Review of Emerging Pharmacotherapy for the Treatment of Coronavirus Disease 2019.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into an emergent global pandemic. Coronavirus disease 2019 (COVID-19) can manifest on a spectrum of illness from mild disease to severe respiratory failure requiring intensive care unit admission. As the incidence continues to rise at a rapid pace, critical care teams are faced with challenging treatment decisions. There is currently no widely accepted standard of care in the pharmacologic management of patients with COVID-19. Urgent identification of potential treatment strategies is a priority. Therapies include novel agents available in clinical trials or through compassionate use, and other drugs, repurposed antiviral and immunomodulating therapies. Many have demonstrated in vitro or in vivo potential against other viruses that are similar to SARS-CoV-2. Critically ill patients with COVID-19 have additional considerations related to adjustments for organ impairment and renal replacement therapies, complex lists of concurrent medications, limitations with drug administration and compatibility, and unique toxicities that should be evaluated when utilizing these therapies. The purpose of this review is to summarize practical considerations for pharmacotherapy in patients with COVID-19, with the intent of serving as a resource for health care providers at the forefront of clinical care during this pandemic.

Phase I trial of concurrent stereotactic body radiotherapy and nelfinavir for locally advanced borderline or unresectable pancreatic adenocarcinoma.

INTRODUCTION: The HIV protease inhibitor nelfinavir (NFV) displays notable radiosensitizing effects. There have been no studies evaluating combined stereotactic body radiotherapy (SBRT) and NFV for borderline/unresectable pancreatic cancer. The primary objective of this phase I trial (NCT01068327) was to determine the maximum tolerated SBRT/NFV dose, and secondarily evaluate outcomes. METHODS: Following initial imaging, pathologic confirmation, and staging laparoscopy, subjects initially received three 3-week cycles of gemcitabine/leucovorin/fluorouracil; patients without radiologic progression received 5-fraction SBRT/NFV. Dose escalation was as follows: (1) 25 Gy/625 mg BID ×3wks; (2) 25 Gy/1250 mg BID ×3wks; (3) 30 Gy/1250 mg BID ×3wks; (4) 35 Gy/1250 mg BID ×3wks; (5) 35 Gy/1250 mg BID ×5wks; and (6) 40 Gy/1250 mg BID ×5wks. Pancreaticoduodenectomy was performed thereafter if resectable; if not, gemcitabine/leucovorin/fluorouracil was administered. RESULTS: Forty-six patients enrolled (10/2008-5/2013); 39 received protocol-directed therapy. Sixteen (41%) experienced any grade ≥2 event during and 1 month after SBRT. Four grade 3 and both grade 4 events occurred in a single patient at the initial dose level. 40 Gy/1250 mg BID ×5wks was the maximum tolerated dose. Five patients had late gastrointestinal bleeding (n = 2 superior mesenteric artery pseudo-aneurysm, n = 1 disease progression, n = 1 lower GI tract, n = 1 unknown location). The median overall survival was 14.4 months. Six (15%) patients recurred locally; median local failure-free survival was not reached. The median distant failure-free survival was 11 months, and median all failure-free survival was 10 months. CONCLUSIONS: Concurrent SBRT (40 Gy)/NFV (1250 mg BID) for locally advanced pancreatic cancer is feasible and safe, although careful attention to treatment planning parameters is recommended to reduce the incidence of late gastrointestinal bleeding.

Study protocol: a multi-centre randomised study of induction chemotherapy followed by capecitabine ± nelfinavir with high- or standard-dose radiotherapy for locally advanced pancreatic cancer (SCALOP-2).

