European Journal of Pharmaceutics and Biopharmaceutics (v.74, #1)

APV Diary (S1).

Imaging tools for pharmaceutical development by Robert Gurny; Karsten Mäder (1).

Utilising atomic force microscopy for the characterisation of nanoscale drug delivery systems by Johannes Sitterberg; Aybike Özcetin; Carsten Ehrhardt; Udo Bakowsky (2-13).
The introduction of atomic force microscopy (AFM) techniques has revolutionised our ability to characterise colloidal objects. AFM allows the visualisation of samples with sub-nanometre resolution in three dimensions in atmospheric or submerged conditions. Nanomedical research is increasingly focused on the design, characterisation and delivery of nano-sized drug carriers such as nanoparticles, liposomes and polyplexes, and this review aims to highlight the scope and advantages of AFM in this area.A significant amount of work has been carried out in drug delivery system (DDS) research in recent years using a large variety of techniques. The use of AFM has enabled us to directly observe very small objects without the need of a cumbersome and potentially contaminating sample preparation. Thus, nanoscale DDS can be investigated in a controlled environment without the necessity of staining or drying. Moreover, intermittent contact mode AFM allows the investigation of soft samples with minimal sample alteration; phase imaging allows accessing information beyond the sample’s topography and also differentiating between different materials, and force spectroscopy experiments help us to understand the intrinsic structure of DDS by recording the elastic or adhesion behaviour of particles. Hence, AFM enables us accessing information which is hardly available by other experimental techniques. It has provided invaluable information about physicochemical properties and helped to shed light on the area of nanoscale drug delivery and will, with more and more sophisticated equipment becoming available, continue to add to our understanding of the behaviour of nanoscale DDS in the future.
Keywords: AFM; SFM; Liposomes; Nanoparticles; Gene vehicle; Drug delivery systems;

Fourier transform infrared (FTIR) microscopy was applied to obtain information on lateral drug diffusion of dithranol in artificial acceptor membranes. Lateral (2D) drug distribution into an artificial membrane was investigated on an area of 300 μm × 1000 μm with a lateral resolution of 25 μm × 25 μm by integrating a specific IR band located at 1430 cm−1. The concentration profiles show a heterogeneous distribution of dithranol particles resulting in non-uniform drug diffusion. Use of the FTIR microscope either in the transmission or in the reflection mode was restricted to a thickness of the DDC membrane <15 μm. The third dimension (depth profile) was analysed by means of confocal Raman microscopy (CRM). In an artificial membrane, the depth range from a minimum of 1.5 μm up to a maximum of 49 μm was analysed for dithranol distribution.
Keywords: FTIR microscopy; Confocal Raman microscopy; Drug diffusion; Semisolid formulations; Non-invasive; Imaging; Mapping; 3D mapping;

Terahertz pulsed imaging, a novel process analytical tool to investigate the coating characteristics of push–pull osmotic systems by Vincent Malaterre; Maireadh Pedersen; Joerg Ogorka; Robert Gurny; Nicoletta Loggia; Philip F. Taday (21-25).
The aim of this study was to investigate coating characteristics of push–pull osmotic systems (PPOS) using three-dimensional terahertz pulsed imaging (3D-TPI) and to detect physical alterations potentially impacting the drug release. The terahertz time-domain reflection signal was used to obtain information on both the spatial distribution of the coating thickness and the coating internal physical mapping. The results showed that (i) the thickness distribution of PPOS coating can be non-destructively analysed using 3D-TPI and (ii) internal physical alterations impacting the drug release kinetics were detectable by using the terahertz time-domain signal. Based on the results, the potential benefits of implementing 3D-TPI as quality control analytical tool were discussed.
Keywords: Controlled drug release; Osmotic pumps; Push–pull osmotic systems; Terahertz pulsed imaging; Coating thickness;

