Chrono-tailored drug delivery systems: recent advances and future directions

Oral administration of drugs is the most widely used route of administration due to its simplicity. However, drugs must contend with the physiological processes involved in the gastrointestinal tract. Physiological processes such as gastrointestinal pH and blood flow along with gastric emptying have been shown to have a circadian fluctuation which all influence drug absorption [23, 24]. The role of the clock has been shown to be interlinked with nutrition, with diurnal variation in various transporters that are involved with lipid, carbohydrate, and protein uptake [25–27]. CLOCK, one of the main components of the core circadian clock, has also been shown to regulate macronutrient absorption [28]. Diurnal variation in nutrient uptake is therefore likely to influence the uptake and absorption of drugs [29]. Lipophilic drugs are shown to be affected by time of day, with faster absorption in the active versus the rest phase. Valproic acid, which is a lipophilic drug used to treat epilepsy, displays time-of-day differences in drug absorption and concentration. A previous study examined the maximum absorption times of valproic acid at different times of day in mice and found that maximum drug concentration in the blood ranged from 386 ± 30.86 mg to 824 ± 39.85 mg during the rest and active phase, respectively [30]. Time of day differences in gastric emptying may also account for variations seen in drug absorption between morning and evening. A previous study with 16 healthy male volunteers showed that gastric emptying in the evening was significantly longer than in the morning [31]. The gastrointestinal tract, which is the most widely used and simplistic method of drug delivery, is influenced by the clock in many aspects. The absorption process should also be considered for the drug delivery to the skin. Factors such as skin pH and perfusion change throughout the day which can influence the absorption of drugs [32, 33]. Diurnal variation has been found in a water transporter in the skin in wild-type mice and HaCaT cells, which suggests the oscillations in epithelial barrier function could play a role in transdermal drug absorption [34]. However, there are no studies directly comparing drug absorption in the skin and the clock.

Drug distribution is an important factor in pharmacokinetics; however, modelling drug distribution is difficult making it the lesser studied of the four pharmacokinetic phases [35]. Distribution concerning the clock is also an understudied area; however, there is indirect evidence of clock involvement. The clock is shown to be involved in both blood flow and plasma protein availability two factors which distribution relies on [36]. In terms of blood flow, cardiac output shows diurnal variation with increased cardiac output and blood flow in the morning and decreasing blood flow and hepatic blood flow in the evening [37]. Distribution also relies on plasma protein availability. Drugs bound to plasma proteins are inactive, while unbound drug exists in the active form. Therefore, the affinity of the drug to bind to plasma proteins will affect its distribution and activity. Circadian variation in the concentration of various plasma proteins has previously been shown [38, 39]. This circadian variation in plasma proteins has also been shown to correlate with drug distribution when using valproic acid and chemotherapeutic agents [30, 40, 41]. Thus, the clock is associated with changes in blood flow and plasma protein levels, two factors which drug distribution relies on. However, currently, there is no strong evidence of direct circadian variation in drug distribution.

The liver and the kidneys are the main organs involved in drug metabolism. Metabolism in the liver of mice has been shown to be under the control of the clock, with 335 transcripts cycling in mouse livers [42] and 50% of metabolites found to be cycling [43]. Within the liver, PAR-domain basic leucine zipper transcription factors DBP, TEF, and HLF are found to accumulate in a circadian manner [44]. These transcription factors interact with genes that control the expression of enzymes which regulate metabolism in the liver. Various metabolic pathways including xenobiotic detoxification, glucose, lipid, and carbohydrate metabolism have all been linked to the clock within the liver [45–47]. Research on metabolism in the kidney has suggested that it is under the control of the renal tubular circadian clock [48]. However, further research is needed to understand the effects of the clock on metabolism in the kidney.

Elimination and excretion of therapeutics are mostly carried out by the kidneys. Diurnal variation in excretion was shown to occur in healthy individuals with rhythmic excretion of proteins such as albumin, transferrin, and immunoglobulin [39, 49, 50]. Renal excretion of electrolytes and minerals such as calcium and magnesium have further been shown to be under the influence of the clock [51, 52]. Urinary pH in humans has been shown to vary with the time of day which may account for variations in drug excretion through the day [53]. Although there are variations in kidney function and elimination of metabolites associated with circadian rhythm, the exact mechanism is unknown. Therefore, to understand the precise role of the clock in drug elimination, we need to further unravel the exact mechanisms by which the clock influences kidney and liver.

