BTS Abstracts October 2005

Toxicology 219 (2006) 230–242

BTS Abstracts October 2005 Available online 13 December 2005

The Royal Society/Royal Academy of Engineering report: Where do we go from here? Anthony Seaton Institute of Occupational Medicine, Edinburgh, Scotland, UK In 2004, the RS/RAEng reported the conclusions of an independent year-long, investigation of the opportunities and possible threats of nanoscience and nanotechnology. The report had been commissioned by the UK Government in the light of expressed anxieties about hazard to human and environmental health and the fear that the development of these technologies might meet with a similar fate as had genetic modification. To that end, the working group was chaired by a mechanical engineer, Prof Ann Dowling FRS, and included examples of the following species: nanoscientists, social scientists, ethicists, doctors, environmentalists and consumer advocates. The report pointed to many areas of technology where nanoscience was already or was likely in the near future to make a significant economic impact. It drew attention to the dangers inherent in overstating both possible benefits, leading to public cynicism, and risks, leading to hindrance to development. In doing this, it pointed to the importance of distinguishing hazard and risk and of the relevance of dose in determining the latter. It acknowledged two particular areas of human health hazard but made clear that their translation into risk was foreseeable and preventable. These related to the development and use of nanotubes, which might behave like other long, thin durable fibres, and the development of nanoparticles. The report discussed analogies with asbestos and with air pollution combustion particles. The report made a large number of recommendations, but did not suggest that a separate or new regulatory body

was necessary; rather, it recommended that all current regulators should turn their attention to the likely impact of nanotechnology in their area, pointing out the specific problems that might be anticipated in the nano-range. In addition, the report recommended a programme of research designed to reduce uncertainty about hazard and risk and to examine methods of risk reduction. The Government welcomed the report (as did most others who read it) and accepted most recommendations, though notably failing to identify research funds. It set up a cross-departmental group to monitor progress on addressing the recommendations and to report back in 2 and 5 years. It also set up an advisory group, chaired by DEFRA, to make recommendations on research; its response is expected soon after this meeting. Where we go now depends on the conclusions of this group, but at present no new UK funds have been made available for this research. In spite of this, some research is being carried out and it seems likely that this will be an area of increasing toxicological and environmental interest. Nanoparticles in drug delivery: Biodistribution, therapeutic and toxicological considerations Martin C. Garnett School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, UK E-mail address: [email protected] Nanoparticles for drug delivery are typically used to refer to particles with a diameter of less than 1 ␮m (compare to 100 nm diameter by the usual nanotechnology definition). However, in practice, useful sizes for intravenous administration of nanoparticles for drug delivery are determined by anatomy and physiology of key compartments. Usually particle sizes less than 250 nm are required to avoid uptake by the spleen, and smaller

0300-483X/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2005.10.021

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particles (theoretically <100 nm, but in practice they may need to be significantly smaller) are able to pass through the liver fenestrations to allow direct access to the microvilli of the liver parenchymal cells. Particles with hydrophobic surfaces become opsonised and recognised by macrophages mainly in the spleen and liver, but can be made to circulate in the bloodstream for extended periods if a suitable hydrophilic coating such as PEG is applied. These basic assertions will be supported by a range of data acquired from studies of model and biodegradable nanoparticles. For some therapies, interaction of drugs with normal tissues give unwanted toxicities, so restriction of access by nanoparticulates could be advantageous. Due to disease-induced changes, we could expect to get increased localisation in tumours and inflammatory diseases. However, it is likely that only a small proportion of the injected dose will reach these sites, and the major dose limiting toxicities will be expected in the spleen, liver and leukocytes. Within those tissues is expected that localisation will be mostly to the macrophages. By using the intradermal rather than the intravenous route, direct injection into tissue can result in accumulation of nanoparticles particles in lymph nodes. From an intravenous injection, it would be expected from size considerations that particulates would be confined to the bloodstream because of the endothelial tight junctions. However, there are well-documented cases of nanoparticles with particular surface characteristics localising in certain tissues. These tissues include bone marrow, gut and even brain. In the case of uptake by the brain, it has been demonstrated that the uptake is mediated by adsorption of specific blood components, which facilitate transcytosis across the endothelium by specific transporters. Non-specific coatings can however lead to adsorption of specific proteins so a ligand attachment is not necessary to access these compartments. Data will be presented from the literature and the presenter’s laboratory illustrating these examples. The extent to which any particular tissue may be accessed by particulates, is currently unknown. Do all tissues have specific receptors for trancytosis or only some? The potential localisation of drug delivery nanoparticles in other tissues means we must now consider potential toxicities to other accessible tissues. Also if particles are transcytosed into tissues, how readily can nanoparticles pass through tissues and which cells within a tissue can take up nanoparticles? Recently acquired data on organotypic cell culture models will be shown addressing these questions. It is believed by many that pinocytosis and receptor mediated uptake effectively restrict uptake to particles less than 100 nm in diameter to most

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normal cells. However, compelling data will be shown that this is not the case. From a toxicological viewpoint, these findings have significant implications. Access of nanoparticles to the body and bloodstream is clearly restricted by the epithelium, but once this barrier has been by-passed, either through broken skin or (in)advertent administration, a range of tissue localisations is potentially available. Localisation of particles will not necessarily be confined to liver spleen and the blood compartment. Depending on the surface characteristics of the particles, it is possible that small but significant amounts of toxic particles could find their way into and cause damage in a range of other, more sensitive tissues. Toxicology of nanoparticles Ken Donaldson ELEGI Colt Laboratory, Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh, Scotland, UK Within the last 10 years there has been an increasing realisation that the adverse health effects of environmental particles (PM10 /PM2.5 ) are very likely driven by the combustion-derived nanoparticle (NP, particles <100 nm diameter) component e.g. diesel soot. For combustion-derived nanoparticles, three properties appear important—surface area, organics and metals. All of these can generate free radicals and so induce oxidative stress and inflammation. Inflammation is a process involved in the diseases exhibited by the individuals susceptible to the effects of PM–development and exacerbations of airways disease and cardiovascular disease. It is therefore possible to implicate combustion–derived NP in the common adverse effects of increased PM. The adverse effects of increases in PM on the cardiovascular system are well documented in the epidemiological literature and, as argued above, these effects are likely to be driven by the combustion-derived NP. The epidemiological findings can be explained in a number of hypotheses regarding the action of NP: (1) inflammation in the lungs caused by NP causes atheromatous plaque development and destabilisation; (2) the inflammation in the lungs causes alteration in the clotting status or fibrinolytic balance favouring thrombogenesis; (3) the inflammation causes stimulation of the autonomic nervous system culminating in alterations in the heart rhythm, leading to fatal dysrhythmia; and (4) the NP themselves or metals/organics released by the particles enter the circulation and have direct effects on the endothe-

