Intranasal administration

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I was wondering whether any one might be able to point me in the right direction of acceptable pH's for intranasal substance administration in hamsters - is pH 4.0 acceptable?

It will depend on the strength of the solution (1M compared to 1mMolar).

Why does it need to be pH 4, as within a few seconds the natural secretions and reactions will raise it to more neutral pH?  The vehicle will also be important as to whether it is buffered or not (I assume it will be in acetate/acetic acid buffer at the pH?) or whether it is some other form of vehicle (liposomes or emulsion).

This paper produced by the Joint Working Group on Refinement may help:

Morton, D.B., Jennings, M., Buckwell, A. Ewbank, R, Godfrey, C, Holgate B., Inglis I, James R, Page, C, Sharman, I, Verschoyle, R, Westall L and A.B. Wilson (2001) Refining procedures for the administration of substances. Third Report of the BVA-AWF/FRAME/RSPCA /UFAW Joint Working Group on Refinement.  Laboratory Animals 35: 1 – 41.

I guess that the volume of solution will be of consideration; a larger volume may present challenges in being neutralised by existing nasal secretions and there may be potential for iatrogenic damage to nasal mucosa. This could be a consideration from a pathological evaluation perspective. Is it possible to include control animals with carrier solution without the test item, which is at the same pH?

Bottom line: pH should be 4.5-6.5 to avoid damaging nasal mucosa, but the pKa of the drug molecule is important.

One of our research groups is planning an experiment on a Covid-19 infected mouse model. For this purpose, the animals will receive intranasal administration under anesthesia every day for almost a week. The constraints are not to use gaseous anaesthesia and to use the shortest anaesthesia possible.

I therefore opted for the use of medetomidine which has the advantage of being flexible in terms of depth of sedation and reversible with atipamezole.

The potential problems are :

- Will the depth of anesthesia be sufficient for effective intranasal administration without causing coughing?

- Is safety in animals with respiratory distress sufficient with this molecule?

Do you have any other ideas for protocols compatible with this type of model, procedure and constraints? Do some of you have any experience with the use of propofol IP (safety, duration of anesthesia...)?

Is there a scientific reason not to use volatiles? I ask because we developed a bell jar protocol for a very similar / identical design. As the animals were in isolators, we decided that on balance this would be the safest way to administer anaesthesia, the best way to prevent coughing, and the quickest recovery.

Regarding medetomidine, I think it’s unlikely you won’t get a cough response but is it possible (according to the PPL) to pilot it with vehicle first, e.g. do a couple of controls?

In previous experiments, they found a higher failure rate to reach the lower respiratory tract especially when comparing with a Xylazine / Ketamine protocol. Beyond that, they absolutely want to avoid using the same route of administration for the anesthetic and future products to be administered.

I don't think I have any flexibility on that side unfortunately...

Do you think that perhaps the administration of midazolam in addition to medetomidine could reduce the risk of coughing?

Sounds like a plan to stick to drug combinations that you can antagonise (alpha 2, benzodiazepines, opioids) because ideally you don’t want any time spent recovering from sedation and having that impact on appetite, locomotion etc.

The medetomidine/midazolam/butorphanol produces general anaesthesia, and reversing gives very fast recovery:

Dexmedetomidine + Midazolam + Butorphanol

0.15mg/kg + 4mg/kg + 5mg/kg

You could also try:

Propofol + Dexmedetomidine + Fentanyl

75mg/kg + 0.5mg/kg + 200ug/kg

(Reversed with atipamezole and butorphanol, the propofol just has to wear off)

Or

Alphaxalone + Dexmed + Butorphanol

(just the atipamezole as an antagonist)

Or

Fentanyl + Dexmedetomidine +Midazolam

50ug/kg + 0.25mg/kg + 5mg/kg ip

With reversal of all 3 components (butorphanol, atipamezole and butorphanol)

If you have medetomidine instead of dexmedetomidine, double the dexmed dose.

Was the intranasal application considered without any anaesthetic?

While working with X at Y we were either not anaesthetising mice or we did but for a very, very short period of time, mainly to immobilised them with Isofluorane. This was a routine procedure used by this group. No problem with infections and of course with the animals.

Yes you can give propofol ip - used by the inventor, at a well known pharmaceutical company many years ago - the snag with ip dosing is that getting a surgical plane is tricky, so the more reliable protocol adds medetomidine then reverses it - but for surgical anaesthesia, propofol/med/fentanyl  - see Alves HC, Valentim AM, Olsson IAS & Antunes LM (2009): Intraperitoneal anaesthesia with propofol, medetomidine and fentanyl in mice.

Propofol/med may be enough for what you aim to do, but its trick to abolish swallowing and coughing without getting close to a surgical plane of anaesthesia with most mixes.

Small update on what you could do in this procedure :

- Indeed, Methomidine alone is not enough

- Medetomidine (0.15mg/kg) + Midazolam (4mg/kg) + Butorphanol (5mg/kg) produced a rapid and sufficient anaesthesia for the procedure, but falling sleep was extremely long (more than 30 min before the first signs of awakening) despite the injection of 1 mg/kg of Atipamezole.

- Propofol (75mg/kg) + Medetomidine (1mg/kg) + Fentanyl (200ug/kg) produced a slower but sufficient anesthesia for the procedure. With the injection of Butorphanol (2mg/kg) and Atipamezole (1mg/kg) the awakening was faster than with the classic Xylazine/Ketamine.

I’m pleased that one of the protocols works. Given the wide strain and sex variation that occurs with all injectable anaesthetics, it may well be that dropping the dose of P/M/F would still be effective. Personally, I would try for a reduction in the propofol dose and leave the medetomiding and fentanyl as is, since those two components are reversible. The reason for me always recommending butorphanol (or nalbuphine) is that if you do surgery with this mix (or the MMF mix), then using naloxone gets rid of all the analgesia from the fentanyl, whereas butorphanol provides some short acting analgesia - in your case it doesn’t matter but I suspect you will also find that naloxone is more expensive and more difficult to obtain rapidly. Onset of reversal with the butorphanol or nalbuphine is just as rapid as with naloxone.