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In this work, an example of analytical method quality by design (AQbD) principles applied to an in-line NIR method to monitor blend potency and uniformity in the manufacturing of an oral solid dosage product is presented. An integrated process analytical technology (PAT) method was developed following the AQbD workflow, including risk assessment, design of experiments (DoE), method control strategy, and method maintenance. Several aspects particular to partial least square (PLS) chemometric method development and validation (such as the combined use of DoE, optimization, and multivariate data analysis) are addressed. Results from the risk assessment showed that the active pharmaceutical ingredient (API) particle size was the most significant risk factor due to the presence of aggregates. This risk was reduced by introducing changes in the API crystallization procedure and by including a wide range of calibration blends within the expected range of particle size. Other potential risks related to the instrumentation, the API and excipient properties, and the environment were further studied and minimized. System suitability tests based on Mahalanobis distance and discriminant analysis (DA) techniques were developed to ensure the quality of the spectral data before blend potency calculation. The use of AQbD provided a robust and rugged method that has the potential to be used as part of the product real-time release (RTR) control strategy.

The compound studied, an antagonist of corticotropin-releasing factor (CRF) receptor 1, was under development for the treatment of major depressive disorder and generalized anxiety disorder. To control the quality of intermediate compounds in the synthetic pathway, the preference is to develop a single HPLC method that could determine impurities of the sequential and structurally similar intermediates. However, the chemical nature of one of those intermediates, Compound C, posed a number of challenges to the development of a suitably rugged impurity method for this isolated intermediate. In the solid state, Compound C is a stable mixture of its trans-isomer and semicarbazone forms. In most solution states, Compound C interchanges between the trans and semicarbazone forms as well as degrades rapidly to impurity X, resulting in sample solution instability. Furthermore, the trans-isomers and semicarbazone exhibited different UV response factors, but reference standards of the two forms were not available to determine the relative response factor. Consequently, irreproducibility was observed during the HPLC analysis when conventional UV detection was applied directly. This article describes the following approaches that were employed to meet these challenges: (1) diluent screening was performed to select a \solvent that afforded adequate sample solution stability; (2) appropriate chromatographic conditions were selected to eliminate or minimize any on-column degradation; (3) alternate HPLC detection, Corona charged aerosol detection, in conjunction with UV analysis, was used to determine relative amounts of the isomeric forms which subsequently permitted the use of conventional UV detection for routine analysis. The validation data of this reversed-phase liquid chromatographic method are also discussed.

A feasibility study was conducted in an analytical laboratory to evaluate implementation of the alternative procedure pr oposed by the FDA for enhanced mechanical calibration of USP dissolution Apparatus 1 and 2. The investigation started with a historical review of qualification data for more than 50 systems, generated through our current practice of combining USP mechanical calibration with the USP Performance Verification Test (PVT) using prednisone tablets. The data were compared with the more comprehensive FDA procedures and more stringent criteria for enhanced mechanical calibration. All the historical results met both USP and FDA criteria for shaft wobble, basket wobble, basket/paddle height check, and rotational speed and, in addition, passed the USP PVT. Shaft verticality was the mechanical parameter that infrequently did not meet the enhanced calibration criterion. Further investigations were conducted on a few representative dissolution systems to examine shaft verticality and determine what factors affect it. System levelness greatly affected whether shaft verticality met the tolerance. It is concluded from the results of the evaluation that it may be difficult for certain older units to consistently meet the criteria for shaft verticality. Therefore, our plan is to continue the current practice of a combination of USP mechanical calibration with PVT and to monitor shaft verticality data.

This is a summary report of the “AAPS Workshop on the Role of Dissolution in QbD and Drug Product Life Cycle” or ganized by the AAPS In Vitro Release and Dissolution Testing (IVDRT) Focus Group. Representatives from the pharmaceutical industry, regulatory authorities, and academia in the U.S. and Europe attended this workshop to discuss the role of dissolution in a Quality by Design (QbD) setting and its relevance in drug product development. Other areas of discussion included IVIVC/R and hot topics like alcohol dose-dumping. Numerous case studies were presented, and issues relevant to the dissolution scientist and areas needing further research were highlighted at this workshop. Views expressed in this paper are those of the participants from the industry and the agency and do not necessarily represent those of the FDA and USP.

