Investigation of protective effects of fullerenol in vivo in acute and chronic cardiomyopathy during doxorubicine therapy of malign neoplasma in rats

Fulereni so posebna oblika ogljika, ki so jih odkrili sredi osemdesetih let devetnajstega stoletja. Najbolj poznan med njimi je buckyball, ki predstavlja zaprto sferično strukture v obliki nogometne žoge. Trenutno se odvijajo številne raziskave, v katerih poskušajo odkriti tisto farmacevtsko obliko, ki bi kar najbolje izkoristila biološke lastnosti fulerenov in njihovih derivatov. Danes že izkoriščajo nekatere lastnosti fulerena, kot sta sposobnost odstranjevanja radikalov ter posebna molekulska zgradba. V raziskavah so pokazali, da je fulerenol učinkovit pri zaviranju metastaziranja, zdravljenju cerebralnih sprememb, kot sta Alzheimerjeva in Parkinsonova bolezen, zdravljenju hepatitisa C in aidsa. Fulerenole C60(OH)n, polihidroksi derivate fulerena, intenzivno proučujejo zaradi dobrih antioksidativnih lastnosti. Domnevajo, da delujejo kot lovilci radikalov v bioloških sistemih, pri oksidativnem stresu, ki ga povzroči radioaktivno obsevanje. Za fulerenol C60(OH)24 so dokazali, da ima zaščitne učinke pred z doksorubicinom povzročeno toksičnostjo v živalskih modelih. Potencialno zaščitno vlogo pred kardio-, hepato-, nefro- in pulmotoksičnostjo, povzročeno z doksorubicinom, so proučevali na modelih podgan s kemijsko povzročenim rakom. Naša raziskovalna skupina je na podlagi rezultatov preliminarnih raziskav na zdravih odraslih podganah vrste Wistar potrdila, da ima fulerenol v odmerku 100 mg/kg intraperitonealno, injiciran 30 minut pred doksorubicinom, zaščitni učinek na srčno in jetrno tkivo. V nadaljnjih raziskavah smo določili, da je tak odmerek učinkovit za zaščito pred z doksorubicinom povzročenima akutno in kronično toksičnostjo. Izvedli smo tudi in vivo ter in vitro raziskavo, s katero smo potrdili potencialno zaščitno vlogo fulerenola C60(OH)24 na z doksorubicinom povzročeno hepatotoksičnost. In vivo rezultati (na podganah vrste Sprague-Dawley) so pokazali, da pride pod vplivom doksorubicina do značilnih sprememb v serumskih koncentracijah alanin-aminotransferaze (ALT), aspartat-aminotransferaze (AST), laktat-dehidrogenaze (LDH) in alfa-hidroksibutirat-dehidrogenaze (α-HBDH) kot tudi sprememb v koncentracijah malondialdehida (MDA), glutationa (GSH), glutation-peroksidaze (GSH-Px), glutation-reduktaze (GR), katalaze (CAT) in superoksid-dizmutaze (SOD) ter spremembe celotnega antioksidativnega statusa (TAS) v jetrnem tkivu. Omenjeni učinki so se izrazito zmanjšali po predhodnem injiciranju fulerenola, in sicer pri vseh preiskovanih dejavnikih, razen pri koncentracijah MDA in GSH. V drugem sklopu poskusov smo celično linijo hepatocelularnega karcinoma (HepG2) izpostavili fulerenolu v koncentracijah 10 ali 44 mg/ml za 12, 24, 48 ali 96 ur. Z namenom, da bi ovrednotili modulirajočo aktivnost fulerenola na z doksorubicinom povzročeno hepatotoksičnost, smo celično linijo istočasno izpostavili doksorubicinu (1 μM5 μM) in fulerenolu (10 μg/ml44 μg/ml) v različnih kombinacijah. Kadar smo celice izpostavili 5 μM doksorubicinu in fulerenolu, smo ohranili celično aktivnost v celotnem času trajanja poskusa. Na osnovi rezultatov lahko zaključimo, da je že sam fulerenol citotoksičen za HepG2, kadar pa je oksidativni stres prevelik, prevladajo nadcitotoksičnimi učinki fulerenola njegove močne zaščitne antioksidativne lastnosti. Poškodbe srčne mišice po vnosu doksorubicina smo potrdili na podlagi ultrastrukturnih patološksprememb, sprememb v koncentracijah SOD, MDA, CAT, GSSG (oksidirani glutation) in GR tsprememb TAS, enako kot protektivni vpliv fulerenola apliciranega pred doksorubicinom v akutfaz. Sam fulerenol v odmerku 100 mg/kg ni imel vliva na poškodbo srca v podganah z rakom dojk. Trezultati nakazujejo, da bi fulerenol lahko uporabili kot potencialni kardioprotektiv pri bolnikih, ji zdravimo z doksorubicinom.V časovnem obdobju treh tednov smo proučevali učinke fulerenola C60(OH)24 v treh odmerkih (250 in 100 mg/kg/teden) na z doksorubicinom povzročeno kardio- in hepatotoksičnost pri podganas kolorektalnim rakom. Uporabili smo in vivo model na podganah samcih vrste Wistar, pri čemsmo ugotavljali, ali je fulerenol sposoben preprečiti z doksorubicinom (1,5 mg/kg/teden v obdobtreh tednov) povzročeni kronični kardio- in hepatotoksičnost. Učinke smo primerjali z učinki dobpoznanega antioksidanta, vitamina C (100 mg/kg/teden v obdobju treh tednov). Na podlagrezultatov makroskopskih, mikroskopskih, hematoloških, biokemijskih, fizioloških, farmakološkin farmakokinetičnih raziskav smo potrdili, da ima fulerenol v vseh preiskovanih odmerkih zaščitnučinke na srčno in jetrno tkivo pred kronično toksičnostjo, inducirano z doksorubicinom.Ključna prednost fulerenola pred ostalimi znanimi antioksidanti je njegovo dvojno delovanje – ščpred učinki sevanja in ščiti pred poškodbami tkiv (srca, jeter, ledvic in pljuč) med