Alper Okyar

Circadian Timing in Cancer Treatments

The circadian timing system is composed of molecular clocks, which drive 24-h changes in xenobiotic metabolism and detoxification, cell cycle events, DNA repair, apoptosis, and angiogenesis. The cellular circadian clocks are coordinated by endogenous physiological rhythms, so that they tick in synchrony in the host tissues that can be damaged by anticancer agents. As a result, circadian timing can modify 2- to 10-fold the tolerability of anticancer medications in experimental models and in cancer patients. Improved efficacy is also seen when drugs are given near their respective times of best tolerability, due to (a) inherently poor circadian entrainment of tumors and (b) persistent circadian entrainment of healthy tissues. Conversely, host clocks are disrupted whenever anticancer drugs are administered at their most toxic time. On the other hand, circadian disruption accelerates experimental and clinical cancer processes. Gender, circadian physiology, clock genes, and cell cycle critically affect outcome on cancer chronotherapeutics. Mathematical and systems biology approaches currently develop and integrate theoretical, experimental, and technological tools in order to further optimize and personalize the circadian administration of cancer treatments.

Dosing-Time Makes the Poison: Circadian Regulation and Pharmacotherapy

Daily rhythms in physiology significantly modulate drug pharmacokinetics and pharmacodynamics according to the time-of-day, a finding that has led to the concept of chronopharmacology. The importance of biological clocks for xenobiotic metabolism has gained increased attention with the discovery of the molecular circadian clockwork. Mechanistic understanding of the cell-autonomous molecular circadian oscillator and the circadian timing system as a whole has opened new conceptual and methodological lines of investigation to understand first, the clocks impact on a specific drugs daily variations or the effects/side effects of environmental substances, and second, how clock-controlled pathways are coordinated within a given tissue or organism. Today, there is an increased understanding of the circadian modulation of drug effects. Moreover, several molecular strategies are being developed to treat disease-dependent and drug-induced clock disruptions in humans.

A Combined Experimental and Mathematical Approach for Molecular-based Optimization of Irinotecan Circadian Delivery

Circadian timing largely modifies efficacy and toxicity of many anticancer drugs. Recent findings suggest that optimal circadian delivery patterns depend on the patient genetic background. We present here a combined experimental and mathematical approach for the design of chronomodulated administration schedules tailored to the patient molecular profile. As a proof of concept we optimized exposure of Caco-2 colon cancer cells to irinotecan (CPT11), a cytotoxic drug approved for the treatment of colorectal cancer. CPT11 was bioactivated into SN38 and its efflux was mediated by ATP-Binding-Cassette (ABC) transporters in Caco-2 cells. After cell synchronization with a serum shock defining Circadian Time (CT) 0, circadian rhythms with a period of 26 h 50 (SD 63 min) were observed in the mRNA expression of clock genes REV-ERBα, PER2, BMAL1, the drug target topoisomerase 1 (TOP1), the activation enzyme carboxylesterase 2 (CES2), the deactivation enzyme UDP-glucuronosyltransferase 1, polypeptide A1 (UGT1A1), and efflux transporters ABCB1, ABCC1, ABCC2 and ABCG2. DNA-bound TOP1 protein amount in presence of CPT11, a marker of the drug PD, also displayed circadian variations. A mathematical model of CPT11 molecular pharmacokinetics-pharmacodynamics (PK-PD) was designed and fitted to experimental data. It predicted that CPT11 bioactivation was the main determinant of CPT11 PD circadian rhythm. We then adopted the therapeutics strategy of maximizing efficacy in non-synchronized cells, considered as cancer cells, under a constraint of maximum toxicity in synchronized cells, representing healthy ones. We considered exposure schemes in the form of an initial concentration of CPT11 given at a particular CT, over a duration ranging from 1 to 27 h. For any dose of CPT11, optimal exposure durations varied from 3h40 to 7h10. Optimal schemes started between CT2h10 and CT2h30, a time interval corresponding to 1h30 to 1h50 before the nadir of CPT11 bioactivation rhythm in healthy cells.

