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biomarker gifThe secretome is a cell free fluid that concentrates the therapeutic components produced by stem cells, giving them healing powers.  The secretome can be produced from a healthy  person or one’s own cells.  The starting material can come from several sources, blood cells or fat cells are favored. The secretome is thought to give stem cells their effectiveness in various diseases.

The secretome is a collection of “paracrine” soluble factors produced by stem cells that are used for inter-cell communications. The paracrine factors also assist in tissue development, regulate normal functions, and regenerate damaged tissues.  Secretomes can inhibit some cancers. The factors are contained in extracellular vesicles and micro vesicles as well as unbound membrane particles, short bits of proteins (peptides) and cytokines.

These are important factors in degenerative, auto-immune and inflammatory diseases. That makes the secretome relevant to many therapeutic uses that include arthritis, chronic lung disease, multiple sclerosis, cancer, and stroke. Secretome is generally considered safe in humans and animals and is in several clinical trials in the United States. In 2017, a preparation of secretome was deployed in Russia for eye and skin injuries in animals.  Not available in the US, you can order Penapuh drops from an online Russian company.  Check out the 4,087 scientific papers on secretomes to really appreciate this expanding field.

One paper referred to cross-talk between the body’s stem cells and the central nervous system (CNS).  If we could decipher this chatter, we would direct CNS healing in diseases that currently have no treatment! The secretome has piqued our interest for both amyotrophic lateral sclerosis (ALS) and equine neurodegenerative disease.  We have gathered 10 research laboratories across the US, each with unique expertise, into one group,to find hope for ALS patients  Sixty-percent of our efforts use stem cell science to understand disease and how to crack the secretome language. We are holding the pedal to the metal as we explore how to get an effective therapy into desperate people and beloved horses.

wbcstem cellThe illustrations are of white blood cells in the peripheral circulation and a picture of stem cells in the flask after processing.  The stem cells are assayed for several specific markers.  And then they are expanded and some are frozen.  A research step for us is determining if starting cells, the factories to produce the secretome, should come from a healthy person or from a patient already afflicted with a disease. Are cells that have been in a diseased environment primed to respond to a curative mode? Or are cells from healthy sources more capable of sending healing messages to tissues? We have two laboratories asking that one question. The consensus opinion of the group is that a healthy cell should be the starting material.

The next unknown is the cell phenotype produced from the undifferentiated cell and how to appropriately induce the cells to communicate with the diseased body.  Stem cells are induced to produce one cell type. In disease, there are multicellular communications that are disturbed between hundreds of other differentiated cell types.  The source of tissue and cell type that are induced are key issues that must be addressed with firm scientific basis. When cells age in the laboratory, they change their secretome.  What’s old in a flask can simply mean 10 or 15 doublings. The change is a change in their phenotype.  If it takes a long time to get enough cells in the flask, their phenotype won’t be the same as the starting cell. You can already envision the changes if autologous cultures are used in some diseases, autologous means the individual donates the cells. There are obvious advantages to a specific cell background that are modified to an individual disease or unique disease process. However, once we can break the language code, we can tell the cells what we’d like them to say.

These are all issues for licensing the ultimate product for the patient.  The goal is a unified line that can become a licensed therapeutic that is safe and effective for the condition that is treated.  That means a safety study (with a price of half a million dollars for horses and more in humans).  Sometimes, if there is literature to support safety and toxicity, time and money are saved.  A dose must be determined from clinical trials.  That dose is used in a toxicity study that is mandated by regulatory agencies.  Rodent toxicity studies are about a quarter of a million dollars.  The dose titration is done by treating patients within a safety study.  If all goes well, the dose is escalated in a second or even a third trial.  Time and money.

