Clinical pharmacology of AMG 181, a gut-specific human anti-α4ß7 monoclonal antibody, for treating inflammatory bowel diseases.

AIMS: AMG 181 pharmacokinetics/pharmacodynamics (PK/PD), safety, tolerability and effects after single subcutaneous (s.c.) or intravenous (i.v.) administration were evaluated in a randomized, double-blind, placebo-controlled study. METHODS: Healthy male subjects (n= 68) received a single dose of AMG 181 or placebo at 0.7, 2.1, 7, 21, 70 mg s.c. (or i.v.), 210 mg s.c. (or i.v.), 420 mg i.v. or placebo. Four ulcerative colitis (UC) subjects (n= 4, male : female 2:2) received 210 mg AMG 181 or placebo s.c. (3:1). AMG 181 concentration, anti-AMG 181-antibody (ADA), α4 ß7 receptor occupancy (RO), target cell counts, serum C-reactive protein, fecal biomarkers and Mayo score were measured. Subjects were followed 3-9 months after dose. RESULTS: Following s.c. dosing, AMG 181 was absorbed with a median tmax ranging between 2-10 days and a bioavailability between 82% and 99%. Cmax and AUC increased dose-proportionally and approximately dose-proportionally, respectively, within the 70-210 mg s.c. and 70-420 mg i.v. ranges. The linear ß-phase t1/2 was 31 (range 20-48) days. Target-mediated disposition occurred at serum AMG 181 concentrations of less than 1 µg ml(-1) . The PD effect on α4 ß7 RO showed an EC50 of 0.01 µg ml(-1) . Lymphocytes, eosinophils, CD4+ T cells and subset counts were unchanged. AMG 181-treated UC subjects were in remission with mucosal healing at weeks 6, 12 and/or 28. The placebo-treated UC subject experienced colitis flare at week 6. No ADA or AMG 181 treatment-related serious adverse events were observed. CONCLUSIONS: AMG 181 has PK/PD, safety, and effect profiles suitable for further testing in subjects with inflammatory bowel diseases.

Similarities in the immunoglobulin response and VH gene usage in rhesus monkeys and humans exposed to porcine hepatocytes.

BACKGROUND: The use of porcine cells and organs as a source of xenografts for human patients would vastly increase the donor pool; however, both humans and Old World primates vigorously reject pig tissues due to xenoantibodies that react with the polysaccharide galactose alpha (1,3) galactose (alphaGal) present on the surface of many porcine cells. We previously examined the xenoantibody response in patients exposed to porcine hepatocytes via treatment(s) with bioartficial liver devices (BALs), composed of porcine cells in a support matrix. We determined that xenoantibodies in BAL-treated patients are predominantly directed at porcine alphaGal carbohydrate epitopes, and are encoded by a small number of germline heavy chain variable region (VH) immunoglobulin genes. The studies described in this manuscript were designed to identify whether the xenoantibody responses and the IgVH genes encoding antibodies to porcine hepatocytes in non-human primates used as preclinical models are similar to those in humans. Adult non-immunosuppressed rhesus monkeys (Macaca mulatta) were injected intra-portally with porcine hepatocytes or heterotopically transplanted with a porcine liver lobe. Peripheral blood leukocytes and serum were obtained prior to and at multiple time points after exposure, and the immune response was characterized, using ELISA to evaluate the levels and specificities of circulating xenoantibodies, and the production of cDNA libraries to determine the genes used by B cells to encode those antibodies. RESULTS: Xenoantibodies produced following exposure to isolated hepatocytes and solid organ liver grafts were predominantly encoded by genes in the VH3 family, with a minor contribution from the VH4 family. Immunoglobulin heavy-chain gene (VH) cDNA library screening and gene sequencing of IgM libraries identified the genes as most closely-related to the IGHV3-11 and IGHV4-59 germline progenitors. One of the genes most similar to IGHV3-11, VH3-11cyno, has not been previously identified, and encodes xenoantibodies at later time points post-transplant. Sequencing of IgG clones revealed increased usage of the monkey germline progenitor most similar to human IGHV3-11 and the onset of mutations. CONCLUSION: The small number of IGVH genes encoding xenoantibodies to porcine hepatocytes in non-human primates and humans is highly conserved. Rhesus monkeys are an appropriate preclinical model for testing novel reagents such as those developed using structure-based drug design to target and deplete antibodies to porcine xenografts.

