Wednesday, April 6, 2011

ANALYTICAL INSTRUMENTATION: Competitiveness of Bioanalytical Laboratories— Technical and Regulatory Perspectives

The discovery and development of a new drug costs around $1 billion, and it may take approximately 10 years for the drug to reach the marketplace. Drug discovery and development are the processes of generating compounds and evaluating all of their properties to determine the feasibility of selecting one new chemical entity (NCE) to become a safe and efficacious drug.
Among many criteria, obtaining experimental pharmacokinetics (PK) data from laboratory animals in the nonclinical stage is critical to evaluating a drug candidate before it can qualify to be tested in the clinical trials for safety and efficacy evaluation.
A key parameter in pharmacokinetics is the plasma or tissue concentration of the new drug after its administration to laboratory animals. Therefore, developing an accurate and fast analytical method for measuring the concentrations of a compound in plasma or tissue is the first step toward yielding the PK of a compound. As the drug candidate moves down in the pipeline, the requirements for PK information differ at the different stages of drug discovery and development, leading to the introduction of “fit for purpose” analytical strategies that provide the appropriate level of bioanalytical support for the purpose at hand, while simultaneously minimizing resource expenditures.
Due to its superior sensitivity and selectivity, liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) has become the analytical technique primarily used by bioanalytical laboratories performing analyses for preclinical and clinical studies. The increased number of biological agents used as therapeutics—in the form of recombinant proteins, monoclonal antibodies, vaccines, and so on—has prompted the pharmaceutical industry to review and refine aspects of the development and validation of bioanalytical methods for the quantification of these therapeutics in biological matrices in support of preclinical and clinical studies. Most of these methodologies are used in quantitative assays supporting PK and toxicokinetic parameters of the therapeutic agents. To date, an alternative approach for dealing with macromolecules is to utilize ligand-binding assays (LBAs) to generate data.
LC–MS/MS has played an incredible role in drug metabolism and pharmacokinetics studies at various drug discovery and development stages since its introduction to the pharmaceutical and biotechnology industries. This section will elaborate on the most recent advances in sample preparation and separation, along with the mass spectrometric aspects of high-throughput quantitative bioanalysis of drugs and metabolites in biological matrices. Recently introduced techniques such as ultra-performance or ultra-speed liquid chromatography (UPLC or USLC), with small particles (sub-2 mm) and monolithic chromatography, offer improvements in speed, resolution, and sensitivity compared to conventional chromatographic techniques. Hydrophilic interaction chromatography (HILIC) on silica columns with low aqueous/high organic mobile phase is emerging as a valuable supplement to the reversed-phase LC–MS/MS.
Sample preparation formatted to 96-well and/or 384-well plates has allowed for semi-automation of off-line sample preparation techniques, significantly improving throughput. On-line solid phase extraction (SPE) utilizing column-switching techniques is rapidly gaining acceptance in bioanalytical applications to reduce both the time and labor required for generating bioanalytical results. Extraction sorbents for on-line SPE extend to an array of media, including large particles for turbulent-flow chromatography, restricted access materials, monolithic materials, and disposable cartridges utilizing traditional packings such as those used in Spark Holland (Symbiosis) systems. In the latter part of this section, recent studies of matrix effect in LC–MS/MS analysis and ways to reduce/eliminate matrix effect in method development and validation will be also discussed.
Bioanalytical laboratories in the pharmaceutical and life science industries are constantly under pressure to reduce overall time for discovery and development. This pressure is often accompanied by an increase in the number of biological samples requiring PK analysis and a decrease in the desired quantitation levels. Hyphenated techniques are examples of new tools adopted for developing fast and cost-effective analytical methods. LC–MS/MS has led to major breakthroughs in the field of quantitative bioanalysis since the 1990s due to its inherent specificity, sensitivity, and speed.1-2 It is now generally accepted as the preferred technique for quantitating small-molecule drugs, metabolites, and other xenobiotic biomolecules in biological matrices. The use of LC–MS/MS has grown exponentially in the last decade due to its unmatched sensitivity, extraordinary selectivity, and rapid rate of analysis. Typical workflow of bioanalysis from sample generation to data delivery is summarized in Figure 6.1.
Figure 6.1: Typical workflow of bioanalysis from sample generation to data delivery
Figure 6.1: Typical workflow of bioanalysis from sample generation to data delivery
Samples from biological matrices are usually not directly compatible with LC–MS/MS methodologies. Sample preparation has traditionally used such approaches as protein precipitation (PPT), liquid–liquid extraction (LLE), or SPE. Manual operations associated with these processes—in particular with LLE and SPE—are fairly labor intensive and time consuming. Parallel sample processing in a 96-well format using robotic liquid handlers has significantly shortened the time analysts have to spend in the laboratory for sample preparation. An alternative sample extraction method that has generated a lot of interest in recent years is the direct injection of plasma using an on-line extraction method. A major advantage of on-line SPE over off-line extraction techniques is that the sample preparation step is embedded into the chromatographic separation and thus eliminates most of the sample preparation time traditionally performed at the bench, potentially resulting in more variability from batch to batch and analyst to analyst. Steep gradients and relatively short columns were first utilized in early applications of high-throughput LC–MS/ MS assays to reduce run times.
A better understanding of how matrix effects can compromise the integrity of bioanalytical methods has re-emphasized the need for adequate chromatographic separation of analytes from endogenous biological components in quantitative bioanalysis using the LC–MS/MS technique. New developments from chromatographic techniques such as UPLC and USLC with sub-2 mm particles and monolithic chromatography show promise in delivering higher speed, better resolution, and increased sensitivity for high-throughput analysis while minimizing matrix effects. LC–MS/MS applications for quantitative bioanalysis have been documented in hundreds of articles in just the past several years, and a number of reviews dealing with one or more aspects of quantitative LC–MS/MS bioanalysis have been published.3-6 Within this section, publications related to technology development for throughput improvement, associated applications, and discussions of key developments in quantitative analysis from 2002 to 2009 will be reviewed and elaborated upon.

