Simultaneous pharmacokinetic modeling of unbound and total darunavir with ritonavir in adolescents: a substudy of the SMILE trial.

Darunavir (DRV) is an HIV protease inhibitor commonly used as part of antiretroviral treatment regimens globally for children and adolescents. It requires a pharmacological booster, such as ritonavir (RTV) or cobicistat. To better understand the pharmacokinetics (PK) of DRV in this younger population and the importance of the RTV boosting effect, a population PK substudy was conducted within SMILE trial, where the maintenance of HIV suppression with once daily integrate inhibitor + darunavir/ritonavir in children and adolescents is evaluated. A joint population PK model that simultaneously used total DRV, unbound DRV, and total RTV concentrations was developed. Competitive and non-competitive models were examined to define RTV's influence on DRV pharmacokinetics. Linear and non-linear equations were tested to assess DRV protein binding. A total of 443 plasma samples from 152 adolescents were included in this analysis. Darunavir PK was best described by a one-compartment model first-order absorption and elimination. The influence of RTV on DRV pharmacokinetics was best characterized by ritonavir area under the curve on DRV clearance using a power function. The association of non-linear and linear equations was used to describe DRV protein binding to alpha-1 glycoprotein and albumin, respectively. In our population, simulations indicate that 86.8% of total and unbound DRV trough concentrations were above 0.55 mg/L [10 times protein binding-adjusted EC50 for wild-type (WT) HIV-1] and 0.0243 mg/L (10 times EC90 for WT HIV-1) targets, respectively. Predictions were also in agreement with observed outcomes from adults receiving 800/100 mg DRV/r once a day. Administration of 800/100 mg of DRV/r once daily provides satisfactory concentrations and exposures for adolescents aged 12 years and older.

Cu(II)-porphyrin molecular dynamics as seen in a novel EPR/Stochastic Liouville equation study.

Copper porphyrin dissolved in CH2Cl2:toluene as fluid and frozen solution was studied as a function of temperature using X-band electron paramagnetic resonance (EPR). Quantitative interpretation was obtained using a recently developed Stochastic Liouville simulation method. For the first time we address the large spin system that translates into a 400,000 dimensional Liouville equation solved under slow-motion conditions. Using a simple three parameter microscopic model, the temperature dependence of porphyrin rotational correlation time is determined to be in the range 1-10 ns and a fast local motion is in the subpico-second regime with an amplitude increasing with temperature. The methodology provides an important tool for arriving at an accurate set of spin Hamiltonian parameters since determining a unique set of parameters from a frozen solution EPR experiment is often difficult. Thus, the proposed method discriminates between parameters proposed from frozen solution EPR experiments or quantum chemistry calculations. The methodology presented is expected to be valuable in obtaining a molecular dynamics picture of metal proteins using EPR as well as in the study of artificial photosynthetic systems.

DNA as supramolecular scaffold for functional molecules: progress in DNA nanotechnology.

Oligonucleotides have recently gained increased attraction as a supramolecular scaffold for the design and synthesis of functional molecules on the nanometre scale. This tutorial review focuses on the recent progress in this highly active field of research with an emphasis on covalent modifications of DNA; non-covalent interactions of DNA with molecules such as groove binders or intercalators are not part of this review. Both terminal and internal modifications are covered, and the various points of attachment (nucleobase, sugar moiety or phosphodiester backbone) are compared. Using selected examples of the recent literature, the diversity of the functionalities that have been incorporated into DNA strands is discussed.

Reconstruction of 3D lung models from 2D planning data sets for Hodgkin's lymphoma patients using combined deformable image registration and navigator channels.

PURPOSE: Late complications (cardiac toxicities, secondary lung, and breast cancer) remain a significant concern in the radiation treatment of Hodgkin's lymphoma (HL). To address this issue, predictive dose-risk models could potentially be used to estimate radiotherapy-related late toxicities. This study investigates the use of deformable image registration (DIR) and navigator channels (NCs) to reconstruct 3D lung models from 2D radiographic planning images, in order to retrospectively calculate the treatment dose exposure to HL patients treated with 2D planning, which are now experiencing late effects. METHODS: Three-dimensional planning CT images of 52 current HL patients were acquired. 12 image sets were used to construct a male and a female population lung model. 23 "Reference" images were used to generate lung deformation adaptation templates, constructed by deforming the population model into each patient-specific lung geometry using a biomechanical-based DIR algorithm, MORFEUS. 17 "Test" patients were used to test the accuracy of the reconstruction technique by adapting existing templates using 2D digitally reconstructed radiographs. The adaptation process included three steps. First, a Reference patient was matched to a Test patient by thorax measurements. Second, four NCs (small regions of interest) were placed on the lung boundary to calculate 1D differences in lung edges. Third, the Reference lung model was adapted to the Test patient's lung using the 1D edge differences. The Reference-adapted Test model was then compared to the 3D lung contours of the actual Test patient by computing their percentage volume overlap (POL) and Dice coefficient. RESULTS: The average percentage overlapping volumes and Dice coefficient expressed as a percentage between the adapted and actual Test models were found to be 89.2 +/- 3.9% (Right lung = 88.8%; Left lung = 89.6%) and 89.3 +/- 2.7% (Right = 88.5%; Left = 90.2%), respectively. Paired T-tests demonstrated that the volumetric reconstruction method made a statistically significant improvement to the population lung model shape (p < 0.05). The error in the results were also comparable to the volume overlap difference observed between inhale and exhale lung volumes during free-breathing respiratory motion (POL: p = 0.43; Dice: p = 0.20), which implies that the accuracies of the reconstruction method are within breathing constraints and would not be the confining factor in estimating normal tissue dose exposure. CONCLUSIONS: The result findings show that the DIR-NC technique can achieve a high degree of reconstruction accuracy, and could be useful in approximating 3D dosimetric representations of historical 2D treatment. In turn, this could provide a better understanding of the biophysical relationship between dose-volume exposure and late term radiotherapy effects.

