Drug delivery and in vitro models of the blood-brain barrier

An understanding of the physiology of the blood-brain barrier (BBB) is crucial when addressing complex issues such as drug delivery, pathogenesis of chronic neurological diseases and bio-defense. Rational central nervous system (CNS) drug design cannot entirely and exclusively rely upon the physicochemical properties of putative neurotherapeutics, since lipophilicity alone is a poor predictor for drug penetration into the CNS. This is particularly true for three large families of CNS drugs: antineoplastics, antivirals and anti-epileptics. For these drugs, in contrast to peripheral acting drugs (eg, antihistamines), negligible penetration across the BBB is preferable in order to avoid CNS side effects. Studies performed using small animals such as rodents cannot be directly extrapolated to human brain tissue, as demonstrated by both clinical and in vitro studies. Furthermore, most of the promising CNS drugs that proved effective in vitro have failed in clinical trials due to misleading predictive permeability data extrapolated from models that were not capable of fully reproducing the functional properties of the BBB in vivo. Therefore, a great effort has been made to develop new in vitro models able to reproduce the physiological, anatomical and functional characteristics of the BBB allowing for a better prediction of drug penetration across the BBB, and enabling the design of new pharmaceutical strategies to bypass the shielding of brain parenchyma. Herein we provide a detailed review and discussion of currently employed in vitro BBB models along with probable future developments.     

The ErbB receptors and their ligands in cancer: an overview

This review article provides an overview on the most recent advances on the role of ErbB receptors and growth factors of the epidermal growth factor (EGF)-family of peptides in cancer pathogenesis and progression. The ErbB tyrosine kinases and the EGF-like peptides form a complex system. In fact, the interactions occurring between receptors and ligands of these families affect the type and the duration of the intracellular signals that derive from receptor activation. Interestingly, activation of ErbB receptors is also driven by different classes of membrane receptor, suggesting that ErbB kinases can amplify growth promoting signals carried by different pathways. The importance of ErbB receptors and EGF-like peptides in development of organs and tissues has been demonstrated by using different mouse models. In vitro and in vivo studies have also shown that ErbB receptors and their ligands can act as transforming genes. However, evidence suggests that cooperation of different receptors and ligands is necessary to induce a fully transformed phenotype. Indeed, co-expression of different ErbB receptors and EGF-like growth factors is a common phenomenon in human primary carcinomas. This observation suggests that the growth and the survival of carcinoma cells is sustained by a network of receptors/ligands of the ErbB family. In this respect, the contemporary expression of different ErbB tyrosine kinases and/or EGF-like growth factors in human carcinomas might also affect tumor response to target based agents directed against the ErbB receptor/ligand system.     

Folding and mis-folding of peptides and proteins: insights from molecular simulations

In this paper, the main achievements and problems of the application of all-atom molecular simulations, with particular attention for Molecular Dynamics (MD), will be critically reviewed. Starting from unfolding simulations, through biased simulations, which require a knowledge of the native state conformation, to folding studies based on the simple knowledge of the protein (or peptide) sequence, the strengths and weaknesses of theoretical approaches to the study of folding and their matching with experimental observations will be discussed. Finally, we will give a critical outlook on the possible developments of this field in the near future.     

Genotoxic effects of extremely low frequency (ELF) magnetic fields (MF) evaluated by the Tradescantia-micronucleus assay

Extremely low frequency (ELF) electric fields (EF) and magnetic fields (MF) are generated during the production, transmission, and use of electrical energy. Although epidemiology studies suggest that there is a cancer risk associated with exposure to ELF-MF, short-term genotoxicity assays with bacteria and mammalian cells have produced inconsistent results. In the present study, we investigated the possible genotoxicity of ELF-MF by using the Tradescantia-micronucleus (Trad-MN) assay, a sensitive, reproducible, well-standardized assay for genotoxicity testing. A 50 Hz ELF-MF was generated by a laboratory exposure system consisting of a pair of parallel coils in a Helmholtz configuration. Exposure of Tradescantia (clone # 4430) inflorescences to the ELF-MF, at a flux density (B) corresponding to 1 mT, for 1, 6, and 24 h resulted in a time-dependent increase in MN frequency. The results indicate that a 50 Hz MF of 1 mT field strength is genotoxic in the Trad-MN bioassay and suggest that this assay may be suitable as a biomonitor for detecting the genotoxicity of ELF-MF in the field.     

