Plasma ANGPTL8 Levels and Risk for Secondary Cardiovascular Events in Japanese Patients With Stable Coronary Artery Disease Receiving Statin Therapy.

BACKGROUND: The ability to predict secondary cardiovascular events could improve health of patients undergoing statin treatment. Circulating ANGPTL8 (angiopoietin-like protein 8) levels, which positively correlate with proatherosclerotic lipid profiles, activate the pivotal proatherosclerotic factor ANGPTL3. Here, we assessed potential association between circulating ANGPTL8 levels and risk of secondary cardiovascular events in statin-treated patients. METHODS: We conducted a biomarker study with a case-cohort design, using samples from a 2018 randomized control trial known as randomized evaluation of high-dose (4 mg/day) or low-dose (1 mg/day) lipid-lowering therapy with pitavastatin in coronary artery disease (REAL-CAD [Randomized Evaluation of Aggressive or Moderate Lipid-Lowering Therapy With Pitavastatin in Coronary Artery Disease])." From that study's full analysis set (n=12 413), we selected 2250 patients with stable coronary artery disease (582 with the primary outcome, 1745 randomly chosen, and 77 overlapping subjects). A composite end point including cardiovascular-related death, nonfatal myocardial infarction, nonfatal ischemic stroke, or unstable angina requiring emergent admission was set as a primary end point. Circulating ANGPTL8 levels were measured at baseline and 6 months after randomization. RESULTS: Over a 6-month period, ANGPTL8 level changes significantly decreased in the high-dose pitavastatin group, which showed 19% risk reduction of secondary cardiovascular events compared with the low-dose group in the REAL-CAD [Randomized Evaluation of Aggressive or Moderate Lipid-Lowering Therapy With Pitavastatin in Coronary Artery Disease] study. In the highest quartiles, relative increases in ANGPTL8 levels were significantly associated with increased risk for secondary cardiovascular events, after adjustment for several cardiovascular disease risk factors and pitavastatin treatment (hazard ratio in Q4, 1.67 [95% CI, 1.17-2.39). Subgroup analyses showed relatively strong relationships between relative ANGPTL8 increases and secondary cardiovascular events in the high-dose pitavastatin group (hazard ratio in Q4, 2.07 [95% CI, 1.21-3.55]) and in the low ANGPTL8 group at baseline (166

A Simple, Fast, Sensitive LC-MS/MS Method to Quantify NAD(H) in Biological Samples: Plasma NAD(H) Measurement to Monitor Brain Pathophysiology.

Nicotinamide adenine dinucleotide (NAD) is a cofactor in redox reactions and an essential mediator of energy metabolism. The redox balance between NAD+ and NADH affects various diseases, cell differentiation, and aging, and in recent years there has been a growing need for measurement techniques with improved accuracy. However, NAD(H) measurements, representing both NAD+ and NADH, have been limited by the compound's properties. We achieved highly sensitive simultaneous measurement of NAD+ and NADH under non-ion pairing, mobile phase conditions of water, or methanol containing 5 mM ammonium acetate. These were achieved using a simple pre-treatment and 7-min analysis time. Use of the stable isotope 13C5-NAD+ as an internal standard enabled validation close to BMV criteria and demonstrated the robustness of NAD(H) determination. Measurements using this method showed that brain NAD(H) levels correlate strongly with plasma NAD(H) levels in the same mouse, indicating that NAD(H) concentrations in brain tissue are reflected in plasma. As NAD(H) is involved in various neurodegenerative diseases and cerebral ischemia, as well as brain diseases such as mitochondrial myopathies, monitoring changes in NADH levels in plasma after drug administration will be useful for development of future diagnostics and therapeutics.

Multi-Omics of Familial Thoracic Aortic Aneurysm and Dissection: Calcium Transport Impairment Predisposes Aortas to Dissection.

