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α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α

Severe α1-antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1-antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high throughput screen to identify small molecules that bind to, and stabilise Z α1-antitrypsin. The lead compound blocks Z α1-antitrypsin polymerisationin vitro, reduces intracellular polymerisation and increases the secretion of Z α1-antitrypsin three-fold in mammalian cells including an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerization pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α1-antitrypsin into the plasma by 7-fold. There was no observable clearance of hepatic inclusions with respect to controls. This study provides proof-of-principle that ‘mutation ameliorating’ small molecules are a viable approach to treat protein conformational diseases.

Fifteen million babies are born preterm every year and a significant number suffer from permanent neurological injuries linked to white matter injury (WMI). A chief cause of preterm birth itself and predictor of the severity of WMI is exposure to maternal-fetal infection-inflammation such as chorioamnionitis. There are no neurotherapeutics for this WMI. To affect this healthcare need, the repurposing of drugs with efficacy in other white matter injury models is an attractive strategy. As such, we tested the efficacy of GSK247246, an H3R antagonist/inverse agonist, in a model of inflammation-mediated WMI of the preterm born infant recapitulating the main clinical hallmarks of human brain injury, which are oligodendrocyte maturation arrest, microglial reactivity, and hypomyelination. WMI is induced by mimicking the effects of maternal-fetal infection-inflammation and setting up neuroinflammation. We induce this process at the time in the mouse when brain development is equivalent to the human third trimester; postnatal day (P)1 through to P5 with i.p. interleukin-1β (IL-1β) injections. We initiated GSK247246 treatment (i.p at 7mg/kg or 20mg/kg) after neuroinflammation was well established (on P6) and it was administered twice daily through to P10. Outcomes were assessed at P10 and P30 with gene and protein analysis. A low dose of GSK247246 (7 mg/kg) lead to a recovery in protein expression of markers of myelin (density of Myelin Basic Protein, MBP & Proteolipid Proteins, PLP) and a reduction in macro- and microgliosis (density of ionising adaptor protein, IBA1 & glial fibrillary acid protein, GFAP). Our results confirm the neurotherapeutic efficacy of targeting the H3R for WMI seen in a cuprizone model of multiple sclerosis and a recently reported clinical trial in relapsing-remitting multiple sclerosis patients. Further work is needed to develop a slow release strategy for this agent and test its efficacy in large animal models of preterm infant WMI.

α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α1-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.

Lp-PLA2 has been explored as a target for a number of inflammation associated diseases, including cardiovascular disease and dementia. This article describes the discovery of a new fragment derived chemotype that interacts with the active site of Lp-PLA2. The starting fragment hit was discovered through an X-ray fragment screen and showed no activity in the bioassay (IC50 > 1 mM). The fragment hit was optimized using a variety of structure-based drug design techniques, including virtual screening, fragment merging, and improvement of shape complementarity. A novel series of Lp-PLA2 inhibitors was generated with low lipophilicity and a promising pharmacokinetic profile.

Elevated levels of human lipoprotein-associated phospholipase A2 (Lp-PLA2) are associated with cardiovascular disease and dementia. A fragment screen was conducted against Lp-PLA2 in order to identify novel inhibitors. Multiple fragment hits were observed in different regions of the active site, including some hits that bound in a pocket created by movement of a protein side chain (approximately 13 Å from the catalytic residue Ser273). Using structure guided design, we optimized a fragment that bound in this pocket to generate a novel low nanomolar chemotype, which did not interact with the catalytic residues.

A series of potent, competitive and highly selective kynurenine monooxygenase inhibitors have been discovered via a substrate-based approach for the treatment of acute pancreatitis. The lead compound demonstrated good cellular potency and clear pharmacodynamic activity in vivo.

