Reversible chemoresistance of pancreatic cancer grown as spheroids.

Better in vitro models are needed to identify active drugs to treat pancreatic adenocarcinoma (PAC) patients. We used 3D hanging drop cultures to produce spheroids from five PAC cell lines and tested nine FDA-approved drugs in clinical use. All PAC cell lines in 2D culture were sensitive to three drugs (gemcitabine, docetaxel and nab-paclitaxel), however most PAC (4/5) 3D spheroids acquired profound chemoresistance even at 10 µM. In contrast, spheroids retained sensitivity to the investigational drug triptolide, which induced apoptosis. The acquired chemoresistance was also transiently retained when cells were placed back into 2D culture and six genes potentially associated with chemoresistance were identified by microarray and confirmed using quantitative RT-PCR. We demonstrate the additive effect of gemcitabine and erlotinib, from the 12 different combinations of nine drugs tested. This comprehensive study shows spheroids as a useful multicellular model of PAC for drug screening and elucidating the mechanism of chemoresistance.

Entinostat as a combinatorial therapeutic for rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. For the alveolar subtype (ARMS), the presence of the PAX3::FOXO1 fusion gene and/or metastases are strong predictors of poor outcome. Metastatic PAX3::FOXO1+ ARMS often responds to chemotherapies initially, only to subsequently relapse and become resistant with most patients failing to survive beyond 8 years post-diagnosis. No curative intent phase II or phase III clinical trial has been available for patients in the past 10 years (ARST0921). Thus, metastatic ARMS represents a significantly unmet clinical need. Chemotherapy resistance in ARMS has previously been attributed to PAX3::FOXO1-mediated cell cycle checkpoint adaptation, which is mediated by an HDAC3-SMARCA4-miR-27a-PAX3::FOXO1 circuit that can be disrupted by HDAC3 inhibition. In this study, we investigated the therapeutic efficacy of combining the epigenetic regulator entinostat, a Class I Histone Deacetylase (HDAC1-3) inhibitor, with RMS-specific chemotherapies in patient derived xenograft (PDX) models of RMS. We identified single agent, additive or synergistic relationships between relapse-specific chemotherapies and clinically relevant drug exposures of entinostat in three PAX3::FOXO1+ ARMS mouse models. This preclinical data provides further rationale for clinical investigation of entinostat, already known to be well tolerated in a pediatric phase I clinical trial (ADVL1513).

High-Dose Intravenous Vitamin C Combined with Docetaxel in Men with Metastatic Castration-Resistant Prostate Cancer: A Randomized Placebo-Controlled Phase II Trial.

High-dose intravenous vitamin C (HDIVC) administered to produce pharmacologic concentrations shows promise in preclinical models and small clinical trials, but larger prospective randomized trials are lacking. We evaluated the clinical benefit of combining HDIVC with docetaxel in patients with progressive metastatic castration-resistant prostate cancer (mCRPC). In this double-blind, placebo-controlled phase II trial, 47 patients were randomized 2:1 to receive docetaxel (75 mg/m2 i.v.) with either HDIVC (1 g/kg) or placebo. Coprimary endpoints were PSA50 response and adverse event rates. Secondary endpoints included overall survival, radiographic progression-free survival, and quality of life measured using the Functional Assessment of Cancer Therapy-Prostate instrument. Correlative analyses included pharmacokinetics and oxidative stress markers. Eighty-nine percent of patients previously had three or more lines of therapy. The PSA50 response rate was 41% in the HDIVC group and 33% in the placebo group (P = 0.44), with comparable adverse event rates in both groups. There were no significant differences in Functional Assessment of Cancer Therapy-Prostate scores. The median radiographic progression-free survival was not significantly different between the HDIVC and placebo groups, with durations of 10.1 and 10.0 months (HR, 1.35; 95% confidence interval, 0.66-2.75; P = 0.40), respectively. The median overall survival was 15.2 months in the HDIVC group and 29.5 months in the placebo group (HR, 1.98; 95% confidence interval, 0.85-4.58; P = 0.11). HDIVC did not decrease F2-isoprostanes, indicators of oxidative stress. The study was suspended after prespecified interim analysis indicated futility in achieving primary endpoints. In this patient population, combining HDIVC with docetaxel did not improve PSA response, toxicity, or other clinical outcomes compared with docetaxel alone. Findings do not support the routine use of HDIVC in mCRPC treatment outside of clinical trials. SIGNIFICANCE: This is the first randomized, placebo-controlled, double-blind trial to evaluate HDIVC in cancer treatment. The addition of HDIVC to docetaxel in patients with mCRPC does not improve PSA response, toxicity, or other clinical outcomes compared with docetaxel alone. The routine use of HDIVC in mCRPC treatment is not supported outside of clinical trials.

