Lycopene, lutein and beta-carotene as determinants of LDL conjugated dienes in serum.

Oxidative modification of low-density lipoprotein (LDL) in the vascular endothelium is considered to be important in the development of early atherosclerosis. The aim of this study was to investigate the main determinants of serum LDL conjugated dienes in women (n=124) and men (n=225). We focused on the influence of fat-soluble vitamins and carotenoids on the concentration of conjugated dienes in LDL. In multivariate linear regression models, including age, body mass index, diastolic blood pressure, symptomatic ischaemic heart disease (IHD) or IHD history, statin medication, leukocytes and serum triglycerides as covariates, plasma lycopene (standardized beta=-0.33; P=0.002) and lutein (standardized beta=-0.22; P=0.027) concentrations were the strongest determinants of serum LDL conjugated dienes in women, whereas plasma beta-carotene (standardized beta=-0.23; P=0.002) was the most important factor in men. Furthermore, statin medication, diastolic blood pressure, age and serum triglycerides were significant determinants of LDL conjugated dienes. The regression model with lycopene contributed to 29% in women and 15% in men with beta-carotene of the variation of serum LDL conjugated dienes. Results of the present study suggest that plasma lycopene, lutein and beta-carotene are the most powerful antioxidants for explaining the content of in vivo oxidatively modified LDL in serum.

Distribution of unique red feather pigments in parrots.

In many birds, red, orange and yellow feathers are coloured by carotenoid pigments, but parrots are an exception. For over a century, biochemists have known that parrots use an unusual set of pigments to produce their rainbow of plumage colours, but their biochemical identity has remained elusive until recently. Here, we use high-performance liquid chromatography to survey the pigments present in the red feathers of 44 species of parrots representing each of the three psittaciform families. We found that all species used the same suite of five polyenal lipochromes (or psittacofulvins) to colour their plumage red, indicating that this unique system of pigmentation is remarkably conserved evolutionarily in parrots. Species with redder feathers had higher concentrations of psittacofulvins in their plumage, but neither feather colouration nor historical relatedness predicted the ratios in which the different pigments appeared. These polyenes were absent from blood at the time when birds were replacing their colourful feathers, suggesting that parrots do not acquire red plumage pigments from the diet, but instead manufacture them endogenously at growing feathers.

High-performance liquid chromatographic assay validation of Manumycin A in mouse plasma.

Manumycin A is a natural antibiotic produced by Streptomyces parvulus that has antineoplastic activity against a variety of human cancers in nude mouse models. We have developed a highly sensitive reverse phase high-performance liquid chromatography (HPLC) method based on ultraviolet (UV) detection for the determination of manumycin A in mouse plasma. Manumycin A was isolated from mouse plasma by solid-phase extraction. A gradient elution of methanol and 0.05 M H(3)PO(4) with 0.2% triethylamine mobile phase was employed and separation was achieved with a C(18) analytical column. Manumycin A was detected by UV absorption at 345 nm. Retention time for manumycin A was 8.9+/-0.2 min. The manumycin A peak was baseline resolved, with the nearest peak at 1.5 min distance and no interfering peaks detected. Inter- and intra-day coefficients of variance were less than 6.1 and 5.1%, respectively. Based on an extracted manumycin A standard plasma sample of 0.25 microg/ml, the assay precision was 99.8% with a mean accuracy of 95.1%. At plasma concentrations of 0.5 and 5 microg/ml, the mean recovery rates of manumycin A were 59.64 and 60.28%, respectively. The lower limit of detection (LLD) for manumycin A was 0.1 microg/ml in mouse plasma. The lower limit of quantification (LLQ) for manumycin A was 0.125 microg/ml. Results of the stability study indicated that when frozen at -80 degrees C, manumycin A was stable in mouse plasma for up to 2 weeks. This method is useful in quantification of manumycin A in mouse plasma for clinical pharmacology studies in mice.

