Effects of a Selective Inhibitor of Secretory Phospholipase A2 on Low Density Lipoproteins

and Inflammatory Pathways

Robert S. Rosenson, MD, FACC1

Colin Hislop, MD2

Daniel McConnell, Ph.D3

Michael Elliott, MA2

Yuri Stasiv, Ph.D2

David Waters, MD4

For the PLASMA Investigators

1 Division of Cardiovascular Medicine, University of Michigan, Ann Arbor, MI2 Anthera Pharmaceuticals, San Mateo, CA

3 Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI4 Division of Cardiology, San Francisco General Hospital, San Francisco, CA

Robert S. Rosenson, M.D. – Consultant/Advisor: Anthera, modest level; Equity Interests/Stock Options: LipoScience, Inc., significant level;Colin Hislop, M.D. – Equity Interests/Stock Options: Anthera, significant level;

Employment: Anthera, significant level;Daniel McConnell, Ph.D. – No DisclosuresMichael Elliott, M.A. –

Equity Interests/Stock Options: Anthera, significant level;Employment: Anthera, significant level;

Yuri Stasiv, Ph.D. – Equity Interests/Stock Options: Anthera, modest level;Employment: Anthera, significant level;

David Waters, M.D. – David Waters, M.D. – Consultant/Advisor: Merck Schering Plough, Atherogenic,

modest level; Pfizer, significant level; Equity Interests/Stock Options: Anthera, modest level; Lecture Fees: Pfizer, significant level;

Disclosure Information

Atherosclerosis, sPLA2 and Inflammation

Macrophage/Foam Cell

Smooth muscle cells Cytokines

Lyso-PC

Small LDLModified LDL

sPLA2

sPLA2

LDL

sPLA2

OX-LDL

Circulating sPLA2

Foam Cells

Monocyte Adhesion

Plaque

Circulating HDL

NEFA

Ox-NEFA

LDL Aggregates

Arachidonic Acid

PLASMA: PLASMA: PPhospholipase hospholipase LLevels evels AAnd nd SSerological erological MMarkers of Atherosclerosis arkers of Atherosclerosis

((PLASMA: PLASMA: NCT00455546))

Varespladib 50 mg BID

Varespladib 100 mg BID

Varespladib 250 mg BID

Varespladib 500 mg BID

Placebo BID

8-week double-blind treatment period

Primary End Point• sPLA2 -IIA mass

Secondary End Points• Lipids• Lipoprotein subclasses• Apo B• Ox-LDL• hs-CRP• Safety in CHD patients

396 Patients• Stable CHD

12wk post MI 6wk post UA

•Therapeutic standard care

(n=79)

(n=77)

(n=78)

(n=80)

(n=79)

Varespladib (A-002), Anthera Pharmaceutical, San Mateo, CA, USAVarespladib (A-002), Anthera Pharmaceutical, San Mateo, CA, USA

Inclusion/Exclusion Criteria

Inclusion

• Men and women > 18 years of age

• Written informed consent from the subject

• Stable CAD

• Stable medical condition, will be compliant and able to comply with the requirements of the protocol

Exclusion

• Active inflammatory disease and drugs to modulate the inflammatory response

MethodsMethods

• sPLAsPLA2 2 mass mass was analyzed by a quantitative two- was analyzed by a quantitative two-

site using a monoclonal antibody specific for site using a monoclonal antibody specific for sPLAsPLA22-Group IIA EIA (Cayman Chemical, Ann -Group IIA EIA (Cayman Chemical, Ann

Arbor, MI). Arbor, MI). • LipoproteinLipoprotein subclassessubclasses were measured by NMR were measured by NMR

spectroscopic assay (LipoScience, Inc., Raleigh, spectroscopic assay (LipoScience, Inc., Raleigh, NC, USA).NC, USA).

• Oxidized LDLOxidized LDL was measured by ELISA using the was measured by ELISA using the specific antibody 4E6 (Mercodia, Uppsula specific antibody 4E6 (Mercodia, Uppsula Sweden).Sweden).

• CRPCRP with a high-sensitivity assay (Dade Behring with a high-sensitivity assay (Dade Behring Nephelometer II, Quest Diagnostics, Van Nuys, Nephelometer II, Quest Diagnostics, Van Nuys, CA, USA).CA, USA).

