Tacrolimus Exposure and Evolution of Renal Allograft Histology in the First Year After Transplantation

Tacrolimus has a narrow therapeutic window and is characterized by a large inter-individual variability in bioavailability. The impact of tacrolimus exposure on subclinical evolution of graft histology has not been studied in renal recipients. This analysis included 239 protocol biopsies (obtained at implantation, 3 and 12 months) of 120 consecutive kidney recipients treated with tacrolimus, mycophenolate mofetil (MMF) and corticosteroids. Biopsies were scored according to the Banff 2001 criteria and a chronicity score was calculated. Prospective pharmacokinetic data were included in the analysis (5544 tacrolimus predose blood concentrations and tacrolimus AUC(0-12) at 3 and 12 months). Higher donor age and higher number of human leukocyte antigen-DR (HLA-DR) mismatches were independent predictors of subclinical acute rejection at 3 months, present in 8.7% of patients. The number of HLA-DR mismatches was independently associated with biopsy-proven clinical acute rejection. Biopsy-proven acute rejection episodes and low mean tacrolimus exposure were independently associated with higher increase in chronicity scores between 3 and 12 months after transplantation. This observational study suggests that rejection phenomena and immune-mediated mechanisms remain important in the early progression of chronic allograft pathology. Tacrolimus doses or systemic exposure were not associated with lesions of calcineurin inhibitor nephrotoxicity, suggesting that other factors determine susceptibility to tacrolimus nephrotoxicity.     

The Evolution of Nonimmune Histological Injury and Its Clinical Relevance in Adult-Sized Kidney Grafts in Pediatric Recipients

To describe the evolution, risk factors and impact of nonimmune histological injury after pediatric kidney transplantation, we analyzed 245 renal allograft protocol biopsies taken regularly from the time of transplantation to 2 years thereafter in 81 consecutive rejection-free pediatric recipients of an adult-sized kidney. Isometric tubular vacuolization was present early after transplantation was not progressive, and was associated with higher tacrolimus pre-dose trough levels. Chronic tubulo-interstitial damage and tubular microcalcifications were already noted at 3 months, were progressive and had a greater association with small recipient size, male donor gender, higher donor age and female recipient gender, but not with tacrolimus exposure. Renal function assessment showed that older recipients had a significant increase in absolute glomerular filtration rate with time after transplantation, which differed from small recipients who showed no increase. It is concluded that progressive, functionally relevant, nonimmune injury is detected early after adult-sized kidney transplantation in pediatric recipients. Renal graft ischemia associated with the donor-recipient size discrepancy appears to be a greater risk factor for this chronic histological injury, suggesting that the exploration of additional therapeutic approaches to increase allograft perfusion could further extend the graft survival benefit of adult-sized kidneys transplanted into small children.     

Transport,Uptake, and Metabolism of the Bis(pivaloyloxymethyl)-Ester Prodrug of 9-(2- Phosphonylmethoxyethyl)Adenine in an In Vitro Cell Culture System of the Intestinal Mucosa (Caco-2)

Purpose. To evaluate intestinal transport, uptake and metabolism characteristics of the bis(pivaloyloxymethyl)-ester [bis(POM)-ester] of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine [PMEA]. Methods. Intestinal transport, uptake and metabolism of bis(POM)-PMEA were studied using an in vitro cell culture system of the intestinal mucosa (Caco-2 monolayers). Concentrations of bis(POM)-PMEA and its metabolites mono(POM)-PMEA and PMEA were determined using a reversed-phase HPLC method. Enzymatic stability of bis(POM)-PMEA was evaluated by incubation with purified liver carboxylesterase, homogenates of Caco-2 cells and scraped pig small intestinal mucosa. Results. The use of bis(POM)-PMEA as a prodrug of PMEA resulted in a significant increase in transport of total PMEA [bis(POM)-PMEA, mono(POM)-PMEA and PMEA] across Caco-2 monolayers. While transepithelial transport of PMEA (500 M) was lower than 0.1% during a 3 hr incubation period, transport of total PMEA after addition of bis(POM)-PMEA (100 M) amounted to 8.8% over the same incubation period. Only 23% of the amount transported appeared as intact bis-ester at the basolateral side, while 33% of this amount was free PMEA and 44% was mono(POM)-PMEA, suggesting susceptibility of the prodrug to chemical and enzymatic degradation. Uptake studies revealed that only negligible amounts of bis(POM)-PMEA (< 0.2%) were present inside the cells. Very high intracellular concentrations of PMEA were found 1.2 mM, after a 3 hr incubation with 50 M bis(POM)-PMEA), which suggests that PMEA was trapped inside the cells probably due to its negative charge. This explains that efflux of PMEA was relatively slow (25% of the intracellular amount in 3 hr). Enzymatic degradation of the prodrug by carboxylesterase was confirmed by incubation of bis(POM)-PMEA with purified enzyme (K_m = 87 M and V_max = 9.5 M/min). Incubation of bis(POM)-PMEA (10 M) with cell homogenate of Caco-2 monolayers and pig small intestinal mucosa produced similar degradation profiles. Conclusions. The use of the bis(POM)-prodrug significantly enhances the intestinal permeability of PMEA. Intracellular trapping of PMEA in the intestinal mucosa may result in slow release of PMEA to the circulation after oral administration of bis(POM)-PMEA.     

