Identification of Potent and Long-Acting Single-Chain Peptide Mimetics of Human Relaxin-2 for Cardiovascular Diseases.

The insulin-like peptide human relaxin-2 was identified as a hormone that, among other biological functions, mediates the hemodynamic changes occurring during pregnancy. Recombinant relaxin-2 (serelaxin) has shown beneficial effects in acute heart failure, but its full therapeutic potential has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. In this study, we report the development of long-acting potent single-chain relaxin peptide mimetics. Modifications in the B-chain of relaxin, such as the introduction of specific mutations and the trimming of the sequence to an optimal size, resulted in potent, structurally simplified peptide agonists of the relaxin receptor Relaxin Family Peptide Receptor 1 (RXFP1) (e.g., 54). Introduction of suitable spacers and fatty acids led to the identification of single-chain lipidated peptide agonists of RXFP1, with sub-nanomolar activity, high subcutaneous bioavailability, extended half-lives, and in vivo efficacy (e.g., 64).

Engineered Histidine-Enriched Facial Lipopeptides for Enhanced Intracellular Delivery of Functional siRNA to Triple Negative Breast Cancer Cells.

Cytosolic delivery of functional siRNA remains the major challenge to develop siRNA-based therapeutics. Designing clinically safe and effective siRNA transporter to deliver functional siRNA across the plasma and endosomal membrane remains a key hurdle. With the aim of improving endosomal release, we have designed cyclic and linear peptide-based transporters having an Arg-DHis-Arg template. Computational studies show that the Arg-DHis-Arg template is also stabilized by the Arg-His side-chain hydrogen bonding interaction at physiological pH, which dissociates at lower pH. The overall atomistic interactions were examined by molecular dynamics simulations, which indicate that the extent of peptide_siRNA assembly formation depends greatly on physicochemical properties of the peptides. Our designed peptides having the Arg-DHis-Arg template and two lipidic moieties facilitate high yield of intracellular delivery of siRNA. Additionally, unsaturated lipid, linoleic acid moieties were introduced to promote fusogenicity and facilitate endosomal release and cytosolic delivery. Interestingly, such protease-resistant peptides provide serum stability to siRNA and exhibit high efficacy of erk1 and erk2 gene silencing in the triple negative breast cancer (TNBC) cell line. The peptide having two linoleyl moieties demonstrated comparable efficacy with commercial transfection reagent HiPerFect, as evidenced by the erk1 and erk2 gene knockdown experiment. Additionally, our study shows that ERK1/2 silencing siRNA and doxorubicin-loaded gramicidin-mediated combination therapy is more effective than siRNA-mediated gene silencing-based monotherapy for TNBC treatment.

The NTCP-inhibitor Myrcludex B: Effects on Bile Acid Disposition and Tenofovir Pharmacokinetics.

Myrcludex B acts as a hepatitis B and D virus entry inhibitor blocking the sodium taurocholate cotransporting polypeptide (SLC10A1). We investigated the effects of myrcludex B on plasma bile acid disposition, tenofovir pharmacokinetics, and perpetrator characteristics on cytochrome P450 (CYP) 3A. Twelve healthy volunteers received 300 mg tenofovir disoproxil fumarate orally and 10 mg subcutaneous myrcludex B. Myrcludex B increased total plasma bile acid exposure 19.2-fold without signs of cholestasis. The rise in conjugated bile acids was up to 124-fold (taurocholic acid). Coadministration of tenofovir with myrcludex B revealed no relevant changes in tenofovir pharmacokinetics. CYP3A activity slightly but significantly decreased by 29% during combination therapy. Myrcludex B caused an asymptomatic but distinct rise in plasma bile acid concentrations and had no relevant impact on tenofovir pharmacokinetics. Changes in CYP3A activity might be due to alterations in bile acid signaling. Long-term effects of elevated bile acids will require critical evaluation.

A brain-targeting lipidated peptide for neutralizing RNA-mediated toxicity in Polyglutamine Diseases.