Locally administered nanosuspension increases delivery of estradiol for the treatment of vaginal atrophy in mice.

Vaginal atrophy affects up to 57% of post-menopausal women, with symptoms ranging from vaginal burning to dysuria. Estradiol hormone replacement therapy may be prescribed to alleviate these symptoms, though many vaginal products have drawbacks including increased discharge and local tissue toxicity due to their hypertonic nature. Here, we describe the development and characterization of a Pluronic F127-coated estradiol nanosuspension (NS) formulation for improved vaginal estradiol delivery. We compare the pharmacokinetics to the clinical comparator vaginal cream (Estrace) and demonstrate increased delivery of estradiol to the vaginal tissue. We utilized ovariectomized (OVX) mice as a murine model of post-menopausal vaginal atrophy and demonstrated equivalent efficacy in vaginal re-epithelialization when dosed with either the estradiol NS or Estrace cream. Further, we demonstrate compatibility of the estradiol NS with vaginal bacteria in vitro. We demonstrate that a Pluronic F127-coated estradiol NS may be a viable option for the treatment of post-menopausal vaginal atrophy.

Nelfinavir inhibition of Kaposi's sarcoma-associated herpesvirus protein expression and capsid assembly.

BACKGROUND: Antiviral therapies that target herpesviruses are clinically important. Nelfinavir is a protease inhibitor that targets the human immunodeficiency virus (HIV) aspartyl protease. Previous studies demonstrated that this drug could also inhibit Kaposi's sarcoma-associated herpesvirus (KSHV) production. Our laboratory demonstrated nelfinavir can effectively inhibit herpes simplex virus type 1 (HSV-1) replication. For HSV-1 we were able to determine that virus capsids were assembled and exited the nucleus but did not mature in the cytoplasm indicating the drug inhibited secondary envelopment of virions. METHODS: For KSHV, we recently derived a tractable cell culture system that allowed us to analyze the virus replication cycle in greater detail. We used this system to further define the stage at which nelfinavir inhibits KSHV replication. RESULTS: We discovered that nelfinavir inhibits KSHV extracellular virus production. This was seen when the drug was incubated with the cells for 3 days and when we pulsed the cells with the drug for 1-5 min. When KSHV infected cells exposed to the drug were examined using ultrastructural methods there was an absence of mature capsids in the nucleus indicating a defect in capsid assembly. Because nelfinavir influences the integrated stress response (ISR), we examined the expression of viral proteins in the presence of the drug. We observed that the expression of many were significantly changed in the presence of drug. The accumulation of the capsid triplex protein, ORF26, was markedly reduced. This is an essential protein required for herpesvirus capsid assembly. CONCLUSIONS: Our studies confirm that nelfinavir inhibits KSHV virion production by disrupting virus assembly and maturation. This is likely because of the effect of nelfinavir on the ISR and thus protein synthesis and accumulation of the essential triplex capsid protein, ORF26. Of interest is that inhibition requires only a short exposure to drug. The source of infectious virus in saliva has not been defined in detail but may well be lymphocytes or other cells in the oral mucosa. Thus, it might be that a "swish and spit" exposure rather than systemic administration would prevent virion production.

Validation of a robust and rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis of VK-2019, a selective EBNA1 inhibitor.

EBNA1 is an Epstein Barr virus (EBV) protein expressed in all EBV-associated cancers. EBNA1 plays a critical role in the replication and maintenance of EBV episomes in latently infected cells. VK-2019 was developed as a highly specific inhibitor of EBNA1 DNA binding activity and is currently in phase 1 development as a treatment for EBV-associated carcinomas. A sensitive and reliable method was developed to quantify VK-2019 in human plasma using liquid chromatography with tandem mass spectrometry to perform detailed pharmacokinetic studies. VK-2019 was extracted from plasma using protein precipitation with acetonitrile. Separation of VK-2019, two purported metabolites, and the internal standard, VK-2019-d6, was achieved with a Zorbax XDB C18 column using a gradient flow over 6 min. VK-2019 was detected using a SCIEX 4500 triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The assay range was 0.5-500 ng/mL and proved to be accurate and precise. Dilutions of 1:10 were accurately quantified. VK-2019 was stable in plasma at -70°C for approximately 18 months. The method was applied to assess the total plasma concentrations of VK-2019 in a patient who received a single and multiple oral daily doses of 120 mg.

