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Effect of captopril on normal and injured hemopoietic stem cells
TOXICOLOGYANDAPPLIEDPHARMACOLOGY
66, 102-108
Effect of Captopril R. Cardeza
Foundation
(1982)
on Normal and Injured Hemopoietic L.
for Hematologic
BOYD,~ Research,
Received
April
J. CARO, Thomas
AND Jeferson
8, 1982; accepted
A.
J. ERSLEV
University,
July
Stem Cells’
Philadelphia,
Pennsylvania
19107
23, I982
Effect of Captopril on Normal and Injured Hemopoietic Stem Cells. BOYD, R. L., CARO. J., AND ERSLEV, A. J. (1982). Toxicol. Appl. Pharmacol. 66, 102-108. In vitro and in vivo studies of the effect of captopril on murine hemopoietic stem cells were performed to elucidate the pathogenic relationship between this drug and agranulocytosis. Up to 20 &ml of drug in vitro or 26 weeks of 450 mg of captopril per kilogram body weight per day by mouth in vivo failed to produce myelotoxicity in normal Swiss mice. However, the same high-dose oral captopril therapy given to Swiss mice with busulfan-induced stem celt damage produced myelosuppression. The hematologic lesion was an isolated, highly significant depression of committed neutrophil-monocyte stem cells (CFU-NMs). These findings suggest that captopril may be selectively toxic for impaired neutrophilic stem cells, producing clinically significant neutropenia in individuals with a previously damaged bone marrow.
sion was observed. Nevertheless, it was noted that in about 5% of patients a decrease occurred in white blood cell count of up to 40% of the pretreatment value. During subsequent trials involving about 7000 hypertensive patients, many of whom were uremic and/or drug resistant, 35 reported cases developed severe neutropenia. Among those, 4 died either as a direct or indirect consequence of their agranulocytosis (Erslev et al., 1982). In all affected patients whose bone marrow was examined (20 of 35) the neutropenia was caused by a potentially reversible marrow suppression. Analysis of these 35 cases revealed that 32 (9 1%) had some degree of renal impairment. Since captopril and its metabolites are primarily excreted by the kidneys (Kripalani et al., 1980) the steady state blood levels at a given captopril dose are higher in these patients suggesting that the pathogenesis is a dose-related toxicity. Furthermore, 10 of these patients (29%) had previously received or were concurrently receiving known myelosuppressive agents, sug-
Captopril, an oral angiotensin I-converting enzyme inhibitor (Ondetti et al., 1977) is an effective therapeutic agent in many types of hypertension (Ferguson and Vlasses, I98 1; Vidt et al., 1982). Initial toxicity studies in rats and mice showed captopril to be well tolerated in amounts up to 100 times the anticipated maximal human dose. In some dogs, however, the ingestion of similar large captopril doses was associated with various cytopenias and bone marrow hypoplasia. Although captopril did not show inhibitory effects on dog neutrophil-monocyte stem cells in vitro, the in vivo observations raised the suspicion that it was potentially myelotoxic (Personal communication, J. C. Alexander, E. R. Squibb and Sons, Inc.). The earliest clinical trials, however, did not support this concern. Statistical analysis of blood counts of the first 2500 treated patients did not disclose any significant changes, and no definite case of bone marrow suppres’ Supported by NIH Grants 04612, AM 29780. and a grant from E. R. Squibb and Sons, Inc. ’ To whom all correspondence should be addressed. 0041-008X/82/130102-07$02.00/0 Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
102
CAPTOPRIL
AND
HEMOPOIETIC
gesting that captopril might be particularly toxic for a partially damaged marrow (Erslev et al., 1982).
