A comparison of major trauma admissions to Christchurch Hospital during and after COVID-19 lockdown in New Zealand.

AIMS: To describe any change in the volume and mechanisms of injury of major trauma admissions during and after COVID-19 lockdown, and in doing so, to provide information for resource planning and identification of priority areas for injury prevention initiatives. METHODS: A retrospective, descriptive study conducted on Canterbury District Health Board trauma registry data. The study population consisted of all major trauma patients of all age groups admitted to Christchurch Hospital over three 33-day periods: before, during and after COVID-19 lockdown in New Zealand. Broadly speaking, major trauma is defined as having an injury severity score 13 or death following injury. RESULTS: There was a 42% reduction in the volume of major trauma admissions during lockdown. Falls were the most common injury during lockdown, and transport-related injuries after lockdown. Alcohol intoxication was associated with 19 to 33% of all injuries across the study periods. CONCLUSION: Major trauma inevitably occurred during lockdown, although at considerably lower volumes. After lockdown, once restrictions were eased, major trauma admissions reverted to pre-lockdown patterns. Injury prevention strategies can reduce avoidable pressures on hospitals at a time of pandemic. In New Zealand, focus should be placed on reducing alcohol- and transport-related injuries and increasing community awareness on falls prevention.

Polymer therapeutics at a crossroads? Finding the path for improved translation in the twenty-first century.

Despite the relatively small early investment, first generation 'polymer therapeutics' have been remarkably successful with more than 25 products licenced for human use as polymeric drugs, sequestrants, conjugates, and as an imaging agent. Many exhibit both clinical and commercial success with new concepts already in clinical trials. Nevertheless after four decades of evolution, this field is arriving at an important crossroads. Over the last decade, the landscape has changed rapidly. There are an increasing number of failed clinical trials, the number of 'copy' and 'generic' products is growing (danger of ignoring the biological rationale for design and suppression of innovation), potential drawbacks of PEG are becoming more evident, and the 'nanomedicine' boom has brought danger of loss of scientific focus/hype. Grasping opportunities provided by advances in understanding of the patho-physiology and molecular basis of diseases, new polymer/conjugate synthetic and analytical methods, as well as the large database of clinical experience will surely ensure a successful future for innovative polymer therapeutics. Progress will, however, be in jeopardy if polymer safety is overlooked in respect of the specific route of administration/clinical use, poorly characterised materials/formulations are used to define biological or early clinical properties, and if clinical trial protocols fail to select patients most likely to benefit from these macromolecular therapeutics. Opportunities to improve clinical trial design for polymer-anticancer drug conjugates are discussed. This short personal perspective summarises some of the important challenges facing polymer therapeutics in R&D today, and future opportunities to improve successful translation.

Polymer therapeutics: Top 10 selling pharmaceuticals - what next?

At the time of the first issue of the Journal of Controlled Release (JCR), polymeric drugs, polymer-drug and protein conjugates and block copolymer micelles carrying bound drugs, i.e. polymer therapeutics, were still regarded as scientific curiosities with little or no prospect of generating practical to use medicines. How this perception has changed. Many major Pharma now have R&D programmes in this area and in 2013 two polymer therapeutics, Copaxone and Neulasta, are featured in the Top 10 US pharmaceutical sales list. Although there are a growing number of marketed products (e.g. PEGylated proteins, a PEG-aptamer and oral polymeric sequestrants), and the first follow-on (generic products) are emerging, the first polymer-drug conjugates and block copolymer micelle products (as covalent conjugates) have yet to enter routine clinical use. Industrial familiarity and recent advances in the underpinning scientific disciplines will no doubt accelerate the transfer of polymer therapeutics into clinically useful medicines and imaging agents. This short personal perspective reflects on the current status of polymer therapeutics and the future opportunities to improve their successful translation. It adds to recent and historical reviews that comprehensively document the evolution of the field since JCR was born.