Nelfinavir Inhibition of Kaposi's sarcoma-associated herpesvirus protein expression and capsid assembly.

Background: Antiviral therapies that target herpesviruses are clinically important. Nelfinavir is a protease inhibitor that targets the human immunodeficiency virus (HIV) infections aspartyl protease. Previous studies demonstrated that this drug could also inhibit Kaposi's sarcoma-associated herpesvirus (KSHV) production. Our laboratory demonstrated nelfinavir can effectively inhibit herpes simplex virus type 1 (HSV-1) replication. For HSV-1 we were able to determine that virus capsids were assembled and exited the nucleus but did not mature in the cytoplasm indicating the drug inhibited secondary envelopment of virions. Methods: For KSHV, we recently derived a tractable cell culture system that allowed us to analyze the virus replication cycle in detail. We used this system to further define the stage at which nelfinavir inhibits KSHV replication. Results: We discovered that nelfinavir inhibits KSHV extracellular virus production. This was seen when the drug was incubated with the cells for 3 days and when we pulsed the cells with the drug for 1-5 minutes. When KSHV infected cells exposed to the drug were examined using ultrastructural methods there was an absence of mature capsids in the nucleus indicating a defect in capsid assembly. Because nelfinavir influences the integrated stress response (ISR), we examined the expression of viral proteins in the presence of the drug. We observed that the expression of many were significantly changed in the presence of drug. The accumulation of the capsid triplex protein ORF26 was markedly reduced. This is an essential protein required for herpesvirus capsid assembly. Conclusions: Our studies confirm that nelfinavir inhibits KSHV virion production by disrupting virus assembly and maturation. Of interest is that inhibition requires only a short exposure to drug. The source of infectious virus in saliva has not been defined in detail but may well be lymphocytes or other cells in the oral mucosa. Thus, it might be that a "swish and spit" exposure rather than systemic administration would prevent virion production.

Engineered peptide-drug conjugate provides sustained protection of retinal ganglion cells with topical administration in rats.

Effective eye drop delivery systems for treating diseases of the posterior segment have yet to be clinically validated. Further, adherence to eye drop regimens is often problematic due to the difficulty and inconvenience of repetitive dosing. Here, we describe a strategy for topically dosing a peptide-drug conjugate to achieve effective and sustained therapeutic sunitinib concentrations to protect retinal ganglion cells (RGCs) in a rat model of optic nerve injury. We combined two promising delivery technologies, namely, a hypotonic gel-forming eye drop delivery system, and an engineered melanin binding and cell-penetrating peptide that sustains intraocular drug residence time. We found that once daily topical dosing of HR97-SunitiGel provided up to 2 weeks of neuroprotection after the last dose, effectively doubling the therapeutic window observed with SunitiGel. For chronic ocular diseases affecting the posterior segment, the convenience of an eye drop combined with intermittent dosing frequency could result in greater patient adherence, and thus, improved disease management.

DNA methyltransferase inhibitor exposure-response: Challenges and opportunities.

Although DNA methyltransferase inhibitors (DNMTis), such as azacitidine and decitabine, are used extensively in the treatment of myelodysplastic syndromes and acute myeloid leukemia, there remain unanswered questions about DNMTi's mechanism of action and predictors of clinical response. Because patients often remain on single-agent DNMTis or DNMTi-containing regimens for several months before knowing whether clinical benefit can be achieved, the development and clinical validation of response-predictive biomarkers represents an important unmet need in oncology. In this review, we will summarize the clinical studies that led to the approval of azacitidine and decitabine, as well as the real-world experience with these drugs. We will then focus on biomarker development for DNMTis-specifically, efforts at determining exposure-response relationships and challenges that remain impacting the broader clinical translation of these methods. We will highlight recent progress in liquid-chromatography tandem mass spectrometry technology that has allowed for the simultaneous measurement of decitabine genomic incorporation and global DNA methylation, which has significant potential as a mechanism-of-action based biomarker in patients on DNMTis. Last, we will cover important research questions that need to be addressed in order to optimize this potential biomarker for clinical use.

