Orally Bioavailable Macrocyclic Peptide That Inhibits Binding of PCSK9 to the Low Density Lipoprotein Receptor.

BACKGROUND: Inhibition of PCSK9 (proprotein convertase subtilisin/kexin type 9)-low density lipoprotein receptor interaction with injectable monoclonal antibodies or small interfering RNA lowers plasma low density lipoprotein-cholesterol, but despite nearly 2 decades of effort, an oral inhibitor of PCSK9 is not available. Macrocyclic peptides represent a novel approach to target proteins traditionally considered intractable to small-molecule drug design. METHODS: Novel mRNA display screening technology was used to identify lead chemical matter, which was then optimized by applying structure-based drug design enabled by novel synthetic chemistry to identify macrocyclic peptide (MK-0616) with exquisite potency and selectivity for PCSK9. Following completion of nonclinical safety studies, MK-0616 was administered to healthy adult participants in a single rising-dose Phase 1 clinical trial designed to evaluate its safety, pharmacokinetics, and pharmacodynamics. In a multiple-dose trial in participants taking statins, MK-0616 was administered once daily for 14 days to characterize the safety, pharmacokinetics, and pharmacodynamics (change in low density lipoprotein cholesterol). RESULTS: MK-0616 displayed high affinity (Ki = 5pM) for PCSK9 in vitro and sufficient safety and oral bioavailability preclinically to enable advancement into the clinic. In Phase 1 clinical studies in healthy adults, single oral doses of MK-0616 were associated with >93% geometric mean reduction (95% CI, 84-103) of free, unbound plasma PCSK9; in participants on statin therapy, multiple-oral-dose regimens provided a maximum 61% geometric mean reduction (95% CI, 43-85) in low density lipoprotein cholesterol from baseline after 14 days of once-daily dosing of 20 mg MK-0616. CONCLUSIONS: This work validates the use of mRNA display technology for identification of novel oral therapeutic agents, exemplified by the identification of an oral PCSK9 inhibitor, which has the potential to be a highly effective cholesterol lowering therapy for patients in need.

Probing the drivers of Staphylococcus aureus biofilm protein amyloidogenesis and disrupting biofilms with engineered protein disaggregases.

Phenol-soluble modulins (PSMs) are the primary proteinaceous component of Staphylococcus aureus biofilms. Residence in the protective environment of biofilms allows bacteria to rapidly evolve and acquire antimicrobial resistance, which can lead to persistent infections such as those caused by methicillin-resistant S. aureus (MRSA). In their soluble form, PSMs hinder the immune response of the host and can increase the virulence potential of MRSA. PSMs also self-assemble into insoluble functional amyloids that contribute to the structural scaffold of biofilms. The specific roles of PSM peptides in biofilms remain poorly understood. Here, we report the development of a genetically tractable yeast model system for studying the properties of PSMα peptides. Expression of PSMα peptides in yeast drives the formation of toxic insoluble aggregates that adopt vesicle-like structures. Using this system, we probed the molecular drivers of PSMα aggregation to delineate key similarities and differences among the PSMs and identified a crucial residue that drives PSM features. Biofilms are a major public health threat; thus, biofilm disruption is a key goal. To solubilize aggregates comprised of a diverse range of amyloid and amyloid-like species, we have developed engineered variants of Hsp104, a hexameric AAA+ protein disaggregase from yeast. Here, we demonstrate that potentiated Hsp104 variants counter the toxicity and aggregation of PSMα peptides. Further, we demonstrate that a potentiated Hsp104 variant can drive the disassembly of preformed S. aureus biofilms. We suggest that this new yeast model can be a powerful platform for screening for agents that disrupt PSM aggregation and that Hsp104 disaggregases could be a promising tool for the safe enzymatic disruption of biofilms. IMPORTANCE Biofilms are complex mixtures secreted by bacteria that form a material in which the bacteria can become embedded. This process transforms the properties of the bacteria, and they become more resistant to removal, which can give rise to multidrug-resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA). Here, we study phenol-soluble modulins (PSMs), which are amyloidogenic proteins secreted by S. aureus, that become incorporated into biofilms. Biofilms are challenging to study, so we have developed a new genetically tractable yeast model to study the PSMs. We used our system to learn about several key features of the PSMs. We also demonstrate that variants of an amyloid disaggregase, Hsp104, can disrupt the PSMs and, more importantly, dissolve preformed S. aureus biofilms. We propose that our system can be a powerful screening tool and that Hsp104 disaggregases may be a new avenue to explore for biofilm disruption agents.

Bone Tumor Suppression in Rabbits by Hyperthermia below the Clinical Safety Limit Using Aligned Magnetic Bone Cement.

