Long-term stability study and evaluation of intact steroid conjugate ratios after the administration of endogenous steroids

The most frequently detected substances prohibited by the World Anti-Doping Agency (WADA) belong to the anabolic steroids class. The most challenging compounds among this class are the endogenous anabolic steroids, which are detected by quantitative measurement of testosterone (T) and its metabolites with a so-called “steroid profiling” method. The current steroid profile is based on the concentrations and ratios of the sum of free and glucuronidated steroids. Recently, our group developed a steroid profiling method for the detection of three free steroids and 14 intact steroid conjugates, including both the glucuronic acid conjugated and sulfated fraction. The study aimed at evaluating the long-term stability of steroid conjugate concentrations and ratios, and the influence of different endogenous steroids on this extended steroid profile. A single dose of oral T undecanoate (TU), topical T gel, topical dihydrotestosterone (DHT) gel, and oral dehydroepiandrosterone (DHEA) was administered to six healthy male volunteers. One additional volunteer with a homozygote deletion of the UGT2B17 gene (del/del genotype) received a single topical dose of T gel. An intramuscular dose of TU was administered to another volunteer. To avoid fluctuation of steroid concentrations caused by variations in urinary flow rates, steroid ratios were calculated and evaluated as possible biomarkers for the detection of endogenous steroid abuse with low doses. Overall, sulfates do not have substantial additional value in prolonging detection times for the investigated endogenous steroids and administration doses. The already monitored glucuronides were overall the best markers and were sufficient to detect the administered steroids.     

Delivering cytokines at tumor site: The immunocytokine-conjugated anti-EDB-fibronectin antibody case

Although considerable efforts have been made in the discovery of new agents for cancer treatment, several promising therapeutics cannot be applied systemically because of their severe side effects. This is the case for various recombinant pro-inflammatory cytokines that, despite their potent anti-cancer activity, can not find their way to clinical exploitation due to their devastating toxicity shown during dose escalation to therapeutically active concentrations. To circumvent these problems, an elegant and efficient way to accumulate therapeutic agents at the tumor site, thus reducing systemic side effects, is their conjugation to tumor-specific antibodies. Here, we review preclinical data about immunocytokines conjugated to a promising single-chain human antibody that selectively targets tumor-associated stroma and blood vessels by binding with high affinity and specificity to the extra domain-B (EDB) of fibronectin.     

Structure-activity relationship of reversibly lipidized peptides: studies of fatty acid-desmopressin conjugates

Purpose. To synthesize a series of reversible fatty acid-desmopressin (DDAVP) conjugates and to study their structure-activity relationship as anti-diuretic drugs. Methods. Seven fatty acid conjugates of DDAVP were prepared using various reversible lipidization reagents as described in our previous reports. All products were purified by acid precipitation and/or size-exclusion chromatography. Reversed-phase HPLC was used to evaluate their purity and lipophilicity. The anti-diuretic efficacy of these fatty acid conjugates was assessed in vasopressin-deficient Brattleboro rats. Four selected conjugates, i.e., DPA, DPH, DPD and DPP (acetic, hexanoic, decanoic, and palmitic acid conjugate, respectively), along with DDAVP itself were used in Caco-2 cell uptake studies and their degradation and the regeneration of active DDAVP were investigated using an in vitro liver slice metabolic system coupled with a HPLC assay. Results. All fatty acid-DDAVP conjugates were more lipophilic than DDAVP as examined by HPLC analyses. When cysteine was used as the linker, the capacity index (k, a measure of lipophilicity) of the conjugates was linearly correlated with the number of carbons in the fatty acid chain. The anti-diuretic activity of the conjugates was correlated with the length of the fatty acid chain, with C10 as the minimal requirement for possessing the enhanced anti-diuretic activity. Among the seven fatty acid conjugates, palmitic acid conjugate was the most potent DDAVP derivative. Removal of carboxyl group from the cysteine linker completely abolished the enhancement of the activity. The extent of cellular uptake also positively correlated with the lipophilicity of the conjugates. The metabolism of DDAVP, DPH, DPD, and DPP by liver slices all followed first order kinetics with half-life of 0.30, 0.01, 0.06 and 3.44 hr, respectively. The degradation rates of DPH and DPD in the liver slice incubation were much faster than that of DDAVP and therefore an accumulation of regenerated DDAVP in the media was observed. In contrast, DPP was metabolized much slower than DDAVP and, consequently, no significant accumulation of regenerated DDAVP could be detected. Conclusion. Conjugation of DDAVP with fatty acids increased the lipophilicity and the anti-diuretic activity of this peptide drug. The anti-diuretic activity of lipidized DDAVP was dependent on the chain length of the fatty acid, as well as the structure of the linker in the conjugate. The preservation and enhancement of the in vivo anti-diuretic activity of the conjugates is most likely due to a combination of an improved pharmacokinetic behavior and a concurrent regeneration of active DDAVP in tissues.     

Absorption, distribution, metabolism, and elimination of 8-2 fluorotelomer alcohol in the rat.

