Lead optimization of cathepsin K inhibitors for the treatment of Osteoarthritis.

Cathepsin K (Cat K) is a cysteine protease involved in bone remodeling. In addition to its role in bone biology, Cat K is upregulated in osteoclasts, chondrocytes and synoviocytes in osteoarthritic (OA) disease states making it a potential therapeutic target for disease-modifying OA. Starting from a prior preclinical compound, MK-1256, lead optimization efforts were carried out in the search for potent Cat K inhibitors with improved selectivity profiles with an emphasis on cathepsin F. Herein, we report the SAR studies which led to the discovery of the highly selective oxazole compound 23, which was subsequently shown to inhibit cathepsin K in vivo as measured by reduced levels of urinary C-telopeptide of collagen type I in dog.

Development of a Meso Scale Discovery ligand-binding assay for measurement of free (drug-unbound) target in nonhuman primate serum.

Aim: To quantify the free form of a protein as a target-engagement biomarker in nonhuman primate serum, a Meso Scale Discovery ligand-binding assay was developed and qualified. Results: The initial assay produced an unexpected artifact when used to measure the free target in study samples dosed with drug. By using incurred study samples dosed with high drug levels to test assay performance, we developed an alternative assay that does not suffer from drug interference. Conclusion: Our work demonstrated that an assay designed to measure free target may not necessarily deliver reliable quantitation. In our case, incurred study samples dosed with drug proved to be useful in developing an alternative free assay that does not suffer from drug interference.

Synthesis and optimization of N-heterocyclic pyridinones as catechol-O-methyltransferase (COMT) inhibitors.

A series of N-heterocyclic pyridinone catechol-O-methyltransferase (COMT) inhibitors were synthesized. Physicochemical properties, including ligand lipophilic efficiency (LLE) and clogP, were used to guide compound design and attempt to improve inhibitor pharmacokinetics. Incorporation of heterocyclic central rings provided improvements in physicochemical parameters but did not significantly reduce in vitro or in vivo clearance. Nevertheless, compound 11 was identified as a potent inhibitor with sufficient in vivo exposure to significantly affect the dopamine metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC), and indicate central COMT inhibition.

Time-Resolved Particle Image Velocimetry Measurements with Wall Shear Stress and Uncertainty Quantification for the FDA Nozzle Model.

We present advanced particle image velocimetry (PIV) processing, post-processing, and uncertainty estimation techniques to support the validation of computational fluid dynamics analyses of medical devices. This work is an extension of a previous FDA-sponsored multi-laboratory study, which used a medical device mimicking geometry referred to as the FDA benchmark nozzle model. Experimental measurements were performed using time-resolved PIV at five overlapping regions of the model for Reynolds numbers in the nozzle throat of 500, 2000, 5000, and 8000. Images included a twofold increase in spatial resolution in comparison to the previous study. Data was processed using ensemble correlation, dynamic range enhancement, and phase correlations to increase signal-to-noise ratios and measurement accuracy, and to resolve flow regions with large velocity ranges and gradients, which is typical of many blood-contacting medical devices. Parameters relevant to device safety, including shear stress at the wall and in bulk flow, were computed using radial basis functions. In addition, in-field spatially resolved pressure distributions, Reynolds stresses, and energy dissipation rates were computed from PIV measurements. Velocity measurement uncertainty was estimated directly from the PIV correlation plane, and uncertainty analysis for wall shear stress at each measurement location was performed using a Monte Carlo model. Local velocity uncertainty varied greatly and depended largely on local conditions such as particle seeding, velocity gradients, and particle displacements. Uncertainty in low velocity regions in the sudden expansion section of the nozzle was greatly reduced by over an order of magnitude when dynamic range enhancement was applied. Wall shear stress uncertainty was dominated by uncertainty contributions from velocity estimations, which were shown to account for 90-99% of the total uncertainty. This study provides advancements in the PIV processing methodologies over the previous work through increased PIV image resolution, use of robust image processing algorithms for near-wall velocity measurements and wall shear stress calculations, and uncertainty analyses for both velocity and wall shear stress measurements. The velocity and shear stress analysis, with spatially distributed uncertainty estimates, highlights the challenges of flow quantification in medical devices and provides potential methods to overcome such challenges.

