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Past Issues
Volume 16, number 1
January 2002
Contents
Express Yourself!
Too much of a good gene may slow recovery
The ABCs of Proteins
A primer on the body's Jack-of-all-trades
P-glycoprotein and AIDS
P-gp's relevance to HIV disease
An Islamic School Tackles HIV
A fascinating report from Nigeria
Start SMART!
Not just another acronym, it's something that's been needed for
years
The Genetic Edge
By Bob Huff
It's well understood that unwavering adherence to one's antiretroviral
therapy (ART) regimen gives the best chance of keeping drug levels
high enough to sustain suppression of viral replication and allow
immune recovery to take root. Yet perfect performance is not a sure
bet. Some people who never miss a dose fail to see their T-cells
rise — even though their viral load stays within moderate levels.
Others have all the luck: their virus goes undetectable and stays
there for years while their CD4 counts hover near quadruple digits
— all with side effects no more serious than occasional diarrhea.
Many clinicians have long thought that some genetic advantage must
be at work, but there's been little proof and no way to tell who's
drawn the lucky DNA.
Now comes a report from researchers associated with the Swiss HIV
Cohort Study identifying a link between the gene for a protein responsible
for pumping toxic interlopers out of cells and the magnitude of
CD4 cell count rise within six months of starting ART. The gene
is called MDR1 (for multi-drug resistance) and it codes for a transporter
protein called P-glycoprotein (P-gp). In the Swiss study, people
with very low amounts of P-gp had a significantly better immunological
response to ART containing nelfinavir or efavirenz than those who
had moderate or high levels of the protein. Specifically, the best
CD4 response was linked to having a double lack (TT) of the MDR1
gene for functional P-gp in one's inherited DNA; poor response was
linked to having a double dose (CC) of the gene, and moderate response
was associated with having a single dose (CT).
P-glycoprotein at The Gates
P-gp is a cell surface protein best known for its ability to evict
a long list of anti-cancer drugs from the interior of a cell. (See
P-glycoprotein and HIV elsewhere in this issue) It seems
to perform an admirably protective function by bouncing unwanted
toxic chemicals from places they shouldn't be, yet it is infamous
because of its propensity to thwart the efficacy of chemotherapeutic
cancer regimens. The HIV protease inhibitors can also be targets
of P-gp's xenophobic urge to purge. P-glycoprotein tends to be found
wherever important and vulnerable tissues need to be sheltered from
poisons. P-gp is on duty at the blood-brain barrier, the placenta,
the testis, and the gut, among other sites. P-gp may also be one
of the factors that allow HIV to replicate within those protected
compartments, safely out of the reach of protease inhibitors. The
reverse transcriptase inhibitors don't seem to be directly affected
by P-gp.
Significant amounts of P-gp have also been detected in stem cells
that eventually give rise to blood cells, including lymphocytes
such as CD4 and macrophages. P-gp is also found on mature T-cells
that have exited the thymus, and are especially common on the so-called
naive subset of CD4 cells that are often quickly depleted by HIV
infection and difficult to replenish. In this study, naive CD4 cells
also recovered at a faster pace in people with stunted P-gp expression.
The authors speculate that "the immunological benefit noted in individuals
with the MDR1 TT genotype and low expression of P-glycoprotein could
suggest enhanced penetration of antiretroviral drugs in cell populations
susceptible to HIV-1 infection, in infected lymphocytes and in pharmacological
sanctuaries." In other words, P-gp may be responsible for trying
to keep drugs out of the very cells and reservoirs that need them
the most. If so, people with the least amount of P-gp may have a
genetic advantage for fighting HIV because their cells put up fewer
barriers to letting drugs in to do their job.
Alleles on Wheels
Sexual reproduction insures that the genetic pot gets stirred with
each generation. Everyone has a duplicate set of chromosomes. At
conception, one set of chromosomes from the egg and another from
the sperm combine to give the embryo a new genome. Depending on
which genes from which chromosome actually become translated into
proteins, an offspring accumulates the unique set of phenotypes
that makes it an individual. Each paired gene in a set of chromosomes
is called an allele. If the same gene for a particular trait resides
on both chromosomes, then that gene product may be doubly expressed.
But if the genes differ and make different proteins, then there
may be a contest for dominance to see which allele is actually expressed
as a phenotype.
In the case of P-gp, there is evidence that if there is a "C" nucleotide
code at position number 3,435 in the MDR1 gene sequence, then normal
copies of functional P-gp can be produced at a normal pace. But
if the normal "C" at that position accidentally turns into a "T"
nucleotide, then the production of P-gp is greatly slowed. Some
people have one normal "C" MDR1 allele in one chromosome and a mutant
"T" allele in the other chromosome. These mixed alleles are represented
as CT and together they produce a moderate amount of P-gp. If the
person has two normal copies (CC) of MDR1, then they produce a large
amount of P-gp. Not surprisingly, if they have a TT genotype, they
produce very little P-gp. In the Swiss study, the people with the
TT genes had the best response to HIV treatment, possibly because
they have very little P-gp at work keeping nelfinavir out of their
cells.
The MDR1 gene is not the first allele identified that has significance
for HIV disease. There are several genetic factors that may affect
the likelihood of becoming infected with HIV or of how virulent
the course of one's disease can be. Another cell surface protein,
CCR5, is used as a coreceptor along with CD4 when HIV attempts to
bind to and enter a new cell. A few individuals carry alleles for
a double lack of CCR5 and it has been proposed that these lucky
people may be highly impervious to HIV infection.
Racial and Ethnic Differences in MDR1 Distribution?
One cautionary note is that the neat 1:2:1 distribution of the
MDR1 alleles shown in this population may be peculiar to European
whites. Worldwide, there seems to be a wide range in the natural
variation of allelic frequencies of MDR1 that may affect P-gp function.
