Design of new oxazaphosphorine anticancer drugs

The oxazaphosphorines including cyclophosphamide (CPA, Cytoxan, or Neosar), ifosfamide (IFO, Ifex) and trofosfamide (Ixoten) represent an important group of therapeutic agents due to their substantial antitumor and immunomodulating activity. However, several intrinsic limitations have been uncounted during the clinical use of these oxazaphosphorines, including substantial pharmacokinetic variability, resistance and severe host toxicity. To circumvent these problems, new oxazaphosphorines derivatives have been designed and evaluated with an attempt to improve the selectivity and response with reduced host toxicity. These include mafosfamide (NSC 345842), glufosfamide (D19575, β-Dglucosylisophosphoramide mustard), S-(-)-bromofosfamide (CBM-11), NSC 612567 (aldophosphamide perhydrothiazine) and NSC 613060 (aldophosphamide thiazolidine). Mafosfamide is an oxazaphosphorine analog that is a chemically stable 4-thioethane sulfonic acid salt of 4-hydroxy-CPA. Glufosfamide is IFO derivative in which the isophosphoramide mustard, the alkylating metabolite of IFO, is glycosidically linked to a β-D-glucose molecule. Phase II studies of glufosfamide in the treatment of pancreatic cancer, non-small cell lung cancer (NCSLC), and recurrent glioblastoma multiform (GBM) have recently completed and Phase III trials are ongoing, while Phase I studies of intrathecal mafosfamide have recently completed for the treatment of meningeal malignancy secondary to leukemia, lymphoma, or solid tumors. S-(-)- bromofosfamide is a bromine-substituted IFO analog being evaluated in a few Phase I clinical trials. The synthesis and development of novel oxazaphosphorine analogs with favourable pharmacokinetic and pharmacodynamic properties still constitutes a great challenge for medicinal chemists and cancer pharmacologists.

Copper-transporting P-Type ATPase, ATP7A, confers multidrug resistance and its expression is related to resistance to SN-38 in clinical colon cancer

We and others have shown that the copper transporters ATP7A and ATP7B play a role in cellular resistance to cisdiaminedichloroplatinum (II) (CDDP).  In this study, we found that ATP7A transfection of Chinese hamster ovary  cells (CHOK1) and fibroblasts isolated from Menkes disease patients  enhanced resistance not only to CDDP but also to various anticancer drugs, such as vincristine, paclitaxel, 7-ethyl-10- hydroxy-camptothecin (SN-38),  etoposide, doxorubicin, mitoxantron, and 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11). ATP7A preferentially localized
doxorubicin fluorescence to the Golgi apparatus in contrast to the more intense nuclear staining of doxorubicin in the parental cells. Brefeldin A   partially and monensin completely altered the distribution of doxorubicin to the nuclei in the ATP7A-expressing cells. ATP7A expression also enhanced the efflux rates of doxorubicin and SN-38 from cells and increased the uptake of SN-38 in membrane vesicles. These findings strongly suggested that   ATP7A confers multidrug resistance to the cells by compartmentalizing drugs in the Golgi apparatus and by enhancing efflux of these drugs, and the trans-Golgi network has an important role of ATP7A-related drug resistance. ATP7A was expressed in 8 of 34 (23.5%) clinical colon cancer specimens but not in the adjacent normal epithelium. Using the histoculture drug response assay that is useful for the prediction of drug sensitivity of clinical cancers, ATP7A-expressing colon cancer cells were significantly more  resistant to SN-38 than ATP7Anegative cells. Thus, ATP7A confers  resistance to various anticancer agents on cancer cells and might be a good index of drug resistance in clinical colon cancers.

Drug-herb interactions: eliminating toxicity with hard drug design.