Duplex stabilization and energy transfer in zipper porphyrin-DNA.

Zip it up: Attachment of porphyrins onto complementary DNA strands leads to zipper-porphyrin arrays and, in the presence of eleven modifications, an increase in the melting temperature of the duplex. Mixed zinc and free-base porphyrin arrays undergo energy transfer from the zinc porphyrin to the free-base porphyrin in the annealed duplex but not in the denatured form (see scheme), giving access to reversible formation of potential photonic wires.

Supramolecular helical porphyrin arrays using DNA as a scaffold.

A diphenyl porphyrin substituted nucleotide was incorporated site specifically into DNA, leading to helical stacked porphyrin arrays in the major groove of the duplexes. The porphyrins show an electronic interaction which is significantly enhanced compared to the analogous tetraphenyl porphyrin (TPP) as shown in the large exciton coupling of the porphyrin B-band absorbance. Analogous to the TPP-DNA, an induced helical secondary structure is observed in the single strand porphyrin-DNA. The modified DNA can be hybridised to an immobilised complementary strand leading to fluorescent beads.

Targeting of a novel fusion protein containing methioninase to the urokinase receptor to inhibit breast cancer cell migration and proliferation.

It has been shown that methionine depletion inhibits tumor cell growth and reduces tumor cell survival. A novel fusion protein targeted specifically to tumor cells was developed. The fusion protein contained two components: the amino terminal fragment of human urokinase (amino acids 1-49) that binds to the urokinase receptor protein expressed on the surface of invasive cancer cells, and the enzyme L-methioninase (containing 398 amino acids) which depletes methionine and arrests the growth of methionine-dependent tumors. The influence of the fusion protein on the growth and motility of human breast cancer cells was examined using a culture wounding assay. It was determined that MCF-7 breast cancer cells, used in this study, were methionine-dependent and that the fusion protein bound specifically to urokinase receptors of the surface of the cancer cells. Further treatment of the cancer cells with fusion protein over the concentration range 10(-8) to 10(-6) M produced a dose-dependent inhibition of both the migration and proliferation index of MCF-7 cells in the culture wounding assay over a period of 1 to 3 days. The results of this study suggest that this novel fusion protein may serve as a prototype for specific targeting of methioninase and perhaps other cytotoxic agents to cancer cells.

Specific and non-specific KGF inhibition of KGF-induced breast cancer cell motility.

BACKGROUND: Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, is a mesenchymally derived mediator of epithelial cell proliferation and migration. In a previous study, we reported that KGF enhanced the motility of human breast cancer cells. The objective of the present study was to examine the influence of specific and non-specific KGF inhibitors on KGF-induced motility and proliferation in ER-positive MCF-7 cells. MATERIALS AND METHODS: In the present study three KGF inhibitors were employed [Heparin, Innohep, a low molecular weight heparin (LMWH) and KGFR2 beta (IIIb)/Fc, a chimeric KGFR fragment]. Heparin and LMWH bind to low affinity sites on KGF and produce non-specific inhibition, while KGFR2 beta (IIIb)/Fc, a soluble chimera of an extracellular KGFR fragment, is a more specific KGF inhibitor. Cellular motility was measured using two methods: culture wounding over a period of 48 hours; and secondly, time-lapse videomicroscopy (TLVM). RESULTS: In these experiments KGF was found to produce a dose-dependent enhancement of MCF-7 cell motility over a dosage range of 5 to 500 ng/ml. In the TLVM experiments, Heparin (30 ng/ml), LMWH (30 ng/ml) and KGFR2 beta (IIIb)/Fc (50 micrograms/ml) completely inhibited KGF-induced motility of MCF-7 cells during the initial 2-hour observation period. In the culture wounding assay, LMWH produced a greater reduction in KGF-induced motility than heparin at 48 hours post-treatment. CONCLUSION: The results of this study indicate that KGF-mediated enhancement of breast cancer cells motility and proliferation is inhibited by both specific and non-specific KGF inhibitors. LMWH appears to produce an inhibition of KGF with a much longer duration of action than Heparin. Our results suggest that KGF inhibition may be a potential new therapeutic approach for the treatment of metastatic breast cancer.

