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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 441-449
The Tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (Goralatide) Protects
From Doxorubicin-Induced Toxicity: Improvement in Mice Survival and
Protection of Bone Marrow Stem Cells and Progenitors
By
A. Massé,
L.H. Ramirez,
G. Bindoula,
C. Grillon,
J. Wdzieczak-Bakala,
K. Raddassi,
E. Deschamps de Paillette,
J.M. Mencia-Huerta,
S. Koscielny,
P. Potier,
F. Sainteny, and
P. Carde
From Institut Gustave Roussy, Villejuif, IPSEN-Biotech, Paris; and
Institut de Chimie des Substances Naturelles, CNRS, Gif-sur-Yvette,
France.
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ABSTRACT |
The tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP or Goralatide), a
physiological regulator of hematopoiesis, inhibits the entry into the
S-phase of murine and human hematopoietic stem cells. It has been shown
to reduce the damage to specific compartments in the bone marrow
resulting from treatment with chemotherapeutic agents, ionizing
radiations, hyperthermy, or phototherapy. The present study was
performed to assess the therapeutic potential of AcSDKP in vivo in
reducing both the toxicity and the hematopoietic damage induced by
fractionated administration of doxorubicin (DOX), a widely used
anticancer drug. Here we showed that AcSDKP could reduce DOX-induced
mortality in mice and could protect particularly the long-term
reconstituting cells (LTRCs) in addition to colony forming
units-spleen, high proliferative potential colony-forming cells, and
colony-forming units-granulocyte-macrophage (CFU-GM) from DOX
toxicity. The protection against DOX-induced mortality in mice was
improved when AcSDKP was administered for 3 days, at a dose of 2.4 µg/d, by continuous subcutaneous (SC) infusion or fractionated SC
injections starting 48 hours before DOX treatment. Moreover, the
recovery of the CFU-GM population in the AcSDKP-DOX-treated mice was
optimized by the subsequent administration of granulocyte colony-stimulating factor (G-CSF). The coadministration of AcSDKP with
DOX may improve its therapeutic index by reducing both acute hematotoxicity on late stem cells and progenitors and long-term toxicity on LTRCs. Optimization of these treatments combined with G-CSF
may provide an additional approach to facilitate hematopoietic recovery
after cancer chemotherapy.
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INTRODUCTION |
MYELOSUPPRESSION IS a major limiting
factor in anticancer chemotherapy. Repeated or high-dose cycles of
chemotherapy or radiotherapy may be responsible for severe stem cell
depletion leading to important long-term hematopoietic sequelae and
marrow exhaustion.1 Growth factors may reduce the
short-term side-effects2 but uncertainties remain about
long-term hematopoietic damage.3,4 Promising results were
obtained in the prevention of short-term myelotoxicity by the use of
negative hematopoietic regulatory factors. It has been proposed that
the use of exogenous inhibitors, which may also prevent quiescent
primitive hematopoietic cells from entering S-phase after chemotherapy
or radiotherapy, could protect this cell population from subsequent
doses of cytotoxic agents.5
In recent years, a number of molecules have been reported to exert a
suppressive effect on hematopoietic stem cell proliferation. These
include transforming growth factor- (TGF-), macrophage inflammatory protein-1 (MIP-1), tumor necrosis factor (TNF-), the pentapeptide pyro-Glu-Glu-Asp-Cys-Lys (pEEDCK), and the
tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP or Goralatide). All of
these factors are potential myeloprotectors because they were shown to
inhibit the proliferation of hematopoietic cells in vivo or in
vitro.6
AcSDKP was isolated and purified from fetal calf bone marrow and
subsequently chemically synthesized.7,8 It is
constitutively produced in vivo and biosynthesized in vitro by bone
marrow cells in murine long-term cultures.9 It has also
been suggested that macrophages produce AcSDKP, whereas bone marrow
stromal cells degrade it.10 It was reported that AcSDKP may
be derived from thymosin 4, which contains the complete N-terminal
AcSDKP amino acid sequence.11 Catabolism studies have shown
that AcSDKP is a physiological substrate of the N-terminal catalytic
site of angiotensin-I-converting enzyme (ACE), which cleaves the
tetrapeptide by a dipeptidyl carboxypeptidase.12-14 It has
recently been suggested that ACE has a role in the regulation of
hematopoiesis.15
Several in vitro inhibitory effects of AcSDKP have been described. The
reversible inhibition of the proliferation of murine and human early