The purpose of this study was to identify two degradation products formed in the parenteral lyophilized formulation of BMS-204352, investigate the possible role of elastomeric closures in their formation, and develop a strategy to minimize/control their formation. The first degradant was identified as the hydroxymethyl derivative (formaldehyde adduct, BMS-215842) of the drug substance formed by the reaction of BMS-204352 with formaldehyde. Structure confirmation was based on liquid chromatography/mass spectroscopy (LC/MS), nuclear magnetic resonance (NMR), and chromatographic comparison to an authentic sample of the hydroxymethyl degradation product, BMS-215842. To confirm the hypothesis that formaldehyde originated from the rubber closure, migrated into the product, and reacted with BMS-204352 drug substance to form the hydroxymethyl degradant, lyophilized drug product was manufactured, the vials were stoppered with two different rubber closure formulations, and its stability was monitored. The formaldehyde adduct degradant was observed only in the drug product vials stoppered with one of the rubber closures that was evaluated. Although formaldehyde has not been detected historically as leachable and is not an added ingredient in the rubber formulation, information obtained from the stopper manufacturer indicated that the reinforcing agent used in the stopper formulation may be a potential source of formaldehyde. The second degradant was identified as the desfluoro hydroxy analog (BMS-188929) based on LC/MS, NMR, and chromatographic comparison to an authentic sample of the desfluoro hydroxy degradation product.

The purpose of this study was to identify a degradation product formed in the clinical parenteral formulation of BMS-204352, investigate the role of excipients in its formation, and develop a strategy to minimize/control its formation. The degradant was identified as the hydroxy methyl derivative (formaldehyde adduct, BMS-215842) of the drug substance based upon liquid chromatography/mass spectroscopy (LC/MS), liquid chromatography/mass spectroscopy/mass spectroscopy (LC/MS/MS), nuclear magnetic resonance (NMR), and chromatographic comparison to an authentic sample of hydroxymethyl degradation product, BMS-215842. An assay method for the detection of formaldehyde based on HPLC quantitation of formaldehyde dinitrophenylhydrazone was developed to quantitate its levels in various Polysorbate 80 and PEG 300 excipient lots. A direct relationship between the levels of formaldehyde in the excipients and the formation of the hydroxymethyl degradant was found. To confirm the hypothesis that the formaldehyde impurity in these two excipients contributed to the formation of the hydroxymethyl degradant, several clinical formulation lots were spiked with formaldehyde equivalent to 1, 10, and 100 mg/g of BMS-204352. A correlation was found between the formaldehyde level and the quantity of the hydroxymethyl degradant formed upon storage at 5 and 25 degrees C. From these experiments, a limit test on the formaldehyde content in polysorbate 80 and PEG 300 can be set as part of a strategy to limit the formation of the degradation product.

