At the 13th Conference on Retroviruses and Opportunistic Infections (CROI) held in Denver USA from Feb 5-8, 2006, one was afforded a snapshot into the world of investigation in HIV (from immunology and vaccines through research on clinical care and scale-up in developing countries). CROI offered plenaries and roundtable symposia, oral presentations, posters and poster discussions, late breakers, and in the last few years have added hands-on computer workshops and a workshop for new investigators and trainees. There were also (5) research overviews, and as this was an anniversary year (25 years of HIV, 10 of HAART), there were good retrospectives on how we have gotten to where we are today in terms of investigation.

Although there are always the camera-nazi shock troops and other non-human policies at work, this year's CROI seemed more laid back and into learning about the science than ever.

Roughly, half of the abstracts (oral, poster or late breaker) were university-driven, one quarter of hospital/medical center origin, and 15% were government-driven. The other 10% was divvied up between industry and foundations/private practices. Geographically, North America led the way (55% of presentations) with Europe second (30%). Africa was third, with close to 6% of the total. Asia, Australia and South America filled out the rest.

Many of the sessions were both webcast and podcast and can be seen at www.retroconference.org for free. To learn more about any drug mentioned here, please look it up by abstract number at the above web-site and you can read the whole abstract and in many cases, the full poster is also available. In this article, I will focus on drugs in the pipeline, from the one that is mere months away from approval (fingers crossed) to many that are just seeing the light of day for the first time here at the conference. Shortly, there will be a new issue of TAGline focusing on oral R5/X4 inhibitors; there will also soon be a more detailed look at drugs in late phases. This article is to give a quick overview of what was presented in Denver.

Phase III

Darunavir/r (Formerly known as TMC114/r)
Darunavir is a PI being studied in an advanced HIV population. People with NNRTI, NRTI, and PI experience, at least 1 primary PI mutation and viral load >1000 copies/mL were randomized to receive an optimized NRTI background /r or a CPI (investigator-selected comparator protease inhibitor) of choice. At week 24, efficacy was analyzed by baseline genotype and sensitivity to the CPI using phenotypic cut-offs; analysis was by viral load (intent-to-treat,non-completer = failure).

Mean baseline viral load was 4.6 log10 copies/mL, with median 8 PI -associated mutations. Viral load reduction for darunavir/r was significantly greater than for any CPI (p <0.0001), sensitive or resistant. Mutations associated with decreased susceptibility to darunavir were identified from over a thousand screening samples from various trials. Among those mutations, the presence of V32I, I47V, or I54M at baseline was associated with a lower mean decrease in viral load at week 24 but still higher than control. Site-directed mutants with these mutations in different combinations did not show a darunavir fold change >4. Analysis of people receiving darunavir/r who responded and then lost their antiviral response showed development of mutations V32I, L33F, I47V, or I54L (abstract 157).

In a related presentation, the two POWER studies are ongoing randomized, controlled, phase IIb/III studies to evaluate dose-response after 24 weeks in triple class-experienced subjects with ?1 primary PI mutation. Darunavir PK parameters, area under the curve and trough concentration in were analyzed in 468 people. Inhibitory quotients (IQ) (the ratio between steady state darunavir trough concentration and baseline darunavir EC50) were shown to be related to efficacy (virologic and response parameters). PK:efficacy, though statistically significant, was strongly influenced by baseline darunavir fold change, baseline viral load and use of sensitive drugs in the optimized background regimen. The IQ was the strongest predictor of virologic response, with the relationship primarily driven by baseline darunavir fold change (FC). IQ values of darunavir were generally high (mean values >200). There was no apparent relationship between darunavir PK and safety.

Although the investigators say that this data implies the dosing choice of 600/100 is justified (abstract 639b), it tells me that a high IQ is helpful although not necessary for a response (sustained?) to darunavir/r. For more efficacy data presented at ICAAC and more recently at BHIVA, there will be a more thorough look at the POWER trials in the next few weeks.

