Chronopharmacology in Drug Development
The human body is highly organized in time and most functions display significant daily [circadian] and seasonal rhythms. Gene expression in the brain as well in the peripheral tissues has been convincingly demonstrated to be circadian phase dependent for many genes. In addition, seasonal gene expression – with inverse rhythms in the Northern and Southern Hemisphere – has been evidenced as well. Taking into account these rhythmic changes in the body, it is conceivable that also the pharmacokinetics and/or the pharmacodynamics can be rhythmic in experimental animals as well in humans. In this review chronopharmacological findings are compiled demonstrating to what an extent rhythmic changes in the composition of the body in health and disease can contribute to variations in time-dependent drug treatments. The results of clinical chronopharmacological studies in hypertension, asthma, and ulcer disease and treatment of antihyperlipidemia are reviewed.
Adequate drug dosing in a given disease/indication is the most important goal in clinical pharmacology. Drug reference books are designed to give advice to the best of scientific knowledge. Both pharmacokinetics and pharmacodynamics of a compound are well-accepted tools to achieve best dosing of a drug. Additional targets in drug dosing are involved: age, disease, gender, food, physical fitness, and family genetics. However, in recent years additional targets were shown to influence dosing, social aspects and living behavior such as shift work and transmeridian flight as well as social jet lag, and most important genetic variation in normal and diseased organ tissues – e.g., in cancer – came into focus in clinical medicine. Finally, the awareness of biological rhythms in organ and tissue functions leads to the introduction of chronopharmacology in clinical medicine. In this review the abovementioned topics will be touched in order to demonstrate the important new “environment” of clinical pharmacology.
The Biological Clock
Rhythmicity is the most ubiquitous feature of nature. Rhythms are found from unicellular to complex multicellular organisms in plants, animals, and men. Living organisms are continuously influenced by external stimuli, many of which have rhythmic patterns. Environmental rhythms in daily and seasonal patterns of light, food availability and temperature, etc. are predictable, and animals – including humans – have the ability to anticipate these environmental events with periodically and predictably changing internal conditions. These rhythmic patterns of anticipation have clear advantages and survival value. The frequencies of rhythms in nature cover nearly every division of time. There are rhythms which oscillate once per second (e.g., in the electroencephalogram), once per several seconds (respiratory rhythm, heart rate), and once per year (circannual rhythm).
The most evident environmental change which results from the regular spin of the earth around its central axis, resulting in the alternation between day and night, seems to have induced the predominant oscillation, the circadian rhythm (the about-24-h rhythm; circa = about, dies = day) described by Jürgen Aschoff (1947). There is sound evidence that living systems including humans are not only organized in space but are also highly organized in time (Albrecht 2012; Lemmer 2009, 2012a). One of the first observations on a rhythmic pattern in man was presented by the famous physiologist Sanctorius Sanctorius in 1664 when he described in a self-experiment daily variation in body weight due to transpiration. A review on biological rhythms described within the last several hundred years is compiled in a review (Lemmer 2009).
Circadian rhythms have been documented throughout the plant and animal kingdom at every level of eukaryotic organization. Circadian rhythms by definition are endogenous in nature, driven by oscillators or clocks, and persist under free-running conditions. In various species (e.g., Drosophila melanogaster, Neurospora, mouse, golden hamster, rhesus macaque, man), the genes controlling circadian rhythms have been identified (genes: per, frq, clock, tau, Rev-erbalpha). In 1971 Konopka and Benzer (1971) were able to identify on the X chromosome of Drosophila a region which controlled the period in the eclosion rhythm of three mutants (per clock gene). Circadian clocks are believed to have evolved in parallel with the geological history of the earth and have undergone selection pressures imposed by cyclic factors in the environment. These clocks regulate a wide variety of behavioral and metabolic processes in many life forms. They enhance the fitness of organisms by improving their ability to efficiently anticipate periodic events in their external environments, especially periodic changes in light, temperature, and humidity.
