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Friday, 26 May 2017

Boosting Bumetanide with an OAT3 Inhibitor?



Today’s post was prompted by our reader Ling, who highlighted research suggesting another way to improve the potency of bumetanide, a drug many readers have found reduces the severity of autism.


Sometime a little extra boost is necessary


There is an ongoing debate in the literature about how poorly bumetanide crosses into the brain and whether the theoretical chloride-lowering benefit can actually take place in humans.  Well for many readers of this blog, we know the answer.

Nonetheless there are efforts underway to improve the potency of bumetanide in neurological disorders. There is a prodrug called BUM5 which has been shown to reverse types of seizure that bumetanide could not, due to much greater potency in the brain.
The French bumetanide researchers are themselves looking to develop a more potent drug.
Ling highlighted a recent paper that suggested using an old drug called Probenecid to increase the concentration of bumetanide in the brain (and plasma) threefold.
This is not a new idea, during World War Two when antibiotics were in short supply, the same drug Probenecid was used to increase the potency of antibiotics to reduce how much you needed to give patients.

Pharmacodynamics
What we want to do is increase the concentration of bumetanide in the brain and ideally increase the half-life.  Both should increase its effect.
The recent research shows that in mice Probenecid does indeed have the effect we want, but humans are not mice.
A very old study looked at the effect in humans of Probenecid on a very similar diuretic called furosemide.


Pharmacodynamic analysis of the furosemide-probenecid interaction in man

The graph above shows that probenecid had a dramatic effect on the potency of the diuretic. Consider the area under the curves lines.  The area is a proxy for the effect of the drug (but it is a log scale).  After eight hours the furosemide alone has gone to zero, whereas when probenecid is added it is as potent as furosemide was alone after 90 minutes.

The recent study highlighted by Ling:-


Bumetanide is increasingly being used for experimental treatment of brain disorders, including neonatal seizures, epilepsy, and autism, because the neuronal Na-K-Cl cotransporter NKCC1, which is inhibited by bumetanide, is implicated in the pathophysiology of such disorders. However, use of bumetanide for treatment of brain disorders is associated with problems, including poor brain penetration and systemic adverse effects such as diuresis, hypokalemic alkalosis, and hearing loss. The poor brain penetration is thought to be related to its high ionization rate and plasma protein binding, which restrict brain entry by passive diffusion, but more recently brain efflux transporters have been involved, too. Multidrug resistance protein 4 (MRP4), organic anion transporter 3 (OAT3) and organic anion transporting polypeptide 2 (OATP2) were suggested to mediate bumetanide brain efflux, but direct proof is lacking. Because MRP4, OAT3, and OATP2 can be inhibited by probenecid, we studied whether this drug alters brain levels of bumetanide in mice. Probenecid (50 mg/kg) significantly increased brain levels of bumetanide up to 3-fold; however, it also increased its plasma levels, so that the brain:plasma ratio (~0.015-0.02) was not altered. Probenecid markedly increased the plasma half-life of bumetanide, indicating reduced elimination of bumetanide most likely by inhibition of OAT-mediated transport of bumetanide in the kidney. However, the diuretic activity of bumetanide was not reduced by probenecid. In conclusion, our study demonstrates that the clinically available drug probenecid can be used to increase brain levels of bumetanide and decrease its elimination, which could have therapeutic potential in the treatment of brain disorders.


Supporting research on organic anion transporters

As is often the case, there is already a wealth of research that we can draw on and it does indeed look like an OAT3 inhibitor should modify the pharmacodynamics of bumetanide in a very helpful way. But questions do remain.


Identification of hOAT1 and hOAT3 inhibitors from drug libraries


The NIH Clinical Collection (NCC) and NIH Clinical Collection 2 (NCC2) drug libraries used for HTS consisted respectively of 446 and 281 small molecules (727 total) approved for clinical use or having a history of use in human clinical trials. The clinically tested compounds in the NCC and NCC2 libraries are highly drug-like with known safety profiles. At the indicated concentrations, 92 compounds resulted in 50 % decrease in hOAT1-mediated 6-CF transport, whereas 262 compounds resulted in 50 % decrease in hOAT3-mediated 6-CF transport (Fig. 2). All of the 92 hOAT1 inhibitors were also inhibitors for hOAT3 but with a different potency. Among the 262 inhibitors for hOAT3, 8 compounds were specific for hOAT3 (Table 1), i.e., they lacked appreciable inhibitory activity for hOAT1. For example, stiripentol inhibited hOAT3 with an IC50 of 27.6 ±1.28 μM, but it barely had any effect on hOAT1 (not shown). These inhibitors for hOAT1 and hOAT3 included classes of anti-inflammatory, antiseptic/anti-infection, antineoplastic, steroid hormones, cardiovascular, antilipemic, CNS, gastrointestinal, respiratory and reproductive control drugs.

