Bone Marrow Transplantation (2002) 29, 313–319
 2002 Nature Publishing Group All rights reserved 0268–3369/02 $25.00
www.nature.com/bmt
Ex vivo purging
Taurolidine: preclinical evaluation of a novel, highly selective, agent
for bone marrow purging
I Ribizzi, JW Darnowski, FA Goulette, MS Akhtar, D Chatterjee and P Calabresi
Department of Medicine, Division of Clinical Pharmacology, Brown University and RI Hospital, Providence, RI, USA
Summary:
Taurolidine has been shown to have remarkable cytotoxic activity against selected human tumor cells at concentrations that spare normal cells. In this study we
have extended this observation and assessed the ability
of Taurolidine to purge tumor cells from chimeric mixtures of bone marrow (BM) and neoplastic cells. Normal
murine BM and human leukemic (HL-60) or ovarian
(PA-1) tumor cell lines were used as models. Exposure
of tumor cells to 2.5 mM Taurolidine for 1 h resulted
in the complete elimination of viable cells. In contrast,
exposure of BM to 5 mM Taurolidine for 1 h reduced
CFU-GM, BFU-E and CFU-GEEM colony formation by
only 23.0%, 19.6% and 25.2%, respectively. Inhibition
of long-term BM culture (LTBMC) growth following a
1 h exposure to 5 mM Taurolidine also was 苲20% compared to untreated LTBMC. Finally, chimeric cultures
were generated from BM and HL-60GR or PA-1GR
cells (tumor cells transfected with the geneticin resistance gene). Exposure of these chimeric cultures to 5 mM
Taurolidine for 1 h totally eliminated viable cancer cells
while minimally reducing viable BM cells. This finding
was confirmed by subsequent positive selection for surviving tumor cells with geneticin. These findings reveal
that Taurolidine holds promise for use in BM purging.
Bone Marrow Transplantation (2002) 29, 313–319. DOI:
10.1038/sj/bmt/1703359
Keywords: bone marrow purging; Taurolidine; positive
selection
Autologous BM transplantation (ABMT) may be a therapeutic option for the treatment of patients with advanced
neoplastic disease. The presence of clonogenic tumor cells
in cell collections, however, appears to contribute to posttransplantation relapse, regardless of whether cells from
marrow or peripheral blood progenitor cells (PBPCs) serve
as the graft. Indeed, ‘mobilization’ of tumor cells along
with HSCs was shown to occur after treatment with chemotherapy and G-CSF.1 Gene-marking studies in patients with
acute myelogenous leukemia (AML) or chronic myelogenous leukemia (CML) following ABMT substantiate these
observations and support this therapeutic concern.2,3
During the past two decades, investigators have
developed a wide range of techniques to remove tumor cells
from hematopoietic cell grafts. These negative purging
techniques include ex vivo treatment of marrow aspirates
or peripheral blood cell collections with chemical
agents (such as 4-hydroperoxycyclophosphamide or
mafosfamide), monoclonal antibodies (MoAbs), toxins, or
chemotherapeutic agents, used alone or in various combinations.4–17 Prospective randomized trials to assess the
potential superiority of purged grafts over unmanipulated
grafts are not available, however, reflecting the high cost,
limited availability and technically demanding nature of the
purging methods. Therefore, the development of effective,
safe and simple purging methods remains a highly desirable goal.
Taurolidine was developed in the 1970s as a broad-spectrum antibiotic18–22 (Figure 1). Its mechanism of action as
an antibiotic appears to be related to a chemical reaction
O2S
H
N
H
N
N
C
H2
SO2
H2O
N
H
N
H
N
+
N
SO2
HN
CH2OH
Methylol
Taurultam
Taurolidine
Taurultam
H2O
H2N
C
H2
H2
C
Taurine
Correspondence: Dr P Calabresi, or Dr JW Darnowski, Rhode Island Hospital, Department of Medicine, Division of Clinical Pharmacology, 593
Eddy Street, Aldrich Bldg Rm 124, Providence, Rhode Island, 02903,
USA
Received 9 July 2001; accepted 9 November 2001
O2S
H2N
SO3H
HOH2C
H
N
SO2
Methylol
Taurinimide
Figure 1 Structure of Taurolidine. Structure of Taurolidine and its major
breakdown products Taurultam, Taurinamide and Taurine. Upon breakdown, each molecule of Taurolidine generates 3 methylol-containing fragments that have been suggested as being responsible for its antibiotic and
endotoxin activities.
