αvβ6 Integrin (pIC50 = 8.4)

28h/GSK-3008348 has high affinity for αvβ6 in both the cell adhesion (pIC50 = 8.4) and the αvβ6 radioligand assay (pKi = 10.4). In a more sensitive version of the radioligand assay with a lower protein concentration (75 pM) and therefore increased sensitivity, GSK-3008348/28h demonstrates an even higher affinity with a pKi = 11.0. While GSK-3008348/28h has weak activity in the in vitro hERG assays, both pIC50 values are <5 and the safety risk was regarded as manageable given the rest of the compound’s profile. Additionally, as the compound is developed, further in vitro and in vivo assessments would be carried out and good safety multiples to predicted human exposures were anticipated (data not shown). 28h/GSK-3008348 has modest to excellent selectivity over the other αv integrins in cell adhesion assays for αvβ6 (see later). Two C-linked pyrazoles 28j and 28k were prepared; the NH analog 28j has high affinity for αvβ6 (pIC50 = 8.6) but reduced selectivity, whereas the demethylated analog 28k is a little less potent (pIC50 = 8.1).

From this work, the very potent and αvβ6 selective dimethylpyrazolo analog GSK-3008348/28h was selected for further profiling (see later) and ultimately became the clinical candidate. This decision was supported by further pharmacological studies including some with IPF human tissue; detailed studies to check the pharmacokinetic profile are commensurate with inhaled dosing and activity in various animal models (see later). However, to understand the SAR around 28h more fully, we continued to explore analogs from both this and related series (46,47) and the profile of these are now described. Some of these analogs have comparable profiles to GSK-3008348/28h but were in fact identified and profiled considerably later. At the time, in the judgment of the project team, these did not offer any significant advantage to GSK-3008348/28h which by that point was progressing through expensive and resource intensive development processes. Five N-linked triazole analogs 28l–p were prepared differing in terms of regiochemistry, linking position, and substitution pattern. The addition of an extra nitrogen into the heteroaromatic over the pyrazole analog reduces the lipophilicity typically by at least a log unit; compare the matched pair dimethylpyrazole GSK-3008348/28h having a chromLogD7.4 of 2.82 with the dimethyltriazole 28n having a chromLogD7.4 of 1.66. All the triazoles have high affinity for the αvβ6 integrin in the cell and in radioligand binding assays although some have less selectivity over some of the other αv integrins compared to 28h. None of them show hERG activity perhaps due to the reduced lipophilicity. Continuing the exploration of various azole analogs, three imidazoles (both C- and N-linked) 28q–s were made based on the ready availability of the building blocks. Despite their isomeric similarity, the matched pair imidazole analogs of the corresponding pyrazoles have reduced lipophilicity, reflecting the obvious differences in basicity; compare dimethylpyrazole 28h having a chromLogD7.4 of 2.82 with the dimethylimidazole 28r having a chromLogD7.4 of 2.09. These changes in lipophilicity between azole matched pairs and isomeric azole matched pairs are in agreement with some of our previous work exploring these effects more generally. Like the triazoles, the imidazoles show no hERG activity in the in vitro assays and have high potency against αvβ6 in the cell adhesion and binding assays with 28r having a similar potency and selectivity profile to 28h. 28q is a little less potent with a pIC50 of 8.2 in the αvβ6 cell adhesion assay but may have a CYP p450 inhibition risk.

The last group to be examined was a range of cyclic ether analogs 28t–z, which are linked to the phenyl ring either via oxygen or carbon atoms. These compounds typically have slightly lower lipophilicity compared to 28h ranging from chromLogD7.4 = 2.75 for 28w to chromLogD7.4 = 2.07 for 28v. Little or no hERG activity is seen in the Barracuda assay, although several have increased activity in the Qpatch assay (see for example the tetrahydropyran 28w, pIC50 < 4.3 in Barracuda but pIC50 = 5.1 in Qpatch). The C4-linked tetrahydropyran 28y (αvβ6 pIC50 = 7.9) has lower affinity in the cell adhesion assay, whereas 28t–w have excellent αvβ6 activity but are typically less selective especially against the αvβ5 integrin having 10-fold more activity against this integrin in comparison to GSK-3008348/28h; see for example the O-linked oxetane 28v with αvβ6 pIC50 = 9.0 (cell adhesion) but αvβ5 pIC50 = 8.3. The C3-linked tetrahydrofuran 28x isomers (described in Table 1 as 28xI1 and 28xI2) are also very potent against αvβ6 (with 28x Isomer 2 (listed in Table 1 as 28xI2) having a pKi of 10.7 in the radioligand binding assay) but a slightly better selectivity profile (almost comparable to 28h). The introduction of a third asymmetric center as in the O- and C-linked tetrahydrofuran-3-yl analogs 28t, 28u, and 28x (isomers 1 and 2) has little effect on the αvβ6 integrin affinity or αv selectivity profile.

Finally, several 3,5-disubstituted phenyl analogs were prepared. Adding a 5-cyclopropyl or a 5-(3,5-dimethyl)pyrazolo substituent to the 3-morpholino analog 28c provided compounds 28bc and 28ch, respectively. Both have comparable αvβ6 potency to GSK-3008348/28h in the radioligand binding assay but show improved selectivity particularly 28ch; compare 28h and 28ch αvβ3 pIC50 of 6.0 and 5.5, respectively, and αvβ5 pIC50 of 6.9 and 5.7, respectively. It is clear the space in the specificity defining loop (SDL) where these substituents bind is more restricted in αvβ3 and αvβ5 compared to αvβ6 (see later). Neither 28bc nor 28ch has any activity in the in vitro hERG assays.

