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Nanotechnology tools in pharmaceutical R&D

Nanotechnology is a new approach to problem solving and can be considered as a collection of tools and ideas which can be applied in pharmaceutical industry. Application of nanotechnology tools in pharmaceutical R&D is likely to result in moving the industry from blockbuster drug model to personalized medicine. There are compelling applications in pharmaceutical industry where inexpensive nanotechnology tools can be utilized. The review explores the possibility of categorizing various nanotechnology approaches to meet the requirements in pharmaceutical R&D.
Challa S.S.R. Kumar1, 2 1 Center for Advanced Microstructures and Devices, Louisiana State University, 6980 Jefferson Hwy, Baton Rouge, LA 70806 2Magnano Technologies, 12538 Frankfurt Ave, Baton Rouge, LA 70816. Email: ckumar1@lsu.edu; challakumar@magnanotechnologies.com

Global pharmaceutical industry is at cross roads. On one hand the big pharmaceutical companies are enjoying enormous profits. The combined annual net income for the top 10 pharmaceutical companies (ranked by market capitalization) is currently around $73 billion. The average price of a new drug has been rising much faster than the rate of inflation. The drug companies continue to charge high retail prices for new drugs that are only incrementally different from older drugs whose prices have fallen. On the other hand, there are increasing costs of R&D spending as the companies are faced with the expiration of the patent protection on their main profit generators. Even though there is a growing knowledge of how diseases work at the genetic and molecular levels opening up new drug targets, there have been relatively few new products or new molecular entities (NME) in the pipeline as the strategy is to continue to follow the blockbuster drug model; which is like searching for a needle in a haystack (Fig. 1).

The blockbuster drug model in itself is questionable as standard drug treatments for a number of therapeutic areas provide a therapeutic benefit only to a limited percentage of patients who receive it

Fig. 1 NME approvals and drug companies spending on research and development (a congressional budget office study on R&D in the pharmaceutical industry, 2006).

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ISSN:1369 7021 Elsevier Ltd 2009

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Fig. 2 The limitations of standard drug treatment as seen from the response rates of patients from selected group of therapeutic areas (Source: TRENDS in Molecular Medicine, 2001).

Fig. 3 Development of a model for application of nanotechnology tools in pharmaceutical industry.

(Fig. 2)1,2. The irony is that medical diagnostics account for just 1% of healthcare spending, yet it is the basis for 60% of all healthcare decision-making. Let us look at the cancer pharmaceutical industry more closely as within the next decade cancer is likely to replace heart disease as the leading cause of U.S. deaths; according to forecasts by the National Cancer Institute (NCI) and the Centers for Disease Control and Prevention. Having approximately published more than 1.5 million scientific publications on cancer and cancer drugs, carried out more than 150 000 studies on mice, with an annual R&D funding of $15 billion and an annual cost of treatment around $65 billion, we are nowhere near to developing drugs that completely cure cancer3. It is quite evident that the pharmaceutical industry has become less innovative. Therefore, new approaches are required not only to change the pace but also the direction of innovation. The emergence of nanoscience and nanotechnology, which is creation and utilization of materials and tools at the nanometer scale, has been a great influence on a number of industries and particularly the pharmaceutical industry. Just as recombinant technology and biotechnology changed the landscape of pharmaceutical industry, nanotechnology is poised to lift the pharmaceutical industry that is at cross roads today, to new levels. However, unlike the biotechnology industry which influenced primarily the pharmaceutical industry, nanotechnology has broader applications and therefore, the nanotechnology tools and materials developed for other industries also have potential opportunities in the pharmaceutical industry as well. In addition, there is a prevalent notion that nanotechnology tools are exotic, too futuristic, disruptive and not suitable for quick commercialization of products. This notion is far from truth as nanotechnology tools are being shown to add value to existing products for existing markets as well as opening opportunities in new markets. This is true not only for the pharmaceutical industry but also for other industries. However, what is clearly lacking is a model for sorting out the plethora of nanotechnology tools that exists and

