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Additional information to provide an insight into the full kinase selectivity profile of AT7519 and full details of its pharmakokinetic parameters

Cyclin-dependent kinases (CDK), and their regulatory cyclin partners, play a central role in eukaryotic cell growth, division, and death. This key role in cell cycle progression, as well as their deregulation in several human cancers, makes them attractive therapeutic targets in oncology. A series of CDK inhibitors was developed using Astex's fragment-based medicinal chemistry approach, linked to high-throughput X-ray crystallography. A compound from this series, designated AT7519, is currently in early-phase clinical development. We describe here the biological characterization of AT7519, a potent inhibitor of several CDK family members. AT7519 showed potent antiproliferative activity (40-940 nmol/L) in a panel of human tumor cell lines, and the mechanism of action was shown here to be consistent with the inhibition of CDK1 and CDK2 in solid tumor cell lines. AT7519 caused cell cycle arrest followed by apoptosis in human tumor cells and inhibited tumor growth in human tumor xenograft models. Tumor regression was observed following twice daily dosing of AT7519 in the HCT116 and HT29 colon cancer xenograft models. We show that these biological effects are linked to inhibition of CDKs in vivo and that AT7519 induces tumor cell apoptosis in these xenograft models. AT7519 has an attractive biological profile for development as a clinical candidate, and the tolerability and efficacy in animal models compare favorably with other CDK inhibitors in clinical development. Studies described here formed the biological rationale for investigating the potential therapeutic benefit of AT7519 in cancer patients. [Mol Cancer Ther 2009;8(2):324–32]

Supplementary Data from Biological characterization of AT7519, a small-molecule inhibitor of cyclin-dependent kinases, in human tumor cell lines

Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.

Introduction HTL0018318 is a selective muscarinic M1 receptor partial agonist under development for the symptomatic treatment of dementias, including Alzheimer’s disease. Clinically, HTL0018318 would likely be used alone or in conjunction with cholinesterase inhibitors (e.g. donepezil). Objective We investigated the safety, tolerability, and pharmacokinetics of HTL0018318 given alone and in combination with donepezil. Methods This was a randomized, double-blind, placebo-controlled trial in 42 (to deliver 36 with combination treatment) healthy elderly subjects investigating the effects of oral HTL0018318 15 and 25 mg given alone and combined with donepezil 10 mg at steady state on adverse events (AEs), vital signs, saliva production, sleep quality, pulmonary function, subjective feelings, and pharmacokinetics. Results AEs were reported by lower percentages of subjects after HTL0018318 alone than after donepezil alone. There was no increase in the percentage of subjects reporting AEs after co-administration than after donepezil alone. Supine systolic blood pressure was 1.6 mmHg (95% confidence interval [CI] −3.1 to −0.1) lower after HTL0018318 alone than after combination treatment. This was comparable with results from placebo alone: 1.7 mmHg (95% CI −3.2 to 0.2) lower than with combination treatment. Supine pulse rate was 3.3 bpm (95% CI 1.5–5.1) higher after HTL0018318 alone than with co-administration. HTL0018318 and donepezil did not meaningfully affect each other’s pharmacokinetics. Conclusion HTL0018318 was well tolerated when given alone and in combination with donepezil. HTL0018318 and donepezil do not demonstrate pharmacokinetic or pharmacodynamic interactions, indicating that HTL0018318 can be safely administered in combination with donepezil. Clinical trial registration Netherlands Trial register identifier NL5915, registered on 28 October 2016. Supplementary Information The online version contains supplementary material available at 10.1007/s40268-021-00352-5.

This study examined the safety and pharmacodynamic effects of selective muscarinic MA randomized, double-blind, placebo-controlled 4-week safety study of HTL0018318 with up-titration and maintenance phases, observing exploratory effects on electrophysiological biomarkers and cognition.Treatment-emergent adverse events (TEAEs) were mild and less frequently reported during maintenance versus titration. Headache was most commonly reported (7-21%); 0 to 13% reported cholinergic TEAEs (abdominal pain, diarrhea, fatigue, nausea) and two patients discontinued due to TEAEs. At 1 to 2 hours post-dose, HTL0018318-related mean maximum elevations in systolic and diastolic blood pressure of 5 to 10 mmHg above placebo were observed during up-titration but not maintenance. Postive effects of HTL0018318 were found on specific attention and memory endpoints.HTL0018318 was well tolerated in mild-to-moderate AD patients and showed positive effects on attention and episodic memory on top of therapeutic doses of donepezil.