To initiate a clinical trial the product is manufactured.  Before clinical trials are contemplated, one must have an idea of the dose, duration, and frequency of treatment.  That will have a direct effect on the formulation, and formulation studies are expensive. The next decision is packaging.  The ultimate package is cast in stone once the initial exploratory batches are used in trials.  Should it be a liquid, lyophilized, or a tablet? That is determined by the expected stability of the product.  Secretome that is liquid in a vial would need refrigeration or freezing, but consider the longevity (stability) of the final product if it was lyophilized!  When dosing a laboratory animal, a vial with a therapeutic volume of 0.1cc is great, but when scaled up to a human, or horse, one would want to avoid 20 mouse vials for one treatment!  And a horse might require 10 times the human volume, 200 mouse-vials.  Lyophilization can overcome that obstacle. All these steps total close to five million dollars…or more.

phone boothFormula and packaging are set. Manufacturing is complete.  Off to regulatory officials to decide on the best path to get into trials.  Phenotype selection is based on cell culture conditions that were worked out when we learned to speak their language. We give them the messages so they can don their superhero costumes and churn out the paracrine factors that are expected to ameliorate disease.  This is what we are working on.  If you’d like to participate, please use the following link. And keep an eye out for our progress, we will report it here. Click here to donate.

ureaA chemist will tell you that molecules changed the world.  The book, Molecules That Changed The World, by KC Nikolaou and T Montagnon will have you believing it by page 333. The synthesis of urea is credited as not only the earliest contribution to organic synthesis, “but as the single most important blow to the vestigial theory” held in the 1700’s. “Despite using the terms daily, chemists often forget that the classifications of inorganic and organic compounds originally arose from the theory of vitalism, which divided matter into two classes based upon the response of the material to the application of heat.” Plants burn, rocks don’t.

What was apparent, and explained by Nobel prize winner (1902) Emil Fischer, is that principles such as  asymmetry, the intrinsic importance of organic chemistry in understanding biological mechanisms, and the art of extracting, identifying, and synthesizing naturally occurring compounds and their analogues for medicinal purposes are important. These concepts have been the foundation of our understanding how to treat horses with equine protozoal myeloencephalitis and polyneuritis equi.  Our story is similar to the story of Aspirin, the most successful medication in history, because treatments we use are steeped in history, synthesis, and hormone chemistry.

Aspirin’s story is using the medicinal properties of a natural product that is optimized through subtle chemical manipulations, and began 3500 years ago.  Egyptian physicians advocated salicin, in herbal preparations of myrtle bark, as a remedy for rheumatism and back pain. Humans are not the only ones that seek this chemical from the bark of trees such as willow.  Supposedly, forest monkeys and apes nibble on the bark of these trees for pain relief.

The first ever clinical trial used pulverized, dried willow bark that was given in a tea or beer, to 50 patients, and published by Edward Stone in 1763. Advances came in 1828 when Johann Andreas Buchner removed tannins and other impurities obtaining a relatively pure sample which he called salicin.  Ten years passed until Raffaele Piria made the next advance by hydrolyzing salicin, splitting it into its sugar and phenolic components.  Later, he succeeded in oxidizing the hydroxymethyl group of the phenolic fragment to make salicylic acid.  In 1853 another chemist, Charles Frederic Gerhardt prepared acetylsalicylic acid and with that step, Aspirin was born. The best was yet to come.

In 1859 Hermann Kolbe synthesized Aspirin by heating the sodium salt of phenol in the presence of carbon dioxide under pressure and commercialized the process, laying the foundation for todays pharmaceutical industry. What came next is the realization that salicylic acid wasn’t a panacea because there were unpleasant side effects.  The effort was on to modify the chemical structure in order to obtain a derivative that might be devoid of the undesirable side effects such as foul taste, mucosal membrane irritation, vomiting, and ulcerations.aspirin

The breakthrough came in 1897 when a chemist at Bayer, Felix Hoffmann, synthesized acetylsalicylic acid. Bayer now owned the miracle drug they trademarked as Aspirin. Aspirin was used in experiments to determine the mode of action  increasing the knowledge of pain and inflammatory mechanisms. Prostaglandins were identified in 1935 and the cascade of biochemical reactions associated with inflammation were revealed. Finally, in 1971 three scientists linked the ability of Aspirin to inhibit a critical enzyme step in the prostaglandin pathway.

