FIRAZYR (icatibant)

1

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

2

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

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

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So what does that long word mean? Well if we break it down to greek we can ease it up to us.

Angio edema vessel swelling

You can see a patient experiencing a mild angioedema attack in his left hand.

Hereditary angioedema is a disease characterized by attacks of inflammation ranging from moderate discomfort to asphyxiation when it occurs in your larynx.

Attacks begin with a tingling sensation. Swelling worsense over the first 24 hours, then gradually subsides over the next few days.

Attacks can occur anywhere in your body and can cause nausea, pain, vomiting, etc.

Can cause severe illness in the GI tract or larynx.

The cause of this disease is a genetic defeciency in a plasma protein known as C1 inhibitor

Now I would like to shift into talking about the biological mechanism of disease.

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Inflammatory Mechanisms

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

Contact activation pathway

Bradykinin-> B2 receptor agonist

Biomaterials. 2009 Apr;30(10):1857-69.

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This is a figure depicting the inflammatory cascade known as the contact activation pathway. Ill come back to this figure a lot, so get used to seeing it. It is termed the contact activation pathway because factor 12 is activated through contact with a particular type of

Talk about contact inhibition pathway: hagemans factor 12 was originally thought to be activated by contact with a negative tissue surface. More recently opinion has shifted as studies have shown it can be activated by other surfaces. Still, this pathway is called the contact activation pathway because it involves autoactivation of factor 12 after factor 12 comes into contact with a surface.

As you can see, the pathway has elements in common with the blood clotting cascade, and begins with an activation of factor 12 from the blood clotting cascade.

The end result here is the production of the inflammatory peptide bradykinin and activation of the B2 receptor, which is a GPCR and follows typical GPCR mediated signaling, which I will cover in the next slide. Note the presence of C1 inhibitor at many of the steps in this cascade and how vital it is to insuring the cascade does not proceed aberrantly. As I mentioned before, lack of operational C1 Inhibitor is the root cause of HAE. The variety of symptoms are caused directly by the final step in this cascade, which involves bradykinin binding to its receptor and the subsequent signaling events. I would like to start by covering some of the signaling events which bradykinin agnoism of B2 receptor causes.

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Bradykinin and the B2 receptor

B2 receptor agonism

Gq activation

DAG

Prostacyclins

IP3

Ca++/calmodulin

NO

PKC also

JOURNAL OF CELLULAR PHYSIOLOGY 193:275286 (2002)

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

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So we begin with bradykinin mediated agonism of the B2 receptor, which causes a change in conformation of receptor and activation of G alpha q subunit. G alpha q is linked with phospholipase C.

Phospholipase C cleaves PIP3 into IP3 and diacylglycerol.

A variety of responses is caused by this cleavage event, including

Calcium is released from intracellular storage in the sarcoplasmic reticulum by the binding of IP3 to its cognate receptor on the SR membrane.

calcium stimulates Nitric oxide synthase. Which produces nitric oxide that can stimulate vasodilation in adjacent smooth muscle cells.

DAG is a substrate for production of inflammatory prostacyclins these molecules can travel to adjacent smooth muscle cells and produce dilation events.

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C1 Esterase Inhibitor

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

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Now that we know a little bit about the signaling events leading up to an angio-edema attack, I would like to backtrack a little bit and talk about the natural suppressor of the contact activation pathway, the C1 inhibitor.

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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)

Arch Intern Med. 2001;161:2417-2429

Immunobiol., vol. 199, pp. 358-365 (1998)

purple: trypsin blue: antitrypsin PDB: 1EZX

Nature 405, 586-590 (2000)

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C1 inhibitor is a member of a class of serine protease inhibitors known as serpins. Named for its original role as discovered in the complement pathway. Serpins such as this one covalently inhibit the protease in question by trapping the active serine residue in the acyl-intermediate state. So one thing to consider, is since the inhibitor acts via suicide, the regulation of its concentration is a bit different than if the inhibition were reverisble.

In hereditary angio- edema, tehre are two types of mutations involving the C1 inhibitor.