Table 1

hOAT3-specific Inhibitors

Stiripentol
Cortisol succinate
Demeclocycline
Penciclovir
Ornidazole
Benazepril
Chlorpropamide
Artesunate

Table 2

Highly potent inhibitors for hOAT1 at peak plasma concentrations

Amlexanox
Telmisartan
Mefenamic Acid
Oxaprozin
Parecoxib Na
Meclofenamic Acid
Nitazoxanide
Ketoprofen
Ketorolac Tromethamine
Diflunisal





Table 3

Highly potent inhibitors for hOAT3 at peak plasma concentrations

Mefenamic Acid
Meclofenamic Acid
Pioglitazone
Oxaprozin
Nateglinide
Amlexanox
Ketorolac Tromethamine
Diflunisal
Nitazoxanide
Irbesartan
Valsartan
Telmisartan
Balsalazide
Ethacrynic Acid



We further increased the stringency of our selection criteria by incorporation of peak unbound plasma concentration of drugs since, for drugs tightly bound to plasma proteins, the free concentration in plasma is a better estimate of the drug level interfering with OAT transport function. Further screening using the peak unbound plasma concentration yielded three inhibitors of hOAT1 (Table 4) and seven inhibitors of hOAT3 (Table 5) with potency >95% inhibition.

Table 4

Highly potent inhibitors for hOAT1 at peak unbound plasma concentrations

Compounds
IC50 in COS-7 cells (μM)
Cmax (μM)
Cmax Unbound (Cu.p) (μM)
Cu.p/IC50
Oxaprozin
0.891±0.292
50116
5.01*
5.62
Mefenamic Acid
1.085±0.124
83.0*
8.30*
7.60
Ketorolac Tromethamine
0.653±0.130
9.5017
0.10017
0.150



Table 5

Highly potent inhibitors for hOAT3 at peak unbound plasma concentrations

Compounds
IC50 in COS-7 cells (μM)
Cmax (μM)
Cmax Unbound (Cu.p) (μM)
Cu.p/IC50
Nateglinide
0.860±0.0953
18.018
0.23019
0.270
Oxaprozin
0.870±0.0704
50116
5.01*
5.76
Nitazoxanide
0.154±0.0711
31.2
0.0300
0.200
Valsartan
0.250±0.143
14.820
0.85021
3.47
Ethacrynic Acid
0.662±0.261
30.922
0.600
0.910
Diflunisal
0.720±0.290
496
0.490
0.680
Mefenamic Acid
1.75±0.258
83.0*
8.30*
4.74


Regulatory Requirements


The FDA and EMA require that the drug interaction liability of this transporter be evaluated in vitro for drug candidates that are renally eliminated. OAT3 contributes to renal drug clearance and transporter – mediated renal drug interactions. Based on the in vitro substrate and inhibition data, decisions are made for OAT transporter–based clinical drug interaction trials, typically with probenecid.

Localization
Endogenous substrates
Substrates used experimentally
Substrate drugs
Inhibitors
Kidney, proximal tubule, basolateral membrane. Brain, choroid plexus and blood–brain barrier
prostaglandin, uric acids, bile acids; conjugated hormones
E3S, furosemide, bumetanide
NSAIDs, cefaclor, ceftizoxime
probenecid, novobiocin




APPENDIX A- Tables

Table 1. Major human transporters

Gene                  Aliases          Tissue                 Drug Substrate                  Inhibitor     

SLC22A6          OAT1       kidney,             acyclovir,                      probenecid

                                                                   adefovir,                      cefadroxil

    methotrexate,             cefamandole

    zidovudine                   cefazolin

SLC22A7          OAT2      liver, kidney    zidovudine                  

SLC22A8          OAT3     kidney, brain   cimetidine,                  probenecid

methotrexate             cefadroxil

zidovudine                  cefamandole

                                   cefazolin


Conclusion
This is a classic case where a little inexpensive experiment could be of huge value.  You just use adult volunteers to test the effect on bumetanide pharmacodynamics of a small number of OAT3 inhibitors.

There are now hundreds of kids in France who take bumetanide, meaning hundreds of parents who are probably more than willing to give up a day to sit in a clinic and give hourly blood samples, so their child might benefit.
Would this common sense approach be followed? Or would it be the case that it needs hundreds of thousands of dollars/euros to do a trial and we wait 3 years for the result?





18 comments:

  1. Peter,

    Eagerly awaiting your post on suramin..effects seem to be dramatic and impacted every child on whom it was tested (5 which does seem pathetic).