strategically correlating with potential opportunities into different segments of pharmaceutical R&D4-6. In addition, there is going to be a paradigm shift in the pharmaceutical industry towards personalized medicine as a new standard of care integrating therapeutics with diagnostics. It is, therefore, important to develop a more scientific approach for strategic implementation of nanotechnology tools in the pharmaceutical industry. The goal of this article is to explore a model for strategic decision making, termed as Innovation Box for Implementation of Nanotech Tools (IBINT) and its utility specifically tailored for the pharmaceutical industry. This approach, as explained below, is likely to be more versatile, valuable and practical than traditional/current classification of nanotechnology in medicine (or nanobiotechnology) into segments such as drug discovery, diagnostics and drug delivery7. The new approach proposed here relies on taking into consideration all the nanotechnology tools, from the more expensive to the less expensive, and sorting them based on their utility into three major categories of pharmaceutical R&D- Process development, product development and personalized medicine (Fig. 3). The concept of IBINT is pictorially represented in Fig. 4. The basic premise of IBINT rests in its broad applicability of nanotechnology tools influencing three major parts of pharmaceutical R&D-Process development, product development and personalized medicine. These three parts have different value propositions which in turn vary depending on the types of targeted markets. While the traditional/ existing model limits the utility of nanotechnology tools to highly complex, costly, risky and time consuming segments of pharmaceutical R&D, IBINT provides a scaffold for strategic development of application of nanotechnology tools both in high cost as well as low cost segments. A number of examples of nanotechnology tools can be classified using IBINT and are described briefly below. However, one needs to keep in mind that the application of IBINT becomes more valuable when it is used for strategic decision making keeping in view specific needs of individual pharmaceutical companies.

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Fig. 4 An approach for strategic implementation of nanotechnology tools in the pharmaceutical industry.

Fig. 5 Some of the nanotechnology tools for process development.

Nanotechnology tools for process development


There are already several nanotechnology tools available that can be utilized in process development. The term process development refers to both synthesis of drugs, drug intermediates and to development of analytical tools for diagnostics. One of the most important tools is miniaturization and automation in organic synthesis and biological screening on a nanoscale. Two examples of such tools are the NanoSynTestTM-system8 and the X-cubeTM system9. The central feature of these systems is the ability to manipulate reactions in nano-size titer plates with a density of about 100 wells/cm2 and the capability to handle nano liter volumes. While the NanoSynTestTM offers several modules for simultaneous drug synthesis and ultra high throughput screening, the X-cubeTM allows organic reactions to be performed in a flow manner at high temperatures and pressures. Such tools in automation and miniaturization coupled with nanotechnological approaches are expected to lead to efficient cost reductions in process development of existing as well as new products. In addition to the miniaturization of synthetic methods, nanomaterials are being developed as efficient catalysts and supports for solid-phase organic synthesis. Magnetic nanoparticle-supported chiral Ru complexes are known to catalyze heterogeneous asymmetric hydrogenation of aromatic ketones with remarkably high activity and enantioselectivity10. Magnesium oxide nanoparticles are utilized for residue-free catalytic process for production of Nabumetone, an anti-inflammatory agent, in high yield and high selectivity11. Recently, functionalized cobalt core carbon shell nanoparticles with excellent magnetic properties and high stability in air and at temperatures up to 190C have been reported12. Such nanobeads with high capacity for ligand binding and ability to rapidly remove them from the reaction mixture make them excellent solid supports in organic synthesis and biotechnological applications. Additionally, futuristic and more expensive nanotechnology tools such as nano devices (nanoelectromechanical resonators) that can

weigh individual molecules13 and ultrafast electron imaging tools for monitoring reactions as they happen14 are going to revolutionize quality control during process development of drug molecules. An overview of some of these nanotechnology tools for process development is depicted in Fig. 5.

