VERSION: 1 2 3 4 5 6 7 8 9
Recent ACS
Trial with CETP inhibitor
A phase III,
double-blind, randomized, placebo-controlled study to evaluate the effects of
RO4607381 on cardiovascular (CV) risk in stable CHD (Coronary Heart Disease)
patients with a documented recent Acute Coronary Syndrome (ACS) (high-risk, non
ST elevation ACS, non ST elevation myocardial infarction {NSTEMI}, or ST
elevation myocardial infarction {STEMI}
Sponsor: Roche
Study Drug: RO4607381 300 mgm tabs (Subjects will take 2 tabs with dinner daily)
Protocol:
Phase: III
CRO: Quintiles
Site #:
Screening #
CRA:
Medical Monitor:
Study Duration: Minimum 2 years
Study
Ends Recruitment: After 1,600 events
occur
Central Lab:
Central EKG:
15,600 Subjects / Sites → Subjects per Site R
Investigator Meeting
VERSION: 1 2 3 4 5 6 7 8 9
Recent ACS
Trial with CETP inhibitor
Target Population: Patients recently hospitalized for ACS, and whose residual CV risk may benefit from an increase in HDL. ACS will be defined by one of the following events:
1. Troponin-I > 2 x ULN plus at least one of the following:
a. Symptoms of ischemia within 48H of the MI
b. New EKG findings
c. Imaging evidence of new wall motion abnormalities
2. High Risk NSTE (Non-ST Elevation) ACS: diagnosis will require Hospital admission with ischemia symptoms with an accelerating tempo in the prior 48 H and/or prolonged (at least 20 min.) rest CP AND either a or b (both OK also):
a. A set of at least 2 Troponins drawn at least 6H apart, with at least one value elevated between 1 and 2x ULN and demonstrating a rising or falling pattern
b. New EKG findings and at least one of the following:
i. 50% stenosis of an epicardial coronary artery
ii. Positive ETT indicating reversible ischemia
iii. Presence of Pathological Q-Waves on EKG
IC
Age:
> 45
Ischemia free x 1 wk prior to R
V2 TG < 400
Statin Use
EC
V2 Hgb
< 10
Index ACS due to Uncontrolled HTN
BP > 180/110 by end of the 4-6 wk Placebo run-in
V2 HgbA1C > 10
V2 LFT > 1.5; Creatinine > 2.2
Niacin, Fibrates or Bile Acid Sequestrants
Prior
exposure to Torcetrapib or RO4607381 within past yr
CA x 3
yrs
VERSION: 1 2 3 4 5 6 7 8 9
Recent ACS
Trial with CETP inhibitor
V1 D -70 to -31 Lab,
Screen
V2 D -42 to -3
Lab
V3 D 1 Lab, R
V4 M 1 Lab
V5 M 3 Lab
V6 M 6 Lab
V7 M 9 Lab
V8 M 12 Lab
V9 M 16 Lab
V10 M 20 Lab
V11 M 24 Lab
V12 M 28 Lab
V13 M 32 Lab
V14 M 36 Lab
V15 EOT (End of Treatment)
VERSION: 1 2 3 4 5 6 7 8 9
Recent ACS
Trial with CETP inhibitor
Questions
- Indemnification: The sponsor likes to
be held harmless for acts of negligence that the PI commits that are
solely the error of the treating physician. For example, if a PI treats a
subject's UTI during a study and the patient suffers an injury from this,
it is squarely the PI’s fault and it would not be proper for the sponsor
to sustain an injury by being held liable for it. However, we are aware of
a BP study where the PI followed the protocol and had the subject wash off
their BP medication and suffer a stroke from the lack of the proper BP
treatment. The sponsor forced the PI to use his medical malpractice
insurance to satisfy the claim for medical malpractice. Clearly, this was
an injustice as the PI was following the protocol precisely and even
though it is unusual for a stroke to occur during wash out, it certainly
does not immunize the subject from having a stroke. When a stroke occurs
during wash out it is important that the sponsor take responsibility for
the liability. This should be true of any injury ex officio of changing a
subject’s treatment while following the protocol. If this drug causes
respiratory depression, are you going to be fully liable?
