Development of Original Analytical Methods for the Determination of Drugs of Abuse in Biological Samples

Drug abuse is a major global problem which has a strong impact not only on the single individual but also on the entire society. Among the different strategies that can be used to address this issue an important role is played by identification of abusers and proper medical treatment. This kind of therapy should be carefully monitored in order to discourage improper use of the medication and to tailor the dose according to the specific needs of the patient. Hence, reliable analytical methods are needed to reveal drug intake and to support physicians in the pharmacological management of drug dependence. In the present Ph.D. thesis original analytical methods for the determination of drugs with a potential for abuse and of substances used in the pharmacological treatment of drug addiction are presented. In particular, the work has been focused on the analysis of ketamine, naloxone and long-acting opioids (buprenorphine and methadone), oxycodone, disulfiram and bupropion in human plasma and in dried blood spots. The developed methods are based on the use of high performance liquid chromatography (HPLC) coupled to various kinds of detectors (mass spectrometer, coulometric detector, diode array detector). For biological sample pre-treatment different techniques have been exploited, namely solid phase extraction and microextraction by packed sorbent. All the presented methods have been validated according to official guidelines with good results and some of these have been successfully applied to the therapeutic drug monitoring of patients under treatment for drug abuse.

Development and performance assessment of a Plasma Focus electron beam generator for Intra-Operative Radiation Therapy

The Plasma Focus is a device designed to generate a plasma sheet between two coaxial electrodes by means of a high voltage difference. The plasma is then driven to collapse into a “pinch”, where thermonuclear conditions prevail. During the “pinch phase” charged particles are emitted, with two main components: an ion beam peaked forward and an electron beam directed backward. The electron beam emitted backward by Plasma Focus devices is being investigated as a radiation source for medical applications, using it to produce x-rays by interaction with appropriate targets (through bremsstrahlung and characteristic emission). A dedicated Plasma Focus device, named PFMA-3 (Plasma Focus for Medical Applications number 3), has been designed, put in operation and tested by the research groups of the Universities of Bologna and Ferrara. The very high dose rate (several gray per discharge, in less than 1 µs) is a peculiarity of this device that has to be investigated, as it might modify the relative biological effectiveness (RBE). Aim of this Ph.D. project was to investigate the main physical properties of the low-energy x-ray beams produced by a Plasma Focus device and their potential medical applications to IORT treatments. It was necessary to develop the optimal geometrical configuration; to evaluate the x-rays produced and their dose deposited; to estimate the energy electron spectrum produced in the “pinch phase”; to study an optimal target for the conversion of the x-rays; to conduct simulations to study the physics involved; and in order to evaluate the radio-biological features of the beam, cell holders had to be developed for both irradiations and cell growth conditions.

Trattamento della malattia paranale complessa di Crohn: rescue therapy dopo fallimento dei farmaci biologici

La malattia paranale di Crohn rappresenta una condizione clinica complessa e invalidante. La chirurgia da sola è efficace nel migliorare i sintomi mediante il controllo della sepsi, ma è raramente associata alla guarigione definitiva. L'introduzione dei farmaci biologici ha aumentato le possibilità di chiusura definitiva delle fistole. Tuttavia molti pazienti non rispondono a questo trattamento bio-chirurgico combinato. Il ruolo del mucosal healing del retto ottenuto con i farmaci non è al momento ancora chiaro. L'obiettivo del presente studio è quello di identificare possibili terapia alternative per pazienti non responsivi ai biologici. Lo studio ha valutato efficacia e sicurezza della chirurgia riparativa, confezionamento di flap mucosi endorettali e posizionamento di protesi biologiche, nei pazienti non responsivi ai biologici ma che grazie ad essi abbiano ottenuto un mucosal healing del retto.

Development and in vitro testing of liposomal gadolinium-formulations for neutron capture therapy of glioblastoma multiforme

For the improvement of current neutron capture therapy, several liposomal formulations of neutron capture agent gadolinium were developed and tested in a glioma cell model. Formulations were analyzed regarding physicochemical and biological parameters, such as size, zeta potential, uptake into cancer cells and performance under neutron irradiation. The neutron and photon dose derived from intracellular as well as extracellular Gd was calculated via Monte Carlo simulations and set in correlation with the reduction of cell survival after irradiation. To investigate the suitability of Gd as a radiosensitizer for photon radiation, cells were also irradiated with synchrotron radiation in addition to clinically used photons generated by linear accelerator.rnIrradiation with neutrons led to significantly lower survival for Gd-liposome-treated F98 and LN229 cells, compared to irradiated control cells and cells treated with non-liposomal Gd-DTPA. Correlation between Gd-content and -dose and respective cell survival displayed proportional relationship for most of the applied formulations. Photon irradiation experiments showed the proof-of-principle for the radiosensitizer approach, although the photon spectra currently used have to be optimized for higher efficiency of the radiosensitizer. In conclusion, the newly developed Gd-liposomes show great potential for the improvement of radiation treatment options for highly malignant glioblastoma.rn

Phage therapy: A software system for phage quantification and kinetic model inference.

