Cryopreserved human hepatocytes help HTS take the next step forwardThe advantages of high-throughput screening have been expounded upon by many for its efficiencies and confirmed by its acceptance in the pharmaceutical and biotechnology industries and government agencies. This has developed from advances in liquid handling systems, diagnostic equipment, and reagents.
The science behind and understanding of the processes involved in deriving useful and accurate data have brought us success with HTS. Growth in HTS will continue as it moves away from simple systems and binary information to more complex models and interpretable data. Two recent publications illustrate these improvements.
HTS started with simple systems such as bacterial cultures and receptor binding. It quickly incorporated cell lines to add to the complexity of biological systems while fulfilling the need for a scalable and renewable source of material. However, cell lines may not fully mimic the in vivo physiology of the cell type they are intended to represent.
A case in point is the HepG2 cell line derived from a human hepatocellular carcinoma. HepG2 cells have been used as a surrogate for hepatocytes, the primary functional cell type of the liver, to study various liver cell functions like metabolism, transport, drug-drug interactions, and cytotoxicity in traditional cell culture and HTS models. However, significant differences, such as lower expression levels of drug-metabolizing enzymes compared to primary human hepatocytes, have limited the ability of HepG2 to predict in vivo situations.
It is well accepted that primary human hepatocytes provide the most apposite in vitro model to predict in vivo function and are, therefore, the gold standard for pharmaceutical and toxicological studies. However, the use of primary human hepatocytes in HTS has been limited by reliance on unscheduled fresh isolations and inability to retest from the same donor. Recent advances in the quality and availability of cryopreserved human hepatocytes have provided the opportunity to exploit them in HTS models for metabolism, drug-drug interaction, and toxicity studies.
The Flexibility of HTSIn a 2011 manuscript, Moeller and colleagues presented the first reported use of cryoplateable human hepatocytes in 1536-well format.1 The validation included confirmation of metabolic activity of cytochrome P450 (CYP) 3A4 in culture and the derivation of toxicity IC50 values from a 16-point concentration response curve after a 40-hour exposure to known cytotoxic compounds. The format provided a balance between a high number of compounds tested and the high number of data points observed. The former was in line with the original purpose of HTS to screen large chemical libraries, while the latter has followed the current trend toward quantitative HTS.
Traditional HTS relied on a single concentration to provide a qualitative assessment. This approach may be acceptable with criteria that are well established to provide actionable and interpretable data. However, qHTS has provided a wider range of concentrations in order to assess the effect when significant concentration thresholds are not known. In this study, the concentration response curve was between 92 µM and 3 nM. The range of IC50 values was between 2 µM for doxorubicin and 90 µM for tamoxifen.
Primary human hepatocytes provide the most apposite in vitro model to predict in vivo function and are the gold standard for pharmaceutical studies.If a single concentration of 10 µM had been employed, about half of the compounds tested would have been negative for cytotoxicity. The significance of the in vitro data from a single concentration or concentration response curve must be vetted with observed toxicity. Although this example doesn’t determine the more significant approach, it does illustrate the flexibility of HTS in meeting the needs of research.
Match Data Quantity, QualityThe quantity of data provided by HTS must be matched by quality. That is, if designed appropriately, the data generated should be able to be interpreted with confidence as to the mechanism of action for the observed event. To achieve this, multiple markers have been used to capture sufficient data in order to discern among multiple pathways in a cell-based screen.
One method is multiplexing, employing two or more signals obtained from a single well. Larson and colleagues presented a model for measuring the induction of metabolic enzymes with drugs using three distinct markers.2 The model was based on the gold standard of measuring enzyme activity for key metabolic enzymes—CYP1A2 and 3A4. The novelty was to perform these measurements, along with a viability marker, from the same well of a 384-well microtiter plate.
Traditional methods relied upon separate incubations between wells to measure the individual event. The data would then be combined to assess the capacity of a test compound for its induction potential. Inherent error, including seeding densities between wells, position of the wells on the plate, microenvironments between wells, and dosing differences, may affect the fidelity of the data when combining separately acquired data into one profile. By measuring these markers from the same well, many of the potential errors may be mitigated, increasing confidence in the data. EC50 values from concentration response curves may be generated due to the capacity of the 384-well format, a process made more difficult in the commonly used 24-well formats.
Two examples highlight the benefits of multiplexing CYP1A2 and 3A4 signals with a viability marker.
Rifampicin is a well-known potent inducer of CYP3A4. The data generated from a concentration response curve provided an EC50 of approximately 2 µM with slight induction of CYP1A2 at the highest concentration (see Study 1A). The viability as measured by ATP content remained stable at 100% compared to vehicle control.
A lower fold induction than the Emax was observed at the highest concentration of rifampicin. If this had been observed with only the CYP3A4 activity as the marker, possible interpretations might be cytotoxicity, inhibition, or suppression, and separate investigations for each possibility would be required to determine its cause. With viability included as a marker, cytotoxicity can be ruled out, leaving inhibition and suppression as possible mechanisms. From the literature, rifampicin has been shown to be an inhibitor of CYP3A4 at high concentration, providing a plausible explanation for the reduced induction observed at the highest concentration.
Lansoprazole presented a different profile and interpretation (see Study 1B). Lansoprazole was confirmed to be an inducer of CYP1A2 from the concentration response curve. It, too, showed lower induction values than the Emax at higher concentrations; however, the viability marker was lower at higher concentrations, indicating lansoprazole-induced cytotoxicity. In this case, cytotoxicity was the cause of the drop in induction at higher concentrations. This was collaborated by the observed reduction of CYP3A4 signal at similar concentrations. Interpretation of the data can be made with confidence, given the multiple markers observed across the concentration response curve.
Tuesday, April 24, 2012
How to overcome low-population assay resultsMost users of biological indicators test incoming BIs for viability and population as part of their acceptance criteria for use. This reasonable or mandated examination prior to routine use or the beginning of a validation is also stressed in U.S. Pharmacopeia:
“Upon initial receipt of the biological indicator from a commercial supplier, the user should verify the purity and morphology of the purchased biological indicator microorganisms. … Also, a microbial count to determine the mean count per biological indicator unit should be conducted.”The USP provides information explaining how to perform a microbial count of BIs in the section titled “Microbiological Tests - Total Viable Spore Count.” According to the USP, after the assay procedure is performed, the total viable CFU count needs to be within minus 50% and 300% of the labeled BI population. The process of performing a total viable spore count on the large variety of BIs being used with differing carrier materials and BI size can be more difficult than it may appear.
Any difficulty encountered in obtaining acceptable results to qualify a BI should in most cases be offset by the wide range of acceptability that the USP allows, minus 50% to 300% of the labeled population. Successful population verification should be easily obtainable; however, even with this wide range, many end users are unable to obtain acceptable assay verification. Nearly all assay result failures are due to low population results, below the acceptable minimum of minus 50%.
Some of the causes for low testing results are easier to locate than others. One challenging part of a population failure investigation is just getting the end user who is actually performing the test to look at what was done. Most lab techs consider the assay procedure to be pretty straightforward and are not aware of the procedure problem areas that can occur to make a test result falsely low. Some find it much easier to just accept the fact that the BI is unacceptable for population and request a replacement than to take a look at the possibility of user error.
During the nearly 19 years I have been addressing complaints of low population on BIs, I have found it is extremely unusual to find a BI complaint where the BI was indeed low in population. Most of those who reported low populations have been able to achieve success by making a few procedural changes and then repeating the assay.
One of the first questions that I ask when investigating a low population complaint is, “Are you following USP procedure?” In most cases the answer is yes. However, many who say they follow the USP have made several unintentional deviations from USP procedure that could account for the low population result.
- You must either follow USP or not. Unless you are using a validated excursion from USP, do not deviate from the procedure as written. Most BI manufacturers follow USP Total Viable Count procedure to make the label claims for the BIs. If you are using a different method from that of the manufacturer, difficulties in population verification can occur.