Appraisal of the extent of chronic allograft vasculopathy in animal models: proposition of a standardized micromorphometric method.

Arterial obstruction due to intimal myproliferation is an outcome used for investigating chronic allograft vasculopathy in animal models. Since harvested tissue may be distorted while processing, discrepancy may happen in the measure of the obstruction. We propose a standardized micromorphometric method for calculating the percentage of obstruction with eliminating the variability in the shape of the vessel cross-section. The mathematical adjustment avoids the overestimation of the percentage of obstruction and improves accuracy.

Effects of JAK3 inhibition with CP-690,550 on immune cell populations and their functions in nonhuman primate recipients of kidney allografts.

BACKGROUND: Janus Kinase (JAK) 3 is a tyrosine kinase essential for proper signal transduction downstream of selected cytokine receptors and for robust T-cell and natural killer cells activation and function. JAK3 inhibition with CP-690,550 prevents acute allograft rejection. To provide further insight into the mechanisms of efficacy, we investigated the immunomodulatory effects of CP-690,550 in vitro and in vivo in nonhuman primates. METHODS: Pharmacodynamic assessments of lymphocyte activation, function, proliferation and phenotype were performed in three settings: in vitro in whole blood isolated from untransplanted cynomolgus monkeys (cynos), in vivo in blood from untransplanted cynos dosed with CP-690,550 for 8 days, and in vivo in blood from transplanted cynos immunosuppressed with CP-690,550. Cell surface activation markers expression, IL-2- enhanced IFN-gamma production, lymphocyte proliferation and immune cell phenotype analyzes were performed with multiparametric flow cytometry. RESULTS: In vitro exposure to CP-690,550 resulted in significant reduction of IL-2-enhanced IFN-gamma production by T-cells (maximum inhibition of 55-63%), T-cell surface expression of CD25 (50% inhibitory concentration (IC50); 0.18 microM) and CD71 (IC50; 1.6 microM), and T-cell proliferative capacities measured by proliferating cell nuclear antigen expression (IC50; 0.87 microM). Similar results were observed in animals dosed with CP-690,550. In addition, transplanted animals displayed significant reduction of NK cell (90% from baseline) and T-cell numbers whereas CD8 effector memory T-cell populations were unaffected. CONCLUSIONS: Potent in vitro and in vivo immunomodulatory effects of the JAK3 inhibitor CP-690,550 likely contribute to its efficacy in the prevention of organ allograft rejection.

Combined use of the JAK3 inhibitor CP-690,550 with mycophenolate mofetil to prevent kidney allograft rejection in nonhuman primates.

BACKGROUND: Immunosuppression via Janus kinase (JAK) 3 inhibition affords significant prolongation of allograft survival. We investigated the effects of an immunosuppressive regimen combining the JAK3 inhibitor CP-690,550 with mycophenolate mofetil (MMF) in nonhuman primates (NHPs). METHODS: Life-supporting kidney transplantations were performed between ABO-compatible, MLR-mismatched NHPs. Animals were treated orally twice a day with CP-690,550 and MMF (n=8) or MMF alone (n=2) and were euthanized at day 90 or earlier due to allograft rejection. RESULTS: Mean survival time (+/-SEM) in animals treated with MMF alone (23+/-1 days) was significantly extended in animals that concurrently received CP-690,550 (59.5+/-9.8 days, P=0.02). Combination animals exposed to higher levels of CP-690,550 had a significantly better survival (75.2+/-8.7 days) than animals that received less CP-690,550 (33.3+/-12.6 days, P=0.02). Three combination therapy animals were euthanized at day 90 with a subnormal renal function and early-stage acute graft rejection. Rejection, delayed by treatment, ultimately developed in other animals. Anemia and gastrointestinal intolerance was seen in combination therapy animals that otherwise did not show evidence of viral or bacterial infection besides signs consistent with subclinical pyelonephritis (n=3). One incidental lymphosarcoma was noted. CONCLUSIONS: Addition of CP-690,550 to MMF significantly improved allograft survival. The observed side effects appear amenable to improvements upon alteration of dosing strategies. Efficacy of this combination regimen suggests that it could become the backbone of calcineurin inhibitor-free regimens.