Introduction: Fiber optic technology for dissolution testing has been a topic of interest for many researchers for some years. Josefson, Johansson, and Tortensson 1 published early research in this field in 1988. They explored the feasibility of using UV fiber optics for in-situ dissolution and to overcome sample turbidity interference without filtration. In 1993, Brown and Lin 2 used a single optical fiber and a photo diode array (PDA) UV/vis spectrometer to track dissolution in a single vessel. Their work was extended thereafter to use six optical fibers and a PDA spectrometer for multiple dissolution vessels 3. In 1995, Cho and coworkers developed a sevenchannel fiber optic dissolution system using a spectrometer with two-dimensional chargecoupled devices (CCD), which allowed the simultaneous monitoring of six dissolution vessels and a seventh reference vessel 4,5. Several other studies around the same time further explored the possibility of using fiber optic technology for special dissolution applications. For example, Chen and his group 6,7 employed a fiber optic based chemical sensor for continuous monitoring of in-situ dissolution. Gemperline et al 8 applied a CCD/fiber optic system for determining two-component dissolution profiles of a pharmaceutical product, which used a full-range spectral principal component regression methodology. Aldridge and coworkers 9 automated a single-probe fiber optic system that used a robot arm to move the probe from vessel to vessel for sequential testing. All these breakthrough studies had great impact on subsequent development of commercial instruments and established the platform for modern in-situ fiber optic dissolution testing. Commercial UV fiber optic dissolution instruments became available in 1999. Several manufacturers in the United States introduced their independently developed instruments one after another. These commercial instruments employed either a spectrometer with multi-channel CCDs 10, 11, multiple PDA spectrometers 12,13, or a scanning spectrometer with a mechanical multiplexer 14,15. Commercialization of this technology and its consequent availability to a broad range of users has attracted much attention from the pharmaceutical industry. Recent publications and research works further described the capability of this technology 16-18 and demonstrated that UV fiber optics is a breakthrough for dissolution testing. A regulatory perspective on this technology has also been discussed in a recent article 19.

A simple generic approach was investigated for the determination of inorganic pharmaceutical counterions in drug substances using conventional high performance liquid chromatographic (HPLC) instruments. An intuitive approach combined Corona charged aerosol detection (CAD) with a polymer-based zwitterionic stationary phase in the hydrophilic interaction chromatography (HILIC) mode. Two generic methods based on this HILIC/CAD technique were developed to quantitate counterions such as Cl-, Br-, SO(4)(2-), K+, Ca2+ and Mg2+ in different pharmaceutical compounds. The development and capability of this HILIC/CAD technique analysis were examined. HILIC/CAD was compared to ion chromatography (IC), the most commonly used methodology for pharmaceutical counterion analysis. HILIC/CAD was found to have significant advantages in terms of: (1) being able to quantitate both anions and cations simultaneously without a need to change column/eluent or detection mode; (2) imposing much less restriction on the allowable organic percentage of the eluents than IC, and therefore being more appropriate for analysis of counterions of poorly water-soluble drugs; (3) requiring minimal training of the operating analysts. The precision and accuracy of counterion analysis using HILIC/CAD was not compromised. A typical precision of

The purpose of this study was to identify two impurities in the parenteral lyophilized formulation of BMS-204352, investigate the role of sanitizing agents as their potential source, evaluate their effect on drug product stability, and develop a strategy to prevent their contamination of the drug product. The two impurities were identified as o-phenylphenol and 4-t-amylphenol based on liquid chromatography/mass spectroscopy (LC/MS) and chromatographic comparison to authentic samples. The LC/MS spectra of commercially available o-phenylphenol and 4-t-amylphenol showed identical patterns of fragmentation and the same retention times as the impurities identified in the BMS-204352 lyophilized product. Levels of these impurities were low and ranged between 0.2-0.3 microg/vial as determined by HPLC and using an authentic external reference standard. To confirm the hypothesis that the commercial sanitizing agents used in the sterile area were the source of these phenolic impurities, several product samples were spiked with the sanitizing agents. Both o-phenylphenol and 4-t-amylphenol were detected in the spiked samples. Further investigation revealed that o-phenylphenol and 4-t-amylphenol are active ingredients of these commercial sanitizing agents. Drug product samples containing the phenolic impurities showed no potency loss following storage at 30, 50, and 70 degrees C indicating these impurities had no adverse effect on product stability. These studies suggest that sanitizing agents used in the sterile area, although may be present at trace levels below typical cleaning procedure detection methods, need to be properly controlled and closely monitored during the manufacturing of injectable products, particularly highly potent drugs. Sanitizing agents, even though not used on product contact surfaces, may potentially contaminate a product through vapor transfer in an open environment.