BACKGROUND: Induction chemotherapy followed by chemoradiation is a treatment option for patients with locally advanced pancreatic cancer (LAPC). However, overall survival is comparable to chemotherapy alone and local progression occurs in nearly half of all patients, suggesting chemoradiation strategies should be optimised. SCALOP-2 is a randomised phase II trial testing the role of radiotherapy dose escalation and/or the addition of the radiosensitiser nelfinavir, following induction chemotherapy of gemcitabine and nab-paclitaxel (GEMABX). A safety run-in phase (stage 1) established the nelfinavir dose to administer with chemoradiation in the randomised phase (stage 2). METHODS: Patients with locally advanced, inoperable, non-metastatic pancreatic adenocarcinoma receive three cycles of induction GEMABX chemotherapy prior to radiological assessment. Those with stable/responding disease are eligible for further trial treatment. In Stage 1, participants received one further cycle of GEMABX followed by capecitabine-chemoradiation with escalating doses of nelfinavir in a rolling-six design. Stage 2 aims to register 262 and randomise 170 patients with responding/stable disease to one of five arms: capecitabine with high- (arms C + D) or standard-dose (arms A + B) radiotherapy with (arms A + C) or without (arms B + D) nelfinavir, or three more cycles of GEMABX (arm E). Participants allocated to the chemoradiation arms receive another cycle of GEMABX before chemoradiation begins. Co-primary outcomes are 12-month overall survival (radiotherapy dose-escalation question) and progression-free survival (nelfinavir question). Secondary outcomes include toxicity, quality of life, disease response rate, resection rate, treatment compliance, and CA19-9 response. SCALOP-2 incorporates a detailed radiotherapy quality assurance programme. DISCUSSION: SCALOP-2 aims to optimise chemoradiation in LAPC and incorporates a modern induction regimen. TRIAL REGISTRATION: Eudract No: 2013-004968-56; ClinicalTrials.gov : NCT02024009.

Metformin combined with nelfinavir induces SIRT3/mROS-dependent autophagy in human cervical cancer cells and xenograft in nude mice.

The molecular mechanisms underlying the antineoplastic properties of metformin combined with nelfinavir remain elusive. To explore this question, transmission electron microscopy (TEM) was used to observe the combinatorial effect of inducing autophagosome formation in human cervical cancer cells. Western blotting respectively assayed protein expression of LC3I, LC3II, Beclin-1, Autophagy-related protein 7 (Atg7), Autophagy-related protein 3 (Atg3), NAD-dependent deacetylase sirtuin-3 (SIRT3) and major histocompatibility complex class I chain-related gene A (MICA). Lactate dehydrogenase (LDH) cytotoxicity assay evaluated natural killer (NK) cell cytotoxicity in the presence of metformin and nelfinavir in combination or each drug alone. Using tumor xenografts in a nude mouse model, antitumor efficacy of the drug combination was assessed. We found that the drug combination could induce autophagosome formation in human cervical cancer cells. The biomarker proteins of autophagy, including Beclin-1, Atg7 and Atg3, decreased, but the ratios of LC3I/II increased. We also found that this drug combination sensitizes human cervical cancer cells to NK cell-mediated lysis by increasing the protein of SIRT3 and MICA. Moreover, this drug combination markedly induced autophagy of SiHa xenografts in nude mice. Therefore, it can be concluded that metformin, in combination with nelfinavir, can induce SIRT3/mROS-dependent autophagy and sensitize NK cell-mediated lysis in human cervical cancer cells and cervical cancer cell xenografts in nude mice. Thus, our findings have revealed the detailed molecular mechanisms underlying the antitumor effects of metformin in combination with nelfinavir in cervical cancer.

A spray dried Nelfinavir Mesylate particles for enhanced oral bioavailability: Systematic formulation optimization and in-vivo performance.

The current endeavor was aimed to fabricate spray dried Nelfinavir Mesylate particles (SDNPs) that upon reconstitution with body fluids releases amorphous Nelfinavir Mesylate with enhanced oral bioavailability. For this purpose, feed mixture was prepared containing solubilized NFM, solid substrate and solvent system. The NFM was solubilized in a mixture of Maisine 35-1 (200 mg), Tween80 (500 mg) and Transcutol HP (300 mg). Three different solid substrates with high specific surface area were used: Neusilin® UFL2, (magnesium aluminometasilicate), Aerosil 200® (colloidal silica) and Syloid 244 FP® (porous silicon dioxide). Central composite design-response surface methodology (CCD-RSM) with three-factor (two numeric and one categorical) at three-level was used to select the appropriate solid substrate and to develop optimized SDNPs. The solid characterization by scanning electron microscopy, differential scanning calorimetry and powder X-ray diffraction studies indicated the absence of crystalline NFM in the formulations. The drug distribution analysis using raman spectroscopy suggested uniform distribution of amorphous NFM in optimized SDNPs-1 formulation. The transmission electron microscopy revealed the spherical structure of reconstituted SDNPs that releases amorphous NFM with globules size less than 110 nm. The solid substrate had significant and positive effect in drug dissolution; the mean dissolution time of NFM loaded in SDNPs was considerably improved. The bioavailability study resulted in enhanced magnitude of Cmax and AUC for SDNPs. The tissue distribution studies exhibited significantly higher brain and lymph nodes distribution as compared to putative form. Overall, the results pointed towards the overwhelming response of the SDNPs to be used for HIV treatment.

