A study of reversibly inactivated lipases for use in a morphine-triggered naltrexone delivery system.

A key component of an implant that can be triggered by external morphine to release naltrexone is an inactivated enzyme that can be activated by morphine and which can then rapidly remove a protective coating surrounding a bioerodible polymer containing dispersed naltrexone. In this article we describe a lipase that has been conjugated with O3-carboxymethylmorphine, morphine-beta-3-glucuronide and O3-carboxypropylmorphine. The enzyme conjugate was then inactivated by complexation with affinity-purified goat polyclonal antimorphine antibodies. Antibody lipase interactions were measured by pH Stat and ELISA techniques. Affinity constants of the antibodies determined by radioimmunoassay using tritium-labeled morphine were 4.10 x 10(6), 3.18 x 10(6) and 3.38 x 10(7), respectively. While a concentration of 10(-5)M morphine was required to restore lipase activity, it is likely that a combination of correct morphine tether and correct affinity-purified antibody can increase sensitivity to the desired 10(-8)10(-9)M morphine level. Thus, a functioning device can almost certainly be constructed. However, it is unlikely that reactivation times of 1-2 h necessary for clinical usefulness in treatment of narcotic addiction can be achieved.

Biocompatibility and in vivo morphine diffusion into a placebo morphine-triggered naltrexone delivery device in rabbits.

Two key factors in developing a clinically useful triggered naltrexone delivery system are device biocompatibility and ability of morphine to diffuse from the blood into the device in concentrations useful to trigger delivery. Two types of devices were implanted subcutaneously into rabbits and their biocompatibility was investigated. One device consisted of the outer semipermeable regenerated cellulose acetate tubing, closed at both ends with double knots and filled with poly(N-vinyl pyrrolidone) as osmotic filler. The other device was a placebo device that contained within the regenerated cellulose acetate tubing, also closed at both ends with double knots, all device components except naltrexone. Both devices were biocompatible. When the regenerated cellulose acetate device filled with osmotic filler was implanted in rabbits which were subsequently dosed at day 7 and day 14 post-implant with 150 mg kg(-1) morphine sulphate, the concentration of morphine in the device was only about 10-fold less than that measured in rabbit blood. Thus, enough morphine diffuses into the device to make triggering in a real-life situation feasible.

Development of a drug delivery system for the treatment of periodontal disease based on bioerodible poly(ortho esters).

Poly(ortho esters) prepared by the condensation of 1,2,6-hexanetriol and an alkyl orthoacetate are viscous, semisolid materials at room temperature that can be injected using a blunt needle. When tetracycline was incorporated into these materials, complete release occurred within about 24 hours, but when small amounts of Mg(OH)2 were incorporated into the polymer release could be extended to many weeks, and a loading of 0.5 wt% resulted in sustained release of about 10 days. When adhesion was tested using bovine teeth, cohesive failure of the pure polymer occurred at a force of about 392 mN cm-2 and cohesive failure of a polymer incorporating 10 wt% tetracycline and 1 wt% (Mg(OH)2 occurred at about 118 mN cm-2. The combination of injectability, dentoadhesiveness and ability to control accurately the release of incorporated antibiotics makes these materials promising candidates for bioerodible delivery systems useful in the treatment of periodontitis. Toxicological studies are currently in progress.

A morphine-triggered delivery system useful in the treatment of heroin addiction.

The ultimate objective of this work is to develop a device that can be triggered by morphine to release naltrexone. Two device configurations are described. In one configuration, naltrexone is dispersed in cellulose acetate phthalate microspheres which are then spray-coated with trilaurin. In the other configuration, naltrexone is dispersed in an n-octyl half ester of methyl vinyl ether and maleic anhydride copolymer and the mixture fabricated into a disk which is then coated with trilaurin. The microspheres are designed to release naltrexone abruptly while the disks are designed to release naltrexone at a constant rate over a two week period. The microspheres, or the disk along with a reversibly inactivated lipase are placed inside a semipermeable membrane that allows free passage of morphine and naltrexone but excludes the higher molecular weight components of the device. Reversible inactivation of lipase is achieved by covalent attachment of morphine and complexing with morphine antibody. Activation of the device occurs by diffusion of morphine into the device and displacing the lipase-morphine conjugate from the antibody. The activated lipase then removes the trilaurin protective coating, thus triggering naltrexone release.

Percutaneous absorption and age. Implications for therapy.

