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ENFUVIRTIDE: THE FIRST TO INHIBIT THE ENTRY OF HIV-1 INTO HOST CD4 LYMPHOCYTES

Tom Matthews*, Miklos Salgo‡,Michael Greenberg*, Jain Chung‡,Ralph DeMasi* and Dani Bolognesi* Highly active antiretroviral therapy (HAART) based on combinations of drugs that target key enzymes in the life-cycle of human immunodeficiency (HIV) has considerably reduced morbidity and mortality from HIV since its introduction in the mid-1990s. However, the growing problem of the emergence of HIV strains that are resistant not only to individual drugs, but to whole drug classes, means that agents with new mechanisms of action are needed. Here, we describe the discovery and development of (Fuzeon), the first drug to inhibit the entry of HIV-1 into host cells.

CASE HISTORY The history of combination antiretroviral therapy, also need to be treated with sequential HAART regimens known as highly active antiretroviral therapy (HAART), progressively, using up the diminishing pool of active for HIV infection has been characterized by an drugs that are left available. aggressive scientific and pharmaceutical response to Mechanistic and in vitro genetic studies of viral the HIV epidemic. The elucidation of the HIV life isolates have signalled considerable cross-resistance cycle (FIG. 1) from the mid-1980s onwards revealed within structural ARV classes, such that resistance to targets for therapeutic intervention, ultimately lead- one member of a class translates to resistance against ing to the development of several classes of antiretro- other members of the class. By 2000, a high proportion of viral (ARV) drugs. Today, ARVs are usually combined patients in the United States and Europe had experienced in specific multidrug regimens, consisting of combi- a lengthy exposure to ARV agents and harboured virus nations of nucleoside (and nucleotide) reverse trans- resistant to one or more drugs4.More worryingly, it is criptase inhibitors (NRTIs), non-nucleoside reverse not unusual for patients to be infected with virus already transcriptase inhibitors (nNRTIs) and/or protease resistant to several ARVs at the time of diagnosis5.With inhibitors (PIs), which are tailored to the patient and chronic ARV use, more patients also experienced a the virus. broadening array of drug toxicities and rates of ade- HAART therapy changed the prognosis for quate compliance inevitably declined. Management of *Trimeris Inc., patients with HIV infection from that of high mor- these advanced patients is a considerable concern for 4727 University Drive, bidity and rapid mortality to, for many at least, a the treating physician6–9. Durham, North Carolina 1–3 27707, USA. chronic, manageable, but still complicated disease . There is therefore a substantial unmet medical need ‡Roche, 340 Kingsland Considering the low fidelity of the HIV reverse trans- for new ARVs with activity against HIV strains resistant Street, Nutley, New Jersey criptase enzyme combined with the high replication to existing ARVs. Enfuvirtide is such an agent, devel- 07110, USA. rate of the virus, it is not surprising that even triple- oped through academic and pharmaceutical partner- Correspondence to D.B. class HAART therapy eventually fails in the vast ships, and it is the first drug in an entirely new ARV e-mail: [email protected] majority of patients and is typically associated with class, the fusion inhibitors. Enfuvirtide was born in the doi:10.1038/nrd1331 the emergence of drug resistance. Patients therefore laboratories of Duke University, nurtured by Trimeris,

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Inc. and developed by the collaboration of Trimeris and for a vaccine, resulting in the rapid identification of these Roche. As the first approved ARV agent to inhibit extra- viral and the investigation of their function in cellular fusion of HIV-1 to host cells, enfuvirtide faced HIV-mediated membrane fusion10,11.The HIV envelope several unique challenges, which are discussed here. glycoprotein () is a type I integral membrane that is translated as a polyprotein (gp160)12–14.Proteolytic Investigation of new drug targets cleavage of gp160 yields the functional glycoproteins HIV fusion. Among the first responses in 1983 to the gp120 and , which are inserted into the HIV mem- identification of HIV as the aetiological agent of acquired brane. In the viral membrane, gp120 and gp41 associate immune deficiency syndrome (AIDS) was the search for as oligomeric trimers that are non-covalently bound. a vaccine. HIV membrane proteins were logical targets HIV-mediated fusion of the virus to the target host is a multi-step cascade involving a series of confor- mational changes in gp120 and gp41 (FIG. 2).The first Envelope event is the binding of gp120 to the CD4 on the core surface of LYMPHOCYTES expressing CD4. This causes Reverse unmasking of a second gp120-binding site for target transcriptase Fusion co-receptors CXCR4 and CCR5 and binding Virus adsorption inhibitors of gp120 to either or both co-receptors. Although CXCR4 and CCR5 are the major co-receptors, other co- RNA receptors can interact with gp120 in some settings11.The Virus–cell fusion completion of co-receptor binding leads to the fusion- active conformation of the viral transmembrane fusion protein gp41 (REF.10).The ectodomain of gp41 contains two heptad repeat regions: HR1 (proximal to the N Receptor and co-receptor proteins terminus) and HR2 (proximal to the C terminus). The hydrophobic fusion region inserts into the host Uncoating , whereas the HR1 regions of gp41 form a trimeric structure. HR2 regions then fold back NRTIs, Reverse nNRTIs within the hydrophobic grooves of the HR1 coiled coil, forming a hairpin structure containing a thermodynam- DNA ically stable six-helix bundle that draws the viral and cellular membranes together for fusion. The exposure of gp41 and conformational changes leading to fusion are Integration not instantaneous processes10.A transient pre-hairpin intermediate stage can last for several minutes, causing exposure of the N terminus of gp41 (REF.15).