- Study Fully Populated
Causing Premature Early Termination: As sites advertise for patients to
become research volunteers, the people that are good enough to step
forward to join, wash off of their existing treatment, come to our site
and devote a significant amount of their time with blood draws, signing
paperwork and allowing the experimental protocol to progress, should be
guaranteed that the sponsor will not prematurely shut down the study
because they have obtained the needed number of subjects. That is, it
should be the sponsor’s responsibility to do the number crunching early
enough not to allow subjects to sign consent and undergo early termination
just because the study is now considered full enough. This is not ethical
or in the patients best interest. All subjects who sign consent must be
allowed to continue thru the protocol if they are continuing to fulfill
criteria regardless of how many subjects are in the trial.
- Reimbursement for
Screen Failures: Sites are taking finincial risk in the name of enrolling only those
subjects who will successfully complete the trial. Sponsors need to
recognize that this significantly slows enrollment. When we see a subject
who may qualify but then again may not, we would be hesitant to enroll in
that study. Since the exclusivity clock starts clicking as soon as the
drug is patented it is imperative that the studies are done as quickly as
possible. Sites that are rewarded for each piece of work they legitimately
perform will have to enroll better. Sites with too high screen failure
rates can be turned off to ensure the proper motivation to enroll high
quality subjects is present.
- Initial Fasting Labs
and Informed Consent: Many trials have visit one with fasting labs and signing informed
consent on the same day. This is less desirable than having a visit one
where the subject presents non-fasting for the purpose of signing the
informed consent form and having the study discussed with them. The
potential problem is with a diabetic. If a diabetic patient answers an ad
and presents fasting for visit one, a research related procedure was done
prior to signing the ICF (Informed Consent Form). They could get a low
sugar reaction while driving to the site and suffer an MVA. They may also
feel pressured to sign the ICF quickly as they know when the do so they
will finally get something to eat. This can be most safely avoided by
allowing subjects to sign the ICF on a pre-visit one or by writing
protocols that don't have fasting labs as a visit one procedure. Although
I understand V1 labs are non-fasting for this trial.
- Recruitment: Too often recruitment is an oversight thought to be the sites problem dealt with at a time other than the beginning of the trial. It becomes the sponsor’s problem after many sites under enroll. Wise sponsors address recruitment up front at the investigator’s meeting and include recruitment agencies. One budget neutral solution is for the sponsor to push to make available an IRB approved ad for those using the central IRB at the very beginning of the trial. In addition use of centralized recruitment agencies like Pi and Acurian can very useful and cost effective. All our databases could be larger. A small minority of people step forward to become research subject volunteers. Planning for recruitment issues at the beginning is much better than catch up at the end.
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Pathway of
cholesterol biosynthesis. Synthesis begins with the transport of acetyl-CoA
from the mitochondrion to the cytosol. The rate limiting step occurs at the
3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, HMGR catalyzed step. The
phosphorylation reactions are required to solubilize the isoprenoid
intermediates in the pathway. Intermediates in the pathway are used for the
synthesis of prenylated proteins, dolichol, coenzyme Q and the side chain of
heme a. |
The acetyl-CoA utilized for cholesterol biosynthesis is derived
from an oxidation reaction (eg, fatty acids or pyruvate) in the mitochondria
and is transported to the cytoplasm by the same process as that described for fatty acid
synthesis (see the Figure below). Acetyl-CoA can also be derived
from cytoplasmic oxidation of ethanol by acetyl-CoA synthetase. All the
reduction reactions of cholesterol biosynthesis use NADPH as a cofactor. The
isoprenoid intermediates of cholesterol biosynthesis can be diverted to other
synthesis reactions, such as those for dolichol (used in the synthesis of N-linked
glycoproteins, coenzyme Q (of the oxidative
phosphorylation) pathway or the side chain of heme a.
Additionally, these intermediates are used in the lipid
modification of some proteins.