Several genetic defects, including a mutation in myosin heavy chain 11 (Myh11), are reported to cause familial thoracic aortic aneurysm and dissection (FTAAD). We recently showed that mice lacking K1256 of Myh11 developed aortic dissection when stimulated with angiotensin II, despite the absence of major pathological phenotypic abnormalities prior to stimulation. In this study, we used a comprehensive, data-driven, unbiased, multi-omics approach to find underlying changes in transcription and metabolism that predispose the aorta to dissection in mice harboring the Myh11 K1256del mutation. Pathway analysis of transcriptomes showed that genes involved in membrane transport were downregulated in homozygous mutant (Myh11ΔK/ΔK) aortas. Furthermore, expanding the analysis with metabolomics showed that two mechanisms that raise the cytosolic Ca2+ concentration-multiple calcium channel expression and ADP-ribose synthesis-were attenuated in Myh11ΔK/ΔK aortas. We suggest that the impairment of the Ca2+ influx attenuates aortic contraction and that suboptimal contraction predisposes the aorta to dissection.

A New Hypothetical Concept in Metabolic Understanding of Cardiac Fibrosis: Glycolysis Combined with TGF-ß and KLF5 Signaling.

The accumulation of fibrosis in cardiac tissues is one of the leading causes of heart failure. The principal cellular effectors in cardiac fibrosis are activated fibroblasts and myofibroblasts, which serve as the primary source of matrix proteins. TGF-ß signaling pathways play a prominent role in cardiac fibrosis. The control of TGF-ß by KLF5 in cardiac fibrosis has been demonstrated for modulating cardiovascular remodeling. Since the expression of KLF5 is reduced, the accumulation of fibrosis diminishes. Because the molecular mechanism of fibrosis is still being explored, there are currently few options for effectively reducing or reversing it. Studying metabolic alterations is considered an essential process that supports the explanation of fibrosis in a variety of organs and especially the glycolysis alteration in the heart. However, the interplay among the main factors involved in fibrosis pathogenesis, namely TGF-ß, KLF5, and the metabolic process in glycolysis, is still indistinct. In this review, we explain what we know about cardiac fibroblasts and how they could help with heart repair. Moreover, we hypothesize and summarize the knowledge trend on the molecular mechanism of TGF-ß, KLF5, the role of the glycolysis pathway in fibrosis, and present the future therapy of cardiac fibrosis. These studies may target therapies that could become important strategies for fibrosis reduction in the future.

Inflammasome-Independent and Atypical Processing of IL-1ß Contributes to Acid Aspiration-Induced Acute Lung Injury.

Inflammation plays a pivotal role in the pathophysiology of gastric aspiration-induced acute lung injury (ALI). However, its mechanism remains unclear. In this study, we investigated the role of NLRP3 inflammasome-driven IL-1ß production in a mouse model of acid aspiration-induced inflammation and ALI. Acid aspiration-induced inflammatory responses and ALI in wild-type mice were significantly attenuated in IL-1ß-/- mice, but not NLRP3-/- mice. In vitro experiments revealed that severe acidic stress (pH 1.75) induced the processing of pro-IL-1ß into its 18-kDa mature form (p18-IL-1ß), which was different from the caspase-1-processed 17-kDa form (p17-IL-1ß), in human THP-1 macrophages and primary murine macrophages. Deficiency of NLRP3 and caspase-1 had no effect on acidic stress-produced IL-1ß. The production of IL-1ß by severe acidic stress was prevented by inhibitors of serine proteases [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride], but not of cysteine proteases (E-64), cathepsin G, or inflammasome. The cathepsin D inhibitor pepstatin A inhibited IL-1ß production induced by mild acidic stress (pH 6.2) or lactic acid, but not severe acidic stress. Using mass spectrometry and processing-site mutants of pro-IL-1ß, we identified D109 as a novel cleavage site of pro-IL-1ß in response to severe acidic stress and calculated the theoretical molecular mass of the mature form to be 18.2 kDa. The bioactivity of acidic stress-produced IL-1ß was confirmed by its ability to promote p38 phosphorylation and chemokine upregulation in alveolar epithelial cells. These findings demonstrate a novel mechanism of acid-induced IL-1ß production and inflammation independent of NLRP3 inflammasome and provide new insights into the therapeutic strategies for aspiration pneumonitis and ALI.

