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The sirtuin class of deacetylases plays important regulatory roles in numerous physiological pathways with examples in all three sub-cellular compartments: nuclear, cytosolic and mitochondria [1, 2]. Of the seven mammalian sirtuin isoforms, the biology of SIRT1 is the most well characterized and its drug discovery platform is also the most advanced . Studies that involve SIRT1 over-expression in mammalian cellular models [4, 5] as well as those that involve the use of transgenic SIRT1 over-expressing mice have demonstrated significant SIRT1-dependent improvements to pathological phenotypes [6–8]. Furthermore, the efficacy induced by small molecule SIRT1 activators in preclinical models of metabolic, inflammatory, cardiovascular and neurodegenerative disease [9–13] suggests that pharmacological activation of SIRT1 may be beneficial for human health. For example, small molecule SIRT1 activators have shown efficacy in ob/ob and diet-induced obesity (DIO) mouse models  with significant effects on weight control, glucose homeostasis and insulin. Moreover, notable observations in additional animal models have included decreased production of inflammatory markers, reduced steatosis, and increased exercise endurance and muscle function, a convergence of effects that promote improved overall health [14–17]. There have been contradictory data published in the literature that link sirtuins to cancer, with both beneficial effects of increased expression  and inhibition or reduced expression [19, 20] of SIRT1 as cancer therapy strategies.
Although the full extent of the biological activity of SIRT1 has yet to be determined, the extraordinary breadth of the pharmacological potential presented by SIRT1 modulation can be more fully appreciated by considering that it has more than 70 known sub-cellular protein substrates, prominent examples of which include p53, PGC1α, FOXO, ACS1 and p65-NFκB, as well as a host of other nuclear and cytosolic proteins with established roles in numerous disease states [21–26] that generally are responsive to periods of cellular stress. Acetylation and deacetylation cycles associated with post translational modification of proteins have been implicated in critical control of cellular processes. Beyond serving as an on/off functional switch, deacetylation can alter protein stability , protein–protein interactions  and sub-cellular localization , thereby controlling downstream biological effects at multiple levels. Furthermore, the unique enzymatic reaction carried out by sirtuins involves NAD+ as a co-substrate, coupling cellular energy status to the regulatory mechanisms. A relatively comprehensive expression analysis of sirtuins in human tissues indicates a high level of SIRT1 expression in skeletal muscle, brain, kidney, thymus and reproductive tissues [30, 31]. Classical drug discovery has predominantly targeted protein classes such as GPCRs, kinases, nuclear receptors, ion channels and enzymes with a narrow, specific function. Their upstream modulation of several protein classes at once makes sirtuin enzymes unique targets for drug development. As such, pharmacological activation of sirtuins has emerged as an approach with great potential for the discovery of therapeutics in multiple disease indications.
The first small scale screen  for SIRT1 activators identified a narrow hit series comprised mostly of polyphenols. The most prominent and well-known hit was resveratrol, a stilbene abundantly available from sources such as grape skin, peanuts and roots of Polygonum cuspidatum. Resveratrol has been investigated in several exploratory studies in man [33, 34] although its lack of selectivity for SIRT1, significant metabolic liabilities and extremely low inherent bioavailability [35, 36] limit its use as a drug targeting SIRT1. Furthermore, some recent studies have proposed alternate mechanisms for the pharmacology of resveratrol  as well as its potential impact on SIRT1 expression levels [38, 39], assertions that are difficult to refute unambiguously. In order to address some of these issues, a search was undertaken for compounds with a high degree of selectivity for SIRT1, and that had improved potency, physiochemical pharmacokinetic properties and no chemical relationship to resveratrol led to SRT2104, a first-in-class activator of SIRT1. Following pharmacology and toxicology assessments, a series of phase 1 clinical studies in normal healthy volunteers were designed to characterize the pharmacokinetics of SRT2104, and to assess whether there were any deleterious effects of dosing a SIRT1 activator in humans. The overall goal of these studies was to identify appropriate doses of SRT2104 for use in future clinical trials designed to probe its safety and efficacy.
