Protease Inhibitors _PIs_

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					                                         Protease Inhibitors (PIs)

         There are seven FDA-approved PIs: amprenavir, indinavir, lopinavir (manufactured in
combination with ritonavir), nelfinavir, ritonavir, saquinavir, and the recently approved
compound atazanavir. The dynamic susceptibility range for each of the PIs is about 100-fold in
most drug susceptibility assays (Hertogs et al., 1998; Hertogs et al., 2000; Petropoulos et al.,
2000; Kempf et al., 2001; Parkin et al., 2003). The spectrum of mutations developing during
therapy with indinavir, nelfinavir, saquinavir, ritonavir, and amprenavir have been well
characterized (Condra et al., 1996; Molla et al., 1996; Schapiro et al., 1996; Boden & Markowitz,
1998; Craig et al., 1998; Patick et al., 1998; Shafer et al., 1999; Atkinson et al., 2000; Maguire et
al., 2002) but fewer data are available for lopinavir (Masquelier et al., 2002; Romano et al., 2002)
and atazanavir (Colonno et al., 2002).

         Pharmacologic factors influence the clinical efficacy of PIs more than that of the other
two classes of HIV drugs (Schapiro et al., 1996; Stein et al., 1996; Burger et al., 1998;
Hoetelmans et al., 1998; Acosta et al., 1999; Murphy et al., 1999; Durant et al., 2000;
Hoetelmans, 2001; van Heeswijk et al., 2001). Virologic response is highly correlated with the
inhibitory quotient (IQ) defined as the trough concentration divided by the inhibitory
concentration of the drug (e.g. the IC50 in a standardized assay) (Hoetelmans, 2001; Shulman et
al., 2002; Marcelin et al., 2003b). Drug levels achieved during PI monotherapy can vary greatly
among individuals, often resulting in low IQs (van Heeswijk et al., 2001). This has led to the
practice of administering sub-therapeutic doses of ritonavir (a cytochrome P450 enzyme
inhibitor) in combination with other PIs to increase, or “boost” their drug levels (van Heeswijk et
al., 2001). Lopinavir is formulated in a fixed combination with ritonavir (Hurst & Faulds, 2000);
and saquinavir, indinavir, and amprenavir are now usually administered with low-dose ritonavir
(van Heeswijk et al., 2001). Boosted PIs require higher levels of resistance than PIs given as
monotherapy before significant loss of antiviral activity and virologic rebound occur (Condra et
al., 2000a; Kempf et al., 2000; Shulman et al., 2002; Marcelin et al., 2003b).

          Protease substrate cleft mutations (Figure 3)
          V82A/T/F/S occur predominantly in HIV-1 isolates from patients receiving treatment
with indinavir or ritonavir (Condra et al., 1996; Molla et al., 1996). V82A also occurs in isolates
from patients receiving prolonged therapy with saquinavir following the development of the
mutation G48V (Winters et al., 1998; Sevin et al., 2000). By themselves, mutations at codon 82
confer reduced susceptibility in vitro to indinavir, ritonavir, and lopinavir (Condra et al., 1996;
Molla et al., 1996; Sham et al., 1998; Kempf et al., 2001) but not to nelfinavir, saquinavir, or
amprenavir. However, when present with other PI mutations, V82A/T/F/S contributes phenotypic
and clinical resistance to each of the PIs (Shafer et al., 1998; Sham et al., 1998; Winters et al.,
1998; Kempf et al., 2001; Colonno et al., 2003; Marcelin et al., 2003b). V82A is the most
common mutation at this position; V82S, the least common. The phenotypic and clinical
significance of the differences between each of these mutations has not been studied. V82I occurs
in about 1% of untreated individuals with subtype B HIV-1 and in 5-10% of untreated individuals
with non-B isolates (Gonzales et al., 2001). Although V82I occasionally emerges during PI
therapy (Maguire et al., 2002), preliminary data suggest that V82I confers minimal or no
resistance to the available PIs (King et al., 1995; Descamps et al., 1998; Brown et al., 2001; Rhee
et al., 2003).

