Pentosan, donkeys and the articular cartilage.

ou guys know I dig the science. No, I'm no PhD and I don't own a lab coat (hmm.. now there is a Christmas

present idea, how about a white lab coat with the insignia embroidered on it?).


I've held off on the sci in the last few weeks, but tonight it's a bit of a science fest!




_{Yeah I had that same expression a few times as I read the main paper (see Ref 1 in Ref section below).}



Tonight, another peer review and this one is very telling. The science is full on but I will break it down so hopefully most of you will get a good understanding with what exactly it means, why it's good for us and what the future might just be for us (not advice, we don't know how this will pan out) (Though I'm getting some pretty sharp clues).

It will be longish so strap yourself in. It won't be all words and definitions and dry paragraphs, there will be quotes and a picture or two. As we wait for milestones to get checked off....here is a nice sampler of what we are really dealing with !


As always, please do enjoy!



INTRO


The peer review starts off with a concise summary of OA. It mentions clearly that OA, particularly in equines, can be classified as a "group of diseases". The progressive deterioration of the articular cartilage is accompanied by changes not only in the joint but the surrounding tissues and fibres, We have heard this a few times in the past referring to the all encompassing nature of this disease.


The intro also covers the advent, in many cases, of synovitis and joint effusion and often clinical symptoms such as pain and dysfunction.

In a subsequent paragraph the researchers go on to postulate that some of the mechanisms of action of PPS appear to include:



Improving the nutritional supply to the joint


Increases the biosynthesis of the extracellular matrix


Inhibition of proteinase activities


Enhances the TMP-3 levels on the cartilage


Inhibits the inflammatory activities if IL-1 and TNF-α




The entire hypothesis of the study was that the outcomes of the donkeys treated with PPS would be more favourable than the control donkeys.

The study protocol was as follows:




So to reiterate, OA was introduced in all donkeys until Day 56 in the right carpal joint. The left carpal joint was left untouched in all donkeys and thus served as a control.At day 70 PPS was introduced to half the donkeys. The other half acted as the control group.


RESULTS

Mate, take a look at these results.


The control donkey's lameness score did decrease somewhat at day 112.What happened to the active (PPS) donkeys? Well at day 77 (7 days after the first course of PPS) this is what took place, I quote directly from the paper:


"In PPS group, lameness begin to decrease from day 77 (1 ± 0) till return to the baseline at day 105".


...here are the words I want you guys to remember... "...till return to the base line...".


P value?

Here is the statement:

"There was a significant difference between the right forelimb of control and PPS-treated groups at study day 77, 84, 91, 98 and 105 (P < 0.001)".


Take a look at the three measures of Lameness, Circumference of the carpal and final Flexion angle:





In terms of Gross Pathology (Think of this as the macro observations of how the disease progresses) the following observations were noted:


"In the PPS group most of the lesions were limited to the C3 region. However, in the the control group partial and full thickness cartilage erosion distributed all over the joint region".



THE SYNOVIUM

One day I will dedicate an entire Mozz weekend post to the synovium, but for the background for the next result observed from the donkey research, let's first take a minute to learn a fraction more about the synovium.

We know that the delicate synovial membrane is highly vascular. To the uninitiated, what does this mean? It means that there is large amount of blood vessels and capillaries that literally feed into the synovium. Why?Because this is how the nutrients are literally fed through from the blood INTO the synovium as well as the other way.


"The highly vascular nature of the synovium allows it to act as the conduit and regulator for inflammatory cells and peripheral mediators that influence metabolic processes, especially in the release of catabolic substances affecting the joint". ²


For further background, a haemorrhage is an escape of blood from a ruptured blood vessel, we don't want this!

How about this for a quote from the researchers:

"Synovial membrane haemorrhage improved in right PPS joints (0.67 ± 0.47) compared to the right control (2.67 ± 0.47). There was a significant difference between the right joints in both groups (P=0.003)".