Transcription HIV fusion targets derived from HR1 and HR2. Initially, synthetic derived from gp41 were not targeted as inhibitors of HIV-1 fusion but were investigated as part of -mapping experiments aimed at evaluat- ing strategies in vaccine development. Serendipitously,

Figure 1 | The life cycle of HIV and targets for therapeutic Translation intervention. Human immunodeficiency virus (HIV) adsorbs to the surface of host CD4 lymphocytes. The HIV envelope Polypeptide glycoproteins undergo a series of interactions with the CD4 receptor and one or more co-receptors (usually CCR5 or CXCR4). The culmination of these interactions is the fusing of Protease Proteolytic processing the HIV outer membrane and the host-cell membrane, inhibitors by viral protease leading to virus–host-cell fusion. The viral capsid core then disassembles and viral nucleic acids enter the cytosol in Viral proteins and association with virion proteins. A reverse-transcription RNA assemble at the cell membrane complex containing the HIV reverse transcriptase catalyses complementary DNA synthesis, and the resulting complex containing viral cDNA is transported to the host-cell nucleus. Budding Here, HIV integrase catalyses integration of viral cDNA into host-cell DNA. Expression of the viral DNA produces a precursor polypeptide that is proteolytically processed by HIV protease to give mature structural and functional viral proteins. These proteins assemble with HIV RNA at the cell membrane, LYMPHOCYTES from which they bud to release new virions. The targets against White blood cells of lymphoid which approved anti-HIV agents act are indicated. nNRTI, non- origin that function as part of nucleoside reverse transcriptase inhibitor; NRTI, nucleoside/ the . tide reverse transcriptase inhibitor. Adapted from REF.88.

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it was observed that when these were incubated with The first synthetic peptide mimetic (DP-107) was human T cells, an antiviral effect was seen. An under- a 38-amino-acid corresponding to gp160 standing of the fusion process, and how envelope glyco- residues 558–595, the N-terminal region of gp41 (REF.17). proteins interact, led to the appreciation of how these DP-107 possessed a highly ordered, stable helical struc- peptides inhibited fusion and thereby were able to inter- ture. In in vitro experiments, DP-107 inhibited HIV rupt the HIV life cycle (FIG. 2)16. replication at concentrations approaching 6 µg per ml.

a b Attachment d Anchorage

HIV gp41 HR2 CD4 receptor gp41 CD4+ HR1 gp120

Co-receptor

d Anchorage c Co-receptor binding g Fusion inhibition

HR2 gp41 HR1 Fusion inhibition

e h Fusion

f Infection i Infection blocked

Figure 2 | HIV entry and the mechanism of action of enfuvirtide. It is thought that HIV enters the host cell through several separate but cooperative steps — typically abbreviated to attachment (b), co-receptor binding (c) and fusion (d–f) — which are shown here. HIV predominantly infects T cells carrying the CD4 antigen (a) through an initial association of the glycoprotein gp120 with the CD4 receptor on the host cell (b). After this initial attachment, a conformational change is believed to occur in gp120 that allows its further association with host-cell chemokine co-receptors CCR5 and CXCR4 (c). Subsequently, a conformational change in the second viral envelope glycoprotein gp41 allows it to insert the hydrophobic N terminus into the host-cell membrane (d). The HR2 domain of gp41 then folds back on itself and associates with the HR1 domain (e); this process (known as gp41 zipping) leads to fusion of the viral and host-cell membranes and infection of the cell (f). However, in the presence of a fusion inhibitor, such as enfuvirtide (shown in yellow), an association between the fusion inhibitor and gp41 (g) prevents the successful completion of gp41 zipping (h), thereby blocking infection (i). Reproduced with permission from F. Hoffmann-La Roche and Trimeris Inc., 2001; copyright owned by Trimeris Inc. An animation of the HIV fusion process and its inhibition by enfuvirtide can be found on http://www.Roche-HIV.com.

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Amino-acid substitutions and chemical modifications Further Phase II studies established the longer-term within DP-107 destabilize secondary structure and safety profile of enfuvirtide-containing regimens of significantly reduce its antiviral activity18. 48–96 weeks26–29, and also investigated dose–response Subsequent studies examined the C terminus of gp41, over 48 weeks of treatment27,28. because it too possessed sequences that were predicted to form an α-helical structure19,20.The 36-amino-acid pep- Formulation and . Human trials with tide DP-178, later to be designated enfuvirtide (Fuzeon, enfuvirtide established predictable pharmacokinetic formerly T-20), corresponded to amino-acid residues indices24,28,30,31.These Phase II trials also demonstrated

643–678 of HIV-1LAI gp160 (FIG. 3).DP-178 blocked that enfuvirtide could be administered by subcutaneous SYNCYTIUM 24 HIV-1LAI-induced formation with an IC90 of 1.5 injection and that exposure to enfuvirtide was inde- ng per ml (100 times more potent than DP-107). DP-178 pendent of the site of injection30. FIGURE 4 shows the contained the epitope for the human monoclonal anti- predicted and mean observed plasma concentration– body 2F5, implying that this region was accessible during time profiles after subcutaneous injection across three the conformational changes leading to fusion16. doses31; the inset plot shows the predicted versus A recombinant gp41 peptide was constructed that observed data. Calculated from model-independent contained both regions, but it had little antiviral activity. analyses, TABLE 1 provides selected pharmacokinetic However, a within the N-terminal (DP-107) parameters after subcutaneous and intravenous region restored activity. The mutation probably caused administration. loss of helical constraint of the N-terminal region, Pharmacokinetic studies also provided confidence thereby liberating the C-terminal (DP-178) region. that sufficient plasma concentrations of enfuvirtide Indeed, alteration or truncation of C-terminal sequences would be available to inhibit HIV-1 during a 12-hour also eliminated activity21.Subsequent studies showed dosing period. Extrapolated from FIG. 4, plasma concen- that the pre-hairpin intermediate is the target for trations of enfuvirtide surpass the upper range of viral enfuvirtide binding22. sensitivity for at least 12 hours. Taking into account area