Comparative population studies of gene frequencies have reported
that the CC double dose of MDR occurs with a frequency exceeding
80 percent among some regional populations of black Africans, while
the TT genotype was correspondingly rare. In another study, African-Americans
had a CC allelic frequency of 84 percent compared to a frequency
of 34 percent among Southwest Asians. The Swiss authors warn, "This
variation could lead to different patterns of HIV-1 disease evolution
and responses to antiretroviral treatment in human populations."
Studies need to be immediately undertaken to determine the impact
that varying levels of MDR1 expression may have on treatment efficacy
as antiretroviral therapies become more available in Africa and
in India.
New Questions
The suggestion that improved intracellular levels of nelfinavir
and efavirenz are responsible for the improved immunological response
raises questions about the possibility of tinkering with nature
to inhibit P-gp artificially so more drug molecules can get inside
and stay inside cells where they are needed. Several common drugs
are effective inhibitors of P-gp, including the HIV protease inhibitors
nelfinavir, ritonavir and saquinavir. But the potential complexity
of interactions with a host of other enzymes calls for much additional
investigation before attempting to leverage P-gp activity in routine
clinical practice.
In their study design, the Swiss researchers were careful to examine
whether other genes coding for other proteins involved in drug metabolism
could be producing the observed responses to treatment. Yet with
the available data, only MDR1 seemed to show the CD4 linkage. In
a seeming paradox, individuals with the TT allele had lower blood
concentrations of the drugs than those with the genes for greater
P-gp production. Furthermore, while MDR1 expression was significantly
associated with T-cell response after six months treatment, the
magnitude of viral suppression achieved during the same time was
roughly equivalent no matter which pairing of genes an individual
had. The authors note, however, that the study lacked the resolution
to track viral decay rates during the first weeks and months of
therapy. Indeed, it remains to be seen if the differences in immune
response will continue to be seen over longer periods of time. Another
nagging question is why immune benefit was also observed for patients
who received efavirenz, a drug not thought to be directly expelled
by P-gp.
Although the impact of P-gp on intracellular drug concentrations
provides an attractive explanation for this study's results, there
may be other explanations for the immunological effect seen. The
authors speculate that P-gp could actually be exerting its influence
by regulating the accumulation of certain chemokine proteins involved
in the process of CD4 cell destruction downstream from direct HIV
activity. It's also been noted that P-glycoprotein tends to localize
in lipid rafts on a cell's surface in close proximity to other proteins.
These rafts also harbor the CD4 and CCR5 proteins that HIV uses
to attach and enter new target cells. Again, more research is needed
to understand if there are interactions among these cell surface
proteins that could possibly inhibit or facilitate HIV infection.
Bottom Line
This latest episode in the unfolding story of P-gp is fascinating
and gives a glimpse into the future potential of pharmacogenetics.
Eventually, genetic tests might be used to guide the selection of
an individual's optimal drug regimen. While there may be no impact
on clinical practice at this point, here is another clear call to
urgently scale up our investigations into the complex pharmacology
of HIV treatment and the implications for treating people from diverse
populations.
Fellay J, et al. Response to antiretroviral treatment in HIV-1-infected
individuals with allelic variants of the multidrug resistance transporter
1: a pharmacogenetics study. Lancet 2002; Jan 5; 359.
Other sources:
Kim RB, et al. Identification of functionally variant MDR1 alleles
among European Americans and African Americans. Clin Pharmacol Ther
2001 Aug, 70(2).
Schaeffeler E, et al. Frequency of C3435T polymorphism of MDR1
gene in African people. Lancet 2001; Aug 4; 358.
Ameyaw M, et al. MDR1 pharmacogenetics: frequency of the C435T
mutation in exon 26 is significantly influenced by ethnicity. Pharmacogenetics
2001;11.
Protein Primer
By Bob Huff
You've heard it said: Amino acids are the building blocks of life.
More literally, amino acids are the building blocks of proteins
— and proteins are the walls, doors, monorails and kitchen
sinks of life. A protein is made from a strand of amino acids strung
together like the various figures on a charm bracelet. Each amino
acid has an identity, properties and charm all its own. Some amino
acids are bulky or rigid while others are small or flexible. Some
amino acids are electrically charged and others are neutral. Some
amino acids prefer to be in water while others shun water and seek
to bundle together or imbed themselves in globs of fat.
With all of these various and competing properties, it's not surprising
that an amino acid in a strand can interact with its neighbors,
perhaps pushing one away which causes the string to bend. But it
can also interact with amino acids far along the string that come
into the neighborhood when the protein string curls back on itself.
To further complicate matters, an amino acid on one protein can
hook up with an amino acid on a completely separate protein, resulting
in multi-protein complexes.
Twist and Shout
All proteins start out as a simple string of amino acids but then,
as the string starts to flop around and interact with itself, it
begins to fold into clumps or twist into braids or take on any number
of shapes. The water-hating amino acids tend to clump together and
push their water-loving neighbors away. Amino acids hundreds of
positions apart on the protein string find themselves married together
in its folded state. These kinds of interactions help give the protein
its working form. To further differentiate the behavior of a protein,
some amino acids might attract and festoon themselves with various
sugars or bits of energized phosphorous molecules. With only 20
different amino acids — each with different properties, all
interacting with each other — proteins can take on an amazing
range of diversity in shape and function.
Proteins are all about function. Some proteins have structural
roles, acting like girders, belts, wires, tracks and zippers. Most
every protein is able to hook up to other molecules in some way,
with some acting specifically as connectors or adapters, similar
to fish hooks, sockets or Velcro. Other proteins act like doorbells
or mail slots that relay signals from one side of a cell to the
other. Enzymes are specialized proteins that behave like machines
and have moving parts such as grippers and cutters. Enzymes perform
their work on various chemicals or other proteins called substrates.
