Multicentre phase II pharmacokinetic and pharmacodynamic study of OSI-7904L in previously untreated patients with advanced gastric or gastroesophageal junction adenocarcinoma.

A two-stage Simon design was used to evaluate the response rate of OSI-7904L, a liposome encapsulated thymidylate synthase inhibitor, in advanced gastric and/or gastroesophageal adenocarcinoma (A-G/GEJA), administered intravenously at 12 mg m(-2) over 30 min every 21 days. Fifty patients were treated. Median age was 64 years (range 35-82), 62% were male and 89% had ECOG PS of 0/1. A total of 252 cycles were administered; median of 4 per patient (range 1-21). Twelve patients required dose reductions, mainly for skin toxicity. Investigator assessed response rate was 17.4% (95% CI 7.8-31.4) with one complete and seven partial responses in 46 evaluable patients. Twenty-one patients (42%) had stable disease. Median time to progression and survival were 12.4 and 36.9 weeks, respectively. NCI CTCAE Grade 3/4 neutropenia (14%) and thrombocytopenia (4%) were uncommon. The main G3/4 nonhaematological toxicities were skin-related 22%, stomatitis 14%, fatigue/lethargy 10%, and diarrhea 8%. Pharmacokinetic data showed high interpatient variability. Patients with higher AUC were more likely to experience G3/4 toxicity during cycle 1 while baseline homocysteine did not predict toxicity. Response did not correlate with AUC. Elevations in 2'-dU were observed indicating target inhibition. Analysis of TS genotype, TS protein and expression did not reveal any correlation with outcome. OSI-7904L has activity in A-G/GEJA similar to other active agents and an acceptable safety profile.

A phase I, first in man study of OSI-7836 in patients with advanced refractory solid tumors: IND.147, a study of the Investigational New Drug Program of the National Cancer Institute of Canada Clinical Trials Group.

PURPOSE: To determine the maximum tolerated dose (MTD), recommended phase II dose (RP2D), safety, tolerability, toxicity profile, dose-limiting toxicities (DLTs), anti-tumor activity and pharmacokinetics of OSI-7836 given IV on day 1 and day 8 every 3 weeks in patients with advanced incurable cancer. METHODS: Twenty-seven previously treated patients with advanced or metastatic solid tumors were enrolled in this phase I study conducted by the National Cancer Institute of Canada Clinical Trial Group (NCIC CTG). OSI-7836 was administered IV on day 1 and day 8 every 3 weeks. The dose was initially escalated from 100 to 600 mg/m2 and finally de-escalated to 200 mg/m2 in seven cohorts of patients. Patients were evaluated every other cycle of treatment for radiological response. Pharmacokinetics were performed on day 1 and day 8 of cycle 1 for all patients. RESULTS: Twenty-six patients were evaluable for toxicity. All patients experienced reversible Grade 3 lymphopenia beginning at cycle 1. The maximal delivered dose was 600 mg/m2. MTD was reached at 400 mg/m2. DLTs included fever, fatigue, rash, herpes simplex infection, nausea and vomiting. The RP2D was 200 mg/m2. No objective responses were seen in 21 evaluable patients. Pharmacokinetics were dose proportional, with a mean half-life of 46.0 min and a clearance of 34 l/(h.m2). CONCLUSION: OSI-7836 given at 200 mg/m2 on day 1 and day 8 every 3 weekly is associated with manageable toxicity and is recommended for further study. While no objective responses were seen, the significant treatment related lymphopenia suggests that hematologic malignancies may warrant further investigation.

Pharmacokinetics and safety of an anti-vascular endothelial growth factor aptamer (NX1838) following injection into the vitreous humor of rhesus monkeys.

PURPOSE: The objective of this study was to determine the pharmacokinetics and safety for NX1838 following injection into the vitreous humor of rhesus monkeys. METHODS: Plasma and vitreous humor pharmacokinetics were determined following a single bilateral 0.25, 0.50, 1.0, 1.5, or 2.0 mg/eye dose. In addition, the pharmacokinetics and toxicological properties of NX1838 were determined following six biweekly bilateral injections of 0.25 or 0.50 mg/eye or following four biweekly bilateral injections of 0.10 mg per eye followed by two biweekly bilateral injections of 1.0 mg per eye. RESULTS: Plasma and vitreous humor NX1838 concentrations were linearly related to the dose administered. NX1838 was cleared intact from the vitreous humor into the plasma with a half-life of approximately 94 h, which was in agreement with the plasma terminal half-life. Vascular endothelial growth factor (VEGF)-binding assays demonstrated that the NX1838 remaining in the vitreous humor after 28 days was fully active. No toxicological effects or antibody responses were evident. CONCLUSIONS: The no observable effect level was greater than six biweekly bilateral 0.50 mg/eye doses or two biweekly bilateral 1.0 mg/eye doses. These pharmacokinetic and safety data support monthly 1 or 2 mg/eye dose regimens in human clinical trials.

