The non-parenteral delivery of peptides via mucosal routes is essential for the enlargement of th... more The non-parenteral delivery of peptides via mucosal routes is essential for the enlargement of the therapeutic benefit of various oligopeptides. Among the routes under discussion the buccal mucosa was found to allow permeation of therapeutically relevant doses, e.g. with insulin, oxytocin, vasopressin analogs, protirelin, and octreotide. However, the overall pe~eability is relatively low, currently still rendering the buccal mucosa a secondary choice in mucosal peptide delivery. The bioavailability of buccal delivery has turned out to be more or less disappointing, usually being in the range of one or more orders of magnitude lower than found with other mucosal sites, in particular with the nasal mucosa. Demonstrations of how the buccal permeability may be raised, mainly concentrate on the use of absorption enhancers. In fact, major success was achieved by simultaneous administration of compounds which have been shown to be effective enhancers with other epithelia as well. More tissuespecific approaches are scarce. Typical are attempts to affect the proteoglycan matrix surrounding the epithelial cells. Another strategy is inhibition of local peptidase activity. Based on permeability considerations alone there would have been f~stration enough to make research on buccal peptide delivery a low priority area. But it is because of the excellent accessibility of the absorption site, the elegance of muco-adhesive dosage forms, the prospects of high patient acceptance, and, in particular, the robustness of this tissue and its fast cellular turnover , that will lead to continuing interest in this area. Under the aspect of its ability to rapid recovery, the toxicological issue of absorption enhancers may be not as dramatic with the buccal epithelium as with other mucosal sites.
Formulation of Silicone Matrix Systems for Long Term Constant Release of Peptides
Drug Development and Industrial Pharmacy, 1991
ABSTRACT Abstract Theoretically, release of drug through the water-filled pores of matrix systems... more ABSTRACT Abstract Theoretically, release of drug through the water-filled pores of matrix systems is expected to show a square-root-of-time dependence, with time exponents of 0.5 and hence continuosly declining release rates. Yet there have been many research groups finding remarkable deviations. The aim of this work was to investigate on factors which lead to deviations from the square-root-of-time law and may be helpful for the development of matrix systems with constant drug release for long time. Matrices of polydimethylsiloxane (PDMS) were prepared incorporating varying amounts of different pore-building, water soluble hydrogels. The hydrophilic model drug was Gly-Tyr. The following essential factors influencing the long-term release profiles were found: (i) total matrix loading, (ii) its dissolution rate and (iii) the viscosity of the pore-building hydrogel. A proper choice of conditions lead to release profiles with time-exponents up to 0.8 for a time period of several weeks.
European Journal of Pharmaceutics and Biopharmaceutics, Sep 1, 2004
Over the past decade, several classes and/or prototypes of cell penetrating peptides (CPP) have b... more Over the past decade, several classes and/or prototypes of cell penetrating peptides (CPP) have been identified and investigated in multiple aspects. CPP represent peptides, which show the ability to cross the plasma membrane of mammalian cells, and may thus give rise to the intracellular delivery of problematic therapeutic cargos, such as peptides, proteins, oligonucleotides, plasmids and even nanometer-sized particles, which otherwise cannot cross the plasma membrane. Most of the currently recognized CPP are of cationic nature and derived from viral, insect or mammalian proteins endowed with membrane translocation properties. The exact mechanisms underlying the translocation of CPP across the cellular membrane are still poorly understood. However, several similarities in translocation can be found. Early studies on CPP translocation mechanisms tended to suggest that the internalization of these peptides was neither significantly inhibited by low temperature, depletion of the cellular adenosine triphosphate (ATP) pool, nor by inhibitors of endocytosis. Moreover, chemical modification of the peptide sequence, such as the synthesis of retro-, enantio-or retroenantio-analogs, appeared not to affect the internalization properties. Therefore, translocation was concluded to result from direct, physical transfer through the lipid bilayer of the cell membrane. Later studies, however, showed convincing evidence for the involvement of endocytosis as the dominating mechanism for cellular internalization. In addition to describing the general properties of the commonly recognized classes of CPP, in this review we will also point out some limitations and typical pitfalls of CPP as carriers for therapeutics. In particular we will comment on emerging discrepancies with the current dogma, on cell-to-cell variability, biological barrier permeability, metabolic fate, toxicity and immunogenicity of CPP.
The study of skin metabolism is of prime importance not only in the field of transdermal drug del... more The study of skin metabolism is of prime importance not only in the field of transdermal drug delivery but also for the safe and efficient local skin treatment with topically applied substances. Since it has become clear that even peptides may be delivered across the permeation barrier of the stratum corneum, e.g. by means of iontophoresis, phonophoresis or electroporation, the enzymatic barrier of the epidermis deserves more attention as another important limiting factor for the dermai delivery of drugs. The purpose of this review is to give a survey of the major aspects concerning the assessment of the metabolism of xenobiotics in the skin. First, this review will focus on the localization of enzyme activity within the skin. Further, important aspects to be considered for the planning and evaluation of skin metabolism studies will be discussed: differences among species and requirements of skin quality. Among the various metabolic pathways within the skin broad interest will be concentrated on the metabolism of peptide drugs. Therefore, exopeptidases in the skin are the subject of an additional section. Also models for skin metabolism studies will be especially considered including the development of suitable cell culture models. Finally, major gap areas in the investigation of skin metabolism will be identified and summarized, namely: classes of xenobiotics, proteolytic enzyme activity in the skin, validity of experimental models, localization of enzyme activity and physical model development.
