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CytoSorb is a Novel Application of Tried and True Technology

|Includes: Nephros, Inc. (NEPH)
Cytosorbents Corporation (OTCBB: CTSO), formerly Medasorb (OTCBB: MSBT), is in the latter stages of its European CytoSorb trial to assess the efficiency of its cytokine filter technology to reduce the levels of cytokines present during a cytokine storm. Cytokines are proteins produced by cells during infections to stimulate and regulate the bodies immunity system. However, this can be a case of more is not necessarily better. During severe infections, a human body may produce an overabundance of cytokines. This is catastrophic in that this triggers an additional product of cytokines hence the term “cytokine storm” potentially leading to sepsis.  According to Cytosorbents CEO Dr. Philip Chan, this massive production of cytokines can directly kill cells and damage organs, ultimately leading to organ failure and death in many cases. This is a major cause of mortality, killing approximately 1,400 people worldwide every day. Approximately 1.0 million patients per year have sepsis in the U.S., with an estimated annual economic cost of $18 billion and approximately 215,000 deaths. Given these numbers, Cytosorbents believes if they receive the European Mark CE and ultimately FDA approval, there will be a large market for CytoSorb™ http://www.medasorb.com/tech.htm.
 
Cytosorbents’ filter, termed CytoSorb, is being used in trials with standard dialysis equipment to removed cytokines, which are actually midsize proteins, from the patient’s bloodstream and then allows the blood to be returned to the patient’s body with a most of the cytokines removed. However, the filter cartridge is a bit more complex than the traditional filters the general population thinks of when it envisions a pretty much two dimensional filter used in air conditioners or automobile oil and air filters (the latter two, though appearing more three dimensional are actually typically pleated, mostly two dimensional filter material). A much more accurate comparison is most likely to that of solid phase extraction (hereafter SPE) cartridges such as those I have my technicians utilize to extract pesticides and other chemicals of interest from various soil, water, plant and animal tissues in the laboratory I manage. In these applications, various physical and chemical mechanisms are utilized in the cartridges to separate the sample matrices from the analytes of interest; in my case pesticides, PCB’s, antibiotics, THM’s and other organic compounds. In most of these separations of these analytes from the original media or matrix, the analytes are retained by the sorbent material(s) in the cartridges while the matrix is allowed to simply pass through the filter. The cartridge’s trapped contents are then washed or eluted from the media by either an organic solvent with a much higher or lower polarity (depending on retention mechanism) or pH adjusted aqueous (water) solution sometimes containing a buffer for subsequent analysis. For Cytosorbents’ cytokine cartridges, the elution of the analytes (here cytokines) is not important only the retention of the cytokines and the passing of the other compounds in the blood’s matrix through the cartridge unscathed.
 
There are several types of mechanisms that SPE cartridges currently in the industry employ. Please note that there are hybrids of these so this is a general guideline only. These mechanisms include, but are not limited to:
1)      Reversed phase in which a polar matrix (usually water containing) has its more nonpolar analytes of interest retained on the sorbent via non-polar interaction with the non-polar sorbent (if you remember in basic chemistry in school, “like likes like” in terms of polarities which is why water (polar) is not a solvent for oil (nonpolar)).
2)      Normal phase in which a nonpolar matrix (IE many oils and solvents) has its polar analytes removed via hydrophilic (polar, usually water soluble) interaction with the sorbent.
3)      Ion exchange in which there is an electrostatic (positive or negative charges) attraction of the sorbet to a group on the compound of interest. For example, common organic acids such as acetic acid or acidic herbicides such as 2,4-D have a positive charge in solution and may be retain by a sorbent with a negative charge.
 
The CytoSorb mechanism, however, is most likely more related to one final type of column or perhaps even chromatography rather than cartridge that I currently utilize to separate compounds of interest form its matrix.  However, I must admit that the column or cartridge we utilize is most likely a much less advanced and less efficient form of that technology. It is called size exclusion chromatography. On November 13th, 2006, Medasorb announced that it had filed a patent covering its Ultra Select™ polymer technology. “The polymer is being developed for use with medical applications requiring long term use and extended treatment times, such as nightly dialysis. Medasorb believes that its Ultra Select(tm) polymer is highly selective in extracting only midsize proteins, such as cytokines and B2-microglobulin, from blood and other physiologic fluids, while keeping the components required for good health such as cells, platelets, albumin, hemoglobin, fibrinogen, and other serum proteins intact.” http://www.medasorb.com/news6.htm. If you would note that this polymer (used, I presume, as the sorbent in their cytokine filters) is selective not via charge, polarity or other chemical mechanism but rather through size as it extracts (or retains) only midsize proteins such as cytokines and B2-microglobulin while allowing higher and lower molecular weight compounds in the biologic matrix to simply pass on through the filter. The CytoSorb resin is specifically designed to remove substances in the molecular weight range of 10-50 KiloDaltons in size, where most cytokines reside, a range that is termed the cytokine ‘sweet spot’. However, the ability also exists to change the porous nature of these sorbent beads to be able to remove other targeted compounds depending on their sizes.  For instance, Dr. Chan notes in a recent interview that they have developed novel, highly efficient resins that can remove a variety of drugs that are common in drug overdoses and other toxins from blood and physiologic fluids.
 