Etravirine (Formerly known as TMC125)
Etravirine is a non-nucleoside reverse transcriptase inhibitor (NNRTI) in Phase II/III with activity against virus with existing NNRTI mutations. This analysis investigated baseline resistance parameters that influenced virologic response. Looking at heavily pretreated people with documented NNRTI resistance and ?3 primary protease inhibitor (PI) mutations at baseline, randomization was to 1 of 2 different doses of etravirine with an optimized background regimen or a standard-of-care control regimen. Intent-to-treat analyses of the etravirine 800 mg (old formulation) BID group is shown in the box. The median PI exposure was 4; 94% had used an NNRTI and 30% T-20. At baseline people had a median of 4 primary PI (83% showed no phenotypic susceptibility to any PI) and 2 NNRTI mutations; 40% were on a treatment interruption at screening. The median baseline fold change in EC50 (FC) for EFV, NVP, and TMC125 was 41, 61, and 1.7, respectively.

In this study of people with extensive NNRTI and PI resistance, a viral load reduction of =1 log was observed at 24 weeks in people using etravirine. (abstract 154). K103N was one of the most frequent mutations, along with Y181C and G190A, but was not always present (personal communication).

In a related PK study, because etravirine is likely to be used in addition to ritonavir (r)-boosted protease inhibitors (PI), it was evaluated in conjuction with a dual-boosted PI-containing regimen. An open-label trial was conducted among HIV+ adults on a stable regimen comprising LPV/r +SQV, and >2 NRTI. Etravirine 800 mg (old formulation) BID was added to the ongoing regimen for 2 weeks starting on day 1. Of 15 subjects, 2 were female. Median baseline CD4 cell count was 321/mm³. Everyone completed the study and 9 (60%) reported >1 adverse event, mainly grade 1. No drug-related rashes or cardiac effects were seen. Etravirine reached steady state by day 7. LPV Cmax and AUC12h were significantly lower, although LPV Cmin and pharmacokinetic parameters for RTV and SQV did not change significantly after 2 weeks additional TMC125. Even though LPV was lowered significantly, the investigators remarked that "The observed changes in plasma PI levels (up to 24#37;) were unlikely to be clinically significant (viral load remained <50 copies/mL throughout the 8 weeks of the study)." We shall see (abstract 575b).

Another PK study evaluated the pharmacokinetic interaction between etravirine (old formulation) and TPV/r (indicated for use in advanced HIV patients with multi-PI experience) in healthy subjects. Etravirine and TPV/r were both administered after food. This study included 24 subjects (19 males, 5 females; mean age 37 years).

Because of the significant and clinically relevant decrease of exposure to etravirine observed (similar to what is seen with LPV, SQV and APV in HIV+ people), the combination of etravirine and TPV/r is not recommended (abstract 583).

Finally, we got a small look at how these two investigational agents of Tibotec might work together. Even though there is a large Phase III program of darunavir + etravirine underway, the drug interactions between the two have not been characterized in HIV+ people. The Chelsea and Westminster Hospital in London did a quick and simple trial to get an idea of the safety and efficacy of this combination, because the data up till now is minimal. In this study, the investigators looked at the pharmacokinetics, safety, and efficacy of darunavir/r 600/100 mg BID and etravirine 200 mg BID (new formulation) plus NRTIs with or without T-20 (ENV). HIV resistance, safety, and efficacy were assessed over the study period. Of 11 subjects, 10 completed the study; median (range) baseline characteristics included age 43 (38 to 56) years; CD4 75 (3 to 490) copies/mm3; viral load 4.6 (3.9 to 5.5); number of mutations (IAS, October 2005) for protease inhibitors, -primary 4 (0 to 5), -associated 11 (2 to 13), for NRTI 7 (2 to 9), and for NNRTI 2 (0 to 6). 6/10 people had prior exposure to TPV/r and to T-20; 2 used T-20 for the first time.

As compared to historical references, these reflect unchanged exposure to darunavir and a > 30% reduced exposure to TMC125 (in HIV- studies, a 33-37% change had been seen). At week 6, all subjects had achieved at least a 2 log₁₀ decrease in viral load with a median of -2.55; 5 and 8/10 had viral load < 40 and 400 copies/mL, respectively (abstract 575c). In sweeping terms, darunavir/r and etravirine together work together, and this small study is a good proof of concept for the larger DUET studies, the registrational program for etravirine now underway.

