Potential Impact of Long-Acting Products on the Control of Tuberculosis: Preclinical Advancements and Translational Tools in Preventive Treatment.

A key component of global tuberculosis (TB) control is the treatment of latent TB infection. The use of long-acting technologies to administer TB preventive treatment has the potential to significantly improve the delivery and impact of this important public health intervention. For example, an ideal long-acting treatment could consist of a single dose that could be administered in the clinic (ie, a "1-shot cure" for latent TB). Interest in long-acting formulations for TB preventive therapy has gained considerable traction in recent years. This article presents an overview of the specific considerations and current preclinical advancements relevant for the development of long-acting technologies of TB drugs for treatment of latent infection, including attributes of target product profiles, suitability of drugs for long-acting formulations, ongoing research efforts, and translation to clinical studies.

Long-Acting Injectable Statins-Is It Time for a Paradigm Shift?

In recent years, advances in pharmaceutical processing technologies have resulted in development of medicines that provide therapeutic pharmacokinetic exposure for a period ranging from weeks to months following a single parenteral administration. Benefits for adherence, dose and patient satisfaction have been witnessed across a range of indications from contraception to schizophrenia, with a range of long-acting medicines also in development for infectious diseases such as HIV. Existing drugs that have successfully been formulated as long-acting injectable formulations have long pharmacokinetic half-lives, low target plasma exposures, and low aqueous solubility. Of the statins that are clinically used currently, atorvastatin, rosuvastatin, and pitavastatin may have compatibility with this approach. The case for development of long-acting injectable statins is set out within this manuscript for this important class of life-saving drugs. An overview of some of the potential development and implementation challenges is also presented.

Assessment of interactions of efavirenz solid drug nanoparticles with human immunological and haematological systems.

BACKGROUND: Recent work has developed solid drug nanoparticles (SDNs) of efavirenz that have been demonstrated, preclinically, improved oral bioavailability and the potential to enable up to a 50% dose reduction, and is currently being studied in a healthy volunteer clinical trial. Other SDN formulations are being studied for parenteral administration, either as intramuscular long-acting formulations, or for direct administration intravenously. The interaction of nanoparticles with the immunological and haematological systems can be a major barrier to successful translation but has been understudied for SDN formulations. Here we have conducted a preclinical evaluation of efavirenz SDN to assess their potential interaction with these systems. Platelet aggregation and activation, plasma coagulation, haemolysis, complement activation, T cell functionality and phenotype, monocyte derived macrophage functionality, and NK cell function were assessed in primary healthy volunteer samples treated with either aqueous efavirenz or efavirenz SDN. RESULTS: Efavirenz SDNs were shown not to interfere with any of the systems studied in terms of immunostimulation nor immunosuppression. Although efavirenz aqueous solution was shown to cause significant haemolysis ex vivo, efavirenz SDNs did not. No other interaction with haematological systems was observed. Efavirenz SDNs have been demonstrated to be immunologically and haematologically inert in the utilised assays. CONCLUSIONS: Taken collectively, along with the recent observation that lopinavir SDN formulations did not impact immunological responses, these data indicate that this type of nanoformulation does not elicit immunological consequences seen with other types of nanomaterial. The methodologies presented here provide a framework for pre-emptive preclinical characterisation of nanoparticle safety.

Synthesis, nanoprecipitation and pH sensitivity of amphiphilic linear-dendritic hybrid polymers and hyperbranched-polydendrons containing tertiary amine functional dendrons.

The combination of linear polymers with dendritic chain-ends has led to numerous studies of linear-dendritic polymer hybrid materials. Interchain branching within the linear segment of these materials has recently extended this concept to the formation of soluble hyperbranched-polydendrons. Here, the introduction of amphiphilicity into hyperbranched-polydendrons has been achieved for the first time through the use of tertiary amine functional dendritic chain-ends and branched hydrophobic polymer segments. The synthesis and aqueous nanoprecipitation of these branched materials is compared with their linear-dendritic polymer analogues, showing that chain-end chemistry/generation, precipitation medium pH and polymer architecture are all capable of influencing the ability to generate nanoparticles, the resulting nanoparticle diameter and dispersity, and subsequent response to changes in pH.

