(M1130-03-20) The Role of MicroCoat™ in Refining Tamoxifen Administration to Mice and Achieving Inducible Cre Recombinase

Purpose: Tamoxifen citrate (TAM) is widely used in animal research to induce Cre recombinase activity and achieve conditional gene knockouts [1]. The commonly used dosing methods are oral gavage or intraperitoneal injection [2], which require specialist staff training and can cause pain, distress and adverse effects to the animal. Tamoxifen-containing rodent chow is commercially available; however, the poor palatability of the diet leads to reduced food intake and weight loss of the mice. The addition of sweeteners to improve palatability is usual but this can affect the metabolic balance of the mice. The aim of this project is to develop palatable TAM-containing micropellets by applying a proprietary MicroCoat^TM technology. The micropellets should effectively mask the taste of TAM without sacrificing gastric release and cre-recombination.

Methods: Micronized TAM (D₅₀ ~ 5µm) was spray layered onto microcrystalline cellulose substrates (MCC, Cellets® 100) to achieve 5-10% drug loading using an aqueous suspension containing talc, hypromellose and optionally, mannitol in a modified bench top fluidized bed coater (Mini-Glatt®). TAM-loaded micropellets were taste mask coated using an aqueous dispersion of ethyl cellulose (Surelease®) and hypromellose (Methocel® E5) as polymer and pore former, respectively. A range of pore former concentrations (15-25% w/w) and coating levels (5-10%) were investigated. The novel MicroCoat^TM technology was applied during taste mask coating whereby powdered magnesium stearate was added as glidant into the coating chamber via an external feeding port. Coating yield was defined as the mass of non-agglomerated particles < 250µm checked by sieve analysis. TAM release (dissolution) from coated micropellets was measured in 1000mL pH 3 HCl (mimicking the stomach pH of mice) using USP II apparatus with a paddle speed of 50rpm at 37°C (n=3). Drug concentration was determined using UV spectrophotometer at 275nm wavelength. Taste masking was determined using a “dispersal” in vitro method whereby the micropellets were dispersed in 7mL of water and TAM release was measured within 30s after 6 inversions of the vial (n=3). The TAM-containing rodent feed was obtained by manually mixing 14mg of taste masked micropellets with 35g of RM3 powder rodent diet (400mg TAM/kg diet) sufficient for feeding 5 mice in one cage) for 2-8min. For in-vivo studies, B6;129S4-Gt(ROSA)26Sortm1Sor/H: het; B6.Cg-Tg(UBC-cre.EsR1)1Ejb/J:het mice were housed into pairs (n=16) and each pair of mice were given 17.5g of RM3 powder diet containing taste masked micropellets at the final concentration of 400mg TAM/kg diet. As a negative control, untreated mice were used (RM3). As positive controls, mice were treated with uncoated TAM micropellets (Tam citrate) or 5 doses of 200mg/kg TAM via oral gavage (oral gavage). Two days prior to initiation of dosing, mice were habituated to powdered diet. Fresh TAM-diet mixture was prepared daily, and mice were dosed for 5 days. To monitor diet consumption, body weight was measured daily from two days prior to dosing until 10 days post dosing. To determine the level of recombination caused by TAM in diet, mice were culled two weeks after the end of dosing period and their tissue (olfactory bulb, cerebellum, liver and testies/overies) were analysed via ddPCR.

Results: TAM was successfully layered onto MCC cores and taste mask coated by applying the MicroCoat^TM technology, with no particle agglomeration and yield for all coating trials >99%. Faster drug release was observed from taste masked micropellets with high pore former concentrations and/or low coating levels. Two formulations were taken forward for in vivo evaluation, F1: 10% drug loaded (no mannitol), 5% taste mask coated, pore former ratio 75:25 and F2: 5% drug loaded (with mannitol), 10% taste mask coated, pore former ratio 85:15. They both showed effective taste masking and acceptable drug release in vitro (Table 1). The in vivo results show comparable weight change between F2 and the negative control (RM3) whereas F1 showed comparable weight change to the positive control (Tam citrate) (Figure 1). Figure 2 shows that the recombination efficiencies vary substantially between different tissues. In liver and testis tissue, there is evidence that suggest that treatment with Tam citrate, F1 and F2 caused recombination that was comparable to the positive control. However, this was not the case in cerebellum, olfactory bulb or in ovary tissue. The evidence for recombination in Cerebellum and Olfactory bulbs is present in the positive controls (Oral gavage).

Conclusion: Taste masking coated TAM micropellets were successfully manufactured by applying the MicroCoat^TM technology with >99% yield. The coating provided effective protection to prevent TAM release in the mouth but immediate drug release in the stomach pH conditions of the mice and effective cre-recombination comparable to oral gavage. The product is flexible in dose adjustment and improves tamoxifen handling safety in animal units, offering an innovative approach of dosing TAM to mice for Cre recombination research via voluntary food intake. The method has the potential to reduce suffering and improve welfare of the mice, promoting 3Rs (replacement, reduction and refinement) in animal research.

References: 1. Kim, H., Kim, M., Im, S. and Fang, S., Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes. Laboratory Animal Research, 34(4), p.147, (2018)
2. Andersson, K., Winer, L., Mørk, H., Molkentin, J. and Jaisser, F., Tamoxifen administration routes and dosage for inducible Cre-mediated gene disruption in mouse hearts. Transgenic Research, 19(4), 715-725, (2009)

Acknowledgements: The project is funded by the United Kingdom National Centre for the Replacement, Refinement and Reduction of Animals in Research (the NC3Rs) through the CRACK IT challenge – Tat-Fit (project number NC/C020S02/1).

Table 1. In vitro performance of F1 and F2 formulations (later used for in vivo testing).

Figure 1: The average body weight of the mice undergoing TAM treatment. F1 and F2 are taste mask coated micropellets. Sample 3 is uncoated tamoxifen citrate and RM3 is diet only control.

Figure 2: ddPCR analysis post TAM treatment in 5 different tissue (Cerebellum, Liver, Olfactory bulb, Testis and Ovary).