Enhancing Selective Antimicrobial and Antibiofilm Activities of Melittin through 6-Aminohexanoic Acid Substitution.

Leucine residues are commonly found in the hydrophobic face of antimicrobial peptides (AMPs) and are crucial for membrane permeabilization, leading to the cell death of invading pathogens. Melittin, which contains four leucine residues, demonstrates broad-spectrum antimicrobial properties but also significant cytotoxicity against mammalian cells. To enhance the cell selectivity of melittin, this study synthesized five analogs by replacing leucine with its structural isomer, 6-aminohexanoic acid. Among these analogs, Mel-LX3 exhibited potent antibacterial activity against both Gram-positive and Gram-negative bacteria. Importantly, Mel-LX3 displayed significantly reduced hemolytic and cytotoxic effects compared to melittin. Mechanistic studies, including membrane depolarization, SYTOX green uptake, FACScan analysis, and inner/outer membrane permeation assays, demonstrated that Mel-LX3 effectively permeabilized bacterial membranes similar to melittin. Notably, Mel-LX3 showed robust antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Furthermore, Mel-LX3 effectively inhibited biofilm formation and eradicated existing biofilms of MDRPA. With its improved selective antimicrobial and antibiofilm activities, Mel-LX3 emerges as a promising candidate for the development of novel antimicrobial agents. We propose that the substitution of leucine with 6-aminohexanoic acid in AMPs represents a significant strategy for combating resistant bacteria.

Enhanced Anti-Tumor Effect of Folate-Targeted FA-AMA-hyd-DOX Conjugate in a Xenograft Model of Human Breast Cancer.

Folate-aminocaproic acid-doxorubicin (FA-AMA-hyd-DOX) was firstly synthesized by our group. It was indicated that FA-AMA-hyd-DOX was pH-responsive, and had strong cytotoxicity on a folate receptor overexpressing cell line (KB cells) in vitro. The aim of our study was to further explore the potential use of FA-AMA-hyd-DOX as a new therapeutic drug for breast cancer. The cellular uptake and the antiproliferative activity of the FA-AMA-hyd-DOX in MDA-MB-231 cells were measured. Compared with DOX, FA-AMA-hyd-DOX exhibited higher targeting ability and cytotoxicity to FR-positive tumor cells. Subsequently, the tissue distribution of FA-AMA-hyd-DOX was studied, and the result confirmed that DOX modified by FA can effectively increase the selectivity of drugs in vivo. After determining the maximum tolerated dose (MTD) of FA-AMA-hyd-DOX in MDA-MB-231 tumor-bearing nude mice, the antitumor effects and the in vivo safety of FA-AMA-hyd-DOX were systematically evaluated. The data showed that FA-AMA-hyd-DOX could effectively increase the dose of DOX tolerated by tumor-bearing nude mice and significantly inhibit MDA-MB-231 tumor growth in vivo. Furthermore, FA-AMA-hyd-DOX treatment resulted in almost no obvious damage to the mice. All the positive data suggest that FA-targeted FA-AMA-hyd-DOX is a promising tumor-targeted compound for breast cancer therapy.

The Importance of 6-Aminohexanoic Acid as a Hydrophobic, Flexible Structural Element.

6-aminohexanoic acid is an ω-amino acid with a hydrophobic, flexible structure. Although the ω-amino acid in question is mainly used clinically as an antifibrinolytic drug, other applications are also interesting and important. This synthetic lysine derivative, without an α-amino group, plays a significant role in chemical synthesis of modified peptides and in the polyamide synthetic fibers (nylon) industry. It is also often used as a linker in various biologically active structures. This review concentrates on the role of 6-aminohexanoic acid in the structure of various molecules.

Catalytic and structural properties of ATP-dependent caprolactamase from Pseudomonas jessenii.

Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence-related to other ATP-dependent enzymes involved in lactam hydrolysis, like 5-oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP-dependent acetone- and acetophenone carboxylases. The caprolactamase was produced in Escherichia coli, isolated by His-tag affinity chromatography, and subjected to functional and structural studies. Activity toward caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6-aminocaproic acid. Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG0 '= 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αß)2 tetramer and revealed an ATP-binding site in CapA and a Zn-coordinating site in CapB. Mutations in the ATP-binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A, and H124A) were inactive and less thermostable than the wild-type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP-dependent lactam hydrolysis.

