Vetrugno, Exp Op Orph Drugs, 15

1. Introduction
2. Past therapeutic attempts and
rationales
3. Past clinical trials: strategies
and difficulties
4. Ongoing or future possible
trials
5. Conclusions
6. Expert opinion
Review
The future for treating
Creutzfeldt--Jakob disease
Vito Vetrugno, Maria Puopolo, Franco Cardone, Fiorentino Capozzoli,
Anna Ladogana & Maurizio Pocchiari†
Istituto Superiore di Sanita, Department of Cell Biology and Neurosciences, Roma, Italy
Introduction: Creutzfeldt--Jakob disease (CJD) is a rare, transmissible and fatal
neurodegenerative illness that affects people worldwide with a prevalence of
about 1 -- 2 cases per million people. Early diagnosis of CJD is still difficult
despite recent development of novel assays for detecting the pathological
prion protein, the only reliable marker of disease found in the cerebrospinal
fluid, urine and mucosa olfactoria.
Areas covered: This analysis covers attempts of therapy in CJD and related dis-
eases. It looks at problems encountered in designing and interpreting the few
available trials with the aim that learning from past experiences would
improve future clinical experimentations.
Expert opinion: The future of therapeutic intervention in CJD should begin
with the identification of novel compounds with strong antiprion effects.
These need validating in animal models of disease before their use in humans.
Improvement of preclinical studies according to internationally recognized
guidelines should cover critical aspects that have been poorly followed in
the past. The use of more than one model of prion infection should also be
encouraged. Novel international diagnostic criteria for including CJD patients
in clinical trials in an early stage are needed and consensus should be reached
for determining the most meaningful criteria to evaluate the progression
of disease.
Keywords: Creutzfeldt--Jakob disease, dementia, neurodegenerative disorders, therapy
Expert Opinion on Orphan Drugs (2015) 3(1):57-74
1. Introduction
Clinical and pathological features of Creutzfeldt--Jakob disease (CJD) were
described about one century ago, but understanding the mechanisms of pathological
processes only begun in the late 60s when the disease was successfully transmitted to
non-human primates and thought to be caused by a slow and unconventional
virus [1]. This finding neared CJD to kuru, an exotic disease of the Fore-speaking
people in Papua New Guinea and now disappearing [2], and scrapie, an endemic dis-
ease affecting herds of sheep and goats in most of the world [3]. However, until the
late 70s to early 80s, there was limited knowledge on clinical and epidemiological
data, and factors influencing disease duration were unknown or poorly under-
stood [4,5]. A better understanding of the natural history of the disease came in
the 90s from the establishment of national surveillance systems in European and
non-European countries [6,7], and from understanding the pathogenic mechanism
of disease, which basically consists in the poorly known process that misfolds the
cellular prion protein (PrPc
) into different pathological conformers (type 1, 2A
and 2B PrPTSE
) with high beta-chain contents [8].
The majority of CJD cases apparently occur spontaneously (sporadic CJD),
about 10 -- 20% within families (genetic CJD; Gerstmann--Stra¨ussler--Scheinker
disease [GSS] and fatal familial insomnia [FFI]) in people carrying a pathogenic
point or insert mutation in one allele of the prion protein gene (PRNP), and a
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few cases are related to medical procedures (iatrogenic
CJD) [7,9] or to the consumption of bovine spongiform
encephalopathy-contaminated food (variant CJD) [10].
Sporadic CJD occurs with different clinical phenotypes and
it is usually difficult to make a correct early diagnosis [11].
Dementia usually occurs in the early phase of disease, but
there are cases where patients present other neurological
symptoms (i.e., ataxia or visual disturbances) at onset. The
disease usually progresses rapidly with death occurring
between 3 and 6 months in about 60% of cases [12]. Support
from clinical diagnosis comes from the electroencephalogram
(EEG), cerebrospinal fluid (CSF) exams and brain MRI, but
so far no disease-specific markers are included in the interna-
tional diagnostic criteria for human prion diseases (Table 1) [13].
EEG shows a characteristic generalized bi- or triphasic peri-
odic sharp wave complexes with a frequency of around
1 -- 2 per second in about 60% of sporadic CJD but it might
appear late in the course of disease and, occasionally, in other
conditions [14,15]. Typical EEG is present in some forms of
genetic prion diseases (genetic CJD), but not in others
(GSS, FFI) nor in variant CJD [16]. Routine CSF analyses
are usually normal, but in the majority of sporadic CJD cases
the 14-3-3 proteins test results positive and tau is highly ele-
vated [17]. The presence of 14-3-3 proteins in the CSF, how-
ever, is not specific for prion diseases and it acquires a
diagnostic value only in combination with CJD-specific
neurological signs [18]. Moreover, in about 50% of variant
CJD this test is negative, therefore of no diagnostic value [19].
Finally, brain MRI shows signal abnormalities in the anterior
basal ganglia (caudate/putamen) and often in the cortex in
sporadic CJD, while signal abnormalities are observed in
about 90% of variant CJD in the posterior thalamic region
(pulvinar) [13,20]. Brain MRI is also useful for excluding other
neurological conditions, some of which might be susceptible
to pharmacological or surgical treatment [21]. The genetic
forms of prion diseases are easily recognized by the combina-
tion of neurological signs in patients presenting specific insert
or point mutations in the open reading frame of the PRNP
gene [22]. The analysis of the PRNP gene in people belonging
to affected families is also a powerful tool for the identifica-
tion of potentially high-risk subjects, although it is usually dif-
ficult to predict when people carrying a PRNP mutation will
develop disease [22,23] and in several occasions carriers do not
develop disease at all [23,24].