progenitors, colony-forming units-granulocyte-macrophage (CFU-GM), and
burst forming units-erythroid (BFU-E) was observed in the presence of
the tetrapeptide.16-19 AcSDKP has also been shown to reduce
in vitro the proliferation of more primitive hematopoietic cells such
as the murine and human high proliferative potential colony-forming
cells (HPP-CFCs) as well as human long-term culture initiating cells
(LTC-ICs).16,20,21 Moreover, it inhibits the proliferative
response of purified human CD34+ cells to a combination of
seven growth factors.21 This inhibitory effect was
dose-dependent being maximal at 10 12 mol/L for
murine and at 109 mol/L for human hematopoietic
cells.16,18 The ability of AcSDKP to maintain the primitive
hematopoietic cells in quiescence is probably responsible for the in
vitro protection of human and murine progenitors from the toxicities of
3 azido-3deoxythymidine,22 mafosfamide,23 phototherapy,24 and
hyperthermy.17
In vivo, the administration of AcSDKP prevents the recruitment of
colony-forming units-spleen (CFU-S) into S-phase in mice submitted to
cytosine-arabinoside treatment.7,25 It was reported that
this activity was specific for cells in G0 or in early
G1.26 The protection of stem cell and
progenitor compartments was observed when AcSDKP administration was
combined with cytosine arabinoside,25 cyclophosphamide,25 5-fluorouracil,27 and
irradiation.28
All these biological properties of AcSDKP suggest possible therapeutic
applications for this molecule, in vivo, as an efficient hemoprotective
agent during repeated and intensive chemotherapeutic and
radiotherapeutic treatments and, in vitro, as an adjuvant to purging
methods.
The present study had two objectives. The first was to investigate
whether the administration of AcSDKP in vivo could protect hematopoietic stem cells in mice given lethal doses of doxorubicin (DOX), a major chemotherapeutic drug. The ability of AcSDKP to improve
the survival of DOX-treated mice and to enhance the recovery of the
differentiated progenitors as well as of the primitive hematopoietic
stem cells (long-term repopulating cells [LTRCs]) was examined. In
addition, dose, mode, and timing of AcSDKP administration relative to
the administration of DOX were investigated to develop a protective
regimen with improved capability. The second objective was to determine
whether the coadministration of AcSDKP with a growth factor,
granulocyte colony-stimulating factor (G-CSF), used routinely to reduce
short-term hematological effects of chemotherapy, could improve
myeloprotection.
Our data showed a protective effect of AcSDKP against DOX-induced
deaths and hematotoxicity, particularly on the most primitive stem
cells, the LTRCs. The effects on CFU-GM were optimized by using this
inhibitor of stem cell proliferation in combination with G-CSF,
suggesting a new approach to improve marrow protection during
chemotherapy.
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MATERIALS AND METHODS |
Animals.
Eight- to 12-week old BALB/c mice (Janvier CERJ, Le Genest-St-lsle,
France), housed under specific pathogen-free conditions, were used in
accordance with French legislation.
Drugs.
DOX was purchased from Pharmacia (Saint Quentin en Yvelines, France).
Synthetic AcSDKP was kindly provided by Ipsen-Biotech (Paris, France).
Recombinant G-CSF was purchased from Rhone Poulenc Rorer (Neuilly sur
Seine, France).
In vivo treatments.
As shown in Fig 1, AcSDKP (in saline) was
administered subcutaneously (SC), 24 hours or 48 hours before the DOX
treatment, either by injection or in a continuous infusion regimen. A
similar dose of AcSDKP (7.2 µg/mouse = 360 µg/kg) was given
according to several schedules: one SC injection 48 hours before DOX
treatment, three injections (48 hours, 24 hours, and 1 hour before
DOX), six injections (twice a day at 9:00 AM and 7:00
PM starting 48 hours before DOX) or nine injections (at 8:00
AM, 4:00 PM, and 12:00 PM each day, starting
48 hours before DOX). The six-injections modality was used to assess a
potential dose-response effect (total doses 0.072, 0.72, 7.2, 72, 720 µg/mouse). In the case of a continuous infusion, the total dose of
AcSDKP (7.2 µg/mouse) was delivered at a constant delivery rate of
100 ng/h for 3 days using minipumps (Alzet osmotic minipump type 1003 D
3 days, Charles Rivers, France) implanted along the dorsal lateral
flank. The pumps were implanted either 24 hours or 48 hours before the
beginning of DOX treatment and were removed 3 days later. Control
animals received saline either SC or through minipumps.
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| Fig 1.
Treatment schedules. (A) Assessment of the survival.
