Syntheses, biological and preclinical evaluations, and nanoparticle formulations of the ROS activatable prodrugs of doxazolidine for drug resistant cancer therapy

Abstract

Doxorubicin (dox) has been used for the treatments of many cancers for more than 50 years since its discovery. Currently, the treatment with dox is often limited by cardiotoxicity and the development of drug resistance. Doxazolidine (doxaz) is a dox-formaldehyde conjugate discovered in 1990s. It bears an extra carbon, linking its daunosamine hydroxyl to its adjacent amino substituent to create an oxazolidine ring. In contrast to dox, which is a topoisomerase inhibitor, doxaz crosslinks DNA to nonspecifically inhibit cell growth. Doxaz is significantly more cytotoxic than dox even against the dox-resistant cancer cells and in spite of its 3 min half-life for hydrolysis to dox. Doxaz has been studied since its discovery but not clinically, yet, due to its cytotoxicity and unsuccessful attempts to generate the prodrugs of doxaz that are activated solely in cancer cells without damaging healthy normal cells. Here, we report the ROS-activatable prodrug of doxaz, named Doxaz-BA formulated as nanoparticle. We synthesized Doxaz-BA and its derivatives and tested them as nanoparticle formulations in vitro in cell cultures and in vivo in mouse xenografts. This technology provides a highly sought after cancer therapy that kills only cancer cells while toxicity to normal tissues is minimal. Doxaz-BA is effective to the drug-resistant cancer cells, and the safety assessments showed no toxicity in mouse models. Therefore, this technology offers a possible solution for the clinical translation of Doxaz in treating the drug-resistant cancers, which are often incurable in the standard clinical settings.

Graphical Abstract

Introduction

Doxorubicin (dox), trade name Adriamycin, is an anthracycline anticancer drug isolated from a mutated soil bacterium, Streptomyces peucetius var. caesius, discovered by Farmitalia Research Laboratories of Milan in Italy in 1969 by a mutagenic treatment to the parental strain, Streptomyces peucetius, producing daunorubicin (DNR) isolated from the ground soil of the famous octagon Castel del Monte in Apulia, Italy. Dox differs from DNR with a hydroxy group at the 14-position of DNR and shows greater anticancer activities and therapeutic indexes than DNR ^{1, 2}. In 1974, dox was approved in the United States and afterward has been used in chemotherapeutics for a wide range of cancers including breast, lung, lymphoma, ovarian, leukemia and others. However, its cardiotoxicity and systemic toxicities hamper the prolonged clinical usage of dox, and the cancer cells eventually acquire the drug resistance ^{1, 3}.

Doxazolidine (doxaz) (Figure 1) is a doxorubicin-formaldehyde conjugate discovered through the sequence of studies to investigate the mechanism of the cytotoxicity caused by dox in 1990s and 2000s ^4–12. One equivalent of formaldehyde reacts with dox at 3’-NH₂ and 4’-OH forming an oxazolidine ring. The toxicity of doxaz is caused by this oxazolidine ring reacting with DNA at 5’-GC-3’ sequence while dox inhibits topoisomerase II. This one carbon difference makes doxaz more cytotoxic than dox from 1 to 4 orders of magnitude to a broad spectrum of cancer cells than the parental drug, dox ¹³. Further, doxaz is effective to drug resistant cancer cells overexpressing p-glycoprotein (p-gp) while dox, paclitaxel and other small molecules are usually ineffective since doxaz covalently alkylates DNA at 5’-GC-3’ sequence making doxaz unaffected by p-gp. The oxazolidine ring in doxaz is unstable and easily hydrolyzed in aqueous buffer with a half-life of 3 min to less toxic drug, dox ¹⁴. The instability of doxaz was overcome by carbamoylation of oxazolidine ring and thereafter the prodrug strategies have been investigated since 2000s. However, none of them reached the clinical investigations yet ^15–19.

Figure 1.

(A) The structure conversions of doxorubicin (dox) and doxazolidine (doxaz). One equivalent of formaldehyde reacts with dox at 3’-NH₂ and 4’-OH positions, producing oxazolidine ring. Doxaz is hydrolyzed to dox in water with a half-life of 3min. (B) The prodrug activation of Doxaz-BA by hydrogen peroxide. Doxaz is released and hydrolyzed to dox with a half-life of 3 min in water.

