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PDF file - 59KB, Rat 2-ME2 and 2-ME2-PD1 dosing summary

Perspectives on This Article from Phase 0 Clinical Chemoprevention Trial of the Akt Inhibitor SR13668

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. Mol Cancer Ther; 12(3); 255–63. ©2012 AACR.

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 CBFβ/RUNX protein-protein interaction on ovarian cancer cell lines. Methods Ovarian cancer cell lines were treated with CBFβ/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 CBFβ/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 CBFβ/RUNX interaction is a viable strategy for the treatment of ovarian cancer.

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 CBFβ. 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.

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 CBFβ binding partner. CBFβ enhances DNA binding of RUNX subunits by relieving auto-inhibition. Both RUNX1 and CBFβ 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 CBFβ 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.

Graphical abstract, This review presents the early history, the motivation, the research and some of the backstories behind the discovery and development of sulfobutylether-β-cyclodextrin as a novel parenterally safe solubilizer and stabilizer. A specific sulfobutylether- β -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 β -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.

Toward drugging the undruggable in cancer Many human cancers are characterized by inappropriate activity of transcription factors. These proteins are attractive drug targets in principle, but normalizing their function requires drugs that modulate specific protein-protein interactions, a goal that has been challenging. In acute myeloid leukemia, a chromosomal translocation creates an aberrant form of the transcription factor CBF-beta, which outcompetes “normal” CBF-beta for binding to another transcription factor called RUNX1, thereby deregulating its activity. Illendula et al. identified and chemically optimized a small molecule that selectively disrupts the interaction between the aberrant CBF-beta and RUNX1 (see the Perspective by Koehler and Chen). This molecule restored normal gene expression patterns and delayed leukemia progression in mice. Thus, transcription factors may not be as undruggable as once thought. Science , this issue p. 779 ; see also p. 713

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.

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.

A Wurster-type coater employing near-critical carbon dioxide as the fluidizing medium and antisolvent was developed for pharmaceutical applications. Suitably designed glass inserts housed in a high-pressure chamber allow for the controlled fluidization and circulation of the substrates with dense CO 2 . The CO 2 also removes the solvent from the coating solution sprayed on the substrates, thereby precipitating the coating as a smooth layer. A variety of substrates were successfully coated with thin polymer films for aesthetic/handling purposes or for delayed release applications such as taste masking. The geometry of substrates include glass and microcrystalline cellulose (MCC) beads ranging in diameter from 0.5 to 2 mm, tablets up to 7.9 mm and cylindrical stents up to 20 mm in length. The demonstrated coating process expands the range of substrate/coating combinations possible with the conventional air-fluidized Wurster coater, making it feasible to coat water-soluble substrates with solutes sprayed from organic solutions at near-ambient temperatures wherein degradation of the active pharmaceutical ingredient is avoided. Coating with dense CO 2 enables the use of traditional organic solvents with no risk of explosion.

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 mid-nanomolar activity against a panel of cancer cell lines and serve as lead compounds that manifest their activities through Hsp90 inhibition.

The human body is continuously exposed to small organic molecules containing one or more basic nitrogen atoms. Many of these are endogenous (i.e., neurotransmitters, polyamines and biogenic amines), while others are exogenously supplied in the form of drugs, foods and pollutants. It is well-known that many amines have a strong propensity to specifically and substantially accumulate in highly acidic intracellular compartments, such as lysosomes, through a mechanism referred to as ion trapping. It is also known that cells have acquired the unique ability to sense and respond to amine accumulation in lysosomes in an effort to prevent potential negative consequences associated with hyperaccumulation. We describe here methods that are used to evaluate the dynamics of amine accumulation in, and egress from, lysosomes. Moreover, we highlight specific proteins that are thought to play important roles in these pathways. A theoretical model describing lysosomal amine dynamics is described and shown to adequately fit experimental kinetic data. The implications of this research in understanding and treating disease are discussed.

The pharmacokinetics of DS-96, an N-alkylhomospermine analog designed to sequester bacterial lipopolysaccharides, has been determined in rodent species. The elimination half-life in mice and rats are about 400 and 500 min, respectively, with other PK parameters being quite similar in the two rodent species. Interestingly, the mouse intravenous plasma concentration time curves exhibit an apparent absorption phase. While the rat intravenous data did not exhibit a pronounced apparent absorption phase immediately following injection, plasma levels did increase between 10 and 30 min following an expected drop from time 0 to 5 min. The data are consistent with first-pass uptake, possibly by the lung, with back diffusion as a function of time. The observed C(max) values of 1.36 microg/mL in the mouse intraperitoneal model suggest that a plasma concentration of 0.5-1 microg/mL corresponds to complete protection for a 200 ng/animal dose of intraperitoneally administered LPS in the D-galactosamine-primed model of endotoxin-induced lethality.

