Simultaneous LC-MS analysis of plasma concentrations of sildenafil, tadalafil, bosentan, ambrisentan, and macitentan in patients with pulmonary arterial hypertension.

Phosphodiesterase-5 (PDE-5) inhibitors and endothelin receptor antagonists (ERAs) are standard therapies for pulmonary arterial hypertension (PAH). The inter-individual variability of these pharmacokinetics is reported remarkably large, and therapeutic drug monitoring (TDM) can be useful to improve the likelihood of the desired therapeutic and safety outcomes. This study aimed to develop a LC-MS method to determine the concentrations of five PAH drugs (PDE-5 inhibitors: sildenafil and tadalafil, ERAs: bosentan, macitentan, and ambrisentan) from plasma samples using a simple process followed by a single mass spectrometric run, and to validate this approach through pharmacokinetic analyses in patients. A solid extraction method was used for sample preparation of the drugs from human plasma. The total run time for a single injection was within 10 min. The calibration curves for all drugs were linear, and the lower limits of quantitation were 1 (sildenafil), 2 (tadalafil), 5 (ambrisentan), and 10 ng/mL (bosentan, macitentan). The accuracy and precision values suggested that the assay had high accuracy and reliability. To prove the utility of this method, the plasma concentrations of the five PAH drugs were determined after their oral administration to nine PAH patients.

Treatment of nonalbumin rats by transplantation of immortalized hepatocytes using artificial human chromosome.

The shortage of organ donors has impeded the development of human hepatocyte transplantation. Immortalized hepatocytes, however, could provide an unlimited supply of transplantable cells. To determine whether immortalized hepatocytes could provide global metabolic support in end-stage liver disease, rat hepatocyte clones were developed by transduction with the gene encoding the simian virus 40 T antigen (SVLT) using the new technique of human artificial mini chromosome (HAC). Immortalized rat hepatocyte clones were developed by transduction with the gene encoding the SV40 using HAC. Many clones were obtained using this technique. From comparison of the properties of all these clones using the normal hepatocytes and reverse transcription-polymerase chain reaction (RT-PCR), the characteristics of the cell clones (at least partially characterized, and assayed for albumin, glucose-6-phosphate and dipeptidyl peptidase-4, gamma-glutamyltranspeptidase, SVLT and beta-actin expression by RT-PCR) showed no differences other than the immortalization. We compared the albumin bands of the first-day (0-day) and 30-day cells by RT-PCR, showing conditions to be stable for at least 1 month. Three experimental animal model groups were used for albumin analysis: nonalbumin rats with 2/3 hepatectomy only (R-NARs; n = 4); R-NARs with intrasplenic transplantation of 3 x 10(7) primary hepatocytes (pHTx; n = 4); and R-NARs with intrasplenic transplantation of 3 x 10(7) immortalized hepatocytes (iHTx; n = 4). All HTx groups produced albumin, but the immortalized hepatocyte group did not generate significantly elevated albumin levels compared with primary hepatocytes. The results presented herein have demonstrated an initial step toward the development of immortalized hepatocytes for transplantable cells or artificial organs using HAC technology.

Continuous, but not occasional, oral ethanol intake reduces the success of intraportal transplanted islets of Langerhans: an experimental study.

BACKGROUND: Islet transplantation is gradually gaining acceptance for the treatment of type 1 diabetes mellitus. One of the unknown questions is alcohol intake; we have prohibited alcohol intake after islet transplantation although there is no solid evidence to support this. MATERIALS AND METHODS: In this study, we employed a mouse model to determine the effect of oral ethanol intake on transplanted islets. Either 500 or 150 islets were infused selectively into the right liver lobe of chemically induced diabetic mice. After transplantation, mice were orally administered either water (as a control) or various concentrations of ethanol for 14 consecutive days occasionally (once per day) or continuously (all intake was alcohol). Blood glucose levels were monitored and oral glucose tolerance tests (OGTT) performed. RESULTS: After 500 islets had been transplanted, all mice were cured from diabetes, but the continuous alcohol intake group showed significantly prolonged time to diabetes reversal and significantly lower glucose clearance rates by OGTT compared with the control group. After 150 islet transplantations, the diabetes cure rate in the continuous alcohol intake group was significantly lower than the control group (continuous alcohol vs control: 3/8 vs 11/12, P < .05). However, the occasional alcohol intake group showed no difference from the control group, even with as few as 150 islets transplanted per mouse. CONCLUSION: The present results demonstrated that continuous but not occasional alcohol intake reduced the success of intraportal islet transplantation.

