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 [email protected]
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
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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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