And speaking of maintaining a lovely synovial plexus (surrounding of synovium with blood vessels):


"Synovial membrane vascularity was increased in right PPS joints (1.67 ± 0.47), and right control joints (3.50 ± 0.50) compared to left control joints (P < 0.001). Right PPS and control joint were statistically different (P=0.012)".


'Subintimal fibrosis' is a new one I'm hearing for the first time tonight....we will get to the definition in a sec. You guys know that the body's many natural process must be in equilibrium, they must be balanced. It applies to nutrients, it applies to blood cells, even inflammation. Processes need to be in balance. Get it out of whack and you will be in trouble. Simple.

Fibrosis is what occurs when there is an excess of connective tissue that accumulates. Of course we need connective tissue., it should be strong, it should be balanced, it should have the right nutrients fed into it and importantly it has to also have the right quantity of nutrients fed into it. But, if there is too much then joint stiffness is what results! ³

Fibrosis not only occurs in the articular cartilage though:

Tissue fibrosis occurs in various tissues, including the liver, kidney, heart, and others, and it eventually leads to organ failure.⁴

But when it does occur in the articular joints, well it plays a critical role:


"Fibrosis also occurs in the articular joint during the progression of OA and plays a critical role in OA pathogenesis and progression as well as cartilage destruction".⁵


As a quick aside, the above reference documents the culprits that all lend their hand into synovial fibrosis, it reads like a who's who in terms of how PPS addresses these culprits...its for another detailed post some time in the future:


"...the following factors are discussed: TGF-β, connective tissue growth factor, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2, tissue inhibitor of metalloproteinase 1, A disintegrin and metalloproteinase domain 12, urotensin-II, prostaglandin F2α and hyaluronan".



Getting too sciency? Just know that PPS has at least some decent amount of efficacy against things such as MMP, TIMPs and certainly the increase in higher weight HA. (HA being a favourite of mine).


Let's get back to the donkeys!


While the PPS donkeys did experience an increase in synovial fibrosis to the tune of 1.33 ± 0.47 , it was less than the control dokeys; 2.33 ± 0.47. The PPS and control joints were statistically different (P=0.014).



MORE?

You want more proof? Guys? Long term holders of Paradigm stock?

The above is nothing compared to the next Mozz^® Paragraph.


Where is my printer and a wall frame...?


Quick primer first!

The researchers conducted Light Microscopy of the articular cartilage. This is the cartilage that displays both viscous and elastic properties. The thin layer provides the perfect structural framework but also has the flexion when stresses are temporarily applied.⁶


Firstly p values, the below observations were seen with the control donkeys -v the PPS donkeys,


"...the histological evaluation revealed a significant difference in OA score for the right control joints compared to its left normal control joints (P < 0.002)'.


In the control joints the lesions were characterised by denudation (fancy word for worn out or wearing out) , a complete erosion of hyaline cartilage to level of subchondral bone accompanied by granulation tissue.


Paradigmers, the researchers observed that some samples of the NON PPS donkeys had changes in the contour of the articular surface characterised by microfractures and deep layer cyst formation.

HOWEVER, in the PPS donkeys, they saw that there was a well-developed layer of typical articular cartilage containing chondrocytes in the deep and middle zone.

In the virtually impossible case that the above paragraph doesn't impress you...how about these quotes directly from the paper:


"Chondrocytes deep within the cartilage layer had continued to undergo the typical sequence of differentiation".

(Mozz Note: remember it's the chondrocytes that are cells that literally spit out new extracellular matrix and collagen that are used to maintain the precious cartilage!). ⁷


"There was evidence of replacement of the dead chondrocytes and resorption and reconstitution of the extracellular matrix within the repair tissue".


"Sections with partial defects (vertical fissures) had no healing evidence in control group, however in PPS group; there was an evidence of minimal extracellular hyaline-like matrix developed between fissures. The right joint was significantly different in histological score between PPS and control groups (P=0.003)".



