FOLATE, VITAMIN B12 and INHIBITORS Katzung (10th ed.) Chapter 33, pg. 531-536

CRITICAL FACTS Folate and vitamin B12 are required for DNA synthesis and for maintenance of neurons and red blood cells. FOLATE is a carbon donor in thymidylate and purine synthesis, as well as amino acid metabolism. Vitamin B12 is a critical cofactor in the generation of tetrahydrofolate from liver stores of tetrahydrofolate. Absence of the reduced folate carrier confers resistance to low doses of folate inhibitors (such as METHOTREXATE), but also allows the design of successful high-dose treatment.  Cancer cells or bacteria that do not have this transporter cannot accumulate reduced folates after inhibitor treatment, while normal cells can protect themselves by accumulating other folates (such as LEUCOVORIN).  This is the fundamental principle underlying rescue therapy. FOLIC ACID and VITAMIN B12 are dietary essentials. Deficiency of either one has catastrophic effects on rapidly growing cells and on neurons. The most prominent and earliest clinical sign of deficiency is megaloblastic anemia. TRIMETHOPRIM (a folic acid inhibitor) is typically combined with SULFAMETHOXAZOLE (which blocks dihydropteroate synthetase). These drugs act synergistically, selectively blocking successive steps in the folate synthesis pathway in bacteria.

LECTURE OBJECTIVES Be able to describe the specific functions of folate and vitamin B12, and relate these functions to the inhibition mediated by METHOTREXATE and TRIMETHOPRIM. Explain the mechanism by which the folate transporter is involved in the differential sensitivity of cancer cells and bacteria to folic acid inhibitors. Know the consequences of folate and vitamin B12 deficiency, and strategies for replacement.

Review of Nucleotide Biochemistry FOLATE BIOCHEMISTRY (METHOTREXATE) Tetrahydrofolate is synthesized by two mechanisms: FOLATE is converted to dihydrofolate and dihydrofolate to tetrahydrofolate by dihydrofolate reductase (DHFR). Methyltetrahydrofolate from liver stores is converted to tetrahydrofolate, a reaction that requires VITAMIN B12. Two steps in the conversion of 5-phosphoribosylamine to IMP (purine synthesis) use tetrahydrofolate as a carbon donor. Tetrahydrofolate is also involved in the generation of dTMP from dUMP (pyrimidine synthesis) – this reaction is catalyzed by thymidylate synthase (see 2A below). PYRIMIDINE SYNTHESIS (5-FLUOROURACIL) The rate limiting step in DNA synthesis is the conversion of UMP to TMP, which is catalyzed by thymidylate synthase. Conversion of UMP to UDP is catalyzed by pyrimidine monophosphate kinase; this reaction is important in the development of resistance to 5-FU. One step in the degradation of thymidine nucleotides is catalyzed by dihydropyrimidine dehydrogenase; an inherited deficiency of this enzyme leads to greatly increased sensitivity to 5-FU.

Review of Nucleotide Biochemistry (cont’d) PURINE SYNTHESIS (6-MP, 6-TG, PENTOSTATIN) De novo purine synthesis begins with the conversion of ribose-5-phosphate to 5‑phosphoribosyl-1‑pyrophosphate (PRPP), a reaction catalyzed by purine nucleoside phosphorylase (PNP). The first committed step in purine synthesis is the formation of 5‑phosphoribosylamine via the enzyme PRPP glutamyl amidotransferase. IMP and GMP can also be created by via the “salvage pathway” whereby PRPP is combined with hypoxanthine or guanine bases (including 6-MP and 6-TG) by the actions of hypoxanthine-guanine phosphoribosyl transferase (HGPRT). 6-MP and 6-TG (and their naturally occurring analogues) inhibit guanylyl kinase, preventing the conversion of GMP to GDP and causing “pseudofeedback inhibition” of PNP and PRPP. One route for the degradation of adenosine nucleotides is conversion of AMP back into IMP, a reaction that is catalyzed by adenosine deaminase. This enzyme is inhibited by PENTOSTATIN, which leads to an increase in dATP levels which reduce the activity of ribonucleotide reductase (see 4 below). One route for the degradation of purine nucleotides (and 6-MP and 6‑TG) occurs via conversion of IMP to uric acid. Two steps in that process, conversion of hypoxanthine to xanthine and xanthine to uric acid, are catalyzed by the enzyme xanthine oxidase. This enzyme is inhibited by ALLOPURINOL.

Review of Nucleotide Biochemistry (cont’d) CONVERSION OF RIBONUCLEOTIDES TO DEOXYRIBONUCLEOTIDES (HYDROXYUREA) This reaction is catalyzed by ribonucleotide reductase.