Nanomedicine strategies to improve therapeutic agents for the prevention and treatment of preterm birth and future directions.

The World Health Organisation (WHO) estimates 15 million babies worldwide are born preterm each year, with 1 million infant mortalities and long-term morbidity in survivors. Whilst the past 40 years have provided some understanding in the causes of preterm birth, along with development of a range of therapeutic options, notably prophylactic use of progesterone or uterine contraction suppressants (tocolytics), the number of preterm births continues to rise. Existing therapeutics used to control uterine contractions are restricted in their clinical use due to pharmacological drawbacks such as poor potency, transfer of drugs to the fetus across the placenta and maternal side effects from activity in other maternal systems. This review focuses on addressing the urgent need for the development of alternative therapeutic systems with improved efficacy and safety for the treatment of preterm birth. We discuss the application of nanomedicine as a viable opportunity to engineer pre-existing tocolytic agents and progestogens into nanoformulations, to improve their efficacy and address current drawbacks to their use. We review different nanomedicines including liposomes, lipid-based carriers, polymers and nanosuspensions highlighting where possible, where these technologies have already been exploited e.g. liposomes, and their significance in improving the properties of pre-existing therapeutic agents within the field of obstetrics. We also highlight where active pharmaceutical agents (APIs) with tocolytic properties have been used for other clinical indications and how these could inform the design of future therapeutics or be repurposed to diversify their application such as for use in preterm birth. Finally we outline and discuss the future challenges.

Understanding the Degradation of Core-Shell Nanogels Using Asymmetrical Flow Field Flow Fractionation.

Nanogels are candidates for biomedical applications, and core-shell nanogels offer the potential to tune thermoresponsive behaviour with the capacity for extensive degradation. These properties were achieved by the combination of a core of poly(N-isopropylmethacrylamide) and a shell of poly(N-isopropylacrylamide), both crosslinked with the degradable crosslinker N,N'-bis(acryloyl)cystamine. In this work, the degradation behaviour of these nanogels was characterised using asymmetric flow field flow fractionation coupled with multi-angle and dynamic light scattering. By monitoring the degradation products of the nanogels in real-time, it was possible to identify three distinct stages of degradation: nanogel swelling, nanogel fragmentation, and nanogel fragment degradation. The results indicate that the core-shell nanogels degrade slower than their non-core-shell counterparts, possibly due to a higher degree of self-crosslinking reactions occurring in the shell. The majority of the degradation products had molecule weights below 10 kDa, which suggests that they may be cleared through the kidneys. This study provides important insights into the design and characterisation of degradable nanogels for biomedical applications, highlighting the need for accurate characterisation techniques to measure the potential biological impact of nanogel degradation products.

Polymer-prodrug conjugates as candidates for degradable, long-acting implants, releasing the water-soluble nucleoside reverse-transcriptase inhibitor emtricitabine.

Circulating, soluble polymer-drug conjugates have been utilised for many years to aid the delivery of sensitive, poorly-soluble or cytotoxic drugs, prolong circulation times or minimise side effects. Long-acting therapeutics are increasing in their healthcare importance, with intramuscular and subcutaneous administration of liquid formulations being most common. Degradable implants also offer opportunities and the use of polymer-prodrug conjugates as implant materials has not been widely reported in this context. Here, the potential for polymer-prodrug conjugates of the water soluble nucleoside reverse transciption inhibitor emtricitabine (FTC) is studied. A novel diol monomer scaffold, allowing variation of prodrug substitution, has been used to form polyesters and polycarbonates by step-growth polymerisation. Materials have been screened for physical properties that enable implant formation, studied for drug release to provide mechanistic insights, and tunable prolonged release of FTC has been demonstrated over a period of at least two weeks under relevant physiological conditions.

Quantitation of tizoxanide in multiple matrices to support cell culture, animal and human research.