Biochemical properties of a Pseudomonas aminotransferase involved in caprolactam metabolism.

The biodegradation of the nylon-6 precursor caprolactam by a strain of Pseudomonas jessenii proceeds via ATP-dependent hydrolytic ring opening to 6-aminohexanoate. This non-natural ω-amino acid is converted to 6-oxohexanoic acid by an aminotransferase (PjAT) belonging to the fold type I pyridoxal 5'-phosphate (PLP) enzymes. To understand the structural basis of 6-aminohexanoatate conversion, we solved different crystal structures and determined the substrate scope with a range of aliphatic and aromatic amines. Comparison with the homologous aminotransferases from Chromobacterium violaceum (CvAT) and Vibrio fluvialis (VfAT) showed that the PjAT enzyme has the lowest KM values (highest affinity) and highest specificity constant (kcat /KM ) with the caprolactam degradation intermediates 6-aminohexanoate and 6-oxohexanoic acid, in accordance with its proposed in vivo function. Five distinct three-dimensional structures of PjAT were solved by protein crystallography. The structure of the aldimine intermediate formed from 6-aminohexanoate and the PLP cofactor revealed the presence of a narrow hydrophobic substrate-binding tunnel leading to the cofactor and covered by a flexible arginine, which explains the high activity and selectivity of the PjAT with 6-aminohexanoate. The results suggest that the degradation pathway for caprolactam has recruited an aminotransferase that is well adapted to 6-aminohexanoate degradation. DATABASE: The atomic coordinates and structure factors P. jessenii 6-aminohexanoate aminotransferase have been deposited in the PDB as entries 6G4B (E∙succinate complex), 6G4C (E∙phosphate complex), 6G4D (E∙PLP complex), 6G4E (E∙PLP-6-aminohexanoate intermediate), and 6G4F (E∙PMP complex).

Effect of dehydrocholic acid conjugated with a hydrocarbon on a lipid bilayer composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine.

The effects of bile acids, dehydrocholic acid (DHA) and DHA conjugated with a hydrocarbon (6-aminohexanoate; 6A-DHA) were evaluated using a lipid bilayer composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). DOPC formed a homogenous thin membrane in presence or absence of the DHA, while 20 mol% 6A-DHA induced phase separation on the DOPC thin membrane. It was observed formation of a stomatocyte-like liposomes when these membranes were suspended in a basic solvent. Generally, liposome formation can be prevented by some bile acids. It was found that DHA and 6A-DHA did not disrupt liposome formation, while DHA and 6A-DHA perturbed the liposomal membrane, resulting in increased local-fluidity due to the bent structure of DHA and 6A-DHA. DHA and 6A-DHA showed completely different effects on the hydrophobicity of the boundary surface of DOPC liposome membranes. The steroidal backbone of DHA was found to prevent the insertion of water molecules into the liposomal membrane, whereas 6A-DHA did not show the same behavior which was attributed to its conjugated hydrocarbon.

Ceria Nanoparticles Fabricated with 6-Aminohexanoic Acid that Overcome Systemic Inflammatory Response Syndrome.

Systemic inflammatory response syndrome (SIRS) is self-destructive and uncontrollable inflammatory response of the whole body triggered by infection, trauma, or a variety of severe injuries. Although reactive oxygen species play a pivotal role in the development of SIRS, the trials with conventional antioxidants have failed to improve patient outcome. Ceria nanoparticles (CeNPs) have potent, autocatalytic reactive oxygen species scavenging activities, which may have sufficient therapeutic effects for SIRS. Herein, 3 nm CeNPs are fabricated totally in aqueous phase by using 6-aminohexanoic acid (6-AHA) and their Ce3+ to Ce4+ ratio is increased to enhance antioxidative properties. The obtained 6-AHA-CeNPs demonstrate strong antioxidative and anti-inflammatory effects in various biofluids and inflammatory cells. In SIRS animal models, 6-AHA-CeNPs are demonstrated to reduce multiple organ injuries and inflammation. Moreover, 6-AHA-CeNPs decrease mortality and improve clinical scores of SIRS models. These findings suggest that 6-AHA-CeNPs have potential as a therapeutic nanomedicine for SIRS.

Ultrasound assisted gene and photodynamic synergistic therapy with multifunctional FOXA1-siRNA loaded porphyrin microbubbles for enhancing therapeutic efficacy for breast cancer.

