Pharvaris N : A novel kinin biomarker assay for characterization of bradykinin-mediated disorders

A Novel Kinin Biomarker Assay for Characterisation of

Bradykinin-Mediated Disorders

Abstract ID 116577

Evangelia Pardali1, Oliver Domenig2, Dan Sexton3, Grit Zahn4, Anne Lesage5

1Pharvaris B.V., Leiden, The Netherlands; 2Attoquant Diagnostics, Vienna, Austria; 3Sexton Bio Consulting, LLC, Melrose, MA, US; 4Globalization Partners, Munich, Germany; 5GrayMatters Consulting, Schilde, Belgium

Introduction

• Bradykinin (BK) is involved in various physiological and pathological processes, including angioedema (AE).1 AE is a predominant manifestation in multiple medical conditions and is generally mediated by BK and/or histamine.2
• Differentiating BK-mediated from histamine-mediated AE and assessing the role of bradykinin in the pathogenesis of other conditions by measuring kinin peptides remains a challenge due to their proteolytic instability and limitations of current analytical assays.3
• In addition, unspecific activation of the plasma kallikrein-kinin system (KKS), resulting in cleavage of high- molecular-weight kininogen (HMWK) and production of kinins, could have a significant impact on the results.3
• Establishment of a method to accurately measure BK and related peptides could aid in identifying, studying, and managing BK-mediated disorders.

Materials and Methods

• To inhibit ex vivo activation of KKS proteases and proteolytic degradation of BK, a protease inhibitor (PI) cocktail was manufactured in a liquid form (Liquid PI).
• Blood samples were collected from healthy volunteers (HV) by Fidelis Research AD (Sofia, Bulgaria) in accordance with the Declaration of Helsinki and approved by The National Bioethics Committee of Medicines and Medical Devices (protocol no. FRT-19101). All participants provided their written informed consent before enrolment.
• Plasma was collected using S-Monovettes (Sarstedt) with a single venipuncture with a 21G x 3/4'' Safety-Multifly® needle using the aspiration technique, into tubes containing either liquid PI or ethylenediaminetetraacetic acid (EDTA) as a control.
• An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC)-MS/MS protocol was optimized to separately measure BK1-9,BK1-8,BK1-7,BK1-5, and kallidin (KD) (Attoquant Diagnostics GmbH, Vienna, Austria).
• Qualification of the UPLC-MS/MS was performed using plasma from HVs collected under different conditions.

Results

• Calibration curves were prepared by spiking different kinin peptides in surrogate blank matrix.
• For all tested kinins, the back-calculated concentrations of the calibrator standards were within ±15% of the nominal value and met qualification criteria (Figure 1).

Figure 1. Calibration curves of BK1-9,BK1-8,BK1-7,BK1-5, and KD	(pg/mL)	100000	BK1-9
	Linearity range of quantification	10000	BK1-8
		BK1-7
Kinin peptide	LLoQ	ULoQ
conc.	1000	BK1-5
BK1-9	5 pg/mL	10240 pg/mL	KD
BK1-8	5 pg/mL	10240 pg/mL	Measured	100
BK1-7	5 pg/mL	10240 pg/mL	10
BK1-5	5 pg/mL	10240 pg/mL
KD	20 pg/mL	10240 pg/mL		1	10	100	1000	10000 100000
				1
LLoQ: lower limit of quantification; ULoQ: upper limit of quantification			Nominal conc. (pg/mL)

• Kinin UPLC-MS/MS assay in blank matrix met qualification criteria for within- and between-run accuracy and precision.
• Accuracy ±15 % deviation from nominal value. Precision Coefficient of variation (CV) <15% for (Figure 2).

Figure 2. Analysis of quality control (QC) samples for BK1-9,BK1-8,BK1-7,BK1-5 and KD in blank matrix

10000

BK1-9

BK1-8

BK1-7

BK1-5

KD

2500 pg/mL

200 pg/mL

50 pg/mL

15 pg/mL

(pg/mL)

1000

conc.