Overall, consideration of the αv selectivity profile played a role in picking GSK-3008348/28h as the clinical candidate and is one of the most selective compounds (Figure 5). Radioligand binding data (see later) confirms 28h has high selectivity for αvβ6 over the other αv integrins[1].

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Affinity and Selectivity [1]
The first steps were to confirm the potency, affinity, and selectivity of GSK-3008348/28h particularly against the other RGD-binding integrins. As described above (Table 1), the 2,5-dimethylpyrazole analog has high affinity for αvβ6. In saturation binding experiments, which provides a more accurate indication of selectivity than the cell adhesion assays, tritiated 28h has a pKD of 10.8 with between 17-fold (α8β1) and 6667-fold (α5β1) selectivity against other RGD binding integrins (Table 4). On the basis of these data, 28h is at least 170-fold selective over the other αv integrins. Importantly minimal activity is seen against αIIbβ3 integrin (the only other RGD binding integrin) up to 10 μM (data not shown) inhibitors of which are used clinically as platelet aggregation inhibitors. While not at antibody selectivity levels (such as the αvβ6 antibody STX-100 in clinical trials), 28h is clearly highly selective and was therefore deemed suitable to test the hypothesis that αvβ6 inhibition might be beneficial in treating IPF.

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Off-Rate [1]
It is important to understand the dissociation of GSK-3008348/28h from αvβ6 because this contributes to estimating the likely size and frequency of clinical dosing. Thus, dissociation binding kinetics of 28h were determined against the αvβ6 protein and compared with those for 28c and a pan αv inhibitor from the literature SC-68448 (Figure 9). (38) Dissociation half-lives of t1/2 ∼ 7 h, 2 h, and 5 min, respectively, were obtained. The half-lives correlate with the affinity of the compounds (Figure 9); the higher is the affinity of the compound, the slower is the dissociation half-life. 28h is clearly superior and therefore more likely to increase duration of action in vivo and minimize clinical dose.

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Receptor Internalization [1]
As has been described elsewhere with the cell surface αvβ6 integrin, igand binding induces rapid receptor internalization (within minutes) and in so doing prevents activation of TGFβ. Having determined the affinity and off-rate for 28h/GSK-3008348, it was next important to characterize its effect on receptor internalization as this also contributes to estimating the size and frequency of the clinical dose. Thus, in primary lung epithelial cells, GSK-3008348/28h causes internalization with a pEC50 of 9.8, and at a concentration of 250 nM, around 80% of the receptor is internalized after 1 h with a t1/2 of 2.6 min. Recycling or re-expression of the receptor is slow with a t1/2 of 11.0 h. This has been shown to be driven by the high affinity engagement of αvβ6 and subsequent slow dissociation of 28h that induces degradation followed by new synthesis of the integrin. As described elsewhere, this internalization translates into a duration of action in vivo. While perhaps unsurprising, it is also encouraging that by substantially removing the αvβ6 driven activation of TGFβ, some reduction in collagen formation is seen in animal models.

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Human Tissue Data [1]
While these results are promising, it is also important to build confidence that similar effects will be observed in the human disease prior to expensive clinical trials. Thus, in precision cut tissue lung slices derived from IPF patients, 1 μM 28h/GSK-3008348 decreases TGFβ levels to those seen with healthy lung slices. It also inhibits phosphoSMAD2 (phosphorylated intracellular signaling molecule Mothers Against Decapentaplegic homolog 2, a marker for TGFβ activation) in a dose dependent manner with an approximate IC50 of 1 nM.

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Summary [1]
These data (together with other pharmacological data published elsewhere) provide confidence GSK-3008348/28h has potential clinically with the diseased human tissue in particular providing reassurance that in vitro assays and in vivo models may be predictive of inhibition of αvβ6 in humans and reduce the effects of TGFβ activation. Given both the usual rate of attrition of clinical candidates and in particular those for IPF, the linking of the target validation, data in vitro assays and in vivo models (54) to efficacy in ex vivo human tissue increases confidence in carrying out clinical studies.

Overall, 28h/GSK-3008348 displays a favorable pharmacokinetic profile in preclinical species, which (combined with its excellent physicochemical properties) suggests full suitability for progression and development as an inhaled medicine. In particular, the likely clinical dose (to be described elsewhere), together with other factors that potentially limit systemic availability, and the anticipated involvement of multiple routes of elimination (metabolic, biliary, and renal) indicate low potential for drug–drug interactions. More specifically for the inhaled route of administration, the combination of high solubility and rapid pulmonary absorption completely minimizes the risk of excessive lung retention or potential for accumulation upon repeated administration.[1]

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Pulmonary Delivery in Rat [1]
Additional in vivo studies were conducted on 28h/GSK-3008348 in order to characterize its pharmacokinetics with pulmonary delivery, to understand any partitioning into lung tissue, and for integration with pharmacodynamic and in vivo efficacy data. Thus, studies conducted in Wistar Han rats by the pulmonary route of administration (by intratracheal instillation and nebulization) suggested a very rapid absorption into the systemic circulation. The observed bioavailability from the pulmonary route was ∼21% from the intratracheal study and ∼54% from the nebulized administration. The lack of complete bioavailability is attributed to the uncertainty regarding the actual dose delivered to the lung in both studies and not to any hypothetical lung retention properties of 28h/GSK-3008348, as demonstrated by the time-course of lung tissue concentration in the nebulized study. After administration by the nebulized route at 0.227 mg/kg, concentrations of 28h/GSK-3008348 in rat lung peaked immediately at the end of the nebulization (716 ng/g), rapidly declined in a biexponential manner, and fell below the limit of quantification (10 ng/lung) at 7 h, suggesting a very low risk of accumulation in the lung upon repeated administration (Figure 10).