One of the most serious problems of the modern medicine is the growing emergence of antibiotic resistance among pathogenic bacteria. In this circumstance, different and innovative approaches for treating infections caused by multidrug-resistant bacteria are imperatively required. Bacteriophage Therapy is one among the fascinating approaches to be taken into account. This consists of the use of bacteriophages, viruses that infect bacteria, in order to defeat specific bacterial pathogens. Phage therapy is not an innovative idea, indeed, it was widely used around the world in the 1930s and 1940s, in order to treat various infection diseases, and it is still used in Eastern Europe and the former Soviet Union. Nevertheless, Western scientists mostly lost interest in further use and study of phage therapy and abandoned it after the discovery and the spread of antibiotics. The advancement of scientific knowledge of the last years, together with the encouraging results from recent animal studies using phages to treat bacterial infections, and above all the urgent need for novel and effective antimicrobials, have given a prompt for additional rigorous researches in this field. In particular, in the laboratory of synthetic biology of the department of Life Sciences at the University of Warwick, a novel approach was adopted, starting from the original concept of phage therapy, in order to study a concrete alternative to antibiotics. The innovative idea of the project consists in the development of experimental methodologies, which allow to engineer a programmable synthetic phage system using a combination of directed evolution, automation and microfluidics. The main aim is to make “the therapeutics of tomorrow individualized, specific, and self-regulated” (Jaramillo, 2015). In this context, one of the most important key points is the Bacteriophage Quantification. Therefore, in this research work, a mathematical model describing complex dynamics occurring in biological systems involving continuous growth of bacteriophages, modulated by the performance of the host organisms, was implemented as algorithms into a working software using MATLAB. The developed program is able to predict different unknown concentrations of phages much faster than the classical overnight Plaque Assay. What is more, it gives a meaning and an explanation to the obtained data, making inference about the parameter set of the model, that are representative of the bacteriophage-host interaction.

Toward cell therapy using placenta-derived cells: disease mechanisms, cell biology, preclinical studies, and regulatory aspects at the round table

Among the many cell types that may prove useful to regenerative medicine, mounting evidence suggests that human term placenta-derived cells will join the list of significant contributors. In making new cell therapy-based strategies a clinical reality, it is fundamental that no a priori claims are made regarding which cell source is preferable for a particular therapeutic application. Rather, ongoing comparisons of the potentiality and characteristics of cells from different sources should be made to promote constant improvement in cell therapies, and such comparisons will likely show that individually tailored cells can address disease-specific clinical needs. The principle underlying such an approach is resistance to the notion that comprehensive characterization of any cell type has been achieved, neither in terms of phenotype nor risks-to-benefits ratio. Tailoring cell therapy approaches to specific conditions also requires an understanding of basic disease mechanisms and close collaboration between translational researchers and clinicians, to identify current needs and shortcomings in existing treatments. To this end, the international workshop entitled "Placenta-derived stem cells for treatment of inflammatory diseases: moving toward clinical application" was held in Brescia, Italy, in March 2009, and aimed to harness an understanding of basic inflammatory mechanisms inherent in human diseases with updated findings regarding biological and therapeutic properties of human placenta-derived cells, with particular emphasis on their potential for treating inflammatory diseases. Finally, steps required to allow their future clinical application according to regulatory aspects including good manufacturing practice (GMP) were also considered. In September 2009, the International Placenta Stem Cell Society (IPLASS) was founded to help strengthen the research network in this field.

