Douglas R. Flanagan

Drug transfer through mucus

Mucus is a complex aqueous mixture of glycoprotein, lipid, salts and cellular debris covering many epithelial surfaces in the human body. It affords protection for the underlying tissues from various environmental insults and the effects of enzymes or other chemical agents. In performing its functions, mucus may adversely affect the absorption or action of drugs administered by the oral, pulmonary, vaginal, nasal or other routes. The nature of mucous in normal and diseased states is summarized and discussed in this review. The study of the permeability of native or purified mucous gels is also important to understanding how it may alter the action or absorption of drugs that come in contact with epithelial surfaces. Various methods for studying mucous permeability and models for analyzing permeation data are discussed. A compilation of drug permeability data through various types of mucus is included. Drug binding to mucus is also important to understanding the relative importance of hindered diffusion versus drug binding to altered permeability through mucous layers. This is discussed with methods for and results of drug-mucus binding studies.

Influence of formulation methods on the in vitro controlled release of protein from poly(ester) microspheres

Abstract Poly ( dl -lactide/glycolide, 50:50) microspheres containing bovine serum albumin (BSA) were pre-pared with and without Carbopol® 951 (a potential adjuvant agent) by o/o, o/w and (w/o) /w emul-sion methods. The protein loading of the microspheres reached 50%–70% of the theoretical amount of protein put into the formulation medium. The microsphere particle size was approximately 500 μm, 25–100μm, 10–20/nn using o/o, o/w, or (w/o)/w emulsion techniques, respectively. The release of BSA was dependent on the preparation method. The greatest burst of release was found for vacuum-dried microspheres formulated using the (w/o)/w method. This burst effect could be eliminated by lyophilizing the microspheres following their preparation. BSA was released at a higher initial rate from microspheres prepared by the o/w emulsion method that contained Carbopol® 951 than from micro-spheres not containing Carbopol® 951. Release studies also suggested that the release of BSA could be sustained for 54, 36, or 34 days for microspheres prepared by o/o, o/w, or (w/o)/w methods, respectively.

Degradation of poly(ester) microspheres

Biodegradable polymeric microspheres have been prepared by spray drying, precipitation, rotary evaporation and press grinding methods. Erosion of microspheres of poly(lactide), poly(3-hydroxybutyrate), copolymers of lactide and glycolide, and copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate at 85 degrees C and 37 degrees C have been studied using ion chromatography, nuclear magnetic resonance, residual mass measurements, viscometry and gel permeation chromatography. Such studies demonstrated that these polyester matrices degraded via (1) random chain scission and (2) release of soluble monomeric and oligomeric products. Protein release from microspheres prepared by these methods indicated that most of the protein is released before the polymer matrix loses weight.

The limiting role of mucus in drug absorption : drug permeation through mucus solution

A purified model in vitro mucus system containing primarily the large, 400 kDa glycoprotein fraction of mucus has been developed for use in drug permeability and drug binding studies. The effect of protein solutions, either bovine serum albumin (BSA) or purified porcine gastric mucus, on the permeability behavior of five drugs was studied. The drugs chosen were isoniazid, pentamidine, rifampicin, p-aminosalicylic acid, and pyrazinamide, all of which can be potentially delivered as pulmonary aerosols. BSA was included in the permeability studies for comparison with previously obtained data regarding their binding behaviors to mucin relative to BSA. A custom membrane holder with a 3 mm chamber for mucin or other solutions was used in a Side-Bi-SideR diffusion apparatus to measure drug permeation through the solutions. Apparent permeability coefficients were calculated for each barrier in the series barrier system, with a protein solution being one of the barriers. The protein solutions significantly reduced the permeability of the drugs studied compared with their permeability through blank buffer solution. Both the lag time and the steady-state flux of the compounds were altered in the presence of protein indicating that there is more than protein binding affecting permeability. Such reductions in permeability coefficients need to be considered for all compounds that must traverse any mucosal surface prior to absorption or action.