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Pathway for
the movement of acetyl-CoA units from within the mitochondrion to the
cytoplasm for use in lipid and cholesterol biosynthesis. Note that the
cytoplasmic malic enzyme catalyzed reaction generates NADPH which can be used
for reductive biosynthetic reactions such as those of fatty acid and
cholesterol synthesis. |
Acetyl-CoA units are converted to mevalonate by a series of
reactions that begins with the formation of HMG-CoA.
Unlike the HMG-CoA formed during ketone body synthesis in the mitochondria,
this form is synthesized in the cytoplasm. However, the pathway and the
necessary enzymes are the same as those in the mitochondria. Two moles of
acetyl-CoA are condensed in a reversal of the thiolase reaction, forming
acetoacetyl-CoA. Acetoacetyl-CoA and a third mole of acetyl-CoA are converted
to HMG-CoA by the action of HMG-CoA synthase. HMG-CoA is converted to mevalonate by HMG-CoA reductase, HMGR (this
enzyme is bound in the endoplasmic reticulum, ER). HMGR absolutely requires
NADPH as a cofactor and two moles of NADPH are consumed during the conversion
of HMG-CoA to mevalonate.
The reaction catalyzed by HMGR is the rate limiting step of
cholesterol biosynthesis, and this enzyme is subject to complex regulatory
controls.
Mevalonate is then activated by three successive
phosphorylations, yielding 5-pyrophosphomevalonate. In addition to activating mevalonate,
the phosphorylations maintain its solubility, since otherwise it is insoluble
in water. After phosphorylation, an ATP-dependent decarboxylation yields isopentenyl pyrophosphate, IPP, an activated isoprenoid
molecule. Isopentenyl pyrophosphate is in equilibrium with its isomer, dimethylallyl pyrophosphate, DMPP. One molecule of IPP condenses with
one molecule of DMPP to generate geranyl pyrophosphate, GPP. GPP further condenses with another IPP molecule to yield farnesyl pyrophosphate, FPP. Finally, the NADPH-requiring enzyme,
squalene synthase catalyzes the head-to-tail condensation of two molecules of
FPP, yielding squalene (squalene synthase also is tightly associated with the
endoplasmic reticulum). Squalene undergoes a two step cyclization to yield lanosterol. The first reaction is catalyzed by
squalene monooxygenase. This enzyme uses NADPH as a cofactor to introduce
molecular oxygen as an epoxide at the 2,3 position of squalene. Through a
series of 19 additional reactions, lanosterol is converted to cholesterol.
Regulating Cholesterol
Synthesis
Normal healthy adults synthesize cholesterol at a rate of
approximately 1g/day and consume approximately 0.3g/day. A relatively constant
level of cholesterol in the body (150 - 200 mg/dL) is maintained primarily by
controlling the level of de novo synthesis. The level of cholesterol
synthesis is regulated in part by the dietary intake of cholesterol.
Cholesterol from both diet and synthesis is utilized in the formation of
membranes and in the synthesis of the steroid hormones and bile acids (see
below). The greatest proportion of cholesterol is used in bile acid synthesis.
The cellular supply of cholesterol is maintained at a steady
level by three distinct mechanisms:
·
1. Regulation of HMGR activity and levels
·
2. Regulation of excess intracellular
free cholesterol through the activity of acyl-CoA:cholesterol acyltransferase,
ACAT
·
3. Regulation of plasma cholesterol
levels via LDL receptor-mediated uptake and HDL-mediated reverse transport.
Regulation of HMGR activity is the primary means for controlling
the level of cholesterol biosynthesis. The enzyme is controlled by four
distinct mechanisms: feed-back inhibition, control of gene expression, rate of
enzyme degradation and phosphorylation-dephosphorylation.
The first three control mechanisms are exerted by cholesterol
itself. Cholesterol acts as a feed-back inhibitor of pre-existing HMGR as well
as inducing rapid degradation of the enzyme. The latter is the result of
cholesterol-induced polyubiquitination of HMGR and its degradation in the
proteosome (see proteolytic
degradation below). This ability of cholesterol is a consequence of
the sterol sensing domain,
SSD of HMGR. In
addition, when cholesterol is in excess the amount of mRNA for HMGR is reduced
as a result of decreased expression of the gene. The mechanism by which
cholesterol (and other sterols) affects the transcription of the HMGR gene is
described below under regulation of
sterol content.
