Maurizio Simmaco, MD UOC Laboratorio Analisi Biochimica Clinica

Diagnostica Molecolare Avanzata

Dipartimento Neuroscienze Salute Mentale Organi di Senso (NESMOS)

Strategie di Medicina personalizzata nella pratica clinica

maurizio.simmaco@uniroma1.it

Personalized Medicine

Società Italiana Medicina Personalizzata www.simep.it

Genomics

Personalized/Precision Medicine

Pharmacogenomic

Risk class definition

safe, effective

safe, not effective

not safe, not effective

No safe, effective

Extensive metabolizers

(EM) Intermediate metabolizers

(IM)

Poor metabolizers

(PM) Ultra-rapid metabolizers

(UM)

The Sant’Andrea experience:

The cultural alliance

2019

Drug use in Italy Database OsMed - AIFA

6

Onder et al., 2014

Adherence to prescription drugs in Europe

• 50% of patients taking medication in a manner different from prescription

• Each year about 200,000 people die in Europe for reasons related to the non-adherence to prescribed treatments

IMPRECISION MEDICINE

7

Nicholas J. Schork, Nature, 2016

The Sant’Andrea experience:

organizational model

BIOPHARMACEUTICAL INDUSTRY AND PERSONALIZED MEDICINE

Tufts Center for the Study of Drug Development. Personalized medicine gains

traction but still faces multiple challenges. Impact Report. 2015;17(3)..

Germinal vs Somatic gene variations

Drug metabolism: Phase I, Phase II and Anti-oxidant enzymes

Dependence on sex of hepatic metabolism

CYP450 Enzymes and their sex-dependent activity

Phase II enzymes and their sex-dependent activity

P450 Family

http://bioinformatics.charite.de/transformer/

2C19 interfering drugs

Interfering profile

Etanolo, THC, Cocaina, Caffeina, Teina....

Warfarin farmacogenomica

Clopidogrel

farmacogenomica

The Italian experience (results)

2nd milestone: NOT just Genomics

Nature Reviews Genetics 11, 855-866 (December

2010)

The Italian experience (results)

2nd milestone: NOT just Genomics

•TDM

•Drug metabolism assay

•Metabolomic fingerprint

•Functional Metabolomics

The Italian experience (results)

2nd milestone: NOT just Genomics

Routine Platforms for therapeutic drug monitoring and Metabolomics

QTrap LC/MS/MS API 3200

QTrap LC/MS/MS 5500

• urine metabolites

• urine and plasma amino acids

• antiepileptics

• antipsychotic drugs

• antiarrhythmic drugs

• test for intestinal permeability

• dosage of the drug 5-fluorouracil

-endocrinology

-pediatrics

-psychiatry

-oncology

-cardiology

-neurology

-infectious diseases

-diabetology

-gastroenterology

-Occupational Medicine

The Italian experience (results)

3st milestone: TDM in clinical routine

Drugs with narrow therapeutic range

Consensus Guidelines for

Therapeutic Drug Monitoring in

Neuropsychopharmacology:

Update 2017.

Hiemke C, et al

Pharmacopsychiatry. 2018

Jan;51(1-02):e1.

Therapeutic Drug Monitoring in Psichiatry Farmaco Metabolita Informazioni per il clinico

(Rapporti delle concentrazioni

metabolita:composto

progenitore

(Media-DS - Media+DS))

Amitriptilina Nortriptilina* 0.2-1.8 (n=83)

Aripiprazolo Deidroaripirazolo(*) 0.3-0.5

PM di CYP2D6: 0.2

Bromperidolo Bromperidolo ridotto 0.11-0.51 (n=31)

Buprenorfina Norbuprenorfina 0.8-2.0 (n=5)

Bupropione Idrossibupropione 5-47 (24 h, n=9)

6-30 (12 h, n=9)

Buspirone 6-Idrossibuspirone 25-53 (n=20)

Carbamazepin

a

Carbamazepina-10,11-

eposside

0.07-0.25 (n=14)

Citalopram N-desmetilcitalopram 0.31-0.60 (n=2330)

Clomipramina Norclomipramina* 0.8-2.6 (n=115)

Clozapina Norclozapina Non fumatori (n=98)

0.5-0.6

Fumatori (n=198)

0.4-0.7

Dotiepina Nordotiepina 0-1.4 (n=50)

