Sample preparation strategy for the detection of steroid-like compounds using MALDI mass spectrometry imaging: pulmonary distribution of budesonide as a case study.

Corticosteroids as budesonide can be effective in reducing topic inflammation processes in different organs. Therapeutic use of budesonide in respiratory diseases, like asthma, chronic obstructive pulmonary disease, and allergic rhinitis is well known. However, the pulmonary distribution of budesonide is not well understood, mainly due to the difficulties in tracing the molecule in lung samples without the addition of a label. In this paper, we present a matrix-assisted laser desorption/ionization mass spectrometry imaging protocol that can be used to visualize the pulmonary distribution of budesonide administered to a surfactant-depleted adult rabbit. Considering that budesonide is not easily ionized by MALDI, we developed an on-tissue derivatization method with Girard's reagent P followed by ferulic acid deposition as MALDI matrix. Interestingly, this sample preparation protocol results as a very effective strategy to raise the sensitivity towards not only budesonide but also other corticosteroids, allowing us to track its distribution and quantify the drug inside lung samples.

Ability of mathematical models to predict human in vivo percutaneous penetration of steroids.

Human skin is a common route for topical steroids to enter the body. To aid with risk management of therapeutic steroid usage, the US Environmental Protection Agency estimates percutaneous penetration using mathematical models. However, it is unclear how accurate are mathematical models in estimating percutaneous penetration/absorption of steroids. In this study, accuracy of predicted flux (penetration/absorption) by the main mathematical model used by the EPA, the Potts and Guy model based on in vitro data is compared to actual human in vivo data from our laboratory of percutaneous absorption of topical steroids. We focused on steroids due to the availability of steroid in vivo human data in our laboratory. For most steroids the flux was underestimated by a factor 10-60. However, within the group itself, there was an association between the Potts and Guy model and experimental human in vivo data (Pearson Correlation = 0.8925, p = 0.000041). Additionally, some physiochemical parameters used in the Potts and Guy equation, namely log Kp (Pearson Correlation = 0.7307, p = 0.0046) and molecular weight (Pearson correlation = -0.6807, p = 0.0105) correlated significantly with in vivo flux. Current mathematical models used in estimating percutaneous penetration/absorption did not accurately predict in vivo flux of steroids. Why? Proposed limitations to mathematical models currently used include: not accounting for volatility, lipid solubility, hydrogen bond effects, drug metabolism, as well as protein binding. Further research is needed in order to increase the predictive nature of such models for in vivo flux.

Pharmacokinetics profiles of polyphyllin II and polyphyllin VII in rats by liquid chromatography with tandem mass spectrometry.

Polyphyllin II (PII) and polyphyllin VII (PVII) are the main active ingredients in Paris Polyphylla with an excellent antitumor effect in vitro and in vivo. In this study, a rapid and precise LC-MS/MS method was developed and validated for the separation and simultaneous determination of PII and PVII in rat plasma, tissues, feces and urine using ginsenoside Rg3 as the internal standard. Positive linearity ranged from 1 to 1,000 ng/ml in samples. At the same time, intra- and inter-day precisions were in range of 1.8-12.0%. The accuracy ranged from 95.9 to 100.8%. Mean extraction recoveries of PII and PVII ranged from 86.6 to 96.4%. The analytical method has been successfully applied to the pharmacokinetic studies of PII and PVII in rats after their i.v. administration. After entering systemic circulation, PII and PVII were rapidly distributed in organs, mainly including liver, lung and spleen. Their elimination rate was slow. All of these data provided a theoretical basis for the application of PII and PVII in the treatment of liver- and lung-related diseases.

Biotransformation of Timosaponin BII into Seven Characteristic Metabolites by the Gut Microbiota.

Timosaponin BII is one of the most abundant Anemarrhena saponins and is in a phase II clinical trial for the treatment of dementia. However, the pharmacological activity of timosaponin BII does not match its low bioavailability. In this study, we aimed to determine the effects of gut microbiota on timosaponin BII metabolism. We found that intestinal flora had a strong metabolic effect on timosaponin BII by HPLC-MS/MS. At the same time, seven potential metabolites (M1-M7) produced by rat intestinal flora were identified using HPLC/MS-Q-TOF. Among them, three structures identified are reported in gut microbiota for the first time. A comparison of rat liver homogenate and a rat liver microsome incubation system revealed that the metabolic behavior of timosaponin BII was unique to the gut microbiota system. Finally, a quantitative method for the three representative metabolites was established by HPLC-MS/MS, and the temporal relationship among the metabolites was initially clarified. In summary, it is suggested that the metabolic characteristics of gut microbiota may be an important indicator of the pharmacological activity of timosaponin BII, which can be applied to guide its application and clinical use in the future.

