Bioanalysis of the Bicycle® toxin conjugate BT5528 and released monomethyl auristatin E via liquid chromatography-tandem mass spectrometry.

Background: The Bicycle® toxin conjugate BT5528 is a novel peptide therapeutic conjugated to the cytotoxic agent monomethyl auristatin E (MMAE). A bioanalytical assay was developed to quantify BT5528 and unconjugated MMAE in human plasma. Methodology: BT5528 quantitation used a protein precipitation procedure followed by LC-MS/MS detection. Quantitation of MMAE required a selective offline and online solid-phase extraction with detection via LC-MS/MS. Results: BT5528 was quantified over the assay range of 5-2500 ng/ml and free MMAE was quantified over the assay range of 0.05-50 ng/ml. Conclusion: Bioanalytical methods were used in the bioanalysis of intact BT5528 and released MMAE, in a phase I/IIa clinical trial; to date, over 2000 human patient samples have been analyzed.

BT8009; A Nectin-4 Targeting Bicycle Toxin Conjugate for Treatment of Solid Tumors.

Multiple tumor types overexpress Nectin-4 and the antibody-drug conjugate (ADC), enfortumab vedotin (EV) shows striking efficacy in clinical trials for metastatic urothelial cancer, which expresses high levels of Nectin-4, validating Nectin-4 as a clinical target for toxin delivery in this indication. Despite excellent data in urothelial cancer, little efficacy data are reported for EV in other Nectin-4 expressing tumors and EV therapy can produce significant toxicities in many patients, frequently leading to discontinuation of treatment. Thus, additional approaches to this target with the potential to extend utility and reduce toxicity are warranted. We describe the preclinical development of BT8009, a "Bicycle Toxin Conjugate" (BTC) consisting of a Nectin-4-binding bicyclic peptide, a cleavable linker system and the cell penetrant toxin mono-methylauristatin E (MMAE). BT8009 shows significant antitumor activity in preclinical tumor models, across a variety of cancer indications and is well tolerated in preclinical safety studies. In several models, it shows superior or equivalent antitumor activity to an EV analog. As a small hydrophilic peptide-based drug BT8009 rapidly diffuses from the systemic circulation, through tissues to penetrate the tumor and target tumor cells. It is renally eliminated from the circulation, with a half-life of 1-2 hours in rat and non-human primate. These physical and PK characteristics differentiate BT8009 from ADCs and may provide benefit in terms of tumor penetration and reduced systemic exposure. BT8009 is currently in a Phase 1/2 multicenter clinical trial across the US, Canada, and Europe, enrolling patients with advanced solid tumors associated with Nectin-4 expression.

Development of a LC-MS/MS method for the quantification of toxic payload DM1 cleaved from BT1718 in a Phase I study.

Background: BT1718 is a novel bicyclic peptide anticancer drug targeting membrane type I matrix metalloproteinase to release its toxic payload DM1. A LC-MS/MS method was validated to quantify DM1 generated from BT1718 in a Phase I/IIa clinical trial. Materials & methods: Plasma samples underwent a reduction reaction to artificially cleave BT1718 into DM1 and its bicycle components. An alkylation step was carried out to stabilize the reaction products, and plasma proteins extracted using acetonitrile. LC-MS/MS analysis utilized a C18 column and Agilent 6460 triple quadrupole mass spectrometer (Agilent, Cheshire, UK). Results: The method was fully validated over a linear range of 200-50,000 ng/ml BT1718, with overall precision ≤10% and accuracy 89-102%. Conclusion: A novel method for quantifying DM1 yielded from BT1718 has been validated and is now being utilized clinically.

MMAE Delivery Using the Bicycle Toxin Conjugate BT5528.

