James Blanchard

Evaluation of the relative efficacy of various techniques for deproteinizing plasma samples prior to high-performance liquid chromatographic analysis

The methods used for treating biological samples prior to their introduction into a high-performance liquid chromatographic (HPLC) system generally fall into one of two categories extraction or direct injection. In the extraction method the compound of interest is removed from the biological matrix (plasma, serum, urine, etc.) using suitable solvent and pH conditions, which selectively extract the desired components and leave behind unwanted materials. The solvent is then removed by gentle evaporation and the dried residue reconstituted in a small volume of the elution solvent (or one quite similar to it) for injection on to the HPLC column. The direct-injection technique is by far the simplest and most rapid of the two methods_ In this procedure the biological sample may be injected directly on to the top of the HPLC column cl] _ However, a number of reports have indicated that this results in a rapid increase in back-pressure -and a deterioration of column performance [Z-5], presumably due to the precipitation of plasma proteins as a result of their contact with the organic solvents and buffer salts commonly utilized in mobile phases [4]_ To alleviate this problem, a number of sample preparation techniques have been described for removing proteins prior to. injection of the sample. These include the use of precolumns [S] , ultrafiltration devices [7, 81, and various protein precipitants such as organic solvents [9-111 and ionic salts [X2-14] _ Only one report dealing -specifically with sample preparation procedures for the direct-injection HPLC technique has appeared to date [15] _ In that study, -six. different methods of deproteinizing plasma were evaluated using the -biuret assay to assess their efficiency. In the present report we describe a number of other potentially useful methods of protein removal,. using the much moresensitive Lowry [ 161 method of protein determination to evaluate :

Cyclodextrins in the treatment of a mouse model of Niemann-Pick C disease

Niemann-Pick type C (NPC) is a neurodegenerative disorder characterized by greatly altered somatic cholesterol metabolism. The NPC1 gene has recently been cloned and shown to have sequence homology to other sterol-sensing proteins. We have used a mouse model with a disrupted npc1 gene to study the effects of the cholesterol-mobilizing compound, 2-hydroxypropyl-beta-cyclodextrins (HPBCD), on the clinical course of this disorder. Treatment with two HPBCDs, with varying levels of 2-hydroxypropyl substitution, had effects in delaying neurological symptoms and in decreasing liver cholesterol storage while a third HPBCD was without effect. The ameliorating effect was not improved by longer exposure times (commencement of exposure in utero), however, it is not known if there is transplacental transfer of HPBCDs. The combination of HPBCD with probucol or nifedipine (which have previously been shown to lower liver cholesterol in this animal model) markedly decreased liver storage of unesterified cholesterol without altering the depressed levels of esterified cholesterol. The slight effects of the HPBCDs on neurological symptoms may be partially due to their apparent non-permeation of the blood-brain barrier (BBB). This non-permeation was assayed with radioactive tracers and was also present in the mdr1a knockout mice which have greatly increased BBB permeability for many drugs. Intrathecal delivery of HPBCD by an Alzet osmotic minipump did not improve its efficacy in ameliorating neurological symptoms.

Comparative pharmacokinetics of caffeine in young and elderly men

The phamacokinetic behavior of caffeine was compared in a group of eight healthy young men aged 20.5±2.0 years (mean ± SD), and in a group of eight healthy, elderly men aged 71.2±3.9 years. Each subject was given a 5 mg/kg dose of caffeine as either an aqueous oral solution or an intravenous infusion over 30 min using a randomized crossover design. Plasma and urine samples were collected for 24 hr following each dose and analyzed for caffeine content using high-performance liquid chromatography.The peak times (tmax), peak concentrations (Cmax), and the percentage of the peroral dose systemically available, F(%), were essentially identical in both age groups, indicating that caffeine was absorbed rapidly and completely after peroral administration. These results also indicated that the first-pass metabolism observed in rats following the peroral administration of caffeine does not occur in either human group studied here. The elimination of caffeine during its terminal disposition phase was log-linear. Several between-group comparisons of other pharmacokinetic parameters were made. Although the average elimination rate constant was greater in the elderly, the difference did not reach statistical significance, possibly because of the considerable intersubject variability in the elimination rate of caffeine, with halflives ranging from 2.27 to 9.87 hr. The average apparent volume of distribution was significantly lower in the elderly subjects while the clearances were slightly, but not significantly, larger in the elderly subjects. It appears that most aspects of the pharmacokinetic behavior of caffeine are very similar in young and elderly men.

Pluronic F127-based ocular delivery system containing biodegradable polyisobutylcyanoacrylate nanocapsules of pilocarpine.

