Innovative analytical methods for Central Nervous System Drug analysis in biological fluids

During recent years a consistent number of central nervous system (CNS) drugs have been approved and introduced on the market for the treatment of many psychiatric and neurological disorders, including psychosis, depression, Parkinson disease and epilepsy. Despite the great advancements obtained in the treatment of CNS diseases/disorders, partial response to therapy or treatment failure are frequent, at least in part due to poor compliance, but also genetic variability in the metabolism of psychotropic agents or polypharmacy, which may lead to sub-therapeutic or toxic plasma levels of the drugs, and finally inefficacy of the treatment or adverse/toxic effects. With the aim of improving the treatment, reducing toxic/side effects and patient hospitalisation, Therapeutic Drug Monitoring (TDM) is certainly useful, allowing for a personalisation of the therapy. Reliable analytical methods are required to determine the plasma levels of psychotropic drugs, which are often present at low concentrations (tens or hundreds of nanograms per millilitre). The present PhD Thesis has focused on the development of analytical methods for the determination of CNS drugs in biological fluids, including antidepressants (sertraline and duloxetine), antipsychotics (aripiprazole), antiepileptics (vigabatrin and topiramate) and antiparkinsons (pramipexole). Innovative methods based on liquid chromatography or capillary electrophoresis coupled to diode-array or laser-induced fluorescence detectors have been developed, together with the suitable sample pre-treatment for interference removal and fluorescent labelling in case of LIF detection. All methods have been validated according to official guidelines and applied to the analysis of real samples obtained from patients, resulting suitable for the TDM of psychotropic drugs.

Bioanalytical applications of multicolour bioluminescence imaging: new tools for drug discovery and development

The subject of this thesis is multicolour bioluminescence analysis and how it can provide new tools for drug discovery and development.The mechanism of color tuning in bioluminescent reactions is not fully understood yet but it is object of intense research and several hypothesis have been generated. In the past decade key residues of the active site of the enzyme or in the surface surrounding the active site have been identified as responsible of different color emission. Anyway since bioluminescence reaction is strictly dependent from the interaction between the enzyme and its substrate D-luciferin, modification of the substrate can lead to a different emission spectrum too. In the recent years firefly luciferase and other luciferases underwent mutagenesis in order to obtain mutants with different emission characteristics. Thanks to these new discoveries in the bioluminescence field multicolour luciferases can be nowadays employed in bioanalysis for assay developments and imaging purposes. The use of multicolor bioluminescent enzymes expanded the potential of a range of application in vitro and in vivo. Multiple analysis and more information can be obtained from the same analytical session saving cost and time. This thesis focuses on several application of multicolour bioluminescence for high-throughput screening and in vivo imaging. Multicolor luciferases can be employed as new tools for drug discovery and developments and some examples are provided in the different chapters. New red codon optimized luciferase have been demonstrated to be improved tools for bioluminescence imaging in small animal and the possibility to combine red and green luciferases for BLI has been achieved even if some aspects of the methodology remain challenging and need further improvement. In vivo Bioluminescence imaging has known a rapid progress since its first application no more than 15 years ago. It is becoming an indispensable tool in pharmacological research. At the same time the development of more sensitive and implemented microscopes and low-light imager for a better visualization and quantification of multicolor signals would boost the research and the discoveries in life sciences in general and in drug discovery and development in particular.

Synthese und Charakterisierung zylindrischer Poly(2-oxazolin)bürsten als Nanocarrier für Drug Delivery Systeme