Immunosuppression by the JAK3 inhibitor CP-690,550 delays rejection and significantly prolongs kidney allograft survival in nonhuman primates.

BACKGROUND: Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the common chain (gammac). Because mutations in genes encoding gammac or JAK3 result in immunodeficiency, we investigated the potential of a rationally designed inhibitor of JAK3, CP-690,550, to prevent renal allograft rejection in nonhuman primates. METHODS: Life-supporting kidney transplantations were performed between mixed leukocyte reaction-mismatched, ABO blood group-matched cynomolgus monkeys. Animals were treated with CP-690,550 (n = 18) or its vehicle (controls, n = 3) and were euthanized at day 90 or earlier if there was allograft rejection. RESULTS: Mean survival time (+/- standard error of mean) in animals treated with CP-690,550 (53 +/- 7 days) was significantly longer than in control animals (7 +/- 1 days, P=0.0003) and was positively correlated with exposure to the drug (r = 0.79, P < 0.01). Four treated animals were euthanized at 90 days with a normal renal function and low-grade rejection at final pathology. Occurrence of rejection was significantly delayed in treated animals (46 +/- 7 days from transplantation vs. 7 +/- 1 days in controls, P = 0.0003). Persistent anemia, polyoma virus-like nephritis (n = 2), and urinary calcium carbonate accretions (n = 3) were seen in animals with high exposure. Natural killer cell and CD4 and CD8 T-cell numbers were significantly reduced in treated animals. Blood glucose, serum lipid levels, and arterial blood pressure were within normal range in treated animals, and no cancers were demonstrated. CONCLUSIONS: CP-690,550 is the first reported JAK3 inhibitor combining efficacy and good tolerability in a preclinical model of allotransplantation in nonhuman primates and thus has interesting potential for immunosuppression in humans.

JAK3 inhibition, a viable new modality of immunosuppression for solid organ transplants.

The field of organ transplantation has had tremendous success because of the availability of immunosuppressive drugs that efficiently prevent acute organ rejection. Numerous and severe side effects are, however, associated with all current immunosuppressive therapies and justify a search for drugs with better efficacy and safety profiles. Janus kinase (JAK) 3, a tyrosine kinase that is crucial for mediating signals from the common gamma-chain of cytokine receptors, is peculiar in that its expression, contrarily to the targets of most current immunosuppressive drugs, is limited to cells that actively participate to the immune response to allografts. The recent demonstration in stringent preclinical models that JAK3 inhibition results in efficacy for the prevention of allograft rejection with a narrow side-effect profile might lead to a new era in the field of immunosuppression.

Improved assessment of graft function by echocardiography in cynomolgus monkey recipients of hDAF-transgenic pig cardiac xenografts.

BACKGROUND: The current practice of evaluating heterotopic heart xenografts by palpation allows only detection of severe graft dysfunction, which indicates terminal graft failure. Therefore, we evaluated whether echocardiography is a better method of detecting early graft dysfunction as a marker of rejection in abdominal pig heart xenografts in cynomolgus monkeys. METHODS: Six cynomolgus monkeys received heterotopic heart transplants from pig donors transgenic for human decay-accelerating factor (hDAF). Induction therapy consisted of either cyclophosphamide or rabbit anti-thymocyte globulin. Maintenance therapy consisted of cyclosporine or tacrolimus, steroids, and sodium mycophenolate or mycophenolate mofetil, GAS914 (alphaGal oligosaccharide containing glycoconjugate), and for some animals TP10 (soluble complement receptor type 1). Echocardiography was performed immediately after transplantation and 3 times a week after surgery. We scored contractility and measured left ventricular wall thickness. Impaired contractility or increased wall thickness were considered graft dysfunction and were treated with pulse steroids. Palpation score was recorded daily. We also obtained myocardial biopsy specimens. RESULTS: Palpation score remained at 4 out of 4 in all animals until 2 to 5 days before final graft failure, whereas echocardiography detected several episodes of impaired graft function, either decreased left ventricular contractility or increased left ventricular wall thickness before graft failure. Treatment with pulse steroids improved graft function only during early episodes of graft impairment. Final graft failure was steroid resistant and caused by severe vascular rejection. CONCLUSIONS: Echocardiography is a better method of assessing graft dysfunction than is palpation. Therefore, echocardiography may detect early rejection episodes of heterotopic heart xenografts in non-human primates.

