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Showing posts with label BUM5. Show all posts
Showing posts with label BUM5. Show all posts

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?