Currently nitazoxanide is being assessed as a candidate therapeutic for SARS-CoV-2. Nitazoxanide is rapidly broken down to its active metabolite tizoxanide upon administration. Unlike many other candidates being investigated, tizoxanide plasma concentrations achieve antiviral levels after administration of the approved dose, although higher doses are expected to be needed to maintain these concentrations across the dosing interval in the majority of patients. Here an LC-MS/MS assay is described that has been validated in accordance with Food and Drug Administration (FDA) guidelines. Fundamental parameters have been evaluated, and these included accuracy, precision and sensitivity. The assay was validated for human plasma, mouse plasma and Dulbecco's Modified Eagles Medium (DMEM) containing varying concentrations of Foetal Bovine Serum (FBS). Matrix effects are a well-documented source of concern for chromatographic analysis, with the potential to impact various stages of the analytical process, including suppression or enhancement of ionisation. Herein a validated LC-MS/MS analytical method is presented capable of quantifying tizoxanide in multiple matrices with minimal impact of matrix effects. The validated assay presented here was linear from 15.6 ng/mL to 1000 ng/mL. The presented assay here has applications in both pre-clinical and clinical research and may be used to facilitate further investigations into the application of nitazoxanide against SARS-CoV-2.

Dual-responsive degradable core-shell nanogels with tuneable aggregation behaviour.

We report the synthesis of core-shell nanogels by sequential addition of thermoresponsive monomers; N-isopropylacrylamide (NIPAM) and N-isopropylmethacrylamide (NIPMAM). The aggregation behaviour of aqueous dispersions of these particles in the presence of salt can be tuned by varying the monomer ratio. The inclusion of degradable cross-linker bis(acryloyl)cystamine (BAC) allows the nanogels to degrade in the presence of reducing agent, with nanogels composed of a copolymer of the two monomers not showing the same high levels of degradation as the comparable core-shell particles. These levels of degradation were also seen with physiologically relevant reducing agent concentration at pH 7. Therefore, it is hoped that the aggregation of these nanogels will have applications in nanomedicine and beyond.

Controlling the pH-response of branched copolymer nanoprecipitates synthesised by transfer-dominated branching radical telomerisation (TBRT) through telogen chemistry and spatial distribution of tertiary amine functionality.

Amine functionality offers the modification of polymer properties to enable stimuli-responsive behaviour, and this feature has been utilised in numerous studies of self-assembly and disassembly. The ability to place amines as pendant groups along linear polymer backbones within distinct blocks, at chain ends or as statistical mixtures with other functionalities, has allowed fine tuning of responses to pH. Here we study and compare the placement of amines within the backbones or as pendant groups within polyesters synthesised by the newly reported transfer-dominated branching radical telomerisation (TBRT). Branched polymers with backbone amines are clearly shown to undergo dissolution that is determined by pH and telogen selection; they undergo nanoprecipitation only when hydrophilic telogens are present within their structure and provide nanoprecipitates that are highly sensitive to the addition of acid. In contrast, TBRT polymers with pendant amines form uniform nanoparticles with remarkable stability to pH changes, under identical nanoprecipitation conditions. The behaviour differences shown here open new avenues of synthetic flexibility for pH-responsive polymer design using TBRT.

Using temperature to modify the reaction conditions and outcomes of polymers formed using transfer-dominated branching radical telomerisation (TBRT).

Transfer-dominated Branching Radical Telomerisation (TBRT) enables the production of branched polymers with step-growth backbones using radical telomerisation chemistry. By conducting identical TBRTs over a broad temperature range, the role of temperature in telomer formation and branching has been evaluated. Elevated temperature limits telomer length, thereby allowing a >10% reduction in the amount of telogen required to produce near identical high molecular weight branched polymers.

Linear and branched polymer prodrugs of the water-soluble nucleoside reverse-transcriptase inhibitor emtricitabine as structural materials for long-acting implants.

Long-acting drug delivery is a growing area of interest as it overcomes many challenges related to patient adherence to therapy and the pill burden associated with chronic illness. Injectable formulations are becoming more common and drug-releasing implants also provide several opportunities. Highly water soluble drug compounds are poor candidates for long-acting delivery. Here, the water-soluble nucleoside reverse transcriptase inhibitor emtricitabine (FTC) has been used as a novel A-B monomer in step-growth polymerisation with chloroformate functional Cn monomers, to produce new poly(carbamate/carbonate) structures with varying architecture. The polymer prodrugs were all solid at ambient temperature and have been shown to release FTC when subjected to mixed gender human plasma. Vacuum compression moulding has been used to form solid rod implants without polymer degradation; the rods show FTC release over long periods in the presence of microsomes, establishing the basis of a polymer prodrug strategy for FTC delivery.