Influence of the aldehyde dehydrogenase 2 polymorphism on the vasodilatory effect of nitroglycerin in infants with congenital heart disease and pulmonary arterial hypertension.

PURPOSE: The influence of the aldehyde dehydrogenase 2 (ALDH2) gene polymorphism on the pharmacokinetics and haemodynamics of nitroglycerin (GTN) was determined in human subjects. METHODS: Eighteen infants (nine each with and without ALDH2 gene polymorphism) with congenital heart disease and pulmonary arterial hypertension participated in this study. GTN treatment started at a dose of 2 µg/kg/min, and the dose was escalated by 1-2 µg/kg/min until pulmonary vascular resistance (PVR) was reduced by more than 30%. The plasma GTN concentration and PVR were measured at the end of each infusion period. RESULTS: Plasma GTN concentrations were significantly higher in patients with the ALDH2 gene polymorphism than in those without the polymorphism. Conversely, the reduction in PVR was smaller in patients with the ALDH2 gene polymorphism than in those without. CONCLUSIONS: These data suggest that the ALDH2 gene polymorphism influences the pharmacokinetics and haemodynamics of GTN in human subjects.

Effects of telmisartan and olmesartan on insulin sensitivity and renal function in spontaneously hypertensive rats fed a high fat diet.

Although telmisartan, an angiotensin II receptor blocker (ARB), has an agonistic action for proliferator-activated receptor (PPAR)-γ in vitro, it remains to be determined whether telmisartan exerts such an action in vivo using a non-toxic dose (<5 mg/kg in rats). To address the issue, telmisartan (2 mg/kg) and olmesartan (2 mg/kg), another ARB without PPAR-γ agonistic action, were given to spontaneously hypertensive rats (SHR) fed a high fat diet (HFD). HFD decreased plasma adiponectin, and caused insulin resistance, hypertriglyceridemia and renal damage, which were improved by ARBs. Protective effects of telmisartan and olmesartan did not significantly differ. In addition, in vitro study showed that 1 µM of telmisartan did not elevate the mRNA expression of adipose protein 2, which is a PPAR-γ-stimulated adipogenic marker gene, in preadipocytes with 3% albumin. To obtain 1 µM of plasma concentration, oral dose of telmisartan was calculated to be 6 mg/kg, which indicates that PPAR-γ agonistic action is negligible with a non-toxic dose of telmisartan (<5 mg/kg) in rats. This study showed that 2 mg/kg of telmisartan and olmesartan ameliorated insulin resistance, hypertriglyceridemia and renal damage in SHR fed a HFD. As beneficial effects of telmisartan and olmesartan did not significantly differ, these were mediated through the PPAR-γ-independent actions.

Metabolomic Profiling of Mice with Tacrolimus-Induced Nephrotoxicity: Carnitine Deficiency in Renal Tissue.

Tacrolimus (TAC)-induced chronic nephrotoxicity (TAC nephrotoxicity) has a detrimental effect on long-term kidney graft survival. However, the pathogenesis of TAC nephrotoxicity remains largely unknown. We explored it by focusing on metabolic changes in renal tissues. In this study, mice were separated into TAC and control groups (n = 5/group). TAC was administered to the TAC group (1 mg/kg/d for 28 days) subcutaneously. The control group was similarly treated with normal saline. Renal tissue metabolomes were evaluated. Renal fibrosis was observed only in the TAC group. Metabolomic analysis showed that carnitine and related metabolites were substantially lower in the TAC group than in the control group, presumably due to impaired biosynthesis and reabsorption. Low carnitine levels impair antioxidation in renal tissues and ß-oxidation in mitochondria, which may lead to renal tissue damage. This metabolomic analysis revealed that carnitine deficiency in renal tissue appears to explain TAC nephrotoxicity.

