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Review Article

Drug Therapy

A

L ASTAIR

J.J. W

OO D

, M.D.,

Editor

DRUG THERAPY

HIV-P

ROTEASE

I

NHIBITORS

C

HARLES

F

LEXNER

, M.D.

From the Division of Clinical Pharmacology, Department of Medicineand Department of Pharmacology and Molecular Sciences, Johns HopkinsUniversity School of Medicine, Baltimore. Address reprint requests to Dr.Flexner at Osler 524, Johns Hopkins Hospital, 600 N. Wolfe St., Balti-more, MD 21287-5554.

1998, Massachusetts Medical Society.

NHIBITORS of human immunodeficiency virus(HIV)encoded protease, combined with nucle-oside analogues with antiretroviral activity, cause

profound and sustained suppression of viral replica-tion, reduce morbidity, and prolong life in patients

with HIV infection.

1-3

Recent guidelines recommendthat initial treatment of all HIV-infected patientsinclude the administration of an HIV-protease inhib-itor.

4

THE HIV-ENCODED PROTEASE

The HIV protease, encoded in the 5

end of the

pol

gene, is expressed as part of the gagpol polypro-tein (Fig. 1). This gene encodes a 99-amino-acidprotein. Homodimers of this protein have the aspar-tyl protease activity that is typical of retroviral pro-teases; monomers are enzymatically inactive.

6

The

enzymes targets are amino acid sequences in the gagand gagpol polyproteins, which must be cleaved be-fore nascent viral particles (virions) can mature.

7-10

Cleavage of the gag polyprotein produces three largeproteins (p24, p17, and p7) that contribute to thestructure of the virion and to RNA packaging, andthree smaller proteins (p6, p2, and p1) of uncertainfunction.

11

Although mammalian cells contain aspar-tyl proteases, none efficiently cleave the gag polypro-tein.

12,13

Three of the HIV-cleavage sites are phenyl-alanineproline or tyrosineproline bonds, which areunusual sites of attack for mammalian proteases.

14

Proteolytic cleavage of the gag polyprotein resultsin morphologic changes in the virion and condensa-

tion of the nucleoprotein core. The protease is pack-aged into virions, and the cleavage events it catalyzes

I

occur simultaneously with or soon after the budding

of the virion from the surface of an infected cell.

15

Proviral DNA lacking functional protease producesimmature, noninfectious viral particles.

9

MECHANISM OF ACTION OFHIV-PROTEASE INHIBITORS

The four approved HIV-protease inhibitors arebased on amino acid sequences recognized andcleaved in HIV proteins. Indinavir (Crixivan), nelfin-avir (Viracept), ritonavir (Norvir), and saquinavir(Invirase and Fortovase) and the investigational pro-tease inhibitor amprenavir are structurally relatedmolecules (Fig. 2). Most contain a synthetic ana-logue of the phenylalanineproline sequence at po-

sitions 167 and 168 of the gagpol polyprotein thatis cleaved by the protease.

14

These drugs are difficultto synthesize in large quantities because of theircomplex structure.

HIV-protease inhibitors prevent cleavage of gagand gagpol protein precursors in acutely and chron-ically infected cells, arresting maturation and therebyblocking the infectivity of nascent virions.

13,16

Themain antiviral action of HIV-protease inhibitors isthus to prevent subsequent waves of infection; theyhave no effect on cells already harboring integratedproviral DNA. These agents are active against clini-cal isolates of HIV types 1 and 2, with the in vitroconcentration of drug required to reduce viral pro-duction by 50 percent (IC

50

) ranging from 2 to 60nM.

16-19

Antiviral activity is correlated with the inhi-bition of enzyme activity, although the drug con-centration required to reduce enzyme activity by 50percent (Ki) is lower than the IC

50

, ranging from0.10 to 2.0 nM.

16-20

These drugs are inactive orweakly active against human aspartyl proteases, witha Ki of at least 10,000 nM for renin and pepsin.

16,17

CLINICAL PHARMACOLOGIC PROPERTIES

The clinical pharmacokinetic properties of thefour approved protease inhibitors are shown in Table1. Oral bioavailability varies because of differences infirst-pass hepatic metabolism, to which saquinavir isthe most susceptible.

27

Food also affects bioavailabil-ity. A high-fat meal increases the bioavailability ofsaquinavir and nelfinavir but reduces that of indina-

vir. The same high-fat meal increases the bioavail-ability of ritonavir capsules but decreases that of ri-tonavir liquid. The ingestion of indinavir capsules

with light meals has no effect on the area under theplasma-concentrationtime curve during an averagedosing interval, but variability is large.