Development and validation of a rapid and sensitive LC-MS/MS method for the determination of polyphyllin II in rat plasma and its application in a pharmacokinetic study.

Polyphyllin II, a major steroidal saponin isolated from Paris polyphylla, exhibits significant pharmacological activities. In this study, a rapid and sensitive liquid chromatography-tandem mass spectrometry method was established and validated for the determination of polyphyllin II in plasma. Polyphyllin II and polyphyllin VII (internal standard) were separated on a Waters Acquity™ HSS T3 column and the mass analysis was performed in a triple quadrupole mass spectrometer equipped with an electrospray ionization ion source. Results showed that the method was sensitive (lower limit of quantitation 0.5 ng/ml), precise (<15%) and linear in the range of 0.5-500 ng/ml (r > 0.99). Interestingly, the sensitivity in current study was ~10 times higher than that in the previous study. The results of the pharmacokinetic study of polyphyllin II in rats suggested that polyphyllin II was poorly absorbed into blood and reached its highest concentration at ~3.67-5.00 h with a slow elimination half-life of 8.34-13.37 h. The bioavailability was 6.1-8.2%. The results indicated that the absorption of polyphyllin II may primarily occur via passive diffusion in rats. This study provides valuable information that can be used as a reference for the pharmacokinetic investigation of other steroidal saponins.

Effect of hepatic impairment on the pharmacokinetics of vilaprisan: An open-label, single-dose, parallel-group study.

AIMS: The study objective was to evaluate the pharmacokinetics of the selective progesterone receptor modulator vilaprisan in participants with hepatic impairment. Additionally, the safety and tolerability of vilaprisan were investigated. METHODS: In this phase 1, open-label, nonrandomised, parallel-group, pharmacokinetic study, men and women with mild or moderate hepatic impairment (Child-Pugh grade A or B) and control participants with normal hepatic function matched by age, weight and sex received a single oral 2 mg dose of vilaprisan. Key pharmacokinetic parameters, relationships between parameters and safety outcomes were measured. RESULTS: Thirty-six participants completed the study: 9 with mild hepatic impairment, 9 with moderate hepatic impairment and 18 matched control participants with normal hepatic function. Vilaprisan reached maximum plasma concentrations after 1-2 hours. Unbound vilaprisan exposure was 1.44-fold higher for participants with mild hepatic impairment vs controls (90% confidence interval: 0.91-2.26), and 1.74-fold higher for participants with moderate impairment vs controls (90% confidence interval: 1.09-2.78). The maximum observed unbound peak concentrations were similar for participants with hepatic impairment and matched controls. Vilaprisan 2 mg was well tolerated and the incidence of treatment-emergent adverse events was similar across cohorts. CONCLUSION: Only mild increases of <1.75-fold in exposure were observed in participants with mild or moderate hepatic impairment compared with control participants. No safety concern was identified. These data, alongside the excellent safety profile observed in phase 1 and 2 studies, do not indicate that a dose adjustment would be required in patients with mild or moderate hepatic impairment.

Quantitative determination of six steroid alkaloids by sensitive hydrophilic interaction liquid chromatography electrospray ionization mass spectrometry and its application to pharmacokinetic study in rats.

In this study, a sensitive hydrophilic interaction liquid chromatography (HILIC) method was developed to determine pseudojervine (PJV), veratrosine (VTS), jervine (JV), veratramine (VTM), veramarine (VA) and veratroylzygadenine (VTG) in rat plasma. Separations were carried out using LC-MS/MS with a Chrom Matrix HP amide column (5 m, 10 cm × 3.0 mm i.d.). The mobile phases were (A) 0.01 mm formic acid and (B) acetonitrile. Good linearity was found for all analytes (R2  > 0.995) in the concentration range from 5 to 1000 µg/L with LLOQ at 5 µg/L for VTM and VTS; and from 1 to 1000 µg/L with LLOQ at 1 µg/L for PJV, JV, VA and VTG. Accuracy of the assay varied from 90.5 to 108.1%. The extraction recovery and matrix effect of six analytes ranged from 72.2 to 95.5% and from 79.2 to 98.4%. According to the stability test, six analytes in rat plasma were stable during the analysis process. On the basis of validation of the assay, the pharmacokinetics of the six steroid alkaloids were investigated after oral administration of Lilu extracts to rats.

