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. Author manuscript; available in PMC: 2024 Jun 5.
Published in final edited form as: Mol Pharm. 2023 May 8;20(6):3049–3059. doi: 10.1021/acs.molpharmaceut.3c00069

Exploration of Verticillins in High-Grade Serous Ovarian Cancer and Evaluation of Multiple Formulations in Preclinical In Vitro and In Vivo Models

Elizabeth N Kaweesa 1,#, Jaqueline M Bazioli 2,#, Herma C Pierre 3, Daniel D Lantvit 4, Samuel K Kulp 5, Kasey L Hill 6, Mitch A Phelps 7, Christopher C Coss 8, James R Fuchs 9, Cedric J Pearce 10, Nicholas H Oberlies 11, Joanna E Burdette 12
PMCID: PMC10405366  NIHMSID: NIHMS1918116  PMID: 37155928

Abstract

Verticillins are epipolythiodioxopiperazine alkaloids isolated from a fungus with nanomolar anti-tumor activity in high-grade serous ovarian cancer (HGSOC). HGSOC is the fifth leading cause of death in women, and natural products continue to be an inspiration for new drug entities to help tackle chemoresistance. Verticillin D was recently found in a new fungal strain and compared to verticillin A. Both compounds exhibited nanomolar cytotoxic activity against OVCAR4 and OVCAR8 HGSOC cell lines, significantly reduced 2D foci and 3D spheroids, and induced apoptosis. In addition, verticillin A and verticillin D reduced tumor burden in vivo using OVCAR8 xenografts in the peritoneal space as a model. Unfortunately, mice treated with verticillin D displayed signs of liver toxicity. Tolerability studies to optimize verticillin A formulation for in vivo delivery were performed and compared to a semi-synthetic succinate version of verticillin A to monitor bioavailability in athymic nude females. Formulation of verticillins achieved tolerable drug delivery. Thus, formulation studies are effective at improving tolerability and demonstrating efficacy for verticillins.

Keywords: verticillin A, natural products, high-grade serous ovarian cancer, drug formulation, tolerability, xenograft

Graphical Abstract

graphic file with name nihms-1918116-f0005.jpg

INTRODUCTION

High-grade serous ovarian cancer (HGSOC) is the most common form of ovarian cancer, accounting for 70% of all cases.1 In 2022, it was estimated that there were approximately 20,000 new cases and 13,000 deaths in the United States, making ovarian cancer the leading cause of gynecological death in women.2 Current treatment strategies include cytoreductive surgery and lifesaving chemotherapy, including the use of many drugs derived from nature.

Natural products and their derivatives have, and continue to be, a great source of drugs, accounting for 50% of FDA-approved drugs used in the clinic today, including many anticancer agents such as Adriamycin and camptothecins.3,4 Of those, the most notable example may be paclitaxel, which is commonly referred to as taxol and was originally isolated from the pacific yew tree (Taxus brevifolia).5,6 This is the most used and most-sold anticancer drug.7–9 Taxol was initially discovered as an effective treatment against refractory ovarian cancer and continues to be used in the treatment of HGSOC in the clinic today.10,11 Unfortunately, chemoresistance to current therapies has led to relapse and eventually death in ovarian cancer patients.12,13 Therefore, new drug treatment and therapeutic strategies are needed for better prognosis and to combat chemoresistance.

Verticillins, which are epipolythiodioxopiperazine (ETP) alkaloids, have drawn interest from both the chemistry community as a synthetic target14,15 and the pharmacology community16,17 due to their potent activity in vitro and in vivo.15–18 More than two dozen verticillins have been isolated from fungal cultures19 since the initial member of the class, verticillin A, was first isolated in 1970.20,21 Recent studies have also probed for structure–activity relationship of the pharmacophore via the generation of analogues.22

In an earlier study, verticillin A was cytotoxic in HGSOC cell lines by inducing DNA damage and blocking histone methyltransferases.23 In addition, an enhancement of efficacy and solubility was achieved by encapsulation of verticillin A in a pH sensitive expansile nano-particle.24

To further explore the pharmacological potential of this class of compounds, a series of semi-synthetic verticillin analogues were generated, where verticillin A succinate showed both nanomolar cytotoxicity and greatly improved solubility.22 Additionally, verticillin D,25 which is an analogue of verticillin A that has two additional hydroxy groups, was also examined, as we recently discovered a fungus that biosynthesizes it in a very high yield.26 Given ongoing studies to further advance both verticillin A succinate and verticillin D, it was important to evaluate the cytotoxicity in HGSOC.27

In this study, we discovered that verticillin A succinate and verticillin D induced nanomolar cytotoxic activity in both 2D and 3D in vitro assays. Also, athymic female nude mice treated with verticillin A and verticillin D displayed a decrease in tumor burden. Dosing and formulation studies of verticillin A were also explored to understand the upper limits of tolerability. The determined doses were well tolerated based on gross appearance and a panel of liver and kidney biomarkers. These findings indicate that verticillin A can be given without inducing toxicity for in vivo efficacy when delivered intraperitoneally (IP) for HGSOC.

MATERIAL AND METHODS

Compound Identification and Purity Verification.

Verticillin A and verticillin D were isolated from fungal strains MSX59553 and MSX51257, respectively, similar to previously described methods;26,28 verticillin A succinate was generated via semi-synthesis.22 Prior to the pharmacological evaluation of these compounds, they were all evaluated by ultra-performance liquid chromatography (UPLC) and NMR, confirming their purity to >95% pure (Supporting Information and Material and Methods).