In the last 10 years, there have been substantial improve-
ments in the detection of the only disease-specific marker of
disease (i.e., PrPTSE
) that finally translated into the develop-
ment of novel diagnostic tools that have achieved enough sen-
sitivity for assessing PrPTSE
in easily accessible tissues or body
fluids [25]. Among them, the in vitro amplification of the mis-
folded prion protein has, in preliminary studies, successfully
detected PrPTSE
in CSF [26,27], in nasal swab samples of spo-
radic CJD [28] and urine of variant CJD [29] with no false-
positive signals, while an adapted solid-state binding matrix
assay is apparently able to detect PrPTSE
in blood samples of
variant CJD patients with over 70% sensitivity and 100%
specificity [30]. As soon as these methods are fully validated,
it would be possible to improve the correctness of an early
diagnosis, thus allowing a faster enrolment of CJD patients
in clinical trials.
Attempts of therapy in patients with CJD and related dis-
eases have been extensively reviewed in recent years [31-37],
with the conclusion that none of the tested drugs has convinc-
ingly shown a significant impact on the natural history of the
disease with the exception of the intraventricular infusion of
the pentosan polysulfate (PPS) in variant CJD patients [38].
To date, this conclusion still holds. This updated review
focuses on the appropriateness of these treatments with the
hope that learning from past weaknesses will improve the
future of therapy in prion diseases.
2. Past therapeutic attempts and rationales
Except for several and inconclusive reports on isolated cases
treated with a variety of drugs (see [31] for a comprehensive
review), there are only five compounds that have been tested
in comparative studies in patients with prion diseases.
2.1 Amantadine
In the early 70s, soon after the successful transmission of CJD
to non-human primates suggested a viral origin of the disease,
Article highlights.
. Four randomized double-blind placebo-controlled clinical
trial studies were attempted in Creutzfeldt--Jakob
disease (CJD) patients showing no efficacy, but proving
that clinical trials are feasible in CJD, though with
some difficulties.
. Analyses of past clinical trials revealed the importance of
recruiting patients at national or international level
because of the rarity of disease, the difficulty in
recruiting patients at an early stage of disease and some
annoyance by patients or caregivers in accepting
randomization because of the rapidly
progressive disease.
. Preclinical studies were often overinterpreted. Most
compounds showed preventive rather than therapeutic
effects in preclinical studies but this finding was often
not fully considered.
. There is only one preventive treatment in people at risk
of developing prion disease. This ongoing trial with
doxycycline is in healthy carriers of the pathogenic
mutation of the PRNP gene responsible for the fatal
familial insomnia.
. Internationally recognized guidelines for performing valid
and relevant preclinical studies should be required for
planning future therapy in humans.
. It is necessary to develop international diagnostic criteria
for including CJD patients in clinical trials in a very early
stage by using new PrPTSE
amplification techniques in
body fluids or easily accessible relevant tissues.
This box summarizes key points contained in the article.
V. Vetrugno et al.
58 Expert Opinion on Orphan Drugs (2015) 3(1)
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Table1.HistoryandfuturedevelopmentsofdiagnosticcriteriaforsporadicCreutzfeldt--Jakobdisease.
*ClinicalsignsaddedinEuropeancriteria.
CJD:Creutzfeldt--Jakobdisease;CSF:Cerebrospinalfluid;EEG:Electroencephalogram;OM:Olfactorymucosa;RT-QuIC:Realtimequackinginducedconversion.
The future for treating Creutzfeldt--Jakob disease
Expert Opinion on Orphan Drugs (2015) 3(1) 59
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therapy with the antiviral drug amantadine was comprehensi-
bly attempted in a bunch of single CJD cases with some
reported beneficial effects [39-41]. The rationale of two observa-
tional studies [42,43] was to clarify the previously questionable
effect of the drug in CJD patients, despite the failure of pro-
longing survival in experimental models of prion disease
were already available (Table 2) [44,45]. Both studies concluded
that treatment did not substantially modify the course of dis-
ease, although Terzano et al. [42] reported some clinical
improvements. Formal clinical trials were likely not per-
formed because of the great difficulties to recruit a reasonable
number of patients in those years when only a small percent-
age of CJD cases were recognized and no neurological centre
had the chance to observe more than a handful number of
patients in years of activity. Because no clinical trials have
ever been performed and preclinical studies with amantadine
in animal models are limited, it is difficult to draw any defin-
itive conclusion on the efficacy of this and other antiviral
drugs, such as acyclovir [46,47], vidarabine [48] and IFN [49] in
CJD patients. However, with the current knowledge of the
pathogenic mechanisms of prions and considering the avail-
able, although scanty clinical studies, it is reasonable to con-
clude that available antiviral therapies are ineffective in the
treatment of CJD patients.
2.2 Flupirtine
In 2004, Otto et al. [50] tested the effect of the non-opioid anal-
gesic flupirtine versus placebo in 28 patients with CJD with the
aim of determining the influence of the drug in the rate of cog-
nitive decline. The study showed a transient beneficial effect of
flupirtine in delaying the cognitive decline of CJD patients but
no changes in survival times (Table 2). In in vitro studies, flupir-
tine prevented apoptosis in neural cell culture exposed to a vari-
ety of toxic agents, including A-beta and a short (21 amino
acids) fragment of PrP (PrP106-126) [51-53], but it had never
been tested in animal models of prion or other neurodegenera-
tive disorders, making disputable its use in CJD patients. The
authors did not comment on the rationale for the possible ben-
eficial effect of flupirtine in delaying cognitive decline in CJD
apart from reporting the in vitro cytoprotective activity.
Otto et al., however, had the great merit to show that double-
blind randomized controlled studies were feasible in CJD,
despite the rarity of disease, the difficulties in getting a correct
clinical diagnosis and the relative short clinical duration.
