Investigación en la Empresa

(Octubre 2022)

Máster en Investigación en
Medicina Traslacional

Dra. Macarena Hernández Jiménez

Directora Científica aptaTargets
Unidad de Inv. Neurovascular
macarenh@ucm.es

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La Investigación en la empresa

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Stroke
Stroke (WHO): the sudden death of brain cells due to lack of oxygen and glucose, caused by blockage of
blood flow or rupture of an artery to the brain. Sudden loss of speech, weakness or paralysis of one side of the
body can be symptoms.

83% 17%

Campbell et al., Nat Rev 2019 3

Stroke
1st cause of death in women and 2nd in men
Leading cause of adult disability
Second cause of dementia
Infections after stroke as unfavourable outcome

Only one therapy available: recanalization

mechanically or pharmacologically (EVT and tPA)

Huge economic burden for health systems: in

the EU = €45bn in 2015
It is estimated an increase of 25% in 2035

Devastating and limiting disease

Huge socio-sanitary expend
Campbell et al., Nat Rev 2019 4
Stroke

One of the main processes involved in the

brain damage after AIS is the inflammation

For patients with successful

recanalization, penumbra loss can
still account for up to 50% of brain
damage

Jung et al., Brain, 2013; Dirnagl et al., TINS, 1999 5

Stroke

En la actualidad, no existen
tratamientos neuroprotectores
aprobados para el tratamiento del ictus

Toll-like receptor type 4 (TLR4)

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TLR4 and Inflammation
Healthy tissue Damaged tissue

STROKE
Tissue
damage

DAMPS

X
ROS
TLR4
Microglial
activation
NFκB

IL-1, IL-6,
TNFα, ... Leukocyte
infiltration

Immune
activation

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ApTOLL
Aptamer
(sequence)
UGAUCC AUUCGGAUCAAGCUAGC

Specific recognition
and stable binding
Aptamer Target of therapeutic
Aptamer (tertiary,
(secondary interest
3D structure)
structure)

ApTOLL TLR4 8
ApTOLL
Healthy tissue Damaged tissue

STROKE
Tissue
damage

DAMPS

X
ROS
TLR4
Microglial
activation
NFκB

IL-1, IL-6,
TNFα, ... Leukocyte
ApTOLL targets one of the infiltration
most upstream receptors
Immune
activation

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aptaTargets
ApTOLL is a TLR4 antagonist anti-inflammatory aptamer (ssDNA)
therapeutic, with acute ischemic stroke (AIS) as the lead indication

aptaTargets ApTOLL AIS

• Myocardial Infarction
v v
• TLR4 antagonist
Biopharmaceutical using • Multiple Sclerosis
aptamer technology for • Anti-inflammatory
targeting acute indications • Hemorrhagic Stroke
• Clinical stage
• COVID-19

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aptaTargets
Investigation

ApTOLL

Preclinical Clinical

Safety Efficacy Safety Efficacy

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aptaTargets
Investigation

ApTOLL is a toll-like receptor 4 (TLR4) antagonist aptamer (ssDNA)

immunomodulator that blocks the inflammatory response after a stroke

Specific recognition
Stable binding
Chemically synthesized
Non-immunogenic
Lyophilized powder formulation

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Preclinical Investigation:
EFFICACY

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ApTOLL: CLINICAL INVESTIGATION

ApTOLL

Pharmacodynamics
Preclinical
In silico PD: aptamer structure, etc.

In vitro PD: receptor binding, affinity,

antagonistic activity, etc.
Safety Efficacy
In vivo PD: efficacy in animal models,
dose-response, administration
regimen, therapeutic window, long-
Centros de Investigación / CROs independientes term efficacy, etc.
Modelos animales dependiendo de la enfermedad
Caracterización del MoA
Planificación de estudios: cegado, condiciones
experimentales, etc.
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ApTOLL: studies in silico

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ApTOLL: in vitro studies

ApTOLL

Confirmed TLR4 inhibition and specificity

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ApTOLL efficacy in experimental ischemic stroke

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ApTOLL efficacy in experimental ischemic stroke
Infarct Footprint
ApTOLL volume ApTOLL test
Reperfusion

tMCAO 60min 70min 24h pMCAO 10min 21d

Ipsi Contra Ipsi Contra

35-66% protection in mice and rats

Better long-term functional outcome

Fernández et al., Mol Ther 2018

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ApTOLL efficacy in experimental ischemic stroke
Cytokine
ApTOLL assessment

pMCAO 10min 24h

400 50 50 50 800 800 150 150

800 300 150 1000

IL-12p70 (pg/mL)
IL-12p70 (pg/mL)

IL-12p70 (pg/mL)
* * *

MCP-1 (pg/mL)
40 40 40 800

TNF (pg/mL)

IL-10 (pg/mL)
IL-10 (pg/mL)
IL-6 (pg/mL)

IL-10 (pg/mL)
300
INF (pg/mL)
INF (pg/mL)
600 600

INF (pg/mL)
600
100 100
200 100

200 *
30 30 30 * * * 600
400 400 400
20 20 20 400
10050 50 50
100 200 200 200
10 10 10 200

