D RUG TH ERAPY
Review Article
Drug Therapy A L A S T A I R J .J . W O O D , M.D., Editor
HIV-P ROTEASE I NHIBITORS CHARLES FLEXNER, M.D.
I
NHIBITORS of human immunodeficiency virus (HIV)–encoded protease, combined with nucleoside analogues with antiretroviral activity, cause profound and sustained suppression of viral replication, reduce morbidity, and prolong life in patients with HIV infection.1-3 Recent guidelines recommend that initial treatment of all HIV-infected patients include the administration of an HIV-protease inhibitor.4 THE HIV-ENCODED PROTEASE
The HIV protease, encoded in the 5! end of the pol gene, is expressed as part of the gag–pol polyprotein (Fig. 1). This gene encodes a 99-amino-acid protein. Homodimers of this protein have the aspartyl protease activity that is typical of retroviral proteases; monomers are enzymatically inactive.6 The enzyme’s targets are amino acid sequences in the gag and gag–pol polyproteins, which must be cleaved before nascent viral particles (virions) can mature.7-10 Cleavage of the gag polyprotein produces three large proteins (p24, p17, and p7) that contribute to the structure of the virion and to RNA packaging, and three smaller proteins (p6, p2, and p1) of uncertain function.11 Although mammalian cells contain aspartyl proteases, none efficiently cleave the gag polyprotein.12,13 Three of the HIV-cleavage sites are phenylalanine–proline or tyrosine–proline bonds, which are unusual sites of attack for mammalian proteases.14 Proteolytic cleavage of the gag polyprotein results in morphologic changes in the virion and condensation of the nucleoprotein core. The protease is packaged into virions, and the cleavage events it catalyzes
From the Division of Clinical Pharmacology, Department of Medicine and Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore. Address reprint requests to Dr. Flexner at Osler 524, Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD 21287-5554. ©1998, Massachusetts Medical Society.
occur simultaneously with or soon after the budding of the virion from the surface of an infected cell.15 Proviral DNA lacking functional protease produces immature, noninfectious viral particles.9 MECHANISM OF ACTION OF HIV-PROTEASE INHIBITORS
The four approved HIV-protease inhibitors are based on amino acid sequences recognized and cleaved in HIV proteins. Indinavir (Crixivan), nelfinavir (Viracept), ritonavir (Norvir), and saquinavir (Invirase and Fortovase) and the investigational protease inhibitor amprenavir are structurally related molecules (Fig. 2). Most contain a synthetic analogue of the phenylalanine–proline sequence at positions 167 and 168 of the gag–pol polyprotein that is cleaved by the protease.14 These drugs are difficult to synthesize in large quantities because of their complex structure. HIV-protease inhibitors prevent cleavage of gag and gag–pol protein precursors in acutely and chronically infected cells, arresting maturation and thereby blocking the infectivity of nascent virions.13,16 The main antiviral action of HIV-protease inhibitors is thus to prevent subsequent waves of infection; they have no effect on cells already harboring integrated proviral DNA. These agents are active against clinical isolates of HIV types 1 and 2, with the in vitro concentration of drug required to reduce viral production by 50 percent (IC50) ranging from 2 to 60 nM.16-19 Antiviral activity is correlated with the inhibition of enzyme activity, although the drug concentration required to reduce enzyme activity by 50 percent (K i) is lower than the IC50, ranging from 0.10 to 2.0 nM.16-20 These drugs are inactive or weakly active against human aspartyl proteases, with a K i of at least 10,000 nM for renin and pepsin.16,17 CLINICAL PHARMACOLOGIC PROPERTIES
The clinical pharmacokinetic properties of the four approved protease inhibitors are shown in Table 1. Oral bioavailability varies because of differences in first-pass hepatic metabolism, to which saquinavir is the most susceptible.27 Food also affects bioavailability. A high-fat meal increases the bioavailability of saquinavir and nelfinavir but reduces that of indinavir. The same high-fat meal increases the bioavailability of ritonavir capsules but decreases that of ritonavir liquid. The ingestion of indinavir capsules with light meals has no effect on the area under the plasma-concentration–time curve during an average dosing interval, but variability is large.21 Nelfinavir, ritonavir, and saquinavir should be taken with meals, Vo l u me 33 8
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The New England Journal of Medicine
A
B Figure 1. Structure and Function of HIV-1 Protease. Panel A shows a ribbon diagram of the protease with an inhibitor molecule in the active site (reproduced from Erickson5 with the permission of the publisher). Panel B shows a horizontal 180-degree rotation of the protease (kindly provided by John W. Erickson, National Cancer Institute, Frederick, Md.). Panel C shows the translational products of the HIV gag–pol gene and the sites at which the gene product is cleaved by the virus-encoded protease. p17 denotes capsid protein, p24 matrix protein, and p7 nucleocapsid; p2, p1, and p6 are small proteins with unknown functions. The arrows denote cleavage events catalyzed by the HIV-specific protease.
