<p>Metotreksat kao antagonista folne kiseline ima &scaron;iroku upotrebu za lečenje brojnih maligniteta, primenjen u visokim dozama i u&nbsp; kombinciji&nbsp; sa leukovorinom. Iako&nbsp; je&nbsp; terapija&nbsp; visokim&nbsp; dozama&nbsp; metotreksata drastično pobolj&scaron;ala&nbsp; prognozu pacijenata sa malignitetom, te&scaron;ki neželjeni efekti terapije predstavljaju stalan klinički problem. Ciljevi istraživanja bili su određivanje serumske koncentracije metotreksata i izračunavanje farmakokinetičkih&nbsp; parametara&nbsp; metotreksata kod dece obolele od malignih&nbsp; bolesti&nbsp; koja su na terapiji visokim dozama metotreksata (2&nbsp; g/m² i&nbsp; 5&nbsp; g/m² ); ispitivanje postojanja uticaja primenjene doze metotreksata, demografskih i kliničkih karakteristika ispitanika&nbsp; na koncentracije i farmakokinetičke parametare. Ispitivano je prisustvo i stepen kliničkih i laboratorijskih znakova toksičnosti metotreksata, kao i uticaj primenjene&nbsp; doze&nbsp; metotreksata&nbsp; i demografskih karakteristika ispitanika&nbsp; na pojavu i stepen toksičnosti . U okviru retrospektivno - prospektivne&nbsp; studije&nbsp; ukjučeno&nbsp; je&nbsp; četrdeset&nbsp; i dva pedijatrijska&nbsp; pacijenta&nbsp; uzrasta od&nbsp; 0,75 do 17,75 godina (medijana 5,75&nbsp; godina). Svi pacijenti&nbsp; su lečeni&nbsp; u&nbsp; Službi&nbsp; za&nbsp; hematologiju i&nbsp; onkologiju&nbsp; Instituta&nbsp; za&nbsp; zdravstvenu za&scaron;titu dece i omladine Vojvodine (Novi Sad, Srbija) u periodu od juna 2004. godine do juna 2012. godine. Trideset i osam ispitanika&nbsp; je lečeno pod dijagnozom akutne limfoblastne leukemije&nbsp; prema dva&nbsp; uzastopna&nbsp; protokola ALL IC - BFM 2002 i ALL IC - BFM 2009 Internacionalne&nbsp; BFM studijske&nbsp; grupe &bdquo;I - BFM - SG&ldquo;&nbsp; (International Berlin -Frankfurt - M&uuml;nster Study Group) za proučavanje i lečenje dečje non-B akutne limfoblastne leukemije. Četvoro je&nbsp; imalo&nbsp; dijagnozu non - Hodgkin limfoma&nbsp; i bili su uključen i u&nbsp; protokol NHL - BFM&nbsp; 95. Istraživanje je obuhvatilo 113 ciklusa terapije metotreksatom (1&ndash; 4 ciklusa po pacijentu) sa 386 izmerenih serumskih koncentracija metotreksata. Raspon primenjenih doza metotreksata kretao se od 800 do 10.000 mg. Koncentracije metotreksata su merene 24, 36 i 42 sata nakon započinjanja infuzije metotreksata, a po potrebi i u dužim vremenskim intervalima. Za izračunavanje farmakokinetičkih parametara kori&scaron;ćen je dvokompartmanskih farmakokinetički&nbsp; model posle obustavljanja&nbsp; intravenske&nbsp; infuzije,&nbsp; gde&nbsp; postoje relacije&nbsp; za&nbsp; farmakokinetičke&nbsp; tačke. Podaci o kliničkim i laboratorijskim znacima toksičnosti metotreksata prikupljani su iz medicinske dokumentacije, a za stepenovanje toksičnosti kori&scaron;ćen je skor sistem - Common Terminology Criteria for&nbsp; Adverse&nbsp; Events (CTCAE), Version 4.0, U.S. Department&nbsp; of&nbsp; health&nbsp; and&nbsp; human services, National Institute of Health, National Cancer Institute. U cilju utvrđivanju uticaja karakteristika ispitanika, primenjene doze i prisustva produžene eliminacije na posmatrane parametre, vr&scaron;eno je poređenje tri grupe&nbsp; pacijenata (doza 2 g/m² bez produžene eliminacije, 5 g/m² bez produžene liminacije i 5 g/m² sa produženom eliminacijom metotreksata). Za celokupnu grupu ispitanika,&nbsp; medijane&nbsp; koncentracije metotreksta&nbsp; bile&nbsp; su 25,82 &mu;mol/l u 24. satu, 0,68 &mu;mol/l u 36. satu i 0,24 &mu;mol/l u 42. satu merenja. Najizraženija interindividualna varijabilnost u koncentracijama metotreksata bila je u 42. satu merenja, dok je&nbsp; intraindividualna varijabilnost bila najizraženija u 36. satu merenja. Medijana&nbsp; klirensa&nbsp;&nbsp; metotreksata&nbsp;&nbsp; bila&nbsp; je 8,32&nbsp;&nbsp; l/h. Farmakokinetički parametri&nbsp; redom bili su:&nbsp; medijana&nbsp; volumena&nbsp; centralnog&nbsp; kompartmana V₁ 28,47&nbsp; l, medijane konstanti k₁₀ 0,206, k₁₂ 0,0245, k₂₁ 0,1114. Najizraženiji uticaj primenjene doze na koncentracije metotreksata pokazan je u 24.