McKenzie, M. W., Marchall, G. L., Netzloff, M. L. & ClufT,L. E. (1976). Adverse drug reactions
leading to hospitalization in children. J. Paediat., 89,487-490 .
Nugent, S. K., Laravuso, R. & Rogers , M. C. (1979). Pharmacology and use ofmuscle relaxants
in infants and children. J. Paediat., 94,481-487.
O'Malley, K. & O'Brien, E. (1980). Management of hypertension in the elderly. New Eng. J.
O'Malley, K., Stevenson, I. H., Ward, C. A., Wood, A. J. & Crooks, J. (1977). Determinants of
anticoagulant control in patients receiving warfarin. Brit. J. c1in. Pharmac., 4, 309-314 .
Rezvani, I. & Di George, A. M. (1977). Reassessment of the daily dose of oral thyroxine for
rep1acement therapy in hypothyroid children. J. Paediat., 90, 291-297 .
Seidl , L. G ., Thomton, G . F., Smith, J. W. & ClufT, L. F. (1966). Studies on the epidemiology of
adverse drug reactions. Bu//. Johns Hopkins Hosp., 119,299-315.
Shocken, D. &Roth, G. (1977). Reduced beta adrenergic receptor concentrations in ageing man .
Tuttle, R. S. (1966). Age related changes in the sensitivity ofrat aortic strips to norepinephrine
and associated chemical and structural alterations. J. Geronto/., 21,510-516.
Vestal , R. E., Wood, A. J. J. & Shand, D. G . (1979). Reduced ß-adrenoceptor sensitivity in the
elderly. Clin. Pharmac. Ther., 26, 181-186.
Alterations in Receptors During
MRC Unit and University Departm ent ofClinical Pharmacolo gy,
RadclifJe Infirma ry, Woodstock Road, Oxford OX2 6H E, UK
Traditionally the effect s of drugs have been monitored in man by careful c1 inic al
observation (exempl ified in the case of digitalis by Withering, 1785). 8edside
observation still holds prime pla ce as a method for monitoring drug effects but more
recently biochemical and pharmacological methods have been developed in attempts
to provide information which might extend the interpretation ofknowledge gained at
the bedside. Principal among the se has been the measurement ofdrug concentrations
in plasma. Such measurements reflect onl y the pharmacokinet ic properties of th e
drugs in question (and therefore their distribution in the bod y) but in some cases have
been shown to correlate weil with the drugs' therapeut ic or, mo re usually, to xic
effects. This approach, however , neglect s the pharmacological effects of the drugs on
body tissues, effects whi ch are eventually translated into therapeutic and toxic effects.
concentrations, in terms ofthe final outcome. This notion is illu strated for th e case of
the cardiac glycosides (for example, digo xin) in Figure I.
Following administration, cardia c glycoside s are distributed Irrst throughout the
plasm a and then to almost all bod y tissues. In erythrocytes cardiac glycosides inhibit
th e cation transport enzyme, Na+,K+-ATPase, and the effects of that inh ibition are
measurable in several ways (see below). Pharmacological effects which occur in the
heart result in therapeutic or tox ic effects and, although it is by no means proven ,
there is a great deal of circumstantial evidence linking those pharmacological effects
either directl y or indirectly to inhibition of the same enzyme, Na+,K+-ATPase
(Schwartz, Lindenmayer & Allen, 1975). Pharmacological effects in other (for
example, central or per ipheral nervou s) tissues ma yaIso be related to the
therapeutic or toxic effects in th e heart but the pharmacol ogical mechanism s ofthose
effects are not weil ch ar acterized. The sequence shown in Figure I thus highlights the
three principal methods of monitoring drug action - pharmacokinetic, pharrnacodynamic and c1inical.
Both c1inical methods and those based on the measurement of pla sm a glyco side
concentrations have been discus sed at length and on numerous occasions by others
and the discussion here will be restricted to methods of pharrnacodynarnic
monitoring with particular regard to digo xin .
