Clinical evaluation of PF614, a novel TAAP prodrug of oxycodone, versus OxyContin in a multi-ascending dose study with a bioequivalence arm in healthy volunteers.

PF614, a trypsin-activated abuse protection oxycodone prodrug designed to reduce recreational drug abuse, was compared to OxyContin for safety and pharmacokinetics (PKs) of plasma oxycodone following oral administration. This study was a two-part design including a multi-ascending dose (part A) and a bioequivalence (BE) study (part B) in healthy volunteers. In part A, 24 subjects were randomized 3:1 to receive PF614 (50, 100, or 200 mg, n = 6/cohort) or OxyContin (20, 40, or 80 mg; n = 2/cohort) in ascending cohorts, delivered every 12 h for a total of nine doses. In part B, 60 subjects randomized in a four-way crossover to evaluate BE, received PF614 100 mg and OxyContin 40 mg in fasted and fed (high-fat diet) states. All subjects were naltrexone blocked prior to first study drug administration to protect against opioid-related adverse effects; repeat doses were provided on days 1-5. In part A, PF614 was well-tolerated following twice daily doses of up to 200 mg for 5 days. Plasma oxycodone maximal plasma concentration and area under the concentration time curve increased linearly with increasing doses. Part B showed that plasma oxycodone BE was achieved following 100 mg PF614 or 40 mg OxyContin under both fasted and fed conditions. Additionally, PF614 provided similar oxycodone exposures following both fasted and fed states. This study confirms findings from our single-ascending dose study, showing that PF614 100 mg releases oxycodone with a PK profile comparable to 40 mg OxyContin under both fasted and fed conditions and with a similar safety profile under naltrexone-blocked conditions.

Multi-Drug/Gene NASH Therapy Delivery and Selective Hyperspectral NIR Imaging Using Chirality-Sorted Single-Walled Carbon Nanotubes.

Single-walled carbon nanotubes (SWCNTs) can serve as drug delivery/biological imaging agents, as they exhibit intrinsic fluorescence in the near-infrared, allowing for deeper tissue imaging while providing therapeutic transport. In this work, CoMoCAT (Cobalt Molybdenum Catalyst) SWCNTs, chirality-sorted by aqueous two-phase extraction, are utilized for the first time to deliver a drug/gene combination therapy and image each therapeutic component separately via chirality-specific SWCNT fluorescence. Each of (7,5) and (7,6) sorted SWCNTs were non-covalently loaded with their specific payload: the PI3 kinase inhibitor targeting liver fibrosis or CCR5 siRNA targeting inflammatory pathways with the goal of addressing these processes in nonalcoholic steatohepatitis (NASH), ultimately to prevent its progression to hepatocellular carcinoma. PX-866-(7,5) SWCNTs and siRNA-(7,6) SWCNTs were each imaged via characteristic SWCNT emission at 1024/1120 nm in HepG2 and HeLa cells by hyperspectral fluorescence microscopy. Wavelength-resolved imaging verified the intracellular transport of each SWCNT chirality and drug release. The therapeutic efficacy of each formulation was further demonstrated by the dose-dependent cytotoxicity of SWCNT-bound PX-866 and >90% knockdown of CCR5 expression with SWCNT/siRNA transfection. This study verifies the feasibility of utilizing chirality-sorted SWCNTs for the delivery and component-specific imaging of combination therapies, also suggesting a novel nanotherapeutic approach for addressing the progressions of NASH to hepatocellular carcinoma.

In vitro and in vivo assessment of the abuse potential of PF614, a novel BIO-MD™ prodrug of oxycodone.