We previously reported the ROS activatable prodrug of doxaz, named Doxaz-Boronic Acid (Doxaz-BA) ²⁰. This prodrug was designed to be activated in cancer cells by endogenous ROS and external stimuli such as radionuclides, ROS inducing drugs, and x-rays. We successfully developed the prodrug system showing in vitro efficacy with human tissue cultures and in vivo with mouse xenografts. Further, we developed the nanoformulation using human serum albumin (HSA) because the prodrug tends to aggregate with serum proteins when intravenously injected into mice due to the hydrophobicity of the molecule and the interactions of the boron moiety of the prodrug with serum proteins. Therefore, we reasoned that pre-forming nanoparticles under controlled conditions could be beneficial, as larger prodrug-serum protein aggregates interfere with mouse blood circulation—though this effect is transient. However, an improved formulation method is essential for future clinical applications. Our pilot formulation method using HSA significantly improved water solubility, drug dosing, and the efficacies in vivo with mouse models. Therefore, we further optimized the chemical structures of prodrugs for improved potencies and pharmacological properties and tested the nanoparticle formulation methods including HSA, liposome and feraheme^® for future clinical applications. By utilizing the unique properties of doxaz, we were able to focus its strong toxicity to cancer cells in the tumor microenvironment where ROS levels are higher than in normal tissues ^{21, 22}. Our prodrug system, Doxaz-BA, has a great potency in vitro with a broad range of cancer cells and in vivo with mouse xenografts with and without external stimuli such as radionuclides including ¹⁸F-FDG and ROS inducing drugs. In this study, we describe the chemical syntheses and optimizations of prodrugs, nanoparticle formulations and biological and preclinical assessments including efficacies and plasma stability, safety for the clinical translation of drug-resistant cancer therapy, and further the investigation of the combination therapy with anti-PD1 immunotherapy.

Experimental Methods

General

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Waltham, MA) unless otherwise noted. All cell lines were purchased from ATCC (Manassas, VA) and authenticated unless otherwise noted. NCI/ADR-RES cell line was purchased from National Cancer Institute (Bethesda, MD). Clinical samples of doxorubicin hydrochloride were gifted by FeRx, Inc. (Aurora, CO). All NMR spectra were taken at 500 or 600 MHz Bruker Avance III spectrometers (Billerica, MA) in deuterated solvents purchased from Cambridge Isotope Laboratories, Inc (Tewksbury, MA). Chemical shifts are reported in δ values of ppm and were standardized by the residual solvent peak in the MestReNova NMR software (Mestrelab Research, Santiago de Compostela, Spain). UPLC-MS and HPLC were performed on Waters Acuity SQD and Waters Alliance 2695/2996 instruments, respectively (Milford, MA). High resolution mass spectrometry was performed on Waters Acuity Premiere XE TOF instrument (Milford, MA). Preparative HPLC was performed on Waters 2545/2996 instrument with SunFire Prep C18 column (Milford, MA). Normal phase column chromatography was performed on CombiFlash NextGen 300+ with RediSep normal phase silica flash column (Teledyne ISCO, Lincoln, NE). The molar extinction coefficient of dox/doxaz is 11500 M⁻¹cm⁻¹ at wavelength of 480 nm. The concentration of Doxaz-BA prodrug was determined by UV-VIS spectrophotometer with a solution of 25% of water in DMSO as a solvent. Optical densities ware measured by SpectraMax iD5 instrument (Molecular Device, San Jose, CA). All tested compounds have more than 95% purity.

Chemical Syntheses

All chemical syntheses are described in the supporting information.

Nanoparticle Formulations

Human Serum Albumin (HSA) Formulation

Human serum albumin (HSA) was purchased from Sigma Aldrich (Product #: A1887). HSA (40 mg) was dissolved in PBS (1 mL). 1 equivalent mole of prodrug in DMSO were added to a solution of HSA in PBS to have a desired concentration. After vertexing, a clear solution appeared. The size distribution was measured by Zetasizer (Malvern Panalytical, UK).

Liposome Formulation

Lipids were purchased from Avanti Polar Lipids (Alabaster, Alabama). Doxaz-BA-Ibu (1 mg), Soy PC (#840054) (100 mg), cholesterol (12.5 mg), and 18:0 PEG 2000-PE (#880120) (14 mg) were dissolved in chloroform. Solvent was evaporated by a rotatory evaporator and the reside was dried in vacuo. The lipid film was dissolved in PBS (2 mL) and the mixture was sonicated until the solution become a homogeneous mixture. The size of liposome was measured by Zetasizer.

Feraheme^® Formulation

A clinical sample Feraheme^® was purchased from a clinical pharmacy at the Memorial Sloan Kettering Cancer Center (New York, NY). Feraheme^® (10 mg/kg) and Doxaz-BA-Ibu (1 mg/kg Dox equivalent weight) in DMSO were mixed and diluted with PBS to have a desired concentration. A clear solution appeared soon after mixing and the particle size was measured by Zetasizer.