         I84V has been reported in patients receiving indinavir, ritonavir, saquinavir, and
amprenavir as their sole PI (Condra et al., 1996; Molla et al., 1996; Craig et al., 1998; Hertogs et
al., 2000; Sevin et al., 2000; Maguire et al., 2002) and causes phenotypic (Partaledis et al., 1995;
Tisdale et al., 1995; Condra et al., 1996; Patick et al., 1996; Carrillo et al., 1998; Palmer et al.,
1999; Kempf et al., 2001; Prado et al., 2002; Colonno et al., 2003) and/or clinical (Zolopa et al.,
1999b; Para et al., 2000; Kempf et al., 2002; Marcelin et al., 2003b) resistance to each of the PIs.
I84V is rarely the first major PI-resistance mutation to develop, usually developing in isolates that
already have the mutation L90M (Kantor et al., 2002; Wu et al., 2003). I84A and I84C are
extremely rare mutations that are also associated with resistance to multiple PIs when present in
combination with other PI-resistance mutations (Mo et al., 2003b).

        G48V occurs primarily in patients receiving saquinavir and rarely in patients receiving
indinavir. This mutation causes 10-fold resistance to saquinavir and about 3-fold resistance to
indinavir, ritonavir, and nelfinavir (Jacobsen et al., 1995; Patick et al., 1996; Hertogs et al., 1998;
Winters et al., 1998). G48V has been reported to cause low-level biochemical resistance to APV
when present in site-directed mutants, but to interfere with APV resistance when present together
with more typical APV-resistance mutations such as M46I, I47V, and I50V (Markland et al.,
2000). Its affect on lopinavir and atazanavir is not known. G48V usually occurs with mutations at
positions 54 and 82 (Shafer et al., 1998; Palmer et al., 1999; Schiffer et al., 2001; Wu et al.,

        D30N occurs solely in patients receiving nelfinavir and confers no in vitro or clinical
cross-resistance to the other PIs (Patick et al., 1996; Markowitz et al., 1998; Winters et al., 1998;
Zolopa et al., 1999a). D30N confers reduces nelfinavir susceptibility by 5-20 fold. D30N is often
followed by the development of N88D and the combination reduces nelfinavir susceptibility by
about 50-fold (Rhee et al., 2003). D30N usually does not develop in isolates containing other
primary PI-resistance mutations (Kantor et al., 2002; Sugiura et al., 2002; Wu et al., 2003).

         I50V has been reported only in patients receiving amprenavir as their first PI (Maguire et
al., 2002). In addition to causing reduced amprenavir susceptibility, it causes reduced
susceptibility to ritonavir and lopinavir (Partaledis et al., 1995; Tisdale et al., 1995; Molla et al.,
2001; Prado et al., 2002; Mo et al., 2003a; Parkin et al., 2003). The development of I50V usually
requires a specific compensatory cleavage site mutation (Maguire et al., 2002; Prado et al., 2002).
I50L occurs in patients receiving atazanavir as their first PI (Colonno et al., 2002). It reduces
atazanavir susceptibility by 5-10 fold and causes hypersusceptibility to each of the remaining PIs
(Colonno et al., 2002).

        V32I occurs in patients receiving indinavir, ritonavir, or amprenavir. It usually occurs in
association with other PI resistance mutations in the substrate cleft or flap and by itself appears to
cause minimal resistance to any one drug. However, in combination with other mutations such as
M46I/L, I47V, V82A, and I84V, high levels of resistance to multiple PIs, including lopinavir,
have been repoted (Parkin et al., 2003).

        R8K and R8Q are substrate cleft mutations that cause high-level resistance to one of the
precursors of ritonavir (A-77003) (Ho et al., 1994; Gulnik et al., 1995) but they have not been
reported with the current PIs.