&nbsp; satu&nbsp; merenja, dok uticaj doze na klirens&nbsp; metotreksata nije&nbsp; pokazan. Prisustvo produžene eliminacije metotreksata dovodi do smanjenih vrednosti konstanta k₁₀ i k₂₁. Nije pokazana statistički značajna&nbsp; interakcija ispitivanih demografskih karakteristika (uzrast, telesna povr&scaron;ina i pol)&nbsp; i koncentracija metotreksata, kao ni klirensa metotreksata. Pokazana je značajna interakcija između koncentracija metotreksata i nivoa laktat dehidrogenaze, kao i klirensa metotreksata i nivoa kreatinina i laktat dehidrogenaze. Većina ispoljenih&nbsp; toksičnosti bila je umerenog stepena (&lt;3 stepena). Najzastupljeniji klinički znak toksičnosti bio je oralni mukozitis, koji je bio većeg stepena u grupi sa većom primenjenom dozom metotreksata&nbsp; (5g/m²). Najzastupljeniji&nbsp; laboratorijski toksični efekti&nbsp; metotreksata bili su leukopenija i anemija. Najteži stepeni laboratorijskih znakova toksičnosti (leukopenija, anemija, porast&nbsp; AST,&nbsp; ALT i GGT) nalazili su se u grupi sa većom dozom&nbsp; (5 g/m²) i&nbsp; sa produženom eliminacijom metotreksata. Osnov&nbsp; za&nbsp; kliničko&nbsp; vođenje&nbsp; pacijenata&nbsp; na&nbsp; terapiji visokim dozama metotreksata je terapijsko praćenje leka (therapeutic drug monitoring &ndash; TDM) zbog velikih&nbsp; interindividualnih i intraindividualnih&nbsp; varijabilnosti&nbsp; u&nbsp; farmakokinetici&nbsp; leka. Rutinsko praćenje koncentracija metotreksata važno je za identifikaciju pacijenata sa povećanim rizikom od razvoja toksičnosti ,&nbsp; te&nbsp; je TDM&nbsp; standardna&nbsp; praksa&nbsp; za smernice spasavanja leukovorinom, naročito za pacijente za koje se zna da imaju smanjen&nbsp;&nbsp; klirens metotreksata ili druge rizike povezane sa prolongiranim citotoksičnim koncentracijama (bubrežna ili jetrena o&scaron;tećenja, kolekcije tečnosti u &ldquo;trećem prostoru&rdquo;, gastrointestinalna opstrukcija). Veliki&nbsp; broj&nbsp; istraživanja&nbsp; kod pedijatrijskih pacijenata pokazao je vezu između sistemskog izlaganja metotreksatu i&nbsp; efikasnosti&nbsp; i&nbsp; toksiĉnosti&nbsp; metotreksata. Ipak, ne postoji dovoljno&nbsp; informacija o farmakokinetici metotreksata kod dece obolele od akutne limfoblastne leukemije. Takođe, ova istraživanja nisu do sada sprovođena kod dece koja su lečena u na&scaron;oj sredini.</p> / <p>Methotrexate&nbsp; is&nbsp; an&nbsp; antifolate&nbsp; drug&nbsp; widely&nbsp; used&nbsp; for&nbsp; treatment&nbsp; of&nbsp; various malignant&nbsp; tumours.&nbsp; It&nbsp; is&nbsp; used&nbsp; at&nbsp; high&nbsp; doses&nbsp; and&nbsp; in&nbsp; combination&nbsp; with leucovorin rescue.&nbsp; Although&nbsp; high - dose&nbsp; MTX&nbsp; therapy&nbsp; dramatically&nbsp; improves&nbsp; the&nbsp; prognosis&nbsp; of patients with malignancies, severe adverse events are constant clinical concern. The&nbsp; aims&nbsp; of&nbsp; this&nbsp; stydy&nbsp; were&nbsp; to&nbsp; determine&nbsp; the&nbsp; serum&nbsp; concentration&nbsp; of&nbsp; methotrexate&nbsp; and&nbsp; to&nbsp; calculate&nbsp; the&nbsp; pharmacokinetic&nbsp; parameters&nbsp; of&nbsp; methotrexate&nbsp; in children&nbsp; suffering&nbsp; from&nbsp; malignant&nbsp; deseases&nbsp; who&nbsp; are&nbsp; treated&nbsp; with&nbsp; high&nbsp; doses&nbsp; of metotrexate&nbsp; (2&nbsp; g/m² i&nbsp; 5&nbsp; g/m² );&nbsp; furthermore,&nbsp; to&nbsp; investigate&nbsp; the&nbsp; effects&nbsp; of&nbsp; the&nbsp; applied doses of methotrexate, and demographic and clinical characteristics of the examinees on&nbsp;&nbsp; the&nbsp;&nbsp; concentration&nbsp;&nbsp; and&nbsp;&nbsp; pharmacokinetic&nbsp;&nbsp; parameters&nbsp;&nbsp; of&nbsp;&nbsp; the&nbsp;&nbsp; drug.&nbsp;&nbsp; The&nbsp;&nbsp; study investigated the&nbsp;&nbsp; presence&nbsp;&nbsp; and&nbsp;&nbsp; the&nbsp;&nbsp; degree&nbsp;&nbsp; of&nbsp;&nbsp; clinical&nbsp;&nbsp; and&nbsp;&nbsp; laboratory&nbsp;&nbsp; signs&nbsp;&nbsp; of metotrexate&nbsp; toxicity,&nbsp; as&nbsp; well&nbsp; as&nbsp; the&nbsp; effect&nbsp; of&nbsp; the&nbsp; applied&nbsp; doses,&nbsp; and&nbsp; demographic characteristics of the examinees on the appearance and the degree of toxicity. The retrospective - prospective study included 42&nbsp; pediatric patients aged from 0.75&nbsp; to&nbsp; 17.75&nbsp; years&nbsp; (median&nbsp; 5.75&nbsp; years).