of both free and total fractions at different times of the day. Changes in ... of time of day on prednisolone binding to ... four experiments were performed at weekly.
Diurnal variation in prednisolone kinetics Plasma concentrations offree and total prednisolone were measured after oral doses at four time points to investigate the possibility of a diurnal variation in the drug's kinetics. There were marked differences in plasma prednisolone concentrations, clearance rates, and bioavailability of both free and total fractions at different times of the day. Changes in the protein binding characteristics of prednisolone with clock time resulted in marked differences between the kinetics of free and total prednisolone. It is recommended that for maximum efficacy and minimum toxicity prednisolone therapy be confined to once-daily dosing in the morning.
Judie English, L.I.Biol.,* Michael Dunne, M.B.Ch.B., and Vincent Marks, M.A. Surrey, England Division of Clinical Biochemistry, Department of Biochemistry, University of Surrey, Guildford
There is considerable evidence that the time of day at which a drug is taken may alter its therapeutic and toxic effects. Diurnal variations in toxicity and cure rate of L1210 leukemic mice have, for example, been reported for cytosine arabinoside,5' adriamycin,'° and a number of cytotoxic drug combinations .17' 18 Diurnal variations in the effects of tolbutamide,sdiazepam," barbiturates ,21 and alcohol2" have also been noted and, in several instances, differences in kinetic behavior have been observed.3' 19' 22 Prednisolone is often taken over long periods for the treatment of a number of conditions including asthma, arthritis, and atopic allergies, and as an immunosuppressant after renal and other transplants. Side effects are common and attempts have been made to minimize one of them, namely adrenal suppression, by taking advantage of the known diurnal variation in adReceived for publication May 26, 1982. Accepted for publication Aug. 6, 1982. Reprint requests to: Judie English, Division of Clinical Biochemistry, Department of Biochemistry, University of Surrey, Guildford, Surrey, England GU2 5X11. *Supported by a grant from Cilag Chemie Stiftung fur Therapeutische Forschung, Switzerland.
renal susceptibility to plasma steroid levels." The ratio between free and protein bound prednisolone in the plasma is known to vary with clock time' and might effect both efficacy and toxicity since it is only the free drug that is thought to be pharmacologically active. Patients with hypoalbuminemia and a low binding capacity are said to be more likely to suffer from side effects during prolonged prednisolone therapy than patients with normal plasma albumin
concentrations." It is currently thought that only free prednisolone is available for either hepatic metabolism or renal disposal, thereby rendering the removal of prednisolone from blood susceptible to variations in plasma binding levels. An investigation into differences in prednisolone kinetics according to time of administration'2 proved inconclusive, but in this study only two time points (8:00 AM. and 8:00 P .M .) were used and no attempt was made to study alterations in the ratio between free and protein bound drug concentrations. We have investigated the matter further with normal subjects to study the effect of time of day on prednisolone binding to plasma proteins and on its kinetics. 381
382
English et al.
Clin. Pharmacol. Titer. March 1983
Table I. Plasma total prednisolone concentrations and kinetic data after prednisolone at different times of the day (mean -± SEM, n = 6) Time of dosing
Parameter Plasma prednisolone concentration (ng/ml) at (mm): 30 60 90 120 180
240 300 360 Cmax (ng/ml) tmax (min) tlh (hr) AUC (ng/ml VD ext
C,.
6:00 P.M.
±
232 -± 26 247 ± 24 266 ± 12 235 ± 13 195 ± 9 152 ± 7 106 ± 7 68 ± 7 280 ± 19t 85 ± 9
75 ± 33 ± 25 156 ± 17 159 ± 13 146 ± 13 126 -± 10
157
33
196 ± 12 207 ± 9 197 ± 11 177 -± 10 153 ± 9 128 ± 8 99 ± 7
± 11 ± 8 ± 0.35* ± 40 0.59 ± 0.07*
(AO
= time to
Noon
214 90 3.40 950
hr)
By Student's t test: *P
6:00 A.M.
> 0.01;
-1-P
> 0.001.
2.00 ± 0.10 1010
-±
35t
0.40 ± 0.03
117
111
±7
96 ± 8 172 -± 18 115 ± 15*
Midnight
138 _± 185 ± 184 -± 176 ± 159 ±
18 13
9 8
9
± 8 124 ± 9 141
99 ± 7 191
± 9
75 ± 10
5.20 ± 0.70t
5.12 ± 0.73t
735 ± 55 0.88 ± 0.11t
0.84 ± 0.08*
865
-±
50
All tests for were conducted relative to the lowest value for the parameter under consideration.