The enzyme stabilizes and speeds up the modification of substrates
by holding them steady while highly specific operations are performed.
Proteins can do every imaginable (and many as yet unimagined) job
in a living organism. Proteins can be described by their characteristic
sequences of amino acids, by their three-dimensional structures
or by the functions they perform. Just to complicate things, two
proteins with very similar activity or structures may have completely
different sequences. Not every protein has to fold up into its perfect
final form; some do their work by just flopping around. Others need
to be embedded into a cell's membrane before they truly come alive
and do their job. Some proteins only exist to guide other proteins
as they fold themselves into useful shapes. Proteins that don't
fold themselves correctly can be a source of disease. Broken or
defective proteins in a cell are usually quickly collected, taken
apart and recycled by — what else? — other proteins. Scientists
are using powerful computers to try to predict the behavior of strings
of amino acids when they start to clump together and curl up, but
the problem is so complex, they have only begun to scratch the surface.
HIV is Made of Proteins
The HIV protease enzyme is made of a protein with two main moving
parts hinged by a flexible sequence of amino acids. The job of the
HIV protease is to hold onto other HIV precursor proteins and cut
them apart between specific amino acids pairs. After the HIV substrate
proteins have been cut, they are free to fold themselves into the
various structural bits and machinery that make up a new HIV virus
particle. These new proteins may include parts of the inner and
outer shell of HIV as well as the HIV-enzymes reverse transcriptase,
integrase and protease itself. If HIV protease can be prevented
from cutting the HIV substrate protein in the right places, the
virus can't replicate.
Of course, this is what protease inhibitors (PI) are designed to
do. These drugs are little molecules that imitate the amino acid
sequence of the HIV substrate protein where protease is supposed
to make it's cut. But instead of cutting, the protease gets stuck
on the decoy molecule and everything stops. These drugs work fine
as long as the protease enzyme is made from its standard, out of
the box, string of amino acids. But if a few of the amino acids
have been changed, or if some amino acids are cut out or different
ones added, the protease starts behaving differently. Maybe the
altered protease starts to grab the drug molecule but then drops
it again before picking up a real HIV substrate and cutting it.
The altered protease is still not working at nearly its normal speed
but it is managing to pump out a small amount of fresh virus. This
process can just limp along until one day a new version of protease
shows up with another switched amino acid that now lets the altered
protease regain its old familiar efficiency by zeroing in on the
HIV substrate while ignoring those pesky decoy drugs completely.
These are scenarios for drug activity and drug resistance. A few
key substituions of the amino acids in the enzyme can let HIV protease
get on with its job despite the drugs. The enzyme may be a little
out of whack, but it can still do its job; it has compensated. It's
like someone who limps after hurting her foot: She may be slower,
but she's still getting around. The enzymes may limp a bit, but
they learn to get by.
The Genius of Genes
Proteins are the products of genes. Proteins are active, messy
things that go out to interact with the rough-and-tumble world and
do things. They can have a limited life span and often get chopped
up and recycled after they have done their jobs. But new proteins
are always waiting to be made. The master recipe for assembling
the string of amino acids that makes up a protein is stored in a
completely different type of chemical structure called genetic material.
Genetic material (made from DNA or RNA) is also arranged in the
form of a string, but one made from nucleotide molecules instead
of amino acid molecules. While there are 20 different types of amino
acids that make up proteins, genes basically use only four different
kinds of nucleotides.
A string of three nucleotides forms a code: Every set of three
nucleotides in a gene represents one amino acid. A sequence of these
nucleotide trios corresponds to a sequence of amino acids in a protein.
Although there are only four nucleotides to work with, as a set
of three (4 times 4 times 4) enough combinations can be made to
code for up to 64 amino acids. Fortunately, we only need 20, so
most amino acids have more than one set of nucleotide codes.
The Seeds of Resistance
There is a set of machinery that is responsible for reading a copied
piece of genetic code and assembling the right amino acids in the
right order to make proteins. This process is called translation
and the translating machinery builds the protein that it's told
to build by the genetic material. There is a different set of cellular
and viral machinery that is responsible for storing, retrieving
and making copies of the genetic material. This is where trouble
creeps in.
If the machinery responsible for copying and storing the HIV gene
skips one of the nucleotides or reads it wrong, several things could
happen. Since the three-letter code for the amino acid has been
changed, it may now code for a different amino acid, the same amino
acid (using one of the alternate codes) or it may code for nothing
at all. The translating machinery doesn't care; it just goes to
work on what it is given. As the protein is assembled, it may stop
short or it may become a useless mess of a mutant. But sometimes,
the substituted amino acid will continue to work just fine. In some
cases this is because the change does not matter, but in special,
rare cases such as results in drug resistance, the changed amino
acid may actually help the enzyme to ignore the drug molecules while
continuing to perform it's duties. This is a drug resistant mutant
and if it is successful, it will thrive.
P-glycoprotein and HIV
By Yvette Delph
Antiviral Project Director, Treatment Action Group (TAG)
P-glycoproteins belong to a family of plasma membrane proteins
encoded by the MDR (multidrug resistance) gene(s) that are well-conserved
in nature. P-glycoprotein (P-gp) functions as a membrane-localized
drug transport mechanism that has the ability to actively pump out
all currently prescribed HIV-protease inhibitors (PIs) from the
intracellular cytoplasm. This effect may result in limited oral
bioavailability of PIs as well as a decreased ability of the drugs
to cross blood-tissue barriers such as the blood-brain barrier (BBB),
the blood-testis barrier (BTB) and the materno-fetal barrier (MFB).
MDR1 encoded P-glycoprotein has also been implicated in the cytotoxicity
process and with the induction of immune responses during HIV infection.