CYP3A4-mediated effects of rifampicin on the pharmacokinetics of vilaprisan and its UGT1A1-mediated effects on bilirubin glucuronidation in humans.

AIMS: The primary aim of the present study was to quantify the effects of rifampicin, a strong cytochrome P450 (CYP) 3A4 inducer, on the pharmacokinetics of the new selective progesterone receptor modulator, vilaprisan. In addition, the effects of rifampicin on the glucuronidation of bilirubin, an endogenous UDP-glucuronosyltransferase family 1 member A1 (UGT1A1) substrate, were explored. METHODS: This was an open-label, two-period study in 12 healthy postmenopausal women. Subjects received a single oral dose of vilaprisan 4 mg in each period. In period 2, administration of vilaprisan was preceded and followed by rifampicin 600 mg day-1 . A subtherapeutic dose of midazolam (1 mg) was coadministered with vilaprisan to monitor CYP3A4 induction. Details of the administration and sampling schedule were optimized by means of a physiologically based pharmacokinetic model. Plasma concentrations of vilaprisan, midazolam, and 1'- hydroxy-midazolam were measured and rifampicin-associated changes in the glucuronidation of bilirubin were determined. RESULTS: As predicted by our model, the coadministration of rifampicin was associated with a substantial decrease in exposure to vilaprisan and midazolam - indicated by the following point estimates (90% confidence intervals) for the area under the plasma concentration-time curve from zero to the time of the last quantifiable concentration ratio with or without rifampicin: 0.040 (0.0325, 0.0505) for vilaprisan and 0.144 (0.117, 0.178) for midazolam. Further, it was associated with an increase in bilirubin glucuronidation, indicating that UGT1A1 was induced. CONCLUSIONS: The exposure to vilaprisan was reduced by 96%. Such a reduction is likely to render the drug therapeutically ineffective. Therefore, it is recommended that the use of strong CYP3A4 inducers is avoided when taking vilaprisan.

Metabolism, pharmacokinetics, and hepatic disposition of xanthones and saponins on Zhimu treatments for exploratively interpreting the discrepancy between the herbal safety and timosaponin A3-induced hepatotoxicity.

Timosaponin A3, a saponin in Zhimu, elicited hepatotoxicity via oxidative stress. However, the clinical medication of Zhimu has been historically regarded as safe, probably associated with the antioxidants it contains. However, the related information on the in vivo levels of timosaponin A3 and antioxidants remained unclear on Zhimu treatments. Therefore, a combination of the in vitro metabolism, including microbiota-mediated and liver-mediated metabolism, and in vivo pharmacokinetics and hepatic disposition, was conducted for three xanthones (neomangiferin, mangiferin, and norathyriol) and three saponins (timosaponin B2, timosaponin B3, and timosaponin A3) on Zhimu treatments. Consequently, following oral administration of Zhimu decoction to rats, those saponins and xanthones were all observed in the plasma with severe liver first-pass effect, where mangiferin was of the maximum exposure. Despite the ignorable content in the herb, timosaponin A3 elicited sizable hepatic exposure as the microbiota-mediated metabolite of saponins in Zhimu. The similar phenomenon also occurred to norathyriol, the microbiota-mediated metabolite of xanthones. However, the major prototypes in Zhimu were of limited hepatic exposure. We deduced the hepatic collection of norathyriol, maximum circulating levels of mangiferin, and timosaponin B2 and mangiferin interaction may directly or indirectly contribute to the whole anti-oxidation of Zhimu, and then resisted the timosaponin A3-induced hepatotoxicity. Thus, our study exploratively interpreted the discrepancy between herbal safety and timosaponin A3-induced hepatotoxicity. However, given the considerable levels and slow eliminated rate of timosaponin A3 in the liver, more attention should be paid to the safety on the continuous clinical medication of Zhimu in the future.

A pilot pharmacokinetic study of miroestrol and deoxymiroestrol on rabbit sera using polyclonal antibody-based icELISA analysis.

