Delivery Of Therapeutic Peptides Research Articles

New pH-Sensitive Glycopolymers for Colon-Specific Drug Delivery

pH-sensitive hydrogels are suitable candidates for oral delivery of therapeutic peptides, proteins, and drugs, due to their ability to respond to environmental pH changes. New pH-sensitive glycopolymers have been developed by free-radical polymerization of methacrylic acid and 6-acryloyl-glucose-1, 2, 3, 4-tetraacetate, using 1, 6-hexandiol diacrylate and 1, 6-hexandiol propoxylate diacrylate as cross-linking agents. The hydrogels were characterized by differential scanning calorimetry and FTIR. Equilibrium swelling studies were carried out in enzyme-free simulated gastric and intestinal fluids (SGF and SIF, respectively). A model drug, olsalazine [3, 3′-azobis (6-hydroxy benzoic acid)] as an azo derivative of 5-aminosalicylic acid, was entrapped in these gels and the in vitro release profiles were established separately in both enzyme-free SGF and SIF. The drug-release profiles indicated that the amount of drug released depended on the degree of swelling. The hydrogels containing polar propoxylate groups were hydrolyzed rather easily.

Design and evaluation of a chitosan–aprotinin conjugate for the peroral delivery of therapeutic peptides and proteins susceptible to enzymatic degradation

One main barrier for the peroral administration of therapeutic peptides and proteins is the enzymatic barrier, that is mediated by luminally secreted and membrane bound proteolytic enzymes. It was the aim of the study to synthesise, characterise and evaluate a novel polymer–inhibitor conjugate in order to improve the bioavailability of orally-administered peptides and proteins. The trypsin/chymotrypsin inhibitor aprotinin was covalently bound to chitosan. The percentage of the inhibitor in the polymer–inhibitor conjugate (m/m) was determined to be between 1.11 ± 0.36 and 1.92 ± 0.05%. In vitro enzyme assays clearly demonstrated the potential of the novel conjugate to inhibit trypsin and chymotrypsin. Moreover, studies in rats were performed to evaluate the efficacy of the conjugate in vivo. Eight hours after oral administration of tablets containing insulin and the novel chitosan–aprotinin conjugate, the mean blood glucose level decreased to 84 ± 6%. In contrast, the mean blood glucose level in the control group increased to 121 ± 8% of the initial measured blood glucose level. In conclusion it was demonstrated that chitosan–aprotinin conjugate represents a novel and promising tool for the oral administration of therapeutic peptides and proteins susceptible to enzymatic degradation caused by trypsin and chymotrypsin.

Targeted Therapeutic Peptide Delivery to Synaptic Junctions as Botulism Countermeasure

Targeted Therapeutic Peptide Delivery to Synaptic Junctions as Botulism Countermeasure

Synthesis and characterization of acrylic type hydrogels containing azo derivatives of 5-amino salicylic acid for colon-specific drug delivery

pH-sensitive hydrogels are suitable candidates for oral delivery of therapeutic peptides, proteins and drugs, due to their ability to respond to environmental pH changes. Terephthalic acid was covalently linked with 2-hydroxyethyl methacrylate (HEMA), abbreviated as cross-linking agent (CA). Acryloyl ester of 5-[4-(hydroxy phenyl) azo] salicylic acid (HPAS) as an azo derivative of 5-amino salicylic acid (5-ASA) was prepared under mild conditions. The HPAS was covalently linked with acryloyl chloride, abbreviated as APAS. Free radical cross-linking copolymerization of polymerizable azo derivative of 5-ASA (APAS) and methacrylic acid (MAA) in two different molar ratios, with the various ratios CA as cross-linking agent were carried out with using 2, 2'-azobisisobutyronitrile (AIBN) as initiator at the temperature range 60-70 degrees C. The composition of the cross-linked three-dimensional polymers was determined by FTIR spectroscopy. Glass transition temperature (Tg) of the network polymers was determined calorimetrically. The hydrolysis of drug-polymer conjugates was carried out in cellophane membrane dialysis bags containing aqueous buffer solutions (pH 7.4 and pH 1) at 37 degrees C. The effect of copolymer composition on the hydrolytic degradation was studied in simulated gastric fluid (SGF, pH 1) and simulated intestinal fluid (SIF, pH 7.4) at 37 degrees C. Monitoring of the hydrolysis process by HPLC and UV spectroscopy shows that the azo prodrug (HPAS) was released by hydrolysis of the ester bond located between the HPAS and the polymer chain. The drug-release profiles indicate that amount drug release dependent on the content of MAA groups and crosslinking.