Potentiation of the leishmanicidal activity of nelfinavir in combination with miltefosine or amphotericin B.

The present work assesses the effect in vitro of combining the antiretroviral drug nelfinavir (NFV), a drug used against HIV but also a strong in vitro inhibitor of the growth of Leishmania promastigotes and amastigotes, with amphotericin B or miltefosine on different strains of Leishmania infantum. The isobolograms revealed a synergistic effect for both combinations, with a fractional inhibitory concentration index (∑FIC) <0.5. The ∑FIC values obtained with reference strain MCAN/ES/98/LLM-724 were 0.25 ± 0.1 (95% CI: 0.178-0.322) for the combination NFV+AMB and 0.48 ± 0.2 (95% CI: 0.377-0.573) for the combination NFV+MTF. The effect of NFV on visceral leishmaniasis induced by L. infantum in BALB/c mice was also examined, and the results confirmed a leishmanicidal effect when administered alone, with approximately 60% (P<0.05) reductions in the liver and spleen parasite burdens. This is the first time this has been reported in an in vivo model. A significant reduction in the liver (77%; P<0.01) and spleen (76%; P<0.01) parasite burdens was also observed for NFV+MTF compared with those obtained when these drugs were used alone. This indicates that such combinations may be useful treatment options in patients with visceral leishmaniasis who are also infected with HIV.

Stomach Dose-Volume Predicts Acute Gastrointestinal Toxicity in Chemoradiotherapy for Locally Advanced Pancreatic Cancer.

AIMS: Gastrointestinal toxicity impedes dose escalation in chemoradiotherapy for hepatobiliary malignancies. Toxicity risk depends on clinical and radiotherapy metrics. We aimed to identify predictive factors using data from two prospective phase II clinical trials of locally advanced pancreatic cancer (LAPC). MATERIALS AND METHODS: Ninety-one patients with available data from the ARCII (59.4 Gy in 33 fractions with gemcitabine, cisplatin and nelfinavir, n = 23) and SCALOP (50.4 Gy in 28 fractions with capecitabine or gemcitabine, n = 74) trials were studied. The independent variables analysed comprised age, sex, performance status, baseline symptoms, tumour size, weight loss, chemotherapy regimen and dose-volume histogram of stomach and duodenum in 5 Gy bins. The outcome measures used were Common Terminology Criteria of Adverse Events (CTCAE) grade and risk of CTCAE grade ≥2 acute upper gastrointestinal toxicity (anorexia, pain, nausea and/or vomiting). The risk of CTCAE grade ≥2 events was modelled using multivariable logistic regression and prediction of severity grade using ordinal regression. RESULTS: CTCAE grade ≥2 symptoms occurred in 38 patients (42%). On univariate analysis, stomach V35-45Gy was predictive of risk (odds ratio 1.035, 95% confidence interval 1.007-1.063) and grade (1.023, 1.003-1.044) of toxicity. The area under the curve was 0.632 (0.516-0.747) with toxicity risk 33/66 (50%) above and 5/25 (20%) below the optimal discriminatory threshold (7.1 cm3). Using a threshold of 30 cm3, risk was 13/20 (65%) versus 25/71 (35%). The optimal multivariable logistic regression model incorporated patient sex, chemotherapy regimen and stomach V35-45Gy. Receiving gemcitabine rather than capecitabine (odds ratio 3.965, 95% confidence interval 1.274-12.342) and weight loss during induction chemotherapy (1.216, 1.043-1.419) were significant predictors for the SCALOP cohort, whereas age predicted toxicity risk in ARCII only (1.344, 1.015-1.780). Duodenum dose-volume did not predict toxicity risk or severity in any cohort. CONCLUSIONS: In chemoradiotherapy for LAPC the volume of stomach irradiated to a moderately high dose (35-45 Gy) predicts the incidence and severity of acute toxicity. Other predictive factors can include age, sex, recent weight loss and concomitant chemotherapy agents.