Enfuvirtide selectively inhibits HIV-1; neither HIV-2 under the curve (AUC)12h, plasma half-life and other nor simian immunodeficiency virus (SIV) are suscepti- pharmacokinetic measures, enfuvirtide clearly possessed ble. On the basis of its distinct extracellular mechanism the pharmacokinetic properties and potency necessary of action, enfuvirtide was expected to be active against for further study in HIV-infected patients. HIV-1 strains with resistance to any of the three other classes of ARVs and to multiple-drug-resistant virus, Drug–drug interactions. Being a peptide, enfuvirtide is and this expectation was confirmed in both subsequent expected to undergo catabolism to its constituent amino in vitro and early clinical studies23–25. acids, followed by retention and recycling of these amino acids in the body pool rather than metabolism by Early clinical studies of enfuvirtide cytochrome P450 (CYP450). Therefore, there is a low At the start of the enfuvirtide clinical programme, there potential for enfuvirtide to affect or be affected by other was some scepticism as to the therapeutic potential of a drugs. To confirm this, several studies have evaluated peptide inhibitor of HIV fusion. Although preclinical the drug– potential of enfuvirtide. data demonstrated substantial viral suppression19,20, The results of the so-called Pittsburgh cocktail enfuvirtide represented the first ARV to act extracellu- study (T20-502) showed that the administration of larly to inhibit and so it was essential to enfuvirtide (90 mg twice daily) to HIV-infected establish definitive proof-of-principle in vivo.The patients was not associated with clinically relevant appropriate choice and management of patients was effects on the pharmacokinetics of several drugs also crucial. The unmet medical need was in very selected because of their known route of metabolism treatment-experienced patients who had already been (caffeine for CYP1A2, chlorzoxone for CYP2E1, dapsone exposed to the existing ARVs and who had failed therapy for CYP3A4, debrisoquine for CYP2D6 and mepheny- because of the emergence of virus resistant to the toin for CYP2C19)32. existing . As enfuvirtide was active against A further three studies have investigated the effect of virus with multiple resistance to other ARVs, it had rifampicin, (Norvir; Abbott) and ritonavir- great potential if it could be used in heavily pre-treated boosted (Invirase/Fortovase; Roche) on patients to meet this unmet medical need. This patient the pharmacokinetics of enfuvirtide33.Rifampicin population had rarely been characterized sufficiently was selected because it is a well-characterized and for large clinical trials and drug registration, so out- potent enzyme inducer and is used in the treatment comes would therefore be unpredictable. of tuberculosis in HIV-infected patients. Ritonavir A simple proof-of-principle result came reassuringly was selected because multiple studies have shown quickly. In a 14-day trial (TRI-001) to establish dose– that this HIV protease inhibitor is the most potent response, enfuvirtide was administered as monotherapy CYP450 inhibitor clinically studied, and ritonavir- by intravenous infusion in the dose range 3–100 mg, boosted saquinavir was selected as a commonly twice daily, to 16 patients. At the highest dose of 100 mg, administered boosted protease-inhibitor regimen. All SYNCYTIUM median viral suppression was –1.96 log copies per ml three studies showed that enfuvirtide was not affected A multi-nucleated mass caused 10 by the fusion of several individual HIV-1 RNA and dose–response was linear within the to a clinically meaningful extent by any of the other cells; often virally induced. entire range23. drugs administered33.

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O NH2 O NH2 O OH HO

HO HO O O O O O O O O H H H H H H H N N N N N N N N N N N N N N N O H H H H H H H H NH O O O O O O O H N H2N 2 HO HO O N O NH O HN O OH OH O NH O

O

H2N HN

O O NH H2N HO NH2 O HO O O HN O

O O O O O H NH HN H H H H N NH N N N N N N N N N O O H H H H H H2N O O O O O HN O OH O OH O NH NH2 O HO O HN O

HN Enfuvirtide C H N O NH NH2 204 301 51 64 O Mr = 4492

Figure 3 | Structure of enfuvirtide.

