December 13, 2012
The rise and fall of pentosan polysulfate in prion disease
Next to quinacrine, pentosan polysulfate (PPS) (above) has probably been
the second-most examined drug in the treatment of prion disease. PPS never made
it to an official clinical trial in the United States, but Japan and the U.K.
each ran clinical trials, both of which failed. PPS has since been pretty much
abandoned as a potential prion treatment, for more than one reason, but we can
learn a lot from the history of research into this compound as an antiprion
therapeutic.
Pentosan polysulfate was tested in mouse models of prion disease as early
as 1984, but the real story begins, in a way, with Caughey & Race 1992, who
observed that Congo red inhibits PrP accumulation in prion-infected cell
culture. Congo red was originally invented as a stain for cellulose but has for
decades been used in laboratories as a stain for amyloid plaques, which, after
all, earned the name “amyloid” for their misleading resemblance to starch
(amylum in Latin), which is composed of the same glucose subunits as cellulose,
under staining. Like Alzheimer’s and some other neurodegenerative diseases,
scrapie results in the deposition of amyloid plaques in brain tissue. Since
Congo red stains (read: binds to) amyloid plaques effectively, Caughey and Race
wondered if it might interfere with the formation of amyloid plaques as well.
And it did – in fact, it pretty much completely abolished the formation of
PrP-res, as shown in Fig 1A:
But Congo red was not exactly ready for prime time as a therapeutic
compound. In fact, according to Wikipedia it’s so toxic that even the textile
and paper industries won’t use it anymore (always a bad sign). So Caughey and
Race’s discovery touched off a search for less toxic compounds with the same
PrP-res-inhibiting property.
That search led ultimately to several hits including cpd-B and curcumin.
But the race had been won before it started: a year before Caughey and Race’s
discovery, another group had already announced that adminstration of the
polyanion compound pentosan polysulfate resulted in a massive extension of
survival in scrapie-infected mice [Diringer & Ehler 1991]. In fact, without
any fanfare at all, these two had reported a significant effect of pentosan
polysulfate as early as 1984, in a study whose primary purpose was to evaluate
the efficacy of another polyanion compound: dextran sulfate 500 [Ehler &
Diringer 1984]. Predictably, the 1984 study had found that DS500 was only
effective against peripheral infection and not intracerebral – “polyanions” by
definition are highly charged compounds, which the BBB‘s border control agents
hate. But Diringer and Ehler’s 1984 and 1991 studies evaluated PPS’s efficacy
only in peripherally infected mice, and only with drug administration either
prophylactically or very early in the disease course. Ladogana 1992 had likewise
shown efficacy against peripheral infection for DS500, pentosan polysulfate
(referred to in that study as SP54) and suramin. In fact, Ladogana even tested
DS500 and suramin (but not PPS) against intracerebral infection and found a
small but significant effect for DS500.
So in fact, lots of work on PPS preceded Caughey’s discovery that Congo red
inhibits PrP-res formation. But I chose to start the story with that oft-cited
paper because it coincided with the beginning of scientific acceptance of the
protein-only hypothesis and, with it, a new perspective on what a compound
needed to do in order to fight prion infection. The studies by Ladogana,
Farquhar, Diringer and Ehler all took place back in the dark ages when people
thought scrapie was an “unconventional virus”, so much of the discussion in
these papers centers on the supposed “antiviral” properties of DS500 and PPS. By
1993, putting together the newly emerging prion hypothesis (described as
“controversial”) with the results from the 1992 Congo red study, Caughey had a
better idea of why PPS might have been effective: perhaps it inhibited PrP-res
formation just like Congo red. PPS isn’t exceptionally similar to Congo red, but
the two do have two sulfate anions in common:
Congo red
And sure enough, Caughey was able to show that PPS inhibited PrP-res
formation “in vivo” [Caughey & Raymond 1993], by which he actually meant ex
vivo: in cell culture, not in a whole organism.
And curiously, no one provided in vivo validation of this finding for over
a decade. Of course, PPS was already known to be effective in vivo – in early or
prophylactic administration in peripherally infected mice. What remained was to
show that PPS was also effective against intracerebral infection, and that it
could abolish the amyloid plaques that characterized scrapie infection in mice
and CJD in humans, plaques composed of a protein beginning to gain acceptance as
being the pathological agent: PrP.