Integrase Inhibitors
MK-0518 is the first HIV-1 integrase inhibitor to get this far. How far? Well,the study presented at CROI evaluated MK-0518 in HIV+ people with viral load level >5000 copies/mL and documented genotypic/phenotypic resistance to each of the 3 classes of oral ART. A multi-center, double-blind, randomized study evaluated the safety and efficacy of MK-0518 (200, 400, or 600 mg BID) vs placebo, all plus optimized background therapy (OBT). Atazanavir (ATV) increases MK-0518 plasma concentrations. The median duration for prior use of ART was ∼10 years. The mean baseline viral load was in the 4.76 log10 copies/mL range. Based on phenotypic resistance testing at baseline, ∼30% of people had no active ART in their OBT.

At week 16, the percentage of people with viral load <400 copies/mL (observed data) is shown in the table below. 56% to 65% of people receiving MK-0518 treatment vs 20% receiving placebo had viral load <50 copies/mL. CD4 cell increases were 100 cells in the two higher doses. The most common drug-related adverse experiences were diarrhea, nausea, vomiting, fatigue, headache, flushing, injection site reaction, and pruritus; these were comparable between the MK-0518 and control groups. There were no dose-related adverse experiences and no discontinuations.

In this early analysis, MK-0518 at all doses in combination with OBT shows potent antiretroviral activity (abstract 159LB). Over the next few weeks, Merck will be going forward with this data into Phase III studies. Although in this study, the trend seemed to be toward a better response with a lower dose, it was not statistically significant, and due to maintaining efficacy and a higher barrier to resistance without increasing side effects, they will move forward with the middle dose, 400 mg BID.

GS-9137 (formerly JTK-303) is a potent (in vitro protein binding-adjusted IC50 < 30 nM) inhibitor of HIV-1 integrase (strand transfer) with favorable human pharmacokinetics (PK), including QD dosing with ritonavir 100 mg (r).

In a late breaker oral presentation, the antiviral activity, safety, pharmacokinetics and pharmacodynamics of GS-9137 were evaluated in a prospective, randomized, double-blind, placebo-controlled monotherapy study in 40 HIV-1-infected treatment-naïve and -experienced subjects. Eligible subjects were not currently on therapy and had HIV-1 RNA between 10,000 and 300,000 copies/mL and CD₄ count ≥ 200 cells/μL. GS-9137 was administered with food for 10 days in dosage cohorts of 200, 400, or 800 mg BID, 800 mg QD, or 50 mg boosted with 100 mg of ritonavir (r) QD (6 active + 2 placebo/cohort). Mean baseline viral load was 4.75 log(sub>10 copies/mL and a CD4 count was 442 cells/μL.

All 40 subjects (30 active -15 experienced / 15 naïve, and 10 placebo) completed the study. There were no study drug discontinuations or serious adverse events. All adverse events related to study drug were grade 1 or 2 in severity, resolved on treatment, and were not associated with GS-9137 dose.

GS-9137 monotherapy demonstrated substantial activity in all BID arms as well a s in the /r boosted arm. QD dosing of GS-9137/r will be evaluated in a dose-ranging Phase II study in treatment-experienced people (abstract 160 LB).

An earlier single blind, randomized, placebo-controlled, single oral dose-escalation study of GS-9137 conducted in 32 healthy male Japanese volunteers was reported on here. Doses of 100, 200, 400, or 800 mg were given to 8 fasting subjects (6 active and 2 placebo) per cohort. It was well tolerated, with no serious adverse events and no grade 3 or 4 adverse events in any cohort. One subject experienced mild anorexia and another experienced mild laboratory abnormalities. No clinically significant ECG changes were noted. Plasma concentrations of JTK-303 attained Cmax at 0.5 to 4 hours post-dose. Cmax and area under the curve (AUC) of GS-9137 increased with dose escalation from 100 to 800 mg. Food significantly increased the Cmax and AUC of GS-9137, approximately 3-fold relative to administration in the fasted state. GS-9137 is orally bioavailable, thus far safe, and well-tolerated following single doses (abstract 580).

