Echinocandins – structure, mechanism of action and use in antifungal therapy

Abstract With increasing number of immunocompromised patients as well as drug resistance in fungi, the risk of fatal fungal infections in humans increases as well. The action of echinocandins is based on the inhibition of β-(1,3)-d-glucan synthesis that builds the fungal cell wall. Caspofungin, micafungin, anidulafungin and rezafungin are semi-synthetic cyclic lipopeptides. Their specific chemical structure possess a potential to obtain novel derivatives with better pharmacological properties resulting in more effective treatment, especially in infections caused by Candida and Aspergillus species. In this review we summarise information about echinocandins with closer look on their chemical structure, mechanism of action, drug resistance and usage in clinical practice. We also introduce actual trends in modification of this antifungals as well as new methods of their administration, and additional use in viral and bacterial infections.


Introduction
Diseases caused by fungi are a serious problem. Currently, the number of people affected by fungal infections worldwide exceeds one billion 1 , and the number of deaths caused by invasive fungal species is comparable to the mortality of tuberculosis À 1.5 million each year 2 . About 90% of mortalities are caused by fungi grouped into four genera: Candida, Aspergillus, Cryptococcus, and Pneumocystis 3 (Table 1). There are at least 17 pathogenic species complexes in the genus Candida, but more than 90% of infections are attributed to Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Candida krusei 4 . The patients suffering from AIDS, cancer (and associated chemotherapy), leukaemia, patients on immune-compromising drug therapy, and after organ transplantation are particularly vulnerable to mycoses 5 .
Until the end of the 20th century, azoles, polyenes, and flucytosine were mainly used to treat mycoses. These classes of drugs can cause serious side effects related to their hepato-and nephrotoxicity. In addition, many fungal strains have developed resistance to these antibiotics, which significantly reduces their efficacy 6 . In rare cases, cross-resistance to polyenes and azoles may occur, raising concerns about the future of antifungals targeting membrane ergosterol (polyens) and sterol synthesis (azoles) 7 . In addition, there are frequent drug-drug reactions associated with interactions of the aforementioned drugs and their metabolites in the body 8 . This has prompted the search for alternative agents to combat fungal infections. Echinocandins are a class of antifungal drugs that are fungicidal against many fungi including Candida species, but are fungistatic to the Aspergillus genus. This class of drugs has been found to cause milder side effects compared to polyenes and azoles 9 . The mechanism of action based on the inhibition of fungal-specific metabolic pathway and limited side effects have resulted in increasing interest and use of this class of drugs 10 .
The purpose of this paper is to introduce echinocandins as antifungal antibiotics for the reader, details of their chemical structure and proposed modifications, mechanism of action as well as their usage in clinical practice are also described. The use of echinocandins has improved patient outcome; however, there are reports about drug resistance of fungi to this class of antibiotics. Therefore, an additional aim of this study is to summarise the latest information on the phenomenon of fungal drug resistance to echinocandins.

History of the discovery of echinocandins
In 1974, in Switzerland the first antifungal drug of the echinocandin classechinocandin B was discovered, which showed good antifungal properties but at the same time strong haemolytic effects. To counteract this, cilofungin, a semi-synthetic analogue of echinocandin B with a 4-octyloxybenzoate side chain, was synthesised. This compound significantly reduced haemolytic activity while retaining antifungal properties 11 . However, cilofungin was withdrawn from Phase II clinical trials due to the poor water solubility and toxicity of its co-solvent 12 . An important step in echinocandin research was the discovery of pneumocandin A 0 and pneumocandin B 0 , of which pneumocandin B 0 was used to synthesise a new antifungal agent 13 (Table 2).
In 1992, caspofungin acetate was first synthesised from pneumocandin B 0 and approved for clinical trials 13 . The U.S. Food and Drug Administration (FDA) approved caspofungin in January 2001 as a drug for the prevention of fungal infections in adult patients, and the first medical preparations containing caspofungin were introduced to the U.S. market the same year. In Europe, the antibiotic was approved for therapy in 2002 under the trade name CANCIDAS (manufacturer: Merck & Co. Inc., USA, MK-0991) 17 . In July 2008, it was approved for the treatment of children over 3 months of age 18 .
The precursor of micafungin, which was FR901379, was discovered in Japan by Fujisawa Pharmaceutical. To stop FR901379induced reticulocyte lysis, it underwent various modifications. This resulted in the compound FK463 called micafungin, which had reduced haemolytic activity and was potent against Candida and Aspergillus species 19 . The drug was approved in the U.S. in 2005, and in Europe in 2008. The compound was also approved for the treatment of invasive candidiasis in patients younger than 4 months of age 20 . The first formulations of micafungin were produced in Japan and sold under the trade name MYCAMINE (manufacturer: Astellas Pharma Inc., Japan, FK-463) 17 .
Another of the echinocandins, anidulafungin was obtained by optimising the chemical structure of echinocandin B. Replacement of the naturally occurring fatty acid side chain with an alkoxytriphenyl side chain reduced the haemolytic effect of echinocandin B 17 . Anidulafungin, as an antifungal compound for the treatment of oesophageal candidiasis, candidemia and deep tissue candidiasis, was approved in the US in 2006 and marketed under the trade name ERAXIS. In Europe, the drug was marketed a year later under the trade name ECALTA (manufacturer: Pfizer Inc. UK) 21 .
The "ReSTORE" study (ClinicalTrials.gov registration number NCT03667690) 16 of rezafungin (CD101), the newest member of the echinocandin class, is currently ongoing. Two post-study Phase III clinical trials are underway to evaluate the efficacy of rezafungin for the treatment of candidemia and invasive candidiasis and for the prevention of invasive fungal infections caused by Candida, Aspergillus and Pneumocystis species (patient recruitment for the trials began in October 2018, with planned completion of the trial set for August 2021) 22 . Rezafungin is a structural analogue of anidulafungin in which the hemiaminal region at the C5 ornithine position has been replaced with a choline amine ether. With this modification, the stability of the drug in solutions and pharmacokinetics are improved, as well as the drug's action in the body is prolonged 10 . It is also possible to treat infections caused by fungal strains resistant to other antibiotics such as azoles with rezafungin 23 .