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lium, plaques, the clotting system or the autonomic nervous system/heart rhythm. Environmental NP are accidentally produced but they provide a toxicological model for a new class of purposely ‘engineered’ NP arising from the nanotechnology industry, whose effects are much less understood. Bridging our toxicological knowledge between the environmental NP and the new engineered NP is a considerable challenge. Impact of nanoparticle-types on respiratory health effects: Some important variables to consider David B. Warheit DuPont Haskell Laboratory for Health and Environmental Sciences, Newark, Delaware, USA The results of several lung toxicology studies in rats have demonstrated that ultrafine or nanoparticles (generally defined as particles in the size range <100 nm) administered to the lungs produce enhanced inflammatory responses when compared to fine-sized particles of similar chemical composition at equivalent doses. However, the common perception that nanoparticles are always more toxic than fine-sized particles is based upon a systematic comparison of only two particle-types, namely, titanium dioxide and carbon black particles. Apart from particle size and corresponding surface area considerations, several additional factors may play more important roles in influencing the pulmonary toxicity of nanoparticles. These include, but are not limited to: (1) surface treatments/coatings of particles; (2) the aggregation/disaggregation potential of aerosolized particles; (3) the method of nanoparticle synthesis—i.e., whether the particle was generated in the gas or liquid phase (i.e., fumed versus colloidal/precipitated); (4) translocation potential of the particle; (5) particle shape; and (6) surface charge. Results of pulmonary bioassay hazard/safety studies will be presented demonstrating that fine-sized quartz particles (1.6 ␮m) may produce greater pulmonary toxicity (inflammation, cytotoxicity, cell proliferation and/or histopathology) in rats when compared to nanoscale quartz particles (50 nm), but not when compared to smaller nanoquartz sizes (e.g., <30 nm). In addition, other studies have demonstrated no measurable difference in pulmonary toxicity indices among particle-types when comparing exposures in rats to: (1) fine-sized TiO2 particles (300 nm–6 m2 /g (surface area); (2) TiO2 nanodots (6–10 nm–169 m2 /g); or (3) TiO2 nanorods (25 m2 /g). Finally, studies will be presented which demonstrate that varying surface treatments on fine-sized TiO2 particles influence lung responses. In

summary, some important take-home messages are the following: (1) Risk is a product of hazard and exposure. (2) In general, one cannot assume that nanomaterials have the same chemistry or biology (i.e., toxicity) as their microscale or macroscale counterparts (i.e., either greater than or less than);therefore, the hazards of each particle-type should be tested on a case-by-case basis.

Evaluation of toxicological properties of nanomaterials: Cellular and organ specificity and correlation with physicochemical characteristics Jacob N. Finkelstein, Gunter Oberdorster D.V.M. Departments of Pediatrics, Environmental Medicine and Radiation Oncology, University of Rochester School of Medicine, UK Nanoparticles are being generated from materials of different chemical composition, range of sizes, shapes, coatings. For many of them, human exposures are expected to occur inadvertently or accidentally by different routes. Inhalation, ingestion, dermal or intravenous exposures can be anticipated, depending on the use of NP. In addition, some are intended for eventual administration to humans as biosensors, bio-imaging. While on the one hand continuing progress in nanotechnology will result in many benefits for diagnostic, therapeutic, biological, industrial and military uses, there is on the other hand an almost complete lack of information about potential adverse effects of nano-sized materials, not only at the level of the whole living organism but also at a more basic cellular level. Toxicological studies with nano-sized particles, indicate that particle size and surface properties for particles <100 nm are important determinants of potential toxic effects. Essentially nothing is known about toxicity for NP whose size is reduced to below 10 nm; it is conceivable that for these smallest NP with an even greater surface to mass ratio, reactivity is much higher than predicted for the larger NP. High reactivity is a desired property for many intended applications of NP, but potentially disastrous for cells. Mechanistic studies at the cellular level to develop models and subsequent extrapolation to the whole organism will be discussed as an approach to developing a paradigm to assess the potential risks of nanomaterials.

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Oncogenesis following delivery of a non-primate lentiviral gene therapy vector to fetal mice

Reference

Mike Themis1 , Simon N. Waddington1 , Manfred Schmidt4 , Christof von Kalle4,5 , Yoahe Wang3 , Faisal Al-Allaf1 , Lisa Gregory1 , Megha Nivsarkar1 , Matthew Themis1 , Maxine Holder1 , Suzanne M.K. Buckley1 , Niraja Dighe1 , Alaine Ruthe1 , Ajay Mistry2 , Brian Bigger1 , Adrian Thrasher2 , Charles Coutelle1

Bienemann, A.S., et al., 2003. Long-term replacement of a mutated nonfunctional CNS gene: reversal of hypothalamic diabetes insipidus using an EIAV-based lentiviral vector expressing arginine vasopressin. Mol. Ther. 7 (5 Pt 1), 588–596.

1 Gene

Therapy Research Group, Section of Cell and Molecular Biology, Imperial College London, SW7 2AZ, UK; 2 Molecular Immunology Unit, Institute of Child Health, London, WC1N 1EH, UK; 3 Cancer Research UK, Queen Mary’s School of Medicine & Dentistry at Barts & The London John Vane Science Centre, London, EC1M 6BQ, UK; 4 Internal Medicine I and Institute for Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany; 5 Division of Experimental Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Gene therapy by use of integrating vectors carrying therapeutic transgene sequences offers the potential for a permanent cure of genetic diseases due to the ability of these vectors to integrate in a stable manner into the patients’ chromosomes. However, three cases of Tcell leukaemia have been identified almost 3 years after retrovirus gene therapy for X-linked severe combined immune deficiency. In the two cases investigated to date, vector insertion into the LMO-2 locus was implicated in leukaemogenesis. The third case is currently under investigation (http://afssaps.sante.fr/ang/indang.htm). This has demonstrated that a more profound understanding is required of the genetic and molecular effects imposed by integrating vectors or transgene expression on the host, and has confirmed the need for in vivo models to test for retro- and lentiviral vector safety prior to clinical application. Our previous work has shown that widespread and efficient gene transfer is achievable with different lentivirus vectors following administration in utero to day 16 mouse fetuses. In the present study, we describe the long-term effects on mice after administration of a third generation EIAV lentivirus originating from Oxford BioMedica Ltd. (Bienemann et al., 2003) with the observation of a high frequency of hepatocellular carcinomas following in utero injection, while no tumours were observed after application of an HIV lentivector. This system provides, for the first time, a sensitive model to investigate the molecular mechanisms of virus induced oncogenesis and to serve as an in vivo system for safety testing of integrating vectors in preparation for clinical applications.