Combining metformin and nelfinavir exhibits synergistic effects against the growth of human cervical cancer cells and xenograft in nude mice.

Human cervical cancer is the fourth most common carcinoma in women worldwide. However, the emergence of drug resistance calls for continuously developing new anticancer drugs and combination chemotherapy regimens. The present study aimed to investigate the anti-cervical cancer effects of metformin, a first-line therapeutic drug for type 2 diabetes mellitus, and nelfinavir, an HIV protease inhibitor, when used alone or in combination. We found that both metformin and nelfinavir, when used alone, were moderately effective in inhibiting proliferation, inducing apoptosis and suppressing migration and invasion of human cervical cell lines HeLa, SiHa and CaSki. When used in combination, these two drugs acted synergistically to inhibit the growth of human cervical cancer cells in vitro and cervical cancer cell xenograft in vivo in nude mice, and suppress cervical cancer cell migration and invasion. The protein expression of phosphoinositide 3-kinase catalytic subunit PI3K(p110α), which can promote tumor growth, was remarkably downregulated, while the tumor suppressor proteins p53 and p21 were substantially upregulated following the combinational treatment in vitro and in vivo. These results suggest that clinical use of metformin and nelfinavir in combination is expected to have synergistic antitumor efficacy and significant potential for the treatment of human cervical cancer.

In-utero exposure to nelfinavir-ethyl methyl sulfone.

Ethyl methyl sulfone contained in nelfinavir between 2007 and 2008 accidentally exposed embryos and fetuses to a powerful mutagen. We report data for 101 HIV-uninfected children exposed in utero included in the French prospective national cohort. The incidence of malformation was similar to that in the cohort as a whole with different drug exposures; no children had developed cancer after 9 years of follow-up.

Quality-by-design based development of a self-microemulsifying drug delivery system to reduce the effect of food on Nelfinavir mesylate.

Poor aqueous solubility and moderate permeability of Nelfinavir mesylate (NFM) leads to high variability in absorption after oral administration. To improve the solubility and bioavailability of NFM, the self microemulsifying drug delivery system (SMEDDS) was developed. For this purpose, Quality by design (QbD) approach employing D-optimal mixture design was used to prepare SMEDDS of NFM. Further, the software generated numerically optimized SMEDDS were developed by utilizing desirability function. Maisine 35-1, Tween 80, and Transcutol HP were identified as oil, surfactant, and co-surfactant that had best solubility for NFM. Ternary phase diagrams were plotted to identify the self-emulsification region. Dissolution of putative NFM in simulated fasted or fed small intestinal conditions, respectively, predicted that there is a positive food effect. However, NFM loaded SMEDDS showed absence of food effect with no significant difference in dissolution performance either in Fasted or fed state simulated intestinal fluid (FaSSIF or FeSSIF) biorelevent dissolution media. The prepared SMEDDS were thermodynamically stable with droplet size (121 nm), poly dispersity index (PDI) (0.198) and emulsification time (<1 min). Transmission electron microscopy (TEM) analysis confirmed the spherical shape of the reconstituted SMEDDS droplets. The ex vivo performance revealed 4.57 fold enhancement in the apparent permeability of NFM as compared to NFM suspension. The animal pharmacokinetic analysis in New Zealand strain rabbits indicated food effect on pure NFM suspension. However, absence of food effect and 3.5-3.6 fold enhancement in the oral bioavailability was observed when NFM was formulated into SMEDDS. Thus, it could be envisaged that development of SMEDDS formulation of NFM could be one of the best alternative to enhance oral bioavailability of NFM.

Is nelfinavir exposure associated with cancer incidence in HIV-positive individuals?

