1 Monitoring Drug Efficacy & Toxicity - along with drug metabolismMichael E. Hodsdon, MD, PhD Associate Professor Departments of Laboratory Medicine & Pharmacology Office: 55 Park Street, 502B, Phone:

2 References Principles of Pharmacology, Golan, et al.Chapter 47, Principles of Toxicology. Chapters 2, 3, 4 on Pharmacodynamics, Pharmacokinetics and Drug Metabolism. My Wiki: Follow ‘Medicine’ link

3 Therapeutic Drug Monitoring (TDM) or “How can you tell if a drug is working or not?”Monitor clinical signs and/or laboratory measures of drug efficacy (and toxicity). Blood pressure monitoring for an anti-hypertensive. Blood coagulation rates (i.e. “prothrombin time” or PT) for coumadin (warfarin) anticoagulation therapy. Very drug specific; not covered in detail here. Measure drug levels. In blood generally (either serum, plasma, whole blood, or a specific cellular component). Other fluids are possible (e.g. saliva or urine). Need to decide what and when to measure.

4 Therapeutic Drug Monitoring (TDM): Basic Principles for ReviewDose-Response Curves, Therapeutic Index & Therapeutic Range (or window) Pharmacokinetics & Pharmacodynamics Relevant because they tells us what to measure, when to measure it, and how to interpret the results. Important concepts: absorption, distribution, metabolism & elimination. A few simple equations are handy (just three).

5 Therapeutic Drug Monitoring (TDM): Important differences between blood and urine.Serum Detection of circulating substances (i.e. drugs and toxins) generally represents a significant, recent exposure. Quantitation of drug/toxin levels (or their metabolites) generally correlates well with current/ongoing drug efficacy and toxicity. Slightly invasive (requires a needlestick) and also less sensitive for detection of previous exposure history. Urine Many drugs, toxins and their metabolites are accumulated in urine over time, thereby increasing the sensitivity of detection for both current and past use. Because urine may remain positive long after the physiological effects of a substance have disappeared, detection of a drug in urine does not always explain a patient’s current clinical condition. Quantitation is not that useful as variability in the timing of collection compared to drug exposure and also variability in the specific gravity of urine (i.e. reflecting a patient’s hydration status) results in a wide range of possible drug concentrations.

6 Dose-Response Curves (part of “pharmacodynamics”

7 Dose-Response Curves Therapeutic ED50 Toxic LD50

8 Dose-Response Curves Therapeutic Range

9 Therapeutic Range Drug levels maintained within the therapeutic range throughout the dosing interval.

10 Therapeutic Range Peak drug levels drift into the toxic range during the dosing interval.

11 Therapeutic Range Trough drug levels fall below the optimal therapeutic range.

12 Simplified PharmacokineticsThree Critical Equations 1) [ ]bolus = dose / Vd 2) [ ]steady state = ratein / Cl 3) Cl = (0.693 * Vd) / t1/2 From the above three equations, it is clear that the critical data to obtain for TDM/pharmacokinetic consultation is the dosage, Vd and either the Cl or t1/2 of the drug. Note that Vd and Cl usually given in weight-based units; hence, it is also necessary to find out the patient’s weight. Of course, knowledge of the references ranges for efficacy and toxicity of the drug is critical as well.

13 Simplified Pharmacokinetics[ ]steady state = Ratein / Cl [ ]bolus = (single dose) / Vd [ ]max = [ ]steady state + ½ * [ ]bolus [ ]min = [ ]steady state - ½ * [ ]bolus

14 Critical Importance of the Half-life!Half life is the key for knowing When a new medication is started, how long until the patient reaches steady state? When a patient stops taking a medication, how long until it is gone? When a drug dosage is changed, how long until a new steady state is achieved?

15 Critical Importance of the Half-life!

16 Simplified PharmacodynamicsStrictly speaking, pharmacodynamics (PD) defines the relationship between drug level and effect (think about the dose-response curves). In practice, it tells us what/when to measure drug levels and what they mean. In some fortunate cases, other measurements are excellent surrogates for drug levels (e.g. the “PT” and coumadin therapy).

17 Main PD ClassificationsMaintain drug levels within the therapeutic range (e.g. phenytoin or digoxin). Maintain drug level above some minimally effective level (e.g. vancomycin and the “MIC”), with or without avoiding toxic (high) levels. For some antibiotics (e.g. gentamicin) efficacy is best measured using a peak level (due to “peak-dependent killing” and the “post-antibiotic effect”) and toxicity is avoided by making sure the trough levels fall below a certain threshold (toxicity due to chronic accumulation). Most complicated situation is where only the “area under the curve” (AUC) correlates with either efficacy or toxicity and the PK are not predictable (e.g. cyclosporine). a combination of trough and peak levels are most commonly used As long as the total dose is adequate, drug monitoring is not necessary (e.g. penicillin). generally applies when EITHER a drug has a very wide therapeutic range/index OR has very predictable pharmacokinetics

18 Classifications of Drug Toxicities - can be overlappingDose-dependent (concentration-dependent) Idiosyncratic (i.e. unpredictable, but strictly defined means genetically determined) Allergic or immunologic Carcinogenic Teratogenic Dependence/addiction etc.

19 Mechanisms of Drug ToxicitiesNonspecific macromolecular damage toxic metabolite of acetaminophen Inflammatory and immune-mediated autoimmune hemolytic anemia (many drugs): methyldopa, penicillin, etc. lupus-like syndrome caused by hydralazine, isoniazid and procainamide (induces antibodies against myeloperoxidase and/or DNA). Enzyme inhibition cellular energy production: salicylate, cyanide, etc. acetylcholinesterase inhibitors (pesticides) Receptor-mediated or “hormonal” glucocorticoids (i.e. “steroids”): prednisone, cortisol, … aryl hydrocarbon receptor: dioxin

20 Acute versus Chronic Most of the acute toxicities are simply dose-dependent and predictable. However, many drugs cause less predictable chronic toxicities. Examples include AZT (mitochondrial toxicity), phenytoin (gum hyperplasia), cyclosporine (nephrotoxicity), …

21 Importance of Drug MetabolismJust a note that it is always important to consider drug metabolism pathways when analyzing drug toxicity issues. Drug metabolites are often responsible for toxicity. This will be illustrated in the upcoming example cases.

22 Important Effects of Drug MetabolismFunctional inactivation Increased water solubility Enhanced excretion Redistribution away from hydrophobic tissue sites Occasionally, functional activation The metabolites of some drugs are also active “Pro-drugs” are activated by metabolic reactions

23 Sites of Drug MetabolismLiver is the most important organ. Heavily perfused. Highest level of drug-metabolizing enzymes. Others sites include skin, lungs, G.I. tract, and the kidneys. “First-pass metabolism” applies to orally administered drugs.

24 Two Major Classes of Enzymatic ReactionsPhase I reactions chemically modify the drug. Phase II reactions conjugate the drug to hydrophilic small molecules.

25 Phase I Reactions Convert the drug to a more polar compound via enzymatic reactions that add small functional groups such as a hydroxyl, sulfhydryl or amino. Oxidation- primarily occur via Cytochrome P450 oxidases (e.g. hydroxylation of barbiturates), but there are also a few P450-independent oxidations (e.g. dehydrogenation of alcohols). Reduction- for a few drugs such as chloramphenicol, methadone, halothane and naloxone . Hydrolysis- Occurs with esterases and amidases (e.g. succinylcholine and indomethicin, respectively).

26 Cytochrome P450 Oxidases Primary Phase I enzyme system and the largest metabolizer of lipid soluble drugs or xenobiotics. Close to 50 family members of P450 enzymes. Membrane bound enzymes in the smooth ER (“microsomal”). Each consists of 2 components: an oxidase and a reductase, which require molecular oxygen and NADPH as electron donor.

