Intranasal absorption of granulocyte-colony stimulating factor (G-CSF) from powder formulations, in sheep.

Granulocyte-colony stimulating factor (G-CSF) was administered to sheep in three different nasal formulations and as a subcutaneous injection. The nasal formulations were: a solution containing L-alpha-lysophosphatidylglycerol (LPG), a powder formulation comprising small starch microspheres (SSMS) and a powder formulation comprising SSMS and LPG. Absorption of G-CSF was assessed directly by quantitation in plasma and indirectly by measurement of the pharmacodynamic response in terms of leucocyte and neutrophil counts. After the nasal delivery of the G-CSF powder formulation containing SSMS and LPG the absorption of G-CSF was significantly higher (P<0.01) than that from the simple nasal solution or the powder without the enhancer, but the resulting pharmacological response was not significantly different. The bioavailability of G-CSF from the powder formulation containing SSMS and LPG relative to the subcutaneous injection was 8.4% (+/-3.4). We also found that at the respective G-CSF doses investigated, the pharmacodynamic response of this nasal formulation, was similar to that obtained after the subcutaneous administration. The study indicates that the powder formulation containing enhancers could offer an alternative delivery route for G-CSF in the form of intranasal administration.

Aggregation pathway of recombinant human keratinocyte growth factor and its stabilization.

Recombinant human keratinocyte growth factor (rhKGF) is prone to aggregation at elevated temperatures. Its aggregation pathway is proposed to proceed initially with a conformational change which perhaps results from repulsion between positively charged residues in clusters forming heparin binding sites. Unfolding of the protein leads to formation of large soluble aggregates. These soluble aggregates then form disulfide cross-linked precipitates. Finally these precipitates are converted to scrambled disulfides and/or non-disulfide cross-linked precipitates. Stabilizers such as heparin, sulfated polysaccharides, anionic polymers and citrate can greatly decrease the rate of aggregation of rhKGF at elevated temperatures. These molecules may all act by reducing charge repulsion on the protein thus stabilizing the native conformation. EDTA, on the other hand, is found to inhibit disulfide formation in aggregates and has only a moderate stabilizing effect on rhKGF.

Poly-DL-lactic acid: polyethylene glycol block copolymers. The influence of polyethylene glycol on the degradation of poly-DL-lactic acid.

ABA block copolymers of polyethylene glycol and poly-DL-lactic acid were prepared by ring-opening polymerization of DL-dilactide with alpha,omega-dihydroxy polyethylene glycol, Mn 1000 or 2000. The morphology of the resulting copolymers, with PEG:PLA ratios(mol/mol) of 1:2, 1:3 and 1:4, was characterized by DSC and ESR spectroscopy. The rate of water uptake was biphasic, reflecting the contribution of two processes: rapid diffusion of water into the initially miscible PEG and PLA blocks; then a slower rate of hydration possibly due to phase separation and hydrolytic cleavage of the PLA blocks. The rate of hydrolytic degradation of the block copolymers in DI water at 37 degrees C was measured by two methods: weight loss and colorimetric analysis of the carboxy end group concentration resulting from chain scission of PLA blocks. As a result of phase separation, the rate of scission of PLA blocks in the copolymers was similar to that of the PLA homopolymer. The more rapid onset of weight loss of the copolymers, relative to PLA, is attributed to the greater water solubility of PEG-PLA oligomers and their greater diffusivity in the more highly hydrated copolymers.

Spin labels as a probe of the molecular environment of covalently bound ligands in an hydrophobic and an hydrophilic polymer.

The molecular motions of spin-labelled ligands covalently bound by spacer groups to an hydrophobic and an hydrophilic polymer matrix were evaluated by ESR spectroscopy. The ligands were prepared by alkylation of 4-N-methylamino-TEMPO with the omega-bromocarboxylic esters, Br(CH2)nCOOEt, n = 1,4 and 10. The hydrolysed esters were coupled to a cross-linked aminoethylated polyacrylamide hydrogel (n = 1,4,10) and to a surface hydroxylated elastomeric polyester (n = 4). The rotational correlation times (tau c) of the nitroxide label in the hydrogels were measured in the dry state and after exposure to water at pH 4, 7.4 and 10.5. The tau c of the nitroxide label was insensitive to the length of the spacer group and to the degree of protonation of the tertiary amino group of the ligand. There was no evidence of self-association of the ligand and spacer, or more than a single phase within the polyacrylamide hydrogel. The tau c of the nitroxide labelled polyester was similarly insensitive to pH, but was sensitive to organic solvents. The low mobility of the spin label and its high concentration were consistent with the spin label being covalently bound within the hydrophobic polyester matrix to a depth of at least 5 microns.

