ROS (reactive oxygen species)-induced agent

RIDR-PI-103 is a novel reactive oxygen species (ROS)-induced drug release prodrug with a self-cyclizing moiety linked to a pan-PI3K inhibitor (PI-103). Under high ROS, PI-103 is released in a controlled manner to inhibit PI3K. The efficacy and bioavailability of RIDR-PI-103 in breast cancer remains unexplored. Cell viability of RIDR-PI-103 was assessed on breast cancer cells (MDA-MB-231, MDA-MB-361 and MDA-MB-453), non-tumorigenic MCF10A and fibroblasts. Matrigel colony formation, cell proliferation and migration assays examined the migratory properties of breast cancers upon treatment with RIDR-PI-103 and doxorubicin. Western blots determined the effect of doxorubicin ± RIDR-PI-103 on AKT activation and DNA damage response. MDA-MB-453, MDA-MB-231 and MDA-MB-361 cells were sensitive to RIDR-PI-103 vs. MCF10A and normal fibroblast. Combination of doxorubicin and RIDR-PI-103 suppressed cancer cell growth and proliferation. Doxorubicin with RIDR-PI-103 inhibited p-AktS473, upregulated p-CHK1/2 and p-P53. [1]

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We have developed a novel technology, called RIDR (ROS-Induced Drug Release) which is a self-cyclizing reagent linked to a PI3K inhibitor (PI-103) to eject PI-103 in a controlled manner under oxidative stress in highly aggressive breast cancers including triple negative breast cancer cell lines (TNBCs) and other ER+, HER2+ breast cancer cell lines with activating PIK3CA mutations. We evaluated the efficacy of RIDR-PI-103 (5-100 µM) or PI-103 (0-5 µM) in normal fibroblasts, T47D, MCF-7, MDA-MB-361, BT474, MDA-MB-453 and MDA-MB-231 breast cancer cell lines. We noted that IC50 of PI-103 is 3.34 µM whereas IC50 of RIDR-PI-103 is >100 µM in normal fibroblasts, indicating the toxicity of the PI-103 alone but not the RIDR-PI-103 in normal fibroblasts. Our data indicated that ~30-40 µM RIDR-PI-103 significantly inhibited T47D, MDA-MB-231, MDA-MB-361 and MDA-MB-453 cell proliferation whereas higher concentrations of the drug were effective in BT474 and MCF-7 breast cancer cell lines. Our quantitative PCR data showed that the antioxidant catalase mRNA levels are statistically lower compared to normal fibroblasts in these cancer cell lines indicating that the efficacy of RIDR-PI-103 could be correlated to catalase expression. [2]

Pharmacokinetics Profile of RIDR-PI-103[1]
In this initial PK study in mice, RIDR-PI-103 (20 mg/kg) was administered intraperitoneally and blood samples were collected over a period of 96 h. The concentration of 20 mg/kg was chosen based on the ability to solubilize RIDR-PI-103 in 40% propylene glycol with 60% injectable saline. The mean plasma concentration-time profile was then analyzed using Phoenix® WinNonlin® v8.2 using both a compartmental and non-compartmental analysis (NCA). As shown (Figure 7A,B), the plasma concentration-time profile fitted a one-compartment model. The key PK parameters are listed in Table 1. As shown, both methods of PK analysis yielded similar results. RIDR-PI-103 has a maximal plasma concentration (Cmax) of 201.5 ng/mL (0.43 µM), which was achieved at the time taken to reach the maximum concentration (Tmax) of 1.44 h. The elimination half-life of RIDR-PI-103 was 9.4 h (Table 2), aligning with the blood sample schedule employed (up to 96 h, approximately a period equivalent to 10 half-lives) facilitating complete characterization of the elimination profile. Based on these studies, RIDR-PI-103 has a large volume of distribution (Vd), 89 L/kg.

