STEADY-STATE PHARMACOKINETICS OF HYDROXYCHLOROQUINE IN RHEUMATOID ARTHRITIS PATIENTS Donald R. Miller, Shoukry K.W. Khalil, and Gloria A. Nygard
ABSTRACT: Steady-state pharmacokinetics of hydroxychloroquine (HC) sulfate (Plaquenil) were studied in five volunteers with rheumatoid arthritis who had taken 6 mg/kg/d of the drug for at least six months. Blood samples were drawn at 0, I, 2, 4, 6, 8, 12, and 24 hours following an oral dose. Both whole blood and plasma were assayed by an HPLC method for HC and its metabolites desethylhydroxychloroquine, desethylchloroquine, and didesethylchloroquine. A 24-hour urine collection was obtained and assayed for the same compounds. The pharmacokinetics of HC and its metabolites conformed to the model predicted by single-dose studies. During the 24-hour period the absorption phase and both early and late distribution phases were seen. Variation in mean maximum/minimum concentration was 40 percent. Renal clearance accounted for only 16 percent of unchanged HC (22 percent of total drug and metabolites) and did not correlate with creatinine clearance. Dlep Ann Pharmacother 1991;25:1302-5.
THE PHARMACOKINETICS of the 4-aminoquinoline drugs chloroquine and hydroxychloroquine (HC) sulfate are extremely unusual and, until recently, there has been confusion over their pharmacokinetic parameters. Widely varied elimination half-lives (t l/2S) were reported (3 hours to 45 days) and even nonlinear kinetics was discussed as a possibility. Tett et al. clarified the reasons for this confusion by demonstrating the importance of assay sensitivity and duration of study period when interpreting kinetic data for these drugs.'? The aminoquinolines are highly tissue bound, with their kinetics conforming to a three-compartment model. The first compartment, consisting of the blood volume, has a rapid distribution phase t l/2 in plasma or blood of about 3 hours. A second distribution phase, as the drug equilibrates into tissues, has a t l / 2 ranging from 40 hours to 5 days.'> Finally, true elimination of the drugs has a t l / 2 of around 40 days because of slow redistribution into blood from tissue repositories. An additional source of pharmacokinetic variation is the fluid used for analysis. Because the drugs accumulate in red blood cells, white blood cells, and platelets, concentrations in whole blood are about five to ten times higher than
DONALD R. MILLER, Pharm.Dc, is an Associate Professor of Pharmacy Practice, College of Pharmacy; SHOUKRY K.W. KHALIL, Ph.D" is a Professor of Pharmaceutical Sciences and the Director, Pharmacokinetics Drug Analysis Laboratory, College of Pharmacy; and GLORIA A. NYGARD, M.S., is a Chemist, Pharmacokinetics Drug Analysis Laboratory, College of Pharmacy, North Dakota State University, Fargo, ND. Reprints: Donald R. Miller, Pharm.D., College of Pharmacy, North Dakota State University, Fargo, ND 58105. Supported by a grant from Winthrop Pharmaceuticals, New York, NY.
1302 • D1CP, The Annals ofPharmacotherapy •
in plasma, and kinetic parameters in blood also differ from those in plasma. Finally, even when the above factors are rigorously controlled, there is large variation in disposition between subjects.P HC is currently used as an antimalarial and antirheumatic drug. However, additional uses, such as improving glucose tolerance in noninsulin-dependent diabetic patients" or controlling hyperlipidemia," have been suggested. Therefore, data on its pharmacokinetic properties are needed. Apart from two single-dose studies by Tett et al. in healthy volunteers.t-' information on human pharmacokinetics of HC has largely been extrapolated from chloroquine. Because limited information is available on HC specifically and no information has been published on its pharmacokinetics in patients at steady-state after oral dosing the following study was conducted to obtain that information.
