Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are a crucial and potent class in our armamentarium of highly active antiretroviral therapy (HAART) agents. With a low pill burden, good tolerability and no association with lipodystrophy, the use of NNRTIs has steadily climbed, especially in the context of first-line therapy.

However, NNRTIs have an Achilles' heel: cross resistance. The cross resistance that exists between the currently available NNRTIs -- nevirapine (NVP, Viramune), delavirdine (DLV, Rescriptor) and efavirenz (EFV, Sustiva, Stocrin) -- gives patients and practitioners only one shot at the use of this class of drugs. As patients live longer on HAART and the pool of NNRTI-resistant viruses increases, so does the need for the development of new, second-generation NNRTIs with antiviral activity against both wild-type and the clinically prevalent NNRTI-resistant strains.

E. Wardrop from Boehringer Ingelheim presented in vitro data on BILR 355 BS, an NNRTI in development that has potent in vitro antiviral activity against a wide range of recombinant NNRTI-resistant viruses (EC₅₀ < 10 nM). Past research has shown excellent pharmacokinetic and safety characteristics for this drug in small phase 1 trials in healthy volunteers.¹ Initial studies with viruses resistant to currently available NNRTIs also support its continued development.² The study presented by Wardrop at this conference further explored the mutational pathways that may reduce a patient's susceptibility to BILR 355 BS. Clade B HIV virus, the most common clade in the developed world, was examined, as were the non-clade B viruses found in the developing world. In addition, HIV-1 groups M and O, as well as HIV-2, were all tested for susceptibility to BILR 355 BS.

Wild-type and NNRTI-resistant HIV-1 were passaged in C8166 cells in the presence of BILR 355 BS. Inhibition of both wild-type and NNRTI-resistant virus containing emergent reverse transcriptase (RT) mutations was determined by p24 antigen assay.

Viral replication capacities were determined by luciferase expression in infected Jurkat-LTR-luciferase cells. ViroLogic PhenoSense methodology or p24 antigen assay was used to determine BILR 355 BS's antiviral activity against a panel of clinical isolates of wild-type group M HIV-1 (including clades A, B, C, D, F, G and H) and HIV-2. All clinical isolates were from treatment-naive patients, in order to minimize the potential for treatment-emergent resistance mutations. Group O isolates harbor naturally occurring polymorphisms, which may confer NNRTI resistance.

The chart below displays the antiviral profile of BILR 355 BS against recombinant viruses harboring the most commonly observed clinical resistance mutations. The data show that these mutations generally have a minimal impact on BILR 355 BS compared to nevirapine and efavirenz (mutations with minimal impact compared to wild-type virus are shown in bold). However, reduced susceptibility was seen with the Y188L and Y181C/ G190A mutations.

Wild-type serum adjusted EC50 (nM)		2.6	11	3.9
Drug		BILR 355 BS	NVP	EFV
Fold change against wild-type HIV	K103N	3X	30X	20X
	Y181C	4X	100X	2X
	Y188L	82X	>800X	120X
	G190A	2X	40X	3X
	K103N L100I	4.5X	35X	>3300X
	K103N V108I	3X	80X	30X
	K103N Y181C	3.5X	>500X	35X
	K103N G190A	3X	175X	125X
	K103N P225H	1X	80X	30X
	Y181C G190A	25X	>1000x	20X

In vitro selection of BILR 355 BS-resistant virus from wild-type virus predominantly yielded the V106A mutation in early passages and V106A/E138K in later passages. Resistance studies on virus already containing one of the two most common mutations (K103N or Y181C) seen in the virus of patients exposed to the currently available NNRTIs found that BILR 355 BS therapy selected for mutations at the V106 position, just as it did in wild-type virus. Interestingly, the original K103N and Y181C mutations persisted throughout the passages. V106I was observed exclusively in viruses already containing the Y181C mutation, and V106A was observed either alone from wild type or in the context of K103N.

High-level resistance to BILR 355 BS was observed with the V106A mutation (60-fold reduction compared to wild type). Double mutants harboring the V106A mutation resulted in substantial reduction (195 to 540-fold) of antiviral activity.

A constructed mutant virus with V106A and Y181C had a 220-fold reduction in susceptibility. Nevirapine and delavirdine had reduced activity against BILR 355 BS-resistant virus with V106A and V106A/E138K mutations, but efavirenz maintained close to wild-type activity.

Viruses with the V106A mutation, especially double mutants, have severely reduced replication capacity (40% to 80% less than wild type). As a result, these viruses may be less fit in vivo, which may explain their low prevalence in NNRTI-treated patients.

PhenoSense assays found no evidence of decreased susceptibility to BILR 355 BS in non-clade B virus, though the biological cutoffs for this new drug have not yet been established. BILR 355 BS did have diminished activity against some HIV-1 group O isolates carrying naturally occurring RT mutations, but this reduction in activity was not as great as for nevirapine or efavirenz. BILR 355 BS had no activity against HIV-2, which is true of other NNRTIs as well.

The preferential selection of V106A over Y188L during in vitro passage may reflect the fact that Y188L requires two nucleotide substitutions. V106A is selected uncommonly by nevirapine (less than 3% of resistant isolates), presumably because it confers less nevirapine resistance than Y181C and results in a greater cost to replicative capacity.

There are now four second-generation NNRTIs in clinical development that have shown improved activity against efavirenz- and nevirapine-resistant virus: In addition to BILR 355 BS, they are GW8248, TMC125 and TMC278. Of those four, BILR 355 BS is most similar to GW8248 in terms of the mutations selected for in drug-exposed virus.

So, to recap, BILR 355 BS displays potent antiviral activity against clinically prevalent, NNRTI-recombinant resistant virus -- both clade B and non-clade B. The principal resistance mutation selected for in vitro is V106A. Some cross resistance to nevirapine and delavirdine was observed with V106A and V106A/E138K mutant virus; however, such cross resistance was not observed with efavirenz or any nucleoside/tide reverse transcriptase inhibitors. The replication capacity of viruses harboring the V106A mutation, especially double mutants, was greatly reduced compared to wild-type virus, which may explain the low prevalence of this mutation in clinical samples despite its ability to confer nevirapine resistance.

Given its antiviral activity against virus with most of today's commonly observed NNRTI mutations and the mutations developed during in vitro passage, BILR 355 BS has considerable potential for good in patients who are failing currently available NNRTIs. This nascent drug deserves further investment in its clinical development.

Footnotes

1. Bonneau P, Robinson P, Duan J, et al. Antiviral characterization and human experience with BILR 355 BS, a novel next-generation non-nucleoside reverse transcriptase inhibitor with a broad anti HIV-1 profile. In: Program and abstracts of the 12th Conference on Retroviruses and Opportunistic Infections; February 22-25, 2005; Boston, Mass. Abstract 558.
2. Bonneau PR, Robinson PA, Lie Y, Parkin N, van Leeuwen R, Bethell R. Antiviral profile of BILR 355 BS against a large panel of clinical isolates with mutations conferring resistance to currently available non-nucleoside reverse transcriptase inhibitors (NNRTI). In: Program and abstracts of the 14th International HIV Drug Resistance Workshop; June 7-11, 2005; Quebéc City, Canada. Abstract 74.
   
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