In vitro blood binding / plasma binding [1]
The blood binding of 28h/GSK-3008348 was measured in vitro in rat, mouse, dog and human blood by rapid equilibrium dialysis at the nominal concentration of 1 μg/mL. The test compound was spiked in blood diluted 1:1 with dialysis buffer (PBS -100mM sodium phosphate + 150mM sodium chloride, pH 6.9 -7.2) and incubated or 4 hours at 37°C in 5 replicates, with aliquots removed at the beginning and end of the incubation period to assess blood stability along with the binding. On one occasion (binding to human blood), undiluted blood was used for the study; as no appreciable difference was observed with the binding value obtained from 1:1 diluted blood, all the results were averaged together. All buffer (PBS) and blood samples (50μL) were matrix matched by diluting 2-fold with either blood or buffer before being taken for analysis. The resultant samples were extracted by protein precipitation and the blood binding of 28h determined by peak area ratio from the LC-MS/MS analysis. The binding of 28h to plasma proteins was measured in vitro in rat, dog and human plasma by rapid equilibrium dialysis at the nominal concentration of 1 μg/mL. The test compound was spiked in neat plasma and incubated for 4 hours at 37°C in 5 replicates, with aliquots removed at the beginning and end of the incubation period to assess plasma stability along with the binding. All buffer (PBS, 100mM sodium phosphate + 150mM sodium chloride, pH 6.9 -7.2) and plasma samples (50μL) were matrix matched by diluting 2-fold with either plasma or buffer before being taken for analysis. The resultant samples were extracted by protein precipitation and the plasma protein binding of 28h determined by peak area ratio from the LC-MS/MS analysis.

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In vitro metabolic stability [1]
The metabolic stability of 28h/GSK-3008348 was tested in male CD mouse, male Wistar Han rat, male Beagle dog and mixed gender pooled human liver microsomes. Microsomes (final protein concentration 0.5mg/mL), 0.1M phosphate buffer pH7.4 and test compound (final substrate concentration = 0.5μM) were pre-incubated at 37°C prior to the addition of NADPH (final concentration = 1mM) to initiate the reaction. The test compound was incubated for 0, 5, 15, 30 and 45min. The control (minus NADPH) was incubated for 45min only. The reactions were stopped by the addition of 50μL methanol containing internal standard at the appropriate time points. Following protein precipitation, the compound remaining in the supernatants was measured using specific LC-MS/MS methods as a ratio to the internal standard in the absence of a calibration curve. Peak area ratios (Compound to IS) were fitted to an unweighted logarithmic decline in substrate. Using the first order rate constant, clearance was calculated by adjustment for protein concentration, volume of the incubation and hepatic scaling factor (52.5mg microsomal protein/g liver for all species). 28h was incubated at 37°C in male CD mouse, male Wistar Han rat, male Beagle dog and mixed gender pooled human hepatocytes. Suspensions of cryopreserved hepatocytes from each species were used. Incubations were performed at a test or control compound concentration of 0.5μM at 37°C, at a cell density of 0.5million viable cells/mL. Control incubations were also performed in lysed cells to reveal any non-enzymatic degradation. Samples (50μL) were removed from the incubation mixture at 0, 5, 10, 20, 40 and 60min (control sample at 60min only) and added to methanol, containing internal standard, (100μL) to stop the reaction. Following protein precipitation, the compound remaining in the supernatants was measured using specific LC-MS/MS methods as a ratio to the internal standard in the absence of a calibration curve. Peak area ratios (Compound to IS) were fitted to an unweighted logarithmic decline in substrate. Using the first order rate constant, clearance was calculated by adjustment for protein concentration, volume of the incubation and hepatic scaling factor (120 million cells / g liver in mouse, rat and human; 240 million cells / g liver in the dog).