Cell Experiments

Growth Inhibition Assays

The cell lines, media and seeding densities on a 96-well plate are summarized in the table below. The cells were cultured in a media supplemented with 10% (v/v) fetal bovine serum (FBS) and 1X penicillin/streptomycin (penicillin: 100 U/mL and streptomycin 100 μg/mL). The cells were incubated at 37 °C in an atmosphere of 95% air and 5% CO₂, unless otherwise noted. The cells were seeded on a 96-well plate and allowed to adhere overnight. The various concentrations of Doxaz-BA prodrugs with/without IKE, sorafenib or cisplatin were added to the cells. The cells were incubated in a cell culture incubator for 72 h.

The cell densities were measured by a crystal violet staining method described in a previous study ²³. In brief, the cells were fixed with 5% formalin in PBS for 10 min and stained with a crystal violet solution (0.1% w/v in water) for 30 min. After the staining solution was removed by pipetting, the cells were washed with water three times carefully and completely, air-dried, and redissolved in a 1:1 mixture of 2-propanol and 2% (w/v) sodium dodecyl sulfate (SDS) in water. The cell densities were measured by the absorptions at 570 nm using a plate reader. The optical densities were normalized to the untreated cells. The dose response curves and the IC₅₀ values were determined using GraphPad Prim (Boston, MA).

Cell line	Organ	Media	Seeding density (96-well plate)
HT-1080	Fibrosarcoma	MEM	1000
CT-26	Mouse colon	RPMI	1000
Miapaca2	Pancreas	DMEM	1500
MDA-MB-231	Breast	DMEM	3000
HepG2	Liver	MEM	5000
NCI/ADR-RES	Drug resistant ovarian	DMEM	5000

Mouse Experiments

General

All mouse experiments were conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center and followed National Institution of Health guidelines for animal care. All mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and husbanded in the cages with foods and water in the animal facilities at Memorial Sloan Kettering Cancer Center. Tumors were implanted subcutaneously at the flank of mice, and the treatments were started when the tumor volume reached 150–200 mm³. An electrical caliper was used to measure the tumor volume, which is calculated by the equation, volume = (width)² x length x 0.5. The tumor volume and body weights were measured twice a week. The endpoint in this study was when the tumor volume reached 800 mm³ and the mice were euthanized by CO₂ gas.

Efficacy Study

Maximum Tolerated Dose (MTD) Determination

Doxaz-BA-Ibu-HSA with different doses (1.5, 1.75 and 2.0 mg/kg dox equivalent weight) were intravenously injected to female nude mice (6 weeks) via tail vein at the days 1 and 5. The body weight changes were monitored twice a week.

Efficacy experiment with the nanoparticle formulation of Doxaz-BA-Ibu (HT-1080)

HT-1080 fibrosarcoma cancer cell line (7.8 × 10⁶ cells) in a 1:1 ratio of matrigel (50 μL) and PBS (50 μL) was implanted on the flank of 6-week-old female nude mice. Mice were randomized and each treatment group had 5 mice. Doxaz-BA-Ibu (1.0 mg/kg dox equivalent weight) with the nanoparticle formulation was dosed intravenously from mouse tail vein. The treatments were conducted on the days 4 and 8 after tumor implantation.

Efficacy experiment with Doxaz-BA-Ibu-HSA (Miapaca2)

Miapaca2 pancreatic cancer cell line (10.5 × 10⁶ cells) in matrigel (100 μL) was implanted on the flank of 6-week-old female NOD scid mice. Mice were randomized and each treatment group had 5 mice. Doxaz-BA-Ibu-HSA (2.0, 1.5 and 1.0 mg/kg dox equivalent weight) was dosed intravenously from tail vein. The treatments were conducted twice a week for 4 weeks on the days 6, 9, 12, 16, 19, 23, 26 and 30 after tumor implantation.

Efficacy experiment with Doxaz-BA-Ibu-HSA (NCI/ADR-RES)

NCI/ ADR-RES drug resistant ovarian cancer cell line (2.0 × 10⁶ cells) in matrigel (150 μL) was implanted on the flank of 6-week-old female NOD scid mice. Mice were randomized and each treatment group had 5 mice. Doxaz-BA-Ibu-HSA (2.0 mg/kg dox equivalent weight), doxorubicin (2.0 mg/kg), and paclitaxel (5.0 mg/kg) were dosed intravenously from tail vein. The treatments were conducted twice a week for 3 weeks plus one dose on the days 43, 46, 50, 53, 57, 60, and 64 after tumor implantation. Due to the severe toxicity caused by doxorubicin, the treatment group with doxorubicin was terminated after the third dose. Paclitaxel was dosed with the same amount of HSA with Doxaz-BA-Ibu-HSA to improve the water solubility.