27 Phase II Reactions Conjugation or addition of polar molecules to a drug to greatly enhance polarity and water solubility in order to facilitate excretion in feces, bile or urine. Important examples include glucuronidation of Tylenol, sulfation of estrogens, acetylation of sulfonamide antibiotics, and glutathione conjugation of Tylenol.

28 Inter-individual Variability in Drug MetabolismAge and gender (and Race?) Diet and drug interactions Co-morbidity (i.e. other diseases) Pharmacogenetics (separate lecture)

29 Age and Gender (Race?) Infants: decreased phase I and II reactionsmature slowly over first two weeks of life bilirubin glucuronidation chloramphenicol toxicity due to deficient phase II reaction (gray baby syndrome) Elderly: general decrease in hepatic capacity Gender: hormones regulate enzyme levels Race: clear differences, part of pharmacogenetics

30 Cytochrome P450 System Induction and InhibitionInducers Inhibitors Barbiturates Cimetidine Phenytoin Omeprazole Carbamazepine Acute Ethanol Toxicity Chronic Ethanol Toxicity Valproic Acid Rifampin Erythromycin Ritonavir Disulfram Griseofulvin Isoniazid (St. John’s Wort) Ciprofloxacin Although all enzyme systems are likely susceptible to both induction and inhibition, this has classically been defined generically for the Cytochrome P450 System given its widespread importance in drug metabolism. Expect to see broader and more precise descriptions of these effects enter into routine clinical practice in the future.

31 Comorbidity (other diseases)Altered hepatic function When hepatic function is compromised, so may drug metabolism. However, this often requires extensive damage before having an effect Altered hepatic perfusion When hepatic tissue is fully intact but not effectively perfused (e.g. from heart failure) drug metabolism may be slowed. Nutritional deficiency Malnutrition depletes sulfation stores, glutathione, and reductive potential (NADH/NADPH levels), hindering drug metabolism. Often “malnutrition” is associated with a chronic illness instead of a restricted diet, a result of catabolic syndromes (e.g. “cancer cachexia”).

32 Resistance to WarfarinA 42-year-old man was admitted to hospital with chills and progressive shortness of breath on exertion. He had received an aortic valve replacement 12 years before presentation and had been taking warfarin (5.5 mg/day) since that time with an INR maintained between 2 and 3. A diagnosis of pneumonia caused by Pneumocystis jiroveci was made. Serologic testing revealed a positive HIV status with a CD4 count of 150 cells/mL. After successful treatment for his pneumonia, the patient was discharged from the hospital and prescribed aggressive antiretroviral therapy (zidovudine, lamivudine and lopinavir/ritonavir).

33 Resistance to WarfarinAt a follow-up visit one month after discharge, the patient’s INR had declined to 1.1 (normal). Patient non-adherence and changes and diet were ruled out as a possible causes of the apparent warfarin resistance. Over a period of six months his warfarin dose was slowly titrated from the initial 5.5 mg/day to a final 13 mg/day in order to maintain an INR between 2 and 3. This most likely represented a drug interaction between the protease inhibitor combination lopinavir/ritonavir and warfarin. Lopinavir/ritonavir both inhibit and powerfully induce the CYP3A4 enzyme complex, as well as, induce other P450 enzymes such as CYP2C9 and CYP1A2, which are both responsible for metabolism of warfarin.

34 Acetaminophen – importance of drug metabolismIllustrative Case #1 HR is a 27 y.o. man with a history of depression who took approximately mg (standard release) acetaminophen tablets around 2 hours ago. His wife found him with the empty pill bottle and brought him into the ED. He is very emotional, describes minor stomach upset, but otherwise has no significant signs or symptoms.

35 How much acetaminophen is too much?For an acute exposure, this is well established: Toxicity is considered possible if maximum potential exposure is > 7.5 g in adults or 150 mg/kg in children. Based on history, our patient’s maximum exposure is 40 x 325 mg = 13,000 mg or 13 g. Note that this is also 13,000mg/70kg = 186 “mg/kg”.

36 Why is Acetaminophen Toxic?Acetaminophen is a strong analgesic and antipyretic with weak anti-inflammatory properties. Its therapeutic effects are attributed to central inhibition of prostaglandin synthetase. However, its toxic effects are unrelated to its therapeutic effects (i.e. for many drugs toxicity is simply “too much therapy”). The toxic effects of acetaminophen are the consequence of an undesired metabolite. This is another common mechanism of drug toxicity.

37 Normally, only 5 – 15% is converted into NAPQI and a vast majority of that is conjugated to glutathione and eliminated. Hence, under normal dosages the toxic effects of NAPQI are avoided. (NAPQI)

38 Four Phases of Acetaminophen PoisoningPhase I (0.5 – 24 h): Consequences of G.I. distress – anorexia, nausea, malaise, pallor, vomiting and diaphoresis. Little or NO signs of hepatotoxicity. The patient may appear normal. Phase II (24 – 72 h): Initial phase of hepatoxicity. RUQ pain may be evident. G.I. distress lessens. Hepatitis – elevated liver enzymes. Decreased hepatic function – elevated PT/INR, elevated unconjugated bilirubin. Possible decreased renal function. Phase III (72 – 96 h): Sequelae of hepatic necrosis – massively elevated liver enzymes, coagulation defects, encephalopathy, jaundice. Renal failure and myocardial dysfunction may be present (multi-system organ failure). Phase IV (4 d – 2 wk): Either death (result of multi-system organ failure) or if hepatic damage is reversible, complete resolution may occur. Liver transplant is another possibility.

39 Back to our patient… Potential toxic exposure (13 g) and symptoms consistent with phase I of aceteminophen toxicity (i.e. he complains of G.I. distress). No physical signs of other drugs (but cannot safely rule them out). What to do next? 1) Laboratory Studies 2) Treatment

40 What lab tests should we get?Serum Overdose Panel (designed to rule out many common and treatable overdoses where laboratory monitoring is particularly valuable): Acetaminophen – 120 mg/ml Salicylate – not detected. Alcohol panel (ethanol, methanol, isopropanol and acetone) – not detected. Barbiturates – not detected. Tricyclic Antidepressants – not detected.

41 What lab tests should we get?Hepatic and Renal Damage/Function Electrolytes (mM): Na – 141, K – 4.1, Cl – 103, Bicarbonate – 28. Basic Renal Function: Cr – 0.8, BUN – 12. Liver Enzymes: AST – 26, ALT – 21, AP – 84, LDH – 120. Evaluation of Liver Function: PT/INR – 11.5s/1.1, Glucose – 95, Total Billirubin – 0.93, Ammonia – 15

42 What about the acetaminophen level? (120 mg/ml)Therapeutic Range is 10 – 20 mg/ml. Toxicity is expected for “acute” peak levels greater than 150 – 200 mg/ml. But, you can’t expect to catch the peak drug level for overdoses. How do you deal with this?

43 Predicting a maximum peak levelWhat information do you need? Dose (13g), Patient Weight (70kg), Volume of distribution (~1 L/kg), Bioavailability (~60 – 98%, but in overdose tends to be on the higher side as metabolism reaches saturation). How do you calculate a maximum peak level? 95% of 13 grams is ~ 12 grams. Volume of distribution = 1 L/kg * 70 kg = 70 Liters. Peak Level = dose/Vd = 12g/70L = 171 mg/ml (multiplied by 1000 to convert g/L into mg/ml). What factors might prevent the patient from actually achieving this theoretical maximum? Delayed absorption due to delayed gastric emptying, formation of “concretions” and intestinal irritation. Ongoing elimination during delayed absorption (peak later and lower). Actual dose less than reported/anticipated. Unfortunate inter-individual variability (often as much as 50 – 100%) in Volume of Distribution, Bioavailability, Elimination Half-life, etc. Hence, although rough PK calculations useful, monitoring of serum drug levels remains essential.