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In vitro and Ex vivo tissue binding [1]
The in vitro binding of 28h/GSK-3008348 to lung homogenate was measured in mouse and human lung tissue by rapid equilibrium dialysis at the nominal concentration of 1 μg/mL (in diluted homogenate). For each in vitro binding study approximately 1g (accurate weight recorded used for subsequent homogenate dilution) of mouse or human lung tissue was weighed out and placed into a 7mL Precellys homogenising tube prefilled with CK28 ceramic beads. 4mL of buffer (PBS -100mM sodium phosphate +150mM sodium chloride, pH 6.9 -7.2) were added before being homogenised (2 x 20s at 6500 rpm). The resulting homogenate was further diluted with buffer to create a 1:10 diluted homogenate, spiked at 1 ug/mL 28h and incubated for 4 hours at 37°C in 5 replicates, with aliquots removed at the beginning and end of the incubation period to assess the stability of the compound in tissue along with the binding. On one occasion (binding to mouse lung homogenate), lung tissue from a mouse previously treated with bleomycin was used for the binding assay; as no appreciable difference was observed with the binding value obtained from lungs of naive mice, all the results were averaged together. Similarly, the binding to human lung homogenate was conducted in tissue aliquots stored under different conditions (fresh, frozen at -20°C, frozen at -80°C), to test the robustness and reproducibility of the assay, so that human tissue from different storage conditions could be potentially used in future assays. On one occasion an aliquot obtained from the lung of a human donor affected by IPF was used for the binding assay; as no appreciable difference was observed in any of these studies, all the results were averaged together. All buffer and lung homogenate samples (50μL) were matrix matched by diluting 2-fold with either lung homogenate or buffer before being taken for analysis. The resultant samples were extracted by protein precipitation and the lung tissue binding of 28h determined by peak area ratio from the LC-MS/MS analysis. The binding of 28h to mouse lung homogenate was also measured ex vivo, using the lungs from an in vivo study (sub-cutaneous administration to the male C57Bl/6J mouse at 15 mg/kg/day by osmotic mini-pumps). Experimental conditions for the preparation of the homogenate, the rapid equilibrium dialysis, the LC-MS/MS analysis and the processing of the results were as described above. The in vitro binding of 28h to kidney and liver homogenates was measured in rat tissue by rapid equilibrium dialysis at the nominal concentration of 1 μg/mL (in diluted homogenate). Experimental conditions for the preparation of the homogenate, the rapid equilibrium dialysis, the LC-MS/MS analysis and the processing of the results were as in the in vitro lung binding studies described above, except for the dilution factor (1:5 instead of 1:10).

In vitro blood distribution [1]
The extent of association of 28h/GSK-3008348 with human, dog and rat blood cells was measured in vitro at a nominal concentration of 1μg/mL. The percentage association of 28h with the cellular fraction of blood was determined from a single donor (dog) or pooled (n=2) donors (rat and human) in triplicate following 30 minutes incubation at 37°C in whole blood. A spiked plasma standard from the same rat, dog or human blood sample was also incubated at the same concentration to generate a value for total blood concentration against the same matrix as the samples. At the end of the incubation blood samples were centrifuged to yield plasma. The concentration of 28h was determined by peak area ratio from the LCMS/MS analysis. Haematocrit values of 44%, 42% and 46% were used for human, dog and rat respectively.

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In vitro passive permeability [1]
The permeability (Pexact) of 28h/GSK-3008348 across an MDCK-MDR1 cell monolayer was measured at a starting concentration of 3μM in the presence of GF120918, an efflux inhibitor. The pH of the donor and receiver compartments was 7.4 (Hanks’ Balanced Salt Solution). Incubations were carried out in an atmosphere of 5% CO2 with a relative humidity of 95% at 37°C for 60 minutes. Apical and basolateral samples were diluted for analysis by LC-MS/MS. The integrity of the monolayers throughout the experiment was checked by monitoring Lucifer yellow permeation using fluorimetric analysis.

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Interaction with P-glycoprotein [1]
The permeability of 28h/GSK-3008348 across an MDCK-MDR1 cell monolayer was measured at a concentration of 2 μM in the presence and absence of GF120918, an efflux inhibitor. The pH of the donor and receiver compartments was 6.9 (simulated lung fluid). All chambers were first pre-incubated for 60 minutes with simulated lung fluid. After 60 minutes, the fluid was removed. Cell monolayers were immediately dosed on the apical side (A-to-B) or basolateral side (B-to-A) with test compound and incubated at 37°C with 5% CO2 in a humidified incubator. The receiver chambers were filled with blank simulated lung fluid. Each determination was performed in triplicate. After 90 minutes aliquots were taken from the receiver and donor chambers. The Lucifer yellow flux was measured for each monolayer after being subjected to the test compound to ensure no damage was inflicted to the cell monolayers during the flux period.

Oral/IV rat pharmacokinetics [1]
The study was conducted as a cross-over design in dual cannulated male Wistar Han rats (n=3, body weight 270-295g; femoral vein for drug administration, jugular vein for blood sampling). GSK-3008348/28h was dosed intravenously at 1 mg/kg as a 30-min infusion at an infusion rate of 10mL/kg/h, dissolved in 100% saline. Following a two-day washout, GSK-300834828h was dosed to the same rats by oral gavage at 3 mg/kg dissolved in 100% saline at a dose volume of 10 mL/kg. Blood samples were collected for both arms of the study up to 24h via a jugular cannula. All blood samples were taken into heparinised containers. Individual aliquots of blood were transferred directly into micronics tubes containing an equal volume of sterile water and stored frozen at -20ºC prior to analysis.

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Oral/IV dog pharmacokinetics [1]
The study was conducted in three male Beagle dogs (body weight 12.1-15.4 kg) with a crossover design including three arms: intravenous administration of vehicle, for the purpose of baseline ECG recording, followed by intravenous and oral administration of the test compound. ECG recording was also conducted during the intravenous arm of the study. The dogs were housed in standard cages. Food was removed at approx 4 p.m. the day prior to dosing and offered 4hr post dose. Water was available ad libitum throughout the study. GSK-3008348/28h was dosed intravenously in the cephalic vein at 1 mg/kg as a 60-min infusion at an infusion rate of 5 mL/kg/h, formulated in 100% saline. Following a six-day washout, GSK-3008348/28h was dosed to the same dogs by oral gavage at 1 mg/kg dissolved in water at a dose volume of 2 mL/kg. Prior to dosing on both occasions, a temporary cannula (angiocath) was inserted into the cephalic vein (opposite leg to the cephalic vein used for intravenous administration) and remained there for the first 2 hours of blood sampling to minimize the number of venipunctures. For each arm of the study, the temporary cannula was removed once the 2h blood samples had been collected. Subsequent samples were collected by direct venipuncture via the jugular vein. Blood samples were collected for both arms of the study up to 24h after dosing. at the following times after dosing. All blood samples were taken into heparinised containers. Individual aliquots of blood were transferred directly into micronics tubes containing an equal volume of 0.02% phosphoric and stored frozen at -20ºC prior to analysis.

