Expression of Concern to: Diet restriction inhibits apoptosis and HMGB1 oxidation and promotes inflammatory cell recruitment during acetaminophen hepatoxicity.

The Editors-in-Chief would like to alert readers that this article [1] is part of an investigation being conducted by the journal following the conclusions of an institutional enquiry at the University of Liverpool with respect to the quantitative mass spectrometry-generated results regarding acetylated and redox-modified HMGB1.

A synthetic gene increases TGFß3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule.

Human transforming growth factor-ß3 (TGFß3) is a new therapeutic protein used to reduce scarring during wound healing. The active molecule is a nonglycosylated, homodimer comprised of 13-kDa polypeptide chains linked by disulphide bonds. Expression of recombinant human TGFß3 in chloroplasts and its subsequent purification would provide a sustainable source of TGFß3 free of animal pathogens. A synthetic sequence (33% GC) containing frequent chloroplast codons raised accumulation of the 13-kDa TGFß3 polypeptide by 75-fold compared to the native coding region (56% GC) when expressed in tobacco chloroplasts. The 13-kDa TGFß3 monomer band was more intense than the RuBisCO 15-kDa small subunit on Coomassie blue-stained SDS-PAGE gels. TGFß3 accumulated in insoluble aggregates and was stable in leaves of different ages but was not detected in seeds. TGFß3 represented 12% of leaf protein and appeared as monomer, dimer and trimer bands on Western blots of SDS-PAGE gels. High yield and insolubility facilitated initial purification and refolding of the 13-kDa polypeptide into the TGFß3 homodimer recognized by a conformation-dependent monoclonal antibody. The TGFß3 homodimer and trace amounts of monomer were the only bands visible on silver-stained gels following purification by hydrophobic interaction chromatography and cation exchange chromatography. N-terminal sequencing and electronspray ionization mass spectrometry showed the removal of the initiator methionine and physical equivalence of the chloroplast-produced homodimer to standard TGFß3. Functional equivalence was demonstrated by near-identical dose-response curves showing the inhibition of mink lung epithelial cell proliferation. We conclude that chloroplasts are an attractive production platform for synthesizing recombinant human TGFß3.

Discovery and development of avotermin (recombinant human transforming growth factor beta 3): a new class of prophylactic therapeutic for the improvement of scarring.

Scarring in the skin following surgery or trauma may be associated with adverse aesthetic, functional, growth and psychological effects, such that both physicians and patients regard it as important to minimize the appearance of scars. The prophylactic improvement of cutaneous scar appearance represents a significant opportunity to improve the well-being of patients. Human recombinant transforming growth factor beta 3 (avotermin) is the first in a new class of therapeutic agents to address this medical need. Herein we describe scar-free healing in early embryonic development, including the identification of the cellular and molecular mechanisms underpinning the scarring process. This understanding has led to the discovery of novel therapeutics such as transforming growth factor beta 3, which can be administered to improve scar appearance in human subjects through pharmacological action. We discuss the pioneering development of transforming growth factor beta 3 in this new therapeutic area showing how it has been possible to translate preclinical concepts into clinical application, namely the improvement of scar appearance following surgery.

Diet restriction inhibits apoptosis and HMGB1 oxidation and promotes inflammatory cell recruitment during acetaminophen hepatotoxicity.

Acetaminophen (APAP) overdose is a major cause of acute liver failure and serves as a paradigm to elucidate mechanisms, predisposing factors and therapeutic interventions. The roles of apoptosis and inflammation during APAP hepatotoxicity remain controversial. We investigated whether fasting of mice for 24 h can inhibit APAP-induced caspase activation and apoptosis through the depletion of basal ATP. We also investigated in fasted mice the critical role played by inhibition of caspase-dependent cysteine 106 oxidation within high mobility group box-1 protein (HMGB1) released by ATP depletion in dying cells as a mechanism of immune activation. In fed mice treated with APAP, necrosis was the dominant form of hepatocyte death. However, apoptosis was also observed, indicated by K18 cleavage, DNA laddering and procaspase-3 processing. In fasted mice treated with APAP, only necrosis was observed. Inflammatory cell recruitment as a consequence of hepatocyte death was observed only in fasted mice treated with APAP or fed mice cotreated with a caspase inhibitor. Hepatic inflammation was also associated with loss in detection of serum oxidized-HMGB1. A significant role of HMGB1 in the induction of inflammation was confirmed with an HMGB1-neutralizing antibody. The differential response between fasted and fed mice was a consequence of a significant reduction in basal hepatic ATP, which prevented caspase processing, rather than glutathione depletion or altered APAP metabolism. Thus, the inhibition of caspase-driven apoptosis and HMGB1 oxidation by ATP depletion from fasting promotes an inflammatory response during drug-induced hepatotoxicity/liver pathology.