Circadian clocks and drug delivery systems: impact and opportunities in chronotherapeutics

Chronotherapeutics aims at the adjustment of treatments to ∼ 24 h rhythms, which result from the moderation of most biological functions by the circadian timing system (CTS). The integration of CTS-related knowledge in drug delivery concepts challenges most current views, where steady-state constant drug levels are synonymous to enhanced tolerability and efficacy. In contrast, robust molecular clocks rhythmically control Phase I, II and III drug metabolism, as well as pharmacodynamics. Thus, circadian timing of medications predictably modifies drug tolerability and/or efficacy up to several-fold in rodents, as well as in patients. Optimal dosing times indeed complement the recommendations for optimal doses of glucocorticoids, NSAIDs, bronchodilators and so on. Clinically-driven in vitro and in silico circadian data now provide mechanistic insights for the effective translation of chronotherapeutic delivery, especially for cancer therapies. Programmable-in-time electronic or polymeric drug delivery systems are being used for improving health in patients with cancer or rheumatoid diseases, respectively. Current research aims at the optimization of circadian amplitude and phase of drug delivery according to CTS biomarkers. Intelligent drug delivery systems could then integrate the critical rhythmic information stemming from the individual patient and achieve a critical leap forward in the safe administration of potentially toxic therapeutic agents.

Strain- and Sex-Dependent Circadian Changes in Abcc2 Transporter Expression: Implications for Irinotecan Chronotolerance in Mouse Ileum

Background ATP-binding cassette transporter abcc2 is involved in the cellular efflux of irinotecan. The drug is toxic for mouse ileum, where abcc2 is highly expressed. Here, we investigate whether circadian changes in local abcc2 expression participate in the circadian rhythm of irinotecan toxicity for ileum mucosa, and further assess whether genetic background or sex modify this relation. Methodology/Principal Findings Ileum mucosa was obtained every 3–4 h for 24 h in male and female B6D2F1 and B6CBAF1 mice synchronized with light from Zeitgeber Time (ZT)0 to ZT12 alternating with 12 h of darkness. Irinotecan (50 mg/kg i.v. daily for 4 days) was administered at the sex- and strain-specific times corresponding to least (ZT11-15) or largest drug-induced body weight loss (ZT23-03-07). Abcc2 expression was determined with qRT-PCR for mRNA and with immunohistochemistry and confocal microscopy for protein. Histopathologic lesions were graded in ileum tissues obtained 2, 4 or 6 days after treatment. Two- to six-fold circadian changes were demonstrated for mRNA and protein mean expressions of abcc2 in mouse ileum (p<0.05). ZT12 corresponded to high mRNA and protein expressions, with circadian waveforms differing according to genetic background and sex. The proportion of mice spared from ileum lesions varied three-fold according to irinotecan timing, with best tolerability at ZT11-15 (p = 0.00003). Irinotecan was also best tolerated in males (p = 0.05) and in B6CBAF1 (p = 0.0006). Conclusions/Significance Strain- and sex-dependent circadian patterns in abcc2 expressions displayed robust relations with the chronotolerance of ileum mucosa for irinotecan. This finding has strong potential implications for improving the intestinal tolerability of anticancer drugs through circadian delivery.

Ondansetron-loaded chitosan microspheres for nasal antiemetic drug delivery: an alternative approach to oral and parenteral routes.