Navigator channel adaptation to reconstruct three dimensional heart volumes from two dimensional radiotherapy planning data.

BACKGROUND: Biologically-based models that utilize 3D radiation dosimetry data to estimate the risk of late cardiac effects could have significant utility for planning radiotherapy in young patients. A major challenge arises from having only 2D treatment planning data for patients with long-term follow-up. In this study, we evaluate the accuracy of an advanced deformable image registration (DIR) and navigator channels (NC) adaptation technique to reconstruct 3D heart volumes from 2D radiotherapy planning images for Hodgkin's Lymphoma (HL) patients. METHODS: Planning CT images were obtained for 50 HL patients who underwent mediastinal radiotherapy. Twelve image sets (6 male, 6 female) were used to construct a male and a female population heart model, which was registered to 23 HL "Reference" patients' CT images using a DIR algorithm, MORFEUS. This generated a series of population-to-Reference patient specific 3D deformation maps. The technique was independently tested on 15 additional "Test" patients by reconstructing their 3D heart volumes using 2D digitally reconstructed radiographs (DRR). The technique involved: 1) identifying a matching Reference patient for each Test patient using thorax measurements, 2) placement of six NCs on matching Reference and Test patients' DRRs to capture differences in significant heart curvatures, 3) adapting the population-to-Reference patient-specific deformation maps to generate population-to-Test patient-specific deformation maps using linear and bilinear interpolation methods, 4) applying population-to-Test patient specific deformation to the population model to reconstruct Test-patient specific 3D heart models. The percentage volume overlap between the NC-adapted reconstruction and actual Test patient's true heart volume was calculated using the Dice coefficient. RESULTS: The average Dice coefficient expressed as a percentage between the NC-adapted and actual Test model was 89.4 ± 2.8%. The modified NC adaptation technique made significant improvements to the population deformation heart models (p = 0.01). As standard evaluation, the residual Dice error after adaptation was comparable to the volumetric differences observed in free-breathing heart volumes (p = 0.62). CONCLUSIONS: The reconstruction technique described generates accurate 3D heart models from limited 2D planning data. This development could potentially be used to retrospectively calculate delivered dose to the heart for historically treated patients and thereby provide a better understanding of late radiation-related cardiac effects.

Adapting population liver motion models for individualized online image-guided therapy.

Respiratory motion varies on a daily basis in abdominal cancer patients, affecting the ability to successfully deliver local therapy and requiring increased treatment margins to account for this variation. Deformable registration techniques can accurately describe respiratory motion, however, online application can be limited by long computational times and user intervention. A technique has been developed to quickly quantify patient breathing motion from respiratory-sorted volumetric images by calculating 1D shifts in image intensities between spatially corresponding regions of interest (navigator channels) on patient's images. The 1D motion at the superior, inferior, anterior, and posterior liver edges was detected and applied to adapt a population liver respiratory motion model. For validation, deformable registration was performed for each patient using a validated technique, MORFEUS, for relative validation, and vessel bifurcations, identified on patient's inhale and exhale images, for absolute validation. The accuracy of the adapted-population model to describe the patient respiratory motion was (absolute mean +/- SD) 0.26 +/- 0.11 cm and 0.30 +/- 0.21 cm in the superior-inferior (SI) and anterior-posterior (AP) directions, respectively. The accuracy of predicting the tumor COM motion was 0.30 +/- 0.22 cm, and 0.34 +/- 0.31, while the absolute validation, based on bifurcations was 0.26 +/- 0.16 cm and 0.13 +/- 0.04 cm in the SI and AP directions, respectively. This technique was developed to complement and quickly adapt a full 3D biomechanical based deformable registration technique, MORFEUS, to be applied in the online setting.

Keratinocyte growth factor stimulates the migration and proliferation of breast cancer cells in a culture wounding model.

Growth factors are known to influence the progression, motility and invasiveness of tumor cells. In a previous study, we reported that conditioned media from NIH 3T3 cells (mouse fibroblast), which contains KGF, increased the motile morphology of estrogen receptor (ER)-positive breast cancer cells and produced no effect on ER-negative cells. The present study examined the influence of human KGF on two estrogen receptor (ER)-positive human breast cancer cell lines (MCF-7 and T-47D) using a culture wounding model to evaluate cell proliferation and migration over a period of 48h. In the present study we observed that KGF enhanced the migration and proliferation of both MCF-7 and T-47D breast cancer cells. In both cell lines the response to KGF was found to be both dose- and time-dependent. However, the total migration and proliferation response of the MCF-7 cells to KGF was much greater than that observed in the T-47D cells. The results of this study demonstrate that human KGF enhances the migration and proliferation of human breast cancer cells. Further, these results support the concept that KGF may be an early signal in the progression of breast cancer to a more motile and metastatic phenotype.