Endothelial endocytic pathways: gates for vascular drug delivery

Vascular endothelium plays strategic roles in many drug delivery paradigms, both as an important therapeutic target itself and as a barrier for reaching tissues beyond the vascular wall. Diverse means are being developed to improve vascular drug delivery including stealth liposomes and polymer carriers. Affinity carriers including antibodies or peptides that specifically bind to endothelial surface determinants, either constitutive or pathological, enhance targeting of drugs to endothelial cells (EC) in diverse vascular areas. In many cases, binding to endothelial surface determinants facilitates internalization of the drug/carrier complex. There are several main endocytic pathways in EC, including clathrin- and caveoli-mediated endocytosis, phagocytosis and macropinocytosis (these two are less characteristic of generic EC) and the recently described Cell Adhesion Molecule (CAM)-mediated endocytosis. The latter may be of interest for intracellular drug delivery to EC involved in inflammation or thrombosis. The metabolism and effects of internalized drugs largely depend on the routes of intracellular trafficking, which may lead to degrading lysosomal compartments or other organelles, recycling to the plasma membrane or transcytosis to the basal surface of endothelium. The latter route, characteristic of caveoli-mediated endocytosis, may serve for trans-endothelial drug delivery. Paracellular trafficking, which can be enhanced under pathological conditions or by auxiliary agents, represents an alternative for transcytosis. Endothelial surface determinants involved in endocytosis, mechanisms of the latter and trafficking pathways, as well as specific characteristics of EC in different vascular areas, are discussed in detail in the context of modern paradigms of vascular drug delivery.     

Reverse geometrical selectivity in glucuronidation and sulfation of cis- and trans-4-hydroxytamoxifens by human liver UDP-glucuronosyltransferases and sulfotransferases

The phenolic active metabolites, cis-4-hydroxytamoxifen (cis-HO-TAM) and trans-4-hydroxytamoxifen (trans-HO-TAM), of the anti-breast-cancer drug, trans-tamoxifen (TAM), were geometrically selectively glucuronidated in the manner of cis>trans by microsomes and sulfated in the manner of trans>cis by cytosol from the liver of 10 human subjects (7 females and 3 males). There was a large individual difference in the microsomal glucuronidation of cis-HO-TAM, which correlated well with glucuronidation of 4-hydroxybiphenyl by human liver microsomes. However, there was only a slight correlation between the glucuronidation of cis-HO-TAM and trans-HO-TAM or 4-nitrophenol (NP). A small individual difference was observed for the human liver cytosolic sulfation of trans-HO-TAM, which correlated well with the sulfation of NP. Recombinant human UDP-glucuronosyltransferase (UGT)2B15 catalyzed the cis-selective glucuronidation of geometrical isomers of HO-TAM. UGTs1A1, 1A4, 1A9 and 2B7 had weak activity toward HO-TAMs with a much smaller cis-selectivity than did UGT2B15. UGTs1A3 and 1A6 had no detectable activity toward these substrates. Among the four known major sulfotransferases (SULTs) occurring in the human liver, SULT1A1 was strongly suggested to play the most important role in the hepatic cytosolic trans-selective sulfation of HO-TAM isomers. A good correlation was observed between the hepatic cytosolic sulfation of trans-HO-TAM and NP, a standard substrate for SULT1A1. SULT1E1 had slight activity toward the HO-TAMs. SULTs1A3 and 2A1 had no detectable activity toward HO-TAMs.     

Pharmacological properties of S1RA, a new sigma-1 receptor antagonist that inhibits neuropathic pain and activity-induced spinal sensitization

BACKGROUND AND PURPOSE

The sigma-1 (σ₁) receptor is a ligand-regulated molecular chaperone that has been involved in pain, but there is limited understanding of the actions associated with its pharmacological modulation. Indeed, the selectivity and pharmacological properties of σ₁ receptor ligands used as pharmacological tools are unclear and the demonstration that σ₁ receptor antagonists have efficacy in reversing central sensitization-related pain sensitivity is still missing.

EXPERIMENTAL APPROACH

The pharmacological properties of a novel σ₁ receptor antagonist (S1RA) were first characterized. S1RA was then used to investigate the effect of pharmacological antagonism of σ₁ receptors on in vivo nociception in sensitizing conditions and on in vitro spinal cord sensitization in mice. Drug levels and autoradiographic, ex vivo binding for σ₁ receptor occupancy were measured to substantiate behavioural data.

