Posts Tagged ‘liposomes’

Self-propelled Liposomes as a Drug Delivery System

Reporter: Irina Robu, PhD

Liposomes are small artificial vesicles of spherical shape that can be created from cholesterol and natural non-toxic phospholipids. As a result of their size and hydrophobic and hydrophilic character, liposomes are promising systems for drug delivery. Liposome properties diverge considerably with lipid composition, surface charge, size, and the method of preparation. Scientists at Penn State developed self-propelled liposomes that migrate away and/or towards chemical signals, making it possible for self-directed drug delivery vehicles that can actively target a specific area of the body. Besides, the choice of bilayer components controls the ‘rigidity’ or ‘fluidity’ and the charge of the bilayer. Countless liposomes proposed for drug delivery are tissue-specific, since of antibodies on their surface bind to the target tissue when they encounter it. The technology may help to enhance efficacy and reduce side-effects of drugs in a variation of applications.

Yet, the key to drug delivery is enhancing the specificity and affinity of a drug delivery vehicle for its target tissue. As the drawbacks of conventional drug therapies, scientists are developing an extensive variability of drug delivery vehicles including nanoparticles, biomaterials, and implantable devices, to increase drug accumulation at a target site in the body and reduce side-effects elsewhere. To address the drawbacks, these researchers developed a type of liposome that can actively propel itself near a chemical signal in the body, such as a chemical attractant released by a target tissue.

The liposomes proposed by Penn researchers are covered in enzymes that react with specific substrates to produce energy, which can help to push the liposomes along, through a phenomenon called chemotaxis. By changing the enzymes coating the liposomes, the investigators can tune this chemotaxis and permit the particles to either move towards or away the chemical signal. This could aid the particles to gravitate near certain tissues, and possibly avoid others in the body.
Currently, the are still developing the liposomes, and hope that they will be able to use them for drug delivery soon

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Targeted Liposome Based Delivery System to Present HLA Class I Antigens to Tumor Cells: Two papers

Reporter: Stephen J. Williams, Ph.D.



Cell-mediated immunotherapies have potential as stand-alone and adjuvant therapies for cancer. However, most current protocols suffer from one or more of three major issues: cost, safety, or efficacy. Here we present a nanoparticle delivery system that facilitates presentation of an immunogenic measles antigen specifically in cancer cells. The delivery system does not contain viral particles, toxins, or biologically derived material. Treatment with this system facilitates activation of a secondary immune response against cancer cells, bypassing the need to identify tumor-associated antigens or educate the immune system through a primary immune response. The delivery system consists of a stealth liposome displaying a cancer-specific targeting peptide, named H1299.3, on its exterior surface and encapsulating H250, an immunogenic human leukocyte antigen class 1 restricted peptide. This targeted-nanoparticle facilitates presentation of the H250 peptide in major histocompatibility complex class I molecules. Activation is dependent on the targeting peptide, previous antigen exposure, and utilizes a novel autophagy-mediated mechanism to facilitate presentation. Treatment with this liposome results in a significant reduction of tumor growth using an aggressive LLC1 model in vaccinated C57BL/6 mice. These data provide proof-of-principle for a novel cell-mediated immunotherapy that is scalable, contains no biologically derived material, and is an efficacious cancer therapy.


Cell-mediated (CM) immunotherapies for cancer treatment are designed to activate the body’s adaptive immune responses against a malignant growth.1,2 Generally, the goal of a CM response is to activate a cytotoxic T-cell response against a tumor to eliminate cancer cells. The principle of these treatments is straightforward, yet current work studying the complexity of the tumor micro-environment2,3 as well as methods that attempt to directly activate T cells against tumor antigens4,5,6 demonstrate the difficulty associated generating an immune response against a tumor.

Several CM cancer immunotherapies exist today. Major examples include PD-1 inhibitors, injection of live virus or viral particles into tumors, and adoptive T-cell therapies.1,6,7,8 However, concerns regarding efficacy, safety, and/or cost have limited the use of many of these treatments. To address these concerns, we sought to develop a novel treatment based on developing a fully synthetic, minimal delivery system that facilitates presentation of human leukocyte antigen (HLA) class I restricted immunogenic peptides specifically on cancer cells without using live virus, viral subunits, or biologically derived material.

Based on these requirements, we developed a liposomal based agent consisting of a neutral, stealth liposome that encapsulates a synthetically manufactured immunogenic HLA class I restricted peptide derived from measles virus.1,2,9 In addition, the liposome has a targeting peptide on the external surface that both specifically accumulates in cancer cells and facilitates presentation of the immunogenic peptide in HLA class I molecules (Figure 1a). Thus, this treatment is designed to generate a secondary CM immune response specifically against the tumor if the patient was previously vaccinated against or infected with measles.