Toxicity is a major concern when drugs are administered to patients. Efforts to reduce toxicity may include reducing dosage, but importantly, research suggests altering the time of day of administration may also influence drug toxicity. It has been shown previously that liver cells’ (hepatocyte) circadian rhythm alters the xenobiotic metabolism [54]. Xenobiotic metabolism is responsible for the removal of foreign or toxic substances that are not normally produced by the body. With disruption of the clock in the liver, xenobiotic metabolism is altered leading to greater or lesser detoxification depending on the clock components altered. Further, hepatocytes’ circadian rhythm has also been shown to affect acetaminophen toxicity [55]. In the kidney, tobramycin administration in rats shows varying nephrotoxicity during excretion depending on the time of day of administration [56].

Large databases show that a number of tissue-specific genes are rhythmically expressed. This data provides further rationale for application to circadian medicine. Interestingly, many key cardiovascular genes are rhythmically expressed, which is of particular importance for drugs which may have off-target cardiovascular effects [1]. Bronchodilators are known to have off-target effects, by interacting with genes that are important in cardiovascular function, causing major adverse cardiac effects, and this is a major issue in their use [57]. Therefore, timing the administration of bronchodilators to coincide with the rhythmic downregulation in off-target cardiovascular genes may prevent cardiotoxicity.

The immune system is one system that is particularly under the influence of the circadian clock and thus may be particularly amenable to a chrono-tailored DDS approach. For example, many immune cells display circadian alterations in number in the body [65]. Members of the innate and adaptive immune system, including dendritic cells (DC) [66, 67], macrophages [68, 69], B cells [70], T Cells [71], and monocytes [72], all display circadian oscillations in clock genes and their outputs. For many years, researchers have been investigating the reasons why immune cells alter their phenotype throughout the time of day. Having immune cells primed for activation during the active phase, when the host is most likely to encounter a pathogen or become injured, versus during the rest phase, when the chance of infection or injury is lower, may be the reason. Whatever the underlying cause for why this mechanism evolved, it is well established that challenging an organism with lethal amounts of lipopolysaccharide (LPS) at varying times of the day leads to different host responses. Indeed, LPS challenge in mice as they approach their active phase is much more lethal than at the start of their rest phase [73]. Furthermore, evidence of rhythmic trafficking of immune cells between the skin, blood, and lymphatics has been elucidated [65, 67]. Circadian expression of chemokine receptors and adhesion molecules aid the circadian movement of immune cells between different organs. Similarly, the metabolism of immune cells varies depending on the time of day [66]. Recent reviews have covered the circadian immune system; in more detail [65, 74], we will discuss further the immune system in relation to chrono-tailored DDS.

One of the most simplified strategies to effectively target drugs to align with circadian rhythms is through direct administration of treatments according to the clock on the wall. An early study by Hamprecht et al. showed that cholesterol synthesis displayed a circadian rhythm [63]. This, in turn, led to Simvastatin, a short-acting cholesterol-reducing agent, being prescribed for patients to be taken in the evening. This is one of the first cases where circadian biology was translated into medical care to increase drug efficacy, with significant differences found in the effectiveness of lowering cholesterol if taken in the evening versus the morning [64].

With respect to immunological diseases, timed delivery of drugs has been shown to be effective in conditions such as asthma. Asthma shows strong circadian variation with symptoms being worse and more prevalent at night than in the morning [75, 76]. In one study, the administration of steroids to patients with moderate and moderate to severe asthma was randomised into two groups. One group received one dose of inhaled steroids at 3 pm, while the second group received four doses at 7 am, 12 pm, 7 pm, and 10 pm. Both groups received a total of 800 µg of steroids per day. They found no increase in overall toxic systemic effects of the drug with both regimens, but that efficacy was still found to be similar between both groups [77]. This is incredibly beneficial as not only is it more cost-effective to reduce the number of dosages per day, but more importantly, it can lead to higher patient compliance [78]. However, one of the main challenges surrounding the timed delivery of drugs is that it requires patients’ consistency with the administration of drugs at the optimal time of day.