         Protease flap mutations (Figure 3)
         The protease flaps (residues 33-62) extends over the substrate-binding cleft and must be
flexible to allow entry and exit of the polypeptide substrates and products (Shao et al., 1997; Scott
& Schiffer, 2000). The flap tips (residues 46-54) are particularly mobile and are the site of many
drug-resistance mutations. In addition to mutations at positions 48 and 50, which extend into the
substrate cleft, mutations at positions 46, 47, 53, and 54 make important contributions to drug
        Mutations at position 54 (generally I54V, less commonly I54T/L/M/S) contribute
resistance to each of the approved PIs (Condra et al., 1996; Molla et al., 1996; Kempf et al., 2001;
Maguire et al., 2002; Colonno et al., 2003) and have been frequently reported during primary
therapy with indinavir, ritonavir, amprenavir, and saquinavir, (Condra et al., 1996; Molla et al.,
1996; Schapiro et al., 1996; Patick et al., 1998; Maguire et al., 2002) and salvage therapy with
lopinavir (Masquelier et al., 2002; Romano et al., 2002; King et al., 2003). I54L and I54M are
particularly common in persons receiving amprenavir and have a greater effect on amprenavir
than does I54V (Maguire et al., 2002).

         Mutations at position 46 (usually M46I/L, rarely M46V) contribute to resistance to each
of the PIs except possibly saquinavir (Condra et al., 1996; Molla et al., 1996; Kempf et al., 2001;
Maguire et al., 2002; Colonno et al., 2003) and have been frequently reported during primary
therapy with indinavir, ritonavir, amprenavir, and nelfinavir (Molla et al., 1996; Schapiro et al.,
1996; Patick et al., 1998; Condra et al., 2000b; Maguire et al., 2002) and during salvage therapy
with lopinavir (Masquelier et al., 2002; Romano et al., 2002).

        I47V has been reported in patients receiving amprenavir, indinavir, and ritonavir, and
often occurs in conjunction with the nearby substrate cleft mutation, V32I (Parkin et al., 2003).
I47A is an uncommon mutation that is associated with high-level resistance to lopinavir and
intermediate resistance to amprenavir (Kagan et al., 2003).

        F53L has been reported rarely in patients receiving PI monotherapy, but it occurs in more
than 10% of patients treated with multiple PIs (Wu et al., 2003). In a multivariate analysis it has
been associated with phenotypic resistance to lopinavir (Kempf et al., 2001). F53Y is a less
commonly occurring substitution at this position which occurs only in treated persons and
probably has a similar role as F53L (Wu et al., 2003).

          Protease mutations at other conserved residues (Figure 3)
          L90M has been reported in isolates from patients treated with saquinavir, nelfinavir,
indinavir, and ritonavir. L90M either contributes to or directly confers in vitro resistance to each
of the seven approved PIs (Condra et al., 1996; Patick et al., 1998; Lawrence et al., 1999; Hertogs
et al., 2000; Para et al., 2000; Dronda et al., 2001; Kempf et al., 2001; Kempf et al., 2002;
Colonno et al., 2003; Marcelin et al., 2003a). Crystal structures with and without the mutant have
shown that the Leu90 side chain lies next to Leu24 and Thr26 on either side of the catalytic
Asp25 (Mahalingam et al., 1999; Olsen et al., 1999; Mahalingam et al., 2001) but the mechanism
by which L90M causes PI resistance is not known.

        Mutations at codon 73, including G73C/S/T, have been reported in 10% of patients
receiving indinavir and saquinavir as their only PI and less commonly in patients receiving
nelfinavir as their only PI (Shafer et al., 1999; Wu et al., 2003). However, this mutation occurs
most commonly in patients failing multiple PIs, usually in conjunction with L90M (Kantor et al.,
2002; Wu et al., 2003).

        Mutations at position 88 (N88D and N88S) commonly occur in patients receiving
nelfinavir and occasionally in patients receiving indinavir. By itself, a mutation at this position
causes low-level resistance to nelfinavir and indinavir. However, mutations at this position causes
high-level nelfinavir resistance in the presence of D30N or M46I (Colonno et al., 2000;
Petropoulos et al., 2000; Ziermann et al., 2000). N88S (but not N88D) has been shown to
hypersensitize isolates to amprenavir (Ziermann et al., 2000).
        L24I has been reported primarily in HIV-1 isolates from patients receiving indinavir
(Condra et al., 2000b) and has not been shown to confer cross-resistance to other PIs, except
possibly lopinavir (Kempf et al., 2001).

