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From: TSS (216-119-132-24.ipset12.wt.net)
Subject: A possible pharmacological explanation for quinacrine failure to treat prion diseases: pharmacokinetic investigations in a ovine model of scrapie
Date: January 19, 2005 at 11:29 am PST

-------- Original Message --------
Subject: A possible pharmacological explanation for quinacrine failure to treat prion diseases: pharmacokinetic investigations in a ovine model of scrapie
Date: Wed, 19 Jan 2005 13:21:55 -0600
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy
To: BSE-L@LISTSERV.KALIV.UNI-KARLSRUHE.DE


##################### Bovine Spongiform Encephalopathy #####################


Paper

Subject Categories: Neuropharmacology


British Journal of Pharmacology advance online publication 10 January
2005; doi: 10.1038/sj.bjp.0706072


A possible pharmacological explanation for quinacrine failure to
treat prion diseases: pharmacokinetic investigations in a ovine
model of scrapie

Véronique Gayrard1
,
Nicole Picard-Hagen1
,
Catherine Viguié1
,
Valerie Laroute1
,
Olivier Andréoletti2

and Pierre-Louis Toutain1


1. 1UMR 181 de Physiopathologie et Toxicologie Expérimentales
INRA/ENVT, Ecole Nationale Vétérinaire de Toulouse, 23 Chemin des
Capelles, BP 87614, 31076 Toulouse Cedex 3, France
2. 2UMR 1225 Interactions hôtes-agents pathogènes INRA/ENVT, Ecole
Nationale Vétérinaire de Toulouse, 31076 Toulouse, France

Correspondence: Pierre-Louis Toutain, E-mail: pl.toutain@envt.fr


Received 25 May 2004; Revised 18 October 2004; Accepted 26 October 2004;
Published online 10 January 2005.


Abstract

1. Quinacrine was reported to have a marked in vitro antiprion action
in mouse neuroblastoma cells. On compassionate grounds, quinacrine
was administered to Creutzfeldt-Jakob disease patients, despite
the absence of preclinical in vivo studies to evaluate efficacy.
Quinacrine failed to provide therapeutic benefit. The aim of the
study was to investigate possible pharmacokinetic and/or
pharmacodynamic explanations for the discrepancy between the
proven action of quinacrine in vitro and its lack of clinical
efficacy.
2. We conducted in vitro experiments reproducing the culture
conditions in which antiprion effects had been previously observed
and recalculated the EC50 by determining the actual extracellular
(120 nM) and intracellular (6713 nM) quinacrine neuroblastoma
concentrations with the reported quinacrine EC50 (300 nM).
3. A randomized clinical trial in scrapie-affected ewes confirmed the
absence of therapeutic benefit of quinacrine. The in vivo
quinacrine exposure was evaluated in a pharmacokinetic
investigation in healthy ewes. Cerebrospinal fluid concentrations
(<10.6 and 55 nM after administration of therapeutic and toxic
quinacrine doses, respectively) were much lower than the
quinacrine extracellular neuroblastoma concentrations
corresponding to the reported EC50. The total brain tissue
concentrations (3556 nM) obtained after a repeated therapeutic
dosage regimen were within the range of the intracellular
neuroblastoma quinacrine concentrations.
4. In conclusion, in order to avoid in vivo trials for which failure
can be predicted, the measurement in vitro of the antiprion EC50
in both intra- and extracellular biophases should be determined.
It can then be established if these in vitro antiprion
concentrations are achievable in vivo.


Keywords:

Prion, therapeutic, quinacrine, pharmacokinetics, neuroblastoma,
Creutzfeldt-Jakob disease (CJD), scrapie


Abbreviations:

AUC, area under the curve; CJD, Creutzfeldt-Jakob disease; Cl/F,
apparent plasma clearance; Cmax, maximum concentration; MRT, mean
residence time; PrP, prion protein; PrPC, normal cellular prion protein;
PrPSc, abnormal form of the PrPC protein; ScN2a, scrapie-infected
neuroblastoma; Tmax, time to maximum concentration; Vss/F, apparent
steady-state volume of distribution

Top of page

Introduction

Prion accumulation is considered to be a key event in the
pathophysiology of Creutzfeldt-Jakob disease (CJD) (Bruce et al., 1989
;
Jendroska et al., 1991
),
and cellular models for prion disorders have been used to screen the
efficacy of many inhibitors of abnormal form of the PrPC protein (PrPSc)
formation. Thus, a 6-day treatment with the anti-malarial drug
quinacrine or with a phenothiazide derivative chlorpromazine cured a
mouse neuroblastoma cell line (ScN2a) that was chronically infected with
prions (Doh-Ura et al., 2000
;
Korth et al., 2001
).
The median effective concentration ('EC50') for quinacrine and
chlorpromazine (i.e. the nominal concentration of the test solution
producing half-maximal inhibition of PrPSc formation) reported in this
study were 300 nM (142 ng ml-1) and 3000 nM, respectively. Based on a
review of the literature, it was anticipated that this effective in
vitro concentration of quinacrine would be attainable in the in vivo
biophase (not precisely identified at present). However, to date all
patients with CJD treated with quinacrine at the dosage usually
recommended in man for other conditions (approximately 300 mg per day)
(Shannon et al., 1944
;
Goodman & Gilman, 1960
)
died without evidence of any retardation in disease progression
(Furukawa et al., 2002
;
Follette, 2003
).