Sancar et al. detail the intricate links of the circadian rhythm coupled with cancer and chemotherapy and conclude that is worth exploring the link between circadian timing of chemo and radiotherapy to try and improve patient outcomes [79].While there are defined links between DNA damage susceptibility and cell cycle changes and the circadian clock, currently, the evidence regarding what time of day is most efficient for chemotherapy is lacking [80–82]. A study looking at B cell lymphoma in female patients found differences in overall survival and progression-free survival between morning and afternoon administration of chemotherapy [83]. Indicating that in this cancer subtype in female patients, there appears to be a benefit to time of day administration, but this cannot be generalised across all cancer subtypes. Some studies have shown that time of day administration of chemotherapy and radiotherapy is more effective for patients in terms of increasing efficacy and decreasing unwanted side effects [60, 84]. However, it is hard to define the exact time of day that chemotherapy should be administered as it appears to depend on several factors including the drugs administered and the cancer subtype. Papers as far back as the early 1990s discuss the implications of chrono-modulation in cancer therapeutics [62, 85]. While some links exist, further studies are warranted for different chemotherapeutic agents and their specific time-of-day effects. Similarly, results may vary based on the patient’s disease status, age, sex, and the different combinations of chemotherapeutic drugs patients are taking. It is a tough endeavour as both circadian interplay with cell cycle and DNA repair needs further exploration, and disruption of the clock within tumours also warrants further studies. There is much research to be done to fully comprehend the complex interplay between cancer, chemotherapy, and the circadian rhythm to precisely define the correct time of day of administration.

One of the most significant barriers to administering not just chemotherapeutic agents but other drugs at the correct time of day in a clinical setting is the scheduling, availability, and coordination with healthcare professionals. Therefore, the main drawbacks of timed delivery of chemotherapeutics are patient compliance and scheduling and, in the healthcare setting, the practicalities of timed delivery.

Delayed release has shown to be beneficial in rheumatoid arthritis, where the modulated release of the glucocorticoid, prednisone, which is used to treat morning stiffness symptoms, was tested in a double-blind, randomised trial. It was found to be more effective at treating symptoms of morning stiffness when given in the modified release form at night versus immediate-release prednisone in the morning [97]. Modified prednisone was given at night for delayed release at 2 am and compared with morning prednisone administration. However, delayed release at night versus regular prednisone treatment at night was not compared; therefore, we cannot conclude that it is the modified delayed release causing an effect or if it is just evening versus morning administration. Nevertheless, this study shows that nighttime dosing of prednisone is more efficacious than morning dosing.

Stroke risk is said to be highest in the morning due to several clinical factors including blood pressure, heart rate, and cerebral blood flow [98, 99]. Therefore, it has been hypothesised that nighttime or evening administration of anti-hypertensives and cardiac therapeutics may be beneficial to counteract the increased incidence of morning stroke. However, there have been no clinical studies to suggest treatment of these morning risk factors will prevent cardiac complications. COER-Verapamil is one of the first delayed-release drug delivery tablets in the USA to be approved by the Food and Drug Administration as a chronotherapeutic for ischemic heart disease and hypertension [100]. COER-Verapamil is an ion channel inhibitor that uses an extended-release system to cause a maximum drug concentration 4–5 h after ingestion. This leads to a high drug concentration in the blood in the early hours of the morning when cardiovascular events are most likely to occur. A large study was carried out longitudinally to compare COER-Verapamil to other cardiovascular drugs, which are usually given as a first line of treatment to patients. This study was terminated early due to sponsor withdrawal; however, after 3 years, it was concluded that COER-Verapamil was not superior to the current recommended first-line cardiovascular treatments [101]. It is essential to note, however, that this study did not include a comparison to treatment at the time of awakening and, as such, cannot be described as a chronotherapy clinical trial. Therefore, more studies are needed to understand COER-Verapamil.