Folate and Vitamin B12

Forms FOLATE FOLIC ACID is the synthetic (more stable) form of folate used in supplements available as an oral formulation, but can also be given IM, IV or SC if GI absorption is impaired LEUCOVORIN (5-formyl-TH4, folinic acid) is a naturally occurring compound that is used to replace folate in rescue therapy

Forms VITAMIN B12 Naturally occurring forms: adenosylcobalamin, methylcobalamin Medicinal forms: CYANOCOBALAMIN: stable, synthetic form used as a supplement given orally, IM, IV, transdermal (patch), spray HYDROXOCOBALAMIN: used for treatment of cyanide poisoning

Function folate and vitamin B12 are required for DNA synthesis and for maintenance of neurons and red blood cells folate is the critical precursor in the neosynthesis of tetrahydrofolate (TH4) TH4 is a 1 carbon donor in several reactions including: thymidylate and purine synthesis methionine synthesis amino acid metabolism

Function vitamin B12 is an intermediate in: a reaction that converts methylTH4 (the primary stored form of TH4) to TH4 in the process, homocysteine is converted to methionine (i.e., homocysteine levels are lowered as a result) the conversion of methylmalonyl CoA to succinyl CoA

Absorption FOLATE found in green leafy vegetables, dried beans and peas, sunflower seeds and citrus fruits, as well as yeast, liver and kidney also found in fortified cereal products (breads, flours, corn meals, pastas and rice) absorbed from the small intestine relatively small quantity is stored in the liver, and there is a high daily requirement  only ~ 1-6 month supply

Absorption VITAMIN B12 original source is bacteria, but most is obtained from animal products or fortified foods in the diet where it is bound to proteins absorption requires acidic pH and intrinsic factor (which is made by the stomach) occurs via a specific transport system in the small intestine ~ 5 year supply is stored in the liver (so deficiency is rare except in the elderly)

Folate Transport and Intracellular Retention Absence of the folate transporter confers differential sensitivity on folate inhibitors (such as METHOTREXATE). Cancer cells or bacteria that do not have this transporter cannot accumulate folates after inhibitor treatment, while normal cells can protect themselves by accumulating other folates (such as LEUCOVORIN). This is the fundamental principle underlying antineoplastic rescue therapy.

Transport

Polyglutamation

Deficiency Deficiency of either folate or vitamin B12 has catastrophic effects on rapidly growing cells and on neurons. The most prominent and earliest clinical sign of deficiency is megaloblastic anemia. deficiency of either folate or vitamin B12 prevents DNA synthesis, with the greatest effect being on rapidly growing cells (esp. bone marrow and GI mucosa), and on neurons FOLATE deficiency can be caused by: insufficient dietary intake high demand (pregnancy) alcoholism intestinal disease (sprue) inhibitors causes megaloblastic anemia also results in neural tube defects in children born to mothers with low folic acid levels (no neurologic damage in adults)

Deficiency Deficiency of either folate or vitamin B12 has catastrophic effects on rapidly growing cells and on neurons. The most prominent and earliest clinical sign of deficiency is megaloblastic anemia. VITAMIN B12 most commonly due to a deficiency in intrinsic factor ( can’t treat with oral doses) – very rarely caused by a nutritional disorder vitamin B12 deficiency will cause a folate deficiency by preventing access to stored tetrahydrofolate

Deficiency VITAMIN B12 (cont’d) Deficiency of either folate or vitamin B12 has catastrophic effects on rapidly growing cells and on neurons. The most prominent and earliest clinical sign of deficiency is megaloblastic anemia. VITAMIN B12 (cont’d) result is megaloblastic anemia (called pernicious anemia if due to impaired B12 absorption) and neurological damage (paresthesias and weakness that progress to spasticity, ataxia and other CNS disorders) correction of the B12 deficiency can stop progression of neurological effects but cannot entirely reverse them folate supplements will reverse the anemia, but not the nervous system damage  critical that B12 status be normal before beginning folic acid supplementation (particularly in people >50 years old) up to 10% of senile dementia is believed to be caused by B12 deficiency This problem is the major source of controversy regarding widespread fortification of foods with folate. Fortification decreases the incidence of neural tube defects (which affect approximately 4000 pregnancies/year in the US), but makes the detection of vitamin B12 deficiency more difficult (it eliminates the anemia which is usually the identifying clinical feature of vitamin B12 deficiency  affecting millions of elderly people)

FOLATE INHIBITORS

METHOTREXATE competitive inhibitor of dihydrofolate reductase (DHFR) therapeutic uses: cancer (1st solid tumour cure – choriocarcinoma) immunosuppressant: rheumatoid arthritis, psoriasis, inflammatory bowel disease/Crohn’s disease antibiotic (TRIMETREXATE is actually the best antibiotic anti-folate): Pneumocystis jirovecii (an AIDS-associated disease) – bug doesn’t have the reduced folate carrier  LEUCOVORIN rescue is effective here also abortifacient: in the 1st trimester – typically combined with MISOPROSTOL (PGE1)

TRIMETHOPRIM selective, competitive inhibitor of BACTERIAL dihydrofolate reductase (DHFR) TRIMETHOPRIM (a folic acid inhibitor) is typically combined with SULFAMETHOXAZOLE (which blocks dihydropteroate synthetase). These drugs act synergistically, selectively blocking successive steps in the folate synthesis pathway in bacteria.

Other Inhibitors OTHER CLINICALLY RELEVANT INHIBITORS include PEMETREXED (related to METHOTREXATE and discussed under antineoplastics) and PYRIMETHAMINE (an antiprotozoal drug related to TRIMETHOPRIM)