Polyglutamine (PolyQ) diseases are progressive neurodegenerative disorders caused by both protein- and RNA-mediated toxicities. We previously showed that a peptidyl inhibitor, P3, which binds directly to expanded CAG RNA can inhibit RNA-induced nucleolar stress and suppress RNA-induced neurotoxicity. Here we report a N-acetylated and C-amidated derivative of P3, P3V8, that showed a more than 20-fold increase in its affinity for expanded CAG RNA. The P3V8 peptide also more potently alleviated expanded RNA-induced cytotoxicity in vitro, and suppressed polyQ neurodegeneration in Drosophila with no observed toxic effects. Further N-palmitoylation of P3V8 (L1P3V8) not only significantly improved its cellular uptake and stability, but also facilitated its systemic exposure and brain uptake in rats via intranasal administration. Our findings demonstrate that concomitant N-acetylation, C-amidation and palmitoylation of P3 significantly improve both its bioactivity and pharmacological profile. L1P3V8 possesses drug/lead-like properties that can be further developed into a lead inhibitor for the treatment of polyQ diseases.

Low penetration of caspofungin into cerebrospinal fluid following intravenous administration of standard doses.

The kinetics of caspofungin (CAS) in cerebrospinal fluid (CSF) following intravenous (i.v.) administration has been studied exclusively in animal models. Human data are missing so far. In this study, 13 CSF samples were obtained at different time points following i.v. infusion of CAS in ten paediatric haemato-/oncological patients (age range 1.0-14.2 years, median 8.6 years) without signs of central nervous system (CNS) infection (n = 10 samples) or with infectious meningitis (n = 3 samples). Serum samples were obtained concurrently. Liquid chromatography-tandem mass spectrometry was used for CAS quantification. Whilst CAS serum levels were in the expected range, varying between 0.6 and 20.3 µg/mL (median 7.0 µg/mL), 11 of 13 CSF levels were below the limit of detection of 0.084 µg/mL at 3.0-48.0 h (median 23.3 h) following i.v. infusion. Only two (of three) levels in patients with bacterial meningitis were above the limit of detection (0.3 µg/mL and 0.09 µg/mL, respectively). These results indicate the low capacity of CAS to penetrate into the CNS even in inflamed meninges. Monotherapy with standard doses of CAS appears not to be suitable for treatment of fungal CNS infections.

Micafungin: A Review in the Prophylaxis and Treatment of Invasive Candida Infections in Paediatric Patients.

Intravenous micafungin (Mycamine®; Funguard®), an echinocandin, is approved in the EU for the treatment of invasive candidiasis in children (including neonates) and adolescents (<16 years of age) and as prophylaxis against Candida infections in patients undergoing haematopoietic stem cell transplantation (HSCT) or who are expected to have neutropenia for ≥10 days. This narrative review focuses on the use of micafungin in paediatric indications approved in the EU, which may vary from those approved elsewhere in the world. Micafungin has a broad spectrum of in vitro activity against clinically relevant isolates of Candida spp. (including fluconazole-resistant Candida glabrata isolates), a low propensity for emergence of resistant isolates and a convenient once-daily regimen. In paediatric substudies and a small multinational, phase 3 trial in neonates with proven invasive candidiasis, intravenous micafungin was effective and generally well tolerated in the treatment of candidaemia and other types of invasive candidiasis and as prophylaxis against fungal infections in patients undergoing HSCT. Hence, micafungin remains an important option for the prophylaxis and treatment of invasive Candida infections in paediatric and adult patients.

Biodegradable stearylated peptide with internal disulfide bonds for efficient delivery of siRNA in vitro and in vivo.

