Evaluation of novel particles as an inhalation system for GLP-1.

AIM: The feasibility of administering native glucagon-like peptide 1 (GLP-1) as GLP-1 Technosphere Inhalation Powder for diabetes therapy has been demonstrated in a rat model. METHODS: GLP-1 Technosphere Inhalation Powders containing 5, 10 and 15% GLP-1 were prepared and administered to healthy female Sprague-Dawley rats and to male Zucker diabetic obese rats. Rats received a single dose of GLP-1 Technosphere Powder by pulmonary insufflation. GLP-1 pharmacokinetic and pharmacodynamic responses were measured. RESULTS: Maximum circulating GLP-1 concentrations were achieved at approximately 10 min after dosing with detectable levels at 40 min. In a food consumption study, Sprague-Dawley rats receiving GLP-1 Technosphere Powder once-daily consumed less food than control rats for up to 24 h after dosing. Cumulative food consumption was decreased approximately 10% after 78 h. In an intraperitoneal glucose tolerance test, Zucker diabetic fatty rats receiving 2 mg GLP-1 Technosphere Powder (0.3 mg GLP-1) by pulmonary insufflation exhibited lower glucose concentrations and higher insulin concentrations than control rats. Pancreatic evaluations showed no differences in apoptotic index or cell proliferation of beta-cells. In addition, a dose-related increase in insulin expression within the pancreas was observed. CONCLUSIONS: These data demonstrate the feasibility of administering native GLP-1 as GLP-1 Technosphere Inhalation Powder for diabetes therapy.

Orally administered unfractionated heparin with carrier agent is therapeutic for deep venous thrombosis.

BACKGROUND: Orally administered heparin (OHEP) is unreliable because of poor absorption. Sodium N-(8[2-hydroxybenzoyl]amino) caprylate (SNAC) is an amido acid that facilitates the gastrointestinal absorption of heparin. We evaluated the effectiveness of OHEP combined with SNAC (OHEP/SNAC) in the treatment of deep-vein thrombosis (DVT). METHODS AND RESULTS: An internal jugular DVT was produced in 54 male Sprague-Dawley rats. Animals were assigned to 6 different groups for 7 days of treatment: untreated control, subcutaneous heparin (SC HEP) (300 U/kg SC TID), SNAC only (300 mg/kg PO TID), OHEP only (30 mg/kg PO TID), low-molecular-weight heparin (LMWH) (enoxaparin 5 mg/kg SC QD), and OHEP/SNAC (30 mg/kg:300 mg/kg PO TID). The activated partial thromboplastin time (aPTT) and anti-factor X (anti-Xa) levels were measured. The incidence of residual DVT after 1 week of treatment was 100% (9 of 9) in the control group versus 10% (1 of 10) in the OHEP/SNAC and 10% (1 of 10) in the LMWH groups (P<0.001). There was also a significant reduction in clot weights between these groups. Compared with controls, there were no significant differences in the residual DVT in the SNAC-only (6 of 6), OHEP-only (9 of 9), or SC HEP (8 of 10) groups. Combination OHEP/SNAC was as effective in the resolution of the clot and reducing clot weight as LMWH. The aPTT levels in the OHEP/SNAC group peaked at 30 minutes and were significantly higher than in all other groups (P<0.01). Anti-Xa levels were elevated at 15 minutes after dosing in the OHEP/SNAC group and remained significantly elevated at 4 hours (P<0.001). CONCLUSIONS: OHEP combined with a novel carrier agent (SNAC) successfully treated DVT in this rat model.

Delivery agents that facilitate the absorption of macromolecular drugs.

Macromolecules comprise a growing group of new therapeutics with great clinical promise. To date, these drugs are limited in their application because they are effective only when administered parenterally. Unfortunately, however, macromolecular drugs are not absorbed following nonparenteral dosing because the human body is designed to exclude them. To overcome the numerous obstacles to the noninvasive delivery of these drugs, various approaches are under investigation including the use of delivery agents to promote drug absorption. This review provides a summary of the novel approaches currently in progress in the areas of transdermal, transmucosal and oral delivery of macromolecular drugs facilitated by delivery agents.

