Adeline Siew is editor for Pharmaceutical Technology Europe. She is also science editor for Pharmaceutical Technology.

Adeline Siew, PhD

While the long-term future of antibody drug conjugates (ADC) market is difficult to predict, the outlook over the next few years is promising. The total market value of ADCs has been projected to be worth $10 billion by 2023. 

The advantages of ADCs due to their ability to offer targeted delivery and therefore reduce side effects of cancer therapies have revolutionized the field of oncology, which can be seen with the recent approvals of Adcetris (brentuximab vedotin) and Kadcyla (ado-trastuzumab emtansine). Many pharma and biotech players are now investing more heavily in ADCs. 

Vijay Shah, executive director and chief operating officer of Piramal Enterprises, noted in the first part of the 

, released earlier this month, that the failures of ADCs in the past was not due to the lack of specificity but rather the lack of activity. “The cytotoxic agents were simply not active enough to kill the target cell with such a small amount of payload (i.e., o.o1%),” he said. However, Vijay Shah believes novel technologies that improve antibody and antigen conjugation could hold the key to unlocking the their potential and the next decade will see ADCs with much higher penetration rates.   

There is still progress to be made in antibody selection, the payload and the linking chemistry to combine the two elements together. Target selection is crucial for a successful conjugate, and the goal is to identify one that is significantly overexpressed on cancer cells but not on healthy cells. The antigen target also needs to be expressed on the cell surface, where it is accessible to the antibody. For an ADC, the antigen needs to be internalized, said Vijay Shah, although this is not a requirement for a radioimmunoconjugate. He noted that this is a key area for research as not all antigens meet these criteria. Nevertheless, we are gradually seeing the light at the end of the tunnel. Today, there is a growing number of companies developing conjugation technologies and linker chemistries, which were once a huge hurdle in the development of ADCs.  

Another major challenge lies in the manufacture of these compounds as it involves the handling of biologics and highly potent small molecule toxins. At the moment, only a few CMOs have such manufacturing capabilities. With more ADCs demonstrating positive outcomes in clinical trials, the industry will soon have to find a better solution to address the strain on ADC manufacturing capacity. 

The success of the recent commercialized products has paved the way for more ADCs to enter the market. It will not only be limited to cancer treatments but extend to other indications such as inflammation as Vijay Shah pointed out. It has been projected that over the next five years, there will be a 50% increase in the number of IND submissions for ADCs, although the majority of them will be for the treatment of lymphoma for the short term. 

Articles

The success of Adcetris and Kadcyla is paving the way for more antibody drug conjugates to enter the market, especially in the treatment of solid tumors.

Unlocking the True Potential of Antibody Drug Conjugates

 Sustained-release formulations are mainly designed to increase the duration of drug action by providing a gradual and continuous release of a drug substance from its dosage form, and thereby, decrease dosing frequency. The advantages of sustained-release drug delivery include enhanced patient compliance, improved treatment efficacy and a lower incidence of adverse reactions due to a more uniform drug levels in the blood or plasma among others. 

 Ethyl cellulose polymer has been used as a coating material for oral sustained-release dosage forms since 1985 (1). “Its primary release mechanism is drug diffusion across a water-insoluble membrane,” explained Rina Chokshi, PhD, project leader, controlled release technology, FMC BioPolymer. “Aquacoat ECD is an aqueous colloidal dispersion of ethyl cellulose polymer, which can be used as a moisture barrier as well as in sustained-release and taste-masking applications.” 

 Ethyl cellulose aqueous dispersion provides a stable, reproducible, pH-independent drug-release profile with similar dissolution profiles in both fed and fasted states (2). “An aqueous dispersion of ethyl cellulose offers various processing advantages over organic ethyl cellulose solution, including reduced coating processing time (higher solid content and low viscosity), avoidance of potential toxicity due to residual solvent, and reduced environmental concerns,” Chokshi told 

 With ethyl cellulose aqueous dispersion, the desired drug release profile can be achieved by adjusting three critical formulations attributes—plasticizer, pore former concentration, and film thickness. “The plasticizer decreases glass-transition and minimum film-forming temperatures while improving film strength and flexibility,” said Chokshi. “The plasticizer can also affect intrinsic film structure. A water-soluble plasticizer such as triethyl citrate (TEC) produces a more hydrophilic film and faster drug dissolution compared with water-insoluble plasticizers such as dibutyl sebacate (DBS) that retain the film’s hydrophobicity.” 