PH-sensitive hydrogel containing acetaminophen silyl ethers for colon-specific drug delivery

pH-sensitive hydrogels are suitable candidates for oral delivery of therapeutic peptides, proteins and drugs, due to their ability to respond to environmental pH changes. Acetaminophen dimethylvinylsilyl ether was prepared under mild conditions. Cubane-1,4-dicarboxylic acid (CDA) linked to two HEMA groups, giving cubane-1,4-bis(methacryloyloxyethyl) carboxylate (CA), was used as cross-linker. The drug linked to dimethylvinylsilyl was abbreviated as ADVSi. Free radical cross-linking co-polymerization of polymerizable ADVSi with methacrylic acid (MAA) or N-vinyl-2-pyrrolidinone (NVP) with various ratios of CA as cross-linking agent was carried out with using AIBN as initiator in the temperature range 60–70°C. The structure of the ADVSi was confirmed by FT-IR and 1H-NMR spectroscopy. The composition of the cross-linked three-dimensional polymers was determined by FT-IR spectroscopy. The glass transition temperature (T g) of the network polymers was determined calorimetrically. The hydrolysis of drug–polymer conjugates was carried out in cellophane membrane dialysis bags containing aqueous buffer solutions (pH 7.4 and pH 1) at 37°C. The effect of co-polymer composition on the hydrolytic degradation was studied in simulated gastric fluid (SGF, pH 1) and simulated intestinal fluid (SIF, pH 7.4) at 37°C. Detection of the hydrolysis product by UV spectroscopy shows that acetaminophen was released by hydrolysis of the ether bond located between the drug and the polymer chain. The drug-release profiles indicate that the amount of drug released depends on the composition of polymers.

Pulmonary delivery of therapeutic peptides via dry powder inhalation: effects of micronisation and manufacturing

Pulmonary drug delivery is increasingly appreciated as a route of administration for systemically acting proteins and peptides. A respirable particle size of the drug is a key requirement, but the fragile nature of many proteins may be a limitation for the application of conventional production processes. The aim of this study was to examine the effect of different micronisation processes on the degradation and aerodynamic properties of the GnRH-antagonist cetrorelix in order to enable its application by a dry powder inhaler (Novolizer ®). A modified pearl mill was used for milling in fluid propellant. Furthermore, a spray drying procedure was established using a novel process of atomisation and drying. Adhesive mixtures of lactose and 5–20% of micronised cetrorelix-acetate were prepared. Analysis by laser light scattering, HPLC, Karl Fischer, cascade impactor and scanning electron microscopy were performed to characterise the manufactured powders. Both micronisation procedures succeeded in producing small range particle size distributions, suitable for deep lung deposition (D50=1.6 μm for milling and 3.3 μm for spray drying). The pearl milled cetrorelix showed promising results when delivered by the Novolizer ®: a reproducible and highly efficient dispersion of the drug was achieved (around 60% of aerosolised drug <5 μm). The spray dried drug was not suitable when processed as adhesive mixture.

Pulmonary delivery of therapeutic peptides via dry powder inhalation: effects of micronisation and manufacturing

Pulmonary drug delivery is increasingly appreciated as a route of administration for systemically acting proteins and peptides. A respirable particle size of the drug is a key requirement, but the fragile nature of many proteins may be a limitation for the application of conventional production processes. The aim of this study was to examine the effect of different micronisation processes on the degradation and aerodynamic properties of the GnRH-antagonist cetrorelix in order to enable its application by a dry powder inhaler (Novolizer ® ). A modified pearl mill was used for milling in fluid propellant. Furthermore, a spray drying procedure was established using a novel process of atomisation and drying. Adhesive mixtures of lactose and 5–20% of micronised cetrorelix-acetate were prepared. Analysis by laser light scattering, HPLC, Karl Fischer, cascade impactor and scanning electron microscopy were performed to characterise the manufactured powders. Both micronisation procedures succeeded in producing small range particle size distributions, suitable for deep lung deposition (D50=1.6 μm for milling and 3.3 μm for spray drying). The pearl milled cetrorelix showed promising results when delivered by the Novolizer ® : a reproducible and highly efficient dispersion of the drug was achieved (around 60% of aerosolised drug <5 μm). The spray dried drug was not suitable when processed as adhesive mixture.