Pharmacokinetics and tissue distribution of a new partricin A derivative (N-dimethylaminoacetyl-partricin A 2-dimethylaminoethylamide diaspartate) in mice.

A single-dose trial in mice (1.25 mg/kg SPA-S-753 or 1 mg/kg amphotericin B [AmB] by intravenous route) was performed to study the pharmacokinetics, tissue distribution, and urinary excretion of a new polyene, SPA-S-753 (N-dimethylaminoacetyl-partricin A 2-dimethylaminoethylamide diaspartate), in comparison with AmB. Antibiotic concentrations were determined by microbiological assay (agar diffusion method). The elimination half-lives in serum were 15.1 and 19.8 h, respectively, for SPA-S-753 and AmB; the area under the curve from 0 to infinity values were 49.3 for SPA-S-753 and 23.6 microg. h/mL for AmB, because of the higher serum levels of SPA-S-753 found just after administration. The tissue concentrations of SPA-S-753 were lower than those of AmB in liver and lungs but higher in the kidneys. The urine concentrations of SPA-S-753 and the percent of the administered dose recovered from the urine were quite low in mice, whereas those of AmB were higher.

Essential fatty acid composition of plasma phospholipids and birth weight: a study in term neonates.

BACKGROUND: Essential fatty acids (EFAs) in umbilical cord blood samples are associated with attained birth weight in premature infants and low-birth-weight neonates. OBJECTIVE: The objective was to investigate relations between the EFA composition of cord and maternal plasma phospholipids and birth weight in term neonates. DESIGN: This was a cross-sectional study in 627 singletons born at term. The plasma phospholipid EFA composition of the mothers was determined by gas-liquid chromatography at study entry (< or = 16 wk gestation), at delivery, and in cord plasma at birth. Birth weights were normalized to SD scores. RESULTS: In cord plasma, the dihomo-gamma-linolenic acid concentration was positively related to weight SD scores. Both arachidonic acid (AA) and docosahexaenoic acid (DHA) were negatively related to weight SD scores. EFA-status indicators showed similar negative associations, whereas eicosatrienoic acid concentrations were positively related to neonatal size. In maternal plasma, proportions of n-3 long-chain polyenes (LCPs) and n-6 LCPs decreased during pregnancy. Larger decreases in AA, DHA, n-3 LCP, and n-6 LCP fractions were observed in mothers of heavier babies. Higher concentrations of LCPs in maternal plasma were, however, not related to a larger infant size at birth. CONCLUSIONS: A lower biochemical EFA status in umbilical cord plasma and a larger decrease in maternal plasma LCP concentrations are associated with a higher weight-for-gestational-age at birth in term neonates. Our findings do not support a growth-stimulating effect of AA or DHA; however, they do suggest that maternal-to-fetal transfer of EFAs might be a limiting factor in determining neonatal EFA status.

Hamycin therapy of murine disseminated candidiasis: efficacy and interaction with fluconazole.

The efficacy of antifungal agent, hamycin, in a murine model of disseminated candidiasis was studied. Mice were intravenously infected with Candida albicans blastoconidia and treated for 14 days with intraperitoneal hamycin, oral fluconazole, or a combination of these two. Hamycin alone was most efficacious in prolonging survival and in decreasing renal colony counts, usually with complete sterilization of the kidneys by the end of the treatment period. Fluconazole improved survival rates and effected a decrease in renal colony counts, but kidneys were not microbiologically sterilized. Combination therapy with hamycin and fluconazole did not result in a decrease in the efficacy of hamycin by either end point (survival or renal colony counts). High-pressure liquid chromatographic analysis of Hamycin concentrations in serum indicated lowlevels of absorption of the drug. From the results of the present study, it can be concluded that hamycin is effective in the treatment of murine invasive candidiasis and that the theoretical concern about adverse interactions between the two drug does not apply to the dosages studied in these experiments.

Maternal and neonatal plasma antioxidant levels in normal pregnancy, and the relationship with fatty acid unsaturation.

