Non-Targeted Therapies

We give hereafter the used non-targeted therapies. They act in different phases of the cell cycle and all have adverse effects that we will detail later.

1. Alkylating and related agents. The alkylating and related agents have a chemical group able to form covalent bonds with the nucleic acids of the DNA chain of the cell and thus inhibit replication. More exactly, the formation of these links entails DNA transcription disordes and replication disorders, base substitutions in DNA and excision of bases and catenary ruptures of DNA. Their main pharmacological activity is manifested during the synthesis phase of DNA.

Indeed, the first metabolic step of alkylating and related agents is the formation of a carbonium ion, a carbon atom comprising only 6 electrons in its outer layer. This ion is extremely reactive and reacts instantaneously with any neighboring electron donor, such as an amine group, -OH or SH. Most alkylating agents used in therapeutics are bifunctional (they possess two alkyl groups) which can bind two adjacent nucleotides. Guanine nitrogen N7 is probably the main molecular target of alkylation, but also N1 and N3 of adenine or N3 of cytosine may be involved. A bifunctional agent is able to react with two nucleotides, it will form intra- or inter-catenary bridges, making DNA transcription or replication impossible. Moreover, the N7 alkylation of the guanine leads to its excision, and thus to the disruption of the DNA chain. In the case of excised guanine repair, the guanine-cytosine pair may be replaced by the adenosine-thymine (mutation) pair.

Alkylation occurs essentially at the time of DNA replication (S phase) when the two strands are separated and thus expose the sites of interest to the alkylating action. The blockade of mitosis in G2 phase leads to the death of the cell. All alkylating agents are myelosuppressive and cause gastrointestinal disorders (diarrhea, nausea, vomiting). After prolonged use, there are gametogenesis disorders and permanent sterility in humans, and an increased risk of acute non-lymphoblastic leukemia. This pharmacological class comprises a very large number of substances used in therapeutics. The most commonly used are presented hereafter.

• Nitrogen mustards. They are all chemically related to the mustard gas used as a com-bat gas during the First World War. Their basic formula is R-N-bis (2-chloroethyl).

In the body, each of the 2-chloroethyl side chains is cyclized with chlore ion release.

The ethylene-ammonium derivative thus formed is highly reactive and can react with DNA or other molecules. The most commonly used alkylating agents are CYCLOPHOS-PHAMIDE (Endoxan) and IFOSFAMIDE (Holoxan). CYCLOPHOSCYCLOPHOS-PHAMIDE is me-tabolized by hepatic cytochrome P450 into two metabolites, one active: phosphoramid-ium mustard, the other one is inactive, the acrolein responsible for an important side effect: hemorrhagic cystitis. This adverse effect can be prevented by increasing diuresis and administration of mesna which antagonizes the effects of acrolein. ISOFOSFAMIDE

is metabolized by hepatic cytochrome P450 into isophosphoramidated mustard. The CY-CLOPHOSPHAMIDE is myelotoxic, relatively more for lymphocyte function. In this respect, it is sometimes used as an immunosuppressant. CYCLOPHOSPHAMIDE may be administered orally, but also parenterally. The main adverse effects include Hemor-rhagic cystitis, myelosuppression, and nausea/vomiting. The chemotherapy with Nitrogen mustards is used for many kind of cancers.

• Aziridines or ethylene imines as THIOTHEPA, MITOMYCINE C and tetrazines as DACARBAZINE (D´etic`ene) and PROCABAZINE (Natulan). They are all used for Hodg-kin’s disease and melanoma.

• Nitrosoureas=the mustines. BCNU, CCNU, FOTEMUSTINE (Muphoran). They are active on a large number of tumors and they need to be metabolized in order to be active (non-enzymatic mechanism), by forming alkylating derivatives. Other metabolites such as organic isocyanates are able to interact with the proteins and can inactivate DNA repair enzymes. They Pass the blood-brain barrier (high fat-solubility), so they are indicated in brain tumors. Nevertheless, the nitrosoureas exhibit a significant myelotoxicity, having two pejorative characteristics, the haematological toxicity is cumulative and is delayed (3 to 6 weeks after start of treatment).

• Platinum salts. The main mechanism of the platine salts action is related to alkylants: for-mation of intra- and inter-strand bridges. Examples of platinum salts are CARBOPLATIN which is used for the treatment of ovarian cancers, small cell lung cancer and cancers of the otolaryngology sphere, OXALIPLATIN which is used for colorectal cancer, and that leads to acute neurological toxicity, and CISPLATIN, which is used for the treat-ment of different cancers such as sarcomas, carcinomas (small cell lung cancer, ovarian cancer, testicular cancer ...), lymphomas. CISPLATIN causes by reaction with the DNA, intracatenary bridges between the adjacent guanine residues. CISPLATIN is administered slowly in intravenous way. After three hours it is concentrated in the kidneys and after 40 hours in the intestine and liver. In plasma it is highly bound to plasma proteins. Its half-life is biphasic with a second phase of several days. It is severely nephrotoxic and re-quires measures of hyperhydration and forced diuresis. It is not very myelosuppressive but causes vomiting of an extreme severity (those may be antagonized by a 5-HT3 inhibitor such as odansetron). CISPLATIN causes peripheral neuropathies, anaphylactic reactions and hyperuricemia. It is a product that has modified the prognosis of solid tumors of lineages germ cells (testicles and ovaries).

