Ischaemic Heart Disease Group D

1. Ischaemic Heart DiseaseGroup D

2. Scenario Mr Pex (55 yrs) has taken these medications for 2 years: • Perhexiline 100mg bd- anti-angina • Gliclazide 80mg bd- hypoglycaemic agent • Fluoxetine 20mg d- antidepressant It is suspected that Mr. Pex has perhexiline toxicity because he has signs and symptoms of hepatotoxicity and peripheral neuropathy • How it should be managed • Place of Monitoring levels of Perhexiline • Explanation of any medications that could be responsible for the abnormalities in the clinical chemistry profile • Whether the abnormalities are likely to be ongoing • Recommended management for this patient with respect to his medication regimen

3. Clinical chemistry profile

4. Perhexiline • indicated for angina • plasma/blood perhexiline concentrations must be maintained within the range 0.15-0.6mg/L (0.5- 2umol/L) • Perhexiline is metabolised via CYP450 2D6 (patient genetic variability- fast, intermediate, slow metabolisers) resulting in variable clearance. • Genetic Polymorphism: approximately 10% of the population are slow metabolisers of perhexiline and are at a higher risk of toxicity, thus requiring decreased doses • Perhexiline and its metabolites undergo extensive hepatic metabolism and are excreted in bile and urine (ratio 1:2 respectively) • It has a low therapeutic index; so therapeutic drug monitoring is essential. Therapeutic Drug Monitoring factors: Individualised dosage; Target range; Concentration related effects (therapeutic and adverse); Narrow Therapeutic Index; Desired therapeutic effect is difficult to monitor.

5. Perhexiline pharmacokinetics • >80% of perhexiline maleate is absorbed from the GIT after oral dosing • Large volume of distribution, that is probably related to the tissue binding of perhexiline and its metabolites • It crosses the BBB (lipophilic) • Highly protein bound (>90%). Some binding to erythrocytes • Saturable rate of hepatic metabolism (genetic polymorphism of CYP2D6) • Several metabolites with unknown pharmacological activity • Racemic mixture- 2 Enantiomers

6. Perhexiline toxicity • long term- usually occurs > 3 months of continued therapy • Directly related to perhexiline blood concentrations • Hepatotoxicity- elevation in serum liver enzymes (AST aspartate aminotransferase, ALT alanine aminotransferase, alkaline phosphatase, LDH lactate dehydrogenase). Monitoring is essential at least every month. In Mr Pex: AST, ALT Alkaline Phosphatase, LDH: elevated. These liver enzymes show that the liver is damaged. They increase with liver dysfunction • Aminotransferases- ALT, AST are sensitive indicators of hepatic inflammation and necrosis. ALT is more specific as it is mainly found in the liver. • Alkaline Phosphatase: primarily made in liver, but also in bones. Sensitive marker of damage. • LDH: LDH4, LDH5 appear mainly in the liver and in skeletal muscles • Total Bilirubin (conjugated + unconjugated): low

7. Hepatotoxicity • Hepatitis and Cirrhosis have been reported. (Higher AST vs. ALT may be indicative of cirrhosis but chronic alcohol consumption must be excluded) • Though, more specifically the non-alcoholic fatty liver disease Non-alcoholic Steatohepatitis (NASH) is associated with perhexiline maleate • NASH is usually asymptomatic and may progress to cirrhosis within 7 years (inflammation and necrosis) • Abnormal liver function test results are present • Markers: Serum aminotransferase activities usually <4 fold above the upper limit of normal; moderately elevated serum ALT and AST • Insulin resistance may occur and may be unrelated to the presence/absence of obesity

8. Non-alcoholic steatohepatitis • No biological test to positively identify it • The following diagnostic approach can be used by health care professionals, to show hepatotoxicity