Taurolidine in bone marrow purging
I Ribizzi et al
314
between the active Taurolidine derivatives, methylol taurinamide and methylol taurultam, and structures on the wall
of bacteria23,24 that results in a disruption of bacterial cell
adhesion accompanied by a prevention of infection. It has
also been reported that Taurolidine can neutralize endotoxins, exotoxins and lipopolysaccharides released by bacteria.25–28 Clinically, intraperitoneal Taurolidine has been
used for the treatment of diffuse peritonitis, either as monotherapy or in combination with systemic antibiotics.18,29 In
this setting, its use has resulted in statistically significant
improvements in postoperative morbidity and mortality and
there have been no observed acute or chronic toxic effects
on hematological and biochemical parameters.
Based on these observations, experiments were initiated
in our laboratory to evaluate the potential ability of Taurolidine to inhibit tumor cell adhesion and growth. The results
of these studies revealed that Taurolidine inhibited the
growth of a variety of human tumor cells and that this effect
was associated with the induction of a potent apoptotic
effect.30 Equally important, this cytotoxic effect was not
observed in ‘normal’ cells such as NIH-3T3 (murine) and
NHLF (human) fibroblasts. In vivo studies confirmed that
Taurolidine exerted a potent antineoplastic effect in nude
mice bearing xenografts of ovarian, melanoma or glioblastoma human tumor cells.30–32 Reflecting this remarkable
tumor cell-specific effect, we hypothesized that Taurolidine
could possess utility as a BM purging agent. We now report
that a 1 h exposure to 2.5 mm Taurolidine completely eliminated viable cells in human leukemic (HL-60) and ovarian
(PA-1) tumor cell cultures but produced only a minimal
growth inhibitory effect against normal murine marrow.
Similarly, exposing chimeric cultures of marrow plus tumor
cells to this Taurolidine regimen also eliminated tumor cells
but only minimally affecting normal marrow viability.
These findings suggest that Taurolidine may represent a
new class of highly selective agents for purging autologous
BM or HSC collection. Preliminary aspects of this work
have been presented.33
Materials and methods
Reagents
Taurolidine was kindly provided by Carter Wallace Inc
(Cranbury, NJ, USA) as a 2% solution in 5% Kollidon
17PF. RPMI 1640, Dulbecco’s modified Eagle’s medium
(DMEM), fetal bovine serum (FBS) and medium supplements were purchased from Gibco/Life Technologies
(Grand Island, NY, USA). Long-term BM culture
(LTBMC) medium (MyeloCult M5300) and methylcellulose for colony assays (MethoCult GF M3434) were purchased from StemCell Technologies (Vancouver, BC,
Canada). All other chemicals were obtained from the Sigma
Chemical Company (St Louis, MO, USA).
Cell lines
HL-60 and HL-60GR cells (HL-60 cells transfected with
the gene conferring resistance to geneticin) used in this
study were kindly provided by Dr Z Han (Department of
Bone Marrow Transplantation
Molecular and Cell Biology, Brown University), and were
maintained in RPMI 1640 medium supplemented with 10%
FBS, 1 mm nonessential amino acids, and 1 mm sodium
pyruvate. The PA-1 and PA-1GR cells were provided by
Dr KA Whartenby (Department of Medicine, Brown
University). The PA-1 cell lines were maintained in DMEM
at high glucose concentration (4.5 g/l) supplemented with
10% FBS. All cell cultures were mantained at 37°C in a
humidified incubator under 5% CO2. Under these conditions the doubling time for all cell lines used in this study
was 苲24 h.
