Effects of chronic cocaine on monoamine levels in discrete brain structures of lactating rat dams.

Chronic gestational cocaine administration has been correlated with high levels of postpartum maternal aggression towards intruders and altered levels of oxytocin in the amygdala. Cocaine may alter both oxytocin and maternal aggression either directly or indirectly through changes in monoamine levels in relevant brain regions. In this study, pregnant female rats were randomly assigned to one of four groups; three cocaine dose groups (7.5, 15 or 30 mg/kg), or a saline-treated group (0.9% normal saline) and given subcutaneous injections twice daily (total volume 2 ml/kg) throughout gestation. Behavioral responses to an inanimate object placed in the homecage were assessed on Postpartum Day (PPD) 6. Immediately following testing, animals were sacrificed and four brain regions implicated in maternal/aggressive behavior (medial preoptic area [MPOA], ventral tegmental area [VTA], hippocampus, and amygdala) were removed for monoamine level analyses using high-performance liquid chromatography. Dams given 30 mg/kg cocaine throughout gestation had significantly higher levels of dopamine (DA) and nonsignificantly elevated serotonin (5-HT) levels relative to saline-treated controls. These dams also exhibited higher frequencies of defensive behavior toward an inanimate object compared to saline-treated controls. Potential mechanisms mediating cocaine-induced increases in responding are proposed.

Alternate drug delivery routes for A-71623, a potent cholecystokinin-A receptor agonist tetrapeptide.

A-71623 (BOC-Trp-Lys(epsilon-N-2-methylphenylaminocarbonyl)- Asp-(N-methyl)-Phe-NH2) is a tetrapeptide which has high affinity and selectivity for cholecystokinin receptors; it is a potent appetite suppresser in animal studies. Because of its low (< 1%) oral bioavailability, studies were performed to assess the feasibility of delivery of A-71623 by pulmonary, sublingual, and transdermal routes of administration. The pKa was determined to be 4.2 by spectrophotometric titration; aqueous solubility is increased by increasing pH and by increasing ethanol content. The solubility of A-71623 in ethanol/propellant mixtures was investigated; solubility ranged from 1.0 to 2.5 mg/mL in mixtures of ethanol, propellant 11 (trichlorofluoromethane), and propellant 12 (dichlorodifluoromethane). The log apparent octanol/water partition coefficient was 2.8 at pH 5 and 1.0 at pH 8. Maximum stability at 70 degrees C was seen in the range of pH values of 5.5-7.5; hydrolysis of the N-terminal BOC group appears to be the primary route of degradation. Increasing ethanol content increases the stability; Arrhenius analysis indicated a t90 of 150 days under ambient conditions in 25% ethanol. Intratracheal delivery of 3 mumol/kg A-71623 in 50% ethanol to rats showed rapid and efficient absorption of drug from the lungs, with a Cmax of 2.7 microM and an AUC of 85 microM*min. Similar studies in dogs showed bioavailabilities of 59% and 46% for 2 and 3 mumol/kg intratracheal doses, respectively, relative to intravenous administration. Sublingual administration of 1 mumol/kg A-71623 in a vehicle of 80% ethanol/2% Klucel/2.5% peppermint oil gave high prolonged plasma levels of A-71623, with a Cmax of 0.37 microM, indicating high bioavailability and favorable partitioning and distribution effects from the sublingual cavity for this formulation. Transdermal delivery was examined by in vitro diffusion through human skin; the permeability coefficient of A-71623 in 40% ethanol was 2.6 x 10(-5) cm/hr, suggesting that transdermal delivery of up to 2 mg/day may be feasible. In conclusion, the results provide preliminary indications that delivery of efficacious doses of A-71623, and perhaps other CCK analogs, by alternate routes of delivery is probably feasible.

Physicochemical properties of A-75998, an antagonist of luteinizing hormone releasing hormone.

