Investigation of pharmacokinetic/pharmacodynamic (PK/PD) relationships for inhaled drugs is challenging because of the limited possibilities of measuring tissue exposure and target engagement in the lung. The aim of this study was to develop a methodology for measuring receptor occupancy in vivo in the rat for the glucocorticoid receptor (GR) to allow more informative inhalation PK/PD studies. From AstraZeneca’s chemical library of GR binders, compound 1 [N-(2-amino-2-oxo-ethyl)-3-[5-[(1R,2S)-2-(2,2-difluoropropanoylamino)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)propoxy]indazol-1-yl]-N-methyl-benzamide] was identified to have properties that are useful as a tracer for GR in vitro. When given at an appropriate dose (30 nmol/kg) to rats, compound 1 functioned as a tracer in the lung and spleen in vivo using liquid chromatography–tandem mass spectrometry bioanalysis. The methodology was successfully used to show the dose-receptor occupancy relationship measured at 1.5 hours after intravenous administration of fluticasone propionate (20, 150, and 750 nmol/kg) as well as to characterize the time profile for receptor occupancy after a dose of 90 nmol/kg i.v. The dose giving 50% occupancy was estimated as 47 nmol/kg. The methodology is novel in terms of measuring occupancy strictly in vivo and by using an unlabeled tracer. This feature confers key advantages, including occupancy estimation not being influenced by drug particle dissolution or binding/dissociation taking place postmortem. In addition, the tracer may be labeled for use in positron emission tomography imaging, thus enabling occupancy estimation in humans as a translatable biomarker of target engagement.
Design of improved drug compounds, formulations, or dosage regimens are generally guided by an understanding of the pharmacokinetic/pharmacodynamic (PK/PD) relationships defining how the drug exposure in blood or target organs relates to efficacy and/or safety. Respiratory diseases are preferably treated with inhaled drugs because pulmonary drug delivery can provide a rapid onset of action and an improved therapeutic index owing to lung selective drug exposure (Olsson et al., 2011). However, PK/PD relationships are not easily established for locally acting inhaled drugs because the drug concentration in blood cannot be assumed to reflect the target site concentration in lung tissue (Cooper et al., 2012), and furthermore, there are no established methodologies to assess this concentration. Because receptor occupancy is driven by the unbound drug concentration at the target site, occupancy measurements would clarify the PK evaluation following topical administration and provide a quantitative estimate of target engagement in the target organ. Occupancy methodologies amenable for targets of inhaled drugs, such as the glucocorticoid receptor (GR), are thus desired.
The pharmacologic effect of corticosteroids is mediated by binding to the cytoplasmic GR, which forms a dimer and translocates to the nucleus, where it either binds to sites on the DNA called glucocorticoid responsive elements (transactivation) or interacts with transcription factors (transrepression) (Barnes, 1998). Hence, it was proposed that receptor-binding properties could be important to link the PK to the PD for this compound class (Derendorf et al., 1993). PK/PD relationships for receptor/gene-mediated effects of intravenously administered corticosteroids have been previously described in the liver (Ramakrishnan et al., 2002).
The receptor occupancy of GRs required to activate and sustain GR-mediated biologic responses in animals and therapeutic efficacy in humans are two important questions that remain unanswered for inhaled GR ligands. Estimates of drug occupancy are typically obtained by assessing the number of free binding sites using a tracer ligand that, when administered at a sufficiently low dose, binds with high affinity and specificity to the target of interest. A tracer for the GR amenable to in vivo occupancy studies has not been previously reported, although numerous attempts have been made to develop positron emission tomography radiotracers for this receptor (Steiniger et al., 2008). Receptor occupancy of GR has been measured in the rat using ex vivo binding assays where the tracer is incubated with the tissue sample after collection from the animal (Hochhaus et al., 1995; Fish et al., 2007). The receptor occupancy profiles were shown to be similar after intravenous and intratracheal instillation of an aqueous solution of triamcinolone acetonide (Hochhaus et al., 1995). It was also demonstrated that intratracheal administration of triamcinolone acetonide phosphate in liposomes with intermediate release rate provided sustained occupancy compared with intratracheal instillation of a solution (Suarez et al., 1998).
While in vitro and ex vivo binding assays rely on radiolabeled tracer ligands, tracer analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS) offers advantages, including simultaneous determination of occupancy and drug concentration. Furthermore, tracer quantification with LC-MS/MS is made without interference of metabolites (Chernet et al., 2005), a feature that is of particular interest for measuring in vivo occupancy in studies where the tracer needs to be administered to the animal.