ART714 is a best-in-class antileukemic 2-carbon-linked dimeric artemisinin derivative.

PURPOSE: It has become increasingly clear that new multiagent combination regimens are required to improve survival rates in acute myeloid leukemia (AML). We recently reported that ART631, a first-in-class 2-carbon-linked artemisinin-derived dimer (2C-ART), was not only efficacious as a component of a novel three-drug combination regimen to treat AML, but, like other synthetic artemisinin derivatives, demonstrated low clinical toxicity. However, we ultimately found ART631 to have suboptimal solubility and stability properties, thus limiting its potential for clinical development. METHODS: We assessed 22 additional 2C-ARTs with documented in vivo antimalarial activity for antileukemic efficacy and physicochemical properties. Our strategy involved culling out 2C-ARTs inferior to ART631 with respect to potency, stability, and solubility in vitro, and then validating in vivo pharmacokinetics, pharmacodynamics, and efficacy of one 2C-ART lead compound. RESULTS: Of the 22 2C-ARTs, ART714 was found to have the most optimal in vitro solubility, stability, and antileukemic efficacy, both alone and in combination with the BCL2 inhibitor venetoclax (VEN) and the kinase inhibitor sorafenib (SOR). ART714 was also highly effective in combination with VEN and the FMS-like tyrosine kinase 3 inhibitor gilteritinib (GILT) against MOLM14 AML xenografts. CONCLUSION: We identified ART714 as our best-in-class antileukemic 2C-ART, based on in vitro potency and pharmacologic properties. We established its in vivo pharmacokinetics and demonstrated its in vitro cooperativity with VEN and SOR and in vivo activities of combinations of ART714, VEN, and GILT. Additional research is indicated to define the optimal niche for the use of ART714 in treatment of AML.

Targeting hypoxia-inducible factors with 32-134D safely and effectively treats diabetic eye disease in mice.

Many patients with diabetic eye disease respond inadequately to anti-VEGF therapies, implicating additional vasoactive mediators in its pathogenesis. We demonstrate that levels of angiogenic proteins regulated by HIF-1 and -2 remain elevated in the eyes of people with diabetes despite treatment with anti-VEGF therapy. Conversely, by inhibiting HIFs, we normalized the expression of multiple vasoactive mediators in mouse models of diabetic eye disease. Accumulation of HIFs and HIF-regulated vasoactive mediators in hyperglycemic animals was observed in the absence of tissue hypoxia, suggesting that targeting HIFs may be an effective early treatment for diabetic retinopathy. However, while the HIF inhibitor acriflavine prevented retinal vascular hyperpermeability in diabetic mice for several months following a single intraocular injection, accumulation of acriflavine in the retina resulted in retinal toxicity over time, raising concerns for its use in patients. Conversely, 32-134D, a recently developed HIF inhibitor structurally unrelated to acriflavine, was not toxic to the retina, yet effectively inhibited HIF accumulation and normalized HIF-regulated gene expression in mice and in human retinal organoids. Intraocular administration of 32-134D prevented retinal neovascularization and vascular hyperpermeability in mice. These results provide the foundation for clinical studies assessing 32-134D for the treatment of patients with diabetic eye disease.

Machine learning-driven multifunctional peptide engineering for sustained ocular drug delivery.

Sustained drug delivery strategies have many potential benefits for treating a range of diseases, particularly chronic diseases that require treatment for years. For many chronic ocular diseases, patient adherence to eye drop dosing regimens and the need for frequent intraocular injections are significant barriers to effective disease management. Here, we utilize peptide engineering to impart melanin binding properties to peptide-drug conjugates to act as a sustained-release depot in the eye. We develop a super learning-based methodology to engineer multifunctional peptides that efficiently enter cells, bind to melanin, and have low cytotoxicity. When the lead multifunctional peptide (HR97) is conjugated to brimonidine, an intraocular pressure lowering drug that is prescribed for three times per day topical dosing, intraocular pressure reduction is observed for up to 18 days after a single intracameral injection in rabbits. Further, the cumulative intraocular pressure lowering effect increases ~17-fold compared to free brimonidine injection. Engineered multifunctional peptide-drug conjugates are a promising approach for providing sustained therapeutic delivery in the eye and beyond.