Mice
C57BL/6 female mice (38–56 days old) were purchased
from Charles River (Wilmington, MA, USA). Mice were
killed by CO2 asphyxiation and the marrow of femurs and
tibias from a single animal (苲4 × 107 cells) was harvested
under sterile conditions by flushing the marrow cavity with
10 ml of RPMI 1640 + 10% FBS, using a 27 gauge needle
fitted to a 1 ml syringe. Cells were washed twice, cell number determined electronically (Coulter Particle Counter;
Beckman Coulter, Miami, FL, USA), resuspended in
medium, and used in experiments as described below.
Assessment of cellular sensitivity to Taurolidine in
normal marrow
LTBMC assay: Aliquots of BM cells (苲2 × 107) were
exposed to high concentrations of Taurolidine (1–10 mm)
for 1 h at room temperature. Matched cell aliquots, obtained
from the same donor mouse, were treated similarly with
supplemented RPMI medium alone or with medium plus
5% Kollidon 17PF and served as control. After this 1 h
incubation period, cells were washed, counted electronically and plated in 35 mm tissue culture dishes (Costar,
Corning Incorporated, NY, USA) at a density of 3.75 × 106
cells/ml, in a final volume of 2 ml MyeloCult M5300 supplemented with 1 ␮m hydrocortisone sodium hemisuccinate. At weekly intervals the cultures were fed by replacing
50% of the supernatant with fresh medium. After 3 weeks,
the feeder/stromal layer was established and clusters of
hematopoietic cells with a cobblestone-like appearance
were recognizable. At this time point, cells taken from the
adherent and nonadherent layers were assayed separately.
The medium containing the nonadherent cells was removed
and reserved. Then the wells were washed with 2 ml of
fresh medium and, after gentle agitation, the washing was
pooled with the reserved non-adherent cell aliquot. Adherent cells were harvested by trypsinization (Sigma Chemical
Company). The cell number in both the adherent and nonadherent cell fractions was determined electronically. From
this differential count and the total number of cells recovered from each well the absolute number of myeloid and
nonmyeloid (adherent) cells/well was determined.
Colony assay: Aliquots of BM cells (苲2 × 107) were
exposed to Taurolidine (1–10 mm) for 1 h, washed, counted
and plated in 35 mm culture dishes in MethoCult GF
M3434 at a density of 2.0 × 104 cells/dish in a final volume
of 1.1 ml. Control cells were treated similarly with RPMI
Taurolidine in bone marrow purging
I Ribizzi et al
medium alone or medium plus 5% Kollidon 17PF. Triplicate cultures were incubated in a humidified atmosphere of
5% CO2 at 37°C. Benzidine-positive colonies were designated erythroid burst-forming units (BFU-E) and benzidinenegative colonies as granulocyte–macrophage colony-forming units (CFU-GM). BFU-E or CFU-GM were scored at
day 7 or 10 while CFU-GEMM were scored at day 12. In
all cases, colonies were defined as aggregates greater than
50 cells.
Assessment of tumor cell sensitivity to Taurolidine
The sensitivity of the HL-60, PA-1, HL-60GR and PA-1GR
cell lines to Taurolidine was assessed by an MTT assay.
Specifically, 1 × 106 tumor cells were exposed to various
concentrations (1–5 mm) of Taurolidine for 1 h at room
temperature and then washed. Cells (1 × 106) were then
added to a 25 cm2 flask in 10 ml of appropiate growth
media. After 21 days the cells were harvested and aliquots
containing 1 × 104 cells were added to each well of 96-well
flat-bottom plate (Costar, Corning Incorporated). Cells not
exposed to Taurolidine, or medium containing no cells,
were used as positive or negative controls, respectively.
Thereafter, 100 ␮l of a 2 mg/ml MTT solution (3–4, 5dimethylthiazol-2, 5-diphenyl tetrazolium biomide) in PBS
was added to each well and incubated for 3 h at 37°C. After
this period, the cells were centrifuged at 200 g for 10 min.
