Tamoxifen information sheet V4.pdf
Refinement of the administration of tamoxifen to mice
Tamoxifen is a selective oestrogen response modifier, protein kinase C inhibitor and anti- angiogenic factor. Tamoxifen is metabolised to active metabolites 4-OHT and endoxifen by cytochrome P450 isoforms CYPD6 and CYP3A4.
Conditional mutations in mice: Used to activate the ERT2-Cre recombinase transgene in vivo – gene of interest is‘floxed’ out. Both tamoxifen and 4-OH tamoxifen are used.
Tamoxifen also acts on oestrogen receptors and is toxic, including to reproductive organs and stomach lining. Itcauses sterility and late abortions.
Biological activity applies also to humans! Appropriate working practices and PPE required to prevent accidental exposure of staff to tamoxifen at all stages of preparation and use.
Precautions should also extend to disposal of bedding, as it’s likely to be contaminated with the
Form of tamoxifen:
Available as citrate and basic salts. Metabolite of tamoxifen (4-OH tamoxifen) is also an active form and has ashorter half-life in plasma. It’s also even harder to dissolve!
Tamoxifen needs to be at the tissue at appropriate concentration and at correct time to induce mutation. Note that if theconditional mutation has deleterious effect on the mouse, then care must be taken with dose and duration of tamoxifen.Dose rates quoted in the literature for gene activation (e.g., up to 100mg/kg) are far higher than those used therapeutically in humans (0.4mg/kg). Note that this high dose may not actually be necessary (both therapeutic and research use requires tamoxifen to act on cell receptors) and will increase the toxicity and adverse effects on the mouse. Cumulativeeffect in tissues on repeat dosing.
Consider what organ is being targeted – e.g., higher doses likely to be required to penetrate say, brain rather thanliver to active the gene.
Recommendation: Carry out pilot studies with lower doses to investigate effectiveness at doses closer totherapeutic levels, as literature doses are extremely high. Check the form of tamoxifen to be used.
Methods of administration: (in order of suggested/ most refined use)
This is the recommended route and should be tried before other routes are attempted.
• Giving tamoxifen in feed reduces peak / variation in plasma levels, as long as mouse is eating well.
• Adverse effects are fewer with this route (e.g. weight loss, nausea).
• Advantages of reduced handling and no restraint/ needlesticks
• Diet is available with tamoxifen pre-mixed in. Needs to be kept in freezer, due to spoilage.
• Best to feed small amounts (what a mouse will eat in 1-2 days) then remove old feed and top up onalternate days as the diet goes rancid quickly at room temperature.
• Palatability: Adding banana milkshake (5 drops on a pellet) improves palatability and helps encourage mouse to eat. [Some sources suggest adding sucrose but our experience is that this isn’t helpful andthere are concerns about dental/metabolic
effects of sugar]. Older mice often more reluctant to eat than older ones. Can be strain specific too.
• Expected weight loss ≤10% with this method.
• By oral pipette or gavage.
• Peak plasma concentration achieved after 3-6 hours following oral dose but depends on solvent used andwhether stomach is empty when dosed. Plasma half-life approx. 12 hours in mice.
• Our experience is more problems with adverse effects (anorexia, gut stasis).
• Also requires restraint; risks associated with OG.
• Possible to dose neonatal pups: use <10mcL and place in mouth by oral pipette to allow mouse to swallow, rather than OG as such. (pups age 5-10 days)
• If repeat OG is proposed, then better to use in-feed route.
• Often in oil – depot effect; effect is detectable for weeks after injection
• Mixture is viscous and may require slight warming to inject and needle size up to 23 gauge.
• Deliver injections at body temperature.
• Usually tamoxifen dissolved in alcohol / oil mixture.
• Higher risk of adverse effects with this route: poor welfare and loss of animals possible. May result in peritonitis; gut damage due to ‘jetting’ of viscous liquid into abdomen via needle; oil itself is a foreign material and has inflammatory effects; also may precipitate in peritoneal region (sterile peritonitis); , general risksassociated with ip injection (penetration of organ, mis-directed injection). Can lead to adhesions.
• Recommend single injection only (if other routes not possible)
• Must use newly prepared sterile solution every time (not previously opened)
• Some groups wish to use tamoxifen in pregnant dams – extreme caution required.