2.3 Quinacrine
Compassionate therapy with quinacrine began 10 years ago
soon after the group of the Nobel Laureate Stanley Prusiner
confirmed a previous study [54], showing that this tricyclic
derivative of acridine potently inhibits PrPTSE
formation in
scrapie-infected neuroblastoma cell cultures [55]. Because
quinacrine was used for decades in humans against malaria
and crosses the blood--brain-barrier, Korth et al. [55] suggested
its immediate use in patients with CJD and related diseases
without waiting for confirmatory preclinical studies. In several
countries, the compassionate use of quinacrine in CJD
patients was initially done under the great pressure of families
with the authorization of competent government authorities;
in the USA, it was advertised in neurology journals, official
websites and by letters to the US neurologists. Overall, the
rationale for these observational studies were based on the
antiprion effect of quinacrine in cell culture, its long and
safety use in humans and because it reaches the CNS. Treat-
ment of isolated CJD or FFI patients with quinacrine showed
either no benefit [56-59] or a transient improvement of clinical
conditions [60-63], and observational studies gave ambiguous
results (Tables 2 and 3). Haik et al. [64] reported neither clinical
improvement nor prolonged survival, Collinge et al. [65]
reported a transient response on neurological rating scales in a
few patients but no effect in survival, while Geschwind et al.
[66] reported a prolonged survival. All observational studies
with quinacrine started to recruit patients between 2000 and
2001, just before the reported failure of quinacrine to prolong
the incubation periods in mice intracerebrally injected with a
CJD-adapted strain [67]. Quinacrine inefficacy in animal models
of prion diseases was further confirmed in the following
years [68-70]. Finally, in 2013 Geschwind et al. reported the
only randomized double-blind placebo-controlled clinical trial
(RCT-DB) to quinacrine or placebo [66]. Patients were recruited
between 2005 and 2009, with the rationale that the same
authors previously observed a positive effect of quinacrine in
prolonging survival in CJD patients in an observational
study [66]. This study finally showed the inefficacy of quinacrine
to improve survival in sporadic CJD patients (Tables 2 and 3)
and likely represents the end of the therapeutic use of quinacrine
in prion diseases.
2.4 Doxycycline
Tetracycline antibiotics revert in vitro the protease resistance
of PrPTSE
extracted from brain tissue of patients with
CJD [71]. Doxycycline, a tetracycline derivative, shares the
same antiprion effect with the advantage of poor toxicity
and of efficiently crossing the blood--brain barrier [72]. Based
on these observations, the group of Fabrizio Tagliavini at
Carlo Besta Neurological Institute in Milan, Italy, pioneered
in 2002 the compassionate therapy with doxycycline for
CJD patients (Tagliavini, pers. commun.). A few years later,
De Luigi et al. [73] reported that doxycycline efficiently pro-
longs the incubation period of hamsters peripherally inocu-
lated with the 263K strain of scrapie and that the
intracerebroventricular infusion of liposome-entrapped doxy-
cycline in hamsters with early clinical signs delays disease
duration confirming its potential use for human therapy.
Data from observational studies in Italy [74] and
Germany [33,75] also showed that treatment with doxycycline
extends between 4 and 7 months the survival of CJD patients
compared with historical controls (Tables 2 and 4). These
findings prompted an Italian-French RCT-DB Phase II study
that, however, did not confirm the positive findings of the
observational studies [76].
V. Vetrugno et al.
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Table2.Attemptsoftherapyinpatientswithpriondiseases.
DrugStudydesignRef.YearCountryDiseaseTreated:
controls
AuthorconclusionAuthors’rationaleComments
AmantadineObservational[42]1983ItalyCJD(likelysporadic)4:5Noeffectonsurvival
Partialclinicalbenefit
Antiviraleffect
Possiblebeneficialeffects
oncasereports
Absenceofefficacyon
incubationperiodsin
animalmodelswas
alreadyavailableatthe
timeofthestudies
[43]1984ItalyCJD(likelysporadic)4:4Noeffectonsurvival
Noclinicalbenefit
UncertainnessofTerzano
etal.dataandpossible
beneficialeffectsoncase
reports
FlupirtineRCT-DB[50]2004GermanySporadicand
geneticCJD
13:15Noeffectonsurvival
Benefitofcognitive
functions
Cytoprotectiveactivityin
vitroandinvivoon
neuronsinducedto
undergoapoptosis
Reductionofinvitro
PrP106-126neurotoxicity
Nopreclinicalstudiesin
animalmodels
QuinacrineObservational[64]2004FranceSporadicand
variantCJD
32:125
(historical)
NoeffectonsurvivalInvitroantiprionactivity
Possibleeffectinpatients
withvariantCJD
Thisstudybegunwhen
preclinicalstudiesin
animalstudieswerenot
yetreported
Open-label,
patient
preferencetrial
(Observational)
[65]2009UKSporadic,iatrogenic,
variant,and
geneticCJD
32:69Noeffectonsurvival
Transientresponseon
neurologicalrating
scalesin4patients
Invitroantiprionactivity
RequestedbytheBritish
ChiefMedicalOfficer
Absenceofefficacyon
incubationperiodsin
animalmodelswas
timeofthestudy
Observational[66]2013USASporadicCJD29:58IncreaseinsurvivalInvitroantiprionactivityThisstudybegunwhen
preclinicalstudiesin
animalstudieswerenot
yetreported
RCT-DB(PhaseIIB)[66]2013USASporadicCJD23:28Noimprovementin
2-monthssurvival
Noclinicalimprovement
Increasesurvivalinthe
observationalstudy
previouslyconductedby
theAuthors
Absenceofefficacyon
incubationperiodsin
animalmodelswas
timeofthestudy
DoxycyclineObservational[74]2008ItalyCJD(likelysporadic)21:78IncreasesurvivalExtendedsurvivalin
experimentalmodels
-
[33]2009GermanySporadicand
geneticCJD
51:not
reported
IncreasesurvivalExtendedsurvivalin
experimentalmodels
Controlsweretakenfrom
historicalcollections
RCT-DB(PhaseII)[76]2014Italyand
France
Sporadic,
iatrogenic,
variantand
geneticCJD
62:59NoeffectonsurvivalExtendedsurvivalin
observationalstudies
-
CJD:Creutzfeldt--Jakobdisease;GSS:Gerstmann--Stra¨ussler--Scheinkerdisease;PPS:Pentosanpolysulfate;RCT-DB:Randomizeddouble-blindplacebo-controlledclinicaltrial.
