Dendrimer biopharmaceutics: toward active dendrimer-cannabinoid drugs

The ultimate aim of the work described in this thesis was to (1) utilise PAMAM dendrimers as a tool to achieve differential transport across the intestinal mucosa and the blood brain barrier, where these dendrimers can be used to achieve oral bioavailability but avoid BBB penetration and CNS access and (2) to create cannabinoid-dendrimer conjugates that are active in their own right and whose penetration to the brain is prevented but whose intestinal activity is afforded for the treatment of IBD. Overall, the work described in this thesis has promoted a strategy whereby an active polymer (dendrimer)-drug conjugate could be formed that is active in its own right and where the polymer can serve to provide differential biological barrier transport which with regard to cannabinoid pharmacology obviates adverse CNS effects. The work in this thesis describes the design and synthesis of novel and active cannabinoid structures that should have commercial interest. These novel compounds served to further elucidate SAR in amino alkyl indole cannabinoids. SAR findings have revealed a site on these cannabinoids that can be functionally altered without loss of pharmacological activity. Additionally, studies in this thesis have led to the development of a novel radiolabelling strategy for anionic polymers that offers a number of distinct advantages over other approaches. Ultimately, a novel stable Dendrimer-cannabinoid conjugate has been synthesised but to date has not shown biological activity in the models utilised in this work

OptiMal-PK: an internet-based, user-friendly interface for the mathematical-based design of optimized anti-malarial treatment regimens.

BACKGROUND The search for highly effective anti-malarial therapies has gathered pace and recent years have seen a number of promising single and combined therapies reach the late stages of development. A key drug development challenge is the need for early assessment of the clinical utility of new drug leads as it is often unclear for developers whether efforts should be focused on efficacy or metabolic stability/exposure or indeed whether the continuation of iterative QSAR (quantitative structure-activity and relationships) cycles of medicinal chemistry and biological testing will translate to improved clinical efficacy. Pharmacokinetic and pharmacodynamic (PK/PD)-based measurements available from in vitro studies can be used for such clinical predictions. However, these predictions often require bespoke mathematical PK/PD modelling expertise and are normally performed after candidate development and, therefore, not during the pre-clinical development phase when such decisions need to be made. METHODS An internet-based tool has been developed using STELLA(®) software. The tool simulates multiple differential equations that describe anti-malarial PK/PD relationships where the user can easily input PK/PD parameters. The tool utilizes a simple stop-light system to indicate the efficacy of each combination of parameters. This tool, called OptiMal-PK, additionally allows for the investigation of the effect of drug combinations with known or custom compounds. RESULTS The results of simulations obtained from OptiMal-PK were compared to a previously published and validated mathematical model on which this tool is based. The tool has also been used to simulate the PK/PD relationship for a number of existing anti-malarial drugs in single or combined treatment. Simulations were predictive of the published clinical parasitological clearance activities for these existing therapies. CONCLUSIONS OptiMal-PK is designed to be implemented by medicinal chemists and pharmacologists during the pre-clinical anti-malarial drug development phase to explore the impact of different PK/PD parameters upon the predicted clinical activity of any new compound. It can help investigators to identify which pharmacological features of a compound are most important to the clinical performance of a new chemical entity and how partner drugs could potentially improve the activity of existing therapies

Intracellular PD Modelling (PDi) for the Prediction of Clinical Activity of Increased Rifampicin Dosing

Increasing rifampicin (RIF) dosages could significantly reduce tuberculosis (TB) treatment durations. Understanding the pharmacokinetic-pharmacodynamics (PK–PD) of increasing RIF dosages could inform clinical regimen selection. We used intracellular PD modelling (PDi) to predict clinical outcomes, primarily time to culture conversion, of increasing RIF dosages. PDi modelling utilizes in vitro-derived measurements of intracellular (macrophage) and extracellular Mycobacterium tuberculosis sterilization rates to predict the clinical outcomes of RIF at increasing doses. We evaluated PDi simulations against recent clinical data from a high dose (35 mg/kg per day) RIF phase II clinical trial. PDi-based simulations closely predicted the observed time-to-patient culture conversion status at eight weeks (hazard ratio: 2.04 (predicted) vs. 2.06 (observed)) for high dose RIF-based treatments. However, PDi modelling was less predictive of culture conversion status at 26 weeks for high-dosage RIF (99% predicted vs. 81% observed). PDi-based simulations indicate that increasing RIF beyond 35 mg/kg/day is unlikely to significantly improve culture conversion rates, however, improvements to other clinical outcomes (e.g., relapse rates) cannot be ruled out. This study supports the value of translational PDi-based modelling in predicting culture conversion rates for antitubercular therapies and highlights the potential value of this platform for the improved design of future clinical trials

Caveolin-1 in renal cell carcinoma promotes tumour cell invasion, and in co-operation with pERK predicts metastases in patients with clinically confined disease