Inspiriert durch natürlich vorkommende Peptide, sind Poly(2-oxazoline) vielversprechende Kandidaten für Anwendungen in Bereichen des kontrollierten Wirkstoff- bzw. Gentransportes, wie die moderne Biomedizin dies fordert. Da Polyoxazoline als strukturisomere Amide von natürlichen Polypeptiden aufgefasst werden können, zeigen diese synthetischen Polymere in direktem Vergleich erhebliche Vorteile etwa hinsichtlich Zytotoxizät und Effizienz, was wesentlich dazu beitragen kann, aktuelle Hürden biomedizinischer Fragestellungen hinsichtlich Transport und Targeting zu überwinden. Darüber hinaus sollten zylindrische Polymerbürsten aufgrund ihrer molekularen, architekturbedingten Formanisotropie und jüngsten Ergebnissen insbesondere zur formabhängigen Endozytose sehr aussichtsreiche Kandidaten für den Einsatz zum Wirkstofftransport sein.rnrnDie vorliegende Arbeit widmete sich deshalb der Synthese und Charakterisierung von biokompatiblen zylindrischen Poly(2-oxazolin)bürsten als potentielle Nanotransporter von Wirkstoffen, Biomolekülen oder genetischem Material. Als Monomer wurde zunächst 2-Isopropyloxazolin gewählt, da das Polymer eine Phasenübergangstemperatur von 37 °C besitzt, was für Konjugatsynthesen wie auch diverse biomedizinische Applikationen interessant sein kann. Durch terminierende Methacrylamid Funktionalisierung der lebenden kationischen Oxazolinpolymerisation bzw. nachfolgende Endgruppen Transferreaktionen sind Makromonomere im Bereich 1000-5000 g/mol zugänglich. Erstmals gelang es so 2-Oxazolin basierte, hochmolekulare zylindrische Bürsten mit Konturlängen im Bereich von 250 nm mittels „Grafting Through“ Technik in freier radikalischer Polymerisation herzustellen.rnrnAusgehend von der entwickelten Syntheseroute konnten so neben Homo- und Blockcopolymerbürsten von 2-Ethyl-2-oxazolin und 2-Isopropyl-2-oxazolin auch Bürstenmoleküle aus statistischen Copolymeren von 2-Ethyl-2-oxazolin und unsubstituiertem 2-Oxazolin hergestellt werden. Während letztere die Einführung kationischer Gruppen durch selektivere Abspaltmethoden der Formylreste erlauben und so etwa DNA/RNA Komplexierungen ermöglichen können, bietet andererseits der in dieser Arbeit erstmalig demonstrierte Einsatz Azid-funktionalisierter Initiatoren zur kationischen Oxazolinpolymerisation unter Beibehaltung aller anderen sonstigen Reaktionsschritte auch die Möglichkeit der Synthese Azid-Endgruppen-funktionalisierter Makromonomere. Die „Grafting Through“ Methodik der freien radikalischen Makromonomer Polymerisation ist selbst bei diesen funktionalisierten Systemen von großem Vorteil, erlaubt sie auch hier den Zugang zu hochmolekularen Substraten mit einem Pfropfungs- bzw. Funktionalisierungsgrad von 100 %, da jede Seitenkette dieser zylindrischen Bürsten die aussenliegende, und damit sterisch leichter zugängliche funktionale Gruppe trägt. Dabei gelang es die Syntheseroute so zu gestalten, dass es möglich war alle vorgestellten Polymerbürsten mittels statischer und dynamischer Lichtstreuung hinsichtlich absoluter Molmasse und molekularer Dimension zu charakterisieren.rnIn weitereren Reaktionen konnten dann reaktive Fluoreszenzfarbstoffe mit Hilfe kupferfreier 1,3 dipolarerer Addition (kupferfreie „Click-Chemie“) an die Azid-funktionalisierten Polymerbürsten angebunden werden, so dass eine wesentliche Voraussetzung für die Detektion in in vivo und in vitro Experimenten erfüllt werden kann. Darüber hinaus gelingt die quantitative polymeranaloge Umsetzung der Azid- zu Aminogruppen durch eine polymeranalog geführte Reduktion nach Staudinger; damit können an diesen Systemen auch etablierte Konjugationstechniken an Aminogruppen durchgeführt werden. Zudem erlauben die Aminogruppen-haltigen Polymerbürsten durch Protonierung schon bei physiologischem pH die Komplexierung von DNA oder RNA. rnrnErste Lichtstreumessungen in Blutserum zeigen im Falle der kationischen Aminogruppen tragenden Polymerbürsten zwar Aggregation, was aber durch entsprechende Umsetzung nach Konjugation wahrscheinlich unterdrückt werden kann, zeigen doch die entsprechenden Precursorpolymerbürsten mit Azidgruppen in Serum keinerlei Aggregation.rnrnZellaufnahmestudien in dendritische Zellen zeigen nur im Falle einer Azid-funktionalisierten Poly(2-isopropyl-2-oxazolin)bürste eine unspezifische Aufnahme. Die hydrophileren Poly(2-oxazolin)bürsten weise keine unspezifische Aufnahme auf, was eine wichtige Anfoderung für die Verwendung als Polymercarrier in der Krebsimmuntherapie ist.rn