Manufacturing of enfuvirtide. Relatively small quantities Efficacy of enfuvirtide of other medical peptides, such as calcitonin, are success- The pivotal Phase III TORO (T-20 versus Optimized fully manufactured synthetically and the early proof-of- Regimen Only) trials were successful in several respects. concept studies with enfuvirtide also used synthetically With ~1,000 treatment-experienced patients enrolled, produced drug. This method of manufacturing was con- they offered definitive proof of the therapeutic value of sidered to have the greatest likelihood of success for enfuvirtide. Because of their inclusiveness, the TORO rapidly scaling up to a commercial scale, although sig- studies enrolled treatment-experienced patients with nificant challenges were anticipated. These challenges broad demographic, disease and virological character- are obvious when one considers that enfuvirtide is a istics (TABLE 2). 36-amino-acid peptide with a complex structure and a TORO 1 was conducted in the United States, molecular weight of 4,492 Da. Due to its large size, the Mexico, Brazil and Canada35 and TORO 2 was con- manufacture of enfuvirtide is very complex, involving ducted in Europe and Australia36.The trial designs were 106 steps in the chemical pathway. In comparison, a almost identical, differing only in the minimum length typical manufacturing process for small-molecule drugs of previous experience for ARVs from each class (six usually involves between eight and twelve steps. It is the months for TORO 1 versus three months for TORO 2), most complex synthetic peptide manufactured at such a and the number of previous PIs that patients must have massive scale (the current manufacturing capacity is >3 received (two for TORO 1 and one for TORO 2). To metric tonnes per year). enter the TORO studies, patients must have been Enfuvirtide, which is supplied as a 100-mg lyo- exposed to, and/or have documented resistance to, each philized, carbonate-buffered powder that must be of the three existing classes of ARVs (NRTIs, nNRTIs reconstituted with 1.1 ml of sterile water at the time of and PIs). FIGURE 5 schematically describes the TORO administration, is given twice daily by a 1.0 ml subcuta- study design. neous injection. The approved dose is 90 mg twice daily, HIV-1 from each patient underwent genotypic and although a dose higher than 180 mg per day has not been phenotypic analysis during screening. Investigators studied clinically. However, the approved dose gave a and patients were instructed to choose an individualized strong pharmacodynamic profile with evidence that ‘Optimized Background’ (OB) ARV regimen of three to the 180 mg per day dose was on the plateau portion of the five drugs on the basis of the viral drug resistance testing exposure–response curve34.Furthermore, higher doses as well as the patient’s previous history and tolerability. would entail a higher injection burden for the patient that Once investigators and patients had committed to this might be expected to impact the acceptance of therapy. OB regimen, they were randomized (at a 2:1 ratio) to

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10 In both TORO trials, therapy with enfuvirtide together with OB for 24 weeks resulted in a further reduction in from baseline, approaching 1.0 68 log copies per ml35,36.Immunological response was also 12 10 greater in the enfuvirtide group, with an additional 3 8 benefit of +44 cells per mm in TORO 1 and +28 cells 6 per mm3 in TORO 2 (REFS 35,36).This was a particularly

g per ml) 4 µ Predicted impressive finding given the advanced stage of disease in

0 the patient group enrolled. 4 0 4 8 12 Combining the data from both TORO studies was Observed

Enfuvirtide ( pre-planned. As the studies were of almost identical design, if the outcomes of the two studies were similar, 2 then the combined database would provide a more accurate estimate of the treatment effect and also pro- vide additional power for subgroup analyses. In this 0 merged data set37,enfuvirtide plus OB again demon- 0 6 12 18 24 30 36 42 48 strated superior virological and immunological activity Time (hours) to OB alone in a 24-week period. Figure 4 | Enfuvirtide plasma concentration–time plots. Predicted (solid line) and mean observed (symbols) plasma concentration–time profiles after administration of a single Accelerated approval was granted to enfuvirtide in subcutaneous bolus dose of 45 mg (squares), 90 mg (circles) and 180 mg (triangles) enfuvirtide. the United States and Europe on the basis of these Phase Inset: plot of predicted versus observed data (180 mg). Adapted from Zhang et al31. III 24-week results. Regulatory authorities and the med- ical community both demand long-term data for con- ventional approval and for reassurance of the durability of response. Data collected after 48 weeks from TORO 1 enfuvirtide plus OB (enfuvirtide group) or to the OB and TORO 2 were combined and analysed by INTENT- regimen alone (control group). If patients experienced TO-TREAT (ITT) methods. Differing from the 24-week virological failure during the TORO trials while receiving analyses, the categorical endpoints were the primary the OB regimen alone, they could revise their OB regi- endpoints for the 48-week analysis of durability of men on the basis of repeat viral resistance testing and response. These analyses confirmed the durability add enfuvirtide (referred to as ‘switch’). of enfuvirtide added to an OB regimen38 (TABLE 3), and Through 24 weeks, the primary efficacy endpoint showed that a high proportion of patients in each treat- was change from baseline in HIV-1 RNA — a departure ment group who responded at week 24 (HIV-1 RNA from most previous Phase III ARV trials evaluating <400 copies per ml) also responded at week 48 (80% in HAART therapies in ARV-naive patients that used a the enfuvirtide group and 70% in the control group39). categorical endpoint of proportion of patients below INTENT TO TREAT limit of assay detection of HIV-1 RNA. Such categorical Subgroup analyses. The principal 48-week results A conservative form of analysis in which the endpoints were secondary endpoints in the TORO extended to demographic and baseline subgroups. numerator is the number of analyses, because of the uncertainty about how many Enfuvirtide worked equally well in men and women, patients in a given arm achieving experienced patients would reach these levels and how irrespective of age or race40.Patients that were healthier an end-point and the well the OB regimen might perform. (If the categorical at the onset of treatment (higher CD4 count, lower viral denominator is the total number response on the control arm was very high or very low, load) derived the greatest benefit from enfuvirtide; how- of patients assigned to that arm, with discontinuations and/or there was a risk that a true treatment difference could ever, patients with advanced disease (viral load >100,000 missing data classed as failures. be missed.) copies per ml and CD4 cell count <100 cells per mm3) also demonstrated significant virological and immuno- logical response. These findings confirmed earlier Table 1 | Enfuvirtide single-dose pharmacokinetic parameters (CV%) analyses based on the TORO 1 and TORO 2 24-week database, in which patients receiving enfuvirtide plus Parameter 45 mg SC 90 mg SC 180 mg SC 90 mg IV OB showed greater virological and immunological C (µg per ml) 2.48 (33%) 4.59 (32%) 8.05 (23%) 22.9 (22%)* max improvement across all subgroups compared with ‡ 41,42 Tmax (h) 5.38 (44%) 6.92 (36%) 6.50 (27%) 0.083 (0%) patients receiving OB alone . •µ AUC∞ (h g per ml) 25.8 (26%) 55.8 (22%) 113 (20%) 67.4 (26%) The TORO patient population had broad treatment experience with a median prior exposure to 12 ARVs and T1/2 (h) 3.46 (12%) 3.80 (15%) 4.35 (11%) 3.16 (13%) CL (l per h) - - - 1.4 (21%) seven years of therapy.Viral suppression was significantly greater with enfuvirtide plus OB treatment than with OB CL/F (%) 1.85 (24%) 1.68 (22%) 1.65 (20%) - abs treatment alone across all subgroups when classified V (l) - - - 5.48 (21%) ss according to the number of active ARVs in the back-

