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.

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.

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.

Robot-assisted laparoscopic pancreaticoduodenectomy.

BACKGROUND: Robotic surgery is the most advanced development in minimally invasive surgery. However, the number of reports on robot-assisted endoscopic gastrointestinal surgery is still very small. In this article, we describe total laparoscopic pancreaticoduodenectomy (PD) undertaken using the da Vinci Surgical System® (Intutive Surgical). METHODS: Three patients underwent robotic PD between November 2009 and February 2010. Following resection of the pancreatic head, duodenum, and the distal stomach, intracorporeal anastomosis was accomplished by Child's method of reconstruction, which includes a two-layered end-to-side pancreaticojejunostomy, an end-to-side choledochojejunostomy, and a side-to-side gastrojejunostomy. RESULTS: The time required for surgery was 703 ± 141 min, and blood loss was 118 ± 72 mL. The average hospital stay period was 26 ± 12 days. As a postoperative complication, pancreatic juice leak occurred in one case, but it was managed with conservative treatment. Of the three patients, one had cancer of the papilla of Vater, one had cancer of the pancreatic head, and one had a solid pseudopapillary neoplasm. In all cases, the surgical margin was negative for tumor. CONCLUSIONS: Robot-assisted PD required a long time, but organ removal with less bleeding was able to be safely performed owing to the high degree of freedom associated with the forceps manipulation and the magnified view. Similarly, pancreatojejunostomy could certainly be conducted. No major postoperative complications were found. Accumulation of da Vinci PD experience in the future will lead to safer and faster PD.

Three-dimensional models of arteries constructed using multidetector-row CT images to perform pancreatoduodenectomy safely following dissection of the inferior pancreaticoduodenal artery.

BACKGROUND/PURPOSE: During a pancreatoduodenectomy (PD) it is important that the anatomy of the arcade of blood vessels in the head of the pancreas is fully understood before the surgery in order to reduce intraoperative bleeding. In most of the patients our group has treated, the inferior pancreaticoduodenal artery (IPDA), one of the efferent arteries of the head of the pancreas, has formed a short common trunk with the first jejunal artery (FJA). Thus, by first locating the origin of the FJA, it was easier to locate the IPDA. There are two ways to locate the IPDA: (1) by measuring the distance between the origin of the superior mesenteric artery (SMA) and that of the FJA; (2) by measuring the distance between the origin of the middle colic artery (MCA) and that of the FJA. Here, we report our measurements of both distances using three-dimensional (3D) models of arteries constructed with multidetector-row computed tomography (MD-CT) images and discuss which is the better measurement to determine the location of the IPDA during PD. METHODS: A total of 140 patients underwent 64-MD-CT imaging to acquire early and late arterial phase scans. The distance between the origin of the SMA and that of the FJA and the distance between the origin of the MCA and that of the FJA origin were measured. RESULTS: In patients whose IPDA formed either a common trunk with the FJA or arose directly from the SMA, the IPDA or the common truck was located in parallel with the SMA at a very short distance of approximately 18 mm from the MCA origin towards the center. The distance between the SMA origin and the IPDA was significantly longer (approximately 36 mm). Therefore, locating the MCA origin during PD helped determine the location of the IPDA. However, in patients whose anterior inferior pancreaticoduodenal artery (AIPDA) and posterior inferior pancreaticoduodenal artery (PIPDA) arose separately, the distance between the AIPDA origin and the MCA origin was approximately 18 mm, the distance between the AIPDA origin and the PIPDA origin was approximately 19 mm, and the distance between the PIPDA origin and the SMA origin was 19 mm. Thus, locating the SMA helped determine the location of the IPDA during PD in these patients. CONCLUSION: Based on our findings that the distance between the IPDA origin and the MCA origin was short, we have shown that it is effective to locate the MCA origin in order to determine the location of the IPDA.

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.

Experimental joint contracture correction with low torque--long duration repeated stretching.

Tension is necessary to maintain and restore the mechanical properties of soft connective tissues. Conversely, reduced tension states such as produced by immobilization weaken mechanical properties and facilitate joint contracture. We assessed the effect of low torque-long duration stretching to increase the range of motion (ROM) and to restore the mechanical properties of contracted joints in 66 rat knees immobilized for 40 days. After remobilization, we randomly divided the contracted knees into four treatment groups treated with repeated stretches of diverse torques and duration: stretching with low-torque and long-duration, high-torque and short-duration, high-torque and long-duration, low-torque and short duration. We included control and natural recovery groups. Phase lag in all treatment groups recovered to the same range as in the normal controls. Dynamic stiffness, which was not altered by joint immobilization, increased in all treatment groups. Deformation and load to failure improved substantially only in the low-torque and long-duration stretching group. Low-torque and long-duration repeated stretching leads to a greater restoration of ROM with more normal mechanical properties compared to high-torque and short duration stretching.

Optimal and effective oral dose of taurine to prolong exercise performance in rat.