- USP procedure for paper strips states that the initial step for BIs to be tested is to “dispense the paper into component fibers by placing the test specimens in a sterile 250 ml cup of a suitable blender containing 100 ml of chilled, sterilized purified water and blend for three to five minutes to achieve a homogeneous suspension.” If you are using something else, you are neither following USP nor following the assay procedure used by this BI manufacturer. The use of a 250 ml cup is not the same as a large blending vessel that is usually used for mixed or blended drinks. It is a 250 ml jar. Such jars are common half pint jars used for canning and can be found in most hardware stores. The threaded lip on the jar fits the same plastic collar and blade setup used with a typical blender. Using this initial 250 ml container is an important part of the procedure and is also required according to USP. The photos below illustrate how easy it is to use these jars. Because they are canning jars, they are also heat treated and autoclavable. They are usually purchased by the dozen and are very reasonably priced.
Case Study: Three Strikes and You Are OutAfter failing to accept three different, consecutive lots of spore strips for use due to low population testing results at a particular company, I was asked to audit the USP assay procedure being used there, because these particular lots of strips had already passed verification efforts at several other sites using the USP procedure. An investigation was requested to look into the assay procedure being used to determine whether there was an error in the procedure or if the strips truly had a low population.
The facility in question followed USP procedures, and lab personnel said that they were following the Total Viable Spore Count procedure as published in USP. When I arrived at the facility, the lab tech had all the necessary tools ready to perform a population assay. The first step was to remove the paper strips from the glassine envelopes and prepare them to be assayed.
STRIKE ONE: In order to macerate the paper, the tech put the strips into a blending vessel that looked much like those used to prepare mixed drinks. The vessel must have been able to hold at least a liter of water, not as specified in USP. The strips were put into the container, and 100 ml of water was added. This large container was put on top of the blender, and it was turned on. Soon the strip/water mixture was churning in the vessel, high enough on the sides of the vessel that liquid could easily spill out.
STRIKE TWO: After the strips were blended in the container, a glass pipette was used to remove a 10 ml aliquot to start the dilution series. There was so much fiber content present that the tip of the pipette drawing up the aliquot got clogged. The lab’s remedy for this problem was to use a large-bore pipette tip that would not clog as easily. This drew even more paper pulp into the 10 ml sample.
STRIKE THREE: Near the end of the dilution series, the last two dilutions were plated out in triplicate petri dishes, with a 1 ml aliquot put into each dish. Approximately 20 ml of TSA was added to each dish, and the dishes were allowed to cool and were inverted as they were placed into the incubator. The TSA used had a high content of dipotassium phosphate and other buffers.
The final assay results for all three lots of strips were over one log low in population and thus failed the verification. On a subsequent assay using a different medium on the same lots of spore strips, and with the above deviations corrected, all three lots passed verification.
Even in what would seem to be a pretty straightforward procedure, you either follow USP or you don’t.—RN
- Blend to a homogenous solution and, while blending, look at the paper material through the glass jar to see when the strips are thoroughly blended. You should be able to draw up a 10 ml aliquot that does not have paper pulp in the sample.
- Make sure, while vortexing between dilutions, that a vortex actually occurs in the dilution tube and that the solution is being mixed well.
In the full study, six groups of log 6 spore strips were processed using the USP Total Viable Spore Count procedure. All strips were treated the same except for the TSA used in final plating. The groups were blended and went through the usual dilution series. Six sterile petri dishes were inoculated with 1 ml aliquots from the last dilution tube. On three of the plates, Brand X TSA was used, and on the other three plates from the same tube, Brand Y TSA was used in making the final pour plates. Colony-forming units recovered with the different brands of TSA are given in Table 1.
The CFU data from above shows that anyone using Brand X TSA in their verification would never confirm or meet USP acceptance criteria even if all the previous steps had been done correctly. Just changing to a different TSA brand can make a huge difference in your CFU recovery.
Based upon the testing results, a recovery medium that is free from additives such as phosphates, diphosphates, or dipotassium phosphate buffers is recommended. In the above study, Brand Y media did not contain phosphate buffers, while Brand X did. Media that contain phosphates and/or buffers are very suitable for a large amount of typical laboratory or micro work, but this study shows that these additives can be inhibitive to the accuracy of a quantitative recovery of injured spores.
Russ Nyberg has worked at the Omaha Biological Indicator manufacturing facility for Mesa Labs (formerly Raven Biological Laboratories) for the past 19 years. He has held the positions of director of manufacturing and production and now works in technical support.
- Sutton S. Counting colonies. Microbiology Network website. Available at: www.microbiol.org/resources/monographswhite-papers/counting-colonies. Accessed Feb. 5, 2012.
- U.S. Pharmacopeia. General chapter 55: Biological indicators — resistance performance tests. U.S. Pharmacopeia. Available at: www.usp.org/usp-nf/notices/retired-compendial-notices/second-supplement-usp-31-nf-26-online-and-cd-includes-incorrect-version-usp-general. Accessed Feb. 5, 2012.
- Shintani H, Sakudo A, McDonnel GE. Methods of rapid microbiological assay and their application to pharmaceutical and medical device fabrication. Biocontrol Sci. 2011;16(1)13-21.
Mark Egerton, PhD
Innovation in the new ecosystem of pharmaceutical R&DBy 2015, a staggering $250 billion of potential pharmaceutical sales will be lost to generic competition.1 The stream of innovative new products that will replenish the industry’s pipeline has failed to appear and, fueled by this R&D productivity gap, the pharmaceutical industry has embarked on a process of reinvention.
Multiple strategic initiatives have been embraced in an attempt to increase R&D productivity. The mega mergers between top tier pharmaceutical companies that occurred during the past 10-15 years were partly designed to consolidate development pipelines and deliver R&D scale. A trend to “externalize” R&D closely followed, with top tier companies looking to supplement internal R&D activities by in-licensing development compounds/projects. Several top-10 pharmaceutical companies have publicly announced an aspiration to license up to 40% to 50% of their development portfolio from external sources, typically smaller biotech/pharma companies. In recent times, the theme of externalization has been further emphasized by a significant shift of pharmaceutical R&D organizations toward outsourcing of operational resource. Consequently, the overall shape and complexity of the pharma R&D ecosystem have changed dramatically as it progresses through the process of reinvention.
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FIGURE 1: The increase in the number of companies actively involved in drug development (A) and the number of molecules in the pharmaceutical industry’s drug development pipeline (B). Data abstracted from the Pharma R&D Annual Review 2010.
The New R&D EcosystemMega mergers and externalization of R&D have driven significant change in the pharma R&D ecosystem. An analysis of top tier pharmaceutical companies reveals that approximately 75% of those companies responsible for drugs receiving FDA approval in the mid-1990s no longer exist.3 Many names well known in the industry, including Ciba-Geigy, Hoechst, Marion-Roussel, Monsanto, Pharmacia, Upjohn, Warner Lambert, Wellcome, and Wyeth, have been relegated to the history books—an occurrence often associated with R&D budget cuts, site closures, and job losses.
The adoption of outsourcing strategies has driven further reorganization of R&D structures within large pharmaceutical organizations, which in recent years have entered into multi-year, multi-compound strategic deals. A number of strategic outsourcing partnerships announced in the public domain—SanofiAventis-Covance, AstraZeneca-Quintiles, and Eli-Lilly-Covance, for example—make it evident that major elements of operational responsibilities and resource have been transferred to the service provider, sometimes in conjunction with R&D facilities. These deals are multiyear partnerships, and it is inconceivable that the pharmaceutical partner could re-create internal competency at some point in the future and remain competitive. The move to outsourcing therefore appears to be irreversible.