Sample Generation, Shipment, and Storage

Figure 6.2: Hamilton STAR
Figure 6.2: Hamilton STAR
From a bioanalytical standpoint, prior to sample analysis, it is important to maintain the quality and integrity of samples to be generated from discovery in nonclinical and clinical programs regardless of whether the studies are nonregulated or regulated. Laboratory management regarding sample handling/custody is key to the success of a bioanalytical operation. These processes generally include but are not limited to:
  • Sample receipt;
  • Login (creating unique identification numbers or ID);
  • Storage (adequate and acceptable conditions);
  • Transfers/relocations from lab to lab and facility to facility; and
  • Sample retention and/or disposal.
Zhang and colleagues described an integrated sample collection and handling process for bioanalysis.7 The sample handling process consumes a significant portion of the resources of a bioanalytical laboratory. One of the primary purposes for analytical automation is the possibility of analyzing a huge number of samples, either simultaneously or sequentially, without any human intervention. When large amounts of samples are analyzed, mainly in routine analyses, the importance of the correct identification of each sample in order to obtain the appropriate correlation between the sample analyzed and the results obtained is obvious.
One of the most efficient and reliable ways to identify a sample, from collection to result, is by using bar codes. A bar code can best be defined as an “optical Morse code.” Series of black bars and white spaces of varying widths are printed on labels to identify items uniquely. The bar code labels are read with a scanner, which measures reflected light and interprets the code into numbers and letters that are passed on to a computer. All bar codes have start/stop characters that allow the bar code to be read from both left to right and right to left.
Unique characters placed at both the beginning and end of each bar code, the stop/start characters provide timing references, symbol identification, and direction of reading information to the scanner. By convention, the unique character on the left of the bar code is considered the “start,” while the character on the right of the bar code is considered the “stop.” Besides the linear bar code, today’s new bar codes are two- dimensional, electronic, or small computer chips, which sort data in inconspicuous places like a credit card. This technology has been included in clinical and bioanalytical analyzers, where large amounts of samples are analyzed daily and a correct assignation of the results obtained is mandatory.
Automation for sample handling meets the business needs of improving productivity and reducing the documentation required for compliance. Procedural elements involved in maintaining bioanalytical data integrity for good laboratory practices and regulated studies are discussed by James and Hill.8 The elements can be divided into three areas.
First, there are elements ensuring the integrity of the analyte until analysis, through correct sample collection, handling, shipment, and storage procedures. Incorrect procedures can lead to loss of analyte due toinstability, addition of analyte through contamination or instability of related metabolites, or changes in the matrix composition that may adversely affect the performance of the analytical method.
Second, the integrity of the sample identity must be maintained to ensure that the result that is reported relates to the individual sample that was taken. Possible sources of error include sample mix-up or mislabeling or errors in data handling.
Finally, there is the overall integrity of the documentation that supports the analysis, along with any pre-study validation of the method. This includes a wide range of information, from paper and electronic raw data through standard operating procedures, analytical procedures, and facility records to study plans and final reports. These are critical, allowing an auditor or regulatory body to reconstruct the study. A laboratory information management system (LIMS) is a preferable database and handling system that maintains a detailed pedigree for each sample by capturing processing parameters, protocols, stocks, tests, and analytical results for the sample’s complete life cycle. Project and study data are also maintained to define each sample in the context of the research tasks it supports. LIMS is required for each analytical pipeline to track all aspects of sample handling.

Sample Preparation

One strategy for high-throughput bioanalytical analysis is to use well-established instrumentation; rigorous, standardized techniques; and automation wherever possible to minimize or replace manual tasks. Automation results in greater performance consistency over time and more reliable methods transfer from site to site. Automated 96-well plate technology is well established and accepted and has been shown to effectively replace manual operations. The 96-well instruments can execute automated off-line extraction and sample cleanups. Automated SPE, LLE, and PPT all can be performed in 96-well format.
Adequate sample preparation is a key aspect of quantitative bioanalysis; without it, bottlenecks can occur during high-throughput analysis. Sample preparation techniques in 96-well format have been well adopted in high-throughput quantitative bioanalysis with a number of applications. Techniques that can use the format include LLE, SPE, and PPT.
Typically, liquid transfer steps, including preparation of calibration standards and quality control samples as well as the addition of the internal standard, have been performed automatically using robotic liquid handling workstations for parallel sample processing (Hamilton STAR, refer to robot liquid handler system in Figure 6.2). The increasing demand for high throughput causes a unique situation of balancing cost versus analysis speed as each sample preparation technique offers unique advantages. Dilute-and-shoot and PPT are among the most popular and effective techniques due to their simplicity.
Sample preparation with PPT is widely used in the bioanalysis of plasma samples. The method has been extended to the quantitation of drug and metabolites from whole blood. Koseki and colleagues developed a sensitive and specific LC–MS/MS method for the simultaneous determination of cyclosporine A (CsA) and its three main metabolites (AM1, AM4N, and AM9) in human blood.9
Mixed-mode polymer-based sorbents (e.g., Waters Oasis MCX cartridge) were introduced in the late 1990’s for the isolation of drugs with ionizable functional groups from biological fluids. The extraction procedures can be a generic protocol or can be optimized if better sample cleanup is desired. The use of SPE often gives superior results to those with a PPT approach but may not be as cost effective as PPT due to the labor and material costs associated with the procedures. Mallet and colleagues described a novel 96-well SPE plate that was designed to minimize the elution volume required for quantitative elution of analytes.10
The plate was packed with 2 mg of a high-capacity SPE sorbent that allows loading of up to 750 mL of plasma. The novel design permitted elution with as little as 25 mL solvent, enabling the plate to offer up to a 30-fold increase in sample concentration. The evaporation and reconstitution step that is typically required in SPE is unnecessary due to the concentrating ability of the sorbent. Yang and colleagues developed a sensitive mElution SPE–LC–MS/MS method for the determination of M+4 stable isotope labeled cortisone and cortisol in human plasma.11 In the method, analytes were extracted from 0.3 mL of human plasma samples using a Waters Oasis HLB 96-well mElution SPE plate with 70 mL methanol as the elution solvent. The lower limit of quantitation was 0.1 ng/mL and the linear calibration range was from 0.1 to 100 ng/mL for both analytes. Several related formats have recently appeared using miniaturized packed-bed formats as well as an adaptation of disk technology for the 96-well format.
Figure 6.3: Spark Holland Symbiosis system
Figure 6.3: Spark Holland Symbiosis system
The ultimate goal for the 96-well approach is to add a higher level of parallel sample preparation using essentially a further miniaturized SPE format. By extracting multiple 96-well format SPE plates, up to four 96-well plates or 384 samples may be prepared and then analyzed within 24 hours by one person with one LC–MS/MS system, a significant improvement in sample throughput using commercially available equipment and supplies. Future developments should further increase throughput significantly.
LLE gives excellent sample cleanup but poses engineering challenges for use in an automated high-throughput fashion. Several groups have developed different approaches to solve the mixing and phase separation problems typically observed in a 96-well LLE method. By using vigorous vortexing after well-controlled heat-sealing, or repeated aspiration and dispensing by robotic liquid handler, common extraction solvents such as methyl-t-butyl ether (MTBE) or ethyl acetate (EA) may be used in the routine extraction of plasma, blood, or tissue samples.
Another approach is on-line SPE for the automated preparation of samples prior to LC–MS/MS analysis. This approach uses a commercial device that combines an auto-sampler and a solvent delivery unit to aliquot multiple liquid samples into a flowing stream of solvent. The solvent has preconditioned an in-line SPE cartridge. After conditioning, the SPE cartridge retains the targeted analytes, while the relatively weak solvent elutes unretained salts and polar matrix components to waste. An empirically optimized sequence of increasingly stronger solvents is then used to further elute weakly retained unwanted sample components. A final elution with high performance liquid chromatography (HPLC) mobile phase elutes the targeted analytes off the SPE cartridge and onto an analytical HPLC column for LC–MS/MS analysis. The on-line SPE technique offers speed, high sensitivity by the preconcentration factor, and low extraction cost per sample, but typically requires the use of program-controlled switch valves and column reconfigurations. The on-line technique can be fully automated, however, offering real-time high-throughput sample analysis.
One commercial automated on-line SPE system is the Symbiosis system, manufactured by Spark Holland (Figure 6.3). It includes an auto-sampler (Reliance), two binary HPLC pumps, an on-line SPE unit with two high-pressure solvent delivery pumps, and a combined valve system to direct fluid for different steps of SPE. At the beginning of each run, an on-line SPE cartridge is loaded into the unit. After a conditioning step with high organic solvent and an equilibrium step with low organic aqueous solution, a sample is injected onto the cartridge and washed with aqueous solution. Proteins and other matrix materials from the sample are removed during the washing step. Analyte of interest is then eluted onto the analytical column and detected by mass spectrometry or tandem mass spectrometries. During the sample elution step, a second sample is loaded to a new on-line SPE cartridge for the next analysis. In this parallel mode, the sample analysis cycle time approximates the LC run time without the time required for the SPE procedures. Because the on-line SPE cartridge is disposable and each sample uses a new cartridge, the carry-over problem from the extraction cartridge is eliminated.