protirakavizdravljenjem (radio- in kemo- terapijo). Potrebne pa bodo še nadaljnje raziskave, in sicer raziskavakutnih in kroničnih učinkov na prašičih in nato klinične raziskave na ljudeh.Fullerenes are a type of carbon molecules first discovered in the mid-1980s. The most well-known of these is the buckyball, which is a closed spherical molecule, shaped like a soccer ball. Many studies are presently in progress to search for pharmaceutical products that can capitalize on the excellent bioactivity of fullerenes and their derivatives. Thus far, different fullerene properties have been put to use, such as its ability to eliminate radicals as well as its particular molecular structure. Studies have found it to be efficient in suppression of metastases, treatment of cerebral conditions such as Alzheimer\u27s and Parkinson\u27s diseases, type-C hepatitis therapy, and HIV treatment. A polyhydroxilated derivative of fullerene, named fullerenols C60(OH)n, is being extensively studied due to its great potential as an antioxidant. It is proposed that fullerenols may act as free radical scavengers in biological systems, in xenobiotics, as well as radioactive irradiation-induced oxidative stress. It has demonstrated protective effects against cytotoxicity of doxorubicin (Dox) in animal models, especially by fullerenol C60(OH)24. Different rat models with chemically induced cancer were used to investigate a potential protective role of fullerenol against cardio-, hepato-, nephro- and pulmotoxicity induced by Dox. According to preliminary studies on healthy adult Wistar rats, our research group confirmed that 100 mg/kg (i.p.) of fullerenol administered 30 min before Dox has a protective influence on heart and liver tissue. Therefore, that dose was chosen as effective against acute and chronic Dox-induced toxicity in further examinations. An in vivo – in vitro study was examined to confirm the potential protective role of fullerenol C60(OH)24 on Dox-induced liver toxicity. The in vivo results (Sprague-Dawley rats) showed that treatment with Dox alone caused significant changes in serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and alpha-hydroxybutyrate dehydrogenase (α-HBDH), as well as in levels of malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-Px), total antioxidant status (TAS), glutathione reductase (GR), catalase (CAT), and superoxide dismutase (SOD) in the liver tissue. These effects were drastically reduced for all investigated parameters by pretreatment with fullerenol, although not for the MDA and GSH level. On the other hand, the human hepatocellular carcinoma (HepG2) cell line was continuously treated with fullerenol for 12, 24, 48 and 96 h, at concentrations of 10 and 44 μg/mL. With the aim of evaluating the modulating activity of fullerenol on Dox-induced hepatotoxicity, the cell line was concurrently treated with Dox (1 μM5 μM) and fullerenol (10 μg/mL44 μg/mL) in different combinations. When the cells are treated with 5 μM Dox along with the fullerenol, a significant development of cell capability during the entire timeline can be seen. It was concluded that fullerenol has cytotoxic effects on HepG2 by itself, but when the oxidative stress is too high, the cytotoxic effects of fullerenol are overcome bits protective role as a strong antioxidant compound. Damage to the heart muscle after Dox administration was also confirmed by changes in the ultstructural pathology results and SOD, MDA, CAT, GSSG (oxidized GSH), GR, and TAS levels, well as potential cardioprotective influence of fullerenol as a pretreatment agent for Dox therapy the acute phase. Fullerenol itself, in a dose of 100 mg/kg, did not affect heart injury in rats witbreast cancer. The presented results suggested that fullerenol might be a potential cardioprotector Dox-treated individuals.Effects of fullerenol C60(OH)24 at three different doses (25, 50, and 100 mg/kg/week) on heart anliver tissue after Dox-induced toxicity in rats with colorectal cancer were investigated durig a timspan of three weeks. An in vivo Wistar male rat model was used to explore whether fullerenocould protect against Dox-induced (1.5 mg/kg/week for three weeks) chronic cardio- andhepatotoxicity, and the effect was compared with a well-known antioxidant, vitamin C (10mg/kg/week for three weeks). According to macroscopic, microscopic, hematological, biochemicaphysiological, pharmacological, and pharmacokinetic results, we confirmed that, at all examinedoses, fullerenol exhibits a protective influence on the heart and liver tissue against chronic toxiciinduced by Dox. The key benefit of fullerenol, in contrast to other known antioxidants, is its dual function as radprotector and organo-protector (heart, liver, kidney, and lung) during the anticancer therapy (radiand chemo-). However, there is a need to carry out further studies, including an acute and chroninvestigation in small pigs, and then after human trials