Concurrently, the total number of organizations active in drug development has almost doubled, rising from 1,167 in 2000 to 2,207 in 2010. An equivalent increase in the total number of drugs in development, from 5,995 in 2000 to 9,737 in 2010, has followed (see Figure 1).4 The majority of the approximately 2,200 organizations that are actively involved in drug development are small- to medium-sized pharma/biotech companies that have development portfolios containing only a few molecules, some with just a single molecule.
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FIGURE C: Pharmacokinetic profile of an optimized SLX-2101 extended-release drug product.
Case Study: Optimizing an Extended-Release Drug Product for a Novel PDE5 InhibitorLx-2101 is a selective PDE5 inhibitor in development for a spectrum of indications, including hypertension. Once-daily dosing was considered essential for the indications being addressed, but the FIH program demonstrated a sub-optimal PK profile.
The maximal concentration (Cmax) exceeded the threshold at which adverse events were observed, and the trough concentration (Cmin) occurred within the 24-hour period, resulting in loss of pharmacodynamic activity. The objective of the program, therefore, was to develop and validate an extended-release
formulation that delivered Cmax below the threshold level for adverse events and Cmin above the threshold level for pharmacodynamic activity for 24 hours, thereby delivering a product suitable for once-a-day dosing in later stage clinical trials.
Program DesignA translational pharmaceutics platform was used to develop a rapid formulation development and clinical testing program (RapidFACT). Hydroxypropylmethylcellulose technology was selected to develop an extended-release matrix tablet as the drug product. The first step was to construct a two-dimensional formulation “design space” (see Figure A) with dosage strength on one axis and HPMC content—and therefore release rate—on the other axis. A clinical trial application was submitted to the UK regulatory agency, which contained supporting chemistry manufacturing and control data for the four prototype compositions at the extreme corners of the design space (FP1-4); however, permission was sought to make and test any formulation within the design space. Regulatory approval was secured within 14 days.
This flexibility of having critical-to-performance formulation components as continuous variables de-risks pharmaceutical development by avoiding the need to pre-select discrete, “locked” formulation compositions based on in vitro or preclinical data alone. Composition and, hence, in vivo performance can be varied within the clinical study in response to arising human data.
The clinical study was a five-period crossover design with 12 healthy volunteers (see Figure B). The interval between each period was 14 days. During this window, clinical safety, pharmacokinetic, and pharmacodynamic data from the previous period(s) were analyzed and a decision made on what formulation composition (dose and release rate) was to be manufactured and tested in the following period given the data from the previous period(s), which guided the project team to the optimal formulation. The clinical protocol was written to allow the option to investigate the effect of food following the selection of the optimal formulation.
ResultsThe RapidFACT program was completed in 21 weeks, from the point of initiation of the formulation development work through completion of the clinical program and delivery of decision-making data. An optimized formulation was identified within the first three extended-release prototypes tested from within the design space. In the fifth period of the study, the selected drug product was then dosed to healthy subjects in the fed state to confirm the absence of a food effect. Compared to a conventional process, the timeline and cost for this program were halved, and drug substance consumption was reduced by approximately 85%. These features, combined with the flexibility of the design space approach and the precision of using clinical data to drive decision making, ensured the program’s success.
From the perspective of a service provider, this new R&D ecosystem presents a broad range of customers with very different requirements and expectations. At one extreme lie the top tier pharmaceutical organizations with significant internal infrastructure and drug development know-how; at the opposite end are the small virtual companies with no/little internal infrastructure and greatly reduced development know-how. The lifespan of many of these smaller companies may depend on the success or failure of a single molecule in development.
Where will the drive to innovate development processes come from within this ecosystem? Historically, pharma R&D personnel have been considered the owners of innovation. As a greater level of development activity is outsourced, however, service organizations are challenged to innovate.
The New Paradigm
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FIGURE 2: Translational pharmaceutics—horizontal integration of “make” and “test” supply chains to drive innovation in early development.
In essence, early development—defined as first-in-human through to proof of concept—has become part of the research continuum. In this process, drug candidates and molecular targets must be carefully tested to remove technical uncertainty and differentiate the “winners” from the “losers.” The conundrum, however, is that later stage attrition has to be reduced at a time when the number of disease targets being explored by the industry has never been higher. The early development process, therefore, must evolve to enable a much more flexible and interrogative approach with shortened cycle times. Early development plans must be tailored to individual molecules and focused on scientific interrogation to address the key questions—risk factors—that must be answered before the molecule progresses into full development. This vision can only be achieved by re-engineering the processes underpinning early development.
Current organizational structures and processes to support early development have evolved from working practices established over several decades in big pharma. Innovators and service providers have consolidated into two vertically integrated supply chains: one focused on the making of test materials (drug substance and product) and the other focused on testing of those materials (preclinical and clinical). In the outsourced industry, these two channels are typically referred to as contract development and manufacturing organizations and contract research organizations, respectively (see Figure 2).
The competitive requirement for innovator companies to deliver first- and best-in-class medicines has increased the emphasis on new molecular targets and disease mechanisms that offer the potential to deliver breakthrough therapies. The challenges of translational science, however, mean that in reality the majority of these projects will fail at or before proof of concept.This strong demarcation of make and test functions has adequately served the industry to date. However, the established processes are sub-optimal when it comes to supporting the new early development paradigm. Transfer of drug product(s) between the two channels is cumbersome and often complex. For example, generation of sufficient stability data to assign an extended drug product shelf life and accommodate the logistical inefficiencies between manufacture and dosing can represent a significant penalty to the overall project timeline. The project team is also confronted with the challenge of investing at risk into drug product development and clinical trial manufacturing of a range of dose strengths at quantities to cover all eventualities.
For an innovator organization that has adopted an outsourcing strategy, these challenges are typically further exacerbated. The provision of a drug product for clinical testing may involve up to four different suppliers to cover drug substance supply, formulation development, clinical trial manufacturing, and packaging. The resulting supply chain presents a significant management burden and timeline risk, especially on those occasions when it breaks down and the incumbent suppliers are not focused on delivering a solution.
Translational pharmaceutics is a new approach in which make and test supply chains are horizontally integrated to create a delivery platform for early development. Such a platform enables the rapid and seamless manufacturing-to-clinic transfer of drug product, with manufacturing often taking place within a 24-72 hour period prior to dosing.
The savings in time and cost delivered by this approach are significant, and reductions by a third to half that of conventional approaches are typical. More importantly, however, it enables innovative thinking within early development, especially for those processes leading up to the FIH study or in subsequent work to optimize the drug product prior to full development. In essence, the make and test philosophy that has been used to drive discovery research can now be applied within clinical research.
First-in-Human ProgramThe FIH program represents a significant milestone for the R&D project team. In reality, it represents the first step into a new phase of investigations to confirm the merits of the drug candidate and molecular target in question.
Traditionally, the FIH program is undertaken with single drug product in a relatively simple format (e.g., drug in capsule). Decisions on this format, such as formulation type and dosage strength, are taken in the pre-clinical phase, often before pivotal toxicology studies have been completed. Significant quantities of drug substance, often in scarce supply at this stage, are consumed by the manufacturing of large batches of multiple dose strengths, to provide sufficient quantities and flexibility of drug product to cover the early development program. Hence, if the molecule fails toxicology or if the chosen drug product format proves sub-optimal following clinical dosing, all of the upfront investment in pharmaceutical drug product development and manufacture—typically $300,000-$500,000—is wasted.
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FIGURE 3: The benefits the can be achieved by using a translational pharmaceutics platform to deliver a FIH program versus a conventional process.
The benefits delivered to the project team are significant (see Figure 3). The approach offers enhanced flexibility in the design of an early development program and equips the project team with a very powerful toolbox to interrogate drug candidates and mechanisms. Perhaps just as importantly for the smaller pharma R&D organizations, these programs can be implemented by working with just one service provider.