Verticillin A Succinate Semi-Synthesis.

Chemical reagents were used as received from vendors. Dimethylformamide was obtained from Thermo Scientific (Thermo Fisher, San Jose, CA, USA), and pyridine, 4-dimethylaminopyridine, and succinic anhydride were obtained from Alfa Aesar (Thermo Fisher, San Jose, CA, USA). All chromatography solvents were obtained from Macron Fine Chemicals (Avantor Sciences, Radnor, PA, USA) and VWR (Avantor Sciences, Radnor, PA, USA).

Verticillin A Succinate Formulation Preparation for UPLC-HRMS Analysis (Samples).

A 0.25 mg aliquot of verticillin A succinate was transferred to a 2 mL high-performance LC (HPLC) vial and dried under nitrogen. To the sample, 250 μL Tween 80:Captex 200P was added, resulting in a 1 mg/mL sample (i.e., 10× concentrated solution). The mixture was vortexed for 30sec, sonicated for 30 min, and placed on a shaker overnight (+16 h, 100 rpm) at room temperature. Then a 100 μL aliquot of the 10× solution was transferred to a 2 mL HPLC vial, diluted to 1 mL with 0.9% saline for a final concentration of 0.1 mg/mL, and vortexed to ensure that the sample dissolved properly. Serial dilutions were carried out to prepare the final two formulations. Once prepared, 20 μL aliquots of each formulation were immediately transferred to a 96 well plate and diluted to 400 μL with acetone. Aliquots were transferred and diluted again at t = 1 and 4 h. The formulations were stored at 4 °C in-between each collection time. Verticillin A succinate standard was prepared at 1000 μg/mL by dissolving 5.90 mg verticillin A succinate in 2.95 mL HPLC-grade acetone. The serial dilution method was used to prepare the working standards.

Cell Culture.

Human melanoma cancer cells (MDA-MB-435), human breast cancer cells (MDA-MB-231), and human ovarian cancer cells (OVCAR3, OVCAR4, OVCAR8) were purchased from the American Type Culture Collection (Manassas, VA). OVCAR8 cells with red fluorescent protein (OVCAR8-RFP) were engineered from OVCAR8 cells. OVCAR3, OVCAR4, and, MDA-MB-435 cells were grown in RPMI1640; OVCAR8 and MDA-MB-231 cells were grown in DMEM, each supplemented with fetal bovine serum (10%), penicillin (100 units/mL), and streptomycin (100 μg/mL). Cells were passaged a maximum of 20 times and maintained in a humidified incubator at 37 °C in a 5% CO2 environment. All lines were STR validated.

Cell Viability Assay.

A total of 5000 cells were seeded per well of a 96-well clear, flat-bottom plate (Microtest 96, Falcon) and incubated overnight (37 °C in 5% CO2). Verticillin A, verticillin A succinate and verticillin D were dissolved in DMSO and then diluted and added to the appropriate wells to give final concentrations of 250, 125, 62.5, 31, 15, 7.8, 3.9, 1.9, and 0.95 nM and a total volume of 100 μL and 0.5% DMSO per well. The cells were incubated in the presence of these compounds for 72 h at 37 °C and evaluated for viability with a commercial absorbance assay (CellTiter-Blue Cell Viability Assay, Promega Corp, Madison, WI) that measured viable cells. IC50 values are expressed in nM relative to the solvent (DMSO) control. Taxol (paclitaxel) was used as a positive control at 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, and 0.39 nM.

2D Foci Assay.

A total of 200 cells were seeded in a 60 mm plate and allowed to attach overnight. Cells were treated with vehicle (DMSO), verticillin A (50 nM), verticillin A succinate (50 nM), verticillin D (50 nM), and chemotherapeutic control Taxol (10 nM) for 8 h. Following 15 days incubation, cells were fixed using 4% paraformaldehyde and stained with 0.05% crystal violet. Plates were washed using distilled water to minimize background. Images were acquired using FluorChem E System (ProteinSimple). Colonies were counted using ImageJ.29

Spheroid Assay.

OVCAR8 cells were trypsinized and 5000 cells were resuspended in 80 μL of media per well in a 96-well round bottom Ultra Low Attachment Plate (Corning 07-201-680). After 4 days, the spheroids were treated with 20 μL of verticillin A, verticillin A succinate, verticillin D at 14,000, 12,000, 10,000, 8000, 4000, 2000, 1000, 500, and 250 nM, chemotherapeutic control Taxol at 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, and 0.39 nM, and vehicle for 7 days at 37 °C. The plate was equilibrated to room temperature for 30 min and 100 μL of CellTiter-Glo 3D Reagent (Promega) was added. The plate was incubated on a shaker for 30 min to obtain complete lysis and luminescence was recorded using Synergy Mx Plate Reader (BioTek).

In Vivo Verticillin A and Verticillin A Succinate Tolerability Study.

