Circadian rhythms in infectious diseases : Do they matter ?

The present paper reviews the importance of the time of day in medicine, with a special emphasis on infectious diseases 
and antibiotics. Data were obtained from scientific journals following a literature search and from studies carried out in 
the authors’ laboratory. Data indicating that the signs and symptoms of disease do not appear at random over a 24 h 
period are now available. For example. asthma occurs usually at night, while myocardial infarction or anginal attacks 
occur in the early morning hours. Very little data are available on infectious diseases, and most studies were carried out 
on the pharmacokinetics and toxicity of antibiotics. These data suggest that antimicrobial agents are eliminated more 
rapidly when injected during the activity period of animals or humans. It is also noteworthy that aminoglycosides and 
amphotericin B induced less toxicity during the activity period of laboratory animals or patients, and highest toxicity 
occurred during the sleeping period. This is particularly evident with data on aminoglycosides. Temporal variations were 
also observed in the effectiveness of aminoglycosides, but these data were found only in animal models of infectious 
diseases. Temporal variations in the pharmacokinetics, toxicity and effectiveness of a ntibiotics may lead to a more 
rational use of medications in patients.


US O y. NOTCGn
T he concept of homeostasis is the basis of our understand- ing of the functions of the human body.It has been taught that a stable 'milieu interieur' must be maintained over time for survival, and that this is achieved by specific feedback mechanisms.Changes in body phys iology between sleep and waking states has been taught, and some clinicians have observed that diseases that typically occur in the spring, such as hay fever and asthma, are different than those that occur in the fall.Howeve r, it was generally thought that the probabili ty of exacerbation of acute and chronic diseases or the occurrence of diseases and mortality was about equal over a 24 h period.It was also assumed that the effects of medi cations and pharmacokinetic processes were constant over day or night.Thus, the idea that time is an important feature in medicine has not been fully considered.
Chronobiology studies the flu ctuation of biological functions and processes over tim e. Biological rhythms have been described in parameters related to physiology, pathology and more recently in the effects and kinetics of medications, including antibiotics.These findings are now being incorporated in clinical medicine.
The present review characterizes biological rhythms and provides examples to illustrate circadian rhythms in different pathologies.Data indicating the importance of time of day in infectious diseases in the effects and pharmacokinetics of antib iotics are presented.

BIOLOGICAL RHYTHMS: DEFINITIONS AND SYNCHRON IZATION
When physiological parameters are monitored repeatedly, it is evident that they are not static over time .On the contrary, most parameters vary in a predictable manner as rhythms of definite periods.The spectral analysis of human biological rhythms indicates that they can be divided into three categories.
• Ultradian rhythms have a range of periods (ie , time between two peaks) of 30 mins to 20 h.Episodic secretion of hormone and the progression of sleep stages during the night are examples of this category of rhythms.
• lnfradian rhythms have a period of low frequency , with monthly or yearly cycles.Menstruation in women and the annual appearance of hay fever are examples of this type of rhythm.
• Circadian rhythms have a period in a range of about 24 h.These rhythms are best known to clinicians.
Examp les include the morning rise of heart rate or blood pressure and the higher plasma cortisol levels observed at the time of daily transition from sleep to activity.
This paper focuses on circadian rhythms because phys icians are most familiar with them and they are the biological rhythms most often encountered in clinical situations.
Biological rhythms are a genetically inherited trait, but they are controlled by environmental time cues termed syn-62C chronizers or zeitbergers.The strongest synchronizers are the wake-sleep routine for humans and the light-dark cycle for laboratory animals.These synchronizers do not create rhythms, but set the inherited pacemaker circadian timekeeping systems to 24 h each day.For example, plasma cortisol peaks at the beginning of the day both in those who work during the day and in those who work durning the night; however, the clock time for peak plasma cortisol differs in each group of individuals because they get up at different times of the day.Thus , it is the biological Lime of the individuals that is pertinent, not the hour by the clock.