Preclinical and clinical toxicological evaluation of polymer-drug conjugates Ruth Duncan Centre for Polymer Therapeutics, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, UK E-mail address: [email protected] There is increasing anticipation that nanotechnology, as applied to medicine, will bring significant advances in the diagnosis and treatment of disease. This has prompted many governmental and funding agencies to strategically review the field (NIH/NCI, 2004; ESF, 2005) with the primary aim of ascertaining the current status of the field, establishing a common terminology, and to assess potential benefits and potential risks of the technology. When a field suddenly becomes fashionable, it is important to keep perspective and to distinguish science-fact from science fiction. Over the last 15 years many novel nano-sized hybrid therapeutics and nanosized drug delivery systems have been approved by Regulatory Authorities for routine clinical use. Products include liposomes (Torchilin, 2005), antibodies and their conjugates (Milenic et al., 2004), nanoparticles (Brigger et al., 2002) and polymer therapeutics (Duncan, 2003). In turn there is also a healthy clinical development pipeline. Stringent Regulatory Authority requirements have ensured that many lessons have already been learnt in respect of the toxicological issues facing each new technology in respect of the frequency of and route of administration and the proposed use. Polymer anticancer conjugates are an interesting example. A growing number of polymer-drug conjugates have entered clinical development (reviewed in Duncan, 2003) and many more systems, including biodegradable polymeric carriers, dendrimers and dendronised polymers, and polymer combinations, are being studied. Using N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers as the carrier, we have now transferred six anticancer conjugates into Phase I/II trials. These compounds contain doxorubicin, paclitaxel, camp-

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tothecin and platinates (Duncan et al., 1998; Duncan, 2003; Duncan, 2005). This presentation will overview the outcome of the preclinical and clinical evaluation. Observation that HPMA copolymers did not elicit toxicity or immunogenicity in man, and moreover when conjugated to cytotoxic chemotherapy were able to reduce drug toxicity markedly has paved the way for its continued development as an anticancer drug carrier (Duncan, 2003), Our recent preclinical studies have been exploring the merits of dendrimers as potential drug carriers, pH-sensitive polymeric drugs, and also examining second-generation HPMA copolymers containing a combination of endocrine and chemotherapy. Experiments conducted to define the safety of these systems will also be presented. References Brigger, I., Dubernet, C., Couvreur, P., 2002. Adv. Drug Del. Rev. 54, 631–651. Duncan, R., 2003. Nat. Rev. Drug Discov. 2, 347–360. Duncan, R., 2005. NanoToday, 16–17. Duncan, R., 2005. In: Kwon, G.S. (Ed.). Polymeric Drug Delivery Systems. Marcel Dekker, New York, pp. 1–92. Duncan, R., 2003. In: Budman D., Calvert, H., Rowinsky, E. (Eds.). Handbook of Anticancer Drug Development. Lippincott, Williams & Wilkins Philadelphia, pp. 239–260. Duncan, R., Coatsworth, J.K., Burtles, S., 1998. Hum. Exp. Toxicol. 17, 93–104. European Science Foundation Forward Look on Nanomedicine Policy Briefing 23, 2005 www.esf.org. Ferrari, M., 2005. Nat. Rev. Cancer 5, 161–171. Malik, N., Evagorou, E.G., Duncan, R., 1999. AntiCancer Drugs 10, 767–776. Milenic, D.E., Brady, E.D., Brechbiel, M.W., 2004. Nat. Rev. Drug Discov. 3, 488–498. NIH/NCI Cancer Nanotechnology Plan, July 2004. http://nano.cancer.gov. Torchilin, V.P., 2005. Nat. Rev. Drug Discov. 4, 145–160. Vicent, M.J., Greco, F., Nicholson, R.I., Duncan, R., 2005. Angew. Chem. Int. Ed. 44, 2–6. Vicent, M.J., Tomlinson, R., Brocchini, S., Duncan, R., 2004. J. Drug Target. 12, 491–501.

The molecular basis of immediate and delayed polycation/polyamine-induced cytotoxicity in gene transfer S Moein Moghimi1 , Peter Symonds2 , Hunter1 , J Clifford Murray2

A

Christy

1 Molecular

Targeting and Polymer Toxicology Group, School of Pharmacy, University of Brighton, Brighton BN2 4GJ, UK; 2 Cancer Research-UK, Tumour Cytokine Biology Group, University Hospital, Nottingham NG7 2UH, UK E-mail address: [email protected] Polycations such as poly(ethylenimine), PEI, and poly(l-lysine), PLL, which are used in transfection protocols, are highly cytotoxic but the molecular basis of their cytotoxicity is poorly understood (Moghimi et al., 2005). We have demonstrated two modes of cytotoxicity with a branched- (25 kDa) and a linear-PEI (750 kDa) molecule as well as two PLL types (Mw 2.9 and 27.4 kDa, respectively) in three clinically relevant human cell lines (Jurkat T-cells, umbilical vein endothelial cells, and THLE3 hepatocyte-like cell line). Phase-I (immediate) toxicity is defined as “early necrotic-like” changes (30–60 min) resulting from compromised membrane integrity, as assessed by considerable lactate dehydrogenase release and phosphatidylserine translocation from the inner plasma membrane to the outer cell surface. These observations were common with all tested polycations in both free form and complexed with mammalian DNA at different weight ratios; however, low Mw PLL was least toxic (Moghimi et al., 2005a; Symonds et al., 2005). Phase-II or delayed cytotoxicity (24 h) was due to activation of a “mitochondrially-mediated apoptotic programme”, resulting from PEI- and PLL (Mw 27.4 kDa)-induced channel formation in the outer mitochondrial membrane, leading to the release of pro-apoptotic cytochrome c (Cyt c), subsequent activation of caspases 9 and 3, and alteration in mitochondrial membrane potential (MMP) due to caspase translocation to mitochondria and cleavage of 75 kDa electron transport protein. Here, cytochrome c release from mitochondria was partly blocked in the presence of Bax channel inhibitor, thus indicating that polycation priming can also facilitate Bax-mediated destabilization/permeabilization of the outer mitochondrial membrane (Symonds et al., 2005). Activation of caspases 8 and 2 was not noted with any of the polycations. Studies with isolated mitochondria demonstrated that PEI-induced Cyt c release was not accompanied by changes in mitochondrial volume, respiration and