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Pharmacological activation of the human SIRT1 enzyme provides a novel therapeutic strategy with potential in a variety of indications including metabolic, inflammation and neurodegenerative diseases. Currently, there exists no validated biomarker to assess directly SIRT1 activation in humans. As such, the dose selected for human efficacy studies will be the dose that achieves the highest exposure whilst maintaining an excellent tolerability profile. The objective of this approach is to maximize the chance of observing a beneficial clinical signal while demonstrating the safety of dosing a SIRT1 activator. The phase I clinical plan reflects the demands of this approach: four separate trials to elucidate safety/tolerability, dose linearity and exposure after single and 7 day repeat dosing as well as formulation/gender/food effect, i.v. PK parameters and absolute bioavailability.
The initial dose escalation study indicated that SRT2104 was poorly absorbed following oral dosing as a suspension. Although some accumulation was observed from 1 to 7 days, there was concern that the drug concentrations achieved might be insufficient to generate a pharmacological response. At that time, it was not known whether the poor exposure was due primarily to poor absorption, high first pass metabolism or a combination of the two. It should be noted, however, that while SRT2104 demonstrates greater than 20 mg ml−1 solubility in simulated gastric fluid (SGF), the solubility in simulated intestinal fluid (SIF) drops to less than 2 μg ml−1 (unpublished data). As such, one explanation for the exposure variability in humans is that it is associated with the variability of gastric emptying times in the fasted state. Regardless, it was assumed that the poor solubility profile in intestinal fluid adversely affected the absorption of SRT2104, and that an optimized formulation, particularly one that improved the intestinal fluid solubility or that was retained for an extended period in the stomach (thereby maximizing drug dissolution in the acidic environment and releasing into the duodenum the dissolved drug over an extended period), might be a viable way to increase exposure and reduce variability. Furthermore, SRT2104 is currently characterized as Biopharmaceutics Classification System (BCS) II, suggesting that solubility, not permeability, is the greatest barrier to absorption.
It was important to determine to what extent clearance of SRT2104 prevented exposure. In the absence of a preclinical toxicology package supporting i.v. administration of SRT2104, an i.v. microdose of 100 μg SRT2104 fortified with 14C-SRT2104 was used. A common criticism of microdose studies is that due to the very small dose, transporters and enzymes involved in the disposition of the drug, which might otherwise be saturated at a pharmacological dose, can exert an influence on the pharmacokinetic parameters. Sirtris employed a microtracer design whereby subjects were administered a 0.25 g oral dose of SRT2104 followed by the microtracer at approximately tmax, thus minimizing the potential for bias resulting from the small i.v. dose. As shown in Table 3, SRT2104 exhibits low to moderate clearance, suggesting that improving the presentation of the drug through formulation would not be made irrelevant by clearance. Further need for an improved formulation was identified when a substantial food effect was observed following a single dose of 0.5 g SRT2104. Indeed, an approximately four fold increase in AUC(0,t) and Cmax was observed following a meal. Thus, further investigation into the causes underlying this observation could lead to an enhanced and improved formulation.
The goal of this phase 1 programme was to determine a dosing strategy to maximize exposure and it has been demonstrated that the food effect and repeat dose accumulation can contribute to such a strategy. The FIH study showed that maximal exposure was observed at the 2.0 g dose and that a further increase in dose provided no additional exposure. SRT2104 was well tolerated after dosing 2.0 g of SRT2104 in the fed state for 7 days, suggesting that this could be an appropriate dosing strategy for further early stage studies. Furthermore, the absence of a substantial gender effect suggests that this can be appropriately used in a mixed gender population. Although there was some evidence of increased exposure in females in the fasted state, this may have been primarily due to variability, which was substantially higher for males in the fasted state in this study (one male subject showed no meaningful exposure after being dosed with SRT2104 in the fasted state). Regardless, the variability and sample size prevents definitive conclusions with regard to gender effect on exposure, but the data suggests that any gender differences would be minimal. Finally, SRT2104 in a capsule does not greatly impact exposure (compared with a suspension formulation), allowing a more facile drug product to be utilized in larger studies.
