Emerging effects of tryptophan pathway metabolites and intestinal microbiota on metabolism and intestinal function.

The metabolism of dietary tryptophan occurs locally in the gut primarily via host enzymes, with ~ 5% metabolized by gut microbes. Three major tryptophan metabolic pathways are serotonin (beyond the scope of this review), indole, kynurenine and related derivatives. We introduce the gut microbiome, dietary tryptophan and the potential interplay of host and bacterial enzymes in tryptophan metabolism. Examples of bacterial transformation to indole and its derivative indole-3 propionic acid demonstrate associations with human metabolic disease and gut permeability, although causality remains to be determined. This review will focus on less well-known data, suggestive of local generation and functional significance in the gut, where kynurenine is converted to kynurenic acid and xanthurenic acid via enzymatic action present in both host and bacteria. Our functional data demonstrate a limited effect on intestinal epithelial cell monolayer permeability and on healthy mouse ileum. Other data suggest a modulatory effect on the microbiome, potentially in pathophysiology. Supportive of this, we found that the expression of mRNA for three kynurenine pathway enzymes were increased in colon from high-fat-fed mice, suggesting that this host pathway is perturbed in metabolic disease. These data, along with that from bacterial genomic analysis and germ-free mice, confirms expression and functional machinery of enzymes in this pathway. Therefore, the host and microbiota may play a significant dual role in either the production or regulation of these kynurenine metabolites which, in turn, can influence both host and microbiome, especially in the context of obesity and intestinal permeability.

Impaired glucose partitioning in primary myotubes from severely obese women with type 2 diabetes.

The purpose of this study was to determine whether intramyocellular glucose partitioning was altered in primary human myotubes derived from severely obese women with type 2 diabetes. Human skeletal muscle cells were obtained from lean nondiabetic and severely obese Caucasian females with type 2 diabetes [body mass index (BMI): 23.6 ± 2.6 vs. 48.8 ± 1.9 kg/m2, fasting glucose: 86.9 ± 1.6 vs. 135.6 ± 12.0 mg/dL, n = 9/group]. 1-[14C]-Glucose metabolism (glycogen synthesis, glucose oxidation, and nonoxidized glycolysis) and 1- and 2-[14C]-pyruvate oxidation were examined in fully differentiated myotubes under basal and insulin-stimulated conditions. Tricarboxylic acid cycle intermediates were determined via targeted metabolomics. Myotubes derived from severely obese individuals with type 2 diabetes exhibited impaired insulin-mediated glucose partitioning with reduced rates of glycogen synthesis and glucose oxidation and increased rates of nonoxidized glycolytic products, when compared with myotubes derived from the nondiabetic individuals (P < 0.05). Both 1- and 2-[14C]-pyruvate oxidation rates were significantly blunted in myotubes from severely obese women with type 2 diabetes compared with myotubes from the nondiabetic controls. Lastly, concentrations of tricarboxylic acid cycle intermediates, namely, citrate (P < 0.05), cis-aconitic acid (P = 0.07), and α-ketoglutarate (P < 0.05), were lower in myotubes from severely obese women with type 2 diabetes. These data suggest that intramyocellular insulin-mediated glucose partitioning is intrinsically altered in the skeletal muscle of severely obese women with type 2 diabetes in a manner that favors the production of glycolytic end products. Defects in pyruvate dehydrogenase and tricarboxylic acid cycle may be responsible for this metabolic derangement associated with type 2 diabetes.

Intestinal serine protease inhibition increases FGF21 and improves metabolism in obese mice.

