*2.3.2 PG strengths*

Personalized medicine has the finality of improving drug safety and efficacy. PG application in clinical practice promises more effective decision-making in relation to diagnostic testing, individualized drug selection, and dosing. This depends on the genetic variations that affect the observed differences in drug response which can be classified into two groups: pharmacokinetic and pharmacodynamic. The genes that influence the pharmacokinetic properties of a drug influence the drug absorption, distribution, metabolism, and excretion. The genes that affect the pharmacodynamic of a drug influence the mechanism of the drug's target and how

it affects the body. The risk-benefit balance of a drug can be evaluated based on PG effects on safety, efficacy, or both. Thus, prior analysis of a patient's genotype may be used to guide clinical decision because the patient may benefit from an alternative drug or reduction in dose of a standard therapy. PG has the potential to reduce the costs (pharmacoeconomy) associated with inappropriate drug or severe adverse drug reactions that require hospitalization [34].

## *2.3.3 PG limitations*

There are several limitations of PG that invariably makes genetic-guided therapies unfavorable in comparison with the standard health care. One of these limitations is the cost of PG test as the additional expense of the genetic test has to be borne as an extra expenditure by the patient, or by healthcare coverage provided by insurance. The second common limitation is the speed of getting genetic test result as the speedy generation of the PG test result is as important as of the genetic information results itself. Imperfect understanding of genetic determinants of drug response is one of the common limitations which in turn affects physician and patient confidence to genetic test [35].

### *2.3.4 Opportunities for growth of PG*

The landscape of PG testing is rapidly evolving. There are several factors that have great impacts on PG development and increase its utility such as accumulations of PG data, the continuity of technological innovations with more accessibility, and costs continuing to drop [35, 36]. Work has been done to suggest that genetic variations may be studied in oral fluid [33]. Oral fluid tests may encourage

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*2.3.5 Uses of PG*

*2.3.6 PG and TR*

functional CYP2D6 alleles [10].

antidepressant activity will be very clear in PM [40].

healthcare professionals to use pharmacogenetic tools more often. Moreover, one of the PG-guided clinical trial principle is to improve and accelerate drug development by correlating genetic patient profile with treatment outcomes in early clinical trial phase, and subsequently extending Phase III only to individuals having the genetic

PG can enhance patient care by applying treatments tailored to genetic makeup and decreasing the risk of severe adverse effects. In 2019, there are 132 PG dosing guidelines for 99 drugs and PG information is included in 309 drug labels. Recently, the genotyping has become more accessible. Next generation sequencing is a cost-effective choice to genotype samples at many PG loci simultaneously [37], and guidelines are available from organizations such as Clinical Pharmacogenetics Implementation Consortium and Dutch Pharmacogenetics Working Group [36]. As mentioned earlier, the major P450 enzyme activities involved in drug metabolism are influenced by genetic variations. Thus, it is not surprising that the currently available examples of the beneficial use of PG in the interpretation of forensic toxicology data predominantly concern drugs that are extensively metabolized by P450s. Many cases exhibited severe intoxication or even death can be attributed to genetic variations in drug-metabolizing enzymes leading to toxic concentrations of either the parent drug or metabolite(s) in the body. Therapeutic opioids (as codeine, TR, oxycodone, hydrocodone, ethylmorphine, and methadone), selective serotonin reuptake inhibitors (as fluoxetine, fluvoxamine, paroxetine, sertraline, and citalopram), and tricyclic antidepressants are commonly implicated in severe adverse effects, as well as drug-related deaths because of

predispositions linked to the safe and effective use of the tested drugs [35].

certain genetic polymorphisms which affect the drug metabolism [38].

The CYP2D6 enzyme is accountable for the formation of M1 (**Figure 1**). Individuals may be classified as PM, IM, EM, or UM according to the metabolic activity of the CYP2D6 enzyme, determined by either phenotyping or predicted from genotyping (**Table 1**) [10]. CYP2D6 genotype data are commonly arranged into star (\*) alleles. The intermediate (extensive/normal commonly divided into fast [NM-F] and slow [NM-S] subgroups) [39]. Individuals with two functional CYP2D6 alleles or one functional and one decreased functional allele were classified as EM. Individuals with one nonfunctional allele and one functional or decreased functional allele are identified as IM [10]. A carrier of two reduced function is also considered IM [22]. Individuals with two nonfunctional alleles as PMs. UMs were defined as individuals with CYP2D6 multiplications, resulting in at least three

Different pharmacological effects can therefore be expected depending on an individual's CYP2D6 phenotype. Both pharmacological properties (opioidergic and monoaminergic) of the drug may also be modified because of modulation of P450 enzymatic activity by genetic polymorphisms and/or drug interactions. The opioidergic properties will be very clear in UM, meanwhile the monoaminergic

The PM, in comparison to EM, have an increased exposure to (+)- and (−)-TR combined with a reduced formation of especially (+)-ODT but also of (−)-ODT. UM, on the contrary, are expected to form higher amounts of (+)-ODT than EM and to be more prone to opioid- related adverse effects. Comparisons between EM and UM are, however, sparse in scientific literature [10]. PMs are experiencing better pain relief

healthcare professionals to use pharmacogenetic tools more often. Moreover, one of the PG-guided clinical trial principle is to improve and accelerate drug development by correlating genetic patient profile with treatment outcomes in early clinical trial phase, and subsequently extending Phase III only to individuals having the genetic predispositions linked to the safe and effective use of the tested drugs [35].