 Water-soluble polymers are used as pore formers to create channels in the film that facilitate drug diffusion. “A higher concentration of pore former increases the rate of drug diffusion. On the other hand, a thicker film can be used to slow down the diffusion process,” Chokshi explained. 

 Coating with an aqueous dispersion poses the challenge of achieving complete film coalescence required to produce a robust dissolution profile and long-term stability. In the case of Aquacoat ECD, the polymer is initially deposited as spheres that fuse or coalesce to form a continuous, dense film as water evaporates during the process. “If polymer particles do not fuse completely during coating, further coalescence may occur during storage, resulting in a denser structure and lower permeability associated with slower drug diffusion rates,” Chokshi noted. “Optimized temperature, time, and humidity are crucial for achieving complete coalescence. A temperature above the minimum film-forming temperature increases macromolecular mobility and facilitates coalescence. Higher humidity also enhances film formation, as water is an efficient plasticizer for many polymers (3).” Various studies have been carried out to optimize time, temperature, and humidity conditions to achieve in-process coalescence; and it has been demonstrated that full film coalescence can be achieved using a fluid-bed process by spraying water and maintaining product temperatures at 55–57 oC for one to two hours (3). 

 1. M.A. Frohoff-Hulsmann, A. Schmitz, B.C. Lippold, 

Formulation and process considerations for ethyl cellulose aqueous dispersion in sustained-release applications.

Ethyl cellulose aqueous dispersion: a coating system for oral sustained-release dosage forms

The global market for biosimilar drugs has been forecasted to be $2.445 billion in 2013 according to a report by the British market-research firm, Visiongain (1). The growth corresponds to a 20% increase from last year’s figures and accounts for approximately 2% of the overall biologics market. Although narrowly focused on only a small number of therapy areas at present, the biosimilars market is set to expand over the next decade and beyond as a result of two major factors: the impending patent expiries on blockbuster biologics and the financial environment that is driving payers to push for wider adoption of biosimilars to manage the escalating costs of healthcare. While many companies are keen on getting a share in the biosimilars market, bringing these complex molecules from bench to launch can be a challenge, not just during the development stage but also in terms of the manufacturing process involved. 
  
  

 spoke with Jonathan Bones, principal investigator at Ireland’s National Institute for Bioprocessing Research and Training (NIBRT); Reg Shaw, PhD, CEO of NIBRT (collectively referred to as NIBRT thereafter); Kamali Chance, PhD, head of global biosimilars regulatory strategy, Biosimilars Strategic Unit; Colin Vose, PhD, vice-president, Centre for Integrated Drug Development; Doris Weilert, PhD, senior research pharmacokineticist, Early Clinical Development, Pharmacokinetic/Pharmacodynamic; and John Patava, PhD, director, head of biosimilar intelligence and capabilities, all at Quintiles (collectively referred to as Quintiles thereafter) about the importance of characterization studies during biosimilars development and related analytical methods.

 Why are biosimilars not considered identical to their 
original biologic products? 

 Biosimilars are not approved using the same pathway as generic medicines because of the sheer size and complexity of the molecules that make up biological medicines. Generic medicines are copies of relatively small, well-characterized molecules with low molecular weights produced using well-defined processes involving chemical synthesis. As such, the active ingredient of generic copies and the original medicines are for all intents and purposes, identical. In contrast, biosimilar medicines are of very high molecular weight and complex structures depending on the product. Biological molecules are also manufactured by processes involving living organisms, such as genetically engineered bacteria, yeast, or mammalian cells, and although these processes can be well-defined, they are subject to the variability inherent in living systems. The active ingredient for biologics can only be considered similar, not identical.

Proteins produced in different mammalian cell lines, or under different environmental conditions, may be expressed with subtle but important differences with respect to characteristics such as glycosylation. For this reason, biosimilar medicines should never be assumed to be exact copies of the originator molecule. In recognition of this inherent variability, regulators have determined that a unique pathway is required for the testing and registration of biosimilar medicines.
  


 Differences regarding the approval process for small molecules as compared to recombinant protein therapeutics are reflected in the complexity that must be considered when comparing small-molecule APIs with biologics, molecules that are often many orders of magnitude larger. For example, the cholesterol-lowering agent atorvastatin has a chemical formula of C

 and a molecular weight of 558.64 g/mol. Compare this compound with the monoclonal antibody trastuzumab, which has a reported chemical formula of C

 and a molecular weight of 145531.50 g/mol, approximately 260 times the size of the small molecule API and ultimately more structurally complex. 

