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sDetermination of the in vivo pharmacokinetics ofpalladium-bacteriopheophorbide (WST09) in EMT6 tumour-bearingBalb/c mice using graphite furnace atomic absorption spectroscopy

Pierre Herve Brun,a Jennifer L. DeGroot,b Eva F. Gudgin Dickson,*b Mohsen Farahanib andRoy H. Pottierb

a Steba Biotech, Avenue de l’Europe, Toussus-le-Noble, Magny-les-Hameaux, Cedex,France 78771

b Department of Chemistry and Chemical Engineering, The Royal Military College of Canada,PO Box 17000, Station Forces, Kingston, Ontario, Canada K7K 7B4.E-mail: dickson-e@rmc.ca; Fax: (613)542-9489; Tel: (613)541-6000 ext. 6360

Received 8th March 2004, Accepted 8th September 2004First published as an Advance Article on the web 30th September 2004

Palladium-bacteriopheophorbide (WST09), a novel bacteriochlorophyll derivative, is currently being investigated foruse as a photodynamic therapy (PDT) drug due to its strong absorption in the near-infrared region and its ability toefficiently generate singlet oxygen when irradiated. In this study, we determined the pharmacokinetics and tissuedistribution of WST09 in female EMT6 tumour-bearing Balb/c mice in order to determine if selective accumulationof this drug occurs in tumour tissue. A total of 41 mice were administered WST09 by bolus injection into the tail veinat a dose level of 5.0 ± 0.8 mg kg−1. Three to six mice were sacrificed at each of 0.08, 0.25, 0.5, 1.0, 3.0, 6.0, 9.0, 12, 24,48, 72, and 96 h post injection, and an additional three control mice were sacrificed without having been administeredWST09. Terminal blood samples as well as liver, skin, muscle, kidney and tumour samples were obtained from eachmouse and analyzed for palladium content (from WST09) using graphite furnace atomic absorption spectroscopy(GFAAS). The representative concentration of WST09 in the plasma and tissues was then calculated. Biphasickinetics were observed in the plasma, kidney, and liver with clearance from each of these tissues being relatively rapid.Skin, muscle and tumour did not show any significant accumulation at all time points investigated. No selective drugaccumulation was seen in the tumour and normal tissues, relative to plasma. Thus the results of this study indicatethat vascular targeting resulting from WST09 in the circulation, as opposed to selective WST09 accumulation intumour tissues, may be responsible for PDT effects in tumours that have been observed in other WST09 studies.

1 IntroductionBacteriochlorophyll-based derivatives are currently being stud-ied as photochemotherapeutic agents, mainly due to their highmolar extinction coefficients in the 760 to 780 nm regionof the electromagnetic spectrum.1–3 The bacteriochlorophyll-based compounds have further benefits of low toxicity and fastclearance from tissues. This results in little to no risk of darktoxicity or lengthy periods of post treatment photosensitivity,a factor which can be a drawback with Photofrin R© basedphotodynamic therapy (PDT).

Palladium bacteriopheophorbide (Fig. 1), code namedWST09, is a novel bacteriochlorophyll derivative that is currentlyin phase II clinical trials for the treatment of recurrent prostatecancer.

To date, investigations concerning the efficacy of WST09-based PDT in in vivo tumour therapy have involved irradiationduring or immediately following intravenous injection of theWST09 chromophore,4,5 an approach that targets WST09 inthe circulation rather than that which might accumulate intumour tissues at times well after administration such asoccurs with other approved PDT drugs.6–8 Results on WST09have shown high cure rates of mice with prostate small cellcarcinoma xenografts,9 and PDT treatment data collected so farprovide preliminary indications that vasculature targeted PDTis effective with this drug.10–12 However the pharmacokinetics ofWST09 accumulation and clearance have yet to be reported inan animal tumour model.

There is mounting evidence that direct cell kill only accountsfor about 1 to 2 logs of direct tumour-cell killing,13 and thatthe required 7 to 8 logs of cell kill required for a tumour cure

Fig. 1 Chemical structure of WST09.

can mostly be related to a microvasculature target as the initialsite of injury. As a result, knowledge of the time dependenttissue and plasma distribution of WST09 in a tumour-bearingmodel can elucidate further the mechanism of PDT action ofthis compound. In addition, knowledge of the pharmacokineticbehaviour of this compound provides valuable informationD

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pertaining to its potential to elicit skin photosensitivity orsystemic toxicity.