Validation of tumour models for use in anticancer nanomedicine evaluation: the EPR effect and cathepsin B-mediated drug release rate.

PURPOSE: Intravenously (i.v.) administered nanomedicines have the potential for tumour targeting due to the enhanced permeability and retention (EPR) effect, but in vivo tumour models are rarely calibrated with respect to functional vascular permeability and/or mechanisms controlling intratumoural drug release. Here the effect of tumour type and tumour size on EPR-mediated tumour localisation and cathepsin B-mediated drug release was studied. METHODS: Evans Blue (10 mg/kg) and an N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer­doxorubicin (Dox) conjugate (FCE28068) (5 mg/kg Dox-equiv) were used as probes and tumour levels (and Dox release) measured at 1 h after i.v. administration in a panel of murine and human xenograft tumours. RESULTS: Evans Blue and FCE28068 displayed similar tumour levels in the range of 2­18 % dose/g at 1 h for B16F10 and L1210. Approximately half of the tumour models evaluated exhibited tumour size-dependent accumulation of FCE28068; smaller tumours had the highest accumulation. Administration of free Dox (5 mg/kg) produced tumour levels of \2.5 % dose/g independent of tumour size. Whereas the degree of EPR-mediated targeting showed *12-fold difference across the tumour models evaluated, Dox release from FCE28068 at 1 h displayed *200-fold variation. CONCLUSIONS: Marked heterogeneity was seen in terms of EPR effect and Dox release rate, underlining the need to carefully calibrate tumour models used to benchmark nanomedicines against known relevant standard agents and for optimal development of strategies for late pre-clinical and clinical development.

Next-generation nanomedicines and nanosimilars: EU regulators' initiatives relating to the development and evaluation of nanomedicines.

Over the last three decades many first-generation nanomedicines have successfully entered routine clinical use and it is now important for medicines regulatory agencies to consider the mechanisms needed to ensure safe introduction of 'follow-on' nanomedicine products, 'nanosimilars'. Moreover, drug regulators need to ensure that 'next'-generation nanomedicines enter clinical development and consequently the market in a safe and timely way for the benefit of public health. Here we review recent European Medicines Agency activities that relate to the effective development and evaluation of nanomedicine products while keeping patient and consumer safety at the forefront.

Polymer therapeutics-prospects for 21st century: the end of the beginning.

The term "polymer therapeutics" was coined to describe polymeric drugs, polymer conjugates of proteins, drugs and aptamers, together with those block copolymer micelles and multicomponent non-viral vectors which contain covalent linkages. These often complex, multicomponent constructs are actually "drugs" and "macromolecular prodrugs" in contrast to drug delivery systems that simply entrap (non-covalently) therapeutic agents. They have also been described as nanomedicines. First polymer-protein conjugates entered routine clinical use in 1990 and a growing number of polymeric drugs/sequestrants and PEGylated proteins/aptamers have since come into the market. Valuable lessons have been learnt over >3 decades of clinical development, especially in relation to critical product attributes governing safety and efficacy, the validated methods needed for product characterisation. Not least there has been improved understanding of polymer therapeutic-specific biomarkers that will in future enable improved selection of patients for therapy. Advances in synthetic polymer chemistry (including control of 3D architecture), the move towards greater use of biodegradable polymers, polymers delivering combination therapy, increased understanding of polymer therapeutic critical product attributes to guide pharmaceutical development, and advances in understanding of endocytosis and intracellular trafficking pathways in health and disease are opening new opportunities for design and clinical use of polymer-based therapeutics in the decades to come.

Endocytosis and intracellular trafficking as gateways for nanomedicine delivery: opportunities and challenges.