Immunocontraception in Wild Horses: One Inoculation Provides Two Years of Infertility

Abstract Previous studies reported one year of contraception associated with a 1-injection porcine zona pellucida (PZP) vaccine. We have subsequently determined contraceptive effectiveness of a presumptive 1-injection, 2-year-duration PZP vaccine in free-roaming wild horses (Equus caballus) in Nevada, USA. In January 2000, we captured, freeze-branded, treated, and subsequently released 96 adult females that received 1) a primary dose of vaccine emulsion consisting of aqueous PZP and Freunds Complete Adjuvant, and 2) booster doses of PZP and adjuvant in controlled-release polymer pellets. We determined PZP release characteristics of pellets in vitro, prior to field use. We determined reproductive success in treated and untreated females through October 2004 via measurement of estrone sulfate and progesterone metabolites in fresh feces collected from the ground and by twice-annual foal counts. Among treated females, annual reproductive success from 2001 though 2004 sequentially was 5.9%, 14.0%, 32.0%, and 47.5%. Untreated females showed average reproductive success of 53.8 ± 1.3% across this period. This study revealed that: 1) PZP acted as an effective contraceptive for 2 years posttreatment; 2) some residual contraceptive effect remained in year 3; and 3) fertility returned to control levels by year 4 posttreatment. It appears that controlled-release technology can replace both the second (1-month) and annual booster injection of PZP vaccine, thereby decreasing cost and increasing efficiency of use of this vaccine in wild horse management.

Salt and cosolvent effects on ionic drug loading into microspheres using an O/W method

Salt effects on aqueous solubility and microsphere entrapment efficiency of a model ionic drug (quinidine sulfate) were studied. Poly-D,L-lactic acid (PLA) microspheres were prepared using an O/W solvent evaporation method with various electrolytes added in different concentrations to the aqueous phase. Salts affect microsphere drug loading by changing the aqueous solubility of both the drug and the organic solvent (dichloromethane, DCM). Quinidine sulfate solubility was depressed by either a common ion effect (Na(2)SO(4)) or by formation of new, less soluble drug salts (e.g., bromide, perchlorate, thiocyanate) for which solubility products (K(sp)) were estimated. Inorganic salts depress DCM aqueous solubility to different extents as described by the Hofmeister series. NaClO(4) and NaSCN depressed drug solubility to the highest extent, resulting in microspheres with high drug loading (e.g., >90%). Other salts such as Na(2)SO(4) did not depress quinidine sulfate solubility to the same extent and did not improve loading. The use of a cosolvent (ethanol) in the organic phase improved microsphere drug loading and resulted in a uniform microsphere drug distribution with smooth release profiles.

Cocrystal intrinsic dissolution behavior using a rotating disk

The aim of this study was to investigate the dissolution characteristics of an acetaminophen/theophylline (AT) cocrystal compared with its pure components and physical mixtures. Intrinsic dissolution studies were conducted by a rotating-disk method. Solubility studies were conducted by collecting transient samples at 5, 30, and 60 min and equilibrium samples after 72 h, both at 37 °C. The AT cocrystal had a faster dissolution rate than AT physical mixtures, and the dissolution profiles were congruent (1:1 mole ratio) under different pH conditions. Thus, the AT cocrystal dissolved congruently at short times and exhibited higher transient solubility compared with its two pure components. Equilibrium solubilities of theophylline from the cocrystal were lower than transient values due to theophylline hydrate precipitation but no precipitation of free acetaminophen occurred. The solubility behavior of acetaminophen and theophylline exhibited typical 1:1 complex formation in physical mixtures, cocrystal, and phase-solubility studies. The Levich equation was used to predict the dissolution behavior of the AT cocrystal as well as that of the single components.

