Rational Design of Amphipathic Antimicrobial Peptides with Alternating L-/D-Amino Acids That Form Helical Structures.

Antimicrobial peptides (AMPs) are promising therapeutic agents against bacteria. We have previously reported an amphipathic AMP Stripe composed of cationic L-Lys and hydrophobic L-Leu/L-Ala residues, and Stripe exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria. Gramicidin A (GA), composed of repeating sequences of L- and D-amino acids, has a unique ß6.3-helix structure and exhibits broad antimicrobial activity. Inspired by the structural properties and antimicrobial activities of LD-alternating peptides such as GA, in this study, we designed Stripe derivatives with LD-alternating sequences. We found that simply alternating L- and D-amino acids in the Stripe sequence to give StripeLD caused a reduction in antimicrobial activity. In contrast, AltStripeLD, with cationic and hydrophobic amino acids rearranged to yield an amphipathic distribution when the peptide adopts a ß6.3-helix, displayed higher antimicrobial activity than AltStripe. These results suggest that alternating L-/D-cationic and L-/D-hydrophobic amino acids in accordance with the helical structure of an AMP may be a useful way to improve antimicrobial activity and develop new AMP drugs.

In Silico Prediction of N-Nitrosamine Formation Pathways of Pharmaceutical Products.

The recent discovery of N-nitrosodimethylamine (NDMA), a mutagenic N-nitrosamine, in pharmaceuticals has adversely impacted the global supply of relevant pharmaceutical products. Contamination by N-nitrosamines diverts resources and time from research and development or pharmaceutical production, representing a bottleneck in drug development. Therefore, predicting the risk of N-nitrosamine contamination is an important step in preventing pharmaceutical contamination by DNA-reactive impurities for the production of high-quality pharmaceuticals. In this study, we first predicted the degradation pathways and impurities of model pharmaceuticals, namely gliclazide and indapamide, in silico using an expert-knowledge software. Second, we verified the prediction results with a demonstration test, which confirmed that N-nitrosamines formed from the degradation of gliclazide and indapamide in the presence of hydrogen peroxide, especially under alkaline conditions. Furthermore, the pathways by which degradation products formed were determined using ranitidine, a compound previously demonstrated to generate NDMA. The prediction indicated that a ranitidine-related compound served as a potential source of nitroso groups for NDMA formation. In silico software is expected to be useful for developing methods to assess the risk of N-nitrosamine formation from pharmaceuticals.

CRBN ligand expansion for hematopoietic prostaglandin D2 synthase (H-PGDS) targeting PROTAC design and their in vitro ADME profiles.

An increasing number of research reports are describing modifications of the E3 ligand, in particular, cereblon (CRBN) ligands, to improve the chemical and metabolic stabilities as well as the physical properties of PROTACs. In this study, phenyl-glutarimide (PG) and 6-fluoropomalidomide (6-F-POM), recently used as CRBN ligands for PROTAC design, were applied to hematopoietic prostaglandin D2 synthase (H-PGDS)-targeted PROTACs. Both PROTAC-5 containing PG and PROTAC-6 containing 6-F-POM were found to have potent activities to induce H-PGDS degradation. Furthermore, we obtained in vitro ADME data on the newly designed PROTACS as well as our previously reported PROTACs(H-PGDS) series. Although all PROTACs(H-PGDS) are relatively stable toward metabolism, they had poor PAMPA values. Nevertheless, PROTAC-5 showed Papp values similar to TAS-205, which is in Phase 3 clinical trials, and is expected to be the key to improving the pharmacokinetics of PROTACs.

Body fluid volume calculated using the uric acid kinetic model relates to the vascular event.