I wasn't going to go into the images but they are too compelling....*clears throat*...ok let's do this:

Fig 1 below depicts the NON PPS group, see that big white line/crevice going all the way down to the bottom? See that number 3 I've circled in red? Well this aint good. This means the cartilage has fractured and its gone all the way down to section 3. What the heck is section 3? The subchondral bone. The donkeys in question aren't in a good place. (That's not my opinion, thats a bl**dy fact)


Figure 1 - Control donkeys - no PPS



Please, let's go to a more cheerful place Mozz...sure, let's take a lil' look at the PPS donkeys, yeah the ones that after a few months have a bit of a skip in their stride (enthusiastic leading statement).



Figure 2 - PPS donkeys


I don't think I can express the above observation (Fig 2 above) any clearly or better than the researchers themselves, here is their interpretation of the Happy Place that the PPS donkeys found themselves in:

This is one of the clearest evidence we have at the micro level of what exactly our pentosan is capable of. I simply cannot express to you what the above means. Pentosan is literally proliferating, or at least assisting, in the growth of new cartilage !!!




Mozz...mate, love your enthusiasm and passion for all things PAR...but this ain't Par my friend...Does Par have any evidence themselves and please don't make an ass out of you and me, I want human evidence!


Gee, you aren't demanding at all are you?


I give you not one but two pieces of HUMAN evidence.

The first (red box below) is Cartilage volume...Placebo continued to DETERIORATE by a massive 4% over 6 months.

Err Mozz, sorry to interrupt, but 4% is not a lot.



Mate, it is huge...it was over just 6 months! 4% degradation of cartilage volume as a mean over the cohort isn't good....

What did our iPPS do?



It improved the cartilage volume ...by what, something like positive 4%?









Try 21%

(See below)⁸



The other second crazy incredible bit of HUMAN evidence was the green box above...yeah yeah we all know the fantastic 4...(new to us? I will re - post the Fab 4 post further below this post if you want to learn more about them and their direct relevance to what our molecule can do).






The green box above gives me TWO incredible markers that are directly relevant to this donkey observation study...namely CTX II and our friend C2C. How are these relevant?

For that I will reference a paper that was co -authored by an assoc. professor, namely Anne Bay-Jensen, I think Dr Bay-Jensen has some association with one of the world's leaders in the OA space, Dr Virginia Kraus. At the very least they have written a number of papers together. Dr Bay-Jensen wrote the following:


"Biomarkers indicative of collagen II degradation are CTXII, Coll2-1, C2C and C2M". ⁹


In other words, CTXII and C2C are markers of cartilage/collagen turnover, yes two of the fabulous four that PAR recently reported on in our Phase 2 study also called 008. Our trends in 008 are aligning nicely here.




A diagram from the Bay-Jensen, Anne C paper. The blue boxes indicate cytokines/markers PAR have reported about in the past to us holders.


Fellow Holders of PAR stock, there are many things I await for in terms of future news from PAR...just one of them is the Peer review where I get to learn just how consistent the drop in CTX II, a most prognostic biomarker of OA, was in our 008 study compared to what we already saw in our 005 study. I want to to take that report, print it off and carry it to every future OA and Rheumatology seminar that I attend, every future Pain seminar and every future Doc meet and physically show it to them. Trust me, I will go to some of these future seminars. I want to meet that French Doc I saw with the lovely accent and show him more MRI's...




EVEN MORE EVIDENCE, AND BACK TO THE DONKEYS...


Yes I got a bit side tracked there, but I think it was worth it...back to the main paper.

The researchers applied a scanning electron microscope to the synovial membrane and found this:



"...control group was characterized by loss of synovial villi, deposition of cartilage fragments and formation of cuboidal crystals adhering to the synovial membrane".

"Right PPS joints shows mild changes includes fibrosis but with presence of synovial villi in some regions. There were significant differences in synovial villi destruction between the both groups (P=0.001)".



