Possibilities Of Prevention And Treatment Of Human Cytomegalovirus Infections Including New Drugs And Compounds With Potential Application

Ganciclovir (GCV) is a synthetic, acyclic analogue of guanosine. After entering the cell, it is transformed into a biologically active phosphate derivative. This process is possible with the participation of the UL97 protein kinase. The next two phosphorylations take place with the participation of cellular kinases. The active drug form, ganciclovir triphosphate (GCV-TP), is a substrate for the viral DNA polymerase. Therefore, the antiviral effect results from the inhibition of the viral DNA synthesis in the mechanism of the competitive incorporation of the deoxyguanosine triphosphate into the DNA chain [14, 16, 19]. Ganciclovir is structurally similar to acyclovir; however, the introduction of an additional hydroxyl group has expanded the drug activity; in addition to alphaherpesviruses (Human herpesvirus 1 and Human herpesvirus 2 i.e. herpes simplex virus 1 and 2; HSV-1 and HSV-2, and Human herpesvirus 3 i.e. Varicella zostaer virus; VZV), ganciclovir inhibits the replication of the Epstein-Barr virus (Human herpesvirus 4; EBV), the cytomegalovirus (CMV), the human herpesviruses 6, 7 and 8 (HHV-6, HHV-7, HHV-8), and the hepatitis type B virus (HBV). Ganciclovir is used parenterally. When administered intravenously, it reaches the maximum concentration after 5 hours. It penetrates well into tissues and the central nervous system. Until 2004, it was also available in the form of an oral drug, but due to the low bioavailability (5%) it was replaced with valganciclovir [5, 14, 19, 20, 33]. The most important and very common side effect of the drug is neutropenia. Among 20–30% of people taking ganciclovir, there is an occurrence of bone marrow suppression, renal hepatic dysfunction and liver dysfunction. The neurotoxic effects of the drug have also been described [5, 11, 14, 18, 33]. Since 1994, ganciclovir has been the drug of choice for the treatment of the cytomegalovirus infection [3]. This pharmaceutical is part of a chemotherapy protocol in stem cell and organ recipients. It has been shown that in cells infected with hCMV ganciclovir reaches a concentration up to 100 times higher than in non-infected cells, and its use in the early stage of infection increases its effectiveness. The drug does not inhibit the latent form of the virus; therefore, 20–30% of patients experience relapses [14]. Long-term exposure to GCV and the use of suboptimal doses may contribute to the selection of resistant strains. The scale of the drug resistance is not precisely understood. It was demonstrated that the problem of resistance to ganciclovir may affect 1.5–10% of organ transplant recipients and 0–14.5% of hematopoietic cells recipients [16, 32]. The risk of drug resistance is higher in the case of high-level immunosuppression and long-term GCV administration [4, 5]. Resistance is most often associated with the UL97 gene mutation (the most common mutations: M460V/I, H520Q, C592G, A549V, L595S, C603W, L595F, L595W), which encodes a kinase necessary for the phosphorylation of the drug. The amino acid substitutions and small deletions do not have a significant effect on the virus’s ability to replicate. Mutations in the UL54 gene encoding DNA polymerase are less common; changes in this area may result in resistance to cidofovir and/or foscarnet, and cross-resistance to ganciclovir, foscarnet and cidofovir. In the in vitro environment, such strains have an attenuated phenotype, i.e. their virulence is reduced and they multiply more slowly when compared to the wild virus strains. Combinations of mutations in the UL97 and UL54 genes multiply the effect of each individual mutation, provide a high level of drug resistance, and can cause an increase in the GCV EC₅₀ values up to 15-fold [3, 5, 11, 19, 21, 30, 32].

Valganciclovir is the L-valyl ester salt of ganciclovir (VGCV). When administered orally, it is well absorbed and is converted into ganciclovir by intestinal and liver esterases.

The bioavailability of the drug is 60%, which is about 10 times higher than that of ganciclovir, and the maximum serum concentration is reached after 1–3 hours [5, 12, 14]. Valganciclovir was first approved by the US Food and Drug Administration (FDA) for the treatment of the hCMV-induced retinitis among AIDS patients. It is used in the prevention of symptoms of the disease associated with hCMV among organ transplantation patients and prophylactically among recipients of the hematopoietic cells. Similarly to ganciclovir, valganciclovir is excreted by the kidneys and has the same mechanism of action and toxicity profile [12, 14, 33].