The resulting solute was aspirated and replaced with 200 ␮l
of DMSO. After gently shaking, the absorbance of each
well at 560 nm was determined using a Bio-Tek EL800
Universal microplate reader. An absorbance 2× that of the
negative control was considered positive of the presence of
viable cells.
Assessment of cellular sensitivity to geneticin (G418)
2 × 106 cells (PA-1, PA-1GR, HL-60, HL-60GR or BM)
in appropriate growth media were exposed to 1 mg/ml of
genticin (G-418) for 28 days. Cells were then harvested,
washed, and plated in a 96-well plate at a density of 1 × 104
cells/well. The presence of viable cells was then determined
by the MTT assay, exactly as described above. Medium
alone or cells unexposed to G-418, were used as negative
or positive controls, respectively.
BM purging
BM cells were mixed 2:1 with HL-60GR or PA-1GR cells
to achieve a final density of 1.5 × 106 cells/ml. The
resulting chimeric mixture was exposed to 5 mm Taurolidine for 1 h at room temperature, washed, and cells plated
in a 12-well plate at a density of 3 × 106 cells/well in 1 ml
of MyeloCult M5300 supplemented with 1 mm hydrocortisone sodium hemisuccinate. After 14 days, the medium of
the resulting cultures was completely discarded and fresh
medium ± 1 mg/ml of G-418 was added. Cells were then
incubated for an additional 28 days, harvested, plated in a
96-well dish and analyzed for viability by the MTT assay,
as previously described. Medium alone, untreated murine
BM alone, untreated tumor cells alone, or tumor cells plus
BM exposed to G-418 were used as negative or positive
controls, respectively.
315
Results
To determine the cytotoxic effect of Taurolidine in the HL60, HL-60GR, PA-1 or PA-1GR lines, cells were incubated
for 1 h in medium containing high concentrations (1–
10 mm) of this agent. Thereafter, Taurolidine was removed
and cell viability determined 3 weeks later by employing
an MTT assay. This 21 day outgrowth period allowed any
surviving cells to repopulate the cultures. The results of
this analysis revealed that Taurolidine, at a concentration
of 2.5 mm, completely eliminated viable cells from each of
the neoplastic cell cultures employed (Table 1).
Importantly, the sensitivity of these tumor cell lines to Taurolidine was not affected by transfection with the gene conferring resistance to G-418. To determine the effect of Taurolidine on normal BM, parallel experiments were
conducted using marrow cells freshly harvested from
C57BL/6 mice and similarly exposed to high concentrations of Taurolidine for 1 h. Thereafter, the ability of
exposed marrow to generate LTBMC or form specific progenitor colonies was assessed (Figures 2 and 3). Exposure
of BM to 5 mm Taurolidine slightly reduced the total number of viable cells recovered 21 days after exposure (苲23%)
as compared to untreated LTBMC controls. This method
assessed all myeloid and adherent marrow cells. To examine more closely fluctuations in specific marrow cell populations as a consequence of Taurolidine exposure, differential cell analysis was also performed. The results showed
that this exposure to Taurolidine induced a decrease in the
growth rate of adherent and myeloid cells by 24% and 18%,
respectively, as compared to controls (Figure 2). Progenitor
colony assays confirmed this slight anti-proliferative effect.
These colony assays, however, also revealed the absence
of a significant inhibitory effect on progenitor-specific
(CFU-GM, BFU-E and CFU-GEMM) colony formation
after Taurolidine exposure. Taurolidine-induced inhibition
of the colony-formation was 23% for CFU-GM, 20% for
BFU-E, and 25% for CFU-GEMM, compared to colony
formation by untreated marrow cells (Figure 3).