For the next quote again we need some background. The term cellular infiltration refers generally to the presence of inflammatory cells. Sure, we need some of these, these cells trigger our body's response. The problem we get is when a chronic inflammatory response is triggered and essentially goes cyclical, ie it doesn't abate. Again, we don't want this....what did the researchers comment about this?


"Synovial membrane cellular infiltration was increased in right control joints".



How much? (3.33 ± 0.47)

And PPS? PPS treated joints (1.33 ± 0.47) both groups were significantly different (P=0.003).


An electron microscope was also used on the actual cartilage surface between the two cohorts of donkeys:

In the Control joints there were partial and full thickness fissures. In some instances these were so deep that they made it all the way through to the subchondral bone.

However, in right PPS treated joints these defects were fully filled with repair tissue. "This repair tissue was rough and acellular however in some areas, evidence of chondrocytes is revealed by the occurrence of pits". There was a significant difference between PPS and control right joints (P=0.001).

Ok lots of words again, time for some more pictures!


Crystallisation in terms of the elements such as Calcium, Phosphorous and Magnesium occurs as a result of broken tidemark and the exposure of the subchondral bone. To me, it is a biomarkers, get these crystals appearing and you know you aren't in a good place...check out the picture below where I have introduced orange circles to highlight the presence of the crystals evident in the non PPS group. These crystals weren't as prolific in the treated group



(Single left click above image to expand if they are small)

The following statement from a different research team again also comes to a similar conclusion:


"Crystals are common components of synovial fluids from degenerated joints and often accompany unusually severe cartilage destruction".¹⁰




(Single left click above image to expand if they are small)

Ok the above might look like a Mozz expedition to The Grand Canyon or perhaps out the backyard looking up at the sky at night...but bare with me....here is my explanation...then we will have the researcher's words...


The left is the Control, no PPS administered...the right is us, PPS administered.
Look for the black arrows in rows A and B...these are pointing out large (macro) cracks...we don't see these in the PPS group
Check out the black arrow heads in Row C, these refer to micro cracks in the Control group.
The white arrows in the PPS group show new filament formation (See Mozz Speak Appendix A) and I wasn't kidding about seeing stars; the black stars in row F incredibly show new tissue repair.


Paradignmers...I want and you want, new tissue repair.



Here is how the researchers put it:

"Representative Series of magnified images of the articular cartilage SEM for right control joints (A-C) and right PPS joints (D-F), Black arrows refers to the large macro cracks, black arrow heads refers to the micro cracks, white arrows refers to new filament formation, black stars refers to new repair tissue, white arrow heads refers to pits with underlying chondrocyte and white stars refers to old cartilage".
The cuboidal crystals are formed due to rising of P and Mg concentration on the control group however it is not found on the PPS group due to lowering of it's concentration with PPS treatment.




CONCLUSION/DISCUSSION

The discussion at the end of the paper is quite a read, the standout parts are as follows:


"In the present study, the dosage administered (3 mg/kg, once weekly for 4 weeks) was effective and elicit a therapeutic response without adverse effects in all clinical variables in this model".



We want safety as a priority, but we are getting efficacy too. This has not been observed to date with any other drug in the OA space.

The researchers propose a MOA such as the following:


"Improvement in the synovial membrane histological score and reduction of cartilage fragments size in PPS treated joints indicates that PPS enhances the phagocytic activities of the mononuclear leukocytes to engulf the foreign bodies".


The authors also allude to the fact that it could be Pentosan's action on the Mesenchymal cells that result in the production and repair of cartilage:


"Neochondrogenesis can be stimulated by PPS as healing of full thickness cartilage defects benefits from the production of granulation tissue by subchondral mesenchymal cells. This tissue matures with time and undergoes metaplasia to fibrocartilage, or hyaline-like cartilage".