AcSDKP was administred SC, either as a continuous infusion delivered by
minipumps (dark ellipses) or as six injections (dark rectangles) beginning either 24 or 48 hours before the first DOX injection. Unless
stated otherwise, the total dose of AcSDKP was 7.2 µg/mouse (approximately 360 µg/kg). DOX was given IP at a dose of 2.65 mg/kg/injection twice on the first day and once on the second, to yield
a total dose of 7.95 mg/kg. (B) Evaluation of the recovery of
progenitors, HPP-CFC and CFU-S. AcSDKP was administered SC as a
continuous infusion delivered by minipump beginning 48 hours before the
first injection of DOX. The total AcSDKP dose was always 7.2 µg/mouse. (C) Assessment of LTRC survival. AcSDKP was delivered by
minipump 48 hours before the first DOX injection. On day 7, male mouse
bone marrow was grafted into irradiated female recipients. (D)
Combination of AcSDKP with G-CSF. AcSDKP was administered as six
injections beginning 48 hours before DOX treatment. G-CSF was injected
IP once a day at either 100, 300, or 500 ng/injection/mouse (approximately 5, 15, or 25 µg/kg) for 4 days, beginning on day 3.
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DOX was injected intraperitoneally (IP) according to a three-injection
protocol: twice a day on day 1 (at 10:00 AM and 5:00 PM) and once on day 2 (at 10:00 AM) at doses of 2.65 mg/kg/injection (total dose of 7.95 mg/kg).
G-CSF was injected IP (100, 300, or 500 ng/injection = 5, 15, or 25 µg/kg) once a day from day 3 to day 6 after the beginning of DOX
treatment.
Survival experiments.
AcSDKP or saline was administered to mice (30 animals/group) either by
an SC continuous perfusion or by SC injection. DOX was injected IP
according to the modalities described previously. In all experiments,
mortality was recorded daily.
Hematologic toxicity experiments.
The kinetics of recovery of three different cellular hematopoietic
compartments were followed after lethal DOX treatment using two
different protocols. In one protocol, AcSDKP was administered alone,
either by injection or as a continuous pump infusion before DOX. Four
or five mice from each group were killed on days 3, 4, 7, 11, 14, and
18. Bone marrow cells (BMCs) from tibias and femurs were collected and
the kinetics of marrow hematopoietic progenitor recovery were
evaluated. In another protocol, AcSDKP administration (6 SC injections
starting 48 hours before DOX) was followed by a subsequent
administration of G-CSF daily from day 3 to day 6 after DOX. BMCs from
tibias and femurs were collected on day 7.
CFU-GM assay.
CFU-GM were assayed as described by Worton.29 BMCs (5 × 104) of treated mice in 1 mL of minimum
essential medium (MEM) containing 100 units/mL penicillin, 100 µg/mL streptomycin, 2 mmol/L L-glutamine (GIBCO, Cergy-Pontoise,
France), 20% fetal calf serum (Eurobio, Les Ulis, France), 0.5 units
of murine recombinant granulocyte-macrophage colony-stimulating factor
(GM-CSF; Valbiotec, Paris, France), and 0.8% methyl-cellulose (Tebu,
Le Perray-en-Yvelines, France) were plated in 35-mm culture dishes.
Quadruplicate cultures were incubated for 7 days at 37°C in a
humidified 5% CO2 atmosphere. CFU-GM colonies consisting
of 50 or more cells were scored using an inverted microscope.
Colony-forming unit-spleen (CFU-S) assay.
CFU-S were studied using the spleen colony assay.30 BMCs of
treated animals were injected intravenously (IV) into eight irradiated
recipient mice (9 Gy from a 60Co source) at appropriate
concentrations to obtain about 12 macroscopic surface colonies per
spleen. Recipients were killed 12 days later and spleens removed and
fixed in Bouin's solution. Macroscopic spleen nodules were scored 24 hours after fixation.
HPP-CFC assay.
HPP-CFCs were monitored using a bilayer semisolid agar
assay.20,31 Two milliliters of complete medium (Dulbecco's
medium containing 20% horse serum, 2 mmol/L L-glutamine, 100 U/mL
penicillin, 100 µg/mL streptomycin) supplemented with 10%
conditioned medium from the WEHI 3B myelomonocytic leukemic cell line,
10% conditioned medium from the L929 fibroblast cell line, and 0.5%
melted agar (Bactoagar; Difco, Detroit, Michigan) were aliquoted into
55-mm diameter non-tissue-culture grade plastic petri dishes as the underlayer. Two milliliters of complete medium supplemented with 0.3%
melted agar and containing 3 × 104 BMC/mL were then
aliquoted over the prepared underlayers. Quadruplicate cultures were
incubated for 14 days at 37°C in a fully humidified atmosphere with
5% CO2. Twelve hours before the end of the culture, 1 mL
of a colorless 1 mg/mL
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT;
Sigma, Saint Quentin Fallavier, France) solution in saline was added,
allowing the staining of viable cells by INT processing into a red
derivative that precipitates inside cells. HPP-CFC macroscopic colonies
defined as those in excess of 2 mm were scored.