The protagonist Aspirin is heroic.  However, the story isn’t complete without considering the antagonist.  New research spawned the new generation of ‘super-analgesic’ drugs such as Celebrex and Vioxx that didn’t have the side effects of Aspirin.  Yet, unexpected side effects were noticed, like heart attacks and strokes with long term use.  The popular drug Vioxx, with $2.5 billion dollar sales, was removed from the market in 2003.  Isn't it ironic that Aspirin is frequently used to reduce the incidence of myocardial infarction and stroke? The positive effects of Aspirin on heart disease isn’t through the prostaglandin pathway, but by inhibition of another product from the arachidonic acid cascade, thromboxane A2, a hormone discovered in 1975.

Aspirin is a synthetic chemical derived from natures molecule that has profound effects on prostaglandin mediated inflammatory pathways and in an alternate pathway, inhibits the clotting cascade driven by a hormone. Our path, clinical trials using hormones and synthetic chemical-mimics to modulate pathologic inflammatory pathways (non-prostaglandin mediated) are similar to Aspirins story.

Presently, we are concerned with chemical modifications of molecules. As you can see from the work on Aspirin, there are unexpected effects with the slightest modification of a molecules structure.  Structural changes can be non-enzymatic (degradation) and/or enzymatic.

Non-enzymatic modifications are made by facilitating chemical breakdown products by manufacturing processes, storage conditions, or compounding.  Non-enzymatic modifications of a molecule can induce anti-inflammatory effects,  make some drugs inflammatory, and in some cases elicit no effect at all.  Outcomes depend on the modification that is made to the molecule, intentional or not.

We know that a simple amino acid change, such as a valine, in the protein sequence of a hormone can elicit profound effects on receptor binding, increasing potency and duration.  Receptor binding affects the outcome of a treatment.  We are also looking a the effects of chemical modifications on hormone receptor binding. We will put all this together in a story and should have quite a story to tell.

vitamineThere are some things to consider when supplementing and testing  vitamin E levels in horses.  Why do it?   The need for supplementation probably stems from deficiencies that are associated with degenerative myeloencephalopathy, equine motor neuron disease, vitamin E-deficient myopathy, and nutritional myodegeneration—or a diagnosis of EPM (unproven).

You decided to test instead of supplementing because conditions that warrant supplementation are rare, supplements are  expensive, and over supplementation may not be benign.  There are high-performance liquid chromatography tests that are as expensive as they sound. There are also enzyme linked immunosorbent assays that are used to capture vitamin E (alpha-tocopherol) from body fluids.  The when and how samples are collected and handled are important to obtain accurate values from ELISA testing. There is a study funded by Kentucky Performance Products, KPP--the makers of Elevate, that determined the effects of feeding different formulations of their products on serum, CSF (cerebrospinal fluid) and muscle tissues.  They used liquid chromatography methods.

It is necessary to test to find out if the horse has a deficiency. How do you test for vitamin E in serum? When using the more common ELISA test on serum, the collected blood should be allowed to clot for 10-20 minutes at room temperature and then removed from the tube carefully without transferring any sediments.  The serum should be protected from light during storage or transport to the lab. When plasma is used, the sample should be collected in an EDTA or sodium citrate tube.  The plasma should be removed from the cells within 30 minutes and no sediment should be removed with the plasma. Samples should be tested within 5 days, stored cold., and protected from light.  Samples stored frozen can be tested within one month.  If samples are stored at –80 the test can be used within 60 days.  Hemolysis will change the results of the vitamin E test.   The time of feeding vitamin E, the horses diet, and other supplements that are added to the diet can affect the bioavailability of  vitamin E and its detection in body fluids.

Horses get natural vitamin E from green forage.  Horses that are confined to a stall or they are required to abstain from fresh grass, are at an increased risk to develop deficiencies.  The normal value of vitamin E in the horse is > 2 micrograms/ml.  A value less than 2 micrograms/ml would mean the horse was deficient in vitamin E. There is no information available for over supplementation and the effects of vitamin E toxicity in horses. Horses show individual variation in the ability to absorb vitamin E from supplements.

The bioavailability of vitamin E when supplemented is important.  Natural vitamin E is composed of one stereoisomer while synthetic vitamin E has several isomers, of these isomers only one is readily available to the horse. If the neurological disease is due to a deficiency of vitamin E in the central nervous system the synthetic acetate form of vitamin E has no impact on the CSF levels according to KPP. The KPP study claims the micellized water-dispensable form, a liquid, is as much as 6 times more bioavailable than other synthetic forms and allows a rapid rise in serum concentrations within 12 hours.  Levels of 10,000 IU/day can increase CSF levels within 2 weeks.  Likewise, serum levels decline rapidly after discontinuing supplementation.

