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Imaging of oily formulations in the gastrointestinal tract
advanced
drugdeliiry reviews
ELSEVIER
Advanced
Drug Delivery
Reviews 25 ( 1997) 91- 101
Imaging of oily formulations
in the gastrointestinal
C.G. Wilsona’*, M. McJuryb, B. O’Mahony”,
tract
M. Frier’, A.C. Perkins”
“Department of Phurmuceuticul Sciences, Royul College, 204 George Street, University qf Strathclyde Gluago~: GI IXW, UK hDepartment of Clinical Physics, The Western InJimmy Glasgow, Scotland, UK ‘Department qf Medical Physics, Queen ‘s Medical Centre Nottingham, UK
Abstract
The use of an imaging technique such as gamma scintigraphy to follow the behaviour of a formulation in the gastrointestinal tract is widely employed in the development of new therapeutic concepts. Such studies are facilitated by the availability of suitable radiolabels, incorporated into the formulation during manufacture. The range of labels for lipophilic excipients is very limited, although new brain-imaging agents may offer interesting possibilities. The use of alternative imaging modalities such as magnetic resonance imaging should also be considered. The difference in the T, - or T,-weighted signal from the oil against the aqueous contents provides a method of visualising an oil-based formulation in vivo without any modification to the formulation. This article reviews approaches in the search for suitable techniques for in vivo imaging of oral formulations containing a high proportion of oil. Keywords:
Oil; Gastrointestinal
tract; Drug delivery; Imaging;
Scintigraphy;
Magnetic
resonance
imaging
Contents 92 Introduction ............................................................................................................................................................................ 92 1.l. Radionuclide techniques ................................................................................................................................................... 92 1.2. Radioiodine complexes.. ................................................................................................................................................... 92 1.3. Technetium complexes ..................................................................................................................................................... 93 1.4. New lipophilic chelates suitable for labelling oily vehicles.. ................................................................................................ 1.5. Neutron activation.. .......................................................................................................................................................... 94 Ultrasound.. ............................................................................................................................................................................ 94 95 Magnetic resonance imaging (MRI). ......................................................................................................................................... 3.1. Contrast................................................................................................................................................................. ._ 95 96 3.1.l. Contrast agents ...................................................................................................................................................... 3.1.2. Positive agents.. ..................................................................................................................................................... 96 3.1 .3. Negative agents. ..................................................................................................................................................... 96 3.1 .4. Other methods: multi-nuclear MRI .......................................................................................................................... 96 3.2. MR sequences for in-viva imaging.. .................................................................................................................................. 96 3.2.1. Spin-echo (SE) sequences.. ..................................................................................................................................... 97 3.22. Gradient-echo (GE) sequences ................. . .............................................................................................................. 97 32.3. Ultra-fast imaging .................................................................................................................................................. 97 98 3.3. In-viva imaging of oils ..................................................................................................................................................... Conclurmns ........................................................................................................................ YY ......................................... ........ Acknowledgments ....................................................................................................................................................................... 100 References .................................................................................................................................................................................. 100
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C.G. Wilson et al. I Advanced
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1. Introduction
labelled materials for the study of the gastrointestinal tract [2].
Oils and fats are essential dietary components and have therefore attracted much clinical and scientific interest. Many gastrointestinal disorders are associated with abnormal fat absorption and therefore it was necessary to develop procedures to aid in clinical diagnosis. Early diagnostic techniques were largely chemical (faecal fats etc.) and required extensive laboratory processing. In the 1950s and 60s tests were developed which orally administered radiotracers, reducing the need for extensive chemical analysis. More recently the development of sophisticated imaging techniques such as radionuelide imaging, ultrasound and magnetic resonance imaging (MRI) have allowed the detailed visualisation of the transit and absorption of lipids and oils in the body. These techniques are of interest to the pharmaceutical industry since they provide valuable information on the gastrointestinal transit, dispersion and absorption of fats and oils in drug formulations.
1.1. Radionuclide
techniques
Traditional measurements of dietary fat absorption were based on the chemical estimation of dietary intake and excretion of fat. Radionuclide techniques for the measurement of fat absorption were first introduced in the late 1940s using ‘3LI-labelled triolein and oleic acid [l]. Other radionuclides included ‘*Br and 14C. These techniques were based on the measurement of activity contained in the blood and faeces and provided some basic metabolic data on fat absorption in health and disease. In particular, conditions in which fat absorption is decreased include idiopathic steatorrhoea, Whipple’s disease, ileitis, diverticulosis and tuberculosis. Fat absorption was also of interest in the study of conditions such as diabetes mellitus, hepatic cirrhosis, pancreatitis and certain cancers. The introduction of radionuclide imaging devices in the 1960s (in particular the gamma camera) provided a means for imaging the gastrointestinal transit of orally administered agents and radiolabelled compounds. The availability of radionuclides with suitable decay characteristics and gamma energies for imaging together with advances in radiopharmaceutical chemistry have resulted in a wide range of radio-
1.2. Radioiodine
complexes
The first documented uses of iodinated oils relate to their use in the investigation of fat malabsorption syndromes. Thus a degree of unsaturation was important in the selection of oils administered, e.g. oleates and myristates. Lubran and Pearson describe a method for the iodination of triolein in the diagnosis of steatorrhoea, which has been adopted as a standard method in clinical investigations [3]. The early imaging investigations of pharmaceutical formulations employed isotopes of iodine, particularly using iodine- 13 1, to label proteins and oils and emulsion formulations including liposomes [4] and emulsions [5]. These early uses are extensively covered in an earlier review to which the reader is referred [6]. The use of iodine-131 is limited by the poor photon yield in relation to the absorbed radiation dose, since beta particles are emitted during decay. Other iodine isotopes are available, of which the most suitable for imaging purposes is cyclotronproduced iodine-123. This can be incorporated into radiopharmaceuticals with little molecular disruption and has a similar energy to technetium-99m. Hardy and co-workers describe the use of iodine-123 in the iodination of arachis oil and Labrophil WL2700, in order to study the spreading of suppository bases delivered in rectal hard gelatin capsules [7]. Long-chain fatty acids such as palmitic and oleic acids are the principal energy source for the myocardium and therefore iodinated derivatives of these fatty acids have received attention as probes for the evaluation of myocardial metabolism. A number of iodine-labelled fatty acids have been used in routine clinical diagnosis and are available from commercial radiochemical suppliers. Potentially, these offer an unexplored and novel range of materials which could be applied to the study of lipophilic excipients. For a review of these materials, the reader is referred to Knapp and Kropp [S]. 1.3. Technetium
complexes
The poor imaging characteristics of I-13 1, and the associated beta radiation encouraged the search for
C.G. Wilson et al. I Advanced Drug Delivery
alternatives, such as ‘231, “3mIn, “‘In and 99mTc. Technetium-99m, which is available at high specific activity, has an ideal energy (141 keV), a short half-life (6.03 h), a higher photon yield and no associated beta radiation. The major difficulty with this element is the high valency state and the need to trap the atom into the tracer molecule by chelation. An unpublished study by our group examined the in vivo behaviour of size 0 soft gelatin capsules. Formulations of varying dispersion characteristics were compared in order to examine the in vitro/in vivo correlation. Batches of the formulations under test were radiolabelled by puncturing the side of the capsule with a hypodermic syringe, expelling the contents, mixing with 99mTc-labelled Amberlite resin to 2% fill weight and refilling. The puncture site was patched with a small gelatin square using a heated spatula to get a smooth seal. Dissolution times of the formulations tested were unaffected by this procedure as measured from in vitro disintegration times. Rates of dispersion in vivo were found to be much lower for poorly dispersible formulations compared to formulations which were highly dispersible in vitro. A typical scintiscan showing the breakup of the formulation is shown in Fig. 1. The low cost and excellent availability of Tc-99m ensure that this radionuclide will remain popular. exametazime such as Radiopharmaceuticals (CeretecB) and tetrofosmin (MyoviewB) have been specifically developed as diagnostic agents for brain and cardiac imaging. However, these agents offer valuable tracers for radiolabelling oily vehicles. As mentioned previously, technetium-99m is usually incorporated into molecules by chelation producing complexes. It is important to note that the entire complex will behave in a manner very different to that of the free ligand. Functional groups are involved in the complexation with technetium, and there is an unavoidable increase in molecular size and an alteration, usually a decrease, in charge. Eckelman has recently produced an excellent review of the technetium complexes available for biochemical studies [9]. He classifies the chelates into groups based on physiological functional: kidneys, bone, liver, heart and brain. It should be noted that this functional classification parallels ascending lipophilicity. Clinically, kidney function generally employs the most water soluble chelates such as 99mTc-labelled diethylenetriaminepentaacetic acid whereas
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Fig. 1. Scintiscan showing disintegration of a soft gelatin capsule in the stomach of a volunteer, 8 min after dosing. The unit was resin in the oil mix as labelled by inclusion of “‘“Tc-labelled described in the text.