Because we’re dealing with relatively simple chemical species when we consider small-molecule API generics, the approval process has been tried and tested effectively over recent decades and is now well established. Rather than requiring full clinical trials, generic drug manufacturers are requested to demonstrate pharmaceutical equivalence to show that their medicine contains the same active pharmaceutical ingredient at the same purity and same dose and with the same administration route as the innovator product. 

Application of the same approval approach for large biological therapeutics is not so straightforward due to the complexity of these large structurally complicated molecules. Despite advances in analytical chemistry and instrumentation, the direct assessment of comparability from analytical data is limited. Using liquid chromatography-mass spectrometry (LC-MS) methods, we can determine and confirm the primary sequence, the presence and identity of post-translational modifications and perform protein structural analysis using advanced experiments such as hydrogen-deuterium exchange MS. Comparison of analytical data forms the case for proving whether two molecules are structurally similar or not; however, data from cell-based potency and bioequivalence assays will also be required. Ultimately, it will be up to the regulatory agency to decide, based upon the presented evidence, whether a biosimlar candidate is similar enough to be approved.

The inherent analytical complexity arises from the fact that recombinant proteins are expressed in cellular systems under defined bioprocess conditions rather than using stepwise chemical synthesis. The cellular machinery that expresses the recombinant protein is sensitive to the physiochemical environment of the cell, the availability of nutrients, and removal of toxicants and inhibitory compounds. All of these factors can affect the quality and quantity of the expressed protein. 

As companies generating biosimilar molecules do not have access to the intellectual property regarding the innovators’ manufacturing process, the process for generating the biosimilar molecule will have, by nature, inherent differences. Therefore, it is inappropriate to say that two molecules are identical; substantive analytical, biochemical, and if necessary, clinical comparison between the biosimilar and the innovator molecules will have to be demonstrated.

 Can you explain the procedures for testing the bioequivalence of biosimilars and how it differs from bioequivalence testing for generic drugs?

The principles of pharmacokinetic bioequivalence testing for proteins are essentially very similar to those for classical medicinal-chemistry small-molecule products and are based on a comparison of peak concentration, time-to-peak concentration, area under the concentration-time curve; however, depending on the mechanism of action of the protein, such a study may need to be done in patients. Although it may be possible to carry out such a study in healthy volunteers, the products are “foreign” proteins that may produce an immune response on repeated administration. In addition, some proteins, for example, monoclonal antibodies, have half-lives of weeks. These proteins would, therefore, require sample collection for many weeks to accurately define the pharmacokinetics and a washout period of some months between treatments with the originator product and biosimilar. Thus, a classical randomized crossover design as used for small molecules is not appropriate on safety and logistics grounds. Such studies, therefore, are most likely to use a parallel-group design, in which each subject only receives one of the products being compared. Such an approach increases the variability and thus, the number of subjects needed to achieve the required power to demonstrate pharmacokinetic bioequivalence.

 Why is it important to characterize amino-acid sequence and carbohydrate structure? 

 Characterization of the amino-acid sequence--normally using LC-MS-based approaches, either top-down or peptide-centric bottom-up methods--is important to verify that the product produced is that as expected from the engineered gene sequence. Data from such experiments facilitate the determination and comparison of the experimental versus the predicted mass of the product. Deviations are indicative of post-translational or other modifications. Such modifications, if present, can then be localized when using peptide-centric LC-MS methods, or sequence variants can also be verified at the peptide level. Data from such studies can then be used to evaluate whether the presence of a particular modification is important with regard to the structure-function relationship of the molecule, or whether for safety or efficacy purposes, it may be necessary to engineer out the modified amino acid.

Similarly for carbohydrates, characterization of the glycosylation present with regard to monosaccharide analysis and structural characterization of the N- and O-linked oligosaccharides present is important to ensure that the desired glycosylation is present and that potentially immunogenic epitopes are absent.

Characterization of the glycosylation present on recombinant proteins is particularly important as the glycans attached to the molecule can modulate the stability and ability of the molecule to elicit its desired effector response, particularly in the case of monoclonal antibodies (1). Because glycosylation is cell-line specific and also affected by the environmental conditions that a cell finds itself in, it is necessary to routinely characterize the oligosaccharides present to make sure that the expressed protein is being produced with consistent and reproducible glycosylation. Furthermore, characterization of the glycosylation present is important to verify the presence or the absence of potentially immunogenic epitopes such as galactose-α1-3-linked galactose motifs and the nonhuman monosaccharide N-glycolyl neuraminic acid.