21

Nelfinavir,ritonavir, and saquinavir should be taken with meals,

Downloaded from www.nejm.org on September 9, 2006 . Copyright 1998 Massachusetts Medical Society. All rights reserved.


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The New England Journal of Medicine

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 Erickson

5

with thepermission 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 gagpol

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 pro-tease.

B

A



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DRUG THERAPY

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and indinavir should be taken after fasting or with alight, low-fat snack.

All these drugs are metabolized by cytochromeP-450 enzymes, mainly the 3A4 isoform.

26,28,29

Theabsorption of the drugs is maximal within fourhours after ingestion. Their elimination half-livesrange from 1.8 to 5 hours. Differences in a drugspharmacokinetic characteristics between patients arelarge, as indicated by a coefficient of variation of 30percent or higher for the mean area under the curve.

All these drugs except indinavir are at least 98 per-cent protein-bound in plasma. The apparent volumeof distribution ranges from 0.4 to 10.0 liters per kil-ogram of body weight.

Alpha

1

-acid glycoprotein, an inducible plasma pro-tein, reduces the in vitro anti-HIV potency of someprotease inhibitors by a factor of 10 or more.

30-33

This effect may be mediated by high-affinity bindingof the drug to the glycoprotein. Since only free drugis available to penetrate cells, plasma concentrationsneed to be high enough to override this effect.

34

There are limited data on central nervous systempenetration of these drugs. The cerebrospinal fluidpenetration of ritonavir and saquinavir, defined asthe ratio of the cerebrospinal fluid concentration tothe simultaneous plasma concentration, is less thanor equal to 1 percent.

23,35

Indinavir has a higher cer-ebrospinal fluid penetration, ranging from 2.2 to 76percent, perhaps because of decreased plasma pro-tein binding.

25

Indinavir, ritonavir, and saquinavir are available ascapsules, and nelfinavir is available as tablets. Sa-quinavir is now also available as soft-gel capsules(Fortovase), which triples oral bioavailability.

36

Nel-finavir powder and liquid ritonavir are pediatric for-mulations. Indinavir must be stored in an airtightcontainer with a desiccant.

21

Ritonavir is heat-sensi-

tive and needs to be refrigerated. It also contains al-cohol and is therefore contraindicated in patientstaking disulfiram or metronidazole.

23

DRUG INTERACTIONS

HIV-protease inhibitors can interact with inhibi-tors or inducers of cytochrome P-450 drug-metab-olizing enzymes.

37

Table 2 shows the magnitude ofinteractions between HIV-protease inhibitors andother drugs commonly used to treat HIV infection.In many cases, inhibitors of P-450 increase plasmaconcentrations of protease inhibitors. For example,concurrent administration of ketoconazole increasesthe area under the plasma-concentrationtime curveby 62 percent with indinavir,

21

by 35 percent withnelfinavir,

22

and by 300 percent with saquinavir.

24

Because the bioavailability of oral saquinavir is poor,this interaction may be advantageous, increasing theamount of drug reaching the systemic circulation.

A more important concern is the effect of concur-rent therapy with inducers of P-450, such as rifam-pin and rifabutin, which accelerate the clearance ofprotease inhibitors. The plasma concentration istherefore decreased, and the efficacy of the drugis likely to be reduced, which may result in the de-

velopment of resistance to the drug. Rifampin re-duces the area under the plasma-concentrationtimecurve by 92 percent with indinavir,

41

by 82 percentwith nelfinavir,

22

by 35 percent with ritonavir,

23

andby 80 percent with saquinavir.

24

Rifampin shouldnot be given to patients who require treatment withHIV-protease inhibitors.

42

HIV-protease inhibitors alter the pharmacokinet-ics of other drugs by acting as P-450 inhibitors orhepatic-enzyme inducers (Table 2). All these drugsinhibit cytochrome P-450 enzymes. Ritonavir is themost potent,

28

indinavir and nelfinavir are less so,

29,37

DNA5

p17 p24 p2 p7 p1 p6 ProteaseReverse

transcriptaseIntegrase

3

RNA

Precursorpolyproteins

Functionalproteins

gag

gagpol

gag

env

pol

C



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and saquinavir is the least potent.

26

The Ki for theinhibition of terfenadine metabolism is 0.017 m

Mfor ritonavir

28

and 0.7 m

M for saquinavir,

26

a 40-folddifference in potency.

Concurrent administration of rifabutin with in-dinavir, nelfinavir, or ritonavir increases the area un-der the rifabutin plasma-concentrationtime curveby 204 percent,

21

207 percent,

22

and 350 percent,

23

respectively. For patients taking indinavir or nelfin-avir who require concurrent therapy with rifabutin,the dose of rifabutin should be decreased to 150 mgdaily.