Figure 1

The minimal antigen delivery system consists of three components. (a) PEGylated stealth liposomes are loaded with an immunogenic human leukocyte antigen (HLA) class 1 restricted peptide derived from measles virus, named H250. The surface of the liposome

In this proof-of-concept study, we synthesized a liposome that encapsulates H250,1 an immunogenic HLA class 1 restricted peptide identified from measles hemagglutinin protein. The liposome is designed to specifically internalize in cancer cells by displaying the recently identified targeting peptide H1299.3 on the exterior surface (Figure 1b).10 H1299.3 is a 20mer, cancer-specific targeting peptide that was recently identified by our group. The peptide was identified using a novel phage display technique that allows for selection of cancer-specific targeting peptides that preferentially internalize in cancer cells via a defined mechanism of endocytosis. This peptide was dimerized on a lysine core and is fully functional outside the context of the phage particle. The H1299.3 peptide accumulates specifically in a panel of non-small cell lung cancer (NSCLC) cell lines compared to a normal bronchial epithelial cell control cell line via a clathrin-dependent mechanism of endocytosis. In this study, we demonstrate that H1299.3 facilitates functional presentation of an immunogenic antigen in both major histocompatibility complex (MHC) and HLA class I molecules as indicated by CD8+-specific interferon (IFN)γ secretion. In addition, H1299.3 facilitated presentation utilizes an autophagy-dependent mechanism. Finally, treatment with H1299.3 targeted liposomes containing H250 substantially reduces the growth rate of subcutaneous LLC1 tumors implanted in vaccinated C57BL/6 mice compared to treatment with vehicle control.

Result summarized:

  1. The H1299.3 targeting ligand specifically accumulates in cancer and facilitates HLA class I presentation: H250 is an immunogenic peptide identified from sequencing peptides present in HLA A*0201 molecules following measles infection. identified two donors that were HLA A*02 positive and had previously been vaccinated against measles virus (the human NSCLC cell line, H1993, which we determined to be HLA A*02 positive)
  2. identified three different cancer-specific targeting peptides that internalize into H1993 that have been previously published: H1299.2, H2009.1, and H1299.3. Each of these peptides specifically internalize in NSCLC cell lines compared to normal bronchial epithelial cells
  3. H1299.3 facilitated HLA class I presentation requires autophagy. H1299.3 peptide colocalizes with Lamp-1 which is a marker of both lysosomes and autolysosomes, therefore it was possible autophagy involved and shown that H1299.3 colocalizes with autophagosomes.  Chlorpromazine, which inhibits clathrin coated mediatated endocytosis, decreased the HLA1 presentation of H250.
  4. H1299.3-targeted liposomes encapsulating H250 reduce tumor burden in vivo. Mice were first vaccinated against H250.  The J1299.3 targeted liposome encapsulation H250 reduced tumor growth of LLC1 s.c. xenograpfts  by 50%.
J Transl Med. 2011 Mar 31;9:34. doi: 10.1186/1479-5876-9-34.

Enhanced presentation of MHC class Ia, Ib and class II-restricted peptides encapsulated in biodegradable nanoparticles: a promising strategy for tumor immunotherapy.



Many peptide-based cancer vaccines have been tested in clinical trials with a limited success, mostly due to difficulties associated with peptide stability and delivery, resulting in inefficient antigen presentation. Therefore, the development of suitable and efficient vaccine carrier systems remains a major challenge.


To address this issue, we have engineered polylactic-co-glycolic acid (PLGA) nanoparticles incorporating: (i) two MHC class I-restricted clinically-relevant peptides, (ii) a MHC class II-binding peptide, and (iii) a non-classical MHC class I-binding peptide. We formulated the nanoparticles utilizing a double emulsion-solvent evaporation technique and characterized their surface morphology, size, zeta potential and peptide content. We also loaded human and murine dendritic cells (DC) with the peptide-containing nanoparticles and determined their ability to present the encapsulated peptide antigens and to induce tumor-specific cytotoxic T lymphocytes (CTL) in vitro.


We confirmed that the nanoparticles are not toxic to either mouse or human dendritic cells, and do not have any effect on the DC maturation. We also demonstrated a significantly enhanced presentation of the encapsulated peptides upon internalization of the nanoparticles by DC, and confirmed that the improved peptide presentation is actually associated with more efficient generation of peptide-specific CTL and T helper cell responses.


Encapsulating antigens in PLGA nanoparticles offers unique advantages such as higher efficiency of antigen loading, prolonged presentation of the antigens, prevention of peptide degradation, specific targeting of antigens to antigen presenting cells, improved shelf life of the antigens, and easy scale up for pharmaceutical production. Therefore, these findings are highly significant to the development of synthetic vaccines, and the induction of CTL for adoptive immunotherapy.