Product OptimizationIn the current industry pipeline, the majority (estimated at 40%-70%) of small molecules for oral administration have sub-optimal solubility and/or permeability properties.6 Prior to transition from early development into full development, these drug products must be optimized so the active drug substance is delivered to the right place, at the right time, at the right concentration, to ensure that its therapeutic benefit can be achieved.
Optimization often requires the use of enabling formulation technology, in a conventional approach that involves iterative investigations in animal models, renowned for producing results with little correlation to humans, prior to testing a limited number of prototypes in the clinic.7 This process, which can take 18 months or more, is expensive and has a high failure rate. Translational pharmaceutics has transformed this process.
Data-driven make-test cycles of drug products in the clinic enable a much more rapid and effective approach to formulation development and clinical testing. Development timelines and costs can be halved—the project presented in the accompanying case study was completed within 21 weeks from formulation initiation to availability of clinical decision-making data. Perhaps more importantly, human clinical data underpinned all selection decisions, ensuring greater precision and accuracy of the drug product selected for full development.
- EvaluatePharma. Evaluate pharma alpha world preview 2014. EvaluatePharma website. May 2009. Available at: www.evaluatepharma.com/worldpreview2014.aspx. Accessed March 15, 2012.
- DiMasi JA, Grabowski HG. The cost of biopharmaceutical R&D: is biotech different? Manage Decis Econ. 2007;28(4-5):469-479.
- LaMattina JL. The impact of mergers on pharmaceutical R&D. Nat Rev Drug Discov. 2011;10(8):559-560.
- Informa Healthcare. Pharma R&D annual review 2010. Pharmaprojects website. Available at: www.pharmaprojects.com/therapy_analysis/annual-review-2010.htm. Accessed March 15, 2011.
- Paul SM, Mytelka DS, Dunwiddie CT, et al. How to improve R&D productivity: the pharmaceutical industry’s grand challenge. Nat Rev Drug Discov. 2010;9(3):203-214.
- Zhang GZ. Crystalline solid dispersions. Formulation strategies for poorly soluble drugs. Paper presented at: AAPS 45th Annual Pharmaceutical Technologies Arden Conference; Feb. 1-5, 2010; West Point, N.Y.
- Grass GM, Sinko PJ. Effect of diverse datasets on the predictive capability of ADME models in drug discovery. Drug Discovery Today 2001;6(12)Suppl:S54–S61.
- Lichtenberg FR. Contribution of pharmaceutical innovation to longevity growth in Germany and France, 2001-7. Pharmacoeconomics. 2012;30(3):197-211.
- Hsieh CR, Liu YM, Chang CL. Endogenous technological change in medicine and its impact on healthcare costs: evidence from the pharmaceutical market in Taiwan [published online ahead of print Dec. 27, 2011]. Eur J Health Econ.
- Seddon G, Lounnas V, McGuire R, et al. Drug design for ever, from hype to hope. J Comput Aided Mol Des. 2012;26(1):137-150.
- Robertson GM, Mayr LM. Collaboration versus outsourcing: the need to think outside the box. Future Med Chem. 2011;3(16):1995-2020.
- Festel G. Outsourcing chemical synthesis in the drug discovery process. Drug Discov Today. 2011;16(5-6):237-243.
Laurie Kronenberg and Brad Beissner
FIGURE 1. Two Points in the Three-Dimensional Color Plane.
Consumer demand for safer products sparks push to eliminate artificial colorantsThe $28 billion nutraceutical market in the United States might face stricter regulation in the near future, some experts say.1 Artificial and genetically modified ingredients, generally shunned by consumers and oral dosage form manufacturers, could become limited in use, and producers of these coating products will likely improve their offerings to remain competitive.
To answer the call for more health-conscious coating choices, companies like Ashland Specialty are creating natural offerings that not only offer a wide variety of colors and palettes but also address the need for safer and more stable products.
Synthetic food colors are rapidly falling out of favor in the European Union, a phenomenon largely spurred by the release of a 2007 British study that identified a list of six particular colorants that showed a link to hyperactivity and attention disorders in children.2 These additives have become known as the “Southampton Six.” The colors in the study are Alurra Red (also called Red 40, E129); Ponceau 4R (E124); Tartrazine (Yellow 5, E102); Sunset Yellow FCF/Orange Yellow S (Yellow 6, E110); Quinoline Yellow (E104); and Carmoisine (E122). These six must now carry a warning label in the EU that states that the colorant “may have an adverse effect on activity and attention in children,” and there is legislation pending in the EU to ban these colors.3 With such an advisory in place, consumers are understandably interested in natural alternatives.
The call for natural colorants is also growing in many other parts of the world. A recent Nielsen poll of 5,000 respondents, commissioned by Chr. Hansen, revealed that 92% were concerned about artificial colors, and many are willing to pay a premium for products made with natural colors.4 Many suppliers of coloring agents to the food, nutraceutical, and pharmaceutical industries are working on more reliable and varied natural color formulations in response to this increased demand. Ashland Specialty Ingredients is working with these suppliers to develop compliant, color-stable coating systems.
The fully formulated Aquarius coating systems natural colors palette contains 72 hues of reds, pinks, oranges, yellows, greens, blues, purples, and browns compliant with current U.S. and EU regulations. Notable differences between the two sets of regulations include iron oxide, which is permitted as a food colorant in the EU but restricted to pharmaceutical use in the U.S., and carmine, which has a strict usage limit in Europe but no restrictions in the U.S. There are numerous other differences in the regulations between the U.S., the EU, and other parts of the world, and these regulations change frequently.
Regulatory ConcernsThe development process for the Aquarius coating systems natural color palette began when Gernot Warnke, Ashland Specialty Ingredients’ Technical Manager for Pharmaceuticals, Europe, scoured the lists of regulations in the U.S. and EU to locate coloring agents that were acceptable under both sets of rules. The list was disappointingly small. As a result, the current natural color offerings include three separate groups of colors: one that complies with regulations in the EU and the U.S., one that meets EU regulatory requirements, and one that meets U.S. regulations. Some shades are EU and U.S. compliant.
Many of the tablet coating formulations are aluminum free. The use of aluminum lakes in food colorants may be banned or at least strictly controlled in the future. Particular colors have been banned previously in some European countries. The aluminum-free colors in the Aquarius coating systems natural colors palette anticipate the possibility of a ban on aluminum lakes.
Color StabilityThe scientists working on this new line of natural colors knew that in addition to the regulatory constraints, good color stability—over time and with exposure to light—would be an important requirement. Natural colors are notoriously unstable under prolonged light exposure. To ensure good quality colors, the natural colors palette began with decisions about what would not be included. Annatto- and chlorophyll-based colorants were not even considered for the new line. The Aquarius natural colors coatings are based on more stable colorants such as carmine, riboflavin, caramel, and anthocyanins.
Each of the natural colors formulations underwent exhaustive light stability testing, using International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use standard protocols for photostability. Samples were exposed to simulated daylight for 1.2 million lux hours under the most stringent conditions, direct light exposure.
A spectrophotometer was used to measure the ΔE of two L*a*b* readings for each sample, a protocol developed by the Color Measurement Committee (CMC) of the Society of Dyers and Colorists (see Figure 1). L* is a reading in the white to black plane, a* is in the red to green plane, and b* is in the yellow to blue plane. The ΔE CMC is simply a Pythagorean distance calculation between the two measured points. A ΔE of 3.0 or less is the amount of color shift for which the average observer will be unable to perceive a color difference. The hues chosen for the current natural colors coatings palette have color stability that ranges from good (ΔE 5.0 to 7.0) to good (ΔE 3.0 to 5.0) to excellent (ΔE < 3.0). Because these testing conditions are extreme, Ashland Specialty Ingredients’ scientists are confident that all the natural colors offerings are color stable under normal lighting conditions.