To further elucidate the pathogenesis of captopril-related marrow suppression, studies are reported here first on the effect of captopril in vitro on normal murine granulocyte-monocyte stem cells, and second on the effect of high-dose captopril in vivo on the hemopoietic stem cells of normal mice and of mice with busulfan-induced latent bone marrow damage (Morely and Blake, 1974a, 1974b; Morely et al., 1976). METHODS Drugs. Captopril was obtained as a white, water-soluble powder from E. R. Squibb and Sons, Inc. Busulfan powder was donated by the Burroughs Wellcome Company and was solubilized in an acetone-water medium (Trainor and Merely, 1976). In vitro studies. Nucleated marrow cells (1 X 10s) from young random-bred Swiss mice (Ace Animals, Boyertown, Pa.) were cultured for CPU-NM colonies in the presence of various concentrations of freshly dissolved captopril (see below). In vivo studies. Initially random-bred, 12. to I6-weekold, female Swiss mice were separated into two groups. Group A mice received four biweekly ip injections of the acetone-water medium while group B mice received four biweekly ip injections of busulfan (20, 20, 20, and 10 mg/kg of body weight). After the fourth injections both groups were rested to allow the acute hemopoietic toxicity of busulfan to resolve. At Week 23 both group A and B mice were randomly subdivided into two subgroups, 1 and 2. Each subgroup I was fed normal chow (Purina Lab Chow) while the chow of each subgroup 2 had 450 mg of captopril/kg of body weight per day added for the next 26 weeks. At Weeks 14-15, 37-38, and 48-49, three to eight mice from each experimental group were used for hematologic studies. Peripheral blood was examined for absolute neutrophil count and hematocrit. After the animals were killed, the marrow from both femurs was aseptically flushed out with (Y-MEM tissue culture medium, and a single cell suspension was mechanically produced. The total number of nucleated marrow cells in each suspension was determined by Coulter Counter, and pluripotential stem cell (CPU-S) and neutrophilic stem cell (CPU-NM) assays were set up. CPU-S assays were by a modification (Jenkins et al., 1969) of the in vivo exogenous spleen colony assay technique developed by Till and McCulloch (McCulloch and
STEM
CELLS
103
Till, 1960; Till and McCulloch, 196 1). Young Swiss mice were prepared as marrow recipients with 850 rads of supralethal, total-body irradiation. Innocula of 2 X 10’ nucleated bone marrow cells harvested from each experimental animal were injected iv into eight irradiated murine recipients. Groups of noninnoculated irradiated recipients were kept as controls. On the ninth day after the innoculation all surviving recipient animals were killed. Their spleens were removed, fixed in Bouin’s fluid, and the number of splenic colonies enumerated. The mean number of splenic colonies for each group was corrected for the mean number of endogenous colonies found in the spleens from the uninnoculated control group. CPU-NM assays were set up in agar (Metcalf, 1977) with an innoculum of 1 X 10’ nucleated marrow cells per culture. Serum from endotoxin-stimulated mice provided a potent source of colony-stimulating factor (Quesenberry et al., 1972). Duplicate cultures were set up for each animal. After a ‘I-day incubation at 37°C in a humidified 5% CO2 atmosphere, each culture was scored under an inverted microscope for its CPU-NM colony number. Data analysis. Statistical analysis was performed through the courtesy of Dr. Patricia Langenberg of Temple University by the two-tailed t-test, the Scheffe multiple comparison test, and/or the analysis of variance.
RESULTS In Vitro Studies
The in vitro studies revealed that captopril in concentrations up to 20 &ml fails to suppress CFU-NM growth from normal murine marrow (Fig. 1). In Vivo Studies
The in vivo studies showed (Table 1) that busulfan given for 8 weeks causes a supprsssion of absolute neutrophils, hematocrit, CFU-NM, and CFU-S. However, the CFUS appeared to recover and numerically move to normal by Weeks 48-49. Figures 2,3, and 4a show that the ingestion by normal mice of 450 mg of captopril/kg/ day for 25 to 26 weeks did not cause a statistically significant decrease in the blood absolute neutrophil count or hematocrit or in the marrow CFU-S growth. Furthermore, the
104
BOYD, CARO, AND ERSLEV
you -p CONTROL
60
40
-
20 -
0
I
1
2.5
5
CAPTOPRIL
IO
20
CONCENTRATION
(yq/ml)
FIG. 1. In vitro effect of captopril on CF’U-NM growth of normal mouse bone marrow (expressed as percent of control).
