Human Cytomegalovirus (hCMV) or human herpesvirus 5 (HHV5) is one of the most common pathogens. Studies indicate the presence of infection in 60–100% of individuals. The ability to cause asymptomatic, infection and a latency promotes the persistence and spread of the virus. hCMV infection is usually asymptomatic and does not require treatment, but in some cases especially in immunocompromised persons (e.g., transplant recipients, patients with hematological malignancies, untreated HIV infected individuals) symptoms can be serious and life-threatening. The paper presents drugs currently used for treatment or prevention of hCMV infection, as well as the prospect of new treatment options. Currently, ganciclovir or valganciclovir are used as the first-line drugs and foscarnet and cidofovir are used alternatively. These drugs usually allow to control hCMV infections, however, there are important limitations. These include the toxicity and the possibility of the development of resistance, including the cross-resistance to all four drugs because they have a common mechanism of action, inhibition of viral DNA polymerase. Therefore, the creation of new drugs, with different mechanisms of action, lower toxicity and better pharmacokinetic parameters is important. Recently, the new drug, letermovir have been registered. Letermovir acts as hCMV DNA terminase inhibitor and due to the different mechanism of action the drug is active against hCMV strains resistant to DNA polymerase inhibitors, and potentially can act synergistically with them. The other drugs that are in the research stage or clinical studies include: brincidofovir, a cidofovir derivative, maribavir, a competitive inhibitor of ATP, cyclopropavir, a guanosine analog and antiviral peptides.
1. Introduction – epidemiology of hCMV infections and prophylaxis schemes. 2. Drugs approved for use in the prevention and treatment of hCMV infections. 2.1. Nucleoside analogues: ganciclovir and valganciclovir. 2.2. Foscarnet. 2.3. Cidofovir. 2.4. Letermovir. 3. Compounds with potential use in the treatment of hCMV infections. 3.1. Brincidofovir. 3.2. Maribavir. 3.3. Cyclopropavir 3.4. Antiviral peptides. 4. Summary
- nucleoside analoques
- analogi nukleozydów
The human cytomegalovirus (hCMV) classified as human herpesvirus 5 (
The ability to cause latent infection (latency of the virus in macrophages, T lymphocytes and the endocrine gland cells) and persistent infection (constant replication and secretion of viruses without any clinical symptoms) are phenomena so epidemiologically unfavourable that they allow the rapid spread of the virus in the population. The primary infection most often occurs in childhood and in most cases, similarly to the secondary infections appearing among immunocompetent persons, has an asymptomatic or mild, and self-limiting course. Infection with this virus is a significant problem for patients with impaired function of the immune system. This group includes: people with primary immunodeficiencies, people with secondary immunodeficiencies caused by diseases of the immune system: leukemia and other hematological malignancies, infection with human immunodeficiency virus (Human immunodeficiency virus 1 or Human immunodeficiency virus 2; HIV-1, HIV-2), as well as people undergoing immunosuppressive treatment, patients on hemodialysis, and newborns due to the immaturity of the immune system [6, 7, 18, 31, 35, 36].
The currently available, approved and certified diagnostic methods allow for a quick and reliable confirmation of the infection, demonstration of viral replication, monitoring of the course of the infection and result in effective antiviral therapy. However, despite the diagnostic and therapeutic solutions in the form of antiviral drugs, the hCMV is still a significant cause of organ infections (most often: pneumonia, liver, retina, and intestinal diseases) as well as systemic disorder. It is also one of the most important viruses associated with the post-transplantation complications, transplant failure, as well as deaths among recipients of allogeneic hematopoietic cells (allo-HCT) and organs [1, 5, 19, 30, 35, 39].
The risk of a full-blown cytomegalovirus disease among transplant recipients depends on the immune status of the donor and the recipient (more often when the donor is seropositive and the recipient is seronegative), the type of transplant, and immunosuppression [2, 5, 10, 11, 19]. It was shown that dsDNA hCMV is detected in the plasm of 44–65% of patients following the allogeneic hematopoietic cell transplantation within 180 days after the transplantation .
Pharmacological prophylaxis is used to prevent the hCMV disease, which is most often carried out as selective prophylaxis (the pre-emptive therapy). It involves the use of sensitive and specific diagnostic methods to systematically monitor the rate of virus replication (quantitative monitoring of DNA levels, less frequently RNA or pp65 antigenemia) before the occurance of any clinical symptoms, and to evaluate the eligibility for chemoprophylaxis in order to prevent direct and indirect consequences of the infection. The advantage of this strategy is that it limits the number of people exposed to the toxic effects of antiviral drugs. In contrast, the universal prophylaxis involves the inclusion of pharmacotherapy within 10 days of the transplantation. This is mainly used among patients who are at a very high risk of developing a disease caused by the CMV infection. The use of chemoprophylaxis allows the reduction of morbidity and mortality as a result of cytomegalovirus infection, especially among patients after hematopoietic cell transplantation [2, 5, 12, 19, 20, 39].
In recent years, numerous antiviral drugs, mainly used in the treatment of the HIV infection and viral hepatitis caused by the Hepatitis B virus (HBV) and Hepatitis C virus (HCV), have been developed and registered [22, 23]. Much attention is also devoted to the viral infections among patients with immunosuppression. Currently, intravenous ganciclovir (GCV) and oral L-valyl ester of ganciclovir-valganciclovir (VGCV) are considered in the control of hCMV infections. Alternatively, in the case of resistance to these inhibitors or the occurrence of unacceptable side effects, the second and third choice drugs are used, i.e. foscarnet (PFA) and cidofovir (CDV). In 1998, the drug indicated for the treatment of cytomegalovirus retinitis among patients with AIDS, fomivirsen, was approved. Fomivirsen is an antisense, intravitreal oligonucleotide with a DNA sequence complementary to mRNA, which hybridises with the mRNA fragments to form an mRNA-DNA structure that ultimately prevents the synthesis of viral proteins.
Fomivirsen was used as a second choice drug when the standard therapies were ineffective or contraindicated. In 2002, due to the presence of adverse side effects (anterior uveitis, cataracts, and toxic retinopathy), fomivirsen was withdrawn from the market at the request of the holder of the marketing authorisation .
The structural formulas of the drugs approved for use in the prevention and treatment of CMV infections can be found in Fig. 1.
Despite the availability of several formulations that inhibit the hCMV replication, there is still a need for new therapeutics. All of the mentioned drugs are DNA polymerase inhibitors. Due to the common mechanism of action, they are not effective in the treatment of patients infected with strains of the virus with a mutation in the
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 (
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 (T1/2) 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
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 . In most cases, the resistance to CDV also means resistance to ganciclovir . 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 .
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
Mutations in the
Less common are mutations in the conserved region of subunit V of the protein encoded by
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.
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 a cell culture study, it was shown that DNA polymerase inhibitors (cidofovir and foscarnet) in combination with maribavir act synergistically . 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 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 .
Currently, phase 1 trials are being conducted to assess the safety and pharmacokinetics of cyclopropavir administered in healthy volunteers at various doses .
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 , HBV  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 . 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 .
The history of antiviral chemotherapy began over 50 years ago. From 1962 to May 2019, 99 antiviral drugs were registered. Some of those were withdrawn due to poor antiviral activity, rapid emergence of resistance or toxicity. A vast majority of approved medications is intended for the treatment of patients infected with HIV and hepatitis B and C viruses (HBV and HCV). Causative antiviral treatment, more or less effective, is also available for infections caused by herpesviruses, among which hCMV requires the most attention. Letermovir was added to the list of anti-cytomegalovirus drugs in 2017, and several other medications are being tested