Effects of the kinase inhibitor CGP41251 (PKC 412) on lymphocyte activation and TNF-alpha production.

CGP41251 is a serine/threonine and tyrosine kinase inhibitor that is a novel anticancer agent. Because the kinases that CGP41251 inhibits play important roles in T lymphocyte activation, we hypothesized that this compound may have useful immunomodulatory properties. Here we characterized the in vitro immunomodulatory effects of CGP41251. The effects of CGP41251 on lymphocyte proliferation, expression of T cell activation surface markers, and intracellular calcium response in peripheral blood mononuclear cells (PBMC's) were measured. Intracellular IL-2, TNF-alpha, IFN-gamma expression in CGP41251-treated T cells stimulated by lectin was measured by flow cytometry. CGP41251 inhibited lectin-induced lymphocyte proliferation and upregulation of activation surface markers with a 50% inhibitory concentration (IC(50)) of 0.1 microM. CGP41251, at micromolar concentrations, blunted the intracellular calcium response during PBMC activation. CGP41251 inhibited TNF-alpha production by T cells with an IC(50) of 0.5 microM and did not significantly inhibit the production of IL-2 or IFN-gamma. In conclusion, CGP41251 potently inhibits T lymphocyte activation and function and interferes with the proximal part of the T cell activation pathway. The ability of CGP41251 to selectively block T cell TNF-alpha production warrants the evaluation of this compound on other, e.g., monocyte, immune cells and in immunological conditions that are characterized by high TNF-alpha levels such as psoriasis and inflammatory bowel diseases.

Prediction of clinical pharmacokinetics of AMG 181, a human anti-α 4 ß 7 monoclonal antibody for treating inflammatory bowel diseases.

The purpose of this study was to predict a safe starting dose of AMG 181, a human anti-α 4 ß 7 antibody for treating inflammatory bowel diseases, based on cynomolgus monkey pharmacokinetic (PK) and pharmacodynamic (PD) data. A two-compartment model with parallel linear and target-mediated drug disposition for AMG 181 PK in cynomolgus monkey was developed. The estimated parameters were allometrically scaled to predict human PK. An E max PD model was used to relate AMG 181 concentration and free α 4 ß 7 receptor data in cynomolgus monkey. AMG 181 clinical doses were selected based on observed exposures at the no adverse effect level of 80 mg·kg(-1) in monkeys, the predicted human exposures, and AMG 181 concentration expected to produce greater than 50% α 4 ß 7 receptor occupancy in humans. The predicted human AMG 181 clearance and central volume of distribution were 144 mL·day(-1) and 2900 mL, respectively. The estimated EC50 for free α 4 ß 7 receptor was 14 ng·mL(-1). At the 0.7 mg starting dose in humans, the predicted exposure margins were greater than 490,000 and AMG 181 concentrations were predicted to only briefly cover the free α 4 ß 7 receptor EC10. Predictions for both C max and AUC matched with those observed in the first-in-human study within the 7 mg subcutaneous to 420 mg intravenous dose range. The developed model aided in selection of a safe starting dose and a pharmacological relevant dose escalation strategy for testing of AMG 181 in humans. The clinically observed human AMG 181 PK data validated the modeling approach based on cynomolgus monkey data alone.

In vitro evaluation of the effects of candidate immunosuppressive drugs: flow cytometry and quantitative real-time PCR as two independent and correlated read-outs.

