OAT3 inhibitors for Bumetanide - Probenecid, but also Aspirin, Chlorogenic acid (Coffee), Epicatechin (Cocoa, Cinnamon) and more.

Today’s post is about OAT3, highlighted by the green lines.
The interventions reduce renal excretion and raise plasma
concentration rather than directly improving transport across the BBB

Today’s post is a collaboration. Our reader Ling pointed out research trying to boost the bioavailability of bumetanide using something clever called an OAT3 inhibitor.  This would reduce the rate at which the body excretes bumetanide and thus potentially improve its therapeutic effect.

Petra, our reader from Greece, pointed out that in her son Bumetanide seemed to work better when taken with Greek coffee and that that Greek Grandpas like to take their diuretics with a steaming Greek coffee.

Most people, me included, automatically think caffeine when someone mentions coffee.

So I assumed that caffeine might be an OAT3 inhibitor and I did make some experiments on that basis. There is no research data to support caffeine as an OAT3 inhibitor.

Recently I was again looking for other potential Bumetanide boosters.  The obvious one is called Probenecid.  Probenecid is used to treat gout because it lowers uric acid.

Aspirin has some odd effects; low dose aspirin will raise uric acid, but high dose aspirin will lower it. Aspirin is an OAT3 inhibitor.

OATs are a very niche subject, to add to the confusion sometimes you are better looking for SLC22A8, the gene that encodes the transporter. 

There was an earlier post on this subject, which showed that many NSAIDs inhibit OAT3, including Knut’s favourite Ponstan. They are not so well suited to continued use.

Boosting Bumetanide with an OAT3 Inhibitor?

At the end of my little investigation I figured it out; there are many OAT3 inhibitors available, including some in your kitchen.  

Key points on OAT3 (Organic Anion Transporter 3)

If you want to increase the peak concentration and indeed the half-life of a drug that is excreted from the body by OAT3 (organic anion transporter 3), an OAT inhibitor is what you need.

The drug Probenecid is by far the best known OAT3 inhibitor and it is very potent. It has long been to boost the performance of penicillin type antibiotics to treat tough bacterial infections.

Probenecid, if available, may very well be the ideal bumetanide booster.

For adults a simple option is Greek/Turkish coffee. I see little downside as long as you can handle the caffeine. The Greeks live a long time and drink plenty of coffee.

For those who do not like caffeine you can go to active components within the coffee, which seem to be the chlorogenic acids (1,3- and 1,5-dicaffeoylquinic acid). They are sold as a weight loss supplement, the long established version is the French-made Svetol, but there are now others. They still contain 2- 3% caffeine.

Epicatechin, found in cinnamon, dark chocolate and high flavanol cocoa is another OAT3 inhibitor. Cocoavia, made by Mars, is used by some readers of this blog. Cocoa flavanols do clever things with nitric oxide (NO) and have been shown to improve mild cognitive impairment (MCI) and heart health by improving blood vessel elasticity.

Catechins are flavanols belonging to a family of closely related compounds, such as epicatechin, epigallocatechin, epicatechin gallate (EGC), and epigallocatechin gallate (EGCG). They are all slightly different. Catechin itself is not an OAT3 inhibitor; EGCG may or may not be.

Low dose aspirin is likely the cheapest OAT3 inhibitor. It also increases peripheral circulation, which could benefit some. Low dose aspirin has the downside of a small bleeding risk, mainly in old people, and there is a risk of Reye’s syndrome if given during/after a viral infection.

I think for adults a Greek coffee may be the best. For people who have a profound benefit from Bumetanide, I think they should look into Probenecid.

Personally I think Svetol is worth a try.

Coffee that has been extensively processed (just as we saw with cocoa) may not have the same chlorogenic acid content as the more gritty coffee used in the Balkans. Coffee consumption is actually associated with many neurological benefits, reducing the incidence of Parkinson’s and Alzheimer’s; the common mistake in research is the assumption that the effect must be from caffeine.

Source: Can coffee consumption lower the risk of Alzheimer’s disease and Parkinson’s disease? A literature review

  

The health effects of decaffeinated Coffee

My eureka moment in this post was reading about gout and coffee and then decaffeinated coffee. 