Six-month effective treatment of corneal graft rejection.

Topical corticosteroid eye drop is the mainstay for preventing and treating corneal graft rejection. While the frequent topical corticosteroid use is associated with risk of intraocular pressure (IOP) elevation and poor patient compliance that leads to graft failure and the requirement for a repeated, high-risk corneal transplantation. Here, we developed dexamethasone sodium phosphate (DSP)-loaded dicarboxyl-terminated poly(lactic acid) nanoparticle (PLA DSP-NP) formulations with relatively high drug loading (8 to 10 weight %) and 6 months of sustained intraocular DSP delivery in rats with a single dosing. PLA DSP-NP successfully reversed early signs of corneal rejection, leading to rat corneal graft survival for at least 6 months. Efficacious PLA DSP-NP doses did not affect IOP and showed no signs of ocular toxicity in rats for up to 6 months. Subconjunctival injection of DSP-NP is a promising approach for safely preventing and treating corneal graft rejection with the potential for improved patient adherence.

Quantitation of navtemadlin in human plasma and brain tissue by liquid chromatography-tandem mass spectrometry.

Navtemadlin is an orally bioavailable small molecule that blocks the protein-protein interaction between murine double minute 2 protein (MDM2) and the tumor suppressor protein p53, leading to p53-mediated cell cycle arrest and apoptosis. It is being evaluated in clinical trials for a variety of malignancies, both as a single agent and in combination regimens. A sensitive, robust LC-tandem mass spectrometry (LC-MS/MS) method was developed to quantitate navtemadlin in plasma, and this method was also validated using brain tissue homogenate. Sample preparation involved protein precipitation of plasma or brain tissue homogenate using acetonitrile. Navtemadlin, navtemadlin glucuronide, and the internal standard, D6 -navtemadlin, were separated from microsomal incubation extracts using gradient elution and a ZORBAX XDB C18 column. Analytes were detected using a SCIEX 5500 triple quadrupole mass spectrometer in positive electrospray ionization mode. The assay range of 1-1000 ng/mL was shown to be accurate (96.1-102.0% and 95.7-104%) and precise (coefficient of variation ≤ 10.6% and ≤ 6.6%) in plasma and brain tissue homogenate, respectively. An 8000 ng/mL navtemadlin sample diluted 1:10 (v/v) with plasma was also accurately quantitated. Navtemadlin has been stable in frozen plasma at -70°C for at least 20 months. This validated LC-MS/MS method was applied to determine navtemadlin concentrations in plasma and brain tissue samples from two separate patients receiving 120 mg/day navtemadlin on protocol ABTC1604.

HIF inhibitor 32-134D eradicates murine hepatocellular carcinoma in combination with anti-PD1 therapy.

Hepatocellular carcinoma (HCC) is a major cause of cancer mortality worldwide and available therapies, including immunotherapies, are ineffective for many patients. HCC is characterized by intratumoral hypoxia, and increased expression of hypoxia-inducible factor 1α (HIF-1α) in diagnostic biopsies is associated with patient mortality. Here we report the development of 32-134D, a low-molecular-weight compound that effectively inhibits gene expression mediated by HIF-1 and HIF-2 in HCC cells, and blocks human and mouse HCC tumor growth. In immunocompetent mice bearing Hepa1-6 HCC tumors, addition of 32-134D to anti-PD1 therapy increased the rate of tumor eradication from 25% to 67%. Treated mice showed no changes in appearance, behavior, body weight, hemoglobin, or hematocrit. Compound 32-134D altered the expression of a large battery of genes encoding proteins that mediate angiogenesis, glycolytic metabolism, and responses to innate and adaptive immunity. This altered gene expression led to significant changes in the tumor immune microenvironment, including a decreased percentage of tumor-associated macrophages and myeloid-derived suppressor cells, which mediate immune evasion, and an increased percentage of CD8+ T cells and natural killer cells, which mediate antitumor immunity. Taken together, these preclinical findings suggest that combining 32-134D with immune checkpoint blockade may represent a breakthrough therapy for HCC.

Serum Concentrations of Losartan Metabolites Correlate With Improved Physical Function in a Pilot Study of Prefrail Older Adults.