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2.5 Intraventricular infusion of PPS
Attempts of therapy with intraventricular infusion of PPS
(iPPS) began in January 2003 [77] soon after Doh-Ura et al.
reported in an international meeting in Paris (2002 [78],
then published in 2004 [69]) the successful effect of this treat-
ment in rodents experimentally infected with different strains
of prions. The antiprion efficacy of intraperitoneal adminis-
tration of PPS and other polyanionic compounds was estab-
lished since 1984 [79] and then confirmed in other studies in
peripherally or intracerebrally scrapie-infected mice or ham-
sters [80], but its efficacy was limited to treatment given just
before or after prion inoculation, a situation non-applicable
for human therapy. Despite these experimental data, therapy
with oral administration of low molecular weight PPS was
reported in eight Japanese CJD patients [81] with no success.
The finding that iPPS was modestly effective even when
given at the beginning of clinical signs in prion-infected
mice reawaken interest in this drug for human therapy [69].
In 2003, Dealler and Rainov [82] reported on the potential
therapeutic use of iPPS for CJD patients based on the exper-
imental findings and on the poor side effects of PPS infusion.
Treatment was then given to several other patients with vari-
ous forms of CJD. In 2007, Rainov et al. [83] in reviewing
26 attempts of therapy with iPPS (4 variant, 8 sporadic,
7 iatrogenic and 1 genetic CJD; 5 GSS and 1 unspecified
case) reported that it appears reasonably safe but survival
was convincingly prolonged only in variant CJD. Bone et al.
[84] and Tsuboi et al. [85] reached similar conclusions in British
(3 variant, 2 iatrogenic CJD; 2 GSS) and Japanese (6 sporadic,
2 iatrogenic and 2 genetic CJD; 1 GSS) patients. A recent
overall picture of the effect of iPPS in prolonging disease
duration in variant British CJD patients showed that four of
five treated patients survived well beyond untreated patients
(Tables 2 and 5) [38].
3. Past clinical trials: strategies and
difficulties
Only 4 RCT-DB studies were attempted in CJD patients
despite 14 drugs were occasionally tested in the last 40 years [31].
All RCT-DB studies showed no efficacy of the investigated
drugs, but they prove that clinical trials are also feasible in
CJD, though with some difficulties. The analysis of the prob-
lems encountered in designing and interpreting the trials
might contribute to improve future clinical experimentations.
In the following pages, we present the major difficulties
that challenged the design of RCT-DB studies for prion dis-
eases, such as the definition of the end points, the approach
for recruiting a significant number of patients, the diagnostic
criteria for including or excluding patients to the study, the
designing of the randomized trial and the ethical considera-
tions for obtaining the consent to the experimentation or for
using a placebo in an otherwise deadly disease.
Table2.Attemptsoftherapyinpatientswithpriondiseases(continued).
DrugStudydesignRef.YearCountryDiseaseTreated:
controls
AuthorconclusionAuthors’rationaleComments
Intraventricular
infusionofPPS
Review[83]2007-Sporadic,iatrogenic,
variantandgenetic
CJD;GSS
26:0Increasesurvivalin
variantCJD
Extendedsurvivalin
experimentalmodelswith
directPPSbraininfusions
Nocontrolpopulation
Observational[84]2008UKIatrogenicand
variantCJD;GSS
7:0Possiblepositive
effectonsurvival
invariantCJD
Extendedsurvivalin
Increasesurvivalinother
observationalstudies
Controlsweretakenfrom
historicalcollections
Observational[85]2009JapanSporadic,iatrogenic
andgeneticCJD;GSS
11:0Noeffecton
neurological
deficits;possible
extendedsurvival
Extendedsurvivalin
Nocontrolpopulation
Casereport
andreview
[38]2014UKVariantCJD5:171Extendedsurvival
on4/5
Extendedsurvivalin
4of5treatedpatients
hadasurvivallongerthat
allotherhistoricalcases
CJD:Creutzfeldt--Jakobdisease;GSS:Gerstmann--Stra¨ussler--Scheinkerdisease;PPS:Pentosanpolysulfate;RCT-DB:Randomizeddouble-blindplacebo-controlledclinicaltrial.
V. Vetrugno et al.
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3.1 End point(s) and minimum clinically relevant
differences
Primary end points represent the bases for determining the
sample size of a clinical trial. Except for the flupirtine trial [50],
which was designed to detect cognitive (ADAS-Cog test) dif-
ferences between baseline and best score under treatment, the
primary end points of the other three trials were time of
survival, though with some differences among studies in terms
of minimum clinically relevant differences (Table 6).
Collinge et al. (quinacrine) [65] were looking at a reduction
in 2-year mortality from 50 to 22%; Geschwind et al. (quin-
acrine) [66] at doubling of mean survival from 0.9 to
1.8 months after 2 months from randomization; Haik et al.
(doxycycline) [76] at doubling survivors after 1 year of ran-
domization from 30 to 60% (Italian patients) or increasing
mean survival from 6.2 to 11 months (French patients). The
calculated sample sizes varied accordingly, but overall it
ranged between 30 and ‡ 160 patients and took between
2 and 4 years for recruiting this significant number of patients
(Table 6). Patients were followed-up in a single neurological
center for the flupirtine [50] and both the US [66] and
UK [65] quinacrine studies, while for the doxycycline study [76]
patients were recruited and followed-up in either three centers
(Italian patients) or by multiple and not predetermined neu-
rological units for French patients. Secondary end points
aim to assess possible clinical improvements related to therapy
were considered in all studies but were finally difficult to eval-
uate mostly because of the serious and advanced clinical phase
of enrolled patients.
