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Drugs 31: 52-63 (1986)
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antidepressants,
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Some drugs are
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adversedrug real
in many other (
tinger and colle
cidence of agran
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of the other ca.
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All rights reserved.
Drug-Induced Aplastic Anaemia and Agranulocytosis
Incidence and Mechanisms
Paul
c.
Vincent
,
The Kanematsu Laboratories, Royal Prince Alfred Hospital, Camperdown, Sydney
Summ,llry
Apia
Aplastic anaemia and agranulocytosis are uncommon but serious adverse effects of
drug therapy. They result from an adverse interaction between the drug and the haemopoietic pathway in certain susceptible individuals. The nature of this idiosyncratic interaction differs for diffefent drugs and possibly for different individuals. In some instances
an immune mechanism might be implicated, in others the patient's cells might carry a
genetic su01:ceptibility to the drug, while yet other patients might metabolise the drug abnormally. The idiosyncratic nature of these effects has made their investigation difficult,
but experimental studies have allowed some progress in our understanding, In a practical
sense, however, responsibility for preventing these problems will remain with clinicians,
who should be alert to the risks and revise their prescribing habits accordingly.
Table
A large number ofhaematological disorders can
follow the administration of a wide variety of drugs,
used in conventional dosage(Young 1984). Drugrelated anaemias, for example, may be due to immune haemolysis, oxidative stress, megaloblastic
change or dyshaemopoiesis with sideroblastosis.
Selective thrombocytopenia can occur following
exposure to a variety of drugs, due usually to peripheral platelet destruction but occasionally to inhibition of megakaryocytopoiesis. However, this
article deals only with drug-induced agranulocytosis and drug-induced aplastic anaemia. Between
them, these are infrequent but justifiably feared
complications of conventional therapy with drugs
which, in the great majority of individuals, are
harmless. These two drug-related disorders have
many features in common but differ in several important respects. Insight into the aetiology and
pathogenesisof aplastic anaemia and agranulocytosis has been gained by examining the similarities
and differences between the two disorders, and by
experimental studies of haemopoietic cells in vitro.
I. Drugs
Vincent
repo
& de Gruc~
Antibacterials
Chloramphenicc
1.
Incidence
The precise incidence of either agranulocytosis
or aplastic anaemia following exposure to a drug
is hard to define accurately, due to difficulty in obtaining reliable information regarding drug usage,
i.e. the denominator of any risk calculation. A literature searchfor the period 1969 to 1979 (Young
& Vincent 1980)found 714 papers reporting agranulocytosis as a complication of drug therapy, and
this almost certainly represents only a small fraction of the numbers of caseswhich occurred in that
time. Many others would have either not been submitted for publication, or not accepted. The drugs
implicated in these papers included antibiotics, antimalarials, sulphonamides, antithyroid drugs, hypoglycaemic agents, antiarrhythmics, antihyper-
Sulphonamides
Anti-inflammatory/a
PhenylbutazonE
Oxyphenbutazc
Gold salts
Indomethacin
Anticonvulsants
Phenytoin
Methoin
Diuretics
Chlorothiazide
a
This
list
is of (
clinical
ground
range
1 : 10.0(
phenbutazone
I
Drug-Induced Aplastic Anaemia and Agranulocytosis
tensives, diuretics, anticoagulants, anticonvulsants,
antidepressants, phenothiazines, tranquillisers,
levodopa, analgesics,anti-inflammatory drugs, allopurinol, levamisole, D-penicillamine, antihistamines and H2-antagonists (tables I and II). Identifying the causative agent is obviously difficult.
Some drugs are more frequently reported in this
context than others, but in the caseof patients being
treated with a variety of agentsthe causative agent
need not necessarilybe the 'most guilty' one on the
list.
The: best data on the overall incidence of druginduced agranulocytosis come from Sweden, where
adversedrug reaction reporting is more reliable than
in many other countries. In a recent survey, Bottinger and colleagues (1979)' found an annual incidence of agranulocytosis of 2.6 per million. Over
a lO-year period there were 199 cases of drug-induced agranulocytosisout ofa total of nearly 12,000
adverse haematological reactions reported. More
deaths (63; 32%) occurred in this group than in any
of the other categories of drug-related blood disorders.
Table I. Drugs reported
Vincent
& de Gruchy
to cause aplastic
1967; Williams
anaemia.
53
The other well-documented estimate of the incidenceof drug-related agranulocytosiscomes from
Pisciotta ( 1973) who screened with serial blood
counts all new patients commencing long term
phenothiazine therapy, and found an incidence of
agranulocytosis of I per 1300. By contrast, there
was a much higher incidence (1/11) of benign transient neutropenia during phenothiazine treatment.