• Literature suggests tamoxifen detectable in plasma around 12 hours
• Variability between operators in adverse effects following ip injection – recommend injection done by technicians and that another operator restrains the mouse to ensure accurate direction of needle.
Experience with this is limited (one group bought pellets from USA and implanted) but it resulted in high incidence of wound breakdown. Release of tamoxifen deemed less reliable.
Making up tamoxifen:
Drug is poorly soluble – usually need to dissolve in alcohol then dilute into vegetable oil before administration byparenteral routes.
Note various forms available: tamoxifen or OH-tamoxifen; basic or citrate salts. (Citrate salt suggested for use in water –dissolved in ethanol first, although our recommendation is not to medicate via water in case of palatability problems).Ensure that correct form is purchased.
All drugs should be pharmaceutical quality (not “reagent” quality) as the former is a higher
standard of purity and quality.
Various “recipes” available eg dissolve 100 mg/ml in 100% ethanol, vortex until completely dissolved, then dilute to afinal concentration of 10 mg/ml into sterile vegetable oil; OR 20mg/mL dissolved into corn oil. Needs to be sonicated and may also require warming to 60C in order to dissolve. Note some mouse strains do not tolerate ethanol, so needto consider this when formulating for administration.
Essential that oil is vegetable origin, pharmaceutical quality (not from Tescos’s!) and sterile filtered before useto make up tamoxifen. Many recipes suggest corn oil but probably more because they originate in USA than anyparticular scientific reason.
Must be kept in the dark, as ingredients degrade rapidly on exposure to heat and light. Can be stored frozen in aliquots (glass bottles) at -20C for up to 4 weeks. Important to reduce air gap in bottle as much as possible to prevent oxidation. Discard once used and do not refreeze thawed mixture.
Allow mixture to stand at room temperature before use, to ensure that it remains in solution and does not precipitate. Precipitation will cause significant adverse effects, including pain, on injection. Any mixture containing clumps should be re-sonicated or discarded.
• As per routes above, including peritonitis, swelling/ infection at site of injection.
• Anorexia, body condition loss and weight loss. Typically around 10% loss but can be up to 20%. Theseeffects are common, and mouse is likely to need additional husbandry support during dosing (see below).
• Gastric stasis/ blockage/ following OG dosing.
• General effects: hunched, piloerection, lethargic.
• Swelling of reproductive organs (e.g. testicles in males). Can be quite long-lasting.
Supporting the mouse
• Consider age, strain and genotype of mouse to be dosed. Young mice often worse affected
• Consider dose rate carefully. Recommend no more than 2mg/ adult mouse.
• Ensure frequent observation and recording of condition, bodyweight, condition, etc
• Consider providing high energy foods, such as condensed milk, transwean, gel or mash if tamoxifen givenby injection
• Provide additional environmental enrichment for nesting to keep mice warm.
• Ensure technical competence of staff carrying out injection
• Carry out pilot study with new dose or gene before dosing all mice.
PPL and other considerations
Dosing generally possible on ‘moderate’ protocol and adverse effects controllable within this framework. Somelicences quote up to 25% weight loss and 20% common on protocols but should aim for well below this.
Quoted from one PPL (most wording similar) for oral gavage dosing -
“Adverse effects of tamoxifen include dose-dependent transient weight loss (typically by 15% and rarely by approximately 20%), which recovers over time. The animals may show signs of general malaise, such as hunched posture, piloerection, inappetence, reduced mobility, unsteady gait and reduced general activity.
Typically these are transient and animals improve over time. Animals displaying unexpected adverse effects of gavage or the signs of general malaise (hunched posture, piloerection, inappetence, reduced mobility) and the loss ofbody weight for >20%, which do not stabilise within 24 h will be killed by a Schedule 1 method. If the animals only experience weight loss up to approximately 20%, and show no obvious signs of malaise, they will be monitored closely to ensure that the weight increases within a few days, while highly palatable food will be provided within easy access throughout the period of gavage treatment.
When a combination of adverse effects, and/or moderate weight loss, is seen, we would seek advice from NVS/NACWO as to what measures should be implemented to monitor and support the animals.”