Characterization of glycosylation is still a significant analytical challenge although recent advances in analytical instrumentation and separation chemistries have benefited the field considerably. Glycans are traditionally analyzed using either liquid-phase separation techniques such as liquid chromatography or capillary electrophoresis with optical or fluorescence detection or mass spectrometry. As oligosaccharides lack inherent chromophores or fluorophores, they must be derivatized to facilitate detection, fluorescent reagents, such as 2-amino-benzamide, 2-aminobenzoic acid, 2-aminopyridie or 2-aminoacridone, have been widely reported as derivatization reagents used in glycan analysis. When using capillary electrophoresis, charged fluorescent labels such as 1-aminopyrene-3,6,8-trisulfonic acid (APTS) or 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) are used to impart electrophoretic mobility to the labeled oligosaccharides and also to facilitate highly sensitive laser induced fluorescence detection.

Mass spectrometry (MS) has also been widely used for glycan analysis, either using matrix-assisted laser-desorption ionization (MALDI) or electrospray ionization (ESI). A caveat with the use of MS is its inability to distinguish isobaric monosaccharide residues or isomeric oligosaccharides; therefore, it is more a compositional analysis. MS/MS based methods can facilitate glycan sequencing when performed on [M+H]+ ions formed during positive ionization. MS/MS of [M-H]- ions formed when using negative ionization is considerably more informative as it can provide diagnostic ions that facilitate the deduction of linkage and positional analysis of the oligosaccharides. There have also been significant advances in associated informatics platforms for the annotation of both LC and MS data in recent years.

Furthermore, the issues of glycan micro- and macroheterogeneity must also be considered. Performing a global glycosylation screen informs us of the identity and relative abundance of the types of oligosaccharides attached to the molecule. However, combination with glycoproteomics is often necessary to identify the sub-populations of glycans present at each glycosylation site.

The combination of analytical techniques in an orthogonal manner is recommended to impart confidence to the analytical data. It is not recommended to analyze the glycosylation using a signal technique; generally, a minimum of two approaches is recommended.

The amino-acid sequence of a biosimilar molecule is one of the starting points in determining similarity to the originator medicine, with the draft guidance from FDA implying that a biosimilar molecule needs to have the same amino-acid sequence as the originator medicine. Some proteins, such as monoclonal antibodies, are glycosylated (i.e., they have carbohydrate molecules attached to them). The extent and the exact structure of carbohydrates attached to a protein may affect its binding to its target receptor, its clearance from the body, and potentially, its immunogenicity. The manufacturing process (e.g., cell line used) can influence the exact nature and extent of glycosylation of the protein, and thus potentially, its activity and safety. It is not possible to definitively determine the structure of a large protein, such as a monoclonal antibody of approximately 150,000 Da, using currently available analytical techniques. This means that confirming similarity of two proteins requires the use of a wide range of analytical techniques.

Functional binding of molecules can be tested using microarray analysis. This technique enables the biosimilar developer to test the binding of the protein to a large number of targets to determine binding and eliminate potential cross reactivity. In many cases, this functional binding may be adequate if the effect of the medicine is simply to neutralize its target. Where the protein-based medicine acts through some cellular signaling pathway, there are tools for assessing these modes of action such as cell-based kinase assays.

 What techniques are used to compare the structure of biosimilars and biologics?

LC-MS is an extremely valuable tool for the analysis of biosimilars and biologics due to its ability to separate many components in complex mixtures and determine the mass of those components in both a qualitative and quantitative manner. LC-MS is routinely used for the characterization of biologics using top-down approaches wherein the intact mass of the molecule is determined, middle-down approaches wherein subunits or domains of the molecule are separated and independently analyzed, and bottom-up approaches following digestion of the therapeutic protein into its constituent peptides through the action of a suitable protease. Such approaches facilitate verification of the primary sequence of the molecule, identification and characterization of post-translation modifications, especially wherein the combined use of collisional-induced dissociation (CID) and/or electron-transfer dissociation (ETD) fragmentation strategies are employed. As mentioned previously, LC, MS, and LC-MS approaches are widely used for the characterization of the glycosylation present.

The combination of generated data from the analysis of the primary sequence with additional analytical platforms is often required to provide an insight into the impact of alterations in the primary sequence or post-translation modifications on the secondary, tertiary, or quaternary structure of the protein. Traditional techniques such as X-ray crystallography and nuclear magnetic resonance (NMR) spectro-scopy can be used for protein structure determination; however, the application to comparability studies can be complicated. Other methods for studying higher order protein structure such as calorimetric methods, analytical ultracentrifugation, circular dichroism, and fluorescence are capable of providing information regarding overall alterations or differences in the protein structure, but generally, are incapable of localizing the area within the sequence of structural difference. Newer methods such as hydrogen-deuterium exchange (HDX) MS are valuable tools for detecting small changes in specific regions of the protein structure. HDX-MS is also more sensitive that other technologies requiring much smaller sample amounts and is also automated.