The mammalian circadian clocks, located in the neurons of suprachiasmatic nuclei (SCN) composed of about 10,000 heterogeneous neurons in the brain and in cells of peripheral tissues, are driven by a self-sustained molecular oscillator, which generates rhythmic gene expression with a periodicity of about 24 h. This molecular oscillator is composed of interacting positive and negative transcription/translation feedback loops in which the heterodimeric transcription activator CLOCK/BMAL1 promotes the transcription of E-box containing Cryptochrome (Cry1 and Cry2) and Period (Per1 and Per2) genes, as well as clock-controlled output genes. After being synthesized in the cytoplasm, CRY and PER proteins feedback in the nucleus to inhibit the transactivation mediated by positive regulators. The mPER2 protein acts at the interphase between positive and negative feedback loops by indirectly promoting the circadian transcription of the Bmal1 gene and by interacting with mCRY proteins However, this is a simplified scheme with additional clock genes and transcription factors involved.
In general, the human endogenous clock does not run at a frequency of exactly 24 h but somewhat slower. The rhythm in human body temperature which is timed by the biological clock has a period of about 24.5-h under free-running conditions, i.e., without environmental time cues or zeitgebers (e.g., light, temperature). The term “zeitgeber” introduced by Jürgen Aschoff at the Max Planck Institute (Aschoff 1947) is now part of the international scientific language. Mammals such as rodents or humans can entrain their activity to regular light cycles not shorter than 22 or longer than 26 h. Zeitgebers entrain the circadian rhythm to a precise 24-h period. Zeitgebers are, therefore, necessary to entrain a living subject to a “normal” period of 24 h! This is of great importance having in mind that seasonal variations not only in the light-dark cycle but also in temperature and other environmental conditions have impact on availability of nutrition, on the onset of diseases, and on time of birth and death, the latter was demonstrated in a single family residing at different latitudes, in Europe (latitude 51°N, 1500–2013 A.C.) and South Africa (30°S, 1750–2013 A.C.) (Lemmer 2014). In this study no seasonal variation was found in birth and death in the data set from South Africa (n = 1.284), whereas a significant peak in winter was detected both in birth and death in the European data (n = 2.361) between 1500 and 1950, which was then lost from 1950 up to 2013, obviously due to changed socioeconomic and environmental conditions (Lemmer 2012b, 2014).
The principles of the pharmacokinetic-pharmacodynamic interactions are well known and the basis for the development of new drugs. In short, pharmacokinetics deals with absorption, distribution, metabolism, and elimination of drugs. The different steps in pharmacokinetics are determined and influenced by physiological functions of the body. Pharmacokinetic parameters such as peak drug concentration [Cmax], time to Cmax [tmax], volume of distribution [Vd], area under the curve [AUC], bioavailability, plasma protein binding, and elimination half-life ([t½] are evaluated during drug development but conventionally not considered to be influenced by the time of day at which a drug is administered. However, an increasing number of recently published studies convincingly gave evidence that this paradigm cannot be maintained any longer (Lemmer and Bruguerolle 1994).
In the following chronokinetics are demonstrated in selected groups of compounds which are of importance in drug treatment.
Chronopharmacology of Antiasthmatic Drugs
Since nocturnal asthma is a common event in asthmatic disease, it is not surprising that antiasthmatic drugs have also been studied in relation to time of day (for review see Lemmer 1991a, 2012a; Smolensky et al. 2007b). Theophylline was one of the first drugs for which daily variations in its pharmacokinetics were reported (for review see Smolensky et al. 2007b), though theophylline is of less importance nowadays. More than 50 studies with different theophylline preparations in different galenic formulations were published demonstrating that in general Cmax was lower and/or tmax was longer after evening than after morning application of theophylline. This observation was supported by studies in which simultaneously the pharmacokinetics and the pulmonary effects of theophylline were compared in asthmatics, demonstrating that the drug might be dosed higher during the night than during daytime hours or even given as a single evening dose in order to adequately overcome the nocturnal symptoms (see Smolensky et al. 2007b). Thus, in contrast to the general belief concerning drug concentration profiles, “the flatter the better,” it seems, therefore, to be advantageous to accept greater fluctuations in drug concentrations throughout 24 h of a day. As a consequence in 1989 for the first time a pharmaceutical company was granted permission from the Food and Drug Administration in the USA and from the Bundesgesundheitsamt in Germany to market a sustained-release theophylline product for once-daily evening administration (see Smolensky et al. 2007b).
