Human immunodeficiency virus type-1 protease inhibitors

Pharmacology & Therapeutics 86 (2000) 145–170

Associate editor: P.K. Chiang

Human immunodeficiency virus type-1 protease inhibitors: therapeutic successes and failures, suppression and resistance Ronald Swanstroma,b,*, Joseph Erona,c a

UNC Center for AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA b Department of Biochemistry and Biophysics, CB7295, Room 22-006 Lineberger Building, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA c Division of Infectious Diseases, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

Abstract The retroviral protease (PR) is responsible for cleaving precursor proteins that contain the virion structural proteins and enzymes. Highly potent inhibitors of the human immunodeficiency virus type-1 PR have been developed, and to date, five of these inhibitors have been approved for clinical use. These inhibitors bind to the active site of the dimeric PR and represent transition state analogs. Combination therapy in which a potent protease inhibitor is combined with inhibitors of the viral DNA polymerase reverse transcriptase can result in the apparent complete suppression of virus replication. Low virus loads associated with suppressed replication are resulting in dramatic reductions in the rate of disease progression. However, incomplete suppression of virus replication results in the selection of resistant variants. Resistance to protease inhibitors is the result of mutations within the PR coding domain, and most of these mutations are able to contribute to cross-resistance among this class of inhibitors. © 2000 Elsevier Science Inc. All rights reserved. Keywords: HIV-1; Protease; Inhibitor; Resistance; Therapy Abbreviations: AIDS, acquired immunodeficiency syndrome; AMV, avian myeloblastosis virus; APV, amprenavir; AUC, area under the time/concentration curve; CA, capsid; d4T, stavudine; ddI, didanosine; Gag, group-specific antigen; HGC, hard gel capsule; HIV-1, human immunodeficiency virus type-1; IDV, indinavir; IN, integrase; MA, matrix; NC, nucleocapsid; NFV, nelfinavir; NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI, nucleoside analog reverse transcriptase inhibitor; PCR, polymerase chain reaction; PI, protease inhibitor; PR, protease; RT, reverse transcriptase; RTV, ritonavir; SGC, soft gel capsule; SQV, saquinavir; 3TC, lamivudine; TPV, tipranavir; ZDV, zidovudine, AZT.

Contents 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Discovery of the retroviral protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Validation of the human immunodeficiency virus type-1 protease as a target. . . . . . . . . . . 4. Development of transition state analog inhibitors of the human immunodeficiency virus type-1 protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Protease inhibitors in clinical use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Ritonavir. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Indinavir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Saquinavir. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Nelfinavir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Amprenavir. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Measuring resistance to human immunodeficiency virus type-1 protease inhibitors. . . . . . 6.1. Biological resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Biochemical resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Primary, secondary, and compensatory mutations. . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Cleavage site mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. Cross-resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Protease inhibitors in combination therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Protease inhibitors and nucleoside analog reverse transcriptase inhibitors . . . . . . . .

* Corresponding author. Tel.: 919-966-5710, fax: 919-966-8212. E-mail address: [email protected] (R. Swanstrom). 0163-7258/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S0163-7258(00)00 0 3 7 - 1

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7.2. 7.3.

The use of two or more protease inhibitors in combination. . . . . . . . . . . . . . . . . . . . Protease inhibitors combined with non-nucleoside reverse transcriptase inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Salvage therapy for protease inhibitor treatment failures . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. New protease inhibitors in development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1. ABT-378. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Tipranavir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The availability of potent inhibitors of the human immunodeficiency virus type-1 (HIV-1) protease (PR) has made it possible to design combinations of inhibitors for therapy that are able to suppress detectable virus replication in infected individuals and to slow, if not suspend, the disease course of HIV-1 infection. In this review, we will discuss the discovery and development of the retroviral PR as a target, the development and use of protease inhibitors (PIs), and the nature of resistance to these inhibitors associated with therapy failure. The dramatic improvements in therapy options and potential long-term outcome have given new importance to issues of compliance, resistance, and salvage therapy strategies. The review will focus mostly on work done with PIs that have been approved for human use. However, work involving other inhibitors will be reviewed where important concepts were first brought to light, and several new inhibitors under development will be presented.

2. Discovery of the retroviral protease The synthesis of large precursor proteins followed by proteolytic processing to generate mature, active proteins is a common theme among many virus families. For some viral protein precursors, host enzymes carry out the proteolytic cleavage events; for example, the cleavage of surface glycoprotein precursors by signal peptidase and the Golgi-localized furin-like PRs. However, in many cases, the virus encodes its own PR to cleave precursor polyproteins. The production of infectious virus invariably requires an active viral PR. For this reason, viral PRs are attracting increasing attention as targets for small molecule inhibitors. The first indication that retroviruses use a strategy of large precursor proteins came from the observation that in cells infected with the avian retrovirus avian myeloblastosis virus (AMV), the initial protein synthesized is much larger than the proteins found in virus particles (Vogt & Eisenman, 1973; Vogt et al., 1975). This precursor contains the major proteins of the core of the virus particle. In the precursor form, the large protein is called the Gag protein, for groupspecific antigen (since these proteins are conserved among groups of related retroviruses). The Gag protein always includes the three major virion proteins: matrix (MA, involved in membrane targeting to the site of particle assem-

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bly); capsid (CA, the major protein involved in the internal core structure); and nucleocapsid (NC, responsible for binding viral genomic RNA as part of the assembly process). These proteins function in the Gag precursor, since virus particles are made in the absence of proteolytic processing, but they require release from the precursor polyprotein to allow the virus particle to be infectious (see Section 3). This general pattern of synthesis of a Gag precursor followed by proteolytic processing was subsequently established for many retroviruses, including murine leukemia virus, mouse mammary tumor virus, human T-cell leukemia virus, and others (reviewed in Swanstrom & Wills, 1997), including HIV-1 (Erickson-Viitanen et al., 1989; Gottlinger et al., 1989; Gowda et al., 1989; Krausslich et al., 1988; Mervis et al., 1988; Robey et al., 1985). The existence of a retroviral precursor protein implied the existence of a PR capable of cleaving the precursor into the mature virion proteins. The first demonstrations of this PR were with AMV and related avian retroviruses (Dittmar & Moelling, 1978; Vogt et al., 1979; von der Helm, 1977) and with murine leukemia virus (Yoshinaka & Luftig, 1977). The PR was found associated with the virus particle, and was shown to have specificity for the correct cleavage of its Gag substrate (reviewed in Oroszlan & Luftig, 1990). Subsequently, virion-associated PR activity has been detected in other retroviruses, including HIV-1 (Lillehoj et al., 1988). A major advance in our understanding of the nature of the retroviral PR came with the identification of a highly conserved sequence within the retroviral genome that encodes a protein sequence representing the active site of an aspartic proteinase (Toh et al., 1985). The aspartic proteinases are a class of eukaryotic enzymes that have the sequence motif Asp-Ser/Thr-Gly at the active site, and include such enzymes as pepsin, renin, and the lysosomal enzyme cathepsin D. The eukaryotic enzymes are single-chained proteins that have two of these motifs comprising the active site; this organization was proposed to be the result of a gene duplication/fusion event (Tang et al., 1978). The retroviral genome encodes a single active-site motif, and this led to the correct prediction that the active form of the viral enzyme would be a homodimer (Pearl & Taylor, 1987). Since the retroviral PR is a sequence-specific processing enzyme, it is more properly called a proteinase. However, its initial appellation of PR is more widely used, and we will follow the popular convention.

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The dimeric form of the retroviral PR and its relationship to the aspartic proteinase family of enzymes were proven with the solution of the crystal structure of the PR from AMV (Miller et al., 1989) and HIV-1 (Lapatto et al., 1989; Navia et al., 1989; Wlodawer et al., 1989). These structures show the two aspartic acid residues coordinating a water molecule, used to hydrolyze the target peptide bond, with the aspartic acid residues lying on the floor of an elongated substrate-binding cleft. The cleft is lined with hydrophobic amino acids that participate in the recognition of substrate. The top of the cleft is covered with a mobile flap, a region of protein from each subunit that forms a ␤ turn over the substrate-binding cleft, but can move away to let substrate enter and products leave. The major elements of the dimer interface are at the active site and a four-stranded ␤ sheet involving the N- and C-terminal strands of each dimer. Fig. 1 shows the dimeric HIV-1 PR with an inhibitor bound in the active site. The PR is encoded in the pro gene of the virus (Fig. 2). The pro gene of different retroviruses is always found between the gag gene, encoding the major structural proteins of the virus particle, and the pol gene, encoding the viral enzymes reverse transcriptase (RT) and integrase (IN). In the case of HIV-1, the pro and pol genes are in the same reading frame. The gag and the pro-pol reading frames overlap by ⵑ200 nucleotides. The Gag protein is synthesized using full-length viral RNA as mRNA. However, ⵑ5% of the time, a ⫺1 frameshifting event occurs at a specific site within the overlap region, allowing translation to continue in the ⫺1 reading frame. This results in the synthesis of a Gag-Pro-Pol fusion protein at about 1/20th the abundance of the Gag precursor (reviewed in Jacks, 1990). The GagPro-Pol precursor is directed to the sites of particle assem-

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bly using the Gag targeting signal. Dimerization of the GagPro-Pol precursor leads to the initial source of PR activity. One model is that a small amount of active dimer PR in the Gag-Pro-Pol precursor cleaves monomer Gag-Pro-Pol precursors to release PR monomers that then dimerize and carry out most of the cleavage events (Swanstrom & Wills, 1997). Like PR, both RT and IN must dimerize to be active.

