Head-to-Head Comparison of Caco-2 Transwell and Gut-on-a-Chip Models for Assessing Oral Peptide Formulations.

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.

Validation of a Caco-2 microfluidic Chip model for predicting intestinal absorption of BCS Class I-IV drugs.

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.

Near UV and Visible Light Photo-Degradation Mechanisms in Citrate Buffer: One-Electron Reduction of Peptide and Protein Disulfides promotes Oxidation and Cis/Trans Isomerization of Unsaturated Fatty Acids of Polysorbate 80.

Citrate is a common buffer for slightly acidic pH ranges of protein formulations. In the presence of iron, citrate buffer undergoes photo-degradation induced by near UV and visible light. Recent studies (Subelzu and Schöneich, Mol. Pharm. 2020, 17, 4163-4179) have documented that such photo-degradation results in the formation of carbon dioxide radical anion (•CO2-), a strong reductant which reduces Fe3+, O2, and disulfide bonds. In the present study we show that near UV and visible light photo-degradation of citrate in the presence of iron can induce reductive peptide and protein disulfide cleavage as well as free radical damage of a surfactant, polysorbate 80 (PS80). Reductive disulfide cleavage is most likely caused by efficient electron transfer from carbon dioxide radical anions to disulfides, resulting in the generation of thiol/thiolate and thiyl radicals. The latter can react with mono- and polyunsaturated fatty acids of PS80 to cause cis/trans isomerization and/or oxidation. Representative products generated by cis/trans isomerization and oxidation of oleic acid esters have been detected by HPLC-MS analysis. Further evidence for reductive disulfide cleavage was obtained through the analysis of free thiols. The oxidation of PS80 can also be the consequence of reactive oxygen species (ROS) generation through the reduction of O2 by carbon dioxide radical anions and/or intermediary Fe2+.

Pharmaceutical Excipients Enhance Iron-Dependent Photo-Degradation in Pharmaceutical Buffers by near UV and Visible Light: Tyrosine Modification by Reactions of the Antioxidant Methionine in Citrate Buffer.

PURPOSE: To evaluate the effect of excipients, including sugars and amino acids, on photo-degradation reactions in pharmaceutical buffers induced by near UV and visible light. METHODS: Solutions of citrate or acetate buffers, containing 1 or 50 µM Fe3+, the model peptides methionine enkephalin (MEn), leucine enkephalin (LEn) or proctolin peptide (ProP), in the presence of commonly used amino acids or sugars, were photo-irradiated with near UV or visible light. The oxidation products were analyzed by reverse-phase HPLC and HPLC-MS/MS. RESULTS: The sugars mannitol, sucrose and trehalose, and the amino acids Arg, Lys, and His significantly promote the oxidation of peptide Met to peptide Met sulfoxide. These excipients do not increase the yields of hydrogen peroxide, suggesting that other oxidants such as peroxyl radicals are responsible for the oxidation of peptide Met. The addition of free Met reduces the oxidation of peptide Met, but, in citrate buffer, causes the addition of Met oxidation products to Tyr residues of the target peptides. CONCLUSIONS: Commonly used excipients enhance the light-induced oxidation of amino acids in model peptides.

Probing Protein Conformation Destabilization in Sterile Liquid Formulations through the Formation of 3,4-Dihydroxyphenylalanine.

This work demonstrates the use of a fluorescent probe to screen protein conformational changes in mixtures of monoclonal antibodies and determine the region of where such changes may originate through a footprinting mass spectrometry approach. The oxidative stress of mixtures of two different immunoglobulins (IgG1, IgG2, or IgG4) performed in the presence of 2,2'-azobis(2-amidinopropane dihydrochloride) results in sequence-specific tyrosine oxidation reactions depending on the time of incubation of the IgG molecules and the nature of the excipients present in the formulation. The combination of a fluorescence assay, based on the detection of 3,4-dihydroxyphenylalanine (DOPA) and mass spectrometry analyses, permits the identification of protein conformation changes. In a mixture of IgG2 and IgG4, a destabilization of IgG4 in the presence of IgG2 is observed. The destabilized region involves the Fab region of IgG4 between Tyr63 and Tyr81 and potentially multiple regions of IgG2.