Chemical structure of selected echinocandins
Echinocandins such as pneumocandin A 0 and B 0 , echinocandin B, and FR901379 are naturally synthesised by filamentous fungi. Caspofungin, micafungin, anidulafungin and rezafungin are semisynthetic cyclic lipopeptides with antifungal activity (Figure 1). They have a core composed of a cyclic hexapeptide and are acylated with a different fatty acid attached to the a-amino group of dihydroxyornithine ( Figure 2). This lipid residue is required to anchor the drug to the cell membrane and is therefore essential for bioactivity 11 .
Echinocandin B, pneumocandin B 0 and synthesis of caspofungin Echinocandin B ( Figure 2) is a major lipopeptide antifungal antibiotic from a complex produced by Aspergillus nidulans and Aspergillus rugulosus. It contains a linoleic acid side chain and a hexacyclic peptide core built by various amino acid residues such as 3,4-dihydroxyhomotyrosine, 3-hydroxy-4-methylproline, 4,5-dihydroxyornithine, 4-hydroxyproline and two threonine residues 24 . These amino acid residues have a significant effect on antifungal activity and determine the physicochemical properties of the echinocandin B nucleus. For  example, modified proline and homotyrosine residues are essential for the antifungal efficacy of echinocandins 25 . Hydroxyl groups at the three amino acid residues forming the cyclic lipopeptide core, improve the solubility of the drug in water and aid its stability in solutions 12 . Taking this into account, during the synthesis of newer echinocandin B derivatives, the core was kept unchanged or underwent minor modifications 26 ( Figure 1). The hydrophobic fatty acid chain attached to the echinocandin B core is crucial for antifungal activity because it acts as a "hook" that allows the drug to anchor in the fungal cell membrane 12 .
Caspofungin is a cyclic, semisynthetic water-soluble lipopeptide ( Figure 3). This compound is a derivative of the naturally occurring hexapeptide in Glarea lozoyensis, pneumocandin B 0 26,28 . This compound was developed as a result of a four-year program of medicinal chemistry to obtained a derivative with improved water solubility that would facilitate the development of an intravenous formulation. Initial attempts to optimise the structure produced derivatives with a cationic aminoethyl ether group, as well as a 3hydroxyornithine in place of the 3-hydroxyglutamine moiety ( Figure 3). These modifications increased water solubility, stability as well as activity against Candida and Aspergillus spp. 27 . Initial studies had suggested that improved activity was caused by the presence of cationic groups that can form ion pairs with the negatively charged phosphate group of the phospholipid. The consequence was an increase in the concentration of lipopeptide in the cell membrane where the target, glucan synthase, is located. Replacement of the aminoethyl ether with ethylene diamine resulted in the identification of caspofungin, which was synthesised via a two-step modification of the peptide core of pneumocandin B 0 . First carboxamide of 3-hydroxyglutamine was selectively reduced to an amine by a two-step method. Second, condensation of the hemiaminal group with ethylenediamine furnished caspofungin 29 ( Figure 4). After improving the methodology, caspofungin acetate -CANCIDASV R was obtained by a very efficient three-step synthesis 29 ( Figure 5). Despite the success of the medicinal chemistry program, new analogues of caspofungin are still under development. To study echinocandins cellular biology in Candida species, some fluorescent caspofungin's derivatives were developed 30,31 ( Figure 6). It should be emphasised that some of new caspofungin derivatives do not show biological activity (such as, for example, derivatives obtained by cross-metathesis with a larger peptide ring size than in naturally occurring echinocandins) 32 (Figure 7). The possibility of designing macrocyclic compounds by total synthesis is very promising. The use of mild conditions in efficient reactions is an interesting alternative to the semi-synthetic method. The application of total synthesis enables to conveniently plan a wide variety of compounds and to study the relationship between structure and biological activity 34 .