Doxepina Nordoxepina 0.6-1.6 (n=12)

PM CYP2C19: 1.8 (n=4)

PM CYP2D6: 0.8 (n=6)

Escitalopram N-Desmetilescitalopram 0.3-1.0 (n=243)

Fluoxetina Norfluoxetina* 0.7-1.9 (n=334)

Fluvoxamina Acido di fluvoxamina 0-1.2 (n= 49)

Aloperidolo Aloperidolo ridotto Media 0.6

Imipramina Desipramina 0.6-3.2 (n=14)

PM CYP2D6 4.1 (n=2)

Maprotilina Desmetilmaprotilina 1.1-3.7 (n=76)

PM CYP2D6 4.9

Mianserina N-Desmetilmianserina 0.5-0.8 (n=182)

Mirtazapina N-Desmetilmirtazapina 0.2-1.2 (n=100)

Moclobemide Moclobemide N-ossido 0.8-2.5 (n= 6)

Olanzapin

a

N-

Desmetilolanzapi

na

Non fumatori: 0.1-0.3

(n=76)

Fumatori: 0.2-0.4

(n=69)

Perazina Desmetilperazina 1.1-3.3 (n= 27)

Perfenazi

na

N-

Dealchilperfenazi

na

0.6-2.8 (n = 54)

Quetiapin

a

Norquetiapina 0.1-3.8 (n=25)

(Calcolato per 400 mg)

Reboxetin

a

O-Desetil-

reboxetina

<0.1

Risperido

ne

9-

Idrossirisperidone

*

2D6:

1.5-10.0

PM CYP2D6: ≤ 1

Risperido

ne depot

9-

Idrossirisperidone

*

EM: 1.2-4.3

Sertindolo Diidrosertindolo 1.1-2.7 (n=6)

1.0 nei PM di CYP2D6

Sertralina Norsertralina 1.7-3.4 (n=348)

Trazodon

e

m-clorofenil-

piperazina

(mCPP)

0.04-0.22 (range totale)

Trimiprami

na

Nortrimipramina* 0-12.0 (n=17)

Venlafaxin

a

O-Desmetil-

venlafaxina*

N-

Desmetilvenlafaxi

na

26:

0.3-5.2

PM CYP2D6: ≤ 0.3

UM CYP2D6: > 5.2

0.46-1.48

Potential Cost-effectiveness of Therapeutic Drug Monitoring for Depressed Patients

Treated With Citalopram.

Ostad Haji E, Mann K, Dragicevic A, Müller MJ, Boland K, Rao ML, Fric M, Laux G, Hiemke C.

Ther Drug Monit. 2013 Jun;35(3):396-401.

Clozapine

Plasma concentration

Ris

po

sta

Toxicity:

Drooling

Hypertension

Convulsions

Tachycardia

Fever

Spontaneous incontinence

Temperature

Malignant neuroleptic syndrom

600 300 ng/ml

Sani et al. - J Clin Psychopharmacol. 2010 Dec;30(6):737-9. Development of asymptomatic pancreatitis with paradoxically high serum clozapine levels in a patient with

schizophrenia and the CYP1A2*1F/1F genotype.

XIC of +MRM (20 pairs): 330.100/192.000 Da ID: paroxetine from Sample 5 (cal) of Dataclozapina 22-03-2011.wiff (Turbo Spray) Max. 7.4e5 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5Time, min

0.0

2.0e5

4.0e5

6.0e5

7.4e5

In

te

n.

..

4.54

Internal Standard

XIC of +MRM (20 pairs): 313.200/270.200 Da ID: norcloz from Sample 5 (cal) of Dataclozapina 22-03-2011.wiff (Turbo Spray) Max. 1.0e4 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5Time, min

0.00

2000.00

4000.00

6000.00

8000.00

1.00e4

In

te

n.

..

4.18

Norclozapina

XIC of +MRM (20 pairs): 327.200/296.200 Da ID: clozapine from Sample 5 (cal) of Dataclozapina 22-03-2011.wiff (Turbo Spray) Max. 1.4e4 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5Time, min

0.0

5000.0

1.0e4

1.4e4

In

te

n.

..

