A phase II/III randomized study to compare the efficacy and safety of rigosertib plus gemcitabine versus gemcitabine alone in patients with previously untreated metastatic pancreatic cancer.

BACKGROUND: Rigosertib (ON 01910.Na), a first-in-class Ras mimetic and small-molecule inhibitor of multiple signaling pathways including polo-like kinase 1 (PLK1) and phosphoinositide 3-kinase (PI3K), has shown efficacy in preclinical pancreatic cancer models. In this study, rigosertib was assessed in combination with gemcitabine in patients with treatment-naïve metastatic pancreatic adenocarcinoma. MATERIALS AND METHODS: Patients with metastatic pancreatic adenocarcinoma were randomized in a 2:1 fashion to gemcitabine 1000 mg/m(2) weekly for 3 weeks of a 4-week cycle plus rigosertib 1800 mg/m(2) via 2-h continuous IV infusions given twice weekly for 3 weeks of a 4-week cycle (RIG + GEM) versus gemcitabine 1000 mg/m(2) weekly for 3 weeks in a 4-week cycle (GEM). RESULTS: A total of 160 patients were enrolled globally and randomly assigned to RIG + GEM (106 patients) or GEM (54). The most common grade 3 or higher adverse events were neutropenia (8% in the RIG + GEM group versus 6% in the GEM group), hyponatremia (17% versus 4%), and anemia (8% versus 4%). The median overall survival was 6.1 months for RIG + GEM versus 6.4 months for GEM [hazard ratio (HR), 1.24; 95% confidence interval (CI) 0.85-1.81]. The median progression-free survival was 3.4 months for both groups (HR = 0.96; 95% CI 0.68-1.36). The partial response rate was 19% versus 13% for RIG + GEM versus GEM, respectively. Of 64 tumor samples sent for molecular analysis, 47 were adequate for multiplex genetic testing and 41 were positive for mutations. The majority of cases had KRAS gene mutations (40 cases). Other mutations detected included TP53 (13 cases) and PIK3CA (1 case). No correlation between mutational status and efficacy was detected. CONCLUSIONS: The combination of RIG + GEM failed to demonstrate an improvement in survival or response compared with GEM in patients with metastatic pancreatic adenocarcinoma. Rigosertib showed a similar safety profile to that seen in previous trials using the IV formulation.

Resistance of Streptococcus pneumoniae to deformylase inhibitors is due to mutations in defB.

Resistance to peptide deformylase inhibitors in Escherichia coli or Staphylococcus aureus is due to inactivation of transformylase activity. Knockout experiments in Streptococcus pneumoniae R6x indicate that the transformylase (fmt) and deformylase (defB) genes are essential and that a def paralog (defA) is not. Actinonin-resistant mutants of S. pneumoniae ATCC 49619 harbor mutations in defB but not in fmt. Reintroduction of the mutated defB gene into wild-type S. pneumoniae R6x recreates the resistance phenotype. The altered enzyme displays decreased sensitivity to actinonin.

A fluorescence-based homogeneous assay for measuring activity of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase.

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is one of the key enzymes of bacterial lipid A biosynthesis, catalyzing the removal of the N-acetyl group of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine. The lpxC gene is essential in Gram-negative bacteria but absent from mammalian genomes, making it an attractive target for antibacterial drug discovery. Current assay methods for LpxC are not suitable for high throughput screening, since they require multiple product separation steps and the use of radioactively labeled material that is difficult to prepare. A homogeneous fluorescence-based assay was developed that uses UDP-3-O-(N-hexyl-propionamide)-N-acetylglucosamine as a surrogate substrate. This surrogate can be prepared from commercially available UDP-GlcNAc by enzymatic conversion to UDP-MurNAc, which is then chemically coupled to n-hexylamine. Following the LpxC reaction, the free amine of the deacetylation product can be derivatized by fluorescamine, thus generating a fluorescent signal. This surrogate substrate has a K(m) of 367 microM and k(cat) of 0.36 s(-1), compared to 2 microM and 1.5 s(-1) for the natural substrate. Since no separation is needed, the assay is easily adaptable to high throughput screening. IC(50)s of LpxC inhibitors determined using this assay method is similar to those measured by traditional method with the natural substrate.