&nbsp; All&nbsp; patients&nbsp; were&nbsp; threated&nbsp; at&nbsp; the&nbsp; Children and Youth Health Care Institute of Vojvodina (Novi Sad, Serbia), Hemathology and Oncology&nbsp; Section,&nbsp; in&nbsp; the&nbsp; period&nbsp; from&nbsp; June&nbsp; 20 04&nbsp; to&nbsp; June&nbsp; 2012.&nbsp; 38&nbsp; examinees diagnosed as acute lymphoblastic leukemia were treated according to two subsequent protocols,&nbsp; ALL&nbsp; IC - BFM&nbsp; 2002&nbsp; and&nbsp; ALL&nbsp; IC - BFM&nbsp; 2009&nbsp; of&nbsp; the&nbsp; International&nbsp; BFM study&nbsp; group &bdquo;I - BFM - SG&ldquo; (International Berlin - Frankfurt - M&uuml;nster&nbsp; Study&nbsp; Group)&nbsp; for management&nbsp;&nbsp; of&nbsp;&nbsp; childhood&nbsp;&nbsp; non - B&nbsp;&nbsp; acute&nbsp;&nbsp; lymphoblastic&nbsp;&nbsp; leukemia.&nbsp;&nbsp; 4&nbsp;&nbsp; examinees diagnosed&nbsp; as&nbsp; non - Hodgkin&nbsp; lymphoma&nbsp; were&nbsp; treated&nbsp; according&nbsp; to&nbsp; the&nbsp; NHL - BFM&nbsp; 95 protocol. The study included 113 cycles of therapy with methotrexate (1-4 cycles per patient)&nbsp; with&nbsp; 3 86&nbsp; measured&nbsp; serum&nbsp; concentrations&nbsp; of&nbsp; methotrexate.&nbsp; The&nbsp; range&nbsp; of&nbsp; the applied doses was between 800 and 10,000 mg. The&nbsp; concentration&nbsp; of&nbsp; methotrexate&nbsp; was&nbsp; measured&nbsp; 24,&nbsp; 36&nbsp; and&nbsp; 42&nbsp; hours&nbsp; after the initiation of the methotrexate infusion, as well as in longer time intervals when needed.&nbsp; To&nbsp; calculate&nbsp; the&nbsp; pharmacokinetic&nbsp; parameters,&nbsp; the&nbsp; study&nbsp; applied&nbsp; the&nbsp; two - compartment&nbsp; pharmacokinetic&nbsp; model&nbsp; after&nbsp; the&nbsp; termination&nbsp; of&nbsp; intravenous&nbsp; infusion, when&nbsp; relations&nbsp; for&nbsp; pharmacokinetic&nbsp; points&nbsp; existed.&nbsp; Data&nbsp; on&nbsp; clinical&nbsp; and&nbsp; laboratory signs of methotrexate toxicity were collected&nbsp; from medical documentation, and the Common&nbsp; Terminology&nbsp; Criteria&nbsp; for&nbsp; Adverse&nbsp; Events&nbsp; (CTCAE),&nbsp; Version&nbsp; 4.0,&nbsp; U.S. Department&nbsp; of&nbsp; health&nbsp; and&nbsp; human&nbsp; services,&nbsp; National&nbsp; Institute&nbsp; of&nbsp; Health,&nbsp; National Cancer&nbsp; Institute, was&nbsp; used&nbsp; as&nbsp; the&nbsp; score&nbsp; system&nbsp; for&nbsp; toxicity&nbsp; ranking.&nbsp; In&nbsp; order&nbsp; to determine&nbsp; the&nbsp; effects&nbsp; of&nbsp; the examinees&rsquo;&nbsp; characteristics, applied&nbsp; doses&nbsp; and&nbsp; the presence&nbsp; of&nbsp; prolonged&nbsp; elimination on&nbsp; the&nbsp; parameters&nbsp; of&nbsp; interest,&nbsp; three&nbsp; groups&nbsp; of patients were&nbsp; compared (2 g/m² dose without prolonged elimination, 5 g/m² without prolonged elimination and 5 g/m² with prolonged elimination of methotrexate). In the&nbsp; entire&nbsp; group of&nbsp; examinees, the median&nbsp; concentration of methotrexate was&nbsp; 25.82 &mu;mol/l in the 24th hour, 0.68 &mu;mol/l in the 36th&nbsp; hour&nbsp; and 0.24 &mu;mol/l in the 42nd hour of&nbsp; observation. The largest inter - individual variability of methotrexate concentration&nbsp; was&nbsp; observed&nbsp; in&nbsp; the&nbsp; 24th&nbsp; hour&nbsp; while&nbsp; the&nbsp; largest&nbsp; intra - individual variability&nbsp; was&nbsp; recorded&nbsp; in&nbsp; the&nbsp; 36th&nbsp; hour&nbsp; of&nbsp; observation.&nbsp; The&nbsp; median&nbsp; clearance&nbsp; of methotrexate&nbsp; was&nbsp; 8.32l/h.&nbsp; Pharmacokinetic&nbsp; parameters&nbsp; were&nbsp; the&nbsp; following:&nbsp; median volume&nbsp; of&nbsp; the&nbsp; central&nbsp; compartment V₁ 28.47&nbsp; l,&nbsp; median&nbsp; constants k₁₀ 0,206, k₁₂ 0,0245, k₂₁ 0,1114, respectively. The&nbsp;&nbsp; strongest&nbsp;&nbsp; influence&nbsp;&nbsp; of&nbsp;&nbsp; the&nbsp;&nbsp; applied&nbsp;&nbsp; dose&nbsp;&nbsp; on&nbsp;&nbsp; the&nbsp;&nbsp; methotrexate concentration was recorded in the 24th hour of observation while no influence on the methotrexate&nbsp; clearance&nbsp; was&nbsp; found.