Experiments that mimicked the clinical setting were next
Table 1
Assessment of cytotoxicity in HL-60 and PA-1 cell lines after
1 h exposure to various concentration of Taurolidine
Cell line
IC100
HL-60
HL-60GR
PA-1
PA-1GR
1 mm
1 mm
2.5 mm
2.5 mm
Cells were exposed to Taurolidine (⬍10 mm) for 1 h. Immediately thereafter, the cells were washed and plated in a 96-well plate (3 × 103
cell/well). After 3 weeks of incubation, cell viability was determined by
an MTT assay. The results were compared with the values of the negative
and positive controls (medium alone and untreated cells, respectively).
Each value represents the minimum concentration of Taurolidine necessary to completely eliminate viable cells. Each condition was tested a
minimum of three times.
Bone Marrow Transplantation
Taurolidine in bone marrow purging
I Ribizzi et al
316
120
Control
+ Taurolidine
Relative percent growth
100
80
60
40
20
0
Myeloid cells
Adherent cells
Figure 2 The effect of a 1 h exposure to 5 mm Taurolidine on normal
murine LTBMC growth. BM cells, harvested from immunocompetent
mice, were exposed to 5 mm Taurolidine for 1 h. Immediately thereafter,
cells were washed, resuspended in fresh Myelocult M5300 supplemented
with hydrocortisone hemosuccynate, and plated at a concentration of
7.5 × 106/well in six-well tissue culture plates. After 3 weeks of incubation, myeloid and adherent cells were harvested separately and counted
electronically. The values are reported as a percentage of the cell number
determined in unexposed BM cell growth (positive controls). Each experiment was repeated a minimum of three times.
Control
120
exposure of BM cells to 1 mg/ml G-418 resulted in the
complete elimination of viable cells. In contrast, neither
transfected cell line was affected by this G-418 selection
regimen (Table 2). Exposure of a chimeric mixture of
BM + tumor cells to only G-418 for 28 days resulted in
viable cultures, presumable containing transfected tumor
cells. Exposing BM alone to 5 mm Taurolidine for 1 h
resulted in cultures containing viable cells when assessed
14 days after drug exposure. In contrast, exposure of transfected tumor cells alone to this Taurolidine regimen completely eliminated viable cells, as measured by the MTT
assay 14 days after drug exposure (Table 2).
Finally, to determine if Taurolidine specifically eliminated tumor cells from the chimeric cell population, these
mixed cultures were exposed to 5 mm Taurolidine for 1 h,
washed, and then incubated in fresh media for 14 days.
After this outgrowth period, only viable BM was expected
to be present. To determine if this was indeed the case, the
resultant cell cultures were positively selected for tumor
cells by exposure to G-418 (1 mg/ml) for 28 days. Any
tumor cells that survived Taurolidine purging would have
had 42 days to recover, resume proliferation and repopulate
the culture under G-418 selection. However, at the end of
the G-418 incubation period MTT analysis revealed no
viable cells following this dual selection regimen
(Table 2) (Figure 4).
Discussion
+Taurolidine
Relative percent growth
100
80
60
40
20
0
CFU-GM
BFU-E
Colony type
CFU-GEMM
Figure 3 The effect of a 1 h exposure to 5 mm Taurolidine on murine
BM colony formation. BM cells, harvested from immunocompetent mice,
were exposed to 5 mm Taurolidine for 1 h. Cells were then washed three
times, resuspended in Methocult GF M3434 and plated at a concentration
of 2 × 104/well in six-well tissue culture plates. After 14 days, BFU-E,
CFU-GM and CFU-GEMM colonies were scored. The values are reported
as percentage of the colony number observed as compared to unexposed
BM colony formation (positive controls). Each experiment was repeated
a minimum of three times.
initiated using fresh murine BM mixed with HL-60GR or
PA-1GR cells. In these studies HL-60 and PA-1 cell lines
transfected with the gene conferring resistance to geneticin
were used to allow the positive selection of surviving,
viable, tumor cells from these chimeric mixtures following
purging with Taurolidine. Initial experiments assessed the
sensitivity of either BM alone or transfected tumor cells
alone to either G-418 or Taurolidine. As expected, a 7 day
Bone Marrow Transplantation
The reinfusion of neoplastic cells has always been a concern in autologous BM transplant protocols. Direct evidence that the reinfusion of malignant cells may contribute
to relapse after autologous marrow transplantation in AML
and CML has been demonstrated by gene-marking studies
that reveal the presence of clonogenic tumor cells after
transplant.2,3 Protocols to purge tumor cells from marrow
or HSCs, while potentially beneficial, are not without risks.