Considering the rarity of CJD, the number of patients that
needs to be recruited requires national, or better international-
based studies for speeding the completion of clinical trials. On
the other hand, the recruitment of patients on national or
international base might prevent simultaneous investigations
of different drugs with delay in testing novel and promising
therapeutic approaches.
Sample size is strongly influenced by the choice of the min-
imum clinically relevant differences; conspicuous clinical
effects require small sample size but might be unrealistic, be
responsible for closing the trial for futility (improbable to
detect prefixed effects) and therefore undermining future eval-
uations of that specific treatment. On the other hand, minor
but realistic clinical improvements would require a large and
unfeasible number of patients. On top of that, the compute
Table 3. Quinacrine.
BSE: Bovine spongiform encephalopathy; CJD: Creutzfeldt--Jakob disease; ic: Intracerebral; ip: Intraperitoneal; RCT-DB: Randomized double-blind placebo-controlled
clinical trial; RML: Rocky mountain laboratory; Ukn: Unknown.
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Table4.Tetracycliccompounds.
BSE:Bovinespongiformencephalopathy;CJD:Creutzfeldt--Jakobdisease;GSH:GoldenSyrianhamster;ic:Intracerebral;im:Intramuscular;IDX:4’-iodo-4’-deoxy-doxorubicin;RCT-DB:Randomizeddouble-blind
placebo-controlledclinicaltrial;sc:Subcutaneous;Ukn:Unknown.
V. Vetrugno et al.
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of the sample size needs to account for multiple testing in
interim analyses for an ethical assessment on whether to ter-
minate the study earlier than planned because of consistent
evidences of futility, efficacy or safety.
3.2 Diagnostic criteria
The presence of sporadic, genetic, iatrogenic and variant
prion diseases as well as criteria for diagnostic accuracy (possi-
ble, probable and definite) based upon clinical, laboratory and
instrumental analyses (Table 1) require the a priori selection of
patients based on disease variability and diagnostic accuracy.
Two studies (UK quinacrine [65] and doxycycline [76])
included all forms of probable or definite prion diseases, though
with minor differences, while the other two (flupirtine [50] and
US quinacrine [66]) selected only probable or definite sporadic
CJD patients. However, in these two last studies, 5% of
enrolled sporadic CJD patients finally were classified as genetic
prion diseases. This is because the result of the genetic screening
requires time and is therefore available only after randomization
and family history is often unremarkable and does not reveal the
suspicion of a genetic trait in these patients.
The authors of the flupirtine study [50] finally decided to
include genetic patients in the analyses of the study while
those of the US quinacrine [66] did not. Because clinical fea-
tures of genetic prion diseases are heterogeneous, mostly
depending on the type of PRNP mutation, it is reasonable
to include only genetic forms of CJD (i.e., the E200K and
V210I mutations) strongly resembling sporadic CJD for clin-
ical presentation, age at onset and disease duration, and
exclude others (i.e., GSS or FFI) that significantly differ
from the sporadic forms. This last approach was adopted in
the doxycycline study [76] for the Italian patients.
All studies included only patients with high ( 90%) diag-
nostic accuracy (i.e., probable or definite cases according to
international classifications). This obligatory strategy, how-
ever, has the great limitation of including most of the patients
in an advanced stage of disease and, therefore, with short nat-
ural survival [86]. In our data set from over 1000 definite spo-
radic CJD cases collected since 1993 during the national
program of surveillance, the median interval between onset
and in vitam diagnosis of ‘probable’ CJD was 3 months
(interquartile range, 3 months) and from the diagnosis of
probable to death only 1 month (interquartile range,
3 months). The two major determinants of survival, that is,
codon 129 polymorphism and PrPTSE
isotype [12], influence
the interval between onset and diagnosis of ‘probable’ but
not from here to death. These data indicate the urgency of
revising diagnostic criteria for the inclusion of CJD patients
in future clinical trials (Table 1).
3.3 Randomization
The golden standard for clinical trials is the allocation of
patients to experimental arms by a randomized procedure
for minimizing biases caused by unknown confounding
Table 5. Intraventricular infusion of pentosan polysulfate.
CJD: Creutzfeldt--Jakob disease; GSS: Gerstmann--Stra¨ussler--Scheinker disease; RML: Rocky mountain laboratory.
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Table 6. Clinical trials in prion diseases.
Drug (vs placebo) Flupirtine Quinacrine Quinacrine Doxycycline
Ref. and year of
publication
[50], 2004 [65], 2009 [66], 2013 [76], 2014
Aim Efficacy Efficacy and safety Efficacy Efficacy and tolerability
Primary end points Difference in cognitive
performance (ADAS-Cog)
between baseline and
treatment
Survival and clinical
improvement or lack of
deterioration
Survival at 2 months
from randomization
Survival time from
randomization
Type of study RCT
National (Germany)
Patient-preference trial-
partially randomized but
finally an observational
study
National (UK)
RCT
National (USA)
RCT
International (Italy and
France)
Inclusion criteria Probable sporadic CJD
who scored ‡ 50% in at
least 2 of 12 subtests of
ADAS-Cog and
Goettingen CJD dementia
tests
All definite or probable
human prion diseases
Definite or probable
sporadic CJD
Italy: Definite or probable
sporadic and genetic CJD
with disease duration
£ 6m;
France: All definite or
probable human prion
diseases
Randomization/blinding Yes/double-blinding Choice between
randomization or no
randomization/double-
blinding
Yes/double-blinding
for the first 2 months,
then open
Yes/double-blinding
Procedure of
randomization
Simple Stratified by type of
disease
Stratified by the
Barthel Index Score
Italy: Minimization
method by sex, age at
onset, months since CJD
clinical onset, codon 129
PRNP genotype.