Another variation of delayed delivery is pulsatile delivery systems. Pulsatile systems have been extensively reviewed by Bussemer et al.; we will review some of the main examples in relation to chronotherapy with a focus on immunological diseases [102]. Many pulsatile systems are aimed to have a lag phase long enough to allow the release of the drug within the colon. Usually, drugs have already been absorbed through the gastrointestinal tract before they reach the colon, but with pulsatile drugs, this can be delayed. Modified Pulsincap of ibuprofen and drugs specific for ulcerative colitis, irritable bowel disease, and other colon-specific diseases have been developed [103–107]. Another pulsatile variation which depends on endogenous signals is a hydrogel for type II diabetics, which is responsive to glucose levels in the body and releases insulin when glucose levels are increased [108]. This system is liquid at room temperature and solid with increasing glucose which causes a burst release of insulin. This is an example of a pulsatile system that is responsive to individual biological signals within the body and thus provides a highly personalised approach.

Delayed and pulsatile systems work on very similar principles of lag phase following the release of the drug at the intended time. However, the manufacture of these tablets is lengthier and requires more steps to ensure that drug release will occur at the correct time and the correct dosage.

Nanoparticles (NP) are small structures that can be loaded with a drug and offer an alternative form of drug delivery in comparison to tablets as previously discussed. NPs are used due to their small size, increased bioavailability, stabilisation of drugs, and modifiable structure to target them to specific tissues. Drug release from NP relies on several factors, including the polymer, ionic charge, drug concentration and interaction with the polymer itself, the ratio of the compositions, and manufacturing methods [109, 110]. Therefore, there are multiple steps in which modifications can be made or adjusted to tailor nanocarriers towards chronotherapeutic purposes.

Mesoporous and hollow silica NPs are examples of NPs that display a delayed-release profile, or their profile can be modified for a pulsatile release [111–113]. Although NP drug delivery systems such as these have demonstrated controlled delivery and can be targeted to specific tissues, they have yet to be widely explored in the context of chrono-therapeutics [114]. Niosomes are non-ionic surfactant-based vesicles that are structurally similar to liposomes. These vesicles have been investigated as a cancer therapeutic delivery system to deliver at the tumour site with less potent cytotoxic side effects [115]. They may pose as a useful chrono-tailored DDS as they appear to enhance drug entrapment and delay the initial burst release of their contents [116]. Nanocarriers offer great potential in vitro, as a desired release profile is easy to obtain; however, elucidating the exact release profile in vivo can be difficult. The mechanisms of polymeric and lipid NPs have been reviewed by Son et al. on the specific mechanisms of release from various NPs [110]. It has been hypothesised that timed drug delivery of NPs in breast cancer could increase the uptake of the drug into cancer cells and improve the treatment [117]. Timed drug release in this instance is related to monthly timing rather than circadian timing. Vascular endothelial growth factor (VEGF) cycles through the menstrual cycle in women with subsequent increase and decrease in vascular permeability [118]. Timing of drug delivery of cytotoxic chemotherapeutics to coincide with the natural increase of VEGF and therefore, vascular permeability leads to higher drug uptake, particularly within the tumour sites, as they are heavily vascularised. However, further evidence is needed in the literature along with the support of clinical trials to confidently conclude the cycling of VEGF can correspond to enhanced cytotoxicity of the tumour.

A novel drug delivery system which became popular in the mid-1990s was the MN drug delivery system. Many detailed reviews exist on MN fabrication and current and future applications of MNs [122, 123]. MN patches contain an array of MNs of lengths of 100–1500 µm that pierce the stratum corneum and can deliver drugs through a microchannel. A microchannel is a small channel in the skin that allows compounds that would otherwise be unable to permeate the stratum corneum to enter the skin at different layers ranging from the epidermis to the dermis depending on the length of the microneedle [124]. MNs can be categorised into separate groups: solid, hollow, coated, dissolving, and hydrogel. Different types of MNs offer additional advantages depending on the desired application. Herein, we provide a brief overview of some dissolvable MNs (dMNs) and their potential to be applied with respect to the host’s circadian rhythm.