The initial hypothesis was that the lack of quinacrine efficacy resulted
from an inability to treat patients with a dosage regimen that would
produce the same biophase concentration in vivo as in vitro. Scrapie is
a naturally occurring disease of sheep, which provides a relevant model
for testing this hypothesis, as scrapie is a prion disease for which the
pathophysiological mechanisms of infection are likely to be similar to
those occurring spontaneously in CJD.

A second hypothesis was that the reported in vitro EC50 (300 nM) was not
the actual in vitro biophase EC50. Indeed, the in vitro EC50 for
quinacrine reported by Korth et al. (2001)

was only the nominal (calculated) concentration of the quinacrine
solution added to the culture medium, which enabled half the maximum
effect on prion replication in neuroblastoma cell lines, to be obtained
with repeated administration in a volume of 4 ml for 2-7 days. It is not
established that this culture medium 'EC50' corresponds to the true EC50
in the in vitro biophase. Indeed, under both in vitro and in vivo
conditions, the drug has to diffuse to the biophase and it can be
removed and/or trapped by the neuroblastoma cellular organelles. Thus,
it cannot be assumed that the concentration in the in vitro biophase is
equal to the nominal concentration of the test solution added to the
culture to obtain half quinacrine efficacy. Therefore, the in vitro EC50
should be determined experimentally from measured in vitro drug
concentrations. This is particularly important when the in vitro effect
of interest develops over several days.

The objectives of the present study were: (i) to determine if quinacrine
together with chlorpromazine is effective in a controlled clinical trial
in a naturally occurring prion disease of sheep; (ii) to recalculate the
in vitro EC50 of quinacrine by establishing its disposition when added
to the culture medium of N2a cells in vitro at the concentration
equivalent to the reported 'EC50'; (iii) to establish the in vivo
exposure to quinacrine after the administration of therapeutic and toxic
dosage regimens; and (iv) to compare the quinacrine concentrations in
the N2a cell model with those obtained experimentally in the central
nervous system (CNS) of ewes treated with quinacrine.

Top of page

Methods


Animal procedures

All procedures involving animals were performed in accordance with
French legal requirements regarding the protection of laboratory animals
and with the authorization for animal experimentation no. 001889 of the
French Ministry of Agriculture. The ewes used in experiments 1 and 3
were maintained under natural photoperiod conditions and they received
daily rations of concentrate. Hay and water were given ad libitum.


Clinical trial

Experiment 1 was designed to evaluate the clinical efficacy of
quinacrine in a randomized clinical trial in naturally infected scrapie
ewes. The trial was carried out in 23 Manech red-faced ewes with
naturally occurring scrapie. The scrapie diagnosis was established from
clinical signs of pruritus, behavioural changes, tremor and locomotion
incoordination, and confirmed by histopathology on brain samples after
necropsy as described previously (Schelcher et al., 1999
).
The ewes were ranked in order of severity of clinical signs, pairs of
ewes with similar clinical signs were formed and, within each pair,
individual ewes were randomly allocated to treated or untreated
(placebo) groups. At the time of inclusion in the clinical trial, the
mean body weights (plusminus s.d.) of 11 treated and 12 untreated
scrapie ewes were 41.6plusminus 6.4 and 41.7plusminus 6.6 kg,
respectively. The 11 ewes in the treated group received 150 mg (317 mu
mol) in toto of quinacrine dihydrochloride and 100 mg (281 mu mol)
chlorpromazine daily by injection into the gluteal muscle, for 7 days in
the case of six ewes and for a nominal period of 30 days (except 1 day
per week) for five ewes. The volumes of quinacrine and chlorpromazine
solutions intramuscularly (i.m.) administered were 5 and 4 ml,
respectively. Chlorpromazine was administered with quinacrine as
recommended for human therapy of CJD (Korth et al., 2001
).
The 12 ewes in the untreated group received the equivalent volume of
vehicle. The dosage regimen of quinacrine administered to the ewes was
consistent with the dose used in humans (300 mg daily; Shannon et al.,
1944
).
Blood samples were collected by venipuncture (jugular vein) from
scrapie-infected ewes once each week during the time course of the
disease. These ewes were killed as soon as they manifested clinical
signs of irreversible recumbency.


Quinacrine disposition in N2a cells

Experiment 2 was designed: (i) to recalculate a posteriori the EC50 of
quinacrine by determining both intra- and extracellular concentrations
obtained when quinacrine was added to the culture medium of N2a cells at
a nominal concentration equivalent to the in vitro EC50 reported by
Korth et al. (2001)
;
and (ii) to determine the relationship between intra- and extracellular
quinacrine concentrations when increasing quinacrine amounts were added
to the culture medium of N2a cells.

