A double-blind, randomized, placebo-controlled, single-center, two-way cross-over study with KH176 in patients with the mitochondrial DNA tRNALeu(UUR) m.3243A>G mutation and clinical signs of mitochondrial disease

This study is a Phase II, Proof of Concept clinical trial with KH176 with the
promise to develop a novel treatment for mitochondrial diseases, specifically
for patients with a m.3243A>G mutation, resulting in oxidative phosphorylation
(OXPHOS) deficiency, for whom currently no clinically relevant treatment is
available.

Inherited Mitochondrial OXPHOS disorders are a group of clinically
heterogeneous diseases defined by lack of cellular energy production due to
defects in the mitochondrial OXPHOS process, affecting ~1/5000 of the
population. Several debilitating mitochondrial OXPHOS diseases exist like
MELAS, LHON, and Leigh syndrome. The disease course depends on the primary
defect, is severely debilitating and progressive, involving dysfunction of
neuromuscular and other organ systems and can result in early death. The
majority of OXPHOS disorders are caused by isolated or combined (involving
other OXPHOS enzyme complexes) OXPHOS Complex I deficiencies.

Despite advances in understanding these disorders, treatment options are
extremely limited and only supportive. Therefore, there is an urgent need for
novel treatments.
Within all cells of the human body, mitochondria act as a powerhouse -
collectively producing energy that is essential for life. When these
mitochondria are defective, the result can take the form of a wide range of
serious and debilitating illnesses. Signs and symptoms of mitochondrial
diseases can include: fatigue, intolerance to exercise, muscle weakness and
loss of muscle coordination, heart failure, diabetes, deafness, blindness,
stunted growth, and learning disabilities. Leigh Disease, MELAS and LHON belong
to the ever-expanding group of mitochondrial diseases.
*Mitochondrial diseases* refers to disorders whose underlying genetic cause
directly or indirectly impairs the composition or function of the 5-complexes
forming the oxidative phosphorylation system (OXPHOS), localized within the
inner mitochondrial membrane in which the end products of intermediary
metabolism are oxidized to generate energy in the form of adenosine
triphosphate (ATP).
Mitochondrial failure is usually the result of genetic defects in either the
mitochondrial DNA or the nuclear DNA, but can also be caused by environmental
factors, including adverse reactions to certain drugs.
Because of the multifactorial genetic background and the interaction of
heteroplasmy, replicative segregation and the threshold effect, mitochondrial
diseases present as a very wide range of phenotypes.
Scharfe et al. (2009) established a clinical phenotype catalogue of 174
mitochondrial disease genes associated with 191 diseases and studied the
association of diseases and genes. They noted that most mitochondrial disease
phenotypes involve several clinical categories including neurologic, metabolic,
and gastrointestinal disorders. All these phenotypes, however, share the common
feature of being based on inadequate OXPHOS within the mitochondria of the
affected tissues.
It has been recognised for more than a decade that cell redox imbalance play a
key role in the pathogenesis of many of the clinical manifestations of
mitochondrial diseases (Atkuri 2009, Hargreaves 2005, Wallace 2010, Kirkinezos
& Moraes, 2001, Scialo, 2016, Koopman 2016). Mitochondrial redox chemistry is
of profound importance to the redox balance of the entire cell and is central
to the signal transduction of many cellular pathways. For example, disruption
of mitochondrial redox circuitry will lead to an increased oxidative stress in
the cells (Jones 2006).



1.2 Mitochondrial DNA 3243A>G mutation, MELAS and MIDD and mixed types
Mitochondrial disorders can be caused by mutations of nuclear DNA or
mitochondrial DNA. The classification of mitochondrial disorders is hampered by
the wide variety in resulting phenotypes, with also a wide variety of clinical
syndromes. In this study, the mitochondrial DNA 3243A>G mutation is chosen as
the clinical syndromes resulting from this mutation are, although variable,
relatively well described. Despite being caused by one mutation, the clinical
syndromes resulting from this mutation are still heterogeneous. In Nijmegen
about 150 carriers of this mutation are involved in a detailed clinical follow
up study.