44 Rumack-Matthew Nomogram

45 What happens with our patient?Treatment is initiated as a follow-up level at ~ 4 hours post-ingestion was > 150 mg/ml. A series of levels measured every 4 hours is consistent with successful treatment as the half-life is less than 4 hours (blue line represents ~ 2.5 – 3 hr half-life).

46 Treatment of Acetaminophen ToxicityGeneric Decontamination: gastric lavage (if early) and activated charcoal (PO or via NG tube) Supportive: not indicated during Phase I, but relevant for other phases. Antidote: N-acetylcysteine (NAC) Maximal effectiveness if given in first 8 hours. Given P.O. in the US (see handout); may require an anti-emetic as reputedly very noxious. However, also demonstrates effectiveness is given after hepatotoxicity begins (i.e. Phase II). Two mechanisms likely. First, replenishes reductive sulfation stores for detoxification of APAP and NAPQI. Second, may have a direct protective and/or regenerative effect on tissue, sometimes used in multi-system organ failure due to other causes.

47 Mechanism for N- acetylcysteine:Provides a substrate for APAP sulfation. Replenishes glutathione stores. Can substitute for glutathione for detoxification of NAPQI.

48 Acetaminophen Case #2 R.W. is a 31 y.o. woman with a PMH of major depression and alcohol abuse. She is currently in the process of divorcing an abusive husband and is separated from her 8 y.o. son. Approximately 24 hours prior to presenting to the YNHH ED, the patient took approximately 48 tablets containing 500 mg Acetaminophen for a total dose of 24 grams. These were taken after approximately 8-10 hours of heavy “binge” drinking by the patient. She subsequently lost consciousness and upon awakening complained of nausea, vomiting and right upper quadrant abdominal pain.

49 Acetaminophen Case #2 Past Medical History: Significant only for major depression and alcohol abuse. She has had two previous suicide attempts and has been under treatment of a psychiatrist. However, she last visited her psychiatrist about one year ago and stopped taking her psychiatric medications at that time. Medications: none Allergies: none Social History: Drinks ½ pint of hard alcohol per day and smokes tobacco occasionally. She has been a Jehovah’s Witness since 1986 and refused all blood products during the admission.

50 Physical Examination Vital Signs: Afebrile, BP 156/93, HR 90, RR 20, SaO2 98% (on room air) She is alert and oriented x 4 and in no acute distress. HEENT: PERRL, EOMI, no scleral icterus, oropharynx clear Neck: Supple, no lymphadenopathy, no cartid bruits, no JVD Heart: sinus tachycardia, regular rhythm, no G/M/R Lungs: clear to auscultation bilaterally Abdomen: soft, nondistended, mild RUQ tenderness Extremities: no asterexis, no C/C/E, no petechiae/ecchymosis Neuro: non-focal Rectal: normal tone, heme negative

51 Initial Laboratory ValuesElectrolytes and Renal Function: Na 137, K3.3, Cl 106, HCO , BUN 9, Cr 1.0 Liver Enzymes: ALT 654, AST 884, Alk Phos 86, Amylase 89 (for the pancreas) Liver Function: Glucose 255, Total Protein 6.4, Albumin 3.6, Bilirubin T/D 1.44/0.37, PT 13.7 (12.2), PTT 26.1 (note that four hours later: PT 16.4 (12.1), PTT 31.9) Serum Overdose Panel: Acetaminophen 19 mg/ml, Salicylates 3 mg/ml, Alcohols/TCAs/Barbiturates - all negative. CBC: WBC 5.0, Hgb 12.8, Hct 40.7, MCV 89, Platelet 60

52 One more example of using the Rumack-Matthew Nomogram…A level measured ~ 24 hours after the overdose was 19 mg/ml. Again, the red bar represents a degree of uncertainty in the timing (arbitrary choice of ± 4 hours). The placement of the level in the region of “probable hepatic toxicity” is clear and also unnecessary as the patient has already elevated liver enzymes and early signs of hepatic failure.

53 Hospital Course The patient did well and never became encephalopathic.Of course, she received a complete treatment of N-acetylcysteine (beginning ~24 hours after the overdose). Her peak liver enzymes occurred about 36 hours after admission: AST 5870, ALT 5440. She never developed signs of multi-organ failure and maintained good ABGs, cardiac function, Cr, urine output, etc. She was discharged on hospital day #4 with normal PT/PTT, AST 219, ALT 1860 and Bilirubin T/D of 0.64/0.26.

54 – more drug metabolism

55 All alcohols and glycols act on the CNS as a “sedative-hypnotic”Respiratory depression (bradypnea) CNS depression (inebriation) Hypothermia Tachycardia and Hypotension Isopropanol is a much stronger CNS depressant than the others and induces coma at around 100 mg/dl (i.e. 0.1 % w/v).

56 Serum Ethanol Level (mg/dL)Clinical effects on the non-tolerant individual < 50 mg/dl Mild muscular incoordination 50-100 Incoordination; driving increasingly dangerous Mood, personality, behavioral changes; driving is dangerous Prolonged reaction time; driving is very dangerous Nausea, vomiting, diplopia, marked ataxia Hypothermia, dysarthria, amnesia Coma, respiratory failure, death

57 “Metabolic” toxicity is a result of metabolism by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH)

58 Metabolism by ADH and ALDH

59 Methanol Poisoning Formaldehyde and formic acid accumulate because there is no endogenous metabolic pathway for detoxification. A minor pathway for elimination can be aided by folate administration. Methanol causes a CNS/respiratory depression like other alcohols and its metabolites poison tissue (oxidative phosphorylation) resulting in an elevated anion gap metabolic acidosis noncardiogenic pulmonary edema Gastritis with N/V, anorexia and abdominal pain Occasional pancreatitis Most common is damage to the retina and optic nerve resulting in “snow fields”, blurred vision, hyperemic optic discs, mydriasis, papilledema and eventually blindness.

60 Ethylene Glycol MetabolismNote that the primary toxic metabolites are glycolic acid, glyoxylic acid, oxalic acid. Precipitation of oxalic acid in tissues causes multisystem organ failure in untreated ingestions Primary treatment is blockade of alcohol dehydrogenase with ethanol or fomepizole. Thiamine and pyridoxine are given therapeutically to detoxify glyoxylic acid and prevent its conversion into oxalic acid.

61 Propylene Glycol Metabolism

62 Stuporous with blurred vision…An 84-year-old woman weighing 121 lb (55 kg) with no previous history of alcoholism was stuporous on presentation at the emergency department. Her family had found her obtunded and reported that she had complained earlier of blurred vision and had had one episode of emesis. On physical examination, her blood pressure was 107/54 mm Hg, pulse rate 60 beats per minute, and respirations 16 per minute. Her lungs were clear and heart rate was regular, with occasional premature beats. Funduscopic examination was unremarkable, and neurologic examination showed no evidence of focal deficits. The remainder of the examination was unremarkable.

63 Laboratory Values Na 146, K 4.2, Cl 107, HCO3- 14 (low)Anion Gap = 146 – ( ) = 25 (high) BUN 10, Cr 1.4, Glucose 148, Lactate 2.1 Liver Enzymes normal Serum Ethanol < 10 mg/dl (enzymatic method) ABG: pH 7.12 (dangerously acidic), PaO2 71, PCO2 30 Measured serum osmolality = 354 Calculated Osmolality = (2*Na + BUN/2.8 + Glc/18) Osmolal Gap = (measured – calculated) Urinalysis revealed calcium oxalate crystals.