Long-term repopulating ability of BMCs.
In an attempt to investigate whether AcSDKP treatment was able to
protect the LTRCs, the repopulating ability of BMCs from AcSDKP-DOX
treated mice (DOX-AcSDKP-BMCs) was assessed32 and compared
with that of BMCs from DOX-treated mice (DOX-BMCs). DOX-AcSDKP-BMCs and
DOX-BMCs were obtained from seven donor male mice on day 7 after the
beginning of DOX administration and injected IV at concentrations of
104, 4 × 104, 6 × 104,
105, or 106 into lethally irradiated female
recipient mice (n > 10). The irradiation dose was 9.5 Gy from a
60Co source. The mortality of recipient mice was followed
for up to 5 months.
Y-chromosome polymerase chain reaction (PCR) analysis.
To determine whether the hematological reconstitution of recipient mice
was endogenous or exogenous,33 genomic DNA from peripheral
blood cells (PBCs) of mice surviving at 5 months posttransplantation was analyzed by PCR, amplifying a fragment of the Y chromosome. Heparinized blood (50 µL) was centrifuged three times for 15 seconds in 500 µL of Tris buffer (10 mmol/L, pH 8) containing 1 mmol/L EDTA
to lyse the red blood cells. Leucocyte membranes were broken by the
addition of 100 µL of Tris-HCl buffer (50 mmol/L) containing 1 mol/L
MgCl2, 50 mmol/L KCl, 0.5% Tween 20 and 10 mg/mL of protease K at
56°C for 45 minutes and then at 95°C for 10 minutes. Ten microliters of the mixture was used in 50 µL of PCR mix, including 140 ng of each: forward 5-TGGGACTGGTGACAATTGTC-3 and
reverse 5-GAGTCAGGTGTGCAGCTCTA-3, Y-chromosome-specific
primers, 0.5 U of Thermophylus aquaticus DNA polymerase (ATGC) and 5 µL of 10x PCR buffer (Perkin-Ellmer Cetus; St Quentin en Yvelines,
France). Samples were amplified for 35 cycles. Each cycle included
denaturation at 94°C for 1 minute, annealing at 55°C for 2 minutes and extension at 72°C for 3 minutes. Actin cDNA fragments
were amplified as a positive control; the forward and reverse actin
primers were 5-GTACCACAGGCATTGTGATG-3 and
5-GCAACATAGCACAGCTTCTC-3, respectively. Amplified cDNA
(10 µL) was run on agarose gel and poststained with ethidium bromide.
Gel photographs were scanned on a ScanMaker E6 (Microtek; International
Computer, Paris, France) and analyzed using the NIH Image 1.54 software.
Statistical analysis.
Results from survival experiments were analyzed using Fisher's exact
test on day 30 or 45. Student's t-test, Wilcoxon's, or Kruskal Wallis rank test were used to compare the results of the clonogenic assays.
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RESULTS |
The effect of AcSDKP on the survival of mice given lethal doses of DOX:
Importance of mode, timing, and schedule of AcSDKP administration.
The effect of AcSDKP on DOX-induced mortality in mice was first
evaluated when AcSDKP was administered using a 3-day continuous infusion starting either 24 or 48 hours before the first injection of
DOX. As shown in Fig 2A, DOX administered
alone at a dose of 2.65 mg/kg/injection induced a 65% lethality in
mice (LD65) with a median survival time (MST) of around 18 days. A
continuous AcSDKP infusion (7.2 µg/mouse) starting 48 hours before
the beginning of DOX administration significantly reduced the
percentage of mortality in DOX-treated animals on day 30 (27%
v 64%, P < .05). In fact, the MST increased from
around 18 days for DOX-treated animals to more than 42 days for the
AcSDKP-DOX-treated group. In contrast, when the AcSDKP infusion
started only 24 hours before DOX administration, mouse survival did not
significantly differ from that observed in the control DOX-treated
group. The optimal survival benefit was observed using the protocol
starting with an AcSDKP infusion 48 hours before DOX.
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| Fig 2.
Improvement of survival with AcSDKP in DOX-treated mice.