BIOLOGICAL RH YTHMS IN HEALTH AND DISEASE
In the past 40 years, many investigators were able to characterize biological rhythms of physiological parameters.Figure 1 presents the time of day where peak levels were found for a series of parameters in laboratory med ici ne.For example, gastric acid secretion, white blood cells, prolactin, melatonin , eosinophils, adrenocorticotropic hormone (ACTH), folliclestimulating hormone and luteinizing hormone, all peak at specific times during the sleeping period.On the other hand , cortisol, aldosterone, testosterone, platelet adhes iveness and blood viscosity are highest early in the morning.Hematocrit, red blood cells and airway ca libre are highest in the afternoon.Finally, insulin, cholesterol, triglycerides, number of platelets, acid phosphatase and uric acid are highest later in the afternoon or during the evening.Thus , it is possible to determine a Can J Infect Dis Vol 10 Suppl C May 1999 temporal organization for the physiological processes.The cell is unable to do everything at the same time; thus , its activities are programmed during the 24 h period to assure the well-being of the body (l). Figure 2 presents the circadian rhythms of diseases and indicates the peak time for signs and symptoms of different diseases.Physicians in the emergency room know that asthma attacks most often occur late in the evening and in the middle of the night.This is supported by a study including 164 l patients that showed that asthma attacks are more prevalent between 01 :00 and 04:00 than at any other time of day (2).Physicians are also becoming aware that myocardial infarction (MI) occurs more frequently during the early morning hours.In fact, the risk of having an MI attack is much higher between 05:00 and 12:00 than at any other time of day (3).Similar data were found with sudden cardiac death and with different cardiovascular diseases, including angina attacks (4)(5)(6) and stroke (7).It is interesting to note that there is a morning surge in blood pressure and heart rate (8), and platelets aggregate more readily in the morning than at any other time of day (3).These examples in cardiology indicate that diseases are influenced by the circadian time structure.This is rarely taken into account in everyday medicine because clinicians believe that the symptoms of disease occur randomly over the 24 h period.Data on biological rhythms in health and disease will change this perception.

BIOLOGICAL RHYTHMS IN INFECTIOUS DISEASES
Seasonal changes in the incidence of infectious diseases are well known to physicians, but very few studies have been done on biological rhythms in infectious processes.Studies Figure 3) Frequency distribution ef the antibody levels efter three ir!}ections qfhepatitis B vaccine in the moming (07:30 to 09:30) and in theefternoon (! 3 :00 to I 5:00) to hospital employees.Reproduced with permissionJrom reference 12 conducted 40 years ago by Halberg (9) indicated that mice were more susceptible to the administration of the same dose of Escherichia coli endotoxin at certa in hours of the day.Endotoxin injection in the middle of the activity period killed 20% of the mice injected, while 80% of the animals died when they were injected at the end of the resting period.The diurnal variations in the human response to Salmonella abotus equi endotoxin were studied recently in healthy volunteers (lO).Results indicated that subjects who received endotoxin in the evening, when endogenous glucocorticoid levels were low, showed abouL twice the increase in rectal temperature, plasma ACTH and cortisol levels as those who received endotoxin 12 h earlier, when endogenous glucocorticoid levels were high.It should be noted that Reinberg et al (l l) reported a circadian rhythm in the mortality produced by an infectious disease; peak mortality occurred at 06:00, at the end of the patients' resting period.
Pollmann and Pollmann ( 12) studied the efficiency of hepatitis B vaccination.Hospital employees and students in medicine or dentistry were vaccinated against hepatitis B with three injections of vaccine available on the German market.The antibody levels were determined four to eight weeks after the third injection.Figure 3 presents the influence of time of injection on these levels and shows that the afternoon injection (13:00 to 15:00) produced significantly higher antibody USEC Y•DO • Black boxes: dark period; white boxes: light period.Laboratory animals are active during the dark period, while are during the light period levels, especially in the patients with antibody levels larger than IOx 10 3 U/L.Pain and swelling at the injection site were also more frequent when the vaccine was given between 13:00 and 15:00.Season, age, sex and site of vaccination did not significantly influence the antibody levels.
The mechanisms of the time-dependent variations in infectious diseases are not known.However, circadian variations were reported in the Bacille Calmette-Guerin-induced migration of polymorphonuclear lymphocytes (13), in the plasma levels of immunoglobulin A (lgA), lgG , IgM and lgE in the different populations ofT total, T helper and T killer lymphocytes (14), as well as in the int1ammato1y reaction (15).