KEY RESULTS

Formalin-induced nociception (both phases), capsaicin-induced mechanical hypersensitivity and sciatic nerve injury-induced mechanical and thermal hypersensitivity were dose-dependently inhibited by systemic administration of S1RA. Occupancy of σ₁ receptors in the CNS was significantly correlated with the antinociceptive effects. No pharmacodynamic tolerance to the antiallodynic and antihyperalgesic effect developed following repeated administration of S1RA to nerve-injured mice. As a mechanistic correlate, electrophysiological recordings demonstrated that pharmacological antagonism of σ₁ receptors attenuated the wind-up responses in spinal cords sensitized by repetitive nociceptive stimulation.

CONCLUSIONS AND IMPLICATIONS

These findings contribute to evidence identifying the σ₁ receptor as a modulator of activity-induced spinal sensitization and pain hypersensitivity, and suggest σ₁ receptor antagonists as potential novel treatments for neuropathic pain.     

Review: Production and functionality of active peptides from milk

In recent years, research on the production of active peptides obtained from milk and their potential functionality has grown, to a great extent. Bioactive peptides have been defined as specific protein fragments that have a positive impact on body functions or conditions, and they may ultimately have an influence on health. Individual proteins of casein or milk-derived products such as cheese and yogurt have been used as a protein source to study the isolation and activity of peptides with several applications. Currently, the milk whey waste obtained in the production of cheese also represents a protein source from which active peptides could be isolated with potential industrial applications. The active properties of milk peptides and the results found with regard to their physiological effects have led to the classification of peptides as belonging to the group of ingredients of protein nature, appropriate for use in functional foods or pharmaceutical formulations. In this study, the main peptides obtained from milk protein and the past research studies about its production and biological activities will be explained. Second, an analysis will be made on the methods to determinate the biological activities, the separation of bioactive peptides and its structure identification. All of these form the base required to obtain synthetic peptides. Finally, we explain the experimental animal and human trials done in the past years. Nevertheless, more research is required on the design and implementation of equipment for the industrial production and separation of peptides. In addition, different authors suggest that more emphasis should therefore be given to preclinical studies, proving that results are consistent and that effects are demonstrated repeatedly by several research human groups.     

Lung resistance-related protein as a predictor of clinical outcome in advanced testicular germ-cell tumours

This study was undertaken to investigate the expression and predictive value for outcome of multidrug resistance-associated (MDR) proteins P-glycoprotein (Pgp), MRP1, BCRP, and LRP, in advanced testicular germ-cell tumours (TGCT). Paraffin-embedded sections from 56 previously untreated patients with metastatic TGCT were immunostained for Pgp, MRP1, BCRP, and LRP. All patients received platinum-based chemotherapy after orchidectomy. Immunostaining was related to clinicopathological parameters, response to chemotherapy, and outcome. Strong and intermediate expressions of the different MDR-related proteins were: 27 and 41% (Pgp), 54 and 37% (MRP1), 86 and 7% (BCRP), and 14 and 29% (LRP). P-glycoprotein and MRP1 associated, respectively, to low AFP (P=0.026) and high LDH levels (P=0.014), whereas LRP expression associated with high beta-hCG levels (P=0.003) and stage IV tumours (P=0.029). No correlation was found between Pgp, MRP1, and BCRP expression and response to chemotherapy and survival. In contrast, patients with LRP-positive tumours (strong or intermediate expression) had shorter progression-free (P=0.0006) and overall survival (P=0.0116) than LRP-negative patients, even after individual log-rank adjustments by statistically associated variables. Our data suggest that a positive LRP immunostaining at the time of diagnosis in metastatic TGCT is associated with an adverse clinical outcome.     

BOMP/EPI intensive alternating chemotherapy for IGCCC poor-prognosis germ-cell tumors: The Spanish Germ-Cell Cancer Group experience (GG)