The objectives of our study were to prepare a biodegradable polyisobutylcyanoacrylate (PIBCA) colloidal particulate system of pilocarpine, to incorporate it into a Pluronic® F127(PF127)based gel delivery system, and to evaluate its ability to prolong the release of pilocarpine. Polyisobutylcyanoacrylate nanocapsules (PIBCA-NC) of pilocarpine were prepared by interfacial polymerization. Physicochemical characterization of the colloidal dispersion of PIBCA-NC of pilocarpine was performed by measuring drug loading, particle size analysis, and scanning electron microscopy. Results indicated that ~13.5% of pilocarpine was loaded onto the PIBCA-NC, the nanocapsules ranged from 370 to 460 nm, the distribution was narrow, and there was no significant effect of stirring speed on particle size. The PIBCA-NC dispersion of 1% pilocarpine alone (I) and after incorporation into the Pluronic® F127 gel delivery system (II) were compared against 1% pilocarpine incorporated into a PF127 gel containing 5% methylcellulose (PF127MC) alone (III) by measuring the miotic response in the albino rabbit eye. Statistical analysis indicated a rank-order for both the duration and intensity of miosis of II > III >> I, with all differences being significant (p < 0.05). Thus, it appears that II increases the contact time of pilocarpine with the absorbing tissue in the eye, thereby improving ocular bioavailability. The PIBCA-NC of pilocarpine dispersed in the PF127MC gel delivery system has considerable potential for achieving a prolonged de livery for such drugs as pilocarpine and other more hydrophobic drugs.The objectives of our study were to prepare a biodegradable polyisobutylcyanoacrylate (PIBCA) colloidal particulate system of pilocarpine, to incorporate it into a Pluronic F127(PF127)-based gel delivery system, and to evaluate its ability to prolong the release of pilocarpine. Polyisobutylcyanoacrylate nanocapsules (PIBCA-NC) of pilocarpine were prepared by interfacial polymerization. Physicochemical characterization of the colloidal dispersion of PIBCA-NC of pilocarpine was performed by measuring drug loading, particle size analysis, and scanning electron microscopy. Results indicated that approximately 13.5% of pilocarpine was loaded onto the PIBCA-NC, the nanocapsules ranged from 370 to 460 nm, the distribution was narrow, and there was no significant effect of stirring speed on particle size. The PIBCA-NC dispersion of 1% pilocarpine alone (I) and after incorporation into the Pluronic F127 gel delivery system (II) were compared against 1% pilocarpine incorporated into a PF127 gel containing 5% methylcellulose (PF127MC) alone (III) by measuring the miotic response in the albino rabbit eye. Statistical analysis indicated a rank-order for both the duration and intensity of miosis of II > III >> I, with all differences being significant (p < 0.05). Thus, it appears that II increases the contact time of pilocarpine with the absorbing tissue in the eye, thereby improving ocular bioavailability. The PIBCA-NC of pilocarpine dispersed in the PF127MC gel delivery system has considerable potential for achieving a prolonged delivery for such drugs as pilocarpine and other more hydrophobic drugs.

Effect of temperature and light on the stability of latanoprost and its clinical relevance.

PurposeTo examine the effect of controlled heat and ultraviolet exposures on the stability of latanoprost (Xalatan, Pharmacia & Upjohn, Kalamazoo, MI) using high-performance liquid chromatography to derive practical recommendations for patients regarding its use and storage. MethodsUsing serial dilution of a latanoprost stock solution, varying concentrations were prepared to obtain a standard curve. The accuracy and precision of the high-performance liquid chromatography assay conditions were validated using between-day and within-day studies. The original latanoprost containers were stored at 4, 25, 50, and 70°C, and the concentration of latanoprost remaining was measured by high-performance liquid chromatography at different times for up to 1 month. In addition, the original latanoprost containers were exposed to known amounts of ultraviolet A and B radiation for 4 hours, and the concentration of latanoprost was measured at 1-hour intervals using high-performance liquid chromatography. ResultsThe increased temperature studies showed that latanoprost remained stable at 4 and 25°C for the 30-day study duration. Analysis of concentration versus time curves for 50 and 70°C yielded a t90 (time for 10% degradation) of 8.25 and 1.32 days, respectively. Ultraviolet B radiation caused a rapid degradation of latanoprost, whereas ultraviolet A radiation was less effective in causing the degradation of latanoprost. ConclusionsLatanoprost exhibits thermal and solar instability and should ideally be stored below room temperature and in the dark. The importance of these storage conditions should be conveyed clearly to the patient.

In vitro characterization and in vivo release profile of a poly (D, L-lactide-co-glycolide)-based implant delivery system for the α-MSH analog, melanotan-I

Abstract Melanotan-I (MT-I) is a superpotent tridecapeptide capable of stimulating melanotropic activity. In order to overcome its short half-life in the systemic circulation, the biodegradable poly( d , l -lactide-co-glycolide) (PLGA) copolymer was used to prepare an implant delivery system for MT-I. The implant was prepared by the hot melt-extrusion method. The surface morphology of the PLGA implant was assessed using scanning electron microscopy. The time-dependent changes in the molecular weight distribution of the copolymer and its erosion were monitored in order to help characterize the hydrolytic degradation processes occurring in vivo. The time required to reduce the weight-average molecular weight of PLGA to 50% of its initial value, as determined by size exclusion chromatography, was about 12 days compared to 5 weeks for 50% erosion of the copolymer mass to occur. The release of lactic acid from PLGA was also quantified simultaneously in order to characterize the degradation, and the onset of increased lactic acid release was found to coincide with the onset of the tertiary phase of the MT-I release profile in vivo in guinea pigs. The MT-I released from the depot implanted subcutaneously in guinea pigs exhibited a release profile extending over one month, in agreement with data from the in vitro studies.