Trypsin is the major serine protease responsible for intestinal protein digestion. An inhibitor, camostat (CS), reduced weight gain, hyperglycemia, and dyslipidemia in obese rats; however, the mechanisms for these are largely unknown. We reasoned that CS creates an apparent dietary protein restriction, which is known to increase hepatic fibroblast growth factor 21 (FGF21). Therefore, metabolic responses to CS and a gut-restricted CS metabolite, FOY-251, were measured in mice. Food intake, body weight, blood glucose, branched-chain amino acids (LC/MS), hormone levels (ELISA), liver pathology (histology), and transcriptional changes (qRT-PCR) were measured in ob/ob, lean and diet-induced obese (DIO) C57BL/6 mice. In ob/ob mice, CS in chow (9-69 mg/kg) or FOY-251 (46 mg/kg) reduced food intake and body weight gain to a similar extent as pair-fed mice. CS decreased blood glucose, liver weight, and lipidosis and increased FGF21 gene transcription and plasma levels. In lean mice, CS increased liver FGF21 mRNA and plasma levels. Relative to pair feeding, FOY-251 also increased plasma FGF21 and induced liver FGF21 and integrated stress response (ISR) transcription. In DIO mice, FOY-251 (100 mg/kg po) did not alter peak glucose levels but reduced the AUC of the glucose excursion in response to an oral glucose challenge. FOY-251 increased plasma FGF21 levels. In addition to previously reported satiety-dependent (cholecystokinin-mediated) actions, intestinal trypsin inhibition engages non-satiety-related pathways in both leptin-deficient and DIO mice. This novel mechanism improves metabolism by a liver-integrated stress response and increased FGF21 expression levels in mice. NEW & NOTEWORTHY Trypsin inhibitors, including plant-based consumer products, have long been associated with metabolic improvements. Studies in the 1980s and 1990s suggested this was due to satiety hormones and caloric wasting by loss of protein and fatty acids in feces. This work suggests an entirely new mechanism based on the lower amounts of digested protein available in the gut. This apparent protein reduction may cause beneficial metabolic adaptation by the intestinal-liver axis to perceived nutrient stress.

Altered tricarboxylic acid cycle flux in primary myotubes from severely obese humans.

BACKGROUND/OBJECTIVE: The partitioning of glucose toward glycolytic end products rather than glucose oxidation and glycogen storage is evident in skeletal muscle with severe obesity and type 2 diabetes. The purpose of the present study was to determine the possible mechanism by which severe obesity alters insulin-mediated glucose partitioning in human skeletal muscle. SUBJECTS/METHODS: Primary human skeletal muscle cells (HSkMC) were isolated from lean (BMI = 23.6 ± 2.6 kg/m2, n = 9) and severely obese (BMI = 48.8 ± 1.9 kg/m2, n = 8) female subjects. Glucose oxidation, glycogen synthesis, non-oxidized glycolysis, pyruvate oxidation, and targeted TCA cycle metabolomics were examined in differentiated myotubes under basal and insulin-stimulated conditions. RESULTS: Myotubes derived from severely obese subjects exhibited attenuated response of glycogen synthesis (20.3%; 95% CI [4.7, 28.8]; P = 0.017) and glucose oxidation (5.6%; 95% CI [0.3, 8.6]; P = 0.046) with a concomitant greater increase (23.8%; 95% CI [5.7, 47.8]; P = 0.004) in non-oxidized glycolytic end products with insulin stimulation in comparison to the lean group (34.2% [24.9, 45.1]; 13.1% [8.6, 16.4], and 2.9% [-4.1, 12.2], respectively). These obesity-related alterations in glucose partitioning appeared to be linked with reduced TCA cycle flux, as 2-[14C]-pyruvate oxidation (358.4 pmol/mg protein/min [303.7, 432.9] vs. lean 439.2 pmol/mg protein/min [393.6, 463.1]; P = 0.013) along with several TCA cycle intermediates, were suppressed in the skeletal muscle of severely obese individuals. CONCLUSIONS: These data suggest that with severe obesity the partitioning of glucose toward anaerobic glycolysis in response to insulin is a resilient characteristic of human skeletal muscle. This altered glucose partitioning appeared to be due, at least in part, to a reduction in TCA cycle flux.

Potent Sodium/Glucose Cotransporter SGLT1/2 Dual Inhibition Improves Glycemic Control Without Marked Gastrointestinal Adaptation or Colonic Microbiota Changes in Rodents.

