The Rho GTPases Rac and Cdc42 are potential targets against metastatic diseases. We characterized the small molecule MBQ-167 as an effective dual Rac/Cdc42 inhibitor that reduces HER2-type tumor growth and metastasis in mice by ∼90%. This study reports the pharmacokinetics and tissue distribution of MBQ-167 following intraperitoneal and oral single-dose administrations. We first developed and validated a bioanalytical method for the quantitation of MBQ-167 in mouse plasma and tissues by supercritical fluid chromatography coupled with electrospray ionization tandem mass spectrometry. MBQ-167 was rapidly distributed into the kidneys after intraperitoneal dosing, whereas oral administration resulted in higher distribution to lungs. The elimination half-lives were 2.17 and 2.6 h for the intraperitoneal and oral dosing, respectively. The relative bioavailability of MBQ-167 after oral administration was 35%. This investigation presents the first analysis of the pharmacokinetics of MBQ-167 and supports further preclinical evaluation of this drug as a potential anticancer therapeutic.

Metastatic breast cancer is the second most common cancer in US women. The mortality rate has decreased approximately 40% in the last 25 years due to advancement in treatment and diagnostics. However, it is estimated that 20–30% of all breast cancer patients will develop metastatic breast cancer. Therefore, there is a need to develop targeted therapeutics to reduce cancer cell migration and subsequent metastasis. The novel small molecule MBQ‐167 inhibits Rac and Cdc‐42, proteins involved in cancer cell growth, migration and invasion. In vivo studies have shown that MBQ‐167 inhibits mammary tumor growth and metastasis in immunocompromised mice by ~90% (Humphries‐Bickley, et al., 2017). However, to continue validating this drug for FDA approval, it is necessary to study the pharmacokinetic profile of the drug, which includes elucidating the tissue distribution of the compound in the body. Hence, it is necessary to develop a rapid and sensitive method to quantify MBQ‐167 in tissues. The purpose of this study was to develop and validate a method for the quantification of MBQ‐167 in mouse tissues using Supercritical fluid chromatography coupled with electrospray ionization tandem mass spectrometry (SFC‐MS/MS). Mouse livers were homogenized, spiked with MBQ‐167, and liquid‐liquid extractions were performed with different organic solvents (heptane, ethyl acetate, 2‐propanol, toluene, and acetonitrile), in order to determine the optimal sample preparation method to selectively separate the drug MBQ‐167 from endogenous material in mouse tissue. Separation was performed on an ACQUITY UPC2BEH (3.0 × 100 mm, 1.7 μm) column at 40 °C with a mobile phase consisting of CO2 and methanol 0.1 % formic acid (95:5, v/v) at a flow rate of 1 mL/min. A tandem triple quadrupole mass spectrometer was operated in multiple reaction monitoring (MRM) mode. High recovery of MBQ‐167 (> 65%) and lower matrix effects were observed upon extraction with heptane:ethyl acetate (1:1). Consistent recovery was observed in other tissues such as kidney, lungs and heart. The method was sensitive with a lower limit of quantification (LLOQ) of 10 ng/mL. Linearity was observed over the concentration range of 10–1000 ng/mL. The data obtained supports the use of SFC‐MS/MS as a highly specific method to evaluate the tissue distribution of MBQ‐167 in mice.Support or Funding InformationThis study is supported by MARC grant NIH/NIGMS 5T34GM007821‐39 (to GRG) and NIGMS‐RISE 25 GM061838 (to MM).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Rho GTPases Rac and Cdc42 are potential targets against metastatic disease. Our group characterized the small molecule MBQ-167 as an effective dual Rac/Cdc42 inhibitor that reduces HER2 positive and triple negative tumor growth and metastasis in mice by ~90%. To continue validating this drug for FDA approval, we recently elucidated the plasma pharmacokinetics and the tissue distribution of MBQ-167 in mice (Maldonado et al., 2019). However, further studies are needed to characterize the tumor pharmacokinetics of MBQ-167 to determine drug exposure, time-to-peak, and the length of time that MBQ-167 remains in tumors after administration. The purpose of this study was to determine the tumor pharmacokinetics of MBQ-167 in a single dose input scheme (10 mg/kg BW) following intraperitoneal (IP) administration. Thirty-five female BALB/c mice (5 mice/group) were injected with 2.5 × 105 4T1 murine metastatic breast cancer cells at the mammary fat pad. After tumor establishment, a single dose of MBQ-167 (10 mg/kg BW) was administered via IP. Tumors were collected at 0.5, 1, 3, 6, 9,12, and 24 hours following drug administration. We used a validated bioanalytical method using supercritical fluid chromatography coupled with tandem mass spectrometry (SFC-MS/MS) to quantify MBQ-167 in tumors. Pharmacokinetic parameters were obtained by non-compartmental analysis using WinNolin® software. Pharmacokinetic analysis revealed that MBQ-167 has an elimination half-life (t1/2) of 8.64 hours in tumors, which is much longer than its half-life in plasma (2.17 hours), indicating preferential absorption and retention of this lipophilic small molecule in tumor tissues. The area under the curve (AUC0-t) was 4.39 %ID⋅hr/g with a maximum concentration (Cmax) of 1268.9 ng/g in tumors. Moreover, the time-to-peak concentration in tumors was 0.5 hours. Hence, this study supports the continued development of MBQ-167 as a potential anti-cancer therapeutic. Citation Format: Maria Del Mar Maldonado, Ailed M. Cruz-Collazo, Luis D. Borrero-García, Gabriela T. Rosado-González, Jean F. Ruiz-Calderon, Jorge Duconge, Suranganie Dharmawardhane. Tumor pharmacokinetics of the metastatic cancer inhibitor MBQ-167 in mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3020.

Nuclear RNA quality control (QC) pathways degrade potentially deleterious transcripts arising from cryptic transcription. These QC pathways also target RNAs that are generated by improper mRNA capping, splicing, 3′ end formation or export. Kaposi’s sarcoma‐associated herpesvirus (KSHV) is a double‐stranded DNA virus whose transcripts avoid many of host RNA QC pathways, and it is used as a model to study mammalian cells RNA decay systems. One of the ways KSHV ensures this is by encoding ORF57 to protect viral transcripts from RNA decay systems. Previous pulse‐chase assay experiments with an unstable nuclear KSHV RNA demonstrated higher steady state KSHV RNA in presence of ORF57. Moreover, we recently found that the host proteins PPP1R10 (PNUTS) and CDK12 play important roles in controlling the steady‐state viral RNA levels. However, it was unknown if these processes work in the same pathway. The purpose of this study was to determine a possible synergistic or independent association between ORF57, PNUTS & CDK12 proteins in RNA stability. To accomplish this, PNUTS or CDK12 were knocked down with siRNAs in 293A‐TOA cells, followed by transient transfection of an ORF57‐responsive ORF59‐GFP reporter. The ORF59‐GFP reporter was co‐transfected with ORF57 or an empty vector control. We assayed RNA levels of the ORF59‐GFP reporter by northern blotting after PNUTS or CDK12 knockdown in the presence or absence of ORF57. Western blot verified that PNUTS and CDK12 were efficiently knocked down in cells treated with siRNA. Depletion of PNUTS or CDK12 resulted in higher steady‐state levels of ORF59 compared to controls. However, depletion of PNUTS or CDK12 in the presence of ORF57 resulted in higher steady‐state levels of ORF59. Our results suggest that PNUTS and CDK12 regulates RNA by a distinct mechanism than ORF57. Because ORF57 protects ORF59 from RNA QC pathways, we conclude that CDK12 and PNUTS do not affect RNA stability, but likely affect transcription or RNA processing.