Kompendium na temat leków biopodobnych

Lek biologiczny to lek, którego substancja aktywna jest białkiem, agregatem białkowym lub polimerem polisacharydowym, który może być wytwarzany jedynie przez żywe komórki. Stosowany w Unii Europejskiej termin „lek biopodobny” oznacza lek biologiczny zarejestrowany na podstawie wykazania biopodobieństwa do odpowiadającego mu leku biologicznego innowacyjnego, zarejestrowanego uprzednio na podstawie przedłożenia pełnej dokumentacji rejestracyjnej. W Unii Europejskiej leki biologiczne zarówno innowacyjne, jak i biopodobne są rejestrowane centralnie na podstawie oceny dokumentacji rejestracyjnej przez ekspertów Europejskiej Agencji Leków (EMA) z siedzibą w Londynie. Dla wykazania biopodobieństwa jest wymagane przedłożenie wyników badań wykazujących, że substancje czynne i formulacje leku biologicznego biopodobnego i odpowiadającego mu leku biologicznego innowacyjnego charakteryzują się takimi samymi właściwościami fizykochemicznymi i farmakologicznymi. Wyniki te muszą wykazywać odpowiednio duży stopień podobieństwa struktury chemicznej i konformacji substancji czynnej, jej porównywalną jakość i trwałość oraz dowodzić takiego samego mechanizmu działania, na przykład identycznego powinowactwa substancji czynnej do receptorów komórkowych. W stosunku do wymagań stawianych lekom biologicznym innowacyjnym zakres badań przedklinicznych (np. toksykologicznych), a zwłaszcza klinicznych, wymaganych dla leku biologicznego biopodobnego jest wówczas ograniczony do minimum uznanego za niezbędne, na przykład tylko do badania farmakokinetycznego i badania immunogenności. Ogólne wytyczne europejskie normujące procedurę rejestracji leków biologicznych biopodobnych wprowadzono w 2005 roku i od tego czasu kilka leków biologicznych biopodobnych zarejestrowano w Unii Europejskiej. Dotychczas nie donoszono o żadnych nieprzewidzianych działaniach niepożądanych lub o niższej skuteczności leczniczej leków biologicznych biopodobnych — potwierdza to trafność przyjętych rozwiązań w tym zakresie. Forum Nefrologiczne 2011, tom 4, nr 3, 193–19

Drug bioavailability from topically applied ocular drops. Does drop size matter?

The application of drops containing ocular medicines to the conjunctival sac is the most common method of drug delivery to the anterior segment of the eye. Although this route of application seemingly displays numerous advantages, obtaining effective drug concentration at its site of action is challenging. The bioavailability of a topically applied drug depends on various factors related to the eye, to the drug and formulation, to the drop, and to the patient. The present article discusses their relative significance. From a drop applied to an eye, at most 5% of a drug dose enters the ocular structures. Of utmost importance for effective ocular drug delivery are patient compliance and the physicochemical properties of the drug. For a given concentration of an active substance, drop size may determine drug adverse effects but does not influence its efficacy.

Application of Western blotting for the detection of uncoupling protein-2 (UCP-2) in mitochondria from smokers and non-smokers