9. Peripheral neuropathy • Occurs when nerves connecting spinal cord and brain to other parts of the body become damaged • 3 different types: mono- (damage to a single nerve); multiple mono- (two or more nerves) poly-neuropathy (many nerves throughout the body) • Most common causes: drugs eg. perhexiline, diabetes Patient signs and Symptoms • (begin in hands or feet; may spread throughout the limbs) • Tingling, prickling, numbness • Sharp pain • Decreased or lack of sensation • Muscle weakness • Lack of muscle control • Burning or freezing sensations • Extreme sensitivity to touch • Loss of balance or coordination

10. Tests for signs and symptoms • Signs of muscle cramping/twitching and muscle weakness • Skin sensitivity to temperature changes, touch, vibration, pinpricks • Observation for clinical signs of hepatic involvement eg. Weakness, loss of appetite, weight loss • Nerve function tests eg. EMG (Electromyography- measures muscle electrical activity) • Analysis of serum enzymes (AST, ALT, alkaline phosphatase, LDH) and bilirubin- abnormalities or persistent elevations • Blood Glucose measurements- persistent/marked hypoglycaemia • Weight- excessive weight loss (>10% initial weight) • Perhexiline plasma levels- therapeutic target range

11. Gliclazide • Risk Factors: • Advanced age • Poor nutrition • Alcohol • Renal disease • Hepatic disease • Concurrent medications Symptoms of hypoglycaemia: • Comfusion • Agitation • Tachycardia • Tremor • Hypothermia • May progress to coma/convulsions Rarely associated with increases in ALP, AST and bilirubin Hypersensitivity can occur with accumulation

12. Fluoxetine (1) • Metabolised by 2D6, as is perhexiline • Clinically significant drug interactions occur when: • Therapeutic index of substrates is narrow (applies to perhexiline) • Isozyme involved in competition is the primary metabolic pathway (applies to perhexiline and fluoxetine; both pathways are saturable at therapeutic concentrations) • Concentration of inhibiting substrate reaches sufficient levels in vivo (demonstrated for both)

13. Fluoxetine (2) • Liver enzyme elevations may occur (reported in about 0.5% of patients receiving SSRIs) • >58,000 reports of hepatic ADRs with SSRIs ± other medications • 493 suspected to be due to fluoxetine • 12 = acute hepatitis (6 were on concurrent medications) • 5 = asymptomatic increase in serum transaminases • 80 with paroxetine • 65 caused by sertraline • 54 attributed to fluvoxamine

14. Management of Mr. Pex • Perhexiline? • Discontinue • Reassess angina (severity, frequency of attacks) and initiate alternative treatment • Fluoxetine? • Reassess the need for this treatment • Phenotyping may be of some value if PM status is suspected • Alternative agent could be used (different class of antidepressant, or try fluvoxamine/sertraline which are less potent CYP2D6 inhibitors and less frequently associated with hepatic ADRs • Gliclazide? • Assess glycaemic control (ongoing) • Determine hepatic and renal function • Change of agent may be necessary

15. Bilirubin • Outline the chemical basis for the spectrophotometric analysis of bilirubin • Look at the various techniques used to obtain values for total and direct bilirubin • Examine the problems and variations of different approaches used to examine patient bilirubin profiles

16. Bilirubin • bile pigment formed from the breakdown of Haemoglobin • found in serum as unconjugated, conjugated and delta • Unconjugated bilirubin is insoluble in aqueous solution, bound to serum albumin and represents bilirubin prior to hepatic processing • Conjugated bilirubin is formed subsequent to hepatic processing and is yielded by unconjugated bilirubins esterification of its two proprionic side groups with glucoronic acid. • Delta bilirubin represents bilirubin which is covalently bound to albumin • Since the liver is involved in the enzymatic modification of bilirubin, the serum plasma concentration is used as a test for hepatic function • Elevation in unconjugated bilirubin can occur as a result of excessive bilirubin production eg. haemolysis, or the inability to conjugate or take up bilirubin from the circulation • Elevations in conjugated bilirubin are commonly seen in hepatocellular or biliary dysfunction

17. Analysis of bilirubin in serum The most widely used methods for the measurement of serum bilirubin are based on the diazo reaction

