Reactivity Under Physiological Conditions

Quantification of intrinsic lipidation products, combined with the newly developed under-standing of the pathway of propranolol 1 intrinsic lipidation, provides a useful tool for further examining membrane reactivity of propranolol 1. Reactivity in vitro under physiological con-ditions provides a biologically relevant starting point. In depth examination of propranolol 1 reactivity within different membrane models provides a route towards understanding reactivity preferences related to phospholipid head group and acyl chain composition. A developed

Chapter 6. Propranolol Intrinsic Lipidation in vitro 169

understanding of propranolol 1 intrinsic lipidation in vitro is vital in order to unravel the biological and pharmaceutical potential. Furthermore, such investigation is expected to aid in directing the future study of propranolol 1 intrinsic lipidation, with particular focus in vivo.

Initially, comparison of propranolol 1 intrinsic lipidation within three different membrane models was attempted. Model membranes were prepared containing phospholipid compositions of DOPC, DOPC:DOPS (4:1), and DOPE:DOPG (3:1) in order to mimic eukaryotic, viral and prokaryotic membranes respectively. Whilst previous analysis shown in Chapter 5 of this thesis indicated reactivity differences between these membrane types, it was hoped that quantification could provide a more definitive comparison. Application of calibration data to the analytical response within each membrane type provides concentration data for propranolol 1 derived species.

Total oleoylated propranolol product concentration ([O-oleoyl propranolol 47] plus [N -oleoyl propranolol 13]) following a 72 hour incubation of propranolol 1 with each membrane type is presented in Table 6.15. Concentration results are obtained as an average of samples prepared and analysed in triplicate. Comparison reveals 68.7 ng mL⁻¹ of oleoylated propranolol product formed in the presence of a DOPC membrane, equivalent to 12 % of the total propranolol 1 derived content of the mixture. Modification of the membrane to contain DOPC:DOPS (4:1) dramatically reduces the oleoylated product concentration to 3.6 ng mL⁻¹, just under 2

% of the total propranolol 1 content. The observed reduction in reactivity is counter-intuitive considering the increased membrane binding affinity of cationic amphiphilic propranolol 1 to a negatively charged PS membrane.²⁷⁰ Considering the increased electrostatic attraction, reduced reactivity can be attributed to a slightly altered membrane binding orientation of propranolol 1 within these negatively charged membranes, enhancing favourable electrostatics but reducing alcohol proximity to the phospholipid ester linkages. Further binding orientation modifications may arise as a result of inherent membrane asymmetry and domain formation, given that PS containing phospholipids preferentially inhabit the membrane inner leaflet in vivo.^1,283,284 Propranolol 1 rich domain formation within the inner leaflet is a potential outcome of this asymmetry, a phenomenon observed for other cationic amphiphilic drugs binding to negatively charged membranes.^27,285

170 6.3. Quantifying Propranolol Intrinsic Lipidation

Membrane Composition

Total Oleoyl Propranolol Concentration

(ng mL⁻¹)

Total Oleoyl Propranolol as % Total Propranolol

Content

DOPC 68.7 12.1

DOPC:DOPS (4:1) 3.6 1.6

DOPE:DOPG (3:1) 16.3 3.1

Table 6.15 Comparison of the total concentration of oleoyl propranolol (O-oleoyl propranolol 47 plus N -oleoyl propranolol 13) after 72 hours under physiological conditions within three membrane types.

The prokaryotic model membrane DOPE:DOPG (3:1) resulted in a total oleoylated product concentration of 16.3 ng mL⁻¹, as shown in Table 6.15, corresponding to just over 3 % of the propranolol 1 derived species. Reduced reactivity compared to the DOPC membrane model supports the determination that increased propranolol 1 membrane binding affinity does not guarantee increased intrinsic lipidation. However, increased reactivity compared to DOPC:DOPS (4:1) suggests more favourable membrane properties such as a lack of domain formation, altered position of the negative charge, or more favourable binding orientation.

Analysis of intrinsic lipidation product formation in eukaryotic, viral and prokaryotic membrane models after 72 hours can be further separated into the component species, as shown in Table 6.16. Interesting differences are evident when comparing the proportion of O-oleoyl propranolol 47 and N -oleoyl propranolol 13 between systems. After 72 hours, DOPC and DOPE:DOPG (3:1) membranes exhibit an increased proportion of N -oleoyl propranolol 13 compared to O-oleoyl propranolol 47. This observation is attributed to initial reactivity at the alcohol moiety of propranolol 1, followed by an intramolecular O to N migration. By contrast, the DOPC:DOPS (4:1) membrane exhibits increased levels of O-oleoyl propranolol 47 compared to N -oleoyl propranolol 13. Insufficient O-oleoyl propranolol 47 production, or reduced rate of O to N migration compared to O-oleoyl propranolol 47 intrinsic lipidation, may cause this. However, it is worth noting that product concentrations within the DOPC:DOPS (4:1) membrane are close to the limit of quantitation. Instrument error associated with these values is thus significant, resulting in reduced confidence in the reported relative proportions of O-oleoyl propranolol 47 and N -oleoyl propranolol 13.

