Yes. It seems so. Please check the ref. (Ref- V. Bocci - ISBN 1402005881)                      Clotrimazole, a specific Ca2+-activated K+ channel (Gardos channel) inhibitor able to reduce the deleterious dehydration of sickle erythrocytes, is being tested and ... human life-span is accompanied by an increase of degenerative diseases

I guess your question is on the impact of RBC volume/hydration on the clearance rate in ... healthy humans, am I right? Inhibition of Gardos channel may have some impact on hydration / RBC volume, although this channel is rather inactive in healthy RBCs, but may be activated by e.g. Ca2+ overload or shear stress. In cells of patients with sickle cell disease, which are dehydrated due to the Ca2+ overload, clotrimazol may support certain re-hydration (see  the old Alper's paper attached). However, there is very little evidence that RBC volume is decisive for selection of healthy human RBCs for clearance. Apart of Mr Higgins and his model (see the second paper attached), not much is supporting this suggestion. Selection for clearance is rather dominated by oxidation stress, band 3 clustering and other well-defined processes (see open-source paper http://www.ncbi.nlm.nih.gov/pubmed/24399969.

Potential usefulness of Gardos chanel inhibitors, mostly Clotrimazole and ICA-17043 (Senicapoc), in sickle cell disease (SCD) has been extensively studied both in SCD mice and patients, most notably by the groups of Carlo Brugnara and Lucia De Franceschi. These two compounds have been shown to reduce sickle cell dehydration and haemolysis and to increase total haemoglobin. Very unfortunately, they were shown also to be inefficient at reducing the occurrence of painful vaso-occlusive crises, the hallmark of the disease. Consequently, research concerning these types of drugs in the field of SCD has considerably slowed down over these past years.

Investigating the role of dehydration on RBC clearance appears somewhat tricky as it is expected that above a certain dehydration threshold, the shrunken cell would become immediately trapped and cleared out from circulation. Below such a threshold, the cell may be pitted during its passage through hepatic and splenic sinusoids or further decreased in volume upon releasing vesicles. Tagging signals for clearance (differing both in quality and quantity) would depend on the amount of Ca load. At high loads (as may occur with sickle cells), additional membrane mechanisms other than Gardos channel activation (for instance PS externalization or diacylglycerol formation) may prevail, contributing to the overall thrombotic pathology. Having this in mind, it seems possible that CLT and its derivatives may become not efficient in ameliorating the vaso-occlusive crises of sickle cell disease while being fully inhibitory of Gardos channel activity.

Very interesting comment Pedro.

Just a precision: yes indeed PS externalization is observed on sickle RBCs, yes indeed there are some biological markers of subintrant activation of the clotting system, but SCD is a vasoocclusive disease and there is no formation physical clot. In other words, SCD is not a thrombotic disease.

Totally agree oncalcium being an important player in SCD pathophysiology. For that very reason we are now preparing a trial to block not Gardos channels, but NMDA receptors in attempt to get some benefit for this group of patients here  in Zurich.

Circulating platelets aggregates have been found concurrent with the severity of vaso-oclusive crisis in some SCD patients, especially at the onset of acute state (for instance see attached file). A thrombus formation may thus expected. Can this be attributed to an altered platelet physiology only?

It is hard to say.  I would say that the alterations in platelet function are in part secondary  to the excessive hemolysis, but not only... They are also responding to teh stimuli activating the NMDA receptors in RBCs; as they also possess some (http://www.ncbi.nlm.nih.gov/pubmed/24593912). So, blocking the receptors will be good for that process as well. 

Intracellular Ca is a key player in SCD pathophysiology. The approach of blocking NMDA receptors in sRBC to ameliorate the vaso-occlusive crisis of SCD patients seems an interesting one. However, other distinct mechanisms (i.e. Psickle and Piezo 1 channel activity) also contribute to the massive Ca entry upon sickling. Combined therapies aimed at targeting not only NMDAR but both Gardos and mechanosensitive channels may appear rather promising.

I agree, targeting more than one channel would be an option, but so far we only have sinecapoc, and I am not aware of any clinically usable Piezo blocker, non-toxic and safe to use without substantial side effects. Do you know any? There are also VDACs, TRPC6 and Cav2.1... Quite an array. I guess they respond to different stimuli to drive Ca2+-dependent response. We have to start somewhere :). 

Dear Anna,

I am afraid I have no knowledge of any blocker of mechanosensitive (MS) channels clinically safe and without side effects, being currently employed for medical care. I am also unaware of pharmacological developments or clinical trials with such a type of compounds. Although a combined therapy with blockers of both Gardos and MS channels (as well as NMDA and voltage-dependent Ca channels) for SCD treatment can be promising, the ubiquitous presence of most of these channels in human tissues is a serious problem to be considered. Moreover, so little is known of the detailed molecular structure of above channels in human erythrocytes, that an approach to employ specific blocking compounds must await further research. An option that seems viable at shorter term may be to encapsulate a cocktail of blockers into liposomes, nanovesicles or nanoparticles and specifically targeting them to sRBC. It may be worth trying this on SDC models.

I agree, the problem with delivery  and specificity is not easy to solve, particularly for such channel as Piezo1 which seems to be of vital importance in a number of cell types. I am not a pharmacologist, they definitely know more about the state of art in this field. For me the brain is the only organ which can be easily protected by making the drug impermeable for the blood-brain barrier. The heart, kidneys and other organs will not be protected that way. Unfortunately, mice have proved to be a useless animal model for us, they just do not have NMDA receptors in their RBCs.Rats do, but there is no model available so far for the SCD. What I think we need now is a proof of principle as to whether the Ca2+ hypothesis will work in vivo in some way.  

Pedro, your suggestion to encapsulate bioactive molecules and targeting them to sRBC specifically is extremely attractive! Any suggestion on how this could be accomplished?

Dear Jacques,

I think very naively that targeting sRBCs may be attained by covering the encapsulating carrier with anti-glycophorin A (monoclonal?) antibody and delivering it intravenously. Various problems seem to arise from such an approach. First, filtering of carrier by spleen and liver sinusoids should be overcome. This, perhaps, may be achieved by reducing the carrier size. Secondly, to what extent would the immunological defense of the recipient interfere? Another important problem concerns the intracellular release of the bioactive compounds. Once attached to sRBCs, how the encapsulating carrier can be induced to discharge its content? Will just suffice the presence a fusogenic molecule (diacyl-glycerol?) on the carrier surface?

Dear Pedro, to avoid filtration can this be done perhaps extracoporeally? For me pegylation of the antibody sounds like an option. Did you try it in an animal model? 

Dear Anna,

I am not quite sure that extracorporeal perfusion may do the job. Carrier filtration by both spleen and liver will still occur.  On the other hand, I believe that using glycophorin A antibodies may be a good option to tag sRBCs. To my knowledge, this glycophorin is present in red but not white blood cells, thus becoming an "ideal" target. Unfortunately, I am not familiar with animal models and have no facilities to work with such models. Still, the idea may be worth trying.