Beta2 agonists are also drugs of first choice in the treatment of asthmatic patients, though inhaled beta agonists are preferable to the oral application in most cases. For oral terbutaline the pharmacokinetics but also the effects on peak expiratory flow were shown to be circadian phase dependent (Lemmer and Bruguerolle 1994) with higher Cmax after morning than evening drug application and with tmax being 3.5 and 6.2 h, resp., thus resembling the daily variations observed with theophylline. A further study with oral terbutaline indicated that doubling the dose in the evening, i.e., unequally dosing during 24 h, can better control the nocturnal fall in peak flow. These studies give further support to the notion that the dose-response relationship of a given drug can be circadian phase dependent, as already demonstrated for theophylline and other compounds (see Lemmer and Bruguerolle 1994; Smolensky et al. 2007b).
Recently, the Commission on Drugs of the German Medical Association included “time of day” into their recommendations (“Arzneiverordnungen,” 22th edition 2009 Allwinn et al. 2009) as an important variable influencing drug efficacy.
Chronopharmacology of H2 Blockers in Peptic Ulcer Disease
This group of compounds are still the drugs of choice in the treatment of peptic ulcer. The chronobiologic finding on a circadian rhythm in gastric pH and acid secretion unanimously led to the recommendation that H2 blockers (ranitidine, cimetidine, famotidine, roxatidine, nizatidine) should be taken once a day in the afternoon when acid secretion is increasing, independently of whether or not the compounds have a short or a long half-life (Lemmer 1991a; Moore 1989). In consequence of this strategy, chronopharmacology helped to improve drug treatment as well as the patient’s compliance.
For both the H2 blocker cimetidine and the proton pump inhibitor omeprazole, significant daily variations in their pharmacokinetics were shown with Cmax being higher and tmax being shorter after morning than evening dosing. However, this does not seem to have an impact on drug efficacy. Daily variation in sensitivity to H2 receptor blockade seems to be of more importance: Recently it has been shown that a continuous infusion of ranitidine over a period of 24 h does not lead to a constant effect, because the increase in gastric pH by ranitidine was less during the nightly than during the daytime hours of drug infusion (Sanders et al. 1988). This may indicate a partial nocturnal resistance to H2 blockade. This interesting finding calls not only for further investigations but could also indicate that drugs with a different mechanism of action may be added to drug treatment with H2 blockers during the nightly hours.
Chronopharmacology of Cardiovascular Active Drugs
The cardiovascular system displays pronounced daily variations in its functions as well as in its hormonal and biochemical regulatory mechanisms (Lemmer 2007b, 2017; Manfredini et al. 2012, 2017). Nearly all groups of cardiovascular active drugs were shown to exert a circadian phase dependency in their effects (Hermida et al. 2007b; Lemmer 2007a; Lemmer and Portaluppi 1997), for a less number of compounds; however, daily variation in pharmacokinetics was reported (Lemmer 2007a).
In the last years an increasing number of reports were published on the effects of cardiovascular active drugs on the 24-h blood pressure profile. It is not possible in this short overview to go into detail. In general, drugs belonging to different classes differently affected the 24-h blood pressure profile.
Beta blockers and calcium channel blockers reduced high blood pressure more pronouncedly during the daytime than during the night. However, only very few studies addressed this question in cross-over studies, i.e., evaluating morning versus evening drug dosing, as well as taking a possible variation in the drugs’ pharmacokinetics or different galenic formulations into account (Lemmer 1991b, 2007c).