Regulation of the alpha-galactosidase activity in Streptococcus pneumoniae: characterization of the raffinose utilization system.

A 10.2-kb gene region was identified in the Streptococcus pneumoniae genome sequence that contains eight genes involved in regulation and metabolism of raffinose. The genes rafR and rafS are transcribed as one operon, and their gene products regulate the raffinose-dependent stimulation of a divergently transcribed second promoter (P(A)) directing the expression of aga, the structural gene for alpha-galactosidase. Raffinose-mediated transcription from P(A) results in a 500-fold increase in alpha-galactosidase activity in the cell. A third promoter within the cluster is responsible for the transcription of the remaining five genes (rafE, rafF, rafG, gtfA, and rafX), whose gene products might be involved in transport and metabolism of raffinose. The presence of additional internal promoters cannot be excluded. The aga promoter P(A) is negatively regulated by the presence of sucrose in the growth medium. Consistent with catabolite repression (CR), a DNA sequence with high homology to the CRE (cis-active element) was identified upstream of the aga promoter. Sucrose-mediated CR depends on the phosphoenolpyruvate: sucrose phosphotransferase system (PTS) but is unaffected by a mutation in a gene encoding a homolog of the CRE regulatory protein CcpA.

Biodegradable polyanhydride devices of cefazolin sodium, bupivacaine, and taxol for local drug delivery: preparation, and kinetics and mechanism of in vitro release.

The overall objective was to design and evaluate biodegradable implants for local drug delivery in clinical conditions and/or diseases described below, which are currently treated with systemic administration of drugs. Local delivery of cefazolin is desired in conditions such as osteomyelitis, soft-tissue infection and for prevention of post-surgical infections. Similarly, implanting a biodegradable device loaded with taxol in the cavity created by tumor resection will provide high local concentrations of taxol killing the malignant cells which may have survived the surgery, thus preventing metastasis and regrowth of the tumor and also prevent the systemic side effects of taxol. Prolonged reversible nerve blockade required in a number of clinical situations involving acute or chronic pain such as post-surgical pain following herniorrhaphy and thoracotomy can be achieved with local delivery of bupivacaine. Therefore, disk-shaped implants of polyanhydride, P(FAD-SA, 50:50 w/w), loaded with 10% w/w of cefazolin sodium, taxol and bupivacaine were prepared and evaluated for content uniformity and in vitro release characteristics for the above mentioned local drug delivery applications. All of cefazolin sodium was released in 14 days while 90% bupivacaine was released in 35 days. In striking contrast, taxol was released very slowly, and only 15% taxol was released in 77 days. The overall release appeared to be following first order kinetics, and the initial linear profile was fitted to zero order kinetics to obtain release parameters. Since cefazolin is highly water soluble and bupivacaine is moderately water soluble, compared to taxol which is extremely lipophilic, the aqueous solubility of the incorporated drug appeared to influence in release characteristics. Very good correlation was observed between release parameters (Ao, ko) and the solubility and intrinsic dissolution rate (IDR) of drugs suggesting that the hydrophilic/hydrophobic nature of the drug influences its release from polyanhydride devices. Since polyanhydrides are believed to undergo pure surface erosion, release of the incorporated drug should be independent of its physicochemical properties, however the results presented in this study suggest otherwise. Therefore, P(FAD-SA, 50:50 w/w) may not be undergoing surface erosion, and the diffusion and dissolution properties of the drug in addition to erosion characteristics of the polyanhydride appear to play a role in drug release. Implants prepared and evaluated in this study released cefazolin, bupivacaine and taxol for a prolonged duration of time; however, depending upon the desired duration of release, an appropriate polyanhydride will have to be selected. For example, taxol was released so slowly that a more hydrophilic polyanhydride may have to be selected to release all the drug in a shorter period of time to be of any therapeutic use. Cefazolin implants released the drug for a sufficient duration for osteomyelitis and soft-tissue infection but the release was more prolonged than required for prevention of post-surgical wound infection.