Chapter 6. Propranolol Intrinsic Lipidation in vitro 171

Membrane Composition

O-oleoyl propranolol 47 as % Total

Propranolol 1 Content

N-oleoyl propranolol 13 as % Total

Propranolol 1 Content

DOPC 3.0 8.9

DOPC:DOPS (4:1) 1.0 0.6

DOPE:DOPG (3:1) 0.9 2.1

Table 6.16 Comparison of the proportion of O-oleoyl propranolol 47 and N -oleoyl propra-nolol 13 as % of total proprapropra-nolol 1 content after 72 hours under physiological conditions within three membrane types.

Study of propranolol 1 intrinsic lipidation under physiological conditions after 72 hours provides informative data regarding the reaction. However, more detailed information can be gleaned through analysis of reaction mixtures at regular time points. As a result, portions of propranolol 1 reaction mixture with each membrane model were taken and analysed in triplicate at time points of 2, 4, 6, 8, 24, 72, 144, and 216 hours. To negate for error associated with fluctuation in sample dilution prior to analysis, values are normalised and reported as a percentage of total unmodified plus modified propranolol 1 content. Data describing total oleoylated propranolol content in each membrane type over the time period studied is presented in Fig. 6.35. Preliminary inspection suggests an increase in oleoylated product formation over time for each membrane composition up to 72 hours. However, later time points 144 and 216 hours indicate a decrease in observed oleoylated product proportion for all membrane types. Data normalisation combined with performance of experiments in triplicate prevents this decrease in oleoylated product being attributed to dilution or sample preparation error.

Furthermore, chemical modification of the oleoylated product, such as diacylation or acyl chain oxidation, which may contribute to oleoylated product depletion have been investigated and eliminated as likely causes. Hydrolysis of O-acylated propranolol derivatives has been noted previously in Section 6.3.2, providing a possible explanation or the observed decrease in oleoylated product proportion. However, observed oleoylated product depletion at later time points is better attributed to the known instability and loss of structural integrity within liposome membrane models beyond 120 hours.²⁴¹ This instability may be further exaggerated by the attainment of a critical mass of oleoylated propranolol derivative, such that further reactivity is prevented and hydrolysis is promoted. Considering this observed complexity in later time points, analyses conducted at 144 and 216 hours were not taken forward for further study.

172 6.3. Quantifying Propranolol Intrinsic Lipidation

0 50 100 150 200 250

0 5 10 15

Time (h)

%TotalPropranololContent

47

Figure 6.35 Comparison of total oleoyl propranolol product over time period up to 216 hours (9 days) within three membrane types: (i) DOPC shown by red circles; (ii) DOPC:DOPS (4:1) shown by green squares; (iii) DOPE:DOPG (3:1) shown by blue triangles.

Closer inspection of membrane models DOPC, DOPC:DOPS (4:1) and DOPE:DOPG (3:1), under physiological conditions over the first 8 hours of study, indicates oleoylated product formation within 2 hours, Fig. 6.35. O-oleoyl propranolol 47 is the sole contributor to total oleoylated product at this time point, as shown in Table 6.17, and continues to be over the initial 8 hours of study. Considering observation of O-oleoyl propranolol 47 only and the lack of ester hydrolysis determined previously, the rate constant attributed to transesterification would be equal to the observed rate constant over the first 2 to 8 hours of study. Relevant plots were therefore prepared, Fig. 6.36, in order to determine rate equation order as zero order (Fig. 6.36 (a)), first order (Fig. 6.36 (b)), or second order (Fig. 6.36 (c)). However, following analysis of the resulting data it remained unclear as to which order of reaction was most relevant. Given challenges in observation and quantification of oleoylated product at these early time points, with low abundance causing issues for both instrument detection and peak modelling, the observed issues in rate constant determination are unsurprising. Furthermore, results are complicated by potential variation in transesterification rate over time, evidenced by reduced DOPC reactivity compared to viral and prokaryotic systems over the first 2 to 8 hours, a trend reversed at 72 hours as demonstrated in Table 6.15. These considerations act to explain some of the discrepancies in oleoylated product content observed during this time period, such as a perceived decrease over time.