Ion-mobility spectrometry-mass spectrometry is also emerging as a new and powerful technology to allow for the elucidation of protein structure based upon comparison of the molecules collisional cross-sectional (CCS) area in the gas phase. If the overall protein structure, say between innovator and biosimilar, is different, the molecules may have differences in drift time and associated CCS value, which may be indicative of a conformational change between the molecules.

With regard to aggregation analysis, size-exclusion chromatography (SEC), often with multi-angle light-scattering detection, is widely used for the determination of aggregates. Other methods employed include asymmetric flow field flow fractionation (A4F) or analytical ultracentrifugation (AUC), often as an orthogonal technique to SEC to increase overall analytical confidence and interpretation.

All of the aforementioned methods provide information regarding the molecules structural similarity; however, to determine similarity of function or efficacious effect bioequivalence assays must be performed.

What are the safety issues that must be considered when developing a biosimilar product? 

The most significant concern, from a safety perspective, with biosimilar medicines, is the risk of eliciting an inappropriate immunogenic response. It is difficult to predict, based on 

 characterization alone, whether a biosimilar product will be more or less immunogenic than the originator molecule. 

 studies in animal models are also not particularly useful for determining immunogenicity of a protein in humans. This is especially so for recombinant human proteins, or ‘humanized’ antibodies (antibodies that have a significant part of their protein amino-acid sequence from human origin), as they are highly immunogenic in most animal models. For this reason also, animal models are not particularly useful in determining pharmacokinetics of biosimilar medicines because the immune response they stimulate accelerates their clearance from the animal.

Pharmacokinetic profile and safety testing of a biosimilar medicine, with the knowledge that this product has been well-characterized and determined to be highly similar to the originator medicine, needs to be conducted in humans. As a starting point to these studies, it should be understood that most biological medicines will lead to an immunogenic response in some patients. Immunogenicity rates of between less than 1% to more than 20% have been reported for human proteins and humanized therapeutic monoclonal antibodies (2). Therefore, clinical studies need to be designed to show, not only the similarity in efficacy of the biosimilar medicine to the originator, but also its immunological profile. This assessment is done by ensuring adequate patient exposure to the biosimilar medicine beyond the initial efficacy testing phase. 

 How do you define ‘similar’ when comparing a biosimilar with a reference product, given there will be differences caused by the manufacturing process? 

The definition of similarity is complicated by nature of the fact that biologic products are expressed in living expression systems and differences regarding the manufacturing processes, be it cell lines used, media used, differences in downstream processing or processing between the innovator and the biosimilar process will undoubtedly exist. Furthermore, there is currently a lack of appropriate reference standard material for the development of analytical methods for the evaluation of comparability and similarity. While batches of drug product are often used, it should be considered that the formulation of the drug product may interfere in the subsequent comparability/similarity study and attempts to deformulate the drug product may unknowingly introduce modifications into the molecule, which complicates the study from the offset. It is inappropriate to say that two molecules are identical due to the inherent complexity of the manufacturing process. Indeed, it has been demonstrated that measureable differences exist between different lots of an innovator product (3). The terms comparable, similar, and highly similar require definition by the regulatory authorities. Analytical chemistry generates the data that forms or backs up the argument regarding comparability or similarity; however, it will always be the decision of the regulators as to whether they believe the data are sufficient to justify such claims. 

 8 (1) 34-47 (2008).
  2.	FDA, “Prescribing Information,” 

www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm,

 accessed Aug. 10, 2013.
3.	M. Schiestl et al., 

Industry experts discuss the importance of characterization studies during biosimilars development and related analytical methods.

Elucidating Biosimilars Characterization

Formulation scientists tasked with developing oral formulations often face many challenges such as drug instability in the acidic and alkaline environments of the gastrointestinal (GI) tract and unsatisfactory absorption variations of compounds with poor physicochemical properties such as low solubility or low permeability. 
  