France: Simple
Randomization ratio 1:1 1:1 1:1 1:1
Informed consent
(provided by)
Yes (unspecified) Yes (patients or family
members or independent
neurologists)
Yes (patients or legally
authorized
representative)
Yes (patients or
caregivers)
Minimum clinical
significant difference
Difference (‡ 4) between
baseline and treatment
(ADAS-Cog)
Reduction in 2 year
mortality from 50 to 22%
Increase in mean
survival time since
randomization from
0.9 to 1.8 months
Italy: Doubling the
percentage of survivors at
1 year (from 30 to 60%);
France Increase in mean
survival time since onset
from 6.2 to 11 months
a; statistical power 0.05; 0.70 0.05; 0.80 0.05; 0.80 0.05; 0.80;
Calculated sample size 30 160 60 Italy: 63
France: sequential design,
interim analysis after first
80 patients enrolled and
every 20 patients
thereafter
Number of patients
assessed for eligibility or
referred to the centres
682 (assessed for
eligibility)
221 (referred to the
national Prion Clinic)
425 (referred to the
UCSF)
103 in Italy and 560 in
France (screened for
eligibility)
Number of patients
enrolled
28 84 54 121 (Italy: 55; France: 66)
% of enrolled patients 5% 38% 47% Italy: 53%; France:12%
Number of patients per
arm
Flupirtine: 13
Placebo: 15
Chose immediate
quinacrine: 24
Chose no quinacrine: 59
Chose random allocation:
1
Quinacrine: 23
Placebo: 28
Doxycycline: 62
Placebo: 59
Status Completed Closed Completed Stopped for futility at first
interim analysis
CJD: Creutzfeldt--Jakob disease; RCT: Randomized controlled trial.
V. Vetrugno et al.
Forpersonaluseonly.

factors, immeasurable variables or both. Informed consent is
asked to patients, often caregivers, when patients have serious
cognitive deficits, who need to accept the gamble of being
assigned to the arm of the putative active drug. This difficult
choice is clearly exemplified in the UK quinacrine study [65],
where patients (or the caregivers) had the choice to take the
drug, not take it or accept randomization to immediate quin-
acrine versus 6-months deferred quinacrine. Only 2 of
84 enrolled patients chose randomization, 23 immediate
quinacrine and 59 no quinacrine resulting in an observational
rather than a RCT study. Interestingly, the most and least
severely affected patients did not choose treatment with quin-
acrine. In the US quinacrine [66], flupirtine [50] and doxycy-
cline [76] studies, however, randomization was successful
suggesting that RCT-DB studies are feasible, though with
some difficulties. In the US quinacrine study [66], 87% of sur-
viving patients in the placebo arm and 100% in the quina-
crine arm returning for the 2-month visit and preferred
treatment with quinacrine in contrast with the trend of the
British study [65], where only 27% of recruited patients asked
for the putative active drug.
A variety of randomization procedures were adopted in
CJD trials; simple random procedures in the flupirtine [50]
and doxycycline (French patients) [76] studies, stratified by
type of disease in the UK quinacrine [65], stratified by perfor-
mance in daily living activities (Barthel Index) in the US quin-
acrine [66], and minimization by sex, age at onset, months
since CJD clinical onset and codon 129 PRNP genotype in
the doxycycline (Italian patients) study [76]. This last random-
ization procedure was adopted for improving the control of
known predictors of survival [12]. Despite differences in the
randomization procedures, all studies adopted the question-
able choice, in a fatale and untreatable disease, of a 1:1 ratio
between the active drug arm and the placebo arm.
Overall, these data indicate caution in oversimplifying the
experience of a single study in terms of feasibility in conduct-
ing an RCT-DB study or in accepting treatment with an
experimental drug, though with uncertain efficacy, by patients
with CJD or their caregivers.
4. Ongoing or future possible trials
Despite the appearance of increasing number of scientific
articles reporting novel therapeutic approaches in in vitro or
in vivo models of prion diseases [35,87,88], the only ongoing clin-
ical trials in human prion diseases is limited to the doxycycline
preventive treatment in healthy members of a large Italian fam-
ily affected by FFI [36,89]. The only other treatments in advanced
stage for a first-in-human safety study are, to the best of our
knowledge, the MRC Prion Unit’s PRN100 antibody [90,91]
and the oligomer modulator Anle138b [92].
4.1 Doxycycline preventive treatment in FFI
FFI is a rare genetic prion disease linked to the presence of the
D178N mutation coupled with methionine at the
polymorphic codon 129 (D178N/M129) of the PRNP gene
and characterized by severe insomnia, autonomic disturbances,
cognitive changes, ataxia and endocrine manifestations [93,94].
Most of mutated subjects develop disease during their lifetime,
usually in the 50s, and inevitably die in about 12 months [93,94].
Because therapy is unavailable, Forloni et al. proposed to
members of one large affected kindred in northern Italy a
life-long preventive therapy with doxycycline [36,89], which in
experimental models of prion diseases efficiently delay onset
of disease if given before onset of clinical signs [73]. Currently,
11 healthy carriers and 19 non-carriers belonging to the above
kindred and born between 1958 and 1969 are blinded treated
with a daily dose of 100 mg doxycycline (mutated carriers) or
placebo (non-mutated subjects) for 11 years. Young family
members will be included in the study as soon as they reach
41 years of age. Participants of the study are requested to
undertake a complete neurological examination every 2 years
including neuropsychological assessment, autonomic nervous
system assessment, polysomnographic EEG recording, actigra-
phy, brain MRI and FDG-PET [95]. The aim of this study is to
assess the delay (or prevention) of disease onset in people who
are predestined to develop FFI in their middle age. The ratio-
nale of this trial is based on the observation in animal models
that therapy with doxycycline and all other antiprion drugs is
much more effective when given before clinical onset [73].