the chelates for heart and brain are neutral or cationic lipophiles. 1.4. New lipophilic chelates suitable for labelling oily vehicles One of the first ligands designed specifically for use with technetium was a lipophilic iminodiacetic acid, which produces a complex with technetium in the + 3 oxidation state and which is cleared rapidly via the liver following intravenous administration [ 101. Mebrofenin, 3-bromo-2,4,6-trimethyl HIDA, shows much improved hepatic clearance, and was developed by Nunn [ 111. Intensive investigations following this period have led to the development of new ligands including exametazime, (RR,SS)-4,8diaza-3,6,6,9-tetramethyl undecane-2, lo-dionebisoxime [12] and ECD [13]. When complexed with technetium-99m, exametazime (Ceretec) has a high log P and distributes into oily vehicles readily. It is however unstable and will slowly decompose to a less lipophilic intermediate. This property is used to label cells since the
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lipophilic complex crosses the cell wall but then becomes internalised as a more hydrophilic complex. The search for monovalent cationic technetium complexes for myocardial studies led to the development of hexakis(t-butylisonitrile)-Technetium(I) [ 141. Subsequent developments produced sestamibi (Cardiolite), the 2-methoxy-2-methyl propyl derivative. This compound binds to p-glycoprotein and it has been suggested that the radiopharmaceutical may be useful in following drug resistance. More recenttetrofosmin, l,Zbis[bis(Zethoxyethyl)phosly* phinolethane, has been developed [15]. Like sestamibi, this produces a monovalent, cationic complex with technetium. In all the above examples, complexes are formed using pre-formed ligands. An alternative way of producing technetium complexes uses the technique known as template synthesis, in which the complex is built around the metal from relatively simple components during formation. The novel complexes known as BATOs (boron adducts of technetium dioxime) are examples of these and are neutral, lipophilic materials with potential for myocardial perfusion studies [ 161. Teboroxime, [bis[ 1,2-cyclohexanedionedioximato l)-O]-[ 1,2-cyclohexanedionedioximato(2)-O]methylborato(2)-N, N’, N”, N”‘, N”“, N”“‘]-chlortechnetium has been used clinically for myocardial imaging, but shows rapid washout. Other neutral compounds of note include the bisaminothiol (BAT) derivatives [17] and the kethoxal bis( thiosemicarbazone) (KTS) complexes
oil and 290 ml of “3mIn60 ml of “““Tc-labelled labelled soup (total 505 kcal) on one day and 280 g “3mIn-minced beef (500 kcal), 60 ml 99mTc-labelled oil and 290 ml soup (505 kcal) on another day [20]. The emptying rate of oil in the oil/soup meal was about twice that for oil consumed in the other meal. These results indicate that major differences in the intragastric distribution of oil occur following incorporation of solids into the meal. The author has commented that [99mTc](V)-thiocyanate olive oil is a better fat marker than [75Se]glycerol triethyl butter.
1.5. Neutron activation The introduction of neutron activation enabled the in situ production of nuclides which proved useful for the labelling of complex preparations including enteric coated tablets and pellets by the incorporation of low amounts of stable isotopes during pilot scale manufacture. Most processes involve the bombardment of the insoluble oxide in the finished product to produce the isotopes 17’Er or ‘53Sm which can be imaged using a gamma camera. Esposito and coworkers have described the synthesis of lanthanide organic salts, including samarium stearate and ytterbium trilaurate, trimyristate etc. [21,22]. However, these organo-metallics are not sufficiently soluble in either organic or aqueous solution and are therefore unsuitable as oil markers [23].
[181. The objective of obtaining a low molecular weight lipophilic technetium-99m complex is difficult because the technetium chelate is bulky, adding a minimum of 250 + Da to the biochemical of interest. The effect of addition of the technetium chelate increases the log P to approximately the same extent as iodination. Some fats are relatively polar and in this case it is possible to use hydrophilic markers for the study of these formulations. Jay et al. utilised 99mTc-technetium hydroxymethyldiphosphonate to label Witepsol W-15 in a study of radiolabelled suppositories and obtained results quantitatively similar to that described by Hardy et al. [19]. Edelbroek describes the use of [ 99mTc]( V)-thiocyanate, to measure the gastric emptying and intragastric distribution of oil in a fatty meal consisting of
2. Ultrasound Ultrasound is an acoustic radiation that occupies a frequency band above the audible range. In medical applications ultrasound frequencies of between 1 and 10 MHz are normally employed. Ultrasound is noninvasive and safe, although at high intensities such as those used for therapy (heat treatment) experiments have shown an association with adverse biological effects produced by thermal damage and cavitation in tissues. The echo information derived from ultrasound investigations depends upon the difference in the characteristic acoustic impedances of the interfaces generating the reflection of the sound. For a given medium (tissue, fluid, air) the acoustic impedance is equal to the product of the density and the velocity
C.G. Wilson et cd. I Advanced
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of sound in that medium. In the case of Doppler investigations the movement of the reflecting interface results in a frequency shift which may be encoded into a colour display to provide additional information on motion within the body. When examining the stomach or GI tract the luminal contents will dictate the nature and strength of the reflected signal. It has been widely appreciated from clinical use that ultrasound examinations of the small and distal bowel have been severely limited due to the presence of gas which produces a very strong reflection and strong attenuation. Such investigations have therefore been restricted to specialist centres and research studies. However, with appropriate preparation the examination of the GI tract may be carried out. In particular, ultrasound imaging of the stomach contents has provided useful data on the gastric emptying of liquids. Sonography has been used to investigate the emptying of fatty meals, for example, Wedman et al. (241 described the use of sonography using a 3.5 MHz curved probe to investigate the gastric emptying, antral motility and gall bladder emptying after a liquid fatty meal in 50 subjects, half of whom had systemic sclerosis. It is not generally considered that fat can be discriminated from protein and carbohydrate components within the GI tract since the presence of gas bubbles would produce an equally strong signal. Sonography per se is therefore of limited use in evaluating the transit of oils in the gut. However, in highly heterogeneous phases in which gas bubbles or oil droplets are uniformly dispersed thorough an aqueous phase the echogenic characteristics of the medium are markedly changed. Fujimura and colleagues described the quantification of duodenal duodeno-gastric reflux and antral motility using colour Doppler shift flow following administration of a consomme meal [25]. The Doppler
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shift flow information was considered to be derived from oil droplets, size range 5-40 pm, in the soup. Brown et al. found that fat quickly layered in the proximal stomach away from the pylorus thus delaying gastric emptying [26].
3. Magnetic
resonance
imaging
(MRI)
MRI is arguably the fastest developing radiological imaging technique in clinical use. Its exquisite sensitivity to soft tissue, the fact that it is noninvasive and does not use ionising radiation, have lead to its being the modality of choice in a growing number of situations. Only certain substances, having protons with non-zero angular momentum and therefore a magnetic moment. are sensitive to the nuclear magnetic resonance phenomenon. Table 1 lists some nuclei observable with NMR. In acquiring good quality in-vivo MR images of fats in the gut, the main aspects of imaging to consider are: optimising image contrast, avoiding motional artefacts and having sufficient resolution to observe the subject of interest.
3. I. Contrast When imaging oils against a background of the watery contents of the gastrointestinal tract, two aspects to the difference in MR signal between fats and water, allow us to manipulated and optimise the contrast: (a) taking advantage of differences in relaxation properties of fat and water protons and (b), using the difference in resonance frequency (or chemical shift) between fat and water protons. Image contrast may be weighted to highlight T, or T, relaxation differences between substances.
