Small molecule inhibition of the CBFß/RUNX interaction decreases ovarian cancer growth and migration through alterations in genes related to epithelial-to-mesenchymal transition.

OBJECTIVE: Ovarian cancer survival and treatment have improved minimally in the past 20years. Novel treatment strategies are needed to combat this disease. This study investigates the effects of chemical inhibition of the CBFß/RUNX protein-protein interaction on ovarian cancer cell lines. METHODS: Ovarian cancer cell lines were treated with CBFß/RUNX inhibitors, and the effects on proliferation, DNA replication, wound healing, and anchorage-independent growth were measured. RNA-Seq was performed on compound-treated cells to identify differentially expressed genes. Genes altered by compound treatment were targeted with siRNAs, and effects on DNA replication and wound healing were measured. RESULTS: Chemical inhibition of the CBFß/RUNX interaction decreases ovarian cancer cell proliferation. Inhibitor treatment leads to an S-phase cell cycle delay, as indicated by an increased percentage of cells in S-phase, and a decreased DNA replication rate. Inhibitor treatment also reduces wound healing and anchorage-independent growth. RNA-Seq on compound-treated cells revealed changes in a small number of genes related to proliferation and epithelial-to-mesenchymal transition. siRNA-mediated knockdown of INHBA and MMP1 - two genes whose expression decreases with compound treatment - slowed DNA replication and impaired wound healing. CONCLUSIONS: Chemical inhibition of the CBFß/RUNX interaction is a viable strategy for the treatment of ovarian cancer.

Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells.

BACKGROUND: The molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells. METHODS: PC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies. RESULTS: KU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model. CONCLUSIONS: Overall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer.

Sulfobutylether-ß-cyclodextrin.

This review presents the early history, the motivation, the research and some of the backstories behind the discovery and development of sulfobutylether-ß-cyclodextrin as a novel parenterally safe solubilizer and stabilizer. A specific sulfobutylether-ß-cyclodextrin with an average degree of 6.5 sulfobutyl-groups variably substituted on the 2-, 3- and 6-hydroxyls of the seven glucopyranose (dextrose) units of ß-cyclodextrin, is known by its commercial name, Captisol®. Today it is in 13 FDA approved injectables and numerous clinical candidates. It is also an example of a novel product discovered and initially preclinically developed at an academic institution.

KU135, a novel novobiocin-derived C-terminal inhibitor of the 90-kDa heat shock protein, exerts potent antiproliferative effects in human leukemic cells.

The 90-kDa heat shock protein (Hsp90) assists in the proper folding of numerous mutated or overexpressed signal transduction proteins that are involved in cancer. Consequently, there is considerable interest in developing chemotherapeutic drugs that specifically disrupt the function of Hsp90. Here, we investigated the extent to which a novel novobiocin-derived C-terminal Hsp90 inhibitor, designated KU135, induced antiproliferative effects in Jurkat T-lymphocytes. The results indicated that KU135 bound directly to Hsp90, caused the degradation of known Hsp90 client proteins, and induced more potent antiproliferative effects than the established N-terminal Hsp90 inhibitor 17-allylamino-demethoxygeldanamycin (17-AAG). Closer examination of the cellular response to KU135 and 17-AAG revealed that only 17-AAG induced a strong up-regulation of Hsp70 and Hsp90. In addition, KU135 caused wild-type cells to undergo G(2)/M arrest, whereas cells treated with 17-AAG accumulated in G(1). Furthermore, KU135 but not 17-AAG was found to be a potent inducer of mitochondria-mediated apoptosis as evidenced, in part, by the fact that cell death was inhibited to a similar extent by Bcl-2/Bcl-x(L) overexpression or the depletion of apoptotic protease-activating factor-1 (Apaf-1). Together, these data suggest that KU135 inhibits cell proliferation by regulating signaling pathways that are mechanistically different from those targeted by 17-AAG and as such represents a novel opportunity for Hsp90 inhibition.

A tool compound targeting the core binding factor Runt domain to disrupt binding to CBFß in leukemic cells.