PURPOSE: We developed a method to measure the extracellular and intracellular fluid volumes using the kinetics of uric acid in the bodies of Japanese patients undergoing dialysis. In this research, we aimed to assess the prognosis of vascular events using this uric acid kinetic model method. METHODS: We conducted a retrospective cohort study of 1,298 patients who were undergoing hemodialysis or predilution online hemodiafiltration at the end of December 2019 at 13 institutions in Japan. Information on vascular events was acquired in 2020. Vascular event prognosis was defined as the new incidence of one or more of the following four types of vascular events: myocardial infarction, cerebral infarction, cerebral hemorrhage, or limb amputation. We measured the extracellular fluid volume and intracellular fluid volume after dialysis using the uric acid kinetic model method and determined the association between ECV, ICV, and vascular event risk. RESULTS: A high extracellular volume was substantially linked to an increased risk of vascular events. In addition, while a crude analysis revealed that a high intracellular volume was associated with a low risk of vascular events, this was not statistically significant after multifactorial adjustment. This result was partly affected by the low measurement accuracy of the serum urea nitrogen level used for the intracellular volume calculation. CONCLUSIONS: Extracellular volume calculated using the uric acid kinetic model method is a prognostic factor for vascular events in patients undergoing hemodialysis.

Comparison of Body Fluid Volumes Determined by Kinetic Modeling and by Bioimpedance Spectroscopy.

INTRODUCTION: The bioimpedance spectroscopy (BIS) method is used in individual patients requiring body fluid volume measurement. In a hemodialysis facility, however, regular screening of body fluid volumes is also necessary. Such screening, by kinetic modeling, may become possible by calculating distribution volumes of urea and uric acid from regular blood test results. OBJECTIVE: The aim is to compare uric acid distribution volumes with BIS-extracellular volume, urea distribution volume with BIS-total body water, and difference between urea and uric acid distribution volumes with BIS-intracellular volume. METHODS: We reanalyzed stored blood test data of 53 hemodialysis patients obtained together with BIS data of the same patients in our previous study. RESULTS: Significant correlations were found between urea distribution volume and total body water predicted by the BIS method, between uric acid distribution volume and extracellular volume predicted by the BIS method, and between the difference of uric acid distribution volume from urea distribution volume and intracellular volume predicted by the BIS method. In Bland-Altman analysis, comparison of each pair showed no systematic error. The mean difference between each pair was minimal. CONCLUSION: Fluid volumes in different body compartments can be estimated by kinetic modeling as well as by the BIS method.

Development of ciclesonide analogues that block SARS-CoV-2 RNA replication.

Ciclesonide is an inhaled corticosteroid used to treat asthma and is currently undergoing clinical trials for treatment of coronavirus disease 2019 (COVID-19). An active metabolite of ciclesonide, Cic2, was recently reported to repress severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genomic RNA replication. Herein, we designed and synthesized a few types of ciclesonide analogues. Cic4 (bearing an azide group) and Cic6 (bearing a chloro group) potently decreased SARS-CoV-2 viral replication and had low cytotoxicity compared with Cic2 (bearing a hydroxy group). These compounds are promising as novel therapeutic agents for COVID-19 that show significant antiviral activity.

Synthesis of Norgestomet and its 17ß-isomer and evaluation of their agonistic activities against progesterone receptor.

Norgestomet is a synthetic progesterone derivative applied in veterinary medicine to control estrus and ovulation in cattle. Norgestomet has been widely used in the livestock industry to promote the synchronization of estrus in cattle and increase pregnancy rates. However, highly reproducible synthetic methods for Norgestomet have been rarely reported. Here, we described a method for the synthesis of Norgestomet and performed quantitative NMR analysis to determine the purity of the products. Moreover, the agonistic activity of the synthesized compounds against progesterone receptors (PRs) was evaluated using an alkaline phosphatase assay. We synthesized Norgestomet with 97.9% purity that exhibited agonistic activity against PR with EC50 values of 4.5 nM. We also synthesized the 17ß-isomer of Norgestomet with 92.7% purity that did not exhibit any PR agonistic activity. The proposed synthetic route of Norgestomet can facilitate the assessment of residual Norgestomet in foods.

User of angiotensin-converting-enzyme inhibitor and/or angiotensin II receptor blocker might be associated with vascular calcification in predialysis chronic kidney disease patients: a retrospective single-center observational study : ACEI/ARB and vascular calcification.