Miroestrol (ME) and deoxymiroestrol (DME) are the most potent phytoestrogens and bioactive markers in Pueraria candollei var. mirifica tuberous roots. To understand their pharmacokinetic profiles, a pharmacokinetic study of ME and DME, at 0.43 and 0.21 mg per kg body weight, respectively, in three rabbits was performed after orally administering a single dose of P. candollei var. mirifica enriched fraction extract. Two established polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assays were validated to determine ME and DME in rabbit sera. In rabbits, the area under the 0- to 48-hr concentration-time curve of ME and DME were 854.92 and 1,692.84 ng·h/ml, respectively. The maximum concentration of ME was measured 1 hr after administration as 69.62 ± 8.28 ng/ml, and the maximum concentration of DME was measured at 3 hr as 81.8 ± 5.43 ng/ml. These results provide an initial approach for designing and studying the relationship between the ME and DME levels and their therapeutic effects based on their pharmacokinetic profiles.

The cellular membrane as a mediator for small molecule interaction with membrane proteins.

The cellular membrane constitutes the first element that encounters a wide variety of molecular species to which a cell might be exposed. Hosting a large number of structurally and functionally diverse proteins associated with this key metabolic compartment, the membrane not only directly controls the traffic of various molecules in and out of the cell, it also participates in such diverse and important processes as signal transduction and chemical processing of incoming molecular species. In this article, we present a number of cases where details of interaction of small molecular species such as drugs with the membrane, which are often experimentally inaccessible, have been studied using advanced molecular simulation techniques. We have selected systems in which partitioning of the small molecule with the membrane constitutes a key step for its final biological function, often binding to and interacting with a protein associated with the membrane. These examples demonstrate that membrane partitioning is not only important for the overall distribution of drugs and other small molecules into different compartments of the body, it may also play a key role in determining the efficiency and the mode of interaction of the drug with its target protein. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

Pharmacokinetic Analysis and in Vivo Antitumor Efficacy of Taccalonolides AF and AJ.

The taccalonolides are microtubule stabilizers that covalently bind tubulin and circumvent clinically relevant forms of resistance to other drugs of this class. Efforts are under way to identify a taccalonolide with optimal properties for clinical development. The structurally similar taccalonolides AF and AJ have comparable microtubule-stabilizing activities in vitro, but taccalonolide AF has excellent in vivo antitumor efficacy when administered systemically, while taccalonolide AJ does not elicit this activity even at maximum tolerated dose. The hypothesis that pharmacokinetic differences underlie the differential efficacies of taccalonolides AF and AJ was tested. The effects of serum on their in vivo potency, metabolism by human liver microsomes and in vivo pharmacokinetic properties were evaluated. Taccalonolides AF and AJ were found to have elimination half-lives of 44 and 8.1 min, respectively. Furthermore, taccalonolide AJ was found to have excellent and highly persistent antitumor efficacy when administered directly to the tumor, suggesting that the lack of antitumor efficacy seen with systemic administration of AJ is likely due to its short half-life in vivo. These results help define why some, but not all, taccalonolides inhibit the growth of tumors at systemically tolerable doses and prompt studies to further improve their pharmacokinetic profile and antitumor efficacy.

Pharmacokinetics and safety of the selective progesterone receptor modulator vilaprisan in healthy postmenopausal women
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OBJECTIVES: Vilaprisan is a novel, potent, and highly selective progesterone receptor modulator, which might offer a promising option for the treatment of uterine fibroids. METHODS AND MATERIALS: In this randomized, placebo-controlled, parallel-group phase 1 study, the pharmacokinetics and safety of vilaprisan were investigated in healthy postmenopausal women. Subjects received a single oral dose of vilaprisan (1, 5, 15, or 30 mg) or placebo and - after a wash-out period - daily doses of the same strength over 28 days. Safety assessments included vital signs, ECGs, clinical laboratory tests, and adverse events. Blood samples for pharmacokinetic (PK) profiles were collected over 14 days after single dose (sd) and multiple dose (md; day 28). RESULTS: Vilaprisan was well tolerated. Mild to moderate adverse events occurred with similar frequency at all dose levels. Following single dose, maximum vilaprisan concentrations were observed 1 - 2 hours post-dose. Terminal half-lives ranged from 31 to 38 hours. Maximum concentrations of vilaprisan (Cmax) and exposure to vilaprisan (AUC) increased roughly dose-proportionally from 3.74 µg/L (1 mg) to 68.6 µg/L (30 mg) and 58.5 µg×h/L to 1,590 µg×h/L, respectively. With daily dosing, accumulation consistent with the long terminal half-life was observed (AUC(0-24)md/AUC(0-24)sd ratios: 1.9 to 3.2). The ratio AUC(0-24)md/AUCsd increased with dose from ~ 1 (1 mg) to 1.5 (30 mg). CONCLUSIONS: Exposure to vilaprisan increased roughly dose-proportionally in the dose range studied and accumulated after multiple dosing as expected based on t1/2, indicating linear pharmacokinetics of vilaprisan in the expected therapeutic dose range.
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Bactericidal Activity of Ceragenin CSA-13 in Cell Culture and in an Animal Model of Peritoneal Infection.