Foscarnet (phosphonoformic acid; PFA) is an anhydride of phosphonoformic acid and an analogue of pyrophosphate [20, 27]. Foscarnet is a broad-spectrum antiviral agent effective against viruses, which have DNA as their genetic material (HSV, VZV, EBV, HHV-6, HBV) and HIV. It was approved for the treatment of infections caused by the cytomegalovirus in 1991. Its mechanism of action is based on the direct inhibition of DNA polymerase by competitively blocking the pyrophosphate binding site, consequently blocking the deoxyribonucleic acid chain extension. In contrast to the previously described inhibitors, foscarnet does not need to be activated by kinases [4, 27, 30]. Due to the fact that the bioavailability of foscarnet is low, the drug is administered intravenously and over 80% of it is excreted in the urine in an unchanged form. The half-life (T_1/2) of PFA is 48 hours. Good penetration through the blood-cerebrospinal fluid barrier allows it to be used in the central nervous system infections. Foscarnet is used in the treatment of HIV-infected patients with symptoms of retinitis with etiology of hCMV. It is also utilised in the treatment of infections caused by the aciclovir-resistant HSV strains and hCMV resistant to ganciclovir. Cross-resistance to ganciclovir and foscarnet is detected less often than the resistance to ganciclovir and cidofovir [3, 19, 32]. An important limitation of using the drug is nephrotoxicity, e.g. interstitial nephritis, acute renal tubular necrosis. Other adverse reactions include liver problems, gastrointestinal disorders (nausea, vomiting), leukopenia, and anaemia. Foscarnet has the ability to chelate divalent metal ions, thus causing electrolyte imbalance, mainly calcium, phosphorus, potassium, and magnesium. Due to the accumulation of the drug in the urine some of the patients suffer from the penile glans ulceration [4, 14, 27, 32].

The foscarnet resistance is associated with mutation(s) within the UL54 gene. The amino acids substitutions in the protein result in limited susceptibility to binding to the drug. Some of these mutations, e.g. N495K, D588E, T700A, V715M, E756D/N/Q and T838A, are resistant only to foscarnet. A number of other amino acid substitutions are responsible for cross-resistance to ganciclovir and cidofovir. Some substitutions or deletions (e.g. Q578, D588N, T700A, V715M, E756D/ N/Q, V781I, V787L, L802M, A809V, V812L, and A834) induce a 3–5-fold decrease in the sensitivity to foscarnet. In the hCMV strains resistant to all polymerase inhibitors, the deletion of del 981–982 has been demonstrated [8, 19, 34]. Currently, there is no data on the incidence of the hCMV resistance to foscarnet; however, it is thought that the resistance index associated with modification in UL54 as a result of long-term treatment with foscarnet is the same as for ganciclovir and cidofovir [5].

Cidofovir (CDV) is an analogue of nucleoside monophosphate (cytidine). Due to the presence of phosphate in the molecule it does not require the first stage of intracellular phosphorylation; hence, it also inhibits the replication of viruses that do not have thymidine kinase. The active metabolite is cidofovir diphosphate. Phosporylation occurs with the participation of cellular kinases (pyrimidine nucleoside monophosphate kinase, pyruvate kinase, creatine kinase, nucleoside diphosphate kinase), whose levels increase in the cells infected with hCMV. CDV is a competitive inhibitor of viral DNA polymerases, thus it inhibits the DNA elongation [4, 30, 20]. The drug is currently used for the treatment of retinitis among patients with AIDS (registered in 1996), for the treatment of infections caused by the aciclovir- and foscarnet-resistant herpes, and hCMV resistant to ganciclovir and foscarnet. It also exhibits antiviral activity against other DNA viruses: VZV, adenoviruses (HAdVs), polyomaviruses, human papillomaviruses (HPVs), as well as orthomyxoviruses belonging to the RNA viruses [4, 14, 32]. The EC₅₀ value of cidofovir towards hCMV is between 0.1 and 0.8 μM [30]. In most cases, the resistance to CDV also means resistance to ganciclovir [19]. The serious restrictions concerning the drug usage are nephrotoxicity and low oral bioavailability. The drug is available in the form of preparations requiring intravenous infusions, which prevents its use in outpatient treatment. The concentration of cidofovir in the kidney cells is 100 times higher than in other tissues, which explains the drug-related proximal tubular injury manifested by proteinuria and glycosuria [18, 30, 32]. For this reason, CDV is the second-line drug used in the treatment of ganciclovir-resistant viral infections [4, 19]. Its use is currently very limited. In many European countries, including Poland, the drug is available for the treatment of individual cases via direct import [16].