I Somenuclei observable with NMR
Table
Nuclei
Natural abundance
‘H “C “N 1‘>F “P
99.98 I.1 I 0.37 I00 100
Source: Adapted
from Ret’. [27]
(%)
Larmor frequency 42.58 IO.71 4.3 40.05 17.24
at
I Telsa (MHz)
Spin 112 I I2 l/2 I/? II2
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3.1.1. Contrast agents In addition to the intrinsic contrast which exists in a sample due to the properties of the protons, it is sometimes necessary to add contrast-agents to enhance image contrast. A detailed discussion of MR contrast agents in beyond the scope of this article and the reader is refered to standard texts in this subject [28]. In the gastrointestinal tract, contrast agents may broadly be classified in a number of ways: based on the magnetic properties of the agents; whether the agent is miscible or immiscible; or into negative (reducing signal from the bowel-lumen) or positive agents (increasing bowel-lumen signal). Obviously care must be taken when using such crude and sweeping definitions. It should be noted that often positive agents can increase image artefacts due to bowel motion, while negative agents produce little artefact.
3.1.2. Positive agents Positive agents include paramagnetic substances which mix with bowel contents. These agents, such as manganese chloride and gadolinium preparations, cause a reduction of relaxation values of bowel contents, and therefore lead to signal enhancement which is greatest in T,-weighted images [29]. Other paramagnetic substances used include ferric ammonium citrate [30]. This produces approximately 20% of the T, shortening generated by gadopentate dimeglumine at equal volumes. Fats and oil emulsions may also be given to replace bowel contents. Although it can be difficult to administer these in sufficient quantities to replace total bowel and small bowel contents, some success has been made in small bowel marking [31].
3.1.3. Negative agents These agents include iron oxides, perfluorocarbons (PFCs), clays, barium sulphate, and gas-evolving pellets. Iron oxide particles increase signal de-phasing (T2 effects) and on T,-weighted images, show negative contrast. PFCs are compounds in which protons are replaced by fluorine atoms. Being immiscible with bowel contents, they are administered to replace bowel contents and produce no MR signal. Gas providing substances also generate contrast by producing regions of low proton density [32]. Of course, two types of contrast, positive and negative
may be used protocols.
simultaneously,
in
double-contrast
3.1.4. Other methods: multi-nuclear MRI As mentioned previously, hydrogen protons in water and lipid are not the only observable nuclei of interest. It is possible to obtain images of the in-vivo distribution of sodium or fluorine. Due to the low concentration of naturally occurring 19F in the body, usually samples to be imaged are labelled with 19F. If double-tuned RF probes are used, it is possible to acquire images of ‘H- and “F-1abelled nuclei simultaneously. The general anatomy of the subject will be shown in the ‘H-image, while the capsule of formulation will be seen in the “F-image. In animal studies, in-vivo imaging of “F-1abelled formulations has been achieved using pentaerythritol trifluoroacetates. These symmetrical structures give a single 19F resonant frequency and yielding a high sensitivity for the lipophile [33,34]. 3.2. MR sequences for in-vivo imaging MR is a unique imaging modality in that MR image contrast depends not on a single parameter (such as electron density in the case of X-ray based imaging), but rather on a complex recipe of parameters including proton density, T,- and TX-values of tissue, diffusion, flow and others. This has resulted in the development of literally thousands of MR imaging sequences with particular contrast characteristics. Depending on application, however, the number of sequences available to a user will shrink dramatically. In particular, when imaging in the thorax, potential artefacts due to respiratory, cardiac, peristaltic and other patient motion imposes severe restrictions on the type of imaging sequences which can be used. Best results for imaging the gastrointestinal tract, therefore, require MR sequences which acquire the image data quickly in relation to the voluntary and involuntary patient motion. These sequences are generally known as fast and ultra-fast imaging sequences. These include three main types: fast lowangle shot (FLASH), Turbo-FLASH gradient echo based sequencessequences and their family of variants; multi-echo spin-echo sequences such as fast spin-echo (FSE) or turbo spin-echo (TSE); and ultrafast variants of the echo-planar imaging (EPI) sequence.
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3.2.1. Spin-echo (SE) sequences The spin-echo sequence was the early work-horse of clinical MR [35]. It acquires a single signal echo from each excitation, and is inherently slow (of the order of min). To produce acceptable images of the thorax, it is necessary to gate the data acquisition to respiration (the sequence is too slow to acquire data in a breath-hold). This will in turn increase the time of data acquisition. Imaging of capsules with a fast disintegration rate in the gastrointestinal tract, for example, is not possible with simple SE methods. In anaesthetised animal models, SE methods have been used to image tablet formulations in the gastrointestinal tract [34]. The techniques have also been used successfully to investigate, for example, gastric-emptying in human volunteers [36]. It is possible to speed up SE sequences by collecting multiple signal echoes from each single excitation. These techniques are called fast or turbo spin-echo methods [37,38]. They can offer significant reduction in scan time (up to an order of around IO). However, they possess a subtle and complex contrast behaviour, and can best be used for T,weighted imaging only. Lipid in TSE images will appear bright, as will water. Images aquired by these methods can be difficult to interpret.
3.2.2. Gradient-echo (GE) sequences Due to the long imaging times needed for early T,-weighted spin-echo sequences, much development has been done on gradient-echo imaging sequences. By using small excitation pulse angles, and GE data acquisitlion, it is possible to dramatically speed up scan times. These sequences have inherently lower signal-to-noise (SNR) ratios than SE sequences, but win in terms of SNR per unit time. In areas of the body where fast imaging is crucial to avoid motion artefacts, they are generally the sequence type of choice. FLASH [39] (closely-related sequences include FISP [40] GRASS [41], CE-FAST [42] is a lowangle GE sequence, and is fast enough to allow acquisition of several 256 X 256 images in a single breath-hold. It is best for Tz-weighted imaging, but satisfactory T,-weighted images may also be obtained. Turbo-FLASH [43] goes further in terms of scan time reduction, allowing a single image to be acquired in around 1 s (Fig. 2).
07
Fig. 2. Shows one image from a set of eight 256 X 256 sagittal T,-weighted breath-hold FLASH images. The images, obtained from a volunteer imaged on a 1-T Siemens Impact MR system, have slice thickness 8 mm, field of view (FOV) 300 mm, and were acquired with a TR of 130 ms, TE 5 ms, with acquisition time (TA) of 16 s. Fat appears bright on the image, with watery gastrointestinal contents dark. The 8 mm slice thickness keeps the SNR high, without significant loss of quality due to partial volume effects, Up to 10 slices may be acquired in a reasonable breathhold time of 16 s, allowing coverage of the entire stomach.
3.2.3. Ultra-fast imaging The gold-standard in terms of real-time NMR imaging is set by echo-planar imaging (EPI) [44,45] and its many variants (Instascan [46,47], Mosiac [21], RARE [38], QUEST [48] and so on). EPI allows acquisition of an image in a fraction of a second ( 128 X 128 pixel matrix in 128 ms) by collecting data for a complete image from a single excitation. This sequence freezes any unwanted patient motion, providing a snap-shot of the body [49]. It has stringent hardware requirements and only recently become available on standard clinical scanners as a retro-fit [50]. EPI is finding application in imaging applications where speed is an important factor in abdominal imaging of the gastrointestinal tract, liver and heart; in functional imaging of the brain, in following gastric-motor function and transit, and in the analysis of the water-content of stomach, intestine and colon [Sl].
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3.3. In-vivo imaging of oils Temporal resolution of around 20 s for a set of breath-hold FLASH images covering the stomach, allows for the possibility of using MR, for example,
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to monitor the disintegration of oil-filled capsules in-vivo. Fig. 3 shows four images from an MR study following the disintegration of oil-filled gelatin capsules in the upper gastrointestinal tract [52]. The recent developments in GE imaging and
a
Fig. 3. Shows four images from an MR study following the disintegration of oil-filled gelatin capsules in the upper .gastrointestinal tract [52]. The images were acquired using a T,-weighted breath-hold FLASH (TR 130, TE 5, FOV 300, thickness 8 mm, TA 16 s, and single average). (a-d) represent 1, 5, 9, and 16 min after capsule intake.