Aims HTL0009936 is a selective M-1 muscarinic receptor agonist in development for cognitive dysfunction in Alzheimer's disease. Safety, tolerability and pharmacokinetics and exploratory pharmacodynamic effects of HTL0009936 administered by continuous IV infusion at steady state were investigated in elderly subjects with below average cognitive functioning (BACF).Methods Part A was a four-treatment open label sequential study in healthy elderly investigating 10-83 mg HTL0009936 (IV) and a 24 mg HTL0009936 single oral dose. Part B was a five-treatment randomized, double-blind, placebo and physostigmine controlled cross-over study with IV HTL0009936 in elderly subjects with BACF. Pharmacodynamic assessments were performed using neurocognitive and electrophysiological tests.Results Pharmacokinetics of HTL0009936 showed dose-proportional increases in exposure with a mean half-life of 2.4 hours. HTL0009936 was well-tolerated with transient dose-related adverse events (AEs). Small increases in mean systolic blood pressure of 7.12 mmHg (95% CI [3.99-10.24]) and in diastolic of 5.32 mmHg (95% CI [3.18-7.47]) were noted at the highest dose in part B. Overall, there was suggestive, but no definitive, positive or negative pharmacodynamic effects. Statistically significant effects were observed on P300 with HTL0009936 and adaptive tracking with physostigmine.Conclusions HTL0009936 showed well-characterized pharmacokinetics and single doses were safe and generally well-tolerated in healthy elderly subjects. Due to physostigmine tolerability issues and subject burden, the study design was changed and some pharmacodynamic assessments (neurocognitive) were performed at suboptimal drug exposures. Therefore no clear conclusions can be made on pharmacodynamic effects of HTL0009936, although an effect on P300 is suggestive of central target engagement.

Funder: Sosei Heptares, BACKGROUND: The cholinergic system and M1 receptor remain an important target for symptomatic treatment of cognitive dysfunction. The selective M1 receptor partial agonist HTL0018318 is under development for the symptomatic treatment of Dementia's including Alzheimer's disease (AD) and dementia with Lewy bodies (DLB). We investigated the safety, tolerability, pharmacokinetics and exploratory pharmacodynamics of multiple doses of HTL0018318 in healthy younger adults and elderly subjects. METHODS: This randomised, double blind, placebo-controlled study was performed, investigating oral doses of 15-35 mg/day HTL0018318 or placebo in 7 cohorts of healthy younger adult (n = 36; 3 cohorts) and elderly (n = 50; 4 cohorts) subjects. Safety, tolerability and pharmacokinetic measurements were performed. Pharmacodynamics were assessed using a battery of neurocognitive tasks and electrophysiological biomarkers of synaptic and cognitive functions. RESULTS: HTL0018318 was generally well-tolerated in multiple doses up to 35 mg/day and were associated with mild or moderate cholinergic adverse events. There were modest increases in blood pressure and pulse rate when compared to placebo-treated subjects, with tendency for the blood pressure increase to attenuate with repeated dosing. There were no clinically significant observations or changes in blood and urine laboratory measures of safety or abnormalities in the ECGs and 24-h Holter assessments. HTL0018318 plasma exposure was dose-proportional over the range 15-35 mg. Maximum plasma concentrations were achieved after 1-2 h. The apparent terminal half-life of HTL0018318 was 16.1 h (± 4.61) in younger adult subjects and 14.3 h (± 2.78) in elderly subjects at steady state. HTL0018318 over the 10 days of treatment had significant effects on tests of short-term (working) memory (n-back) and learning (Milner maze) with moderate to large effect sizes. CONCLUSION: Multiple doses of HTL0018138 showed well-characterised pharmacokinetics and were safe and generally well-tolerated in the dose range studied. Pro-cognitive effects on short-term memory and learning were demonstrated across the dose range. These data provide encouraging data in support of the development of HTL0018138 for cognitive dysfunction in AD and DLB. TRIAL REGISTRATION: Netherlands Trial Register identifier NTR5781 . Registered on 22 March 2016.