21,22,42 Concurrent administration of rifabutinwith ritonavir or saquinavir is not recommended.

23,24

The combination of ritonavir and rifabutin is associ-ated with an increased incidence of rifabutin-associ-ated toxicity (uveitis).

43

Clinicians must be aware ofpotentially toxic drug interactions involving HIV-protease inhibitors and avoid prescribing such drugsas terfenadine, astemizole, cisapride, ergotamines,and potent benzodiazepines such as midazolam andtriazolam to patients receiving an HIV-protease in-hibitor.

Cytochrome P-450 pharmacokinetic interactionsmay be beneficial when two protease inhibitors aregiven simultaneously. For example, ritonavir inhibitsthe hepatic first-pass metabolism of saquinavir, in-

Figure 2.

Structures of Five HIV-Protease Inhibitors with Antiretroviral Activity in Clinical Trials.

NHtBU denotes amino-tertiary butyl, and Ph phenyl.

Indinavir

Nelfinavir

Ritonavir

Saquinavir

Amprenavir

N

N

N N

N

N

N

N

N NN

O

O

OO

OO

CH3

O

O

O

H

H

H

H

H

S

S

S

S

OH

NHtBu

HO

OH

OH

Ph

Ph

Ph

Ph

O

O

O

O

N

H

N

H

N

H

N

H

N

H

N

H

CH3SO3H

CH3SO3H

NH2

NH2

H2SO4

O

N

H

N

H

OH

OH

OH

O NHtBu

OO



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DRUG THERAPY

Vo lu me 3 38 N um be r 18

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creasing steady-state plasma concentrations of sa-quinavir by a factor of 20 to 30.

44

Nelfinavir increas-es the area under the plasma-concentrationtimecurve of saquinavir by 392 percent and increases thatof indinavir by 51 percent.

22

Indinavir increases thearea under the curve of saquinavir by about 500 per-cent.

38

Treatment with two protease inhibitors takesadvantage of this pharmacokinetic enhancement andincreases antiviral activity.

Nelfinavir and ritonavir also reduce the plasmaconcentrations of other drugs, presumably becauseof hepatic enzyme induction (Table 2). Nelfinavirand ritonavir decrease the area under the plasma-concentrationtime curve of ethinyl estradiol by 47percent

22

and 40 percent,

23

respectively. These twoprotease inhibitors should not be given to womentaking a combination oral contraceptive. Nelfinavirand ritonavir reduce the area under the plasma-con-centrationtime curve of zidovudine by 35 percent

22

and 25 percent,

23

respectively, presumably becauseof the induction of glucuronyl transferases. Howev-er, the intracellular concentration of zidovudine tri-phosphate (the active drug) is not usually affected bysuch a reduction in the plasma concentration of zi-dovudine,

45

and therefore no adjustment of the dose

of zidovudine is recommended when it is given withnelfinavir or ritonavir. The pharmacokinetics of oth-er nucleoside analogues, which are mainly eliminat-ed by the kidneys, are not affected by protease inhib-itors.

Ritonavir can induce its own metabolism, a proc-ess known as autoinduction. Steady-state troughplasma concentrations fall by a factor of two to threeduring the first two weeks of therapy in patients giv-en a fixed dose of ritonavir.

46

Therefore, the doseshould be higher during the first two weeks of ther-apy. The current recommendations are to start witha dose of 300 mg every 12 hours for three days, fol-lowed by a dose of 400 mg every 12 hours for threedays, then 500 mg every 12 hours for three days, andthen 600 mg every 12 hours, if tolerated.

23

Such adose escalation is not recommended for nelfinavir.

22

SIDE EFFECTS

Protease inhibitors have important side effects(Table 3). All approved protease inhibitors have gas-trointestinal side effects. High serum aminotransfer-ase concentrations have been reported in association

with these drugs, but hepatitis is rare.

50

Hyperlipide-mia,

51

glucose intolerance, and abnormal fat distri-

*Data are mean values and ranges in adults without hepatic or renal dysfunction, as reported by Merck (for indinavir),

21Agouron Pharmaceuticals (fornelfinavir),

22

Abbott Laboratories (for ritonavir),

23

and Roche Laboratories (for the Invirase formulation of saquinavir).