Representative experiments showing the survival rates of mice (n = 30) treated with DOX either alone (closed squares) or in conjunction with AcSDKP given either 24 (open circles) or 48 hours (open squares) before the first DOX injection. (A) Continuous administration of AcSDKP
(7.2 µg) by minipumps at a rate of 100 ng/h for 72 hours. (B)
Discontinuous administration of AcSDKP in six injections (1.2 µg/injection) given twice daily beginning at the time indicated. The
asterisk denotes a significant difference in day-30 survival compared
with the control group treated with DOX alone (P < .05; Fisher's exact test).
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A protocol using SC injections of AcSDKP was developed to investigate
whether a more simple treatment protocol would also result in
protection. AcSDKP was given 48 hours before DOX. When AcSDKP was
administered by SC injection, the results were similar to those
obtained with AcSDKP given by minipump (Fig 2B).
With the aim of optimizing the protocol of AcSDKP administration,
comparative studies were performed using the same total dose of AcSDKP
given in one or in multiple injections starting 48 hours before the
beginning of DOX treatment. Results (not shown) indicated that 7.2 µg/mouse of AcSDKP given in six or nine injections significantly
increased the survival of the mice both on day 30 (4% to 6% v
28% deaths; P < .05) and on day 45 (22% to 25%
v 67%; P < .05). The six- and nine-injection AcSDKP
treatment almost doubled the MST from 35 days to up to 60 days for the
AcSDKP-DOX-treated group. Survival improvement was not observed on day
30 when the same dose of AcSDKP was administered in only 1 or 3 injections.
The effect of AcSDKP on the recovery of CFU-GM, HPP-CFC, and CFU-S of
mice given lethal doses of DOX: Importance of mode and dose of AcSDKP
administration.
The kinetics of recovery were studied in parallel for three different
cell populations (CFU-GM, HPP-CFC, and CFU-S) in mice given lethal
doses of DOX alone or preceded by AcSDKP. As shown in
Fig 3, DOX-alone treatment led to a nadir
in all the cellular systems studied, which occurred around days 3 to 4, depending on the cell type. The number of cells returned progressively
to normal values by day 18.
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| Fig 3.
Improvement of the recovery of hematopoietic cells with
AcSDKP in DOX-treated mice. AcSDKP was administered continuously by minipumps at a rate of 100 ng/h for 72 hours beginning 48 hours before
the first DOX injection. In each experiment, mice (n = 5) were killed
at different times, bone marrow was collected and pooled, and colony
assays were performed: (A) CFU-GM, (B) HPP-CFC, (C) CFU-S. Results are
expressed as the mean ± standard deviation of three (B and C) or four
(A) experiments. Asteriks indicate significant differences between
experimental and control groups: *P < .05;
**P < .01 (Student's t-test).
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In mice given DOX plus AcSDKP, the recovery of CFU-GM (Fig 3A) was
improved on day 7 and on day 11 (P < .01) as compared with that obtained with DOX alone. Recovery was more pronounced and faster
when the tetrapeptide was given 48 hours rather than 24 hours before
DOX. The same improvement in CFU-GM number was observed on day 7 when
AcSDKP was given in six SC injections (results not shown). The highest
doses of AcSDKP (7.2, 72, and 720 µg/mouse) were shown to
significantly enhance CFU-GM recovery at day 7, whereas 0.072 and 0.72 µg/mouse were ineffective (results not shown).
A significant improvement of HPP-CFC recovery (Fig 3B) was observed on
both days 14 and 18 or only on day 14 when AcSDKP was given
respectively 48 or 24 hours before DOX. It should be pointed out that
the number of HPP-CFCs was significantly increased on day 3 (day of the
nadir) when AcSDKP was given 48 or 24 hours before DOX. Such a trend
could also be observed for CFU-GM. Overall, the rate of recovery was
faster when AcSDKP was given 48 hours before DOX.
The continuous 3-day perfusion of AcSDKP led to a faster recovery of
the CFU-S compared with that observed with DOX alone (Fig 3C). A
significant protective effect of AcSDKP against DOX toxicity on CFU-S
was evidenced on day 7 and on day 11 (P < .01) when AcSDKP
administration was started respectively 48 and 24 hours before DOX.
Protection of the primitive stem cells LTRC by AcSDKP.
The in vivo ability of AcSDKP to protect LTRC in DOX-treated mice was
evaluated when the tetrapeptide was given in a continuous regimen
starting 48 hours before DOX. As shown in
Fig 4A, the survival of lethally irradiated
mice (5 months postgrafting), injected with DOX-AcSDKP-BMCs was higher
than that of mice injected with similar numbers of DOX-BMCs. In fact, 6 × 104 control BMCs must be grafted to achieve a 100%
survival of recipient mice; a 16-fold number of DOX-BMCs
(106 cells) was necessary to induce a similar survival of
recipients, whereas only 105 AcSDKP-DOX-BMCs were required.