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Preliminary studies to investigate route of elimination in vivo (rat and dog) [1]
A preliminary study was conducted in a single male Wistar Han rat, in order to investigate renal and biliary elimination of GSK-3008348/28h in this species. The rat (260g) was housed in a standard holding cage and maintained in a controlled environment with free access to food and water. The animal was surgically prepared according to local procedures and practises: following general anaesthesia, the rat was cannulated via the femoral vein into the vena cava (for drug administration), jugular vein (for blood sampling) and bile duct (for bile collection) whilst under anaesthesia. The rat was allowed to recover for ca. 1 h prior to dosing. GSK-3008348/28h was dosed intravenously as a 30-min infusion at 1 mg/kg free base eq. at an infusion rate of 10 mL/kg/h, formulated in 100% saline. Serial blood samples were collected from the jugular cannula up to 7h from the start of the infusion. Blood concentration-time data were used to determine PK parameters. Urine and bile were collected over the study duration (7h). Urine and bile concentration data were used to determine the contribution of renal and biliary clearance respectively. Following the final sample (7h) the rat was culled and lung, liver and kidneys collected to determine blood:tissue ratios. All blood samples were taken into heparinised containers. Individual aliquots of blood were transferred directly into micronics tubes containing an equal volume of 0.02% phosphoric and stored frozen at -20ºC prior to analysis. The bile was collected in two separate aliquots (0-4h and 4h-7h) on dry ice into pre-weighed tubes containing 40μL of 1% phosphoric acid to result in a nominal final concentration of 0.01% (assuming 4mL bile). Urine samples were collected on dry ice over two intervals (0-4h and 4h-7h) into labelled tubes containing 10μL of 1% phosphoric acid to result in a nominal final concentration of 0.01% (assuming 1mL urine). A preliminary study was conducted in a single male Beagle dog, in order to investigate renal elimination of 28h in this species. All animal work was carried out following routine animal husbandry methods. The dog (16.9kg) was kept in a sling for the first two hours after the start of the infusion and subsequently housed in a metabolism cage between 2-8h of the study. Food was removed at approx 4 p.m. the day prior to dosing and offered 4h post dose. Water was available ad libitum throughout the study. 28h was dosed intravenously in the cephalic vein at 1 mg/kg as a 60-min infusion at an infusion rate of 5 mL/kg/h, formulated in 100% saline. Prior to dosing a temporary cannula (angiocath) was inserted into the cephalic vein (opposite leg to the cephalic vein used for intravenous administration) and remained there for the first 2 hours of blood sampling to minimize the number of venipunctures. The temporary cannula was removed once the 2h blood sample had been collected. Subsequent samples were collected by direct venipuncture via the jugular vein. Blood samples were collected up to 8h from the start of the infusion. All blood samples were taken into heparinised containers. Individual aliquots of blood were transferred directly into micronics tubes containing an equal volume of 0.02% phosphoric and stored frozen at -20ºC prior to analysis. Urine was collected on dry ice as a single aliquot (2h-8h) in a tube containing 100μL of 10% phosphoric acid to result in a final concentration of 0.01% (assuming 100mL urine).

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Intratracheal and nebulised pharmacokinetics in the rat [1]
Intratracheal pharmacokinetics was investigated in the male Wistar Han rat (n=3, 280-315g). Rats, housed in standard holding cages and maintained in a controlled environment with free access to food and water, received an intratracheal dose of GSK-3008348/28h at the nominal dose level of 1 mg/kg. The compound was dosed at a fixed volume of 200 μL (assuming a 250g BW) as a solution of 1.25 mg/mL in 100% saline. Blood samples were collected up to 12h after dosing via a temporary cannula in the tail vein (direct tail venipuncture at 12h). All blood samples were taken into heparinised containers. Individual aliquots of blood were transferred directly into micronics tubes containing an equal volume of sterile water and stored frozen at -20ºC prior to analysis. Pharmacokinetics by the nebulised route was investigated in the female Wistar Han rat. The study was conducted to generate systemic pharmacokinetics and a lung retention time course for 28h after nebulisation in an ADG inhalation tower. Preliminary work was conducted to characterise the aerosol delivered by the ADG system in anticipation of the target dose level for the study. On the day of the nebulised PK study, fourteen rats (280- 330g) were placed in a restrainer and randomly attached to ports on an inhalation tower prior to the start of the nebulisation (using an LC Sprint jet nebuliser). All rats were exposed for 10 minutes to an aerosol generated from a solution containing 28h at 10 mg/mL, formulated in 100% saline adjusted to pH 7.0. The target dose level for the study was 1 mg/kg, but subsequent filter analysis allowed the estimation of the actual dose level as 0.227 mg/kg. Rats 11-14 provided serial blood samples collected up to 7h from the beginning of the nebulisation from a temporary cannula in the tail vein into a heparinised microtainer. All rats in the study were culled (n=2 rats/timepoint, up to 12h) with an overdose of pentobarbitone. A terminal blood sample was collected for all rats from a severed jugular vein into a heparinised microtainer and the lungs were removed and placed in Eppendorf tubes, both being kept on wet ice. Individual aliquots of blood were transferred directly into micronics tubes containing an equal volume of sterile water and stored frozen, together with the lung tissue, at -20ºC prior to analysis.