BACKGROUND: Immune monitoring may use flow cytometry or molecular biology techniques. Flow cytometry assays cells that are phenotypically characterized, whereas TaqMan RT-PCR starts with RNA extraction from unfractionated heterogeneous cell populations. We therefore wondered how the effects of immunosuppressive drugs on cytokine production in stimulated whole blood, as determined by flow cytometry, would correlate with those obtained with quantitative real-time PCR (TaqMan RT-PCR). METHODS: Blood drawn from naive cynomolgus monkeys was exposed to incremental amounts of cyclosporine (CsA; 300, 600, 900 and 1200 ng/ml) or tacrolimus (TRL; 8, 20, 40 and 80 ng/ml) before lectin stimulation in vitro. Blood was then either stained for CD3, IFN-gamma, IL-2, IL-4, and TNF-alpha and analyzed on a flow cytometer with various gating strategies, or submitted to RNA extraction for analysis of the above mentioned cytokines mRNA transcripts using TaqMan RT-PCR. RESULTS: Both methods revealed a parallel dose-dependent inhibition of cytokine production in stimulated blood. The 50% inhibitory concentrations (IC(50)'s) ranged from 511-771 ng/ml (CsA) and 15-29 ng/ml (TRL) with flow cytometry, and from 275-529 ng/ml (CsA) and 11-48 ng/ml (TRL) with TaqMan RT-PCR for T-helper 1 cytokines. Both assays correlated well with a Pearson product moment correlation of 0.76. Extending gating from a CD3(+) gate to a lymphocyte gate improved correlation (r = 0.85) for all cytokines investigated (except IL-2; unchanged) whereas further extending gating resulted, to the contrary, in lower correlations. Independent of gating strategy a high correlation (r = 0.97) was observed when drug IC(50)'s were considered. CONCLUSIONS: Flow cytometry and TaqMan RT-PCR may be used interchangeably to monitor the effects of candidate immunosuppressive drugs on cytokine mRNA production in lectin-stimulated whole blood.

Compared with cyclosporine, ISATX247 significantly prolongs renal-allograft survival in a nonhuman primate model.

BACKGROUND: ISATX247 is a novel calcineurin inhibitor that has shown more potency than cyclosporine in vitro. This is the first study to compare the survival times of renal allografts in nonhuman primates treated with either ISATX247 or cyclosporine. METHODS: Adult, male cynomolgus monkeys were divided into blood-group compatible and mixed-lymphocyte, stimulation-mismatched, donor-recipient pairs. Heterotopic renal transplantation and bilateral native nephrectomies were performed. The monkeys were placed into either an ISATX247 or cyclosporine treatment group. Both groups were dosed twice daily to maintain a 12-hour drug-trough level of 150 ng/mL. Whole-blood concentrations of ISATX247 and cyclosporine, complete blood counts, and serum chemistry profiles were performed three times a week. Euthanasia was performed if the serum creatinine concentration became 7 or more mg/dL or a serious complication developed. RESULTS: The group receiving ISATX247 (n=8) survived significantly (P=0.0036) longer than the group receiving cyclosporine (n=7). The mean trough blood concentration of ISATX247 was 120 +/- 32 ng/mL and cyclosporine was 189 +/- 130 ng/mL. The average area under the curve 0-12 for ISATX247 was 6045 +/- 1679 ng/mL/hr and for cyclosporine was 4919 +/- 823 ng/mL/hr. The average calcineurin inhibition at trough blood concentrations was 80 +/- 11% for ISATX247 and 48 +/- 12% for cyclosporine. CONCLUSIONS: Allografts in monkeys treated with ISATX247 survived significantly longer than those treated with cyclosporine. On the basis of survival times and degree of calcineurin inhibition, ISATX247 is a more potent immunosuppressive agent than cyclosporine in this nonhuman primate model of renal-allograft transplantation.

JAK3 inhibition as a new concept for immune suppression.

Although current immunosuppressive drugs are effective, they have numerous severe side effects that mandate the search for new agents. Mutations in the gene for janus kinase (JAK)3 result in severe combined immune deficiency with severely impaired humoral and cellular immunity, an observation that has prompted the development of JAK3 inhibitors. Due to its central role in lymphocyte activation, proliferation and homeostasis, targeting the JAK/signal transducer and activator of transcription (STAT) pathway may provide the required efficacy, without the toxicities associated with current therapies. Several studies conducted in rodents have validated the proof-of-concept, with a variety of JAK3 inhibitors demonstrating efficacy for immune suppression. In addition, the selective JAK3 inhibitor CP-690550 (Pfizer Inc) significantly improved allograft survival in a stringent preclinical model in primates and exhibited a good safety profile in non-human primates. This, along with studies of protein kinase inhibitors for cancer treatment, could demonstrate that development of effective, safe and selective kinase inhibitors for immunosuppression is possible.

Outbreak of Mycobacterium bovis in a conditioned colony of rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques.