The core binding factor (CBF) gene RUNX1 is a target of chromosomal translocations in leukemia, including t(8;21) in acute myeloid leukemia (AML). Normal CBF function is essential for activity of AML1-ETO, product of the t(8;21), and for survival of several leukemias lacking RUNX1 mutations. Using virtual screening and optimization, we developed Runt domain inhibitors which bind to the Runt domain and disrupt its interaction with CBFß. On-target activity was demonstrated by the Runt domain inhibitors' ability to depress hematopoietic cell formation in zebrafish embryos, reduce growth and induce apoptosis of t(8;21) AML cell lines, and reduce progenitor activity of mouse and human leukemia cells harboring the t(8;21), but not normal bone marrow cells. Runt domain inhibitors had similar effects on murine and human T cell acute lymphocytic leukemia (T-ALL) cell lines. Our results confirmed that Runt domain inhibitors might prove efficacious in various AMLs and in T-ALL.

Preliminary evaluation of polymer-based drug composite microparticle production by coacervate desolvation with supercritical carbon dioxide.

Drug/polymer particles incorporating phenytoin in polyvinylpyrrolidone (PVP) were prepared by desolvation of coacervates sprayed through an ultrasonic converging-diverging nozzle into supercritical (SC) carbon dioxide. The mean diameter of the particles produced and the crystallinity of phenytoin in the drug/polymer particles were evaluated with an Aerosizer DSP Particle Size Analyzer and powder X-ray diffraction, respectively. The drug release properties from the composite particles were evaluated using the USP 24 Method 2 rotational paddle method with UV detection. Spraying PVP in ethanol solution into SC carbon dioxide did not produce particles. However, a PVP coacervate in a mixture of ethanol and hexanes had lower viscosity than the solution, and spraying the coacervate into SC carbon dioxide through an ultrasonic converging-diverging nozzle produced micron sized particles. The use of a coacervate containing phenytoin and PVP likely led to increased interaction between drug and polymer and the composite particles contained amorphous phenytoin. The drug content in the composite particles approached theoretical values. The drug release rates from the composite particles produced from the coacervate were faster than those from particles produced by conventional SC methods and complete release was observed.

Small Molecule Inhibitor of CBFß-RUNX Binding for RUNX Transcription Factor Driven Cancers.

Transcription factors have traditionally been viewed with skepticism as viable drug targets, but they offer the potential for completely novel mechanisms of action that could more effectively address the stem cell like properties, such as self-renewal and chemo-resistance, that lead to the failure of traditional chemotherapy approaches. Core binding factor is a heterodimeric transcription factor comprised of one of 3 RUNX proteins (RUNX1-3) and a CBFß binding partner. CBFß enhances DNA binding of RUNX subunits by relieving auto-inhibition. Both RUNX1 and CBFß are frequently mutated in human leukemia. More recently, RUNX proteins have been shown to be key players in epithelial cancers, suggesting the targeting of this pathway could have broad utility. In order to test this, we developed small molecules which bind to CBFß and inhibit its binding to RUNX. Treatment with these inhibitors reduces binding of RUNX1 to target genes, alters the expression of RUNX1 target genes, and impacts cell survival and differentiation. These inhibitors show efficacy against leukemia cells as well as basal-like (triple-negative) breast cancer cells. These inhibitors provide effective tools to probe the utility of targeting RUNX transcription factor function in other cancers.

Chemical biology. A small-molecule inhibitor of the aberrant transcription factor CBFß-SMMHC delays leukemia in mice.

Acute myeloid leukemia (AML) is the most common form of adult leukemia. The transcription factor fusion CBFß-SMMHC (core binding factor ß and the smooth-muscle myosin heavy chain), expressed in AML with the chromosome inversion inv(16)(p13q22), outcompetes wild-type CBFß for binding to the transcription factor RUNX1, deregulates RUNX1 activity in hematopoiesis, and induces AML. Current inv(16) AML treatment with nonselective cytotoxic chemotherapy results in a good initial response but limited long-term survival. Here, we report the development of a protein-protein interaction inhibitor, AI-10-49, that selectively binds to CBFß-SMMHC and disrupts its binding to RUNX1. AI-10-49 restores RUNX1 transcriptional activity, displays favorable pharmacokinetics, and delays leukemia progression in mice. Treatment of primary inv(16) AML patient blasts with AI-10-49 triggers selective cell death. These data suggest that direct inhibition of the oncogenic CBFß-SMMHC fusion protein may be an effective therapeutic approach for inv(16) AML, and they provide support for transcription factor targeted therapy in other cancers.

In vitro and in vivo evaluation of a sulfobutyl ether beta-cyclodextrin enabled etomidate formulation.