The aim of this study was to identify a tracer molecule for GR that is amenable to a fully in vivo–based receptor occupancy methodology using LC-MS/MS tracer quantification, and to establish experimental procedures that allow occupancy to be assessed in the lung and other organs of individual animals. Occupancy measurements in organs other than the lung describe the systemic exposure of drug, which is of interest after inhalation where the focus is typically on attaining low systemic concentrations in plasma and other tissues to avoid unwanted side effects and toxicity. The methodology is applied to study the dose dependence and time profile of GR occupancy of fluticasone propionate (FP) in the lung and spleen, demonstrating an opportunity for drug discovery to obtain key information in order to understand inhalation PK/PD, select the best candidate drug for clinical development, and underpin the predicted dosing regimen for patients.
Materials and Methods
Bovine serum albumin Fraction V was purchased from Roche (Penzberg, Germany). KCl, MgSO4, HEPES, NaHCO3, NaOH, glucose, N,N-dimethylacetamide, dexamethasone, 5,5-diethyl-1,3-diphenyl-2-iminobarbituric acid, and polyethylene glycol 400 were obtained from Sigma-Aldrich (St. Louis, MO). NaCl, CaCl2, KH2PO4, and ascorbic acid were purchased from Merck (Darmstadt, Germany). FP, R-budesonide, compound 1 [N-(2-amino-2-oxo-ethyl)-3-[5-[(1R,2S)-2-(2,2-difluoropropanoylamino)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)propoxy]indazol-1-yl]-N-methyl-benzamide] (Fig. 1A), and compound 2 [2,2-difluoro-N-((1R,2S)-1-(1-(6-fluoropyridin-3-yl)-1H-indazol-5-yloxy)-1-(4-(methylsulfonyl)phenyl)propan-2-yl)propanamide] (Fig. 1B) were synthesized at AstraZeneca R&D according to experimental descriptions provided in Supplemental Schemes 1 and 2. Compounds 1 and 2 were discovered as part of the collaboration between AstraZeneca and Bayer HealthCare in the field of selective GR modulators. Chemicals were of analytical grade and all solvents were of high-performance liquid chromatography grade.
Male Wistar Han rats (Harlan, Horst, The Netherlands) weighing 275–325 g were used for the in vitro and the in vivo studies. The animals were group housed at 18–22°C under 12-hour light/dark cycles with free access to food and water for at least five days prior to the experiments. The studies were approved by the Animal Ethics Committee of Gothenburg (234-2011, 190-2010, and 71-2013). Studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health.
Development of a Pulmonary In Vivo Receptor Occupancy Methodology for GR
The following section describes the sequence of steps taken for developing the in vivo receptor occupancy methodology with regard to tracer identification in vitro and protocol development.
Tracer Identification In Vitro.
AstraZeneca’s internal chemical library was searched for compounds with measured values of both lipophilicity (i.e., the distribution coefficient, log D7.4) and functional cellular activity from a reporter gene assay system in which transrepression activity was determined (Edman et al., 2014). The in vitro potency (EC50) from this assay was used as a surrogate for the dissociation constant, Kd. Compounds were ranked after their differences between the potency EC50 and log D7.4 because a large difference indicates a compound with high potency and a low degree of nonspecific binding in tissue. This combination is predicted to give a high target bound fraction, ftb, which is an important property for successful tracer molecules (Fridén et al., 2014). Based on this criterion a first selection of compounds was made and these were evaluated for the degree of nonspecific binding in slices of agarose-inflated rat lungs using a methodology developed for brain slices (Fridén et al., 2009). Two compounds with low measured values of EC50 and nonspecific binding were subsequently identified from this selection. To experimentally determine ftb for these compounds in vitro, lung slices were incubated at subnanomolar concentrations of the potential tracer either in the absence (n = 3–6) or presence of an excess of R-budesonide (100 nM, n = 3–6). Lung slice concentrations of the tracer candidate from incubations with excess R-budesonide corresponded to nonspecific binding (Cslice,nonspec) and the concentrations from incubations containing only the tracer candidate gave the total binding (Cslice,total). The ftb value is calculated using the following equation, given by(1)and these values, as well as the analytical sensitivity, were subsequently used for ranking of the compounds.
Studies for In Vivo Protocol Development.
