High wavenumber Raman spectroscopy for intraoperative assessment of breast tumour margins.

Optimal oncological results and patient outcomes are achieved in surgery for early breast cancer with breast conserving surgery (BCS) where this is appropriate. A limitation of BCS occurs when cancer is present at, or close, to the resection margin - termed a 'positive' margin - and re-excision is recommended to reduce recurrence rate. This is occurs in 17% of BCS in the UK and there is therefore a critical need for a way to assess margin status intraoperatively to ensure complete excision with adequate margins at the first operation. This study presents the potential of high wavenumber (HWN) Raman spectroscopy to address this. Freshly excised specimens from thirty patients undergoing surgery for breast cancer were measured using a surface Raman probe, and a multivariate classification model to predict normal versus tumour was developed from the data. This model achieved 77.1% sensitivity and 90.8% specificity following leave one patient out cross validation, with the defining features being differences in water content and lipid versus protein content. This demonstrates the feasibility of HWN Raman spectroscopy to facilitate future intraoperative margin assessment at specific locations. Clinical utility of the approach will require further research.

Quantitative Evaluation of Dendritic Nanoparticles in Mice: Biodistribution Dynamics and Downstream Tumor Efficacy Outcomes.

A physiologically based pharmacokinetic model was developed to describe the tissue distribution kinetics of a dendritic nanoparticle and its conjugated active pharmaceutical ingredient (API) in plasma, liver, spleen, and tumors. Tumor growth data from MV-4-11 tumor-bearing mice were incorporated to investigate the exposure/efficacy relationship. The nanoparticle demonstrated improved antitumor activity compared to the conventional API formulation, owing to the extended released API concentrations at the site of action. Model simulations further enabled the identification of critical parameters that influence API exposure in tumors and downstream efficacy outcomes upon nanoparticle administration. The model was utilized to explore a range of dosing schedules and their effect on tumor growth kinetics, demonstrating the improved antitumor activity of nanoparticles with less frequent dosing compared to the same dose of naked APIs in conventional formulations.

ADME and DMPK considerations for the discovery and development of antibody drug conjugates (ADCs).

 The therapeutic concept of antibody drug conjugates (ADCs) is to selectively target tumour cells with small molecule cytotoxic drugs to maximise cell kill benefit and minimise healthy tissue toxicity.An ADC generally consists of an antibody that targets a protein on the surface of tumour cells chemically linked to a warhead small molecule cytotoxic drug.To deliver the warhead to the tumour cell, the antibody must bind to the target protein and in general be internalised into the cell. Following internalisation, the cytotoxic agent can be released in the endosomal or lysosomal compartment (via different mechanisms). Diffusion or transport out of the endosome or lysosome allows the cytotoxic drug to express its cell-killing pharmacology. Alternatively, some ADCs (e.g. EDB-ADCs) rely on extracellular cleavage releasing membrane permeable warheads.One potentially important aspect of the ADC mechanism is the 'bystander effect' whereby the cytotoxic drug released in the targeted cell can diffuse out of that cell and into other (non-target expressing) tumour cells to exert its cytotoxic effect. This is important as solid tumours tend to be heterogeneous and not all cells in a tumour will express the targeted protein.The combination of large and small molecule aspects in an ADC poses significant challenges to the disposition scientist in describing the ADME properties of the entire molecule.This article will review the ADC landscape and the ADME properties of successful ADCs, with the aim of outlining best practice and providing a perspective of how the field can further facilitate the discovery and development of these important therapeutic modalities.

Obesity impacts the regulation of miR-10b and its targets in primary breast tumors.

