Pergamon Press
Life Sciences, Vol . 24, pp . 2467-2474 Printed in the U.S .A .
ANTISCHIZOPHRENIC DRUGS: DIFFERENTIAL PLASMA PROTEIN BINDING AND THERAPEUTIC ACTIVITY Kenneth A. Freedberg,
Robert B . Innil,
Ian Creese *aad Solomon H. Snyder
Departments of Pharmacology and Experimental Therapeutics and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine Baltimore, Maryland 21205 (Received in final form May 7,
1979)
Summary
The percentage of free neuroleptic drug unbound to plasma protein is much higher for the respective metabolites of chlorpromazine and thiori~azine, 7-hydroxychlorpromazine and mesoridazine, than for the parent drugs. The therapeutic activities of chlorpromazine and thioridazine may be mediated to a major extent by 7-hydroxychlorpromazine and mesoridazine respectively . Measuring free levels of the active metabolites of neuroleptics as well as the parent drugs may facilitate regulation of neuroleptic doses to secure optimal therapeutic responses. Bioavailability of drugs is a major determinant of therapeutic activity . Blood levels of several drugs, ouch as theophylline, digoxin, diphenylhydantoin, lithium and phenobarbital are routinely assayed to adjust dosage for During the past decade optimal therapeutic response with minimal side effects . various techniques have been employed to measure plasma levels of drugs in psychiatry, especially tricyclic antidepressants and the neuroleptic, antiachizophrenic drugs . Poor clinical response to neuroleptics may be associated not only with abnormally low blood levels but with abnormally high blood levels of the drugs (1-4) . Difficulties in predicting clinical status from blood levels of neuroleptics in some studies (5, 6) may reflect human biological variability For lipophilic drugs such as the or varying plasma protein binding of drugs . tricyclic antidepressants and the neuroleptics, high proportions of the total For the neuroleptic chlorpromaplasma content are bound to plasma proteins . zine, the percentage of drug bound to plasma protein can range between 90 and 99% in different individuals so that with identical total plasma levels of the drug the concentration of free chlorpromazine could vary ten fold (7) .Recently McDevitt _et _al . (S) showed that the extent of clinically evident ß-noradrenergic receptor blockade elicited by the ß-blocking drug propranolol did not correlate with total plasma levels of propranolol, was only modestly related to red cell levels of the drug but closely paralleled free levels of propranolol in patient plasma . These findings confirm the supposition that only the free drug is available to target organs ouch as the heart or brain .
*Permanent Address : Dr . Ian Creese, Department of Neurosciences, University of California at San Diego, La Jolla, California 92093 . 0024-3205/79/262467-0702 .00/0 Copyright (c) 1979 Pergamon Press Ltd
2468
Antischizophrenic Drugs
Vol . 24, No . 26, 1979
The major circulating metabolite of the neuroleptic chlorpromazine is In pharmacological tests which predict clin7-hydroxychlorpromazine (9,10) . ical antiachizophrenic effects, 7-hydroxychlorpromazine has about the same po tency as chlorpromazine (11-15) . Moreover, 7-hydroxychlorpromazine and chlorpromazine have similar affinities for dopamine receptors (16) the presumed sites for the therapeutic actions of neuroleptica (17,18) . In several clinical studies levels of 7-hydroxychlorpromazine correlated better with therapeutic responses of schizophrenics than did levels of chlorpromazine, despite the fact that the total plasma chlorpromazine levels were as high or higher than levels of 7-hydroxychlorpromaziae (19-22) . If total plasma levels of the drugs determined therapeutic response, then the clinical status of patients should have been at least as closely related to chlorpromazine as to 7-hydroxychlorpromazine levels . Recently we developed a simple, sensitive and specific radioreceptor assay for neuroleptic drugs based on their ability to compete for the binding of 3H-spiroperidol to dopamine receptors in corpus striatum membranes (23) . Using this assay, in the present study we have measured the proportion of various neuroleptice which are bound to plasma protein . We show that in fresh human plasma the percentage of unbound drug is 10 times greater for 7-hydroxychlorpromazine than for chlorpromazine . After treatment with thioridazine, blood levels of its metabolite meaoridazine are 40200% of total thioridazine levels (24-28), but at equal plasma levels the percent of free meaoridazine is 50-100 times that of free thioridazine . Thus chlorpromazine and thioridazine, two of the most widely employed antischizophrenic drugs, may not exert antischizophrenic effects themselves, but instead may act through their respective metabolites 7-hydroxychlorpromazine and meaoridazine . Methods Plasma from healthy adult male and female laboratory personnel (ages 2040) was freshly drawn into glass syringes containing about 200 U heparin. Neuroleptics dissolved in 0.1% ascorbic acid were added to plasma along with 0 .5 mg sodium metabisulfite per ml plasma and incubated for 30 minutes at 37 ° C to allow binding to plasma proteins, Sodium metabisulfite had no effect on plasma protein binding and was added to stabilize certain neuroleptics (29) . One ml plasma samples were dialysed against one