1282 !
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DRUG THERAPY
DNA
env
gag 5’
3’
pol
RNA
gag–pol
Precursor polyproteins gag
Functional proteins C
p17
p24
p2
p7
p1
and indinavir should be taken after fasting or with a light, low-fat snack. All these drugs are metabolized by cytochrome P-450 enzymes, mainly the 3A4 isoform.26,28,29 The absorption of the drugs is maximal within four hours after ingestion. Their elimination half-lives range from 1.8 to 5 hours. Differences in a drug’s pharmacokinetic characteristics between patients are large, as indicated by a coefficient of variation of 30 percent or higher for the mean area under the curve. All these drugs except indinavir are at least 98 percent protein-bound in plasma. The apparent volume of distribution ranges from 0.4 to 10.0 liters per kilogram of body weight. Alpha1-acid glycoprotein, an inducible plasma protein, reduces the in vitro anti-HIV potency of some protease inhibitors by a factor of 10 or more.30-33 This effect may be mediated by high-affinity binding of the drug to the glycoprotein. Since only free drug is available to penetrate cells, plasma concentrations need to be high enough to override this effect.34 There are limited data on central nervous system penetration of these drugs. The cerebrospinal fluid penetration of ritonavir and saquinavir, defined as the ratio of the cerebrospinal fluid concentration to the simultaneous plasma concentration, is less than or equal to 1 percent.23,35 Indinavir has a higher cerebrospinal fluid penetration, ranging from 2.2 to 76 percent, perhaps because of decreased plasma protein binding.25 Indinavir, ritonavir, and saquinavir are available as capsules, and nelfinavir is available as tablets. Saquinavir is now also available as soft-gel capsules (Fortovase), which triples oral bioavailability.36 Nelfinavir powder and liquid ritonavir are pediatric formulations. Indinavir must be stored in an airtight container with a desiccant.21 Ritonavir is heat-sensi-
p6
Protease
Reverse transcriptase
Integrase
tive and needs to be refrigerated. It also contains alcohol and is therefore contraindicated in patients taking disulfiram or metronidazole.23 DRUG INTERACTIONS
HIV-protease inhibitors can interact with inhibitors or inducers of cytochrome P-450 drug-metabolizing enzymes.37 Table 2 shows the magnitude of interactions between HIV-protease inhibitors and other drugs commonly used to treat HIV infection. In many cases, inhibitors of P-450 increase plasma concentrations of protease inhibitors. For example, concurrent administration of ketoconazole increases the area under the plasma-concentration–time curve by 62 percent with indinavir,21 by 35 percent with nelfinavir,22 and by 300 percent with saquinavir.24 Because the bioavailability of oral saquinavir is poor, this interaction may be advantageous, increasing the amount of drug reaching the systemic circulation. A more important concern is the effect of concurrent therapy with inducers of P-450, such as rifampin and rifabutin, which accelerate the clearance of protease inhibitors. The plasma concentration is therefore decreased, and the efficacy of the drug is likely to be reduced, which may result in the development of resistance to the drug. Rifampin reduces the area under the plasma-concentration–time curve by 92 percent with indinavir,41 by 82 percent with nelfinavir,22 by 35 percent with ritonavir,23 and by 80 percent with saquinavir.24 Rifampin should not be given to patients who require treatment with HIV-protease inhibitors.42 HIV-protease inhibitors alter the pharmacokinetics of other drugs by acting as P-450 inhibitors or hepatic-enzyme inducers (Table 2). All these drugs inhibit cytochrome P-450 enzymes. Ritonavir is the most potent,28 indinavir and nelfinavir are less so,29,37 Vo l ume 33 8
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The New England Journal of Medicine
N
Indinavir
Ph H
OH
N
N
N
H2SO4
O
O
N
OH
H
O HO
Nelfinavir
S
O
N
N
H
NHtBu CH3SO3H
H
OH H
H
N
Ritonavir
S
O
N O
O NH2
H N
O
O
CH3
N
N
Ph
O N
N O
H
S N
O
O
N
O
H
H
Ph
N
Amprenavir
O
N
H
Saquinavir
Ph
OH
OH
NHtBu
H
CH3SO3H
H
NH2 OH N O
S O
Figure 2. Structures of Five HIV-Protease Inhibitors with Antiretroviral Activity in Clinical Trials. NHtBU denotes amino-tertiary butyl, and Ph phenyl.
and saquinavir is the least potent.26 The Ki for the inhibition of terfenadine metabolism is 0.017 mM for ritonavir28 and 0.7 mM for saquinavir,26 a 40-fold difference in potency. Concurrent administration of rifabutin with indinavir, nelfinavir, or ritonavir increases the area under the rifabutin plasma-concentration–time curve by 204 percent,21 207 percent,22 and 350 percent,23 respectively. For patients taking indinavir or nelfinavir who require concurrent therapy with rifabutin, the dose of rifabutin should be decreased to 150 mg daily.21,22,42 Concurrent administration of rifabutin with ritonavir or saquinavir is not recommended.23,24 1284 !