          L33F has been reported primarily in persons treated with ritonavir or amprenavir (Molla
et al., 1996; Maguire et al., 2002). Its effect on PI susceptibility levels has not been studied.
However, it has gained attention recently because of its association with lack of response to the
experimental PI, tipranavir (McCallister et al., 2003). In contrast, L33I/V are polymorphisms in
untreated persons and their effect, if any, on drug resistance is not known.

         Polymorphic sites contributing to resistance (Figure 3)
         Amino acid variants at several polymorphic positions also make frequent contributions to
drug resistance but only in combination with drug-resistance mutations at non-polymorphic
positions. Mutations at positions 10, 20, 36, and 71 each occur in up to 5 to 10% of untreated
persons infected with subtype B viruses. However, in heavily treated patients harboring isolates
with multiple other PI-resistance mutations, the prevalence of mutations at these positions
increases dramatically. Mutations at positions10 and 71 increase to 60 to 80%, whereas mutations
at positions 20 and 36 increase to 30 to 40% (Hertogs et al., 2000; Wu et al., 2003). Position 63 is
the most polymorphic protease position. In untreated persons about 45% of isolates have 63L
(considered the subtype B consensus), about 45% have 63P, and about 10% have other residues at
this position. However, the prevalence of amino acids other than L increases to 90% in heavily
treated patients (Yahi et al., 1999; Wu et al., 2003). Mutations at positions 77 and 93 increase in
prevalence from about 25% in untreated persons to about 40% in heavily treated persons (Wu et
al., 2003). I93L is statistically associated with multiple PIs; whereas V77I is statistically
associated only with nelfinavir.

          In some HIV-1 subtypes, mutations at codons 20, 36, and 93 occur at higher rates than
they do in subtype B isolates (Cornelissen et al., 1997; Pieniazek et al., 2000; Gonzales et al.,
2001). In contrast, mutations at positions 63 and 77 usually occur more commonly in subtype B
than in non-B isolates. It has been hypothesized that individuals harboring isolates containing
multiple accessory mutations may be at a greater risk of virologic failure during PI therapy (Perez
et al., 2001; Perno et al., 2001). However, most studies have not supported this hypothesis (Bossi
et al., 1999; Harrigan et al., 1999; Kuritzkes et al., 2000; Alexander et al., 2001; Frater et al.,
2001; Perez et al., 2001; Perno et al., 2001; Servais et al., 2001).

        Additional treatment-associated mutations
        In a recent analysis of 2,244 protease isolates from 1,919 persons, 45 protease positions
were more likely to be mutant in isolates from treated compared with untreated persons, 17
positions exhibited polymorphisms that were unrelated to treatment, and 37 positions rarely, if
ever varied (Wu et al., 2003). The 45 treatment-associated positions included 23 positions
previously associated with drug resistance that are described above and 22 positions that had not
previously been associated with drug resistance. Thirteen of the 22 newly described treatement-
associated positions (positions 11, 22, 23, 45, 58, 66, 74, 75, 76, 79, 83, 85, 85) were highly
conserved in untreated persons. Nine of the newly described treatment-associated positions
(positions 13, 34, 35, 43, 45, 55, 62, 72, 89) were polymorphic in untreated persons. Several of
these mutations have also been described in other recent publications containing analyses of large
databases (Parkin et al., 2003; Wang et al., 2003). The phenotypic and clinical impact of these
mutations is not yet known because they rarely occur in the absence of other known drug-
resistance mutations and have not been studied in vitro (Wu et al., 2003).

        PI cross-resistance patterns and salvage therapy
         In a study of over 6000 HIV-1 isolates tested for susceptibility to indinavir, nelfinavir,
ritonavir, and saquinavir, 59% to 80% of isolates with a 10-fold decrease in susceptibility to one
PI also had a 10-fold decrease in susceptibility to at least one other PI (Hertogs et al., 2000). In a
study of 3000 HIV-1 isolates, resistance to indinavir, ritonavir, and lopinavir were highly
correlated (Parkin et al., 2001). Isolates that were resistant to these drugs were generally also
resistant to nelfinavir; however, isolates resistant to nelfinavir due to D30N were not resistant to
other drugs.