Apomorphine Suppresses the Progression of Steatohepatitis by Inhibiting Ferroptosis.

The role of ferroptosis in steatohepatitis development is largely unknown. We investigated (1) whether hepatocyte ferroptosis occurs in a gene-modified steatohepatitis model without modifying dietary components, (2) whether ferroptosis occurs at an early stage of steatohepatitis, and (3) whether apomorphine, recently reported as a ferroptosis inhibitor, can ameliorate steatohepatitis. Hepatocyte-specific PTEN KO mice were used. Huh 7 and primary cultured hepatocytes isolated from the mice were used in this study. The number of dead cells increased in 10-week-old PTEN KO mice. This cell death was suppressed by the administration of ferroptosis inhibitor ferrostatin-1 for 2 weeks. Apomorphine also ameliorated the severity of steatohepatitis. Treatment with ferroptosis inhibitors, including apomorphine, decreases the level of lipid peroxidase. Apomorphine suppressed cell death induced by RSL-3 (a ferroptosis inducer), which was not suppressed by apoptosis or necroptosis inhibitors. Apomorphine showed a radical trapping capacity with much more potent activity than ferrostatin-1 and Trolox, a soluble form of vitamin E. In addition, apomorphine activated nrf2 and its downstream genes, including HO-1 and xCT. In conclusion, ferroptosis occurs in steatohepatitis from an early stage in PTEN KO mice. In addition, apomorphine ameliorates the severity of steatohepatitis by inhibiting ferroptosis.

Peptide mimetic NC114 induces growth arrest by preventing PKCδ activation and FOXM1 nuclear translocation in colorectal cancer cells.

The peptide mimetic, NC114, is a promising anticancer compound that specifically kills colorectal cancer cells without affecting normal colon epithelial cells. In our previous study, we observed that NC114 inhibited the Wnt/ß-catenin pathway, with significant downregulation of both Ser 675-phosphorylated ß-catenin and its target genes, cyclin D1 and survivin. However, the molecular mechanism responsible for its cytotoxic effect has not yet been fully characterized. In the present study, we demonstrated that NC114 prevented cell cycle progression from S to G2/M phase by downregulating cell cycle-related gene expression, and also induced growth arrest in SW480 and HCT-116 colorectal cancer cells. A novel covariation network analysis combined with transcriptome analysis revealed a series of signaling cascades affected by NC114 treatment, and identified protein kinase C-δ (PKCδ) and forkhead box protein M1 (FOXM1) as important regulatory factors for NC114-induced growth arrest. NC114 treatment inhibits the activation of PKCδ and its kinase activity, which suppresses MEK/ERK signaling. Attenuated MEK/ERK signaling then results in a reduction in FOXM1 phosphorylation and subsequent nuclear translocation of FOXM1 and ß-catenin. Consequently, formation of a T-cell factor-4 (TCF4)/ß-catenin transcription complex in the nucleus is inhibited and transcription of its target genes, such as cell cycle-related genes, is downregulated. The efficacy of NC114 on tumor growth was confirmed in a xenograft model. Collectively, elucidation of the mechanism by which NC114 induces growth arrest in colorectal cancer cells should provide a novel therapeutic strategy for colorectal cancer treatment.

Processed B-type natriuretic peptide is a biomarker of postinterventional restenosis in ischemic heart disease.

