High Dose "HDR-Like" Prostate SBRT: PSA 10-Year Results From a Mature, Multi-Institutional Clinical Trial.

Purpose/Objectives: Although ample intermediate-term prostate stereotactic body radiotherapy (SBRT) outcomes have been reported, 10-year results remain relatively sparse. Materials/Methods: Eighteen institutions enrolled 259 low- and intermediate-risk patients. Median follow-up is 5.5 years, with 66 patients followed ≥ 10 years. This SBRT regimen specifically emulated an existing HDR brachytherapy dose schedule and isodose morphology, prescribed to 38 Gy/4 fractions, delivered daily by robotic SBRT, mandating > 150% dose escalation in the peripheral zone. Androgen deprivation therapy was not allowed, and a hydrogel spacer was not available at that time. Results: Median pre-SBRT PSA 5.12 ng/mL decreased to 0.1 ng/mL by 3.5 years, with further decrease to a nadir of < 0.1 ng/mL by 7 years, maintained through 10 years. Ten-year freedom from biochemical recurrence measured 100% for low-risk, 84.3% for favorable intermediate risk (FIR), and 68.4% for unfavorable intermediate (UIR) cases. Multivariable analysis revealed that the UIR group bifurcated into two distinct prognostic subgroups. Those so classified by having Gleason score 4 + 3 and/or clinical stage T2 (versus T1b/T1c) had a significantly poorer 10 year freedom from biochemical recurrence rate, 54.8% if either or both factors were present, while UIR patients without these specific factors had a 94.4% 10-year freedom from biochemical recurrence rate. The cumulative incidence of grade 2 GU toxicity modestly increased over time - 16.3% at 5 years increased to 19.2% at 10 years-- while the incidence of grade 3+ GU and GI toxicity remained low and stable to 10 years - 2.6% and 0%, respectively. The grade 2 GI toxicity incidence also remained low and stable to 10 years - 4.1% with no further events after year 5. Conclusion: This HDR-like SBRT regimen prescribing 38 Gy/4 fractions but delivering much higher intraprostatic doses on a daily basis is safe and effective. This treatment achieves a median PSA nadir of <0.1 ng/mL and provides high long-term disease control rates without ADT except for a subgroup of unfavorable intermediate-risk patients.

Continuous synthesis of nano-drug particles by antisolvent crystallization using a porous hollow-fiber membrane module.

The synthesis of nano-size drug particles by antisolvent crystallization using a porous hollow fiber membrane provides promising benefits such as the capability of continuous operation, low energy input, and ease of scale-up for a variety of industrial processes. Porous hollow fiber membranes have also been shown to produce more efficient mixing than conventional mixing equipment mostly because in mixing binary fluids, they provide sufficient mixing time, retention time, and a large contact interface for the drug solution and the antisolvent, allowing for the precise control of nucleation and crystal growth necessary to form nano-size particles. This study reports an experimental and numerical approach to obtain a further understanding of the fundamental principles of antisolvent crystallization using a porous hollow fiber membrane. This includes producing a particle size-controlled drug nanosuspension experimentally using a commercial microfiltration (MF) pencil scale module, and a numerical analysis of mixing behavior using a computational fluid dynamics (CFD) simulation. From the results obtained, a nanosuspension of a model drug, Indomethacin, with particles of average diameter 0.320 µm was prepared. Furthermore, this nanosuspension has higher stability and a much lower tendency to agglomerate as compared to simple mixing of the anti-solvent and drug solution. Results from the numerical simulation showed that micromixing is possible using the porous hollow fiber membrane even under the most compromising conditions.

Membrane-Based Technologies in the Pharmaceutical Industry and Continuous Production of Polymer-Coated Crystals/Particles.

BACKGROUND: Membrane technologies are of increasing importance in a variety of separation and purification applications involving liquid phases and gaseous mixtures. Although the most widely used applications at this time are in water treatment including desalination, there are many applications in chemical, food, healthcare, paper and petrochemical industries. This brief review is concerned with existing and emerging applications of various membrane technologies in the pharmaceutical and biopharmaceutical industry. METHODS: The goal of this review article is to identify important membrane processes and techniques which are being used or proposed to be used in the pharmaceutical and biopharmaceutical operations. How novel membrane processes can be useful for delivery of crystalline/particulate drugs is also of interest. RESULTS: Membrane separation technologies are extensively used in downstream processes for bio-pharmaceutical separation and purification operations via microfiltration, ultrafiltration and diafiltration. Also the new technique of membrane chromatography allows efficient purification of monoclonal antibodies. Membrane filtration techniques of reverse osmosis and nanofiltration are being combined with bioreactors and advanced oxidation processes to treat wastewaters from pharmaceutical plants. Nanofiltration with organic solvent-stable membranes can implement solvent exchange and catalyst recovery during organic solvent-based drug synthesis of pharmaceutical compounds/intermediates. Membranes in the form of hollow fibers can be conveniently used to implement crystallization of pharmaceutical compounds. The novel crystallization methods of solid hollow fiber cooling crystallizer (SHFCC) and porous hollow fiber anti-solvent crystallization (PHFAC) are being developed to provide efficient methods for continuous production of polymer-coated drug crystals in the area of drug delivery. CONCLUSION: This brief review provides a general introduction to various applications of membrane technologies in the pharmaceutical/biopharmaceutical industry with special emphasis on novel membrane techniques for pharmaceutical applications. The method of coating a drug particle with a polymer using the SHFCC method is stable and ready for scale-up for operation over an extended period.