0 0 0 0 0 0 0 0 0 0 0 0
A veh

LL

A veh

A veh
A veLhL
A veh

A vehL
LL

A OvLeh

A OvLeLh
LL
A OvLeh

LL
LL

A eh

LL
A TOveh
pT L
A veh

pT L
pT L

L
pT L
O

O
O
v

O
O

O
pT

pT

pT
pT

pT

pT
pT

p
Confirmed mechanism of action: ApTOLL interferes in the inflammatory response after ischemia

Fernández et al., Mol Ther 2018

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ApTOLL efficacy in experimental ischemic stroke

Fernández et al., Mol Ther 2018

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ApTOLL efficacy in experimental myocardial infarction
Infarct
ApTOLL
50
80 assessment
Vehicle Vehicle
ApTOLL
45 min 75 ApTOLL

% Fractional Shortening
70 40
*

% Ejection fraction
10 min
LAD R65 7d
60
30
55
50
20
45
40
35 10
0.4 0.6 0.4 Basal Post-Infar 3 days 7 days Basal Post-Infar 3 days 7 days
TNF (ng/mL)

IL1A (ng/mL)
IL6 (ng/mL)

0.3 0.3
0.4 50
80
* Vehicle
0.2 0.2
* * 75 ApTOLL

% Fractional Shortening
0.2
0.1 0.1 70 * 40

% Ejection fraction
0.0
65
0.0 0.0
Vehicle ApTOLL Vehicle ApTOLL Vehicle ApTOLL 60
30
0.4 2.0 0.5 55
60% reduction of infarct
CXCL10 (ng/mL)

IL17A (ng/mL)

0.4 50
IL8 (ng/mL)

0.3 1.5

0.2
*
1.0
0.3 volume and improved 45
20

0.1 0.5 *
0.2
* functionality 40

0.1 35 10
Basal Post-Infar 3 days 7 days
0.0 0.0 0.0
Vehicle ApTOLL Vehicle ApTOLL Vehicle ApTOLL

ApTOLL causes a strong downstream reduction in

nearly every proinflammatory cytokine evaluated
Ramírez-Carracedo et al., Biomolecules 2020 21
ApTOLL efficacy in experimental hemorrhagic stroke
Hemorrhagic
ApTOLL volume

Collagenase 30 min 24h

Injection

175
*

Hemorraghe (mm3)
150

125

100

75

50
Vehicle ApTOLL

ApTOLL reduces >20% the hemorrhagic

volume in the ICH model

Unpublished data 22
ApTOLL efficacy in experimental multiple sclerosis

ApTOLL improves functional outcome reducing

demyelinated area in mice

Fernandez-Gómez et al., 2022 (under review) 23

Preclinical Investigation:
SAFETY

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ApTOLL: PRECLINICAL INVESTIGATION

ApTOLL

Preclinical Clinical

Safety Efficacy Safety Efficacy

CROs independientes --- GLPs

Farmacocinética y toxicidad a todos los niveles
Planificación de estudios: cegado, condiciones
experimentales, etc.
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ApTOLL safety in animals

Ongoing 2021 26
 ApTOLL safety in animals
ApTOLL shows good pharmacokinetic profile
Pharmacokinetics with no off-target reactions or drug-to-drug
interactions
In silico
In vitro approaches: half-life in presence of ✓ ApTOLL fits perfectly with acute indications
nucleases, half-life in plasma ex-vivo, etc.
✓ T1/2 10min rats and 1,4h monkeys
In vivo pharmacokinetics: distribution of drug in
target organs, half-life in plasma, exposure, etc.

ApTOLL does not show any toxicological effect

Toxicity even 100-fold the therapeutic dose

In silico toxicology ✓ Wide range of dosing strategies

In vitro toxicology: cell toxicity assays ✓ Excellent candidate to be combined with
In vivo toxicology: toxicokinetics, histopathology, other drugs
immunotoxicity, CNS respiratory and
cardiovascular toxicity, genotoxicity, etc. ✓ 13.9 mg/kg/day ApTOLL = NOAEL

ApTOLL is safe in rats and non-human primates up to 100-fold the therapeutic dose
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ApTOLL safety and efficacy in animals

• Follow the guidelines

• Confirm the results in different
disease models
• Use different animal species
• Try to reproduce the results in
other laboratories
• Confirm the drug MoA
• Be blinded
• Show your results to people

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ApTOLL: CLINICAL INVESTIGATION

ApTOLL

Preclinical Clinical

Safety Efficacy Safety Efficacy

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ApTOLL: CLINICAL INVESTIGATION

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ApTOLL Clinical Investigation

ApTOLL

Clinical

Safety Efficacy

SAFETY IN HUMANS
PHARMACOKINETICS

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 Clinical Investigation:
SAFETY in healthy volunteers

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ApTOLL Phase Ia clinical study
First-in-Human Dose Ascending, Randomized, Placebo-Controlled Clinical Trial to Assess
Tolerability and Pharmacokinetics of ApTOLL in Healthy Volunteers (NCT04742062)

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 ApTOLL Phase Ia clinical study
First-in-Human Dose Ascending, Randomized, Placebo-Controlled Clinical Trial to Assess
Tolerability and Pharmacokinetics of ApTOLL in Healthy Volunteers (NCT04742062)

OBJECTIVES

1. To evaluate the tolerability and pharmacokinetic characteristics of ApTOLL in healthy volunteers,

after single dose administration in fasting conditions, following an ascending dosing scheme.