By searching the literatures, it was found that a total of 32 drugs interacting with herbal medicines in humans. These drugs mainly include anticoagulants (warfarin, aspirin and phenprocoumon), sedatives and antidepressants (midazolam, alprazolam and amitriptyline), oral contraceptives, anti-HIV agents (indinavir, ritonavir and saquinavir), cardiovascular drug (digoxin), immunosuppressants (cyclosporine and tacrolimus) and anticancer drugs (imatinib and irinotecan). Most of them are substrates for cytochrome P450s (CYPs) and/or P-glycoprotein (PgP) and many of which have narrow therapeutic indices. However, several drugs including acetaminophen, carbamazepine, mycophenolic acid, and pravastatin did not interact with herbs. Both pharmacokinetic (e.g. induction of hepatic CYPs and intestinal PgP) and/or pharmacodynamic mechanisms (e.g. synergistic or antagonistic interaction on the same drug target) may be involved in drug-herb interactions, leading of altered drug clearance, response and toxicity. Toxicity arising from drug-herb interactions may be minor, moderate, or even fatal, depending on a number of factors associated with the patients, herbs and drugs. Predicting drug-herb interactions, timely identification of drugs that interact with herbs, and therapeutic drug monitoring may minimize toxic drug-herb interactions. It is likely to predict pharmacokinetic herb-drug interactions by following the pharmacokinetic principles and using proper models that are used for predicting drug-drug interactions. Identification of drugs that interact with herbs can be incorporated into the early stages of drug development. A fourth approach for circumventing toxicity arising from drug-herb interactions is proper design of drugs with minimal potential for herbal interaction. So-called ”hard drugs” that are not metabolized by CYPs and not transported by PgP are believed not to interact with herbs due to their unique pharmacokinetic properties. More studies are needed and new approached are required to minimize toxicity arising from drug-herb interactions.

Insights into exazaphosphorine resistance and possible approaches to its circumvention

The oxazaphosphorines cyclophosphamide, ifosfamide and trofosfamide remain a clinically useful class of anticancer drugs with substantial antitumour activity against a variety of solid tumors and hematological malignancies. A major limitation to their use is tumour resistance, which is due to multiple mechanisms that include increased DNA repair, increased cellular thiol levels, glutathione S-transferase and aldehyde dehydrogenase activities, and altered cell-death response to DNA damage. These mechanisms have been recently re-examined with the aid of sensitive analytical techniques, high-throughput proteomic and genomic approaches, and powerful pharmacogenetic tools. Oxazaphosphorine resistance, together with dose-limiting toxicity (mainly neutropenia and neurotoxicity), significantly hinders chemotherapy in patients, and hence, there is compelling need to find ways to overcome it. Four major approaches are currently being explored in preclinical models, some also in patients: combination with agents that modulate cellular response and disposition of oxazaphosphorines; antisense oligonucleotides directed against specific target genes; introduction of an activating gene (CYP3A4) into tumor tissue; and modification of dosing regimens. Of these approaches, antisense oligonucleotides and gene therapy are perhaps more speculative, requiring detailed safety and efficacy studies in preclinical models and in patients. A fifth approach is the design of novel oxazaphosphorines that have favourable pharmacokinetic and pharmacodynamic properties and are less vulnerable to resistance. Oxazaphosphorines not requiring hepatic CYP-mediated activation (for example, NSC 613060 and mafosfamide) or having additional targets (for example, glufosfamide that also targets glucose transport) have been synthesized and are being evaluated for safety and efficacy. Characterization of the molecular targets associated with oxazaphosphorine resistance may lead to a deeper understanding of the factors critical to the optimal use of these agents in chemotherapy and may allow the development of strategies to overcome resistance.