It also plays a role in oxidative and inflammatory processes and
it may be involved in lipid transport and metabolism.
What is P-Glycoprotein?
P-gp is a phosphorylated and glycosylated plasma membrane protein
belonging to the ATP-binding cassette superfamily of transport proteins.
MDR1 P-gp (referred to simply as P-gp in this article) is a transmembrane
protein that is 1280 amino acids long and consists of two homologous
halves of 610 amino acids joined by a flexible linker region of
60 amino acids.
When viewed from above the plasma membrane, P-gp is donut shaped
with 6-fold symmetry, a diameter of about 10nm and a large central
pore of about 5nm in diameter. It has a thickness in the plane of
the plasma membrane of about 8nm. Since the depth of the plasma
membrane lipid bilayer is about 4nm, about half of the molecule
is within the plasma membrane.
Mechanism of Action, P-Gp Substrates and Inhibitors
The majority of published data suggest that P-gp acts as a transmembrane
pump which removes drugs from the cell membrane and cytoplasm. ATP
hydrolysis provides the energy for active drug transport, which
can occur against steep concentration gradients. It was initially
hypothesized that P-gp forms a hydrophilic pathway and that drugs
are transported from the cytoplasm to the extracellular medium through
the central pore, thereby shielding the substrate from the hydrophobic
lipid phase. It has more recently been proposed that P-gp acts like
a hydrophobic vacuum cleaner or flippase. In this model P-gp intercepts
the drug as it moves through the lipid membrane and flips the drug
from the inner leaflet of the plasma membrane lipid bilayer to the
outer leaflet and into the extracellular medium.
Molecules interacting with P-gp may be classified as substrate
or antagonist (inhibitor). Cancer drugs in the substrate group are
characterized by a >4-fold increase in cytotoxicity in MDR cells.
Compounds in the antagonist group increase the intracellular accumulation
of the P-gp substrates and display reversal of drug cytotoxicity.
Inhibitors may bind P-gp more tightly and, failing to be transported,
prevent the transport of other compounds, while substrates, in being
transported, do not block the transport of other substrates.
A partial list of P-gp substrates would include cancer drugs, such
as doxorubicin, daunorubicin, vinblastine, and vincristine; immunosuppressive
drugs, including cyclosporin A; steroids like aldosterone, hydrocortisone,
and cortisol; HIV PIs, such as amprenavir (APV), indinavir (IDV),
nelfinavir (NFV), ritonavir (RTV) and saquinavir (SQV); the antihistamine
terfenadine; cardiac drugs, such as digoxin and quinidine; the lipid
lowering agent lovastatin; the antibiotic erythromycin; and the
anti-tuberculous agent rifampicin. P-gp activity decreases the intracellular
concentration of cancer drugs, thus enabling resistance to develop;
the same may be true for PIs.
P-gp inhibitors include the immunosuppressant cyclosporin A; the
calcium channel blocker verapamil; the PIs RTV, SQV, NFV and possibly
IDV; the progesterone antagonist mifepristone (RU486); the sedative
midazolam; the anti-estrogen tamoxifen; the antibiotic erythromycin;
and the antifungal ketoconazole.
P-gp-dependent drug transport activity depends on the level of
expression of the MDR1 gene as well as on the functionality of the
MDR1-encoded P-gp. Induction of intestinal P-gp by rifampicin has
been shown to be the major mechanism responsible for reduced digoxin
levels during concomitant rifampicin therapy.
Gene amplifications, rifampicin induction and probably other factors
cause MDR1 over-expression. Polymorphism in exon 26 (C3435T) of
MDR1 is significantly correlated with levels of expression and function
of MDR1. Individuals homozygous for this polymorphism (TT allele)
showed significantly lower duodenal MDR1 expression and higher digoxin
plasma levels than volunteers with the CC genotype. Evaluation of
maximum plasma concentrations (Cmax) during steady state conditions
of digoxin administration showed a mean difference of 38% in digoxin
Cmax in the homozygous TT genotype compared with the CC genotype.
Where Is P-Gp Found?
Monoclonal antibody MRK16 was used to localize P-gp in normal human
tissues. Most tissues examined revealed very little P-gp. However,
P-gp was found in the liver, pancreas, kidney, colon and jejunum
and the adrenal gland. P-gp is also found in the epithelium of the
choroid plexus of the brain (which forms the blood-cerebrospinal
fluid (CSF) barrier) as well as on the luminal surface of blood
capillaries of the brain (the blood-brain barrier). In mice, MDR
mRNA expression levels increase dramatically during pregnancy and
are expressed at extremely high levels in the gravid compared with
the non-gravid uterus. P-gp is also expressed in the testis and
ovaries of mice and in the steroid-producing endometrial glands
of the pregnant uterus.
P-gp has been found in normal bone marrow in hematopoietic stem
cells and in peripheral blood mononuclear cells (PBMCs), mature
macrophages, natural killer (NK) cells, antigen-presenting dendritic
cells (DCs) and T- and B-lymphocytes. P-gp and MDR1 are expressed
to different levels in normal leukocytes. The presence of P-gp has
been demonstrated at relatively high levels in CD56+ cells (NK cells),
high to moderate levels in CD4+, CD8+ and CD15+ cells (T-helper
cells, T-suppressor cells and granulocytes, respectively) and lower
levels in CD19+ and CD14+ cells (B-lymphocytes and monocytes, respectively).
What is the Function of P-Gp?
The normal physiological function of P-gp in the absence of therapeutics
or toxins is unclear. Studies of MDR1 knock out (KO) mice (mice
lacking the MDR1 genes) show that they have normal viability, fertility
and a range of biochemical and immunological parameters. Predictably,
they do have delayed kinetics and clearance of vinblastine and they
accumulate high levels of certain drugs (vinblastine, cyclosporin
A, dexamethasone, loperamide and digoxin) in their brains. These
mice also demonstrated marked increases in the levels of these drugs
in the testis, ovary and adrenal gland compared with wild type mice.