Synthesis of micafungin
Another group of echinocandins are compounds with a sulphate moiety instead of a hydroxyl group in dihydroxyhomotyrosine. Micafungin ( Figure 8) was formed by enzymatic deacylation of a naturally occurring hexapeptide derived from Coleophoma empetri (FR901379), to which an optimised N-acylated side chain was then introduced 35 . The side chain contains an isoxazole ring substituted by 3,5-diphenyl 36 . This modification reduces the haemolytic activity of the drug compared to FR901379 while retaining the antifungal properties of the precursor 19 . The sulphate group in the dihydroxyhomotyrosine side chain in the compound structure increase the water solubility of micafungin 37 .

Modification of echinocandin Banidulafungin and rezafungin
Anidulafungin is a semi-synthetic derivative of echinocandin B 9 ( Figure 9). Anidulafungin, as well as other echinocandins, consists of a peptide nucleus of echinocandin B, composed of amino acid residues. Enzymatical deacylation of echinocandin B using Actinoplanes utahensis culture introduced hydrochloride salt in place of the linoleoyl side chain. Next, this HCl salt was reacted with the activated ester to form anidulafungin 38 ( Figure 10). The introduction of an alkoxytriphenyl side chain in place of the alkyl chain of echinocandin B reduces the haemolytic properties of the drug and has a key effect on the intercalation of anidulafungin with the fungal cell membrane, but reduces the solubility of the drug in water 12,38 . Poor solubility and low oral bioavailability of anidulafungin cause a necessity of administration by intravenous injection 40,41 . To increase the solubility, derivatives with a modified side chain have recently been prepared, and one of them is particularly promising 42 ( Figure 11). According to primary tests, it shows high activity against C. albicans and C. krusei with better water solubility and lower toxicity in vitro in murine macrophages (RAW264.7) than anidulafungin 41 . The observed effect may be particularly important considering the therapy of immunocompromised patients.
Anidulafungin is the precursor of rezafungin (CD101) with an additional choline ether 15 ( Figure 12). The choline amine ether moiety at the C5 position of ornithine increased the stability of the compound (reduced degradation of the compound in the hemiaminal region), half-life, solubility 10,22,23 . During degradation processes, anidulafungin may undergo a ring-opening process in which the hemiaminal cleaves to form a linear peptide having a terminal amide and aldehyde from the hydroxyl C5 ornithine residue forming reactive intermediates that undergo further metabolism in the body and persist until eliminated in the faeces. The increased chemical stability of rezafungin prevents ring opening resulting in the absence of reactive metabolites that may contribute to toxicity 15,43 .  45 . This enzyme uses UDP-glucose as a reaction substrate to form b-(1,3)-D-glycosidic bonds 46 . The enzyme is a transmembrane heteromeric glycosyltransferase consisting of at least two subunits. The Fks1p subunit (encoded by  the FKS1, FKS2, and FKS3 genes) has a catalytic function, while the Rho1p subunit (belonging to the GTPase family) has a regulatory function. Echinocandins binding non-competitively to the Fks1p subunit of the enzyme inhibits its activity 47,48 . Blocking b-(1,3)-Dglucan biosynthesis leads to structural abnormalities of the fungal cell wall (Figure 13), resulting in growth inhibition or death by imbalance in osmotic pressure 49 . The fungicidal or fungistatic effects of echinocandins have been confirmed for most species of the Candida and Aspergillus genera 50 .