24

C

max

denotes maximal concentra-tion during a dosing interval, T

max

time to the maximal concentration, T

1/2

half-life of the principal elimination (

b

) phase, V

d

volume of distribution, CSF

cerebrospinal fluid, P-450 cytochrome P-450 drug-metabolizing enzymes (with induction denoting a significant increase and inhibition a significantdecrease 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-concentrationtime curve after a standard (high-fat) breakfast as comparedwith 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

T

ABLE

1. P

HARMACOKINETICS

OF

A

PPROVED

HIV-P

ROTEASE

I

NHIBITORS

.*

D

RUG

D

OSE

A

PPROXIMATE

O

RAL

B

IO

-

AVAILABILITY

E

FFECT

OF

F

OOD

C

max

T

max

T

1

/

2

VARIABILITYPROTEINBINDING Vd

CSFCONCEN-TRATION

CLEARANCE(ROUTE) P-450

INDUC-TION

INHIBI-TION

mg % % mg/ml hr hr % % liters/kg % %

Indinavir 800 every8 hr

6065 77 7.7 0.8 1.8 2247 6065 NR 2.276** 8890(hepatic)

No Yes

Nelfinavir 750 threetimesa day

78 200 to 300 3.04.0 2.04.0 3.55.0 NR 98 2.07.0 NR 78(hepatic)

Yes Yes

Ritonavir 600 twicea day

6675 7 (liquid);15 (capsules)

11.2 2.04.0 3.05.0 3036 9899 0.4 1 95(hepatic)

Yes Yes

Saquinavir 600 threetimesa day

4 670 0.2 NR NR 4684 98 10.0 1 97(hepatic)

No No



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bution (buffalo hump) may also occur (Table 3).Hemorrhage has been reported in patients with he-mophilia taking protease inhibitors,21-24 but the roleof the drug is uncertain. Current information on theside effects of protease inhibitors is based on treat-ment of only a few thousand patients per drug. Oth-er toxic effects may be recognized with wider use.

Each of these drugs has distinct dose-limiting tox-ic effects. Nephrolithiasis is the most important side

effect of indinavir and can occur within a few daysafter the start of treatment.21 The incidence of flankpain and nephrolithiasis in clinical studies rangesfrom 3 to 15 percent.21,52,53 Indinavir is poorly watersoluble; it is associated with crystalluria, and neph-rolithiasis may result from the precipitation of indin-avir in the renal tubules.53 Patients taking indinavirshould drink at least 1.4 liters (48 oz) of fluid dailyin addition to their normal fluid intake.21 Indinavircauses fewer gastrointestinal problems than the oth-er drugs. Reversible unconjugated hyperbilirubine-mia is frequent in patients taking indinavir but is notusually associated with high serum aminotransferaseconcentrations or overt liver disease.21 Several casesof hemolytic anemia have also been associated withthe use of indinavir.21

Diarrhea is the dose-limiting side effect of nelfin-avir.22,54,55 It can usually be controlled with antidiar-rheal drugs such as loperamide or fiber supplements.

Nausea, vomiting, and abdominal pain occur fre-quently with ritonavir, especially during the first few

weeks of therapy.23 Patients starting treatment shouldbe told to expect these side effects. Ritonavir alsocauses circumoral paresthesias in up to 25 percent of

*Except as otherwise indicated, data are mean values reported by Merck(for indinavir),21 Agouron Pharmaceuticals (for nelfinavir),22 Abbott Lab-oratories (for ritonavir),23 and Roche Laboratories (for the Invirase formu-lation of saquinavir).24 AUC denotes area under the plasma-concentrationtime curve during an average dosing interval, NR not reported, and NCno 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