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Fig. 4. The upper image is a I-cm transverse section through the abdomen of a volunteer, lying right side down soon after consumption of an Olive oil/Beef consomme soup meal using the Echo-Planar (MBEST) technique, acquisition time 130 ms. The bright area (B) corresponds to the soup lying in the dependent part of the gastric body. The horizontal greyish layer (A), apparently floating above the soup is the olive oil. The fat component of the image is displaced owing to the difference in resonance frequency between protons in water and fat, which is approximately 72 Hz at 0.5 T. The lower image taken immediately after the upper image corresponds to the same region of interest. However, the signal from the water has been suppressed, leaving an Image which includes a layer of extracellular fat surrounding the volunteer as well as the olive oil layer in the stomach.
particularly in EPI, have revived interest in imaging the gastrointestinal tract. Boulby and his co-workers used echo planar imaging to assess the effect of posture on the emptying of an oil-water meal [53] (Fig. 4).
4. Conclusions The application of new imaging techniques in the pharmaceutical sciences has evolved steadily from clinical diagnostic imaging procedures. The number of modalities and of imaging agents for the study of
formulations is increasing, although not always directly in gastroenterological sciences. It is necessary therefore to maintain acquaintance with developments in parallel disciplines, which provide new tools for the study of the behaviour of fats and oils. In the immediate future, gamma scintigraphy will provide the widest range of probes and the availability of these instruments to academic and commercial organisations, ensures progress in the understanding of the behaviour of oil-based delivery systems. MRI will provide adjunct information, but the cost and availability of these instruments is as yet prohibitive for wide-scale use.
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Acknowledgments The authors thank Mr Philip Boulby and Dr Penny Gowland of The Magnetic Resonance Centre Nottingham for helpful comments and the provision of the Echo-Planar Images.
References [I] Hofer, R. (1971) Studies of absorption of fats and protein. In: E.H. Belcher and H. Vetter (Eds.), Radioisotopes in Medical Diagnosis, Butterworths, London, pp. 437-449. [2] Frier, M. and Perkins, AC. (1994) Radiopharmaceuticals and the gastrointestinal tract. Eur. J. Nucl. Med. 21, 123442. [31 Lubran, M. and Pearson, J.D. (1958) A screening test for steatorrhoea using ‘3’I-labelled triolein. J. Clin. Pathol. 11, 165-9. [41 Hardy, J.G., Kellaway, I.W., Rogers, J. and Wilson, C.G. ( 1980) The distribution and fate of I-l 3 I-labelled liposomes. J. Pharm. Pharmacol. 32, 309-13. [51 D’Amico, K.M., Davis, S.S., Frier, M., Hansrani, P., Hardy, J.G. and Wilson, C.G. (1981) Effects of emulsifiers on the elimination of iv-administered fat emulsions measured by gamma scintigraphy. Br. J. Pharmacol. 72, 18OP [61 Hardy, J.G. and Wilson, C.G. (1981) Radionuclide imaging in pharmaceutical, physiological and pharmacological research. Clin. Phys. Physiol. Meas. 2, 71-121. [71 Hardy, J.G., Feely, L.C., Wood, E. and Davis, S.S. (1987) The application of gamma-scintigraphy for the evalua:ion of the relative spreading of suppository bases in rectal hard gelatin capsules. Int. J. Pharm. 38, 103-108. PI Knapp, J.F.F. and Kropp, J. (1995) Iodine-123 labelled fatty acids for myocardial SPECT: current staus and future perspectives. Eur. J. Nucl. Med. 22, 361-381. with technetium-99m [91 Eckelman, W.C. (1995) Radiolabeling to study high capacity and low capacity biochemical systems. Eur. J. Nucl. Med. 22, 249-263. [101 Loberg, M.D., Cooper, M., Harvey, E., Gallery, P. and Faith, W. (1976) Development of new radiopharmaceuticals based on N-substitution of iminodiacetic acid. J. Nucl. Med. 17, 633-638. Nunn, A.D., Loberg, M.D. and Conley, (1983) A structuredistribution-relationship approach to the development of Tc99m Mebrofenin: an improved cholescintigraphic agent. J. Nucl. Med. 24, 423-30. [12j Nowotnik, D.P., Canning, L.R., Cumming, S.A., Harrison, R.C., Higley, B., Nechvatal, G., Pickett, R.D., Piper, I.M., Bayne, V.J., Forster, A.M., Weisner, P.S., Neirinckx, R.D., Volkert, W.A., Troutner, D.E. and Holmes, R.A. (1985) Development of a Tc-99m labelled radiopharmaceutical for cerebral blood flow imaging. Nucl. Med. Commun. 6, 499506. chemistry and radiophar[I31 Tulip, T. (1987) Technetium
maceuticals: a prospectus. In: 194th National Meeting of the American Chemical Society Procedings. [14] Jones, A.G., Davison, A. and Abrams, M.J. (1982) Investigations on a new class of technetium cations. J. Nucl. 1Med. 23, P16. [I51 1Higley, B., Smith, F.W., Smith, T., Gemmell, H.G., Gupta, P.D., Gvozdanovic, D.V., Graham, D., Hinge, D., Davidson, J. and Lahiri, A. (1993) Human biodistribution, dosimetry and safety of a new myocardial perfusion agent. J. Nucl. Med. 34, 30-38. [I61 NUM, A.D., Treher, E.N. and Feld, T. (1986) Boronic acid adducts of technetium oxime complexes (BATOs): a new class of neutral complexes with myocardial imaging capabilities. J. Nucl. Med. 27, 893A. [I71 Bums, H.D., Dannals, R.F., Dannals, T.E., Kramer, A.V and Marzilli, L.G. (1981) Synthesis of tetradentate aminothiol ligands and their technetium complexes. J. Label. Comp. Radiopharm. 18, 54-55. [I81 Yokoyama, A., Terauchi, Y., Horiuchi, K., Tanaka, H., Odori, K. (1976) T., Morita, R., Mori, T. and Torizuku, Technetium-99m-kethoxal-bis(thiosemicarbazone), an uncharged complex with a tetravalent Tc-99m state, and its excretion into bile. J. Nucl. Med. 17, 816-819. P91 Jay, M., Beihn, R.M., Digenis, G.A., DeLand, F.H. and Caldwell, L. (1985) Disposition of radiolabelled suppositories in humans. J. Pharm. Pharmacol. 37, 266-268. [201 Edelbroek, M., Horowitz, M., Maddox, A. and Bellen, J. (1992) Gastric-emptying and intragastric distribution of oil in the presence. J. Nucl. Med. 33, 1283-1290. [211 Esposito, P., Dobetti, L., Rabaglia, L., Boltri, L. and Carli, F. (1995) New lanthanide organic salts suitable for neutron activation 2. Solid state incorporation into polymeric microcarriers and feasibility as markers for scintigraphic imaging. Eur. J. Pharm. Biopharm. 41, 88-93. WI Esposito, P., Dobetti, L., Boltri, L. and Rabaglia, L. (1994) Use of a novel neutron activable excipient (samarium stearate) as a lubricant in tablets. Eur. J. Pharm. Biopharm. 40, 341-343. V31 Dobetti, L., Esposito, P. and Boltri, L. (1994) New lanthanide organic salts suitable for neutron activation. Eur. J. Pharm. Biopharm. 40, 161-167. ~241 Wedmann, B., Schmidt, G., Wegener, M., Coenen, C., Ricken, D. and Althoff, J. (1991) Effects of age and gender on fat-induced gallbladder contraction and gastric-emptying of a caloric liquid meal - a sonographic study. Am. J. Gastroenterol. 86, 1765- 1770. ~251 Fujimura, J., Haruma, K., Hata, J., Yamanka, H., Sumii, K. and Kajiyama, G. (1994) Quantitation of duodenogastric reflux and antral motility by color doppler ultrasonography. Stand. J. Gastroenterol. 29, 897-902. K., Carmichael, D.C., WI Brown, B.P., Gross, J., Schulzedehieu, Barloon, T.J. and Abuyousef, M.M. (1994) Layering of fat and intermittent cessation of antral contractions delay liquid fat emptying in the human stomach. Gastroenterology 106, A472-A472. ~271 Harris, R.K. (1987) NMR Spectroscopy, Longman, London. P81 Saini, S., Medic, M.T., Hamm, B. and Hahn, P.F. (1991) Advances in contrast-enhanced MR imaging. Am. J. Roentgenol. 156, 235-254.
