Phase I trial of oral 2'-deoxy-2'-methylidenecytidine: on a daily x 14-day schedule.

2'-deoxy-2'-methylidenecytidine (DMDC) is a potent deoxycytidine analogue. Preclinical studies of DMDC demonstrated activity against a variety of murine and human tumors in cell cultures and murine models and indicate enhanced antitumor activity of DMDC when it was administered in a manner that provided prolonged systemic exposure. In view of this observation, this study was designed to determine the toxicities, maximum-tolerated dose, and pharmacokinetic profile of DMDC. DMDC was given p.o. under fasting conditions for 14 consecutive days every 4 weeks in patients with advanced solid tumors. The starting dose was 12 mg/m2/day. Pharmacokinetic studies were carried out on days 1 and 14 of the first cycle. Fourteen patients received 22 courses of DMDC. The dose-limiting toxicities were anorexia, leukopenia, thrombocytopenia, and anemia. General fatigue was the common nonhematological toxicity. The maximum-tolerated dose was 18 mg/m2/day, at which two of six patients developed grade 3 toxicities. This dose level could also be considered for Phase II testing with this schedule. At the 18-mg/m2/day dose level, the mean terminal half-life, maximum plasma concentration (Cmax), the area under the plasma drug concentration-time curve (AUC(0-infinity)) on day 1 were 1.7496 h, 112.9 ng/ml, and 399.8 ng x h/ml, respectively. Forty to 50% of the administered dose was recovered in the urine, indicating a good bioavailability and resulting significant systemic exposure to the drug, which may enable chronic oral treatment.

Application of homogeneous time-resolved fluorescence (HTRFTM) to monitor poly-ubiquitination of wild-type p53.

Rapid degradation of wild-type p53 in the human uterine cervix is induced by the infection of high-risk human papilloma virus (HPV) types 16 and 18. HPV-E6 protein plays a critical role in the poly-ubiquitination of wild-type p53 by mediating the association of p53 with E6-associated protein (E6AP). As a result, the poly-ubiquitinated p53 is rapidly and selectively degraded by the 26S proteasome. We have established a high throughput assay system to monitor poly-ubiquitination of wild-type p53 using a new fluorescence homogeneous technology known as Homogeneous Time-Resolved Fluorescence (HTRFTM). The Europium Cryptate [Eu(K)]-labeled ubiquitins are incorporated into poly-ubiquitin chains conjugated with the biotinylated p53. In the HTRF assay, Europium cryptate-labeled ubiquitin and streptavidin-labeled allophycocyanin (XL665) are used as the fluorescence donor and acceptor, respectively. The biotinylated p53 is ubiquitinated by ubiquitination enzymes, then by the addition of streptavidin-labeled XL665, the donor and acceptor molecules are brought in close proximity, thereby generating fluorescent signals. This time-resolved fluorescence assay system shows a sufficient signal for its application in synthetic compound screening and having almost the same level of sensitivity as that monitored by the scintillation proximity assay (SPA) using 125I-labeled ubiquitin. The detection of poly-ubiquitination of wild-type p53 by using the HTRFTM or SPA systems described here is much easier and quicker than by using conventional methods. Therefore, these new systems would be appropriate for high throughput screening of compounds for the discovery of new inhibitors of poly-ubiquitination of wild-type p53.

Screening of an inhibitor of the tetracycline efflux pump in a tetracycline-resistant clinical-isolate of Staphylococcus aureus 743.

Clinically-isolated methicillin-resistant Staphylococcus aureus (MRSA) strain 743 exhibited resistance to tetracycline as judged from the active efflux of the drug. The efflux of tetracycline was inhibited by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and minocycline. Inhibitors of the efflux pump were examined in this strain to determine the cellular accumulation of tetracycline. Out of seven compounds examined, three caused a significant increase in the cellular concentration of tetracycline by inhibiting the efflux pump. Two of them seem to be energy inhibitors. Ro 07-3149 inhibited the efflux pump without affecting the energy state, and exhibited very low antibacterial activity but showed weak synergy with tetracycline.

[Immune intervention by peptides having a MHC class II binding motif--application of MHC blockers to autoimmune disease models].