Objective To evaluate and compare the effects of the aqueous sulfobutyl ether β -cyclodextrin (SBE-CD) etomidate formulation and the commercial etomidate formulation on mean arterial pressure (MAP), heart rate, and sympathetic outflow using neuraxis-intact and baro-denervated rabbits. Study design Prospective experimental study. Animals Twenty-seven male New Zealand white rabbits. Methods Under basal anesthesia (urethane) and ventilation with intermittent positive pressure (IPPV), the New Zealand white rabbits underwent surgical preparation including isolation of the left renal sympathetic nerve and, in the baro-denervated models, additional combined denervation of bilateral carotid sinus, aortic, and vagal nerves. After hemodynamic stabilization, both neuraxis-intact and baro-denervated animals received bolus intravenous (IV) injection (0.6 mg kg −1 ) of either the SBE-CD or commercial etomidate formulation over 5 seconds ( n = 6 animals per group). Results Mean arterial pressure decreased significantly in all four groups to the same extent. However, the rate of MAP decrease was lower in the SBE-CD-treated groups relative to the commercial formulation. Renal sympathetic nerve activity was decreased significantly less in the SBE-CD group in the baro-denervated studies. Independent pharmacokinetic evaluation demonstrated that the two formulations had comparable plasma concentration–time profiles. Conclusions and Clinical relevance Etomidate in the commercial drug product is solubilized with propylene glycol, a cosolvent associated with adverse side effects on injection. An aqueous formulation of etomidate has been developed, which utilizes SBE-CD as a solubilizing agent. The data suggest that the SBE-CD etomidate formulation may be a safer IV induction formulation than the commercial etomidate drug product.

Most drug discovery efforts for Alzheimer's disease (AD) have focused on prevention or clearance of beta-amyloid (Abeta) fibrils or oligomers, with far less attention to prevention of tau abnormalities that lead to neurofibrillary tangles (NFTs). Much evidence now indicates that Abeta multimers can trigger neurodegenerative changes that involve formation of dystrophic neurites and cytoskeletal collapse, possibly due loss of microtubule (MT) stabilization by the tau protein. We have found that several MT-stabilizing agents such as Taxol significantly enhanced neuronal survival in the presence of Abeta and identified agents that enter the brain, a necessity for in vivo testing in animal models of tau pathology. Studies were designed to test two agents in the tau mutant (JNPL3) mouse that develops severe motor deficits at about seven months of age, accompanied by neuropathological markers of tau pathology. In addition to using motor performance tests through the planned period of drug administration, we designed a simple appetitive memory test that required a reduction in ad lib food intake. Although the neurochemical data are still being analyzed, we were surprised to find that all of the JNPL3 mice, whether receiving the drug or not, developed no signs of motor impairment up to 10 months of age. This is considerably beyond the age at which free-fed mice survived and suggests that the food restriction alone may have delayed the pathological process. A study is ongoing with free-fed mice to determine if the drug interventions do have any beneficial effects in these mutant mice.

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.

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.

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.

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 CBFβ binding partner. CBFβ enhances DNA binding of RUNX subunits by relieving auto-inhibition. Both RUNX1 and CBFβ 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 CBFβ 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., Graphical Abstract Image 10, Highlights • Small molecule inhibitors of CBFβ-RUNX protein-protein interaction developed. • Inhibitors alter occupancy of RUNX1 on target genes and alter their expression. • Inhibitors show efficacy against leukemia cell lines and basal-like (triple negative) breast cancer cell lines. Transcription factors are proteins that bind to DNA and regulate how much of other proteins are made. We describe the development of inhibitors of the interaction between two transcription factors, CBFβ and RUNX. Both these transcription factors are the targets of alterations in human leukemia as well as in a number of solid tumors. The inhibitor changes the behavior of the RUNX transcription factor and alters the levels of proteins it regulates. We show these inhibitors may have potential utility for leukemia as well as one specific type of breast cancer which has a very poor prognosis.