  The primary function of the GI tract is to digest and absorb nutrients and water while at the same time impede the entry of potentially noxious luminal contents such as harmful pathogens and toxins. This function is accomplished by the establishment of intestinal barriers formed by a continuous sheet of polarized columnar epithelial cells that separate the external luminal environment from the internal environment of the body and exhibit selective absorption (1-3). Oral formulations must, therefore, be able to withstand the hostile acidic environment of the stomach and the high enzymatic activity in the small intestine, circumvent the inherently impermeable intestinal epithelial barrier and the binding capacities of the resident mucus and 
  luminal contents, escape the efflux system that pumps substances back into the lumen, and evade first pass metabolism by the liver (3). Pharmaceutical Technology spoke with Anshul 
  Gupte, PhD, senior formulation scientist, and Michael DeHart, PhD, development scientist, from the CDMO Metrics, about the strategies used to enhance oral absorption and the challenges in developing oral formulations for poorly permeable drugs. 

 What factors influence drug absorption in the GI tract? 

Characteristics of the drug that influence absorption include particle size, solubility, lipophilicity, stability, ionization state (pKa), and polymorphism. Sometimes these 
characteristics work against each other to improve absorption. For example, a drug that is more lipophilic may increase permeability, but may result in lower solubility, thereby, decreasing the overall absorption. To overcome deficiencies of absorption due to drug properties, the dosage form may help improve absorption by altering the disintegration and dissolution time, increasing residence time in the intestine, and providing delayed release in the lower intestine instead of the stomach. 

The absorption of the drug through the GI tract is governed by either a simple passive diffusion or active transport with the aid of transporters located in the GI tract. For most drugs, passive diffusion is the mode of transport. The amount of drug absorbed is dictated by the gradient created across the membranes in the stomach and the small intestine as well as the chemical structure of the drug molecule itself such as the size and degree of ionization. Size and absorption usually have an inverse relationship. Most drugs are weak acid or bases, therefore, the pH of the stomach and the intestinal tract affects the degree of ionization of the drug, which directly affects the solubility of the drug in the GI tract. The stomach has a pH of between 1 and 2 while the intestine has a pH ranging from 4 to 8. With this environment, the intestine provides a wide pH range for drugs to exist in their ionized state, which increases their solubility compared to the more lipid-soluble, un-ionized state. 

Most of the absorption takes place in the intestine mainly because the intestine offers much greater surface area for drug uptake. The residence time of the drug in the stomach may vary depending on the time for gastric emptying. The latter is affected by food uptake, other drugs, and other factors. Due to the acidic nature of the stomach mucosa and the presence of enzymes such as pepsin, drugs may be degraded before they are absorbed. The presence of fatty foods can also lead to increased gastric emptying time, which can lead to degradation or reduced absorption of some drugs. 

Lipinski’s rule of five has been used as a preclinical tool to assess the bioavailability of a drug. According to Lipinski’s rule of five, the molecular weight of the drug has to be below 500 Da for it to be a good candidate for oral absorption. Additionally, Lipinski’s rule of five states that the drug molecule should have less than five hydrogen-bond donors and not less than 10 hydrogen-bond acceptors. The log P for the drug should ideally be less than 5. A balance between the polarity and lipophilicity of the drug is necessary so that the drug has sufficient solubility in the gastric media as well as permeability through the gastric mucosa. 

To optimize drug absorption, pharmaceutical scientists employ formulation strategies such as salt formation to increase drug solubility, micronization or particle size reduction to increase surface area, inclusion of surfactants and emulsion system to alter wetting and dispersibility, and addition of buffer systems that alter the microenvironment pH. Spray drying and conversion of the drug into its amorphous, and thus, more soluble form is well established. In the case of a moderately or highly permeable drug, the disintegration and subsequent dissolution from the formulation are critical rate-limiting steps. In the case of an immediate-release formulation, the disintegration of the dosage form is the critical step for the drug to be in solution and subsequently absorbed. The dissolution of the formulation in the fed and fasted gastric media and intestinal media is usually studied in vitro to establish a relationship for the drug 


. Additionally, the drug could be formulated into sustained-release, delayed-release dosage forms. 

What strategies are used to increase oral absorption of poorly permeable drugs?

Creating an esterified prodrug is the most common way of increasing permeability for a highly soluble drug. Prodrugs are reversible derivatives of drug molecules that undergo an enzymatic and/or chemical transformation in vivo to release the active parent drug. Common functional groups that are employed for prodrug formation are amine, hydroxyl, sulfhydryl, and carboxyl moieties. A few examples of the drug/esterified prodrugs are acyclovir/valacyclovir and melagatran/ximelagatran. Esterification of these drugs eliminates an ionized functional group and increases the lipophilicity of the drug molecule. When developing prodrugs, the pharmaceutical scientist must be careful that the increased permeability due to increased lipophilicity doesn’t result in reduced solubility, whichcould offset any increase in absorption due to increased permeability.