RNA-based delivery system for cancer therapy remains a challenge. In this study, a stearyl-peptide (SHR) was synthesized using arginine, histidine, cysteine, and stearyl moieties. Further, the stearyl-peptides were cross-linked by disulfide bonds to obtain cross-linked polypeptides (SHRss) with different molecular weight (SHRss1, SHRss2, SHRss3, SHRss4). The SHRss could effectively condense small interfering RNA (siRNA) into polyplexes with a hydrodynamic size of 100-300 nm and zeta potential of 20-40 mV. Flow cytometry and confocal laser scanning microscope studies revealed high cellular uptake and rapid dissociation behavior of SHRss2/siRNA complexes. Long-lasting high concentration of siRNA in cytoplasm was observed even at 24 h after SHRss2/Cy3-siRNA transfection. Compared with SHR, the SHRss showed much improved siRNA interference efficiency targeting luciferase on Luc-Hela cells. Moreover, SHRss2 exhibited higher interference efficiency and slower decay rate on Luc-Hela cells than Lipofectamine 2000 and SHR. In addition, much weaker expression of red fluorescence protein was also observed on SHRss2/simCh-treated mCherry-HEK293 cells than Lipofectamine 2000 and SHR. The SHRss did not induce cytotoxicity at siRNA concentrations of 25-200 nM under transfection. The in vivo studies demonstrated the gene interference efficiency of SHRss2/siRNA complexes. Our studies indicated that the SHRss are promising and efficient nonviral vectors for siRNA delivery.

Micafungin twice weekly as antifungal prophylaxis in paediatric patients at high risk for invasive fungal disease.

BACKGROUND: Current guidelines recommend antifungal prophylaxis for children at high risk for invasive fungal disease (IFD), but the use of polyenes and triazoles may not be feasible in some patients due to toxicities and drug-drug interactions. Micafungin is well tolerated, with intravenous daily dosing being the current standard. Recent reports indicate safety and efficacy of intermittent dosing of micafungin. METHODS: We analysed safety, efficacy and micafungin serum concentrations of children at high risk for IFD receiving prophylactic micafungin between 3 and 4 mg/kg twice weekly. All children were intolerant or had contraindications to polyenes and triazoles. RESULTS: A total of 21 children (median age = 9 years) at high risk for IFD were included in the analysis. No significant clinical adverse event occurred, and end of treatment values of parameters of renal and hepatic function in serum were not different from baseline. Proven or probable breakthrough IFD did not occur in any of the patients. In 9 out of 11 patients in whom plasma micafungin concentrations were assessed, the first trough concentration exceeded 150 ng/mL, a concentration proposed to be effective for prophylaxis. CONCLUSIONS: Our data indicate that micafungin administered twice weekly at a dosage of 3-4 mg/kg of bodyweight could be a convenient, safe and efficient alternative for antifungal prophylaxis in children at high risk for IFD.

Pharmacokinetic and safety profiles of repeated-dose prophylactic micafungin in children and adolescents undergoing hematopoietic stem cell transplantation.

Micafungin is a potent echinocandin antifungal that can be used for both prophylaxis and treatment of Candida infections. This open-label study assessed the pharmacokinetics and safety profile of prophylactic micafungin in children and adolescents (aged 4 mo to 16 y) undergoing hematopoietic stem cell transplantation. Patients received once-daily doses of either 1 or 1.5 mg/kg micafungin, based on their body weight, for 10 to 14 days. In total, 40 patients received micafungin. Area under the plasma micafungin concentration-time curve was highest in patients aged 6 to 11 years in the 1.5 mg/kg treatment group. Peak plasma micafungin concentration displayed no age-related differences, but was higher in the 1.5 mg/kg versus the 1 mg/kg group. Clearance at steady state by weight and volume of distribution by weight were considerably higher in patients aged 4 months to 5 years. Results from this study show that age and body weight affect micafungin pharmacokinetics in pediatric patients undergoing hematopoietic stem cell transplantation.

Pharmacokinetics of micafungin in subjects with severe hepatic dysfunction.