The combination of ritonavir and rifabutin is associated with an increased incidence of rifabutin-associated toxicity (uveitis).43 Clinicians must be aware of potentially toxic drug interactions involving HIVprotease inhibitors and avoid prescribing such drugs as terfenadine, astemizole, cisapride, ergotamines, and potent benzodiazepines such as midazolam and triazolam to patients receiving an HIV-protease inhibitor. Cytochrome P-450 pharmacokinetic interactions may be beneficial when two protease inhibitors are given simultaneously. For example, ritonavir inhibits the hepatic first-pass metabolism of saquinavir, in-
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DRUG TH ERA PY
TABLE 1. PHARMACOKINETICS
DRUG
DOSE†
mg
Indinavir
800 every 8 hr Nelfinavir 750 three times a day Ritonavir 600 twice a day Saquinavir 600 three times a day
APPROXIMATE ORAL BIOAVAILABILITY
EFFECT
OF
FOOD‡
%
%
60–65
"77!
#78
$200 to $300
66–75†† %4
"7 (liquid); $15 (capsules)†† $670
Cmax
mg/ml
7.7
OF
APPROVED HIV-PROTEASE INHIBITORS.*
Tmax
T 1/ 2
0.2
Vd
CSF CONCEN-
TRATION¶
CLEARANCE (ROUTE)
hr
hr
%
%
liters/kg
%
%
0.8
1.8
22–47
60–65
NR
2.2–76**
NR
#98
2.0–7.0
88–90 (hepatic) #78 (hepatic)
30–36
98–99
0.4
46–84
98
10.0
3.0–4.0 2.0–4.0 3.5–5.0 11.2
PROTEIN VARIABILITY§ BINDING
2.0–4.0 3.0–5.0 NR
NR
NR 1†† %1
P-450 INDUC-
INHIBI-
TION
TION
#95 (hepatic) #97 (hepatic)
No
Yes
Yes
Yes
Yes
Yes
No
No‡‡
*Data are mean values and ranges in adults without hepatic or renal dysfunction, as reported by Merck (for indinavir),21 Agouron Pharmaceuticals (for nelfinavir),22 Abbott Laboratories (for ritonavir),23 and Roche Laboratories (for the Invirase formulation of saquinavir).24 Cmax denotes maximal concentration during a dosing interval, Tmax time to the maximal concentration, T1/2 half-life of the principal elimination (b) phase, Vd volume of distribution, CSF cerebrospinal fluid, P-450 cytochrome P-450 drug-metabolizing enzymes (with “induction” denoting a significant increase and “inhibition” a significant decrease in the metabolism of other P-450 substrates), and NR not reported. †Recommended doses and regimens are listed. ‡The effect of food is expressed as the change in the area under the plasma-concentration–time curve after a standard (high-fat) breakfast as compared with the area under the curve during fasting. §Variability is expressed as the coefficient of variation (the standard deviation divided by the mean) for the area under the curve during a dosing interval. ¶The values shown are the ratio of the CSF concentration to the simultaneous plasma concentration. !Lighter meals (toast and coffee or corn flakes with skim milk) have no significant effect on the area under the curve. **Data are from Collier et al.25 ††Data are on file at Abbott Laboratories. ‡‡In vitro studies show that saquinavir can act as a P-450 inhibitor at higher concentrations than those usually achieved with the Invirase formulation.26
creasing steady-state plasma concentrations of saquinavir by a factor of 20 to 30.44 Nelfinavir increases the area under the plasma-concentration–time curve of saquinavir by 392 percent and increases that of indinavir by 51 percent.22 Indinavir increases the area under the curve of saquinavir by about 500 percent.38 Treatment with two protease inhibitors takes advantage of this pharmacokinetic enhancement and increases antiviral activity. Nelfinavir and ritonavir also reduce the plasma concentrations of other drugs, presumably because of hepatic enzyme induction (Table 2). Nelfinavir and ritonavir decrease the area under the plasmaconcentration–time curve of ethinyl estradiol by 47 percent22 and 40 percent,23 respectively. These two protease inhibitors should not be given to women taking a combination oral contraceptive. Nelfinavir and ritonavir reduce the area under the plasma-concentration–time curve of zidovudine by 35 percent22 and 25 percent,23 respectively, presumably because of the induction of glucuronyl transferases. However, the intracellular concentration of zidovudine triphosphate (the active drug) is not usually affected by such a reduction in the plasma concentration of zidovudine,45 and therefore no adjustment of the dose