same amount of captopril given to mice with busulfan-induced marrow damage (Figs. 2, 3, 4b) did not cause an additional decrease in these three parameters. Data in Fig. 5a demonstrate that captopril ingestion had no consistent suppressive effect on CFU-NMs from normal mouse marrow. However, data in Fig. 5b show that mice with busulfandamaged bone marrow had a highly significant depression of CFU-NM colony formation after both 15 and 26 weeks of 450 mg of captopril orally per kilogram body weight per day. Furthermore two by two TABLE MURINE
HEMOFQIESIS
analysis of variance analysis demonstrates that while busulfan and captopril show only additive myelosuppression after 15 weeks of captopril, the two drug treatments are exerting synergistic CFU-NM depression after 26 weeks of captopril ingestion. DISCUSSION The administration of captopril, a new antihypertensive agent, has been associated with the development of myeloid suppres1
AFTER BUSULFAN
TREATMENT
Busulfan, weeks after treatment Hematologic parameters Absolute neutrophils (lO’/~l) Hematocrit (%) CPU-NM colonies Per 1X lo5 nucleated marrow cells 96 of control CPU-S colonies Per 2 X 10’ nucleated marrow cells % of control
Controls (no busulfan)
14-15
37-38
2.2 + 0.1” 52 +1
2.3 f 0.4 52 +1
1.1 f 0.2 47 *2
1.0 + 0.2 46 +3
l49b 100
73
+8
54
+8
53
*8
2lb 100
48
+5
65
*5
108
f4
’ Mean of data from 11 animals in three separate experiments,
48-49
CAPTOPRIL
AND HEMOPOIETIC
STEM
105
CELLS
ABSOLUTE NEUTROPHILS (X103)
1 0
10
20
I 30
TIME
I
I
40
50
(weeks)
FIG. 2. The effect of prolonged captopril ingestion on the absolute neutrophil count of blood from normal mice and mice pretreated with busulfan.
sion and severe neutropenia in 0.3 to 0.5% of treated patients (Vidt et al., 1982; Erslev et al., 1982). Since captopril is primarily eliminated by the kidneys and since most of the affected patients had renal insufficiency, it was suspected that the neutropenia was SHAM INJECTIONS
caused by a dose-related toxic suppression of marrow precursors. In an attempt to examine this possibility, various amounts of captopril were added to normal murine marrow in vitro. In this system, however, no drug suppression of murine CFU-NM was observed even
t 111
BUSULFAN INJECTIONS
1 111 60 c
HEMATOCRIT
30
-
20
-
WO)
10 -
0.
I
I
10
20
I 30 TIME
1
1
40
50
(weeks)
FIG. 3. The effect of prolonged captopril ingestion on the hematocrit of blood from normal mice and mice pretreated with busulfan.
106
BOYD, CARO, AND ERSLEV
INJECTIONS
CFU-S ‘0 OF CONTROLS
100
-
60
-
60
-
40
-
20
-
iA
----rJ-
-
;/4
CONTROL
10 . 0
10
----i---f
BUSULFAN
1 20
1 30
1 40
1 50 TIME
0
1 10
I 20
I 30
40
I 50
(weeks)
FIG. 4. (a) The effect of prolonged captopril ingestion on the CFU-S growth of bone marrow from normal mice. (b) The effect of prolonged captopril ingestion on the CFW-S growth of the bone marrow of mice pretreated with busulfan.