How will this integrase inhibitor work with others? GS-9137 has a low molecular weight. Its in vitro activity was evaluated in combination with 6 approved HIV drugs. Pharmacokinetic parameters were determined in rats and dogs. GS-9137 inhibits the DNA strand transfer activity of HIV-1 integrase with an IC50 of 8.8 nM. It also inhibited replication of HIV-1 clinical isolates, including 8 subtypes (A, B, C, D, E, F, G, O) and 8 drug-resistant viruses, with EC50 ranging from 0.02 to 1.26 nM. In vitro, it exhibited synergy with 3TC and AZT/3TC. It was additive with AZT, TDF, TDF/3TC, EFV, IDV, and NFV. Bioavailability and total body clearance of GS-9137 was 34.1 and 29.6%, and 0.5 and 1.0 L/hour/kg in non-fasting rats and dogs, respectively (abstract 508).

Maturation inhibitors
PA-457 is the first in a new class of compounds that inhibit viral maturation, a process essential for virus infectivity. It was administered as monotherapy to HIV+ subjects in a Phase II trial. 32 HIV+ adults received 10 days of placebo or oral PA-457 once daily at 25, 50, 100, or 200 mg. A loading dose (double the maintenance dose) was administered on day 1 in all but the 200-mg cohort. Plasma viral load and drug concentrations were determined over 21 days. Consistent with previous studies, PA-457 exhibited linear pharmacokinetics, with a long mean half-life of 62.7 hours. The mean oral clearance was 0.21 L/h. The AUC and trough concentrations were highly correlated , and could not be differentiated as predictors of response. PA-457 demonstrated significant antiviral activity at the higher dose levels. Additional antiviral activity is likely with doses higher still, as E_max was not reached at 200 mg. PK / PD appears to be an important determinant of activity (abstract 52).

Viral maturation requires the sequential proteolytic cleavage of Gag to matrix, capsid (CA), nucleocapsid, and p6 domains, and spacer peptides SP1 and SP2. PA-457 disrupts the cleavage of CA-SP1 (p25) to CA (p24), resulting in the release of immature, non-infectious viral particles. Looking at resistance, an in vitro selection identified 5 amino acid substitutions that independently confer PA-457 resistance. The first (4), mutations H226Y, L231M, L231F, and A1V, had little effect on viral replication kinetics in the absence of drug or at various drug concentrations. But, the A3V change is the one that greatly decreased viral fitness in the absence of drug and at a suboptimal drug concentration. A3V fitness increased at a high drug concentration. The replication defect imposed by A3V was reversed by a second-site change in CA (G225S). The A3V/G225S mutant was highly fit and fully drug resistant. None of the changes associated with resistance were observed in patient samples collected during, or following completion of, dosing. The clustering of PA-457-resistance-conferring mutations at the CA/SP1 junction confirms that this region is the major target of PA-457 activity. The drug dependence observed for A3V (the third residue of SP1) suggests multiple distinct mechanisms of resistance. All the amino acid positions to which PA-457 resistance maps in vitro are highly conserved among HIV-1 isolates, suggesting that there may be a fitness cost to PA-457 resistance (abstract 156).

How strong is it, and can it work well with others? PA-457 retains activity against drug resistant isolates (including multi-drug and fusion inhibitor resistant viruses) and shows synergy with a comprehensive panel of approved drugs. Against a panel of resistant viruses, it retained wild type activity while the approved drugs exhibited decreases in activity ranging from several fold to >100-fold. Synergy or additivity was observed with representatives of all classes of approved HIV drugs. At the 90% inhibitory concentrations, combination index (CI) values ranging from 1 (nearly additive) to 0.2 (strongly synergistic) were obtained. PA-457 inhibits wild type virus isolates with an IC50 comparable to currently approved drugs. Importantly, PA-457 is a potent inhibitor of drug resistant HIV strains, and exhibits synergy or additivity when combined with approved HIV drugs (abstract 509).