Ceragenins constitute a novel family of cationic antibiotics characterized by a broad spectrum of antimicrobial activities, which have mostly been assessed in vitro. Using a polarized human lung epithelial cell culture system, we evaluated the antibacterial activities of the ceragenin CSA-13 against two strains of Pseudomonas aeruginosa (PAO1 and Xen5). Additionally, the biodistribution and bactericidal activity of a CSA-13-IRDye 800CW derivate were assessed using an animal model of peritoneal infection after PAO1 challenge. In cell culture, CSA-13 bactericidal activities against PAO1 and Xen5 were higher than the activities of the human cathelicidin peptide LL-37. Increased CSA-13 activity was observed in polarized human lung epithelial cell cultures subjected to butyric acid treatment, which is known to increase endogenous LL-37 production. Eight hours after intravenous or intraperitoneal injection, the greatest CSA-13-IRDye 800CW accumulation was observed in mouse liver and kidneys. CSA-13-IRDye 800CW administration resulted in decreased bacterial outgrowth from abdominal fluid collected from animals subjected to intraperitoneal PAO1 infection. These observations indicate that CSA-13 may synergistically interact with antibacterial factors that are naturally present at mucosal surfaces and it maintains its antibacterial activity in the infected abdominal cavity. Cationic lipids such as CSA-13 represent excellent candidates for the development of new antibacterial compounds.

Determination of Caudatin in Rat Plasma by UPLC-MS/MS: Application to a Preclinical Pharmacokinetic Study.

In this study, a simple, sensitive, and robust analytical method based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed for the determination of caudatin in rat plasma using carbamazepine as internal standard (IS). This method was linear over the concentration range 2.5-300 ng/ml with a lower limit of quantification (LLOQ) of 2.5 ng/ml. Inter- and intra-day precision (RSD%) were all within 10% and the accuracy (RE%) was equal or lower than 5%. Recoveries of caudatin and IS were more than 80% and matrix effects were not significant. Stability studies showed that caudatin was stable under a variety of storage conditions. The method was successfully applied to a pharmacokinetic study involving oral administration of caudatin to rats.

Clinical efficacy and tolerability of a novel selective corticosteroid in atopic dermatitis--two randomised controlled trials.

Topical corticosteroids remain the first-line therapy for atopic dermatitis (AD). Hence, we investigated the efficacy and safety profile of a novel selective corticosteroid, GW870086. We performed 2 randomised, double-blind, controlled studies with 25 AD patients and 20 healthy subjects. The changes in the Three-Item Severity (TIS) score and the skin thickness were the primary end points, respectively. The adjusted TIS score (day 22) shows that the novel corticosteroid resulted in a non-significant, but dose-dependent reduction compared to placebo (GW870086 0.2% vs. placebo = -0.38, GW870086 2% vs. placebo = -0.89). Significant skin thinning was observed in the second study on days 14 and 21 when patients were treated with the comparator but not with the novel corticosteroid compared to placebo. The clinical efficacy of the new selective corticosteroid was not superior to placebo, although a dose-dependent improvement upon treatment was noticed without the onset of skin thinning.

Pharmacokinetics and tissue distribution study of caudatin in normal and diethylnitrosamine-induced hepatocellular carcinoma model rats.