  1. Bakhtiar R, Ramos L, Tse FLS. High-throughput mass spectrometric analysis of xenobiotics in biological fluids. J Liq Chromatrogr Related Technol. 2002;25(4):507–540.
  2. Zhou S, Song Q, Tang Y, et al. Critical review of development, validation, and transfer for high throughput bioanalytical LC-MS/MS methods. Curr Pharm Anal. 2005;1(1):3–14.
  3. Ackermann BL, Berna MJ, Murphy AT. Recent advances in use of LC/MS/MS for quantitative high-throughput bioanalytical support of drug delivery. Curr Top Med Chem. 2002;2(1):53–66.
  4. Jemal M, Xia YQ. LC-MS development strategies for quantitative bioanalysis. Curr Drug Metab. 2006;7(5):491–502.
  5. Naidong W. Bioanalytical liquid chromatography tandem mass spectrometry methods on underivatized silica columns with aqueous /organic mobile phases. J Chromatogr BAnalyt Technol Biomed Life Sci. 2003;796(2):209–224.
  6. Hsieh Y, Korfmacher WA. Increasing speed and throughput when using HPLC-MS/MS systems for drug metabolism and pharmacokinetic screening. Curr Drug Metab. 2006;7(5):479–489.
  7. Zhang NY, Rogers K, Gajda K, et al. Integrated sample collection and handling for drug discovery bioanalysis. J Pharm Biomed Anal. 2000;23(2-3):551–560.
  8. James CA, Hill HM. Procedural elements involved in maintaining bioanalytical data integrity for good

CONTROLLED RELEASE: Challenges and New Technologies of Oral Controlled Release

Significant advances have been attained in developing and commercializing oral controlled release products. Many platforms are available for delivering small molecule drugs with good aqueous solubility in prolonged or delayed release forms.
However, there are significant challenges in developing controlled release formulations for drugs with poor aqueous solubility, which require both solubilization and engineering of release profile. To deliver drugs at zero-order release rate, preferably independent of the gastrointestinal (GI) tract environment, many efforts and achievements have been made besides osmotic pump drug delivery systems.
Moreover, many of the new therapeutics under development are large molecules like peptides, proteins, oligonucleotides, and vaccines. Their physical, chemical, and biopharmaceutical attributes, distinct from small molecule drugs, demand novel controlled-release technologies to diminish barriers for oral delivery like instability in the GI tract and poor absorption. Those unmet technology needs create great opportunities for research, development, and innovation. Breakthroughs in controlled oral delivery for water-insoluble drugs and biopharmaceuticals are likely to have a significant impact on the pharmaceutical and biotechnology industries.
On the other hand, the continuous improvement of current delivery technologies is also important when it comes to decreasing cost and increasing efficiency. Those advancements include novel excipients, processes, and equipment as new tools formulation scientists can use to develop oral controlled-release formulations.

Oral Controlled Delivery for Water-Insoluble Drugs

Table 16.1
click for larger version
With few exceptions, for small molecule drugs, drug products have to be dissolved in GI fluids in order to be absorbed. For a water-insoluble drug, the absorption and bioavailability could be restricted by dissolution rate and solubility in the GI tract. There are many established approaches to formulating water-insoluble drugs as oral dosage forms.
Strategies include salt formation, microenvironmental pH control, solubilization by surfactants, complexation with cyclodextrins, solid dispersion, lipid-based formulation, and nanoparticles formulation (Table 16.1). A strategy is chosen based on the molecular and physical properties of a drug. For a water-insoluble drug, substantial formulation and process development are necessary to formulate a drug product with enhanced bioavailability.
Developing a controlled-release formulation for a water-insoluble drug is very challenging. A controlled oral delivery may be needed to achieve prolonged exposure or time-based release under certain circumstances. This approach could improve efficacy, reduce side effects, or achieve a more desirable dose regimen. However, many platforms for controlled release have been established for drugs with acceptable aqueous solubility. (These platforms have been thoroughly reviewed in previous chapters.) The release rate of a soluble drug in a solid dosage form is slowed down in a certain mode to achieve controlled release. It is clear that direct plug-in of current matrix-based or coating-based delivery systems without technology fabrication will fail to achieve acceptable controlled release of a water-insoluble drug. On the other hand, in vitro dissolution methods based on a sink condition generated by surfactants may provide misleading correlation for in vivo behaviors.
A combination of solubilization and release modulation is needed to achieve controlled release for a water insoluble drug. If a drug can be solubilized by a surfactant or a complex agent, inclusion of a solubilizing agent in polymer-based matrix tablets may provide a solution. Rao and colleagues studied the matrix tablet formulation of prednisolone, a sparingly water-soluble drug, using sulfobutylether-b-cyclodextrin (SBEBCD) as a solubilizing agent. SBEBCD promotes a sustained and complete release in a hydroxypropyl methylcellulose-based tablet formulation.1 Another study also demonstrated that SBEBCD worked as a solubilizing agent and an osmotic agent for controlled porosity osmotic pump pellets of prednisolone.2 A complete and sustained release of prednisolone has been observed.
It is clear that direct plug-in of current matrix-based or coating-based delivery systems without technology fabrication will fail to achieve acceptable controlled release of a water-insoluble drug.
It has been reported that controlled release felodipine tablets have been effectively prepared using Poloxamer as a solubilizing agent and Carbopol as a controlled release matrix.3 Many drugs need more complicated formulation approaches to enhance the dissolution, such as amorphous solid dispersion, emulsion, microemulsion, self-emulsifying, and nanoparticles. Among these, amorphous solid dispersion is the most popular approach to enhancing solubility and dissolution.
There are numerous publications about the development of controlled release formulations of water-insoluble compounds using solid dispersion as a solubilization approach. For a solid dispersion, drug molecules are stabilized in a high-energy state with hydrophilic polymers such as polyethylene glycol, polyvinyl povidone, and polyvinyl alcohol. Solid dispersions could be prepared by spray drying or melting extrusion. Mehramizi reported that an osmotic pump tablet of glipizide has been developed using glipizide/polyvinylpyrrolidone dispersion as the core, where the solid dispersion enhanced the solubility and ensured the complete release.4
Figure 16.1: Proposed mechanism of drug release from DCMT
Figure 16.1: Proposed mechanism of drug release from DCMT.6
Hong and Oh studied the dissolution kinetics and physical characterization of three-layered tablets of nifedipine solid dispersion with poly(ethylene oxide) matrix capped by Carbopol.5 They discovered that the swelling and morphological change of Carbopol layers minimized the release of rapidly erodible PEO200K (MW 200,000) and changed the nifedipine release to a diffusion-controlled process. However, the physical stability of solid dispersions must be monitored for polymer-based matrix systems, membrane coatings, or osmotic systems during prolonged release. All three approaches need water to diffuse inside formulation to solubilize drugs and advance the release. Drugs may crystallize out during the prolonged exposure to water due to supersaturation inside dosage form or change of glass transition temperature because of interaction with water.
Nanoparticle formulation can be used to formulate poorly soluble drugs to enhance bioavailability. The drug dissolution rate is increased due to the increase of surface area. However, little literature exists regarding the controlled release of poorly soluble drugs with nanoparticles as carriers. It is thought that an erosion-based system may be more suitable because drug solubility is not changed. Diffusion-controlled matrix or membrane coating system is challenging to achieve the goal.

