Oxidative metabolism of the anti-cancer agent mitoxantrone by horseradish, lacto-and lignin peroxidase

Mitoxantrone (MH₂X), an anthraquinone-type anti-cancer agent used clinically in the treatment of human malignancies, is oxidatively activated by the peroxidase/H₂O₂ enzyme system. In contrast to the enzymatic mechanisms of drug oxidation, the chemical transformations of MH₂X are not well described. In this study, MH₂X metabolites, produced by the horseradish, lacto- or lignin peroxidase (respectively HRP, LPO and LIP)/H₂O₂ system, were investigated by steady-state spectrokinetic and HPLC-MS methods. At an equimolar mitoxantrone/H₂O₂ ratio, the efficacy of the enzyme-catalyzed oxidation of mitoxantrone decreased in the following order: LPO > HRP > LIP, which accorded with the decreasing size of the substrate access channel in the enzyme panel examined. In all cases, the central drug oxidation product was the redox-active cyclic metabolite, hexahydronaphtho-[2,3-f]-quinoxaline-7,12-dione (MH₂), previously identified in the urine of mitoxantrone-treated patients. As the reaction progressed, data gathered in this study suggests that further oxidation of the MH₂ side-chains occurred, yielding the mono- and dicarboxylic acid derivatives respectively. Based on the available data a further MH₂ derivative is proposed, in which the amino-alkyl side-chain(s) are cyclised. With increasing H₂O₂ concentrations, these novel MH₂ derivatives were oxidised to additional metabolites, whose spectral properties and MS data indicated a stepwise destruction of the MH₂ chromophore due to an oxidative cleavage of the 9,10-anthracenedione moiety. The novel metabolites extend the known sequence of peroxidase-induced mitoxantrone metabolism, and may contribute to the cytotoxic effects of the drug in vivo. Based on the structural features of the proposed MH₂ oxidation products we elaborate on various biochemical mechanisms, which extend the understanding of mitoxantrone’s pharmaceutical action and its clinical effectiveness with a particular focus on peroxidase-expressing solid tumors, such as breast carcinoma.     

Evacetrapib is a novel, potent, and selective inhibitor of cholesteryl ester transfer protein that elevates HDL cholesterol without inducing aldosterone or increasing blood pressure

Cholesteryl ester transfer protein (CETP) catalyses the exchange of cholesteryl ester and triglyceride between HDL and apoB containing lipoprotein particles. The role of CETP in modulating plasma HDL cholesterol levels in humans is well established and there have been significant efforts to develop CETP inhibitors to increase HDL cholesterol for the treatment of coronary artery disease. These efforts, however, have been hampered by the fact that most CETP inhibitors either have low potency or have undesirable side effects. In this study, we describe a novel benzazepine compound evacetrapib (LY2484595), which is a potent and selective inhibitor of CETP both in vitro and in vivo. Evacetrapib inhibited human recombinant CETP protein (5.5 nM IC(50)) and CETP activity in human plasma (36 nM IC(50)) in vitro. In double transgenic mice expressing human CETP and apoAI, evacetrapib exhibited an ex vivo CETP inhibition ED(50) of less than 5 mg/kg at 8 h post oral dose and significantly elevated HDL cholesterol. Importantly, no blood pressure elevation was observed in rats dosed with evacetrapib at high exposure multiples compared with the positive control, torcetrapib. In addition, in a human adrenal cortical carcinoma cell line (H295R cells), evacetrapib did not induce aldosterone or cortisol biosynthesis whereas torcetrapib dramatically induced aldosterone and cortisol biosynthesis. Our data indicate that evacetrapib is a potent and selective CETP inhibitor without torcetrapib-like off-target liabilities. Evacetrapib is currently in phase II clinical development.     

A PCSK9-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.     

Modelling HIV/AIDS in the presence of an HIV testing and screening campaign

Preventing and managing the HIV/AIDS epidemic in South Africa will dominate the next decade and beyond. Reduction of new HIV infections by implementing a comprehensive national HIV prevention programme at a sufficient scale to have real impact remains a priority. In this paper, a deterministic HIV/AIDS model that incorporates condom use, screening through HIV counseling and testing (HCT), regular testing and treatment as control strategies is proposed with the objective of quantifying the effectiveness of HCT in preventing new infections and predicting the long-term dynamics of the epidemic. It is found that a backward bifurcation occurs if the rate of screening is below a certain threshold, suggesting that the classical requirement for the basic reproduction number to be below unity though necessary, is not sufficient for disease control in this case. The global stabilities of the equilibria under certain conditions are determined in terms of the model reproduction number R₀. Numerical simulations are performed and the model is fitted to data on HIV prevalence in South Africa. The effects of changes in some key epidemiological parameters are investigated. Projections are made to predict the long-term dynamics of the disease. The epidemiological implications of such projections on public health planning and management are discussed.     