Methods Five volunteers with rheumatoid arthritis (three women and two men) were enrolled in the study (Table I). All were white and nonsmokers, Each had been taking HC sulfate (Plaquenil) 6 mg/kg/d for at least six months, Informed consent was obtained from each subject after routine medical and ophthalmologic assessment to rule out serious concomitant conditions, The subjects were asked to take their HC at 0900 for the week prior to the study. On the day of the study heparin locks were inserted into each patient's forearm and a blood sample was taken at 0900. All patients emptied their bladders and then took their regular daily dose of HC with 200 mL of water, Additional blood samples were drawn at I, 2, 4, 6, 8, 12, and 24 hours following the dose. A 24-hour urine collection was begun with the start of HC administration for determining renal clearance, A sample aliquot was retained for analysis. The exact sampling times for both blood and urine collection were recorded and used in the phannacokinetic analysis of data, The volunteers fasted for 12 hours before their dose and for 2 hours afterward. All were served a meal after 2 hours. Subjects were ambulatory throughout the study, From each patient, 5 mL of blood was collected into heparinized polypropylene tubes at each sampling point; 2 mL was saved as whole blood and 3 mL was spun down to plasma within 30 minutes by centrifugation at 1200 g for 7 minutes, Blood and urine samples were immediately frozen at -20°C until analysis. HC and its three major metabolites (desethylhydroxychloroquine [DEHC], desethylchloroquine [DEC], and didesethylchloroquine [DDEC)) were analyzed in all samples using a sensitive HPLC method with fluorescence detection. Whole blood samples were diluted with water and sonicated to disrupt the red blood cells, then centrifuged and an aliquot of the supernatant extracted using ethylacetate/2-propanol (90/10) under basic conditions. Plasma samples were rendered basic and extracted similarly. The organic phase was evaporated to dryness and the residue reconstituted in methanol for injection into the HPLC. Protein in urine
1991 December. Volume 25
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Research/Practice samples was precipitated by the addition of acetonitrile containing the internal standard, followed by centrifugation. An aliquot of the supernatant was chromatographed. Propranolol was the internal standard for all three types of samples analyzed. Chromatography was performed on a Hewlett Packard Model 1090 equipped with a Beckman Ultrasphere Cyano column and a Perkin Elmer Model LS-4 fluorescence spectrometer. A mobile phase consisting of 30% dibasic potassium phosphate (0.02 mol/L, pH 7.0), 56% acetonitrile, and 14% methanol was used to elute the drug, its metabolites, and the internal standard. The flow rate was 3 mlzmin, the injection volume ranged from 2 to 15 mL, and the column compartment was maintained at 50°C. The effluent was monitored using an excitation wavelength of 330 nm and emission of 380 nm. The standard curves were linear and reproducible over the following concentration ranges: plasma, 5-1000 ng/mL; whole blood, 20-5000 ng/mL; and urine, 100-50000 ng/mL. Peak-height ratios (parent or metabolites to internal standard) for each standard and sample were calculated and a least-squares regression analysis was performed on the peak-height ratio versus nominal concentration. The concentrations of HC and its metabolites were determined by inverse prediction from the linear regression. Coefficients of variation were 6-10 percent. For reporting purposes the concentrations were converted to nanomoles per liter using the conversion factor of I ng/mL = 2.98 nmol/L. Areas under the concentration versus time curve (AUCo-24) were calculated by the trapezoidal rule. The maximum HC concentration and time of maximum concentration were obtained by visual inspection of individual patient concentration versus time curves. Renal clearances were determined by dividing the amount of HC or metabolite excreted in the urine for the 24-hour period after drug administration by the respective AUCo.24. Distribution t l /2 was estimated from the slope of the mean log HC concentration versus time curve between 12 and 24 hours.
Results
1000
:::J :::::.
• HC o DEHC • DEC
0
E 800
.s, 0
z
600
a:
~
400
I-
jr--...../\--_ .... C1\
•
_--------------0
.P,
'\0-_------0-----
\'_0---0//
Z
LU
oZ
200
o
0
---------.
~-.-..........
0
o
12
8
4
16
24
20
TIME (h) Figure I. Mean plasma concentration versus timecurvefor hydroxychloroquine and metabolites. HC = hydroxychloroquine; DEHC = desethylhydroxychloroquine; DEC = desethylchloroquine.
~ o
4000 - , - - - - - - - - - - - - - - - - - - - ,
.sE 3000 Z
Q ~
a:
I
• HC c DEHC DEC ... DDEC
I\.
•
"-
--
2000
oP'o-__ -o
I-
Z
~
{
1000
Z
8
o---o--------o------------------------o
-.- -.
....... -..... -..........
k-
.
---.------------------.
-,
' .... - -
O-'------.---,-----,.---,---,---------,---,---J 4
Peak concentrations were late and extremely variable, occurring at one to six hours in plasma and two to eight hours in blood. As shown in Figures I and 2, the variation from trough to peak mean concentration of parent drug in plasma was 37 percent, and in whole blood was 39 percent. This is much less variability than in the patient-to-patient mean concentration, which was 71 percent in plasma and 150 percent in blood (reflected in the AUCO-24 reported in Table 2). The t l/ 2 of the last distribution phase observable was 95 hours in plasma and 86 hours in whole blood. True elimination rate constants and t l /2S could not be calculated because of the limited sampling period during a single dosing interval and the long elimination phase of the drug. Because of the fluctuation in individual patient data during the short sample collection period, it also was not possible to fit the data into a compartmental pharmacokinetic model. However, the data generally are similar to what would be expected from single-dose studies.P
_
12
16
20
24
TIME (h) Figure 2. Mean whole blood concentration versus timecurveforhydroxychloroquine and metabolites. HC = hydroxychloroquine; DEHC = desethylhydroxychloroquine; DEC =desethylchloroquine, DDEC =didesethylchloroquine.
Steady-state concentrations of DEHC, DEC, and DDEC were 64, 17, and 15 percent, respectively, of parent drug in plasma and 54, 19, and II percent of parent drug in whole blood (Figures I and 2). Fluctuation in concentrations of the metabolites was small and appeared unrelated to the peak of the parent drug. Because of a technical problem DDEC could not be assayed in all plasma samples and therefore these concentrations are not shown in Figure I. Whole blood HC concentrations were an average of 5.0 times higher than simultaneous plasma concentrations. However, the ratios varied from 2.5 to 10.2. A similar range of blood to plasma ratios was found for the HC metabolites. ANOVA showed that the mean ratios were
Table I. Patient Characteristics HC CREATININE (nmol/L)
DOSAGE (mg/kg)
PATIENT
SEX
AGE
WEIGHT (kg)
I 2 3
M F F
82 65 65
94.2 79.5 65.9
168 88 62
6.4 6.3 6.1
4 5
M F
51 67
87.5 81.8
106 80
5.7 6.1
OTHER MEDICATIONS prednisone, methylphenidate aspirin prednisone, auranofin, levothyroxine, aspirin, diclofenac, amitriptyline naproxen indomethacin, levothyroxine, hydrochlorothiazide
He = hydroxychloroquine.
