Pankaj Karande

Discovery of transdermal penetration enhancers by high-throughput screening

Although transdermal drug delivery is more attractive than injection, it has not been applied to macromolecules because of low skin permeability. Here we describe particular mixtures of penetration enhancers that increase skin permeability to macromolecules (∼1–10 kDa) by up to ∼100-fold without inducing skin irritation. The discovery of these mixtures was enabled by an experimental tool, in vitro skin impedance guided high-throughput (INSIGHT) screening, which is >100-fold more efficient than current tools. In vitro experiments demonstrated that the mixtures delivered macromolecular drugs, including heparin, leutinizing hormone releasing hormone (LHRH) and oligonulceotides, across the skin. In vivo experiments on hairless rats with leuprolide acetate confirmed the potency and safety of one such mixture, sodium laureth sulfate (SLA) and phenyl piperazine (PP). These studies show the feasibility of using penetration enhancers for systemic delivery of macromolecules from a transdermal patch.

Enhancement of transdermal drug delivery via synergistic action of chemicals

Transdermal drug delivery is an attractive alternative to conventional techniques for administration of systemic therapeutics. One challenge in designing transdermal drug delivery systems is to overcome the natural transport barrier of the skin. Chemicals offer tremendous potential in overcoming the skin barrier to enhance transport of drug molecules. Individual chemicals are however limited in their efficacy in disrupting the skin barrier at low concentrations and usually cause skin irritation at high concentrations. Multicomponent mixtures of chemicals, however, have been shown to provide high skin permeabilization potency as compared to individual chemicals without necessarily causing irritation. Here we review systems employing synergistic mixtures of chemicals that offer superior skin permeation enhancement. These synergistic systems include solvent mixtures, microemulsions, eutectic mixtures, complex self-assembled vesicles and inclusion complexes. Methods for design and discovery of such synergistic systems are also discussed.

Relationships between skin's electrical impedance and permeability in the presence of chemical enhancers.

Stratum corneum, the outermost layer of the skin, offers a strong barrier to the movement of solutes as well as ions. We report on the existence of a relationship between skins electrical impedance and its permeability to hydrophilic (mannitol and inulin) as well as hydrophobic (corticosterone and estradiol) solutes in the presence of 33 distinct chemical penetration enhancer formulations. The correlation between impedance and permeability was excellent (r2=0.8) for hydrophilic solutes and moderate, yet significant (r2 approximately 0.5), for hydrophobic solutes. The possibility of using skins electrical impedance to choose potent formulations was quantitatively assessed. Percentile ranking of penetration enhancers based on their effect on skins electrical impedance matched well with the ranking based on their effect on solute permeability (r2>0.9 for both mannitol and estradiol). These studies demonstrate the feasibility of using skins electrical impedance to screen potent chemical enhancers.

High throughput screening of transdermal formulations.

AbstractPurpose. Applications of transdermal drug delivery are limited by low skin permeability. Many chemicals have been used to enhance skin permeability, however, only a handful are actually used in practice. Combinations of chemicals are likely to be more efficient in enhancing skin permeability compared to individual enhancers. However, identification of efficient enhancer combinations is quite challenging because many chemical enhancers interact with each other and with the skin in a complex manner. In the absence of a fundamental knowledge of such interactions, we need to rely on rapid methods to screen various enhancer combinations for their effectiveness. In this paper, we report a novel high throughput (HTP) method that is at least 50-fold more efficient in terms of skin utilization and up to 30-fold more efficient in terms of holdup times than the current methods for formulation screening (Franz diffusion cells). Methods. A high throughput method was developed based on skin conductivity and mannitol penetration into the skin. This method was used to perform at least 100 simultaneous tests per day. Detailed studies were performed using two model enhancers, sodium lauryl sulfate (SLS) and dodecyl pyridinium chloride (DPC). The predictions of the high throughput method were validated using Franz diffusion cells. Results. High throughput screening revealed that mixtures of SLS and DPC are significantly more effective in enhancing transdermal transport compared to each of them alone. Maximum efficiency was observed with near-equimolar mixtures of SLS: DPC. The predictions of the HTP method compared well against those made using Franz diffusion cells. Specifically, the effect of surfactant mixtures on skin conductivity and mannitol permeability measured using Franz cells also showed a maximum at near-equimolar mixtures of SLS: DPC. Conclusions. The novel HTP method allows rapid screening of enhancer formulations for transdermal applications. This method can be used to discover new and effective enhancer mixtures. At the same time, these data may also broaden our understanding of the effect of enhancers on skin permeability.