The ability to use magnetic nanoparticles for cell tracking, or for the delivery of nanoparticle-... more The ability to use magnetic nanoparticles for cell tracking, or for the delivery of nanoparticle-based therapeutic agents, requires a detailed understanding of probe metabolism and transport. Here we report on the development and metabolism of a dual fluorochrome version of our tat-CLIO nanoparticle termed Tat(FITC)-Cy3.5-CLIO. The nanoparticle features an FITC label on the tat peptide and a Cy3.5 dye directly attached to the cross-linked coating of dextran. This nanoparticle was rapidly internalized by HeLa cells, labeling 100% of cells in 45 min, with the amount of label per cell increasing linearly with time up to 3 h. Cells loaded with nanoparticles for 1 h retained 40-60% of their FITC and Cy3.5 labels over a period of 72 h in label-free media. Over a period of 144 h, or approximately 3.5 cell divisions, the T2 spin-spin relaxation time of cells was not significantly changed, indicating retention of the iron oxide among the dividing cell population. Using confocal microscopy and unfixed cells, both dyes were nuclear and perinuclear (broadly cytoplasmic) after Tat(FITC)-Cy3.5-CLIO labeling. Implications of the rapid labeling and slow excretion of the Tat(FITC)-Cy3.5-CLIO nanoparticle are discussed for cell tracking and drug delivery applications.
New aspects of pharmaceutical dosage forms for controlled drug delivery of peptides and proteins
European Journal of Pharmaceutical Sciences, Sep 1, 1994
Drug delivery’s quest for polymers: Where are the frontiers?
European Journal of Pharmaceutics and Biopharmaceutics, Nov 1, 2015
Since the legendary 1964 article of Folkman and Long entitled "The use of silicone rubbe... more Since the legendary 1964 article of Folkman and Long entitled "The use of silicone rubber as a carrier for prolonged drug therapy" the role of polymers in controlled drug delivery has come a long way. Today it is evident that polymers play a crucial if not the prime role in this field. The latest boost owes to the interest in drug delivery for the purpose of tissue engineering in regenerative medicine. The focus of this commentary is on a selection of general and personal observations that are characteristic for the current state of polymer therapeutics and carriers. It briefly highlights selected examples for the long march of synthetic polymer-drug conjugates from bench to bedside, comments on the ambivalence of selected polymers as inert excipients versus biological response modifiers, and on the yet unsolved dilemma of cationic polymers for the delivery of nucleic acid therapeutics. Further subjects are the complex design of multifunctional polymeric carriers including recent concepts towards functional supramolecular polymers, as well as observations on stimuli-sensitive polymers and the currently ongoing trend towards natural and naturally-derived biopolymers. The final topic is the discovery and early development of a novel type of biodegradable polyesters for parenteral use. Altogether, it is not the basic and applied research in polymer therapeutics and carriers, but the translational process that is the key hurdle to proceed towards an authoritative approval of new polymer therapeutics and carriers.
Current research and development of antigens for vaccination often center on purified recombinant... more Current research and development of antigens for vaccination often center on purified recombinant proteins, viral subunits, synthetic oligopeptides or oligosaccharides, most of them suffering from being poorly immunogenic and subject to degradation. Hence, they call for efficient delivery systems and potent immunostimulants, jointly denoted as adjuvants. Particulate delivery systems like emulsions, liposomes, nanoparticles and microspheres may provide protection from degradation and facilitate the co-formulation of both the antigen and the immunostimulant. Synthetic double-stranded (ds) RNA, such as polyriboinosinic acid-polyribocytidylic acid, poly(I:C), is a mimic of viral dsRNA and, as such, a promising immunostimulant candidate for vaccines directed against intracellular pathogens. Poly(I:C) signaling is primarily dependent on Toll-like receptor 3 (TLR3), and on melanoma differentiation-associated gene-5 (MDA-5), and strongly drives cell-mediated immunity and a potent type I interferon response. However, stability and toxicity issues so far prevented the clinical application of dsRNAs as they undergo rapid enzymatic degradation and bear the potential to trigger undue immune stimulation as well as autoimmune disorders. This review addresses these concerns and suggests strategies to improve the safety and efficacy of immunostimulatory dsRNA formulations. The focus is on technological means required to lower the necessary dosage of poly(I:C), to target surface-modified microspheres passively or actively to antigen-presenting cells (APCs), to control their interaction with non-professional phagocytes and to modulate the resulting cytokine secretion profile.
groups, between the alkyl groups, or between the alkyl and phenyl groups of the interacting speci... more groups, between the alkyl groups, or between the alkyl and phenyl groups of the interacting species. Also, in this context, the degree of effect of higher body temperature (fever) on the tertiary structure of vital biopolymers (enzymes), and consequently on their physiological activities, could be expected to depend on the nature of hydrophobic groups involved in hydrophobic bonding responsible for maintaining the tertiary structure of the biopolymers. REFERENCES (1) A. Korolkovas, "Essentials of Molecular Pharmacology," (2) W. Kauzmann, Aduu/i.