The mouse neuroblastoma cell line (N2a) was stably transfected as
previously described (Lehmann & Harris, 1995
).
Transfected cells were plated at a density of 40,000 cm-2 into 25 cm2
flasks of 5 ml of OptiMEM (GIBCO, BRL, Cergy Pontoise, France)
containing 10% foetal calf serum, and penicillin-streptomycin. These
culture conditions were almost the same as those previously used (Korth
et al., 2001
),
except that the cells were split at day 4 using 0.05 (w v-1)
trypsin-EDTA (GIBCO, BRL) and were not infected with scrapie. The medium
was changed every 48 h together with quinacrine, except on the 7th day
of culture when the medium was collected after 24 h. The viability of
neuroblastoma cells was assessed by counting in satellite flasks. The
media from the final 24 or 48 h of culture were stored at -20°C until
assayed for quinacrine content. At the end of the treatment period,
cells were washed four times with isotonic saline solution, scraped and
resuspended in 1 ml of deionized water and stored at -20°C until
assayed. Intracellular concentrations of quinacrine were assayed after
sonication of cells.

In a first series of in vitro experiments reproducing the culture
conditions in which antiprion effects were observed (Korth et al., 2001
),
the time courses of intra- and extracellular quinacrine concentrations
were measured over a period of 2-7 days in the presence of a nominal
quinacrine concentration of 300 nM.

In a second set of experiments, the N2a cells were cultured in the
presence of different concentrations of quinacrine (from 0 to 850 nM)
for 2 days. At the end of the culture period, the quinacrine
concentrations in the media and in the cells were determined.


In vivo quinacrine disposition

The objectives in Experiment 3 were: (i) to determine the overall in
vivo quinacrine exposure in the animal model to enable comparison to
exposure in man, and (ii) to compare the concentrations of quinacrine in
the CNS of healthy ewes, treated with either a therapeutic dose or a
toxic dose of quinacrine, with quinacrine concentrations that were
effective in vitro.

The in vivo disposition of quinacrine was investigated in seven healthy
Lacaune ewes, under conditions reproducing the therapeutic dosage
regimen of quinacrine in the clinical trial. The ewes received 8 daily
(day-0 to -7) i.m. injections of a therapeutic dose of 3 mg kg-1 day-1
of quinacrine. The daily injections were administered in turn in the
gluteal, vastus lateris, brachiocephalicus or longissimus dorsi muscles,
and in the right or left sides in an 8 times 8 Latin square design. One
ewe has to be excluded from the experiment for an unrelated reason.

On day-0 and -7, blood samples were serially collected by direct
venipuncture (jugular vein) within the hour preceding quinacrine i.m.
administration and 10, 30, 60, 90, and 120 min after quinacrine
administration, then at 2-h intervals until 10 h post-administration and
finally at 23-24 h post-administration. From day-2 to -6, a blood sample
was collected daily within the hour preceding quinacrine i.m.
administration to determine trough plasma concentration. At 24 h after
the final injection of quinacrine (day-8), cerebrospinal fluid (CSF) was
sampled from the cisterna magna of four ewes. These ewes were then
euthanized with intravenous (i.v.) pentobarbitone and exsanguinated. The
brains were immediately removed and homogenized at 4°C in 15% (w v-1)
deionized water and stored at -20°C until assayed.

Jugular venous blood samples were collected from the three remaining
ewes daily until day-15, then every 2 or 3 days until day-21 and on
day-28, when plasma quinacrine concentrations were no longer detectable.
On day-28, CSF was sampled from the cisterna magna (two ewes) or from
the lombosacral space (one ewe). The ewes were immediately euthanized
and the brains homogenized at 4°C in 30% (w v-1) deionized water and
stored at -20°C until assayed.

A second series of experiments was performed using three healthy ewes to
determine CNS exposure to quinacrine over a wide range of quinacrine
doses. Two ewes received an i.m. injection of a single therapeutic dose
of 150 mg in toto of quinacrine and one ewe received a slow 5.4 h i.v.
quinacrine infusion of a toxic dose of 2600 mg in toto. This i.v. dose
was injected into the right jugular vein. At 24 h after the
administration of quinacrine, a blood sample was taken from the left
jugular vein via an indwelling catheter and CSF was sampled from the
cisterna magna of all three ewes. The ewes were immediately euthanized
and the brains homogenized at 4°C in 15% (w v-1) deionized water and
stored at -20°C until assayed.


Sampling

Blood samples (5 ml) were collected in heparinized tubes and centrifuged
for 10 min at 3000 times g. The plasma was removed and stored at -20°C
until assayed. CSF was collected from anaesthetized animals (sodium
pentobarbitone, Nesdonal®, Merial, Lyon, France) by puncture of the
cisterna magna or the lombosacral space with a 20-gauge needle. A volume
of 1-9 ml of CSF was gently withdrawn and centrifuged for 10 min at 1500
times g to remove cells and stored at -20°C until assayed.


