Prospective Validation of Machine Learning Algorithms for Absorption, Distribution, Metabolism, and Excretion Prediction: An Industrial Perspective.

Absorption, distribution, metabolism, and excretion (ADME), which collectively define the concentration profile of a drug at the site of action, are of critical importance to the success of a drug candidate. Recent advances in machine learning algorithms and the availability of larger proprietary as well as public ADME data sets have generated renewed interest within the academic and pharmaceutical science communities in predicting pharmacokinetic and physicochemical endpoints in early drug discovery. In this study, we collected 120 internal prospective data sets over 20 months across six ADME in vitro endpoints: human and rat liver microsomal stability, MDR1-MDCK efflux ratio, solubility, and human and rat plasma protein binding. A variety of machine learning algorithms in combination with different molecular representations were evaluated. Our results suggest that gradient boosting decision tree and deep learning models consistently outperformed random forest over time. We also observed better performance when models were retrained on a fixed schedule, and the more frequent retraining generally resulted in increased accuracy, while hyperparameters tuning only improved the prospective predictions marginally.

Depigmented-polymerised allergoids favour regulatory over effector T cells: enhancement by 1α, 25-dihydroxyvitamin D3.

BACKGROUND: Allergen immunotherapy (SIT) is the only treatment for allergic disease capable of modifying disease long term. To reduce the risk of anaphylaxis from SIT, allergen-extracts have been modified by polymerisation with glutaraldehyde to reduce IgE binding. It is suggested that these allergoid extracts also have reduced T cell activity, which could compromise clinical efficacy. Effective SIT is thought to act through regulatory T cells (Tregs) rather than activation of effector T cells. There is no published data on the activity of modified extracts on Tregs. RESULTS: We compared the capacity of modified (depigmented-polymerised) versus unmodified (native) allergen extracts of grass pollen and house dust mite to stimulate proliferation/cytokine production and to modulate Treg/effector T cell frequency in cultures of peripheral blood mononuclear cells (PBMC), from volunteers sensitised to both allergens in vitro. Depigmented-polymerised allergen extracts stimulated less proliferation of PBMC, and reduced effector cell numbers after 7 days in culture than did native extracts. However, the frequency of Foxp3+ Tregs in cultures were similar to those seen with native extract so that ratios of regulatory to effector T cells were significantly increased in cultures stimulated with depigmented-polymerised extracts. Addition of 1α, 25-dihydroxyvitamin D3 further favoured Treg, and reduced effector cytokine production, but not interleukin-10. CONCLUSIONS: Depigmented-polymerised allergen extracts appear to favour Treg expansion over activation of effector T cells and this may relate to their demonstrated efficacy and safety in SIT. 1α, 25-dihydroxyvitamin D3 further reduces effector T cell activation by allergen extracts and may be a useful adjuvant for SIT.

Defective IL-10 expression and in vitro steroid-induced IL-17A in paediatric severe therapy-resistant asthma.

BACKGROUND: Understanding of immune mechanisms underpinning asthma has emerged from studies in adults. It is increasingly recognised, both immunologically and in the development of novel therapies, that adult responses cannot be used accurately to predict those of children. METHODS: Using a well-defined paediatric cohort of severe therapy-resistant asthma (STRA) patients, we investigated cytokine profiles in the airway by analysis of bronchoalveolar lavage fluid. The in vitro capacity of peripheral blood mononuclear cells (PBMCs) for cytokine production was also assessed following polyclonal T cell activation in culture, in the absence or presence of dexamethasone and 1α,25-dihydroxyvitamin D3. RESULTS: Children with both moderate and STRA had significantly diminished levels of anti-inflammatory interleukin (IL)-10 in airway lavage samples when compared with non-asthmatic controls (p<0.001). Their PBMCs also demonstrated significantly impaired capacity to secrete IL-10 in culture (p<0.001). Dexamethasone regulated the balance between PBMC IL-10 and IL-13 production, increasing IL-10 secretion (p<0.001) and decreasing IL-13 (p<0.001) but unexpectedly enhanced IL-17A production in all groups-most strikingly in the STRA cohort (p<0.001). The inclusion of the active form of vitamin D, 1α,25-dihydroxyvitamin D3, in culture enhanced dexamethasone-induced IL-10 (p<0.05) without marked effects on IL-13 or IL-17A production. Furthermore, systemic vitamin D status directly correlated with airway IL-10 (r=0.6, p<0.01). CONCLUSIONS: These findings demonstrate reduced peripheral and local IL-10 synthesis in paediatric asthma, and support therapeutic augmentation of low circulating vitamin D in severe, difficult-to-treat asthma, in order to correct impaired IL-10 levels. Conversely, steroids enhanced IL-17A levels, and therefore any steroid-sparing properties of vitamin D may have additional benefit in STRA.