In the current study we have determined the in vivo phar-macokinetics and tissue distribution of WST09 in an EMT6tumour-bearing Balb/c mouse model after a single intravenousinjection. The analysis was carried out using a graphite furnaceatomic absorption spectroscopy (GFAAS) method in order toselectively detect the Pd in WST09. This is a complementarymethod to those reported previously for monitoring biologicalsamples containing Pd-organometallics.14–16

2 Materials and methods2.1 Materials and injection formulation

Palladium-bacteriopheophorbide (WST09) was obtained bothin powder form and as a 5 mg mL−1 injection formulation inan alcohol/Cremophor EL R©/NaOH based proprietary vehiclefrom Steba Biotech (Toussus-le-Noble, France). Stock solutionwas diluted in the solvent in order to obtain a WST09 concen-tration of 0.5 mg mL−1 suitable for injection into the mice. Thestock and diluted solutions were protected from light at all times;the dilutions were performed in dim light conditions and allsolution containers were wrapped in aluminum foil. The dilutedconcentrations were verified by measuring the absorbance ofWST09 at 758 nm.

All solvents were commercially available spectroscopic orHPLC grade. Solvable R© digestion solution was obtained fromCanberra-Packard. Cremophor EL R© was obtained from Sigma.

2.2 Animal model

A total of 44 female Balb/c mice were obtained from CharlesRiver, Canada. At the time of injection the mice were between6 and 16 weeks old and had an average body mass of 19.8 ±1.6 g. Each mouse carried an EMT6 mammary adenocarcinomatumour (NCI Frederick Cancer Research and DevelopmentCenter CDT Tumour Repository). This animal/tumour modelhas been shown in our laboratory to selectively accumulate otherPd-containing drugs suitable for photoactivation.14 The tumourswere maintained by serial transplantation of homogenizedtumour tissue subdermally into the right flank 3 to 15 days priorto WST09 administration. Under these conditions, the tumoursstudied were of roughly equal size, with an average weight atthe time of sacrifice of 100 to 150 mg. Animals were maintainedon an ad libitum diet of Agribrands Purina Rodent Chow 5001and tap water. Animal care was performed in accordance withthe guidelines set forth by both the Queens University AnimalCare Committee and the Canadian Council for Animal Care.All procedures as well as the experimental protocol were peer-reviewed and approved by the Queens University Animal CareCommittee prior to the commencement of this study.

2.3 Absorption spectroscopy

The UV-Visible spectra of WST09 dissolved in Solvable R©, orprepared in injection formulation as described below, wererecorded on a UV-Vis spectrophotometer (Shimadzu UV-160,Kyoto, Japan).

2.4 Tissue injection and extraction procedure

The photosensitizer drug was introduced in the mice via bolusinjections of the WST09 formulation into the tail vein at a doselevel of 5.0 ±1.0 mg kg−1. The mice were kept in the dark,with food and water ad libitum, until the time of sacrifice. Priorto sacrifice the back of each mouse was depilated using Nair R©

Lotion Hair Remover (Carter Horner Corp., Mississauga, Ont.,Canada) in order to provide a hairless skin sample; animals wererinsed thoroughly in warm water in order to ensure that all theNair R© lotion was removed. Three to four mice (six in the caseof the 1 and 48 h time points) were sacrificed by euthanizing

with natural gas (propane) at each of 0.08, 0.25, 0.5, 1.0, 3.0,6.0, 9.0, 12, 24, 48, 72, and 96 h post injection. An additionalthree mice were sacrificed without having been administeredWST09 in order to provide WST09-free baseline values. Termi-nal blood samples were obtained via cardiac puncture usinga syringe coated with 0.1 mL of 3.2% (0.105 M) bufferedsodium citrate solution (obtained from a sterile Vacutainer R©

tube, Becton-Dickinson Vacutainer Systems). The volume ofthe blood obtained in each case was noted and the sampleswere centrifuged for 10 minutes at 2500 rpm at 4 ◦C. 100 lL ofplasma was then added to 1 mL of Solvable R©. Samples of liver,kidney, depilated skin, leg adductor muscle, and tumour wereobtained by dissection and rinsed in sterile saline. Accuratelyweighed samples of each tissue were added to Solvable R© in avolume ratio of 2 mL Solvable R© per 100 mg of tissue and these,as well as the plasma samples, were placed in a 55 ◦C oven andallowed to digest for 24 h. After digestion, the samples werestored in the dark at room temperature until analysis. The entireanalysis procedure was carried out under subdued light in orderto prevent photodegradation of the WST09.

