the primary efflux inhibition assay is a direct indication of the mode of action for the compounds discussed. Further studies with other fluorescent substrates could be used to further indicate site of action with each given transporter depending on the substrate selectivity profiles.

4.3. Planned Future Studies

Probe Enhancement

This project will be submitted for approval as a candidate for probe enhancement via the “Further Development of a Probe with Extended Characterization” outlined by the NIH and MLP. We have defined a scaffold with efflux activity for both targets as well as reversal of drug resistance conferred on both targets with the identification of an ABCG2 selective probe SID 88095709. There are > 30 compounds in this subclass to support the SAR discussion. Furthermore, we have identified a late stage, structurally-related, but SAR-distinct analogue of the probe that has an improved selectivity toward ABCG2 in the efflux assessment, demonstrates sub-nanomolar potentiation activity in the chemoreversal assays, and is devoid of toxicity (> 100 μM). The emergence of this compound occurred after extensive supporting SAR had been established for SID 88095709, thus limiting the expansion of another arm of SAR in support of SID 97301789 as the probe in this report; however, this development provides an exciting opportunity for refinement in an extended project and the candidate for preliminary animal studies. The probe extension proposal consists of: 1) in vitro testing characterization of the compound selectivity against other transporters in the portfolio of the UNMCMD Center Driven Project on Transporters: 2) evaluation of the in vivo characteristics and lead-like properties of the probe in vitro; and 3) medicinal chemistry to establish SAR around analogue SID 97301789 and overcome liabilities that are identified through the current studies.

Chemistry (KUSCC)

As previously discussed, the combination of R1-R4 has been found to radically influence the profile of the compounds towards ABCB1 or ABCG2, and distinct lines of SAR seemed to result depending on the nature of this substitution. As analogue SID 97301789 represents a line of SAR which was sidelined before the impact of the benzyl R4 head group was discovered, the team proposes to investigate the nature of this R4 substituent and others like it in concert with the disubstituted core (R2 = aryl, R3 = Me, Figure 15).

Figure 15

SAR enhancement of SID 97301789.

Enhancement of the probe or one of the analogues such as SID 97301789 will also require resynthesis of compound(s) as well as potential medicinal chemistry optimization for liabilities in solubility, permeability, and toxicity. Along with this synthetic effort, lead-like properties will be assessed via chemical characterization, compound profiling, and in vivo analysis.

Specifically, during the probe enhancement phase the optimized probe will be provided in sufficient quantities and purity for the proposed studies and be further refined structurally to tune physiochemical and pharmacokinetic parameters necessary for adequate analysis in rodent models. The probe would be optimized to: possess suitable aqueous solubility, stability, and cellular permeability; possess reasonable microsomal stability, plasma protein binding and plasma stability; be assessed for in vivo pharmacokinetics; show minimal off target effects as determined by a broad spectrum profiling panel such as Ricerca panel and PDSP GPCR panel. The probe will also be compared to the state of the art for ABCG2 relevant efflux inhibitors/potentiators.

Biology (UNMCMD, Target Provider at UNM)

Further selectivity profiling across related, cancer relevant transporter families will be performed both in the efflux inhibition screen as well as in the drug potentiation assays available for each of the transporter expressing cell lines. Preliminary in vivo mouse studies will be done on the most promising in vitro compound(s).

Specifically, in the probe enhancement phase the optimized probe will profile the existing SAR series against a SupT1-Vin ABCC1 over-expressing cell line in: 1) the original HTS flow cytometric efflux inhibition screen as compared to ABCB1 and ABCG2; 2) a modified cell-based potentiation assay using the CellTiter-Glo® (Promega) luminescent cell viability assay as a pre-screen for compounds going into the traditional direct viability count potentiation assay; and 3) the traditional trypan blue viability count potentiation assay to determine inhibition mediated cell killing as well as compound toxicity.

The enhancement will also initiate pilot studies of compound efficacy in mouse cancer models, specifically: 1) determine the optimal dose of the inhibitor via small scale dose response; 2) apply optimized inhibitor dose in larger scale survival study; 3) assess acute toxicity; and 4) test expression effects by measuring RNA expression of ABCG2 by RT-PCR in the tumors. Probe compound SID 88095709 is currently in process for preliminary studies and the data will likely be included in resubmission of the enhancement proposal.

Details for the proposed biological experimentation in the enhancement are located in Appendix C.

In vitro methods

Efflux inhibition primary screen protocol: The assay is conducted in 384-well format in a total volume of 15.1 μL. Cells and reagents are added sequentially as follows: 1) PBS buffer (5 μL/well); 2) test compound (0.1 μL/well); 3) drug-resistant cells (10 μL/well) pre-stained with the JC-1 substrate at 1 μM. Final in-well concentration of test compound ranges from 50 μM to 69 nM over an 18 point dose response and the cell concentration is 1 million cells/mL. Nicardipine (50 μM) is added to each plate as a pan-inhibition positive control. The plate is rotated end-over-end at 4 RPM and 25 °C for 30 minutes and then cell samples are analyzed via the HyperCyt® high throughput flow cytometry platform. Flow cytometric data for light scatter and fluorescence emission at 530 +/− 20 nm (FL1) are collected via CyAn flow cytometer (Beckman Coulter). The resulting time-resolved single data file per plate is analyzed by HyperView software to determine the compound activity in each well. Methods previously published.⁵²

Chemoreversal assay in efflux pump over-expressing cells (trypan blue): Cells are incubated with test compound (concentration range of 3 logs) over 3 and 7-day periods in the presence of selective agent such that a cell concentration of at least 1 X 10⁵ cells/mL was maintained. Cell viability was determined by trypan blue staining and enumeration under light microscopy. A chemoreversal index (Chemoreversal 50, CR₅₀) is determined from the viability assessment. Using a similar approach, direct cytotoxicity index (Toxic Dose 50, TD₅₀) was determined by assessment of cell death of cells grown in media alone. Results were compared with the survival of parental cells in the presence of the selective agent (DNR/MTX/Vin/etc.; 100% cell death), as well as survival of drug-resistant cells in the presence of the chemotherapeutic drug (control yields 100% viability). The difference between the CR₅₀ and the TD₅₀ give an estimation of the in vitro therapeutic index for the test compound. Sub-micromolar potency is desired along with a TD₅₀/CR₅₀ ratio of at least 10.