A high throughput platform for systematic evolution of ligands by exponential enrichment (SELEX).

The systematic evolution of ligands by exponential enrichment (SELEX) process is a combinatorial chemistry method for the isolation of nucleic acid ligands (aptamers) that bind to a desired target molecule with high affinity. In order to increase throughput via automation, we have adapted the SELEX process for protein targets to a robotics-compatible microtiter plate format. A remarkable feature of the platform is that targets are immobilized by hydrophobic adsorption onto the plate surface. Hydrophobic immobilization procedures are simple and require no specialized modification of the protein target. This format was tested by manually performing four independent SELEX experiments. All were concluded within 8 rounds of selection and yielded aptamers that bind in solution to their respective protein target, calf intestinal alkaline phosphatase, human alpha-thrombin or human platelet derived growth factor, with equilibrium dissociation constants below 3 x 10-10 M.

Detection and plasma pharmacokinetics of an anti-vascular endothelial growth factor oligonucleotide-aptamer (NX1838) in rhesus monkeys.

Aptamers are oligonucleotide ligands selected, in vitro, to bind a specified target protein. The first aptamer to reach human clinical testing is NX1838, a polyethylene glycol conjugated aptamer that inhibits vascular endothelial growth factor. This paper describes the validation of a high-performance liquid chromatographic anion-exchange method for the determination of NX1838 in plasma. Measurements of intact NX1838 had a coefficient of variation of less than 8% and an accuracy between 107% and 115%. The assay was utilized to determine NX1838 plasma pharmacokinetics in rhesus monkeys following a single 1 mg/kg intravenous or subcutaneous dose. Following intravenous administration, the maximum achieved plasma concentration was 25.5 microg/ml with a terminal half-life of 9.3 h and clearance rate of 6.2 ml/h. After subcutaneous administration, the fraction of the dose absorbed into the plasma compartment was 0.78 with a time to peak concentration (4.9 microg/ml) of 8 to 12 h.

Aptamer affinity chromatography: combinatorial chemistry applied to protein purification.

The systematic evolution of ligands by exponential enrichment process is a combinatorial chemistry method that allows the identification of specific oligonucleotide sequences, known as aptamers, that bind to a desired target molecule with high affinity and specificity. Here, a DNA-aptamer specific for human L-selectin was immobilized to a chromatography support to create an affinity column. This column was effectively applied as either the first or second step in the purification of a recombinant human L-selectin-Ig fusion protein from Chinese hamster ovary cell-conditioned medium. The fusion protein was efficiently bound to the column and efficiently eluted by gentle elution schemes. Application of the aptamer column as the initial purification step resulted in a 1500-fold purification with an 83% single step recovery. These results demonstrate that oligonucleotide aptamers can be effective affinity purification reagents.

Purification of a highly modified RNA-aptamer. Effect of complete denaturation during chromatography on product recovery and specific activity.

To evaluate RNA-aptamers as potential drug candidates, efficient and scaleable purification protocols are needed. Because aptamers are highly structured and rigid molecules, denaturation during the purification process is a critical aspect to obtain a pure and active product. A two-step chromatographic procedure was developed to purify a synthetic anti-VEGF aptamer at the preparative scale. A reversed-phase chromatographic step was optimized with a highly hydrophobic ion pairing reagent, followed by ion-exchange chromatography in which heat and a chaotropic salt were used. Because of the presence of 2'-modified ribose, denaturation conditions had to be optimized in both chromatographic steps to achieve a fully active molecule.

Expression patterns of the hepatic leukemia factor gene in the nervous system of developing and adult mice.