Chapter 6. Propranolol Intrinsic Lipidation in vitro 173

Total Oleoylated Product = O-Oleoyl Propranolol 47

Time (h) DOPC PC:PS PE:PG

2 0.06 0.06 0.19

4 0.02 0.11 0.46

6 0.05 0.46 1.32

8 0.08 0.36 2.51

Table 6.17 Comparison of the proportion of oleoyl propranolol equal to O-oleoyl propranolol 47 as % of total propranolol 1 content over the first 8 hours of study plus under physiological conditions within three membrane types: (i) DOPC; (ii) DOPC:DOPS (4:1); (iii) DOPE:DOPG (3:1).

47Figure 6.36 Plots prepared for the determination of rate equation order within DOPC (red circles), DOPC:DOPS 4:1 (green squares) and DOPE:DOPG 3:1 (blue triangles) membrane models: (a) zero order; (b) first order; (c) second order.

Between 24 hours and 72 hours, analytical results obtained from the study of propranolol 1 incubation under physiological conditions with three membrane models, prove most informative.

A linear increase in proportion of total oleoylated propranolol product is evident for each membrane type, as shown in Fig. 6.37. The gradient attributed to each increase represents the rate of oleoylated product formation, Table 6.18, however care must be taken considering the low number of data points available. The steepest gradient of 0.17 h⁻¹, attributed to

174 6.3. Quantifying Propranolol Intrinsic Lipidation

the fastest rate of oleoylated propranolol formation, is credited to the eukaryotic DOPC membrane. The prokaryotic model DOPE:DOPG (3:1) exhibits the second fastest rate with a gradient of 0.04 h⁻¹, and the viral model DOPC:DOPS (4:1) the slowest rate with a gradient of 0.02 h⁻¹. Variation in rate of oleoylated product formation presents a clear preference towards propranolol 1 intrinsic lipidation in a eukaryotic membrane model, despite reduced electrostatic attraction compared to DOPC:DOPS (4:1) and DOPE:DOPG (3:1) membranes.

It is impossible to attribute the increased reactivity of the eukaryotic DOPC membrane to a single factor, such as binding orientation, hydration levels and nucleophile properties. This conclusion highlights the complex nature of intrinsic lipidation, and supports the assumption that numerous factors contribute to reactivity. Further, analysis of the rate of oleoylated product formation must be considered in light of the broader and more complex picture of propranolol 1 intrinsic lipidation. Several distinct reactions contributed to the production of total oleoylated propranolol content. These reactions, which include transesterification, hydrolysis, and intramolecular O to N migration, each have a unique associated rate constant.

It is unclear as to whether these individual rate constants remain consistent over time or are altered by environmental factors such as membrane integrity or accumulation of a critical proportion of oleoylated product.

0 20 40 60 80

0 5 10 15

Time (h)

%TotalPropranololContent

47

Figure 6.37 Comparison of total oleoyl propranolol product over time period up to 72 hours within three membrane types: (i) DOPC shown by red circles and modelled with a solid red line; (ii) DOPC:DOPS (4:1) shown by green squares and modelled by a green dahsed line; (iii) DOPE:DOPG (3:1) shown by blue triangles and modelled by a blue dotted and dashed line.

Membrane Type Rate (h⁻¹)

DOPC 0.17

DOPC:DOPS (4:1) 0.02 DOPE:DOPG (3:1) 0.04

Table 6.18 Comparison of the rate of oleoyl propranolol formation within three membrane types, determined from gradients of trend fitted data shown in Fig. 6.37.

Chapter 6. Propranolol Intrinsic Lipidation in vitro 175

Separation of total oleoylated product proportion into its contributing species, O-oleoyl propranolol 47 and N -oleoyl propranolol 13, aids in understanding the relative rates of formation. Despite variation in absolute quantities over 72 hours, Fig. 6.38 indicates that the three membrane models exhibit similar trends. As previously demonstrated in Table 6.17, O-oleoyl propranolol 47 production via transesterification from a membrane phospholipid, is observed within two hours. O-oleoyl propranolol 47 production then continues to increase before starting to plateau as a result of competitive O to N migration. Considering both the observed plateau in O-oleoyl propranolol 47 concentration reached over time, and the determination of O to N migration as the primary mechanism of O-oleoyl propranolol 47 decomposition, the rate of initial intrinsic lipidation via transesterification is predicted to be comparable to the rate of O to N migration.

0 20 40 60

Figure 6.38 Comparison of concentrations of O-oleoyl propranolol 47 (experimental data1 as green circles) and N -oleoyl propranolol 13 (experimental data as red squares and trend fitted by dashed red line) products over time period 24 and 72 hours within three membrane types: (a) DOPC; (b) DOPC:DOPS (4:1); (c) DOPE:DOPG (3:1).