OBJECTIVE: The need for pain medication which will not lead to abuse is well recognized. Ensysce has designed prodrug analogs of the commonly used pain medications including hydromorphone, oxycodone (OC), hydrocodone, and morphine that limit their use to oral delivery, two of which are in clinical development. This study was undertaken to demonstrate that PF614, an extended-release prodrug of OC, allows the release of OC as designed when delivered orally, yet it resists ex vivo extraction with household chemicals and is pharmacologically inactive when administered by nonoral routes (nasal and parenteral), thereby substantially reducing its intravenous (IV) and intranasal abuse potential. METHODS: In vitro and in vivo methods were used to determine release of OC from PF614 and to show potential µ-opioid receptor activity. Plasma and cerebral spinal fluid levels of OC were evaluated following in vivo IV administration of PF614 in rats. In vitro extraction of OC from PF614 was explored using enzymes, common solvents, and household chemicals at room temperature and elevated temperature over time to determine release of OC from the prodrug. RESULTS: PF614 was stable with in vitro exposure to human plasma, saliva, and liver microsomes or culinary enzyme preparations. PF614 was stable (≥90 percent remaining as intact prodrug) under all room temperature conditions evaluated for 24 hours. At 80°C for 1 hour, no OC was released. Incubation at 80°C for 24 hours in vinegar or vodka produced a conversion to OC of 6 percent. Incubation with trypsin at 37°C converted PF614 approximately stoichiometric to OC with half-life of 4 hours. PF614's penetration of the central nervous system was 83-fold lower than OC and it had a 6.5-fold reduced potency as a µ-opioid agonist. Finally, oral PF614 delivers OC into plasma with an extended-release profile in dogs (reduced Cmax; delayed Tmax). CONCLUSIONS: The Bio-Activated Molecular Delivery prodrug design limits the use of PF614 to the intended oral route of delivery with reduced potential for IV or nasal abuse, as it cannot be activated intravenously or nasally to provide an active opioid. Unlike existing opioid formulations, the extended-release profile of PF614 cannot be accelerated by chewing or ex vivo extraction to pharmacologically active substances.

Clinically Evaluated Cancer Drugs Inhibiting Redox Signaling.

SIGNIFICANCE: There are a number of redox-active anticancer agents currently in development based on the premise that altered redox homeostasis is necessary for cancer cell's survival. Recent Advances: This review focuses on the relatively few agents that target cellular redox homeostasis to have entered clinical trial as anticancer drugs. CRITICAL ISSUES: The success rate of redox anticancer drugs has been disappointing compared to other classes of anticancer agents. This is due, in part, to our incomplete understanding of the functions of the redox targets in normal and cancer tissues, leading to off-target toxicities and low therapeutic indexes of the drugs. The field also lags behind in the use biomarkers and other means to select patients who are most likely to respond to redox-targeted therapy. FUTURE DIRECTIONS: If we wish to derive clinical benefit from agents that attack redox targets, then the future will require a more sophisticated understanding of the role of redox targets in cancer and the increased application of personalized medicine principles for their use. Antioxid. Redox Signal. 26, 262-273.

Carbon Nanotubes: Solution for the Therapeutic Delivery of siRNA?

Carbon nanotubes have many unique physical and chemical properties that are being widely explored for potential applications in biomedicine especially as transporters of drugs, proteins, DNA and RNA into cells. Specifically, single-walled carbon nanotubes (SWCNT) have been shown to deliver siRNA to tumors in vivo. The low toxicity, the excellent membrane penetration ability, the protection afforded against blood breakdown of the siRNA payload and the good biological activity seen in vivo suggests that SWCNT may become universal transfection vehicles for siRNA and other RNAs for therapeutic applications. This paper will introduce a short review of a number of therapeutic applications for carbon nanotubes and provide recent data suggesting SWCNT are an excellent option for the delivery of siRNA clinically.

Molecular pharmacology and antitumor activity of PHT-427, a novel Akt/phosphatidylinositide-dependent protein kinase 1 pleckstrin homology domain inhibitor.