Photothermal imaging in 3D surface analysis of membrane drug delivery by B. Gotter; W. Faubel; R.H.H. Neubert (26-32).
Various methods exist for research into the penetration process in the human nail plate and for investigation of dermal drug delivery. Application of spectroscopic methods in this scientific field is gaining importance. However, no method meets all demands of the large variety of applications. An alternative optical technique for the characterisation of samples is the photothermal spectroscopy. Photoacoustic techniques, photothermal radiometry, and photothermal beam deflection spectroscopy (PDS) are non-destructive analytical techniques that take advantage of the so-called photoacoustic and photothermal phenomena. PDS, in conjunction with an appropriate scanner, allows for depth profiling and is a promising technique for studies of three-dimensional drug diffusion into artificial and biological membranes. The objective of this article is to demonstrate the use of PDS imaging for pharmaceutical applications and drug delivery studies, with two experiments being used as examples: the follow-up of lateral dithranol penetration into an artificial membrane and depth-resolved measurement of the distribution of a model drug within a keratin membrane from bovine hoof.
Keywords: Photothermal deflection spectroscopy; Photothermal imaging; Depth profiling; Drug penetration; Bovine hoof; Methyl orange; Dithranol;

Since its seminal introduction 50 years ago, confocal microscopy has been applied in numerous fields in life sciences. This review presents the different key elements of confocal microscopes, in particular scanning techniques, light sources and especially laser sources are described in this review. Furthermore, an overview of the different image processing systems coupled with confocal microscopy is provided. The chapter closes with the applications of confocal microscopy in drug delivery to the eye.
Keywords: Confocal microscopy; Scanning techniques; Laser; Image processing; Ocular drug delivery;

Micro-CT in drug delivery by Yiwei Wang; David F. Wertheim; Allan S. Jones; Allan G.A. Coombes (41-49).
Micro-computed tomography (micro-CT) has not to date been fully exploited in the area of controlled drug delivery despite its capability for providing detailed, 3-D images of morphology and the opportunity this presents for exploring the relationships between delivery device formulation, structure and performance. Micro-CT was used to characterize the internal structure of polycaprolactone (PCL) matrix-type devices incorporating soluble particulates (lactose Mw 342.30, gelatin Mw 20–25 kDa) as models of hydrophilic bioactives or pore-forming excipients. Micro-CT images confirmed that the lactose and gelatin particles were uniformly dispersed throughout the PCL phase and that efficient delivery of 95–100% of each species in 9 days involved transport from the matrix core. Quantitative analysis of micro-CT images provided values for matrix macroporosity, which were within 15% of the theoretical value and revealed uniform porosity throughout the samples. Total release of protein occurred in 9 days (PBS, 37 °C) from matrices containing a high protein load (44% w/w) and was independent of particle size. Measurements of equivalent pore diameter and frequency distribution identified a large population of sub-40 μm pores in each material, indicative of a high density of connecting channels between particles which facilitates protein transport through the matrices.
Keywords: Micro-CT; Drug delivery; Matrix;

Small-animal PET: A promising, non-invasive tool in pre-clinical research by Uta Schnöckel; Sven Hermann; Lars Stegger; Marilyn Law; Michael Kuhlmann; Otmar Schober; Klaus Schäfers; Michael Schäfers (50-54).
Today, non-invasive imaging techniques are significantly contributing to the understanding of molecular processes in vivo. Positron emission tomography (PET) is a scintigraphic medical imaging modality that uses radiolabelled molecules (tracers), provides quantitative tomographic images and allows non-invasive assessment of the biodistribution of radioactive substances in vivo. The assessment of pathological glucose metabolism is the clinically best-established application of PET today; however, a multitude of different tracers are available to assess diverse physiological processes. The growing interest in pre-clinical imaging studies, in biological and medical basic research, as well as in pharmaceutical research, has fostered the recent growth in small-animal PET. Small-animal PET can be applied to enable the transfer from molecular findings in vitro to in vivo applications in humans, from bench to bed side.
Keywords: Small-animal PET; Nuclear medicine; Pre-clinical research; Molecular imaging;

Electron Paramagnetic Resonance (EPR) spectroscopy is a powerful technique to study chemical species with unpaired electrons. Since its discovery in 1944, it has been widely used in a number of research fields such as physics, chemistry, biology and material and food science. This review is focused on its application in drug delivery research. EPR permits the direct measurement of microviscosity and micropolarity inside drug delivery systems (DDS), the detection of microacidity, phase transitions and the characterization of colloidal drug carriers. Additional information about the spatial distribution can be obtained by EPR imaging. The chances and also the challenges of in vitro and in vivo EPR spectroscopy and imaging in the field of drug delivery are discussed.
Keywords: EPR; ESR; Electron Spin Resonance; Electron Paramagnetic Resonance; Drug delivery; EPR imaging; In vivo;