&nbsp; The&nbsp; presence&nbsp; of&nbsp; prolonged&nbsp; elimination&nbsp; of methotrexate&nbsp;&nbsp; causes&nbsp;&nbsp; lower&nbsp; constants k₁₀ and&nbsp;&nbsp; k₂₁. There&nbsp;&nbsp; was&nbsp;&nbsp; no&nbsp;&nbsp; statistically significant&nbsp; interaction&nbsp; between&nbsp; the&nbsp; investigated&nbsp; demographic&nbsp; characteristics&nbsp; (age, body&nbsp; surface&nbsp; and&nbsp; gender)&nbsp; and&nbsp; the&nbsp; methotrexate&nbsp; concentration,&nbsp; nor&nbsp; between&nbsp; the demographic&nbsp;&nbsp; characteristics&nbsp;&nbsp; and&nbsp;&nbsp; the&nbsp;&nbsp; methotrexate&nbsp;&nbsp; clearance.&nbsp;&nbsp; A&nbsp;&nbsp; significant interaction was found between methotrexate concentration and lactat dehydrogenase level, as&nbsp;&nbsp; well&nbsp;&nbsp; as&nbsp;&nbsp; between&nbsp;&nbsp; methotrexate&nbsp;&nbsp; clearance&nbsp;&nbsp; and&nbsp;&nbsp; creatinine&nbsp;&nbsp; and&nbsp;&nbsp; lactate dehydrogenase level, respectively. Most of the observed toxicities were of moderate degree (&lt; 3 degrees). Oral mucositis&nbsp; was&nbsp; the&nbsp; most&nbsp; represented&nbsp; clinical&nbsp; sign&nbsp; of&nbsp; toxicity,&nbsp; and&nbsp; it&nbsp; was&nbsp; of&nbsp; higher degree&nbsp; in&nbsp; the&nbsp; group&nbsp; where&nbsp; the&nbsp; applied&nbsp; dose&nbsp; of&nbsp; methotrexate&nbsp; was&nbsp; higher&nbsp; (5&nbsp; g/m² ). Leucopenia&nbsp; and&nbsp; anemia&nbsp; were&nbsp; the&nbsp; most&nbsp; represented&nbsp; laboratory&nbsp; toxic&nbsp; effects.&nbsp; The most severe laboratory signs of toxicity&nbsp; (leucopenia, anemia, increase in AST, ALT and&nbsp; GGT&nbsp; activity)&nbsp; were&nbsp; observed&nbsp; in&nbsp; the&nbsp; group&nbsp; with&nbsp; the&nbsp; higher&nbsp; dose&nbsp; (5&nbsp; g/m² )&nbsp; and prolonged methotrexate elimination. Due to high inter- and&nbsp;&nbsp; intra-individual&nbsp;&nbsp;&nbsp; variability&nbsp; of&nbsp; the drug pharmacokinetics,&nbsp; the&nbsp; basis&nbsp; for&nbsp; the&nbsp; clinical&nbsp; care&nbsp; of&nbsp; patients&nbsp; on&nbsp; high&nbsp; methotrexate dosage&nbsp; therapy&nbsp; is&nbsp; therapeutic&nbsp; drug&nbsp; monitoring &ndash; TDM.&nbsp; Routine&nbsp; monitoring&nbsp; of methotrexate serum concentration is important for the identification of patients with a high&nbsp; risk&nbsp; of&nbsp; toxicity,&nbsp; and&nbsp; thus&nbsp; TDM&nbsp; is&nbsp; used&nbsp; as&nbsp; a&nbsp; standard&nbsp; procedure&nbsp; which&nbsp; provides guidelines&nbsp; for&nbsp; leucovorin&nbsp; rescue,&nbsp; particularly&nbsp; for&nbsp; patients&nbsp; with&nbsp; a&nbsp; lower&nbsp; methotrexate clearance or other risks associated with prolonged cytotoxic concent rations (kidney or liver&nbsp; damage,&nbsp; body&nbsp; fluid&nbsp; accumulation&nbsp; in&nbsp; the&nbsp; &ldquo;third&nbsp; space&rdquo;,&nbsp; gastrointestinal obstruction). Numerous studies involving pediatric patients have documented the link between&nbsp; a&nbsp; systemic&nbsp; methotrexate&nbsp; exposure&nbsp; on&nbsp; one&nbsp; hand,&nbsp; and&nbsp; the&nbsp; efficiency&nbsp; and toxicity of&nbsp; ethotrexate on the other hand. However, there is no sufficient data on the methotrexate&nbsp;&nbsp;&nbsp; pharmacokinetics&nbsp;&nbsp; in&nbsp;&nbsp; children&nbsp;&nbsp; suffering&nbsp;&nbsp; from&nbsp;&nbsp; acute&nbsp;&nbsp; lymphoblastic leukemia.&nbsp;&nbsp; Moreover,&nbsp;&nbsp; this&nbsp;&nbsp; type&nbsp;&nbsp; of&nbsp;&nbsp; research,&nbsp;&nbsp; involving&nbsp;&nbsp; children&nbsp;&nbsp; treated&nbsp;&nbsp; in&nbsp;&nbsp; the geographical region of this study, have not been conducted.</p>

The ochratoxins, metabolites of certain Aspergillus and Penicillium species are the first group of mycotoxins discovered subsequent to the epoch-making discovery of the aflatoxins. Ochratoxin A (OTA) is a very important mycotoxin owing to its frequent occurrence in nature, its established role in Danish porcine nephropathy and in poultry mycotoxicoses and its implicated role in Balkan endemic nephropathy and urinary system tumors among population groups in North Africa. Chapters 2 and 3 highlight the importance of OTA and the research currently being done on mycotoxins. These efforts are focused on the molecular genetics of toxinogenic fungi; the mechanism of their action; species