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Notes: Abstract  The pharmacokinetics of 1, 19-bis(ethylamino)-5, 10, 15-triazanonadecane (BE-4-4-4-4) were determined in CD2F1 female mice after administration of i.v. bolus doses of 20 mg/kg (approximately the dose lethal to 10% of the study animals, ∼LD10) as well as 15, 10, and 5 mg/kg and after s.c., i.p., or p.o. doses of 20 mg/kg. BE-4-4-4-4 in plasma and urine was derivatized with dansyl chloride and measured by gradient high-performance liquid chromatography (HPLC) with fluorescence detection. Data were modeled by noncompartmental and compartmental methods. The declines observed in plasma BE-4-4-4-4 concentrations after i.v. delivery of 20, 15, 10, and 5 mg/kg were modeled simultaneously using an interval of 2000 min between doses and were best approximated by a two-compartment, open, linear model. The time courses of plasma BE-4-4-4-4 concentrations after i.p. and s.c. delivery were fit best by a two-compartment, open, linear model with first-order absorption. Peak plasma concentrations of BE-4-4-4-4 measured following an i.v. dose of 20 mg/kg ranged between 30 and 33 μg/ml, the terminal elimination half-life was 94 min, and the volume of distribution (Vdss) was 850 ml/kg. The plasma pharmacokinetics of BE-4-4-4-4 were linear with dose. BE-4-4-4-4 (0.5 and 2.0 μM) in mouse plasma was approximately 67% protein-bound. Bioavailabilities after i.p., s.c., and p.o. delivery were 40%, 50%, and approximately 3%, respectively. Urinary excretion of parent BE-4-4-4-4 in the first 24 h after dosing accounted for less than 30% of the delivered dose. As BE-4-4-4-4 proceeds toward and undergoes clinical evaluation, the data and analytical method presented herein should prove useful in formulating a dose-escalation strategy and, possibly, evaluating toxicities encountered.

Notes: Abstract Purpose: Pc4 is a silicone phthalocyanine photosensitizing agent that is entering clinical trials. Studies were undertaken in mice to develop a suitable formulation and analytical methodology for use in pharmacokinetic studies and to define the plasma pharmacokinetics, tissue distribution, and urinary excretion of Pc4 after i.v. delivery. Methods: An HPLC method suitable for separation and quantification of Pc4 was developed and validated for use in mouse plasma, tissues, and urine. The stability of Pc4 was characterized in a variety of formulations as well as in mouse plasma. Before pursuing pharmacokinetic studies, preliminary toxicity studies were undertaken. These studies utilized Pc4 formulated in diluent 12:0.154 M NaCl (1:3, v:v). Pharmacokinetic studies involved Pc4 doses of 40 mg/kg, 10 mg/kg and 2 mg/kg administered as i.v. boluses to female, CD2F1 mice . Doses of 40 mg/kg, 10 mg/kg, and 2 mg/kg were studied with drug formulated in diluent 12:0.154 M NaCl (1:3, v:v). Doses of 10 mg/kg and 2 mg/kg were also studied with drug formulated in a vehicle consisting of polyethylene glycol:Tween 80:0.01 M sodium phosphate buffer, pH 7.0 (40:0.2:59.8, v:v:v). Compartmental and non-compartmental analyses were applied to the plasma concentration-versus-time data. Concentrations of Pc4 were also determined in a variety of tissues, including brain, lung, liver, kidney, skeletal muscle, skin, heart, spleen, and abdominal fat. Urine was collected from animals treated with each of the doses of Pc4 mentioned above, and daily, as well as cumulative drug excretion was calculated until 168 h after treatment. Results: At a dose of 80 mg/kg, two of five male and two of five female mice were dead by 24 h after injection. Pathologic examination revealed gross findings of blue discoloration affecting many tissues, with lungs that were grossly hemorrhagic and very blue-black. Microscopic examination of the lungs revealed mild acute interstitial pneumonia, with perivascular edema and inflammation, and a detectable margination of neutrophils around larger pulmonary blood vessels. Animals sacrificed 14 days after treatment showed mild granulomatous pneumonia, characterized by clusters of multi-nucleated giant cells, with fewer macrophages and neutrophils. The giant cells frequently contained phagocytized particles, which were clear and relatively fusiform. All mice treated with 40 mg/kg or 20 mg/kg survived and returned to pretreatment weight during the 14 days after treatment. Intravenous bolus delivery of Pc4, at a dose of 40 mg/kg, produced “peak” plasma Pc4 concentrations between 7.81 and 8.92 μg/ml in mice killed at 5 min after injection (the earliest time studied after drug delivery). Sequential reduction of the Pc4 dose to 10 mg/kg in diluent 12:0.154 M NaCl (1:3, v:v), 10 mg/kg in polyethylene glycol:Tween 80:sodium phosphate buffer (40:0.2:59.8, v:v:v), 2 mg/kg in diluent 12:0.154 M NaCl (1:3, v:v), and, finally, 2 mg/kg in polyethylene glycol:Tween 80:sodium phosphate buffer (40:0.2:59.8, v:v:v) resulted in “peak” plasma Pc4 concentrations between 2.07 and 3.24, 0.68 and 0.98 μg/ml, and 0.29 and 0.41 μg/ml, respectively. Pc4 persisted in plasma for prolonged periods of time (72–168 h). Non-compartmental analysis of plasma Pc4 concentration-versus-time data showed an increase in area under the plasma Pc4 concentration-versus-time curve (AUC) when the dose of Pc4 increased from 2 mg/kg to 40 mg/kg. Across the 20-fold range of doses studied, total body clearance (CLtb) varied from 376 to 1106 ml h−1 kg−1. Compartmental modeling of plasma Pc4 concentration versus time data showed the data to be fit best by a two-compartment, open, linear model. Minimal amounts of Pc4 were detected in the urine of mice. After i.v. bolus delivery to mice, Pc4 distributed rapidly to all tissues and persisted in most tissues for the duration of each pharmacokinetic study. Tissue exposure, as measured by AUC, increased in a dose-dependent fashion. Conclusions: The HPLC method developed for quantification of Pc4 in plasma, urine, and tissues should be suitable for clinical studies of the drug. Pc4 is widely distributed and persists in plasma and tissues of mice for prolonged periods of time. These data are relevant to the design of forthcoming clinical trials of Pc4.