OBJECTIVE: Nelfinavir exhibits potent anticancer properties against a range of tumours. However, in 2006/2007, nelfinavir supplies were accidently contaminated with a carcinogen. This analysis investigated the association between nelfinavir use and cancer risk in HIV-positive persons. DESIGN: Observational cohort study. METHODS: D:A:D study data was analysed using Poisson regression models to examine associations between cancer incidence and cumulative nelfinavir exposure, current nelfinavir exposure, and exposure to nelfinavir between 1 July 2006-30 June 2007. RESULTS: A total of 42 006 individuals (50% white, 73% male) contributed 303 005 person-years of follow-up between 1 January 2004 and 1 February 2014. At study enrolment, median age was 40 [interquartile range (IQR) 33-46] years and 8305 individuals had a history of nelfinavir use [median duration 1.7 (IQR 0.7-3.4) years]. During follow-up, nelfinavir was used by 2476 individuals for a median of 1.7 (IQR 0.7-3.8) years; 1063 were exposed to nelfinavir between 1 July 2006 and 30 June 2007. Overall, 2279 cancers were diagnosed at a rate of 0.75 [95% confidence interval (95% CI) 0.72-0.78] per 100 person-years. Neither greater cumulative exposure to nelfinavir [adjusted risk ratio (aRR) 0.93 for every additional 5 years, 95% CI 0.82-1.06, P = 0.26] nor current use of nelfinavir (aRR 0.98 vs other protease inhibitor use, 95% CI 0.68-1.41, P = 0.92) were associated with cancer risk. The adjusted risk of cancer for participants exposed to nelfinavir between 1 July 2006 and 30 June 2007 compared to those receiving other treatment over this period was 1.07 (95% CI 0.78-1.46, P = 0.68). CONCLUSION: Nelfinavir use was not associated with a lower cancer incidence than other protease inhibitor regimens. As of February 2014, exposure to the 2006/2007 contamination of nelfinavir does not appear to be associated with increased cancer incidence.

Clinical Trial of Oral Nelfinavir before and during Radiation Therapy for Advanced Rectal Cancer.

PURPOSE: Nelfinavir, a PI3K pathway inhibitor, is a radiosensitizer that increases tumor blood flow in preclinical models. We conducted an early-phase study to demonstrate the safety of nelfinavir combined with hypofractionated radiotherapy (RT) and to develop biomarkers of tumor perfusion and radiosensitization for this combinatorial approach. EXPERIMENTAL DESIGN: Ten patients with T3-4 N0-2 M1 rectal cancer received 7 days of oral nelfinavir (1,250 mg b.i.d.) and a further 7 days of nelfinavir during pelvic RT (25 Gy/5 fractions/7 days). Perfusion CT (p-CT) and DCE-MRI scans were performed pretreatment, after 7 days of nelfinavir and prior to the last fraction of RT. Biopsies taken pretreatment and 7 days after the last fraction of RT were analyzed for tumor cell density (TCD). RESULTS: There were 3 drug-related grade 3 adverse events: diarrhea, rash, and lymphopenia. On DCE-MRI, there was a mean 42% increase in medianKtrans, and a corresponding median 30% increase in mean blood flow on p-CT during RT in combination with nelfinavir. Median TCD decreased from 24.3% at baseline to 9.2% in biopsies taken 7 days after RT (P= 0.01). Overall, 5 of 9 evaluable patients exhibited good tumor regression on MRI assessed by tumor regression grade (mrTRG). CONCLUSIONS: This is the first study to evaluate nelfinavir in combination with RT without concurrent chemotherapy. It has shown that nelfinavir-RT is well tolerated and is associated with increased blood flow to rectal tumors. The efficacy of nelfinavir-RT versus RT alone merits clinical evaluation, including measurement of tumor blood flow.

Plasma Mitochondrial DNA Levels as a Biomarker of Lipodystrophy Among HIV-infected Patients Treated with Highly Active Antiretroviral Therapy (HAART).