For color storage stability testing, we again chose to follow ICH guidelines. We used an accelerated stability test that is run at 40 degrees C and 75% relative humidity. Coated tablets were placed in high-density polyethylene bottles for up to three months and measurements were taken with a Hunter LabScan spectrophotometer at one-, two-, and three-month intervals. Again, ΔE values of less than 3.0 were considered stable, and those colors were included as part of the natural colors palette.
Additional QualitiesAs with the entire line of Aquarius coating systems colors, natural colors are sprayable at 20% solids, making them an efficient and economical choice, with shorter tablet coating times. This has the added benefit of allowing effective coating of friable or moisture- and temperature-sensitive cores. The formulations in the natural colors palette are color stable, label friendly, and easy to use. In addition, they can be combined with Aquarius barrier coating technology for protection against moisture and odor.
All of the Aquarius coating systems natural colors palette offerings have been formulated from Ashland Specialty Ingredients’ line of cellulose polymers. Ashland is the only film coating supplier that is back integrated in the manufacturing of hydroxy propyl methylcellulose, the cellulose polymer raw ingredient in many film coatings. This polymer know-how gives Ashland the ability to make specific polymer grades for coating applications and leads to a strong technical advantage in delivering film coating products. Ashland Specialty Ingredients’ technical experts work on customized coatings, color matching, and troubleshooting, and can even provide tablet development at state-of-the-art technical centers across the globe.
- Nutrition Business Journal. NBJ supplement business report. NewHope360 website. Sept. 1, 2011. Available at: http://newhope360.com/2010-supplement-business-report-0. Accessed March 19, 2012.
- Food Standards Agency. FSA advice to parents on food colours and hyperactivity. FSA website. Available at: www.food.gov.uk/safereating/chemsafe/additivesbranch/colours/hyper. Accessed Sept. 28, 2011.
- Fusaro D. When it comes to synthetic food colors: beware the “Southampton Six.” Food Processing.com. July 6, 2010. Available at: www.foodprocessing.com/articles/2010/colorants.html. Accessed March 19, 2012.
- Byrne J. Nielsen poll indicates global preference for natural food colors. FoodNavigator.com. Oct. 6, 2011. Available at: www.foodnavigator.com/Financial-Industry/Nielsen-poll-indicates-global-preference-for-natural-food-colours. Accessed March 19, 2012.
- May RK, Evans MJ, Zhong S, et al. Terahertz in-line sensor for direct coating thickness measurement of individual tablets during film coating in real-time. J Pharm Sci. 2011;100(4):1535-1544.
- Maurer L, Leuenberger H. Terahertz pulsed imaging and near infrared imaging to monitor the coating process of pharmaceutical tablets. Int J Pharm. 2009;370(1-2):8-16.
- Ho L, Müller R, Gordon KC, et al. Terahertz pulsed imaging as an analytical tool for sustained-release tablet film coating. Eur J Pharm Biopharm. 2009;71(1):117-123.
‘Stealth’ and stimuli methods explored as ways to bolster deliveryLiposomes are spherical phospholipid vesicles, typically ranging in size from 50 to 1,000 nanometers (nm), that serve as delivery systems for a wide variety of drugs. Liposomes are made of one or more concentric lipid bilayers surrounding an aqueous inner compartment with aqueous phases also occurring between the lipid bilayers. Hydrophilic, or water-soluble, drugs can be loaded into the aqueous compartments, while hydrophobic, or water-insoluble, drugs can be loaded into the lipid bilayers.1
Liposomes are classified in various ways, including size (small, intermediate, or large) and lamellarity (unilamellar, oligolamellar, and multilamellar). Unilamellar vesicles are generally 50 to 250 nm, with one lipid bilayer around an aqueous core that can encapsulate water-soluble drugs. Multilamellar vesicles measure one to five µm and contain several concentric lipid bilayers. Their high lipid content facilitates encapsulating lipid-soluble drugs.2
A Neat PackageFirst developed around 1980, the potential of liposomes as a drug delivery system has been recognized for more than 20 years. In addition to the fact that they can incorporate both hydrophilic and hydrophobic compounds, liposomes display good biocompatibility and low toxicity. They tend not to activate the immune system and can deliver a drug directly to the site of action.3
Liposomes are especially useful in the delivery of protein and peptide drugs. These categories include potent and life-saving therapeutics, including enzymes and insulin. The use of proteins and peptides is limited by their instability at physiological pH and temperature. Incorporating these compounds into liposome delivery systems improves their pharmacological properties by delivering:
- Increased stability;
- Prolonged activity;
- Decreased total amount of active ingredient needed;
- Possibility of a single dose administration; and
- Decreased immune system activation.
Liposomes are especially useful in the delivery of protein and peptide drugs. These categories include potent and life-saving therapeutics, including enzymes and insulin.On the other hand, there are definite drawbacks to the use of liposomes. They cannot achieve sustained drug delivery over a long period of time, and liposomes with a positive charge carry the risk of toxicity. Also, as a result of their rapid opsonization (the process by which phagocytes eliminate pathogens from the system), liposomes are quickly eliminated from the blood by the reticuloendothelial system, particularly the liver. These factors have limited the use of liposomes in pharmaceuticals.
Sneak Up on Target TissueOne of the most widely used methods to overcome the downsides of liposome drug delivery is stealth liposomes. This technology involves altering the surface of the liposome by coating it with either a natural or synthetic polymer conjugate. The most commonly used polymer is polyethylene glycol (PEG), a linear polyether diol with a number of beneficial properties:
- Lack of toxicity.
- Low immunogenicity;
- Solubility in aqueous or organic vehicles; and
- Good excretion profile.
But does the addition of PEG have any other effect on the drug payload? To answer this question, researchers at Seoul National University’s College of Pharmacy evaluated the circulation longevity of methotrexate, a chemotherapy compound, when delivered in a PEG-coated liposome. They discovered that plasma levels of methotrexate increased as the concentration of PEG increased, up to 5%. From these results, investigators concluded that PEG-coated liposomes show promise for targeted delivery of anti-cancer drugs.
Case Study: Liposome-Hydrogel Hybrid NanoparticlesAmong the disadvantages of liposomes as a drug delivery system is their instability in the blood stream and their tendency to rupture easily if the environment changes.1,2 One vehicle that addresses these issues combines the biocompatibility and targeting dexterity of liposomes with the strength of a hydrogel—a network of polymer chains filled with water.
Researchers at the University of Maryland and the National Institute of Standards and Technology have developed a method to manufacture these hybrid vesicles, or rather, to help them self-assemble. The technique is called COMMAND: controlled microfluidic mixing and nanoparticle determination.
The process uses a microscopic fluidic device made by etching “channels” into a silicon wafer. Phospholipid molecules and cholesterol are dissolved in isopropyl alcohol. This solution is forced through the miniscule center channel (21 micrometers, approximately three times the size of a yeast cell). On either side of this channel are two channels in which a water-based solution containing hydrogel particles flows. The water-based solution “focuses” the central stream, housing the lipids as they all flow into a “mixer” channel. At the point of intersection, the lipids encapsulate the hydrogen particles, forming hybrid vesicles.3,4
The size of the vesicles can be controlled by varying the volumetric flow rate ratio between the central stream and the focusing streams. If the lipid-alcohol stream is tightly focused and slow flowing, small vesicles form. A fast flowing, highly focused stream produces larger vesicles. The dimensions of the microfluidic device and the geometry of the channels also affect vesicle size.
Researchers believe that this process, which facilitates mass production of uniform vesicles, will pave the way for the use of liposome-hydrogel hybrid nanoparticles in a wide variety of future clinical applications.
- Mufamadi MS, Pillay V, Choonara YE, et al. A review on composite liposomal technologies for specialized drug delivery. J Drug Deliv. 2011;2011:939851.
- National Institute of Standards and Technology. Liposome-hydrogel hybrids: no toil, no trouble for stronger bubbles. NIST website. June 9, 2010. Available at: www.nist.gov/pml/div683/bubbles_060910.cfm. Accessed Jan. 24, 2012.