N-acylated alpha-amino acids as novel oral delivery agents for proteins.

A series of N-acylated alpha-amino acids were synthesized and shown to improve the oral delivery of two protein drugs, salmon calcitonin (sCT) and interferon-alpha. Forty-five compounds in this series were tested in vivo in rats and primates. A significant positive correlation was found between the log P of the acylated amino acids and the decrease in serum calcium following oral dosage of sCT in rats. Such a correlation was not found for interferon-alpha. These derivatized amino acids only weakly inhibited the activity of trypsin or leucine aminopeptidase. Histological examinations of rat intestinal tissue after oral dosing of acylated amino acid/protein combinations revealed no detectable pathology.

Microsphere formation in a series of derivatized alpha-amino acids: properties, molecular modeling, and oral delivery of salmon calcitonin.

A series of benzoylated and phenylsulfonylated amino acids are novel, low molecular weight, self-assembling molecules. At low pH, these compounds form microspheres that dissolve readily under neutral conditions. In a given synthetic series, those molecules with low aqueous solubility formed microspheres more readily than did the molecules possessing high water solubility, suggesting that the hydrophobicity of these compounds contributes to the ability to form microspheres. In addition, molecular modeling studies on selected compounds have shown that microsphere formation may depend also on various aromatic ring and dipole-dipole interactions, which could effect the extent and types of favorable stacking conformations between molecules. The microspheres prepared from these compounds have been used to effect the oral delivery of salmon calcitonin, a model protein drug, in both rodents and primates.

Studies directed at the use of a parallel synthesis matrix to increase throughput in an in vivo assay.

Heparin is the anticoagulant of choice for hospitalized patients, but it is dosed only by injection because it is not absorbed following oral administration. We have discovered and prepared compounds (delivery agents) that facilitate the gastrointestinal absorption of heparin in rats, monkeys, and humans when given orally. We are currently developing a parallel synthesis approach to increase our delivery agent screening throughput in vivo. This approach has been used to produce micromolar quantities of compounds for testing in rats in a 5 x 5 parallel synthesis array. Using an amine benzoylation reaction sequence, 10 mixtures were prepared. These mixtures contained equal weight quantities of five N-substituted, non-alpha, amino acid delivery agents. Each of these mixtures was orally administered to rats in combination with heparin, and plasma clotting times (APTT) were measured to determine activity. Deconvolution of the data accurately identified the most active individual components. Independent synthesis of these compounds verified their activity. This parallel synthesis approach is an effective tool for the screening of oral heparin delivery agents and has increased screening throughput significantly.

4-[4-[(2-Hydroxybenzoyl)amino]phenyl]butyric acid as a novel oral delivery agent for recombinant human growth hormone.

A series of N-acetylated, non-alpha, aromatic amino acids was prepared and shown to promote the absorption of recombinant human growth hormone (rhGH) from the gastrointestinal tract. Seventy compounds in this family were tested in vivo in rats. Of the compounds tested, 4-[4-[(2-hydroxybenzoyl)amino]phenyl]butyric acid was identified as a preclinical candidate and was used to demonstrate the oral delivery of rhGH in primates. A significant positive correlation was found between the relative log k' of the delivery agents, as determined by HPLC on an immobilized artificial membrane (IAM) column, and serum rhGH concentrations following oral or colonic dosing in rats. Structure-activity relationships have also been developed on the basis of electronic effects and hydrogen-bonding characteristics of the aromatic amide substituents.

Synthesis and evaluation of compounds that facilitate the gastrointestinal absorption of heparin.

A family of novel compounds (delivery agents) that promote the gastrointestinal absorption of USP heparin in rats and primates has been discovered. The delivery agents in combination with heparin were administered either orally or intracolonically in an aqueous propylene glycol solution and caused dramatic increases in both plasma heparin concentrations (anti-Factor Xa) and clotting times (APTT). Using one of the most effective delivery agents in this series, an estimated relative bioavailability of 8% can be achieved following oral administration to cynomolgus monkeys. To establish a correlation between the in vivo data and an in vitro parameter, immobilized artificial membrane (IAM) chromatography was performed. Log relative k' values were correlated to the efficiency of oral heparin delivery.