Experimental methods
Protocol. Our subjects were five men and one woman between 20 and 34 yr old who were studied on each of 4 occasions in the Clinical Investigation Unit of St. Luke's Hospital, Guildford. None drank alcohol excessively and none had taken corticosteroids for any reason in the previous year. All subjects were fasted for 6 hr before and for the duration of the experiment. It has been shown that circadian variations in aminopyrine disposition after oral doses were related to the time of meals in relation to the time of taking the drug and that fasting ablated such time related changes." The subjects were given prednisolone tablets, which they took with a glass of tap water to facilitate ingestion, at a dose of 2 mg/10 kg body weight. The environmental conditions were kept as constant as possible on each of the four occasions. Blood samples (10 ml) were collected through an indwelling cannula in the antecubital vein, kept patent with 3.8% sodium citrate, at 0, 30, 60, 90, 120, 180, 240, 300, and 360 min after drug ingestion. The plasma was separated immediately and kept frozen at 20° until assayed. The four experiments were performed at weekly intervals with drug being taken at 6:00 A.M., noon, 6:00 P M . , and midnight.
Analytical procedures. Plasma total prednisolone concentrations (Table I) were measured by radioimmunoassay2' 7 and free prednisolone concentrations (Table II) were measured by dialysis in Teflon cells after the addition of tracer levels of 3H-prednisolone, as previously described.4 Plasma total and free prednisolone concentrations were used to calculate the plasma half-life (t1/2), apparent area of distribution (VD ext), and area under the plasma concentration time curve (AUC) of both the total and the free drug by conventional methods.23 The application of kinetic methods designed for use after intravenous injection to data obtained after oral prednisolone dosing has recently been discussed.24 Values of much the same order for AUC, elimination rate constant, and VD ext were found after oral and intravenous prednisolone, the latter route being further complicated by the need to use an ester of prednisolone. Since the drug is frequently given orally to patients, it was felt that the findings would be of greater clinical relevance if this route was followed. The results when plasma free prednisolone concentrations were measured (Table II) showed a similar pattern. The longest t1/2, lowest maximum concentration (Cm), and smallest AUC all occurred after the 6:00 P.M. dose.
Diurnal variation in prednisolone kinetics 383
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Table II. Plasma free prednisolone and kinetic data after prednisolone at different times of the day (mean ± SEM, n = 6) Time of dosing
Parameter Plasma free prednisolone concentrations (ng/ml) at (min): 30 60 90 120 180
A.M.
27 32
7
31
2 2 2
25 19 12 8 5 35 55
240 300 360
(ng/ml)
tn,, (mm) (hr) AUC (ng/ml
6:00
1.75
t1/2
hr)
110
3
Noon
27 ± 7 30 ± 2 32 ± 2 25 ± 2 17
1
10
1
6
1
3t 12
0.10t 10t
3
± ± ± ± ± ±
1 1
0 0 5*
36 10 75 1.30 ± 0.05 100 ± 10*
6:00
P.M.
Midnight
7
4
12
11
3
16 19 14 12 8 6
2 2
23 24 20
3
16
-±- 1
2
11
±
20 100
2.30 70
1
0 2
10 0.2017
3 3
2 1
1
9
0
6 26 75
1
2.30 90
2
7 0.1516*
By Students t test: 5P> 0.01; tP > 0.001. All tests for significance were conducted relative to the lowest value for the parameter under consideration. time to Cmax.
Discussion
is reached. If the situation is the same in man,
The results appear to indicate an indirect correlation between peak plasma prednisolone concentrations after oral dosing and its rate of disappearance from the circulation (Fig. 1); the two extremes occurred at noon and 6:00 P.M. The t1/2 was a consequence of the combined effect of binding of prednisolone to the tissues, its excretion through the kidneys, and metabolism in the liver, although which of these is the major determinant is not revealed by our experiment. After dosing at noon, was greater than at any other time of day, but the t1/2 is at its shortest and the reverse is true at 6:00 P.M., when the minimum Cn,a), coincides with the maximum t1/2. It seems unlikely, however, that an increase in peak plasma concentration of only 39% could account for the more than doubling of prednisolone t½, so a further explanation must be sought. Studies in rats and mice have shown, in vitro, that the rate of hepatic drug metabolism follows a diurnal rhythm,9' 15' 21 with maximal activity around midnight, which corresponds with the middle of the waking period in these nocturnal animals Under normal lighting conditions this is the middle of the dark period and about 6 hr after peak plasma corticosteroid concentration
C.
maximum hepatic drugmetabolizing enzyme activity would be expected to occur in the middle of the day, or about 6 hr after the peak plasma cortisol concentration, and to result in maximum clearance of prednisolone from circulation. This, in fact, corresponds with findings reported here and may partially explain them. The amount of free prednisolone in the plasma at any time is governed, not only by the total concentration, but also by the binding capacity of the transcortin or corticosteroid binding globulin. In the case of prednisolone, this in turn depends on the concentration of endogenous corticosteroid in the plasma, since it and the synthetic steroid compete for the same binding sites. Plasma cortisol concentration is itself subject to a diurnal rhythm and consequently affects prednisolone binding capacity. The highest plasma cortisol concentration occurs at about 6:00 A.M. and is reflected in a slightly (P> 0.001) lower binding capacity for prednisolone (trace concentrations of 92.2 -± 0.5% against a mean value of 95.2 ± 0.2% bound at the other three time points). The corresponding values for free prednisolone of 7.8% and 4.8% represent a 60% higher level at 6:00 A.M. than during the rest of the day.
384
Clin. Pharmacol. Ther. March 1983
English et al.