Micafungin is an echinocandin with potent activity against a broad range of fungal species, including Candida species. The pharmacokinetic and safety profiles of micafungin have been evaluated in individuals with mild-to-moderate hepatic dysfunction, but not in individuals with severe hepatic dysfunction. Therefore, the present study assessed the pharmacokinetics and safety of a single 100 mg dose of micafungin in healthy subjects (n = 8) and subjects with severe hepatic dysfunction (n = 8). Mean maximum plasma concentration of micafungin and mean area under the plasma micafungin concentration-time curve extrapolated to infinity were lower in subjects with severe hepatic dysfunction (7.3 ± 2.4 µg/mL and 100.1 ± 34.5 h·µg/mL, respectively) than in subjects with normal hepatic function (10.3 ± 2.5 µg/mL and 142.4 ± 28.9 h·µg/mL, respectively). Mean clearance was higher in subjects with severe hepatic dysfunction (1,098 ± 347 mL/h) than in subjects with normal hepatic function (728 ± 149 mL/h). Concentrations of albumin in subjects with severe hepatic dysfunction were lower. Assessments of micafungin plasma protein binding suggested that the higher clearance in subjects with severe hepatic dysfunction may be due to higher unbound concentrations. However, the magnitude of the differences was not considered clinically meaningful and is comparable with exposures reported elsewhere for a 100-mg dose in patients treated for invasive candidiasis. Thus, dose adjustment in subjects with severe hepatic dysfunction is not warranted. Micafungin was well tolerated in all subjects throughout the study.

Recent advances in characterization of nonviral vectors for delivery of nucleic acids: impact on their biological performance.

INTRODUCTION: Nucleic acid delivery is a complex process that requires transport across numerous extracellular and intracellular barriers, whose impact is often neglected during optimization studies. As such, the development of nonviral vectors for efficient delivery would benefit from an understanding of how these barriers relate to the physicochemical properties of lipoplexes and polyplexes. AREAS COVERED: This review focuses on the evaluation of parameters associated with barriers to delivery such as blood and immune cells compatibility which, as a collective, may serve as a useful prescreening tool for the advancement of nonviral vectors in vivo. An outline of the most relevant rationally developed polyplexes and lipoplexes for clinical application is also given. EXPERT OPINION: The evaluation of scientifically recognized parameters enabled the identification of systemic delivered nonviral vectors' behavior while in blood as one of the key determinants of vectors function and activity both in vitro and in vivo. This multiparametric approach complements the use of in vitro efficacy results alone for prescreening and improves in vitro-in vivo translation by minimizing false negatives. Further, it can aid in the identification of meaningful structure-function-activity relationships, improve the in vitro screening process of nonviral vectors before in vivo use and facilitate the future development of potent and safe nonviral vectors.

Safety, tolerability, and feasibility of antifungal prophylaxis with micafungin at 2 mg/kg daily in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation.

INTRODUCTION: Micafungin (MCFG) is used for the prophylaxis of invasive fungal disease (IFD) after allogeneic hematopoietic stem cell transplantation (HSCT). However, the safety, efficacy, or optimal dosage/blood levels as prophylaxis is uncertain in pediatric HSCT-patients. METHODS: We prophylactically administered MCFG at 2 mg/kg once daily to 38 children and adolescents undergoing allogeneic HSCT. RESULTS: During MCFG prophylaxis, infusion reactions or adverse events (grades 2-5) related to MCFG use were not found in all the patients. Thus, MCFG prophylaxis was not discontinued and other antifungal agents were not added except for 2 patients in whom probable or possible IFDs developed (completion rate, 94.7 %). To elucidate the influence of HSCT-related complications/drugs on blood concentration of MCFG, we determined the plasma trough and peak levels in 13 and 10 among 38 patients, respectively. The mean trough and peak levels were 3.04 ± 1.21 µg/mL (569 samples) and 9.63 ± 3.62 µg/mL (44 samples), respectively. The peak levels were moderately correlated to the trough levels (R (2) = 0.466). In a patient, the trough level of MCFG transiently increased up to 10.21 µg/mL during hepatic dysfunction due to acute graft-versus-host disease. The MCFG trough levels strongly correlated with T-Bil value (R (2) = 0.894). There was no relationship between the trough levels of MCFG and the circulating concentrations of tacrolimus (R (2) = 0.040). Additionally, MCFG levels were not influenced by treatment with cyclophosphamide or corticosteroids. CONCLUSIONS: Prophylaxis with MCFG at 2 mg/kg once daily may be safe, tolerable, and feasible in pediatric HSCT-patients.