Downregulation of a functional protein through the promoter region

Loss of function mutations such as truncations or key residue mutations

Its not a surprise that the first medication for acute angio-edema was a purified C1 inhibitor delivered directly to the circulation intravenously. Ill talk about that soon.

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Treatment options

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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

Int. J. lmmunopharmac., Vol. 17, No. I I, pp. 857-863, 1995

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

Biochimica et Biophysica Acta, 673 (1981) 75--85

Ann. Rev. Immunol. 1988. 6: 595-628

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Of course, stress being a primary instigator of an angio-edema attack, doctors will recommend that patients seek to reduce at least some of the stress in their lives. In addition, since many patients seem to have certain external triggers, doctors recommend that patients identify these triggers so that prophylactic treatment can be more effective.

17 alpha alkylated androgens are used as a prophylactic treatment option, they provoke a general immunosuppressive response and lead to increased amount of endogenous C1 inhibitor production by the liver. This therapy is pretty successful in preventing attacks, however it is not without side effects. Here I have listed a few of the side effects that can occur from prolongated use of alpha androgens.

Another class of prophylactic drugs are the anti fibrinolytics. These are both mimics of the amino acid lysine, and they serve to block lysine binding sites on plasminogen, preventing it being cleaved by a its activating protease. Antifibrinolytics are able to inhibit the processing of plasminogen into plasmin which is responsible for producing an amount of factor 12 and also activating the complement cascade which is a non-specific immuno inflammatory response.

These drugs can have side effects such as fatigue and cramps. Neither of these drugs are desirable to use in the long term due to their side effects.

Since this condition manifests as attacks, the focus of most drug development program was to find either a preventative measure or a drug that would instantly relieve an attack at the onset. And now I will transition to talking about treatments for acute attacks.

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Acute Treatments

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

C1 Inhibitor

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The first idea to treat an attack was to give patients exactly what they were missing, C1 inhibitor. C1 inihibitor can be derived from a vareity of sources.

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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-d6asv54.png

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C1 inhibitor can be derived from a variety of sources. the FDA approved drugs, cinryze and berinert, are isolated from human plasma.

There are also recombinant C1 inhibitors available, such as Rhucin. Interestingly enough, rhucin is produced in the milk of transgenic rabbits.

So C1 inhibitor can be given as an acute prophylaxis, is relatively free of side effects, and works great.

So why even develop a new drug?

One of the main issues is that as icatibant was being developed, most of these C1 inhibitors werent approved for at home treatment, leading to significant symptom onset for most patients before the drug could be administered at a clinic.

Since then, I believe all of these drugs are approved for at home use, resolving this problem.

To note, the recombinant C1 inhibitor has been reported to cause an immunogenic response in a low frequency of patients.

But the biggest issue is that the C1 inhibitor has to be given intravenously, which can be a problem in terms of at home dosing and patient compliance. Improper IV injection can be a problem.

In addition, since the c1 inhibitor inhibits a protease cascade and the resulting receptor mediated signaling cascade, immediate relief is not seen.

Also, money. Less of an issue since the recombinant one, but still producing a whole protein is pretty expensive compared to our final peptidomimetic product.

So from here I would like to talk about a few other treatments approved for acute attacks.

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Acute Treatments

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

Ecallantide(2009)

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So as Ive highlighted here, ecallantide blocks the final step where kallikrein liberates bradykinin peptide from HMWK. In addition, it can also block further activation of hagemans factor and increased pathway transmission.

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Ecallantide (Kalbitor)

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

http://www.kalbitor.com/patient/about-kalbitor/how-kalbitor-works.html

http://www.drugdevelopment-technology.com/projects/dyax-corps-kalbitor/images/1-kalibator.jpg

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

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So in 2009 the first subcutaneous treatment for HAE attacks was approved. Known as ecallantide or kalbitor, as it acts as an inhibitor of plasma kallikrein, the protease responsible for liberating bradykinin from HMWK.

This treatment is given in 3 subsequent subq shots, and must be given by a doctor or nurse due to potential immunogenicity.

This drug is a small protein identified by phage display which consists of a small segment of an endogenous serpin.

It is given subcutaneously and thus has a distinct advantage over c1 esterase inhibitor. Symptoms are marked to improve in a couple hours.

