HPLC determination of imatinib in plasma and tissues after multiple oral dose administration to mice.

Imatinib inhibits Bcr-Abl, c-KIT and PDGFR kinases. It is approved for the treatment of chronic myeloid leukemia (CML), gastrointestinal stromal tumors (GIST) and has further therapeutic potential. Male ICR mice were given imatinib PO (50 or 25 mg/kg, 5 doses every 2 h); euthanized 2 h after the last dose administration; plasma, liver, brain, spleen and kidney were collected and imatinib concentration measured by an optimized HPLC method for quantification in tissues. Methanol (1:1 v/v plasma) and pH 4, 40:30:30 (v/v/v) water-methanol-acetonitrile at 5 ml/g (brain) and 10 ml/g (spleen, kidney, liver) ratio was added to the samples, homogenized, sonicated, centrifuged (15,000 rpm, 5 min, 2 degrees C) and the supernatant injected into an Inertsil CN-3 column (4.6 mm x 150 mm, 5 microm) using 64:35:1 (v/v/v) water-methanol-triethylamine (pH 4.8), flow rate 1 ml/min, 25 degrees C. Imatinib eluted at 7.5 min (268 nm). Linearity: 0.1-50 microg/ml; precision, accuracy, inter- and intra-day variability was within 15%. Recovery was above 95% (plasma), 80% (brain) and 90% (kidney, liver, spleen). Imatinib tissue concentrations were 6-8 folds higher than plasma except brain, where the ratio decreased from 0.24 to 0.08 suggesting limited brain penetration, likely due to blood brain barrier efflux transporters. The extensive distribution supports the expansion of therapeutic applications.

Coadministration of neoral and the novel rapamycin analog, SDZ RAD, to rat lung allograft recipients: potentiation of immunosuppressive efficacy and improvement of tolerability of staggered versus simultaneous treatment.

BACKGROUND: Neoral and rapamycin derivative (RAD) have complementary mechanisms for inhibition of lymphocyte activation and are substrates for the same pathways of drug metabolism. Therefore, we investigated treatment regimens designed to minimize pharmacokinetic interactions and to potentiate immunosuppressive efficacy in a highly stringent rat lung allograft model. METHODS: Lewis recipients of Brown Norway lungs received the following daily oral doses: (A) RAD at 2.5 mg/kg (n=9); (B) Neoral at 7.5 mg/kg (n=8); (C) RAD at 2.5 mg/kg + Neoral at 7.5 mg/kg simultaneously (n=8); or (D) RAD at 2.5 mg/kg + Neoral at 7.5 mg/kg (n=6) staggered 6 hr apart. Rats were assessed by daily weights, chest radiographs, drug trough levels (high-performance liquid chromatography/mass spectrometry), and blinded scoring of graft histology at death (day 21). RESULTS: Radiographs were completely opacified in all grafts of control and RAD monotherapy groups on days 7 and 14, respectively. Grafts were mildly opacified (Neoral monotherapy) and completely clear (both RAD + Neoral groups) on day 21. Simultaneous or staggered combined treatment dramatically reduced histologic rejection compared with treatment with either drug alone. Simultaneous treatment caused poor tolerability (poor grooming, lethargy) and significantly higher day-14 RAD and cyclosporine (CsA) trough levels (49+/-5 and 638+/-106 ng/ml; P<0.04) than in the staggered group (28+/-3 and 318+/-25 ng/ml) in which all animals were clinically normal. RAD and CsA day-14 trough levels in the staggered group were the same or lower than trough levels in animals treated with either drug alone (RAD 27+/-3/Neoral 815+/-67 ng/ml). CONCLUSIONS: (1) Administration of RAD + Neoral suppressed lung rejection more effectively than treatment with either drug alone. (2) Trough levels did not differ between monotherapy and staggered combination therapy for RAD but were lower for CsA. These results suggested that pharmacological, rather than pharmacokinetic, interactions between the parent drugs were responsible for the potentiation of immunosuppression when these drugs were coadministered. 3) Staggered administration of RAD+Neoral avoided the pharmacokinetic interactions that caused the elevated drug blood levels and poor tolerability caused by simultaneous administration. Thus, we could potentiate efficacy and improve tolerability by staggering administration of RAD and Neoral.

Suppression of acute rejection in allogeneic rat lung transplantation: a study of the efficacy and pharmacokinetics of rapamycin derivative (SDZ RAD) used alone and in combination with a microemulsion formulation of cyclosporine.