Pharmacokinetics of micafungin in patients undergoing allogeneic hematopoietic stem cell transplantation.

OBJECTIVES: Micafungin (MCFG) is an antifungal agent that is widely used for the treatment of invasive fungal infection. Although the pharmacokinetics of MCFG is considered to depend on the hepatic metabolism, the impact of hepatic function on the pharmacokinetics of MCFG has been inconsistent among previous studies. The object of this study was to evaluate the relationship between plasma MCFG concentration and clinical and laboratory data. PATIENTS AND METHODS: We examined the plasma concentration of MCFG in 10 patients who underwent allogeneic hematopoietic stem cell transplantation (HSCT). MCFG at 150 mg/day was administered intravenously a median of 58.5 days after HSCT. Trough and peak concentrations of MCFG (Cmin and Cmax) were measured at a median of 5.5 days after the first administration of MCFG. RESULTS: The presence of graft-versus-host disease involving the liver at blood sampling was associated with significantly higher Cmin and Cmax of MCFG. Among the laboratory data, Cmin and Cmax were significantly higher in patients with severely impaired hepatic function defined as serum total bilirubin (TBi) level >5 mg/dL and/or serum gamma-glutamyltransferase (γ-GTP) level >500 IU/L, but the presence of mildly impaired hepatic function defined as serum TBi level >2 mg/dL and/or serum γ-GTP level >200 IU/L did not affect Cmin and Cmax. Renal function did not show significant impact on Cmin and Cmax. CONCLUSION: These findings suggest that the pharmacokinetics of MCFG is affected only by severely impaired liver function.

Hepatitis B virus hepatotropism is mediated by specific receptor recognition in the liver and not restricted to susceptible hosts.

UNLABELLED: The human hepatitis B virus (HBV) causes acute and chronic infections in humans and chimpanzees. HBV infects its hosts at minimal inoculation doses and replicates exclusively in hepatocytes. The viral determinants for the pronounced species specificity and the high efficacy to address hepatocytes in vivo are unknown. Previous findings showed that N-terminally myristoylated peptides constituting a receptor binding domain of the HBV large envelope (L)-protein block HBV entry in vitro and in vivo. Here we investigate the ability of such peptidic receptor ligands to target the liver. Injection of radioactively labeled HBVpreS-lipopeptides resulted in rapid accumulation in livers of mice, rats, and dogs but not cynomolgus monkeys. Without lipid moiety the peptide was excreted by renal filtration, indicating its possible retention through the lipid by serum factors. Organ distribution studies of 26 HBVpreS peptide variants revealed a correlation of HBV infection inhibition activity and the ability to target mouse livers. Together with complementary studies using primary hepatocytes of different species, we hypothesize that HBV hepatotropism is mediated through specific binding of the myristoylated N-terminal preS1-domain of the HBV L-protein to a hepatocyte specific receptor. Moreover, the restricted infectivity of HBV to human primates is not generally determined by the absence of this binding receptor in nonsusceptible hosts (e.g., mice) but related to postbinding step(s) (e.g., membrane fusion). CONCLUSION: HBVpreS-lipopeptides target to the liver. This observation has important clinical implications regarding the pharmacokinetic properties of Myrcludex B, the first entry inhibitor for HBV/HDV. In addition, this provides the basis for the application of the peptides as vehicles for hepatocyte-specific drug targeting.

An inhibitor of neuronal exocytosis (DD04107) displays long-lasting in vivo activity against chronic inflammatory and neuropathic pain.