ml 0.9% NaCl for 18 hours (equilibrium was attained in 6-15 hr) at 4° C in the dark in an EMO dialysis apparatus (Hoefer Scientific) using 12,000-14,000 dalton molecular weight cut-off membranes . The radioreceptor sassy for unlabeled neuroleptica was essentially Fresh rat caudate was homogenized with a Brinkas described previously (23) . man Polyfron PT10 in 70 volumes 50 mM Tris/CH1 pH 7 .7 and centrifuged at 50,000 After an intermediate reauspenaion and centrifugation in x g for 10 minutes . fresh buffer, the final pellet was reauspended in 70 volumes of ice cold 50 mM TripTria/HC1 pH 7 .6 containing O .1X ascorbic acid, 5 mM EDTA and 100 mM NaCl . licate assay tubes received in order : 250 ul dialysate, appropriately diluted retentate (material retained in the plasma chamber after dialysis), control plasma, or saline ; 50 ul O .1X ascorbic acid or 50 ul 10-5 (+)-butaclamol to measure nonspecific binding ; 25 ul 3H-spiroperidol (24 .6 Ci/mmol, New England Nuclear) for a final concentration of 0 .5 nM ; 175 ul caudate tissue for a total volume of 0.5 ml . Tubes were incubated at 37° C for 15 minutes and rapidly filtered through Whatman GF/B filters with three 5 ml rinses of ice cold buffer . Filters were counted by liquid scintillation spectrometry in 10 ml Formula 947 (New England Nuclear) . Concentrations of drugs in the dialyeate and retentate The percentage free drug was were determined from standard displacement curves . calculated as : ~di~ x free ~ retentate a 100
Vol . 24, No . 26, 1979
Antischizophrenic Drugs
2469
In our initial studies blood had been drawn into VacutainerR (BectonDickinson) tubes and stored at 4°C for 1-20 days, similar to conditions for most plasma protein binding studies . Since we found that use of VacutainerR tubes as well as storage increased the percent free for moat neuroleptics 1080 fold,all experiments whose results are reported here employed freshly drawn plasma using heparinized glass syringes . These technical difficulties may account for the substantially higher free levels measured previously for chlorpromazine and thioridazine (7,30) . However, our free levels for thiroida~ine and meaoridazine are equal to those recently determined by Axelaeon and Martensson (31) and that of haloperidol determined by Foreman and~ffhman (32) . By dialysing aeuroleptics is normal saline against this same solution, we have determined that a small amount (< 5X) of each drug adsorbs to the chamber and membrane . One might initially think that this adsorption would signifi cantly affect the free level, which is less than 1X for some neuroleptica . However, this adsorption would act transiently as a "sink" and should not affect the percentage of free drug in equilibrium with plasma proteins . Results and Discussion The percent free neuroleptic varies with concentration for certain of the drugs . Thus with chlorpromazine at respective concentrations of 8 yM, 80 uM and 400 yM the free levels are 0 .13X, 0.24X and 0 .79X (Figure 1, Table 1) . This pattern is also evident with ciethiothixene and meaoridazine, somewhat less with thioridazine and promazine and not apparent for 7-hydroxychlorpromazine . These differences might reflect varying drug-protein binding constants . The percent free drug at the lowest plasma concentration is probably most relevant to therapeutic practice, since therapeutic total plasma levels of chlorpromazine (0 .1 -1 .0 uM) are less than the lowest concentration of chlorpromazine examined here . The neuroleptic concentrations we employed were selected to facilitate measuring the fairly low concentrations of free drug . For comparisons among the different aeuroleptics, we will refer primarily to the data obtained at the lowest concentrations examined . At 8 uM only about 0 .13X of plasma chlorpromazine is free, while more than ten times as much 7-hydroxychlorpromazine is free . EV~en at the highest concentration examined, 0.4 mM, more than twice ae much 7-hydroxychlorpromazine is free than chlorpromazine . After chronic chlorpromazine treatment, total plasma levels of 7-hydroxychlorpromazine are 20 - 120X of the chlorpromazine levels (19-22, 33) . Assuming that the percentage of free drug at therapeutic doses corresponds to values we obtained at 8 uM, then free 7-hydroxychlorpromazine would be 2 - 12 times the levels of chlorpromazine . Thus after chronic treatment with chlorpromazine, the metabolite 7-hydroxychlorpromazine may be responsible for therapeutic effects, though with acute treatment with very low levels of 7-hydroxychlorpromazine the parent drug may be the primary active agent (9, 10) . These observations might explain the better correlation of 7-hydroxychlorpromazine than chlorpromazine plasma levels with therapeutic response (19-22) . Meaoridazine, a metabolite o£ thioridazine, has about the same potency as the parent drug in blocking dopamine receptors (34) and, after therapeutic doses of thioridazine, total plasma levels of meaoridazine are similar to those of thioridazine (11) . At all free concentrations examined the percentage of free meaoridazine exceeds that of thioridazine by 50-100 times, confirming the findings of Nyberg et al (31), and after thioridazine treatment, meaoridazine is the major therapeutically active agent . Mesoridazine itself is a marketed, clinically effective antipsychotic drug .