In general terms, in vivo receptor occupancy is measured by tracer administration, usually via the intravenous route, and subsequent quantification of tracer concentration in the tissue(s) of interest. Receptor occupancy is reflected by the difference between tissue tracer concentrations in treated animals and drug naïve control animals. A low tracer dose is used to ensure that the tracer binds only a small portion of available binding sites, and thus that the measured tracer concentration will be sensitive to changes in occupancy by the drug. The maximum concentration of tracer is derived from drug naïve control animals and is denoted by the tissue concentration of tracer in control animals, Ccontrol, which represents the sum of the nonspecific tissue concentration of the tracer, Cnonspec, and the specific binding of tracer and corresponds to 0% occupancy (i.e., all receptors are available to the tracer). Thus, Cnonspec has to be taken into account to be able to estimate the level of specific binding of tracer to derive the occupancy by drug from the tracer concentration in animals pretreated with test compound, i.e., the total tissue tracer concentration in treated animals, Ctest. A second group of animals is dedicated to determination of Cnonspec. Pretreatment with a high dose of another ligand, with the purpose of occupying all receptors of interest, is made prior to tracer administration. Hence, the lower tissue concentration of tracer measured in these animals is only reflective of Cnonspec and is assigned to a value of 100% occupancy. An appropriate time period, a so-called post-tracer survival interval, is needed after tracer administration for the tracer to bind the receptor and for the nonspecific binding to decrease such that a high ratio of total to nonspecific binding is achieved (Need et al., 2006). Consequently, once a promising tracer candidate has been identified, several tracer doses—and sometimes also post-tracer survival intervals—are tested to develop experimental procedures that yield a high ratio of total to nonspecific binding.
Evaluation of Tracer Dose.
Three different dose levels of the tracer candidate compound 1 were assessed (40, 400, and 4000 nmol/kg). In the following, compound 1 is referred to as the tracer. At each dose level, the animals were divided into two groups to investigate if Ccontrol was distinguishable from Cnonspec. A post-tracer survival interval of 30 minutes was tested since it has been proven successful for several in vivo receptor occupancy methodologies (Need et al., 2007; Nirogi et al., 2013). For determination of tissue concentrations of tracer, the animals were injected via the tail vein with their group-specific dose of tracer (n = 3). Animals dedicated to determination of Cnonspec were pretreated with a high intravenous dose of compound 2 via the tail vein 30 minutes prior to the tracer administration (compound 2, 5.0 µmol/kg and n = 2, 3, and 2 for the 40, 400, and 4000 nmol/kg dose groups). A high dose of compound 2 was used as pretreatment since it is a highly potent compound (transrepression EC50 = 0.476 nM) with a long half-life in blood, and was thus expected to give a high occupancy over a long time period. This study was complemented with an additional dose level (4 nmol/kg, n = 3) using an identical protocol, except that the animals dedicated to determination of Cnonspec were pretreated with dexamethasone (20 mg/kg, n = 3). The vehicle used for all three compounds was N,N-dimethylacetamide, polyethylene glycol 400, and water (1:1:1, w/w/w). Thirty minutes after administration of the potential tracer, a blood sample was taken and the lung was dissected for concentration determination.
Evaluation of Nonspecific Binding.
To confirm that only nonspecifically bound tracer remained after pretreatment with compound 2, Cnonspec was measured after pretreatment with dexamethasone (20 mg/kg, n = 3). Dexamethasone was injected via the tail vein 30 minutes prior to intravenous administration of tracer (40 nmol/kg). Thirty minutes after the tracer administration, a terminal blood sample was collected and the lung was dissected for concentration determination.
Evaluation of Tracer Function in Other Organs.
To investigate the functionality of the tracer in various organs, Ccontrol and Cnonspec was compared in several organs. Animals dedicated to measurements of Cnonspec were pretreated intravenously with dexamethasone (20 mg/kg, n = 5), whereas animals dedicated to measurements of Ccontrol did not get any pretreatment (n = 5). Dexamethasone was injected via the tail vein 30 minutes prior to intravenous administration of tracer (30 nmol/kg). All animals were sacrificed 30 minutes after tracer administration, a terminal blood sample was collected, and organs (lung, liver, brain, kidney, and spleen) were dissected for concentration determination.
General Procedures and Final Protocol for In Vivo Receptor Occupancy Measurements.