Background: Patients with poor-prognosis germ-cell tumors according to the IGCCC have a poor long-term survival. This study evaluates the efficacy and toxicity of the intensive alternating chemotherapy regimen BOMP/EPI in these patients.Patients and methods: Patients with IGCCC poor-prognosis germ-cell tumors treated at 13 centres were studied. Treatment consisted of bleomycin 30 mg, vincristine 2 mg, methotrexate 300 mg/m2 and cisplatin 100 mg/m2 (BOMP), alternating after a 14-day interval with etoposide 120 mg/m2 day 1–4, ifosfamide 1.3 g/m2 day 1–4 and cisplatin 25 mg/m2 day 1–4 (EPI). BOMP was administered 21 days after the EPI. Bleomycin was administered weekly per 12 weeks.Results: Thirty-eight patients were treated. The median number of cycles administered was 7 (1–10 cycles). Eighteen patients achieved complete responses with chemotherapy alone (12 had necrosis and 2 mature teratoma at postchemotherapy resection), and four achieved complete responses with chemotherapy and surgical resection of viable cancer. Thus, an overall favorable response was achieved in 22 patients (60%). Four additional patients had marker-negative non-resected residual masses. Eleven patients were considered treatment failures, including one who died early and another who succumbed to granulocytopenic sepsis and renal failure. Hematologic toxicity was the most common, with 26 patients (70%) having grade 4 granulocytopenia. After a median follow-up of 41 months, the actuarial two-year overall survival and progression-free survival were 64% and 58%, respectively.Conclusion: BOMP/EPI is active in poor-prognosis germ-cell tumors according to the IGCCC criteria. The results obtained compare favorably with those expected with conventional chemotherapy, and justify further studies.     

Anti-annexin V antibodies and digital ischemia in patients with scleroderma.

OBJECTIVE: To elucidate the frequency and clinical significance of anti-annexin V antibodies in patients with scleroderma. METHODS: The study population consisted of 66 patients with scleroderma. Their sera were examined for IgG anti-annexin V antibodies by ELISA and immunoblotting. RESULTS: IgG anti-annexin V antibodies were detected by ELISA in 12 patients (18.2%) with scleroderma. Anti-annexin V antibodies were associated with digital ischemia in the patients with scleroderma. Only one patient of the 12 had anticardiolipin antibodies. Although 6 patients with anti-annexin V antibodies were also examined for activated partial thromboplastin time (APTT), none showed prolonged APTT. No IgG anti-annexin V antibodies were detected by immunoblotting. CONCLUSION: Anti-annexin V antibodies in scleroderma are related to digital ischemia. These antibodies may be associated with the pathogenesis of vascular involvement in scleroderma.     

Ultrasound diagnosis of ruptured hydatid cyst of the liver with biliary obstruction

Communication between a hydatid cyst of the liver and biliary tract results in a clinical picture of obstructive jaundice because of occupation of the extrahepatic biliary tract by intracystic material. Five cases of this complication are presented. Ultrasound criteria for diagnosis are analyzed, based on previous publications as well as our own experience.     

The management of chest injuries

The mortality from chest injuries is so high due to severe physiologic imbalance that an immediate and accurate diagnosis of the injured organ and prompt treatment can salvage the patient from the strategy. This study comprises 1329 injured cases including 145 patients with crushed chests. The cause of injury was traffic accident in 537 cases (40.5%), fall or degradation in 332 cases (25%). There was a correlation between the cause of injury and age, as that traffic accident was a major one in young aged and fall in elders. Treatment against crushed patients included 150 surgical operations, 206 plaster bandages, 56 drainage of thoracic, peritoneal and cranial cavities. Thoracotomies performed in patients with flail chest (2), lung contusion (4), rupture of the bronchi and diaphragm (each 1) and for evacuation of clotted hemothorax in 3 patients. The prognosis of all these patients was good. Lastly we conclude that since the prognosis of injured patients depends on how fast the patient can be carried to the hospital and how quickly the physician or surgeon can evaluate the trauma and institute a prompt treatment, the education of the primary staff is the most important.     

Analysis of the phenotypic distribution of HLA class I and class II in atopic and non-atopic asthma patients.

In several studies the HLA system has been implicated in the development of asthma, but the importance of the associations between HLA genes and asthma remains unclear. The aim of this study was to determine the HLA class I and II phenotypic frequencies in a population of asthmatics, and to analyse the relationship between these phenotypes and any type of asthma. We typed HLA class I and II antigens in a series of 189 asthmatic individuals (102 atopic and 87 non-atopic), and in a control population of 150 unrelated healthy Caucasoid donors. When the HLA phenotypic frequencies were compared, no statistical differences were found. Therefore, no definitive HLA association could be established with atopic or non-atopic asthma in the studied population. Abbreviations AA, atopic asthma; FEV1, forced expiratory volume in 1 s; NAA, non-atopic asthma; PCR, polymerase chain reaction; SSOP, sequence-specific oligonucleotide probes; SPT, skin prick test.