Quinacrine assay

Quinacrine concentrations were determined using a validated
high-performance liquid chromatography (HPLC) method in which the
internal standard and all biological samples were extracted by
liquid/liquid extraction. The HPLC apparatus consisted of a pump system
equipped with an automatic injector and a variable-wavelength
fluorescence monitor. The separation was achieved by reverse phase
column (Inertsil ODS3 column, 3 mu m, 150 times 4.0 mm2). The column was
equilibrated at a flow rate of 0.3 ml min-1, with a mobile phase
consisting of methanol : 25 mM citrate buffer (pH=4.0) (60 : 40, v v-1)
containing 0.1 mM benzamidine. As far as possible, polypropylene was
used for collecting, storing and assaying samples. The adsorption of
drug on to materials during the assay was minimized by including a
competing hydrophobic molecule, benzamidine in the mobile phase. The
fluorescence detector was set at 300 nm (excitation) and 495 nm
(emission). The sample volumes used in the assay were 200 mu l for the
plasma and 100 mu l for the other biological samples. Quinacrine was
extracted from biological samples with 1 ml (3 ml for the plasma
samples) of 1,2 dichloroethane and 100 mu l of 0.2 M sodium hydroxide
and 100 mu l of 277 nM ethodin as internal standard. Dichloroethane
extracts of the matrices were evaporated under nitrogen at 40°C and
resuspended in 100 mu l DL-lactic acid (0.85%) before injection of a
volume of 50 mu l on to the column. The mean recoveries of quinacrine
from the culture media and from the plasma were 85 and 50%,
respectively. The quinacrine assay was performed accurately and
reproducibly in the range of 21-254 nM. Within- and between-day
precision was less than 15%. The validated quantification limit of the
assay was 5.3 nM for the plasma and 10.6 nM for the CSF and culture media.


Pharmacokinetic analysis

Both compartmental and statistical moment approaches were used for the
pharmacokinetic analysis of quinacrine concentrations, using WinNonlin
4.0 (Pharsight Corporation, Mountain View, CA, U.S.A.). The maximum
concentration (Cmax) and time to maximum concentration (Tmax) were
determined directly from plasma concentrations obtained after the first
quinacrine i.m. administration, for each animal. The area under the
curve (AUC0-24 h) for plasma quinacrine concentrations was calculated
from t=0 to 24 h, after the first and eighth quinacrine administrations
using the arithmetic trapezoidal rule. The AUC0-inf and the area under
the first moment curve (AUMC) for quinacrine plasma concentrations after
the first administration were calculated using the linear trapezoidal
rule with extrapolation to infinity. The mean residence time (MRT, h) of
quinacrine for a single administration was calculated using the equation

Unfortunately we are unable to provide accessible alternative text for
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help@nature.com or the author

where AUMC is the area under the moment curve observed after i.m.
administration of quinacrine.

The plasma concentration versus time curves after the eight i.m.
quinacrine injections at the dose rate of 3 mg kg-1 day-1 were fitted
with a polyexponential equation to assess possible dose and time
dependencies of quinacrine disposition. The parameters were estimated by
nonlinear regression. The number of exponents was determined by
application of the Akaike's Information Criterion (Yamaoka et al., 1978
).
The data points were weighted with the inverse of the squared fitted
value. The goodness of fit of the described model was assessed using
least-squares criteria. A triexponential equation describing a
bicompartmental open model with first-order absorption was selected

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where C(t) represents the plasma quinacrine concentration at time t; Yi
(nM) the coefficient of the ith exponential term; lambda 1 and lambda 2
the first-order rate constants of the initial and terminal phases; and
K01(h-1) the first-order absorption rate constant.

The plasma half-life for the terminal phase was calculated using the
equation

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this. If you require assistance to access this image, please contact
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with lambda 2 as defined above.

The apparent steady-state volume of distribution (Vss/F) was obtained
with equation (4)


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where F is the relative bioavailability and Vc/F is the apparent volume
of the central compartment, K12 is the first-order rate constant between
central and peripheral compartments and K21 is the first-order rate
constant between peripheral and central compartments.

The apparent plasma clearance (Cl/F) was obtained with equation (5)


Unfortunately we are unable to provide accessible alternative text for
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with Vc/F as defined above and K10 the rate constant of quinacrine
elimination.


Materials

Drug solutions were freshly prepared for in vitro and in vivo use.
Quinacrine dihydrochloride (molecular weight: 472.9 Da),
chlorpromazine-HCl, ethodin, benzamidine and 1,2 dichlororethane were
obtained from Sigma-Aldrich (Saint Quentin Fallavier, France).
Quinacrine was dissolved in saline to produce a concentration of 63.3
mM, except for the seven ewes in Experiment 3 for which quinacrine was
dissolved in saline and dimethyl sulphoxide (50 : 50, v v-1).
Chlorpromazine-HCl was dissolved at the concentration of 70.4 mM in
vehicle containing 11.4 mM ascorbic acid, 5.3 mM sodium bisulphite, 7.9
mM sodium sulphite, 17.1 mM sodium chloride and 2% benzyl alcohol. Stock
solutions of quinacrine were filtered throughout a 0.2 mu m syringe
filter for in vitro use.