1. Protection from Drugs and Toxins
Expression of P-gp on the luminal surfaces of the epithelial cells
of the small and large intestine, biliary ductules, and proximal
tubules of the kidney suggest a role in decreasing the absorption
from the gut and/or the excretion of endogenous and exogenous hydrophobic
amphipathic toxins. P-gp plays a role in the intestinal excretion
and, hence, in the reduced bioavailability after oral ingestion
of several drugs, including digoxin, paclitaxel, and HIV PIs. The
plasma concentrations after oral administration of IDV, NFV and
SQV were 2- to 5-fold higher in MDR1a KO mice compared with wild-type
(WT) or normal mice.
Expression in the capillary endothelial cells of the brain, nerves,
testis and placenta suggest a barrier function to keep toxins out
of the nervous system, gonads and fetus. Many relatively hydrophobic
drugs that were expected to diffuse easily across lipid membranes
did not readily enter the brain. P-gp has been found in hematopoietic
stem cells and probably contributes significantly to the removal
of drugs and toxins from the bone marrow. Studies have also noted
that P-gp can be detected in human placental trophoblasts from the
first trimester of pregnancy to full term, making it very likely
that placental P-gp protects the developing embryo and fetus from
toxic insult in humans as well.
2. Steroid Metabolism
The presence of P-gp in the adrenal and in steroid-producing cells
of the endometrium suggest it may also have a role in the handling
of steroids, possibly providing a protective function for the plasma
membranes of steroid-producing cells. Furthermore, it has been found
that P-gp-expressing epithelial monolayers of cells are able to
transport steroids and that some lymphoid cells expressing P-gp
are resistant to the cytotoxic effects of steroids.
3. Cholesterol Metabolism
P-gp appears to have a role in cholesterol metabolism. Cholesterol
esterification is one of the mechanisms that cells use to control
the amount of toxic free cholesterol. Under conditions of excess
cholesterol, cholesterol is transported from the plasma membrane
to the endoplasmic reticulum (ER) where it is esterified. P-gp functions
to increase esterification of cholesterol derived from plasma membrane
by facilitating the movement of cholesterol from the plasma membrane
to the ER.
4. Immune System
Immune responses in a peripheral organ like skin are initiated
when antigen-presenting cells, especially dendritic cells (DCs),
capture antigens locally. The DCs then migrate via lymphatic vessels
to draining lymph nodes where they select T lymphocytes that bear
receptors for the presented antigen. In vitro models have demonstrated
that P-gp facilitates this migration of DCs and that in the presence
of P-gp antagonists, DCs are retained in the epidermis.
There is also evidence that P-gp may be involved in the transport
of some cytokines (CKs), particularly interleukin-1 (IL-1), IL-2,
IL-4 and interferon-gamma (IFN-y) out of activated normal lymphocytes.
However, P-gp does not seem to transport IL-6. The biological importance
of P-gp to CK secretion during an immune response is still to be
clarified.
5. Cell Death and Cell Differentiation
The high level of P-gp expression on NK and CD8+ T cells has raised
the question of the role of P-gp in the function of these cells.
Inhibition of P-gp results in a reduction in NK and CD8+ T cell
cytolytic activity. The majority of physiological cell death pathways
appear to involve cystein-aspases (caspases). Cell death due to
membrane and cytosolic perturbations by cytotoxic granules occurs
in the absence of activation of the caspase pathway, whereas nuclear
damage requires caspase activation. NK and cytotoxic CD8+ T cells
bind their target cells and induce death either via the Fas/Fas
ligand system or, usually, by release of cytotoxic granules, such
as perforin and granzymes, into the target cell. In tumor cells,
P-gp confers resistance to Fas-mediated apoptosis. It has been demonstrated
that tumor cells expressing P-gp are resistant to a wide range of
stimuli that activate the caspase apoptotic cascade, but are not
resistant to caspase-independent cell death mediated by pore-forming
proteins and granzyme B. Inhibition of P-gp completely reverses
this resistance to caspase-dependent cell death.
6. Chloride channels
P-gp does not seem to have intrinsic channel activity, but may
regulate an endogenous chloride channel that is yet to be identified.
The physiological importance of this indirect modulation of chloride
channels is controversial.
7. Cytochromes
Many of the same drugs that are transported by P-gp are metabolized
by some of the cytochromes (CYPs), especially CYP450 3A. CYP3A substrates
include HIV PIs, the antibiotic erythromycin, and rifampicin. CYP3A
can be induced by many agents, including glucorticoids, barbiturates
and rifampicin. Human variation in CYP3A expression is believed
to influence drug response for up to one-third of all drugs.
It is likely that because P-gp can influence the intracellular
concentration of many CYP3A substrates, it may also affect the availability
of those substrates to CYP3A and therefore the extent of CYP3A metabolism
of those substrates. P-gp thus plays an important role in modulating
expression of CYP3A and this is likely to complicate the prediction
of drug interactions among drugs that are substrates for both P-gp
and CYP3A systems.
P-gp and HIV
All HIV PIs currently in use (IDV, SQV, NFV, RTV and APV) are transported
by P-gp, which can actively expel these PIs from cells. Transport
of HIV PIs can be inhibited by P-gp inhibitors like cyclosporin
A, quinidine, verapamil, and PSC833. PIs interact with P-gp with
affinities in the order RTV>NFV>IDV>SQV. RTV, SQV, NFV and possibly
IDV have also been shown to inhibit transport of some of the known
P-gp substrates. Except for RTV and maybe SQV, their inhibitory
effects are weaker than established inhibitors like verapamil or
cyclosporin A.