BACKGROUND: Obesity increases breast cancer (BC) risk in post-menopausal women by mostly unknown molecular mechanisms which may partly be regulated by microRNAs (miRNAs). METHODS: We isolated RNA from paired benign and malignant biopsies from 83 BC patients and determined miRNA profiles in samples from 12 women at the extremes of the BMI distribution by RNA-seq. Candidates were validated in all samples. Associations between miR-10b expression and validated target transcript levels, and effects of targeted manipulation of miR-10b levels in a primary BC cell line on proliferation and invasion potential, were explored. RESULTS: Of the 148 miRNAs robustly expressed in breast tissues, the levels of miR-21, miR-10b, miR-451a, miR-30c, and miR-378d were significantly associated with presence of cancer. Of these, miR-10b showed a stronger down-regulation in the tumors of the obese subjects, as opposed to the lean. In ductal but not lobular tumors, significant inverse correlations were observed between the tumor levels of miR-10b and miR-30c and the mRNA levels of cancer-relevant target genes SRSF1, PIEZO1, MAPRE1, CDKN2A, TP-53 and TRA2B, as well as tumor grade. Suppression of miR-10b levels in BT-549 primary BC-derived cells increased cell proliferation and invasive capacity, while exogenous miR-10b mimic decreased invasion. Manipulation of miR-10b levels also inversely affected the mRNA levels of miR-10b targets BCL2L11, PIEZO1 and NCOR2. CONCLUSIONS: Our findings suggest that miR-10b may be a mediator between obesity and cancer in post-menopausal women, regulating several known cancer-relevant genes. MiR-10b expression may have diagnostic and therapeutic implications for the incidence and prognosis of BC in obese women.

Intratracheal and oral administration of SM-276001: a selective TLR7 agonist, leads to antitumor efficacy in primary and metastatic models of cancer.

Topical TLR7 agonists such as imiquimod are highly effective for the treatment of dermatological malignancies; however, their efficacy in the treatment of nondermatological tumors has been less successful. We report that oral administration of the novel TLR7-selective small molecule agonist; SM-276001, leads to the induction of an inflammatory cytokine and chemokine milieu and to the activation of a diverse population of immune effector cells including T and B lymphocytes, NK and NKT cells. Oral administration of SM-276001 leads to the induction of IFNα, TNFα and IL-12p40 and a reduction in tumor burden in the Balb/c syngeneic Renca and CT26 models. Using the OV2944-HM-1 model of ovarian cancer which spontaneously metastasizes to the lungs following subcutaneous implantation, we evaluated the efficacy of intratracheal and oral administration of SM-276001 in an adjuvant setting following surgical resection of the primary tumor. We show that both oral and intratracheal TLR7 therapy can reduce the frequency of pulmonary metastasis, and metastasis to the axillary lymph nodes. These results demonstrate that SM-276001 is a potent selective TLR7 agonist that can induce antitumor immune responses when dosed either intratracheally or orally.

The regulation of ATP release from the urothelium by adenosine and transepithelial potential.

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Stretch of the urothelium, as occurs during bladder filling, is associated with a release of ATP that is postulated to act as a sensory neurotransmitter. The regulation of ATP release is poorly understood and in particular if there is a feedback mechanism provided by ATP itself. Adenosine, a breakdown product of ATP, is a potent inhibitor of stretch-induced ATP release, acting through and A1 receptor; endogenous levels are about 0.6µM. Data are consistent with ATP release relying on the rise of intracellular Ca2+. Transepithelial potential also controls ATP release, also acting via an A1 receptor-dependent pathway. OBJECTIVES: To test the hypothesis that distension-induced ATP release from the bladder urothelium is regulated by adenosine as well as changes to transurothelial potential (TEP). To examine the role of changes to intracellular [Ca(2+) ] in ATP release. MATERIALS AND METHODS: Rabbit urothelium/suburothelium membranes were used in an Ussing chamber system. Distension was induced by fluid removal from the chamber bathing the serosal (basolateral) membrane face. The TEP and short-circuit current were measured. ATP was measured in samples aspirated from the serosal chamber by a luciferin-luciferase assay. Intracellular [Ca(2+) ] was measured in isolated urothelial cells using the fluorochrome Fura-2. All experiments were performed at 37°C. RESULTS: Distension-induced ATP release was decreased by adenosine (1-10 µm) and enhanced by adenosine deaminase and A1- (but not A2-) receptor antagonists. Distension-induced ATP release was reduced by 2-APB, nifedipine and capsazepine; capsaicin induced ATP release in the absence of distension. ATP and capsaicin, but not adenosine, generated intracellular Ca(2+) transients; adenosine did not affect the ATP-generated Ca(2+) transient. ATP release was dependent on a finite transepithelial potential. Changes to TEP, in the absence of distension, generated ATP release that was in turn reduced by adenosine. CONCLUSION: Adenosine exerts a powerful negative feedback control of ATP release from the urothelium via A1 receptor activation. Distension-induced ATP release may be mediated by a rise of the intracellular [Ca(2+) ]. Modulation of distension-induced ATP release by adenosine and TEP may have a common pathway.