The EphA2 receptor is found at high levels in tumors and low levels in normal tissue and high EphA2 expression in biopsies is a predictor of poor outcome in patients. Drug discovery groups have therefore sought to develop EphA2-based therapies using small molecule, peptide, and nanoparticle-based approaches (1-3). However, until now only EphA2-targeting antibody-drug conjugates (ADC) have entered clinical development. For example, MEDI-547 is an EphA2-targeting ADC that displayed encouraging antitumor activity in preclinical models and progressed to phase I clinical testing in man. Here we describe the development of BT5528, a bicyclic peptide ("Bicycle") conjugated to the auristatin derivative maleimidocaproyl-monomethyl auristatin E to generate the Bicycle toxin conjugate BT5528. The report compares and contrasts the Pharmacokinetics (PK) characteristics of antibody and Bicycle-based targeting systems and discusses how the PK and payload characteristics of different delivery systems impact the efficacy-toxicity trade off which is key to the development of successful cancer therapies. We show that BT5528 gives rise to rapid update into tumors and fast renal elimination followed by persistent toxin levels in tumors without prolonged exposure of parent drug in the vasculature. This fast in, fast out kinetics gave rise to more favorable toxicology findings in rats and monkeys than were observed with MEDI-547 in preclinical and clinical studies.Graphical Abstract: http://mct.aacrjournals.org/content/molcanther/19/7/1385/F1.large.jpg.

Identification and Optimization of EphA2-Selective Bicycles for the Delivery of Cytotoxic Payloads.

Bicycles are constrained bicyclic peptides that represent a promising binding modality for use in targeted drug conjugates. A phage display screen against EphA2, a receptor tyrosine kinase highly expressed in a number of solid tumors, identified a number of Bicycle families with low nanomolar affinity. A Bicycle toxin conjugate (BTC) was generated by derivatization of one of these Bicycles with the potent cytotoxin DM1 via a cleavable linker. This BTC demonstrated potent antitumor activity in vivo but was poorly tolerated, which was hypothesized to be the result of undesired liver uptake caused by poor physicochemical properties. Chemical optimization of a second Bicycle, guided by structural biology, provided a high affinity, metabolically stable Bicycle with improved physicochemical properties. A BTC incorporating this Bicycle also demonstrated potent antitumor activity and was very well tolerated when compared to the initial BTC. Phage display selection followed by chemical optimization of Bicycles can deliver potent drug conjugates with favorable pharmaceutical properties.

Investigation of Fugitive Aerosols Released into the Environment during High-Flow Therapy.

BACKGROUND: Nebulised medical aerosols are designed to deliver drugs to the lungs to aid in the treatment of respiratory diseases. However, an unintended consequence is the potential for fugitive emissions during patient treatment, which may pose a risk factor in both clinical and homecare settings. METHODS: The current study examined the potential for fugitive emissions, using albuterol sulphate as a tracer aerosol during high-flow therapy. A nasal cannula was connected to a head model or alternatively, a interface was connected to a tracheostomy tube in combination with a simulated adult and paediatric breathing profile. Two aerodynamic particle sizers (APS) recorded time-series aerosol concentrations and size distributions at two different distances relative to the simulated patient. RESULTS: The results showed that the quantity and characteristics of the fugitive emissions were influenced by the interface type, patient type and supplemental gas-flow rate. There was a trend in the adult scenarios; as the flow rate increased, the fugitive emissions and the mass median aerodynamic diameter (MMAD) of the aerosol both decreased. The fugitive emissions were comparable when using the adult breathing profiles for the nasal cannula and tracheostomy interfaces; however, there was a noticeable distinction between the two interfaces when compared for the paediatric breathing profiles. The highest recorded aerosol concentration was 0.370 ± 0.046 mg m-3 from the tracheostomy interface during simulated paediatric breathing with a gas-flow rate of 20 L/min. The averaged MMAD across all combinations ranged from 1.248 to 1.793 µm by the APS at a distance of 0.8 m away from the patient interface. CONCLUSIONS: Overall, the results highlight the potential for secondary inhalation of fugitive emissions released during simulated aerosol treatment with concurrent high-flow therapy. The findings will help in developing policy and best practice for risk mitigation from fugitive emissions.

Comparison of aerosol delivery across combinations of drug delivery interfaces with and without concurrent high-flow nasal therapy.