64 Summary and Differential Diagnosis84 y.o. woman with severe CNS depression, blurred vision, a metabolic acidosis with an elevated anion gap (possible respiratory contribution), an osmolal gap of 46.2 and a finding of calcium oxalate crystals in the urine. A toxic alcohol or glycol ingestion is highly suspected. Although blurred vision could suggest methanol poisoning, oxalate crystals in the urine is most consistent with ethylene glycol. Note that with an osmolal gap of 46.2, expect a high ethylene glycol level (46.2 * 5.8 = 268).

65 An explanation… On further questioning, the family reports keeping a 2 L soda bottle containing antifreeze diluted with water in the kitchen. Suspect that patient’s poor baseline eyesight may have led to mistaken ingestion. Confirmed when a sample from the patient’s bedside drinking glass contained antifreeze and an ethylene glycol level was confirmed in the patient’s blood (217 mg/dl).

66 Treatment Gastric lavage and oral activated charcoal.5% dextrose and sodium bicarbonate administered IV. An intravenous IV loading dose of 10% ethanol given for a target of 100 mg/dl EtOH: Vd = 0.54 L/kg * 55 kg = 29.7 L (297 dl) (100 mg/dl * 297 dl)/(1000 mg/g * g/ml) 37.4 ml of 100% EtOH or 374 ml of 10% EtOH A maintenance dose of 75 ml/hr was required to maintain a constant 100 mg/dl (frequently monitoring of EtOH level necessary). (20 mg/dl/hr * 297 dl) / (1000 mg/g * g/ml) Requires 7.5 ml/hr of 100% EtOH or 75 ml/hr of 10% EtOH Patient also given IV thiamine and pyridoxine to encourage non-toxic metabolism of ethylene glycol. Because of extremely high ethylene glycol level, hemodialysis also performed to speed removal.

67 Chronic Toxicity of Anti-Retroviral TherapyL.R., a 31 y.o. HIV+ female, presents to the ED with 3 day h/o fever, tachycardia, and general malaise. Fever not associated with chills, cough or dysuria. She reports intermittent watery diarrhea and abdominal cramping over the past two months. Her diarrhea is associated with early satiety and bloating after PO intake; these abdominal symptoms are often accompanied by chest pressure and sweats. She also reports episodes of aching leg pain progressing to involve her lower back, generally lasting 1/2 to 1 day, over the last two months. L.R. has been on an anti-retroviral regimen of d4T, ddI, and nelfinavir for the past six months.

68 Physical Exam Vitals: T 100.7; HR 115; RR 20; BP 130/90; O2 98 % on room air HEENT: within normal limits Lungs: clear to auscultation bilaterally Heart: regular but tachycardic; III/VI systolic ejection murmur radiating to LUSB. Abdomen: mildly distended but soft and non-tender, no palpable masses, no hepatosplenomegaly, normal active bowel sounds, guaiac negative stool. Pelvic: WNL except single non-tender, movable inguinal lymph node.

69 Lab Tests Na+ 136, K+ 3.4, Cl- 98, HCO3- 11.8BUN 11, Cr 0.8, Glucose 138 WBC 17.9 (66N 14B 3Meta 11L 5M) Hb 14.8, Hct 42.5, Plt 236,000 ABG: pH 7.31, pCO2 32, pO2 111 Lactic Acid 10.3 (nml = ) LDH 441 ( ), ALT 63 (0-40), AST 78 (7-40), Alk Phos 86 (70-230) Normal values: Na , K , Cl , HCO BUN 10-26, Cr , Glu WBC x 103 (Neutrophils 57-67%, Lymphocytes 23-33%, Monocytes 3-7%, Eosinophils 1-3%, Basophils 0-1%) Hgb 12-16, Hct 35-45, Plt x 103

70 What’s Going On? The initial leading diagnosis was infection and dehydration, most likely G.I. in origin. This conclusion was supported by the patient’s malaise and G.I. symptoms, fever and elevated WBC. The negative chest x-ray, negative urinalysis and a lack of cough or dysuria ruled out other potential sites of infection. However, this diagnosis failed to account for several of her other findings including mild lactic acidosis and elevated LFTs (evidence of hepatic injury).

71 Could LR have liver damage?Since the introduction of antiretroviral therapy, there have been numerous reports of hepatotoxicity and lactic acidosis related to Nucleoside Analogue Reverse Transcriptase Inhibitors (nRTIs), e.g. AZT, d4T, ddI, etc.. Early papers documented several severe cases of rapidly progressing liver disease and acidosis that has in some cases proven fatal. Olana et al Massive Hepatic Steatosis and Lactic Acidosis in a Patient with AIDS who was receiving Zidovudine. Clinical Infectious Diseases 21:973-6. Sundar K et al Zidovudine-induced fatal lactic acidosis and hepatic failure in patients with acquired immunodeficiency syndrome: report of two patients and review of the literature. Fortgang I Hepatomegaly and Steatosis in HIV-Infected Patients Receiving Nucleoside Analog Antiretroviral Therapy. Am J of Gastroenterology 90:

72 How Might this Happen? nRTIs are designed to inhibit HIV reverse transcriptase by terminating growing DNA transcripts. However, nRTIs also inhibit human -polymerase, found in mitochondria. Chronic inhibition of -pol leads to massive mitochondrial dysfunction and a severe energy crisis throughout the body. we can’t survive without our mitochondria! Famously “predicted” by Tommy Cheng (Pharm., YMS) from in vitro studies provided a relative “risk” ranking of nRTIs based on activity against -pol that correlated nearly perfectly with clinical experience

73 The Always Important Cori CycleWhen the muscle switches to anaerobic metabolism, lactate is secreted into the bloodstream. The excess circulating lactate is taken up by the liver and converted back into reduced sugar chains by gluconeogenesis, which requires a lot of NADH.

74 Physiologic Consequences of Chronic Mitochondrial DysfunctionEnergy starvation in tissue due to poor aerobic oxidation. lactic acid produced due to anaerobic metabolism of glucose. results in weakness and muscle pain (myopathy). can also result in a peripheral neuropathy. The liver has to metabolize all this lactic acid and attempt to keep up with glucose demands of the body. for a variety of reasons, triglyceride production by the stressed liver dramatically increases resulting in severely elevated blood triglyceride levels, hepatic steatosis (fat deposits) which ultimately damages the liver (hepatitis) and eventually the liver begans to fail (lactic acidosis, hypoglycemia, etc.). Dyslipidemia (fat deposits) are often seen in other tissues.