Specifically, purging with chemotherapeutic agents may
result in varying losses of hematopoietic stem cells and thus
may add to the hematologic toxicity of ABMT. For this
reason there is a continuous need for new compounds that
selectively remove cancer cells, while sparing normal hematopoietic progenitor cells.
In this study we determined that highly selective and
effective BM purging could be achieved with Taurolidine,
an agent that, when given systemically at high doses, is
without significant toxicity. In the cancer cell lines used in
this study, ablative tumor cell killing was observed following 1 h exposure to Taurolidine at a concentration of
2.5 mm. This exposure regimen induced a total depletion
of cancer cells (6 log reduction in viable cell number) in a
chimeric mixture of marrow and neoplastic cells. The magnitude of this observed depletion in tumor cell number is
clinically acceptable. Indeed, harvested BM from leukemic
patients in remission may contain 106 leukemic cells/ml.
Therefore, any ‘prospective’ purging agent should be able
to eliminate selectively at least 6 logs of contaminating neoplastic cells.34,35 Of interest, this same exposure condition
induced only a 苲20% depletion of normal marrow
elements, supporting our previous observation that Tauroli-
Taurolidine in bone marrow purging
I Ribizzi et al
317
Table 2
Assessment of viable cells in culture containing BM alone, HL-60GR alone, PA-1GR alone,
BM + HL-60GR or BM + PA-1GR following purging with Taurolidine and/or subsequent selection with G-418
No treatment
G-418
Taurolidine
Taurolidine + G-418
No cells
BM
HL-60GR
PA-1GR
BM+HL-60GR
BM+PA1GR
−
−
−
−
+
−
+
−
+
+
−
−
+
+
−
−
+
+
+
−
+
+
+
−
Cells were exposed to 5 mm Taurolidine for 1 h. Immediately thereafter the cells were washed and plated in
a six-well plate (3 × 106 cell/well). After 2 weeks the cells were incubated in medium containing G-418
(1 mg/ml) for an additional 28 days. Finally, cells were transferred in a 96-well plate at a density of 104
cells/well and viability was determined by the MTT assay. The results were compared with the values of the
negative and positive controls (medium alone and untreated cells, respectively). Each condition was tested a
minimum of three times. (+) denotes conditions in which the absorbance/well is 2× that of the negative control.
(−) corresponds to a condition in which the absorbance/well is ⬍2× that of the negative control.
Percent positive wells
120
100
80
60
40
20
0
0 cell
1 cell
10 cells
Seeded cells/well
Figure 4 Macroscopic comparison of BM alone (a), HL-60GR alone (b),
untreated chimeric cultures of BM+HL-60R (c) and Taurolidine purged
chimeric cultures of BM + HL-60GR (d). (a) BM was harvested from
C57BL/6 mice and 3.75 × 106 were plated in a six-well plate. The two
layers of adherent and myeloid cells are recognizable in the culture. (b)
HL-60GR cells were plated in a six-well plate at a density of 1 × 106 in
LTBMC medium. (c) Unexposed chimeric cultures with BM (2 × 106) and
HL-60GR cells (1 × 106) were constituted and incubated for 14 days. BM
adherent elements are not clearly recognizable and are presumably masked
by proliferating neoplastic and non-adherent marrow cells. (d) BM
(2 × 106) was mixed with HL-60GR cells (1 × 106) and exposed to 5 mm
Taurolidine for 1 h. Then, cells were incubated in a six-well plate for 14
days. BM adherent and myeloid cells are recognizable in the picture.
dine exerted a selective cytotoxic effect in various human
cancer cell lines.