Background: The aim of this study was to develop chitosan microspheres for nasal delivery of ondansetron hydrochloride (OND). Method: Microspheres were prepared with spray-drying method using glutaraldehyde as the crosslinking agent. Microspheres were characterized in terms of morphology, particle size, zeta potential, production yield, drug content, encapsulation efficiency, and in vitro drug release. Results: All microspheres were spherical in shape with smooth surface and positively charged. Microspheres had also high encapsulation efficiency and the suitable particle size for nasal administration. In vitro studies indicated that all crosslinked microspheres had a significant burst effect, and sustained drug release pattern was observed until 24 hours following burst drug release. Nasal absorption of OND from crosslinked chitosan microspheres was evaluated in rats, and pharmacokinetic parameters of OND calculated from nasal microsphere administration were compared with those of both nasal and parenteral administration of aqueous solutions of OND. In vivo data also supported that OND-loaded microspheres were also able to attain a sustained plasma profile and significantly larger area under the curve values with respect to nasal aqueous solution of OND. Conclusion: Based on in vitro and in vivo data, it could be concluded that crosslinked chitosan microspheres are considered as a nasal delivery system of OND.

Circadian Variations in Exsorptive Transport: In Situ Intestinal Perfusion Data and In Vivo Relevance

The circadian timing system (CTS) governs the 24-h rhythm of the organism and, hence, also main pathways responsible for drug pharmacokinetics. P-glycoprotein (P-gp) is a drug transporter that plays a pivotal role in drug absorption, distribution, and elimination, and temporal changes in its activity may affect input, output, activity, and toxicity profile of drugs. In the current study, the influence of different circadian stages on the overall intestinal permeability (Peff) of the P-gp substrates talinolol and losartan was evaluated in in situ intestinal perfusion studies in rats. Additionally, in vivo studies in rats were performed by employing the P-gp probe talinolol during the day (nonactive) and night (active) period in rats. Effective intestinal permeabilities of talinolol and losartan were smaller in studies performed during the night (p < .05), indicating that P-gp–dependent intestinal secretion is greater during the nighttime activity span than daytime rest span of the animals. P-gp modulators vinblastine and PSC833 led to a significant decrease of talinolol and losartan exsorption in the intestinal segments as compared with control groups. Strikingly, the permeability-enhancing effect of vinblastine and PSC833 was higher with night perfusions, for both talinolol and losartan. In vivo studies performed with talinolol revealed—consistent with the in situ studies (Peff day > night)—a day vs. night difference in the oral availability of talinolol in the group of male rats in terms of the area under the curve (AUC) data (AUCday > AUCnight). The P-gp modulator vinblastine significantly increased talinolol AUCday (p < .05), whereas only a weak vinblastine effect was seen in night. According to the in situ data, the functional activity of P-gp was regulated by the CTS in jejunum and ileum, which are major intestinal segments for energy-dependent efflux. In conclusion, circadian rhythms may affect carrier-mediated active efflux and play a role in the absorption process. In addition to daily rhythms in P-gp activity in rat intestine, the in vivo studies indicate that absorption-, distribution-, metabolism-, and elimination-relevant rhythms may be involved in the circadian kinetics of the drug, besides transporter-dependent efflux, such well-known aspects as metabolic or renal clearance or motility. Since this also holds true for a potentially interacting second compound (modulator), modulator effects should be evaluated carefully in transporter related drug-drug interactions. (Author correspondence: aokyar@istanbul.edu.tr)

In Vitro and Ex Vivo Permeation Studies of Etodolac from Hydrophilic Gels and Effect of Terpenes as Enhancers

Etodolac, a highly lipophilic anti-inflammatory drug, is widely used in rheumatoid arthritis usually at an oral dose of 200 mg twice daily. The commonest side effects during therapy with etodolac is generally gastrointestinal disturbances these are usually mild and reversible but in some patients are peptic ulcer and severe gastrointestinal bleeding. To eliminate these side effects and obtain high drug concentration at the application side, dermal application of etodolac seems to be an ideal route for administration. Hydrophilic gel formulations of etodolac were prepared with carboxymethylcellulose sodium. The effect of different terpenes (anethole, carvacrol, and menthol) as an enhancer on the percutaneous absorption of etodolac was also investigated. Permeation studies were carried out with unjacketed modified horizontal diffusion cells through cellulose membrane and rat skin. In vitro studies with cellulose membrane showed that all formulations presented the same drug release profile (p > 0.05). Ex vivo studies with excised rat skin revealed that etodolac was released and penetrated into rat skin quickly. Anethole, a hydrophobic terpene, enhanced the absorption of etodolac significantly (p < 0.05). This result is consistent with the fact that hydrophobic terpenes are effective on the percutaneous absorption of lipophilic drugs. Menthol and carvacrol, hydrophilic terpenes, did not enhance the absorption of etodolac. The lipophilicity of the enhancers seems an important factor in promoting penetration of etodolac through the skin. Since etodolac creates gastrointestinal disturbances, topical formulations of etodolac in gel form including 1% anethole could be an alternative.

