Systems Pharmacology Modeling of Prostate-Specific Antigen in Patients With Prostate Cancer Treated With an Androgen Receptor Antagonist and Down-Regulator.

First-in-human (FIH) studies with AZD3514, a selective androgen receptor (AR) down-regulator, showed decreases of >30% in the prostate-specific antigen (PSA) in some patients. A modeling approach was adopted to understand these observations and define the optimum clinical use hypothesis for AZD3514 for clinical testing. Initial empirical modeling showed that only baseline PSA correlated significantly with this biological response, whereas drug concentration did not. To identify the mechanistic cause of this observation, a mechanism-based model was first developed, which described the effects of AZD3514 on AR protein and PSA mRNA levels in LNCaP cells with and without dihydrotestosterone (DHT). Second, the mechanism-based model was linked to a population pharmacokinetic (PK) model; PSA effects of clinical doses were subsequently simulated under different clinical conditions. This model was used to adjust the design of the ongoing clinical FIH study and direct the backup program.

AZD3514, an oral selective androgen receptor down-regulator in patients with castration-resistant prostate cancer - results of two parallel first-in-human phase I studies.

BACKGROUND: AZD3514 is a first-in-class, orally bio-available, androgen-dependent and -independent androgen receptor inhibitor and selective androgen-receptor down-regulator (SARD). METHODS: In study 1 and 2, castration-resistant prostate cancer (CRPC) patients (pts) were initially recruited into a once daily (QD) oral schedule (A). In study 1, pharmacokinetic assessments led to twice daily (BID) dosing (schedule B) to increase exposure. Study 2 explored a once daily schedule. RESULTS: In study 1, 49 pts were treated with escalating doses of AZD3514 (A 35 pts, B 14 pts). Starting doses were 100 mg (A) and 1000 mg (B). The AZD3514 formulation was switched from capsules to tablets at 1000 mg QD. 2000 mg BID was considered non-tolerable due to grade (G) 2 toxicities (nausea [N], vomiting [V]). No adverse events (AEs) met the dose-limiting toxicity (DLT) definition. Thirteen pts received AZD3514 in study 2, with starting doses of 250 mg QD. The most frequent drug-related AEs were N: G1/2 in 55/70 pts (79 %); G3 in 1 pt (1.4 %); & V: G1/2 in 34/70 pts (49 %) & G3 in 1 pt (1.4 %). PSA declines (≥50 %) were documented in 9/70 patients (13 %). Objective soft tissue responses per RECIST1.1 were observed in 4/24 (17 %) pts in study 1. CONCLUSION: AZD3514 has moderate anti-tumour activity in pts with advanced CRPC but with significant levels of nausea and vomiting. However, anti-tumour activity as judged by significant PSA declines, objective responses and durable disease stabilisations, provides the rationale for future development of SARD compounds.

Transition state theory and the dynamics of hard disks.

The dynamics of two- and five-disk systems confined in a square has been studied using molecular dynamics simulations and compared with the predictions of transition state theory. We determine the partition functions Z and Z(‡) of transition state theory using a procedure first used by Salsburg and Wood for the pressure. Our simulations show this procedure and transition state theory are in excellent agreement with the simulations. A generalization of the transition state theory to the case of a large number of disks N is made and shown to be in full agreement with simulations of disks moving in a narrow channel. The same procedure for hard spheres in three dimensions leads to the Vogel-Fulcher-Tammann formula for their alpha relaxation time.

Do gastrointestinal transit parameters influence the pharmacokinetics of gefitinib?

The selective EGFR tyrosine kinase inhibitor, gefitinib has been shown to be active against certain human carcinomas. It had been noted that a proportion of volunteers consistently had lower gefitinib exposure following oral administration. The shape of the elimination profile in this subset was also different, showing a monophasic elimination pattern rather than the biphasic pattern observed in the majority of subjects. A gamma scintigraphic study was conducted to examine the relationship of gastrointestinal transit and drug absorption in a cohort of rapid clearance subjects (n=5) and normal profile volunteers (n=7). The fasted volunteer panel received a 250 mg gefitinib tablet labelled with [(111)In]-DTPA together with 240 mL [(99m)Tc]-labelled water. The rapid clearance cohorts were shown to have a faster mean gastric emptying T90 (37 min vs 74 min) and shorter small intestinal transit time (156 min vs 204 min), resulting in an earlier colonic arrival time (181 min vs 244 min). Mean plasma C(max) was lower (99.2 ng/mL vs 116 ng/mL) and AUC almost half in the rapid clearance group (2162+/-81 ngh/mL vs 4996+/-64 ngh/mL). These data suggest that gastrointestinal transit parameters play a role in the differences in the rapid clearance profile group, also contributing to the biphasic to monophasic switch. However, historical data show, at the recommended dose of 250 mg/day steady-state plasma concentrations adequate for clinical benefit are achieved in patients with non-small cell lung cancer.