BACKGROUND: Restenosis, a condition in which the lesion vessel renarrows after a coronary intervention procedure, remains a limitation in management. A surrogate biomarker for risk stratification of restenosis would be welcome. B-type natriuretic peptide (BNP) is secreted in response to pathologic stress from the heart. Its use as a biomarker of heart failure is well known; however, its diagnostic potential in ischemic heart disease is less explored. Recently, it has been reported that processed forms of BNP exist in the circulation. We hypothesized that circulating processed forms of BNP might be a biomarker of ischemic heart disease. METHODS: We characterized processed forms of BNP by a newly developed mass spectrometry-based detection method combined with immunocapture using commercial anti-BNP antibodies. RESULTS: Measurements of processed forms of BNP by this assay were found to be strongly associated with presence of restenosis. Reduced concentrations of the amino-terminal processed peptide BNP(5-32) relative to BNP(3-32) [as the index parameter BNP(5-32)/BNP(3-32) ratio] were seen in patients with restenosis [median (interquartile range) 1.19 (1.11-1.34), n = 22] vs without restenosis [1.43 (1.22-1.61), n = 83; P < 0.001] in a cross-sectional study of 105 patients undergoing follow-up coronary angiography. A sensitivity of 100% to rule out the presence of restenosis was attained at a ratio of 1.52. CONCLUSIONS: Processed forms of BNP may serve as viable potential biomarkers to rule out restenosis.

Takotsubo cardiomyopathy: Japanese perspective.

Takotsubo cardiomyopathy is likely induced by stress, and postmenopausal women account for the majority of patients. Several questions about takotsubo cardiomyopathy remain unanswered, including why it is induced by stress, what differentiates the stress that induces this disease from other types of stress that do not induce it, why women are more likely to develop this disease, why the apical part of the heart is affected, and how it is different from the subtype in which the base and middle parts are affected. Although takotsubo cardiomyopathy is uncommon, clinicians should be aware that this disease can develop in wider areas.

Anatomical Targeting of Anticancer Drugs to Solid Tumors Using Specific Administration Routes: Review.

Despite remarkable recent progress in developing anti-cancer agents, outcomes of patients with solid tumors remain unsatisfactory. In general, anti-cancer drugs are systemically administered through peripheral veins and delivered throughout the body. The major problem with systemic chemotherapy is insufficient uptake of intravenous (IV) drugs by targeted tumor tissue. Although dose escalation and treatment intensification have been attempted in order to increase regional concentrations of anti-tumor drugs, these approaches have produced only marginal benefits in terms of patient outcomes, while often damaging healthy organs. To overcome this problem, local administration of anti-cancer agents can yield markedly higher drug concentrations in tumor tissue with less systemic toxicity. This strategy is most commonly used for liver and brain tumors, as well as pleural or peritoneal malignancies. Although the concept is theoretically reasonable, survival benefits are still limited. This review summarizes clinical results and problems and discusses future directions of regional cancer therapy with local administration of chemotherapeutants.

Polypharmacy-Related Shock Symptoms and Complications Associated with Phenothiazine: A Case Report.

This report describes a case of shock symptoms in a 72-year-old woman with epilepsy who had been in a state of polypharmacy, taking multiple antipsychotic drugs. After receiving a normal dose of periciazine, she exhibited impaired consciousness, hypothermia, and hypotension and was admitted to hospital. Despite poor response to vasopressors, conservative treatment led to gradual improvement. Subsequent pharmacokinetic analysis showed non-toxic blood concentrations of periciazine, suggesting that even small doses of phenothiazines could result in toxic symptoms. This case highlights the importance of monitoring for adverse reactions when prescribing multiple antipsychotic drugs, particularly in older polypharmacy patients.

A Potent PDK4 Inhibitor for Treatment of Heart Failure with Reduced Ejection Fraction.

Heart failure with reduced ejection fraction (HFrEF) is characterized not only by reduced left ventricular ejection fraction (EF) but is also combined with symptoms such as dyspnea, fatigue, and edema. Several pharmacological interventions have been established. However, a treatment targeting a novel pathophysiological mechanism is still needed. Evidence indicating that inhibition of pyruvate dehydrogenase kinase 4 (PDK4) may be cardioprotective has been accumulating. Thus, we focused on vitamin K3 and used its framework as a new PDK4 inhibitor skeleton to synthesize new PDK4 inhibitors that show higher activity than the existing PDK4 inhibitor, dichloroacetic acid, and tested their cardioprotective effects on a mouse heart failure model. Among these inhibitors, PDK4 inhibitor 8 improved EF the most, even though it did not reverse cardiac fibrosis or wall thickness. This novel, potent PDK4 inhibitor may improve EF of failing hearts by regulating bioenergetics via activation of the tricarboxylic acid cycle.