Demographic and Baseline CharacteristicsDemographic and Baseline CharacteristicsVariableVariable VarespladibVarespladib

50 mg 50 mg (n=79)(n=79)

VarespladibVarespladib

100 mg 100 mg (n=80)(n=80)

VarespladibVarespladib

250 mg 250 mg (n=78)(n=78)

VarespladibVarespladib

500 mg 500 mg (n=77)(n=77)

VarespladibVarespladib

Overall Overall (n=314)(n=314)

PlaceboPlacebo

(n=79)(n=79)

Age (y)Age (y) 62 62 ++ 10 10 64 64 ++ 10 10 63 63 ++ 10 10 61 61 ++ 11 11 62 62 ++ 10 10 62 62 ++ 11 11

Sex (%M/F)Sex (%M/F) 78.5/21.578.5/21.5 72.5/27.572.5/27.5 83.3/16.783.3/16.7 81.8/18.281.8/18.2 79.0/21.079.0/21.0 75.9/24.175.9/24.1

Race (% Caucasian)*Race (% Caucasian)* 100100 7575 94.994.9 98.798.7 97.197.1 94.994.9

BMI (kg/mBMI (kg/m2)2) 30.4 30.4 ++ 5.9 5.9 30.6 30.6 ++ 6.3 6.3 29.9 29.9 ++ 5.4 5.4 30.4 30.4 ++ 5.1 5.1 30.3 30.3 ++ 5.7 5.7 30.5 30.5 ++ 5.3 5.3

MI (%)MI (%) 52 (66)52 (66) 54 (68)54 (68) 47 (60)47 (60) 54 (70)54 (70) 207 (66)207 (66) 53 (67)53 (67)

CABG (%)CABG (%) 54 (68)54 (68) 56 (70)56 (70) 55 (70) 55 (70) 53 (69)53 (69) 218 (69)218 (69) 52 (66)52 (66)

HTN (%)HTN (%) 70 (89)70 (89) 64 (80)64 (80) 67 (86)67 (86) 61 (79)61 (79) 262 (83)262 (83) 57 (72)57 (72)

Diabetes (%)Diabetes (%) 16 (20)16 (20) 28 (35)28 (35) 20 (26)20 (26) 18 (23)18 (23) 82 (26)82 (26) 20 (25)20 (25)

Aspirin (%)Aspirin (%) 74 (94)74 (94) 74 (92)74 (92) 68 (87)68 (87) 72 (94)72 (94) 288 (92)288 (92) 67 (85)67 (85)

Thienopyridine (%)Thienopyridine (%) 20 (25)20 (25) 12 (15)12 (15) 26 (32)26 (32) 29 (38)29 (38) 86 (27)86 (27) 22 (28)22 (28)

Beta Blockers (%)Beta Blockers (%) 52 (66)52 (66) 66 (82)66 (82) 60 (77)60 (77) 54 (70)54 (70) 232 (74)232 (74) 47 (59)47 (59)

ACE/ARB (%)ACE/ARB (%) 45 (57)45 (57) 42 (53)42 (53) 47 (60)47 (60) 41 (53)41 (53) 175 (56) 175 (56) 43 (54)43 (54)

Statin (%)Statin (%) 49 (62)49 (62) 54 (68)54 (68) 53 (68)53 (68) 52 (68)52 (68) 207 (66)207 (66) 51 (65)51 (65)

Data are expressed as means Data are expressed as means ++ standard deviations standard deviations* Race was determined by the investigator* Race was determined by the investigator

VariableVariable VarespladibVarespladib

50 mg 50 mg

(n=79)(n=79)

VarespladibVarespladib

100 mg 100 mg

(n=80)(n=80)

VarespladibVarespladib

250 mg (n=78)250 mg (n=78)

VarespladibVarespladib

500 mg (n=77)500 mg (n=77)

VarespladibVarespladib

Overall Overall (n=314)(n=314)

PlaceboPlacebo

(n=79)(n=79)

sPLAsPLA22 Mass Mass

(ng/mL)*(ng/mL)*

2.10 2.10

(0.2-8.80)(0.2-8.80)

2.20 2.20

(0.05-(0.05-22.30)22.30)

2.00 2.00

(0.20-6.00)(0.20-6.00)

2.402.40

(0.40-5.40)(0.40-5.40)

2.20 2.20

(0.05-22.30)(0.05-22.30)

2.202.20

(0.08-6.80)(0.08-6.80)

TC (mg/dL)TC (mg/dL) 175 175 ++ 6.0 6.0 178 178 ++ 5.7 5.7 173 173 ++ 5.1 5.1 178 178 ++ 6.6 6.6 176 176 ++ 2.9 2.9 174 174 ++ 5.9 5.9