18. Analysis of bilirubin in serum Jendrassik Grof Assay • Uses caffeine-benzoate which acts as an accelerator • Caffeine benzoate displaces unconjugated bilirubin from albumin perhaps making it more water soluble by disruption of the internal hydrogen bonds → making bilirubin more readily available for reaction with the diazo reagent • Performed at alkaline pH Azobilirubins produced in these reactions are measured spectrophotometry at 600nm Evelyn Malloy Assay • Uses methanol to dissociate albumin • Performed at acidic pH • Absorbances are measured at 560nm producing red or purple colour

19. Direct bilirubin Conjugated Bilirubin + Diazotized sulfanilic acid → Azobilirubin B (Isomers 1 & 2) • Measures the majority of conjugated and delta bilirubin and a variable but small percentage of unconjugated bilirubin • To prevent measurement of unconjugated bilirubin, the serum should be diluted with HCl first

20. Total bilirubin DIRECT BILIRUBIN Conjugated Bilirubin + Diazotized sulfanilic acid → Azobilirubin B (Isomers 1 & 2) INDIRECT BILIRUBIN Total Bilirubin – Direct Bilirubin = Indirect Bilirubin

21. Other methods used to obtain bilirubin fractions High Performance Liquid Chromatography Measures four bilirubin fractions in serum Unconjugated bilirubin Delta bilirubin Bilirubin monocongugate Bilirubin diconjugate

22. Other methods (cont.) Direct Spectrophotometric Method • Measures conjugated and unconjugated bilirubin and calculates delta bilirubin as the difference between the sum of these and total bilirubin • Based on the absorbance of unconjugated bilirubin at 454nm and Hb at 540nm Enzymatic Methods BOX Bilirubin + ½ 0² → Biliverdin + H2O • Based on enzyme bilirubin oxidase • Oxidation rates depend on the pH of the reaction mixture Maximum oxidation Conjugated pH 4.5 & 10 Unconjugated pH 6 Delta pH 4

23. Problems with techniques Despite the advances of these methods, in the clinical laboratory they still have limitations. Some of these are related to bilirubin's instability and insolubility in water, but there are also problems of assay interference, lack of pure conjugated bilirubin standards, and interpretation of bilirubin fractions depending on the method on use

24. Diazo method Limitations include: The assumption that direct and indirect bilirubins represent conjugated and unconjugated bilirubins, respectively although in several assays this may be incorrect. The lack of adequate standards for calibration of the direct bilirubin assay. Direct bilirubin assays have used unconjugated bilirubin for assay calibration, not a particularly stable compound and is not ideal because assays should be calibrated with the analyte that they are designed to measure. Many manufacturers now use synthetic forms of bilirubin in their calibration and control material, and it is hoped that use of these synthetic variants will help improve the accuracy of the direct bilirubin assay Direct bilirubin assay is dependent on reaction conditions, especially pH and often underestimates conjugated bilirubin , this leads to inaccuracies in indirect bilirubin

25. Direct spectrophotometry The assay is only suitable for serum neaonates (usually less than 2 - 3 weeks of age) because other pigments, notably carotene, start to appear as infants get older and cause interference at 454nm. Studies have shown that such spectrophotmeteric methods are not only rapid, easy to carry out, requiring small samples but also are less influenced by factors like hemoglobin concentration and hemolysis

26. HPLC Originally limited by inadequate fraction quantitation and the large sample size requirement Modifications allowed measurement of all four fractions using small sample size. More recently developed procedures do not precipitate albumin and lose delta bilirubin Delta bilirubin represents bilirubin covalently bound to plasma proteins, predominately albumin. This binding unlike that of unconjugated bilirubin, is resistant to physical, chemical, and enzymatic treatments HPLC is considered the gold standard as it measures all four fractions, however, it is extremely expensive, elaborate and time consuming for routine clinical use. It is also labor intensive and requires specialized equipment and therefore not a method suited for a laboratory required to perform bilirubin 7 days per week, often on a 24-hour basis. It does however remain a method to which the more commonly used procedures can be compared