Background: Up to 40% of patients initially diagnosed with clinically-confined renal cell carcinoma (RCC) and who undergo curative surgery will nevertheless relapse with metastatic disease (mRCC) associated with poor long term survival. The discovery of novel prognostic/predictive biomarkers and drug targets is needed and in this context the aim of the current study was to investigate a putative caveolin-1/ERK signalling axis in clinically confined RCC, and to examine in a panel of RCC cell lines the effects of caveolin-1 (Cav-1) on pathological processes (invasion and growth) and select signalling pathways. Methods: Using immunohistochemistry we assessed the expression of both Cav-1 and phosphorylated-ERK (pERK) in 176 patients with clinically confined RCC, their correlation with histological parameters and their impact upon disease-free survival. Using a panel of RCC cell lines we explored the functional effects of Cav-1 knockdown upon cell growth, cell invasion and VEGF-A secretion, as well Cav-1 regulation by cognate cell signalling pathways. Results: We found a significant correlation (P = 0.03) between Cav-1 and pERK in a cohort of patients with clinically confined disease which represented a prognostic biomarker combination (HR = 4.2) that effectively stratified patients into low, intermediate and high risk groups with respect to relapse, even if the patients’ tumours displayed low grade and/or low stage disease. In RCC cell lines Cav-1 knockdown unequivocally reduced cell invasive capacity while also displaying both pro-and anti-proliferative effects; targeted knockdown of Cav-1 also partially suppressed VEGF-A secretion in VHL-negative RCC cells. The actions of Cav-1 in the RCC cell lines appeared independent of both ERK and AKT/mTOR signalling pathways. Conclusion: The combined expression of Cav-1 and pERK serves as an independent biomarker signature with potential merit in RCC surveillance strategies able to predict those patients with clinically confined disease who will eventually relapse. In a panel of in-vitro RCC cells Cav-1 promotes cell invasion with variable effects on cell growth and VEGF-A secretion. Cav-1 has potential as a therapeutic target for the prevention and treatment of mRCC

Human Direct Skin Feeding Versus Membrane Feeding to Assess the Mosquitocidal Efficacy of High-Dose Ivermectin (IVERMAL Trial)

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Suboptimal Exposure to Anti-TB Drugs in a TBM/HIV+ Population is not Related to Anti-retroviral Therapy.

A placebo-controlled trial that compares the outcomes of immediate versus deferred initiation of antiretroviral therapy in HIV+ve Tuberculous Meningitis (TBM) patients was conducted in Vietnam in 2011. Here, the pharmacokinetics of Rifampicin, Isoniazid, Pyrazinamide and Ethambutol were investigated in the presence and absence of anti-HIV treatment in 85 patients. Pharmacokinetic analyses show that HIV therapy has no significant impact upon the pharmacokinetics of TB drugs in this cohort. The same population, however, displayed generally low CSF and systemic exposures to rifampicin compared to previously reported HIV –ve cohorts. Elevated CSF concentrations of pyrazinamide on the other hand were strongly and independently correlated with increased mortality and neurological toxicity. The findings suggest that the current standard dosing regimens may put the patient at risk of treatment failure from suboptimal rifampicin exposure, and potentially increasing the risk of adverse CNS events which are independently correlated with pyrazinamide CSF exposure

Pharmacokinetic-Pharmacodynamic modelling of intracellular Mycobacterium tuberculosis growth and kill rates is predictive of clinical treatment duration

Tuberculosis (TB) treatment is long and complex, typically involving a combination of drugs taken for 6 months. Improved drug regimens to shorten and simplify treatment are urgently required, however a major challenge to TB drug development is the lack of predictive pre-clinical tools. To address this deficiency, we have adopted a new high-content imaging-based approach capable of defining the killing kinetics of first line anti-TB drugs against intracellular Mycobacterium tuberculosis (Mtb) residing inside macrophages. Through use of this pharmacokinetic-pharmacodynamic (PK-PD) approach we demonstrate that the killing dynamics of the intracellular Mtb sub-population is critical to predicting clinical TB treatment duration. Integrated modelling of intracellular Mtb killing alongside conventional extracellular Mtb killing data, generates the biphasic responses typical of those described clinically. Our model supports the hypothesis that the use of higher doses of rifampicin (35?mg/kg) will significantly reduce treatment duration. Our described PK-PD approach offers a much needed decision making tool for the identification and prioritisation of new therapies which have the potential to reduce TB treatment duration

Intracellular Pharmacodynamic Modeling Is Predictive of the Clinical Activity of Fluoroquinolones against Tuberculosis