Cell Viability Assay[1]
Growth kinetics of fibroblasts, MCF10A, MDA-MB-231, MDA-MB-361 and MDA-MB-453 cells was determined by the 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 2 × 104 cells/well were seeded in 96 well plates in triplicate. After 72 h of treatment with RIDR-PI-103 (0–110 µM) and PI-103 (0–5 µM), media was substituted with 50 mg/mL MTT solution and absorbance was recorded at 570 nm using SPECTRAmax PLUS Microplate Spectrophotometer Plate Reader and expressed as the mean of triplicates relative to vehicle (DMSO) control together with standard error of mean (SEM). In separate experiments, MDA-MB-231, MDA-MB-361 and MDA-MB-453 cells were treated with a series of concentrations using doxorubicin in the presence or absence of RIDR-PI-103 (10–30 µM). MDA-MB-231 cells were treated with 500–5000 nM, MDA-MB-361 with 50–450 nM and MDA-MB-453 with 100–4000 nM of doxorubicin for 72 h. MDA-MB-231, MDA-MB-361 and MDA-MB-453 cells were treated with a series of concentrations using docetaxel in the presence or absence of RIDR-PI-103 (10, 15 or 30 µM). MDA-MB-231 cells were treated with 50–50,000 pM docetaxel. MDA-MB-361 cells were treated with 10–90 nM of docetaxel. MDA-MB-453 cells were subjected 50–10,000 pM of docetaxel. The cell proliferation was analyzed after 72 h and represented as mean of triplicate values relative to DMSO control. The bar graph generated using graph pad prism 7.

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Cell Proliferation Assay[1]
MDA-MB-231, MDA-MB-361 and MDA-MB-453 cells were seeded at a density of 5 × 104 cells/well in 6 well plates in triplicate. Complete media containing 125 nM doxorubicin or 10 µM RIDR-PI-103 alone or combination of both respectively was replaced every alternate day and cells were stained with 0.5% crystal violet in methanol within 7–10 days. The intensities were measured using an Odyssey infrared system. The values are expressed as mean of intensities obtained from 3 independent experiments.

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Cell Migration Assay[1]
MDA-MB-231, MDA-MB-361 and MDA-MB-453 cells were seeded at a density of 5 × 104 cells/well in 6 well plates and treated with vehicle (DMSO), 125 nM doxorubicin and 10 µM RIDR-PI-103 or combination of both for 8 h. After 8 h, cells were counted and 2 × 104 cells/well were added to the upper chamber of transwell and incubated for 24–48 h. After 24–48 h, migrated cells in the lower chamber were stained with 0.5% crystal violet and images were captured from three areas under phase contrast microscope. The intensities from three areas collected from three independent experiments were measured using ImageJ and expressed as % of control and represented graphically.

Female C57BL/6J mice at 4 weeks were used with n = 3 per time point. Time points for blood collection were based on previous findings regarding the in vitro microsomal metabolic stability of RIDR-PI-103. RIDR-PI-103 was formulated using a mixture of 40% propylene glycol with 60% injectable saline in which RIDR-PI-103 was soluble in solution and not a suspension. The stability of RIDR-PI-103 in this formulation was ascertained by measuring drug content over 7 days. RIDR-PI-103 (dose = 20 mg/kg) was injected intraperitoneally in all mice at the start of the experiment. Blood collection was done via cardiac puncture under anesthesia at 0, 0.5, 4, 6, 24, 48, 72, 96 h post injection. Plasma was isolated from the blood samples by centrifugation and was stored at −80 °C until further analysis.

[1]. Phosphoinositide 3-Kinase (PI3K) Reactive Oxygen Species (ROS)-Activated Prodrug in Combination with Anthracycline Impairs PI3K Signaling, Increases DNA Damage Response and Reduces Breast Cancer Cell Growth. Int J Mol Sci. 2021;22(4):2088. Published 2021 Feb 19.

[2]. Efficacy of RIDRPI103, a reactive oxygen species (ROS) activated prodrug in treatment of breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-04.

RIDR-PI-103 is a novel reactive oxygen species (ROS)-induced drug release prodrug with a self-cyclizing moiety linked to a pan-PI3K inhibitor (PI-103). Under high ROS, PI-103 is released in a controlled manner to inhibit PI3K. The efficacy and bioavailability of RIDR-PI-103 in breast cancer remains unexplored. Cell viability of RIDR-PI-103 was assessed on breast cancer cells (MDA-MB-231, MDA-MB-361 and MDA-MB-453), non-tumorigenic MCF10A and fibroblasts. Matrigel colony formation, cell proliferation and migration assays examined the migratory properties of breast cancers upon treatment with RIDR-PI-103 and doxorubicin. Western blots determined the effect of doxorubicin ± RIDR-PI-103 on AKT activation and DNA damage response. Pharmacokinetic (PK) studies using C57BL/6J mice determined systemic exposure (plasma concentrations and overall area under the curve) and T1/2 of RIDR-PI-103. MDA-MB-453, MDA-MB-231 and MDA-MB-361 cells were sensitive to RIDR-PI-103 vs. MCF10A and normal fibroblast. Combination of doxorubicin and RIDR-PI-103 suppressed cancer cell growth and proliferation. Doxorubicin with RIDR-PI-103 inhibited p-AktS473, upregulated p-CHK1/2 and p-P53. PK studies showed that ~200 ng/mL (0.43 µM) RIDR-PI-103 is achievable in mice plasma with an initial dose of 20 mg/kg and a 10 h T1/2. (4) The prodrug RIDR-PI-103 could be a potential therapeutic for treatment of breast cancer patients.[1]