Method for producing 4-amino-5-chloro-1-phenyl pyridazinone-(6)
Fluorescence study of human calcitonin fibrillation kinetics using the hydrophobic probe Nile red
Peptides 1992, 1993
(D) Routes of delivery: Case studies
Advanced Drug Delivery Reviews, 1992
Abstract The columnar epithelium of the gastrointestinal tract, nasal and pulmonary airways is vi... more Abstract The columnar epithelium of the gastrointestinal tract, nasal and pulmonary airways is virtually impermeable to most peptides, proteins and other macromolecules. However, the very thin (0.1–0.2 μm) alveolar epithelium of the deep lung appears to be unique among the body's ports of entry in that it is permeable to some peptides and proteins. A dense network of endocytic vesicles in the type-1 alveolar epithelial cells offers a possible mechanism to explain how the airsacs remain nearly free of fluid and how macromolecules are subsequently absorbed. Three therapeutic peptides (leuprolide: nine amino acids, insulin: 51 amino acids and growth hormone: 192 amino acids) are absorbed in biologically active form from the lungs with bioavailabilities of 10%–25%. Inefficient aerosol delivery, aggregation and enzymatic degradation reduce bioavailability. Long-term safety remains a concern, although no serious effects have been observed in studies to date.
Serum protein adsorption to the surface of particulate synthetic drug carrier systems has a major... more Serum protein adsorption to the surface of particulate synthetic drug carrier systems has a major influence on their uptake by phagocytes. The influence of a2-human serum glycoprotein (a2GP) on the phagocytosis of various surface modified microparticles was studied in dendritic cells (DC) and was compared with a potent opsonin, IgG, and a dysopsonin, human serum albumin (HSA). The microparticles were administered to DC before and after the incubation with a2GP, IgG and HSA in single, binary or ternary protein systems and in whole blood serum. Phagocytosis of microparticles was vastly affected by the surface character of the microparticles themselves and by the adsorption of the proteins. Poly-l-lysine (PLL)-modified microparticles were under all conditions internalized with highest efficiency which is suggested to be mediated by their positive surface charge. The adsorption of commonly phagocytosis promoting proteins reduced the uptake of PLL-modified particles and is explained by compensation of the positive surface charge by the adsorbed negatively charged proteins. In all other particle types tested, freshly adsorbed a2GP was found to exhibit a strong phagocytosis promoting activity which was comparable to that of adsorbed IgG. Interestingly, this opsonic activity was lost already 2 h after adsorption to the particle surface. Protein adsorption from binary and ternary protein systems and from whole blood serum occurred in a competitive manner. Significant inhibition of phagocytosis was observed, even when HSA was combined with strong opsonins such as a2GP or IgG or in mixtures of all three proteins, indicating the importance of studying the influence of protein adsorption in protein mixtures.
The support of adenosine release from adenosine kinase deficient ES cells by silk substrates
Biomaterials, Sep 1, 2006
Adenosine kinase deficient (Adk-/-) embryonic stem cells (ESCs) encapsulated in synthetic polymer... more Adenosine kinase deficient (Adk-/-) embryonic stem cells (ESCs) encapsulated in synthetic polymers have previously been shown to provide therapeutic adenosine release and transient seizure suppression in epileptic rats. Here we explored the utility of biopolymer-substrates to promote long-term adenosine release from Adk-/- ESCs. Three different substrates were studied: (1) type I collagen (Col-1), (2) silk-fibroin (SF), and (3) poly(L-ornithine) (PO) coated tissue culture plastic. Adk-/- or wild type (wt) ESC-derived glial precursor cells were seeded on the substrates and cultured either in proliferation medium containing growth factors or in differentiation medium devoid of growth factors. In proliferation medium cell proliferation was higher and metabolic activity lower on Col-1 and PO substrates as compared to SF. Cells from both genotypes readily differentiated into astrocytes after growth factor removal on all substrates. Adk-/- cells cultured on biopolymers released significantly more adenosine than their wt counterparts at all developmental stages. Adenosine release was similar on SF and PO substrates and the amounts released from Adk-/- cells (>20 ng/ml) were considered to be of therapeutic relevance. Taken together, these results suggest that silk matrices are particularly suitable biomaterials for ESC encapsulation and for the design of adenosine releasing bioincubators for the treatment of epilepsy.
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Papers by Hans P Merkle