In his address at the opening session of the British Pharmaceutical Conference on September 13, the Science Chairman presented evidence that, contrary to the opinion of many, pharmaceutics as a science is not dead but active. He illustrated his argument by describing examples from work undertaken and supported by Astra Zeneca in the field of molecular pharmaceutics - specifically, the structure and stability of antiseptic creams and the structure and properties of crystals. Based on his own experience, he concluded that, although pharmaceutics is an applied science and requires the development of new technology in order for it to progress, solving problems in the field is both difficult and rewarding, and requires as much, if not more, knowledge and commitment than a pure science
Pharmaceutics as a term is rapidly disappearing from the letter headings of many pharmacy schools at our universities and is being replaced by terms such as "drug delivery" and "materials science". The reason would appear to be that many regard pharmaceutics as unworthy of being called a science. This paper will look at pharmaceutics and in particular some work that colleagues and I have undertaken at Astra Zeneca to illustrate the fact that, contrary to the opinion of many, pharmaceutics is not a dead or even dying science but is very active indeed. The paper is based around four elements - molecules, microscopy, crystals and chromatography - in an attempt to show how all four are relevant and alive in the field of pharmaceutics.
The definition of pharmaceutics - the application of the physical, chemical and biological sciences to the formulation, design, preparation and presentation of dosage forms - is widely known. Indeed, nowadays it has been divided into the subsets of biopharmaceutics, physical pharmaceutics, and even chronopharmaceutics. However, few will have heard of a new subset called molecular pharmaceutics, defined as the research and interpretation of the properties and processes in pharmaceutics at the molecular level.
Why molecules? Molecules represent the smallest fundamental unit of all chemical compounds and hence the study of how molecules arrange themselves and pack to form secondary structures at interfaces and in bulk should promote the understanding and even enable the prediction of the properties and behaviour of materials, formulations and medicines. Indeed, I am a great believer in the philosophy expounded by Jean Baptiste Lamarck, the great pioneer biologist in the formative era of that science, who is reported as saying in the early 19th century: "The most important discoveries of the laws, methods and progress of nature have always sprung from the examination of the smallest objects which she contains."
Admittedly, Lamarck was referring to cells seen under a microscope. I would like to extend this concept to even smaller objects, namely, molecules, using examples of antiseptic creams and crystals.
In their simplest form, antiseptic creams have four basic ingredients: an antiseptic surfactant, in this case cetrimide; a cosurfactant, in this case cetostearyl alcohol; an emollient, in this case liquid paraffin at varying concentration depending on use; and water as the continuous phase (Table 1). They are prepared by heating both the aqueous (cetrimide and water) and oil phases (liquid paraffin and cetostearyl alcohol) to 80C, followed by mixing, homogenisation and cooling. The cream formed is a white, aqueous-based semisolid. These creams, although simple in terms of formulation, have a complicated structure at the molecular level, a knowledge of which is essential in understanding their properties and stability.1 |
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If the cream is spread thinly on a microscope slide and examined under a standard light microscope (Figure 1), some interesting features can be seen: some spherical/round objects, some polyhedral shaped objects and a filamentous network. If the same sample is examined under a light microscope fitted with differential interference contrast optics, small changes in the refractive index and thickness within the sample can be highlighted, providing a detail of relief-like appearance. Although the same three features can be seen, their detail is enhanced. If then the sample is examined using polarised light, the polyhedral shaped objects display the "Maltese cross" indicative of a crystalline material.
Differential interference contrast |
Transmitted light |
Polarised light |
Figure 1: The structure of antiseptic creams as seen under a light microscope |
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Although it is possible to make some inferences from these observations, for instance, it is quite possible that the spherical/round objects are droplets of liquid paraffin, it is impossible to be confident without some form of analysis. In this respect it is possible to couple a microscope with either a Raman spectrometer or a Fourier transform infrared spectrometer, the microscopist gaining the molecular information available from the spectroscopy for the identification of the visual image.