of zidovudine is recommended when it is given with nelfinavir or ritonavir. The pharmacokinetics of other nucleoside analogues, which are mainly eliminated by the kidneys, are not affected by protease inhibitors. Ritonavir can induce its own metabolism, a process known as autoinduction. Steady-state trough plasma concentrations fall by a factor of two to three during the first two weeks of therapy in patients given a fixed dose of ritonavir.46 Therefore, the dose should be higher during the first two weeks of therapy. The current recommendations are to start with a dose of 300 mg every 12 hours for three days, followed by a dose of 400 mg every 12 hours for three days, then 500 mg every 12 hours for three days, and then 600 mg every 12 hours, if tolerated.23 Such a dose escalation is not recommended for nelfinavir.22 SIDE EFFECTS
Protease inhibitors have important side effects (Table 3). All approved protease inhibitors have gastrointestinal side effects. High serum aminotransferase concentrations have been reported in association with these drugs, but hepatitis is rare.50 Hyperlipidemia,51 glucose intolerance, and abnormal fat distriVo l ume 33 8
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TABLE 2. SELECTED PHARMACOKINETIC DRUG INTERACTIONS INVOLVING APPROVED HIV-PROTEASE INHIBITORS.*
TABLE 3. MAJOR SIDE EFFECTS SIDE EFFECT
DRUG
INDINAVIR
NELFINAVIR
RITONAVIR
SAQUINAVIR
% change in AUC
Effect of other drugs on HIV-protease inhibitors HIV-protease inhibitors Indinavir — $83 Nelfinavir $51 — Ritonavir NR $152 Saquinavir NR $18 P-450 inhibitors $35 $62 Ketoconazole NR $29 Clarithromycin NR "19 Fluconazole NR NR Fluoxetine P-450 inducers Rifabutin "32 "32 Rifampin "92 "82 Nucleoside analogues Didanosine NR NC Lamivudine NC NR Stavudine NC NR Zidovudine $13 NC Nonnucleoside reversetranscriptase inhibitors Delavirdine‡ $72 NR Nevirapine§ "28 NR
NR $9 — NR
$500† $392 $#2000 —
NR $12 $12 $19
$300 NR NR NR
NR "35
"40 "80
NC NR NR NC
NR NR NR NC
$2 NC
$520 "27
Effect of HIV-protease inhibitors on other drugs Anti-infective drugs Clarithromycin $53 NR $77 Isoniazid $13 NR NR Ketoconazole $68 NR NR Rifabutin $204 $207 $350 Sulfamethoxazole NC NR NR Nucleoside analogues Didanosine NR NR "13 Lamivudine "6 $10 NR Stavudine $25 NC NR Zalcitabine NR NR NR Zidovudine $17 to $36 "35 "25 Other drugs Desipramine NR NR $145 Ethinyl estradiol $24 "47 "40 Norethindrone $26 "18 NR Theophylline NR NR "43 Trimethoprim $19 NR $20
NR NR NC NR NR NR NR NR NC NC NR NR NR NR NR
*Except as otherwise indicated, data are mean values reported by Merck (for indinavir),21 Agouron Pharmaceuticals (for nelfinavir),22 Abbott Laboratories (for ritonavir),23 and Roche Laboratories (for the Invirase formulation of saquinavir).24 AUC denotes area under the plasma-concentration– time curve during an average dosing interval, NR not reported, and NC no statistically significant change. †The value is reported by McCrea et al.38 ‡Data are from Cox et al.39 §Data are from Murphy et al.40
bution (buffalo hump) may also occur (Table 3). Hemorrhage has been reported in patients with hemophilia taking protease inhibitors,21-24 but the role of the drug is uncertain. Current information on the side effects of protease inhibitors is based on treatment of only a few thousand patients per drug. Other toxic effects may be recognized with wider use. 1286 !