Clinical studies of new antitubercular drugs are costly and time-consuming. Owing to the extensive tuberculosis (TB) treatment periods, the ability to identify drug candidates based on their predicted clinical efficacy is vital to accelerate the pipeline of new therapies. Recent failures of preclinical models in predicting the activity of fluoroquinolones underline the importance of developing new and more robust predictive tools that will optimize the design of future trials. Here, we used high-content imaging screening and pharmacodynamic intracellular (PDi) modeling to identify and prioritize fluoroquinolones for TB treatment. In a set of studies designed to validate this approach, we show moxifloxacin to be the most effective fluoroquinolone, and PDi modeling-based Monte Carlo simulations accurately predict negative culture conversion (sputum sterilization) rates compared to eight independent clinical trials. In addition, PDi-based simulations were used to predict the risk of relapse. Our analyses show that the duration of treatment following culture conversion can be used to predict the relapse rate. These data further support that PDi-based modeling offers a much-needed decision-making tool for the TB drug development pipeline

Population Pharmacokinetics of Liposomal Amphotericin B in Immunocompromised Children

BACKGROUND Liposomal amphotericin B (LAmB) is widely used in the treatment of invasive fungal disease (IFD) in adults and children. There are relatively limited PK data to inform optimal dosing in children that achieves systemic drug exposures comparable to those of adults. OBJECTIVES To describe the pharmacokinetics of LAmB in children aged 1-17 years with suspected or documented IFD. METHODS Thirty-five children were treated with LAmB at dosages of 2.5-10 mg kg(-1) daily. Samples were taken at baseline and at 0.5-2.0 hourly intervals for twenty-four hours after receipt of the first dose (n=35 patients) and on the final day of therapy (n=25 patients). LAmB was measured using high performance liquid chromatography (HPLC). The relationship between drug exposure and development of toxicity was explored. RESULTS An evolution in PK was observed during the course of therapy resulting in a proportion of patients (n=13) having significantly higher maximum serum concentration (Cmax) and area under the concentration time curve (AUC0-24) later in the course of therapy, without evidence of drug accumulation (Cmin accumulation ratio, AR < 1.2). The fit of a 2-compartment model incorporating weight and an exponential decay function describing volume of distribution best described the data. There was a statistically significant relationship between mean AUC0-24 and probability of nephrotoxicity (OR 2.37; 95% CI 1.84-3.22, p=0.004). CONCLUSIONS LAmB exhibits nonlinear pharmacokinetics. A third of children appear to experience a time-dependent change in PK, which is not explained by weight, maturation or observed clinical factors

Efficacy and Safety of High-Dose Ivermectin for Reducing Malaria Transmission (IVERMAL): Protocol for a Double-Blind, Randomized, Placebo-Controlled, Dose-Finding Trial in Western Kenya

Background: Innovative approaches are needed to complement existing tools for malaria elimination. Ivermectin is a broad spectrum antiparasitic endectocide clinically used for onchocerciasis and lymphatic filariasis control at single doses of 150‐200 mcg/kg. It also shortens the lifespan of mosquitoes that feed on individuals recently treated with ivermectin. However, the effect after a 150‐200 mcg/kg oral dose is short‐lived (6‐11 days). Modelling suggests higher doses, that prolong the mosquitocidal effects, are needed to make a significant contribution to malaria elimination. Ivermectin has a wide therapeutic index and previous studies have shown doses up to 2,000 mcg/kg, i.e. 10x the US Food and Drug Administration approved dose, are well tolerated and safe; the highest dose used for onchocerciasis is single‐dose 800 mcg/kg. Objective: To determine the safety, tolerability, and efficacy of ivermectin 0, 300, 600 mcg/kg/day for 3 days, when provided with a standard 3‐day course of the antimalarial dihydroartemisinin‐piperaquine, on mosquito survival. Methods: This is a double‐blind, randomised, placebo‐controlled, parallel‐group, 3‐arm, dose‐finding trial in adults with uncomplicated malaria. Monte Carlo simulations based on pharmacokinetic modelling were performed to determine the optimum dosing regimens to be tested. Modelling showed that a 3‐day regimen of 600 mcg/kg/day achieves similar median (5‐95 percentiles) Cmax concentrations of ivermectin to single‐dose of 800 mcg/kg, while increasing the median time above the LC50 (16 ng/mL) from 1.9 days (1.0‐5.7) to 6.8 (3.8‐13.4) days. The 300 mcg/kg/day dose was chosen at 50% of the higher dose to allow evaluation of the dose response. Mosquito survival will be assessed daily up to 28 days in laboratory‐reared Anopheles gambiae s.s. populations fed on patients’ blood taken at days 0, 2 (Cmax), 7 (primary outcome), 10, 14, 21, and 28 after the start of treatment. Safety outcomes include QT‐prolongation and mydriasis. The trial will be conducted in 6 health facilities in western Kenya and requires a sample size of 141 participants (47 per arm). Sub‐studies include: (1) rich pharmacokinetics and (2) direct skin vs membrane feeding assays. Results: Recruitment started July 20th, 2015. Data collection was completed on July 2nd, 2016. Unblinding and analysis will commence once the database has been completed, cleaned and locked. Discussion: High‐dose ivermectin, if found to be safe and well tolerated, might offer a promising new tool for malaria elimination. Trial registration: ClinicalTrials.gov: NCT02511353 (July 15, 2015)