A tolerability study of verticillin A and verticillin A succinate was performed in female athymic nude (nu/nu) mice (CrTac:NCr-Foxn1nu, 7–8 weeks, Taconic, Germantown, NY, USA) to inform dose selection for follow-up efficacy studies in xenograft models of ovarian cancer. Mice were group-housed under conditions of constant photoperiod (12 h light/12 h dark), temperature, and humidity with ad libitum access to water and standard pelleted chow. Mice were randomized to treatment groups (n = 5 per group) that received verticillin A at 0.1, 0.25, 0.5, and 1.0 mg/kg, verticillin A succinate at the molar equivalent doses of 0.114, 0.285, 0.570, and 1.14 mg/kg, or vehicle (Tween80:Captex200:0.9% saline, 8:2:90 by volume). Treatments were administered by IP injection every 2 days for 14 days. Mouse weights were measured every 2 days. Mice that reached the study endpoint were euthanized approximately 2–4 h after the last dose. Blood was collected by cardiac puncture immediately after CO2 euthanasia. At necropsy, livers and tissues exhibiting grossly visible lesions were fixed in 10% neutral buffered formalin. Whole blood, serum, and fixed tissues were submitted to the Comparative Pathology and Digital Imaging Shared Resource at The Ohio State Comprehensive Cancer Center (OSUCCC, Columbus, OH, USA) for determinations of complete blood counts and serum chemistry, and histopathological evaluation of H&E-stained tissue sections. This study was conducted according to protocols approved by The Ohio State University Institutional Animal Care and Use Committee.

Mouse Pharmacokinetics.

The pharmacokinetics of verticillin A and verticillin A succinate after IV, IP, and PO administration were assessed in female C57BL/6 mice. The inlife portion of this study was performed at Charles River Laboratories, Inc. (Wilmington, MA, USA). Quantitative analysis of plasma samples was performed by the OSUCCC Pharmacoanalytical Shared Resource (Columbus, OH, USA). Mice (approximately 30 grams, n = 3 per group) were administered a single dose of verticillin A at 0.25 mg/kg (IV, IP) or 2.5 mg/kg (PO), or verticillin A succinate at 0.285 mg/kg (IV, IP) or 2.85 mg/kg (PO). Blood samples were collected serially into Li-heparin tubes at 0.083, 0.25, 0.5, 1, 2, 6, and 24 h after IV dosing, and at 0.25, 0.5, 1, 2, 4, 8, and 24 h after IP and PO dosing. Plasma samples were stored at −80 °C until shipped on dry ice to The Ohio State University for analysis.

LC/MS Detection of Verticillin A and Verticillin A Succinate.

HPLC grade methanol and acetonitrile and LC/MS grade formic acid were purchased from Fisher Scientific (Thermo Fisher Scientific, Fairlawn, NJ, USA). Deionized water was acquired from a Elga Purelab Flex 2 water purification system (Woodridge, IL, USA). ACS grade (≥99.9%) DMSO and hesperetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Blank mouse plasma lithium heparin was purchased from Innovative Research, Inc. (Novi, MI, USA).

Verticillin A and verticillin A succinate concentrations in mouse plasma were measured using a liquid chromatographytandem mass spectrometry (LC–MS/MS) method based on Wang et al. with modifications.30 Stock solutions of verticillin A and verticillin A succinate were prepared at 0.5 and 2 mg/mL in a mixture of acetonitrile and DMSO, respectively. An intermediate working stock solution was prepared containing 10,000 nM of each analyte. The calibrators and quality control (QC) samples were prepared by spiking blank mouse plasma with two times working stock solutions and 500 nM hesperetin internal standard solution for linear ranges of 1–1000 nM for verticillin A and 3–1,000 nM for verticillin A succinate. The QC concentrations were 3, 450, and 750 nM for verticillin A and 9, 450, and 750 nM verticillin A succinate. Briefly, 10 μL of sample was prepared with protein precipitation using cold methanol after initially spiking the sample with internal standard solution. The supernatant was dried under a stream of nitrogen and then reconstituted with an acetonitrile/water solution (1:1 v/v). The supernatant was transferred to 96-well autosampler plates, where 5 μL were injected by a Thermo Vanquish UHPLC (Waltham, MA, USA). The chromatographic separation was performed on an Agilent Zorbax Extend-C18 3.5 μm, 2.1 mm × 50 mm column (Santa Clara, CA, USA) coupled with a Thermo Betabasic C8 5 μm Javelin Guard at 40 °C using a 6 min gradient containing water and acetonitrile modified with formic acid. The flow rate was maintained at 0.4 mL/min and the autosampler temperature was set at 4 °C. The Vanquish UHPLC was paired with a TSQ Altis mass spectrometer equipped with a heated electrospray ionization source. Nitrogen was used for the sheath and auxiliary gases, while argon was utilized as the collision gas. The analytes were measured by selected reaction monitoring in positive ion mode at m/z of 697.212 → 615.208 for verticillin A, 797.212 → 615.208 for verticillin A succinate, and 303.138 → 177.125 for hesperetin.

Animal Xenograft Experiments.

All animals were treated in accordance with NIH Guidelines for the Care and Use of Laboratory Animals and the established Institutional Animal Use and Care protocol at the University of Illinois, Chicago. Xenograft studies utilized NCr nu/nu athymic female mice 6–8 weeks in age (Taconic). Mice were housed in a temperature and light-controlled environment (12 h light and 12 h dark) and provided with food and water ad libitum. For xenograft experiments, OVCAR8-RFP cells (5 × 106) were injected intraperitoneally (IP) per mouse and tumor growth was monitored using LagoX in vivo imaging system as previously described. Once all the mice formed tumors (~4 weeks), the mice were dosed with compounds solubilized in DMSO; verticillin A and verticillin D (dosage: 0.25 mg/kg), Taxol (0.5 mg/kg), and vehicle control (DMSO). Mice were imaged twice weekly (535 nm excitation, and 620 nm emission, Exposure time: 2sec, F stop: 2). Aura Living Image 4.0 software was used to quantify the total flux and normalization was performed using the vehicle control mice. After 4 weeks of treatment, all the animals were sacrificed.