Nanotechnology tools for product development


Some of the tools described above can also be utilized for product development. The term product development encompasses drug discovery as well as development of diagnostic tools. One of the most obvious and important nanotechnology tools for product development is the opportunity to convert existing drugs having poor water solubility and dissolution rate into readily water soluble dispersions by converting them into nano-size drugs15,16. Simply by reducing the particle size of drugs to the nanometer range, the exposed surface area of the drug is increased and hence its ability to be absorbed. Once the drug is in nano form, it can be converted into different dosage forms such as oral, inhalation, nasal and injectable. Key implication of this approach is the possibility of life cycle extension for existing drugs, for existing markets. There are a number of well known drugs that have already been commercialized using this approach for existing markets. For example, immunosuppressant drug Rapamune (sirolimus), Emend (aprepitant, MK 869), a substance P antagonist (SPA) for prevention of acute and delayed chemotherapy-induced nausea and vomiting (CINV) and for prevention of postoperative nausea and vomiting, have nano-size drugs made by Elan Corporation using nanocrystal technology17. Using micro emulsions as templates for solid nanoparticles, NanoMed Pharmaceuticals is currently developing a number of nano-size drugs using its patented Nanotemplate Engineering technology. The insoluble drug delivery (IDD) technology platform from SkyePharma is utilized in several drugs currently on the market such as SOLARAZE for skin cancer and antidepressant PAXIL CR. Finally, RBC Life Sciences is developing a new

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line of nutritional and skincare supplements called NanoCeuticals with nanoscale ingredients. This allows RBC to create products which, when consumed, reduce the surface tension of foods and supplements to increase the wetness and absorption of nutrients. As emphasis in drug development is towards controlled delivery and release, nanotechnology offers a number of tools in order to transform current drugs with these capabilities15,18-20. Nanotechnology tools for developing drug delivery systems and devices are valued at around $300 billion capturing significant applications in the health care market. As controlled release and delivery technologies are patentable, old blockbuster drugs can have continued protection at a relatively low cost. This is immensely attractive for the pharmaceutical industry in order to ward off the generics industry away from their hard earned profits. Additionally, drugs with these new capabilities will likely find applications in new markets. Simplest in this category are polymer encapsulated drugs such as MutliSal, DermaSal and MatrixSal from the company Salvona. Such polymer encapsulated drugs can be fine tuned to deliver the drugs using a number of stimuli such as pH, temperature, and water. In addition to simple polymer encapsulation, more sophisticated nanotechnology tools, such as nano core-shell designs, are also being developed in order to further fine tune delivery and release. For example, in a recent work Sengupta et al.21 demonstrated a novel approach for treating angiogenesis using a core-shell architecture wherein the polymer core has cytotoxic agent with the encapsulation of the anti-angiogenesis agent in the surrounding phospholipid block-copolymer22 (Fig. 6). Such a design enables temporal targeting of the tumor vasculature, resulting in the intra-tumoural trapping of the nanoparticles. This resulted in slow release and focal build-up of the cytotoxic agent within the tumor thereby prolonging exposure and an increase in the apoptotic potential, which can overcome hypoxia-induced reactive resistance. Yet another interesting example is gene delivery using multi-segment Au-Ni nanorods23. By attaching selectively plasmids to the nickel segment and a cell-targeting protein, transferrin, to spatially separated Au region, the gene delivery system provides precise control of composition, size and multi-functionality. Such nanotechology tools have potential applications in genetic vaccination.