Phosphatidylinositol 3-kinase/phosphatidylinositide-dependent protein kinase 1 (PDPK1)/Akt signaling plays a critical role in activating proliferation and survival pathways within cancer cells. We report the molecular pharmacology and antitumor activity of PHT-427, a compound designed to bind to the pleckstrin homology (PH) binding domain of signaling molecules important in cancer. Although originally designed to bind the PH domain of Akt, we now report that PHT-427 also binds to the PH domain of PDPK1. A series of PHT-427 analogues with variable C-4 to C-16 alkyl chain length were synthesized and tested. PHT-427 itself (C-12 chain) bound with the highest affinity to the PH domains of both PDPK1 and Akt. PHT-427 inhibited Akt and PDPK1 signaling and their downstream targets in sensitive but not resistant cells and tumor xenografts. When given orally, PHT-427 inhibited the growth of human tumor xenografts in immunodeficient mice, with up to 80% inhibition in the most sensitive tumors, and showed greater activity than analogues with C4, C6, or C8 alkyl chains. Inhibition of PDPK1 was more closely correlated to antitumor activity than Akt inhibition. Tumors with PIK3CA mutation were the most sensitive, and K-Ras mutant tumors were the least sensitive. Combination studies showed that PHT-427 has greater than additive antitumor activity with paclitaxel in breast cancer and with erlotinib in non-small cell lung cancer. When given >5 days, PHT-427 caused no weight loss or change in blood chemistry. Thus, we report a novel PH domain binding inhibitor of PDPK1/Akt signaling with significant in vivo antitumor activity and minimal toxicity.

Cellular and in vivo activity of a novel PI3K inhibitor, PX-866, against human glioblastoma.

The phosphatidylinositol-3-kinase (PI3K)/Akt oncogenic pathway is critical in glioblastomas. Loss of PTEN, a negative regulator of the PI3K pathway or activated PI3K/Akt pathway that drive increased proliferation, survival, neovascularization, glycolysis, and invasion is found in 70%-80% of malignant gliomas. Thus, PI3K is an attractive therapeutic target for malignant glioma. We report that a new irreversible PI3K inhibitor, PX-866, shows potent inhibitory effects on the PI3K/Akt signaling pathway in glioblastoma. PX-866 did not induce any apoptosis in glioma cells; however, an increase in autophagy was observed. PX-866 inhibited the invasive and angiogenic capabilities of cultured glioblastoma cells. In vivo, PX-866 inhibited subcutaneous tumor growth and increased the median survival time of animals with intracranial tumors. We also assessed the potential of proton magnetic resonance spectroscopy (MRS) as a noninvasive method to monitor response to PX-866. Our findings show that PX-866 treatment causes a drop in the MRS-detectable choline-to-NAA, ratio and identify this partial normalization of the tumor metabolic profile as a biomarker of molecular drug action. Our studies affirm that the PI3K pathway is a highly specific molecular target for therapies for glioblastoma and other cancers with aberrant PI3K/PTEN expression.

Mutations in the phosphatidylinositol-3-kinase pathway predict for antitumor activity of the inhibitor PX-866 whereas oncogenic Ras is a dominant predictor for resistance.

The novel phosphatidylinositol-3-kinase (PI3K) inhibitor PX-866 was tested against 13 experimental human tumor xenografts derived from cell lines of various tissue origins. Mutant PI3K (PIK3CA) and loss of PTEN activity were sufficient, but not necessary, as predictors of sensitivity to the antitumor activity of the PI3K inhibitor PX-866 in the presence of wild-type Ras, whereas mutant oncogenic Ras was a dominant determinant of resistance, even in tumors with coexisting mutations in PIK3CA. The level of activation of PI3K signaling measured by tumor phosphorylated Ser(473)-Akt was insufficient to predict in vivo antitumor response to PX-866. Reverse-phase protein array revealed that the Ras-dependent downstream targets c-Myc and cyclin B were elevated in cell lines resistant to PX-866 in vivo. Studies using an H-Ras construct to constitutively and preferentially activate the three best-defined downstream targets of Ras, i.e., Raf, RalGDS, and PI3K, showed that mutant Ras mediates resistance through its ability to use multiple pathways for tumorigenesis. The identification of Ras and downstream signaling pathways driving resistance to PI3K inhibition might serve as an important guide for patient selection as inhibitors enter clinical trials and for the development of rational combinations with other molecularly targeted agents.

Molecular mechanisms for the activity of PX-478, an antitumor inhibitor of the hypoxia-inducible factor-1alpha.