The combination therapy with anti-PD1 and Doxaz-BA-Ibu-FH

CT-26 murine colorectal cancer cell line (0.5 × 10⁶ cells) in a 1:1 ratio of matrigel (50 μL) and PBS (50 μL) was implanted on the flank of 8-week-old female BALB/c mice. Mice were randomized and each treatment group had 5 mice. Doxaz-BA-Ibu-FH (1.5 mg/kg dox equivalent weight) was dosed intravenously from tail vein at the days of 4, 6, 8, 11, 13 and 15 after tumor implantation. Anti-mouse PD1 antibody purchased from Bio-X Cell Inc. (West Lebanon, NH) (100 μg/mouse) was dosed intraperitoneally on the days of 3, 5, 7, 9, and 12 after tumor implantation. Mouse blood was analyzed 3 days after the final treatment.

Pharmacokinetics (PK)

PK study was performed at the Antitumor Assessment Core in the Memorial Sloan Kettering Cancer Center. Athymic nude female mice (6–8 weeks) were purchased from the Jackson Laboratory (Bar Harbor, ME). Doxaz-BA-Ibu-HSA (3.5 mg/kg real weight, 2.0 mg/kg dox equivalent weight) in PBS was dosed intravenously. Following a single dose of the drug, 3 mice from each group were bled at the following timepoints: 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h. At the timepoint, the mice were sacrificed and 250 μL of whole blood was collected into an EDTA tube, and the drug concentration was measured by LC-MS with a method developed by the antitumor assessment core.

Necropsy

Necropsy study was performed at the Antitumor Assessment Core and the Laboratory for Comparative Pathology at the Memorial Sloan Kettering Cancer Center. Athymic nude female mice (6–8 weeks) were purchased from Jackson Laboratory (Bar Harbor, ME). Doxaz-BA-Ibu-HSA (3.5 mg/kg real weight, 2.0 mg/kg dox equivalent weight) in PBS was dosed intravenously twice a week for four weeks (total 8 doses). The mice were euthanized, and necropsy was performed after 24 hours or 2 weeks later. The comments from the pathologist are as follow,

“This study is the safety assessment for an anticancer agent developed in Grimm lab. The agent is an anthracycline small molecule prodrug, formulated in human serum albumin and activated in cancer cells. Presumably, cardiotoxicity and liver toxicity are the possible, but a previous study using a similar compound did not show any toxicity. There was no significant body weight change in these animals over time. Submitted slides include tissue from the heart, liver, gallbladder, lungs, spleen, kidneys, and brain.

There are three distinct experimental groups:

1. A vehicle control group (time of euthanasia and vehicle components not provided)

2. Anti-cancer agent administered via tail vein 2x per week for 4 weeks (8 doses total); euthanized 24 hrs after final dose

3. Anti-cancer agent administered via tail vein 2x per week for 4 weeks (8 doses total); euthanized 14 days after final dose

There are no adverse events related to drug administration in the heart, kidney, liver, gallbladder, spleen, or brain. Some animals in the 24-hour group have small granulomas or neutrophilic aggregates occasionally containing foreign material. The foreign material is colorless, birefringent, and intravascular or intra-septal. I suspect this lesion is not directly related to the agent itself, but possibly due to the mechanism of drug formulation and delivery with dissemination of larger particles lodging within lung vasculature and alveolar septae. Per the investigator, these particles may be larger nanoparticles consisting of human serum albumin and drug. Another etiology is intravenous administration of non-pharmaceutical grade compounds that have not been adequately filtered or have contaminated components. Strict adherence to the IACUC policy on the “Use of Non-Pharmaceutical Grade Compounds” may result in resolution of these lesions. The lung lesions are small and not adverse. The vehicle group and 14-day group do not have similar lesions in the lung, indicating possible clearance of this material by 2 weeks in the latter. Remaining lesions are considered background/incidental. Images are available upon request.”

Mouse blood cell counting

Mouse blood was collected from mouse retroorbital using a capillary blood collection tube coated with EDTA and analyzed by Element HT5 (HESKA, Loveland, Colorado).

