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.

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.

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.

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.

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.

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.

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.

A hypotonic gel-forming eye drop provides enhanced intraocular delivery of a kinase inhibitor with melanin-binding properties for sustained protection of retinal ganglion cells.

While eye drops are the most common ocular dosage form, eye drops for treating diseases of the posterior segment (retina, choroid, optic nerve) have yet to be developed. In glaucoma, eye drops are used extensively for delivering intraocular pressure (IOP)-lowering medications to the anterior segment. However, degeneration of retinal ganglion cells (RGCs) in the retina may progress despite significant IOP lowering, suggesting that a complementary neuroprotective therapy would improve glaucoma management. Here, we describe a hypotonic, thermosensitive gel-forming eye drop for effective delivery of sunitinib, a protein kinase inhibitor with activity against the neuroprotective targets dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK), to enhance survival of RGCs after optic nerve injury. Further, binding of sunitinib to melanin in the pigmented cells in the choroid and retinal pigment epithelium (RPE) led to prolonged intraocular residence time, including therapeutically relevant concentrations in the non-pigmented retinal tissue where the RGCs reside. The combination of enhanced intraocular absorption provided by the gel-forming eye drop vehicle and the intrinsic melanin binding properties of sunitinib led to significant protection of RGCs with only once weekly eye drop dosing. For a chronic disease such as glaucoma, an effective once weekly eye drop for neuroprotection could result in greater patient adherence, and thus, greater disease management and improved patient quality of life.

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 robust and rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis of navitoclax.

The Bcl-2 family small molecule inhibitor navitoclax is being clinically evaluated to treat multiple cancers including lymphoid malignancies and small cell lung cancer. A sensitive and reliable method was developed to quantitate navitoclax in human plasma using liquid chromatography with tandem mass spectrometry with which to perform detailed pharmacokinetic studies. Sample preparation involved protein precipitation using acetonitrile. Separation of navitoclax and the internal standard, navitoclax-d8, was achieved with a Waters Acquity UPLC BEH C18 column using isocratic flow 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 navitoclax. The assay range was 5-5,000 ng/ml and proved to be accurate (89.5-104.9%) and precise (CV ≤ 11%). Long-term frozen plasma stability for navitoclax at -70°C was at least 34 months. The method was applied for the measurement of total plasma concentration of navitoclax in a patient receiving a 250 mg daily oral dose.

Ion-Complex Microcrystal Formulation Provides Sustained Delivery of a Multimodal Kinase Inhibitor from the Subconjunctival Space for Protection of Retinal Ganglion Cells.

Glaucoma is the leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP) is one of the major risk factors for glaucoma onset and progression, and available pharmaceutical interventions are exclusively targeted at IOP lowering. However, degeneration of retinal ganglion cells (RGCs) may continue to progress despite extensive lowering of IOP. A complementary strategy to IOP reduction is the use of neuroprotective agents that interrupt the process of cell death by mechanisms independent of IOP. Here, we describe an ion complexation approach for formulating microcrystals containing ~50% loading of a protein kinase inhibitor, sunitinib, to enhance survival of RGCs with subconjunctival injection. A single subconjunctival injection of sunitinib-pamoate complex (SPC) microcrystals provided 20 weeks of sustained retina drug levels, leading to neuroprotection in a rat model of optic nerve injury. Furthermore, subconjunctival injection of SPC microcrystals also led to therapeutic effects in a rat model of corneal neovascularization. Importantly, therapeutically relevant retina drug concentrations were achieved with subconjunctival injection of SPC microcrystals in pigs. For a chronic disease such as glaucoma, a formulation that provides sustained therapeutic effects to complement IOP lowering therapies could provide improved disease management and promote patient quality of life.

Ultra-thin, high strength, antibiotic-eluting sutures for prevention of ophthalmic infection.

