Mutual inhibition of the insulin absorption-enhancing properties of dodecylmaltoside and dimethyl-beta-cyclodextrin following nasal administration.

PURPOSE: To determine if a nasal insulin formulation containing two distinct absorption-enhancing agents exhibits an additive or synergistic increase in the rate of systemic insulin absorption. METHODS: The pharmacokinetics and pharmacodynamics of insulin absorption were measured in hyperglycemic anesthetized rats following nasal insulin administration with formulations containing two different types of absorption-promoting agents, dimethyl-beta-cyclodextrin (DMBCD) and dodecylmaltoside (DDM). RESULTS: When either DDM (0.1-0.5%) or DMBCD (1.0-5.0%) was added to the nasal insulin formulation, a significant and rapid increase in plasma insulin levels was observed, with a concomitant decrease in blood glucose concentration. A combined preparation containing 0.25% DDM (0.005 M) and 2.5% DMBCD (0.019M), however, failed to cause an increase in plasma insulin levels or a decrease in blood glucose concentration. Increasing concentrations of DDM added to an insulin formulation with a fixed DMBCD concentration caused a decrease, rather than an increase, in systemic absorption of insulin. CONCLUSIONS: Mixing DMBCD and DDM resulted in mutual inhibition of their ability to enhance systemic absorption of insulin following nasal delivery. The results are consistent with the formation of an inclusion complex between DDM and DMBCD which lacks the ability to enhance nasal insulin absorption.

Enhanced bioavailability of calcitonin formulated with alkylglycosides following nasal and ocular administration in rats.

PURPOSE: The purpose of this study was to characterize the effects of alkylglycosides on the bioavailability of calcitonin following nasal and ocular administration. METHODS: A salmon calcitonin specific radioimmunoassay kit was used to measure calcitonin levels in anesthetized rats at various times after nasal or ocular administration of calcitonin formulated with saline or with octylmaltoside, a medium chain length alkylglycoside or tetradecylmaltoside, a long chain alkylglycoside. The extent of calcitonin absorption was determined directly from the plasma calcitonin level-time curve and the bioavailability of calcitonin was determined from the area under the plasma calcium level-time curve. The calcium level was determined using a colorimetric method. RESULTS: When the nasal formulation contained calcitonin plus saline or 0.125% octylmaltoside, little or no calcitonin was absorbed. However, plasma calcitonin levels were increased and plasma calcium levels were decreased when the nasal formulation contained calcitonin plus 0.125% or 0.25% tetradecylmaltoside. Maximal calcitonin levels were observed 7.5-10 min after nasal administration of the formulation. Ocular administration of calcitonin formulated with tetradecylmaltoside also resulted in calcitonin absorption, but less calcitonin absorption was found after ocular administration than after nasal administration. CONCLUSION: The experimental data indicate that tetradecylmaltoside, but not octylmaltoside. can be effectively used to enhance the bioavailability of nasally and ocularly administered calcitonin.

Sleep apnea symptoms, nocturia, and diabetes in African-American community dwelling older adults.

The primary aim of this exploratory study was to examine the association between obstructive sleep apnea (OSA) symptoms, nocturia and diabetes in African-American community-dwelling older adults. A pencil and paper survey was used to collect symptoms of OSA, nocturia, excessive daytime sleepiness (EDS), lower urinary tract symptoms (LUT), and self-rated health. The convenience sample of community dwelling African-Americans (n = 87) included a majority of women (61%) and obese persons (60% with BMI > 30). The mean age was 64 years (range 50-91), and 40% were self reported Type 2 diabetics. Diabetics had significantly more OSA symptoms, more nocturia, and decreased self-rated health. Results of a stepwise logistic regression showed that persons with diabetes and those who reported EDS experienced significant three to four-fold risks for OSA. Similarly, diabetics, women, persons with LUT symptoms and those reporting EDS, had significant three to six-fold risks for nocturia > or = 2/night.