To improve the non-invasive therapeutic efficacy for ER positive breast cancer (ER+ BC), we fabricated a multifunctional FOXA1 loaded porphyrin microbubble to combine photodynamic therapy (PDT) and gene therapy of FOXA1 knockdown (KD) with ultrasound targeted microbubble destruction (UTMD) technology under the guidance of contrast enhanced ultrasound (CEUS). Cationic porphyrin microbubbles (CpMBs) were firstly fabricated from a porphyrin grafted lipid with two cationic amino groups (PGL-NH2) and fluorocarbon inert gas of C3F8. Porphyrin group in the CpMBs monolayer could be used as a photosensitizer for PDT, while amino groups could adsorb siRNA through electrostatic interaction for FOXA1 KD, which could inhibit the proliferation of estrogen-dependent ER+ BC. This system showed high photosensitizer and gene loading content. Moreover, CpMBs/siRNA can be converted into nanoparticles with low-frequency pulsed ultrasound (LFUS) exposure, which increase the transfection efficiency of siRNA (∼4 fold) and the porphyrin uptake (∼8 fold) in MCF-7 (a human breast cancer cell line, ER+) by sonoporation effect. In vivo, UTMD was performed under the guidance of CEUS, and the fluorescence intensity of CpMBs/siRNA at the tumour site reached a peak value at 6 h after injection and it was retained in the following 24 h. Furthermore, there was no tumour recurrence during the observation period (21 days) in the group of PDT combined with FXOA1 KD. Compared to the PDT or FOXA1 KD alone group, the combination of these two methods was much more efficient in inhibiting ER+ breast cancer, showing a good synergistic effect. CpMBs/siRNA combined with UTMD dramatically increased the local accumulation of porphyrin and siRNA through ultrasound-induced sonoporation effect under the guidance of CEUS, showing excellent therapeutic effect for estrogen-dependent ER+ breast cancer.

Expanding lysine industry: industrial biomanufacturing of lysine and its derivatives.

L-Lysine is widely used as a nutrition supplement in feed, food, and beverage industries as well as a chemical intermediate. At present, great efforts are made to further decrease the cost of lysine to make it more competitive in the markets. Furthermore, lysine also shows potential as a feedstock to produce other high-value chemicals for active pharmaceutical ingredients, drugs, or materials. In this review, the current biomanufacturing of lysine is first presented. Second, the production of novel derivatives from lysine is discussed. Some chemicals like L-pipecolic acid, cadaverine, and 5-aminovalerate already have been obtained at a lab scale. Others like 6-aminocaproic acid, valerolactam, and caprolactam could be produced through a biological and chemical coupling pathway or be synthesized by a hypothetical pathway. This review demonstrates an active and expansive lysine industry, and these green biomanufacturing strategies could also be applied to enhance the competitiveness of other amino acid industry.

Effects of the incorporation of ε-aminocaproic acid/chitosan particles to fibrin on cementoblast differentiation and cementum regeneration.

Cementum formation on the exposed tooth-root surface is a critical process in periodontal regeneration. Although various therapeutic approaches have been developed, regeneration of integrated and functional periodontal complexes is still wanting. Here, we found that the OCCM30 cementoblasts cultured on fibrin matrix express substantial levels of matrix proteinases, leading to the degradation of fibrin and the apoptosis of OCCM30 cells, which was reversed upon treatment with a proteinase inhibitor, ε-aminocaproic acid (ACA). Based on these findings, ACA-releasing chitosan particles (ACP) were fabricated and ACP-incorporated fibrin (fibrin-ACP) promoted the differentiation of cementoblasts in vitro, as confirmed by bio-mineralization and expressions of molecules associated with mineralization. In a periodontal defect model of beagle dogs, fibrin-ACP resulted in substantial cementum formation on the exposed root dentin in vivo, compared to fibrin-only and enamel matrix derivative (EMD) which is used clinically for periodontal regeneration. Remarkably, the fibrin-ACP developed structural integrations of the cementum-periodontal ligament-bone complex by the Sharpey's fiber insertion. In addition, fibrin-ACP promoted alveolar bone regeneration through increased bone volume of tooth roof-of-furcation defects and root coverage. Therefore, fibrin-ACP can promote cementogenesis and osteogenesis by controlling biodegradability of fibrin, implicating the feasibility of its therapeutic use to improve periodontal regeneration. STATEMENT OF SIGNIFICANCE: Cementum, the mineralized layer on root dentin surfaces, functions to anchor fibrous connective tissues on tooth-root surfaces with the collagenous Sharpey's fibers integration, of which are essential for periodontal functioning restoration in the complex. Through the cementum-responsible fiber insertions on tooth-root surfaces, PDLs transmit various mechanical responses to periodontal complexes against masticatory/occlusal stimulations to support teeth. In this study, periodontal tissue regeneration was enhanced by use of modified fibrin biomaterial which significantly promoted cementogenesis within the periodontal complex with structural integration by collagenous Sharpey's fiber insertions in vivo by controlling fibrin degradation and consequent cementoblast apoptosis. Furthermore, the modified fibrin could improve repair and regeneration of tooth roof-of-furcation defects, which has spatial curvatures and geometrical difficulties and hardly regenerates periodontal tissues.