The sodium/glucose cotransporters (SGLT1 and SGLT2) transport glucose across the intestinal brush border and kidney tubule. Dual SGLT1/2 inhibition could reduce hyperglycemia more than SGLT2-selective inhibition in patients with type 2 diabetes. However, questions remain about altered gastrointestinal (GI) luminal glucose and tolerability, and this was evaluated in slc5a1-/- mice or with a potent dual inhibitor (compound 8; SGLT1 Ki = 1.5 ± 0.5 nM 100-fold greater potency than phlorizin; SGLT2 Ki = 0.4 ± 0.2 nM). 13C6-glucose uptake was quantified in slc5a1-/- mice and in isolated rat jejunum. Urinary glucose excretion (UGE), blood glucose (Sprague-Dawley rats), glucagon-like peptide 1 (GLP-1), and hemoglobin A1c (HbA1c) levels (Zucker diabetic fatty rats) were measured. Intestinal adaptation and rRNA gene sequencing was analyzed in C57Bl/6 mice. The blood 13C6-glucose area under the curve (AUC) was reduced in the absence of SGLT1 by 75% (245 ± 6 vs. 64 ± 6 mg/dl⋅h in wild-type vs. slc5a1-/- mice) and compound 8 inhibited its transport up to 50% in isolated rat jejunum. Compound 8 reduced glucose excursion more than SGLT2-selective inhibition (e.g., AUC = 129 ± 3 vs. 249 ± 5 mg/dl⋅h for 1 mg/kg compound 8 vs. dapagliflozin) with similar UGE but a lower renal glucose excretion threshold. In Zucker diabetic fatty rats, compound 8 decreased HbA1c and increased total GLP-1 without changes in jejunum SGLT1 expression, mucosal weight, or villus length. Overall, compound 8 (1 mg/kg for 6 days) did not increase cecal glucose concentrations or bacterial diversity in C57BL/6 mice. In conclusion, potent dual SGLT1/2 inhibition lowers blood glucose by reducing intestinal glucose absorption and the renal glucose threshold but minimally impacts the intestinal mucosa or luminal microbiota in chow-fed rodents.

Synthesis and biological evaluation of benzocyclobutane-C-glycosides as potent and orally active SGLT1/SGLT2 dual inhibitors.

Synthesis and biological evaluation of benzocyclobutane-C-glycosides as potent and orally active SGLT1/SGLT2 dual inhibitors are described. Compound 19 showed high inhibitory potency at SGLT1 (IC50 = 45 nM), and excellent potency at SGLT2 (IC50 = 1 nM). It also displayed excellent PK profiles in mice, rats, dogs and monkeys (F = 78-107%). In SD rats, compound 19 treatments significantly reduced blood glucose levels in a dose-dependent manner. In ZDF rats, compound 19 displayed anti-hyperglycemic effect up to 24 h. Therefore, compound 19 may serve as valuable pharmacological tool, and potential use as a treatment for metabolic syndrome.

Intestinal SGLT1 in metabolic health and disease.

The Na(+)-glucose cotransporter 1 (SGLT1/SLC5A1) is predominantly expressed in the small intestine. It transports glucose and galactose across the apical membrane in a process driven by a Na(+) gradient created by Na(+)-K(+)-ATPase. SGLT2 is the major form found in the kidney, and SGLT2-selective inhibitors are a new class of treatment for type 2 diabetes mellitus (T2DM). Recent data from patients treated with dual SGLT1/2 inhibitors or SGLT2-selective drugs such as canagliflozin (SGLT1 IC50 = 663 nM) warrant evaluation of SGLT1 inhibition for T2DM. SGLT1 activity is highly dynamic, with modulation by multiple mechanisms to ensure maximal uptake of carbohydrates (CHOs). Intestinal SGLT1 inhibition lowers and delays the glucose excursion following CHO ingestion and augments glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) secretion. The latter is likely due to increased glucose exposure of the colonic microbiota and formation of metabolites such as L cell secretagogues. GLP-1 and PYY secretion suppresses food intake, enhances the ileal brake, and has an incretin effect. An increase in colonic microbial production of propionate could contribute to intestinal gluconeogenesis and mediate positive metabolic effects. On the other hand, a threshold of SGLT1 inhibition that could lead to gastrointestinal intolerability is unclear. Altered Na(+) homeostasis and increased colonic CHO may result in diarrhea and adverse gastrointestinal effects. This review considers the potential mechanisms contributing to positive metabolic and negative intestinal effects. Compounds that inhibit SGLT1 must balance the modulation of these mechanisms to achieve therapeutic efficacy for metabolic diseases.