In Raman microscopy, a laser is used and focused into the part of the image to be identified. The Raman scattered light is directed into a monochromator by a prism and the spectra formed analysed. Very small areas down to 1μm in diameter can be examined. The three features present in the cream can now be analysed (Figure 2). The spectra of the spherical droplet can be identified as liquid paraffin containing an impurity (possibly cetostearyl alcohol), the filamentous network can be identified as primarily cetostearyl alcohol and water, and the polyhedral particles can be identified as cetostearyl alcohol.2
If a Fourier transform infrared microscope is used, it is possible to extend the analysis further. In the case of the Fourier transform infrared microscope, an infrared beam is directed through the sample and analysed by an infrared spectrometer. In this case it is possible to compare the spectra produced by the spherical droplet against a series of standards prepared by dissolving cetostearyl alcohol in liquid paraffin at various concentrations. Hence it is possible to say with certainty that the droplets are indeed liquid paraffin containing 20 per cent w/w cetostearyl alcohol. The filamentous network is indeed cetostearyl alcohol as are the polyhedral particles.3
So much for the direct examination of these creams using light microscopy. If more detail is required then it is necessary to use electron microscopy.4 However, for this technique it is necessary to prepare the samples by first freezing them quickly in liquid nitrogen followed by fracturing under vacuum. The fracture surface is then heated to sublime the water of the continuous phase. For the scanning electron microscope this surface is then sputter-coated with gold and directly examined, while for the transmission electron microscope the surface is first coated with platinum and the replica of the surface is examined. Under the scanning electron microscope it is possible to see the features in great detail as three-dimensional images. Under the transmission electron microscope the detail is flatter but higher magnifications are possible (Figure 3).
Combining all of this information collected by microscopy, it is possible to concoct a schematic structure of the cream. The filamentous network consists of bilayers of cetostearyl alcohol containing interlamellar water. The oil droplets contain an interfacial layer of cetostearyl alcohol as well as free cetostearyl alcohol and the polyhedral particles are cetostearyl alcohol crystalline monohydrate. The thickness of both the bilayers and interlamellar water can be determined using X-ray scattering.5
An interesting feature of these creams is their instability, especially after prolonged storage at low temperatures (below 4C). The creams lose structure and become fluid. Samples before and after storage when examined using scanning electron microscopy show a change in the structure of the network. More detailed examination yields the presence of platey crystals consisting of layer upon layer of plates (Figure 4). These are thought to be produced by the dehydration and fusion of the bilayers of cetostearyl alcohol as a result of point defects or dislocations in the bilayers on cooling.6
Probably more important than the mechanism is the reason behind this effect, and here we must look to the ocean depths and to the sperm whale Physeter macrocephalus with its enormous head. It was to collect the material within its head that this beautiful creature was almost hunted to extinction earlier in this century. Within the giant head is a long sac, called the spermaceti organ, filled with oil, known as spermaceti. Until the 1960s this oil was the prime source of cetostearyl alcohol used in cream manufacture and it is interesting to note that thinning of creams on storage only became common when the moratorium on the hunting of sperm whales was introduced in the late 1970s, for it was at this time that cetostearyl alcohol began to be made using other sources, notably tallow. Cetostearyl alcohol is not a pure compound but consists of a mixture of a number of fatty alcohols and it is interesting to compare the composition of the two sources (Table 2). The most interesting difference is not primarily in the proportion of the C16 (cetyl) and the C18 (stearyl) alcohols but the fact that the sperm whale source contains a relatively large proportion of "odd chain" (C15 and C17) alcohols, especially the branched chain C17 cetostearyl alcohol.7 These are only found in the sperm whale, probably to help keep the spermaceti at the correct consistency during its plunge to depths of 1,000m for food where the temperature is a constant 2C. It is believed that the same alcohols keep the bilayers in the cream more fluid and less likely to fuse during prolonged storage. |
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Crystals are ordered molecules in the solid state. They are a regular repetition in three dimensional space of a unit cell (a parallelipiped containing the smallest complete structural unit) extending over a distance corresponding to thousands of molecular dimensions. Figure 5A shows the molecular structure of the anticonvulsant primidone. The unit cell consists of four molecules in a specific arrangement governed by the laws of physics (Figure 5B) and this unit cell is extended over many thousands of molecular dimensions to show the packing of the molecules in the solid state (Figure 5C).
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Figure 5: The molecular structure of primidone (A) together with its unit cell (B) and packing in the solid state (C) |
Polymorphism is the phenomenon that involves different packing arrangements of the same molecule in the solid state. Approximately 33 per cent of all molecule solids show polymorphism. Dr Walter
McCrone is purported to have said that "every compound has different polymorphic forms and the number of forms known for a given compound is proportional to the time and energy spent in research on that compound", and there is no doubt that there is a great deal of truth in this.