Insect repellent formulations of N,N-diethyl-m-toluamide (deet) in a liposphere system: efficacy and skin uptake.

Novel formulations for a deet in liposphere microdispersion in the form of lotion were prepared from natural solid triglycerides and phospholipids dispersed in buffer solution. The formulations containing 6.5, 10, and 20% deet were effective as a repellent against the common aggressive biting mosquitoes, Aedes aegypti and Anopheles stephensi, for up to 6 h. The acute dermal absorption of the 10% loaded formulation was conducted in rabbits using 14C-labeled deet. 14C-labeled deet, 10% in alcohol solution or in liposphere microdispersion was applied to the intact rabbit skin under a porous nonirritating cover for 7 days. Plasma levels of radioactivity were determined for 24 h, and daily for a total of 7 days. The 14C-deet blood levels following intravenous bolus administration were also measured. The bioavailability of deet from 10% ethanol solution was 45%, whereas the bioavailability of deet from lipospheres was 16%, a 3-fold reduction in the amount of deet absorbed. Examination of the rabbits during the experiment and after necropsy showed no evidence of toxicity or irritation. The 10% deet-liposphere formulation was stable at room temperature for at least 1 year.

Alkaline hydrolysis of oligomers of tartrate esters: effect of a neighboring carboxyl on the reactivity of ester groups.

The importance and the effect of neighboring groups on the hydrolysis of polymeric esters were demonstrated. Several oligomers of poly(butylene tartrate) were synthesized, and their kinetic behavior was studied under alkaline conditions. The oligomers and their degradation products were monitored by HPLC and identified by fast atom bombardment mass spectrometry. The rates of hydrolysis measured at pH 5-8 and at 75 degrees C indicate that the degradation of the oligomers obeyed pseudo-first-order kinetics. The rate of hydrolysis among the homologous series of oligomers increased as the molecular weight increased. However, the increase in hydrolysis rate was not proportional to the number of ester linkages in the oligomers. This deviation was explained on the basis of electrostatic repulsion of the neighboring carboxylate group toward the hydroxide ion. The calculations revealed that the electrostatic effect was so great that the ester linkage adjacent to the carboxylate anion did not contribute to the overall rate of hydrolysis. The technique presented here can be extended to any multifunctional-group compound that has repeat units and can undergo a specific reaction that can be accurately measured.

Anesthetic activity of the lipospheres bupivacaine delivery system in the rat.

The Lipospheres Bupivacaine Delivery System (bupivacaine-lipospheres) is a novel sustained-release local anesthetic preparation that has recently been made available for research purposes. This investigation compared the local anesthetic efficacy and safety of 2% bupivacaine-lipospheres, 0.5% bupivacaine plus 1:200,000 epinephrine, lipospheres plain, and physiologic saline following subcutaneous tail injection in the rat. A modified tail-flick paradigm was used to assess local anesthetic efficacy. Animals treated with 2% bupivacaine-lipospheres or 0.5% bupivacaine with epinephrine displayed significant antinociception (P < 0.05) compared to saline or lipospheres plain with 5 min of injection. Bupivacaine with epinephrine had an anesthetic duration of 30 min, whereas 2% bupivacaine-lipospheres had a duration of 3 hr. The local anesthetic blockade produced by both active solutions was completely reversible. All animals gained weight normally during the 1-wk course of the study, and there were no signs of local tissue toxicity at the injection sites. We conclude that 2% bupivacaine-lipospheres is a safe and efficacious local anesthetic preparation in this particular animal model. It possesses an onset of action that is a rapid as 0.5% bupivacaine with 1:200,000 epinephrine, and a duration that is six times longer.

Determination of specific rate constants of specific oligomers during polyester hydrolysis.