Letermovir (LTV, Prevymis") is a novel antiviral drug, belonging to a new class of chemical compounds – quinazolines. It has a narrow spectrum of activity, focused on CMV, and a mechanism of action, which is different from the drugs used so far [16, 21]. The target site of letermovir is the terminase complex composed of proteins, gene products of UL51, UL56 and UL89. The pUL56 subunit exhibits ATPase activity and is the main site of action of the drug. pUL89 exhibits ATPase and endonuclease activity, and pUL51 has not been well characterised so far. As a terminase complex inhibitor, letermovir inhibits the replication of hCMV at the stage of the viral DNA maturation and its packaging into the capsid. The first stage of this process is catalysed by pUL89 and involves binding of the terminase complex to the pac sequence and cleavage of the concatemeric DNA. The viral DNA/terminase complex subsequently binds to the apex of the formed procapsid, through which the DNA of a single unit length, obtained after cleaving the fragment from the DNA chain, moves. The process of genetic translocation requires energy, which is generated by ATPase [5, 11, 30, 32]. A specific indication for administering letermovir is the prevention of hCMV infection and cytomegalovirus disease in adult hCMV-seropositive haematopoietic cell recipients. The drug is available in an oral form (240 mg or 480 mg tablets) and as a concentrate for preparing an infusion solution. The recommended dose is 480 mg administered once a day, orally or intravenously, for a period of one hour up to a hundred days after the transplant [19, 24, 32]. Letermovir is tolerated well and its oral bioavailability is 35%. The drug reaches maximum concentration in the serum approximately 1.5 hours following the oral administration, and its half-life is 10 hours. Letermovir is excreted with bile in an unchanged form [30]. The most commonly reported adverse reactions include nausea, diarrhoea and vomiting, which are found in 7.2%, 2.4% and 1.9% of those taking LTV, respectively. Clinical trials have not demonstrated any haemotoxic or nephrotoxic effects of the drug [11, 23]. Letermovir shows high efficacy against cytomegalovirus. It is about 1000 times more active than ganciclovir [30]. Phase 3 clinical trials were conducted using the double-blind method and involved 570 CMV-seropositive adult haemopoietic cell recipients, randomly (2:1) assigned the letermovir or placebo therapies. The drug was found to be more effective than placebo in preventing CMV from reactivating for up to 14 weeks after transplantation. Within 24 weeks from the transplantation, symptoms of infection with the virus occurred in 37.5% of patients receiving letermovir and in 61% of those receiving the placebo. Moreover, a lower mortality rate was observed within 24 weeks from the transplantation in patients receiving letermovir (10%) compared to those receiving the placebo (16%) [16, 24, 30]. In hCMV-infected cell culture studies, it was observed that administration of letermovir together with ganciclovir or cidofovir resulted in synergistic effects. The combination of these medications may be an interesting strategy in the prevention and treatment of cytomegalovirus disease [30]. Letermovir is also active against hCMV strains that are resistant to other anti-cytomegaloviral drugs [3, 30]. However, mutations associated with the drug resistance have been described. Most often, they occur in the UL56 gene, much less frequently in the UL89 and UL51 genes [30]. Resistance to letermovir has been evaluated using the results of in vitro tests and clinical trials. The substitutions responsible for the resistance were located in codon 25 and in between codons 229–369 of the pUL56 subunit [19, 30]. In the AD169 hCMV strain, the following amino acid substitutions were described: V231L, V236M, L241P, C325Y, R369G, R369M and R369S, which correlated with an increase in the EC₅₀ of the drug by 5, 45, 160, 8796, 4, 13 and 38 times, respectively [30].

Mutations in the UL56 gene conferring resistance to LMV have minimal or no effect on the efficiency of the virus replication compared to the wild-type hCMV viruses.

