In publication II, the intra-brain distribution of prodrug 1 selected from the previous study (publication I) was investigated using brain-slice method combined with homogenate method and compared to that of parent drug ketoprofen. In addition, the involvement of LAT1 to brain cellular barrier transport of prodrug 1 was studied using LAT1 inhibitor (Huttunen et al., 2016a). The study was conducted in mice and rats to detemine intespecies difference in intra-brain distribution of prodrug. These data were integrated to PK parameters of prodrug 1 using the combinatory mapping approach (CMA) for estimation of brain intracellular delivery of parent drug via prodrug approach.
4.8.1 Evaluation of intra-brain distribution
The study brain slice protocol was adapted from work of (Friden et al., 2009a, Loryan et al., 2013). Briefly, six and ten 300-μm coronal slices from drug-naïve rat or mouse, respectively, were cut using a vibrating blade microtome Leica VT1200 (Leica Microsystems AB, Sweden) and placed into an 80-mm diameter beaker. The dish was filled with 15 mL or 12 mL (for rat and mouse slices, respectively) of pre-oxygenated aECF with either ketoprofen (0.2 μM) or prodrug 1 (0.2 μM). The aECF constituted a buffer, which composition is described in publication II. The slices were incubated in a shaker (MaxQ4450, Thermo Fisher Scientific, Sweden) for 5 h at 37 °C, 45 rpm under a constant oxygenation of the incubation chamber to ensure slice viability. In order to investigate the compound uptake dynamics, aECF (200 μL) was collected from the beakers at designated time points (at the beginning, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h and 5 h) of incubation to Eppendorf tubes, containing 200 μL of drug-free brain homogenate in aECF (1:4, w/v). After 5 h incubation, the brain slices were dried on filter paper and homogenized in aECF (1:9, w/v) individually with an ultrasonic processor (VCX-130, Sonics, Chemical Instruments AB, Sweden). The obtained homogenates were stored at −20 °C until bioanalysis. The viability of the slices was studied using a dynamic pH measurement and the release of lactate dehydrogenase activity with a cytotoxicity detection kit (Roche Diagnostics GmbH, Germany).
The intra-brain distribution was evaluated by calculating Vu,brain (mL/g brain) according Eq. 11 (Friden et al., 2009a, Loryan et al., 2013):
𝑉𝑢,𝑏𝑟𝑎𝑖𝑛 =𝐴𝑏𝑟𝑎𝑖𝑛− 𝑉𝑖× 𝐶𝑏𝑢𝑓𝑓𝑒𝑟
𝐶𝑏𝑢𝑓𝑓𝑒𝑟× (1 − 𝑉𝑖) (11)
where Abrain (nmol/g brain) is the drug amount quantified in the brain slice, Cbuffer is the concentration of investigated compounds in aECF after 5 h incubation; Vi (mL/g brain) is the volume of the aECF layer surrounding the brain slices, which was determined previously using [14C]-inulin marker and equal 0.094 mL/g brain (Friden, DMD, 2009).
The Kp,uu,cell for unbound ketoprofen and prodrug 1 was estimated using Eq.12 as a modified Eq. 7 (Friden et al., 2007):
𝐾𝑝,𝑢𝑢,𝑐𝑒𝑙𝑙=𝑉𝑢,𝑏𝑟𝑎𝑖𝑛− 𝑉𝑏𝑟𝑎𝑖𝑛,𝐸𝐶𝐹 𝑉𝐼𝐶𝐹× 𝑉𝑢,𝐼𝐶𝐹
(12)
where Vbrain,ECF is the brain ECF physiological fractional volume, estimated to be 0.2 mL/g brain (Nicholson and Sykova, 1998); VICF is the brain intracellular physiological fractional volume, estimated to be 0.8 mL/g brain (Reinoso et al., 1997); Vu,ICF is the unbound volume of distribution of drug in the ICF. The latter reflects the drug intracellular binding and can be calculated using Eq. 13 (Friden et al., 2007):
𝑉𝑢,𝐼𝐶𝐹= 1 + 𝐷
𝑉𝐼𝐶𝐹× ( 1 𝑓𝑢,ℎ𝑜𝑚𝑜𝑔𝑒𝑛𝑎𝑡𝑒
− 1) (13)
4.8.2 LAT1 contribution to intra-brain distribution of prodrug
The contribution of LAT1 to intra-brain distribution of the prodrug 1 in mice and rats was investigated in the presence of the selective LAT1 inhibitor. After 30 min pre-incubation of brain slices in aECF containing 0.8 µM of LAT1-inhibitor, prodrug 1 was added to the beaker to achieve the final concentration of 0.2 µM. In order to investigate uptake dynamics of prodrug 1 in present of LAT1 inhibitor, the aECF samples were collected at 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h and 4.5 h after adding the prodrug 1. After 4.5 h incubation with prodrug 1, the brain slices were collected and stored at −20 °C prior to the bioanalysis. The prodrug 1 Vu,brain and Kp,uu,cell were calculated using Eq. 11-13. In the calculations, the fu, homogenate of the prodrug1 was measured without the LAT1 inhibtor assuming the absence of the effect of the inhibitor on the prodrug 1 nonspecific brain tissue binding properties.