Coffee Itself, Not Caffeine, Reduces Risk of Gout 

Dietary supplementation with decaffeinated green coffee improves diet-induced insulin resistance and brain energy metabolism in mice. 

Decaffeinated Green Coffee Bean Extract Attenuates Diet-Induced Obesity and Insulin Resistance in Mice

So then it was a question of finding what in coffee could be the OAT3 inhibitor. At which point I found a very insightful paper that tells you everything, once you realise that:

Coffee = chlorogenic acids  = 1,3- and 1,5-dicaffeoylquinic acid

Interaction of Natural Dietary and Herbal Anionic Compounds and Flavonoids with Human Organic Anion Transporters 1 (SLC22A6), 3 (SLC22A8), and 4 (SLC22A11)

Five compounds, 1,3- and 1,5-dicaffeoylquinic acid, ginkgolic acids (15 : 1) and (17 : 1), and epicatechin, significantly inhibited hOAT3 transport under similar conditions

3.2. Inhibition of hOAT3 by Natural Anionic Compounds and Flavonoids

Human OAT3 expressing cells showed about 4-fold greater accumulation of ES as compared to background control cells ( versus  pmol mg 10  , resp.). Similar to hOAT1, hOAT3-mediated ES uptake was completely (>96% inhibition) blocked by probenecid (Figure 4). Five of the compounds, 1,3- and 1,5-dicaffeoylquinic acid, epicatechin, and ginkgolic acids (15 : 1) and (17 : 1), significantly inhibited hOAT3-mediated transport at 50-fold excess (Figure 4). 1,3-Dicaffeoylquinic acid and ginkgolic acid (17 : 1) exhibited 41% inhibition, while 30–35% reduction of hOAT3-mediated ES uptake was observed for 1,5-dicaffeoylquinic acid, epicatechin, and ginkgolic acid (15 : 1). Catechin, 18β-glycyrrhetinic acid, and ursolic acid failed to produce significant inhibition. Based on the level of inhibition observed, values for all of these compounds would be greater than 50 μM, much higher than clinically relevant concentrations (Table 1). Therefore, further dose-response studies were not performed.

Lay off the Lycopene?

Lycopene does the opposite of what we want. Too much lycopene may lower the effectiveness of a drug that is excreted via OAT3. 

2.29. Lycopene

Lycopene is a carotenoid pigment found in tomato [94]. Lycopene from dietary sources has been shown to reduce the risk of some chronic diseases including cancer and cardiovascular disorders [95]. The administration of lycopene significantly normalized the kidney function and antioxidant status of CSP-treated animals. Furthermore, lycopene also increased the expression of the organic anion and cation transporters (OAT and OCT, resp.) including OAT1, OAT3, OCT1, and OCT2 in the renal tissues [96–98]. In addition, lycopene also decreased the renal efflux transporters (multidrug resistance-associated protein [MRP]-2 and MRP4) levels and induced Nrf2 activation, which activated the antioxidant defense system [99]. Furthermore, lycopene protected against CSP-induced renal injury by modulating proapoptotic Bax and antiapoptotic Bcl-2 expressions and enhancing heat shock protein (HSP) expression [97].

https://www.hindawi.com/journals/omcl/2016/4320374/                                                                                                                  

Aspirin

I actually started out this post by looking at what dose of aspirin might be effective in inhibiting OAT3.  We do know that Aspirin is indeed an OAT3 inhibitor.  

Aspirin and probenecid inhibit organic anion transporter 3

I did find the answer, but along the way you do end up having to look at uric acid. 

Uric acid is taken up by OAT1 and OAT3 from the blood and reabsorbed into renal tubular cells via URAT1 Uric acid is taken up by OAT1 and OAT3 from the blood and reabsorbed into renal tubular cells via URAT1Uric acid is taken up by OAT1 and OAT3 from the blood and reabsorbed into renal tubular cells via URAT1. 

Uricosuric drugs increase the excretion of uric acid in the urine, thus reducing the concentration of uric acid in blood plasma. 

In general, uricosuric drugs act on as urate transporter 1 (URAT1). URAT1 is the central mediator in the transport of uric acid from the kidney into the blood.  By their mechanism of action, some uricosurics (such as  probenecid) increase the blood plasma concentration of certain other drugs and their metabolic products  – this is their effect on OAT3.