Hepatic leukemia factor (HLF) is a bZIP transcription factor related to the CES-2 protein, which controls apoptosis of the NSM serotoninergic neurons in Caenorhabditis elegans. Ectopic expression of HLF as an E2A-HLF fusion protein in t(17;19)-positive human pro-B cell acute lymphoblastic leukemias is believed to promote malignancy by interfering with apoptosis. While HLF has been linked to malignancies of the lymphoid system, it is not normally expressed in these cells. Rather, HLF transcripts are detected in the liver, kidney, lung and adult nervous system by Northern blotting. Despite the links to cell death, little is known of the distribution or function of HLF in the adult and developing mammalian nervous system. Therefore, we cloned mouse Hlf and studied its expression by in situ hybridization. During embryonic brain development, Hlf expression was restricted to the anterior pituitary and meninges. By early postnatal life, Hlf was highly expressed in somatosensory cortex, thalamic nuclei, and structures arising from ectodermal placodes. Subsequently, Hlf expression increased in the central nervous system and was found throughout the brain by adulthood. In the developing pituitary gland, Hlf was highly expressed in the rostral tip of the anterior lobe. This pattern is similar to that of Tef, an Hlf-related bZIP protein. However, while Tef is expressed in the anterior pituitary of the adult mouse, Hlf was detected in both the anterior and posterior pituitary. Hlf expression was not associated with cells undergoing programmed cell death in the nervous system. Hlf expression increased markedly with synaptogenesis and was coincident with barrel formation revealed by cytochrome oxidase staining. Together, these data suggest that Hlf plays a role in the function of differentiated neurons in the adult nervous system rather than programmed cell death.

Reduced fertility in mice deficient for the POU protein sperm-1.

Members of the POU-homeodomain gene family encode transcriptional regulatory molecules that play important roles in terminal differentiation of many organ systems. Sperm-1 (Sprm-1) is a POU domain factor that is exclusively expressed in the differentiating male germ cell. We show here that the Sprm-1 protein is expressed in the haploid spermatid and that 129/Sv Sprm-1(-/-) mice are subfertile when compared with wild-type or heterozygous littermates yet exhibit normal testicular morphology and produce normal numbers of mobile spermatozoa. Our data suggest that the Sprm-1 protein plays a discrete regulatory function in the haploid spermatid, which is required for the optimal function, but not the terminal differentiation, of the male germ cell.

An enzyme-linked oligonucleotide assay.

The recent development of in vitro methods to select high-affinity ligands by combinatorial chemistry methodologies promises unique and theoretically unlimited supplies of novel therapeutic and diagnostic reagents. One such combinatorial chemistry process, systematic evolution of ligands by exponential enrichment (SELEX), allows rapid identification, from large random sequence pools, of the few oligonucleotide sequences that bind to a desired target molecule with high affinity and specificity. We describe an enzyme-linked sandwich assay that uses a SELEX-derived oligonucleotide. This assay demonstrates that these oligonucleotides can be effective and useful analytical reagents.

Pituitary ontogeny of the Snell dwarf mouse reveals Pit-1-independent and Pit-1-dependent origins of the thyrotrope.

The anterior pituitary provides a model to study the molecular mechanisms responsible for emergence of distinct cell types within an organ. Dwarf mice (Snell) that express a mutant form of the tissue-specific POU-domain transcription factor Pit-1 fail to generate three cell types, including the thyrotrope (S. Li, E. B. Crenshaw, E. J. Rawson, D. S. Simmons, L. Swanson and M. G. Rosenfeld (1990), Nature 347, 528-533). Analyses of wild-type and Pit-1-defective mice, presented here, have revealed that thyrotropes unexpectedly arise from two independent cell populations. The first population is Pit-1-independent and appears on e12 in the rostral tip of the developing gland, but phenotypically disappears by the day of birth. The second is Pit-1-dependent and arises subsequently in the caudomedial portion of the developing gland (e15.5), following the initial expression of Pit-1 in this region. The failure of caudomedial thyrotrope cells to appear in the Snell dwarf, and the observation that Pit-1 can bind to and transactivate the TSH beta promoter, apparently enhanced by its phosphorylation, suggests that Pit-1 is directly required for the appearance of this distinct population that serves as the precursors of the mature thyrotrope cell type. These data suggest that different molecular mechanisms, based on the actions of distinct transcription factors, can serve to independently generate a specific cell phenotype during mammalian organogenesis.

POU-domain proteins: structure and function of developmental regulators.

POU-domain proteins are a group of developmental regulators found in organisms as distant as worm and man. The sequence conservation of the POU-domain has allowed the characterization of increasing numbers of proteins containing the domain, many of which act to control the generation and maintenance of differentiated cell phenotypes in organs as diverse as skin and brain. Analysis of the means by which POU-domain proteins regulate transcription has led to a further understanding of how this group initiates specific developmental programs.

Synergistic interactions between Pit-1 and other elements are required for effective somatotroph rat growth hormone gene expression in transgenic mice.