2.5 Calibration standards

Calibration standards were made by dissolving WST09 inSolvable R© to obtain a standard solution whose concentrationwas verified by UV-Vis spectroscopy. 2 mL calibration standardswere then made by adding 100 mg of chicken breast (storedfrozen and thawed just prior to use) to appropriate volumes ofstandard solution and Solvable R©. The standards were placedin a 55 ◦C lab oven for 24 h in order to allow for completetissue digestion. Calibration standards were used to constructcalibration plots during GFAAS analysis. Since tissue causes alight scattering effect, the inclusion of tissue in the calibrationstandards was deemed necessary. The use of chicken breast tomimic the scattering effect of mouse tissues has been previouslyvalidated.15

2.6 Sample stability

In order to check stability, the standards were analysed forpalladium content on days 1, 2, 3, and 7 post preparation.Standards were stored in the dark, at room temperature forthe duration of the study. Absorbance peak area of palladiumin solutions of 50 mg chicken tissue per mL of Solvable R© wasfound to vary linearly with Pd concentration in the region of 5 to95 ng mL−1 and the solutions as well as the instrument readingswere found to be stable for up to 7 days. A calibration curve wasobtained each day sample analysis was performed.

2.7 GFAAS analysis

Calibration standards and tissue samples were analyzed us-ing a Unicam 939 Graphite Furnace Atomic AbsorptionSpectrometer with a Unicam 247.6 nm (palladium atomicabsorption line) hollow cathode lamp and a coated, ridged,graphite cuvette. A deuterium source was used for backgroundcorrection and absorbance peak area was measured. The coatedgraphite cuvette was changed every 110 firings because ofcuvette aging effects noted during previous method validation.15

Twenty microlitres of unknown sample or calibration standardwere injected directly into the graphite cuvette without furtherpreparation. Three replicate measurements of the absorbancepeak area were routinely obtained from each sample, fromwhich the mean absorbance value and standard deviation wereobtained. Calibration curves were obtained as described abovein order to permit determination of palladium content (ng mL−1

Solvable R©) of the unknowns based on the equation of the bestlinear fit line of the calibration plot. The concentration ofpalladium (and corresponding concentration of WST09 basedon the mass fraction of palladium in WST09) per gram tissueor mL of plasma were then calculated from the concentration of

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palladium in each sample and known amount of tissue addedto the digestion solution (molar ratio of palladium atom to theWST09 macrocycle is 1 : 1). The limit of detection of WST09by this method has been determined elsewhere to be 330 ng g−1

in skin, 360 ng mL−1 in plasma, and 610 ng g−1 in liver, kidney,tumour, and muscle respectively.15

2.8 Determination of pharmacokinetic parameters

The mean concentration of WST09 in each tissue was plottedlogarithmically versus time. Assuming a two-compartment openmodel, the bi-exponential curves that best fit the data weredetermined using the method of residuals.17 In each case,the distribution and elimination rate constants (ka and kb)were obtained graphically from each drug concentration versustime plot. The highest drug concentration observed followingadministration, and the time at which it is observed (Cmax andtmax), were determined directly from the concentrations measuredat each time point. The area under the drug concentration versustime curve, both from time zero to infinity (AUC0-∞), and fromtime zero to the time at which the last quantifiable value wasmeasured (AUC0-tlast), were calculated through application of thetrapezoidal rule.17 Infinity was taken to be the clearance time, orthe time at which 99.9% of the initial concentration of the drugwas cleared from the tissues (as determined by extrapolation ofthe terminal elimination phase). The rate of total clearance ofthe drug (Cl) was calculated as the dose (lg WST09 injected)divided by the area under the drug concentration versus timeplot (AUC0-∞). The apparent volume initial distribution (V d)was calculated as the dose (lg WST09 injected) divided by theconcentration of WST09 in the plasma when the concentrationversus time plot was extrapolated back to zero..

3 Results and discussionThe pharmacokinetic distribution of WST09 in the plasma andtissues at 0.08, 0.25, 0.5, 1.0, 3.0, 6.0, 9.0, 12.0, 24.0, 48.0,72.0 and 96.0 h post intravenous injection was determinedusing gFAAS. The mean and standard deviation of the con-centration of WST09 present in plasma and each tissue at eachtime point tested are given below in Table 1.