Potential Medical Use of Fullerenols After Two Decades of Oncology Research

Fullerenes are carbon molecules that are found in nature in various forms. They are composed of hexagonal and pentagonal rings that create closed structures. Almost 4 decades ago, fullerenes were identified in the form of C 60 and C 70 , and following the award of the Nobel Prize in Chemistry for this discovery in 1996, many laboratories started working on their water-soluble derivatives that could be used in different industries, including pharmaceutical industries. One of the first fullerene forms that was the focus of different research groups was fullerenol, C 60 (OH) n ( n   =  2-44). Both in-vitro and in-vivo studies have shown that polyhydroxylate fullerene derivatives can potentially be used as either antioxidative agents or cytostatics (depending on their co-administration, forms, and concentration/dose) in biological systems. The current review aimed to present a critical view of the potential applications and limitations of fullerenols in oncology, as understood from the past 2 decades of research

Potential medical use of fullerenols after two decades of oncology research

Fullerenes are carbon molecules that are found in nature in various forms. They are composed of hexagonal and pentagonal rings that create closed structures. Almost 4 decades ago, fullerenes were identified in the form of C$_{60}$ and C$_{70}$, and following the award of the Nobel Prize in Chemistry for this discovery in 1996, many laboratories started working on their water-soluble derivatives that could be used in different industries, including pharmaceutical industries. One of the first fullerene forms that was the focus of different research groups was fullerenol, C$_{60}$(OH)$_n$ (n = 2-44). Both in-vitro and in-vivo studies have shown that polyhydroxylate fullerene derivatives can potentially be used as either antioxidative agents or cytostatics (depending on their co-administration, forms, and concentration/dose) in biological systems. The current review aimed to present a critical view of the potential applications and limitations of fullerenols in oncology, as understood from the past 2 decades of research

Spermatogenesis recovery treatment in less than four months from zero to almost 16 million sperms per milliliter after several cycles of steroids in 44‐year‐old healthy man