Aims HTL0018318 is a selective M1 receptor partial agonist currently under development for the symptomatic treatment of cognitive and behavioural symptoms in Alzheimer's disease (AD) and other dementias. We investigated safety, tolerability, pharmacokinetics (PK) and exploratory pharmacodynamics (PD) of HTL0018318 following single ascending doses. Methods This randomized, double-blind, placebo-controlled study in 40 healthy younger adult and 57 healthy elderly subjects, investigated oral doses of 1-35 mg HTL0018318. PD assessments were performed using a battery of neurocognitive tasks and electrophysiological measurements. CSF concentrations of HTL0018318 and food effects on PK of HTL0018318 were investigated in an open label and partial cross-over design in 14 healthy subjects. Results Pharmacokinetics of HTL0018318 were well-characterized showing dose proportional increases in exposure from 1-35 mg. Single doses of HTL0018318 were associated with mild dose-related adverse events of low incidence in both younger adult and elderly subjects. The most frequently reported cholinergic AEs included hyperhidrosis and increases in blood pressure up to 10.3 mm Hg in younger adults (95% CI [4.2-16.3], 35 mg dose) and up to 11.9 mm Hg in elderly subjects (95% CI [4.9-18.9], 15 mg dose). There were no statistically significant effects on cognitive function but the study was not powered to detect small to moderate effect sizes of clinical relevance. Conclusions HTL0018318 showed well-characterized pharmacokinetics and following single doses were generally well tolerated in the dose range studied. These provide encouraging data in support of the development for HTL0018318 for AD and other dementias. Netherlands Trial Register identifier NTR5648.

Aims HTL0018318 is a selective M1 receptor partial agonist currently under development for the symptomatic treatment of cognitive and behavioural symptoms in Alzheimer's disease and other dementias. We investigated safety, tolerability, pharmacokinetics and exploratory pharmacodynamics (PD) of HTL0018318 following single ascending doses. Methods This randomized, double‐blind, placebo‐controlled study in 40 healthy younger adult and 57 healthy elderly subjects, investigated oral doses of 1–35 mg HTL0018318. Pharmacodynamic assessments were performed using a battery of neurocognitive tasks and electrophysiological measurements. Cerebrospinal fluid concentrations of HTL0018318 and food effects on pharmacokinetics of HTL0018318 were investigated in an open label and partial cross‐over design in 14 healthy subjects. Results Pharmacokinetics of HTL0018318 were well‐characterized showing dose proportional increases in exposure from 1–35 mg. Single doses of HTL0018318 were associated with mild dose‐related adverse events of low incidence in both younger adult and elderly subjects. The most frequently reported cholinergic AEs included hyperhidrosis and increases in blood pressure up to 10.3 mmHg in younger adults (95% CI [4.2–16.3], 35‐mg dose) and up to 11.9 mmHg in elderly subjects (95% CI [4.9–18.9], 15‐mg dose). There were no statistically significant effects on cognitive function but the study was not powered to detect small to moderate effect sizes of clinical relevance. Conclusion HTL0018318 showed well‐characterized pharmacokinetics and following single doses were generally well tolerated in the dose range studied. These provide encouraging data in support of the development for HTL0018318 for Alzheimer's disease and other dementias.