Development and evaluation of buccoadhesive propranolol hydrochloride tablet formulations: effect of fillers

The buccal mucosa has been investigated for local and systemic delivery of therapeutic peptides and other drugs that are subjected to first-pass metabolism or are unstable within the rest of the gastrointestinal tract. Propranolol hydrochloride (propranolol HCl) is subjected to first-pass effect, therefore formulation of buccal-adhesive dosage form can circumvent this effect. The effect of lactose (a soluble excipient) and dicalcium phosphate (DCP) (an insoluble excipient) on dissolution rate, kinetic of release and adhesion force of buccal-adhesive tablets of propranolol HCl were evaluated. Each tablet composed of 80 mg propranolol HCl, 80 mg hydroxypropylmethylcellulose (HPMC) K4M, polycarbophil AA1 and lactose or DCP with different ratios. The results showed that the presence of the fillers increased dissolution rate of the drug. The release data also showed that the effect of lactose on the dissolution rate was greater than the DCP. Kinetic release of propranolol HCl from buccal-adhesive matrices was affected by the different ratios of polymers and fillers. The fillers reduced the bioadhesion force and this effect was more considerable in formulation containing DCP. In order to determine the mode of release, the data were analyzed based on the equation Q = kt n . The results showed that an increase in the concentration of HPMC K4M resulted in a reduction in the value of n. The value of n was not significantly affected by an increase in the concentration of lactose or DCP. The values of n in this study were calculated to be between 0.461 and 0.619, indicating both diffusional release and erosional mechanism.

Antennapedia transduction sequence promotes anti tumour immunity to epicutaneously administered CTL epitopes

The identification of tumor antigens has spurred the development of efficient adjuvants and novel delivery systems for cancer immunotherapy. To this end, a peptide-based vaccine consisting of the Antennapedia transduction sequence (ANTP) attached to an antigenic peptide was designed to enhance per-cutaneous delivery into cells of the epidermis and dermis. Here we show that the topical application of OVA 257–264 linked to ANTP in mice onto tape-stripped skin resulted in enhanced delivery of the antigen through the skin whereas OVA 257–264 alone remained distributed uniformly on the skin surface. This delivery correlated with an increase in the CTL response against OVA. When mixed with CpG oligodinucleotides (ODN), the recombinant antigen protected mice from tumor challenge. These data provide the first indication that in vivo use of a translocation sequence can enhance delivery of therapeutic peptides and increase anti-tumor immunity through a simple and safe mechanism involving enhanced penetration of the skin barrier.

Poly(ethylene glycol)-containing Hydrogels for Oral Protein Delivery Applications

Novel pH-sensitive hydrogels were developed as suitable candidates for carriers in bioMEMS devices as well as for oral delivery of therapeutic peptides and proteins due to their ability to respond to environmental pH change. Macromonomers containing various PEG molecular weights were synthesized and used to prepare P(MAA-g-EG) hydrogels were by photopolymerization. P(MAA-g-EG) hydrogels showed a drastic change of the equilibrium swelling ratio between pH 2.2 and 7.0. At pH 7.0, hydrogels with PEGMA2000 exhibited higher swelling ratio than hydrogels with PEGMA1000. For both hydrogels with PEGMA1000 and PEGMA2000, the swelling mechanism became more relaxation-controled as the environmental pH changed from 2.2 to 7.0 due to the ionization of the functional groups in polymer networks at high pH. In vitro release studies of insulin were conducted. P(MAA-g-EG) hydrogels exhibited drastic increase of insulin release as the pH of the medium was changed from acidic to basic. Insulin release from P(MAA-g-EG) hydrogels with PEGMA2000 was slower than from hydrogels with PEGMA1000 at both low and high pH. These results were used to design and improve protein release behavior from these carriers.