Near UV and Visible Light Induce Iron-Dependent Photodegradation Reactions in Pharmaceutical Buffers: Mechanistic and Product Studies.

Near UV (λ = 320-400 nm) and visible light (λ = 400-800 nm) can lead to the oxidation of pharmaceutical proteins, which can affect efficiency and promote immunogenicity. However, no concise mechanism has been established for the photo-oxidation of pharmaceutical proteins under near UV and visible light. Here, we show that carboxylic acid buffer-Fe3+ complexes can function as photosensitizers, causing peptide degradation via the formation of various radicals and oxidants. Three pharmaceutical relevant carboxylic acid buffers (citrate, acetate, and succinate) were tested under near UV and visible light. Oxidation reactions were monitored for model peptides containing readily oxidizable amino acids, such as methionine- or leucine-enkephalin and proctolin peptide. Oxidation products were evaluated by RP-HPLC coupled to UV or fluorescent detection and RP-HPLC-MS/MS. Specifically for citrate buffer, the light-induced formation of H2O2, •OH, •CO2-, and formaldehyde was demonstrated. The peptides displayed oxidation of Met, hydroxylation of Tyr and Phe, as well as the formation of novel products from Tyr. Experiments with 18O2 resulted in the incorporation of 18O into various reaction products, consistent with a metal-catalyzed activation of O2 into reactive oxygen species. The addition of EDTA and DTPA did not prevent the oxidation of the peptides and, in some cases, enhanced the oxidation. Our results demonstrate that pharmaceutical buffer-Fe3+ complexes, exposed to UV and visible light, can promote various pathways of oxidation reactions in pharmaceutical formulations.

Glatiramer acetate persists at the injection site and draining lymph nodes via electrostatically-induced aggregation.

Glatiramer acetate (GA) is widely prescribed for the treatment of relapsing-remitting multiple sclerosis, however, the mechanism of action is still not fully understood. We investigated the structural properties of GA and examined alterations to the drug upon injection into the subcutaneous space. First, a variety of biophysical characterization techniques were employed to characterize GA in solution. GA was found to exist as alpha helices in solution with a hydrodynamic radius of ~3 nm in size. To simulate GA behavior at the site of injection, GA was injected into a solution of 1.5 MDa hyaluronic acid (HA). Visible aggregates were observed immediately upon injection and subsequent testing indicated aggregation was driven by electrostatic interactions between the positively-charged GA and negatively-charged HA. In vivo testing confirmed GA formed spherical particles in the nano- to micrometer size range, suggesting this mechanism contributes to persistence at the injection site and in draining lymph nodes. The aggregates were found to associate with glycosaminoglycans, suggesting an electrostatic mechanism of induced aggregation like the simulated injection. These novel observations may help explain the complex immunomodulatory mechanisms of GA and adverse injection site reactions seen in patients.

Identification of D-Amino Acids in Light Exposed mAb Formulations.

PURPOSE: We previously demonstrated that D-amino acids can form as a result of photo-irradiation of a monoclonal antibody (mAb) at both λ = 254 nm and λ > 295 nm (λmax = 305 nm), likely via reversible hydrogen transfer reactions of intermediary thiyl radicals. Here, we investigate the role of various excipients (sucrose, glucose, L-Arg, L-Met and L-Leu) on D-amino acid formation, and specifically the distribution of D-amino acids in mAb monomers and aggregates present after light exposure. METHODS: The mAb-containing formulations were photo-irradiated at λ = 254 nm and λmax = 305 nm, followed by fractionation of aggregate and monomer fractions using size exclusion chromatography. These aggregate and monomer fractions were subjected to hydrolysis and subsequent amino acid analysis. RESULTS: Both aggregate and monomer fractions collected from all formulations showed the formation of D-Glu and D-Val, whereas the formation of D-Ala was limited to the aggregate fraction collected from an L-Arg-containing formulation. Interestingly, quantitative analysis revealed higher yields of D-amino acids in the L-Arg-containing formulation. CONCLUSIONS: Generally, D-amino acids accumulated to similar extents in monomers and aggregates.