Dried Blood Spot Sampling to Assess Rifampicin Exposure and Treatment Outcomes among Native and Non-Native Tuberculosis Patients in Paraguay: An Exploratory Study.

The aim of this study was to evaluate the difference in drug exposure of rifampicin in native versus non-native Paraguayan populations using dried blood spots (DBS) samples collected utilizing a limited sampling strategy. This was a prospective pharmacokinetic study that enrolled hospitalized tuberculosis (TB) patients from both native and non-native populations receiving oral rifampicin 10 mg/kg once-daily dosing. Steady-state DBS samples were collected at 2, 4, and 6 h after intake of rifampicin. The area under the time concentration curve 0-24 h (AUC0-24) was calculated using a Bayesian population PK model. Rifampicin AUC0-24 < 38.7 mg*h/L was considered as low. The probability of target attainment (PTA) was calculated using AUC0-24/MIC > 271 as a target and estimated MIC values of 0.125 and 0.25 mg/L. In total, 50 patients were included. Native patients (n = 30) showed comparable drug exposure to the non-natives (n = 20), median AUC0-24 24.7 (17.1-29.5 IQR) and 21.6 (15.0-35.4 IQR) mg*h/L (p = 0.66), respectively. Among total patients, only 16% (n = 8) had a rifampicin AUC0-24 > 38.7 mg*h/L. Furthermore, PTA analysis showed that only 12 (24%) of the patients met a target AUC0-24 /MIC ≥ 271, assuming an MIC of 0.125 mg/L, which plummeted to 0% at a wild-type MIC of 0.25 mg/L. We successfully used DBS and limited sampling for the AUC0-24 estimation of rifampicin. Currently, our group, the EUSAT-RCS consortium, is preparing a prospective multinational, multicenter phase IIb clinical trial evaluating the safety and efficacy of high-dose rifampicin (35 mg/kg) in adult subjects using the DBS technique for AUC0-24 estimation.

Safety of Rifampicin at High Dose for Difficult-to-Treat Tuberculosis: Protocol for RIAlta Phase 2b/c Trial.

Previous clinical trials for drug-susceptible tuberculosis (DS-TB) have shown that first-line treatment with doses of rifampicin up to 40 mg/kg are safe and increase the early treatment response for young adults with pulmonary tuberculosis. This may lead to a shorter treatment duration for those persons with TB and a good baseline prognosis, or increased treatment success for vulnerable subgroups (age > 60, diabetes, malnutrition, HIV, hepatitis B or hepatitis C coinfection, TB meningitis, stable chronic liver diseases). Here, we describe the design of a phase 2b/c clinical study under the hypothesis that rifampicin at 35 mg/kg is as safe for these vulnerable groups as for the participants included in previous clinical trials. RIAlta is an interventional, open-label, multicenter, prospective clinical study with matched historical controls comparing the standard DS-TB treatment (isoniazid, pyrazinamide, and ethambutol) with rifampicin at 35 mg/kg (HR35ZE group) vs. rifampicin at 10 mg/kg (historical HR10ZE group). The primary outcome is the incidence of grade ≥ 3 Adverse Events or Severe Adverse Events. A total of 134 participants will be prospectively included, and compared with historical matched controls with at least a 1:1 proportion. This will provide a power of 80% to detect non-inferiority with a margin of 8%. This study will provide important information for subgroups of patients that are more vulnerable to TB bad outcomes and/or treatment toxicity. Despite limitations such as non-randomized design and the use of historical controls, the results of this trial may inform the design of future more inclusive clinical trials, and improve the management of tuberculosis in subgroups of patients for whom scientific evidence is still scarce. Trial registration: EudraCT 2020-003146-36, NCT04768231.

Semisynthesis, biological activity, and molecular modeling studies of C-ring-modified camptothecins.

The synthesis, biological activity, and molecular modeling studies of C-ring-modified camptothecins are reported. A general synthetic protocol, based on "C-5 camptothecin (C-5-CPT) enolate chemistry", allows one to obtain various C5-substituted analogues. All new compounds, obtained as 1:1 epimeric mixtures, were tested for their antiproliferative activity. Experimental data showed that all novel derivatives are less active than the reference compounds and that one of the two epimers is more active than the other. Molecular docking simulations were performed to achieve more insight into the interactions between the new C5-modified CPTs and Topo I. A good correlation was observed when the data of cytotoxicity and the values calculated for the free binding energy were combined.

The role of polyamine architecture on the pharmacological activity of open lactone camptothecin-polyamine conjugates.