De novo sequencing of highly modified therapeutic oligonucleotides by hydrophobic tag sequencing coupled with LC-MS.

Correct sequences are prerequisite for quality control of therapeutic oligonucleotides. However, there is no definitive method available for determining sequences of highly modified therapeutic RNAs, and thereby, most of the oligonucleotides have been used clinically without direct sequence determination. In this study, we developed a novel sequencing method called 'hydrophobic tag sequencing'. Highly modified oligonucleotides are sequenced by partially digesting oligonucleotides conjugated with a 5'-hydrophobic tag, followed by liquid chromatography-mass spectrometry analysis. 5'-Hydrophobic tag-printed fragments (5'-tag degradates) can be separated in order of their molecular masses from tag-free oligonucleotides by reversed-phase liquid chromatography. As models for the sequencing, the anti-VEGF aptamer (Macugen) and the highly modified 38-mer RNA sequences were analyzed under blind conditions. Most nucleotides were identified from the molecular weight of hydrophobic 5'-tag degradates calculated from monoisotopic mass in simple full mass data. When monoisotopic mass could not be assigned, the nucleotide was estimated using the molecular weight of the most abundant mass. The sequences of Macugen and 38-mer RNA perfectly matched the theoretical sequences. The hydrophobic tag sequencing worked well to obtain simple full mass data, resulting in accurate and clear sequencing. The present study provides for the first time a de novo sequencing technology for highly modified RNAs and contributes to quality control of therapeutic oligonucleotides. Copyright © 2016 John Wiley & Sons, Ltd.

Production of calcitonin, adrenocorticotropic hormone, and beta-melanocyte-stimulating hormone in tumors derived from amine precursor uptake and decarboxylation cells.

The tumor production of human calcitonin (CT) was examined by radioimmunoassay, and it was found that 50 of 85 (59%) tumor tissues collected at random contained immunoreactive CT. These tumors were grouped as to whether they were derived from the amine precursor uptake and decarboxylation (APUD) series. The group that was derived from APUD cells showed appreciable amounts of CT in 30 of 31 (97%) of these tumors or in 20 of 21 (95%) when the medullary carcinomas of the thyroid were excluded. However, of the non-APUD group of tumors only 20 of 54 (37%) were found to contain CT, so that the difference between these two groups was highly significant (p less than 0.001). Of the tumors with ectopic adrenocorticotropic hormone-melanocyte-stimulating hormone production, 12 of 14 were shown to contain CT. These data indicate that CT is a common product of the APUD tumors and that tumor production of CT is often associated with that of adrenocorticotropic hormone and beta-melanocyte-stimulating hormone.

Pharmacokinetic and Pharmacodynamic Comparison of Sildenafil-Bosentan and Sildenafil-Ambrisentan Combination Therapies for Pulmonary Hypertension.

To elucidate whether the pharmacokinetics (PK) and pharmacodynamics (PD) of sildenafil are influenced differently when it is coadministered with bosentan (S+B) or with ambrisentan (S+A), we evaluated the PK and PD profiles of sildenafil before and after 4-5 weeks of S+A or S+B treatment in patients with pulmonary arterial hypertension. The area under the plasma concentration-time curve of sildenafil was significantly higher in S+A treatment than in S+B treatment (165.8 ng•h/mL vs. 396.8 ng•h/mL, P = 0.018) and the oral clearance of sildenafil was significantly lower after S+A treatment than after S+B treatment (120.6 L/h/kg vs. 50.4 L/h/kg, P = 0.018). In the PD study, incremental shuttle walking distance was superior during treatment with S+A than during treatment with S+B (S+B; 280 m vs. S+A; 340 m, P = 0.042). There were no concerns about safety with either combination therapy regime.

