Hi Theodore,

It depends on many things but in your case I would think about an hour. When we inject paracetamol into rodents we notice the effect by 30min. However, it would be best to check the plasma levels, which should not be too difficult to do.

It would be difficult to notice any changes in plasma levels as Dex or Annexin will be injected pre-treatment and then I will administer the stimulant. In your experiments, do u mean you saw an effect of paracetamol at 30min after delivery? Did u give it to the animals pre- or post- stimulation?

Hi Theodore,

I Igree with Chukwuemeka Nworu about absorbition in peritoneal area.

About my experience I inject 18F-FDG IP in rats and in 2 minute i can start to have radioactivity countin in the brain, but of course it takse around more or less 15 minute total absorbition of 0.6/0,8 ml .. in your case you have mice so more then 0,2 ml you should not inject .. and absorbition of your drugs depend also about dilution with saline.. because .. some times i use 18F FDG concentrate without dilution and rimain there in peritoneal area ... to stimulate vessel absorbition i advice to you to use total volume of 0.2 in mice, massage the area after injection, and put the animal in stimulate environmet to stimulate the movement and consequent absorbition

goodluck

Ketamine enters the blood stream via absorption probably between 10-15 minutes. However, as stated above, it depends on the drug. Smaller molecules would be absorbed more quickly than larger molecules. From experience, IP injection of antibodies (for example anti-platelet) leads to acute thrombocytopenia within 2 hours. I think one of the key factors is to deliver the drug in the smallest volume possible without jeopardizing accuracy. Remember there will be some diluting in the peritoneal cavity because of the peritoneal fluid.

As others have stated above, it depends not only on the drug and vehicle, but volume and frequency of administration. Is this going to be a single-dose, short-term acute or longer term treatment protocol? I'm mostly curious because Dex is a steroid, and lipophilic, and I'm wondering if it may partition to and remain longer in the fat pad than more hydrophilic compounds (especially following repeated doing). For example, when we inject 1g/kg ethyl carbamate in saline in 0.1mL i.p., we know that it has reached the bloodstream (and brain) within 1-2 minutes or so, because the anesthetic activity of that drug causes the animals to fall asleep. This is one example of a very small, very water-soluble compound. Your question pertains to not only a steroid, but a protein agent as well. As others have suggested, if the PK information regarding i.p. bioavailability of your compounds of interest are not generally available in the literature, the best way to know that your agents are in the blood is to measure plasma levels in a pilot study with a few animals. Then you will know how long it takes to get a X concentration in plasma, with X being the minimal concentration that you will accept for agent activity. You could also determine the time-to-peak and trough levels, and AUC, to find out what your therapeutic window will be. Many "inflammatory" stimulators work in phase, with immediate and delayed effects- will Dex affect both? Either? Will your planned experimental duration extend long enough to measure durable effects?

The model we will use is an acute inflammation murine model with a terminal point of 2 hours after stimulant delivery. Dex or Annexin will serve as positive anti-inflammatory controls and will have to be added before the stimulant to repress inflammation as far as possible.

We have not decided yet on the diluent yet but it will probably ethanol for Dex or PBS for Annexin.

TheodoreKapellos

if you use Ethanol as diluent, pay attention, itravenusly, in rats you cant inject more then 10%, maybe by IP you can go bit more

The reason I want it to reach the bloodstream is because the inflammation markers I want to study the expression of are blood-bourne. Therefore, I want the drug to be in the circulation so that subsequent stimulant will not induce the markers overexpression in the blood as much as in non drug-treated mice.

Theodore: The bioavailability (F) of a drug that is injected I.P is particularly considered as close to 1.0 as you can (the F=1 is that of i.v drug administration). I.P provide a vast absorption area for the drug and fast availability compared with other routes such as P.O or SC. but you also will have with some drugs that are metabolized by the liver (such as Dexa) a first pass problem where the drug after absorption will go through the portal vein and undergo liver metabolism, which will make the proportion of the drug that reach the blood stream (or final target) lower. Off course if you want to ensure fast delivery and all the drug in the blood you can try the tail vein injection for mice or rats (more challenging for mice)

Tail vein injection into mice are not as easy. Will need lots of practice!!! I personally hate doing it while I do not have problem with ip injection. So if you can avoid it, do not do tail vein.

Theodore, we did not stimulate the immune system, I was just stating how long it takes for the paracetamol to be absorbed.

I would do a pilot study to ensure that your experimental set up is sound. If you carefully plan your experiment, by measuring inflammatory markers you can get an idea of what is happening. So if you inject your drugs at different time points, and then administer inflammatory stimulators and measure inflammatory markers, you would know what is happening. Also have animals just with the stimulators.

Cheers

Ethanol as a solvent will have an effect on the immune response. There are a number of papers that look specifically at what ethanol does to immune responses in vitro and in vivo. If you are using ethanol as your solvent, you will need to run an "ethanol only" control group. You might want to do a pilot study comparing PBS (or saline) vs. ethanol.