Tissue engineering strategies for biomaterial-based anticancer drug delivery and enhancement of angiogenesis in tumor-resected bone

Patienten, die an Osteosarkom leiden werden derzeit mit intravenös applizierten krebstherapeutischen Mitteln nach Tumorresektion behandelt, was oftmals mit schweren Nebenwirkungen und einem verzögerten Knochenheilungsprozess einhergeht. Darüber hinaus treten vermehrt Rezidive aufgrund von verbleibenden neoplastischen Zellen an der Tumorresektionsstelle auf. Erfolgreiche Knochenregeneration und die Kontrolle von den im Gewebe verbleibenden Krebszellen stellt eine Herausforderung für das Tissue Engineering nach Knochenverlust durch Tumorentfernung dar. In dieser Hinsicht scheint der Einsatz von Hydroxyapatit als Knochenersatzmaterial in Kombination mit Cyclodextrin als Medikamententräger, vielversprechend. Chemotherapeutika können an Biomaterial gebunden und direkt am Tumorbett über einen längeren Zeitraum freigesetzt werden, um verbliebene neoplastische Zellen zu eliminieren. Lokal applizierte Chemotherapie hat diverse Vorteile, einschließlich der direkten zytotoxischen Auswirkung auf lokale Zellen, sowie die Reduzierung schwerer Nebenwirkungen. Diese Studie wurde durchgeführt, um die Funktionsfähigkeit eines solchen Arzneimittelabgabesystems zu bewerten und um Strategien im Bereich des Tissue Engineerings zu entwickeln, die den Knochenheilungsprozess und im speziellen die Vaskularisierung fördern sollen. Die Ergebnisse zeigen, dass nicht nur Krebszellen von der chemotherapeutischen Behandlung betroffen sind. Primäre Endothelzellen wie zum Beispiel HUVEC zeigten eine hohe Sensibilität Cisplatin und Doxorubicin gegenüber. Beide Medikamente lösten in HUVEC ein tumor-unterdrückendes Signal durch die Hochregulation von p53 und p21 aus. Zudem scheint Hypoxie einen krebstherapeutischen Einfluss zu haben, da die Behandlung sensitiver HUVEC mit Hypoxie die Zellen vor Zytotoxizität schützte. Der chemo-protektive Effekt schien deutlich weniger auf Krebszelllinien zu wirken. Diese Resultate könnten eine mögliche chemotherapeutische Strategie darstellen, um den Effekt eines zielgerichteten Medikamenteneinsatzes auf Krebszellen zu verbessern unter gleichzeitiger Schonung gesunder Zellen. Eine erfolgreiche Integration eines Systems, das Arzneimittel abgibt, kombiniert mit einem Biomaterial zur Stabilisierung und Regeneration, könnte gesunden Endothelzellen die Möglichkeit bieten zu proliferieren und Blutgefäße zu bilden, während verbleibende Krebszellen eliminiert werden. Da der Prozess der Knochengeweberemodellierung mit einer starken Beeinträchtigung der Lebensqualität des Patienten einhergeht, ist die Beschleunigung des postoperativen Heilungsprozesses eines der Ziele des Tissue Engineerings. Die Bildung von Blutgefäßen ist unabdingbar für eine erfolgreiche Integration eines Knochentransplantats in das Gewebe. Daher ist ein umfangreich ausgebildetes Blutgefäßsystem für einen verbesserten Heilungsprozess während der klinischen Anwendung wünschenswert. Frühere Experimente zeigen, dass sich die Anwendung von Ko-Kulturen aus humanen primären Osteoblasten (pOB) und humanen outgrowth endothelial cells (OEC) im Hinblick auf die Bildung stabiler gefäßähnlicher Strukturen in vitro, die auch effizient in das mikrovaskuläre System in vivo integriert werden konnten, als erfolgreich erweisen. Dieser Ansatz könnte genutzt werden, um prä-vaskularisierte Konstrukte herzustellen, die den Knochenheilungsprozess nach der Implantation fördern. Zusätzlich repräsentiert das Ko-Kultursystem ein exzellentes in vitro Model, um Faktoren, welche stark in den Prozess der Knochenheilung und Angiogenese eingebunden sind, zu identifizieren und zu analysieren. Es ist bekannt, dass Makrophagen eine maßgebliche Rolle in der inflammatorisch-induzierten Angiogenese spielen. In diesem Zusammenhang hebt diese Studie den positiven Einfluss THP-1 abgeleiteter Makrophagen in Ko-Kultur mit pOB und OEC hervor. Die Ergebnisse zeigten, dass die Anwendung von Makrophagen als inflammatorischer Stimulus im bereits etablierten Ko-Kultursystem zu einer pro-angiogenen Aktivierung der OEC führte, was in einer signifikant erhöhten Bildung blutgefäßähnlicher Strukturen in vitro resultierte. Außerdem zeigte die Analyse von Faktoren, die in der durch Entzündung hervorgerufenen Angiogenese eine wichtige Rolle spielen, eine deutliche Hochregulation von VEGF, inflammatorischer Zytokine und Adhäsionsmoleküle, die letztlich zu einer verstärkten Vaskularisierung beitragen. Diese Resultate werden dem Einfluss von Makrophagen zugeschrieben und könnten zukünftig im Tissue Engineering eingesetzt werden, um den Heilungsprozess zu beschleunigen und damit die klinische Situation von Patienten zu verbessern. Darüber hinaus könnte die Kombination der auf Ko-Kulturen basierenden Ansätze für das Knochen Tissue Engineering mit einem biomaterial-basierenden Arzneimittelabgabesystem zum klinischen Einsatz kommen, der die Eliminierung verbliebener Krebszellen mit der Förderung der Knochenregeneration verbindet.