Drug bioactivation, covalent binding to target proteins and toxicity relevance

A number of therapeutic drugs with different structures and mechanisms of action have been reported to undergo metabolic activation by Phase I or Phase II drug-metabolizing enzymes. The bioactivation gives rise to reactive metabolites/intermediates, which readily confer covalent binding to various target proteins by nucleophilic substitution and/or Schiff's base mechanism. These drugs include analgesics (e.g., acetaminophen), antibacterial agents (e.g., sulfonamides and macrolide antibiotics), anticancer drugs (e.g., irinotecan), antiepileptic drugs (e.g., carbamazepine), anti-HIV agents (e.g., ritonavir), antipsychotics (e.g., clozapine), cardiovascular drugs (e.g., procainamide and hydralazine), immunosupressants (e.g., cyclosporine A), inhalational anesthetics (e.g., halothane), nonsteroidal anti-inflammatory drugs (NSAIDSs) (e.g., diclofenac), and steroids and their receptor modulators (e.g., estrogens and tamoxifen). Some herbal and dietary constituents are also bioactivated to reactive metabolites capable of binding covalently and inactivating cytochrome P450s (CYPs). A number of important target proteins of drugs have been identified by mass spectrometric techniques and proteomic approaches. The covalent binding and formation of drug-protein adducts are generally considered to be related to drug toxicity, and selective protein covalent binding by drug metabolites may lead to selective organ toxicity. However, the mechanisms involved in the protein adduct-induced toxicity are largely undefined, although it has been suggested that drug-protein adducts may cause toxicity either through impairing physiological functions of the modified proteins or through immune-mediated mechanisms. In addition, mechanism-based inhibition of CYPs may result in toxic drug-drug interactions. The clinical consequences of drug bioactivation and covalent binding to proteins are unpredictable, depending on many factors that are associated with the administered drugs and patients. Further studies using proteomic and genomic approaches with high throughput capacity are needed to identify the protein targetsof reactive drug metabolites, and to elucidate the structure-activity relationships of drug's covalent binding to proteins and their clinical outcomes.

Substrates and inhibitors of human multidrug resistance associated proteins and the implications in drug development

Human contains 49 ATP-binding cassette (ABC) transporter genes and the multidrug resistance associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP4/ABCC4, MRP5/ABCC5, MRP6/ABCC6, MRP7/ABCC10, MRP8/ABCC11 and MRP9/ABCC12) belong to the ABCC family which contains 13 members. ABCC7 is cystic fibrosis transmembrane conductance regulator; ABCC8 and ABCC9 are the sulfonylurea receptors which constitute the ATP-sensing subunits of a complex potassium channel. MRP10/ABCC13 is clearly a pseudo-gene which encodes a truncated protein that is highly expressed in fetal human liver with the highest similarity to MRP2/ABCC2 but without transporting activity. These transporters are localized to the apical and/or basolateral membrane of the hepatocytes, enterocytes, renal proximal tubule cells and endothelial cells of the blood-brain barrier. MRP/ABCC members transport a structurally diverse array of important endogenous substances and xenobiotics and their metabolites (in particular conjugates) with different substrate specificity and transport kinetics. The human MRP/ABCC transporters except MRP9/ABCC12 are all able to transport organic anions, such as drugs conjugated to glutathione, sulphate or glucuronate. In addition, selected MRP/ABCC members may transport a variety of endogenous compounds, such as leukotriene C(4) (LTC(4) by MRP1/ABCC1), bilirubin glucuronides (MRP2/ABCC2, and MRP3/ABCC3), prostaglandins E1 and E2 (MRP4/ABCC4), cGMP (MRP4/ABCC4, MRP5/ABCC5, and MRP8/ABCC11), and several glucuronosyl-, or sulfatidyl steroids. In vitro, the MRP/ABCC transporters can collectively confer resistance to natural product anticancer drugs and their conjugated metabolites, platinum compounds, folate antimetabolites, nucleoside and nucleotide analogs, arsenical and antimonial oxyanions, peptide-based agents, and in concert with alterations in phase II conjugating or biosynthetic enzymes, classical alkylating agents, alkylating agents. Several MRP/ABCC members (MRPs 1-3) are associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. Drug targeting of these transporters to overcome MRP/ABCC-mediated multidrug resistance may play a role in cancer chemotherapy. Most MRP/ABCC transporters are subject to inhibition by a variety of compounds. Based on currently available preclinical and limited clinical data, it can be expected that modulation of MRP members may represent a useful approach in the management of anticancer and antimicrobial drug resistance and possibly of inflammatory diseases and other diseases. A better understanding of their substrates and inhibitors has important implications in development of drugs for treatment of cancer and inflammation.

