HDL Review: Translating new findings on LCAT into novel ... · HDL Review: Translating new findings on LCAT into novel therapies Alan T. Remaley, MD, PhD Lipoprotein Metabolism Section

HDL Review: Translating new findings on LCAT into novel therapies

HDL Review: Translating new findings on LCAT into novel therapies

Alan T. Remaley, MD, PhD

Lipoprotein Metabolism SectionCardiopulmonary Branch, NHLBI

National Institutes of Health

Alan T. Remaley, MD, PhD

Lipoprotein Metabolism SectionCardiopulmonary Branch, NHLBI


DisclosuresDisclosures

Patent: NIH patent licensed to Alpha Core Pharma/AstraZeneca

Research Support: CRADA grant with Alpha Core Pharma/AstraZeneca

OverviewOverview

HDL Biology

LCAT Biology

Phase I Clinical trial of rLCAT

Single Patient IND Treatment FLD

HDL Biology

LCAT Biology



HDL Structure and Composition

Pre-beta Discoidal Shaped HDL

Alpha Spherical Shaped HDL

Initial Cholesterol Acceptor Delivers Cholesterol to Liver

HDL Structure and Composition

Pre-beta Discoidal Shaped HDL

Alpha Spherical Shaped HDL

LCAT

Initial Cholesterol Acceptor Delivers Cholesterol to Liver

Vaisar T, et al JCI 2007

HDL-Proteome HDL-Proteome

HDL-LipidomeHDL-Lipidome

SterolsHDL

Pleiotropic Anti-AtherogenicEffects Of HDL (“Good Cholesterol”)


AntiinflammatoryActivity

ReverseCholesterolTransport

AntithromboticActivity

AntiapoptoticActivity

AntioxidativeActivity

AntiinfectiousActivity

EpithelialRepair

VasodilatoryActivity

Chapman MJ, et al Curr Med Res Opin. 2004:20:1253-1268Assmann G, et al Annu Rev Med 2003:53:321-341

AntiinflammatoryActivity

ReverseCholesterolTransport

AntithromboticActivity

AntiapoptoticActivity

AntioxidativeActivity

AntiinfectiousActivity

EpithelialRepair

VasodilatoryActivity

Chapman MJ, et al Curr Med Res Opin. 2004:20:1253-1268Assmann G, et al Annu Rev Med 2003:53:321-341


Reverse Cholesterol Transport Pathway

Anti-Atherogenic Effects of HDL

HDL

LDL

LDL

OxLDL

Inhibition ofLDL-oxidation

Inhibition of monocyte-adhesion

Cholesterol-efflux

HDL

HDL

HDL

Endothelium

mono-cyte

macro-phage foam

cell

Therapeutic lifestyle changes

Pharmacologic therapy

Statins

Fibrates

Niacin

CETP-inhibitors ?

Acute HDL Therapy ?

Management of low HDL-C

ApoA-I Milano Infusion StudiesApoA-I Milano Infusion StudiesLimone sul Garde, Italy

Free SH groupImproved anti-oxidant

ApoA-I Milano Infusion StudiesApoA-I Milano Infusion StudiesLimone sul Garde, Italy

Circ. 103(2001) 3047-50

Pre-clinical Animal Exp.

Free SH groupImproved anti-oxidant

Figure adapted from PLATO Trial Cornel JH et al. Am Heart J 2012;164:334‐342

Recurrent CVD in ACS Patients

Statins

Clinical trials of HDL Infusion TherapyClinical trials of HDL Infusion Therapy

Nissen, S., et al. JAMA 290 (2003) 2292-2300

Tardif, J., et al. J Clin Lipidol 4 (2010) 399-404

CSL-111 (Purified ApoA-I) TrialCSL-111 (Purified ApoA-I) TrialApoA-I Milano (recombinant) TrialApoA-I Milano (recombinant) Trial

OverviewOverview

HDL Biology

LCAT Biology



HDL Biology

LCAT Biology



Vanloo B et al. J. Lipid Res. 2000;41:752-761

Predicted Structure of LCAT ProteinAlpha/Beta Fold Structure

Plasma protein produced by liver

Concentration 5 ug/mL

Mostly resides on HDL but also LDL

Over 75 mutations described

FLD mutations tend to decrease LCAT to a greater extent and affect esterification on HDL and LDL

FED mutations tend to affectesterification just on HDL

LCAT Features

mature HDL

nascent HDL

LCAT ReactionLCAT Reaction

Potential Clinical Indications for rLCATPotential Clinical Indications for rLCAT

Acute coronary syndrome

For the rapid stabilization of ACS patients

Familial LCAT deficiency (FLD)

For the prevention and or treatment renal disease.

