Medical Pharmacology Chapter 36:  Antiviral Drugs

Non-nucleoside/Nucleotide Reverse Transcriptase Inhibtors (NNRTI)

• Antiretroviral Drugs Used in Treating HIV Infection

  • →Non-nucleoside/Nucleotide Reverse Transcriptase Inhibitors (NNRTI):  Introduction

    • Sites of Antiretroviral Drug Actions¹⁴

      a: The lifecycle of HIV-1, showing the step at which enfuvirtide acts, and also the steps targeted by other drugs.14  b:  "Simplified schematic of the mechanism by which enfuvirtide inhibits viral entry into host cells.14    The envelope glycoprotein of HIV-1 consists of two non-covalently associated subunits, gp120 and gp41. After attachment of HIV-1 to its target cells carrying the CD4 receptor, gp120 interacts with the CD4 receptor, which initiates a series of conformational changes in gp41 and gp120 that lead to the insertion of a region of gp41 into the membrane of the host cell, and formation of a ' pre-hairpin intermediate' (top). Further changes in the confirmation of gp41 bring the viral and cellular membranes into close enough proximity for membrane fusion (bottom left). Abbreviations: NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; PI, protease inhibitor" Attribution:  Slight modification of Figure 1 from:  LaBonte J Lebbos J Kirkpatrick nature Reviews Drug Discovery 2, 345-346 (May 2003).14

    •  

      NNRTI:  Site of Action in HIV Viral Replication

      Optigan13 "Non-nucleoside reverse transcriptase inhibitors (NNRTIs) work by inhibiting the reverse transcriptase enzyme."

    • Non-nucleoside reverse transcriptase inhibitors bind to a "hydrophobic pocket" in the "p66" subunit of the heterodimeric protein.^1,11 

      • This site is about 10 Å removed from the enzyme catalytic site. The binding of an NNRTI induces a conformational change which rotates specific side chains in the protein towards the catalytic site.¹¹  

      • This structural change causes additional structural changes directly affecting three catalytic aspartic acid residues.

      • The magnitude of the change at the catalytic site appears to be of the order of about 2 Å.

        • These confirmation alterations caused by NNRTI binding to the retroviral reverse transcriptase are likely to be the inhibitory mechanisms exhibited by drugs belonging to this class.¹¹

           

          • Ribbon Representation of the Viral Reverse Transcriptase Active Domain (the p66 monomer)¹⁰

            Madrid M, Pittsburgh Supercomputing Center "This ribbon representation of the RT (reverse transcriptase) active domain illustrates its hand-like structure, showing fingers (blue), palm (pink) and thumb (green). "The active site (red atoms), where DNA is elongated, is the palm region. "Also shown is an NNRTI drug (yellow) in the pocket where it binds.10 http://en.wikipedia.org/wiki/File:RTenzyme-hand.jpg

      • Non-nucleoside reverse transcriptase inhibitors are classified as noncompetitive, acting by inducing a conformational change in the enzyme as opposed to competing with other molecules at the binding site (competitive inhibition).¹ 

        • Binding site specificity for these drugs results in their activity against HIV-1 but these agents are not active against HIV-2 or other retroviruses.

        • Furthermore the non-nucleoside reverse transcriptase inhibitors did not exhibit activity with respect to cell DNA polymerases.¹ 

      • Efavirenz and nevirapine are examples of drugs in this category and represents the two agents most frequently administered.¹  

        • Employed as monotherapy, administration of these drugs may decrease in HIV RNA plasma concentrations by a factor of 100 or more, although resistance typically rapidly develops.¹³ 

          • For example, a clinical study utilizing nevirapine involved asymptomatic patients with CD4⁺ T cell counts >500 cells/cc.

          • Using a dosage of 400 mg of nevirapine once a day, HIV plasma RNA decreased with a mean reduction  of 0.73 log₁₀ and a median reduction of 0.51 log₁₀ copies/cc.

          • The study involved 17 seropositive individuals.

          • After 12 weeks of nevirapine administration, all HIV viral isolates exhibited drug resistance.¹³ 

      • Non-nucleoside reverse transcriptase inhibitors (presently approved) utilize the liver microsomal metabolizing system for elimination.¹ 

        • The cytochrome P450-dependent system describes a variety of cytochrome P450 similar enzymes or "isoforms".

        • For example, delavirdine and nevirapine are primarily metabolized by an important drug metabolizing isoform, CYP3A4.

        • By contrast, efavirenz is mainly metabolized by CYP2B6 and CYP3A4.

        • Elimination half-times for efavirenz and nevirapine extend from 1 to 3 days which permits daily dosing.

          • Sometimes drugs are also able to induce cytochrome P450 enzymes, resulting usually and elevated enzyme cellular concentrations.

            • In this instance, efavirenz, etravirine, and nevirapine are classified as moderately potent inducers of the cytochrom P450 system.¹   

              • In particular, these agents elevate CYP3A4 levels; however, delavirdine has been shown to be a CYP3A4 inhibitor.