In general, the use of a 2.0 g dose for target validation in the clinic presents a risk that pharmacological effects resulting from interaction with targets other than SIRT1 could confound any SIRT1-specific efficacy signals that might be observed. In the absence of a valid biomarker for SIRT1 activation, one approach to discharge the liability of a large dose with respect to target engagement in vivo is to evaluate extensively the selectivity of SRT2104. Indeed, SRT2104 displays minimal non-SIRT1 related activity as assessed by an in vitro profiling study including over 180 targets representing kinases, GPCRs, nuclear recptors, ion channels, enzymes and transporters (unpublished results). In vitro characterization of this favourable selectivity profile, along with its safety and tolerability profile in healthy volunteers, suggested the evaluation of high doses of SRT2104 in exploratory clinical trials in patient populations is acceptable.
The clinical development for SRT2104 will include studies designed to demonstrate that administration of this compound can exert a pharmacological effect in humans and help to validate SIRT1 as a target of therapeutic value. Increased SIRT1 activity by overexpression of both the gene and protein has been reported to have beneficial effects in inflammation and metabolic disease [10, 44, 45]. Preclinical testing (unpublished data) has shown SRT2104 to be efficacious in murine models of inflammation [lipopolysaccharide (LPS)-induced tumour necrosis factor-alpha (TNF-α) production, dextran sulphate sodium (DSS) and trinitrobenzesulphonic (TNBS) acid-induced colitis, caecal ligation and puncture-induced sepsis, experimental autoimmune encephalomyelitis (EAE)] and diabetes (improved glucose and insulin homeostasis in DIO mice and ob/ob mice). The potential for SRT2104 to exert an anti-inflammatory signal could be assessed in human subjects with LPS-induced endotoxaemia. Intravenous injection of LPS can activate multiple inflammatory cascades resulting in a potent pro-inflammatory response [46–49]. Attenuation of the immune response can be evaluated through the measurement of cytokines and other pro-inflammatory markers, as well as by clinical assessment of symptoms known to be associated with acute LPS administration (fever, headache, generalized malaise, and myalgia) . While a favourable result in this setting does not necessarily ensure a clinical benefit, it does provide confidence in the anti-inflammatory potential of SRT2104. Such a result could be followed up with studies in patients with psoriasis, ulcerative colitis, Crohn's disease and a variety of other diseases known to be mediated by inflammation. Further to inflammation, there exists preclinical rationale for beneficial SIRT1 activation in the areas of diabetes [51–54], exercise tolerance and disuse atrophy , all of which could be assessed in a series of small, focused exploratory medicine studies.
The current formulation of SRT2104 is extremely minimal and does not circumvent the inherently poor physical properties of the molecule. Of particular concern is the low bioavailability and the significant inter-subject variability in exposure. Although there appears to be opportunity for formulation optimization, this may be a significant challenge should a high dose be required. In addition, reliance on a food effect to maximize exposure, while useful in exploratory studies, would present some challenges later in development since patients would be required to take SRT2104 with food. In fact, some patient populations (type II diabetics, ulcerative colitis patients) may be unable to comply with instructions to take SRT2104 following a standardized meal based on dietary restrictions. Despite these issues, the remarkable pharmacology associated with SIRT1 modulation in preclinical models makes assessment of this target in humans of paramount importance. Indeed, SRT2104 is the first, highly selective small molecule activator of SIRT1 to progress to human subjects. Demonstration of pharmacological activity in humans, and correlation of this activity with preclinical efficacy data, will provide support for the tractability of SIRT1 as a drug target. Despite some of the challenges highlighted above, SRT2104 is well-tolerated and has limited non-SIRT1 activity, making it a useful molecule for interrogating SIRT1 activation in the clinic during early stage development. Future clinical studies, with comprehensive pharmacokinetic assessments, may allow for detection of a clinical signal as well as associated pharmacodynamic drivers, thereby allowing for a better understanding of the biological impact of selected SIRT1 activation in a variety of human diseases.
The bioanalysis was performed by Simbec, Ltd, UK under contract by the sponsor, with special thanks to Tracy Thomas. Quotient Clinical Ltd and Dr Lloyd Stevens, in particular, assisted with the bioavailability study under contract by the sponsor. The quantification of the 14C-SRT2104 was performed by Vitalea Inc (Davis, CA) under contract by the sponsor. In addition, the authors are grateful to Rachel D. Collier, Steve Corless, Clive Felgate, Sian Hilton, Andy Roberts, Rita Tailor, Graeme Young and Claire Beaumont for the metabolite identification.