We have reported previously that PX-478 (S-2-amino-3-[4'-N,N,-bis(chloroethyl)amino]phenyl propionic acid N-oxide dihydrochloride) has potent antitumor activity against a variety of human tumor xenografts associated with the levels of the hypoxia-inducible factor-1alpha (HIF-1alpha) within the tumor. We now report that PX-478 inhibits HIF-1alpha protein levels and transactivation in a variety of cancer cell lines. Hypoxia-induced vascular endothelial growth factor formation was inhibited by PX-478, whereas baseline levels of vascular endothelial growth factor in normoxia were unaffected. Studies of the mechanism of PX-478 action showed that HIF-1alpha inhibition occurs in both normoxia and hypoxia and does not require pVHL or p53. In addition, PX-478 decreases levels of HIF-1alpha mRNA and inhibits translation as determined by 35S labeling experiments and reporter assays using the 5' untranslated region of HIF-1alpha. Moreover, to a lesser extent, PX-478 also inhibits HIF-1alpha deubiquitination resulting in increased levels of polyubiquitinated HIF-1alpha. The inhibitory effect of PX-478 on HIF-1alpha levels is primarily due to its inhibition of translation because HIF-1alpha translation continues in hypoxia when translation of most proteins is decreased. We conclude that PX-478 inhibits HIF-1alpha at multiple levels that together or individually may contribute to its antitumor activity against HIF-1alpha-expressing tumors.

Thioredoxin signaling as a target for cancer therapy.

Thioredoxin (Trx) family members play critical roles in the regulation of cellular redox homeostasis. Cancer cells exist in a stressed environment and rely on the Trxs for protection against stress-disregulated redox signaling. The most extensively studied member of the family is Trx-1 whose levels are increased in many human cancers most likely in direct response to stress. Trx-1 contributes to many of the hallmarks of cancer including increased proliferation, resistance to cell death and increased angiogenesis. Trx-1 is a validated cancer drug target associated with aggressive tumor growth, resistance to standard therapy and decreased patient survival. A surrogate target for Trx-1 may be thioredoxin reductase (TR). Drugs that inhibit Trx-1 and TR are in clinical development with early promising results.

A Phase I pharmacokinetic and pharmacodynamic study of PX-12, a novel inhibitor of thioredoxin-1, in patients with advanced solid tumors.

PURPOSE: Thioredoxin-1 (Trx-1) is a cellular redox protein that promotes tumor growth, inhibits apoptosis, and up-regulates hypoxia-inducible factor-1alpha and vascular endothelial growth factor. Objectives of this study were to determine safety, tolerability, pharmacodynamics, and pharmacokinetics of PX-12, a small-molecule inhibitor of Trx-1. EXPERIMENTAL DESIGN: Thirty-eight patients with advanced solid tumors received PX-12 at doses of 9 to 300 mg/m(2), as a 1- or 3-h i.v. infusion on days 1 to 5, repeated every 3 weeks. RESULTS: At the 300 mg/m(2) dose level, one patient experienced a reversible episode of pneumonitis during the first cycle, and a second patient developed pneumonitis after the second cycle. Doses up to 226 mg/m(2) were well tolerated, and grade 3/4 events were uncommon (<3% of patients). The limiting factor on this dosing schedule was pungent odor caused by expired drug metabolite, 2-butanethiol. The best response was stable disease in seven patients (126-332 days). Whereas PX-12 was not detectable following the infusion, the C(max) of its inactive metabolite, 2-mercaptoimidazole, increased linearly with dose. PX-12 treatment lowered plasma Trx-1 concentrations in a dose-dependent manner. CONCLUSIONS: PX-12, the first Trx-1 inhibitor to enter clinical trials, was tolerated up to a dose of 226 mg/m(2) by a 3-h infusion. Based on pharmacodynamic and pharmacokinetic data, a trial of prolonged infusion schedule of PX-12 has been initiated.

Molecular pharmacology and antitumor activity of palmarumycin-based inhibitors of thioredoxin reductase.