Caudatin is a potential antitumor agent isolated from the traditional Chinese medicine "baishouwu", which was the root tuber of Cynanchum auriculatum Royle ex Wight. In our previous studies, caudatin showed selectivity on human hepatoma cell line SMMC7721 among several different tumor cell lines, and further in vivo tests validated the inhibitory action of caudatin against hepatic cancer using an H22 solid tumor model in mice, but to our knowledge, the biopharmaceutical properties of caudatin are largely unknown. In this study, a simple, rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of caudatin in rat plasma and tissues, which kept the run time to detect one sample within 4 min, was developed and validated. Pharmacokinetics and tissue distribution studies of caudatin in conventional rats and hepatocellular carcinoma (HCC) model rats were then conducted for the first time. Statistically significant differences were observed between conventional rats and diethylnitrosamine (DEN)-induced HCC rats with respect to pharmacokinetic parameters, including maximum concentration (Cmax), time to reach Cmax (Tmax), half-life (t1/2), area under the concentration-time curve (AUC0-t, AUC0-∞), mean residence time (MRT0-t and MRT0-∞), and oral clearance (CL/F). Increased exposures of caudatin were found in the plasma and livers of HCC model rats, which would be helpful for a better understanding of pharmacological effect of caudatin in treating HCC disease.

Simultaneous determination of six C21 steroids of Xiao-Ai-Ping injection in rat plasma by LC-MS/MS.

Xiao-Ai-Ping injection (XAPI) is a traditional Chinese medicine that has been widely used to treat cancer. Modern pharmacological studies have demonstrated that C21 steroids are the main active compounds in XAPI. In this study, a sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated the first time for simultanenous determination of three isomeric pregnane genins (17ß-tenacigenin B, tenacigenin B and tenacigenin A) and their corresponding glycosides (tenacigenoside A, tenacissoside F and marsdenoside I) from XAPI in rat plasma. A simple liquid-liquid extraction technique was used after the addition of dexamethasone acetate as internal standard. The chromatography separation of analytes was achieved on an Agilent Zorbax Eclipse XDB-C18 column (3.5 µm, 150 × 3 mm i.d.) using methanol-water as mobile phase in a gradient elution program. Detection was performed in multiple reaction monitoring mode using electrospray ionization in the negative ion mode. The method showed satisfactory linearity over a concentration range 5.00-2000.00 ng/mL for tenacigenin B, tenacigenin A, marsdenoside I and tenacissoside F (r(2) > 0.99), 10.00-4000.00 ng/mL for 17ß-tenacigenin B and tenacigenoside A (r(2) > 0.99). Intra- and inter-day precisions (valued as relative standard deviation) were <9.00% and accuracies (as relative error) in the range -6.31 to 7.23%. Finally, this validated method was successfully applied to the pharmacokinetic study of XAPI after intravenous administration to rats.

A novel LC-MS/MS method for determination of tissue distribution and excretion of timosaponin B-II in rat biological matrices.

Timosaponin B-II (TB-II) is a natural bioactive steroid glycoside extracted from the Chinese medicinal herb Anemarrhena asphodeloides Bge. (Fam. Liliaceae). It has been demonstrated to have a good anti-inflammatory effect and a low bioavailability (1.1%). Clinical research has focused on developing it into a completely new medicine. In this study, a rapid and sensitive analytical method based on LC-MS/MS has been developed for the determination of TB-II in rat biological matrices (tissues, bile, urine and feces samples). The analytes and internal standard were isolated from 100 µL samples by solid-phase extraction and then separated using a DIKMA Inertsil ODS-3 column (5 µm, 2.1 × 150 mm) with an isocratic mobile phase consisting of acetonitrile-0.05% formic acid (35:65) at a flow rate of 0.25 mL/min. Calibration curves (1/χ(2) -weighted) offered satisfactory linearity (r(2) ≥ 0.990) within the test range. The accuracy, precision, recoveries and matrix effects were satisfactory in all the biological matrices examined. The assay was successfully applied to a tissue distribution and excretion study in rats. The preclinical data are useful for the design of clinical trials of TB-II.

Macrolactonolides: a novel class of anti-inflammatory compounds.

A new concept in design of safe glucocorticoid therapy was introduced by conjugating potent glucocorticoid steroids with macrolides (macrolactonolides). These compounds were synthesized from various steroid 17ß-carboxylic acids and 9a-N-(3-aminoalkyl) derivatives of 9-deokso-9a-aza-9a-homoeritromicin A and 3-descladinosyl-9-deokso-9a-aza-9a-homoeritromicin A using stable alkyl chain. Combining property of macrolides to preferentially accumulate in immune cells, especially in phagocyte cells, with anti-inflammatory activity of classic steroids, we designed molecules which showed good anti-inflammatory activity in ovalbumin (OVA) induced asthma in rats. The synthesis, in vitro and in vivo anti-inflammatory activity of this novel class of compounds are described.