In Vivo Mechanism of Action of Sodium Caprate for Improving the Intestinal Absorption of a GLP1/GIP Coagonist Peptide.

Sodium caprate (C10) has been widely evaluated as an intestinal permeation enhancer for the oral delivery of macromolecules. However, the effect of C10 on the intestinal absorption of peptides with different physicochemical properties and its permeation-enhancing effect in vivo remains to be understood. Here, we evaluated the effects of C10 on intestinal absorption in rats with a glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GIP-GLP1) dual agonist peptide (LY) and semaglutide with different enzymatic stabilities and self-association behaviors as well as the oral exposure of the LY peptide in minipigs. Furthermore, we investigated the mechanism of action (MoA) of C10 for improving the intestinal absorption of the LY peptide in vivo via live imaging of the rat intestinal epithelium and tissue distribution of the LY peptide in minipigs. The LY peptide showed higher proteolytic stability in pancreatin and was a monomer in solution compared to that in semaglutide. C10 increased in vitro permeability in the minipig intestinal organoid monolayer to a greater extent for the LY peptide than for semaglutide. In the rat jejunal closed-loop model, C10 increased the absorption of LY peptide better than that of semaglutide, which might be attributed to higher in vitro proteolytic stability and permeability of the LY peptide. Using confocal live imaging, we observed that C10 enabled the rapid oral absorption of a model macromolecule (FD4) in the rat intestine. In the duodenum tissues of minipigs, C10 was found to qualitatively reduce the tight junction protein level and allow peptide uptake to the intestinal cells. C10 decreased the transition temperature of the artificial lipid membrane, indicating an increase in membrane fluidity, which is consistent with the above in vivo imaging results. These data indicated that the LY's favorable physicochemical properties combined with the effects of C10 on the intestinal mucosa resulted in an ∼2% relative bioavailability in minipigs.

Identification of a Multi-Component Formulation for Intestinal Delivery of a GLP-1/Glucagon Co-agonist Peptide.

PURPOSE: Oral delivery of therapeutic peptides has been challenging due to multiple physiological factors and physicochemical properties of peptides. We report a systematic approach to identify formulation compositions combining a permeation enhancer and a peptidase inhibitor that minimize proteolytic degradation and increase absorption of a peptide across the small intestine. METHODS: An acylated glucagon-like peptide-1/glucagon co-agonist peptide (4.5 kDa) was selected as a model peptide. Proteolytic stability of the peptide was investigated in rat and pig SIF. Effective PEs and multiple component formulations were identified in rats. Relative bioavailability of the peptide was determined in minipigs via intraduodenal administration (ID) of enteric capsules. RESULTS: The peptide degraded rapidly in the rat and pig SIF. Citric acid, SBTI, and SBTCI inhibited the enzymatic degradation. The peptide self-associated into trimers in solution, however, addition of PEs monomerized the peptide. C10 was the most effective PE among tested PEs (DPC, LC, rhamnolipid, C12-maltosides, and SNAC) to improve intestinal absorption of the peptide in the rat IJ-closed loop model. A combination of C10 and SBTI or SBTCI increased the peptide exposure 5-tenfold compared to the exposure with the PE alone in the rat IJ-cannulated model, and achieved 1.06 ± 0.76% bioavailability in minipigs relative to subcutaneous via ID administration using enteric capsules. CONCLUSION: We identified SBTI and C10 as an effective peptidase inhibitor and PE for intestinal absorption of the peptide. The combination of SBTI and C10 addressed the peptide physiochemical properties and provides a formulation strategy to achieve intestinal delivery of this peptide.

Influence of FcRn binding properties on the gastrointestinal absorption and exposure profile of Fc molecules.

The neonatal Fc receptor (FcRn) represents a transport system with the potential to facilitate absorption of biologics across the gastrointestinal barrier. How biologics interact with FcRn to enable their gastrointestinal absorption, and how these interactions might be optimized in a biological therapeutic are not well understood. Thus, we studied the absorption of Fc molecules from the intestine using three IgG4-derived Fc variants with different, pH-dependent FcRn binding and release profiles. Using several different intestinal models, we consistently observed that FcRn binding affinity correlated with transcytosis. Our findings support targeting FcRn to enable intestinal absorption of biologics and highlight additional strategic considerations for future work.