Mouse Plasma Stability

Lyophilized mouse plasma purchased from Sigma Aldrich were dissolved in Milli-Q water (1 mL). The plasma was pre-warmed to 37 °C and Doxaz-BA prodrug was added to the plasma (300 μL) in an eppendorf tube to prepare a 50 μM solution. The mixture was incubated at 37 °C and a 50 μL aliquot was isolated at each time point (0, 15, 30, 60 and 120 min) to a new eppendorf tube containing acetonitrile (50 μL) to precipitate plasma proteins. The eppendorf tube was centrifuged at 14,000 rpm for 5 min and the supernatant was analyzed by a reverse phase HPLC with a C18 column. The peaks of Doxaz-BA prodrug and dox at 480 nm absorption were integrated. The natural log of % component remaining was plotted as a function of time and the slope was calculated using GraphPad Prism. The half-life was calculated using the equation: t(1/2) = Ln2/−slope.

Results

Syntheses

The syntheses of Doxaz, Doxaz-BA and the intermediate (6) are described in our previous report ²⁰. Doxaz-BA forms the complex with HSA improving the drug dosing and the efficacies in vivo with mouse xenografts. To further improve the efficacy, Doxaz-BA was modified to increase the affinity to HSA by incorporating hydrophobic moieties such as lipids and ibuprofen known to bind HSA ^{24 25}. We synthesized several Doxaz-BA derivatives (compounds 1–5) (Figure 2). Doxaz-BA-Hex (1) and Doxaz-BA-Deca (2) have lipid moieties, hexanamide and decanamide, respectively. Doxaz-BA-Ibu (3) and Doxaz-BA-Ibu-Me (5) have ibuprofen, (5) has an extra methyl group. Doxaz-BA-IP (4) has an isopropyl group. The syntheses of the Doxaz-BA derivatives are described in supporting information (Synthetic Schemes S1–S5). In brief, starting from the intermediate (6), lipids and ibuprofen with NHS ester were coupled to the amine of aryl borate. Doxaz was coupled to the para-nitrophenyl ester using HOBt. The final product was purified by a preparative HPLC. All tested compounds have more than 95% purity. UPLC-MS analysis showed Doxaz-BA-Hex is the most polar and Doxaz-BA-IP is the next. Doxaz-BA-Ibu with and without methyl group and Doxaz-BA-Deca were similarly more hydrophobic than others.

Figure 2.

The structures of Doxaz-BA and its derivatives.

Nanoparticle Formulations

The prodrug HSA complexes were prepared by mixing the prodrug and HSA with 1:1 molar ratio. The final complexes were completely water soluble. We also tested additional formulation methods by either loading Doxaz into liposomes or feraheme^®. In brief, the drug was encapsulated into the lipid bilayer of liposomes and the iron oxide core of feraheme^®. The sizes of HSA complex analyzed by dynamic light scattering (DLS) are in the range between 150 and 230 nm, compared to Doxaz-BA-HSA (120 nm) (Figure S1) ²⁰. The larger molecules tend to make larger particles. The liposome formulation of Doxaz-BA-Ibu gave the size of 120 nm. The feraheme^® formulation gave two peaks at 15 and 87 nm.

Cytotoxicity Assessment

We tested the cytotoxicity of Doxaz-BA and its derivatives with and without HSA formulation in the HT-1080 fibrosarcoma cell line (Figure 3 and Table S1). All derivatives of Doxaz-BA are more cytotoxic than the parental drug, Doxaz-BA, and no significant difference in the toxicity between the HSA formulation and the drug alone was observed. The larger and more hydrophobic molecules showed the stronger potency. We advanced Doxaz-BA-Ibu as an exploratory molecule for further studies because ibuprofen is FDA approved and the pharmacological effects are well studied ²⁶. Doxaz-BA-Ibu was tested for other cancer cell lines (CT-26 murine colorectal cancer, Miapaca2 human pancreatic cancer and MDA-MB-231 human breast cancer) (Figure 4A and Table S2). Doxaz-BA-Ibu showed an excellent potency with low nM IC₅₀ values. Further, we tested the nanoparticle formulations for HT-1080 fibrosarcoma cell (Figure 4B and Table S3). The liposomal and feraheme^® formulations showed similar potency, but both were less potent than the HSA formulation. Presumably, this lower potency was caused by the stronger protection of prodrug by liposome and feraheme^® nanoparticles. Further, we tested the prodrugs in HepG2 hepatocellular carcinoma to investigate the liver toxicity (Figure 5 and Table S4). Interestingly, the smaller molecules showed better potency than the larger and more hydrophobic molecules. Doxaz-BA-Ibu showed the highest IC₅₀ value. This lower toxicity would be beneficial to minimize liver toxicity in animal models.

Figure 3.

The cytotoxicity of Doxaz-BA and its derivatives with and without HSA formulation to HT-1080 fibrosarcoma. The prodrugs were incubated for 72 hours. The experiments were at least duplicated. The error bar is the 95% confidence interval.