In contrast to O-oleoyl propranolol 47, production of N -oleoyl propranolol 13 is characterised by an initial lag phase followed by a linear increase, shown in Fig. 6.38. The lag phase is attributed to time designated for the accumulation of O-oleoyl propranolol 47, required prior to N -oleoyl propranolol 13 formation via O to N migration. Extrapolating the rate of N-oleoyl propranolol 13 production back from 24 hours, the lag phase can be estimated to last approximately 4 to 5 hours, as highlighted by dashed vertical lines in Fig. 6.38. However, at

176 6.3. Quantifying Propranolol Intrinsic Lipidation

experimental time points close to this, 6 and 8 hours, N -oleoyl propranolol 13 is not observed by MS due to low abundance (<1 ng mL⁻¹) preventing detection. This observation further supports the issues raised previously, associated with validity of results obtained by mass spectrometry at the limit of instrument detection. The subsequent linear increase in N -oleoyl propranolol 13, irrespective of precursor concentration, indicates both a consistent rate of formation and stability of N -oleoyl propranolol 13. The gradient associated with the linear increase in N -oleoyl propranolol 13 can be correlated with rate of its production. In a now familiar trend, the membrane model DOPC exhibits the fastest rate of product formation, followed by DOPE:DOPG (3:1), and then DOPC:DOPS (4:1). It is unclear whether this rate of N -oleoyl propranolol 13 formation continues indefinitely until no precursor propranolol 1 or O-oleoyl propranolol 47 remains, or whether production is capped at a critical proportion of N -oleoyl propranolol 13.

Propranolol 1 intrinsic lipidation under physiological conditions clearly differs between model membranes containing phospholipids with differing phosphate head groups. Reactivity varia-tion between membranes containing different acyl chain types is also of interest. In particular, investigation of preferential transfer of an acyl chain, either due to chemical composition or position on the phosphate backbone, is required. Phospholipids POPC and OPPC, which contain the same acyl chain moieties in alternate positions on the phosphate backbone, provide an excellent means of probing preferential transfer. Additionally, both POPC and OPPC contain a eukaryotic PC phosphate head group, optimising both predicted reactivity towards intrinsic lipidation, and pharmaceutical relevance.

Comparison of total palmitoylated product and total oleoylated product observed within POPC and OPPC membranes following 72 hours under physiological conditions, is presented in Table 6.19. Liposomes containing POPC produced an increased quantity of palmitoylated product, whereas liposomes containing OPPC present with increased oleoylated product.

These observations correspond to preferential transfer of the acyl chain at the sn-1 position of the phosphate backbone. Acyl transfer dictated by backbone position rather than chemical nature highlights the importance of membrane binding orientation, and suggests reactivity facilitated by increased proximity between the alcohol of propranolol 1 and the sn-1 position of the phosphate backbone.²⁶⁷An alternative interpretation is that increased lability promotes transesterification at the sn-1 position over the sn-2 position.

Chapter 6. Propranolol Intrinsic Lipidation in vitro 177

POPC 100.7 36.8 49.8 18.2

OPPC 13.3 5.4 20.9 8.5

Table 6.19 Comparison of the total concentration of palmitoyl propranolol (O-palmitoyl propranolol 62 plus N -palmitoyl propranolol 12) and the total concentration of oleoyl propra-nolol (O-oleoyl proprapropra-nolol 47 plus N -oleoyl proprapropra-nolol 13) after 72 hours under physiological conditions within POPC and OPPC membrane types.

Differences in total acylated product formation, the sum of palmitoylated product plus oleoylated product, are also evident between POPC and OPPC membrane types, Table 6.19.

Intrinsic lipidation within a POPC membrane attributes 55 % of the total propranolol 1 derived species to acylated propranolol, compared to 14 % within an OPPC membrane. The reason for this variation is unclear, but is hypothesised to be due to either minor differences in propranolol 1 binding orientation, or to membrane fluidity variation considering the transition temperatures of -2^◦C and -9 ^◦C for POPC and OPPC respectively. The conversion level of 55 % observed within the POPC membrane contrasts with earlier observations for lipidation reactions in DOPC (Chapter 5) which suggested that acylated propranolol production is capped at a final critical concentration.

In parallel to experiments conducted to test the influence of phosphate head group upon propranolol 1 intrinsic lipidation, regular reaction monitoring was executed utilising POPC and OPPC membranes. Analysis of total palmitoylated propranolol and total oleoylated propranolol at time points of 2, 4, 6, 8, 24, 72, 144 and 216 hours, is presented in Fig. 6.39.