Instead, the next decade saw a series of basic science papers on PPS and
related molecules such as heparin and heparan sulfate, using these molecules as
tools to understand the biological and biochemical properties of PrP [Caughey
1994a, Caughey 1994b, Shyng 1995, Brimacombe 1999]. The most surprising of these
was the confirmation that although PPS inhibits PrP-res formation in cell
culture, it stimulates PrP-res formation in cell-free conversion [Wong 2001].
One other paper testing PPS in vivo did emerge after several years [Farquhar
1999], but it didn’t cite Caughey’s work and managed to remain wholly agnostic
as to the identity of what it called the “bovine spongiform encephalopathy
agent”, avoiding calling it either a ”prion” or a “virus”. Farquhar successfully
duplicated the earlier in vivo results, showing that a single dose of PPS could
dramatically delay disease onset or prevent it altogether, if administered
intraperitoneally within several hours of the initial peripheral infection. The
results replicated across several different mouse strains, but the issue of
PPS’s possible interference with PrP and efficacy against CNS infections was
left untouched.
When evidence on the in vivo efficacy of PPS against CNS prion infections
finally did surface after more than a decade, it did so in a spectacular
fashion. In 2004, Katsumi Doh-Ura demonstrated that continuous ventricular
infusion of PPS of mice could extend survival by as much as 2.4-fold [Doh-Ura
2004]. His primary model was Tg7 mice expressing hamster PrP, infected
intracerebrally with 263K hamster prions. Doh-Ura tested not only PPS but also
quinacrine (no effect), chloroquine (no effect), E-64d (no effect), and
amphotericin B (some effect). The whole experiment was exquisitely designed to
test the drugs’ efficacy against CNS infections without bumping up against
issues of blood-brain barrier permeability. It must have been an impossibly
difficult experiment to pull off: Doh-Ura implanted miniature devices in the
backs of the mice, just under the skin, with tubes leading to their left
ventricles to continuously drip tiny amounts of PPS for four weeks after
implantation until the chemical ran out. And it wasn’t prophylactic, either: the
mice were treated beginning at 7, 21, 35 or 42 days post-infection (dpi),
meaning the surgery had to be done on already scrapie-infected mice. Perhaps
this explains what one prion researcher recently told me: Doh-Ura’s result was
the best result there’s ever been for prion therapeutics; it’s just that no one
has managed to reproduce it.
In the published literature, at least, no one has even tried to reproduce
it. That’s probably in part because of the technical difficulty involved and in
part because Doh-Ura had already left no stone unturned. The incredibly thorough
study varied both the dose and the date that drug administration was started, as
summarized beautifully in Fig. 2:
The results are very clear: PPS is most effective at a medium dose of 230
ug/kg/day and when administered as early as possible, though it still has a
significant effect when administered late in the disease. At its best, it
delayed death from 51 dpi for controls to 123 dpi for mice treated with 460
ug/kg/day starting from 7 dpi.
Not satisfied to show an effect on just one model, Doh-Ura also varied the
mouse model and prion strain in question. His primary experiment used Tg7 mice,
which overexpress hamster PrP and no mouse PrP, infected with 263K hamster
prions; subsequently he also tested PPS by the same protocol in Tga20 mice
(which overexpress mouse PrP) infected with Fukuoka-1 and RML prions. Those
experiments showed smaller (regression to the mean, perhaps?) but still quite
large, extensions of survival – 117% and 49% respectively when treatment was
begun at 14 dpi.
Doh-Ura also tried subcutaneous administration of PPS with, as would be
expected, no effect: PPS could not cross the blood-brain barrier.
The study didn’t skimp on follow-up experiments to figure out how PPS had
worked, either. Doh-Ura did immunohistochemistry on the treated mouse brains and
found a remarkable reduction in PrP deposits. In fact, the neurodegeneration
that had killed the mice was almost wholly concentrated in the right hemisphere
of the brain, while the left hemisphere, where the infusion site was, was spared
the worst of it. Doh-Ura also assayed the infectivity of the mouse brains in the
same blunt way that it’s still done today – by injecting their brain homogenate
into other mice and comparing the survival of those mice to a standard table.
Last but not least, Doh-Ura did toxicology experiments on mice, rats, and dogs –
again, with intraventricular infusion – to determine the tolerated dose and
assess what adverse effects might be anticipated.
The overall picture was of a drug of fairly low toxicity that reduced
infectivity through direct action on the infectious protein itself, largely
regardless of prion strain, and could more than double the incubation time of
the disease if administered early enough. It was, and probably still is, the
strongest result that’s ever been shown for a potential antiprion drug. But
there were two catches.
Two big catches. First, it had to be administered directly into the brain.