An investigation of the disintegration of tablets in biorelevant media.

The purpose of the study was to examine the disintegration of tablets in media designed to simulate conditions pertaining in the stomach. Although many studies have been performed to determine dissolution rates in these media, little work has been undertaken on the preliminary step in dissolution, namely disintegration. Two tablet formulations were prepared. One disintegrated rapidly (under 25 s in water) and the other more slowly (8 min in water). The disintegration times were measured by the BP 2000 test using discs. For the rapidly disintegrating tablets, disintegration times were similar in all media except for whole milk. This media is used to simulate the fed stomach and disintegration times were over five times longer than in the other media (P < 0.05). A similar effect was seen with the poorly disintegrating tablets in milk, and prolonged times were also observed in some of the other media. For these latter media, there was a good correlation between the penetration rate of the fluid into the tablet and the disintegration time. Penetration rates for milk were also slow which may be a reflection of its relatively high viscosity and low surface tension.

Lung surfactant phospholipids inhibit the uptake of respirable microspheres by the alveolar macrophage NR8383.

Fluorescent poly(lactic-co-glycolic acid) microspheres of a respirable size were fabricated for use in a fluorescent activated cell sorting assay utilizing the continuous alveolar macrophage NR8383. This is a suitable model of alveolar phagocytosis, which permitted an investigation of the influence of phospholipid structure on the inhibition of phagocytosis of microspheres. Phospholipid inhibition was found to be independent of phosphatidylcholine alkyl chain length. Head group effects were investigated by studies employing phosphatidyl-choline, -serine, and -ethanolamine, and inhibition was shown to be independent of head group. Closer modelling of the lung environment by co-culturing NR8383 on A549 alveolar epithelium showed type II secretions to also down-regulate phagocytosis. In addition, pre-incubation with microspheres coated with dipalmitoylphosphatidylcholine reduced the uptake of a second microsphere (fluorescein isothiocyanate-labelled latex).

The inhibition of phagocytosis of respirable microspheres by alveolar and peritoneal macrophages.

Respirable poly(lactic co-glycolic acid) (PLGA) microspheres (2-3 microm diameter), were fabricated as a model drug delivery system whose uptake by macrophages could be quantified by fluorescent activated cell sorting. The microspheres exhibited minimal release of the entrapped flourophore (rhodamine B) and thus avoided possible fluid phase uptake of the flourophore. Externally bound microspheres were removed from the cell membrane by acid washing. The fluorescent intensity associated with the cells arose, therefore, from the internalised microspheres. NR8383 continuous culture alveolar macrophages were verified against primary cultures as a good model of alveolar phagocytosis. Peritoneal macrophages were also isolated and systemic and alveolar phagocytosis compared. Poloxamer 338 adsorbed at the microsphere surface did not reduce phagocytosis by NR8383 macrophages. It did, however, reduce the number of microspheres contained in primary alveolar macrophages but did not reduce the percentage of phagocytic cells. Poloxamer coatings did not reduce phagocytosis by peritoneal macrophages once the ratio of five microspheres per cell was exceeded. Dipalmitoylphosphatidylcholine (DPPC), the major component of lung surfactant, was added to cultures to model the alveolar environment where it was observed to reduce phagocytosis. In light of this finding, microspheres were coated in DPPC, which reduced their uptake by all cell types at all microsphere to cell ratios.

Novel nanoparticles for pulmonary drug administration.

A novel one-step, low energy method, which avoids harsh processing conditions including potentially toxic and chemically reactive cross-linking agents, for the production of hydrophilic drug nanoparticles suitable for dispersion in the hydrofluoroalkane propellants was investigated. Reverse-phase microemulsions were used as the template for the production of nanoparticles. Two microemulsion systems were investigated: water/sodium bis(2-ethylhexyl) sulphosuccinate (AOT)/iso-octane and water/lecithin/propan-2-ol/iso-octane. Nanoparticles were captured by snap freezing with subsequent freeze-drying. Nanoparticles were dispersed in 1,1,1,2,3,3,3-heptafluoropropane (HFA-227) and the aerosol performance of the pressurised metered dose inhaler (pMDI) assessed by cascade impaction. Spherical nanoparticles less than 300 nm in size were produced. Nanoparticles produced using AOT as the surfactant could not be dispersed in HFA-227. However lecithin based nanoparticles could be dispersed in co-solvent modified HFA-227 and produced fine aerosols (Mass Median Aerodynamic Diameter < or = 1.5 microns, fine particle fraction > 58%). This data suggests that a high fraction of the nanoparticles would be deposited (targeted) within the lung with the deposition being mainly alveolar. That is the ideal deposition profile for the systemic delivery of drugs via the lungs.