Continuous preparation of polymer coated drug crystals by solid hollow fiber membrane-based cooling crystallization.

A facile way to continuously coat drug crystals with a polymer is needed in controlled drug release. Conventional polymer coating methods have disadvantages: high energy consumption, low productivity, batch processing. A novel method for continuous polymer coating of drug crystals based on solid hollow fiber cooling crystallization (SHFCC) is introduced here. The drug acting as the host particle and the polymer for coating are Griseofulvin (GF) and Eudragit RL100, respectively. The polymer's cloud point temperature in its acetone solution was determined by UV spectrophotometry. An acetone solution of the polymer containing the drug in solution as well as undissolved drug crystals in suspension were pumped through the tube side of the SHFCC device; a cold liquid was circulated in the shell side to rapidly cool down the feed solution-suspension in the hollow-fiber lumen. The polymer precipitated from the solution and coated the suspended crystals due to rapid temperature reduction and heterogeneous nucleation; crystals formed from the solution were also coated by the polymer. Characterizations by scanning electron microscopy, thermogravimetric analysis, laser diffraction spectroscopy, X-ray diffraction, Raman spectroscopy, and dissolution tests show that a uniformly coated, free-flowing drug/product can be obtained under appropriate operating conditions without losing the drug's pharmaceutical properties and controlled release characteristics.

Continuous synthesis of polymer-coated drug particles by porous hollow fiber membrane-based antisolvent crystallization.

Using porous hollow fiber membranes, this study illustrates a novel technique to continuously synthesize polymer-coated drug crystals by antisolvent crystallization. The synthesized polymer-coated drug crystals involve crystals of the drug Griseofulvin (GF) coated by a thin layer of the polymer Eudragit RL100. The process feed, an acetone solution of the drug GF containing the dissolved polymer, was passed through the shell side of a membrane module containing many porous hollow fibers of Nylon-6. Through the lumen of the hollow fibers, the antisolvent water was passed at a higher pressure to inject water jets through every pore in the fiber wall into the shell-side acetone feed solution, creating an extremely high level of supersaturation and immediate crystallization. It appears that the GF crystals are formed first and serve as nuclei for the precipitation of the polymer Eudragit, which forms a thin coating around the GF crystals. The polymer-coated drug crystals were collected by a filtration device at the shell-side outlet of the membrane module, and the surface morphology, particle size distribution, and the polymer coating thickness were then characterized by scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM), laser diffraction spectroscopy (LDS), and thermogravimetric analysis (TGA). To study the properties of the coated drug crystals, X-ray diffraction (XRD), Raman spectroscopy, and dissolution tests were implemented. These results indicate that a polymer-coated, free-flowing product was successfully developed under appropriate conditions in this novel porous hollow fiber antisolvent crystallization (PHFAC) method. The coated drug particles can be potentially used for controlled release. The molecular and the crystal structures of GF were not affected by the PHFAC method, which may be easily scaled up.

Continuous polymer nanocoating on silica nanoparticles.

Continuous polymer coating of nanoparticles is of interest in many industries such as pharmaceuticals, cosmetics, food, and electronics. Here we introduce a polymer coating/precipitation technique to achieve a uniform and controllable nanosize polymer coating on nanoparticles in a continuous manner. The utility of this technique is demonstrated by coating Aerosil silica nanoparticles (SNPs) of diameter 12 nm with the polymer Eudragit RL 100. Both hydrophilic and hydrophobic SNPs were successfully coated. After determining the cloud point of an acetone solution of the polymer containing a controlled amount of the nonsolvent water, the solid hollow fiber cooling crystallization (SHFCC) technique was employed to continuously coat SNPs with the polymer. A suspension of the SNPs in an acetone-water solution of the polymer containing a surfactant was pumped through the lumen of solid polypropylene hollow fibers in a SHFCC device; cold liquid was circulated on the shell side. Because of rapid cooling-induced supersaturation and heterogeneous nucleation, precipitated polymers will coat the nanoparticles. The thickness and morphology of the nanocoating and the particle size distribution of the coated SNPs were analyzed by scanning transmission electron microscopy (STEM) with electron energy loss spectroscopy (EELS), thermogravimetric analysis (TGA), and dynamic light scattering (DLS). Results indicate that uniformly polymer-coated SNPs can be obtained from the SHFCC device after suitable post-treatments. The technique is also easily scalable by increasing the number of hollow fibers in the SHFCC device.

