This article provides a note on the antimetabolites, explained with the help of suitable diagrams.
Folate: MOA
By structure folate consists of a pteridine nucleus. PABA and glutamic acid. These folates are taken up into cell actively and converted in presence of DHFR from FH2 into FH4.
FH4 acts as a cofactor in the transfer of single carbon fragment via 5,10- methylene FH4 to 2-de-oxy-uridylate (dUMP), which under the catalysis of thymidylate synthetase forms 2-deoxythymidylate (DTMP) and FH2 is recirculated. Inhibitor like methotrexate (structural analogue of folate) is actively taken up like folate in cells and is converted to polyglutamate.
Methotrexate has high affinity (50000 times than folate) for DHFR, thus DHFR is restricted to act and form FH4, producing acute intracellular deficiency of FH4 and toxic accumulation of FH2 polyglutamate.
In addition, methotrexate polyglutamate inhibit DHFR and thymidylate synthetase. These poly-glutamates remain entrapped in cells for longer due to its larger size and greater -ve charge even in absence of the extra cellular methotrexate.
The one carbon transfer reaction is equally critical for de novo synthesis of purine nucleotide and thus DNA and RNA synthesis is interrupted by methotrexate. Rapidly proliferating (S phase) cells as neoplasm, GIT epithelium, fetal cells are more sensitive (due to inhibition of DNA synthesis).
Rescue of Non Malignant Cells:
Leucovorin (Folinic acid), carry a form of FH4 which bypasses the blocked enzyme and replenishes the folic acid pool, and thus facilitate normal cells to form thymidylate even in presence of Methotrexate. Albeit tumor cells seem to be deficient in transport of folinic acid, remains starved of folinic acid.
Resistance to Anti-folate:
1. Impaired active transport of methotrexate into cells.
2. Altered DHFR, with reduced affinity for methotrexate.
3. Increased concentration of DHFR in cells due to altered gene.
4. Little synthesis of methotrexate polyglutamate.
5. Reduced thymidylate synthase activity.
Salicylate, phenylbutazone, Sulfonamides, dicumarol displaces it (methotrexate) from protein binding sites and aspirin & sulfonamide reduces its renal tubular secretion, thus enhances its toxicity.
Methotrexate has been used for treatment of lymphoreticular neoplasia, myeloproliferative disorders and other carcinomas & sarcomas. It is useful as an immunosuppressive agent in the management of rheumatoid arthritis @ 2.5 mg/m2 PO daily for 5 day/week 2 day gap or 10-25mg iv wkly. Methotrexate metabolites 7-OH metholrexate is less water soluble, may accumulate in kidney causes crystal urea & nephrotoxicity.
Purine Antagonist:
Mercaptopurine is a structural analogue of natural purine (hypoxanthine) and is highly effective antineoplastic agent. After entry into cells, mercaptopurine is converted by hypoxanthine guanine phosphoribosyl transferase (HGPRT) into 6- mercaptopurine ribose phosphate (6MPRP) also known as 6-inosinic acid or 6 thio MP, which is later dehydrogenated to 6 thio GTP (fradulent nucleotide) and incorporated into RNA.
The deoxyribonucleotide analogue are also formed, gets imbedded into DNA. These lead to synthesis of dysfunctional RNA & DNA which ultimately inhibits cell division and may lead to cell death (Apoptosis).
Deficiency of HGPRT may confer resistance to tumor cells.
Azathioprine (6MP derivative) is metabolized by xanthine oxidase. So concomitant administration of xanthine oxidase inhibitor (Allopurinol) and 6 MP/Azathioprine require reduction of dose of 6 MP and Azathioprine up to 1 /4 to 1/2.
Commonly utilized against lymphoreticular tumors @ 50mg/m2 PO daily.
Thioguanine/6TG is a structural analogue of guanine, has similar mode of action as 6MP but undergo different metabolic pathway, used against acute lymphocytic leukemia @ 50 mg /m2 PO every 1-2 day & 1-2mg/kg bwt too.
Other purine antagonists are pentostatin, Cladribine. fludarabine etc.
Pentostatin:
A transition state analogue of the intermediate stage in adenosine deaminase reaction utilized in leukemia.
Cladribine:
An adenosine deaminase resistant purine analogue, has potent activity against hairy cell leukemia, lymphocytic leukemia etc.
Pyrimidine Analogue:
5-Fluorouracil and Floxuridine (deoxy analogue 5 FU) 5-FU requires enzymatic activation (ribosylation and phosphorylation).