The non-R5/X4 entry inhibitors
TNX-355A, from Tanox, is a humanized monoclonal antibody that competes with CD4-binding antibodies. It is a large molecule that requires intravenous infusion, probably once per week or, after eight weeks, once every two weeks. It binds to domain 2 of the CD4 receptor. It is being studied in treatment-experienced persons who have been on at least one failing triple regimen (n = 82). Tanox previously released 24-week data from a study that showed that 10mg works better than 15mg (ICAAC 2005). The difference between 10mg and placebo (both + OBR) was significant (-1.19log vs -0.32 log). One concern is that CD4 cells in this advanced population were not raised any more than in the placebo group (Tanox notes that the study was not powered to be able to see that, even though it was a secondary endpoint), and that only 55% of patients on the 10mg dose were undetectable at 24 weeks (once again, Optimised Background Regimen is not Optimum Background Regimen — ie, more than one active agent). In vitro, something called synergy was seen with the combination of T-20 and TNX-355 (low combination indices showed <0.5, where anything below 0.9 is considered synergistic). A real life demonstration of this is called for, although one agent is via BID SC injection and the other is QW or Q2W via transfusion. In another study presented at ICAAC, they showed activity against CCR5, CXCR4 and dual/mixed strains of HIV in vivo.

In their CROI presentation of the same study, they tried to characterize each person's virus. 78/82 viruses were successfully amplified (all sub-type B): 49 viruses (62.8%) were CCR5 tropic, 2 (2.5%) exhibited tropism for CXCR4 expressing cells only, and 27 (34.6%) were dual/mixed tropic. Susceptibility to TNX-355 was similar for all viruses, regardless of their tropism, possibly limiting worries regarding tropism shifts (abstract 158LB). "Tropism independence"? Stay tuned.

Progenics let it be known that PRO542 (a recombinant CD4-IgG2, a soluble CD4 receptor ) will not be going forward, but PRO140 will, a similar monoclonal antibody that binds at gp120, also administered by infusion. In the dose-ranging study presented at CROI, they saw concentrations rise dose proportionally. The serum half-life is in the order of (2) weeks! Cells stayed coated with PRO140 for out to 60 days post-treatment. Development of anti-PRO 140 antibodies was not observed (abstract 515).

Pre-clinical entry
CCR5mAb004 inhibits HIV-1 R5 isolates in vitro. To determine the pattern and rate of emergence of resistance to mAb004, R5 clinical isolates were passed through in the presence drug for up to 6 months. 'Passage' of one viral isolate was halted after 19 to 20 weeks because of the emergence of X4 viruses. One other isolate continued to week 24 (passage 33) before there was suggestion of a tropism switch, again observed both in the absence and presence of drug. Resistance to mAb004 may thus require multiple mutations and may be slow to emerge in vivo. As always, this lovely pre-clinical work gives an proof-of-concept to move forward, but resistance and its significance will always be best seen and characterized in real life (abstract 505).

According to the investigators at Sangamo, blocking HIV co-receptors (R5 or X4) is attractive, but no current methods permit their efficient therapeutic disruption, although there are many drugs that are trying to do just that. Zinc-finger protein nucleases (ZFN) are designed to target the CCR5 gene and create a double-strand break (DSB) at predetermined sequences. Natural DNA repair pathways subsequently repair the break, but the repair is error prone and results in a permanent disruption of the target gene. ZFN can generate high-efficiency target gene disruption in transiently transfected cells even in the absence of selective pressure. The frequency of gene disruption observed supports its examination as a possible method to generate HIV-resistant T cells (abstract 51). Sangamo hopes to move forward with a Phase I study sometime in 2006.