American Society of Clinical Oncology/American Society of Hematology clinical practice guideline update on the use of epoetin and darbepoetin in adult patients with cancer

Purpose To update American Society of Clinical Oncology/American Society of Hematology recommendations for use of erythropoiesis-stimulating agents (ESAs) in patients with cancer. Methods An Update Committee reviewed data published between January 2007 and January 2010. MEDLINE and the Cochrane Library were searched. Results The literature search yielded one new individual patient data analysis and four literature-based meta-analyses, two systematic reviews, and 13 publications reporting new results from randomized controlled trials not included in prior or new reviews. Recommendations For patients undergoing myelosuppressive chemotherapy who have a hemoglobin (Hb) level less than 10 g/dL, the Update Committee recommends that clinicians discuss potential harms (eg, thromboembolism, shorter survival) and benefits (eg, decreased transfusions) of ESAs and compare these with potential harms (eg, serious infections, immune-mediated adverse reactions) and benefits (eg, rapid Hb improvement) of RBC transfusions. Individual preferences for assumed risk should contribute to shared decisions on managing chemotherapy-induced anemia. The Committee cautions against ESA use under other circumstances. If used, ESAs should be administered at the lowest dose possible and should increase Hb to the lowest concentration possible to avoid transfusions. Available evidence does not identify Hb levels � 10 g/dL either as thresholds for initiating treatment or as targets for ESA therapy. Starting doses and dose modifications after response or nonresponse should follow US Food and Drug Administration–approved labeling. ESAs should be discontinued after 6 to 8 weeks in nonresponders. ESAs should be avoided in patients with cancer not receiving concurrent chemotherapy, except for those with lower risk myelodysplastic syndromes. Caution should be exercised when using ESAs with chemotherapeutic agents in diseases associated with increased risk of thromboembolic complications. Table 1 lists detailed recommendations. This guideline was developed through a collaboration between the American Society of Clinical Oncology and the American Society of Hematology and has been published jointly by invitation and consent in both Journal of Clinical Oncology and Blood.

American Society of Hematology/American Society of Clinical Oncology clinical practice guideline update on the use of epoetin and darbepoetin in adult patients with cancer