Background The cholinergic system and M1 receptor remain an important target for symptomatic treatment of cognitive dysfunction. The selective M1 receptor partial agonist HTL0018318 is under development for the symptomatic treatment of Dementia’s including Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB). We investigated the safety, tolerability, pharmacokinetics and exploratory pharmacodynamics of multiple doses of HTL0018318 in healthy younger adults and elderly subjects. Methods This randomised, double blind, placebo-controlled study was performed, investigating oral doses of 15–35 mg/day HTL0018318 or placebo in 7 cohorts of healthy younger adult (n = 36; 3 cohorts) and elderly (n = 50; 4 cohorts) subjects. Safety, tolerability and pharmacokinetic measurements were performed. Pharmacodynamics were assessed using a battery of neurocognitive tasks and electrophysiological biomarkers of synaptic and cognitive functions. Results HTL0018318 was generally well-tolerated in multiple doses up to 35 mg/day and were associated with mild or moderate cholinergic adverse events. There were modest increases in blood pressure and pulse rate when compared to placebo-treated subjects, with tendency for the blood pressure increase to attenuate with repeated dosing. There were no clinically significant observations or changes in blood and urine laboratory measures of safety or abnormalities in the ECGs and 24-h Holter assessments. HTL0018318 plasma exposure was dose-proportional over the range 15–35 mg. Maximum plasma concentrations were achieved after 1–2 h. The apparent terminal half-life of HTL0018318 was 16.1 h (± 4.61) in younger adult subjects and 14.3 h (± 2.78) in elderly subjects at steady state. HTL0018318 over the 10 days of treatment had significant effects on tests of short-term (working) memory (n-back) and learning (Milner maze) with moderate to large effect sizes. Conclusion Multiple doses of HTL0018138 showed well-characterised pharmacokinetics and were safe and generally well-tolerated in the dose range studied. Pro-cognitive effects on short-term memory and learning were demonstrated across the dose range. These data provide encouraging data in support of the development of HTL0018138 for cognitive dysfunction in AD and DLB. Trial registration Netherlands Trial Register identifier NTR5781. Registered on 22 March 2016. Supplementary Information The online version contains supplementary material available at 10.1186/s13195-021-00816-5.

Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.

Current therapies for improving memory in Alzheimer’s disease are focused on correcting for defective cholinergic transmission by increasing acetylcholine levels through inhibition of acetylcholinesterase enzymes. These treatments are however associated with limited clinical efficacy and dose-limiting adverse effects. Here we describe agonist-bound crystal structures of the M1 muscarinic acetylcholine receptor (M1 mAChR) and its application in supporting the rational drug design of a selective M1-orthosteric agonist, HTL0009936. On the basis of pro-cognitive effects across a range of pre-clinical animal models and a favourable safety and toxicology profile HTL0009936 was progressed to first in human trials where the agonist activated central processes corresponding to learning and memory circuitry. Hence, our study not only describes the pharmacological profile of a M1 mAChR agonist with the potential to improve memory in Alzheimer’s disease but also demonstrates how to rationally progress the development of a G protein-coupled receptor ligand from structure to clinical candidate.

Gradient-based optimization for large-scale, multidisciplinary design problems requires accurate and efficient sensitivity analysis to compute design derivatives. The local continuum shape sensitivity method with spatial gradient reconstruction is a nonintrusive design sensitivity analysis method that was previously published for linear and nonlinear structural analysis. The numerical behavior, accuracy, and convergence for this method are studied using benchmark problems. The benchmark problems include finite element analysis of an axial bar and finite element analysis of a rectangular membrane using unstructured grids. The results are used to establish general guidelines for conducting mesh refinement and selecting the parameters associated with spatial gradient reconstruction calculations. Lastly, design derivatives are calculated for the potential flow solution around a Joukowsky airfoil. The analytic design derivative solutions are used to calculate the true errors associated with the local continuum...