Fabs (%) 77.3 (17%) 84.3 (18%) 85.6 (15%) - ground regimen, including those patients with no active 40 Data are mean values, with coefficients of variations given in parentheses. AUC∞, area under the plasma ARVs in their background regimen .Response in both concentration–time curve extrapolated to infinity; CL, systemic clearance; CL/F , apparent clearance; abs treatment groups improved with increasing numbers of C , maximum observed plasma concentration; Fabs, absolute ; IV, intravenous; MRT, mean max active drugs in the background regimen (FIG. 6), and residence time; SC, subcutaneous; T1/2, half-life; Tmax, time to reach Cmax; VSS, steady-state volume of distribution. *Mean concentration at first sampling time. ‡First sampling time. response to enfuvirtide plus OB in patients with one

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Table 2 | TORO demographic and baseline data (ITT) insight and suggests that although enfuvirtide will bene- fit all patients, earlier use in the treatment algorithm, Parameter Enfuvirtide Control group N = 661 group N = 334 combined with other fully active ARVs, can provide the greatest benefit. Gender, male (%) 90 90 Race, white (%) 89 89 Virological failure. Median time to protocol-defined Age (median, years) 41 42 failure during 48 weeks of therapy was three times as

HIV-1 RNA (median, log10 copies per ml) 5.2 5.1 long in the enfuvirtide group (32 weeks) as in the con- 38 CD4 count (median, cells per mm3)8897 trol group (11 weeks, P <0.0001) .Patients who were Prior ADEs (%) 79 86 characterized as failing therapy were more likely to be on the OB arm (71% versus 46%). Patients that failed GSS* (mean) 1.7 1.8 over the course of the trials tended to have fewer active PSS* (mean) 1.6 1.6 drugs in their background regimen, lower baseline CD4 *The number of antiretrovirals in the OB regimen to which the virus is genotypically (GSS) or pheno- cell counts and had been exposed to more ARVs before typically (PSS) sensitive. ADE, AIDS-defining event; GSS, genotypic sensitivity score; ITT, intent to 44 treat; PSS, phenotypic sensitivity score. enrolling in the TORO trials .

Resistance to enfuvirtide active ARV in their background regimen was similar to, Although enfuvirtide is active in patients whose virus is or greater than, response to OB alone in patients with resistant to the other classes of ARVs, as is the case with two, three, four or five active ARVs in the background all other antiretrovirals, continued exposure, especially regimen. Not surprisingly, patients who had been previ- if the virus is not completely suppressed, can lead to ously exposed to fewer ARVs also responded better to emergence of viral resistance to enfuvirtide. enfuvirtide plus OB, presumably an indication of less advanced disease and a reflection of the increased In vitro studies. The N-terminal region of gp41 was options that these patients had to include fully active evaluated with laboratory and clinical viral isolates ARVs in their background regimen. with reduced susceptibility to enfuvirtide. Rimsky et al. A simplified model investigating prognostic factors identified susceptibility loci by SERIAL PASSAGE of viral

that might predict response to ARV therapy including strains HIV-1IIIB and NL4-3 in increasing concentra- enfuvirtide has identified four factors that are associated tions of enfuvirtide45.All escape mutants mapped to with greater response to enfuvirtide: first, CD4 count the HR1, N-terminus of gp41 and in particular to a >100 cells per mm3;second, HIV-1 RNA <100,000 contiguous sequence within the heptad-repeat motif. copies per ml; third, experience of ≤10 prior ARVs; and This sequence is a deep cavity within the HR1 trimer last, combining enfuvirtide with at least two active and spanned positions 32–41 within the HR1 region of ARVs43.For patients that have all four positive prognostic gp41, with positions 36 (G36D) and 38 (V38M) being SERIAL PASSAGE factors, the chance of achieving a viral load <400 copies most sensitive46,47. Innoculation of a chain of in vitro cell cultures with samples of an per ml at week 24 when receiving enfuvirtide plus OB is infectious agent drawn from the 80%, compared with 50% for patients receiving OB Resistance to enfuvirtide in clinical studies. An early clini- previously infected culture. alone43.For the treating physician, this provides valuable cal trial exposed patients to short courses of enfuvirtide monotherapy to evaluate dose–response relationships23. Viral isolates were sequenced by amplifying the HR1 and HR2 regions of gp41 (REF.48).The lowest (10 mg twice Randomized 2:1, then start enfuvirtide + OB or OB daily) and highest (100 mg twice daily) doses of enfuvir- tide did not select for resistance, but virus isolated during exposure to, and rebound from, intermediate doses Screening period Enfuvirtide + OB showed amino-acid substitutions within the HR1 region. Amino-acid substitutions occurred at gp41 positions 32 Sample for Stable (Q to R/H), 36 (G to S), 38 (V to A) and 39 (Q to R). GT/PT regimen Switch permitted at virological failure* after Subsequent analysis of sequences derived from clinical –6 –4 week eight isolates continued to highlight the crucial core region of residues 36–45 within HR1 (REF.49,50). OB The evolution of resistance to enfuvirtide at amino- BL 8 16 24 48 acid positions 36–45 was analysed from virus isolated before and during treatment51.At baseline, virus from Weeks 177 enfuvirtide-naive patients was sequenced with 80% Figure 5 | TORO 1 and TORO 2 study design. The novel TORO study design accommodated showing the consensus sequence at positions 36–45, treatment-experienced patients by analysing virus at screen genotype (GT) and phenotype (PT) and and 15% of the viral isolates showing an N42S poly- selecting an optimized treatment background (OB) that patients began at baseline (BL). After week morphism. The remaining 5% had substitutions other eight, patients who failed OB therapy could switch to revised OB regimens and receive enfuvirtide. The initial randomization was 2 (enfuvirtide + OB): 1 (OB alone). First planned analysis was at week than N42S. At protocol-defined virological failure, the 24 and the second was at week 48. *Criteria for virological failure based on two consecutive values: virus from 78% (31 out of 40) of patients had substitu- tions associated with resistance at positions 36–45. <0.5 log10 decrease from BL starting at week six and eight; <1.0 log10 decrease from BL starting at week fourteen and sixteen; and ≥ 2 log response and and >1 log rebound at any time. Investigation of the replicative capacity of virus with

