R

I
F

R
Y
Z

(I

B
I
T
A
C

T)

OUTLINE

Background on Hereditary Angioedema (HAE) + existing

therapies

Rationale for new drug + mechanism of action

Source of lead

Summary of optimization + metabolic stability

Testing funnel

Pharmacokinetics

HEREDITARY ANGIO-EDEMA
angio- vessel
edema- swelling
Acute inflammatory attacks
anywhere
GI tract- hypotension
Larynx- asphyxiation
30% of deaths
Cause: deficiency of functional C1
esterase inhibitor

3
N Engl J Med 2008;359:1027-36.

INFLAMMATORY MECHANISMS
Contact
activation
pathway
Bradykinin->
B2 receptor
agonist

N Engl J Med 2008;359:1027-36.

Biomaterials. 2009

BRADYKININ AND THE B2 RECEPTOR

B2 receptor agonism
Gq activation
DAG
Prostacyclins
IP3
Ca++/calmodulin
NO
PKC also

http://flipper.diff.org/app/pathways/Bradykinin

OURNAL OF CELLULAR PHYSIOLOGY 193:275286 (2002)

C1 ESTERASE INHIBITOR

6
N Engl J Med 2008;359:1027-36.

C1 ESTERASE INHIBITOR

Serpin-class serine protease inhibitor

-Locks substrate covalently
-inhibition by distortion
Two types of HAE:
1. downregulation of C1INH
2. mutated C1INH (loss of
function)

Nature 405, 586-590 (2000)

h Intern Med. 2001;161:2417-2429
Immunobiol., vol. 199, pp. 358-365 (1998)

purple: trypsin blue: antitrypsin PDB: 1EZX

TREATMENT OPTIONS

PROPHYLACTIC TREATMENT OPTIONS

Stress reduction
Identification of Triggers
17-Alkylated androgens- general
immunosuppresants
jauncdice
Peliosis hepatis
Hepatocellular adenoma
Antifibrinolytics- inhibit activation of
factor 12
Tranexamic acid
-aminocaproic acid
Fatigue
cramps

N Engl J Med 2008;359:1027-36.

Ann. Rev. Immunol. 1988. 6: 595-628
Biochimica et Biophysica Acta, 673 (1981) 75--85
Int. J. lmmunopharmac., Vol. 17, No. I I, pp. 857-863, 1995

ACUTE TREATMENTS

C1 Inhibitor

10
N Engl J Med 2008;359:1027-36.

C1 INHIBITOR AND RATIONALE FOR NEW

DRUG
Human derived
Cinryze (2008)
Berinert (2009)
Recombinant
Ruconest (2014)
cheaper
Rationale for new drug
Need immediate relief
Timing (home therapy)
Immunogenicity of full proteins
IV
$$$- producing full protein

N Engl J Med 2008;359:1027-36.

TRANSFUSION 2014;54:2566-2573.
Nature Reviews Drug Discovery 7, 801-802 (October 2008)
http://fc01.deviantart.net/fs70/i/2013/177/a/7/scared_bugs_bunny_by_yetioner-

11

ACUTE TREATMENTS

Ecallanti
de
(2009)

12
N Engl J Med 2008;359:1027-36.

ECALLANTIDE (KALBITOR)
Small protein inhibitor of plasma
kallikrein
Approved in 09
Given in 3 subq shots
Identified by phage display
Small segment of endogenous
protein inhibitor of factor 10a
Produced recombinantly in yeast
Significant reduction of symptoms
in 4 hours
Problems:
Nurse/doctor required to
administer
Still need immediate relief

N Engl J Med 2010;363:523-31.

http://www.kalbitor.com/patient/about-kalbitor/how-kalbitorworks.html
http://www.drugdevelopment-technology.com/projects/dyax-corps-kalbitor/images/1-kalibator.jpg

13

ACUTE TREATMENTS

Icatibant
(2011)

14
N Engl J Med 2008;359:1027-36.

 Approved in 2011 by FDA

Subcutaneous
At home single use injection
Stable for 2 years at 4C

ICATIBANT

Peptidomimetic
unnatural amino acids
bioisosteres
conformational restriction
Direct B2 antagonist (nM)
immediate relief
symptoms can reoccur
peptidomimetic strategy proves useful:
high bioavailability- immediate relief
fast clearance- few side effects
No appreciable immune response
Compared to full protein precursors

N Engl J Med 2010;363:532-41.