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•
1991 December, Volume 25 •
1303
significantly different between patients and chemical compounds (DEC> HC + DEHC) but that time of collection was not a significant factor influencing the ratios. Renal clearances were calculated for each subject (Table 3). The mean amount of HC excreted unchanged was 61 mg (about 16 percent of the daily dose). The mean amount of total drug plus metabolites excreted renally was only 84 mg (22 percent of the daily dose). There was no correlation between HC clearance and creatinine clearance (p>O.05).
Discussion The steady-state pharmacokinetics of HC were similar to what could have been expected from single-dose studies. Because of the long elimination t'/2 of the drug, steady-state concentrations undergo limited fluctuation over a daily interval. The observed variation is attributable to the processes of absorption and distribution. Absorption in our patients was slow, as in other studies.v The advanced age of our patients also may have caused slow gastric emptying. The extent of accumulation of HC metabolites during chronic dosing has not been previously documented. The HC metabolites accumulated in blood cells similarly to the parent drug as determined by similar blood:plasma ratios. Concentrations of DEHC were high enough to be of possible clinical importance. However, the implications of metabolite accumulation are unknown, as the antirheumatic and toxicologic properties of these chemicals have not been determined. The large variability in plasma concentrations of 4aminoquinolines is well known'- and thought to be related to the difficulty in separating plasma from the cell components of blood (red cells, white cells, and platelets), each of which harbor the drug. I However, various authors have found a wide range of blood:plasma ratios even after following recommendations for thorough centrifugation of Table 2. Pharmacokinetic Parameters of
HydroxychloroquinePARAMETER
AUCo-24 (nrnol-h/L) C max (nmol/L) T max (h)
WHOLE BLOOD
PLASMA
14 706± 3057 (11437-19591) 858 ±221 (605-1132) 3.6 ± 2.5 (l---Q)
77 140 ± 27 386 (47740-121363) 3794± 1296 (2265-5719) 4.4 ± 3.3 (2-8)
"Values are given as mean ± SD (range). AUCo-24 = area under the concentration versus time curve for 24 hours; C max =maximum concentration; T max =time to maximum concentration.
plasma.t-" In practice, plasma concentrations of aminoqui no lines reach fairly stable values at centrifugation forces over 500 g or at times over 10 minutes. Completely platelet-free plasma would require centrifugation force (20000 g for 30 minutes) not practical for routine use." The most reasonable procedure is to standardize centrifugation force. Whole blood concentrations are probably subject to similar variability secondary to variation in white cell and platelet count from patient to patient and time to time. The blood:plasma ratios obtained in this study were no more variable than in other investigations. The ANOVA suggests that ratios depend partly on individual patient variability; however, most of the variability in blood:plasrna ratios is still unaccounted for. Tett et al. have found more patient-to-patient variability in plasma concentrations than in blood and recommended that whole blood concentrations be followed to avoid the problem with plasma separation.P Our results do not support that conclusion, although the choice of whole blood over plasma for analysis does have the advantage of yielding much higher concentrations for all compounds and therefore the sensitivity of assay long after dosing is greater if blood is used. The estimates of renal clearance in this study were similar to those reported after single doses, I which suggests that no change occurs with chronic dosing. However, the proportion of drug eliminated renally (16 percent) is less than estimated previously. Only 22 percent (84 mg/d) of the daily dose could be recovered in urine. Although renal clearance is high, its contribution to overall rate of elimination is minimal because of the huge tissue stores of the drug. Nonrenal clearance, via enterohepatic recycling and shedding of pigmented tissue such as skin, accounts for most of the elimination." The steady-state concentrations for our subjects in both plasma and blood were lower than predicted by Tett et al. from single-dose data. Also, the fraction of drug eliminated by renal clearance was less and the concentrations of DEHC were higher than previously reported.' Together, these observations suggest that the rate of metabolism may be induced by chronic dosing. Concentrations should have been at steady-state because even with the very slow elimination of HC, concentrations should reach 96 percent of steady-state by 180 days.' All of our patients had taken the drug for longer than that and were believed to be good compliers. It is also possible that elimination of HC was influenced by the concomitant drugs our patients were taking, or by their fairly advanced age. In conclusion, during chronic dosing the processes that determine daily variation in blood or plasma concentrations of HC are those of absorption and distribution rather
Table 3. Renal Clearances WHOLE BLOOD (mL/min)
PLASMA (mL/min) PATIENT
HC
DEHC
DEC
HC
DEHC
DEC
DDEC
1 2 3 4 5
271.3 136.5 205.2 174.2 213.8 200.2
45.8 28.3 26.3 49.3 88.6 47.7
219.7 100.1 153.2 161.8 209.9 168.9
44.0 25.8 43.9 28.4 59.0 40.2
9.9 6.7 7.7 5.6 38.5 13.7
34.5 17.2 28.3 16.6 51.4 29.6
55.0 18.5 29.8 20.0 52.9 35.2
MEAN
DDEC
=didesethylchloroquine; DEC =desethylchloroquine; DEHC =desethylhydroxychloroquine; HC =hydroxychloroquine.
