Repeated Chlamydia trachomatis infections are associated with lower bacterial loads.

Chlamydia trachomatis (CT) infections remain highly prevalent. CT reinfection occurs frequently within months after treatment, likely contributing to sustaining the high CT infection prevalence. Sparse studies have suggested CT reinfection is associated with a lower organism load, but it is unclear whether CT load at the time of treatment influences CT reinfection risk. In this study, women presenting for treatment of a positive CT screening test were enrolled, treated and returned for 3- and 6-month follow-up visits. CT organism loads were quantified at each visit. We evaluated for an association of CT bacterial load at initial infection with reinfection risk and investigated factors influencing the CT load at baseline and follow-up in those with CT reinfection. We found no association of initial CT load with reinfection risk. We found a significant decrease in the median log10 CT load from baseline to follow-up in those with reinfection (5.6 CT/ml vs. 4.5 CT/ml; P = 0.015). Upon stratification of reinfected subjects based upon presence or absence of a history of CT infections prior to their infection at the baseline visit, we found a significant decline in the CT load from baseline to follow-up (5.7 CT/ml vs. 4.3 CT/ml; P = 0.021) exclusively in patients with a history of CT infections prior to our study. Our findings suggest repeated CT infections may lead to possible development of partial immunity against CT.

Assessing and minimizing time-dependent inhibition of cytochrome P450 3A in drug discovery: a case study with melanocortin-4 receptor agonists.

1-[(2R)-2-([[(1S,2S)-1-amino-1,2,3,4-tetrahydronaphthalen-2-yl]carbonyl]amino)-3-(4-chlorophenyl)propanoyl]-N-(tert-butyl)-4-cyclohexylpiperidine-4-carboxamide (1) is a potent melanocortin-4 receptor agonist that exhibited time-dependent inhibition of cytochrome P450 (P450) 3A in incubations with human liver microsomes. In incubations fortified with potassium cyanide, a cyano adduct was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis as a cyanonitrosotetrahydronaphthalenyl derivative. The detection of this adduct suggested that a nitroso species was involved in the formation of a metabolite intermediate (MI) complex that led to the observed P450 inactivation. Further evidence supporting this hypothesis derived from incubations of 1 with recombinant P450 3A4, which exhibited a lambda(max) at approximately 450 nm. The species responsible for this absorbance required the presence of beta-nicotinamide adenine dinucleotide phosphate reduced form (NADPH), increased with increasing incubation time and decreased following the addition of potassium ferricyanide to the incubation mixture, suggestive of an MI complex. Similar results were obtained with rat liver microsomes and with recombinant P450 3A1. When rats were dosed with indinavir as a P450 3A probe substrate, plasma exposure to indinavir increased three-fold following pretreatment with 1, consistent with drug-drug interaction projections based on the k(inact) and K(I) parameters for 1 in rat liver microsomes. A similar approach was used to predict the magnitude of the corresponding drug-drug interaction potential in humans dosed with a drug metabolized predominantly by P450 3A, and the forecast area under the curve (AUC) increase ranged from four- to ten-fold. These data prompted a decision to terminate further evaluation of 1 as a development candidate, and led to the synthesis of the methyl analogue 2. Methyl substitution alpha to the amino group in 2 was designed to reduce the propensity for formation of a nitroso intermediate and, indeed, 2 failed to exhibit time-dependent inhibition of P450 3A in human liver microsomal incubations. This case study highlights the importance of mechanistic studies in support of drug-discovery and decision-making processes.

4-Methyl-3-oxo-4-aza-5alpha-androst-1-ene-17beta-N-aryl-carboxamides: an approach to combined androgen blockade [5alpha-reductase inhibition with androgen receptor binding in vitro].

4-Aza-5alpha-androstan-3-one 17beta-(N-substituted carboxamides) are potent human type 2 5alpha-reductase (5aR) inhibitors with generally poor binding to the human androgen receptor (hAR). When the 17-amide N-substituent included an aromatic residue, potent dual inhibitors of both type 1 and 2 5aR are produced, but hAR binding remained poor. Tertiary-substituted-17-amides have reduced inhibition of both 5aR isozymes. The addition of an N4-methyl substitutent to the A-ring profoundly increased hAR affinity and the addition of unsaturation to the A-ring (delta1) modestly augmented hAR binding. The unsubstituted carbanilides in the delta1-N4-methyl series show some selectivity for type 1 5aR over the type 2 isozyme, whereas addition of aryl substituents, particularly at the 2-position, increased type 2 5aR binding to provide dual inhibitors with excellent hAR binding, e.g. N-(2-chlorophenyl)-3-oxo-4-methyl-4-aza-5alpha-androst-1-ene-17bet a-carboxamide (9c). Compounds of this type exhibit low nanomolar IC50s for both human 5aR isozymes as well as the human androgen receptor. Kinetic analysis confirms that the prototype 9c displays reversible, competitive inhibition of both human isozymes of 5aR with K(i) values of less than 10 nM. Furthermore, this compound binds to the androgen receptor with an IC50 equal to 8 nM. Compounds in this series are projected to be powerful antagonists of testosterone and dihydrotestosterone action in vivo, with potential utility in the treatment of prostatic carcinoma (PC).

MK386: a potent, selective inhibitor of the human type 1 5alpha-reductase.

Steroid 5alpha-reductase is required for the conversion of testosterone to dihydrotestosterone. Localization of type 1 5alpha-reductase in the sebaceous gland of skin offers the possibility for selective inhibition of this isozyme as a treatment for acne. The goals of these studies are to demonstrate the mechanism of inhibition of MK386 and its selectivity for type 1 5alpha-reductase. The apparent potency of MK386 differed depending on the source of the enzyme (i.e. recombinant vs. native), yet selectivity for type 1 5alpha-reductase was unchanged. Our results indicate that the apparent potency of MK386 is modulated by the membrane concentration of the assay. These results suggest that MK386 has a high affinity for the lipid-rich membrane environment of 5alpha-reductase. MK386 was also found to be a slow binding inhibitor of type 1 5alpha-reductase. However, the cause of this time-dependent inhibition is unrelated to partitioning of the inhibitor into the membrane because similar studies with type 2 5alpha-reductase indicate that MK386 is a reversible, competitive inhibitor. A number of counterscreens were developed to demonstrate that MK386 is a poor inhibitor of other steroid metabolizing enzymes.

Modeling directed design and biological evaluation of quinazolinones as non-peptidic growth hormone secretagogues.

Quinazolinone derivatives were synthesized and evaluated as non-peptidic growth hormone secretagogues. Modeling guided design of quinazolinone compound 21 led to a potency enhancement of greater than 200-fold compared to human growth hormone secretagogue affinity of a screening lead 4.