Acylated non-alpha-amino acids as novel agents for the oral delivery of heparin sodium, USP.

Ten N-acylated, non-alpha-amino acids have been prepared as oral delivery agents and used to demonstrate the oral delivery of heparin in vivo in rats and primates. Following the oral administration of solutions containing a combination of heparin and a delivery agent to rats or primates, significant plasma heparin concentrations were evidenced by APTT and anti-Factor Xa assays. The estimated pharmacodynamic equivalence for an oral dosing solution containing heparin and a delivery agent is 39% in primates. In vitro experiments based on heparin affinity chromatography or heparin/methylene blue complexation were also performed to begin investigation of the mechanism by which these compounds facilitate heparin oral delivery. Results of in vitro studies suggest that absorption of the drug across the gastrointestinal membrane is the result of a non-covalent interaction between heparin and the delivery agent.

Partially unfolded proteins efficiently penetrate cell membranes--implications for oral drug delivery.

We have previously reported on the biological activity of members of a library of low molecular weight compounds (carriers) that enable the oral delivery of proteins (Milstein, Proceedings of the 1995 Miami Bio/Technology Winter Symposium on Protein Engineering and Structural Biology, IRL Press at Oxford University Press, 1995, p. 13; Leone-Bay et al., J. Med. Chem. 38 (1995) 4263-4269; Leone-Bay et al., J. Med. Chem. 39 (1996) 2571-2578; [1-3]). When rats or primates are orally administered a solution of carrier and either recombinant human alpha-interferon (rhIFN), insulin or recombinant human growth hormone (rhGH) significant serum concentrations of the proteins are detectable. The transport activity of these compounds is positively correlated with their structural effects on the protein molecules. Direct measurement of the interaction of these carrier molecules with the proteins indicates that they reversibly destabilize the native state of the molecule favoring a partially unfolded conformation. Apparently these intermediate protein conformations are transport competent and are able to be absorbed through the intestinal tissue and into the bloodstream. Since the measured binding of the carriers to the partially unfolded proteins is relatively weak (Kb = 100 M(-1)) and the systemic activity of the proteins appears to be unaffected, the changes in the structure of the proteins are manifestly reversible.

Orally administered heparin for preventing deep venous thrombosis.

BACKGROUND: Sodium N-(8-[2-hydroxybenzoyl] amino) caprylate (SNAC) is an acetylated amino acid molecule that facilitates the gastrointestinal absorption of heparin. This study was undertaken to evaluate the efficacy of orally administered combination SNAC:heparin in preventing deep venous thrombosis in a standard rat model. METHODS: Forty-four adult male Sprague-Dawley rats were randomly divided into five groups: group I control, group II SNAC, group III oral heparin, group IV combination SNAC:heparin, and group V intravenous heparin. Thirty minutes after drug administration, the internal jugular vein was bathed in a sclerosant mixture for 2 minutes and reexplored at 120 minutes. Activated partial thromboplastin times (aPTT) were measured in 30 rats equally divided into three groups: group I SNAC, group II oral heparin, and group III combination SNAC:heparin. Forty-five minutes posttreatment, blood was obtained for aPTT levels. RESULTS: The incidence of deep venous thrombosis in the control group was 89% (8 of 9) versus 25% (2 of 8) in the combination SNAC:heparin group (p < 0.01). There was also a significant reduction in clot weight among groups. Combination SNAC:heparin significantly increased aPTT levels compared with SNAC or oral heparin alone. CONCLUSION: In a rat model of venous thrombosis, combination of orally administered heparin:SNAC elevated aPTT levels and significantly reduced the formation of deep venous thrombosis.

Receptor-mediated inhibition of small bowel migrating complex by GLP-1 analog ROSE-010 delivered via pulmonary and systemic routes in the conscious rat.

