Bioreductive GDEPT using cytochrome P450 3A4 in ... - Nature

Cancer Gene Therapy (2003) 10, 40 – 48 D 2003 Nature Publishing Group All rights reserved 0929-1903 / 03 $25.00 www.nature.com / cgt

Bioreductive GDEPT using cytochrome P450 3A4 in combination with AQ4N Helen O McCarthy,1,a Anita Yakkundi,1,a Verna McErlane,1,a Ciara M Hughes,1 Gillian Keilty,1 Margaret Murray,1 Laurence H Patterson,2 David G Hirst,1 Stephanie R McKeown,1 and Tracy Robson1 1

Radiation Science Research Group, School of Biomedical Sciences, University of Ulster at Jordanstown, Newtownabbey, County Antrim, UK; and 2Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, University of London, London, UK.

The bioreductive drug, AQ4N, is metabolized under hypoxic conditions and has been shown to enhance the antitumor effects of radiation and chemotherapy drugs. We have investigated the role of cytochrome P450 3A4 ( CYP3A4 ) in increasing the metabolism of AQ4N using a gene - directed enzyme prodrug therapy ( GDEPT ) strategy. RIF - 1 murine tumor cells were transfected with a mammalian expression vector containing CYP3A4 cDNA. In vitro AQ4N metabolism, DNA damage, and clonogenic cell kill were assessed following exposure of transfected and parental control cells to AQ4N. The presence of exogenous CYP3A4 increased the metabolism of AQ4N and significantly enhanced the ability of the drug to cause DNA strand breaks and clonogenic cell death. Cotransfection of CYP reductase with CYP3A4 showed a small enhancement of the effect in the DNA damage assay only. A single injection of CYP3A4 into established RIF - 1 murine tumors increased the metabolism of AQ4N, and when used in combination with radiation, three of nine tumors were locally controlled for >60 days. This is the first demonstration that CYPs alone can be used in a GDEPT strategy for bioreduction of the cytotoxic prodrug, AQ4N. AQ4N is the only CYP - activated bioreductive agent in clinical trials. Combination with a GDEPT strategy may offer a further opportunity for targeting radiation - resistant and chemo - resistant hypoxic tumor cells. Cancer Gene Therapy ( 2003 ) 10, 40 – 48 doi:10.1038/sj.cgt.7700522 Keywords: GDEPT; cytochrome P450; AQ4N; radiation; bioreductive prodrug; CYP3A4

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rodrug activating systems operate on the principle that a nontoxic prodrug is converted to a cytotoxic product. Research indicates that prodrug systems, together with gene therapy, may provide the desired specificity for cancer treatment.1 Gene - directed enzyme prodrug therapy ( GDEPT ) delivers a gene that codes for an enzyme that is capable of converting a prodrug to its cytotoxic product.2 Several GDEPT strategies have been tested to date: herpes simplex virus thymidine kinase ( HSV-TK ) converts the nontoxic prodrug ganciclovir (GCV ) into a metabolite that kills replicating cells; cytosine deaminase (CD ), which converts 5- fluorocytosine (5 -FC ) to 5 -fluorouracil ( 5- FU ); nitroreductase (NTR ), which reduces the mustard prodrug, CB1954, to a toxic bifunctional alkylating agent; carboxypeptidase G2, which converts CMDA to a cytotoxic DNA cross - linking mustard and horseradish peroxidase that converts indole -3 -acetic acid to a number of toxic products ( reviewed in Ref. [ 1 ]). Cytochrome P450s (CYPs) have also been utilized. For example, the CYP2B6 isoform converts the Received August 8, 2002. Address correspondence and reprint requests to: Dr Tracy Robson, Radiation Science Research Group, School of Biomedical Sciences, University of Ulster, Newtownabbey, County Antrim BT37 0QB, Northern Ireland, UK. E - mail: [email protected] a These authors contributed equally to this manuscript.