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MALDI imaging [1]
Two male Crl:WI(Han) rats (ID: 099 & 100) were dosed with GSK-3008348/28h by the inhaled route as part of a 14-day safety assessment study. One male Crl:WI(Han) rat (ID: 098) was dosed with vehicle. Animals 099 and 100 received a single, 60 minute, snout only exposure of nebulised 28h dissolved in 0.9% aqueous sodium chloride (pH adjusted to 7) at a dose level of 6.23 mg/kg. Animal 098 received a single, 60 minute, snout only exposure of saline alone (pH adjusted to 7). Following death by exsanguination via abdominal aorta under isoflurane anaesthesia, whole lungs with trachea intact were collected immediately after dosing for animals 098 and 099, and 1 hour after completion of dosing for animal 100. Lungs were stored frozen at -80°C. Rat lungs were sectioned frozen at a thickness of 12 μm. MALDI-MS (Matrix Assisted Laser Desorption / Ionisation Mass Spectrometry) Imaging was performed using a Bruker UltrafleXtreme instrument and a spatial resolution of 200 μm.

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Lung tissue partitioning in mouse [1]
Eight male C57Bl/6J mice were housed in standard holding cages and maintained in a controlled environment with free access to food and water. Animals were surgically prepared according to local procedures and practises. Mice (n=4 per dose level) were implanted with an osmotic mini pump (Alzet 1002 model, 100 μL volume reservoir, 0.25 μL/h flow rate) that had been fully loaded with formulated drug solution and primed overnight in sterile 0.9% saline at 37º C. The minipumps were loaded with clear solutions of GSK-3008348/28h formulated in DMSO:PEG200:water 5:45:50 (v/v/v) at a concentration of 6 and 60 mg/mL, in order to achieve the desired delivery of 1.5 and 15 mg/kg/day respectively. Blood samples were collected daily up to 96h after the minipumps were implanted from the tail vein. All blood samples were taken into heparinised containers. Individual aliquots of blood were transferred directly into micronics tubes containing an equal volume of sterile water and stored frozen at -20ºC prior to analysis. At the end of the study (96h) termination was carried out for all mice by an appropriate Schedule 1 method and lungs collected for quantitative analysis (tissue drug levels). Lungs from the mice dosed at 15 mg/kg/day were also used to measure the ex vivo binding of 28h in lung tissue homogenate, based on the same assay developed for in vitro tissue binding.

Pharmacokinetic Profile [1]
In order to predict its pharmacokinetic (PK) profile in human, evaluate its tractability as an inhaled drug candidate, and assist with the interpretation of pharmacokinetic/pharmacodynamic studies, 28h was profiled in a range of in vivo studies in preclinical species (Table 5). In particular, intravenous (iv) studies in rat and dog were conducted to support allometric scaling and predictions of clearance and volume of distribution in human, while characterization of oral pharmacokinetics is essential to predict the fate of the fraction of an inhaled dose in human that is swallowed into the gastrointestinal tract. The pharmacokinetic parameters obtained for rat and dog contributed to the prediction of the clinical PK for GSK-3008348/28h, which is described elsewhere.

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ntravenous/Oral PK in Rat and Dog [1]
The selection of rat and dog as main species for testing the preclinical safety of GSK-3008348/28h was driven by (a) knowledge that the pharmacological target is actively expressed in these two species and (b) the considerable in-house knowledge of the general pathology in these species and their response to a wide variety of drugs developed by the inhaled route. In terms of ADME properties, metabolic stability data in hepatocytes and liver microsomes were generated across species, and the results are described later.

In a crossover iv infusion/oral PK study in male Wistar Han rat, the pyrazole GSK-3008348/28h showed moderate–high blood clearance (56 mL min–1 kg–1, ∼70% liver blood flow (LBF)), moderate volume of distribution (2.1 L/kg), short terminal half-life (0.8 h), and negligible oral bioavailability (<2%). In a crossover iv infusion/oral PK study in male Beagle dog, GSK-3008348/28h showed moderate–high blood clearance (28 mL min–1 kg–1, ∼50% LBF), moderate volume of distribution (3.6 L/kg), moderate terminal half-life (3.6 h), and moderate oral bioavailability (30%). In preliminary studies (n = 1 animal/species) to investigate the routes of elimination in vivo, after intravenous administration at 1 mg/kg, 28h showed a low–moderate renal clearance (15% and 21% of the total blood clearance in rat and dog, respectively). 28h also showed moderate biliary clearance (as unchanged parent) in the rat (24% of the total blood clearance). The unaccounted fraction of the total clearance (∼55% in the rat) is likely to be due to metabolism as the chemical structure of 28h offers several potential sites for both phase I (oxidation) and phase II (conjugation) metabolism. However, a formal metabolite identification study has not yet been conducted.