TABLE 2. SELECTED PHARMACOKINETIC DRUG INTERACTIONSINVOLVING APPROVED HIV-PROTEASE INHIBITORS.*

DRUG INDINAVIR NELFINAVIRRITONA-

VIR SAQUINAVIR

% change in AUC

Effect of other drugs on HIV-protease inhibitors

HIV-protease inhibitorsIndinavirNelfinavirRitonavirSaquinavir

51NRNR

83

15218

NR9NR

500392

2000

P-450 inhibitorsKetoconazoleClarithromycinFluconazoleFluoxetine

622919NR

35NRNRNR

NR121219

300NRNRNR

P-450 inducersRifabutinRifampin

3292

3282

NR35

4080

Nucleoside analoguesDidanosineLamivudine

StavudineZidovudine

NRNC

NC13

NCNR

NRNC

NCNR

NRNC

NRNR

NRNC

Nonnucleoside reverse-transcriptaseinhibitors

DelavirdineNevirapine

7228

NRNR

2NC

52027

Effect of HIV-protease inhibitors on other drugs

Anti-infective drugsClarithromycinIsoniazidKetoconazoleRifabutinSulfamethoxazole

531368

204NC

NRNRNR

207NR

77NRNR

350NR

NRNRNCNRNR

Nucleoside analoguesDidanosineLamivudineStavudine

ZalcitabineZidovudine

NR6

25

NR17 to 36

NR10NC

NR35

13NRNR

NR25

NRNRNR

NCNC

Other drugsDesipramineEthinyl estradiolNorethindroneTheophyllineTrimethoprim

NR2426NR

19

NR4718NRNR

14540NR

4320

NRNRNRNRNR

*One plus sign indicates toxicity of moderate or severe intensity reported

in less than 10 percent of treated patients but occurring at least twice asoften as in concurrently treated patients not taking the protease inhibitor.Two plus signs indicate toxicity in at least 10 percent of treated patientsand occurring at least twice as often as in control patients. A minus signindicates toxicity occurring less than twice as often in treated patients as incontrol patients and in less than 3 percent of treated patients. NR denotesnot reported. Data are for the doses listed in Table 1. Data are from Merck(for indinavir),21 Agouron Pharmaceuticals (for nelfinavir),22 Abbott Lab-oratories (for ritonavir),23 and Roche Laboratories (for the Invirase andFortovase formulations of saquinavir).24,36

Reported by Keruly et al.47 and Dong et al.48

Reported by Mann et al.49

TABLE 3. MAJORSIDE EFFECTSOF HIV-PROTEASE INHIBITORS.*

SIDE EFFECT INDINAVIR NELFINAVIR RITONAVIR SAQUINAVIR

Nausea

Vomiting NR

Diarrhea

Asthenia or fatigue

Nephrolithiasis orflank pain

NR NR NR

Hyperbilirubinemia NR

High serum amino-transferase con-centrations

High serum triglyc-eride concentra-tions

NR NR NR

Hyperglycemia

Fat redistribution

Paresthesias NR NR



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patients and, less commonly, paresthesias of thearms and legs.23 The cause is unclear. In most pa-tients, these symptoms resolve during continuedtreatment and are not severe enough to cause dis-continuation of the drug. Ritonavir causes hypertri-glyceridemia in up to 5 percent of patients; serum

triglyceride concentrations can exceed 1000 mg perdeciliter (11 mmol per liter).23 This side effect hasnot been accompanied by complications such aspancreatitis.

In its original formulation, saquinavir was associ-ated with occasional diarrhea but few serious sys-temic toxic effects.24 The new formulation, For-tovase, has improved bioavailability; it is much morelikely to cause nausea and diarrhea36 and may havemore frequent systemic toxicity than the older for-mulation, Invirase.

CLINICAL ANTIVIRAL ACTIVITY

Monotherapy

HIV-protease inhibitors rapidly and profoundlyreduce the viral load, as indicated by a decline inplasma HIV RNA concentrations within a few daysafter the start of treatment.56,57 Monotherapy withindinavir, nelfinavir, or ritonavir causes plasma HIVRNA concentrations to be reduced by a factor of100 to 1000 in 4 to 12 weeks.58,59 The rate of de-cline in the viral load suggests that the half-life ofcirculating plasma virus is 6 to 24 hours and thehalf-life of circulating virus-infected cells is 2 to3 days.56,57,60 Reductions in the viral load are paral-leled by mean increases in the CD4 count of 100to 150 cells per cubic millimeter.3,52,58,59

The magnitude and duration of the reduction inthe viral load with protease-inhibitor monotherapyare directly related to the dose and dosing regimen.In a study of indinavir, doses of less than 2400 mgper day caused only short-lived suppression of the vi-ral load.61 With ritonavir, twice-daily doses of 300,400, 500, or 600 mg resulted in equivalent initial re-ductions in the viral load, but only the 600-mg dosecaused a sustained suppression of the viral load and asustained increase in the CD4 count.58,59 The doseresponse effects of nelfinavir and amprenavir weresimilar.54,62 Because of the poor oral bioavailability ofsaquinavir in the older formulation (Invirase), onlydoses of 3600 to 7200 mg per day, which exceeded

the approved dose by a factor of 2 to 4, caused re-ductions in the viral load approaching those achievedwith indinavir, nelfinavir, or ritonavir.63

In an occasional patient, improvements in the vi-ral load and CD4 count may persist for more thanone year with the use of only one protease inhibi-tor.64 Monotherapy is no longer recommended,however, because the duration of the antiviral re-sponse is usually limited and resistance to the drugmay develop.

Combination Therapy

Phase 3 clinical trials have evaluated the adminis-tration of protease inhibitors in combination withnucleosides. Current clinical guidelines recommendcombining a protease inhibitor with two nucleosideanalogues (e.g., zidovudine and lamivudine or stav-udine and lamivudine4,65).