prodrug cyclophosphamide (CPA ) to an alkylating toxic phosphoramide mustard that results in apoptosis.3 – 5 More recently, macrophages have been used to deliver a CYP2B6 / CPA gene combination to hypoxic regions of tumor.6 Also, a CYP2B6/ P450 reductase combination has been used to enhance the antitumor effects of CPA in combination with the bioreductive drug tirapazamine ( TPZ ).7 In the present study, we used a CYP isoform in a GDEPT strategy to metabolize the bioreductive prodrug AQ4N [1,4-bis[2( dimethylamino -N - oxide )ethyl]amino -5,8 -di -hydroxyanthracene -9,10 -dione ] to its cytotoxic product, AQ4. AQ4N prodrug activation displays a high selectivity towards hypoxic cells,8,9 which are a specific feature of tumors and are known to limit the success of conventional treatments.10 We have already demonstrated in vivo that AQ4N can significantly improve antitumor efficacy in combination with oxic cell cytotoxins such as radiation, CPA, and cisplatin.9,11 – 15 Clinical trials of AQ4N have now been initiated. CYP activation of AQ4N as a GDEPT strategy has several advantages over other GDEPT strategies currently in clinical trial. Firstly, AQ4N does not require actively replicating cells for activation unlike GCV; AQ4N targets hypoxic cells, which occur almost exclusively in solid tumors. Hypoxic cells are resistant to many of the current GDEPT strategies mentioned above; activation of CPA is an oxygen -dependent

Bioreductive GDEPT utilising CYP3A4 and AQ4N HO McCarthy et al

41 reaction and there is a loss of cytotoxicity of 5 -FU and CD / 5 -FC in hypoxic tumor cells.1 Furthermore, in other systems, the activated prodrug requires further metabolism by endogenous enzymes ( e.g., HSV- TK, CD / 5- FC, and NTR / CB1954 ) and, in many cases, the active drugs are not membrane - permeable and depend on cell -to -cell contact for transfer into neighbouring cells to induce bystander effects ( HSV /TK ). AQ4N does not have any of these drawbacks, making it ideally suited for use in combination with an oxic cell killer such as radiation or CPA, providing targeted drug activation in the normally radio - and chemo -resistant hypoxic cells. Furthermore, there is already evidence that an AQ4N - mediated bystander effect will be independent of gap junctions as the toxic prodrug appears to move by passive diffusion in cellular systems observed by confocal microscopy.16 AQ4N is reduced under hypoxic conditions to its toxic product, AQ4, by a process that is inhibited by molecular oxygen.17 AQ4N does not bind to DNA, but AQ4 has a high DNA affinity and is a potent topoisomerase II inhibitor.16,18 Studies using ketoconazole and carbon monoxide, global inhibitors of the CYP superfamily, implicate CYP enzymes in the metabolism of AQ4N to AQ4 in rat liver microsomes.19 Furthermore, AQ4N metabolism is correlated with CYP3A activity as determined using phenotyped human liver microsomes.20 It follows that for AQ4N to be successful in the clinic, both hypoxia and the correct CYP will need to be present in the tumor. Clearly, tumors with high levels of hypoxia would be appropriate for treatment with AQ4N. The rationale behind this investigation is that a significant enhancement of AQ4N activation in tumors may be achieved using CYP -mediated GDEPT. This is especially relevant in those tumors where the appropriate natural CYP isoform expression required for AQ4N activation is either absent or present at low levels. This study investigated a novel gene therapy strategy utilizing the human cytochrome 3A4 gene to activate the bioreductive prodrug AQ4N in tumor cells. In addition, the role of P450 reductase was assessed, as it has previously been described as a rate - limiting component of P450dependent drug activation.21,22

Materials and methods Cell culture

RIF - 1 murine fibrosarcomas were routinely maintained by the protocol of Twentyman et al.23 In culture, cells were maintained in RPMI 1640 with L -glutamine (10 mM ) ( Gibco BRL, Paisley, UK ) supplemented with fetal bovine serum (15% ) (Globe Pharm, Surrey, UK ) and penicillin / streptomycin (1% ) (Sigma, Poole, UK ). Cells were grown as monolayers in tissue culture flasks at 378C under 5% CO2 /95% air and passaged every 3 – 4 days to maintain exponential growth. Transgene constructs

The cytochrome P450 3A4 (CYP3A4 ) cDNA was a kind gift from Dr J Horbach (Research Institute of Toxicology,