75 Many of the following slides were “borrowed” from Greg Howe, PhDpharmacogenetics

76 Pharmacogenetics Genetic variation that personalizes an individual’s response to a drug. Two flavors Variation in drug response (pharmacodynamics) e.g. drug receptor polymorphisms Variation in drug disposition (pharmacokinetics) Drug metabolizing enzymes (vast majority to date) Drug transporters

77 Evans WE, McLeod HL. Pharmacogenomics--drug disposition, drug targets, and side effects.N Engl J Med Feb 6;348(6):

78 Molecular DiagnosticsEvans WE, Relling MV. Pharmacogenomics: translating functional genomics into rational therapeutics. Science Oct 15;286(5439):

79 Drug Development General public supports medical research. They want cures. Cures are generally in the form of drugs. Pharmaceutical companies discover 90% of these drugs. It takes 12 years on average for an experimental drug to travel from lab to medicine chest. Only five in 5,000 compounds that enter preclinical testing make it to human testing. Only one of the five tested in people is approved. The cost of this development has been estimated to be in the hundreds of millions (>800 million in 2003). Only half is out of pocket- the rest is lost investment. Only 3 out 10 drugs generate enough profits to cover R&D costs. DiMasi et.al. Journal Health Economics 22: (2003)

80 Adverse Drug ReactionsThe overall incidence of serious and fatal adverse drug reactions (ADR) in the hospitalized patient populations was 6.7% and 0.32% or 2,200,000 serious and 106,000 fatal ADRs in 1994. Deaths from ADRs ranks somewhere between the 4th and 6th most common cause of death in the US. JAMA 279: 1200; 1998

81 Vioxx – Cox-2 Painkiller from MerckInhibits the activity of the enzyme cyclooxygenase which mediates the synthesis of endogenous prostaglandins which causes the joint pain of arthritis. Dollars spent to develop the drug ($800 million) Annual sales of Vioxx before taken off market ($2.5 billion) Market of Cox-2 drugs ($9 billion by 2010) Useful painkillers on market to replace Cox-2 painkiller (aspirin- problems of GI bleeding). 1-2% of people are susceptible to cardiovascular problems when taking Cox-2 painkillers. A state jury found Merck liable on April 28, 2006 for the death of a 71-year-old man who had a fatal heart attack within a month of taking Vioxx and ordered the company to pay $7 million in non-economic compensatory damages and $25 million in punitive damages. In a prior loss, Merck was ordered to pay one plaintiff million dollars -- reduced to 26 million dollars under Texas caps on punitive damages. In Nov. 2007, Merck announced an agreement to pay $4.85 billion to settle about 27,000 lawsuits. Total amount earned by plaintiffs’ firms will be nearly $2 billion in fees at their standard rates of 33 to 40%. New York Times ; ; Washington Post 11/9/07

82 Pharmacogenetics – Drug ResponseAngiotensin converting enzyme (ACE inhibitors, e.g. enalapril) Adrenergic receptors (e.g. albuterol) Dopamine receptors (e.g. haloperidol) Serotonin Transporter (antidepressants) Glycoprotein IIb/IIIa (e.g. aspirin)

83 Pharmacogenetics of the human beta-adrenergic receptors The Pharmacogenomics Journal (2007) 7, 29–37. M R G Taylor Beta receptor agonists (e.g. albuterol – asthma) and beta receptor antagonists (e.g. propanolol for hypertension) are a widely prescribed and critical set of medication. Location of reported ADRB1 and ADRB2 polymorphisms. The location of the reported (literature and in National Center of Biotechnology Information database) polymorphisms is shown for the ADRB1 and ADRB2 genes and the ADRB2 leader peptide sequence. Amino-acid positions are numbered and the wild-type and polymorphic variant are indicated. Red diamonds indicate sites of missense polymorphisms that alter the amino-acid sequence of the protein. Yellow squares indicate polymorphisms of the DNA sequence that do not translate into an alteration of the amino-acid residue (silent polymorphisms).

84 Polygenic Drug ResponseDrug-response phenotype is a complex trait. (a) The HT3 antagonist tropisetrone is a CYP2D6 substrate. After receiving the same dose, patients with high enzyme activity due to gene duplication will not achieve effective drug concentrations. (b) Because the drug is a Pgp substrate, transfer from blood to central nervous system will be influenced by the level of Pgp expression, an additional source of variability, at the blood-brain barrier (BBB). (c) The magnitude of response at the HT3 receptor is influenced not only by drug concentration but also by genetic polymorphisms in the receptor and concentration of neutrotransmitter in the synaptic cleft. Serotonin concentration is influenced by proteins involved in biosynthesis (TPH2, tryptophane hydroxylase 2), transport (SERT, high-affinity serotonin reuptake transporter), and catabolism (MAO, monoamine oxidase). Genetic polymorphisms that affect function have been described for all of the genes encoding these proteins. A pharmacogenetic analysis of nonresponse or poor response (observed in 30% of patients) should include all of these candidate genes.

85 Cancer PharmacogenomicsClinical Pharmacology & Therapeutics (2011) 90 3, 461–466. doi: /clpt S W Paugh, G Stocco, J R McCorkle, B Diouf, K R Crews and W E Evans “In cancer pharmacogenomics, there are at least two genomes of importance: the patient's germline genome (inherited genome variation) and the tumor genome (inherited genome variation plus acquired genome variation). Moreover, there may be additional acquired genome variations in metastatic or recurrent tumor cells that influence drug response and treatment outcome. A comprehensive pharmacogenomic strategy interrogates multiple mechanisms of genome variation in both germline and tumor genetic material (blue box), assessing their influence on multiple drug-response phenotypes (green box). GWAS, genome-wide association studies.” Also, check out what Jeffrey Sklar is doing…

86 Pharmacogenetics – Drug MetabolismMost drug metabolizing enzymes are genetically polymorphic in humans (gene frequencies generally range from 1 – 10%). Probably confers an evolutionary advantage Diversity promotes adequate response to a new environmental toxin (xenobiotic). Good correlation of P450 genetics with diet Complicates drug therapy. Pharmacogenetic Testing (Molecular Diagnostics Labs) Take a look at “The Pharmacogenetics and Pharmacogenomics Database”:

87 Important Pharmacogenetic ExamplesEnzyme Medication Thiopurine S-methyltransferase (TPMT) 6-thioguanine, mercaptopurine, azathioprine Plasma (pseudo)cholinesterase Succinylcholine N-acetyl transferase (NAT1) Isoniazid UDP glucuronosyltransferase 1A (UGT1A1) Irinotecan Dihydropyrimidine Dehydrogenase 5-Fluorouracil CYP2D6 (cytochrome P450 isoenzyme) Codeine

88 Pharmacogenetic Case HPI: 56 y.o. female, 140 lbs., presents with bleeding gums, epistaxis, and bloody stools. Also describes excessive fatigue and mild chest pain on exertion. PMH: Previously diagnosed with psoriasis and put on azathioprine (100 mg/day PO) about one month ago. Lab: Hct 18%, Hb 6, WBC 800, Platelets 1,000: “Pancytopenia” – all blood cell levels strongly suppressed.

89 Role of TPMT in Metabolism of 6-thiopurine (6-TP) MedicationsBoth efficacy and toxicity are dependent on the level of 6-TGNs.

90 Inter-individual Heterogeneity in TPMT Activity Recognized by Weinshilboum over 20 Years Ago.

91 Relationship between Inherited Variations in TPMT Actvity and Serum Levels of (active) 6-TGNs

92 Pharmacogenetics of TPMTHeterogeneity in tissue TPMT activities associated with a limited number of genetic polymorphisms. “Zygosity” for these polymorphisms correlates very well with 6-TGN levels in tissue Risk for toxicity Efficacy, indirectly TPMT genotyping is becoming routine clinically and is recommended before starting any patient on any 6-TP medication. Genotype-based dosing regimens have been established

93 Polymorphisms in the Human TPMT Gene

94 Clinical Consequences of Inherited Polymorphisms in the TPMT Protein SequenceMultiple studies have demonstrated the increased risk of toxicity from 6-TP medications due to TPMT deficiency: increased risk of life-threatening myelosuppression risk for a secondary brain tumor after irradiation greater risk for chemotherapy-associated acute myelogenous leukemia On the other hand, unusually high TPMT activities and consequent low 6-TGN levels have been associated with a higher rate of leukemic relapse (i.e. decreased efficacy). Recent studies have demonstrated a relationship between clinical resistance to 6-MP therapy for inflammatory bowel disease (IBD) and decreased concentrations of 6-TGNs

95 6-TP TDM and TPMT TestingBefore 6-TP drugs are started, TPMT genotype and/or RBC TPMT activity can be assessed and used to guide 6-TP dosage. During treatment, RBC 6-MP, 6-MMP and 6-TGN levels can be measured to guide therapy. TPMT was one of the first enzymes whose levels were tested by DNA-based methods.