Acute coronary syndrome

For the rapid stabilization of ACS patients

Familial LCAT deficiency (FLD)

For the prevention and or treatment renal disease.

LDLLDL

Macrophages and peripheral

tissues

Macrophages and peripheral

tissues

SR-BI

LDL-RLDL-R CETP

mature HDL(α-HDL)

mature HDL(α-HDL)

nascent HDL (Preβ-HDL)

nascent HDL (Preβ-HDL)

LCATABCA1

ABCG1

LCAT Potentiates the ReverseCholesterol Transport PathwayLCAT Potentiates the ReverseCholesterol Transport Pathway

Cholesteryl Ester Free Cholesterol

Clinical Features

Cloudy cornea “fish eye”

Lipoprotein ProfileLow HDL-C (<10 mg/dL)Low apoA-I (20-30 mg/dL)<25% Cholesteryl estersLow LDL-CPresence of Lp-X particles

Normochromic AnemiaEnd stage renal diseaseComplete Deficiency FLDPartial Deficiency FED

Features of LCAT DeficiencyFeatures of LCAT Deficiency

Rare autosomal recessive

LpX Features

Lp-XLp-X

Bilayer or multi-lamellar complex of phospholipids


Aqueous core

Aqueous core

LpX and Renal DiseaseLpX and Renal Disease

± 60% PL± 30% Free Cholesterol≥ 5% AlbuminNo apoB or neutral lipidsSome exchangeale apo’s30 – 70 nm1.038-1.058 g/mL

LpX Features

Lp-XLp-X



Aqueous core

Aqueous core


Glomerulus in FLD “Foamy” Mesangial cells Accumulation Lipid in BM

± 60% PL± 30% Free Cholesterol≥ 5% AlbuminNo apoB or neutral lipidsSome exchangeale apo’s30 – 70 nm1.038-1.058 g/mL

LpX Cell Uptake StudiesLpX Cell Uptake StudiesElectron microscopy of LpX

Mesangial cell uptake of LpX

Lipoprotein Electrophoresis

LpX Activates the InflammasomeLpX Activates the InflammasomeInflammasome Activation Pathway





Proteinuria (Albumin/creat. ratio) Fold Increase in Nephrotoxic Genes

LpX injection in LCAT KO mice causes proteinuria and inducesgenes associated with nephrotoxicity.

Increased Pre-beta HDL andDecreased LCAT in CHD Subjects

Sethi A. Clin Chem: 2009;56:1128

From Copenhagen City Heart Study

CHD-RedNO-CHD Green

High HDL>62 mg/dLLow HDL<33 mg/dL

LCAT Activity low in CHD



CHD-RedNO-CHD Green


LCAT Activity low in CHD Pre-beta HDL high in CHD




CHD-RedNO-CHD Green


LCAT Activity low in CHD Pre-beta HDL high in CHD

pre-beta HDL inversely related to LCAT


Carotid MRI of LCAT HeterozygotesCarotid MRI of LCAT Heterozygotes

Similar lumen areas

Approx. 30-fold increase plaque volume in FLD compared to controls

No significant difference in CIMT

Dulvenoorden R J Am Coll Cardiol (2011) 6:2481

Similar lumen areas




Carotid MRI

http://yourimt.com/images/707_PLAQUE.JPG

Carotid MRI of LCAT HeterozygotesCarotid MRI of LCAT Heterozygotes

Similar lumen areas




Similar lumen areas




Carotid MRI

http://yourimt.com/images/707_PLAQUE.JPG

Production of Human Recombinant LCATProduction of Human Recombinant LCAT

AlphaCore PharmaLicense/CRADA 2008

HEK transfected cells

4-step purification following ZnCl ppt.

> 99.5% purity

Yield 15 mg/L fromconditioned media

AlphaCore PharmaLicense/CRADA 2008

HEK transfected cells

4-step purification following ZnCl ppt.