Probenecid is a medication that increases uric acid excretion in the urine.

Atorvastatin is a so-called secondary uricosuric. High dose aspirin should also be called a secondary uricosuric.

Antiuricosuric drugs raise serum uric acid levels and lower urine uric acid levels. These drugs include all diuretics and low dose aspirin. 

Low dose aspirin inhibits OAT1 and OAT3 which reduces urate secretion, but high dose aspirin inhibits URAT1 and reduces urate re absorption. This is sometimes known as the biphasic effect.

So low dose aspirin will increase plasma uric acid, but high dose aspirin has the same effect as Probenecid, it lowers plasma uric acid levels.

So Aspirin and Probenecid both affect URAT1 and OAT3. 

Source: Effects on Uric Acid Metabolism of the Drugs except the Antihyperuricemics

At what dose is Aspirin an OAT3 inhibitor?

If we just want aspirin to inhibit OAT3 and not inhibit URAT1, what dose is effective? Fortunately this has been answered in the research. The typical low dose of aspirin (75mg) used preventatively in older people is OAT3 inhibiting, it raises plasma uric acid.  

THE EFFECT OF MINI-DOSE ASPIRIN ON RENAL FUNCTION AND URIC ACID HANDLING IN ELDERLY PATIENTS

Salicylate

Salicylic acid and its derivatives are the most prescribed analgesic, antipyretic, and anti-inflammatory agents. Salicylates have a “paradoxical effect” on the handling of uric acid by the kidney. The action of salicylates on uric acid excretion depends on the dose of salicylates. At doses of less than 2.5 g/day, salicylates cause the retention of uric acid by blocking the tubular secretion of uric acid, while at dose of higher than 3 g/day, they cause increased urinary excretion of uric acid [70]. Mini-dose aspirin, even at a dosage of 75 mg/day, caused a decrease in uric acid excretion and raised serum uric acid level [71]. It has been suggested that the “paradoxical effect” of salicylate can be explained by two modes of salicylate interaction with URAT1: (1) acting as an exchange substrate to facilitate uric acid reabsorption, and (2) acting as an inhibitor for uric acid reabsorption [72]. Low dose of salicylate interact with OAT1/OAT3, the uric acid secreters [73].

Low dose aspirin leads to decreased renal excretion of uric acid and raised serum uric acid levels, which can cause a gout attack in those predisposed to this condition.

High doses of aspirin lower serum uric acid concentration.

Reye’s Syndrome

In children aspirin is very rarely used because of the risk of Reye’s syndrome. Reye’s syndrome causes severe liver and brain damage. It is a type of severe mitochondrial failure that can occur after a viral infection like flu or chickenpox, but it almost only occurs when aspirin has been prescribed. Nobody knows for sure the exact mechanism of the disease.

So do not give aspirin to children with a viral infection.  We already know to avoid paracetamol/acetaminophen (Tylenol in the US) in babies/children and people with autism. Paracetamol/acetaminophen depletes the body’s key antioxidant GSH. 

If someone overdoses on Paracetamol/acetaminophen you give them a high dose of NAC to prevent death. 

Conclusion

Given how long it takes to develop new drugs, I think that improving the pharmokinetics of bumetanide is a pretty obvious thing to do. 

Diamox is an OAT3 inhibitor and our reader Agnieszka found it beneficial only when administered along with Bumetanide.

Strong coffee is an OAT3 inhibitor and this was found to enhance bumetanide by Petra’s son with Asperger’s.

Cinnamon which contains epicatechin, another OAT3 inhibitor, did seem to be helpful in Monty who also takes bumetanide.

I suspect Diamox may be the most potent OAT3 inhibitor of those three

The interesting OAT3 inhibitors seem to be:-

·        Probenecid

·        Low dose aspirin

·        Epicatechin (cocoa, cinnamon ..)

·        Chlorogenic acids (coffee and decaffeinated green coffee extracts) 

Cinnamon, high flavanol cocoa and indeed coffee (minus the caffeine) have numerous health benefits.

Note that Catechin has no effect on OAT3. EGCG was not tested but in other studies has been shown it does affect.

Interactions of green tea catechins with organic anion-transporting polypeptides.