BIOLOGICAL RHYTHMS IN THE PHARMACOKINETICS OF ANTIBIOTICS
In recent years, investigators looked at the effect of time of day on the pharmacokinetics and renal toxicity of antibiotics .The objective of these studies was to use the time of antibiotic administration as a tool to reduce the side effects of potentially toxic drugs , such as aminoglycosides.When reviewing data obtained by many investigators, it is necessary to keep in mind that laboratory anim als have an activity pattern that is 64( 12 h out of phase from that of humans.In other words, rats and mice are active during the night (ie, during the dark phase of the light-dark cycle) and are sleeping during the light phase, when lights are on .Thus, humans are sleeping when rats are active and vice-versa.
Very few investigators have looked specifically at the temporal changes in the pharmacokinetics of antimicrobial agents.Table 1 presents the chronopharmacokinetics of different antibiotics in both laboratory animals and humans.For example, higher peak serum levels of gentamicin ( 16,17) and isepamicin (18) were found when these drugs were injected in the middle of the rest period of animals.Similar results were observed in the levels of gentamicin (16, l 9 ,20) and isepamicin (18,21) in the renal cortex, while the largest renal cortical content of amphotericin B was found early in the resting period of the animals (22) .Table 1 also shows that the maximal serum clearance of gentamicin (17,23), tobra mycin (24) and amikacin (25) was found when these antibiotics were injected in the middle of the activity period of rodents.In the cases of ampicillin (26) and liposomal ampicillin (27), maximal clearance was reported to occur during the activity period of animals.In agreement with these data , the area under the serum Can J Infect Dis Vol 10 Suppl C May 1999 *Black boxes: dark period; white boxes: light period.Laboratory an imal are active during the dark period, while humam are active during the light period curve (AUC) was largest when gentamicin (17,23), tobramycin (24) and amikacin (25) were injected in the middle of the resting period .The pharmacokinetic data from patients and volunteers are presented in Table 1.Higher trough serum levels were found in the early morning in patients who had normal renal function , but who were suffering from acute severe infections (28).In neutropenic patients receiving continuous infusion of amikacin therapy for infection, levels of amikacin obtained in the early hours of the day were significantly higher than those obtained for the same patient in the evening (29).Table 1 shows that the clearance of gentamicin (23) and isepamicin (30) was higher when these drugs were injected during the activity period, while a longer serum half-life and higher AUC in serum were found after the evening dose.Similar data were found for cefodizime (31) and sulfamethoxazole (32).Other observations suggest that the time to reach peak concentration in serum was longer when rifampicin was injected during the rest period (33).Finally, a higher rate of excretion in urine was found when ciprofloxacin (34) or sulfamethoxazole (32) was injected in the beginning of the activity period .These data are in agreement with previous observations in experimental animals suggesting that the clearance of these drugs is lower during the rest period and higher during the activity period of animals.
Can J In fect Dis Vol 10 Suppl C May 1999