A series of camptothecin open-ring lactone tripartate conjugates were synthesized, in which polyamine side chains with different architecture (ethane-1,2-diamine, spermidine, homospermidine, spermine, and 4,8,13,17-tetraza-icosane-1,20-diamine) are linked to the 21-carboxylic function through an amidic bond, while the 17-CH(2)OH is acetylated. The rationale for the synthesis of these compounds was to explore the influence of the polyamine architecture on the activity of these CPT conjugates into cells, since the positively charged ammonium cations would favor interaction through electrostatic binding to the negatively charged DNA backbone. Topoisomerase I-mediated DNA cleavage assay was used to investigate the ability of these compounds to stimulate the DNA damage. The cleavage pattern was found to be similar to that of SN38 for all the new CPTs. The CPT tripartates were tested for growth inhibition ability against the human non-small-cell lung cancer carcinoma NCI-H460 cell line. Although these compounds were less potent than topotecan, SN38, and CPT after 1 h of treatment, the antiproliferative effects greatly increased after 72 h of exposure. The growth inhibition potency during long-term exposure is correlated with the number of charges of the 21-amide substituent. Both cleavage assay and in vitro effects support the interpretation that the compounds may have inhibitory activity also in the open-ring form. The architecture of the polyamine moiety is important for antiproliferative activity, and a balance between the hydrophilic and lipophilic properties of the polyamine is critical for CPT potency.

Thiocamptothecin.

The synthesis and the X-ray structure of 16a-thiocamptothecin (TCPT), the thiopyridone analog of camptothecin (CPT), are accomplished. The crystal contains two structurally identical, yet independent molecules. Both of them are connected to other molecules via two intermolecular hydrogen bonds. S-methylation of TCPT leads to the cleavage of the C-ring. The cytotoxic activity of TCPT was evaluated against different human tumor cell lines using CPT as reference compound.

Biological properties of IDN5174, a new synthetic camptothecin with the open lactone ring.

A series of water-soluble camptothecins obtained by linking a spermidine moiety to the 21-position of the open form through an amidic bond have been tested for their biochemical and biological activities. Growth inhibition assay on the human non-small cell lung cancer carcinoma NCI-H460 cell line revealed that the camptothecin analogues were less potent than topotecan and SN38 after 1 hour of treatment. The potency increased after 72 hours of exposure, being similar to that of reference camptothecins. The analysis of topoisomerase I-mediated DNA cleavage using the purified enzyme indicated that the novel camptothecin analogues retained ability to poison topoisomerase I and displayed the same cleavage pattern of SN38. Persistence of the DNA cleavage was comparable with that of SN38. Stabilization of the cleavable complex was not the result of hydrolysis of the N-C bond between polyamine and the drug because no free camptothecin was recovered at the end of DNA cleavage in presence of IDN5174, the analogue selected for detailed studies. IDN5174 exhibited an antitumor activity comparable with that of topotecan and irinotecan against NCI-H460 tumor xenograft. The pharmacokinetics in mice showed a favorable disposition in tumor tissue with low amount of camptothecin detectable in plasma and tumor (around 5-10%), thus supporting the efficacy of intact IDN5174. In conclusion, we found that IDN5174 maintained the biological and antitumor properties, in spite of lack of the closed E ring. The available results support the interpretation that the polyamine linked at the 21-position may allow a favorable drug interaction in the ternary complex.

Stereoselective one-pot synthesis of constrained N,O-orthogonally protected C-glycosyl norstatines [C(1')-aminoglycosyl-1,3-dioxolan-4-ones].

A procedure for the synthesis of conformationally constrained C-glycosyl norstatines has been developed. The key step of the reaction is the addition of (S)-N-sulfinyl azomethines to chiral (2S)-enolates of dioxolanones which exploits Seebach's "SRS" principle. The trisubstituted C-glycosyl-alpha-hydroxy-beta-amino acids are formed as N,O-orthogonally protected 1'-glycosyl-sulfinylamino-dioxolan-4-ones, usually with high diastereomeric excesses. Both the sulfinyl group at the nitrogen atom and the acetal moiety of the dioxolanone ring were selectively removed, thus demonstrating the orthogonality of the two protecting groups. In fact, the MeO(-) induced methanolysis of the acetal group gave the corresponding methyl C-glycosyl-sulfinylamino-isoserinates, while the acid induced removal of the sulfinyl group gave the Nu-deprotected 1'-glycosylamino-dioxolan-4-ones, which were in several cases subjected to a one-pot base-induced cyclization yielding the corresponding glycosyl-beta-lactams. This allowed the stereochemical configuration assessment of the parent 1'-glycosyl-sulfinylamino-dioxolan-4-ones by chemical correlation methods or by NOE experiments performed on the beta-lactams.

Synthesis of deserpidine from reserpine.

The Rauwolfia alkaloids reserpine (1) and deserpidine (2), two alkaloids from Rauwolfia species, have been widely used for their antihypertensive action. Deserpidine (2) is a compound with limited availability from natural sources, and its synthesis from 1 in six steps (41% overall yield) is reported here.

Advances in the universal NMR database approach. 2'-substituted taxanes as probes for an improved protocol of diastereomeric differentiation.

[structure: see text] The configuration of the alpha-substituted alpha-hydroxy-beta-aminoester moiety in a series of 2'-substituted taxanes was analyzed according to the recently proposed Universal NMR Database (UDB) approach. A critical analysis of the results showed that modifications regarding chemical shift adjustment (so as to render the shifts virtually connectivity independent) were necessary to get consistent stereoassignments in this set of compounds. On this basis, a modified UDB-based strategy, especially tailored to the configurational assignment of densely substituted diastereomeric fragments, is proposed.