Pharmaceutical applications of AC Biosusceptometry by Luciana A. Corá; Madileine F. Américo; Fernando G. Romeiro; Ricardo B. Oliveira; José Ricardo A. Miranda (67-77).
AC Biosusceptometry offers an alternative to investigate noninvasively and without ionizing radiation the behavior of solid dosage forms in vitro and in the human gastrointestinal tract. This versatility allowed applying this technique in a wide field ranging from characterization of the disintegration process to elucidation of how the physiological parameters can interfere with pharmaceutical processes. It is increasingly important to understand how oral solid dosage forms behave in the human gastrointestinal tract. Once labelled, magnetic dosage forms provide an excellent opportunity to investigate complexes’ interactions between dosage form and gastrointestinal physiology. In this paper, basic principles of this biomagnetic instrumentation and of the quantification based on magnetic images are reviewed. Also will be presented are some of the most recent applications of AC Biosusceptometry in the pharmaceutical research including oesophageal transit, gastric emptying and transit time of multiparticulate dosage forms, hydrophilic matrices and disintegration of tablets.
Keywords: Biosusceptometry; Gastrointestinal tract; Solid dosage forms; Imaging; Disintegration;

Magnetic resonance imaging (MRI) is the technique of choice for measuring hydration, and its effects, during dissolution of tablets since it non-invasively maps 1H nuclei associated with ‘mobile’ water. Although most studies have used MRI systems with high-field superconducting magnets, low-field laboratory-based instruments based on permanent magnet technology are being developed that provide key data for the formulation scientist. Incorporation of dissolution hardware, in particular the United States Pharmacopeia (USP) apparatus 4 flow-through cell, allows measurements under controlled conditions for comparison against other dissolution methods. Furthermore, simultaneous image acquisition and measurement of drug concentration allow direct comparison of the drug release throughout the hydration process. The combination of low-field MRI with USP-4 apparatus provides another tool to aid tablet formulation.
Keywords: NMR; MRI; Magnetic resonance imaging; Low field; Tablet; Dissolution; USP apparatus 4;

Assessing gastrointestinal motility and disintegration profiles of magnetic tablets by a novel magnetic imaging device and gamma scintigraphy by Kirsteen Goodman; Lee Ann Hodges; Janet Band; Howard N.E. Stevens; Werner Weitschies; Clive G. Wilson (84-92).
To validate Magnetic Moment Imaging (MMI) for the investigation of gastrointestinal transit and disintegration of solid dosage forms and to correlate the MMI findings with the corresponding gamma scintigraphic data.Three magnetic tablets (MTs) were investigated using in vitro and in vivo tests. The clinical study was a four-way, crossover study with the following arms: (a) immediate-release tablets administered in fasted state; (b) immediate-release tablets administered after 400 mL of Clinutren® ISO; (c) enteric-coated tablets administered in the fasted state; and (d) non-disintegrating tablets studied in the lightly fed state (100 mL of Clinutren® ISO).In both the in vitro and in vivo studies, tablets were detected successfully by MMI and scintigraphy. There was a good correlation between gastric residence times and positional data (in the x, y and y, z-axes). In addition, MMI revealed early swelling behaviour of the tablet matrix. There was excellent agreement for the disintegration times of MT(A) in the fasted arm (scintigraphy 12.0 ± 4.4 min, MMI 11.8 ± 4.4 min). In the MT(A)-fed arm, onset times determined by scintigraphy were delayed in three subjects when compared to the corresponding MMI results. Delayed disintegration was observed with MT(A) administered after food (p  < 0.01) in both the techniques.The MMI device is a reliable imaging tool for tracking the transit and disintegration of a magnetic tablet through the gastrointestinal tract.
Keywords: Gastrointestinal motility; Magnetic tablets; Magnetic imaging; Gamma scintigraphy;

Knowledge about the performance of dosage forms in the gastrointestinal tract is essential for the development of new oral delivery systems, as well as for the choice of the optimal formulation technology. Magnetic Marker Monitoring (MMM) is an imaging technology for the investigation of the behaviour of solid oral dosage forms within the gastrointestinal tract, which is based on the labelling of solid dosage forms as a magnetic dipole and determination of the location, orientation and strength of the dipole after oral administration using measurement equipment and localization methods that are established in biomagnetism. MMM enables the investigation of the performance of solid dosage forms in the gastrointestinal tract with a temporal resolution in the range of a few milliseconds and a spatial resolution in 3D in the range of some millimetres. Thereby, MMM provides real-time tracking of dosage forms in the gastrointestinal tract. MMM is also suitable for the determination of dosage form disintegration and for quantitative measurement of in vivo drug release in case of appropriate extended release dosage forms like hydrogel-forming matrix tablets. The combination of MMM with pharmacokinetic measurements (pharmacomagnetography) enables the determination of in vitro–in vivo correlations (IVIC) and the delineation of absorption sites in the gastrointestinal tract.
Keywords: Magnetic Marker Monitoring; Oral dosage forms; Gastric emptying; Small intestinal transit time; Colon transit; IVIVC; In vivo dissolution; Review;