Structure-function relationship among Quillaja saponins serving as excipients for nasal and ocular delivery of insulin.

The purpose of this investigation was to explore the structure-function relationship among naturally occurring Quillaja saponins and derivatives for their ability to stimulate insulin delivery from nosedrops and eyedrops and to test the hypothesis that stimulation of peptide drug delivery was correlated with surfactant strength. Native saponins, including QS-21, were purified from an aqueous extract of Quillaja saponaria bark by adsorption chromatography and HPLC. Native saponins were then deacylated by mild alkaline hydrolysis to form DS-1 and DS-2, derivatives that are smaller and more hydrophilic than their parent compounds. DS-1 was further treated either to reduce an aldehyde residue to form DS-1(R) or to remove the fucose-containing oligosaccharide to form QH-957. Rats receiving eyedrops or nosedrops formulated with insulin, but without any Quillaja saponins, showed no hypoglycemic response. Rats receiving eyedrops or nosedrops formulated with insulin plus saponins showed a dose-dependent hypoglycemic response, with the following rank order: QS-21 > DS-1 > DS-1(R) > DS-2 > QH-957. Surfactant strength was determined by measurement of the critical micellar concentration (cmc) and hemolysis of sheep erythrocytes. The cmc was lowest for the parent saponins QS-21 and QS-18, and increased for the deacylated saponin derivatives DS-1, DS-2, and QH-957; hemolysis of sheep erythrocytes was observed at low concentrations (approximately 0.006 mM) of the parent saponins, QS-21 and QS-18, at intermediate concentrations (0.06-0.08 mM) of DS-1 and DS-2, and at higher concentrations of DS-1(R) (0.45 mM) and QH-957 (1.5 mM). Hence, efficacy as an absorption-enhancing agent was greatest in those saponins with the lowest hemolytic titers and cmc values. However, this relationship was not a strict one, because DS-1, which differs from DS-2 only in the absence of one glucose residue, was significantly more potent than DS-2 in stimulating the absorption of insulin. DS-1 and DS-2 share a similar cmc and hemolytic titer, so this difference in efficacy must be due to some specificity beyond simple surfactant strength. Furthermore, DS-1 does not trigger an immune response when administered to animals, whereas QS-21 is a strong immune system activator. Therefore, DS-1 has emerged as an interesting candidate for inclusion in an eyedrop or nosedrop formulation.

DS-1, a modified Quillaja saponin, enhances ocular and nasal absorption of insulin.

The purpose of this study was to test DS-1, a modified Quillaja saponin, for its efficacy as an absorption enhancer. Anesthetized rats receiving eyedrops or nosedrops formulated with regular pork insulin in saline showed no hypoglycemic response, indicating no systemic absorption of insulin. However, rats receiving eyedrops or nosedrops formulated with insulin plus 0.025-0.10% DS-1 showed rapid absorption of insulin and a concomitant decrease in serum D-glucose levels. No response was observed following sublingual or buccal delivery of insulin. In conclusion, the modified saponin DS-1 was efficacious at enhancing nasal or ocular insulin delivery at extremely low concentrations. The mechanism of DS-1 action is not yet known.

Systemic absorption of insulin and glucagon applied topically to the eyes of rats and a diabetic dog.

Nondiabetic rats were anesthetized with xylazine/ketamine to induce hyperglycemia and systemic insulin absorption from eyedrops formulated with dodecylmaltoside was quantitated by both a decrease in serum levels of D-glucose and an increase in immunoreactive insulin levels. When insulin eyedrop administration was delayed until 60 minutes after the administration of eyedrops containing 0.25% dodecylmaltoside, the enhanced systemic absorption of insulin was maintained, suggesting that dodecylmaltoside had an effect directly on the permeability of the nasal sinus epithelium. When glucagon was formulated in eyedrops or nosedrops containing dodecylmaltoside, systemic absorption of glucagon could be measured in the form of an increase in the serum D-glucose concentration following nasal application, but not after ocular application. Eyedrops containing insulin plus 0.125% dodecylmaltoside were administered to a diabetic dog; a dose of 20 units of regular insulin caused a modest decrease in serum D-glucose concentration and a concomitant increase in serum immunoreactive insulin content. These results provide evidence that peptide drugs such as insulin can be formulated in eyedrops with low concentrations of dodecylmaltoside, a mild nonionic surfactant.

