Approximately half of the drugs included in Appendix № 9 belong to the Antineoplastic agents (37 drugs). They refer to the following groups of antineoplastic agents: alkylating agents, antimetabolites, plant alkaloids, cytotoxic antibiotics and other antineoplastic agents. These drugs have different mechanism of action and therapeutic indications, but most of them have serious adverse reactions that may be even life-threatening in some patients. This is due to the fact that antineoplastic agents are toxic to cancer cells as well as to the normal cells (Colvin 2003).

In Table 2 are shown alkylating agents that are listed in the Appendix. The mechanism of action of alkylating agents is based on a covalent interaction between an alkyl group of the drug and DNA (usually the N7 position of guanine). This result in cross-linking of double-stranded DNA and inhibition of DNA replication in the tumor cells. However, alkylating agents also lead to DNA damage in the normal cells that divide frequently and this is the reason for their cytotoxic effects to the gastrointestinal tract, bone marrow, testicles, ovaries and other tissues. The nitrogen mustard analogues are the most frequently used alkylating agents. Some of the alkylating agents require activation by a microsomal P450 enzyme or undergo microsomal metabolism. As a result, drug interactions with CYP450 substrates may occur leading to an increased risk of toxic effects (Colvin 2003). Another alkylating agent included in Appendix № 9 is mitolactol (Dibromodulcitol; DBD) but it doesn‘t have an ATC code. It’s a conformational isomer of mitobronitol (Dibromomannitol; DBM) which refers to the group of other alkylating agents. Mitolactol is under clinical investigation for treatment of different types of cancer (Simonetti et al. 2014; Jeney et al. 2017).

Antimetabolites are anticancer drugs that substitute the actual metabolites in the DNA synthesis and thus inhibit DNA replication, cancer cell growth and survival. Antimetabolite agents are folic acid analogues, purine analogues and pyrimidine analogues. Methotrexate is folic acid analogue that competitively inhibits dihydrofolate reductase, an essential enzyme in the synthesis of tetrahydrofolic acid (THFA). The latter is the active form of folic acid in humans and is needed as a cofactor in the synthesis of thymidylate, purine nucleotides and several amino acids. Therefore, reduced production of THFA by methotrexate leads to an inhibition of DNA/RNA and protein synthesis in cancer cells (Howard et al. 2016). Purine and pyrimidine antimetabolites diffuse into cells and are metabolized respectively into purine and pyrimidine analogues that inhibit enzymes essential for DNA synthesis. Therefore, purine and pyrimidine analogues inhibit DNA replication, resulting in DNA damage and cell death (Parker 2009). Purine analogues have also an immunosuppressive effect and are used in the treatment of autoimmune diseases (LiverTox 2014). Antimetabolites listed in Appendix № 9 are presented in Table 3.

Some plant alkaloids and other natural products have also significant anticancer effect in different types of tumors. Anticancer drugs of plant origin included in Appendix № 9 belong to the class of Vinca alkaloids, Podophyllotoxin derivatives, Taxanes and Topoisomerase 1 (TOP1) inhibitors (Table 4). Vinca alkaloids are derived from Catharanthus roseus and other plants of the genus Vinca. Natural vinca alkaloids and their analogues are cell cycle–specific cytotoxic drugs. They bind to tubulin dimers and inhibit their polymerization and microtubules formation thus block cancer cell growth and division (Martino et al. 2018; Madsen et al. 2019). Etoposide and teniposide are semisynthetic derivatives of podophyllotoxin, the active ingredient of the resin Podophyllin, derived from the rhizomes of Podophyllum peltatum. They inhibit topoisomerase II enzyme that is essential for DNA replication, resulting in single- and double-strand breaks in DNA and promotes apoptosis (LiverTox 2020). Topoisomerase I inhibitors are also used as anticancer drugs. Member of this group is irinotecan that is a semisynthetic derivative of camptothecin, a cytotoxic alkaloid extracted from the plant Camptotheca acuminata. Irinotecan binds to the Topoisomerase I – DNA complex, leading to DNA damage, inhibition of DNA replication and cell death (de Man et al. 2018; LiverTox 2020). Taxanes are natural antineoplastic drugs extracted from the bark of Taxus brevifolia or their semisynthetic derivatives. They bind to tubulin, stabilize the microtubules and block their disassembly. In result, taxanes stop the cell cycle and induce apoptosis (Farrar and Jacobs 2023).

The most widely used cytotoxic antibiotics are anthracyclines and their derivatives. Cytotoxic antibiotics included in the Appendix are shown on Table 5. Anthracyclines are natural compounds, produced by different strains of Streptomyces bacterium. The mechanism of action of anthracyclines is not fully understood. However, two main mechanisms of anthracyclines cytotoxic activity have been proposed. These are formation and accumulation of reactive oxygen species that cause oxidative stress and DNA intercalation and inhibition of Topoisomerase II isoenzymes, leading to double-strand breaks in DNA. In result, anthracyclines cause DNA damage, inhibit DNA and RNA synthesis and initiate programmed cell death (McGowan et al. 2017; Radeva-Ilieva et al. 2020). Bleomycin and mitomycin are the other cytotoxic antibiotics, included in Appendix № 9. Bleomycin is a member of the family of natural glycopeptide antibiotics (bleomycins), produced by the Gram-positive bacteria Streptomyces verticillus while mitomycin is a member of mitomycins, aziridine-containing natural antibiotics isolated from Streptomyces caespitosus. The mode of action of bleomycin is associated mostly with signle- stranded DNA breaks and inhibition of DNA replication. The anticancer activity of mitomycin is due to induction of DNA cross-linking and blockade of DNA synthesis (LiverTox 2017; Sinawe and Casadesus 2022).

