LSD1 inhibition suppresses ASCL1 and de-represses YAP1 to drive potent activity against neuroendocrine prostate cancer.

Lysine-specific demethylase 1 (LSD1 or KDM1A ) has emerged as a critical mediator of tumor progression in metastatic castration-resistant prostate cancer (mCRPC). Among mCRPC subtypes, neuroendocrine prostate cancer (NEPC) is an exceptionally aggressive variant driven by lineage plasticity, an adaptive resistance mechanism to androgen receptor axis-targeted therapies. Our study shows that LSD1 expression is elevated in NEPC and associated with unfavorable clinical outcomes. Using genetic approaches, we validated the on-target effects of LSD1 inhibition across various models. We investigated the therapeutic potential of bomedemstat, an orally bioavailable, irreversible LSD1 inhibitor with low nanomolar potency. Our findings demonstrate potent antitumor activity against CRPC models, including tumor regressions in NEPC patient-derived xenografts. Mechanistically, our study uncovers that LSD1 inhibition suppresses the neuronal transcriptional program by downregulating ASCL1 through disrupting LSD1:INSM1 interactions and de-repressing YAP1 silencing. Our data support the clinical development of LSD1 inhibitors for treating CRPC - especially the aggressive NE phenotype. Statement of Significance: Neuroendocrine prostate cancer presents a clinical challenge due to the lack of effective treatments. Our research demonstrates that bomedemstat, a potent and selective LSD1 inhibitor, effectively combats neuroendocrine prostate cancer by downregulating the ASCL1- dependent NE transcriptional program and re-expressing YAP1.

Targeted delivery of cytotoxic proteins to prostate cancer via conjugation to small molecule urea-based PSMA inhibitors.

Prostate cancer cells are characterized by a remarkably low proliferative rate and the production of high levels of prostate-specific proteases. Protein-based toxins are attractive candidates for prostate cancer therapy because they kill cells via proliferation-independent mechanisms. However, the non-specific cytotoxicity of these potent cytotoxins must be redirected to avoid toxicity to normal tissues. Prostate-Specific Membrane Antigen (PSMA) is membrane-bound carboxypeptidase that is highly expressed by prostate cancer cells. Potent dipeptide PSMA inhibitors have been developed that can selectively deliver and concentrate imaging agents within prostate cancer cells based on continuous PSMA internalization and endosomal cycling. On this basis, we conjugated a PSMA inhibitor to the apoptosis-inducing human protease Granzyme B and the potent Pseudomonas exotoxin protein toxin fragment, PE35. We assessed selective PSMA binding and entrance into tumor cell to induce cell death. We demonstrated these agents selectively bound to PSMA and became internalized. PSMA-targeted PE35 toxin was selectively toxic to PSMA producing cells in vitro. Intratumoral and intravenous administration of this toxin produced marked tumor killing of PSMA-producing xenografts with minimal host toxicity. These studies demonstrate that urea-based PSMA inhibitors represent a simpler, less expensive alternative to antibodies as a means to deliver cytotoxic proteins to prostate cancer cells.

In vitro induction of cartilage-specific macromolecules by a bone extract.

An in vitro system has been developed to study the onset of chondrogenesis. Embryonic rat muscle mesenchymal cells, when treated in suspension culture with an extract of bovine bone matrix, synthesized cartilage-specific proteoglycan and type II collagen. The synthesis of these two macromolecules was assayed by the enzyme-linked immunosorbent assay inhibition technique. Further evidence of chondrogenesis was demonstrated by morphological changes of treated cells when cultured in firm agarose and stained for metachromatic matrix. Even with crude bone matrix extracts, the assay was sensitive at the microgram level and significant differences in cartilage macromolecules compared with controls were observed in 2-3 d. In vivo the same extract induced first cartilage and then bone.

Cellular pharmacology of N1- and N8-aziridinyl analogues of spermidine.