Systems pharmacology modeling of drug-induced modulation of thyroid hormones in dogs and translation to human.

PURPOSE: To develop a systems pharmacology model based on hormone physiology and pharmacokinetic-pharmacodynamic concepts describing the impact of thyroperoxidase (TPO) inhibition on thyroid hormone homeostasis in the dog and to predict drug-induced changes in thyroid hormones in humans. METHODS: A population model was developed based on a simultaneous analysis of concentration-time data of T4, T3 and TSH in dogs following once daily oral dosing for up to 6-months of a myeloperoxidase inhibitor (MPO-IN1) with TPO inhibiting properties. The model consisted of linked turnover compartments for T4, T3 and TSH including a negative feedback from T4 on TSH concentrations. RESULTS: The model could well describe the concentration-time profiles of thyroid hormones in dog. Successful model validation was performed by predicting the hormone concentrations during 1-month administration of MPO-IN2 based on its in vitro dog TPO inhibition potency. Using human thyroid hormone turnover rates and TPO inhibitory potency, the human T4 and TSH concentrations upon MPO-IN1 treatment were predicted well. CONCLUSIONS: The model provides a scientific framework for the prediction of drug induced effects on plasma thyroid hormones concentrations in humans via TPO inhibition based on results obtained in in vitro and animal studies.

Discovery of a potent respiratory syncytial virus RNA polymerase inhibitor.

Targeting viral polymerases has been a proven and attractive strategy for antiviral drug discovery. Herein we describe our effort in improving the antiviral activity and physical properties of a series of benzothienoazepine compounds as respiratory syncytial virus (RSV) RNA polymerase inhibitors. The antiviral activity and spectrum of this class was significantly improved by exploring the amino substitution of the pyridine ring, resulting in the discovery of the most potent RSV A polymerase inhibitors reported to date.

Immunosuppressive properties of a series of novel inhibitors of the monocarboxylate transporter MCT-1.

We have recently described the immunosuppressive properties of AR-C117977 and AR-C122982, representatives of a group of compounds identified as inhibitors of lactate transporters (monocarboxylate transporters; MCTs). These compounds demonstrate the potential therapeutic usefulness of inhibiting MCT-1, but their physical and metabolic properties made them unsuitable for further development. We have therefore tried to find analogues with similar immunosuppressive efficacy and a more suitable profile for oral administration. Five analogues of AR-C117977 were synthesised and screened for binding to the transporter, for inhibition of proliferation of both human and rat lymphocytes, for in vivo activity in a model of graft-versus-host (GvH) response in the rat, and in high- and low-responder cardiac transplant models in the rat. There was a good correlation between levels of binding of the five analogues to MCT and their inhibition of lymphocyte proliferation in human and rat cells. Furthermore, activity in both the GvH response and the cardiac transplant models correlated well with the determined concentrations of test compound in plasma. These findings on new analogues of MCT-1 inhibitors have taken us further towards defining the pharmacokinetic properties that may help to identify future drug candidates among inhibitors of MCT-1.

Practical use of the regression offset approach for the prediction of in vivo intrinsic clearance from hepatocytes.