BACKGROUND: Current clinical practice during high-flow nasal therapy (HFNT) involves utilization of a nasal cannula to provide humidification, with a facemask placed over the cannula to deliver aerosol. Few studies have compared aerosol delivery across various delivery interfaces during HFNT. The objective of this study was to address this gap in the literature and evaluate aerosol delivery using two nebulizer types across different drug delivery interfaces, nasal cannula, facemask, and mouthpiece, during simulated adult HFNT. METHODS: A facemask or mouthpiece and/or a nasal cannula were positioned on an anatomically correct adult head model. The head model was connected to a breathing simulator via a collection filter. Both healthy breathing pattern and distressed breathing patterns were utilized. Aerosol dose was determined by quantifying the mass of drug captured on a filter positioned distal to the trachea. RESULTS: During simulated healthy breathing, a significantly greater aerosol dose was observed when the vibrating mesh nebulizer (VMN) was integrated with HFNT alone, supplying aerosol and humidified air simultaneously (2.88 ± 0.15%), as opposed to using with a facemask (0.33 ± 0.07%, 1.62 ± 0.46%, and 1.07 ± 0.25% at 0 L/min (LPM), 2LPM, and 6LPM, respectively) or mouthpiece (0.56 ± 0.13%, 2.16 ± 0.06%, and 1.82 ± 0.41% at 0LPM, 2LPM, and 6LPM). In addition, aerosol delivery was also significantly greater when the VMN was integrated into simulated HFNT (2.88 ± 0.15%), in comparison with using the jet nebulizer (JN) with a facemask (0.82 ± 0.16%) or a mouthpiece (0.86 ± 0.11%). During simulated distressed breathing, a significantly greater aerosol dose was observed when the VMN was integrated with HFNT, supplying aerosol and humidified air simultaneously (6.81 ± 0.45%), compared with using a facemask (0.86 ± 0.04%, 2.96 ± 0.26%, and 4.23 ± 0.93% at 0LPM, 2LPM, and 6LPM) or mouthpiece (0.73 ± 0.37%, 0.97 ± 0.20%, and 3.11 ± 0.53% at 0LPM, 2LPM, and 6LPM, respectively). Aerosol delivery was also greater when the VMN was integrated into HFNT (6.81 ± 0.45%), in comparison with using the JN with a facemask (5.72 ± 0.71%) or a mouthpiece (0.69 ± 0.53%). Furthermore, across all drug delivery interfaces, and in line with previous reports, aerosol delivery was greater during simulated distressed breathing, in comparison with simulated healthy adult breathing. CONCLUSIONS: This article will be of considerable benefit in enhancing the understanding of aerosol delivery during HFNT, an increasingly adopted therapeutic intervention by healthcare professionals.

Investigation of the Quantity of Exhaled Aerosols Released into the Environment during Nebulisation.

BACKGROUND: Secondary inhalation of medical aerosols is a significant occupational hazard in both clinical and homecare settings. Exposure to fugitive emissions generated during aerosol therapy increases the risk of the unnecessary inhalation of medication, as well as toxic side effects. METHODS: This study examines fugitively-emitted aerosol emissions when nebulising albuterol sulphate, as a tracer aerosol, using two commercially available nebulisers in combination with an open or valved facemask or using a mouthpiece with and without a filter on the exhalation port. Each combination was connected to a breathing simulator during simulated adult breathing. The inhaled dose and residual mass were quantified using UV spectrophotometry. Time-varying fugitively-emitted aerosol concentrations and size distributions during nebulisation were recorded using aerodynamic particle sizers at two distances relative to the simulated patient. Different aerosol concentrations and size distributions were observed depending on the interface. RESULTS: Within each nebuliser, the facemask combination had the highest time-averaged fugitively-emitted aerosol concentration, and values up to 0.072 ± 0.001 mg m-3 were recorded. The placement of a filter on the exhalation port of the mouthpiece yielded the lowest recorded concentrations. The mass median aerodynamic diameter of the fugitively-emitted aerosol was recorded as 0.890 ± 0.044 µm, lower the initially generated medical aerosol in the range of 2⁻5 µm. CONCLUSIONS: The results highlight the potential secondary inhalation of exhaled aerosols from commercially available nebuliser facemask/mouthpiece combinations. The results will aid in developing approaches to inform policy and best practices for risk mitigation from fugitive emissions.

Bicyclic Peptides as a New Modality for Imaging and Targeting of Proteins Overexpressed by Tumors.