Plasma predniso lone
Time of administration
concentrations (ng/ml) 0600 hr 1200 hr
1800 hr 2400 hr
1
3
2
4
5
6
Time after administration (hr)
Fig. I. Plasma prednisolone concentration after oral dosing (2 mg/kg) at different times of day. Each point is mean for six subjects.
The peak free prednisolone concentration of 35 ± 3 ng/ml at 6:00 A.M. represents a con-
siderably higher proportion of the peak total prednisolone concentration of 214 ± 11 ng/ml than the corresponding figures at other times, again reflecting the relative saturation of protein binding sites for prednisolone in the presence of high plasma cortisol concentrations. A higher concentration of free prednisolone during the day than in the evening and at night after identical dosing has been noted by others.' This relatively high concentration of free drug undoubtedly contributes to its high disposal rate and short t1/2. The combination of low enzyme activity and high protein binding capacity at 6:00 P.M. and midnight accounts for the long prednisolone t1/2 observed at these times. There is a direct relationship (P > 0.005) between Cmax of prednisolone after oral dosing and the AUC, as would be expected and, although the differences are reduced by the dif-
ferences in disappearance rate, there is still more drug available in the plasma after dosing at noon than at other times. VD tS of prednisolone show considerable variation with clock time, but it is not possible from these data to suggest explanations. The differences probably result from a combination of factors, including the rapidity and completeness of absorption from the gut and the extent of its binding to plasma proteins and peripheral tissues. The data derived from measurement of plasma free prednisolone concentrations and those obtained from analysis of total drug concentrations follow a similar pattern. It seems on the evidence that time-related differences in prednisolone kinetics in man are due to complex interactions of diurnal rhythms in endogenous corticosteroid production, plasma corticosteroid binding capacity, and hepatic enzyme activities, although their clinical relevance, if any, is not known. Adrenal suppres-
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sion by exogenous steroids is reduced when they are given in the morning rather than at night,"' 16 even though our work and that of Angeli et al.' indicates that there is a greater proportion of the drug in the blood in the free active form after morning than after evening dosing. This, together with the evidence for its greater bioavailability between 6:00 A.M. and noon, suggests that maximum efficacy can be achieved with minimal suppressive effect on the hypothalamic-pituitary-adrenal function when prednisolone is taken early rather than late in the day.
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adriamycin by Bragg albino mice and Fischer rats depends on circadian timing of injection. Int J Chronobiol 1:335, 1973. Lewis GP, Jusko WJ, Burke CW, Graves L: Prednisone side-effects and serum-protein levels. Lancet 1:778-781, 1971. McAllister WAC, Mitchell DM, Collins J V: Prednisolone pharmacokinetics compared between night and day in asthmatic and normal subjects. Br J Clin Pharmacol 11:303-304, 1981. Nichols T, Nugent CA, Tyler FH: Diurnal variation in suppression of adrenal function by glucocorticoids. J Clin Endocrinol 25:343-349, 1965. Owasoyo JO, Walker CA: Circadian variations of diazepam acute toxicity. Experientia 32:7273, 1981. Radzialowski FM, Bousquet WF: Circadian rhythm in hepatic drug metabolising activity in the rat. Life Sci 6:2545-2548, 1967. Seigre EJ, Klaiber EL: Therapeutic utilisation of the diurnal variation in pituitary adrenocortical activity. Calif Med 104:363-365, 1966. Scheving LE, Burns ER, Pauly JE, Halberg F, Haus E: Survival and cure of leukaemic mice after circadian optimisation of treatment with cyclophosphamide and 1-0-13-arabinofuranosyl cytosine. Cancer Res 37:3648-3655, 1977. Scheving LE, Burns ER, Halberg F, Pauly JE: Combined chronochemotherapy of L1210 leukaemic mice using 1-0-o-arabino furanosyl cytosine, cyclophosphamide, vincristine, methyl prednisolone and cis-diaminedichloro-platinum. Chronobiologia 7:33-40, 1980. Shively CA, Vesell ES: Temporal variations in acetaminophen and phenacetin half-life in man. CLIN PIIARMACOL THER 18:413-424, 1975. Shively CA, Simons RJ, Passananti GT, Dvorchik BH, Vesell GS: Dietary patterns and diurnal variations in aminopyrine disposition. CLIN PHARMACOL THER 29:65-73, 1981. Vesell ES: Factors altering the responsiveness of mice to hexabarbital. Pharmacology 1:81-97, 1968. Vesell ES, Shively MS, Passananti GT: Temporal variations in antipyrine half-life in man. CLIN PHARMACOL THER 22:843-852, 1977. Wagner JG: Fundamentals of clinical pharmacokinetics. Hamilton, III., 1975, Drug Intelligence Publications Inc. Wagner JG, DiSanto AR, Gillespie WR, Albert KS: Reversible metabolism and pharmacokinetics: Application to prednisone-prednisolone. Res Comm Chem Pathol Pharmacol 32:387-405, 1981. Wilson RH, Newman EJ, Newman HW: Diurnal variations in the rate of alcohol metabolism. J Appl Physiol 8:556-558, 1956.