1,605 ± 175
1,588 ± 173
509 ± 31
530 ± 26
0.9 ± 0.3
2.1 ± 0.4**
5.2 ± 0.7
4.9 ± 0.3
9,539 ± 827
10,959 ± 707
6,729 ± 375
6,418 ± 199
t1/2 ß (h)
4.6 ± 0.4
4.2 ± 0.4
6.4 ± 0.6
6.1 ± 0.5
Tmax SBP decrease (mmHg)
0.7 ± 0.1
1.1 ± 0.1
5.0 ± 0.6
2.8 ± 0.5*
Tmax DBP decrease (mmHg)
0.4 ± 0.1
0.6 ± 0.2
6.0 ± 0.7
2.9 ± 0.5**
Tmax HR decrease (S/min)
0.8 ± 0.3
0.9 ± 0.2
5.2 ± 1.0
3.8 ± 0.6
Chronokinetics of cardiovascular active drugs, * at least <0.05 (Data from and references in Lemmer 2006)
Dose (mg) and duration
0.5, single dose
Bruguerolle et al. (1988)
Witte et al. (1993)
60, single dose
60, single dose
Lemmer et al. (1991a)
8, single dose
Nold and Lemmer (1998)
10, single dose
Lemmer et al. (1991a)
2 × 20, 1 week
50, Single dose
Shiga et al. (1993)
80, single dose
Koopmans et al. (1993)
80, single dose
Langner and Lemmer (1988)
80, single dose
Semenowicz-Siuda et al. (1984)
360, 2 weeks
Jespersen et al. (1989)
80, single dose
Hla et al. (1992)
Conventionally, different galenic formulations are only considered in that way that the duration of drug action is modified. For two drugs, however, the pharmacokinetics of both an immediate-release and a sustained-release formulation have been studied at different times of day giving evidence that circadian time can influence the kinetics depending on the formulation (Table 2): In contrast to the i.r. nifedipine mentioned above, a s.r. formulation did not display significant daily variations in its pharmacokinetics in hypertensive patients. A sustained-release preparation of the calcium channel blocker verapamil, on the other hand, did show a significant longer tmax after evening than morning dosing in healthy subjects.
In the following cross-over studies (morning vs. evening) are presented which demonstrate that the time of day of drug application can have an effect on the drug response on the 24-h blood pressure profile. Fortunately, those studies were performed for those drugs which are drugs of choice in treating hypertension, i.e., calcium channel blockers, ACE inhibitors, and AT1 receptor blockers. These studies clearly demonstrate that those clinical studies are of importance for drug development by the pharmaceutical industry in order to better treat chronic diseased patients with hypertension.
Cross-Over Studies with Calcium Channel Blockers
Calcium channel blockers are also not a homogenous group of drugs. Vasodilatation by calcium channel blockers occurs at lower concentrations than the cardiodepressant effects. However, the difference between vasodilating and cardiodepressant effects is greater with the 1,4-dihydropyridines (e.g., nifedipine, nitrendipine, isradipine, amlodipine) than with the verapamil- and diltiazem-like compounds. Moreover, these drugs differ in their kinetics with amlodipine having a long half-life per se.
A single morning dose of a sustained-release verapamil showed a good 24-h blood pressure control, whereas a sustained-release formulation of diltiazem was less effective at night. Dihydropyridine derivatives, differing in pharmacokinetics, seem to reduce blood pressure to a varying degree during day and night; drug formulation and dosing interval may play an additional role.
Up to now, 12 studies using a cross-over design [morning vs. evening] have been published (Table 3). In primary essential hypertensives with a dipper profile, amlodipine, isradipine, nifedipine GITS, and nisoldipine did not differently affect the 24-h blood pressure profile after once morning or once-evening dosing, whereas with nitrendipine and lacidipine, the profile remained unaffected or slightly changed after evening dosing. Most interestingly, the greatly disturbed blood pressure profile in secondary hypertensives [non-dippers] due to renal failure was only normalized after evening but not after morning dosing of isradipine. Similarly, amlodipine and nisoldipine ER transformed non-dippers into dippers, but both after evening and morning dosing (Table 3), which might be due to the longer “apparent” half-life of these drugs. These findings demonstrate that time of drug dosing of a dihydropyridine calcium channel blocker can be advantageous in not only reducing the elevated blood pressure but also normalizing the disturbed blood pressure profile.