Comparative in vitro dissolution studies were performed on several tablet batches of zofenopril calcium, an ACE inhibitor, to determine if they could be differentiated on the basis of their release rates. The samples included six batches produced at Site 1 and one batch produced at Site 2. Using regular dissolution conditions (USP paddle method at a 50-rpm agitation speed in phosphate buffer, pH 7.5, at 37 degrees C), release rates of all the tablet batches were similar. By independently altering one of the dissolution test parameters, either a lower pH or a slower agitation rate, discrimination between the Site 1 and Site 2 tablets was enhanced. Discrimination was only slightly enhanced when a lower dissolution medium temperature was used. Tablets made from different polymorphs of zofenopril calcium could not be differentiated by their dissolution profiles, even with the more discriminating conditions. The dissolution profiles of certain other zofenopril calcium tablets (including film-coated vs uncoated tablets, and tablets made with micronized vs unmicronized drug particles) were indistinguishable using a 50-rpm agitation rate, but they could be clearly differentiated using a 40-rpm agitation rate.

The levels of both free and conjugated ecdysteroids, maternally labeled from [14C]cholesterol, of six different age groups of Manduca sexta eggs were quantitatively determined. Eggs 0–1-h old contain about 2.5 and 35 μ/g of the 2- and 26-phosphates of 26-hydroxyecdysone, respectively, and 1 μg/g of 26-hydroxyecdysone. During embryogenesis of 26-hydroxyedcdysone 26-phosphate is hydrolyzed to 26-hydroxyecdysone, which reaches a peak titer in 1–18-h-old eggs; the level of 26-hydroxyecdysone 2-phosphate remains rather constant. Additionally, other metabolic modifications such as hydroxylation, conjugation, epimerization, and oxidation are occurring; and as the level of the 26-hydroxyecdysone 26-phosphate decreases there is a progression of other ecdysteroids. C-20 hydroxylation first appears in 24–40-h-old eggs and reaches peak activity in 48–64-h-old eggs, where 20-hydroxyecdysone and 20, 26-dihydroxyecdysone are both present at peak titer but the latter is the major free ecdysteroid. Ecdysone is observed at measurable levels only in the three age groups of eggs between 1 and 64 h-old. C-3 epimerase activity also appears at 24–40 h and continually increases throughout embryogenesis to the point that 3-epi-26-hydroxyecdysone and 3-epi-20, 26-dihydroxyecdysone are the major free ecdysteroids in 96-h-old eggs. A new ecdysteroid conjugate, 26-hydroxyecdysone 22-glucoside, first appears at 24–40h and becomes the major conjugate in 72–80-h-old eggs; it represents an apparent end-product as its peak titer is reached and maintained throughout the final embryonic stages. 20-Hydroxyecdysonoic acid occurs in 48–64-h-old eggs, and along with 3-epi-20-hydroxyecdysonoic and ecdysonoic acids in 72–88-h-old eggs. 20-Hydroxyecdysonoic acid peaks during the latter time interval, and as its titer subsequently falls, there is a concurrent increase in the level of 3-epi-20-hydroxyecdysonoic which was identified as the second major component of the ecdysteroid conjugate fraction of 0–1-h-old larvae. Our results indicate that there is little or no biosynthesis of ecdysteroids during embryogenesis; that the materal ecdysteroid conjugate 26-hydroxyecdysone 26-phosphate serves as source for 26-hydroxyecdysone and the numerous metabolites; that 26-hydroxyecdysone and 20,26-dihydroxyecdysone may be the active hormones during embryonic development; and that glucosylation, epimerization, and formation of acids cosntitute inactivation processes. A scheme of the proposed pathways involved in the metabolism of 26–hydroxyecdysone 26-phosphate in the developing eggs of m. sexta is presented.