at concentrations as high as 20 pg/ml. Although the blood level of captopril in patients with renal failure rarely exceeds 5 pgfml, the in vitro results do not rule out that murine CFU-NMs would have been suppressed if exposed to captopril for a longer period of time. Zn vivo the administration of 450 mg/kg/ day of captopril to normal mice for a period of 26 weeks did not result in a significant neutropenia or a reduction in the neutrophilic stem cells in the marrow. In comparison, all cases of neutropenia in humans have occurred in the first 12 weeks of the administration of captopril in doses not exceeding 10 mgf kg/day. The observation that 29% of patients who developed neutropenia had previous or concurrent exposure to myelosuppressive drugs raised the possibility that an already damaged marrow may be more susceptible to the toxicity of captopril (Lohrmann and Schreml, 1982). This conclusion appears to be the case for chloramphenicol which as shown by Mo-
rely et al (1976) has little effect on the marrow of normal mice but a suppressive effect on marrow stem cells in mice previously exposed to busulfan. In the present study with captopril, a latent marrow hypoplasia was induced by giving busulfan every 2 weeks for 8 weeks and then having the animals recover for another 17 weeks. In accord with the observations of Morely and co-workers (1976), the mice had a sustained decrease in the number and/or function of neutrophilic stem cells (CF’UNM). However, the pluripotential stem cells (CFU-S), after an initial suppression, did return to normal. When these mice received large amounts of captopril in their feed for the next 26 weeks, a significant decrease was observed in the number of functional neutrophilic stem cells (CFIJ-NM). This observation is in contradistinction to the findings with chlormaphenicol in which there was suppression of the pluripotential stem cells (CF’U-S). This observation suggests strongly that the induction of neutropenia by capto-
CAPTOPRIL
AND HEMOPOIETIC
STEM CELLS
107
b
+
REST
a
NO CAPTOPRIL
.
CAPTOPRIL
--
100 -
-
450mqlk9ld
-p---o I
80-
CFU- NM % OF CONTROLS
60
-
40
-
20
-
I
10 I 0
‘-“\
BUSULFAN
CONTROL
I 10
I 20
1 30
1 40
I 50 TIME
0
10
20
30
40
3”
Weeks)
FIG. 5. (a) The effect of prolonged captopril ingestion on the CPU-NM growth of bone marrow from normal mice. (b) The effect of prolonged captopril ingestion on the CPU-NM growth of bone marrow from mice pretreated with busulfan.
pril in some dogs and in occasional humans is not completely capricious but probably is caused by a specific action of the drug on early neutrophilic progenitor cells. In the present study selective myelosuppression could only be demonstrated in mice fed large amounts of captopril(50 times the therapeutic dose for humans) and having an already damaged or depleted neutrophilic stem cell compartment. It seems likely that in humans the same combination of an excessive plasma concentration of captopril or its metabolites (due to renal insufficiency) and a previous or concurrent exposure to myelotoxic agents will aggrevate the myelotoxic potential of captopril and may lead to clinically significant neutropenia. Such a combination should be looked for in any hypertensive patient scheduled for captopril therapy and if identified should call for careful hematologic surveillance.
REFERENCES ERSLEV, A. J., ALEXANDER, J. C., CARO, J., ANDBOYD, R. L. (1982) Hematologic side effects of captopril and associated risk factors. Cardiovasc. Rev. Rep. 3, 660670. FERGUSON,R. K., AND VLASSES, P. H. (1981). Clinical pharmacology and therapeutic applications on the new oral angiotensin converting enzyme inhibitor: captopril. Amer. Hear1 J. 101, 650-656. GRAVRAS, H., BRUNNER, H. R., AND GRAVRAS, I. (1981). Editorial. Captopril in the treatment of hypertension. Ann. Intern. Med. 95, 505-506. JENKINS, V. K., UPTON, C. A., AND ODELL, T. T., JR. (1969). Differences between exogenous and endogenous hematopoietic spleen colonies. J. Cell Physiol. 73, 141-148. KRIPALANI, K. J., MCKINSTRY, D. N., SINGHVI, S. M., WILLARD, D. A., VUKOVICH, R. A., AND MIG-DALOF, B. H. (1980). Disposition of captopril in normal subjects. Clin. Pharmacol. Ther. 27, 636-641. LOHRMANN, H.-P., AND SCHREML, W. (1982). C.vrotoxic Drugs and the Granulopoietic System, Chaps. I6. Springer-Verlag, New York. MCCULLOCH, E. A., ANDTILL, J. E. (1960). The radia-
BOYD, CARO, AND ERSLEV
108
tion sensitivity of normal mouse bone marrow cells. determined by quantitative marrow transplantation in irradiated mice. Rudiut. Rex 13, 1 15-125. METCALF, D. (1977). Hemopoietic Colonies, Chap. 3. Springer-Verlag, New York. MORELY, A., AND BLAKE, J. (1974a). An animal model of chronic aplastic marrow failure. 1. Late marrow failure after busulfan. Blood 44, 46-49. MORELY, A., AND BLAKE, J. (1974b). Haematopoietic precursor cells in experimental hypoplastic marrow failure. Aust. J. Exp. Biol. Med. Sci. 52, 909-914. MORELY, A., TRAINOR, K., AND REMES, J. (1976). Residual marrow damage: Possible explanation for idiosyncrasy to chloramphenicol. Brit. J. Haematol. 32, 525-531.