During pregnancy, maternal plasma concentrations of the peroxidation-susceptible polyunsaturated fatty acids (polyenes) increase. In addition, the proportion of polyenes is higher in neonatal plasma than in maternal plasma. To study whether these increased amounts of polyenes affect antioxidant levels, we measured lipid-soluble antioxidants in maternal and neonatal plasmas obtained during thirty-five normal pregnancies. These values were then related to the degree of phospholipid-fatty acid unsaturation. Maternal plasma levels of tocopherols and lutein increased during pregnancy, as assessed at 14, 22, and 32 weeks of gestation. However beta-carotene levels decreased, and levels of other carotenoids remained unchanged. Retinol levels were only decreased at 32 weeks of gestation. The value for alpha-tocopherol: phospholipid-polyene unsaturation index (UI) also increased during pregnancy, despite the observed increase in UI. Corresponding ratios for several carotenoids and retinol, however, decreased during pregnancy. After delivery, maternal plasma levels of delta-tocopherol and beta + gamma-tocopherol, as well as beta + gamma-tocopherol: UI values, were lower than values at 32 weeks of gestation. Umbilical-cord plasma antioxidant levels and antioxidant: UI values, except retinol: UI, were significantly lower than maternal values. Significant and consistent cord v. maternal correlations were observed for plasma levels of beta + gamma-tocopherol, lutein and beta-carotene, but not for delta-tocopherol, alpha-tocopherol, lycopene, alpha-carotene, and retinol. In conclusion, although during pregnancy maternal plasma tocopherol levels increased concurrently with, or more than, fatty acid unsaturation in plasma phospholipids, the decrease in carotenoid: UI values during gestation, the decrease in maternal plasma levels of delta-tocopherol and beta + gamma-tocopherol after delivery, and the low neonatal antioxidant levels merit further investigation.

Automated simultaneous quantification of the immunosuppressants 40-O-(2-hydroxyethyl) rapamycin and cyclosporine in blood with electrospray-mass spectrometric detection.

A new analytical method to quantify 40-O-(2-hydroxyethyl)rapamycin (SDZ RAD) and cyclosporine (Cs) simultaneously in blood is presented. The combination of an on-line solid-phase extraction step with an HPLC system coupled to an electrospray mass spectrometer gave excellent specificity, sensitivity, and reproducibility. Aliquots of deproteinized blood samples were injected into the HPLC system and extracted on-line, using a conventional C18 guard column. The extract was eluted from the guard column in the backflush mode and injected into the liquid chromatography-mass spectrometry system. The calibration functions for SDZ RAD and Cs extracted from blood with added analyte were linear from 0.15 to 30 microg/L (r2 = 0.999) and from 1.5 to 1000 microg/L (r2 = 0.999), respectively. The CVs of peak areas were 6.2% at 10 microg/L SDZ RAD (n = 6) and 6.2% at 100 microg/L Cs (n = 6). Recovery ranged from 84.3% to 102.3% for SDZ RAD and from 81.7% to 92.2% for Cs. The lower limit of detection for both drugs was 0.05 microg/L. A rate of four samples per hour was maintained during the consecutive analysis of SDZ RAD and Cs in >500 blood samples with one single extraction and analytical column. The method described is a powerful tool for the simultaneous determination of SDZ RAD and Cs in blood. It works without time-consuming sample preparation steps and with excellent reproducibility. Because of the detection performance of electrospray mass spectrometry, this system offers flexibility in the working range, which is essential for therapeutic drug monitoring under different conditions.

Relative tissue distributions of cyclosporine and sirolimus after concomitant peroral administration to the rat: evidence for pharmacokinetic interactions.