Figure 4.

(A) The cytotoxicity assessment of Doxaz-BA-Ibu to various cancer cells. The prodrugs were incubated for 72 hours. The experiments were triplicated. The error bar is the 95% confidence interval. (B) The cytotoxicity assessment of the nanoparticle formulation of Doxaz-BA-Ibu with HT-1080 fibrosarcoma. The prodrugs were incubated for 72 hours. The experiments were at least duplicated. The error bar is the 95% confidence interval.

Figure 5.

The cytotoxicity assessments of Doxaz-BA prodrugs with HepG2 liver cancer cells. The prodrugs were incubated for 72 hours. The experiments were at least duplicated. The error bar is the 95% confidence interval.

As doxaz is effective to the drug-resistant cancer cell line (NCI/ADR-RES ovarian cancer cell), which overexpresses p-gp and in which paclitaxel and doxorubicin are not effective because of the elevated level of p-gp enhancing the efflux of small molecules ^{27, 28}. We previously reported that Doxaz-BA is effective in NCI/ADR-RES cells and the ROS inducing drugs such as IKE and sorafenib further enhance the toxicity of Doxaz-BA ²⁰. We tested Doxaz-BA-Ibu in conjunction with sorafenib and cisplatin, also known to enhance the ROS level in cells (Figure 6 and Table S5) ^{29, 30}. We found Doxaz-BA-Ibu alone is more potent than Doxaz-BA, and the toxicity was further enhanced in conjunction with sorafenib and cisplatin.

Figure 6.

The cytotoxicity assessment with the drug-resistant cancer cell (NCI/ADR-RES). The drugs were incubated for 72 hours. The experiments were triplicated except for Doxaz-BA, doxorubicin and paclitaxel with duplication. For the combination therapy, the IC₅₀ values are the toxicity from only Doxaz-BA prodrug activations. The error bar is the 95% confidence interval. The concentrations of IKE, sorafenib and cisplatin are as follow, IKE with Doxaz-BA is 100 nM, sorafenib with Doxaz-BA is 1μM, sorafenib with Doxaz-BA-Ibu is 5 μM, and cisplatin with Doxaz-BA-Ibu is 2.5 μM.

Plasma Stability

We tested the stabilities of Doxaz-BA and its derivatives in mouse plasma (Figure 7). Doxaz-BA prodrugs have an ester moiety, which can be hydrolyzed by mouse esterases. The stabilities of prodrugs were measured by a reverse phase HPLC. The smaller molecules, Doxaz-BA and Doxaz-BA-IP, were more susceptible to esterases than the larger molecules, presumably this is due to easier access to the carboxyl moiety. Doxaz-BA-Ibu-Me was synthesized and tested to learn if the methyl group would enhance the plasma stability: it showed better stability than Doxaz-BA-Ibu. Doxaz-BA-Deca was the most stable in mouse plasma.

Figure 7.

The stabilities (half-life) of Doxaz-BA prodrugs in mouse plasma, measured by a reverse phase HPLC. The experiments were duplicated.

In vivo Assessment

First, we assessed the nanoparticle formulations of Doxaz-BA-Ibu in the mouse xenografts of HT-1080 fibrosarcoma (Figure 8). The drugs with nanoparticles (1 mg/kg dox equivalent weight) were injected into the mouse tail vein on the days 4 and 8 after the tumor implantation. All nanoparticle formulations showed tumor growth inhibitions compared to the control (PBS) group with the HSA formulation providing more potency than the liposome and feraheme^® formulations. Therefore, the HSA formulation of Doxaz-BA-Ibu was used as the lead agent for further preclinical investigations. The maximum tolerated dose (MTD) of Doxaz-BA-Ibu-HSA was determined to be 2.0 mg/kg dox equivalent weight (3.5 mg/kg real weight) (Figure S2).

Figure 8.

In vivo efficacy assessment of the nanoparticle formulations of Doxaz-BA-Ibu with the mouse xenografts implanted with HT-1080 fibrosarcoma. The nanoparticle formulations of Doxaz-BA-Ibu (1.0 mg/kg dox equivalent weight) were dosed from mouse tail vein at the days 4 and 8 after the tumor implantation (5 mice per group). The error bar is the standard error of the mean.