Prophylactic administration of avotermin for improvement of skin scarring: three double-blind, placebo-controlled, phase I/II studies.

BACKGROUND: Research into mechanisms of skin scarring identified transforming growth factor beta3 (TGFbeta3) as a potential antiscarring therapy. We assessed scar improvement with avotermin (recombinant, active, human TGFbeta3). METHODS: In three double-blind, placebo-controlled studies, intradermal avotermin (concentrations ranging from 0.25 to 500 ng/100 microL per linear cm wound margin) was administered to both margins of 1 cm, full-thickness skin incisions, before wounding and 24 h later, in healthy men and women. Treatments (avotermin and placebo or standard wound care) were randomly allocated to wound sites by a computer generated randomisation scheme, and within-participant controls compared avotermin versus placebo or standard wound care alone. Primary endpoints were visual assessment of scar formation at 6 months and 12 months after wounding in two studies, and from week 6 to month 7 after wounding in the third. Investigators, participants, and scar assessors were blinded to treatment. Efficacy analyses were intention to treat. These studies are registered with ClinicalTrials.gov, numbers NCT00847925, NCT00847795, and NCT00629811. RESULTS: In two studies, avotermin 50 ng/100 microL per linear cm significantly improved median score on a 100 mm visual analogue scale (VAS) by 5 mm (range -2 to 14; p=0.001) at month 6 and 8 mm (-29 to 18; p=0.0230) at month 12. In the third, avotermin significantly improved total scar scores at all concentrations versus placebo (mean improvement: from 14.84 mm [95 % CI 5.5-24.2] at 5 ng/100 microL per linear cm to 64.25 mm [49.4-79.1] at 500 ng/100 microL per linear cm). Nine [60%] scars treated with avotermin 50 ng/100 microL per linear cm showed 25% or less abnormal orientation of collagen fibres in the reticular dermis versus five [33%] placebo scars. After only 6 weeks from wounding, avotermin 500 ng/100 microL per linear cm improved VAS score by 16.12 mm (95% CI 10.61-21.63). Adverse events at wound sites were similar for avotermin and controls. Erythema and oedema were more frequent with avotermin than with placebo, but were transient and deemed to be consistent with normal wound healing. INTERPRETATION: Avotermin has potential to provide an accelerated and permanent improvement in scarring.

The potential of cytokines as safety biomarkers for drug-induced liver injury.

Drug-induced liver injury (DILI) is an event that has a detrimental impact on drug development and patient safety; therefore the identification of novel biomarkers that are both sensitive and specific to the liver would have great benefit. Inflammation is known to be associated with human cases of DILI, and given the role of cytokines in modulating the inflammatory response, changes in cytokine expression patterns certainly show promise as potential biomarkers of DILI. Cytokines are interesting candidates for novel biomarkers as they are relatively accessible (by blood sampling) and accurately quantifiable. In particular, recent interest has developed in mechanism-specific, rather than tissue-specific, biomarkers. However, without fully understanding the role of inflammation in DILI and the role of cytokines in modulating the inflammatory response, cytokines may be limited in their use, being either diagnostic of the type of injury that has occurred and/or prognostic of outcome (recovery from DILI, cirrhosis, acute liver failure). Intracellular components released by damaged hepatocytes, although inaccessible and currently difficult to quantify, may be better biomarkers for the prognosis of severity of injury. In both cases there is a pressing need for the development and validation of assays sensitive enough and with a sufficient dynamic range to detect changes upon drug treatment. Although promising candidates are appearing in the literature, much remains to be done to understand the role of inflammation in DILI and the role that a given cytokine has in the inflammatory cascade associated with DILI before cytokines are viewed as biomarkers for DILI.

The Innovative Medicines Initiative -10 Years of Public-Private Collaboration.

The Innovative Medicines Initiative (IMI) is a public-private partnership between the European Union and the European pharmaceutical industry. Born of the necessity to foster collaboration between different stakeholders in order to address growing challenges in bringing new medicines to market and the rapidly evolving healthcare landscape, IMI has successfully delivered the radical collaboration needed to address these challenges. In this article we reflect on some of the major achievements of the programme by highlighting a few of the key projects funded and the progress they have made, as well as some of the lessons learnt in delivering such an ambitious partnership. Those that drove the foundation of IMI recognized that to address these challenges required not just ambitious scientific approaches, but also an awareness of societal needs. Therefore, actors from beyond the traditional pharmaceutical research communities would be needed. One of the key successes of IMI has been to foster radical collaboration between diverse public and private partners of all types, including large pharmaceutical companies, SMEs, regulators, patient organizations and public research institutions. It has achieved this by being a neutral platform where all partners are bound by the same rights and responsibilities. Since it began there has been an evolution in the understanding of what is considered "pre-competitive," resulting in IMI projects now addressing all of the steps within the pharmaceutical development value chain. With this expansion in the types of projects supported by IMI, different actors from beyond the traditional pharmaceutical research family have been attracted to participate, enriching further the collaboration at the heart of the programme. Finally, such a complex programme brings with it challenges, and we reflect on some of the important learnings that should be applied to future collaborative models to ensure that they are as successful as possible and deliver the expected impact.