Nausea Vomiting Diarrhea Asthenia or fatigue Nephrolithiasis or flank pain Hyperbilirubinemia High serum aminotransferase concentrations High serum triglyceride concentrations Hyperglycemia† Fat redistribution‡ Paresthesias
OF
HIV-PROTEASE INHIBITORS.*
INDINAVIR
NELFINAVIR
RITONAVIR
SAQUINAVIR
$$ $ $ " $
$ NR $$ " NR
$$ $$ $$ $$ NR
$$ $ $$ " NR
$ $
NR $
" $
$ $
NR
NR
$
NR
$ $ NR
$ $ NR
$ $ $$
$ $ "
*One plus sign indicates toxicity of moderate or severe intensity reported in less than 10 percent of treated patients but occurring at least twice as often as in concurrently treated patients not taking the protease inhibitor. Two plus signs indicate toxicity in at least 10 percent of treated patients and occurring at least twice as often as in control patients. A minus sign indicates toxicity occurring less than twice as often in treated patients as in control patients and in less than 3 percent of treated patients. NR denotes not reported. Data are for the doses listed in Table 1. Data are from Merck (for indinavir),21 Agouron Pharmaceuticals (for nelfinavir),22 Abbott Laboratories (for ritonavir),23 and Roche Laboratories (for the Invirase and Fortovase formulations of saquinavir).24,36 †Reported by Keruly et al.47 and Dong et al.48 ‡Reported by Mann et al.49
Each of these drugs has distinct dose-limiting toxic effects. Nephrolithiasis is the most important side effect of indinavir and can occur within a few days after the start of treatment.21 The incidence of flank pain and nephrolithiasis in clinical studies ranges from 3 to 15 percent.21,52,53 Indinavir is poorly water soluble; it is associated with crystalluria, and nephrolithiasis may result from the precipitation of indinavir in the renal tubules.53 Patients taking indinavir should drink at least 1.4 liters (48 oz) of fluid daily in addition to their normal fluid intake.21 Indinavir causes fewer gastrointestinal problems than the other drugs. Reversible unconjugated hyperbilirubinemia is frequent in patients taking indinavir but is not usually associated with high serum aminotransferase concentrations or overt liver disease.21 Several cases of hemolytic anemia have also been associated with the use of indinavir.21 Diarrhea is the dose-limiting side effect of nelfinavir.22,54,55 It can usually be controlled with antidiarrheal drugs such as loperamide or fiber supplements. Nausea, vomiting, and abdominal pain occur frequently with ritonavir, especially during the first few weeks of therapy.23 Patients starting treatment should be told to expect these side effects. Ritonavir also causes circumoral paresthesias in up to 25 percent of
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D RUG TH ERA PY
patients and, less commonly, paresthesias of the arms and legs.23 The cause is unclear. In most patients, these symptoms resolve during continued treatment and are not severe enough to cause discontinuation of the drug. Ritonavir causes hypertriglyceridemia in up to 5 percent of patients; serum triglyceride concentrations can exceed 1000 mg per deciliter (11 mmol per liter).23 This side effect has not been accompanied by complications such as pancreatitis. In its original formulation, saquinavir was associated with occasional diarrhea but few serious systemic toxic effects.24 The new formulation, Fortovase, has improved bioavailability; it is much more likely to cause nausea and diarrhea36 and may have more frequent systemic toxicity than the older formulation, Invirase. CLINICAL ANTIVIRAL ACTIVITY Monotherapy
HIV-protease inhibitors rapidly and profoundly reduce the viral load, as indicated by a decline in plasma HIV RNA concentrations within a few days after the start of treatment.56,57 Monotherapy with indinavir, nelfinavir, or ritonavir causes plasma HIV RNA concentrations to be reduced by a factor of 100 to 1000 in 4 to 12 weeks.58,59 The rate of decline in the viral load suggests that the half-life of circulating plasma virus is 6 to 24 hours and the half-life of circulating virus-infected cells is 2 to 3 days.56,57,60 Reductions in the viral load are paralleled by mean increases in the CD4$ count of 100 to 150 cells per cubic millimeter.3,52,58,59 The magnitude and duration of the reduction in the viral load with protease-inhibitor monotherapy are directly related to the dose and dosing regimen. In a study of indinavir, doses of less than 2400 mg per day caused only short-lived suppression of the viral load.61 With ritonavir, twice-daily doses of 300, 400, 500, or 600 mg resulted in equivalent initial reductions in the viral load, but only the 600-mg dose caused a sustained suppression of the viral load and a sustained increase in the CD4$ count.58,59 The dose– response effects of nelfinavir and amprenavir were similar.54,62 Because of the poor oral bioavailability of saquinavir in the older formulation (Invirase), only doses of 3600 to 7200 mg per day, which exceeded the approved dose by a factor of 2 to 4, caused reductions in the viral load approaching those achieved with indinavir, nelfinavir, or ritonavir.63 In an occasional patient, improvements in the viral load and CD4$ count may persist for more than one year with the use of only one protease inhibitor.64 Monotherapy is no longer recommended, however, because the duration of the antiviral response is usually limited and resistance to the drug may develop.