The physicochemical properties of A-75998, a synthetic antagonist of luteinizing hormone releasing hormone with potential for treatment of hormone-sensitive cancers and endometriosis, are described. An accelerated solution stability study indicated that the compound is relatively stable and showed a U-shaped pH-rate profile, with maximum stability between pH 4.5 and 6.5. The acid dissociation behavior of A-75998 was examined by UV-visible spectrophotometry at 25 degrees C in a series of buffers ranging from pH 1 to 13. The data were fit to a model in which the dissociations of all four ionizable groups contributed to changes in the absorbance. The estimated macroscopic acid dissociation constants were p beta 1 = 3.230 +/- 0.022, p beta 2 = 4.885 +/- 0.030, p beta 3 = 9.871 +/- 0.022, and p beta 4 = 11.026 +/- 0.157. The corresponding microscopic dissociation constants were pk1 = 3.24 (nicotinyl), pk2 = 4.88 (pyridyl), pk5 = 9.91 (tyrosyl), and pk6 = 10.99 (isopropyllysyl). The apparent n-octanol/water partition coefficients were measured from pH 2 to 13, and the profile was consistent with the expected acid-dissociation behavior. While appearing fairly water-soluble at pH < 5, dynamic light scattering of A-75998 in pH 4.5 buffer indicated the formation of aggregates of nonuniform size distribution. A-75998 exhibited reverse or thermal gelation; sodium chloride exacerbates this gel formation and self-association. Surface activity was pH-dependent, but no evidence was found for micelle formation. Based on the results, development of a parenteral formulation of A-75998 appears feasible, provided that aggregation can be minimized.

The effect of aggregation inhibitors and antioxidants on the stability of hemin solutions.

Despite their importance as infusions for the treatment of porphyrias, aqueous solutions of hemin can be quite unstable, with a reported half-life of a few hours. We have examined factors which affect the stability of hemin solutions in order to identify possible excipients and conditions which would increase the stability. In agreement with previous reports, we have found that human serum albumin leads to stabilization of hemin solutions; polyvinylpyrrolidone is also an effective stabilizer of hemin. Imidazole, caffeine, and niacinamide were also found to stabilize hemin, apparently by complexing to hemin and preventing the formation of hematin dimers. Addition of certain antioxidants, e.g., butylated hydroxyanisole and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), led to stabilization of hemin, suggesting that radicals are involved in the degradation process. A comparison was also made by HPLC analysis of the hemin autooxidation products with those from the reaction of hemin with hydrogen peroxide; the results indicate that the products are similar but not identical. The implications of the results for clinical use of hemin solutions are discussed.

Targeted delivery of a heme oxygenase inhibitor with a lyophilized liposomal tin mesoporphyrin formulation.

Tin mesoporphyrin (SnMP) is a competitive inhibitor of heme oxygenase being examined clinically for the treatment of hyperbilirubinemia. Since liposomes have been shown to target SnMP to the spleen and increase its efficacy (S. A. Landaw, G. S. Drummond, and A. Kappas, Pediatrics 84, 1091-1096, 1989), we began investigating the feasibility of the preparation and scaleup of a liposomal SnMP formulation for clinical use. SnMP liposomes were prepared by high-pressure homogenization of a suspension of SnMP and egg phosphatidylcholine (1:20, w/w) in lactose-phosphate buffer, resulting in SnMP liposomes that were less than 200 nm in diameter and had encapsulation efficiencies of up to 90% at pH 5. The SnMP liposomes could be sterile filtered and lyophilized in a 1-day cycle with retention of the encapsulation efficiency and particle size. Following injection into rats, the distribution of liposomal SnMP to spleen at 2 and 6 hr after dosing was 5-20 times higher than for aqueous SnMP. Lyophilized SnMP liposomes were also more effective than aqueous SnMP in decreasing bilirubin production in bile-cannulated rats. The results suggest the potential for producing a safe, sterile, and effective lyophilized formulation of SnMP liposomes for targeting of heme oxygenase inhibitors to the spleen.

Pharmaceutics and drug delivery aspects of heme and porphyrin therapy.