Utrecht, The Netherlands ). CYP3A4 was excised from the original construct and directionally cloned into the mammalian expression vector, pcDNA 3.1 (Invitrogen, Groningen, NL ), using NotI and HindIII linkers. CYPRed was provided by Prof CR Wolf (University of Dundee, UK ) and also directionally cloned into the pcDNA 3.1 vector using HindIII and XbaI linkers. Stable transfections of RIF - 1 tumor cells

RIF- 1 cells were plated at 8105 per 60 -mm Petri dish ( Nunc, Paisley, UK ). After 24 hours, 8 g of CYP 3A4 was mixed with 39 L of calcium phosphate maximizer solution ( Clontech, Oxford, UK ) and 32 L of 12 M calcium solution and brought to a total volume of 260 L with sterile water. The solution was subsequently added drop wise to an equal volume of 2 HBS buffer (provided in the kit). After 15 -minute incubation at room temperature, the solution was added to the cells. Cells were incubated at 378C under 5% CO2 /95% air for 4 hours. The calcium phosphate and medium were removed, cells washed in PBS, and fresh medium added. After 36 hours, the cells were trypsinized, seeded 1:5 into 60- mm Petri dishes, and placed under selective pressure by addition of geneticin G418 ( 600 g /mL) (Sigma ). An antibiotic -resistant CYP3A4 -overexpressing clone was selected for in vitro metabolism studies with AQ4N. Transient transfections of RIF - 1 tumor cells

RIF- 1 cells were plated at 1105 per flask ( T25 ) ( Nunc ). After 24 hours, the cells were transfected with 12 g of CYP3A4 and CYPRed constructs using Lipofectamine Plus ( Gibco BRL ) according to the manufacturer’s instructions. The controls included parental nontransfected cells and cells treated with lipofectamine alone. After 24 hours, the cells were harvested for use in the comet or clonogenic assays. In control experiments using the pEGFP reporter gene construct ( Clontech), we were able to obtain transfection efficiencies of 35% (data not shown). Determination of AQ4N - induced DNA damage using the comet assay

At 24 hours after transfection, the RIF -1 cells were incubated under anoxic conditions ( 95% N2 /5% CO2 ) for 30 minutes prior to addition of 20 M AQ4N. Cells were consistently undamaged under oxic ( 95% air /5% CO2 ) conditions for all groups (data not shown). The controls included parental control with and without drug, empty vector transfected cells, and lipofectamine alone. Following a 90 -minute incubation with AQ4N, the drug was removed and the cells washed in PBS and then a single cell suspension was prepared. The alkaline single cell gel (comet ) electrophoresis assay24 was used to assess DNA damage in transfected RIF- 1 cells at 0 and 24 hours after drug treatment. Frosted microscope slides ( Labcraft, Dakin, UK ) were coated with 90 L of 0.6% normal melting point ( NMP ) agarose and allowed to solidify under a coverslip. Fifty microliters of cell suspension ( 1105 ) in PBS was added to 50 L of 1.2% low melting point ( LMP ) agarose

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42 and gently mixed; this was immediately pipetted on top of the NMP layer and allowed to harden on ice with coverslips. Coverslips were removed and the slides were immersed in lysing solution ( 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10, and 1% Triton X - 100) at 48C in the dark for at least 1 hour. Slides were then placed in electrophoresis buffer ( 300 mM NaOH and 1 mM Na2EDTA ) for 20 minutes. Horizontal gel electrophoresis was performed for 20 minutes at 0.83 V / cm ( 25 V, 300 mA). Slides were then washed three times in neutralization buffer (0.4 M Tris, pH 7.5 ) for 15 minutes, stained with 40 L of ethidium bromide ( 20 g/ mL), and analysed immediately. Comets were analysed under a 40 objective Olympus BH - 2 epifluorescence microscope equipped with a 100 -W mercury lamp, a 515 - to 560- nm excitation filter, and a 590 -nm barrier filter. The microscope was attached to a Pulnix MT765 intensified camera and a Hewlett Packard PC486 /33 u. DNA was assessed using the ‘‘tail moment’’ parameter defined as the product of the percentage DNA in the tail and the tail length. Tail moment was found to be the most reliable indicator for measuring DNA damage.25 The mean tail moment was calculated by evaluating 50 cells from each of the three slides for each treatment group. Each experiment was repeated three times. The data were checked for normality and a three - way ANOVA was performed with mean tail moment as the y -variable and time, RIF - 1 transfectant, and experiment replicate as factors. Determination of AQ4N induced cell kill using the in vitro clonogenic assay