Dissolvable MNs can offer rapid or slow release depending on the polymer and drug composition, concentration, and ratio. The active ingredient and the polymer must be biodegradable in the dissolving MNs and easily excreted from the body as the entire microneedle is dissolved into the skin. Dissolving MNs, therefore, offer a distinct advantage as they do not produce biohazardous waste. When choosing polymers for dissolvable microneedles, various properties of the polymer and the interactions between the polymer and active ingredient need to be considered. While dissolving MNs require strength to penetrate the skin, they must also readily dissolve. Polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP) are two polymers used in dMN applications due to their strength, biodegradability, and overall tolerability in patients. However, a wide range of polymers are being explored for MN applications. Drug loading capabilities of dissolvable MN polymers are an important quality to consider to ensure the correct dosage through intradermal delivery. Recent reviews attached detail the manufacturing protocols of MNs, qualities to consider, and current challenges faced in the field [125–127]. MN and hydrogel MN patches for immunotherapy and chemotherapeutic agents have been described by Wang et al. and Courtenay et al. [128, 129]. Delivering chemotherapeutic agents trans-dermally with microneedles not only offers more comfort than a needle and syringe method of application but, as previously mentioned, there is a potential benefit to time-of-day administration of chemotherapeutics that could be achieved. There are some clinical trials of dissolving MNs indicating good efficacy in the treatment of keloids and influenza vaccination [130–132]. A clinical trial investigating insulin delivery through dissolving MNs showed faster insulin delivery and higher patient compliance when compared to traditional routes of administration [133, 134]. MNs may also offer a unique function as a bio-responsive agent. Bio-responsive agents act with biological stimuli or abnormalities and therefore could act in accordance with circadian rhythm fluctuations in cytokines, hormones, etc. In terms of microneedles, the release of an active ingredient in line with an increase or decrease in a particular biological marker that is circadian would lead to smart and highly personalised administration of chronotherapeutic drugs. Bio-responsive MNs and polymers that work to release insulin to control glucose levels are a new and interesting area of research [108, 135–137]. The expansion of research into bio-responsive polymers and particles provides an opportunity for the expansion of microneedles as bio-responsive agents, allowing the delivery of drugs at the correct time of day and aiding in chrono-tailoring drug delivery.

Transdermal patches, medicated adhesive patches, can also be used to deliver drugs through the skin. Transdermal patches have been widely used for asthma treatment. As previously mentioned, asthma has been shown to have a strong circadian variation with symptoms being worst in the early hours of the morning. Transdermal delivery of tulobuterol, the beta2-adrenoceptor agonist and bronchodilator, is delivered through a crystal reservoir system. The tulobuterol is in crystal form surrounded by a polymer which controls the release of the drug depending on the polymer properties. The tulobuterol patch has a slow nighttime release to target early morning asthmatic symptoms [138]. The patch is applied at night and provides effective bronchodilation as drug concentration slowly builds over time to be effective during the early hours of the morning when nocturnal symptoms peak. The tulobuterol patch has shown good safety and efficacy in clinical trials, along with greater patient compliance versus inhaled drugs [139]. Thus, this is a good example of the beneficial effects of transdermal delivery to coincide with the circadian rhythm and onset of asthmatic symptoms. The tulobuterol patch is currently on the market in seven countries. This drug delivery system is preferred by patients showing higher patient adherence over inhaled drugs, proving to be an excellent circadian-tailored drug delivery system [140].

As discussed, a wide range of immune parameters exist which display circadian rhythms and are affected by circadian gene or environmental perturbation. Thus, the circadian clock plays a significant role in terms of immune cell effector function. At the cellular level, the circadian clock influences immune cell metabolism and morphology. In haematopoietic stem cells, circadian rhythm has been shown to influence migration and release [141, 142]. Within the immune cell populations, macrophage metabolism and immunophenotype have been linked to the circadian clock [18, 68, 143]. Cervantes et al. recently uncovered that DC mitochondrial morphology and metabolism change throughout the 24-h cycle and these changes are dependent on Bmal1 [66]. This impacts the timing of when to process and present antigens to T cells.