Measured

100

10

R1 R2 R3

R1 R2 R3

R1 R2 R3

R1 R2 R3

R1 R2 R3

R1 R2 R3

R1 R2 R3

R1 R2 R3

R1 R2 R3

R1 R2 R3

R1 R2 R3

R1 R2 R3

R1 R2 R3

R1 R2 R3

R1 R2 R3

R1 R2 R3

R1 R2 R3

R1 R2 R3

R1 R2 R3

• KD levels were below the limit of detection in all types of human plasma analysed (not shown).
• Liquid PI efficiently inhibited KKS activation and stabilized levels of all BK peptides compared with EDTA alone, resulting in low inter-run and intra-run variability (CV <15%) for BK1-9,BK1-8,BK1-7,BK1-5 and total BK levels (Figure 3).
  Figure 3. Inter- and intra-run variability in EDTA plasma and plasma with Liquid PI

Results

• EDTA plasma presented higher levels of all BK peptides and high intra-individual variability in plasma from HVs.
• Low intra-individual variability, CV <15 % for all BK peptides in Liquid PI plasma samples from all HVs (Figure 4).
• Liquid PI efficiently inhibited ex vivo KKS activation and kinin degradation.

Figure 4. Acceptable intra-individual variability for Liquid PI plasma samples, high for EDTA

BK1-9

800

BK1-8

BK1-7

EDTA

Liquid PI

EDTA

Liquid PI

200

800

600

EDTA

Liquid PI

[pg/mL]9-BK1

[pg/mL]8-BK1

[pg/mL]7-BK1

150

600

Det 1

Det 1

Det 1

400

100

400

200

50

20

Det 2

8

Det 2

8

Det 2

10

Det 3

4

Det 3

4

Det 3

0

HV1 HV2

HV3

HV4

HV1

HV2 HV3

HV4

0

HV1 HV2

HV3

HV4 HV1 HV2 HV3 HV4

0

HV1

HV2

HV3

HV4

HV1

HV2

HV3

HV4

BK1-5

Total BK levels

80

EDTA

Liquid PI

1000

Liquid PI

EDTA

[pg/mL]5-

60

[pg/mL]BK

800

600

BK1

40

Total

400

20

Det 1

30

Det 1

Det 2

15

Det 2

0

Det 3

0

Det 3

HV1

HV2 HV3

HV4 HV1 HV2 HV3 HV4

HV1

HV2 HV3 HV4

HV1 HV2 HV3 HV4

EDTA

BK1-9

BK1-8

BK1-7

BK1-5

Total BKs

Mean±SD

CV%

Mean±SD

CV%

Mean±SD

CV%

Mean±SD

CV%

Mean±SD

CV%

(pg/mL)

(pg/mL)

(pg/mL)

(pg/mL)

(pg/mL)

HV1

291.1±87.7

30.1

87.0±10.0

11.5

43.9±5.2

11.8

22.1±1.6

7.3

444.0±101.2

22.8

HV2

2323.6±2440.4 105.0

345.3±301.2

87.2

105.7±80.4

76.1

38.4±17.0

44.1

2813.0±2839.1 100.9

HV3

679.8±130.5

19.2

245.1±70.7

28.8

85.9±11.0

12.8

49.1±10.2

20.7

1059.9±222.4

21.0

HV4

1230.5±992.7

80.7

193.3±129.1

66.8

58.5±15.8

27.0

20.9±9.4

45.1

1503.2±1147.1

76.3

Liquid

BK1-9

BK1-8

BK1-7

BK1-5

Total BKs

Mean±SD

Mean±SD

Mean±SD

Mean±SD

Mean±SD

PI

CV%

CV%

CV%

CV%

CV%

(pg/mL)

(pg/mL)

(pg/mL)

(pg/mL)

(pg/mL)

HV1

13.1±1.4

11.0

3.4*±0.4

na

1.5*±0.4

na

9.8±0.5

5.2

27.7±1.4

4.9

HV2

7.7±0.3

3.8

2.6*±0.1

na

1.6*±0.0

na

10.2±0.9

9.1

21.7±1.1

4.9

HV3

6.2±0.7

11.2

2.3*±0.6

na

1.1*±0.2

na

12.4±0.6

4.7

22.0±1.4

6.2

HV4

4.1±0.4

8.6

2.2*±0.2

na

1.0*±0.2

na

6.2±0.7

11.0

13.2±0.7

5.5

• • Concentrations are below LLoQ; na: not applicable
• Liquid PI efficiently inhibited ex vivo KKS activation and stabilized BK peptides following 2 cycles of freeze and thaw (FT) as compared with EDTA (Figure 5).
• FT induced KKS activation in EDTA plasma resulting in high levels and r variability in BK levels (Figure 5).