We describe a tuberculosis outbreak caused by Mycobacterium bovis in a conditioned colony of rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques. Animals in five rooms were exposed, but most (16/27) infections were confined to the room that housed a mixed population of cynomolgus and rhesus macaques. In this room, rhesus (8/8) and cynomolgus (10/11) macaques naturally exposed to M. bovis were infected at nearly identical rates (Fisher exact test, 2-tailed P = 1). The clinical signs of disease and pathologic lesions in infected macaques, however, were moderately different between the two species. Rhesus macaques were more likely (5/8) to exhibit clinical signs of persistent coughing and inappetance, and had more severe pulmonary lesions. By contrast, clinical signs of disease were seen in only 1 of 19 cynomolgus macaques, and overall, the pulmonary lesions were often focal and less severe, although some still had severe involvement of the lungs similar to that seen in rhesus macaques. These differences should be taken into consideration when developing or evaluating a tuberculosis-screening program. On the basis of observations made during this outbreak, we recommend that alternative screening methods such as the PRIMAGAM test and the ESAT-6 ELISA, be incorporated into the screening program to aid in the identification of infected animals.

Janus kinase 3 inhibition with CP-690,550 prevents allograft vasculopathy.

Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the common gamma chain. The rationally designed inhibitor of JAK3, CP-690,550, prevents acute allograft rejection in rodents and in nonhuman primates. Here we investigated the ability of CP-690,550, to prevent allograft vasculopathy in a rodent model of aorta transplantation. Aortas from AxC Irish (RT1(a)) or Lewis (RT1(l)) rats were heterotopically transplanted into the infra-renal aorta of Lewis recipients and harvested at 28 or 56 days. Treated recipients received CP-690,550 by osmotic pumps (mean drug exposure of 110 +/- 38 ng/ml). Significant intimal hyperplasia was demonstrated in untreated allografts when compared with isografts at 28 days (2.08 +/- 0.85% vs. 0.43 +/- 0.2% luminal obliteration, respectively, P = 0.001) and 56 days (5.3 +/- 2.4% vs. 0.38 +/- 0.3%, P = 0.002). Treatment caused a 51% reduction in intimal hyperplasia at day 56. CP-690,550-treated animals also had a significant reduction of donor-specific IgG production and of the gene expression for suppressor of cytokine signaling-3 and with unchanged levels of expression of RANTES, IP-10 and transforming growth factor-beta1. These results are the first to show that JAK3 blockade by CP-690,550 effectively prevents allograft vasculopathy in this rat model of aorta transplantation.

Inhibition of lymphocyte activation and function by the prenylation inhibitor L-778,123.

Prenylated Ras GTPases transduce signals from the T cell receptor, CD28 costimulatory receptor and IL-2 receptor. Since signals from these receptors mediate T cell activation, proliferation and survival, we hypothesized that the prenylation inhibitor L-778,123 would impart immunomodulation. The effect of L-778,123 on T cell activation (CD71 or CD25 surface expression) was determined by flow cytometry. Peripheral blood mononuclear cell (PBMC) proliferation in the presence of L-778,123 and/or cyclosporine (CsA) was determined by [3H]thymidine incorporation. The ability of L-778,123 to inhibit IL-2 receptor signaling was investigated by measuring IL-2 induced proliferation in CTLL-2 cells and IL-2 prevention of apoptosis in activated human PBMC. L-778,123 inhibited lectin induced expression of CD71 and CD25 with IC50's of 6.48 +/- 1.31 microM and 84.1 +/- 50.0 microM, respectively. PBMC proliferation was inhibited by L-778,123 with an IC50 of 0.92 +/- 0.23 microM, and addition of CsA did not increase the potency. L-778,123 did not inhibit IL-2 and IFN-gamma production by T cells. L-778,123 abrogated IL-2 induced proliferation of CTLL-2 cells with an IC50 of 0.81 +/- 0.44 microM. However, L-778,123 minimally reversed the prosurvival effect of IL-2 in activated lymphocytes. IL-2 ligand and receptor production during T cell activation are relatively unaffected by L-778,123. However, the activation and proliferative effects of IL-2 on T cells are potently blocked by L-778,123. These results reveal a selective blockade of the IL-2 cytokine axis distal to the IL-2 receptor by the L-778,123 and warrant evaluation of prenylation inhibitors in treating transplant rejection and autoimmune diseases.

Quantitative analysis of the immunosuppressant CP-690,550 in whole blood by column-switching high-performance liquid chromatography and mass spectrometry detection.