Molecular imaging of cancers using probes specific for tumor-associated target proteins offers a powerful solution for providing information regarding selection of targeted therapy, patient stratification, and response to therapy. Here we demonstrate the power of bicyclic peptides as targeting probes, exemplified with the tumor-overexpressed matrix metalloproteinase MT1-MMP as a target. A bicyclic peptide with subnanomolar affinity towards MT1-MMP was identified, and its radioconjugate showed selective tumor uptake in an HT1080 xenograft mouse model. Proteolytic stabilization of the peptide by chemical modification significantly enhanced the in vivo tumor signal [from 2.5%ID/g to 12%ID/g at 1 hour post injection (p.i.)]. Studies using mouse xenograft models with different cell lines show a robust correlation between tumor signals and in vivo MT1-MMP expression levels. Fatty acid modification of the bicyclic peptide extended its circulating half-life, resulting in increased tumor signals (36%ID/g at 6 hours p.i.). Comparative work with an equipotent radiolabeled MT1-MMP targeting antibody demonstrated starkly differential biodistribution and tumor accumulation properties, with the tumor signal slowly increasing to 6.2%ID/g within 48 hours. The rapid tumor penetration characteristics of bicyclic peptides, coupled with high potency and chemical versatility, thus offer high-contrast imaging probes for clinical diagnostics with compelling additional potential in targeted therapy.Significance: This work demonstrates the potential of bicyclic peptides as a platform for the development of high-contrast imaging probes for potential use in clinical cancer diagnostics and molecularly targeted therapeutics.

In Vitro Study of the Effect of Breathing Pattern on Aerosol Delivery During High-Flow Nasal Therapy.

INTRODUCTION: The use of concurrent aerosol delivery during high-flow nasal therapy (HFNT) may be exploited to facilitate delivery of a variety of prescribed medications for inhalation. The study assessed the effect of tidal volume, breath rate, and inspiratory:expiratory (I:E) ratio on the quantity of aerosol captured at the level of the trachea during simulated HFNT. METHODS: Testing was completed according to a factorial statistical design of experiments (DOE) approach. Tracheal dose was characterized with a vibrating mesh nebulizer (Aerogen Solo, Aerogen Ltd) using simulated adult, small child, and infant HFNT models. Furthermore, aerosol delivery was evaluated across a range of adult patient profiles with clinically representative test setups. RESULTS: Aerosol delivery increased with a large tidal volume, a rapid breath rate, and a long inspiratory time. Tidal volume, breath rate, and I:E ratio each had a significant effect on tracheal dose across simulated adult, small child, and infant breathing. CONCLUSION: The main trends that were identified in the statistical DOE predicted aerosol delivery across adult patient breathing profiles, in terms of tidal volume, breath rate, and I:E ratio. Therefore, patients with distressed breathing profiles may be expected to receive a larger aerosol dose than those with normal breathing rates. FUNDING: Aerogen Limited.

Stable and Long-Lasting, Novel Bicyclic Peptide Plasma Kallikrein Inhibitors for the Treatment of Diabetic Macular Edema.

Plasma kallikrein, a member of the kallikrein-kinin system, catalyzes the release of the bioactive peptide bradykinin, which induces inflammation, vasodilation, vessel permeability, and pain. Preclinical evidence implicates the activity of plasma kallikrein in diabetic retinopathy, which is a leading cause of visual loss in patients suffering from diabetes mellitus. Employing a technology based on phage-display combined with chemical cyclization, we have identified highly selective bicyclic peptide inhibitors with nano- and picomolar potencies toward plasma kallikrein. Stability in biological matrices was either intrinsic to the peptide or engineered via the introduction of non-natural amino acids and nonpeptidic bonds. The peptides prevented bradykinin release in vitro, and in vivo efficacy was demonstrated in both a rat paw edema model and in rodent models of diabetes-induced retinal permeability. With a highly extended half-life of ∼40 h in rabbit eyes following intravitreal administration, the bicyclic peptides are promising novel agents for the treatment of diabetic retinopathy and diabetic macular edema.

In Vitro Determination of the Main Effects in the Design of High-Flow Nasal Therapy Systems with Respect to Aerosol Performance.