Synthesis of optically active constrained 2-substituted norstatines: a straightforward application of Seebach's "SRS" synthetic principle.

A straightforward two-step methodology of synthesis of optically active (2R)-substituted norstatines via addition of N-tert-butoxycarbonyl-substituted aldimines to (2S)-chiral enolates of 1,3-dioxolan-4-ones has been developed. In particular, the use of the natural (2S)-malic acid is examined for the synthesis of potential GABAergic spirocyclic gamma-lactams.

New taxane derivatives: synthesis of baccatin[14,1-d]furan-2-one nucleus and its condensation with the norstatine side chain.

New taxanes 15 and 18, containing the unsaturated and saturated baccatin[14,1-d]furan-2-one nucleus, respectively, were prepared starting from the readily available 13-oxo-7-Tes-baccatin III (3). Sequential formation of the enolate of 3 and reaction with ethyl glyoxylate gave the 13-oxo-7-Tes-baccatin[14,1-d]-3,4-dehydrofuran-2-one 4. The reduction of 4 can result in the formation of a mixture of compounds corresponding to 13-hydroxy alcohol 5 and 13-enol derivative 6. Both 5 and 6 were transformed into 13-oxo-7-Tes-baccatin[14,1-d]furan-2-one 8 by treatment with a base. Further reduction of 8 gave 13-hydroxy compound 9. Esterification of 6 and 9 with N,O-protected norstatine 12, followed by deprotection, gave the new promising anticancer taxanes 15 and 18, respectively.

Diastereoselective 14beta-hydroxylation of baccatin III derivatives.

14beta-Hydroxybaccatin III, a compound with limited availability by natural sources, is the starting material for the synthesis of the second-generation anticancer taxoid ortataxel. The 7-tert-butoxycarbonyl (1a) and 7-triethylsilyl (1b) derivatives of 14beta-hydroxybaccatin III 1,14-carbonate were synthesized from 10-deacetylbaccatin III (3). The crucial steps were (a) the C(14)beta hydroxylation of the corresponding 13-oxobaccatin III derivatives by oxaziridine-mediated electrophilic oxidation and (b) the reduction of the C(13) carbonyl group with sodium or alkylammonium borohydrides. This protocol provides a practical way for the semisynthesis of ortataxel from 10-deacetylbaccatin III, a compound readily available from various yews.

Synthesis and biological evaluation of 2'-methyl taxoids derived from baccatin III and 14beta-OH-baccatin III 1,14-carbonate.

A series of 2'-methyl taxoids were synthesized and essayed for growth inhibition experiments conducted in human ovarian cancer cell line A2780wt and its counterparts A2780cis, A2780tax, and A2780adr, resistant to cisplatin, paclitaxel, and doxorubicin, respectively, to test the effect of this substituent on the antitumor activity. Additional experiments were performed on MCF-7 human breast cancer cell line and MCF7-R resistant to doxorubicin. In several cases these taxoids were more active than paclitaxel showing subnanomolar IC(50) values.

Synthesis and HSA binding characterisation of the water soluble 7-succinylpaclitaxel.

A water soluble paclitaxel analogue, the 7-hemisuccinylpaclitaxel, was synthesised and its binding to human serum albumin (HSA) was characterised by difference circular dichroism and optical biosensor methodologies. The carboxylate group was introduced at paclitaxel C-7 position to improve the drug water solubility without significantly changing the biological activity. The paclitaxel analogue showed a relatively low affinity to HSA (3.5x10(4) M(-1)), while no significant interactions were evidenced with selective markers for the most characterised binding sites on the carrier, suggesting a non-selective binding to low affinity binding sites.

(3R)-Chiral Control of 3-Alkyl-3-hydroxy-beta-lactams via Addition Reaction of Imines to Enolates of 1,3-Dioxolan-4-ones.

A method is described for the chiral construction of (3R)-3-alkyl-3-hydroxy-beta-lactams in a versatile and predictable manner. This protocol follows Seebach's synthetic principle of self-regeneration of stereocenters and has been applied to addition reactions among a selected number of imines and (2S)-chiral enolates of 1,3-dioxolan-4-ones. These reagents are easily available from the acetalization of (S)-alpha-hydroxy acids (lactic, mandelic, isovaleric, malic) and pivalaldehyde or pinacolone. In several cases, the addition of the enolate to the imine, the cyclization, and the removal of the auxiliary center occur in a one-step sequence, affording the corresponding beta-lactams as (3R,4S)-Z and (3R,4R)-E diastereomeric mixtures with high enantiomeric excesses. Four N-unsubstituted (3R,4S)-3-hydroxy-3-methyl-beta-lactams bearing 2-furyl (4e), phenylethenyl (4h), methoxycarbonyl (4i), and 2-thienyl (4l) substituents at C4 were obtained as major diastereomers and were purified by crystallization. The simultaneous presence of these substituents at C3 and C4 make these beta-lactams useful intermediates for the synthesis of new taxoids with interesting structural modifications at the isoserine moiety.