BACKGROUND: Vascular calcification is a prominent feature in chronic kidney disease (CKD) and diabetes mellitus. A recent report suggests that angiotensin II is protective to vascular calcification. Therefore, we investigated the relationship between vascular calcification and use of angiotensin-converting-enzyme inhibitor (ACEI) and/or angiotensin II receptor blocker (ARB) from a cross-sectional view. METHODS: A total of 121 predialysis CKD patients (age 71 ± 12 y; male 72; estimated glomerular filtration rate (eGFR) 20.2 (11.8 - 40.3) mL/min/1.73 m2) who underwent thoracoabdominal plain computed tomography scan were included in this study. The total vascular calcification volume (Calc) was calculated with a three-dimensional imaging software and standardized by body surface area (BSA). The relevance between log [Calc/BSA] and ACEI/ARB use was investigated by multivariate linear regression analyses with or without a time-duration factor of ACEI/ARB use. RESULTS: The Calc/BSA was 5.62 (2.01 - 12.7) mL/m2 in 121 patients. In multivariate analyses adjusted with age, sex, ACEI/ARB and log [eGFR], ACEI/ARB use is significantly and positively associated with log [Calc/BSA] (ß = 0.2781, p = 0.0007). Even after the adjustment by age, sex, log [eGFR], phosphate, diabetes mellitus, systolic blood pressure, warfarin, hypertension, dyslipidemia, low-density lipoprotein cholesterol, diuretics and ACEI/ARB, ACEI/ARB use is significantly and positively associated with log [Calc/BSA] (ß = 0.1677, p = 0.0487). When 90 patients whose time-duration of ACEI/ARB use was clear in medical records were studied, a multivariate analysis adjusted with age, sex, log [eGFR], and ACEI/ARB duration factors showed that the longer use of ACEI/ARB more than 2 years was significantly, independently and positively associated with log [Calc/BSA] (ß = 0.2864, p = 0.0060). CONCLUSIONS: ACEI/ARB user was associated with vascular calcification in predialysis patients with low eGFR. Prospective studies with larger numbers of patients or more in vitro studies are needed to confirm whether this phenomenon is due to the use of ACEI/ARB itself, the underlying disease condition or the prescription bias.

Peptide Stapling Improves the Sustainability of a Peptide-Based Chimeric Molecule That Induces Targeted Protein Degradation.

Peptide-based target protein degradation inducers called PROTACs/SNIPERs have low cell penetrability and poor intracellular stability as drawbacks. These shortcomings can be overcome by easily modifying these peptides by conjugation with cell penetrating peptides and side-chain stapling. In this study, we succeeded in developing the stapled peptide stPERML-R7, which is based on the estrogen receptor alpha (ERα)-binding peptide PERML and composed of natural amino acids. stPERML-R7, which includes a hepta-arginine motif and a hydrocarbon stapling moiety, showed increased α-helicity and similar binding affinity toward ERα when compared with those of the parent peptide PERML. Furthermore, we used stPERML-R7 to develop a peptide-based degrader LCL-stPERML-R7 targeting ERα by conjugating stPERML-R7 with a small molecule LCL161 (LCL) that recruits the E3 ligase IAPs to induce proteasomal degradation via ubiquitylation. The chimeric peptide LCL-stPERML-R7 induced sustained degradation of ERα and potently inhibited ERα-mediated transcription more effectively than the unstapled chimera LCL-PERML-R7. These results suggest that a stapled structure is effective in maintaining the intracellular activity of peptide-based degraders.

Doublecortin marks a new population of transiently amplifying muscle progenitor cells and is required for myofiber maturation during skeletal muscle regeneration.