In this study, we report the formulation and in vivo evaluation of etomidate in an aqueous solution using sulfobutyl ether-7 beta-cyclodextrin (SBE-CD, Captisol) as a solubilizing agent. The phase-solubility behavior of etomidate as a function of SBE-CD concentration was evaluated, and accelerated solution stability studies of 2 mg/mL etomidate in a 5% w/v SBE-CD solution were conducted. The intravenous administration of the SBE-CD etomidate formulation in dogs was compared with Amidate, the commercial etomidate drug product formulated with propylene glycol as a cosolvent. The etomidate plasma concentration-time data were fit to a three-compartment mamillary model and the derived standard pharmacokinetic parameters were not statistically different between the two formulations (n = 4, p > 0.050). Concurrent pharmacodynamic analysis provided statistically equivalent maximum effects and median inhibitory concentrations for the two formulations. In vivo hemolysis after intravenous administration of Amidate was 10-fold higher than the SBE-CD formulation. Whereas Amidate cannot be given subcutaneously because of the cosolvent in the formulation, a 12 mg/mL aqueous solution of etomidate in 20% (w/v) SBE-CD was well tolerated by this route. The results suggest that the SBE-CD formulation is a viable clinical drug product with a reduced side-effect profile.

Micronization of insulin from halogenated alcohol solution using supercritical carbon dioxide as an antisolvent.

Insulin was precipitated from solution in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) using supercritical carbon dioxide (CO2) as an antisolvent. Biosynthetic human insulin crystals were dissolved in HFIP and the solution was sprayed through an ultrasonic nozzle into supercritical CO2. The factors in the 2(3) factorial experimental design with a center point replicate included pressure (83.7 and 97.5 bar), solution concentration (15 and 30 mg/mL), and solution flow rate (2 and 4 mL/min). Temperature (37 degrees C), CO2 mass flow rate (137 g/min), and volume of solution sprayed (20 mL) were held constant. High-performance liquid chromatography, circular dichroism spectroscopy, infrared and Raman spectroscopy, scanning electron microscopy, dry powder size distribution analysis, thermogravimetric analysis, and atomic absorption spectroscopy were used to characterize the processed insulin powder. The processed insulin retained its potency, was slightly degraded chemically, and exhibited reversible structural changes. The precipitated powder consisted of physical aggregates of 50-nm spheres. Through deagglomeration of these aggregates, it may be possible to obtain discrete uniform particles (1-5 microm) suitable for pulmonary therapy. Over the ranges of operating variables studied, the factors chosen for the experimental design had little effect on the product characteristics.

Applicability of (SBE)7m-beta-CD in controlled-porosity osmotic pump tablets (OPTs).

The purpose of this study was to investigate the general application of a controlled-porosity osmotic pump tablet (OPT) utilizing (SBE)7m-beta-CD as both a solubilizer and an osmotic agent for drugs with varying physical properties. OPTs utilizing (SBE)7m-beta-CD were prepared for five poorly soluble and two highly water-soluble drugs. The Japanese Pharmacopoeia dissolution method was used to study the drug and (SBE)7m-beta-CD release from the OPTs. The drug concentration in the OPT core after the OPT was placed in the release medium for two hours was assayed gravimetrically and by HPLC. An appropriate composition ratio (ACR) of (SBE)7m-beta-CD to drug at which drug release from the OPT was complete and pH-independent within the physiological pH range of the GI tract was determined for each drug. The ACR values correlate to the drug concentration in the OPT core when the OPTs were placed in the release medium for two hours. The release profiles of prednisolone (a poorly water-soluble drug) and sodium chloride (a water-soluble compound) from the OPTs were almost the same as that of (SBE)7m-beta-CD. Also, the release rate of each drug per unit membrane surface area from the OPTs was similar, regardless of the differences in drug solubility. The present results confirmed that (SBE)7m-beta-CD serves as both a solubility modulator and as an osmotic pumping agent for OPTs, from which the release rate of both water-soluble and poorly water-soluble drugs can be controlled.

A second-generation 2-Methoxyestradiol prodrug is effective against Barrett's adenocarcinoma in a mouse xenograft model.

2-Methoxyestradiol (2-ME2) is an endogenous metabolite of estradiol. In preclinical models, 2-ME2 is effective against different types of tumors. Unfortunately, only low systemic concentrations of 2-ME2 can be achieved following oral administration, even after very high doses are administered to patients. In an effort to solve this problem, we have now synthesized and tested a new prodrug of 2-ME2 that is water-soluble due to a bioreversible hydrophilic group added at the 3-position and that more effectively resists metabolic inactivation due to an ester moiety added to mask the 17-position alcohol. We are reporting here for the first time that this double prodrug of 2-ME2 is effective as an antiproliferative and anticancer agent for both in vitro and in vivo studies against Barrett esophageal adenocarcinoma (BEAC) and provided greater potency than 2-ME2 in inhibiting the growth of BEAC xenografts. Finally, studies indicate that, like 2-ME2, the 2-ME2-PD1 exhibits anticancer effect through possible disruption of microtubule network.