In three large clinical trials, protease inhibitorscombined with nucleoside analogues slowed the pro-gression of disease and improved survival. In a trialinvolving 1090 patients with base-line CD4 countsof less than 100 per cubic millimeter, ritonavir add-ed to nucleoside therapy reduced the combined endpoints of new opportunistic diseases and death by53 percent and reduced the end point of death aloneby 43 percent, as compared with placebo.1,23 Themedian duration of follow-up in this study was sixmonths. Scores for the quality of life declined dur-ing the first four weeks of treatment with ritonavir,

probably because of side effects, but then improvedsignificantly as compared with base-line values. Thepatients in the placebo group had a gradual declinein the quality of life.66

Saquinavir in combination with zalcitabine signif-icantly improved survival and slowed the progressionof disease, as compared with saquinavir or zalcita-bine alone, in a randomized, double-blind trial in-

volving 978 patients treated for a minimum of 16weeks. Combination therapy reduced the combinedclinical end points of disease progression and deathby 40 percent and reduced the single end point ofdeath by 68 percent, as compared with either drugalone.2

In a randomized, double-blind trial involving1156 patients with base-line CD4 counts of lessthan 200 per cubic millimeter who were followedfor a median of 38 weeks, the combination of indin-avir, zidovudine, and lamivudine reduced the com-bined end points of clinical progression and deathby 50 percent and reduced the end point of deathalone by 57 percent, as compared with the results inpatients treated only with zidovudine and lamivu-dine.3 Studies of nelfinavir and amprenavir with theuse of clinical end points have not been completed.

Combinations of protease inhibitors and nucleo-side analogues can suppress HIV for long periods oftime. The combination of indinavir with zidovudine

and lamivudine reduced the plasma viral load to un-detectable concentrations (50 copies per milliliter)in 70 percent of patients after 24 weeks52 and in 60percent after 2 years.67 The combination of nelfin-avir with zidovudine and lamivudine produced sim-ilar results, with the viral load reduced to less than500 copies per milliliter in 80 percent of patients af-ter one year of treatment.68 The response rate wassimilar in patients given ritonavir combined with zi-dovudine and lamivudine.69



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With 11 antiretroviral drugs currently approved inthe United States, clinicians and investigators are us-ing a variety of two-, three-, and even four-drugcombinations to treat HIV infection. For example, acombination of two protease inhibitors suppressesthe viral load in patients who have not previously re-

ceived protease inhibitors. The combination of ri-tonavir (400 mg twice daily) and saquinavir (400mg twice daily) was tolerated by most patients andreduced the plasma viral load to a level of less than500 copies per milliliter for more than 16 weeks.44

These results are similar to those of studies usingeither drug in combination with nucleoside ana-logues. The effects of combination therapy withritonavir and saquinavir were less impressive in pa-tients in whom prior treatment with a protease in-hibitor had failed. In one small study, only 45 per-cent of patients who did not have responses toindinavir had viral loads of less than 500 copies permilliliter 12 weeks after switching to treatment with

ritonavir and saquinavir.70 In contrast, all 12 patients who did not have responses to nelfinavir had viralloads of less than 500 copies per milliliter when theyreceived the four-drug regimen of ritonavir, saquin-avir, stavudine, and lamivudine.71

Combining a protease inhibitor with a nonnucleo-side reverse-transcriptase inhibitor can also profound-ly suppress the viral load. All 12 patients treated withindinavir and the nonnucleoside reverse-transcrip-tase inhibitor nevirapine had viral loads of less than500 copies per milliliter after an average of 24 weeksof treatment.40 Combined treatment with indinavirand the investigational nonnucleoside reverse-trans-criptase inhibitor efavirenz reduced the viral load to

less than 400 copies per milliliter in 91 percent ofpatients after 60 weeks.72

RESISTANCE AND TREATMENT FAILURE

The limited duration of the anti-HIV responsethat occurs in most patients treated with protease-inhibitor monotherapy is associated with the appear-ance of drug-resistant virus.73,74 The major aminoacid mutations associated with clinical resistancehave been mapped.75 A substitution of phenylalaninefor valine at position 82 is the initial mutation asso-ciated with resistance to indinavir and ritonavir, anaspartate-to-asparagine mutation at position 30 re-sults in initial resistance to nelfinavir, and mutationsin glycine at position 48 and leucine at position 90result in initial resistance to saquinavir.75