Drug-induced aplastic anaemia is, fortunately,
much less common. In different series, from onethird to two-thirds of all casesof aplastic anaemia
have been drug related, the others of course being
viral or idiopathic (Heimpel & Heit 1980; Tso et
al. 1977; Vincent & De Gruchy 1967; Williams et
al. 1973). Chloramphenicol and the pyrazolones
phenylbutazone and oxyphenbutazone have been
the drugs most frequently responsible. The risk for
chloramphenicol has been estimated at between
1/10,000 and 1/40,000, while that for phenylbutazone or oxyphenbutazone is between 1/30,000
and 1/300,000. These figures are much less than
the risk of aplasia with cytotoxic drugs, which occurs inevitably ifa large enough dose is given. Idio-
(after Alter et al. 1978; Australian
Drug Evaluation
Antithyroid
Antibacterials
Propylthiouracil
Sul~,honamides
Methylthiouracil
Carbimazole
/antirheumatic
agents
Phenylbutazone
Potassium
perchlorate
Thiocyanate
Oxyphenbutazone
Gold salts
Oral hypoglycaemic
Chlorpropamide
Indomethacin
Psychotherapeutic
Anticonvulsants
agents
and other
sulphonylureas
drugs
Chlorpromazine
Phenytoin
Methoin
Chlordiazepoxide
Antimalarials
Diuretics
Chlorothiazide
1981; Niewig 1974;
drugs
Chloramphenicol
Anti-inflammatory
Committee
Elt al. 1973, 1983)
and other thiazides
Amodiaquine
Chloroquine
Mepacrine
(quinacrine)
This list is of drugs for which an association with aplastic anaemia, as an idiosyncratic reaction, has been well established on
clinical grounds. The frequencies with which these reactions occur have not been defined for most drugs, but estimates in the
range 1 : 10,000 to 1 : 40,000 have been made for chloramphenicol and 1 : 30,000 to 1 : 300,000 for phenylbutazone and oxyphenbutazone.
54
Analgesics and anti-inflammatory/
antirheumatic agents
Amidopyrine
Allopurinol
Colchicine
D-Penicillamine
Fenoprofen
Gold salts
Ibuprofen
Indomethacin
Levamisole
Oxyphenbutazone
Pentazocine
Phenylbutazone
Psychotherapeutic drugs
Chlorpromazine
Chlordiazepoxide
Clomipramine
Clozapine
Desipramine
Diazepam
Fluphenazine
Imipramine
Mepazine
Meprobamate
Methylpromazine
Prochlorperazine
Promazine
Thioridiazine
Trimeprazine
Sulphonamides
CephaJexin
Aplasti
~
Cephalothin
Chloramphenicol
Clindamy~in
Doxycycline
Flucytosine
Gentamicin
Griseofulvin
Isoniazid
lincomycin
Metronidazole
Nitrofurantoin
P-Aminosalicylic acid
Rifampicin
Streptomycin
Antimalarials
AmOdiaquine
Dapsone
Hydroxychloroquine
auinine
Pyrimethamine
H2-Receptor entegonists
Cimetidine
Metiamide
Antithyroid
drugs
Carbimazole
Methylthiouracil
Propylthiouracil
Potassium
perchlorate
Oral hypoglycaemic
-..J
agents
Chlorpropamide
Tolbutamide
Diuretics
Acetazolamide
Bumetanide
Chlorothiazide
Chlorthalidone
Ethacrynic
acid
Hydrochlorothiazide
Methazolamide
Anticonvulsants
Carbamazepine
Ethosuximide
Methoin
Phenytoin
Primidone
Troxidone (trimethadione)
Antiparkinsonian drugs
Levodopa (L-dopa)
Antibacterial/antifungal agents
Penicillins (high dose)
Drug-Induced
Antihistamines
Brompheniramine
Mebhydrolin
Promethazine
Thenalidine
Spironolactone
Cardiovascular
Captopril
Diazoxide
Disopyramide
Hydralazine
Methy!dopa
Pindolol
Procainamide
Propranolol
drugs
Fig. 1. Schematic
repl
(clu-E). granulomono
stem cells maintain ti
primitive
stem cells ~
Recognisable precur
critical stage of cell n
The circulation
ti'11e
time 7 hours). The IE
the blood. Decrease,
from damage to cfu-
syncratic aplasti
aplasia differ in
the idiosyncrati.
pendent of dose
after drug admi
taneous recover
duced aplasia is
ally develops
administration,
supported thro\
usually reversit
1.
Normal
Although dl
drug-induced ;
I
I
54
Drug-Induced
Aplastic Anaemia and Agranulocytosis
55
1981
.14
Marrow
I~
Blood
1
--
/
bcfu-~
<
~
~M) 0--.<
A-P/
HPSC
~
"
+
,---
::J -+<C=:::J
1
cfu-Meg
i-
Committed
stem cells
precursors
Fig. 1. Schematic representation
of haemopoiesis.