> 99.5% purity

Yield 15 mg/L fromconditioned media

kDakDa

250150100

250150100

7575

5050

3737

2525

Std 1 µg 2 µg 5 µgStd 1 µg 2 µg 5 µgW. BlotW. BlotSDS-PAGESDS-PAGE

Rousset X, et al. J Pharmacol Exp Ther. 2010;335(1):140-148.

0

5

10

15

20

10 20 30

T0

LDL

LP-X

Cho

lest

erol

(ug/

mL)

Volume of Elution (mL)Volume of Elution (mL)

Intravenous rLCAT Rapidly CorrectsLipoprotein Profile in LCAT KO MiceIntravenous rLCAT Rapidly Corrects

Lipoprotein Profile in LCAT KO Mice


0

5

10

15

20

10 20 30

0

5

10

15

20

10 20 30

T0 1h

LDL

LP-XIncrease

HDL LDL

DecreaseLP-X

Cho

lest

erol

(ug/

mL)





0

5

10

15

20

10 20 30

0

5

10

15

20

10 20 30

0

5

10

15

20

10 20 30

T0

4h

1h

LDL

LP-XIncrease

HDL

HDL

LDL

DecreaseLP-X

LDL

LP-XCho

lest

erol

(ug/

mL)


0

5

10

15

20

10 20 30 40




24hLDLLP-X

In Vitro Treatment of Serum with rLCAT Rapidly Normalizes FLD Lipoprotein Profile

In Vitro Treatment of Serum with rLCAT Rapidly Normalizes FLD Lipoprotein Profile

Rousset X, et al. J Pharmacol Exp Ther. 2010;335(1):140-148.Rousset X, et al. J Pharmacol Exp Ther. 2010;335(1):140-148.

FLD serum incubated 1 hour at 37o C with or without rLCAT

rLCAT Decreases Tissue Cholesterol Levels in LCAT Ko x hapoA-I Tg Mice

rLCAT Decreases Tissue Cholesterol Levels in LCAT Ko x hapoA-I Tg Mice

4 daily IP injections of rLCAT injection in LCAT KO x apoA-I Tg mice (N=5)

** *

* P<0.05

Aortic Gene Expression Changes after rLCAT Treatment

Aortic Gene Expression Changes after rLCAT Treatment

Favorable gene expression changes consistent with increased mobilization of cholesterol from arterial wall

Synergistic Effect of rLCAT and HDL in Plasma Cholesterol Esterification (CER)Synergistic Effect of rLCAT and HDL in Plasma Cholesterol Esterification (CER)

HDL(CSL-111) dose response response

rLCAT dose response

**



rLCAT dose response

Both HDL and LCAT are rate limiting

(* P<0.05 above baseline)

Both HDL and LCAT are rate limiting


**

**

*



rLCAT dose response

CSL-111+ rLCAT dose response

Both HDL and LCAT are rate limiting and show a synergistic effecton cholesterol esterification.


Both HDL and LCAT are rate limiting and show a synergistic effecton cholesterol esterification.


**

*

**

**

*

Co-infusion of rHDL and rLCAT IncreasesCholesterol Efflux and Esterification in MiceCo-infusion of rHDL and rLCAT Increases

Cholesterol Efflux and Esterification in Mice

*

020406080100120140160180200

0 1 2 3 4 5 6

Concen

tration (m

g/dL)

Time (h)

Total Cholesterol

*

0

50

100

150

200

CSL111 CSL111+LCAT

AUC (R.U.)

Increased Total Cholesterol

(* P<0.05)

Co-infusion of rHDL and rLCAT IncreasesCholesterol Efflux and Esterification in MiceCo-infusion of rHDL and rLCAT Increases

Cholesterol Efflux and Esterification in Mice

* * *

020406080

100120140160180200

0 1 2 3 4 5 6

Concen

tration (m

g/dL)

Time (h)

Cholesteryl EsterCSL111 (60mg/kg); n=4

CSL111 (60mg/kg) + hLCAT (30mg/kg); n=4

*

020406080100120140160180200

0 1 2 3 4 5 6

Concen

tration (m

g/dL)

Time (h)

Total Cholesterol

*

0

50

100

150

200

CSL111 CSL111+LCAT

AUC (R.U.)

