Arginine and its Derivatives Suppress the Opalescence of an Antibody Solution.

Opalescence is a problem concerned with the stability of an antibody solution. It occurs when a high concentration of a protein is present. Arginine (Arg) is a versatile aggregation suppressor of proteins, which is among the candidates that suppress opalescence in antibody solutions. Here, we investigated the effect of various types of small molecular additives on opalescence to reveal the mechanism of Arg in preventing opalescence in antibody solution. As expected, Arg suppressed the opalescence of the immunoglobulin G (IgG) solution. Arg also concentration dependently inhibited the formation of microstructures in IgG molecules. Interestingly, the intrinsic fluorescence spectra of highly concentrated IgG solutions differed from those having low concentrations, even though IgG retained a distinct tertiary structure. Arginine ethylester was more effective in suppressing the opalescence of IgG solutions than Arg, whereas lysine and γ-guanidinobutyric acid were less effective. These results indicated that positively charged groups of both α-amine and guanidinium actively influence Arg as an additive for suppressing opalescence. Diols, which are the suppressors of the liquid-liquid phase separation of proteins were also effective in suppressing the opalescence. These results therefore provide insight into the control of opalescence of antibody solutions at high concentrations using solution additives.

Arginine suppresses opalescence and liquid-liquid phase separation in IgG solutions.

Antibody formulation often necessitates the protein concentration to be increased above 100 mg/ml, because of the large therapeutic doses of antibodies required and the volume limitations of subcutaneous injections. However, high concentrations of antibody lead to opalescent states in solution, resulting in safety and application problems. In this study, we investigated the effect of additives on opalescence in IgG solutions. Arginine (Arg) was observed to most effectively suppress opalescence in IgG solutions among the additives tested, which included guanidine hydrochloride, NaCl, and other amino acids. Moreover, Arg also suppressed liquid-liquid phase separation (LLPS) of highly concentrated IgG solutions during incubation at low temperature. Comparative analysis showed that the effects of Arg on opalescence and LLPS in IgG solutions result from its unique structure, which comprises an amino acid main chain, a guanidinium group, and a counter ion. These results indicate that Arg has high potency as an excipient in antibody drug formulations for the suppression of opalescence and LLPS as well as protein aggregation.

A Novel p.L145Q Mutation in the HNF1B Gene in a Case of Maturity-onset Diabetes of the Young Type 5 (MODY5).

Maturity-onset diabetes of the young (MODY) is an autosomal dominant form of early onset diabetes. The hepatocyte nuclear factor-1-beta (HNF1B) gene is responsible for MODY type 5 (MODY5) with distinctive clinical features, including pancreatic atrophy and renal disease. We herein report a Japanese case of young-onset diabetes with typical phenotypes of MODY5 and a novel heterozygous missense mutation (p.L145Q) in the HNF1B gene. The mutation was located in the Pit-Oct-Unc (POU)-specific domain, and the amino acid residue L145 was highly conserved among species. It is strongly suggested that this mutation explains the phenotypes of MODY5.

Specific solubilization of impurities in culture media: Arg solution improves purification of pH-responsive tag CspB50 with Teriparatide.

Protein purification using non-chromatographic methods is a simple technique that avoids costly resin. Recently, a cell surface protein B (CspB) tag has been developed for a pH-responsive tag for protein purification by solid-liquid separation. Proteins fused with the CspB tag show reversible insolubilization at acidic pH that can be used in solid-liquid separation for protein purification. However, brown-color impurities from co-precipitation hamper further analysis of the target proteins. In this study, we investigated the effect of additives on the co-precipitation of CspB-tagged Teriparatide (CspB50TEV-Teriparatide) expressed in Corynebacterium glutamicum and associated impurities. Arginine (Arg) at 1.0 M was found to be the most effective additive for removing impurities, particularly carotenoids and nucleic acids. Furthermore, all impurities detected in the fluorescence and absorbance spectra were successfully removed by the repetition of precipitation-redissolution in the Arg solution. The precipitation yield of the CspB50TEV-Teriparatide did not change with the addition of Arg and the repetition of the precipitation-redissolution process. Collectively, our findings indicate that the specific desorption of π-electron rich compounds by Arg may be useful in conjunction with the pH-responsive CspB tag for solid-liquid protein purification.