2470
Antischizophrenic Drugs
Vol,
SOr
24, No, 26, 1979
e 0
a
T
a
,m
lo
t a
a
.8
tiL 0
m~ .
0
a
â
.o
. ~
.o
.o
m
i .
0.10
oa
.
0
i
0
m
v
.oni .oe .oe woemon i . i W W o$g omo e~ o a °m8 ~S~ oog ~r9fn ~,9,D O ~ d N ô
O ~
~ i
O
n
O_ ~
O J i
J
O
û
~DRUG~ pM FIG . 1 Plasma neuroleptics not bound to plasma protein (free) . Fresh human plasma samples were incubated with neuroleptics, dialysed and bound and free drug concentration assayed as described in the text . Each point represents the mean value of triplicate assays from a separate human volunteer . Short horizontal bars indicate the means of the samples for each total drug concentration. Drug abbreviations are the same as in Table 1 .
Vol . 24, No . 26, 1979
0 0 ~
H a
o
Antischizophrenic Drugs
2471
~o 0 0 ~O r, O
F
'-I
N
d w W A ~ ~ ~ W
~ N
r1 ~ N A W b A TJ ~ q
tl N
p
N
O
v1
Ô r~l
ao ~
b
O
O
1
rl
O O
â F
aov â 7.~ Fd+
u
o y
+i
N
N
r~l a0
~;
~
z °~
+1 u'f ~D
+1 "O
O ~
a^
~
~
~
O~ O
~
ao +~1
O
~
N
m
~ d N W O v
Ô/' MI
O M
c
F,
Ç tl ~O .-i
~
,a
C +I M ~
C
Ô v1 .-a
ü aD n1
.C
a+
d
N ÛN
~
wW
q~ m~ N LL `O ~I ~ ^ ~ O d q I ~ a+ O 00 N m W o ."
^ d adl $ m Ir .w ~ W ~ q ~ q a
a~l
a~+
rl
~ ~
d
W rl .O O
~~H
wâ "d oâl+ m d N u ~ q W O ~ q W Ô .C m d U rl 1~ d M .C vl ra ~ W U ~ rl
~ a d a
ri
M
~
ô ^
~ ri
N
Ô +. a+
I
ao
rl
..
U
m
ô ~
â
,~
â~
O r~.l
ô
a° o
o
ao
ri , . .,
+i
~ N
q W a+~
~
d
~ ~
o
~ +.
o ~Y
a ^
..
â Û
wp ~: ai q m
~
ô ~
â
~
ô ~
Ô
.. w6 w ai q m
Gl a+ R7 .C p, al a+ C~ ~ .-1 m .. .~ d N
N
~
P"
â
x
CJ
b O W W N ~7
F] u
d
'i
r-1 d ~
.d
H
~ ~ rpl
d D,~ W H
u
a+
ao ~ N d
W
W
m
â~
ci ~ ~ â
adie 1~ ai ~ m ô
~
â~
N ,~-1
W
m ô ,qa c~voa+âx~ ~i o a+ ,i rl ~" aaa4 a ,i TI o q
W ai
~ 3 o m .~ ao u
m a+ "-1 a m
~ Id+û ~~w
ql ~ U ~ Hr
u
a47 m m
,5
r ql
0 orl q oe o ~ ~ D q ~ F+ ,~-1-1 u d a w ~° w~c"
ô ad+ uml .q a .é
2472
Antischizophrenic Drugs
Vol . 24, No . 26, 1979
What factors might account for the markedly differing protein binding of various neuroleptics? Lipophilic drugs tend to be most tightly bound to plasma proteins . 7-Hydroxylation of chlorpromazine and oxidation of the sulfur-2 substituent comrertiag thioridazine to meaoridazine both decrease lipophilicity. A chlorine-2 aubatituent transforms promazine to chlorpromazine, increasing lipophilicity and plasma protein binding . Trifluoperazine, the moat tightly protein bound neuroleptic, possesses both a lipophilic trifluomethyl ring aubstituent and piperazine side chain . Fluphenaziae displays about twice the percent free drug observed with chlorpromazine . Molindone and haloperidol, whose chemical structures are less lipophilic than the phenothiazines, display the highest percentage of free drug . The value of 9X free drug for haloperidol we observe confirms findings of Forsman and Ohman (1977) . A role of free drugs and metabolites in therapeutic activities has implications for attempts to relate clinical response to blood levels . Free levels of neuroleptic and active metabolites may provide better predictors of clin ical responses than total plasma levels of parent drugs assayed in earlier investigations . Acknowledgements Robert B . lams is the recipient of a fellowship from the Insurance Medical Scientist Training Fund sponsored by the Mutual of Omaha Insurance Co . References 1. 2. 3. 4.
5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 .