More than 40 nanomedicines are already in routine clinical use with a growing number following in preclinical and clinical development. The therapeutic objectives are often enhanced disease-specific targeting (with simultaneously reduced access to sites of toxicity) and, especially in the case of macromolecular biotech drugs, improving access to intracellular pharmacological target receptors. Successful navigation of the endocytic pathways is usually a prerequisite to achieve these goals. Thus a comprehensive understanding of endocytosis and intracellular trafficking pathways in both the target and bystander normal cell type(s) is essential to enable optimal nanomedicine design. It is becoming evident that endocytic pathways can become disregulated in disease and this, together with the potential changes induced during exposure to the nanocarrier itself, has the potential to significantly impact nanomedicine performance in terms of safety and efficacy. Here we overview the endomembrane trafficking pathways, discuss the methods used to determine and quantitate the intracellular fate of nanomedicines, and review the current status of lysosomotropic and endosomotropic delivery. Based on the lessons learned during more than 3 decades of clinical development, the need to use endocytosis-relevant clinical biomarkers to better select those patients most likely to benefit from nanomedicine therapy is also discussed.

Nanomedicine(s) under the microscope.

Depending on the context, nanotechnologies developed as nanomedicines (nanosized therapeutics and imaging agents) are presented as either a remarkable technological revolution already capable of delivering new diagnostics, treatments for unmanageable diseases, and opportunities for tissue repair or highly dangerous nanoparticles, nanorobots, or nanoelectronic devices that will wreak havoc in the body. The truth lies firmly between these two extremes. Rational design of "nanomedicines" began almost half a century ago, and >40 products have completed the complex journey from lab to routine clinical use. Here we critically review both nanomedicines in clinical use and emerging nanosized drugs, drug delivery systems, imaging agents, and theranostics with unique properties that promise much for the future. Key factors relevant to the design of practical nanomedicines and the regulatory mechanisms designed to ensure safe and timely realization of healthcare benefits are discussed.

Polymer therapeutics as nanomedicines: new perspectives.

A growing number of polymer therapeutics have entered routine clinical use as nano-sized medicines. Early products were developed as anticancer agents, but treatments for a range of diseases and different routes of administration have followed--recently the PEGylated-anti-TNF Fab Cimzia® for rheumatoid arthritis and the PEG-aptamer Macugen® for age related macular degeneration. New polymer therapeutic concepts continue to emerge with a growing number of conjugates entering clinical development, for example PEGylated-aptamers and a polymer-based siRNA delivery system. 'Hot' topics of the past 2 years include; emerging issues relating to polymer safety, the increasing use of biodegradable polymers, design of technologies for combination therapy, potential biomarkers for patient individualisation of treatment and Regulatory challenges for 'follow-on/generic' polymer therapeutics.

Cell uptake and trafficking behavior of non-covalent, coiled-coil based polymer-drug conjugates.

This paper reports on the cell uptake and trafficking properties of a series of non-covalent polymer-drug conjugates. These nanomedicines are composed of a poly(N-(2-hydroxypropyl)methacrylamide) backbone functionalized with multiple copies of a drug. The drug moieties are attached to the polymer via a non-covalent, so called coiled coil motif, which is formed by heterodimerization of two complementary peptide strands, one of which is attached to the polymer carrier and the other to the drug. Cytotoxicity and FACS experiments, which were carried out with model anticancer drug or fluorophore conjugates, provided insight into the cell uptake and trafficking behavior of these conjugates.

The effect of dextrin-rhEGF on the healing of full-thickness, excisional wounds in the (db/db) diabetic mouse.