In the present study, we used benchtop magnetic resonance imaging (BT-MRI) for non-invasive and continuous in vivo studies of in situ forming poly(lactide-co-glycolide) (PLGA) implants without the use of contrast agents. Polyethylene glycol (PEG) 400 was used as an alternative solvent to the clinically used NMP. In addition to BT-MRI, we applied electron paramagnetic resonance (EPR) spectroscopy to characterize implant formation and drug delivery processes in vitro and in vivo.We were able to follow key processes of implant formation by EPR and MRI. Because EPR spectra are sensitive to polarity and mobility, we were able to follow the kinetics of the solvent/non-solvent exchange and the PLGA precipitation. Due to the high water affinity of PEG 400, we observed a transient accumulation of water in the implant neighbourhood. Furthermore, we detected the encapsulation by BT-MRI of the implant as a response of the biological system to the polymer, followed by degradation over a period of two months. We could show that MRI in general has the potential to get new insights in the in vivo fate of in situ forming implants. The study also clearly shows that BT-MRI is a new viable and much less expensive alternative for superconducting MRI machines to monitor drug delivery processes in vivo in small mammals.
Keywords: In situ forming implants; MRI; Benchtop NMR; EPR; Poly(lactide-co-glycolide); In vitroin vivo correlation;

Magnetic resonance imaging and X-ray microtomography studies of a gel-forming tablet formulation by P.R. Laity; M.D. Mantle; L.F. Gladden; R.E. Cameron (109-119).
The capabilities of two methods for investigating tablet swelling are investigated, based on a study of a model gel-forming system. Results from magnetic resonance imaging (MRI) were compared with results from a novel application of X-ray microtomography (XμT) to track the movements of embedded glass microsphere tracers as the model tablets swelled. MRI provided information concerning the movement of hydration fronts into the tablets and the composition of the swollen gel layer, which formed at the tablet surface and progressively thickened with time. Conversely, XμT revealed significant axial expansion within the tablet core, at short times and ahead of the hydration fronts, where there was insufficient water to be observed by MRI (estimated to be around 15% by weight for the system used here). Thus, MRI and XμT may be regarded as complementary methods for studying the hydration and swelling behaviour of tablets.
Keywords: X-ray microtomography (XμT); Magnetic resonance imaging (MRI); Tablet; Swelling; Hydroxypropyl-methyl-cellulose (HPMC); Gel-forming;

Non-invasive MRI detection of individual pellets in the human stomach by Manfred Knörgen; Rolf Peter Spielmann; Ahmed Abdalla; Hendrik Metz; Karsten Mäder (120-125).
MRI is a powerful and non-invasive method to follow the fate of oral drug delivery systems in humans. Until now, most MRI studies focused on monolithic dosage forms (tablets and capsules). Small-sized multi-particulate drug delivery systems are very difficult to detect due to the poor differentiation between the delivery system and the food. A new approach was developed to overcome the described difficulties and permit the selective imaging of small multi-particulate dosage forms within the stomach. We took advantage of the different sensitivities to susceptibility artefacts of T 2-weighted spin-echo sequences and T 2-weighted gradient echo pulse sequences. Using a combination of both methods within a breath hold followed by a specific mathematical image analysis involving co-registration, motion correction, voxel-by-voxel comparison of the maps from different pulse sequences and graphic 2D-/3D-presentation, we were able to obtain pictures with a high sensitivity due to susceptibility effects caused by a 1% magnetite load. By means of the new imaging sequence, single pellets as small as 1 mm can be detected with high selectivity within surrounding heterogeneous food in the human stomach. The developed method greatly expands the use of MRI to study the fate of oral multi-particulate drug delivery systems and their food dependency in men.
Keywords: MRI; NMR-imaging; Pellets; In vivo; Stomach; Oral drug delivery; Multi-particulates; Magnetite;