A time-of-day effect was also described for the kinetics of various calcium channel blockers (Table 2). The bioavailability of an immediate-release formulation of nifedipine was found to be reduced by about 40% after evening compared to morning dosing with Cmax being higher and tmax being shorter after morning dosing. No such circadian time-dependent kinetics were observed with a sustained-release formulation of nifedipine. Also regular as well as sustained-release verapamil displayed higher Cmax and/or shorter tmax values after morning dosing. Similar chronokinetics have been reported after oral dosing of other cardiovascular active drugs such as enalapril, propranolol, and others. Conversely, intravenously infused nifedipine did not display daily variations in its pharmacokinetics indicating that gastrointestinal mechanisms must be involved in the drug’s chronokinetics after oral application.
Cross-Over Studies with Converting Enzyme Inhibitors
Captopril is the representative of a short-acting converting enzyme inhibitor; longer acting ones are enalapril, benazepril, quinapril, and ramipril, and these prodrugs are hydrolyzed to active metabolites. Converting enzyme inhibitors are not only effective antihypertensive drugs but can also increase the life expectancy in congestive heart failure.
Several studies with converting enzyme inhibitors dosed once in the morning or twice daily showed that these drugs did not greatly modify the 24-h blood pressure pattern. However, intra-arterial studies with enalapril or ramipril have shown that while causing sustained daytime reduction in blood pressure, these drugs had only marginal effects on nighttime pressures. Thus, the findings obtained with converting enzyme inhibitors in conventional, i.e., not time specific, clinical studies are controversial.
Cross-Over Studies with AT1 Receptor Blockers
There are four studies published with the AT1 receptor blockers in dippers, valsartan, irbesartan, telmisartan, and olmesartan, which similarly reduced the blood pressure after both morning and bedtime dosing (Hermida et al. 2003, 2007a; Smolensky et al. 2007a; Pechere-Bertschi et al. 1998). In patients with chronic renal disease, olmesartan restored the nightly decline in blood pressure (Fukuda et al. 2008). In non-dippers valsartan had only a slightly more pronounced effect at night (Hermida et al. 2003).
Diuretics and Other Antihypertensive Drugs
Antihypertensives of other classes have rarely been studied in relation to possible circadian variation. Once-daily morning dosing of the diuretics such as indapamide or xipamide reduced blood pressure in essential hypertensives without changing the 24-h blood pressure pattern. In salt-sensitive hypertensive patients (dippers and non-dippers), an interesting study was performed with diuretics: Uzu and Kimura (1999) could demonstrate that diuretics did not affect the circadian blood pressure profile in dippers but transformed the non-dippers into dippers.
In conclusion, there is sound evidence that the treatment of hypertension by various groups of antihypertensive drugs is dependent on the underlying rhythmic organization of the cardiovascular system. Since we are not inbred strains of rats, individualization of drug therapy is the choice of treatment; fortunately, the ability of a great number of drugs from various antihypertensive groups allows us to treat a patient according to his individual setting. As a rule non-dipping hypertensive patients seem to be best treated when the drug is given at bedtime. In dipping hypertensives morning dosing seems to be best, and it seems not justified to treat all hypertensive patients with an antihypertensive drug given in the evening. Thus, the underlying rhythmic pattern of blood pressure regulation has impact on drug treatment. In order to achieve this goal, it is essential that a 24-h blood pressure profile by ABPM is used in each patient.
However, unfortunately only a small group of patients by about 25% is really controlled by ABPM as shown in epidemiological studies in Germany and worldwide (Lemmer et al. 2008).
Chronopharmacology of CSE Inhibitors
a.m. (n = 12)
p.m. (n = 12)
a.m. (n = 32)
p.m. (n = 29)
a.m. (n = 15)
p.m. (n = 15)
Chronokinetics of atorvastatin after morning and evening application (Cilla et al. 1996)
Chronokinetics of pravastatin after morning and evening application (Triscari et al. 1995)
Summary of drugs for which chronokinetic and/or chronodynamic studies were performed
Morning stiffness: evening dosing of NSAR
Taking into account rhythm in pain perception
Morning dosing of glucocorticosteroids
Lithium increases circadian rhythm in physiological functions
Sleep deprivation, light therapy
24-h blood pressure monitoring [ABPM],white coat effect
Antihypertensives: morning dosing
Altered BP rhythm in pregnancy:
High BP evening, at night
Asthma with nightly symptoms
Symptoms mainly at night and morning
Day-night rhythm in FEV1
Long-acting beta agonists in nightly asthma
Theophylline evening dosing in nightly asthma
Proton pump inhibitors: morning dosing
Statins evening dosing
High morning glucocorticoid concentration: Evening dosing of slow-release preparations
Prednisolone single dose in the morning
Pituitary insufficiency/male sexual disturbances
Somatotropin evening dosing
Testosterone-TTS evening dosing
Melatonin and sleep-wake rhythm
Melatonin in blind persons
Melatonin in jet-lag symptoms
In conclusion, this review sheds a light on the importance of the time of day at which a drug is given to a patient and how the pharmacokinetics and/or the effects can vary with time of day. Thus, it is of utmost importance to include this chronopharmacological paradigm into the design of a clinical study. In the last years chronopharmacological findings were included in the recommendations of the German Medical Association to the doctors when prescribing drugs in certain entities of diseases (Table 8).