3. Validation of the human immunodeficiency virus type-1 protease as a target The clearest demonstration of the critical role the viral PR plays in the replication cycle has come from genetic approaches. When mutations were placed in the murine leukemia virus genome (Crawford & Goff, 1985; Katoh et al., 1985) or the HIV-1 genome (Gottlinger et al., 1989; Kohl et al., 1988) that inactivated the PR, virus particles were produced, but these particles were not infectious. This genetic approach validated the concept that an inhibitor of the viral PR would be effective at blocking the virus life cycle. Furthermore, an inhibitor of the PR blocks a late step in the virus life cycle, i.e., the production of infectious virions. This is distinct from the inhibitors of the viral DNA polymerase, RT, which block the synthesis of viral DNA during an early stage of the virus life cycle. Thus, PR inhibitors have the potential to block the production of infectious virus after a cell is infected, which DNA synthesis inhibitors cannot do. However, given the rapid turnover of infected cells in the body (Perelson et al., 1996) this distinction has not had a significant practical impact on therapeutic strategies. The genetic knockout experiment represents the extreme case where all PR activity is lost, a very high standard to

Fig. 1. Dimeric structure of the HIV-1 PR with a bound PI. The two subunits of the dimer are shown in amber and lavender. The active site aspartic acid residues are shown below the inhibitor in stick form. This figure was created using Ribbons from the structure determined by Erickson et al. (1990).

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Fig. 2. Organization of the HIV-1 genome. The cis-acting replication signals are shown along with the mature protein products generated from the Gag and Gag-Pro-Pol precursors. The positions of the splice donor (SD) and splice acceptor (SA) sites used to generate subgenomic mRNAs are shown. The polypurine tract at the boundary of U3 (PPT) and the internal polypurine tract (cPPT), both used in the priming of the second strand of viral DNA synthesis, are indicated. The position of the Rev response element (RRE) is shown also.

reach with small molecule therapy. It has been of interest to understand the extent to which PR activity must be inhibited to effect loss of virus particle infectivity. This question has been approached in two ways. First, PR mutants with varying levels of residual activity have been tested for their ability to support virus replication. Mutant PRs retaining only 25% enzyme activity will still support virus replication, while a mutant with only 2% remaining activity cannot (Konvalinka et al., 1995; Rose et al., 1995). The second approach involved the titration of a PI to determine the extent to which cleavage events had to be inhibited to affect virion infectivity. When total processing events in Gag were inhibited ⵑ50%, there was an 80-fold drop in particle infectivity (Kaplan et al., 1993). Taken together, these results suggest that PR activity is in excess over its required cleavage events and that a large excess of PR activity must be inhibited to lead to a loss in virion infectivity. In addition, the large effect on infectivity after only moderate inhibition of processing events suggests that the proteolytic processing/ virion assembly pathway is more sensitive to inhibition than the PR itself. What is the mechanism of the loss of infectivity through inhibition of PR activity? The answer to this question will ultimately be found through an understanding of how virion proteins interact in the context of the Gag and Gag-Pro-Pol precursors versus their interactions as mature proteins. In the absence of processing of Gag, virus particles are made that have an immature morphology (Fig. 3). These structures are interpreted as the entire Gag protein being anchored in the membrane envelope through interactions of the MA protein domain at the N-terminus of Gag. With processing, the CA and NC proteins are released from the Gag precursor and reorganized into a condensed core in a structure that is associated with infectious virus. One of the determinants that regulates this change in structure is likely to

be a ␤ hairpin that forms with the N-terminal region of the CA protein (Gitti et al., 1996). This hairpin forms when the N-terminal amino acid of CA, which is generated by proteolytic cleavage, forms a salt bridge with an internal aspartic acid. This salt bridge cannot form in the Gag precursor, which suggests that the ␤ hairpin is a structure that is unique to the processed form of CA. A role for this hairpin is further suggested by the observation that the presence or absence of N-terminal extensions of the CA protein mediate the formation of two different forms of particle structures in vitro (Gross et al., 1998). Other differences between immature and mature virions are less well characterized. There are differences in the stability of the dimeric RNA genome, with immature virions having a less stable association between the dimeric RNAs (Fu et al., 1994; Fu & Rein, 1993).

Fig. 3. HIV-1 virion forms. (a) Particles assembling and budding at the cell membrane. (b) An immature virus particle. (c) Mature forms of HIV-1.

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Finally, infection of cells with immature virus particles shows an early block in replication, with significant reductions in viral DNA synthesis (Kaplan et al., 1994a). These comparisons represent the extreme case of no PR activity, again a circumstance that is hard to achieve with inhibitors in vivo. An examination of the virus structure under circumstances where cleavage events were partially inhibited showed that these virus particles, which were largely noninfectious, had an aberrant internal core structure (Kaplan et al., 1993) (Fig. 4). Thus, the partial inhibition of processing had a dramatic impact on the nature of the core structure that appeared. Presumably the aberrant core structure contributes to the loss of particle infectivity.

4. Development of transition state analog inhibitors of the human immunodeficiency virus type-1 protease The identification of the viral PR as an aspartic proteinase led to an initial screening of existing inhibitors of this class of enzymes. While it was possible to demonstrate some sensitivity to some of these inhibitors (Katoh et al., 1987; Richards et al., 1989; Seelmeier et al., 1988), again supporting the designation as an aspartic proteinase, the initial molecules did not have sufficient potency to warrant their development as clinical agents. The aspartic proteinase designation also brought an important strategy to drug design borrowed from efforts to inhibit renin: the placement of a hydroxyl group in the inhibitor at the position equivalent to the scissile bond at the cleavage site. The hydroxyl group displaces the active site water molecule and engages the active site aspartic acids in interactions mimicking the transition state of water adding across the peptide bond. All of the potent HIV-1 PIs developed to date use this strategy. The hydroxyl group is presented in the form of hydroxyethylene,

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hydroxyethylamine, or hydroxyethylamino sulfonamide in the inhibitors that are approved or in clinical evaluation. While the hydroxyl substituent provides a specific mechanism for inhibiting an aspartic proteinase, it does not contain the specificity needed to target the HIV-1 PR. This specificity comes from a consideration of its protein target. The naturally occurring substrates of the HIV-1 PR represent a diverse set of protein sequences (Fig. 5). The substrate of the PR must be at least seven amino acids long for efficient cleavage (Darke et al., 1988; Moore et al., 1989). The extreme heterogeneity in cleavage site sequences implies either that the PR has multiple discrete modes of interaction or that there are many combinations of possible interactions between the PR and its target sequence. Another feature of these cleavage sites is that not all sites are cleaved with equal efficiency (Krausslich et al., 1988), which indicates that some of the cleavage site sequences may be suboptimal, perhaps as part of the regulation of ordered cleavage. An analysis of cleavage site sequences has led to the appreciation of the general features of increased hydrophobicity flanking the cleavage site (scissile bond) and the potential for some flanking polar interactions (Henderson et al., 1988; Pettit et al., 1991). Analysis of crystal structures with bound inhibitors has provided the structural basis for these interactions, and has shown that bound inhibitors (and presumably substrate) assume a ␤-sheet conformation that includes interactions with the peptide backbone (reviewed in Wlodawer & Erickson, 1993). The ␤-sheet results in the placement of alternating side chains of the bound inhibitor pointing into the same side of the substrate-binding cleft. These side chains extend into pockets or subsites in the enzyme, and it is the sum of these side chain interactions with the subsites that determines the PR specificity. An example of inhibitor side chains extending into the enzyme subsites on one wall of the substrate-binding cleft is shown in Fig. 6.

Fig. 4. Aberrant virion morphology with partial inhibition of PR activity. Virus particles made in the absence (a) or the presence of increasing inhibitor concentration are shown. At the highest concentration, immature forms of virions are seen (c, d, g, h). At intermediate concentrations, novel forms with eccentric cores are seen (b, f). Reproduced from Kaplan et al. (1993).

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large hydrophobic side chains near the active hydroxyl of the inhibitor to fill the enzyme subsites, and the potential for some peptide backbone interactions between the enzyme and the inhibitor. These inhibitors are all much shorter than the length of peptide required for cleavage, and they all have incorporated features that improve the solubility of these otherwise poorly soluble molecules.

5. Protease inhibitors in clinical use Fig. 5. Sequence heterogeneity of the processing sites found in the HIV-1 Gag and Gag-Pro-Pol protein precursors. The amino acid sequences of the individual processing sites are given in single letter code from the P4 though the P3⬘ positions. The scissile bond is represented by the slash. The protein products are indicated in capital letters. TF, transframe; RH, RNase H. Adapted from Pettit et al. (1993).