Antifungal activity, metabolism and medicinal use of echinocandins
The range of MIC values of echinocandins varies from 0.007 lg/ml to 32 lg/ml depending on the Candida species. Overall, anidulafungin shows the most potency against most Candida pathogens 51 (Table 3).
Echinocandins are recommended as a treatment for patients suffering from an invasive infection caused mainly by Candida, Aspergillus species and some other pathogenic fungi 50 (Table 4).
These antibiotics also act on biofilm-forming yeasts especially on the Candida genus 64 . Relative to Candida species, echinocandins exhibit fungicidal activity manifested by significant cell enlargement and distortion, which contributes to inhibition of cell proliferation. Against Aspergillus species (A. fumigatus, A. flavus, A. niger, and A. terreus), echinocandins exert fungistatic effects by causing irregular growth of the hyphae with multiple branched tips and distended cells, preventing the pathogen from spreading beyond the initial site of infection [65][66][67] . Echinocandins are also active against some species of Penicillium and Paecilomyces. To a lesser extent, they show activity against Madurella, Wangiella, Sporothrix, Exophiala, Scedosporium, Pseudallescheria and Fonsecaea genera 68 . These antibiotics used without additional antifungal compounds are not effective for the treatment of mycoses caused by Mucorales, Cryptococcus, Fusarium, Rhizpous and Trichosporon genera 69,70 . The cell wall of the above-mentioned fungal genera contains mainly b-(1,6)-D-glucans, which limits their sensitivity to echinocandins 69 . The activity of these antibiotics against representatives of the genera Histoplasma, Blastocystis and Coccidioides is also limited 71 . Echinocandins are given to patients intravenously (over 1 h infusion), because they are poorly absorbed in the gastrointestinal tract (less than 3% of the antibiotic is absorbed after oral administration). They also cannot penetrate the central nervous system or the eyes, and for this reason are not used to treat intraocular inflammation or fungal meningitis 72 . These antibiotics bind strongly to proteins (97-99%), leading to lower concentrations of the drug available in serum and tissues 36,59 . The half-life ranges from 9 to 133 h (Table 5), so they are administered once daily (for FDA approved ones) or once weekly (rezafungin). The clinical Completely synthetic caspofungin derivatives. The differences in the structure of new compounds relative to caspofungin are shown in green (II., III., IV. according to 25 ; I. according to 32 ; V. according to 33 ). response of the patient affects the length of treatment. Most patients should be treated 14 days or no more than 7 days after the resolution of symptoms 5 . Echinocandins are metabolised mostly in the liver as well as in the adrenal glands and spleen through hydrolysis and N-acetylation. Their metabolites are excreted mainly in the bile and faeces 18,36 . Echinocandins are found in the highest concentrations in the liver, spleen, intestine, kidney, and lung. In these tissues, their concentrations can be two to sixteen times higher than in plasma 79 . Echinocandins do not affect the P-glycoprotein family and are weak substrates for cytochrome P450 72 .
The administered dose in geriatric patients does not require appropriate adjustment. The elderly do not exhibit changes in pharmacokinetics in comparison to younger patients (dose adjustment is not required) 80 . Echinocandins are not removed renally, so they can be safely administered to elderly patients with renal impairment and patients undergoing dialysis. If a patient is taking several pharmaceuticals during echinocandins therapy, it is not necessary to adjust these medications because echinocandins have little drug-drug interaction. The exception is the special dosage adjustment of caspofungin in patients with hepatic impairment 80,81 .

Caspofungin
Caspofungin besides many Candida and Aspergillus species, also has activity against Pneumocystis jirovecii. Its activity against C. parapsilosis and C. guilliermondii, as well as Trichosporon beigelii, Rhizopus arrhizus and Fusarium spp. is moderate 17 . Studies have also shown antibacterial activity of caspofungin against Enterococcus faecium 82 . Caspofungin has nonlinear  pharmacokinetics, has an affinity for plasma proteins (97%) and has a half-life of 9-11 h in the human body (Table 5), allowing for once-daily dosing. The drug is metabolised in the liver by hydrolysis and N-acylation, and the metabolites are excreted mainly in the faeces (35%) and urine (41%) 72,83 . Some caspofungin undergoes spontaneous chemical degradation caused by peptide ring-opening 84 . A small amount of caspofungin is excreted unchanged in the urine (2%) 83 . In clinical practice, caspofungin is used in patients with invasive aspergillosis (caused by A. fumigatus) and associated neutropenia, as well as in cancer patients, HIVinfected patients, allogeneic haematopoietic stem cell transplant patients, and organ transplant patients, for preventing of fungal infections 85,86 . It may also be used to treat pleural infections, candidemia, oesophageal candidiasis, peritonitis, intra-abdominal abscesses, and abdominal infections caused by Candida species 13 . Caspofungin is used for treatment against voriconazole-and polyene-resistant Aspergillus fumigatus 9,49 . The recommended dosing regimen in adults consists of a single saturating dose of 70 mg on the first day, followed by 50 mg daily, administered over 1 h, reaching an initial therapeutic plasma concentration of 1 lg/ ml 87,88 (Table 6). Paediatric patients receive a saturating dose of 70 mg and a maintenance dose of 50 mg 14,72 . Caspofungin may reduce concentrations of rifampicin and tacrolimus in human serum 88 . In the absence of treatment response or concomitant use of cytochrome P450 inducing drugs (e.g. rifampin, efavirenz, dexamethasone), the daily dose of caspofungin should be increased to 70 mg 18 . In patients with hypoalbuminemia and liver failure, caspofungin doses should be individually adjusted 91 . Inhalation administration of the drug is also being studied, which according to data from experiments shows better antifungal properties. Additionally, the inhaled form can be used once a week and has lower hepatotoxicity than intravenous form administered once at high concentrations 92 . It is possible to treat animals such as cats suffering from invasive fungal mucositis and sinusitis caused by Aspergillus fumigatus with caspofungin 93 .