Enhanced production of IL-17A in patients with severe asthma is inhibited by 1α,25-dihydroxyvitamin D3 in a glucocorticoid-independent fashion.

BACKGROUND: TH17 cells are proposed to play a role in the pathology of asthma, including steroid-resistant (SR) disease. We previously identified a steroid-enhancing function of vitamin D in patients with SR asthma in restoring the impaired response to steroids for production of the anti-inflammatory cytokine IL-10. OBJECTIVE: We sought to investigate the production of the TH17-associated cytokines IL-17A and IL-22 in culture in patients with moderate-to-severe asthma defined on the basis of their clinical response to steroids and the susceptibility of this response to inhibition by steroids and the active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25[OH]2D3). METHODS: PBMCs were stimulated in culture with or without dexamethasone and 1,25(OH)2D3. A cytometric bead array, ELISA, and intracellular cytokine staining were used to assess cytokine production. The role of CD39 in inhibition of the TH17 response was studied by using quantitative real-time PCR, flow cytometry, and addition of the antagonist POM-1 to culture. RESULTS: Asthmatic patients synthesized much higher levels of IL-17A and IL-22 than nonasthmatic control subjects, with patients with SR asthma expressing the highest levels of IL-17A. Glucocorticoids did not inhibit IL-17A cytokine expression in patients and enhanced production in cultures from control subjects. Treatment with 1,25(OH)2D3 with or without dexamethasone significantly reduced both IL-17A and IL-22 levels. An antagonist of the ectonucleotidase CD39 reversed 1,25(OH)2D3-mediated inhibition of the IL-17A response. CONCLUSION: Patients with severe asthma exhibit increased levels of TH17 cytokines, which are not inhibited by steroids. 1,25(OH)2D3 inhibits TH17 cytokine production in all patients studied, irrespective of their clinical responsiveness to steroids, identifying novel steroid-enhancing properties of vitamin D in asthmatic patients.

Cerebrospinal fluid can be used as a surrogate to assess brain exposures of breast cancer resistance protein and P-glycoprotein substrates.

The objectives of the study were to characterize the selectivity of dantrolene to breast cancer resistance protein (Bcrp) and to evaluate whether cerebrospinal fluid (CSF) can be used as a surrogate to assess brain exposures of BCRP and P-glycoprotein (Pgp) substrates. The impact of Bcrp and Pgp on dantrolene exposures in brain and CSF was examined in Bcrp and Mdr1a/1b knockout mice and was further investigated in wild-type mice in the presence of the Bcrp inhibitor (3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1',2':1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester (Ko143), the Pgp inhibitor 6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-l-valine-cyclosporine A (PSC833), and the dual inhibitor N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). The effect of Bcrp and Pgp on digoxin exposures in brain and CSF was investigated in wild-type mice in the presence of the inhibitors. In vivo studies showed dantrolene exposures in brain and CSF, but not the blood, increased in Bcrp(-/-) and Mdr1a/1b(-/-)/Bcrp(-/-) mice, or in the presence of the Bcrp inhibitors Ko143 or GF120918. Inhibition of Pgp by GF120918 and PSC833 significantly increased digoxin exposures in brain, CSF, and blood to a lesser extent. Results from the present study demonstrated that inhibition of Bcrp and Pgp increased not only the exposures of dantrolene and digoxin in brain, but also the exposures in CSF. In addition, the change of exposures in CSF reflected the changes in brain. The present study strongly suggests that the dantrolene and digoxin exposures in CSF are primarily determined by the rapid transport from brain to CSF, and inhibition of Bcrp and Pgp exhibits little impact on using CSF as surrogates to assess brain exposures of Bcrp and Pgp substrates.

Synthesis, SAR and biological evaluation of 1,6-disubstituted-1H-pyrazolo[3,4-d]pyrimidines as dual inhibitors of Aurora kinases and CDK1.