The authors sought to determine the effect of concomitant peroral (PO) administration of cyclosporine (CsA) and sirolimus (SRL, rapamycin) on the tissue distributions of CsA and SRL in the rat. Groups of four adult male Wistar-Furth rats were treated for 14 days with 2.5, 5.0, or 10.0 mg CsA/kg x day. Other groups of four adult male Wistar-Furth rats were treated for 14 days with a 1-to-6.25 weight-to-weight ratio of SRL to CsA at SRL doses of 0.4, 0.8, or 1.6 mg/kg x day. Concentrations of CsA and SRL in homogenates of heart, intestinal, kidney, liver, lung, muscle, spleen, and testes were compared to those in whole blood (WB). There was a large, dose-dependent, distinctive distribution of CsA among rat tissues, as has previously been well documented. At a constant molar dose ratio, concomitant oral administration of SRL produced an approximately two-fold increase in the concentrations of CsA in rat tissues, although SRL did not change the CsA tissue-to-WB partition coefficients. Concomitant oral CsA administration produced dose-dependent increases in SRL tissue concentrations and decreases in the SRL tissue-to-WB partition coefficients. The increases in tissue and WB concentrations on coadministration of both agents may be explained either by an increase in absorption caused by competition between the two agents for binding sites on P-glycoprotein in the gut, a reduced rate of metabolism, or to an as yet unidentified elimination mechanism. The dose-independent and unchanged CsA tissue-to-WB partition coefficients suggest that SRL does not affect the equilibrium of CsA between the central and tissue compartments, namely the tissue uptake or intracellular binding. Altered values of the SRL tissue-to-WB partition coefficients suggest that, under the conditions studied, CsA disturbs the equilibrium of SRL between the central and tissue compartments.

Species differences in sirolimus stability in humans, rabbits, and rats.

Species differences in the in vitro stability of sirolimus was assessed in plasma and whole blood in relation to red blood cell distribution. Fresh blood and plasma samples obtained from humans, rabbits, and rats were aliquoted and spiked with sirolimus. After incubation from 0 to 144 hr in a shaking water bath maintained at 37 degrees C, sirolimus concentrations were quantified by a specific high-liquid performance chromatographic method. Sirolimus was unstable in both plasma and whole blood. Sirolimus degradation half-life in whole blood was 135 hr (vs. 7.2 hr in plasma) in humans, 62 hr (vs. 3.1 hr) in rabbits, and 15 hr (vs. 2.2 hr) in rats. Sirolimus stability is greater in whole blood compared with plasma in proportion to the whole blood/plasma ratio and hematocrit. In vivo instability may account for up to 36% of sirolimus clearance in humans and 13% in rabbits, and this is an important factor in the pharmacokinetics of this drug.

Kinetics and dynamics of single oral doses of sirolimus in sixteen renal transplant recipients.

Sirolimus is a new immunosuppressive drug that has been evaluated in animal experiments. The current study was conducted on humans with reformulated sirolimus in doses from 3 mg/m2 to 15 mg/m2. Sixteen renal transplant recipients were included in this phase I study to determine the safety, tolerance, and preliminary pharmacokinetics of increasing single doses of orally administered sirolimus. All 16 patients had stable renal graft function after a renal transplant at least 6 months before the study. Basal immunosuppression consisted of cyclosporine and prednisolone (n = 10) or cyclosporine, azathioprine, and prednisolone (n = 6). Four groups (I, 3 mg/m2; II, 5 mg/m2; III, 10 mg/m2; IV, 15 mg/m2) of four patients were assigned randomly to receive sirolimus (n = 3) or placebo (n = 1). Among the 12 patients who received sirolimus, five had mild transient study events such as headache, nausea, mild dizziness, hypoglycemia, epistaxis, and decrease in platelets. No serious adverse events occurred and no nephrotoxic effects could be related to the single dose administration of sirolimus. The only study event that was judged as probably related to sirolimus was the single case of thrombocytopenia. The other events were evaluated as possibly related. Thrombocytopenia occurred at the highest dose level (15 mg/m2 sirolimus). In two of the patients in the placebo group, slight elevations of liver enzymes and serum amylase were seen. Blood and plasma sirolimus concentrations were analyzed by an electrospray-high performance liquid/mass spectrophotometric (ESP-HPLC/MS) method Sirolimus showed an extensive red blood cell distribution with a mean blood/ plasma ratio of 49.1. The elimination half-life ranged from 43.8 to 86.5 hours (mean 56.9 hours). The Cmax and the area under the concentration versus time curves (AUC) correlated reasonably with doses from 3 to 15 mg/m2. The oral dose clearance ranged from 42 to 339 ml/h.kg. No clinically significant differences were seen in the trough concentrations of cyclosporine or the AUCs before and after the administration of sirolimus. Administration of single oral doses of sirolimus from 3 to 15 mg/m2 was safe and well tolerated in stable renal transplant recipients. Thrombocytopenia may be the dose-limiting toxicity. Additional phase II and phase III clinical trials will define the immunosuppressive efficacy of sirolimus.

