Today many proteins and peptides are potential drugs, but problems related to their physical/chemical instability hamper their therapeutic applicability. Association of these drugs with carriers, such as liposomes or micro/nanoparticles, is envisioned to allow effective delivery. After several years of protein/peptide DDS research, there is still a lack of suitable systems. Issues such as local reactions and toxicity or long-term accumulation and immunogenicity may have been overlooked, and there is a need for suitable models to address these problems.
by Dr Prof. Sophia G. Antimisiaris
The evolution of recombinant DNA technology and the emergence of bioactive peptides and proteins opened a new era in pharmaceutical science. More than half of the 30,000 human genes encode proteins, many of which are valuable drug candidates, or can serve as targets for protein or peptide-based drug research and development. Many proteins and peptides are recognised as potential leads for new therapeutics. The main consequence of the evolution of DNA technology in pharmacy was the biotechnological synthesis of bulk quantities of peptide and protein drugs, making the manufacturing of such products possible. In 2008, more than 500 new biotechnology medicines (including proteins and peptides) were linked to serious diseases such as cancer, AIDS, or autoimmune and infectious diseases. From this pool, more than 50 protein or peptide derived molecules are undergoing human clinical testing or review by the FDA [1].
One of the main advantages of protein and peptide drugs is that due to their specific mode of action, low doses can be delivered to the respective sites of action. However, their fast and economical production in bulk quantities is offset by various obstacles related mainly to their delivery, which frequently hamper their therapeutic potential and clinical application. Currently there are many examples of drugs that are not small molecules, but are large peptides or proteins; a few examples are given in Table 1 [2]. The increasing recent developments in the field of advanced Drug Delivery Systems (DDSs) offer a number of new technologies that may be utilised for the delivery of protein and peptide drugs.
In this article, the problems encountered when trying to formulate a peptide or protein drug will be summarised and possible solutions will be discussed, mainly in relation to the application of particulate DDSs, such as liposomes or polymeric micro- or nanoparticles.
Specific problems associated with manufacture and administration
A peptide or protein is comprised of several amino acid residues and thus contains multiple reactive sites for different types of chemical reactions to occur, such as hydrolysis, oxidation, disulphide exchange, b-elimination or racemisation. The occurrence of such reactions will involve the formation of new bonds -or cleavage of existing ones- leading to covalent modification of the protein, and potential decrease or even total loss of its activity. In addition to chemical instability, physical instability is also a problem for peptides and proteins, since several procedures may result in modification of their secondary, tertiary or quaternary structure, or in denaturation, aggregation, precipitation and/or adsorption to surfaces. All of these possible alterations of chemical structure or physical characteristics can lead to serious problems during the formulation of such drugs, since several formulation/manufacturing procedures (as sterilisation, lyophilisation, agitation, shearing, kneading etc), could cause drug inactivation. In addition to manufacturing procedures, other factors may also trigger drug inactivation, including environmental factors (pH, ionic strength, temperature and high pressure), adsorption, or contact with non-aqueous solvents, metal ions or detergents.
Due to their chemical and physical instability and degradation by enzymes and proteases (present at the site of administration or on the way to their site of action), most protein/peptide drugs exhibit poor bioavailability and short biological half lives. Frequent and high dosing is therefore required for therapy, posing a risk of undesirable side-effects, such as immune responses to the drug. Furthermore, the delivery of proteins and peptides in vivo can be hindered by their three-dimensional structure and molecular size, due to which their transport by diffusion is slow
(compared to small molecule drugs).
Novel drug delivery systems for peptide and protein drugs
For all these reasons, a carrier or delivery system for therapeutic proteins and peptides would be ideal. In general, DDSs provide a number of important advantages for the delivery of protein and peptide therapeutics such as:
(1) Improved bio distribution and bioavailability
(2) Protection against degrading enzymes and/or detrimental biological environments
(3) Controlled and/or sustained drug release rate
(4) Avoidance of high drug levels, and related undesirable side-effects
(5) Increase of solubility
(6) Possibility to target specific tissues
Ideally, the administration of protein/peptides within DDSs or carriers should decrease the drug clearance rate, so lower volumes or concentrations of drug can be administered, and unwanted side effects or toxicity can be avoided. Depending on the specific therapeutic need and drug characteristics, the DDS should be selected and designed in a way which overcomes the most serious, if not all, of the problems encountered when the drug is administered as a free molecule. Additionally the manufacturing technique should not include procedures that will eventually lead to drug inactivation. The carrier should retain the drug so that its bioactivity is not affected, yet be robust enough to deliver the drug to its site of action and release it in a controlled, sustained manner. In this instance, DDSs formulated with biocompatible and biodegradable materials would certainly be advantageous so as to minimise any adverse host response to the DDSs.
Numerous examples of development of protein/peptide particulate drug delivery systems can be found in the recent literature [3], some of which are summarised in Table 2. The suggested systems are liposomes, or polymeric particles of micro- or nano-scale size, but other types of delivery systems such as micro or nano emulsions, hydrogels, micelles etc, are also currently under intensive investigation for delivery of protein drugs. In most of the cases studied, the results of developing liposomal or particulate formulations for protein/peptide drugs lead to a much better therapeutic outcome. Furthermore, in most cases, the therapeutic benefit observed was mainly attributed to the protection of the drug from degrading agents or media at the site of administration and/or to the prolonged retention of the delivery system at the site, which eventually leads to improved drug bioavailability. Nevertheless, in some of these studies, in vivo experiments and tests were not carried out, and while in most of them several types of polymers or coating materials were used for the first time for the specific application, the toxicity and biocompatibility of the empty DDS was not evaluated. In fact in most of the studies, toxicity and biocompatibility/haemocompatibility tests were not performed under the appropriate conditions [4]. Importantly, in most of the cases in which the drug delivered with the newly developed delivery system did not function any better than the free drug (or perhaps some simpler system to which it was compared), the problem was related to adverse effects or toxicities of one or more sof the DDS.