The levels of individual free and conjugated ecdysteroids and ecdysteroid acids, labeled from [14C]cholesterol, in five different age groups of male Manduca sexta during pupal-adult development were determined by HPLC. Eight free ecdysteroids, eight ecdysteroid phosphates, and two ecdysteroid acids were identified. Newly ecdysed pupae contained predominantly 3-epiecdysteroids in each of the free, conjugated, and acidic ecdysteroid fractions. The titer of each ecdysteroid fraction rose sharply by day 4, and this was particularly noteworthy with respect to free ecdysone and 3-epi-20-hydroxyecdysonoic acid. This stage demonstrated high degrees of ecdysone biosynthesis, oxidative catabolism, and phosphorylation. As development proceeded to day 16, total ecdysteroid titer remained constant; a decreasing free ecdysteroid titer was accompanieid by increasing titers of both conjugates and acids resulting from the metabolic processes of hydroxylation, oxidation, epimerization, and phosphorylation. The predominant metabolites throughout development were 3-epi-20-hydroxyecdysonoic acid and the phosphate conjugates of 3-epi-20-hydroxyecdysone and 3-epi-20,26-dihydroxyecdysone. The ultimate inactivation of the ecdysteroids of M. sexta during pupal-adult development is possibly mediated by two pairs of metabolically-linked processes, one leading to a 3-epiecdysteroid acid, and the other to 3-epiecdysteroid phosphates.

The metabolism of 4 dietary 24-alkylsterols was investigated in the free-living nematodeCaenorhabditis elegans. The major unesterified sterols ofC. elegans in media supplemented with either campesterol, 22-dihydrobrassicasterol or stigmasterol included cholesta-5,7-dienol, cholesterol, cholest-7-enol, and 4α-methylcholest-8(14)-enol. Dietary stigmastanol yielded cholest-7-enol, cholestanol, cholest-8(14)-enol, and 4α-methylcholest-8(14)-enol as major unesterified sterols. Esterified sterols comprised less than 22% of the total sterol. Removal of a C-24 ethyl substituent of sterols was neither hindered by the presence of a Δ22-bond in the sterol side chain nor was it depedent on unsaturation in ring B of the steroid nucleus.C. elegans reduced a Δ22-bond during its metabolism of stigmasterol; it did not introduce a Δ22-bond during stigmastanol metabolism.C. elegans was capable of removing a C-24 methyl substituent regardless of its stereochemical orientation. Metabolic processes involving the steroid ring system of cholesterol (C-7 dehydrogenation, Δ5-bond, 4α-methylation, Δ8(14)-isomerization inC. elegans were not hindered by the presence of a 24-methyl group; various 24-methylsterol metabolites from campesterol were detected, mostly 24-methylcholesta-5,7-dienol. In contrast, no 24-ethylsterol metabolites from the dietary ethylsterols were found. More dietary 24-methylsterol remained unmetabolized than did dietary 24-ethylsterol. A 24α-ethyl group and a 24β-methyl group were dealkylated to a greater extent byC. elegans than was a 24α-methyl group, perhaps reflecting the substrate specificity of the dealkylation enzyme system, or suggesting different enzymes altogether.

Radiolabeled polar ecdysteroids were isolated from Manduca sexta pupae and applied to a column of the ion-exchange adsorbent DEAE-Sephadex. Elution with a NaCl gradient solvent system efficiently separated the polar ecdysteroids into three groups, eluted in order of their increasing ionic strength: free ecdysteroids, ecdysteroid acids, and ecdysteroid conjugates. The separation method afforded excellent sample recovery and purification, and facilitated HPLC analysis of the ecdysteroid acids which may be key inactivation products of molting hormones in M. sexta . Isolation procedures are described in detail.

1. An analogous series of dimethylalkyl compounds, consisting of four amines, an amide, and a phosphonate ester, inhibited motility and reproduction of the nematode Caenorhabditis elegans. 2. Dimethylamines with straight-chain lengths of 12, 14, or 16 carbon atoms were equally active nematicides, causing greater than 80% population growth inhibition at a concentration of 25 ppm. 3. The C 12 straight-chain amine and its corresponding amide produced similar inhibition and were much more potent than either the corresponding C 12 phosphonate or a C 12 branched-chain amine. 4. Inhibition of the Δ 24 -sterol reductase system was exhibited by all four amines, but not by the amide or phosphonate, in the following order of activity: C 12 branched-ehain amine > C 12 straight-chain amine > C 14 amine > C 16 amine. 5. The C 12 branched amine also blocked the C-24(28)-dehydrogenase system in the conversion of sitosterol to fucosterol, the initial step in sitosterol dealkylation.