BACKGROUND: ROSE-010, a Glucagon-Like Peptide-1 (GLP-1) analog, reduces gastrointestinal motility and relieves acute pain in patients with irritable bowel syndrome (IBS). The rat small bowel migrating myoelectric complex (MMC) is a reliable model of pharmacological effects on gastrointestinal motility. Accordingly, we investigated whether ROSE-010 works through GLP-1 receptors in gut musculature and its effectiveness when administered by pulmonary inhalation. MATERIALS AND METHODS: Rats were implanted with bipolar electrodes at 5, 15 and 25 cm distal to pylorus and myoelectric activity was recorded. First, intravenous or subcutaneous injections of ROSE-010 or GLP-1 (1, 10, 100 µg/kg) with or without the GLP-1 receptor blocker exendin(9-39)amide (300 µg/kg·h), were studied. Second, ROSE-010 (100, 200 µg/kg) Technosphere® powder was studied by inhalation. RESULTS: The baseline MMC cycle length was 17.5±0.8 min. GLP-1 and ROSE-010, administered intravenously or subcutaneously, significantly inhibited myoelectric activity and prolonged MMC cycling; 100 µg/kg completely inhibited spiking activity for 49.1±4.2 and 73.3±7.7 min, while the MMC cycle length increased to 131.1±11.4 and 149.3±15.5 min, respectively. Effects of both drugs were inhibited by exendin(9-39)amide. Insufflation of ROSE-010 (100, 200 µg/kg) powder formulation totally inhibited myoelectric spiking for 52.6±5.8 and 70.1±5.4 min, and increased MMC cycle length to 102.6±18.3 and 105.9±9.5 min, respectively. CONCLUSIONS: Pulmonary delivery of ROSE-010 inhibits gut motility through the GLP-1R similar to natural GLP-1. ROSE-010 causes receptor-mediated inhibition of MMC comparable to that of intravenous or subcutaneous administration. This suggests that ROSE-010 administered as a Technosphere® inhalation powder has potential in IBS pain management and treatment.

The development of delivery agents that facilitate the oral absorption of macromolecular drugs.

Macromolecules comprise a growing group of new drugs with great clinical promise. To date, the therapeutic application of these drugs has been limited, because they are effective only when administered parenterally. Unfortunately, macromolecular drugs are not absorbed following nonparenteral dosing, because the mechanisms of the human body are designed to degrade and/or exclude them. To overcome the numerous obstacles to the noninvasive delivery of these drugs, various approaches are under investigation including the use of delivery agents to promote drug absorption. This review provides a summary of the novel approaches currently in progress in the areas of transdermal, transmucosal, and oral delivery of macromolecular drugs facilitated by delivery agents. We review our own novel work in this area in some detail, including the methods developed for the synthesis of the delivery agents, in vitro screening techniques developed to select compounds for in vivo testing, and the results of in vivo screening in both rats and primates, including preliminary safety and efficacy studies. Finally, the results of Phase I clinical studies showing the oral delivery of heparin are presented.

Heparin absorption across the intestine: effects of sodium N-[8-(2-hydroxybenzoyl)amino]caprylate in rat in situ intestinal instillations and in Caco-2 monolayers.