Synthesis and characterization of pH-sensitive glycopolymers for oral drug delivery systems

pH-sensitive hydrogels are suitable candidates for oral delivery of therapeutic peptides and proteins, due to their ability to respond to environmental pH changes. New pH-sensitive glycopolymers have been developed by free-radical photopolymerization of methacrylic acid and 2- methacryloxyethyl glucoside, using tetra(ethylene glycol) dimethacrylate as a cross-linking agent. To determine the suitability of these hydrogels as carriers for oral drug delivery devices, their swelling behavior was investigated as a function of the pH and copolymer compositions, and various structural parameters such as the number-average molecular weight between cross-links, M c, the mesh size, ξ, and the cross-linking density, ρ x, were calculated. The transition between the swollen and the collapsed states of these hydrogels was at a pH of 5. The swelling ratios of the hydrogels increased at pH values above 5. The mesh sizes of the hydrogels were between 18 and 35 Å in the collapsed state (at pH 2.2) and between 70 and 111 Å in the swollen state (at pH 7.0). Finally, as the cross-linking ratio of the copolymer increased, the swelling ratio of the hydrogels decreased at both pH 2.2 and 7.0.

Inhalation Delivery of Therapeutic Peptides and Proteins

Inhalation Delivery of Therapeutic Peptides and Proteins

In vivo delivery of recombinant human growth hormone from genetically engineered human fibroblasts implanted within Baxter immunoisolation devices.

Continuous delivery of therapeutic peptide to the systemic circulation would be the optimal treatment for a variety of diseases. The Baxter TheraCyte system is a membrane encapsulation system developed for implantation of tissues, cells such as endocrine cells or cell lines genetically engineered for therapeutic peptide delivery in vivo. To demonstrate the utility of this system, cell lines were developed which expressed human growth hormone (hGH) at levels exceeding 1 microgram per million cells per day. These were loaded into devices which were then implanted into juvenile nude rats. Significant levels of hGH of up to 2.5 ng/ml were detected in plasma throughout the six month duration of the study. In contrast, animals implanted with free cells showed peak plasma levels of 0.5 to 1.2 ng four days after implantation with no detectable hGH beyond 10 days. Histological examination of explanted devices showed they were vascularized and contained cells that were viable and morphologically healthy. After removal of the implants, no hGH could be detected which confirmed that the source of hGH was from cells contained within the device. The long term expression of human growth hormone as a model peptide has implications for the peptide therapies for a variety of human diseases using membrane encapsulated cells.

Chitosans for enhanced delivery of therapeutic peptides across intestinal epithelia: in vitro evaluation in Caco-2 cell monolayers

The aim of the study was to evaluate the transport enhancing effects of two chitosan salts, chitosan hydrochloride and chitosan glutamate (1.5% w/v), and the partially quaternized chitosan derivative, N-trimethyl chitosan chloride (TMC) (1.5 and 2.5% w/v), in vitro in Caco-2 cell monolayers. The transport of the peptide drugs buserelin, 9-desglycinamide, 8-arginine vasopressin (DGAVP) and insulin was followed for 4 h at pH values between 4.40 and 6.20. All the chitosans (1.5%) were able to increase the transport of the peptide drugs significantly in the following order: chitosan hydrochloride>chitosan glutamate>TMC. Due to its quaternary structure, TMC is better soluble than the chitosan salts and further increases in peptide transport were found at higher concentrations (2.5%) of this polymer. The better solubility of TMC may compensate for its lower efficacy at similar concentrations. The increases in peptide drug transport are in agreement with a lowering of the transepithelial electrical resistance (TEER) measured in the cell monolayers. No deleterious effect to the cell monolayers could be detected with the trypan blue exclusion technique. The enzyme inhibitory effect of chitosan hydrochloride (1.5%) was compared with carbomer (1.5%) [Carbopol® 934P] in transport studies with buserelin in the presence of the endoprotease, α-chymotrypsin. In the presence of α-chymotrypsin the transport of buserelin was decreased markedly (from 4.3 to 1.3% of the total dose applied) with chitosan hydrochloride (1.5%), in contrast with carbomer (1.5%) where the transport remained constant (1.4% of the total dose applied). Also the chitosan derivative TMC was not able to inhibit α-chymotrypsin. It is concluded from this study that chitosans are potent absorption enhancers, and that the charge, charge density and the structural futures of chitosan salts and N-trimethyl chitosan chloride are important factors determining their potential use as absorption enhancers for peptide drugs, but that they are unable to prevent degradation from proteolytic enzymes. Structural modification of the chitosan molecule may compensate for this shortcoming.