We have previously described the synthesis and cytotoxic properties of 2 polyamine analogues in which either the N1- or N8-amino group of spermidine was replaced by the alkylating moiety, aziridine. However, the mechanisms by which these aziridinyl analogues of spermidine inhibit cell growth remain unknown. As a result, we have studied: (a) the effect of pretreatment with difluoromethyl ornithine (DFMO) and coincubation with exogenous spermidine on cytotoxicity induced by the aziridinyl spermidines; (b) the reversibility of the cytotoxicity induced by the aziridinyl spermidines; (c) the accumulation of N1- and N8-aziridinyl spermidine by cells and the effects of DFMO on this process; and (d) the impact of N1- and N8-aziridinyl spermidine on cellular polyamine pools and on cellular accumulation of spermidine. The cytotoxicity induced by these 2 aziridinyl derivatives of spermidine [concentration required to inhibit cell growth or incorporation of radiolabeled precursor into trichloroacetic acid-precipitable material by 50% (IC50) N1 = 0.2 microM, IC50 N8 = 0.4 microM)] was potentiated by pretreatment of L1210 cells for 24 h with 100 microM DFMO (IC50 N1 = 0.05 microM, IC50 N8 = 0.15 microM) and was prevented by coincubation with 3.7 microM spermidine (IC50 N1 = 1.1 microM, IC50 N8 = 2.4 microM). In contrast, similar pretreatment with DFMO or coincubation with spermidine had no effect on the cytotoxicity induced by the aziridine-containing alkylating agent, N,N',N"-triethylenethiophosphoramide (thiotepa) (IC50 = 2.4 microM). The cytotoxicity induced by 24-h incubation with either N1- or N8-aziridinyl spermidine was not altered by removal of those compounds and incubating treated cells in medium augmented with 3.7 microM spermidine. However, and as expected, similar maneuvers did not reverse the cell growth-inhibitory effect induced by 24-h incubation with 100 microM DFMO. Cellular accumulation of both N1- and N8-aziridinyl spermidine increased with increasing extracellular concentrations. N1-Aziridinyl spermidine was accumulated to a greater degree than was the N8-analogue, achieving up to 6-fold higher intracellular concentrations at the same extracellular concentration. Cellular accumulation of both aziridinyl compounds was greatly enhanced by 24-h pretreatment with DFMO. Both N1- and N8-aziridinyl spermidine inhibited the uptake of spermidine in a dose-dependent manner. The perturbation of polyamine biochemistry by the test compounds was characterized by their ability to deplete cellular putrescine, as well as spermidine and spermine. These results imply that the cytotoxic mechanism of the aziridinyl spermidine analogues is, to a great extent, dependent on their polyamine nature and may imply selectivity for rapidly growing and neoplastic cells.

Comparative molecular field analysis-based predictive model of structure-function relationships of polyamine transport inhibitors in L1210 cells.

Maintenance of intracellular polyamine concentrations necessary for cell growth and proliferation is regulated in part by an energy-dependent polyamine uptake system. To obtain information on the characteristics of the polyamine uptake system in L1210 leukemia cells, we have applied computational chemistry techniques to the study of relationships between structure and function of 57 polyamine analogues. Ki values of polyamine analogues, derived from competitive inhibition of [3H]spermidine transport into L1210 cells, were chosen as the measure of biological activity. Using comparative molecular field analysis (CoMFA), a model was constructed to relate molecular structure with biological activity. The model was based on 4 monocationic, 8 dicationic, 14 tricationic, and 20 tetracationic polyamine analogues with a range of Ki values for the inhibition of [3H]spermidine uptake of 0.97-521 microM. The CoMFA model successfully predicted the inhibitory potency of 11 polyamines that had not previously been tested for polyamine uptake inhibitory activity. The 11 values predicted were within 33 +/- 62% of the actual Ki values. The test group included aziridinyl diamines, acetylated spermidines, two new oxazolidinonyl spermidines, monoaziridinyl spermidines, and a diaziridinyl spermine. Several of the compounds from this test group have been shown to have anticancer activity in mice. Consistent with the CoMFA model, certain basic functional groups, such as aziridines that have pKa values in the range of 6-7, seem to interact with the polyamine transporter in a cationic form. The results suggest that the CoMFA model is useful in drug design strategies as a predictive tool for the discovery of new anticancer agents that utilize a polyamine transporter for cellular uptake.

Metabolism of 17-(allylamino)-17-demethoxygeldanamycin (NSC 330507) by murine and human hepatic preparations.