Through their function as epigenetic readers of the histone code, the BET family of bromodomain-containing proteins regulate expression of multiple genes of therapeutic relevance, including those involved in tumor cell growth and inflammation. BET bromodomain inhibitors have profound antiproliferative and anti-inflammatory effects which translate into efficacy in oncology and inflammation models, and the first compounds have now progressed into clinical trials. The exciting biology of the BETs has led to great interest in the discovery of novel inhibitor classes. Here we describe the identification of a novel tetrahydroquinoline series through up-regulation of apolipoprotein A1 and the optimization into potent compounds active in murine models of septic shock and neuroblastoma. At the molecular level, these effects are produced by inhibition of BET bromodomains. X-ray crystallography reveals the interactions explaining the structure-activity relationships of binding. The resulting lead molecule, I-BET726, represents a new, potent, and selective class of tetrahydroquinoline-based BET inhibitors.

The connection between Netherton syndrome and overactivation of epidermal/dermal proteases, particularly Kallikrein 5 (KLK5) has been well established and it is expected that a KLK5 inhibitor would improve the dermal barrier and also reduce the pain and itch that afflict Netherton syndrome patients. One of the challenges of covalent protease inhibitors has been achieving selectivity over closely related targets. In this paper we describe the use of structural insight to design and develop a selective and highly potent reversibly covalent KLK5 inhibitor from an initial weakly binding fragment.

A key objective of the drug discovery project team is to discharge as much risk as possible for the selected candidate such that the best possible molecule can be progressed.

During the development of a dermal drug, much efforts tend to be placed on improving its delivery through skin. These efforts are driven by the known risk of not engaging the target. Indeed to increase comfort that the target will be engaged one need to try fullfill as best as it can evidence of (1) sufficient compound on board for a desired time (i.e., PK within skin) and (2) demonstration of pharmacological activity [1].

Over the past decades, the drug discovery process of the pharmaceutical industry has vastly improved.

This chapter reviews the most commonly used topical pharmacokinetic techniques. Numerous other techniques (e.g., the non-invasive spectroscopic techniques like confocal, fluorescence techniques as well as the NMR technique) are not described here as they will have one or more of the following characteristics: very specific for a drug or disease, possibly impractical, possibly not quantitative, possibly lacking sensitivity or possibly at a too early stage of development. They cannot, therefore, be applicable as versatile techniques for studying topical pharmacokinetics.

As seen in Chap. 1, the pharma industry nowadays works on more therapeutic targets than it used to do thanks to the advances in the understanding of the molecular basis of diseases as well as the push from payors to deliver better medicines compared to standard of care. This presents a strong opportunity for pharma companies who want to invest into dermal drug development projects as more assets and more therapeutic targets are becoming available.

In the preceding chapters, several elements and concepts have emerged. In order to put in perspectives some of them in the industrial context, some quotations and rules are listed in this chapter. Extra comments met during the course of various topical drug development programs are added too. Overall these quotations and rules, sometimes considered provocative, may be found helpful for program teams developing a topical molecule.

As seen previously, a large part of the selection of a candidate process for an oral/systemic application relies on the use of plasma exposure and in vitro potency. This allows translating reachable plasma exposure (pharmacokinetics) into reachable in vivo pharmacodynamic effect: This is PharmacoKinetic/Pharmacodynamic (PK/PD) (Fig. 6.1).

This chapter focuses on lessons learned from the success and failure of topical drug development over the last 60 years. First, the skin barrier quality for a particular skin disease will be compared to the easiness of topical drug development. The importance of the candidate molecule potency will be considered as well in comparison to its clinical efficacy.

One key advantage of a topical administration is the decreased risk of toxicological findings. This perceived element is generally the driving rationale for developing a topical medicine.

Historically most topical drug classes seem to have been originally developed following the pragmatic principle that, “if an existing drug with an interesting pharmacology is effective orally/systemically, and the target is in the skin, it could well work topically without giving side effects and, therefore, would deserve to be tried topically”.