New Designs for Desired Release Profiles

Many formulation designs have been pursued to achieve controlled release and minimize the impact of the GI environment. Osmotic pump drug delivery systems pioneered by Alza have many proven successes in those two areas, as covered in previous chapters. The disintegration-controlled matrix tablet (DCMT) and erodible molded multilayer tablet by Egalet take an erosion approach and show some promise. On the other hand, bioadhesive polymers offer advantages in improving gastroretentive delivery and enhancing localized therapy in the GI tract. Moreover, significant progress has been made in the use of computer modeling to design controlled-release formulations.
Disintegration-Controlled Matrix Tablet: The DCMT is an erosion-based controlled-release platform. It was developed for the sustained release of solid dispersions by Tanaka and colleagues.6-7 DCMTs contain hydrogenated soybean oil as the wax matrix, with solid dispersion granules uniformly distributed in the wax. The solid dispersion granules are formulated with low-substituted hydroxypropylcellulose as a disintegrant.
Figure 16.2: Plasma concentration profiles of nilvadipine after oral administration of DCMTs to beagle dogs under fasting condition.
Figure 16.2: Plasma concentration profiles of nilvadipine after oral administration of DCMTs to beagle dogs under fasting condition.
Drug release is controlled by the process of tablet erosion. The wax only allows the penetration of water to the surface layer of the tablet, water triggers the swelling of the disintegrant on the surface, and, subsequently, tablet erosion results in the separation of solid dispersion granules from the tablet. A constant rate of tablet disintegration/erosion can be achieved by repeating the processes of water penetration and swelling/separating of solid dispersion granules (Figure 16.1).
DCMT has been successfully applied to the sustained-release formulation of nilvadipine, a poorly soluble drug with an aqueous solubility of 1 mg/mL.6 The release profile of nilvadipine from DCMT has been modified by balancing the amount of wax and disintegrant. The wax matrix prevented water penetration into the tablet and ensured the amorphous state of solid dispersion during the dissolution process. Sustained-release profiles of nilvadipine from DCMT were nearly identical in several dissolution media with varying pH and agitation speed. An in vivo study in dogs revealed that DCMTs successfully sustained the absorption of nilvadipine without reducing the bioavailability compared with IR coprecipitate tablets (Figure 16.2).7 The results suggest that DCMT is able to achieve the complete dissolution and absorption of a poorly water-soluble drug by maintaining the physical stability of solid dispersion in the GI tract.
Erodible Molded Multilayer Tablet (Egalet): Similar to DCMTs, Egalet’s erodible molded tablet is an erosion-based platform. It has the advantage of delivering zero-order or delayed release with minimal impact from gastrointestinal conditions. Egalet’s is a more sophisticated engineered delivery system, however, with erosion occurring in one dimension, while DCMT erosion takes place in all three dimensions. It is obvious that Egalet could achieve a better zero-order release. Drug is dispersed in the matrix, and the release is controlled by the rate of erosion in the tablet’s two ends. The surface area for erosion is constant.
Figure 16.3: Egalet delivery for a zero-order release.
Figure 16.3: Egalet delivery for a zero-order release.
Egalet erodible molded multilayered tablets are prepared by injection molding (IM).8 As shown in Figure 16.3, a tablet produced using Egalet technology has a coat and a matrix. Drug release is controlled through the gradual erosion of the matrix part. The mode and rate of release are designed and engineered by altering the matrix, the coat, and the geometry to achieve either a zero-order release or a delayed release. For a zero-order release, a drug is dispersed through the matrix. The coat is biodegradable but has poor water permeability to prevent its penetration. The matrix tends to erode when in contact with available water.
The erosion of the matrix is caused by GI fluids and promoted by gut movements in the GI tract. The drug release is mediated almost wholly by erosion, because the dosage form is designed to slow down the water diffusion into the matrix. The method is definitely more desirable for drugs that have chemical and physical stability issues after contacting with water. For example, if the drug is solubilized as a solid dispersion and the solid dispersion tends to crystallize after interacting with water, the erosion-based delivery will ensure the drug’s stability. It is clear that the Egalet-based delivery system is suitable for the controlled delivery of water-insoluble compounds. The unique delivery system will also prevent hydrolysis and reduce luminal enzymatic activity.
The Egalet delivery system is easily fabricated for delayed release. Delayed release is gaining popularity for the enhancement of local effect or chronotherapy.
As illustrated in Figure 16.3, the release rate of Egalet prolonged release is dependent on the erosion rate and drug concentration. It is clear that a zero-order release can be easily achieved with a uniform drug concentration in the matrix and a constant erosion rate. The erosion rate could be tailored by altering the composition of the matrix. For example, addition of polyethylene glycol could speed up the erosion.9 The in vivo erosion rate may be affected by GI mobility, however. Due to the erosion-controlled delivery, the burst release effect should be minimized in the Egalet system.
On the other hand, the Egalet delivery system is easily fabricated for delayed release. Delayed release is gaining popularity for the enhancement of local effect or chronotherapy. The release of drug is delayed for a certain period of time in the GI tract and released in a bolus dose or a designated modified release. One area of delayed release where this is applicable is in achieving colonic delivery for some therapeutic agents. On the other hand, the delayed release may provide the advantages of a time release. The release of the drug can be timed to match the natural rhythms of a disease, such as the morning stiffness and pain experienced by arthritis patients on waking.
A delayed release can be accomplished through three compartment tablets, including a coat, a drug release matrix, and a lag component. The lag component provides a predetermined delay for the drug release. After the lag component is eroded, the release drug is initiated in a designed mode as depicted in Figure 16.4. Egalet delivery technology is developed based on standard plastic injection molding to ensure accuracy, reproducibility, and low production cost. It is being actively evaluated for the development of numerous controlled-release formulations by various companies.
Bioadhesive Oral Delivery: Bioadhesive delivery could be applied to oral controlled release. Bioadhesive polymers tend to adhere to the mucin/epithelial surface and find applications in buccal, ocular, nasal, and vaginal drug delivery. These polymers could also help increase the residential time of solid dosage forms in the GI tract to improve gastroretentive delivery. On the other hand, bioadhesive polymers enable oral dosage forms to stay close to the epithelial layer and allow the quick flux of drugs after dissolution. Oral absorption or localized therapy could be improved if the disease is in the GI tract.
Bioadhesion is an interesting phenomenon that involves the attachment of a synthetic or biological polymer to a biological tissue.10 Adhesion can occur either with the epithelial cell layer or with the mucus layer. Adhesion to the mucus layer—namely, mucoadhesion—is more applicable to oral delivery. The GI tract is covered by a layer of mucus. Polymers containing hydrogen bonding groups tend to bind to the mucus layer. The mechanism of mucoadhesion is not fully understood, but it is thought that attraction forces such as hydrogen bonding, van der Waals, and charges bring polymers into close contact with the mucus. This contact further promotes the penetration of polymers and formulation of entanglements with the mucin. Many natural polymers and pharmaceutical ingredients show bioadhesive properties. Those polymers are carbomers, chitosan, starch, polymethacrylic acid, hydroxypropylcellulose, hydroxypropyl methylcellulose, and sodium carboxy-methylcellulose. Bioadhesive polymers could be formulated with drugs in monolithic or multiparticulate forms to achieve controlled release.
Figure 16.4: Egalet delivery for a delayed release
Bioadhesive delivery could benefit the controlled release of drugs with narrow absorption windows. Many drugs have a narrow absorption window from the proximal part of the GI tract due to transporter-mediated absorption. Increasing residence time in the upper GI tract could extend and enhance the absorption. It has been reported that mucoadhesive microspheres of acyclovir made from ethylcellulose and Carbopol achieved a better bioavailability than a suspension formulation.11 The mucoadhesive micro-spheres had an AUC0–t of 6055.7 ng h/mL and a mean residence time (MRT) of 7.2 hours, whereas the suspension had an AUC0–t of 2335.6 ng h/mL and an MRT of 3.7 hours. Combination of bioadhesive polymers with another mechanism might improve the degree of success of gastroretention, a challenging goal to achieve. Chavanpatil and colleagues have discussed the development of a novel sustained-release, swellable, and gastroretentive drug delivery system with additional bioadhesive properties for ofloxacin.12
Varshosaz and colleagues reported the design and in vitro test of a bioadhesive and floating drug delivery system of ciprofloxacin.13 Bioadhesive delivery is advantageous in providing sustained release for localized therapy. Deshpande and colleagues published a study about the design and evaluation of oral bioadhesive-controlled release formulations of miglitol, intended for the prolonged inhibition of intestinal a-glucosidases and the enhancement of plasma glucagon like peptide-1 levels.14 Pectin-based microspheres for the colon-specific delivery of vancomycin have been developed by Bigucci and colleagues.15 The microspheres made of pectin and chitosan show desirable mucoadhesive properties.