Micafungin
Micafungin has potent antifungal activity against a broad spectrum of Candida species and azole-resistant Aspergillus species 19 . It binds significantly (>99%) to plasma proteins, mainly albumin and, to a lesser extent, a1-acid glycoprotein, and has a half-life of Figure 10. Synthesis of anidulafungin (according to 39 ). Figure 11. Structure of new semi-synthetic derivative of anidulafungin (according to 42 ).
11 to 17 h (Table 5). Micafungin is not a substrate for P-glycoprotein and does not affect its activity. It is metabolised by arylsulfatase, catechol-O-methyltransferase, and several cytochrome P450 (CYP) isoenzymes (3A4, 1A2, 2B6, and 2 C). It degrades to at least eleven metabolites that are excreted in the bile over many days.
Human organism also disposes of them in the urine. Less than one percent of the drug is excreted unchanged via this route 94,95 . Micafungin is used to treat patients suffering from invasive candidiasis, candidemia, oesophageal candidiasis, abdominal abscesses, and peritonitis. It is used in the prophylaxis of Candida  infections in patients undergoing haematopoietic stem cell transplantation or when a patient is expected to have granulocytopenia 96 . Studies show that it is possible to use micafungin as alternative antifungal prophylaxis in patients with acute leukaemia and myelodysplastic syndrome 97 . Micafungin also affects the immune system by potentiating its response by improving human macrophage activation 98 . A saturating dose is not required; doses of 100-150 mg daily provide minimum plasma concentrations of approximately 2-2.5 mg/ml on the first day of therapy 95 . Dosing in adults is 150 mg for oesophageal candidiasis, 100 mg for invasive candidiasis, and 50 mg for prevention of Candida infection (Table  6). In paediatric patients, the administered dose ranges from 1 to 4 mg/kg body weight depending on the disease present 53 .

Anidulafungin
Anidulafungin is active against a wide range of Candida species, including those resistant to azoles, amphotericin B, and other echinocandins. It is less active against C. guilliermondii and C. parapsilosis 99 . Out of the echinocandins used, anidulafungin has the greatest activity against species of the genus Aspergillus even in the case of Aspergillus lentulus, which has reduced sensitivity to most antifungal drugs 100 . This antibiotic is highly bound to plasma proteins (99%) and has a half-life of 24-26 h in the human body ( Table 5). Anidulafungin is non-enzymatically metabolised in human plasma during biotransformation processes to an open ring peptide lacking antifungal activity, which is excreted mainly in the faeces 99,101 . Since the degradation of the drug does not occur in the liver and the metabolites are mainly excreted biliary, the compound is safe for the treatment of individuals with liver and kidney failure 102 . As an antifungal antibiotic, anidulafungin is used to treat oesophageal candidiasis, candidemia, abdominal abscesses, and peritonitis in patients with or without neutropenia [103][104][105] . It is possible to treat pneumonia caused by Pneumocystis jirovecii with anidulafungin in patients who cannot tolerate trimethoprim or sulfamethoxazole 106 . Adult patients with invasive candidiasis receive a dose of 100 mg of anidulafungin per day after a 200 mg saturating dose, and for oesophageal candidiasis, 50 mg per day after a 100 mg saturating dose ( Table 6).
Infusion of the saturating dose should last approximately 3 h and the maintenance dose approximately 1.5 h 99 . Altered clearance and volume of distribution have been noted in patients weighing more than 140 kg which may result in less exposure to echinocandins in infected tissues. In that cases it is recommended to increase the antibiotic dose by 25% compared to the standard amount 95 . The plasma concentration of anidulafungin is maintained below 1 mg/l throughout the dosing period. Paediatric patients receive a saturating dose of 3 mg/kg body weight followed by a maintenance dose of 1.5 mg/kg body weight 5,21 . Liposome formulations of anidulafungin show greater efficacy compared to administration of the drug in free form. Such formulations could potentially (so far not approved by FDA) support treatment and reduce the occurrence of drug resistance 107 .

Rezafungin
Rezafungin is the first representative of the second generation class of echinocandins 10 . It is effective against Candida spp., Aspergillus spp., Trichophyton mentagrophytes, Trichophyton rubrum, and Microsporum gypseum 50 . Rezafungin is also effective against isolates in which there is confirmed resistance to other  "þ" fungi susceptible to a specific antibiotic; "À" resistant species; "1/2" organisms showing variable response indicated drugs. echinocandins or azoles (C. auris, C. parapsilosis, C. glabrata) 48,73 . Rezafungin is currently in Phase III of clinical trials in patients with candidemia and invasive candidiasis 48 . The antibiotic is also effective in preventing Pneumocystis spp. pneumonia 23 . A study in a mouse model showed that 3 weeks of rezafungin prophylaxis was as effective as the standard treatment of pneumocystis pneumonia with trimethoprim or sulfamethoxazole 22 . Rezafungin also shows very potent activity against A. fumigatus, even against cryptic multidrug-resistant strains carrying a mutation in the CYP51A gene (causing azole resistance) which may suggest an effective treatment for azole-resistant aspergillosis 10,108 . Rezafungin binds strongly to proteins (99%), has a long half-life (80 h after the first dose, after subsequent dose 150 h), and its high safety profile allows for high doses (400 mg first dose, subsequent 200 mg) once a week 22,23,108,109 (Table 5). The pharmacokinetics of rezafungin are relatively linear regardless of dose and its prolonged action enhanced drug penetration into infected tissues 22,110 . Rezafungin has a better safety profile compared to other echinocandins 10 . The solubility in water is above 150 mg/ml and in acetate, lactate and tris buffers is 45 mg/ml 15 . Rezafungin is rapidly distributed to the kidney and liver and its urinary excretion is negligible. It shows better distribution to hepatic abscess areas compared to micafungin (the concentration for rezafungin was 44.5 mg/g and for micafungin was 3.4 mg/g 24 h after drug administration) 111 .
Rezafungin at a concentration of 0.25 mg/ml inhibits biofilm formation in C. albicans by deformation of the cells and release of their contents 10,112 . Lyophilised rezafungin powder shows much less degradation during storage compared to other echinocandins. Storage of rezafungin at 40 C, at room temperature in 5% dextrose, 0.9% NaCl solution or sterile water for more than 1 year showed minimal degradation of the compound (less than 7%) and no epimerization 10,15,23 . Infusion solutions can be stored without stabilisers and without fear of photolysis or spontaneous degradation 15 . Rezafungin can potentially be topical and subcutaneously applied 10,113 .