LDL-C (mg/dL)LDL-C (mg/dL) 93.3 93.3 ++ 4.8 4.8 98.8 98.8 ++ 4.7 4.7 93.2 93.2 ++ 4.5 4.5 95.1 95.1 ++ 4.8 4.8 95.1 95.1 ++ 2.3 2.3 96.2 96.2 ++ 5.1 5.1

HDL-C ( mg/dL)HDL-C ( mg/dL) 47.4 47.4 ++ 1.6 1.6 46.0 46.0 ++ 1.2 1.2 48.0 48.0 ++ 1.7 1.7 48.9 48.9 ++ 1.7 1.7 47.6 47.6 ++ 0.8 0.8 50.1 50.1 ++ 1.5 1.5

TG (mg/dL)TG (mg/dL) 169 169 ++ 12.4 12.4 161 161 ++ 10.1 10.1 161 161 ++ 10.6 10.6 171 171 ++ 18.0 18.0 165 165 ++ 6.5 6.5 152 152 ++ 11.5 11.5

LDL-P (nmol/L)*LDL-P (nmol/L)* 1076.51076.5

(344-2531)(344-2531)

1105.01105.0

(502-2443)(502-2443)

1043.51043.5

(451-2422)(451-2422)

1076.01076.0

(445-2410)(445-2410)

1085.01085.0

(344-2531)(344-2531)

1183 1183 ++ 47.447.4

Small LDL-P (nmol/L)*Small LDL-P (nmol/L)* 755.0 755.0

(16-2251)(16-2251)

779.0779.0

(57-2143)(57-2143)

741.5741.5

(29-2185)(29-2185)

838.0838.0

(48-1980)(48-1980)

777.0777.0

(16-2251)(16-2251)

1065.01065.0

(509-2641)(509-2641)

Apo B, (mg/dL)Apo B, (mg/dL) 93.1 93.1 ++ 4.6 4.6 96.4 96.4 ++ 4.1 4.1 96.3 96.3 ++ 3.9 3.9 98.0 98.0 ++ 4.5 4.5 96.0 96.0 ++ 2.1 2.1 98.4 98.4 ++ 4.3 4.3

Ox-LDL (mU/L)Ox-LDL (mU/L) 60.8 60.8 ++ 2.5 2.5 60.1 60.1 ++ 2.1 2.1 59.5 59.5 ++ 2.3 2.3 60.2 60.2 ++ 2.2 2.2 60.2 60.2 ++ 1.1 1.1 61.8 61.8 ++ 2.4 2.4

Baseline Plasma Levels of Lipid, Lipoprotein and Baseline Plasma Levels of Lipid, Lipoprotein and Inflammatory MarkersInflammatory Markers

*sPLA2 Mass, LDL-P and Small LDL-P in medians and interquartile ranges

0 weeks

2 weeks

4 weeks

8 weeks*

0

0.5

1

1.5

2

2.5

sPL

A2

mas

s (n

g/m

L)

50 mg 100 mg 250 mg 500 mg Placebo

Varespladib dosage

* **

* * * * ** * *

Secretory PLASecretory PLA2 2 Mass in Placebo and Mass in Placebo and Varespladib Treatment GroupsVarespladib Treatment Groups

* p<0.0001Data are expressed as medians

Baseline

Week 2

Week 4

Week 8

50

60

70

80

100

110

50 mg 100 mg

250 mg 500 mg Placebo

Varespladib dosage

LD

L c

ho

les

tero

l (m

g/d

L)

** *

* † †‡‡

§

LDL-C in Placebo and Varespladib LDL-C in Placebo and Varespladib Treatment GroupsTreatment Groups

* p<0.05; † p<0.005; ‡ p<0.001; § p=0.0005Data are expressed as means

90

40

LDL-C Levels in Patients Taking Varespladib Monotherapy

80

90

110

120

140

150

50 mg 100 mg

250 mg 500 mg Placebo

Varespladib dosage

LD

L c

ho

les

tero

l (m

g/d

L)

130

70

Baseline

Week 8

Data are expressed as means

**

* p<0.05

BaselineWeek 2Week 4Week 8

50

60

70

80

100

110

120

50 mg 100 mg 250 mg 500 mg Placebo

Varespladib dosage

LD

L c

ho

lest

ero

l (m

g/d

L)