Quantification of branching within high molecular weight polymers with polyester backbones formed by transfer-dominated branching radical telomerisation (TBRT).

New branched polymerisations offer previously inaccessible macromolecules and architectural understanding is important as it provides insight into the branching mechanism and enables the determination of structure-property relationships. Here we present a detailed inverse gated 13C NMR characterisation of materials derived from the very recently reported Transfer-dominated Branching Radical Telomerisation (TBRT) approach to quantify branching and provide an insight into cyclisation.

Evaluating the impact of systematic hydrophobic modification of model drugs on the control, stability and loading of lipid-based nanoparticles.

A significant number of new chemical entities in the drug development pipeline are poorly soluble, therefore routes that facilitate effective administration is of considerable value. Lipid nanoparticles have proved an attractive approach for drug delivery; however, challenges that include optimising drug loading and understanding the impact of drug physiochemical parameters on nanoparticle properties have limited progression. In this work, we investigate the effect of modifying the log P of a model drug on the formation and stability of lipid-based nanoparticles. A range of model drug analogues with systematically varying alkyl chains were produced using a lamivudine (nucleoside analog reverse transcriptase inhibitor) scaffold and processed into lipid nanoparticles by nanoprecipitation. Characterisation included evaluation of particle diameter, size distribution, drug loading and nanoformulation stability. A distinct correlation with the LaMer model of nucleation was observed and log P appeared to strongly influence rates of nucleation. Model drugs with high log P were uniform in particle size and distribution and offered enhanced stability. In addition, various model drug/lipid blends were produced and their physical properties were investigated using dynamic light scattering (DLS) and differential scanning calorimetry (DSC). Complex mixtures of lipids were shown to influence formulation crystallinity and strategies to form uniform and stable lipid based nanoparticles of high drug loading- through manipulation of log P are discussed.

Quantitation of tizoxanide in multiple matrices to support cell culture, animal and human research.

Currently nitazoxanide is being assessed as a candidate therapeutic for SARS-CoV-2. Unlike many other candidates being investigated, tizoxanide (the active metabolite of nitazoxanide) plasma concentrations achieve antiviral levels after administration of the approved dose, although higher doses are expected to be needed to maintain these concentrations across the dosing interval in the majority of patients. Here an LC-MS/MS assay is described that has been validated in accordance with Food and Drug Administration (FDA) guidelines. Fundamental parameters have been evaluated, and these included accuracy, precision and sensitivity. The assay was validated for human plasma, mouse plasma and Dulbeccos Modified Eagles Medium (DMEM) containing varying concentrations of Foetal Bovine Serum (FBS). Matrix effects are a well-documented source of concern for chromatographic analysis, with the potential to impact various stages of the analytical process, including suppression or enhancement of ionisation. Therefore, a robustly validated LC-MS/MS analytical method is presented capable of quantifying tizoxanide in multiple matrices with minimal impact of matrix effects. The validated assay presented here was linear from 15.6ng/mL to 1000ng/mL. Accuracy and precision ranged between 102.2% and 113.5%, 100.1% and 105.4%, respectively. The presented assay here has applications in both pre-clinical and clinical research and may be used to facilitate further investigations into the application of nitazoxanide against SARS-CoV-2.

Development of a highly sensitive bioanalytical assay for the quantification of favipiravir.

Favipiravir (FAV; T-705) has been approved for use as an anti-influenza therapeutic and has reports against a wide range of viruses (e.g., Ebola virus, rabies and norovirus). Most recently FAV has been reported to demonstrate activity against SARS-CoV-2. Repurposing opportunities have been intensively studied with only limited success to date. If successful, repurposing will allow interventions to become more rapidly available than development of new chemical entities. Pre-clinical and clinical investigations of FAV require robust, reproducible and sensitive bioanalytical assay. Here, a liquid chromatography tandem mass spectrometry assay is presented which was linear from 0.78-200 ng/mL Accuracy and precision ranged between 89% and 110%, 101% and 106%, respectively. The presented assay here has applications in both pre-clinical and clinical research and may be used to facilitate further investigations into the application of FAV against SARS-CoV-2.