Muscle satellite cells are indispensable for muscle regeneration, but the functional diversity of their daughter cells is unknown. Here, we show that many Pax7(+)MyoD(-) cells locate both beneath and outside the basal lamina during myofiber maturation. A large majority of these Pax7(+)MyoD(-) cells are not self-renewed satellite cells, but have different potentials for both proliferation and differentiation from Pax7(+)MyoD(+) myoblasts (classical daughter cells), and are specifically marked by expression of the doublecortin (Dcx) gene. Transplantation and lineage-tracing experiments demonstrated that Dcx-expressing cells originate from quiescent satellite cells and that the microenvironment induces Dcx in myoblasts. Expression of Dcx seems to be necessary for myofiber maturation because Dcx-deficient mice exhibited impaired myofiber maturation resulting from a decrease in the number of myonuclei. Furthermore, in vitro and in vivo studies suggest that one function of Dcx in myogenic cells is acceleration of cell motility. These results indicate that Dcx is a new marker for the Pax7(+)MyoD(-) subpopulation, which contributes to myofiber maturation during muscle regeneration.

The role of acylated-ghrelin in the regulation of sucrose intake.

The octanoyl modification of ghrelin by ghrelin O-acyltransferase (GOAT) is essential for exerting its physiologic actions. Since exogenous acylated-ghrelin has shown to stimulate food intake in humans and rodents, GOAT has been regarded as a promising target for modulating appetite, thereby treating obesity and diabetes. However, GOAT-knockout (KO) mice have been reported to show no meaningful body weight reduction, when fed a high-fat diet. In this study, we sought to determine whether GOAT has a role in the regulation of body weight and food intake when fed a dietary sucrose. We found that GOAT KO mice showed significantly reduced food intake and marked resistance to obesity, when fed a high-fat + high-sucrose diet. In addition, GOAT KO mice fed a medium-chain triglyceride (MCT) + high-sucrose diet showed a marked resistance to obesity and reduced feed efficiency. These results suggest that blockade of acylated-ghrelin production offers therapeutic potential for obesity caused by overconsumption of palatable food.

The Superiority of AN69ST Membrane in the Adsorption of Fibroblast Growth Factor-23.

BACKGROUND: The most frequent cause of acute kidney injury (AKI) needing renal replacement therapy is sepsis, with the prognosis of patients with septic AKI worse than for other causes of this disease. Recent studies have shown that fibroblast growth factor-23 (FGF-23) levels, one of the phosphaturic and prognostic factors in chronic kidney disease, are also elevated in patients with AKI and correlate with an increased risk of death or a need for dialysis. In addition, FGF-23 was found to inhibit the extra-renal synthesis of 1,25-dihydroxyvitamin D by human monocytes. Therefore, the elevated expression of FGF-23 may play a crucial role in defining the immune response to vitamin D and this, in turn, may be a key determinant of infection in patients. Continuous renal replacement therapy (CRRT) is often essential for the treatment of septic AKI. However, reports related to the influence of CRRT on serum FGF-23 levels are lacking. In this study, we undertook preliminary in vitro investigations evaluating the effect of different types of CRRT membranes on FGF-23 adsorption. METHODS: To study how FGF-23 is adsorbed by hemofiltration fiber, in vitro experiments were performed based on a batch method using three types of fiber: polysulfone (PS), polymethyl methacrylate (PMMA), and acrylonitrileco-methallyl sulfonate surface treatment (AN69ST) fiber. RESULTS: The adsorptive properties of the various membranes were determined from measuring changes in the concentration of FGF-23 in a solution containing the membrane after incubation for 60 or 240 minutes. The amount of FGF-23 adsorbed by an AN69ST membrane was significantly more than for other membranes (P < 0.01). The amounts adsorbed by PS and PMMA membranes were similar. CONCLUSIONS: We found an AN69ST membrane has a greater capacity for FGF-23 adsorption than two other membranes tested. Although this is an in vitro study, we believe the present findings indicate an exciting new direction in the treatment of septic AKI and highlight the necessity of acute clinical investigations using AN69ST-CRRT.

Abnormal Diurnal Patterns of Parathyroid Hormone are Associated with Sustained Mild Hypercalcemia in Non-Dialyzed Chronic Kidney Disease.