Next, we tested the different doses of Doxaz-BA-Ibu-HSA in the mouse xenografts implanted with MIA PaCa-2 pancreatic cancer cells (Figure 9 A–C). Doxaz-BA-Ibu-HSA (1.0, 1.5, and 2.0 mg/kg dox equivalent weight) was dosed from mouse tail vein twice a week for four weeks. The highest dose (2.0 mg/kg dox equivalent weight) showed the strongest tumor growth inhibition and the longest survival. There was no difference between 1.5 and 1.0 mg/kg on the tumor growth though 1.5 mg/kg showed better survival. Further, no toxicity was observed from mouse body weight changes. Then, we investigated the highest dose (2.0 mg/kg dox equivalent weight) in the mouse xenografts implanted with the drug-resistant cancer cell (NCI/ADR-RES ovarian cancer cells) (Figure 10 A–B). Doxorubicin (2.0 mg/kg) and paclitaxel (5.0 mg/kg) were also tested. The drugs were dosed twice a week for four weeks at the mouse tail vein. Doxaz-BA-Ibu-HSA showed the strongest tumor growth inhibition. The dox treated mice showed a significant body weight reduction with more than 20 % body weight loss and were severely moribund. Therefore, the dox treated group was terminated after the third dose. The paclitaxel treated group showed mild tumor growth inhibition. Further, the group of Doxaz-BA-Ibu-HSA did not show any body weight change. After this study, Doxaz-BA-Ibu-HSA was tested for more in-depth toxicity assessment with the antitumor assessment core and the laboratory of comparative pathology at the Memorial Sloan Kettering Cancer Center. Doxaz-BA-Ibu-HSA (2.0 mg/kg dox equivalent weight) were dosed to female nude mice twice a week for four weeks from mouse tail vein. Doxaz-BA-Ibu-HSA did not show any toxicity from the pathological analysis and mouse body weight changes (Figure S3). Further, we assessed the plasma half-life of Doxaz-BA-Ibu-HSA in mice, and it was 24.19 hours (Figure S4).

Figure 9.

(A) In vivo efficacy experiment of Doxaz-BA-Ibu-HSA with the mouse xenografts implanted with Miapaca2 pancreatic cancer cells. Doxaz-BA-Ibu-HSA (2.0, 1.5 and 1.0 mg/kg dox equivalent weight) was dosed intravenously from mouse tail vein. The treatments were performed twice a week for 4 weeks on the days 6, 9, 12, 16, 19, 23, 26 and 30 after the tumor implantation (5 mice per group). The error bar is the standard error of the mean. (B) Survival analysis. (C) The body weight change. The error bar is the standard deviation.

Figure 10.

(A) In vivo efficacy experiment of Doxaz-BA-Ibu-HSA with the mouse xenografts implanted with the drug-resistant cancer cell line (NCI/ADR-RES). Doxaz-BA-Ibu-HSA (2.0 mg/kg dox equivalent weight), doxorubicin (2.0 mg/kg), and paclitaxel (5.0 mg/kg) were dosed intravenously from mouse tail vein. The treatments were performed twice a week for 3 weeks plus one dose on the days 43, 46, 50, 53, 57, 60, and 64 after the tumor implantation. Due to the severe toxicity caused by doxorubicin, the treatment group with doxorubicin was terminated after the third dose. The error bar is the standard error of the mean. (B) The body weight change. The error bar is the standard deviation.

Lastly, we tested the combination therapy of Doxaz-BA-Ibu with anti-PD1 antibody (Figure 11 A–E). As HSA and liposome potentially activate mouse immune system, we used the feraheme^® formulation method. Doxaz-BA-Ibu-FH showed the synergistical tumor growth inhibition with anti-PD1 antibody. Furthermore, the combination therapy did not show any hematological toxicities.

Figure 11.

The combination therapy with anti-PD1 immunotherapy with Doxaz-BA-Ibu-FH in the mouse xenografts implanted with CT26 murine colorectal cancer cells. Doxaz-BA-Ibu-FH (1.5 mg/kg dox equivalent weight) was dosed intravenously from mouse tail vein at the days of 4, 6, 8, 11, 13 and 15 after the tumor implantation. Anti-mouse PD1 antibody purchased from Bio-X Cell Inc. (West Lebanon, NH) (100 μg/mouse) was dosed intraperitoneally on the days of 3, 5, 7, 9, and 12 after tumor implantation (5 mice per group). The error bar is the standard error of the mean. (B) The body weight change. The error bar is the standard deviation. (C)-(E) Hematological toxicity assessments. The error bar is the standard deviation. The 95% confidence intervals reported by Charles River Laboratories International, Inc are as follow, WBC: 5.69–14.84 (K/μL), RBC: 8.16–11.69 (M/μL), and PLT: 476–1611 (K/μL).