'Big data' in pharmaceutical science: challenges and opportunities.

Future Medicinal Chemistry invited a selection of experts to express their views on the current impact of big data in drug discovery and design, as well as speculate on future developments in the field. The topics discussed include the challenges of implementing big data technologies, maintaining the quality and privacy of data sets, and how the industry will need to adapt to welcome the big data era. Their enlightening responses provide a snapshot of the many and varied contributions being made by big data to the advancement of pharmaceutical science.

Assessing the safety of stem cell therapeutics.

Unprecedented developments in stem cell research herald a new era of hope and expectation for novel therapies. However, they also present a major challenge for regulators since safety assessment criteria, designed for conventional agents, are largely inappropriate for cell-based therapies. This article aims to set out the safety issues pertaining to novel stem cell-derived treatments, to identify knowledge gaps that require further research, and to suggest a roadmap for developing safety assessment criteria. It is essential that regulators, pharmaceutical providers, and safety scientists work together to frame new safety guidelines, based on "acceptable risk," so that patients are adequately protected but the safety "bar" is not set so high that exciting new treatments are lost.

Managing the challenge of chemically reactive metabolites in drug development.

The normal metabolism of drugs can generate metabolites that have intrinsic chemical reactivity towards cellular molecules, and therefore have the potential to alter biological function and initiate serious adverse drug reactions. Here, we present an assessment of the current approaches used for the evaluation of chemically reactive metabolites. We also describe how these approaches are being used within the pharmaceutical industry to assess and minimize the potential of drug candidates to cause toxicity. At early stages of drug discovery, iteration between medicinal chemistry and drug metabolism can eliminate perceived reactive metabolite-mediated chemical liabilities without compromising pharmacological activity or the need for extensive safety evaluation beyond standard practices. In the future, reactive metabolite evaluation may also be useful during clinical development for improving clinical risk assessment and risk management. Currently, there remains a huge gap in our understanding of the basic mechanisms that underlie chemical stress-mediated adverse reactions in humans. This review summarizes our views on this complex topic, and includes insights into practices considered by the pharmaceutical industry.

Effects of avotermin (transforming growth factor ß3) in a clinically relevant pig model of long, full-thickness incisional wounds.

BACKGROUND: The pig is an accepted species for evaluating the safety of molecules in dermal wound healing indications; however, the sizes of wounds assessed have not always been comparable to large incisions encountered clinically. OBJECTIVE: To develop a clinically relevant model of incisional wounding in the Göttingen minipig for assessing the safety and tolerance of compounds in development to improve scarring. METHODS: Intradermal avotermin (recombinant transforming growth factor ß3 [TGFß3]) up to 6,000 ng/100µL was administered twice to 20 cm full-thickness incisions. RESULTS: Incisions were well tolerated in the minipig. Avotermin treatment was not associated with adverse changes in a range of clinical parameters, including wound healing and strength. Plasma TGFß3 levels were transient with ≈0.1% bioavailability. CONCLUSION: A clinically relevant model of long, full-thickness, sutured surgical incisions in the minipig is achievable. Avotermin is well tolerated in this model and does not adversely affect normal wound healing at levels that significantly exceed those doses to be used clinically in humans.

Prevention and reduction of scarring in the skin by Transforming Growth Factor beta 3 (TGFbeta3): from laboratory discovery to clinical pharmaceutical.

Scarring in the skin after trauma, surgery, burn or sports injury is a major medical problem, often resulting in adverse aesthetics, loss of function, restriction of tissue movement and/or growth and adverse psychological effects. Current treatments are empirical and unpredictable, and there are no prescription drugs for the prevention or treatment of dermal scarring. We have investigated the cellular and molecular differences between scar-free healing in embryonic wounds and scar-forming healing in adult wounds. We have identified Transforming Growth Factor beta 3 (TGFbeta3) as a key regulator of the scar-free phenotype in embryonic healing. Exogenous addition of TGFbeta3 to cutaneous wounds in pre-clinical (adult) in vivo models reduces early extracellular matrix deposition and these molecules are deposited with a markedly improved architecture in the neodermis, resembling that of normal skin. This improvement of structural organisation in the healing wound is self-propagating and leads to a reduction of subsequent scarring. TGFbeta3 has completed safety studies and entered human clinical trials. Data from these studies have demonstrated that TGFbeta3 (Juvista) in humans is safe and well tolerated. Acute, local administration of TGFbeta3 (Juvista) significantly reduces dermal scarring in a dose responsive manner resulting in the regeneration of a skin structure that is permanently improved.