Key Clinical Message The most common external factors that men can have influence on, and improve its own spermatogenesis, are related to lifestyle, habits, stress level, sport activities, nutrition quality, and medications. Steroids became one of the key issues in young men during reproductive stage of life. More and more teenagers who are not even developed yet naturally start using supplements very early to build up body and strengths of muscles in the shortest possible time. In this report is presented the case of 44‐year‐old (November 2022), healthy, and sporty man, who is 1–2 times per year on steroid cycles since he turned 40th. He had intention to become a father; however after 6 months of the last steroid cycle, sperm count was zero. This case will show what was needed and successfully done within less than 4 months after last steroids administration and the moment when sperm count reached almost 16 million sperms per milliliter. However, it has to be clear that this is unique case and additional tests would be needed on bigger population to confirm fast post steroid sperm count recovery in healthy subjects, using approach from this manuscript

Optimized Method for Determination of Amoxicillin, Ampicillin, Sulfamethoxazole, and Sulfacetamide in Animal Feed by Micellar Electrokinetic Capillary Chromatography and Comparison with High-Performance Liquid Chromatography

Antibiotics as additives in animal feedstuffs are forbidden in many countries in the world, but they are still abused. A micellar electrokinetic capillary chromatography method was performed at 25 °C and 30 kV (under pressure 15 mbar) using 25 mmol dm–3 phosphate buffer (pH = 8.0) containing 70 mmol dm–3 sodium dodecysulfate (SDS) and 10 % (volume fraction) methanol as the background electrolyte for separation and determination of amoxicillin, ampicillin, sulfamethoxazole, and sulfacetamide. UV detection was set at 210 nm. The method was validated and antibiotics were quantitatively determined as additives in spiked animal feedstuffs. Results were compared with a new HPLC method for the evaluation of four antibiotics in real samples. Both developed methods can be used for routine analysis of amoxicillin, ampicillin, sulfamethoxazole, and sulfacetamide as additives in animal feedstuffs. </p

SPE and large-volume sample stacking in MEKC for determination of doxycycline in biological fluids: Comparison of direct injection to SPE-MEKC

A novel and simple method has been developed for the determination of doxycycline (DOX) in biological fluids. The method is based on SPE, large-volume sample stacking (LVSS) and MEKC with UV-DAD detection. Six SPE cartridges have been used in investigation for sample clean up and pre-concentration (Supelco (R) LC-8, LC-18, LC-SCX, and LC-WCX, as well as Strata (TM)-X and X-C). DOX was determined on a 56 cm. (effective length 50 cm) x 50 mu m id fused-silica capillary. The BGE was 20 mM borate buffer, pH 9.3, containing 80 mM SDS and 7.5% v/v of methanol (30 s x 50 mbar), and the temperature and voltage were 25 degrees C and 30 kV, respectively. The analytical wavelength was set at 210 nm. Under optimized conditions it is possible to determine DOX in human serum, urine, semen, tears and saliva with recovery of 97.5% (RSD 2.5%). The method was shown to be sensitive (LOD is 1 mu g/L) and precise (intra-day RSD 0.2 and 2.4%; inter-days 0.4 and 3.5% for migration time and peak area, respectively). Results for developed SPE-LVSS-MEKC were compared with LVSS-MEKC method with direct sample injection. The new LVSS-MEKC method is presented as a useful technique for rapid determination without extraction procedure of DOX in human urine and serum, using 80mM of SDS, 10% v/v of methanol and 40 mM borate buffer (pH 9.3; 30 s x 50 mbar; 25 degrees C; 30 kV; 350 nm), but not for the other biological fluids, according to lower sensitivity of the method and because of the sample composition

Application of micellar electrokinetic capillary chromatography for routine analysis of different materials

Micellar electrokinetic capillary chromatography (MEKC) has become a popular mode among the several capillary electro-migration techniques. Most drug analysis can be performed by using MEKC because of its wide applicability. Separation of very complex mixtures, determination of drugs in the biological materials, etc., can be successfully achieved by MEKC. This review surveys typical applications of MEKC analysis. Recent advances in MEKC, especially with solid-phase extraction and large-volume sample stacking, are described. Modes of electrokinetic chromatography including MEKC, a separation theory of MEKC, environmental friendly analysis, and selectivity manipulation in MEKC are also briefly mentioned