[PubMed – indexed for MEDLINE]

Free PMC Article

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follow-on complex drugs

Larry H. Bernstein, MD, FCAP, Curator




Clinical development, immunogenicity, and interchangeability of follow-on complex drugs

Generics and Biosimilars Initiative Journal (GaBI Journal). 2014;3(2):71-8.       DOI: http://dx.doi.org:/10.5639/gabij.2014.0302.020


Although not derived from living sources, non-biological complex drug (NBCD) products have the immunogenicity and molecular complexity of biological drugs. NBCDs typically contain heterogenous mixtures of closely related nanoparticulate components that cannot be isolated, quantified, or entirely characterized physicochemically. Development of follow-on versions of NBCDs poses many of the same scientific challenges associated with biosimilar drugs. Like biologicals, the manufacturing methods used by the innovator to produce NBCDs ensure their identity, and consistent quality and activity. Some variation in alternate-sourced products is inevitable. Because of their complexity and because biological activity is often not correlated with serum pharmacokinetics, follow-on NBCDs can be shown to be similar, but not identical, to the originator product. Even slight variations in a follow-on NBCD can increase the risk of unwanted immunogenicity, safety problems, and/or reduced therapeutic effects. Issues related to follow-on versions of liposomal formulations, iron-carbohydrate complexes, and glatiramoids are described here to illustrate aspects of NBCDs that render the abbreviated pathway for approval of small-molecule drugs unsuitable for follow-on NBCDs. The US Food and Drug Administration has made ‘equivalence of complex drugs’ a Generic Drug User Fee Amendment priority initiative for fiscal year 2014. Experience suggests the same enhanced pre-approval scrutiny of biosimilar drugs should be applied to follow-on NBCDs. Preclinical and/or clinical data may be required to establish similar quality, immunogenicity, safety, and efficacy between a follow-on NBCD and a reference drug, and automatic switching or substitution of a follow-on NBCD for the originator should be contingent on demonstration of therapeutic equivalence.


The Biologics Price Competition and Innovation (BPCI) Act of 2009 was instituted to create an abbreviated pathway for approval of biosimilar drugs [1]. In 2014, the biosimilar pathway is still evolving; at this writing, the US Food and Drug Administration (FDA) has issued three draft guidelines for manufacturers seeking approval of biosimilar drugs [24]. Regulatory authorities agree that pre-approval evaluation of biosimilar drugs must be held to a higher standard than generic versions of small-molecule drugs because of their complexity and immunogenicity [57]. Non-biological complex drugs (NBCDs) have the molecular complexity of biological drugs, are immunogenic, and developing follow-on NBCDs poses many of the same scientific challenges associated with biosimilar drugs [810]. NBCDs typically contain heterogenous mixtures of closely related, macromolecular, nanoparticulate components that cannot be isolated, quantified, and/or entirely characterized physicochemically using available analytical technology [11]. As is true for biological drugs, consistent NBCD activity and quality typically rely on strictly controlled manufacturing procedures [1214], such that even small differences in the manufacture of a follow-on NBCD from that of the originator product can increase the risk of safety problems or reduced therapeutic efficacy [13, 15, 16].

Currently, follow-on NBCDs can be approved under the generics pathway established for traditional small-molecule drugs via an abbreviated new drug application (ANDA [505(j) application]), or under section 505(b)(2) of the Federal Food, Drug, and Cosmetic Act (FFDCA) [17]. Acknowledging that this pathway may not address the scientific challenges of ensuring the safety and efficacy of follow-on NBCDs [11, 18], FDA has made ‘equivalence of complex drugs’ a Generic Drug User Fee Amendment (GDUFA) Regulatory Science Priority Initiative for fiscal year 2014 [19].

A key aspect of pending legislation for biosimilars and follow-on NBCDs will be the development of science-based policies for interchangeability and drug substitution. The BPCI Act makes clear that biosimilarity does not imply interchangeability or substitutability [1]. Unlike generic copies of small-molecule drugs, biosimilars and follow-on NBCDs will not be identical to the innovator products. Because of their complexity and because the manufacturing method used to produce the innovator drug is often proprietary, some variation in alternate-sourced products is inevitable. Slight but clinically meaningful differences between originator and follow-on NBCDs may make interchangeability unfeasible. By law, to gain approval for interchangeability for biological drugs, the risk in terms of safety or diminished efficacy of alternating or switching between the generic product and the reference product must be no greater than continuing to use the reference product [1].

Scientific issues related to therapeutic equivalence of NBCDs

The ANDA generic drug pathway for small-molecule drugs requires proof of therapeutic equivalence of the generic to the innovator product, i.e. pharmaceutical equivalence (identical active substances, dosage form, strength, route of administration, labelling, quality, performance characteristics, and intended use), and bioequivalence (comparable pharmacokinetics in healthy humans) [17]. For some NBCDs, full proof of pharmaceutical equivalence is impossible, since two drugs cannot be shown to have identical active substances if the active substance has not been identified and the mechanism of action of the reference drug remains unknown [8, 10, 13]. Gross characterization of drug composition showing similarities in certain vectors, e.g. molar ratio or molecular weight distribution of constituents, does not guarantee similarity of other product characteristics [13, 20]. Similarly, bioequivalence cannot be established for many NBCDs because their biological activity is not correlated to serum pharmacokinetics [10, 21, 22]. These drugs typically comprise nanoparticle-size substructures that release or form the active ingredient, which is then transported to the targeted tissue.