Trimethylamine N-oxide (TMAO) is a counteracting solute of benzyl alcohol for multi-dose formulation of immunoglobulin.

Multi-dose formulation of biopharmaceuticals contains an antimicrobial preservative, which may facilitate aggregation of the proteins due to their hydrophobic protein binding. Here, we have investigated the effects of co-solvents on heat-induced protein aggregation in the presence of an antimicrobial preservative. Human immunoglobulin G (IgG) and benzyl alcohol were chosen because of their wide usage as a pharmaceutical protein and a preservative. Trimethylamine N-oxide (TMAO) was found to be the most effective additive in suppressing benzyl alcohol-induced IgG aggregation among the additives tested. Interestingly, TMAO was less effective in increasing the thermal-unfolding temperature of IgG than trehalose, indicating that the observed suppression of IgG aggregation by TMAO in the presence of benzyl alcohol is not simply due to its stabilization effect of the protein structure. The solubility of benzyl alcohol increased with increasing concentrations of TMAO, suggesting that the hydrophobic interaction of TMAO with benzyl alcohol is responsible for the observed aggregation suppression. Thus, TMAO may be a potential aggregation suppressor in multi-dose formulation of biopharmaceuticals.

Mechanism of co-aggregation in a protein mixture with small additives.

Co-aggregation plays an important role in processing protein-rich food materials under heterogeneous conditions. The main cause of co-aggregation is an electrostatic attraction between oppositely charged molecules. This study investigated thermal aggregation of ß-lactoglobulin (BLG) (pI=5.1) and lysozyme (LYZ) (pI=10.7) as a model for the heterogeneous conditions of a protein solution. BLG and LYZ were more aggregated in the mixture than in the single solutions. Co-aggregation of the BLG-LYZ mixture was not observed below 60°C at which temperature BLG and LYZ retained their native structures. Adding sugars, salts, or amino acids to the BLG-LYZ mixture during the heat treatment revealed the co-aggregation process as follows. (i) All additives tested suppressed both the nucleation and growth of aggregates. (ii) Salts affected nucleation stage to the same degree, except arginine hydrochloride (Arg). (iii) Arg specifically suppressed both nucleation and growth of aggregates. These results indicate that co-aggregation in a protein mixture is more sensitive to the partial unfolding of proteins than that in a single protein solution, due to the presence of electrostatic attraction between different molecules. These results provide new insight into protein aggregation as well as the molecular mechanism of additives under heterogeneous conditions.

Challenging Differential Diagnosis of Hypergastremia and Hyperglucagonemia with Chronic Renal Failure: Report of a Case with Multiple Endocrine Neoplasia Type 1.

A 53-year-old woman developed end-stage renal failure during a 15-year clinical course of primary hyperparathyroidism and was referred to our hospital for evaluation of suspected multiple endocrine neoplasia type 1 (MEN1). Genetic testing revealed a novel deletion mutation at codon 467 in exon 10 of the MEN1 gene. Systemic and selective arterial calcium injection (SACI) testing revealed hyperglucagonemia and hypergastrinemia with positive gastrin responses. A pathological examination revealed glucagonoma and a lymph node gastrinoma. The findings in this case indicate the importance of early diagnosis of MEN1 and demonstrate the utility of systemic and SACI testing in renal failure cases.

Syndrome of Inappropriate Antidiuretic Hormone Secretion Associated with Amyotrophic Lateral Sclerosis in a Patient Developing Carbon Dioxide Narcosis.

We report a rare case of syndrome of inappropriate antidiuretic hormone secretion (SIADH) associated with amyotrophic lateral sclerosis (ALS). A 69-year-old man was admitted to our hospital with sustained hyponatremia. Hyposmolality with elevated urinary osmolality and sodium excretion was observed, which indicated SIADH. The treatment for SIADH was challenging; the patient developed carbon dioxide narcosis, which led to the diagnosis of ALS. After the initiation of noninvasive positive-pressure ventilation, the patient's serum sodium concentration normalized and became stable. Thus, ALS should be recognized as a possible cause of SIADH in the clinical setting.

Successful Glycemic Control Decreases the Elevated Serum FGF21 Level without Affecting Normal Serum GDF15 Levels in a Patient with Mitochondrial Diabetes.