MELAS (Mitochondrial Epilepsy Lactic Acidosis and Stroke like episodes) is a
well-known syndrome that can be caused by the m.3243A>G mutation. The
encephalopathy is characterized by epilepsy or dementia, often with additional
neurological symptoms like migraine or psychiatric problems. Elevated lactic
acid levels are present in most patients. Stroke like episodes may arise at any
age (typically before the age of 40 years) and are characterized by the same
neurological abnormalities as seen in strokes (hemianopia, hemiparesis,
aphasia, hemineglect), associated with additional symptoms like seizures,
ataxia, migraine-like headaches, impaired vision or hearing, or disturbances in
consciousness. After stroke-like episodes, some patients retain a normal
intelligence, whereas others have severe mental and physical disabilities.

MIDD (Maternally Inherited Diabetes and Deafness) is the most common syndrome
caused by the m.3243A>G mutation and is a syndrome of early onset sensorineural
hearing loss and a defect in insulin secretion. The mean age at onset is 30
years for diabetes and 40 years for sensorineural hearing loss. MIDD typically
presents as early onset diabetes without obesity, rapid progression to insulin
requirement, high likelyhood of microvascular complications, and family history
suspicious of maternal inheritance. Other symptoms associated with the mutation
may also occur. These symptoms include: focal segmental glomerulosclerosis,
hypertrophic cardiomyopathy, macular retinal dystrophy, short stature, ptosis,
myopathy, neuropathy, and also MELAS syndrome.

The study will allow both phenotypes as well as mixed types to be included.
Although different in phenotype and clinical syndrome, several symptoms seem a
common denominator for all phenotypes. Fatique and tiredness is a clear symptom
present in all patients and disturbances in Gait appears to be present in the
vast majority.

CPEO (Chronic Progressive External Ophthalmoplegia) is another syndrome caused
by the m.3243A>G mutation. It can occur as an isolated ophthalmologic syndrome,
which would mean that most assessments done in this trial would not be
affected. Therefore, patients with CPEO can only participate if associated with
other features, such as abnormal gait.

1.3 Mitochondrial dysfunction and redox/ROS-imbalance
Mitochondria constantly metabolize oxygen thereby producing reactive oxygen
species (ROS) as a by-product. These organelles have their own ROS scavenging
mechanisms that are required for cell survival. It has been shown, however,
that mitochondria produce ROS at a rate higher than their scavenging capacity,
resulting in the incomplete metabolism of approximately 1-3% of the consumed
oxygen.
The by-products of incomplete oxygen metabolism are superoxide (O2-), hydrogen
peroxide (H2O2), and the hydroxyl radical (OH•).
The formation of superoxide occurs via the transfer of a free electron to
molecular oxygen. This reaction occurs at specific sites of the electron
transport chain (ETC), which resides in the inner mitochondrial membrane. ETC
complexes I (NADH: ubiquinone oxidoreductase) and III (cytochrome bc1 complex)
produce most of the superoxide, which is then scavenged by the mitochondrial
enzyme manganese superoxide dismutase (MnSOD) to produce H2O2.
Since mitochondria do not contain catalase, their only defence against the
potentially toxic properties of H2O2 is the enzyme glutathione peroxidase
(GSPx). GSPx requires reduced glutathione (GSH) as a coenzyme and converts H2O2
to water, thus completely detoxifying ROS. However, in the presence of reduced
transition metals, H2O2 can produce the highly reactive OH•, which can cause
extensive damage to DNA, proteins, and lipids.
Two other important radical species are nitric oxide (NO) and peroxynitrite
(ONOO*). Mitochondria possess their own nitric oxide synthase (mtNOS) and can
produce endogenous NO and ONOO*. Mitochondrial NO decays mainly via ONOO*
formation, ubiquinol oxidation, and reversible binding to cytochrome c oxidase.
Under normal conditions, the effects of ROS are counteracted by a variety of
antioxidants, by both enzymatic and non-enzymatic mechanisms. Oxidative stress
is considered to be the result of an imbalance of two opposing and antagonistic
forces, ROS and antioxidants, in which the effects of ROS are more potent than
the compensatory capacity of antioxidants. In the case of mitochondrial-derived
ROS, superoxide is the first radical produced. It is a highly reactive species
and does not diffuse easily throughout the cell. H2O2 is the next key player in
mitochondrially derived ROS, as it is the product of superoxide detoxification
by MnSOD.
H2O2 is not a free radical by definition because it lacks free electrons.
Nevertheless, its role in ROS-mediated damage is extremely significant by
virtue of chemical versatility and diffusibility. H2O2 is a substrate in many
physiological and abnormal chemical reactions both intracellularly and
extracellularly. Due to its small size and relatively benign reactivity,
compared to the rest of the ROS, H2O2 can diffuse freely across several cell
radii; therefore, it is able to mediate toxic effects far from the site of ROS
production. Although by itself H2O2 is not a major source of oxidative damage,
it can react with free transition metals via the Fenton reaction (Fe2+ + H2O2 *
Fe3+ + OH + OH•), producing the extremely reactive hydroxyl radical. OH• has a
very short half-life and reacts with virtually any molecules in close
proximity. There is no known scavenger for OH•, but OH• toxicity can be avoided
by minimizing the levels of H2O2 and most importantly the availability of free
transition metals (e.g. Fe2+, Cu+).
Nitric oxide has also been implicated in ROS mediated damage. NO has a dual
personality, which is both beneficial, and detrimental. In some instances,
different intracellular levels of NO can mediate diverse effects. For example,
physiological levels of NO inhibit the opening of the mitochondrial
permeability transition pore (PTP), whereas high NO concentrations promote PTP
opening. High levels of NO are cytotoxic, although the exact mechanism
associated with this effect is still unclear. It may be involved in
inflammatory, neurodegenerative, and cardiovascular pathological processes.