Development of a high-throughput screening cancer cell-based luciferase refolding assay for identifying Hsp90 inhibitors.

The 90 kDa heat-shock protein (Hsp90) and other cochaperones allow for proper folding of nascent or misfolded polypeptides. Cancer cells exploit these chaperones by maintaining the stability of mutated and misfolded oncoproteins and allowing them to evade proteosomal degradation. Inhibiting Hsp90 is an attractive strategy for cancer therapy, as the concomitant degradation of multiple oncoproteins may lead to effective anti-neoplastic agents. Unfortunately, early clinical trials have been disappointing with N-terminal Hsp90 inhibitors, as it is unclear whether the problems that plague current Hsp90 inhibitors in clinical trials are related to on-target or off-target activity. One approach to overcome these pitfalls is to identify structurally diverse scaffolds that improve Hsp90 inhibitory activity in the cancer cell milieu. Utilizing a panel of cancer cell lines that express luciferase, we have designed an in-cell Hsp90-dependent luciferase refolding assay. The assay was optimized using previously identified Hsp90 inhibitors and experimental novobiocin analogues against prostate, colon, and lung cancer cell lines. This assay exhibits good interplate precision (% CV), a signal-to-noise ratio (S/N) of ≥7, and an approximate Z-factor ranging from 0.5 to 0.7. Novobiocin analogues that revealed activity in this assay were examined via western blot experiments for client protein degradation, a hallmark of Hsp90 inhibition. Subsequently, a pilot screen was conducted using the Prestwick library, and two compounds, biperiden and ethoxyquin, revealed significant activity. Here, we report the development of an in-cell Hsp90-dependent luciferase refolding assay that is amenable across cancer cell lines for the screening of inhibitors in their specific milieu.

Comparative canine pharmacokinetics-pharmacodynamics of fospropofol disodium injection, propofol emulsion, and cyclodextrin-enabled propofol solution following bolus parenteral administration.

The pharmacokinetics and pharmacodynamics of fospropofol (FP) disodium injection, propofol emulsion (PE), and cyclodextrin-enabled propofol (CDP) solution following bolus parenteral administration in dogs was evaluated. Three healthy male beagle dogs were treated in a three-way cross-over study (14 day washout period) with 6 mg/kg propofol equivalents. Blood samples were collected predose and at 16 points postdose through 1440 min and analyzed for propofol and FP, when appropriate. From 5 min predose to 30 min postdose, brain electrical activity [electroencephalography (EEG)] was recorded and analyzed by power spectrum analysis techniques. Each formulation appeared to be well tolerated with transient discomfort observed in the PE and CDP animals and minor excitability in the FP animals prior to loss of consciousness. Blood propofol followed three-compartment pharmacokinetic behavior and derived parameters were not statistically different except for elimination half-life from the CDP formulation and onset, and duration of anesthesia from the FP formulation. The effect site concentrations at 50% the maximum EEG effect for the FP and CDP formulations were approximately one-half that of the PE formulation. Onset and duration of anesthesia are correlated with modeled effect site propofol concentrations. The implications of formulation on pain on injection and propofol activity are discussed.

Hemodynamic profile in rabbits of fospropofol disodium injection relative to propofol emulsion following rapid bolus injection.

The effects of aqueous fospropofol disodium (FP) and propofol emulsion (PE) on hemodynamics and sympathetic nerve activity in rabbits following bolus injection were evaluated. Barodenervated and neuraxis-intact rabbits received PE at 4 mg/kg (PE(4)) or FP equal to 4 or 8 mg/kg propofol equivalents (FP(4) and FP(8), respectively) intravenously as a rapid bolus injection, and mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were recorded for 20 min. The plasma propofol pharmacokinetic behavior from FP and PE was evaluated to support the pharmacodynamic observations. In barodenervated animals, MAP and RSNA decreased significantly in all groups (PE(4) > FP(8) > FP(4)). HR decreased only in the PE(4) group. The time for the maximum reduction of MAP was significantly longer with FP(8) compared with PE(4). MAP decreased, and RSNA and HR increased significantly in the neuraxis-intact animals (PE(4) > FP(8) > FP(4)). The time for maximum reduction of MAP was essentially the same in all neuraxis-intact groups. Plasma propofol levels from FP were lower than those from PE in the first 4 min following administration. The results suggest that the tachycardia observed in humans following injection of FP is not a direct physiological effect of the agent.