Intervention of T cell activation and the treatment of autoimmune disease models by MHC class II binding peptides was reviewed in this article. Analog peptides derived from antigenic peptides were shown to inhibit T cell activation in vitro as well as in vivo either by T cell antagonism, T cell tolerance induction, or by MHC blockade. The induction of immune suppression by MHC blocker peptides was discussed in detail. Successful application of MHC blocker peptides in the treatment of experimental allergy encephalomyelitis (EAE), non-obese diabetic mice and collagen-induced arthritis models indicated that in vivo blocking of MHC class II molecules represents a promising approach for the prevention and possibly treatment of human autoimmune diseases. An approach in identifying non-peptidic MHC blockers was also described.

Induction of class II major histocompatibility complex blockade as well as T cell tolerance by peptides administered in soluble form.

Peptides binding to a particular class II major histocompatibility complex (MHC) molecule can inhibit the activation of T cells by other peptides binding to the same molecule, a phenomenon termed class II MHC blockade. All class II-binding peptides exert MHC blockade in vivo in depot form with adjuvant, and some also retain their blocking properties in soluble form. We demonstrate here that soluble peptides, when used at doses causing short-term MHC blockade, can also induce long-term antigen-specific T cell tolerance to themselves. The tolerogenicity of soluble peptides correlates with their antigenicity in adjuvant, but it is not necessarily related to their capacity to act as class II blockers in vivo. The tolerant state is manifested in a decreased production of both T helper cell 1 (Th1)-type and Th2-type lymphokines, and it cannot be reversed by interleukin-2. Once T cells are primed with a peptide in complete Freund's adjuvant, they are resistant to tolerization with the same peptide applied in soluble form. Tolerance induction is partially impaired in B cell-deficient mu MT-/- mice, suggesting a role for B cell antigen presentation in this process. The results suggest that the potential immunogenicity of class II MHC blockers could be circumvented by choosing a tolerogenic mode of application.

Relationship of urinary sodium and potassium and of urinary sodium/creatinine, potassium/creatinine and sodium/potassium ratios to stomach cancer and cerebrovascular disease mortalities in Japanese women.

To test Joossens's hypothesis that salt is a common cause of both cerebrovascular disease (CVD) and stomach cancer (SC), the standardized mortality ratios (SMRs) of SC and CVD were related to urinary Na and K and urinary Na/creatinine (Cr), K/Cr, and Na/K ratios. Fifty spot urine of females between 40 to 69 years was collected from each of 169 municipalities (88 urban and 81 rural) covering all prefectures in Japan using the filter paper sampling technique in 1985. While the SMR levels of CVD correlated significantly with Na, Na/Cr and Na/K, the SMR levels of SC showed no correlation with Na, K, Na/Cr, K/Cr or Na/K. It was therefore concluded that the geographical distribution of female SMR for SC could not be explained using only the urinary Na excretion as an index of Na intake, and that multiple risk factors and risk reducing factors should be taken into account to explain the distribution.

Evaluation of the individual effects of a health education program for sodium restriction by a simple method for measuring 24-hour urinary sodium excretion.

A simple method for measuring 24-hr urinary sodium excretion was applied to the evaluation of the individual effects of a health education program for sodium restriction in a rural community in Japan. Eighteen subjects (6 males and 12 females) between 35 and 72 years of age were advised to reduce their sodium intake. Twenty-four-hour urinary sodium, potassium excretion, and sodium/potassium ratio were measured using the simple method for seven consecutive days in three periods which were before the sodium restriction, 6 months after the sodium restriction, and 6 months after the end of the program. Mean sodium excretion of 18 subjects significantly decreased (p less than 0.05) after the end of the program. The reduced level was maintained until six months after the end of the program. Within individual cases, sodium excretion decreased significantly in subjects who had levels higher than 170 mmol at the initial stage. The reduced levels of sodium excretion were maintained until six months after the end of the program except for one subject. The subjects who had an initial level lower than 170 mmol of sodium, which is the upper limit of present recommendation for Japanese adults, did not change their levels.

A new hematopoietic cell line, KMT-2, having human interleukin-3 receptors.