Mitochondrial diabetes mellitus is a subtype of diabetes linked to mutations in mitochondrial DNA. In patients with mitochondrial diabetes mellitus, the effect of glycemic control on the serum concentrations of fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15) has not been evaluated. FGF21 and GDF15 have been reported to be useful biomarkers for the diagnosis and severity assessment of mitochondrial diseases like mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Recent studies have shown FGF21 acts in an endocrine fashion to regulate glucose and lipid metabolism in type 2 diabetes mellitus, while the exact biological functions of GDF15 remain unknown. Although mitochondrial diabetes mellitus is commonly found in cases with mitochondrial diseases, the comparison of FGF21 and GDF15 levels between those with and without diabetes has not been performed. Here, we report a 24-year-old woman with mitochondrial diabetes mellitus, who showed a high level of serum FGF21, but not serum GDF15, at diagnosis. In our case, liraglutide, a glucagon-like peptide-1 receptor agonist, added to insulin glargine was effective for her glycemic control and showed no adverse effects, including gastrointestinal symptoms and hypoglycemia, during a 14-week observation. The successful glycemic control caused a decrease in the FGF21 level, without affecting the GDF15 level. Thus, we should consider patients' glycemic control levels in using FGF21 values for the diagnosis of mitochondrial diseases. In addition, sustained GDF15 levels during glycemic treatment in our case suggest the usefulness of GDF15 as a marker for clinical severity of muscle-manifested mitochondrial diseases.

Early Onset of Diabetes Mellitus Accelerates Cognitive Decline in Japanese Patients with Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-Like Episodes.

Approximately 80% of patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) carry the A3243G mutation in the mitochondrial tRNALeu (UUR) gene. Conversely, this mutation has also been identified as one of the most prevalent genetic abnormalities in patients with diabetes mellitus. Mitochondrial diabetes mellitus complicated with MELAS is relatively common, and 12.5% of patients with the A3243G mutation develop MELAS after being diagnosed with diabetes mellitus. However, the clinical impact of diabetes mellitus in MELAS patients remains unclear. Therefore, we retrospectively studied 14 Japanese MELAS patients with the A3243G mutation: three men and eleven women, with the mean age of 48.0 (± 15.4) years. Eight patients had been diagnosed with diabetes mellitus prior to the diagnosis of mitochondrial disease, and all of them were treated with insulin. The other six included four patients with concurrent diagnosis of diabetes and mitochondrial disease, one patient diagnosed with diabetes after the diagnosis of mitochondrial disease, and one patient without developing diabetes currently. We thus compared the patients' characteristics between those with and without early onset of diabetes mellitus. Cognitive decline (75.0% vs. 0%; p = 0.03) and poor glycemic control with severe hypoglycemic events (75.0% vs. 16.7%; p = 0.05) were more common in MELAS patients with a prior diagnosis of diabetes than in those without the prior diagnosis of diabetes. Our data suggest that the latent progress of cognitive decline is accelerated because of early onset of diabetes mellitus in MELAS patients.

A Novel Missense Mutation of the MEN1 Gene in a Patient with Multiple Endocrine Neoplasia Type 1 with Glucagonoma and Obesity.

A 35-year-old obese diabetic man presented with recurrent primary hyperparathyroidism during a three-year outpatient follow-up. He was clinically diagnosed with multiple endocrine neoplasia type 1 (MEN1) due to the presence of a pituitary adenoma and multiple glucagonomas. The glucagonomas may have affected his glycemic control. However, he did not demonstrate weight loss, suggesting that the patient's obesity could have obscured the early diagnosis of a glucagonoma. Genetic testing revealed a novel missense mutation at codon 561 in exon 10, resulting in an amino acid substitution from methionine to arginine (M561R) in the MEN1 gene. This mutation appeared to be responsible for the MEN1 pathogenicity.

Diffuse thyroid enlargement following metastasis of lung adenocarcinoma.