(cfu-C GM) or megakaryocytopoiesis
Plu~ipotential
by re-entrant
stem c~lIs (HPSC) give rise to committed
arrows).
primitive stem cells which also give rise to B-Iymphocytes.
Re.:ognisabie precursors of each cell series proliferate (shown
critical stage of cell maturity
The circulation
is reached, but differentiation
ti'11e span in the blood for erythrocytes
time 7 hours). The levels of haemoglobin,
the blood. Decreased
production
platelets
myeloid
precursors,
Normal
Although drug-induced aplastic anaemia and
drug-induced agranulocytosis are different dis-
stem cells for erythropoiesis
to regulators.
of HPSC, not shown
lines) and differentiate.
HPSC and committed
in this figure,
Proliferation
for several days before mature cells are released
9 to 11 days, and for granulocytes
are determined
by the rate of production
to the HPSC, as in aplastic
or circulating
Haemopoiesis
which respond
Antecedents
is 120 days, for platelets
and granulocytes
syncratic aplastic anaemia and cytotoxic-induced
aplasia differ in several other important respects:
the idiosyncratic disorder is unpredictable, independent of dose, frequently delayed in onset (even
after drug administration has ceased), and spontaneous recovery is rare. By contrast, cytotoxic-induced aplasia is predictable, dose-dependent, usually develops 14 to 21 days after drug
administration, and (provided the patient can be
supported through the period of pancytopenia) is
usually reversible.
2.
(cfu-Meg)
by expanding
continues
of all 3 series will follow damage
from damage to cfu-C (GM), recognisable
Mature
forms
Recognisable
(cfu-E), granulomonocytopoiesis
stem cells maintain their own number (shown
D
anaemia.
are even more
ceases
when a
into the blood.
10 hours (half-
and survival
Agranulocytos\s
time in
can result
granulocytes.
eases,they have in common an adverse reaction
of some sort between the drug and part of the haemopoietic pathway. Recent studies,reviewed below,
have helped define the site and nature of these interactions, but it is necessary first to review our
current understanding of normal hilemopoiesis [fig.
1]. The system is fed by a series of haemopoietic
pluripotential stem cells (HPSC), distinguished
functionally by their ability to maintain their own
number, to provide an input of cells into the next
most mature compartment and to respond to regulatory signals. The antecedents of HPSC, not
shown in the figure, are even more primitive progenitors capable of differentiating also into B lymphocytes.The progeny of the HPSC are committed
stem cells which are restricted to one line of dif-
Drug-Induced Aplastic Anaemia and Agranulocytosis
ferentiation and are more susceptible to regulatory
controls but still retain the ability to self-replicate.
The committed stem cell compartments lead
into erythropoiesis, granulomonocytopoiesis and
megakaryocytopoiesis,respectively, to give rise to
the morphologically recognisable forms seen in
bone marrow aspirates. Initially, cellular prollferation and maturation occur together, but a point
is reached in each of the series where cellular maturation stops further division (Cronkite 1979;
Cronkite & Vincent 1969; Stohlman et al. 1968;
Yataganas et al. 1970). Maturation continues for
some time in the marrow, after which cells are released into the blood where they circulate for periods ranging from 120 days (for erythrocytes) to
as short as 7 hours (half-time for granulocytes). The
total production of cells by the haemopoietic system is considerable, being of the order of 4 to 7 X
107cells/kg/h for granulocytes, and 10 to 14 x 107
cells/kg/h for erythrocytes (Vincent 1977).
In man, normal haemopoiesis proceeds only in
the bone marrow, in a unique microenvironm~nt
consisting of stroma, microvasculature, fibroblasts,
fat cells, endosteal cells and bone. In this setting,
the HPSC pool provides an orderly flow of cells,
through committedstt:m cells, into erythropoiesis,
granulomonocytopoiesis,megakaryocytopoiesisand
B-celllymphopoiesis. The system is under the control of humoral regulators, of which the best-defined is erythropoietin (Fried 1979;Stohlman 1968),
and short-range cellular regulators, including particularly substancesreleased by monocytes, granulocytes and lymphocytes (Boyurn et al. 1980;
Broxmeyer et al. 1977; Cline & Golde 1979;
Cronkite et al. 1977; Dexter et al. 1977; Kurland
& Moore 1977; Lajtha 1975; Lord 1979; Peluset
al. 1979; Mangan & Desforges 1980).