Thus, researchers have begun to explore the impact of the circadian clock within immune cells and its rhythms on the response to vaccination. Herein, we discuss some of the key findings and research which has been carried out to date with respect to circadian timing and vaccination. Time-of-day studies in vaccination have shown differences in response depending on when the influenza or BCG vaccine was administered [144, 145]. A cluster-randomised trial using influenza vaccination compared morning vaccination to that of evening vaccination in older adults. The study demonstrated that morning vaccination induced a higher antibody response in older adults [145]. Enhancing antibody responses in older adults is particularly important as the efficacy of vaccines deteriorates with age [146, 147]. However, the time of sample collection was not noted in this study. As Kurupati et al. recently discussed, the time of sample collection can also influence antibody titres [148]. Similarly, studies assessing the Bacille Calmette-Guerin (BCG) vaccination response demonstrated that the circadian rhythm influenced the trained immune response [144]. De Bree et al. observed this through two studies, a large cohort of 302 individuals and a smaller study of 54 healthy volunteers. In the large cohort, they looked at vaccinations between 8 am and 12 pm, while in the small cohort, vaccination was carried out in the morning and evening. In both cases, peripheral blood mononuclear cells (PBMC) were taken from volunteers before and after vaccination and stimulated with Staphylococcus aureus and tuberculosis, and cytokine production was measured. Overall, they found that the trained immune response to be higher in PBMCs isolated from the morning cohorts versus the evening cohorts. Further studies have investigated an inactivated SARS-CoV-2 vaccine against Covid-19, with respect to the time of day. Inactivated SARS-CoV-2 vaccination in the morning in health care workers induced higher antibody titres and induced stronger B cell and follicular helper T cells, versus what time of day [149]. An observational study of healthcare workers found significant differences in the anti-spike antibodies against Covid-19 when comparing the Pfizer vaccination versus AstraZeneca at the same times of day [150]. Pfizer’s Covid-19 vaccination is mRNA based, while AstraZeneca is an adenoviral vaccine, thus, highlighting how different vaccination types may be affected by the clock to a greater or lesser extent. Further studies have found that men mount a higher antibody response to the hepatitis A vaccine and influenza vaccine, while females do not have an enhanced antibody response in comparison to men depending on the time of day [151]. Perhaps another cycle which influences the vaccination in women other than the circadian rhythm is the menstrual cycle. There is very little published regarding the effects of the menstrual cycle on vaccination. However, there are studies showing immune fluctuations through the menstrual cycle; therefore, this should also be considered in the effects of circadian rhythm and vaccination in women [152]. Overall, there is evidence which would suggest that the vaccination response is influenced by the circadian clock. Further studies are warranted that take into consideration gender, age, and sample time collection to assess the effects of the circadian clock and what other variables exist which determine the response.

Timing of both drugs and vaccination with respect to circadian rhythm is an important consideration. While further research is warranted to understand the exact time of day of administration of both drugs and vaccination, another key factor to be explored is the best method to administer medications and vaccinations at the correct time of day. If vaccination strategies were to adapt to circadian timing, the traditional route of administration of vaccines would require adaptation. Traditional methods of vaccination through intramuscular injection must be carried out by a healthcare professional, and therefore, it would be difficult to implement vaccination at the correct time of day should that time fall out of hours. Alternative drug delivery systems should be considered for time-of-day vaccination. In clinical trials, CD8 effector cell responses were shown to be increased through intradermal vaccination versus intramuscular route [153]. Fluzone Intradermal Quadrivalent is an intradermal injection influenza vaccination that is currently on the market in the USA for persons aged over 65. Fluzone has shown equivocal or increased immunogenicity when compared to intramuscular vaccination [154–156]. Similar studies and meta-analyses have also shown that intradermal vaccination offers an excellent alternative to the traditional routes of vaccination [157–161]. Clinical trials have also shown self-administration of vaccine patches to be similar to alternative vaccination routes [162–164]. Furthermore, intradermal routes of administration such as MN patches are shown to be preferred by individuals versus an intramuscular route [134, 164]. MNs offer an alternative route of immunisation that is comparable in efficacy to intramuscular which can be self-administered at the correct time of day. Therefore, MN systems may be a useful drug delivery system when considering future strategies to target the circadian rhythm in vaccination.