Since the early 2000s, the Aurora kinases have become major targets of oncology drug discovery particularly Aurora-A and Aurora-B kinases (AKA/AKB) for which the selective inhibition in cells lead to different phenotypes. In addition to targeting these Aurora kinases involved in mitosis, CDK1 has been added as a primary inhibition target in hopes of enhancing the cytotoxicity of our chemotypes harboring the pyrazolopyrimidine core. SAR optimization of this series using the AKA, AKB and CDK1 biochemical assays led to the discovery of the compound 7h which combines strong potency against the 3 kinases with an acceptable microsomal stability. Finally, switching from a primary amide to a two-substituted pyrrolidine amide gave rise to compound 15a which exhibited the desired AKA/CDK1 inhibition phenotype in cells but showed moderate activity in animal models using HCT116 tumor cell lines.

Discovery and Preclinical Characterization of BIIB091, a Reversible, Selective BTK Inhibitor for the Treatment of Multiple Sclerosis.

Multiple Sclerosis is a chronic autoimmune neurodegenerative disorder of the central nervous system (CNS) that is characterized by inflammation, demyelination, and axonal injury leading to permeant disability. In the early stage of MS, inflammation is the primary driver of the disease progression. There remains an unmet need to develop high efficacy therapies with superior safety profiles to prevent the inflammation processes leading to disability. Herein, we describe the discovery of BIIB091, a structurally distinct orthosteric ATP competitive, reversible inhibitor that binds the BTK protein in a DFG-in confirmation designed to sequester Tyr-551, an important phosphorylation site on BTK, into an inactive conformation with excellent affinity. Preclinical studies demonstrated BIB091 to be a high potency molecule with good drug-like properties and a safety/tolerability profile suitable for clinical development as a highly selective, reversible BTKi for treating autoimmune diseases such as MS.

Stereochemistry-activity relationship of orally active tetralin S1P agonist prodrugs.

Modifying FTY720, an immunosuppressant modulator, led to a new series of well phosphorylated tetralin analogs as potent S1P1 receptor agonists. The stereochemistry effect of tetralin ring was probed, and (-)-(R)-2-amino-2-((S)-6-octyl-1,2,3,4-tetrahydronaphthalen-2-yl)propan-1-ol was identified as a good SphK2 substrate and potent S1P1 agonist with good oral bioavailability.

2-Aminoimidazoles inhibitors of TGF-beta receptor 1.

The 4-(5-fluoro-6-methyl-pyridin-2-yl)-5-quinoxalin-6-yl-1H-imidazol-2-ylamine 3 is a potent and selective inhibitor of TGF-betaR1. Substitution of the amino group of 3 typically led to a slight decrease in the affinity for the receptor and in TGF-beta-inducted PAI-luciferase reporter activity. However, 2-acetamidoimidazoles were identified as attractive candidates for further optimization as a result of their significant activity combined to their superior pharmacokinetic profile.

Differential Effects of Depleting versus Programming Tumor-Associated Macrophages on Engineered T Cells in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to therapies, including immune-checkpoint blockade. We investigated two distinct strategies to modulate tumor-associated macrophages (TAM) to enhance cellular therapy targeting mesothelin in an autochthonous PDA mouse model. Administration of an antibody to colony-stimulating factor (anti-Csf1R) depleted Ly6Clow protumorigenic TAMs and significantly enhanced endogenous T-cell intratumoral accumulation. Despite increasing the number of endogenous T cells at the tumor site, as previously reported, TAM depletion had only minimal impact on intratumoral accumulation and persistence of T cells engineered to express a murine mesothelin-specific T-cell receptor (TCR). TAM depletion interfered with the antitumor activity of the infused T cells in PDA, evidenced by reduced tumor cell apoptosis. In contrast, TAM programming with agonistic anti-CD40 increased both Ly6Chigh TAMs and the intratumoral accumulation and longevity of TCR-engineered T cells. Anti-CD40 significantly increased the frequency and number of proliferating and granzyme B+ engineered T cells, and increased tumor cell apoptosis. However, anti-CD40 failed to rescue intratumoral engineered T-cell IFNγ production. Thus, although functional modulation, rather than TAM depletion, enhanced the longevity of engineered T cells and increased tumor cell apoptosis, ultimately, anti-CD40 modulation was insufficient to rescue key effector defects in tumor-reactive T cells. This study highlights critical distinctions between how endogenous T cells that evolve in vivo, and engineered T cells with previously acquired effector activity, respond to modifications of the tumor microenvironment.

Developmental changes in upper airway dynamics.