"Our study is the first to examine the healing of partial and full thickness cartilage defects. Examination of the articular cartilage with SEM was able to detect improvement in PPS joints. Disappearance of cartilage erosion in PPS treated joints and the appearance of a pitted surface suggests that a newly layer of matrix is largely formed".



Finally they conclude with the following statement:


"These data clearly demonstrate the ability of PPS to stimulate the biosynthesis of components of the extracellular matrix accompanied by limiting their degradation by its anticatabolic effects. These beneficial pharmacological activities of PPS have resulted in its widespread use for the treatment of osteoarthritis in both veterinary, and human practice".



I, for one indeed want to see our iPPS used wide-spread in society, but for new applications like OA, inflammatory based indications and of course, pain in general. To see the articular cartilage embody some degree of repair to me is revolutionary. I feel we are approaching the final pivotal time where even more data will finally stimulate a direction and step change for us all. Yeah that's not advice.





Mozz






APPENDIX A




Mozz Speak


Sometimes I start a Mozz post and it's on topic that initially seems straightforward, I tell myself I have an entire week to research and write, the problem is that when you start you often come across just one term and it literally expands and expands.


The term cartilage filament you would think is like a one sentence explanation and we can move on?Dang, it's more like a few paragraphs. I'm not going to go into that detail but I wanted to give you a high level glimpse of just how important this stuff is and what your drug is capable of. Getting some repair done in the articular cartilage is one thing, to show this in the depths of these fissures and cracks is quite something else. The repair work that I believe iPPS is capable of (Perhaps I need to caveat it and say in some or many instances)...is quite amazing. Let's have a closer look at these filaments.¹¹


The cartilage filaments comprise of a number of different subunits, some names include vimentin, cytokeratins, and glial fibrillary acidic protein. The interesting part is that some of these occur at different depths and in different concentrations and actually a lot of them are governed by the mechanical loading that takes place. The cells in these areas respond to the external forces. Vimentin, as an example, is responsible for not only mechanical strength of the cartilage as a whole but also provides the flexibility to resist stress.

Researchers from another study found "Disruption of vimention was found to reduce stiffness approximately 2.8-fold in normal chondrocytes".¹²


We don't want the brittleness to seep in when these fibres are under load. We want that cartilage FRESH...we want that cartilage REPAIRED....and the ultimate natural stimulant from within to do this may indeed be the one drug you and I own.



DYOR!







REFERENCES


MAIN REFERENCE

1] https://www.iomcworld.org/open-access/pentosan-polysulfate-as-a-disease-modifier-of-cartilage-degeneration-inexperimental-osteoarthritis-2167-7921-1000199.pdf



OTHER REFERENCES

2] Synovial Joint Biology and PathobiologyDavid D. Frisbie, Sherry A. Johnson, in Equine Surgery (Fifth Edition), 2019.
3] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822172/
4] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822172/
5] Remst D.F., Blaney Davidson E.N., van der Kraan P.M. Unravelling osteoarthritis-related synovial fibrosis: A step closer to solving joint stiffness. Rheumatology. 2015;54:1954–1963. doi: 10.1093/rheumatology/kev228.
6] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3445147/
7] https://www.ncbi.nlm.nih.gov/books/NBK557576/#:~:text=Chondrocytes%20are%20mainly%20responsible%20for,the%20fifth%20week%20of%20development.
8] https://app.sharelinktechnologies.com/announcement/asx/63a249bdb0b4e5e1dc93c8ee2644f3a2
9] https://www.sciencedirect.com/science/article/pii/S1877065716300185#:~:text=Collagen%20type%20II%20is%20the,PIIBNP%20and%20CPII%20%5B11%5D.
10] https://www.researchgate.net/publication/301337819_In_vivo_Model_of_Crystal-Associated_Osteoarthritis
11] https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/jemt.1070280503
12] https://www.healio.com/news/orthopedics/20120331/study-characterizes-protein-filament-s-role-in-cartilage-strength