Vascular Health and Risk Management 2010:6 795802
Maurer M, Aberer W, Bouillet L, Caballero T, Fabien V, et al. (2013) Hereditary Angioedema Attacks Resolve Faster and Are
Shorter after Early Icatibant Treatment. PLoS ONE 8(2): e53773.

15

OUTLINE

Background on Hereditary Angiodeema (HAE) + existing

therapies

Rationale for new drug + mechanism of action

Source of lead

Summary of optimization + metabolic stability

Testing funnel

Pharmacokinetics

16

T
D
N
A
A
E N

F DYK
OA

IN

IS

R
E
B
C OF
R
U RY
SOI S T O
H

17

BRADYKININ RECEPTORS

Bradykinin is an agonist for the

GPCRs bradykinin receptor 1
and 2 (BR1 and BR2), which
are responsible for controlling
pain and inflammation.
BR1 is inducible, and only
present in localized areas at a
given time
BR2 is responsible for localized
vasodilation and increased
vascular edema associated
with hereditary angioedema.
BR2 is widespread and
constitutive

ano, Masaoki, and Shogo Matsuyama. "Intracellular and Nuclear Bradykinin B2 Receptors."European Journal of Pharmacology732
14): 169-72. Web
nghurst, Hilary J. Management of Acute Attacks of Hereditary Angioedema: Potential Role of Icatibant.Vascular Health and Risk Managem
10): 795802. Print.
p://structbio.vanderbilt.edu/sanders/Research_Julia_Ver_1/temp.html
18
ias, Ch et al. The Kinin System - Bradykinin: Biological Effects and Clinical Implications. Multiple Role of the Kinin System - Bradykinin.
ppokratia11.3 (2007): 124128. Print.

BRADYKININ AS A LEAD
Structure expresses high
efficacy in tissue culture
Compound is agonist for these
effects, an antagonist is
necessary to combat these.
Compound is rapidly degraded
in vivo, stability is an issue.

19

F
O N ITY
Y IO I L
R
A ATS T A B
M IZ C
I
M
L
M
SU PTIT A B O
O ME
+

20

EVALUATING BRADYKININ ACTIVITY

The are no crystal structures to
evaluate binding conformation of
BR2.
Bradykinin analogs were originally
evaluated in tissue cultures,
leading to optimization based on
efficacy, although with variability
depending on tissue type.
Agonism is measure by pD2 , or the
negative log of the concentration
of compound required to induce 50
% of the maximal response in the
tissue
Antagonism is measured by pA2, or
the negative log of the
concentration of antagonist
necessary to double the
concentration of agonist in order
to induce 50 % of the maximal
response

21
Regoli, D., J. Barabe, and Paula H. Stern.Pharmacology of Bradykinin and Related Kinins. Baltimore: Williams & Wilkins, 1980

THE ALANINE STUDY

Tissue
Culture

Bradyki Ala- Ala-2 Ala-3 Ala-4 Ala-5 Ala-6 Ala-7 Ala-8 Ala-9
nin
1

<4.3
5.52
7

Cat Ileum
(pD2)

8.47

5.27 6.8

8.37 5.17 5.69 7.92

Rabbit
Jugular
Vein (pD2)

8.46

5.07 6.1

7.19 4.73 5.41 6.94 5.66 4.37 3.73

Dog
Carotid
Artery
(pD2)

8.64

5.97 6.33 8.34 6.78

4.4

6.7

6.49 6.16

5.4

Regoli, D., J. Barabe, and Paula H. Stern.Pharmacology of Bradykinin and Related Kinins. Baltimore: Williams &

5.38
22

POSITIONS 5 AND 8

Tissue Culture

Cat Ileum
(pD2)

Bradykinin
Position 5

Position 8

Rabbit
Dog
Jugular
Carotid
Vein (pD2) Artery
(pD2)

8.47

8.46

8.64

Ala

5.69

5.41

4.4

Tyr

6.84

6.21

7.55

Cha

8.06

8.15

7.92

Ala

>4.37

4.37

5.4

Tyr(Me)

8.51

8.59

8.64

Regoli, D., J. Barabe, and Paula

H. Stern.Pharmacology
and Related Kinins.7.82
Baltimore: Williams &
Cha
8.47 of Bradykinin8.15
Wilkins, 1980.

23

FROM AGONIST TO ANTAGONIST AND

INCREASING POTENCY

Antagonism!!
Compound
Bradykinin (pD2)

Rabbit Jugular Vein

Dog Carotid Artery

8.46

8.64

[D-Phe7]-BK (pA2)