,
HPSC can be assayedin the mouse as those cells
capable of giving rise to colonies in the spleens of
irradiated recipient mice (cfu-S) [Till ct al. 1964],
and in man, possibly as cells forming mixed colonies in culture (Messner & Fauser 1980). Stem
cells committed to erythropoiesis, to granulomonocytopoiesis, to megakaryocytopoiesis and to
lymphopoiesis can be assayed in man and in the
mouse by cultures in which colonies of differen-
56
tiated cells can be recognised after 7 to 21 days,
depending on the system. It is well-established that
these colonies are clonal in origin, and the cells
from which they arise are known as colony-forming units, abbreviated for example as cfu-E or bfuE for erythropoietic progenitors, cfu-GM for the
precursor of granulocytes and monocytes and so on
(Metcalf et al. 1982; Vincent 1977).
Other culture systems which have contributed
to our understanding of haemopoietic stem cells
include diffusion chamber cultures in mice (E6yum et al. 1972; Chikkappa et al. 1980) and long
term cultures of murine (Dexter et al. 1977; Williams et al. 1977) or human (Gartner & Kaplan
1980) marrow.
A family of glycoproteins, designated colonystimulating activity (CSA), synthesised by monocytes, stimulate the proliferation and differentiation of cfu-GM (Cronkite 1977; Kurland & Moore
1977). 'Switching' between granulocytopoiesis and
monocytopoiesis is determi~ed by which class of
CSA is produced (Metcalf et al. 1982). Lactoferrin
from granulocytes inhibits production of granulocyte-CSA by monocytes, providing a negative feedback loop, and prostaglandin E, produced by monocytes, inhibits proliferation and differentiation of
cfu-GM (Pelus et al. 1979). Normal non-E, non-T
lymphocytes inhibit cfu-GM (Morris et al. 1980),
and erythropoiesis appearsto be modulated by balanced T-Iymphocyte stimulation and monocyte inhibition (Mangan & Desforges 1980).
3. Experimental
Aplastic
Studies
in
Anaemia
Techniques which have been devised for evaluating the functional integrity of the microenvironment include the support of long term marrow
cultures, the formation of fibroblast colonies (cfuF) in vitro (Kaneko et al. 1982), the capacity to
support sustained marrow regeneration (Dexter et
al. 1977;Zipori et al. 1982), or the growth oftransplanted syngeneic marrow in experimental animals.
In experimental animals, microenvironmental
damage is responsible for the persistent aplasia in
Drug-Induced Apia
1-4--
p
I~\
"
Fig.
2.
Schema
maturation
--'
I
of
through
bones exposed1
than 4000 rads)
tium-89 (Klassl
tributes to the ;
or busulphan (
for the latter is
argued for the
damagein the ~
(Camitta et al.
clusive evidenc
coming. Despi
there is no con
microenvironn
Indeed, the el
plantation in
anaemia, the 1
mix in the ma
of patients (B
Kern et al. 19~
to demonstra'
in the macro
(Samson et a)
microenviron
56
Drug-Induced Aplastic Anaemia and Agranulocytosis
days,
d that
: cells
Marrow
I~
formr bfuIr the
,.
buted
PQ
I fdJ )- +~ ~
,
/
,-/
cells
(B6-
I?//A'
I~I
\
/
~
,,"
I ~
,
valnVlrow
cfur to
r et
msini-
:ltal
1 in
--i
J
L.J
HPSC
'
J -~///////////////////////////////;,
/
cf~Meg
180),
balem-
+I
J
iplan
mof
m-T
Blood
~cfu-E
cfu-C (GM)
long
Wil-
errin
lulofeednon-
--.I~
C)
soon
lonylono'ntia[oore
:and
ss of
-
~/////////////////////////////~
'-L
Committed
stem cells
I
T
J
r--,
-
~
L- -J
Mature
forms
Recognisable
precursors
---J
Fig. 2. Schema
rnaturation
of haemopoiesis.
through
committed
as in figure
1, showing
stem cells and recognisable
reduced
HPSC numbers
precursors,
bones exposedto high dosesof irradiation (greater
than 4000 rads) or to internal radiation with strontium-89 (Klassen et al. 1972), and possibly contributes to the aplasia in mice exposed to benzene
or busulphan (Haak 1980), although the evidence
for the latter is unconvincing. Although some have
argued for the importance of microenvironmental
damagein the aetiology of human aplastic anaemia
(Camitta et al. 1982; Knospe & Crosby 1971), conclusive evidence fOl this view has not been forthcoming. Despite its importance in haemopoiesis,
there is no conclusive evidence that damage to the
microenvironment causesaplastic anaemia in man.