    Since summer vacations are on, I am toying with the idea of a bumetanide retrial...still toying as I am at my wits end on how to manage bumetanide side effects. My son cannot tolerate potassium supplements, did bad on magnesium and even a regular junior multi vitamin/mineral (pure encapsulations) given at half the specified dose does strange things to him. Vitamins, minerals, probiotics even at low dose may cause complex effects and interactions leading to gut dysbiosis, bacterial overgrowth, histamine issues and what not. Very low dose, pulse dosing, one supplement at a time, nothing can pre empt eventualities. And how can you give one at a time when supplemnts acting synergistically or one creating deficiency or need for another is suggested in bold letters. Things can downspiral bigtime and very rapidly. We had our first SIB and rage behaviour yesterday and I am shocked..it was triggered by lack of sleep and a gi infection..painful erections which are a sign of excitoxicity and stress in my child. Peter, it was too painful to see my son trying to beat down his erection, tears streaming down his face, contorted in pain. He was trying hard not to hit me but I still ended up with scratches. Cognitively, he is d doing well but huge sensory issues...all the yime trying to cool himself off. I had assumed that a little attitude, defiance, sleep issues and hyperactivity are accepted side effects of mb 12 and folimic suplementation. But seems there is nothing like a non toxin. These biochemicals do not just enter your body, do tgeir job and leave decently...they interact, build up, affect your biochemical pathways, feed bacteria.

    In short, I am really distraught as I feel my son who now and then runs and puts his arms around me calling me 'mama' could have done wonderfully with the correct drug/supplement.








    ReplyDelete
    Replies
    1. Kritika,the suramin post will be the next one.

      The problem is that the trial was tiny and very weak in terms of behavioral as opposed to biological changes.

      If all the studies used the same autism rating scales you could compare the magnitude of the improvement. So we do not know much more now than before the trial was published.

      Everyone thought it helped, but the important thing is the magnitude. In the CARS scale, was it a change of 4, 10 or 15?

      I think in many cases you can add enough potassium via diet. Only about 20% of people will have a big issue with potassium. You may not need supplements, but you need to measure potassium in his blood to be sure.

      SIB is indeed shocking for parents.

      Unfortunately it is not easy to find effective therapies.

      Delete
  2. ALERT!

    I'm at work and can't write much on my iPhone but Naviaux paper just came out!

    AJ

    ReplyDelete
  3. Has anybody tried this combination out? Also since the half life will be increased will the diuretic effect be prolonged?

    ReplyDelete
    Replies
    1. I doubt anyone has yet tried this, but if/when someone does please leave a comment with the effect.

      Delete
  4. Will the diuretic effect be prolonged due to the prolonged half life?

    ReplyDelete
    Replies
    1. Good question, the only way to find out the effect on diuresis and potassium loss would be to do an experiment.

      Delete
  5. What dose of probenecid was given in the furosemide-probenecid study?

    ReplyDelete
    Replies
    1. The study say:
      " Subjects ingested 1 g of probenecid at bedtime the night before and on arising the morning of the study (30 to 60 mm before administration of furosemide)."

      Delete
  6. Old study from 1981:
    "We administered 0.5- and 1.0-mg doses of bumetanide intravenously to eight normal subjects with and without pretreatment with probenecid. Probenecid did not affect either the cumulative response or the time course of response of bumetanide. These results are in contrast to results reported in dogs but consistent with similar studies in cats. The data imply that probenecid and potentially other exogenous or endogenous organic acids do not affect the renal handling of bumetanide in normal man."
    http://onlinelibrary.wiley.com/doi/10.1002/j.1552-4604.1981.tb01772.x/abstract

    On the other hand:
    "Probenecid has been found to decrease the intensity of the natriuretic activity of (...), furosemide, (..) confirming that these diuretics require tubular secretion for their natriuretic action. Some confusion on the role of tubular secretion in the response to bumetanide occurred initially because several studies demonstrated no or partial attenuation of bumetanide diuresis during probenecid administration. Thus it was hypothesized that in man a significant amount of bumetanide entered the proximal tubule lumen through passive diffusion. However, subsequent animal studies have shown that bumetanide has greater affinity than most other diuretics for the basolateral anion transporter. Consequently the failure to observe antagonism between bumetanide and probenecid in humans may have been due administration of inadequate doses of prebenecid to completely inhibit bumetanide secretion."
    (Diuretic Agents: Clinical Physiology and Pharmacology)
    https://books.google.se/books?id=VHcsrw6unuAC&pg=PA265&lpg=PA265&dq=bumetanide+probenecid&source=bl&ots=X0q3i61Prn&sig=Ep2062uIAkrPOgLHDGIMg5A_sec&hl=sv&sa=X&ved=0ahUKEwifq7Sh-I_UAhUDuBoKHdo9BwIQ6AEIYzAJ#v=onepage&q=bumetanide%20probenecid&f=false

    I think that this simply means that we can't compare bumetanide to furosemide, nor compare humans with animals in this case.