The in vitro clonogenic assay was used to determine clonogenic cell kill following treatment with AQ4N. Twentyfour hours following transfection, RIF -1 cells were plated in triplicate into six -well plates (Sarsted) with increasing cell numbers plated for higher drug doses; 200 cells / well were plated for the lower drug doses and 2000 cells /well for the higher drug doses. Cells were allowed to attach at 378C under 5% CO2 / 95% air for 4 hours and then incubated for 30 minutes in either oxic (95% air /5% CO2 ) or anoxic ( 95% N2 / 5% CO2 ) conditions. The cells were then treated with AQ4N ( 0– 12 M) for 90 minutes in either anoxic or oxic conditions. The drug was aspirated off, the cells washed twice in PBS, fresh media added, and the plates incubated at 378C under 5% CO2 /95% air to allow colony formation. Fourteen days later, colonies were fixed and stained with 0.4% crystal violet solution dissolved in 70% methanol and counted. The plating efficiencies (PE ± SE ) were as follows: Parental Control (oxic ) =0.46 ( ± 0.032 ); Lipofectamine ( oxic ) = 0.41 ( ±0.035); CYPReductase ( oxic ) =0.35 ( ± 0.032); CYP3A4 (oxic )= 0.28 ( ± 0.025 ); CYP3A4 /Red ( oxic ) = 0.35 ( ± 0.033 ); Parental Control (anoxic )= 0.33 ( ±0.030); Lipofectamine (anoxic )= 0.35 ( ± 0.029 ); CYPReductase ( anoxic )= 0.28( ± 0.023 );CYP3A4( anoxic )= 0.38( ± 0.029 ); CYP3A4 /Red ( anoxic )= 0.33 ( ± 0.034 ). Three independent experiments were performed. The data were arcsintransformed to improve normality and a three - way ANOVA test was performed. Surviving fraction was the y -variable with drug concentration, RIF - 1 treatment, group and oxic /anoxic status as factors.

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Preparation of S9 fraction from CYP3A4 transfectants

These fractions consist of cytosolic and microsomal enzymes. In vitro CYP3A4 RIF -1 transfectants were scraped into ice -cold homogenization buffer ( 0.25 M sucrose, 0.01 M Tris – HCl, 15% glycerol at pH 7.4) with 0.5% protease inhibitor cocktail ( Protease Inhibitor Cocktail Set III; Calbiochem, San Diego, USA ). Samples were then homogenized at low frequency, centrifuged at 9000g at 48C for 20 minutes. The resultant supernatant was assayed for protein content using the Bradford method (Bio -Rad Protein Assay; Biorad Laboratories, Herts, UK ) and stored at 708C until they were assayed. Metabolism of AQ4N by the S9 fraction of CYP3A4 transfectants

Anoxic incubations were performed in an airtight perspex glovebox at 378C. All reagents were purged of oxygen by bubbling oxygen -free nitrogen through them and an anoxic environment was confirmed using an O2 electrode (Hanna H1 9143 microprocessor dissolved O2 meter ). Each reaction mix consisted of 15 mM NADPH, 50 M AQ4N, and 0.1 M potassium phosphate buffer (pH 7.4 ). Incubates were prewarmed for 5 minutes and the reaction started with the addition of S9 fraction prepared from in vitro cells (10 or 5 mg / mL protein ). The reaction was quenched at various time points by the addition of an equal volume of ice cold 80:20 ammonium formate buffer (0.5 M, pH 2.5) and acetonitrile. Detection and quantification of AQ4N and its metabolites by HPLC

Samples were centrifuged at 13,000 rpm for 10 min. The supernatant was collected and the protein pellet resuspended in 50 L of 80% ammonium formate ( 0.5 M pH 4.2):20% acetonitrile. This procedure was repeated twice and supernatants were pooled and analysed using HPLC essentially as previously described.26 In vivo transgene delivery in RIF - 1 murine tumors and assessment of AQ4N induced tumor growth delay when used in combination with radiation