Notes: Abstract  The purpose of the present study was to define the plasma pharmacokinetics, bioavailability, and tissue distribution in mice of halomon, a halogenated monoterpene from Portieria hornemanii that is active in vitro against brain-, renal-, and colon-cancer cell lines. Halomon formulated in cremophor : ethanol : 0.154 M NaCl (1 : 1 : 6, by vol.) was injected i.v. at 20, 60, 90, or 135 mg/kg into female CD2F1 mice. Doses of 135 mg/kg were also given i.p., s.c., and by enteral gavage to female CD2F1 mice and i.v. to male CD2F1 mice. Plasma halomon concentrations were measured with a gas-chromatography system using electron-capture detection. Halomon concentrations were also determined in the brains, hearts, lungs, livers, kidneys, spleens, skeletal muscles, fat, red blood cells, and, if present, testes of mice given 135 mg/kg i.v. Halomon plasma pharmacokinetics were well fit by a two-compartment, open linear model and were linear between 20 and 135 mg/kg. Population estimates of parameters describing halomon plasma pharmacokinetics in female CD2F1 mice were developed with a standard two-stage technique and also by simultaneous modeling of data from 20-, 60-, 90-, and 135-mg/kg i.v. studies in female mice. Halomon bioavailability was 45%, 47%, and 4% after i.p., s.c., and enteral dosing, respectively. Urinary excretion of the parent compound was minimal. Halomon was distributed widely to all tissues studied but was concentrated and persisted in fat. Halomon concentrations measured in the brain were comparable with concomitant concentrations detected in plasma and most other tissues. These data and models are helpful in the simulation and evaluation of conditions produced by preclinical screening and toxicology studies.

Notes: Δ1-Pyrroline is a putrescine metabolite that is biotransformed by rabbit liver preparations to 4-aminobutanoic acid and its lactam, 2-pyrrolidinone. Analysis of dilute aqueous solutions of Δ1-pyrroline by proton nuclear magnetic resonance indicated that the predominating species in the liver incubation preparations was Δ1-pyrroline monomer, although other species, such as 4-aminobutyraldehyde and Δ1-pyrroline trimer, may exist in equilibrium with the monomer. [2H12]-Δ1-Pyrroline trimer was synthesized from [2H5]pyrrolidine by conversion to the N-chloro derivative followed by dehydrohalogenation. 4-Aminobutanoic acid was measured by a gas chromatographic mass spectrometric assay after derivatization with dimethylformamide dimethyl acetal. The 4-aminobutanoic acid homologue, 5-aminovaleric acid, served as internal standard. 2-Pyrrolidinone was hydrolyzed and measured as 4-aminobutanoic acid. A comparison of the amounts of product formed following incubation of labeled and unlabeled Δ1-pyrroline indicated a significant isotope effect in the formation of 2-pyrrolidinone. The influence of the label was much less on 4-aminobutanoic acid production. The results suggest that there are two separate pathways involved in the reaction.