Lipodystrophy is a common complication in HIV-infected patients taking highly active antiretroviral therapy. Its early diagnosis is crucial for timely modification of antiretroviral therapy. We hypothesize that mitochondrial DNA in plasma may be a potential marker of LD in HIV-infected individuals. In this study, we compared plasma mitochondrial DNA levels in HIV-infected individuals and non-HIV-infected individuals to investigate its potential diagnostic value. Total plasma DNA was extracted from 67 HIV-infected patients at baseline and 12, 24 and 30 months after initiating antiretroviral therapy. Real-time quantitative PCR was used to determine the mitochondrial DNA levels in plasma. Lipodystrophy was defined by the physician-assessed presence of lipoatrophy or lipohypertrophy in one or more body regions. The mitochondrial DNA levels in plasma were significantly higher at baseline in HIV-infected individuals than in non-HIV-infected individuals (p<0.05). At month 30, 33 out of 67 patients (49.2%) showed at least one sign of lipodystrophy. The mean plasma mitochondrial DNA levels in lipodystrophy patients were significantly higher compared to those without lipodystrophy at month 24 (p<0.001). The receiver operating curve analysis demonstrated that using plasma mitochondrial DNA level (with cut-off value <5.09 log10 copies/ml) as a molecular marker allowed identification of patients with lipodystrophy with a sensitivity of 64.2% and a specificity of 73.0%. Our data suggest that mitochondrial DNA levels may help to guide therapy selection with regards to HIV lipodystrophy risk.

Impact of CYP2C19 polymorphism on the pharmacokinetics of nelfinavir in patients with pancreatic cancer.

AIM: This study evaluated the influence of CYP2C19 polymorphisms on the pharmacokinetics of nelfinavir and its metabolite M8 in patients with pancreatic cancer. METHODS: Nelfinavir was administered orally to patients for over 10 days. The plasma concentrations of nelfinavir and M8 were measured by HPLC. The genotypes of CYP2C19*1, CYP2C19*2 and CYP2C19*3 were determined by the polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: Pharmacokinetic profiles of nelfinavir and M8 were characterized by wide interindividual variability. The mean Cmax of nelfinavir in CYP2C19*1/*1 patients was 3.89 ± 0.40 (n = 3) and 5.12 ± 0.41 (n = 30) µg ml(-1) , while that of CYP2C19*1/*2 patients was 3.60 (n = 1) and 6.14 ± 0.31 (n = 5) µg ml(-1) at the doses of 625 and 1250 mg nelfinavir twice daily, respectively. For the M8 metabolite, the mean Cmax of CYP2C19*1/*1 patients was 1.06 ± 0.06 (n = 3) and 1.58 ± 0.27 (n = 30) µg ml(-1) , while those of CYP2C19*1/*2 patients were 1.01 (n = 1) and 1.23 ± 0.15 (n = 5) µg ml(-1) at the doses of 625 and 1250 mg nelfinavir twice daily, respectively. The area under the plasma concentration-time curve (AUC(0,12 h)) values of nelfinavir for CYP2C19*1/*1 patients were 28.90 ± 1.27 and 38.90 ± 4.99 µg ml(-1) ·h and for CYP2C19*1/*2 patients, AUC(0,12 h) was 28.20 (n = 1) and 40.22 ± 3.17 (n = 5) µg ml(-1) ·h at the doses of 625 and 1250 mg nelfinavir twice daily, respectively. The Cmax of nelfinavir was significantly higher (P <0.05) in CYP2C19*1/*2 patients but there was no statistical difference in AUC(0,12 h). CONCLUSION: CYP2C19*1/*2 genotype modestly affected the pharmacokinetic profiles of nelfinavir and M8 in patients with locally advanced pancreatic cancer.

A phase I trial of the HIV protease inhibitor nelfinavir in adults with solid tumors.

Nelfinavir is an HIV protease inhibitor being repurposed as an anti-cancer agent in preclinical models and in small oncology trials, yet the MTD of nelfinavir has not been determined. Therefore, we conducted a Phase Ia study to establish the maximum tolerated dose (MTD) and dose limiting toxicities (DLT) of nelfinavir in subjects with advanced solid tumors. Adults with refractory cancers were given oral nelfinavir twice daily with pharmacokinetic and pharmacodynamic analyses. Twenty-eight subjects were enrolled. Nelfinavir was generally well tolerated. Common adverse events included diarrhea, anemia, and lymphopenia, which were mostly mild. The DLT was rapid-onset neutropenia that was reversible. The MTD was established at 3125 mg twice daily. In an expansion cohort at the MTD, one of 11 (9%) evaluable subjects had a confirmed partial response. This, plus two minor responses, occurred in subjects with neuroendocrine tumors of the midgut or pancreatic origin. Thirty-six percent of subjects had stable disease for more than 6 months. In peripheral blood mononuclear cells, Nelfinavir inhibited AKT and induced markers of ER stress. In summary, nelfinavir is well tolerated in cancer patients at doses 2.5 times the FDA-approved dose for HIV management and showed preliminary activity in tumors of neuroendocrine origin.