The importance of porphyrins and metalloporphyrins as therapeutic drugs has increased significantly over the last decade. This review highlights some of the challenges faced by pharmaceutical scientists in formulating these drugs into stable, effective, and safe dosage forms. Most activity in the clinic has focused on three areas: photodynamic therapy of cancer (e.g., hematoporphyrin derivatives), porphyrias and hematological diseases (e.g., heme), and various forms of jaundice (e.g., tin porphyrins). The biodistribution, stability, aggregation, toxicology, and analytical methodology of porphyrin drugs are all important considerations in the pharmaceutical development of porphyrin drugs. The utility of delivery systems such as liposomes hold promise of increasing the therapeutic potential of these drugs. Future prospects for therapeutic applications of porphyrin drugs are also discussed.

The effect of metalloporphyrins and heme liposomes on delta-aminolevulinate synthase activity in rat liver.

Heme administration causes inhibition of delta-aminolevulinate synthase (ALAS), best tested in the allylisopropylacetamide (AIA)-treated rat, a model for hepatic porphyrias. Because heme suspended in aqueous media (for injection) is unstable and has adverse effects on coagulation, alternate therapeutic modalities are being explored. The present study tries to answer two questions: 1) are any heme analogs as effective inhibitors of ALAS as heme is; and 2) does heme administration in the form of liposomes increase its effectiveness? None of the liposome compositions tested, even if containing lactosylceramide for preferential hepatocyte uptake, was more effective in inhibiting AIA-induced ALAS activity than heme in buffer. As for the function of the heme analogs, although deuteroheme and heme dimethyl ester proved ineffective, mesoheme and cobalt protoporphyrin were nearly as effective as heme itself, indicating that both hydrophobic side chains in positions 2 and 4 and free propionate groups at 6 and 7 are essential for ALAS inhibition, as is the presence of a central cobalt or iron atom.

Kinetics of the interaction of hemin liposomes with heme binding proteins.

As a model for the transport of hemin across biological membranes, sonicated phosphatidylcholine liposomes with incorporated hemin were characterized. The interaction of the hemin liposomes with the heme binding proteins albumin, apomyoglobin, and hemopexin was examined as a function of liposome charge and cholesterol content. In all cases, there was an almost complete transfer of hemin from liposome to protein; a rapid phase and a slow phase were observed for the transfer. For negatively charged liposomes (with 11% dicetyl phosphate), the rapid and slow phases showed observed rates of transfer of ca. 2 and 0.01 s-1, respectively, for all three proteins. The presence of cholesterol in the liposomes decreased the observed rates by a factor of 2, and positively charged liposomes (with 11% stearylamine) showed about one-fifth the observed rates of negatively charged liposomes. The observed rates were independent of protein concentration, indicating that the rate-determining step is hemin efflux from the lipid bilayer. The hemin interaction with the phospholipid bilayer is suggested to be primarily hydrophobic with some electrostatic character. The two phases are suggested to arise from two different populations of hemin within the liposomes and are interpreted as arising from two different orientations of hemin within the bilayer.

Determination of the equilibrium constant for the binding of ferricytochrome c to phospholipid vesicles and the effect of binding on the reduction rate of cytochrome c.

The rate of reduction of cytochrome c by 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine was examined as a function of binding to liposomes prepared from mixed soybean phospholipids, asolectin, and from various purified phospholipids. Binding of cytochrome c to asolectin liposomes caused an increase in the rate of reduction by the pteridine derivative from 2900 to 16 000 M-1 x s-1 at pH 7. At low ionic strength (0.003 M) the binding stoichiometry between cytochrome c and asolectin vesicles is 15 +/- 2 phosphospolipid/cytochrome c (mole ratio), determined by monitoring the change in reduction rate of cytochrome c by pteridine as cytochrome c is bound to the vesicles. A stoichiometry of 14 phospholipid/cytochrome c was obtained from gel filtration studies. Equilibrium association constants for the binding of cytochrome c to sites on the asolectin vesicles varies from 2.2 x 10(6) to 1.8 x 10(3) M-1 between 0.02 and 0.10 M ionic strength, respectively. In general, liposomes prepared from purified phospholipids resulted in less binding of cytochrome c per mole of phospholipid and lower reduction rates than those prepared from asolectin.