ProfK is setting me up


ProfK went mouse fishing following one of the comments. He threw me a carrot, to see if I would bite and comment on this paper .

Jørgensen LØ, Hyrlov KH, Elkjaer ML, Weber AB, Pedersen AE, Svenningsen ÅF, Illes Z. C2020 Sep;201(3):328.

Cladribine (CdA), an oral prodrug approved for the treatment of relapsing multiple sclerosis, selectively depletes lymphocytes. CdA passes the blood-brain barrier, suggesting a potential effect on central nervous system (CNS) resident cells. We examined if CdA modifies the phenotype and function of naive and activated primary mouse microglia, when applied in the concentrations 0·1-1 μM that putatively overlap human cerebrospinal fluid (CSF) concentrations. Primary microglia cultures without stimulation or in the presence of proinflammatory lipopolysaccharide (LPS) or anti-inflammatory interleukin (IL)-4 were treated with different concentrations of CdA for 24 h. Viability was assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Phagocytotic ability and morphology were examined by flow cytometry and random migration using IncuCyte Zoom and TrackMate. Change in gene expression was examined by quantitative polymerase chain reaction (qPCR) and protein secretion by Meso Scale Discovery. We found that LPS and IL-4 up-regulated deoxycytidine kinase (DCK) expression. Only activated microglia were affected by CdA, and this was unrelated to viability. CdA 0·1-1 μM significantly reduced granularity, phagocytotic ability and random migration of activated microglia. CdA 10 μM increased the IL-4-induced gene expression of arginase 1 (Arg1) and LPS-induced expression of IL-1β, tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS) and Arg1, but protein secretion remained unaffected. CdA 10 μM potentiated the increased expression of anti-inflammatory TNF receptor 2 (TNF-R2) but not TNF-R1 induced by LPS. This suggests that microglia acquire a less activated phenotype when treated with 0·1-1 μM CdA that putatively overlaps human CSF concentrations. This may be related to the up-regulated gene expression of DCK upon activation, and suggests a potential alternative mechanism of CdA with direct effect on CNS resident cells.

The blood concentration of cladribine after is about 80ng/ml maximum (Hermans et al. 2018). 1M = 286g/Litre = 286mg/ml. 1mM= 286microg/ml 1 micro molar = 286ng/ml, so 80mg = about 0.3 micro molar. As brain levels are about 25% the blood levels suggest a maximum concentration of less than 0.1micro molar.

On first view you say great news for controlling progressive MS with cladribine. This may be the case but…..

It casts my mind back to when beta interferon was discovered and the EAEers couldnt wait and soon we had human beta interferon inhibiting the mousers…..What do I think of that?

Answer: A pile of pants…why?…human beta interferon does not react with the mouse beta interferon receptor. So the results were artefact.

Clofarabine - Wikipedia
Cladribine - Wikipedia

Fast forward a few years and we were asked to study with chlofarabine and it did nothing to our surprise. This is cladribine with a fluorine on it, which is a trick to make it hang around in the body for longer. Cladribine is deoxyadenosine with a chlorine on it a trick to stop it beiing degraded and guess what?…..It does not work in mice!…We found out the hard way back in the early noughties.

So when I see cladribine on mouse and get asked a response is “Next Question!”.

Here is the one reason why it does not work in mice and it is because cladribine needs a protein call deoxycytotine kinase to enact its killing mechanisms.

You can see the gene expression below

As you can see mice do not have it in their T and B cells. They also do not have much adenosine diaminase. By contrast humans have loads of it notably on B cells and this is why the B cells get killed with cladribine in MS. It also looks like marmosets dont have a functional killing system either so it is not worth doing cladribine studies in animals

However, fear not it has been done and they claim it works in EAE….

Em………Should we believe their other stuff or put them on the EAE-dreamer list…where the results are typically made out of fanasy. Surprisingly this group say “Cladribine failed to interfere with EAE-induced microglial and astroglial activation…”

However macrophages do express deoxycytidine kinases and so maybe there is an inhibitory effect on microglial activity. We say Great but does the data pass the “Smack you in the eye” test often no effect at 0.01micromolar and 1omicromolar …. a tough call.

However, I guess we can wait to see what happens in humans as there is a study called CLADPET. (NCT04239820) where they will be looking microglial activity after cladribine using imaging. The human experiment will say it all…or will it?. If cladribine is active it will surely get rid of the inflammatory response and so there will be less microglial activity…Less microglial activity does not mean that cladribine works on microglia.

Now I see why ProfK batted the question to me…he doesn’t want mud on his shoes:-)

COI: Multiple. I have recieved compensation from Merck for different activities.

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  • Could this exam be also added to ChariotMS and/or to the proteasome inhibitor trial at least for a subset of patients to see if microglia is switching off with the treatment to try to get some data in advance? These trials could be the right settings to generate this type of relevant data to flank clinical observations.

    I would expect that microglia switches off in a much shorter time than clinical differences take to appear.

  • Hello Mouse Doctor,

    I thought this could be interesting for you as well as for professor gavin giovannoni. I do not see in my search any articles on the barts ms blog about this. Here is the title : Simvastatin induces apoptosis of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines and delays development of EBV lymphomas

    Link 1 :

    Here is the title to the second article : Epstein-Barr virus subverts mevalonate and fatty acid pathways to promote infected B-cell proliferation and survival

    Link 2 :

    Here is a citation for the article in link number 2 .

    Notably, 2 μM simvastatin significantly impaired EBV-driven primary B-cell outgrowth (Fig 3A), and dose response analysis identified 1.6 μM as the simvastatin EC50 (S4C Fig), suggesting on-target activity as the basis for this effect. Propidium iodide cell cycle analysis demonstrated that treatment with either simvastatin or the related HMGCR antagonist atorvastatin significantly increased the sub-2N population and decreased the 2N population, indicative of cell death (Fig 3B). Atorvastatin impaired EBV-driven B-cell outgrowth (S4D Fig), even though it has not been shown to share the LFA-1 off-target effect.

    • Thanks EBV is profGs realm

      But this paper was done in 2004 long before the blog started.
      The human dose is I think about 20mg so that is about 0.3mg/kg

      “Mean concentration of statins in human serum (at therapeutic doses) is only 1–15 nmol L−1. In addition, the protein binding of statins in human blood is high, 95–99%, and it is only the free fraction (0.01–0.5 nmol L−1) that is pharmacologically active, so 2 micromolar is 2000nM so that is 4000-200,000 times the human drug levels in the mouse they used 250mg/kg so that is 750 times the human dose.

  • OK cladribine kills B cells that respond to pro-inflammatory LPS, IL-4 etc. that upregulate DCK expression. But is it initially microglia driving the immune response or is it lymphocytes? Does it matter the sequence of immune activation? Would innate immunity trump adaptive immunity sequentially? IDK

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