It has been demonstrated that plasma levels of oral IDV, SQV and
NFV were 2 to 5 times higher in MDR1a KO mice compared with WT mice.
This strongly suggests that P-gp transport at the intestinal and/or
hepatic level limits the systemic bioavailability of these drugs.
Studies in Caco-2 cells, which exhibit many of the morphological
and biochemical characteristics of human small intestine, suggest
that P-gp transports absorbed PIs (APV, RTV, IDV, NFV and SQV) back
into the intestinal lumen, thus limiting oral bioavailability.
Studies have reported that the rank order of in vitro intracellular
accumulation of PIs was SQV>RTV>IDV. P-gp, MRP (multidrug resistance
protein, another drug transporter), protein binding and HIV infection
all decreased the intracellular accumulation of PIs. Compared with
human erythroleukemia cells that don't express P-gp, cells over-expressing
P-gp demonstrated a 10-fold reduction in APV and IDV intracellular
concentrations, 3- and 6-fold reductions for RTV and SQV, respectively,
while there was no difference in NFV intracellular concentrations.
P-gp may also limit the penetration of PIs into several tissue
compartments in the body, thereby possibly creating sanctuary sites,
such as the brain and gonads. Brain penetration of IDV, SQV and
NFV were increased 7-, 10- and 36-fold respectively in MDR1a KO
mice compared with WT mice. A study using an in vitro BBB model
demonstrated that APV, RTV and IDV are actively transported by P-gp
across the BBB. Another study demonstrated that brain and testis
levels of NFV, APV, IDV and SQV were significantly increased in
mice when the potent P-gp inhibitor LY335797 was administered intravenously
and that this increase was not due to increased PI plasma levels.
While IDV achieves good penetration into the semen (seminal plasma
(SP): blood plasma (BP) ratio = 0.9), RTV and SQV penetrated very
poorly (SP:BP were 0.02 and 0.03, respectively).
The effect of P-gp on limiting oral bioavailability and tissue
distribution of PIs has obvious implications for the effectiveness
of PI-containing regimens. Poor penetration of PIs into the brain,
testis and other sanctuary sites may result in de facto compartmental
mono- or dual antiretroviral therapy with ongoing HIV replication
and development of resistance.
HIV PIs do not cross the placental barrier appreciably and placental
P-gp may be an important factor in this low penetration. PIs are
therefore generally considered unsuitable for prevention of mother-to-infant
transmission. After intravenous administration of SQV to pregnant
mice, the ratios of SQV concentration in fetal tissue to that in
maternal plasma were 5-7 fold higher in MDR1a/1b KO mice than in
WT mice. P-gp fetal and blood-brain barriers are not abolished by
co-administration of high doses of RTV.
The effects of P-gp on the distribution, metabolism and excretion
of drugs, including PIs, in the body is great. Blockage of P-gp
may prove useful in facilitating greater intestinal absorption,
bioavailability and penetration of PIs into HIV sanctuary sites
as well as in reducing PI excretion. It may also simplify PI containing
regimens by reducing the oral doses of PIs and the frequency at
which they are taken. Higher PI levels in these sites may result
in greater suppression of viral replication in these sanctuary sites,
but they may also result in unwanted adverse effects. The effects
of P-gp inhibition may not be limited to PIs but may extend to other
co-administered drugs. For example, the antidiarrheal agent loperamide
is a peripherally acting opiate which penetrates the brain poorly.
However, in MDR1a KO mice, loperamide exhibits strong morphine-like
effects on the central nervous system.
In HIV infected cells with high P-gp expression, both accumulation
and antiviral efficacy of IDV, SQV and RTV are diminished. One study
found that 90% of all peripheral blood lymphocyte subsets (CD4+,
CD8+, CD56+ and CD10+ cells) expressed surface P-gp in both HIV-infected
patients and controls. However, P-gp function was significantly
reduced in CD16+ NK cells and CD19+ B-cells from HIV+ patients compared
with controls. This reduced function significantly correlated with
decreased NK cytotoxicity observed in HIV+ patients. P-gp can also
be detected on an intracellular level in different peripheral blood
monocyte subpopulations, mainly CD8+ T cells, CD16+ NK cells and
CD14+ monocytes. This intracellular expression was decreased in
CD8+ T cells and CD16+ NK cells from HIV-infected patients.
In addition, a significantly increased proportion of CD4+ T-cells
from HIV-infected patients expressed P-gp compared with controls.
This resulted in a significantly increased ratio of the proportions
of CD4+/P-gp+ to CD8+/P-gp+ cells. This ratio was significantly
higher in patients with CD4+ cell counts of <200/mL than in those
with CD4+ cell counts >200/mL. However, both CD4+ and CD8+ T-cells
from HIV-infected patients accumulated more of the P-gp substrate
rhodamine 123 compared with controls. P-gp inhibitors failed to
increase further this intracellular accumulation in HIV+ patients.
This suggests that in HIV infection there is increased expression
of a functionally defective P-gp in CD4+ and CD8+ T-cells that appears
to increase with disease progression.
P-gp expression may affect HIV-infectivity. Studies have demonstrated
a reduction in virus production when P-gp was over-expressed at
the surface of 12D7, a continuous CD4+ human T cell leukemia cell
line, infected with a laboratory strain of HIV-1. Reduction in infectivity
occurred both during the fusion of viral and plasma membranes and
at subsequent steps in the HIV life cycle. P-gp over-expression
did not significantly alter the surface expression or distribution
of either the CD4 receptor or the CXCR4 coreceptor.
PIs can cause hypercholesterolemia and, as explained earlier, P-pg
plays a role in cholesterol metabolism and possibly in atherogenesis.
Whether P-gp plays any role in PI-mediated dyslipidemia is not known.
Further Research on P-gp and HIV
The P-gp transport system clearly has major implications for HIV
infection and its treatment. There is still much left to be understood.