The logical next step would be to improve bumetanide transport across the blood brain barrier.

Inflammation Leading to Cognitive Dysfunction

Today’s post highlights a paper with some very concise insights into how microglial cells become “activated” resulting in the “exaggerated inflammatory response” that many people with autism experience on a daily basis.  

This is very relevant to treatment, which is not usually the objective of much autism research.

I recall reading a comment from John’s Hopkins about neuroinflammation/activated microglia in autism; they commented that no known therapy currently exists and that, of course, common NSAIDs like ibuprofen will not be effective.  But NSAIDs are effective.

As we see in today’s paper, there a least 4 indirect cytokine-dependent pathways leading to the microglia, plus one direct one.

NSAIDs most definitely can reduce cytokine signaling and thus, indirectly, reduce microglial activation.

The ideal therapy would act directly at the microglia, and as Johns Hopkins pointed out, that does not yet exist with today's drugs.  If you read the research on various natural flavonoids you will see that “in vitro” there are known substances with anti-neuroinflammatory effects on microglial activation.  The recurring “problem” with such substances is low bioavailability and inability to cross the blood brain barrier.

Back to Today’s Paper

It was a conference paper at the 114th Abbott Nutrition Research Conference - Cognition and Nutrition

The full conference proceedings can be downloaded

The paper is not about autism, it is about more general cognitive dysfunction.  It is from mainstream science (I checked).

It explains how inflammation anywhere in the body can be translated across the BBB (Blood Brain Barrier) to activate the microglia.  This of course allows you to think of ways to counter these mechanisms.

It also raises the issue of whether or not anti-inflammatory agents really need to cross the BBB.  While you might think that ability to cross the BBB is a perquisite to mitigate the activated microglia, this may not be the case.  Much can be achieved outside the BBB, and we should not rule out substances that cannot cross the BBB.

Very many known anti-inflammatory substances do not cross the BBB.   

  

From Inflammation to Sickness and Cognitive Dysfunction: When the Immune System Subjugates the Brain

extracts from the above paper ...

Example – Influenza and Cognition

Neurological and cognitive effects associated with influenza infection have been reported throughout history.

The simplest explanation for these neurocognitive effects is that influenza virus makes its way to the brain, where it is detected by neurons.

However, most influenza strains, including those responsible for pandemics, are considered non-neurotropic, neurological symptoms associated with influenza infection are not a result of direct viral invasion into the CNS.

Moreover, neurons do not have receptors to detect viruses (or other pathogens) directly.

Cells of the immune system do, however, as the immune system’s primary responsibility is to recognize infectious pathogens and contend with them. For example, sentinel immune cells such as monocytes and macrophages are equipped with toll-like receptors (TLR) that recognize unique molecules associated with groups of pathogens (i.e., pathogen-associated molecular patterns). Stimulation of TLRs that recognize viruses (TLR3 and TLR7) and bacteria (TLR4) on immune sentinel cells can have profound neurological and cognitive effects, suggesting the immune system conveys a message to the brain after detecting an infectious agent. This message is cytokine based.

Macrophages and monocytes produce inflammatory cytokines (e.g., interleukin [IL]-1β, IL-6, and tumor necrosis factor-α [TNF-α]) that facilitate communication between the periphery and brain.

Cytokine-dependent Pathways to the Brain

Several cytokine-dependent pathways that enable the peripheral immune system to transcend the blood-brain barrier have been dissected.

Inflammatory cytokines present in blood can be actively transported into the brain.

But there are also four indirect pathways:-

1.     Cytokines produced in the periphery need not enter the brain to elicit neurocognitive changes. This is because inflammatory stimuli in the periphery can induce microglial cells to produce a similar repertoire of inflammatory cytokines. Thus, brain microglia recapitulates the message from the peripheral immune system.