Increased Total Cholesterol

*

0

50

100

150

200

CSL111 CSL111+LCAT

AUC (R.U.)

Increased Cholesteryl Ester

(* P<0.05) (* P<0.05)

OverviewOverview

HDL Biology

LCAT Biology



HDL Biology

LCAT Biology



rLCAT Phase 1 Study DesignrLCAT Phase 1 Study Design

Sex: 14 males and 2 females

Dx: (Pos. hx CHD or radiology or dyslipidemia)

Race: 12 Caucasian and 4 other races

Mean age: 67.6 years

Mean HDL-C: 37.6 mg/dL

Summary of rLCAT Safety DataSummary of rLCAT Safety Data

All subjects completed the study

No Serious Adverse Events

No infusion reactions

No clinically significant changes in clinical lab

tests, PE, vital signs, EKG attributable to drug

Two mild rashes possibly attributable to drug

All subjects completed the study

No Serious Adverse Events

No infusion reactions

No clinically significant changes in clinical lab

tests, PE, vital signs, EKG attributable to drug

Two mild rashes possibly attributable to drug

Dose Response rLCAT vs. HDL-CDose Response rLCAT vs. HDL-C

rLCAT acutely increased HDL-C in dose dependent mannerrLCAT acutely increased HDL-C in dose dependent manner

rLCAT Increases HDL-C and CErLCAT Increases HDL-C and CE

rLCAT treatment increases HDL-C and CE, which peaks at 12-24 h

but remains partially elevated after 7 days

rLCAT treatment increases HDL-C and CE, which peaks at 12-24 h

but remains partially elevated after 7 days

Non-denaturing Gel Electrophoresisof HDL after rLCAT Treatment

Non-denaturing Gel Electrophoresisof HDL after rLCAT Treatment

rLCAT transiently decreases pre-beta HDL and increases larger size HDL species

rLCAT increases apoA-I but noeffect on TG

rLCAT increases apoA-I but noeffect on TG

rLCAT treatment also increases apoA-I but is delayed comparedto HDL-C and persists longer

rLCAT transiently lowers LDL-C and then increases it by day 3

rLCAT transiently lowers LDL-C and then increases it by day 3

Peak in HDL-C levels following rLCAT precedes rise in LDL-C consistent with CETP mediated transfer of CE from HDL to LDL

Peak in HDL-C levels following rLCAT precedes rise in LDL-C consistent with CETP mediated transfer of CE from HDL to LDL

LCAT Mass vs. ABCA1 Dependent Cholesterol Efflux

LCAT Mass vs. ABCA1 Dependent Cholesterol Efflux

rLCAT transiently decreases ABCA1 dependent cholesterol efflux from plasma and then later increases cholesterol efflux

rLCAT transiently decreases ABCA1 dependent cholesterol efflux from plasma and then later increases cholesterol efflux

rLCAT vs. rHDL Infusion TherapyrLCAT vs. rHDL Infusion Therapy

Compared to rHDL, ACP-501 caused a higher and more sustained increase in HDL-C and more CE formation

Compared to rHDL, ACP-501 caused a higher and more sustained increase in HDL-C and more CE formation

OverviewOverview

HDL Biology

LCAT Biology

Pre-clinical animal models



HDL Biology

LCAT Biology

Pre-clinical animal models



Single Patient IND StudySingle Patient IND Study52‐yo male FLD patient recommended for dialysis

Stage 4/5 renal disease BUN 159 mg/dL Creatinine 5.56 mg/dL cystatin‐C 4.16 mg/L Urine protein 2307 mg/24hr

Very low HDL‐C < 5 mg/dLAnemia

Hgb 8.2 g/dLHct 24.7%

Severe Corneal opacities

52‐yo male FLD patient recommended for dialysisStage 4/5 renal disease BUN 159 mg/dL Creatinine 5.56 mg/dL cystatin‐C 4.16 mg/L Urine protein 2307 mg/24hr

Very low HDL‐C < 5 mg/dLAnemia

Hgb 8.2 g/dLHct 24.7%

Severe Corneal opacities

Safety TolerabilitySafety Tolerability

rLCAT was well‐tolerated with 1 non‐

attributable serious adverse event of recurrent

atrial fibrillation and no injection site

reactions/toxicities.

rLCAT was well‐tolerated with 1 non‐

attributable serious adverse event of recurrent

atrial fibrillation and no injection site

reactions/toxicities.