Population pharmacokinetics of sirolimus in kidney transplant patients.

OBJECTIVE: To characterize the dose-related pharmacokinetics of the immunosuppressant agent sirolimus (formerly rapamycin) in kidney transplant patients by use of two-stage and nonlinear mixed-effect model population methods. METHODS: Patients (n = 36) from three centers (Germany, the United Kingdom, and Sweden) who received steady-state oral doses of cyclosporine (ciclosporin) were assessed after single oral administration of sirolimus at doses of 3, 5, 10, and 15 mg/m2. Plasma and whole blood sirolimus samples were analyzed by a high-performance liquid chromatographic/mass spectrophotometric method. Simultaneous fitting used biexponential functions with intercept/slope or clearance/volume terms, as well as first-order absorption (ka) and a lag-time. RESULTS: The nonlinear mixed-effect model method (P-Pharm) provided a better characterization of sirolimus kinetics, especially for the absorption and distribution phases where fewer data were available per patient. Sirolimus distribution between whole blood and plasma was concentration-independent, with a mean blood/plasma ratio (coefficient of variation) of 30.9 (48.5%). Elimination was not influenced by dose, as shown by estimates of the terminal half-life of 63 hours (27.5%) and apparent oral blood clearance of 8.9 L/hr (38.2%). Sirolimus distribution parameters were influenced by body weight and surface area. Sirolimus was rapidly absorbed, as shown by the absorption lag-time of 0.27 hour (35.1%), and ka of 2.77 hr-1 (48.4%). The concomitant administration of sirolimus and cyclosporine did not reveal any pharmacokinetic interactions. CONCLUSION: This report provides an initial population pharmacokinetics of sirolimus in kidney transplant recipients receiving cyclosporine concurrently. Sirolimus blood and plasma pharmacokinetics were biexponential and linear for doses from 3 to 15 mg/m2. No pharmacokinetic interaction was found between sirolimus and cyclosporine.

Distribution of sirolimus in rat tissue.

OBJECTIVES: To examine the distribution of sirolimus (SRL, rapamycin), an immunosuppressive macrolide antibiotic, in the tissues of adult male Wistar-Furth rats following continuous intravenous infusion (CIVI) and repeated daily peroral gavage (PO). DESIGN AND METHODS: Animals received 14-day courses of SRL by either CIVI (0.04-0.4 mg/kg/day) or PO (0.4-1.6 mg/kg/day) administration. Samples of whole blood and homogenates of five solid organs (heart, kidney, liver, lung and spleen), and portions of intestinal, muscle and testicular tissues were prepared on day 13 of CIVI treatment or 24 hours after administration of the 14th PO dose. SRL concentrations were determined by high performance liquid chromatography with reference to calibration curves produced from SRL-spiked whole blood or tissue homogenates prepared from drug-free animals. RESULTS: Following PO but not CIVI administration, SRL concentrations in whole blood and all tissues increased linearly in relation to dose. SRL was extensively distributed among most tissues tested (tissue partitions coefficients of > 40 were observed in some cases). Comparatively, SRL whole blood concentrations were low. The ratio between the SRL whole blood concentrations after PO versus after CIVI administration (at like doses of 0.4 mg/kg/day) was 0.04. Therefore, we inferred that the oral bioavailability of SRL was low. CONCLUSIONS: The linear relationships between PO dose and SRL concentrations in whole blood and tissues may be attributed to the low oral bioavailability of SRL, which is indicated by the low levels of SRL observed in whole blood and tissues after PO administration. The nonlinear relationships between CIVI dose and SRL concentrations in whole blood and tissues may result because although whole blood depots may be saturated with SRL, the tissues continue to absorb SRL as the dose of SRL increases. Thus, because a high percentage of SRL is widely distributed into tissues stores, caution must be used when administering this drug in humans.