Figure1 The sequence of events following the administration of a cardiac glycoside (for
Pharmacodynamic monitoring of digoxin therapy
Because cardiac glycosides have effects in tissues (for example, erythrocytes) other
than those at which therapy is aimed, the usefulness of measuring those peripheral
effects and comparing them with the observed c1inical effects has been explored.
The course of events following the exposure of digoxin to erythrocytes is presumed
I) Digoxin binds to the erythrocyte membrane.
2) Membrane-bound Na+,K+-ATPase activity is inhibited with consequent
inhibition oftransmembrane sodium and potassium fluxes.
3) Intracellular sodium and potassium concentrations alter accordingly.
The following measurements may be carried out related to each part of this
I) The ability ofthe erythrocyte membrane to bind 12-a -[3H]-digoxin specifically in
vitro ('[3H]-digoxin binding').
2) The ability of the erythrocyte membrane to transport potassium in vitro. In
practice, it is simpler to measure rubidium transport ('86Rb uptake') using
radioactive rubidium (86Rb) which is handled in the same way as 42K by the
erythrocyte membrane and which has a longer half-life .
3) Intraerythrocytic sodium and potassium concentrations.
The following methods have been used in making these measurements:
I) PHJ-Digaxin binding (Ford, Aronson, Grahame-Smith & Rose , 1979a)
Erythrocytes are prepared from whole venous blood by centrifugation and separation
of plasma and buffy coat followed by washing three times by alternate suspension in
112 rnv MgCb and recentrifugation at 4T. The erythrocytes are then incubated at a
haematocrit of 10% in a pota ssium-free Ringer solution at 37°C for 2 h in the
presence or absence of 12-a -[3H]-digoxin in concentrations varying between 0 and
1000 ng mi-I. After further washes at 40C the erythrocytes are haemolysed with a
phosphate buffer. The membranes are prepared by centrifugation at 4°C, washed in
RED CELL DIG ITALIS RECEPTORS 137
the buffer , solubili zed and then bleached with hydrogen peroxide. The a mount of
12-a-(lH]-digoxin bound to the membran es is det ermined by liquid scintilla tion
The characteristics of th e membran e binding of (lH] -digo xin a re: time- and
ternperature-dependency; saturability (maximum binding after a 2 h incubation
occurring at a digo xin concentration of 100 ng ml" ); slow reve rsib ility (T'I, of
disso ciation at 37 'C = 17 h); stoichiome tric inhibition by other ca rdiac glycosides and
pot assium; onl y one d ass ofbinding sites is demonstrable.
By measuring th e ability of pati ent s' red cells to bind (lH]-digoxin befor e and then
during th erapy with digoxin it is possible to assess th e extent of occupan cy of red cell
receptor sites by therapeuticall y admi nistered digoxin. This conce pt is illustrated in
1 Er yt h r ocyt e Irom unt re at ed parti_en_t_, _"\ .1" _
2 Er ythrocyt e l r om pat ient t aki ng dig oxin in tn e snor t - t er m.
Figure 2 A schematic representation of digitalis receptor sites on a portion of erythrocyte
membrane. Before treatment with digoxin(l) all the sites are available for binding (lH]-digoxin
in vitro. After the administration of digoxin(2) some of the sites are occupied, resulting in a
lowered binding of (lH]-digoxin in vitro. The characteristics of the binding (see text) ensure
that the digoxin already bound in vivo is not removed by the procedure preparatory to the
measurement of(lH]-digoxin binding.
2) 86Rb upta ke (Aronson , Grahame-Smith , Hallis, Hibble & Wigley, 1977)
Erythrocytes are prepared as described under '[3H]-digoxin binding' above, incubated
with 86RbC l for 1 h at 37'C , washed th ree tim es at 4'C and th e accumulat ed
rad ioactivity in the erythrocy tes detected by a y-counter . 86Rb upt ak e is then
expressed as the amount accum ulated with in the cells as a per cen t oftotal 86Rb in the
3) In traerythrocytic cation concentrations (Ford, Aro nso n, Grah am e-Smith &
Erythrocytes, prepared as described above, are haemolysed in distilled water and
cation conce ntrations mea sured by flarne photometry or atomic absorption spectrophotometry.