To check whether the hematopoietic reconstitution of mice grafted with
DOX-AcSDKP-BMCs was of donor origin, the Y-chromosome fragment was
amplified on DNA extracted from PBCs of surviving recipient mice,
because sex-mismatched grafting (male donor/female recipient) was
performed. As shown in Fig 4B, the presence of a 400-bp
Y-chromosome-specific amplicon in seven of seven recipients grafted
with DOX-AcSDKP-BMCs was observed. Comparison of the intensity of these
bands to that of controls consisting of DNA extracted from mixtures of
male and female BMCs (from 0% to 100% male cells) established that,
on average, more than 75% of the PBCs were of donor origin.
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| Fig 4.
LTRC protection with AcSDKP in DOX-treated mice. (A) Male
donor mice given saline, DOX alone, or in association with AcSDKP administred as a continuous infusion beginning 48 hours before the
first DOX injection were killed on day 7. Various numbers of their BMCs
were then injected into lethally irradiated female recipients.
(A) Recipient mice survival at 5 months postgrafting, as a
function of the dose of cells injected, *P < .02 (Student's t-test) between AcSDKP-DOX- and DOX-alone-treated
mice. (B) Left panel: Y chromosome PCR analysis of peripheral leukocyte
DNA from 7 female mice grafted with bone marrow from male donors given both AcSDKP and DOX. Right panel: Controls consisted of Y chromosome PCR analysis of DNA from a mixture of male and female BMCs (from 0% to
100% male cells) and were used for quantification of the data. Mixed
chimerism (an average of 75% donor cells) was seen in all long-term
survivors tested.
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Enhancement of the AcSDKP response on CFU-GM recovery by G-CSF.
The impact of a combined administration of G-CSF after DOX in the
sequence of the AcSDKP-DOX protocol was next investigated. The recovery
of CFU-GM in mice given AcSDKP in six SC injections initiated 48 hours
before DOX administration was compared with that evaluated in mice
receiving the combined AcSDKP-DOX administration and the subsequent
four IP injections of G-CSF on days 3, 4, 5, and 6 after DOX treatment.
The results presented in Table 1 show that
AcSDKP or G-CSF given independently, in association with DOX, allowed
the recovery of a higher number of CFU-GM. When compared with
AcSDKP-DOX or G-CSF-DOX, the combination of AcSDKP with G-CSF resulted
in an enhanced recovery of CFU-GM. This significant effect was observed
at all G-CSF doses studied.
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DISCUSSION |
Previous studies have shown the myeloprotective effect of the
tetrapeptide AcSDKP against two cytotoxic drugs, cytosine arabinoside and cyclophosphamide.25 Moreover, clinical trials with this peptide (Goralatide) in patients undergoing monochemotherapy with similar drugs led to an improvement of the neutrophil
recovery.34 These results prompted the initiation of the
present studies to assess the potential of AcSDKP as a protector
against marrow damage induced by doxorubicin, a widely used anticancer
agent. In addition, the effect of the combined administration of AcSDKP
and G-CSF on the CFU-GM recovery of DOX-treated mice was evaluated.
Preliminary experiments with DOX (results not shown) showed a
dose-response curve for mouse survival. Based on these results, the
total DOX dose of 2.65 mg/kg has been chosen to provide a mortality
greater than 60% on day 30. AcSDKP administration before DOX appeared
to protect mice from lethal doses of DOX and improved significantly
their survival. Such an improvement in mice survival, because of the
coadministration of AcSDKP and high doses of cytosine arabinoside or
cyclophosphamide has already been observed.25 TGF-,
another negative regulator of hematopoiesis was also shown to be
effective in vivo in protecting mice from acutely toxic doses of
DOX.35 Interestingly, the mortality was not reversed by a subsequent bone marrow graft, suggesting that TGF-
protection was at least partially mediated by nonhematopoietic
mechanisms.