1304 • Dlep, The Annals ofPharmacotherapy •
1991 December, Volume25
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Research/Practice
than elimination. The timing of peak concentrations is quite variable. Therefore, the daily fluctuation in concentrations for a particular patient is fairly small, particularly in comparison with patient-to-patient variability. This has implications for attempts at monitoring therapeutic blood concentrations, in that the exact timing of blood draws will not be of critical importance. ~ We wish tothank Keith Rodvold, Phann.D., forhishelpful review ofthemanuscript, Connie Kluender, Kathy Cota, and Justus Fiechtner, M.D" forrecruiting thepatients, and Gwen Lunde fortyping themanuscript.
References I, TETI SE, CUTLER OJ, DAYRO, BROWN KF. A dose-ranging studyof the pharmacokinetics of hydroxychloroquine following intravenous administration to healthy volunteers, Br J Clin PharmacoI1988;26:303-13, 2, TETISE, CUTLER OJ, DA YRO, BROWN KF. Bioavailability of hydroxychloroquine tablets in healthyvolunteers, Br J Clin Pharmacol1989; 27:771-9. 3. CUTLER OJ, MACINTYRE AC, TETI SE. Pharmacokinetics and cellular uptakeof 4-aminoquinoline antimalarials. Agents Actions 1988;24(suppl): 142-57, 4. QUATRARO A, CONSOLI G,MAGNO M, et aI. Hydroxychloroquine in decompensated, treatment-refractory noninsulin-dependent diabetes mellitus, Ann Intern Med 1990;//2:678-81. 5. WALLACE OJ, METZGER AL, STECHER V. Antihyperlipidemic actions of hydroxychloroquine (Plaquenil)(abstract). Arthritis Rheum 1990;33 (Maysuppl): R7. 6. MCCHESNEY EW. Animal toxicity and pharmacokineticsof hydroxychloroquine sulfate, Am J Med 1983;75(suppl 6A):11-8, 7. GUSTAFSSON LL, WALKER 0, ALVAN G,et al. Dispositionof chloroquine in man after single intravenous and oral doses, Br J Clin PharmacoI1983;15:471-9.
8. ROMBO L, ERICSSON O. ALVAN G, LINDSTROM B,GUSTAFSSON LL, SJOQVIST F.Chloroquineand desethylchloroquine in plasma, serum and whole blood: problems in assay and handling of samples. Ther Drug Monit 1985;7:211-5.
9, MACKENZIE AH. Pharmacologic actions of 4-aminoquinoline compounds. Am J Med 1983;75(suppI6A):5-1O,
EXTRACTO
Estudiode los parametres farmacocineticosdel sulfato de hidroxicloroquina (HCQ) en cinco pacientes con artritis reumatoide que
tomaban HCQ 6 mg/kg/d durante aI menos seis meses antes, Se recogieronmuestras de sangre a las 0, I, 2, 4, 6, 8, 12, Y 24 horas despues de una dosis oral. HCQ y sus metabolitos,desetilhidroxicloroquina, desetilcloroquina, y didesetilcloroquina, fueron determinados en sangre total, plasma, y orina de 24 horas. EI comportamientofarmacocineticode HCQ y sus metabolitosrespondi6 aI modele previsto tras adrninistraci6n de dosis unica, Durante las 24 horas se pudo ver la fase de absorci6n y dos fases de distribuci6n,una inicial y otra tardfa, EI porcentaje de variaci6nde la concentraci6n media maxima/minima fue del 40 por ciento, S610 el 12 por ciento de HCQ inalterada (17 por ciento HCQ mas metabolitos) fue recuperadaen orina. No hubo correlaci6nentre el aclaramientode farrnaco y el de creatinina. CARMENCAO
RESUME
La pharmacocinetique du sulfate d'hydroxychloroquine (Plaquenil, Winthrop) 11 I' etat d' equilibre a ete etudiee chez cinq patients souffrant d'arthrite rhumatoide, Ces patients avaient recu 6 mg/kg/jour du medicament en question pour une duree minimale de six mois avant de participerdans cette etude. Des echantillons sanguins furent preleves 11 0, 1,2,4,6,8, 12, et 24 heures suivant l'administration d'une dose orale du medicament.Le sang et Ie plasma furent analyses par chromatographie liquide pour determiner les concentrationssanguineset plasmatiquesd'hydroxychloroquine (HCQ) ainsi que ses metabolites: desethylhydroxychloroquine, desethylchloroquine et didesethylchloroquine, Une collection urinairede 24 heures a egalernent ete effectuee suivant la prise orale d'une dose d'HCQ. Les concentrations urinairesde l'HCQ et des metabolitesont ete determinees par la meme methode. La pharmacocinetique de I'HCQ et des metabolites, 11 l'etat d'equilibre, etait conforme au modele pharmacocinetique estime 11 partir d'une dose orale unique. Cependant, la nature des donnes n' a pas permis de determiner la constanteet la demi-vie d'elimination, Durant une periode de 24 heures,la phase d' absorption ainsi que les phases rapide et lente de distributionfurent observees. Le degre de variationentre les concentrationsmoyennes minimales et maximales etait de 40 pourcent. L' elimination renale ne representait que 12 pourcent de la clairance de I'HCQ et 17 pourcent de la clairance totale (HCQ et ses metabolites).De plus, aucune correlation entre la clairance de la creatinine et la clairance renale du medicament n' a pu etre demontree.