The cytosolic thioredoxin redox system composed of thioredoxin-1 and the NADPH-dependent thioredoxin reductase-1 reductase is an important regulator of cell growth and survival. Thioredoxin-1 is overexpressed in many human tumors where it is associated with increased cell proliferation, decreased apoptosis, and decreased patient survival. We hypothesized that thioredoxin reductase-1 provides a target to inhibit the activity of overexpressed thioredoxin-1 for the development of novel anticancer agents. We found that the naphthoquinone spiroketal fungal metabolite palmarumycin CP1 is a potent inhibitor of thioredoxin reductase-1, but attempts to exploit the activity of palmarumycin CP1 analogues as antitumor agents in vivo were hampered by their insolubility. We have therefore developed PX-916, a water-soluble prodrug of a palmarumycin CP1 analogue. PX-916 rapidly releases the parent compound at physiologic pH and in plasma but is stable at acid pH, allowing its i.v. administration. PX-916 is a potent inhibitor of purified human thioredoxin reductase-1 and of thioredoxin reductase-1 activity in cells and tumor xenografts when given to mice and inhibits the downstream targets of thioredoxin-1 signaling, hypoxia-inducible factor-1alpha, and vascular endothelial growth factor in tumors. PX-916 showed excellent antitumor activity against several animal tumor models with some cures. Thus, the study shows that water-soluble inhibitors of thioredoxin reductase-1, such as PX-916, can block thioredoxin-1 signaling in tumors producing marked inhibition of tumor growth.

The antitumor thioredoxin-1 inhibitor PX-12 (1-methylpropyl 2-imidazolyl disulfide) decreases thioredoxin-1 and VEGF levels in cancer patient plasma.

Thioredoxin-1 (Trx-1) is a small redox protein that is overexpressed in many human tumors, where it is associated with aggressive tumor growth and decreased patient survival. Trx-1 is secreted by tumor cells and is present at increased levels in the plasma of cancer patients. PX-12 is an irreversible inhibitor of Trx-1 currently in clinical development as an antitumor agent. We have used SELDI-TOF mass spectroscopy to measure plasma Trx-1 from patients treated with PX-12 during a phase I study. Mean plasma Trx-1 levels at pretreatment were significantly elevated in the cancer patients at 182.0 ng/mL compared with 27.1 ng/mL in plasma from healthy volunteers. PX-12 treatment significantly lowered plasma Trx-1 in cancer patients having the highest plasma Trx-1 pretreatment levels. High-plasma vascular endothelial growth factor (VEGF) levels have been correlated to decreased patient survival. PX-12 treatment also significantly lowered plasma VEGF levels in cancer patients with high pretreatment VEGF levels. SELDI-TOF mass spectrometry identified seven additional plasma proteins whose levels decreased after PX-12 administration, one of which was identified as a truncated form of transthyretin. The results of this study suggest that the lowering of elevated levels of plasma Trx-1 in cancer patients may provide a surrogate for the inhibition of tumor Trx-1 by PX-12. Furthermore, PX-12 decreases plasma VEGF levels that may contribute to the antitumor activity of PX-12.

The phosphatidylinositol-3-kinase inhibitor PX-866 overcomes resistance to the epidermal growth factor receptor inhibitor gefitinib in A-549 human non-small cell lung cancer xenografts.

Epidermal growth factor receptor (EGFR) inhibitors such as gefitinib show antitumor activity in a subset of non-small cell lung cancer (NSCLC) patients having mutated EGFR. Recent work shows that phosphatidylinositol-3-kinase (PI3-K) is coupled to the EGFR only in NSCLC cell lines expressing ErbB-3 and that EGFR inhibitors do not inhibit PI3-K signaling in these cells. The central role PI3-K plays in cell survival suggests that a PI3-K inhibitor offers a strategy to increase the antitumor activity of EGFR inhibitors in resistant NSCL tumors that do not express ErbB-3. We show that PX-866, a PI3-K inhibitor with selectivity for p110alpha, potentiates the antitumor activity of gefitinib against even large A-549 NSCL xenografts giving complete tumor growth control in the early stages of treatment. A-549 xenograft phospho-Akt was inhibited by PX-866 but not by gefitinib. A major toxicity of PX-866 administration was hyperglycemia with decreased glucose tolerance, which was reversed upon cessation of treatment. The decreased glucose tolerance caused by PX-866 was insensitive to the AMP-activated protein kinase inhibitor metformin but reversed by insulin and by the peroxisome proliferator-activated receptor-gamma activator pioglitazone. Prolonged PX-866 administration also caused increased neutrophil counts. Thus, PX-866, by inhibiting PI3-K signaling, may have clinical use in increasing the response to EGFR inhibitors such as gefitinib in patients with NSCLC and possibly in other cancers who do not respond to EGFR inhibition.