P-gp expression and function in HIV-infection needs to be studied
— both in different tissues as well as in various stages of
disease. The possibility of using P-gp function and expression as
another surrogate marker for HIV disease progression should be explored.
The effects of P-gp expression — and alterations in P-gp expression
on HIV infectivity, on the immune and other systems of HIV-infected
individuals and on HIV therapy should be fully evaluated. Does P-gp
play a role in the failure of antiretroviral therapy and the development
of resistance to PIs?
The safety and efficacy of P-gp modulation in the management of
HIV disease, especially in the use of PI-containing regimens, require
further study. This includes the use of recognized P-gp inhibitors
like PSC833, LY335979, and PIs like RTV, as well as the possibility
of using P-gp maturation inhibitors (proteasome inhibitors). The
optimal therapeutic dose of RTV required to inhibit P-gp; its effects
on intracellular concentrations of PIs in HIV infected cells and
on tissue penetration of PIs; its effects on concomitantly administered
drugs; and the clinical value of using RTV as a P-gp inhibitor in
the treatment of HIV disease remain to be evaluated. Are the different
P-gp modulators site-specific; do they inhibit P-gp to different
degrees depending on location?
The interactions and interdependence of the P-gp transport and
the cytochrome metabolic systems need further elucidation. Both
are important causes of drug-drug interactions and HIV PIs interact
with both systems. It may be necessary in the future to determine
the interactions of HIV drugs not only with the cytochrome system,
but also with the P-gp transport system.
It is necessary to investigate properly whether the co-administration
of P-gp inhibitors with PIs is safe and effective for prophylaxis
of mother-to-child transmission. Administration of P-gp inhibitors
may be best done in later pregnancy to minimize the adverse effects
of drugs and toxins on the developing fetus.
Studies also need to be undertaken to discern the mechanism of
action of PI-induced dyslipidemias and what role, if any, P-gp plays.
Does HIV disease itself affect the role of P-gp in cholesterol metabolism?
The P-gp transport system is complex and poorly understood. It
is even less well understood in HIV disease, in which it may play
a significant role. The role of P-gp in HIV disease pathogenesis
and its effect on HIV drugs are undoubtedly deserving of greater
study. It may become routine in the future to determine the interactions
of HIV drugs not only with the cytochrome system, but also with
the P-gp transport system.
An extended, fully referenced version of this article entitled,
P-glycoprotein: A tangled web waiting to be unraveled is available
at the TAG website: www.treatmentactiongroup.org
Areas for Further Research
Further research on P-gp is neeeded in the following areas
1. Related to HIV Disease and Progression
- P-gp expression and function in HIV infection, both in different
tissues of the body as well as in various stages of disease
- The possibility of using P-gp function and expression as one
of the surrogate markers for HIV disease progression
- The effects of P-gp expression — and alterations in P-gp
expression
- on HIV infectivity
- on the immune systems of HIV-infected individuals
- on HIV therapy
- The role of P-gp in viral failure and the development of resistance
to PIs
2. Related to Treatment for HIV
- The safety and efficacy of P-gp modulation in the management
of HIV disease, especially during the use of PI-containing regimens
- do the modulators affect P-gp differently in different tissues?
- Inhibition of P-gp transport by RTV (and possibly other PIs)
- Clinical value of using RTV (and other PIs) as a P-gp inhibitor
- Optimal therapeutic dose(s) of RTV (and of other PIs)
- Effects of P-gp on intracellular concentrations of PIs in HIV
infected cells and on tissue penetration of PIs
- Effects of P-gp on concomitantly administered drugs
3. Related to the Cytochrome System
- Pharmokinetic interactions and interdependence of the P-gp transport
and the cytochrome metabolic systems
4. Mother-to-Child Transmission
- Safety and Efficacy of co-administration of P-gp inhibitors
with PIs for prophylaxis of vertical transmission
- The optimal time to administer P-gp inhibitors to minimize the
adverse effects of drugs and toxins on the developing fetus
5. Cholesterol Metabolism and Dyslipidemias
- The mechanism of action of PI-induced dyslipidemias and what
role, if any, P-gp plays.
- Whether HIV disease itself affects the role of P-gp in cholesterol
metabolism.
HIV Campaign in an Islamic School
Courtesy of Nigeria-AIDS eForum, a project of Journalists Against
AIDS (JAAIDS) Nigeria
http://www.nigeria-aids.org/eforum.cfm
I am pleased to share with you and others a brief report of the
HIV/AIDS Awareness program held at Ansar-Ud-Deen High School, Liberty
road, Oke Ado, Ibadan. The school is an Islamic school with 98%
of the students being Muslim and has a student population of about
3,000. The receptivity of the school to the program is contrary
to the views expressed in many circles that schools that are predominantly
Muslim are opposed to such programs. The program was sponsored by
the Association for Reproductive and Family Health (ARFH) and was
part of the UNICEF-Massive Awareness Raising Activity (UNICEF-MARC)
on HIV/AIDS in Ibadan, the capital city of Oyo State, Nigeria.
A carnival along Liberty road enabled students and teachers wearing
fez caps with appropriate messages to create awareness on HIV/AIDS
and sensitize people to prevention strategies around and beyond
the school's immediate environment. They also distributed HIV/AIDS
leaflets. The carnival was very entertaining as the students and
teachers danced to the admiration of the people. This drew the attention
of the people who listened to the messages and collected the leaflets.
Questions asked by these passers-by were answered by available ARFH
staff on the spot.
A Program Officer from ARFH, Mrs. Stella Akinso gave a talk on
"Roles of youth in preventing the spread of HIV/AIDS". At the end
of the lecture, students and teachers asked questions with appropriate
answers provided by the resource person. Most of the students who
could not openly ask questions wrote them on slips and passed them
over.