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[29] Laniado, M., Kornmeisser, W., Hamm, B., Clauss, W., Weinmann, H.J. and Felix, R. (1988) MR imaging of the gastrointestinal tract: value of Gd-DTPA. Am. J. Roentgenol. 150, 817-821. [30] Wesbey, G.E., Brasch, R.C., Engelstad, B.L., Moss, A.A., Crooks, L.E. and Brito, A.C. (1983) Nuclear magnetic resonance contrast enhancement study of the gastrointestinal tract of rats and a human volunteer using nontoxic oral iron solutions. Radiology 149, l75- 180. 1311 Ang, P.G.P., Li, K.C.P., Tart, R.P., Storm, B. and Rolfes, R. (1989) Geritol oil emulsion: ideal positive oral contrast agent for MR imaging. Radiology 172(P), 522. [32] Mattrey. R.F., Hajek, P.C., Gylys-Morin, VM., Baker, L.L., Martin, J., Long, D.C. and Long, D.M. (1987) Perlluorochemicals as gastrointestinal contrast agents for MR imaging: preliminary studies in rats and humans, Am. J. Roentgenol. 148, 125991263. [33] Anie, S.J., Fell, J.T. and Waigh, R.D. (1991) Visualization of a fluorinated pharmaceutical formulation in the anesthetized rat using F-19-NMR and H-I-NMR imaging. Int. J. Pharm. 69, 181-186. [34] Anie, S.J., Fell, J.T., Waigh, R.D. and Wood, B. (1991) Gastrointestinal transit studies in the rat using F-19-NMR. Int. J. Pharm. 76, 183-185. [35] Edelstein, W.A., Hutchison, J.M.S., Johnson G. and Redpath, T. (1980) Spin-warp NMR imaging and applications to human whole-body imaging. Phys. Med. Biol. 25, 751-6. [36] Schwizer, W., Maecke, H. and Freed, M. (1992) Measurement of gastric emptying by magnetic resonance imaging in humans, Gastroenterology 103, 369-76. [37] Mehlkopf, A.F., Van-der-Meulen, P. and Smidt, J. (1984) A multiple-echo and multiple-shot sequence for fast NMR fourier imaging. Magn. Reson. Med. 1, 295-297. [38] Hennig, J., Nauerth, A. and Friedburg, H. (1986) RARE imaging: a fast method for clinical MR. Magn. Reson. Med. 3, 823-33. [39] Haase, A., Frahm, J., Mattael, W., Hanicke, W. and Merboldt, K.D. (1986) FLASH imaging. Rapid NMR imaging, using low flip-angle pulses. J. Magn. Reson. 67, 258-66. [40] Oppelt, A., Graumann, R., Barfuss, H., Fischer, H., Hard, W.
[41]
[42]
[43]
[44] [45] [46] [47] [48] [49]
[50]
[5l]
[52]
[53]
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and Schajor, W. (1986) FISP - a new fast MRI sequence. Electromedica 54. 15-I 8. Glover, G.H. and Pelt, N.J. (1988) A rapid gated tine MRI technique. In: H.Y. Kressel (Ed.), Magnetic Resonance Annual, Raven, New York, pp. 299-333. Gyngell, M.L. (1987) Steady-state free precession sequences. In: Topical Conference on Fast Magnetic Resonance Imaging Technology Proceedings. Haase, A. (1990) Snap-shot-FLASH MR imaging: applications to T,, Tz and chemical-shift imaging. Magn. Reson. Med. 13, 77-89. Mansfield, P. ( 1977) Multi-planar image formation using NMR spin echoes. J. Phys. CIO, L55-58. Mansfield, P. and Pykett, IL. ( 1978) Biological and medical imaging by NMR. J. Magn. Reson. 29, 355-73. Rredzian, R.R. and Pykett, I.L. (1986) An instant technique for real-time MR imaging. Radiology 161(P). 333. Pykett, I.L. and Rzedzian, R.R. (1987) Instant images of the body by magnetic resonance. Magn. Reson. Med. 5, 563-7 I. Heid, 0. and Huk, D.W. (1993) QUEST - a quick echo split NMR imaging technique. Magn. Reson. Med. 29, 280-83. Stehling, M.K., Evans, D.F., Lamont, G.. Ordidge, R.J., Howseman, A.M., Chapman, B., Coxon, R., Mansfield, P., Hardcastle, J.D. and Coupland, R.E. (1989) Gastrointestinaltract-dynamic MR studies with echo-planar imaging. Radiology 171, 41-46. Reimer, P., Saini, S., Hahn, P., Brady, T. and Cohen, M.S. ( 1994) Clinical application of abdominal echo planar imaging (EPI): optimization using a retrofitted EPI system. J. Comput. Assist. Tomogr. 18, 673-79. Hykin, J., Freeman, A., Gowland, P.R., Worthington, B., Spiller, R. and Mansfield, P. (1994) Measurement of GI water content using EPI at 0.5 Tesla. MAGMA 2, 471-73. O’Mahony, B., McJury, M. and Wilson, C.G. (1995). Use of MRI to study the behaviour of soft gelatin capsules in the upper gastrointestinal tract. Sot. Magn. Reson. 3, 1483. Boulby, P., Gowland, P.A., Adams, V and Spiller, R. (1995) The use of EPI to study the fate of stomach contents: an assessment of the affect of posture on the emptying of an oil/water meal. Sot. Magn. Reson. 3. 1475.
drugdeliiry reviews
ELSEVIER
Advanced
Drug Delivery
Reviews 25 ( 1997) 91- 101
Imaging of oily formulations
in the gastrointestinal
C.G. Wilsona’*, M. McJuryb, B. O’Mahony”,
tract
M. Frier’, A.C. Perkins”
“Department of Phurmuceuticul Sciences, Royul College, 204 George Street, University qf Strathclyde Gluago~: GI IXW, UK hDepartment of Clinical Physics, The Western InJimmy Glasgow, Scotland, UK ‘Department qf Medical Physics, Queen ‘s Medical Centre Nottingham, UK
Abstract
The use of an imaging technique such as gamma scintigraphy to follow the behaviour of a formulation in the gastrointestinal tract is widely employed in the development of new therapeutic concepts. Such studies are facilitated by the availability of suitable radiolabels, incorporated into the formulation during manufacture. The range of labels for lipophilic excipients is very limited, although new brain-imaging agents may offer interesting possibilities. The use of alternative imaging modalities such as magnetic resonance imaging should also be considered. The difference in the T, - or T,-weighted signal from the oil against the aqueous contents provides a method of visualising an oil-based formulation in vivo without any modification to the formulation. This article reviews approaches in the search for suitable techniques for in vivo imaging of oral formulations containing a high proportion of oil. Keywords:
Oil; Gastrointestinal
tract; Drug delivery; Imaging;
Scintigraphy;
Magnetic
resonance
imaging
Contents 92 Introduction ............................................................................................................................................................................ 92 1.l. Radionuclide techniques ................................................................................................................................................... 92 1.2. Radioiodine complexes.. ................................................................................................................................................... 92 1.3. Technetium complexes ..................................................................................................................................................... 93 1.4. New lipophilic chelates suitable for labelling oily vehicles.. ................................................................................................ 1.5. Neutron activation.. .......................................................................................................................................................... 94 Ultrasound.. ............................................................................................................................................................................ 94 95 Magnetic resonance imaging (MRI). ......................................................................................................................................... 3.1. Contrast................................................................................................................................................................. ._ 95 96 3.1.l. Contrast agents ...................................................................................................................................................... 3.1.2. Positive agents.. ..................................................................................................................................................... 96 3.1 .3. Negative agents. ..................................................................................................................................................... 96 3.1 .4. Other methods: multi-nuclear MRI .......................................................................................................................... 96 3.2. MR sequences for in-viva imaging.. .................................................................................................................................. 96 3.2.1. Spin-echo (SE) sequences.. ..................................................................................................................................... 97 3.22. Gradient-echo (GE) sequences ................. . .............................................................................................................. 97 32.3. Ultra-fast imaging .................................................................................................................................................. 97 98 3.3. In-viva imaging of oils ..................................................................................................................................................... Conclurmns ........................................................................................................................ YY ......................................... ........ Acknowledgments ....................................................................................................................................................................... 100 References .................................................................................................................................................................................. 100
*Corresponding
author. Tel.:
016Y-4OYX/97/$32.00 0 PII SOl69-409X(96)00493-0
+ 44 141 5524400;
19Y7 Elsevier Science
fax:
+ 44 141 5526443;
e-mail: pharmacy@.\trath.ac.uk.