Statistical Analysis.

Data presented are mean ± standard error of the mean (SEM) and represent at least three independent biological replicates. Statistical analysis was carried out using GraphPad Prism software 9.4.0. Statistical significance was determined by one-way analysis of variance (ANOVA), with Dunnett’s multiple comparisons to DMSO test as mentioned in figure legends. p < 0.05 was considered statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

RESULTS

In Vivo Tolerability of Verticillin A and Verticillin A Succinate.

Our previous work with verticillin A required encapsulation into an expansile nanoparticle (eNP) for optimal delivery in vivo and this also provided efficacy against the OVCAR8 model grown as an IP xenograft.24 However, our group also developed several semi-synthetic analogues of verticillin A, with the succinate version showing the most promise in terms of solubility.22 Therefore, sufficient verticillin A succinate was generated to perform a tolerability study in female athymic nude mice to identify tolerated doses to be used in subsequent efficacy studies in an ovarian cancer xenograft model and in an assessment of mouse pharmacokinetics. Dose levels were informed by previous efficacy and pharmacokinetic studies with verticillin A with molar equivalent doses chosen for the succinate form.30

The two lower dose levels of each compound (0.100 and 0.250 mg/kg of verticillin A and 0.114 and 0.285 mg/kg of verticillin A succinate) were well tolerated. Mice in these groups maintained their body weights throughout the study (Figure 1) with no remarkable findings at necropsy. In contrast, the two higher dose levels of each compound were poorly tolerated. These mice exhibited weight loss, poor body condition, and prominent abdominal distension secondary to accumulation of copious ascites fluid observed at necropsy, requiring early removal of mice in the 1.0 and 1.14 mg/kg groups at 8 days of treatment and those in the 0.5 and 0.570 mg/kg groups at day 13 of treatment. In addition to ascites, the primary finding at necropsy for these high dose groups was diffuse abdominal adhesions attaching abdominal viscera together and to the abdominal musculature and diaphragm.

Figure 1.

Figure 1.

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Changes in mouse body weight during the 14 day tolerability study. Female athymic nude mice were treated with verticillin A (left panel) or verticillin A succinate (right panel) at the indicated dose levels by IP injection every two days. The data for the single vehicle control group is shown in both graphs. Body weights (expressed as % change from initial [day 0] body weight) were measured every two days immediately before agent administration. Data are expressed as mean ± SD (n = 5 per treatment group).

Major histopathological findings were fibrin adhesions, fibrinosuppurative peritonitis, and pancreatitis associated with the two highest dose levels of each compound, consistent with the gross necropsy findings of abdominal adhesions and ascites fluid. Mice at the lower dose levels exhibited less severe effects with mildly increased neutrophils along the hepatic capsule in the 0.250 and 0.285 mpk dose groups and no histologic lesions at the lowest dose levels. The major finding from complete blood counts was a mild neutrophilia in animals treated with the higher doses, which was consistent with the inflammation observed in the peritoneal cavities of high-dose-treated mice (Table 1). Compound-related findings on the serum chemistry panel revealed minor elevations in γ-glutamyl transferase (GGT) and aspartate aminotransferase (AST), along with inconsistent increases in total bilirubin among the compound-treated mice (Table 2). The highest elevations in AST were associated with the higher, non-tolerated doses of the compounds and, in the absence of biochemical changes reflecting kidney and skeletal muscle, changes in GGT and bilirubin may be related to extrahepatic biliary obstruction linked to the observed peritoneal inflammation in compound-treated mice. Other biochemical findings revealed compound-associated decreases in several parameters relative to the vehicle control group (Table 2) that were also limited to the non-tolerated, higher dose levels of the compounds. Alkaline phosphatase (ALP), however, was significantly decreased in nearly all compound-treated groups compared to vehicle controls. The greatest decreases in ALP were associated with the higher doses, which were, nonetheless, within the reference range for mouse serum ALP (23–181 U/L) provided by the core that analyzed the samples.

Table 1.