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Table 3 | Percent responders at week 48 (intent to treat population) would enfuvirtide cause local inflammatory or more serious immunological reactions?; and last, would the Parameter Enfuvirtide Control group N = 661 group N = 334 human immune system eventually reject enfuvirtide? Analysis of clinical data from Phase II and the Phase III HIV RNA copies/ml (% patients): <50 18 8 HIV RNA copies/ml (% patients): <400 30 12 TORO trials addressed these questions. Overall surveillance during the TORO trials included HIV RNA copies/ml (% patients): reduction of >1.0 log10 37 17 663 patients randomized to enfuvirtide plus OB and Change from baseline log10 HIV-1 RNA –1.48 –0.63 (mean, copies per ml) 334 patients randomized to OB alone. The combined Time to protocol-defined 32 11 safety record of the TORO trials through 24 weeks has virological failure (median, weeks) been described37.During the 48 weeks, 26.5% of Least squares mean increase in CD4 cell 91 45 patients in the enfuvirtide group and 36.6% in the count (median cells per mm3) control group discontinued therapy. Discontinuation All differences P <0.0001. due to adverse events occurred in 8.9% of patients in the enfuvirtide group and 3.6% of patients in the con- trol group. Injection-site reactions (ISRs) were tabu- enfuvirtide confirmed that enfuvirtide- lated separately and accounted for a discontinuation resistance mutations in HR1 identified in patient isolates rate of 4.4%. obtained after treatment with enfuvirtide conferred a During the 48-week period, the exposure to enfu- significant growth disadvantage in the absence of drug virtide plus OB, switch, combined enfuvirtide [(enfuvir- as compared with wild type52,53. tide plus OB) plus switch] and OB was 557, 120, 677 Viral preference for chemokine co-receptor varies and 162 patient years, respectively. The most com- with the phase of HIV infection; during acute infection, monly reported events were diarrhoea (37.1 per 100 CCR5-tropic HIV variants predominate, whereas patient-years on combined enfuvirtide versus 73.4 on CXCR4-tropic variants emerge during later stages of OB), nausea (26.2 versus 50.0) and fatigue (25.0 versus disease. In vitro evidence from laboratory virus and 37.6)60.Other common constitutional signs/symptoms from a small number of clinical isolates indicated that reported at a lower frequency on combined enfuvirtide sensitivity to enfuvirtide might be influenced by than on OB were headache (15.8 per 100 patient years chemokine co-receptor TROPISM54–56. Enfuvirtide clinical on combined enfuvirtide versus 24.1 on OB), trials provided the opportunity to examine a larger (16.6 versus 19.7) and vomiting (15.8 versus 26.5). number of clinical isolates that expressed tropism for Among the events reported more commonly on the dual or individual co-receptors. Using several cell-based combined enfuvirtide arms were peripheral neuro- assays, Greenberg et al. evaluated co-receptor pheno- pathy (16.3 per 100 patient years on combined enfuvir- type and syncytium formation of 151 clinical trial iso- tide versus 13.6 on OB), weight decrease (12.1 versus lates and 8 isolates from the National Institute of Allergy 10.5), sinusitis (9.3 versus 6.2), appetite decrease (8.3 and Infectious Disease Repository. CCR5-, CXCR4- and versus 4.9) and lymphadenopathy (7.1 versus 1.2). An dual-tropic virus exhibited similar sensitivities57. imbalance in the rate of , primarily bacterial, Whitcomb et al. examined 612 baseline clinical-trial was also observed at 48 weeks. In the combined enfu- isolates using a recombinant assay to target cells that virtide group, 6.7 patients per 100 patient years expressed CD4 and either CCR5 or CXCR4 co- showed pneumonia, in contrast to 0.6 patients per 100 receptors58. CCR5 isolates comprised 62% of the popu- patient years in the control group60.Ofnote, the rate lation, 34% of isolates were dual-tropic and only 4% of observed in the enfuvirtide arm was within ranges

baseline isolates used CXCR4. The geometric mean IC50 reported for this patient population in the literature. A was 44 ng per ml for CXCR4 and 25 ng per ml for CCR5 review of data from the Adult Spectrum of Disease

isolates. Although the IC50 for CXCR4 isolates was study (Seattle) indicated that in the post-HAART-era slightly higher than for the other two types, mean viro- pneumonia rates have declined, but are still in the logical response (change from baseline) to enfuvirtide at range seen on the enfuvirtide arm in the TORO studies 24 weeks (ITT, last observation carried forward and that the risk of pneumonia is closely linked with (LOCF)) was generally similar among patients who CD4 count.