A drama titled "HIV/AIDS is Dead" was presented by members of the
Youth Rescue Club of the Association for Reproductive and Family
Health. The drama presentation portrayed youth as a most vulnerable
group but who also have the power to wage war and conquer HIV/AIDS.
The drama started with a scene showing youth drinking, partying,
smoking and engaging in sexual escapades. The outcome was that they
played into the hands of HIV/AIDS who had also enticed them with
his "humane posture" and eventually clubbed them to death. Some
managed to escape and decided to learn more preventive measures
from a wise old man whose counsel they had initially rejected.
Amongst the youth were two young persons who eventually emerged
as the hero and heroine. They are in a relationship and had received
information and education on HIV/AIDS from the "Wise Old Man." As
a result of the counseling, they agreed to keep to all HIV/AIDS
preventive strategies. They promised to be faithful and abstain
from sex till they are matured and ready for marriage. With this
information, they challenged HIV/AIDS to a war of wits and an open
display of strength.
HIV/AIDS challenged their suitability to confront him seeking to
know what weapons they have and the secret of their boldness. HIV/AIDS
became weak when confronted with what they had learnt. The boy and
the girl joined efforts to physically engage HIV/AIDS in a fight
and in the end AIDS was killed and carried off the stage by the
young persons. The students and teachers, including the Principal
of the school, openly applauded the story line and its ending.
In other recent activities, a lecture on HIV/AIDS was presented
at School for the Handicapped (HLA, Agodi Gate, Ibadan). Highlights
were a film show and distribution of leaflets. This Oyo State Government-owned
school has a population of about 250 students. The program was at
the instance of one of the female teachers who volunteered information
about the sexual activities of the students and requested for a
health talk on HIV/AIDS and unwanted pregnancy. She reported that
the students are sexually abused by their colleagues (who are equally
disabled) and other able bodied people within the school's immediate
environment. Some of them become pregnant by persons they are unable
to identify which further compounds their problems. It was the first
time such activity is taking place in the school.
— Reported by Grace E. Delano, Executive Director/VP, Association
for Reproductive and Family Health
SMART Starts! (and not a moment
too soon) The Community Programs
for Clinical Research on AIDS (CPCRA) has opened a new clinical
trial of a size and duration designed to finally provide high quality
data about what happens to people who have been on antiretroviral
therapy for more than a couple of years. The SMART study (Strategies
for Management of Antiretroviral Therapy) plans to enroll 6,000
people and follow them for as long as seven years. The trial will
be the first randomized comparison of two viable but competing strategies
for how to treat HIV. The study is open to both treatment-naive
and treatment-experienced HIV-positive people over the age of 13
who currently have CD4 counts over 350. The only other requirement
is a willingness to admit an open mind (or just plain confusion)
about the optimum way to use antiretroviral therapy for the best
long-term outcome.
SMART participants will be randomly assigned to one of two camps.
One group will follow the classic path of making every effort to
keep their viral loads undetectable at all times. This is the VS
(viral suppression) group. They will face off against the DC (drug
conservation) group who will follow a strategy of avoiding treatment
(despite detectable viral load numbers) as long as their CD4 counts
stay above 250. People in this group will probably tend to go on
and off drugs as needed, while the folks in the VC group will tend
to go on drugs and stay on, switching parts of their regimens whenever
viral load bounces back. The trial has been characterized as a comparison
of continuous versus episodic therapy or the "hit hard" versus the
"go slow" schools of thought.
Although recent versions of treatment guidelines recommend starting
therapy later, there is little evidence other than some observational
studies and expert opinion for either treatment strategy. Many clinicians
reason that some drug toxicity is an acceptable price to pay compared
to the risk of becoming resistant to antiviral drugs, losing immune
competency and developing AIDS. Other HIV physicians increasingly
worry that long-term toxicity from the drugs may eventually erase
the life-extending benefits of HAART. Both of these views are reasonable
given an individual's training and experience, but there is little
objective data to convincingly support one over the other. This
is what SMART will help provide.
Seven years ago when the promise of protease inhibitors was first
coming into focus, the idea of launching a seven-year trial was
unthinkable to many people. Most were thrilled to be given another
six months of life; seeing the next century seemed like an impossible
dream. But expectations have changed, although after many years
we still lack answers to fundamental questions about the best way
to treat the disease. With the realization that no truly dramatic
therapeutic breakthrough is on the horizon, setting out on a seven-year
long study doesn't seem as impractical these days.
The trial also incorporates several substudies within the main
objective. One particularly overdue investigation will examine the
effects of treatment on the heart. Another will examine how treatment
causes changes to body fat distribution and bone density. Additional
substudies will compare quality-of-life measures, cost-effectiveness,
drug resistance and HIV transmission.
Dr. Wafaa El-Sadr, principal investigator at Harlem Hospital and
Columbia University in New York and co-chair of the study commented,
"A trial of this scope and length will be a challenge. (But) the
SMART study will address questions that are uppermost in the minds
of people with HIV and the clinicians who treat them. While significant
advances have been made in the treatment of HIV, after two decades
we still do not know for certain that the current method of treating
HIV, with continuous therapy to maximally suppress viral load, is
the best way to manage HIV in the long-run."
SMART is one of the most important research undertakings to come
out of the government trials system since the early 1990s. It has
the historic potential to produce a body of information with broad
and lasting significance, not only for the health of the participants,
but for the millions of HIV-positive people in the world who will
eventually face the need to begin treatment.
For more information about the trial, visit the SMART Study web
site at www.smart-trial.org
. Information about SMART and other AIDS clinical trials and how
to enroll is available at the AIDS Clinical Trials Information Service
(ACTIS) Web site www.actis.org
or 1-800-874-2572 (1-800-TRIALS-A).
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