1.4 KH176

Khondrion has identified a class of highly active compounds that are capable of
preventing the negative cellular consequences of OXPHOS dysfunction. The most
promising lead compound is KH176, a redox modulator, currently under
development. KH176 acts as a potent intracellular redox-modulating agent
targeting the reactive oxygen species as demonstrated by a number of in vitro
and in vivo assays.

Khondrion has recently received Orphan Drug Designation (ODD) status for KH176
to treat Leigh and MELAS syndrome by the European Medicines Agency (EMA) and by
the FDA for the treatment of all inherited mitochondrial respiratory chain
disorders. Leigh syndrome is actually a pediatric indication and it should be
noted that translation of a successful proof of concept in this study to a
pediatric dosing regimen is foreseen.

KH176 underwent pre-clinical testing to ensure its activity and safety before
entering clinical studies. Further details can be found in the Investigator*s
Brochure. In brief KH176 showed a potent intracellular redoxmodulating capacity
to target the reactive oxygen species that have been shown in different in
vitro and in vivo models to play an important role in the pathogenesis of the
above described conditions. The metabolite KH183 has been shown to have a
pharmacological activity that is (in vitro) at least comparable to the activity
of KH176.
The genotoxicity studies (Ames test, Chromosomal Aberration test and in vivo
Micronucleus test) revealed no mutagenic nor clastogenic effects. Also the
safety pharmacology studies showed no relevant KH176-related effects, i.e. for
both the CNS rat study and the respiratory rat study, the No Observed Adverse
Effect Levels (NOAELs) were indicated to be higher than the highest dose level
administered (250 mg/kg/dose BID). For the dog cardiovascular study the NOAEL
was set at the highest dose level of 45 mg/kg/dose BID. Moderate adverse
effects (vomiting, salivation and tremors in dogs, and histopathological
findings in the rat, including phospholipidosis in a broad range of organs
(high dose only) and follicular cell hypertrophy in the thyroid glands up to
the lowest dose level tested (25 mg/kg/dose BID)) were recorded in the repeated
dose toxicity studies. Effects as described in these studies were mainly
showing reversibility in the 2 week treatment free period.
A Phase I study in healthy male participants was performed to evaluate the
safety, tolerability, pharmacokinetics and pharmacodynamics in a Single
Ascending Dose (SAD) and Multiple Ascending Dose (MAD) design.

