Phase 1/2 Study to Investigate the Safety, Pharmacokinetics and Efficacy of Tinostamustine, a First-in-Class Alkylating Histone Deacetylase Inhibition (HDACi) Fusion Molecule, in Patients with Advanced Solid Tumors

Initially regarded as *epigenetic modifiers* acting predominantly through
chromatin remodeling by maintaining histone acetylation, histone deacetylase
(HDAC) inhibitors (HDACi) are recognized to exert multiple cytotoxic actions in
cancer cells, often through acetylation of non-histone proteins. Some
well-recognized mechanisms of HDACi lethality include, in addition to
relaxation of DNA and de-repression of gene transcription, interference with
chaperone protein function, free radical generation, induction of
deoxyribonucleic acid (DNA) damage, up-regulation of endogenous
inhibitors of cell cycle progression, e.g., p21, and promotion of apoptosis.
This class of agents is relatively selective for transformed cells, at least in
nonclinical trials. In recent years, additional mechanisms of action of these
agents have been uncovered. For example, HDACi compounds interfere with
multiple DNA repair processes, as well as disrupt cell cycle checkpoints,
critical to the maintenance of genomic integrity in the face of diverse
genotoxic insults. Despite their nonclinical potential, the clinical use
of HDAC inhibitors remains restricted to certain subsets of T-cell
lymphoma. Currently, it appears likely that the ultimate role of these agents
will lie in rational combinations, only a few of which have been pursued in the
clinic to date.

Multiple lines of recent data have begun to suggest that there is biologically
important synergy that exists between alkylating agents and HDAC inhibitors.
For example, in one trial the combination of bendamustine, an alkylating agent,
and entinostat, a HDAC inhibitor, synergistically inhibits proliferation of
multiple myeloma (MM) cells via induction of apoptosis and DNA damage response.
In this trial, cell growth assays showed that bendamustine or entinostat
inhibited proliferation in a dose-dependent manner, and their combinations
synergistically induced growth inhibition in all MM cells tested. An apoptotic
enzyme-linked immunoassay (ELISA) and western blot assays on poly (ADP-ribose)
polymerase (PARP) cleavage and caspase-8 and caspase-3 revealed that
bendamustine in combination with entinostat exhibited a much more potent
activity than either agent alone to promote the MM cells undergoing apoptosis
in a dose-dependent manner. Flow cytometric analysis found that entinostat
exhibited distinct effects on cell cycle progression in different lines
and bendamustine mainly arrested the cells at S phase, whereas their
combinations dramatically blocked the S cells entering G2/M phase.
Furthermore, trials on DNA damage response indicated that phosphohistone
H2A.X (P-H2A.X), a hallmark of DNA double strand break, along with
phosphorylated CHK2 (P-CHK2) was significantly enhanced by the combinations of
bendamustine and entinostat as compared to either agent alone. These molecular
changes were correlated with the increases in mitotic catastrophe.

Tinostamustine is a first in class alkylating HDAC inhibitor that is being
developed for the treatment of relapsed/refractory hematologic malignancies and
solid tumors. The compound underwent broad evaluation in nonclinical models for
human cancer. In in vitro and in vivo trials demonstrate efficacy in models of
Hodgkin lymphoma, aggressive non-Hodgkin lymphoma, multiple myeloma, T-cell
lymphoma and acute myeloid leukemia. In solid tumors activity was seen in
models of sarcoma, small cell lung cancer (SCLC), non-small cell lung cancer,
breast cancer, ovarian cancer and glioblastoma. The efficacy was independent
from p53 status and cell lines resistant to other chemotherapy agents still
responded to tinostamustine, including cell lines particularly resistant
to bendamustine. Mechanistically, tinostamustine induces a strong DNA
damage response, evidenced by a rise of *-pH2AX and p53, while DNA damage
response was suppressed.

Consequently, in vitro experiments showed synergy with DNA repair influencing
agents such as
PARP inhibitors.