96 Biochemical mechanism of decreased tissue TPMT activity (W. Evans at St. Jude’s and R. Weinshilboum at Mayo): Decreased tissue TPMT activities are a result of decreased steady-state enzyme levels and not an inherent catalytic defect of the enzyme. Experiments performed in yeast, COS-1 cells and reticulocyte lysates have demonstrated that deficiency is a consequence of increased protein degradation (synthesis is unchanged). Increased TPMT protein degradation requires association with protein folding chaperones (hsp70, hsp90 and hop), polyubiquitylation of TPMT, an intact proteasome, and is stabilized by addition of excess SAM, suggesting the importance of native-state stabilization in targeting of the polymorphs for intracellular degradation.

97 A General Phenomenon? Intermediate frequency (~ 1 – 10%) genetic polymorphisms appear to exist in the protein sequences of many “small molecule” enzymes. A large number of these polymorphisms reduce tissue enzyme levels by similarly destabilizing the protein and targeting it for proteasomal degradation.

98 A General Phenomenon? Aromatase (cytochrome P450 19) [CYP19]involved in estrogen biosynthesis potential importance in breast cancer pathogenesis and treatment Reduced (NADPH)/quinone oxidoreductase I [NQO1] reduces oxidized metabolites of xenobiotic quinones involved in metabolism of various endogenous quinones including vitamin E generates “antioxidants” seen as a “chemoprotective” agent Phenylethanolamine N-methyltransferase [PNMT] involved in epinephrine synthesis implicated in numerous psychiatric and neurological disorders. Catechol O-methyltransferase [COMT] degradation of catecholamines and pharmaceutical L-dopa, cSNPs assciated with development of estrogen-based cancers and a wide spectrum of mental disorders Histamine N-methyltransferase [HNMT] involved in degradation of histamine (a major neurotransmitter) cSNP has been associated with schizophrenia and asthma Nicotinamide N-methyltransferase [NNMT] suggested involvement in idiopathic Parkinson's and hepatic cirrhosis Multiple Sulfotransferases [SULT]: 1A1, 1A2, 1A3, 1E1 sulfation of steroids, catecholamines, Implicated in breast cancer

99 Uridine Diphosphate Glucuronyl Transferase UGT1AUridine diphosphate glucuronyl transferase (UGT) catalyzes a phase II conjugation step that increases the solubility of drug metabolities. Its normal activity is the glucuronidation of bilirubin. At least 15 transcripts exist from two loci, UGT1A and UGT2A. UGT1 gene locus in humans is located on chromosome 2. There are 5 exons, of which exons 2-5 are at the 3' end of all isoforms of UGT. Exon 1 can be encoded by multiple first exons (at least 13 exist).

100 UGT1A Gene CLINICAL PHARMACOLOGY & THERAPEUTICS 2004;75(6):

101 Clinical Significance- UGT1A1*28Pharmacokinetic studies of irinotecan, an anticancer drug derived from camptothecin, and used with ovarian and colon cancer patients, have shown large inter-individual variability to its exposure. Adverse events for patients receiving irinotecan-based therapy are diarrhea, neutropenia, nausea, vomiting and alopecia (hair loss)- found in 20% to 35% of patients. SN-38 is an active metabolite of irinotecan and is responsible for the pharmacological and toxic effect of irinotecan. SN-38 is glucuronidated by UGT1A1 isoenzyme. The addition of 2 extra TA bases to the TATAA box, that is found in the variant allele UGT1A1*28, is the cause of this variability. The TA addition interferes with binding of the transcription factor IID and results in 30% reduction in expression of UGT1A. Patients who are homozygous for the UGT1A1*28 polymorphism should be considered for a reduced initial dose of irinotecan.

102 CYP2D6 The human cytochrome P450 2D6 (CYP2D6) gene produces a debrisoquine 4-hydroxylase involved in the metabolism of endogenous compounds and drugs. This enzyme is classified as polypeptide 6, subfamily D, family 2, superfamily cytochrome P450. The CYP2D6 gene is located on chromosome 22q13 and is transcribed into a mRNA containing nine exons. A 9432 base pair genomic sequence containing the entire gene and several kilobases of intergenic sequence serves as a full-length gene. CYP2D6 has at least 70 allelic variants

103 Cytochrome P-450 2D6 Mutations Detected CYP2D6 allele Nucleotide change Effect on Enzyme Metabolism *1 None (wildtype) Normal *2 2850C>T *3 2549A>del Inactive *4 1846G>A *5 Gene Deletion *6 1707T>del *7 2935A>C *8 1758G>T *9 delAGA Partially active *10 100C>T *11 883G>C *12 124G>A *17 1023C>T Gene Duplication Increased/decreased (depends on gene)

104 CYP2C19 The human cytochrome P450 2C19 (CYP2C19) gene encodes the enzyme mephenytoin 4-hydroxylase), which is involved in the metabolism of compounds from classes of anticonvulsants and others. This enzyme is classified as polypeptide 19, subfamily C family 2, of superfamily cytochrome P450. The CYP2C19 gene is located on chromosome 10q24 and is transcribed into mRNA containing eight exons and a protein of 490 amino acids. CYP2C19 has two major variant alleles but also other minor variants that result in enzyme deficiency.

105 Effect on Enzyme MetabolismCytochrome P-240 2C19 Mutations Detected CYP2C19 allele Nucleotide change Effect on Enzyme Metabolism *1 None (wildtype) Normal *2 681G>A Inactive *3 636G>A *4 1A>G *5 1297C>T *6 395G>A *7 IVS5+2T>A *8 358T>C Caucasian 85% 15% minor

106 Clinical Significance- CYP2D6 and CYP2C19CYP2D6 (cytochrome P450 2D6) is the best studied of the CYP genes and approximately 10% of the population has a slow acting form of this enzyme and 7% a super-fast acting form. Thirty-five percent are carriers of a non-functional 2D6 allele, especially elevating the risk of ADRs when these individuals are taking multiple drugs. Drugs that CYP2D6 which metabolizes 25% of all prescription drugs includes Prozac, Zoloft, Paxil, Effexor, hydrocodone , amitriptyline, Claritin, cyclobenzaprine, Haldol, metoprolol, Rythmol, Tagamet, tamoxifen, and the over-the-counter diphenylhydramine drugs, Allegra, Dytuss, and Tusstat. CYP2C19 (cytochrome P450 2C19) is associated with the metabolism of carisoprodol, diazepam (Valium), Dilantin, and Prevacid (Ulcers).

107 Four Metabolizing GroupsPeople are divided into four groups: Poor metabolizer (PM)- lack functional enzymes Intermediate metabolizer (IM)- heterogeneous for one deficient allele or carry 2 alleles with reduced activity Extensive metabolizer (EM)- two normal alleles Ultraextensive metabolizer (UEM)- multiple gene copies of functional alleles. Among Caucasian populations most people are EM with 5-10% being PM and a similar amount are UEM. Among African and Asian populations PMs are infrequent

108 Pharmacogenetic Effect of Cytochrome p450 GenotypesPM IM EM UM A. PM poor metabolizer, absent or greatly reduced ability to clear or activate drugs. B. IM intermediate metabolizer. Heterozygotes for normal and reduced activity genes. C. EM extensive metabolizer. The norm. D. UM Ultra Metabolizer. Greatly increased activity accelerating clearance or activation Genelex

109 Roche AmpliChip CYP450 First FDA Approved Microarray Dec. 2004Tests for a patient’s CYP2D6 and CYP2C19 genotype from genomic DNA extracted from whole blood. Tests for 76 CYP2D6 variants Tests for 2 CYP2C19 variants GeneChip® System 3000Dx Microfluidics Reader Affymetrix

110 Preliminary Average Dose Recommendations for Antidepressant DrugsDrug Usual Dose UM% EM% IM% PM% CYP2D6-dependent (10-100) mg Desipramine (des-IP-ra-meen) Mianserin (30-70) mg CYP2C19- dependent 50 (50-200) mg Clomipramine Roche

111 CYP2C9 The human cytochrome P450 2C9 (CYP2C9) gene produces a mephenytoin hydroxylase involved in the metabolism of endogenous compounds and toxic chemicals. This enzyme is classified as polypeptide 9, subfamily IIC, family 2. The CYP2C9 gene is located on chromosome 10q24 and is transcribed into mRNA containing ten exons. A base pair genomic sequence containing the entire gene and several kilobases of intergenic sequence serves as a full-length gene. There are 5 major allelic variants.