4.28

Clozapina

TDM clozapine concentration

Risperidone

Plasma concentration

Ris

po

sta

Toxicity

68,2 15 ng/ml

- Dystonia

Extrapyramidal Symptoms

Hypotension

Tachycardia

Fever

QTc elongation with possible heart arrhythmias

2-6 mg/die

Aripiprazole

Ris

po

sta

300 150 ng/ml

Plasma concentration

Toxicity - Lethargy

Hypertension

Tachycardia

Extrapyramidal Symptoms

Nausea, vomiting, diarrhea

5-30 mg/die

Paroxetina

Plasma concentration

Ris

po

sta

Toxicity

120 40 ng/ml

- Diarrhea

He retched

Tremor

Nausea

Irritability

Sweating

Lethargy

20-40 mg/die

NOT just Genomics

Drug metabolism assay

Roberto M, et al. Evaluation of 5-fluorouracil degradation rate and Pharmacogenetic profiling to predict

toxicity following adjuvant Capecitabine. Eur J Clin Pharmacol. 2017

Onesti CE, et al. 5-Fluorouracil degradation rate could predict toxicity in stages II-III colorectal cancer

patients undergoing adjuvant FOLFOX. Anticancer Drugs. 2017

Borro M, et al. Pre-treatment assay of 5-fluorouracil degradation rate (5-FUDR) to improve prediction of 5-

fluorouracil toxicity in gastro-esophageal cancer. Oncotarget. 2017

Botticelli A, et al. Degradation Rate of 5-Fluorouracil in Metastatic Colorectal Cancer: A New Predictive

Outcome Biomarker? PLoS One. 2016

Lostia AM, et al. A liquid chromatography-tandem mass spectrometry method for the determination of 5-

Fluorouracil degradation rate by intact peripheral blood mononuclear cells. Ther Drug Monit. 2009

5-FU Metabolism

5-FU is rapidly degraded to a-fluoro-b-alanine by the enzyme

dihydropyrimidine dehydrogenase (DPD).

The major active metabolites of 5-FU, essential for its

chemotherapeutic action, are:

- 5-Fluoro-uridine-5 'monophosphate (FUTP) RNA

- 5-Fluoro-2 'deoxyuridine-5'-monophosphate (FdUMP) DNA

w3.uniroma1.it/biocmed2/dima

Prevention of 5-FU-induced toxicities

using pre-treatment DPD deficiency screening

37

Michèle Boisdron-Celle, GOCCI, Firenze, 2013

Early severe toxicities (25% after the first cycle) or death (0.3% =

200 patients/year in France or in Italy; 1,300 in USA (Bamat M,

ASCO 2011)

Related to an asymptomatic DPD deficiency

Complete DPD deficiency = multi-organ toxicity (diarrhea,

mucositis, deH2O, coma,…)

Autosomal Codominant (DPD deficient families)

SEVERE TOXICITY: 30%

TANDEM MASS METHOD

DEVELOPMENT

• Selection of the molecular ion

• Selection of derived fragments

• Optimization of mass spectrometry parameters

METHOD VALIDATION:

Linearity, LLOD, LOQ, precision and accuracy within-day and between-day, sample stability

Ex vivo assay for the determination of the rate of degradation of

5-FU by LC-MS/MS technology

HPLC METHOD DEVELOPMENT

Chromatographic column choice

Mobile phase choice

Optimization of chromatography gradient

Lostia AM, et al. Ther Drug Monit. 2009

5-FU degradation rate distribution (n = 1010; n = 518, females; n = 492, males)

PFS according 5-FU degradation rate

5-FUDR test allows to define 3 metabolic classes

POOR-metabolizer < 0,85

NORMAL-metabolizer 0,85 – 2,2

ULTRA-metabolizer > 2,2

Mazzuca et al ONCOTARGET 2016

Botticelli et al PLoS One. 2016 Sep 22;11(9):e0163105

NOT JUST GENOMICS

METABOLOMICS FINGER PRINTING

Serum xanthurenic acid levels: Reduced in subjects at ultra high risk for psychosis.

Curto M, Lionetto L, Fazio F, Corigliano V, Comparelli A, Ferracuti S, Simmaco M, Nicoletti F,

Baldessarini RJ. Schizophr Res. 2019

Mitochondrial myopathy and comorbid major depressive disorder: effectiveness of dTMS on gait

and mood symptoms.

Rapinesi C, et al Gen Hosp Psychiatry. 2015

Levels of the Rab GDP dissociation inhibitor (GDI) are altered in the prenatal restrain stress mouse

model of schizophrenia and

are differentially regulated by the mGlu2/3 receptor agonists, LY379268 and LY354740.