Gradient-based optimization for large-scale, multidisciplinary design problems requires accurate and efficient sensitivity analysis to compute design derivatives. Presented here is a nonintrusive analytic sensitivity method, that is relatively easy to implement. Furthermore, it can be as accurate as conventional analytic sensitivity methods, which are intrusive and tend to be difficult, if not infeasible, to implement. The nonintrusive local continuum shape sensitivity method with spatial gradient reconstruction (SGR) is formulated for nonlinear systems. This is an extension of the formulation previously published for linear systems. SGR, a numerical technique used to approximate spatial derivatives, can be leveraged to implement the sensitivity method in a nonintrusive manner. The method is used to compute design derivatives for a variety of applications, including nonlinear static beam bending, nonlinear transient gust response of a 2-D beam structure, and nonlinear static bending of rectangular plates. To demonstrate that the method is nonintrusive, all analyses are conducted using black box solvers. One limiting requirement of the method is that it requires the converged Jacobian or tangent stiffness matrix as output from the analysis tool. For each example the design derivatives of the structural displacement response are verified with finite difference calculations.

Sitaxentan (Thelin®), an endothelin receptor antagonist with a long duration of action and high specificity for the endothelin receptor A subtype, was used to treat pulmonary arterial hypertension. It was withdrawn from the market due to an idiosyncratic risk of drug-induced liver injury identified from emerging clinical trial data and clinical case reports. The preclinical safety profile of sitaxentan is presented, including single- and repeat-dose toxicity in mice, rats, and dogs and carcinogenicity in mice and rats. Sitaxentan-related adverse effects included coagulopathy in rats and dogs, increased serum alkaline phosphatase activity in mice and dogs, and hepatic hypertrophy in all species. Decreased albumin, erythrocyte count, hemoglobin concentration and hematocrit, and increased coagulation times and liver weight were also noted. These effects generally occurred at systemic exposures (AUC(0-24)) that were substantially greater than those seen in humans. Twice-daily (vs. once daily) dosing resulted in increased toxicity, which correlated with increased trough plasma sitaxentan concentrations. Sitaxentan appeared to have a low potential for testicular and hepatic toxicity and was not carcinogenic. These studies suggested that sitaxentan would have a reasonable margin of safety when used as directed in humans and supported a positive benefit:risk assessment at the time of marketing approval.

Sitaxentan sodium (Thelin) is a once daily, orally bioavailable, highly selective endothelin A receptor antagonist. Initially approved for the treatment of pulmonary arterial hypertension, sitaxentan was withdrawn in 2010 following the recognition of a pattern of idiosyncratic liver injury. During development of this drug, a series of nonclinical studies investigated the effects of orally administered sitaxentan on fertility, embryofetal development, and pre- and postnatal development in the rat; results of these studies are reported here. In the fertility study, sitaxentan did not affect mating behavior, fertility, sperm morphology, or estrous cycle. Sitaxentan was teratogenic in the embyrofetal development study, which was expected based on its pharmacologic mechanism of action. Teratogenic effects included malformations of the head, mouth, face, and large blood vessels. In the pre- and postnatal study, sitaxentan administration was associated with reduced pup survival, large or abnormally shaped livers, and delays in markers of auditory and sexual development. Sitaxentan was detected in plasma of suckling pups receiving milk from females dosed with sitaxentan. These nonclinical study findings were reflected in the sitaxentan product label warnings.

A ubiquitously expressed chaperone, heat shock protein 90 (HSP90) is of considerable interest as an oncology target because tumor cells and oncogenic proteins are acutely dependent on its activity. AT13387 (2,4‐dihydroxy‐5‐isopropyl‐phenyl)‐[5‐(4‐methyl‐piperazin‐1‐ylmethyl)‐1,3‐dihydro‐isoindol‐2‐yl] methanone, l‐lactic acid salt) a novel, high‐affinity HSP90 inhibitor, which is currently being clinically tested, has shown activity against a wide array of tumor cell lines, including lung cancer cell lines. This inhibitor has induced the degradation of specific HSP90 client proteins for up to 7 days in tumor cell lines in vitro. The primary driver of cell growth (mutant epidermal growth factor receptors) was particularly sensitive to HSP90 inhibition. The long duration of client protein knockdown and suppression of phospho‐signaling seen in vitro after treatment with AT13387 was also apparent in vivo, with client proteins and phospho‐signaling suppressed for up to 72 h in xenograft tumors after treatment with a single dose of AT13387. Pharmacokinetic analyses indicated that while AT13387 was rapidly cleared from blood, its retention in tumor xenografts was markedly extended, and it was efficacious in a range of xenograft models. AT13387's long duration of action enabled, in particular, its efficacious once weekly administration in human lung carcinoma xenografts. The use of longer‐acting HSP90 inhibitors, such as AT13387, on less frequent dosing regimens has the potential to maintain antitumor efficacy as well as minimize systemic exposure and unwanted effects on normal tissues. (Cancer Sci, 2012; 103: 522–527)