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depolarization, thus corroborating that the loss of MMP in cells occurred after Cyt c release and was mediated by activated executioner caspases (Moghimi et al., 2005a). PLL (Mw 27.4 kDa)-mediated Cyt c release from isolated mitochondria was associated with mitochondrial depolarization and partial suppression in respiration. Although, low Mw PLL was able to induce apoptosis in cells via Cyt c release and subsequent apoptosome formation, Cyt c release was independent of Bax channel formation. Interestingly, low Mw PLL did not induce Cyt c release from isolated mitochondrial preparations; all other mitochondrial functions also remained intact. We speculate that “early necrotic-like” PLL (2.9 kDa)-mediated trauma may trigger stress-induced apoptosis. For instance, PLL (2.9 kDa)-mediated cell damage may induce phospholipase activation through involvement of protein kinases. Indeed, PLL was shown recently to induce phospholipase D activation in bovine pulmonary artery endothelial cells through the involvement of protein kinase C. Nevertheless, these processes may lead towards insertion of lysolipids into the outer mitochondrial membrane, presumably through the recently identified lipid transfer capacity of full-length Bid, thereby changing the physical state of the outer membrane and/or priming mitochondria for the release of apoptogenic factors (Goonesinghe et al., 2005). These possibilities are currently under investigation. In summary, we believe that understanding of the molecular basis of polycation-mediated cytotoxicity can eventually help to design safer materials with high transfection efficiencies for clinical gene therapy. References Goonesinghe, A., Mundy, E.S., Smith, M., KhosravaniFar, R., Martinou, J.C., Esposti, M.D., 2005. Biochem. J. 387, 109–118. Moghimi, S.M., Hunter, A.C., Murray, J.C., 2005. FASEB J. 19, 311–330. Moghimi, S.M., Symonds, P., Murray, J.C., Hunter, A.C., Debska, G., Szewczyk, A., 2005a. Mol. Ther. 11, 990–995. Symonds, P., Murray, J.C., Hunter, A.C., Debska, G., Szewczyk, A., Moghimi, S.M., 2005. FEBS Lett. 579, 6191–6198. Antibody directed enzyme prodrug therapy: Issues of mechanism, toxicity and effect Richard Begent Cancer Research UK Targeting and Imaging Group, Department of Oncology, Royal Free and University

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College Medical School, University College London, UK Common cancers are a major health burden and curative options are not available if the disease is deemed non-not respectable. Response to treatment is limited due to drug resistance, lack of selectivity and inherent pathway redundancy favouring survival of the cancer cell. Antibody-directed enzyme prodrug therapy (ADEPT) aims to overcome these shortcomings by selective generation of high concentration of drug in the tumor while sparing normal tissues. A tumor-targeting antibody linked to an enzyme is given intravenously. When cleared from normal tissue prodrug is given and converted to active drug in tumor by targeted enzyme. The essential requirements for effective ADEPT include rapid normal tissue clearance of enzyme, sufficient tumour localisation, effective prodrug conversion and low immunogenicity of the antibody-enzyme. To address this, an ADEPT system with the antibodyenzyme fusion protein MFECP1 and the bis-iodo phenol prodrug ZD2767P was designed. MFECP1 is a recombinant fusion protein of carboxypeptidase G2 (CPG2) directly linked to the N-terminus of the anti-CEA scFv antibody MFE-23 and has a hexahistidine tag on the Cterminus. Expression in Pichia pastoris added branched mannose at two sites on each enzyme monomer. CPG2 converts a bis-iodo phenol mustard prodrug to active drug by cleavage of a glutamate moiety. The design criteria of ADEPT with MFECP1 and ZD2767P were met in studies of ADEPT in nude mice bearing human colon carcinoma xenografts. Sustained tumor regression without significant toxicity was obtained with repeated therapy given over 3 weeks (Sharma et al., 2005). A phase I clinical trial of a single administration of MFECP1 and ZD2767P will be described. Unusual requirements on the trial design were imposed since ADEPT requires delivery of effective concentrations of enzyme to the tumor followed by administration of a potentially effective dose of prodrug at a time when enzyme levels in the blood are low enough to avoid systemic prodrug activation. This was addressed by dividing the trial into three phases. Firstly, the concentration of enzyme in blood, which was safe for prodrug administration was determined. Secondly, a dose escalation of prodrug was conducted and thirdly, the dose of MFECP1 and timing of prodrug administration were investigated. This design facilitated investigation of toxicity of each component and the combinations. The function of each component of the system was tested to support conclu-

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sions and an optimal regimen for ADEPT was defined for investigation of efficacy in a subsequent trial of repeated therapy. Acknowledgement This work is supported by Cancer Research UK, The Royal Free Cancer Research Trust and NTRAC. Reference Sharma, S.K., Pedley, R.B., Bhatia, J., Boxer, G.M., ElEmir. E., Qureshi, U., Tolner, B., Lowe, H., Michael, N.P., Minton, N., Begent, R.H., Cheste, K.A., 2005. Sustained tumor regression of human colorectal cancer xenografts using a multifunctional mannosylated fusion protein in antibody-directed enzyme prodrug therapy. Clin. Cancer Res. 11, 814–825.

Overcoming barriers: Challenges and opportunities in macromolecular delivery H. Oya Alpar Centre for Drug Delivery Research, The School of Pharmacy, University of London, 29-39 Brunswick Sq., London, WC1N 1AX, UK E-mail address: [email protected] Modern vaccines and therapeutics are based on molecules, which exhibit a variety of challenging physicochemical properties. Mucosal routes of administration are preferred due to increased patient compliance and avoidance of first-pass metabolism but suffer such drawbacks as low permeability and harsh environmental conditions that reduce drug stability and therapeutic effect. Although in the 1980s dogma maintained that biological membranes were impermeable to particulates, there has been considerable evidence amassed since, particularly for the gastrointestinal tract, of particulate uptake and translocation by intestinal epithelial cells, microfold cells, M-cells which are believed to play a specific role in luminal content sampling, as well as luminal phagocytes. Therefore, the use of absorption and penetration enhancers such as surfactants, fatty acids and colloidal delivery systems (nano/microspheres) has been proposed to overcome limitations in mucosal drug delivery. Of these, colloidal delivery systems based on biodegradable and biocompatible polymers such as poly (lactide-co-glycolide) and chitosan have attracted much attention as they achieve the desired effects, and controlled drug release both locally and systemically without the toxic side effects associated to other systems.