Gamma knife radiosurgery for typical trigeminal neuralgia: An institutional review of 108 patients.

BACKGROUND: In this study, we present the previously unreported pain relief outcomes of 108 patients treated at Gamma Knife of Spokane for typical trigeminal neuralgia (TN) between 2002 and 2011. METHODS: Pain relief outcomes were measured using the Barrow Neurological Institute (BNI) pain intensity scale. In addition, the effects gender, age at treatment, pain laterality, previous surgical treatment, repeat Gamma Knife radiosurgery (GKRS), and maximum radiosurgery dose have on patient pain relief outcomes were retrospectively analyzed. Statistical analysis was performed using Andersen 95% confidence intervals, approximate confidence intervals for log hazard ratios, and multivariate Cox proportional hazard models. RESULTS: All 108 patients included in this study were grouped into BNI class IV or V prior to GKRS. The median clinical follow-up time was determined to be 15 months. Following the first GKRS procedure, 71% of patients were grouped into BNI class I-IIIb (I = 31%; II = 3%; IIIa = 19%; IIIb = 18%) and the median duration of pain relief for those patients was determined to be 11.8 months. New facial numbness was reported in 19% of patients and new facial paresthesias were reported in 7% of patients after the first GKRS procedure. A total of 19 repeat procedures were performed on the 108 patients included in this study. Following the second GKRS procedure, 73% of patients were grouped into BNI class I-IIIb (I = 44%; II = 6%; IIIa = 17%, IIIb = 6%) and the median duration of pain relief for those patients was determined to be 4.9 months. For repeat procedures, new facial numbness was reported in 22% of patients and new facial paresthesias were reported in 6% of patients. CONCLUSIONS: GKRS is a safe and effective management approach for patients diagnosed with typical TN. However, further studies and supporting research is needed on the effects previous surgical treatment, number of radiosurgery procedures, and maximum radiosurgery dose have on GKRS clinical outcomes.

G proteins and autocrine signaling differentially regulate gonadotropin subunit expression in pituitary gonadotrope.

Gonadotropin-releasing hormone (GnRH) acts at gonadotropes to direct the synthesis of the gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH). The frequency of GnRH pulses determines the pattern of gonadotropin synthesis. Several hypotheses for how the gonadotrope decodes GnRH frequency to regulate gonadotropin subunit genes differentially have been proposed. However, key regulators and underlying mechanisms remain uncertain. We investigated the role of individual G proteins by perturbations using siRNA or bacterial toxins. In LßT2 gonadotrope cells, FSHß gene induction depended predominantly on Gα(q/11), whereas LHß expression depended on Gα(s). Specifically reducing Gα(s) signaling also disinhibited FSHß expression, suggesting the presence of a Gα(s)-dependent signal that suppressed FSH biosynthesis. The presence of secreted factors influencing FSHß expression levels was tested by studying the effects of conditioned media from Gα(s) knockdown and cholera toxin-treated cells on FSHß expression. These studies and related Transwell culture experiments implicate Gα(s)-dependent secreted factors in regulating both FSHß and LHß gene expression. siRNA studies identify inhibinα as a Gα(s)-dependent GnRH-induced autocrine regulatory factor that contributes to feedback suppression of FSHß expression. These results uncover differential regulation of the gonadotropin genes by Gα(q/11) and by Gα(s) and implicate autocrine and gonadotrope-gonadotrope paracrine regulatory loops in the differential induction of gonadotropin genes.

Fluidization of nanopowders: a review.

Nanoparticles (NPs) are applied in a wide range of processes, and their use continues to increase. Fluidization is one of the best techniques available to disperse and process NPs. NPs cannot be fluidized individually; they fluidize as very porous agglomerates. The objective of this article is to review the developments in nanopowder fluidization. Often, it is needed to apply an assistance method, such as vibration or microjets, to obtain proper fluidization. These methods can greatly improve the fluidization characteristics, strongly increase the bed expansion, and lead to a better mixing of the bed material. Several approaches have been applied to model the behavior of fluidized nanopowders. The average size of fluidized NP agglomerates can be estimated using a force balance or by a modified Richardson and Zaki equation. Some first attempts have been made to apply computational fluid dynamics. Fluidization can also be used to provide individual NPs with a thin coating of another material and to mix two different species of nanopowder. The application of nanopowder fluidization in practice is still limited, but a wide range of potential applications is foreseen. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11051-012-0737-4) contains supplementary material, which is available to authorized users.

Polymeric nanocomposites of functionalized carbon nanotubes synthesized in supercritical CO2.