Statistical analysis

Results are reported as meanplusminus s.d. (or median). Statistical
analyses were performed using SYSTAT 8.0 (SPSS Inc., Chicago, IL,
U.S.A.). In Experiment 1, the median delay between inclusion in the
clinical trial and death was determined for both treated and control
groups. The percentage of ewes that died from the beginning of treatment
was compared for the two treated and untreated groups with the log-rank
test for equality of survival (Kaplan-Meier test).

Top of page

Results


Experiment 1

The median survival time of untreated scrapie-affected control ewes (36
days, range 13-72 days, n=12) did not differ from that of ewes treated
for 7 days (45 days, range 9-90 days, n=6) or for a nominal period of 30
days (22 days, range 6-91 days, n=5, Figure 1
).
Plasma quinacrine concentrations of the five treated diseased ewes
varied from 47 to 721 nM (22-341 ng ml-1) 7 or 9 days after the
beginning of the 7-day quinacrine treatment.


Figure 1.


Figure 1 - Unfortunately we are unable to provide accessible alternative
text for this. If you require assistance to access this image, please
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Survival curves for scrapie-infected ewes (days after commencing a
combined quinacrine and chlorpromazine treatment). There was no
significant difference in survival between the two groups (11 treated
ewes versus 12 untreated ewes).

Full figure and legend (16K)


Experiment 2

The time course of intra- and extracellular quinacrine concentrations
was determined during a 2- to 7-day period of N2a cell culture in the
absence and presence of a nominal quinacrine concentration of 300 nM
(Figure 2
).
A 34plusminus 8% loss of quinacrine occurred after the dilution and
filtration of a standard quinacrine solution of 21 mu M through a 0.2 mu
m filter and 16plusminus 3% loss was due to adsorption to the culture
disk. The quinacrine concentrations measured in the culture media
remained relatively constant during the period of culture (mean: 120 nM;
range: 100-140 nM) and were approximately 60% lower than the nominal
concentration of the added solution.


Figure 2.


Figure 2 - Unfortunately we are unable to provide accessible alternative
text for this. If you require assistance to access this image, please
contact help@nature.com or the author

Distribution of quinacrine between extracellular and intracellular
compartments in N2a neuroblastoma cells during the 7-day culture. Cells
were cultured in the absence or presence of a nominal quinacrine
concentration of 300 nM. At every change of medium, the concentration of
quinacrine remaining in the medium and present in cells after 24-48 h of
culture was determined. Values represent the meanplusminus s.d. of an
experiment performed in duplicate. Note that the scales of quinacrine
concentration differ for the cells (left ordinate) and the culture media
(right ordinate).

Full figure and legend (16K)

The mean intracellular quinacrine concentrations were calculated from an
estimated 50 mu l volume of subconfluent cells (i.e. a layer of 25 cm2
and 20 mu m depth) to range from 2057 to 6713 nM (973-3175 ng ml-1)
during the 7 days of culture. The ratio between the intra- and
extracellular concentrations of quinacrine varied between 18 and 58 and
tended to remain constant between day-2 and -6 of culture.

In the second in vitro experiment, the measured extracellular and
intracellular quinacrine concentrations increased linearly with the
added quinacrine concentrations (slope 0.43 and 13.8, for intra- and
extracellular concentrations, respectively; R2=0.99, coefficient of
determination, Figure 3
).
For a nominal medium concentration of 300 nM (i.e. the reported EC50),
the intra- and extracellular quinacrine concentrations calculated from
the regression line were 3761 nM (1779 ng ml-1) and 120 nM (57 ng ml-1),
respectively. The ratio between the intra- and extracellular
concentrations of quinacrine varied between 25 and 47.


Figure 3.


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Distribution of quinacrine between extracellular and intracellular
compartments in N2a neuroblastoma cells as a function of the quinacrine
concentration, added to the culture medium. The N2a cells were cultured
in the presence of seven concentrations of quinacrine (from 0 to 850 nM)
for 2 days. At the end of the culture period, quinacrine concentration
in the media and cells was determined. Values represent the
meanplusminus s.d. of an experiment performed in triplicate. Note that
the scales of quinacrine concentration differ for the cells (left
ordinate) and the culture media (right ordinate).