112 CYP2C9 Variants Genelex

113 Clinical Significance CYP2C9CYP2C9 (cytochrome P450 2C9) is the primary route of metabolism for Coumadin (warfarin) and Dilantin (phenytoin-antiepileptic). Approximately 10% of the population are carriers of at least one allele for the slow-metabolizing form of CYP2C9 and may be treatable with 50% of the dose at which normal metabolizers are treated. Other drugs metabolized by CYP2C9 include Amaryl, isoniazid, sulfa, ibuprofen, amitriptyline, Hyzaar, THC (tetrahydrocannabinol), naproxen, and Viagra. Approximately 5-10% of all drugs are metabolized by this enzyme

114 Warfarin Reduced Vitamin K Prothrombin Oxidized Vitamin KWarfarin impairs the hepatic enzymes vitamin K-epoxide reductase (VKOR1) and vitamin K reductase, which are required for the "recycling" of oxidized vitamin K into reduced vitamin K. Reduced vitamin K is required for the normal posttranslational gamma-carboxylation of select glutamic acid residues in the N-terminal domain of coagulation factors II (prothrombin), VII, IX, and X. Warfarin Reduced Vitamin K Prothrombin vitamin K-epoxide reductase VKORC1 Oxidized Vitamin K gamma-carboxylation

115 Thrombosis and WarfarinWarfarin is the most widely prescribed oral anticoagulant drug in the United States, with approximately 30 million prescriptions per year. Warfarin has a very narrow therapeutic range, outside of which the patient can suffer from clotting or bleeding events. The drug is responsible for 29,000 emergency room visits for bleeding events every year and responsible for the largest number of drug-related fatalities. Warfarin is given to people with an increased tendency for thrombosis or to people that have already formed a blood clot (thrombus). Common clinical indications for warfarin use are artificial heart valves, deep venous thrombosis, and pulmonary embolism. Dosing of warfarin is complicated because it interacts with many commonly used medications and chemicals present in food. Therapeutic effect monitoring of the degree of anticoagulation is required by blood testing and determining the internal normalized ratio (INR). During the initial stage, the INR is checked as often as every day; the intervals can be lengthened if the patient manages stable therapeutic INR levels on an unchanged warfarin dose. The target (INR) level tends to be 2-3.

116 CYP2C9 and VKORC1 GenotypesCYP2C9 polymorphisms Frequency Caucasians CYP2C9*1- wild type allele cys144/leu359 CYP2C9* arg144/leu CYP2C9* cys144/Ile VKORC1 polymorphisms Frequency Bodin et al (2005) Blood 106:

117 Distribution of Warfarin Dose by CYP2C9 and VKORC1 genotypeBoxes indicate the median and interquartile ranges. Vertical lines above and below boxes indicate the minimum and maximum values. The numbers above whiskers show mean values. Each outlier is shown by an asterisk. Sconce et al (2005) Blood 106:

118 Contribution to Warfarin Dose Variability Regression AnalysisAge 17% Height 16% CYP2C9 polymorphisms 18% % VKORC gene mutations 15% % A= Sconce et al (2005). Blood 106, 2329 B= Rieder et al (2005). NEJM 352, 2285

119 Warfarin Drug Labeling Revision Medicare reimbursementThe warfarin drug labeling was revised on August 16, 2007 by the FDA to include genomic information. It states that lower initial doses should be considered for patients with genetic variations in CYP2C9 and VKORC1.  Physicians are not required to perform genetic testing before initiating warfarin therapy, nor delay the initiation of warfarin therapy. Labeling is intended to inform physicians that up to 30% of their patients (i.e. those who carry the CYP2C9 and/or VKORC1 genetic variations) may be at risk for an adverse response to warfarin. Centers for Medicare & Medicaid Services reviewed the evidence and determined that the test is insufficient to guide health decisions and will not be reimbursed . Genetics and Warfarin Dosing: revision to warfarin labeling. AMA website

120 Camptothecin Derived from Chinese tree Camptotheca acuminata.Wani and Wall Derived from Chinese tree Camptotheca acuminata. Mansukh Wani and Monroe Wall reported the structure in 1966 at Research Triangle Institute in North Carolina. Inhibits DNA topoisomerase I which prevents DNA replication. Same group reported the structure of Taxol (Pacific yew tree) in 1971.

121 SN-38

122 DNA Sequencing of UGT1A1 DNA S Normal6 TAs Normal 15 13 7 DNA S Allele frequency %-40% depending on ethnic background 6 TAs 15 13 7 Heterozygous Mutant 7 TAs 13 15 7 Homozygous Mutant

123 Extra cases (for fun)

124 Toxicities of Autonomic Drugs

125 Autonomics Case #1 16 m.o. girl brought to ED by parents after becoming unresponsive. On way to hospital, had brief episode of eye rolling, produced large mucous stool, then became limp. On presentation to ER, suffers seizure lasting 30 minutes, which is eventually controlled with diazepam and phenytoin.

126 Autonomics Case #1 cont’dVS: RR 35, P 135; pupils 3 mm, sluggish response to light. Child passed a large bloody stool, then resumed seizure activity. Severe respiratory distress required intubation. IV phenobarbital and midazolam given to control seizures; stabilized with Glasgow Coma Score 3. normal pulse for child > 1 year is ~ 100. GCS is on scale of , 3 being worst. Points in areas of eye response (4 possible points), verbal response (5 possible points), motor response (6 possible points).

127 Symptoms--Summary Unresponsive Constricted pupilsEpisodes of bowel incontinence ...what could this be?

128 ACh Overdose! Excessive ACh stimulation at central and peripheral receptors can lead to a diversely presenting cholinergic toxidrome. Classic presentation is unresponsive patient, pinpoint pupils, muscle fasciculations, diaphoresis, emesis, diarrhea, salivation, lacrimation, urinary incontinence (S.L.U.D.G.E.).

129 Causes of ACh Overdose Exogenous ACh ingestion is almost never a cause of overdose because the compound is unstable outside neurons. Most cases of ACh toxicity are due to acetylcholinesterase inhibitors (AChEIs). Common non-pharmaceutical sources of AChEIs include organophoshates and carbamates (common pesticide ingredients).

130 Insecticide Poisoning?Child’s pseudocholinesterase level was 0 (3 - 9 u/ml normal). Parents reported garden had recently been sprayed with pesticide that was found to contain organophosphates. Mother had used pesticide to treat girl’s head lice on day of admission. Moral: don’t use poison as shampoo! Pseudocholinesterase degrades a number of exogenous substances that act at the NMJ (suxamethonium, mivacurium), as well as cocaine, heroin, chlorprocaine, tetracaine, etc. Assaying level is clinically useful since a low level of PCE indicates increased activity in degrading one of these substances.