  1. Rao VM, Haslam JL, Stella VJ. Controlled and complete release of a model poorly water-soluble drug, prednisolone, from hydroxypropyl methylcellulose matrix tablets using (SBE)(7m)-beta-cyclodextrin as a solubilizing agent. J Pharm Sci. 2001;90(7):807-816.
  2. Sotthivirat S, Haslam JL, Stella VJ. Controlled porosity-osmotic pump pellets of a poorly water-soluble drug using sulfobutylether-beta-cyclodextrin, (SBE) 7M-beta-CD, as a solubilizing and osmotic agent. J Pharm Sci. 2007;96(9):2364-2374.
  3. Lee KR, Kim EJ, Seo SW, et al. Effect of poloxamer on the dissolution of felodipine and preparation of controlled release matrix tablets containing felodipine. Arch Pharm Res. 2008;31(8):1023-1028.
  4. Mehramizi A, Alijani B, Pourfarzib M, et al. Solid carriers for improved solubility of glipizide in osmotically controlled oral drug delivery system. Drug Dev Ind Pharm. 2007;33(8):812-823.
  5. Hong SI, Oh SY. Dissolution kinetics and physical characterization of three-layered tablet with poly(ethylene oxide) core matrix capped by Carbopol. Int J Pharm. 2008;356(1-2):121-129.
  6. Tanaka N, Imaia K, Okimoto K, et al. Development of novel sustained-release system, disintegration-controlled matrix tablet (DCMT) with solid dispersion granules of nilvadipine. J Control Release. 2005;108 (2-3):386-395.
  7. Tanaka N, Imai K, Okimoto K, et al. Development of novel sustained-release system, disintegration-controlled matrix tablet (DCMT) with solid dispersion granules of nilvadipine (II): in vivo

API Update: Heparin Scare a Lesson in Safeguarding APIs

By Catherine Shaffer In 2007 and 2008, patients—primarily those undergoing hemodialysis—began having severe, life-threatening allergic reactions to a drug that is normally very safe—heparin. The reactions were at first quite mysterious.1 As adverse-event reports piled up and some patients died, drug manufacturers, suppliers, and regulatory agencies joined forces to track down and identify the contaminant.
In addition to increased testing of heparin and inspection of factories, the FDA has taken a number of steps to prevent contamination, not only with heparins but also with other active pharmaceutical ingredients vulnerable to contamination.
In addition to increased testing of heparin and inspection of factories, the FDA has taken a number of steps to prevent contamination, not only with heparins but also with other active pharmaceutical ingredients vulnerable to contamination.
Early in 2008, heparin manufacturer Baxter International Inc. issued a voluntarily recall of nine lots of heparin.2 Because heparin is a life-saving drug for some patients and the contamination was so widespread, it was not possible to immediately remove all affected product, and doctors were put in the agonizing position of dispensing a drug with known contamination.
Low molecular weight heparin products were also affected, leading to recalls of Lovenox (enoxaparin sodium) by Sanofi-Aventis and heparins made by France’s Rotexmedica and Italy’s Opocrin S.p.A.
Ultimately, the primary contaminant was identified as oversulfated chondroitin sulfate (OCS).3-5 Because that chemical does not occur in nature, and because it shares some properties with heparin, it is thought to be an example of economically motivated adulteration (EMA).6 The discovery of melamine in milk products and pet foods is another example. Perpetrators of EMA add a foreign substance in order to counterfeit a product or enhance a poor one. EMA is a huge problem for products sourced from China, as is increasingly the case in the drug industry.
Three years after the recall, the drug industry and the FDA have developed regulations and protocols for testing heparin sources. While steps have been taken to make it more unlikely that contaminated heparin will slip past U.S. agencies again, the greater question is how many other products could be affected by economically motivated adulteration. In theory, any complex biological mixture could contain difficult-to-detect contaminants.