It get converted to concerned deoxynucleotide, 5-fluoro-2-deoxyuridine-5 monophosphate (FdUMP; FRADULENT NUCLEOTIDE) in the target tumor cells.
Absorption of FdUMP thus GIT is erratic & variable but after absorption, distributed widely (except CSF & Brain). Vd is good even in CNS when administered intravenously.
This FdUMP inhibits thymidylate synthetase, affects DNA synthesis and forms dysfunctional nucleic acid (Fig. 37.7).
It affects all phases but preferably S and G1.
5 FU is used in various carcinoma and sarcoma @ 200mg/m2 i.v. weekly.
ADR – Severe ulceration of oral and GI mucosa in human & dog.
C/I – Severe neurotoxicity in cats.
Cytarabine/Ara C/Cytosine Arabinside (1 -β-D- arabinofuranocylecytocytosine):
It is an analogue of natural nucleoside 2-deoxy cytidine. Enter into cell through similar carrier (as natural nucleosides) Ara C undergo phosphorylation (activation) in cells and form
These phosphorylated Ara C competes with deoxycytidine 5′ triphosphate (dCTP), gets incorporated into DNA by DNA polymerase.
Incorporated Ara C (in DNA) is potent inhibitor of DNA polymerase.
Substituted DNA chain fails to elongate, replicate and or repair, may even lead to its fragmentation followed by apoptosis. It interferes with late S phase of cycle.
In addition Ara C alters membrane structure function and antigenicity by:
1. Its conversion to Ara CDP choline thus synthesis of altered membrane glycoproteins and glycolipids.
2. Inhibits transfer of galactose, sialic acid and N-acetyglucosamine to cell surface glycoproteins.
3. Inhibits the synthesis of CMP acetyl muramic acid
Resistance may be due to:
1. Increased concentration of 2- deoxycytidine nucleoside.
2. Change in phosphorylating enzyme.
3. Enhanced biotransformation to inactive metabolite.
Use- Lymphoreticular neoplasia and lymphosarcoma in canine and feline @ 100 mg/m2 i.v. daily for 4 days repeat cycle at 3 week interval.
Gemcitabine/2, 2 di-fluoro-deoxycytidine/dFdC:
It has similar activity as cytarabine with little difference i.e.:
1. Penetrate via active nucleoside transfer into cells.
2. The cytotoxicity is not confined to S phase, rather equally effective against confluent cells and cells in log phase growth.
3. The phosphorylated gemcitabine dFdCTP competes with dCTP which may terminate the DNA strand, as well as inhibits DNA polymerase.
4. dFdCDP is a potent ribonucleotide reductase inhibitor which facilitate depletion of ribonucleotide pool necessary for DNA synthesis.
Used in human metastatic pancreatic cancer, non small cell lung cancer, breast cancer, ovarian cancer and cervical cancer etc. @ 1000 to 1200 mg/m2 for 30 minutes infusion on 1st, 8th and 15th day for each month /28 days.
Mitosis Inhibitor:
Vinca alkaloids- Vincristine, vinblastine, vinleurosine, vinrosidine
Source – Vinca rosea/Catheranthus roseus.
MOA:
1. Vinca alkaloids bind specifically to tubulin → block the ability of tubulin protein to polymerize into microtubules → disrupts mitotic apparatus i.e. spindle formation (known as spindle poison) chromosome fails to segregate, metaphase gets arrested.
2. Failure of chromosome segregation chromosome disperses in cytoplasm – called exploded mitosis → apoptosis.
3. Unlike vincristine, vinblastine also blocks purine synthesis and citric acid cycle in tumor cells other phases of cell cycle are also affected.
4. Due to interference with tubulin function, vinca alkaloids interfere with phagocytosis, chemo taxis and axonal transport in neurons.
Resistance – May be due to:
1. Altered tubulin, escape binding with vinca alkaloids.
2. Greater production of P-glycoprotein → more efflux of drugs (Ca++ channel blocker: verapamil can reverse it).
Toxicity:
Myelosupression and neurological toxicity.
Vincristine is preferred over vinblastine usually for lymphoproliferative disorder, transmissible venereal tumor and solid carcinoma in dogs and cats @ 0.3 to 0.75 mg/ m2 i.v. each week/fortnight. Vinblastine is used @ 2-2.5 mg/m2 i.v. wkly/fortnightly. Other two i.e. vinleurosine and Vinrosidine are of human oncology interest.