Lp-PLA

Acute pancreatitis (AP) is a common and devastating inflammatory condition of the pancreas that is considered to be a paradigm of sterile inflammation leading to systemic multiple organ dysfunction syndrome (MODS) and death1,2. Acute mortality from AP-MODS exceeds 20% (ref. 3), and the lifespans of those who survive the initial episode are typically shorter than those of the general population4. There are no specific therapies available to protect individuals from AP-MODS. Here we show that kynurenine-3-monooxygenase (KMO), a key enzyme of tryptophan metabolism5, is central to the pathogenesis of AP-MODS. We created a mouse strain that is deficient for Kmo (encoding KMO) and that has a robust biochemical phenotype that protects against extrapancreatic tissue injury to the lung, kidney and liver in experimental AP-MODS. A medicinal chemistry strategy based on modifications of the kynurenine substrate led to the discovery of the oxazolidinone GSK180 as a potent and specific inhibitor of KMO. The binding mode of the inhibitor in the active site was confirmed by X-ray co-crystallography at 3.2 Å resolution. Treatment with GSK180 resulted in rapid changes in the levels of kynurenine pathway metabolites in vivo, and it afforded therapeutic protection against MODS in a rat model of AP. Our findings establish KMO inhibition as a novel therapeutic strategy in the treatment of AP-MODS, and they open up a new area for drug discovery in critical illness.

Topical aciclovir cream (ACV, Zovirax Cream) containing 40% propylene glycol (PG), the optimum found for skin penetration, is clinically effective in the treatment of recurrent herpes labialis. One hundred and thirty-nine ACV generic creams were analysed and 80% of these contained less than 20% PG. From this, we hypothesised that these generics might be bioinequivalent to the innovator cream. A pilot in vitro skin permeation study compared the innovator cream with two generics containing about 15% PG. Next, 10 generics containing 0-15% PG were tested in an independent laboratory. Finally, a PG dose-ranging study was conducted in Zovirax cream base. In all studies, human skin was used and ACV analysed by LC-MS-MS. In the pilot study, the innovator cream delivered 7.5-fold more ACV than the two generics. Superiority was confirmed in the second study against all 10 ACV generic creams. By grouping the creams according to PG content, a relationship to ACV skin permeation was suggested. The PG dose effect was confirmed in the third study. These studies suggest that not all marketed ACV creams are bioequivalent to the clinically proven innovator. Given the magnitude of the differences seen, there is concern over therapeutic inequivalence of generic ACV creams to the innovator cream.

The aim of this study was to test the hypothesis that the most appropriate model for studying the diffusional release of an active from a topical formulation is one in which the membrane offers minimal resistance to release and involves a receptor phase that presents the least possible interfacial discontinuity. Using ketoprofen as the active, a series of simple gels were prepared consisting of PEG400 thickened with Cabosil M5. Using Franz-type diffusion cells, three different types of membrane (two porous and one non-porous) were compared, as were receptor phases of PEG400 (component of formulation) and PBS. Of the membranes tested only 0.2 microm nylon provided consistent first order kinetics for a range of gel consistencies, indicating negligible influence of the membrane. The non-porous silicone membrane did not show first order kinetic profile confirming the diffusional nature of such a membrane. From the non-thickened formulations, diffusional release into a receptor phase of PEG400 was some 3x that into PBS, whereas from the formulation thickened with 5% Cabosil M5, diffusional release into a receptor phase of PEG400 was 6x lower than that into PBS. Diffusional release into PBS did not follow first order kinetics while diffusion into PEG400 did, suggesting that the existence of a discontinuity affected the release process. Although the importance of zero-resistance membranes is of perhaps obvious importance, it is often not stated in the literature. The existence of phase/hydrodynamic boundaries in release studies can be a source of significant inaccuracy.

Bromodomains (BRDs) are small protein domains found in a variety of proteins that recognize and bind to acetylated histone tails. This binding affects chromatin structure and facilitates the localisation of transcriptional complexes to specific genes, thereby regulating epigenetically controlled processes including gene transcription and mRNA elongation. Inhibitors of the bromodomain and extra-terminal (BET) proteins BRD2-4 and T, which prevent bromodomain binding to acetyl-modified histone tails, have shown therapeutic promise in several diseases. We report here the discovery of 1,5-naphthyridine derivatives as potent inhibitors of the BET bromodomain family with good cell activity and oral pharmacokinetic parameters. X-ray crystal structures of naphthyridine isomers have been solved and quantum mechanical calculations have been used to explain the higher affinity of the 1,5-isomer over the others. The best compounds were progressed in a mouse model of inflammation and exhibited dose-dependent anti-inflammatory pharmacology.