There is sufficient evidence, however, to suggest physiological and biological similarities between the gastrointestinal and nasal epithelia and subepithelial associated structures (e.g. immunological tissues), with the added benefit of less harsh luminal conditions, high vascularisation, access to the common mucosal immune system and avoidance of hepatic clearance, encouraging the use of the nose as a vaccine and drug delivery site. We and others have developed nano and microparticulate vaccines for intranasal immunisation; in our laboratories, we have achieved local and systemic, fully protective immunity against lethal challenge to virulent pathogens such as Yersinia pestis, Bacillus anthracis and botulinum toxin using polymeric microsphere formulations. To be effective particulate carriers will need to reach the target cells in the nasal lymphoid tissue and be transferred across the nasal membrane by some forms of transport mechanism. Therefore, it is important to study the fate of nano and microspheres after nasal application to nasal mucosae. Only a limited number of publications have studied the fate of nano-microparticles following nasal administration and their transport across the nasal mucosae. We investigated the transport of fluorescently labelled, carboxylated polystyrene particles from the nasal cavity into the systemic circulation in rats and rabbits (Almeida et al., 1993; Alpar et al., 1994) and showed that small amounts of particles were detected in the blood of animals. We have also recently investigated (Eyles et al., 2001) the transfer of nasally administered fluorescent latex particles and Scandium 46 labelled nonbiodegradable particles from the nasal cavity to the NALT, lungs, liver, spleen and draining lymph nodes. A substantial accumulation of the particles was found in NALT. In addition, the olfactory mucosa of the nasal epithelium is interspersed with neurons that provide direct access to the central nervous system and cerebrospinal fluid via the olfactory bulb. This suggests an alternative route for the delivery of therapeutics to the brain, circumventing the blood brain barrier. This concept is supported by a number of studies, which have successfully investigated this administration route in comparison to intravenous dosing, including differences in distribution patterns across the brain. References Almeida, A.J., Alpar, H.O., Brown, M.R., 1993. Immune response to nasal delivery of antigenically intact tetanus toxoid associated with poly(l-lactic acid) microspheres in rats, rabbits and guinea-pigs. J. Pharm. Pharmacol. 45 (3), 198–203.

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Alpar, H.O., Almeida, A.J., Brown, M.R., 1994. Microsphere absorption by the nasal mucosa of the rat. J. Drug Target. 2 (2), 147–149. Eyles, J.E., Spiers. I.D., Williamson. E.D., Alpar. H.O., 2001. Tissue distribution of radioactivity following intranasal administration of radioactive microspheres. J. Pharm. Pharmacol. 53 (5), 601–607. Oral and Poster Abstracts Proinflammatory and toxicological effects of zinc and nanoparticle interactions are induced via nonoxidative mechanisms Martin R. Wilson1 , Martin Clift1 , Peter Barlow1 , Gary Hutchison2 , Keith Guy1 , Alex Griffiths1 , Rickie Simpson1 , Jill Sales3 , Vicki Stone1 1 Biomedicine

Research Group, School of Life Sciences, Napier University, 10 Colinton Road, Edinburgh EH10 5DT, UK; 2 M.R.C. Human Reproductive Sciences Unit, Centre for Reproductive Biology, 49 Little France, Old Dalkeith Road, Edinburgh EH16 4SB, UK; 3 Biomathematics and Statistics Scotland, James Clerk Maxwell Building, The King’s Buildings, Edinburgh EH9 3JZ, UK E-mail address: [email protected] A number of newly derived man-made nanomaterials include zinc within their chemical make-up. A DEFRA funded study conducted in our laboratory indicated that the zinc content of particulate air pollution is associated with its ability to induce inflammation in the rat lung (Lightbody et al., 2003). The zinc content of welding fumes is also implicated with the induction of the respiratory illness metal fume fever (Kuschner et al., 1997). The aims of this study were to investigate the potential of zinc (zinc chloride; ZnCl2 ) and ultrafine or nanoparticle carbon black (14 nm diameter; 14 nm CB; ‘Printex 90’, Degussa, Germany) to interact in both cell-free and biological systems and hence explain some of the biological reactivity observed for welding fumes and air pollution. The murine macrophage cell line, J774 was treated for 4 h with 14 nm CB at concentrations from 1.9 to 31 ␮g/ml. Cells were treated in the absence or presence of ZnCl2 (0 to 100 ␮M) before measuring the TNF-␣ production of the cells by ELISA. Cell-free and intracellular reactive oxygen species (ROS) production was examined using the probe 2
,7-dichlorofluorescein diacetate (DCFH-DA; Wilson et al., 2002). Changes in the cytoskeleton of the cells were investigated using antibodies and probes for F-actin and tubulin and analysed

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using a Zeiss LSM 510 META laser scanning confocal microscope. A concentration-dependant increase in TNF-␣ production was observed following treatment of the cells with up to 100 ␮M ZnCl2 . With the further addition of 31 ␮g/ml 14 nm CB, there was a synergistic increase in TNF-␣ production (P < 0.001). If both treatments were acting independently, the TNF-␣ production would have been 218 pg/ml, whereas the actual concentration was 1081 pg/ml. Using a single concentration of ZnCl2 (20 ␮M), the effect of an increasing concentration of 14 nm CB was examined from 0 to 31 ␮g/ml. Although 14 nm CB alone induced TNF-␣ production in cells, this was synergistically enhanced by the addition of ZnCl2 (P < 0.01 and P < 0.05). In the cell-free system (cell medium and DCFH-DA only), particle treatments alone generated a significant increase in ROS when compared to controls (P < 0.001). ZnCl2 treatments did not induce significant changes in ROS when compared to the controls and, when added to 14 nm CB, ZnCl2 actually decreased the ROS production of the particles. The intracellular ROS production following treatment with 14 nm CB was not enhanced by ZnCl2 . Morphological changes in the cytoskeleton were observed with increasing concentrations of ZnCl2 . Tubulin and F-actin condensing was observed in some cells in addition to nuclear condensation and fragmentation. ZnCl2 synergistically enhanced the ability of ultrafine or nanoparticles to stimulate macrophages to produce TNF-␣. The nuclei condensation and fragmentation are indicative of necrosis or apoptosis however, when considering the DCFH results, the increased proinflammatory cytokine production and toxicological effects induced by ZnCl2 do not appear to be via ROS production. This work will be continued by investigating the possible routes of uptake and fate of nanoparticles in macrophages using fluorescent quantum dots. References Kuschner, W.G., D’Alessandro, A., Wong, H., Blanc, P.D., 1997. Environ. Res. 75, 7–11. Lightbody, J., Hutchison, G., Donaldson, K., Stone, V., 2003. DEFRA Report EPG 1/3/147 Part 1. Wilson, M.R., Lightbody, J.H., Donaldson, K., Sales, J., Stone, V., 2002. Toxicol. Appl. Pharmacol. 184, 172–179. Does the size of zinc oxide particles affect their cutaneous toxicity? Natalya Hanlon, Julie A. Howarth, Richard H. Hinton, Andrew Taylor, Shirley C. Price