A conceptually new treatment approach for relapsed glioblastoma: coordinated undermining of survival paths with nine repurposed drugs (CUSP9) by the International Initiative for Accelerated Improvement of Glioblastoma Care.

To improve prognosis in recurrent glioblastoma we developed a treatment protocol based on a combination of drugs not traditionally thought of as cytotoxic chemotherapy agents but that have a robust history of being well-tolerated and are already marketed and used for other non-cancer indications. Focus was on adding drugs which met these criteria: a) were pharmacologically well characterized, b) had low likelihood of adding to patient side effect burden, c) had evidence for interfering with a recognized, well-characterized growth promoting element of glioblastoma, and d) were coordinated, as an ensemble had reasonable likelihood of concerted activity against key biological features of glioblastoma growth. We found nine drugs meeting these criteria and propose adding them to continuous low dose temozolomide, a currently accepted treatment for relapsed glioblastoma, in patients with recurrent disease after primary treatment with the Stupp Protocol. The nine adjuvant drug regimen, Coordinated Undermining of Survival Paths, CUSP9, then are aprepitant, artesunate, auranofin, captopril, copper gluconate, disulfiram, ketoconazole, nelfinavir, sertraline, to be added to continuous low dose temozolomide. We discuss each drug in turn and the specific rationale for use- how each drug is expected to retard glioblastoma growth and undermine glioblastoma's compensatory mechanisms engaged during temozolomide treatment. The risks of pharmacological interactions and why we believe this drug mix will increase both quality of life and overall survival are reviewed.

The antiretroviral protease inhibitor ritonavir accelerates glutathione export from cultured primary astrocytes.

Antiretroviral protease inhibitors are a class of important drugs that are used for the treatment of human immunodeficiency virus infections. Among those compounds, ritonavir is applied frequently in combination with other antiretroviral protease inhibitors, as it has been reported to boost their therapeutic efficiency. To test whether ritonavir affects the viability and the glutathione (GSH) metabolism of brain cells, we have exposed primary astrocyte cultures to this protease inhibitor. Application of ritonavir in low micromolar concentrations did not compromise cell viability, but caused a time- and concentration-dependent loss of GSH from the cells which was accompanied by a matching increase in the extracellular GSH content. Half-maximal effects were observed for ritonavir in a concentration of 3 µM. The ritonavir-induced stimulated GSH export from astrocytes was completely prevented by MK571, an inhibitor of the multidrug resistance protein 1. In addition, continuous presence of ritonavir was essential to maintain the stimulated GSH export, since removal of ritonavir terminated the stimulated GSH export. Ritonavir was more potent to stimulate GSH export from astrocytes than the antiretroviral protease inhibitors indinavir and nelfinavir, but combinations of ritonavir with indinavir or nelfinavir did not further stimulate astrocytic GSH export compared to a treatment with ritonavir alone. The strong effects of ritonavir and other antiretroviral protease inhibitors on the GSH metabolism of astrocytes suggest that a chronic treatment of patients with such compounds may affect their brain GSH metabolism.

Phase I study of nelfinavir in liposarcoma.