         Susceptibilities to saquinavir and amprenavir are less well correlated with one another
and with susceptibilities to the other PIs (Race et al., 1999; Schmidt et al., 2000a; Kemper et al.,
2001; Parkin et al., 2001), although isolates that are highly resistant to amprenavir are often cross-
resistant to lopinavir (Parkin et al., 2003). Atazanavir selects for a unique protease mutation in
previously untreated persons, I50L, but most of the mutations that confer resistance to other PIs,
appear to also confer atazanavir resistance (Colonno et al., 2003).

         Patients in whom nelfinavir-resistant isolates arise after nelfinavir treatment often
respond to a regimen containing a different PI because D30N and N88D/S confer little cross-
resistance to other PIs (Zolopa et al., 1999b; Kemper et al., 2001). But because as many as 15%
of nelfinavir failures may be associated with mutations at positions 46 and/or 90, virologic failure
during nelfinavir does not guarantee susceptibility to other PIs (Patick et al., 1998; Atkinson et
al., 2000; Saah et al., 2003). Nelfinavir is usually unsuccessful as salvage therapy because most of
the mutations that confer resistance to other PIs confer cross-resistance to nelfinavir (Lawrence et
al., 1999; Hertogs et al., 2000; Schmidt et al., 2000b; Walmsley et al., 2001).

         In a study of ritonavir/saquinavir salvage therapy using the hard gel capsule formulation
of saquinavir (400-600 mg twice daily), the number of mutations at positions 46, 48, 54, 82, 84,
and 90 predicted the virologic response at 4, 12, and 24 weeks. Patients with three or more of
these mutations had no virologic response to salvage therapy (Zolopa et al., 1999b). Decreased
phenotypic susceptibility also predicted a reduced virologic response in this cohort (Zolopa et al.,
1999b). However, nine patients with isolates having mutations at positions 82 and 90 and at either
or both positions 46 and 54 had no virologic response to ritonavir/saquinavir salvage despite the
fact that their isolates were found to be phenotypically susceptible to saquinavir or to have only
low-level reductions of saquinavir susceptibility (Zolopa et al., 1999b; Zolopa et al., 2001).

        There are few data on the genotypic predictors of response to indinavir/ritonavir salvage
therapy. In two small published studies, adherence, indinavir levels, and the number of PI-
resistance mutations at positions 46, 48, 54, 82, 84, 90 were predictive of virologic response
(Shulman et al., 2002; Campo et al., 2003).

         In vitro susceptibility studies suggest that patients failing other PIs often have isolates
that retain susceptibility to amprenavir (Race et al., 1999; Schmidt et al., 2000a). Data on the
utility of amprenavir for salvage therapy, however, are limited (Falloon et al., 2000; Klein et al.,
2000; Descamps et al., 2001; Duval et al., 2002). In the NARVAL ANRS 088 trial, the presence
of fewer than four of the following mutations -- L10I, V32I, M46IL, I47V, I54V, G73S,
V82A/T/F/S, I84V, L90M -- was associated with a 1.6 log10 RNA reduction 12 weeks after the
administration of an amprenavir-containing regimen (Descamps et al., 2001). The presence of
exactly four mutations was associated with a 0.6 log10 RNA reduction. In another study,
suppression of plasma HIV-1 RNA levels to <400 copies/ml during treatment with
amprenavir/ritonavir was associated with having fewer than 6 of the following mutations
(L10FIV, K20MR, E35D, R41K, I54V, L63P, V82AFTS, I84V) (Marcelin et al., 2003b). Of
note, the mutations at positions 35 and 41 are common polymorphisms and have not been
associated with PI resistance in any previous analyses.