Systematic under-prediction of clearance is frequently associated with in vitro kinetic data when extrapolated using physiological scaling factors, appropriate binding parameters and the well-stirred model. The present study describes a method of removing this systematic bias through application of empirical correction factors derived from regression analyses applied to the in vitro and in vivo data for a defined set of reference compounds. Linear regression lines were established with in vivo intrinsic clearance (CLint), derived from in vivo clearance data and scaled in vitro intrinsic clearance from isolated hepatocyte incubations. The scaled CLint was empirically corrected to a predicted in vivo CLint using the slope and intercept from a uniform weighted linear regression applied to the in vitro to in vivo extrapolation. Cross validation of human data demonstrated that 66% of the reference compounds had a predicted in vivo CLint within two-fold of the observed value. The average absolute fold error (AAFE) for the in vivo CLint predictions was 1.90. For rat, 54% of the compounds had a predicted value within two-fold of the observed and the AAFE was 1.98. Three AstraZeneca projects are used to exemplify how a two-sided prediction interval, applied to the rat regression corrected reference data, can form the basis for assessing the likelihood that, for a given chemical series, the in vitro kinetic data is predictive of in vivo clearance and is therefore appropriate to guide optimisation of compound metabolic stability.

Challenges and Opportunities for Improved Drug-Drug Interaction Predictions for Renal OCT2 and MATE1/2-K Transporters.

Transporters contribute to renal elimination of drugs; therefore drug disposition can be impacted if transporters are inhibited by comedicant drugs. Regulatory agencies have provided guidelines to assess potential drug-drug interaction (DDI) risk for renal organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 and 2-K (MATE1/2-K) transporters. Despite this, there are challenges with translating in vitro data using currently available tools to obtain a quantitative assessment of DDI risk in the clinic. Given the high number of drugs and new molecular entities showing in vitro inhibition toward OCT2 and/or MATE1/2-K and the lack of translation to clinically significant effects, it is reasonable to question whether the current in vitro assay design and modeling practice has led to unnecessary clinical evaluation. The aim of this review is to assess and discuss available in vitro and clinical data along with prediction models intended to provide clinical context of risk, including static models proposed by regulatory agencies and physiologically-based pharmacokinetic models, in order to identify best practices and areas of future opportunity. This analysis highlights that different in vitro assay designs, including substrate and cell systems used, strongly influence the derived concentration of drug producing 50% inhibition values and contribute to high variability observed across laboratories. Furthermore, the lack of sensitive index substrates coupled with specific inhibitors for individual transporters necessitates the use of complex models to evaluate clinical DDI risk.

Discovery of a Series of 7-Azaindoles as Potent and Highly Selective CDK9 Inhibitors for Transient Target Engagement.

Optimization of a series of azabenzimidazoles identified from screening hit 2 and the information gained from a co-crystal structure of the azabenzimidazole-based lead 6 bound to CDK9 led to the discovery of azaindoles as highly potent and selective CDK9 inhibitors. With the goal of discovering a highly selective and potent CDK9 inhibitor administrated intravenously that would enable transient target engagement of CDK9 for the treatment of hematological malignancies, further optimization focusing on physicochemical and pharmacokinetic properties led to azaindoles 38 and 39. These compounds are highly potent and selective CDK9 inhibitors having short half-lives in rodents, suitable physical properties for intravenous administration, and the potential to achieve profound but transient inhibition of CDK9 in vivo.

Multiscale Model Identifies Improved Schedule for Treatment of Acute Myeloid Leukemia In Vitro With the Mcl-1 Inhibitor AZD5991.

Anticancer efficacy is driven not only by dose but also by frequency and duration of treatment. We describe a multiscale model combining cell cycle, cellular heterogeneity of B-cell lymphoma 2 family proteins, and pharmacology of AZD5991, a potent small-molecule inhibitor of myeloid cell leukemia 1 (Mcl-1). The model was calibrated using in vitro viability data for the MV-4-11 acute myeloid leukemia cell line under continuous incubation for 72 hours at concentrations of 0.03-30 µM. Using a virtual screen, we identified two schedules as having significantly different predicted efficacy and showed experimentally that a "short" schedule (treating cells for 6 of 24 hours) is significantly better able to maintain the rate of cell kill during treatment than a "long" schedule (18 of 24 hours). This work suggests that resistance can be driven by heterogeneity in protein expression of Mcl-1 alone without requiring mutation or resistant subclones and demonstrates the utility of mathematical models in efficiently identifying regimens for experimental exploration.