differences in metabolism and pharmacokinetics; quantification of mycotoxins; risk assessments on the exposure of man and animals to mycotoxins and regulations for the control of mycotoxin contamination. Methods developed to analyse OTA in different matrices by using reversed phase high performance-liquid chromatography with fluorescence detection and tandem liquid chromatography-mass spectrometry techniques are described in Chapter 10. Amino propyl solid phase extraction columns were used for the first time in cleanup steps of ochratoxin analysis. These techniques and methods were applied to the first survey on the levels of OTA in coffee on the South African retail market (Chapter 5). The results suggest that the levels of OT A in the coffee on the South African market are somewhat higher than the levels of OTA in coffees on the European market. The possibility to biologically produce different halogen-ochratoxins by supplementing the growth medium of Aspergillus ochraceus with halogen salts was investigated. Bromoochratoxin A was produced for the first time in this way. Supplementation of inoculated wheat with potassium iodide and -fluoride resulted in the poisoning of the yeast and no iodoor fluoro-ochratoxin B was produced. It was found that Aspergillus ochraceus produced OTA in higher yields at elevated levels of potassium chloride. This finding has important commercial applications in the production ofOTA (Chapter 4). The ochratoxins are hydrolyzed in vivo by carboxypeptidase A. The hydrolysis of the ochratoxins and analogues by carboxypeptidase A was measured in vitro in a structurefunction relation study by employing mass spectrometric techniques. The kinetic data of the ochratoxins were compared to the values of a number of synthesized structural analogues. It was found that the halogen containing analogues had lower turnovers than their des-halo analogues. There were no substantial differences in the kinetic data between the different halogen containing analogues (Chapter 8). The toxicokinetics of OTA in vervet monkeys were determined for the first time. The clearance of OTA from the plasma suggested a two-compartment model and the elimination half-life was determined to be 19-21 days. The half-life of OTA in humans was determined by allometric calculations to be 46 days. We came to the conclusion that the long term consumption of OT A contaminated foods will lead to potentially hazardous levels of the toxin in the body (Chapter 9). This hypothesis can be substantiated by the incidence of OTA in the blood of various population groups. Possible ways to decontaminate OT A contaminated foods by degrading the compound biologically with yeast; moulds or lipases to non-toxic compounds were investigated. Eight moulds, 323 yeasts and 23 lipases were screened for ochratoxin degradation. A lipase from Aspergillus niger is the first lipase that was proven to degrade OTA (Chapter 7). Four yeasts were found to degrade OT A of which one, Trichosporon mucoides degraded OTA substantially within 48 hours in a growing culture (Chapter 6). In addition to this first report of yeasts which have the ability to degrade OTA, the fungi Cochliobolus sativus, Penicillium islandicum and Metarhizium anispoliae also proved to degrade OT A. OT A was degraded in all instances to the non-toxic ochratoxin a and the amino acid phenylalanine. / Thesis (PhD (Chemistry))--Potchefstroom University for Christian Higher Education, 2000

The ochratoxins, metabolites of certain Aspergillus and Penicillium species are the first group of mycotoxins discovered subsequent to the epoch-making discovery of the aflatoxins. Ochratoxin A (OTA) is a very important mycotoxin owing to its frequent occurrence in nature, its established role in Danish porcine nephropathy and in poultry mycotoxicoses and its implicated role in Balkan endemic nephropathy and urinary system tumors among population groups in North Africa. Chapters 2 and 3 highlight the importance of OTA and the research currently being done on mycotoxins. These efforts are focused on the molecular genetics of toxinogenic fungi; the mechanism of their action; species