As mentioned earlier, medical diagnostics account for just 1% of the total health care costs, even though at least 60% of all healthcare decision-making is based on diagnosis. Limitations in existing medical detection technologies complexity, cost and time consuming - have led to the development of several nanotechnology tools for ultra-sensitive and fast diagnosis of disease conditions and biomarkers20. Such nanotechnology diagnostic tools are driving the product development not only in drug discovery process but also in sensitive and timely disease detection. The majority of the commercialized nanotechnology diagnostic tools involve either quantum dots or gold nanoparticles. For example, biological barcode assay utilizing gold nanoparticles has become a powerful analytical tool for detection of both protein and nucleic acids in zepto molar concentrations, much higher than those possible using the PCR or ELISA techniques24. The company, Nanosphere, is using these tools to develop rapid, multiplex clinical tests for some of the most common inherited genetic disorders, including certain types of thrombophilia, alterations of folate metabolism, and cystic fibrosis25. Similarly, hitherto undetectable pathogenic Alzheimers disease markers in cerebral spinal fluid are being detected using these tools26. Quantum dot-based nanotechnology tools have been instrumental in visualizing not only cells but also specific components in the cell such as proteins and to follow the growth of an organism through live cell imaging7. They are also utilized for multiplexed and quantitative immunohistochemistry for evaluating biomarkers on intact cells and tissue specimens27. The companies, Invitrogen and Evident technologies, offer a wide range of quantum dots for a variety of applications in product development. High throughput synthesis and screening is yet another important requirement in product development and nanotechnology is offering several effective tools. The aim is to go beyond the current ultrahigh-throughput screening levels of more than 100 000 assays per day covering not only larger hits but also qualified leads through simultaneous screening for ADME (adsorption, distribution, metabolism, and excretion) and toxicity testing. Efforts towards miniaturization in high throughput parallel and combinatorial synthesis, as described earlier, coupled with novel technological tools for screening and bioinformatics for process integration are likely to bring out a paradigm shift in product development. Some of the exciting nanotechnology tools for such ultrahigh throughput screening are described below. Dip Pen Lithography is one such tool. The challenge of precision nanoscale deposition of a wide range of molecules onto diverse surfaces is overcome by Dip Pen Nanolithography (DPN), a high resolution scanning probe-based direct-write technology. It satisfies and exceeds these fundamental requirements through massive scalability of the process with two dimensional probe arrays. The researchers at Northwestern University28 demonstrated massively parallel nanoscale deposition with a 2D array of 55 000 pens on a

Fig. 6 Polymer core-lipid shell nanoparticles carrying drug for anti-angiogenesis within the lipid shell and a chemotherapeutic drug within the polymer core. (Reprinted from22 with permission from Macmillan Publishers Ltd).

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thereby reducing the reaction volumes at least 200 times smaller than in current micro plate screening, making PCR an inexpensive option in ultrahigh throughput screening. An overview of some of these nanotechnology tools for product development is depicted in Fig. 8.

Nanotechnology tools for personalized medicine


Fig. 7 SEM image of gold dot arrays on a silicon substrate. (Reprinted from28 with permission from Macmillan Publishers Ltd)

Scientific advances in genetic engineering, identification of biomarkers, and mapping of molecular pathways for diseases coupled with explosive growth of nanotechnology tools are driving the pharmaceutical industry towards personalized medicine31-33. The pharmaceutical industry today is already practicing the personalized medicine in a limited way. For example, selective prescription of antidepressant drugs, anticoagulants and proton pump inhibitors based on Cytochrome P450 (CYP450) test. Similarly Herceptin, a breast cancer drug made by Genentech, is only given to patients that test for unusual gene called HER2, which has been implicated in cancer growth. As more and more drugs are getting personalized, there is more pressure for pharmaceutical industries to move from blockbuster business model to tailored therapies. This in turn is leading to the development of novel nanotechnology tools for personalized medicine. The model IBINT, therefore, considers personalized medicine as an important segment for the strategic growth of pharmaceutical industry. There is a number of exciting nanotechnology tools currently finding applications in the development of personalized therapies16,19 and some of them are given below. One of the most important nanotechnology tools for personalized medicine is ultrasensitive molecular imaging. There are several novel techniques that are currently under development. One such technique is HYPER-CEST molecular magnetic resonance imaging (MRI) which allows for detection of signals from individual molecules within a cell at about 10 000 times lower concentration than conventional MRI techniques. The technique uses chemical exchange saturation transfer (CEST) between the biosensor-encapsulated Xe and the easily detectable pool of free Xe34. The various components of the Xe biosensor are cryptophane-A cage, the linker, the targeting moiety (biotin in this case), and the peptide chain. The 129Xe NMR spectrum of this construct through chemical exchange with free Xe outside the cage (resonance d1) enables sensitivity enhancement by depolarizing the d3 nuclei and detecting at d1. Yet another tool is in situ atomic force microscopy (AFM) with simultaneous confocal and epifluorescence microscopes. It has been recently utilized to investigate the interactions of enzymes and proteins within lipid bilayers35. Surface-enhanced Raman spectroscopy (SERS) using nanoparticle tags for detection and identification of biomarkers is gaining a lot of prominence. Recently, PEGylated gold nanoparticles coupled with targeting agents and small-molecule Raman reporters such as organic dyes were found to be >200 times brighter than near infrared-emitting