Purpose: To update American Society of Hematology/American Society of Clinical Oncology recommendations for use of erythropoiesis-stimulating agents (ESAs) in patients with cancer. Methods: An Update Committee reviewed data published between January 2007 and January 2010. MEDLINE and the Cochrane Library were searched. Results: The literature search yielded one new individual patient data analysis and four literature-based meta-analyses, two systematic reviews, and 13 publications reporting new results from randomized controlled trials not included in prior or new reviews. Recommendations: For patients undergoing myelosuppressive chemotherapy who have a hemoglobin (Hb) level less than 10 g/dL, the Update Committee recommends that clinicians discuss potential harms (eg, thromboembolism, shorter survival) and benefits (eg, decreased transfusions) of ESAs and compare these with potential harms (eg, serious infections, immune-mediated adverse reactions) and benefits (eg, rapid Hb improvement) of RBC transfusions. Individual preferences for assumed risk should contribute to shared decisions on managing chemotherapy-induced anemia. The Committee cautions against ESA use under other circumstances. If used, ESAs should be administered at the lowest dose possible and should increase Hb to the lowest concentration possible to avoid transfusions. Available evidence does not identify Hb levels 10 g/dL either as thresholds for initiating treatment or as targets for ESA therapy. Starting doses and dose modifications after response or nonresponse should follow US Food and Drug Administration-approved labeling. ESAs should be discontinued after 6 to 8 weeks in nonresponders. ESAs should be avoided in patients with cancer not receiving concurrent chemotherapy, except for those with lower risk myelodysplastic syndromes. Caution should be exercised when using ESAs with chemotherapeutic agents in diseases associated with increased risk of thromboembolic complications. Table 1 lists detailed recommendations.

Impact of stent overlap on angiographic and long-term clinical outcome in patients undergoing drug-eluting stent implantation

Objectives We compared the angiographic and long-term clinical outcomes of patients with and without overlap of drug-eluting stents (DES). Background DES overlap has been associated with delayed healing and increased inflammation in experimental studies, but its impact on clinical outcome is not well established. Methods We analyzed the angiographic and clinical outcomes of 1,012 patients treated with DES in the SIRTAX (Sirolimus-Eluting Versus Paclitaxel-Eluting Stents for Coronary Revascularization) trial according to the presence or absence of stent overlap and the number of stents per vessel: 134 (13.2%) patients with multiple DES in a vessel with overlap, 199 (19.7%) patients with multiple DES in a vessel without overlap, and 679 (67.1%) patients with 1 DES per vessel. Results Angiographic follow-up at 8 months showed an increased late loss in DES overlap patients (0.33 ± 0.61 mm) compared with the other groups (0.18 ± 0.43 mm and 0.15 ± 0.38 mm, p < 0.01). The smallest minimal lumen diameter was located at the zone of stent overlap in 17 (68%) of 25 patients with stent overlap who underwent target lesion revascularization. Major adverse cardiac events were more common in patients with DES overlap (34 events, 25.4%) than in the other groups (42 events, 21.1% and 95 events, 14.0%) at 3 years (p < 0.01). Both the risk of target lesion revascularization (20.2% vs. 16.1% vs. 9.7%, p < 0.01) and the composite of death or myocardial infarction (17.2% vs. 14.1% vs. 9.1%, p = 0.01) were increased in patients with DES overlap compared with the other groups. Conclusions DES overlap occurs in >10% of patients undergoing percutaneous coronary intervention in routine clinical practice and is associated with impaired angiographic and long-term clinical outcome, including death or myocardial infarction. (Sirolimus-Eluting Versus Paclitaxel-Eluting Stents for Coronary Revascularization; NCT00297661).

Cross-reactivity in drug hypersensitivity reactions to sulfasalazine and sulfamethoxazole

Sulfonamides are generally classified into 2 groups: antibiotics and non-antibiotics. Recent studies showed that patients allergic to sulfonamide antibiotics do not have a specific risk for an allergy to sulfonamide non-antibiotic. However, the anti-inflammatory drug sulfasalazine represents an important exception. Used in rheumatic diseases, it is classified as a non-antibiotic sulfonamide, but is structurally related to antibiotic sulfonamides. Therefore, we aimed to analyze in vitro the cross-reactivity between the antimicrobial sulfamethoxazole and the anti-inflammatory drug sulfasalazine.