The tracer, compound 1, was given intravenously via the tail vein (30 nmol/kg). Animals dedicated to determination of Cnonspec had been pretreated intravenously with dexamethasone (20 mg/kg) 30 minutes prior to the tracer administration. In contrast, animals set aside for determination of Ccontrol did not get any pretreatment. Animals were anesthetized by inhalation of isoflurane during the administration(s) and were then allowed to wake up. Anesthesia during tail vein administrations was made a standard procedure since the tail is sensitive. In conjunction with the termination, the rats were anesthetized once more by inhalation of isoflurane. The fur was rinsed with 70% ethanol and laparatomy was conducted. Heparin (2000 IU) was injected into vena cava, a terminal blood sample was collected (3–4 ml) in a heparinized tube from the abdominal aorta and the rat was euthanized by cutting the abdominal aorta. The terminal blood sample was taken 30 minutes after the tracer administration. The diaphragm was then removed and the apex was incised along its transverse axis. The pulmonary circulation was perfused with room-tempered bovine serum albumin solution (40 ml, 2.5% bovine serum albumin) via the pulmonary artery to clear the tissue of blood. The heart and lung were removed from the thorax en bloc. The trachea was removed from the lung and a predefined section of the spleen (∼200 mg) was dissected. Lung and spleen samples were weighed and stored separately in tissue tubes and Eppendorf tubes, respectively, at −20°C until sample handling and analysis. A detailed description of these procedures is provided in the section on analytical procedures. Receptor occupancy was then calculated for each animal (eq. 4) (see the section on data and statistical analyses).
Application of the Methodology
Dose-Receptor Occupancy Relationship.
The dose-receptor occupancy relationship for FP was assessed by measuring GR occupancy 1.5 hours after intravenous administration of three escalating doses of FP: 20 nmol/kg (n = 4), 150 nmol/kg (n = 6), and 750 nmol/kg (n = 6). The intravenous route of administration was favored versus other routes since it provides the lowest possible variability between animals in lung tissue exposure. Prior to both the administration of FP and tracer, plasma samples were taken for measurements of corticosterone to investigate if the endogenous ligand was a potential source of variability. To allow for interindividual differences in plasma corticosterone, the intravenous administrations of FP were spread out over the time course of a day (8:00 AM–7:00 PM). Receptor occupancy was calculated for each animal according to eq. 4 (see the section on data and statistical analyses).
Receptor Occupancy Time Profile.
The time course of receptor occupancy was studied after intravenous administration of FP (90 nmol/kg). Tracer was administered intravenously at the following time points after drug administration: 0.5, 1, 2, 4, 7, 24, and 48 hours (three animals/time point). Receptor occupancy was calculated for each animal according to eq. 4 (see the section on data and statistical analyses).
The blood samples were collected in heparinized tubes, which were centrifuged for 10 minutes at 4000g (Rotanta 46R; Hettich, Germany). The plasma samples were subsequently transferred to Eppendorf tubes and stored at −20°C until analysis.
The tissue tubes containing lung samples were separately flash-frozen in liquid nitrogen and then immediately placed into a Covaris CryoPrep (Covaris Inc., Woburn, MA) for pulverization. This freeze-fracture procedure was repeated until pulverization was judged as being sufficient, generally three times. The pulverized samples were then transferred to separate glass tubes and Ringer buffer was added to each lung sample (4.0 ml). Covaris E210 and SonoLab software version 4.2.0 (intensity, 10.0; cycles, 1000; treatment time, 180 seconds; bath degree, 40°C; Covaris Inc.) were used for dissolution of the samples. Predefined parts of the liver, brain, spleen, and kidney were weighed in Eppendorf tubes and homogenized in 3 volumes of distilled water (w/v) with an ultrasonic probe. The samples were stored at −20°C until analysis.
Concentrations of tracer and FP in tissues and plasma were quantified using reversed phase LC-MS/MS with the electrospray ionization source set in positive mode. The LC system used was a LC Agilent 1200 Series gradient pump (Agilent Technologies, Waldbronn, Germany), a CTC Autosampler (CTC Analytics, Zwingen, Switzerland), a HALO C18 column (30 × 2.1 mm, 2.7 µm; Advanced Material Technology, Wilmington, DE), and an Agilent 6460 detector (Santa Clara, CA). Gradient elution over 1.5 minutes time with acetonitrile and 0.2% formic acid and a flow rate of 0.7 ml/min was performed.
Protein precipitation was used for all samples except lung homogenates. A defined volume (50 µl) of each sample was added in triplicate to a NUNC 96-deep well plate (Nalge Nunc International, Rochester, NY) and was then protein precipitated by addition of cold acetonitrile containing 0.2% formic acid and 100 nmol/l of 5,5-diethyl-1,3-diphenyl-2-iminobarbituric acid as the internal standard (200 µl) to each sample. After two minutes of mixing with a VWR VX-2500 Multi-Tube Vortexer (VWR International, West Chester, PA) and 20 minutes of centrifugation at 4000 rpm at 4°C (Centrifuge 5810 R; Eppendorf, Hamburg, Germany) the supernatants (75 µl) were transferred to a new plate, where each sample was diluted by addition of 0.2% formic acid (75 µl). The diluted samples were then injected (20 µl) to the high-performance liquid chromatography system.