Hematology valuesa of Athymic Nude Mice Treated with Verticillins

testb vehicle verticillin A (mg/kg) verticillin A succinate (mg/kg)
0.10 0.25 0.50 1.00 0.114 0.285 0.570 1.14
WBC # (K/μL) 5.18 ± 1.35 6.01 ± 1.29 4.22 ± 1.68 n.d.c 5.97 ± 1.67 6.52 ± 1.73 5.99 ± 1.11 n.d.c 6.80 ± 1.14
neutrophil # (K/μL) 1.62 ± 0.38 1.65 ± 0.15 2.30 ± 0.98 4.09 ± 1.30 2.33 ± 0.63 2.64 ± 0.54 4.26 ± 0.56
lymphocyte # (K/μL) 3.16 ± 0.90 3.94 ± 1.16 1.72 ± 0.76 1.63 ± 1.19 3.81 ± 1.09 3.12 ± 1.25 2.19 ± 0.61
monocyte # (K/μL) 0.38 ± 0.14 0.37 ± 0.09 0.18 ± 0.06 0.21 ± 0.29 0.36 ± 0.10 0.20 ± 0.08 0.18 ± 0.04
eosinophil # (K/μL) 0.01 ± 0.01 0.02 ± 0.02 0.02 ± 0.01 0.03 ± 0.01 0.01 ± 0.01 0.02 ± 0.02 0.10 ± 0.16
basophil # (K/μL) 0.01 ± 0.01 0.02 ± 0.01 0.01 ± 0.01 0.01 ± 0.01 0.01 ± 0.01 0.01 ± 0.01 0.07 ± 0.10
nucleated RBCs # (K/μL) 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
hematocrit (%) 42.9 ± 2.0 44.1 ± 1.4 43.5 ± 4.0 44.6 ± 3.4 39.7 ± 1.4 40.9 ± 1.2 44.7 ± 2.7
RBC (M/μL) 9.39 ± 0.49 9.89 ± 0.33 9.28 ± 0.81 9.79 ± 0.75 8.95 ± 0.15 9.15 ± 0.22 9.49 ± 0.49
hemoglobin (g/dL) 14.2 ± 0.8 14.4 ± 0.3 14.2 ± 1.3 15.0 ± 1.0 13.0 ± 0.2 13.5 ± 1.8 14.5 ± 0.42
MCV (fL) 45.7 ± 0.5 44.6 ± 0.2 46.8 ± 1.0 45.5 ± 0.6 44.4 ± 1.2 44.7 ± 0.9 47.2 ± 1.6
MCH (pg) 15.1 ± 0.3 14.6 ± 0.2 15.2 ± 0.4 15.4 ± 0.3 14.6 ± 0.2 14.7 ± 0.2 15.3 ± 0.4
MCHC (g/dL) 33.1 ± 0.7 32.7 ± 0.7 32.6 ± 1.2 33.7 ± 0.7 32.8 ± 0.6 32.9 ± 0.8 32.5 ± 1.1
RDW (%) 14.3 ± 0.4 13.8 ± 0.5 14.2 ± 0.4 16.2 ± 0.9 13.9 ± 0.4 14.1 ± 0.6 15.1 ± 0.2
Reticulocyte # (K/μL) 9.6 ± 14.4 0 ± 0 8.6 ± 8.3 0 ± 0 11.3 ± 12.9 6.3 ± 13.7 0 ± 0
reticulocyte (%) 0.11 ± 0.16 0 ± 0 0.10 ± 0.09 0 ± 0 0.13 ± 0.14 0.07 ± 0.15 0 ± 0
platelet # (K/μL) 996 ± 141 892 ± 74 964 ± 203 1057 ± 146 954 ± 118 938 ± 98 947 ± 271
PCT (%) 0.769 ± 0.080 0.701 ± 0.065 0.735 ± 0.147 0.815 ± 0.096 0.754 ± 0.093 0.722 ± 0.072 0.701 ± 0.202
MPV (fL) 7.8 ± 0.4 7.9 ± 0.1 7.6 ± 1.1 7.7 ± 0.2 7.9 ± 0.2 7.7 ± 0.07 7.4 ± 0.1
PDW (%) 47.2 ± 2.4 47.4 ± 1.9 48.7 ± 0.8 47.3 ± 1.7 47.5 ± 2.5 48.2 ± 2.2 51.5 ± 1.4
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a

Values represent means ± SD (n = 3–5).

b

Abbreviations: WBC: white blood cells; RBC: red blood cells; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; RDW: Red cell distribution width; PCT: plateletcrit; MPV: mean platelet volume; PDW: platelet distribution width; n.d.: not determined.

c

Sample quality inadequate for analysis.

Table 2.