         In a study of salvage therapy with a regimen containing lopinavir and efavirenz, the
number of mutations at positions 10, 20, 24, 46, 53, 54, 63, 71, 82, 84, and 90 predicted the level
of phenotypic resistance and the virologic response after 24 weeks of therapy (Kempf et al., 2000;
Kempf et al., 2001). A decreased response to therapy was observed only in those patients that had
six or more of the listed mutations. Subsequent analyses have suggested that mutations at
positions 10, 20, 46, 54, and 82 may be more predictive than the other mutations listed (Calvez et
al., 2001; Molla et al., 2001) and that other mutations, including V32I, I47V/A, I50V, and G73S
may contribute to resistance in patient cohorts with different antiretroviral treatment experience
(Harrigan et al., 2001; Prado et al., 2002; Bongiovanni et al., 2003; Parkin et al., 2003). Lopinavir
has also proven highly effective as salvage therapy when combined with nevirapine in NNRTI-
naive patients failing their first PI regimen (Benson et al., 2002).

        During in vitro passage experiments atazanavir-resistant isolates develop mutations at
positions 32, 50, 84, and/or 88, a pattern of mutations that differs from but overlaps with the
mutations developing in patients treated with other PIs (Gong et al., 2000). In patients receiving
atazanavir as their first PI, the most common drug resistance mutation to develop, I50L, causes
resistance to atazanavir alone, while hypersensitizing to other PIs. However, two of eight
atazanavir failures had mutations at positions 46 and/or 82 in addition to I50L (Colonno et al.,
2002) suggesting that susceptibility to other PIs may not be guaranteed. The usefulness of
atazanavir for salvage therapy is currently being studied in phase III clinical trials (Badaro et al.,

        These studies suggest that because of the high cross-resistance between the approved PIs,
the choice of a PI for salvage therapy depends primarily on the drug levels that are likely to be
achieved. The presence of mutations known to preferentially affect one drug (e.g. G48V and
saquinavir, I50V and amprenavir), will occasionally also influence the choice of salvage therapy.
However, many combinations of mutations produce only subtle differences in susceptibility
between available drugs. Clinical studies are needed to determine the usefulness of the protease
genotype or phenotype at pointing to a preferred boosted PI for salvage.

         Investigational PIs
         Tipranavir is the investigational PI at the most advanced state of clinical development.
The potency of tipranavir in vitro (i.e. IC50 of wildtype isolates in a standard susceptibility assay)
and in vivo (reduction in plasma HIV-1 RNA levels in previously untreated individuals) has not
been well described (Plosker & Figgitt, 2003). However, tipranavir has a remarkably high genetic
barrier to resistance. After prolonged in vitro passage, mutations at positions 32, 33, 45, 82, and
84 have been selected leading to a virus with 14-fold reduced susceptibility (Doyon et al., 2002).
However, most PI-resistant clinical isolates, even those with >10-fold resistance to the original
four PIs (saquinavir, indinanvir, ritonavir, and nelfinavir) rarely have more than 2-fold resistance
to tipranavir (Larder et al., 2000).

         Reduced susceptibility of clinical isolates obtained from persons with other PIs appears to
require three of the following four mutations: L33F/I/V, V82A/F/L/T, I84V, L90M (Cooper et al.,
2003). Phase II salvage therapy studies have shown that the optimal response to tipranavir occurs
when 500 mg of tipranavir is administered with 200 mg of ritonavir twice daily (Yeni et al.,
2003). In heavily treated persons harboring viruses resistant to most other PIs, 14 days of boosted
tipranavir reduced plasma HIV-1 RNA levels by 1.2 logs provided baseline tipranavir
  susceptibility was reduced by <2-fold (Yeni et al., 2003). No virologic suppression was observed
  with viruses having >2-fold reduction in susceptibility.

  Figure 1. Structural Model of HIV-1 Protease Homodimer Labeled with Protease Inhibitor
  Resistance Mutations

The polypeptide backbone of both protease subunits (positions 1-99) is shown. The active
site, made up of positions 25-27 from both subunits, is displayed in ball and stick mode. The
protease inhibitor resistance mutations are shown for the subunit on the left but not for the
mirror-image subunit on the right. The protease was co-crystallized with indinavir which is
displayed in space-fill mode. This drawing is based on a structure published by Chen et al.
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Figure 2. Schematic Representation of How the Protease Recognizes Nine Cleavage Sites to
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