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Lung Tissue Partitioning in Mouse [1]
An important aspect of inhaled drug discovery is understanding the lung tissue partitioning of the candidate regardless of any specific route of administration. The pulmonary uptake of GSK-3008348/28h was therefore investigated in the mouse by the subcutaneous route of administration. A steady state was achieved within 24 h by continuous infusion of 28h/GSK-3008348 using osmotic minipumps at 1.5 and 15 mg kg–1 day–1 which showed linear increase in exposure in the dose range investigated. Lung to blood ratios of 8:1 (15 mg kg–1 day–1) to 11:1 (1.5 mg kg–1 day–1) were measured in the mouse at steady state (96 h) indicating favorable distribution to the site of action irrespective of the route of administration. As no intravenous PK study was conducted in the mouse, the high lung-to-blood partition measured in this species cannot be related to the observed volume of distribution in the same species. Nonetheless the data suggest the findings are not specific to the mouse. In a preliminary (n = 1) distribution and elimination study conducted in the rat after a 30 min intravenous infusion of 28h at 1 mg/kg, a lung-to-blood ratio of 12 was measured at 7 h. Other studies (e.g., toxicokinetics from early safety investigations) also confirm the favorable lung distribution.

The high values of the lung to blood partition coefficients might be explained by GSK-300834828h having higher affinity for lung tissue proteins compared to plasma proteins or by association to cell membrane phospholipids or intracellular uptake. However, when the unbound concentrations of 28h at steady state in lung tissue and blood were compared, values within 2-fold were obtained, suggesting there is no active uptake mechanism in the lung that could subvert the “free fraction hypothesis”. Although the high tissue partitioning observed with total drug concentrations was not confirmed by the unbound drug ratio, the partitioning is still a favorable condition in terms of PK/PD modeling, as it suggests that the easily measurable systemic unbound drug levels are representative of free drug at the site of action.

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Supporting in Vitro Studies [1]
To complete the characterization as an inhaled drug candidate, assist with the interpretation of PK/PD results (reported elsewhere), help building relevant physiologically based PK models, characterize the safety margins, and enable the pharmacokinetic scaling to human, in vitro studies on GSK-3008348/28h were also conducted. These included in vitro metabolic stability in hepatocytes and liver microsomes, in vitro blood, plasma and tissue binding, in vitro blood distribution, in vitro passive permeability, and preliminary assessment of P-glycoprotein. The results are summarized in Table 6.

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Physicochemical Properties of GSK-3008348/28h [1]
The measured pKa’s of GSK-3008348/28h are 4.1, 7.4, and 9.5 for the carboxylic acid, the tetrahydronaphthyridine, and pyrrolidine, respectively. The zwitterionic form of GSK-3008348/28h is an amorphous solid, whereas the corresponding monohydrochloride is crystalline. The hydrochloride is stable in the solid state for 4 weeks at a range of elevated temperatures and relative humidity. The solubility of free base 28h in saline (pH 7) and simulated lung fluid (SLF) at pH 6.9 is >71 and >34 mg/mL, respectively. The solubility of hydrochloride 28h in saline at pH 4, 5, 6, and 7 is >55 mg/mL in all cases. In saline at pH 7.5 the solubility is 31 mg/mL and in SLF at pH 6.6 is >55 mg/mL. Compound 28h thus demonstrated acceptable solubility for inhaled dosing by nebulization. 28h at a concentration of 1 mg/mL is stable in saline at pH 4 at 50 °C for up to 6 weeks, while solutions prepared at pH 5, 6, and 7 are stable for 6 weeks when refrigerated.

In Vitro Safety Profile [1]
The in silico assessment of direct acting mutagenicity was conducted according to the guidelines of ICH M7 and included the expert rule-based structure–activity relationship model Derek Nexus version 4.0.5 (KB version 2014 1.0) developed by Lhasa Ltd. and the statistical based SAR model Leadscope version 1.8 (Salmonella version 3, E. coli-Sal 102 version 1) developed by Leadscope Inc. GSK-3008348/28h was not predicted to be mutagenic in Derek Nexus, version 4.0.5, but was out of the domain of applicability of the Leadscope model. The mutagenic and clastogenic potential of 28h hydrochloride was assessed in the AMES and mouse lymphoma screening assays and was not genotoxic in these assays up to the recommended maximum concentrations tested.

[1] J Med Chem . 2018 Sep 27;61(18):8417-8443. doi: 10.1021/acs.jmedchem.8b00959.

[2] https://clinicaltrials.gov/ct2/show/NCT02612051.

A series of 3-aryl(pyrrolidin-1-yl)butanoic acids were synthesized using a diastereoselective route, via a rhodium catalyzed asymmetric 1,4-addition of arylboronic acids in the presence of ( R)-BINAP to a crotonate ester to provide the ( S) absolute configuration for the major product. A variety of aryl substituents including morpholine, pyrazole, triazole, imidazole, and cyclic ether were screened in cell adhesion assays for affinity against αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8 integrins. Numerous analogs with high affinity and selectivity for the αvβ6 integrin were identified. The analog ( S)-3-(3-(3,5-dimethyl-1 H-pyrazol-1-yl)phenyl)-4-(( R)-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)butanoic acid hydrochloride salt was found to have very high affinity for αvβ6 integrin in a radioligand binding assay (p Ki = 11), a long dissociation half-life (7 h), very high solubility in saline at pH 7 (>71 mg/mL), and pharmacokinetic properties commensurate with inhaled dosing by nebulization. It was selected for further clinical investigation as a potential therapeutic agent for the treatment of idiopathic pulmonary fibrosis. [1]