KD-247 is a broadly neutralizing monoclonal antibody. At passage 8 in culture, 1 amino acid substitution, G314E, in the V3-tip of gp120 was identified. G314E was completely resistant to KD-247. Interestingly, virus that contained G134E maintained sensitivity to CCR5 inhibitors, RANTES, rsCD4, and anti-CCR5 monoclonal antibodies, although not to anti-CD4 monoclonal antibody compared with wild type virus. In vitro, combinations of KD-247 with the CCR5 inhibitors showed highly synergistic interactions. Also in vitro, a very high concentration of KD-247 is needed for the virus to acquire resistance, and this resistant virus is less dependent on CD4 binding for entry (abstract 506).

Nukes
For new nukes (NRTIs), as for most new drugs coming out in existing classes, a primary emphasis is on their activity in the presence of virus that already has resistance to existing nukes. TAMs are thymidine analog mutations, and are those that come up in the presence of AZT or d4T selective pressure. The M184V/I is the major 3TC/FTC mutation. The K65R is a mutation that comes up from the use of TDF. There are some cross-class mutations, notably the 69 and the 151. When there were fewer NRTIs , and fewer combinations of them, resistance was more straightforward. Now that there are seven widely used nukes (let's forget about ddC), there are various levels of resistance to most of them in their various combinations, and many people depend on the use of new NRTIs that do not share mutations with the current drugs. Recently, it has been seen that the presence of some mutations (the M184) may not be a bad thing (in black and white terms) - it may keep the resulting virus less fit, less able to replicate, which may be not as good as having an active drug, but not as bad as having no drug at all. And while investigators do tremendous amounts of work in vitro (before getting to humans) regarding the activity of the drug in the presence different cell lines with different resistance patterns, the early results on all these new nukes (and to a large extent, new PIs) below, are at best a guide — they rarely correlate directly to clinical results. So, below are some hopeful candidates but the truth will not fully out until we see longer-term data in people.

May the company find a follow-up compound soon: RVT-203 was a randomized, placebo-controlled, 24-week study to examine efficacy and safety of 50-, 100-, and 200-mg once-daily doses of dexelvucitabine (DFC), in nucleoside reverse transcriptase inhibitor -experienced people. The CROI assessed the role of specific genotype in predicting virological response, and the emergence of DFC resistance at 16 weeks. Screening genotypes from 199 subjects were examined, and stratified according to the number of TAMs; 58% of the study population had either 3 or 4 TAMs. The K65R was present in 11 people, Q151M was present in 5 people, and M184V in 129 people. For subjects with TAMs ? M184V, after 16 weeks of therapy, mean change in viral load was almost twice as great for 200 mg DFC (n = 22, -1.01) vs placebo (n = 23, -0.6). Of subjects taking 200 mg DFC without concurrent 3TC/FTC, 71% had viral load decline >1 log by week 16. The mutations elicited upon failure were the W88X in (4) people and the L210S in (2) people. No DFC signature mutations have been characterized (abstract 632).

An interesting and very complicated drug that might have antagonism with 3TC/FTC but works very well in the presence of the major 3TC/FTC mutation. They will move forward with a study of DTC (without 3TC / FTC) vs 3TC, all plus optimized background regimen, to determine signature mutations and/or a clinically relevant cut-off predictive of viral response.

On 3 April 2006, Incyte announced the discontinuation of dexelvucitabine due to unacceptably high rates of hyperlipasemia, a marker of pancreatic inflammation.

This drug epitomizes the difficulty of designing drugs that work in various real world scenarios, teasing out exactly when it works, and when it won't, as well as the resistances that this drug may cause upon failure. Also, the numbers of people studied are very small and it may not be fair to make huge assumptions (like that it isn't affected by the K65R or the Q151M) from small numbers of studied subjects. And that's not getting into adverse events!