Other antineoplastic agents include platinum compounds, methylhydrazines and drugs with different structure (Table 6). The platinum anticancer drugs contain a platinum ion (Pt) linked to different ligands (-Cl, -NH3 or other). Their mechanism of action is similar to that of alkylating agents. They form covalent cross-links in DNA that prevent DNA replication and cause cell death. Procarbazine is a member of Methylhydrazines that acts like the alkylating agents (Colvin 2003). Amsacrine is an inhibitor of Topoisomerase II enzyme, that cause double-strand breaks in DNA, similar to anthracyclines (Cassileth and Gale 1986). Asparaginase metabolizes the amino acid asparagine that plays an important role in proteins biosynthesis. Unlike normal cells, some tumor cells have a limited ability to synthesize asparagine, which is why it diffuses from the extracellular fluid. Asparaginase treatment leads to reduced asparagine levels in serum, resulting in inhibition of protein synthesis in cancer cells and induction of apoptosis (Lopes et al. 2017). Hydroxycarbamide, also known as hydroxyurea, blocks ribonucleotide reductase, an enzyme involved in deoxyribonucleotides production. Thus, it inhibits DNA synthesis in cancer cells (Jinna and Khandhar 2022). Estramustine is used as estramustine phosphate which is dephosphorylated in the gastrointestinal tract. Estramustine is a normustine ester of estradiol that acts as an agonist of the estrogen receptors and leads to suppression of androgens production, such as testosterone. In addition, еstramustine phosphate has a direct cytotoxic effect due to tubulin and microtubule-associated proteins binding and induction of microtubules depolymerization. The result is inhibition of mitosis and initiation of apoptosis in tumor cells (Qin et al. 2016).

In Tables 7, 8 is summarized information about the abovementioned anticancer drugs that are authorized in Bulgaria and their brand names. Defined daily doses (DDDs) have not been established in this group because of highly individualized use and wide dosage ranges.

Immunosuppressants included in Appendix № 9 are ciclosporin and azathioprine. Ciclosporin is a calcineurin inhibitor (L04AD01, DDD = 0.25 g O/P; S01XA18), while azathioprine (L04AX01, DDD = 0.15 g O/P) refers to the group of other immunosuppressants (WHO, Calcineurin inhibitors; WHO, Other immunosuppressants). Ciclosporin blocks the activity of calcineurin, a protein which activates the T cells of the immune system and stimulates the production of cytokines. Azathioprine is a purine analog and inhibits purine synthesis, leading to production of less DNA and RNA for the synthesis of white blood cells. In result, both drugs lead to immunosuppression and are used in transplanted patients to prevent rejection and to treat autoimmune diseases (rheumatoid arthritis, granulomatosis, Crohn’s disease, ulcerative colitis and systemic lupus erythematosus). Ciclosporin is also authorized for use in the European Union to treat severe vernal keratoconjunctivitis (VKC) and severe keratitis in adult patients with dry eye disease. In these cases, it is applied locally in the eye. The most serious side effects of these immunosuppressants are bone marrow suppression, anemia, an increased risk of infection and lymphoma, kidney toxicity is characteristic for ciclosporin while azathioprine can lead to severe hepatic impairment (Mohammadi and Kassim 2023; Tapia et al. 2023). Ciclosporin is metabolized extensively in the liver mainly by CYP3A4 enzyme and inhibits the activity of CYP3A4 and P- glycoprotein. Thus, there is an increased risk of drug interactions if taken together with other drugs especially CYP3A4 substrates (Tapia et al. 2023). In Bulgaria ciclosporin is registered under the brand name Sandimmun Neoral (soft capsules, 25, 50 and 100 mg and oral solution, 100 mg/ml) and azathioprine is sold under the brand name Imuran 50 mg film-coated tablets (BDA, Register of pharmaceutical products).

In the present study, we analyzed and summarized the available information about the anticancer drugs and immunosuppressants included in Appendix № 9 to Art. 17, para. 1 of Ordinance № 28/9.12.2008, issued by the Minister of Health. In result, we established that some of them are not registered for use by the Bulgarian Drug Agency (BDA) to date and are not used in clinical practice in Bulgaria. Furthermore, there is a number of antineoplastic agents that have a marketing authorization granted by the BDA or are authorized for use under a centralized procedure under Regulation (EC) №726 / 2004 of the European Parliament and of the Council of 31 March 2004 but are not included in Appendix № 9. An example is the anticancer agent capecitabine, a pro-drug of 5-fluorouracil (5-FU), that is widely used to treat colorectal carcinoma. Capecitabine is a pyrimidine analogue, such as fluorouracil but unlike it capecitabine is not included in Appendix № 9 although it is metabolized in the body to 5-FU (Stoeva et al. 2020). More examples are given in Table 9.

It remains unclear why these anticancer drugs and immunosuppressants shown in table 8 are not included in the Appendix. Moreover, in Appendix № 9 is not included any representative of Protein kinase inhibitors (ATC code: L01E), Monoclonal antibodies and antibody drug conjugates (ATC code: L01F) as well as other antineoplastic agents (ATC code: L01X) that are the main drugs in the targeted cancer therapy and are widely used in recent years. In addition, many protein kinase inhibitors as well as monoclonal antibodies have marketing authorization in Bulgaria via the Centralized procedure pursue to Regulation (EC) No 726/2004 of the European Parliament and of the Council of 31 March 2004 (BDA, Register of pharmaceutical products).