Residual Agonism

6.25

[Hyp3, D-Phe7]-BK (pA2)

Residual Agonism

8.01

7.93

6.7

6.55

[Hyp3,Thi5,8,D-Phe7]-BK (pA2)

6.96

7.01

D-Arg[Thi5,8,D-Phe7]-BK (pA2)

7.86

7.86

D-Arg[Hyp3,D-Phe7]-BK (pA2)
[Thi5,8,D-Phe7]-BK (pA2)

Regoli, Domenico, Suzanne Nsa Allogho, Anna Rizzi, and Fernand Junior Gobeil. "Bradykinin Receptors and Their
Antagonists."European Journal of Pharmacology348.1 (1998): 1-10.
Vavrek, Raymond J., and John M. Stewart. "Competitive Antagonists of Bradykinin."Peptides6.2 (1985): 161-64.
Rhaleb, N. E., S. Telemaque, N. Rouissi, S. Dion, D. Jukic, G. Drapeau, and D. Regoli. "Structure-activity Studies of Bradykinin and
Related Peptides. B2- Receptor Antagonists."Hypertension17.1 (1991): 107-15.
Dunn, Floyd W., and John M. Stewart. "Analogs of Bradykinin Containing P-2-Thienyl- L-alanine1."Journal of Medicinal
Chemistry14.9 (1971): 779-81

24

TO ICATIBANT

Compound

Guinea Pig Pulmonary

Artery (pD2)

DArg[Hyp3-DTic7-Oic8]-BK

4.92
8.16

DArg[Hyp3-Thi5-DTic7Oic8]-BK

8.27

DArg[Hyp3-Thi5-DPhe7Oic8]-BK

7.85

DArg[Hyp3-DTic7]-BK

25
Henke, Stephan, Hiristo Anagnostopulos, Gerhard Breipohl, Jochen Knolle, Jens Stechl, Bernward Scholkens, Hans-Wolfram Fehlhaber,
Hermann Gerhards, and Franz Hock. Peptides Having Bradykinin Antagonist Action. Hoechst Aktiengesellschaft., assignee. Patent 5,648,
15 July 1997. Print.

METABOLIC STABILITY OF BRADYKININ

Bradykinin destruction during

first pass through vasculature: 98%

Sidorowicz, W., J. Szechinski, P. C. Canizaro, and F. J. Behal. "Cleavage of the ARG-PRO Bond of Bradykinin by a Human Lung
Peptidase: Isolation, Characterization, and Inhibition by Several -Lactam Antibiotics." Experimental Biology and Medicine
175.4 (1984): 503-09.
Kaplan, A. P. (2008) Bradykinin Pathways and Allergic Disease, in Allergy and Allergic Diseases, Volume 1, Second Edition
26
(eds A. B. Kay, A. P. Kaplan, J. Bousquet and P. G. Holt), Wiley-Blackwell, Oxford, UK. doi:10.1002/9781444300918.ch20
Regoli, Domenico, Suzanne Nsa Allogho, Anna Rizzi, and Fernand Junior Gobeil. "Bradykinin Receptors and Their

ICATIBANT ANTAGONIST STABILITY

T1/2=1.2-1.5 hrs

Ghazi, Aasia, and J Andrew Grant. Hereditary Angioedema: Epidemiology, Management, and Role of Icatibant.Biologics: Targets & Thera
(2013): 103113.PMC. Web. 17 Apr. 2015.
Kaplan, A. P. (2008) Bradykinin Pathways and Allergic Disease, in Allergy and Allergic Diseases, Volume 1, Second Edition (eds A. B. Kay, A
27
Kaplan, J. Bousquet and P. G. Holt), Wiley-Blackwell,
Oxford, UK. doi:10.1002/9781444300918.ch20

BRADYKININ AND ICATIBANT

28

OUTLINE

Background on Hereditary Angioedema (HAE) + existing

therapies

Rationale for new drug + mechanism of action

Source of lead

Summary of optimization + metabolic stability

Testing funnel

Pharmacokinetics

29

I
T
S
E
T
B

IO

G
N

IC

N
N
FU
A

IM

EL
L

IN

IO

30

IN VITRO STUDIES
RECEPTOR BINDING

Radioligand
competition binding
assays in:
Guinea-pig ileum
(crude membrane)
Rat uterus
Guinea-pig
pulmonary artery
Rabbit aorta