BACKGROUND: The novel immunosuppressant SDZ RAD, 40-0 (2-hydroxyethyl)rapamycin, is an orally active rapamycin analogue developed for use in combination with cyclosporine (Neoral). The present study was designed to evaluate the efficacy of SDZ RAD, Neoral, or a combination of both drugs for suppression of acute rejection in an allogeneic, unilateral rat lung transplant model. METHODS: Brown-Norway (RT1n) donor lungs were implanted into Lewis (RT1l) recipients that were observed for 21 days. Postoperative evaluation included daily weights, serial chest radiographs, drug trough levels, and histology scores of the transplanted lung on the day of sacrifice. Treatment groups were comprised of rats treated orally with the RAD vehicle as controls (n = 6); SDZ RAD 2.5 mg/kg/day (n = 9); Neoral 7.5 mg/kg/day (n = 8); Neoral 2.5 mg/kg/day (n = 6); SDZ RAD 2.5 mg/kg/day plus Neoral 7.5 mg/kg/day (n = 7); and Neoral 2.5 mg/kg/day plus SDZ RAD 2.5 mg/kg/day (n = 6). RESULTS: The results of this study showed that neither monotherapy with 2.5 mg/kg/day of Neoral, nor 2.5 mg/kg/day of SDZ RAD prevented severe acute rejection in unilateral lung transplant recipients. Furthermore, despite high dose (7.5 mg/kg/day) Neoral treatment, graft histology showed moderate rejection. However, addition of 2.5 mg/kg/day of SDZ RAD to 7.5 mg/kg/day of Neoral completely prevented histologic rejection in four of seven grafts, although the remaining 3 grafts showed minimal rejection. This combination resulted in significantly higher RAD trough levels when compared to SDZ RAD treatment alone. Combining a lower dose of Neoral (2.5 mg/ kg/day) with 2.5 mg/kg/day of SDZ RAD resulted in less weight loss and improved animal health; however, the histology of lung grafts in these rats showed mild rejection. CONCLUSIONS: This is the first study on the efficacy of the novel rapamycin derivative SDZ RAD for the control of acute lung allograft rejection. Results showed that acute unilateral rat lung allograft rejection is refractory to monotherapy with either high dose Neoral or SDZ RAD. The two regimens of combined treatment with Neoral plus SDZ RAD used in these studies produced either minimal rejection and reduced tolerability or mild rejection and better tolerability and showed potentiation of immunosuppression when both drugs were used together. Additional investigation of these two drugs is needed, however, to devise regimens that produce both high immunosuppressive efficacy and good tolerability.

Granulocyte-specific monoclonal antibody technetium-99m-BW 250/183 and indium-111 oxine-labelled leucocyte scintigraphy in inflammatory bowel disease.

Thirty-three patients suspected of suffering from inflammatory bowel disease were studied. Autologous leucocytes were labelled with indium 111 oxine and re-injected simultaneously with 0.3-0.5 mg of technetium 99m granulocyte-specific monoclonal antibody BW 250/183. Two scans were obtained, the early scan 3-4 h postinjection (p.i.) and the late scan 18-24 h p.i. Using the endoscopy study as standard, the diagnostic accuracy of both agents was determined. Sensitivity, specificity and accuracy of 111In scans was 88.8%, 100.0% and 93.7% at 4 h and 94.7%, 100.0% and 96.9% at 24 h, respectively. Concerning the results using antibodies, the values were 61.1%, 100.0% and 78.1% at 4 h and 78.9%, 92.8% and 84.8% at 24 h, respectively. Segmental analysis showed concordance in 89.3% and 93.3% of the cases at 4 and 24 h, respectively. Though less sensitive and less accurate than scanning employing indium 111 leucocytes, BW 250/183 granulocyte-specific scintigraphy can be used for inflammatory bowel disease diagnosis and localization.

Development of a high-performance liquid chromatographic-electrospray mass spectrometric assay for the specific and sensitive quantification of the novel immunosuppressive macrolide 40-O-(2-hydroxyethyl)rapamycin.

It was our objective to develop a rapid, sensitive and specific assay to quantify the immunosuppressive macrolide 40-O-(2-hydroxyethyl)rapamycin (SDZ-RAD) in blood of transplant patients. SDZ-RAD was extracted from blood by solid-liquid extraction. SDZ-RAD and its internal standard 28,40-diacetyl rapamycin were quantified using HPLC-electrospray MS. The assay was linear from 0.1 to 100 microg/l (r2 = 0.99). The mean recovery was 83% for SDZ-RAD and 80.5% for the internal standard. The mean day-to-day precision was 8.0%. Extracted samples were stable at 20 degrees C for at least 48 h and SDZ-RAD blood samples at -80 degrees C for at least six months.