PURPOSE: HIV protease inhibitors are associated with HIV protease inhibitor-related lipodystrophy syndrome. We hypothesized that liposarcomas would be similarly susceptible to the apoptotic effects of an HIV protease inhibitor, nelfinavir. METHODS: We conducted a phase I trial of nelfinavir for liposarcomas. There was no limit to prior chemotherapy. The starting dose was 1,250 mg twice daily (Level 1). Doses were escalated in cohorts of three to a maximally evaluated dose of 4,250 mg (Level 5). One cycle was 28 days. Steady-state pharmacokinetics (PKs) for nelfinavir and its primary active metabolite, M8, were determined at Levels 4 (3,000 mg) and 5. RESULTS: Twenty subjects (13 males) were enrolled. Median (range) age was 64 years (37-81). One subject at Level 1 experienced reversible, grade 3 pancreatitis after 1 week and was replaced. No other dose-limiting toxicities were observed. Median (range) number of cycles was 3 (0.6-13.5). Overall best responses observed were 1 partial response, 1 minor response, 4 stable disease, and 13 progressive disease. Mean peak plasma levels and AUCs for nelfinavir were higher at Level 4 (7.3 mg/L; 60.9 mg/L × h) than 5 (6.3 mg/L; 37.7 mg/L × h). The mean ratio of M8:nelfinavir AUCs for both levels was ~1:3. CONCLUSIONS: PKs demonstrate auto-induction of nelfinavir clearance at the doses studied, although the mechanism remains unclear. Peak plasma concentrations were within range where anticancer activity was demonstrated in vitro. M8 metabolite is present at ~1/3 the level of nelfinavir and may also contribute to the anticancer activity observed.

Preferential killing of triple-negative breast cancer cells in vitro and in vivo when pharmacological aggravators of endoplasmic reticulum stress are combined with autophagy inhibitors.

The cellular processes of autophagy and endoplasmic reticulum stress (ERS) appear to be interconnected, and it has been proposed that autophagy may serve to reduce ERS via removal of terminally misfolded and aggregated proteins. Conversely, there are indications that blockage of autophagy may increase ERS. Based on earlier work demonstrating that pharmacologically aggravated ERS can result in tumor cell killing, we investigated whether blockage of autophagy would enhance this effect in a therapeutically useful manner. We therefore combined chloroquine (CQ), a pharmacological inhibitor of autophagy, with other drugs known to act as ERS aggravators (ERSA), namely nelfinavir (an HIV protease inhibitor) and celecoxib (a cyclooxygenase-2 inhibitor) or its non-coxib analog 2,5-dimethyl-celecoxib (DMC), and investigated combination drug effects in a variety of breast cancer cell lines. We found that the addition of CQ resulted in synergistic enhancement of tumor cell killing by ERSA compounds, particularly in triple-negative breast cancer (TNBC) cells. This combination effect could also be confirmed in an in vivo model, where CQ boosted low-dose ERSA effects, resulting in rapid deterioration of xenografted tumors in mice. Altogether, our results indicate that combinations of an autophagy inhibitor with pharmacological ERSA (i.e. compounds that lead to ER stress aggravation) should be further explored for potential therapy of otherwise difficult-to-treat TNBC.

Complex drug interactions of the HIV protease inhibitors 3: effect of simultaneous or staggered dosing of digoxin and ritonavir, nelfinavir, rifampin, or bupropion.

As part of a larger clinical drug-drug interaction (DDI) study aimed at in vitro to in vivo prediction of HIV protease inhibitor metabolic and transporter-based DDIs, we measured the inductive (staggered administration) and inductive plus inhibitory (simultaneously administered) effect of multiple dose ritonavir (RTV), nelfinavir (NFV), or rifampin (RIF) on the pharmacokinetics of the P-glycoprotein probe, digoxin (DIG), when administered simultaneously or staggered with the protease inhibitors or RIF. In both cases, NFV did not significantly affect DIG disposition. RTV decreased DIG renal clearance (Cl(renal)) when administered simultaneously or staggered but significantly increased DIG area under the curve from time zero to 24 h (AUC(0-24 h)) only when administered simultaneously. RIF decreased DIG AUC(0-24 h) only when RIF and DIG administration was staggered. When RIF and DIG were administered simultaneously, DIG maximal observed plasma concentration and area under the curve from time zero to 4 h were significantly increased, and DIG Cl(renal) was decreased. An unexpected and potentially clinically significant DDI was observed between DIG and the CYP2B6 probe, bupropion, which decreased DIG AUC(0-24 h) 1.6-fold and increased Cl(renal) 1.8-fold. Because this was an unexpected DDI and our studies were not specifically designed to quantify this interaction, further studies are required to confirm the interaction and understand the mechanistic basis of the DDI. In summary, RTV or NFV do not induce P-glycoprotein activity measured with DIG, and RIF does so only under staggered administration.