- Hong JS, Stavis SM, DePaoli Lacerda SH, Locascio LE, Raghavan SR, Gaitan M. Microfluidic directed self-assembly of liposome-hydrogel hybrid nanoparticles. Langmuir. 2010;26(13):11581-11588.
- National Institute of Standards and Technology. NIST, Maryland researchers COMMAND a better class of liposomes. NIST website. April 27, 2010. Available at: www.nist.gov/pml/div683/command_042710.cfm. Accessed Jan. 24, 2012.
The Cancer ChallengeThe use of conventional chemotherapy drugs is often limited by their inability to target diseased tissue and the severe toxic effects on healthy organs and tissues that result. The “holy grail” is, therefore, a drug that targets tumors exclusively. Stealth liposomes have made great strides in this direction.
PEG-coated stealth liposomes have a passive targeting effect not yet fully understood. They collect in the interstitial spaces in the tumor cells, but in order for the drug to be effective, it must be released from the liposome into the tumor extracellular fluid and then diffuse into the tumor cells. Current research has concentrated on achieving this action with the use of pH-sensitive liposomes. These vesicles are designed to release their drug payload in an acidic environment. In general, tumor tissue has a lower pH than healthy tissue, triggering drug release.
Among the arenas in which PEG-coated liposomes have achieved clinical success is the treatment of Kaposi’s sarcoma and recurrent ovarian cancer with PEGylated liposomal doxorubicin (PLD). Taking the drug in this format, patients experience significantly less myelosuppression, alopecia, and nausea compared with an equally effective dose of conventional doxorubicin.
Another new technology to increase the accumulation of liposomes at the target site is immunoliposomes. Immunoglobulins, especially those of the IgG class, are attached to the surface of the liposomes. They act as ligands—molecules that connect to a site on a receptor protein—capable of recognizing and binding to tissue at sites of interest. However, most immunoliposomes are still eliminated by the liver before they can deliver significant results. One way to meet this challenge is to use stealth liposome technology: Coat the immunoliposomes with PEG to create long-circulating liposomes. Currently, one immunoliposome formulation is in clinical trials, a PEGylated DXR formulated to recognize gastric, colon, and breast cancer cells.
Triggering ReleaseRecent developments in liposomal drug delivery include stimuli-type liposomes. This technology employs various environmental factors to trigger drug delivery. The stimuli can include light, temperature, magnetism, and ultrasound waves. Once exposed to the proper stimulus, the liposome delivers its drug to the cytoplasm of the targeted cell through cell membrane pores.
An example of this technology is superparamagnetic iron oxide particles embedded in the shells of liposomes, making the shell leaky when exposed to an alternating current electromagnetic field. The drug is then released through the shell into the site of action. The rate of release can be controlled by altering the magnetic field strength and adjusting how the nanoparticles are loaded.5
Nanoparticles made of gold can deliver efficient, targeted delivery of multiple drug compounds. When exposed to infrared light, gold nanoparticles melt and release their payload. Different shaped nanoparticles melt at different wavelengths. Therefore, to release multiple compounds, each drug can be encased in a unique nanoparticle shape. As the wavelength of the external light source varies, each drug is released into the patient’s system.6
Polymer VesiclesOver the years, the terms “liposome” and “vesicle” have been used almost interchangeably. However, vesicle formation occurs in many materials other than conventional lipids.7
Vesicles made of polymers offer exciting possibilities for drug delivery. Polymer bilayers are thicker than lipids, so permeation is slower. Also, the release rate can be tailored by altering the thickness of the bilayers during preparation. Additionally, by using polymers that respond to various environmental stimuli, including pH and temperature, drug release can be further controlled.
- Torchilin VP. Lipid-based parenteral drug delivery systems: biological implications. In: Wasan KM, ed. Role of Lipid Excipients in Modifying Oral and Parenteral Drug Delivery: Basic Principles and Biological Examples. Hoboken, N.J.: John Wiley & Sons; 2007:48-87.
- Immordino ML, Dosio F, Cattel L. Stealth liposomes: review of the basic science, rarionale, and clinical applications, existing and potential. Int J Nanomedicine. 2006:1(3):297-315.
- Mufamadi MS, Pillay V, Choonara YE, et al. A review on composite liposomal technologies for specialized drug delivery. J Drug Deliv. 2011;2011:939851.
- Hong MS, Lim SJ, Oh YK, Kim CK. pH-sensitive, serum-stable and long-circulating liposomes as a new drug delivery system. J Pharm Pharmacol. 2002;54(1):51-58.
- Chin Y, Bose A, Bothun GD. Controlled release from bilayer-decorated magnetoliposomes via electromagnetic heating. ACS Nano. 2010;4(6):3215-3221.
- Trafton A. Gold particles for controlled drug delivery. Massachusetts Institute of Technology website. Dec. 30, 2008. Available at: http://web.mit.edu/newsoffice/2008/nanorods-1230.html. Accessed Jan. 24, 2012.
- Antonietti M, Förster S. Vesicles and liposomes: a self-assembly principle beyond lipids. Adv Mater. 2003;15(16):1323-1333.
- Drummond DC, Noble CO, Hayes ME, Park JW, Kirpotin DB. Pharmacokinetics and in vivo drug release rates in liposomal nanocarrier development. J Pharm Sci. 2008;97(11):4696-4740.
- Kan P, Tsao CW, Wang AJ, Su WC, Liang HF. A liposomal formulation able to incorporate a high content of Paclitaxel and exert promising anticancer effect. J Drug Deliv. Available at: www.hindawi.com/journals/jdd/2011/629234/ref. Accessed Jan. 24, 2012.
- Jesorka A, Orwar O. Liposomes: technologies and analytical applications. Annu Rev Anal Chem. 2008;1:801-832.
Platforms prove elusive, but research persistsWhat the pharmaceutical industry needs is a plug-and-play nanotechnology plat- form, a suite of constituent parts and instructions that can facilitate the realization of new oral drug formulations. Just don’t hold your breath waiting for it.
“Even if all parts remain the same, the API may affect how the whole process works,” warned Ravi Kumar, PhD, professor of drug delivery, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, U.K.
Dr. Kumar spoke from knowledge of recent efforts: The first attempt looked at nanoparticle formulation of the immunosuppressant drug cyclosporine A, with a multi-block copolymer of lactic acid and ethylene glycol; the second used a similar approach with the chemotherapy agent paclitaxel and a charged surfactant.1,2 His most recent work was with amphotericin B (AMB), a polyene antibiotic administered intravenously to treat invasive aspergillosis, a life-threatening infection, and leishmaniasis, a condition caused by a parasite endemic to many developing countries. An oral formulation could increase bioavailability, potentially reducing the amount of drug needed and the cost due to ease of drug manufacture and administration.
In the most recent investigation, AMB was combined with poly(lactide-co-glycolode), dimethyl sulfoxide, and vitamin E TPGS, a non-ionic surfactant, to create nanoparticles of 113±20 nm in size with 70% entrapment efficiency at 30% AMB w/w of polymer.3
The resulting formulation was effective in murine models of aspergillosis; however, data for leishmaniasis was not as encouraging. Dr. Kumar suspects there are issues with the formulation’s release profile. “With leishmaniaisis, you would probably require a very fast release initially to combat the protozoa’s growth rate,” and to accommodate this would require a separate balancing act between drug loading and particle size—thus, not quite a platform.
For now, Dr. Kumar advises gaining a true understanding of the properties of the drug molecule itself first, and then studying what the drug is being used for. Once you have an idea about the delivery vehicle, “the simpler you can keep the formulation the better.” And always keep an eye on scale up—for instance, using TPGS facilitated the use of filters, rather than centrifugation, to harvest large quantities of nanoparticles.
Dr. Kumar also requested scientists working in the field to be more comprehensive in their reporting. “This is a key issue. Lots of publishing on this work doesn’t really describe how they are freeze drying these formulations.” Stating that the material was lyophilized is not enough; all players need to know the myriad tweaks that got you there.