4.8.3 Estimation of neuroPK parameters of prodrug
The evaluation of BBB transport, intra-brain distribution of ketoprofen and prodrug 1 and intra-brain release of parent drug in mouse brain was performed using the CMA. The AUCtotal,plasma and AUCtotal,brain evaluated in PK study in mice (chapter 4.7, publication I) were combined with parameters obtained from the intra-brain distribution study and equilibrium dialysis and used for calculations of other neuroPK parameters. The extent of delivery of investigated ketoprofen and prodrug 1 across the BBB was evaluated by calculating the total concentration ratio in brain
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and plasma (Kp,brain, Eq. 4), and the unbound concentration ratio in brain ECF and plasma concentration ratio (Kp,uu,brain, Eq. 14):
𝐾𝑝,𝑢𝑢,𝑏𝑟𝑎𝑖𝑛= 𝐾𝑝,𝑏𝑟𝑎𝑖𝑛
𝑉𝑢,𝑏𝑟𝑎𝑖𝑛× 𝑓𝑢,𝑝𝑙𝑎𝑠𝑚𝑎
(14)
where the fu,plasma is the unbound fraction of ketoprofen or prodrug 1 in plasma.
The AUCu,brainECF for unbound ketoprofen and prodrug 1 after crossing the BBB was calculated using Eq. 15:
𝐴𝑈𝐶𝑢,𝑏𝑟𝑎𝑖𝑛𝐸𝐶𝐹 =𝐴𝑈𝐶𝑡𝑜𝑡𝑎𝑙,𝑏𝑟𝑎𝑖𝑛
𝑉𝑢,𝑏𝑟𝑎𝑖𝑛
(15)
The AUCu,brainICF for unbound ketoprofen and prodrug 1 in the brain intracellular compartment was estimated according to Eq. 16:
𝐴𝑈𝐶𝑢,𝑏𝑟𝑎𝑖𝑛𝐼𝐶𝐹=𝐴𝑈𝐶𝑡𝑜𝑡𝑎𝑙,𝑏𝑟𝑎𝑖𝑛× (𝑉𝑢,𝑏𝑟𝑎𝑖𝑛− 𝑉𝑏𝑟𝑎𝑖𝑛𝐸𝐶𝐹) 𝑉𝐼𝐶𝐹× 𝑉𝑢,𝐼𝐶𝐹× 𝑉𝑢,𝑏𝑟𝑎𝑖𝑛
(16)
As AUCtotal,brain of ketoprofen covers both ketoprofen released in the brain and ketoprofen that has distributed from plasma, the AUCu,brainECF and AUCu,brainICF of unbound ketoprofen, which was quantified in plasma and further distributed into the brain ECF after crossing the BBB was estimated using Eq. 17 and 18:
𝐴𝑈𝐶𝑢,𝑏𝑟𝑎𝑖𝑛𝐸𝐶𝐹 = 𝐾𝑝,𝑢𝑢,𝑏𝑟𝑎𝑖𝑛× 𝐴𝑈𝐶𝑡𝑜𝑡𝑎𝑙,𝑝𝑙𝑎𝑠𝑚𝑎× 𝑓𝑢,𝑝𝑙𝑎𝑠𝑚𝑎 (17) 𝐴𝑈𝐶𝑢,𝑏𝑟𝑎𝑖𝑛𝐼𝐶𝐹= 𝐾𝑝,𝑢𝑢,𝑐𝑒𝑙𝑙× 𝐴𝑈𝐶𝑢,𝑏𝑟𝑎𝑖𝑛𝐸𝐶𝐹 (18) where AUCtotal,plasma of released ketoprofen had been quantified previously after single dose of prodrug 1 (publication I). The calculations were based on assumption that ketoprofen quantified in plasma after prodrug dosing would follow similar kinetics as ketoprofen itself. Therefore, the Kp,uu,brain and Kp,uu,cell estimated for ketoprofen administration were used.
AUCtotal,brain of ketoprofen after prodrug 1 dosing does not provide quantitative information about the intra-brain bioconversion. Therefore, Eq. 15 and 16 were used for estimation of AUCu,brainECF and AUCu,brainICF of ketoprofen released after prodrug 1 dosing, respectively, which included both ketoprofen released in the brain and delivered from plasma after prodrug 1 administration. Finally, AUCu,brainICF was corrected by AUCu,brainICF of ketoprofen entered the ICF from plasma to estimate AUCu,brainICF of unbound ketoprofen released in the brain ICF. These calculations assume the fact that unbound ketoprofen reaches an equilibrium between brain ECF and ICF. However, if the release of ketoprofen occurs in the brain intracellular compartment, the equilibrium can be achieved only when the rate of elimination from the brain ICF, or binding or distribution to cell organelles of released unbound
ketoprofen, is equal to the bioconversion rate inside the cells. Thus, this approach may provide AUCu,brainICF underestimation and AUCu,brainECF overestimation.