Ireland I.td. All rights reserved
92
C.G. Wilson et al. I Advanced
Drug Delivery Reviews 25 (1997) 91-101
1. Introduction
labelled materials for the study of the gastrointestinal tract [2].
Oils and fats are essential dietary components and have therefore attracted much clinical and scientific interest. Many gastrointestinal disorders are associated with abnormal fat absorption and therefore it was necessary to develop procedures to aid in clinical diagnosis. Early diagnostic techniques were largely chemical (faecal fats etc.) and required extensive laboratory processing. In the 1950s and 60s tests were developed which orally administered radiotracers, reducing the need for extensive chemical analysis. More recently the development of sophisticated imaging techniques such as radionuelide imaging, ultrasound and magnetic resonance imaging (MRI) have allowed the detailed visualisation of the transit and absorption of lipids and oils in the body. These techniques are of interest to the pharmaceutical industry since they provide valuable information on the gastrointestinal transit, dispersion and absorption of fats and oils in drug formulations.
1.1. Radionuclide
techniques
Traditional measurements of dietary fat absorption were based on the chemical estimation of dietary intake and excretion of fat. Radionuclide techniques for the measurement of fat absorption were first introduced in the late 1940s using ‘3LI-labelled triolein and oleic acid [l]. Other radionuclides included ‘*Br and 14C. These techniques were based on the measurement of activity contained in the blood and faeces and provided some basic metabolic data on fat absorption in health and disease. In particular, conditions in which fat absorption is decreased include idiopathic steatorrhoea, Whipple’s disease, ileitis, diverticulosis and tuberculosis. Fat absorption was also of interest in the study of conditions such as diabetes mellitus, hepatic cirrhosis, pancreatitis and certain cancers. The introduction of radionuclide imaging devices in the 1960s (in particular the gamma camera) provided a means for imaging the gastrointestinal transit of orally administered agents and radiolabelled compounds. The availability of radionuclides with suitable decay characteristics and gamma energies for imaging together with advances in radiopharmaceutical chemistry have resulted in a wide range of radio-
1.2. Radioiodine
complexes
The first documented uses of iodinated oils relate to their use in the investigation of fat malabsorption syndromes. Thus a degree of unsaturation was important in the selection of oils administered, e.g. oleates and myristates. Lubran and Pearson describe a method for the iodination of triolein in the diagnosis of steatorrhoea, which has been adopted as a standard method in clinical investigations [3]. The early imaging investigations of pharmaceutical formulations employed isotopes of iodine, particularly using iodine- 13 1, to label proteins and oils and emulsion formulations including liposomes [4] and emulsions [5]. These early uses are extensively covered in an earlier review to which the reader is referred [6]. The use of iodine-131 is limited by the poor photon yield in relation to the absorbed radiation dose, since beta particles are emitted during decay. Other iodine isotopes are available, of which the most suitable for imaging purposes is cyclotronproduced iodine-123. This can be incorporated into radiopharmaceuticals with little molecular disruption and has a similar energy to technetium-99m. Hardy and co-workers describe the use of iodine-123 in the iodination of arachis oil and Labrophil WL2700, in order to study the spreading of suppository bases delivered in rectal hard gelatin capsules [7]. Long-chain fatty acids such as palmitic and oleic acids are the principal energy source for the myocardium and therefore iodinated derivatives of these fatty acids have received attention as probes for the evaluation of myocardial metabolism. A number of iodine-labelled fatty acids have been used in routine clinical diagnosis and are available from commercial radiochemical suppliers. Potentially, these offer an unexplored and novel range of materials which could be applied to the study of lipophilic excipients. For a review of these materials, the reader is referred to Knapp and Kropp [S]. 1.3. Technetium
complexes
The poor imaging characteristics of I-13 1, and the associated beta radiation encouraged the search for
C.G. Wilson et al. I Advanced Drug Delivery
alternatives, such as ‘231, “3mIn, “‘In and 99mTc. Technetium-99m, which is available at high specific activity, has an ideal energy (141 keV), a short half-life (6.03 h), a higher photon yield and no associated beta radiation. The major difficulty with this element is the high valency state and the need to trap the atom into the tracer molecule by chelation. An unpublished study by our group examined the in vivo behaviour of size 0 soft gelatin capsules. Formulations of varying dispersion characteristics were compared in order to examine the in vitro/in vivo correlation. Batches of the formulations under test were radiolabelled by puncturing the side of the capsule with a hypodermic syringe, expelling the contents, mixing with 99mTc-labelled Amberlite resin to 2% fill weight and refilling. The puncture site was patched with a small gelatin square using a heated spatula to get a smooth seal. Dissolution times of the formulations tested were unaffected by this procedure as measured from in vitro disintegration times. Rates of dispersion in vivo were found to be much lower for poorly dispersible formulations compared to formulations which were highly dispersible in vitro. A typical scintiscan showing the breakup of the formulation is shown in Fig. 1. The low cost and excellent availability of Tc-99m ensure that this radionuclide will remain popular. exametazime such as Radiopharmaceuticals (CeretecB) and tetrofosmin (MyoviewB) have been specifically developed as diagnostic agents for brain and cardiac imaging. However, these agents offer valuable tracers for radiolabelling oily vehicles. As mentioned previously, technetium-99m is usually incorporated into molecules by chelation producing complexes. It is important to note that the entire complex will behave in a manner very different to that of the free ligand. Functional groups are involved in the complexation with technetium, and there is an unavoidable increase in molecular size and an alteration, usually a decrease, in charge. Eckelman has recently produced an excellent review of the technetium complexes available for biochemical studies [9]. He classifies the chelates into groups based on physiological functional: kidneys, bone, liver, heart and brain. It should be noted that this functional classification parallels ascending lipophilicity. Clinically, kidney function generally employs the most water soluble chelates such as 99mTc-labelled diethylenetriaminepentaacetic acid whereas
Reviews 25 (1997) 91-101
93
Fig. 1. Scintiscan showing disintegration of a soft gelatin capsule in the stomach of a volunteer, 8 min after dosing. The unit was resin in the oil mix as labelled by inclusion of “‘“Tc-labelled described in the text.