Liposome-mediated peptide loading of MHC-DR molecules in vivo

Amino acid residues 3–15 of mycobacterial HSP60 define a dominant T-cell epitope for HLA-DR3+ve humans and Mamu-DR3+ve rhesus monkeys. Our results show that Mamu-DR3 molecules on PBMC can be efficiently loaded in vivo with the above-mentioned peptides when they are intravenously injected encapsulated in liposomes, but not in the free form. Mamu-DR3 loading is abolished by encapsulation of a nonstimulatory peptide. These results have implications for the delivery of therapeutic peptides in vivo.

Utilization of an endogenous cellular transport system for the delivery of therapeutics across the blood–brain barrier

The delivery of non-lipophilic compounds to the brain is severely limited by the presence of the blood–brain barrier (BBB). This barrier exists at the level of the capillary endothelial cells which form the vascular network within the brain. However, brain capillary endothelial cells do express specific receptors on their surface which are essential for the transport of nutrients and signaling molecules from the bloodstream to the brain. These endogenous transport pathways are potential portals for the transport of therapeutic molecules to the brain. The transferrin receptor is expressed at unusually high levels on the surface of brain capillary endothelial cells and is believed to be the primary, if not only, means for the transport of iron into the brain. It has been shown that antibodies that bind to the transferrin receptor selectively target BBB endothelium due to the high levels of this receptor expressed by these cells. These antibodies are then transported across the capillary endothelial cells into the brain and can function as carriers for the delivery of therapeutic peptides and proteins to the CNS. In particular, this drug delivery system has been used to transport nerve growth factor (NGF) across the BBB in a biologically active form and at levels sufficient to prevent the degeneration of NGF-dependent neurons.

Pulmonary Drug Delivery: Physiologic and Mechanistic Aspects

Peptide and protein drugs are being used increasingly in major research and development programs in the pharmaceutical industry and are also an important class of therapeutic agents due to advances in genetic engineering and biotechnology. Systemic delivery of these macromolecular drugs, however, has been limited to the parenteral route largely because of their extensive presystemic elimination when taken orally. Faced with this dilemma concerning the systemic delivery of these macromolecules with their unique conformational complexity for therapeutic activity, pharmaceutical scientists have evaluated the potential of various non-oral routes of administration as alternatives. Despite the tremendous efforts that have been devoted to this problem, only limited success has been achieved--mostly with small peptides. Growing attention has been given to the potential of a pulmonary route as an alternative non-invasive means for systemic delivery of peptide/protein-based therapeutic agents due to the fact that the lung provides a huge but extremely thin absorptive mucosal membrane. Although current studies show great promise, pulmonary delivery of therapeutic peptides and proteins is complicated by the complexity of the anatomic structure of the human respiratory system and the influence on drug deposition exerted by respiration. This review discusses the fundamental structure and physiology of the human respiratory system, current methodology used to study pulmonary drug absorption, approaches of drug delivery to the distal lung, and recent progress in pulmonary drug delivery by case studies. The mechanisms of pulmonary drug absorption are also discussed.