Structural and functional properties of human multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug ABC transporters such as P-glycoprotein (P-gp/MDR1/ABCB1) and multidrug resistance protein 1 (MRP1/ABCC1) play an important role in the extrusion of drugs from the cell and their overexpression can be a cause of failure of anticancer and antimicrobial chemotherapy. Recently, the mouse P-gp/Abcb1a structure has been determined and this has significantly enhanced our understanding of the structure-activity relationship (SAR) of mammalian ABC transporters. This paper highlights our current knowledge on the structural and functional properties and the SAR of human MRP1/ABCC1. Although the crystal structure of MRP1/ABCC1 has yet to be resolved, the current topological model of MRP1/ABCC1 contains two transmembrane domains (TMD1 and TMD2) each followed by a nucleotide binding domain (NBD) plus a third NH2-terminal TMD0. MRP1/ABCC1 is expressed in the liver, kidney, intestine, brain and other tissues. MRP1/ABCC1 transports a structurally diverse array of important endogenous substances (e.g. leukotrienes and estrogen conjugates) and xenobiotics and their metabolites, including various conjugates, anticancer drugs, heavy metals, organic anions and lipids. Cells that highly express MRP1/ABCC1 confer resistance to a variety of natural product anticancer drugs such as vinca alkaloids (e.g. vincristine), anthracyclines (e.g. etoposide) and epipodophyllotoxins (e.g. doxorubicin and mitoxantrone). MRP1/ABCC1 is associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. However, most compounds that efficiently reverse P-gp/ABCB1-mediated multidrug resistance have only low affinity for MRP1/ABCC1 and there are only a few effective and relatively specific MRP1/ABCC1 inhibitors available. A number of site-directed mutagenesis studies, biophysical and photolabeling studies, SAR and QSAR, molecular docking and homology modeling studies have documented the role of multiple residues in determining the substrate specificity and inhibitor selectivity of MRP1/ABCC1. Most of these residues are located in the TMs of TMD1 and TMD2, in particular TMs 4, 6, 7, 8, 10, 11, 14, 16, and 17, or in close proximity to the membrane/cytosol interface of MRP1/ABCC1. The exact transporting mechanism of MRP1/ABCC1 is unclear. MRP1/ABCC1 and other multidrug transporters are front-line mediators of drug resistance in cancers and represent important therapeutic targets in future chemotherapy. The crystal structure of human MRP1/ABCC1 is expected to be resolved in the near future and this will provide an insight into the SAR of MRP1/ABCC1 and allow for rational design of anticancer drugs and potent and selective MRP1/ABCC1 inhibitors.

Dynamic analysis of drug-induced cytotoxicity using chip-based dielectrophoretic cell immobilization technology

Quantification of programmed and accidental cell death provides useful end-points for the anticancer drug efficacy assessment. Cell death is, however, a stochastic process. Therefore, the opportunity to dynamically quantify individual cellular states is advantageous over the commonly employed static, end-point assays. In this work, we describe the development and application of a microfabricated, dielectrophoretic (DEP) cell immobilization platform for the realtime analysis of cancer drug-induced cytotoxicity. Microelectrode arrays were designed to generate weak electro-thermal vortices that support efficient drug mixing and rapid cell immobilization at the delta-shape regions of strong electric field formed between the opposite microelectrodes. We applied this technology to the dynamic analysis of hematopoietic tumor cells that represent a particular challenge for real-time imaging due to their dislodgement during image acquisition. The present study was designed to provide a comprehensive mechanistic rationale for accelerated cell-based assays on DEP chips using real-time labeling with cell permeability markers. In this context, we provide data on the complex behavior of viable vs dying cells in the DEP fields and probe the effects of DEP fields upon cell responses to anticancer drugs and overall bioassay performance. Results indicate that simple DEP cell immobilization technology can be readily applied for the dynamic analysis of investigational drugs in hematopoietic cancer cells. This ability is of particular importance in studying the outcome of patient derived cancer cells, when exposed to therapeutic drugs, as these cells are often rare and difficult to collect, purify and immobilize.