Specificity of Growth Inhibitors and their Cooperative Effects in Calcium Oxalate Monohydrate Crystallization

The molecular recognition and interactions governing site-specific adsorption of growth inhibitors on crystal surfaces can be tailored in order to control the anisotropic growth rates and physical properties of crystalline materials. Here we examine this phenomenon in calcium oxalate monohydrate (COM) crystallization, a model system of calcification with specific relevance for pathological mineralization. We analyzed the effect of three putative growth inhibitors--chondroitin sulfate, serum albumin, and transferrin--using analytical techniques capable of resolving inhibitor-crystal interactions from interfacial to bulk scales. We observed that each inhibitor alters surface growth by adsorbing on to distinct steps emanating from screw dislocations on COM surfaces. Binding of inhibitors to different crystallographic faces produced morphological modifications that are consistent with classical mechanisms of layer-by-layer crystal growth inhibition. The site-specific adsorption of inhibitors on COM surfaces was confirmed by bulk crystallization, fluorescent confocal microscopy, and atomic force microscopy. Kinetic studies of COM growth at varying inhibitor concentrations revealed marked differences in their efficacy and potency. Systematic analysis of inhibitor combinations, quantified via the combination index, identified various binary pairings capable of producing synergistic, additive, and antagonistic effects. Collectively, our investigation of physiologically relevant biomolecules suggests potential roles of COM inhibitors in pathological crystallization and provides guiding principles for biomimetic design of molecular modifiers for applications in crystal engineering.

Transcutaneous Immunization: An Overview of Advantages, Disease Targets, Vaccines, and Delivery Technologies

Skin is an immunologically active tissue composed of specialized cells and agents that capture and process antigens to confer immune protection. Transcutaneous immunization takes advantage of the skin immune network by inducing a protective immune response against topically applied antigens. This mode of vaccination presents a novel and attractive approach for needle-free immunization that is safe, noninvasive, and overcomes many of the limitations associated with needle-based administrations. In this review we will discuss the developments in the field of transcutaneous immunization in the past decade with special emphasis on disease targets and vaccine delivery technologies. We will also briefly discuss the challenges that need to be overcome to translate early laboratory successes in transcutaneous immunization into the development of effective clinical prophylactics.

Erythropoietin receptor contributes to melanoma cell survival in vivo

Erythropoietin (Epo) is widely used clinically to treat anemia associated with various clinical conditions including cancer. Data from several clinical trials suggest significant adverse effect of Epo treatment on cancer patient survival. However, controversy exists whether Epo receptor (EpoR) is functional in cancer cells. In this study, we demonstrated that EpoR mRNA expression was detectable in 90.1% of 65 melanoma cell lines, and increased copy number of the Epo and EpoR loci occurred in 30 and 24.6% of 130 primary melanomas, respectively. EpoR knockdown in melanoma cells resulted in diminished ERK phosphorylation in response to Epo stimulation, decreased cell proliferation and increased response to the inhibitory effect of hypoxia and cisplatin in vitro. EpoR knockdown significantly decreased melanoma xenograft size and tumor invasion in vivo. On the contrary, constitutive activation of EpoR activated cell proliferation pathways in melanoma cells and resulted in increased cell proliferation and resistance to hypoxia and cisplatin treatment in vitro. EpoR activation resulted in significantly larger xenografts with increased tumor invasion of surrounding tissue in vivo. Daily administration of recombinant Epo fails to stimulate melanoma growth in vivo, but the treatment increased vascular size in the xenografts. Increased local recurrence after excision of the primary tumors was observed after Epo treatment. Epo induced angiogenesis in Matrigel plug assays, and neutralization of Epo secreted by melanoma cells results in decreased angiogenesis. These data support that EpoR is functional in melanoma and EpoR activation may promote melanoma progression, and suggest that Epo may stimulate angiogenesis and increase survival of melanoma cells under hypoxic condition in vivo.