The ability to fuse discrete small molecules with different types of
pharmacologic activity has created remarkable opportunities in drug discovery
and development. Bendamustine itself is a fusion molecule of the nitrogen
mustard mechlorethamine and a purine analog based on fludarabine that exhibits
unique activity where cancer cells have become resistant to conventional
alkylating agents. In fact, randomized clinical trials have established that
the combination of rituximab and bendamustine exhibits less toxicity and
greater efficacy compared to a conventional R-CHOP based regimen. This concept
opens the prospect that other rational fusion molecules could exhibit activity
even greater than that seen in the parent molecule.

Tinostamustine is a unique new chemical entity. In the current and ongoing
first-in-human dose escalation trial, some patients with relapsed or refractory
hematological malignancies, for which there are no available approved
therapies, benefited from treatment. The benefit risk assessment is
in favour of further development of this molecule in humans.

The trial is designed as an open label, Phase 1/2 trial of single agent
tinostamustine. The Phase 1 portion of the trial was designed to define the
MTD by evaluating toxicities during dose escalation until MAD. The Phase 2
portion of the trial is designed to evaluate ORR of the RP2D (80 mg/m2 of
tinostamustine administered over 1 hour on Day 1 and 15 of each 4-week
treatment cycle) at 4 or 6 months, depending on the type of solid tumor.
Secondary objectives are evaluation of safety and tolerability of the RP2D in
selected solid tumors. Patients will be eligible for this trial if they have a
histologically confirmed solid tumor, sign informed consent and meet the
inclusion/exclusion criteria. After enrollment, patients will be screened, and
all
procedures will be performed as per protocol.

Patients will receive 80 mg/m2 of tinostamustine administered over 1 hour on
Day 1 and 15 of each 4-week treatment cycle.

There are currently no known drug interactions with Tinostamustine, however not
all possible side effects are known. Furthermore, the risk described below may
occur more often or more severely than previously seen. In addition to the
possible side effects mentioned below, there is always the possibility of
unexpected side effects that you may experience.

Risks Related to Tinostamustine:

Side effects associated with Tinostamustine are summarized below. Side effects
with grade 3 or 4 incidence (severe or life-threatening occurrences) are
indicated with an asterisk (*). Furthermore, side effects with serious
occurrences (serious adverse events) are presented in bold font.

The following adverse events are very common (>=1 in 10 patients):

• Low number of red blood cells that can cause tiredness and shortness of
breath* (anemia)
• Condition in which the number of white bloods cells called neutrophils is
abnormally low. This increases the risk of infection, which may be serious or
life-threatening* (neutropenia)
• Low number of platelets, which may cause bleeding and bruising. Bleeding may
be serious or life threatening and may require a blood transfusion*
(thrombocytopenia)
• Diarrhea
• Nausea
• Vomiting
• Fatigue*
• Abnormal electrical conduction within the heart which may lead to arrhythmias
or irregular heartbeat*
• Loss of appetite
• Headache
• Fever
• Cough


The following adverse events are common (>=1 in 100 and <1 in 10 patients):