harboured CCR5 (–1.55 log10 copies per ml), dual tropic Clinical laboratory findings identified only increased × 9 (–1.64 log10 copies per ml) or CXCR4 (–1.34 log10 copies eosinophila (>0.7 10 cells per l) as imbalanced per ml) at baseline (P = 0.4)59.These data would between treatment groups, occurring in 11.1 per 100 therefore indicate that viral tropism has no clinically patient years in combined enfuvirtide patients and 3.7 relevant effect on treatment response to an enfuvirtide- per 100 patient years in OB patients. However, when containing regimen. using the higher (>1.4 × 109 per l) threshold for marked , similar rates per 100 patient years were The clinical safety of enfuvirtide seen for both treatment groups. Eosinophila was not As enfuvirtide entered the clinic, several safety questions associated with hypersensitivity in either treatment were foremost: what would be the overall safety profile group. Overall, Grade 4 treatment-emergent laboratory TROPISM in treatment-experienced patients on various combina- abnormalities were seen in 15.5 per 100 patient years of The affinity of a virus for a particular biological feature that tions of ARVs?; what would be the consequences of combined enfuvirtide patients and in 25.9 per 100 affects its infectivity or growth. chronic, twice-daily injections?; as a foreign peptide, patient years of patients on OB regimen alone.

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The presence of anti-gp41 did not seem to ENF + OB (N = 661) influence the activity of enfuvirtide. Mean change from 60 OB (N = 334) baseline in HIV-1 RNA at 24 weeks was identical among P ≤ 0.05 * enfuvirtide groups with negative, non-quantifiable and 50 48 positive antibody levels (1.6 log10 copies per ml) and was 39 in a narrow range (0.6–0.8 log copies per ml) in the 40 10 33 OB group. Rates of adverse events, hypersensitivity reac- 29 29 29 tions and Grade 3 or Grade 4 laboratory toxicities were 30 comparable, regardless of baseline or on-treatment * * antibody status or magnitude of antibody level. 20 18 15 12 * Quality of life 8 10 7 The TORO studies included a Subcutaneous Injection * Survey (SIS) to evaluate patient attitude to self- 0 0 63,64 Percentage of patients (viral load <400 copies per ml) Percentage injection .Patients were surveyed at weeks 8, 24 and 0 1 2 3 4 5 48. The majority of patients assessed all items relating to Genotypic sensitivity score (GSS) ease of injection as ‘very easy’ or ‘easy’ and assessed their ENF + OB 112 194 174 108 44 17 daily functioning as ‘not at all’ or ‘a little’ limited by enfu- OB 53 95 93 61 21 8 virtide self-injection. The results of the SIS indicated that Figure 6 | TORO subgroup analysis by genotypic sensitivity score. Percent responders with most patients with appropriate education and training HIV-1 RNA <400 copies per ml, week 48 (intent-to-treat, discontinuation or virological failure = were able to learn to integrate enfuvirtide dosing into failure). GSS is the actual number of antiretrovirals that the baseline virus is sensitive to as indicated their daily routines. Patients were also evaluated using the by standard primary mutations that each virus possesses. ENF, enfuvirtide; OB, optimized Medical Outcomes Study (MOS)-HIV questionnaire65. treatment background. This is a well-established and comprehensive measure of Heath Related Quality of Life (HRQoL). Patients were evaluated at 24 and 48 weeks. The results of the MOS- Throughout 48 weeks, almost all patients (98.3%) HIV showed that for most parameters evaluated there receiving enfuvirtide cumulatively reported ISRs. At any was a slight improvement in HRQoL during the 48 weeks given visit, 46–53% of patients reported mild tender- of therapy in both patients receiving enfuvirtide plus OB ness, 15–20% moderate pain without limitation of and those receiving OB alone65. usual activities and 0.9–3.3% severe pain requiring anal- gesics or limiting usual activities. The most frequent The future of entry inhibitors signs and symptoms of a local ISR were pain/discomfort Enfuvirtide ushered in a new class of ARVs that can (96%), erythema (91%), induration (90%) and nodules suppress multidrug-resistant virus by virtue of a distinct and cysts (80%)60.Importantly, there was no evidence of mechanism of action. Entry inhibitors also have the an increase in severity over time for any of the signs and potential for marked synergism with, and lack of cross- symptoms of a local ISR and few patients experienced resistance to, NRTIs, nNRTIs and PIs66.Each step in the infection at the injection site. entry process is a potential target, and there are a Several histopathology and histochemistry studies number of compounds in development directed against have been conducted to help elucidate possible mecha- each step that show promise, some of which are now in nisms of ISRs. Ball and Kinchelow61 reported on exci- clinical trials. The initial step in HIV entry is the recog- sional biopsy findings from seven patients with ISRs in nition and binding of viral gp120 to host CD4 receptors, enfuvirtide clinical trials. Although responses varied, an interaction that is the target of several small mole- inflammatory infiltrates were found that were consistent cules67,monoclonal antibodies68 and recombinant with a localized reaction. Conspicuous eosinophils and that link CD4 receptors to IgG2 constant histocytes were present, but lymphocytes and plasma regions69–73.The CD4-IgG2 tetramer seems to be highly cells were rare. Multinucleated forms occurred that in synergistic in vitro with enfuvirtide73.A single-dose, two patients were similar to a granulomatous response. dose-escalation study of the intravenously administered There was no relationship between inflammatory TNX-355 demonstrated antiviral response and clinical findings. activity at all doses68. Questions of antibody neutralization of enfuvirtide CCR5 and CXCR4 co-receptor inhibitors have also and induction of inflammatory responses or other been intensely investigated. CCR5 inhibitors include adverse events have been addressed40,62.Serum samples RANTES antagonists and other small molecules, some of for assaying of anti-gp41 antibody that crossreacted to which have already demonstrated early clinical activity enfuvirtide were taken during the TORO trials. Across and also in vitro synergism with enfuvirtide. The TAK both treatment arms, approximately 75% of patients series has good in vitro activity74,75 and synergism with were positive for anti-gp41 antibody and 23% of patients enfuvirtide has been reported76,77.A monoclonal anti- had non-quantifiable levels of antibody at baseline; less body (PRO-140; Progenics) inhibits viral entry without than 2% of patients were antibody-negative at baseline. influencing CCR5 function in vitro,and antiviral activity At 24 weeks, 65% of enfuvirtide patients and 13% of OB was seen in a therapeutic mouse model78.The RANTES patients had decreases of 30% or more in antibody levels. antagonist SCH-C (Schering-Plough) performed well in