Rapid Tests for OCS

German researchers combined a two-step fluorescence assay and a two-step anti-Factor Xa assay to detect counterfeit heparin and protein in heparin. And researchers from Sanofi-Aventis in Paris developed a polymerase chain reaction method for quality control of heparins.
One of the scientific community’s first responses was to develop workable methods for testing heparin sources to confirm the purity of the product. During the crisis, a number of labs worked together to use sophisticated methods like nuclear magnetic resonance (NMR), heteronuclear single quantum coherence (HSQC), total correlation spectroscopy (TOCSY), and high performance liquid chromatography (HPLC) to analyze the complex biological samples. Unfortunately, those methods are not easily available to the agencies charged with inspecting factories or testing product.
German researchers combined a two-step fluorescence assay and a two-step anti-Factor Xa assay to detect counterfeit heparin and protein in heparin.7 And researchers from Sanofi-Aventis in Paris developed a polymerase chain reaction method for quality control of heparins.8
Researchers at Rensselaer Polytechnic Institute are developing hyphenated methods of analyzing heparins. They studied combinations of numerous analytical techniques, including mass spectrometry, HPLC, capillary electrophoresis, and NMR spectroscopy to find the best methods for analyzing heparin, concluding that a combination of separation and spectra techniques is necessary for the best results.9

The FDA Acts

In addition to increased testing of heparin and inspection of factories, the FDA has taken a number of steps to prevent contamination incidents, not only with heparins but also with other active pharmaceutical ingredients (APIs) vulnerable to contamination.
The FDA’s inspection process now takes into account traceability, testing, verification of controls, and supplier qualification. The agency is also developing risk models to predict which drugs and ingredients are most at risk of adulteration in order to develop a response before a crisis emerges.
In a letter to the FDA, Martin VanTrieste, vice president of quality and commercial operations at Amgen, encouraged the FDA to “think like a criminal would” to counter attempts at deliberate adulteration motivated by opportunities such as supply shortages (M. VanTrieste [mvantrie], e-mail, May 1, 2009).
“We know that the shortage of pigs in China provided an opportunity for unethical players and criminals to introduce economically motivated adulterated heparin into the supply chain. Learning from this lesson, does it apply to today, where we face a potential pandemic flu with a shortage of effective anti-viral agents that governments around the world are trying to stockpile?” wrote VanTrieste.
The FDA has ranked more than 1,000 APIs in order of risk of EMA, based on their multifactorial model, and have targeted a subset of high-risk ingredients for additional sampling and testing at the border.
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Case study: The Hunt for a Killer Contaminant

A large team of experts assembled to carry out the analysis of contaminated lots of heparin, including scientists from the Massachusetts Institute of Technology in Boston, Momenta Pharmaceuticals Inc. in Cambridge, Mass., Rensselaer Polytechnic Institute in Troy, N.Y., and the G. Ronzoni Institute for Chemical and Biochemical Research in Milan.
The lots involved in the reactions had already been tested for typical contaminants such as proteins, lipids, and DNA, and nothing suspicious had been found in them. Tests for atypical contaminants such as lead and dioxin also revealed nothing. “My first instinct in looking at it was that it was an accidental, not a deliberate, contamination,” said Robert Linhardt, PhD, a professor of chemistry at Rensselaer Polytechnic Institute and a noted heparin expert. “We didn’t assume instantly that there was some nefarious activity here ... . Most problems with pharmaceuticals are just that—accidental.”
Because heparin is a complex mixture of biomolecules, the strategy for analyzing it involved a combination of orthogonal techniques. Nuclear magnetic resonance (NMR) analysis revealed a string of unusual N-acetyl signals, which suggested O-substituted N-acetylgalactosamine. Analysis by heteronuclear single quantum coherence (HSQC), total correlation spectroscopy (TOCSY), correlation spectroscopy (COSY), and rotating-frame nuclear Overhauser enhancement spectroscopy (ROSEY) supported that finding. It was now known that the contaminant was a polymeric repeat of N-acetylgalactosamine linked to glucuronic acid exclusively through beta linkages.
High performance liquid chromatography (HPLC) analysis of digests with heparinases or heparinases plus delta-4,5 glycuronidase and 2-O sulfatase were consistent with the NMR and other results. Further analysis of the isolated contaminant by NMR and other methods yielded a match for oversulfated chondroitin sulfate.
Once the unknown contaminant was identified as oversulfated chondroitin sulfate, it was positively connected with the adverse clinical symptoms that had been observed. Because oversulfated chondroitin sulfate is not found in nature, researchers concluded that its presence in the lots of heparin was no accident, as Dr. Linhardt at first assumed. It was a case of deliberate product adulteration.

Companies’ Efforts

“Mostly, after the recall, people didn’t want to deal with China at all,” Brian James, PhD, director of strategic development at the pharmaceutical consulting firm Rondaxe, told PFQ. “They were going to stay away from China at all costs.” Rondaxe, in Syracuse, N.Y., advises companies in the pharmaceutical and biotechnology industry on issues related to manufacturing, business development, and information technology. In the aftermath of the heparin recalls, Dr. James worked with a number of companies to secure API supplies sourced from overseas.
“Product is the process. When you globalize those types of drugs, it’s difficult to be certain that the process is well controlled. You can’t just do it by depending on analysis.”
—Robert Linhardt, professor of chemistry, Rensselaer Polytechnic Institute, Troy, N.Y.
Dr. James cited language as one of the biggest issues in working with a supplier in China. He advised bringing your own translators rather than relying on those provided by the company you are inspecting. A fluent speaker of Mandarin can pick up on sketchy conversations or inaccurate documentation.
Another factor that threatens purity of APIs is the intertwining of food supply and drug supply chains. Heparin is sourced from pig intestines. Pig farming in China is in no way regulated or inspected by the FDA, so even the most scrupulous supplier can’t guarantee that the pigs have been handled in a manner consistent with American current good manufacturing practice standards.
“Product is the process,” said Robert Linhardt, PhD, a professor of chemistry at Rensselaer Polytechnic Institute in Troy, N.Y., and a noted heparin expert. “When you globalize those types of drugs, it’s difficult to be certain that the process is well controlled. You can’t just do it by depending on analysis.”
Dr. Linhardt’s lab is working to develop a non-animal source of heparin.
Experts agree that responsibility for purity of product and source ingredients falls on the manufacturer, not the FDA or any other intermediary. In an increasingly globalized economy where the rules of engagement vary drastically among cultures, it is important to think ahead and not get caught flat-footed by an instance of EMA. The best defense is to “think like a criminal” and anticipate how a product could be adulterated or counterfeited before it happens.