Ondansetron-loaded biodegradable microspheres as a nasal sustained delivery system: In vitro/in vivo studies

The aim of this study was to prepare ondansetron-loaded biodegradable microspheres as a nasal delivery system. Microspheres were prepared with emulsification/spray-drying technique using poly(d,l-lactide) (PLA) and two different types of poly(d,l-lactide-co-glycolide) (PLGA). The effect of the type of organic solvent (dichloromethane (DCM) or a mixture of DCM and ethyl acetate) on the microsphere characteristics was also examined. The prepared microspheres were evaluated with respect to the morphological properties, particle size, zeta potential, drug loading efficiency, and in vitro drug release. The mean particle size (d50) of microsphere formulations was ranged from 11.67–25.54 μm, indicating suitable particle size for nasal administration. All microspheres had low drug loading efficiency in the range of 12.28–21.04%. The results indicated that particle size of microspheres were affected by both type of polymer and organic solvent, however drug loading efficiency of microspheres were affected by only the type of organic solvent used. All microspheres were negatively charged due to the polymers (PLA or PLGA) used. A prolonged in vitro drug release profile was observed for 96 h. Based on in vitro data, the selected microsphere formulation has been applied via nasal route to rats in vivo. Following nasal administration of ondansetron-loaded microsphere to rats, ondansetron plasma levels were within a range of 30–48 ng/mL during 96 h, indicating a sustained drug delivery pattern and relatively a constant plasma drug concentration level. The results suggested that biodegradable microspheres prepared with emulsification/spray-drying technique could be considered to deliver ondansetron via nasal route to obtain a prolonged release.

Clock gene Per2 as a controller of liver carcinogenesis

Environmental disruption of molecular clocks promoted liver carcinogenesis and accelerated cancer progression in rodents. We investigated the specific role of clock gene Period 2 (Per2) for liver carcinogenesis and clock-controlled cellular proliferation, genomic instability and inflammation. We assessed liver histopathology, and determined molecular and physiology circadian patterns in mice on chronic diethylnitrosamine (DEN) exposure according to constitutive Per2 mutation. First, we found that Per2m/m liver displayed profound alterations in proliferation gene expression, including c-Myc derepression, phase-advanced Wee1, and arrhythmic Ccnb1 and K-ras mRNA expressions, as well as deregulated inflammation, through arrhythmic liver IL-6 protein concentration, in the absence of any DEN exposure. These changes could then make Per2m/m mice more prone to subsequently develop liver cancers on DEN. Indeed, primary liver cancers were nearly fourfold as frequent in Per2m/m mice as compared to wild-type (WT), 4 months after DEN exposure. The liver molecular clock was severely disrupted throughout the whole carcinogenesis process, including the initiation stage, i.e. within the initial 17 days on DEN. Per2m/m further exhibited increased c-Myc and Ccnb1 mean 24h expressions, lack of P53 response, and arrhythmic ATM, Wee1 and Ccnb1 expressions. DEN-induced tumor related inflammation was further promoted through increased protein concentrations of liver IL-6 and TNF-α as compared to WT during carcinogenesis initiation. Per2 mutation severely deregulated liver gene or protein expressions related to three cancer hallmarks, including uncontrolled proliferation, genomic instability, and tumor promoting inflammation, and accelerated liver carcinogenesis several-fold. Clock gene Per2 acted here as a liver tumor suppressor from initiation to progression.