PROBLEM SET 1: THE SULFONAMIDES

1. Several bacteria are uneffected by the sulfonamides even though the drugs are capable ofpenetrating their cell walls and membranes and they do not contain a resistantdihydropteroate synthetase or increased levels of PABA. Also, these bacteria require folateslike sulfonamide-sensitive bacteria. Postulate an explanation for these observations.

SEVERAL ANSWERS ARE ACCEPTABLE. Most commonly bacteria that fit this descriptionare those that can take up folic acid (like human cells) and reduce it directly to dihydro- and thentetrahydrofolate. These bacteria don't have dihydropteroate synthetase, much less a resistant formof the enzyme, and therefore are not affected by the sulfonamides.

OTHER POSSIBLE ANSWERS:

• It would be possible for bacteria that require the dihydropteroate synthetase pathway tosynthesize large quantities of this enzyme to competitively overcome sulfonamide inhibition.Thus while the sulfonamides can inhibit the enzyme, it is produced in such large quantities thatthe sulfonamides cannot effectively shut down the pathway.

• Bacteria may produce enzymes (like acetylases) that inactivate the drug. This pathway ofresistance has not been detected in resistant bacteria, but is possible.

• It is at least theoretically possible that bacteri may not require the folates for DNA, protein,etc. biosynthesis, although very unlikely.

2. The American Academy of Pediatrics has stated that "breast feeding and sulfonamideuse are compatible in healthy, full-term neonates, but not premature infants or those withhyperbilirubinemia or glucose-6-phosphate deficiency". Explain the chemical/biochemicalrationale for this precaution.

This clearly relates to fact that the sulfonamides distribute to breast milk, although in lowconcentrations, and can be effectively transferred to (absorbed by) breast-fed offspring. The keyto this precaution involves the inability of newborn to effectively clear the drug by conjugativemetabolism. Healthy, full-term neonates typically have sufficient metabolic capacity to conjugate(acetylate, glucuronidate) the small quantity of drug transferred in the breast milk. Prematureinfants cannot. As a result, even these low sulfonamide concentrations may displace bilirubin (seethe answer to question 6 elow) and interfer with glucose-6-phosphate dehydrogenase mediatedformation of NADPH and GSH (see answer to question 3 below).

ALSO, the N-acetyl conjugates of the sulfonamides are more efficiently transferred in breast milkthan the parent sulfonamides, and this may contribute to greater "apparent sulfonamide sensit-ivity" (toxicity) in premature breast-fed neonates.

3. Patients with sulfonamide-associated hemolytic anemia have reduced erythrocyte levelsof glutathione (reduced form) compared to "normals" who do not display drug-inducedanemia. Explain the chemical/biochemical reason for this and its significance relative tohemolytic anemia.

See class notes on sulfonamides and GSH levels in RBCs. Glutathione (GSH) is a key"protective" peptide for red blood cells (and other cells). It detoxifies electrophilic toxins bysulfhydryl atttack (as you learned in PY 419) and quenches free radicals (formed largely under theoxidative conditions in which the red blood cell exists). When glutatione levels are below thoserequired for red blood cell protection, lysis of the cell occurs more readily (hemolytic anemia). Glutathione is formed from oxidized glutathione (GS-SG) by glutathone reductase, an reactionthat requires the cofactor NADPH. NADPH is generated by the phosphate shunt which isinitiated by glucose-6-phosphate. Individuals with a genetic deficiency of this enzyme haveminimal capacity to generate NADPH. Thus they also have minimal NADPH reserve to sustainadequate levels of GSH. The sulfonamides, or more likely "aniline radicals" (see below) orelectrophilic oxidation products (see class notes) formed from the sulfonamides, may eitherdirectly inhibit the already impaired dehydrogenase system, or directly place an excess oxidativestress on cells with minimal free GSH (the radicals themselves cause cellular injury and death). Thus individuals with a deficiency of the dehydrogenase will not produce "normal" NADPHlevels, and this will lead to a decline in GSH levels.

4. Facts and Comparisons states that "aniline radicals" are responsible for the effects ofsulfonamides on the blood and the hematopoietic system. What is meant by this, what arethese radicals and how are they formed? (THE ANSWER IS NOT IN FACTS ANDCOMPARISONS).