AZD4573 Is a Highly Selective CDK9 Inhibitor That Suppresses MCL-1 and Induces Apoptosis in Hematologic Cancer Cells.

PURPOSE: Cyclin-dependent kinase 9 (CDK9) is a transcriptional regulator and potential therapeutic target for many cancers. Multiple nonselective CDK9 inhibitors have progressed clinically but were limited by a narrow therapeutic window. This work describes a novel, potent, and highly selective CDK9 inhibitor, AZD4573. EXPERIMENTAL DESIGN: The antitumor activity of AZD4573 was determined across broad cancer cell line panels in vitro as well as cell line- and patient-derived xenograft models in vivo. Multiple approaches, including integrated transcriptomic and proteomic analyses, loss-of-function pathway interrogation, and pharmacologic comparisons, were employed to further understand the major mechanism driving AZD4573 activity and to establish an exposure/effect relationship. RESULTS: AZD4573 is a highly selective and potent CDK9 inhibitor. It demonstrated rapid induction of apoptosis and subsequent cell death broadly across hematologic cancer models in vitro, and MCL-1 depletion in a dose- and time-dependent manner was identified as a major mechanism through which AZD4573 induces cell death in tumor cells. This pharmacodynamic (PD) response was also observed in vivo, which led to regressions in both subcutaneous tumor xenografts and disseminated models at tolerated doses both as monotherapy or in combination with venetoclax. This understanding of the mechanism, exposure, and antitumor activity of AZD4573 facilitated development of a robust pharmacokinetic/PD/efficacy model used to inform the clinical trial design. CONCLUSIONS: Selective targeting of CDK9 enables the indirect inhibition of MCL-1, providing a therapeutic option for MCL-1-dependent diseases. Accordingly, AZD4573 is currently being evaluated in a phase I clinical trial for patients with hematologic malignancies (clinicaltrials.gov identifier: NCT03263637).See related commentary by Alcon et al., p. 761.

Author Correction: On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.

The inaccessibility of living bone marrow (BM) hampers the study of its pathophysiology under myelotoxic stress induced by drugs, radiation or genetic mutations. Here, we show that a vascularized human BM-on-a-chip (BM chip) supports the differentiation and maturation of multiple blood cell lineages over 4 weeks while improving CD34+ cell maintenance, and that it recapitulates aspects of BM injury, including myeloerythroid toxicity after clinically relevant exposures to chemotherapeutic drugs and ionizing radiation, as well as BM recovery after drug-induced myelosuppression. The chip comprises a fluidic channel filled with a fibrin gel in which CD34+ cells and BM-derived stromal cells are co-cultured, a parallel channel lined by human vascular endothelium and perfused with culture medium, and a porous membrane separating the two channels. We also show that BM chips containing cells from patients with the rare genetic disorder Shwachman-Diamond syndrome reproduced key haematopoietic defects and led to the discovery of a neutrophil maturation abnormality. As an in vitro model of haematopoietic dysfunction, the BM chip may serve as a human-specific alternative to animal testing for the study of BM pathophysiology.

Discovery of AZD4573, a Potent and Selective Inhibitor of CDK9 That Enables Short Duration of Target Engagement for the Treatment of Hematological Malignancies.