differences in metabolism and pharmacokinetics; quantification of mycotoxins; risk assessments on the exposure of man and animals to mycotoxins and regulations for the control of mycotoxin contamination. Methods developed to analyse OTA in different matrices by using reversed phase high performance-liquid chromatography with fluorescence detection and tandem liquid chromatography-mass spectrometry techniques are described in Chapter 10. Amino propyl solid phase extraction columns were used for the first time in cleanup steps of ochratoxin analysis. These techniques and methods were applied to the first survey on the levels of OTA in coffee on the South African retail market (Chapter 5). The results suggest that the levels of OT A in the coffee on the South African market are somewhat higher than the levels of OTA in coffees on the European market. The possibility to biologically produce different halogen-ochratoxins by supplementing the growth medium of Aspergillus ochraceus with halogen salts was investigated. Bromoochratoxin A was produced for the first time in this way. Supplementation of inoculated wheat with potassium iodide and -fluoride resulted in the poisoning of the yeast and no iodoor fluoro-ochratoxin B was produced. It was found that Aspergillus ochraceus produced OTA in higher yields at elevated levels of potassium chloride. This finding has important commercial applications in the production ofOTA (Chapter 4). The ochratoxins are hydrolyzed in vivo by carboxypeptidase A. The hydrolysis of the ochratoxins and analogues by carboxypeptidase A was measured in vitro in a structurefunction relation study by employing mass spectrometric techniques. The kinetic data of the ochratoxins were compared to the values of a number of synthesized structural analogues. It was found that the halogen containing analogues had lower turnovers than their des-halo analogues. There were no substantial differences in the kinetic data between the different halogen containing analogues (Chapter 8). The toxicokinetics of OTA in vervet monkeys were determined for the first time. The clearance of OTA from the plasma suggested a two-compartment model and the elimination half-life was determined to be 19-21 days. The half-life of OTA in humans was determined by allometric calculations to be 46 days. We came to the conclusion that the long term consumption of OT A contaminated foods will lead to potentially hazardous levels of the toxin in the body (Chapter 9). This hypothesis can be substantiated by the incidence of OTA in the blood of various population groups. Possible ways to decontaminate OT A contaminated foods by degrading the compound biologically with yeast; moulds or lipases to non-toxic compounds were investigated. Eight moulds, 323 yeasts and 23 lipases were screened for ochratoxin degradation. A lipase from Aspergillus niger is the first lipase that was proven to degrade OTA (Chapter 7). Four yeasts were found to degrade OT A of which one, Trichosporon mucoides degraded OTA substantially within 48 hours in a growing culture (Chapter 6). In addition to this first report of yeasts which have the ability to degrade OTA, the fungi Cochliobolus sativus, Penicillium islandicum and Metarhizium anispoliae also proved to degrade OT A. OT A was degraded in all instances to the non-toxic ochratoxin a and the amino acid phenylalanine. / Thesis (PhD (Chemistry))--Potchefstroom University for Christian Higher Education, 2000

<p>Sukcinimidi su jedinjenja koja pokazuju vi&scaron;estruke farmakolo&scaron;ke efekte uključujući i antiproliferativnu aktivnost, zahvaljujući prisustvu farmakofore sa dva hidrofobna regiona i dva regiona bogata elektronima. Savremeni dizajn lekova ima za cilj da se modifikacijama u strukturi (promena vrste, položaja i orijentacije supstituenata) i in silico računarskim metodama predvide i optimizuju farmakokinetske osobine i bezbednosni profil kandidata za lek. U ranoj fazi razvoja lekova se koriste postojeće baze podataka o molekulskim, farmakokinetskim i toksikolo&scaron;kim parametrima već ispitanih jedinjenja i pomoću matematičkih modela i algoritama predviđaju se osobine novih