Alkylglycosides enhance systemic absorption of insulin applied topically to the rat eye.

A series of alkylglycosides with various alkyl chain lengths and carbohydrate residues were tested for their ability to enhance systemic absorption of insulin after topical ocular delivery in anesthetized rats. Several reagents, including tetradecyl-, tridecyl- and dodecylmaltoside and dodecylsucrose, were potent stimulators of insulin absorption when used at concentrations as low as 0.125%. Other alkylglycosides such as decylsucrose, decylmaltoside, nonylglucoside, octylmaltoside, heptylglucoside and hexylglucoside were less potent or ineffective as absorption-enhancing reagents. By comparison, the bile salt sodium glycocholate was effective only when used at concentrations of 0.5% or greater. All of the reagents were effective only when used at concentrations above their critical micelle concentration and the most hydrophobic alkylglycoside reagents were the most efficacious at promoting systemic insulin absorption. The possible utilization of eyedrops containing insulin plus an absorption-enhancing alkylglycoside reagent in humans is discussed.

Insulin delivery in nosedrops: new formulations containing alkylglycosides.

Insulin was administered to rats via nosedrops in the presence or absence of various alkylglycosides; systemic insulin absorption was measured as a fall in blood D-glucose concentration in animals made hyperglycemic by xylazine/ketamine anesthesia. Nosedrops (0.04 ml) containing alkylglycosides or regular porcine insulin alone were without effect. Nosedrops containing both a small amount of alkylglycoside (0.03-0.50%) and insulin (2 U regular porcine) caused a rapid decrease in blood D-glucose levels and a concomitant increase in serum immunoreactive insulin levels. The maximal hypoglycemic response was observed between 60 and 120 min after delivery of nosedrops. Decylmaltoside was less effective at enhancing systemic insulin absorption than dodecylmaltoside, tridecylmaltoside, or tetradecylmaltoside, whereas octylmaltoside was totally ineffective. Dodecylsucrose, a compound which differs from dodecylmaltoside only in one carbohydrate residue, had a similar effect on blood D-glucose values when it was included in the nosedrop formulation with insulin. Decylsucrose was considerably less potent than dodecylsucrose at enhancing systemic absorption of insulin. Nonylglucoside was effective at promoting insulin absorption from nosedrops only when used at higher doses (0.25-0.50%), whereas heptylglucoside and hexylglucoside were ineffective. These results indicated that nosedrops containing insulin plus an extremely low concentration (0.03%) of an absorption-enhancing agent such as tetradecylmaltoside can be used to lower blood D-glucose values.

Protein crystal growth in microgravity-temperature induced large scale crystallization of insulin.

One of the major stumbling blocks that prevents rapid structure determination using x-ray crystallography is macromolecular crystal growth. There are many examples where crystallization takes longer than structure determination. In some cases, it is impossible to grow useful crystals on earth. Recent experiments conducted in conjunction with NASA on various Space Shuttle missions have demonstrated that protein crystals often grow larger and display better internal molecular order than their earth-grown counterparts. This paper reports results from three Shuttle flights using the Protein Crystallization Facility (PCF). The PCF hardware produced large, high-quality insulin crystals by using a temperature change as the sole means to affect protein solubility and thus, crystallization. The facility consists of cylinders/containers with volumes of 500, 200, 100, and 50 ml. Data from the three Shuttle flights demonstrated that larger, higher resolution crystals (as evidenced by x-ray diffraction data) were obtained from the microgravity experiments when compared to earth-grown crystals.