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initial dose-range trials56.SCH-C is synergistic with enfu- The subgroup analyses uncovered clues to the optimal virtide79, and along with enfuvirtide is active in multiple use of enfuvirtide and the management of treatment- cell types, including peripheral blood mononuclear experienced patients. Regardless of baseline status, all cells, dendritic cells and macrophages80.To date, escape patient groups benefited from enfuvirtide therapy. mutants do not seem to switch to a CXCR4 pheno- Multiple- and logistic-regression analyses identified four type81,82.The development of inhibitors of CXCR4 key prognostic factors for virological and immunological binding has been somewhat less successful and there is response: lower baseline viral load, higher baseline CD4, concern about the consequences of blocking a receptor more active ARVs in the OB and a fewer number of that is widely present in different cell types66.Results from prior ARVs. Although recognition of such patterns a clinical study with the AMD-3100 might also be thought of as common-sense logic in (AnorMed) did not show consistent antiviral activity83. treating HIV patients, it takes on additional gravity Similarly to enfuvirtide, T-1249 is a peptide fusion when demonstrated for heavily treatment-experienced inhibitor that interacts with the gp41 HR1 region of patients and provides confidence when devising treat- HIV-1. Its sequence overlaps with, but is not identical to, ment strategies. enfuvirtide and contains short sequences derived from The TORO studies succeeded with the utilization of SIV and HIV-2. Owing to these differences, T-1249 is state-of-the-art patient management, incorporating active against laboratory and clinical viral isolates that both physician and nursing support. An often over- are resistant to enfuvirtide84.Although not presently in looked finding is that patients receiving the background clinical development, the results of a dose-ranging trial therapy alone demonstrated an uncharacteristically in 115 patients during a 14-day period demonstrated good clinical response. This probably reflects the use of dose-dependent viral suppression with a median HIV-1 antiviral-resistance testing before study treatment. Most

RNA suppression of –1.96 log10 copies per ml at the patients could construct an OB regimen with at least highest dose (200 mg per day) during monotherapy. No partial activity. The fact that the addition of enfuvirtide dose-limiting toxicity occurred85.Furthermore, T-1249 to this OB regimen significantly enhanced the response activity was not affected by ARV resistance or prior in this patient population highlights the additional treatment history 86,87. benefits of adding an ARV agent from a new class. Obviously at the start of the enfuvirtide clinical pro- Discussion gramme the impact of chronic daily injections was a Enfuvirtide is the first ARV fusion inhibitor approved major concern; injection burden, the routine of assem- for the treatment of HIV-1-infected patients. It is by far bling supplies and preparing and administering injec- the most complex ARV ever manufactured at such a tions could have affected the utility of enfuvirtide large scale, which provided both production and eco- therapy. The experience of study patients for as long as nomic challenges. four years indicates that injection burden or fatigue was The target population for enfuvirtide is treatment- not insurmountable and did not unduly affect activities experienced patients with limited remaining therapeutic of daily living. options. This population is steadily rising as increasing The synergistic effects of investigational ARVs, such numbers of patients progress through the treatment as CCR5 inhibitors with enfuvirtide, are now being algorithm. The most gratifying aspect of the develop- investigated. Long-term data from these studies will be ment of enfuvirtide is the hope it has given to these important in determining future treatment strategies individuals, who are some of the most difficult to treat and, once again, prolonging and enhancing the life of HIV-infected patients. patients infected with HIV.

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Analysis of virological failure through 24 and antiviral activity of the human immunodeficiency virus AIDS: understanding HIV transmission | HIV life cycle and weeks of treatment in the TORO pivotal studies. 2nd Int. (HIV) type 1 PRO 542 in HIV-infected adults. inherited co-receptors | human immunodeficiency viruses (HIV) | AIDS Soc. Conf. HIV Pathog. Treat. A574 (2003). J. Infect. Dis. 182, 326–329 (2000). Access to this interactive links box is free online.

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