BACKGROUND: Plasma PTH levels are normally high during the night and early morning and lowest at approximately 10 am (the PTH circadian rhythm). Our objective was to examine the relationship between the PTH circadian rhythm and calcium-phosphorus metabolism in non-dialyzed, chronic kidney disease (CKD) patients. METHODS: The characteristics of twenty-eight subjects comprised: male, 23; diabetic patients, 16; mean age, 71.1 +/- 10.5 years; mean eGFR, 18.3 +/- 8.1 mL/min/1.73 m2. Under a protein-restricted diet, plasma intact PTH (iPTH) was measured at 7 am (iPTH7), 10 am (iPTH10), and 10 pm (iPTH22). Serum concentrations of calcium (Ca), phosphate (Pi), 25-hydroxyvitamin D (25-OHD), 1,25-dihydroxyvitamin D (1,25[OH]2D), and fibroblast growth factor (FGF)-23 were measured at 7 am. A normal iPTH rhythm was defined as when both iPTH7 and iPTH22 exceeded iPTH10. When iPTH10 was equal to, or exceeded either iPTH7 or iPTH22, or both, the rhythm was considered abnormal. RESULTS: Median levels of iPTH7, iPTH10, and iPTH22 were 92.5 [IQR: 60.8-152.01, 85.5 [61.0-144.5], and 95.5 164.3-160.5] pg/mL, respectively. Sixteen subjects showed an abnormal iPTH rhythm. There was no significant difference between groups in age, eGFR, iPTH7, iPTH10, iPTH22, Pi, 25-OH1D, 1,25(OH)2D, or FGF-23. However, the abnormal group showed significantly higher mean levels of corrected Ca as compared to the normal group (9.50 +/- 0.42 vs. 9.18 +/- 0.28; p = 0.032). CONCLUSIONS: Abnormal diurnal patterns of PTH are associated with sustained mild hypercalcemia in nondialyzed chronic kidney disease patients. This abnormal rhythm was not associated with Pi or FGF-23, and this may be an independent risk factor for CKD-mineral and bone disorder.

Continuous Hemodiafiltration with an AN69ST Hemofilter (AN69ST-CHDF) as FGF-23-Lowering Therapy.

BACKGROUND: Recent studies have shown that fibroblast growth factor-23 (FGF-23) is elevated not only in chronic kidney disease (CKD), but also in acute illnesses such as acute kidney injury, septic shock, and acute heart failure. FGF-23 would be not only a simple biomarker but also a direct toxic factor in acute illness. Therefore, lowering circulating FGF-23 levels in clinical practice would be an exciting and valuable interventional strategy. Continuous hemodiafiltration (CHDF) is often performed for the treatment of the aforementioned acute illnesses. We have previously reported that an acrylonitrile-co-methallyl sulfonate surface-treated (AN69ST) membrane has a greater capacity for in vitro FGF-23 adsorption than polysulfone and polymethyl methacrylate membranes. However, reports related to the influence of AN69ST-CHDF on serum FGF-23 levels in acute illness are lacking. In this study, we investigated the effect of AN69ST-CHDF on circulating FGF-23 concentrations in clinical practice. METHODS: Subjects comprised six inpatients who underwent AN69ST-CHDF for an acute illness. Blood samples for the measurement of serum FGF-23 were collected at 0, 3, and 12 hours post-treatment. Blood samples were also drawn from the extracorporeal circuit at the inlet and outlet of the hemofilter 3 hours after CHDF initiation, in order to calculate the clearance of serum FGF-23. RESULTS: Three and 12 hours after the start of AN69ST-CHDF, circulating FGF-23 levels decreased from baseline values with a marginal statistical significance (p = 0.0625 and 0.0938, respectively). An FGF-23 clearance of 27.5 [interquartile range: 19.4 - 29.2] mL/minute 3 hours after the initiation of AN69ST-CHDF was achieved. CONCLUSIONS: Our results suggest that AN69ST-CHDF can be a novel FGF-23 lowering therapy for acute illnesses requiring acute blood purification.