Chronic wounds, such as ulceration of the lower limb, represent a significant clinical challenge in today's ageing society. With the aim of identifying improved therapeutics, we have previously described a bioresponsive, dextrin-recombinant human epidermal growth factor conjugate (dextrin-rhEGF), that (i) protects rhEGF against proteolytic degradation by human chronic wound fluid; and (ii) mediates rhEGF release by α-amylase, capable of stimulating increased proliferation/migration in normal dermal and chronic wound fibroblasts; and keratinocytes, in vitro. The aim of this study was to extend these findings, by investigating the effects of dextrin-rhEGF on wound healing in the (db/db) diabetic mouse, a widely used in vivo model of delayed wound healing. Standardised, full-thickness excisional wounds, created in the dorsal flank skin, were treated topically with succinoylated dextrin (50 µg/mL), rhEGF (10 µg/mL) or dextrin-rhEGF (1 or 10 µg/mL). Treatments were applied immediately after injury and subsequently on post-wounding, days 3 and 8. Wound healing was assessed macroscopically, in terms of initiation of neo-dermal tissue deposition and wound closure (including wound contraction and re-epithelialisation), over a 16 day period. Wound healing was assessed histologically, in terms of granulation tissue formation/maturity; cranio-caudal wound contraction and wound angiogenesis (CD31 immuno-staining), using tissues harvested at day 16. Blood samples were also analysed for α-amylase and rhEGF concentrations. In this established impaired wound healing model, the topically-applied dextrin-rhEGF significantly accelerated wound closure and neo-dermal tissue formation at the macroscopic level; and significantly increased granulation tissue deposition and angiogenesis at the histological level (p<0.05), relative to untreated, succinoylated dextrin and rhEGF alone controls. Overall, these findings support the further development of bioresponsive polymer conjugates, for tissue repair.

Impact of polymer tacticity on the physico-chemical behaviour of polymers proposed as therapeutics.

Although water-soluble polymers are finding increasing use as polymer therapeutics, there has been little consideration of the effect of polymer stereochemistry on their physico-chemical and biological properties. The aim of this study was to investigate these properties using polymethacrylic acids (PMAs) of similar molecular weights with a difference in syndiotacticity of about 20% of rr triad content. Experiments to characterize the solution behaviour were conducted at pHs encountered during the transport, endocytic uptake and intracellular trafficking (7.4-3.0). These showed that with increasing rr triads, the polymer become less hydrophobic, a stronger acid, displayed a locally ordered solution conformation at pH 5.5, and interacted more strongly with dodecyl trimethylammonium bromide (DTAB) micelles. Preliminary cytotoxicity experiments using B16F10 melanoma cells showed lower toxicity in the concentration range of 1-100 µg/mL with increased rr triads. These observations indicate that the higher content of rr triads could drive a chain organization that minimize the influence of negative charges and so underline the importance of further, systematic studies to investigate the effect of tacticity on the behaviour of polymers in respect of their pharmacokinetics, toxicity and efficacy.

Polymer coiled-coil conjugates: potential for development as a new class of therapeutic "molecular switch".

Polymer therapeutics, including polymeric drugs and polymer-protein conjugates, are clinically established as first-generation nanomedicines. Knowing that the coiled-coil peptide motif is fundamentally important in the regulation of many cellular and pathological processes, the aim of these studies was to examine the feasibility of designing polymer conjugates containing the coiled-coil motif as a putative therapeutic "molecular switch". To establish proof of concept, we prepared a mPEG-FosW(C) conjugate by reacting mPEG-maleimide (M(w) 5522 g mol(-1), M(w)/M(n) 1.1) with a FosW peptide synthesized to contain a terminal cysteine residue (FosW(C)). Its ability to form a stable coil-coil heterodimer with the target c-Jun sequence of the oncogenic AP-1 transcription factor was investigated using 2D (15)N-HSQC NMR together with a recombinantly prepared (15)N-labeled c-Jun peptide ([(15)N]r-c-Jun). Observation that heterodimerization was achieved and that the polymer did not sterically disadvantage hybridization suggests an important future for this new family of polymer therapeutics.

Interaction of an endosomolytic polyamidoamine ISA23 with vesicles mimicking intracellular membranes: A SANS/EPR study.