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Figure 6: Unit cells of form I and form III of sulphathiazole showing the differences in packing arrangement of the two polymorphs | |
However, this is not the whole story. Sulphathiazole has, in fact, four polymorphs, all produced by the same synthetic pathway. This involves an amine substitution of an ethamidobenzenesulphonyl chloride followed by hydrolysis of the ethamido group to the aniline functional group. The final hydrolysis is performed in aqueous media. One of the main impurities in the final compound is the ethamidosulphathiazole and we have found that when the sulphathiazole is crystallised in the presence of this impurity it is possible to produce all the polymorphic forms of sulphathiazole, the percentage of each present being dependent on the concentration of the impurity present (Figure 7). At high concentrations of impurity, form I is produced exclusively, while at low concentrations form IV is produced exclusively. Using computer simulations it is possible to demonstrate how this happens in terms of the structure of the impurity entering the growing faces of the crystals of the polymorphs. Such are the subtleties of the solid state.13
Of course, such subtleties as the part of a molecule that is present at the face of a crystal can and do affect the properties of that crystal. An analytical method that can probe these effects is inverse gas chromatography.14
In essence, this is a relatively simple procedure. Crystals are packed into a column through which is passed dry nitrogen. Various organic probes in their vapour state are passed over the crystals and the selective adsorption of the molecules assessed by the net retention volume of each probe. The data from alkanes can be used to determine what is known as the dispersive component of the surface free energy and data from the other probes, the specific interactions. Each molecule will thus have a different data set dependent on the molecular arrangement within the crystal and the specific groups that lie on the crystal surfaces.
To illustrate this effect further I have used, as a model, the milling of propranolol hydrochloride crystals.15 Inverse gas chromatography data as the material is milled down show that the dispersive component of the surface energy rises and then levels off, while the specific interactions also show similar behaviour, the data for tetrahydrofuran (an electron donating molecule) being the inverse of that for dichloromethane (an electron accepting molecule). The critical inflections all occur at the same particle size (20μm). For the reasons behind these effects it is necessary to look at the propranolol hydrochloride molecule and how it packs to form a crystal. In the unit cell there are four molecules and this unit cell like all others is repeated in the crystal structure. Using computer simulation it is possible to predict the plane along which the crystal will fracture under stress in a mill as shown by the green plane (Figure 8). It can be seen that dominant on this surface is the naphthalene ring rich in p electrons. Hence, as the crystal is fractured during milling more and more of this surface will be exposed to provide the inverse gas chromatography results. However, at the critical size of 20μm, this process stops and the crystal does not fracture any more. Size reduction processes that occur below this point are associated with attrition rather than fracture. |
Figure 8: A computer simulation of the fracture plane along which a crystal of propranolol hydrochloride will break during milling |
On a pragmatic note inverse gas chromatography can be and is being used to investigate batch variation in both drugs16 and excipients.17
The theme for this year's conference is "New technology - a catalyst for change" and I hope have illustrated how it applies to the examples I have described. In the case of cream structure, we could not have progressed without, at that time, the technologies of electron microscopy and Raman spectroscopy. Yet both these technologies had been invented many years earlier. Ernst Ruska designed the first electron microscope in the early 1930s and Chandrasekhara Raman had discovered Raman scattering some 10 years previously. But it was the development of the technologies using freeze fracture electron microscopy and Raman microscopy that really were important and both these were only developed in the mid 1970s. Incidentally, the work on the antiseptic creams was undertaken on one of the first Raman microscopes built.18
If we look at the new technology involved in crystal structure measurements, by far the most important is that of the computer. The increasing power of the computer coupled with its decreasing cost make it possible to do things now that would have been inconceivable a decade or two ago. In fact we now have computers that can reason. Artificial intelligence is defined as the capacity of a computer to perform tasks commonly associated with the higher intellectual processes characteristic of humans, and such computers are being used in pharmaceutics to produce expert systems, especially in the field of formulation.19
The problem of formulation is one at which the pharmaceutical formulator excels. In all cases of product formulation, whether it be for tablets, capsules, parenterals or any one of the numerous other dosage forms, the process is generically the same, beginning with the presentation of some form of product specification and ending with the generation of one or more formulations that fulfil these requirements. In many cases, the specification has potentially conflicting requirements and it is left to the highly skilled formulator with his broad experience to balance these and produce the optimum compromise formulation.