A total of 2105 RIF - 1 tumor cells were injected intradermally into the rear dorsum of lightly anesthesized 8- to 10- week -old C3H mice. Established tumors (150– 225 V3 ) were injected with 25 g of CYP3A4/ liposomal mix ( Mirus polymer, TransIT1 in vivo gene delivery kit; Clontech, Cambridge, UK ). Twenty -four hours later, mice were injected intraperitoneally with 100 mg /kg AQ4N and then tumors were irradiated 30 minutes later with 20 -Gy X -rays. For irradiation, mice were immobilized in lead jigs with the dorsal tumor exposed to the radiation beam. X - rays were delivered using a Pantak 250- kV X -ray machine. Tumors were measured three times weekly and the volume quadrupling time (VQT ) was used as the end point unless otherwise stated; all animals were killed when the tumors reached their VQT. Animal experiments were carried out in accordance with the Animals (Scientific Procedures) Act 1986. It should be noted that Mirus was selected as the


43 delivery agent in this study based on a preliminary experiment using a GFP reporter construct. Following direct intratumoral injection, transgene expression was seen to be most widely distributed in the tumor when this agent was used, as compared to other liposomal delivery agents. Detection of CYP3A4 protein levels in injected tumors using Western blot analysis

The animals were killed 24 hours after injection of the tumor with the CYP3A4 construct. The tumors were removed and homogenized in PBS using a sonicator (MSE, Amersham, Bucks, UK ). Protein concentration was measured as above. Thirty micrograms of each sample in 1 Laemlii buffer (Sigma, UK ) was electrophoresed through a 10% SDS polyacrylamide gel, transferred to a nitrocellulose membrane (Hybond C; Amersham, Bucks, UK ), and probed with a rabbit monoclonal antihuman CYP3A4 antibody (Gentest, Cambridge Bioscience, Cambridge, UK). Levels of gene expression were assessed using an ECL detection kit and horseradish peroxidase – conjugated anti -rabbit antibody ( Amersham, UK ). Blots were assessed for loading using a b - actin antibody ( Sigma, UK ). Results In vitro metabolism of AQ4N in cell lines stably expressing CYP3A4

RIF - 1 clones stably transfected with the CYP3A4 gene and resistant to geneticin ( G418 ) were isolated. The ability of the CYP3A4- overexpressing cells to increase AQ4N metabolism compared to nontransfected parental control cells was confirmed by in vitro metabolism studies (Fig 1). AQ4N and

Figure 2 DNA damage ( mean tail moment ) assessed using the alkaline comet assay in parental nontransfected RIF - 1 cells, a range of experimental controls, and cells transiently transfected with CYP3A4, CYP3A4 / CYPRed. Cells were assessed for DNA damage after exposure of all groups to 20 M AQ4N under anoxia / hypoxia ( except one of the parental control groups ) ( a ) immediately and ( b ) 24 hours later. The results are the mean ± SE of three independent experiments.

50

its metabolites were quantified using HPLC analysis. The rate of AQ4N metabolism was calculated from the initial velocity of the substrate concentration /time plots. The rate of metabolic reduction of AQ4N to AQ4 for parental RIF- 1 cells was 229.12 nM /min /mg S9 protein and for CYP3A4 transfected RIF - 1 cells was 518.16 nM /min / mg S9 protein. This represents a 2.3- fold increase in AQ4N metabolism in CYP3A4 stably transfected cells when compared to controls. Unfortunately, further experiments using the stably transfected CYP3A4 cell line were precluded as CYP3A4 activity was rapidly silenced and unstable in vivo. For the remainder of the study, transiently transfected cells were used.

AQ4N Conc ( M)

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AQ4N induced DNA damage in cells transiently transfected with CYP3A4

RIF-1 parental control cells CYP3A4 RIF-1 stable transfectants

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Time (min) Figure 1 In vitro metabolism of AQ4N in S9 protein ( 10 mg / mL ) fractions prepared from stable CYP3A4 - transfected and parental RIF - 1 cells. AQ4N loss over time was determined by HPLC.