An investigation of the solubility of various compounds in the hydrofluoroalkane propellants and possible model liquid propellants.

The aims of this study were to investigate descriptive parameters that may predict the solubility of compounds in the hydrofluoroalkane (HFA) propellants and to identify a model HFA propellant that is liquid at room temperature and atmospheric pressure. The solubility of 32 and 20 compounds chosen to give a wide range of physicochemical properties in HFA-134a and HFA-227, respectively, was measured. The Fedors solubility parameter and a computed log octanol water partition coefficient (CLOGP) were compared with the compounds' solubility in the HFA propellants. A total of 19 and 15 solutes had finite solubilities for HFA-134a and HFA-227, respectively, although the remaining solutes were miscible in all proportions. There was no apparent relation between solubility in HFA and the Fedors solubility parameter. This was not improved by considering the hydrogen-bonding potential of the compounds. When log solubility versus CLOGP was plotted, there was a linear relation for 16 and 12 of the compounds exhibiting a finite solubility in the HFA propellants, although four solutes (phenols) were displaced to the left of the linear relation. The remaining 3 compounds had much lower solubilities than was predicted from their CLOGPs, possibly as a consequence of their crystallinity (high melting points). Of the putative model propellants investigated (i.e., perfluorohexane (PFH), 1H-perfluorohexane [1H-PFH], and 2,2,2-trifluoroethanol), 1H-PFH was the most promising, with a linear relation between solubility in 1H-PFH and solubility in HFA propellant being observed. The solubilities in 1H-PFH were approximately 11 and 26% of those in HFA-134a and HFA-227.

Improved nasal bioavailability of FITC-dextran (Mw 4300) from mucoadhesive microspheres in rabbits.

The nasal bioavailability of fluorescein isothiocyanate-dextran (FITC-dextran) (Mw = 4300) encapsulated in non-mucoadhesive and mucoadhesive microspheres in New Zealand White rabbits was investigated. FITC-dextran was administered nasally encapsulated in carbopol 934P, chitosan and lactose microspheres and the bioavailability compared to intravenous administration of FITC-dextran solution. Administration of FITC-dextran as carbopol microspheres produced a significantly greater bioavailability (33%) than after administration as chitosan (13%) and non-mucoadhesive rapidly dissolving control lactose microspheres (9%). The FITC-dextran terminal plasma half-lives after carbopol 934P and chitosan microsphere administration were significantly longer than after intravenous administration of FITC-dextran. The FITC-dextran terminal plasma half-life after carbopol 934P microspheres administration was significantly longer than after lactose microsphere administration. This data suggested that the increase in FITC-dextran bioavailability after carbopol 934P microspheres administration was due to increased residence at the absorptive site via mucoadhesion and reduced mucociliary clearance. A change in mucosal permeability could not however be discounted especially for the chitosan microspheres.

Prodrug to probe solution HFA pMDI formulation and pulmonary esterase activity.

A novel salbutamol prodrug was synthesised. Solubility in HFA-134a and susceptibility to rat lung homogenate, blood and plasma esterase enzymes were investigated. Whereas salbutamol had a very low solubility in HFA-134a, the prodrug was found to be miscible in all proportions. In lung homogenate, the prodrug hydrolysed with a half-life of 45 min, re-generating approximately 17% of expected salbutamol after 8 h incubation. The use of a solution pMDI for pulmonary delivery of the salbutamol prodrug is predicted to result in liberation of salbutamol in the lungs following in vivo hydrolysis by lung esterases.

Microcalorimetry does not predict the cellular phagocytosis of latex microspheres.

Current literature highlights the potential suitability of microcalorimetry for the investigation of cell-drug interactions. Previous work using bacteria or antigens derived from infectious organisms yielded conclusions that heat production is a quantitative means of measuring phagocytosis. In this study we evaluated the potential of flow-through microcalorimetry as a method of quantifying the phagocytosis of microsphere particulates. The technique avoids the need to incorporate radioactive or fluorescent markers into the particulate formulation, and would be widely applicable in biopharmaceutical research. Using the monocyte cell line Mono Mac 6 a power output of 9.00 microW per million cells was increased significantly on addition of zymosan, lipopolysaccaride (LPS) and phorbol myristate acetate but not following exposure to FITC labelled latex microspheres (LM). TNFalpha production increased on exposure to zymosan, LPS and LPS-phorbol myristate acetate, though not on exposure to LB. An assay was developed which allowed the quantification of internalised particulates in phagocytic cells using fluorescent activated cell sorting (FACS). In contrast to the microcalorimetric and TNFalpha data FACS revealed that 20% of the MM6 population phagocytosed a mean of 1.35 LM. Microcalorimetry and measurements of TNFalpha production are assays of cellular activation a phenomenon not necessarily associated with phagocytosis. FACS, however, serves as a specific and quantitative measure of phagocytosis. Microcalorimetry may not be a suitable technique for the quantitative assessment of the phagocytosis of drug delivery particulates.

