Refined tamoxifen administration in mice by encouraging voluntary consumption of palatable formulations

lab animal Article https://doi.org/10.1038/s41684-024-01409-z Refined tamoxifen administration in mice by encouraging voluntary consumption of palatable formulations Check for updates Dominique Vanhecke    1, Viola Bugada1, Regula Steiner    2, Bojan Polić3 & Thorsten Buch    1 Drug administration in preclinical rodent models is essential for research and the development of novel therapies. Compassionate administration methods have been developed, but these are mostly incompatible with water-insoluble drugs such as tamoxifen or do not allow for precise timing or dosing of the drugs. For more than two decades, tamoxifen has been administered by oral gavage or injection to CreERT2–loxP gene-modified mouse models to spatiotemporally control gene expression, with the numbers of such inducible models steadily increasing in recent years. Animal-friendly procedures for accurately administering tamoxifen or other water-insoluble drugs would, therefore, have an important impact on animal welfare. On the basis of a previously published micropipette feeding protocol, we developed palatable formulations to encourage voluntary consumption of tamoxifen. We evaluated the acceptance of the new formulations by mice during training and treatment and assessed the efficacy of tamoxifen-mediated induction of CreERT2–loxP-dependent reporter genes. Both sweetened milk and syrup-based formulations encouraged mice to consume tamoxifen voluntarily, but only sweetened milk formulations were statistically noninferior to oral gavage or intraperitoneal injections in inducing CreERT2-mediated gene expression. Serum concentrations of tamoxifen metabolites, quantified using an in-house-developed cell assay, confirmed the lower efficacy of syrup- as compared to sweetened milk-based formulations. We found dosing with a micropipette to be more accurate than oral gavage or injection, with the added advantage that the method requires little training for the experimenter. The new palatable solutions encourage voluntary consumption of tamoxifen without loss of efficacy compared to oral gavage or injections and thus represent a refined administration method. The increased efforts to conduct more humane animal research (3R principle1,2) include the development and application of animal-friendly drug administration methods. Unlike water-soluble drugs, most water-insoluble drugs cannot be mixed with drinking water or chow. Instead, they are typically administered via oral gavage (OG) or intraperitoneal (IP) or subcutaneous injection. These invasive interventions can induce stress-related responses in rodents, as reflected by increased stress hormone levels or heart rates3–8. Furthermore, repeated OG can increase the risk of unintentional injuries, including perforation of the trachea, esophagus or stomach, introduction of fluids into the trachea or lung, and hemorrhage9. Repeated IP injections have been reported to cause local irritation, pain, infection and damage to surrounding tissue10. Recently, a procedure for drug administration in mice that aims to minimize the above-mentioned disadvantages has been proposed as an alternative to OG or IP injections7,8. This so-called micropipette-guided drug administration makes use of a sweetened condensed milk solution as a vehicle to motivate mice to voluntarily consume drug solutions. This noninvasive procedure was shown to achieve pharmacokinetic profiles similar to those obtained by OG7 or IP injections8. However, until now, similar pipette feeding has not been successfully adopted for the administration of water-insoluble compounds such as tamoxifen (TAM). TAM is a selective estrogen receptor (ER) modulator that is widely used in clinical and basic research applications. For more than 20 years, TAM has been utilized in research to induce spatiotemporal modifications 1 Institute of Laboratory Animal Science, University of Zurich, Zurich, Switzerland. 2Institute of Clinical Chemistry, University and University Hospital of Zurich, Zurich, Switzerland. 3Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia. e-mail: Lab Animal | Volume 53 | August 2024 | 205–214 205 Article https://doi.org/10.1038/s41684-024-01409-z a c b Training d2 d3 d4 MOE 75 50 25 0 Training TAM treatment d1 – d2 – d3 – – d4 100 75 50 25 0 d1 – d2 – d3 – e SOE Percentage of mice 100 Percentage of mice Percentage of mice Oil – d4 120 100 Consumption time (seconds per 80 µl) d1 d 1× TAM 75 50 25 0 d1 – d2 – d3 – – d4 MOE 100 SOE 80 60 40 20 0 d1 d2 d3 d1 d2 d3 Training (days) Fig. 1 | Sweetened oil emulsions and training improve voluntary drug consumption. a, Adult male and female mice were trained for 3 days to consume 80 μl of formulations from a disposable plastic micropipette tip and on the fourth day were offered the same formulation containing TAM. b, Feeding time was recorded and considered voluntary if the mouse, held only slightly by the proximal tail section, drank the offered volume in less than 60 s. c, Any formulation not consumed after 60 s was offered again after gently restraining the mice by the scruff, and the extra time required to drink the remaining solution was recorded. d, The fraction of mice that voluntarily consumed (<60 s) oil (n = 21), sweetened MOE (n = 52) or SOE (n = 33) during 3 days of training (green bars) or TAM-containing formulations on the fourth day. Yellow bar, OIL-TAM (n = 12); blue bar, MOE-TAM (n = 17); orange bar, SOE-TAM (n = 17). The oil used for the formulations in d was sterilized by heat treatment. e, Total consumption time for three training days, recorded from the moment the MOE or SOE formulation was offered (n = 13 per group). Indicated is the time before restraining (white zone) as described in b and after restraining (red zone) as described in c. The oil used for the emulsions in e was sterilized by filtration. Minimum and maximum values, interquartile range and median are depicted as Tukey box plots with individual data points shown as gray circles. in gene expression in CreERT1/T2–loxP transgenic mouse models11–14. The number of different CreERT1 and CreERT2 mouse strains generated for research exceeded 1,400 by May 2023, of which more than 200 new strains were generated since May 2021 (refs. 15–17). Despite its routine use in research, there is no consensus on the best method for TAM delivery11. In most cases, TAM is administered via OG or IP injections but also occasionally by subcutaneous injections or medicated diets18,19. While injections allow controlled dosing and timed treatments, the oil- or ethanol-based vehicles required to dissolve TAM can cause local adverse reactions at injection sites10,11,20. Oral administration is more physiologically relevant for TAM because TAM first needs to be metabolized by the liver into the biologically active metabolites 4-hydroxytamoxifen and endoxifen21,22. However, of the existing oral admini (...truncated)