ONDETTI, M. A.. RUBIN, B.,
AND
CUSHMAN, D. W.
(1977). Design of specific inhibitors of angiotensinconverting enzyme: New class of orally active antihypertensive agents. Science 196, 44 l-449. QUESENBERRY, P.. MORELY. A.. STOHLMAN, F., JR., RICKARD, K., HOWARD. D., AND SMITH, M. ( 1972). Effect of endotoxin on granulopoiesis and colonystimulating factor. N. Engl. J. Med. 286, 227-232. TILL, J. E., AND MCCULLOCH, E. A. ( 196 1). A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Rex 14, 2 13-222. TRAINOR, K. J., AND MORELY, A. A. (1976). Screening of cytotoxic drugs for residual bone marrow damage. J. Nat. Cancer Inst. 57, 1237-1239. VIDT, D. G.. BRAVO. E. L., AND FOUAD, F. M. (1982). Drug therapy: Captopril. N. Engl. J. Med. 306, 214219.
66, 102-108
Effect of Captopril R. Cardeza
Foundation
(1982)
on Normal and Injured Hemopoietic L.
for Hematologic
BOYD,~ Research,
Received
April
J. CARO, Thomas
AND Jeferson
8, 1982; accepted
A.
J. ERSLEV
University,
July
Stem Cells’
Philadelphia,
Pennsylvania
19107
23, I982
Effect of Captopril on Normal and Injured Hemopoietic Stem Cells. BOYD, R. L., CARO. J., AND ERSLEV, A. J. (1982). Toxicol. Appl. Pharmacol. 66, 102-108. In vitro and in vivo studies of the effect of captopril on murine hemopoietic stem cells were performed to elucidate the pathogenic relationship between this drug and agranulocytosis. Up to 20 &ml of drug in vitro or 26 weeks of 450 mg of captopril per kilogram body weight per day by mouth in vivo failed to produce myelotoxicity in normal Swiss mice. However, the same high-dose oral captopril therapy given to Swiss mice with busulfan-induced stem celt damage produced myelosuppression. The hematologic lesion was an isolated, highly significant depression of committed neutrophil-monocyte stem cells (CFU-NMs). These findings suggest that captopril may be selectively toxic for impaired neutrophilic stem cells, producing clinically significant neutropenia in individuals with a previously damaged bone marrow.