Please note before we continue on with this discussion that Cytosorbents Corporation has a myriad of patents covering this polymeric technology employing different pore size polymer beads in fairly narrow and discreet particle sizes which means it can target specific molecular weight compounds of interest in theory from drugs to cytokines. http://www.Cytosorbents.com/property.htm. The trick to removing the cytokines while allowing the other components in the bloodstream is the narrow range of pore sizes in the beads that comprise the CytoSorb sorbent. Typical filters, either two or three dimensional in nature, can become clogged up fairly quickly with impurities equal to or greater than the size of the pores in the filter rapidly if the concentration of the particles is great enough. However, the CytoSorb sorbent massive capacity to retain cytokines before it is “spent” is due to its large surface area available in each cartridge to retain them in its many pores. According to Dr. Chan, a single CytoSorb cartridge has the surface area of seven football fields. This is rivaled perhaps only by activated charcoal. One gram of activated charcoal has a surface area of 500 square meters. This is about 1/10th the size of a football field. However, activated charcoal (which is found in many filtering applications such as drinking water filters) is non-selective and absorbs many types of compounds and would not be suitable for a biological matrix such as blood. 
 
How familiar am I with this size exclusion chromatography? Like the three other types of SPE cartridges I mentioned earlier, I also utilize a GPC or Gel Permeation Chromatograph additionally in my laboratory as a mechanism of separating organochlorine and organophosphate pesticides from animal tissue to determine if the meat or fatty tissue has acceptable or detectable levels of these pesticides as analyzed via Gas Chromatography. In my application, the pesticides are typically nonpolar which make them fat soluble, and they bioaccumulate in fatty tissues of animals and humans. The fatty tissue sample is rendered under heat, and then filtered to remove any remaining solids. The filtrate is then dissolved in either a nonpolar solvent such as methylene chloride or a 70/30 mixture of ethyl acetate and cyclopentane. Formerly, the dilemma would have been “how do I separate non-charged AND nonpolar pesticides from a nonpolar matrix when the most common SPE methods utilize polarity differences or ion charges to separate out the analytes from the matrix?” The reverse phase, normal phase and ionic mechanisms of separating the pesticides from the matrix would not have worked especially for a broad screen of 30-40 pesticides that my technicians would screen for. Rather, we choose to employ a size-exclusion methodology in which the relatively low molecular weight and size pesticides are separated from the high molecular weight dissolved fats/oils in the matrix. The solvent mixture containing the dissolved fats and potential pesticides present is injected into the GPC and allowed to flow at the rate of about five milliliters per minute through a 15 inch glass column packed with porous glass beads. The solution flows through the cartridge where the separation of the fats from the pesticides occur in an almost reverse logic manner. The larger (and higher molecular weight) fats are allowed to pass around the beads while the lower molecular weight pesticides find themselves going THROUGH the porous beads, which is a substantially slower path. This phenomenon allows me to set the parameters on the GPC such that the first eluting compounds in the solvent matrix, which are the fats, are simply dumped to a waste container. The next eluting compounds, which are lagging by as much as 10 minutes are collected in a separate container that will contain the carrier solvent and any pesticides that may be present. This solution can either be concentrated down to aid in lowering the detection limits or injected directly into the gas chromatograph of choice depending on the analyte to be identified and quantified. Samples spiked with pesticides as a control are analyzed via this method every day with typical results of 80 to 110% recovery of pesticides. The chromatograms of the fatty tissue extractions are almost indiscernible from the solvent blanks that are analyzed as a baseline control. Meanwhile, the chromatograms of the pesticide-spiked samples have very minimal to no interference from any remaining matrix impurities and are as easy to analyze as a standard in a pure solvent matrix. In other words, almost all interfering higher and lower molecular weight compounds have been removed leaving only the molecular weight compounds in the range of the pesticide weights. It is for this reason that Gel permeation chromatography is also known more generally as size exclusion chromatography. This is the very efficient methodology as is, I assume, of the CytoSorb filtering process.
 
In conclusion, I do not pretend to know the exact mechanisms of retention the CytoSorb cartridges employ. However, it appears, the CytoSorb technology has the compactness of the aforementioned SPE cartridges and the particle size differentiation of GPC or size exclusion chromatography. These are very robust technologies utilized elsewhere for other applications on an everyday basis, and the great minds at Cytosorbents have a truly profound application that may soon revolutionize the medical field. It has the potential to save millions of lives every year due to the cytokine storms or severe sepsis caused by a myriad of infections, most notably all types of flu. In the future, it can also perhaps be used to filter out other discreet particle size or molecular weight compounds such as drugs. The current European sepsis trial should wrap up in the latter part of 2010 or early in 2011 at the latest at the current enrollment rate (though the coming flu season will likely accelerate enrollment). This will give them a stronger chance at FDA approval and perhaps even allow them fast-track designation for their trials here in the United States. If or when a flu pandemic whether H1N1, Avian or other unknown type hits; this will be another weapon in the world’s arsenal against a killer of potentially millions. I anxiously await the completion of the European trials, the CE Mark approval, United States trial initiation and/or partnerships along the way. This technology is certainly bigger than the company and geniuses that created it!


Disclosure: Long

Disclosure: Long