    /Ling

    ReplyDelete
    Replies
    1. Ling, I think as your study says:-

      "failure to observe antagonism between bumetanide and probenecid in humans may have been due administration of inadequate doses of prebenecid to completely inhibit bumetanide secretion"

      We know that OAT3 is the transporter for bumetanide. We do not know if probenecid is more/less potent than the other OAT3 inhibitors.

      The question seems to be is there a safe OAT3 inhibitor that can boost bumetanide in humans? None of the FDA's OAT3 inhibitors appear in the list of most potent OAT3 inhibitors in the Tables 1-5 above. Mefenamic Acid (Ponstan) is available as a cheap OTC pain killer in some countries. I have already suggested the potential benefit of angiotensin receptor blockers (Telmisartan, Valsartan)

      Delete
    2. Ling, it looks like the classic paper is:-

      In Vitro and In Vivo Evidence of the Importance
      of Organic Anion Transporters (OATs) in Drug
      Therapy

      But the full text is not in the public domain.

      It seems that Acetazolamide and Aspirin are potent OAT3 inhibitors. The most potent are some antibiotics
      Cefadroxil, Cefoselis and Ceftriaxone.

      Delete
    3. http://libgen.io/book/index.php?md5=9576727A47382F85B9CA13DF20D20AB2

      Delete
  7. Peter, started with Miyairi, in the label says that 18 tablets contain 180 mg c.butyricum. Started with 4 tablets a day, 2 after main meals. Compared to sodium butyrate that comes in 500 mg tablets Miyarisan is very low.I don´t know if it is the same thing. You have to take 18 tablets to get less than half the dose of sodium butyrate. What do you think? Miyarisan tablets contain sucrose,this could be an issue?.
    Valentina

    ReplyDelete
    Replies
    1. I do not know the amount of c.butyricum that will equal 500mg of sodium butyrate. I also would like to know.

      The Miyarisan bacteria reacts with fiber in the intestines and produces butyric acid.

      I would gradually increase the dose to the maximum suggested. The product has been used for more than 50 years.

      Delete
  8. Hi Peter,
    I have mentioned before how my son's anxiety has not improved on any medications we have tried -- the usual suspects SSRI's. I keep pushing his doctor to allow him a trial of low dose Clonazepam to no avail. Today he got irritated and told me that he absolutely won't do it and suggested that he give me a referral (which I am inclined to take). He did offer me a trial of an anti viral drug called Amantadine which is used for ADHD. After reading about it it says that it is a dopamine promoter. I thought high dopamine (which I would think my son has) would increase anxiety. Am I wrong? Please educate me and let me know if this sounds like a viable option or if I should move on to a new doctor. Thanks so much for your thoughts.
    --Christine

    ReplyDelete
    Replies
    1. Christine, Amantadine has numerous different effects. One of which is to increase dopamine. One common side effect is anxiety.

      You would expect it might make your son worse, but it affects many things and so you cannot be sure.

      Delete
  9. Hi Christine
    I've Aspergers and luckily I once tried Amantadine. The effect was somewhat unclear. It made me feel dissociated from the world. Like when there was a stimuli coming, I didn't really think it's my perception. It was just perceived, but in thoughts I couldn't connect to the stimuli. Stimuli - Thinking connection was not really there anymore. I didn't continue with this med and I wouldn't recommend it. It didn't really help my concentration nor my anxiety.
    Low-dose Clonazepam is actually quite incredible, it helps a lot with anxiety and sensory overload. For example: I have no problems now with wearing lenses or putting sunscream on my skin.
    The other option would be a Neuroleptic. I was on Seroquel now for a year. It clearly slows down perception, and thus it makes the world less frightening. Then I could react more appropriately to thoughts or stimuli and in turn social anxiety was reduced.
    I used 50mg Seroquel, which is very low, but worked wonders. I think people with Autism are much more Perception-dependent (driven). This said, Seroquel lets you handle sensory stimuli more dynamically. For example: Before Seroquel I was playing the piano very strong. With Seroquel I gained the ability to just give in to the sensory stimuli and accept it. 3 days later my neighbour complimented me on my improvement in playing more dynamically. Same for goes for social situations.

    Greetings Stefan

    ReplyDelete

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