Human and non-human primate intestinal FcRn expression and immunoglobulin G transcytosis.

PURPOSE: To evaluate transcytosis of immunoglobulin G (IgG) by the neonatal Fc receptor (FcRn) in adult primate intestine to determine whether this is a means for oral delivery of monoclonal antibodies (mAbs). METHODS: Relative regional expression of FcRn and localization in human intestinal mucosa by RT-PCR, ELISA & immunohistochemistry. Transcytosis of full-length mAbs (sandwich ELISA-based detection) across human intestinal segments mounted in Ussing-type chambers, human intestinal (caco-2) cell monolayers grown in transwells, and serum levels after regional intestinal delivery in isoflurane-anesthetized cynomolgus monkeys. RESULTS: In human intestine, there was an increasing proximal-distal gradient of mucosal FcRn mRNA and protein expression. In cynomolgus, serum mAb levels were greater after ileum-proximal colon infusion than after administration to stomach or proximal small intestine (1-5 mg/kg). Serum levels of wild-type mAb dosed into ileum/proximal colon (2 mg/kg) were 124 ± 104 ng/ml (n = 3) compared to 48 ± 48 ng/ml (n = 2) after a non-FcRn binding variant. In vitro, mAb transcytosis in polarized caco-2 cell monolayers and was not enhanced by increased apical cell surface IgG binding to FcRn. An unexpected finding in primate small intestine, was intense FcRn expression in enteroendocrine cells (chromagranin A, GLP-1 and GLP-2 containing). CONCLUSIONS: In adult primates, FcRn is expressed more highly in distal intestinal epithelial cells. However, mAb delivery to that region results in low serum levels, in part because apical surface FcRn binding does not influence mAb transcytosis. High FcRn expression in enteroendocrine cells could provide a novel means to target mAbs for metabolic diseases after systemic administration.

Identification of a dual δ OR antagonist/µ OR agonist as a potential therapeutic for diarrhea-predominant Irritable Bowel Syndrome (IBS-d).

A small set of acyclic analogs 5 were prepared to explore their structure-activity relationships (SARs) relative to heterocyclic core, opioid receptor (OR) agonists 4. Compound 5l was found to have very favorable OR binding affinities at the δ and µ ORs (r K(i) δ=1.3 nM; r K(i) µ=0.9 nM; h K(i) µ=1.7 nM), with less affinity for the κ OR (gp K(i) κ=55 nM). The OR functional profile for 5l varied from the previously described dual δ/µ OR agonists 4, with 5l being a potent, mixed dual δ OR antagonist/µ OR agonist [δ IC(50)=89 nM (HVD); µ EC(50)=1 nM (GPI); κ EC(50)=1.6 µM (GPC)]. Compound 5l has progressed through a clinical Phase II Proof of Concept study on 800 patients suffering from diarrhea-predominant Irritable Bowel Syndrome (IBS-d). This Phase II study was recently completed successfully, with 5l demonstrating statistically significant efficacy over placebo.

Design, synthesis and preclinical evaluation of bio-conjugated amylinomimetic peptides as long-acting amylin receptor agonists.

Pramlintide is an equipotent amylin analogue that reduces food intake and body weight in obese subjects and has been clinically approved as an adjunctive therapy for the treatment of adult diabetic patients. However, due to its extremely short half-life in vivo, a regimen of multiple daily administrations is required for achieving clinical effectiveness. Herein is described the development of prototypical long-acting pramlintide bioconjugates, in which pramlintide's disulfide-linked macrocycle was replaced by a cyclic thioether motif. This modification enabled stable chemical conjugation to a half-life extending antibody. In contrast to pramlintide (t1/2 < 0.75 h), bioconjugates 35 and 38 have terminal half-lives of ∼2 days in mice and attain significant exposure levels that are maintained up to 7 days. Single dose subcutaneous administration of 35 in lean mice, given 18-20 h prior to oral acetaminophen (AAP) administration, significantly reduced gastric emptying (as determined by plasma AAP levels). In a separate study, similar administration of 35 in fasted lean mice effected a reduction in food intake for up to 48 h. These data are consistent with durable amylinomimetic responses and provide the basis for further development of such long-acting amylinomimetic conjugates for the potential treatment of obesity and associated pathologies.