Regulation of HMGR through covalent modification occurs as a
result of phosphorylation and dephosphorylation. The enzyme is most active in
its unmodified form. Phosphorylation of the enzyme decreases its activity. HMGR
is phosphorylated by AMP-activated protein kinase, AMPK (this is not the same as cAMP-dependent protein kinase,
PKA). AMPK itself is activated via phosphorylation. The phosphorylation of AMPK
is catalyzed by one or more AMPK kinases (AMPKKs). Visit AMPK: The Master Metabolic Regulator
for more detailed information on the role of AMPK in regulating metabolism.
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Regulation
of HMGR by covalent modification. HMGR is most active in the dephosphorylated
state. Phosphorylation is catalyzed by AMP-activated protein kinase, AMPK,
(used to be termed HMGR kinase), an enzyme whose activity is also regulated
by phosphorylation. Phosphorylation of AMPK is catalyzed by AMPK kinase
(AMPKK). Hormones such as glucagon and epinephrine negatively affect
cholesterol biosynthesis by increasing the activity of the inhibitor of phosphoprotein
phosphatase inhibitor-1, PPI-1. Conversely, insulin stimulates the removal of
phosphates and, thereby, activates HMGR activity. Additional regulation of
HMGR occurs through an inhibition of its' activity as well as of its'
synthesis by elevation in intracellular cholesterol levels. This latter
phenomenon involves the transcription factor SREBP described below. |
The activity of HMGR is additionally controlled by the cAMP
signaling pathway. Increases in cAMP lead to activation of cAMP-dependent protein
kinase, PKA. In the context of HMGR regulation, PKA phosphorylates
phosphoprotein phosphatase inhibitor-1 (PPI-1) leading to an increase in its'
activity. PPI-1 can inhibit the activity of numerous phosphatases including
protein phosphatase 2C and HMG-CoA reductase phosphatase which remove
phosphates from AMPK and HMGR, respectively. This maintains AMPK in the
phosphorylated and active state, and HMGR in the phosphorylated and inactive
state. As the stimulus leading to increased cAMP production is removed, the
level of phosphorylations decreases and that of dephosphorylations increases.
The net result is a return to a higher level of HMGR activity.
Since the intracellular level of cAMP is regulated by hormonal
stimuli, regulation of cholesterol biosynthesis is hormonally controlled.
Insulin leads to a decrease in cAMP, which in turn activates cholesterol
synthesis. Alternatively, glucagon and epinephrine, which increase the level of
cAMP, inhibit cholesterol synthesis.
The ability of insulin to stimulate, and glucagon to inhibit,
HMGR activity is consistent with the effects of these hormones on other
metabolic pathways. The basic function of these two hormones is to control the
availability and delivery of energy to all cells of the body.
Long-term control of HMGR activity is exerted primarily through
control over the synthesis and degradation of the enzyme. When levels of
cholesterol are high, the level of expression of the HMGR gene is reduced.
Conversely, reduced levels of cholesterol activate expression of the gene.
Insulin also brings about long-term regulation of cholesterol metabolism by
increasing the level of HMGR synthesis.
Proteolytic Regulation
of HMG-CoA Reductase
The stability of HMGR is regulated as the rate of flux through
the mevalonate synthesis pathway changes. When the flux is high the rate of
HMGR degradation is also high. When the flux is low, degradation of HMGR
decreases. This phenomenon can easily be observed in the presence of the statin drugs.
HMGR is localized to the ER and like SREBP (see below) contains
a sterol-sensing domain, SSD. When sterol levels increase in cells there is an
concomitant increase in the rate of HMGR degradation. The degradation of HMGR
occurs within the proteosome, a multiprotein complex dedicated to protein
degradation. The primary signal directing proteins to the proteosome is
ubiquitination. Ubiquitin is a 7.6kDa protein that is covalently attached to
proteins targeted for degradation by ubiquitin ligases. These enzymes attach
multiple copies of ubiquitin allowing for recognition by the proteosome. HMGR
has been shown to be ubiquitinated prior to its degradation. The primary sterol
regulating HMGR degradation is cholesterol itself. As the levels of free
cholesterol increase in cells, the rate of HMGR degradation increases.