Synthesis and Evaluation of Heterocyclic Catechol Mimics as Inhibitors of Catechol-O-methyltransferase (COMT).

3-Hydroxy-4-pyridinones and 5-hydroxy-4-pyrimidinones were identified as inhibitors of catechol-O-methyltransferase (COMT) in a high-throughput screen. These heterocyclic catechol mimics exhibit potent inhibition of the enzyme and an improved toxicity profile versus the marketed nitrocatechol inhibitors tolcapone and entacapone. Optimization of the series was aided by X-ray cocrystal structures of the novel inhibitors in complex with COMT and cofactors SAM and Mg(2+). The crystal structures suggest a mechanism of inhibition for these heterocyclic inhibitors distinct from previously disclosed COMT inhibitors.

Commissioning results of an automated treatment planning verification system.

A dose calculation verification system (VS) was acquired and commissioned as a second check on the treatment planning system (TPS). This system reads DICOM CT datasets, RT plans, RT structures, and RT dose from the TPS and automatically, using its own collapsed cone superposition/convolution algorithm, computes dose on the same CT dataset. The system was commissioned by extracting basic beam parameters for simple field geometries and dose verification for complex treatments. Percent depth doses (PDD) and profiles were extracted for field sizes using jaw settings 3 × 3 cm2 - 40 × 40 cm2 and compared to measured data, as well as our TPS model. Smaller fields of 1 × 1 cm2 and 2 × 2 cm2 generated using the multileaf collimator (MLC) were analyzed in the same fashion as the open fields. In addition, 40 patient plans consisting of both IMRT and VMAT were computed and the following comparisons were made: 1) TPS to the VS, 2) VS to measured data, and 3) TPS to measured data where measured data is both ion chamber (IC) and film measurements. Our results indicated for all field sizes using jaw settings PDD errors for the VS on average were less than 0.87%, 1.38%, and 1.07% for 6x, 15x, and 18x, respectively, relative to measured data. PDD errors for MLC field sizes were less than 2.28%, 1.02%, and 2.23% for 6x, 15x, and 18x, respectively. The infield profile analysis yielded results less than 0.58% for 6x, 0.61% for 15x, and 0.77% for 18x for the VS relative to measured data. Analysis of the penumbra region yields results ranging from 66.5% points, meeting the DTA criteria to 100% of the points for smaller field sizes for all energies. Analysis of profile data for field sizes generated using the MLC saw agreement with infield DTA analysis ranging from 68.8%-100% points passing the 1.5%/1.5 mm criteria. Results from the dose verification for IMRT and VMAT beams indicated that, on average, the ratio of TPS to IC and VS to IC measurements was 100.5 ± 1.9% and 100.4 ± 1.3%, respectively, while our TPS to VS was 100.1 ± 1.0%. When comparing the TPS and VS to film measurements, the average percentage pixels passing a 3%/3mm criteria based gamma analysis were 96.6 ± 4.2% and 97 ± 5.6%, respectively. When the VS was compared to the TPS, on average 98.1 ± 5.3% of pixels passed the gamma analysis. Based upon these preliminary results, the VS system should be able to calculate dose adequately as a verification tool of our TPS.

Limitations of using synthetic blood clots for measuring in vitro clot capture efficiency of inferior vena cava filters.