GLP-2 receptor agonism ameliorates inflammation and gastrointestinal stasis in murine postoperative ileus.

Glucagon-like peptide 2 (GLP-2) is a pleiotropic intestinotrophic hormone that we hypothesized could lessen gastrointestinal inflammation associated with postoperative ileus (POI). To test this idea, the prophylactic timing and dose of a long-acting variant of human GLP-2 linked to the Fc portion of murine immunoglobulin G (IgG) (GLP-2/IgG) was optimized in a murine model of POI. Surgically treated mice received a single dose of GLP-2/IgG, IgG isotype control, or phosphate-buffered saline 1 to 48 h before small bowel surgical manipulation. The distribution of orally fed fluorescein isothiocyanate-dextran and histological analyses of myeloperoxidase-positive immune cells were determined 24 and 48 h postoperatively. TaqMan quantitative polymerase chain reaction was used to determine early changes in mRNA expression in the muscularis or mucosa. In normal mice, prolonged exposure to GLP-2 increased upper gastrointestinal (GI) transit and mucosal weight. When administered 1 or 3 h before surgery, GLP-2/IgG reduced the leukocyte infiltrate 24 and 48 h postoperatively and improved GI transit 48 h postoperatively. Surgical manipulation rapidly increased gene expression of proinflammatory cytokines and enzymes for kinetically active mediators in the mucosa and muscularis. GLP-2/IgG2a affected the expression of genes associated with mucosal inflammation and barrier function. We conclude that prophylactic treatment with a long-acting GLP-2 agonist ameliorates inflammation and improves intestinal dysmotility associated with surgical manipulation of the bowel. The action of GLP-2 is consistent with a lessening of inflammation, leading to a more rapid recovery.

TLR3 activation stimulates cytokine secretion without altering agonist-induced human small airway contraction or relaxation.

Respiratory infections exacerbate chronic lung diseases promoting airway inflammation and hyperreactivity. Toll-like receptor 3 (TLR3) recognizes viral double-stranded (ds) RNA such as polyinosinic-polycytidylic acid [poly(I:C)] and stimulates innate immune responses. The objective of this study was to test the hypothesis that dsRNA promotes lung inflammation and alters airway responsiveness to cholinergic and beta-adrenergic receptor agonists in human lung slices. Human airway smooth muscle (ASM) was incubated for 24 h in poly(I:C) +/- TNFalpha and a TLR3 monoclonal antibody. Precision-cut lung slices (PCLS; 250-microm thickness) from healthy human lungs containing a small airway were incubated in 0, 10, or 100 microg/ml poly(I:C) for 24 h. Intravital microscopy of lung slices was used to quantify contractile and relaxation responsiveness to carbachol and isoproterenol, respectively. Supernatants of ASM and PCLS were analyzed for cytokine secretion using a 25-multiplex bead assay. In human ASM, poly(I:C) (0.5 microg/ml) increased macrophage inflammatory protein-1alpha (MIP-1alpha) and RANTES that was prevented by a TLR3 monoclonal receptor antibody. Incubation of human PCLS with poly(I:C) (10 and 100 microg/ml) had little effect on the log EC(50) or maximum drug effect (E(max)) for contraction and relaxation in response to carbachol and isoproterenol, respectively. The responsiveness of the same human PCLS to poly(I:C) incubation was confirmed by the robust increase in chemokines and cytokines. In separate experiments, incubation of PCLS with IL-13 or TNFalpha (100 ng/ml) increased airway sensitivity to carbachol. Poly(I:C) promotes inflammatory mediator release that was not associated with enhanced bronchoconstriction or attenuated bronchodilation in normal healthy human lung slices. Transduction at the TLR3 initiated by dsRNA stimulates downstream innate immune responses.

Triazinediones as prokineticin 1 receptor antagonists. Part 1: SAR, synthesis and biological evaluation.