Chemoreversal assay in efflux pump over-expressing cells (luminescence): Compound dilution plates are made containing DMSO control wells and duplicate rows of 100x concentrations of compounds half-fold diluted from 1 mM to 3.91 μM. All 96-well assay plates contain media only, 1% DMSO, and 1% DMSO + potentiating agent control wells along with dose response compound wells (assay well concentration range 100 μM to 39.1 nM). Cells are plated in opaque white 96-well plates at 2500–5000 cells (depending on cell line) in 50 μL RPMI media/well with sub-inhibitor concentrations of the corresponding chemotherapeutic drug. Then, 1 μL of compounds from dilution plates was added to applicable wells. Plates are allowed to incubate ~72 hr, 37°C, 5% CO₂. CellTiter-Glo® is then added to plates following manufacturer’s instructions, and plates are read on a plate reader to collect fluorescence data. Curve fits were normalized based on viability of the control wells containing chemotherapeutic killing agent and a comparison was then made similar to what is described for the trypan blue methods. This secondary assay has been applied as a means of quickly profiling compounds and determining those that will be taken into the more labor intensive trypan blue potentiation assay.

In vivo animal study methods

Due to the defined nature of this study, we will focus on the in vivo efficacy of our ABCG2 inhibitor. Our studies will demonstrate that we can effectively administer an ABCG2 inhibitor in a manner that will lead to in vivo killing of a drug-resistant tumor and increased survival. We will also perform limited acute toxicity studies and verify that in vivo growth does not alter ABCG2 expression.

Model and study design: For the in vivo studies, we employ the same cell lines that were used in our in vitro screening assays and secondary confirmatory assay. Specific-pathogen-free adult CB-17 female SCID mice (5–6 weeks of age) weighing 20–25 g, are purchased. Briefly, ABCG2 over-expressing Igrov1/T8 cells are injected (5 x 10⁶ cells/mouse). Prior to injection, Igrov1/T8 cells are maintained in topotecan in order to assure continuous expression of ABCG2. Tumors are allowed to grow for 3–4 weeks or until tumor volumes are greater than ~100 cubic millimeters, at which time they are stratified into treatment groups. Treatment is administered and we observe the size of the tumor and weight of the mouse over a 7 day period. At the end of 7 days, we: 1) sacrifice the animal; 2) measure and weigh the tumor (if present); 3) analyze the histology of the tumor as well as other organs for evidence of toxicity; and 4) measure the expression of ABCG2 receptors on the tumor to determine if in vivo growth has altered ABCG2 expression. In our initial studies, we have generally observed high survival at 4 weeks (>90%) which escalates rapidly with near 100% mortality by 6 weeks. This approach is well described elsewhere.⁵⁶

In the proposed experimentation, three groups of mice will be studied: 1) mice with Igrov11T8 tumors and injected with ABCG2 inhibitor; 2) mice with Igrov11T8 tumors that are sham injected; and 3) mice with parental cells that are not resistant to topotecan. In the first set of studies, we will determine the optimal dose of the ABCG2 inhibitor using primarily Group 1 mice. Using the in vitro cell killing data as a guide, we will test 6 doses of the ABCG2 inhibitor (SID 103911215) over 2 logs of final blood concentration (10 nM to 1 μM). In order to assure statistical significance, we will study 5 mice in each group. Results will be compared against one group of mice from Groups 2 and 3. The reduction of the size of the tumor will be used as the principal factor in determining the optimal dose, although toxicity will also be considered.

In the second set of studies, we will employ all three groups of mice using the optimal dose of ABCG2 inhibitor for Group 1. We will examine mice for overall survival at 6 weeks (including tumor size and body weight), acute toxicity due to the administration of the ABCG2 inhibitor, and tumors to make sure that the level of ABCG2 expression is not altered by in vivo growth.

Acute toxicity: Since prior information regarding the acute toxicity of the ABCG2 inhibitor is not available, body weight and histology of liver, kidneys, spleen, lungs, and heart will be obtained and analyzed. In addition to standard histologic stains, immunohistochemical stains to detect apoptosis will be obtained in order to compare organs and toxicity from sham and ABCG2 injected animals.

ABCG2 expression by RT-PCR: We will also measure the RNA expression of ABCG2 by RT-PCR in 10 tumors from treated and sham-injected mice in order to measure the effects of in vivo growth and treatment on the expression of ABCG2 in these tumors. Finally, we will perform limited toxicity studies and any acute toxicity leading to cell necrosis or other histologic change will be observed. Pharmacokinetic sampling and analysis will be performed in subsequent studies outside of this proposal.

Regulatory: Mice will be studied and maintained in accordance with guidelines of our Institutional Animal Research Committee at UNM HSC. We have already obtained IACUC approval.