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School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey, UK E-mail address: [email protected] There has been increasing concern about the possible toxicity of very small particles. Animal experiments have demonstrated that small nanoparticles (<50 nm) have greater inflammatory potential (Oberdorster et al., 1992) and this is consistent with their physiochemical properties (Jefferson, 2000). Zinc oxide and similar particles are increasingly being used in cosmetics although there has been very little work on the cutaneous toxicity of these particles (Tan et al., 1996; Lademann et al., 1999). The present study was designed to investigate whether there was a significant difference in the penetration of 1000 and 20 nm zinc oxide particles into the intact skin or skin partially permeabilised by treatment with ethanol and whether there were differences in response to particles injected subcutaneously. Nine groups of three Wistar Albino rats (Bantin and Kingman, UK) were randomly assigned to treatment groups. The zinc oxide particles (1000 and 20 nm) were either applied suspended in a cream or in a permeablising agent (ethanol) daily for 3 days, or injected once only into the subcutaneous tissue. Appropriate controls received the corresponding vehicles only. All areas of administration were marked for ease of identification. The animals were sacrificed by exposure to rising levels of CO2 after 14 days following treatment. Zinc concentrations were analysed in the dorsal skin of topically treated rats, in the muscles of the body wall taken from underneath the injection site of the subcutaneously treated animals and in the liver and spleen of all animals using Flame Atomic Absorption Spectrophotometry. Further samples of skin from these groups were taken and stained with haematoxylin and eosin for histological examination. Neither topical nor subcutaneous administration of conventional or nanosized zinc oxide had a biologically significant effect of the body weights, spleen, kidney and liver weights. There were no treatment related changes in the macroscopic or microscopic appearance of the skin of rats treated topically. However, granulomas were induced by subcutaneous injection of both conventional and nanosized zinc oxide powder. Granulomas induced by nanosized zinc oxide particles appeared encapsulated, larger and had more extensive areas of necrosis compared to lesions produced following injection of conventional sized powders of zinc oxide. The skin concentrations of zinc remained unchanged following application of zinc oxide in a cream, while

significantly increased after administration in ethanol although there were no significant variations of zinc levels between the treatments (conventional versus nanosized). Following subcutaneous administration, zinc levels were significantly increased in the body wall taken below the injection site, however there was no difference in the extent of diffusion between the large and small particulates.

In summary there may be some pathological changes occurring following exposure to nano particles of zinc oxide but further work is needed to substantiate this finding. References Jefferson, D.A., 2000. Philos. Trans. R. Soc. Lond. 358, 2683–2692. Lademann, J., Weigman, H.J., Rickmeyer, C., Barthelmes, H., Schaefer, G.M., Sterry, W., 1999. Skin Pharmacol. Appl. 12, 247–256. Oberdorster, G., Ferin, J., Gelein, R., Soderholm, S.C., Finklestein, J., 1992. Environ. Health Perspect. 97, 193–197. Tan, M.H., Conmere, C.A., Burnett, L., Snitch, P.J., 1996. Aust. J. Dermatol. 37, 185–187.

Neurotoxic effects of monodispersed colloidal gold nanoparticles December S.K. Ikah1 , Charles. V. Howard1 , W. Graham McLean2 , Mathias Brust3 , Robert Tshikhudho3 1 Department

of Human Anatomy and Cell Biology, University of Liverpool, UK; 2 Department of Pharmacology, University of Liverpool, UK; 3 Department of Chemistry, University of Liverpool, UK E-mail address: [email protected] Particles have been shown to have adverse effects on cellular function and morphology by reason of their size (Maynard and Howard, 1999). In this study we set out to examine the effect of particle size and surface chem-

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Table 1 Mean percentage neurite length outgrowth following 24 h exposure to gold nanoparticles Conc. no/ml

Mean % control neurite outgrowth 5 nm BSA

1.0 × 104 1.0 × 106 1.0 × 108 1.0 × 1010 1.0 × 1012 1.1 × 1012 2.2 × 1012 3.3 × 1012 4.2 × 1012 5.4 × 1012

89 63 70 70 94 77 96 117 135 116

± ± ± ± ± ± ± ± ± ±

31 20 28 34 33 23 26 42 28 8

10 nm BSA#

14 nm BSA#

5 nm PEG#

10 nm PEG#

123 ± 45 143 ± 43 105 ± 24 143 ± 47 138 ± 51 115 ± 48 120 ± 46 114 ± 49 124 ± 31 120 ± 27

104 ± 27 103 ± 32 101 ± 45 118 ± 52 114 ± 34 111 ± 43 150 ± 34* 133 ± 28 136 ± 25 131 ± 38

111 ± 33 111 ± 24 120 ± 38 103 ± 21 141 ± 42

98 130 100 100 164

± ± ± ± ±

14 nm PEG 46 41 27 33 44*

109 134 146 102 136

± ± ± ± ±

51 34 40 64 43

14 nm PEG-OH# 109 102 94 100 76

± ± ± ± ±

41 8 9 22 19*

14 nm PEG-NH2# 65 107 105 115 130

± ± ± ± ±

22 32 26 44 34

5 nm PEG-OH 95 92 89 109 91

± ± ± ± ±

19 31 20 23 28

N = 6 ± S.D., * P < 0.05 ANOVA, Bonferroni correction, # P < 0.05 Cuzick’s trend analysis.

istry on the toxicity of gold nanoparticles. Conventional colloidal gold particles of 5, 10 or 14 nm size and at concentrations of 1.0 × 104 to 5.4 × 1012 particles/ml were characterised by transmission electron microscope (TEM) and UV–vis spectroscopy. Particles were modified for stability and solubility by adsorbing neutral polymer, polyethylene glycol (PEG), or cationic polymer, bovine serum albumin (BSA). Monolayer-protected clusters (MPCs) consisted of 14 nm size PEGylated alkanethiol ligand-stabilized particles with neutral (OH) or cation (amine, NH2 ) function. Taking into account the physical instability of particles in a biological milieu, particle interaction in media was examined with observation of the classical plasmon resonance band of gold nanoparticles at 528–530 nm wavelength. TEM images showed that particle uptake into the cells depended on their dispersity in media. We employed an in vitro neurite outgrowth assay with mouse neuroblastoma NB2a cells to assess the neurotoxicity of particles (Flaskos et al., 2001). Cells were plated on to 48-well plates in serum-containing medium. Medium was removed after 24 h incubation and replaced with serum-free, medium supplemented with 0.5 mM dibutyryl cyclic AMP, with or without particles at various known concentrations. Neurite outgrowth was measured by phase contrast microscopy linked to automated image analysis to determine the length of growing neurites. Results were variable (shown in Table 1). We suggest that the inhibitory effect is due to particle internalisation and subsequent interaction, while the observed enhancement of neurite outgrowth could be an effect of particles acting extracellularly to support neurite adhesion and or motility on the culture surface.