the chelates for heart and brain are neutral or cationic lipophiles. 1.4. New lipophilic chelates suitable for labelling oily vehicles One of the first ligands designed specifically for use with technetium was a lipophilic iminodiacetic acid, which produces a complex with technetium in the + 3 oxidation state and which is cleared rapidly via the liver following intravenous administration [ 101. Mebrofenin, 3-bromo-2,4,6-trimethyl HIDA, shows much improved hepatic clearance, and was developed by Nunn [ 111. Intensive investigations following this period have led to the development of new ligands including exametazime, (RR,SS)-4,8diaza-3,6,6,9-tetramethyl undecane-2, lo-dionebisoxime [12] and ECD [13]. When complexed with technetium-99m, exametazime (Ceretec) has a high log P and distributes into oily vehicles readily. It is however unstable and will slowly decompose to a less lipophilic intermediate. This property is used to label cells since the
94
C.G. Wilson et al. I Advanced Drug Delivery Reviews 25 (1997) 91-101
lipophilic complex crosses the cell wall but then becomes internalised as a more hydrophilic complex. The search for monovalent cationic technetium complexes for myocardial studies led to the development of hexakis(t-butylisonitrile)-Technetium(I) [ 141. Subsequent developments produced sestamibi (Cardiolite), the 2-methoxy-2-methyl propyl derivative. This compound binds to p-glycoprotein and it has been suggested that the radiopharmaceutical may be useful in following drug resistance. More recenttetrofosmin, l,Zbis[bis(Zethoxyethyl)phosly* phinolethane, has been developed [15]. Like sestamibi, this produces a monovalent, cationic complex with technetium. In all the above examples, complexes are formed using pre-formed ligands. An alternative way of producing technetium complexes uses the technique known as template synthesis, in which the complex is built around the metal from relatively simple components during formation. The novel complexes known as BATOs (boron adducts of technetium dioxime) are examples of these and are neutral, lipophilic materials with potential for myocardial perfusion studies [ 161. Teboroxime, [bis[ 1,2-cyclohexanedionedioximato l)-O]-[ 1,2-cyclohexanedionedioximato(2)-O]methylborato(2)-N, N’, N”, N”‘, N”“, N”“‘]-chlortechnetium has been used clinically for myocardial imaging, but shows rapid washout. Other neutral compounds of note include the bisaminothiol (BAT) derivatives [17] and the kethoxal bis( thiosemicarbazone) (KTS) complexes
oil and 290 ml of “3mIn60 ml of “““Tc-labelled labelled soup (total 505 kcal) on one day and 280 g “3mIn-minced beef (500 kcal), 60 ml 99mTc-labelled oil and 290 ml soup (505 kcal) on another day [20]. The emptying rate of oil in the oil/soup meal was about twice that for oil consumed in the other meal. These results indicate that major differences in the intragastric distribution of oil occur following incorporation of solids into the meal. The author has commented that [99mTc](V)-thiocyanate olive oil is a better fat marker than [75Se]glycerol triethyl butter.
1.5. Neutron activation The introduction of neutron activation enabled the in situ production of nuclides which proved useful for the labelling of complex preparations including enteric coated tablets and pellets by the incorporation of low amounts of stable isotopes during pilot scale manufacture. Most processes involve the bombardment of the insoluble oxide in the finished product to produce the isotopes 17’Er or ‘53Sm which can be imaged using a gamma camera. Esposito and coworkers have described the synthesis of lanthanide organic salts, including samarium stearate and ytterbium trilaurate, trimyristate etc. [21,22]. However, these organo-metallics are not sufficiently soluble in either organic or aqueous solution and are therefore unsuitable as oil markers [23].
[181. The objective of obtaining a low molecular weight lipophilic technetium-99m complex is difficult because the technetium chelate is bulky, adding a minimum of 250 + Da to the biochemical of interest. The effect of addition of the technetium chelate increases the log P to approximately the same extent as iodination. Some fats are relatively polar and in this case it is possible to use hydrophilic markers for the study of these formulations. Jay et al. utilised 99mTc-technetium hydroxymethyldiphosphonate to label Witepsol W-15 in a study of radiolabelled suppositories and obtained results quantitatively similar to that described by Hardy et al. [19]. Edelbroek describes the use of [ 99mTc]( V)-thiocyanate, to measure the gastric emptying and intragastric distribution of oil in a fatty meal consisting of
2. Ultrasound Ultrasound is an acoustic radiation that occupies a frequency band above the audible range. In medical applications ultrasound frequencies of between 1 and 10 MHz are normally employed. Ultrasound is noninvasive and safe, although at high intensities such as those used for therapy (heat treatment) experiments have shown an association with adverse biological effects produced by thermal damage and cavitation in tissues. The echo information derived from ultrasound investigations depends upon the difference in the characteristic acoustic impedances of the interfaces generating the reflection of the sound. For a given medium (tissue, fluid, air) the acoustic impedance is equal to the product of the density and the velocity
C.G. Wilson et cd. I Advanced
Drug
of sound in that medium. In the case of Doppler investigations the movement of the reflecting interface results in a frequency shift which may be encoded into a colour display to provide additional information on motion within the body. When examining the stomach or GI tract the luminal contents will dictate the nature and strength of the reflected signal. It has been widely appreciated from clinical use that ultrasound examinations of the small and distal bowel have been severely limited due to the presence of gas which produces a very strong reflection and strong attenuation. Such investigations have therefore been restricted to specialist centres and research studies. However, with appropriate preparation the examination of the GI tract may be carried out. In particular, ultrasound imaging of the stomach contents has provided useful data on the gastric emptying of liquids. Sonography has been used to investigate the emptying of fatty meals, for example, Wedman et al. (241 described the use of sonography using a 3.5 MHz curved probe to investigate the gastric emptying, antral motility and gall bladder emptying after a liquid fatty meal in 50 subjects, half of whom had systemic sclerosis. It is not generally considered that fat can be discriminated from protein and carbohydrate components within the GI tract since the presence of gas bubbles would produce an equally strong signal. Sonography per se is therefore of limited use in evaluating the transit of oils in the gut. However, in highly heterogeneous phases in which gas bubbles or oil droplets are uniformly dispersed thorough an aqueous phase the echogenic characteristics of the medium are markedly changed. Fujimura and colleagues described the quantification of duodenal duodeno-gastric reflux and antral motility using colour Doppler shift flow following administration of a consomme meal [25]. The Doppler
DeliveT
Re\?ews 25 (1997)
9.5
91- 101
shift flow information was considered to be derived from oil droplets, size range 5-40 pm, in the soup. Brown et al. found that fat quickly layered in the proximal stomach away from the pylorus thus delaying gastric emptying [26].
3. Magnetic
resonance
imaging
(MRI)
MRI is arguably the fastest developing radiological imaging technique in clinical use. Its exquisite sensitivity to soft tissue, the fact that it is noninvasive and does not use ionising radiation, have lead to its being the modality of choice in a growing number of situations. Only certain substances, having protons with non-zero angular momentum and therefore a magnetic moment. are sensitive to the nuclear magnetic resonance phenomenon. Table 1 lists some nuclei observable with NMR. In acquiring good quality in-vivo MR images of fats in the gut, the main aspects of imaging to consider are: optimising image contrast, avoiding motional artefacts and having sufficient resolution to observe the subject of interest.
3. I. Contrast When imaging oils against a background of the watery contents of the gastrointestinal tract, two aspects to the difference in MR signal between fats and water, allow us to manipulated and optimise the contrast: (a) taking advantage of differences in relaxation properties of fat and water protons and (b), using the difference in resonance frequency (or chemical shift) between fat and water protons. Image contrast may be weighted to highlight T, or T, relaxation differences between substances.
I Somenuclei observable with NMR
Table
Nuclei
Natural abundance
‘H “C “N 1‘>F “P
99.98 I.1 I 0.37 I00 100
Source: Adapted
from Ret’. [27]
(%)
Larmor frequency 42.58 IO.71 4.3 40.05 17.24
at
I Telsa (MHz)
Spin 112 I I2 l/2 I/? II2
96
C.G. Wilson et al. I Advanced Drug Delivery Reviews 25 (1997) 91-101
3.1.1. Contrast agents In addition to the intrinsic contrast which exists in a sample due to the properties of the protons, it is sometimes necessary to add contrast-agents to enhance image contrast. A detailed discussion of MR contrast agents in beyond the scope of this article and the reader is refered to standard texts in this subject [28]. In the gastrointestinal tract, contrast agents may broadly be classified in a number of ways: based on the magnetic properties of the agents; whether the agent is miscible or immiscible; or into negative (reducing signal from the bowel-lumen) or positive agents (increasing bowel-lumen signal). Obviously care must be taken when using such crude and sweeping definitions. It should be noted that often positive agents can increase image artefacts due to bowel motion, while negative agents produce little artefact.
3.1.2. Positive agents Positive agents include paramagnetic substances which mix with bowel contents. These agents, such as manganese chloride and gadolinium preparations, cause a reduction of relaxation values of bowel contents, and therefore lead to signal enhancement which is greatest in T,-weighted images [29]. Other paramagnetic substances used include ferric ammonium citrate [30]. This produces approximately 20% of the T, shortening generated by gadopentate dimeglumine at equal volumes. Fats and oil emulsions may also be given to replace bowel contents. Although it can be difficult to administer these in sufficient quantities to replace total bowel and small bowel contents, some success has been made in small bowel marking [31].