Xylosyl transfer to an endogenous renal acceptor. Characteristics of the reaction and properties of the product.

In the course of a study of UDP-xylose:proteoglycan core protein xylosyltransferase (EC 2.4.2.26), another xylosyltransferase was discovered in the soluble fraction of a rat kidney homogenate. The latter enzyme catalyzed [3H]xylosyl transfer from UDP-[3H]xylose to an endogenous acceptor and yielded a product in which the xylose was bound by an alkali-stable linkage. It was therefore concluded that the acceptor was not the core protein of one of the proteoglycans containing a xylose-->serine linkage, since this linkage is cleaved by alkali. The [3H]xylose-labeled product emerged with the void volume when chromatographed on Sephadex G-50, it was precipitated by trichloroacetic acid, and it had a mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of about 32,000 Da. Digestion with trypsin or alpha-amylase degraded the labeled product to small fragments, as determined by gel chromatography, suggesting that it was a glycoprotein related to glycogen. A product of similar characteristics was formed when UDP-[3H]glucose was substituted for UDP-[3H]xylose as the glycosyl donor, and the two nucleotide sugars were mutually competitive in the respective transfer reactions, indicating that they were substrates for the same enzyme. On the basis of these findings, it was tentatively concluded that the xylosyltransferase and its acceptor were the renal form of glycogenin.

Insulin administration to the eyes of normoglycemic human volunteers.

Animal studies have shown that insulin eyedrops containing an absorption-enhancing agent can have a significant effect on blood glucose levels. When formulated as a topical solution, insulin might potentially be used to treat or augment the treatment of diabetes mellitus in humans. We sought to investigate the feasibility of using insulin eyedrops in humans by studying the local toxicity and efficacy of insulin administered without surfactant to the eyes of healthy volunteers. A prospective, randomized, placebo-controlled, single-masked study was conducted in which 8 subjects were given 50 microliters of sterile normal saline containing varying insulin concentrations randomized to one eye, and 50 microliters of placebo (sterile normal saline) to the fellow eye. Subjective ocular irritation was evaluated, and the eyelids, conjunctiva, cornea, and anterior chamber were examined objectively with slit lamp biomicroscopy. Subjects were evaluated for 2 hours following administration of a single dose of insulin. There was no statistically significant difference (P > 0.05) in toxicity observed by any parameter evaluated between eyes receiving insulin and placebo. No systemic absorption of insulin was observed; blood glucose levels and serum immunoreactive insulin levels were unchanged. The results of this study suggest that single-dose insulin in concentrations up to 100 U/ml formulated in saline has no detectable clinical toxicity to the anterior structures of the human eye.

Efficacy of insulin eyedrops.

Systemic absorption of insulin delivered via eyedrops has been studied in rats made transiently hyperglycemic by anesthesia with xylazine/ketamine. Insulin at a concentration of 2 mg/ml was not absorbed significantly when saline alone was used as the formulation for the eyedrops (0.04 ml). When various emulsant agents were added to the eyedrop formulation, systemic insulin levels were increased and concomitantly, blood D-glucose levels were decreased. Saponin, Brij-78, BL-9 and several alkylglycosides all increased the systemic absorption of insulin following delivery in eyedrops. Not all surfactant agents were effective in promoting systemic insulin absorption from eyedrops, as evidenced by the failure of some non-ionic surfactants to increase insulin absorption. Similar results were obtained when nosedrops containing insulin plus non-ionic surfactants were administered to rats. In conclusion, systemic insulin absorption was greatly accelerated by the addition of certain emulsants to the eyedrop formulation and physiologically important levels of insulin could be delivered systemically following eyedrop administration.

Toxicity of insulin administered chronically to human eye in vivo.