The mechanism of ISA23 · HCl interaction with model membrane vesicles (80-100 nm in diameter) was investigated using EPR in conjunction with SANS. For EPR, 16-DSE was dissolved in the vesicle membrane to measure its dynamics and polarity, whereas a spin-labeled (Tempo)-ISA 23 analogue was used to give a measure of the polymer flexibility. When ISA23 was added to the external vesicle surface, no interaction was found. This observation conflicts with the reported ability to lyse RBC, but is in agreement with recent studies that showed no effect on membrane permeability when a PAA was added to an incubation medium containing isolated lysosomal vesicles. The vesicle-mimetic models used here provide a new and useful tool for studying endosomolytic polymer/membrane interactions.

Hybrid polymer therapeutics incorporating bioresponsive, coiled coil peptide linkers.

This article reports the design, synthesis and results of first in vitro model studies of a conceptually novel class of polymer therapeutics in which the cargo is attached to a polymer backbone via a noncovalent, biologically inspired coiled coil linker, which is formed by heterodimerization of two complementary peptide sequences that are linked to the polymer carrier and the cargo, respectively. In contrast with the polymer-drug conjugates prepared so far, in which the drug is typically attached via an enzymatically or hydrolytically cleavable linker, the noncovalent polymer therapeutics proposed in this article offer several potential advantages, including facile access to combination therapeutics and rapid production of compound libraries to screen for structure-activity relationships. Furthermore, the coiled coil based peptide linkers may not only be useful to bind and release guests but may also play an active role in enhancing and directing intracellular transport and trafficking, which may make these constructs of particular interest for the cytosolic delivery of biomolecular therapeutics.

Studies on the mechanism of action of dextrin-phospholipase A2 and its suitability for use in combination therapy.

The bioresponsive conjugate dextrin-phospholipase A2 (PLA2) is a novel anticancer polymer therapeutic. Dextrin conjugation decreases PLA2 bioactivity, but this can be restored following triggered degradation by alpha-amylase. The conjugate displays reduced hemolytic activity but retains, or shows enhanced, cytotoxicity in vitro that partially correlates with epidermal growth factor receptor (EGFR) expression. Here, we investigate further the mechanism of action of dextrin-PLA2 with the aim of judging its potential for combination with tyrosine kinase inhibitors (TKI) and/or chemotherapy and selecting the first models for in vivo evaluation. The endocytic fate of Oregon Green (OG)-labeled probes was assessed in MCF-7 cells. Whereas PLA2-OG showed greatest membrane binding, the dextrin-PLA2-OG conjugate displayed higher internalization. Moreover, cells incubated with PLA(2)-OG and dextrin-PLA2-OG showed an altered pattern of intracellular vesicle distribution compared to dextrin-OG. When cell lines known to express different levels of EGFR were used to assess cytotoxicity, free PLA2 activity was enhanced by addition of EGF whereas the conjugate was less cytotoxic, perhaps due to differences in their PK/PD profile. Co-incubation of cells with the TKI inhibitor, gefitinib, led to reduced cytotoxicity of both PLA2 and dextrin-PLA2 suggesting a TK-mediated PLA2 mechanism of action. However, the enhanced cytotoxicity seen in the presence of doxorubicin suggested potential for development of a dextrin-PLA2/doxorubicin combination therapy.

Bioresponsive dextrin-rhEGF conjugates: in vitro evaluation in models relevant to its proposed use as a treatment for chronic wounds.