Formulation of vanillin cross-linked chitosan nanoparticles and its characterization.

Chitosan nanoparticles were successfully prepared by chemical cross-linking with vanillin. The nanoparticles were spherical in shape with smooth surface, and the average particle size of chitosan nanoparticles was 141 nm. The formulation of chitosan nanoparticles is based on Shiff reaction between aldehyde group of vanillin and amino group of chitosan. Chitosan nanoparticles prepared by crosslinking with vanillin are promising vehicle for the drug delivery of various anticancer drugs in the chemotherapy of cancers.

Role of multidrug resistance associated proteins in drug development

The Multidrug Resistance Associated Proteins (MRPI, MRP2, MRP3, MRp4, MRp5, MRP6, MRP7, MRPS and MRP9) belong to the ATP-binding cassette superfamily (ABCC family) of transporters expressed differentially in the liver, kidney, intestine and blood-brain barrier. MRps transport a structurally diverse array of endo- and xenobiotics and their metabolites (in particular conjugates) and are subject to induction and inhibition by a variety of compounds. An increased efflux of natural product anticancer drugs and other anticancer agents by MRPs in cancer cells is associated with tumor resistance. These transporting proteins play a role in the absorption, distribution and elimination of various compounds in the body. There are increased reports on the clinical impact of genetic mutations of genes encoding MRP1-9. Therefore, MRPs have an important role in drug development, since a better understanding of their function and regulating mechanism can help minimize and avoid drug toxicity, unfavorable drug-drug interactions, and to overcome drug resistance.

Multifunctional and multitargeted nanoparticles for drug delivery to overcome barriers of drug resistance in human cancers

The recurrence and metastatic spread of cancer are major drawbacks in cancer treatment. Although chemotherapy is one of the most effective methods for the treatment of metastatic cancers, it is nonspecific and causes significant toxic damage. The development of drug resistance to chemotherapeutic agents through various mechanisms also limits their therapeutic potential. However, as we discuss here, the use of nanodelivery systems that are a combination of diagnostics and therapeutics (theranostics) is as relatively novel concept in the treatment of cancer. Such systems are likely to improve the therapeutic benefits of encapsulated drugs and can transit to the desired site, maintaining their pharmaceutical properties. The specific targeting of malignant cells using multifunctional nanoparticles exploits theranostics as an improved agent for delivering anticancer drugs and as a new solution for overriding drug resistance.

Development of drug-loaded chitosan-vanillin nanoparticles and its cytotoxicity against HT-29 cells

Chitosan as a natural polysaccharide derived from chitin of arthropods like shrimp and crab, attracts much interest due to its inherent properties, especially for application in biomedical materials. Presently, biodegradable and biocompatible chitosan nanoparticles are attractive for drug delivery. However, some physicochemical characteristics of chitosan nanoparticles still need to be further improved in practice. In this work, chitosan nanoparticles were produced by crosslinking chitosan with 3-methoxy-4-hydroxybenzaldehyde (vanillin) through a Schiff reaction. Chitosan nanoparticles were 200-250 nm in diameter with smooth surface and were negatively charged with a zeta potential of - 17.4 mV in neutral solution. Efficient drug loading and drug encapsulation were achieved using 5-fluorouracil as a model of hydrophilic drug. Drug release from the nanoparticles was constant and controllable. The in vitro cytotoxicity against HT-29 cells and cellular uptake of the chitosan nanoparticles were evaluated by methyl thiazolyl tetrazolium method, confocal laser scanning microscope and flow cytometer, respectively. The results indicate that the chitosan nanoparticles crosslinked with vanillin are a promising vehicle for the delivery of anticancer drugs.