The absorption, distribution, metabolism, and elimination of [3-14C] 8-2 fluorotelomer alcohol (8-2 FTOH, C7F1514CF2CH2CH2OH) following a single oral dose at 5 and 125 mg/kg in male and female rats have been determined. Following oral dosing, the maximum concentration of 8-2 FTOH in plasma occurred by 1 h postdose and cleared rapidly with a half-life of less than 5 h. The internal dose to 8-2 FTOH, as measured by area under the concentration-time curve to infinity, was similar for male and female rats and was observed to increase in a dose-dependent fashion. The majority of the 14C 8-2 FTOH (> 70%) was excreted in feces, and 37-55% was identified as parent. Less than 4% of the administered dose was excreted in urine, which contained low concentrations of perfluorooctanoate (approximately 1% of total 14C). Metabolites identified in bile were principally composed of glucuronide and glutathione conjugates, and perfluorohexanoate was identified in excreta and plasma, demonstrating the metabolism of the parent FTOH by sequential removal of multiple CF2 groups. At 7 days postdose, 4-7% of the administered radioactivity was present in tissues, and for the majority, 14C concentrations were greater than whole blood with the highest concentration in fat, liver, thyroid, and adrenals. Distribution and excretion of a single 125-mg/kg [3-14C] 8-2 FTOH dermal dose following a 6-h exposure in rats was also determined. The majority of the dermal dose either volatilized from the skin (37%) or was removed by washing (29%). Following a 6-h dermal exposure and a 7-day collection period, excretion of total radioactivity via urine (< 0.1%) and feces (< 0.2%) was minor, and radioactivity concentrations in most tissues were below the limit of detection. Systemic availability of 8-2 FTOH following dermal exposure was negligible.     

Hydrolysis of soy isoflavone conjugates using enzyme may underestimate isoflavone concentrations in tissue

Objective: To investigate the differences of using enzymatic hydrolysis and acid hydrolysis for identification and quantification of isoflavone aglycones from biomatrices. Methods: β-glucuronidase/sulfatase isolated from Helix pomat/a for routine enzy-matic hydrolysis or 6N HC1 was used to release glucuronide and sulfate conjugates in the serum, urine and tissue samples. Profiles of soy isoflavones after enzymatic hydrolysis or acid hydrolysis in several tissues of rat fed with diets containing soy protein isolate were also com-pared using I.C/MS and HPLC-ECD. Results: Acid hydrolysis released more aglycone than enzymatic digestion (P＜0.05) in liver tissue. The total genistein, daidzein and other metabolites were 20% to 60% lower in samples from enzymatic hydrolysis than in acid hydrolysis. Conclusion: These results indicated that unknown factors in tissues reduced the enzymatic hydrolytic efficiency for releasing isoflavone aglycones even in op optimized condition. This would underestimate isoflavone tissue concentrations up to 60%.     

Hepatotoxicity of 3,4-methylenedioxyamphetamine and α-methyldopamine in isolated rat hepatocytes: formation of glutathione conjugates

The amphetamine designer drugs 3,4-methylenedioxymethamphetamine (MDMA or ecstasy) and its N-demethylated analogue 3,4-methylenedioxyamphetamine (MDA or love) have been extensively used as recreational drugs of abuse. MDA itself is a main MDMA metabolite. MDMA abuse in humans has been associated with numerous reports of hepatocellular damage. Although MDMA undergoes extensive hepatic metabolism, the role of metabolites in MDMA-induced hepatotoxicity remains unclear. Thus, the aim of the present study was to evaluate the effects of MDA and -methyldopamine (-MeDA), a major metabolite of MDA, in freshly isolated rat hepatocyte suspensions. The cells were incubated with MDA or -MeDA at final concentrations of 0.1, 0.2, 0.4, 0.8, or 1.6 mM for 3 h. The toxic effects induced following incubation of hepatocyte suspensions with these metabolites were evaluated by measuring cell viability, the extent of lipid peroxidation, levels of glutathione (GSH) and glutathione disulfide (GSSG), the formation of GSH conjugates, and the activities of GSSG reductase (GR), GSH peroxidase (GPX), and GSH S-transferase (GST). MDA induced a concentration- and time-dependent GSH depletion, but had a negligible effect on lipid peroxidation, cell viability, or on the activities of GR, GPX, and GST. In contrast, -MeDA (1.6 mM, 3 h) induced a marked depletion of GSH accompanied by a loss on cell viability, and decreases in GR, GPX and GST activities, although no significant effect on lipid peroxidation was found. For both metabolites, GSH depletion was not accompanied by increases in GSSG levels; rather, 2-(glutathion-S-yl)--MeDA and 5-(glutathion-S-yl)--MeDA were identified by HPLC-DAD/EC within cells incubated with MDA or -MeDA. The results provide evidence that one of the early consequences of MDMA metabolism is a disruption of thiol homeostasis, which may result in loss of protein function and the initiation of a cascade of events leading to cellular damage.     

Cytotoxic effect of conjugates of doxorubicin and human chorionic gonadotropin (hCG) in breast cancer cells

Cytotoxic activity of drug conjugates of human chorionic gonadotropin (hCG) and doxorubicin alone was investigated compared to doxorubicin in breast cancer cells with and without expression of hCG receptors. Expression of hCG receptor was determined in MCF-7 and MB231 breast cancer cell line using a multiplex nested rt-PCR approach. The entire sequence of mRNA encoding for hCG receptor was detected in MCF-7 but not in MB231 breast cancer cell line. Cytostatic effect of doxorubicin–hCG conjugates was investigated in these cell lines in comparison to unconjugated doxorubicin. The number of viable cells was determined after 24, 48, 72, 96, and 120 h. To exclude non-specific uptake of the carrier hCG from the culture media, a similar experiment was performed with albumin–doxorubicin conjugates. The number of viable cells decreased in a concentration depending manner after doxorubicin and hCG–doxorubicin conjugate treatment. However, the cytotoxic effect of hCG–doxorubicin conjugate was 10-fold increased compared to unconjugated doxorubin in hCG-receptor positive MCF-7 but not in hCG-receptor negative MB231 cells. Albumin–doxorubicin conjugates showed no increased toxicity compared to doxorubicin. We conclude that the cytotoxic effect of hCG–doxorubicin conjugates is mediated specifically via the hCG receptor. By using hCG conjugates, the development of more selective cytostatics can be achieved.