Engineering an antibiotic to fight cancer: optimization of the novobiocin scaffold to produce anti-proliferative agents.

Development of the DNA gyrase inhibitor, novobiocin, into a selective Hsp90 inhibitor was accomplished through structural modifications to the amide side chain, coumarin ring, and sugar moiety. These species exhibit ∼700-fold improved anti-proliferative activity versus the natural product as evaluated by cellular efficacies against breast, colon, prostate, lung, and other cancer cell lines. Utilization of structure-activity relationships established for three novobiocin synthons produced optimized scaffolds, which manifest midnanomolar activity against a panel of cancer cell lines and serve as lead compounds that manifest their activities through Hsp90 inhibition.

Phase 0 clinical chemoprevention trial of the Akt inhibitor SR13668.

SR13668, an orally active Akt pathway inhibitor, has demonstrated cancer chemopreventive potential in preclinical studies. To accelerate the clinical development of this promising agent, we designed and conducted the first-ever phase 0 chemoprevention trial to evaluate and compare the effects of food and formulation on SR13668 bioavailability. Healthy adult volunteers were randomly assigned to receive a single, 38-mg oral dose of SR13668 in one of five different formulations, with or without food. On the basis of existing animal data, SR13668 in a PEG400/Labrasol oral solution was defined as the reference formulation. Blood samples were obtained pre- and post-agent administration for pharmacokinetic analyses. Area under the plasma concentration-time curve (AUC(0-∞)) was defined as the primary endpoint. Data were analyzed and compared using established statistical methods for phase 0 trials with a limited sample size. Participants (n = 20) were rapidly accrued over a 5-month period. Complete pharmacokinetic data were available for 18 randomized participants. AUC(0-∞) values were highest in the fed state (range = 122-439 ng/mL × hours) and were statistically significantly different across formulations (P = 0.007), with Solutol HS15 providing the highest bioavailability. SR13668 time to peak plasma concentration (3 hours; range, 2-6 hours) and half-life were (11.2 ± 3.1 hours) were not formulation-dependent. Using a novel, highly efficient study design, we rapidly identified a lead formulation of SR13668 for further clinical testing. Our findings support application of the phase 0 trial paradigm to accelerate chemoprevention agent development.

Inhibiting heat-shock protein 90 reverses sensory hypoalgesia in diabetic mice.

Increasing the expression of Hsp70 (heat-shock protein 70) can inhibit sensory neuron degeneration after axotomy. Since the onset of DPN (diabetic peripheral neuropathy) is associated with the gradual decline of sensory neuron function, we evaluated whether increasing Hsp70 was sufficient to improve several indices of neuronal function. Hsp90 is the master regulator of the heat-shock response and its inhibition can up-regulate Hsp70. KU-32 (N-{7-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-2-yloxy]-8-methyl-2-oxo-2H-chromen-3-yl}acetamide) was developed as a novel, novobiocin-based, C-terminal inhibitor of Hsp90 whose ability to increase Hsp70 expression is linked to the presence of an acetamide substitution of the prenylated benzamide moiety of novobiocin. KU-32 protected against glucose-induced death of embryonic DRG (dorsal root ganglia) neurons cultured for 3 days in vitro. Similarly, KU-32 significantly decreased neuregulin 1-induced degeneration of myelinated Schwann cell DRG neuron co-cultures prepared from WT (wild-type) mice. This protection was lost if the co-cultures were prepared from Hsp70.1 and Hsp70.3 KO (knockout) mice. KU-32 is readily bioavailable and was administered once a week for 6 weeks at a dose of 20 mg/kg to WT and Hsp70 KO mice that had been rendered diabetic with streptozotocin for 12 weeks. After 12 weeks of diabetes, both WT and Hsp70 KO mice developed deficits in NCV (nerve conduction velocity) and a sensory hypoalgesia. Although KU-32 did not improve glucose levels, HbA1c (glycated haemoglobin) or insulin levels, it reversed the NCV and sensory deficits in WT but not Hsp70 KO mice. These studies provide the first evidence that targeting molecular chaperones reverses the sensory hypoalgesia associated with DPN.