Total and reduced/oxidized forms of coenzyme Q10 in fibroblasts of patients with mitochondrial disease.

Coenzyme Q10 (CoQ10) is involved in ATP production through electron transfer in the mitochondrial respiratory chain complex. CoQ10 receives electrons from respiratory chain complex I and II to become the reduced form, and then transfers electrons at complex III to become the oxidized form. The redox state of CoQ10 has been reported to be a marker of the mitochondrial metabolic state, but to our knowledge, no reports have focused on the individual quantification of reduced and oxidized CoQ10 or the ratio of reduced to total CoQ10 (reduced/total CoQ10) in patients with mitochondrial diseases. We measured reduced and oxidized CoQ10 in skin fibroblasts from 24 mitochondrial disease patients, including 5 primary CoQ10 deficiency patients and 10 respiratory chain complex deficiency patients, and determined the reduced/total CoQ10 ratio. In primary CoQ10 deficiency patients, total CoQ10 levels were significantly decreased, however, the reduced/total CoQ10 ratio was not changed. On the other hand, in mitochondrial disease patients other than primary CoQ10 deficiency patients, total CoQ10 levels did not decrease. However, the reduced/total CoQ10 ratio in patients with respiratory chain complex IV and V deficiency was higher in comparison to those with respiratory chain complex I deficiency. Measurement of CoQ10 in fibroblasts proved useful for the diagnosis of primary CoQ10 deficiency. In addition, the reduced/total CoQ10 ratio may reflect the metabolic status of mitochondrial disease.

Comparison of analytical performance of two single-step measurement devices of B-type natriuretic Peptide.

Rapid measurement of B-type natriuretic peptide (BNP) plays a practical role in the diagnosis of congestive heart failure. Analytical evaluation of a new small-footprint immunochromatography reader of BNP (Rapidpia®) was performed and compared with the commercially available SHIONOSPOT® Reader as the index. The new BNP assay had a within-run coefficient of variation (CV) of 9.0% and a between-run CV of 2.1%. Correlations between whole blood and plasma samples and those with the index SHIONOSPOT® Reader were y = 0.93x + 0.88, R2 = 0.98 and y = 1.08x - 6.67, R2 = 0.93, respectively. Based on our findings, the two point-of care (POC) assays for BNP, Rapidpia® and SHIONOSPOT® Reader, showed comparable results.

Prevalence of Asymptomatic SARS-CoV-2 Infection in Japan.

Importance: Real-world evidence of SARS-CoV-2 transmission is needed to understand the prevalence of infection in the Japanese population. Objective: To conduct sentinel screening of the Japanese population to determine the prevalence of SARS-CoV-2 infection in asymptomatic individuals, with complementary analysis for symptomatic patients as reported by active epidemiologic surveillance used by the government. Design, Setting, and Participants: This cross-sectional study of a sentinel screening program investigated approximately 1 million asymptomatic individuals with polymerase chain reaction (PCR) testing for SARS-CoV-2 infection between February 22 and December 8, 2021. Participants included children, students, employed adults, and older individuals, as well as volunteers to broadly reflect the general Japanese population in the 14 prefectures of Japan that declared a state of emergency. Saliva samples and a cycle threshold (Ct) value of approximately 40 as standard in Japan were used. Polymerase chain reaction testing for symptomatic patients was separately done by public health authorities, and the results were obtained from the Ministry of Health, Labour, and Welfare of Japan to complement data on asymptomatic infections from the present study. Main Outcomes and Measures: Temporal trends in positivity and prevalence (including surges of different variants) and demographic associations (eg, age, geographic location, and vaccination status) were assessed. Results: The positive rate of SARS-CoV-2 infection in 1 082 976 asymptomatic individuals (52.08% males; mean [SD] age 39.4 [15.7] years) was 0.03% (95% CI, 0.02%-0.05%) during periods without surges and a maximum of 0.33% (95% CI, 0.25%-0.43%) during peak surges at the Japanese standard Ct value of approximately 40; however, the positive rate would have been 10-fold less at a Ct value of 25 as used elsewhere in the world (eg, UK). There was an increase in patients with a positive PCR test result with a Ct value of 25 or 30 preceding surges in infection and hotspots of asymptomatic infections. Conclusions and Relevance: In this cross-sectional study of asymptomatic SARS-CoV-2 infection in the general population of Japan in 2021, as investigated by sentinel surveillance, a low rate of infection was seen in the Japanese population compared with reported levels elsewhere in the world. This finding provides real-world data on the state of infection in Japan.