Introduction: Uncoupling proteins (UCPs) are a family of transmembrane anion transporters present in the inner mitochondrial membrane. UCP-2, which exhibits the widest distribution in various tissues, plays an important role in many physiological processes. Human UCP-2 studies have been hampered by the lack of a method for measuring this protein in an easily accessible human tissue, e.g. blood. The aim of this study was to develop such a method and test its utility by comparing UCP-2 levels in smokers and non-smokers. Material and methods: Venous blood samples from 10 smoking and seven non-smoking volunteers were used for the study; lymphocytes were isolated employing Lymphoprep. UCP-2 levels were measured by Western blotting combined with chemoluminescence detection. Results: Total lymphocyte homogenates were found useless for measuring UCP-2 levels, but it was possible to measure UCP-2 in homogenates of purified lymphocyte mitochondria. There was a significant, though moderate, linear correlation between UCP-2 level and daily cigarette use. UCP-2 level in peripheral blood lymphocytes from smokers was higher than that in non-smokers. Conclusion: The method for measuring UCP-2 in peripheral blood lymphocytes opens the possibility of UCP-2 screening studies in humans and thus may be useful for studying the role of the protein in human physiology and pathology.Wstęp: Białka rozprzęgające (UCPs) stanowią rodzinę białek transportujących aniony przez wewnętrzną błonę mitochondrialną. Białko UCP-2 - które jest najszerzej rozpowszechnione w tkankach - odgrywa ważną rolę w wielu procesach fizjologicznych. Badanie funkcji tego białka u ludzi było utrudnione z powodu braku prostych metod jego pomiaru w łatwo dostępnych tkankach, na przykład we krwi. Celem tej pracy było opracowanie takiej metody i wykonanie pilotowego badania jej przydatności przez porównanie ekspresji białka UCP-2 u osób palących i niepalących. Materiał i metody: W badaniu wzięło udział 17 ochotników (10 osób palących i 7 niepalących), od których pobrano krew żylną. Z krwi izolowano limfocyty za pomocą Lymphoprepu. Ekspresję UCP-2 oceniano metodą Western blot z detekcją chemoluminescencyjną. Wyniki: Homogenat całych limfocytów krwi obwodowej okazał się nieprzydatny do mierzenia poziomu UCP-2, wykazano zaś, że dobrym materiałem do takich oznaczeń jest homogenat oczyszczonych mitochondriów z tych komórek. Stwierdzono wyższą ekspresję UCP-2 u osób palących oraz istotną, chociaż umiarkowaną, liniową korelację między ekspresją UCP-2 a liczbą dziennie wypalanych papierosów. Wnioski: Opisana metoda oznaczania UCP-2 umożliwia prowadzenie badań przesiewowych nad rolą tego białka w stanach fizjologicznych i patologicznych u ludzi

High-Dose Testosterone Propionate Treatment Reverses the Effects of Endurance Training on Myocardial Antioxidant Defenses in Adolescent Male Rats

This study was aimed at evaluation of changes in activities of selected antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase) and contents of key nonenzymatic antioxidants (glutathione, protein thiol groups, and α- and γ-tocopherols) in the left heart ventricle of young male Wistar rats subjected to endurance training (treadmill running, 1 h daily, 5 days a week, for 6 weeks) or/and testosterone propionate treatment (8 or 80 mg/kg body weight, intramuscularly, once a week, for 6 weeks) during adolescence. The training alone increased the activities of key antioxidant enzymes, but lowered the pool of nonenzymatic antioxidants and enhanced myocardial oxidative stress as evidenced by elevation of the lipid peroxidation biomarker malondialdehyde. The lower-dose testosterone treatment showed mixed effects on the individual components of the antioxidant defense system, but markedly enhanced lipid peroxidation. The higher-dose testosterone treatment decreased the activities of the antioxidant enzymes, lowered the contents of the nonenzymatic antioxidants, except for that of γ-tocopherol, reversed the effect of endurance training on the antioxidant enzymes activities, and enhanced lipid peroxidation more than the lower-dose treatment. These data demonstrate the potential risk to cardiac health from exogenous androgen use, either alone or in combination with endurance training, in adolescents

Survival rates of homozygotic Tp53 knockout rats as a tool for preclinical assessment of cancer prevention and treatment