Postoperative intravenous administration of lysine acetylsalicylate: effect on pain relief and platelet function.

The effects of lysine acetylsalicylate, a new injectable salicylate, on postoperative pain relief and platelet function were studied in ten men who had surgery for peptic ulcer. Four of five patients receiving lysine acetylsalicylate had satisfactory pain relief. One patient required an additional injection of 15 mg of pentazocine. Of the five patients in the control group, an average (+/- SD) dose of 90 +/- 23.7 mg of pentazocine was required to achieve adequate postoperative pain relief. Lysine acetylsalicylate decreased platelet aggregability but without resulting in hemorrhage. We concluded that this new salicylate administered intravenously to patients in the postoperative period provided adequate analgesia while allowing effective hemostasis despite its inhibitory effect on platelet aggregation.

Intraductal papillary adenocarcinoma with mucin hypersecretion and coexistent invasive ductal carcinoma of the pancreas with apparent topographic separation.

We report a 66-year-old man who had a cystic intraductal papillary adenocarcinoma containing a papillary adenoma, in the head of the pancreas and a coexistent invasive, well differentiated solid tubular adenocarcinoma in the tail of the pancreas. He was hospitalized with acute epigastralgia. Computed tomography demonstrated a multilocular cystic mass in the head of the pancreas and a solid tumor in the tail. Endoscopic retrograde pancreatography showed mucin secretion from an enlarged papilla of Vater, marked dilatation of the main pancreatic duct in the head and body, cystic dilatation of the uncinate branch, and irregular narrowing of the main pancreatic duct in the tail. Total pancreatectomy was performed. Between the cystic tumor and the solid tumor there was a distance of 4.8 cm of normal pancreatic parenchyma and duct, recognized both grossly and microscopically. The patient died 35 months after the operation. At autopsy, peritonitis carcinomatosa was found in the abdominal cavity. Microscopically, disseminated nodules were also well differentiated tubular adenocarcinoma. The apparent anatomic separation of these two tumors within the pancreas is extremely unusual.

Long-term implantation of zinc-releasing calcium phosphate ceramics in rabbit femora.

Zinc is an essential trace element that has stimulatory effects on bone formation. Recently, we developed zinc-releasing calcium phosphate ceramics in order to add the pharmacologic effect of zinc to calcium phosphate ceramics. In our previous study, we showed that the optimum zinc content for promoting bone formation was 0.316 wt %. Therefore a zinc composite ceramic of zinc-containing beta-tricalcium phosphate and hydroxyapatite, with a zinc content of 0.316 wt %, was chosen for long-term implantation. Cylindrical rods of the zinc composite ceramic were implanted in rabbit femora for 2 to 60 weeks. Using computer-aided image analysis, a histomorphometric study was carried out to investigate bone formation and resorption around the implants. The control was a composite ceramic of beta-tricalcium phosphate and hydroxyapatite without zinc. The addition of zinc to the implant demonstrated both favorable and unfavorable effects on bone remodeling. The favorable effect was enhanced bone apposition to the implant surface, demonstrated by a significant increase in intramedullary bone apposition rate at 6 weeks and in cortical bone apposition rate at 24 and 60 weeks (p < 0.05). The unfavorable effect was increased bone resorption, demonstrated by a significant increase in medullary cavity area at 60 weeks (p < 0.05). In order to utilize the favorable effect and avoid the unfavorable effect of zinc, either a reduction in zinc content in the zinc composite ceramic or the selection of implantation sites that do not have excessive exposure to bone marrow are required.