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Described here is the discovery and profile of an αvβ6 inhibitor clinical candidate 28h. The candidate came from lead optimization of a series of novel RGD αvβ6 integrin inhibitors possessing the 1,8-tetrahydronaphthyridine guanidine isostere as a replacement of arginine, a pyrrolidine ring as a constrained glycine replacement, and 3-arylbutanoic acid as a replacement of aspartic acid. Compounds were synthesized using a diastereoselective route that involved rhodium catalyzed asymmetric 1,4-addition of arylboronic acids. Thus, a variety of aryl substituents including cycloalkyl, heterocyclic, heteroaryl, and cyclic ether were screened in cell adhesion assays for affinity against αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8 integrins, and several analogs with high aqueous solubility, high affinity, and selectivity for the αvβ6 integrin (over the other αv integrins) were identified. (S)-3-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl)-4-((R)-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)butanoic acid hydrochloride salt (28h) has physicochemical properties commensurate with nebulized delivery (71 mg/mL saline solubility at pH 7), a molecular weight of 487, and moderate lipophilicity (chromLogD7.4 = 2.77). The measured pKa for the carboxylic acid group was 4.07, for the tetrahydronaphthyridine 7.38, and for the pyrrolidine 9.50. 28h is exceptionally potent with a pKi of 11 for αvβ6 integrin in a radioligand binding assay and a long dissociation half-life (7 h). It inhibits the release of active TGFβ in primary human cellular assays and exhibits a long duration of action as a result of rapid induction of αvβ6 endocytosis (internalization), which occurs in minutes, and subsequent slow recycling (over hours). The high solubility of 28h and overall pharmacokinetic properties including the rapid pulmonary absorption, multiple routes of elimination, and potentially limited systemic availability are commensurate with inhaled administration. The hydrochloride salt of 28h was therefore selected for further investigation as a potential therapeutic agent for the treatment of idiopathic pulmonary fibrosis and is currently undergoing clinical investigations. [1]

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GSK3008348 is an investigational drug, being developed by GlaxoSmithKline Research and Development Limited (the Sponsor, a pharmaceutical company based in the UK) for the treatment of Idiopathic Pulmonary Fibrosis (IPF). IPF is a rare and poorly understood disease that causes scarring of the lungs. The main symptoms are shortness of breath and a dry cough. Symptoms generally worsen over time and in some subjects may prove fatal. The cause of IPF is unknown. This is a First Time in Human, Phase 1, 3-part study which is being carried out on behalf of the Sponsor by Quintiles. The primary purpose of Part A is to examine the safety and tolerability of single nebulised (a medicated spray) doses of GSK3008348 following inhalation in healthy volunteers. The secondary objective is to determine how and at what rate the body absorbs, distributes, breaksdown and eliminates the drug. Parts B and C of this study will be in-patients with Idiopathic Pulmonary Fibrosis (IPF). The purpose of Part B and C is to examine the safety and tolerability, and how much of the drug binds to its target, following single nebulised (a medicated spray) doses of GSK3008348 following inhalation in patients with Idiopathic Pulmonary Fibrosis (IPF). The secondary objective is to determine how and at what rate the bodies of these patients absorbs, distributes, breaksdown and eliminates the drug. The total duration of Part A will be 65 - 87 days, Part B 62 days and Part C 43 days.[2]

C29H38CLN5O2

524.0973

487.294

C, 71.43; H, 7.65; N, 14.36; O, 6.56

1629249-33-7

122553774

Brown to orange solid powder

1.3±0.1 g/cm3

716.2±60.0 °C at 760 mmHg

387.0±32.9 °C

0.0±2.4 mmHg at 25°C

1.659

3.57

3

6

9

37

722

0

Cl[H].O([H])C(C([H])([H])C([H])(C1C([H])=C([H])C([H])=C(C=1[H])N1C(C([H])([H])[H])=C([H])C(C([H])([H])[H])=N1)C([H])([H])N1C([H])([H])C([H])([H])C([H])(C([H])([H])C([H])([H])C2C([H])=C([H])C3C([H])([H])C([H])([H])C([H])([H])N([H])C=3N=2)C1([H])[H])=O

ZMXBIIQMSGOIRZ-WIOPSUGQSA-N

InChI=1S/C29H37N5O2/c1-20-15-21(2)34(32-20)27-7-3-5-24(16-27)25(17-28(35)36)19-33-14-12-22(18-33)8-10-26-11-9-23-6-4-13-30-29(23)31-26/h3,5,7,9,11,15-16,22,25H,4,6,8,10,12-14,17-19H2,1-2H3,(H,30,31)(H,35,36)/t22-,25+/m0/s1

(3S)-3-[3-(3,5-Dimethylpyrazol-1-yl)phenyl]-4-[(3R)-3-[2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl]pyrrolidin-1-yl]butanoic acid

GSK3008348; GSK 3008348; Integrin Antagonist 1 hydrochloride; 1629249-40-6; GSK3008,348; Integrin Antagonist 1 (hydrochloride); 1629249-33-7; 3-[3-(3,5-dimethylpyrazol-1-yl)phenyl]-4-[3-[2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl]pyrrolidin-1-yl]butanoic acid;hydrochloride; GSK-3008348 HCl; SCHEMBL18092915;GSK-3008348

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)