Full figure and legend (21K)


Experiment 3

The mean plasma quinacrine concentration versus time profile after the
first i.m. quinacrine injection at the dose rate of 3 mg kg-1 is
presented in Figure 4
.
The mean (plusminus s.d.) quinacrine AUC0-24 h after the first i.m.
injection was 898plusminus 593 nM h (425plusminus 280 ng ml-1 h), giving
an average plasma quinacrine concentration of 37.4plusminus 24.7 nM
(17.7plusminus 11.7 ng ml-1) over the first 24 h. The quinacrine MRT for
a single dose was 13.43plusminus 5.90 h. The mean plasma maximum
quinacrine concentration was 189plusminus 152 nM (89plusminus 72 ng
ml-1) and mean time to maximal plasma concentration was 0.79plusminus
0.39 h.


Figure 4.


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Semilogarithmic plot of mean plasma quinacrine concentration (nM) versus
time (h) after a single i.m. quinacrine injection at the dose rate of 3
mg kg-1 in seven ewes.

Full figure and legend (9K)

After the eighth quinacrine administration, the mean (plusminus s.d.)
AUC0-24 h was 2958plusminus 1918 nM h (1399plusminus 907 ng ml-1 h),
giving an average plasma quinacrine concentration of 123plusminus 80 nM
(58plusminus 38 ng ml-1) and indicating an accumulation ratio of
approximately 3 between the first and the eighth injections.

Figure 5

illustrates the time courses of observed and fitted plasma quinacrine
concentrations for a representative healthy ewe for an 8-day i.m.
quinacrine treatment of 3 mg kg-1 day-1. Visual inspection of Figure 5

indicates that plasma quinacrine concentrations increased almost
linearly from day-0 to -6 and tended to remain constant from day-6 to
-8. Table 1

presents the mean values of the quinacrine pharmacokinetic parameters as
obtained by fitting the plasma quinacrine concentrations after an 8-day
i.m. quinacrine treatment. After the final injection, the plasma
concentration decreased, with a terminal half-life (meanplusminus s.d.)
of 52plusminus 11 h, to a value similar to the quantification limit of
the assay at day-16. The apparent quinacrine clearance (Cl/F) was
3.04plusminus 0.70 l h-1 kg-1. The apparent steady-state volume of
distribution (Vss/F) was 185plusminus 42 l kg-1.


Figure 5.


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Observed (point) and fitted (line) plasma quinacrine concentrations (nM)
in a representative ewe during 8 days of i.m. quinacrine administration
at the dose rate of 3 mg kg-1 day-1 and for 5 further days after
termination of dosing. Horizontal bar indicates the period of the
quinacrine treatment. The good fitting supports the linearity (dose and
time) of quinacrine disposition in our experimental conditions.

Full figure and legend (14K)


Table 1 - Pharmacokinetic parameters (meanplusminus s.d.)
describing the plasma disposition of quinacrine after an 8-day
i.m. quinacrine treatment at the dose of 3 mg kg-1 day-1 in
three ewes.


Table 1 - Pharmacokinetic parameters (mean[plusmn]s.d.) describing the
plasma disposition of quinacrine after an 8-day i.m. quinacrine
treatment at the dose of 3[thinsp]mg[thinsp]kg-1[thinsp]day-1 in three
ewes - Unfortunately we are unable to provide accessible alternative
text for this. If you require assistance to access this image, please
contact help@nature.com or the author
Full
table ()

At 20 days after the 8-day i.m. quinacrine treatment, quinacrine
concentrations in the plasma, brain tissue and CSF were below the limit
of quantification of the assay, except in the nervous tissue of one ewe,
for which the value obtained was 63 nM.

Quinacrine concentrations were much higher in the nervous system than in
plasma and CSF (Table 2
).
The ratios of the mean brain tissue concentration to mean plasma
quinacrine concentration were 76, 53 and 24, 24 h after a single
therapeutic quinacrine dose, repeated therapeutic doses and a toxic
dose, respectively. The brain tissue/CSF ratio was 979 after the toxic
dose of quinacrine and more than 335 after the chronic therapeutic
dosage regimen.


Table 2 - Quinacrine concentrations (nM, meanplusminus s.d.)
in plasma, CSF and brain tissue in ewes for different
conditions.


Table 2 - Quinacrine concentrations (nM, mean[plusmn]s.d.) in plasma,
CSF and brain tissue in ewes for different conditions - Unfortunately we
are unable to provide accessible alternative text for this. If you
require assistance to access this image, please contact help@nature.com
or the author
Full
table ()

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Discussion

The results of this investigation failed to show any therapeutic benefit
of a combined quinacrine and chlorpromazine treatment regimen in a
controlled clinical trial in naturally infected scrapie ewes. Using 12
animals per arm, the power of this trial was theoretically sufficient to
demonstrate a significant decrease in mortality rate of 24% for a
unilateral risk of 5% and a power of 80%. This is satisfactory for this
type of exploratory trial designed to demonstrate the existence or not
of drug efficacy. This negative result is consistent with observations
made in a model of mouse-adapted CJD (Collins et al., 2002
)
and in humans (Furukawa et al., 2002
;
Follette, 2003
).
However, the two mortality curves in our investigation were not
parallel. Therefore, it cannot be totally excluded that treatment
accelerated the death in the most affected ewes, while slowing disease
progression in the less affected ewes.

