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Oral delivery of potent peptide drugs provides key formulation challenges in the pharmaceutical industry: stability, solubility, and permeability. Intestinal permeation enhancers (PEs) can overcome the low oral bioavailability by improving the drug permeability. Conventional in vitro and ex vivo models for assessing PEs fail to predict efficacy in vivo. Here, we compared Caco-2 cells cultured in the conventional static Transwell model to a commercially available continuous flow microfluidic Gut-on-a-Chip model. We determined baseline permeability of FITC-Dextan 3 kDa (FD3) in Transwell (5.3 ± 0.8 × 10-8 cm/s) vs Chip (3.2 ± 1.8 × 10-7 cm/s). We screened the concentration impact of two established PEs sodium caprate and sucrose monolaurate and indicated a requirement for higher enhancer concentration in the Chip model to elicit equivalent efficacy e.g., 10 mM sodium caprate in Transwells vs 25 mM in Chips. Fasted and fed state simulated intestinal fluids (FaSSIF/FeSSIF) were introduced into the Chip and increased basal FD3 permeability by 3-fold and 20-fold, respectively, compared to 4-fold and 4000-fold in Transwells. We assessed the utility of this model to peptides (Insulin and Octreotide) with PEs and observed much more modest permeability enhancement in the Chip model in line with observations in ex vivo and in vivo preclinical models. These data indicate that microfluidic Chip models are well suited to bridge the gap between conventional in vitro and in vivo models.

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Oral delivery is considered the most patient preferred route of drug administration, however, the drug must be sufficiently soluble and permeable to successfully formulate an oral formulation. There have been advancements in the development of more predictive solubility and dissolution tools, but the tools that has been developed for permeability assays have not been validated as extensively as the gold-standard Caco-2 Transwell assay. Here, we evaluated Caco-2 intestinal permeability assay in Transwells and a commercially available microfluidic Chip using 19 representative Biopharmaceutics Classification System (BCS) Class I-IV compounds. For each selected compound, we performed a comprehensive viability test, quantified its apparent permeability (Papp), and established an in vitro in vivo correlation (IVIVC) to the human fraction absorbed (fa) in both culture conditions. Permeability differences were observed across the models as demonstrated by antipyrine (Transwell Papp: 38.5 ± 6.1 × 10-8 cm/s vs Chip Papp: 32.9 ± 11.3 × 10-8 cm/s) and nadolol (Transwell Papp: 0.6 ± 0.1 × 10-7 cm/s vs Chip Papp: 3 ± 1.2 × 10-7 cm/s). The in vitro in vivo correlation (IVIVC; Papp vs. fa) of the Transwell model (r2 = 0.59-0.83) was similar to the Chip model (r2 = 0.41-0.79), highlighting similar levels of predictivity. Comparing to historical data, our Chip Papp data was more closely aligned to native tissues assessed in Ussing chambers. This is the first study to comprehensively validate a commercial Gut-on-a-Chip model as a predictive tool for assessing oral absorption to further reduce our reliance on animal models.

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Microphysiological systems (MPS) are powerful predictive tools for assessing drug-induced kidney injuries. Previous MPS have examined single regions of the nephron, but lack simultaneous filtration, reabsorption, and secretion functionality. Here, we developed a partially open MPS that structurally and functionally recapitulated the glomerular filtration barrier, proximal tubular reabsorption, and secretion for seven days. The system introduced a recirculation circuit and an open filtrate output as a source of functional testing. As a proof-of-concept, a tri-culture of immortalized podocytes, umbilical vein endothelial cells, and proximal tubule (PCT) cells were housed in a single MPS: T-junction, glomerulus housing unit, and PCT chip. The MPS successfully retained blood serum protein, reabsorbed glucose, secreted creatinine, and expressed cell-type specific proteins (VE-cadherin, nephrin, and ZO-1). To simulate drug-induced kidney injuries, the system was perfused with cisplatin and adriamycin, and then tested using serum albumin filtration, glucose clearance, and lactate dehydrogenase release. The glomerulus and PCT MPS demonstrated a complex, dynamic microenvironment and recreated some in vivo-like functions in basal and drug-induced conditions, offering a novel prototype for preclinical testing.

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Kidney microphysiological systems (MPS) serve as potentially valuable preclinical instruments in probing mechanisms of renal clearance and osmoregulation. Current kidney MPS models target regions of the nephron, such as the glomerulus and proximal tubule (PCT), but fail to incorporate multiple filtration and absorption interfaces. Here, we describe a novel, partially open glomerulus and PCT microdevice that integrates filtration and absorption in a single MPS. The system equalizes pressure on each side of the PCT that operates with one side "closed" by recirculating into the bloodstream, and the other "opened" by exiting as primary filtrate. This design precisely controls the internal fluid dynamics and prevents loss of all fluid to the open side. Through this feature, an in vitro human glomerulus and proximal tubule MPS was constructed to filter human serum albumin and reabsorb glucose for seven days of operation. For proof-of-concept experiments, three human-derived cell types-conditionally immortalized human podocytes (CIHP-1), human umbilical vein endothelial cells (HUVECs), and human proximal tubule cells (HK-2)-were adapted into a common serum-free medium prior to being seeded into the three-component MPS (T-junction splitter, glomerular housing unit, and parallel proximal tubule barrier model). This system was optimized geometrically (tubing length, tubing internal diameter, and inlet flow rate) using in silico computational modeling. The prototype tri-culture MPS successfully filtered blood serum protein and generated albumin filtration in a physiologically realistic manner, while the device cultured only with proximal tubule cells did not. This glomerulus and proximal convoluted tubule MPS is a potential prototype for the human kidney used in both human-relevant testing and examining pharmacokinetic interactions.

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BACKGROUND: Transposon mutagenesis is highly valuable for bacterial genetic and genomic studies. The transposons are usually delivered into host cells through conjugation or electroporation of a suicide plasmid. However, many bacterial species cannot be efficiently conjugated or transformed for transposon saturation mutagenesis. For this reason, temperature-sensitive (ts) plasmids have also been developed for transposon mutagenesis, but prolonged incubation at high temperatures to induce ts plasmid loss can be harmful to the hosts and lead to enrichment of mutants with adaptive genetic changes. In addition, the ts phenotype of a plasmid is often strain- or species-specific, as it may become non-ts or suicidal in different bacterial species. RESULTS: We have engineered several conditional suicide plasmids that have a broad host range and whose loss is IPTG-controlled. One construct, which has the highest stability in the absence of IPTG induction, was then used as a curable vector to deliver hyperactive miniTn5 transposons for insertional mutagenesis. Our analyses show that these new tools can be used for efficient and regulatable transposon mutagenesis in Escherichia coli, Acinetobacter baylyi and Pseudomonas aeruginosa. In P. aeruginosa PAO1, we have used this method to generate a Tn5 insertion library with an estimated diversity of ~ 108, which is ~ 2 logs larger than the best transposon insertional library of PAO1 and related Pseudomonas strains previously reported. CONCLUSION: We have developed a number of IPTG-controlled conditional suicide plasmids. By exploiting one of them for transposon delivery, a highly efficient and broadly useful mutagenesis system has been developed. As the assay condition is mild, we believe that our methodology will have broad applications in microbiology research.

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