The bottom line for supplementation is that horses without clinical signs of deficiency can be supplemented with a synthetic form at 10 IU/kg body weight per day and serum levels are expected to  increase after 47 days or so.

If neurological disease is present, a diagnosis of equine motor neuron disease or vitamin E deficient myopathy (diseases that are responsive to supplementation), are expected to respond to vitamin E.  A regime of supplementation of 5000 to 10,000 IU vitamin E given daily is the standard protocol.  Remember, the water dispersible form is more appropriate for these conditions when it is desirable to increase levels quickly.  There is a rapid decline in serum values when horses are removed from some forms of vitamin E supplementation and a tapering regime may be appropriate. Another point is that there is no correlation between serum and muscle levels of vitamin E. If the disease is vitamin E-deficient myopathy an alternate protocol may be required.

To summarize, if a horse is suspected of a low vitamin E level due to disease or diet, the serum and/or CSF should be tested.  The normal vitamin E value is >2 micrograms per ml of serum.  Abnormal values will be returned if the sample wasn’t collected and handled properly or if the sample was held too long, even if it was frozen. Light is detrimental to vitamin E in the serum and in supplements, samples need to be protected from inactivation as soon as they are collected.  Samples that contain microparticles or are hemolyzed will give an abnormal value.  It is important to select the most appropriate dose and formulation to achieve normal and sustained values when supplementing.  The rate of decline in the serum may depend on the form of the supplement that is given. And finally, there is an individual variation in the response to supplements so individual protocol should be designed to achieve a therapeutic response.

 

funny rat We predict that in the year 2050 there will be no animal experimentation.  That includes animal testing for drug licensing.  Today, 100 million animals are used in experiments, about 13% of those are involved in studies that are required to license drugs.

Happily, a large step toward the goal of providing alternatives to terminal animal studies was published recently.  The FDA commissioner Scott Gottlieb, MD, stated that the FDA is trying to reduce, replace, or refine the use of animals in research. This is heartening news. We hate the current standards for new drug development that require euthanizing animals in studies. Even if a drug is commonly used in other species—if a new indication is under investigation, terminal studies are required. We’ve repeatedly argued against this position.  We lost the debates. But FDA is not insensitive to the issue.

On the horizon are simulation models that include in vitro (non-animal) dissolution tests and computer modeling that will replace animals in some studies.   A first step to animal-alternative testing is the validation of simulation methods designed to show the equivalence of a proposed generic formulation to an approved drug. We can predict a future that allows these same models for new drug development.  Currently, FDA requires data to understand how a drug performs in a live animal.  In our case, mountains of data obtained from goats and dogs and cats won’t substitute for horses.

A part of the generic licensing process involves an animal drug developer to perform “bioequivalence studies”.  These live animal studies compare the originally approved product and a proposed generic version to see if they are similar enough to link them in terms of safety and effectiveness. These are expensive studies and these studies are ignored by compounders.  Another subject for another day.

Back to the good news. There are studies afoot at FDA that will create a physical model to simulate properties of a specific drug when tested in live animals. The proposed studies use dogs. The live animal data will validate the simulations for anti-parasite formulations that are currently used in dogs.  If the studies are successful, animal drug sponsors may use these data to aid in designing in vitro studies.  And that will save the lives of animals.  The initial studies used to equate the simulation to the live animal are designed to use a small number of anima.  The animals are subjected to minimally invasive procedures, perhaps as simple as a blood draw. And more good news, the lab-dogs will be adopted as pets.

The FDA is encouraging drug companies to develop and validate alternatives to animal testing, including in vitro dissolution tests and computer modeling. For now some drugs may still require live animal studies, and these studies may require euthanizing the test subjects to support a new animal drug. We’d like to get back to the future.  We developed a simulation method that is designed to test the equivalence of a proposed generic anti-protozoal medication against approved drugs.  As licensed EPM drugs age out of patents there will be an opportunity for licensed generics. We are refining an animal model that can prevent excessive horse use and will design and test the validation parameters.  I suspect that it will take a year or so to get the rubber stamp on a validated model.  We would like to be instrumental in the movement to decrease animal experimentation.

