Antimicrobial Chemotherapy, 4th Edition
General properties of antimicrobial agents
- L. Irving
The first human immunodeficiency virus (HIV) was identified and characterized as a retrovirus in 1983. Long-term infection with HIV leads to inexorable destruction of the host immune system, and the development of the acquired immune deficiency syndrome (AIDS). In the short time since that momentous discovery, considerable progress has been made in devising novel antiretroviral agents which, by inhibiting HIV replication, can prolong the time to the development of AIDS. Unfortunately, the concept of ‘curing’ a patient of HIV infection remains an elusive and as yet unattainable goal.
The fact that HIV belongs to the retroviral family identified the first and most obvious target in the search for anti-HIV drugs—the enzyme reverse transcrip-tase. This enzyme, which copies an RNA template into DNA, is not present within host cells, which have no need to perform this ‘backwards’ synthetic process. The enzyme is encoded within the HIV genome, and the protein itself is an essential component of infectious viral particles.
Initially, anti-HIV drugs were all nucleoside analogues. Subsequently, it became clear that compounds with other structures could also act as reverse tran-scriptase inhibitors. A further advance came with the development of drugs that target a completely different virally-encoded enzyme—HIV protease. Thus, although they exploit only two targets within the HIV replicative cycle, there are currently three categories of antiretroviral drugs licensed for clinical use: the nucleoside analogues, the non-nucleoside reverse transcriptase inhibitors, and the protease inhibitors (Table 7.1). Various other potential targets have been iden-tified, and it is hoped that the number of anti-HIV drugs will continue to expand.
Table 7.1 Antiretroviral agents in clinical use in the UK and/or US (1999)
Zidovudine (azidothymidine; often simply called AZT), was originally investigated for use as an anticancer agent. However, when the HIV epidemic arose in the 1980s, it was tested along with many other drugs for activity against the virus. It was found to inhibit the reverse transcriptase activity of retroviruses at concentrations considerably lower than those needed to interfere with synthesis of host cell DNA.
AZT is a dideoxynucleoside in which the 3′ hydroxyl group of thymidine has been replaced by an azido (N3) group (Fig. 7.1). Molecules such as AZT are referred to as 2′–3′-dideoxy nucleoside analogues, as they lack hydroxyl groups at both the 2′ and 3′ positions of the deoxyribose ring. Like other nucleoside analogues, AZT is activated by phosphorylation to the triphosphate form, these steps being carried out by cellular enzymes. As with aciclovir (p. 83), incorporation of AZT-triphosphate into a growing DNA chain will result in chain termination, as there is no 3′ hydroxyl group available for formation of the next 5′–3′ phosphodiester linkage.
Fig. 7.1 Structure of azidothymidine (AZT).
Non-specific side-effects such as headache, anorexia, and nausea are common with AZT therapy, but these effects often abate after 2–3 weeks. More serious toxicity to bone marrow cells is dose-related, and hence much effort has been directed at defining the minimum dose that exhibits effective antiviral action. Temporary cessation of therapy usually results in restoration of marrow function, but some patients on AZT become transfusion dependent. Combination therapy with ganciclovir, although desirable, since many HIV-infected patients also suffer serious CMV infection, is complicated by an additive effect on the bone marrow.
Other nucleoside analogues
The success of AZT, albeit with limitations, at least showed that it was possible for anti-HIV drugs to be therapeutically useful. The pharmaceutical industry was therefore encouraged to design and test other potential nucleoside analogues. To date, five further drugs have been licensed for use in the USA and some other countries (Table 7.1). These newer agents either belong to the general category of 2′–3′ -dideoxynucleoside analogues, or have a similar structure—lamivudine has an oxathiolanyl group, and abacavir a cyclopentene ring, rather than a ribose group. Their mode of action is similar to that of AZT. They all require triphos-phorylation, and in their triphosphate form they act as chain terminators and competitive inhibitors of HIV-derived reverse transcriptase.
The most frequent adverse reaction with all of these drugs is gastrointestinal disturbance, including nausea, vomiting, abdominal pain, and diarrhoea. More serious and dose-limiting side-effects include peripheral neuropathy (didanosine, zalcitabine, stavudine), pancreatitis (didanosine, zalcitabine), elevation of liver transaminases (stavudine), and hypersensitivity reactions (abacavir).
Non-nucleoside analogue reverse transcriptase inhibitors
The recognition that compounds with a range of dissimilar structures could also act as inhibitors of reverse transcriptase led to the categorization of a new class of anti-HIV agents: the non-nucleoside analogue reverse transcriptase inhibitors. In contrast to the nucleoside analogues, these agents bind specifically to a non-substrate binding site of reverse transcriptase located close to the substrate-binding site. These drugs inhibit the reverse transcriptase of HIV-1 but not that of HIV-2. Since the original report, more than 20 chemical classes of non-nucleoside compounds that act as reverse transcriptase inhibitors have been described,
but only three drugs have so far been used extensively in the treatment of HIV infection (Table 7.1). Figure 7.2 shows the structure of one of these agents, nevirapine. Many other drugs in this category are at various stages of clinical and preclinical testing.
Fig. 7.2 Structure of nevirapine.
The most serious adverse events related to nevirapine therapy include rash and abnormal liver function tests. Rashes are usually mild and occur within a few weeks of starting therapy. Occasionally, life-threatening hypersensitivity reactions, including Stevens–Johnson syndrome, may occur.