Less common are mutations in the conserved region of subunit V of the protein encoded by UL89 (N320H, N329S, D344E and T350M); individual changes cause low-level resistance to letermovir. However, the coexistence of a D344E substitution in pUL89 with one of the substitutions, such as E237D, F261L, M329T or Q204R, in pU56 increases the EC₅₀ of the drug. A mutation in the UL51 (P91S) gene associated with letermovir resistance has also been observed [21, 20]. The first clinical LTV-resistant isolate was obtained from a patient in phase 2b clinical trials. The patient received a suboptimal dose of the drug (60 mg) and the failure of the treatment was associated with the mutation in UL56. The presence of the V236M substitution, as well as the C325W, C325F and C325Y subtitutions demonstrated in strains isolated from other patients was associated with a high level of resistance to LMV. The results suggest that mutations associated with letermovir resistance occur relatively easily and quickly, therefore, the drug should be used with caution outside of the indicated scope and most importantly, the treatment should be monitored to check for drug resistance [30].

Brincidofovir (BCV; CMX001) is a prodrug containing a synthetic, acyclic monophosphate nucleotide analogue (cidofovir) conjugated to a lipid (3-hexadecyloxy-1-propanol) via a phosphonate group. The structures of brancidofovir and other compounds with potential use in the treatment of CMV infections are shown in Fig. 2.

Following the entry into the cell and cleavage of the lipid chain, the released cidofovir monophosphate is phosphorylated by intracellular kinases to cidofovir diphosphate (CDV-DP). The drug is an alternative substrate for DNA polymerase. The incorporation of cidofovir into the viral DNA blocks the deoxyribonucleic acid chain elongation. Brincidofovir has a broad spectrum of antiviral activity. Cell culture studies were used to evaluate its activity against various DNA viruses: herpesviruses (VZV, CMV, EBV, HHV-6), polyomaviruses (BK virus, BKV), adenoviruses, and papilomaviruses [15, 16, 37, 38]. Brincidofovir is characterised by a better bioavailability when administered orally in comparison with cidofovir. It achieves an over 100 times higher intracellular concentration than CDV, and its antiherpesvirus activity (HSV, CMV, VZV) is up to 1000 times higher in comparison to cidofovir, as well as ganciclovir and foscarnet.

Fig. 2.

In vitro tests and studies using animal models have demonstrated a synergistic effect of brincidofovir and acyclovir on inhibiting HSV replication [32].

Administered intravenously, the drug has no myelotoxic activity. It is also not nephrotoxic, probably because unlike cidofovir, it is not a substrate for human organic anion transporters (OAT1), located in the proximal convoluted tubule [30, 37]. Brincidofovir showed promising outcomes in preclinical trials; however, the disappointing results of phase 3 clinical trials, which evaluated its efficacy in preventing diseases associated with hCMV infection in seropositive patients with allogeneic hematopoietic cell transplantation, have slowed down its introduction into the clinic. Nevertheless, the initial enthusiasm for the drug has not waned, and the tests using brincidofovir in the prevention of hCMV infection in hematopoietic stem cell (HSC) patients continue, as the control of infection in this group of patients is still severely limited [29, 32]. Currently, recruitment is planned for a randomised, controlled, open, multi-centre clinical trial evaluating tolerance, pharmacokinetics, and anti-adenovirus activity of BCV administered intravenously at different doses (NCT03532035). In addition, an open, randomised, multi-centre parallel study has commenced with a goal of assessing the safety, tolerability and antiviral activity of brincidofovir. Furthermore, evaluation of the effectiveness of this drug compared to the standard care for paediatric patients receiving allogeneic haematopoietic cells and being at risk of adenovirus infection has also taken place (NCT03339401). Adverse effects of the drug include gastrointestinal disorders [16, 25, 30].