DICP, The Annals ofPharmacotherapy • Downloaded from aop.sagepub.com at Elsevier Scirus on May 23, 2015
ERJcMASSON
1991December, Volume 25 •
1305
ABSTRACT: Steady-state pharmacokinetics of hydroxychloroquine (HC) sulfate (Plaquenil) were studied in five volunteers with rheumatoid arthritis who had taken 6 mg/kg/d of the drug for at least six months. Blood samples were drawn at 0, I, 2, 4, 6, 8, 12, and 24 hours following an oral dose. Both whole blood and plasma were assayed by an HPLC method for HC and its metabolites desethylhydroxychloroquine, desethylchloroquine, and didesethylchloroquine. A 24-hour urine collection was obtained and assayed for the same compounds. The pharmacokinetics of HC and its metabolites conformed to the model predicted by single-dose studies. During the 24-hour period the absorption phase and both early and late distribution phases were seen. Variation in mean maximum/minimum concentration was 40 percent. Renal clearance accounted for only 16 percent of unchanged HC (22 percent of total drug and metabolites) and did not correlate with creatinine clearance. Dlep Ann Pharmacother 1991;25:1302-5.
THE PHARMACOKINETICS of the 4-aminoquinoline drugs chloroquine and hydroxychloroquine (HC) sulfate are extremely unusual and, until recently, there has been confusion over their pharmacokinetic parameters. Widely varied elimination half-lives (t l/2S) were reported (3 hours to 45 days) and even nonlinear kinetics was discussed as a possibility. Tett et al. clarified the reasons for this confusion by demonstrating the importance of assay sensitivity and duration of study period when interpreting kinetic data for these drugs.'? The aminoquinolines are highly tissue bound, with their kinetics conforming to a three-compartment model. The first compartment, consisting of the blood volume, has a rapid distribution phase t l/2 in plasma or blood of about 3 hours. A second distribution phase, as the drug equilibrates into tissues, has a t l / 2 ranging from 40 hours to 5 days.'> Finally, true elimination of the drugs has a t l / 2 of around 40 days because of slow redistribution into blood from tissue repositories. An additional source of pharmacokinetic variation is the fluid used for analysis. Because the drugs accumulate in red blood cells, white blood cells, and platelets, concentrations in whole blood are about five to ten times higher than
DONALD R. MILLER, Pharm.Dc, is an Associate Professor of Pharmacy Practice, College of Pharmacy; SHOUKRY K.W. KHALIL, Ph.D" is a Professor of Pharmaceutical Sciences and the Director, Pharmacokinetics Drug Analysis Laboratory, College of Pharmacy; and GLORIA A. NYGARD, M.S., is a Chemist, Pharmacokinetics Drug Analysis Laboratory, College of Pharmacy, North Dakota State University, Fargo, ND. Reprints: Donald R. Miller, Pharm.D., College of Pharmacy, North Dakota State University, Fargo, ND 58105. Supported by a grant from Winthrop Pharmaceuticals, New York, NY.
1302 • D1CP, The Annals ofPharmacotherapy •
in plasma, and kinetic parameters in blood also differ from those in plasma. Finally, even when the above factors are rigorously controlled, there is large variation in disposition between subjects.P HC is currently used as an antimalarial and antirheumatic drug. However, additional uses, such as improving glucose tolerance in noninsulin-dependent diabetic patients" or controlling hyperlipidemia," have been suggested. Therefore, data on its pharmacokinetic properties are needed. Apart from two single-dose studies by Tett et al. in healthy volunteers.t-' information on human pharmacokinetics of HC has largely been extrapolated from chloroquine. Because limited information is available on HC specifically and no information has been published on its pharmacokinetics in patients at steady-state after oral dosing the following study was conducted to obtain that information.