Liquid-liquid extraction was performed before analysis of the lung homogenates. A certain volume (50 µl) of each sample was added in triplicate to glass vials to which a carbonate buffer pH 10 (50 mM Na2CO3, 50 mM NaHCO3) containing 100 nmol/l of 5,5-diethyl-1,3-diphenyl-2-iminobarbituric acid as the internal standard was added (50 µl). Each sample was extracted with methyl tert-butyl ether (600 µl). After sealing the vials with a silicon cover, the vials were shaken horizontally for 5 minutes (150 min−1; Edmund Bühler Swip, POCD Scientific, Artarmon, NSW, Australia), followed by 15 minutes of mixing with a VWR VX-2500 Multi-Tube Vortexer (speed 5; VWR International). A defined volume of the organic layer (450 µl) was then transferred to glass vials, which were vaporized with a Techne Sample Concentrator (Techne Incorporated, Staffordshire, UK) for approximately 20 minutes. Samples were reconstituted in 33% acetonitrile and 0.2% formic acid in water (150 µl) and placed in an ultrasonic bath (5210 Branson; Gemini, Apeldoorn, The Netherlands) for 10 minutes. Except for the sample preparation technique for the lung homogenates, the analytical procedure was identical to that used for the other samples.
The compounds were quantified using Mass Hunter Workstation Software for Quantitative Analysis (Agilent Technologies, Santa Clara, CA). A standard curve was created for each sample type: plasma, lung, spleen, liver, brain, and kidney. The standard curve samples contained matrix (blank plasma, blank lung homogenate, blank spleen homogenate, blank liver homogenate, blank brain homogenate, and blank kidney homogenate, respectively) in a 1:9 volume ratio to match the samples. Corticosterone plasma levels were analyzed using an ELISA Kit (ab108821; Abcam, Cambridge, UK).
Data and Statistical Analyses
Modeling of Tissue Concentrations of the Tracer.
The four different tracer doses were evaluated by a modeling approach where the tissue concentration of the tracer was assumed to consist of two parts: (1) linear nonspecific binding determined by the plasma tracer concentration, Cp, and a partitioning constant for nonspecific binding, Kp,ns; and (2) nonlinear binding to the target determined by Cp, the dissociation constant, Kd, and the receptor density, Bmax, given by(2)(3)The model was implemented in MATLAB R2013a (MathWorks Inc., Natick, MA) using a nonlinear least-squares algorithm, lsqnonlin, and iterative reweighting using predicted data.
Calculation of Receptor Occupancy.
Receptor occupancy calculations were made for each animal using a previously established method (Farde et al., 1988; Wadenberg et al., 2000; Need et al., 2007; Nirogi et al., 2013) described as follows:(4)where(5)(6)Ratiot refers to the ratio of total tracer concentration in tissue from animals treated with the test compound (Ctest) divided by the Cnonspec obtained from the group of animals pretreated with a high dose of another ligand. Ratioc represents the average ratio of Ccontrol to Cnonspec, where Ccontrol is obtained from a drug naïve control group. Accordingly, when Ratiot is equal to Ratioc this corresponds to an occupancy of 0% and when the Ratiot is 1 (e.g., tracer concentration in the treated animal is equal to Cnonspec) this corresponds to an occupancy of 100%.
Modeling of the Dose-Receptor Occupancy Relationship.
A nonlinear Emax model with a baseline is used to describe the dose-receptor occupancy relationship in the following equation:(7)where Ci is the tissue tracer concentration in animal i; Cnonspec is the nonspecific binding; Cspec,max is the maximum specific binding; D is the dose; and ED50 is the dose that gives 50% occupancy at 1.5 hours. A high dose (10,000 nmol/kg) was assigned to the animals pretreated with dexamethasone, and D was set to 0 for drug naïve control animals.
The model was implemented in MATLAB R2013a using the nonlinear least-squares algorithm, lsqnonlin. An exhaustive-search algorithm was used to obtain initial estimates for Cnonspec, Cspec,max, and ED50. The Ccontrol value for the drug naïve control animals was then calculated as follows:(8)
Statistical comparisons between two groups were made using a two-tailed Student’s t test. The level of significance was set at P ≤ 0.05. Data are presented as mean ± S.D., and the precision of the parameter estimates is presented as S.E. For each dose group or investigated time point, the S.E. for receptor occupancy was assessed using the propagation of error method assuming independent variables (Kendall and Stuart, 1987).