Practical considerations, such as sample selection and data analysis, associated with local norms development are discussed. A local norming project involving administering Form L of the PPVT-R to ...

Caenorhabditis elegans possesses a unique sterol methylation pathway not reported to occur in any other organism and also removes the C-24 ethyl group of sitosterol (a plant sterol). This nematode produced substantial quantities of 4 alpha-methyl-5 alpha-cholest-8(14)-en-3 beta-ol and smaller amounts of lophenol from dietary cholesterol, desmosterol or sitosterol. When C. elegans was propagated in media containing sitosterol plus 25-azacoprostane hydrochloride (25-aza-5 beta-cholestane hydrochloride), an inhibitor of delta 24-sterol reductase in insects, its 4 alpha-methylsterol fraction largely consisted of equal amounts of 4 alpha-methyl-5 alpha-cholesta-7,24-dien-3 beta-ol and 4 alpha-methyl-5 alpha-cholesta-8(14),24-dien-3 beta-ol. Thus 25-azacoprostane hydrochloride inhibited both a delta 24-sterol reductase and a delta 7-sterol isomerase in C. elegans.

Panagrellus redivivus produced 24-methyl-23-dehydrocholesterol as 4.0% of the 4-desmethylsterols when propagated in a medium containing campesterol as the dietary sterol. The re-examination of previous data revealed that Caenorhabditis elegans produced 1.8% 24-methyl-23-dehydrocholesterol when propagated in medium containing campesterol. 24-Methyl-23-dehydrocholesterol was not detected when the nematodes were propagated in medium containing 22-dihydrobrassicasterol or 24-methylenecholesterol. This may be a result of the greater efficiency of dealkylation of the latter two sterols. This is the first report of the natural occurrence of this sterol in a non-photosynthetic organism, and the first report in organisms that dealkylate 24-alkylsterols.

Following injection into female Manduca sexta pupae, [14C]cholesterol is converted to a radiolabeled C21 nonecdysteroid conjugate as well as ecdysteroid conjugates, which in ovaries and newly-laid eggs consist mainly of labeled 26-hydroxyecdysone 26-phosphate. During embryogenesis, as the level of 26-hydroxyecdysone 26-phosphate decreases there is a concurrent increase in the amount of a new, labeled ecdysteroid conjugate. This conjugate, which is the major ecdysteroid conjugate (9.4 μg/g) in 0- to 1-hour-old larvae was identified as 26-hydroxyecdysone 22-glucoside by nuclear magnetic resonance and chemical ionization mass spectrometry. This is the first ecdysteroid glucoside to be identified from an insect. The disappearance of 26-hydroxyecdysone 26-phosphate in 0- to 1-hour-old larvae indicates that the 26-hydroxyecdysone 22-glucoside is derived from 26-hydroxyecdysone 26-phosphate. 3-Epi-26-hydroxyecdysone was the major free ecdysteroid isolated from these larvae and 3-epi-20,26-dihydroxyecdysone was the next most abundant ecdysteroid isolated. Interestingly, the 0- to 1-hour-old larvae contained the highest levels of 3α-ecdysteroids per gram of insect tissue (8.7 μg/g) to be isolated from an insect, yet there was a complete absence of the corresponding free 3β-epimers. The ecdysteroid conjugate profiles of ovaries and 0- to 1-hour-old larvae are discussed. Methodology is presented that permits the efficient separation of free and conjugated ecdysteroids and nonecdysteroid conjugates (C21-steroid conjugates).