References Flaskos, J., Fowler, M.J., McLean, W.G., Hargreaves, A.J. (2001): J. Neurochem. 76, 671–678. Maynard, R.L., Howard, C.V., 1999. Particulate Matter: Properties and Effects upon Health. BIOS Scientific Publishers Oxford. Infrared imaging for the non-invasive assessment of chemical burns: A preliminary study Robert P. Chilcott1 , Christopher H. Dalton2 , Ian J. Hattersley2 , Stephen J. Rutter2 , John S. Graham3 1 Chemical

Hazards & Poisons Division HQ, Health Protection Agency, Chilton, Oxfordshire, OX11 0RQ, UK; 2 Department of Biomedical Sciences, Dstl Porton Down, Salisbury, Wiltshire, SP4 0JQ, UK; 3 United States Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, Aberdeen Proving Ground, MD 21010-5400, USA E-mail address: [email protected] Infrared (IR) imaging can be used to measure local variations in skin temperature and the use of quantitative thermal imaging (QTI) has potential application as a prognostic indicator for the clinical management of skin burns (Jones, 1998). The purpose of this study was to investigate the thermal properties of normal and chemically damaged skin using a commercially available IR camera in order to evaluate IR imaging as a prognostic indicator for chemical burns. Superficial-dermal skin lesions were induced on the abdominal flank of six pigs by controlled exposure to sulphur mustard using a standard dosing procedure involving the application of undiluted agent for up to 8 min

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(Graham et al., 2000). Two days post-exposure, the animals were placed under (terminal) anaesthesia and the end of a brass cylinder (300 g; 3 cm diameter; ∼21 ◦ C) was manually placed onto an area of normal or damaged skin for 5 s to induce cooling. The restitution of skin surface temperature was subsequently monitored using a digital IR camera (ThermaCam P25, FLIR, Kent, UK). Normal skin was more sensitive to cooling and warmed at a faster rate than damaged skin: Table 1 Thermal properties of normal and chemically damaged skin Parameter


Tmax

dT/dt (◦ C s−1 )

E (W m−2 s−1 )

Control Damaged

−6.83 ± 0.44 −4.67 ± 0.54

0.20 ± 0.11 0.12 ± 0.08

336 ± 3 329 ± 3

where
Tmax is the maximum decrease in temperature (measured 10 s after cooling), dT/dt is the rate of warming (measured 10–30 s after cooling) and E is the equivalent thermal energy absorption rate (measured 10–30 s after cooling) of control (normal) or chemically damaged skin. Control and damaged values were significantly different (P < 0.05) for all three parameters. This preliminary study has demonstrated that there are significant differences in the thermal properties of normal and chemically damaged skin that may have clinical utility for the assessment of skin lesions. In particular, this study has demonstrated how a relatively simple method may reproducibly elicit such differential thermal behaviour. Clearly, further work is required to ascribe specific biological endpoints to the observed changes in the thermal properties of damaged skin. Acknowledgements This work was sponsored by the US Department of Defense (DoD) and the UK Ministry of Defence (MoD) and was conducted at facilities operated by the Defence Science and Technology Laboratory (Dstl). References Jones, B.F., 1998. IEEE Trans. Med. Imaging 17, 1019–1027. Graham, J.S., Reid, F.M., Smith, J.R., Stotts, R.R., Tucker, F.S., Shumaker, S.M., Niemuth, N.A., Janny, S.J., 2000. J. Appl. Toxicol. 20, s161–172. A novel model of image acquisition and processing for precise quantification of angiogenesis disrupted by application of mainstream and sidestream cigarette smoke solutions Sohail Ejaz, Lim Chae Woong

Department of Pathology, College of Veterinary Medicine, Chonbuk National University, Jeonju 561756, South Korea E-mail address: [email protected] Angiogenesis is a vital process in the growth of new blood vessels from pre-existing vasculature (Folkman, 2001). Among several approaches being used for studies related to angiogenesis, chicken chorioallantoic membrane assay (CAM) is an excellent model system (Ribatti et al., 1995). However, its utility has been limited due to difficulty in quantifying putative angiogenic and antiangiogenic response to an experimental compound in an objective and quantifiable manner. Herein, we report a novel approach of image acquisition and processing for better evaluation of neovascularization. The effects of mainstream cigarette smoke solutions (MSCSS) and sidestream cigarette smoke solutions (SSCSS) from different commercially available cigarettes on angiogenesis were quantified, using CAM assay. Two hundred and ten CAMs (day-5 old), divided in seven groups were exposed to MSCSS and SSCSS with different nicotine concentration: 0.2 mg (group B), 0.3 mg (group C), 0.5 mg (group D), 0.6 mg (group E), 0.7 mg (group F) and 1 mg (group G). After 24 h of exposure, different gross and nanometer scale topographies of CAMs were quantified using novel “scan probing image processing” method. A highly significant decrease (P < 0.01) in pattern formation, diameter, and area of the blood vessels was observed among all treated groups, with fluctuations depending upon nicotine concentration. Investigations of 3D and nanometer scale topographies of blood vessels revealed very highly significant decrease (P < 0.001) in the height of the abbot curve, angular spectrum, Sa, Sq, Sk, Sy, Sz, Spk, and Svk values (Table 1).

Table 1 A comparison of different parameters of 3D surface topographies of CAMs between control and group G Parameters Sa Sq Sk Sy Sz Spk Svk

Control (nm) 2692 3457 8081 22503 17453 6349 3762

± ± ± ± ± ± ±

115 205 313 850 1102 112 86

MSCSS (nm) 2.07 2.74 4.23 20.0 15.1 6.28 2.97

± ± ± ± ± ± ±

0.08*** 0.32*** 0.17*** 0.8*** 1.1*** 0.19*** 0.5**

SSCSS (nm) 1.92 2.41 4.02 20.0 13.2 6.07 1.21

± ± ± ± ± ± ±

0.05*** 0.21*** 0.8*** 0.7*** 0.2*** 0.03*** 0.02***

Sa: average roughness, Sq: root mean square deviation, Sk: Skewness of the surface, Sy: lowest valley, Sz: maximum height of the surface, Spk: reduced summit height, Ssk: Skewness of the surface (** P < 0.01, *** P < 0.001 2-way ANOVA).

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Moreover, SSCSS appeared significantly more toxic than MSCSS with respect to their effects on angiogenesis. This imaging technique combined with other modalities may provide a novel solution for precise 3D quantification of angiogenesis. The advantage of this system is that it easily and quickly provides reproducible results, making it highly suitable for drug screening and toxicity evaluation. References Folkman, J., 2001. A new family of mediators of tumor angiogenesis. Cancer Invest. 19 (7), 754–755 Ribatti, D., Urbinati, C., Nico, B., Rusnati, M., Roncali, L., Presta, M., 1995. Endogenous basic fibroblast growth factor is implicated in the vascularization of the chick embryo chorioallantoic membrane. Dev. Biol. 170, 39–49.