BIOLOGICAL RHYTHMS IN THE RENAL TOXICITY OF ANTIBIOTICS
The effect of time of day on the toxicity of antibiotics was determined mainly in laboratory animals.In a first series of studies, the mortality rate produced by a single injection of antibiotics was investigated.Nakano and Ogawa (35) reported that very large doses of gentamicin killed more mice when injected in the middle of the resting period of the rodents (ie, at 13:00) than at any other time of the day.The data with gentamicin were confirmed by other investigators (23,35 ,36), and Table 2 demonstrates that temporal variations in the mortality rate were also found after high doses of netilmicin (36) , dibekacin (36) and amikacin (25).On the other hand, the mortality rate produced by amphotericin B was maximal when this drug was injected at the beginning of the activity period of laborato1y animals (37).Finally, the lethality produced by 3'azido-3'-deoxythymidine (AZT) was significantly higher when it was injected at 24:00, which is the middle of the activity period of rodents, but the largest bone marrow toxicity induced by AZT injection was found 4 h later at 04:00 (38).
The temporal variations in the acute and chronic renal toxicity produced by low doses of aminoglycosides were investigated in recent years by following the urinary excretio n of enzymes such as N-acetyl-beta-o-glucosaminidase and betagalactosidase.For example, maximal enzyme excretion in

Period of Injection
Figure 4) Per cent increase in serum creatinine levels over day I ef therapy in 54 hospitalized patients treated with djfferent aminog(ycosides at djfferent times ef the day.SEM Standard error ef mean urine was obtained when gentamicin (16,19,20,39,40) , tobramycin (24), dibekacin (40) and amikacin (41 ,42) were injected between 13:00 and 20:00.The postnecrotic cellular regeneration measured in the renal cortex was maximal when gentamicin (20) , tobramycin (24,43) and isepamicin (21) were injected at 14:00 compared with any other time of day.In contrast, the cellular regeneration in the renal cortex was found to be maximal 07:00 in animals given amphotericin B (22).Similarly, the increase in serum creatinine was found to be maximal in rats treated with amphotericin Bat 07:00 compared with any other time of day (22).Fujimura et al (44) reported a higher decrease of renal function (decrease creatinine clearance) when amikacin was injected at 16:00 than at other times of the day.
In humans, the effects of the ti me of day on the renal toxicity of aminoglycosides are difficult to investigate.Thus , a retrospective review of the changes in renal function in 54 hospitalized patients treated with different aminoglycosides was carried out.The percentage increase of serum creatinine over day 1 was used as an index of reduced renal function in patients treated with aminoglycosides at different hours of the day.Patients younger than 16 years of age, those receiving aminoglycosides for less than four days and those receiving a combined therapy with other nephrotoxic drugs were all excluded from the study.Figure 4 shows that the serum creatinine levels increased in patients treated in the evening compared with any other time of day.These data are in agreement with the data obtained in laboratory animals, although no significant difference was found between groups.As this retrospective study was being completed, Prins et al (45) published a prospective clinical trial on th e temporal variations in the renal toxicity of aminoglycosides.Patients were randomly separated into three groups with respect to the time of gentamicin or tobramycin therapy.Treatment with aminoglycosides between 00:00 and 07: 30 resulted in a significantly higher incidence of renal dysfunction (35%) compared with any other time of day.

MECHANISMS ASSOCIATED WITH TEMPORAL VARIATIONS IN THE RENAL TOXICITY OF AMINOGLYCOSIDES
The mechanisms associated with temporal variations in the renal toxicity of aminoglycosides are not completely understood.Table 3 shows the different potential mechanisms that have been investigated recently.First, temporal variations in the pharmacokinetics of aminoglycosides have been determined by several investigators both in laboratory animals (17 ,23-25) and in humans (23,30).An increase in serum clearance of these antibiotics observed during the activity period may have contributed significantly to the reduced renal toxicity of aminoglycosides observed during this period of the day (Table 2).Other experiments have shown that the subcellular distribution of tobramycin (24) and isepamicin (21) in proximal tubular cells was similar in animals treated at night and those treated during the day.Furthermore, temporal variations in the serum levels of adrenal gland hormones had no effect on the temporal variations in the renal toxicity of tobramycin (46).In addition, the role of food intake on the circadian variations in the nephrotoxicity of aminoglycosides has not been considered a factor.This potential mechanism was investigated by two different approaches.First, the circadian variations in the renal toxicity of gentamicin was compared in fasted rats versus in rats fed ad libitum.Interestingly, fasting abolished the 24 h variations in the nephrotoxicity of gentamicin (20,4 7) .Second , the circadian variations in the renal toxicity of gentamicin was studied in three groups of rats that had access to food at different periods.For a brief time , Can J Infect Dis Vol 10 Suppl C May 1999 th e toxicity was minimal when ge ntami cin was injected during th e feeding peri od, while the maximal to xicity w as always fo und when ge ntamicin wa s administered during the fas ting peri od independently of th e li g ht-dark cyc le (48 ).Son g et al ( 17) also show ed th at the manipulation of feedin g sc hedul es ca n modify th e rhythm of ge ntami cin tox icity by chang ing th e rh y thm of its kin eti cs.

