Background
to drug resistance
The genes of living organisms contain the information necessary for their growth and
survival. These genes are carried as sequences of chemical "bases" (the chemical
"letters" of the genetic code) in strands of deoxyribonucleic acid (DNA) in the
chromosomes (Appendix 1).
Proteins are the workhorses of living systems, forming structural components of cells
and tissues, or acting as enzymes (biological catalysts) to drive essential reactions in
the body. Each protein is specified ("encoded") by a gene, the information in
which is read end to end by using the genetic code to join amino acids in a chain. The
amino acid sequence of a protein determines its three-dimensional structure, for example
to create the "active site" of an enzyme - the space into which the molecules
processed by the enzyme will fit. Inhibitors can often be made which will fit into an
active site and block the enzyme, allowing their use as drugs. Mutations in the gene
(changes in one or more of the bases) may lead to the incorporation of wrong amino acids,
distorting the active site and crippling the enzyme, or preventing the binding of an
inhibitory drug while leaving the enzyme active, so that therapy fails (Appendix 2).
Viruses are not living organisms, but depend on infection of cells for their survival
and multiplication. Consequently viruses must play the game by the same rules, albeit
sometimes with variations such as the use of ribonucleic acid (RNA) rather than DNA for
their main genetic material. Human immunodeficiency virus (HIV) particles contain RNA,
which on infection of a cell (in this case in the immune system) must be copied first of
all to DNA in order to take over control of the cells processes for the production
of new virus particles. This copying is performed by the virus enzyme reverse
transcriptase (RT) which was the initial (and continuing) target of some anti-HIV drugs
such as AZT. Roche, and later other companies, attacked a second target through the
powerful inhibition of protease a viral enzyme which cuts up newly-made viral
proteins to allow the formation of infectious particles. Because of their different
targets, it has become commonplace to use inhibitors of these two enzymes in combination
therapy, resulting in much-improved suppression of the "virus load" (circulating
concentration of virus) to levels below the current limits of detection.
Though it is a very vigorous enzyme, RT is unable to correct frequent errors in copying
the RNA, creating large numbers of viral variants, some of which cannot survive. Those
that do, however, generate the highly mixed population of
subtypes("quasi-species") which are present in an infected person. This gives
the virus a potential head start over any change in its environment, such as the
introduction of antiviral drugs, since a variant may already be present which will survive
the new conditions better than the typical virus ("wild type") of the original
population, and can take over under the "selective pressure" of the drug. As a
result, drugs such as reverse transcriptase inhibitors and protease inhibitors will tend
to select for variants in their respective target enzymes, which can be detected as
mutations in either gene. In combination therapy, each gene may change independently.
Resistance to an RT inhibitor will have no direct effect on a protease inhibitor and vice
versa, though it will reduce the potency of a drug combination. Within each class of
compound, the mutations which bring about resistance to a particular drug depend on the
chemical shape of that drug and its interaction with the enzyme. To a greater or lesser
extent, therefore, compounds will have a "genetic signature" of resistance
mutations, and depending on the similarity of their shape some compounds (eg indinavir and
ritonavir) will share mutations and therefore cross-resistance. Saquinavir has a
distinctive signature, marked by changes at protease amino acids 48 ("G48V") or
90 ("L90M"), the former being the less common of the two in the clinic, using
either the original, hard gelatin formulation (Invirase®)
or the soft gelatin formulation (Fortovase®).
Though they may enable some growth in the presence of a selecting drug
("resistance" to that drug), mutations may also lower the efficiency of the
enzyme and slow the multiplication of the virus. This is true of G48V and L90M, which is
why they are virtually absent from untreated populations and (particularly G48V and the
double mutation) arise late and infrequently during saquinavir treatment. Following the
appearance of mutations to drug resistance, enzymes (hence, their genes) will undergo
further selection for changes which will correct the distortion and so improve their
efficiency and viral "fitness" for example, changes at amino acids 63 and
82 tend to associate with G48V. In the case of protease, changes to improve fitness may
also occur at the specific points in the virus structural ("gag" and
"pol") proteins which are cleaved by the protease, to tailor them to the altered
enzyme.
Testing in the clinic
Resistance in viral isolates obtained during treatment may be defined by
"genotyping" (looking for particular mutations associated with relevant drugs
i.e. "probe-based genotyping" - or analysing gene sequences for all
possibly relevant changes i.e. "sequence-based genotyping") or by
"phenotyping" (looking for what may be defined as a significant change in viral
drug sensitivity in tissue-culture). These are still matters for further research and
clarification, particularly as they apply in a clinical context. Unfortunately,
"fitness" changes have often been labelled "resistance mutations", and
the definition of phenotypic change is variable, adding to the complexity of an already
difficult area. This confusion has hindered the wider understanding of HIV drug resistance
and its role in progressing disease, and most importantly decision-making on the best
application of the drugs available to the treating physician which drug(s) to use,
and when and how to change therapy in the best interests of the subject and limitations in
the drugs budget, especially for multiple therapy. A precedent has been set for the use of
single mutations to "define" drug resistance as a basis for clinical management,
without taking account of other mutations, patient history and ongoing circumstances
(viral load, CD4 count, etc.).
Roche collaborative surveillance programme
Roche has embarked on a strategy to apply genotyping in conjunction with new,
customisable software for the interpretation of genetic changes in viral isolates, which
will allow powerful analysis of resistance trends and offer appropriate guidance on
clinical management (Appendix 3).
The GREAT (Genotypic Resistance Evaluation to Aid Therapy-switching) Study, in
collaboration with Virology Networks of Utrecht and Perkin-Elmer Corporation of Norwalk,
Connecticut, will test the prototype RetroGram Decision Support Software in
realistic settings which allow a variety of antiviral treatment regimens. RetroGram
employs an electronic rule-based algorithm to interpret genotype data and guide physicians
and patients in treatment decisions, and will be constantly updated as new information
emerges on the type and clinical significance of resistance mutations in HIV genes.
GREAT is a randomized, international, parallel group, open label, 48-week trial
enrolling up to 360 patients who are currently failing their first-line protease inhibitor
anti-HIV combination regimens failure being defined by at least 24 consecutive
weeks' experience with a PI-containing combination regimen and a viral load of at least
5,000 copies/ml (or 3.7 log10) at screening. Participants will be randomized to
a new regimen by one of two methods: either best clinical judgment or best clinical
judgment in conjunction with real-time HIV resistance genotyping. This will provide a
clinically useful tool to physicians and patients, and will also be an advance in rational
combination drug regimen design, intended to maximize individuals' therapeutic options
over the long term.
While Roche is a major GREAT sponsor, the trial has been designed to include the entire
armamentarium of approved HIV treatments from a wide array of manufacturers. Roche has
purposely left data analysis in the hands of Virology Networks, the investigators and
community advocates involved in the trial. This will ensure that the GREAT data are
thorough, accurate and impartial and can be applied to the broadest range of patients in
the real world.