The development and validation of an effective and simplified LC assay for the quantitation of etomidate in beagle plasma is described. The methodology employs a rapid and simple protein precipitation procedure in combination with previously reported chromatographic conditions. Using a 0.3 ml aliquot of plasma, the assay is linear in the concentration range of 50 to 5000 ng/ml, with an extraction efficiency between 97 to 104% and accuracy between 98 and 105%.

The purpose of this study was to evaluate and compare the potential use of two parenterally safe β‐cyclodextrins derivatives, (SBE) 7m ‐β‐CD and HP‐β‐CD, as solubilizers and stabilizers for melphalan and carmustine, two very unstable antineoplastic agents. Phase solubility and chemical stability of the compounds in the presence of the cyclodextrins were studied. UV, fluorescence, and several NMR techniques were used to probe the potential causes for the differences observed. The phase solubility method was found to provide only qualitative data on the binding of melphalan to the cyclodextrins since rapid degradation and the presence of products of degradation complicated the interpretation of the results. Qualitatively, however, the solubilizing potential was similar for the two cyclodextrins. The chemical stability studies indicate that both of the drugs had similar binding constants for both cyclodextrins; however, the intrinsic reactivities in the complexes were significantly lower with (SBE) 7m ‐β‐CD than for HP‐β‐CD. The main cause for this distinct difference appeared to correlate with differences in the site of binding and the polarity of the binding site. © 2000 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: 275–287, 2000

Purpose. To understand the role of degree of substitutionon binding of molecules to β-Cyclodextrins (β-CDs) with varyingdegrees of sulfobutyl ether (SBE) substitution.

Purpose: The purpose of this study was to define membrane controlling factors responsible for drug release from a controlled-porosity osmotic pump tablet (OPT) that utilizes a sulfobutyl ether-β-cyclodextrin, (SBE)7m-β-CD, as both a solubilizing and osmotic agent. Method: The OPT was spray coated with cellulose acetate solutions varying the amount and size of micronized lactose, the amount of triethyl citrate (TEC) and the composition ratio of dichlormethane to ethanol. Chlorpromazine (CLP) was used as a model drug. The release of CLP from the OPTs was studied using the Japanese Pharmacopoeia dissolution method. The membrane surface area of the OPTs were measured with multi-point analysis by the gas absorption method. Results: The release rate of CLP from OPTs containing (SBE)7m-β-CD increased with increasing amounts of micronized lactose and decreasing amounts of TEC and lactose particle size in the membrane. Also, the CLP release rates from the spray-coated OPTs using mixtures of varying ratios of dichlormethane to ethanol were almost identical. The membrane surface area of the OPTs following release of membrane components had a linear relationship to CLP release rates from the OPTs. Conclusion: The present results confirmed that the membrane controlling factors responsible for the drug release were the amount and size of micronized lactose and the amount of TEC in the membrane.

A controlled porosity osmotic pump system for poorly water soluble drugs has been developed using sulfobutyl ether-beta-cyclodextrin sodium salt, (SBE)7m-beta-CD, which can act as both a solubilizing and an osmotic agent. The release of testosterone, a poorly water soluble drug (0.039 mg/ml at 37 degrees C), was evaluated using a new model device. The effect of (SBE)7m-beta-CD as the solubilizing and osmotic pump agent was compared with hydroxypropyl-beta-cyclodextrin (HP-beta-CD), a neutral cyclodextrin, and a sugar mixture (osmotic agent only). Testosterone release from the device was significantly faster with (SBE)7m-beta-CD than with HP-beta-CD or the sugar mixture. The solubility of testosterone in the device increased to 76.7 mg/ml through complexation with (SBE)7m-beta-CD in the imbibed water. It appears that testosterone release from the device in the presence of (SBE)7m-beta-CD was mainly due to osmotic pumping while for HP-beta-CD the major contribution appears to be due to diffusion. In the case of the sugar mixture, testosterone was poorly released, presumably due to the absence of a solubilizer. Therefore, it was concluded that (SBE)7m-beta-CD provides novel properties for the development of controlled- porosity osmotic pump tablets for poor solubility drugs.