The Utilization of
Cholesterol
Cholesterol is transported in the plasma predominantly as
cholesteryl esters associated with lipoproteins.
Dietary cholesterol is transported from the small intestine to the liver within
chylomicrons. Cholesterol synthesized by the liver, as well as any dietary
cholesterol in the liver that exceeds hepatic needs, is transported in the
serum within LDLs. The liver synthesizes VLDLs and these are converted to LDLs
through the action of endothelial cell-associated lipoprotein lipase.
Cholesterol found in plasma membranes can be extracted by HDLs and esterified
by the HDL-associated enzyme LCAT. The cholesterol acquired from peripheral
tissues by HDLs can then be transferred to VLDLs and LDLs via the action of
cholesteryl ester transfer protein (Apo-D) which is associated with HDLs. Reverse cholesterol transport allows peripheral cholesterol to be
returned to the liver in LDLs. Ultimately, cholesterol is excreted in the bile
as free cholesterol or as bile salts following conversion to bile acids in the
liver.
Regulation of Cellular
Sterol Content
The continual alteration of the intracellular sterol content
occurs through the regulation of key sterol synthetic enzymes as well as by
altering the levels of cell-surface LDL receptors. As cells need more sterol
they will induce their synthesis and uptake, conversely when the need declines
synthesis and uptake are decreased. Regulation of these events is brought about
primarily by sterol-regulated transcription of key rate limiting enzymes and by
the regulated degradation of HMGR. Activation of transcriptional control occurs
through the regulated cleavage of the membrane-bound transcription factor sterol regulated element binding protein,
SREBP. As discussed
above, degradation of HMGR is controlled by the ubiquitin-mediated pathway for
proteolysis.
Sterol control of transcription affects more than 30 genes
involved in the biosynthesis of cholesterol, triacylglycerols, phospholipids
and fatty acids. Transcriptional control requires the presence of an octamer
sequence in the gene termed the sterol regulatory element, SRE-1. It has been shown that SREBP is the transcription factor
that binds to SRE-1 elements. It turns out that there are 2 distinct SREBP
genes, SREBP-1 and SREBP-2. In addition, the SREBP-1 gene encodes 2 proteins,
SREBP-1a and SREBP-1c as a consequence of alternative exon usage. All 3
proteins are proteolytically regulated by sterols. Full-length SREBPs have
several domains and are embedded in the membrane of the endoplasmic reticulum
(ER). The N-terminal domain contains a transcription
factor motif of the basic helix-loop-helix (bHLH) type that is
exposed to the cytoplasmic side of the ER. There are 2 transmembrane spanning
domains followed by a large C-terminal domain also exposed to the cytosolic
side. When sterols are scarce, cleavage of the full-length SREBP takes place
with the result being that the N-terminal bHLH motif is released into the
cytosol. The bHLH domain then migrates to the nucleus to direct transcription.
Conversely, when sterols are abundant, cleavage of SREBP is inhibited. To
control the level of SREBP-mediated transcription, the soluble bHLH domain is
itself subject to rapid proteolysis.
The cleavage of SREBP is carried out by 2 distinct enzymes, one
of which is regulated by sterols. The regulated cleavage occurs in the lumenal
loop between the 2 transmembrane domains. This cleavage is catalyzed by site-1 protease, S1P. High sterol content blocks the
activity of S1P. The second cleavage, catalyzed by site-2 protease, S2P, occurs in the first transmembrane
span, leading to release of active SREBP. In order for S2P to act on SREBP,
site-1 must already have been cleaved.