Small peptides patterned after the N terminus of the synaptosomal protein of 25 kDa, a member of the protein complex implicated in Ca(2+)-dependent neuronal exocytosis, inhibit in vitro the release of neuromodulators involved in pain signaling, suggesting an in vivo analgesic activity. Here, we report that compound DD04107 (palmitoyl-EEMQRR-NH(2)), a 6-mer palmitoylated peptide that blocks the inflammatory recruitment of ion channels to the plasma membrane of nociceptors and the release of calcitonin gene-related peptide from primary sensory neurons, displays potent and long-lasting in vivo antihyperalgesia and antiallodynia in chronic models of inflammatory and neuropathic pain, such as the complete Freund's adjuvant, osteosarcoma, chemotherapy, and diabetic neuropathic models. Subcutaneous administration of the peptide produced a dose-dependent antihyperalgesic and antiallodynic activity that lasted ≥24 h. The compound showed a systemic distribution, characterized by a bicompartmental pharmacokinetic profile. Safety pharmacology studies indicated that the peptide is largely devoid of side effects and substantiated that the in vivo activity is not caused by locomotor impairment. Therefore, DD04107 is a potent and long-lasting antinociceptive compound that displays a safe pharmacological profile. These findings support the notion that neuronal exocytosis of receptors and neuronal algogens pivotally contribute to chronic inflammatory and neuropathic pain and imply a central role of peptidergic nociceptor sensitization to the pathogenesis of pain.

Intracellular concentrations of micafungin in different cellular compartments of the peripheral blood.

Whilst micafungin serum concentrations are well studied, little is known about its concentrations within cellular compartments of the peripheral blood. Hence, in this study blood samples were collected from patients receiving micafungin (n=26). These samples were separated by double-discontinuous Ficoll-Hypaque density gradient centrifugation. Intracellular concentrations within the obtained cells, i.e. peripheral blood mononuclear cells (PBMCs), polymorphonuclear leukocytes (PMNs) and red blood cells (RBCs), were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Within PBMCs and PMNs, the intracellular micafungin concentration was significantly increased compared with the concentration in plasma (P<0.001). The intracellular concentration within RBCs did not significantly differ from the plasma concentration. Micafungin reaches high concentrations in human PBMCs and PMNs and is present in RBCs. In vitro data showed that intracellular uptake of micafungin by PBMCs depends on the albumin concentration of the surrounding medium, but only at non-physiological protein concentrations.

CBLB613: a TLR 2/6 agonist, natural lipopeptide of Mycoplasma arginini , as a novel radiation countermeasure.

To date, there are no safe and effective drugs available for protection against ionizing radiation damage. Therefore, a great need exists to identify and develop non-toxic agents that will be useful as radioprotectors or postirradiation therapies under a variety of operational scenarios. We have developed a new pharmacological agent, CBLB613 (a naturally occurring Mycoplasma-derived lipopeptide ligand for Toll-like receptor 2/6), as a novel radiation countermeasure. Using CD2F1 mice, we investigated CBLB613 for toxicity, immunogenicity, radioprotection, radiomitigation and pharmacokinetics. We also evaluated CBLB613 for its effects on cytokine induction and radiation-induced cytopenia in unirradiated and irradiated mice. The no-observable-adverse-effect level of CBLB613 was 1.79 mg/kg and 1 mg/kg for single and repeated doses, respectively. CBLB613 significantly protected mice against a lethal dose of (60)Co γ radiation. The dose reduction factor of CBLB613 as a radioprotector was 1.25. CBLB613 also mitigated the effects of (60)Co γ radiation on survival in mice. In both irradiated and unirradiated mice, the drug stimulated induction of interleukin-1ß (IL-1ß), IL-6, IL-10, IL-12, keratinocyte-derived chemokine, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-1α. CBLB613 also reduced radiation-induced cytopenia and increased bone marrow cellularity in irradiated mice. Our immunogenicity study demonstrated that CBLB613 is not immunogenic in mice, indicating that it could be developed as a radioprotector and radiomitigator for humans against the potentially lethal effects of radiation exposure.

Pharmacokinetics of micafungin in HIV positive patients with confirmed esophageal candidiasis.