• Fever with dangerously low white blood cell count* (febrile neutropenia)
• Condition in which the number of white blood cells circulating in the blood
is abnormally low* (leukopenia)
• Decreased number of a type of white blood cells. This is associated with an
increased risk of infection* (lymphopenia)
• Irregular heartbeat
• Dry eye
• Blurred vision
• Abdominal pain
• Constipation
• Dry mouth
• Indigestion
• Chronic heartburn and/or acid reflux
• Inflammation of the mouth/mouth sores*
• Feeling weak and having no energy
• Chills
• Influenza-like illness
• Injection site reaction, build-up of fluid in the body or extremities causing
swelling
• Fever
• Life-threatening allergic reaction (such as difficulty breathing, low blood
pressure, and/or organ failure)*
• Allergic reaction that may include a rash, hives, fever, difficulty
breathing, and low blood pressure. Although usually reversible with treatment,
it can be severe or life threatening
• Lung inflammation
• Increased risk of infection. This infection may occur anywhere. It may become
life-threatening. Symptoms of infection may include fever, pain, redness,
and/or difficulty breathing. Infections may include: urinary tract infection,
infection of the bone*, infection of the lungs (pneumonia)*, and sepsis
(widespread inflammation resulting in poor blood supply to vital organs)*.
• Infusion-related reaction*
• Wound bleeding
• Increased blood level of creatinine, a substance normally eliminated by the
kidneys into the urine. This may mean that your kidneys are not functioning
properly
• Weight loss
• Low levels of a blood protein called albumin. This can cause generalized
swelling (edema)
• Decreased blood calcium level that usually does not cause any symptoms but
when severe can cause muscle twitching and/or contractions, abnormal heart
beats or seizures
• Low levels of potassium in the blood, which can cause an abnormal heart rate.
This could cause an irregular heartbeat, which can be serious and life
threatening, Decreased levels of sodium in the blood, which can cause
confusion, seizures, fatigue and low levels of consciousness
• Muscle spasms
• Arm/leg pain
• Mental status change (such as memory loss and impaired thinking)
• Dizziness
• Tingling, pricking, chilling, burning, or numb sensation on the skin
• Vision, hearing, taste and smell disturbance
• Frequent urination
• Bloody nose
• Nasal congestion
• Mouth or throat pain
• Collection of fluid around the lungs in the chest cavity, which can cause
shortness of breath and may require treatment
• Redness of the skin
• Itching
• Flushing
• Irritation or inflammation of a vein
• Sepsis (also known as infection of the blood leading to weakness,
lightheadedness, confusion, uncontrollable shakes) which can lead to organ
failure)

The following rare but serious effects have been reported in two drugs like the
ones that are combined to make the study drug but have not been seen so far
with EDO-S101:
• Release of large amounts of dying tumor cells into the blood which can cause
serious problems
• Blood clots in the lungs
• Increased sugar in the blood

Use of allopurinol concomitantly with study drug may cause skin reactions which
could be severe and therefore an alternative drug with a different mode of
action will be prescribed, if necessary.

When looking at data across all studies with the study drug, the occurrence of
some specific ECG abnormalities has been identfied, so called QTc
prolongations, which are now closely monitored across the studies. QTc
prolongation means a lengthening of the time between certain waves on an ECG.
Cardiac monitoring using Holter monitor will be used to determine if there is
any significant prolongation. The patient will be continuously checked to see
if ECGs show any abnormalities during the infusion and up to 6 hours after the
start of infusion. In case of any ECG abnormalities, the doctor may decide to
stop the infusion of the drug.

As certain drugs commonly given for side effects of chemotherapy may cause QTc
prolongation, the patient may not be able to receive some drugs (such as some
anti-nausea drugs, but may be prescribed alternative drugs that do not cause
QTc prolongation) during a period of at least 24 hours prior to administration
of study drug.

Reproductive risks: The patient should not get pregnant, breastfeed, or father
a baby while in this study. The Tinostamustine used in this study could be very
damaging to an unborn baby.

The patient should not engage in hazardous activities (like driving a car or
operating machinery) requiring mental alertness and motor coordination
following drug administration until the patient knows how the study drug will
affect him/her.

As a participant, the patient has to come to the clinic for all scheduled
visits as requested by the study staff.
4-6 hours per visit per cycle of 28 days, a total of 6 cycles 24-36 hours.
(Cycly 1; 6 visits, following cycles 5 visits).

The patients undergo the following actions during participation:
- physical examination and medical history (weight and height)
- measuring vital functions (pulse, temperature, blood pressure and breathing)
- evaluation of daily activities
- ECG
- Holter ECG
- blood and urine collection and pregnancy test
- Tumor determination by CT scan / MRI
- Infusion research agent

Radiation risk as a result of CT scan / MRI
Blood samples: some known risks, while rare, are pain, bleeding, post-bleeding,
burning, discomfort, or bruising or infection where the needle is inserted
Infusion: same as for blood draws
Blood pressure measurement: The cuff that is being inflated may cause discomfort
ECG: The adhesion and removal of the ECG patches (small sticky pads) can cause
a transient skin reaction, such as red skin or itching. local skin discomfort
and / or hair loss can also be prevented by applying the electrodes.
Side effects of the administered drug / infusion of Tinostamustine (side
effects as cytostatics) with special attention to ECG abnormalities, QT
prolongation (see above)