Demonstrating highly efficient alternating current (AC) magnetic field heating of nanoparticles in physiological environments under clinically safe field parameters has remained a great challenge, hindering clinical applications of magnetic hyperthermia. In this work, exceptionally high loss power of magnetic bone cement under the clinical safety limit of AC field parameters, incorporating direct current field-aligned soft magnetic Zn0.3 Fe2.7 O4 nanoparticles with low concentration, is reported. Under an AC field of 4 kA m-1 at 430 kHz, the aligned bone cement with 0.2 wt% nanoparticles achieves a temperature increase of 30 °C in 180 s. This amounts to a specific loss power value of 327 W gmetal-1 and an intrinsic loss power of 47 nHm2 kg-1 , which is enhanced by 50-fold compared to randomly oriented samples. The high-performance magnetic bone cement allows for the demonstration of effective hyperthermia suppression of tumor growth in the bone marrow cavity of New Zealand White Rabbits subjected to rapid cooling due to blood circulation, and significant enhancement of survival rate.

Functional analysis of proposed substrate-binding residues of Hsp104.

Hsp104 is a hexameric AAA+ yeast disaggregase capable of solubilizing disordered aggregates and amyloid. Hsp104 couples ATP hydrolysis to polypeptide translocation through its central channel. Substrate binding by Hsp104 is mediated primarily by two conserved tyrosine residues in nucleotide binding domain (NBD) 1 and NBD2. Recent structural studies have revealed that an additional tyrosine residue (Y650) located in NBD2 appears to contact substrate and may play an important role in Hsp104 function. Here, we functionally analyze the properties of this proposed Hsp104 -substrate interaction. We find that Y650 is not essential for Hsp104 to confer thermotolerance. Supporting these findings, in a potentiated Hsp104 variant background, the Y650A mutation does not abolish potentiation. However, modulation of this site does have subtle effects on the activity of this potentiated Hsp104 variant. We therefore suggest that while Y650 is not essential for Hsp104 function, its modulation may be useful for fine-tuning Hsp104 properties.

Performance of the cone beam computed tomography-based patient positioning system on the Gamma Knife Icon™.

PURPOSE: Cone beam computed tomography (CBCT) imaging has been implemented on the Leksell Gamma Knife® Icon™ for assessing patient positioning in mask-based Gamma Knife radiosurgery. The purpose of this study was to evaluate the performance of the CBCT-based patient positioning system as a tool for frameless Gamma Knife radiosurgery. METHODS: Daily quality assurance (QA) CBCT precision test results from a 12-month period were analyzed for the geometric accuracy and the stability of the imager. The performance of the image acquisition module and the image registration algorithm was evaluated using an anthropomorphic head phantom (CIRS Inc., Norfolk, VA) and a XYZR axis manual positioning stage (TOAUTO Inc., Guangdong, China). The head phantom was fixed on a mask adaptor and manually translated in the X, Y, Z directions or rotated around the X, Y, Z axes in the range of ±10 mm or ±10º. A CBCT scan was performed after each manual position setup followed by an image registration to the reference scan. To assess the overall setup uncertainties in fractionated treatment, two cylindrical Presage phantoms (Heuris Inc., Skillman, NJ) of 15 cm diameter and 10 cm height were irradiated with identical prescription dose and shot placement following standard mask-based treatment workflow according to two different fraction schedules: a single fraction treatment of 7.5 Gy and a 5-fraction treatment with 1.5 Gy per fraction. RESULTS: The averaged vector deviations of the four marks from their preset values are 0.087, 0.085, 0.095, and 0.079 mm from the 212 daily QA tests. The averaged displacements in the X, Y, Z coordinates and the pitch, yaw, roll angles from the image registration tests are 0.23, 0.27, 0.14, 0.32º, 0.19º, 0.31º from the manual setup. The corresponding maximum differences are 0.41, 0.33, 0.29 mm, 0.45º, 0.31º, and 0.43º, respectively. Compared to the treatment plan using the 2% & 1 mm criteria, the averaged 2D Gamma passing rate is 98.25% for the measured dose distribution from the Presage phantom with 1-fraction irradiation and 95.12% for the 5-fraction irradiation. The averaged Gamma passing rates are 99.53% and 98.16% for the 1-fraction and 5-fraction irradiations using the 2% & 2 mm criteria. CONCLUSIONS: The CBCT imager and the image registration algorithm can reproduce phantom position with <0.5 mm/0.5º uncertainty. A systematic contribution from the interfraction phantom repositioning procedure was observed in the Gamma analysis over the irradiated volumes of two end-to-end test phantoms.

Failure modes and effects analysis (FMEA) for Gamma Knife radiosurgery.