Combination Therapy
Phase 3 clinical trials have evaluated the administration of protease inhibitors in combination with nucleosides. Current clinical guidelines recommend combining a protease inhibitor with two nucleoside analogues (e.g., zidovudine and lamivudine or stavudine and lamivudine4,65). In three large clinical trials, protease inhibitors combined with nucleoside analogues slowed the progression of disease and improved survival. In a trial involving 1090 patients with base-line CD4$ counts of less than 100 per cubic millimeter, ritonavir added to nucleoside therapy reduced the combined end points of new opportunistic diseases and death by 53 percent and reduced the end point of death alone by 43 percent, as compared with placebo.1,23 The median duration of follow-up in this study was six months. Scores for the quality of life declined during the first four weeks of treatment with ritonavir, probably because of side effects, but then improved significantly as compared with base-line values. The patients in the placebo group had a gradual decline in the quality of life.66 Saquinavir in combination with zalcitabine significantly improved survival and slowed the progression of disease, as compared with saquinavir or zalcitabine alone, in a randomized, double-blind trial involving 978 patients treated for a minimum of 16 weeks. Combination therapy reduced the combined clinical end points of disease progression and death by 40 percent and reduced the single end point of death by 68 percent, as compared with either drug alone.2 In a randomized, double-blind trial involving 1156 patients with base-line CD4$ counts of less than 200 per cubic millimeter who were followed for a median of 38 weeks, the combination of indinavir, zidovudine, and lamivudine reduced the combined end points of clinical progression and death by 50 percent and reduced the end point of death alone by 57 percent, as compared with the results in patients treated only with zidovudine and lamivudine.3 Studies of nelfinavir and amprenavir with the use of clinical end points have not been completed. Combinations of protease inhibitors and nucleoside analogues can suppress HIV for long periods of time. The combination of indinavir with zidovudine and lamivudine reduced the plasma viral load to undetectable concentrations (%50 copies per milliliter) in 70 percent of patients after 24 weeks52 and in 60 percent after 2 years.67 The combination of nelfinavir with zidovudine and lamivudine produced similar results, with the viral load reduced to less than 500 copies per milliliter in 80 percent of patients after one year of treatment.68 The response rate was similar in patients given ritonavir combined with zidovudine and lamivudine.69
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With 11 antiretroviral drugs currently approved in the United States, clinicians and investigators are using a variety of two-, three-, and even four-drug combinations to treat HIV infection. For example, a combination of two protease inhibitors suppresses the viral load in patients who have not previously received protease inhibitors. The combination of ritonavir (400 mg twice daily) and saquinavir (400 mg twice daily) was tolerated by most patients and reduced the plasma viral load to a level of less than 500 copies per milliliter for more than 16 weeks.44 These results are similar to those of studies using either drug in combination with nucleoside analogues. The effects of combination therapy with ritonavir and saquinavir were less impressive in patients in whom prior treatment with a protease inhibitor had failed. In one small study, only 45 percent of patients who did not have responses to indinavir had viral loads of less than 500 copies per milliliter 12 weeks after switching to treatment with ritonavir and saquinavir.70 In contrast, all 12 patients who did not have responses to nelfinavir had viral loads of less than 500 copies per milliliter when they received the four-drug regimen of ritonavir, saquinavir, stavudine, and lamivudine.71 Combining a protease inhibitor with a nonnucleoside reverse-transcriptase inhibitor can also profoundly suppress the viral load. All 12 patients treated with indinavir and the nonnucleoside reverse-transcriptase inhibitor nevirapine had viral loads of less than 500 copies per milliliter after an average of 24 weeks of treatment.40 Combined treatment with indinavir and the investigational nonnucleoside reverse-transcriptase inhibitor efavirenz reduced the viral load to less than 400 copies per milliliter in 91 percent of patients after 60 weeks.72 RESISTANCE AND TREATMENT FAILURE
The limited duration of the anti-HIV response that occurs in most patients treated with proteaseinhibitor monotherapy is associated with the appearance of drug-resistant virus.73,74 The major amino acid mutations associated with clinical resistance have been mapped.75 A substitution of phenylalanine for valine at position 82 is the initial mutation associated with resistance to indinavir and ritonavir, an aspartate-to-asparagine mutation at position 30 results in initial resistance to nelfinavir, and mutations in glycine at position 48 and leucine at position 90 result in initial resistance to saquinavir.75 In general, initial single amino acid mutations yield only a slight change (by less than a factor of 5) in drug sensitivity.18,20,76,77 However, secondary mutations accumulate in the virus and can lead to high-level drug resistance. Unlike the initial mutations, the secondary mutations in virus from patients with resistance to different protease inhibitors overlap.73-75 HIV protease can tolerate a substantial 1288 !