Observations discussed during comparison of eukaryotic, viral, and prokaryotic membrane models regarding data complexity at time points of 144 and 216 hours also hold for the POPC and OPPC models tested. The validity of results obtained at early time points, 8 hours and prior, is also noted here given that low abundance causes instrumental challenges, and thus results exhibit significant error. Despite these caveats, preferential sn-1 transfer is evident for both membrane compositions as early as 2 hours into the study. The preference is maintained for both membrane compositions throughout the time period up to 72 hours. Application of linear trend fitting to data presented in Fig. 6.39 at time points up to 72 hours informs upon the relative reaction rates, summarise in Table 6.20. Gradients of formation up to 72 hours for the species associated with sn-1 acyl transfer are steeper than those of their sn-2

178 6.3. Quantifying Propranolol Intrinsic Lipidation

counterparts, indicating a faster associated rate. Beyond 72 hours, at time points 144 and 216 hours, preferential transfer becomes less obvious, attributed to an increased lysolipid contribution to membrane composition and associated variation in intrinsic lipidation rate.

0 50 100 150 200 250

0 20 40 60 80

Time (h)

%TotalPropranololContent (a)

0 50 100 150 200 250

0 5 10 15

Time (h)

%TotalPropranololContent (b)

47Figure 6.39 Comparison of concentrations of total palmitoyl propranolol (green circles) and total oleoyl propranolol (red squares) intrinsic lipidation products formed under physiological conditions over a time period up to 216 hours (9 days) within two membrane models: (a) POPC; (b) OPPC.

Membrane Composition Rate sn-1 Transfer (h⁻¹) Rate sn-2 Transfer (h⁻¹)

POPC 0.48 0.24

OPPC 0.13 0.08

Table 6.20 Rates of sn-1 and sn-2 transfer within POPC and OPPC membrane systems under physiological conditions. Rates were calculated as the gradient of plots of relevant product concentration vs. time up to 72 hours.

Data presented in Table 6.21 separates total palmitoylated product and total oleoylated product formed within POPC and OPPC membranes after 72 hours under physiological conditions into the constituent O-acylated and N -acylated species. Preferential sn-1 transfer is exhibited for both POPC and OPPC membranes within both O-acylated and N -acylated propranolol species. Irrespective of membrane composition and acyl chain chemistry, the N-acylated propranolol analogue is identified as the major reaction product. The variation between O-acylated propranolol and N -acylated propranolol concentration is striking, with the latter present at four to eight times the abundance. This observation is consistent with the behaviour of propranolol 1 within DOPC and DOPE:DOPG (3:1) membrane models, previously presented in Table 6.16.

Chapter 6. Propranolol Intrinsic Lipidation in vitro 179

Membrane Composition

% O-palmitoyl

propranolol 62 % N -palmitoyl

propranolol 12 % O-oleoyl

propranolol 47 % N -oleoyl propranolol 13

POPC 4.1 32.8 2.1 16.1

OPPC 1.0 4.4 1.3 7.2

Table 6.21 Comparison of the % of O-palmitoyl propranolol 62, N -palmitoyl propranolol 12, O-oleoyl propranolol 47, and N -oleoyl propranolol 13 after 72 hours under physiological conditions within POPC and OPPC membranes.

Separation of acylated propranolol 1 derivatives into constituent species can also be achieved utilising by performing reaction monitoring at regular time points, as demonstrated in Fig. 6.40.

Production of O-palmitoyl propranolol 62 and O-oleoyl propranolol 47 within the POPC membrane, Fig. 6.40 (a) and (b), increases initially from time point zero before reaching a plateau. Comparison to data obtained for DOPC suggests a similar mechanism of action, in which the rate of transesterification is similar to rate of O to N migration. By contrast, proportion of O-palmitoyl propranolol 62 and O-oleoyl propranolol 47 within an OPPC membrane exhibits an initial increase followed by a fall, Fig. 6.40 (c) and (d). The decrease observed between 24 and 72 hours suggests that rate of O to N migration plus hydrolysis outstrips rate of transesterification. An increased rate of breakdown of the O-acylated products may be attributed to facilitation of intramolecular transfer or hydrolysis by an increase in membrane fluidity, or binding orientation of the small molecule.

As shown in Fig. 6.40, N -palmitoyl propranolol 12 and N -oleoyl propranolol 13 formation

As shown in Fig. 6.40, N -palmitoyl propranolol 12 and N -oleoyl propranolol 13 formation

In document Reactivity at the membrane interface (Page 177-190)