[14C]Cholesterol was injected into fifth-instar larvae of Manduca sexta, and the metabolites were isolated and identified from 8-day-old male and female pupae. A major portion of the metabolized cholesterol was esterified either with a sulfate group or with fatty acids. The predominant ecdysteroid metabolites were 20-hydroxyecdysone, 20,26-dihydroxyecdysone, 20-hydroxyecdysonoic acid, and 3-epi-20-hydroxyecdysonoic acid. Smaller amounts of ecdysteroids were identified as conjugates of 26-hydroxyecdysone, 3-epi-20-hydroxyecdysone, 20,26-dihydroxyecdysone, and its 3α-epimer. The metabolic profiles were similar for both male and female pupae. The two ecdysteroid acids were identified by nuclear magnetic resonance spectroscopy and chemical ionization mass spectrometry and by mass spectral analyses of their methyl esters. Detection of 3-epi-20-hydroxyecdysonoic acid as a major metabolite is significant, as its occurrence has been scarcely reported. 3-Epiecdysteroid acid formation is discussed as a possible ecdysteroid-inactivating pathway that may be operating specifically in lepidopterous insects or in particular developmental stages such as eggs or pupae.

The metabolism of three dietary 4,4-desmethylsterols and two 4 alpha-methylsterols was investigated in the free-living nematode Caenorhabditis elegans. Dietary cholestanol was converted mostly to lathosterol. Dietary lathosterol, 7-dehydrocholesterol, 4 alpha-methylcholest-7-enol and 4 alpha-methylcholest-8(14)-enol each remained largely unchanged. An absolute requirement for a substantial quantity of 7-dehydrocholesterol in C. elegans did not exist. C. elegans was unable to remove a 4 alpha-methyl group or introduce a double bond at C-5 and also demonstrated the lack of a delta 7-reductase. Its nutritional sterol requirement was satisfied by cholestanol, lathosterol or 7-dehydrocholesterol; growth was comparable to that obtained previously in media containing delta 5-sterols. However, the two 4 alpha-methylsterols appeared to be unsatisfactory sterol nutrients. The possible physiological importance of 4 alpha-methylsterols is discussed briefly.

Effects on the metabolism of campesterol and stigmasterol in Caenorhabditis elegans were investigated using N,N-dimethyldodecanamine, a known inhibitor of growth, reproduction and the delta 24-sterol reductase of this nematode. 7-Dehydrocholesterol was the predominant sterol (51%) of C. elegans grown in stigmasterol-supplemented media, whereas addition of 25 ppm amine resulted in a large decrease in the relative percentage of 7-dehydrocholesterol (23%) and the accumulation of a substantial proportion (33%) of delta 24-sterols (e.g., cholesta-5,7,24-trienol) and delta 22,24-sterols (e.g., cholesta-5,7,22, 24-tetraenol) but yielded no delta 22-sterols. Dealkylation of stigmasterol by C. elegans proceeded in the presence of the delta 22-bond; reduction of the delta 22-bond occurred prior to delta 24-reduction. Addition of 25 ppm amine to campesterol-supplemented media altered the sterol composition of C. elegans by increasing the percentage of unmetabolized dietary campesterol from 39 to 60%, decreasing the percentage of 7-dehydrocholesterol from 26 to 12%, and causing the accumulation of several delta 24-sterols (6%). C. elegans also was shown to be capable of dealkylating a delta 24 (28)-sterol as it converted 24-methylenecholesterol to mostly 7-dehydrocholesterol. The proposed role of 24-methylenecholesterol as an intermediate between campesterol and 7-dehydrocholesterol was supported by the results.

Cholesterol, (E)- but not (Z)-17(20)-dehydrocholesterol, 5 alpha-cholestan-3 beta-ol, sitosterol, and certain other sterols lacking a 24 beta-methyl group will replace (spare) most but not all of the 24 beta-methylsterol which has recently been found to be absolutely necessary for growth of oxygen-deprived wild type Saccharomyces cerevisiae in the presence of 2,3-iminosqualene. The results imply the existence of two stereochemically distinct roles for sterol in this organism. One of them (perhaps regulatory) requires, whereas the other (probably playing the so-called "bulk" membranous role) does not require the presence of the 24 beta-methyl group. The latter function, for which most of the sterol is needed, can be performed by various 24-alkyl- and 24-desalkylsterols.