The biodegradability of polyester blends.

Blends of poly(epsilon-caprolactone) (PCL) and poly(L-lactic acid) (PLLA) with polyglycolic acid-co-L-lactic acid (PGLA) were prepared by three methods: compression moulding, coprecipitation, and solvent evaporation of a methylene chloride-in-water emulsion of the polymers. The rates of hydrolytic chain scission of each component of the blends were determined by deconvolution of GPC traces of samples maintained in phosphate buffer, pH 7.4, 37 degrees C, for up to 3000 h. The observed rates were dependent on the method of blending. For compression moulded blends, the rate of chain scission of PGLA was decreased and that of PCL and PLLA increased. A corresponding delay in the onset of weight loss was also observed. There was no evidence of blend miscibility.

Esters and lactones of phenolic amino carboxylic acids: prodrugs for iron chelation.

The new iron chelator N,N'-bis(2-hydroxyphenyl)ethylenediamine-N,N'-diacetic acid (1), its dilactone 2, N,N'-bis(2-hydroxybenzyl)-2-hydroxypropylene-1,3-diamine-N,N'- diacetic acid (3), and its methyl ester lactone 4 and a series of esters of N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (5) were prepared and their iron chelating efficacy and toxicity determined by using the hypertransfused mouse model of iron overload. The biological activities were compared with results obtained with use of the hypertransfused rat. Esterification enhanced the oral iron chelating activity but also increased toxicity. The diisopropyl ester of 5 exhibited the highest therapeutic index. In vitro measurements showed that the rate of ester hydrolysis at pH 7.5 increased by a factor of 10(4) in the presence of 5 X 10(-4) M ferric ion, which may account for the utility of esters and lactones as prodrugs. Seventeen other chelating agents were screened but showed no intraperitoneal or oral activity.

High-affinity cannabinoid binding sites in brain membranes labeled with [3H]-5'-trimethylammonium delta 8-tetrahydrocannabinol.

The binding of [3H]-5'-trimethylammonium delta 8-tetrahydrocannabinol (THC) [( 3H]TMA) to rat neuronal membranes was studied. TMA is a positively charged analog of delta 8THC modified on the 5' carbon, a portion of the molecule not important for its psychoactivity. Unlabeled TMA inhibits field-stimulated contractions of the guinea-pig ileum (IC50 = 1 microM) in the same presynaptic manner as delta 9THC. [3H]TMA binds saturably and reversibly to brain membranes with high affinity (KD = 89 nM) to apparently one class of site (Hill coefficient, 1.1). Highest binding site density occurs in the brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacological properties. Molecular sieve chromatography reveals a bimodal peak of [3H]TMA binding activity of approximately 60,000 daltons apparent molecular weight. delta 9THC competitively inhibits [3H]TMA binding potently (Ki = 27 nM) and stereoselectively. For some cannabinoids potency in behavioral and physiological tests parallels their affinity for the [3H]TMA binding site. However, several nonpsychotropic cannabinoids are active at the binding site.

The intracellular degradation of poly(epsilon-caprolactone).

Poly(epsilon-caprolactone) [PEC], a biodegradable aliphatic polyester, undergoes a two-stage degradation process: The first lengthy phase involves nonenzymatic hydrolytic cleavage of ester groups, the second phase beginning when the polymer is more highly crystalline, and of low molecular weight. The cellular events of the second phase were examined by implanting gelatin capsules containing 25 mg of low molecular weight (Mn 3000) PEC powders, 106 to 500 micron, in rats. PEC fragments ultimately were degraded in phagosomes of macrophages and giant cells, the process requiring less than 13 days for completion at some sites. PEC was also identified within fibroblasts. These studies support the intracellular degradation of PEC as the principal pathway of degradation once the molecular weight of the aged polymer is reduced to 3000 or less.

The effect of a biodegradable contraceptive capsule (Capronor) containing levonorgestrel on gonadotropin, estrogen, and progesterone levels.