Development of a Pulmonary In Vivo Receptor Occupancy Methodology for GR
Tracer Identification In Vitro.
Fifteen GR binders were selected from AstraZeneca’s internal chemical library on the basis of having large numerical differences between the values of the potency EC50 and log D7.4. Based on measured nonspecific binding in lung slices and analytical sensitivity, a refined selection of two compounds was made. The chemical structures of compounds 1 and 2 are shown in Fig. 1. Incubation of lung slices in the absence and presence of an excess of R-budesonide resulted in statistically significant differences between Cslice,total and Cslice,nonspec for both compounds. The ftb values were calculated to be 0.56 ± 0.071 and 0.48 ± 0.069 for compounds 1 and 2, respectively. The ftb value and analytical sensitivity were subsequently used for ranking. Based on these criteria, compound 1 was identified as the most promising tracer candidate.
Evaluation of Tracer Dose.
As illustrated in Fig. 2, which shows Ccontrol and Cnonspec for each tracer dose, there was a separation between Ccontrol and Cnonspec at the second lowest dose level (40 nmol/kg); Ccontrol was 9.2 ± 2.1 nM and Cnonspec was 3.8 ± 0.32 nM, resulting in a Ccontrol/Cnonspec ratio of 2.4, which corresponded to a value of 0.59 for ftb. The ratio between Ccontrol and Cnonspec decreased with ascending tracer doses (2.4, 1.5, and 1.3 for 40, 400, and 4000 nmol/kg, respectively). The Cnonspec value was below the lower limit of quantification at the lowest dose level (4 nmol/kg), and therefore the ratio could not be calculated. The Cnonspec value was proportional to Cp, and thus they were found to be highly correlated (r2 = 0.99).
The dependence of Ccontrol and Cnonspec on Cp was captured by the model. The parameter estimates and S.E. values for the model are presented in Table 1. According to simulations of the system, the ratio consistently increased with lower Cp levels of tracers and was judged as sufficiently high when Cp < 3.5 nM. The PK simulations showed that this corresponded to a tracer dose of approximately 55 nmol/kg or less (data not shown).
Evaluation of Nonspecific Binding.
The same degree of nonspecific binding remained when dexamethasone was used as the pretreatment; Cnonspec/Cp was of the same magnitude when the animals had been pretreated with dexamethasone instead of compound 2 (0.76 ± 0.12 and 0.69 ± 0.11 after pretreatment with dexamethasone and compound 2, respectively). As was given in the previous section on the evaluation of tracer dose, Cnonspec was proportional to Cp (r2 = 0.99).
Evaluation of Tracer Function in Other Organs.
There were statistically significant differences between Ccontrol and Cnonspec in the lung, liver, spleen, and kidney (Table 2). The difference was especially pronounced in the lung and spleen (P < 0.001). All brain samples were below the lower limit of quantification. The ratio of Ccontrol/Cnonspec in the spleen was found to be higher than the corresponding ratio in the other organs.
Application of the Methodology
Dose-Receptor Occupancy Relationship.
There was a dose-dependent decrease in tracer concentrations in the lung and spleen, which is illustrated in Fig. 3, A and B, respectively. A dose-receptor occupancy relationship was thus observed after intravenous administration of FP (Fig. 4). An Emax model with a baseline captured how tissue concentrations of tracer depend on the dose of FP. The parameter estimates and S.E. values obtained from the model are presented in Table 3. The ED50 value was estimated to be 47 ± 8.6 nmol/kg. Although the measurements of corticosterone, both before the administration of FP (27–330 µg/l) and tracer (25–260 µg/l), varied between the animals, no relationship between corticosterone and the estimates of receptor occupancy was observed in the treated animals. This is shown in a representative graph (Fig. 5).
Receptor Occupancy Time Profile.
As can be seen in Fig. 6, a high initial occupancy was observed (88 ± 7.5% and 85 ± 3.9% at t = 0.5 hours in the lung and spleen, respectively), which was followed by a time-dependent decline between 0.5 and 7 hours. Receptor occupancy in the spleen had returned to baseline 7 hours after dosing, whereas there still remained some occupancy in the lung. The occupancy profiles initially followed each other closely, while the occupancy was estimated to be slightly higher in the lung at t = 4 and 7 hours after intravenous administration of FP.
Concentration-Receptor Occupancy Relationship in the Spleen.
When data from both studies (see the sections on the dose-receptor occupancy relationship and receptor occupancy time profile) were used, a relationship between occupancy in the spleen and the spleen concentration of FP was observed (Fig. 7). Since the analytical sensitivity for FP in lung tissue was not equally good, a corresponding graph could not be created for the lung.