Methods Five volunteers with rheumatoid arthritis (three women and two men) were enrolled in the study (Table I). All were white and nonsmokers, Each had been taking HC sulfate (Plaquenil) 6 mg/kg/d for at least six months, Informed consent was obtained from each subject after routine medical and ophthalmologic assessment to rule out serious concomitant conditions, The subjects were asked to take their HC at 0900 for the week prior to the study. On the day of the study heparin locks were inserted into each patient's forearm and a blood sample was taken at 0900. All patients emptied their bladders and then took their regular daily dose of HC with 200 mL of water, Additional blood samples were drawn at I, 2, 4, 6, 8, 12, and 24 hours following the dose. A 24-hour urine collection was begun with the start of HC administration for determining renal clearance, A sample aliquot was retained for analysis. The exact sampling times for both blood and urine collection were recorded and used in the phannacokinetic analysis of data, The volunteers fasted for 12 hours before their dose and for 2 hours afterward. All were served a meal after 2 hours. Subjects were ambulatory throughout the study, From each patient, 5 mL of blood was collected into heparinized polypropylene tubes at each sampling point; 2 mL was saved as whole blood and 3 mL was spun down to plasma within 30 minutes by centrifugation at 1200 g for 7 minutes, Blood and urine samples were immediately frozen at -20°C until analysis. HC and its three major metabolites (desethylhydroxychloroquine [DEHC], desethylchloroquine [DEC], and didesethylchloroquine [DDEC)) were analyzed in all samples using a sensitive HPLC method with fluorescence detection. Whole blood samples were diluted with water and sonicated to disrupt the red blood cells, then centrifuged and an aliquot of the supernatant extracted using ethylacetate/2-propanol (90/10) under basic conditions. Plasma samples were rendered basic and extracted similarly. The organic phase was evaporated to dryness and the residue reconstituted in methanol for injection into the HPLC. Protein in urine
1991 December. Volume 25
Downloaded from aop.sagepub.com at Elsevier Scirus on May 23, 2015
Research/Practice samples was precipitated by the addition of acetonitrile containing the internal standard, followed by centrifugation. An aliquot of the supernatant was chromatographed. Propranolol was the internal standard for all three types of samples analyzed. Chromatography was performed on a Hewlett Packard Model 1090 equipped with a Beckman Ultrasphere Cyano column and a Perkin Elmer Model LS-4 fluorescence spectrometer. A mobile phase consisting of 30% dibasic potassium phosphate (0.02 mol/L, pH 7.0), 56% acetonitrile, and 14% methanol was used to elute the drug, its metabolites, and the internal standard. The flow rate was 3 mlzmin, the injection volume ranged from 2 to 15 mL, and the column compartment was maintained at 50°C. The effluent was monitored using an excitation wavelength of 330 nm and emission of 380 nm. The standard curves were linear and reproducible over the following concentration ranges: plasma, 5-1000 ng/mL; whole blood, 20-5000 ng/mL; and urine, 100-50000 ng/mL. Peak-height ratios (parent or metabolites to internal standard) for each standard and sample were calculated and a least-squares regression analysis was performed on the peak-height ratio versus nominal concentration. The concentrations of HC and its metabolites were determined by inverse prediction from the linear regression. Coefficients of variation were 6-10 percent. For reporting purposes the concentrations were converted to nanomoles per liter using the conversion factor of I ng/mL = 2.98 nmol/L. Areas under the concentration versus time curve (AUCo-24) were calculated by the trapezoidal rule. The maximum HC concentration and time of maximum concentration were obtained by visual inspection of individual patient concentration versus time curves. Renal clearances were determined by dividing the amount of HC or metabolite excreted in the urine for the 24-hour period after drug administration by the respective AUCo.24. Distribution t l /2 was estimated from the slope of the mean log HC concentration versus time curve between 12 and 24 hours.
Results
1000
:::J :::::.
• HC o DEHC • DEC
0
E 800
.s, 0
z
600
a:
~
400
I-
jr--...../\--_ .... C1\
•
_--------------0
.P,
'\0-_------0-----
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Z
LU
oZ
200
o
0
---------.
~-.-..........
0
o
12
8
4
16
24
20
TIME (h) Figure I. Mean plasma concentration versus timecurvefor hydroxychloroquine and metabolites. HC = hydroxychloroquine; DEHC = desethylhydroxychloroquine; DEC = desethylchloroquine.
~ o
4000 - , - - - - - - - - - - - - - - - - - - - ,
.sE 3000 Z
Q ~
a:
I
• HC c DEHC DEC ... DDEC
I\.
•
"-
--
2000
oP'o-__ -o
I-
Z
~
{
1000
Z
8
o---o--------o------------------------o
-.- -.
....... -..... -..........
k-
.
---.------------------.
-,
' .... - -
O-'------.---,-----,.---,---,---------,---,---J 4
Peak concentrations were late and extremely variable, occurring at one to six hours in plasma and two to eight hours in blood. As shown in Figures I and 2, the variation from trough to peak mean concentration of parent drug in plasma was 37 percent, and in whole blood was 39 percent. This is much less variability than in the patient-to-patient mean concentration, which was 71 percent in plasma and 150 percent in blood (reflected in the AUCO-24 reported in Table 2). The t l/ 2 of the last distribution phase observable was 95 hours in plasma and 86 hours in whole blood. True elimination rate constants and t l /2S could not be calculated because of the limited sampling period during a single dosing interval and the long elimination phase of the drug. Because of the fluctuation in individual patient data during the short sample collection period, it also was not possible to fit the data into a compartmental pharmacokinetic model. However, the data generally are similar to what would be expected from single-dose studies.P
_
12
16
20
24
TIME (h) Figure 2. Mean whole blood concentration versus timecurveforhydroxychloroquine and metabolites. HC = hydroxychloroquine; DEHC = desethylhydroxychloroquine; DEC =desethylchloroquine, DDEC =didesethylchloroquine.
Steady-state concentrations of DEHC, DEC, and DDEC were 64, 17, and 15 percent, respectively, of parent drug in plasma and 54, 19, and II percent of parent drug in whole blood (Figures I and 2). Fluctuation in concentrations of the metabolites was small and appeared unrelated to the peak of the parent drug. Because of a technical problem DDEC could not be assayed in all plasma samples and therefore these concentrations are not shown in Figure I. Whole blood HC concentrations were an average of 5.0 times higher than simultaneous plasma concentrations. However, the ratios varied from 2.5 to 10.2. A similar range of blood to plasma ratios was found for the HC metabolites. ANOVA showed that the mean ratios were
Table I. Patient Characteristics HC CREATININE (nmol/L)
DOSAGE (mg/kg)
PATIENT
SEX
AGE
WEIGHT (kg)
I 2 3
M F F
82 65 65
94.2 79.5 65.9
168 88 62
6.4 6.3 6.1
4 5
M F
51 67
87.5 81.8
106 80
5.7 6.1
OTHER MEDICATIONS prednisone, methylphenidate aspirin prednisone, auranofin, levothyroxine, aspirin, diclofenac, amitriptyline naproxen indomethacin, levothyroxine, hydrochlorothiazide
He = hydroxychloroquine.