Replacement of traditional solvents with "environmentally benign" carbon dioxide is receiving increased attention in pharmaceutical processing. Among the reported applications, particle formation with dense carbon dioxide and the "clean" synthesis of drug compounds using carbon dioxide as a reaction medium hold immense potential for large-scale application in the pharmaceutical industry. This paper provides an overview of these rapidly emerging technologies along with examples of the wide variety of relatively contaminant-free pharmaceutical compounds that have been processed via these technologies on a laboratory scale. Challenges facing successful implementation in practice include demonstration of continuous production and harvesting of particles with desired and reproducible product characteristics. Mathematical models aimed at a better fundamental understanding of the underlying thermophysical phenomena are essential for rational design and scale-up of these technologies.

The substitution profile of 4-sulfobutyl ether derivatives of cyclomaltoheptaose (β-cyclodextrin) (SBE-β-CD) prepared in our laboratories has been previously described. However, in those studies, no attempt was made to characterize the positional or regional isomers of this material. SBE-β-CD derivatives with degrees of substitution of two or higher represent a large number of possible isomers dependent on this positional and regional substitution. The monosubstituted SBE derivative, however, cannot have regional isomers and, therefore, has only three possible substitution products related to the 2-, 3-, and 6-hydroxyl groups of a glucose unit. In this study the isomers were fractionated by preparative anion-exchange chromatography with the progress of the elution being followed by a capillary electrophoretic (CE) method that resolved these isomers. The eluent containing the isomers was processed, and the pure materials were characterized by nuclear magnetic resonance spectroscopy ( 1 H NMR, DEPT, HETCOR, HOHAHA). Through this analysis the assignment of the positional isomers was made. Three isomeric monosubstituted 4-sulfobutyl ether derivatives of cyclomaltoheptaose (β-cyclodextrin) were separated by anion-exchange chromatography and characterized by 2D NMR spectroscopy.

This study was designed to test how the sulfoalkyl ether (SAE) modification of beta-cyclodextrin (beta-CD) affects the binding capacity of testosterone and progesterone, thereby enhancing their solubility. The SAE-beta-CD derivatives contain either sulfopropyl ether (SPE) or sulfobutyl ether (SBE) groups on the 2-, 3-, and 6-hydroxyl positions of the dextrose moieties. SAE-beta-CDs are a mixture of positional and regional isomers containing from one to as many as 12 SAE groups per CD. The effect of chain length and the degree of substitution on complexation behavior was investigated by the phase-solubility method. The results were compared with those obtained with beta-CD, where possible, and with hydroxypropyl-beta-CD (HP-beta-CD). To determine the effect of degree of substitution (DS) on the binding, mixtures of SAE-beta-CDs with multiple substitution levels and varying average degrees of substitution were studied as well as mixtures of SAE-beta-CDs that contained the same degree of substitution. Mixtures that contained SAE-beta-CDs of the same degree of substitution were isolated from the multiple substitution level mixtures by ion-exchange chromatography and purified for investigation. Unlike the parent beta-CD, linear increases in the apparent solubilities of testosterone and progesterone were observed, and the binding potentials were comparable to those of beta-CD or better. The results demonstrate that the binding potentials of the SAE-beta-CD derivatives were dependent on the guest molecule, the degree of substitution, and the alkyl ether chain length. Our previous study showed the inhibition of complexation by direct sulfonation of the beta-CD. However, in the present work, interferences with the charged sulfonate groups were avoided by repositioning them away from the cavity. Increasing the degree of substitution assisted in complex formation; however, its effects were limited. Reduction of the alkyl chain length, as in the case of SPE-beta-CD compared with SBE-beta-CD, decreased the complexation potential. This decrease in complexation potential was further suppressed with an increase in the number of substituents placed on the CD torus. Generally, the binding potential of SAE-beta-CD derivatives increased with increasing alkyl chain length. However, placement of more than an optimum number of SAE groups on the CD torus resulted in inhibition of complexation.

Since their discovery, cyclodextrins and their ability to form inclusion complexes have fascinated chemists, formulators and recently, entrepreneurs. This mini-review has as its objective, a critical assessment of the current status of cyclodextrins in the formulation and delivery of pharmaceuticals and commentary on their potential future uses. The emphasis will be on answers to common questions often asked of pharmaceutical scientists working in this area. Why use cyclodextrins for drug solubilization and stabilization when alternative techniques are available? Why the greater interest in modified cyclodextrins and not the parent cyclodextrins? If a drug forms a strong cyclodextrin inclusion complex, how is the drug releasedin vivo? Does the injection of a cyclodextrin/drug complex alter the pharmacokinetics of the drug? Are there drug products on the market which contain cyclodextrins? What is the regulatory status of cyclodextrins? Although definitive answers to all these questions are not possible at this time, many of these questions are answerable, and educated and informed responses are possible for the rest.