Role of somatostatins in gastroenteropancreatic neuroendocrine tumor development and therapy

The incidence and prevalence of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) have increased in the past 20 years. GEP-NETs are heterogeneous tumors, in terms of clinical and biological features, that originate from the pancreas or the intestinal tract. Some GEP-NETs grow very slowly, some grow rapidly and do not cause symptoms, and others cause hormone hypersecretion and associated symptoms. Most GEP-NETs overexpress receptors for somatostatins. Somatostatins inhibit the release of many hormones and other secretory proteins; their effects are mediated by G protein-coupled receptors that are expressed in a tissue-specific manner. Most GEP-NETs overexpress the somatostatin receptor SSTR2; somatostatin analogues are the best therapeutic option for functional neuroendocrine tumors because they reduce hormone-related symptoms and also have antitumor effects. Long-acting formulations of somatostatin analogues stabilize tumor growth over long periods. The development of radioactive analogues for imaging and peptide receptor radiotherapy has improved the management of GEP-NETs. Peptide receptor radiotherapy has significant antitumor effects, increasing overall survival times of patients with tumors that express a high density of SSTRs, particularly SSTR2 and SSTR5. The multi-receptor somatostatin analogue SOM230 (pasireotide) and chimeric molecules that bind SSTR2 and the dopamine receptor D2 are also being developed to treat patients with GEP-NETs. Combinations of radioactive labeled and unlabeled somatostatin analogues and therapeutics that inhibit other signaling pathways, such as mammalian target of rapamycin (mTOR) and vascular endothelial growth factor, might be the most effective therapeutics for GEP-NETs.

Biological determinants of responsiveness to growth hormone: pharmacogenomics and personalized medicine

It is becoming most clear that many genes are involved in controlling the regulation of growth. Ultimately however, at the level of growth hormone (GH), the relevant question may be not whether a patient is GH-deficient, but whether he is GH-responsive. As these disturbances can be divided into two gross categories, namely alterations causing subnormal GH secretion and/or those presenting with subnormal GH sensitivity/responsiveness, the main aim of this review is to focus on genes involved in growth regulation leading to short stature caused by an alteration of GH insensitivity/GH responsiveness; in other words, clinical circumstances where individually adapted GH replacement therapy may help to increase height velocity and eventually final height.

Comparison of biological stability and metabolism of CCK2 receptor targeting peptides, a collaborative project under COST BM0607

Stability of radiolabelled cholecystokinin 2 (CCK2) receptor targeting peptides has been a major limitation in the use of such radiopharmaceuticals especially for targeted radionuclide therapy applications, e.g. for treatment of medullary thyroid carcinoma (MTC). The purpose of this study was to compare the in vitro stability of a series of peptides binding to the CCK2 receptor [selected as part of the COST Action on Targeted Radionuclide Therapy (BM0607)] and to identify major cleavage sites.

Biologicals and fetal cell therapy for wound and scar management

Few biopharmaceutical preparations developed from biologicals are available for tissue regeneration and scar management. When developing biological treatments with cellular therapy, selection of cell types and establishment of consistent cell banks are crucial steps in whole-cell bioprocessing. Various cell types have been used in treatment of wounds to reduce scar to date including autolog and allogenic skin cells, platelets, placenta, and amniotic extracts. Experience with fetal cells show that they may provide an interesting cell choice due to facility of outscaling and known properties for wound healing without scar. Differential gene profiling has helped to point to potential indicators of repair which include cell adhesion, extracellular matrix, cytokines, growth factors, and development. Safety has been evidenced in Phase I and II clinical fetal cell use for burn and wound treatments with different cell delivery systems. We present herein that fetal cells present technical and therapeutic advantages compared to other cell types for effective cell-based therapy for wound and scar management.

Systemic therapy of atopic dermatitis in children and adults

Systemic therapy is required in patients with severe atopic dermatitis (AD) refractory to adequate topical therapy. The aim of a systemic therapy is the rapid and efficient improvement of skin symptoms and pruritus in acute exacerbation and/or the long-term control of severe chronic disease. A number of immunosuppressive and immunomodulating substances are available that may efficiently reduce skin inflammation and thus lead to a relief of symptoms including pruritus. The excellent effects of cyclosporine as short-term as well as maintenance therapy have been documented in several studies in children and adults. Furthermore, other immunosuppressive substances such as azathioprine, mycophenolate mofetil and methotrexate are effective in patients with moderate to severe AD. Intravenous immunoglobulins and γ-interferon exert immunomodulatory effects and thus may improve severe AD. Biological agents are a new approach in AD therapy since they may specifically target cells, cytokines or mediators involved in the pathogenesis of AD.