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significantly different between patients and chemical compounds (DEC> HC + DEHC) but that time of collection was not a significant factor influencing the ratios. Renal clearances were calculated for each subject (Table 3). The mean amount of HC excreted unchanged was 61 mg (about 16 percent of the daily dose). The mean amount of total drug plus metabolites excreted renally was only 84 mg (22 percent of the daily dose). There was no correlation between HC clearance and creatinine clearance (p>O.05).
Discussion The steady-state pharmacokinetics of HC were similar to what could have been expected from single-dose studies. Because of the long elimination t'/2 of the drug, steady-state concentrations undergo limited fluctuation over a daily interval. The observed variation is attributable to the processes of absorption and distribution. Absorption in our patients was slow, as in other studies.v The advanced age of our patients also may have caused slow gastric emptying. The extent of accumulation of HC metabolites during chronic dosing has not been previously documented. The HC metabolites accumulated in blood cells similarly to the parent drug as determined by similar blood:plasma ratios. Concentrations of DEHC were high enough to be of possible clinical importance. However, the implications of metabolite accumulation are unknown, as the antirheumatic and toxicologic properties of these chemicals have not been determined. The large variability in plasma concentrations of 4aminoquinolines is well known'- and thought to be related to the difficulty in separating plasma from the cell components of blood (red cells, white cells, and platelets), each of which harbor the drug. I However, various authors have found a wide range of blood:plasma ratios even after following recommendations for thorough centrifugation of Table 2. Pharmacokinetic Parameters of
HydroxychloroquinePARAMETER
AUCo-24 (nrnol-h/L) C max (nmol/L) T max (h)
WHOLE BLOOD
PLASMA
14 706± 3057 (11437-19591) 858 ±221 (605-1132) 3.6 ± 2.5 (l---Q)
77 140 ± 27 386 (47740-121363) 3794± 1296 (2265-5719) 4.4 ± 3.3 (2-8)
"Values are given as mean ± SD (range). AUCo-24 = area under the concentration versus time curve for 24 hours; C max =maximum concentration; T max =time to maximum concentration.
plasma.t-" In practice, plasma concentrations of aminoqui no lines reach fairly stable values at centrifugation forces over 500 g or at times over 10 minutes. Completely platelet-free plasma would require centrifugation force (20000 g for 30 minutes) not practical for routine use." The most reasonable procedure is to standardize centrifugation force. Whole blood concentrations are probably subject to similar variability secondary to variation in white cell and platelet count from patient to patient and time to time. The blood:plasma ratios obtained in this study were no more variable than in other investigations. The ANOVA suggests that ratios depend partly on individual patient variability; however, most of the variability in blood:plasrna ratios is still unaccounted for. Tett et al. have found more patient-to-patient variability in plasma concentrations than in blood and recommended that whole blood concentrations be followed to avoid the problem with plasma separation.P Our results do not support that conclusion, although the choice of whole blood over plasma for analysis does have the advantage of yielding much higher concentrations for all compounds and therefore the sensitivity of assay long after dosing is greater if blood is used. The estimates of renal clearance in this study were similar to those reported after single doses, I which suggests that no change occurs with chronic dosing. However, the proportion of drug eliminated renally (16 percent) is less than estimated previously. Only 22 percent (84 mg/d) of the daily dose could be recovered in urine. Although renal clearance is high, its contribution to overall rate of elimination is minimal because of the huge tissue stores of the drug. Nonrenal clearance, via enterohepatic recycling and shedding of pigmented tissue such as skin, accounts for most of the elimination." The steady-state concentrations for our subjects in both plasma and blood were lower than predicted by Tett et al. from single-dose data. Also, the fraction of drug eliminated by renal clearance was less and the concentrations of DEHC were higher than previously reported.' Together, these observations suggest that the rate of metabolism may be induced by chronic dosing. Concentrations should have been at steady-state because even with the very slow elimination of HC, concentrations should reach 96 percent of steady-state by 180 days.' All of our patients had taken the drug for longer than that and were believed to be good compliers. It is also possible that elimination of HC was influenced by the concomitant drugs our patients were taking, or by their fairly advanced age. In conclusion, during chronic dosing the processes that determine daily variation in blood or plasma concentrations of HC are those of absorption and distribution rather
Table 3. Renal Clearances WHOLE BLOOD (mL/min)
PLASMA (mL/min) PATIENT
HC
DEHC
DEC
HC
DEHC
DEC
DDEC
1 2 3 4 5
271.3 136.5 205.2 174.2 213.8 200.2
45.8 28.3 26.3 49.3 88.6 47.7
219.7 100.1 153.2 161.8 209.9 168.9
44.0 25.8 43.9 28.4 59.0 40.2
9.9 6.7 7.7 5.6 38.5 13.7
34.5 17.2 28.3 16.6 51.4 29.6
55.0 18.5 29.8 20.0 52.9 35.2
MEAN
DDEC
=didesethylchloroquine; DEC =desethylchloroquine; DEHC =desethylhydroxychloroquine; HC =hydroxychloroquine.