27. Specimen requirements Because both conjugated and unconjugated forms of bilirubin are photo-oxidised on exposure to UV light, it is recommended that sample should be protected from light. Bilirubin is unstable and light sensitive and therefore the assay should be carried out within 2 hours of sample collection. If a longer delay is unavoidable, refrigerate the sample. Bilrubin is stable in the refrigerator (40C) for 3 days. Samples can be frozen at -700C, to keep bilirubin stable for 3 months

28. Measurement in urine Because conjugated but not unconjugated bilirubin is excreted in urine, uniary examination may be used as a simple screen to determine whether high levels of bilirubin is due to prehepatic causes or to hepatic or post hepatic disorders. The urine specimen to be investigated should be fresh. If delays are anticipated, the urine container should be protected from light and refrigerated. Urine bilirubin measurements are often made using qualatative methods such as dipsticks impregnated with diazo reagent, which reacts with bilirubin to produce a colour change. The conjugated forms of bilirubin can be isolated from bile however, are not suitable for mass isolation and use in calibrators and control material and therefore unstable

29. References • Australian Medicines Handbook 2004 (p241, 244) • Birkett D. J., Therapeutic Drug Monitoring Australian Prescriber 1997; 20:9-11 • Shargel L, Mutnick AH, Souney PF, Swanson LN. Comprehensive Pharmacy Review 4th ed. 2001 Lippincott Williams & Wilkins • Australian Pharmaceutical Formulary and Handbook 18th edition • Campbell T J; Williams KMTherapeutic drug monitoring: antiarrhythmic drugs 46(4):307-3191998 • Davis TM; Daly F et al. Pharmacokinetics and pharmacodynamics of gliclazide in Caucasians and Australian Aborigines with type 2 diabetes. Br J Clin Pharmacol 49(3):223-30 • Chitturi, S; Le, V; Kench, J; Loh, C; George, J. Gliclazide-Induced Acute Hepatitis with Hypersensitivity Features. Dig Dis and Sci 47(5):1107-1110 • Spigset O; Hägg S; Bate A. Hepatic injury during treatment with SSRIs. Int Clin Psychopharm 18(3);157-61 • Lustman PJ; Griffith LS; Freedland KE, Clouse RE. The Course of Major Depression in Diabetes. Gen Hosp Psych 19:138-143 • Alfaro CL; Lam Y; Francis W; Simpson J; Ereshefsky L. CYP2D6 Status of Extensive Metabolizers After Multiple-Dose SSRIs. J Clin Psychopharmacol 19(2):155-163 • Davies B; Coller JK; James HM; Gillis D; Somogyi AA; Horowitz JD; Morris Raymond; Sallustio BC. Clinical inhibition of CYP2D6-catalysed metabolism by the antianginal agent perhexiline. Br J Clin Pharmacol 57(4):456-463 • Ahlofors, C. Measurement of Plasma Unbound Unconjugated Bilirubin, Analytical Biochemistry, 2000, vol. 279, 130-135. • Doumas, B. & Tai-Wing, W. The Measurement of Bilirubin Fractions in Serum, Critical Reviews in Clinical Laboratory Sciences, 1991, vol. 28(5), 415-445. • Parviainen, M. A Modification of the acid-diazo Coupling Method (Malloy-Evelyn) for the Determination of Serum Total Bilirubin, Scand J Clin Lab Invest, 1997, vol. 57, 275-280. • Jianxin, Z. Analysis of Unconjugated Bilirubin in Serum by Reverse-Phase High Performance Liquid Chromatography, Scand J Clin Lab Invest. 1992, vol. 52, 565-569 • Sykes E, Epstein E. Laboratory Measurement of Bilirubin. Clin Perinatol 1990 Jun; 17: 397-416. • Vreman HJ, Verter J, Oh W, et al. lnterlaboratory variability of bilirubin measurements. Clin Chem 1996 Jun; 42: 869