Hi,

There are two points to consider here: 1. toxicity of clodronate; 2. toxicity of liposomes.

Lets take these factors one by one.

1. There are literature claiming that Clodronate is not a toxic drug (e.g. see:

Immediate mineralocorticoid receptor blockade improves myocardial infarct healing by modulation of the inflammatory response

D Fraccarollo, P Galuppo, S Schraut, S Kneitz… - …, 2008 - Am Heart Assoc

and:

Macrophage depletion by clodronate liposomes suppresses neointimal formation after carotid artery injury in apolipoprotein e-deficient mice

SJ Hong, TH Ahn, WJ Shim, SM Park… - Korean Circulation …, 2008 - synapse.koreamed.org

and:

http://www.clodronateliposomes.org/ashwindigital.asp?docname=mechanism ).

2. Liposome toxicity:

There are reports that cationic vesicles show some cytotoxicity whereas anionic vesicles prepared without using toxic solvents are completely safe and non-toxic (e.g. see:

Cytotoxicity evaluation of anionic nanoliposomes and nanolipoplexes prepared by the heating method without employing volatile solvents and detergents

MR Mozafari, CJ Reed… - Die Pharmazie-An …, 2007 - ingentaconnect.com). Therefore, if you use natural ingredients in your liposome formulation and avoid using toxic solvents or detergents in the preparation method, your liposomes will be non-toxic. Let me know if you need more clarification or references.

Thanks for the response and info!

We order our reagent from clodronateliposomes.org. Multiple groups have published repeated treatment with 200ul for a total of 5-10 treatments spaced 72hrs apart. However, when I inject 100ul, administer the second treatment 72hrs, the mice dispaly high amounts of toxicity within a day or two, not immediately after. Gross pathological evaluation shows no signs of infection.

Given that the reagent is prepared and acquired from the same source, do you have any ideas what I might be doing on my end that would result in toxicity issues that other groups have either not encountered, or found a solution.

Thanks!

Todd

Are you running control groups in parallel of empty liposomes?  The website says they are composed of Phosphatidylcholine and chol. So they are non-toxic but if you do see toxicity it could be your technique.

Are you using an animal strain that has been used before with these liposomes?

We are having the same problem with CLs from the same source. A previous batch (november 2016) gave no tox issues and worked fine, but after that, we've tried two batches which both have produced severe toxicity 2-5 days after a single i.p. injection (both with 100uL and 200uL volumes). No issues with the control liposomes. Contacting the company has not yielded any new hints as to what could be the issue. We are therefore looking into alternative sources of CLs. If you find a solution I'd be grateful if you send me an update.

Hi, just wondering whether either of you have made any progress with these issues or found solutions? We recently started using clodronate liposomes from the same source. Initial tests with a single injection of 200 ul iv were well-tolerated by the mice, gave very effective depletion of Kupffer cells from liver and also reduced spleen macrophages as expected. However, most mice receiving 3 doses of 200 ul 6 days apart lost a significant amount of weight, and some required euthanasia. At necropsy, there was a consistent finding of pale spleens, occasionally with a nodular appearance. Histology showed a dramatic reduction of cellularity in the white pulp and a replacement of much of the red pulp by amorphous pink material, consistent with widespread splenic necrosis. Has anyone else seen anything like this?

We have continued to struggle. We have always done IP injections. We tried two sources, one that isn’t as potent. We even tried 100ul of the less potent one and still have mice die. It’s been frustrating. We thought IV might have less toxicity, but it sounds like that route also has issues. We are going to test 100ul via RO to see if that makes a difference. I contact groups who published long term treatment and they claim no toxicity. But even the site we get ours from says to have extra mice due to toxicity. Please keep me updated if anyone makes progress

Hi, we are currently facing the same issue. We are using the same supplier as Todd A Triplett. We injected 200µl i.p. and 20% of the treated animals died (10% after the 1st injection, 10% after the 2nd injection). We used DiI-liposomes for a control group - all animals were fine. Thus toxicity is not due to the Liposomes but due to the clodornate.

The necropsy pointed towards a lung emphysema but was not clear.

We had this issue with two batches of Clodronate.

We didn't see anything abnormal within the spleen.

If there are any new information regarding your experiments or may be a solution for the problem, please share it.

Yea, it is weird though because other groups have injected strait clodronate as controls without issues (10.1208/s12248-013-9501-7), though they also don't report issues with ClodLip. I think most publications get toxicity, but they don't mention it.

We found that hematopoiesis was greatly affected in BM due to depletion of island MACs (0.1016/j.exphem.2014.03.009 ). However, we have found how fast the injection is greatly impacts toxicity and that slow is better, which doesn't quite fit with the anemia theory.

So it might be accumulating too quickly and causing organ failure. Respiratory seems to make the most sense. Did you evaluate pulmonary edema?

Anyone not have toxicity issues?! Please help.

After the experiment was finished, we send the body of one of the treated mice to an institute for pathology (this mouse survived the clodronate treatment but was in the same cohort like the deceased animal).

In the lung they found a low-grade, acute, diffuse, alveolar edema and emphysema. In addition they found multiple thrombi. This might be an indication for respiratory organ side effect.

It is somehow reassuring to read that we are not the only to experience toxicity with clodronate.

A month ago, we just started an experiment and here is what we did :

- Liposomes purchased from liposoma.

- We injected 0.2 mg of clodronate liposomes i.p. into 5 C57/bl6 mice, in 200 µL.

- We realized 2 injections per week during 4 weeks.

One mouse died after the second injection. Another one lost 6g (!!!!) so we sacrified her. A third one died after the fourth injection.

That means that 3 mice over 5 died during the experiment. Hopefully, that was just a trial experiment but now, we don't know if we wil do it again. That would mean starting with a lot of mice plus the fact that I will never be sure of the results (the living mice will maybe suffer a lot, which could affect the results).

Did you inject "pure" clodronate (5 mg/mL)?

I wonder whether anyone has still experienced problems, especially deaths or toxicity, more recently? We are planning on using the clodronate liposomes in the next few months. Company has now this link: https://clodronateliposomes.com/?v=79cba1185463

thanks

I am having an opposite problem with clodronate liposomes. It did not seem to really deplete any macrophages although 2 of my mice died 24 hours after the injection. Could there have been something wrong with my technique?