BIOLOG ICAL RH YTHMS IN THE EFFECTIVENESS OF AMINOGLYCOSIOES
Very few investiga tors have looked at the temporal va riations in th e effecti ve ness of antibiotics.ln an experim ental model of pyelon ephriti s in rats, it was found th at gen ta micin , given for three and seven days at th e time of th e least toxicity (i e, 01 :00), showed a better effi cacy (ie, lowe r bacterial counts in the kidn eys and higher percentage of steril e kidneys) th an at oth er hours of the day (49) .By contrast, Hosokawa et al (25) demonstrated that the medi an effecti ve dose of amikacin in mi ce infected with Pseudomonas aerug inosa was signifi ca ntly lower wh en amikacin was injected in e middle of th e rest period (13:00) , with lower amikacin clea ran ce, than in the middle of the acti vity period (01 :00) , with higher amikacin clea ran ce .Funh er resea rch is needed in thi s area.

CONCLUSIONS
Th e data presented in thi s revi ew illustrate that temporal va ri ation s ca n be detected in the pharma cokineti cs, toxicity and effectiveness of antimicrobial age nts.These age nts are more rapidly eliminated and are less toxi c to laboratory animals wh en they are injected during the activity peri od of laboratory anim als.Th e human data are in agree ment with data obtained in laboratory animal s.
The study of th e temporal va ri ati ons in the pharm acokin etics , to xicity and effecti ve ness of antimicrobial agents may co ntribute significa ntly to increase their effi cacy and to reduce tox icity.Moreove r, beca use different likely age nts to be given in combinati on are fo und to be more toxic al different hours of the day, the injection of these drugs at their res pecti ve hours of lowest toxicity should be use ful to decrease their toxici ty as we ll as potenti al additi ve toxiciti es.

Cutaneous AUergic Reactions Oennatoses Osteoarthritis Perforated Ulcer Crises Anemia Crises Peptic Ulcer Exacerbation Hypertension Congestive Heart Failure Prinzmetal's Angina BP Surge Migraine Headache Allergic Rhinitis Rheumatoid Arthritis Angina, Myocardial Infarction Sudden Cardiac Death Figure
2) Peak time in the signs and symptoms qf diseases.

Using the ONLY•DONOT Frequency Distribution 30 PY Circadian rhythms and infectious diseases AM Vaccination. n= 267 .005.01 .0 15 .02 .03 .04 .06 .08 .12 .16 .25 .30 .45 .60 .80 1 1.5 2 3.25 S 7. 5 10 > 10 Frequency Distribution
1s many diseases are likely to be worst at a specjfic time ef day or night.For example, asthma is ivorst during the sleeping period, 1vhile cardio-,o vascular events are moreJrequent in the morning, and osteoarthritic pain peaks early in the evening.BP Blood pressure.Reproduced with permission.fromreference I

TABLE 1
Effects of time of day on the pharmacokinetics of antibiotics

TABLE 2
Effects of time of day on the toxicity of antibiotics

TABLE 3
Mechanisms associated with the temporal variations in the renal toxicity of aminoglycosides