A series of guanidine triazinediones were identified as potent PK1 receptor antagonists. A compound in this series inhibited the PK1 invoked prosecretory response in rat ileum tissue.

The pharmacology and therapeutic applications of monoclonal antibodies.

Monoclonal antibodies (mAbs) have emerged as a major class of therapeutic agents on the market. To date, approximately 80 mAbs have been granted marketing approval. In 2018, 12 new mAbs were approved by the FDA, representing 20% of the total number of approved drugs. The majority of mAb therapeutics are for oncological and immunological/infectious diseases, but these are expanding into other disease areas. Over 100 monoclonal antibodies are in development, and their unique features ensure that these will remain a part of the therapeutic pipeline. Thus, the therapeutic value and the elucidation of their pharmacological properties supporting clinical development of these large molecules are unquestioned. However, their utilization as pharmacological tools in academic laboratories has lagged behind their small molecule counterparts. Early therapeutic mAbs targeted soluble cytokines, but now that mAbs also target membrane-bound receptors and have increased circulating half-life, their pharmacology is more complex. The principles of pharmacology have enabled the development of high affinity, potent and selective small molecule therapeutics with reduced off-target effects and drug-drug interactions. This review will discuss how the same basic principles can be applied to mAbs, with some important differences. Monoclonal antibodies have several benefits, such as fewer off-target adverse effects, fewer drug-drug interactions, higher specificity, and potentially increased efficacy through targeted therapy. Modifications to decrease the immunogenicity and increase the efficacy are described, with examples of optimizing their pharmacokinetic properties and enabling oral bioavailability. Increased awareness of these advances may help to increase their use in exploratory research and further understand and characterize their pharmacological properties.

Synthesis and SAR of pyridazinone-substituted phenylalanine amide alpha4 integrin antagonists.

Structural modification and cellular adhesion inhibition activities of pyridazinone-substituted phenylalanine amide alpha(4) integrin antagonists are described. Functionality requirements for the arylamide moiety and the carboxylic acid group were demonstrated. The study also revealed novel structure-activity relationships (SAR) for arylated pyridazinones. A correlation between bioavailability and permeability was also explored. A selected compound showed effectiveness in a mouse leukocytosis study.

Synthesis and characterization of 5,6,7,8-tetrahydroquinoline C5a receptor antagonists.

A novel series of substituted 2-aryl-5-amino-5,6,7,8-tetrahydroquinoline C5a receptor antagonists is reported. Synthetic routes were developed that allow the substituents on the tetrahydroquinoline core to be efficiently varied, facilitating determination of structure-activity relationships. Members of the series display high binding affinity for the C5a receptor and are potent functional antagonists.

Design and optimization of aniline-substituted tetrahydroquinoline C5a receptor antagonists.

A series of aniline-substituted tetrahydroquinoline C5a receptor antagonists were discovered. A functionality requirement of ortho substitution on the aniline was revealed. Secondary anilines, in general, outperformed tertiary analogs in inhibition of C5a-induced calcium mobilization. Further enhancement of activity was realized in the presence of an ortho hydroxyalkyl side chain. The functional IC(50) of selected analogs was optimized to the single-digit nanomolar level.

Functional effects and characteristics of cecum-projecting neurons in the dorsal motor nucleus of the vagus of rats.