Insulin administered in eyedrop from with a surfactant agent has been shown to be clinically effective in treating diabetes in animal models. Concentrations of insulin as high as 100 U/ml in saline were shown to produce no detectable clinical toxicity to human eyes in single-dose administration. We sought to investigate the local toxicity of insulin in human eyes during long-term, multidose administration. A prospective, randomized, placebo controlled, double-masked study was conducted involving eight healthy volunteers. Subjects were given 50 microliters sterile saline containing 100 U/ml crystalline porcine insulin randomized to one eye and 50 microliters placebo (sterile saline) to the fellow eye administered twice daily for 8 weeks. Subjective ocular irritation and visual acuity and objective assessment of the eyelids, conjunctiva, cornea, anterior chamber, crystalline lens, pupil size, and intraocular pressure were evaluated. Blood D-glucose levels were monitored to assess glycemic levels. There was no statistically significant difference (p > 0.05) observed between insulin-treated and placebo-treated eyes. Eyedrops containing insulin were subjectively as comfortable and objectively as clinically innocuous as sterile saline alone. The results of this study demonstrate that insulin (100 U/ml) in saline is nontoxic to the human eye after long-term, multi-dose exposure. Topical administration of insulin combined with an absorption-promoting agent may be a practical and feasible alternative to multiple daily subcutaneous injections or implanted pump devices currently used in the long-term treatment of diabetes mellitus if a nonirritating absorption-promoting agent can be identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of IGF-II/Man-6-P receptors on rat, rabbit, and human colon epithelial cells.

Previous experiments from this laboratory have established the presence of receptors for insulin and insulin-like growth factor I (IGF-I) on apical membranes prepared from rabbit colon epithelial cells; however, no receptors for multiplication-stimulating activity (MSA), the rat peptide hormone equivalent of human IGF-II, were found in this tissue. In the current studies, radioligand binding assays, covalent cross-linking experiments, and immunoblot analyses using a polyclonal rabbit antiserum that recognizes the IGF-II/mannose 6-phosphate (Man-6-P) receptor, all confirmed the presence of IGF-II/Man-6-P receptors on membranes prepared from rat and human colon epithelial cells. Exposure of rat colon epithelial cell membrane fractions to 5 mM Man-6-P before incubation with 125I-labeled IGF-II increased radioligand binding. Immunoblot analysis indicated that IGF-II/Man-6-P receptors were present in both unfractionated rat colon membranes and fractions enriched with apical membranes. Rabbit and human colon epithelial cells displayed a different pattern of receptor distribution than rat colon epithelial cells, with more insulin receptors but relatively few IGF-II/Man-6-P receptors. Immunohistochemical studies using a rabbit polyclonal antiserum confirmed that IGF-II/Man-6-P receptors were present on both the apical and the basolateral surfaces of colon epithelial cells.

Glucagon administration to the rat via eye drops.

Systemic absorption of glucagon from eye drops containing an emulsant caused an elevation in blood D-glucose concentrations in anesthetized rats. Glucagon (0.03 mg) delivered to the eye in buffered saline had no significant hyperglycemic action. However, delivery of glucagon in eye drops containing 0.25% saponin caused a rapid, dose-dependent increase in blood D-glucose values. Maximal absorption was observed 20 minutes after eye drop administration and values returned to baseline after 60 minutes. Immunoreactive glucagon levels measured 20 minutes after administration of eye drops containing saponin plus glucagon were increased by 2.4-fold compared to basal values. Glucagon administration via eye drops was ineffective when 0.5% Brij78 or BL-9 was substituted for saponin. These results demonstrate that in the rat, systemic absorption of glucagon delivered in eye drops is possible, but the choice of an emulsant may be critical.

Insulin signal transduction: the role of protein phosphorylation.

Recent evidence suggests that the mechanism of insulin action depends in part on protein phosphorylation on tyrosine residues. A cascade of phosphorylation/dephosphorylation reactions is proposed to modulate multiple enzymes involved in metabolism, protein synthesis, and cell growth. Direct evidence is presented for the phosphorylation of myelin basic protein and microtubule-associated protein 2 on tyrosine residues by the insulin receptor.