sion was observed. Nevertheless, it was noted that in about 5% of patients a decrease occurred in white blood cell count of up to 40% of the pretreatment value. During subsequent trials involving about 7000 hypertensive patients, many of whom were uremic and/or drug resistant, 35 reported cases developed severe neutropenia. Among those, 4 died either as a direct or indirect consequence of their agranulocytosis (Erslev et al., 1982). In all affected patients whose bone marrow was examined (20 of 35) the neutropenia was caused by a potentially reversible marrow suppression. Analysis of these 35 cases revealed that 32 (9 1%) had some degree of renal impairment. Since captopril and its metabolites are primarily excreted by the kidneys (Kripalani et al., 1980) the steady state blood levels at a given captopril dose are higher in these patients suggesting that the pathogenesis is a dose-related toxicity. Furthermore, 10 of these patients (29%) had previously received or were concurrently receiving known myelosuppressive agents, sug-
Captopril, an oral angiotensin I-converting enzyme inhibitor (Ondetti et al., 1977) is an effective therapeutic agent in many types of hypertension (Ferguson and Vlasses, I98 1; Vidt et al., 1982). Initial toxicity studies in rats and mice showed captopril to be well tolerated in amounts up to 100 times the anticipated maximal human dose. In some dogs, however, the ingestion of similar large captopril doses was associated with various cytopenias and bone marrow hypoplasia. Although captopril did not show inhibitory effects on dog neutrophil-monocyte stem cells in vitro, the in vivo observations raised the suspicion that it was potentially myelotoxic (Personal communication, J. C. Alexander, E. R. Squibb and Sons, Inc.). The earliest clinical trials, however, did not support this concern. Statistical analysis of blood counts of the first 2500 treated patients did not disclose any significant changes, and no definite case of bone marrow suppres’ Supported by NIH Grants 04612, AM 29780. and a grant from E. R. Squibb and Sons, Inc. ’ To whom all correspondence should be addressed. 0041-008X/82/130102-07$02.00/0 Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
102
CAPTOPRIL
AND
HEMOPOIETIC
gesting that captopril might be particularly toxic for a partially damaged marrow (Erslev et al., 1982).
To further elucidate the pathogenesis of captopril-related marrow suppression, studies are reported here first on the effect of captopril in vitro on normal murine granulocyte-monocyte stem cells, and second on the effect of high-dose captopril in vivo on the hemopoietic stem cells of normal mice and of mice with busulfan-induced latent bone marrow damage (Morely and Blake, 1974a, 1974b; Morely et al., 1976). METHODS Drugs. Captopril was obtained as a white, water-soluble powder from E. R. Squibb and Sons, Inc. Busulfan powder was donated by the Burroughs Wellcome Company and was solubilized in an acetone-water medium (Trainor and Merely, 1976). In vitro studies. Nucleated marrow cells (1 X 10s) from young random-bred Swiss mice (Ace Animals, Boyertown, Pa.) were cultured for CPU-NM colonies in the presence of various concentrations of freshly dissolved captopril (see below). In vivo studies. Initially random-bred, 12. to I6-weekold, female Swiss mice were separated into two groups. Group A mice received four biweekly ip injections of the acetone-water medium while group B mice received four biweekly ip injections of busulfan (20, 20, 20, and 10 mg/kg of body weight). After the fourth injections both groups were rested to allow the acute hemopoietic toxicity of busulfan to resolve. At Week 23 both group A and B mice were randomly subdivided into two subgroups, 1 and 2. Each subgroup I was fed normal chow (Purina Lab Chow) while the chow of each subgroup 2 had 450 mg of captopril/kg of body weight per day added for the next 26 weeks. At Weeks 14-15, 37-38, and 48-49, three to eight mice from each experimental group were used for hematologic studies. Peripheral blood was examined for absolute neutrophil count and hematocrit. After the animals were killed, the marrow from both femurs was aseptically flushed out with (Y-MEM tissue culture medium, and a single cell suspension was mechanically produced. The total number of nucleated marrow cells in each suspension was determined by Coulter Counter, and pluripotential stem cell (CPU-S) and neutrophilic stem cell (CPU-NM) assays were set up. CPU-S assays were by a modification (Jenkins et al., 1969) of the in vivo exogenous spleen colony assay technique developed by Till and McCulloch (McCulloch and
STEM
CELLS
103
Till, 1960; Till and McCulloch, 196 1). Young Swiss mice were prepared as marrow recipients with 850 rads of supralethal, total-body irradiation. Innocula of 2 X 10’ nucleated bone marrow cells harvested from each experimental animal were injected iv into eight irradiated murine recipients. Groups of noninnoculated irradiated recipients were kept as controls. On the ninth day after the innoculation all surviving recipient animals were killed. Their spleens were removed, fixed in Bouin’s fluid, and the number of splenic colonies enumerated. The mean number of splenic colonies for each group was corrected for the mean number of endogenous colonies found in the spleens from the uninnoculated control group. CPU-NM assays were set up in agar (Metcalf, 1977) with an innoculum of 1 X 10’ nucleated marrow cells per culture. Serum from endotoxin-stimulated mice provided a potent source of colony-stimulating factor (Quesenberry et al., 1972). Duplicate cultures were set up for each animal. After a ‘I-day incubation at 37°C in a humidified 5% CO2 atmosphere, each culture was scored under an inverted microscope for its CPU-NM colony number. Data analysis. Statistical analysis was performed through the courtesy of Dr. Patricia Langenberg of Temple University by the two-tailed t-test, the Scheffe multiple comparison test, and/or the analysis of variance.