So again, this is an improvement. But its not ideal. We are still not seeing immediate relief of symptoms, which is absolutely essential in the case of a laryngeal attack.

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Acute Treatments

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

Icatibant

(2011)

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And that brings us to our drug for this presentation, Icatibant. Icatibant as a receptor antagonist for the bradykinin B2 receptor at a roughly nanomolar ec50 in vitro. It directly quells the exponential signaling events that occur upon agonism of the B2 receptor by BK.

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icatibant

Approved in 2011 by FDA

Subcutaneous

At home single use injection

Stable for 2 years at 4C

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

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.

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

Vascular Health and Risk Management 2010:6 795802

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So since icatibant directly agonises the B2 receptor, it should result in the most immediate relief out of any of the drugs ive shown before. And it does, with a response time of blank. Icatibant is a single subcutaneous injection at 30mg. This is easy to do at home, and because of the ease of use, patients can quell the attack at early onset.

so here on the top we see the 9-AA peptide bradykinin, and underneath it the 10 AA icatibant. It is a peptido mimetic, mimicking bradykinin, with a few unnatural modifications that sean will go into more detail on later.

I would like to note that the peptidomimetic response is very useful in terms of an acute treatment not only due to the ease of development using bradykinin as lead.

Ideally, we would like to have a compound that is instantly bidning whos effefcts last until the attack Is over, and is then rapidly cleared to avoid side effects. And we see with a subcutaneous peptidomimetic strategy we achieve all of those points. Peptide is highly soluble and has great bioavailability (need percent here), enters systemic distribution and reaches its target quickly. Its potency is on par with the endogenous ligand bradykinin. Finally, since it is peptidomimetic, it is cleared after one pass throughout the circulature, leading to largely decreased side effets. Also, since it is relatively small, there is less of a risk of immune response than those full protein drugs I mentioned earlier.

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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

Source of lead

History of Bradykinin Antagonists

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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

Takano, Masaoki, and Shogo Matsuyama. "Intracellular and Nuclear Bradykinin B2 Receptors."European Journal of Pharmacology732

(2014): 169-72. Web

Longhurst, Hilary J. Management of Acute Attacks of Hereditary Angioedema: Potential Role of Icatibant.Vascular Health and Risk Management6

(2010): 795802. Print.

http://structbio.vanderbilt.edu/sanders/Research_Julia_Ver_1/temp.html

Golias, Ch et al. The Kinin System - Bradykinin: Biological Effects and Clinical Implications. Multiple Role of the Kinin System - Bradykinin.

Hippokratia11.3 (2007): 124128. Print.

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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.

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Summary of optimization

+ metabolic stability

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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

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

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The Alanine Study

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

Tissue CultureBradykininAla-1Ala-2Ala-3Ala-4Ala-5Ala-6Ala-7Ala-8Ala-9Cat Ileum (pD2)8.475.276.88.375.175.697.926.7190000

3020

3.47

8510

794

64.6

18600

3890

115

2190

42700

186000

2.29

1070

468

4.57

166

39800

324

692

3980

4170

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Positions 5 and 8

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

Tissue CultureCat Ileum (pD2) Rabbit Jugular Vein (pD2) Dog Carotid Artery (pD2) Bradykinin8.478.468.64Position 5Ala5.695.414.4Tyr6.846.217.55Cha8.068.157.92Position 8Ala>4.374.375.4Tyr(Me)8.518.598.64Cha8.478.157.82

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3.39E-09 3.47E-09 2.29E-09 2.04E-06 3.89E-06 3.98E-05 1.45E-07 6.17E-07 2.82E-08 8.71E-09 7.08E-09 1.20E-08 >4.27E-05 4.27E-05 3.98E-06 3.09E-09 2.57E-09 2.29E-09 3.39E-09 7.08E-09 1.51E-08

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From Agonist to Antagonist and Increasing Potency

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

CompoundRabbit Jugular VeinDog Carotid ArteryBradykinin (pD2)8.468.64[D-Phe7]-BK (pA2)Residual Agonism6.25[Hyp3, D-Phe7]-BK (pA2)Residual Agonism7D-Arg[Hyp3,D-Phe7]-BK (pA2)8.017.93[Thi5,8,D-Phe7]-BK (pA2)6.76.55[Hyp3,Thi5,8,D-Phe7]-BK (pA2)6.967.01D-Arg[Thi5,8,D-Phe7]-BK (pA2)7.867.86

Antagonism!!