Second, and more importantly, the study provided no evidence for – indeed, some
evidence against – the idea that PPS would work in already-symptomatic animals.
As shown in Fig 2B above, Doh-Ura tested PPS at 7, 21, 35, and 42 dpi in mice
that showned “definite symptoms” at 49 dpi (though Doh-Ura allowed for the
possibility that more subtle earlier symptoms could have gone unnoticed) and
died at 51 dpi. The effect of the drug was huge at 7 dpi, small by 35 dpi, and
so tiny as to be statistically insignificant by 42 dpi. To help you see this,
Doh-Ura has actually drawn line segments connecting through the means of the
different groups:
The the lack of a ‘day 14′ experiment to evenly space the X axis makes the
lines a bit misleading (the group means aren’t actually collinear as they appear
in this figure), but the trend is pretty obvious. Based on this figure you can
have a pretty good guess that mice not treated until they exhibited symptoms at
day 49 or day 50 would not experience any increase in survival.
And Doh-Ura fully recognized this, noting that “our data do not guarantee
similar effectiveness in human patients who already have signs and symptoms of
the disease”. Acknowledging this, and acknowledging as well the personal and
public health risks of installing intraventricular catheters in prion dsiease
patients, Doh-Ura still chose to end the paper on an optimistic note. Though the
United States was yet to start its own clinical trial of quinacrine, PRION-1,
Doh-Ura had read the literature on quinacrine in mice and hamsters and had
(correctly) concluded that quinacrine would not prove effective in humans. So he
put forth PPS as the next candidate for clinical trials:
As an immediately applicable remedy, however, continuous intraventricular
PPS administration with an infusion device may be a candidate for a clinical
trial, with a view to preventing the disease in those people categorized as
being at extremely high risk or to improving the prognosis of diseased people
with TSEs.
PPS was already approved in the U.S. (and presumably in Japan too, though I
can’t find any confirmation of that online) for interstitial cystitis, a.k.a.
painful bladder syndrome, which is basically a diagnosis of exclusion meaning
“we can’t identify any other problem to explain your symptoms, so we’ll call it
this”. For a review of pentosan polysulfate in painful bladder syndrome, see
Teichman 2002. The bladder connection seems at first interesting in light of the
known connections between prion disease and bladder problems (see posts on
dapsone and ibuprofen), though it’s almost surely a coincidence. In any event,
here you have an already approved drug which might be effective against a
sudden, deadly disease with no known treatment or cure. Almost immediately,
neurologists started trying PPS in their prion disease patients.
Over the next five years, ambiguous reports of human results trickled in
from several places. In the peer-reviewed journals, a handful of case reports
arrived, some of which reported considerably extended survival [Todd 2005, Parry
2007, Rainov 2007] and some of which did not [Whittle 2006], though in no case
was the patient’s condition seen to actually improve.
Other reports came from outside the traditional journal setting. A series
of short articles in BMJ [Dyer 2003, Gould 2003, Mayor 2003] and a subsequent
BBC report documented one family’s battle to get PPS (which is not an approved
drug in Britain) approved for their son Jonathan Simms – they eventually
prevailed and the young man, though still sick, remained alive with vCJD for 10
years (he passed away in 2011) while being treated with PPS – possibly the same
patient from Todd 2005. BBC also reported that MRC had announced the drug “had
appeared to help several people live longer than expected.” And in a powerpoint
apparently presented at a meeting in Glasgow organized by CJD Alliance, Dr. Ian
Bone reported on eight U.K. patients who had, again, lived slightly longer than
expected, though with multiple complications related to the ventricular
catheters and one possible adverse reaction to the drug itself [Bone
2006].
That powerpoint is actually a more accessible discussion of the issues
involved in the study design than many of the published papers are. Bullets on
slides 2 and 23 show a key part of Bone’s thought process: there are too few
prion disease patients, and their diseases are too urgent, for there to ever be
a large, statistically powered case/control study as is the standard in
medicine. Instead, we are stuck with an observational study of a handful of
patients – but if we can use a “surrogate marker” to assess disease progression,
maybe we can tell if the drug is working in each individual. It is this same
thought process that motivates our early biomarkers research goal at Prion
Alliance.