In general, initial single amino acid mutations yield only a slight change (by less than a factor of5) in drug sensitivity.18,20,76,77 However, secondarymutations accumulate in the virus and can lead tohigh-level drug resistance. Unlike the initial muta-tions, the secondary mutations in virus from pa-tients with resistance to different protease inhibitorsoverlap.73-75 HIV protease can tolerate a substantial

amount of mutation; at least one third of its 99 ami-no acids can deviate from the wild-type sequence

without altering function.73,74 In patients with resist-ance to protease inhibitors, plasma viral loads andCD4 cell counts have returned to pretreatment

values, indicating that resistant mutants are viru-

lent.73,74,78,79Prolonged treatment with one protease inhibitor

can result in the emergence of virus with both pri-mary and secondary resistance mutations. These vi-ruses are resistant not only to the drug being givenbut also to other protease inhibitors that the patienthas never received. Monotherapy with indinavir, forexample, can result in the development of virus thatis resistant to indinavir and to other protease inhib-itors.73 This pattern also occurs with ritonavir mono-therapy74 and could presumably be caused by mono-therapy with any HIV-protease inhibitor. Once apatient has resistant virus, it is retained even aftertreatment stops. In patients with indinavir-resistant

virus who discontinued monotherapy with indinavirand then received indinavir combined with nucleo-side analogues, an initial small reduction in the viralload was followed by an increase within four weeks.The failure of this treatment was associated with therapid reemergence of indinavir-resistant virus.79

In some circumstances, the development of virusthat is resistant to protease inhibitors may be a func-tion of plasma drug concentrations. Higher drugdoses, with higher plasma concentrations, were asso-ciated with a more prolonged antiviral response andpresumably a lower propensity for the developmentof resistant virus.54,58,59,61,62 A concentrationresponserelation for indinavir was delineated in a small num-

ber of patients in whom the maximal reduction inthe viral load occurred with trough plasma concen-trations of more than 0.4 mmol per liter; the reduc-tion was minimal if the trough plasma concentration

was less than 0.2 mmol per liter.80 In addition, therate of accumulation of mutations causing resistanceto ritonavir was inversely proportional to the troughplasma concentration of the drug during an averagedosing interval.74

Drug regimens that maintain high plasma concen-trations may be more effective in preventing theemergence of resistant virus than regimens with low-er plasma concentrations. The currently recommend-ed regimens for ritonavir, indinavir, and nelfinavir re-sult in plasma concentrations greater than or equalto the concentration required to reduce virus pro-duction by 90 percent (IC90) throughout an averagedosing interval.27 Although it seems reasonable tomaintain trough concentrations at a level higher thanthe IC90 value, saquinavir in combination with zal-citabine was clinically beneficial with a dosing regi-men that resulted in trough concentrations far be-low the IC90 value.

2,27 Similarly, nevirapine, whichinduces cytochrome P-450, reduced plasma indina-



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vir concentrations by 28 percent, on average, but thecombination of these two drugs resulted in a reduc-tion in the viral load of the same magnitude as thatassociated with regimens that did not affect theclearance of indinavir.40

However, because resistance may be a conse-

quence of suboptimal plasma drug concentrations,strict adherence to recommended regimens shouldbe encouraged. In a study of compliance in a smallnumber of patients, genotypic resistance was associ-ated with a high plasma viral load or episodes ofpoor compliance.81 Patients who have difficulty tol-erating a high dose of a protease inhibitor may bebetter advised to stop taking all antiretroviral drugsthan to take a lower dose of the protease inhibitor.Physicians should be cautious when prescribing pro-tease inhibitors for patients with a history of poorcompliance.

Because of the problem of cross-resistance, a switchfrom one protease inhibitor to another should take

place, if possible, before high-level resistance to theinitial drug occurs (Fig. 3). The sequential additionof single drugs to an ineffective regimen is associated

with a poor outcome and may result in the sequen-tial selection of drug-resistant virus. For example,

when indinavir was added after long-term combina-tion therapy with zidovudine and lamivudine, only45 percent of patients had plasma viral loads that

were less than 500 copies per milliliter67 abouthalf the expected response rate in patients withoutprior antiretroviral therapy who are given the samethree drugs. In patients without responses to initialregimens, salvage therapy should be started at theearliest sign of failure (e.g., a sustained rise in the

plasma viral load 1.0 log10 copies per milliliter) andshould include two or three drugs the patient hasnever received.4,65

Combining a potent protease inhibitor with twonucleoside analogues appears to prevent the emer-gence of resistance to either class of drugs.82 Occa-sional lapses in compliance may also be less danger-ous in patients taking combinations of antiretroviraldrugs, because of the ability of one type of drug tosuppress the replication of virus resistant to the oth-er type of drug.