2. Topoisomerase inhibitors=intercalating agents. The topoisomerases are enzymes which en-sure the spiralization/desiccation of DNA after transient breaks of one (topoisomerase 1) or 2 (topoisomerase 2) DNA strands. They then ensure the repair of these cuts, especially at the tip of the chromosomes that alter during successive divisions which leads to apoptosis.

• Those of type 1 are derived from Camptothecin, as IRINOTECAN (Campto) and

TOPOTE-CANT (Hycamtin). The action of anti-topoisomerase type 1 is to prevent the reconstitu-tion of the DNA strand after cleavage, inhibiting the correct synthesis of DNA. They have specific cytotoxic effect in Phase S of the cycle and they have haematological toxicity.

• Those of type 2 are Anthracyclines= the rubicines (DOXORUBICIN, ADRIAMYCIN, EPIRUBICIN), Anthracenediones (MITOXANTRONE=Novantrone) and Epipodophyl-lotoxins (VP-16 = VEPESIDE). The presence of an anti-topoisomerase type 2 leads to the rupture of the strands of the DNA, since they can not be stuck together after the passage of a double strand of the DNA. They prevent reconstitution of DNA strand (re-pair) after cleavage and then lead to the definitive cut of DNA strands and to apoptosis.

They also make troubles in the replication and transcription of DNA in phases G1 and G2. The Antracyclines, but not the Antracenediones, contribute to the formation of free radicals at the origin of membrane lesions (cardiotoxicity of anthracyclines). They fre-quently cause severe alopecia. The Antracyclines have a major biliary elimination, it is necessary to adapt the doses in case of disruption of the hepatic balance. They also have low plasma protein binding The Anthracyclines and in particular the Anthracenediones use the intercalation in the double helix of DNA between 2 adjacent bases: they stabi-lize the double-stranded cuts. The Anthracenediones have leukemogenic potential. The Etoposide (CELLTOP, VEPESIDE) are indicated for lung cancers, testis, breast, placen-tal choriocarcinoma and other kind of cancers. They have strong plasma protein binding (94%.) The perfusion should be slow and under supervision (anaphylactic reaction). They also present cross-resistance with anthracyclines and periwinkle alkaloids. The Etoposide is a plant derivative. It is derived from the root of podophyllum peltatum. Its mode of antimitotic action is not completely determined; however, it inhibits mitochondrial activ-ity and intracellular transport of nucleosides, and interacts with topoisomerase 2. It is administered by oral or intra-veinous way, widely distributed in the body, does not pass the hemato-encephalic barrier and is eliminated mainly by urine. Its adverse effects are mainly myelosuppression, nausea vomiting, and alopecia.

The anthracyclines are also part of cytotoxic antibiotics.

3. Cytotoxic antibiotics. Cytotoxic antibiotics generally produce their antimitotic effects through interactions With DNA.

• Anthracyclines.

• Bleomycins. Bleomycins are metal chelating glycopeptides that degrade the DNA in-ducing the fragmentation of the chain and release of bases (splitting agent, “Chemical scissors”). Their action on DNA is believed to be related to the release of free radicals by chelation of the ferrous ion and then oxidation generating superoxide ions. Bleomycin is active in G2 phase during mitosis but also on cells that are not in division (G0). The product is administered in intravenous, rapidly distributed and renally eliminated with-out metabolization, with a half-life of 2 hours. Bleomycin is one of the rare antimitotics without myelotoxicity. Its most serious adverse effect is irreversible pulmonary fibrosis, occurring in 10 per cent of cases and fatal in one per cent of cases. Immunoallergic reac-tions are also observed, as well as mucocutaneous toxicity and melanodermia.

• Dactinomycin: an antibiotic derived from streptomyces, it is an intercalating agent block-ing the transcription of DNA by disruptblock-ing the movement of RNA polymerase along the DNA chain. Also acts via topoisomerase 2. It is a particularly active product on cancer

cells at fast rate of division. Its adverse effects are representative of those of most an-timitotics. It is administered in intraveinous, rapidly eliminated, and does not pass the blood-brain barrier.

4. Inhibitors of the biosynthesis of nucleic acids = Antimetabolites. They are structural ana-logues of the purine and pyrimidine bases. They block or divert one or more pathways of synthesis of DNA. They then have direct interaction with DNA, so they lead to the inhibition synthesis of DNA.