The mechanism(s) responsible for this selective cytotoxic
effect is unknown but may be unrelated to its proposed
mechanism of antibiotic action. As an antibiotic, Taurolidine was shown to interfere with bacterial adherence. We
have observed that Taurolidine is cytotoxic against hematological tumor cells, cells that grow in suspension. Presumably these cells would be much less affected by a drug with
anti-adherence activity. Furthermore, experiments in our
laboratory have revealed that Taurolidine-induced tumor
cell death was associated with the induction of apoptosis.30
In contrast, a 72 h exposure of ‘normal’ murine or human
fibroblasts to Taurolidine did not induce apoptosis and
resulted in only a temporary cell growth arrest, with full
Figure 5 The ability of a 42 day outgrowth period to generate detectable
tumor cell clones by the MTT assay. Zero, one or 10 HL-60GR or PA1GR cells/well were plated in a volume of 200 ␮l in 96-well plates. After
42 days the cell growth per well was quantified by the MTT assay. The
results are reported as percentage of positive wells detected. The experiments were repeated a minimum of three times.
recovery when Taurolidine was removed from the medium.
Of interest, preliminary mechanistic evaluation of Taurolidine in cultures of HL-60 cells has shown that cleavage of
procaspase 8, 7 and 3 occurred within 3 h of drug
exposure.36 This finding suggests that the apoptotic cascade
triggered by Taurolidine may involve surface signaling
events. Studies to elucidate the mechanism of action of
Taurolidine are in progress.
The ability of Taurolidine to efficiently purge tumor cells
from marrow was evaluated in chimeric cultures using normal murine bone marrow and human cancer cells transfected with the gene conferring resistance to G418. These
transfected cancer cell lines allowed the positive selection
of surviving, clonogenic, tumor cells after Taurolidine
purging. Chimeric cell cultures were maintained in drugfree medium for 14 days after Taurolidine purging and then
incubated for an additional 28 days in tumor cell selection
medium containing G-418. In this setting, a single cancer
cell that survived Taurolidine purging would have had sufficient time to repopulate the chimeric culture (Figure 5).
Indeed, we observed that both HL-60GR and PA-1GR cells
alone survived and proliferated during this G-418 selection
regimen. However, neither viable tumor nor BM was
detected following dual selection with Taurolidine and G418. Thus, Taurolidine was able to selectively and comBone Marrow Transplantation
Taurolidine in bone marrow purging
I Ribizzi et al
318
pletely purge contaminating tumor cells from the chimeric
culture. The choice to use this biologic method as an alternative to PCR-based methods was made because of its
ability to detect viable, clonogenic, cancer cells in the
purged chimeric cultures. Indeed, while PCR can reproducibly detect a limited number of tumor cells this analytical
method cannot identify cells with clonogenic potential. This
limitation of qualitative PCR in the detection of minimal
residual disease after BM transplant has already been highlighted in previous studies37–39 that underlined the clinical
finding that a PCR-negative result is not predictive of complete eradication of the leukemic clone. For these reasons
we chose to use this functional method to assess the
efficiency of Taurolidine as a purging agent.
Our findings reveal that a 5–6 log depletion in the number of cancer cells from a chimeric mixture of BM and
cancer cells can readily be achieved with this agent. Since
PSCs have replaced marrow as source of stem cells for
autotransplantation, we are planning to repeat these studies
using PBSC. Indeed, preliminary studies have shown that
Taurolidine exposure of human T cells, obtained from the
peripheral blood of healthy donors, does not significantly
affect their viability or ability to be activated. Concomitant
studies are also underway to assess the in vivo marrow
purging potential of Taurolidine in a murine model system.
In conclusion, our present results reveal that efficient and
highly selective purging of infiltrated BM is possible with
this agent. Based on this finding, and the observed low toxicity associated with the clinical use of this agent, further
evaluation of the purging potential of Taurolidine is
warranted.
Acknowledgements
This work was supported by Carter Wallace, Inc., Rhode Island
Hospital, Associazione Cristina Bassi contro le Leucemie Acute
dell’Adulto and PhARMA Foundation.
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