To determine the precursors and the fate of 26-hydroxyecdysone in eggs, the fate of labeled putative ecdysteroid precursors was examined in the tobacco hornworm,Manduca sexta. Following injection of [(14)C]cholesterol, 22,25[(14)C]dideoxyecdysome or [(3)H]ecdysone into female pupae (day 16), only [(14)C]cholesterol was incorporated and metabolized. It was converted to labeled nonecdysteroid and ecdysteroid conjugates, of which the latter in ovaries and 48- to 64-hr-old eggs is mainly 26-hydroxyecdysone 26-phosphate (85% in ovaries). Quantitation of the ecdysteroid conjugate by reversed-phase high performance liquid chromatography (RP-HPLC) showed that the levels of 26-hydroxyecdysone 26-phosphate were 31 μg/g of ovaries from 4-day-old females and 25 μg/g and 17 μg/g of 48- to 64-hr-old and 72- to 88-hr-old eggs, respectively. The RP-HPLC of the conjugate fraction of 48- to 64-hr-old eggs showed an additional peak of radioactive material eluting about three min before the 26-hydroxyecdysone 26-phosphate. The quantity of this material increased in the 72-to 88-hr-old eggs, though it was not detected in the analyses of the conjugate fraction from ovaries. Additional peaks of radioactive material eluting before the 26-hydroxyecdysone 26-phosphate peak were observed in the chromatogram of the conjugates of 72- to 88-hr-old eggs. These radioactive materials need to be identified to determine the ultimate fate of ecdysteroids in the developing embryos of the tobacco hornworm. No radioactive free ecdysteroids were detected in either egg age group.

The metabolism of various dietary sterols and the effects of an azasteroid on sitosterol metabolism in the free-living nematodeCaenorhabditis elegans was investigated. The major unesterified sterols ofC. elegans in media supplemented with sitosterol, cholesterol or desmosterol included 7-dehydrocholesterol (66.5%, 40.5%, 31.2%, respectively), cholesterol (6.7%, 52.3%, 26.9%), lathosterol (4.4%, 3.6%, 1.7%) and 4α-methylcholest-8(14)-en-3β-ol (4.2%, 2.1%, 3.8%). Esterified sterols, representing less than 20% of the total sterols, were somewhat similar except for a significantly higher relative content of 4α-methylcholest-8(14)-en-3β-ol (23.3%, 23.4%, 10.6%). ThusC. elegans not only removes the substituent at C24 of dietary sitosterol but possesses the unusual ability to produce significant quantities of 4α-methylsterols. WhenC. elegans was propagated in medium supplemented with sitosterol plus 5 μg/ml of 25-azacoprostane hydrochloride, the azasteroid strongly interfered with reproduction and motility ofC. elegans and strongly inhibited the Δ24-sterol reductase enzyme system; excluding sitosterol, the major free sterols of azacoprostane-treatedC. elegans were cholesta-5, 7, 24-trien-3β-ol (47.9%), desmosterol (9.4%), fucosterol (2.1%) and cholesta-7,24-dien-3β-ol (2.0%). These 4 sterols are likely intermediates in the metabolism of sitosterol inC. elegans.

After injection into male and female fifth-instar larvae of Manduca sexta , [14C]cholesterol was converted to C21 steroids, [14C]pregn-5-ene-3β, 20-diols. These metabolites were isolated from 8-day-old pupae and were identified by TLC, HPLC, and GC-MS as the C-20 isomers of pregnene-3β, 20-diol. They also were isolated from male and female meconium fluid (of 16-day-old pupae) following injection of [14C]cholesterol into 14-day-old pupae.

When accumulation of squalene was used as a measure of the flow of carbon into the sterol pathway in whole cells of semi-anaerobic Saccharomyces cerevisiae, both ergosterol and cholesterol were found to be inhibitory. However, at equivalent concentrations in the medium ergosterol was substantially the more potent inhibitor. Marked differences found in the absorption and esterification of the two sterols failed to account for the observed difference in their capacities to act as feedback agents. Cholesterol was much more effectively absorbed as well as esterified, but, when the abilities of the two sterols to lower the squalene level were calculated on the basis of free sterol in the cells, ergosterol remained more effective by a factor of four.