The aim of this study was to determine the effective and optimum dose of taurine for exercise performance and to maintain tissue taurine concentration. Rats received a respective daily dose of 0, 20, 100, and 500 mg/kg body weight of taurine (EC and ET-1, -2, -3 groups, respectively) for two weeks, and then, were subjected to treadmill until exhaustion. The running time to exhaustion was significantly prolonged by 25% and 50% in the ET-2 and -3 groups, respectively, compared to that in the EC group accompanied with maintenance of taurine tissue concentrations. Furthermore, the oxidative glutathione per total glutathione ratio in tissues was inhibited in the ET-2 and -3 groups whereas it was higher in the EC group than in both the no exercise and taurine-administered groups. Therefore the effective and optimal doses of oral taurine administration for two weeks on a transient exercise performance were between 100 and 500 mg/kg/day.

A new technique of laparoscopic surgery for rectal disease.

Laparoscopic rectal surgery is a technique that has to be done in a narrow space: the pelvis. If an immoderate operation is performed with a difficult view, for example in female where the field is disturbed by uterus, it is possible to produce organ trauma or an unexpected bleeding. Taking these problems in mind, we performed laparoscopic surgery in 44 cases of rectal disease with several techniques which we have invented. In these cases either the uterus or the rectum was retracted in the narrow space, and if an anterior resection was to be done, the tape was tied tightly around the rectum below the tumor to avoid touching the tumor and leaving adequate vascular irrigation to the remnant rectum. With the use of our techniques, we did not have female intestinal injury or unexpected bleeding. In addition laparoscopic anterior resection of rectum did not cause any intestinal injury, or unexpected bleeding or anastomotic leakage; also we did not have any local tumor recurrence. It is our belief that these techniques can decrease complications that traumatize the grasping intestine with intestinal forceps and prevent implantation in the anastomosis. This technical report validate that our technique modifications for rectal laparoscopic surgery are useful when a surgeon has to work in a narrow space.

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.

Clinical and histopathological spectrum of cutaneous vasculitis in rheumatoid arthritis.

BACKGROUND: Cutaneous manifestations are the most frequent, and often the initial feature of extra-articular involvement in patients with rheumatoid vasculitis. OBJECTIVES: The purpose of the study was to evaluate the clinical and histological spectrum of cutaneous vasculitis and the associated systemic involvement in patients with rheumatoid vasculitis. METHODS: Among 525 patients with rheumatoid arthritis, 20 tissue specimens with histologically proven cutaneous necrotizing vasculitis from 11 patients were investigated by studying the types and levels of affected vessels and related clinical features. RESULTS: Small-vessel vasculitis identified as dermal necrotizing venulitis was found in 10 patients, clinically characterized by palpable purpura, maculopapular erythema, erythema elevatum diutinum and haemorrhagic blisters. Arteritis histologically resembling cutaneous polyarteritis nodosa, clinically characterized by subcutaneous nodules, livedo reticularis, atrophie blanche and deep ulcers was identified in four patients all with systemic complications. Coexistence of venulitis and arteritis was identified in three patients. Different cutaneous vasculitic manifestations often coexisted and recurred in the same patient. Three patients with systemic complications of mononeuritis multiplex (two of three), interstitial pulmonary fibrosis (two of three) and abdominal microaneurysms (one of three) died within 1 year of onset of the cutaneous vasculitis. Immunofluorescence demonstrated vessel wall deposition of IgM and/or complement in six of the seven patients examined. CONCLUSIONS: Features of cutaneous rheumatoid vasculitis overlapping both the characteristics of cutaneous necrotizing venulitis and cutaneous polyarteritis nodosa together with coexistence of these different type of vasculitis in the same or different lesional skin account for the associated diverse cutaneous vasculitic manifestations. Although dermal venulitis (leucocytoclastic vasculitis) was the most common presentation, the presence of leucocytoclastic vasculitis in rheumatoid patients did not necessarily indicate a favourable prognosis. Associations with mononeuritis multiplex and bowel involvement had a fatal prognosis, while patients with superficial dermal venulitis without other extra-articular involvement may follow a favourable prognosis.

Coexistence of beta2 microglobulin and lambda light chain in amyloid fibrils of dialysis-unrelated plasma cell dyscrasia-associated systemic amyloidosis.

BACKGROUND: Systemic amyloidosis occurs as a result of amyloid deposition in various tissues. The amyloid fibrils in systemic amyloidosis have been reported to originate from immunoglobulin light chains. OBJECTIVE: We studied the composition of amyloid fibrils from two patients with plasma cell-associated systemic amyloidosis (PASA). METHODS: A double immunofluorescence study of the lesional skin of PASA was undertaken. Amyloid proteins were extracted with distilled water from one case of PASA. RESULTS: The double immunofluorescence study showed that anti-lambda light chain and anti-beta2 microglobulin antibodies mostly reacted with the same area of amyloid deposit. Amyloid deposits from two patients with PASA who had never undergone haemodialysis showed a positive reaction with the antibodies for beta2 microglobulin as well as immunoglobulin lambda light chain. By the use of immunoblot assay of amyloid fibril proteins, polypeptides immunoreactive with antigamma light chain antibody (29 kDa) and with anti-beta2 microglobulin antibody (12 kDa) were detected. CONCLUSIONS: These results indicate that beta2 microglobulin is a component of amyloid fibrils in PASA.