Another approach is to use polymers that, in general, mask the inherent hydrophilic nature of the API and hence, lead to enhanced absorption. Liposomes, nanoparticles, and microspheres are all polymer-based delivery systems that generally incorporate a hydrophilic compound inside a lipid membrane. Mucoadhesive polymers can be used to increase the residence time in the GI tract. Specific molecular weights and combinations of polyethylene oxide, for example, have been shown to exhibit excellent 
adhesive characteristics 

Other methods include the formation of bile salt complexes or using cyclodextrin inclusion complexes, which allows poorly soluble drugs to be combined with cyclodextrins, thereby, creating a soluble complex. Developing self-emulsifying drug-delivery systems (SMEDDS) is another strategy to increase oral absorption of a poorly soluble drug. These systems are typically administered as soft gelatin capsules. The drug is dissolved in the oil phase, and through careful selection of surfactants, SMEDDS are able to promote GI absorption by inhibiting bile secretion. In addition, the surface area of cells exposed to certain surfactants is increased.

What are the challenges when developing oral formulations for poorly permeable drugs? 

One challenge is that a drug would be soluble in the gastric media, but would not get absorbed along the GI tract and is subsequently eliminated from the body. One must consider the permeability change over the entire length of GI tract. Another factor to consider is whether the drug is a substrate for the active transport system in the gastric mucosa. The 
  strategy of creating SMEDDS can be undertaken by those companies that possess soft gelatin capsule technology. In some cases, there may be benefit of a delayed-release formulation. 

 The two biggest hurdles in formulation development for a poorly permeable drug are cost and time. Using newer technologies or equipment that have not been fully tested and validated as a commercial option poses a risk. Therefore, proven methodologies such as the addition of functional excipients, particle size reduction, modified-release tablets, or enteric coatings may offer the most practical option to increasing permeability. 

What are some recent advances in oral absorption enhancement strategies? 

Spray drying to generate amorphous material to increase bioavailability is becoming more prevalent. Spray drying is a fast process where the drug molecule and a stabilizer, usually a polymer, can generate a wide range of particle sizes to improve solubility (BCS II and IV) or permeability (BCS III and IV). The spray-dried amorphous material generated from spray drying can then be processed into tablets or capsules with specific release profiles. Other techniques that generate amorphous material to improve absorption are hot-melt extrusion, freeze drying, and supercritical fluid drying. Each of these techniques has their own advantages and disadvantages. 

A more recent advancement in increasing oral absorption is the advent of chronotherapeutics. Chronotherapy uses the body’s natural, circadian rhythms to improve absorption of drugs. For example, blood flow, active versus resting state, and gastric mobility are a few things that change throughout the course of the day that could influence drug absorption. To effectively target the circadian rhythms of the body, several techniques have been developed to offer pulsatile drug release. To achieve the pulsatile-release profile, tablets are generally formulated with multiple layers that contain a combination of immediate-release, extended-release, and delayed-release matrixes. The one-step drug coating (OSDrC) Optidose technology, a 
  proprietary technology of Catalent, can manufacture multiple layer tablets and a tablet-in-tablets with multiple core placement to achieve almost any desired pulsatile-release profile. In a similar fashion, capsules containing beads with various polymer coatings can effectively deliver a drug using the body’s circadian rhythms. 

, 4 (2) 144-151 (1999).
  2. 		P.D. Ward, T.K. Tippin, and D.R. Thakker,

, 3 (10) 346-358 (2000). 
  3. 		T.Y. Ma and J.M. Anderson, “Tight Junctions and the Intestinal Barrier,” in 

, L.R. Johnson, Ed. (Academic Press, Burlington, MA, 4th ed., 2006) pp 1559-1594.

Scientists from the CDMO Metrics talk about the challenges in developing oral formulations for poorly permeable drugs and the strategies used to enhance oral absorption in the gastrointestinal tract.

Evaluating Strategies for Oral Absorption Enhancement

 Most drugs exhibit polymorphism. The pharmaceutical implications of this phenomenon have received a good deal of attention from both academia and the industry given that polymorphism can have significant effects on drug stability, bioavailability and manufacturability. The challenge for formulation scientists, therefore, is to develop the most thermodynamically stable polymorph of the drug to ensure reproducible bioavailability of the product over its shelf life under various storage conditions (1). 