Case Study: Scaling Down for Faster Pain Relief?A recent experiment performed by end Research in Bend, Ore., in collaboration Pfizer Pharmaceuticals, demonstrated the potential of drug/nanoparticle constructs for increasing the rate and extent of oral absorption of low solubility, high-permeability drugs—in this case, the anti-inflammatory agent celecoxib.1
Dr. Morgen and his team are creating drug/nanoparticles using high surface area dispersions of celecoxib, in combination with ethyl cellulose and either sodium caseinate or bile salt. This approach has previously been shown to be particularly well suited to rapid-release, rapid-onset applications.2
The architecture of the particle is such that the drug molecule is dispersed within the polymer matrix, with the stabilizing excipients located primarily on the exterior (see Figure 1).
“We used a variety of surface-stabilizing agents to help keep the particles from aggregating—for the in vivo experiments we used casein, which is a naturally occurring, charged polymer.”
Once prepared, Dr. Morgen’s nanoparticles proved in his in vivo study to have higher systemic exposure and faster attainment of peak plasma concentrations than commercial celecoxib capsules.
The technique, though successful, does not suggest a clear path forward. “The raw technical performance was pretty good, but we had a lot of difficulty trying to scale up just to do a small clinical study,” said Dr. Morgen. And he sees this problem in broader terms. “The particles could be broadly applicable, but there isn’t the developed infrastructure yet in the industry to handle the process development.” To perform large-scale trials, more emphasis must be placed on the commercial-scale manufacture of the small.
- Morgen M, Bloom C, Beyerinck R, et al. Polymeric nanoparticles for increased oral bioavailability and rapid absorption using celecoxib as a model of a low-solubility, high-permeability drug. Pharm Res. 2012;29(2):427-440.
- Yang W, Tam J, Miller DA, et al. High bioavailability from nebulized itraconazole nanoparticle dispersions with biocompatible stabilizers. Int J Pharm. 2008;361(1-2):177–188.
Sticky SituationUnlike most of those working with oral formulations, Kevin Pojasek, PhD, vice president of corporate development for Kala Pharmaceuticals in Waltham, Mass., isn’t looking to increase systemic bioavailability. He would rather the drug’s activity remain on site.
In a recent investigation, AMB was combined with poly(lactide-co-glycolode), dimethyl sulfoxide, and vitamin E TPGS, a non-ionic surfactant, to create nanoparticles of 113±20 nm in size with 70% entrapment efficiency at 30% AMB w/w of polymer.“At Kala, we’re looking at how can we better treat diseases of the GI tract through topical administration—essentially trapping the API at the site of infection or inflammation.” That goal first requires a thorough understanding of the mucosal lining of the GI tract, a knowledge foundation revealed and recently reviewed by Kala investigators.4 “We started with the engineering principles of mucus, the rheology,” said Dr. Pojasek, and then combined that with the observed properties of certain viruses that are able to penetrate a mucosal barrier. Taken together, these data guided the researchers to an effective GI-targeting nanoparticle design.
Leaving out proprietary details, proof-of-principle for this approach, described by Yang and colleagues, used Pluronic block copolymers to enable membrane translocation. Mindful of eventual marketing, the techniques evolving from this approach are only incorporating pre-FDA approved moieties, minimizing regulatory hurdles by skirting the definition of new chemical entities.5
Though still in early development, there is something resembling a platform in this work. “In essence, the secret sauce of what we do is engineering the coating on the outside of the particles,” said Dr. Pojasek. As for the core, “we can tailor the release kinetics to meet the challenge of whatever mucosal disease we’re trying to treat.”
- Ankola DD, Battisti A, Solaro R, Kumar MN. Nanoparticles made of multi-block copolymer of lactic acid and ethylene glycol containing periodic side-chain carboxyl groups for oral delivery of cyclosporine A. J R Soc Interface. 2010;7 Suppl 4:S475-S481.
- Bhardwaj V, Plumb JA, Cassidy J, Ravi Kumar MNV. Evaluating the potential of polymer nanoparticles for oral delivery of paclitaxel in drug-resistant cancer. Cancer Nanotechnol. 2010;1(1-6)29-34.
- Italia JL, Sharp A, Carter KC, Warn P, Kumar MN. Peroral amphotericin B polymer nanoparticles lead to comparable or superior in vivo antifungal activity to that of intravenous Ambisome® or Fungizone™. PLoS One. 2011;6(10):e25744.
- Ensign LM, Cone R, Hanes J. Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers [published online ahead of print Dec. 24, 2011]. Adv Drug Deliv Rev.
- Yang M, Lai SK, Wang YY, et al. Biodegradable nanoparticles composed entirely of safe materials that rapidly penetrate human mucus. Angew Chem Int Ed Engl. 2011;50(11):2597-2600.
- Batrakova EV, Kabanov AV. Pluronic block copolymers: evolution of drug delivery concept from inert nanocarriers to biological response modifiers. J Control Release. 2008;130(2):98-106.
- Cai Z, Wang Y, Zhu LJ, Liu ZQ. Nanocarriers: a general strategy for enhancement of oral bioavailability of poorly absorbed or pre-systemically metabolized drugs. Curr Drug Metab. 2010 Feb;11(2):197-207.
- Wawrezinieck A, Péan JM, Wüthrich P, Benoit JP. Oral bioavailability and drug/carrier particulate systems [in French]. Med Sci (Paris). 2008;24(6-7):659-664.
- Roger E, Lagarce F, Garcion E, Benoit JP. Biopharmaceutical parameters to consider in order to alter the fate of nanocarriers after oral delivery. Nanomedicine (Lond). 2010;5(2):287-306.
- Yamanaka YJ, Leong KW. Engineering strategies to enhance nanoparticle-mediated oral delivery. J Biomater Sci Polym Ed. 2008;19(12):1549-1570.
Reversible and noncovalent methods enhancing protein and peptide performancePEGylation—the covalent attachment of polyethylene glycol groups to proteins and peptides—is a strategy commonly used to improve the performance of these therapeutic molecules in vivo. However, successful drug development using PEGylation can pose a number of challenges, not least of which is that the process of attaching PEG covalently can alter the activity of the protein or peptide itself.
In response to these challenges, a number of novel variations on PEGylation have been developed in recent years, including reversible PEGylation and noncovalent PEGylation. There has also been the realization that there is “nothing magical” about PEG, in the words of one researcher, and that other biomolecules, such as starches, can be used to change pharmacokinetic qualities.
Challenges of FormulationWhile PEGylation can extend the in vivo circulatory half-life and medicinal effect of proteins or peptides, any such chemical modification can also alter these molecules’ physical properties. These changes introduce challenges in creating a drug, said Mark C. Manning, PhD, chief scientific officer of Legacy BioDesign, a contract formulation service in Johnstown, Colo.
At least eight PEGylated protein and peptide therapeutics have been developed and reached the market, beginning with Adagen in 1990.“If you take a protein and chemically modify it, whether it’s by PEGylation or something else, then you’ve introduced new chemistry, and that then affects the manufacturing, the approval process, and so on. It complicates the analytical methods you have to use, it changes the evaluation of [active pharmaceutical ingredient] purity, it affects formulation, and so on,” he said.
Dr. Manning and colleagues recently reviewed the challenges to drug development with PEGylated proteins.1 They noted that the chemistry of attaching PEG to the protein is the critical issue affecting product development. In addition, the quality of the starting materials and the coupling process must be carefully controlled, and these issues become exacerbated as the scale is increased and the chemistry must be performed under current good manufacturing practice (cGMP) conditions. Further, a suitable purification method must be selected for the potentially complex mixture of multiple isomers of the PEGylated compound. Once the purified drug substance is obtained, proper analytical methods must be chosen to characterize the material.