Two-dimensional capillary zone electrophoresis-mass spectrometry for the characterization of intact monoclonal antibody charge variants, including deamidation products.

Capillary zone electrophoresis (CZE) is a powerful tool that is progressively being applied for the separation of monoclonal antibody (mAb) charge variants. Mass spectrometry (MS) is the desired detection method concerning identification of mAb variants. In biopharmaceutical applications, there exist optimized and validated electrolyte systems for mAb variant quantification. However, these electrolytes interfere greatly with the electrospray ionization (ESI) process. Here, a heart-cut CZE-CZE-MS setup with an implemented mechanical four-port valve interface was developed that used a generic ε-aminocaproic acid based background electrolyte in the first dimension and acetic acid in the second dimension. Interference-free, highly precise mass data (deviation less than 1 Da) of charge variants of trastuzumab, acting as model mAb system, were achieved. The mass accuracy obtained (low parts per million range) is discussed regarding both measured and calculated masses. Deamidation was detected for the intact model antibody, and related mass differences were significantly confirmed on the deglycosylated level. The CZE-CZE-MS setup is expected to be applicable to a variety of antibodies and electrolyte systems. Thus, it has the potential to become a compelling tool for MS characterization of antibody variants separated in ESI-interfering electrolytes. Graphical Abstract Two-dimensional capillary zone electrophoresis mass spectrometry for the characterization of intact monoclonal antibody (mAb) charge variants. A generic, but highly electrospray-interfering electrolyte system was used as first dimension for mAb charge variant separation and coupled to a volatile electrolyte system as second dimension via a four-port nanoliter valve. In this way, interference-free and precise mass spectrometric data of separated mAb charge variants, including deamidation products, were obtained.

Carbon nanotubes functionalized with folic acid attached via biomimetic peptide linker.

AIM: Anchoring folic acid (FA) with a biomimetic peptidic linker resistant to proteolytic degradation to act as a homing device on functionalized carbon nanotubes. MATERIALS & METHODS: Ethylenediamine was attached to oxidized multiwalled carbon nanotubes (MWNTs) using 4-(4,6-dimethoxy-[1,3,5]triazin-2-yl)-4-methylmorpholinium tetrafluoroborate. FA was coupled with 6-aminohexanoic acid and derivatives of ß-alanine, affording four intermediates, which connected to the MWNTs via peptidic linkers of various lengths. RESULTS: Biomimetic nanomaterials were produced with FA as a homing molecule. The structure and properties of the nanomaterials were analyzed, confirming the versatility of the peptides used as linkers. CONCLUSION: Conjugates of FA attached to MWNTs via peptide linkers prepared from ß-alanine residues are resistant to proteolytic degradation. Viability in colon cancer cells and normal colonocytes confirmed their lack of cytotoxicity.

Structural Dimorphism of Achiral α,γ-Hybrid Peptide Foldamers: Coexistence of 12- and 15/17-Helices.