centimeter square probe chip. Fig. 7 shows SEM image of a portion of an 88 000 000 gold dot array (40 40 within each block) on an oxidized silicon substrate. On the right-hand side is a representative AFM topographical image of part of a block28. The technique, therefore, enables direct-writing of flexible patterns with a variety of molecules; simultaneously generating 55 000 duplicates at the resolution of single-pen DPN. To date, there is no other way to accomplish this kind of patterning at this unprecedented nano-resolution. The 2D nano PrintArray from the company Nanoink is based on the DPN technology. Such advances enable ultrahigh throughput screening for biological interactions at single molecular level. Similarly, the company Nanostream offers a wide range of nanotechnology-based tools for high throughput design and conduct of challenging biological assays, screening compounds for purity and solubility, profiling of ADME properties, accelerate formulation studies and perform comparative stability screening29. The company Nanosyss nanowirebased microarrays is providing a 100-fold higher binding area without reducing binding kinetics. While such nanostructured microarrays have the flexibility of being compatible with todays microarray platforms, the technology can be used to create, in principle, arrays of any dimensions. Similarly, the company Bio Trove30 offers microarrays with over 24 000 nano-liter reactions chambers for PCR amplifications

Fig. 8 Some of the nanotechnology tools for product development.

quantum dots, and allowed spectroscopy imaging of tumors as small as

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Fig. 9 In-vivo SERS spectra obtained from PEGylated gold nanoparticles injected into subcutaneous and deep muscular sites in a nude mouse. (Reprinted from36 with permission from Macmillan Publishers Ltd).

Fig. 10 Multi-reservoir devices for detecting a soluble cancer biomarker (Reproduced from42 with permission of The Royal Society of Chemistry).

300 m3 at a penetration depth of 1-2 cm36. Fig. 9 shows in-vivo SERS spectra obtained from PEGgylated gold nanoparticles injected into subcutaneous and deep muscular sites in a nude mouse. In addition to cancer diagnosis, SERS is also proving to be a valuable technique for carrying out a number of sensitive immune assays37, for example in the diagnosis of musculoskeletal diseases38. Yet another promising tool is the Sensation Detection Platform from Nanomix39. This tool enables direct, electronic, label-free detection of gases and biomarkers based on the changes in the electronic characteristics of random networks of carbon nanotubes. These devices are arrayed on silicon and plastic substrates and provide multi-plexed, multi-analyte detection and pattern analysis for ultrasensitive, specific, and reproducible bio-assays. For example, the Nanomix asthma monitor in development is a small, inexpensive unit that measures the level of nitric oxide (NO) in exhaled breath. Such devices could reduce needed medicines such as inhaled corticosteroids, reduction of traumatic and costly asthma attacks. Also, monitoring of the progression and regression of a disease pre-symptomatically enables a whole range of new medical decision-making. In addition to carbon nanotubes, semiconducting nanowires have been shown to be promising as highly sensitive and selective sensors for labelfree detection40. Similarly, analysis of various biomarkers in human breath is anticipated to lead to point of care diagnosis of diseases such as virus, lung or breast cancers, liver disease, gastrointestinal problems, and many others. In general, explosion of highly sensitive nanotechnology diagnostic tools is driving a change from empirically based, trial-and-error medicine, to more evidence-based, personalized solutions. These techniques enable simple, cost-effective, point-of-care results for critical decision-making. Novel concepts such as body-on-a-chip which can mimic human cells and human body and their reaction to different therapeutic conditions help reduce the need for testing ADME in animals and