• The antipurines: Adenine or guanine substitution, as FLUDARABINE (Fludara) and THIOGUANINE (Lanvis). FLUDARABINE Interferes with DNA synthesis by inhibit-ing DNA polymerase and ribonucleotide reductase, this leads to apoptose. It mainly has haematological indications.

• Antipyrimidines: Substitution with cytosine, Thymine or Uracile, as CAPECITABINE (Xeloda), CYTARABINE (ARA-C), 5-FU (Fluoro-uracile). Fluorouracil inhibits the syn-thesis of thymidilates and thus blocks DNA synsyn-thesis. It is metabolized into fluoxuridine by thymidine phosphorylase and then into fluorodeoxyuridine monophosphate under the action of thymidine kinase (5FdUMP). 5FdUMP inhibits the thymidilate synthetase en-zyme essential to the synthesis of pyrimidines. 5FdUMP may also be triphosphorylated and incorrectly incorporated into the DNA chain which deteriorates its metabolism and function. Its plasma half-life is short: 11 min, so the administration is done parenterally by continuous perfusion. Fluorouracil can also be administered orally. It passes well the hemato-encephalic barrier. The main adverse effects are lesions of the digestive tract ep-ithelium and myelosuppression. Neurological disorders with cerebellar involvement can also be observed. The elimination is renal for 5-FU. Another antipyrimidines is GEMC-ITABINE (Gemzar), which is analogue of the pyrimidine bases where 2 fluorine atoms have replaced two hydrogen atoms of deoxyribose. It is metabolized to mono, di and triphosphate derivatives, at the end, it makes perturbation of DNA synthesis. TEGUFAR URACILE (UFT)is also another kind of antipyrimidines.

• The antifolates: substitution of folic acid, The main folic antagonist is METHOTREX-ATE. It is the most common antimetabolite used in cancer chemotherapy. Folates are essential for the synthesis of purines bases and thymidine, which are essential for DNA synthesis and cellular division . Folate to act as a coenzyme must be reduced to tetrahydro-folate by the dihydrotetrahydro-folate reductase. The METHOTREXATE analogous of tetrahydro-folates has an affinity higher for dihydrofolate reductase than the folates themselves. This competition rapidly leads to depletion of tetrahydrofolate in the cell and thus to the interruption of synthesis of DNA.

Other antifolates are RALTITREXED (Tomudex) and PERMETREXED (Alimta.) 5. Tubulo-affine plant derivatives, also called The spindle poisons. Two groups of molecules

are represented in this class, the alkaloid derivatives of vinca rosea (periwinkle of Madagascar) or vinca-alkaloids and the taxanes derived from taxus atlanticus (if). Their common molecular target is the cytoplasmic tubulin whose polymerization is necessary for the construction of the mitotic spindle. They act on the spindle cell by blocking the mitosis and are specific to phase M

“phase dependent”. More precisely, their common targets are the microtubules. The induction of apoptosis is mainly due to mitosis inhibition at metaphase/anaphase transition and cell cycle blocking at the mitosis control point.

• Periwinkle alkaloids as Vinca-alkaloids: VINORELBINE (Navelbine), VINCRISTINE (Oncovin), VINBLASTINE (Velbe). Another Periwinkle alkaloid is VINDESINE (El-disine). The vinca alkaloids block mitosis by inhibiting the polymerization of tubuline.

Their elimination is 90 per cent by biliary route, they are also metabolized by cytochrome P450, this means that there is a high level of risk of interactions. Their half-life is long.

There is also the possibility of treatment per os with vinorelbine for which the bioavail-ability is 50 per cent. Similarly, VINCRISTINE is a derivative of periwinkle, it is an alkaloid that causes a sequestration of tubulin by irreversible crystallization of the intra-cellular tubulin, depleting the cell of tubulin capable of integrating the mitotic spindle.

The product is administered intravenously (strictly intra-veinous way because the product is caustic, and venous rinse because of venous toxicity), it is eliminated mainly by biliary route with a plasma half-life of 2 hours and tissular of 48 hours. The derivatives of the periwinkle are relatively non-toxic, and does not cause myelosuppression. However, they are frequently neurotoxic (sensitivo-motor neuropathy.)

• Taxanes as PACLITAXEL (Taxol), and DOCETAXEL (Taxotere). During anaphase, the chromosomes separate. Microtubules shorten by depolymerization. This process is in-hibited by taxanes. More exactly, the taxanes are microtubule stabilizing agents as they inhibit their depolymerization, the cells are then blocked in metaphase. The taxanes in-duce hepatic metabolism, so it is necessary to adapt the doses or contraindication in case of major disturbances of liver function. In the case where the action of the taxanes, as for Vinca, occurs during mitosis, their action induces apoptosis. When the action is non-mitotic, in the transition from G0 to S or G2 to mitosis, there is an increase in oxidative stress and tubulin involvement in the cell membrane.