The development of drugs acting on a viral target other than reverse transcrip-tase represented a major breakthrough in therapy for HIV-infected patients. When HIV replicates, it produces polycistronic mRNA, which is translated into a series of polyproteins that must be cleaved to yield the component proteins for infectious viral particles. This function is performed by a virally-encoded aspartyl protease. Although mammalian cells also contain aspartyl proteases, these do not cleave HIV polyproteins efficiently.
Several drugs have been developed that specifically inhibit HIV protease. Five are currently licensed for use and more are in advanced stages of clinical trials (Table 7.1). Most are structurally related. All have important side-effects. Besides gastrointestinal problems (particularly prominent with ritonavir), the most worrying adverse effect of protease inhibitors arises from their interference with fat and carbohydrate metabolism, leading to hyperlipidaemia, glucose intolerance (and even frank diabetes mellitus), and peripheral lipodys-trophy (abnormal body fat distribution) with increased abdominal fat, buffalo humps, and breast hypertrophy. The frequency and pathophysiology of these reactions are unclear, as is the long-term risk of other complications such as ischaemic heart disease. Indinavir may cause nephrolithiasis, by precipitation in the renal tubules.
Resistance to anti-HIV drugs
Unfortunately, optimism encouraged by the emergence of effective anti-HIV drugs has had to be tempered by the realization that the virus is rapidly able to acquire resistance to these agents. The enzyme reverse transcriptase is considerably more error-prone than other DNA polymerases. Thus, generation of large numbers of viral mutants is part of the natural replication cycle of HIV: it has been estimated that, within a single patient, every possible base substitution could occur at every possible nucleotide position in the viral genome every day! Clearly, the vast majority of these mutations will be deleterious and result in non-viable virus. However, the potential is there for mutations to occur which do confer benefit to the virus, especially if those mutations result in decreased efficacy of an antiviral drug. Experience with all anti-HIV drugs used thus far indicates that resistance is an inevitable consequence of drug usage, at least if the suppression of virus replication is not absolute.
Isolates of HIV derived from patients who have been taking AZT for at least 6 months are invariably less sensitive to the drug in vitro than isolates taken from the same patient at the initiation of therapy. This arises from mutations in the gene coding for reverse transcriptase, leading to reduced binding of AZT triphosphate. Detailed molecular studies have demonstrated that resistance arises as a series of five sequential point mutations within the reverse transcriptase gene, each additional mutation resulting in an increase in the dose of AZT necessary to inhibit the virus. This relative resistance is of considerable clinical importance. Trials of AZT monotherapy conducted in the late 1980s showed early promise, increasing CD4 cell counts and prolonging survival in treated patients. However, as these patients were followed for longer periods, it became clear that the therapeutic efficacy of the drug was limited to a period of about 6 months, after which it was of no benefit. This loss of efficacy coincided with emergence of highly resistant viruses in treated patients.
Resistance to other nucleoside analogues similarly arises through mutations in the reverse transcriptase gene. In general, cross-resistance between AZT and the other nucleoside analogues is not a problem, as the positions of the mutations conferring resistance to AZT differ from those giving rise to resistance to, for instance, didano-sine or zalcitabine. In contrast, mutations causing resistance to didanosine and zalcitabine are similar, and also overlap with those causing lamivudine resistance.
The news concerning resistance mutations may not be all bad, however. Mutation at position 184 in the reverse transcriptase gene results in increasing resistance to lamivudine, but paradoxically causes an increase in sensitivity to AZT in previously AZT-resistant virus. Thus, in theory at least, a combination regimen of AZT plus lamivudine should be of benefit, a hope reflected by the availability of a tablet that contains both agents.
Yet another set of mutations in the reverse transcriptase gene confers resistance to the non-nucleoside reverse transcriptase inhibitors, and several such
changes have been reported. Some, but not all, lead to cross-resistance among the different drugs of this type, but they are distinct from those leading to resistance to the nucleoside analogues.
As with the reverse transcriptase inhibitors, resistance to the protease inhibitors may arise through point mutations in the gene coding for the target protein. Various mutations in the protease gene have been described. Many of these map at similar points in the genome of virus from patients receiving different protease inhibitors, indicating that cross-resistance between these drugs is common. Continued use of a given protease inhibitor leads to the accumulation of mutations, with a concomitant increase in resistance.
Anti-HIV drugs in development
Despite the clinical successes achieved by use of the above agents, there is a desperate need for new drugs with different sites of action, and different toxicity profiles in the fight against HIV infection. Agents under investigation include new nucleoside and non-nucleoside reverse transcriptase inhibitors; nucleoside phosphonates (see p. 86), including adefovir and phosphonylmethoxypropylade-nine (known, not surprisingly, as PMPA); and protease inhibitors such as tipranavir, which are being developed to counteract protease-resistant strains.
Two novel targets for anti-HIV drugs are of particular interest: a virus-encoded integrase, which is responsible for integration of the DNA provirus into host cell chromosomes; and the process of fusion whereby viral particles gain entry into a target cell following the initial attachment process. Inhibition of the inte-grase would prevent virus from becoming latent in resting T cells. The most promising fusion inhibitor at present is a synthetic peptide corresponding to part of the HIV gp41 envelope protein. This blocks viral entry in vitro by disrupting the conformational change in gp41 triggered by binding of the HIV gp120 molecule to host cell CD4 and chemokine co-receptors.
Optimal therapy for patients with HIV infection may involve treatment with other drugs which do not necessarily exhibit antiviral activity. Thus, there are encouraging reports of the use of hydroxyurea, which blocks cellular activation necessary for viral replication in resting CD4 positive T cells. Similarly, immunomodulatory agents such as interleukin-2 also have their advocates.