Maribavir (MBV; 1263W94) is a competitive inhibitor of ATP. It binds specifically to the serine/threonine protein kinase UL97, which mediates one of the final stages of viral replication, inhibiting its encapsidation and releasing viruses from infected cells. The drug has been studied in vitro for many years and is a subject of clinical trials [4, 16, 30, 32]. Maribavir does not require intracellular phosphorylation. In vitro studies have shown that it is over 10 times more active against CMV than ganciclovir. The EC₅₀ of the preparation is approximately 0.3 μM [30]. The antiviral activity of the formulation also applies to CMV strains resistant to ganciclovir and cidofovir. Maribavir is characterised by good bioavailability following oral administration [30, 32]. Results of clinical trials demonstrated that the drug is well tolerated, and its haematoxicity is lower than that of ganciclovir and valganciclovir. It also displays no nephrotoxicity [16, 30, 32]. A randomised, placebocontrolled, double-blind clinical trial investigated the efficacy of maribavir in the prophylaxis against hCMV in adults after allogeneic stem cell transplantation (allo-HSCT). MBV (100 mg, BID) and placebo were administered for a period of 12 weeks. After 6 months of observation, the incidence of cytomegalovirus disease in the first 6 months following the transplantation was 4% in those taking maribavir and 5% in those taking placebo. These observations resulted in the discontinuation of the phase 2 trial, which tested the effectiveness of the drug in the prevention of hCMV disease in the liver transplant recipients [13, 32]. Despite these disappointing results, it is recognised that maribavir may have other applications in transplant patients; therefore, studies involving this preparation continue. Currently, two groups are being recruited for phase 3 clinical trials, in which the efficacy and safety of maribavir and valganciclovir will be compared in the treatment of cytomegalovirus infection in asymptomatic haematopoietic cell transplant recipients (NCT02927067). The efficacy and safety of using maribavir to treat transplant recipients with infections caused by cytomegalovirus resistant to ganciclovir, valganciclovir, foscarnet or cidofovir will also be evaluated (NCT02931539) [32].

In a cell culture study, it was shown that DNA polymerase inhibitors (cidofovir and foscarnet) in combination with maribavir act synergistically [30]. Similarly, an enhancement of antiviral activity was observed after co-administration of letermovir and maribavir in the cell culture. In contrast, maribavir and ganciclovir show antagonistic activity. This is because the inhibition of UL97 kinase caused by maribavir negatively influences the phosphorylation of ganciclovir [9, 16, 32]. Maribavir also inhibits the EBV replication in vitro [1].

In addition to maribavir, other UL97 kinase inhibitors with potential anti-cytomegalovirus activity have been synthesised. Included in them are: indolocarbazoles, quinazolines and benzimidazole analogues; however, so far none of the preparations have been investigated in as much detail as maribavir [3, 4].

Cyclopropavir (CPV; MBX-400) is a second generation nucleoside analogue of 2-deoxyguanosine (guanosine nucleoside analogue). The first generation drugs are bioisosteric analogues of aciclovir. The new generation of nucleoside analogues arose as a result of a structural change, i.e. the replacement of the acyclic C-O-C group with methylcyclopropane. Cyclopropavir is structurally most similar to ganciclovir [4]. In vitro tests have shown that CPV has approximately 10 times higher (EC₅₀ 0.46 (iM) anti-cytomegalovirus activity than ganciclovir, probably as a result of the accumulation of the drug in the infected cells at a higher concentration without causing toxicity. CPV has also been demonstrated to be active in vitro against other human herpesviruses including VZV, HHV-6A, HHV-6B and HHV-8. Similarly to ganciclovir, cyclopropavir requires phosphorylation to monophosphate by a viral kinase (pUL97), as well as further phosphorylations, in which cellular kinases are involved. The drug inhibits the replication of viral DNA by inhibiting DNA polymerase and/or incorporation into the viral DNA, resulting in the termination of DNA elongation [4, 29]. As with other nucleoside analogues, it appears that valylated cyclopropavir ester may have improved pharmacokinetic properties [7, 17, 29, 40].

Currently, phase 1 trials are being conducted to assess the safety and pharmacokinetics of cyclopropavir administered in healthy volunteers at various doses [4].

It is believed that peptide drugs may be an alternative for the treatment of viral infections. So far, studies have been concerned with their mechanism of action against viruses causing respiratory infections [41], HBV [28] and HIV (fusion inhibitors: enfuvirtide, sifuvirtide, albuvirtide, and other peptides showing structural similarity to peptides found in the HIV capsid proteins) [18, 23, 26]. Antiviral proteins act as inhibitors, interfering with various stages of virus replication, e.g. they can inhibit viral attachment, cell entry, replication or release from the cell [18]. hCMV attaches to the host cell via heparan sulfate proteoglycans (HSPG). The viral gB and gM/gN glycoproteins interact with negatively charged sulfate residues, resulting in attachment of the hCMV virion to the host cell. This triggers a cascade of signals, which in effect allows the virus to enter the cell. HSPGs are present on most host cells, which explains the fact that hCMV can infect many types of human cells. HS binding peptides can, therefore, effectively inhibit hCMV infection [18].