Current state of evidence on 'off-label' therapeutic options for systemic lupus erythematosus, including biological immunosuppressive agents, in Germany, Austria and Switzerland--a consensus report

Systemic lupus erythematosus (SLE) can be a severe and potentially life-threatening disease that often represents a therapeutic challenge because of its heterogeneous organ manifestations. Only glucocorticoids, chloroquine and hydroxychloroquine, azathioprine, cyclophosphamide and very recently belimumab have been approved for SLE therapy in Germany, Austria and Switzerland. Dependence on glucocorticoids and resistance to the approved therapeutic agents, as well as substantial toxicity, are frequent. Therefore, treatment considerations will include 'off-label' use of medication approved for other indications. In this consensus approach, an effort has been undertaken to delineate the limits of the current evidence on therapeutic options for SLE organ disease, and to agree on common practice. This has been based on the best available evidence obtained by a rigorous literature review and the authors' own experience with available drugs derived under very similar health care conditions. Preparation of this consensus document included an initial meeting to agree upon the core agenda, a systematic literature review with subsequent formulation of a consensus and determination of the evidence level followed by collecting the level of agreement from the panel members. In addition to overarching principles, the panel have focused on the treatment of major SLE organ manifestations (lupus nephritis, arthritis, lung disease, neuropsychiatric and haematological manifestations, antiphospholipid syndrome and serositis). This consensus report is intended to support clinicians involved in the care of patients with difficult courses of SLE not responding to standard therapies by providing up-to-date information on the best available evidence.

A phase IIa randomized controlled pilot study evaluating the safety and clinical outcomes following the use of rhGDF-5/β-TCP in regenerative periodontal therapy

To present the safety profile, the early healing phase and the clinical outcomes at 24 weeks following treatment of human intrabony defects with open flap debridement (OFD) alone or with OFD and rhGDF-5 adsorbed onto a particulate β-tricalcium phosphate (β-TCP) carrier. Twenty chronic periodontitis patients, each with at least one tooth exhibiting a probing depth ≥6 mm and an associated intrabony defect ≥4 mm entered the study. Ten subjects (one defect/patient) were randomized to receive OFD alone (control) and ten subjects OFD combined with rhGDF-5/β-TCP. Blood samples were collected at screening, and at weeks 2 and 24 to evaluate routine hematology and clinical chemistry, rhGDF-5 plasma levels, and antirhGDF-5 antibody formation. Plaque and gingival indices, bleeding on probing, probing depth, clinical attachment level, and radiographs were recorded pre- and 24 weeks postsurgery. Comparable safety profiles were found in the two treatment groups. Neither antirhGDF-5 antibody formation nor relevant rhGDF-5 plasma levels were detected in any patient. At 6 months, treatment with OFD + rhGDF-5/β-TCP resulted in higher but statistically not significant PD reduction (3.7 ± 1.2 vs. 3.1 ± 1.8 mm; p = 0.26) and CAL gain (3.2 ± 1.7 vs. 1.7 ± 2.2 mm; p = 0.14) compared to OFD alone. In the tested concentration, the use of rhGDF-5/β-TCP appeared to be safe and the material possesses a sound biological rationale. Thus, further adequately powered, randomized controlled clinical trials are warranted to confirm the clinical relevance of this new approach in regenerative periodontal therapy. rhGDF-5/β-TCP may represent a promising new techology in regenerative periodontal therapy.

Reduced neuronal activity in language-related regions after transcranial magnetic stimulation therapy for auditory verbal hallucinations

Transcranial magnetic stimulation (TMS) is a novel therapeutic approach, used in patients with pharmacoresistant auditory verbal hallucinations (AVH). To investigate the neurobiological effects of TMS on AVH, we measured cerebral blood flow with pseudo-continuous magnetic resonance-arterial spin labeling 20 ± 6 hours before and after TMS treatment.

Intervertebral disc regeneration or repair with biomaterials and stem cell therapy--feasible or fiction?

The "gold standard" for treatment of intervertebral disc herniations and degenerated discs is still spinal fusion, corresponding to the saying "no disc - no pain". Mechanical prostheses, which are currently implanted, do only have medium outcome success and have relatively high re-operation rates. Here, we discuss some of the biological intervertebral disc replacement approaches, which can be subdivided into at least two classes in accordance to the two different tissue types, the nucleus pulposus (NP) and the annulus fibrosus (AF). On the side of NP replacement hydrogels have been extensively tested in vitro and in vivo. However, these gels are usually a trade-off between cell biocompatibility and load-bearing capacity, hydrogels which fulfill both are still lacking. On the side of AF repair much less is known and the question of the anchoring of implants is still to be addressed. New hope for cell therapy comes from developmental biology investigations on the existence of intervertebral disc progenitor cells, which would be an ideal cell source for cell therapy. Also notochordal cells (remnants of the embryonic notochord) have been recently pushed back into focus since these cells have regenerative potential and can activate disc cells. Growth factor treatment and molecular therapies could be less problematic. The biological solutions for NP and AF replacement are still more fiction than fact. However, tissue engineering just scratched the tip of the iceberg, more satisfying solutions are yet to be added to the biomedical pipeline.