Impaired viability of muscle precursor cells in muscular dystrophy with glycosylation defects and amelioration of its severe phenotype by limited gene expression.

A group of muscular dystrophies, dystroglycanopathy is caused by abnormalities in post-translational modifications of dystroglycan (DG). To understand better the pathophysiological roles of DG modification and to establish effective clinical treatment for dystroglycanopathy, we here generated two distinct conditional knock-out (cKO) mice for fukutin, the first dystroglycanopathy gene identified for Fukuyama congenital muscular dystrophy. The first dystroglycanopathy model-myofiber-selective fukutin-cKO [muscle creatine kinase (MCK)-fukutin-cKO] mice-showed mild muscular dystrophy. Forced exercise experiments in presymptomatic MCK-fukutin-cKO mice revealed that myofiber membrane fragility triggered disease manifestation. The second dystroglycanopathy model-muscle precursor cell (MPC)-selective cKO (Myf5-fukutin-cKO) mice-exhibited more severe phenotypes of muscular dystrophy. Using an isolated MPC culture system, we demonstrated, for the first time, that defects in the fukutin-dependent modification of DG lead to impairment of MPC proliferation, differentiation and muscle regeneration. These results suggest that impaired MPC viability contributes to the pathology of dystroglycanopathy. Since our data suggested that frequent cycles of myofiber degeneration/regeneration accelerate substantial and/or functional loss of MPC, we expected that protection from disease-triggering myofiber degeneration provides therapeutic effects even in mouse models with MPC defects; therefore, we restored fukutin expression in myofibers. Adeno-associated virus (AAV)-mediated rescue of fukutin expression that was limited in myofibers successfully ameliorated the severe pathology even after disease progression. In addition, compared with other gene therapy studies, considerably low AAV titers were associated with therapeutic effects. Together, our findings indicated that fukutin-deficient dystroglycanopathy is a regeneration-defective disorder, and gene therapy is a feasible treatment for the wide range of dystroglycanopathy even after disease progression.

Hesr1 and Hesr3 are essential to generate undifferentiated quiescent satellite cells and to maintain satellite cell numbers.

Satellite cells, which are skeletal muscle stem cells, divide to provide new myonuclei to growing muscle fibers during postnatal development, and then are maintained in an undifferentiated quiescent state in adult skeletal muscle. This state is considered to be essential for the maintenance of satellite cells, but their molecular regulation is unknown. We show that Hesr1 (Hey1) and Hesr3 (Heyl) (which are known Notch target genes) are expressed simultaneously in skeletal muscle only in satellite cells. In Hesr1 and Hesr3 single-knockout mice, no obvious abnormalities of satellite cells or muscle regenerative potentials are observed. However, the generation of undifferentiated quiescent satellite cells is impaired during postnatal development in Hesr1/3 double-knockout mice. As a result, myogenic (MyoD and myogenin) and proliferative (Ki67) proteins are expressed in adult satellite cells. Consistent with the in vivo results, Hesr1/3-null myoblasts generate very few Pax7(+) MyoD(-) undifferentiated cells in vitro. Furthermore, the satellite cell number gradually decreases in Hesr1/3 double-knockout mice even after it has stabilized in control mice, and an age-dependent regeneration defect is observed. In vivo results suggest that premature differentiation, but not cell death, is the reason for the reduced number of satellite cells in Hesr1/3 double-knockout mice. These results indicate that Hesr1 and Hesr3 are essential for the generation of adult satellite cells and for the maintenance of skeletal muscle homeostasis.

Fibrosis and adipogenesis originate from a common mesenchymal progenitor in skeletal muscle.