It is not surprising, therefore, that this field has become a popular target for the application of expert systems, and nearly all of the major companies have projects in this area. However, it has been reported that up to 40 per cent of computer-based projects fail to deliver. In addition, there are the now common warnings from eminent people in IT against over-reliance on computers.
An example is a remark made by Ian Angell (professor of information systems at the London School of Economics). In 1991, he was reported to have said about artificial intelligence: "If you turn to a computer to solve a problem you don't understand all you're doing is transferring your lack of understanding to a technology you don't understand."
Surely this is true of all new technology and hence it is necessary to counter this lack of understanding by working with colleagues who are expert in the technology.
The following two quotations by eminent scientists in the early part of this century are highly relevant to my theme. The first by Sir Henry Tizard: "The secret of science is to ask the right question and it is the choice of problem more than anything else that marks the man of genius in the scientific world."
This is true for all the examples I have discussed. In all, the most important aspect in the solution of the problem was in asking the right question. Who would have thought at the outset that the stability of antiseptic creams would be related to the sperm whale?
The second quotation is by Sir William Hardy, a friend of Tizard: "Applied science is just as interesting as pure science and, what's more, it's a damned sight more difficult."
Pharmaceutics is an applied science and does require the development of technology for it to progress. However, solving problems in the field is both difficult and rewarding, and it requires as much, if not more, knowledge and commitment than a pure science. Why then should we ignore it?
Acknowledgments My thanks must go to my many friends and colleagues in ICI, Zeneca, Astra Zeneca and the Universities of Bradford and London and UMIST who have collaborated with me. However, there are two people to which I must express eternal gratitude, the late Peter Elworthy (Conference Science Chairman in 1971), my mentor who taught me to ask the right question at the right time, and David Ganderton (Conference Science Chairman in 1979), my first manager at ICI Pharmaceuticals, who gave me the freedom to undertake research in pharmaceutics and set me on my way.
Professor Ray Rowe is a company research associate at Astra Zeneca in the United Kingdom. He supervises a team engaged in research and problem solving in all areas of physical sciences (including colloid science, powder technology and knowledge engineering) involved in the formulation of medicines. He has been with Astra Zeneca (formerly Zeneca/ICI Pharmaceuticals) since 1973, having received his BPharm degree from the University of Nottingham in 1969 and his PhD from the University of Manchester in 1973.
Professor Rowe's research interests lie in the the areas of polymer coating, powder technology (including compaction and granulation), the structural characterisation of complex colloid systems and, more recently, applications of knowledge engineering in formulation and analysis. He has published over 300 research papers and reviews, including eight patents, and a book. In 1982 he was joint recipient of the British Pharmaceutical Conference award. In 1992, he was designated a fellow of the Royal Pharmaceutical Society for distinction in the science of pharmacy and, in 1993, he was awarded a DSc from the University of Manchester. In 1998, he was awarded the Chiroscience award for industrial achievement. Professor Rowe is a chartered chemist and fellow of the Royal Society of Chemistry and a chartered physicist and member of the Institute of Physics.
Professor Rowe has lectured widely in Europe and in the United States and he referees for 12 scientific journals. He is currently a member of the Chemical Engineering College of the EPSRC and is a member of the editorial boards of the International Journal of Pharmaceutics, the European Journal of Pharmaceutical Sciences and Pharmaceutical Science and Technology Today. He maintains close ties with various universities where he has co-supervised some 24 postdoctoral, PhD and MSc students. He has been an adjunct professor at the University of Illinois at Chicago and a visiting professor at the University of Strathclyde and at the University of Santiago de Compostela in Spain. He is currently a visiting professor of industrial pharmaceutics at the University of Bradford.
Professor Rowe has been a member of the Group 12 working party of the European Pharmacopoeia, the scientific programme committee of the Third European Congress of Pharmaceutical Sciences, and a past chairman of Interpharm. He is currently a member of the board of advisers standing panel of experts in pharmaceutics at the University of London, and of the Science Committee, the Pharmaceutical Sciences Group Committee and the Conference Committee of the Royal Pharmaceutical Society. He is also a member of the steering committee of the ‘Handbook of pharmaceutical excipients', published by the Pharmaceutical Press.