A novel human cell line, KMT-2, from umbilical cord blood cells was established based on the selection of cultures in the presence of recombinant human interleukin-3 (IL-3) and the sorting of cells with anti-My 10 antibody. Morphologic and cytochemical studies (peroxidase negative, Sudan-black negative, chloroacetate esterase negative, PAS positive, nonspecific esterase positive) and phenotyping (HLA-DR, My7 = CD13, My9 = CD33, My10 = CD34, MCS-2, LeuM1 positive, glycophorin A negative, and P2 negative) suggest that the KMT-2 cells are myelomonocytic cells, probably of immature progenitor origin. Besides IL-3, granulocyte-macrophage colony-stimulating factor supported the growth of the KMT-2 cells, but IL-1 alpha, IL-2, IL-4, IL-5, and erythropoietin did not. IL-6 showed only slight activity. Binding studies with 125I-labeled recombinant human (rh) IL-3 indicated that IL-3 bound to a single class of high affinity receptors (approximately 4,000 receptors/cell) on KMT-2 cells with a kd of approximately 200 pmol/L. The chemical cross-linking assay demonstrated that radiolabeled hIL-3 bound three molecules with molecular masses of 170, 130, and 70 Kd. Present data suggest that the newly established human cell line will be a valuable tool for the biologic assay of hIL-3, and a model for biochemical studies of IL-3 receptors.

Familial mental retardation associated with balanced chromosome rearrangement rcp t(8;11)(q24.3;p15.1).

We report three sisters and their father with a reciprocal balanced translocation, rcp t(8;11)(q24.3;p15.1) and the same abnormal phenotypes, including mental retardation, growth disturbance, and amblyopia. It is considered that the abnormal phenotypes in our four cases might result from a tiny deletion or gene mutation at the breakpoints in 8q or 11p or both. Our cases showed no resemblance, apart from mental retardation, to Langer-Giedion syndrome, which is caused by the deletion of 8q23.3 and 8q24. Furthermore, our patients did not have the cardinal features of Beckwith-Wiedermann syndrome or WAGR which are caused by deletion of 11p. It is suggested that the amblyopia in our four cases might have resulted from the breakpoints at 11p15.1.

Relationship of blood pressure to sodium and potassium excretion in Japanese women.

The cross-sectional association of blood pressure with urinary sodium and potassium excretion was investigated with a stepwise regression analysis. Spot urine of 7441 females between 40 and 69 years was collected from 169 municipalities (88 urban and 81 rural) covering all prefectures in Japan. The filter paper sampling technique for urine was used to collect samples of subjects from March to December in 1985. Spot urine samples were analyzed for sodium, potassium and creatinine. In addition, 24-hr sodium and potassium excretions were estimated by predictive equations. Blood pressure, sodium excretion and sodium potassium ratios were higher in rural areas than in urban areas. Consistent positive correlations between urinary sodium and blood pressure, and negative correlations between urinary potassium and blood pressure were observed in the whole country of Japan, in both urban and rural areas, and also in separate observations of twelve regions in Japan with some exceptions. When compared in standardized partial regression coefficients, relative effects of potassium on systolic blood pressure were higher than those of sodium in the whole of Japan, in urban and rural areas, and in five among the twelve regions. The present Japanese study confirmed a positive within-population relationship between sodium excretion and blood pressure and a negative relationship between potassium excretion and blood pressure.

Thymosin-alpha 1 increases the capability to produce interleukin-3 but not interleukin-2 in nu/nu mice.

Thymosin-alpha 1 increased the capability of nu/nu mice to produce interleukin (IL)-3 but not IL-2 when administered intraperitoneally at doses of 0.5 and 5.0 micrograms/kg twice a week for 3-6 weeks. Thy-1-positive cells were responsible for the production of IL-3, which was determined by proliferation of the IL-3-dependent cell line (FDC-P2) as well as by augmentation of the in vitro growth of hematopoietic stem cells (colony-forming unit S [CFU-S]). The IL-3 activity released by nu/nu splenocytes emerged in fractions coincidentally with IL-3 released by WEHI-3 cells on Mono Q anion-exchange chromatography. However, IL-2 activity determined by proliferation of the IL-2-dependent cell line was not detected in any fractions of the same chromatography. These findings indicate that IL-2 is not always coproduced with IL-3 by Thy-1-positive cells. This suggests, taken together with results from previous studies about the cells producing IL-2 and IL-3 by other investigators, that thymosin-alpha 1 exerts its effect at an early stage of T cell differentiation to induce a T cell subpopulation capable of producing IL-3 (but not yet IL-2). Based on the present findings, we discuss the mechanism of action by which thymosin-alpha 1 exerts its diverse effects in immunosuppressed hosts but not a potentiating effect in healthy normal animals or in in vitro assays of T cell function.