L . RIVERA-CALLMLIM, H. NASRALLAH, J. STRAUSS and L . LASAGNA, Am . J . Psychiatry, _133 646-652 (1976) . D . L. GARVER, H . DEKIRMENJIAN, J. M. DAVIS, R. CASPER and S . ERICRSEN, Am . J. Psychiatry, _134 304-307 (1977) . R. C. SMITH, A. DERIRMENJIAN, J . M. DAVIS, J . CRAYTON and H. EVANS, Comm . Psychopharmacol . _1 319-324 (1977) . D. L. GARVER, H . DERIRMENJIAN and J. M . DAMS, Pharmacokinetics and Clinical Response : Further Studies, L . A . Gottschalk Eds ., Spectrum Publications, is press, 1979 . D. A. WILES, T. ROLAKOWSKA, A. S . McNEILLY, B . M. MANDELBROTE and M. G. GELDER, Psychological Med ., _6 407-415 (1976) . G. SARALIS, S . H. CURRY, G . P . MOULD and M. H. LADER, Clin . Pharmacol . Ther ., _13 931-946 (1972) . S . A. CURRY, J . Pharm . Pharmacol., _22 193-197 (1970) . D. G. McDevitt, M. FRISR-HOLMBERG, J . W. HOLLIFIELD and D. G. SAAND, Clin . Pharmacol . Ther ., _20 152-157 (1976) . S, H. CURRY, Anal . Chem ., _40 1251-1255(1968) . S . A. CURRY and J . A. L. MARSHALL, Life Sciences _7 9-17 (1968) . A. BARRY, M. L. STEINBERG, A . A . MAMAN and J. P . BUCRLEY, Paychopharmacologia, _34 351-360 (1974) . A. A. MAMAN, D . H . EFRON and M . E . GOLDBERG, Life Sciences _4 2425-2438 (1965) . H. NYBACK and G . SEDVALL, Paychopharmacologia _26 155-160 (1972) . H. H. MELTZER, V. S . FANG, M. SINONOVICA and S . M. PAUL, Eur . J. Pharmacol . 41 431-436 (1977) . B. S . BUNNEY and G . K. AGHAJANIAN, Life Sciences _15 309-318 (1974) . I . CREESE, A . A . MAMAN, T. D. PROSSER and S . H . SNYDER, Eur . J. Pharmacol . _47 291-296 (1978) . L. L. IVERSEN, Science _188 1084-1089 (1975) . I . CREESE, D. R . BURT and S . H . SNYDER, Handbook of Psychopharmacology , Vol . 10, L. L. Iveraen, S . D . Iversen and S . H . Snyder, Eds ., pp . 37-88, Plenum Press (1978) .
Vol . 24, No . 26, 1979
19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 . 28 . 29 . 30 . 31 . 32 . 33 . 34 . 35 .
Antischizophrenic Drugs
2473
A . V . P . MACKAY, A . F . HEALEY and J . BARER, Br . J . Clin . Pharmacol . _1 425-430 (1974) . 0 . T . PHILLIPSON, J . M . McReown, J . BAKER and A . F . HEALEY, Br . J . Psychiat . _131 172-184 (1977) . G . SARALIS, T . L . CHAN, G . SATHANANTAAN, N . SCHOOLER, S . GOLDBERG and S . GERSHON, Comet . Peychopharmacol . _1 157-166 (1977) . G . SARALIS, T . L . CHAN, S . GERSHON and S . PARK, Psychopharmacologie 32 279-284 (1973) . I . CREESE and S . H . SNYDER, Nature _270 180-182 (1977) . F . A . J . VANDERHEEREN and R . G . MWSZE, Eur . J . Clin . Pharmacol . 1 1 135140 (1977) . E . MARTENSSON and B . E . ROOSE, Eur . J . Clin . Pharmacol _6 181-186 (1973) . E . MARTENSSON, G . NYBERG, R . ARELSSON and R . SERCR-HANSEN, Curr . Ther . Res . _18 687-700 (1975) . E . DINOVO, L . A . GOTTSCHALR, E . P . NOBLE and R . BIENER, Res . Comet . Chem . Pathol . Pharmacol . _7 489-496 (1974) . G . SAKALIS, S . J . TRAFICANTE and S . GERSHON, Curr . Ther . Res . _21 720-724 (1977) . GLASSMAN, A ., J . PEREL, J . Clin . Pharmacol . Psych . Newsletter _1 5 (1978) . F . M . BELPAIRE, F . A . J . VANDERHEEREN and M . G . BOGAERT, Arzneim . Forsch . (Drug Res .), _25 1969-1971 (1975) . G . NYBERG, R . ASELSSON, E . MARTENSSON, Eur . J . Clin . Pharmacol . _14 341350 (1978) . A . FORSMAN, R . OHMAN, Curr . Ther . Res . _21 245-255 (1977) . G . ALFREDSSON, B . WODE-HELGODT and G . SEDVALL, Psychopharmacology 48 123131 (1976) . I . CREESE, D . R . BURT aad S . H . SNYDER, Science _192 481-483 (1976) . I . CREESE, R . FREEDBERG and S . H . SNYDER, in preparation .