Can this paper help?

The anti-inflammatory potency of dexamethasone is determined by the route of application in vivo.

Weichhart T, Brandt O, Lassnig C, Müller M, Hörl WH, Stingl G, Säemann MD.

Immunol Lett. 2010 Mar 10;129(1):50-2. doi: 10.1016/j.imlet.2009.12.025. Epub 2010 Jan 12

I agree with Peter as well. Do take into account how ethanol or any other solvent will affect your results in general, the immune response and whatever else you want to measure later on (ie expression levels of certain things).

I dont work with any of the drugs you are using. But you should consider whether the drug requires bioactivation and if so, which route would help you best achieve this. You might also have to do preliminary dose testing to ensure your steady plasma levels are within the range you want.

A good relatively recent reference is attached. Tail vein injections are much more reliable than IP in my experience. I never had the nerve to do the retro orbital injection, but you can find references and video how-to's online. The key to master tail vein injections is less practice than finding a competent instructor. I didn't have much luck until I hired a technician who had learned it from someone in his past lab - he taught me. You need to warm the mice (not too warm!). If no one in your lab can do it, try your animal facility or put out a request in your organization - nearby vet school?

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3189662/

I agree with all of the above. If I want to guarantee the drug enters the bloodstream rapidly then I go I.V. via tail vein. This is not a procedure for those who haven't done this before though as it really does take practice and some just can't manage it (particularly in pigmented mice). As regards ip injections, when I inject cannabinoids I see effects after 10-15 minutes as a general rule. I don't see a problem with dexamethasone but wonder whether annexin will gain entry to the bloodstream (I'd go iv with this).

My experience with i.v. injection in mice is, that is is usually not that easy for most scientists or even rather difficult and needs lots of experience - especially compared with i.p. injections. For the beginner, many things can go wrong - the vein gets easily damaged and the incection can go anywhere but the vein - leading to unwanted results. I remember it sometimes worked surprisingly good with having a heating lamp, or ethanol (isopropanol ?) on the vein for a few minutes.

But do you really need to have that short time of only 5-10 min. What would happen, if you wait just longer, for example, 1 hour after i.p. injection - is there a risk of having too low levels then? I understand the "working" concentration should be achieved in the tissue of interest, but is it in your case really (only) the blood? This may differ a lot from tissue to tissue (brain vs. liver vs. joints).

I think 5 min is enough. I did sodium fluorescein i.p. injection. This dye circulates into retinal blood stream less than 5min.

I really do not know how fast Dexamethasone or other drugs may need to reach their full pharmacological potential, but I could imagine that even a satisfactory blood level of dexamethasone after a few minutes may not reflect blockig of all pathways in all cells and tissues. I also could Imagine that just your two windows of 5 and 10 minutes could lead to different results. I could be wrong, but would check how fast it gets into the cells (and their nucleus) of interest - and then interfere with genes. Blood levels may be important for drug-monitoring AFTER the working level is reached.

Some of the anti inflammatory responses of corticosteroids depend on the effect on intranuclear transcription pathways. This could take longer than the rapid maximal bloodstream concentration. A recent study of the time course of such effects in humans called "Prednisone affects inflammation, glucose tolerance, and bone turnover within hours of treatment in healthy individuals" was published in European Journal of Endocrinology at: http://www.eje-online.org/content/166/3/459.full

The timing of administration of cytokines and inflammatory mediators could possibly change the observed clinical and biomarker responses.

Peritoneal transport is highly variable across species and from individual to individual, and can be affected by infection, inflammation and other factors, such as fibrosis. Drug transport will also be affected by molecular weight but also lipohilicity and active transport mechanism. So it is often hard to predict. We measured urea (MW 60), creatinine (113) and cyanocobalamin (1355) in peritoneal dialysis patients and equilibration with serum levels of all three taking between 2 hours (urea) to 4 hours (cyano) in one individual and reaching 90% equilibration at 3 hours for urea and only about 15% for cyano at 3 hours in another (Randerson & Farrell. ASAIO J 3:140, 1980). Count on dexamethasone taking a few hours and annexin unlikely in 24 hours, unless by other than passive diffusion. I'd go tail vein if you want rapid and reproducible results.

Keep the route of administration as i.p and try different intervals [5, 10 and 15 mts] and choose the best response. lalitha kabilan

Theodore, could you give some technical feedback (without telling us any critical or secret results) - could you use any of the suggestions from here?

We proceeded with i.p. injections which worked quite well for us. We also administered some animals with Dex i.p. which was effective when given as a 30min pre-treatment. We now consider the sc route for administration of our drugs but I would be a little reluctant because of the discussion here and the fact that we would use different routes of administration for our stimulant and drug (raises additional parameters in an already complex system).

I do not want to use oral administrations at all because of technical difficulties and scientific caveats (e.g. pH in the stomach, degradation till absorption by the circulation, variability because of the microbiota in each lab s animals' gut.