A fast and accurate method to quantify the new immunosuppressive JAK3 inhibitor CP-690,550 in whole blood using a dual-pump liquid chromatography-liquid chromatography-mass spectrometry (LC/LC-MS) system was developed and validated in nonhuman primate blood. Before injection, blood samples were prepared by precipitation with a reagent that included methanol and acetonitrile (30:70, vol/vol) along with the internal standard (CP-istd). Column-switching LC/LC-MS analysis used online extraction followed by separation on a C8 analytic column and MS detection of the [M + H] CP-690,550 (m/z = 313.1) and CP internal standard (m/z = 288.1). Linearity was always better than r = 0.99 (n = 7) for CP-690,550 (range 2.5-750 ng/mL), with a lower limit of quantification (LLOQ) of 2.5 ng/mL. The intrarun accuracy and precision ranged from 103.0% to 105.4% and 2.7% to 4.3%, respectively (n = 5), and the interday precision ranged from 8.7% to 11.1%, and the interday accuracy ranged from 98.1% to 103.8% of nominal values (n = 14). The injection repeatability for the method was 1.3% (n = 7). Except for the LLOQ, the intraday accuracy and precision in human blood were also within 15% (n = 5). The combination of simple sample preparation and short analytic run time of this sensitive procedure makes it effective for monitoring the concentration of CP-690,550 in whole blood in organ-transplant recipients.

Immunosuppressive effects of surgery assessed by flow cytometry in nonhuman primates after nephrectomy.

Despite previous studies suggesting that surgery cause immune suppression, the underlying biologic mechanisms have not been studied using advanced immune function assays. Unilateral nephrectomy was performed in nonhuman primates. Blood was collected before surgery and at different time-points through 14 days after surgery. Lymphocyte proliferation (expression of proliferating cell nuclear antigen in cells in S/G(2)M-phase), production of intracellular cytokines [interleukin (IL)-2, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha] and expression of surface-activation antigens (CD25, CD71) on T-lymphocytes were assessed in whole blood using flow cytometry. Results were compared with nonoperated control animals. The procedure caused a decrease of 25% in absolute lymphocyte count on postoperative day 3. Inhibition of lymphocyte proliferation was maximal on postoperative day 1 (55% normalized to preoperative values) and was detectable until postoperative day 7, when it was 25%. Expression of T-cell activation antigens was decreased during the first postoperative week with a maximum on postoperative day 1 for CD71 (29%) and on postoperative day 3 for CD25 (49%). Intracellular production of cytokines by T cells was decreased only on postoperative day 1 (50% for IL-2, 29% for IFN-gamma and 22% for TNF-alpha). Immune functions returned to presurgery values by day 14. A major surgical procedure severely inhibits lymphocyte proliferation and various T-cell functions up to 1 week postoperatively.

A technique of bone marrow collection from vertebral bodies of cynomolgus macaques for transplant studies.

BACKGROUND: Strategies to induce donor-specific allograft tolerance are best tested in preclinical models developed in nonhuman primates (NHPs). Most protocols prepare the recipient by infusing hematopoietic cells from the donor. We report here a procedure to isolate and characterize large numbers of bone marrow cells (BMCs) from cynomolgus monkeys (cynos) that can then successfully be transplanted into conditioned recipients. MATERIALS AND METHODS: Vertebral columns of five cynos were excised en bloc and separated into individual vertebrae. The cancelous bone was extracted with a core puncher, fractionated, filtered, centrifuged, and resuspended in transplantation media before being analyzed by flow cytometry. In two instances, the collected BMCs were reinfused into allogeneic recipients preconditioned with a nonmyeloablative regimen. Chimerism was monitored using short-tandem repeat analysis. RESULTS: The mean total BMCs yield was 25.5 x 10(9) (range of 4.00 x 10(9) to 59 x 10(9)) with mean cell viability of 93.4% (range: 90-96%). CD34+ cells and CD3+ cells averaged 0.34 and 3.91% of total BMCs, respectively. This resulted in absolute cell number yields of 1.02 x 10(8) and 1.15 x 10(9) for CD34+ and CD3+ cells, respectively. Graft-versus-host disease was absent in both bone marrow infused animals, and a maximum level of chimerism of 18% was detected at 3 weeks after BMCs infusion. CONCLUSION: We present here the first detailed report of a procedure to retrieve and characterize large numbers of BMCs from vertebral bodies of cynos and demonstrate that cells collected with this technique have the capability of engrafting in allogenic recipients.