Microvolume Analysis of Aflibercept in Aqueous Humor Using Mass Spectrometry.

Purpose: To develop a microvolume analytical method for measurement of the aflibercept concentration in human intraocular fluid and plasma. Methods: We analyzed trace amounts of aflibercept in human aqueous humor using Fab-selective proteolysis and nano-surface and molecular-orientation limited (nSMOL) proteolysis, coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Patients with age-related macular degeneration or diabetic macular edema were recruited. Just after an injection of 50 µL of aflibercept, regurgitate from needle holes was collected with a micropipette pressed to the side of the injection hole within 10 seconds. The median amount of regurgitate was 4 µL (range, 1-18 µL). Results: In human plasma, the aflibercept concentration ranged between 0.195 and 50 µg/mL when using the quantitative signature peptide IIWDSR (aa. 56-61) present on the vascular endothelial growth factor receptor 1 domain of aflibercept. The method was validated by evaluating its linearity, carryover, selectivity, accuracy and precision, dilution effect, and sample/processing stability. As only a minimal amount of regurgitate through needle holes can be sampled, we performed and verified the aflibercept assay using patient samples after 1:10 dilution with control human plasma, a recognized diluent. The median concentration of aflibercept in the regurgitate was 240 µg/mL (range, 13-4300 µg/mL). Conclusions: Our findings indicate that the aflibercept assay using human intraocular fluid can be reliably performed using nSMOL coupled with LC-MS/MS. Translational Relevance: This technique for quantifying aflibercept in the regurgitate suggests that the amount of drug lost post-injection can be ignored, even in patients with a relatively large leak after vitreous injection. This new methodology suggests possible therapeutic responses and may be employed as a general analytical method for trapping many biologics, such as vascular endothelial growth factor, in various types of clinical samples, unaffected by proteinaceous or small organic pharmaceuticals.

Novel Drug Delivery Method Targeting Para-Aortic Lymph Nodes by Retrograde Infusion of Paclitaxel into Pigs' Thoracic Duct.

Gastrointestinal cancer with massive nodal metastases is a lethal disease. In this study, using a porcine model, we infused the anti-cancer drug Paclitaxel (PTX) into thoracic ducts to examine the efficiency of drug delivery to intra-abdominal lymph nodes. We established a technical method to catheterize the thoracic duct in the necks of pigs. We then compared the pharmacokinetics of PTX administered intrathoracically with those of systemic (intravenous) infusion. Serum, liver, and spleen concentrations of PTX were significantly lower following thoracic duct (IT) infusion than after intravenous (IV) administration approximately 1-8 h post-infusion. However, PTX levels in abdominal lymph nodes were maintained at relatively high levels up to 24 h after IT infusion compared to after IV infusion. Concentrations of PTX in urine were much higher after IT administration than after IV administration. After IT infusion, the same concentration of PTX was obtained in abdominal lymph nodes, but the serum concentration was lower than after systemic infusion. Therefore, IT infusion may be able to achieve higher PTX doses than IV infusion. IT delivery of anti-cancer drugs into the thoracic duct may yield clinical benefits for patients with extensive lymphatic metastases in abdominal malignancies.