3.1.3. Negative agents These agents include iron oxides, perfluorocarbons (PFCs), clays, barium sulphate, and gas-evolving pellets. Iron oxide particles increase signal de-phasing (T2 effects) and on T,-weighted images, show negative contrast. PFCs are compounds in which protons are replaced by fluorine atoms. Being immiscible with bowel contents, they are administered to replace bowel contents and produce no MR signal. Gas providing substances also generate contrast by producing regions of low proton density [32]. Of course, two types of contrast, positive and negative
may be used protocols.
simultaneously,
in
double-contrast
3.1.4. Other methods: multi-nuclear MRI As mentioned previously, hydrogen protons in water and lipid are not the only observable nuclei of interest. It is possible to obtain images of the in-vivo distribution of sodium or fluorine. Due to the low concentration of naturally occurring 19F in the body, usually samples to be imaged are labelled with 19F. If double-tuned RF probes are used, it is possible to acquire images of ‘H- and “F-1abelled nuclei simultaneously. The general anatomy of the subject will be shown in the ‘H-image, while the capsule of formulation will be seen in the “F-image. In animal studies, in-vivo imaging of “F-1abelled formulations has been achieved using pentaerythritol trifluoroacetates. These symmetrical structures give a single 19F resonant frequency and yielding a high sensitivity for the lipophile [33,34]. 3.2. MR sequences for in-vivo imaging MR is a unique imaging modality in that MR image contrast depends not on a single parameter (such as electron density in the case of X-ray based imaging), but rather on a complex recipe of parameters including proton density, T,- and TX-values of tissue, diffusion, flow and others. This has resulted in the development of literally thousands of MR imaging sequences with particular contrast characteristics. Depending on application, however, the number of sequences available to a user will shrink dramatically. In particular, when imaging in the thorax, potential artefacts due to respiratory, cardiac, peristaltic and other patient motion imposes severe restrictions on the type of imaging sequences which can be used. Best results for imaging the gastrointestinal tract, therefore, require MR sequences which acquire the image data quickly in relation to the voluntary and involuntary patient motion. These sequences are generally known as fast and ultra-fast imaging sequences. These include three main types: fast lowangle shot (FLASH), Turbo-FLASH gradient echo based sequencessequences and their family of variants; multi-echo spin-echo sequences such as fast spin-echo (FSE) or turbo spin-echo (TSE); and ultrafast variants of the echo-planar imaging (EPI) sequence.
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3.2.1. Spin-echo (SE) sequences The spin-echo sequence was the early work-horse of clinical MR [35]. It acquires a single signal echo from each excitation, and is inherently slow (of the order of min). To produce acceptable images of the thorax, it is necessary to gate the data acquisition to respiration (the sequence is too slow to acquire data in a breath-hold). This will in turn increase the time of data acquisition. Imaging of capsules with a fast disintegration rate in the gastrointestinal tract, for example, is not possible with simple SE methods. In anaesthetised animal models, SE methods have been used to image tablet formulations in the gastrointestinal tract [34]. The techniques have also been used successfully to investigate, for example, gastric-emptying in human volunteers [36]. It is possible to speed up SE sequences by collecting multiple signal echoes from each single excitation. These techniques are called fast or turbo spin-echo methods [37,38]. They can offer significant reduction in scan time (up to an order of around IO). However, they possess a subtle and complex contrast behaviour, and can best be used for T,weighted imaging only. Lipid in TSE images will appear bright, as will water. Images aquired by these methods can be difficult to interpret.
3.2.2. Gradient-echo (GE) sequences Due to the long imaging times needed for early T,-weighted spin-echo sequences, much development has been done on gradient-echo imaging sequences. By using small excitation pulse angles, and GE data acquisitlion, it is possible to dramatically speed up scan times. These sequences have inherently lower signal-to-noise (SNR) ratios than SE sequences, but win in terms of SNR per unit time. In areas of the body where fast imaging is crucial to avoid motion artefacts, they are generally the sequence type of choice. FLASH [39] (closely-related sequences include FISP [40] GRASS [41], CE-FAST [42] is a lowangle GE sequence, and is fast enough to allow acquisition of several 256 X 256 images in a single breath-hold. It is best for Tz-weighted imaging, but satisfactory T,-weighted images may also be obtained. Turbo-FLASH [43] goes further in terms of scan time reduction, allowing a single image to be acquired in around 1 s (Fig. 2).
07
Fig. 2. Shows one image from a set of eight 256 X 256 sagittal T,-weighted breath-hold FLASH images. The images, obtained from a volunteer imaged on a 1-T Siemens Impact MR system, have slice thickness 8 mm, field of view (FOV) 300 mm, and were acquired with a TR of 130 ms, TE 5 ms, with acquisition time (TA) of 16 s. Fat appears bright on the image, with watery gastrointestinal contents dark. The 8 mm slice thickness keeps the SNR high, without significant loss of quality due to partial volume effects, Up to 10 slices may be acquired in a reasonable breathhold time of 16 s, allowing coverage of the entire stomach.
3.2.3. Ultra-fast imaging The gold-standard in terms of real-time NMR imaging is set by echo-planar imaging (EPI) [44,45] and its many variants (Instascan [46,47], Mosiac [21], RARE [38], QUEST [48] and so on). EPI allows acquisition of an image in a fraction of a second ( 128 X 128 pixel matrix in 128 ms) by collecting data for a complete image from a single excitation. This sequence freezes any unwanted patient motion, providing a snap-shot of the body [49]. It has stringent hardware requirements and only recently become available on standard clinical scanners as a retro-fit [50]. EPI is finding application in imaging applications where speed is an important factor in abdominal imaging of the gastrointestinal tract, liver and heart; in functional imaging of the brain, in following gastric-motor function and transit, and in the analysis of the water-content of stomach, intestine and colon [Sl].
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3.3. In-vivo imaging of oils Temporal resolution of around 20 s for a set of breath-hold FLASH images covering the stomach, allows for the possibility of using MR, for example,
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to monitor the disintegration of oil-filled capsules in-vivo. Fig. 3 shows four images from an MR study following the disintegration of oil-filled gelatin capsules in the upper gastrointestinal tract [52]. The recent developments in GE imaging and
a
Fig. 3. Shows four images from an MR study following the disintegration of oil-filled gelatin capsules in the upper .gastrointestinal tract [52]. The images were acquired using a T,-weighted breath-hold FLASH (TR 130, TE 5, FOV 300, thickness 8 mm, TA 16 s, and single average). (a-d) represent 1, 5, 9, and 16 min after capsule intake.
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Fig. 4. The upper image is a I-cm transverse section through the abdomen of a volunteer, lying right side down soon after consumption of an Olive oil/Beef consomme soup meal using the Echo-Planar (MBEST) technique, acquisition time 130 ms. The bright area (B) corresponds to the soup lying in the dependent part of the gastric body. The horizontal greyish layer (A), apparently floating above the soup is the olive oil. The fat component of the image is displaced owing to the difference in resonance frequency between protons in water and fat, which is approximately 72 Hz at 0.5 T. The lower image taken immediately after the upper image corresponds to the same region of interest. However, the signal from the water has been suppressed, leaving an Image which includes a layer of extracellular fat surrounding the volunteer as well as the olive oil layer in the stomach.
particularly in EPI, have revived interest in imaging the gastrointestinal tract. Boulby and his co-workers used echo planar imaging to assess the effect of posture on the emptying of an oil-water meal [53] (Fig. 4).
4. Conclusions The application of new imaging techniques in the pharmaceutical sciences has evolved steadily from clinical diagnostic imaging procedures. The number of modalities and of imaging agents for the study of
formulations is increasing, although not always directly in gastroenterological sciences. It is necessary therefore to maintain acquaintance with developments in parallel disciplines, which provide new tools for the study of the behaviour of fats and oils. In the immediate future, gamma scintigraphy will provide the widest range of probes and the availability of these instruments to academic and commercial organisations, ensures progress in the understanding of the behaviour of oil-based delivery systems. MRI will provide adjunct information, but the cost and availability of these instruments is as yet prohibitive for wide-scale use.
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Acknowledgments The authors thank Mr Philip Boulby and Dr Penny Gowland of The Magnetic Resonance Centre Nottingham for helpful comments and the provision of the Echo-Planar Images.
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