Abstract Background The gene that encodes tumor protein p53, Tp53, is mutated or silenced in most human cancers and is recognized as one of the most important cancer drivers. Homozygotic Tp53 knockout mice, which develop lethal cancers early in their lives, are already used in cancer prevention studies, and now Tp53 knockout rats have also been generated. This study assessed feasibility of using homozygous Tp53 knockout rats to evaluate the possible outcome of cancer chemoprevention. Methods A small colony of Tp53 knockout rats with a Wistar strain genetic background was initiated and maintained in the animal house at our institution. Tp53 heterozygotic females were bred with Tp53 homozygous knockout males to obtain a surplus of knockout homozygotes. To evaluate the reproducibility of their lifespan, 4 groups of Tp53 homozygous knockout male rats born during consecutive quarters of the year were kept behind a sanitary barrier in a controlled environment until they reached a moribund state. Their individual lifespan data were used to construct quarterly survival curves. Results The four consecutive quarterly survival curves were highly reproducible. They were combined into a single “master” curve for use as a reference in intervention studies. The average lifespan of untreated male Tp53 homozygous knockout rats was normally distributed, with a median of 133 days. Sample size vs. effect calculations revealed that confirming a 20% and 30% increase in the lifespan would respectively require a sample size of 18 and 9 animals (when assessed using the t-test with a power of 80% and alpha set at 0.05). As an example, the Tp53 homozygous knockout rat model was used to test the chemopreventive properties of carnosine, a dipeptide with suspected anticancer properties possibly involving modulation of the mTOR pathway. The result was negative. Conclusion Further evaluation of the Tp53 homozygous knockout male rat colony is required before it can be confirmed as a viable tool for assessing new methods of cancer prevention or treatment

Cytotoxic effects of cladribine and tezacitabine toward HL-60.

The aim of the study was to determine the relation between the cytotoxic and cytostatic effects of tezacitabine and cladribine on a HL-60 cell line and the time of exposure of cells to these drugs. Cell viability and induction of apoptosis were assessed using flow cytometry methods. Apoptosis was confirmed by direct microscopic observation. Growth inhibition was examined by cell counting. After 24 h incubation tezacitabine was equally or less toxic compared to cladribine. However, toxicity of tezacitabine strongly rose after 48 h incubation leading to massive cell death at doses much lower than those of cladribine. Assessment of the effect of increased exposure time on the clinical efficacy of tezacitabine is indicated

Cytostatic and cytotoxic effects of (E)-2'-deoxy-2'-(fluoromethylene)-cytidine on a solid tumor and a leukemia cell line.

(E)-2'-deoxy-2'-(fluoromethylene)-cytidine (FMdC), a deoxycytidine analog displaying a very high toxicity toward a variety of solid tumor cell lines and xenografts, is activated intracellularly by deoxycytidine kinase (dCK). We have compared cytotoxicity of FMdC towards a human promyeolocytic leukemia line HL-60 and a human colorectal carcinoma line COLO-205. Despite dCK activity being by far the highest in cells of lymphoid origin, the effects of FMdC were detectable at the lowest drug concentration only in a solid tumor cell line, and at higher concentrations they were qualitatively similar in the two tumor lines (increased cell protein content, cell cycle block and apoptosis). Apparently, low dCK activity in solid tumor cells sufficiently activates FMdC to yield cytotoxic effects, while high dCK activity in leukemia cells does not increase its cytotoxicity

Sonodynamic Therapy for Gliomas. Perspectives and Prospects of Selective Sonosensitization of Glioma Cells

Malignant glial tumors (gliomas) are the second (after cerebral stroke) cause of death from diseases of the central nervous system. The current routine therapy, involving a combination of tumor resection, radio-, and chemo-therapy, only modestly improves survival. Sonodynamic therapy (SDT) has been broadly defined as a synergistic effect of sonication applied in combination with substances referred to as “sonosensitizers”. The current review focuses on the possibility of the use of tumor-seeking sonosensitizers, in particular 5-aminolevulinic acid, to control recurring gliomas. In this application, SDT employs a principle similar to that of the more widely-known photodynamic therapy of superficially located cancers, the difference being the use of ultrasound instead of light to deliver the energy necessary to eliminate the sensitized malignant cells. The ability of ultrasound to penetrate brain tissues makes it possible to reach deeply localized intracranial tumors such as gliomas. The major potential advantage of this variant of SDT is its relative non-invasiveness and possibility of repeated application. Until now, there have been no clinical data regarding the efficacy and safety of such treatment for malignant gliomas, but the preclinical data are encouraging