CXCR4 inhibition in human pancreatic and colorectal cancers induces an integrated immune response.

Inhibition of the chemokine receptor CXCR4 in combination with blockade of the PD-1/PD-L1 T cell checkpoint induces T cell infiltration and anticancer responses in murine and human pancreatic cancer. Here we elucidate the mechanism by which CXCR4 inhibition affects the tumor immune microenvironment. In human immune cell-based chemotaxis assays, we find that CXCL12-stimulated CXCR4 inhibits the directed migration mediated by CXCR1, CXCR3, CXCR5, CXCR6, and CCR2, respectively, chemokine receptors expressed by all of the immune cell types that participate in an integrated immune response. Inhibiting CXCR4 in an experimental cancer medicine study by 1-wk continuous infusion of the small-molecule inhibitor AMD3100 (plerixafor) induces an integrated immune response that is detected by transcriptional analysis of paired biopsies of metastases from patients with microsatellite stable colorectal and pancreatic cancer. This integrated immune response occurs in three other examples of immune-mediated damage to noninfected tissues: Rejecting renal allografts, melanomas clinically responding to anti-PD1 antibody therapy, and microsatellite instable colorectal cancers. Thus, signaling by CXCR4 causes immune suppression in human pancreatic ductal adenocarcinoma and colorectal cancer by impairing the function of the chemokine receptors that mediate the intratumoral accumulation of immune cells.Stand Up 2 Cancer, Lustgarten Foundation, NIH

Phase I clinical trial repurposing all-trans retinoic acid as a stromal targeting agent for pancreatic cancer

Abstract: Pre-clinical models have shown that targeting pancreatic stellate cells with all-trans-retinoic-acid (ATRA) reprograms pancreatic stroma to suppress pancreatic ductal adenocarcinoma (PDAC) growth. Here, in a phase Ib, dose escalation and expansion, trial for patients with advanced, unresectable PDAC (n = 27), ATRA is re-purposed as a stromal-targeting agent in combination with gemcitabine-nab-paclitaxel chemotherapy using a two-step adaptive continual re-assessment method trial design. The maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D, primary outcome) is the FDA/EMEA approved dose of gemcitabine-nab-paclitaxel along-with ATRA (45 mg/m2 orally, days 1–15/cycle). Dose limiting toxicity (DLT) is grade 4 thrombocytopenia (n = 2). Secondary outcomes show no detriment to ATRA pharmacokinetics.. Median overall survival for RP2D treated evaluable population, is 11.7 months (95%CI 8.6–15.7 m, n = 15, locally advanced (2) and metastatic (13)). Exploratory pharmacodynamics studies including changes in diffusion-weighted (DW)-MRI measured apparent diffusion coefficient after one cycle, and, modulation of cycle-specific serum pentraxin 3 levels over various cycles indicate stromal modulation. Baseline stromal-specific retinoid transport protein (FABP5, CRABP2) expression may be predicitve of response. Re-purposing ATRA as a stromal-targeting agent with gemcitabine-nab-paclitaxel is safe and tolerable. This combination will be evaluated in a phase II randomized controlled trial for locally advanced PDAC. Clinical trial numbers: EudraCT: 2015-002662-23; NCT03307148. Trial acronym: STARPAC

Development of a sensitive immunosensor for the detection of cardiac Troponin T in cardiovascular disease

Cardiovascular disease (CVD) is currently globally the biggest cause of mortality, with rising figures, especially now in the developing world. Early and accurate diagnosis of CVD, (especially acute myocardial infarction (AMI) is important in being able to provide appropriate, timely and cost effective treatment, or to take preventative action. Biomarkers and biosensors are playing an increasingly important role in this diagnosis, especially those based on immunoassays. As technology improves and becomes cheaper, there is the potential to develop immunosensors which use optical techniques such as surface plasmon resonance (SPR) for biomarker measurement which could be used effectively in point-of-care diagnostics for real-time detection. This thesis describes the development and optimisation of a sensitive immunosensor for the AMI specific biomarker, cardiac Troponin T (cTnT), on an SPR platform. Early diagnosis of AMI requires an assay methodology which can determine very low concentrations of cTnT in human serum. The work conducted includes the development of a set of optimised conditions for the immobilisation of the capture antibody (anti-cardiac Troponin T 1C11 antibody) onto a gold surfaced SPR sensor chip, to which a self-assembled monolayer of 11-mercaptoundecanoic acid has been applied. A direct immunoassay for cTnT in buffer was examined and a limit of detection (LOD) of 25 ng ml-1 cTnT was achieved. A sandwich immunoassay format was then developed to enhance the sensitivity of the assay. The use of a detection antibody (anti-cardiac Troponin T 7G7 antibody) was shown to successfully amplify the SPR response five-fold, with the LOD improving to 5 ng ml-1 cTnT. The second stage of the project involved examining the extent of non-specific binding of the cTnT and of serum proteins, and investigating how best to minimise and control for it. Non-specific binding of cTnT was eliminated, and serum protein binding was reduced by 93% in 10% serum and 73% in 50% serum. To achieve greater sensitivity, amplification of the signal through the use of detector antibodies conjugated to gold nanoparticles (AuNPs) for the sandwich assay was investigated. The performance of the cTnT immunosensor sandwich assay in human serum was evaluated using non-modified and AuNP modified detector antibodies. The LOD of the immunosensor in 50% serum was assessed as 5 ng ml-1 cTnT for the standard sandwich assay, and 0.5 ng ml-1 cTnT when using AuNP conjugated detector antibodies to enhance the sensitivity

Determination of codeine and its metabolites in microsomal incubates by high-performance liquid chromatography

A rapid and sensitive HPLC method has been developed for the determination of codeine, norcodeine and morphine in small volumes of a biological matrix, using a cyanopropyl column and a combination of coulometric and UV detection. The compounds were isolated using C18 solid-phase extraction cartridges prior to quantitative analysis. The limit of detection was 250 pg/ml for morphine and 5 ng/ml for both norcodeine and codeine. Recovery of each compound was greater than 90% and intra- and inter-assay precision was better than 10%. The method has been used to study the metabolism of codeine in microsomal incubations

An improved extraction method for the HPLC determination of morphine and its metabolites in plasma

A new, simple and rapid extraction procedure coupled with a combined coulometric-fluorescence HPLC assay is described for the simultaneous determination of morphine (M) and morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G), and normorphine (NM) in plasma. The effect of concentration and pH of selected ion-pairing agents on the extraction of these compounds from plasma by solid-phase extraction was investigated. The extraction procedure was optimized in terms of recovery, reproducibility and lack of interference from endogenous materials. The optimized method uses tetrabutylammonium hydrogen sulphate (TBAHS) at pH 10 followed by separation on a single C18 solid-phase extraction cartridge. For routine analysis the procedure provides high and reproducible recoveries over a concentation range of 1.0-1000 ng ml-1 for morphine, M6G and normorphine and 20-1000 ng ml-1 for M3G. The method was used successfully to analyse plasma samples from a pharmacokinetic study in which sheep had received an intravenous dose of 0.015 mg kg-1 of M6G