We recently developed a bioresponsive dextrin-recombinant human epidermal growth factor (rhEGF) conjugate as a polymer therapeutic with potential for use in the promotion of tissue repair. The aim of these studies was to use patient-derived wound fluid and fibroblasts to evaluate its potential for further development as a treatment for chronic wounds, such as venous leg ulceration, a growing clinical challenge in the aging population. First, the levels of EGF (ELISA assay), alpha-amylase and elastase (enzyme assays) were measured in patient-derived acute and chronic wound fluid. EGF was detected in acute, but not in chronic wound fluid. alpha-Amylase concentrations were higher in acute (188 IU/L), compared to chronic wound fluid (52 IU/L), but both were in the range of human serum levels. Although elastase was present in chronic wound fluid (2.1 +/- 1.2 RFU/min), none was detected in acute wound fluid. Dextrin-rhEGF incubation in chronic wound fluid led to endogenous alpha-amylase-mediated release of rhEGF (ELISA) that was maximal at 48 h. When the migration of HaCaT keratinocytes and of human fibroblasts (isolated from patient-matched, normal skin and chronic dermal wounds) was studied in vitro using the scratch wound assay, enhanced cell migration was observed in response to both free rhEGF and alpha-amylase-activated dextrin-rhEGF conjugate compared to controls. In addition, fibroblasts displayed increased proliferation (normal dermal fibroblasts approximately 160%; chronic wound fibroblasts approximately 140%) following incubation (72 h) with dextrin-rhEGF that had been exposed to physiological levels of alpha-amylase (93 IU/L). These results suggest further preclinical in vivo evaluation of dextrin-rhEGF is warranted to determine whether conjugate pharmacokinetics and rhEGF liberation into such a complex and aggressive environment can still lead to bioactivity.

Do HPMA copolymer conjugates have a future as clinically useful nanomedicines? A critical overview of current status and future opportunities.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer conjugates containing doxorubicin designed in the late 1970s/early 1980s as anticancer polymer therapeutics were the first synthetic polymer-based anticancer conjugates to enter clinical trial beginning in 1994. Early clinical results were promising, confirming activity in chemotherapy refractory patients and the safety of HPMA copolymers as a new polymer platform in this setting. Subsequent Phase I/II trials have investigated conjugates containing paclitaxel (PNU 166945), camptothecin (PNU 166148) (both failed in clinical trials underlining the importance of rational design), and most recently HPMA-copolymer platinates (AP5280 and then AP5346-ProLindac(TM)) entered Phase II clinical development. There are a growing array of second generation HPMA copolymer-based systems involving combination therapy, incorporating putative targeting ligands, having an ever more complex architecture, and both drug and protein conjugates are being proposed as novel treatments for diseases other than cancer. Despite their promise, and the success of polymeric drugs and polymer-protein conjugates, no polymer-drug conjugate (HPMA copolymer-based or otherwise) has yet entered routine clinical use. It is timely to reflect on the progress made over the last 30 years, the relative merits of HPMA copolymers as a platform compared to other polymeric carriers, and comment on their future potential as polymer-based nanomedicines into the 21st century in comparison with the many alternative strategies now emerging for creation of nanopharmaceuticals.

Intracellular fate of bioresponsive poly(amidoamine)s in vitro and in vivo.

Linear poly(amidoamine)s (PAAs) have been designed to exhibit minimal non-specific toxicity, display pH-dependent membrane lysis and deliver genes and toxins in vitro. The aim of this study was to measure PAA cellular uptake using ISA1-OG (and as a reference ISA23-OG) in B16F10 cells in vitro and, by subcellular fractionation, quantitate intracellular trafficking of (125)I-labelled ISA1-tyr in liver cells after intravenous (i.v.) administration to rats. The effect of time after administration (0.5-3h) and ISA1 dose (0.04-100mg/kg) on trafficking, and vesicle permeabilisation (N-acetyl-b-D-glucosaminidase (NAG) release from an isolated vesicular fraction) were also studied. ISA1-OG displayed approximately 60-fold greater B16F10 cell uptake than ISA23-OG. Passage of ISA1 along the liver cell endocytic pathway caused a transient decrease in vesicle buoyant density (also visible by TEM). Increasing ISA1 dose from 10mg/kg to 100mg/kg increased both radioactivity and NAG levels in the cytosolic fraction (5-10 fold) at 1h. Moreover, internalised ISA1 provoked NAG release from an isolated vesicular fraction in a dose-dependent manner. These results provide direct evidence, for the first time, of PAA permeabilisation of endocytic vesicular membranes in vivo, and they have important implications for potential efficacy/toxicity of such polymeric vectors.