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The Phosphatidylinositol-3 kinases (PI3Ks) is a family of lipid kinases encoded by PIK3C isoform genes. PI3K phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3), leading to AKT phosphorylation along with other proteins containing a PH domain. Phosphorylation of AKT stimulates protein synthesis and cancer cell growth. PI3K pathway is hyper-activated in >60% of clinical breast cancer patients due to aberrations in the genes encoding HER2, PTEN, PIK3CA, or AKT1-3 leading to de novo and acquired treatment resistance. There has been intense interest in developing drugs that target PI3K. However, drugs targeting PI3K activity are toxic, due to the physiological roles of PI3Ks in basic cellular processes. We have developed a novel technology, called RIDR (ROS-Induced Drug Release) which is a self-cyclizing reagent linked to a PI3K inhibitor (PI-103) to eject PI-103 in a controlled manner under oxidative stress in highly aggressive breast cancers including triple negative breast cancer cell lines (TNBCs) and other ER+, HER2+ breast cancer cell lines with activating PIK3CA mutations. We evaluated the efficacy of RIDR-PI-103 (5-100 µM) or PI-103 (0-5 µM) in normal fibroblasts, T47D, MCF-7, MDA-MB-361, BT474, MDA-MB-453 and MDA-MB-231 breast cancer cell lines. We noted that IC50 of PI-103 is 3.34 µM whereas IC50 of RIDR-PI-103 is >100 µM in normal fibroblasts, indicating the toxicity of the PI-103 alone but not the RIDR-PI-103 in normal fibroblasts. Our data indicated that ~30-40 µM RIDR-PI-103 significantly inhibited T47D, MDA-MB-231, MDA-MB-361 and MDA-MB-453 cell proliferation whereas higher concentrations of the drug were effective in BT474 and MCF-7 breast cancer cell lines. Our quantitative PCR data showed that the antioxidant catalase mRNA levels are statistically lower compared to normal fibroblasts in these cancer cell lines indicating that the efficacy of RIDR-PI-103 could be correlated to catalase expression. Doxorubicin is a clinically relevant chemotherapy known to induce reactive oxygen species (ROS) in breast cancer cell lines. We are currently assessing the levels of biomarkers for ROS in formalin-fixed paraffin embedded breast tumors treated without or with chemotherapy including 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) and 4-hydroxy-2-noneal (4HNE). Our data indicated that doxorubicin significantly sensitized MDA-MB-453, MDA-MB-361 and MDA-MB-231 cells to RIDR-PI-103 indicated by significant lower IC50 values of the combined drug treatment versus the single agent. Doxorubicin and RIDR-PI-103 showed a synergistic effect in MDA-MB-361 and MDA-MB-231 cells to inhibit cancer cell proliferation. Doxorubicin was more effective than docetaxel, another chemotherapeutic drug, in sensitizing these cells to growth inhibition. Thus, this novel combination of the ROS-activatable PI3K inhibitor prodrug and chemotherapy provides strong justification for its continued development and future clinical trials for patients stricken with PI3K driven tumors. In other experiments, we show that a recent FDA-approved CDK4/6 inhibitor, palbociclib, sensitized ER+ breast cancer cell lines (T47D and MDA-MB-361) to RIDR-PI103 using cell viability assays. Experiments are ongoing to determine the mechanism of action of these novel drug combinations that could be potentially translated to clinic for treatment of breast cancer patients.[2]

C27H25N7O4

511.531904935837

511.196

2581114-71-6

163196423

Typically exists as Light yellow to yellow solid at room temperature

2.2

3

10

6

38

801

0

MTALGWVUOBSKIC-UHFFFAOYSA-N

InChI=1S/C27H25N7O4/c28-15-22(35)31-20-14-17(29)6-7-21(20)37-18-4-1-3-16(13-18)25-32-23-19-5-2-8-30-27(19)38-24(23)26(33-25)34-9-11-36-12-10-34/h1-8,13-14H,9-12,15,28-29H2,(H,31,35)

2-amino-N-[5-amino-2-[3-(6-morpholin-4-yl-8-oxa-3,5,10-triazatricyclo[7.4.0.02,7]trideca-1(9),2(7),3,5,10,12-hexaen-4-yl)phenoxy]phenyl]acetamide

RIDR-PI-103; 2581114-71-6

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~195.49 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)