Here, novel 12-helices in α,γ-hybrid peptides composed of achiral α-aminoisobutyric acid (Aib) and 4-aminoisocaproic acid (Aic, doubly homologated Aib) monomers in 1:1 alternation are reported. The 12-helices were indicated by solution and crystal structural analyses of tetra- and heptapeptides. Surprisingly, single crystals of the longer nonapeptide displayed two different helix types: the novel 12-helix and an unprecedented 15/17-helix. Quantum chemical calculations on both helix types in a series of continuously lengthened Aib/Aic-hybrid peptides confirm that the 12-helix is more stable than the 15/17-helix in shorter peptides, whereas the 15/17-helix is more stable in longer sequences. Thus, the coexistence of both helix types can be expected within a definite range of sequence lengths. The novel 15/17- and 12-helices in α,γ-hybrid peptides with 5→1 and 4→1 hydrogen-bonding patterns, respectively, can be viewed as backbone-expanded analogues of native α- and 310 -helices.

Study on mutual interactions and electronic structures of hyaluronan with Lysine, 6-Aminocaproic acid and Arginine.

Interactions between polyelectrolytes and oppositely charged surfactants have been in a great interest for several decades, yet the conventional surfactants may cause a problem in medical applications. Interactivity between polysaccharide hyaluronan (HA) and amino acids Lysine, 6-Aminocaproic acid (6-AcA), and Arginine as an alternative system is reported. The interactions were investigated by means of rheology and electric conductance and the electronic structures were explored by the density functional theory (DFT). Lysine exhibits the strongest interaction of all, which was manifested, e.g. by nearly 6-time drop of the initial viscosity comparing with only 1.3-time lower value in the case of 6-AcA. Arginine interaction with HA was surprisingly weaker in terms of viscosity than that of Lysine due to a lower and delocalized charge density on its guanidine group. According to the DFT calculations, the binding of Lysine to HA was found to be more flexible, while Arginine creates more rigid structure with HA.

A bio-injectable algin-aminocaproic acid thixogel with tri-stimuli responsiveness.

In this article a novel bio-injectable algin-aminocaproic acid (Alg-ACA) tri-stimuli responsive thixogel system is reported. The designed soft thixotrophic hydrogel (thixogel) was characterized using various analytical techniques such as FT-IR, NMR, SEM, AFM and DSC. The soft thixogel system was further investigated for stress responsiveness using different rheological studies which confirmed the thixotropic nature of the gel [Thixotropic area (Ar) of Alg-ACA (1:0.5), Alg-ACA (1:1) and Alg-ACA (1:2), were 23.5%, 43.1%, and 27.59%, respectively, which were higher than that of Na-Alg (2.08%)]. The thixogel also demonstrated temperature and ultrasonication responsiveness. This tri-stimuli responsive soft thixogel system was rendered flowable (fluid) on applying the described physical stimuli and recovered its "rigid" gel structure upon removal of the applied stimuli. This approach of synthesizing a thixogels may be applicable to a broad variety of other natural polymers and has the potential for use in biomedical applications.

Development of CdS Nanostructures by Thermal Decomposition of Aminocaproic Acid-Mixed Cd-Thiourea Complex Precursor: Structural, Optical and Photocatalytic Characterization.

The present work deals with two different CdS nanostructures produced via hydrothermal and solvothermal decompositions of aminocaproic acid (ACA)-mixed Cd-thiourea complex precursor at 175 °C. Both nanostructures were extensively characterized for their structural, morphological and optical properties. The powder X-ray diffraction characterization showed that the two CdS nanostructures present a wurtzite morphology. Scanning electron microscopy and energy-dispersive X-ray characterizations revealed that the hydrothermal decomposition produced well-shaped CdS flowers composed of six dendritic petals, and the solvothermal decomposition produced CdS microspheres with close stoichiometric chemical composition. The UV-vis absorption and photoluminescence spectra of CdS dendritic flowers and microsphere nanostructures showed that both nanostructures present a broad absorption between 200 and 700 nm and exhibit strong green emissions at 576 and 520 nm upon excitations at 290 nm and 260 nm, respectively. The transmission electron microscopy (TEM) and Brunauer-Emmett-Teller (BET) characterizations confirmed that CdS microspheres were mesoporous and were composed of small nanocrystals. A possible growth mechanism in the formation of the CdS nanostructures was proposed based on morphology evolution as a function of the reaction time. Furthermore, the as-synthesized CdS nanostructures were found to exhibit highly efficient photocatalytic activities for the degradation of methyl orange (MeO) and rhodamine B (RhB) dyes.

Evaluation of a specific diagnostic marker for rheumatoid arthritis based on cyclic citrullinated peptide.