Indeed, the effectiveness of bone marrow transplantation in untransfused patients with aplastic
anaemia, the low levels of cfu-GM, cfu-E or cfumix in the marrow and blood of the great majority
of patients (Barrett et al. 1979; Hara et al. 1980;
Kern et al. 1977;Morris et al. 1984),andthe failure
to demonstrate any ultrastructural abnormalities
in the marrow parenchyma in aplastic anaemia
(Samson et al. 1972) all argue against the role of
microenvironmental damage in the genesis of
as postulated
(shaded)
by Boggs
in aplastic
anaemia,
but persistent
and Boggs (1976).
aplastic anaemia in man (Vincent 1984). Failure of
syngeneic marrow transplantation has often been
quoted as evidence of microenvironmental insufficiency, but in most situations where this has occurred in man a second marrow transplant following immunosuppressivetherapy has been successful
(Gordon 1979).
The most likely target for damage in drug-induced aplastic anaemia is the HPSC itself, but the
nature of the damage remains unexplained. As
noted above, acute HPSC damage following cytotoxic drug administration is reversible provided th.e
patient can be supported, while in drug-induced
aplastic anaemia the HPSC pool appears to be
damagedpermanently, or at least for prolonged periods. Patients who recover may show blood count
abnormalities for years afterwards (Vincent & de
Gruchy 1967). On the other hand, even patients
with severe aplastic anaemia show some persistence of haemopoiesis, so by inference a small population of HPSC must remain intact. Boggs and
Boggs(1976) have proposed a hypothesis which is
consistent with these observation,s'(fig. 2). In mice;
Drug-Induced Aplastic Anaemia and Agranulocytosis
58
reduction of the HPSC pool to approximately 10%
normally causesa block of differentiation until selfreplication has acted partly to replenish the HPSC
pool size. Boggs and Boggs suggest that, by contrast, HPSC in aplastic anaemia are qualitatively
abnormal and cbntinue to differentiate even when
the pool size is severely decreased,thus accounting
for the continued trickle of cell production when
the HPSC pool is almost non-existent. If this hypothesis is correct, the reasons for the decreased
numbers of HPSC in the first place, and for the
qualitative abnormality, have to be explained. Does
one insult, for example, both reduce the HPSC pool
and render the survivors abnormal, or are the
HPSC in susceptibleindividuals abnormal to begin
with?
An alternative possibility which has not been
canvassed is that the abnormality of HPSC in
aplastic anaemia blocks their differentiation rather
than their ability to self-replicate. This is the converse of the hypothesis outlined above, but is not
inconsistent with the meagredata available in man.
For example, reduced numbers of second-generation stem cells in aplastic anaemia could result from
afailure ofHPSC to differentiate. In viewofrecent
interest in the possiblity that acute leukaemia results from abnormal differentiation of HPSC, the
possibility that aplastic anaemia results from
blocked differentiation might be worthwhile exploring.
.
A variety of animal models which are intended
to simulate various aspects of clinical aplastic anaemia have been described, including the use of
external or internal radiat~on,and the study of mice
with congenital defects of stem cells or the marrow
microenvironment. None of these animal models,
however, is very helpful in trying to understand the
pathogenesisof aplastic anaemia in man. The animal model which most closely mimics the pathogenesisof human aplastic anaemia is the residually
marrow-damaged mouse model developed by
Morley and colleagues (Morley 1980; Morley &
Black 1974; Morley et al. 1975, 1976, 1978; Trainor et al. 1979, 1980). These workers showed that
mice which recovered from moderately large doses
ofbusulphan (or other agents, such as mitomycin,
chlorambucil, melphalan or carmustine) entered a
latent phase during which their marrow stem cell
populations were significantly depleted but their
haematology was essentially normal. After this latent phase,just under half the animals went on to
develop aplastic marrow failure. Marrow taken
from mice during the latent phase was defective in
its ability to repopulate the marrow of irradiated
syngeneic recipients; conversely, normal marrow
cells infused into mice in the latent phase were
largely able to correct the defect. The fact that normal marrow did not completely restore marrow
function to normal has been interpreted as showing microenvironmental as well as stem cell damage (Camitta et al. 1982). By contrast, Morley and
co-workers felt that proliferative demands placed
on re-populating stem cells would more probably
have accounted for the observation. Mice with residual marrow damage were more sensitive to
chloramphenicol than normal mice, suggestingthe
possibility that chloramphenicol-induced aplastic
anaemia in man might be more likely in subjects
with pre-existing stem cell damage.
4. Mechanism
of Drug-Induced
in Aplastic Anaemia
Damage
By definition, the occurrence of aplastic anaemia or agranulocytosis following administration of
a drug is an idiosyncratic reaction. The label
'idiosyncratic' is not the same as 'unpredictable',
although the two terms are often used
interchangeably. An idiosyncratic reaction should
be predictable in a small susceptible population;
the problem, however, is to define that population.