Transport of a series of D-phenylalanine-glycine hexapeptides across rat alveolar epithelia in vitro.

The effect of lipophilicity on the absorption of peptides from the lungs was investigated. D-phenylalanine (F)-glycine (G) hexapeptides were synthesised to differ, predominantly, only in their lipophilicity. Rat alveolar type II cells were isolated and cultured on plastic, or polycarbonate filters; by day 6 they had de-differentiated to an alveolar type I-like epithelium. The permeability of the monolayers to the hexapeptides was determined. The hexapeptides were metabolically and chemically stable for greater than 24h in the presence of the cells. They did not adhere to the cell culture plastic and were associated only to a low extent with the cell monolayer. The apical to basolateral permeability coefficients for D-F1G5, D-F2G4, and D-F3G3 were 2.19+/-0.53, 1.75+/-0.42 and 2.20+/-0.56 x 10(-7) cm s(-1) respectively. The permeability of the monolayers to D-F1G5 and D-F2G4 was concentration and direction independent, however for D-F3G3 the monolayer was more permeable in the basolateral to apical direction. There was no correlation between the lipophilicity of the hexapeptides and permeability coefficients: other physicochemical parameters did not predict hexapeptide transport. Lipophilicity does not appear to control the transport of hexapeptides across the alveolar epithelium probably as a consequence of the peptides being transported via the paracellular route.

Route dependent pulmonary first-pass metabolism of a series of biphenylacetic acid esters in rats.

The influence of route of administration of a series of biphenylacetic acid aliphatic esters on their pulmonary first-pass metabolism in rats was investigated. The results were compared with in vitro data and with the physicochemical properties of the esters. Pre- and post-absorptive first-pass metabolism was assessed by comparing area under blood concentration-time curves after intra-arterial administration of the esters with those seen after intravenous (to assess post-absorptive) and intratracheal (to assess pre-absorptive) administration. The extent of pulmonary metabolism was dependent on the route of administration. For all the esters studied there was insignificant post-absorptive first-pass extraction, however there was a large pre-absorptive first-pass extraction (greater than 80%). Enzyme kinetics derived in vitro using the 'well-stirred' model (Dickinson and Taylor, 1995. Int. J. Pharm. 116, 231-236) predicted the extent of post-absorptive but not the pre-absorptive, first-pass metabolism.

Putrescine uptake by alveolar epithelial cell monolayers exhibiting differing transepithelial electrical resistances.

Rat alveolar type II cells were isolated following elastase digestion and cultured on polycarbonate filters at various densities and in different media. Two days after seeding, the cells formed a monolayer on the filters which consisted predominantly of type II cells, these then de-differentiated to a alveolar type I-like cell monolayer by day 6. The seeding density and media utilized affected the transepithelial electrical resistance (TEER) generated by the monolayer. Only certain culture conditions allowed the production of a monolayer that mimics, putatively, the in vivo alveolar epithelium (TEER greater than 1000 omega cm2). Vmax and K(m) values for the uptake of putrescine by monolayers exhibiting low and high TEERs on day 6 were determined. The capacity of the putrescine uptake mechanisms was greater in cell monolayers exhibiting a high TEER than those exhibiting a low TEER, suggesting that the TEER does not only measure the "tightness" of the monolayer but contains an element representative of the viability of the cell monolayer. The selection of appropriate TEERs for cell culture investigations is discussed.

Pulmonary first-pass and steady-state metabolism of phenols.

PURPOSE: To study (A) the effect of the administration route (i.v. and i.t.) on pre- and post-absorptive metabolism of phenol and 1-naphthol by the lung and (B) pulmonary extraction of phenol at steady-state. METHODS: Phenols were administered intra-arterially, intravenously and intratracheally and the pre- and post-absorptive metabolism assessed by comparing the AUCs in arterial blood. Phenol was infused to steady-state and the pulmonary extraction assessed by measuring jugular vein and carotid artery blood concentrations. RESULTS: In contrast to previously published data (e.g., Mistry and Houston, Drug Metab.Dispos.l3:740-745 (1985)) we could not detect pulmonary first-pass metabolism of the phenols; reasons for this disparity are discussed. An apparent negative pulmonary extraction of phenol at steady-state was observed, probably as a consequence of extraction by organs which are in series, and not parallel, with the lungs (e.g. liver, kidneys and GIT). CONCLUSIONS: (A) Phenol and 1-naphthol do not undergo pulmonary first-pass metabolism. (B) Traditional methods of assessing organ extraction and clearance at steady-state cannot be utilised when metabolising organs are in series.