amount of mutation; at least one third of its 99 amino acids can deviate from the wild-type sequence without altering function.73,74 In patients with resistance to protease inhibitors, plasma viral loads and CD4$ cell counts have returned to pretreatment values, indicating that resistant mutants are virulent.73,74,78,79 Prolonged treatment with one protease inhibitor can result in the emergence of virus with both primary and secondary resistance mutations. These viruses are resistant not only to the drug being given but also to other protease inhibitors that the patient has never received. Monotherapy with indinavir, for example, can result in the development of virus that is resistant to indinavir and to other protease inhibitors.73 This pattern also occurs with ritonavir monotherapy74 and could presumably be caused by monotherapy with any HIV-protease inhibitor. Once a patient has resistant virus, it is retained even after treatment stops. In patients with indinavir-resistant virus who discontinued monotherapy with indinavir and then received indinavir combined with nucleoside analogues, an initial small reduction in the viral load was followed by an increase within four weeks. The failure of this treatment was associated with the rapid reemergence of indinavir-resistant virus.79 In some circumstances, the development of virus that is resistant to protease inhibitors may be a function of plasma drug concentrations. Higher drug doses, with higher plasma concentrations, were associated with a more prolonged antiviral response and presumably a lower propensity for the development of resistant virus.54,58,59,61,62 A concentration–response relation for indinavir was delineated in a small number of patients in whom the maximal reduction in the viral load occurred with trough plasma concentrations of more than 0.4 mmol per liter; the reduction was minimal if the trough plasma concentration was less than 0.2 mmol per liter.80 In addition, the rate of accumulation of mutations causing resistance to ritonavir was inversely proportional to the trough plasma concentration of the drug during an average dosing interval.74 Drug regimens that maintain high plasma concentrations may be more effective in preventing the emergence of resistant virus than regimens with lower plasma concentrations. The currently recommended regimens for ritonavir, indinavir, and nelfinavir result in plasma concentrations greater than or equal to the concentration required to reduce virus production by 90 percent (IC90) throughout an average dosing interval.27 Although it seems reasonable to maintain trough concentrations at a level higher than the IC90 value, saquinavir in combination with zalcitabine was clinically beneficial with a dosing regimen that resulted in trough concentrations far below the IC90 value.2,27 Similarly, nevirapine, which induces cytochrome P-450, reduced plasma indina-
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D RUG TH ERA PY
PATHOPHYSIOLOGIC CONSEQUENCES
Despite potential problems with resistance, treatment with combination antiretroviral regimens containing protease inhibitors has a number of beneficial pathophysiologic consequences. Besides increasing the overall CD4$ cell count, combination therapy may increase naive and memory T cells, enhance lymphoproliferative responses, and reduce plasma concentrations of harmful cytokines such as tumor necrosis factor.83 Early intervention with combination regimens in a small number of patients with recent seroconversion forestalled the loss of HIV-spe-
200,000 20,000
Successful Therapy
2,000 200 20
Plasma HIV RNA (copies/ml)
vir concentrations by 28 percent, on average, but the combination of these two drugs resulted in a reduction in the viral load of the same magnitude as that associated with regimens that did not affect the clearance of indinavir.40 However, because resistance may be a consequence of suboptimal plasma drug concentrations, strict adherence to recommended regimens should be encouraged. In a study of compliance in a small number of patients, genotypic resistance was associated with a high plasma viral load or episodes of poor compliance.81 Patients who have difficulty tolerating a high dose of a protease inhibitor may be better advised to stop taking all antiretroviral drugs than to take a lower dose of the protease inhibitor. Physicians should be cautious when prescribing protease inhibitors for patients with a history of poor compliance. Because of the problem of cross-resistance, a switch from one protease inhibitor to another should take place, if possible, before high-level resistance to the initial drug occurs (Fig. 3). The sequential addition of single drugs to an ineffective regimen is associated with a poor outcome and may result in the sequential selection of drug-resistant virus. For example, when indinavir was added after long-term combination therapy with zidovudine and lamivudine, only 45 percent of patients had plasma viral loads that were less than 500 copies per milliliter 67 — about half the expected response rate in patients without prior antiretroviral therapy who are given the same three drugs. In patients without responses to initial regimens, salvage therapy should be started at the earliest sign of failure (e.g., a sustained rise in the plasma viral load &1.0 log10 copies per milliliter) and should include two or three drugs the patient has never received.4,65 Combining a potent protease inhibitor with two nucleoside analogues appears to prevent the emergence of resistance to either class of drugs.82 Occasional lapses in compliance may also be less dangerous in patients taking combinations of antiretroviral drugs, because of the ability of one type of drug to suppress the replication of virus resistant to the other type of drug.