In vitro testing for idiosyncratic reactions of any
kind is extremely difficult. By definition, the effect
should not be demonstrable in normal cells at therapeutic or near-therapeutic concentrations of the
drug, and yet considerable efforts have been expended in studies of this type. There are several
reasons why an idiosyncratic drug reaction might
cause aplastic anaemia or agranulocytosis: (a) abnormal drug metabolism; (b) a genetically determined HPSC abnormality and/or previous damage
to HPSC; (c) an immune reaction involving the
Drug-Induced
Apia
drug; or (d) a cO'
ceptible to the d
reactions are thc
tabolism or excr
cumulation in e>
ious study by C
this idea. These ,
nilide and pheny
the same oxidat
subjects are cle
showed that acet
8 patients with
anaemia but ru
aplastic anaemi!
of phenylbutazo
Chlorampher
studied than an
aemia, largely b)
1974; Yunis & j
Chloramphenicc
benzene ring, w'
is substituted b)
amphenicol and
dependentinhit
thesis with dec
sponsible for th,
seen in viva (W
Zelkowitz, 1968
and cfu-E.
The idiosync
causing aplastic
quite independ,
has proved mol
has focused 01
present in chlol
icol, and has le;
trosochlorampl
sis at signific
chloramphenicc
effect is not re'
Nitrosochloran
cfu-GM than c
loss of cell viab
of rat chloroma
, sequent death,
1980). The cytc
58
red
a
1 cell
their
is la)n
to
aken
veIn
lated
rrow
were
norTOW
IOWamand
Iced
Ibly
reto
the
stic
~cts
aeof
bel
ed
lId
m;
ly
'ct
'Tle
)(-
Drug-Induced Aplastic Anaemia and Agranulocytosis
drug; or (d) a coincident insult making HPSC susceptible to the drug. In many cases, idiosyncratic
reactions are thought to be due to abnormal metabolism or excretion of a drug, leading to its accumulation in excessiveconcentrations. An ingenious study by Cunningham et al. (1974) supports
this idea. These workers utilised the fact that acetanilide and phenylbutazone are both metabolised by
the same oxidative liver enzymes and in normal
subjects are cleared at comparable rates. They
showed that acetanilide clearancewas prolonged in
8 patients with phenylbutazone-induced aplastic
anaemia but not in 5 patients with idio~athic
aplastic anaemia, implying an impaired oxidation
of phenylbutazone in the affected patients.
Chloramphenicol has been more extensively
studied than any other drug causing aplastic anaemia, largely by Yunis and colleagues(Yunis 1973,
1974; Yunis & Adamson 1977; Yunis et al. 1980).
Chloramphenicol contains a paranitro group on the
benzene ring, which in its analogue thiamphenicol
is substituted by a methyl sulphonyl group. Chloramphenicol and thiamphenicol both causethe dosedependent inhibition of mitochondrial protein synthesis with decreased ferrochelatase activity responsible for the predictable erythroid suppression
seen in viva (Weisberger et al., 1964; Yunis, 1973;
Zelkowitz, 1968),and both inhibit normal cfu-GM
and cfu-E.
The idiosyncratic action of chloramphenicol in
causing aplastic anaemia, however, appears to be
quite independent of the above mechanisms and
has proved more difficult to unravel. Recent work
has focused on the nitrobenzene ring uniquely
present in chloramphenicol but not in thiamphenicol, and has lead to the reduction synthesis of nitrosochloramphenicol. This inhibits DNA synthesis at significantly lower concentrations than
chloramphenicol, and unlike chloramphenicol the
effect is not reversible when the drug is removed.
Nitrosochloramphenicol is also more inhibitory to
cfu-GM than chloramphenicol, causes significant
loss of cell viability, and in flow cytometric studies
of rat chloroma leads to an accumulation, and subsequent death, of cells in G2 and M (Yunis et al.
1980).The cytotoxic effect of nitrbsochloramphen-
59
icol has yet to be defined, but by analogy with the
nitrosoureas the possibility exists that it is due to
interaction with DNA. Yunis and colleagues suggest that chloramphenicol aplasia might occur in
individuals who provide the milieu for the reduction synthesis of nitrosochloramphenicol.
There are reports suggesting a genetic predisposition to marrow injury by chloramphenicol. The
best known of these is the occurrence of aplastic
anaemia in one pair of identical twins (Nagao &
Mauer 1969) and the finding that cells from the
fathers of patients with aplastic anaemia show a
sensitivity to chloramphenicol (measured by impaired DNA synthesis) similar to that in their
children (Yunis 1974).