This study demonstrates the first methodology that is capable of simultaneous measurements of both pulmonary and systemic in vivo occupancy of the GR. It is novel both in terms of measuring GR occupancy strictly in vivo and by the analytical technique used for tracer quantification (LC-MS/MS). By applying the methodology, a dose-receptor occupancy relationship could be successfully characterized for a well known GR agonist (Fig. 4). The main incentive for developing this method was to provide a conclusive biomarker of target engagement that can be used to increase our understanding of PK and PK/PD for inhaled drugs, which is currently held back by the lack of relevant exposure measurements.
We reasoned that a fully in vivo–based receptor occupancy methodology would carry advantages over ex vivo binding assays for the study of inhalation drugs. This reasoning was primarily based on the anticipated risk of additional particle dissolution and thus subsequent drug-receptor association post sacrifice during the processing steps of tissues, which could lead to overestimation of pulmonary occupancy if the tracer is added ex vivo. Because several inhaled corticosteroids are poorly soluble, and hence retained in a solid state in the lung for a considerable time period after inhalation, the advantage of in vivo tracer administration is perhaps even more pronounced for this drug class and route of administration. Moreover, the reliability of ex vivo binding assays has been questioned since dissociation of drug from receptors during incubations could lead to underestimation of occupancy (Kapur et al., 2001; Miller et al., 2009).
Compound 1 proved to be a successful tracer in vivo as an appropriate Ccontrol/Cnonspec ratio was obtained at a low tracer dose. As expected, this ratio decreased with ascending tracer doses since the relative contribution of Cnonspec increased. This behavior was successfully captured by the model, which described the receptor-bound and nonspecifically-bound tracer concentrations as a function of Cp (Fig. 2). It is well known that the dose of a tracer should be low to allow its function as a tracer of a specific binding site. By using both predictions from the model as well as PK simulations and information about the analytical sensitivity, an appropriate dose was identified. The lowest tracer dose that could be reliably quantified by LC-MS/MS was chosen (30 nmol/kg). A post-tracer survival interval of 30 minutes was tested since it is commonly used in other preclinical occupancy experiments (Need et al., 2007; Nirogi et al., 2013). As it proved to give an appropriate ratio of total to nonspecific binding, this post-tracer survival interval was chosen. It was also shown that normal variation of corticosterone plasma concentrations had no relevant impact on the estimates of occupancy after drug administration (Fig. 5).
There is no tissue devoid of GR that can be used as a reference region to estimate nonspecific tissue binding for the tracer. Instead, the nonspecifically bound tracer concentration must be estimated in separate animals. As expected, it was shown that Cnonspec was proportional to Cp. The partitioning constant for nonspecific binding (Kp,ns) was of the same magnitude when high doses of two structurally different GR binders, a steroid and a selective nonsteroidal GR modulator, respectively, were used as pretreatment. Based on these results, the tested intravenous dose of dexamethasone (20 mg/kg) was considered to be an appropriate pretreatment for measurements of Cnonspec.
A general aim of inhalation therapy is to achieve a high local drug concentration at the target site in the lung, whereas the systemic concentration is low to avoid unwanted side effects. It would thus be interesting to simultaneously measure receptor occupancy in the lung and in another organ that could be used as a reference organ for systemic exposure following inhalation. The functionality of the tracer was therefore investigated in several organs. The difference between Ccontrol and Cnonspec was found to be statistically significant in three tested organs (liver, kidney, and spleen). A higher ratio of total to nonspecific binding allows for more precise measurements. Since this ratio was considerably higher in the spleen than in the two other organs, the spleen was judged as the most appropriate organ to use as a reference organ. Moreover, because it is a well perfused organ, fast equilibrium between the unbound drug concentration in plasma and tissue can be assumed, given that the distribution of drug is perfusion-rate limited. This property also contributed to the choice of reference organ.
As expected, when the dose-receptor occupancy relationship for FP was assessed, a dose-dependent decrease in tracer concentrations was observed in the lung and spleen (Fig. 3, A and B). Hence, the methodology proved to be able to successfully show a dose-receptor occupancy relationship after intravenous administration of FP (Fig. 4), and ED50 was found to be 47 nmol/kg.
GR occupancy has previously not been determined after intravenous administration of FP. Since it is a highly potent substance (Kd = 0.49 nM) (Högger and Rohdewald, 1994), the highest dose level (750 nmol/kg) was expected to give very high occupancy. Indeed, complete occupancy was observed in both organs. It should not be possible to exceed 100% occupancy; however, because Cnonspec was obtained from a separate group of animals, interindividual variability can explain that some estimates were slightly higher than 100%. Measurements of occupancy were therefore made in treatment groups of 4–6 animals to reduce the impact of interindividual variability.