In vitro cell-free synthesis of bullous pemphigoid polypeptide.

We investigated the genetic expression of bullous pemphigoid (BP) antigen (230 kD) synthesized by Pam cells using an in vitro cell-free translation assay followed by immunoprecipitation. Prior to the study, we showed that (1) the 230 kD polypeptide does not undergo further processing after it is produced by cells in the short pulse experiment; (2) the 230 kD polypeptide is not degraded at least within the 18 h of the chase experiment; and (3) with tunicamycin treatment the underglycosylated BP antigen still interacts specifically with BP serum. The polypeptide of molecular size slightly greater than 230 kD was identified in the translated proteins directed by ribonucleic acid (RNA) isolated from Pam cells. The size of the mRNA was estimated to be approximately 34S-40S (7.7-10.9 kilobase) using the fractionation method with sucrose density gradient ultracentrifugation of RNA. These results indicate that bullous pemphigoid antigen (230 kD) is a primarily translated product, genetically expressed by Pam cells.

Thiolactomycin, a new antibiotic. IV. Biological properties and chemotherapeutic activity in mice.

The new thiolactone antibiotic, thiolactomycin, is rapidly absorbed in rats when administered either orally or by intramuscular injection. A peak in concentration of the drug is reached in the blood and in various visceral organs within 15 minutes after administration. The concentration decreases rather rapidly and about 51-69% of the drug is excreted in urine during the first 24 hours. Though the in vitro effect of thiolactomycin is moderate, it effectively protected mice challenged intraperitoneally with several strains of S. marcescens and K. pneumoniae and more effective than carbenicillin in treating experimental acute urinary tracts infected with S. marcescens. Also, in mice whom immunodefense was decreased by treatment with cyclophosphamide, thiolactomycin was more effective than carbenicillin against S. marcescens challenge.

Thiolactomycin, a new antibiotic. III. In vitro antibacterial activity.

Thiolactomycin is a new broad-spectrum antibiotic, active in vitro against many species of Gram-positive cocci, Enterobacteriaceae, Haemophilus, acid-fast bacteria and anaerobic bacteria. However, the activity is generally moderate and bacteriostatic in action. This antibiotic eludes cross resistance with any of the known antibacterial drugs such as ampicillin, carbenicillin or cycloserine. The effect on Gram-negative bacilli in vitro is little affected by the inoculum size, the presence of horse serum, the pH of the medium or the type of medium. When thiolactomycin was added to the assay medium, the minimum inhibitory concentration of ampicillin against inducible beta-lactamase producing strains of Staphylococcus aureus and Pseudomonas aeruginosa was reduced. Thiolactomycin inhibited the production of inducible beta-lactamase in several organisms.

Athletes as health testing examinees.

Health-testing examination data of 91 male and 54 female athletes were studied, together with age-matched controls, on serum biochemistry, ECG findings, hematology, and some data on gynecological physiology. Analysis of serum biochemical values revealed statistically significant differences in 14 of 18 routine test items as compared with the age-matched controls. In ECG findings, the combination of bradycardia and left ventricular hypertrophy was most frequently found in male athletes, whereas that of bradycardia and sinus arrhythmia was observed predominantly in female athletes. The incidence of anemia and menstrual dysfunction was higher in female athletes than in controls, especially in the basketball team. In the same team, a high rate of deviant ECG records and a different distribution of the age at menarche were also observed.

The use of arteriovenous anastomosis for replantation of the distal phalanx of the fingers.

Microvascular replantation at the distal phalangeal level has recently been reported by several authors, but as yet the rate of success has not been constant owing to the technical difficulties associated with small vessels. To solve this problem, over the last 4 years we have used arteriovenous anastomosis to reestablish either the arterial system or the venous drainage system in the 33 digits of our 23 patients. The results have been excellent, with a 91 percent success rate. Such results for replantation of the distal phalanx may be maintained and improved if a small venous graft with several branches is also utilized.