The purpose of this study was first to evaluate the clot capture efficiency and capture location of six currently-marketed vena cava filters in a physiological venous flow loop, using synthetic polyacrylamide hydrogel clots, which were intended to simulate actual blood clots. After observing a measured anomaly for one of the test filters, we redirected the focus of the study to identify the cause of poor clot capture performance for large synthetic hydrogel clots. We hypothesized that the uncharacteristic low clot capture efficiency observed when testing the outlying filter can be attributed to the inadvertent use of dense, stiff synthetic hydrogel clots, and not as a result of the filter design or filter orientation. To study this issue, sheep blood clots and polyacrylamide (PA) synthetic clots were injected into a mock venous flow loop containing a clinical inferior vena cava (IVC) filter, and their captures were observed. Testing was performed with clots of various diameters (3.2, 4.8, and 6.4 mm), length-to-diameter ratios (1:1, 3:1, 10:1), and stiffness. By adjusting the chemical formulation, PA clots were fabricated to be soft, moderately stiff, or stiff with elastic moduli of 805 ± 2, 1696 ± 10 and 3295 ± 37 Pa, respectively. In comparison, the elastic moduli for freshly prepared sheep blood clots were 1690 ± 360 Pa. The outlying filter had a design that was characterized by peripheral gaps (up to 14 mm) between its wire struts. While a low clot capture rate was observed using large, stiff synthetic clots, the filter effectively captured similarly sized sheep blood clots and soft PA clots. Because the stiffer synthetic clots remained straight when approaching the filter in the IVC model flow loop, they were more likely to pass between the peripheral filter struts, while the softer, physiological clots tended to fold and were captured by the filter. These experiments demonstrated that if synthetic clots are used as a surrogate for animal or human blood clots for in vitro evaluation of vena cava filters, the material properties (eg, elastic modulus) and dynamic behavior of the surrogate should first be assessed to ensure that they accurately mimic an actual blood clot within the body.

Characterization of non-nitrocatechol pan and isoform specific catechol-O-methyltransferase inhibitors and substrates.

Reduced dopamine neurotransmission in the prefrontal cortex has been implicated as causal for the negative symptoms and cognitive deficit associated with schizophrenia; thus, a compound which selectively enhances dopamine neurotransmission in the prefrontal cortex may have therapeutic potential. Inhibition of catechol-O-methyltransferase (COMT, EC 2.1.1.6) offers a unique advantage, since this enzyme is the primary mechanism for the elimination of dopamine in cortical areas. Since membrane bound COMT (MB-COMT) is the predominant isoform in human brain, a high throughput screen (HTS) to identify novel MB-COMT specific inhibitors was completed. Subsequent optimization led to the identification of novel, non-nitrocatechol COMT inhibitors, some of which interact specifically with MB-COMT. Compounds were characterized for in vitro efficacy versus human and rat MB and soluble (S)-COMT. Select compounds were administered to male Wistar rats, and ex vivo COMT activity, compound levels in plasma and cerebrospinal fluid (CSF), and CSF dopamine metabolite levels were determined as measures of preclinical efficacy. Finally, novel non-nitrocatechol COMT inhibitors displayed less potent uncoupling of the mitochondrial membrane potential (MMP) compared to tolcapone as well as nonhepatotoxic entacapone, thus mitigating the risk of hepatotoxicity.

Quantitative estimation of ultrasound beam intensities using infrared thermography-Experimental validation.

Infrared (IR) thermography is a technique that has the potential to rapidly and noninvasively determine the intensity fields of ultrasound transducers. In the work described here, IR temperature measurements were made in a tissue phantom sonicated with a high-intensity focused ultrasound (HIFU) transducer, and the intensity fields were determined using a previously published mathematical formulation relating intensity to temperature rise at a tissue/air interface. Intensity fields determined from the IR technique were compared with those derived from hydrophone measurements. Focal intensities and beam widths determined via the IR approach agreed with values derived from hydrophone measurements to within a relative difference of less than 10%, for a transducer with a gain of 30, and about 13% for a transducer with a gain of 60. At axial locations roughly 1 cm in front (pre-focal) and behind (post-focal) the focus, the agreement with hydrophones for the lower-gain transducer remained comparable to that in the focal plane. For the higher-gain transducer, the agreement with hydrophones at the pre-focal and post-focal locations was around 40%.