Formal reports on PPS’s efficacy finally came out in 2008-9. Bone reported
that the seven patients formally included in the observational study in the U.K.
had indeed lived longer than the mean for their respective prion diseases, but
clinical symptoms as well as MRI – his “surrogate marker” – both showed
continued disease progression after drug treatment began [Bone 2008]. The
slightly longer survival might be due to PPS but Bone felt he couldn’t rule out
several other possible explanations:
chance alone and biases such as lead-time bias from attentive carers
diagnosing onset early; selection bias from included patients having prolonged
survival whilst awaiting PPS or bias from increased use of active interventions
for complications in more actively managed PPS patients amongst others
The report doesn’t take a firm stance for or against continued use of PPS
in humans, instead concluding that “More experimental work in animal models is
clearly needed” and that “Until then, all patients with prion diseases
considering PPS therapy should be informed of existing evidence and, if opting
for treatment, managed and monitored in a standardized manner”.
The following year, Doh-Ura and his colleagues reported on their use of PPS
in eleven patients in Japan [Tsuboi 2009] with almost identical results. Again,
the patients survived longer than mean for their diseases, but not out of the
plausible range, and showed “continued deterioration” after drug treatment
commenced.
Both of these formal reports discuss the possibility that different doses
might be more effective, and neither rules out the possibility of further use of
PPS. But not much has been published on PPS since then: a Google scholar search
for more recent papers on prion pentosan polysulfate since 2011 turned up only
one more case report [Terada 2010] and a good review of all the human case
reports and observational studies [Appleby 2011].
In reading all these reports, perhaps the most interesting thing I noticed
was in Bone’s Table 1 introducing the seven patients from the U.K. observational
study, including their dates of disease onset and treatment and their clinical
state [Bone 2008]. The patients had started PPS treatment anywhere from 6 months
to 2.5 years after disease onset. At the time that the official observational
period commenced (which was for most patients 1-2 years after they had already
begun receiving PPS), three were bed-bound and two were chair-bound.
We don’t know whether the patients were already bed-bound or chair-bound
when they started PPS treatment, but given the length of time between disease
onset and treatment, it does seem likely that they were very sick by the time
they started receiving the drug.
In the mouse study that touched off all this interest in PPS, Doh-Ura
stated that the first noted symptom in the mice was “ambiguous signs of reduced
activity” two days prior to death, so on average 49 dpi, while his experiments
showed no significant effect of PPS even when started at 42 dpi. Since
investigators won’t notice subtle symptoms in a mouse, it is very hard to know
what day post infection in a mouse corresponds to the moment, in a human
patient, that a diagnosis would be made. But given the disease state of the
patients and the length of time between disease onset and initiation of
treatment, it seems they were much closer to the equivalent of 49 dpi than they
were to 35 dpi (the latest treatment date for which Doh-Ura observed a
significant effect), by the time they received treatment.
Some of the delay in starting treatment for these patients was surely due
to the experimental nature of the PPS treatment. But given the swift and
unexpected nature of prion diseases, it seems that even under the best of
conditions, it is very likely that patients will decline severely before any
treatment can be started. Patients will need to exhibit a significant decline
before it even triggers a hospital visit, referrals to specialists at tertiary
care facilities will have to be made, and diagnosis will not always be quick or
easy and tests will not always give immediate confirmation. The only exception
to this rule is the handful of genetic prion disease carriers who have chosen to
be tested and know their status – but healthy asymptomatic individuals will
never go in for intraventricular catheterization.
Hence the inherent contradiction in pentosan polysulfate: it’s only
effective at times when the patient’s condition is not desperate enough to merit
such drastic measures.
Possibly this all could have been recognized at the outset: Doh-Ura’s study
was extremely thorough and extremely honest about the fact that PPS required
intraventricular infusion and didn’t work late in the disease. But given the
complete fatality of prion diseases and the utter lack of any treatments,
desperation gave way, and the story of Jonathan Simms as documented by BBC and
BMJ shows that even when medical authorities are duly skeptical of a potential
treatment, families will actively campaign to be able to use it. After all,
they’ve got nothing to lose.
Wednesday, March 28,
2012
VARIABLY PROTEASE-SENSITVE PRIONOPATHY IS TRANSMISSIBLE, price
of prion poker goes up again $
http://prionopathy.blogspot.com/2012/03/variably-protease-sensitve-prionopathy.htmlhttp://sporadicffi.blogspot.com/
Friday, November 23, 2012
sporadic
Creutzfeldt-Jakob Disease update As at 5th November 2012 UK, USA, AND CANADA
http://creutzfeldt-jakob-disease.blogspot.com/2012/11/sporadic-creutzfeldt-jakob-disease.html
http://transmissiblespongiformencephalopathy.blogspot.com/
Terry S. Singeltary Sr. P.O. Box 42
Bacliff, Texas USA 77518
MOM DOD 12/14/97 CONFIRMED
hvCJD...