In order to avoid the generation of debatable data with the in vitro
test system, a second aim of our study was to determine why an antiprion
drug (quinacrine) that was active in vitro was not clinically effective
when administered to human patients and sheep. It was hypothesized that
one explanation for the lack of clinical efficacy is the impossibility
of treating patients with a quinacrine dosage regimen that enables
similar quinacrine biophase concentrations to be achieved in vivo as in
vitro. A second possible explanation is that while the reported
quinacrine in vitro EC50 (300 nM) is the medium concentration required
to obtain an effect on a neuroblastoma test system, this may not be the
concentration required in the target biophase in vivo for clinical
efficacy. The in vitro quinacrine disposition study showed first that
the nonspecific quinacrine loss was approximately 50%, in agreement with
the potential of quinacrine to be adsorbed on to surfaces (Björkman &
Elisson, 1987
).
Secondly, the study demonstrated that quinacrine was relatively stable
over 7 days of N2a cell culture. In addition, it is estimated that
approximately 25% of the quinacrine loss is attributable to uptake of
drug by the cells.

In the presence of a nominal concentration of quinacrine equivalent to
the reported in vitro quinacrine EC50 (300 nM), the intracellular
quinacrine concentrations (2057-6713 nM) attained values that were
approximately 30 to 50 times higher than the extracellular levels
(100-140 nM). This ratio is not very different to that which could be
predicted from the pH partitioning hypothesis. Indeed, considering that
quinacrine is a weak base with pKa=10.4 and assuming that the pH values
of the culture medium, the neuroblastoma cytosol and the intralysosomal
space are 7.4, 7.2 and 5, respectively, and that the lysosomial space
represents about 5% of the cell volume, the predicted theoretical ratio
of quinacrine concentrations between the intra- and extracellular spaces
was about 13. Thus, the present experiment suggests that the antiprion
in vitro EC50 may lie between 2000 and 7000 nM if the biophase is
intracellular, but be only 120 nM if the biophase is extracellular. This
extracellular concentration is almost identical to the free quinacrine
concentration, as the binding to protein is very limited in the culture
medium, whereas the intracellular concentration like the total tissue
concentration comprises both free and bound quinacrine.

The effective quinacrine concentrations in the neuroblastoma cell model
were compared to those obtained in the two putative biophases of the
CNS, that is the brain tissue (representative of an intracellular
biophase) and CSF (representative of an extracellular biophase) (De
Lange & Danhof, 2002
).
The comparison was made under similar conditions to those prevailing in
the sheep clinical trial. When quinacrine was administered at the
recommended therapeutic dosage regimen (3 mg kg-1 day-1), plasma
quinacrine concentrations increased progressively and attained an
apparent steady-state level of approximately 120 nM after the eighth
quinacrine administration. This is consistent with a quinacrine terminal
half-life of 52 h. These results indicated that the ewes had been
appropriately exposed during the clinical trial, despite considerable
interindividual variability. Moreover, our data are consistent with
findings in man. In humans, the recommended dosage regimen (800 mg per
os the first day, then 100 mg three times daily) produces a similar
quinacrine exposure, with plasma quinacrine concentrations in the range
of 100-200 nM (Shannon et al., 1944
;
Nakajima et al., 2004
).
In addition, in sheep, the data indicate that the blood/plasma ratio of
quinacrine concentrations was approximately 1.15 (unpublished
observations) demonstrating that quinacrine is poorly accumulated in red
blood cells as previously reported by Shannon et al. (1944)
.

Despite the variability in exposure to quinacrine, after both a single
therapeutic and a toxic quinacrine dose, the relationship between the
plasma and brain tissue quinacrine concentrations remained similar
(range of brain tissue to plasma quinacrine concentrations ratio of
24-76). This confirms that plasma concentration is the driving
concentration influencing drug distribution to and accumulation in the
tissue compartment (Gibaldi & Perrier, 1982
).
Another fundamental tenet in pharmacokinetics is that it is the free
drug concentration (and not the total drug concentration) which is the
driving concentration for distribution. Therefore, it is the free drug
plasma concentration which should be taken into account when considering
drug efficacy and also when comparing in vitro and in vivo conditions.