A paragonimiasis patient with allergic reaction to praziquantel and resistance to triclabendazole: successful treatment after desensitization to praziquantel

Paragonimiasis is an infectious disease caused by trematodes of the genus Paragonimus. This trematode can be treated successfully with praziquantel in more than 90% of the cases. Although praziquantel is generally well tolerated, anaphylactic reactions to this drug have been reported in a few cases. We report here a 46-year-old Korean female with paragonimiasis, presumed to be due to Paragonimus westermani, who displayed an allergic reaction to praziquantel and resistance to triclabendazole treatment. The patient was successfully treated with praziquantel following a rapid desensitization procedure. Desensitization to praziquantel could be considered when no alternative drugs are available.     

The effects of calcium channel blockade on proliferation and differentiation of cardiac progenitor cells

Cardiogenesis depends on a tightly regulated balance between proliferation and differentiation of cardiac progenitor cells (CPCs) and their cardiomyocyte descendants. While exposure of early mouse embryos to Ca²⁺ channel antagonists has been associated with abnormal cardiac morphogenesis, less is known about the consequences of Ca²⁺ channel blockade on proliferation and differentiation of CPCs at the cellular level. Here we showed that at embryonic day (E) 11.5, the murine ventricles express several L-type and T-type Ca²⁺ channel isoforms, and that the dihydropyridine Ca²⁺ channel antagonist, nifedipine, blunts isoproterenol induced increases in intracellular Ca²⁺. Nifedipine mediated Ca²⁺ channel blockade was associated with a reduction in cell cycle activity of E11.5 CPCs and impaired assembly of the cardiomyocyte contractile apparatus. Furthermore, in cell transplantation experiments, systemic administration of nifedipine to adult mice receiving transplanted E11.5 ventricular cells (containing CPCs and cardiomyocytes) was associated with smaller graft sizes compared to vehicle treated control animals. These data suggest that intracellular Ca²⁺ is a critical regulator of the balance between CPC proliferation and differentiation and demonstrate that interactions between pharmacological drugs and transplanted cells could have a significant impact on the effectiveness of cell based therapies for myocardial repair.     

Database of protein complexes with multivalent binding ability: Bival‐bind

Phenomena of multivalent binding of ligands with receptors are ubiquitous in biology and of growing interest in material sciences. Multivalency can enhance binding affinity dramatically. To understand the mechanism of multivalent binding in more detail model systems of bi‐ and multivalent receptors are needed, but are difficult to find. Furthermore it is useful to know about multivalent receptors, which can serve as targets to design multivalent drugs. The present contribution tries to close this gap. The Bival‐Bind database ( http://agknapp.chemie.fu‐berlin.de/bivalbind ) provides a relatively complete list – 2073 protein complexes with less than 90% sequence identity – out of the protein database, which can serve as bi‐ or multivalent receptors. Steric clashes of molecular spacers – necessary to connect the monomeric ligand units – with the receptor surface can diminish binding affinity dramatically and, thus, abolish the expected enhancement of binding affinity due to the multivalency. The potential multivalent receptors in the Bival‐Bind database are characterized with respect to the receptor surface topography. A height profile between the receptor binding pockets is provided, which is an important information to estimate the influence of unfavorable spacer receptor interaction. Proteins 2014; 82:744–751. © 2013 Wiley Periodicals, Inc.     

A review of advanced oral drug delivery technologies facilitating the protection and absorption of protein and peptide molecules

The oral delivery of proteins and peptides is a dynamic research field despite the numerous challenges limiting their effective delivery. Successful oral delivery of proteins and peptides requires the accomplishment of three key tasks: protection of the macromolecules from degradation in the gastrointestinal tract (GIT), permeation through the intestinal barrier and absorption of molecules into the systemic circulation. Currently, no clinically useful oral formulations have been developed but several attempts have been made to overcome the challenges of low oral bioavailability resulting from poor absorption, poor permeation and enzymatic degradation of the proteins and peptides in the GIT. Present strategies attempt to provide structural protection of the proteins and peptides and improved absorption through the use of enzyme inhibitors, absorption enhancers, novel polymeric delivery systems and chemical modification. However, each of these technologies has their limitations despite showing positive results. This review attempts to discuss the physical and chemical barriers of the GIT with particular emphasis on the current approaches employed to overcome these barriers, including the evaluation of other non-parenteral routes of protein and peptide delivery. In addition, this review assimilates oral formulation strategies under development and within the clinical trial stage in relation to their benefits and drawbacks with regard to facilitating optimal protection and absorption of proteins and peptides, as well as pertinent future challenges and opportunities governing oral drug delivery.