In vitro evaluation of Poly(d,l-lactide-co-glycolide) polymer-based implants containing the α-melanocyte stimulating hormone analog, Melanotan-I

Abstract The release of the melanotropic peptide, Melanotan-I (MT-I), from biodegradable implants of poly( d , l lactide-co-glycolide) (PLGA) copolymer was studied. The implants were prepared by a melt-extrusion method. The in vitro release of MT-I exhibited a triphasic profile with an initial rapid release followed by a secondary phase of slow release, then a tertiary phase of rapid release due to erosion of the polymer. The initial rapid release observed with PLGA (50:50 molar ratio of lactic/glycolic acid) polymers was less than 5% of the drug load and the tertiary phase commenced after about 3 weeks. The factors controlling the drug release are degradation and erosion of the polymer which may, in turn, be controlled by the physical properties of the polymer such as molecular weight and viscosity. The influence of viscosity (0.2–1.08 dl/g) of the polymer, on the release kinetics of MT-I were analyzed and the polymer having a viscosity of 0.6 dl/g was selected for preparing a 1-month implant system. Molecular weight distribution analysis indicated a biphasic rate of molecular weight reduction and within 12 days, the molecular weight had decreased to 50% of the initial value. The release rate was examined at different drug loading levels and in the presence of some hydrophilic additives. The effect of gamma radiation on the release kinetics of the peptide was analyzed to determine the optimal radiation sterilization dose for the PLGA implants. There was no significant difference in the total duration of MT-I release between the implants exposed to no radiation and the 2.5 Mrad dose selected.

Skin pigmentation and pharmacokinetics of melanotan-I in humans

A comparative pharmacokinetic trial was performed with a superpotent synthetic melanotropic peptide, [Nle4‐D‐Phe7]‐α‐MSH1–13 (melanotan‐I or MT‐I) given by three routes of administration. Plasma levels were measured by RIA and tanning was quantitated using serial reflectometry. Doses of 0·16 mg kg−1 were administered intravenously (IV) and orally (PO), and doses from 0·08 to 0·21 mg kg−1 subcutaneously (SC), in a randomized crossover fashion to three male volunteers over five consecutive days for 2 weeks (ten doses). The results indicate that the SC dose is completely bioavailable compared to the IV dose. No detectable drug levels were observed following PO dosing. The plasma half‐lives following SC dosing ranged from 0·07 to 0·79 h for the absorption phase and from 0·8 to 1·7 h for the β‐phase. Clearance ranged from 0·12 to 0·19 L kg−1 h−1 and 3·9% or less of the dose was recovered in the urine. Side‐effects were minimal, consisting of occasional gastrointestinal upset and facial flushing. Significant tanning of the forehead, arms, and neck was noted following IV or SC dosing. This effect peaked at 1 week following drug administration but was still present 3 weeks after completing the ten‐dose regimen. It is concluded that SC administration is an efficacious method of delivering melanotan‐I.

Calcium absorption in man: Some dosing recommendations

The absorption of calcium involves a saturable (active) and a nonsaturable (passive) component. The work of several investigators indicates that an inverse relationship exists between calcium intake and absorption efficiency. Human calcium absorption data from the literature were analyzed using a model which included both an active and a passive absorption component. Simulations were provided to illustrate the suitability of this model, and another previously reported model, to fit the data and to estimate the absorption efficiency of calcium when using different dosing regimens. Comparisons of the values predicted in this study with some literature values are provided and some assumptions and potential limitations associated with the use of this method are discussed. The division of the daily dose into equal increments taken at equally spaced intervals over the course of the day is recommended as a useful procedure for increasing the absorption efficiency and efficacy of calcium.

Phase 1 Study of Topical Perillyl Alcohol Cream for Chemoprevention of Skin Cancer

Perillyl alcohol (POH) is a natural product derived from plants such as cherry and lavendin. Previous studies have indicated that topical POH inhibits ultraviolet (UV) B-induced skin carcinogenesis in vivo, and it may be an effective chemopreventive agent for skin cancer. We performed a 1-mo, first-in-man, Phase 1 trial of topically administered POH cream in human subjects. Endpoints included safety and evaluation of any histopathological changes in skin after 1 mo use of POH cream. We randomized 25 subjects with normal, healthy skin with little or no sun damage and no history of skin cancer in a double-blind fashion to receive topical POH (0.76% wt/wt) on 1 forearm with placebo cream applied to the other forearm twice daily for 30 days. Subjects were monitored for toxicity, and a 4 mm punch biopsy in the treated area was performed at the end of study for histopathological evaluation. The topical cream was well tolerated. No serious cutaneous toxicities, systemic toxicities, or histopathological abnormalities were observed. A total of 8 subjects (32%) reported mild adverse events possibly or probably related to use of cream including reversible appearance of 1 to 2 small papules. However, there was no significant difference between lesions appearing on the POH treated forearm vs. the placebo-treated forearm.