Using the alkaline comet assay, a minimal amount of DNA damage was observed immediately following treatment with AQ4N ( 20 M ), under hypoxia ( Fig 2a ), with no discernible difference between RIF - 1 parental controls and cells transiently transfected with CYP3A4. However, 24 hours after treatment with AQ4N, under hypoxia, significantly increased DNA damage was evident in all cells transiently transfected with CYP3A4 compared to nontransfected controls ( Fig 2b ) (three - way ANOVA; df =6, F

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44 ratio = 4.9125, P= .0012 ). CYP3A4 -transfected cells showed a mean tail moment of 5.86, whereas those cells cotransfected with CYP3A4 /CYPRed had a mean tail moment of 8.17. This would suggest that cotransfection of CYP3A4 /CYPRed results in greater DNA damage than CYP3A4 alone in AQ4N - treated RIF -1 cells. Overall, when compared to con-

A

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Control 20Gy X-rays 20Gy X-rays+AQ4N+ Liposomes 20Gy X-rays+AQ4N+ Liposomes +empty vector 20Gy X-rays+AQ4N+ Liposomes+CYP 3A4

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trols at 24 hours, the DNA damage was increased 2 -fold in CYP3A4 transfectants and 2.5 -fold in cells cotransfected with CYP3A4/ CYPRed. No significant differences occurred among the results obtained from the three experimental replicates (df =2, F ratio = 0.0554, P= .9462 ).

0.100

parental control lipofectamine alone CYPRed alone CYP3A4 alone CYP3A4/CYPRed

0.010 Concentration (µM) Figure 3 Surviving fraction in cells transiently transfected with CYP3A4, CYP3A4 / CYPRed, and various controls following treatment with AQ4N ( 0 – 12 M ) ( A ) under oxic ( 95% air / 5% CO2 ) conditions and ( B ) anoxic ( 95% N2 / 5% CO2 ) conditions. Surviving fraction was assessed using a clonogenic assay. The results are the mean ± SE of three independent experiments.

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Figure 4 Tumor growth in RIF - 1 murine tumors in vivo following injection of CYP3A4 in combination with AQ4N and radiation. A: Kaplan - Meier survival curve showing the percent of mice with tumors reaching their VQT at various times following injection with CYP3A4 / liposomal mix and exposed to AQ4N ( 100 mg / kg ) and radiation ( 20 Gy ) 24 hours later. Controls included mice with no tumor treatment, mice with tumors exposed to X - rays alone, mice with tumors exposed to X - rays in combination with AQ4N and injected with liposomal agent only, and mice with tumors exposed to X - rays in combination with AQ4N and injected with liposomal agent and empty vector. Each symbol represents a different animal. There were five to nine animals in each group ( see Table 1 ). B: Photograph of two mice on day 20 of the experiment. Both mice 1 and 2 were treated with AQ4N and radiation; however, mouse 1 was also injected with the CYP3A4 construct.

Clonogenic survival in cells transiently transfected with CYP3A4

There was a clear difference in clonogenic cell kill between oxic (Fig 3A ) and anoxic ( Fig 3B ) conditions, confirming previous reports that AQ4N is metabolized only under hypoxic /anoxic conditions7,17 (three -way ANOVA; df = 1, F ratio = 58.8834, P < .0001 ). AQ4N drug concentration also had a significant effect on surviving fraction (df =7, F ratio= 58.8834, P < .0001 ). In oxic conditions, AQ4N caused little cytotoxicity; however, under anoxic conditions, there was a significant decrease in cell survival with increasing AQ4N concentration. Under anoxic conditions, cells transiently transfected with CYP3A4 or CYP3A4 / CYPRed exhibited a marked reduction (by a factor of 10 )


45 in colony survival compared to nontransfected and lipofectamine controls (df =1, F ratio = 19.7845, P 53 days. Three tumors in this group never reached their VQT (indicated by the arrows; Fig 4A ); the original injected tumors shrank completely to the point where they were not detectable by gross examination, but the mice had to be killed because of metastasis to other sites. Fig 4B shows a photograph of two mice on day 20 of the experiment. It is clear that the tumor injected with CYP3A4 (mouse 1) has shrunk to nondetectable levels, whereas the tumor treated with AQ4N and radiation alone is about 1000 mm3. Detection of CYP3A4 protein levels in injected tumors using Western blot analysis

Levels of CYP3A4 protein were increased 24 hours following injection of the CYP3A4 construct into the RIF1 tumor, compared to control noninjected tumors as assessed by Western blot analysis (Fig 5).