An allosteric Akt inhibitor effectively blocks Akt signaling and tumor growth with only transient effects on glucose and insulin levels in vivo.

The PI3K-Akt pathway is dysregulated in the majority of solid tumors. Pharmacological inhibition of Akt is a promising strategy for treating tumors resistant to growth factor receptor antagonists due to mutations in PI3K or PTEN. We have developed allosteric, isozyme-specific inhibitors of Akt activity and activation, as well as ex vivo kinase assays to measure inhibition of individual Akt isozymes in tissues. Here we describe the relationship between PK, Akt inhibition, hyperglycemia and tumor efficacy for a selective inhibitor of Akt1 and Akt2 (AKTi). In nude mice, AKTi treatment caused transient insulin resistance and reversible, dose-dependent hyperglycemia and hyperinsulinemia. Akt1 and Akt2 phosphorylation was inhibited in mouse lung with EC50 values of 1.6 and 7 µM, respectively, and with similar potency in other tissues and xenograft tumors. Weekly subcutaneous dosing of AKTi resulted in dose-dependent inhibition of LNCaP prostate cancer xenografts, an AR-dependent tumor with PTEN deletion and constitutively activated Akt. Complete tumor growth inhibition was achieved at 200 mpk, a dose that maintained inhibition of Akt1 and Akt2 of greater than 80% and 50%, respectively, for at least 12 hours in xenograft tumor and mouse lung. Hyperglycemia could be controlled by reducing C(max), while maintaining efficacy in the LNCaP model, but not by insulin administration. AKTi treatment was well tolerated, without weight loss or gross toxicities. These studies supported the rationale for clinical development of allosteric Akt inhibitors and provide the basis for further refining of pharmacokinetic properties and dosing regimens of this class of inhibitors.

A novel study of mechanical heart valve cavitation in a pressurized pulsatile duplicator.

Submission of data regarding the cavitation potential of a mechanical heart valve is recommended by the U.S. Food and Drug Administration in the device-review process. An acoustic method has long been proposed for cavitation detection. However, the question as to whether such a method can differentiate the cavitation noise from the mechanical closing sound has not been sufficiently addressed. In this study, cavitation near a Medtronic Hall tilting disc valve was investigated in a pressurized pulsatile duplicator. The purpose of pressurizing the testing chambers was to prevent cavitation under a normally cavitating loading condition to isolate the mechanical closing sound. By comparing the sound signals before and after pressurization, some noticeable differences were found between them. In the time domain, the intensity of the sound under a cavitating condition was much higher. In the frequency domain, the energy distribution of a sound signal was distinctively different depending on whether cavitation occurred or not. The valve closing sound had a large amount of energy in the low-frequency range (less than about 25 kHz). When cavitation took place, the sound energy shifted toward the high-frequency range (from 25 to 500 kHz).

Allosteric inhibitors of Akt1 and Akt2: discovery of [1,2,4]triazolo[3,4-f][1,6]naphthyridines with potent and balanced activity.

A series of [1,2,4]triazolo[3,4-f][1,6]naphthyridine allosteric dual inhibitors of Akt1 and 2 have been developed. These compounds have been shown to have potent dual Akt1 and 2 cell potency. The representative compound 13 provided potent inhibitory activity against Akt1 and 2 in vivo in a mouse model.

Effects of thrombosed vena cava filters on blood flow: flow visualization and numerical modeling.