Characterization of the activation pathway of phosphoramidate triester prodrugs of stavudine and zidovudine.

The phosphoramidate triester prodrugs of anti-human HIV 2', 3'-dideoxynucleoside analogs (ddN) represent a convenient approach to bypass the first phosphorylation to ddN 5'-monophosphate (ddNMP), resulting in an improved formation of ddN 5'-triphosphate and, hence, higher antiviral efficacy. Although phosphoramidate derivatization markedly increases the anti-HIV activity of 2',3'-didehydro-2', 3'-dideoxythymidine (d4T) in both wild-type and thymidine kinase-deficient CEM cells, the concept is far less successful for the 3'-azido-2',3'-dideoxythymidine (AZT) triesters. We now investigated the metabolism of triester prodrugs of d4T and AZT using pure enzymes or different biological media. The efficiency of the first activation step, mediated by carboxylesterases, consists of the formation of the amino acyl ddNMP metabolite. The efficiency of this step was shown to be dependent on the amino acid, alkyl ester, and ddN moiety. Triesters that showed no conversion to the amino acyl ddNMP accumulated as the phenyl-containing intermediate and had poor, if any, anti-HIV activity. In contrast to the relative stability of the triesters in human serum, carboxylesterase-mediated cleavage of the prodrugs was found to be remarkably high in mouse serum. The subsequent conversion of the amino acyl ddNMP metabolite to ddNMP or ddN was highest in rat liver cytosolic enzyme preparations. Although L-alaninyl-d4TMP was efficiently converted to d4TMP, the main metabolite formed from L-alaninyl-AZTMP was the free nucleoside (AZT), thus explaining why d4T prodrugs, but not AZT prodrugs, retain anti-HIV activity in HIV-infected thymidine kinase-deficient cell cultures. The rat liver phosphoramidase responsible for the formation of ddNMP was shown to be distinct from creatine kinase, alkaline phosphatase, and phosphodiesterase.     

2-Chloro-3-pyridin-3-yl-5,6,7,8-tetrahydroindolizine-1-carboxamide (CMV423), a new lead compound for the treatment of human cytomegalovirus infections

Human cytomegalovirus (HCMV) remains one of the major pathogens in immunocompromised patients (AIDS and transplants) and the main cause for congenital infections leading from slight cognitive defects up to severe mental retardation. The drugs that are currently available for the treatment of HCMV infections, i.e. ganciclovir, foscarnet and cidofovir, are all acting at the level of the viral DNA polymerase. Here we describe an entirely new molecule, the 2-chloro-3-pyridin-3-yl-5,6,7,8-tetrahydroindolizine-1-carboxamide (CMV423), that shows very potent in vitro activity against HCMV. CMV423 is highly active against HCMV reference strains and clinical isolates, but also against those strains, isolated from patients or emerging after in vitro selection, that are resistant to either ganciclovir, foscarnet or cidofovir. CMV423 also showed activity in two ex vivo models, that are both highly relevant for the pathophysiology of HCMV, the retinal pigment epithelial and the bone marrow stromal cell assays. Viral antigen expression analysis by flow cytometry, as well as time of addition experiments, confirmed that CMV423 acts on a step of the viral replicative cycle that precedes the DNA polymerase step and, most likely, coincides with the immediate early (IE) antigen synthesis. Finally, CMV423 combined with either ganciclovir, foscarnet or cidofovir in checkerboard experiments demonstrated a highly synergistic activity.     