Determination of rapamycin in whole blood by HPLC.

Sirolimus (rapamycin, RAPA) is a natural fermentation product (macrolide antibiotic) that has demonstrated potent immunosuppressive activity. A reverse-phase high-performance liquid chromatographic (HPLC) method is described for analysis of the drug in whole blood. The samples are purified by using liquid-liquid extraction with butyl chloride/diethyl ether combined with reversed-phase solid-phase extraction. RAPA and internal standard were traced by UV-detection at 278 nm. Linear calibration curves with correlation coefficients > 0.999 were obtained (range, 1-50 ng/ml). Minimum detectable concentration was approximately 0.4 ng/ml and recovery approximately 45% for both RAPA and internal standard. Coefficient of variation (day to day) was 9.8% at 5 ng/ml (n = 6) and 5.6% at 40 ng/ml (n = 6). The chromatography requires < 10 min per sample. The assay has proved to be free of interference peaks from cyclosporine or tacrolimus. The method has been used to determine the whole blood concentrations in samples from healthy volunteers and renal transplant recipients receiving single and multiple doses of oral rapamycin.

Lipophilic benzamide and anilide derivatives as high-performance liquid chromatography internal standards: application to sirolimus (rapamycin) determination.

Finding a suitable internal standard in reversed-phase high-performance liquid chromatography is often difficult. A general approach for selecting and synthesizing the proper internal standard is presented and applied to a validated method for quantitation of sirolimus in several biological matrices. A series of fifteen N-alkylbenzamides, N-alkyltoluamides and N-alkanoylanilines with a log P range of 3.51 to 6.68 were synthesized as internal standards; N-undecyl-o-toluamide was evaluated most extensively. Sirolimus quantitation involves a simple sample clean-up procedure followed by isocratic chromatography on a heated C18 analytical column with an 70% methanol-water mobile phase and ultraviolet detection at 278 nm. This method was linear from 2.5 to 200 ng with a limit of quantitation of 2.5 ng using a 1-ml blood sample. Sirolimus recovery was above 72.1%. The intra-day and inter-day coefficients of variation were less than 11.7%. Several drugs and sirolimus metabolites do not interfere with the analysis. This method was used to measure sirolimus in blood from rats, rabbits and humans.

Routine clinical monitoring of sirolimus (rapamycin) whole-blood concentrations by HPLC with ultraviolet detection.

During phase I/II clinical trials of sirolimus (rapamycin; SRL), therapeutic drug monitoring was performed with a multistep liquid-liquid extraction of 1-mL aliquots of whole blood followed by reversed-phase HPLC with ultraviolet detection. Blood was sampled according to a standardized protocol and clinical status. SRL concentrations were interpolated from calibration curves with a linear range of 0-50 micrograms/L and 1 microgram/L lower limit of quantification. Quality control was monitored over 68 consecutive analytical runs by using frozen aliquots of SRL-supplemented pooled whole blood at 4, 12, and 32 micrograms/L. These samples showed mean concentrations of 3.7 +/- 0.6, 10.9 +/- 1.1, and 29.6 +/- 2.6 micrograms/L, respectively. This method for therapeutic drug monitoring of SRL permits one full-time technician to analyze 100 clinical specimens per week with a 24-h turnaround time. With this method, a strong linear relation (r2 = 0.946, Sy/x = 0.41, n = 115) between the average SRL concentration over a 24-h period and the SRL concentration at the 24th h was revealed.