Life Sciences, Vol . 24, pp . 2467-2474 Printed in the U.S .A .
ANTISCHIZOPHRENIC DRUGS: DIFFERENTIAL PLASMA PROTEIN BINDING AND THERAPEUTIC ACTIVITY Kenneth A. Freedberg,
Robert B . Innil,
Ian Creese *aad Solomon H. Snyder
Departments of Pharmacology and Experimental Therapeutics and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine Baltimore, Maryland 21205 (Received in final form May 7,
1979)
Summary
The percentage of free neuroleptic drug unbound to plasma protein is much higher for the respective metabolites of chlorpromazine and thiori~azine, 7-hydroxychlorpromazine and mesoridazine, than for the parent drugs. The therapeutic activities of chlorpromazine and thioridazine may be mediated to a major extent by 7-hydroxychlorpromazine and mesoridazine respectively . Measuring free levels of the active metabolites of neuroleptics as well as the parent drugs may facilitate regulation of neuroleptic doses to secure optimal therapeutic responses. Bioavailability of drugs is a major determinant of therapeutic activity . Blood levels of several drugs, ouch as theophylline, digoxin, diphenylhydantoin, lithium and phenobarbital are routinely assayed to adjust dosage for During the past decade optimal therapeutic response with minimal side effects . various techniques have been employed to measure plasma levels of drugs in psychiatry, especially tricyclic antidepressants and the neuroleptic, antiachizophrenic drugs . Poor clinical response to neuroleptics may be associated not only with abnormally low blood levels but with abnormally high blood levels of the drugs (1-4) . Difficulties in predicting clinical status from blood levels of neuroleptics in some studies (5, 6) may reflect human biological variability For lipophilic drugs such as the or varying plasma protein binding of drugs . tricyclic antidepressants and the neuroleptics, high proportions of the total For the neuroleptic chlorpromaplasma content are bound to plasma proteins . zine, the percentage of drug bound to plasma protein can range between 90 and 99% in different individuals so that with identical total plasma levels of the drug the concentration of free chlorpromazine could vary ten fold (7) .Recently McDevitt _et _al . (S) showed that the extent of clinically evident ß-noradrenergic receptor blockade elicited by the ß-blocking drug propranolol did not correlate with total plasma levels of propranolol, was only modestly related to red cell levels of the drug but closely paralleled free levels of propranolol in patient plasma . These findings confirm the supposition that only the free drug is available to target organs ouch as the heart or brain .
*Permanent Address : Dr . Ian Creese, Department of Neurosciences, University of California at San Diego, La Jolla, California 92093 . 0024-3205/79/262467-0702 .00/0 Copyright (c) 1979 Pergamon Press Ltd
2468
Antischizophrenic Drugs
Vol . 24, No . 26, 1979
The major circulating metabolite of the neuroleptic chlorpromazine is In pharmacological tests which predict clin7-hydroxychlorpromazine (9,10) . ical antiachizophrenic effects, 7-hydroxychlorpromazine has about the same po tency as chlorpromazine (11-15) . Moreover, 7-hydroxychlorpromazine and chlorpromazine have similar affinities for dopamine receptors (16) the presumed sites for the therapeutic actions of neuroleptica (17,18) . In several clinical studies levels of 7-hydroxychlorpromazine correlated better with therapeutic responses of schizophrenics than did levels of chlorpromazine, despite the fact that the total plasma chlorpromazine levels were as high or higher than levels of 7-hydroxychlorpromaziae (19-22) . If total plasma levels of the drugs determined therapeutic response, then the clinical status of patients should have been at least as closely related to chlorpromazine as to 7-hydroxychlorpromazine levels . Recently we developed a simple, sensitive and specific radioreceptor assay for neuroleptic drugs based on their ability to compete for the binding of 3H-spiroperidol to dopamine receptors in corpus striatum membranes (23) . Using this assay, in the present study we have measured the proportion of various neuroleptice which are bound to plasma protein . We show that in fresh human plasma the percentage of unbound drug is 10 times greater for 7-hydroxychlorpromazine than for chlorpromazine . After treatment with thioridazine, blood levels of its metabolite meaoridazine are 40200% of total thioridazine levels (24-28), but at equal plasma levels the percent of free meaoridazine is 50-100 times that of free thioridazine . Thus chlorpromazine and thioridazine, two of the most widely employed antischizophrenic drugs, may not exert antischizophrenic effects themselves, but instead may act through their respective metabolites 7-hydroxychlorpromazine and meaoridazine . Methods Plasma from healthy adult male and female laboratory personnel (ages 2040) was freshly drawn into glass syringes containing about 200 U heparin. Neuroleptics dissolved in 0.1% ascorbic acid were added to plasma along with 0 .5 mg sodium metabisulfite per ml plasma and incubated for 30 minutes at 37 ° C to allow binding to plasma proteins, Sodium metabisulfite had no effect on plasma protein binding and was added to stabilize certain neuroleptics (29) . One ml plasma samples were dialysed against one ml 0.9% NaCl