Lowering of elevated ambulatory blood pressure by HMG-CoA reductase inhibitors

Controversial results were reported as to a possible blood pressure-lowering effect of statins. This may relate to methodological limitations (blood pressure measuring techniques) or to putative different effects of statins in different biologic conditions (cholesterol or blood pressure levels, age, etc). Patients with cholesterol>200 mg/dL and no previous statin treatment underwent 24-hour ambulatory blood pressure (ABP) monitoring and were classified as normotensives or hypertensives according to their ABP. They were randomized to statin (n=51, simvastatin or pravastatin, 10-20 mg/d; atorvastatin, 5-10 mg/d) or control treatment (n=23, soy lecithin, 20 g/d) for 2 months, after which ABP assessment was repeated. No consistent treatment-related reduction in ABP was observed in lecithin-treated patients (either hypertensives or normotensives) or in statin-treated normotensive patients (-0.7+/-5.1/-1.0+/-4.6 mm Hg, both P=ns). In contrast, statin-treated hypertensive patients showed lower systolic and diastolic blood pressure (-5.7+/-5.8/-3.5+/-3.9 mm Hg, both P<0.001), the effect was entirely accounted for by reduced daytime values with no change in nighttime values, and it was unrelated to the concomitant statin-induced cholesterol reduction. Statins moderately but significantly lower blood pressure in patients with high (but not with normal) ABP; the effect is confined to the daytime period and is unrelated to the extent of the cholesterol lowering.     

A mutated p53 status did not prevent the induction of apoptosis by sulforaphane,a promising anti-cancer drug

Summary We investigated apoptosis induction by sulforaphane on three cell lines characterized by a different p53 status. In particular, we used p53-knock-out fibroblasts from newborn mice transfected with the p53-Ser220 mutation, observed in Li-Fraumeni Syndrome patients, as a model of mutated p53 status. Moreover, immortalized fibroblasts from newborn mice expressing or lacking p53 (p53 +/+ andp53-/-, respectively) have been used to verify whether mutated p53 status could prevent sulforaphane-induced apoptotic events. Sulforaphane was able to induce apoptosis on all three cell lines. Indeed, the caspase-3 assays and poly(ADP-ribose)polymerase (PARP) cleavage data indicated that sulforaphane stimulated caspase-3-like activity and degradation of PARP. However, cells with a wild-type or mutated p53 appeared to be more sensitive to the effects of sulforaphane than cells lacking p53. Taken together, our results suggest that sulforaphane could act by a p53-independent pathway. For this reason, sulforaphane can be viewed as a novel agent useful not only in the treatment of Li-Fraumeni-associated tumors but also drug resistant tumors where p53 dysregulation is a feature.     

Combined chemotherapy with carmustine, doxorubicin, etoposide, vincristine, and cyclophosphamide plus mitoxantrone, cytarabine and methotrexate with citrovorum factor for the treatment of aggressive non-Hodgkin lymphoma: a long-term follow-up study

BACKGROUND: The standard treatment for patients with aggressive non-Hodgkin lymphoma (NHL) is cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP). Since 1989, the authors have used a new chemotherapy regimen with combined carmustine, doxorubicin, etoposide, vincristine, and cyclophosphamide plus mitoxantrone, cytarabine and methotrexate with citrovorum factor called BAVEC-MiMA. The objective of the current study was to explore, after a long follow-up period, the impact of this third-generation regimen for the treatment of aggressive NHL. METHODS: One hundred and one consecutive patients (median age, 41 years) with either B-cell (n=94 patients) or non-B-cell (n=7 patients), aggressive lymphoma were diagnosed and treated between 1989 and 1999 with the BAVEC-MiMA regimen. RESULTS: The complete response rate was 74%, and the overall response rate was 89%. Eleven patients with refractory disease died rapidly after a median period of 5 months. The major toxicity was Grade 4 neutropenia (according to World Health Organization criteria), which was observed in 15 patients (15%). There were four toxicity-related deaths. The overall survival rate was 63% at 9 years. In multivariate analysis, factors that were associated with advantage in overall survival were response to induction therapy, bulky disease, and high score on the International Prognostic Index (IPI). The disease-free survival rate was 77% at 9 years. In multivariate analysis, the IPI was the most important variable for the definition of disease-free survival. CONCLUSIONS: The BAVEC-MiMA regimen was feasible on an outpatient basis, it was tolerated well, and it showed a low toxicity-related mortality. The long follow-up in patients with NHL, which is a rapidly fatal disease, led the authors to observe that, with this regimen, a cure was obtained in > 50% of patients who had low-risk or low-to-intermediate-risk, aggressive NHL.     

Achilles tendon for sternal synthesis in the treatment of mediastinitis

Surgical approaches to postoperative mediastinitis that imply wire removal achieve earlier infection recovery but leave the patient with sternal instability. In 10 patients after wound surgical debridement, my colleagues and I achieved sternal synthesis by using Achilles tendons retrieved from multiorgan donors and stored in glutaraldehyde. Three tendons were used in each patient; they were passed through the bone at the manubrium and parasternally at the midsternum and the lower sternum. Thirty-day computed tomographic scan results, infection recovery, and quality of life were satisfactory.