In this review it was only possible to outline the contribution of circadian rhythms to the pharmacokinetics and the effects of drugs used in the treatment of various diseases. There is no doubt that the rhythmic organization of the human body has an impact on drug treatment.
It is plausible to assume that this time-dependent expression of genes can/must be involved in daily as well as seasonal variation in disease entities as well as in metabolism and effects of drug.
In the light of the tendency for an increased individualization of drug therapy, drug development should consider an additional aspect of drug efficacy, i.e., the contribution of physiological-biological rhythms – down to the expression of genes – to the kinetics and effects of drugs. As demonstrated in this review, this is possible when the underlying rhythmic pattern in health and disease is regarded as a tool for improving drug development.
The Nobel Prize award in 2017 for the molecular biology of the body clock underlines also the importance of biological rhythms for medicine and this award can further stimulate the incorporation of this branch of science into clinical research.
The author has no conflict of interest.
References and Further Reading
- Allwinn R et al (2009) Arzneiverordnungen. In: Arzneimittelkommission, der, deutschen, Ärzteschaft (eds). 22 edn. Medizinische Medien Informations GmbH, Neu IsenburgGoogle Scholar
- Lemmer B, Behne S, Becker HJ (1989) Chronopharmacology of oral nifedipine in healthy subjects. Eur J Clin Pharmacol 36 Suppl:A 177Google Scholar
- Lemmer B (2007b) Chronobiology and chronopharmacology of hypertension: importance of timing of dosing. In: White WB (ed) Blood pressure monitoring in cardiovascular medicine and therapeutics, 2nd edn. Humana Press, Totowa, pp 410–435Google Scholar
- Lemmer B (2012a) Chronopharmakologie, 4th edn. Wiss. Verlagsges, StuttgartGoogle Scholar
- Lemmer B (2017) Signal transduction and chronopharmacology of regulation of circadian cardiovascular rhythms in animal models of human hypertension. Heart Fail Clin. https://doi.org/10.1016/j.hfc.2017.05.009
- Manfredini R et al (2012) Twenty-four-hour patterns in occurrence and pathophysiology of acute cardiovascular events and ischemic heart disease. Chronobiol Int. https://doi.org/10.3109/07420528.2012.715843
- Manfredini R, Salmi R, Cappadona R, Signani F, Basili S, Katsiki N (2017) Sex and circadian periodicity of cardiovascular diseases. Heart Fail Clin. https://doi.org/10.1016/j.hfc.2017.05.008
- Moore JG (1989) Circadian rhythmicity in gastric emptying, acid secretion, and mucosal damage by drugs: implications for drug therapy. In: Lemmer B (ed) Chronopharmacology – biochemical and cellular interactions, Chronopharmacology – cellular and biochemical interactions. Marcel Dekker, New York, pp 631–654Google Scholar
- Nold G, Lemmer B (1998) Pharmacokinetics of sustained-release molsidomine after morning versus evening application in healthy subjects. Naunyn Schmiedebergs Arch Pharmacol 357:R173.Google Scholar
- Sanders SW, Moore JG, Buci KN, Bishop AL (1988) Circadian variation in the pharmacodynamic effects of intravenous ranitidine. Annu Rev Chronopharmacol 5:335–338Google Scholar