INTRODUCTION: The use of concurrent aerosol delivery during high-flow nasal therapy (HFNT) may be exploited to facilitate the delivery of a variety of prescribed medications for inhalation. Until now, a systematic approach to determine the conditions required to yield an optimal emitted dose has not been reported. The aim of this study was to establish the effects of gas flow rate, input droplet size, and nebulizer position on the amount of aerosol exiting the nasal cannula during HFNT and thus becoming available for inhalation. METHODS: Testing was completed according to a factorial statistical design of experiments (DOE) approach. Emitted dose was characterized with a vibrating mesh nebulizer (Aerogen Solo, Aerogen Ltd) for an adult model of HFNT at three clinically relevant gas flow rates, using three nebulizers producing varying input droplet sizes and placed at two different nebulizer positions. RESULTS: Increasing the gas flow rate significantly lowered the emitted dose, with a dose of 7.10% obtained at 10 LPM, 2.67% at 25 LPM, and 1.30% at 40 LPM (p < 0.0001). There was a significant difference in emitted dose between nebulizers with different input droplet sizes, with increasing input droplet size associated with a reduced emitted dose (6.11% with an input droplet size of 3.22 µm, 2.76% with 4.05 µm, and 2.38% with 4.88 µm, p = 0.0002, Pearson's r = - 0.2871). In addition, the droplet size exiting the nasal cannula interface was lower than that produced by the aerosol generator for all devices under test. Positioning the nebulizer immediately after the humidification chamber yielded a marginally greater emitted dose (3.79%) than when the nebulizer was placed immediately upstream of the nasal cannula (3.39%). Flow rate, input droplet size, and nebulizer position were at the 0.10 level of significance, indicating that all three factors had significant effects on emitted dose. According to the DOE model, flow rate had the greatest influence on emitted dose, followed by input droplet size and then nebulizer position. CONCLUSION: Our findings indicate that in order to optimize the amount of aerosol exiting the nasal cannula interface during HFNT, it is necessary for gas flow rate to be low and the input droplet size to be small, while the nebulizer should be positioned immediately after the humidification chamber. FUNDING: Aerogen Limited.

CZ415, a Highly Selective mTOR Inhibitor Showing in Vivo Efficacy in a Collagen Induced Arthritis Model.

CZ415, a potent ATP-competitive mTOR inhibitor with unprecedented selectivity over any other kinase is described. In addition to a comprehensive characterization of its activities in vitro, in vitro ADME, and in vivo pharmacokinetic data are reported. The suitability of this inhibitor for studying in vivo mTOR biology is demonstrated in a mechanistic mouse model monitoring mTOR proximal downstream phosphorylation signaling. Furthermore, the compound reported here is the first ATP-competitive mTOR inhibitor described to show efficacy in a semitherapeutic collagen induced arthritis (CIA) mouse model.

A rapid and transient peripheral inflammatory response precedes brain inflammation after experimental stroke.

Increasing evidence suggests that peripheral inflammatory responses to stroke and other brain injuries have an important role in determining neurological outcome. The mediators of this response and the temporal relationships between peripheral and central inflammatory alterations are poorly understood. In this study, we show that experimental stroke in mice induces a peripheral inflammatory response that peaks 4 h after stroke, and precedes the peak in brain inflammation 24 h after stroke. This peripheral response is dominated by the induction of the chemokine CXCL-1 and the proinflammatory cytokine interleukin-6 and could serve as an accessible target for therapy and as a source of biomarkers predictive of prognosis.

Optimization of a series of multi-isoform PI3 kinase inhibitors.

Optimization of the cellular and pharmacological activity of a novel series of PI3 kinase inhibitors targeting multiple isoforms is described.

Achieving multi-isoform PI3K inhibition in a series of substituted 3,4-dihydro-2H-benzo[1,4]oxazines.

The SAR and pharmacokinetic profiles of a series of multi-isoform PI3K inhibitors based on a 3,4-dihydro-2H-benzo[1,4]oxazine scaffold are disclosed.

Chondroitinase ABC promotes functional recovery after spinal cord injury.

The inability of axons to regenerate after a spinal cord injury in the adult mammalian central nervous system (CNS) can lead to permanent paralysis. At sites of CNS injury, a glial scar develops, containing extracellular matrix molecules including chondroitin sulphate proteoglycans (CSPGs). CSPGs are inhibitory to axon growth in vitro, and regenerating axons stop at CSPG-rich regions in vivo. Removing CSPG glycosaminoglycan (GAG) chains attenuates CSPG inhibitory activity. To test the functional effects of degrading chondroitin sulphate (CS)-GAG after spinal cord injury, we delivered chondroitinase ABC (ChABC) to the lesioned dorsal columns of adult rats. We show that intrathecal treatment with ChABC degraded CS-GAG at the injury site, upregulated a regeneration-associated protein in injured neurons, and promoted regeneration of both ascending sensory projections and descending corticospinal tract axons. ChABC treatment also restored post-synaptic activity below the lesion after electrical stimulation of corticospinal neurons, and promoted functional recovery of locomotor and proprioceptive behaviours. Our results demonstrate that CSPGs are important inhibitory molecules in vivo and suggest that their manipulation will be useful for treatment of human spinal injuries.