PURPOSE: Gamma Knife radiosurgery is a highly precise and accurate treatment technique for treating brain diseases with low risk of serious error that nevertheless could potentially be reduced. We applied the AAPM Task Group 100 recommended failure modes and effects analysis (FMEA) tool to develop a risk-based quality management program for Gamma Knife radiosurgery. METHODS: A team consisting of medical physicists, radiation oncologists, neurosurgeons, radiation safety officers, nurses, operating room technologists, and schedulers at our institution and an external physicist expert on Gamma Knife was formed for the FMEA study. A process tree and a failure mode table were created for the Gamma Knife radiosurgery procedures using the Leksell Gamma Knife Perfexion and 4C units. Three scores for the probability of occurrence (O), the severity (S), and the probability of no detection for failure mode (D) were assigned to each failure mode by 8 professionals on a scale from 1 to 10. An overall risk priority number (RPN) for each failure mode was then calculated from the averaged O, S, and D scores. The coefficient of variation for each O, S, or D score was also calculated. The failure modes identified were prioritized in terms of both the RPN scores and the severity scores. RESULTS: The established process tree for Gamma Knife radiosurgery consists of 10 subprocesses and 53 steps, including a subprocess for frame placement and 11 steps that are directly related to the frame-based nature of the Gamma Knife radiosurgery. Out of the 86 failure modes identified, 40 Gamma Knife specific failure modes were caused by the potential for inappropriate use of the radiosurgery head frame, the imaging fiducial boxes, the Gamma Knife helmets and plugs, the skull definition tools as well as other features of the GammaPlan treatment planning system. The other 46 failure modes are associated with the registration, imaging, image transfer, contouring processes that are common for all external beam radiation therapy techniques. The failure modes with the highest hazard scores are related to imperfect frame adaptor attachment, bad fiducial box assembly, unsecured plugs/inserts, overlooked target areas, and undetected machine mechanical failure during the morning QA process. CONCLUSIONS: The implementation of the FMEA approach for Gamma Knife radiosurgery enabled deeper understanding of the overall process among all professionals involved in the care of the patient and helped identify potential weaknesses in the overall process. The results of the present study give us a basis for the development of a risk based quality management program for Gamma Knife radiosurgery.

Two-year experience with the commercial Gamma Knife Check software.

The Gamma Knife Check software is an FDA approved second check system for dose calculations in Gamma Knife radiosurgery. The purpose of this study was to evaluate the accuracy and the stability of the commercial software package as a tool for independent dose verification. The Gamma Knife Check software version 8.4 was commissioned for a Leksell Gamma Knife Perfexion and a 4C unit at the University of Pittsburgh Medical Center in May 2012. Independent dose verifications were performed using this software for 319 radiosurgery cases on the Perfexion and 283 radiosurgery cases on the 4C units. The cases on each machine were divided into groups according to their diagnoses, and an averaged absolute percent dose difference for each group was calculated. The percentage dose difference for each treatment target was obtained as the relative difference between the Gamma Knife Check dose and the dose from the tissue maximum ratio algorithm (TMR 10) from the GammaPlan software version 10 at the reference point. For treatment plans with imaging skull definition, results obtained from the Gamma Knife Check software using the measurement-based skull definition method are used for comparison. The collected dose difference data were also analyzed in terms of the distance from the treatment target to the skull, the number of treatment shots used for the target, and the gamma angles of the treatment shots. The averaged percent dose differences between the Gamma Knife Check software and the GammaPlan treatment planning system are 0.3%, 0.89%, 1.24%, 1.09%, 0.83%, 0.55%, 0.33%, and 1.49% for the trigeminal neuralgia, acoustic neuroma, arteriovenous malformation (AVM), meningioma, pituitary adenoma, glioma, functional disorders, and metastasis cases on the Perfexion unit. The corresponding averaged percent dose differences for the 4C unit are 0.33%, 1.2%, 2.78% 1.99%, 1.4%, 1.92%, 0.62%, and 1.51%, respectively. The dose difference is, in general, larger for treatment targets in the peripheral regions of the skull owing to the difference in the numerical methods used for skull shape simulation in the GammaPlan and the Gamma Knife Check software. Larger than 5% dose differences were observed on both machines for certain targets close to patient skull surface and for certain targets in the lower half of the brain on the Perfexion, especially when shots with 70 and/or 110 gamma angles are used. Out of the 1065 treatment targets studied, a 5% cutoff criterion cannot always be met for the dose differences between the studied versions of the Gamma Knife Check software and the planning system for 40 treatment targets.

Gamma Knife radiosurgery with CT image-based dose calculation.