Knowledge of substrate sequences and of the mode of binding based on the initial PR/inhibitor structures (see, e.g., Miller et al., 1989) led to a “structure-informed” strategy of drug development that led to the design of an initial set of inhibitors that proved the concept of inhibition of the HIV-1 PR (Ashorn et al., 1990; Erickson et al., 1990; Meek et al., 1990). This knowledge has ultimately led to successful drug discovery by a number of research groups. These inhibitors are highly potent in cell culture assays and highly specific for the HIV-1 PR. While their initial designs were based on peptidomimetics, few natural features of the peptide substrate remain. They share several features, including

The potential of HIV-1 PIs suggested from in vitro experiments was quickly tested in clinical trials. In initial studies, the PIs were given as single agents in single- or multiple-dose studies to define pharmacokinetic parameters of each of the agents and to establish initial antiretroviral activity. The clinical development stage of PIs coincided with the development of assays based on quantitative amplification using the polymerase chain reaction (PCR) to measure the amount or copy number of HIV RNA in blood plasma (Mellors et al., 1995, 1996). Unlike previous assays that measured HIV levels (such as p24 antigen assays or culture of virus from peripheral blood mononuclear cells or plasma, which were positive only in a minority of infected individuals), HIV-1 RNA levels were measurable in most patients by these newer techniques. These assays proved to be sensitive measures of both HIV replication and the inhibition of HIV replication via antiretroviral agents (Ho et al., 1995; Wei et al., 1995). The range of HIV RNA levels in untreated infected individuals is remarkable. In the absence of therapy, a small number of individuals may have levels

Fig. 6. The side wall of the substrate binding cleft. The inhibitor A-77003 is visualized extending alternating side chains into the side of the substrate-binding cleft. The residues are identified and color coded to indicate interactions with specific inhibitor side chains or placement between side chains and interaction with both. This figure was created with MacImdad using the structure determined by Erickson et al. (1990).

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⬍1000 copies of viral RNA per mL or even below measurable limits, and these individuals tend to progress slowly over time (Mellors et al., 1997). Other individuals may have HIV RNA levels of 500,000 copies/mL or more and rapid disease progression. On average, HIV RNA levels in plasma tend to rise slowly over time (0.1 log10/year), although the rate of rise is also variable between individuals and may be more precipitous prior to a clinical decline (Gange et al., 1998). Currently, the lower limits of detection for the most widely available assays are between 400 and 500 copies per mL. More sensitive assays with limits of detection of 50 copies per mL are now available. HIV-1 RNA values are usually analyzed after log10 transformation. Because of individual variation and variation in the assays, changes of up to 3-fold (0.5 log10) may not be significant and changes of 10- to 1000-fold (1.0–3.0 log10) or more can be seen with potent therapy. The measurement of HIV RNA levels in blood plasma became an essential tool for the assessment of activity of new antiretroviral compounds. PIs that were developed initially had poor oral bioavailability and were administered through intravenous infusion. However, improvements in the solubility of agents within this class of compounds led to the development of agents that had enough oral bioavailability to allow for larger scale clinical development. Currently, five compounds—ritonavir (RTV), saquinavir (SQV), indinavir (IDV), nelfinavir (NFV), and amprenavir (APV) (Kempf et al., 1995; Patick et al., 1996; Roberts et al., 1990; Vacca et al., 1994; Partaledis et al., 1995) (Fig. 7)—have been approved by the United States Food and Drug Administration for the treatment of HIV-1 infection. As noted in Section 4, these inhibitors share the important hydroxyl group and large hydrophobic substituents. Several factors are important determinants of potency in vivo of these highly active compounds. The oral bioavailability of the currently available PIs ranges from 4% for SQV (in its original hard gel capsule [HGC/Invirase] formulation) to greater than 70% for RTV and NFV (Flexner, 1998). In addition to the challenges in the development of PIs placed by the potential for low oral bioavailability, protein binding of PIs offered another obstacle. Plasma protein binding, which occurs to predominantly two proteins, albumin and ␣-1-acid glycoprotein, ranges from 60% with IDV to greater than 98% for RTV, NFV, and SQV (Flexner, 1998). The extent and avidity of protein binding influence the amount of free drug that is available for entry into cells and for clearance by metabolic pathways. One compound, SC52151, which showed substantial antiretroviral activity in vitro, had limited antiretroviral activity when given orally to HIV-infected individuals because, at least in part, of tight protein binding (Fischl et al., 1997). The evaluation of PIs includes not only antiretroviral activity, but also pharmacokinetics, which may help predict antiviral activity and durability, and toxicity of these agents. Peak, trough, and overall blood concentrations over time (or area under the time/concentration curve, AUC) are mea-

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Fig. 7. Structures of HIV-1 PIs.

sured both with a single dose and with chronic dosing (steady state). Trough concentrations are frequently compared with an arbitrary measure of in vitro activity for a given agent, such as the concentration in vitro that produces a 50 or 90% inhibitory effect (IC50 or IC90), usually determined in the presence of human plasma proteins (Molla et al., 1997). Trough levels or the ratio of trough levels to in vitro IC50 or IC90 may be one way to compare different compounds with different pharmacokinetics, protein binding, and in vitro activity. Trough levels or AUC measurement may correlate with durability of activity and the development of resistance in vivo (Molla et al., 1998). These parameters need to be explored with larger numbers of patients and with other PIs. 5.1. Ritonavir Initial studies of RTV (ABT-538, Norvir) administered as a single agent demonstrated that the compound had antiretroviral activity over a range of doses from 200 to 600 mg twice daily (Danner et al., 1995; Markowitz et al., 1995b). A clear dose-response relationship was observed, with the highest dose level (600 mg twice daily) having the most du-

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rable antiretroviral response. Lower dosing schemes resulted in substantial antiretroviral effects, but loss of suppression of HIV-1 levels in plasma over a relatively brief period of time (Fig. 8). Doses of RTV higher than 600 mg twice daily proved intolerable (Hicks et al., 1996), and the dose of 600 mg twice daily was chosen for further development. RTV has relatively high (⬎98%) protein binding, but remains active in vitro in the presence of human plasma proteins (Flexner, 1998). 5.2. Indinavir Initial clinical studies of IDV sulfate (MK-639, Crixivan) demonstrated that plasma concentrations measured by AUC increased with increasing doses, and trough concentrations exceeded the IC95 for HIV-1 in cell culture (Teppler et al., 1993). The agent is not tightly bound by human plasma, with the unbound fraction of the compound equal to 40–60% (Vacca et al., 1994). HIV-1-infected subjects given 600 mg of IDV three times a day achieved mean peak concentrations and mean trough concentrations ⵑ50 and 3 times the in vitro IC95 for clinical isolates (Stein et al., 1996). The most potent antiretroviral activity as measured by changes in HIV-1 RNA levels was observed with 2400 mg/day (Steigbigel et al., 1996; Stein et al., 1996). Daily doses higher than 2400 mg/day did not appear to increase antiretroviral effect (Steigbigel et al., 1996). Multiple larger studies including an IDV monotherapy arm (Gulick et al., 1997; Hirsch et al., 1997; Massari et al., 1995, 1996) showed similar findings. IDV monotherapy resulted in declines in HIV-1 RNA ranging from 1.5 to 3.1 log10 over 12–

24 weeks. Sustained responses, i.e., HIV-1 RNA level below quantifiable limits for 6 months, were seen in ⵑ40% of the subjects in each study. While use of PI monotherapy is not recommended (Carpenter et al., 1998), this proportion of success with IDV as a single agent speaks to the overall potency of this agent. 5.3. Saquinavir In its original formulation, SQV (Ro 31-8959, Invirase, SQV-HGC) has a bioavailability of ⵑ4% when given with a high fat meal. Concentrations are decreased ⵑ5-fold if SQV is given in the fasting state. The low bioavailability is due in part to extensive first-pass metabolism in the intestinal wall and in the liver by the cytochrome P450 enzyme system. SQV-HGC was studied as monotherapy in HIV-1-infected men who had minimal symptoms related to their disease, CD4 counts ⬍500 cells/␮L, and no previous antiretroviral therapy (Kitchen et al., 1995). In this trial, patients received up to 600 mg three time a day for 16 weeks, which produced a peak median percent decrease in HIV-1 RNA of 80% (or ⵑ0.7 log10). Higher doses of SQV-HGC have been tested. A dose of 3600 mg/day resulted in a maximal mean decrease in plasma HIV RNA levels of 1.06 log10 RNA copies per mL of plasma, and 7200 mg/day produced a mean maximal decrease in the plasma HIV RNA level of 1.34 log10 RNA copies per mL of plasma. Higher plasma drug concentrations in individual patients correlated with greater reductions in plasma HIV RNA levels over the two doses (Schapiro et al., 1996). A more bioavailable formulation of SQV has been developed, SQV-soft gel capsules (SGC) (Fortovase), although this formulation has not been tested as monotherapy. SQV-SGC not only has a greater bioavailability than SQV-HGC, but the recommended dose is twice as high at 1200 mg three times daily, resulting in substantially higher plasma concentrations with the newer formulation. 5.4. Nelfinavir

Fig. 8. Dose response of virus load to increasing concentrations of RTV. This figure shows the change in blood plasma HIV-1 RNA levels observed with four different doses of RTV, each given twice daily. Also shown is the number of subjects from whom results were obtained at each time point. Although antiretroviral effects are seen with each dose, the most potent effects are seen with 600 mg twice daily. Only the highest dose demonstrates activity that is beyond 28 weeks. Adapted from Danner et al. (1995). Patient groups (at twice daily dosing): 䊉, 300 mg; 䊏, 400 mg; 䊊, 500 mg; 䊐, 600 mg.