In studies ofthe use ofdigoxin in the short term (up to about two weeks ofcontinuous
daily therapy following a loading dose) in 25 patients in atrial fibrillation and 21
patients in heart failure in sinus rhythm the following observations have been made
(Aronson et al., 1977; Ford et al., 1979b):
a) During the first few days oftreatment red cell digitalis receptor sites are occupied
in vivo by digoxin (as assessed by a fall in in vitro [3H]-digoxin binding), membrane
Na+,K +-ATPase is inhibited (as assessed by a fall in 86Rb uptake) and consequently
intraerythrocytic Nat concentrations rise . Others have shown similar changes in
intraerythrocytic Na" concentrations during the first few days of digitalis therapy
(KettleweIl, Nowers & White, 1972; Astrup, 1974; Funder & Wieth, 1974) but in
a single patient in Figure 3 (for discussion ofthis ca se see below).
QS 2 I . 520 540~~ Im sec) 500
(ng ml-I) O---._----- ....I....l...--l.......I-_L.....L---l....J
I , I I I I , I : f---I---fI---l--{ f---l-{f--l
Figure 3 Changes in erythrocytic 86Rb uptake and [3H]-digoxin binding, intraerythrocytic Na"
concentrations, QS21 and plasma digoxin concentrations during short- and long-terrn digoxin
therapy in the patient discussedin the text.
RED CELL DIGITALIS RECEPTORS 139
b) These effeets in the three erythrocyte funetions occur eonsistently during digoxin
therapy and eorrelate weil with some observed therapeutie effeets of digoxin (slowing
of ventricular rate in artrial fibrillation, shortening ofsystolie time intervals in heart
failure in sinus rhythm). The correlations between the systolie time interval QS2I (the
total eleetromeehanical systole correeted for heart rate) (Weissler, Lewis & Leighton,
1972» and the three erythrocyte measurements are shown in Figure 4. In eontrast
plasma digoxin eoneentrations either do not eorrelate with the c1inical effects (in
sinus rhythm) or correlate less weil (in atrial fibrillation) (not iIIustrated) .
c) After a few (3-11) days of daily digoxin therapy, fluetuations occur in both
[3H]-digoxin binding and 86Rb uptake in parallel (Figure 3). No adequa te explanation
for these fluctuations is available at present but they may be related in some way to
homoeostatie meehanisms to maintain intraeellular Na- eoncentrations, whieh do
1I0t fluctuate during the period of fluctuation of the other two measurements
So mueh for the effeets of short-terrn administration. The effeets of long-term
digoxin treatment have been studied in four patients and the results are exernplified
The patient was in eardiae failure in sinus rhythm and when digoxin therapy was
started the ehanges discussed above oceurred in in vitro [3H]-digoxin binding, 86Rb
uptake, intraerythroeytie sodium eoneentrations and the systolic time interval, QS2I.
Note that the maximum ehanges oeeurred whilst the plasma digoxin eoneentrations
were less than I ng mi-I. After three days fluetuations in [3H]-digoxin binding and
86Rb uptake began. The patient improved c1inieally and after discharge from hospital
was seen intermittently over the next four months, during whieh time measurements
of[3H]-digoxin binding, 86Rb uptake and intraerythroeytie sodium coneentration all
returned to pre-treatment values despite plasma digoxin eoncentrations of about
I ng ml:', One measurement of QS2I made four months after discharge had also
returned to the pre-treatment level. The patient had remained c1inieally weil during
this time but suddenly died of a coronary oeclusion four months after the initial
Similar observations in four patients initiated a cross-sectional study of patients at
different stages ofdigoxin therapy, the results ofwhich are summarized in Figure 5.
Measurements ofthe three red cell funetions were made in four groups ofpatients: 69
control patients, 38 patients who had been taking digoxin for less than ten days, 46
patients who had been taking digoxin eontinuously for at least two months, and 13
patients who had c1inical digoxin toxieity.