molekula, elimini&scaron;u se neodgovarajući kandidati i postiže se u&scaron;teda u vremenu i materijalnim sredstvima. Da se ispitaju fizičko-hemijske karakteristike 11 novosintetisanih metil-etil-N-aril-sukcinimida na osnovu strukture, primenom različitih softverskih paketa; da se na osnovu strukture odrede farmakokinetski i toksikolo&scaron;ki parametri, primenom različitih softverskih paketa; da se ispita retenciono pona&scaron;anje, odnosno odrede retencione konstante za svako jedinjenje primenom visokoefikasne hromatografije na tankom sloju (HP-TLC) i ispita mogućnost primene retencionih konstanti kao mere lipofilnosti ispitivanih jedinjenja; da se ispita antiproliferativna aktivnost na odabranim kulturama ćelija karcinoma i na zdravim ćelijama fibroblasta pluća; da se analizom molekulskog dokinga ustanovi vezivanje za estrogene receptore. Ispitano je retenciono pona&scaron;anje 11 novosintetisanih derivata sukcinimida primenom visokoefikasne hromatografije na tankom sloju (HP-TLC) obrnute faze uz primenu dvokomponentne sme&scaron;e vode i organskog rastvarača (metanola, acetonitrila ili acetona), sa odgovarajućim zapreminskim udelom organskog rastvarača kao mobilne faze. Iz razvijenih hromatograma su izračunate retencione konstante RM0 i S. Logaritam podeonog koeficijenta (logP) određen je in silico, kori&scaron;ćenjem različitih računarskih programa. In silico su određene fizičko-hemijske karakteristike, farmakokinetski parametri, toksikolo&scaron;ki parametri, akvatična toksičnosti i afinitet vezivanja za estrogene receptore. Izračunate su vrednosti afiniteta za 4 vrste receptora (G-protein spregnuti receptori, jonski kanali, inhibitori kinaza, nuklearni receptori). Antiproliferativna aktivnost ispitivanih derivata sukcinimida određena je primenom kolorimetrijskog testa sa tetrazolijum solima (MTT testa) na komercijalnim kulturama ćelija (MRC-5, A549, HeLa, MDA-MB-231, MCF-7, HT-29) i izračunate su IC50 vrednosti. Urađena je i doking analiza sukcinimida prema ERA (estrogen receptor alfa) i ERB (estrogen receptor beta) i dobijene su vrednosti energije formiranja kompleksa sa posmatranim receptorima (MolDock Score). Statistički najznačajnije linearne korelacije dobijene su između eksperimentalno određenih hromatografskih parametara (RM0 i S) i in silico parametara lipofilnosti MlogP i ClogP. Ispitivanjem uticaja promene RM0 i S na farmakokinetske karakteristike dobijeni su rezultati koji pokazuju paraboličnu zavisnost konstante apsorpcije (Ka) i procenta vezivanja za proteine plazme (PPB) od posmatranih retencionih konstanti, dok je zavisnost sa volumenom distribucije (Vd) i sposobno&scaron;ću prolaska kroz krvno-moždanu barijeru (logBBB) bila linearnog tipa. Toksičnost ispitivanih jedinjenja, procenjena na osnovu in silico dobijenih LD50 vrednosti, nije bila vi&scaron;a od toksičnosti već registrovanih lekova sa strukturom sukcinimida, i dala je parabolične zavisnosti u odnosu na RM0 i S vrednosti. Eksperimentalno nijedno od ispitivanih jedinjenja nije pokazalo aktivnost u odnosu na zdrave fibroblaste pluća. Najznačajniju antiproliferativnu aktivnost (najniže IC50) su pokazala jedinjenja 6 i 7 u odnosu na ćelije linije MCF-7 i jedinjenje 11 u odnosu na A549 ćelijsku liniju. Doking analiza je pokazala niže energije formiranja kompleksa sa ERA, u odnosu na ERB. Eksperimentalno određeni parametri RM0 i S se mogu koristiti kao alternativne i pouzdane mere lipofilnosti analiziranih sukcinimida. Ispitivana jedinjenja pokazuju povoljne fizičko-hemijske karakteristike, predviđene in silico metodama i povoljne farmakokinetske karakteristike: male vrednosti konstante apsorpcije, umeren volumen distribucije, povoljan afinitet vezivanja za proteine plazme, favorizovan prolazak kroz krvno-moždanu barijeru za lipofilnija jedinjenja. Procenjuje se da sva ispitivana jedinjenja, izuzev derivata sa &ndash;CN supstituentom, imaju zahtevani nizak stepen toksičnosti. Po antiproliferativnoj aktivnosti u odnosu na ćelije ER-zavisnog karcinoma dojke (MCF-7) izdvajaju se jedinjenja sa metil i nitro supstituentom u para položaju. Na osnovu malih energija formiranja kompleksa sa ERA, koji su eksprimirani na ćelijama MCF-7 linije, pretpostavlja se da bi mehanizam njihovog delovanja delimično mogao biti obja&scaron;njen uticajem na ERA, ali su potrebna dodatna istraživanja na tom polju.