Nelfinavir mesylate (NFV, Viracept) has an oral bioavailability of ⬎75% and high protein binding (Flexner, 1998). NFV has a half-life of ⵑ4 hr, and when given at a dose of 750 or 1000 mg three times a day, trough concentrations remain above the IC95 of NFV for the wild-type HIV measured in the presence of ␣-1-acid glycoprotein (Markowitz et al., 1998). There is a dose response in HIV-1 RNA effects and CD4 effects at NFV doses from 500 mg twice daily to 1000 mg three times daily. A total daily dose of 1500 mg or less resulted in a decline in antiviral activity over 28 days, after a peak effect at 14 days (Markowitz et al., 1998). Subjects treated with 750 or 1000 mg three times daily had an antiviral effect of 1.5 log10 or greater over 28 days, and 60% of the subjects had RNA levels fall below the level of detection (500 copies per mL). However, unlike with IDV, virtually all the subjects who remained on NFV monotherapy after 28 days had HIV RNA levels return to or

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exceed the HIV-1 RNA level they had prior to starting therapy (Markowitz et al., 1998). NFV currently is administered at either 750 mg three times daily or 1250 ␮g twice daily. 5.5. Amprenavir APV (GW-141, Vertex-478) is the most recently approved PI in the United States. This agent has a long halflife of 7–9.5 hr, and its oral bioavailability is unaffected by food (Painter et al., 1995). In some studies, the protein binding of this compound in the presence of physiologic concentrations of plasma proteins has been shown to be relatively slight (Lazdins et al., 1997; Livington et al., 1995) and the in vitro potency of APV high (St Clair et al., 1996). Higher concentrations of ␣-1-acid glycoprotein may have a substantial negative effect on APV activity (Lazdins et al., 1997). APV monotherapy resulted in an almost 2 log10 decrease in HIV-1 RNA levels in plasma over 28 days of therapy (Schooley, 1997). However, in a larger trial that compared APV monotherapy with APV/lamivudine (3TC)/ zidovudine (ZDV, AZT) the antiretroviral effect of APV monotherapy was not sustained. After a median follow-up of 88 days, greater than one-third of the subjects had a return in their HIV RNA levels to baseline or to 1 log10 or higher than their nadir value (Murphy et al., 1999). Only 26% of the subjects who reached 12 weeks of APV monotherapy had HIV-1 RNA levels below the limit of quantification. Because of these results, treatment with APV monotherapy was ceased and, therefore, long-term data from subjects treated for prolonged periods with APV monotherapy are not available.

6. Measuring resistance to human immunodeficiency type-1 protease inhibitors Two approaches have been used to document the effect of resistance-associated mutations on drug sensitivity. The most commonly used approach is to test a virus carrying relevant mutations for a change in sensitivity relative to the parental (non-mutated) virus. In this case, a requirement for more inhibitor to block virus replication is used as a measure of resistance, with the readout being the IC50 or IC90. The second approach involves producing recombinant PRcarrying mutations of interest and measuring a change in Ki. In theory, these two approaches should give comparable answers. In general, mutations that confer resistance in enzymatic assays can also be demonstrated to confer resistance in the context of virus replication. However, the magnitude of the effect in these two systems is not always the same (Klabe et al., 1998). 6.1. Biological resistance In the strictest sense, the term “a resistant virus” or “drug resistance” should refer to a virus that is not affected by the drug in a therapeutic setting. A virus may have reduced sensitivity to a drug, but still be impacted by that drug, if suffi-

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ciently high drug levels can be attained in a patient. However, for ease of discussion, we will refer to a reduction in sensitivity as resistance, here in the mechanistic sense, not as a clinical outcome. The measurement of virus sensitivity to inhibition of replication is made in one of two ways. The most common approach is to take a virus isolate, or pool, after selection (either in vitro or in vivo) and test its sensitivity. Virus pools can represent mixtures of sequences that can complicate attempts to draw conclusions about the effects of specific mutations. However, after strong selective pressure, a virus pool is usually sufficiently homogeneous to have a predominant species. The alternative approach is to reconstruct the mutations of interest into a molecular clone of the viral genome and generate a virus stock from the DNA clone. In this case, it is important to maintain the virus stock at a low passage number to limit further evolution. Commercial tests that combine features of the ease of working with virus generated from recombinant DNA with the ability to sample patient virus populations directly are becoming available. One assay involves generating RTPCR products from a patient sample and cloning the mixture into a viral vector that can be assayed for drug sensitivity in a single-step replication cycle (Parkin et al., 1999, Petropoulos et al., 2000). Another assay recombines the PCR product with a deleted viral genome to generate a virus stock that is then assayed for drug sensitivity in a virus growth assay (Hertogs et al., 1998). These types of assays, along with the analysis of sequence changes associated with resistance, are being used retrospectively and prospectively to evaluate the utility of this information in making clinical decisions (e.g., see Harrigan et al., 1998; Baxter et al., 1999; Condra et al., 1999; Deeks et al., 1999; Durant et al., 1999; Hammer et al., 1999; Zolopa et al., 1999). The initial reports of selection for resistance were with several symmetric PIs, designed to take advantage of the near symmetry of the PR dimer (Erickson et al., 1990). Otto et al. (1993) reported that clonal virus isolates carrying the active site mutation V82A were 6- to 8-fold less sensitive to the inhibitor P9941, and that a recombinant PR with this mutation also showed reduced sensitivity. Several groups (Ho et al., 1994; Kaplan et al., 1994b; Tisdale et al., 1995) carried out selections with A77003 and obtained virus stocks between 10- and 30-fold resistant. Ho et al. (1994) and Tisdale et al. (1995) also reported reduced sensitivity for virus generated from several mutated molecular clones, showing the impact of mutations at positions 8, 32, and 46, while Kaplan et al. (1994b) showed changes in enzyme sensitivity in the range of 7- to 50-fold for active site mutations at 32 and 82. Selection with a nonsymmetrical inhibitor demonstrated a potential role for the active site residue 84 in contributing to a resistance phenotype (El-Farrash et al., 1994). Initial selections with the inhibitor SQV demonstrated the potential to select for reduced sensitivity in the range of 10- to 30-fold. However, the nature of the mutations in-

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volved was not examined (Craig et al., 1993; Dianzani et al., 1993). In a subsequent study, a selected virus pool with G48V, I54V, and L90M mutations showed an increase in resistance of greater than 50-fold (Eberle et al., 1995). Another resistant virus pool selected with SQV had predominant mutations of M36I, G48V, L63P, and L90M, and showed a 30-fold change in the IC50. In addition, the mutational pattern was stable to 20 passages in the absence of inhibitor (Jacobsen et al., 1995). The single mutations of G48V and L90M were 8- and 3-fold resistant, respectively, when placed in a molecular clone, while the double mutant was 20-fold resistant (Jacobsen et al., 1995). Similar, although slightly different, values were reported by other workers for viruses with these mutations generated from molecular clones (Maschera et al., 1995; Tisdale et al., 1995). Patients failing the initial hard capsule formulation of SQV frequently have sensitive virus, probably due to poor drug bioavailability, but some patients will have virus that is resistant up to 160-fold (Ives et al., 1997; Jacobsen et al., 1996; Winters et al., 1998; Schapiro et al., 1999a; Vaillancourt et al., 1999), and high drug doses can select for more complex resistance patterns (Schapiro et al., 1999b). An initial selection with IDV to 15-fold resistance occurred with 4 mutations, including a V82A mutation. A molecular clone with these 4 mutations was 6-fold resistant (Tisdale et al., 1995). Patients failing IDV therapy show a wide range of mutations (Condra et al., 1995, 1996). Patient isolates show resistance to 30-fold and greater. Viruses generated from mutated molecular clones showed more modest resistance, in the range of 4- to 8-fold, with up to 5 mutations, including the active site mutations V82T/I84V (Condra et al., 1995). The work with molecular clones showed a strong correlation between the number of mutations, the ability to measure any resistance, and increasing resistance. Selection with RTV to ⵑ30-fold resistance gave a virus pool with predominant mutations M46I, V82F, and I84V, while the latter two mutations as isolated substitutions had changes in IC50 values of 5- and 10-fold, respectively (Markowitz et al., 1995a). In a test of a series of viruses generated from mutated molecular clones, with the mutations selected based on sequences that appeared in patients failing RTV, Molla et al. (1996) showed resistance in the range of 6- to 32-fold. This work demonstrated two important points: (1) the importance of mutations at positions 82 and 54 and (2) the enhanced effect of multiple mutations, including those lying outside of the substrate binding cleft. Patients failing RTV therapy have levels of resistance in the range of 5- to several hundred-fold (Molla et al., 1996; Schmit et al., 1996). It has been possible to select for higher levels of resistance with longer selection schemes. In this work, virus pools showing resistance of several hundred-fold to over 1000-fold have been selected (T. Smith and R. Swanstrom, in preparation). In addition, a molecular clone containing 8 resistance-associated mutations displayed resistance to PIs in the range of 10- to 1000-fold.