for 18 hours (equilibrium was attained in 6-15 hr) at 4° C in the dark in an EMO dialysis apparatus (Hoefer Scientific) using 12,000-14,000 dalton molecular weight cut-off membranes . The radioreceptor sassy for unlabeled neuroleptica was essentially Fresh rat caudate was homogenized with a Brinkas described previously (23) . man Polyfron PT10 in 70 volumes 50 mM Tris/CH1 pH 7 .7 and centrifuged at 50,000 After an intermediate reauspenaion and centrifugation in x g for 10 minutes . fresh buffer, the final pellet was reauspended in 70 volumes of ice cold 50 mM TripTria/HC1 pH 7 .6 containing O .1X ascorbic acid, 5 mM EDTA and 100 mM NaCl . licate assay tubes received in order : 250 ul dialysate, appropriately diluted retentate (material retained in the plasma chamber after dialysis), control plasma, or saline ; 50 ul O .1X ascorbic acid or 50 ul 10-5 (+)-butaclamol to measure nonspecific binding ; 25 ul 3H-spiroperidol (24 .6 Ci/mmol, New England Nuclear) for a final concentration of 0 .5 nM ; 175 ul caudate tissue for a total volume of 0.5 ml . Tubes were incubated at 37° C for 15 minutes and rapidly filtered through Whatman GF/B filters with three 5 ml rinses of ice cold buffer . Filters were counted by liquid scintillation spectrometry in 10 ml Formula 947 (New England Nuclear) . Concentrations of drugs in the dialyeate and retentate The percentage free drug was were determined from standard displacement curves . calculated as : ~di~ x free ~ retentate a 100
Vol . 24, No . 26, 1979
Antischizophrenic Drugs
2469
In our initial studies blood had been drawn into VacutainerR (BectonDickinson) tubes and stored at 4°C for 1-20 days, similar to conditions for most plasma protein binding studies . Since we found that use of VacutainerR tubes as well as storage increased the percent free for moat neuroleptics 1080 fold,all experiments whose results are reported here employed freshly drawn plasma using heparinized glass syringes . These technical difficulties may account for the substantially higher free levels measured previously for chlorpromazine and thioridazine (7,30) . However, our free levels for thiroida~ine and meaoridazine are equal to those recently determined by Axelaeon and Martensson (31) and that of haloperidol determined by Foreman and~ffhman (32) . By dialysing aeuroleptics is normal saline against this same solution, we have determined that a small amount (< 5X) of each drug adsorbs to the chamber and membrane . One might initially think that this adsorption would signifi cantly affect the free level, which is less than 1X for some neuroleptica . However, this adsorption would act transiently as a "sink" and should not affect the percentage of free drug in equilibrium with plasma proteins . Results and Discussion The percent free neuroleptic varies with concentration for certain of the drugs . Thus with chlorpromazine at respective concentrations of 8 yM, 80 uM and 400 yM the free levels are 0 .13X, 0.24X and 0 .79X (Figure 1, Table 1) . This pattern is also evident with ciethiothixene and meaoridazine, somewhat less with thioridazine and promazine and not apparent for 7-hydroxychlorpromazine . These differences might reflect varying drug-protein binding constants . The percent free drug at the lowest plasma concentration is probably most relevant to therapeutic practice, since therapeutic total plasma levels of chlorpromazine (0 .1 -1 .0 uM) are less than the lowest concentration of chlorpromazine examined here . The neuroleptic concentrations we employed were selected to facilitate measuring the fairly low concentrations of free drug . For comparisons among the different aeuroleptics, we will refer primarily to the data obtained at the lowest concentrations examined . At 8 uM only about 0 .13X of plasma chlorpromazine is free, while more than ten times as much 7-hydroxychlorpromazine is free . EV~en at the highest concentration examined, 0.4 mM, more than twice ae much 7-hydroxychlorpromazine is free than chlorpromazine . After chronic chlorpromazine treatment, total plasma levels of 7-hydroxychlorpromazine are 20 - 120X of the chlorpromazine levels (19-22, 33) . Assuming that the percentage of free drug at therapeutic doses corresponds to values we obtained at 8 uM, then free 7-hydroxychlorpromazine would be 2 - 12 times the levels of chlorpromazine . Thus after chronic treatment with chlorpromazine, the metabolite 7-hydroxychlorpromazine may be responsible for therapeutic effects, though with acute treatment with very low levels of 7-hydroxychlorpromazine the parent drug may be the primary active agent (9, 10) . These observations might explain the better correlation of 7-hydroxychlorpromazine than chlorpromazine plasma levels with therapeutic response (19-22) . Meaoridazine, a metabolite o£ thioridazine, has about the same potency as the parent drug in blocking dopamine receptors (34) and, after therapeutic doses of thioridazine, total plasma levels of meaoridazine are similar to those of thioridazine (11) . At all free concentrations examined the percentage of free meaoridazine exceeds that of thioridazine by 50-100 times, confirming the findings of Nyberg et al (31), and after thioridazine treatment, meaoridazine is the major therapeutically active agent . Mesoridazine itself is a marketed, clinically effective antipsychotic drug .