Optimisation and validation of a sensitive bioanalytical method for niclosamide.

The SARS-CoV-2 pandemic has spread at an unprecedented rate, and repurposing opportunities have been intensively studied with only limited success to date. If successful, repurposing will allow interventions to become more rapidly available than development of new chemical entities. Niclosamide has been proposed as a candidate for repurposing for SARS-CoV-2 based upon the observation that it is amongst the most potent antiviral molecules evaluated in vitro . To investigate the pharmacokinetics of niclosamide, reliable, reproducible and sensitive bioanalytical assays are required. Here, a liquid chromatography tandem mass spectrometry assay is presented which was linear from 31.25-2000 ng/mL (high dynamic range) and 0.78-100 ng/mL (low dynamic range). Accuracy and precision ranged between 97.2% and 112.5%, 100.4% and 110.0%, respectively. The presented assay should have utility in preclinical evaluation of the exposure-response relationship and may be adapted for later evaluation of niclosamide in clinical trials.

Scalable nanoprecipitation of niclosamide and in vivo demonstration of long-acting delivery after intramuscular injection.

The control of COVID-19 across the world requires the formation of a range of interventions including vaccines to elicit an immune response and immunomodulatory or antiviral therapeutics. Here, we demonstrate the nanoparticle formulation of a highly insoluble drug compound, niclosamide, with known anti SARS-CoV-2 activity as a cheap and scalable long-acting injectable antiviral candidate.

Safety assessment of a new nanoemulsion-based drug-delivery system reveals unexpected drug-free anticoagulant activity.

Aim: A preclinical safety assessment of a novel nanoemulsion drug-delivery system, initially developed to improve the posology of efavirenz (EFV), was conducted with a specific focus on possible immunological and hematological complications. Materials & methods: Assessment of common acute toxicities, such as complement activation and cytokine secretion, was performed using validated assays known to have good correlation with in vivo end points. Results & conclusion: Compared with a standard aqueous solution of EFV, the EFV nanoemulsion showed no significant effect on immune cell function or phenotype. Prolongation of activated partial thromboplastin time was observed for EFV-loaded nanoemulsions (88% at 4 µg/ml) as well as unloaded nanoemulsions (52%) highlighting the potential for drug-free anticoagulant activity and warranting further investigation of the mechanism and utility of these materials.

Mucus-responsive functionalized emulsions: design, synthesis and study of novel branched polymers as functional emulsifiers.

Mucus lines the moist cavities throughout the body, acting as barrier by protecting the underlying cells against the external environment, but it also hinders the permeation of drugs and drug delivery systems. As the rate of diffusion is low, the development of a system which could increase retention time at the mucosal surface would prove beneficial. Here, we have designed a range of branched copolymers to act as functional mucus-responsive oil-in-water emulsifiers comprising the hydrophilic monomer oligo(ethylene glycol) methacrylate and a hydrophobic dodecyl initiator. The study aimed to investigate the importance of chain end functionality on successful emulsion formation, by systematically replacing a fraction of the hydrophobic chain ends with a secondary poly(ethylene glycol) based hydrophilic initiator in a mixed-initiation strategy; a decrease of up to 75 mole percent of hydrophobic chain ends within the branched polymer emulsifiers was shown to maintain comparative emulsion stability. These redundant chain ends allowed for functionality to be incorporated into the polymers via a xanthate based initiator containing a masked thiol group; thiol groups are known to have mucoadhesive character, due to their ability to form disulfide bonds with the cysteine rich areas of mucus. The mucoadhesive nature of emulsions stabilised by thiol-containing branched copolymers was compared to non-functional emulsions in the presence of a biosimilar mucosal substrate and enhanced adherence to the mucosal surface was observed. Importantly, droplet rupture and mucus triggered release of dye-containing oil was seen from previously highly-stable thiol-functional emulsions; this observation was not mirrored by non-functional emulsions where droplet integrity was maintained even in the presence of mucus.