Rational Design, Synthesis and Binding Affinity Studies of Anthraquinone Derivatives Conjugated to Gonadotropin-Releasing Hormone (GnRH) Analogues towards Selective Immunosuppression of Hormone-Dependent Cancer.

Gonadotropin-releasing hormone (GnRH) is pivotal in regulating human reproduction and fertility through its specific receptors. Among these, gonadotropin-releasing hormone receptor type I (GnRHR I), which is a member of the G-protein-coupled receptor family, is expressed on the surface of both healthy and malignant cells. Its presence in cancer cells has positioned this receptor as a primary target for the development of novel anti-cancer agents. Moreover, the extensive regulatory functions of GnRH have underscored decapeptide as a prominent vehicle for targeted drug delivery, which is accomplished through the design of appropriate conjugates. On this basis, a rationally designed series of anthraquinone/mitoxantrone-GnRH conjugates (con1-con8) has been synthesized herein. Their in vitro binding affinities range from 0.06 to 3.42 nM, with six of them (con2-con7) demonstrating higher affinities for GnRH than the established drug leuprolide (0.64 nM). Among the mitoxantrone based GnRH conjugates, con3 and con7 show the highest affinities at 0.07 and 0.06 nM, respectively, while the disulfide bond present in the conjugates is found to be readily reduced by the thioredoxin (Trx) system. These findings are promising for further pharmacological evaluation of the synthesized conjugates with the prospect of performing future clinical studies.

The effect of four hemostatic gene polymorphisms on the outcome of septic critically ill patients.

Genetic variants of hemostatic factors leading to prothrombotic phenotypes of hypercoagulability and hypofibrinolysis might affect prognosis of septic critically ill patients. Our aim was to evaluate the effect of four hemostatic genetic variants, namely fibrinogen-beta-455G/A, factor XIII (FXIII) V34L, plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphisms and factor V Leiden (FVL) mutation on survival of critically ill patients with severe sepsis or septic shock. A prospective, observational study in an 18-bed general ICU included 73 patients with severe sepsis or septic shock. Epidemiological, laboratory data and comorbidities along with severity scores were recorded. Genotyping for fibrinogen-beta-455G/A, FXIII V34L and PAI-1 4G/5G polymorphism and FVL mutation was carried out in all patients. The primary outcomes were the 28-day and the 90-day survival. Age, septic shock, severity indexes, prior steroid use and arterial pH were identified as predictors of the 28-day and 90-day survival in both the univariate and the multivariate models. On the contrary, none of the examined polymorphisms was found to significantly affect either the 28-day or the 90-day survival. Our data suggest that the importance of these hemostatic polymorphisms as predictors of the prognosis of sepsis in critically ill patients is probably very small.

Evaluation of a reverse-hybridization StripAssay for the detection of genetic polymorphisms leading to acenocoumarol sensitivity.

Acenocoumarol is mainly catabolized by CYP2C9 isoform of cytochrome P450 (CYP) liver complex and exerts its anticoagulant effect through the inhibition of Vitamin K Epoxide Reductase (VKOR). The most important genetic polymorphisms which lead to an impaired enzymatic activity and therefore predispose to acenocoumarol sensitivity, are considered to be CYP2C9*2 (Arg144Cys), CYP2C9*3 (Ile359Leu) and VKORC1-1639G>A, respectively. In this study we compared the results of the PGXThrombo StripAssay kit (ViennaLab Diagnostics,Vienna, Austria) with direct DNA sequencing and in house Restriction Fragment Length Polymorphisms (RFLP) for the detection of the aforementioned Single Nucleotide Polymorphisms (SNPs). The reverse hybridization StripAssay was found to be equally effective with RFLP and direct DNA sequencing for the detection of CYP2C9*2 and CYP2C9*3 polymorphisms, respectively. The comparison of the RFLP reference method with the reverse hybridization StripAssay for the detection of VKORC1-1639 G>A polymorphism showed that the reverse hybridization StripAsssay might misclassify some A/A homozygotes as heterozygotes. Optimization of the hybridization procedures may eliminate the extra low signal band observed in some samples at the reverse hybridization StripAssay and improve its diagnostic value.

Prevalence of prothrombotic polymorphisms in Greece.

The aim of this study was to assess the prevalence of several polymorphisms in genes that are involved in several pathways such as hemostasis, fibrinolysis, platelet membrane receptor activity, endothelial integrity and function, lipid metabolism, and regulation of blood pressure in healthy subjects of Greek origin. Most of these polymorphisms are mainly associated with conditions such as venous thromboembolism and atherothrombosis, and their prevalence has not been studied yet in Greece. We tested 140 healthy individuals for factor V (FV)1691G/A, FV4070G/A, FII 20210G/A, factor XIII (FXIII) exon 2G/T, fibrinogen beta-455G/A, plasminogen activator inhibitor-1 (PAI-1)-675 4G/5G, human platelet antigens 1 (HPA1) a/b, apolipoprotein B (ApoB) 10708 G/A, apolipoprotein E (ApoE) E2, E3, and E4, angiotensin-converting enzyme (ACE) D/I, 5,10 methylenetetrahydrofolate reductase (MTHFR) 677C/T, and MTHFR 1298A/C polymorphisms using a PCR and reverse hybridization technique that detects all of them simultaneously. The allele frequencies observed are in accordance with those reported in other Caucasian populations and almost identical to those of East Mediterranean populations. This first report from Greece may serve as a baseline for planning further investigations of these polymorphisms in association with several clinical entities and for launching guidelines for patient testing of various disease settings in this population.

VKORC1 and CYP2C9 allelic variants influence acenocoumarol dose requirements in Greek patients.

AIM: To identify the frequencies of the polymorphisms CYP2C9*2, CYP2C9*3 and VKORC1-1639 G>A in the Greek population and investigate whether these polymorphisms and patient demographics (age, sex and comedication) could explain the interindividual variability of acenocoumarol dose requirements for efficient anticoagulation. MATERIALS & METHODS: CYP2C9*2 (Arg144Cys), CYP2C9*3 (Ile359Leu) and VKORC1-1639G>A allelic variants were analyzed in 98 patients treated with acenocoumarol. RESULTS: Allelic frequencies of CYP2C9*2, CYP2C9*3 and VKORC1A were found to be 0.155, 0.075 and 0.485, respectively. Carriership of at least one CYP2C9*3 allele led to the most pronounced reduction in the required mean dose (p<0.0001). In contrast, the CYP2C9*2 allele played a minor role (p=0.3). VKORC1 A/A patients needed approximately a third of the dose required by wild-type patients to achieve the target INR (p<0.0001). Age was the only demographical factor significantly affecting acenocoumarol dose (p<0.0001). In a multivariable regression model, CYP2C9, VKORC1 genotypes and age explained 55% of acenocoumarol dosing variability. CONCLUSION: VKORC1-1639G>A, CYP2C9*2 and CYP2C9*3 polymorphisms were found to predispose to acenocoumarol sensitivity in Greeks. Other hereditary and nongenetic parameters must be incorporated in an individualized dosing algorithm to achieve a safer anticoagulant effect.