AMP-activated protein kinase (AMPK) is an evolutionarily conserved fuel-sensing enzyme that is activated in shortage of energy and suppressed in its surfeit. AMPK activation stimulates fatty acid oxidation, enhances insulin sensitivity, alleviates hyperglycemia and hyperlipidemia, and inhibits proinflammatory changes. Thus, AMPK is a well-received therapeutic target for type 2 diabetes and other metabolic disorders. Here, we will report the discovery of pyrrolopyridone derivatives as AMPK direct activators. We will illustrate the synthesis and structure–activity relationships of the series as well as some pharmacokinetic results. Some compounds exhibited encouraging oral exposure and were evaluated in a mouse diabetic model. Compound 17 showed oral activity at 30 mg/kg on blood glucose.

The discovery, synthesis and biological evaluation of a novel series of 7-isoxazoloquinolines is described. Several analogs are shown to increase ApoA1 expression within the nanomolar range in the human hepatic cell line HepG2.

In vitro measurements of skin absorption are an increasingly important aspect of regulatory studies, product support claims, and formulation screening. However, such measurements are significantly affected by skin variability. The purpose of this study was to determine inter‐ and intralaboratory variation in diffusion cell measurements caused by factors other than skin. This was attained through the use of an artificial (silicone rubber) rate‐limiting membrane and the provision of materials including a standard penetrant, methyl paraben (MP), and a minimally prescriptive protocol to each of the 18 participating laboratories. “Standardized” calculations of MP flux were determined from the data submitted by each laboratory by applying a predefined mathematical model. This was deemed necessary to eliminate any interlaboratory variation caused by different methods of flux calculations. Average fluxes of MP calculated and reported by each laboratory (60 ± 27 μg cm −2 h −1 , n = 25, range 27–101) were in agreement with the standardized calculations of MP flux (60 ± 21 μg cm −2 h −1 , range 19–120). The coefficient of variation between laboratories was approximately 35% and was manifest as a fourfold difference between the lowest and highest average flux values and a sixfold difference between the lowest and highest individual flux values. Intralaboratory variation was lower, averaging 10% for five individuals using the same equipment within a single laboratory. Further studies should be performed to clarify the exact components responsible for nonskin‐related variability in diffusion cell measurements. It is clear that further developments of in vitro methodologies for measuring skin absorption are required. © 2005 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:632–638, 2005

The migration of ketoprofen through a series of simple gels that varied in solvent composition to simulate snapshots of a dynamically drying topical formulation was studied. Firstly, the release rate of ketoprofen was determined from formulations based on Cabosil and PEG 400, the proportion of which was varied to mimic progressively dryer states. Secondly, the apparent permeability of ketoprofen across the corresponding blank Cabosil gels was determined. Thirdly, the effect of macro viscosity on these data was probed by comparing permeation of ketoprofen across Cabosil and hydroxypropyl cellulose (HPC) gels of equal viscosity. Linear release profiles were produced for all formulations suggesting first-order release and the rate of ketoprofen liberated was inversely to the proportion of Cabosil, suggesting that the drier the film, the slower the rate of release. At the lowest level of thickener used (5%) the release rate was reduced to 45% of the control. At 25% the release rate was reduced to 24% of the control. The presence of the Cabosil had an even more dramatic effect on the apparent permeability of ketoprofen across the gels. At 5% Cabosil the apparent steady state flux was reduced to 4% of the control. At 25% the apparent steady state flux was reduced to < 1% of the control. Although the 0.5% HPC gel and the 1% Cabosil gel possessed identical macro viscosities, the permeation of ketoprofen through the HPC gel was almost double that of the Cabosil gel. The data from these experiments demonstrated that migration of active molecules through a gel is significantly affected by the amount of solvent present in, or lost from, the system. It is proposed that increased adsorption of active to the thickener plays a more important role than increased macro viscosity for reduced active release as the formulation becomes increasingly dry. Furthermore, such affects are profoundly influenced by the chemical nature of the thickener.