Normal children have a less collapsible upper airway in response to subatmospheric pressure administration (P(NEG)) during sleep than normal adults do, and this upper airway response appears to be modulated by the central ventilatory drive. Children have a greater ventilatory drive than adults. We, therefore, hypothesized that children have increased neuromotor activation of their pharyngeal airway during sleep compared with adults. As infants have few obstructive apneas during sleep, we hypothesized that infants would have an upper airway that was resistant to collapse. We, therefore, compared the upper airway pressure-flow (V) relationship during sleep between normal infants, prepubertal children, and adults. We evaluated the upper airway response to 1). intermittent, acute P(NEG) (infants, children, and adults), and 2). hypercapnia (children and adults). We found that adults had a more collapsible upper airway during sleep than either infants or children. The children exhibited a vigorous response to both P(NEG) and hypercapnia during sleep (P < 0.01), whereas adults had no significant change. Infants had an airway that was resistant to collapse and showed a very rapid response to P(NEG). We conclude that the upper airway is resistant to collapse during sleep in infants and children. Normal children have preservation of upper airway responses to P(NEG) and hypercapnia during sleep, whereas responses are diminished in adults. Infants appear to have a different pattern of upper airway activation than older children. We speculate that the pharyngeal airway responses present in normal children are a compensatory response for a relatively narrow upper airway.

Proof of principle and first cases using preimplantation genetic haplotyping--a paradigm shift for embryo diagnosis.

Preimplantation genetic haplotyping (PGH) proof-of-principle was demonstrated by multiple displacement amplification (MDA) of single buccal cells from a female donor and genotyping using 12 polymorphic markers within the dystrophin gene; the known paternal genotype enabled identification of the paternal haplotype in the MDA products despite 27% allele dropout. MDA amplified DNA from 49 single human blastomeres with 100% success. The MDA products were genotyped using a total of 57 polymorphic markers for chromosomes 1, 7, 13, 18, 21, X and Y; 72% of alleles amplified providing results at 90% of the loci tested. A PGH cycle was carried out for Duchenne muscular dystrophy. One embryo was biopsied: PGH showed a non-carrier female, which was transferred with no resulting pregnancy. A PGH cycle was carried out for cystic fibrosis. Seven embryos were biopsied and PGH allowed the exclusion of 2 affected embryos; a carrier and a non-carrier embryo were transferred resulting in an on-going twin pregnancy. PGH represents a paradigm shift in embryo diagnosis, as one panel of markers can be used for all carriers of the same monogenic disease, bypassing the need for development of mutation-specific tests, and widening the scope and availability of preimplantation genetic testing.

Comparison of oxycodone and hydrocodone for the treatment of acute pain associated with fractures: a double-blind, randomized, controlled trial.

BACKGROUND: Previous studies have demonstrated the efficacy of oxycodone and hydrocodone for the treatment of acute pain. However, to the best of the authors' knowledge, no previous reports have compared the efficacies of these commonly prescribed agents. OBJECTIVES: To compare the efficacies of oxycodone and hydrocodone for the treatment of acute pain associated with fractures in emergency department (ED) patients. METHODS: This prospective, double-blind, randomized, controlled trial was conducted at an urban trauma center with an annual census of 65,000. Eligible participants included ED patients over the age of 12 years with fractures who consented to participate. Subjects were randomized to receive either oxycodone (5 mg orally [po]) with acetaminophen, or hydrocodone (5 mg po) with acetaminophen. Measurements included demographic information; pain scores on a verbal numeric rating scale at baseline and at 30 and 60 minutes; vital signs at baseline and at 30 and 60 minutes; and adverse effects. Ninety-five-percent confidence intervals (95% CIs) constructed about means and proportions were used to assess differences between the oxycodone and hydrocodone groups in analgesic efficacy and side effects. RESULTS: Seventy-three subjects were randomized to receive oxycodone or hydrocodone. Sixty-seven subjects completed the ED study period (n = 35, oxycodone; n = 32, hydrocodone). There was no difference between the two groups in age, weight, gender, ethnicity, diagnoses, baseline pain scores, or vital signs. Patients in both groups had pain relief from baseline to 30 minutes (oxycodone mean change 3.7, 95% CI = 2.9 to 4.6; hydrocodone mean change 2.5, 95% CI = 1.7 to 3.3), and from baseline to 60 minutes (oxycodone mean change 4.4, 95% CI = 3.2 to 5.6; hydrocodone mean change 3.0, 95% CI = 2.1 to 3.9). There was no difference in pain between the patients treated with oxycodone and hydrocodone at 30 minutes (mean difference between groups -0.6, 95% CI = -1.8 to 0.5) or at 60 minutes (mean difference -0.5, 95% CI = -2.0 to 1.0). There was no difference between the groups in nausea, vomiting, itching, or drowsiness; however, the hydrocodone patients had a higher incidence of constipation (oxycodone 0%, hydrocodone 21%, difference in proportions 21%, 95% CI = 3% to 39% more with hydrocodone). CONCLUSIONS: Treatment with acetaminophen and either oxycodone, 5 mg po, or hydrocodone, 5 mg po, resulted in pain relief among ED patients with acute fractures, and there was no difference between the two agents at 30 and 60 minutes. Adverse effect profiles were similar, with the exception of a higher incidence of subsequent constipation with the use of hydrocodone. These results suggest that oxycodone and hydrocodone have similarly potent analgesic effects in the first hour of treatment for ED patients with acute fractures.