The Leksell GammaPlan software version 10 introduces a CT image-based segmentation tool for automatic skull definition and a convolution dose calculation algorithm for tissue inhomogeneity correction. The purpose of this work was to evaluate the impact of these new approaches on routine clinical Gamma Knife treatment planning. Sixty-five patients who underwent CT image-guided Gamma Knife radiosurgeries at the University of Pittsburgh Medical Center in recent years were retrospectively investigated. The diagnoses for these cases include trigeminal neuralgia, meningioma, acoustic neuroma, AVM, glioma, and benign and metastatic brain tumors. Dose calculations were performed for each patient with the same dose prescriptions and the same shot arrangements using three different approaches: 1) TMR 10 dose calculation with imaging skull definition; 2) convolution dose calculation with imaging skull definition; 3) TMR 10 dose calculation with conventional measurement-based skull definition. For each treatment matrix, the total treatment time, the target coverage index, the selectivity index, the gradient index, and a set of dose statistics parameters were compared between the three calculations. The dose statistics parameters investigated include the prescription isodose volume, the 12 Gy isodose volume, the minimum, maximum and mean doses on the treatment targets, and the critical structures under consideration. The difference between the convolution and the TMR 10 dose calculations for the 104 treatment matrices were found to vary with the patient anatomy, location of the treatment shots, and the tissue inhomogeneities around the treatment target. An average difference of 8.4% was observed for the total treatment times between the convolution and the TMR algorithms. The maximum differences in the treatment times, the prescription isodose volumes, the 12 Gy isodose volumes, the target coverage indices, the selectivity indices, and the gradient indices from the convolution and the TMR 10 calculations are 14.9%, 16.4%, 11.1%, 16.8, 6.9%, and 11.4%, respectively. The maximum differences in the minimum and the mean target doses between the two calculation algorithms are 8.1% and 4.2% of the corresponding prescription doses. The maximum differences in the maximum and the mean doses for the critical structures between the two calculation algorithms are 1.3 Gy and 0.7 Gy. The results from the two skull definition methods with the TMR 10 algorithm agree either within ± 2.5% or 0.3 Gy for the dose values, except for a 4.9% difference in the treatment times for a lower cerebellar lesion. The imaging skull definition method does not affect Gamma Knife dose calculation considerably when compared to the conventional measurement-based skull definition method, except in some extreme cases. Large differences were observed between the TMR 10 and the convolution calculation method for the same dose prescription and the same shot arrangements, indicating that the implementation of the convolution algorithm in routine clinical use might be desirable for optimal dose calculation results.

Dose differences between the three dose calculation algorithms in Leksell GammaPlan.

The purpose of this study was to evaluate the dose differences introduced by the TMR 10 and the convolution dose calculation algorithms in GammaPlan version 10, as compared to the TMR classic algorithm in the previous versions of GammaPlan. Computed axial tomographic images of a polystyrene phantom and a human head were acquired using a GE LightSpeed VCT scanner. A treatment target with a prescription dose of 20 Gy to 50% isodose line was defined in the phantom or the head CT set. The treatment times for single collimator, single shot placements were calculated using the three dose calculation algorithms in GammaPlan version 10. Four comparative studies were conducted: i) the dose matrix position was varied every 10 mm along the x-, y-, z-axes of the stereotactic coordinate system inside the phantom and the treatment times were compared on each matrix for the three collimators of the Gamma Knife Perfexion and the four collimators of the 4C;ii) the study was repeated for the human head CT dataset; iii) the matrix position was varied every 20 mm in the X and the Y directions on the central slice (Z = 100mm) of the head CT and the shot times were compared on each matrix for the 8 mm collimator of both units; a total of 51 matrix positions were identified for each unit; iv) the above comparison was repeated for the head CT transverse slices with Z = 20, 40, 60, 80, 120, 140, and 160 mm. A total of 271 matrix positions were studied. Based on the comparison of the treatment times needed to deliver 20 Gy at 50% isodose line, the equivalent TMR classic dose of the TMR 10 algorithm is roughly a constant for each collimator of the 4C unit and is 97.5%, 98.5%, 98%, and 100% of the TMR 10 dose for the 18 mm, 14 mm, 8 mm, and the 4 mm collimators, respectively. The numbers for the three collimators of the Perfexion change with the shot positions in the range from 99% to 102% for both the phantom and the head CT. The minimum, maximum, and the mean values of the equivalent TMR classic doses of the convolution algorithm on the 271 voxels of the head CT are 99.5%, 111.5%, 106.5% of the convolution dose for the Perfexion, and 99%, 109%, 104.5% for the 4C unit. We identified a maximum decrease in delivered dose of 11.5% for treatment in the superior frontal/parietal vertex region of the head CT for older calculations lacking inhomogeneity correction to account for the greater percentage of the average beam path occupied by bone. The differences in the inferior temporal lobe and the cerebellum/neck regions are significantly less, owing to the counter-balancing effects of both bone and the air cavity inhomogeneities. The dose differences between the TMR 10 and the TMR classic are within ± 2.5% for a single shot placement on both Perfexion and 4C. Dose prescriptions based on the experiences with the TMR classic may need to be adjusted to accommodate the up to 11.5% difference between the convolution and the TMR classic.