Cyclin-dependent kinases (CDK), and their regulatory cyclin partners, play a central role in eukaryotic cell growth, division, and death. This key role in cell cycle progression, as well as their deregulation in several human cancers, makes them attractive therapeutic targets in oncology. A series of CDK inhibitors was developed using Astex's fragment-based medicinal chemistry approach, linked to high-throughput X-ray crystallography. A compound from this series, designated AT7519, is currently in early-phase clinical development. We describe here the biological characterization of AT7519, a potent inhibitor of several CDK family members. AT7519 showed potent antiproliferative activity (40-940 nmol/L) in a panel of human tumor cell lines, and the mechanism of action was shown here to be consistent with the inhibition of CDK1 and CDK2 in solid tumor cell lines. AT7519 caused cell cycle arrest followed by apoptosis in human tumor cells and inhibited tumor growth in human tumor xenograft models. Tumor regression was observed following twice daily dosing of AT7519 in the HCT116 and HT29 colon cancer xenograft models. We show that these biological effects are linked to inhibition of CDKs in vivo and that AT7519 induces tumor cell apoptosis in these xenograft models. AT7519 has an attractive biological profile for development as a clinical candidate, and the tolerability and efficacy in animal models compare favorably with other CDK inhibitors in clinical development. Studies described here formed the biological rationale for investigating the potential therapeutic benefit of AT7519 in cancer patients. [Mol Cancer Ther 2009;8(2):324–32]

Here, we describe the identification of a clinical candidate via structure-based optimization of a ligand efficient pyrazole-benzimidazole fragment. Aurora kinases play a key role in the regulation of mitosis and in recent years have become attractive targets for the treatment of cancer. X-ray crystallographic structures were generated using a novel soakable form of Aurora A and were used to drive the optimization toward potent (IC(50) approximately 3 nM) dual Aurora A/Aurora B inhibitors. These compounds inhibited growth and survival of HCT116 cells and produced the polyploid cellular phenotype typically associated with Aurora B kinase inhibition. Optimization of cellular activity and physicochemical properties ultimately led to the identification of compound 16 (AT9283). In addition to Aurora A and Aurora B, compound 16 was also found to inhibit a number of other kinases including JAK2 and Abl (T315I). This compound demonstrated in vivo efficacy in mouse xenograft models and is currently under evaluation in phase I clinical trials.

The application of fragment-based screening techniques to cyclin dependent kinase 2 (CDK2) identified multiple (>30) efficient, synthetically tractable small molecule hits for further optimization. Structure-based design approaches led to the identification of multiple lead series, which retained the key interactions of the initial binding fragments and additionally explored other areas of the ATP binding site. The majority of this paper details the structure-guided optimization of indazole (6) using information gained from multiple ligand−CDK2 cocrystal structures. Identification of key binding features for this class of compounds resulted in a series of molecules with low nM affinity for CDK2. Optimisation of cellular activity and characterization of pharmacokinetic properties led to the identification of 33 (AT7519), which is currently being evaluated in clinical trials for the treatment of human cancers.

Accurate and efficient computation of sensitivities are critical to the success of implementing gradient based optimization algorithms for large-scale, multi-disciplinary design problems. Traditional methods for computing design sensitivities, such as the finite difference, complex step, discrete semi-analytic, and discrete analytic methods often yield unsatisfactory results. Presented here is a local continuum shape sensitivity method with spatial gradient reconstruction. This method is accurate, efficient, and easy to implement. Most importantly, it is formulated as a general approach to sensitivity analysis, which makes it amenable to use with black box analyses. The method has previously been implemented for beam models, and here it is implemented for linear static bending of rectangular stiffened plate models. Among the examples presented are rectangular plates analysed with a variety of pressure loads, boundary conditions, plate theories, and finite element formulations. The final implementation is for a beam-stiffened rectangular plate, which is representative of a built-up structure. The local continuum sensitivity solutions are compared to either analytically derived sensitivities or finite difference sensitivities.