hence lower the cost of developing new drugs. The new body-on-achip device, being developed by Cornell biomedical engineers, is lined with human cells and reacts the way tissues and organs would do to drugs41. In the same way, implantable micro chips such as those being developed by MIT researchers will ensure continuous monitoring of diseases and detection of biomarkers42. The concept is based on what is called magnetic relaxation switches wherein shortening of transverse relaxation time (T2) on aggregation of magnetic nanoparticles was observed using MRI. By combining the sensing capabilities of nanoscale magnetic relaxation switches (MRS) within multi-reservoir micro structures made from Polydimethylsiloxane (PDMS), they have recently demonstrated possible in vivo detection of soluble biomarkers for ovarian cancer such as -hCG. A prototype of such an implantable chip is schematically represented in the figure below. In addition to bodyon-a-chip and implantable microchips, a new class of nanostructured hybrid systems such as those consisting of neuronal cells grown on nanoporous membranes coupled with microfluidic devices are likely to lead to the development of artificial chemical synapse interface43. Similarly, artificially modulated neuronal connections between the neuronal cells and silicon nanowires have been recently reported to have potential opportunities to develop artificial neural networks44. Finally, the most promising of all the nanotechnology tools for personalized medicine is the ability to carry out integrated imaging and therapy. The nanotechnology tools based on both optical and magnetically tunable systems are currently under development. For example, immunotargeted nanoshells consisting of dielectric silica core gold shell nanoparticles coupled to targeting agents that have the ability to either scatter or absorb light in the near infrared (NIR) region are being developed to detect tumors using optical imaging and destroy them using photo thermal therapy45. An overview of some of these nanotechnology tools for personalized medicine is depicted in Fig. 11.

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are compelling applications in the pharmaceutical industry where inexpensive nanotechnology tools can be utilized. While it is true that many tools in the nanotechnology toolbox are still at the concept level, there are also a number of them that can be applied to process and product development of pharmaceuticals. In order to strategically bring together available nanotechnology solutions to pharmaceutical R&D, the concept of Innovation Box for Implementation of Nanotech Tools (IBINT) is proposed. The ultimate application of nanotechnology tools in pharmaceutical R&D is to move the industry from blockbuster drug model to personalized medicine. Huge commitments to the development of nanotechnology tools have already been made by both governmental and private agencies all over the world46-49. It is, therefore, important to continue to find models to strategically match nanotechnology tools with appropriate requirements in the pharmaceutical industry.
Fig. 11 Some of the nanotechnology tools for personalized medicine

Acknowledgements
The author gratefully acknowledges support from the Center for Advanced Microstructures and Devices (CAMD), Louisiana State University, Baton Rouge. The author is also thankful to Pharma IQ, a division of international quality and productivity center (IQPC) for the invitation to deliver a lecture during the meeting on The Future of Nanotechnology for Targeted Drug Delivery, February 25 27, 2008 at Hyatt Harborside, Boston, MA. The inspiration for this article is based on that lecture.

Conclusions
Nanotechnology is all about an innovative approach to problem solving and is simply a collection of tools and ideas which have potential applications in the pharmaceutical industry. Unlike the widespread notion that nanotechnology tools are exotic, too futuristic, disruptive and not suitable for quick commercialization of products, there

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