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Activity of analogs compared wit,h bradykinin, based on the dose required to cause a half-maximal isotonic contraction. Dose

(in pg) required to effect a depressor response of 20-25 mm of Hg by the intraaortic (ia) and iv routes. Percentage of an iv dose destroyed

by a single passage through the pulmonary circulation in the rat.

3.47E-09 2.29E-09 1.00E+00 5.62E-07 1.00E+00 1.00E-07 9.77E-09 1.17E-08 2.00E-07 2.82E-07 1.10E-07 9.77E-08 1.38E-08 1.38E-08

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To Icatibant

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 5,648,333.

15 July 1997. Print.

CompoundGuinea Pig Pulmonary Artery (pD2) DArg[Hyp3-DTic7]-BK4.92DArg[Hyp3-DTic7-Oic8]-BK8.16DArg[Hyp3-Thi5-DTic7-Oic8]-BK8.27DArg[Hyp3-Thi5-DPhe7-Oic8]-BK7.85

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1.20E-05 6.92E-09 5.37E-09 1.41E-08

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Metabolic Stability of Bradykinin

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 (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 Antagonists."European Journal of Pharmacology348.1 (1998): 1-10.

Bradykinin destruction during

first pass through vasculature: 98%

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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 & Therapy7

(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. P.

Kaplan, J. Bousquet and P. G. Holt), Wiley-Blackwell,

Oxford, UK. doi:10.1002/9781444300918.ch20

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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

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Testing funnel

Biochemical and animal models of inflammation

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In Vitro Studies

Receptor binding

Downstream effects

Bradykinin induced releases of:

EDRF

Guanylate cyclase activation

32P quantification

Ca2+

indo-1 Fluorescent probe

Prostacyclin

radioimmunoassay

31

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

Radioligand competition binding assays in:

Guinea-pig ileum (crude membrane)

Rat uterus

Guinea-pig pulmonary artery

Rabbit aorta

Receptor binding: essentially a scintillation proximity assay using tritiated compound. In brief, crude membranes from guinea-pig ileum were incubated for 1 h at 25C in a buffer solution with [3H]-BK (1 nmolI 1-) and various concentrations of the test compounds (11 concentrations; range: 1 x 10' to 1 x 10-10moll-'). Non specific binding was determined in the presence of 1 ,umol 1-unlabelled BK.

Bradykinin induced releases: all cell based assays. Calcium and EDRF (endothelium derived relaxing factor) in endothelial pig aorta. EDRF itself is not stable, but is known to active guanylate cyclase. So cyclic GMP is a good indicator of this activation. Of note, in the 90s it was pretty well accepted that EDRF was the free radical NO (nitric oxide). Prostacyclin from endothelial bovine aorta. About EDFRs, now it is accepted that there are a few and this is a class of factors that include NO, endothelin-1, prostaglandin H2, and endothelium derived hyperpolarizing factor.

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Inhibitory effect based on Ca2+ release

Summary of radioligand binding results

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

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+

As Sean discussed earlier, there was a BK antagonist developed in the mid 80s that the Hoest company was using for their lead. In a pair of papers published in 1991, they reported their best antagonist, Hoe 140, as compared to this 80s antagonist in all of the testing.

And as Sumit showed us, BK binding to the receptor activates a g protein, and then activates phospholipase C invokes an enhanced production of inositol tris (1,4,5) phosphate increases free calcium.

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EDRF release

prostacyclin release

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

Endothelium- derived relaxing factor: measured as the formation of cyclic GMP. This was a plate assay coupled with the calcium probe shown previously. Briefly, 24 well plates of pig aorta endothelial cells were incubated with drugs and superoxide dismutase (SOD is an anti-inflammatory agent, the enzyme reacts with reactive oxygen species) and then dosed with BK to induce inflammation. Supernatant was removed at certain time points and the cGMP assay was performed. The left graph shows the time points comparing control (vehicle) to differing concentrations of Hoe140. Starting with 0.1 nM going to 1 uM.