Thymosin alpha 1 enhances haemopoietic colony formation by stimulating the production of interleukin 3 in nu/nu mice.

We have studied the action mechanism of thymosin alpha 1 in modulation of haemopoietic system. The present study demonstrated that thymosin alpha 1 enhanced the colony formation as determined by CFU-s, GM-CFU and T-CFU, as well as the production of IL-3, when administered twice a week for a total of six or twelve times into nu/nu mice. The incubation of bone marrow cells with thymosin alpha 1 in vitro did not cause an increase of CFU-s, in contrast to IL-3 which caused a marked increase of CFU-s during the incubation of 72 h. These experiments indicate that thymosin alpha 1 exerts its effect on colony formation of haemopoietic stem cells (CFU-s) indirectly through the enhancement of the IL-3 production. The present finding may support clinical applications of thymosin alpha 1 in a wide range, since IL-3 is known to be the growth factor for many kinds of haemopoietic precursor cells including not only CFU-s but also GM-CFU, BFU-E, Eos-CFU, Meg-CFU, Mast cells, Pre B-cells and Pre T-cells.

Analysis of proteases involved in phagocytic activity of macrophages through the use of various amino acid esters.

Inhibitory effects of various amino acid esters on the phagocytic activity of guinea pig peritoneal macrophages were studied with sensitized 51Cr-sheep erythrocytes (51Cr-EAb) as well as 125I-alpha-amylase complexed with homologous IgG2 antibody (Ag-Ab complex). The intracellular uptake of 51Cr-EAb was markedly inhibited by N-acetyl-L-phenylalanine ethyl ester (Ac-Phe-OEt), N-acetyl-L-tryptophan ethyl ester (Ac-Trp-OEt) and N-benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt), but not by N-acetyl-L-tyrosine ethyl ester (Ac-Tyr-OEt), N-alpha-acetyl-L-arginine methyl ester (Ac-Arg-OMe), N-alpha-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt) or N-alpha-acetyl-L-lysine methyl ester (Ac-Lys-OMe). When phagocytosis of the Ag-Ab complex was assayed by measuring the amount of digested products released from macrophage cells, Ac-Tyr-OEt also inhibited it as markedly as Ac-Phe-OEt, Ac-Trp-OEt, and Bz-Tyr-OEt did, whereas Bz-Arg-OEt again did not show any effect. The results of analysis of the intracellular fate of the Ag-Ab complex taken up by macrophages through the use of analytical density gradient fractionation of the homogenized cells suggest that Ac-Phe-OEt inhibits the ingestive process since the distribution of Ag-Ab complex showed a single peak, closely accompanying the plasma membrane. Ac-Tyr-OEt, on the other hand, caused a marked accumulation of Ag-Ab complex in the lysosome fraction, reflecting the inhibition of intralysosomal digestion of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of protease inhibitors on ingestion and digestion of soluble antigen-antibody complexes by macrophages.

The inhibitory effects of diisopropyl fluorophosphate (DFP) and p-tosyl-L-lysine chloromethyl ketone (TLCK) on the ingestion and digestion of 125I-alpha-amylase [B alpha A, EC 3.2.1.1] complexed with antibody by guinea pig peritoneal macrophages were studied by sucrose density gradient fractionation. When macrophages were exposed to the complex at 37 degrees C and homogenized after washing, most of the trichloroacetic acid (TCA)-insoluble radioactivity was recovered in two fractions which behaved like the plasma membrane and lysosomes in the density gradient, and the TCA-soluble radioactivity was found in the fraction not moving into the gradient. The complex bound to Fc receptors on the cell surface, therefore, was shown to be internalized, transported to the lysosomes, and finally digested to amino acid catabolites. The inhibition with DFP caused an accumulation of the complex in a region between the plasma membrane and lysosomes. This distribution of radioactivity resembled that of cytochalasin B-inhibited macrophages. TLCK also inhibited the digestion of the complex, but the distribution of radioactivity revealed a marked accumulation of the complex in the region of lysosomes. These results provide strong support to the hypothesis that DFP blocks the ingestion of the complex, as in the case of cytochalasin B, whereas TLCK inhibits the intralysosomal proteolysis of the complex.