Taxanes:
It includes Paclitaxel and its structural analogue-Docetaxel. These are complex semi-synthetic diterpene synthesized from bark of western yew tree.
MOA:
Taxanes binds to b-tubulin, promotes polymerization and stabilizes microtubules, following which de-polymerization is prevented, leads to inhibition of normal dynamic reorganization of microtubule network. Thus blocks the vital interphase and mitotic function. Abnormal arrays of bundles disperse in cells. Docetaxel has similar MOA and indications.
Indication:
Ovarian and metastatic breast cancer, small cell lung cancer, esophageal adenocarcinoma, prostrate cancer (hormone refractory) in humans. Yet to be used in animals.
Epipodophyllotoxin:
It includes Etoposide and Teniposide. These are semisynthetic glycosides, contains active principle podophyllotoxin, obtained from Podophyllum peltatum.
MOA:
Etoposide and Teniposide complexes with DNA and Topoisomerase. This leads to double strand DNA break. Teniposide and Etoposide prevents Topoisomerase II binding to DNA. Thus usual resealing of broken DNA is inhibited leads to accumulation of DNA fragment followed by apoptosis. S and G phase of cell cycle is most vulnerable to Teniposide/Etoposide. Used in human testicular neoplasia.
Resistance mode:
1. P- glycoprotein mediated efflux due to over expression of mdr gene.
2. Reduce expression of Topoisomerase II.
3. Mutation of p53 tumor suppressor gene.
Topotecan and Irinotecan:
These are semi-synthetic less toxic analogues of campothecins, isolted from Camptotheca acuminata a Chinese tree in 1966.
Irinotecan/CPT-2 is a pro-drug, yields SN-38, and is 1000 times more potent metabolite.
MOA:
Topoisomerase I & II are enzymes which engineers nicking and resealing of DNA strands. Unlike Topoisomerase II (which nicks both strands of DNA) Topoisomerase I nicks single strand of DNA, forms a cleavable complex and relaxes torsion of DNA. This (Topoisomerase I) mediates DNA replication, recombination, repair and transcription, followed by religation/resealing.
Camptothecins gets hooked (reversibly) to DNA Topoisomerase I cleavable complex. This impairs religation/resealing (nicking is not affected) leads to accumulation of single strand breaks (S phase) in DNA. This can be reversed by camptothecins removal.
But longer exposure of camptothecins mediated single strand break ultimately causes irreversible double strand break followed by apoptosis. Camptothecin has been observed to arrest G2 phase. These analogues have been used in colorectal, ovarian and small cell lung cancer in human.
Dactinomycin/Actinomycin D:
It is one of antitumor chroma-peptides (yellow-red) antibiotic obtained from Streptomyces spp.
MOA:
It intercalates between Guanine-Cytosine base pairs of DNA and forms a stable dactinomycin DNA complex. This complex interferes with transcription of DNA by RNA polymerase. It also produce single strand break in DNA.
Dactinomycin is cell cycle phase non specific drug, it inhibits rapidly proliferating neoplastic as well as normal cells.
Dactinomycin has radiation recall effect i.e. produces inflammation at the site of radiation earlier.
P glycoproteins mediated efflux may confer resistance in cancerous tissues.
It is used as adjunctive therapy for sarcoma of soft and hard tissues, malignant melanoma and lymphoreticular neoplasm in small animals @ 0.5-1 mg/m2, i.v. infusion along with dextrose or NSS weekly.
Anthracycline Antitumor antibiotic:
This antibiotic consists of tetracycline ring attached with daunosamine sugar by glycosidic linkage. This group includes daunorubicin, doxorubicin, idarubicin and epirubicin, obtained from a fungus Streptomyces peucetius var caesius. These are S-phase specific antitumor antibiotics, having multiple mode of action i.e.
1. Non specific intercalation in DNAs’ adjacent base pair thus affect DNA template, thereby affect synthesis of DNA and RNA. It also produce nick in one or both strands of DNA (mediated by anthracycline binding to DNA & Topoisomerases).
2. It contains quinone group which may induce/generate free radical and ultimately oxidize DNA bases.
3. It also bind and interfere with cell membrane transport
Doxorubicin is used in lymphoproliferative and myeloproliferative disorders as well as sarcoma of soft and hard tissues @ 30 mg/m2 slow infusion at every 21 days in dogs and @ 20 mg/m2 slow i.v. once in 21 to 42 days.