Accelerated Degradation of Poly(ε-caprolactone) by Organic Amines

The solid-state degradation of poly(ε-caprolactone) catalyzed by primary, secondary and tertiary alkylamines was investigated. The degradation process was monitored by weight loss and molecular weight change measured by gel permeation chromatography. Degradation studies were conducted at 37°C in methanol solutions of the alkylamines. Primary alkylamines caused rapid weight loss (i.e., ~90% weight loss in 30 days) that depended on alkylamine concentration, molar ratio of alkylamine to poly(ε-caprolactone) monomer and alkyl chain length. The secondary alkylamines caused less rapid polymer weight loss (i.e., ~90%) weight loss within 80 days). One tertiary alkylamine (N,N-diisopropylethylamine) showed little catalytic effect while a bicyclic tertiary alkylamine (quinuclidine) was about as catalytic as the primary alkylamines. The degradation products isolated when primary alkylamines were used include both esters and amides indicating that nucleophilic attack by the alkylamines competed with the amine-catalyzed methanolysis reaction. Only ester moieties could be identified in the products from reactions containing secondary and tertiary alkylamines, which indicated that they acted as nucleophilic catalysts. All of the primary alkylamines reduced poly(ε-caprolactone) molecular weight from about 25,000 to 10,000 within 10 days after which the molecular weight of the remaining solid leveled off even though weight loss continued.

Controlled-release components of PZP contraceptive vaccine extend duration of infertility

Successful immunocontraception of wildlife relying on repeated access to individuals for boosters has highlighted the need to incorporate primer and booster immunisations into one injection. We have investigated use of controlled-release polymers (lactide–glycolide) in small pellets to provide delayed in vivo delivery of booster porcine zona pellucida (PZP) antigen and adjuvant. This report reviews pellet-making methodology, in vitro testing of controlled-release pellets and in vivo effects of controlled-release PZP vaccine. We assessed 3 different manufacturing approaches for producing reliable, cost-effective pellets: (1) polymer melting and extrusion; (2) solvent evaporation from polymer solution; and (3) punch and die polymer moulding. In vitro testing of release patterns of controlled-release formulations, towards development of a 3-year duration vaccine, provided estimates for in vivo use of pellet preparations. These in vitro studies demonstrated protein release delay up to 22 months using 100% l-lactide or polycaprolactone polymers. For in vivo tests, pellets (1-, 3-, and 12-month release delay) serving as boosters were administered intramuscularly with PZP/adjuvant liquid primer to wild horses (Equus caballus), white-tailed deer (Odocoileus virginanus) and African elephants (Loxodonta africana). Horse field studies assessed fertility via offspring counts and/or faecal-hormone pregnancy testing. Treatment decreased fertility 5.3–9.3-fold in Year 1 and 3.6-fold in Year 2. In preliminary testing in deer, offspring counts revealed treatment-associated fertility reduction of 7.1-fold Year 1 and 3.3-fold Year 2. In elephants, treatment elevated anti-PZP titres 4.5–6.9-fold from pretreatment (no fertility data).

Immunocontraception in feral horses: One inoculation provides one year of infertility

We determined contraceptive effectiveness of a 1-inoculation, 1-year porcine zona pellucida (PZP) vaccine in free-roaming feral horses (Equus caballus) in Nevada. We captured, freeze-branded, treated, and subsequently released 267 adult feral mares given (1) 2 inoculations (13-17 days apart) of vaccine emulsion consisting of aqueous PZP and Freunds Complete Adjuvant (FCA; injection 1) or Freunds Incomplete Adjuvant (FIA; injection 2), (2) same 2 inoculations as in (1) except also containing a carbomer adjuvant in inoculation 1, or (3) a single inoculation of an emulsion of PZP and FCA containing a second dose of PZP and carbomer adjuvant in controlled-release polymer microspheres. We administered inoculations in January 1996, and monitored the mares via fecal analysis for pregnancy and via ground survey for foal production through October 1997. We determined pregnancy via measurement of estrone sulfate and progesterone metabolites in fresh feces collected from the ground. Among 2-inoculation mares, reproductive success across 1 year was 12.8% (carbomer adjuvant absent) and 10.6% (carbomer adjuvant present). In mares given 1 injection containing microspheres, reproductive success was 11.3%. The concurrent rate in 72 untreated mares was 62.5%. This study revealed marked and equivalent 1-year contraceptive efficacy in 1-and 2-inoculation PZP vaccine, indicating that controlled-release technology can replace a second inoculation and thereby increase PZP vaccine cost effectiveness and potential for use in feral horse management.