A 55-year-old man presented with a rapidly enlarging thyroid. He had been diagnosed with lung adenocarcinoma nine months earlier. Computed tomography (CT) and ultrasound (US) detected reticular cord-like structures, but no nodules, in the thyroid. A fine-needle aspiration biopsy (FNAB) of the thyroid revealed thyroglobulin-negative adenocarcinoma cells, thus establishing the diagnosis of diffuse thyroid metastases of lung cancer. Moreover, the fluid demonstrated milky chyliform effusion. This case suggests that the presence of reticular cord-like structures on US and CT may be a key imaging finding for the clinical diagnosis of diffuse thyroid metastases and that chyliform effusion may contribute to rapid goiter formation.

Add-on treatment with mitiglinide improves residual postprandial hyperglycemia in type 2 diabetic patients receiving the combination therapy with insulin glargine and sitagliptin.

The combination of a dipeptidyl peptidase-4 (DPP-4) inhibitor and a long-acting insulin analogue is widely used in clinical practice. However, some patients fail to achieve lower postprandial hyperglycemia. Mitiglinide, a short-acting insulinotropic sulfonylurea receptor ligand, is effective for postprandial hyperglycemia. Recently, it has been reported that the combination therapy of mitiglinide with a DPP-4 inhibitor could improve glycemic control. However, the efficacy of those under long-acting insulin analogue therapy remains to be investigated. Thus, we conducted a prospective single-center study of eight Japanese patients with type 2 diabetes mellitus receiving mitiglinide added to the combination therapy of sitagliptin and insulin glargine, and evaluated its efficacy and safety by continuous glucose monitoring (CGM). Participants' (four men and four women) mean age was 70.3 ± 10.6 years. Their mean body mass index, HbA1c level, and urinary C-peptide level were 22.0 ± 2.8 kg/m(2), 9.2 ± 1.2%, and 50.0 ± 31.4 µg/day, respectively. CGM showed that as compared with the combination of only sitagliptin and insulin glargine, mitiglinide in combination with sitagliptin and insulin glargine significantly reduced glycemic fluctuation indices, total area for the range of 24-h glycemic fluctuations (p = 0.04), mean amplitude of glycemic excursions (p = 0.03), and the proportion of time in hyperglycemia (p = 0.02) without significant difference in the proportion of time in hypoglycemia (p = 0.18). Hence, we have demonstrated the efficacy and safety of the add-on treatment with mitiglinide in type 2 diabetic patients, receiving the combination therapy of sitagliptin and insulin glargine.

Prolonged disturbance of consciousness caused by severe hypophosphatemia: a report of two cases.

We herein describe two patients with a prolonged disturbance of consciousness due to severe hypophosphatemia. Case one presented with pneumococcal infection and acute exacerbation of chronic obstructive pulmonary disease and asthma. Case two presented with diabetic foot infections and diabetic ketoacidosis. Both patients responded to initial therapy for their primary diseases, but consciousness became worse in both cases. Their test results for impaired consciousness revealed severe hypophosphatemia; therefore, phosphate replacement therapy was administered, thus resulting in complete alertness. These cases demonstrate that we should consider the possibility of hypophosphatemia in critically ill patients with an altered consciousness.

Desmopressin orally disintegrating tablet in Japanese patients with central diabetes insipidus: a retrospective study of switching from intranasal desmopressin.

Central diabetes insipidus (CDI) is a rare disease characterized by polyuria and polydipsia. Patients with CDI have been successfully treated with desmopressin administered either by intranasal instillation or oral tablets. Recently, a desmopressin orally disintegrating tablet (ODT) was approved as the first oral desmopressin tablet for CDI treatment in Japan. We conducted a retrospective single-center study of 15 Japanese CDI patients treated with desmopressin ODT therapy, which aimed to evaluate the efficacy and safety of switching to desmopressin ODT and to analyze the clinical factors that affect the desmopressin ODT dose in Japanese patients. The daily mean dose of desmopressin ODT was 104 ± 46.30 µg and the mean ratio of oral to nasal desmopressin dose was 17.0 ± 7.6, both of which are considerably smaller than those of previous dose-titration study. Moreover, the nasal spray group needed significantly smaller ratios of nasal to oral desmopressin than the nasal drop group (11.7 ± 6.5 vs 21.0 ± 5.5, p = 0.02). The ratio of oral to nasal desmopressin dose had a significant inverse correlation with the required nasal desmopressin dose. Multiple regression analysis demonstrated the ratios of nasal to oral desmopressin dose depended on intranasal formulations. In conclusion, desmopressin ODT was safe and effective in the treatment of Japanese adult CDI patients. When switching to ODT, we should care about the possibility that patients require smaller ODT doses than what was initially expected based on previously published data and also nasal formulations in terms of their differences of expected switching ratio.