Notes: A limiting factor in the application of mass spectrometry to biochemically important molecules has been the inability to analyze compounds without inducing thermal decomposition. Glutamine, which readily cyclizes to 2-pyrrolidone-5-carboxylic acid with loss of ammonia, is one of the more difficult of these non-volatile, thermolabile biomolecules to determine. Using a heated, direct probe, no molecular ion for glutamine was observed with electron impact ionization. A protonated molecular ion was detected with direct probe chemical ionization and thermospray. In both cases, the major ion formed was derived from the thermolysis product, 2-pyrrolidone-5-carboxylic acid. Analysis by fast atom bombardment and 252Cf plasma desorption mass spectrometry yielded a molecular ion as base peak with a small ion from the 2-pyrrolidone-5-carboxylic acid. Ion evaporation produced only the molecular cations, and collisionally activated dissociation demonstrated that glutamine does not cyclize appreciably once protonated. These results suggest that glutamine can be used as a reference compound when analysis requires the optimization of conditions to produce a molecular ion. Furthermore, the relative intensity of the pyrolytic products, 2-pyrrolidone-5-carboxylic acid derived molecules, divided by the relative intensity of the molecular ions of glutamine provides a numerical evaluation of ionization conditions.

Notes: Determination of the molecular weight for an unknown by thermospray liquid chromatographic/mass spectrometric analysis using ammonium acetate mobile phase can be both confusing and misleading. This is due to the uncertainty of whether an ion is either a protonated species or an ammonium adduct or both. Deuterium oxide and ammonium acetate buffers have been used to elucidate molecular species of unknown compounds with some success. We have investigated an alternative method which utilizes diamine reagents. This method simplifies the identification of unknown molecular species in an inexpensive manner. Diamines form adduct ions readily with many substances. A comparison of spectra obtained for an unknown using ammonium acetate or diaminoethane in separate analyses is useful for identifying molecular species. Using ammonium acetate as a mobile phase, a molecule is often detected as a protonated molecular species. However, using diaminoethane mobile phase, this same molecule is most often detected as a diaminoethane adduct. Comparison of each spectrum reveals a mass difference of 60 for a molecule detected as a protonated species and a diaminoethane adduct. In addition, a mass difference of 43 will be obtained in a similar spectral comparison if a molecule forms ammonium adducts using ammonium acetate mobile phase and diaminoethane adducts using diaminoethane. An analysis of an unknown substance with several molecular species is presented as an example of the utility of diamine reagents in thermospray mass spectrometry. By observing mass spectral differences, molecular species can be identified as either a protonated ion or an adduct ion.

Notes: A packed-column supercritical-fluid chromatograph was interfaced with a mass spectrometer via a modification of a thermospray probe. This modification allowed a capillary restrictor for the supercritical fluid (CO2) and reagent gas for chemical ionization to be introduced directly into a thermospray source. Chemical ionization conditions were observed when either the filament or discharge electrode was used and the source pressure was above 0.5 torr. The discharge electrode produced more efficient ionization, resulting in approximately a tenfold larger signal than that observed in the filament mode. The usefulness of this instrumentation was demonstrated on several anticancer drugs. Methanol positive ion chemical ionization (PICI) spectra were recorded for cyclophosphamide, diaziquone, mitomycin C and thiotepa. Methane PICI spectra of thiotepa were obtained in the absence of methanol as a mobile-phase modifier. A 50 ng on-column injection of diaziquone produced approximately a 6:1 signal to noise ratio in the scanning mode.

Notes: Labeled γ-aminobutyric acid was detected in mouse brain following intravenous injections of deuterium labeled 2-pyrrolidinone. [2H6]Pyrrolidinone was prepared by the reduction of [2H4]succinimide with lithium aluminum deuteride. Quantification was accomplished by a gas chromatography mass spectrometry assay method. γ-Aminobutyric acid and internal standard, 5-aminovaleric acid, were converted to volatile derivatives by treatment with N,N-dimethylformamide dimethyl acetal. Quantitative estimates were derived from peak area measurements obtained from monitoring the parent ions of the γ-aminobutyric acid and internal standard derivatives by repetitive scanning during the GC run. The conversion of pyrrolidinone to γ-aminobutyric acid may provide a method for labeling central γ-aminobutyric acid pools.

Notes: The methane negative-ion chemical ionization (NCI) mass spectrum of chlorprothixene shows an unusual MH- ion. This ion can be accounted for by electron capture followed by H· transfer from the reagent gas. The most probable site of electron attachment was concluded to be related to the sulfur atom of the thioxanthene ring based on the observation of analogous ions for structurally related compounds, all containing a heterocyclic sulfur. The MH- ion observed with methane as the reagent gas was shifted to MD- when tetradeuteromethane was used in place of methane. The ratio of [M — H]- to MH- did not change with emission current suggesting that the process is independent of the radical concentration in the CI plasma. Consistent with this observation is the lack of CH3· or C2H5· adduct ions in the NCI mass spectrum and the fact that gold-plating the ion source did not decrease the proportion of MH-. Also, this mechanism is consistent with thermochemical considerations of reactions of a phenyl radical with various alkanes and observations of ions formed by methane NCI from model compounds. Therefore, unlike other MH- ions observed in methane NCI mass spectra, the mechanism of formation does not appear to involve a hydrogen radical addition followed by electron capture.