Selection with NFV in vitro gives an initial mutation of D30N with ⵑ7-fold resistance, while further selection to 30-fold resistance led to reversion at position 30 and the appearance of mutations at 46 and 84 (Patick et al., 1996). An I84V mutation by itself confers 5-fold resistance (Patick et al., 1996). Isolates from patients failing therapy show resistance in the range of 5- to 80-fold, predominantly with the D30N mutation and a variety of nonactive site mutations, including positions 35, 36, 46, 71, 77, and 88 (Markowitz et al., 1998; Patick et al., 1998). Another study has noted that up to 30% of patients failing NFV do so with an L90M mutation, and this predicts a poorer outcome on subsequent IDV therapy (Condra et al., 1999). Selection with APV to a level of 150-fold resistance gave a virus pool with mutations at 10, 46, and 84, and the unusual mutations I47V and I50V (Partaledis et al., 1995; Pazhanisamy et al., 1998). Virus generated from molecular clones with these two mutations, along with M46I, gave only 10-fold resistance. Variants with the I47V and I50V mutations are selected for in vivo, but other mutations outside of the active site and also at the active site residues 32, 82, and 84 are also seen (De Pasquale et al., 1998; Maguire et al., 1999). A summary of the observed positions of the most common mutations within the PR coding domain that appear in vivo and in vitro (including T. Smith and R. Swanstrom, unpublished) is shown in Fig. 9. It should be noted that the available data for RTV and IDV are significantly greater than for the other inhibitors. In the case of SQV, the potential for observing the full range of resistance mutations has been limited by limited drug exposure with the initial hard capsule formulation. In the case of NFV and APV, the data are limited by the number of patients examined and the length of time on drug. Conversely, our sense of the potential for resistance with IDV and RTV is more complete since in their initial use, patients were maintained on failed therapy for longer periods of time than is currently done. The accumulation of resistance mutations is frequently associated with a decrease in viral fitness or growth capacity. Zennou et al. (1998) have noted a loss in fitness associated with mutations in the protease when comparing patient viruses before treatment with a protease inhibitor with after therapy failure and virus outgrowth. It has been shown previously that selection of virus in vitro for resistance to a protease inhibitor can lead to attenuation (Markowitz et al., 1995a; Croteau et al., 1997). Recently, the contributions of individual mutations of attenuation of replication in vitro has been described (Martinez-Picado et al., 1999), with D30N being more attenuating than L90M, the latter of which was compensated, at least in part, with an L63P mutation, and multiply mutated proteases having more wildtype-like replication properties. One important area of research is whether attenuation of growth can account for persistent improvements in CD4 counts, which are seen in a subset of patients even after experiencing treatment failure and virus rebound (Faye et al., 1999).

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Fig. 9. Summation of residues that are frequently mutated after therapy failure or selection with HIV-1 PIs. Data for sequence changes after treatment with RTV are taken from Molla et al. (1996). Data for sequence changes after treatment with IDV are taken from Condra et al. (1996). Data for sequence changes after treatment with SQV are included from Jacobsen et al. (1996), Schapiro et al. (1996), and R. Swanstrom (unpublished). Data for sequence changes after treatment with NFV are taken from Markowitz et al. (1998). Data for sequence changes after treatment with APV are taken from De Pasquale et al. (1998).

6.2. Biochemical resistance Recombinant PRs have been used to test directly the effect of specific mutations on enzyme activity and sensitivity to inhibitors. Several studies have looked at large collections of mutant PRs for the effect of specific mutations on sensitivity to a variety of inhibitors. In general, single mutations outside of the active site have little effect on inhibitor sensitivity, although the presence of multiple nonactive site mutations can have a measurable and significant effect (Olsen et al., 1999). The two most common active site mutations, at positions 82 and 84, do have measurable effects on sensitivity. An I84V mutation increases the Ki for RTV, IDV, and SQV by 5- to 10-fold (Gulnik et al., 1995; Partaledis et al., 1995; Vacca et al., 1994), although higher values for SQV and RTV and a lower value for IDV have been reported (Wilson et al., 1997). Position 82 mutations (especially A and F) have much larger effects on sensitivity to IDV and RTV than SQV (Gulnik et al., 1995; Wilson et al., 1997). An I50V mutation increases the Ki for APV 80-fold, and affects the Ki for SQV and IDV in the range of 10- to 20-fold (Partaledis et al., 1995). Several groups have studied the effects of the L90M and G48V mutations on sensitivity to SQV. Ki increases of 3- to 20-fold have been reported for the L90M change, 13- to 200-fold for the G48V change, and 400- to 1000-fold for the double mutant (Ermolieff et al., 1997; Maschera et al., 1996; Wilson et al., 1997). Maschera et al. (1996) also documented that the basis of resistance was reduced affinity of the enzyme for the inhibitor manifested by an increase in the rate of dissociation of the inhibitor from the enzyme. Some structural information is available about the nature of resistance. A V82A mutation results in changes in the enzyme backbone to retain some elements of hydrophobic interaction with the shorter 82A side chain (Baldwin et al.,

1995). A V82T substitution creates an unfavorable hydrophilic interaction, while an I84V substitution creates an unfilled space in an enzyme carrying these two active site mutations (Chen et al., 1995). In the latter example, the PR also carried mutations at positions 46 (M to I) and 63 (L to P), which affected the conformation of the flap. In comparing the structure of an unliganded simian immunodeficiency virus PR, Rose et al. (1998) noted that many nonactive site resistance mutations lie at the interface of five rigid domains of the protease, suggesting that these mutations may function in altering the movement of these domains relative to each other. In general, the changes in structure are subtle; for example, the single mutant proteases R8Q, K45I, and L90M all bind a substrate analog through similar interactions (Mahalingam et al., 1999). Another effect of some resistance mutations is a reduction in the stability of the protease dimer, perhaps suggesting another mechanism of resistance (Mahalingam et al., 1999; Xie et al., 1999). 6.3. Primary, secondary, and compensatory mutations Primary mutations are considered those that appear first in the course of selection for resistance. Secondary mutations are considered to be those mutations that further decrease drug sensitivity, but appear subsequent to the primary mutation. Compensatory mutations have no effect on drug sensitivity, but increase enzyme efficiency to compensate for the deleterious effects of the primary and secondary mutations on enzyme activity. While these definitions are simple and convenient, the small magnitude of the effect of any single mutation makes the unequivocal determination of mechanism challenging. Primary mutations have been described for several of the inhibitors. Mutations at position 90 (L90M) are frequently seen as the only mutation for patients receiving low dose

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SQV (Jacobsen et al., 1996). Mutations at position 82 are seen initially in patients failing RTV therapy (Eastman et al., 1998; Molla et al., 1996). Patients failing NFV usually have a D30N mutation (Markowitz et al., 1998; Patick et al., 1998). However, the concept of primary mutations must be viewed in the context of the level of selective pressure. Selections in vitro with SQV can also lead to the initial appearance of an I84V mutation (Vaillancourt et al., 1999) or a G48V mutation (Tisdale et al., 1995). Thus, higher selective pressure may change the temporal pattern of mutations seen with SQV. Similarly, the D30N mutations seen in patients taking NFV represents an intermediate level of resistance since selection in vitro can proceed to higher levels of resistance with the loss of the D30N mutations (Patick et al., 1996). In the cases of L90M and D30N, it is likely that viruses with these mutations give the best replication properties for the level of selection that is applied. In this sense, they are not primary mutations along a longer mutational pathway, but rather, an initial stopping point. In contrast to RTV, evolution of resistance to IDV appears to be able to follow a greater variety of starting paths (Condra et al., 1995, 1996). It is not clear whether this variety of paths is due to differences in the starting PR sequence that can restrict the range of useful mutations or due to stochastic effects among a series of mutations that can each contribute a moderate level of resistance. A secondary mutation either adds to the resistance of the primary mutations (enhancing its effect) or adds another level of resistance in its own right. The former appears to be the case for APV resistance with the I47V mutation in conjunction with an I50V mutation (Partaledis et al., 1995), although the I47V mutation has been observed, linked to V32I, in the absence of I50V (Maguire et al., 1999). Inference of a secondary mutation has been made with the addition of an I54V mutation to a position 82 mutation, which resulted in further virus load rebound in a patient failing RTV (Eastman et al., 1998). Given that there are a large number of mutations associated with resistance outside of the active site, it is tempting to attribute them to compensatory effects. However, the range of effects compensatory mutations might have is largely unexplored, and in only a few cases has a compensatory effect been documented. In one case, a position 10 substitution had a clear phenotype in enhancing replication of a virus carrying several resistance-associated mutations (Rose et al., 1996). In another case, the effect of two common mutations, M46I and L63P, was shown to be an improvement in catalytic efficiency (Schock et al., 1996), although a direct effect of a 46I substitution on drug sensitivity has also been observed (Condra et al., 1995). In general, active-site mutations at positions 82 and 84 have been shown to be deleterious to enzyme activity (Gulnik et al., 1995; Schock et al., 1996; Vacca et al., 1994; Wilson et al., 1997), as have mutations at position 48 (Ermolieff et al., 1997; Maschera et al., 1996; Wilson et al., 1997; Mahalingam et al., 1999), providing the selective pressure for compensatory muta-