Orlando R, et al Neuropharmacology. 2014

Head-to head comparison of mGlu1 and mGlu5 receptor activation in chronic treatment of absence

epilepsy in WAG/Rij rats.

D'Amore V, et al Neuropharmacology. 2014

Cinnabarinic acid, an endogenous agonist of type-4 metabotropic glutamate receptor, suppresses

experimental autoimmune encephalomyelitis in mice.

Fazio F, et al Neuropharmacology. 2014

THE KYNURENINE PATHWAY

LC/MS-MS method for the assay of serum levels of all kynurenine metabolites

Fazio F, Lionetto L, et al. Sci Rep. 2015

HPLC-MS/MS MEASUREMENTS OF ALL KYNURENINE METABOLITES IN THE

SERUM AND BRAIN TISSUE

LEVELS OF XANTHURENIC ACID ARE ALSO REDUCED IN SERUM OF THEIR FIRST-DEGREE RELATIVES

p < 0.001

p = 0.004

Sci Rep. 2015 Dec 8;5:17799. doi: 10.1038/srep17799.

Xanthurenic Acid Activates mGlu2/3 Metabotropic Glutamate Receptors and is a Potential Trait Marker for Schizophrenia.

Fazio F, Lionetto L, Curto M, Iacovelli L, Cavallari M, Zappulla C, Ulivieri M, Napoletano F, Capi M, Corigliano V, Scaccianoce S, Caruso A, Miele J, De Fusco A, Di

Menna L, Comparelli A, De Carolis A, Gradini R, Nisticò R, De Blasi A, Girardi P, Bruno V, Battaglia G, Nicoletti F, Simmaco M.

Curto et al. The Journal of Headache and Pain (2016) 17:27

Altered serum levels of kynurenine metabolites in patients affected by

cluster headache

Serum levels of all kynurenine

metabolites are altered in patients

with chronic migraine.

Reduction ofKYNA levels, in

context of an enhanced

release of glutamate might

contribute to a hyperactivity of

NMDA receptors resulting into

nociceptive sensitization

in CH.

Trp metabolites as

Aryl hydrocarbon receptor (AhR) ligands

AhR, a cytosolic ligand-activated transcription factor that mediates xenobiotic metabolism, is a critical regulator of immunity and inflammation, involving fine-tuning of adaptive immunity and mucosal barrier function, maintenance of intestinal homeostasis, and carcinogenesis.

Endogenous Trp metabolites have been proven to act as AhR ligands, and their binding activates AhR to regulate intestinal immunity..

Kynurenine and immune suppression

Botticelli A, et al. J Transl Med. 2018

IDO enzyme induces Treg both directly and by induction of kynurenin expression

Tregactive in peri-

tumoral area

Metabolomics and Gut microbiota The gut microbiota can influence the scope and quality of the immune system response; in turn, the immune system participates in regulating the localization and composition of the gut microbiota

Recent studies have emphasized the profond effects of diet and nutrients on the localization and composition of the gut microbiota as well as on the connection between the gut microbiota and immunological pathways (Thorburn et al.,2014)

Endogenous and bacterial Trp metabolism The microbiota can directly and indirectly modulate host endogenous Trp metabolism, and variations

in Trp metabolism can influence microbial proliferation and microbiota diversity.

Microbiota-associated tryptophan metabolism in the gut. Kynurenine pathway

Gao J,Xu K, Liu H et all. Impact of the Gut Microbiota on Intestinal Immunity Mediated by Tryptophan Metabolism. Front Cell Infect Microbiol. 2018 6;8:13.

Lyer et al., 2018, Cell 173, 1123–1134

Dietary and Microbial Oxazoles Induce Intestinal Inflammation by

Modulating Aryl Hydrocarbon Receptor Responses

Oxazole compounds (class of bacterial-

derived toxins, termed thiazole-/oxazole-

modified microcins (TOMMs)) directly

enhance the ability of IDO1 to metabolize

tryptophan.

Mucosal Immunology (April, 2018)

Host genetic factors

• NOD 2 • ATG16L1 • IL-

23R • FUT2 • CARD9 • ...

Environmental factors •

Diet • Antibiotic • Stress •

Microbiota….