Additional studies on sterol-regulated gene expression
demonstrated that cleavage of SREBP by S1P is controlled by the level and
action of an additional protein termed, SREBP cleavage-activating protein, SCAP. SCAP is a large protein also found in
the ER membrane and contains at least 8 transmembrane spans. The C-terminal
portion, which extends into the cytosol, has been shown to interact with the
C-terminal domain of SREBP. This C-terminal region of SCAP contains 4 motifs
called WD40 repeats. The WD40 repeats are required for
interaction of SCAP with SREBP. Interestingly, the N-terminus of SCAP,
including membrane spans 2-6, resembles HMGR which itself is subject to
sterol-stimulated degradation (see above). This shared motif is called the sterol sensing domain, SSD. Several proteins whose functions
involve sterols also contain the SSD. These include patched, an important development regulating receptor whose
ligand, hedgehog, is modified by attachment of
cholesterol and the Neimann Pick C1
(NPC1) protein which is involved in cholesterol transport in the
secretory pathway. NPC1 is one of several genes whose activities, when
disrupted, lead to severe neurological dysfunction.
The function of SCAP is to positively stimulate S1P-mediated cleavage
of SREBP. The function of sterols is to inhibit this positive action of SCAP.
The activity of SCAP involves movement from the ER to the Golgi and back.
Because the C-terminus of SCAP interacts with SREBP, movement of SCAP takes
SREBP along for the ride. When sterols are low, SCAP and SREBP move to the
Golgi. This transit is required for SREBP cleavage as S1P is Golgi-localized.
When sterols are high, movement of SCAP is halted. Thus, the overall effect of
sterols is to regulate the ability of SCAP to present SREBP to S1P.
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Diagrammatic representation of the
interactions between SREBP and SCAP in the membrane of the ER |
Bile
Acids Synthesis and Utilization
The end products of cholesterol utilization are the bile acids,
synthesized in the liver. Synthesis of bile acids is one of the predominant
mechanisms for the excretion of excess cholesterol. However, the excretion of
cholesterol in the form of bile acids is insufficient to compensate for an
excess dietary intake of cholesterol.
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Synthesis of
the 2 primary bile acids, cholic acid and chenodeoxycholic acid. The reaction
catalyzed by the 7a-hydroxylase
is the rate limiting step in bile acid synthesis. Conversion of 7a-hydroxycholesterol to the bile acids
requires several steps not shown in detail in this image. Only the relevant
co-factors needed for the synthesis steps are shown. |
The most abundant bile acids in human bile are chenodeoxycholic acid (45%) and cholic acid (31%). These are referred to as the primary bile acids. Within the intestines the primary
bile acids are acted upon by bacteria and converted to the secondary bile acids, identified as deoxycholate (from
cholate) and lithocholate (from chenodeoxycholate). Both primary and secondary
bile acids are reabsorbed by the intestines and delivered back to the liver via
the portal circulation.
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Structure of the conjugated cholic
acids |
Within the liver the carboxyl group of primary and secondary
bile acids is conjugated via an amide bond to either glycine or taurine before
their being resecreted into the bile canaliculi. These conjugation reactions
yield glycoconjugates and tauroconjugates, respectively. The bile canaliculi join with the bile ductules,
which then form the bile ducts. Bile acids are carried from the liver through
these ducts to the gallbladder, where they are stored for future use. The
ultimate fate of bile acids is secretion into the intestine, where they aid in
the emulsification of dietary lipids. In the gut the glycine and taurine
residues are removed and the bile acids are either excreted (only a small
percentage) or reabsorbed by the gut and returned to the liver. This process of
secretion from the liver to the gallbladder, to the intestines and finally
reabsorbtion is termed the enterohepatic
circulation.
Clinical Significance
of Bile Acid Synthesis
Bile acids perform four physiologically significant functions:
·
1. Their synthesis and subsequent
excretion in the feces represent the only significant mechanism for the
elimination of excess cholesterol.
·
2. Bile acids and phospholipids
solubilize cholesterol in the bile, thereby preventing the precipitation of
cholesterol in the gallbladder.
·
3. They facilitate the digestion of
dietary triacylglycerols by acting as emulsifying agents that render fats
accessible to pancreatic lipases.
·
4. They facilitate the intestinal
absorption of fat-soluble vitamins.