tions. However, the magnitude of the deleterious effect that has been measured varies, perhaps due to the use of different substrates (Ridky et al., 1998). 6.4. Cleavage site mutations A novel type of compensatory mutation that has been observed is at PR cleavage sites. Multiple active site mutations might be expected to result in a PR with altered specificity, which could lead to the subsequent evolution or co-evolution of the target cleavage site sequences. However, this is not what appears to have happened in the present circumstance. Rather, two sites have been observed to evolve to make them “better” sites, rather than “different” sites. The effect has been to improve their rate of cleavage, perhaps to keep them in sync with the ordered cleavage that occurs in the Gag precursor as part of the assembly/maturation process. Mutations have appeared at the two processing sites in the NC-p1-p6 Gag processing intermediate p15. After extensive selection with the inhibitor BILA 2185 BS, mutations appeared at both the NC-p1 site (RQAN-FLG to RRVN-FLG) and the p1-p6 site (PGNF-LQS to PGNFFQS) (Doyon et al., 1996). These changes improved cleavage by both the mutant and the wild-type enzymes, and they represented the only cleavage site mutations found. In addition, the cleavage site mutations improved the replication kinetics of viruses with resistance mutations in the PR. Zhang et al. (1997) have detected similar mutations in patients failing IDV therapy, and have shown that the presence of cleavage site mutations enhances the replication of resistant virus in vitro. Similar results have been reported studying patients who have failed RTV or SQV therapy (Mammano et al., 1998). 6.5. Cross-resistance The issue of cross-resistance is complex. Ultimately, the most important measure of cross-resistance, or lack of it, is shown by the sequential treatment of patients with two different PIs with an assessment of the potency and duration of effect of the second inhibitor compared with its effect in drug-naive patients. While such studies are only now beginning, the initial impression is that sequential therapy with a regimen containing only one PI after therapeutic failure with a regimen containing a different PI will be difficult. Finding exceptions to this trend represents one of the highest priorities of HIV clinical research. The use of regimens in which two PIs are combined may improve antiretroviral response in individuals that have already received one PI (Gulick et al., 1999a; Tebas et al., 1999). The general observation is that when several mutations are present, there is usually a moderate level of cross-resistance. Most claims for an absence of cross-resistance have been made when only one mutation is present, usually one that by itself confers only a moderate level of resistance to the selecting inhibitor. With moderate numbers of mutations, cross-resistance is not complete, i.e., the change in

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IC50 is usually greater for the selecting inhibitor than for the inhibitors being tested for cross-resistance. Is the glass half full or half empty? Incomplete cross-resistance would imply residual potency with other inhibitors. However, this potency is clearly somewhat reduced, and the constellation of mutations needed to give high-level resistance to the second inhibitor invariably includes many that were initially selected. Thus, there is likely to be a reduction in both potency and the pathway to resistance. The potential for cross-resistance even after only moderate selection in vitro was demonstrated for a wide range of inhibitors (Tisdale et al., 1995). Subsequent work with selection to very high levels of resistance has shown that increasing selection confers increasing resistance and cross-resistance (T. Smith and R. Swanstrom, unpublished). Similarly, the potential for cross-resistance has been demonstrated for patient isolates from patients failing IDV (Condra et al., 1995), RTV (Molla et al., 1996), and SQV (Winters et al., 1998; Dulioust et al., 1999). The level of resistance and cross-resistance varies greatly between patients, and the predictors of subsequent therapy success are just starting to be identified (Deeks et al., 1999; Lorenzi et al., 1999; Piketty et al., 1999). In one small study, three patients failing NFV therapy had virus isolates that remained sensitive to the other three approved PIs (Markowitz et al., 1998). However, patients failing NFV may have different responses to the subsequent protease inhibitor, based on the predominant mutation selected (Condra et al., 1999). Important issues that need to be explored are the role of the predominant mutations in determining outcome, the potential role of minor mutations in the population, and the utility of developing more potent protease inhibitors or ones that are less affected by resistance mutations. In an initial use, resistance testing to determine appropriate HIV treatment appears to improve treatment response (Durant et al., 1999).

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ing this result (Carpenter et al., 1998; Centers for Disease Control and Prevention, 1998a). 7.1. Protease inhibitors and nucleoside analog reverse transcriptase inhibitors In a large trial designed to show the clinical benefit of RTV, RTV was added to existing therapy of subjects who had advanced HIV-1 disease (as defined by CD4 T-cell counts ⬍100 cells/␮L) (Cameron et al., 1998). Although for most subjects RTV was given with a combination of other agents, some subjects received RTV as monotherapy. Because RTV was added to existing therapy, the combination of this PI and nucleosides in this trial would not be considered optimal. Despite this suboptimal use, the addition of RTV resulted in dramatic clinical effects. Progression to new acquired immunodeficiency syndrome (AIDS)-defining illnesses was significantly delayed when the addition of RTV was compared with the addition of placebo (Cameron et al., 1998). In addition, survival was prolonged in the RTV-treated group (Fig. 10). The use of a potent PI in combination with two nucleoside agents has become one of the recommended standards for the therapy of previously treatment-naive HIV-infected individuals (Carpenter et al., 1998; Centers for Disease Control and Prevention, 1998a). A substantial body of information now exists on the effect of PI combination regimens on HIV viral load and on clinical improvement and survival (Palella et al., 1998). One of the first studies to

7. Protease inhibitors in combination therapy The use of combinations of nucleoside analogs and the use of PIs as monotherapy or when added to a failing regimen have been shown to have substantial antiretroviral activity and clinical benefit (Anonymous, 1997; Cameron et al., 1998; Eron et al., 1995; Hammer et al., 1996; Katzenstein et al., 1996). However, therapy that only partially suppresses HIV-1 load can be expected to result in rebound of viral load, resistance to antiretroviral agents, and eventual loss of clinical effect. Currently, the goal of antiretroviral therapy is to suppress HIV-1 replication and load to the greatest extent possible, that is, to below the detectable limits of the most sensitive assays available (Carpenter et al., 1998; Centers for Disease Control and Prevention, 1998b). Aggressive inhibition of replication is associated with more durable suppression of plasma HIV-1 RNA levels. The initiation of therapy with multiple agents, usually including a potent PI, is one of the recommended standards for achiev-

Fig. 10. Extended survival due to therapy with a potent protease inhibitor. This figure is a Kaplan-Meier analysis demonstrating the proportion of subjects who survived and remained free of a new AIDS-defining diagnosis while being treated with either RTV or placebo. The number of subjects observed at each time point is also shown. The addition of RTV to ongoing antiretroviral therapy significantly delays the onset of new AIDS-defining conditions or death compared with placebo. The hazard ratio for an AIDSdefining event or death for RTV relative to placebo was 0.53 (95% CI 0.42–0.66, P ⬍ .0001). After 16 weeks, subjects receiving placebo who experienced an AIDS-defining event were allowed to receive RTV. Adapted from Cameron et al. (1998).

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demonstrate the profound effects of a PI plus two nucleoside analog RT inhibitors (NRTIs) on HIV-1 RNA levels compared IDV, 3TC, and ZDV to IDV alone and to ZDV/ 3TC (Gulick et al., 1997). In this study, the 3-drug combination resulted in over 80% of the subjects achieving HIV RNA levels below the limit of quantification at 6 months of treatment. This level of effectiveness has persisted in the subjects initially treated with 3-drug therapy for ⬎100 weeks (Fig. 11) (Gulick et al., 1998) and now ⬎148 weeks (Gulick et al., 1999b). IDV, 3TC, and ZDV have also been shown to have potent effects on HIV-1 RNA levels and CD4 cell counts in very advanced patients with CD4 cell counts ⬍50 cells/␮L (Hirsch et al., 1997). Similar potent results were seen using IDV with didanosine (ddI) and ZDV (Massari et al., 1996), IDV with 3TC and stavudine (d4T) (Squires et al., 1998), and IDV with ddI and d4T (Eron et al., 1998b). In treatment of naive subjects, NFV in combination with ZDV and 3TC also resulted in suppression of HIV RNA in 75% of the subjects over a 6-month period (Saag et al., 1997), and this activity also seems to persist over time. SQV-SGC (Fortovase) appears to be more potent that SQVHGC (Thompson, 1998) and to have similar potency and antiretroviral efficacy compared with IDV when combined with ZDV and 3TC (Boucher & Borleffs, 1998). When given in combination with ZDV and 3TC, APV gives relatively similar antiviral results when compared with the results achieved with other PIs combined with these nucleosides (Murphy et al., 1999). In combination with nucleoside analogs, IDV has also been shown to improve survival and to delay HIV-1 disease progression (Hammer et al., 1997). In this trial, subjects with CD4 cell counts ⬍200 cells/␮L, who had received ZDV and other nucleosides, but not 3TC or PIs, were randomized to receive either ZDV plus 3TC or the 3-drug regimen of ZDV, 3TC, and IDV. The addition of 3TC to ZDV-containing regimens had been shown to improve survival and delay disease progression in one trial (Anonymous, 1997). Despite this fact, the 3-drug regimen of ZDV/3TC/IDV resulted in a marked improvement in survival and a significant delay in HIV-1 disease progression compared with ZDV/3TC. The relative risks of death and disease progression were both improved by over 50% (Table 1) (Hammer et al., 1997). A cautionary note to this trial was added when the virology data were examined (Demeter et al., 1998). Demeter and colleagues demonstrated that only approximately one-half of the subjects in this trial treated with IDV/3TC and ZDV had HIV RNA levels fall and remain below detectable limits after 48 weeks. In the subgroup of subjects who entered the study with CD4 cell counts ⬍50 cells/␮L, the proportion of subjects with HIV RNA levels below quantifiable limits at 48 weeks approached 40%. In approximately one-half of those subjects, HIV-1 could still be cultured from their peripheral blood mononuclear cells using a simple co-culture assay. Recent reports also demonstrate that even in patients who have had their HIV-1 RNA levels in plasma suppressed below quantifiable limits by PI-containing combination regimens,