2470
Antischizophrenic Drugs
Vol,
SOr
24, No, 26, 1979
e 0
a
T
a
,m
lo
t a
a
.8
tiL 0
m~ .
0
a
â
.o
. ~
.o
.o
m
i .
0.10
oa
.
0
i
0
m
v
.oni .oe .oe woemon i . i W W o$g omo e~ o a °m8 ~S~ oog ~r9fn ~,9,D O ~ d N ô
O ~
~ i
O
n
O_ ~
O J i
J
O
û
~DRUG~ pM FIG . 1 Plasma neuroleptics not bound to plasma protein (free) . Fresh human plasma samples were incubated with neuroleptics, dialysed and bound and free drug concentration assayed as described in the text . Each point represents the mean value of triplicate assays from a separate human volunteer . Short horizontal bars indicate the means of the samples for each total drug concentration. Drug abbreviations are the same as in Table 1 .
Vol . 24, No . 26, 1979
0 0 ~
H a
o
Antischizophrenic Drugs
2471
~o 0 0 ~O r, O
F
'-I
N
d w W A ~ ~ ~ W
~ N
r1 ~ N A W b A TJ ~ q
tl N
p
N
O
v1
Ô r~l
ao ~
b
O
O
1
rl
O O
â F
aov â 7.~ Fd+
u
o y
+i
N
N
r~l a0
~;
~
z °~
+1 u'f ~D
+1 "O
O ~
a^
~
~
~
O~ O
~
ao +~1
O
~
N
m
~ d N W O v
Ô/' MI
O M
c
F,
Ç tl ~O .-i
~
,a
C +I M ~
C
Ô v1 .-a
ü aD n1
.C
a+
d
N ÛN
~
wW
q~ m~ N LL `O ~I ~ ^ ~ O d q I ~ a+ O 00 N m W o ."
^ d adl $ m Ir .w ~ W ~ q ~ q a
a~l
a~+
rl
~ ~
d
W rl .O O
~~H
wâ "d oâl+ m d N u ~ q W O ~ q W Ô .C m d U rl 1~ d M .C vl ra ~ W U ~ rl
~ a d a
ri
M
~
ô ^
~ ri
N
Ô +. a+
I
ao
rl
..
U
m
ô ~
â
,~
â~
O r~.l
ô
a° o
o
ao
ri , . .,
+i
~ N
q W a+~
~
d
~ ~
o
~ +.
o ~Y
a ^
..
â Û
wp ~: ai q m
~
ô ~
â
~
ô ~
Ô
.. w6 w ai q m
Gl a+ R7 .C p, al a+ C~ ~ .-1 m .. .~ d N
N
~
P"
â
x
CJ
b O W W N ~7
F] u
d
'i
r-1 d ~
.d
H
~ ~ rpl
d D,~ W H
u
a+
ao ~ N d
W
W
m
â~
ci ~ ~ â
adie 1~ ai ~ m ô
~
â~
N ,~-1
W
m ô ,qa c~voa+âx~ ~i o a+ ,i rl ~" aaa4 a ,i TI o q
W ai
~ 3 o m .~ ao u
m a+ "-1 a m
~ Id+û ~~w
ql ~ U ~ Hr
u
a47 m m
,5
r ql
0 orl q oe o ~ ~ D q ~ F+ ,~-1-1 u d a w ~° w~c"
ô ad+ uml .q a .é
2472
Antischizophrenic Drugs
Vol . 24, No . 26, 1979
What factors might account for the markedly differing protein binding of various neuroleptics? Lipophilic drugs tend to be most tightly bound to plasma proteins . 7-Hydroxylation of chlorpromazine and oxidation of the sulfur-2 substituent comrertiag thioridazine to meaoridazine both decrease lipophilicity. A chlorine-2 aubatituent transforms promazine to chlorpromazine, increasing lipophilicity and plasma protein binding . Trifluoperazine, the moat tightly protein bound neuroleptic, possesses both a lipophilic trifluomethyl ring aubstituent and piperazine side chain . Fluphenaziae displays about twice the percent free drug observed with chlorpromazine . Molindone and haloperidol, whose chemical structures are less lipophilic than the phenothiazines, display the highest percentage of free drug . The value of 9X free drug for haloperidol we observe confirms findings of Forsman and Ohman (1977) . A role of free drugs and metabolites in therapeutic activities has implications for attempts to relate clinical response to blood levels . Free levels of neuroleptic and active metabolites may provide better predictors of clin ical responses than total plasma levels of parent drugs assayed in earlier investigations . Acknowledgements Robert B . lams is the recipient of a fellowship from the Insurance Medical Scientist Training Fund sponsored by the Mutual of Omaha Insurance Co . References 1. 2. 3. 4.
5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 .