In January of 2017 CBS Evening News (Lee Cowan) reviewed a book by Doug Preston…The Lost City of the Monkey God.  I won’t spoil the story for you--it’s a must read if you are obsessed with parasitic protozoa as we are.

The Lost City is a captivating true story and reveals a curse bestowed on man.  Legend has it that this ancient and magnificent Lost City, located in the rain-forested mountains of Mosquita was cursed.  Mosquita is a 20,000 square mile section of Honduras and Nicaragua.  The inhabitants lived in the area between 2600 B.C. and 1800 B.C and somehow incurred the ire of the Gods.  The angry Gods brought on a series of catastrophes.  Diseased and devastated, defeated inhabitants melted into the jungle, leaving everything behind, perhaps to appease the Gods.  The Lost City was not Mayan, yet it existed at the same time as the Mayans. These prosperous civilizations vanished at the apex of their existence.

Since the Lost City was first reported by Cortés in 1526, explorers and charlatans searched for them in vain.  That is, until the invention of LIDR in 2010.  The modern three-dimensional radar technology enabled mapping of the ruins beneath the dense canopy. It wasn’t the ruins that revealed the curse--the explorers may be cursed.

If you ask bestselling author Doug Preston, he’ll say he doesn’t believe in curses. And yet, here he is, being treated for leishmaniasis that he contracted while on expedition to The City. Half of the expedition party have contracted leishmaniasis--I believe at least one member died and some are not able to get treatment. The parasite is transmitted by a sand fly, the bite releases the protozoa into the skin and causes lesions that may take years to heal--cutaneous leishmania. Effective treatment at this stage may be life-saving but there are no current satisfactory treatments for cutaneous leishmaniasis.

As disease progresses, the parasite migrates to the mucous membranes of the mouth and the nose, and eats them away; this is mucosal leishmaniasis. The nose falls off, the lips fall off, and eventually the face becomes a gigantic open sore.  Organisms move on to the the organs, and visceral leishmaniasis is often fatal. Visceral leishmania is the second leading cause of parasitic death worldwide. The treatment is rough because it poisons the patients organs.  Patients die from treatment complications of renal or liver failure. Dogs get leishmania and are often euthanized when the diagnosis is made. Horses get leishmania, two cases were reported in Florida. When you read the book, you will realize why the curse reached Western Civilizations. We lead lives that prevent us developing immunity to this developing 20th century threat.

We are invigorated because there are some novel studies to report, and they parallel our investigations.  A promising combination therapy showed a complete clinical cure in 75% of the patients (human) with cutaneous leishmaniasis! The study also reported that 10% of the patients had a partial improvement and alas, the remaining 15% had an underlying chronic diseases and they had no response to the treatment. There were no cytotoxic effects associated with the drugs in the range of the experiments.  The mechanism of action of the drugs predicted promotion of some cytokine gene expression levels and reduced others.  The experiments supported the anticipated changes in cytokines. This is great news because researchers are understanding how to target parasitic protozoal diseases by modulating the response to infection. This has been our mantra for years.

What gives us satisfaction is revealing that the patients were unresponsive to the anti-leishmania drug, Glucantime, but responded when when Glucantime was combined with the immune modulating drug levamisole. Here is some evidence that end-stage unresponsive parasitic protozoal disease in patients is treatable when combined with levamisole.  The absence of cytotoxicity in the treated patients when given the combination of drug/levamisole is also highly noteworthy.

In the last 100 pages of The Lost City, Doug Preston suggests the Curse may be the downfall of Western Civilization--a result of invading parties driving civilizations to extinction by not so unintended consequences and bringing back malady-in-kind.  While we doubt the impact of our studies with levamisole (in horses with EPM and polyneuritis equi) will save the world, we think our efforts are worthy. Support us in our research.  You never know where it will lead.

leishmanialeishmania 2

Leishmania parasites are shown in a host cell and in tissues.

Photos were taken from goedleven.be.