Aryl hydrocarbon receptor and intestinal immunity

Trp metabolites as

Aryl hydrocarbon receptor (AhR) ligands

AhR, a cytosolic ligand-activated transcription factor that mediates xenobiotic metabolism, is a critical regulator of immunity and inflammation, involving fine-tuning of adaptive immunity and mucosal barrier function, maintenance of intestinal homeostasis, and carcinogenesis.

Endogenous Trp metabolites have been proven to act as AhR ligands, and their binding activates AhR to regulate intestinal immunity..

NOT JUST GENOMICS FUNCTIONAL METABOLOMICS (INTESTINAL PERMEABILITY & MICROBIOTA)

A liquid chromatography/mass spectrometry method for the evaluation

of intestinal permeability.

Lostia AM, et al Clin Biochem. 2008

Intestinal permeability is increased in children with non-alcoholic fatty liver disease, and

correlates with liver disease severity.

Giorgio V, et al Dig Liver Dis. 2014

Intestinal permeability in children with recurrent respiratory and gastrointestinal symptoms.

Barreto M, et al J Paediatr Child Health. 2015

Altered intestinal permeability in patients with relapsing-remitting multiple sclerosis: A pilot

study.

Buscarinu MC, et al. Mult Scler. 2017

The selective permeability of the intestinal barrier

is altered in many diseases

• Celiac disease

• Crohn’s

• Parasitosis

• Cystic fibrosis

• Pancreatitis

• Rheumatoid Arthritis

• Inflammatory bowel diseases

• Bacterial and viral gastroenteritis

• Spondyloarthropathies

• AIDS / HIV

• Food Allergies

• Asthma, eczema, urticaria

• Autism / Schizophrenia

I.P. Evaluation

Società Italiana Medicina Personalizzata www.simep.it

Intestinal permeability

Approximately 10–15% of adults

in the U.S. suffer from irritable

bowel syndrome (IBS), which

yields an excess of eight billion

dollars in medical costs annually

Transporters in the intestinal barrier

Società Italiana Medicina Personalizzata www.simep.it

Società Italiana Medicina Personalizzata www.simep.it

Mass spectrum and ion fragmentation

Turbo Ion Spray

Negative modality Pre Post

Società Italiana Medicina Personalizzata www.simep.it

A liquid chromatography/mass spectrometry method for the

evaluation of intestinal permeability.

Lostia AM, et al Clin Biochem. 2008

Intestinal permeability is increased in children with non-

alcoholic fatty liver disease, and correlates with liver disease

severity.

Giorgio V, et al Dig Liver Dis. 2014

Intestinal permeability in children with recurrent respiratory

and gastrointestinal symptoms.

Barreto M, et al J Paediatr Child Health. 2015

4 sugar probes

Clinica Chimica Acta 418 (2013) 97–101

Courtesy from Dominga Maio

Intestinal permeability evaluation

Conditions for digestion and absorption of SUCRALOSE

Sucralose is not fermented by numerous colon bacteria and percentage of urinary recovery must be <1.5% of

the administered dose. Used for assessing the condition of the large intestine

mucosa.

Urinary reduced recovery Mannitol

& Lactulose

Normal intestinal mucosa

Colon Alterated permeability

Urinary sucralose high recovery

Normal urinary recovery

Lactulose & sucrose

Urinary sucralosy high recovery

Small intestine Alterated permeability

Major pathways of drug metabolism

and the role of microbiota

60

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61

JL Alexander, Nature Reviews | Gastroenterology & Hepatology 14 | 2017 | 357

Microbiota–host interactions:

modulation of chemotherapy efficacy and toxicity

Microbiota-triggered innate immune receptors S Roy, G Trinchieri, Nature Reviews | Cancer Vol 17, may 2017, 271

PM Concept Agreement

The Precaution Principle as

the leading rule to guarantee

protection of patients and doctors

Curr Pharm Biotechnol. 2016;17(10):926-9

The Precaution Principle

Image from http://www.cmbi.ru.nl/redock/Glossary.php

Design of genotyping panels Detect “crash points”

Choose alternative ways and

get your destination

Image from google maps

Image from http://www.infoblu.it/it/traffico+roma

Application in real-world:

The Genomic road map and the MIFAR strategy

Score’s logic:

the landscape approach

The Sant’Andrea experience Workflow in polytherapy regimen by “Precaution principle”

www.esptnet.eu