Serum Chemistry valuesa of Athymic Nude Mice Treated with Verticillins

testb vehicle verticillin A (mg/kg) verticillin A succinate (mg/kg)
0.10 0.25 0.50 1.00 0.114 0.285 0.570 1.14
ALT (U/L) 48 ± 13 38 ± 6 46 ± 13 55 ± 23 44 ± 8 39 ± 6 47 ± 13 48 ± 25 121 ± 145
AST (U/L) 102 ± 15 98 ± 16 112 ± 19 201 ± 51 225 ± 68 88 ± 10 125 ± 18 254 ± 138 420 ± 438*
ALP (U/L) 201 ± 19 163 ± 27 141 ± 14** 92 ± 19**** 70 ± 61**** 139 ± 9** 133 ± 19*** 89 ± 13**** 74 ± 12****
GGT (U/L) 2 ± 0 5 ± 0 3 ± 1 7 ± 2** 7 ± 3** 8 ± 0** 0 ± 1 4 ± 3 6 ± 4
total bilirubin (mg/dL) 0.3 ± 0.1 0.4 ± 0.1 0.3 ± 0.0 0.4 ± 0.2 0.3 ± 0.0 0.3 ± 0.1 0.3 ± 0.0 0.4 ± 0.1 0.5 ± 0.3
BUN (mg/dL) 23 ± 1 18 ± 3 17 ± 4 26 ± 3 25 ± 4 20 ± 1 15 ± 1 24 ± 8 24 ± 4
creatinine (mg/dL) 0.48 ± 0.06 0.56 ± 0.03 0.21 ± 0.7 0.36 ± 0.03* 0.30 ± 0.12*** 0.43 ± 0.02 0.42 ± 0.8 0.29 ± 0.05*** 0.26 ± 0.05****
calcium (mg/dL) 11.3 ± 0.6 11.8 ± 0.4 11.6 ± 0.5 11.7 ± 0.4 11.4 ± 0.4 11.8 ± 0.3 11.2 ± 0.7 11.5 ± 0.9 7.2 ± 5.3**
phosphorous (mg/dL) 14.3 ± 2.3 12.8 ± 1.1 14.0 ± 1.0 16.4 ± 2.3 10.6 ± 6.8 11.7 ± 0.8 11.9 ± 0.8 15.5 ± 1.4 12.2 ± 1.0
albumin (g/dL) 3.6 ± 0.1 3.5 ± 0.3 3.3 ± 0.2 3.2 ± 0.2 2.1 ± 0.9*** 3.7 ± 0.2 3.4 ± 0.2 2.6 ± 0.9* 2.4 ± 0.8**
total protein (g/dL) 5.7 ± 0.3 5.6 ± 0.4 5.5 ± 0.4 5.3 ± 0.3 5.0 ± 0.4* 5.8 ± 0.3 5.4 ± 0.3 5.2 ± 0.4 4.9 ± 0.4**
globulin (g/dL) 2.1 ± 0.2 2.2 ± 0.4 2.1 ± 0.3 2.1 ± 0.2 2.9 ± 1.0 2.1 ± 0.2 2.0 ± 0.2 2.6 ± 1.1 2.5 ± 0.8
cholesterol (mg/dL) 132 ± 14 144 ± 16 141 ± 10 124 ± 22 90 ± 26** 141 ± 12 146 ± 13 115 ± 23 111 ± 10
glucose (mg/dL) 197 ± 27 191 ± 42 187 ± 33 176 ± 19 116 ± 17* 225 ± 28 235 ± 10 172 ± 49 141 ± 62
lipase (U/L) 95 ± 21 43 ± 15 71 ± 4 n.d. 52 ± 22 55 ± 44 74 ± 5 n.d 84 ± 64
amylase (U/L) 1339 ± 468 1153 ± 176 1189 ± 89 1397 ± 318 1213 ± 149 1039 ± 215 1208 ± 75 1203 ± 240 1221 ± 520
creatine kinase (U/L) 265 ± 48 241 ± 85 237 ± 60 302 ± 65 498 ± 184 160 ± 37 214 ± 28 1916 ± 1915** 582 ± 384
triglyceride (mg/dL) 223 ± 48 202 ± 42 236 ± 52 187 ± 139 114 ± 20 247 ± 70 262 ± 68 128 ± 38 266 ± 160
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a

Values represent means ± SD (n = 4–5).

Differences from the Vehicle group are significant at *p < 0.05

**

p < 0.01

***

p < 0.001, and

****

p < 0.0001 by one-way ANOVA followed by Dunnett’s multiple comparisons test.

b

Abbreviations: ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase; GGT: γ-glutamyl transferase; BUN: blood urea nitrogen; n.d.: not determined.

Pharmacokinetics of Verticillin A and Verticillin A Succinate.

Unfortunately, verticillin A succinate showed poor stability in mouse plasma lithium heparin. For initial PK time points with IV dosing, all sample results were below the limit of quantitation (BLOQ) or 3 nM. Additionally, verticillin A succinate QC extracts were not stable in the 4 °C autosampler after approximately 19 h. Supplemental QCs were prepared with 500 nM of verticillin A and verticillin A succinate in mouse plasma to account for samples with inadequate volume (less than 10 μL). The 500 nM QCs were prepared by combining 5 μL of the QC with 5 μL of blank mouse plasma to give a final in-well concentration of 250 nM. The verticillin A succinate concentration only met method criteria of ±15% accuracy on the day of QC preparation or when analyte was spiked at low volume into mouse plasma. When the supplemental QCs were extracted and analyzed in the subsequent day(s), which included freezing and thawing, the verticillin A succinate concentration did not meet the method accuracy criteria.

The verticillin A mouse plasma samples were within the linear range (1–1000 nM) for mice dosed by IV and IP, but most results were just above the lower limit, which was broadly consistent with prior evaluations of verticillin A pharmacokinetics30 given the lower dose levels administered in our studies. Oral gavage samples were BLOQ for verticillin A. The additional LC–MS/MS method development required to measure verticillin A across these lower dose levels was not pursued given the limits of tolerability at these doses.

Additional Exploration of Verticillin A-Succinate Stability.

Given the translational limitations of administering DMSO-based formulations, we explored an aqueous microemulsion but found issues with the stability of the succinate form.31 Verticillin A succinate formulation samples at final concentrations of 0.1, 0.05, and 0.025 mg/mL, based approximately on the dosing solution concentrations used in the above tolerability and pharmacokinetic studies, were prepared and analyzed using a UPLC-PDA-HRMS. These data indicated that verticillin A succinate is not stable in the Tween80:Captex200P:0.9% saline formulation, as it degrades almost immediately to verticillin A (Supporting Information Figure S1). When assessed immediately after preparation, the verticillin A succinate levels were either below the quantitation limit (BQL) or not found/detected (NF) at all concentration levels tested. These readings persisted even up to 4 h after preparation, when all sample readings were below the detection limit (BDL) or NF (Supporting Information Figure S2). These data support the hypothesis that the mice treated with verticillin A succinate formulations were, in fact, treated with the formulation breakdown product, verticillin A. This would explain the similar findings for tolerated doses and toxicities of the two verticillin-based formulations, as both treatment groups were technically administered the same active compound.

Verticillin D Induces Cytotoxicity in HGSOC Cell Lines In Vitro.