Table 1 Time ( days ) for RIF - 1 tumors to reach their VQT Treatment Control 20 - Gy X - rays 20 - Gy X - rays + AQ4N + liposomes 20 - Gy X - rays + AQ4N + liposomes / empty vector 20 - Gy X - rays + AQ4N + liposomes / CYP3A4

Mean VQT ( days )

SEM

7.85 30.32 34.50

1.104 ( n = 6 ) 3.830 ( n = 9 ) 3.830 ( n = 5 )

41.30

2.735 ( n = 7 )

> 53.30*

2.480 ( n = 9 )

*P < .024 compared to the empty vector group receiving AQ4N, radiation, and liposomes. It should be noted that the value is an underestimate of the mean as three tumors were cured and, therefore, failed to reach their VQT before mice were sacrificed due to disseminated disease.

Figure 5 Levels of CYP3A4 protein in control and CYP3A4 - injected RIF - 1 tumors. Western blot showing CYP3A4 levels ( top panel ) in established control and treated RIF - 1 tumors, 24 hours following a single injection of CYP3A4. Protein loading was assessed using a b - actin antibody ( lower panel ).

Discussion

GDEPT strategies using CYP450s have been shown to be highly effective for targeting prodrugs to tumor cells. The CYPs 2B1, 2C6, and 2C11 have been shown to be involved in the activation of CPA,27 and CYP3A has been shown to activate ifosphamide.28 In addition, tumors overexpressing CYP2B6 in vivo have been shown to increase the metabolism of CPA.3,4,21 When CYP reductase was also overexpressed, increased antitumor efficacy was observed following administration of both CPA (presumed to be metabolized by CYP2B6) and TPZ (presumed to be metabolized by CYP reductase ).7 In this study, we aimed to increase the metabolism of the bioreductively activated drug, AQ4N, which targets hypoxic cells. The evidence for hypoxic regions in tumors is now generally well accepted.29 These hypoxic cells are a significant problem in the control of solid tumors because they are two to three times more resistant to radiotherapy and are less sensitive to most chemotherapeutic agents.29 The concept of metabolic activation of a drug to a cytotoxic agent within the hypoxic environment makes direct use of this property to target treatment- resistant hypoxic cells. As AQ4N requires both the appropriate CYP and hypoxia for its activation, the expression of this transgene in normal tissue should not cause systemic toxicity. The data described here using oxic conditions support this hypothesis.

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46 Generally, CYP activity is low or even absent in cells cultured in vitro.30 Previously, this has led to difficulties in unequivocally establishing the potential of AQ4N as a metabolically activated antitumor agent.18 Several studies have demonstrated indirectly the importance of CYPs in the bioreductive activation and cytotoxicity of AQ4N.18 – 20 However, this study has, for the first time, allowed effective assessment of CYP -mediated AQ4N cytotoxicity in vitro and in vivo using delivery of specific CYPs by molecular approaches. Our results show that stable transfection of CYP3A4 significantly increases AQ4N metabolism compared to the parental cell line. This supports previous inhibitor studies, which show that AQ4N metabolism correlates with CYP3A activity.19,20 Interestingly, the in vitro metabolism study demonstrated that the parental cell line was also able to metabolize AQ4N, although to a much lesser extent than the stable transfectant. As CYP activity is known to be down - regulated in vitro, it is possible that this activity may be due to metabolism by other endogenous enzymes. The stable CYP3A4 transfectant was found to lose CYP activity within a few weeks — a phenomenon not uncommon with cells stably transfected with CYPs.22 We do not know the mechanism by which this activity was lost, but other evidence in our laboratory suggests that it is not a mechanism involving gene regulation, rather that enzyme activity is decreased. The loss of CYP activity in the stable transfectant made it difficult to perform in vitro studies; this was not perceived to be a problem in vivo because CYPs activity is stable in vivo. Our subsequent in vivo experiments show that CYP activity was effective following direct injection. For all subsequent in vitro experiments, transient transfections were performed. There was a significant increase in DNA damage in CYP3A4- transfected cells in comparison to parental control cells, 24 hours after treatment with AQ4N (Fig 2b ); importantly, this was observed only in hypoxic/ anoxic conditions. This confirms the requirement for hypoxia to allow CYP - mediated AQ4N reduction to proceed. A further increase in damage could be achieved by cotransfection with CYPRed. Single strand breaks, as measured using the alkaline comet assay, have previously been shown to be a mechanism of AQ4N -induced cell death.31 It is likely that the DNA strand breaks observed are indicative of topoisomerase inhibition by AQ4, produced following reduction of AQ4N through a CYP -mediated process.32 Therefore, the increase in DNA damage found in the cells transfected with CYP3A4 implicates this isoform in AQ4N activation leading to tumor cell kill. No increase in DNA damage ( tail moment < 2 ) was evident in cells exposed to AQ4N for 90 minutes ( in anoxia /hypoxia ) and immediately assessed using the comet assay (Fig 2a ). This supports previous work, which showed that AQ4 does not cause strand breaks per se, but compromises the ability of cells to proceed through the cell cycle.31 This is evidenced in the comet assay by increasing numbers of damaged cells with time because the binding of AQ4 to DNA /topoisomerase II results in DNA damage /cell death. This is consistent with the bioreductive mechanism of activation proposed by Patterson.18 DNA damage also correlated with clonogenic cell kill. Cells transiently transfected with CYP3A4 showed signifi-