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.

A capillary electrophoresis (CE) method for the analysis of a sulfobutyl ether beta-cyclodextrin (SBE-beta-CD) mixture is described. The SBE-beta-CD has been prepared as a parenterally safe solubilizing agent and has historically been characterized by elemental analysis and nuclear magnetic resonance spectroscopy. While these methods provide gross values for the degree of substitution, the CE method described resolves the mixture of positional and regional isomers based on the degree of SBE substitution. The method uses benzoic acid in the running buffer and detects the CD by a decrease in background absorbance of the benzoic acid due to complexation. The necessity of a defined column was sequence between injections was evaluated. The reproducibility of migration times and peak areas/heights for 10 components of the mixture was determined. The modular CE system gave a relative standard deviation of 2.5% (n = 3) for six of the 10 peaks. Further refinements (pH buffer effects) were explored to improve the reproducibility with remaining components. The method was used to evaluate the reproducibility of the synthesis (21 different lots) and the effect of reaction variables (time, temperature and base) on the composition pattern of the modified CD.

Purpose. The objective of this work was to determine the role that charge might play in the interaction of charged and uncharged drugs with neutral (2-hydroxypropyl-β-cyclodextrin, HP-β-CD) and anionically charged (SBE7-β-CD) modified β-cyclodextrins. SBE7-β-CD is a sulfobutyl ether, sodium salt, derivative variably substituted on the 2-, 3- and the 6-positions of β-cyclodextrin. The number seven refers to the average degree of substitution.

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. Mol Cancer Ther; 12(3); 255–63. ©2012 AACR.

SPC-100270 drug substance ((2S,3S)-2-aminooctadecane-1,3-diol) is a synthetic sphingosine analogue possessing limited ultraviolet absorbance (UV) and two chiral centres. Analytical methodology employing derivatization of SPC-100270 with o-phthaldialdehyde (OPA) and a chiral thiol, N-acetyl-D-penicillamine, was developed to assess the isomeric purity of SPC-100270 in the presence of two of its three stereoisomers. Separation of the isoindole derivatives was achieved with isocratic reversed-phase column chromatography with detection at 330 nm. Under the conditions studied, the derivatization was complete within 3 h at 50 degrees C. The standard and derivatized solutions were stable for 7 days under refrigerated conditions. The limit of detection was 0.036 micrograms SPC-100270 per ml and the limit of quantitation was 0.237 micrograms SPC-100270 per ml. The assay response was linear over a concentration range of 4.7-376 micrograms SPC-100270 per ml with a coefficient of determination of greater than 0.9999. The precision, ruggedness and specificity of the assay were acceptable for determination of SPC-100270 in the presence of its stereoisomers and under forced degradation conditions. The method has been applied successfully in two independent laboratories for quality control release and stability assessment of SPC-100270 drug substance for early clinical studies.

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.

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-[(2 R, 3 R, 4S, 5 R)-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.

A capillary electrophoresis method which characterizes the degrees of substitution of heterogeneous sulphoalkyl ether β-cyclodextrin derivatives is described. The separation is based on the different electrophoretic mobilities observed from changes in the overall charge of the molecule as a result of substitution. Individual peaks of the electropherogram then provide a measure of each degree of substitution of the parent β-cyclodextrin. Detection of these β-cyclodextrin derivatives is performed by indirect UV detection.

SR13668 (2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo-(2,3-b)carbazole), is a new candidate cancer chemopreventive agent under development. It was designed using computational modeling based on a naturally occurring indole-3-carbinol and its in vivo condensation products. It showed promising anti-cancer activity and its preclinical toxicology profile (genotoxicity battery and subchronic rat and dog studies) was unremarkable. However, it exhibited a very poor oral bioavailability (

Amifostine is a prodrug of its free thiol, active metabolite, WR-1065. The thiophosphate promoiety protects the free thiol group of WR-1065 from oxidation to the symmetrical disulfide, WR-33278, while providing a handle for high levels of WR-1065 production at the target tissue. The thiophosphate promoiety of amifostine is cleaved by alkaline phosphatase to produce WR-1065, which accumulates in non-cancerous tissues to a greater extent than in cancerous tissues (Capizzi, 1996).