Change in regimen
200,000 20,000
Salvage Therapy
2,000 200 20
Change in regimen
200,000 20,000
Unsuccessful Therapy
2,000 200 20 0
4 8 12 16 20 24 28 32 36 40 44 48 52
Week Figure 3. Changes in HIV Viral Load over Time in Three Patients Treated with Combination Antiretroviral Regimens Including HIV-Protease Inhibitors. Viral load was measured every four weeks. A change of both the protease inhibitor and the nucleoside analogues used in the regimen occurred at the times indicated by the arrows. The dotted lines indicate the lower limit of detection in an ultrasensitive polymerase-chain-reaction assay for HIV-1 RNA.
cific CD4$ lymphocyte responses that characterizes the natural history of this disease.84 Unfortunately, protease-inhibitor therapy may not correct deficiencies in the T-cell repertoire already induced by HIV, and lost lymphocyte clones may not be replaced.85 These observations may explain why infections caused by opportunistic pathogens such as cytomegalovirus developed in some patients during the late stage of the acquired immunodeficiency syndrome (AIDS) even though their CD4$ counts increased from less than 50 to more than 200 cells per cubic millimeter while they were receiving treatment with highly active regimens containing protease inhibitors.86,87 Finally, despite the prolonged suppression of HIV viremia in large numbers of patients, no one has yet been cured of HIV infection. Patients with undeVo l u me 33 8
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tectable plasma viral loads and greatly reduced numbers of viral particles in lymph nodes still had detectable proviral DNA in cells even after 6 to 12 months of therapy.88,89 After at least two years of combination therapy consisting of a protease inhibitor and nucleoside analogues, patients with undetectable plasma viral loads still had HIV in their lymph nodes and peripheral-blood cells that was capable of replication.90-92 Eradication of HIV with current combination regimens may be impossible. Most if not all infected patients have a small amount of virus in long-lived cellular reservoirs, and eliminating HIV in even a small percentage of such patients may require at least three to five years of continuous treatment.93,94 Furthermore, among patients with undetectable viral loads who are receiving indinavir combined with zidovudine and lamivudine, those who stopped taking one or two of the drugs after three to six months of therapy had relapses up to six times as often as those who continued to take all three drugs.95,96 CONCLUSIONS
Protease-inhibitor therapy is the most important recent advance in the treatment of HIV infection. All infected patients with symptomatic disease, CD4$ counts of less than 500 cells per cubic millimeter, or plasma HIV RNA concentrations of more than 5000 to 10,000 copies per milliliter should receive a protease inhibitor plus two nucleoside analogues unless contraindicated.4,64 Although these combinations substantially reduce the plasma viral load, increase the CD4$ cell count, reduce the progression of disease, and prolong life, they have side effects. The emergence of drug-resistant strains of HIV may limit the long-term effectiveness of treatment with protease inhibitors. Monotherapy, subtherapeutic drug concentrations, and noncompliance may promote resistance to this class of drugs. Using protease inhibitors in combination with nucleoside analogues or nonnucleoside reverse-transcriptase inhibitors reduces the development of resistance but requires substantial cooperation on the patient’s part. Protease inhibitors are expensive, with an annual wholesale price ranging from $4,320 to $8,010 per patient.97 Combination regimens that include protease inhibitors cost about $10,000 per year of life saved.97 This figure compares favorably with the cost of other prescription drugs given to prevent morbidity and mortality in patients with other chronic conditions such as hypertension and ischemic heart disease.97 To maximize the long-term benefit of HIV-protease inhibitors, several issues need to be resolved. What are the most appropriate regimens for infants, children, and pregnant women? Can we develop simplified dosing schemes to promote adherence to 1290 !
the regimen? Will combinations of two protease inhibitors be as effective over time as a single protease inhibitor combined with reverse-transcriptase inhibitors? What role should nonnucleoside reverse-transcriptase inhibitors play in these regimens? Important questions about protease inhibitors remain. Much of the information in this article has not been published in peer-reviewed journals, and the results of important new studies are reported monthly. The complexity of decisions involving antiretroviral therapy now rivals that of decisions involving chemotherapy for cancer. Physicians should maintain a healthy skepticism while awaiting clarification of the optimal use of protease inhibitors, and whenever possible, specialists in the care of patients with AIDS should be involved in treatment decisions. I am indebted to Ms. Laura Rocco for editorial assistance, to Ms. Anne Capriotti for assistance with the figures, and to Ms. Joann Nicolette and Dr. Carol Trapnell for assistance with the preparation of the manuscript.
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