  1. Centers for Disease Control and Prevention. Acute allergic-type reactions among patients undergoing hemodialysis—multiple states, 2007-2008. MMWR Morb Mortal Wkly Rep. 2008;57(5)5:124-125. Available at: Accessed February 13, 2011.
  2. U.S. Food and Drug Administration (FDA). Baxter issues urgent nationwide voluntary recall of heparin 1,000 Units/ml 10 and 30ml multi-dose vials. FDA website. January 25, 2008. Available at: Accessed February 13, 2011.
  3. Guerrini, M, Beccati D, Shriver Z, et al. Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events. Nat Biotechnol. 2008;26(6):669-675. Available at: Accessed February 13, 2011.
  4. Kishimoto, TK, Viswanathan K, Ganguly T, et al. Contaminated heparin associated with adverse clinical events and activation of the contact system. N Engl J Med. 2008;358(23):2457-2467. Available at: Accessed February 13, 2011.
  5. McMahon AW, Pratt RG, Hammad TA, et al. Description of hypersensitivity adverse events following administration of heparin that was potentially contaminated with oversulfated chondroitin sulfate in early 2008. Pharmacoepidemiol Drug Saf. 2010;19(9): 921-933. Available at: http://onlinelibrary. Accessed February 13, 2011.
  6. U.S. Food and Drug Administration (FDA). Margaret A. Hamburg, M.D., Commissioner of Food and Drugs - remarks at the partnership for safe medicines interchange 2010. FDA website. October 8, 2010. Available at: Accessed February 13, 2011.
  7. Alban S, Lühn S, Schiemann S. Combination of a two-step fluorescence assay and a two-step anti-Factor Xa assay for detection of heparin falsifications and protein in heparins. Anal Bioanal Chem. 2011;399(2): 681-690. Available at: Accessed February 13, 2011.
  8. Auguste C, Dereux S, Martinez C, et al. New developments in quantitative polymerase chain reaction applied to control the quality of heparins. Anal Bioanal Chem. 2011; 399(2):747-755. Available at: Accessed February 13, 2011.
  9. Yang B, Solakyildirim K, Chang Y, et al. Hyphenated techniques for the analysis of heparin and heparin sulfate. Anal Bioanal Chem. 2011;399(2):541-557. Available at: Accessed F

New Insights into Ophthalmic Drug Delivery

By Neil Canavan
New Insights into Ophthalmic Drug Delivery
Drug delivery to the eye might seem simple because it is a readily accessible target. However, the structure, function, and biochemistry of the eye render it highly impervious to foreign substances.
The barriers to drug penetration and retention represent significant therapeutic challenges met by less-than-optimal methods. Topical administrations, which often require frequent applications over indefinite periods of time, or direct injection of a drug, which risks adverse events related to the procedure, represent profound obstacles to patient compliance. Noninvasive, depot, and/or targeted therapies are needed. (For a detailed discussion of ocular dynamics in relation to drug delivery, see Das and colleagues.1)

Innovation Starts to Gel

Recently improved topical solutions are gels, used to treat outer-eye infections or irritations. This advance addresses issues of patient comfort—compared with ointments—and greater drug retention relative to liquid drops. These new viscous preparations may be made up of polysaccharides, carbomers, cellulose derivatives, and recently, hyaluronic acid. Optimization efforts for this approach include the addition of tamarind seed polysaccharide to hyaluronic acid, which, in one investigation, was found to result in a 3/2 synergistic enhancement of either extra- or intra-ocular drug availability.2
Hyaluronic acid has also been used in the preparation of dexamethasone nanoparticles intended for intravitreal delivery. Devising a method for enhanced drug entrapment, Gomez-Gaete and colleagues spray dried 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, hyaluronic acid, and different concentrations of dexamethasone-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticle suspensions, producing a so-called Trojan particle.3 In vitro studies of Trojan demonstrated a depot drug release profile, which the investigators hypothesized stems from the properties of the excipient matrix and surface irregularities of the resultant spherical particle.
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Case study: Iontophoresis to the Fore

Iontophoresis, a process long used for transdermal drug delivery, has only recently been adapted for ocular use. “Prototypes of the current model were first created by the ophthalmic scientist David Maurice,” according to Michael Patane, PhD, chief scientific officer, Eyegate
Pharmaceuticals, Waltham, Mass. “Then Jean-Marie Parel, a biophysical engineer at the Bascom Palmer Eye Institute, designed versions of an applicator to encompass the drug and the electrode (for ocular use).”
EyeGate Pharma’s corticosteroid EGP-437. The system, in Phase III trials, delivers drug with a special applicator.
The current optimized device addresses effectiveness of delivery and, perhaps more importantly, patient comfort.1-2 With this successful clinical translation, Dr. Patane and colleagues set out to validate the process for ocular drug delivery, specifically to characterize transport-structure relationships dependant on permeability and selectivity
differentials. Using rabbit sclera, Dr. Patane looked at the electro-transport of model compounds of neutral, cationic, and anionic charges. The transport of vancomycin, a structurally complex positively charged glycopeptide antibiotic, was also explored.3
Having established efficacy of transport—at superior concentrations as compared with eye drops—investigation continued with a focus on antibiotics alone, with EyeGate’s device highlighted in two poster presentations at the 2010 Association for Research in Vision and Ophthalmology meeting. The first investigation used mounts of ocular tissue in in vitro studies. Data collected for these comparisons of electro-transport vs. passive diffusion (a topical eye drop) for six antibiotic drug classes were then correlated with values observed in anesthetized rabbits. Results showed marked differences in transport rates between molecules of like charge and a lesser efficacy overall for cathodic delivery. This suggested the need for protocols tailored to the given agent to be transported. Also demonstrated was the ability of iontophoresis to push drug into the anterior chamber and the vitreous humor of the posterior segment, suggesting this method could replace the need for some ocular injections.4
The special applicator that delivers EyeGate Pharma’s corticosteroid EGP-437.
The special applicator that delivers EyeGate Pharma’s corticosteroid EGP-437.
A second poster discussed a phase 1/2, randomized, dose-ranging study of an iontophoresis-optimized formulation of dexamethasone (EGP-437) in patients with non-infectious anterior uveitis. Results of this trial established an optimum dosing charge of 1.6mA-min at 0.4mA over four minutes, for which a resolution of infection was observed in as few as two weeks after a single treatment—a treatment that did not cause an elevation in intraocular pressure typically seen with standard-of-care injections.5
As progress continues toward the first iontophoresis ocular indication,
Dr. Patane and colleagues are working on the transport of proteins and further device optimization. “We need to trim the reservoir and enhance the buffering capacity of the system. Right now we’re loading almost 0.5 ml of drug product,” which is fine for an antibiotic. But to reach the holy grail of
penetrating the posterior segment to treat macular degeneration, loading 0.5 ml of a drug like Lucentis would be cost-prohibitive.


