I am currently inducing knockout in my Cre-Flox mice. My i.p. injection of tamoxifen (Sigma) was dissolved in 1/10vol EtOH and 9/10 vol sunflower oil. However, now I want to do some experiments where I administer tamoxifen through drinking water, because I need to observe some effects during the process of administration and also need to administer TAM for a continuous period of time.
Since I know that tamoxifen is difficult to dissolve, is normal tamoxifen applicable in drinking water? (i found protocols where 100µg are dissolved in100µl to 1ml of EtOH and then diluted to an end concentration of 0.1 to 1 mg/ml in an end concentration of 0.05 to 1% EtOH), or do I need to use for example tamoxifen citrate (for which on the Sigma site no use for genetic recombination induction was specified)
I think you can add tamoxifen citrate instead of tamoxifen base to drinking water to overcome solubility problem. However, you should consider following issues:
1- First in a pilot study be sure that mice drink water containing desirable concentration of tamoxifen citrate
2- In obtaining desirable concentration of tamoxifen citrate adjust the concentration based on molecular weight of tamoxifen base
3- You should find evidence for oral absorption of tamoxifen in mice or measure its blood concentration to prove it
whether u want to give Tam/Tam Cit depends on your experimental requirement. Tam /TC do not dissolve in water. The powder/powdered pills have to be sonicated in water. U have to weigh the base/salt according to the dose u want to give and then orally feed with a feeding tube the volume according to the weight of the animal. As for proof of oral absorption, u need not to worry because TC is an oral pill.
"Since I know that tamoxifen is difficult to dissolve, is normal tamoxifen applicable in drinking water? (i found protocols where 100µg are dissolved in100µl to 1ml of EtOH and then diluted to an end concentration of 0.1 to 1 mg/ml in an end concentration of 0.05 to 1% EtOH), or do I need to use for example tamoxifen citrate (for which on the Sigma site no use for genetic recombination induction was specified)"
Karin, pure Tam is used for in vitro studies and this protocol for solubilizing it is fine. U can also use any alternative solvent which will solubilize Tam and is miscible with an aqueous medium and essentially is not harmful to cultured cells/tissues. Otherwise the drug will precipitate out of the aqueous medium /cells may die in either case the experiment is bound to fail. The problem is the same for all compounds/drugs that are insoluble in any aqueous medium.
Karin, once Tam/TC enters the biosystem it is metabolized to so many metabolites and that too in a species specific manner. Each of these metabolites has its own effects ranging form more to less to ineffective. All these metabolites can occupy the same receptors and compete for this site in vivo. There are very few chances of the parent drug remaining as Tam molecule for long. Once Tam enters the system it is not excreted and keeps circulating via the hepatic portal circulation and is concentrated particularly in the gall bladder. I dont know if these facts have any bearing to your experiment but u certainly discuss with your mentors. U can read about Tam in the earlier papers of Dr Jordan.
Thank you a lot, Hossein and Manjeet, I am at the moment discussing with my collegues and supervisor and we are considering the use of Tamoxifen diets or to find a way to repeatedly use i.p. injections.
OK. Still, I would want u to keep in mind that Tam is a SERM as it has dual estrogenic as well as antiestrogenic properties thanks to its hepatic metabolites. For your experiments estrogenic metabolite would be more useful because it will turn on the ERE on the DNA wheras an antiestrogen/metabolite will block it. If you could search for the estrogenic metabolite in mice and buy it/get it synthesized whichever, it would be more fruitful to use it instead of/in parallel for your knockout study. It is essential to transcribe the relevant proteins which only estrogenic metabolite can achieve. U will also be avoiding competition from several circulating molecules which,depending upon their serum conc and affinities, will occupy the ER. Good luck Karin
I have the same question / problem, but I was thinking of using tamoxifen pellets implanted under the skin. But also here: Should I use citrate or the base? Thank you!!
Mathias, if tamoxifen citrate is available in the tablet form. Is pure tamoxifen too available in tablet form? If pure tam is not available in tablet form than you will have to go for powder inplants. In any case, carry out a comparative study in 2 animals and monitor the effect on serum LH, FSH, Prl, estradiol and in particular TESTOSTERONE hormone profile as end point. Then you can decide after comparing their efficacity.
is there any reference for oral administration for mice? I only found injection references.
JRF 99:395-402 (1993). Effect of tamoxifen on the fertility of male rats.