The time course of occupancy after intravenous administration of FP (90 nmol/kg) was shown both in the lung (the target tissue for FP treatment) and in the spleen (the reference organ for systemic exposure) (Fig. 6). As expected, a high initial occupancy was observed in both organs after an intravenous bolus of this potent GR agonist. The initial occupancy peak was followed by a relatively fast decline, where the occupancy in the spleen had returned to 0% after 7 hours. This is in line with the behavior observed after intravenous administration of another corticosteroid, triamcinolone acetonide, where the level of free receptors was found to return to baseline within 6 hours using an ex vivo binding assay (Hochhaus et al., 1995). Receptor occupancy measured at 1 and 2 hours fell within the occupancy range that was expected from the results of the dose-receptor occupancy relationship study where occupancy measurements were made at 1.5 hours after a lower (20 nmol/kg) and higher dose (150 nmol/kg), respectively. The estimated occupancy at 24 hours after dosing (42 ± 8.9%) was higher than after 7 hours and is most likely not reflective of the drug-receptor interaction. Rather, the observation may reflect the dynamics of the GR with a drug-induced downregulation of the receptor population, which has been demonstrated to take place after administration of GR agonists both in vitro and in vivo (Schaaf and Cidlowski, 2002). Either way, the estimated occupancy can be viewed as a reflection of free binding sites.
By combining the results from these two studies, a relationship appeared between GR occupancy in the spleen and total organ concentration of FP (Fig. 7). A plateau of occupancy (100%) was reached at a total organ concentration of 40 nM, which is in close proximity to the measured GR density of 31 nM in the spleen (Miller et al., 1990). It was also noted that the rise in occupancy below 40 nM showed a linear behavior; these two indices suggest that the nonspecific concentration of FP is low relative to the receptor-bound concentration. Due to a relatively poor analytical sensitivity for FP, it could not be investigated whether the same relationship was present in the lung, since several samples were below the lower limit of quantification.
By using this methodology, a dose-receptor occupancy relationship was established and the occupancy time profile after intravenous administration of a well known GR agonist was captured. This method can thus be used henceforth to increase the fundamental understanding of inhalation PK and PK/PD by investigating receptor occupancy for different GR binders. Since receptor occupancy is a biomarker of target engagement, it elucidates lung retention more correctly than the commonly used measurement of total drug amounts in the lung. Several intriguing issues and questions can be addressed using this methodology, which include the following: (1) the study of different mechanisms of lung retention; (2) providing conclusive evidence of target engagement; and (3) investigating which occupancy profiles of the GR are required for activation and sustaining of relevant GR-mediated biologic responses in animals. Information on the aforementioned issues would be extremely valuable in making strategic decisions in inhalation discovery programs. Furthermore, the ability to simultaneously assess pulmonary and systemic GR occupancy can also allow an early assessment of the therapeutic index of inhalation drugs because it provides an estimate of pulmonary targeting; in the end, compound 1 can potentially be used as a positron emission tomography tracer for drug occupancy estimation in humans, thus providing a translatable biomarker of drug response.
In conclusion, this paper presents the development of the first methodology that enables simultaneous measurements of pulmonary and systemic in vivo GR occupancy. By application in PK/PD studies, this method has the potential to provide a better understanding of PK/PD for inhaled drugs and an improved ability to translate this to humans.
The authors thank Susanne Arlbrandt for the invaluable contribution to the in vivo studies and Gunilla Jerndal for the significant contribution to the bioanalysis. Martin Hemmerling and Thomas Hansson are acknowledged for their valuable input. Finally, gratitude is also extended to Henrik Johansson and Philip Thorne for the chemical synthesis of compounds 1 and 2, respectively.
Participated in research design: Boger, Ewing, Fridén.
Conducted experiments: Boger, Fihn, Fridén.
Performed data analysis: Boger, Eriksson, Ewing, Fridén.
Wrote or contributed to the writing of the manuscript: Boger, Chappell, Eriksson, Evans, Ewing, Fihn, Fridén.
- Received November 3, 2014.
- Accepted February 12, 2015.
This work was supported by the Marie Curie FP7 People ITN European Industrial Doctorate (EID) [Project No.316736], Innovative Modelling for Pharmacological Advances through Collaborative Training (IMPACT).
- compound 1
- compound 2
- fluticasone propionate
- glucocorticoid receptor
- liquid chromatography–tandem mass spectrometry
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics