Studies In Cynomolgus Monkeys Research Articles

Characterization and Optimization of AMG 517 Supersaturatable Self‐Emulsifying Drug Delivery System (S‐SEDDS) for Improved Oral Absorption

Supersaturatable self‐emulsifying drug delivery systems (S‐SEDDS) were explored to improve the oral absorption of AMG 517, a poorly water‐soluble drug candidate. In vitro characterizations indicate the level of Tween 80 in the formulation dictates the initial degree of supersaturation of AMG 517, and, therefore, its precipitation kinetics. The presence of a small amount of cellulosic polymer (e.g., HPMC) effectively sustained a metastable supersaturated state by retarding precipitation kinetics. Precipitates from the S‐SEDDS formulations (with HPMC) from in vitro test media were identified as amorphous AMG 517 while crystalline AMG 517 precipitates were found when either HPMC was absent or PVP was present in the formulation. In vivo pharmacokinetic study in Cynomolgus monkeys reveals that the S‐SEDDS formulation showed ∼30% higher mean Cmax and comparable exposure (AUC) of AMG 517 as compared to an aqueous suspension at a dose of 12.5 mg. The rapid absorption characteristics of AMG 517 from the S‐SEDDS formulation as evidenced by high Cmax and short Tmax are attributed to a high free drug concentration in vivo, implying a supersaturated state. This case demonstrates that S‐SEDDS technology is an effective approach for improving the rate and extent of absorption of poorly soluble drugs. © 2008 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:516–528, 2009

Preclinical Support for Combination Therapy in the Treatment of Autoimmunity with Atacicept

Atacicept, a fully human recombinant fusion protein that blocks the activity of BLyS (B-Lymphocyte Stimulator) and APRIL (a proliferation-inducing ligand), is undergoing clinical evaluation in B-cell-mediated diseases, including autoimmune disorders. Nonclinical studies in mice and cynomolgus monkeys demonstrate dose-dependent, reversible decreases in circulating Ig concentrations and reductions in mature B cells in the peripheral blood and lymphoid tissues. However, the combination of atacicept with purine synthesis inhibitors (e.g., mycophenolate mofetil [MMF]) and anti-B-cell monoclonal therapy (e.g., rituximab) has not been evaluated. Atacicept does not augment hematological toxicities associated with MMF, including anemia or thrombocytopenia. Combination of atacicept with MMF or rituximab reduced B cells in the periphery (MMF) or tissues (MMF and rituximab) further than did monotherapy, as was the case with atacicept-MMF combination therapy and serum Ig concentrations. Overall, atacicept appears to augment the pharmacologic activity toward B cells of current immunosuppressive therapies without increasing the hematological toxicities associated with MMF. Enhanced reduction in B cells and Ig concentrations associated with atacicept combination therapy may increase therapeutic activity or allow dose reduction in autoimmune patients. Findings from nonclinical safety studies support clinical evaluation of atacicept combination therapies.

Evaluation of the Pharmacokinetics, Immunogenicity, and Safety of XmAb®2513 in the Ongoing Study XmAb2513-01: A Phase 1 Study of Every Other Week XmAb2513 to Evaluate the Safety, Tolerability, and Pharmacokinetics in Patients with Hodgkin Lymphoma or Anaplastic Large Cell Lymphoma

XmAb2513 is a novel second-generation humanized monoclonal antibody (mAb) directed against the human cell surface antigen CD30, with an Fc region engineered to enhance cell killing activity via recruitment of effector cells through increased binding affinity to Fcγ receptors (FcγRs). The CD30 antigen (Ki-1) is a member of the tumor necrosis factor superfamily and was originally identified as a cell surface antigen on primary and cultured Reed-Sternberg cells of Hodgkin Lymphoma (HL). Expression of CD30 is a hallmark for the identification of HL and a subset of T-cell lymphomas, including Anaplastic Large Cell Lymphoma (ALCL). Preclinical in vitro data demonstrated XmAb2513 to be more potent with regards to antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) than the first generation anti- CD30 antibodies MDX-060 and SGN-30. Studies in cynomolgus monkeys demonstrated that XmAb2513 exposure was proportional to dose with half-lives (t½) ranging from 5 to 9 days in single dose studies and 12 to 17 days in repeat dose studies. The exposure and t½ data from these cynomolgus monkey studies supported an every other week dosing interval in clinical trials. Based on these favorable features, a Phase 1 study has been initiated to examine the safety and efficacy of XmAb2513 in patients with relapsed and refractory classical HL and ALCL. This ongoing Phase 1 Study is currently evaluating every other week dosing of XmAb2513 using a dose-escalation study design to establish the maximum tolerated dose (MTD). The dose levels under evaluation are 0.3, 1.0, 3.0, 6.0, 9.0, and 12.0 mg/kg, given intravenously over two hours. To date, seven patients are enrolled on the study in the first three dose levels. Pre-study simulation of human exposure based on the pharmacokinetic (PK) parameters obtained from cynomolgus monkeys predicted that 0.3 mg/kg XmAb2513 in humans would result in serum trough levels of 1–1.5 μg/mL after four infusions given every other week. Interim PK and immunogenicity results from the treated patients have been analysed and the results observed thus far are on track with these simulations. Additionally, immunogenicity assessment for human anti-XmAb2513 (HAHA) showed that the treated patients are so far negative for HAHA. Importantly XmAb2513 was well tolerated. As dose escalation continues, results for human PK, immunogenicity, and safety for additional dose cohorts of HL patients, and preliminary safety and response assessment data will be presented.

Histologic Processing of the Eye for Obtaining the Macular Region of the Retina of the Cynomolgus Monkey

Microscopic evaluation of the macular region of the retina is essential in preclinical drug safety studies in cynomolgus monkeys. A number of therapeutic agents are suspected of causing damage to the macula in humans; therefore, the macular region of the retina should be evaluated in preclinical drug safety studies in monkeys. Histopathologic examination of the fovea may be required for preclinical drug safety evaluation of certain ocular drugs for human risk assessment. Using the inferior oblique muscle as a landmark, the macula including the fovea was obtained in 100% of 20 eye sections. It is easier to obtain just the macular region of the retina than the fovea in the section. Measurements for the inferior oblique muscle (point of insertion), fovea, macula, and the optic disc for 20 eyes of untreated cynomolgus monkeys were taken. In the cynomolgus monkey, the macula was 2.01 mm in diameter and the fovea was 0.53 mm in diameter. The distance from the center of the optic disc to the center of the fovea was 3.23 mm. In the fixed eyes, the point of insertion of the inferior oblique muscle to the sclera was 5.96 mm. (The J Histotechnol 31:113, 2008)Submitted January 7, 2008; accepted with revisions June 22, 2008

Hepatic irradiation augments engraftment of donor cells following hepatocyte transplantation

Engraftment of donor hepatocytes is a critical step that determines the success of hepatocyte transplantation. Rapid and efficient integration of donor cells would enable prompt liver repopulation of these cells in response to selective proliferative stimuli offered by a preparative regimen. We have earlier demonstrated that hepatic irradiation (HIR) in combination with a variety of hepatotrophic growth signals, such as partial hepatectomy and hepatocyte growth factor, can be used as a preparative regimen for liver repopulation of transplanted hepatocytes. In this study, we investigated the effects of HIR on engraftment of transplanted dipeptidyl peptidase IV (DPPIV)-positive hepatocytes in congeneic DPPIV-deficient rats. HIR-induced apoptosis of hepatic sinusoidal endothelial cells (SEC) within 6 hours of HIR resulted in dehiscence of the SEC lining in 24 hours. Although there was no change of the number of Kupffer cells after HIR, colloidal carbon clearance decreased 24 hours post HIR, indicating a suppression of phagocytic function. DPPIV+ donor cells were transplanted 24 hours after HIR (0-50 Gy). There was an HIR dose-dependent increase in the donor hepatocyte mass engrafted in the liver parenchyma. The number of viable transplanted hepatocytes present in hepatic sinusoids or integrated in the parenchyma was greater in the HIR-treated group at 3 and 7 days after transplantation compared with the sham controls. Finally, we validated these rodent studies in cynomolgus monkeys, demonstrating that a single 10-Gy dose of HIR was sufficient to enhance engraftment of donor porcine hepatocytes. These data indicate that transient disruption of the SEC barrier and inhibition of the phagocytic function of Kupffer cells by HIR enhances hepatocyte engraftment and the integrated donor cell mass. Thus, preparative HIR could be potentially useful to augment hepatocyte transplantation.

Quantitative determination of humanized monoclonal antibody rhuMAb2H7 in cynomolgus monkey serum using a Generic Immunoglobulin Pharmacokinetic (GRIP) assay

Preclinical pharmacokinetic (PK) assays are important to help evaluate the safety and efficacy of a potential biotherapeutic before clinical studies. The assay typically requires a biotherapeutic-specific reagent to minimize matrix effects especially when the host species are non-human primates such as cynomolgus monkeys and the biotherapeutic is a humanized monoclonal antibody (MAb). Recombinant humanized mAb 2H7 (rhuMAb2H7) binds to the extracellular domain of CD20 that is expressed on B cells and results in B cell depletion. It is currently being evaluated for its therapeutic potential in rheumatoid arthritis (RA) in clinical studies. During the early development of rhuMAb2H7, a cynomolgus monkey PK assay was needed to help assess the pharmacokinetic parameters of rhuMAb2H7 in a pilot cynomolgus monkey study. However, development of a cynomolgus monkey PK assay was challenging due to lack of rhuMAb2H7-specific reagents. Here we describe an alternative method for detection of rhuMAb2H7 in cynomolgus monkey serum using polyclonal antibodies against human IgGs. This assay quantifies rhuMAb2H7 in 10% cynomolgus monkey serum with high sensitivity, accuracy, and precision. This assay successfully supported the rhuMAb2H7 development, and has the potential to be used to quantify other humanized MAb biotherapeutics in serum from a variety of non-human species.

Toxicological Protein Biomarker Analysis—An Investigative One-week Single Dose Intravenous Infusion Toxicity and Toxicokinetic Study in Cynomolgus Monkeys using an Antibody–cytotoxic Conjugate against Ovarian Cancer

Antibody-cytotoxic conjugates are complex novel therapeutic agents whose toxicological properties are not presently well understood. The objective of this study was to identify toxicological markers in serum that correlate with MLN8866 (an antibody-cytotoxic conjugate) exposure and related pathological events in monkeys. Cynomolgus monkeys were treated once with 5, 15, or 30 mg/kg MLN8866 via a 20 min intravenous infusion. MLN8866 exposure (Cmax and AUCO-4 day) was determined by quantifying MLN8866 levels in serum. The increase in MLN8866 exposure was approximately dose proportional. Two acute phase proteins in serum (serum amyloid A and haptoglobin) were correlated with MLN8866 exposure and toxicological outcomes (e.g., erythropoiesis and leucopoiesis).

Antibody-Drug Conjugates with Uncleavable Linkers for the Treatment of Non-Hodgkin Lymphoma.

Antibody-drug conjugates (ADCs), potent cytotoxic drugs linked to antibodies via specialized chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Our research has focused on ADCs that contain the maytansinoid DM1, monomethylauristatin E (MMAE), or monomethylauristatin F (MMAF), all of which are potent inhibitors of microtubule polymerization. These drugs are attached to the antibody via several different linker formats (Table 1).Table 1Linker-drugLinker MechanismLinked viaDrug Cell PermeablePAB-vc-MC-MMAECleavage of peptide bondCysteineYesMC-MMAFUncleavableCysteineNoSPP-DM1Reduction of disulfide bondLysineYesMCC-DM1UncleavableLysineYesWe identified seven cell surface proteins expressed in non-Hodgkin lymphoma (NHL) with limited expression patterns in normal tissue (CD19, CD20, CD21, CD22, CD72, CD79, and CD180), as potential therapeutic targets for ADCs. ADCs directed to any of these seven targets are effective in xenograft models when containing linkers that can be cleaved in the tumor microenvironment or when internalized by tumor cells. However, ADCs with uncleavable linkers, i.e., linkers that are active when antibody is internalized and degraded within cells, are effective only when targeted to CD22 or CD79b. This suggests that these antigens provide the desired intracellular targeting of the cytotoxic drug. Further, we demonstrated that the uncleavable-linker anti-CD22 and anti-CD79b ADCs result in complete tumor regression with no recurrence in some xenograft models of NHL. In vitro experiments revealed that sensitivity of a given cell line to the ADCs anti-CD22-MCC-DM1 and anti-CD79b-MCC-DM1, correlated more with sensitivity of the cell line to the corresponding free drug, than to the amount of surface expression of the target or the amount of internalized ADC. We conducted pilot safety studies in rats (non-binding species) and cynomolgus monkeys (binding species). In rats, ADCs with uncleavable linkers caused less target-independent hepatic and hematologic toxicity than their counter parts with cleavable linkers, likely due to their decreased systemic release of free drug. Because of these results, we evaluated three ADCs with uncleavable linkers (anti-CD22-MCC-DM1, anti-cynomolgus monkey CD79b-MCC-DM1, and anti-CD22-MC-MMAF) at ∼30 mg/kg q3 weeks for 2 doses in cynomolgus monkeys. Normal B-cells were depleted in circulation and in tissue, particularly in germinal centers of lymphoid tissues; an expected pharmacological effect. Other findings included clinically tolerated and reversible elevations in liver enzymes (MCC-DM1 and MC-MMAF conjugates) and decreases in platelet counts (MCC-DM1 conjugates); and minimal to mild sciatic nerve degeneration (MCC-DM1 conjugates). The data demonstrate that anti-CD22 and anti-CD79b ADCs with uncleavable linkers have potent efficacy in xenograft models of NHL, favorable safety profiles in pilot animal studies, and suggest that for specific targets, uncleavable linkers provide a promising mechanism to improve margins of safety in humans.

IL-13 is a novel therapeutic target in allergic asthma

Evaluation of: Bree A, Schlerman FJ, Wadanoli M et al. IL-13 blockade reduces lung inflammation after Ascaris suum challenge in cynomolgus monkeys. J. Allergy Clin. Immunol. 119, 1251–1257 (2007).There is abundant evidence supporting IL-13 as having a central role in the pathology of allergic asthma. IL-13 induces immunological responses relevant to allergic asthma and exerts effects on airway structural cells, leading to remodeling and enhanced contractility of airway smooth muscle. This paper evaluates the results from a recent preclinical study in cynomolgus monkeys, showing that treatment with an antibody to IL-13 significantly attenuates airway inflammation induced by segmental allergen challenge. Efficacy of anti-IL-13 in proof-of-concept human studies will be critical to evaluate the potential of this therapy for treatment of allergic asthma.

Selective clearance of glycoforms of a complex glycoprotein pharmaceutical caused by terminal N-acetylglucosamine is similar in humans and cynomolgus monkeys

To understand how the carbohydrate moieties of a recombinant glycoprotein affected its pharmacokinetic (PK) properties, the glycan distribution was directly assessed from serial blood samples taken during PK studies in cynomolgus monkeys and humans. The protein studied was an immunoadhesin (lenercept), containing an Fc domain from human immunoglobulin G (IgG-1) and two copies of the extensively glycosylated extra cellular domain of tumor necrosis factor receptor p55. The protein was recovered in pure form using a dual column, immunoaffinity-reversed-phase high-performance liquid chromatography method. The glycans were released and analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Alternatively, trypsin was used to obtain glycopeptides, and these were analyzed by MALDI-TOF. The composition versus time profiles show that the distribution of glycans in the Fc domain was not altered over 10 days of circulation, consistent with their sequestration in the interior of the protein. However, the glycan composition in the receptor domain was changed dramatically in the first 24 h and then remained relatively constant. Analysis of the acidic glycans (derived exclusively from the receptor domain) showed that, in the rapid initial phase of clearance, glycans carrying terminal N-acetylglucosamine (tGlcNAc) were selectively cleared from the circulation. This phenomenon occurred similarly in humans and cynomolgus monkeys. Sialic acid content and terminal galactose showed only small changes. These data confirm the correlation of tGlcNAc and half-life of the molecule, and support the hypothesis that the mannose receptor (which can also bind tGlcNAc) causes the variable clearance of this molecule.

Tandem optimization of target activity and elimination of mutagenic potential in a potent series of N-aryl bicyclic hydantoin-based selective androgen receptor modulators

Pharmacokinetic studies in cynomolgus monkeys with a novel prototype selective androgen receptor modulator revealed trace amounts of an aniline fragment released through hydrolytic metabolism. This aniline fragment was determined to be mutagenic in an Ames assay. Subsequent concurrent optimization for target activity and avoidance of mutagenicity led to the identification of a pharmacologically superior clinical candidate without mutagenic potential.

Candidate HIV-1 Tat vaccine development: from basic science to clinical trials

Over the past 20 years most of the efforts in HIV vaccinedevelopment have focused on sterilizing immunity bytargeting the Envelope protein (Env). However, resultsfrom preclinical and clinical trials have been largelydisappointing [1–11]. Therefore, current vaccine strat-egies are not only aimed at preventing virus infection butalso at blocking virus replication and disease onset. Inparticular, the control of virus replication should provideprotection from disease development and reduce virustransmission, halting the HIV epidemic. This objectivemay be achieved by targeting virus regulatory genes,which are expressed early after infection, are essential forvirus replication and pathogenesis, and are moreconserved among HIV clades. This approach may beeffective for both preventive and therapeutic vaccinestrategies [12–68]. In this article we review thecharacteristics of Tat and why it was selected for use in avaccine.Wealsocitethelessonlearnedinthedevelopmentof this anti-Tat vaccine for use in human clinical trials.

In vitro characterization and pre-clinical pharmacokinetics of CP-870,893, a human anti-CD40 agonist antibody

2539 Background: CD40 is expressed on B-cells, monocytes, dendritic cells, other normal tissues and tumors. Previous studies showed that CD40 stimulation enhances antigen presentation, breaks tolerance, bypasses T-cell help, and induces apoptosis in CD40pos tumor cells. We report the in vitro activity and primate pharmacokinetics of a human anti-CD40 agonist antibody, CP-870,893, currently in clinical trials for the treatment of cancer. Methods: CP-870,893 was identified as a CD40 agonist antibody by screening lead molecules generated through the Abgenix Xenomouse® platform. Agonist activity was determined using upregulation of B-cell and monocytes derived dendritic cell surface markers, as well as dendritic cell IL-12 induction. BIAcore and equilibrium binding were utilized to determine affinity, and competition studies with CD40L were conducted on BIAcore. CP-870,893 was administered to cynomolgus monkeys i.v. at various doses, serum antibody levels were evaluated over time in an ELISA assay, and B-cell markers were monitored by FACS. Results: CP-870,893 (IgG2, kappa) binds CD40 with sub-nanomolar affinity, and does not block binding of CD40L. When human whole blood is incubated with CP-870,893, upregulation of key surface molecules involved in antigen presentation (MHC Class II, CD80, CD86, CD23 and ICAM-1) is observed with an EC50 of 5–50 ng/ml. Human monocytes derived dendritic cells, when stimulated with CP-870,893, upregulate activation markers (MHC Class II, CD80 and CD83) with an EC50 of 100–300 ng/ml, and secrete high levels of IL-12p40. In the presence of a second stimulus, such as LPS, human dendritic cells also secreted bioactive IL12-p70 when stimulated with CP-870,893 (EC50 ∼ 150 ng/ml). In addition, a CD40 positive human B-cell tumor line, when stimulated with CP-870,893, becomes susceptible to killing by human CTLs. In cynomolgus monkey studies, the clearance of CP-870,893 decreased with increasing dose. Circulating B-cell numbers decreased, and surface molecules were upregulated on B-cells. Conclusions: These data support the potential utility of CP-870,893 as an immune enhancing agent in cancer immunotherapy, by activating antigen presenting cells, and by enhancing the immunogenicity of CD40 positive tumor cells. [Table: see text]

IL-21 Improves Rituximab-Mediated B Cell Depletion.

IL-21 is a cytokine produced by activated CD4+ T cells that augments the growth, survival, and function of NK, T and phagocytic cells. IL-21 also plays an important role in regulating B cell proliferation and differentiation. In a previous study of cynomolgus monkeys treated with recombinant human IL-21, we demonstrated increased numbers of NK cells and phagocytic cells expressing Fcγ RI and III, enhancement of ex vivo antibody dependent cellular cytotoxicity (ADCC) activity, altered lymphocyte trafficking, and improved B cell depletion with subclinical doses of rituximab. The current study aimed to extend these findings to a clinically relevant rituximab dose regimen, and to evaluate the safety and tolerability of IL-21 administration in this setting. Three groups of cynomolgus monkeys (n=8) were treated by IV injection once weekly for four weeks with rituximab (10 mg/kg), concurrently with vehicle or IL-21 (0.3 or 1.5 mg/kg). Standard toxicology data were collected. The number and activation state of peripheral blood leukocyte subsets were determined by flow cytometry. Effects on B cells within spleen and lymph nodes were evaluated by immunohistochemistry. Co-administration of rituximab and IL-21 was well tolerated at both dose levels of IL-21 tested. The primary toxicological responses to the combination treatment were moderate anemia and thrombocytopenia, which were IL-21 dose dependent and similar in magnitude to those observed in previous IL-21 toxicology studies. In animals treated with rituximab alone, circulating B cells were depleted to a nadir 0.3–3 % of baseline on Day 11 of the study (Figure 1). In contrast, significantly lower B cell nadirs were observed following the first treatment with rituximab and IL-21 combination therapy (p< 0.01, ANOVA). In animals receiving the 1.5 mg/kg dose of IL-21, circulating B cells were depleted to a nadir 0 −1 % of baseline on Day 4, followed by partial recovery and further depletion to 0.3 – 2% of baseline on Day 11. More importantly, B cell recovery was significantly delayed in the group treated with rituximab and 1.5 mg/kg IL-21, compared to rituximab treatment alone (p< 0.05, ANOVA/PLSD). Thirty days following the fourth and final treatment, B cell numbers had recovered to only 6% of baseline, compared to 22% in the rituximab monotherapy treated group. Additionally, coadministration of IL-21 with rituximab increased circulating activated monocytes, granulocytes and NK cells compared with rituximab monotherapy. In conclusion, addition of IL-21 to a standard weekly, 4-dose regimen of IV rituximab was well-tolerated in cynomolgus monkeys and resulted in more complete, rapid and durable depletion of circulating CD20-expressing target cells. As depletion of B cells in this model serves as a surrogate for clearance of CD20-expressing malignant cells, these data support the evaluation of IL-21 and rituximab combination therapy in patients with advanced CD20 positive malignancies. [Display omitted]

Population pharmacokinetics of recombinant factor XIII in cynomolgus monkeys

Hemostasis in humans and other animals is a complex process that controls blood loss after a vascular injury. Factor XIII (FXIII) stabilizes clots primarily by cross-linking fibrin, thus protecting a newly formed clot from fibrinolysis by plasmin. Congenital deficiencies in humans involving FXIII are associated with delayed bleeding and wound healing and severe spontaneous hemorrhaging. These symptoms can be alleviated by intravenous administration of enriched FXIII plasma fractions. Circulating plasma FXIII is found as a heterotetramer that dissociates in the presence of calcium and thrombin into an active dimer and 2 inactive monomers. The recombinant FXIII under investigation is the active dimer alone. A 3-compartment, nonlinear population pharmacokinetic model was implemented in NONMEM V and then used to analyze data from preclinical studies in cynomolgus monkeys. The model simultaneously describes endogenous production of dimer (0.622 microg kg(-1) hr(-1)) and monomer (12.1 microg kg(-1) hr(-1)), and the administration of recombinant dimer. The model incorporates the rate and extent of complexation of recombinant dimer with available endogenous monomer (6.59 mg(-1) kg hr(-1)) to form the heterotetramer. Half-lives for dimer, heterotetramer, and monomer (3.33 hours, 2.83 days, and 3.94 hours for A(2), A(2)B(2), and B, respectively) were estimated, along with their variability in the population studied.

Tolerability of cyclosphosphamide and methotrexate induction immunosuppression in nonhuman primates

Transplantation in nonhuman primates, in particular using solid organs from porcine donors, requires an efficacious induction immunosuppression. Besides biologicals, the low molecular weight drugs used include cyclophosphamide (CyP) and methotrexate (MTX). As these compounds generally have a narrow therapeutic window, we performed tolerability studies in baboons and cynomolgus monkeys, with/without maintenance immunosuppressants such as cyclosporine A, everolimus, mycophenolate sodium and FTY720. In both species, a four-dose CyP regimen of 40, 20, 30 and 30 mg/kg i.v. on days 1, 2, 4 and 6 is not tolerated, but the regimen is tolerated upon individual adjustment of the third and fourth dose to 18–25 and 8–20 mg/kg, respectively, based on white blood cell count. In cynomolgus monkeys, a 5-day course of MTX i.v. at 0.5 mg/(kg d) is well tolerated, but not MTX at 1.0 mg/(kg d); in combination with maintenance immunosuppression, the 0.5 mg/(kg d) dose can cause adverse effects. Combinations of CyP and MTX are tolerated using the 5-day course of MTX at 0.25 mg/(kg d) and a four-dose regimen of CyP at 10, 2.5, 7.5 and 7.5 mg/kg. These regimens are tolerated in combination with maintenance immunosuppressants. The data provide base values for investigators using nonhuman primates in experimental studies, particularly in xenotransplantation requiring effective induction immunosuppression that is close to maximum tolerated dose levels.

Norethisterone enanthate has neither a direct effect on the testis nor on the epididymis: a study in adult male cynomolgus monkeys (Macaca fascicularis)

Norethisterone enanthate (NETE) is evaluated in trials of hormonal male contraception. It has been speculated that progestins may exert their contraceptive effects not only by suppressing gonadotropins but also by direct effects on male organs. NETE was given to monkeys in which endogenous gonadotropin secretion was suppressed by a gonadotropin releasing hormone (GnRH) antagonist, and replaced by human follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). If NETE has a direct effect on spermatogenesis and/or epididymal function, some changes in testicular histology, sperm motility and/or morphology should occur soon after exposure to NETE. Fifteen adult intact male monkeys were grouped and treated for a 38-day period. Group I received GnRH antagonist, FSH, hCG and NETE while group II received a regime identical to group I without NETE and group III received only NETE and vehicle. Ejaculates, body weight, testicular biopsies and volume, and hormones were evaluated. There was a similar pattern of serum FSH and testosterone in groups I and II. Testicular volume and the proportion of tubuli exhibiting spermatids was significantly decreased in group III. There were no significant differences between group I and group II in any parameters measured. The forward progression of sperm was not affected by NETE treatment. The consistently low percentages of grade c sperm indicated no sign of hyperactivation. No changes in the gross morphology of the acrosome were detected. Short-term NETE treatment has neither a direct effect on the testis nor on the epididymis in this nonhuman primate model and its contraceptive effects appear to be exerted exclusively through gonadotropin suppression.

Norethisterone enanthate has neither direct effect on the testis nor on the epididymis: A study in adult male cynomolgus monkeys (Macaca fascicularis)