PURPOSE: The effects of sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC) on heparin intestinal absorption were studied using rat in situ ileal and colonic instillations and Caco-2 monolayers. METHODS: The flux of heparin was tested in the following groups: i) heparin alone, ii) heparin in the presence of SNAC, iii) heparin in the presence of propylene glycol (PG), and iv) heparin in the presence of SNAC and PG. Heparin absorption was measured by the APTT assay in the in situ models and by the anti-Factor Xa assay in Caco-2. SNAC and [3H]-SNAC fluxes were assessed by HPLC and by scintillation counting respectively. RESULTS: In the rat ileal and colonic in situ instillations SNAC (17-35 mg) promoted heparin absorption in the presence and absence of PG without damaging the tissue. PG alone did not alter heparin absorption in situ, but it amplified the effect of SNAC. In Caco-2, enhanced heparin fluxes were variable in the presence of non-cytotoxic concentrations of SNAC (< 10 mg/ml) and these effects could not be discriminated from those of PG. Papp values for SNAC alone were 2.2 x 10(-5) cm/s and 2.0 x 10(-5) cm/s in the mucosal-to-serosal and serosal-to-mucosal directions respectively, suggesting a substantial passive transcellular flux. Transport of SNAC was significantly reduced in the presence of heparin and/or PG, perhaps indicating physical association between the agents. CONCLUSIONS: SNAC augmented heparin absorption alone and in combination with PG in the rat in situ models without causing toxicity. Caco-2 had limitations for testing increased heparin absorption due to cytotoxic effects of high concentrations of SNAC and PG. However, SNAC itself was well absorbed across Caco-2 and its mechanism of permeation was determined.

Oral immunization with a model protein entrapped in microspheres prepared from derivatized alpha-amino acids.

A model antigen, ovalbumin (OVA), was encapsulated in microspheres prepared from derivatized alpha-amino acids and administered orally to mice. These microspheres are quickly and easily prepared, without the use of organic solvents, high temperatures, or complex purification techniques. Immunological responses included induction of OVA-specific antibodies in both sera (IgG) and in intestinal secretions (sIgA), as well as antigen-dependent proliferation of splenic CD4+ T cells following, in some cases, as little as a single oral priming dose containing 0.1 mg OVA. Oral administration of microspheres was also found to be effective as a secondary immunization following a subcutaneous prime with soluble antigen. In addition, the protective effect of co-encapsulation of cholera toxin, a mucosal adjuvant, was demonstrated in a whole virus model (infectious bursal disease in chickens). These results indicate that oral administration of antigen-loaded derivatized alpha-amino acid microspheres can induce local and systemic antibody production and/or stimulation of effector cells.

Oral delivery of sodium cromolyn: preliminary studies in vivo and in vitro.

PURPOSE: Herein we report the discovery of a group of derivatized alpha-amino acids that increase the oral bioavailability of sodium cromolyn. METHODS: We prepared three N-acylated alpha-amino acids and used these compounds to demonstrate the oral delivery of cromolyn in an in vivo rat model. In vitro experiments, including permeation studies and near infrared spectroscopy, were also performed to initiate an understanding of the mechanism by which these compounds facilitate cromolyn oral delivery. RESULTS: Following oral administration to rats of solutions containing a combination of cromolyn and the delivery agent, significant systemic plasma concentrations of the drug were detected. In vitro studies suggest that absorption of the drug across the gastrointestinal membrane is a passive process. CONCLUSION: The absolute oral bioavailability of sodium cromolyn in the rat model is estimated to be approximately 5%. Preliminary mechanistic studies suggest that a complex of the cromolyn/delivery agent facilitates permeation across/through the membrane.

Oral low-molecular weight heparin and delivery agent prevents jugular venous thrombosis in the rat.

PURPOSE: Sodium N-[10-(2-hydroxybenzoyl)amino]decanoate (SNAD) is a novel carrier that allows the gastrointestinal absorption of low-molecular weight heparin (LMWH). The purpose of this experiment was to evaluate oral LMWH with SNAD for the prevention of deep venous thrombosis. METHODS: Sixty Sprague-Dawley rats were equally assigned to five experimental groups: group 1 (control), oral saline solution; group 2, oral LMWH (15 mg/kg); group 3, oral SNAD (300 mg/kg); group 4, subcutaneous LMWH (5 mg/kg); and group 5, oral LMWH (15 mg/kg) and SNAD (300 mg/kg). After treatment, the jugular vein was isolated, occluded, and bathed in an ethanol and formalin solution for 2 minutes. Two hours later, the vessel was examined for patency, presence of thrombus, and thrombus weight. Serum measurement of anti-factor Xa activity was performed in a separate set of 30 rats, which were placed into the following four groups: group A, LMWH (5 mg/kg); group B, oral LMWH (15 mg/kg) and SNAD (300 mg/kg); group C, oral LMWH (15 mg/kg); and group D, SNAD (300 mg/kg). RESULTS: The animals that underwent oral LMWH/SNAD therapy had a statistically significant decrease in visible thrombi. The thrombus weight of the oral LMWH/SNAD group was significantly less than the weights of all other groups, except the subcutaneous LMWH group. Anti-factor Xa levels were significantly elevated in the LMWH/SNAD group. There was no statistically significant difference between the data for the oral LMWH/SNAD group and the subcutaneous LMWH group. CONCLUSION: The combination of oral LMWH and SNAD prevented deep venous thrombosis. The oral LMWH and SNAD therapy effected an increase in levels of anti-factor Xa.