The transport of (free) quinacrine across the blood-brain barrier was
shown to involve both an influx system (organic cation transporter-like
machinery) and an efflux system via the P-gp, which might restrict the
entry of quinacrine into the brain (Dohgu et al., 2004
).
In the present experiment, the free drug concentration in vivo was not
directly measured but can be estimated from the plasma/CSF
concentrations ratio. Indeed, the CSF is an ultrafiltrate of plasma,
virtually devoid of plasma protein and the drug concentration in the CSF
represents the maximal value of the plasma free drug concentrations. The
CSF quinacrine concentration was lower than the level of quantification
of the analytical technique (10.6 nM) 24 h after repeated i.m.
administration of 3 mg kg-1 day-1 of quinacrine, suggesting that the
free quinacrine concentration was also less than 10.6 nM. After
administration of a toxic quinacrine dose, quinacrine in CSF was
measurable (55 nM) and the estimated plasma (total) to CSF (free)
concentration ratio was 40, suggesting that the free quinacrine fraction
in the plasma was greater than or equal to 2.5%. This latter value is of
the same order as that reported for the free fraction in human plasma
(Shannon et al., 1944
;
Goodman & Gilman, 1960
),
suggesting that the quinacrine concentrations in the CSF are similar to
or slightly lower than the plasma free quinacrine concentrations.
Considering this free fraction (2.5%), the estimated free plasma
quinacrine concentration after a therapeutic dose of quinacrine (3 mg
kg-1 day-1) for 8 days ranges from 0.5 to 3 nM, that is, much less than
the reported in vitro EC50 (120 nM). Hence, if the biophase for
antiprion activity is extracellular, that is, if the CSF quinacrine
concentration is the relevant active quinacrine concentration, the
current quinacrine dosage regimen will be wholly unable to achieve an in
vivo therapeutic antiprion concentration.

On the other hand, it has been suggested that quinacrine interacts with
the prion protein, PrPC within the endolysosomes (Doh-Ura et al., 2000
),
and that this interaction could prevent its conversion into the
pathogenic form, PrPSc, in the endocytic pathway (Vogtherr et al., 2003
).
In this investigation, the total quinacrine concentration in the brain
tissue was much higher (about 1000-fold) than the estimated CSF
quinacrine concentrations (0.5-3 nM), attaining a concentration of 3556
nM after an 8-day treatment, that is, a tissue/CSF ratio much greater
than the ratio of 13 predicted solely from equilibrium pH-pKa partition
considerations, and assuming that the pH of CSF is equivalent to that of
the culture medium (7.4). This finding is consistent with tissue
trapping of the drug (Dubin et al., 1982
)
and with previous reports that quinacrine is concentrated in tissues,
with only low concentrations in the CSF (Shannon et al., 1944
).
Lysosomal trapping of quinacrine (O'Neill et al., 1998
)
might account for most of the concentration of this drug in nervous
tissue, but the extensive in vivo uptake of quinacrine by cells may also
involve binding to other cell organelles or macromolecules. The
lysosomes might be a privileged site of action for quinacrine, but this
possibility does not exclude a plasma membrane biophase (Shyng et al.,
1993
).

Assuming that there is a lysosomal biophase, the total neuroblastoma and
total brain tissue concentrations may be considered as relevant in
relation to efficacy. As the total brain quinacrine concentration was of
the same order as the in vitro measured EC50 (2000-7000 nM), attaining
3556 nM after a therapeutic treatment of quinacrine (3 mg kg-1 day-1),
it can be reasonably assumed that the present dosage regimen sufficed to
achieve an appropriate in vivo lysosomal quinacrine concentration.
Consequently, it is likely that the lack of in vivo quinacrine efficacy
in the clinical trial was of pharmacodynamic rather than pharmacokinetic
origin. In agreement with this hypothesis, Barret et al. (2003)

demonstrated that quinacrine could interact with PrPC to inhibit PrPSc
formation in ScN2a cells, but was unable to affect the protease
resistance of pre-existing PrPSc from brain homogenates of BSE-infected
mice.

In conclusion, if the quinacrine biophase is located in the
extracellular compartment (or intracellularly in the cytosol), the range
of measured quinacrine concentrations required to obtain a 50% efficacy
level in the neuroblastoma will clearly never be achieved in vivo, even
with a toxic dose of quinacrine. In contrast, if the in vivo quinacrine
biophase is lysosomal, appropriate quinacrine exposure can be achieved
with a currently recommended therapeutic dosage regimen. In this
circumstance, the lack of quinacrine clinical efficacy suggests that the
in vitro quinacrine action of recovery of the neuroblastoma is not a
relevant in vivo mechanism of action to obtain clinical improvement in
treated patients.

Finally, from these experiments it can be recommended that in future
investigations of putative antiprion drugs, the in vitro drug potency
should be determined by measuring the actual in vitro drug concentration
in the potential biophases. It should not be assumed that the in vitro
biophase concentration is equal to the nominal concentration in the
culture system. In vivo pharmacokinetic investigations are also required
to predict whether a dosage regimen that has both antiprion effect and
is safe will achieve an appropriate in vivo concentration in the
possible biophases.


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Top of page

Acknowledgements

We thank S. Lehmann for providing the N2a cell clone, A. Chabert and G.
Costes for technical assistance and P. Lees for comments on the
manuscript. This study was supported by grants from the French National
Institute for Agricultural Research (INRA), from GIS prion and from the
General Direction for Education and Research (DGER) of the French
Ministry of Agriculture.

http://www.nature.com/bjp/journal/vaop/ncurrent/full/0706072a.html


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