Conclusiones del diagnóstico de la situación actual

The urine metabolomic profiles of individuals taking the two cART regimes were compared to HIV positive and negative cART naïve patients. The scores plots for these data in positive and negative ESI are shown in Figures 6.2 and 6.3 respectively, both display tight grouping of the QC samples further supporting the high quality nature of the metabolomic analysis. As with the previous study in Chapter 5 there was no discrimination detected between the metabolomics profiles of urine from cART naïve HIV positive and negative patients. However, patients on cART regimes containing either atazanavir or darunavir clustered separately and away from the cART naïve patients. In positive ESI, the two treatment groups are separated from the cART naïve groups on the first component and from each other on the second component. However, in negative ESI discrimination is only observed on the first component, which suggests that the two nESI modes are detecting different metabolomics profiles associated with the two types of cART intervention.

Figure 6.2: PCA scores plot analysis of HIV status and cART intervention in positive ionization mode

The four patient groups are given in the legend, and patients on the two cART medications also received NRTIs tenofovir and emtricitabine, and the ritonavir protease inhibitor. No discrimination was detected between the HIV positive and negative patients. However, both cART groups discriminate from the cART naïve patients on the first component and cART groups cluster separately from each other on the second component.

Figure 6.3: PCA scores plot analysis of HIV status and cART intervention in negative ionization mode

The four patient groups are given in the legend. No discrimination was observed between the HIV positive and negative patients. However, both cART groups discriminate from the cART naïve patients on the first component and cART groups cluster separately from each other also on the first component.

In order to identify discriminating markers between cART with cART naïve patients, S-plots from OPLS-DA models were constructed for Atazanavir versus cART naïve HIV positive patients and for Darunavir versus cART naïve HIV positive patients (see example Figures 6.4 and 6.5 respectively for positive nESI mode data sets). The majority of the discrimination observed in the PCA scores plots is driven by ions associated with the cART drugs and their metabolites. This is evident from the large amount of markers present at the top right of both S-plots which are markers of darunavir, atazanavir, ritonavir or their metabolites. In total > 70 ions were positively identified as either the parent compounds, metabolites of cART or their associated fragments all of which are detailed in Table 6.3. The identified metabolites given in Table 6.3, are supported by significant fragmentation patterns which were compared to reported fragments of genuine standards in rodent urine and human blood product and hepatocytes studies (Lin et al., 2013, ter Heine et al., 2009, Vermeir et al., 2009, Zheng et al., 2014). Unlike the PIs, only parent compounds for NRTIs tenofovir or emtricitabine were detected. This is due to the minimal metabolism these drugs experience in the body, and the majority of the drug is excreted as the parent compound (Kearney et al., 2004, Gallant and Deresinski, 2003). All but one of these compounds, Darunavir M4, are highly prevalent in the positive mode dataset, and as such only S-plots from the positive nESI mode are shown. This is the first time these metabolites have been reported in a human urine metabolomic study. The table does not

include any of the significant number of adducts of the molecular ions also identified from the S-plot analysis; some of these are however labelled in Figures 6.4 and 6.5. When adducts and dimers are taken into account there are >100 ions associated with the 3 PIs taken in this study. The ability to detect these metabolites of the 3 PI drugs highlights the sensitivity of the nLC- nESI-TOFMS and SPE sample preparation technique, as less than 20% of the total parent compound and its metabolites are excreted via the urine (see Table 6.1) (Rittweger, 2007, Le Tiec et al., 2005, Vermeir et al., 2009). The main route of excretion for these compounds is via biliary excretion (Rittweger, 2007, Le Tiec et al., 2005). By using conventional sample preparation and LC-MS analysis, several of these metabolites such as ritonavir metabolites M1 and M2 were unlikely to have been detected. The ability to detect the parent drug and a wide range of its metabolites suggest that in future studies, links between pharmacogenetics, the role of genetics in an individual’s drug response, and metabolomics effects could be investigated. This may lead to a more personalised approach to pharmaceutical interventions for a wide range of conditions where individual variation in the extent of drug metabolism and persistence may lead to different treatment options being implemented (Song et al., 2012, Trupp et al., 2012, Agúndez et al., 2009).

In addition to investigating the metabolism and/or the effect of pharmaceuticals on the urinary metabolome, it is also possible to identify any individuals who either have not taken the medication, or are particularly high metabolisers. Indeed, this is evident in patient 284 from the darunavir group where neither darunavir nor any of its metabolites or other cART drugs were detected in their urine sample (Figure 6.6). The BPI chromatograms of patient 284 and 302 (Figure 6.7) clearly show that both darunavir and ritonavir are not present in the urine sample of patient 284. The fact that no other cART drug or metabolites are detected makes it unlikely that the patient took their medication prior to providing the urine sample.