  1. Behar-Cohen FF, El Aouni A, Gautier S, et al. Transscleral Coulomb-controlled iontophoresis of methylprednisolone into the rabbit eye: influence of duration of treatment, current intensity and drug concentration on ocular tissue and fluid levels. Exp Eye Res. 2002;74(1):51-59. Available at: Accessed February 12, 2011.
  2. Hughes L, Maurice DM. A fresh look at iontophoresis. Arch Ophthalmol. 1984;102(12):1825-1829. Available at: Accessed February 12, 2011.
  3. Güngör S, Delgado-Charro MB, Ruiz-Perez B, et al. Trans-scleral iontophoretic delivery of low molecular weight therapeutics. J Control Release. 2010;147(2):225-231. Available at: Accessed February 12, 2011.
  4. Ruiz-Perez B, Dowie T, Schubert W, Isom P, Moslemy P, Patane M.. A non-invasive ocular drug delivery system that delivers substantially greater antibiotic levels than topical administration. Abstract presented at: Association for Research in Vision and Ophthalmology Annual Meeting; May 6, 2010; Fort Lauderdale, Fla. Abstract #5713. Available at: Accessed February 13, 2011.
  5. Patane MA, Cohen A, Sugarman J, From S. Randomized, double-masked study of four iontophoresis dose levels of EGP-437 in non-infectious anterior segment uveitis subjects. Abstract presented at: Association for Research in Vision and Ophthalmology Annual Meeting; May 6, 2010; Fort Lauderdale, Fla. Abstract #5263. Available at: Accessed February 13, 2011.
Indeed, the promise of a nanoparticle approach is being aided by collaborations between the physiochemical bench and the bedside. A case in point: the co-development of a depot functioning, biodegradable delivery system by Tueng Shen, MD, associate professor of ophthalmology, and Buddy Ratner, PhD, a biomaterials pioneer and professor of bioengineering and chemical engineering, both at the University of Washington, Seattle.
“Dr. Shen related to me a need for such a polymer,” said Dr. Ratner. “My group had been working with the NIPAM (N-isopropyl acrylamide) polymer, a thermosensitive material. But it wasn’t degradable. Then, recently, we developed a strategy to make another well-known biostable polymer into a degradable form. We applied this strategy to the polyNIPAM.”4 The result is a biodegradable particle that self-constructs at body temperature after injection into the eye.
“The injectable is a viscous liquid, and when it hits a 37 degrees C surface (the body), it becomes a solid,” Dr. Ratner said.
Over time, via hydrolysis and possibly enzymatic activity, the polyNIPAM solid breaks down into minute, oligomer fragments that are soluble and nontoxic and can be cleared by the kidney. In principle, according to Dr. Ratner, any molecule can be carried in the polyNIPAM. It is electrically neutral and can accommodate hydrophobic molecules if they are emulsified. For varied depot formulation needs, the degradation time of polyNIPAM can be tuned from days to months.
To demonstrate proof of principle for this sustained ocular drug delivery strategy, Drs. Ratner, Shen, and colleagues incorporated the antibiotic norfloxicin within polyNIPAM. For in vivo assays using 3T3 NIH cells, spectrophotometry revealed that the polymer-drug solution exhibited rapid reversible phase transition at body temperature from liquid to solid, and that after an initial burst of drug release over 48 hours, gradual release persisted over two weeks. Results of in vivo rabbit studies were consistent with these observations, while at the same time exhibiting no significant toxicity. The investigators concluded that the delivery platform has the potential to be customized for a wide range of ocular treatment needs.5

Eye Cells, Micelles

No less exotic than phase-shifting polyNIPAM are drug encompassing micelles, developed by Ashim Mitra, PhD, vice provost for interdisciplinary research and director of translational research at the University of Missouri-Kansas City, in collaboration with Lux Biosciences of Jersey City, N.J. The project began with the goal of developing a formulation for treating dry-eye symptoms caused by an inflammatory response, and the drug candidate under consideration was voclosporin, a novel immunosuppressive compound.6 “My idea was to develop a topical aqueous eye drop,” said Dr. Mitra, “but it’s very difficult to do that because voclosporin is completely water insoluble—like a brick.” Dr. Mitra decided to try to deliver it in an outwardly hydrophobic, inwardly hydrophilic micellular package.
Once in the choroidal area, says Dr. Mitra, micelles reach the Bruch’s membrane, fuse with it, and release the therapeutic cargo.
The resulting mixed nanomicelles comprise two non-ionic surfactants, D-alpha-tocopheryl polyethylene glycol 1000 succinate (Vitamin E TPGS) stabilized with octyl phenol ethoxylate (octoxynol-40) in a defined ratio.7 “This is a highly stable particle and is not temperature or pressure sensitive,” said Dr. Mitra, adding that it has a shelf life as long as two years. The stability of the drug formulation, now known as LUX 214, was not entirely unexpected. However, during pharmacokinetic (PK) studies of topical administration in a rabbit model, the micelles exhibited rapid voclosporin distribution in both the anterior and, much to Dr. Mitra’s surprise, the posterior ocular segment.8
“We found that a reasonable quantity of drug—higher than therapeutic levels—are achieved in retina and on the choroid in the back of the eye,” said Dr. Mitra. “We never expected that. With cyclosporine you don’t see that.” Other laboratories later validated these observations. In explaining the mechanism, Dr. Mitra has surmised that the drug is traversing a conjunctival, scleral pathway to the back of the eye. “We see very little drug in the vitreous humor.” Once in the choroidal area, micelles reach the Bruch’s membrane, fuse with it, and release its therapeutic cargo into the cell.
This observation is tantalizing to Dr. Mitra, as it may be the answer to the elimination of some, if not all, ocular injections. Such an application is not a near-term goal, however. In focus for the moment is the recent clinical data for LUX 214, a first-in-man study of 25 healthy individuals and five subjects with keratoconjctivitis sicca (KCS), which demonstrated overall safety of the voclosporin formulation, and, though not controlled or powered for significance, a documented improvement in both sign and symptom for the KCS cohort.
Progress in ocular drug delivery is somewhat reined in by lack of accurate PK measures. Attaining drug levels in the various compartments of the eye is problematic at best. Dr. Mitra is eager to at least provide would-be formulators with methods for drug detection in ocular delivery in rabbits without the need to sacrifice the animal; recent approaches include LC-MS/MS and microdialysis.9-10


  1. Das S, Suresh P. Drug delivery to eye: special reference to nanoparticles. Int J Drug Deliv. 2010;2:12-21.