The ambitious result of the study is to observe a 50% reduc-
tion of mortality in treated carriers over 10 years of treatment
with respect to historical familial data. The success of this
study will open the possibility of preventive treatment in
genetic prion diseases [22] and eventually in people at high
risk of developing iatrogenic CJD [9].
4.2 MRC Prion Unit’s PRN100 antibody
In 2003, White et al. [96] showed that ICSM35 and
ICSM18 mAbs directed against the a-helix-1 of PrP [97]
strongly delay onset of scrapie disease in intraperitoneally
infected mice. Although treatment was only effective if started
before clinical onset, the finding that these antibodies did not
cause any brain damages fostered the possibility of their use for
human therapy [98]. The MRC Pion Unit followed this line of
thought and developed a human analogue of ICSM18 (named
PRN100), which showed no CNS toxicity in cynomolgus
macaques at intravenous doses (up to 200 mg/kg) suggesting
a safe use for human studies [90]. With these bases, the MRC
Prion Unit (UK) has designed a clinical trial, waiting for final
approvals, to simultaneously evaluate safety and efficacy on a
small number of CJD patients [91]. There are, however, issues
that need clarification. One is on the real efficacy of passive
immunization therapy in prion diseases. Petsch et al. [99]
showed that treatments with the mAb W226, which binds to
the same epitopes of ICSM18, is unable to protect mice
from an intraperitoneal prion infection, arguing that the ther-
apeutic efficacy likely depends also on the genetic background
of the host (Petsch et al. [99] and White et al. [96] used different
strains of mice) or differences in the antibody sequences
Forpersonaluseonly.

(ICSM18 and W226 show significant differences in the
sequence of the hypervariable complementarity determining
regions), which might influence the half-life of the compound
or its efficacy to reach the CNS. The other issue is safety,
which remains unsettled because of contrasting experimental
data between mAbs binding overlapping epitopes on a-helix-1
of PrP. ICSM18 [100], D18 [101] and PRN100 [90] look safe
while POM1 shows severe neuronal loss [102]. Understanding
the mechanisms that differentiate mAbs with the same binding
epitopes on PrP in terms of efficacy and safety is mandatory
before starting any clinical trial in patients with CJD.
4.3 3-(1,3-Benzodioxol-5-yl)-5-(3-bromophenyl)-1H-
pyrazole (anle138b)
Anle138b is a synthetic compound with a di-phenyl-pyrazole
structure that selectively inhibits pathological oligomer accu-
mulation of PrPTSE
and a-synuclein without interfering
with the physiological functions of the non-pathological
monomeric isoforms [103]. The finding that anle138b is effec-
tive in preventing PrPTSE
formation from different prion
strains and a-synuclein suggests that it targets common
structure-dependent epitopes responsible for the oligomeric
formation of pathological proteins. Experimental data also
showed that anle138b prolongs the incubation period of
scrapie-infected mice and inhibits disease progression in a
Parkinson mouse model, even when treatment is started after
disease onset [103,104]. The spin-off company (MODAG
GmbH) from the Ludwig-Maximilians-Universita¨t Mu¨nchen
and the Max Planck Society plans to develop anle138b up to
market maturity for a Phase I/II clinical trial in CJD and
other neurodegenerative diseases [105]. Although the com-
pound has an excellent oral bioavailability, efficiently bypasses
the blood--brain barrier and shows no toxicity at therapeutic
doses, it would be wise to improve preclinical data (i.e., prov-
ing efficacy in mice with different genetic backgrounds and
inoculated with a variety of prion strains, including sporadic
CJD) before its use in human therapy.
5. Conclusions
In the last few years, treatment of patients with CJD has been
successfully approached with RCTs [50,66,76]. Although the
outcome of these initial trials was substantially negative,
they establish the feasibility of RCTs in rare and rapidly pro-
gressive diseases such as CJD. The failure of these trials
depends on several factors, which include the difficulty of an
early clinical diagnosis and the consequent short interval
between treatment and death [86], the variability of clinical
duration of illness [12], the absence of reliable disease-modifier
markers [25] and validated clinical scales [106,107] and often the
overinterpretation of preclinical data.
Patients are usually recruited when clinical signs and instru-
mental features fulfill the diagnosis of probable CJD (Table 1),
which occurs relatively late in the course of illness and it is
inevitably accompanied by an irreversible brain damage.
Novel diagnostic procedures, based on the signal amplifica-
tion of the misfolded prion protein in body fluids, that is,
CSF [26,27] and urine [29], or easily accessible tissues, such as
nasal olfactory mucosa [28] would likely anticipate the time
for an accurate diagnosis of CJD. Whether PrPTSE
in body
fluids and nasal olfactory mucosa would be a useful marker
for monitoring the effect of future therapy is still unknown.
Variability in disease duration depends on known factors
(codon 129 polymorphism, type of PrPTSE
deposition in the
CNS, gender and age at onset) [12] that were partially consid-
ered during randomization [76], but survival significantly varies
even within these categories [12] for yet unknown reasons,
likely including the poorly controllable supportive care of fam-
ily members [108]. A boost in searching for such factors is com-
ing from genome-wide association studies, which might soon
identify novel genes able to modulate disease duration [109,110].
Finally, the rationales leading to the use of compounds
entering into human therapy were either poorly supported
by preclinical data or these studies were overinterpreted. Quin-
acrine, for example, was initially given to CJD patients based
only on in vitro studies [54,55] without waiting for confirmatory
efficacy in animal models, which finally did not confirm the
antiprion effect [67,68] and doxycycline was given to patients
late in the course of disease despite preclinical studies showed
only modest, if any, therapeutic effect in early diseased
prion-infected rodents [73].