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Research/Practice
than elimination. The timing of peak concentrations is quite variable. Therefore, the daily fluctuation in concentrations for a particular patient is fairly small, particularly in comparison with patient-to-patient variability. This has implications for attempts at monitoring therapeutic blood concentrations, in that the exact timing of blood draws will not be of critical importance. ~ We wish tothank Keith Rodvold, Phann.D., forhishelpful review ofthemanuscript, Connie Kluender, Kathy Cota, and Justus Fiechtner, M.D" forrecruiting thepatients, and Gwen Lunde fortyping themanuscript.
References I, TETI SE, CUTLER OJ, DAYRO, BROWN KF. A dose-ranging studyof the pharmacokinetics of hydroxychloroquine following intravenous administration to healthy volunteers, Br J Clin PharmacoI1988;26:303-13, 2, TETISE, CUTLER OJ, DA YRO, BROWN KF. Bioavailability of hydroxychloroquine tablets in healthyvolunteers, Br J Clin Pharmacol1989; 27:771-9. 3. CUTLER OJ, MACINTYRE AC, TETI SE. Pharmacokinetics and cellular uptakeof 4-aminoquinoline antimalarials. Agents Actions 1988;24(suppl): 142-57, 4. QUATRARO A, CONSOLI G,MAGNO M, et aI. Hydroxychloroquine in decompensated, treatment-refractory noninsulin-dependent diabetes mellitus, Ann Intern Med 1990;//2:678-81. 5. WALLACE OJ, METZGER AL, STECHER V. Antihyperlipidemic actions of hydroxychloroquine (Plaquenil)(abstract). Arthritis Rheum 1990;33 (Maysuppl): R7. 6. MCCHESNEY EW. Animal toxicity and pharmacokineticsof hydroxychloroquine sulfate, Am J Med 1983;75(suppl 6A):11-8, 7. GUSTAFSSON LL, WALKER 0, ALVAN G,et al. Dispositionof chloroquine in man after single intravenous and oral doses, Br J Clin PharmacoI1983;15:471-9.
8. ROMBO L, ERICSSON O. ALVAN G, LINDSTROM B,GUSTAFSSON LL, SJOQVIST F.Chloroquineand desethylchloroquine in plasma, serum and whole blood: problems in assay and handling of samples. Ther Drug Monit 1985;7:211-5.
9, MACKENZIE AH. Pharmacologic actions of 4-aminoquinoline compounds. Am J Med 1983;75(suppI6A):5-1O,
EXTRACTO
Estudiode los parametres farmacocineticosdel sulfato de hidroxicloroquina (HCQ) en cinco pacientes con artritis reumatoide que
tomaban HCQ 6 mg/kg/d durante aI menos seis meses antes, Se recogieronmuestras de sangre a las 0, I, 2, 4, 6, 8, 12, Y 24 horas despues de una dosis oral. HCQ y sus metabolitos,desetilhidroxicloroquina, desetilcloroquina, y didesetilcloroquina, fueron determinados en sangre total, plasma, y orina de 24 horas. EI comportamientofarmacocineticode HCQ y sus metabolitosrespondi6 aI modele previsto tras adrninistraci6n de dosis unica, Durante las 24 horas se pudo ver la fase de absorci6n y dos fases de distribuci6n,una inicial y otra tardfa, EI porcentaje de variaci6nde la concentraci6n media maxima/minima fue del 40 por ciento, S610 el 12 por ciento de HCQ inalterada (17 por ciento HCQ mas metabolitos) fue recuperadaen orina. No hubo correlaci6nentre el aclaramientode farrnaco y el de creatinina. CARMENCAO
RESUME
La pharmacocinetique du sulfate d'hydroxychloroquine (Plaquenil, Winthrop) 11 I' etat d' equilibre a ete etudiee chez cinq patients souffrant d'arthrite rhumatoide, Ces patients avaient recu 6 mg/kg/jour du medicament en question pour une duree minimale de six mois avant de participerdans cette etude. Des echantillons sanguins furent preleves 11 0, 1,2,4,6,8, 12, et 24 heures suivant l'administration d'une dose orale du medicament.Le sang et Ie plasma furent analyses par chromatographie liquide pour determiner les concentrationssanguineset plasmatiquesd'hydroxychloroquine (HCQ) ainsi que ses metabolites: desethylhydroxychloroquine, desethylchloroquine et didesethylchloroquine, Une collection urinairede 24 heures a egalernent ete effectuee suivant la prise orale d'une dose d'HCQ. Les concentrations urinairesde l'HCQ et des metabolitesont ete determinees par la meme methode. La pharmacocinetique de I'HCQ et des metabolites, 11 l'etat d'equilibre, etait conforme au modele pharmacocinetique estime 11 partir d'une dose orale unique. Cependant, la nature des donnes n' a pas permis de determiner la constanteet la demi-vie d'elimination, Durant une periode de 24 heures,la phase d' absorption ainsi que les phases rapide et lente de distributionfurent observees. Le degre de variationentre les concentrationsmoyennes minimales et maximales etait de 40 pourcent. L' elimination renale ne representait que 12 pourcent de la clairance de I'HCQ et 17 pourcent de la clairance totale (HCQ et ses metabolites).De plus, aucune correlation entre la clairance de la creatinine et la clairance renale du medicament n' a pu etre demontree.
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