Therapeutic Polypeptides Research Articles

Sustainable systemic delivery via a single injection of lentivirus into human skin tissue.

The skin offers a tissue site accessible for delivery of gene-based therapeutics. To develop the capability for sustained systemic polypeptide delivery via cutaneous gene transfer, we generated and injected pseudotyped HIV-1 lentiviral vectors intradermally at a range of doses into human skin grafted on immune-deficient mice. Unlike Moloney murine leukemia virus (MLV)-based retrovectors, which failed to achieve detectable cutaneous gene transfer by this approach, lentivectors effectively targeted all major cell types within human skin tissue, including fibroblasts, endothelial cells, keratinocytes, and macrophages. After a single injection, lentivectors encoding human erythropoietin (EPO) produced dose-dependent increases in serum human EPO levels and hematocrit that increased rapidly within one month and remained stable subsequently. Delivered gene expression was confined locally at the injection site. Excision of engineered skin led to rapid and complete loss of human EPO in the bloodstream, confirming that systemic EPO delivery was entirely due to lentiviral targeting of cells within skin rather than via spread of the injected vector to visceral tissues. These findings indicate that the skin can sustain dosed systemic delivery of therapeutic polypeptides via direct lentivector injection and thus provide an accessible and reversible approach for gene-based delivery to the bloodstream.

Inhibition of a model protease — pyroglutamate aminopeptidase by a natural oligosaccharide gum from Hakea gibbosa

The purpose of this study was to investigate the effect of the oligosaccharide gum from Hakea gibbosa on the activity of a model protease enzyme pyroglutamate aminopeptidase (5-oxoprolyl peptidase; EC 3.4.19.3) and to elucidate the mechanism responsible for the decreased activity. Enzyme kinetic studies were conducted at 37°C in 100 mM potassium phosphate buffer with 10 mM EDTA, 5% (v/v) glycerol, and 5 mM DTT (pH 8) for 15 min and were performed both in the presence and absence of the gum. Enzymatic activity was determined by a colorimetric assay using the specific substrate l-pyroglutamic acid β-napthylamide. The enzyme kinetics was studied at various substrate and gum concentrations. The velocity of the reaction was determined by the amount of the product (β-napthylamine) liberated at each substrate and gum concentration. The K s and V max of the enzyme in the absence of the gum were 24.40±2.14 μM and 502.95±28.90 nmoles·min −1·mg protein −1, respectively. As the concentration of the gum was gradually increased from 0.1 to 2%, the value of the V max decreased from 318.94±21.46 to 158.83±24.51 nmoles·min −1·mg protein −1 while K s increased from 17.42±4.6 to 63.03±1.89 μM. The mechanism for the inhibition of the enzyme by Hakea appeared to be a mixed-linear type (a type of non-competitive inhibition) as suggested from Hanes-Woolf, Dixon and Cornish-Bowden plots. The turnover number, k cat, calculated for the enzyme also decreased from 14.09±0.81 to 4.45±0.69 min −1 as the concentration of the inhibitor was incrementally increased from 0 to 2% (w/v). The K i and αK i calculated from Dixon and Cornish-Bowden plots were found to be 0.31±0.11% (w/v) and 1.33±0.42% (w/v), respectively. The natural gum from Hakea gibbosa was effective in non-competitively inhibiting the enzyme pyroglutamate aminopeptidase. Thus, the natural gum may be a promising additive not only for its sustained-release and mucoadhesive properties as shown previously, but also for its ability to slow the enzymatic degradation of therapeutic polypeptides incorporated in dosage forms.

Polycarbophil–Cysteine Conjugates as Platforms for Oral Polypeptide Delivery Systems

The purpose of the present study was to evaluate the potential of polycarbophil–cysteine conjugates as carrier systems for orally administered peptide and protein drugs. Mediated by a carbodiimide, cysteine was covalently attached to polycarbophil. The properties of resulting conjugates, displaying 35–50 μM thiol groups per gram of polymer, to bind polypeptides and to inhibit pancreatic proteases was evaluated in vitro. Results demonstrated that only some polypeptides are immobilized to the polycarbophil–cysteine conjugate. Due to the covalent attachment of cysteine to polycarbophil, the inhibitory effect of the polymer toward carboxypeptidase A (EC 3.4.17.1) and carboxypeptidase B (EC 3.4.17.2) could be significantly (p < 0.05) improved. As the zinc binding affinity of polycarbophil could be improved by the covalent attachment of cysteine, the raised inhibitory effect seems to be based on the complexation of this divalent cation from the enzyme structure. Whereas the covalent attachment of cysteine on polycarbophil had no influence on the enzymatic activity of trypsin (EC 3.4.21.4) and elastase (EC 3.4.21.36), the inhibitory effect of the polymer–cysteine conjugate toward chymotrypsin (EC 3.4.21.1) was significantly (p < 0.05) higher than that of the unmodified polymer. Because of these inhibitory features, polycarbophil–cysteine conjugates seem to be a promising tool in protecting orally administered therapeutic polypeptides, which are not bound to the polymer, from presystemic metabolism in the intestine. © 2000 Liss‐Wiley Inc. and the American Pharmaceutical Association J Pharm Sci 89: 901–909, 2000

Rapid identification of all known retroviral reverse transcriptase sequences with a novel versatile detection assay.