#### *2.3.5 Uses of PG*

*Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

it affects the body. The risk-benefit balance of a drug can be evaluated based on PG effects on safety, efficacy, or both. Thus, prior analysis of a patient's genotype may be used to guide clinical decision because the patient may benefit from an alternative drug or reduction in dose of a standard therapy. PG has the potential to reduce the costs (pharmacoeconomy) associated with inappropriate drug or severe adverse

There are several limitations of PG that invariably makes genetic-guided therapies unfavorable in comparison with the standard health care. One of these limitations is the cost of PG test as the additional expense of the genetic test has to be borne as an extra expenditure by the patient, or by healthcare coverage provided by insurance. The second common limitation is the speed of getting genetic test result as the speedy generation of the PG test result is as important as of the genetic information results itself. Imperfect understanding of genetic determinants of drug response is one of the common limitations which in turn affects physician and

The landscape of PG testing is rapidly evolving. There are several factors that have great impacts on PG development and increase its utility such as accumulations of PG data, the continuity of technological innovations with more accessibility, and costs continuing to drop [35, 36]. Work has been done to suggest that genetic variations may be studied in oral fluid [33]. Oral fluid tests may encourage

drug reactions that require hospitalization [34].

patient confidence to genetic test [35].

*2.3.4 Opportunities for growth of PG*

*2.3.3 PG limitations*

**Figure 1.**

*Tramadol metabolism.*

**164**

PG can enhance patient care by applying treatments tailored to genetic makeup and decreasing the risk of severe adverse effects. In 2019, there are 132 PG dosing guidelines for 99 drugs and PG information is included in 309 drug labels. Recently, the genotyping has become more accessible. Next generation sequencing is a cost-effective choice to genotype samples at many PG loci simultaneously [37], and guidelines are available from organizations such as Clinical Pharmacogenetics Implementation Consortium and Dutch Pharmacogenetics Working Group [36].

As mentioned earlier, the major P450 enzyme activities involved in drug metabolism are influenced by genetic variations. Thus, it is not surprising that the currently available examples of the beneficial use of PG in the interpretation of forensic toxicology data predominantly concern drugs that are extensively metabolized by P450s. Many cases exhibited severe intoxication or even death can be attributed to genetic variations in drug-metabolizing enzymes leading to toxic concentrations of either the parent drug or metabolite(s) in the body. Therapeutic opioids (as codeine, TR, oxycodone, hydrocodone, ethylmorphine, and methadone), selective serotonin reuptake inhibitors (as fluoxetine, fluvoxamine, paroxetine, sertraline, and citalopram), and tricyclic antidepressants are commonly implicated in severe adverse effects, as well as drug-related deaths because of certain genetic polymorphisms which affect the drug metabolism [38].

### *2.3.6 PG and TR*

The CYP2D6 enzyme is accountable for the formation of M1 (**Figure 1**). Individuals may be classified as PM, IM, EM, or UM according to the metabolic activity of the CYP2D6 enzyme, determined by either phenotyping or predicted from genotyping (**Table 1**) [10]. CYP2D6 genotype data are commonly arranged into star (\*) alleles. The intermediate (extensive/normal commonly divided into fast [NM-F] and slow [NM-S] subgroups) [39]. Individuals with two functional CYP2D6 alleles or one functional and one decreased functional allele were classified as EM. Individuals with one nonfunctional allele and one functional or decreased functional allele are identified as IM [10]. A carrier of two reduced function is also considered IM [22]. Individuals with two nonfunctional alleles as PMs. UMs were defined as individuals with CYP2D6 multiplications, resulting in at least three functional CYP2D6 alleles [10].

Different pharmacological effects can therefore be expected depending on an individual's CYP2D6 phenotype. Both pharmacological properties (opioidergic and monoaminergic) of the drug may also be modified because of modulation of P450 enzymatic activity by genetic polymorphisms and/or drug interactions. The opioidergic properties will be very clear in UM, meanwhile the monoaminergic antidepressant activity will be very clear in PM [40].

The PM, in comparison to EM, have an increased exposure to (+)- and (−)-TR combined with a reduced formation of especially (+)-ODT but also of (−)-ODT. UM, on the contrary, are expected to form higher amounts of (+)-ODT than EM and to be more prone to opioid- related adverse effects. Comparisons between EM and UM are, however, sparse in scientific literature [10]. PMs are experiencing better pain relief


*UM, ultra-rapid metabolizer; EM, extensive metabolizer; IM, intermediate metabolizer; PM, poor metabolizer.*

#### **Table 1.**

*CYP2D6 phenotypes in relation to their genotypes.*

than IMs who may experience insufficient relief. UMs are being more likely to experience adverse effects from TR because of the more rapid release of M1 [9].