Additionally, follow-on NBCDs cannot be presumed to have the same immunogenic profiles as innovator complex drugs [23]. The ability to predict drug-induced immunogenicity of uncharacterized NBCDs is limited, because immunogenicity is subject to influence by many variables. Patient-related factors such as genetic background, immune status, and the disease under treatment will influence the immunogenic response to treatment [24]. Autoimmune diseases can augment the immune response to immunogenic drugs. Product- and manufacturing-related factors also influence immunogenicity [5, 25, 26]; minor but key changes to the synthesis or manufacture of follow-on protein- and peptide-based NBCDs can lead to formation of aggregates or other impurities that can enhance drug-related immunogenicity and be immunogenic in their own right [26, 27].

Two products purported to be the same drug can produce antibodies with varying specificity such that one drug produces neutralizing antibodies (NABs) and the other does not [14]. When switching between a follow-on drug and the reference product (or among follow-on products), pre-existing antibodies to one NBCD could neutralize the efficacy of an analogous product. NABs that decrease drug efficacy can develop months or years after beginning treatment [28, 29]. For example, clinically important NABs associated with interferon-beta (IFNβ) therapy for MS generally develop after 12 to 18 months of treatment [30], and the clinical effects of decreased efficacy may take years to detect, resulting in irreversible disability progression that might have been avoided by performing regular antibody assessments [31].

Liposomes, iron-carbohydrate complexes, and glatiramoids

The 2014 GDUFA initiative regarding equivalence of complex drugs specifically mentions generic versions of (among others) liposomal drug formulations, e.g. Doxil (doxorubicin HCl liposome); iron-carbohydrate complexes, e.g. Venofer (iron sucrose); and products that contain complex peptide mixtures and peptides, e.g. Copaxone (glatiramer acetate) [19]. These NBCD classes exemplify challenges to the classic abbreviated pathway for generic drug approval and indicate a need for increased pre-approval assessment for follow-on NBCDs.

Liposomal drug formulations
Liposomal drug formulations act as carrier vehicles to deliver active agents to a specific body site. Nanoparticles of the bioactive agent encapsulated in vesicles composed of a phospholipid bilayer act as targeted antigen delivery systems to induce therapeutic humoral and cell-mediated immune responses [22]. As vaccines, synthetic antigenic peptides in liposomal formulation induce autoantibodies for prophylaxis of chronic conditions, such as hypertension [32]. Liposomal formulations of anticancer drugs allow antibody- or ligand-mediated targeting specifically to tumour cells, to increase therapeutic effects while reducing toxicity [33].

The physicochemical properties of liposomal vaccines – method of antigen attachment, lipid composition, bilayer fluidity, particle charge, and other properties – strongly influence the immune responses to them [22, 34], see Table 1. Thus, small differences in particles or complex attributes in follow-on versions of liposomal drugs could alter the activity of the drug, its distribution profile, and/or its persistence at tissue/cellular or subcellular levels to a clinically meaningful extent [35]. Currently, little is known about the cellular distribution of lipid-modified
peptides [22].


Consistent plasma concentrations of the active substance in two liposomal formulations does not guarantee similar efficacy or safety of the two products, since nanoparticles of active drug may distribute differently in tissues and cells [13, 35]. To investigate whether a conventional bioequivalence approach could ensure therapeutic equivalence of liposomal products, the pharmacokinetics, efficacy, and toxicity of six formulation variants of the originator PEGylated liposomal doxorubicin product (Doxil/Caelyx, Janssen-Cilag Pty Ltd) were prepared differing in composition and liposome size and evaluated in preclinical models for antitumour activity and toxicity [36]. Although some formulations demonstrated similar plasma pharmacokinetics and systemic exposure of doxorubicin, they exhibited different antitumour activity and toxicity profiles. Investigators concluded that a conventional bioequivalence approach is not appropriate for establishing therapeutic equivalence of a generic product.

Augmenting immunogenicity is key to the therapeutic activity of many liposomal preparations. However, some therapeutic liposomes are recognized by the immune system as foreign, likely because the phospholipid vesicles of the liposome mimic the size and shape of pathogenic microbes [37], leading to a variety of adverse immune reactions. Hypersensitivity reactions to liposomal drugs appear to be primarily mediated through complement activation triggered by an immune reaction to liposome surface charge or topography [37].

Detecting clinically meaningful differences in the therapeutic activity, toxicity, and immunogenicity of a follow-on liposomal drug may require nonclinical and clinical studies. A reflection paper issued by the European Medicines Agency (EMA) on data requirements for follow-on versions of liposomal products indicates clinical data for these products will be considered on a case-by-case basis [38]. Currently, EMA has not approved any follow-on versions of liposomal drugs.