Losartan is an oral antihypertensive agent that is rapidly metabolized to EXP3174 (angiotensin-subtype-1-receptor blocker) and EXP3179 (peroxisome proliferator-activated receptor gamma [PPARγ] agonist), which was shown in animal studies to reduce inflammation, enhance mitochondrial energetics, and improve muscle repair and physical performance. We conducted an exploratory pilot study evaluating losartan treatment in prefrail older adults (age 70-90 years, N = 25). Participants were randomized to control (placebo) or treatment (daily oral losartan beginning at 25 mg per day and increasing every 8 weeks) for a total of 6 months. Fatigue, hyperkalemia, and hypotension were the most observed side effects of losartan treatment. Participants in the losartan group had an estimated 89% lower odds of frailty (95% confidence interval [CI]: 18% to 99% lower odds, p = .03), with a 0.3-point lower frailty score than the placebo group (95% CI: 0.01-0.5 lower odds, p = .04). Frailty score was also negatively associated with serum losartan and EXP3179 concentrations. For every one standard deviation increase in EXP3179 (ie, 0.0011 ng/µL, based on sample values above detection limit) and EXP3174 (ie, 0.27 ng/µL, based on sample values above detection limit), there was a 0.0035 N (95% CI: 0.0019-0.0051, p < .001) and a 0.0027 N (95% CI: 0.00054-0.0043, p = .007) increase in average knee strength, respectively.

Exosome-Encased Nucleic Acid Scaffold Chemotherapeutic Agents for Superior Anti-Tumor and Anti-Angiogenesis Activity.

Extracellular vesicles (EVs), or exosomes, play a pivotal role in tumor growth and metastasis, such as in the case of Kaposi Sarcoma. By loading tumor-derived EVs with chemotherapeutic drugs, we noted that their pro-tumor/pro-angiogenic phenotype was converted into an anti-tumor phenotype in vivo. Drug concentration in EVs was significantly higher than in clinically approved liposome formulation, as retention was facilitated by the presence of miRNAs inside the natural EVs. This demonstrates a new mechanism by which to increase the payload capacity of nanoparticles. By exploiting the targeting preferences of tumor-derived EVs, chemotherapeutics can be directed to specifically poison the cells and the microenvironment that enables metastasis.

Quantitation of terameprocol in human plasma by liquid chromatography-tandem mass spectrometry.

The global transcription inhibitor terameprocol is being evaluated clinically as an oral formulation to treat high-grade glioma. A sensitive, reliable method was developed to quantitate terameprocol using LC-MS/MS to perform detailed pharmacokinetic studies. Sample preparation involved protein precipitation using acetonitrile. Separation of terameprocol and the internal standard, Sorafenib-methyl-d3, was achieved with a Zorbax XDB C18 column (2.1 × 50 mm, 3.5 µm) and gradient elution over a 2-minute total analytical run time. A SCIEX 4500 or SCIEX 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for terameprocol detection. The assay range of 5-1000 ng/mL was demonstrated to be accurate (92.7-107.4%) and precise (CV ≤ 11.3%). A sample diluted 1:10 (v/v) was accurately quantitated. Terameprocol in plasma has been proven stable for at least 20 months when stored at -70 °C. The method was applied to the measurement of total plasma concentrations of terameprocol in a patient with a high-grade glioma receiving a 300 mg oral dose.

Validation of a rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis of vistusertib.

Vistusertib is an orally bioavailable mTOR inhibitor that is being studied in clinical trials. A novel reliable method was developed to quantitate vistusertib using LC-MS/MS to explore drug exposure-response relationships. Sample preparation involved protein precipitation using acetonitrile. Separation of vistusertib and the internal standard, AZD8055, was achieved with a Waters Acquity UPLC BEH C18 column utilizing isocratic elution over a 3 min total analytical run time. A SCIEX 4500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of vistusertib. The assay range was 5-5000 ng/mL and proved to be accurate (98.7-105.7%) and precise (CV ≤ 10.5%). A 40,000 ng/mL sample that was diluted 1:10 (v/v) with plasma was accurately quantitated. Long-term frozen plasma stability for vistusertib at -70 °C has been determined for at least 29 months. The method was applied for the measurement of plasma concentrations of vistusertib in a patient a solid tumor receiving 35 mg twice daily dose orally.