L . RIVERA-CALLMLIM, H. NASRALLAH, J. STRAUSS and L . LASAGNA, Am . J . Psychiatry, _133 646-652 (1976) . D . L. GARVER, H . DEKIRMENJIAN, J. M. DAVIS, R. CASPER and S . ERICRSEN, Am . J. Psychiatry, _134 304-307 (1977) . R. C. SMITH, A. DERIRMENJIAN, J . M. DAVIS, J . CRAYTON and H. EVANS, Comm . Psychopharmacol . _1 319-324 (1977) . D. L. GARVER, H . DERIRMENJIAN and J. M . DAMS, Pharmacokinetics and Clinical Response : Further Studies, L . A . Gottschalk Eds ., Spectrum Publications, is press, 1979 . D. A. WILES, T. ROLAKOWSKA, A. S . McNEILLY, B . M. MANDELBROTE and M. G. GELDER, Psychological Med ., _6 407-415 (1976) . G. SARALIS, S . H. CURRY, G . P . MOULD and M. H. LADER, Clin . Pharmacol . Ther ., _13 931-946 (1972) . S . A. CURRY, J . Pharm . Pharmacol., _22 193-197 (1970) . D. G. McDevitt, M. FRISR-HOLMBERG, J . W. HOLLIFIELD and D. G. SAAND, Clin . Pharmacol . Ther ., _20 152-157 (1976) . S, H. CURRY, Anal . Chem ., _40 1251-1255(1968) . S . A. CURRY and J . A. L. MARSHALL, Life Sciences _7 9-17 (1968) . A. BARRY, M. L. STEINBERG, A . A . MAMAN and J. P . BUCRLEY, Paychopharmacologia, _34 351-360 (1974) . A. A. MAMAN, D . H . EFRON and M . E . GOLDBERG, Life Sciences _4 2425-2438 (1965) . H. NYBACK and G . SEDVALL, Paychopharmacologia _26 155-160 (1972) . H. H. MELTZER, V. S . FANG, M. SINONOVICA and S . M. PAUL, Eur . J. Pharmacol . 41 431-436 (1977) . B. S . BUNNEY and G . K. AGHAJANIAN, Life Sciences _15 309-318 (1974) . I . CREESE, A . A . MAMAN, T. D. PROSSER and S . H . SNYDER, Eur . J. Pharmacol . _47 291-296 (1978) . L. L. IVERSEN, Science _188 1084-1089 (1975) . I . CREESE, D. R . BURT and S . H . SNYDER, Handbook of Psychopharmacology , Vol . 10, L. L. Iveraen, S . D . Iversen and S . H . Snyder, Eds ., pp . 37-88, Plenum Press (1978) .
Vol . 24, No . 26, 1979
19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 . 28 . 29 . 30 . 31 . 32 . 33 . 34 . 35 .
Antischizophrenic Drugs
2473
A . V . P . MACKAY, A . F . HEALEY and J . BARER, Br . J . Clin . Pharmacol . _1 425-430 (1974) . 0 . T . PHILLIPSON, J . M . McReown, J . BAKER and A . F . HEALEY, Br . J . Psychiat . _131 172-184 (1977) . G . SARALIS, T . L . CHAN, G . SATHANANTAAN, N . SCHOOLER, S . GOLDBERG and S . GERSHON, Comet . Peychopharmacol . _1 157-166 (1977) . G . SARALIS, T . L . CHAN, S . GERSHON and S . PARK, Psychopharmacologie 32 279-284 (1973) . I . CREESE and S . H . SNYDER, Nature _270 180-182 (1977) . F . A . J . VANDERHEEREN and R . G . MWSZE, Eur . J . Clin . Pharmacol . 1 1 135140 (1977) . E . MARTENSSON and B . E . ROOSE, Eur . J . Clin . Pharmacol _6 181-186 (1973) . E . MARTENSSON, G . NYBERG, R . ARELSSON and R . SERCR-HANSEN, Curr . Ther . Res . _18 687-700 (1975) . E . DINOVO, L . A . GOTTSCHALR, E . P . NOBLE and R . BIENER, Res . Comet . Chem . Pathol . Pharmacol . _7 489-496 (1974) . G . SAKALIS, S . J . TRAFICANTE and S . GERSHON, Curr . Ther . Res . _21 720-724 (1977) . GLASSMAN, A ., J . PEREL, J . Clin . Pharmacol . Psych . Newsletter _1 5 (1978) . F . M . BELPAIRE, F . A . J . VANDERHEEREN and M . G . BOGAERT, Arzneim . Forsch . (Drug Res .), _25 1969-1971 (1975) . G . NYBERG, R . ASELSSON, E . MARTENSSON, Eur . J . Clin . Pharmacol . _14 341350 (1978) . A . FORSMAN, R . OHMAN, Curr . Ther . Res . _21 245-255 (1977) . G . ALFREDSSON, B . WODE-HELGODT and G . SEDVALL, Psychopharmacology 48 123131 (1976) . I . CREESE, D . R . BURT aad S . H . SNYDER, Science _192 481-483 (1976) . I . CREESE, R . FREEDBERG and S . H . SNYDER, in preparation .