We have developed a highly sensitive, universal assay that allows detection as well as identification of all known retroviral reverse transcriptase (RT)-related nucleic acids in a biological sample by a single two-step experiment. The assay combines polymerase chain reaction (PCR) and reverse dot-blot hybridization (RDBH), using an array of immobilized synthetic retrovirus-specific oligonucleotides and two sets of mixed oligo primers (MOPs). These primers were derived from highly conserved motifs found in all known reverse transcriptase genes. The PCR/RDBH assay was used for qualitative analyses of human endogenous retrovirus (HERV) transcription in peripheral blood mononuclear cells (PBMCs) and in particles released by the human mammary carcinoma-derived cell line T47D. Sensitivity was further demonstrated by detection of down to 10 copies of pig endogenous retrovirus (PERV) DNA in human cDNA samples. Therefore, this assay is particularly useful for the identification of retroviral sequences in xenografts as well as in recipients of xenografted tissues and organs. Moreover, it is a valuable tool to detect retroviral transcripts and particles in cell cultures used for production of therapeutic polypeptides. The assay is further suitable for monitoring vector preparation used in human gene therapy to exclude transfer of copackaged endogenous retroviruses into target cells.

Evaluation of a novel, natural oligosaccharide gum as a sustained-release and mucoadhesive component of calcitonin buccal tablets

The objective of this study was to evaluate the gum from Hakea gibbosa (hakea) as a sustained-release and mucoadhesive component in buccal tablets for a model peptide, namely, salmon calcitonin. Flat-faced core tablets containing either 12 or 32 mg of hakea and 40 µg (200 IU) of salmon calcitonin (sCT) per tablet were formulated using a direct compression technique and were coated with Cutina on all but one face. The in vitro release profiles were sigmoidal in nature and according to a mathematical model indicated super Case II transport as the primary mechanism of release. The resulting plasma sCT and calcium concentrations were determined following both intravenous administration and buccal application of mucoadhesive tablets in rabbits. Following intravenous administration, the mean values determined for t1/2 (α), t1/2 (β), Vd, and CL for sCT were 0.76 ± 0.06 min, 67 ± 18 min, 1484 ± 454 mL/kg, and 19 ± 2 mL/min·kg, respectively. Following the application of the mucoadhesive buccal tablets which contained 40 µg of sCT and either 12 or 32 mg of hakea, the calculated apparent bioavailability (F) and clearance (CL) were 37 ± 6% and 19 ± 3.3 mL/min·kg and 16 ± 8% and 18 ± 0.4 mL/min·kg, respectively. Serum calcium concentrations indicated that biologically active sCT was delivered across the rabbit buccal mucosa. The strength of mucoadhesion of the tablets was also quantitated in terms of the force of detachment as a function of time. The force of detachment for the mucoadhesive buccal tablets containing either 12 or 32 mg of hakea and 40 µg of sCT increased from 4.47 ± 0.68 to 8.41 ± 1.0 N and 8.23 ± 1.62 to 14.98 ± 1.63 N, respectively, from 5 to 90 min following application to excised rabbit intestinal mucosa. These results demonstrate that the novel, natural gum from Hakea gibbosa may be used to sustain the release of sCT from a unidirectional-release buccal tablet. The mechanism of in vitro release is likely to involve peptide diffusion/polymer dissolution. The mucoadhesive strength, as measured by the force of detachment, can be modulated by altering the amount of hakea in the tablet. The mucoadhesive buccal tablets described in this paper represent an improved transbuccal delivery system for therapeutic polypeptides.

Simplified pepstatins: synthesis and evaluation of N-terminally modified analogues.

