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Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 441-449
By
From Institut Gustave Roussy, Villejuif, IPSEN-Biotech, Paris; and
Institut de Chimie des Substances Naturelles, CNRS, Gif-sur-Yvette,
France.
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.
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- 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 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 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.
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.
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 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 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.
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.
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.
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.
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.
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.
Submitted January 28, 1997;
accepted September 10, 1997.
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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Introduction
Abstract
(TGF-
(MIP-1
12 mol/L for
murine and at 10
azido-3
The possible role of inhibitors.
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