Human skin changes dramatically with increasing age. Morphological, physiological, and biochemical changes within the tissue have been investigated and documented. Considerable interest in transdermal drug delivery to produce systemic effect has occurred in recent years. However, it is not known whether the penetration barrier of aged skin changes. Morphological and physiological changes in aged skin may affect the percutaneous absorption of compounds and thus their potential for localised, as well as systemic, efficacy. This article reviews the published literature on skin aging changes from adulthood to old age, collates these changes with clinical implications pertinent to the practising dermatologist, reviews the existing data supporting a change in the barrier function of human skin with increasing age, and comments on the relevance of conclusions previous investigators have drawn from their studies.

The effect of lipase on the release of naltrexone from triglyceride-coated cellulose acetate phthalate microspheres.

The ultimate objective of this work is to develop a device that can be triggered by morphine to release naltrexone. In this device, naltrexone is dispersed in cellulose acetate phthalate microspheres which are then spray-coated with a trilaurin protective coating. The microspheres are contained within a macroporous cylinder which also contains a reversibly inactivated lipase. This enzyme in its inactive state is unable to remove the protective coating but in its active state is able to do so. Inactivation is achieved by the covalent attachment of morphine followed by complexation with a morphine antibody. Triggering is accomplished by the displacement of the lipase-morphine conjugate from the antibody. In this phase we have investigated the effect of lipase on the release of naltrexone from trilaurin-coated microspheres and found that the coated microspheres are stable in a pH 7.4 phosphate buffer at 37 degrees C for at least 1 month, but release 80% of the incorporated naltrexone in one hour when 100 mg of capsules in 5 mL buffer are exposed to 25 micrograms of lipase.

Development of enzymatically degradable protective coatings for use in triggered drug delivery systems: derivatized starch hydrogels.

Starch is a common polysaccharide which consists of glucopyranose residues in an alpha-D-(1-4) linkage that yields D-glucose upon hydrolysis. Saturated aqueous solutions of soluble starch are easily reacted in the presence of glycidyl methacrylate to produce methacrylate grafted starch. Grafted starch solutions were polymerized to produce hydrogels, with and without the addition of an unsaturated acid. The grafted starch solutions and the resulting hydrogels are both shown to be degraded by the enzyme alpha-amylase. These acidic hydrogels are potentially useful as enzymatically degradable protective coatings in self-regulated drug delivery system applications. The pH of an acidic starch hydrogel required by this self-regulated drug delivery system does indicate that an adequately acidic hydrogel can be produced.

Controlled drug release from bioerodible hydrophobic ointments.

A new type of poly (ortho ester) was prepared by the condensation of a triol and an alkyl ortho ester. The condensation produces polymers that are viscous pastes at r.t. even though molecular weights in excess of 50,000 dalton can be produced. Such materials are useful as bioerodible drug delivery devices because rate of release of an incorporated therapeutic agent can be controlled by the addition of small amounts of acidic excipients to the polymer. A significant advantage of these materials is that therapeutic agents can be incorporated by simple mixing at r.t. without the use of solvents.

Pharmacodynamic measurements of methyl nicotinate percutaneous absorption: the effect of aging on microcirculation.

The penetration of drugs through aged skin is important both in terms of transdermal delivery to elicit systemic pharmacological effects, and for topical treatment. Cutaneous microcirculation efficiency, an integral parameter in the overall process of percutaneous absorption, was studied in young (20-34 years) and old (64-86 years) individuals. Cutaneous erythema as induced by topical administration of methyl nicotinate to the ventral forearm, was monitored non-invasively using laser-Doppler flowmetry. Dose-response behaviour was characterized by five parameters: (i) the time of onset of action; (ii) the time to reach maximum response; (iii) the magnitude of the maximum response; (iv) the area under the response-time curve; and (v) the time to decay to 75% of the maximum response. Additionally, the sensitivity and efficiency of the cutaneous microcirculation in both age groups was evaluated using a pharmacokinetic-pharmacodynamic model. Statistical analysis of all data showed no significant differences between the age groups for the same concentrations. The results indicate that microvessel reactivity to the applied stimulus is comparable in the ventral forearm of both young and old populations.

The effect of aging on percutaneous absorption in man.