The promising strategy of gastric ulcer healing with perorally administered epidermal growth factor (EGF) is so far strongly limited by the pepsinic degradation of this therapeutic polypeptide in the stomach. The incorporation of EGF in a bioadhesive polymer-pepsin inhibitor conjugate used as drug carrier matrix, however, might provide sufficient protection toward pepsinic degradation. The synthesis of appropriate pepsin inhibitors represents a prerequisite for the development of such polymer-inhibitor conjugates. The presented study demonstrates that modifications at the N-terminus of simplified analogues of pepstatin which can be synthesized in a simple and straight way result only in slight variations of the inhibitory activity. These analogues display only 10-fold reduced inhibitory activity, compared to pepstatin A, when bearing a greater N-terminal group like isovaleryl, Boc, or Cbz. Compounds which are substituted at the N-terminus by a shorter N-acyl group like propionyl or cyclopropylcarbonyl show further reduced activity (0.01, compared to pepstatin A). The presence of an amide or a urethane moiety at the N-terminus has no considerable effect on enzyme inhibition. Therefore, the N-terminus of these analogues is able to be modified forming a covalent bond to various bioadhesive polymers via a suitable functionality.

CUTANEOUS GENE THERAPY

Gene therapy efforts in a variety of tissues have foundered on fundamental technologic barriers, such as difficulties in achieving high-efficiency gene transfer to diseased tissues and in sustaining delivered transgene production. The skin offers an attractive tissue for development of approaches to therapeutic gene delivery by virtue of its accessibility for regulation by topical agents, the ease of gene transfer into cutaneous tissues, and the ready ability to monitor the impact of somatic gene transfer. With the ability of the skin to deliver therapeutic polypeptides to the systemic circulation and the recent molecular characterization of monogenic skin diseases, efforts to target genes to the skin are expected to accelerate. The current status of gene therapy efforts is reviewed, with a special focus on the skin.

The pharmaceutics and delivery of therapeutic polypeptides and proteins

Proteins and peptides with immunoregulatory properties are a rapidly growing class of pharmacophores for a wide variety of indications in a number of clinical disciplines. Despite the rapid development of these new drugs, they retain severe pharmacologic deficiencies due to their rapid degradation and elimination. This results in their administration at superpharmacological doses and subsequent clinical toxicity. Further, because these compounds often act indirectly, their clinical development path is less obvious than that of other therapeutics. Thus, their development has become focused on a pharmacologic approach predicated on the preclinical identification of therapeutic surrogates and the subsequent testing of a clinical hypothesis. Immunoregulatory peptides and proteins, including cytokines and growth factors, have been used as single agents and in combinations with one another or other therapeutic modalities. Due to the challenge associated with the targeting and delivery of these therapeutics, novel strategies to achieve the desired therapeutic end-points have been developed. This includes the use of peptide inducers or antagonists as well as delivery strategies via gene and cellular therapy. These therapeutic strategies of the future are in response to the pharmacologic deficiencies of proteins and provide exciting new directions for their development in both the near and long term. This review addresses the pharmacologic deficiencies of proteins, preclinical and clinical developmental strategies, and future strategies based on the use of molecular therapeutics. The primary emphasis has been placed on preclinical and clinical studies of immune and hematopoietic augmenting agents. However, we also discuss proteins that are still in animal models, agents whose profile of activity is associated with the induction of a cytokine cascade and the targeted delivery of a gene via hematopoietic stem cells, lymphocytes, or other autologous tissues or tumors.

Design of site-specific protein drugs

The ability to clone and express proteins at high levels of abundance and purity has led to their increasing consideration as therapeutic agents. The use of various protein engineering and recombinant DNA techniques has enabled progression to where proteins can be tailored so that they have not only the ability to interact with a unique (extra)cellular component in order to produce a pharmacological effect, but their structure can be altered so as to achieve a selective biological disposition. This ability to control the biological dispersion of proteins is becoming a key element in the design of therapeutic polypeptides and proteins, finding application in modalities intended for treating HIV infections, accessible tumor masses, and diseases of the hematopoietic system, etc. For recombinant site-specific systems to be successfully introduced into the clinic, radically different approaches are required during their development and clinical testing. These relate not only to the pharmacokinetics and receptor availability of the drug, but also its pharmacodynamics, its analytics and its safety pharmacology. This contribution examines how the control of the biological disposition of proteins may be achieved using both site-directed mutagenesis, sequence re-ordering, hybrid protein formation, protein resurfacing, as well as synthetic adduction to polymers.

Selective delivery of therapeutic polypeptides and proties

Selective delivery of therapeutic polypeptides and proties

Selective of therapeutic polypeptides and proteins: 24th–26th September, 1990, Montreaux, Switzerland

Selective of therapeutic polypeptides and proteins: 24th–26th September, 1990, Montreaux, Switzerland

Selective delivery of therapeutic polypeptides and proteins: 24–26 September 1990 Montreux, Switzerland

Selective delivery of therapeutic polypeptides and proteins: 24–26 September 1990 Montreux, Switzerland