Synergistic action of echinocandins with other antibiotics
Combinations of echinocandins along with other antifungal antibiotics are currently being investigated. Results of recent studies indicate an additive effect of caspofungin and voriconazole against an echinocandin-resistant strain of C. glabrata. Moreover, using the checkerboard test, combinations of caspofungin with azoles and amphotericin B showed an increase in fungicidal effect from 17.65% to 29.41% against this species 114 . The combination of anidulafungin and isavuconazole increases efficacy against azole-resistant A. fumigatus, which may benefit patients with invasive aspergillosis when azole therapy does not work 115 . Nikkomycin Z, along with caspofungin or micafungin, has shown enhanced activity against biofilms produced by C. albicans and C. parapsilosis. More effective activity against biofilms was shown by the combination of nikkomycin Z with micafungin compared to the combination with caspofungin 116 . The combination of colistin, which has no antifungal properties, with caspofungin resulted in increased potency against C. auris. This is presumed to be due to the alteration of the fungal cell wall structure by echinocandins, making it easier for colistin to interact with the fungal cell membrane 117 . Combination therapy with voriconazole and anidulafungin has also been shown to be equally effective and less costly in patients with a haemolytic disease or haematopoietic cell transplantation compared to voriconazole monotherapy 118 . New research suggests a synergistic effect of caspofungin and isavuconazole, but further studies are required 119 . Farnesol, which is a quorum-sensing molecule that enhances the effects of antifungal drugs, exhibits synergistic effects with echinocandins against C. auris biofilms 120 .
During studies on echinocandin-resistant species, compounds that potentiate their effects were also discovered. The most promising was DTPA (pentetic acid), which modulates echinocandin resistance phenotypes by chelating metal cations, especially magnesium and zinc excluding iron. This compound yielded beneficial relationships in mouse models of candidiasis. A chelator that Table 6. Characteristics of echinocandins as medical preparations 5,10,17,18,22,51,89,90  potentiates the effects of echinocandins may represent a novel therapeutic strategy for combating drug-resistant fungi 121 .

Side effects of echinocandins
Side effects of treatment with echinocandins are much milder compared to other antifungal antibiotics. Troublesome adverse reactions that force withdrawal occur less frequently with echinocandins than with other systemic antifungal drugs 18 . For example, amphotericin B administered intravenously can cause seizures, violent fevers, chills, myalgia, and hyperkalemia. In addition, the use of this antibiotic poses the likelihood of permanent kidney and liver damage due to its hepato-and nephrotoxicity 122 . The most common side effects associated with intravenous infusion of echinocandins are facial flushing, edoema, rash, pruritus, thrombophlebitis, bronchospasm, dyspnoea, decreased blood pressure, and fever 9 . The symptoms listed above can be seen with all echinocandins, but the incidence varies depending on the drug administered. Fever is a common side effect reported in approximately 35% of caspofungin-treated patients, whereas it is reported in only 1% of micafungin-treated patients. To reduce side effects, the rate of antibiotic infusion may be reduced 36,123 . Common disordered effects such as nausea, vomiting, and diarrhoea occur in 7% of patients, and 3-25% of patients treated with caspofungin are diagnosed with phlebitis. Less than 2% of patients experience these complaints after treatment with anidulafungin and micafungin 14 . Caspofungin shows a higher frequency of liver dysfunction (1-15%) compared to other echinocandins. Micafungin may increase risk of liver cancer 72 . Complications such as anaemia, neutropenia, leukopoenia, and thrombocytopenia comprise less than 10% of all adverse effects of echinocandins 9 . Echinocandins should be avoided during pregnancy because they exhibit embryotoxic and teratogenic effects 124 .