Inferior vena cava (IVC) filters are used to prevent pulmonary embolism (PE) in patients with deep vein thrombosis for whom anticoagulation is contraindicated. IVC filters have been shown to be effective in trapping embolized clots and preventing PE; however, among the commercially available designs, the optimal balance of clot capture efficiency, clot dissolution, and prevention of to vena cava occlusion is unknown. Clot capture efficiency has been quantified in numerous in vitro studies, in which model clots are released into a mock circulation system, with the relative capture efficiency of various IVC filters analyzed statistically. In general, two-stage filters have been found to be more efficient than one-stage filters. However, other factors may play a role in the ultimate dissolution of clots and in the overall effect of the resulting blood flow on caval vasculature. Clot dissolution has been shown to increase with increasing wall shear stress, while low and oscillating wall shear stresses are known to have a deleterious effect on vessel walls, causing intimal hyperplasia. This paper describes the effect of IVC filters on blood flow, velocity patterns, and wall shear stress by flow visualization and computational fluid dynamics.

Discovery of potent and cell-active allosteric dual Akt 1 and 2 inhibitors.

This paper describes the improvement of cell potency in a class of allosteric Akt 1 and 2 inhibitors. Key discoveries include identifying the solvent exposed region of the molecule and appending basic amines to enhance the physiochemical properties of the molecules. Findings from the structure-activity relationships are discussed.

The design and synthesis of potent and cell-active allosteric dual Akt 1 and 2 inhibitors devoid of hERG activity.

This letter details the attenuation of hERG in a class of Akt inhibitors through heteroatom insertions into aromatic rings. The development of a cell-active dual Akt 1 and 2 inhibitors devoid of hERG activity is discussed using structure-activity relationships.

Allosteric inhibitors of Akt1 and Akt2: a naphthyridinone with efficacy in an A2780 tumor xenograft model.

A series of naphthyridine and naphthyridinone allosteric dual inhibitors of Akt1 and 2 have been developed. These compounds have been optimized to have potent dual activity against the activated kinase as well as the activation of Akt in cells. One molecule in particular, compound 17, has potent inhibitory activity against Akt1 and 2 in vivo in a mouse lung and efficacy in a tumor xenograft model.

Characterization of high intensity focused ultrasound transducers using acoustic streaming.

A new approach for characterizing high intensity focused ultrasound (HIFU) transducers is presented. The technique is based upon the acoustic streaming field generated by absorption of the HIFU beam in a liquid medium. The streaming field is quantified using digital particle image velocimetry, and a numerical algorithm is employed to compute the acoustic intensity field giving rise to the observed streaming field. The method as presented here is applicable to moderate intensity regimes, above the intensities which may be damaging to conventional hydrophones, but below the levels where nonlinear propagation effects are appreciable. Intensity fields and acoustic powers predicted using the streaming method were found to agree within 10% with measurements obtained using hydrophones and radiation force balances. Besides acoustic intensity fields, the streaming technique may be used to determine other important HIFU parameters, such as beam tilt angle or absorption of the propagation medium.

Development of pyridopyrimidines as potent Akt1/2 inhibitors.

This communication reports a new synthetic route of pyridopyrimidines to facilitate their structural optimization in a library fashion and describes the development of pyridopyrimidines that have excellent enzymatic and cell potency against Akt1 and Akt2. This series also shows a high level of selectivity over other closely related kinases and significantly improved caspase-3 activity with the more optimized compounds.

Optimization of 2,3,5-trisubstituted pyridine derivatives as potent allosteric Akt1 and Akt2 inhibitors.

This letter shows inhibitor SAR on a pyridine series of allosteric Akt inhibitors to optimize enzymatic and cellular potency. We have optimized 2,3,5-trisubstituted pyridines to give potent Akt1 and Akt2 inhibitors in both enzyme and cell based assays. In addition, we will also highlight the pharmacokinetic profile of an optimized inhibitor that has low clearance and long half-life in dogs.

Rapid assembly of diverse and potent allosteric Akt inhibitors.

This paper describes the rapid assembly of four different classes of potent Akt inhibitors from a common intermediate. Among them, a pyridopyrimidine series displayed the best intrinsic and cell potency against Akt1 and Akt2. This series also showed a promising pharmacokinetic profile and excellent selectivity over other closely related kinases.