Effect rLCAT on FLD LipidsEffect rLCAT on FLD Lipids

rLCAT treatment normalized lipoproteins and % CE normalizedfor more about 1 week after single treatment at higher doses.

Effect rLCAT on HDL SubfractionsEffect rLCAT on HDL Subfractions

Longitudinal effect of rLCAT on FLD LipidsLongitudinal effect of rLCAT on FLD Lipids

One rLCAT treatment every 2 weeks significantly raised HDL-C above baseline andmaintained cholesteryl esters above threshold which should prevent LpX formation.

Longitudinal effect of rLCAT on FLD LipidsLongitudinal effect of rLCAT on FLD Lipids

One rLCAT treatment every 2 weeks significantly raised HDL-C above baseline andmaintained cholesteryl esters above threshold which should prevent LpX formation.

Effect rLCAT on Renal FunctionEffect rLCAT on Renal Function

rLCAT treatment appeared to modestly improve renal function.

Effect of rLCAT on AnemiaEffect of rLCAT on Anemia

rLCAT improves significantly improves anemia by decreasing

cholesterol content on RBCs

rLCAT improves significantly improves anemia by decreasing

cholesterol content on RBCs

Summary rLCAT Clinical TrialsSummary rLCAT Clinical Trials

rLCAT was safe and well-tolerated :

• No Serious adverse events

• 2 possibly attributable AEs: mild skin rashes

• No clinically significant attributable changes in clinical lab tests, physical exam, ECG parameters or vital signs

Plasma LCAT mass and activity increased with dose of rLCAT and returned to baseline by 48 hours

rLCAT was safe and well-tolerated :

• No Serious adverse events

• 2 possibly attributable AEs: mild skin rashes

• No clinically significant attributable changes in clinical lab tests, physical exam, ECG parameters or vital signs

Plasma LCAT mass and activity increased with dose of rLCAT and returned to baseline by 48 hours

Summary rLCAT Phase I Clinical TrialSummary rLCAT Phase I Clinical Trial

Evidence that RCT pathway is stimulated by rLCAT

• Rapid increases in HDL-C and cholesterol esterification

• Increased in vitro cholesterol efflux by plasma

• Transient decrease in LDL-C followed by and increase suggestive of CETP transfer of esterifiedcholesterol

rLCAT reverses biochemical abnormalities in FLD and support its use in future clinical trials to prevent or reverse renal disease in FLD

Evidence that RCT pathway is stimulated by rLCAT

• Rapid increases in HDL-C and cholesterol esterification

• Increased in vitro cholesterol efflux by plasma

• Transient decrease in LDL-C followed by and increase suggestive of CETP transfer of esterifiedcholesterol

rLCAT reverses biochemical abnormalities in FLD and support its use in future clinical trials to prevent or reverse renal disease in FLD

51th Year Anniversary of LCAT Discovery

51th Year Anniversary of LCAT Discovery

CollaboratorsCollaborators

Robert Shamburek

Marcelo Amar

Boris Vaisman

Lita Freeman

Xavier Rousset

Amar Sethi

Seth Thacker

Lusana Aslan

Alice Ossoli

Robert Shamburek

Marcelo Amar

Boris Vaisman

Lita Freeman

Xavier Rousset

Amar Sethi

Seth Thacker

Lusana Aslan

Alice Ossoli


Bruce Auerbach

Brian Kraus

Reyn Homan

Becky Baker

Bruce Auerbach

Brian Kraus

Reyn Homan

Becky Baker

AstraZeneca(Alpha Core Pharma)

Tufts Univ. Ernie Schaeffer

Bella Azlos

Vascular Strategies

Steve Adelman

Questions?

Alan T. Remaley M.D., Ph.D.National Institutes of HealthBuilding 10, Room 2C-433Bethesda, MD 20892-1508 USATel: 301-402-9796Fax: 301-402-1885email: [email protected]

Why not just clean out your pipeswith rLCAT Therapy?

Documents

HDL Review: Translating new findings on LCAT into novel ... · HDL Review: Translating new findings on LCAT into novel therapies Alan T. Remaley, MD, PhD Lipoprotein Metabolism Section