Pseudohypoparathyroidism type II in a woman with a history of thyroid surgery.

We herein describe the case of a woman with pseudohypoparathyroidism (PHP) type II. She had a history of subtotal thyroidectomy against Graves' disease without levothyroxine supplementation and presented with stiffness, numbness and muscle cramps. Her surgical history suggested the possibility of secondary hypoparathyroidism; however, the serum intact parathyroid hormone level and results of a Ellsworth-Howard test led to the diagnosis of PHP type II. In the present case, making the differential diagnosis was challenging because two distinct disorders, such as PHP and secondary hypoparathyroidism, may exist simultaneously. This case demonstrates the need to consider the possibility of PHP type II in patients exhibiting hypocalcemia.

Evaluation of the effects of exenatide administration in patients with type 2 diabetes with worsened glycemic control caused by glucocorticoid therapy.

Glucocorticoid-induced hyperglycemia is common in patients with or without known diabetes mellitus. Exenatide, a glucagon-like peptide-1 receptor agonist, improves glycemic control without causing weight gain or hypoglycemia and is currently widely used in patients with type 2 diabetes mellitus. We herein report four cases of patients with type 2 diabetes with worsened glycemic control due to glucocorticoids who were successfully treated with exenatide administration.

Liraglutide administration in type 2 diabetic patients who either received no previous treatment or were treated with an oral hypoglycemic agent showed greater efficacy than that in patients switching from insulin.

AIMS/INTRODUCTION: Liraglutide, a glucagon-like peptide-1 receptor agonist, is expected to provide a new treatment option for diabetes. However, the suitable timing of liraglutide administration in type 2 diabetic patients has not yet been clarified. MATERIALS AND METHODS: We reviewed type 2 diabetic patients (n = 155) who visited the Osaka Red Cross Hospital for glycemic control, with administration of liraglutide at a dose of 0.6 mg (average glycated hemoglobin [HbA1c] level, 8.7 ± 0.1%). The effect of liraglutide based on the pretreatment status was compared. We also analyzed the background factors of both a successful and failed group of patients who switched to liraglutide from insulin. RESULTS: An improvement in blood glucose levels was confirmed in 122 of 155 patients. During the 4-month observation period, the improvement in HbA1c levels was significantly greater in the group of drug-naïve/previous oral hypoglycemic agent (9.1 ± 0.2 to 7.2 ± 0.2%) than that in the group switching from insulin (8.6 ± 0.2 to 7.8 ± 0.2%). In addition, C-peptide immunoreactivity levels (fasting > 2.2 ng/mL; delta >1.6 ng/mL; urine > 70 µg/day), younger age and a smaller number of insulin units used per day were considered important when deciding on switching to liraglutide from insulin. CONCLUSIONS: Liraglutide was more effective in patients who had not been treated previously or received oral hypoglycemic agents than in patients switching from insulin. With respect to switching to liraglutide from insulin, the most important factors to be considered were C-peptide immunoreactivity levels, age, and the number of insulin units used per day.

Distal renal tubular acidosis that became exacerbated by proton pump inhibitor use.

Acid-base imbalances and electrolyte disorders induced by proton pump inhibitors (PPIs) are extremely rare. However, under certain conditions, PPIs may cause metabolic acidosis or hypokalemia, probably due to an inhibitory action on the proton pump that contributes to H(+) and K(+) homeostasis in the kidney. We herein present a case of marked hypokalemia accompanied by distal renal tubular acidosis in which a PPI appeared to contribute to the pathophysiology of metabolic acidosis.

Diabetic ketoacidosis accompanied by hypothermia: a case report.

Diabetic ketoacidosis (DKA) is an acute, life-threatening complication of diabetes mellitus and is caused by insulin insufficiency. Hypothermia is defined as a core temperature of less than 35°C and is sometimes accompanied by DKA. We report two patients with diabetes who were admitted for DKA accompanied by hypothermia.