Solid state properties of pure UC-781 and solid dispersions with polyvinylpyrrolidone (PVP K30)

The purpose of this study was to elucidate the physical structure of solid dispersions of the antiviral agent UC-781 (N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]-2-methyl-3-furancarbothioamide) with polyvinylpyrrolidone (PVP K30). Solid dispersions were prepared by coevaporating UC-781 with PVP K30 from dichloromethane. The physicochemical properties of the dispersions were evaluated in comparison with the physical mixtures by differential scanning calorimetry (DSC), X-ray powder diffraction, and FT-IR spectroscopy. We investigated the single crystal structure of pure UC-781. The data from single crystal analysis showed that UC-781 crystallized with orthorhombic symmetry in the space group Pcab. Its cell parameters were found to be; a = 8.1556(7) A,b = 17.658(2) A and c = 23.609(2) A; the unit cell was made up of eight molecules of UC-781. The molecules formed intermolecular hydrogen bonds between NH and thio groups, and were packed in a herringbone-like structure. The data from X-ray powder diffraction showed that crystalline UC-781 was changed into the amorphous state by co-evaporating it with PVP K30. From differential scanning calorimetry analysis, UC-781 peaks were observed in the DSC curves of all physical mixtures, while no peaks corresponding to the drug could be observed in the solid dispersions with the same drug composition up to the concentration of 50% w/w. The data from FT-IR spectroscopy showed the distortions and disappearance of some bands from the drug, while other bands were too broad or significantly less intense compared with the physical mixtures of the crystalline drug in PVP K30. Furthermore, the results from IR spectroscopy demonstrated that UC-781 interacted with PVP K30 in solid dispersions through intermolecular H-bonding.     

Evaluation of the potential of ion pair formation to improve the oral absorption of two potent antiviral compounds, AMD3100 and PMPA.

9-(2-phosphonyomethoxypropyl)adenine (PMPA) and AMD3100 are highly potent and selective antiretroviral agents. Since PMPA is negatively charged and AMD3100 positively charged at physiological pH, their transepithelial transport and their potential for oral drug delivery is very low. In this study, ion pair formation was evaluated as a possible strategy to enhance transepithelial transport of PMPA and AMD3100. Positively charged counter ions such as t-hexyl-, t-heptyl-, t-octylammonium bromide and dodecyl-, tetradecyl-, hexadecyltrimethylammonium bromide were used to form ion pairs with PMPA, while sodium taurodeoxycholate (in vitro experiments) and sodium taurocholate (in vivo experiments) were used as counter ions for AMD3100. The effect of counter ions on transepithelial transport of PMPA (1 mM) and AMD3100 (1 mM) was investigated by measuring the flux across Caco-2 monolayers. An enhancement in drug transport could be observed at a concentration of 2 mM of hexadecyltrimethylammonium bromide (counter ion for PMPA) and 10 mM of sodium taurodeoxycholate (counter ion for AMD3100), but at the concentrations used, the absorption enhancing effect could be attributed to a reduction of the integrity of the monolayers. When AMD3100 transport was tested at a concentration of 200 microM, no flux was observed, even in the presence of relatively high concentrations of counter ion (20 times the concentration of AMD3100). Results obtained from partitioning studies of the drugs in the presence or absence of counter ion revealed that competition by other ions was responsible for the absence of an effect: when pure water was used as the aqueous phase, a reduction up to 24.4+/-1.4% and 17.0+/-1.3% of the initial aqueous concentration was observed for PMPA and AMD3100, respectively; however, as soon as other ions were present in the aqueous phase, the effect of the counter ion was diminished (25-50 mOsm) or completely abolished (270-305 mOsm). The absorption enhancing effect of counter ions was also studied in vivo: pharmacokinetic studies in rabbits showed that the oral bioavailability of AMD3100 in the presence of 4 equivalents of taurocholic acid remained very low and was only 3.2-fold better (i.e. 3.6%) in comparison to pure AMD3100. In view of the results obtained in the Caco-2 system, this absorption enhancement can be attributed to an effect on monolayer integrity rather than to the potential to form ion pairs. We can conclude that the formation of ion pairs may not be very efficient as a strategy to enhance transepithelial transport of charged hydrophilic compounds, as competition by other ions may abolish the beneficial effect of counter ions.