RESULTS In Vitro Studies
The in vitro studies revealed that captopril in concentrations up to 20 &ml fails to suppress CFU-NM growth from normal murine marrow (Fig. 1). In Vivo Studies
The in vivo studies showed (Table 1) that busulfan given for 8 weeks causes a supprsssion of absolute neutrophils, hematocrit, CFU-NM, and CFU-S. However, the CFUS appeared to recover and numerically move to normal by Weeks 48-49. Figures 2,3, and 4a show that the ingestion by normal mice of 450 mg of captopril/kg/ day for 25 to 26 weeks did not cause a statistically significant decrease in the blood absolute neutrophil count or hematocrit or in the marrow CFU-S growth. Furthermore, the
104
BOYD, CARO, AND ERSLEV
you -p CONTROL
60
40
-
20 -
0
I
1
2.5
5
CAPTOPRIL
IO
20
CONCENTRATION
(yq/ml)
FIG. 1. In vitro effect of captopril on CF’U-NM growth of normal mouse bone marrow (expressed as percent of control).
same amount of captopril given to mice with busulfan-induced marrow damage (Figs. 2, 3, 4b) did not cause an additional decrease in these three parameters. Data in Fig. 5a demonstrate that captopril ingestion had no consistent suppressive effect on CFU-NMs from normal mouse marrow. However, data in Fig. 5b show that mice with busulfandamaged bone marrow had a highly significant depression of CFU-NM colony formation after both 15 and 26 weeks of 450 mg of captopril orally per kilogram body weight per day. Furthermore two by two TABLE MURINE
HEMOFQIESIS
analysis of variance analysis demonstrates that while busulfan and captopril show only additive myelosuppression after 15 weeks of captopril, the two drug treatments are exerting synergistic CFU-NM depression after 26 weeks of captopril ingestion. DISCUSSION The administration of captopril, a new antihypertensive agent, has been associated with the development of myeloid suppres1
AFTER BUSULFAN
TREATMENT
Busulfan, weeks after treatment Hematologic parameters Absolute neutrophils (lO’/~l) Hematocrit (%) CPU-NM colonies Per 1X lo5 nucleated marrow cells 96 of control CPU-S colonies Per 2 X 10’ nucleated marrow cells % of control
Controls (no busulfan)
14-15
37-38
2.2 + 0.1” 52 +1
2.3 f 0.4 52 +1
1.1 f 0.2 47 *2
1.0 + 0.2 46 +3
l49b 100
73
+8
54
+8
53
*8
2lb 100
48
+5
65
*5
108
f4
’ Mean of data from 11 animals in three separate experiments,
48-49
CAPTOPRIL
AND HEMOPOIETIC
STEM
105
CELLS
ABSOLUTE NEUTROPHILS (X103)
1 0
10
20
I 30
TIME
I
I
40
50
(weeks)
FIG. 2. The effect of prolonged captopril ingestion on the absolute neutrophil count of blood from normal mice and mice pretreated with busulfan.
sion and severe neutropenia in 0.3 to 0.5% of treated patients (Vidt et al., 1982; Erslev et al., 1982). Since captopril is primarily eliminated by the kidneys and since most of the affected patients had renal insufficiency, it was suspected that the neutropenia was SHAM INJECTIONS
caused by a dose-related toxic suppression of marrow precursors. In an attempt to examine this possibility, various amounts of captopril were added to normal murine marrow in vitro. In this system, however, no drug suppression of murine CFU-NM was observed even
t 111
BUSULFAN INJECTIONS
1 111 60 c
HEMATOCRIT
30
-
20
-
WO)
10 -
0.
