hdl review: translating new findings on lcat into novel ... · hdl review: translating new findings...
TRANSCRIPT
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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
National Institutes of Health
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DisclosuresDisclosures
Patent: NIH patent licensed to Alpha Core Pharma/AstraZeneca
Research Support: CRADA grant with Alpha Core Pharma/AstraZeneca
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OverviewOverview
HDL Biology
LCAT Biology
Phase I Clinical trial of rLCAT
Single Patient IND Treatment FLD
HDL Biology
LCAT Biology
Phase I Clinical trial of rLCAT
Single Patient IND Treatment FLD
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HDL Structure and Composition
Pre-beta Discoidal Shaped HDL
Alpha Spherical Shaped HDL
Initial Cholesterol Acceptor Delivers Cholesterol to Liver
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HDL Structure and Composition
Pre-beta Discoidal Shaped HDL
Alpha Spherical Shaped HDL
LCAT
Initial Cholesterol Acceptor Delivers Cholesterol to Liver
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Vaisar T, et al JCI 2007
HDL-Proteome HDL-Proteome
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HDL-LipidomeHDL-Lipidome
SterolsHDL
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Pleiotropic Anti-AtherogenicEffects Of HDL (“Good Cholesterol”)
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
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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
Pleiotropic Anti-AtherogenicEffects Of HDL (“Good Cholesterol”)
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Reverse Cholesterol Transport Pathway
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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
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Therapeutic lifestyle changes
Pharmacologic therapy
Statins
Fibrates
Niacin
CETP-inhibitors ?
Acute HDL Therapy ?
Management of low HDL-C
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ApoA-I Milano Infusion StudiesApoA-I Milano Infusion StudiesLimone sul Garde, Italy
Free SH groupImproved anti-oxidant
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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
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Figure adapted from PLATO Trial Cornel JH et al. Am Heart J 2012;164:334‐342
Recurrent CVD in ACS Patients
Statins
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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
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OverviewOverview
HDL Biology
LCAT Biology
Phase I Clinical trial of rLCAT
Single Patient IND Treatment FLD
HDL Biology
LCAT Biology
Phase I Clinical trial of rLCAT
Single Patient IND Treatment FLD
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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
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mature HDL
nascent HDL
LCAT ReactionLCAT Reaction
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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.
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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
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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
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LpX Features
Lp-XLp-X
Bilayer or multi-lamellar complex of phospholipids
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
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LpX Features
Lp-XLp-X
Bilayer or multi-lamellar complex of phospholipids
Bilayer or multi-lamellar complex of phospholipids
Aqueous core
Aqueous core
LpX and Renal DiseaseLpX and Renal Disease
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
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LpX Cell Uptake StudiesLpX Cell Uptake StudiesElectron microscopy of LpX
Mesangial cell uptake of LpX
Lipoprotein Electrophoresis
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LpX Activates the InflammasomeLpX Activates the InflammasomeInflammasome Activation Pathway
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LpX Activates the InflammasomeLpX Activates the InflammasomeInflammasome Activation Pathway
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LpX Activates the InflammasomeLpX Activates the InflammasomeInflammasome Activation Pathway
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LpX Activates the InflammasomeLpX Activates the InflammasomeInflammasome Activation Pathway
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LpX and Renal DiseaseLpX and Renal Disease
Proteinuria (Albumin/creat. ratio) Fold Increase in Nephrotoxic Genes
LpX injection in LCAT KO mice causes proteinuria and inducesgenes associated with nephrotoxicity.
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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
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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 Pre-beta HDL high in CHD
Increased Pre-beta HDL andDecreased LCAT in CHD Subjects
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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 Pre-beta HDL high in CHD
pre-beta HDL inversely related to LCAT
Increased Pre-beta HDL andDecreased LCAT in CHD Subjects
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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
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
Carotid MRI
http://yourimt.com/images/707_PLAQUE.JPG
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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
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
Carotid MRI
http://yourimt.com/images/707_PLAQUE.JPG
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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.
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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
Rousset X, et al. J Pharmacol Exp Ther. 2010;335(1):140-148.
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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)
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
Rousset X, et al. J Pharmacol Exp Ther. 2010;335(1):140-148.
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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)
Volume of Elution (mL)Volume of Elution (mL)
0
5
10
15
20
10 20 30 40
Intravenous rLCAT Rapidly CorrectsLipoprotein Profile in LCAT KO MiceIntravenous rLCAT Rapidly Corrects
Lipoprotein Profile in LCAT KO Mice
Rousset X, et al. J Pharmacol Exp Ther. 2010;335(1):140-148.
24hLDLLP-X
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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
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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
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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
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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
**
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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
Both HDL and LCAT are rate limiting
(* P<0.05 above baseline)
Both HDL and LCAT are rate limiting
(* P<0.05 above baseline)
**
**
*
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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
CSL-111+ rLCAT dose response
Both HDL and LCAT are rate limiting and show a synergistic effecton cholesterol esterification.
(* P<0.05 above baseline)
Both HDL and LCAT are rate limiting and show a synergistic effecton cholesterol esterification.
(* P<0.05 above baseline)
**
*
**
**
*
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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)
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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)
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OverviewOverview
HDL Biology
LCAT Biology
Phase I Clinical trial of rLCAT
Single Patient IND Treatment FLD
HDL Biology
LCAT Biology
Phase I Clinical trial of rLCAT
Single Patient IND Treatment FLD
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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
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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
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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
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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
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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
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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
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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
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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
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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
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OverviewOverview
HDL Biology
LCAT Biology
Pre-clinical animal models
Phase I Clinical trial of rLCAT
Single Patient IND Treatment FLD
HDL Biology
LCAT Biology
Pre-clinical animal models
Phase I Clinical trial of rLCAT
Single Patient IND Treatment FLD
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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
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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.
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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.
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Effect rLCAT on HDL SubfractionsEffect rLCAT on HDL Subfractions
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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.
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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.
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Effect rLCAT on Renal FunctionEffect rLCAT on Renal Function
rLCAT treatment appeared to modestly improve renal function.
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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
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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
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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
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51th Year Anniversary of LCAT Discovery
51th Year Anniversary of LCAT Discovery
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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
National Institutes of Health
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
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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?