Figure 5. Effects of Freeze and thaw on BK peptide levels and stability

BK1-9

BK1-8

BK1-7

1500

EDTA

Liquid PI

250

EDTA

Liquid PI

200

EDTA

Liquid PI

1250

[pg/mL]

[pg/mL]

200

[pg/mL]

150

1000

150

100

-9

750

-8

100

-7

BK1

BK1

BK1

50

Det 1

50

Det 1

50

Det 1

25

Det 2

Det 2

Det 2

0

Det 3

0

Det 3

0

Det 3

No FT 1xFT

2xFT No FT 1xFT

2xFT

No FT 1xFT

2xFT No FT 1xFT

2xFT

No FT 1xFT

2xFT No FT 1xFT

2xFT

BK1-5

Total BK levels

60

2000

EDTA

Liquid PI

Total BKs [pg/mL]

EDTA

Liquid PI

BK1-5 [pg/mL]

40

1500

1000

20

Det 1

Det 1

100

Det 2

Det 2

50

Det 3

Det 3

0

0

No FT

1xFT 2xFT No FT

1xFT 2xFT

No FT 1xFT

2xFT No FT 1xFT

2xFT

EDTA plasma

Liquid PI plasma

Mean (pg/mL)

Deviation %

Mean (pg/mL)

Deviation %

Kinin peptide

No FT

1xFT

2xFT

1xFT

2xFT

No FT

1xFT

2xFT

1xFT

2xFT

BK1-9

582.4

806.4

962.9

38.5

65.3

34.7

35.6

37.9

2.5

8.9

BK1-9 [pg/mL]

1250

1000

750

50

25

0

BK1-9

EDTA							Liquid PI

Det 1

Det 2

Det 3

Run 1

Run 2

Run 1

Run 2

BK1-5

60

EDTA

Liquid PI

-5 [pg/mL]

40

20

BK1

0

Run 1

Run 2

Run 1

	250
[pg/mL]8-	200
150
BK1	100
50
	0

Run 2

BK1-8

EDTA					Liquid PI

Run 1	Run 2		Run 1
		[pg/mL]	1600
		1400
		1200
		BKs
		1000
		Total	800
Det 1		80
Det 2			40
Det 3			0

				BK1-7
		200		Liquid PI
		EDTA
	[pg/mL]	150
	100
	BK1-7
Det 1	50
Det 2
Det 3		0
Run 2		Run 1	Run 2	Run 1	Run 2

Total BK levels

EDTA							Liquid PI

Det 1

Det 2

Det 3

Run 1

Run 2

Run 1

Run 2

Det 1

Det 2

Det 3

BK1-8	151.7	177.7	183.4	17.1	20.9	10.0	11.3	10.0	13.5	0.5
BK1-7	114.2	139.6	134.5	22.2	17.7	6.5	6.7	6.4	1.6	-3.0
BK1-5	41.6	48.3	46.4	16.1	11.6	8.9	10.3	10.4	15.7	16.8
Total BK	889.9	1172.0	1327.2	31.7	49.1	60.2	63.9	64.6	6.2	7.4

No FT, no freeze/thaw; 1xFT, 1 freeze/thaw cycle; 2xFT, 2 freeze/thaw cycle

Conclusions

•The Liquid PI cocktail was shown to be effective in inhibiting non-specific activation of KKS and

generation of BK1-9 as compared to EDTA without PI.

•The established qualified kinin UPLC-MS/MS biomarker assay can be used to reliably measure

KKS biomarkers in human plasma.

		EDTA plasma		Liquid PI plasma
Kinin peptide	Mean ± SD (pg/mL)	CV%	Mean ± SD (pg/mL)	CV%
BK1-9	748.9	± 81.3	10.9	35.9 ± 0.4	1.0
BK1-8	169.4	± 11.7	6.9	10.4 ± 1.3	12.8
BK1-7	129.3	± 14.6	11.3	6.8 ± 0.3	3.9
BK1-5	45.8	± 3.5	7.6	10.3 ± 0.0	0.1
Total BK	1093.5	± 111.1	10.2	63.4 ± 0.7	1.1

•The assay could become a key tool for identifying, studying, and managing BK-mediated

diseases, including BK-mediated AE.

References

1. Kaplan AP, et al. Adv Immunol. 2014;121:41-89.2. Maurer M, et al. Clin Rev Allergy Immunol. 2021;61:40-9.3. Kaplan AP, et al. Front Med (Lausanne).

2017;4:206.

COI: E.P.: employee of Pharvaris, holds stock in Pharvaris; O.D.: CEO of Attoquant Diagnostics GmbH; D.S.: principle at Sexton Bio Consulting, LLC, consultant to Pharvaris; G.Z.: employee of Globalization Partners GmbH, consultant to Pharvaris, holds RSU; A.L.: employee of GrayMatters Consulting and consultant to Pharvaris, holds stocks/stock options in Pharvaris; advisor to Kosa Pharma.

Funding statement: Funding towards this study was provided by Pharvaris.