Eight ovulatory women participated in the preliminary evaluation of a new method of contraception. The technique consisted of subdermal implantation of a biodegradable capsule capable of controlled release of levonorgestrel. The study spanned five menstrual cycles: three observation cycles to confirm ovulation, a cycle with the capsule implanted, and a follow-up observation cycle after its removal. All subjects who were sexually active relied on barrier contraception during the study. Basal body temperature determinations were made throughout all five cycles, and the last three cycles included serum assays of luteinizing hormone, follicle-stimulating hormone, estradiol, progesterone, and levonorgestrel on days 5, 8 to 18, and 22. All subjects except one experienced suppression of ovulation while the capsule was in place. No serious adverse effects were encountered. These results would seem to justify a larger clinical trial to assess the actual efficacy of this contraceptive method.

Radioimmunoassays for cannabinoids.

The simplicity, sensitivity, and specificity of radioimmunoassay have made it an attractive procedure for the analysis of delta-9-tetrahydrocannabinol (THC) in biological fluids or tissues. The presence of closely related compounds such as metabolites may interfere with radioimmunoassay results. Appropriate design of immunogens may diminish such interference. This work has been directed towards the use of the amyl side chain for linking cannabinoid compounds to proteins to form immunogens. Although the amyl side chain is metabolized to some extent, the metabolites are not quantitatively significant in most cases. 5'-Carboxy-delta-8-THC and 5'-carboxy-delta-9-THC were linked to bovine serum albumin. Immunization of rabbits with the resulting conjugates resulted in the formation of antisera with high selectivity for delta-9-THC vs. its carboxylic acid metabolite, 11-nor-9-carboxy-delta-9-THC. Delta-8-THC radioligands (4',5'-tritium and 5'-iodine-125) could be used with these antisera for analysis of delta-9-THC in plasma. Sensitivity with tritium-labeled material is about 2.5 ng/ml. 5'-Oxo-11-nor-9-carboxy-delta-8-THC was used to prepare an immunogen which led to the generation of an antiserum highly specific for 11-nor-9-carboxy-delta-9-THC. This antiserum and iodine-125-5'-iodo-11-nor-9-carboxy-delta-8-THC were used to develop a highly specific assay for 11-nor-9-carboxy-delta-9-THC in plasma.

Aliphatic polyesters II. The degradation of poly (DL-lactide), poly (epsilon-caprolactone), and their copolymers in vivo.

The mechanisms of biodegradation of poly (DL-lactide), poly (epsilon-caprolactone), and copolymers of epsilon-caprolactone with DL-dilactide, delta-valerolactone, and DL-epsilon-decalactone in rabbit were shown to be qualitatively similar. However, the rate of the first stage of the degradation process, non-enzymatic random hydrolytic chain scission, varied by an order of magnitude and was dependent on morphological as well as chemical effects. Weight loss was generally not observed until the molecular weight had decreased to 15,000 or less. Poly (DL-lactide) differed from the other polyesters studied, the rate of chain scission increasing after the commencement of weight loss. The rate of weight loss was greater and the period prior to weight loss was shorter when the comonomer content of copolymers of epsilon-caprolactone was sufficient to reduce the melting point of epsilon-caproate sequences to body temperature.

Biodegradable drug delivery systems based on aliphatic polyesters: application to contraceptives and narcotic antagonists.

The classes of polymer which form the basis of different types of drug delivery systems are discussed, and the relationships between the chemical structure of the polymer and its permeability, morphology, biodegradability, and mechanical properties are considered, using polyesters as specific examples. Studies of the permeability and biodegradability of poly epsilon-caprolactone), poly(DL-lactic acid), and various copolymers are described and used to illustrate how these properties may be varied by the choice of polymer structure. An induction period prior to bioerosion of these polymers, coupled with high permeability, permits their use as reservoir devices (capsules) which exhibit constant, diffusion-controlled drug release rates and which erode after the drug is exhausted. The applications of this approach to the long term delivery (1 year) of levonorgestrel, a contraceptive agent, and the short term delivery (1-2 months) of naltrexone, a narcotic antagonist, are described.

Sustained drug delivery systems II: Factors affecting release rates from poly(epsilon-caprolactone) and related biodegradable polyesters.