PATHOPHYSIOLOGIC CONSEQUENCES

Despite potential problems with resistance, treat-ment with combination antiretroviral regimens con-taining protease inhibitors has a number of beneficialpathophysiologic consequences. Besides increasingthe overall CD4 cell count, combination therapymay increase naive and memory T cells, enhancelymphoproliferative responses, and reduce plasmaconcentrations of harmful cytokines such as tumornecrosis factor.83 Early intervention with combina-tion regimens in a small number of patients with re-cent seroconversion forestalled the loss of HIV-spe-

cific CD4 lymphocyte responses that characterizesthe natural history of this disease.84

Unfortunately, protease-inhibitor therapy may notcorrect deficiencies in the T-cell repertoire alreadyinduced by HIV, and lost lymphocyte clones maynot be replaced.85 These observations may explain

why infections caused by opportunistic pathogenssuch as cytomegalovirus developed in some patientsduring the late stage of the acquired immunodefi-ciency syndrome (AIDS) even though their CD4counts increased from less than 50 to more than 200cells per cubic millimeter while they were receivingtreatment with highly active regimens containingprotease inhibitors.86,87

Finally, despite the prolonged suppression of HIVviremia in large numbers of patients, no one has yetbeen cured of HIV infection. Patients with unde-

Figure 3. Changes in HIV Viral Load over Time in Three PatientsTreated 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 inthe regimen occurred at the times indicated by the arrows. Thedotted lines indicate the lower limit of detection in an ultrasen-

sitive polymerase-chain-reaction assay for HIV-1 RNA.

20

200,000

20,000

2,000

200

SuccessfulTherapy

20

200,000

20,000

2,000

200

PlasmaHIVRNA(copies/ml)

SalvageTherapy

Changein regimen

20

200,000

20,000

2,000

200

0 4 8 12 16 20 24 28 32 36 40 44 48 52

Week

UnsuccessfulTherapy

Changein regimen



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tectable plasma viral loads and greatly reduced num-bers of viral particles in lymph nodes still had detect-able proviral DNA in cells even after 6 to 12 monthsof therapy.88,89 After at least two years of combina-tion therapy consisting of a protease inhibitor andnucleoside analogues, patients with undetectable

plasma viral loads still had HIV in their lymph nodesand peripheral-blood cells that was capable of repli-cation.90-92

Eradication of HIV with current combinationregimens may be impossible. Most if not all infectedpatients have a small amount of virus in long-livedcellular reservoirs, and eliminating HIV in even asmall percentage of such patients may require at leastthree to five years of continuous treatment.93,94 Fur-thermore, among patients with undetectable viralloads who are receiving indinavir combined with zi-dovudine and lamivudine, those who stopped takingone or two of the drugs after three to six months oftherapy 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 importantrecent advance in the treatment of HIV infection.

All infected patients with symptomatic disease,CD4 counts of less than 500 cells per cubic milli-meter, or plasma HIV RNA concentrations of morethan 5000 to 10,000 copies per milliliter should re-ceive a protease inhibitor plus two nucleoside ana-logues unless contraindicated.4,64 Although thesecombinations substantially reduce the plasma viralload, increase the CD4 cell count, reduce the pro-gression of disease, and prolong life, they have side

effects. The emergence of drug-resistant strains ofHIV may limit the long-term effectiveness of treat-ment with protease inhibitors. Monotherapy, sub-therapeutic drug concentrations, and noncompli-ance may promote resistance to this class of drugs.Using protease inhibitors in combination with nu-cleoside analogues or nonnucleoside reverse-trans-criptase inhibitors reduces the development of resist-ance but requires substantial cooperation on thepatients part.

Protease inhibitors are expensive, with an annualwholesale price ranging from $4,320 to $8,010 perpatient.97 Combination regimens that include pro-tease inhibitors cost about $10,000 per year of lifesaved.97 This figure compares favorably with the costof other prescription drugs given to prevent morbid-ity and mortality in patients with other chronic con-ditions such as hypertension and ischemic heart dis-ease.97

To maximize the long-term benefit of HIV-pro-tease inhibitors, several issues need to be resolved.

What are the most appropriate regimens for infants,children, and pregnant women? Can we developsimplified dosing schemes to promote adherence to

the regimen? Will combinations of two protease in-hibitors be as effective over time as a single proteaseinhibitor combined with reverse-transcriptase inhib-itors? What role should nonnucleoside reverse-trans-criptase inhibitors play in these regimens?

Important questions about protease inhibitors re-

main. Much of the information in this article has notbeen published in peer-reviewed journals, and the re-sults of important new studies are reported monthly.The complexity of decisions involving antiretroviraltherapy now rivals that of decisions involving che-motherapy for cancer. Physicians should maintain ahealthy skepticism while awaiting clarification of theoptimal use of protease inhibitors, and wheneverpossible, specialists in the care of patients with AIDSshould 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 preparationof the manuscript.

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