Because the succinate form was unstable, we recognized the need to ultimately develop new semi-synthesis or formulation approaches to improve the in vivo efficacy of verticillins. Verticillin D is produced in high abundance from the fungus and could provide a starting material for synthesis; due to the two additional hydroxy moieties, relative to verticillin A, verticillin D is also more soluble. Verticillin D was shown previously to inhibit the growth of the murine lymphoma L5178Y and A2780 human ovarian cancer cell lines.32 To explore its specificity for HGSOC, verticillin D was tested in OVCAR3, MDA-MB-435, and MDA-MB-231 as well as OVCAR4 and OVCAR8 cell lines (Supporting Information Figure S3) where it was cytotoxic to ovarian cancer cells at nanomolar concentrations. In these experiments, taxol was used as positive control and dimethylsulfoxide (DMSO) as vehicle control. Verticillin D was found to inhibit the growth of all three cell lines with average IC50 values of 34.2, 35.3, and 12.2 nM in OVCAR3, MDA-MB-435, and MDA-MB-231, respectively (Figure 2A). Furthermore, to determine if verticillin D reduced viability of tumor spheroids, OVCAR8 spheroids were generated and treated with vehicle (DMSO), verticillin A, verticillin A succinate, verticillin D, and taxol. A spheroid viability assay was performed, and it was found that verticillin D reduced spheroid viability similar to verticillin A, with an IC50 value of 1166 nM (Figure 2B).

Figure 2.

Figure 2.

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(A) OVCAR3, MDA-MB-435, and MDA-MB-231 cells were treated with vehicle (DMSO), verticillin A, verticillin A succinate, verticillin D, and chemotherapeutic control taxol for 72 h. Dose–response curves were generated and normalized to vehicle control. IC50 values are denoted in the table. (B) OVCAR8 spheroids were treated with vehicle, verticillin A, verticillin A succinate, verticillin D, and chemotherapeutic control taxol for 7 days. Spheroid viability was determined using CellTiter-Glo Cell Viability assay. IC50values are denoted in the table. Data represents mean ± SEM from three biological replicates.

In order to determine whether the growth inhibition was due to cytotoxic effects exerted by verticillin D, a 2D foci assay was performed. Cells were treated with verticillin D for 8 h, media washed away, and cells without drug were incubated for 2 weeks to form colonies. As shown in Figure 3, treatment with verticillin A, verticillin A succinate, and verticillin D completely abrogated foci formation in OVCAR4 and OVCAR8 cells, relative to vehicle control, suggesting a cytotoxic effect and not a cytostatic effect.

Figure 3.

Figure 3.

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OVCAR8 and OVCAR4 cells were treated with vehicle and verticillin A (50 nM), verticillin A succinate (50 nM), verticillin D (50 nM), and chemotherapeutic control taxol (10 nM) for 8 h. Following drug incubation, media was changed, and cells were incubated for two weeks to form colonies. Representative images of 2D foci assay performed in OVCAR4 and OVCAR8 cells are shown. Each experiment was performed in three biological replicates, and data represent mean ± SEM. Significance was tested by Dunnett’s multiple comparisons test in comparison to vehicle control.

Verticillin D Did Not Demonstrate Reduced Liver Toxicity Compared to Verticillin A.

OVCAR8-RFP cells were xenografted IP into female athymic nude mice to determine whether verticillin D affected tumor burden in vivo, in comparison to verticillin A as described in previous studies.24 Tumors were allowed to form for 4 weeks and once all the mice displayed detectable tumors, the animals were treated with verticillin D (0.25 mg/kg), verticillin A (0.25 mg/kg), and vehicle control DMSO twice weekly for four weeks. Animals were weighed weekly and imaged to quantify fluorescent intensity from the RFP stably integrated into the OVCAR8 model. Animals treated with verticillin D presented gross morphological liver damage and fused organs relative to verticillin A and vehicle control, suggesting a non-specific cytotoxic effect in vivo (Figure 4A). Tumor burden was reduced in verticillin A, verticillin D, and taxol treated mice compared to DMSO (Figure 4B).

Figure 4.

Figure 4.

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(A) Representative images show liver damage in verticillin D treated animals. OVCAR8-RFP cells were xenografted IP to form tumors. Mice were dosed twice weekly with verticillin A and verticillin D (dosage: 0.25 mg/kg), Taxol (0.5 mg/kg), and vehicle control (DMSO). (B) Quantification of tumor burden (total flux as measured with Aura imaging) normalized to vehicle control. Statistics were performed using One-way ANOVA with Dunnett’s posthoc relative to vehicle control.

DISCUSSION

HGSOC is the fifth leading cause of cancer-related female deaths and the most common and lethal form of ovarian cancer due to its asymptomatic nature and late-stage diagnosis.33 One strategy to identify new chemotherapies is by exploring novel natural product anticancer agents, such as the verticillins.34,35 These fungal metabolites belong to a class of ETP alkaloids and were first described >50 years ago with the discovery of verticillin A.21 More recently, they have gained attention due to their potent activity against cancer cells.20,24

Recently, our collaborators developed methods to supply these compounds via optimized fermentations28 and the mechanism of action of verticillin A has been actively investigated. Verticillin A demonstrated cytotoxicity in HGSOC cell lines in a dose-dependent manner with a low nanomolar IC50. However, due to its low solubility, a nanoparticle-based drug delivery system was used resulting in better solubility and reduced liver toxicity with verticillin A treatment.24,36,37 In addition, verticillin A was then used as a feedstock to generate semi-synthetic analogues, such as verticillin A succinate, with the goals of improving solubility and pharmacodynamic properties.22

Our assessment of verticillin A and verticillin A succinate tolerability identified a maximal tolerated IP dose of verticillin A (0.25 mg/kg) that was used for the evaluation of efficacy in the OVCAR-8 xenograft model, in which it was tolerated. It is worth noting that the dose-limiting toxicities observed after IP dosing of verticillin A could feasibly be attributed to the route of administration and consequent local inflammation within the peritoneal cavity. Thus, it is worth considering whether non-IP routes of administration would permit the use of higher dose levels to achieve higher tissue level exposures and potentially enhanced antitumor efficacy. In addition, the limited pharmacokinetic data we generated following extravascular verticillin A dosing suggests that dose limiting toxicities prevented dosing high enough to achieve therapeutic levels of verticillin A in our studies.