A specific peptide marker for diagnosing rheumatoid arthritis (RA) was found based on cyclic citrullinated peptide (CCP) using the following three steps: (1) analysis of the binding epitope of autoimmune antibodies using ϵ-aminocaproic acid-modified peptides; (2) RA diagnosis using sequence-modified peptides; and (3) evaluation of the peptides' diagnostic performance for RA diagnosis. Ninety-five serum samples were analyzed by ELISA and compared using MedCalc (version 15.2.1). Microplate binding ϵ-aminocaproic acid was added to the N- or C-terminal of the CCP sequence. The N-terminal anchoring peptide assay showed 15% higher specificity compared with the C-terminal anchoring peptide assay. Based on this result, the hydrophilic C-terminal sequence of CCP was substituted with a hydrophobic amino acid. Among the sequence-modified peptides, CCP11A (in which alanine was substituted for the 11th amino acid of CCP) assay showed the highest sensitivity (87%) and specificity (100%) for RA diagnosis. Thus, CCP11A was selected as a possible specific marker peptide for RA diagnosis and further analyzed. The results of this analysis indicated that CCP11A showed better specificity than the CCP assay in both healthy individuals (11% better) and OA cohort (20% better). From these results, CCP11A was evaluated as a specific marker for diagnosing RA with higher diagnostic performance.

Evaluation of microwave oven heating for prediction of drug-excipient compatibilities and accelerated stability studies.

Microwave ovens have been used extensively in organic synthesis in order to accelerate reaction rates. Here, a set up comprising a microwave oven combined with silicon carbide (SiC) plates for the controlled microwave heating of model formulations has been applied in order to investigate, if a microwave oven is applicable for accelerated drug stability testing. Chemical interactions were investigated in three selected model formulations of drug and excipients regarding the formation of ester and amide reaction products. In the accelerated stability studies, a design of experiments (DoE) approach was applied in order to be able to rank excipients regarding reactivity: Study A: cetirizine with PEG 400, sorbitol, glycerol and propylene glycol. Study B: 6-aminocaproic acid with citrate, acetate, tartrate and gluconate. Study C: atenolol with citric, tartaric, malic, glutaric, and sorbic acid. The model formulations were representative for oral solutions (co-solvents), parenteral solutions (buffer species) and solid dosage forms (organic acids applicable for solubility enhancement). The DoE studies showed overall that the same impurities were generated by microwave oven heating leading to temperatures between 150°C and 180°C as compared to accelerated stability studies performed at 40°C and 80°C using a conventional oven. Ranking of the reactivity of the excipients could be made in the DoE studies performed at 150-180°C, which was representative for the ranking obtained after storage at 40°C and 80°C. It was possible to reduce the time needed for drug-excipient compatibility testing of the three model formulations from weeks to less than an hour in the three case studies. The microwave oven is therefore considered to be an interesting alternative to conventional thermal techniques for the investigation of drug-excipient interactions during preformulation.

Structure elucidation and quantification of impurities formed between 6-aminocaproic acid and the excipients citric acid and sorbitol in an oral solution using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy.

Concentrated solutions containing 6-aminocaproic acid and the excipients citric acid and sorbitol have been studied at temperatures of 50°C, 60°C, 70°C and 80°C as well as at 20°C. It has previously been reported that the commonly employed citric acid is a reactive excipient, and it is therefore important to thoroughly investigate a possible reaction between 6-aminocaproic acid and citric acid. The current study revealed the formation of 3-hydroxy-3,4-dicarboxy-butanamide-N-hexanoic acid between 6-aminocaproic acid and citric acid by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance spectroscopy (NMR). Less than 0.03% of 6-aminocaproic acid was converted to 3-hydroxy-3,4-dicarboxy-butanamide-N-hexanoic acid after 30 days of storage at 80°C. Degradation products of 6-aminocaproic acid were also observed after storage at the applied temperatures, e.g., dimer, trimer and cyclized 6-aminocaproic acid, i.e., caprolactam. No reaction products between D-sorbitol and 6-aminocaproic acid could be observed. 3-Hydroxy-3,4-dicarboxy-butanamide-N-hexanoic acid, dimer and caprolactam were also observed after storage at 20°C for 3 months. The findings imply that an oral solution of 6-aminocaproic acid is relatively stable at 20°C at the pH values 4.00 and 5.00 as suggested in the USP for oral formulations. Compliance with the ICH guideline Q3B is expected.