I
I
10
20
I 30 TIME
1
1
40
50
(weeks)
FIG. 3. The effect of prolonged captopril ingestion on the hematocrit of blood from normal mice and mice pretreated with busulfan.
106
BOYD, CARO, AND ERSLEV
INJECTIONS
CFU-S ‘0 OF CONTROLS
100
-
60
-
60
-
40
-
20
-
iA
----rJ-
-
;/4
CONTROL
10 . 0
10
----i---f
BUSULFAN
1 20
1 30
1 40
1 50 TIME
0
1 10
I 20
I 30
40
I 50
(weeks)
FIG. 4. (a) The effect of prolonged captopril ingestion on the CFU-S growth of bone marrow from normal mice. (b) The effect of prolonged captopril ingestion on the CFW-S growth of the bone marrow of mice pretreated with busulfan.
at concentrations as high as 20 pg/ml. Although the blood level of captopril in patients with renal failure rarely exceeds 5 pgfml, the in vitro results do not rule out that murine CFU-NMs would have been suppressed if exposed to captopril for a longer period of time. Zn vivo the administration of 450 mg/kg/ day of captopril to normal mice for a period of 26 weeks did not result in a significant neutropenia or a reduction in the neutrophilic stem cells in the marrow. In comparison, all cases of neutropenia in humans have occurred in the first 12 weeks of the administration of captopril in doses not exceeding 10 mgf kg/day. The observation that 29% of patients who developed neutropenia had previous or concurrent exposure to myelosuppressive drugs raised the possibility that an already damaged marrow may be more susceptible to the toxicity of captopril (Lohrmann and Schreml, 1982). This conclusion appears to be the case for chloramphenicol which as shown by Mo-
rely et al (1976) has little effect on the marrow of normal mice but a suppressive effect on marrow stem cells in mice previously exposed to busulfan. In the present study with captopril, a latent marrow hypoplasia was induced by giving busulfan every 2 weeks for 8 weeks and then having the animals recover for another 17 weeks. In accord with the observations of Morely and co-workers (1976), the mice had a sustained decrease in the number and/or function of neutrophilic stem cells (CF’UNM). However, the pluripotential stem cells (CFU-S), after an initial suppression, did return to normal. When these mice received large amounts of captopril in their feed for the next 26 weeks, a significant decrease was observed in the number of functional neutrophilic stem cells (CFIJ-NM). This observation is in contradistinction to the findings with chlormaphenicol in which there was suppression of the pluripotential stem cells (CF’U-S). This observation suggests strongly that the induction of neutropenia by capto-
CAPTOPRIL
AND HEMOPOIETIC
STEM CELLS
107
b
+
REST
a
NO CAPTOPRIL
.
CAPTOPRIL
--
100 -
-
450mqlk9ld
-p---o I
80-
CFU- NM % OF CONTROLS
60
-
40
-
20
-
I
10 I 0
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BUSULFAN
CONTROL
I 10
I 20
1 30
1 40
I 50 TIME
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10
20
30
40
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Weeks)
FIG. 5. (a) The effect of prolonged captopril ingestion on the CPU-NM growth of bone marrow from normal mice. (b) The effect of prolonged captopril ingestion on the CPU-NM growth of bone marrow from mice pretreated with busulfan.
pril in some dogs and in occasional humans is not completely capricious but probably is caused by a specific action of the drug on early neutrophilic progenitor cells. In the present study selective myelosuppression could only be demonstrated in mice fed large amounts of captopril(50 times the therapeutic dose for humans) and having an already damaged or depleted neutrophilic stem cell compartment. It seems likely that in humans the same combination of an excessive plasma concentration of captopril or its metabolites (due to renal insufficiency) and a previous or concurrent exposure to myelotoxic agents will aggrevate the myelotoxic potential of captopril and may lead to clinically significant neutropenia. Such a combination should be looked for in any hypertensive patient scheduled for captopril therapy and if identified should call for careful hematologic surveillance.
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ONDETTI, M. A.. RUBIN, B.,
AND
CUSHMAN, D. W.
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