The leukemia fusion protein CBFβ-SMMHC, associated with acute myeloid leukemia (AML) with chromosome inversion inv(16)(p13q22), is a driver mutation in leukemia development. Studies by our laboratory and others have established that CBFβ-SMMHC outcompetes CBFβ for binding to RUNX1, deregulates RUNX1 transcription factor activity in hematopoiesis, and induces AML. Studies in mice and patient AML cells support the concept that CBFβ-SMMHC generates pre-leukemic myeloid progenitors, which acquire cooperating mutations to progress to leukemia. Current inv(16) AML treatment using non-selective cytotoxic chemotherapy results in a good initial response, but long-term survival is approximately 60%. This suggests that additional efforts are necessary for the development of improved therapeutic response for CBF AML patients. We have identified AI-4-57 as the active compound that inhibits CBFβ-SMMHC/RUNX1 binding (IC50= 22 uM), using a screen of the NCI diversity set library. AI-4-57 specifically binds to the CBFβ portion of CBFβ-SMMHC, as determined by NMR. This compound was modified in order to improve its potency and stability, and identified the divalent derivative AI-10-49 for further characterization. AI-10-49 showed increased potency (IC50= 0.26 µM) improved in vivo pharmacokinetics (serum t1/2 = 380 min), and enhanced activity in inv(16) positive ME-1 cells (IC50 = 0.6 uM). Importantly, AI-10-49 showed negligible activity (IC50>25 μM) in normal human bone marrow, defining a robust potential therapeutic window. Co-immunoprecipitation assays of ME-1 cells demonstrated that AI-10-49 (1µM for six hours) effectively and specifically dissociated RUNX1/CBFβ-SMMHC when compared to CBFβ/RUNX1 binding (Meandiss: 90% and 15%, respectively). Expression of RUNX1 target genes RUNX3, CSF1R, and CEBPA is repressed by CBFβ-SMMHC in inv(16) AML. The occupancy of RUNX1 in their promoters was significantly increased by chromatin-immunoprecipitation (8, 2.2, and 8 fold, respectively) in 6 hour treated (1µM AI-10-49) ME-1 cells, suggesting that CBFβ-SMMHC represses RUNX1 targets by blocking RUNX1 binding to target regulatory sites. In addition, RUNX3, CSF1R, and CEBPA expression increased 2 to 8 fold when compared to DMSO treated ME-1 cells. Importantly, RUNX1 occupancy and target expression changes were not observed in inv(16)-negative U937 cells. These data establish AI-10-49 selectivity in inhibiting CBFβ-SMMHC binding to RUNX1 and validate our approach of using bivalent inhibitors to achieve this specificity. To test AI-10-49 activity in vivo, mice were transplanted with leukemic cells expressing CBFβ-SMMHC and NrasG12D (from Cbfb+/MYH11:Ras+/G12Dknock-in mice), and treated, starting at day five post transplantation, with vehicle (DMSO) or 200 mg/kg AI-10-49 for ten days. The median latency of leukemia was delayed one fold in AI-10-49 treated mice (MLAI-10-49= 61 days, MLDMSO= 33.5 days, P=2.7x10-6; Log-rank test). In addition, toxicity assays revealed no detectable cumulative toxicity in mice treated with AI-10-49 for seven days. To test the efficacy of AI-10-49 in human inv(16) AML, the survival of four inv(16) and four normal karyotype AML patient samples were tested in 48 hour dose response assays. The viability of inv(16) patient cells was clearly reduced by AI-10-49 (viability: 50%, 10 μM AI-10-49/DMSO). In contrast, the viability of normal karyotype AML samples was unaffected at concentrations below 20µM. These studies show that AI-10-49 selectively inhibits viability in inv(16) AML blasts, while having negligible effects on AML blasts with normal karyotype or on normal human hematopoietic progenitors. Dysregulated gene expression is a hallmark of cancer and is particularly important for the maintenance of cancer stem cells, such as self-renewal, leading to relapse. The targeting of proteins that drive transcriptional dysregulation, so called “transcription therapy”, represents an avenue for drug development with immense potential. This study reports the development of a small molecule with high efficacy and specific in the inhibition of CBFβ-SMMHC activity while having a minimal effect on CBFβ function. In summary, AI-10-49 is a potent first generation CBFβ-SMMHC inhibitor that induces cell death in inv(16) AML cells and establishes a proof-of-principle that transcription factor fusion oncoproteins can be directly targeted for leukemia treatment. Disclosures No relevant conflicts of interest to declare.