Becauseof the grossly decreased numbers, or
complete absence,of detectable cfu-GM in aplastic
anaemia it has not been possible to evaluate the
effects of drugs in vitro in the acute phase. The
studies which have been reported have used cfuGM from the marrows of patients who have recovered from aplastic anaemia secondary to chloramphenicol or phenylbutazone. These have shown
no greater sensitivity of the cfu-GM to the causative drug when compared with normal subjects
(Firkin & Moore 1976; Firkin et al. 1974; Kern et
al. 1975; Morley et al. 1974; Ratzan et al. 1974).
Indeed, in some studies there has even been a suggestion that the cfu-GM from the patient's marrow
were relatively resistant to the causative drug
(Howell et al. 1975a).
5. Experimental
Studies
in Drug-Induced
Agranulocytosis
The differing clinical features of aplastic anaemia and agranulocytosis suggestdifferent mechanisms in the two disorders. A drug might cause
agranulocytosisby damaging committed stem cells,
proliferating precursors, or mature granulocytes
(Pisciotta 1973; Young & Vincent 1980). In cases
resulting from damage to committed stem cells
there is a delay between drug exposure and the onset of agranulocytosis, as cells in the proliferating
and storagepools move through the maturation sequence and enter the blood. In this type of damage
Drug-Induced
the marrow at presentation is typically hypocellular and almost totally devoid of granulocytic precursors. Recovery, when it occurs, is heralded by
a wave of cells moving through the maturation
stages.This will often given the appearance of an
excessof early forms and a relative absenceof mature cells -the so-called 'maturation arrest'. Marrow culture studies in patients with this type of
agranulocytosis may demonstrate the toxic effect
of the drug under suspicion (Howell et al. 1975;
Kelton et al. 1979;Lind et aI. 1973;Singer & Brown,
1978; Sutherland et al. 1977; Smith et al. 1977;
Young et al. 1982).
However, not all patients with agranulocytosis
can be studied by these techniques. A proportion
will have low or undetectable numbers of cfu-GM
and in these, obviously, in vitro drug effects cannot
be evaluated. In others the drug cannot be dissolved in physiological media -indeed, it is amazing how many drugs are insoluble. In a third group,
a metabolite of the drug might be responsible,while
in a fourth the drug may be associatedwith an immune-mediatedeffect. Evaluation of these last two
groups may be facilitated by testing acute phase
plasma, with and without complement (Kelton et
al. 1979;Young et al. 1982). In our experience with
in vitro cfu-GM cultures in 33 patients with druginduced agranulocytosisover the 9-year period 1974
to 1982, it was possible to culture marrow from 23
patients with the suspect drug, and inhibition (in
excessof control) was seen in 8. In 10 of the remaining patients, acute phase plasma was tested,
and in 2 of these it was inhibitory (Young et al.
1984).
Drug damage to the proliferating pool of granulopoietic precursors can also result in agranulocytosis. The best-documented drugs causing this
type of damage are the phenothiazines, especially
chlorpromazine. Pisciotta and colleagueshave made
an important series of obserVations of this phenomenon (Pisciotta, 1973). They have shown that
chlorpromazine itself is toxic to normal proliferating systemsat concentrations of 0.1 to 0.01 mmol/
L. In addition, they have shown that marrow cells
from patients who have recovered from chlorpromazine-induced agranulocytosis are unduly
sensitive to the drug in vitro, as measured by DNA
synthesisand metabolic activity. Acute phase marrow taken from patients with agranulocytosisof this
type shows almost complete absence of granulopoietic precursors and is morphologically indistinguishablefrom that seenfollowing committed stem
cell damage. Recovery also follows the same pattern, with a wave of regeneration moving through
the granulopoietic pathway.
While these two mechanisms -a drug effect on
committed stem cells or on recognisableprecursors
-have been presented as distinct entities, it is possible that many drugs could damage both compartments. If committed stem cells share similar
antigens with those on more mature cells, or have
similar metabolic pathways, it is conceivable that
the drug could adversely affect both the granulopoietic stem cell and recognisable precursors. On
the other hand, some patients with drug-induced
agranulocytosisresulting from damage to recognisable myeloid precursorshave shown normal or even
increasednumbers of cfu-GM, suggestinga sparing
of the committed stem cell compartment (Heit et
al. 1977).
The third type of mechanism involves the destruction of mature granuloctyes in the blood as
well as those in the marrow storage compartment.
It is exemplified by the dramatic agranulocytosis
which used to be seen with amidopyrine (aminopyrine). Amidopyrine-induced agranulocytosis has
all the hallmarks of an immune-mediated disorder
with destruction of circulating granulocytes, but
leucocyte agglutinins have been found only infrequently (Moeschlin & Wagner 1952;Pisciotta 1973).