The metabolites detected at the other end of the OPLS-DA S-plots such as the highlighted carnitine suggest that cART intervention has led to changes in the endogenous metabolome. These findings are detailed and their biological implications are discussed in the following section.

Figure 6.4: OPLS-DA S-plot analysis for patients taking atazanavir therapy compared to cART naive HIV positive patients

Samples were analysed in positive nESI mode. Many of the main discriminating markers are metabolites of the cART therapy. Many metabolites have only been described in non-primate species or cell lines prior to this study.

Figure 6.5: OPLS-DA S-plot analysis for patients taking darunavir therapy compared to cART naive HIV positive patients

Samples were analysed in positive ESI mode. Many of the main discriminating markers are metabolites of the cART therapy.

Table 6.3: Detailed identity of cART and related metabolites identified by comparison of cART patient metabolomes with that of cART naive patients Metabolite Metabolite ID Rt Exp. mass Thr. mass Formula Fragments

Ritonavir Parent compound 24.09 721.3186 721.3191 C37H48N6O5S2 551.2606, 533.2429, 426.1936, 296.1448, 268.1936,

197.0763, 167.086

Ritonavir M1 Hydroxylation 17.96 737.3163 737.3155 C37H48N6O6S2 719.3021, 426.1872, 312.1411, 284.1420

Ritonavir M2 Deacylation 17.28 580.3333 580.3321 C32H45N5O3S 410.2411, 295.1519, 285.1958, 268.162, 250.1606, 171.1004

Darunavir Parent compound 18.41 548.2439 548.2430 C27H37N3O7S 392.2005, 241.1039, 202.1623, 156.0143, 113.0626

Darunavir M1 Carbamate hydrolysis 12.00 392.2014 392.2008 C20H29N3O3S 241.1021, 156.0147

Darunavir M2 Carbamate hydrolysis and hydroxylation

10.09 408.1971 408.1957 C20H29N3O4S 390.1872, 156.0143

Darunavir M3 Glucuronide of M2 9.67 584.2279 584.2278 C26H37N3O10S 348.0390, 257.0811, 172.0091

Darunavir M4 Glucuronide of parent 14.11 722.2585* 722.2595* C33H45N3O13S 175.0238, 157.0137, 113.0239

Atazanavir Parent compound 19.43 705.3964 705.3976 C38H52N6O7 534.3081, 335.1984, 168.034, 144.1045, 120.0834

Atazanavir M1 Deacylation 14.49 538.3255 538.3241 C26H43N5O7 367.2264

Atazanavir M2 Carbamate hydrolysis 12.59 647.3929 647.3921 C36H50N6O5 534.3082, 192.1040, 168.0835

Atazanavir M3 Carbamate hydrolysis 13.06 647.3912 647.3921 C36H50N6O5 534.3082, 335.1956 168.0835

Atazanavir M4 Hydroxylation 15.41 721.3910 721.3925 C38H52N6O8 534.3086, 351.1931, 168.0836

Atazanavir M5 Keto metabolite 21.18 719.3770 719.3768 C38H50N6O8 701.3637, 530.2915, 363.1786, 197.0829, 168.0824

Tenofovir Parent compound 5.27 288.0884 288.0862 C9H14N5O4P 176.0958, 159.0730

Emtricitabine Parent compound 5.16 248.0522 248.0505 C8H10FN3O3S 130.0507, 114.0944

*Refers to m/z of M-H ion, all other ions reported are M+H. RT refers to retention time and Exp. And Thr. mass to experimental and theoretical masses respectively. Details of fragmentation and order of the relative retention time for ritonavir metabolites, atazanavir metabolites and darunavir metabolites were acquired from pharmacokinetic studies in mice and human blood products and hepatocytes (Vermeir et al., 2009, Lin et al., 2013, ter Heine et al., 2009, Zheng et al., 2014)

Figure 6.6: Loadings plot for darunavir parent compound

It is evident that one individual (red circle) did not take any cART medication on the day of providing the urine sample. The same pattern is present for metabolites of darunavir or any other cART pharmaceutical they were prescribed.

Figure 6.7: Positive ESI BPI chromatogram of urine extracts obtained from patients 302 and 284 with darunavir and ritonavir peaks highlighted in patient 302

The missing peaks corresponding to both darunavir and ritonavir in patient 284 compared to 302 clearly indicate that the cART interventions were not taken in patient 284.