WST09 accumulation was not observed in skin, muscle,or tumour at any time. This lack of accumulation indicatesthat direct tumour cell kill would not be possible using thisdrug. However, local irradiation of the tumour area shortlyfollowing iv administration of the drug will potentially targetthe tumour vasculature. This treatment approach has beendemonstrated with positive results using bacteriochlorophyll-serine18,19 as well as uncoupled bacteriochlorophyll.3 The lack

of accumulation in the peritumoural skin and muscle maycontribute to the selectivity of the resulting tumour destruction ifsuch a technique is employed, especially if the tumour is the mosthighly vascularized of the three tissues. The lack of accumulationin the skin also makes unwanted phototoxicity unlikely.

The level of WST09 in the plasma (Fig. 2) was measured tobe maximal (19 lg mL−1) at the earliest time point tested, i.e.5 minutes post injection. Considering the small size of the mouseand the fact that circulation time is rapid, the level of WST09 inthe plasma is actually very likely maximized within seconds ofthe delivery of the bolus dose. Furthermore, clearance from theplasma is rapid, with the concentration of WST09 decreasing tozero within three hours of injection. Therefore, if the vasculatureis the intended target, the plasma data support an irradiationtime immediately post iv injection of the WST09 in order totake advantage of maximal intravascular concentrations of thedrug.

Fig. 2 Semi-logarithmic plot of mean WST09 concentration inplasma versus time after intravenous injection of WST09 formula-tion–analysis by GFAAS. Dotted line: bi-exponential fit to data,C = 107721e−22t + 2532e−0.55t, determined by method of residuals. Singleexponential construction lines are presented as solid lines. Three to sixsamples included in each data point.

Initially high levels of WST09 (Cmax equal to 43 lg g−1 at5 minutes post injection) are present in the liver that steadilydecrease up to 24 h post injection (Fig. 3). This affinity for thereticuloendothelial organs is typical of lipophilic drugs and rep-resents the binding of the drug to high density lipoprotein (HDL)in the serum and its clearance via the bile-gut pathway.20–24

WST09 is also found to be present in the kidney (Cmax equal to8.1 lg g−1 at 5 minutes post injection) with the levels decreasing tozero at 9 h post injection (Fig. 4). The concentration of WST09 in

Table 1 Mean concentration of WST09 in the plasma and tissues (lg mL−1 plasma or lg g−1 tissue) after iv injection in EMT6 tumour-bearingfemale Balb/c micea

Time point/h Plasma Kidney Liver Skin Muscle Tumour

Control 0 0 0 0 0 00.08 19 ± 5 8.1 ± 1.2 43 ± 5 0 0 00.25 2.6 ± 0.6 6.0 ± 1.5 36 ± 5 0 0 00.50 2.1 ± 0.7 5.8 ± 2.5 33 ± 6 0 0 01.0 1.0 ± 0.4 3.5 ± 1.3 18 ± 8 0 0 03.0 0.53 ± 0.14 2.4 ± 0.4 17 ± 2 0 0 06.0 0 0.93 ± 0.16 6.1 ± 2.1 0 0 09.0 0 0.87 ± 0.10 5.0 ± 3.8 0 0 012 0 0 4.2 ± 1.4 0 0 024 0 0 1.1 ± 0.1 0 0 048 0 0 0 0 0 072 0 0 0 0 0 096 0 0 0 0 0 0.62 ± 0.59b

a All values below the limit of detection of the GFAAS instrument are reported as zero. n = 3 to 6 mice per time point. b Due to the high standarddeviation associated with this value, and its nearness to the limit of detection (LD), it is considered to be statistically insignificant and is not includedin the pharmacokinetic analysis.

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Fig. 3 Semi-logarithmic plot of mean WST09 concentration inliver versus time after intravenous injection of WST09 formula-tion–analysis by GFAAS. Dotted line: bi-exponential fit to data,C = 39994e−2.8t + 17864e−0.12t, determined by method of residuals. Singleexponential construction lines are presented as solid lines. Three to sixsamples included in each data point.