Esophageal candidiasis (EC) is a common and serious complication in patients infected with the human immunodeficiency virus (HIV). Micafungin has been shown to have dose-related efficacy and to be well tolerated in patients with HIV and EC. This analysis of data from a randomized, double-blind study examined pharmacokinetic parameters of micafungin (dosed at 50, 100, and 150 mg/day) and its metabolites in a subset of patients with HIV and EC. Micafungin exhibited linear, predictable pharmacokinetics, similar to the previous observations in healthy control subjects. Micafungin peak plasma concentration and exposure were increased with dose, while half-life and clearance remained consistent with increasing dose. Plasma concentrations of the metabolites M-1, M-2, and M-5 remained low throughout the study (24 h exposure ≤14% relative to micafungin at end of therapy for each). No differences in micafungin pharmacokinetic parameters were observed according to the sex or race of the patients. The high systemic exposures associated with micafungin 100 and 150 mg/day relative to micafungin 50 mg/day were found to directly correlate with endoscopic clearance. These data provide evidence that the pharmacokinetics of micafungin underlie the dose-related efficacy in patients with HIV and EC.

Targeting protease-activated receptor-1 with cell-penetrating pepducins in lung cancer.

Protease-activated receptors (PARs) are G-protein-coupled receptors that are activated by proteolytic cleavage and generation of a tethered ligand. High PAR1 expression has been documented in a variety of invasive cancers of epithelial origin. In the present study, we investigated the contribution of the four PAR family members to motility of lung carcinomas and primary tumor samples from patients. We found that of the four PARs, only PAR1 expression was highly increased in the lung cancer cell lines. Primary lung cancer cells isolated from patient lung tumors migrated at a 10- to 40-fold higher rate than epithelial cells isolated from nonmalignant lung tissue. Cell-penetrating pepducin inhibitors were generated against the first (i1) and third (i3) intracellular loops of PAR1 and tested for their ability to inhibit PAR1-driven migration and extracellular regulated kinase (ERK)1/2 activity. The PAR1 pepducins showed significant inhibition of cell migration in both primary and established cell lines similar to silencing of PAR1 expression with short hairpin RNA (shRNA). Unlike i1 pepducins, the i3 loop pepducins were effective inhibitors of PAR1-mediated ERK activation and tumor growth. Comparable in efficacy with Bevacizumab, monotherapy with the PAR1 i3 loop pepducin P1pal-7 provided significant 75% inhibition of lung tumor growth in nude mice. We identify the PAR1-ERK1/2 pathway as a feasible target for therapy in lung cancer.

Higher clearance of micafungin in neonates compared with adults: role of age-dependent micafungin serum binding.

Micafungin, a new echinocandin antifungal agent, has been used widely for the treatment of various fungal infections in human populations. Micafungin is predominantly cleared by biliary excretion and it binds extensively to plasma proteins. Micafungin body weight-adjusted clearance is higher in neonates than in adults, but the mechanisms underlying this difference are not understood. Previous work had revealed the roles of sinusoidal uptake (Na(+) -taurocholate co-transporting peptide, NTCP; organic anion transporting polypeptide, OATP) as well as canalicular efflux (bile salt export pump, BSEP; breast cancer resistance protein, BCRP) transporters in micafungin hepatobiliary elimination. In the present study, the relative protein expression of hepatic transporters was compared between liver homogenates from neonates and adults. Also, the extent of micafungin binding to serum from neonates and adults was measured in vitro. The results indicate that relative expression levels of NTCP, OATP1B1/3, BSEP, BCRP and MRP3 were similar in neonates and in adults. However, the micafungin fraction unbound (f(u) ) in neonatal serum was about 8-fold higher than in the adult serum (0.033±0.012 versus 0.004±0.001, respectively). While there was no evidence for different intrinsic hepatobiliary clearance of micafungin between neonates and adults, our data suggest that age-dependent serum protein binding of micafungin is responsible for its higher clearance in neonates compared with adults.