Norethisterone enanthate (NETE) is currently evaluated in trials of hormonal male contraception. NETE acts suppressing gonadotropin secretion. In this study we analysed whether NETE has a direct effect on the testis and/or on the epididymis in adult monkeys. NETE was given to cynomolgous monkeys in which endogenous gonadotropin secretion was suppressed by a GnRH antagonist (Cetrorelix), and replaced by injecting recombinant human FSH and human chorionic gonadotropin (hCG). We postulated that, if NETE has a direct effect on spermatogenesis and/or epididymal function, some changes in testicular histology, sperm motility and/or morphology should be found to occur soon after exposure to NETE. Fifteen adult intact male monkeys were allocated to three groups and treated for a 38 day period. Group I received GnRH antagonist, recombinant FSH, hCG and NETE while group II received a regime identical to group I without NETE and group III received only NETE and vehicle. Ejaculates, body weight, testicular volume and hormones were evaluated. Testicular biopsies were taken before and after treatment. There was a similar pattern of serum FSH and testosterone in group I and II throughout the study period, whereas serum FSH and testosterone dropped below the limit of detection in group III. Testicular volume and the proportion of tubuli exhibiting elongated and round spermatids was significantly decreased in group III. No significant differences were found between group I and group II in any parameters measured. The forward progression of sperm was not affected by NETE treatment. The low percentages of grade c sperm indicated no sign of NETE-induced hyperactivation. No changes in the acrosome gros morphology were detected among the different treatment groups. These results suggest that short-term NETE treatment has neither direct effects on the testis nor on the epididymis in this non-human primate model.

Simultaneous Inhibition of Epidermal Growth Factor Receptor (EGFR) Signaling and Enhanced Activation of Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Receptor-mediated Apoptosis Induction by an scFv:sTRAIL Fusion Protein with Specificity for Human EGFR

Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific tyrosine kinase inhibitors has shown clinical efficacy in cancer by restoring susceptibility of tumor cells to therapeutic apoptosis induction. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent with tumor-selective apoptotic activity. Here we present a novel approach that combines EGFR-signaling inhibition with target cell-restricted apoptosis induction using a TRAIL fusion protein with engineered specificity for EGFR. This fusion protein, scFv425:sTRAIL, comprises the EGFR-blocking antibody fragment scFv425 genetically fused to soluble TRAIL (sTRAIL). Treatment with scFv425:sTRAIL resulted in the specific accretion to the cell surface of EGFR-positive cells only. EGFR-specific binding rapidly induced a dephosphorylation of EGFR and down-stream mitogenic signaling, which was accompanied by cFLIP(L) down-regulation and Bad dephosphorylation. EGFR-specific binding converted soluble scFv425:sTRAIL into a membrane-bound form of TRAIL that cross-linked agonistic TRAIL receptors in a paracrine manner, resulting in potent apoptosis induction in a series of EGFR-positive tumor cell lines. Co-treatment of EGFR-positive tumor cells with the EGFR-tyrosine kinase inhibitor Iressa resulted in a potent synergistic pro-apoptotic effect, caused by the specific down-regulation of c-FLIP. Furthermore, in mixed culture experiments binding (L)of scFv425:sTRAIL to EGFR-positive target cells conveyed a potent apoptotic effect toward EGFR-negative bystander tumor cells. The favorable characteristics of scFv425:sTRAIL, alone and in combination with Iressa, as well as its potent anti-tumor bystander activity indicate its potential value for treatment of EGFR-expressing cancers.

Preparation of highly specific radioactivity [ 18F]flumazenil and its evaluation in cynomolgus monkey by positron emission tomography

A straightforward method for the preparation of no-carrier-added (n.c.a.) [ 18F]flumazenil via standard nucleophilic radiofluorination of the corresponding nitro-analog Ro 15-2344 has been developed. The labeling was performed by employing the K 18F/kryptofix complex in DMF at 160°C for 30 min and equimolar ratio [K/K2.2.2] +18F −/precursor. Under these conditions, an 18F incorporation rate into flumazenil was in the range of 55–60%. The final product was isolated by HPLC purification within a total synthesis time of 75 min and a radiochemical yield of about 30% (EOB). Human post-mortem whole-hemisphere autoradiography of brain sections demonstrated selective uptake of the radioligand in the areas of high density of the central benzodiazepine receptors (BZR). PET studies in a cynomolgus monkey and metabolite studies by HPLC demonstrated similar results by [ 18F]flumazenil as for [ 11C]flumazenil. In blocking experiments, almost all radioactivity was inhibited by the addition of unlabeled flumazenil. [ 18F]Flumazenil is a suitable radioligand for PET assessment of the BZR.