Portal vein resection without reconstruction during Appleby operation in a patient with pancreatic body carcinoma with cavernous transformation.

The prognosis of pancreatic body carcinoma has been poor due to cancerous invasion of major vessels. Resection of the involved vessels may improve resectability and prognosis. We report a patient who had a pancreatic body carcinoma with cavernous transformation of the portal vein, in whom the portal vein was resected without reconstruction during an Appleby operation. A 67 year-old man was admitted for evaluation of back pain. Enhanced computed tomography showed no main trunk of the portal vein but a developed collateral circulation. Celiac angiography revealed encasement of the common hepatic, splenic and celiac artery. Venous angiography revealed obstruction of the portal and splenic veins with cavernous transformation surrounding these veins. Pre-operative diagnosis was carcinoma in the pancreatic body, which invaded the portal vein, the celiac and common hepatic arteries. The Appleby operation combined with resection of the portal vein without reconstruction could be performed, by preserving collateral vessels and monitoring hepatic venous oxygen saturation (ShvO2) to prevent hepatic ischemia caused by occlusion of the portal vein. The post-operative course was uneventful.

Arterial reconstruction of the posterior segment after central bisegmentectomy and caudate lobectomy.

A 71 year old woman was admitted with jaundice and found to have a tumor originating from the cystic and common hepatic ducts, which infiltrated the origins of the right bile duct and duct of Spiegel. The tumor was also shown to encase the right hepatic artery. A central bisegmentectomy with concurrent caudate lobectomy was performed using the right gastroepiploic artery for reconstruction of the posterior segmental branch of the right hepatic artery. Celiac angiography conducted 3 weeks postoperatively confirmed the patency of the hepatic segmental arterial anastomosis. The patient had an uneventful recovery with no evidence of liver failure, and was discharged home thirty-two days. Tumors infiltrating the right hepatic artery, which have traditionally been treated with extensive liver resection, can be managed with hepatic segmentectomy and reconstruction of the segmental hepatic artery.

New hydroxybenanomicins produced by Actinomadura.

A soil microorganism, Actinomadura sp. MH193-16F4, produces an antifungal antibiotic benanomicin A and several related compounds. Among them, benanomicin A is the best candidate as a chemotherapeutic agent in terms of antifungal activity, toxicity and water-solubility. Three novel hydroxyl congeners, 3'-hydroxybenanomicin A, 7-hydroxybenanomicin A and 7-hydroxybenanomicinone have been isolated from the culture broth of the MH193-16F4 strain or its mutant. Interestingly, 3'-hydroxy-benanomicin A was as effective as benanomicin A, but the 7-hydroxy congeners were inactive. The inactive congeners differ from benanomicin A and 3'-hydroxyenanomicin A in their conformational structures at C-5 and C-6.

Complete transection of the left hepatic duct due to blunt abdominal trauma.

A 21-year-old man was transferred to our hospital after a traffic accident. He had chief complaint of right upper abdominal pain. Abdominal computed tomography scan at admission showed fluid collection in the abdominal cavity and linear low density area in the hepatic hilus including segment V of the liver. Operative findings revealed a hepatic laceration extending from the boundary between segment IV and segment V to the hepatic hilus, and a completely transection of the left hepatic duct near the caudate lobe without ischemic change and contusion. The bile duct of the caudate lobe communicated with the left hepatic bile duct at the distal portion of the torn wedge, and was not injured. The transected bile duct was primarily repaired with an interrupted end-to-end anastomosis using a retrograde transhepatic bile duct tube (RTBD tube) as stent. Intra- and postoperative cholangiography via the tube revealed no leakage or narrowing. He is in good health for 60 months after surgery. The case illustrates the condition of the transectional wedges for end-to-end anastomosis, and the usefulness of RTBD tube for the anastomotic stent and the diagnosis of bile leakage after anastomosis.