Iron-carbohydrate drugs
Intravenous (IV) iron products are used to treat iron deficiency anaemia in patients undergoing chronic haemodialysis and receiving supplemental EPO therapy and in people with iron-deficiency anaemia associated with chronic blood loss or impaired iron absorption. The chemical structures of parenteral iron agents have not been characterized in full detail. Venofer (iron sucrose, Vifor Inc) and Ferrlecit (iron gluconate, Sanofi) comprise nanoparticle-sized iron cores surrounded by a complex carbohydrate layer. Because the physicochemical and biological properties of iron-carbohydrate compounds depend on their manufacturing processes, subtle structural modifications during manufacture may affect drug stability; if weakly bound iron dissociates prematurely it can catalyse the generation of reactive oxygen species leading to oxidative stress and inflammation [39]. Moreover, any variation in mean/median size and size distribution of the iron-carbohydrate nanoparticles can result in a generic product with different physicochemical properties and different biopharmaceutical profile with respect to pharmacokinetics and biodistribution compared with the originator drug [21]. In fact, animal studies show differences between the originator iron sucrose product (Venofer) and iron sucrose similar (ISS) products with increased markers of inflammation and increased serum iron and transferrin saturation levels in animals receiving the ISS [13, 40].

Despite these differences and the inability to completely characterize these drugs, and with no nonclinical or clinical studies to establish their therapeutic equivalence to the innovator drug, ISS products gained marketing approval via the small-molecule drug generic pathway [41]. Subsequently, in controlled trials in anaemic patients undergoing haemodialysis, ISS use was associated with reduced efficacy and the potential for increased safety risk related to iron overload [42, 43]. Clinically meaningful differences have been demonstrated when patients were switched to an ISS from Venofer. A switching study in which 75 stable haemodialysis patients taking Venofer for at least six months switched to an ISS product for six months resulted in decreased haemoglobin levels and reduced iron indices despite higher doses of the ISS [42], see Figure 1.


Iron-carbohydrate products can cause life-threatening or fatal hypersensitivity reactions, especially in pregnant women [44, 45]. The immunologic basis of allergic hypersensitivity to iron agents remains unknown [44]. Substitution of Venofer with an ISS at the pharmacy level (without physician or patient knowledge) was associated with hypersensitivity reactions and hospitalization in subjects who previously tolerated the originator drug [41]. Safety concerns surrounding all IV iron products led to recommendations of stronger measures to manage and minimize the risk of hypersensitivity [45]. The recommendations state that every dose of IV iron administered should be monitored for potential hypersensitivity reactions, even if previous administrations were well tolerated.

Both FDA and EMA have indicated that follow-on versions of iron sucrose (FDA) and nanoparticulate iron medicinal products (EMA) are not suitable for approval through the classic generic approval pathway [21, 46]. Neither agency has indicated what clinical evaluation will be required for approval of follow-on products.

The prototype glatiramoid, Copaxone, (Teva Pharmaceutical Industry) is a complex heterogenous mixture of synthetic proteins and polypeptide nanoparticles with immunomodulatory activity approved for treatment of relapsing-remitting multiple sclerosis (RRMS) [10,4750]. The active ingredient in Copaxone, glatiramer acetate, comprises a potentially incalculable number of unidentified active peptide moieties that are not characterizable with available technology [10], although the amino acid sequences in Copaxone are not entirely random [8]). The mechanism of action of Copaxone is not fully elucidated but the drug is thought to act as an antigen-based therapeutic vaccine [5153]. Pharmacokinetic data are uninformative for glatiramoids because the polypeptides in a glatiramoid mixture are hydrolysed at the drug injection site into unidentifiable peptide fragments that stimulate proliferation of glatiramoid-specific immune cells, which migrate to the central nervous system where they ameliorate auto-immune destruction of myelin [54, 55]. Therefore, blood levels of the glatiramoid or its hydrolysis products are not indicative of drug activity.

Glatiramoids appears to act as altered peptide ligands (APL) of encephalitogenic epitopes within myelin basic protein (MBP), an autoantigen implicated in MS [56]. Decades of clinical use demonstrate that Copaxone does not contain encephalitogenic epitopes and does not induce auto-reactive antibodies [48]. However, the same cannot be assumed for a follow-on glatiramer acetate product. In the last two decades, other APLs of MBP epitopes have been studied for use as therapeutic vaccines in MS. Clinical development of at least two APLs of MBP antigenic peptides was halted due to adverse events indicative of auto-reactive antibodies (i.e. immediate-type hypersensitivity reactions [57]) or substantial expansion of pro-inflammatory T cells that were cross-reactive with the MBP autoantigens [58].

Because Copaxone works as a therapeutic vaccine, anti-drug antibodies are detectable in all treated patients [48, 49, 52]. These antibodies, however, do not neutralize biological activity or clinical efficacy and are not associated with local or systemic adverse effects in RRMS patients receiving chronic treatment [49]. Anti-Copaxone antibody titers and isotypes change over time with repeated drug administration [48, 49, 59]. Although anti-Copaxone antibodies are predominantly of the IgG subclasses over time [48, 49, 60, 61], there have been rare reports of anti-Copaxone IgE antibodies associated with anaphylactic reactions that can arise up to 10 months to a year after treatment initiation, with no symptomology beforehand to signal hypersensitivity [62, 63].

A purported follow-on product of glatiramer acetate is currently marketed in India and the Ukraine (Glatimer, Natco Pharma Ltd, Hyderabad, India). There are no published data of the safety, efficacy, or immunogenicity of this product at this writing. In analytical tests, this product demonstrated physicochemical differences from Copaxone and poor batch-to-batch reproducibility [8, 20, 64]. When activatedex vivo with Glatimer, splenocytes from GA-treated mice showed distinctly different gene transcription profiles among different batches, and between Glatimer and Copaxone, see Figure 2 [64].