Accurate modeling of the aeroelastic response due t o gust loads is becoming a design necessity for many novel concepts. Both the high a spect ratio wings of high altitude long endurance (HALE) aircraft and flexible membrane wings of micro aerial vehicles (MAVs) are susceptible to large deformations, geometric no nlinearities, and rigid body motion when gust loads are encountered. Using gradient based o ptimization for aeroelastic shape design of such configurations can be computationally expensive. Furthermore, multidisciplinary design optimization frameworks that use gradient-based algorithms are becoming increasingly popular. A major challenge in developing such frameworks is implementing accurate and efficient sensitivity analyses for the various disciplines. This becomes especially challenging when a “black box” analysis tool is used and access to the source code is not possible. Presented in this paper is a cont inuum shape sensitivity method which does not require access to or knowledge of source code f ormulation. A static nonlinear beam bending problem and a static linear plate bending p roblem are presented as benchmarks. In addition, continuum shape sensitivities are compute d for an aeroelastic gust response of both a one- and two-dimensional structure. In all cases , the analysis tool is treated as a “black box”.

Pulmonary arterial hypertension (PAH) is characterized by increasing pulmonary vascular resistance leading to right heart failure and death. Sitaxentan (Thelin®) demonstrated efficacy in adult PAH; however, PAH therapy for children is critically needed. To support development for pediatric patients, sitaxentan (10, 30, or 60mg/kg/day) toxicity was assessed in juvenile (postnatal day 22-14weeks) rats. Sitaxentan did not affect survival, clinical signs, or body weight; no target organ of toxicity was identified. Hematologic changes were decreased erythrocyte parameters, prothrombin time, and activated partial thromboplastin time. Reproductive development and function in both sexes was unaffected, as assessed by mating performance; fertility, estrous cyclicity, and maintenance of normal pregnancy up to mid-gestation; sperm count, morphology, and motility; and testicular changes. The no-observed-adverse-effect level (NOAEL) on reproductive development and function was 60mg/kg/day; for toxicity, the NOAEL was 30mg/kg/day (coagulation parameter changes). Sitaxentan did not adversely affect physical development, cognitive ability, or reproductive function at exposures that were 58- and 61-fold higher than those found in adults after therapeutic exposure (100mg/day). This study is discussed in the context of evolving European pediatric drug legislation and guidance.

Anidulafungin, an echinocandin, is currently approved for treatment of fungal infections in adults. There is a high unmet medical need for treatment of fungal infections in neonatal patients, who may be at higher risk of infections involving bone, brain, and heart tissues. This in vivo preclinical study investigated anidulafungin distribution in plasma, bone, brain, and heart tissues in neonatal rats. Postnatal day (PND) 4 and PND 8 Fischer (F344/DuCrl) rats were dosed subcutaneously once with anidulafungin (10 mg/kg) or once daily for 5 days (PND 4-8). Plasma and tissue samples were collected and anidulafungin levels were measured by liquid chromatography-tandem mass spectrometry. The mean plasma Cmax and AUC0-24 values were consistent with single-dose plasma pharmacokinetics (dose normalized) reported previously for adult rats. Observed bone concentrations were similar to plasma concentrations regardless of dosing duration, with bone-to-plasma concentration ratios of approximately 1.0. Heart concentrations were higher than plasma, with heart to plasma concentration ratios of 1.3- to 1.8-fold. Brain concentrations were low after single dose, with brain-to-plasma concentration ratio of approximately 0.23, but increased to approximately 0.71 after 5 days of dosing. Tissue concentrations were nearly identical after single-dose administration in both PND 4 and PND 8 animals, indicating that anidulafungin does not appear to differentially distribute in this period in neonatal rats. In conclusion, anidulafungin distributes to bone, brain, and heart tissues of neonatal rats; such results are supportive of further investigation of efficacy against infections involving bone, brain, and heart tissues.