Preganglionic neurons in the dorsal motor nucleus of the vagus (DMV) innervate most of the gastrointestinal tract; with the stomach and the cecum/proximal colon having a greater proportion of vagal input. Cecum-projecting neurons have been thought to be distinct from other preganglionic neurons due to their location within the DMV, but it is unknown whether these neurons innervate the cecum exclusively or what effect their activation has on cecal motor activity. Therefore, we investigated the extent of coinnervation of cecum and stomach by vagal neurons, their neurochemistry, and the effect of DMV stimulation on intracecal and intragastric volumes. Fluorescent retrograde tracers injected into the serosa of the cecum and stomach revealed that in the DMV 49+/-5% CTB-labeled cecum-projecting neurons also innervated the stomach. Immunocytochemical staining for nitric oxide (NO) synthase and tyrosine hydroxylase indicated that only 3+/-1% and 4+/-1% of cecum-projecting neurons contained these markers, respectively. In anesthetized rats gastric and cecal volumes were measured by prototypic miniaturized dual barostats that were developed for use in rodents. Microinjection of l-glutamate into the DMV increased gastric contractile activity and tone, and reduced on-going cecum contractile activity (2.6+/-0.7 contractions/2 min after injection versus 8.2+/-0.4 contractions/2 min before injection, N = 5). The barostat was able to detect decreases (-0.88+/-0.13 ml) and increases (0.25+/-0.05 ml) in cecum volume in response to carbachol and sodium nitroprusside, respectively. In summary, cecum-projecting neurons are not an entirely exclusive population within the DMV because a percentage of these also innervate the stomach. Central vagal stimulation can modulate both gastric and cecum contractile activity. Together, these data support a role of the vagus in neural reflexes involving gastric and large bowel motor function, such as the immediate phase of the gastrocolonic reflex.

Synthesis and biological evaluation of novel pyridazinone-based alpha4 integrin receptor antagonists.

A novel series of pyridazinone-functionalized phenylalanine analogues was prepared and evaluated for inhibition of cellular adhesion mediated by alpha4beta1/VCAM-1 and alpha4beta7/MAdCAM-1 interactions. Concise syntheses were developed and applied for exploration of structure-activity relationships pertaining to the pyridazinone ring as well as the N-acyl phenylalanine scaffold. Potent dual antagonists of alpha4beta1 and alpha4beta7 were generated from an amide subseries; antagonists selective for alpha4beta7 were identified from urea and carbamate-based subseries. The pharmacokinetic properties of selected members of the series have been determined in rats and demonstrate that the use of ester prodrugs and alterations to the amide linkage can lead to improved oral bioavailability in this series. An alpha4beta7-selective member of the carbamate subseries (36c), upon oral administration, demonstrated in vivo efficacy in the mouse DSS colitis model.

FcRn binding is not sufficient for achieving systemic therapeutic levels of immunoglobulin G after oral delivery of enteric-coated capsules in cynomolgus macaques.

Although much speculation has surrounded intestinally expressed FcRn as a means for systemic uptake of orally administered immunoglobulin G (IgG), this has not been validated in translational models beyond neonates or in FcRn-expressing cells in vitro. Recently, IgG1 intestinal infusion acutely in anesthetized cynomolgus resulted in detectable serum monoclonal antibody (mAb) levels. In this study, we show that IgG2 has greater protease resistance to intestinal enzymes in vitro and mice in vivo, due to protease resistance in the hinge region. An IgG2 mAb engineered for FcRn binding, was optimally formulated, lyophilized, and loaded into enteric-coated capsules for oral dosing in cynomolgus. Small intestinal pH 7.5 was selected for enteric delivery based on gastrointestinal pH profiling of cynomolgus by operator-assisted IntelliCap System(®). Milling of the lyophilized IgG2 M428L FcRn-binding variant after formulation in 10 mmol/L histidine, pH 5.7, 8.5% sucrose, 0.04% PS80 did not alter the physicochemical properties nor the molecular integrity compared to the batch released in PBS. Size 3 hard gel capsules (23.2 mg IgG2 M428L ~3 mg/kg) were coated with hydroxypropyl methylcellulose acetate succinate for rapid dissolution at pH 7.5 in small intestine and FcRn binding of encapsulated mAb confirmed. Initial capsule dosing by endoscopic delivery into the small intestine achieved 0.2 + 0.1 ng/mL (n = 5) peak at 24 h. Weekly oral capsule dosing for 6 weeks achieved levels of 0.4 + 0.2 ng/mL and, despite increasing the dose and frequency, remained below 1 ng/mL. In conclusion, lyophilized milled mAb retains FcRn binding and molecular integrity for small intestinal delivery. The low systemic exposure has demonstrated the limitations of intestinal FcRn in non-human primates and the unfeasibility of employing this for therapeutic levels of mAb. Local mAb delivery with limited systemic exposure may be sufficient as a therapeutic for intestinal diseases.