Cancer Gene Therapy

cantly reduced clonogenicity as compared to parental controls. Furthermore, the fraction of cells killed was greater than the fraction of cells transfected. Using a GFP reporter gene, we were able to measure transfection efficiencies of 25 –35% (data not shown ), and yet the cell kill observed using this assay was greater than 97%. This phenomenon may be explained by a bystander effect. Further studies are now underway to verify this. In other GDEPT strategies, transfection efficiencies as low as 4– 10% in solid tumors resulted in eradication.33 A number of studies suggest that tumor tissues down - regulate intercellular GAP junctions, adversely affecting the bystander effect of some agents, e.g., GCV.34,35 However, there is evidence that an AQ4 -mediated bystander effect may occur independently of gap junctions by passive transportation in cellular systems.36 – 38 Other studies support a role for CYPRed in the activation of prodrugs through a CYP - mediated mechanism. For example, cotransfection with CYPRed decreased the surviving fraction of CYP2B1 transfected 9L cells in vivo by a factor of 10.21 In our study, the metabolism of AQ4N through CYP3A4 had only a slight dependence in the presence of cotransfected CYPRed; DNA damage induction was only slightly increased in CYP3A4 /CYPRed – transfected cells and there was no difference in clonogenicity in cells containing both CYP and CYPRed. It may be that the levels of endogenous CYP reductase are sufficient in the RIF- 1 tumor cell line, although evidence for this is only circumstantial. In vivo growth delay studies in the RIF - 1 murine tumor look promising. Clearly, the data show that a single CYP3A4 injection using a simple nonoptimized approach can increase the metabolism of AQ4N and, when used in combination with radiation, three of nine tumors are locally controlled for >60 days. This implies that the bioreduction of AQ4N by CYPs in this tumor system is suboptimal and this strategy could, therefore, be very promising for clinical use where CYP levels are known to be variable. As CYP3A4/ AQ4N / radiation treatment provided complete control in three of nine tumors, a true growth delay estimation was impossible; the animals had to be killed because of distant metastasis. This is often seen in studies where extensive cell kill occurs in the primary tumor. The use of repeated transgene injections or injections at multiple sites along with clinically fractionated radiotherapy plus AQ4N treatment might enhance this GDEPT strategy further. For any drug to be a candidate for a GDEPT strategy, there must be a significant therapeutic gain. Ideally, the activated drug should be at least 100- fold more toxic than the prodrug and have the ability to diffuse to surrounding cells.1 This was apparent in this study. Furthermore, AQ4N has the advantage of being selectively activated under hypoxic /anoxic conditions, whereas its active metabolite can diffuse to adjacent oxygenated tumor cells, affording a bystander effect. Importantly, tumor selectivity should be maintained because AQ4N should not be activated to its toxic metabolite in well - oxygenated nontumor tissues that express CYPs. In conclusion, this study demonstrates that CYPs can be used in a GDEPT strategy for bioreduction of the cytotoxic prodrug, AQ4N. AQ4N is the only


CYP -activated bioreductive agent in clinical trials and, as such, may offer a clinical opportunity for GDEPT in the future.

Acknowledgment

We would like to thank the Cancer Research, UK (Grant no. SP2423/ 0101 ), for supporting this research.

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