PGF assay: RIGHT. C is baseline. Log scale X axis is concentrations of BK. Open circles are Hoe140, open triangles are 1980s BK antagonist. Solid circles are BK alone. *= significance to baseline. They had a radioimmunoassay for the hydrolysis product of PGI2, = 6-keto PGF 1 alpha. So it is actually kind of the same assay as EDRF release, except in bovine aorta.

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In vivo studies

Comparison of BK antagonists

Duration of action

Anesthetized rats

BK induced bronchoconstriction

anesthetized guinea-pig

Standard Inflammation Model

Carrageenin-induced rat paw oedema

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

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

Goal of these studies was to test the efficacy of Hoe140 in vivo in models where BK served as an agonist For this purpose, models of BK-induced hypotensive reaction and BK-induced bronchoconstriction were used to demonstrate its high potency and long duration of action.

Comparison: 1985 class of antagonists synthesized by Vavrek & Stewart 1985, D-Arg-[Hyp2, Thi5,8, D-Phe7] BK for the blood pressure experiments

Carrageenin: class of natural products from seaweds, linear sulfated polysacharides.

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Duration of action

Effect on BK-induced bronchoconstriction

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

Right: systemic blood pressure was measured in anesthetized rats; BK was given as an intra-arterial bolus injection 3 times. Subcutaneous administration of the antagonist (Hoe140). Control was vehicle. Shown in the figure is time versus %change in mean arterial pressure (MAP). Solid columns are Hoe140, open= vehicle. You see an immediate change in MAP at 1 hour, with it not quite returning at 5 hours.

Left: Effect of bronchoconstriction in guinea-pigs shows dose dependence of the antagonist. RL (top)= pulmonary resistance. Cdyn (bottom)=dynamic compliance. BK-induced bronchoconstriction normally does two things compared to baseline, it increases RL 42-58% and decreases Cdyn 33-39%. This graph shows the effect of increasing concentrations of Hoe140. Open columns are before the i.v infusion of Hoe140, solid columns are after.

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

So, this study is exactly what you think it is. They initiate inflammation in a rats paw, drain it and measure the volume.

Note about Patent life: two patents are most easily tied to this drug, the original 1995 one from Hoescht Aktiengesellscaft. Initially they report the same in vitro type radioligand binding data from the 1991 papers. There is a 2014 paper from Shire, which includes more updated experiments like knockout mice, and null mice in addition to the radioligand binding assay. Because if it aint broke, dont fix it.

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Pharmacokinetics

+ final product formulation

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Icatibant

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PropertiesLog P-2.3# Hydrogen Acceptors23# Hydrogen Donors15MW1304.522 Da

Icatibant (DB06196) [online]. Available from URL:

http://www.drugbank.ca/drugs/DB06196#admet

Not going to be orally bioavailable, but who cares! As you can see, this is a huge drug and breaks the rule of 5 in multiple ways. First of all, it is a very hydrophilic decapeptide. The logP is -2.3, so incredibly soluble, but membrane permeability is a huge issue! Other faults in the rule of 5 are its many hydrogen bond donors and acceptors, and huge molecular weight. The polarity is also a big problem for permeability. When this drug was tested using the Caco-2 cell layer assay, it was found to not be cell permeable. However, the target is in the blood so it really didnt matter. There might be some reabsorption in the kidneys by OATP1, (it was found to not be an inhibitor).

If they had cared about permeability, they should have increased LogP, decreased ionizable groups, decreased size, and decreased polarity.

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Icatibant acetate (active) + NaCl, glacial acetic acid, NaOH, H20

Available as a 3mL injection (10 mg/ml) subcutaneously

Requires no mixing or measuring

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Formulation

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

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

PharmacokineticS

Metabolism/ elimination

Proteolytic enzymes (2 inactive metabolites)

CYPs: non-substrate, non-inhibitor

Elimination: urine (