A comparability trial of a generic glatiramer acetate product (GTR, Synthon VB) and Copaxone is currently underway in RRMS patients [65]. The GATE trial (ClinicalTrials.gov NCT01489254) is a 24-month study comprising a 9-month, placebo-controlled, active-comparator phase followed by a 15-month open-label phase in which all participants remaining in the study receive GTR. Characterizing the immunogenicity of GTR is not an objective of the study because the protocol suggests that anti-GTR antibodies will be the same as anti-Copaxone antibodies; specifically, that because anti-Copaxone antibodies are not neutralizing, anti-GTR antibodies will not be neutralizing either [65]. This assumption requires verification: GTR may be shown to be a close approximation to Copaxone at best and small differences in amino acid sequences or of protein folding in GTR could generate an antibody repertoire with different, isotopes, specificities, and affinities from those of anti-Copaxone antibodies, with variable consequences on patient safety and response to therapy [10, 13, 66, 67]. Accordingly, experts in the field of MS agree that the immunogenicity of follow-on versions of Copaxone cannot be assumed and should be established for each formulation [23].

According to the study protocol, the assessment of the immunogenicity of GTR is to compare proportions of subjects who develop anti-drug antibodies after receiving GTR or Copaxone [65]. As antigen-based therapeutic vaccines, antibody development to either drug would be expected in 100% of treated participants. Thus, it will not be possible to compare efficacy outcomes in patients free from anti-GTR antibodies with outcomes in GTR-antibody-positive patients to determine potential formation of NABs.

In the US, at least three manufacturers have filed ANDAs for follow-on glatiramer acetate products with FDA under the small-molecule generic pathway [6871]. The manufacturers maintain that these products will be interchangeable with Copaxone, despite the fact that the first clinical exposure to these products in MS patients will occur post-approval. Given the complexity, unknown mechanism of action, uncharacterized epitopes, and strong immunogenicity of Copaxone; the variable nature of RRMS disease activity; and the inter- and intra-patient variability of antibody responses to immunogenic drugs; adequate testing of the immunogenicity of uncharacterized follow-on glatiramoid products in MS patients should precede approval and marketing of these products.

Considerations for approval of follow-on NBCDs

The generic approach should be limited to products that can be fully characterized and allow prediction of biological effects with pharmacokinetics data as surrogates for clinical efficacy [11]. Because NBCDs have many of the same features as biologicals, it seems prudent to extend guidelines for biosimilar products to follow-on NBCDs [2, 5, 24, 72]. When it is not possible to prove bioequivalence of follow-on NBCDs, requiring non-clinical and clinical testing can ensure therapeutic equivalence between NBCDs and the reference drug. Comparability evaluations for a follow-on NBCD should include physicochemical properties, impurities, biological activity, pharmacokinetics, efficacy, and safety, see Table 2. The extent of testing needed to establish adequate similarity between an originator drug and a follow-on product will likely depend on NBCD complexity, mode of action (if known), and the potential for toxicity. The risks of free substitution between an uncharacterized, immunogenic NBCD and a follow-on product will remain unknown without a clinical crossover study that provides direct evidence that repeated switching between the reference and the generic drug has no negative impact.


For immunogenic NBCDs, it may be necessary to ensure that the immunologic and immunogenic safety of the follow-on NBCD is comparable to that of the reference drug in clinical studies in patients with the disease under study. Considerable inter-individual variability in antibody responses warrants assessment in a sufficient number of patients to characterize variability in antibody responses. Additionally, evaluation of a follow-on NBCD should ensure that anti-drug antibodies do not neutralize drug efficacy or bind to endogenous proteins; and characterize the immunologic effects of switching between a reference NBCD and a generic product.

In many countries generic approval of a follow-on product allows automatic substitution at the pharmacy level. While there is continued pressure worldwide to reduce drug costs, a major concern is whether patient safety and well-being are compromised by automatic substitution or interchange with follow-on products [73]. Commonly, clinicians, caregivers and patients are not aware of the change in medication [11], often to the frustration of prescribing physicians [7477]. At minimum, substitution of NBCDs without the involvement of a healthcare professional should be discouraged. Generally, patients should not be automatically switched to a generic NBCD if they are doing well. If a switch is unavoidable, the safety and efficacy of the new product should be monitored [78].

In some instances, substituting a lower priced generic for an innovator drug has resulted in higher healthcare utilization and overall costs [16, 17, 25] due to decreased efficacy or adverse events. Overall, drug product replacement that is guided by acquisition cost only may increase other costs and not be cost-effective from the patient’s and payer’s perspective [11].


Patients, physicians, and third-party payers expect generic products to be equally safe and comparably effective to the reference drug. For follow-on NBCDs, this will likely require more thorough assessment than the current generic drug approval process. Ultimately, regulatory requirements for approval and interchangeability of follow-on NBCDs will probably require a ‘case-by-case’ approach.