Furthermore, with the initial discovery of verticillin D, moderate cytotoxic activity against the mouse lymphoma cells (L5178Y) was reported in 2012.32,38 However, based on our studies, the cytotoxic properties of verticillin D were evaluated against OVCAR3, MDA-MB-435, and MDA-MB-231, retaining IC50 values in the nanomolar range, comparable to verticillin A and verticillin A succinate, which also showed pronounced cytotoxic activities. Further, these nanomolar activities are closer to what has been reported from multiple analogs and in other cell lines.39–41

To evaluate the ability of verticillin D to reduce in vivo tumor burden, OVCAR8-RFP cells were xenografted following previous protocols.24 Both verticillin A and D reduced tumor burden compared to control. Verticillin D exhibited liver toxicity causing organ fusion. Verticillin A did not demonstrate changes in liver morphology. An adequate supply of verticillin D now exists to explore the pharmacologic potential of this drug lead more fully as well as for the semi-synthesis of other analogs. We hypothesize that the higher solubility of verticillin D, which serves to benefit the delivery of the compound, may have contributed to the morphological damages that were observed. Thus, next generation analogues will explore developing ways to meter the dosing of this analogue, such that cytotoxicity to tumor cells can be improved, which concomitantly minimizes general toxicity.

This study confirmed that verticillin D has cytotoxic activity; however, further toxicology studies are required. In our studies, eNPs have been used successfully to deliver verticillin A. Verticillin A-eNP treated animals showed significant reduction in tumor burden in comparison to the eNP treated animals.24 Similarly, to overcome the toxic effects also induced by verticillin D, a well-characterized nanoparticle-based drug delivery system could be tested to determine if this would overcome the toxicity observed, enabling an optimized effective formulation worthy of large animal pharmacokinetic studies.

CONCLUSIONS

In conclusion, verticillin D shows potent nanomolar cytotoxic activity against HGSOC cell lines comparable to verticillin A. In addition to in vitro cytotoxic activity, in vivo experiments with these compounds show reduced tumor burden compared to vehicle. However, verticillin D treated mice had fused organs and showed liver damage while verticillin A did not detect any toxicity. Verticillin A and verticillin A succinate were tested in a unique formulation to monitor bioavailability in vivo and the 0.25 mg/kg dosing was found to be well tolerated in verticillin A. Verticillin A succinate was unstable in vitro based on LCMS data and pharmacokinetic studies showed similar clearance and expression of serum biomarkers for liver and kidney toxicity to verticillin A. The semi-synthetic version lost the succinate functional group in solution as observed in the stability studies and the mice were likely exposed primarily to the breakdown product, verticillin A. Therefore, formulation and stability studies are effective ways to improve tolerability and demonstrate efficacy of verticillins.

Supplementary Material

supplemental

ACKNOWLEDGMENTS

This work was supported in part by the National Institutes of Health via grants P01 CA125066 awarded to J.E.B. and N.H.O., K12 GM139186 to E.N.K., and T32 AT008938 to H.C.P. We also acknowledge Coordenação de Aperfeiçoamento de Pessoal de NívelSuperior (CAPES) [Finance Code 001], Fundação de Amparo à Pesquisa no Estado de São Paulo (FAPESP) [grant numbers 2019/11563-2, 2021/08535-7 to J.M.B. Additional support for the research was provided by the Comparative Pathology and Digital Imaging Shared Resource and the Pharmacoanalytical Shared Resource at The Ohio State University Comprehensive Cancer Center, Columbus, OH (NIH P30 CA016058).

Footnotes

The authors declare no competing financial interest.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.3c00069.

(PDF)

Complete contact information is available at: https://pubs.acs.org/10.1021/acs.molpharmaceut.3c00069

Contributor Information

Elizabeth N. Kaweesa, Department of Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, Illinois 60607, United States.

Jaqueline M. Bazioli, Department of Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, Illinois 60607, United States.

Herma C. Pierre, Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, North Carolina 27412, United States

Daniel D. Lantvit, Department of Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, Illinois 60607, United States

Samuel K. Kulp, Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, Ohio 43210, United States

Kasey L. Hill, Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, North Carolina 27412, United States

Mitch A. Phelps, Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, North Carolina 27412, United States

Christopher C. Coss, Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, North Carolina 27412, United States

James R. Fuchs, Division of Medicinal Chemistry and Pharmacognosy, The Ohio State University, Columbus, Ohio 43210, United States

Cedric J. Pearce, Mycosynthetix, Inc., Hillsborough, North Carolina 27278, United States

Nicholas H. Oberlies, Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, North Carolina 27412, United States

Joanna E. Burdette, Department of Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, Illinois 60607, United States

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