HIV-1 can be cultured using sophisticated techniques from their peripheral blood mononuclear cells (Finzi et al., 1997; Wong et al., 1997). These results suggest that even though HIV-1 replication can be profoundly suppressed by PI-containing combination regimens in many patients, the virus is not eradicated from these individuals. 7.2. The use of two or more protease inhibitors in combination Each of the currently available PIs has limitations that affect the effectiveness of these agents or at best, make longterm adherence to them challenging. Some of these limitations include low bioavailability, short half-lives, toxicity, food requirements for administration, and potentially limited potency. The use of two PIs in combination has been proposed as a way to overcome some of these limitations. In order to explore more potent regimens and/or to take advantage of pharmacokinetic interactions, combinations of PIs are under active study in the laboratory and in the clinic. Current limitations on PI potency may be predominantly due to inadequate exposure of HIV to the inhibitor. The best example of this concept is the PI SQV, with its bioavailability of 4–20%, depending on the drug formulation. Optimal exposure to SQV and, indeed, probably to any of the currently available PIs is limited by bioavailability, protein binding, drug half-life, formulation (i.e., pill number, size, and dose frequency), and/or toxicity. Combining PIs may improve bioavailability and reduce dosing frequency through pharmacokinetic interaction. The use of two PIs may increase overall exposure to PI concentrations by administering two PIs that do not have substantial overlapping toxicity, even if full doses of each inhibitor are required. Multiple PI combinations are under active study in clinical research trials. The combination of RTV and SQV has been most extensively studied. RTV enhances the pharmacokinetic profile of SQV dramatically by inhibiting the P450-mediated metabolism of SQV in the gut and liver. Administration of SQV with 400 mg of RTV increases SQV levels by 20- to 50-fold (Kempf et al., 1996). Several different dosing combinations of RTV and SQV have been studied in a randomized trial in PI-naive patients (Cameron et al., 1999). Over 72 weeks, fully 90% of the subjects who remained on therapy had HIV RNA levels ⬍200 copies per mL. Approximately one-quarter of these subjects added nucleoside analogues to their regimen after 12 weeks on RTV/ SQV alone. The dose that was best tolerated was 400 mg of RTV and 400 mg of SQV, both given twice daily. These doses have been the ones most commonly used in subsequent studies. Diarrhea, asthenia, and nausea were the most common adverse events at these doses. This combination is also being used extensively in protease inhibitor-experienced patients. Some investigators have been able to demonstrate reasonable antiviral responses, depending on previous PI experience and timing of institution of the RTV/SQV regimen (Tebas et al., 1999; Hall et al., 1999).

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Fig. 11. Apparent suppression of virus replication with combination therapy. This figure shows the proportion of subjects with HIV RNA levels in blood ⬍500 copies per mL (a) and ⬍50 copies per mL over the 100-week duration of study treatment. The number of subjects available for analysis at each time point is also listed. At study initiation, subjects previously had been treated with ZDV, but were receiving IDV, 3TC, or both for the first time. After 6 months of therapy, all subjects receiving IDV or ZDV/3TC received therapy with all three agents. Adapted from Gulick et al. (1998).

RTV has also been studied in combination with NFV and IDV. When 400 mg of RTV twice daily is given in combination with NFV at either 500 or 750 mg twice daily, the AUC concentration for NFV is similar to what is seen with the approved dose of NFV (750 mg three times daily). With the higher dose of NFV, the AUC concentration of M8 (the active metabolite of NFV) is raised even further (Flexner et al., 1998). The antiretroviral effect of this combination was substantial in the small number of subjects studied (Gallant et al., 1998). Of the 10 subjects who received the higher dose of NFV, 6 out of the 7 subjects who remained on treatment had HIV RNA levels below 400 copies per mL at 16 weeks of study. Moderate to severe diarrhea was a common side effect seen in almost one-half of the 20 subjects treated. Recently, RTV has also been combined with IDV. IDV, although potent, has practical limitations because currently recommended dosing is 800 mg every 8 hr on an empty or near-empty stomach. However, when 400 mg of IDV is given with 400 mg of RTV, both twice daily, AUC measurements are almost identical, and higher trough concen-

trations and lower peak concentrations are seen than when IDV is given alone at 800 mg every 8 hr. These results are seen even when the RTV/IDV is given with a meal (Hsu et al., 1998). This combination has demonstrated potent antiretroviral effects in treatment-naive subjects receiving RTV/ IDV plus 3TC and d4T (Workman et al., 1998). Adverse events seen with this combination appear mild, but the follow-up period of observation is short. No episodes of nephrolithiasis were observed. IDV, 800 mg twice daily with either 100 or 200 mg of RTV twice daily, is also being studied (Saah et al., 1999; Burger et al., 1999). IDV has also been studied with NFV in a small Phase I/II pharmacokinetic trial. Given with 1000 mg of IDV twice daily, doses of NFV at 750 mg and 1000 mg twice daily resulted in low NFV trough concentrations. At 24 weeks of study, 11 of 18 subjects who received study medications had plasma HIV RNA levels below 500 copies per mL, 6 were below 50 copies per mL. Diarrhea was reported in one-third of the study subjects and kidney stones were also seen (Saah et al., 1998). IDV at 1200 mg twice daily and

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Table 1 Rates of disease progression in ACTG 320

All subjects AIDS or death Death CD4 ⭐ 50 cells/␮L AIDS or death Death CD4 51–200 cells/␮L AIDS or death Death

ZDV/IDV/3TC [N (%)]

ZDV/3TC [N (%)]

577 33 (6) 8 (1) 219 23 (11) 5 (2) 358 10 (3) 3 (1)

579 63 (11) 18 (3) 220 44 (20) 13 (6) 359 19 (5) 5 (1)

Hazard ratioa

P Valueb

0.50 (0.33–0.76) 0.43 (0.19–0.99)

.001 .042

0.49 (0.30–0.82) 0.37 (0.13–1.04)

.005 .51

0.51 (0.24–1.10) 0.59 (0.14–2.46)

.08 .46

a

Numbers in parentheses are 95% confidence interval. Log-rank test. Adapted from Hammer et al. (1997).

b

1250 mg twice daily are the doses now being used to achieve optimal concentrations of both agents. NFV combined with SQV has been studied in small pharmacokinetic trials and in a larger antiretroviral efficacy trial. Single-dose studies suggested that NFV would increase SQV levels by ⵑ5-fold (Merry et al., 1997), although the effect in different individuals was quite variable. However, because of the inductive effects of NFV on the P450 liver enzyme system, the effects of NFV on SQV concentrations over the long term may be less marked. In a larger study of 157 subjects, 54 of whom were randomized to SQV-SGC 800 mg three times daily plus NFV 750 mg three times daily, this combination without added nucleosides had a relatively modest antiretroviral effect (Moyle, 1998). The proportion of subjects with HIV-1 RNA levels ⬍50 copies over time was similar or less than the proportion seen with either PI alone with two nucleosides. This dual PI combination given with two NRTIs had the most potent antiretroviral effect in this study, as measured by the proportion of subjects below 50 copies. The NFV/SQV combination, given twice daily as 1250 mg and 1200 mg, respectively, along with 1 NRTI, has similar activity to SQV-SGC plus 2 nucleosides (Cohen et al., 1999). APV has been studied in combination with IDV, NFV, or SQV to obtain multi-dose pharmacokinetic data and preliminary antiretroviral activity. Each compound was dosed 3 times daily. IDV and NFV were given at their approved doses, and SQV-SGC was administered at 800 mg three times daily, as was APV. Preliminary results show potent activity for each of the combinations, although patient numbers were small and follow-up was short (Eron et al., 1998a). Only one subject withdrew from this study due to an adverse event over the first 24 weeks of study. Overall, combinations of two PIs appear to increase potency over single PI therapy, although head-to-head comparisons of two PI regimens with single PI regimens have yet to show dramatic differences (Moyle, 1998; Pedersen et al., 1998; Cohen et al., 1999). However, many of these combinations require large numbers of pills each day and the

side-effect profile and cost of some of these regimens may become significant considerations. RTV-based dual PI combinations seem to have the most favorable pharmacokinetics, allowing decreased pill numbers and cost without reducing potency. 7.3. Protease inhibitors combined with non-nucleoside reverse transcriptase inhibitors PIs have been combined with non-NRTIs (NNRTIs), both with and without co-administration of NRTIs. The combination of IDV and the NNRTI efavirenz is the most extensively studied. This combination results in a substantial proportion of subjects achieving HIV RNA levels below 400 copies per mL (⬎80%) in two relatively large clinical trials (Riddler et al., 1998; Staszewski et al., 1999). NFV has also been combined with efavirenz and similar results were seen with ⬎70% of the subjects achieving an HIV RNA level below 400 copies per mL after 16 weeks (Eyster et al., 1998). The use of combination PI/NNRTI regimens with or without NRTIs as initial therapy may be somewhat limited by the concern that individuals who do not achieve plasma HIV RNA levels below detectable limits or who have a relapse of detectable virus may have limited subsequent treatment options.