Educational case series: Mechanisms of drug allergy

Once administered, a drug can activate the immune system by various mechanisms and lead to a large range of clinical manifestations closely related to the type of immune reaction elicited. Administration of the drug can classically result in an immunoglobulin E (IgE)-type sensitization, but can also result in more complex activation of the immune system potentially resulting in severe syndromes, such as the drug-induced hypersensitivity syndrome (DIHS). Although there has been a major increase in our knowledge over the last years, the exact mechanisms of drug allergy are not well understood for most clinical manifestations. A complex interaction between individual characteristics, environmental factors, and the drug itself is usually responsible for adverse reactions to drugs. In this educational review series, we described three cases of drug allergy: first, a child with a typical IgE-mediated drug allergy, second, a child with a non-immediate reaction to penicillin, and in the third patient, we will discuss the drug-induced hypersensitivity syndrome, which is rare but potentially fatal. These cases are correlated to the immune mechanism potentially involved.

Human leukocyte antigens (HLA) associated drug hypersensitivity: consequences of drug binding to HLA

Recent publications have shown that certain human leukocyte antigen (HLA) alleles are strongly associated with hypersensitivity to particular drugs. As HLA molecules are a critical element in T-cell stimulation, it is no surprise that particular HLA alleles have a direct functional role in the pathogenesis of drug hypersensitivity. In this context, a direct interaction of the relevant drug with HLA molecules as described by the p-i concept appears to be more relevant than presentation of hapten-modified peptides. In some HLA-associated drug hypersensitivity reactions, the presence of a risk allele is a necessary but incomplete factor for disease development. In carbamazepine and HLA-B*15:02, certain T-cell receptor (TCR) repertoires are required for immune activation. This additional requirement may be one of the 'missing links' in explaining why most individuals carrying this allele can tolerate the drug. In contrast, abacavir generates polyclonal T-cell response by interacting specifically with HLA-B*57:01 molecules. T cell stimulation may be due to presentation of abacavir or of altered peptides. While the presence of HLA-B*58:01 allele substantially increases the risk of allopurinol hypersensitivity, it is not an absolute requirement, suggesting that other factors also play an important role. In summary, drug hypersensitivity is the end result of a drug interaction with certain HLA molecules and TCRs, the sum of which determines whether the ensuing immune response is going to be harmful or not.

Long-lasting reactivity and high frequency of drug-specific T cells after severe systemic drug hypersensitivity reactions

BACKGROUND: Drug-reactive T cells are involved in most drug-induced hypersensitivity reactions. The frequency of such cells in peripheral blood of patients with drug allergy after remission is unclear. OBJECTIVE: We determined the frequency of drug-reactive T cells in the peripheral blood of patients 4 months to 12 years after severe delayed-type drug hypersensitivity reactions, and whether the frequency of these cell differs from the frequency of tetanus toxoid-reactive T cells. METHODS: We analyzed 5 patients with delayed-type drug hypersensitivity reactions, applying 2 methods: quantification of cytokine-secreting T cells by enzyme-linked immunospot (ELISpot), and fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) intensity distribution analysis of drug-reactive T cells. RESULTS: Frequencies found were between 0.02% and 0.4% of CD4(+) T cells reacting to the respective drugs measured by CFSE analysis, and between 0.01% and 0.08% of T cells as determined by ELISpot. Reactivity was seen neither to drugs to which the patients were not sensitized nor in healthy individuals after stimulation with any of the drugs used. CONCLUSION: About 1:250 to 1:10,000 of T cells of patients with drug allergy are reactive to the relevant drugs. This frequency of drug-reactive T cells is higher than the frequency of T cells able to recognize recall antigens like tetanus toxoid in the same subjects. A substantial frequency could be observed as long as 12 years later in 1 patient even after strict drug avoidance. Patients with severe delayed drug hypersensitivity reactions are therefore potentially prone to react again to the incriminated drug even years after strict drug avoidance.

Adverse side-effects to biological agents

Biological agents-like cytokines, monoclonal antibodies and fusion proteins are widely used in anti-inflammatory and tumour therapy. They are highly efficient in certain diseases, but can cause a great variety of adverse side-effects. Based on the peculiar features of biological agents a new classification of these adverse side-effects of biological agents is proposed - related but clearly distinct from the classification of side-effects observed with chemicals and drugs. This classification differentiates five distinct types, namely clinical reactions because of high cytokine levels (type alpha), hypersensitivity because of an immune reaction against the biological agent (beta), immune or cytokine imbalance syndromes (gamma), symptoms because of cross-reactivity (delta) and symptoms not directly affecting the immune system (epsilon). This classification could help to better deal with the clinical features of these side-effects, to identify possible individual and general risk factors and to direct research in this novel area of medicine.