                • A further complication is that a drug may inhibit some cytochrome P450  isoforms while inducing others.

                  • This effect, "mix metabolism", describes the case noted for etravirine.¹   

        • Changes in concentrations or activities of cytochrome P450 isoforms may result in drug-drug interactions.¹  

          • For example, if one drug increases the concentration of the cytochrome P450 isoform mainly responsible for the metabolism of the second drug, the plasma concentration of the second drug may decrease to below therapeutic levels.

            • Should one drug decrease the concentration of a cytochrome P450 isoform which metabolizes a second drug, plasma concentration of the second drug may be increased resulting in toxicities.

            • Microsomes have been used to study mixed function oxidases

              • Drug metabolizing enzymes are located in lipophilic, hepatic endoplasmic reticulum membranes.  Smooth endoplasmic reticulum contains those enzymes responsible for drug metabolism.

            • The reaction:

              • One molecule oxygen is consumed per substrate molecule

              • One oxygen atom -- appears in the product; the other in the form of water

              • Oxidation-Reduction Process:

                • Two important microsomal enzymes:

                  1. Flavoprotein  is NADPH cytochrome P450 reductase

                  2. Cytochrome P450, a terminal oxidase:

                     1. Consists of multiple forms

                     2. Named cytochrome P450 because:

                     3. Note in the Figure below the conversion of RH to ROH represents drug oxidation.

        •  

          Mixed Function Oxidase System (Cytochrome P450 System):  Phase I Reactions

          The reaction:  One molecule oxygen is consumed per substrate molecule One oxygen atom appears in the product; the other in the form of water Oxidation-Reduction Process: Two important microsomal enzymes: Flavoprotein  is NADPH cytochrome P450 reductase Cytochrome P450, a terminal oxidase: Consists of multiple forms Named cytochrome P450 because: Note in the Figure below the conversion of RH to ROH represents drug oxidation. Cytochrome p450 cycle (diagram by  Matthew Segall, 1997) 1.  "The binding of a substrate to a P450 causes a lowering of the redox potential by approximately 100mV, which makes the transfer of an electron favourable from its redox partner, NADH or NADPH. 2.  The first reduction -The next stage in the cycle is the reduction of the Fe3+ ion by an electron transfered from NAD(P)H via an electron transfer chain. 3.  Oxygen binding An O2 molecule binds rapidly to the ion Fe2+ forming Fe2+-O2 4.  Second reduction A second reduction is required by the stoichiometry of the reaction. This has been determined to be the rate-determining step of the reaction 5.  O2 cleavage: The O2 reacts with two protons from the surrounding solvent, breaking the O-O bond, forming water and leaving an Fe-O3+ complex. 6.  Product formation The Fe-ligated O atom is transferred to the substrate forming an hydroxylated form of the substrate. 7.  Product release The product is released from the active site of the enzyme which returns to its initial state."--Matthew Segall, 1997

       

      • Currently, FDA-approved non-nucleoside reverse transcriptase inhibitors include:⁹

        • Delavirdine (DLV)

        • Efavirenz (EFV)

        • Etravirine (ETR)

        • Nevirapine (NVP) and

        • Rilpivirine (RPV).

      • Genotypic testing is suggested prior to initiating NNRTI treatment because of relatively high resistance rate: 2%-8%.⁷   

      • NNRTI resistance, as noted, develops quickly with monotherapy and may occur following a single mutation.⁷   

        • Examples of mutations include K103N, a substitution of lysine by asparagine, noted in resistance to nevirapine and Y181C, a substitution of a tyrosine by a cysteine.

          • These mutations appear more likely to induce resistance to the first-generation NNRTI agents while not affecting newer drugs such as rilpivirine and etravirine.

          • Some mutations induce cross-resistance to drugs belonging to the NNRTI drug group.⁷

      • With the exception of etravirine, other NNRTI agents may exhibit significant drug resistance due to single-amino-acid substitutions in the drug-binding allosteric region.¹  

        • By contrast, other HIV-1 antiretroviral drugs belonging to the nucleoside analog or protease inhibitor classes, efavirenz or nevirapine resistance and associated virologic relapse may occur quickly, within a few days or weeks, if the drug is administered as monotherapy.

          • As monotherapy, a single dose of nevirapine may be associated with mutations resulting in drug resistance in as many as 33% of patients.¹  

        • As a result of these considerations, NNRTI antiretroviral drugs must be administered in combination with a minimum of two other active drugs in order to avoid drug resistance.¹  

          • Efavirenz or nevirapine administration along with other antiretroviral agents results in long-term suppression of viremia and increases in CD4⁺ T lymphocyte counts.¹  

            • Efavirenz is frequently used as a component of the first drug regimen in treatment-naïve patients.

              • Efavirenz is favored in this setting as a result of convenience of use, patient's ability to tolerate the drug, and drug potency.

                • A common side effect associated with NNRTIs, most likely occurring within the first month of treatment is a mild, self-limited rash.

                • Rare cases of potentially fatal Stevens-Johnson syndrome have been documented with nevirapine, efavirenz, and etravirine.¹