The significant mortality induced by DOX administration in our
experiments was shown to be associated with a marked hematological toxicity. The myelosuppressive effect of DOX was evaluated by ex vivo
measurement of the level and duration of the nadir of primitive bone
marrow cells. The recovery of pluripotent stem cells as well as of
progenitors was faster and improved in AcSDKP-pretreated mice. The
variable effects on the different progenitors and stem cells may be
related to their specific cell cycle length. Because of the burden of
our experiments, no attempt has been made to assess the megakaryocytic
lineage, which has recently been reported to also be
protected.27,28 Our present observations are in agreement
with the protection of neutrophil, lymphocytes, CFU-GM, and
colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM) induced by AcSDKP in DOX-treated primates.36
The remarkable positive effect of AcSDKP observed on the protection of
LTRC is worth emphasizing because, in the long- term, effects on these
cells are of greater significance than protection of any other stem
cells and progenitors. In fact, a bone marrow graft containing only
105 cells obtained from AcSDKP-DOX-treated donors allowed
the survival of 100% of lethally irradiated recipients whereas
106 BMCs from DOX-alone-treated mice were required to
achieve the same effect. At 5 months posttransplantation, the
recipients had a hematopoietic reconstitution with cells from donor
origin. Therefore, the increased survival of mice grafted with BMCs
from AcSDKP-DOX-treated animals is probably because of the protection
of the LTRC after administration of AcSDKP. These findings confirm the
fact that primitive stem cell exhaustion, which often prevents the
continuation of chemotherapy, can be circumvented both in terms of
number and of function. Such a prevention has previously been shown for
pre-CFU-S, an undefined cell population consisting of a mixture of
CFU-S and long-term repopulating cells and for LTC-ICs with another negative marrow regulator, the pentapeptide pEEDCK in AraC-treated mice.37 Conversely, the chemokine MIP-1, a potent
inhibitor of hematopoietic stem cell proliferation, which does not
protect stem cells more primitive than CFU-S and MIP-1, given twice
daily for 7 days, did not prevent the 10-fold LTRC depletion induced by
5FU in mice.38 Thus, whereas the protection reported for MIP-1,38,39 TGF-,35
TNF-,40 and pEEDCK37,41 appears to be
restricted to certain hematopoietic compartments, such a preservation
of various hematopoietic cell compartments, including LTRCs, in
correlation with a survival improvement, as we describe with AcSDKP,
has never been reported with any other negative regulator. AcSDKP
appears to be quite unique in its ability to protect bone marrow.
To examine the protection achieved by AcSDKP several experiments
varying the dose, timing, and route of administration of AcSDKP were
undertaken. The best results were obtained with 7.2 µg of AcSDKP
given by a continuous 3-day infusion initiated 48 hours before the
first DOX injection. These conditions provided the best recovery of
CFU-GM, CFU-S, and HPP-CFC as well as a significant improvement of
mouse survival. Conversely, adequate protection was achieved against
some other toxic agents with different modalities of AcSDKP
administration. In vivo protective effect of AcSDKP against the
hematological toxicity of sublethal irradiation was reported only when
the tetrapeptide was given 24 hours before the
irradiation.28 In monkeys, a 16-hour interval between
AcSDKP and drug administration appeared to be sufficient to obtain
protection against DOX-induced toxicity.36 The variable
effects of AcSDKP associated with the use of different toxic agents and
modalities of administration may be linked with the specificity of the
mechanism of action of AcSDKP. In fact, the better mouse survival and
progenitor recovery observed when AcSDKP was started 48 rather than 24 hours before DOX could be related with the longer interval allowed
between the end of AcSDKP administration and the moment when stem cells return into cycle. A prolonged presence of AcSDKP may interfere negatively with the initiation of marrow recovery. The delay of the
onset and duration of AcSDKP activity and, consequently, the duration
of the quiescence of the hematopoietic target cells depend in part on
the catabolism of the tetrapeptide in defined experimental conditions.
Indeed, AcSDKP, a circulating natural peptide, is continuously degraded
in vivo by ACE.12,13 It has been shown that both
administration of cytotoxic drugs (unpublished results) and
irradiation15 are followed by an accelerated catabolism of
AcSDKP and a consequent transient decrease of its plasma level. Moreover, significant changes of plasma AcSDKP levels have been reported in patients with acute myeloid leukemia undergoing
chemotherapy.42 In such a context, repeated administration
of AcSDKP might lead to a brief but important increase of its
concentration in vivo and be more effective than the continuous
administration of low doses. To verify this hypothesis and to provide
the simplest protocol for AcSDKP administration, the efficacy of the
same total dose was evaluated when given in one, three, six, or nine
injections. AcSDKP given through six or nine SC injections was as
effective on survival as the continuous infusion, whereas the same dose given in one or three injections provided less or no protective effect.
CFU-GM recovery was also improved no matter what mode of AcSDKP
administration was used. This observation suggests that CFU-GM
protection alone is unable to account for the survival improvement of
DOX-treated mice.