Pre-clinical nukes
In vitro, nucleotide analog GS9148 shows improved pharmacological and resistance profiles over current NRTIs regarding antiviral potency, toxicity, and resistance. The activity of GS9148 was compared to 6 approved NRTIs-AZT, ddI, d4T, FTC, ABC, and TDF-against variants with all major NRTI-resistance mutations. A prodrug version of GS9148 showed a favorable in vivo pharmacokinetic profile. The prodrug exhibits potent antiretroviral activity (EC50 = 25 to 200 nM). Unlike ddI, d4T, or d4FC, neither GS9148 nor its prodrug showed an effect on mitochondrial DNA or lactate production in HepG2 liver cells. The activity of GS9148 was not affected by the presence of K65R, L74V, M184V or their various combinations. Viruses with 4 or more TAMs showed 0.74- to 2.0-fold change in susceptibility to GS9148, a shift that was smaller than any other tested NRTI. Passaging of HIV-1 in the presence of GS9148 selected for a primary K70E mutation in RT that conferred <3-fold reduced susceptibility to GS9148. In dogs, the oral bioavailability of the prodrug exceeded 20%. This amidate prodrug has potential for QD dosing, and activity in both treatment-naïve and NRTI-experienced people (abstract 45).

Amidate prodrug technology (previously explored with TDF) was applied to GS9148 to maximize the intracellular accumulation of its active diphosphate (DP) metabolite. This study explored whether the oral administration of GS9148 amidate can deliver GS9148-DP at levels sufficient for a clinical antiviral effect. While delivering similar amounts of GS9148 into plasma, a single dose of the prodrug administered orally to dogs (3 mg/kg) resulted in >90-fold higher intracellular concentrations of GS9148 and GS9148-DP compared to intravenous administration of GS9148 itself (0.5 mg/kg). In the oral arm, the prodrug showed a bioavailability of >20%. The levels of GS9148-DP persisted for >24 hours. Clinically effective concentrations of GS9148-DP in human PBMC are expected to be readily achieved following QD oral administration of the prodrug at doses <2 mg/kg (abstract 498).

Nucleotide-competing reverse transcriptase inhibitors (NcRTI) represent a novel class of compounds that has been shown to block the DNA polymerase activity of HIV-1 RT. Previous data suggested that one prototype compound (Compound X) can compete with natural dNTP substrates, despite significant structural differences, and block DNA synthesis (Jochmans, 2005). Antiviral inhibition studies have shown that the M184V mutation is associated with lowered susceptibility to Compound X (increases in IC50), while the K65R mutation confers hypersusceptibility (decreases in the IC50). The presence of ATP facilitates the formation of the inhibitory complex. Compound X can partially occupy the nucleotide-binding site of HIV-1 RT. This traps the RT-primer/template complex in a dead-end configuration, preventing the incorporation of dNTP, although this is diminished in the presence of the M184V (abstract 47).

Resistance-wise, the susceptibility of > 6000 clinical isolates toward Compound X (EC50 for wild type HIV-1 = 30 nM) was looked at. More than 80% of the profiled clinical isolates remained susceptible to Compound X (fold change in EC50 [FC] <4). No cross-resistance was observed between it and EFV, NVP, TDF, AZT, d4T, ABC, ddC, or ddI. Some cross-resistance with 3TC and FTC was seen. Analysis of the genotype of >1700 of these viruses showed that the combination of M184V + Y115F correlated most with reduced susceptibility to Compound X. They showed a FC of 5.0 and 7.9, respectively. The combination of both resulted in a FC of 39. Analysis of the dataset also indicates that the K65R mutation is associated with hypersusceptibility to Compound X, and that it reverses M184V-induced resistance, which was confirmed using an M184V + K65R strain (FC = 0.63). Also, in contrast to 3TC or ABC, Compound X prevents the selection of K65R. Its activity is not influenced by the presence of TAMs, the 69ins-MDR complex, the Q151M-MDR complex, or mutations associated with NNRTI-resistance (abstract 500).