Particularly if the DDS has to be administered for prolonged periods, or perhaps frequently for a chronic condition such as diabetes, it may be a problem for the corresponding amounts of excipients to be cleared at appropriate rates from the administration site, and thus adverse reactions may occur. Of course this will be closely related to the amount of excipients needed for delivery of the required amount of drug (related with the drug loading capacity), and also with the specific
physiology at the site of administration.
For systemic administration, since the circulatory system will deliver the drug carriers, it is very important that all the DDSs used are blood-compatible and do not induce any blood toxicity (haemolysis) [4]. When topical administration is used, then the specific site characteristics of the site of delivery may impose other types of restrictions. For example, in the case of pulmonary delivery, which is currently under intensive reasearch as a route for administration of many drugs, many natural or synthetic materials used for carrier preparation or as targeting moieties may be incompatible with lung tissue. While the safety of some carriers has been examined (eg, conventional liposomes), for many other carriers it has not. Cationic liposomes, for example, that have gained popularity for gene delivery have been found to induce oxygen radical-mediated pulmonary toxicity.
For carriers that are used to prolong drug release, there is a danger that with long-term use the carrier material may accumulate in the lung, especially in the lung periphery, which is not served by mucociliary clearance. Another source of toxicity may be residual solvents, which remain after formulation processes for microencapsulation or liposome preparation. Excipients used in dry powder formulations to promote the stability of proteins, such as salts and sugars, may induce bronchoconstriction in hypersensitive patients.
Another possibility that may have been overlooked in some studies is the inactivation of the drug during the formulation procedure, since some of the methods used for preparation of DDSs cannot be considered as “gentle”. Processing of excipients and/or excipients that denature the protein/peptide in addition to drug inactivation, may lead to increased immunogenicity, so processing techniques and formulation components must be considered carefully. Indeed, most of the techniques involved in DDS formation, such as vigorous mixing or homogenising, sonication steps, contact with organic solvents, high temperatures etc, may lead to drug inactivation. In the case of liposomal carriers, the DRV (dried rehydrated vesicle) method is a gentle protocol for development of protein formulations, without any loss of drug activity, which has been recently reviewed in detail [5].
When comparing liposomes with other types of particulate drug carriers for protein/peptide drugs, it can be seen that liposomes have many advantages. These are mainly because of the ability of liposomes to entrap large amounts of different types of drugs (lipid- or aqueous-soluble), and also because there are several manufacturing techniques that are gentle enough to protect them from inactivation during the encapsulation procedure. Furtherore, as mentioned previously, conventional liposomes have been proven to be biocompatible and non-toxic when administered by a number of different routes.
Another concern might be the size of carriers, which are typically around or above the micrometre range, as the larger the DDS, the higher the possibility of triggering an opsonisation effect by the macrophages or other components of the immune system. For this reason nanosystems are preferable.
Conclusions and possible future directions
In summary, it is envisioned that DDSs can provide protection to proteins/peptide drugs until they have reached their site of action. However, after several years of protein/peptide DDS research, there is still a lack of good systems. In order for more efficient protein and peptide DDS to be designed, researchers must prioritise the following issues:
(1) Biocompatibility, biodegradability and safety profile of DDS
(2) Drug encapsulation efficiency and stability
(3) Maintenance or enhancement of protein/peptide activity
(4) Ease of preparation/manufacturing and administration (5) Ability to control/sustain drug release
(6) Cost
When designing a DDS for a specific protein drug, factors such as the biochemistry of the protein or peptide, the intended administration route for a desired treatment, and also the action site of the drug should be taken into consideration. Researchers should first characterise their drug of interest for properties such as structure, surface charge and solubility before identifying a formulation method which is the most appropriate. To prevent undesirable effects and negative interactions with the host, safer materials that are natural, biocompatible, biodegradable and less likely to incite an adverse reaction should be utilised. Finally, issues regarding local reactions and toxicity, and long-term accumulation and immunogenicity, will all have to be addressed using suitable models.
References
1. Tauzin B. Report: biotechnology medicines in development. Washington, DC: Pharmaceutical Research and Manufacturers Association; 2008.
2. Crommelin DJA, Sindelar RD. Pharmaceutical Biotechnology: An Introduction for pharmacists and pharmaceutical scientists, Taylor & Francis, 2nd edition, 2002.
3. Tan ML, Choong PFM, Dass CR. Recent drug delivery strategies for PPd Recent developments in liposomes, microparticles and nanoparticlesfor protein and peptide drug delivery. Peptides 2010; 31: 184–193.
4. Michanetzis GPAK, Missirlis YF. Antimisiaris SG. Hemocompatibility of Nanosized Drug Delivery Systems: Has It Been Adequately Considered? J. Biomed. Nanotechnol 2008; 4: 218-233.
5. Antimisiaris SG. Preparation of DRV liposomes, from Methods in molecular biology, Edited by V. Weissig (Clifton NJ), 2010; 605: 51-75.
The author
Prof. Sophia G. Antimisiaris, University of Patras & ICE-HT/FORTH
Rio 26510 • Patras • Greece