Influence of physiochemical properties on the subcutaneous absorption and bioavailability of monoclonal antibodies.

Many therapeutic monoclonal antibodies (mAbs) were initially developed for intravenous (IV) administration. As a means to improve mAb drug-ability and the patient experience, subcutaneous (SC) administration is an increasingly important delivery route for mAbs. Unlike IV administration, bioavailability limitations for antibodies have been reported following SC injection and can dictate whether a mAb is administered via this parenteral route. The SC bioavailability of antibodies has been difficult to predict, and it can be variable and partial, with values ranging from ~50% to 100%. The mechanisms leading to the incomplete bioavailability of some mAbs relative to others are not well understood. There are some limited data that suggest the physiochemical properties inherent to a mAb can contribute to its SC absorption, bioavailability, and in vivo fate. In this study, we evaluated the integrated influence of multiple mAb physiochemical factors on the SC absorption and bioavailability of six humanized mAbs in both rats and cynomolgus monkeys. We demonstrate the physiochemical properties of mAbs are critical to their rate and extent of SC absorption. The combination of high positive charge and hydrophobic interaction significantly reduced the rate of the evaluated mAb's SC absorption and bioavailability. Reduction or balancing of both these attributes via re-engineering the mAbs restored desirable properties of the molecules assessed. This included reduced association with SC tissue, improvements in mAb absorption from the SC space and overall SC bioavailability. Our findings point to the importance of evaluating the relative balance between various physiochemical factors, including charge, hydrophobicity, and stability, to improve the SC drug-ability of mAbs for selecting or engineering mAbs with enhanced in vivo absorption and bioavailability following SC administration.

Hyperactive RAS/PI3-K/MAPK Signaling Cascade in Migration and Adhesion of Nf1 Haploinsufficient Mesenchymal Stem/Progenitor Cells.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in the NF1 tumor suppressor gene, which affect approximately 1 out of 3000 individuals. Patients with NF1 suffer from a range of malignant and nonmalignant manifestations such as plexiform neurofibromas and skeletal abnormalities. We previously demonstrated that Nf1 haploinsufficiency in mesenchymal stem/progenitor cells (MSPCs) results in impaired osteoblastic differentiation, which may be associated with the skeletal manifestations in NF1 patients. Here we sought to further ascertain the role of Nf1 in modulating the migration and adhesion of MSPCs of the Nf1 haploinsufficient (Nf1(+/-)) mice. Nf1(+/-) MSPCs demonstrated increased nuclear-cytoplasmic ratio, increased migration, and increased actin polymerization as compared to wild-type (WT) MSPCs. Additionally, Nf1(+/-) MSPCs were noted to have significantly enhanced cell adhesion to fibronectin with selective affinity for CH271 with an overexpression of its complimentary receptor, CD49e. Nf1(+/-) MSPCs also showed hyperactivation of phosphoinositide 3-kinase (PI3-K) and mitogen activated protein kinase (MAPK) signaling pathways when compared to WT MSPCs, which were both significantly reduced in the presence of their pharmacologic inhibitors, LY294002 and PD0325901, respectively. Collectively, our study suggests that both PI3-K and MAPK signaling pathways play a significant role in enhanced migration and adhesion of Nf1 haploinsufficient MSPCs.

Mutational survey of the PHEX gene in patients with X-linked hypophosphatemic rickets.

X-linked hypophosphatemic rickets (XLH) is a dominantly inherited disorder characterized by renal phosphate wasting, aberrant vitamin D metabolism, and abnormal bone mineralization. XLH is caused by inactivating mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome). In this study, we sequenced the PHEX gene in subjects from 26 kindreds who were clinically diagnosed with XLH. Sequencing revealed 18 different mutations, of which thirteen have not been reported previously. In addition to deletions, splice site mutations, and missense and nonsense mutations, a rare point mutation in the 3'-untranslated region (3'-UTR) was identified as a novel cause of XLH. In summary, we identified a wide spectrum of mutations in the PHEX gene. Our data, in accord with those of others, indicate that there is no single predominant PHEX mutation responsible for XLH.

Neurofibromin-deficient Schwann cells have increased lysophosphatidic acid dependent survival and migration-implications for increased neurofibroma formation during pregnancy.