Sutures are applied almost universally at the site of trauma or surgery, making them an ideal platform to modulate the local, postoperative biological response, and improve surgical outcomes. To date, the only globally marketed drug-eluting sutures are coated with triclosan for antibacterial application in general surgery. Loading drug directly into the suture rather than coating the surface offers the potential to provide drug delivery functionality to microsurgical sutures and achieve sustained drug delivery without increasing suture thickness. However, conventional methods for drug incorporation directly into the suture adversely affect breaking strength. Thus, there are no market offerings for drug-eluting sutures, drug-coated, or otherwise, in ophthalmology, where very thin sutures are required. Sutures themselves help facilitate bacterial infection, and antibiotic eye drops are commonly prescribed to prevent infection after ocular surgeries. An antibiotic-eluting suture may prevent bacterial colonization of sutures and preclude patient compliance issues with eye drops. We report twisting of hundreds of individual drug-loaded, electrospun nanofibers into a single, ultra-thin, multifilament suture capable of meeting both size and strength requirements for microsurgical ocular procedures. Nanofiber-based polycaprolactone sutures demonstrated no loss in strength with loading of 8% levofloxacin, unlike monofilament sutures which lost more than 50% strength. Moreover, nanofiber-based sutures retained strength with loading of a broad range of drugs, provided antibiotic delivery for 30 days in rat eyes, and prevented ocular infection in a rat model of bacterial keratitis.

A randomized, placebo-controlled, double-blinded clinical trial of colchicine to improve vascular health in people living with HIV.

OBJECTIVES: People living with HIV (PWH) experience an increased burden of coronary artery disease (CAD) believed to be related, in part, to an interplay of chronically increased inflammation and traditional risk factors. Recent trials suggest cardiovascular benefits of the anti-inflammatory, colchicine, in HIV-seronegative CAD patients. However, the impact of colchicine on impaired vascular health, as measured by coronary endothelial function (CEF), an independent contributor to CAD, has not been studied in PWH. We tested the hypothesis that colchicine improves vascular health in PWH. DESIGN: This was a randomized, placebo-controlled, double-blinded trial in 81 PWH to test whether low-dose colchicine (0.6 mg daily) improves CEF over 8-24 weeks. METHODS: Coronary and systemic endothelial function and serum inflammatory markers were measured at baseline, and at 8 and 24 weeks. The primary endpoint was CEF, measured as the change in coronary blood flow from rest to that during an isometric handgrip exercise, an endothelial-dependent stressor, measured with non-invasive MRI at 8 weeks. RESULTS: Colchicine was well tolerated and not associated with increased adverse events. However, there were no significant improvements in coronary or systemic endothelial function or reductions in serum inflammatory markers at 8 or 24 weeks with colchicine as compared to placebo. CONCLUSIONS: In PWH with no history of CAD, low-dose colchicine was well tolerated but did not improve impaired coronary endothelial function, a predictor of cardiovascular events. These findings suggest that this anti-inflammatory approach using colchicine in PWH does not improve vascular health, the central, early driver of coronary atherosclerosis.

Enhanced drug delivery to the reproductive tract using nanomedicine reveals therapeutic options for prevention of preterm birth.

Inflammation contributes to nearly 4 million global premature births annually. Here, we used a mouse model of intrauterine inflammation to test clinically used formulations, as well as engineered nanoformulations, for the prevention of preterm birth (PTB). We observed that neither systemic 17a-hydroxyprogesterone caproate (Makena) nor vaginal progesterone gel (Crinone) was sufficient to prevent inflammation-induced PTB, consistent with recent clinical trial failures. However, we found that vaginal delivery of mucoinert nanosuspensions of histone deacetylase (HDAC) inhibitors, in some cases with the addition of progesterone, prevented PTB and resulted in delivery of live pups exhibiting neurotypical development. In human myometrial cells in vitro, the P4/HDAC inhibitor combination both inhibited cell contractility and promoted the anti-inflammatory action of P4 by increasing progesterone receptor B stability. Here, we demonstrate the use of vaginally delivered drugs to prevent intrauterine inflammation-induced PTB resulting in the birth of live offspring in a preclinical animal model.