Aliphatic polyesters have great utility as temporary prosthetics and surgical aids, and in drug delivery systems. Knowledge of the mechanism and pathways of hydrolysis can form a basis for the design and selection of a controlled-release system. Because of the importance of hydrolysis to the properties of such a system, a technique was developed to accurately determine the specific rate constants and relevant kinetic parameters for the specific oligomers from the reaction mixture of polyesters undergoing hydrolysis. A detailed kinetic analysis of the acid hydrolysis of well-characterized oligomers of polyesters is presented. Several oligomers of poly(butylene tartrate)s were synthesized and their kinetic behavior was studied under acidic conditions. The oligomers and their degradation products were monitored by HPLC and identified by fast-atom bombardment mass spectrometry. From the rates of hydrolysis measured from pH 1 to 3.0 and at temperature of 75 degrees C, it becomes evident that the degradation of the oligomers obeys pseudo-first-order kinetics. The rate of hydrolysis among the homologous series of oligomers increases as the molecular weight increases. The hydrolysis was catalyzed by hydrogen ion; the catalytic rate constant increased predictably with the number of ester linkages present in the molecule. The reaction is first order with respect to the catalyst concentration and the number of ester linkages. Good agreement was obtained with a model in which the individual rate of bond cleavage depends only on the statistical factor. The technique described in this paper is unique for the determination of polyester hydrolysis pathways and isolation of any structural effects on the rates of hydrolysis.

Drug delivery to the brain using polymers.

The delivery of drugs to the brain has been a major challenge to the scientist developing drugs designed for central nervous system (CNS) activity. One of the obstacles to the progress is the transport of drug through the blood brain barrier (BBB). The criteria for effective drug delivery to the CNS include the following: (a) the drug must have access to the brain, (b) the effect of the drug should be localized, (c) the drug must be stable, and (d) the effective dose should be sustained and controlled. To meet some of the above criteria, two approaches have been used: systemic administration of drugs, and direct delivery of drugs into the brain. The systemic administration of drugs relies on passive diffusion of drug through the BBB, formation of lipid soluble prodrugs and the use of monoclonal antibodies for targeting the drug to the CNS. The other approach includes the use of implantable polymer systems and infusion pumps. Both of the approaches have some advantages and disadvantages. Because of the enormous amount of literature on drug delivery to the brain, the following review focuses on the use of polymer-based implantable systems. The review includes nondegradable and biodegradable polymer implants from the conceptual phase to the clinic.

Polyanhydrides. V. Branched polyanhydrides.

The objective of the present study was to investigate the properties of branched polyanhydrides and compare them to the corresponding linear polymers. Sebacic acid was polymerized with 1,3,5 benzenetricarboxylic acid and poly(acrylic acid) to yield random and graft-type branched polyanhydrides. The polymerization was followed until the gel point and the resulting polymers were evaluated for their physico-chemical properties and degradation behaviour. Drug release from these polymers was studied using morphine as a model drug. The experiment showed that the molecular weights of branched polyanhydride were significantly higher (mol wt 250,000) than the molecular weight of linear poly(sebacic anhydride) (mol wt 80,000). In the case of poly(acrylic acid) branched polymers, the molecular weight increased linearly with increasing concentration of poly(acrylic acid). The specific viscosities of the branched polyanhydrides were lower than linear polyanhydrides with similar molecular weights. Except for the difference in molecular weights, there were no noticeable changes in the physico-chemical or thermal properties of the branched polymers and the linear poly(sebacic anhydride). The degradation of the branched polyanhydride was triphasic and the degradation rates were faster than for linear poly(sebacic anhydride). The release of morphine from the branched polymers was lower than the corresponding poly(sebacic anhydride). Release of morphine was much higher from the poly(acrylic acid) branched polymers compared to the 1,3,5 benzenetricarboxylic acid branched polymers and increased with increasing concentrations of the branching agent. However, in both cases the release rates and the total amounts of morphine released approached that of poly(sebacic anhydride).

Use of liquid chromatography and mass spectroscopy to select an oligomer representative of polyester hydrolysis pathways.

An analytical method has been developed for the simultaneous determination of a series of low molecular weight biodegradable polyesters and its degradation products. The separation is accomplished using a strong anion exchange HPLC column. The polyester and its degradation products are identified by fast atom bombardment mass spectrometry. This technique will enable one to establish the polyester hydrolysis pathways and determine accurate kinetic parameters.