KH176 was well tolerated by healthy subjects up to 800 mg as a single dose and
up to 400 mg BID for 7 days, with mild to moderate headache as the only
possibly drug related adverse event. A dose of 2000 mg, which was administered
as a single dose, was however not tolerated. At 2000 mg participants
experienced dizziness, nausea and vomiting, oral paraesthesia and psychiatric
symptoms (depersonalisation, bradyphrenia and visual hallucinations).
Furthermore, telemetry and ECG recordings post-dosing showed a marked QTc
prolongation and to a lesser extent a QRS prolongation. To follow up on these
findings, an extensive evaluation of the ECG*s and an analysis of the different
intervals were performed and a PKPD evaluation was performed to understand the
relation of the ECG changes with dose and exposure of KH176. In addition, an in
vitro study was conducted to determine the ion channel-blocking profile of
KH176 and KH183. The following currents were examined; hERG, Nav1.5 (peak and
late), Cav1.2, and KvLQT1/mink. These currents are known to be relevant for
conduction and repolarization. The results of this analysis are described in
paragraph 1.4.4. dose justification.

The pharmacokinetics of KH176 shows a rapid absorption profile with a time to
reach maximum concentrations (Tmax) of around 2 hours. Half live of KH176 was
around 9 h for KH176 and around 15 hours for its active metabolite KH183. Which
results in an accumulation factor of 2.5 for KH176 and a factor 2 for KH183
with 100 mg BID dosing in steady state conditions. Steady state conditions were
reached within 3 days of dosing.

With increasing dose, the plasma-exposure increased slightly more than
proportional, although half-live remained the same across doses, indicating a
potential difference in bioavailability with dose. This seems in line with the
observation that KH176 is a substrate for Cytochrome P450 3A4 and the efflux
pump P-glycoprotein. Saturation of this efflux pump typically results in this
type of disproportional increases of plasma concentrations with increasing dose.

The intake of food with the administration of KH176 had no relevant influence
on the pharmacokinetics of KH176 and KH183 and therefore administration of
KH176 in this study will be done just prior to a meal for compliance reasons.

Pharmacodynamic evalutions in these healthy participants did not reveal any
relevant signs related to mitochondrial functioning, however, it should be
noted that the lack of impairment in mitochondrial functioning in this
population hampers any conclusions.

Objectives:
Primary Objective
1. To evaluate the effect of KH176 on gait (Gaitrite®) parameters: step-length
and variability in step-time and step-width in patients with a m.3243A>G
mutation

Secondary Objectives
2. To explore the effect of KH176 on biomarkers of mitochondrial functioning in
patients with an m.3243A>G mutation.
3. To explore the effect of KH176 on functional clinical measures of
mitochondrial disease in patients with an m.3243A>G mutation.
4. To investigate the tolerability and safety of KH176 following 28 days of
oral administration to patients with an m.3243A>G mutation.
5. To investigate the multiple dose pharmacokinetics of KH176 following 28 days
of oral administration in patients with an m.3243A>G mutation.

Methodology:
The trial will be a double blind, randomized, placebo-controlled,
single-centre, two-way cross-over trial. Twenty patients, with a confirmed
mitochondrial DNA tRNALeu(UUR) m.3243A>G mutation and with clinical signs of
mitochondrial disease, will be randomized over 2 groups (active or placebo
first). After a screening period and a training session, each group will have 2
dosing periods of 28 days, with a washout period of at least 28 days in
between. On these occasions, patients will receive 100 mg KH176 twice daily
(treatment A) or a matching placebo (treatment B) twice daily for 28 days.