Apart from M1, there are two additional primary TR metabolites, M2 and M5. M2 is an inactive metabolite which its formation is mediated by presence of two enzymes (CYP2B6 and CYP3A4), while M5 has some opioid effects and its metabolic route is less assured. However, all three enzymes (CYP2D6, CYP2B6, and CYP3A4) are involved in TR metabolism (**Figure 1**) [10].

The CYP2B6 gene is highly polymorphic as well as CYP2D6, but the relevance of CYP2B6 polymorphisms in TR has been less studied. Many CYP3A4 polymorphisms have been identified, although it is not well associated with the phenotypical variability. The CYP3A4\*22 allele is associated with reduction of the enzymatic activity. Recent study tried to explain if the interindividual differences in enantioselective metabolic profiles could be explained by CYP2D6, CYP2B6, and/or CYP3A4 genotype. This item hypothesized that interindividual differences are better explained by the combined genotype of all three enzymes involved in the metabolism of the drug, rather than by CYP2D6 itself [10].

Rudaz et al. showed that enantiomer ratios of all four compounds in urine changed over time in one individual administered 100 mg TR [41]. The largest increase in enantiomer ratio was observed for M2 in CYP2D6 EM and IM, rising from about two to almost seven during 24 h following drug intake. Homozygosity of CYP2B6\*5 and \*6 indicated a reduced enzyme function, although further studies are required to confirm it. The significance of CYP2B6 polymorphisms in TR pharmacokinetics has not been carefully investigated. The same study concluded that consideration for the time is important when you assess enantiomer ratios as it might possibly be used to distinguish a recent TR intake from a past one [10].

Of additional relevance is the significant interethnic differences in CYP2D6 allele frequencies demonstrated across many countries [9]. For example, the CYP2D6\*1 seems to be the most prevalent in studied groups from Egyptians. In contrast, CYP2D6\*4, CYP2D6\*10 and CYP2D6\*DUP showed minor occurrence [42].

Chronic treatment with TR induced hepatotoxicity in all patients with duplicated or normal function (CYP2D6\*DUP or \*1) allele in UMs and EMs displayed higher blood levels of M1, but in none of the patients with impaired or reduced functioning allele \*4 or \*10 [42].

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mass spectrometry, and chromatography.

periods >1 month [45].

samples, urine, and tablets [47].

**3.2 Interpretations of toxicological and genetic analyses**

and PM. The M2/M1 ratio in PM is almost above 7 [48].

Toxicological analysis of TR is based on chemical spot tests, immunoassays,

• Preliminary urine multidrug screen tests as well as automated immunoassay systems which are commonly used with a 0.3 mg/L cut-off value [43].

• Gas chromatography-mass spectrometry (GC-MS) can be used to analyze blood samples for detection of TR and its metabolites. Blood concentrations are ranging from 0.03 to 22.59 mg/L, from 0.02 to 1.84 mg/L and from 0.01 to

• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantitation of TR and its main metabolites in hair. The Lower detected Limits were in the range 0.010–0.030 ng/mg hair. The TR, M1, and M2 concentrations were markedly lower in the nonabuse cases (3.3–20.1 ng/mg, 0.3–1.9 ng/mg, 0.5–4.3 ng/mg, respectively) compared to the abuse cases (63.4–107 ng/mg, 3.8–6.3 ng/mg, 24.9–45.7 ng/mg hair, respectively); also the M2/M1 ratio differed significantly. Hair has become an important biomarker owing to the possibility of detecting target analytes for

• Liquid chromatography coupled to fluorescence detection was also used for TR and its main metabolites M1 and M2 and M5 simultaneous determinations in human plasma, oral fluid, and urine. The lower limit of detection was 2.5 ng/mL for all compounds. The assay was applied to assess the pharmacokinetics of TR and its main metabolites following administration of a single oral dose (100 mg) TR to healthy volunteers [46]. Maltodextrin-modified capillary electrophoresis method was reported to detect the stereoisomers of TR for a single-run chiral separation of TR with detection limit of 2 mg/L. This method was approved to measure the concentration of drug in plasma

• HPLC (linear dual column-MS/MS) was successfully used on oral fluid for the simultaneous detection of TR and its metabolite (M1, M2, and M5) [33].

Different studies tried to correlate concentrations of TR and its metabolites with different genotyping of CYP2D6. The best correlation was obtained for M2/M1 ratio

Levo et al. calculated the TR/M1 and TR/M2. When the number of functional alleles increased, the median TR/M1 decreased. They also showed that median TR/M2 also correlated with the number of functional alleles, but in the reverse direction, as

The literature reported the value of M1/M2 ratio in case of massive TR intoxica-

The metabolic profiles of CYP2D6 PM showed large area under curve (AUCs) of the M2 enantiomers with low corresponding values of the M1 and M5 enantiomers. The (+)-enantiomers of M1 and M5 were affected to a larger extent than the

can be expected based on the complementary nature of two pathways [49].

tion. A quick death has been expected in case of M1/M2 is more than 1 [50].

2.08 mg/L for TR, M1, and M2, respectively [9, 44].

**3. Detection of TR**

**3.1 Techniques**