Adverse drug reaction:
Anthra cycline has cardio toxic effect specifically related to generation of free radical in presence of iron in heart. Epirubicin has less cardio toxic and myelosuppressive action, although used at similar dose rate for similar indication. Idarubicin can be used orally. Daunorubicin is used in human oncology. Other antibiotics of this group are Adarubicin and Valrubicin. Recent addition similar to this group is Mitoxantrone.
Bleomycin:
It is an amalgamation of metal binding basic glycopeptides: bleomycin A2 and B2 (fermentation product of Streptomyces verticillus.
MOA:
Metallobleomycin complex ultimately (generate free radical) causes oxidative damage to the deoxyribose of thymidylates and other nucleotide leading to single and double stranded break in DNA and seizes the cells in G2 phase of cell cycle (in vitro study) results into fragmentation of chromatids.
The degradation of bleomycin depends upon specific hydrolase enzyme in normal tissues. Lungs and skin has lesser hydrolase activity (therefore indicated as well as prone to ADR). Higher content of hydrolase may render cancer cells resistant. Added DNA reparability may aid to this resistance.
Bleomycin has little myelosuppression but may cause skin ulcers and pulmonary fibrosis. It is indicated for combination chemotherapy in squamous cell cancer and lymphoma in dogs and cats @ 10-20 units/m2 i.v. or s.c. weekly.
Mitomycin:
Obtained from Streptomyces caespitosus contains mitosine, quinone and aziridine ring, produce bi and/or tri functional alkylating agent which inhibits DNA synthesis as well as induce cross linking in DNA at N6 of adenine and O6 & N7 of guanine. Used in combination chemotherapy in humans.
L-Asparaginase:
Kidd observed anti-leukemic activity of L-asparagine (amino acid) in 1953, now a day it is obtained from Escherichia coli.
MOA:
Neoplastic cells lack asparagine (amino acid) thus require it from exogenous sources for certain protein synthesis. L-asparaginase catalyses hydrolysis of circulating asparagines to aspartic acid and ammonia, thus tumor cells is starved off waiting for exogenous asparagine and later undergo apoptosis. This leads to inhibition of protein synthesis. Myelosuppression and alopecia is rare.
In order to observe synergism, combination chemotherapy methotrexate is used earlier followed by L-asparaginase. Reverse order will have no impact of methotrexate due to L-Asparaginase induced protein synthesis inhibition in the neoplasm, used in lymphosarcoma in dogs.
Pesparagine is modified form of L- asparagines
Platinum complexes:
These diffuse into cells gets hydrolyzed → produce activated species → interacts with DNA → forms intra & inter-strand cross link e.g. between N7 guanine (of same DNA) helix and cross link between Guanine-Adenosine, produces bend in DNA helix. Platinum to Guanine-Adenosine cross link is critically cytotoxic. It can interact with thiol group (-SH) in proteins.
It has radiomimetic properties.
Nucleotide excision repair pathway can produce resistance in tumor cells.
E.g. Cisplatin, Carboplatin. Oxaliplatin.
Cisplatin:
Used in metastatic ovarian, testicular and many other carcinoma in dogs. In cats it produces pulmonary edema, hydrothorax and dyspnoea which is lethal @ 60 mg/m2 i.v. over 20-25 minutes once in 20 days with proper hydration before and after cisplatin administration. Carboplatin is safer in cats @ 150 mg/m2 slow i.v. once in a month. In dogs @ 250 mg/m2 slow i.v. once in a month.
Hydroxy urea:
It interferes in reductive conversion of ribonucleotide to deoxyribonucleotide by inhibiting enzyme ribonucleoside diphosphate reductase (RDR), the rate limiting enzyme in DNA biosynthesis. Since concentration of (RDR) is higher in S phase of cell cycle therefore affected to its most. The cell gets seized in Gi-S interface. It also facilitates DNA interfering drugs (Ara C, gemcitabine etc.) incorporation into the DNA.
Indication:
Polycythemia vera, leukemia, mastocytoma in dogs and cats @ 50mg/ kg orally.
Procarbazine:
It is activated in vivo and generate cytotoxic reactant which methylate DNA thus damages chromosomes, used in human oncology.
Mitotane:
It is chemically like DDT used in human oncology in the treatment of adrenocortical cancer.
Future:
Now a days cancer chemotherapy is employed as a part of a multimodality approach changing to the initial treatment of many other tumors. The basic approach to cancer treatment is constantly changing. The clinical protocol are now exploring genetic therapy, manipulation of immune system, stimulation of normal hematopoietic elements, induction of differentiation in tumor tissues and inhibition of angiogenesis.