Leucocyteagglutinins have been reported in the sera
of patients with agranulocytosis associated with a
variety of other drugs, including sulphonamides,
quinidine, propylthiouracil, phenytoin and semisynthetic penicillins (Bilezikian et al. 1976; Eisner
et al. 1977; Pentz & Fudenberg 1975; Weitzmann
et al. 1978).As well as these immune mechanisms
directed at mature cells, there have been reports of
some patients with what could have been drug-dependent antibodies directed against cfu-GM (Kelton et al. 1979; Taetle et al. 1979). In patients withl
immune-mediated drug-induced agranulocytosis, a
Apl
variety of mech
& Vincent 1980
drug (at high do'
ing of immune
Fc receptors on
protein comple,
ations of the ce
of new antigen!
esis of Niewig (
An illustrati,
lism as a cause
tosis comes fro
agonists. Metia
in man caused
agranulocytosis
which succeede
the substitutior
a thiourea, yet
idine, very few
reported (Frest
block the H2thought to be il
(Byron 1976).
labelled comp'
metiamide, bUl
marrow cells (
difference couJ
cimetidine cor
6.
Screenil
Anaemia
£
The practi(
vitro testing to
encing a drug
aemia or agra
thetica1drug ~
of 1110,000,a
devised to ide
test costs -at
then up to 1 n
to detect tha'
more cost-effe
the decision ,
known to be
agranu1ocyto~
60
rA
Irlis
0n-
Drug-Induced
variety of mechanisms could be involved (Young
& Vincent 1980). These include absorption of the
drug (at high doses)on to the cell membrane, binding of immune (drug and antibody) complexes to
Fc receptors c;>n
the cell surface, binding of a drug/
protein complex to the cell, or drug-induced alterations of the cell membrane, with the production
of new antigens [the 'spoiled membrane' hypothesis of Niewig ( 1974)].
An illustration of differences in drug metabolism as a cause of marrow damage in agranulocytosis comes from the development of the H2-antagonists. Metiamide, the first H2-antagonist used
in man caused an unacceptably high incidence of
agranulocy.tosis and was withdrawn. Cimetidine,
which succeededit, differs from metiamide only in
the substitution of a cyanoguanidine side chain for
a thiourea, yet despite the extensive use of cimetidine, very few casesof agranulocytosis have been
reported (Freston 1979). In vitro, both drugs will
block the H2-receptor on stem cells, which is
thought to be involved in triggering them into cycle
(Byron 1976). Studies in the rat with isotopically
labelled compounds, however, have shown that
metiamide, but not cimetidine, is taken up by bone
marrow cells (Brimblecombe et al. 1978) and this
difference could account for the relative safety of
cimetidine compared with the earlier analogue.
6. Screening
,4naemia
61
Aplastic Anaemia and Agranulocytosis
for
Drug-
I nduced
Aplastic
or Agranulocytosis
The practical aspects are rather daunting of in
vitro testing to detect patients at risk before commencing a drug possibly associatedwith aplastic anaemia or agranulocytosis. Let us consider a hypothetical drug with a risk of causing aplastic anaemia
of 1/10,000, and let us assume that a test has been
devised to identify the 1 individual at risk. If each
test costs -at a conservative estimate -about $100,
then up to 1 million dollars would need to be spent
to detect that individual. It would obviously be
more cost-effectiveto changeprescribing habits, and
the decision to commence treatment with a drug
known to be associated with aplastic anaemia or
agranulocytosis must rest on the indications for its
use, whether alternatives are available, and the extent of the risk. A strong case could be made, for
example, for using thiamphenicol rather than
chloramphenicol (Baumelon & Najean 1983) where
one of these agents is clearly indicated, and newer
non-steroidal anti-inflammatory agents instead of
phenylbutazone or oxyphenbutazone.
In vitro screening is potentially helpful, however, in trying to identify the agent responsible in
an individual being treated with multiple drugs,
who presents with aplastic anaemia or agranulocytosis. In patients such as this, one cannot automatically assume that the drug best known for its
myelosuppressiveeffect is necessarilythe culprit in
that individual. In a recent patient of ours, for example, co-trimoxazole was thought to be responsible for an episode of agranulocytosis at a time
when she was also taking mebhydrolin, quinine,
dextropropoxyphene, diazepam, chlorothiazide,
aspirin and paracetamol (acetaminophen). She represented with agranulocytosis 4 years later after
self-medication with mebhydrolin, and in vitro
studies then confirmed this drug as the culprit
(Young et al. 1982).
No practical system has been devised to screen
new drugs for their potential to cause idiosyncratic
haemopoietic damage. Normal animals will not
develop myelosuppressionat pharmacological doses
with any of the drugs known to cause aplastic anaemia or agranulocytosis in man. However, mice
with residual marrow damage are abnormally sensitive to chloramphenicol (Morley et al. 1976) and
this might be a model which could be used to screen
new drugs prior to clinical testing.
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