Discussions

We successfully synthesized the derivatives of Doxaz-BA and tested them in vitro with the cell cultures and in vivo with the mouse xenografts. The syntheses are straightforward and the scale up to a larger quantity is viable. The potencies of Doxaz-BA derivatives in vitro with the tissue culture are better than the parental prodrug, Doxaz-BA. Doxaz-BA and its derivatives are effective in the drug-resistant cancer cell line (NCI/ADR-RES) and further the ROS inducing drug such as IKE, sorafenib and cisplatin synergistically improve the potencies of the Doxaz-BA prodrugs. As the molecule becomes more hydrophobic, the prodrugs showed better potency than Doxaz-BA. However, this trend flips in HepG2 hepatocellular carcinoma to which Doxaz-BA-Ibu and Doxaz-BA-Deca are less effective than Doxaz-BA. This result may suggest Doxaz-BA-Ibu and Doxaz-BA-Deca are more protective to liver-toxicity and -damage.

The potential problem of Doxaz-BA and its derivatives is the aggregation with serum proteins caused by the boron-protein interactions and the hydrophobicity of the molecule. This problem was overcome by developing the nanoparticle formulations with HSA, liposome and feraheme^®. The nanoparticle formulations of Doxaz-BA derivatives are completely water soluble, and no aggregation was observed. The HSA formulation is more potent than the liposome and feraheme^® formulations and as potent as the small molecule alone.

We advanced Doxaz-BA-Ibu for in vivo mouse studies because Ibuprofen is FDA approved and known to bind HSA ²⁴. The HSA formulation of Doxaz-BA-Ibu is more potent than the liposome and Feraheme^® formulations, consistent with the in vitro cell culture studies. The HSA formulation of Doxaz-BA-Ibu was further tested for the mouse xenografts implanted with pancreatic and drug-resistant cancer cell lines with good efficacy. Doxaz-BA-Ibu inhibited tumor growth and extended the survival without any toxicities. FDA approved drugs, dox and paclitaxel, did not work well in the drug-resistant cancer and the toxicity caused by dox was very severe while Doxaz-BA-Ibu did not show any toxicities. Further the safety assessment and necropsy studies confirmed no adverse effect caused by the HSA formulation of Doxaz-BA-Ibu.

We previously reported that Doxaz-BA can synergistically activated by radionuclides such as ¹⁸F-FDG, x-rays and ROS inducing drugs ²⁰. Besides them, we tested the combination therapy with anti-PD1 antibody in this study. We used the feraheme^® formulation of Doxaz-BA-Ibu because HSA and liposome potentially activate the immune system in more immunocompetent mouse than nude mice like balb-c mice. Our study clearly showed the synergistical tumor growth inhibition by the combination therapy of the feraheme^® formulation of Doxaz-BA-Ibu and anti-PD1 antibody. More importantly, this therapy did not show any toxicities and hematological damages in the mouse xenografts.

Conclusions

In this study, we described the syntheses, and biological and preclinical evaluations of the derivatives of Doxaz-BA with nanoparticle formulations. The syntheses are scalable to a large quantity and the therapeutic effects are remarkable in the cell cultures and the mouse xenografts. Doxaz-BA-Ibu-HSA is effective to the drug-resistant cancer without any adverse effects. Therefore, further preclinical development is expected for the treatments of the drug-resistant cancers using this technology.

Supplementary Material

Supporting Information

Supporting information (SI) includes the supporting figures and tables, synthetic schemes, chemical syntheses, and sets of NMR spectra.

Abbreviations

ACN

Acetonitrile

DCM

Dichloromethane

DIEA

N,N-diisopropylethylamine

DMSO

dimethyl sulfoxide

Dox

doxorubicin

DMAP

4-(dimethylamino)pyridine

Doxaz

doxazolidine

DoxF

doxoform

EM

exact mass

ESI-MS

Electrospray Ionization Mass Spectrometry

TOF

Time of Flight

FBS

Fetal Bovine Serum

HOBt

hydroxybenzotriazole

HPLC

High Performance Liquid Chromatography

UPLC

Ultra Performance Liquid Chromatography

IC₅₀

half maximal inhibitory concentration

MeOH

methanol

NMR

Nuclear Magnetic Resonance

DLS

Dynamic Light Scattering

PBS

Phosphate Buffered Saline

RT

room temperature

SDS

Sodium dodecyl sulfate

TFA

Trifluoroacetic acid

TLC

Thin Layer Chromatography

EDC-HCl

N-(3 -Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride

NHS

N-hydroxysuccinimide

NaHCO₃

Sodium bicarbonate

Na₂SO₄

Sodium sulfate

Rf

Retention Factor

TEA

Triethyl amine

IKE

Imidazole Ketone Erastin