ock, et al.Br. J. Pharmacol. 1991,102, 769

DOWNSTREAM EFFECTS

Bradykinin induced
releases of:
EDRF
Guanylate cyclase activation

32

P quantification

Ca2+
indo-1 Fluorescent probe

Prostacyclin
radioimmunoassay

31

Summary of radioligand binding results

Hoe 140s IC50 is 3x lower in
each model
Both compounds exhibit agonist
activity at low concentrations
(20 M)
Hoe 140 shown to block
downstream effect of increased
free Ca2+

ock, et al.Br. J. Pharmacol. 1991,102, 769

Inhibitory effect based on Ca2+ releas

32

EDRF RELEASE

P R O S TA C Y C L I N
RELEASE

ock, et al.Br. J. Pharmacol. 1991,102, 769

33

IN VIVO STUDIES
C O M PA R I S O N O F
B K A N TA G O N I S T S

S TA N D A R D
I N F L A M M AT I O N M O D E L

Duration of
action
Anesthetized rats

BK induced
bronchoconstri
ction

Carrageenininduced rat paw

oedema

anesthetized guineapig

irth, et al. Br. J. Pharmacol. 1991, 102, 774

inter, et al. Exp. Biol. Med. 1962, 111, 544

34

D U R AT I O N O F A C T I O N

rth, et al. Br. J. Pharmacol. 1991, 102, 774

E F F E C T O N B KINDUCED
BRONCHOCONSTRICTI
ON

35

rth, et al. Br. J. Pharmacol. 1991, 102, 774

36

E
N

KI F O R

O
T
C
C
A DU
O
M
R
RL P
A
H A
+

S
N
C
O
TI T I
M

IN

37

ICATIBANT

Properties
Log P

-2.3

# Hydrogen
Acceptors

23

# Hydrogen
Donors

15

MW
1304.522
atibant (DB06196) [online].
Available from URL:
Da
ttp://www.drugbank.ca/drugs/DB06196#admet

38

FORMULATION
Icatibant acetate
(active) + NaCl,
glacial acetic
acid, NaOH, H20
Available as a
3mL injection (10
mg/ml)
subcutaneously
Requires no
mixing or
measuring

yr (Icatibant Injection) Prescribing information [online]. Available from URL:

/pi.shirecontent.com/PI/PDFs/Firazyr_USA_ENG.pdf

39

PHARMACOKINETICS
M E TA B O L I S M /
E L I M I N AT I O N

Proteolytic
enzymes (2
inactive
metabolites)
CYPs: nonsubstrate, noninhibitor

P K PA RA M ETE R S

SC
bioavailability:
97%
VD= 29 L
44% Plasma
bound

Elimination: urine
(<10%
unchanged)

Terminal
elimination half
life= ~2hr

P OT EN TI A L DD I S / TOX .

Pgp: substrate

hERG predictor:
weak inhibitor

non-AMES toxic

LD50= 2.42 mol/kg

(rat)

Jerini AG. Firazyr 30mg solution for injection in pre-filled syringe: summary of
product characteristics [online]. Available from URL:
http://www.emea.europa.eu/humandocs/ PDFs/EPAR/firazyr/emea-combinedh889en.pdf
European Medicines Agency. CHMP assessment report for Firazyr [online].40
Available from URL: http://www.emea.

E
V
R
E
S
E

D
I
L
S

ES

41

ANIMAL TISSUES- AN EXPLANATION

Guinea pig
ileum
Rat uterus
Guinea pig
pulmonary
artery
Rabbit aorta

pig aorta
endothelial
cells
Bovine aorta
endothelial
cells

BK1-receptor
(Regoli & Barabe,
1980)

42

FURTHER WORK IN DRUG DISCOVERY

WITH BK ANTAGONISTS

uang, et al. J. Med. Chem. 2010, 53, 53835399

43

INDO-1 FLUORESCENCE PROBE

nkiewicz, G. et al. J. Biol. Chem. 1985, 260 (6), 3440-3450.

44

CLINICAL TRIALS- PHASE III

FA S T- 1 A N D FA S T- 2

Single dose not

significantly faster
than placebo
More rapid symptom
relief than oral
(tranexamic acid)
No rescue therapy
required of icatibant
patients

Deeks, E.D. Drugs, 2010, 70 (1), 73-81

45

SMALL MOLECULE B2
ANTAGONIST DEVELOPMENT
Oral drug usually
desired
Clinical trials
Easier dosing
Could be prophylactic
measures if patient
has prior knowledge
of oncoming trigger

46