Upper airway dynamic responses in children with the obstructive sleep apnea syndrome.

Normal children have a smaller upper airway than adults, but, nevertheless, snore less and have less apnea. We have previously shown that normal children have an upper airway that is resistant to collapse during sleep. We hypothesized that this resistance to collapse is due to preservation of upper airway neuromotor responses during sleep. Furthermore, we hypothesized that upper airway responses would be diminished in children with the obstructive sleep apnea syndrome (OSAS). We therefore compared the upper airway pressure-flow relationship during sleep between children with OSAS and controls. Measurements were made by correlating maximal inspiratory airflow with the level of nasal pressure applied via a mask. Neuromotor upper airway activation was assessed by evaluating the upper airway response to 1) hypercapnia and 2) intermittent, acute negative pressure. We found that children with OSAS had no significant response to either hypercapnia or negative pressure during sleep, compared with the normal children. After treatment of OSAS by tonsillectomy and adenoidectomy, there was a trend for normalization of upper airway responses. We conclude that upper airway dynamic responses are decreased in children with OSAS but recover after treatment. We speculate that the pharyngeal airway neuromotor responses present in normal children are a compensatory response for a relatively narrow upper airway. Further, we speculate that this compensatory response is lacking in children with OSAS, most likely due to either habituation to chronic respiratory abnormalities during sleep or to mechanical damage to the upper airway.

Generation of a human embryonic stem cell line encoding the cystic fibrosis mutation deltaF508, using preimplantation genetic diagnosis.

Human embryonic stem (hES) cells are pluripotent cells isolated from early human embryos. They can be grown in vitro and made to differentiate into many different cell types. These properties have suggested that they may be useful in cell replacement therapy for many degenerative diseases. However, if hES cells could also be manufactured with mutations significant in human disease, they could provide a powerful in-vitro tool for modelling disease processes and progression in a number of different cell types, as well as providing an ideal system for studying in-vitro toxicity and efficacy of drugs and other therapeutic systems such as gene therapy. Embryos with such mutations are generated as part of routine genetic testing during preimplantation genetic diagnosis, providing the opportunity to generate cell lines with significant mutations. A human embryonic stem cell line homozygous for the most common mutation leading to cystic fibrosis in humans (delta F508) has been generated and characterized. This cell line has the same morphology and expresses proteins typical of other unaffected hES cell lines. This cell line represents an important in-vitro tool for understanding the pathophysiology of cystic fibrosis, and presents exciting opportunities to test the efficacy and toxicity of new therapies relevant to CF.

Pulse transit time as a measure of arousal and respiratory effort in children with sleep-disordered breathing.

The upper airway resistance syndrome (UARS) is associated with neurobehavioral morbidity in children. The diagnostic gold standard for UARS is esophageal manometry. However, this is invasive. Furthermore, upper airway obstructive events in sleeping children frequently terminate without visible electrocortical (EEG) arousal. The pulse transit time (PTT) is a noninvasive marker of blood pressure and, therefore, subcortical arousal. Blood pressure elevation, associated with respiratory arousal from sleep, results in a drop in the PTT. We hypothesized that: 1) the PTT is a more sensitive measure of respiratory arousal than EEG; and 2) the PTT arousal index can distinguish children with UARS from those with primary snoring. Polysomnography, including esophageal manometry and PTT, was measured prospectively in 24 symptomatic children and 10 normal controls. Apnea, hypopnea, and respiratory effort-related arousal events terminated in a PTT arousal 91%, 83%, and 80% of the time, and in an EEG arousal in 55%, 51%, and 43% (all p < 0.05), respectively. The PTT arousal index was significantly greater in children with UARS (6.8 events/h) than primary snoring (2.2 events/h) (p < 0.05). We conclude that, in children, PTT arousals are a more sensitive measure of obstructive events than visible EEG arousals.