Hepatotoxicity of cannabinoids in vitro Colin White, Karen McArdle, Gabrielle Hawksworth Department of Medicine and Therapeutics and School of Medical Sciences, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen AB25 2ZD, UK E-mail address: [email protected]
9 -Tetrahydrocannabinol (
9 -THC) and cannabidiol (CBD) are toxic to primary cultures of rat hepatocytes, with an IC50 value of 40 ± 5 ␮M at 24 h when viability was determined by the MTT assay. Both compounds induced apoptosis in a dose-dependent manner, with approximately 15% of adherent cells showing characteristics of apoptosis after cannabinoid treatment (200 ␮M for 6 h). The toxicity did not appear to be dependent on the production of reactive metabolites (Tayloret al., in press). The endogenous cannabinoid, anandamide, an arachidonic acid derivative, also causes apoptosis in hepatocytes and the mechanism was suggested to be cannabinoid (CB)-receptor independent (Biswas et al., 2001). The aim of this study was to determine the mechanism of cell death caused by CBD and anandamide, using the human hepatoma cell line, HepG2. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1 g/l glucose, GlutaMAXTM , pyruvate and 10% FBS in 96 well plates. Cells were treated with cannabinoids in DMEM for 24 h. The IC50 values were 12 ␮M for CBD and 200 ␮M for anandamide, determined using the MTT assay. Arachidonic acid, a polyunsaturated fatty acid structurally similar to anandamide, did not exhibit significant toxicity to

241

HepG2 cells at concentrations up to 300 ␮M. In a separate series of experiments cells were treated for 2–6 h with CBD and anandamide, then the extent of apoptosis in adherent cells was determined from morphological changes (nuclear fragmentation) visualised using fluorescence microscopy after staining with Hoechst 33342 and propidium iodide for 30 min prior to fixing. There was a dose-, but not time-, dependent increase in apoptosis, with CBD being significantly more toxic to HepG2 cells than anandamide at 2, 4 and 6 h. When HepG2 cells were incubated with 1 mM methyl-␤-cyclodextrin (MCD), which specifically removes membrane cholesterol, or 1 ␮M mevastatin, an inhibitor of HMG-CoA reductase, MCD and mevastatin significantly prevented anandamide-induced apoptosis at 300 ␮M for MCD and concentrations equal to and exceeding 100 ␮M for mevastatin (P < 0.05). For CBD there was no significant difference in cell death between cells dosed with CBD only and cells dosed with CBD together with MCD or mevastatin. Both CBD and anandamide induce apoptosis in hepatocytes and HepG2 cells, via CB receptor-independent mechanisms. For anandamide there appears to be a direct interaction with membrane cholesterol and with lipid rafts in the cell membrane, resulting in the hydrolysis of sphingomyelin to yield ceramide (Sarker and Maruyama, 2003). The mechanism of CBD-induced apoptosis in hepatocytes and HepG2 cells differs from that of anandamide. It is CB receptor independent, but does not involve direct interaction with membrane cholesterol. References Biswas, K.K., Sarker, K.P., Abeyama, K., Kawahara, K., Iino, S., Otsubo, Y., et al., 2003. Hepatology 38, 1167–1177. Sarker, K.P., Maruyama, I., 2003. Cells Mol. Life Sci. 60, 1200–1208. Taylor, A., Cameron, G., Hawksworth, G., in press. Toxicology.

Polyamine depletion protects from simvastatin toxicity in human cancer cell lines Yashka R. Manning, Claire S. Whyte, Abhilash Sadasivan, Heather M. Wallace Department of Medicine and Therapeutics, College of Life Sciences and Medicine, University of Aberdeen, Foresterhill, Aberdeen AB24 2ZD, UK E-mail address: [email protected]

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Fig. 1. Effect of 1 and 5 mM DFMO pre-treatment and treatment with simvastatin. (A) MDA-MB-231 cells (n = 7 ± S.E.M. for sim, n = 3 ± S.E.M. for DFMO, all values from 5 ␮M onwards p < 0.01) and (B) PC3 cells (n = 6 ± S.E.M. for sim, n = 2 ± S.E.M. for DFMO).

Breast and prostate cancers are the second most common types of cancer in women and men, respectively. Current treatments are limited by their toxicity but combination therapy may provide a safer and more effective treatment. The polyamines, putrescine, spermidine and spermine, are essential for cell growth and function and are found at increased levels in cancer cells. ␣-difluoromethylornithine (DFMO) induces polyamine depletion, by irreversibly inhibiting the rate limiting enzyme of the polyamine pathway, ornithine decarboxylase (Wallace et al., 2003). DFMO is a non-toxic drug that depletes polyamine content and slows the growth of cancer cells. It is therefore, an ideal drug to use in combination therapies. 3-Hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase inhibitors (statins) inhibit the ratelimiting step of the mevalonate pathway. Statins inhibit cell growth and cause cell death, making them ideal for cancer treatment. Dose-related myotoxicity has hindered progress and studies are now focusing on combination therapy with other anti-cancer agents (Graaf et al., 2004). The aim of this study was to determine whether a synergistic effect would be seen with combination treatment of polyamine depletion and simvastatin in human cancer cell lines. Cell lines used were; MCF-7, MDA-MB-231, PC3 and LNCaP. The lactone form of simvastatin was used

throughout. Sensitivity was measured using 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assays and various cell growth measurements. Apoptosis was measured by DAPI staining, polyamine content determination by dansylation and HPLC analysis. All cell lines tested were sensitive to increasing concentrations of simvastatin. Pre-treatment with both 1 mM and 5 mM DFMO appeared to have a protective effect against simvastatin treatment (Fig. 1) preventing cytotoxicity as determined by MTT assay. This effect was most pronounced in MDA-MB-231 cells and to a lesser extent in MCF-7 and PC3 cell lines. DFMO pre-treatment resulted in polyamine depletion and in inhibition of the growth of both breast and prostate cancer cells (result not shown). Pre-treatment with DFMO appeared to attenuate the actions of simvastatin. This suggests that statins have growth inhibitory activity only in proliferating cancer cells and not in cells whose growth is already compromised. References Graaf, M.R., Richel, D.J., van Noorden, C.J.F., Guchelaar, H., 2004. Cancer Treat. Rev. 30 (7), 609–641. Wallace, H.M., Fraser, A.V., Hughes, A., 2003. Biochem. J. 376 ( Pt 1), 1–14.