Below the design of the trial has been presented
Week
Group -3 - -1 -1 1-4 5-8 9-12 14
I Screening Training Treatment A Washout Treatment B Follow up
II Screening Training Treatment B Washout Treatment A Follow up

Clinical assessments will be performed once in a training session prior to
baseline, at baseline and in week 4 post dosing during each treatment phase (A
and B), details can be found in the flow chart (paragraph 3.2). Testing
conditions and circumstances, with respect to timing of the assesments,
hospitilization and meals, will be standardized for each assessement period.
Furthermore, assessments of biomarkers for mitochondrial functioning,
pharmacokinetics and specific safety assessments will be performed weekly
according to the time points indicated in the flowchart.
In each treatment phase (A and B), patients will be hospitalized for the
baseline assessments and for safety monitoring for the first 3 days of
treatment (Day*s 1, 2 completely and Day 3 until discharge in the afternoon).
As an interim evaluation for safety monitoring, and as a part of the
pharmacokinetic evaluation, 3 plasma samples will be taken on Day 1 after the
morning dose, shipped and analysed immediately, with the aim to have results
prior to discharge on Day 3. The results will not be reported to the study team
(as the study team should remain blinded), but only the conclusion whether or
not the safety treshold (1000 ng/mL) was exceeded will be reported. The
objective of this interim evaluation is to compare plasma exposure in these
patients with the plasma exposure of KH176 and its metabolite KH183 in healthy
participants. In case the treshold of 1000 ng/mL is exceeded, investigators and
sponsor will discuss options for continuation, including a dose reduction,
taking all safety and pharmacokinetic results into account.
For logistical reasons of the intense safety monitoring on Days 1 to 3
participants will be scheduled in groups of maximally 3 participants.
Participants will be randomized in such a way that no more than 2 participants
will have active treatment at the same time during those first 3 days of
intense monitoring.

Investigational Drug:
Treatment A: Twice daily dosing of 100 mg (100 mg BID/ 200 mg per day).
KH176 will be administered as an oral liquid, just before a meal.
Comparative Drug:
Treatment B: Twice daily dosing of a matching placebo as an oral liquid, just
before a meal.

Side effects/risks as described in the subjects Information

When we administered KH176 in very high doses (1 x 800 mg or 2 x daily 400 mg)
in healthy men, changes were seen on the heart tracing (ECG). It concerns a
change in the conduction of the electric stimulation of the heart. More
specifically, an extension of the so-called QT-time (the time between the
control of the contraction of the heart to the recovery phase of the
conduction) was observed. This change will not be noticed by the patient.
However, if this change is larger (the QT interval is longer), this could lead
to arrhythmia. The arrhythmia that can occur if you receive too high of a dose
can be fatal if they are not observed or treated.

There are drugs that are prescribed by doctors that affect the conduction of
the heart, such as anti-psychotic, some antidepressants and anti-vomiting
medication. In high doses, these frequently prescribed drugs can also lead to
arrhythmia. Persons with a sensitive heart muscle, for example, after a
myocardial infarct, are hereby more sensitive to the onset of arrhythmia of the
heart.

Despite the fact that we will investigate a lower dose of KH176 in this study
and we estimate the risk is very low for these changes, we will take strict
security measures to prevent KH176 to cause arrhythmia. Below are the steps we
have taken:
- You will receive 2 x daily 100 mg KH176. To illustrate: The first changes in
the conduction of the heart were observed at 2 x 400 mg per day.
- We know that patients with abnormal cardiac muscle are more sensitive to
cardiac arrhythmia. Therefore only in this study m.3243A> G carriers
participate with no or very mild abnormalities of the heart muscle. This will
be assessed on the basis of your medical information. If necessary, we will
perform an additional cycling test to assess the function of your heart during
exercise.
- The transformation of a normal conduction to arrhythmia is gradual. If we see
that your ECG shows any change, we will monitor you extra well and possibly
stop the medication so that the conduction can recover. This is to prevent you
from getting heart arrhythmia. You will be at the medium or intensive care
during the first period of treatment to ensure maximum monitoring.
- We determine the amount of KH176 in your blood after the first dose to see if
you have an amount of KH176 in your blood that we would expect
- We will make multiple heart movies during the entire treatment period, a few
per day during the first doses and subsequently 1 x weekly at home
- You will receive a box (Holter) to take home on which your heart rhythm is
registered.

KH176 is ingested only by 30 healthy men for 7 days. They did not give more
complaints than healthy men who received a placebo. The study drug could have
side effects which are unknown.