2.     in a second pathway, inflammatory cytokines in the periphery can bind receptors on blood-brain barrier endothelial cells and induce perivascular microglia or macrophages to express cytokines that are released into the brain

3.     In a third pathway, cytokines in the periphery convey a message to the brain via the vagus nerve. After immune challenge, dendritic cells and macrophages that are closely associated with the abdominal vagus have been shown to express IL-1β protein; IL-1 binding sites have been identified in several regions of the vagus as well. When activated by cytokines, the vagus can activate specific neural pathways that are involved in neurocognitive behavior. However, activation of the vagus also stimulates microglia in the brain to produce cytokines via the central adrenergic system 

4.     A fourth pathway provides a slower immune-to-brain signaling mechanism based on volume transmission.  In this method of immune-to-brain communication, production of IL-1β by the brain first occurs in the choroid plexus and circumventricular organs—brain areas devoid of an intact blood-brain barrier. The cytokines then slowly diffuse throughout the brain by volume transmission, along the way activating microglia, neurons, and neural pathways that induce sickness behavior and inhibit cognition.

Can Flavonoids Reduce Neuroinflammation and Inhibit Cognitive Aging?

Flavonoids are naturally occurring polyphenolic compounds present in plants. The major sources of flavonoids in the human diet include fruits, vegetables, tea, wine, and cocoa.  Significant evidence has emerged to indicate that consuming a diet rich in flavonoids may inhibit or reverse cognitive aging

Flavonoids may improve cognition in the aged through a number of physiological mechanisms, including scavenging of reactive oxygen and nitrogen species and interactions with intracellular signaling pathways. Through these physiological mechanisms, flavonoids also impart an anti-inflammatory effect that may improve cognition. This seems likely for the flavone luteolin, which is most prominent in parsley, celery, and green peppers.

Whereas luteolin inhibits several transcription factors that mediate inflammatory genes (e.g., nuclear factor kappa B [NF-κB]and activator protein 1 [AP-1]), it is a potent activator of nuclear factor erythroid 2-related factor 2 (Nrf2), which induces the expression of genes encoding antioxidant enzymes. A recent study of old healthy mice found improved learning and memory and reduced expression of inflammatory genes in the hippocampus when luteolin was included in the diet. Thus, dietary luteolin may improve cognitive function in the aged by reducing brain microglial cell activity.

Hence, the flavonoid luteolin is a naturally occurring immune modulator that may be effective in reducing inflammatory microglia in the senescent brain.

Conclusion

In light of the recent evidence suggesting microglial cells become dysregulated due to aging and cause neuroinflammation, which can disrupt neural structure and function, it is an interesting prospect to think dietary flavonoids and other bioactives can be used to constrain microglia. But how can flavonoids impart this anti-inflammatory effect? Although in vitro studies clearly indicate that several flavonoids can act directly on microglial cells to restrict the inflammatory response, results from in vivo studies thus far do not prove that dietary flavonoids access the brain to interact with microglia in a meaningful way. This is a complicated question to dissect because flavonoids reduce inflammation in the periphery and microglia seem to act like an “immunostat,” detecting and responding to signals emerging from immune-to-brain signaling pathways. Thus, whether dietary flavonoids enter the brain and impart an anti-inflammatory effect on microglia is an interesting question but one that is more theoretical than practical because what is most important is how the immunostat is adjusted, whether that is via a direct or indirect route. However, because flavonoids are detectable in the brain they most likely affect microglia both directly and by dampening immune-to-brain signaling.

Interesting Natural Substances

In no particular order, these are several very interesting flavonoids/carotenoids.  In the lab, they all do some remarkable things.

In humans, they also do some interesting things; how helpful they might be in autism remains to be seen.

Being “natural” does not mean they are good for you and have no side-effects.

Some of the following are very widely used and so you can establish if there are issues with long term use.  It also makes them accessible.

Quercetin (found in many fruits, numerous interesting effects)

and two Quercetin-related flavonoids:-

Kaempferol (widely used in traditional medicine)

Myricetin (has good and bad effects)

Lycopene  (from tomatoes, potent anti-cancer, does not cross the BBB)

  

Luteolin(in many vegetables, like broccoli) 

Apigenin (from chamomile, stimulates neurogenesis, PAM of GABA_A, block NDMA receptors, antagonist of opioid receptors …)

Tangeretin (from tangerines, does cross the BBB, has potent effects in vitro)

Nobiletin (from tangerines)

Hesperidin (from tangerines)

Naringin (from Grapefruit, contraindicated with many prescription drugs)

Epicatechin/Catechin  (the chocolate/cocoa flavonoids, do cross the BBB, well researched)