Packaging biological cargoes in mesoporous materials: opportunities for drug delivery

Introduction: Confinement of biomolecules in structured nanoporous materials offers several desirable features ranging from chemical and thermal stability, to resistance to degradation from the external environment. A new generation of mesoporous materials presents exciting new possibilities for the formulation and controlled release of biological agents. Such materials address niche applications in enteral and parenteral delivery of biologics, such as peptides, polypeptides, enzymes and proteins for use as therapeutics, imaging agents, biosensors, and adjuvants. Areas covered: Mesoporous silica Santa Barbara Amorphous-15 (SBA-15), with its unique, tunable pore diameter, and easily functionalized surface, provides a representative example of this new generation of materials. Here, we review recent advances in the design and synthesis of nanostructured mesoporous materials, focusing on SBA-15, and highlight opportunities for the delivery of biological agents to various organ and tissue compartments. Expert opinion: The SBA-15 platform provides a delivery carrier that is inherently separated from the active biologic due to distinct intra and extra-particle environments. This permits the SBA-15 platform to not require direct modification of the active biological therapeutic. Additionally, this makes the platform universal and allows for its application independent of the desired methods of discovery and development. The SBA-15 platform also directly addresses issues of targeted delivery and controlled release, although future challenges in the implementation of this platform reside in particle design, biocompatibility, and the tunability of the internal and external material properties. Examples illustrating the flexibility in the application of the SBA-15 platform are also discussed.

Transcutaneous immunization using common chemicals

Transcutaneous immunization, topical application of vaccines on skin, provides several advantages over needle based immunization. However, simple topical application of vaccines does not generate sufficient immune response due to limited transport of vaccines across the stratum corneum of skin. Here we report that chemicals used in common skin products can enhance the immunogenicity of topically applied antigens. Six hundred formulations of commonly used chemicals were screened systematically for their potency (delivery of antigen) in vitro. A selected subset of these formulations was subsequently tested for their adjuvanticity (activation of immune response) in vitro. Lead formulations were tested in vivo for their ability to generate antibody titers against topically applied ovalbumin, a model antigen. Lead formulations were significantly more effective in generating anti-ovalbumin IgG titers. Our results demonstrate that chemical formulations can be successfully used to deliver antigens and that such formulations can be rationally designed by combinatorial screening of individual chemical components.

Natural Promoters of Calcium Oxalate Monohydrate Crystallization

Crystallization is often facilitated by modifiers that interact with specific crystal surfaces and mediate the anisotropic rate of growth. Natural and synthetic modifiers tend to function as growth inhibitors that hinder solute attachment and impede the advancement of layers on crystal surfaces. There are fewer examples of modifiers that operate as growth promoters, whereby modifier-crystal interactions accelerate the kinetic rate of crystallization. Here, we examine two proteins, lysozyme and lactoferrin, which are observed in the organic matrix of three types of pathological stones: renal, prostatic, and pancreatic stones. This work focuses on the role of these proteins in the crystallization of calcium oxalate monohydrate (COM), the most prominent constituent of human kidney stones. Using a combination of experimental techniques, we show that these proteins, which are rich in l-arginine and l-lysine amino acids, promote COM growth. The synthesis and testing of peptides derived from contiguous segments of lysozymes primary amino acid sequence revealed subdomains within the protein that operate either as an inhibitor or promoter of COM growth, with the latter exhibiting efficacies that nearly match that of the protein. We observed that cationic proteins promote COM growth over a wide range of modifier concentration, which differs from calcification promoters in the literature that exhibit dual roles as promoters and inhibitors at low and high concentration, respectively. This seems to suggest a unique mechanism of action for lysozyme and lactoferrin. Possible explanations for their effects on COM growth and crystal habit are proposed on the basis of classical colloidal theories and the physicochemical properties of peptide subdomains, including the number and spatial location of charged or hydrogen-bonding moieties.