As the concentration of AcSDKP was shown to be critical in vitro in
obtaining an effect on hematopoietic progenitor cells, a broad range of
doses was investigated. Only the highest doses of AcSDKP, varying from
7.2 µg to 720 µg, were able to improve significantly the recovery
of CFU-GM. A bell-shaped response has been previously observed in
several in vitro models, indicating an activity for AcSDKP at doses
greater than 106 mol/L and less than
1016 mol/L.16,18 Such a dose-dependent
effect was also suggested by the results of a phase II clinical study
in which AcSDKP appeared to better protect hematopoiesis at
intermediate doses (results not published). This response profile
suggests that accessory cells and/or release of other mediators
may be involved. Cashman et al43 reported that adherent
cells were required for the expression of the inhibitory effect of
AcSDKP and that the activity of AcSDKP could be blocked by the addition
of MIP-1, an antagonist of MIP-1 and of other hematopoietic
negative-regulating chemokines. However, the mechanism of AcSDKP
activity cannot be entirely explained through the action of MIP-1,
because AcSDKP possesses a broader range of activities as shown by its
protective effect on LTRCs. In vivo investigations on the effect of
AcSDKP on hematopoiesis in a canine model indicate that AcSDKP exerts
its inhibitory effect not only on hematopoietic precursors but also on
the function of marrow-derived stromal cells.44 This
finding is consistent with the earlier reports on the modulation of the
adherence of hematopoietic stem cells to a stromal cell line in the
presence of AcSDKP.8,45 Moreover, it has been shown in an
in vitro purging model that human stromal cells could be selectively
protected by AcSDKP from the toxicity of mafosfamide.46
These observations concur to suggest that AcSDKP can protect very
primitive hematopoietic stem cells in their natural environment and may
decrease the long-term sequelae caused by chemotherapy.
Finally, we have been interested in determining whether the biological
effect achieved by AcSDKP was additive to other types of
myeloprotection, namely, to the use of G-CSF as suggested using GM-CSF
(Bogden et al, unpublished data). The use of positive growth factors
postchemotherapy may lead to stem cell depletion either by inducing the
regenerating marrow to differentiate to such an extent that it would
exhaust the pool of the primitive stem cells or by driving the
primitive cells into cell cycle, making them more vulnerable to the
subsequent courses of chemotherapy.4 Therefore, in a set of
experiments AcSDKP preceded DOX administration where G-CSF was given
after DOX. In these experiments, the administration of
G-CSF resulted in a significant improvement of the CFU-GM recovery compared with that obtained with AcSDKP alone. In fact, the benefit from the combined sequence AcSDKP-G-CSF may be interpreted, first, as
if AcSDKP ensured protection of the stem cell and progenitor compartments from the toxicity of chemotherapy and, second, as if G-CSF
would induce more efficient recovery after progenitor depletion.
In summary, our preclinical studies using an inhibitor of hematopoietic
stem cell proliferation, the tetrapeptide AcSDKP, have clearly shown
that in vivo pretreatment with AcSDKP protects a broad range of stem
cells and progenitor cells from the toxicity of DOX lethal doses,
improves the survival of treated mice, and reduces the long-term toxic
effects of chemotherapy as evidenced by LTRC studies. Ultimately, the
combined administration of AcSDKP and G-CSF enhances the
myeloprotection achieved by the application of either AcSDKP or G-CSF
alone, suggesting that the use of a negative and positive regulator may
be helpful for the prevention of the acute chemotherapy toxicity on
hematopoiesis. All these results show the ability of AcSDKP alone or in
combination with stimulating factors to reduce both short-term and
long-term myelosupression. The results of phase I to II clinical trials
have already showed a reduced period of neutropenia in cancer patients
receiving Goralatide and either cytosine arabinoside or
ifosfamide.34 Therefore, if applied to the clinics, the use
of Goralatide should improve in the short term the therapeutic index of
anticancer chemotherapy, and, in the long term, it should decrease the
marrow sequelae.
| |
FOOTNOTES |
Submitted January 28, 1997;
accepted September 10, 1997.
Supported by Ipsen-Biotech, CNRS, INSERM, Jacques and Monique Roboh,
Henry and Louise Marchal, Asclepios and Suzanne Axel funds, grants 4/95
from the Association pour la Recherche sur le Cancer (ARC), and Contrat
de Recherche Clinique No. 95-6 from the Institut Gustave Roussy.
Address reprint requests to P. Carde, MD, Institut Gustave
Roussy, 39, rue Camille Desmoulins, 94800 Villejuif, France.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Drs E. Frindel, A. Bogden, F. Hérodin, L.L.
Pritchard, and A. Riches for their helpful suggestions. We also thank
P. Ardouin and A. Rouches for their excellent technical expertise. A
special acknowledgment is directed to Prof M. Tubiana and Dr M. Guigon
for the stimulating discussion and continuous support in the conduct of
these studies.
| |
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Abstract
Introduction
Abstract