Fig. 4 Semi-logarithmic plot of mean WST09 concentration inkidney versus time after intravenous injection of WST09 formula-tion–analysis by GFAAS. Dotted line: bi-exponential fit to data,C = 6115e−3.1t + 3938e−0.19t, determined by method of residuals. Singleexponential construction lines are presented as solid lines. Three to sixsamples included in each data point.

the kidney is substantially less than that of the liver, consistentwith clearance via the bile gut pathway. The relatively rapidclearance of the drug from the liver and kidney reduces the riskof unwanted systemic toxicity.

The kinetic results indicate biphasic kinetics for plasma,kidney, and liver. The existence of a distribution phase in theplasma data was postulated based on only two data points(0.08 and 0.25 h) and therefore the delineation of this phasemay be subject to considerable error. The application of a two-compartment open model was assumed to be correct based onthe biphasic kinetics exhibited in the liver and kidney. However,due to the dynamic equilibrium that exists between the amountof drug present in the plasma and tissues, biphasic kinetics arealso expected in the plasma. Therefore, discounting the existenceof the initial fast distribution phase in the plasma would likelyhave introduced a greater margin of error than that incurredthrough the determination of the elimination phase using sucha small number of data points. Analysis of the pharmacokineticand distribution characteristics in each tissue was performedusing a two compartment open model. The results of thisanalysis are shown in Table 2.

In each of plasma, kidney, and liver, tmax was observed at theearliest time point taken, i.e., 5 minutes post injection. Relativelyrapid clearance was observed in plasma, kidney and liver. Thetotal rate of clearance and the apparent volume of distributionwere calculated to be 11 mL h−1 and 0.9 mL respectively. This

Table 2 Pharmacokinetic and distribution parameters of WST09 inplasma, kidney and liver after iv injection into EMT6 tumour-bearingfemale BALB/c mice

Parameter Plasma Kidney Liver

AUC0-tlast 8.0 lg h mL−1 21 lg h g−1 1.7 × 102 lg h g−1

AUC0-∞ 9.1 lg h mL−1 28 lg h g−1 1.9 × 102 lg h g−1

Cmax 19 lg mL−1 8.1 lg g−1 43 lg g−1

tmax 5 min 5 min 5 minka 22 h−1 3.1 h−1 2.8 h−1

kb 0.55 h−1 0.19 h−1 0.12 h−1

t1/2a 0.031 h 0.23 h 0.25 ht1/2b 1.3 h 3.7 h 5.7 hCl 11 mL h−1 N/a N/aV d 0.90 mL N/a N/aClearancetime

5.7 h 33 h 47 h

small volume of initial distribution is consistent with the druginitially distributing only within the plasma and blood-richtissues. This is what was expected based on the lack of drugfound in the non-blood-rich tissues, and supports the view thatthe initial linear portion of the drug concentrations versus timecurve for plasma was extrapolated correctly despite the smallnumber of data points present.

The goal of this study was the determination of the phar-macokinetic profile of WST09 in an EMT6 tumour-bearingBalb/c mouse model for the purpose of determining its tumourselectivity. Based on the results obtained by GFAAS, WST09appears to be efficiently cleared from the plasma and tissues ofthis model via the liver and kidney. The drug does not appearto accumulate in the skin, muscle, or tumour indicating thatvascular targeting is most likely necessary for the effective useof this drug in PDT. Furthermore, due to the rapid clearanceof WST09 from the plasma, irradiation should be carriedout immediately after intravenous injection in order to takeadvantage of maximal intravascular drug concentration.

AbbreviationsGFAAS, graphite furnace atomic absorption spectroscopy;WSTO9/TOOKAD, palladium-bacteriopheophorbide; PDT,photodynamic therapy; AUC0-tlast, area under the drug concen-tration vs. time plot from 0 h to the last quantifiable value;AUC0-∞, area under the drug concentration vs. time plot from0 h to infinity; Cl, rate of total clearance of the drug; Cmax, highestdrug concentration measured following administration; ka, drugdistribution rate constant; kb, drug elimination rate constant;t1/2a, drug distribution half-life; t1/2b, drug elimination half-life;tmax, time at which the highest drug concentration is measuredfollowing administration; V d, apparent volume of initial drugdistribution; IR, infrared; UV-Vis, ultraviolet-visible; HPLC,high pressure liquid chromatography; rpm, revolutions perminute; HDL, high density lipoproteins.

AcknowledgementsFinancial support for this research was provided by StebaBiotech and by the Canadian Department of National DefenceAcademic Programme (FDG66).

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