6. Expert opinion
The future of therapeutic intervention in CJD starts from the
identification of novel compounds with strong antiprion
effects. Based on the only undisputed pathogenic mechanism
of disease, novel drugs are usually selected for their action on
halting PrPTSE
formation or increasing its clearance by
in silico technology [111] or through the screening of large
chemical libraries containing hundreds of thousands of
compounds [111-115]. These ongoing activities have already
identified a number of potential antiprion compounds that
would now being further tested for determining their efficacy
in animal models of prion disease, CNS bioavailability and
toxicity before their possible use in humans. Other potential
antiprion candidates are biological products, either in terms
of passive or active immunization with mAbs targeting spe-
cific PrPC
epitopes [116-118], by using small interfering RNAs
that downregulate PrPC
expression [119,120], or targeting novel
pathway of neurodegeneration [121,122]. Immunological thera-
pies in Alzheimer’s disease, another protein misfolding CNS
pathology, yield some positive results, which were, however,
so far overcome by serious safety issues [123,124]. The future
of immunological treatments in neurodegenerative diseases
is promising but some adjustments of the molecules are likely
needed to minimize adverse effects.
Prion diseases have the great advantage over other neurode-
generative diseases of excellent animal models that mimic clin-
ical, pathological and biochemical features of human diseases,
V. Vetrugno et al.
Forpersonaluseonly.

but their use for the design and interpretation of preclinical
studies were often poorly performed or misinterpreted.
A boost in getting valid and relevant preclinical studies for plan-
ning future therapy in humans would come from the develop-
ment of an ad hoc internationally recognized guideline for
preclinical studies in prion diseases based on available check-
lists [125]. This guideline should cover critical aspects that have
been poorly followed in past preclinical prion studies, such as
the adoption of a randomized procedure for the allocation of
animals, the use of appropriate controls, the blindness of the
investigators during the experiment and a power calculation
performed a priori to determine sample size. Other important
aspects should include the use of more than one model of prion
infection considering that available data showed that drugs
might be effective in one model and not in others [126-129], the
choice of relevant models, for example, mice [130] or bank
voles [131] infected with and reproducing human PrPTSE
type 1 or type 2 [8] rather than animal strains, the route of infec-
tion and the need of confirming efficacy by independent
research groups. Finally, it is important to define the relevant
timing and route of treatment administration in relation to
the aim of the study. Researchers often inferred that drugs
with strong preventive effects in preclinical studies, that is,
given during the preclinical phase of disease, would have thera-
peutic effects despite countless evidences that treatments are
ineffective or poorly effective when given at early clinical phases
or even close to onset of disease [69,73,80]. Overall, these preclin-
ical studies show that brain damage is already too severe at onset
of clinical signs for attempting any possible therapy and there-
fore suggest that any potential treatment in CJD patients
should begin as soon as possible. This is in contrast with past
clinical trials where patients were enrolled only when they ful-
filled the diagnostic criteria of probable CJD, which inevitably
occurs at a late stage of disease [86]. Thus, it would be necessary
to develop novel international diagnostic criteria for including
CJD patients in clinical trials in a very early stage by using the
new approaches of PrPTSE
amplification and identification in
the CSF [26,27], urine [29] or olfactory mucosa (Table 1) [28].
Although these assays need further validations before their use
in diagnostic criteria for surveillance, they might be adequate
for the selection and inclusion of patients in clinical trials at
an early stage of disease. Autopsy should be performed in all
enrolled patients to confirm clinical diagnosis and for deter-
mining PrPTSE
type accumulation, which is an important
determinant of survival in sporadic CJD [12].
Future clinical studies in CJD need some rethinking in
terms of the choice of end points, the design of randomized
trials and the ethical considerations for obtaining the consent
to the experimentation or for using a placebo in an otherwise
deadly disease [132,133].
Survival is a robust end point in prion diseases but drugs,
such as antibiotics (e.g., doxycycline), able to improve the
general conditions of terminally ill patients, might give false-
positive outcomes. The identification of disease-modified bio-
markers, the use of internationally validated prion rating
scales or both might represent valid alternatives [106,107]. How-
ever, consensus is needed on the minimal clinically important
changes because a high expectancy [134] might be unrealistic
and will make future trials ending for futility, or be ethically
controversial [132,133].
Improvement in the design of trials for rare, fatal and rap-
idly progressive diseases such as CJD is strongly needed, espe-
cially if treatments with striking efficacy become available. In
such circumstances, study designs should rapidly prove or dis-
prove efficacy. Randomized trials have the advantage of pre-
venting biases but there are still uncertainties whether
randomization is always necessary [135] or ethically
acceptable [133,136-138]. It is of note that 33% of the pivotal tri-
als of orphan drugs for neurological diseases approved by the
US FDA did not use placebo controls, 27% were not double
blind and 12% were not randomized [139]. Properly collected
historical controls might be a valid alternative [133], but biases
in terms of selection of patients based on those who consent to
take the active drugs and assessment of primary end points
should be considered.
Acknowledgments
We acknowledge funding from the Ministry of Health,
National Centre for Disease Prevention and Control, Central
Actions ‘Sorveglianza della Malattia di Creutzfeldt--Jakob’.
We thank D Meloni for critical reading of the manuscript
and A Garozzo for technical support.
Declaration of interest
The Italian Ministry of Health has supported this work. The
authors have no other relevant affiliations or financial involve-
ment with any organization or entity with a financial interest
in or financial conflict with the subject matter or materials dis-
cussed in the manuscript. This includes employment, consultan-
cies, honoraria, stock ownership or options, expert testimony,
grants or patents received or pending or royalties.
Forpersonaluseonly.

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Affiliation
Vito Vetrugno PhD, Maria Puopolo DStat,
Franco Cardone PhD, Fiorentino Capozzoli MD,
Anna Ladogana MD Maurizio Pocchiari†
MD
†
Author for correspondence
Istituto Superiore di Sanita, Department of Cell
Biology and Neurosciences, Viale Regina Elena
299, Roma 00161, Italy
E-mail: maurizio.pocchiari@iss.it
V. Vetrugno et al.
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