The following observations were made in these subjeets:
a) As in the longitudinal studies, the values of[3H]-digoxin binding and 86Rb uptake
were significantly lower and those of intraerythroeytie Nat eoneentrations
significantly higher in patients treated in the short term than in untreated subjects.
b) However, and as suggested by the long-terrn studies in four patients (Figure 3),
there were no differenees for any of four measurements when comparing patients on
long-term treatment with untreated subjeets. Although some of these patients
supposedly taking long-term digoxin treatment had plasma digoxin eoneentrations
less than 0.8 ng mi-I, omission oftheir values does not alter the relationships shown
here. Thus the apparent pharmacologieal 'tolerance' which was observed in patients
on long-term treatment is independent of plasma digoxin eoneentrations. It is also
independent of the amount of digoxin bound to the red cell membrane in viva,
measured by extraeting the digoxin bound to patients' erythroeyte membranes during
therapy and subjecting the extraet to radioimmunoassay.
.' • .', .. • • • • , • • "- .. '. • ·.', . • • .. , • •
, •• •• • ..", • • • e .... • ... • •• '" ,
, • • 0 •• .?-,• • • :. •• • , •
Figure 4 Relationships between QSlI during short -term digoxin the rap y and (a) [JH)-digoxin
binding, (b) 86Rb upt ake , (c) int raeryth rocytic (RBC) Na" concentra tion.
RED CELL DIGIT AllS RECEPTORS 141
on long-term treatment before the on set of aeute toxieity and would therefore have
been expeeted, before the onset oftoxieity, to have had values ofthe three erythroeyte
measurements no different to those of untreated subjeets. When toxieity supervened
the y would then ha ve been expeeted to have shown similar ehanges to those who had
been tre ated in the short term. The faet that the ehanges in tox ie patients are no
different from those in patients on short-term treatment (who were not clinieally
tox ie) suggests that the re ma y be meehanism s of digo xin to xieity whieh are distinet
from inhibition ofNat.K+-ATPase.
o Sh orl te r m t reat ment I< 10 day, '
l ong ler m t reat me nt '>2 monln\ 1
Figure 5 Mean +s.d. values of intraerythrocytic (RBC) Na" concentration, 8f>Rb uptake and
(lH]-digoxin binding in the four groups of subjects shown (see text for discussion). The number
ofsubjects studied isshown in each case.
It has been shown that during short-term treatment with digoxin, for either at rial
ftbrillation or eard iae failure in sinus rhythm, changes oeeur in erythroeytie
ATPase. After treatment for about one week , fluctuations oeeur in the first two
ofNatK+-ATPase inhibition are not seen .
Before eonsidering the therapeutie implieations ofthese pharmaeologieal fmdings,
the relationship between the changes observed in the erythroeyt e and ehanges whieh
might be oeeurring in the heart mu st be eonsidered.
The link between the pharmaeological events in.the erythrocyte and those in the
heart has not yet been sub stantiated. The therapeutie and toxi e effects of cardiae
glyeosides are widel y eonsidered to be mediated, either direetl y or indirectly, by
inhibition of Na+,K+-ATPase (Sehwartz et al., 1975) and th e kin etie eharaeteristies of
the Na +,K+-ATPase of erythroeyte membranes are similar to those of the Na+,K+-
ATPase of eardiae membranes (Erdmann & Hasse, 1975). Furthermore, as diseussed
(OS a6e 01 papaJJo)) e ro as pajap JnOloJ
Figure6 The relation ships between colour vision scores and each of the three erythrocyte
measurements in patients during digoxin toxicity and following digoxin withdrawal.
(Reproduced by permission, Oxford University Press)(Aronson , J. K. & Ford, A. R., 1980).
RED CELL DIGITALIS RECEPTORS 143
above, the changes which occur during the earl y stages of digoxin therapy are
accompanied by concomitant and correlating changes in cardiac performance.
Comments
Post a Comment
اكتب تعليق حول الموضوع