Identification of macrophage cell-surface binding sites for cationized bovine serum albumin.

Autoimmune diseases are characterized by the presence of autoantibodies often restricted to host proteins exhibiting charge rich domains. Charged polypeptides elicit strong immune responses, and cationized bovine serum albumin and other cationic proteins are significantly more immunogenic than their less charged counterparts. These phenomena may involve enhanced protein uptake by macrophages, resulting in greater processing and presentation of antigenic peptide-MHC complexes to T-cells. We compared macrophage cell-surface binding and uptake of native and cationized bovine serum albumin. Specific binding of [125I]cationized bovine serum albumin to THP-1 macrophages in vitro was 11-16 fold greater than for native albumin. Half-maximal inhibition of [125I]cationized albumin binding was observed at 10-7M ligand. The specificity of [125I]cationized bovine serum albumin binding and uptake was further studied in terms of competitive inhibition of proteolysis by proteins of varying charge content. Cationized bovine serum albumin, but not native albumin, inhibited proteolysis of [125I]cBSA. Calf thymus histones also inhibited cBSA degradation. High concentration of myelin basic protein was moderately effective at blocking cBSA degradation, while myoglobin and beta lactalbumin showed no inhibition. These results indicate that specific cell-surface binding sites which occur on macrophages may mediate selective uptake of certain proteins with highly charged domains including some autoantigens.

Apolipoprotein A-I decreases neutrophil degranulation and superoxide production.

Neutrophils participate in the acute phase response and are often associated with tissue injury in a number of inflammatory disorders. The acute phase response is accompanied by alterations in the metabolism of apolipoprotein A-I and high density lipoprotein (HDL). Structural considerations led to studies investigating the effect of purified HDL and apolipoprotein A-I on neutrophil degranulation and superoxide production. Apolipoprotein A-I but not HDL inhibited IgG-induced neutrophil activation by about 60% as measured by degranulation and superoxide production. This suggests that the lipid-associating amphipathic helical domains of apolipoprotein A-I mediate this effect. In support of this was finding inhibitory effects with two synthetic model lipid-associating amphipathic helix peptide analogs. Apolipoprotein A-I, containing tandem repeating amphipathic helical domains, was approximately ten times more effective than the two peptide analogs and inhibited neutrophil activation at well below physiologic concentrations. Competitive binding studies indicate that resting neutrophils have approximately 190,000 (Kd = 1.7 x 10(-7)) binding sites per cell for apolipoprotein A-I, consistent with a ligand-receptor interaction. These observations suggest that apolipoprotein A-I may play an important role in regulating neutrophil function during the inflammatory response.

Systemic absorption of insulin delivered topically to the rat eye.

Insulin administration in eye drops containing 1% saponin caused a rapid and reproducible reduction in blood levels of D-glucose in anesthetized rats; insulin eye drops lacking saponin were ineffective. Systemic insulin absorption also was observed when Brij 78 or BL-9 was substituted for saponin in eye drops containing insulin. Nonanesthetized rats displayed lower initial D-glucose levels than anesthetized rats, and little hypoglycemic response to insulin eye drops could be observed, suggesting that counter-regulatory hormones could effectively counterbalance the influence of exogenous insulin on glycemic control. Streptozotocin-diabetic rats displayed elevated blood D-glucose values (greater than 400 mg/dl), and these values were decreased to 100-200 mg/dl after the administration of insulin in eye drop solutions containing saponin. The site of insulin absorption appears to be the nasolacrimal drainage system of the rat because administration of a solution containing insulin plus saponin directly into the punctum caused a rapid, significant decrease in blood D-glucose levels. The observation that insulin could be absorbed from the rat eye is consistent with the possibility that insulin eye drops containing an absorption-enhancing surfactant agent could be of potential benefit in the treatment of diabetes mellitus in humans.