Drugs compounds possessing an aniline moiety (acetaminophen, aminoquinoline antimalarials, andSULFONAMIDES) or a nitro group that can be reduced to an aniline-type moiety(chloramphenicol, nitrofurantoin) are notorious "blood toxins". The precise mechanism of theeffects of anilines on blood cells or the hematopoietic system are not clearly understood, but itappears to involve OXIDATION of the aniline nitrogen to a hydroxylamine and then nitrosometabolite. These species may may give rise to the free radicals by redox cycles that produceoxidative damage to blood cell enzymes and structures. See class notes on sulfonamides andGSH.

5. A prominent pharmacology text states that the "volume of distribution of mostsulfonamides approaches that of total body water and the drugs penetrate cells. Oneexception to this is sulfisoxazole which distributes only in extracellular water". What ismeant by this statement and what is the chemical basis for this?

Sulfisoxazole is one of the most polar sulfonamides employed as a systemic antibacterial. It isrelatively acidic, but even it's unionized form is relatively water soluble; it contains an isoxazole"R" group with two polar atoms and polarized double bonds. Thus this compound distributespreferentially to aqueous environments. Of course this polarity does not limit kidney distributionsince capillary membranes of these tissues are relatively porous. Naturally the acidic sulfonamidesalso tend to be more extensively bound by plasma proteins and this may contribute as well.

6. Some sulfonamides are reported to “cause fatal kernicterus when administered toneonates”. What is "kernicterus" and what is the chemical/biochemical rationale for thisreaction with sulfonamides?

The plasma concentration of the bile pigment bilirubin is maintained (kept low) by metabolicconjugation (glucuronidation) and plasma protein (albumin) binding; plasma proteins bind theunconjugated form of bilirubin. In neonates conjugative metabolism is not completely developed,thus protein binding represents the principle mechanism of control of free bilirubin levels. This isimportant because free bilirubin can penetrate the CNS of neonates and impair brain development,a condition known as KERNICTERUS. When sulfonamides are administered they, as a result oftheir acidic nature, displace bilirubin from albumin, thereby elevating plasma levels of free bilirubinand penetration into the CNS. Also, there are some reports that sulfonamides can inhibit theglucuronidation of bilirubin. This would also contribute to the development of fatal kernicterus.

7. Why is sulfasalazine used to treat inflammatory bowel disease and NOTphthalylsulfacetamide or phthalylsulfathiazole?

The principle difference between these three sulfonamide prodrugs is that sulfasalazine releases asulfonamide and 5-AMINOSALICYLIC ACID upon metabolic activation (reduction), while theother two release phthalic acid and a sulfonamide. In inflammatory bowel disease5-aminosalicylic acid is proposed to be the ACTIVE moiety. 5-Aminosalicylic acid inhibitsprostaglandin synthesis, reduces leukotriene production, inhibits leukocyte mobility and inhibitsmast cell degranulation (and other anti-inflammatory actions). Phthalic acid (released by the othertwo sulfonamide prodrugs) does not produce these anti-inflammatory effects. The sulfonamidemoiety of sulfasalazine (sulfapyridine) may contribute to the overall activity of this compound as aresult of its antibacterial actions.

8. The sulfonamides are reported to increase the bone marrow depressant effects ofmethotrexate (A DHFR inhibitor). Provide the biochemical/chemical rationale for thisobservation

This may result from pharmacokinetic or pharmacologic drug interactions or both. Of the twotype of interactions, the pharmacokinetic appear more likely to result in the observed increased inbone marrow toxicity. Pharmacokinetic interactions may involve:

a. The sulfonamides are capable of displacing methotrexate and it's 17-hydroxy metabolite fromplasma proteins. This would increase free levels of these compounds and could contribute toincreased toxicity via inhibition of mammalian DHFR.

b. A small fraction of methotrexate is metabolized by oxidation to the 17-hydroxy metabolite. Some evidence suggests the sulfonamides may inhibit this reaction and thereby block thiscontribution to clearance

c. Competition for tubular secretion. A significant fraction of methotrexate is eliminated renallyby tubular secretion. Secretion may play (although probably lesser) a role in sulfonamideelimination. Thus any ability of sulfonamides to competitively inhibit secretion of methotrexatewould delay it's clearance, elevate plasma levels and thereby enhance toxicity.

A pharmacologic contribution to this interaction may result from the ability of both drugs todepress folate biosynthesis. Methotrexate is an effective inhibitor of DHFR and thereby reducestetrahydrofolate levels. The sulfonamides themselves or sulfonamide-dihydropteridine conjugatesmay also inhibit tetrahydrofolate formation (possible inhibition of DHFR) and this would result inan apparent increase in methotrexate activity, particularly versus blood cell lines.