LDL-C in Statin-Treated Subjects with Baseline LDL-C > 70 LDL-C in Statin-Treated Subjects with Baseline LDL-C > 70 mg/dL in Placebo and Varespladib Treatment Groupsmg/dL in Placebo and Varespladib Treatment Groups

* p<0.05; † p<0.01; ‡ p<0.005

*‡

‡

‡*

Data are expressed as means

90

40

† †

‡

Effects of Varespladib on LDL SubclassesEffects of Varespladib on LDL Subclasses

• In varespladib-treated subjects, median LDL In varespladib-treated subjects, median LDL particle concentration was reduced by 80 particle concentration was reduced by 80 nmol/L (p <0.05) primarily due to lowering of nmol/L (p <0.05) primarily due to lowering of small LDL particles (58 nmol/L, p<0.01).small LDL particles (58 nmol/L, p<0.01).

• Overall LDL particle size increased in the Overall LDL particle size increased in the varespladib-treated subjects by 0.02 nm varespladib-treated subjects by 0.02 nm compared to a decrease in placebo=treated compared to a decrease in placebo=treated subjects (-0.17 nm), p = 0.015).subjects (-0.17 nm), p = 0.015).

• Oxidized LDL was reduced in varespladib Oxidized LDL was reduced in varespladib treated patients (p = 0.011). treated patients (p = 0.011).

hs-CRP Levels in Placebo and Varespladib hs-CRP Levels in Placebo and Varespladib Treatment GroupsTreatment Groupsh

s-C

RP

(m

g/L

)

0.1

0.3

0.4

0.5

0.6

0

0.2

50 mg 100 mg 250 mg 500 mg Placebo

Baseline

8 Weeks

Varespladib dosageData are expressed as means

Safety and Adverse Events Safety and Adverse Events

• The proportion of subjects reporting TEAEs and The proportion of subjects reporting TEAEs and receiving varespladib (47%) was no different from receiving varespladib (47%) was no different from placebo (44%).placebo (44%).

• Most frequent amongst the varespladib treatment Most frequent amongst the varespladib treatment groups were headache 6.4%; nausea 5.4%; groups were headache 6.4%; nausea 5.4%; diarrhea 3.8%; ALT increase 3.2%; dizziness diarrhea 3.8%; ALT increase 3.2%; dizziness 2.9%; AST increased 2.2%; back pain 2.2%.2.9%; AST increased 2.2%; back pain 2.2%.

• There was one serious adverse event (SAE) in the There was one serious adverse event (SAE) in the varespladib 50 mg BID treatment group resulting varespladib 50 mg BID treatment group resulting

in discontinuation.in discontinuation. An exacerbation of COPD An exacerbation of COPD was reported in a subject who had a viral was reported in a subject who had a viral

prodrome (fever, headache) 2 days earlierprodrome (fever, headache) 2 days earlier..

ConclusionsConclusions

1.1. Varespladib treatment markedly reduced Varespladib treatment markedly reduced median sPLAmedian sPLA22 mass by 69% to 96% in a mass by 69% to 96% in a dose-dependent manner.dose-dependent manner.

2.2. Compared to placebo, varespladib Compared to placebo, varespladib reduced LDL-C by 8.0 to 11.9% primarily reduced LDL-C by 8.0 to 11.9% primarily mediated by a reduction in small LDL mediated by a reduction in small LDL particles.particles.

3.3. The maximal reduction in LDL-C was The maximal reduction in LDL-C was larger among statin-treated patients with larger among statin-treated patients with baseline LDL-C >70 mg/dL ranging from baseline LDL-C >70 mg/dL ranging from 6.1 to 17.1%.6.1 to 17.1%.

Plasma Investigators

• The investigators that participated in the PLASMA study are as follows – United States: M. Matsumura, M. Imburgia, S. Chrysant, J. Rozanski, S. Smith, R. Weiss, P. Underwood, D. Ende, D. Brown, C. Brown, V. Nadar, R. Carlson, D. Wombolt, F. Matar, P. Rossi, F. Eder, B. Bowling, R. Pellegrino, N. Ferrier, D. Hotchkiss, S. Broadwater. Ukraine: K. Amosova, O. Korkushko, V. Kovalenko, O. Kupchynska, M. Lutay, V. Lizogub, B. Mankovsky, O. Mitchenko, Y. Sirenko, L. Yena, V. Volkov, I. Kraiz, M. Perepelytsya, V. Vizir, G. Dzyak.