Antiviral activity of echinocandins
Studies on micafungin have demonstrated antiviral activity against enterovirus 71 (EV71), chikungunya virus (CHIKV) and dengue virus serotype 2 (DENV-2) [125][126][127] . For the first time, micafungin as an antiviral agent was used against the proliferation and replication of enterovirus 71 (EV71) replicons with an IC50 (concentration of drug that reduce viral activity by 50%) value of 6.35 mg/ml 126 . Micafungin also has antiviral activity against other enteroviruses such as group B coxsackievirus type 3 (CVB3) and human rhinovirus (HRV). Studies on the effect of micafungin against CHIKV have provided information on the binding of the antibiotic to the virus envelope proteins. Micafungin may affect the later stages of viral infection. The drug reduces the cytopathic effect of the virus, its replication and impairs cell-to-cell transmission. The IC50 value of micafungin against CHIKV S27 ranged from 17.2-20.63 mM, which was higher than the IC50 value against EV71. Micafungin is also effective against other alphaviruses such as SINV and SFV 127 .
Recently, a pioneering study on the action of micafungin against dengue virus serotype 2 (DENV-2) has emerged. It is speculated that the mechanism of antiviral action of micafungin may be related to the degradation of the virion by binding the antibiotic to the envelope protein of DENV-2. The action of micafungin on the dengue virus results in the reduction of viral RNA levels. Micafungin mainly acts during the first stages of dengue virus infection. It inhibits DENV-2 binding and entry at doses ranging from 12.5-100 mM. Moreover, micafungin showed activity against other serotypes of the dengue virus (DENV-1, DENV-3 and DENV-4). Other echinocandins have also been shown to act on the dengue virus. Micafungin has the ability to suppress infections caused by arboviruses (CHIKV and DENV). The data presented by Chen et al. 2021 in the in vitro cellular model cannot be directly applied in clinical practice at this time therefore further studies require in vivo testing 125 . Resistance of fungi to echinocandins and "eaglelike effect" Fungi possess adaptive mechanisms by which they compete with other microorganisms for the resources of the ecological niche they occupy. These adaptations can result in increased resistance to antifungal drugs 128 . Multidrug-resistant Candida species have spread worldwide, which in the future may affect methods used to treat infections 129 . Acquired resistance to echinocandins of various strains of C. albicans, C. dubliniensis, C. kefyr, C. glabrata, C. krusei, C. tropicalis and C. lusitaniae has been increasingly described. It is speculated that prolonged or repeated exposure to these antibiotics is a major factor in the acquisition of resistance by Candida spp. to echinocandins 130 .
In response to echinocandins, fungi activate adaptive mechanisms that induce cell wall repair. Signals of cell wall instability are transmitted to the Rho1 subunit of b-(1,3)-D-glucan synthase, whose function is to control glucan synthase and coordinate the activity of protein kinase C (PKC) 131 . PKC controls the activity of other proteins responsible for maintaining the integrity of the fungal cell by synthesising a compensatory cell wall, which is mainly composed of chitin ( Figure 14) 132 . Increased levels of chitin synthesis in response to echinocandins can also be controlled by the mitogen-activated protein kinase (MAPK), high-osmolarity glycerol response (HOG), and calcineurin pathways 46 . Calcineurin, upon calcium activation, causes dephosphorylation of the protein transcription factor Crz1 (Calcineurin-Responsive Zinc Finger), which, upon translocation to the cell nucleus, induces FKS2 expression by binding to calcium-dependent response elements (CDREs) in promoter sequences 46,131 .
Many studies on echinocandin resistance involve mutations in the catalytic subunit of glucan synthase (Fks). Three genes encoding this subunit are known: FKS1, FKS2, and FKS3. Variations in the gene encoding the Fks3 subunit affect the enzyme activity more weakly compared to the other genes in this group and therefore are not essential for the development of drug resistance 133 . Most Candida species acquire resistance to echinocandins through mutations in the Fks1 subunit gene, but in C. glabrata, resistancecausing mutations can also occur in the FKS2 gene 134 . Acquired changes in the structure of the Fks subunit are mostly amino acid substitutions, but deletions and alternative "stop" codons are also found in C. glabrata 130 . Mutations that determine fungal resistance to echinocandins are located in two highly conserved regions of genes encoding the Fks subunit of glucan synthase called "hot spots" (HS). The point mutations are grouped into two "hot spot" regions: HS1 encoding amino acids at positions 641-649 of the enzyme and HS2 responsible for encoding amino acid residues 1345-1365 in the Fks1 protein in Candida albicans and homologous regions of Fks2 in C. glabrata. The FKS2 "hot spot" mutation was also detected in echinocandin-resistant S. cerevisiae strains 133 . All members of the C. parapsilosis group (C. parapsilosis, C. orthopsilosis, and C. metapsilosis) have a natural DNA polymorphism at position Pro 649 of glucan synthase, resulting in a proline substitution by alanine, making them less susceptible to caspofungin. In C. guilliermondii these mutations are located at Met 663 and Ala 634 and serve similar results 135 . Glucan synthase gene sequence changes associated with the phenotype with the strongest resistance to echinocandins occur at positions encoding Ser 645 and Phe 641 and account for 80% of all mutations detected in C. albicans in the Fks1 subunit 136 . The most common and strongest phenotype substitution involves a change in serine at position 645 to phenylalanine, proline, or tyrosine 129 . The Ser 663 change in the Fks2 subunit in C. glabrata that is equivalent to Ser 645 in C. albicans is the most relevant amino acid substitution from the perspective of acquiring echinocandin resistance in this species 137 . Mutations in A. fumigatus occur in the AfFKS1 gene of glucan synthase, which, along with increased chitin production, develops resistance of Aspergillus to echinocandins 138 .
Single amino acid substitutions in the glucan synthase sequence in various fungi reduce drug sensitivity by 50-3000-fold and increase MIC values by 5-100-fold. Amino acid substitutions in C. albicans Fks1 resulted in altered cell wall morphology 139 . C. albicans strains with a homozygous FKS1 "hot spot" mutation exhibit thicker cell walls, which may be partially attributed to a compensatory increase in wall chitin content. Mutants with high chitin content in the cell wall show reduced growth rates in a liquid medium and impaired ability to transform blastospores into hyphae 140 . The increase in MIC values for echinocandins depends on the position and specific amino acid substitution in the glucan synthase structure. The most significant increase in MIC values was found for changes involving the first and fifth amino acids (phenylalanine and serine, respectively) in the "hot spot 1" regions of the genes encoding FKS1 or FKS2. In most cases, FKS sequence changes cause cross-resistance to all echinocandins 130 . Resistance to echinocandins can change with the level of FKS gene expression in C. albicans 133,140 . FKS2 expression in C. glabrata is calcineurin-dependent, meaning that echinocandin resistance can be reduced by including calcineurin inhibitors FK506 in treatment 141 .
The observation of the specific strain of C. albicans which was able to grow in the presence of very high concentrations of caspofungin, significantly exceeding the MIC values, prompted attempts to explain this phenomenon 142 . It was found that a similar phenomenon can also be observed for other species such as C. parapsilosis, C. glabrata, C. tropicalis and C. krusei, as well as for other antibiotics from the echinocandins 143 . This paradoxical effect of fungal growth at very high concentrations of echinocandins is referred to as "Eagle-like effect" and is defined as the reduced activity of echinocandins against fungi when exposed to a dose well above the MIC. This effect is suspected to be due to adaptation as a result of stimulation of calcineurin pathways and chitin synthesis to maintain cell wall integrity 144 . Another hypothesis is the overproduction of polysaccharides that may complement b-(1,3)-D glucan deficiencies in the cell wall. "Eagle-like effect" is often observed in Aspergillus species (e.g. A. fumigatus) during exposure to high concentrations of caspofungin (above 1 mg/l) 143 . The described effect was found in vitro and during clinical caspofungin treatment of patients with invasive pulmonary aspergillosis 67 . The study showed that high doses of caspofungin on the order of 150 mg per day did not significantly improve treatment outcomes compared to patients treated with standard therapy (50 mg caspofungin per day). In addition, in cases of infections caused by C. glabrata and C. tropicalis, more failed treatments were reported at the 150 mg daily dose of caspofungin compared to the 50 mg daily dose, which may confirm the occurrence of the "Eagle-like effect" in clinical practice 145 .