</p> / <p>Succinimides have exhibited various pharmaceutical effects including antiproliferative activity due to an important structural fragment (a pharmacophore) presented in form of two hydrophobic regions and two electron-rich centers. Current development of new drugs involves modifications in structure (type, position and orientation of substituents) and usage of in silico computational programs to predict and optimize pharmacokinetic and safety profile of drug candidates. In early phase of drug development, databases regarding the molecular, pharmacokinetic and toxicological parameters of already tested compounds are used, mathematical models and algorithms are applied for predicting the properties of new molecules and inadequate candidates are eliminated saving time and resources. Determination of physico-chemical properties of the analyzed methyl-ethyl-N-phenilsuccinimide derivatives by software packages; virtual pharmacokinetic and toxicology screening; investigation of retention behavior of the compounds by the reversed-phase HPTLC analysis and calculation of retention constants and their correlation with lipophilicity; in vitro evaluation of antiproliferative activity toward five carcinoma cell lines and normal fetal lung cell line; molecular behavior study on target estrogen receptors by molecular docking and correlation of antiproliferative activity toward ER+ breast carcinoma cell lines and in silico estrogen receptor affinity binding. Retention behavior of 11 newly synthesized succinimide derivatives was determined by reversed phase high performance thin layer chromatography (RP HPTLC) with the application of two-component mixtures water - organic solvent (methanol, acetonitrile or acetone) with adequate volume fractions of the organic modifier. After chromatographic development RM0 and S parameters were calculated. The logarithm of partition coefficient, logP for the analyzed compounds were calculated by different softwares. Physico-chemical properties, pharmacokinetic and toxicological parameters, aquatic toxicity and relative affinity to estrogen receptors were predicted in silico. The affinity toward 4 types of receptors (G-proteine coupled receptors, ion channels, kinase inhibitors, nuclear receptors) were calculated as well. Standard MTT assay was applied to evaluate cytotoxic activities of the analyzed succinimides after cells were exposed. Antiproliferative activity were investigated toward commercial MRC-5, A549, HeLa, MDA-MB-231, MCF-7, HT-29 cell lines and IC50 values were calculated for each compound. MolDock Score that represents energy of binding to estrogen alfa and estrogen beta receptors was determined by molecular docking. Statistically significant linear correlations were determined between the chromatographic retention constants (RM0 and S) and calculated logP, and the best two were obtained in correlation of retention constants with MlogP and ClogP. The examination of RM0 and S influence on pharmacokinetics indicated parabolic dependence of the absorption constant (Ka) and plasma protein binding predictor (PPB) from the observed constants while the volume of distribution (Vd) and the ability to cross the brain blood barrier (logBBB) had linear association with the retention parameters. The toxicity of the analysed compounds evaluated in silico as LD50 on rodents was lower in comparison with the drugs with succinimide structure that are on the market and had parabolic correlation with the RM0 and S values. The experiments indicated that none of the compounds examined had cytotoxic activity toward the healthy lung fibroblast cells. The results of the in vitro assay shown that none of the investigated compounds demonstrated antiproliferative activity toward fetal lung cells. The most potent antiproliferative agents were compounds 6 and 7 toward MCF-7 cell line, and compound 11 toward A549 cell line. Molecular docking shown lower energy for binding to ERA in comparison to ERB.</p>