The role of the pituitary-specific POU-domain protein, Pit-1, in GH gene activation has been established by in vitro analyses and by the observation that mutations affecting the Pit-1 genomic locus result in genetically transmitted dwarfism. To define the quantitative contribution of the two Pit-1 response elements and the potential role of other factors in GH gene activation, we systematically assessed the ability of a series of GH promoter regions to activate transgenes in the mouse anterior pituitary gland. These studies revealed that the two GH Pit-1 binding sites are necessary, but not sufficient, for efficient transcriptional activation. Transgenes containing information including only these cis-active regions are expressed at extremely low levels in the pituitary glands of transgenic mice. The addition of 35 base pairs of 5'-flanking information, contributing other elements including a thyroid hormone/retinoic acid response element, results in much higher levels of transgene expression. Sequences located upstream of this segment contribute a further 5- to 10-fold activation. Thus, while Pit-1 is required for GH gene activation, it alone can only direct minimal expression in transgenic animals. Rather, synergistic interactions between other promoter elements and Pit-1 appear to be required for expression of the transgenes at approximately the 100-fold higher levels that are characteristic of somatotrophs, and are therefore likely to be critical components of somatotroph-specific expression of the GH gene.

Regulation of JC virus by the POU-domain transcription factor Tst-1: implications for progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy results from an opportunistic infection of myelin-producing oligodendrocytes by the glia-specific human papovavirus JC. In this report, evidence is presented that the glial transcription factor Tst-1, a member of the POU-domain family, stimulates transcription of both early and late viral genes. Stimulation was dependent on site-specific binding of Tst-1 to the JC viral regulatory region and on the presence of an intact amino-terminal transactivation domain within Tst-1. Because of its ability to increase the expression of viral large tumor antigen, Tst-1 stimulated viral DNA replication, without participating directly in the replication event. Our results suggest that Tst-1 is one of the determining factors in the glia specificity of JC virus.

TEF, a transcription factor expressed specifically in the anterior pituitary during embryogenesis, defines a new class of leucine zipper proteins.

We have identified and characterized a new member of the leucine zipper (bZIP) gene family of transcription factors, thyrotroph embryonic factor (TEF). Analysis of the ontogeny of TEF gene expression reveals the presence of TEF transcripts, beginning on embryonic day 14, only in the region of the rat anterior pituitary gland in which thyrotrophs arise. This pattern of gene expression corresponds temporally and spatially to the onset of thyroid-stimulating hormone (TSH beta) gene expression, which defines the thyrotroph phenotype. Coupled with this observation, we find that TEF can bind to and trans-activate the TSH beta promoter. In contrast to this restricted pattern of expression during embryogenesis, TEF transcripts appear in several tissues in the mature organism. We propose that TEF belongs to a new class of bZIP proteins on the basis of the unique homology between TEF and another member of the bZIP gene family, the albumin D box-binding protein (DBP). TEF and DBP transcripts are coexpressed in a pituitary cell line, and these two proteins can readily form heterodimers. The DNA-binding and dimerization domains of TEF correspond to those found in other bZIP proteins. We have however, identified a cluster of basic amino acids, found only in TEF and DBP, that is necessary for the proper DNA-binding site specificity of TEF. A major trans-activation domain of TEF resides outside the region of homology to other bZIP proteins. These data are consistent with a role for a member of a new class of bZIP transcription factors in activating gene expression in the developing thyrotroph.

Increase in cyclic AMP levels by relaxin in newborn rhesus monkey uterus cell culture.

A novel relaxin sensitive cell line of apparent smooth muscle origin has been established from a newborn rhesus monkey uterus (NRMU). NRMU cells respond to relaxin, in the presence of 1 microM forskolin, by producing intracellular adenosine 3', 5'-cyclic monophosphate (cAMP). The increase in cAMP levels is dose, time and cell density dependent, reaching peak levels at 10 min when cells are seeded at 1 X 10(5) cells/well. Specificity was demonstrated by neutralization of the relaxin activity with anti-relaxin monoclonal and polyclonal antibodies, degradation of cAMP in the presence of phosphodiesterase, and confirmation of the absence of cGMP. Three synthetic analogs of human relaxin generated a dose-related cAMP response as did synthetic native human relaxin. Natural relaxin purified from human corpora lutea tissue also generated a response similar to synthetic human relaxin. Porcine and rat relaxins also increased levels of cAMP. Insulin, but not IGF I or IGF II, was capable of increasing cAMP levels in NRMU cells, however, 200 ng/mL were required to achieve cAMP levels comparable to 6.25 ng/ml relaxin. Combinations of relaxin with insulin, IGF I or IGF II did not increase cAMP levels above levels obtained with relaxin alone. The effect on NRMU cells of other hormones, growth factors and drugs potentially present in cell culture systems or serum samples was evaluated. In combination with relaxin, oxytocin significantly decreased the cAMP production below the levels induced by relaxin alone, whereas progesterone and prostaglandin E2 resulted in additive increases in cAMP. These data suggest that the NRMU cell line is an appropriate target tissue for studying relaxin-mediated biological responses in vitro as well as functioning as the primary component of a relaxin in vitro bioassay.