Effects of the pharmacokinetic interaction between orally administered sirolimus and cyclosporine on the synergistic prolongation of heart allograft survival in rats.

Oral administration, but not continuous intravenous infusion, of sirolimus (SRL) in combination with cyclosporine (CsA) produces a pharmacokinetic interaction, namely increases in the whole blood trough concentrations of SRL ([SRL(WB)]) and CsA ([CsA(WB)]). The effects of this pharmacokinetic interaction on the synergism between SRL and CsA was examined in Wistar Furth (RT1u) recipients of Buffalo (RT1b) heart allografts. A 14-day course of oral SRL produced dose-dependent prolongation of heart allografts: in untreated controls, 0.5 mg/kg SRL per day extended the mean survival time (MST) from 6.4+/-0.5 days to 12.3+/-3.8 days (P<0.05); SRL at 1.0 mg/kg per day prolonged the MST to 18.0+/-5.5 days (P<0.01); at 2.0 mg/kg SRL per day, MST was extended to 52.5+/-13.2 days (P<0.01); and 4.0 mg/kg SRL per day prolonged MST to 90.0+/-41.1 days (P<0.01). Comparison of the in vivo effects after oral versus continuous intravenous SRL administration suggested that the oral bioavailability of SRL is less than 10%. Combinations of oral SRL and CsA synergistically prolonged heart allograft survival, as documented by combination index values of 0.01-0.64 (combination index <1 indicates synergistic interaction). In rats treated with dual drug combinations, CsA increased the bioavailability of SRL by two- to elevenfold, and SRL increased the bioavailability of CsA by two- to threefold, thereby significantly decreasing the oral effective dose (ED) values for each drug. The ED50 for SRL alone is 2.4 mg/kg per day, which produces an average [SRL(WB)] of 13.2 ng/ml. The ED50 for CsA alone is 8.0 mg/kg per day, which produces an average [CsA(WB)] of 1642 ng/ml. However, when the two drugs are combined, the ED50 effect is achieved with only 0.34 mg/kg SRL per day ([SRL(WB)]=1.1 ng/ml) and 2.1 mg/kg CsA per day ([CsA(WB)] =326 ng/ml). Individually, 0.34 mg/kg SRL per day produces an ED9 with an average [SRL(WB)] of 0.6 ng/ml, and 2.1 mg/kg CsA per day produces an ED22 with an average [CsA(WB)] of 174 ng/ml. Thus, the pharmacokinetic interaction between oral SRL and CsA contributes to the in vivo synergism between the two drugs.

Therapeutic blood levels of sirolimus (rapamycin) in the allografted rat.

A study was conducted to determine the relationship among oral dose, trough whole blood levels, graft survival, and side effects in sirolimus-treated allografted rats. The heterotopic heart allograft model using Brown Norway donors and Lewis rat recipients was used. Rats were dosed daily with sirolimus or vehicle until graft failure or up to a maximum of 28 days. Upon graft failure, rats were bled for measurement of trough blood levels of drug and tissues sent for histopathologic analysis. Sirolimus blood concentration correlated positively with dose and graft survival. Significant graft survival occurred at whole blood trough levels of 0.5 ng/ml achieved at the 0.3 mg/kg/day dose. Analysis of the concentration-effect data using a sigmoidal Emax model calculated a whole blood EC50 of 2.0 ng/ml for graft survival. With mean trough concentrations of 7 ng/ml and higher, grafts survived after cessation of drug treatment. At the 0.8 mg/kg/day dose, there was a significant decrease in body weight gain in the rats. Histopathologic examination of sirolimus-treated animals detected thymic and lymphoid atrophy, both considered pharmacologic extensions of sirolimus's immunosuppressive activity and focal myocardial degeneration, an exacerbation of a spontaneous occurring lesion. These results demonstrate that sirolimus prolongs graft survival in rat in a concentration dependent manner with therapeutic whole blood levels of about 10 ng/ml.