 In some instances, particularly for poorly soluble drugs, the amorphous or metastable crystalline form is preferred and justified by the higher dissolution rate needed for rapid absorption and so that sufficient therapeutic concentrations can be achieved. It is, however, important that the risks associated with these less stable forms are properly evaluated to ensure that the largest possible form change would have no detrimental effect on product quality and bioavailability; and/or a change would not occur under all reasonable storage conditions; and/or analytical methodology and sampling procedures are in place to detect problems before dosage forms with compromised quality and bioavailability reach the end users (1). 

 Pharmaceutical scientists have been exploring the use of metastable polymorphs with the most stable form; however, the most stable form often has poor solubility, low dissolution rate and insufficient bioavailability apart from manufacturing difficulties and IP issues. A team of researchers from the University of Bradford, UK, recently developed a solvent-free, continuous method using high-temperature extrusion to produce the metastable form of the antimalarial drug, artemisinin. The technique is similar to hot-melt extrusion but operates below the melting point of the API (2). “This is the first commercial application of high-temperature extrusion to control polymorphic transformation,” Professor Anant Paradkar, director of the University’s Centre for Pharmaceutical Engineering Science, who led the research, told 

 Artemisinin is known to have two polymorphic forms: orthorhombic and triclinic. The commercially available orthorhombic form is more thermodynamically stable but suffers from low water solubility, which results in poor bioavailability. Consequently, large amounts of the drug have to be administered to achieve sufficient therapeutic concentrations. The metastable triclinic form, on the other hand, is more soluble, and therefore, has a higher dissolution rate with better bioavailability profiles (3). 

 According to Professor Anant Paradkar, previous attempts have been made to produce the triclinic form of artemisinin but these methods involve the use of solvents. “The triclinic form is unstable in the presence of even small traces of solvents, making it problematic to manufacture,” explained Professor Paradkar. “Very often, you will find that the presence of solvents causes the triclinic form to transform into the more thermodynamically stable orthorhombic form.” Producing metastable crystals in a solvent-free environment is, therefore, a potential solution and Professor Paradkar and his team explored the application of high-temperature extrusion to produce the metastable triclinic form of artermisinin. 

 High-temperature extrusion applies mechanical shear stress that can disrupt the crystalline structure of the drug, increase the surface area and result in solid deformation. The elevated temperature can produce local melting of a crystal or melting at the interface between crystals and solid-state transformation may occur in the melt phase before solidification (2). 

 According to the report published in the Royal Society of Chemistry journal, CrystEngComm, the researchers were able to demonstrate that the triclinic form obtained by high-temperature extrusion is more stable than triclinic crystals made using solvent-based techniques (2). “The triclinic form produced using this solvent-free technique has remained stable for two years,” Professor Paradkar told 

. “It is four times more soluble than the orthorhombic form, with a two-fold increase in bioavailability.” 

 More importantly, Paradkar believes that this method is capable of being scaled-up for high throughput. “The method is not specific to artemisinin, but can be applied to many other pharmaceutical drugs, not only making drugs that are more effective in smaller doses, but making drug manufacture more cost-effective,” said Paradkar. 

Researchers from the University of Bradford, UK, have developed a solvent-free, continuous method to manufacture the more soluble and bioavailable form of artemisinin. The metastable form produced using high-temperature extrusion has been shown to have greater stability and longer shelf-life.

Novel Solvent-Free Method to Manufacture Metastable Drug Forms

 that the European Commission (EC) has approved its new subcutaneous formulation of Herceptin (trastuzumab) for the treatment of early- and late-stage HER2-positive breast cancer. The new subcutaneous formulation can be administered six times faster, taking only 2 min to 5 min compared with the standard intravenous formulation, which is given over 30 min to 90 min. This advantage enables patients to spend less time receiving Herceptin treatment in hospital. 

Roche’s new subcutaneous Herceptin is a ready-to-use liquid formulation that is administered as a 600 mg/5 mL fixed dose every three weeks. There is no need for reconstitution or dose calculation based on the patient’s body weight, hence making healthcare procedures simpler. Moreover, a loading dose is not required for subcutaneous administration.  

The subcutaneous formulation uses a technology developed by Halozyme Therapeutics, which temporarily and reversibly degrades hyaluronan, a gel-like substance that forms a barrier between cells under the skin. By doing so, the 5 mL volume of the subcutaneous formulation of Herceptin can be rapidly dispersed and absorbed over a greater area. 

Approval was based on results from the HannaH study, in which the subcutaneous formulation demonstrated comparable safety and efficacy to the intravenous formulation in women with HER2-positive breast cancer. 

The new subcutaneous formulation of Herceptin can be administered six times faster than the standard intravenous formulation.