8. Salvage therapy for protease inhibitor treatment failures PIs are clearly an important component of effective salvage therapy for subjects who have not responded to nucleoside analog therapy. IDV in combination with 3TC and ZDV has been shown to be highly effective in subjects who have had extensive ZDV treatment experience (Gulick et al., 1997, 1998) and in subjects who have had experience with multiple nucleoside analogs, as long as the subjects were naive to 3TC (Hammer et al., 1997). The combination of PI with NNRTIs and nucleosides has also been used suc-

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cessfully to salvage individuals who have had extensive NRTI treatment (Albrecht et al., 1998; Harris et al., 1997). Combination therapy including a PI also seems to be an effective salvage therapy for subjects who have persistent or recurrent evidence of HIV replication on therapy with NNRTIs in combination with nucleoside analogs. The number of studies is small and the duration of follow-up is short (Curry et al., 1998; Para & Weinstock, 1998). However, a good response to PI-based therapy following therapy directed only at RT seems theoretically quite plausible. Strategies for the treatment of individuals who have not had their HIV-1 RNA levels fall below detectable levels or who have had a rebound of HIV-1 RNA levels above detectable levels on their initial PI-containing regimens are currently being evaluated. Little definitive information exists in the literature. The use of IDV or RTV following prolonged SQV therapy had only modest antiretroviral effect (Mayers et al., 1998; Para et al., 1998). The use of SQV in a regimen that does not result in suppression of HIV-1 RNA below detectable limits appears to predispose to IDV resistance, even if resistance mutations cannot be documented at the time when the therapy is changed (Schapiro et al., 1999b). The substantial degree of cross-resistance between PIs (Condra et al., 1995, 1996), especially if the virus has a high level of resistance to one inhibitor, makes the failure of single-PI therapy following failure of another PI not surprising. Current salvage therapies for HIV-1-infected individuals who have failed a PR-containing regimen involve the use of dual PI therapy with one or more agents to which virus from the individual would be expected to be sensitive. The combination of RTV and SQV has been used to treat individuals who have failed single-PI-containing regimens (Cassano et al., 1998; de Truchis et al., 1998; Sampson et al., 1997). The successes of this combination, reported in predominantly small, non-randomized trials, has been quite variable. Following NFV-containing regimens, treatment with RTV/SQV with 3TC and d4T resulted in HIV RNA levels below 400 copies per mL in about two-thirds of the subjects after 6 months of therapy (Tebas et al., 1999). Initial resistance to NFV may result predominantly from a single point mutation that conveys limited cross-resistance to other PIs (Markowitz et al., 1998; Patick et al., 1996). RTV plus SQV-based regimens appear to be less successful following initial failure of an IDV- or RTV-containing regimen (Cassano et al., 1998; Sampson et al., 1997). However, changing from the initial therapy early after the detection of HIV-1 RNA levels in plasma (i.e., early virologic failure) may enhance the response to subsequent dual PI regimens. The level of resistance to PIs appears to increase over time if replication continues in the presence of the agent as additional mutations accumulate (Condra et al., 1995; Molla et al., 1996). Earlier switching to a salvage regimen may limit the degree of cross-resistance between the initial PI regimen and the salvage regimen. Adding a non-nucleoside RT inhibitor to a dual PI salvage regimen may be another way to improve response rates when treating individuals who have

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had virologic failure on their initial PI-containing regimen in those individuals who are naive to NNRTI therapy (Deeks et al., 1999; Farthing et al., 1998).

9. New protease inhibitors in development Although the current PIs that are approved by the United States Food and Drug Administration have demonstrated potent antiretroviral activity and/or clinical benefits, collectively and individually they have limitations. These limitations for some or all of these agents include limited bioavailability, large pill numbers, dosing frequency, dosing schedule with meals, and toxicity. There is also substantial cross-resistance between the currently available PI (as outlined in Section 6.5), and successful salvage treatment of individuals who have failed treatment with a PI is by no means guaranteed. For these and other reasons, additional PIs are needed. 9.1. ABT-378 ABT-378 is a peptidomimetic PI than is ⵑ10 times more active than RTV in vitro (Stewart et al., 1997). ABT-378 has also shown relatively significant in vitro activity against HIV variants that have decreased susceptibility to RTV (Chen et al., 1997; Korneyeva et al., 1997). This agent is extensively metabolized by the P450 3A4 system, but its catabolism is substantially inhibited by RTV. When ABT-378 is administered with RTV in rats, the AUC of ABT-378 is increased 13-fold and the half-life is increased substantially (Kumar et al., 1997). In HIV-1 seronegative volunteers, ABT-378, given over a dose range of 200–600 mg twice daily with RTV either 50 or 100 mg twice daily, resulted in trough drug concentrations 20- to 80-fold greater than the IC50 of ABT-378 against wild-type HIV-1 in vitro in the presence of plasma proteins (Lal et al., 1998). ABT-378 is greater than 98% protein-bound at protein concentrations in the physiologic range (Lal et al., 1998). ABT-378 is being developed only in combination with RTV. Of 100 HIV-1infected persons who were naive to previous antiretroviral therapy and who were administered ABT-378/RTV (100 mg twice daily) plus nucleoside analogues 3TC and d4T, HIV-1 RNA levels in plasma were ⬍ 50 copies per mL of plasma in 79 at 36 weeks of therapy (Eron et al., 1999). No subject discontinued due to ABT-378/RTV toxicity. Because of its activity against RTV-resistant isolates (Korneyeva et al., 1997), ABT-378 may be an effective component of salvage therapy for individuals who have had virologic failure while being treated with a PI. Selection with ABT-378 in vitro has given rise to highly resistant virus (Carrillo et al., 1998). Active site mutations at positions 32, 47, and 84 were seen in addition to mutations outside of the active site. The selected virus showed moderate crossresistance to RTV, but surprisingly low cross-resistance to SQV. HIV-infected individuals with ⬎ 3 months of experience on a single PI-containing regimen, and at least 1000

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copies per mL of HIV RNA, were switched to ABT378/ RTV, NVP, and at least 1 new NRTI. After 36 weeks, 67% of the subjects had HIV RNA levels ⬍ 400 copies per mL plasma on this regimen (Eron et al., 1999). 9.2. Tipranavir Tipranavir (PNU-140690, TPV) is the first nonpeptidic HIV PI to reach clinical development. This agent has been given to HIV seronegative subjects at doses ranging from 300 to 2000 mg as a single dose and then three times a day. Doses of 900 mg three times a day or greater produced trough concentrations above 1 ␮M, the IC90 for wild-type HIV-1 to PNU-140690 in vitro (Borin et al., 1998). This agent has good activity in vitro, is synergistic with NRTIs and NNRTIs, and appears to have activity in vitro against HIV-1 variants that have decreased susceptibility to the currently approved PIs (Poppe et al., 1997). TPV also shows additive to synergistic activity with RTV in vitro, even against RTV-resistant isolates (Chong & Pagano, 1997). Preliminary data in individuals who had never received a PI showed substantial activity of TPV as a single agent, with decreases in HIV-1 RNA in plasma in the range of 1.0 to 1.5 log10 over an 11-day period. A dose response was seen with the highest dose, 1500 mg three times daily, having the greatest activity. No serious adverse events were seen in this early Phase I/II study. An analysis after 12 weeks revealed no distinct mutational pattern suggestive of resistance development. This agent appears to require three times a day dosing, and with the current formulation, the 1500-mg dose requires the ingestion of 10 capsules three times daily. The pharmacokinetics of TPV are significantly improved by the administration of RTV (Baldwin et al., 1999), and a recent in vitro study demonstrated activity of TPV against a broad range of protease inhibitor-resistant clinical variants (Larder et al., 1999). Additional PIs are in clinical development. BMS 232632 and AG-1776 appear active in vitro against at least some PIresistant variants (Gong et al., 1999; Patick et al., 1999). A prodrug of APV, which should improve pill burden and pharmacokinetics, is also in development.

10. Summary PI therapy of HIV-1-infected individuals represents a triumph of basic virology and targeted drug development. Five PIs are widely available in North America and Europe. With the development of PIs, a better understanding of HIV pathogenesis and a better understanding of how to use antiretroviral agents have also developed. This convergence of factors led to the implementation of highly active combination antiretroviral therapy, centered on the potent activity of PIs. These treatments dramatically altered the clinical course of HIV for many infected individuals. Unfortunately, the plasticity of the HIV genome coupled with the high er-

ror rate of HIV RT and the potential for high levels of replication has led to emergence of resistance to PIs both in vitro and in vivo. The consequences of resistance to PIs (and other antiretrovirals) are not yet fully explored, but loss of treatment effects and progression of clinical disease have been noted. Resistance to PIs develops in two loci, either mutations within the PR coding domain that alter amino acids in the PR or mutations that result in changes in one or two of the target or cleavage sites of the PR. Initial mutations in the PR may be selected more commonly with one PI over another, but the pathway to high-level resistance appears to have many common mutational steps. Cross-resistance between currently available PI is common. One important focus of current antiretroviral strategies is the treatment of individuals who have persistent detectable viral replication while on potent PI-containing combination therapy, which frequently involves attempts to inhibit PI-resistant HIV-1 variants. New PIs in development may have increased activity against variants resistant to the first generation of PIs. Development of PIs that have high potency, favorable pharmacokinetics, low daily pill numbers, and reduced side effects is an important goal of drug development and clinical research.

Acknowledgments R.S. is supported in this work by NIH award R01 AI32892. Both authors acknowledge the support of the UNC CFAR (P30-HD37260). The efforts of Dr. Steve Pettit, Dr. John Erickson, and Dr. Ed Collins in preparing the figures is appreciated. The authors appreciate the efforts of Sumathy Arunachalam in compiling the references.

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