As FDA approaches the challenge of developing guidelines for follow-on NBCDs, it will be important to include a variety of constituents in the process. Members of the medical community have expressed concerns about the safety and efficacy of biosimilar drugs that indicate an increasing lack of trust of the drug regulatory process, primarily due to ‘an absence of the organized medical community in the public process of creating and updating the guidelines’ [74]. The same may hold true for follow-on NBCDs. Regulators must operate in different worlds to balance legal, scientific, and public health considerations as legislation for approval of follow-on NBCD products evolves. Scientific discussion and multidisciplinary research between experts from academia, industry, the medical community, and regulatory bodies; and consensus discussions with all stakeholders on an international level will aid in development of meaningful regulatory guidelines to ensure the safety and effectiveness of follow-on NBCD products.


1. Biologics Price Competition and Innovation Act of 2009, Public Law 111-148, Sec. 7001-7003, 124 Stat. 119. Mar. 23, 2010.
2. U.S. Department of Health and Human Services. Guidance for industry scientific considerations in demonstrating biosimilarity to a reference product. Draft guidance. Februray 2012 [homepage on the Internet]. 2012 Feb [cited 2014 Feb 24]. Available from: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM291128.pdf
3. U.S. Department of Health and Human Services. Guidance for industry biosimilars questions and answers regarding implementation of the Biologics Price Competition Act of 2009. Draft guidance. Februray 2012 [homepage on the Internet]. 2012 Feb [cited 2014 Feb 24]. Available from: http://www.fda.gov/downloads/Drugs/Guidances/UCM273001.pdf
4. U.S. Department of Health and Human Services. Guidance for industry quality considerations in demonstrating biosimilarity to a reference protein product. Draft guidance. Februray 2012 [homepage on the Internet]. 2012 Feb [cited 2014 Feb 24]. Available from: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM291134.pdf
5. European Medicines Agency. Guideline on immunogenicity assessment of biotechnology-derived therapeutic proteins. EMEA/CHMP/BMWP/14327/2006. 13 December 2007 [homepage on the Internet]. 2008 Jan [2014 Feb 24]. Available from: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003946.pdf
6. European Medicines Agency. Guideline on similar biological medicinal products. CHMP/437/04. 30 October 2005 [homepage on the Internet]. 2005 Nov [cited 2014 Feb 24]. Available from: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003517.pdf
7. European Medicines Agency. Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substance: non-clinical and clinical issues. EMEA/CHMP/BMWP/42832/2005 Rev. 1. 3 June 2013 [homepage on the Internet]. 2013 Jun [cited 2014 Feb 24]. Available from: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2013/06/WC500144124.pdf
8. Nicholas JM. Complex drugs and biologics: scientific and regulatory challenges for follow-on products. Drug Inf J. 2012;46(2):197-206.
9. Schellekens H, Klinger E, Mühlebach S, Brin JF, Storm G, Crommelin DJ. The therapeutic equivalence of complex drugs. Regul Toxicol Pharmacol. 2011;59(1):176-83.
10. Varkony H, Weinstein V, Klinger E, et al. The glatiramoid class of immunomodulator drugs. Expert Opin Pharmacother. 2009;10(4):657-68.




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Author: Tilda Barliya PhD

Ocular drug delivery is a very challenging field for pharmaceutical scientists.  The unique structure of the eye restricts the entry of drug molecules at the required site of action. The eye and its drugs are classically divided into : Anterior and Posterior segments (1).

Conventional systems like eye drops, suspensions and ointments cannot be considered optimal in  the treatment of vision threatening ocular diseases yet  more than 90% of the marketed ophthalmic formulations are in the form of eye drops.

In the majority of these topical  formulations which target the anterior chamber (the front of the eye) are washed off from the eye by various mechanisms:

  • lacrimation,
  • tear dilution
  • tear turnover
  • Moreover, human cornea comprising of epithelium, substantia propria and endothelium also restricts the ocular entry of drug molecules

Under normal condition the human eye can hold about 25–30 μl of an ophthalmic solution; however after a single blink the volume is reduced to 7–10 μl through nasolacrimal drainage which cause the drug to be systemically absorbed across the nasal mucosa or the gastrointestinal tract. A significant systemic loss from topically applied drugs also occurs from conjunctival absorption into the local circulation (4)

Thus resulting in low ocular  bioavailability of drugs with less than 5% of the drugs entering the eye.   Recently many drug efflux pumps have been identified and significant  enhancement in ocular drug absorption was achieved following their inhibition or evasion. But prolonged use of such inhibitors may result in undesirable effects.

Targeting the posterior chamber is even more difficult due to the tight junctions  of blood retinal barrier (BRB) restrict the entry of systemically administered drugs into the retina. Drugs are therefore delivered to the posterior chamber via:

  • Intravitreal injections
  • Implants
  • periocular injections

Here’s an illustration of the several ocular drug and their administration path

The success of nanoparticle systems for ocular drug delivery may depend on optimizing lipophilic-hydrophilic properties of the polymer-drug system, optimizing rates of biodegradation, and safety. Polymers used for the preparation of nanoparticles should be mucoadhesive and biocompatible. The choice of polymer plays an important role in the release kinetics of the drug from a nanoparticle system (4).