A CDK9 inhibitor having short target engagement would enable a reduction of Mcl-1 activity, resulting in apoptosis in cancer cells dependent on Mcl-1 for survival. We report the optimization of a series of amidopyridines (from compound 2), focusing on properties suitable for achieving short target engagement after intravenous administration. By increasing potency and human metabolic clearance, we identified compound 24, a potent and selective CDK9 inhibitor with suitable predicted human pharmacokinetic properties to deliver transient inhibition of CDK9. Furthermore, the solubility of 24 was considered adequate to allow i.v. formulation at the anticipated effective dose. Short-term treatment with compound 24 led to a rapid dose- and time-dependent decrease of pSer2-RNAP2 and Mcl-1, resulting in cell apoptosis in multiple hematological cancer cell lines. Intermittent dosing of compound 24 demonstrated efficacy in xenograft models derived from multiple hematological tumors. Compound 24 is currently in clinical trials for the treatment of hematological malignancies.

Modeling Dose and Schedule Effects of AZD2811 Nanoparticles Targeting Aurora B Kinase for Treatment of Diffuse Large B-cell Lymphoma.

Barasertib (AZD1152), a pro-drug of the highly potent and selective Aurora B kinase inhibitor AZD2811, showed promising clinical activity in relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients administered as a 4-day infusion. To improve potential therapeutic benefit of Aurora B kinase inhibition, a nanoparticle formulation of AZD2811 has been developed to address limitations of repeated intravenous infusion. One of the challenges with the use of nanoparticles for chronic treatment of tumors is optimizing dose and schedule required to enable repeat administration to sustain tumor growth inhibition. AZD2811 gives potent cell growth inhibition across a range of DLBCL cells lines in vitro In vivo, repeat administration of the AZD2811 nanoparticle gave antitumor activity at half the dose intensity of AZD1152. Compared with AZD1152, a single dose of AZD2811 nanoparticle gave less reduction in pHH3, but increased apoptosis and reduction of cells in G1 and G2-M, albeit at later time points, suggesting that duration and depth of target inhibition influence the nature of the tumor cell response to drug. Further exploration of the influence of dose and schedule on efficacy revealed that AZD2811 nanoparticle can be used flexibly with repeat administration of 25 mg/kg administered up to 7 days apart being sufficient to maintain equivalent tumor control. Timing of repeat administration could be varied with 50 mg/kg every 2 weeks controlling tumor control as effectively as 25 mg/kg every week. AZD2811 nanoparticle can be administered with very different doses and schedules to inhibit DLBCL tumor growth, although maximal tumor growth inhibition was achieved with the highest dose intensities.

A new species of Hypoprepia from the mountains of central Arizona (Lepidoptera, Erebidae, Arctiinae, Lithosiini).

A new firefly-mimicking lichen moth of the genus Hypoprepia, H.lampyroides Palting & Ferguson, sp. n., is described from the mountains of east-central Arizona and the Sierra Madre Occidental of Mexico. Hypoprepia Hübner, 1831 is a North American genus of lithosiine tiger moths, previously consisting of five species: H.fucosa Hübner, 1831 and H.miniata (Kirby, 1837), both of eastern and central North America; H.cadaverosa Strecker, 1878 from the Rocky Mountains into New Mexico and west Texas; H.inculta H. Edwards, 1882, a widespread western USA species and H.muelleri Dyar, 1907 from the vicinity of Mexico City. The latter is herein synonymized under H.inculta (= H.muelleri syn. n.), resulting in the total number of taxa in the genus unchanged at five.

Translational model to predict pulmonary pharmacokinetics and efficacy in man for inhaled bronchodilators.

Translational pharmacokinetic (PK) models are needed to describe and predict drug concentration-time profiles in lung tissue at the site of action to enable animal-to-man translation and prediction of efficacy in humans for inhaled medicines. Current pulmonary PK models are generally descriptive rather than predictive, drug/compound specific, and fail to show successful cross-species translation. The objective of this work was to develop a robust compartmental modeling approach that captures key features of lung and systemic PK after pulmonary administration of a set of 12 soluble drugs containing single basic, dibasic, or cationic functional groups. The model is shown to allow translation between animal species and predicts drug concentrations in human lungs that correlate with the forced expiratory volume for different classes of bronchodilators. Thus, the pulmonary modeling approach has potential to be a key component in the prediction of human PK, efficacy, and safety for future inhaled medicines.