“These are all things that drug developers should think about as they embark along these lines,” Dr. Manning said. “We say this technology is well-established and can provide benefit, which is true, but it comes at a cost, a price in terms of the time required to characterize these materials.”
Despite these challenges, at least eight PEGylated protein and peptide therapeutics have been developed and reached the market, beginning with Adagen (pegademase bovine, Enzon) in 1990, and more than two dozen PEGylated peptides and proteins of interest have been described in the literature.1 A recently introduced PEGylated product, Cimzia (certolizumab pegol, UCB), is discussed in the case study.
Case Study: PEGylation Increases Half-Life of TNF BlockerDespite the challenges of using PEGylation to create useful therapeutic entities, at least eight PEGylated therapeutic products have reached the market, and many more molecules of interest have been described in the literature. One of the most recently approved PEGylated therapeutics is Cimzia (certolizumab pegol, UCB), a tumor necrosis factor blocker indicated for treatment of Crohn’s disease and rheumatoid arthritis.1
Certolizumab pegol is a recombinant, humanized antibody Fab’ fragment with specificity for human TNF-alpha, conjugated to an approximately 40 kDa polyethylene glycol. The Fab’ fragment is manufactured in E. coli and subsequently subjected to purification and conjugation to the PEG to generate certolizumab pegol. The molecular weight of certolizumab pegol is approximately 91 kDa.1
PEGylation is an essential factor in the formulation of certolizumab pegol because of the instability of the active molecule, said Mark C. Manning, PhD.
“Without PEGylation, the active ingredient would be cleared in minutes, in vivo. It’s a very unstable molecule,” he said. “Once it is PEGylated, it can reside in the body for hours.”
Even though many PEGylated proteins exhibit compromised efficacy—a possible side effect of PEGylation—this could be an acceptable tradeoff in return for longer in vivo stability, Dr. Manning said.
“Even if you compromise the intrinsic activity a bit, you’re willing to pay that price because the overall effectiveness for the patient is going to increase significantly,” he said. —TD
- UCB, Inc. Cimzia [package insert]. Available at: http://cimzia.com/?v=GOOG&WT.srch=1&gclid=COurlfa35a4CFWYJRQodISNRvQ. Accessed March 13, 2012.
Reversible PEGylationOne of the potential drawbacks of protein or peptide PEGylation is that the attachment of the PEG polymer may block the active sites of these therapeutic molecules. Linking a PEG or another polymer to protein drugs often yields derivatives that are largely or completely devoid of biologic potency, and therefore pharmacologically ineffective, said Matityahu Fridkin, PhD, the Lester B. Pearson Professorial Chair of Protein Research in the department of organic chemistry at the Weizmann Institute of Science in Rehovot, Israel.
“Classical PEGylation of peptides and proteins often leads to prevention of binding and activation of certain specific receptors, as for instance is known to occur with interferon,” Dr. Fridkin said.
In order to overcome this phenomenon, Dr. Fridkin and colleagues have used a process called reversible PEGylation, in which chain-like spacers are used to link PEG to the protein drug. This attachment turns the short-acting protein or peptide drug into a long-acting prodrug, which maintains circulating levels for extended periods after administration. The reversible chemical bonds dissolve slowly under physiologic conditions, releasing the active drug slowly over time, Dr. Fridkin and colleagues have shown.
“Reversible PEGylation leads to the slow release of the intact parent drug with full bioactivity,” Dr. Fridkin said.
He noted that the group’s work with insulin has demonstrated the potential advantages of reversible PEGylation. In a 2008 publication, the researchers engineered a long-acting prodrug of insulin that released biologically active insulin with a half-life of 30 hours under physiologic conditions.2 By contrast, conventional PEGylation of insulin led to inactivation of the hormone.
In addition to PEG, other molecules can be used to improve the PK of proteins and peptides in vivo. A number of biomolecules are being investigated and used in this way, including polyglycine and, increasingly, several types of starches.The preparation of large-scale amounts of reversibly PEGylated conjugates remains beyond the researchers’ current capability, Dr. Fridkin said. “This will be the major direction in our near future research,” he said. “We are attempting to simplify the current synthetic methodology to achieve this goal.”
Noncovalent PEGylationAnother approach to obtaining stable biologically active compounds in vivo is to use noncovalent bonds to attach PEG to proteins and peptides.
Researchers at the University of Kansas, led by Cory J. Berkland, PhD, recently used noncovalent PEGylation by polyanion complexation to improve the in vivo stability of keratinocyte growth factor-2.3
“Normally, we use a covalent chemical bond to conjugate the PEG onto the protein, and that can affect the activity of the protein. We thought that if we can use a noncovalent bond to put the PEG on the protein, when the formulation goes into the body the protein can be released from the PEG quite easily, and that would not interfere with the protein activity. That was the starting point of our work,” said Supang Khondee, PhD, first author of the paper on noncovalent PEGylation of KGF-2.3 Now a research fellow in internal medicine at the University of Michigan Medical School, she was a graduate student at the University of Kansas under Dr. Berkland at the time of this research.
Using the polyanions pentosan polysulfate and dextran sulfate, the researchers attached PEG noncovalently to KGF-2, a heparin-binding protein with regenerative properties. This increased the melting temperature and improved the stability of the compound. The researchers suggested that this approach can be used with other heparin-binding proteins.
Related TechnologiesIn addition to PEG, other molecules can be used to improve the PK of proteins and peptides in vivo, Dr. Manning noted.
“There’s nothing magical about PEG,” he said. “In general, if you attach another polymer to a protein, you will increase its circulating half-life. So we’re seeing a lot of people exploring whether other biomolecules that are safe can chemically attach to proteins and therefore change their PK properties.”
A number of biomolecules are being investigated and used in this way, including polyglycine and, increasingly, several types of starches, Dr. Manning said.
The starch derivative hydroxyethyl starch has been used in this manner in a proprietary process called HESylation. HESylation allows targeted modification of drugs and their characteristics by site-specific coupling to HES molecules, according to Boehringer Ingelheim, of Ingelheim, Germany.4
In a collaboration between Fresenius Kabi, of Bad Homburg, Germany, and Boehringer Ingelheim, HES was coupled to a therapeutic protein, and the resulting HESylated pharmaceutical was produced at industrial scale with quality and yield comparable to product made in the laboratory, a press release from Boehringer Ingelheim stated. The press release did not name the compound, but it stated that the two companies will continue to pursue their collaboration.
- Payne RW, Murphy BM, Manning MC. Product development issues for PEGylated proteins. Pharm Dev Technol. 2011;16(5):423-440.
- Shechter Y, Mironchik M, Rubinraut S, et al. Reversible pegylation of insulin facilitates its prolonged action in vivo. Eur J Pharm Biopharm. 2008;70(1):19-28.
- Khondee S, Olsen CM, Zeng Y, Middaugh CR, Berkland C. Noncovalent PEGylation by polyanion complexation as a means to stabilize keratinocyte growth factor-2 (KGF-2). Biomacromolecules. 2011;12(11):3880-3894.
- Boehringer Ingelheim. Boehringer Ingelheim RCV and Fresenius Kabi successfully coupled HES to a therapeutic protein in an industrial scale applying Fresenius Kabi’s HESylation Technology [press release]. Boehringer Ingelheim website. November 11, 2010. Available at: www.boehringer-ingelheim.com/news/news_releases/press_releases/2010/11_november_2010fresenius.html. Accessed March 13, 2012.
- Gokarn YR, McLean M, Laue TM. Effect of PEGylation on protein hydrodynamics [published online ahead of print Feb. 21, 2012]. Mol Pharm.
- Vasudev SS, Ahmad S, Parveen R, et al. Formulation of PEG-ylated L-asparaginase loaded poly (lactide-co-glycolide) nanoparticles: influence of pegylation on enzyme loading, activity and in vitro release. Pharmazie. 2011;66(12):956-960.
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