Accumulation of adipocytes and collagen type-I-producing cells (fibrosis) is observed in muscular dystrophies. The origin of these cells had been largely unknown, but recently we identified mesenchymal progenitors positive for platelet-derived growth factor receptor alpha (PDGFRα) as the origin of adipocytes in skeletal muscle. However, the origin of muscle fibrosis remains largely unknown. In this study, clonal analyses show that PDGFRα(+) cells also differentiate into collagen type-I-producing cells. In fact, PDGFRα(+) cells accumulated in fibrotic areas of the diaphragm in the mdx mouse, a model of Duchenne muscular dystrophy. Furthermore, mRNA of fibrosis markers was expressed exclusively in the PDGFRα(+) cell fraction in the mdx diaphragm. Importantly, TGF-ß isoforms, known as potent profibrotic cytokines, induced expression of markers of fibrosis in PDGFRα(+) cells but not in myogenic cells. Transplantation studies revealed that fibrogenic PDGFRα(+) cells mainly derived from pre-existing PDGFRα(+) cells and that the contribution of PDGFRα(-) cells and circulating cells was limited. These results indicate that mesenchymal progenitors are the main origin of not only fat accumulation but also fibrosis in skeletal muscle.

[Changes in Test Methods for Internationalization in the Japanese Pharmacopoeia (Part 3): Establishment of a Quantitative Method for Sodium Cromoglicate and Trihexyphenidyl Hydrochloride Using HPLC Analysis].

The Japanese Pharmacopoeia (JP) is an official normative publication for establishing the authenticity and properties and maintaining the quality of pharmaceutical products in Japan. The JP is revised every five years and partially revised in order to respond to the progress of science and technology, the demand for medical care, and international harmonization. Thus, "Internationalization of the JP" is one of the most important issues to address for the revision of the JP, which is also referred to the basic principles for the preparation of the JP 19th edition. For instance, the incorporation of the test methods that have been used in other pharmacopeias, such as the European Pharmacopoeia (EP) and the United States Pharmacopeia (USP), into the JP is one of promising approaches. From this perspective, we have recently reported changes in test methods, establishment of a quantitative test method for the JP-listed clonidine hydrochloride as well as lorazepam from using a potentiometric titration method to using HPLC method. As our ongoing study to change test methods for internationalization, we selected sodium cromoglicate and trihexyphenidyl hydrochloride. Each pharmaceutical product is analyzed using a potentiometric titration method as listed in the 18th JP; however, both the EP and the USP use HPLC method for quantitative analysis of these drugs. In this study, we synthesized the related impurities of sodium cromoglicate and trihexyphenidyl hydrochloride listed in the EP and determined their purities using quantitative NMR. The separation conditions of these compounds were examined using HPLC and simultaneous analyses were performed.

Enhancing Chemical Stability through Structural Modification of Antimicrobial Peptides with Non-Proteinogenic Amino Acids.

Multidrug-resistant bacteria (MDRB) remain a significant threat to humanity, resulting in over 1.2 million deaths per year. To combat this problem effectively, the development of therapeutic agents with diverse mechanisms of action is crucial. Antimicrobial peptides (AMPs) have emerged as promising next-generation therapeutics to combat infectious diseases, particularly MDRB. By targeting microbial membranes and inducing lysis, AMPs can effectively inhibit microbial growth, making them less susceptible to the development of resistance. Numerous structural advancements have been made to optimize the efficacy of AMPs. Previously, we developed 17KKV-Aib, a derivative of the Magainin 2 (Mag2) peptide, by incorporating a,a-disubstituted amino acids (dAAs) to modulate its secondary structure. 17KKV-Aib demonstrated potent antimicrobial activity against Gram-positive and Gram-negative bacteria, including multidrug-resistant Pseudomonas aeruginosa (MDRP), with minimal hemolytic activity against human red blood cells. However, 17KKV-Aib faces challenges regarding its susceptibility to digestive enzymes, hindering its potential as an antimicrobial agent. In this study, we designed and synthesized derivatives of 17KKV-Aib, replacing Lys residues with 4-aminopiperidine-4-carboxylic acid (Api), which is a cyclized dAA residue possessing cationic properties on its side chain. We investigated the impact of Api substitution on the secondary structure, antimicrobial activity, hemolytic activity, and resistance to digestive enzymes. Our findings revealed that introducing Api residues preserved the helical structure and antimicrobial activity and enhanced resistance to digestive enzymes, with a slight increase in hemolytic activity.