Pulmonary delivery of therapeutic peptides and proteins

There have been many attempts to improve systemic delivery of peptide and protein drugs by routes of administration other than by injection. Studies in this area of drug delivery have included nasal, rectal, buccal and the transdermal route of administration. There is little research in the area of pulmonary delivery of these compounds. Yet, the lung is unquestionably the most relevant site for delivering anti-asthmatic and mucolytic drugs. The human lung is a complex organ with high enzymatic activity. Yet it is effective for the delivery of locally acting drugs some of which are unstable to metabolizing enzymes. This makes it probable for the lung to become a feasible port for entry of peptide and protein drugs to the body. Being labile and refractory, survival to metabolizing enzymes and influence of airway filtration mechanisms make quantization of lung bioavailability of peptide and protein drugs a formidable effort. Furthermore, the effect of complex biophysical (e.g., inertial impaction) and biochemical factors (e.g., size, charge, permeability, lung clearance mechanisms) causes significant reduction of lower airway drug deposition. Subsequently, the efficiency of drug absorption from the lung is often orders of magnitude lower than it is for other routes. This manuscript reviews data on lung delivery of several peptidic compounds administered for either local or systemic effects. Leuprolide, a non-apeptide and analog of LH-RH is used as a model drug for those peptides administered via lung for a systemic pharmacological effect. A mechanistic approach to describe the bioavailability of leuprolide is described. The paper presents descriptive terms (intrinsic, extrinsic and absolute bioavailability) used to describe the fractional bioavailability of this peptide. Reported data include the estimated bioavailable fractions from bolus dosages of either instillations or inhalation dosages of this nonapeptide, F, based on comparable injectable dosages (i.v. or s.c.). The extent of lung absorption correlated well with depth of drug deposition in the airways. There was quantitative absorption of drug deposited at the bifurcation of the trachea. This peptide has both the C and N terminals blocked. Therefore, it is stable to lung metabolizing enzymes. The discussion includes clinical perspectives and implications for using this method to estimate peptide absorption from the airways.

Clearance of different-sized proteins from the alveolar space in humans and rabbits.

Investigation of the clearance of proteins from the air spaces is important for an understanding of the resolution of pulmonary edema and also because of current interest in delivery of therapeutic peptides via the distal air spaces. Few experimental studies have examined the size dependence for alveolar clearance of large macromolecules; there have been no human studies. In anesthetized rabbits, we measured clearance of cyanocobalamin and different-sized human proteins instilled into the air spaces. After 8 h, the amounts of instilled tracer recovered in the lungs were [57Co]cyanocobalamin, 19.4 +/- 3.0% (Stokes radius 0.65 nm); 125I-labeled insulin, 64.6 +/- 3.9% (1.2 nm); 131I-labeled albumin, 87.0 +/- 4.0% (3.5 nm); and 125I-labeled immunoglobulin G, 91.8 +/- 3.3% (5.5 nm) (P < 0.05). Sieving of different-sized proteins occurred across the alveolar epithelial barrier because tracer concentrations in air space lavage fluid after 8 h were decreased more for the smaller tracers than the larger ones. Size selectivity for alveolar protein clearance in humans with resolving alveolar edema was investigated by measuring the changes in albumin and total protein concentration. The fraction of total protein concentration made up of albumin was greater in the edema fluid than in the plasma initially. The albumin fraction decreased with time in 9 of 10 patients with resolving edema, from 0.62 +/- 0.2 to 0.58 +/- 0.10 (P < 0.05) after 10 +/- 5 h. Thus both rabbit studies and human studies provide evidence for size-dependent clearance of protein from the air spaces of the lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Facilitated transdermal delivery of therapeutic peptides and proteins by iontophoretic delivery devices

Over the years, extensive research effort has been devoted to the development of noninvasive drug delivery techniques for controlled systemic delivery of therapeutic peptides and proteins through various easily accessible organs/tissues. Iontophoresis has recently been discovered as a potential drug delivery technique for noninvasive, programmed systemic delivery of peptide and protein drugs. Several iontophoretic delivery devices have been developed. In this article, the feasibility of using iontophoretic delivery devices to facilitate the transdermal transport of hydrophilic charged macromolecules of peptides, such as vasopressin and proteins, such as insulin, across the skin (which has the lipophilic stratum corneum as the permeation barrier) is demonstrated. In vitro skin permeation studies and in vivo pharmacokinetic and pharmacodynamic studies in diabetic animals suggested that the systemic bioavailability of peptides and proteins, as well as the pharmacodynamic responses, are dependent upon the electronic variables of the iontophoretic delivery device, e.g. waveform, frequency, on/off ratio and intensity of the current applied, physiochemical parameters, e.g. pH and ionic strength, as well as physiological variables, such as treatment of the stratum corneum. The pharmacokinetics-pharmacodynamics relationship is established and the mechanism of iontophoresis-facilitated transdermal transport of peptide-based macromolecules are analyzed.