Conclusion
Echinocandins have become a good alternative to azoles and polyenes in the treatment of severe fungal infections caused in particular by Candida and Aspergillus species due to their unique mechanism of action and relatively mild side effects compared to other antifungal drugs. These antibiotics block the synthesis of one of the main components of the fungal cell wall (b-(1,3)-D-glucan) by binding to the Fks subunit of b-(1,3)-D-glucan synthase. Their action results in cell wall defects and the death of fungal cells. The metabolic pathway that echinocandins targets does not occur in human cells, which limits side effects. They are also administered to patients with a weakened immune system, e.g. people suffering from AIDS, cancer, neutropenia, as well as to transplant patients who are particularly at risk of fungal infections due to immunosuppressive therapy. On the other hand, echinocandins are administered only intravenously, which limits the treatment process only to hospital conditions. They are also embryotoxic so they cannot be administered to pregnant women. They are also not effective in the treatment of fungal infections caused by species with limited content of b-(1,3)-D-glucan in the cell wall.
However, taking into account the limited number of available antifungal agents and the characteristic chemical structure of echinocandins, the road opens up to various structural modifications, and thus to obtain new and effective drugs. An example is rezafungin, which is currently in Phase III clinical trials. Due to its improved pharmacokinetics as well as better stability in solutions, it is a good example of the development of this class of antibiotics. Despite the relatively short history of medical use, echinocandins have been approved by the FDA and EMA as first-line drugs for oesophageal candidiasis, invasive candidemia and the prevention of mycoses in transplant patients, which undoubtedly confirms their high usefulness in the fight against mycoses. The cost of one dose of the drug varies between ± $50 (caspofungin) and ± $100 (anidulafungin, micafungin), which makes the therapy relatively expensive, but the increasing use of these drugs as well as the introduction of new preparations from this class may reduce the cost of therapy in the near future. Fungal resistance to echinocandins is found relatively rarely compared to other antifungal antibiotics, yet we already know the basic mechanisms of this phenomenon, which may help in the work on the synthesis of new representatives of more effective echinocandins.
Summarising this basic review of information on echinocandins, it can be stated that despite the relatively short period of their use, these drugs have already found a significant place in clinical practice and are now a very promising and developing class of antifungal antibiotics.