1-(ß-D-dioxolane) thymine (DOT) is a potent inhibitor of HIV-1 with excellent pharmacological properties including good oral bioavailability in rats and monkeys, rapid intracellular phosphorylation to DOT-TP, and low toxicity in vitro. In culture, DOT has shown selective activity against viruses containing TAMs and M184V. DOT-TP was evaluated against a large panel of purified HIV-1 reverse transcriptases (RT) with multi-nucleoside resistance mutations. Enzymatic studies were designed to explore the mechanism of resistance. Site-directed RT mutants were constructed containing TAMs, a 69-mutant, a151-mutant, or K65R-the last 2 representing a non-excision mechanism of resistance, i.e., binding discrimination. The M184V mutation was studied separately and also added to the TAM, 69- and 151-mutant. Compared with other RT inhibitors, DOT-TP was overall more effective against RT-containing TAMs, M184V, and K65R. The 69-mutant demonstrated a lower level of resistance to DOT-TP than AZT-TP and TFV-DP. However, as expected for dioxolane nucleosides, an increased non-ATP-dependent resistance was found with the 151-mutant. These enzymatic studies indicate that DOT resistance mainly involves binding discrimination (abstract 46).

A series of 4'-C-ethynyl nucleoside analogs was previously shown to be active against a wide spectrum of HIV-1 isolates, including a variety of laboratory and drug-resistant HIV-1 strains in vitro. Among such analogs, E2FdA represents one of the most potent agents against HIV-1 with IC50 values of ∼1nM. In this study, the amounts of intracellular E2FdA-monophosphates (MP), -diphosphates (DP), and -triphosphates (TP) increased proportionately with increased E2FdA concentrations. When exposed to AZT, only AZT-MP levels markedly increased compared with AZT-DP and AZT-TP. Intracellular T1/2 of E2FdATP (∼18 hours) was significantly greater than T1/2 of AZT-TP (∼3 hours). E2FdA exerts minimal inhibition to DNA polymerase-?. QD dosing of E2FdA is possible (abstract 499).

Pre-clinical PIs
SPI-256 is a novel PI with potency against wild type and MDR viruses. It was evaluated in a panel of wild type strains of clades A, B, C, and D (n = 7) and PI-resistant (n = 44) HIV-1 strains. The antiviral potency was compared to that of 7 FDA-approved PI-SQV, RTV, IDV, NFV, LPV, APV and ATV. Viruses contained as many as 6 primary mutations in a background of 5 to 21 total mutations (median = 13). SPI-256 exhibited an average IC50 value of 0.3 nM against wild type viruses (both clades B and non-B), and in vitro was 4- to 50-fold more potent than the FDA-approved PIs. Importantly, it was nearly 100-fold more potent against MDR viruses, compared to the same PIs. For a subset of isolates with >50-fold phenotypic resistance to reference PI or 6 primary PI mutations (n = 12) — 'worst-case MDR', the average IC50 value for SPI-256 was 12.9 nM compared to >937 nM for the FDA-approved PIs. In vitro, it shows hypersensitivity to the ATZ I50L mutation and maintains potency against I50L + primary mutations (abstract 501).

The potency is there, and it will be very important to see if this potency is matched with a gentleness regarding adverse events, something very much needed in the salvage population.

GRL-02031 is a novel peptidic PI that exerts potent activity against a wide spectrum of HIV-1 isolates including multi-drug-resistant HIV-1 variants. We attempted to select GRL-02031-resistant variants by propagating a laboratory X4 HIV-1 strain. Upon the selection of GRL-02031-resistant HIV-1 in the presence of as many as 5 nM of GRL-02031, mutations in the protease-encoding region as wellas in the p17, the p24/p2 cleavage site, p7 and the p7/p1 cleavage site in the gag-encoding region emerged. GRL-02031 was potent against molecular clones with IC50 value differences by 0.7- to 1.7-fold. HIV-1 develops substantial resistance to GRL-02031 only when it acquires >4 mutations in its protease (abstract 503).

Pre-clinical maturation
Searching for inhibitors targeting new mechanisms, UK-201844 specifically inhibits the production of infectious virions, dependent on the Env protein. UK201844 acts during the late stages of HIV replication and Env sequences are critical determinants of compound susceptibility, thus confirming that UK-201844 targets Env function. UK-201844 interferes with gp160 processing in infected cells, resulting in the production of non-infectious virions. UK-201844 is another maturation inhibitor, this one directly or indirectly inhibiting gp160 processing, resulting in the production of virions with non-functional Env proteins (abstract 50LB).