An enzyme-linked immunosorbent assay to study human relaxin in human pregnancy and in pregnant rhesus monkeys.

A sensitive and specific double-antibody enzyme-linked immunoassay, using a synthetic analogue of human relaxin for standard and immunogen, was developed for the measurement of human relaxin (hRLX) in serum and plasma. No cross-reactivity was observed for human insulin, human insulin-like growth factor-I, hGH, human chorionic gonadotropin, hFSH, hLH or human prolactin. The assay was used to monitor RLX concentrations in samples from men, non-pregnant and pregnant women, and in pregnant rhesus monkeys infused with hRLX. RLX was not detected in serum from men nor from non-pregnant women, while a concentration of 600 ng/l was measured in pooled sera from two pregnant women (pregnancies achieved by in-vitro fertilization). Immunoreactive RLX (1.1 micrograms/g) was found in human corpora lutea taken from ectopic pregnancies at 7 weeks. In an experiment with a pregnant rhesus monkey infused with human RLX analogue, less than 1.5% of the maternal concentration was measured in the fetal circulation. Even though preliminary, these data suggest a low level of transfer of human analogue relaxin across the placenta in a rhesus monkey. Further studies of the physiology of RLX in human pregnancy will be facilitated by the availability of this immunoassay.

The effect of changes in muscle function and bone growth on muscle migration.

The elastic sleeve model of the periosteum of a long bone presents the periosteum as a structure which, because it is attached to the epiphyses rather than the diaphysis, expands interstitially and equally at all points as the bone grows at its ends. Structures attached to the periosteum are seen as essentially passive hitchhikers on the expanding periosteum. Two corollaries of this model are tested here. First, that changes in the magnitude or direction of the force that an attached structure exerts on the periosteum do not affect the migration of the structure. Second, that changes in the proportion of growth that occur at each end of the bone do not affect the migration of attached structures. Experiments performed on rabbits to test these corollaries include muscle paralysis, muscle transection, changes in the direction pull of a muscle, and epiphysiodesis. The results are in agreement with the hypotheses. This model should have applicability to functional and comparative anatomy, since it postulates that differences in positions of attachment of muscles and ligaments to bones reflect underlying genetic differences (phylogeny) rather than the effects of differences in behavior of the animal (ontogeny).

Invariance of the relative positions of structures attached to long bones during growth: cross-sectional and longitudinal studies.

This article reports the results of cross-sectional and longitudinal studies on the positions of muscles relative to bones. The cross-sectional study reports on the positions of 20 muscles attached to long bones in the chicken from prehatching ages to maturity. The results show that these muscles maintain constant positional relationships during growth as was reported in an earlier study by the authors using rabbits. The longitudinal study reports on the migration of the semitendinosus muscle in the rabbit during growth. The results show that this muscle migrates the distance predicted by the regression equations derived from the cross-sectional study. These results are related to the model of the periosteum as an expanding elastic sleeve attached at its ends to the epiphyses and put under tension by the activity of the epiphyseal plates. In this model the soft structures attached to bones are seen as attached only to the periosteum in growing animals and thus carried along as hitchhikers by the expanding periosteum.

Positional relationships of structures attached to long bones during growth. Cross-sectional studies.

The various soft structures attached to bones maintain relatively constant relationships during growth. The exact number of these relationships has, however, never been studied. As part of our ongoing research into the factors controlling muscle migration, we have determined these relationships for some structures. We studied the positions of 37 muscles from 24 New Zealand white rabbits ranging in age from birth to maturity. The muscles were selected to illustrate different kinds of attachments: fleshy and tendonous, restricted and extensive, and at both ends of a bone. The proximal and distal edges of attachments were carefully exposed and measured in a device that allowed us to measure the position relative to the ends of the bones without parallax distortion. We used the data to compute correlation coefficients and regression equations for position vs. length of bone. Results show that the correlation coefficient was above 0.9 for most cases and was significant at the 0.5 level for all but 4 cases. Slopes of the regression equations varied considerably, but in all cases they indicated that the closer to the end of a bone, the greater the distance migrated. The significance of these results is discussed.