The thioredoxin redox inhibitors 1-methylpropyl 2-imidazolyl disulfide and pleurotin inhibit hypoxia-induced factor 1alpha and vascular endothelial growth factor formation.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays a critical role in tumor growth by increasing resistance to apoptosis and the production of angiogenic factors such as vascular endothelial growth factor (VEGF). HIF-1 is a heterodimer comprised of oxygen-regulated HIF-1alpha and constitutively expressed HIF-1beta subunits. The redox protein thioredoxin-1 (Trx-1), which is found at high levels in many human cancers, increases both aerobic and hypoxia-induced HIF-1alpha protein in cells leading to increased expression of HIF-regulated genes. We have investigated whether two cancer drugs that inhibit Trx-1 signaling, PX-12 (1-methylpropyl 2-imidazolyl disulfide) and pleurotin, decrease HIF-1alpha protein levels and the expression of downstream target genes. Treatment of MCF-7 human breast cancer and HT-29 human colon carcinoma cells with PX-12 and pleurotin prevented the hypoxia (1% oxygen)-induced increase in HIF-1alpha protein. HIF-1-trans-activating activity, VEGF formation, and inducible nitric oxide synthase were also decreased by treatment with PX-12 and pleurotin under hypoxic conditions. PX-12 and pleurotin also decreased HIF-1alpha protein levels and HIF-1 trans-activation in RCC4 renal cell carcinoma cells that constitutively overexpress HIF-1alpha protein because of loss of the pVHL gene, indicating that HIF-1alpha is inhibited independently of the pVHL pathway. HIF-1alpha and VEGF protein levels in MCF-7 tumor xenografts in vivo were decreased by PX-12 treatment of mice. The results suggest that inhibition of HIF-1alpha by Trx-1 inhibitors may contribute to the growth inhibitory and antitumor activity of these agents.

Solubility, ionization, and partitioning behavior of unsymmetrical disulfide compounds: alkyl 2-imidazolyl disulfides.

Alkyl 2-imidazolyl disulfide compounds are novel antitumor agents, one of which is currently being evaluated in Phase I clinical trials. These molecules contain an unsymmetrical disulfide fragment, the lipophilic and electronic contributions of which are still not defined in the literature. Lipophilicity, ionization, and solubility of a number of alkyl 2-imidazolyl disulfides were studied. Based on the additivity of lipophilicity and ionization properties, the contribution of the unsymmetrical disulfide fragment to lipophilicity and ionization was elucidated. The unsymmetrical disulfide fragment contributed a Rekker's hydrophobic constant of 0.761 to the lipophilicity of these compounds and an approximated Hammett constant (sigma) of 0.30 to their ionization. The applicability of the general solubility equation (GSE) proposed by Jain and Yalkowsky in predicting the aqueous solubility of these analogs was evaluated. The GSE correctly ranked the aqueous solubilities of these compounds and estimated their log molar solubilities with an average absolute error of 0.35.

Normal-phase and stability-indicating reversed-phase high-performance liquid chromatographic methods for the determination of the novel antitumor agent: 1-methylpropyl-2-imidazolyldisulfide.

1-Methylpropyl-2-imidazolyl disulfide (MID) is a novel antitumor agent currently in Phase I clinical trials. The chromatographic behavior of MID and its potential impurity, degradation product, and metabolite 2-mercaptoimidazole (2MI) was studied under reversed-phase (RP) and normal-phase (NP) conditions. Both RP- and NP-HPLC separation methods were developed. RP-HPLC was validated as a stability-indicating assay for MID. NP-HPLC retained both MID and 2MI and pending further validation, could prove useful in the study of MID pharmacokinetics.