Elevated interleukin-12 in pseudosynovial fluid in patients with aseptic loosening of hip prosthesis.

Loosening of a prosthesis is a major problem in total joint arthroplasty. To assess levels of cytokines in patients with such loosening, we measured the pseudosynovial fluid concentration of the following cytokines; tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-1beta, IL-4, IL-6, IL-10, IL-12, matrix metalloproteinase (MMP)-1, and MMP-3. We examined the pseudosynovial fluid in patients with a loose hip prosthesis (group A; n = 8) and the synovial fluid in patients with osteoarthritis (OA) of the hip (group B; n = 18) using enzyme-linked immunosorbent assays. The mean concentration of IL-12 was significantly higher in group A than in group B (P < 0.01). Also, we found a significant (P < 0.05) correlation between the concentration of IL-12 and the concentration of MMP-1 in the patients with prosthesis loosening. The concentrations of TNF-alpha, IL-1beta, IL-4, IL-6, IL-10, IFN-gamma, MMP-1, and MMP-3 appeared to be similar in the two groups, although the small number of samples available precluded us from determining that there was no significant difference. The present study is the first to report elevated IL-12 levels in the pseudosynovial fluid of patients with a loose prosthesis. The immunoregulatory effect of IL-12 against wear particles could play an important role in causing loosening of the prosthesis.

Stimulatory effect of zinc-releasing calcium phosphate implant on bone formation in rabbit femora.

Although hydroxyapatite (HAP) and tricalcium phosphate (TCP) are currently used as bone graft substitutes or coatings on metallic prostheses because of their excellent biocompatibility and osteoconductivity, they do not stimulate bone formation or inhibit bone resorption. Zinc, an essential trace element in many animals, has a direct specific proliferative effect on osteoblastic cells and has a potent and selective inhibitory effect on osteoclastic bone resorption in vitro. Therefore, zinc-containing beta-tricalcium phosphate (ZnTCP) ceramics and composite ceramics of ZnTCP and HAP (ZnTCP/HAP) were implanted in the femora of New Zealand White rabbits for 4 weeks to promote bone formation. The implants were sintered ceramics with zinc contents of 0 (control), 0.063, 0.316 and 0.633 wt %. Histological and histomorphometrical investigation of the undecalcified sections revealed an increase by 51% (p =.0509) in the area of newly formed bone around the ZnTCP/HAP implants of 0. 316 Zn wt % compared with the control. Plasma zinc concentration was unchanged. An increased bone resorption on the endosteal surface was observed when ZnTCP and ZnTCP/HAP of 0.633 Zn wt % were implanted. To promote bone formation, the optimum zinc content of the calcium phosphate ceramics was therefore 0.316 wt %.

Fat absorption after pylorus-preserving pancreatoduodenectomy reconstructed with Billroth II pancreaticojejunostomy or Billroth I pancreaticogastrostomy.

BACKGROUND/AIMS: The aim of this study was to determine whether Billroth I pancreaticogastrostomy (PG-I) or Billroth II pancreaticojejunostomy (PJ-II) after pylorus-preserving pancreatoduodenectomy is associated with better postoperative fat absorption, based on residual pancreatic exocrine function. Several reconstructive operations have been employed after pylorus-preserving pancreatoduodenectomy to maximize postoperative nutrition. However, no single-institution study has been published comparing the reconstructive procedures with respect to digestion and absorption of fat. METHODOLOGY: Fat absorption was studied using the 13C-trioctanoin breath test in patients who were grouped according to the degree of fibrosis of the pancreatic remnant, which was determined by histologic examination of the resection specimen. The fibrosis was graded: grade 0, < 10% fibrosis; grade 1, 10-30% fibrosis; and grade 2, > 30% fibrosis. There were 22 patients in the PG-I group and 22 patients in the PJ-II group. RESULTS: There were no significant differences between the PG-I and PJ-II groups in the cumulative excretion of labeled carbon dioxide in the patients with grade 0 pancreatic fibrosis. The cumulative excretion in the PG-I group was better than in the PJ-II group in the patients with grade 1 and grade 2 pancreatic fibrosis. CONCLUSIONS: Fat absorption after PG-I is superior to that after PJ-II in patients with disordered exocrine function of the pancreatic remnant. Billroth I pancreaticogastrostomy allows more effective utilization of the exocrine enzymes of the pancreatic remnant due to elimination of the blind loop characteristic of the Billroth II pancreaticojejunostomy.

Abiotic synthesis of guanine with high-temperature plasma.

The origin of guanine has been unknown, though there are some reports concerning its abiotic synthesis. We show here that guanine, as well as uracil and cytosine, are synthesized from a 90%N2-10%CO-H2O gas mixture via a complex organic product produced with the high-temperature and rapid quenching technique. This result implies that a large amount of complex organic matter including precursors of bioorganic compounds might have been produced on the primitive earth after cometary impacts.