The choice of polymer plays an important role in the release kinetics of the drug from a nanoparticle system. Ocular bioavailability from a mucoadhesive dosage form will depend on the polymer’s bioadhesion characteristics, which are affected by its swelling properties, hydration time, molecular weight, and degree of crosslinking. The binding of drug depends on the physicochemical properties of the molecule as well as of the nanoparticle polymer, and also on the manufacturing process for these nanoparticle systems (4).

Other areas in which nanotechnology may be used is the use as biosensors, cell delivery and scaffolds etc (2)

Delivery of a drug via nanotechnology based product fulfills mainly three  objectives as follows:

  1. enhances drug permeation
  2. controls the release of drug
  3. targets drug

Tiwari et al (1) nicely covered different ocular delivery systems available. In this section we’ll review only few of the these drug products:

Viscosity improver:

The viscosity enhancers used are hydrophilic polymers such as cellulose, polyalcohol and polyacrylic acid. Sodium carboxy methyl cellulose is one of the most important mucoadhesion polymers having mono adhesive strength. Viscosity vehicles increases the contact time and no marked sustaining effect are seen.


Prodrugs enhance comeal drug permeability through modification of the hydrophilic or lipophilicity of the drug . The method includes modification of chemical structure of the drug molecule, thus making it selective, site specific and a safe ocular drug delivery system. Drugs with increased penetrability through prodrug formulations are epinephrine1, phenylephrine, timolol, and pilocarpine. The main indication of these drugs is to treat glaucoma thought epinephrine1 and phenylephrine are also being used to treat redness of the eye  and/or part of dialing eye-drops.

Colloidal Carriers:
Nanoparticles  provide sustained release-and prolonged therapeutic activity when retained in the cul-de-sac after  topical administration and the entrapped drug must be released from the particles at an appropriate rate. Most commonly used polymers are venous poly (alkyl cyanoacrylates), poly Scaprolactone and polylactic-co-glycolic acid, which undergo hydrolysis in tears. Enhanced permeation across the cornea was also observed when poly (epsilon-caprolactone) nanoparticles were coated with polyethylene glycol.


Liposomes are lipid vesicles containing aqueous core which have been widely exploited in ocular delivery for various drug molecules.Liposomes are favorable for lipophilic drugs and not for-hydrophilic drugs. liposomes has an affinity to bind to, ocular surfaces, and release contents at optimal rates. Coating with bioadhesive polymers to liposomes, prolong the  precomea retention of liposomes. Carbopol 1342-coated pilocarpine containing liposomes were  shown to produce a longer duration of action. Ciprofloxacin (CPFX) was also formulated in  liposomal environmental which lowered tear-driven dilution in the conjunctival sac.  Multilamellar vesicles from lecithin and alpha-L-dipalmithoyl-phosphatidylcholine were used to prepare liposome containing CPFX. This approach produced sustained release of the drug  depending on the nature of the lipid composition selected.

There are many other known forms used in the industry to enhance drug penetration and bioavailability such as dendrimers, bioadhesive polymers, niosomes and microemulsions which will be discussed elsewhere.


Drug delivery by topical and intravitreal routes cannot always be considered safe, effective and patient friendly. Drug delivery by periocular route can potentially overcome many of these limitations and also can provide sustained drug levels in  ocular pathologies affecting both segments. Transporter targeted delivery can be a promising  strategy for many drug molecules. Colloidal carriers can substantially improve the current therapy and may emerge as an alternative following their periocular administration. Ophthalmic drug delivery, more than any other route of administration, may benefit to a full extent from the characteristics of nano-sized drug particles. Other aspect of nanotechnology and ocular drug delivery will be discussed in the next chapter.


1. Tiwari A and Shukla KR. Novel ocular drug delivery systems: An overview. J. Chem. Pharm. Res., 2010, 2(3):348-355

Click to access JOCPR-2010-2-3-348-355.pdf

2. Kalishwaralal K., Barathmanikanth S., Pandian SR, Deepak V and Gurunathan S.  Silver nano-a trove for retinal therapies. J Control Release  2010 Jul 14;145(2):76-90http://www.ncbi.nlm.nih.gov/pubmed/20359511

3.Cholkar K., Patel SP., Vadlapudi AD and Mitra AK. Novel Strategies for Anterior Segment Ocular Drug Delivery. J Ocul Pharmaco Ther  2012 Dec 5. [Epub ahead of print]

4. Bucolo C., Drago F and Salomone S. Ocular drug delivery: a clue from nanotechnology. Front Pharmacol. 2012; 3: 188.


5. Vega E., Gamisans F., García M. L., Chauvet A., Lacoulonche F., Egea M. A. (2008). PLGA nanospheres for the ocular delivery of flubiprofen: drug release and interactions. J. Pharm. Sci.97, 5306–5317.

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