Anidulafungin, an echinocandin, is currently approved for treatment of fungal infections in adults. There is a high unmet medical need for treatment of fungal infections in neonatal patients, who may be at higher risk of infections involving bone, brain, and heart tissues. This in vivo preclinical study investigated anidulafungin distribution in plasma, bone, brain, and heart tissues in neonatal rats. Postnatal day (PND) 4 and PND 8 Fischer (F344/DuCrl) rats were dosed subcutaneously once with anidulafungin (10 mg/kg) or once daily for 5 days (PND 4-8). Plasma and tissue samples were collected and anidulafungin levels were measured by liquid chromatography-tandem mass spectrometry. The mean plasma Cmax and AUC0-24 values were consistent with single-dose plasma pharmacokinetics (dose normalized) reported previously for adult rats. Observed bone concentrations were similar to plasma concentrations regardless of dosing duration, with bone-to-plasma concentration ratios of approximately 1.0. Heart concentrations were higher than plasma, with heart to plasma concentration ratios of 1.3- to 1.8-fold. Brain concentrations were low after single dose, with brain-to-plasma concentration ratio of approximately 0.23, but increased to approximately 0.71 after 5 days of dosing. Tissue concentrations were nearly identical after single-dose administration in both PND 4 and PND 8 animals, indicating that anidulafungin does not appear to differentially distribute in this period in neonatal rats. In conclusion, anidulafungin distributes to bone, brain, and heart tissues of neonatal rats; such results are supportive of further investigation of efficacy against infections involving bone, brain, and heart tissues.

BACKGROUND: Anidulafungin, an echinocandin antifungal marketed for adult use, is being considered for use in pediatric populations, including neonates. The evolution of the nonclinical pediatric safety strategy for anidulafungin serves as an example of case-by-case negotiation through the European Medicines Agency pediatric investigation plan process, resulting in an acceptable juvenile rat toxicity study. METHODS: Study design challenges included animal selection, route, dose, age, and duration of dosing in relation to brain maturity, and appropriate study endpoints. The definitive study consisted of subcutaneous dosing at 0, 3, 10, and 30 mg/kg/day from postnatal day 4 to 62 (preterm infant to adulthood) with a 5-week recovery period. Study endpoints evaluated the potential for increased juvenile sensitivity to liver toxicity (seen in adults) and for novel toxicities in the central nervous system. RESULTS: Anidulafungin-related effects included slightly reduced body weight, increased liver weight, and a mild decrease in red blood cell mass with increased reticulocyte count. There was no liver pathology and in the posttreatment phase there were no effects on neurological function. Following recovery, effects on body weight, hematology, and liver weight were reversing or reversed. CONCLUSIONS: Therefore, the juvenile rat no-adverse-effect-level was 30 mg/kg/day. Exposures at this dose are similar to those achieved at the adult rat no-adverse-effect-level, suggesting that the juvenile rat is no more sensitive to anidulafungin than the adult rat. In conclusion, dialog and negotiation between the sponsor and the European Medicines Agency allowed for successful execution of a nonclinical safety strategy that enabled further clinical investigation of anidulafungin in pediatric populations. Birth Defects Res (Part B) 92:333–344, 2011. © 2011 Wiley-Liss, Inc.

Historically, the study of drug metabolism [1] in the pharmaceutical industry has of necessity concentrated on the practical issues concerning the toxicological response of selected animal species to candidate drug compounds, and a balancing of the presence of metabolites in one species against those found in another. The responses in terms of metabolite production have been compared to those expected or demonstrated in man. Although understanding the mechanism underlying metabolite formation was erstwhile the province of the academic world, advances in technology, especially molecular biology of the cytochrome P450 (CYP) enzymes, have significantly influenced the pragmatic industrial approach. It is now widely recognized that in vitro screening for metabolism and/or specific CYP interactions can foster a rational approach to the selection of drug molecules.