A novel method to synthesize single-wall carbon nanotube (SWNT)/poly(methyl methacrylate) (PMMA) nanocomposite by in-situ polymerization in supercritical CO2 is presented. The surfaces of the SWNT bundles were first functionalized with amino ethyl methacrylate (AEMA) followed by co-polymerization with methyl methacrylate. Supercritical fluid enhanced the diffusivity of monomer and facilitated the growth of tethered PMMA chains near the entanglement area and the interstitial space of the SWNT bundles. Partial debundling and disentanglement of the SWNT bundles and an enhanced dispersion in the polymer matrix were observed under SEM and TEM. After the removal of the polymer matrix physically attached to the nanotubes, it is found that the nanotubes were covered by tethered PMMA chains, which were a few nanometers in thickness. This work creates a route for improving impregnation and dispersion in SWNT composites; the same process can be extended to other vinyl polymers.

Freeze-dried sperm fertilization leads to full-term development in rabbits.

To date, the laboratory mouse is the only mammal in which freeze-dried spermatozoa have been shown to support full-term development after microinjection into oocytes. Because spermatozoa in mice, unlike in most other mammals, do not contribute centrosomes to zygotes, it is still unknown whether freeze-dried spermatozoa in other mammals are fertile. Rabbit sperm was selected as a model because of its similarity to human sperm (considering the centrosome inheritance pattern). Freeze- drying induces rabbit spermatozoa to undergo dramatic changes, such as immobilization, membrane breaking, and tail fragmentation. Even when considered to be "dead" in the conventional sense, rabbit spermatozoa freeze-dried and stored at ambient temperature for more than 2 yr still have capability comparable to that of fresh spermatozoa to support preimplantation development after injection into oocytes followed by activation. A rabbit kit derived from a freeze-dried spermatozoon was born after transferring 230 sperm-injected oocytes into eight recipients. The results suggest that freeze-drying could be applied to preserve the spermatozoa from most other species, including human. The present study also raises the question of whether rabbit sperm centrosomes survive freeze-drying or are not essential for embryonic development.

Coupling of GnRH concentration and the GnRH receptor-activated gene program.

The initial waves of gene induction caused by GnRH in the LbetaT2 gonadotrope cell line have recently been identified using microarrays. We now investigate the relationship of the concentration of GnRH to the level of biosynthesis induced. Using an optimized custom cDNA microarray, we show that a large number of genes are induced in a concentration-dependent fashion. Detailed time course studies of the induction of six induced transcripts using quantitative real-time PCR suggest that the amplitude, but not the temporal pattern, depends on the concentration of GnRH. The early genes appear to show a delay in gene induction, followed by a linear phase of increase. The relationship of rate of synthesis and GnRH concentration was studied by mathematical modeling of the induction of two genes, gly96 and tis11. In both cases, only the rates of increase, but not the lag times, are influenced by the concentration of GnRH exposure. Western blot analyses for c-Jun and Egr1 show that the levels of nuclear protein for these transcription factors also depend on the concentration of GnRH. These studies indicate that, despite the complex signaling network connecting the receptor to the activated genes, the biosynthetic rate of RNA polymerase at induced genes is correlated with the concentration of GnRH at the GnRH receptor.

Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays.

We compared the accuracy of microarray measurements obtained with oligonucleotide arrays (GeneChip, Affymetrix) with a laboratory-developed cDNA array by assaying test RNA samples from an experiment using a paradigm known to regulate many genes measured on both arrays. We selected 47 genes represented on both arrays, including both known regulated and unregulated transcripts, and established reference relative expression measurements for these genes in the test RNA samples using quantitative reverse transcriptase real-time PCR (QRTPCR) assays. The validity of the reproducible (average coefficient of variation = 11.8%) QRTPCR measurements were established through application of a new mathematical model. The performance of both array platforms in identifying regulated and non-regulated genes was identical. With either platform, 16 of 17 definitely regulated genes were correctly identified, and no definitely unregulated transcript was falsely identified as regulated. Accuracy of the fold-change measurements obtained with each platform was assessed by determining measurement bias. Both platforms consistently underestimate the relative changes in mRNA expression between experimental and control samples. The bias observed with cDNA arrays was predictable for fold-changes <250-fold by QRTPCR and could be corrected by the calibration function F(c) = F(a(cDNA))(q), where F(a(cDNA)) is the microarray-determined fold-change comparing experimental with control samples, q is the correction factor and F(c) is the calibrated value. The bias observed with the commercial oligonucleotide arrays was less predictable and calibration was unfeasible. Following calibration, fold-change measurements generated by custom cDNA arrays were more accurate than those obtained by commercial oligonucleotide arrays. Our study demonstrates systematic bias of microarray measurements and identifies a calibration function that improves the accuracy of cDNA array data.