Despite much research into the mechanisms of cutaneous aging and the identification of significant age-associated biological and biophysical changes within the skin, the question "How does aging affect percutaneous absorption (PA) in vivo?" remains unanswered. We have made in vivo measurements of PA in young (18-40 years) and old (greater than 65 years) subjects. Standard radiotracer methodology was employed and PA was quantified from the urinary excretion profiles of 14C radiolabel (corrected for incomplete renal elimination). Testosterone (TST), estradiol (EST), hydrocortisone (HC), benzoic acid (BA), acetylsalicylic acid (ASA), and caffeine (CAFF) have been studied. Permeation of HC, BA, ASA, and CAFF was significantly (p less than 0.01, 0.01, 0.01, and 0.05, respectively) lower in aged subjects, whereas the absorption of TST and EST was similar in the two groups. Thus it appears that aging can affect PA in vivo and that relatively hydrophilic compounds are particularly sensitive. The diminished surface lipid content of "old" skin implies a diminished dissolution medium for compounds administered topically. It is reasonable to speculate that this physiologic change will impact most severely upon those permeants whose lipid solubility is lowest (that is, HC, BA, ASA, CAFF). Furthermore, the typically reduced hydration of aged stratum corneum will compound this effect for these chemicals. Conversely, highly lipid-soluble chemicals (TST and EST) may still be able to dissolve readily into the stratum corneum even when the available lipid medium is reduced.

Assessment of skin barrier function using transepidermal water loss: effect of age.

To probe age-related changes in skin barrier function, transepidermal water loss (TEWL) rates have been measured in "young" (19-42 years) and "old" (69-85 years) subjects. TEWL was determined at ventral forearm skin sites, which had been occluded for 24 hr with polypropylene chambers. Baseline TEWL rates (J infinity), which showed no dependence on age, were measured for each subject before and after the experiment. Following removal of the occlusive chamber, TEWL was monitored continuously from t = 0.5 min until its return to the baseline (preocclusion) level, which was typically in the range of 2-7 g/m2/hr. Initial TEWL rates (mean +/- SD) were found to differ significantly between young (28.6 +/- 7.5 g/m2/hr; n = 26) and old (36.9 +/- 10.5 g/m2/hr; n = 18) subjects (P less than 0.01). Relaxation of TEWL to J infinity was significantly slower in the aged cohort, such that the characteristic time for diffusion of water in the stratum corneum was estimated to be (mean +/- SD) 176 +/- 59 min for the young subjects, compared to 360 +/- 76 min for the old (P less than 0.001.). Thus, the initial TEWL value following removal of occlusion is significantly greater, and the excessive stratum corneum hydration produced by occlusion is dissipated more slowly, in old skin than in young. A hypothesis to explain the slower relaxation of perturbed TEWL in old skin is proposed.

Percutaneous absorption in the aged.

The work described in this article reveals a remarkable lack of consensus as to whether percutaneous absorption changes as humans grow older. The data that have been recorded point to possible significant alterations in the barrier function with age. The importance of these observations with respect to dermatopharmacology and dermatotoxicology is clear. The absence of a clearly defined relationship between aging, percutaneous penetration, and the properties of the molecules crossing the skin barrier represents an unacceptable gap in fundamental dermatologic knowledge. With the changing demographic pattern of Western civilization and the increasing awareness of human subjects for the condition of their skin, and the potential for drug delivery via their skin, it is crucial that we begin to establish precisely how the barrier function alters with increasing age. The answer to this question may permit unique improvements in the quality of both local and systemic health in aging populations.

Biologically active products of complement and acute lung injury in patients with the sepsis syndrome.

To determine if biologically active products of complement appear during sepsis and to establish the relationship of these components to the respiratory and hemodynamic complications of sepsis, we measured C5a des Arg and C3a des Arg (radioimmunoassay), neutrophil chemotaxis, and neutrophil-aggregating activity in plasma obtained from 40 patients at the time sepsis was suspected clinically. Levels of C3a des Arg and C5a des Arg were elevated in 35 and 38 patients, respectively, and in all 25 with positive blood cultures. Highest C5a des Arg levels occurred in patients with hypotension (less than 90 mmHg) and/or acidemia. The C5a des Arg concentrations were significantly higher in patients with than in those without neutrophil-chemotactic activity. Neutrophil-aggregating activity was less sensitive an index of complement activation, as it was positive in only 8 patients and correlated poorly with C5a des Arg and C3a des Arg values. Using a composite scoring system to quantify sepsis-related pulmonary abnormalities, we found that neither biologic nor immunologic assays of complement activation products correlated with the initial severity nor predicted the development or worsening of associated acute lung injury.