2. To evaluate the tolerability and pharmacokinetic characteristics of ApTOLL in healthy volunteers,

after multiple dose administration in fasting conditions.

DESIGN

First part: a single dose, i.v. administration (slow infusion), dose escalation with 7 single dose levels,
randomized, double-blind, placebo-controlled (saline solution), in healthy subjects.

Second part: a multiple dose, i.v. administration (slow infusion), randomized, double-blind, placebo-
controlled (saline solution), in healthy subjects.
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 ApTOLL Phase Ia clinical study
First-in-Human Dose Ascending, Randomized,
Placebo-Controlled Clinical Trial to Assess
Tolerability and Pharmacokinetics of ApTOLL in
Healthy Volunteers (NCT04742062)

Hernández-Jiménez et al., MTNA 2022 35

ApTOLL Completed Phase Ia clinical study

Hernández-Jiménez et al., MTNA 2022 36

ApTOLL Clinical Investigation

ApTOLL

Clinical

Safety Efficacy

SAFETY IN PATIENTS
PHARMACOKINETICS
BIOLOGICAL EFFECT

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 Clinical Investigation:
SAFETY and EFFICACY in patients

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ApTOLL Completed Phase Ib/IIa clinical trial
A Double Blind, Placebo-Controlled, Randomized, Phase Ib/IIa Clinical Study of ApTOLL
for the Treatment of Acute Ischemic Stroke (APRIL) (NCT04734548)

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ApTOLL Phase Ib/IIa clinical trial in progress
OBJECTIVE:
To evaluate if administration of ApTOLL at different doses is safe and well tolerated compared to
placebo when administered with endovascular therapy (EVT), with or without i.v. rt-PA, in the
acute ischemic stroke (AIS) target population (patients with confirmed Large Vessel Occlusion
(LVO) who are candidates to receive reperfusion therapies including endovascular treatment with
or without i.v. rt-PA (recombinant tissue Plasminogen Activator).

Early recanalization
Thrombolysis
Mechanical Thrombectomy

ApTOLL

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 ApTOLL Phase Ib/IIa clinical trial in progress
DESIGN:
Phase Ib/IIa trial where 2 doses selected, based on safety criteria, on Phase Ib will be
administered in the following Phase IIa

Phase Ib
Phase IIa
D1 D2 D3 D4
Dose A x35
Two doses
+ + + + selected
Dose B x35

D1-P D2-P D3-P D4-P

Placebo x49

0.025mg/kg - 0.2mg/kg

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In Summary….

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 Summary

APTABOLUS

2010 2014 2017 2018 2019 2020 2021 2022

Preclinical (AIS) Preclinical Clinical Clinical Clinical Clinical ApTOLL

TLR4 aptamer Discovery Prelim. PC Results APRIL APRIL APRIL
Regulatory studies Phase Ia
MCAO model (First-in-Human) Phase Ib Phase IIa Patient enrollment
ApTOLL first GMP process finished
Fernández et al., batch Hernández-Jiménez et APTACOVID
Mol. Ther. 2018 al., MTNA, 2022 Phase IIb APTABOLUS
Phase Ia
Patent application Preclinical Hemorrhagic
ES201430955 stroke
Myocardial Infarct
ApTOLL Multiple sclerosis

Ramirez-Carracedo et
al., Biomolecules, 2020

ApTOLL second GMP

batch

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Summary
ApTOLL

Preclinical Clinical

Consistent animal
efficacy results FIH with an
across species and excellent
disease models ApTOLL is a safety profile
confirmed anti- Active APRIL
inflammatory and APTACOVID
Pipeline myocardial drug clinical trials
infarction, multiple
sclerosis and
hemorrhagic stroke

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To take home...

• En investigación preclínica se utilizan modelos in silico, in vitro e in vivo para

determinar la seguridad y la eficacia de las moléculas candidatas a fármacos

• Los ensayos clínicos se realizan con el objetivo de evaluar la farmacocinética,

la seguridad y la eficacia de los fármacos en humanos

• ApTOLL es un fármaco que se ha desarrollado para reducir la inflamación

mediante la inhibición del TLR4

• ApTOLL es un fármaco que actualmente está en fase clínica de desarrollo

• Las empresas biotecnológicas realizan diferentes tipos de investigación.

AptaTargets, por ejemplo, realiza tanto investigación básica (preclínica) como
clínica, además de actividades de gestión y divulgación

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¡¡¡GRACIAS!!!