The release rates of several steroids from films and capsules of homopolymers and copolymer of epsilon-caprolactone, DL-lactic acid, and glycolic acid were measured in vitro and in vivo for up to 200 days. Relatively constant release rates from capsules (reservoir devices) were observed only under certain conditions. Factors that influence the drug release kinetics were evaluated. Release from poly(epsilon-caprolactone) and poly(epsilon-caprolactone-co-DL-lactic acid) was diffusion controlled. Release from poly(DL-lactic acid-co-glycolic acid) was associated with polymer degradation. Release from poly(DL-lactic acid) was very slow when diffusion controlled.

Synthesis of 11 beta,13 beta- and 13 beta,16 beta-propano steroids: probes of hormonal activity.

Syntheses of 11 beta,13 beta- and 13 beta,16 beta-propano derivatives of 17 alpha-ethynyl-17 beta-hydroxygon-4-en-3-one are described. The 13 beta,16 beta bridge was constructed by intramolecular alkylation of the C-16 enolate anion from 3-methoxy-13 beta-[3'-(tosyloxy)propyl]gona-3,5-dien-17-one, the latter being obtained via Birch reduction of both aryl groups of 17 beta-hydroxy-3-methoxy-13 beta-(3'-phenoxypropyl)gona-1,3,5(10),8-tetraene (1). The 11 beta,13 beta bridge was constructed by Prins cyclization of 17 beta-acetoxy-3-methoxy-13 beta-(3'-oxopropyl)gona-1,3,5(10),9(11)-tetraene, itself obtained via Birch reduction of only the side-chain aryl group of 1. Binding affinities of certain of these compounds and substituted 13 beta-propyl derivatives of 17 alpha-ethynyl-17 beta-hydroxygon-4-en-3-one for the uterine cytosol receptor of progesterone are reported, and the origin of the high progestational activity of norgestrel and 11 beta-substituted progestins is discussed.

Sustained drug delivery systems. I. The permeability of poly(epsilon-caprolactone), poly(DL-lactic acid), and their copolymers.

The maximum steady state flux, diffusion coefficients, and solubilities of five contraceptive steroids in homopolymers and copolymers of epsilon-caprolactone and DL-lactic acid were determined. The permeabilities of polymers of epsilon-caprolactone were comparable to silicone rubber and, by inference, are suitable for the construction of drug delivery devices. Poly(DL-lactic acid) was 10(4) times less permeable, although its permeability was significantly enhanced by additives.

The selection and evaluation of new chelating agents for the treatment of iron overload.

A large-scale systematic evaluation of potential iron chelators for the treatment of hemosiderosis was conducted. The compounds were identified and evaluated using a hypertransfused mouse screen in which deferrioxamine B was a standard. This screen was designed to measure iron depletion in the tissues as well as iron excretion. Groups of 10 previously hypertransfused BDF1 male mice received a single daily i.p. injection of either vehicle, standard, or test compound for 7 days. Iron in daily urine pools and individual spleen and liver homogenates was determined by atomic absorption. More than 70 chelators were evaluated, including natural and synthetic hydroxamic acids, phenols, catechols and tropolones known to have a high affinity for iron (III) in vitro. Ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid) was shown to be considerably more effective than deferrioxamine B (i.p.) and, in addition, was orally active. Factors determining the efficacy of this and other chelating agents are discussed.

Synthesis, antifertility activity, and protein binding affinity of 7(8 leads to 11 alpha)abeo steroids.

A series of 7(8 leads to 11 alpha)abeo steroids was synthesized by a modification of the previously described total synthesis of this class of compounds and evaluated for biological activity. In general, there was a marked reduction in the relative binding affinities of these compounds for the rabbit uterus estrogen and progestin receptor proteins. None of the compounds which were subjected to uterotropic, antiuterotropic, postcoital, progestational, antiprogestational, or antiandrogenic assays showed any significant activity.

PIP: Synthesis, antifertility activity, and protein binding afinity of 7(8 to 11alph) abeo-estranes and -pregnanes are described. There was a marked reduction in the relative binding affinities of these compounds for the rabbit uterus estrogen and progestin receptor proteins. None of the compounds subjected to uterotropic, antiuterotropic, postcoital, progestational, antiprogestational, or antiandrogenic assays revealed any marked activity.