Neurofibromas are the clinical hallmark of neurofibromatosis Type 1 (NF1), a genetic disorder caused by mutations of the NF1 tumor suppressor gene, which encodes neurofibromin that functions as a GTPase activating protein (GAP) for Ras. During pregnancy, up to 50% of existing neurofibromas enlarge and as many as 60% of new neurofibromas appear for the first time. Lysophosphatidic acid (LPA) is a prototypic lysophospholipid that modulates cell migration and survival of Schwann cells (SCs) and is made in increasing concentrations throughout pregnancy. We addressed the influence of LPA on the biochemical and cellular functions of SCs with a homozygous mutation of the murine homologue of the NF1 gene (Nf1-/-). LPA promoted F-actin polymerization and increased migration and survival of Nf1-/- SCs as compared to wild type (WT) SCs. Furthermore, LPA induced a higher level of Ras-GTP and Akt phosphorylation in Nf1-/- SCs as compared to WT cells. Pharmacologic inhibition or siRNA for the p85beta regulatory subunit of Class I A PI3-K significantly reduced LPA-induced Schwann cell survival and migration. Introduction of NF1-GRD reconstitution was sufficient to normalize the LPA-mediated motility of Nf1-/- SCs. As LPA modulates excessive cell survival and motility of Nf1-/- SCs, which are the tumorigenic cells in NF1, targeting PI3-K may be a potential therapeutic approach in diminishing the development and progression of neurofibromas in pregnant women with NF1.

Neurofibromin plays a critical role in modulating osteoblast differentiation of mesenchymal stem/progenitor cells.

Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1, a pandemic autosomal dominant genetic disorder with an incidence of 1:3000. Individuals with NF1 have a variety of malignant and non-malignant manifestations, including skeletal manifestations, such as osteoporosis, scoliosis and short statures. However, the mechanism(s) underlying the osseous manifestations in NF1 are poorly understood. In the present study, utilizing Nf1 haploinsufficient (+/-) mice, we demonstrate that Nf1+/- mesenchymal stem/progenitor cells (MSPC) have increased proliferation and colony forming unit-fibroblast (CFU-F) capacity compared with wild-type (WT) MSPC. Nf1+/- MSPC also have fewer senescent cells and have a significantly higher telomerase activity compared with WT MSPC. Nf1+/- MSPC have impaired osteoblast differentiation as determined by alkaline phosphatase staining, and confirmed by single CFU-F replating assays. The impaired osteoblast differentiation in Nf1+/- MSPC is consistent with the reduced expression of osteoblast markers at the mRNA level, including osteocalcin and osteonectin. Importantly, re-expression of the full-length NF1 GTPase activating related domain (NF1 GAP-related domain) is sufficient to restore the impaired osteoblast differentiation in Nf1+/- MSPC. Taken together, our results suggest that neurofibromin plays a crucial role in modulating MSPC differentiation into osteoblasts, and the defect in osteoblast differentiation may contribute at least in part to the osseous abnormalities seen in individuals with NF1.

Nf1+/- mast cells induce neurofibroma like phenotypes through secreted TGF-beta signaling.

Neurofibromas are common tumors found in neurofibromatosis type 1 (NF1) patients. These complex tumors are composed of Schwann cells, mast cells, fibroblasts and perineurial cells embedded in collagen that provide a lattice for tumor invasion. Genetic studies demonstrate that in neurofibromas, nullizygous loss of Nf1 in Schwann cells and haploinsufficiency of Nf1 in non-neuronal cells are required for tumorigenesis. Fibroblasts are a major cellular constituent in neurofibromas and are a source of collagen that constitutes approximately 50% of the dry weight of the tumor. Here, we show that two of the prevalent heterozygous cells found in neurofibromas, mast cells and fibroblasts interact directly to contribute to tumor phenotype. Nf1+/- mast cells secrete elevated concentrations of the profibrotic transforming growth factor-beta (TGF-beta). In response to TGF-beta, both murine Nf1+/- fibroblasts and fibroblasts from human neurofibromas proliferate and synthesize excessive collagen, a hallmark of neurofibromas. We also establish that the TGF-beta response occurs via hyperactivation of a novel Ras-c-abl signaling pathway. Genetic or pharmacological inhibition of c-abl reverses fibroblast proliferation and collagen synthesis to wild-type levels. These studies identify a novel molecular target to inhibit neurofibroma formation.