Design of fucoidan functionalized - iron oxide nanoparticles for biomedical applications

This research aims to develop an iron oxide nanoparticle drug delivery system utilizing a recent material discovered from ocean, fucoidan. The material has drawn much interest due to many biomedical functions that have been proven for human health. One interesting point herein is that fucoidan is not only a sulfated polysaccharide, a polymer for stabilization of iron oxide nanoparticles, but plays a role of an anticancer agent also. Various approaches were investigated to optimize the high loading efficiency and explain the mechanism of nanoparticle formations. Fucoidan was functionalized on iron oxide nanoparticles by a direct coating or via amine groups. Also, a hydrophobic part of oleic acid was conjugated to the amine groups for a more favorable loading of poorly water-soluble anticancer drugs. This study proposed a novel system and an efficient method to functionalize fucoidan on iron oxide nanoparticle systems which will lead to a facilitation of a double strength treatment of cancer.

Mechanism-based inhibition of cytochrome P450 3A4 by therapeutic drugs

Consistent with its highest abundance in humans, cytochrome P450 (CYP) 3A is responsible for the metabolism of about 60% of currently known drugs. However, this unusual low substrate specificity also makes CYP3A4 susceptible to reversible or irreversible inhibition by a variety of drugs. Mechanism-based inhibition of CYP3A4 is characterised by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-, time- and concentration-dependent enzyme inactivation, occurring when some drugs are converted by CYP isoenzymes to reactive metabolites capable of irreversibly binding covalently to CYP3A4. Approaches using in vitro, in silico and in vivo models can be used to study CYP3A4 inactivation by drugs. Human liver microsomes are always used to estimate inactivation kinetic parameters including the concentration required for half-maximal inactivation (K(I)) and the maximal rate of inactivation at saturation (k(inact)).Clinically important mechanism-based CYP3A4 inhibitors include antibacterials (e.g. clarithromycin, erythromycin and isoniazid), anticancer agents (e.g. tamoxifen and irinotecan), anti-HIV agents (e.g. ritonavir and delavirdine), antihypertensives (e.g. dihydralazine, verapamil and diltiazem), sex steroids and their receptor modulators (e.g. gestodene and raloxifene), and several herbal constituents (e.g. bergamottin and glabridin). Drugs inactivating CYP3A4 often possess several common moieties such as a tertiary amine function, furan ring, and acetylene function. It appears that the chemical properties of a drug critical to CYP3A4 inactivation include formation of reactive metabolites by CYP isoenzymes, preponderance of CYP inducers and P-glycoprotein (P-gp) substrate, and occurrence of clinically significant pharmacokinetic interactions with coadministered drugs.Compared with reversible inhibition of CYP3A4, mechanism-based inhibition of CYP3A4 more frequently cause pharmacokinetic-pharmacodynamic drug-drug interactions, as the inactivated CYP3A4 has to be replaced by newly synthesised CYP3A4 protein. The resultant drug interactions may lead to adverse drug effects, including some fatal events. For example, when aforementioned CYP3A4 inhibitors are coadministered with terfenadine, cisapride or astemizole (all CYP3A4 substrates), torsades de pointes (a life-threatening ventricular arrhythmia associated with QT prolongation) may occur.However, predicting drug-drug interactions involving CYP3A4 inactivation is difficult, since the clinical outcomes depend on a number of factors that are associated with drugs and patients. The apparent pharmacokinetic effect of a mechanism-based inhibitor of CYP3A4 would be a function of its K(I), k(inact) and partition ratio and the zero-order synthesis rate of new or replacement enzyme. The inactivators for CYP3A4 can be inducers and P-gp substrates/inhibitors, confounding in vitro-in vivo extrapolation. The clinical significance of CYP3A inhibition for drug safety and efficacy warrants closer understanding of the mechanisms for each inhibitor. Furthermore, such inactivation may be exploited for therapeutic gain in certain circumstances.