Treatment of experimentally induced caval thrombosis with oral low molecular weight heparin and delivery agent in a porcine model of deep venous thrombosis.

OBJECTIVE: This experiment evaluated enterally administered low molecular weight heparin (LMWH) combined with sodium N-[10-(2-hydroxybenzoyl)amino] decanoate (SNAD) for the treatment of induced venous thrombosis. SUMMARY BACKGROUND DATA: SNAD is a delivery agent that potentiates the gastrointestinal absorption of LMWH. METHODS: Forty female pigs were equally assigned to four groups: control (saline); enteral LMWH, 2,000 IU/kg; enteral SNAD, 50 mg/kg; and enteral LMWH, 2,000 IU/kg and SNAD, 50 mg/kg. Under fluoroscopic guidance, the infrarenal vena cava was occluded with a balloon catheter. Two milliliters of ethanol was injected into the distal vena cava. The inflated balloon catheter remained in situ for 5 days, at which time animals angiographically exhibiting thrombus were randomly assigned to the four groups. Study medications were dosed at 12-hour intervals by means of a gastrostomy tube placed previously. After 7 days of treatment, thrombus was extracted. A separate group of 10 animals was used to measure plasma antifactor Xa levels for 6 hours after enteral dosing of LMWH/SNAD. RESULTS: The amount of residual thrombus after treatment with enteral LMWH/SNAD was significantly decreased. Antifactor Xa levels were significantly elevated in the LMWH/SNAD group versus baseline. CONCLUSION: The combination of enterally administered LMWH and SNAD given for 7 days appeared to decrease caval thrombosis in this model of deep vein thrombosis. Enteral LMWH/SNAD effected an increase in plasma levels of antifactor Xa.

Oral delivery of biologically active parathyroid hormone.

PURPOSE: Parathyroid hormone (PTH), the only drug known to stimulate bone formation. is a peptide therapeutic indicated in the treatment of osteoporosis. Unfortunately, PTH is only effective when dosed by injection because it has no oral bioavailability. Herein we report the oral absorption of PTH in rats and monkeys facilitated by the novel delivery agent, N-[8-(2-hydroxy-4-methoxy)bensoyl]amino caprylic acid (4-MOAC). METHODS: 4-MOAC was selected from a group of 100 delivery agents based on in vitro chromotography studies and in vivo screening studies in rats. The PTH/4-MOAC combination was then tested in monkeys. The interaction of 4-MOAC and PTH was evaluated by nuclear magnetic resonance (NMR) spectroscopy. RESULTS: Monkeys were administered an aqueous solution containing 4-MOAC and PTH and mean peak serum PTH concentrations of about 3000 pg/mL were obtained. The relative bioavailability of oral PTH was 2.1% relative to subcutaneous administration. The biological activity of the orally-delivered PTH was further evaluated in a rat model of osteoporosis. These studies showed that the bone formed following oral PTH/4-MOAC administration was comparable to that formed following PTH injections. The 4-MOAC mediated absorption of PTH is hypothesized to be the result of a noncovalent interaction between 4-MOAC and PTH. The preliminary evaluation of this interaction by NMR is described. CONCLUSIONS: 4-MOAC facilitates the absorption of PTH following oral administration to both rats and monkeys. The orally-absorbed PTH is biologically active as demonstrated in a rat model of osteoporosis.