131 Autonomics Case #2 65 y.o. man presents to ED after an episode of syncope and emesis at his birthday party. On presentation, he is anxious and diaphoretic and complains of non-pleuritic chest pain and headache. PMH: HTN, CAD (CABG x 2, 5 years ago), Type II DM, depression. Meds: Cozaar, Aspirin, Paxil. Drug Intell Clin Pharm Nov;15(11): Hypertensive crisis resulting from avocados and a MAO inhibitor.Generali JA, Hogan LC, McFarlane M, Schwab S, Hartman CR. Cozaar = Losartan (ATRB).

132 Physical Exam VS: T 98.5, P 115, RR 22, BP 210/150General: Anxious, diaphoretic, slightly overweight man HEENT: Pupils dilated to 9 mm. Skin: warm, moist. Chest: Lungs CTA. Tachycardic, bounding pulse. Abdomen: absent bowel sounds.

133 Fight or Flight... What can cause unrestrained sympathetic activation?Cocaine, Methamphetamine, PCP... Dopamine? Norepinephrine? What else?

134 Monoamine Oxidase InhibitorsMAOIs were introduced in 1960s as therapy for depression. Classic interaction between old MAOIs and tyramine (ingredient in fermented foods like wine and cheese). Tyramine displaces NE, and in absence of degradation leads to sympathetic activation.

135 Other Causes The newer MAOIs are more selective and avoid this interaction. In addition, SSRIs (like Paxil) are not associated with irreversible sympathetic interaction by themselves. SSRI + MAOI can lead to “Serotonin Syndrome,” characterized by muscle rigidity, hyperthermia, changes in mental status.

136 Course of Action Patient was treated with IV Fenoldopam (D1/ 2 agonist), which relieved hypertension and gradually slowed heart rate. Once stabilized, he admitted that he had run out of Paxil the day before and had taken “some of my old pills” that he found in his medicine cabinet. These proved to be iproniazid, an old non-selective MAOI.

137 Autonomics Case #3 39 y.o. man presents to ED with orthopnea and altered mental status (confusion). PMH notable for CHF, PAF, panic disorder, GERD, and EtOH abuse. In last 3 days had become fatigued and lost appetite; stopped alcohol. Meds: digoxin, ticlopidine hydrochloride, disopyramide phosphate, paroxetine. Ticlopidine = platelet aggregation inhibitor; disopyramide = IA antiarrythmic; paroxetine = Paxil

138 Case #3 - Physical Exam VS: P 140, BP 115/65, RR 35, T 37.3 CHEENT: WNL Chest: Lungs CTA, heart sounds distant Abdomen: Liver tender to palpitation G/U: small amount of tea-colored urine Extremities: cold; pretibial edema Neuro: somnolent, agitated; unable to follow commands; hyperreflexic.

139 Selected Labs Hct 43.6, HbG 14.5, WBC 13.1, Platelets 4.3BUN 54; Creatinine 4.1 Troponin T neg CK 10,776 (43-239); CK-MB 25.9 (0-3.6) ALT 4375 (4-41); AST 12,283 (10-29) PTT 24 (11-15; INR 11.9); FSP 108 (<5) Myoglobin 181 (<60) Drugs: Paroxetine 70 (<23); Ticlopidine 160 (<50)

140 Lab indications CK, Myoglobin levels w/ no Troponin elevation ==> Rhabdomyolysis BUN, Creatinine ==> Renal Failure ALT, AST ==> Hepatic Failure FSP ==> DIC Drug levels ==> overdose or ???

141 Cytochrome Testing Patient was tested for abnormalities of CYP2D6 and CYP2C19, isoforms of cytochrome P450 that metabolize paroxetine and ticlopidine, respectively. Patient was found to have severely reduced amount of functional CYP2D6. Slow metabolism of paroxetine proposed to have caused symptoms of serotonin syndrome.

142 Immunosuppressants Cytotoxic Agents CorticosteroidsMethotrexate, Azathioprine, Cyclophosphamide Corticosteroids Prednisone, Dexamethasone Signal Transduction Inhibitors Calcineurin Inhibitors (Cyclosporin A), FK506 (Tacrolimus) “Biologic Modifiers” (monoclonal antibodies) Anti- CD25, Anti-TNF IgG1

143 Calcineurin InhibitorsMany immunospressive regimens are based on calcineurin inhibitors (e.g. cyclosporin A and tacrolimus). Side effects include nephrotoxicity, neurotoxicity, diabetogenicity, increased susceptibility to opportunistic infections, and certain de novo malignancies.

144 Immuno Case #1 A year after having a renal transplant, a 76 year-old woman on cyclosporin A develops nausea and mild diarrhea. Her daughter stops by her home to check on her and finds her her confused and extremely lethargic. She is immediately taken to the ER. Physical Exam: Temp = 98.7 BP = 120/80 Edema in lower extremities

145 Lab Tests Blood and urine cultures negativeWBC = 6,000 (no sign of infection) Cr = 5 (normal ) BUN = 45 (normal 8-25) BUN/Cr < 10 (indicates “intrinsic” renal failure) Na+ = 130 (normal = ), Cl- = 88 (normal ), HCO3- = 18, K+ = 5.5 (normal ) Summary: Acute renal failure (intrinsic) with mild acidosis, elevated anion gap and abnormal electrolytes. No overt signs of infection.

146 Differential DiagnosisInfection due to excess immunosuppression (not likely). Transplant Rejection due to subtherapeutic cyclosporine therapy. Nephrotoxicity due to chronically excessive cyclosporine therapy (toxicity).

147 Cyclosporine PK and PD OverviewCyclosporine is usually given once a day PO. G.I. absorption is incomplete and variable. Peak levels seen between 2 – 4 hours. Extensively metabolized by many pathways. Approximately 10 – 15 major metabolites with varying functional activity. Excreted primarily through the biliary system. Terminal half-life between 10 – 24 hours. Both toxicity and efficacy seem to be a consequence of long term “overall exposure” and does not correlate well with acute levels.

148 Monthly Cyclosporine LevelsTrough levels taken just before morning dose. Therapeutic range (trough): 100 – 500 ng/ml. This patient’s levels were between 150 and 350 ng/ml for the past few months, though a slight upwards trends is apparent. Today’s level is 375 ng/ml. Next step is usually to do a renal (kidney) biopsy.

149 Renal Biopsy Shows Classic Cyclosporine-Induced ToxicityTacrolimus and cyclosporine toxicity are associated with a fine isometric vacuolization of the renal tubular epithelium.

150 Serum [Cyclosporine] How did she become toxic?Were her drug levels too high? Trough levels, measured just before a regular dose, were monitored monthly and always within the therapeutic range. So, then why toxic?

151 Efficacy/Toxicity vs. the AUC, Trough (C0) and Peak (C2) LevelsThe efficacy and toxicity of cyclosporine are best estimated by the total “area under the curve” (AUC) of drug concentration during the recurring period of administration. However, because cyclosporine has variable absorption, distribution and metabolism, trough levels do NOT correlate very well with the AUC (reflected in the typical wide reference ranges for trough levels). Next best approach is to combine trough levels (just before next dose) with the serum concentration approximately two hours after each dose, known as a “C2” level. However, this has really only been demonstrated for the immediate post-transplant period (first two weeks), when the patient is still hospitalized and under more controlled conditions. For this patient we can only speculate that over the past few months she has had a higher than usual “overall exposure” or AUC. Possibly, this could have been detected by “C2” levels, though we can’t know that for certain. A common scenario is that a patient is started on another medication (or over the counter “supplement”) that alters cyclosporine metabolism (St. Johns Wort!!!)

152 Use of AUC and C2 Levels for Cyclosporine TDM