17-(Allylamino)-17-demethoxygeldanamycin (17AAG), a compound that is proposed for clinical development, shares the ability of geldanamycin to bind to heat shock protein 90 and GRP94, thereby depleting cells of p185erbB2, mutant p53, and Raf-1. Urine and plasma from mice treated i.v. with 17AAG contained six materials with absorption spectra similar to that of 17AAG. Therefore, in vitro metabolism of 17AAG by mouse and human hepatic preparations was studied to characterize: (a) the enzymes responsible for 17AAG metabolism; and (b) the structures of the metabolites produced. These materials had retention times on high-performance liquid chromatography of approximately 2, 4, 5, 6, 7, and 9 min. When incubated in an aerobic environment with 17AAG, murine hepatic supernatant (9000 x g) produced each of these compounds; the 4-min metabolite was the major product. This metabolism required an electron donor, and NADPH was favored over NADH. Metabolic activity resided predominantly in the microsomal fraction. Metabolism was decreased by approximately 80% in anaerobic conditions and was essentially ablated by CO. Microsomes prepared from human livers produced essentially the same metabolites as produced by murine hepatic microsomes, but the 2-min metabolite was the major product, and the 4-min metabolite was next largest. There was no metabolism of 17AAG by human liver cytosol. Metabolism of 17AAG by human liver microsomes also required an electron donor, with NADPH being preferred over NADH, was inhibited by approximately 80% under anaerobic conditions, and was essentially ablated by CO. Liquid chromatography/mass spectrometry analysis of human and mouse in vitro reaction mixtures indicated the presence of materials with molecular weights of 545, 601, and 619, compatible with 17-(amino)-17-demethoxygeldanamycin (17AG), an epoxide, and a diol, respectively. The metabolite with retention time of 4 min was identified as 17AG by cochromatography and mass spectral concordance with authentic standard. Human microsomal metabolism of 17AAG was inhibited by ketoconazole, implying 3A4 as the responsible cytochrome P450 isoform. Incubation of 17AAG with cloned CYP3A4 produced metabolites 4 and 6. Incubation of 17AAG with cloned CYP3A4 and cloned microsomal epoxide hydrolase produced metabolites 2 and 4, with greatly decreased amounts of metabolite 6. Incubation of 17AAG with human hepatic microsomes and cyclohexene oxide, a known inhibitor of microsomal epoxide hydrolase, did not affect the production of metabolite 4 but decreased the production of metabolite 2 while increasing the production of metabolite 6. These data imply that metabolite 2 is a diol and metabolite 6 is an epoxide. Mass spectral fragmentation patterns and the fact that 17AG is not metabolized argue for the epoxide and diol being formed on the 17-allylamino portion of 17AAG and not on its ansamycin ring. These data have implications with regard to preclinical toxicology and activity testing of 17AAG as well as its proposed clinical development because: (a) production of 17AG requires concomitant production of acrolein from the cleaved allyl moiety; and (b) 17AG, which was not metabolized by microsomes, has been described as being as active as 17AAG in decreasing cellular p185erbB2.

Plasma concentrations and pharmacokinetics of dimethylsulfoxide and its metabolites in patients undergoing peripheral-blood stem-cell transplants.

PURPOSE: Dimethylsulfoxide (DMSO) is used to cryopreserve hematopoietic stem cells and is obligatorily infused into patients who receive stem-cell transplants. This study characterized the plasma concentrations and pharmacokinetics of DMSO and its metabolites in patients who underwent peripheral-blood stem-cell transplants. MATERIALS AND METHODS: Plasma concentrations of DMSO, dimethylsulfone (DMSO2), and dimethylsulfide (DMSH2) were assessed in 10 patients who underwent autologous transplants with stem cells, cryopreserved in 10% DMSO (vol/vol). Blood was sampled at multiple times after the stem-cell infusion. Urine was pooled during the 24 hours postinfusion. DMSO, DMSO2, and DMSH2 were assayed simultaneously by gas chromatography. A one-compartment model with saturable elimination proved most suitable for fitting plasma DMSO concentration-versus-time data. RESULTS: Stem-cell volumes infused ranged between 180 and 585 mL (254 to 824 mmol DMSO). Infusions lasted between 20 and 120 minutes. Peak plasma DMSO concentrations were 19.1 +/- 6.3 mmol/L (mean +/- SD). Pharmacokinetic parameters for volume of the central compartment (Vc), maximum velocity (Vmax), and Michaels-Menten constant (Km) were 37.3 +/- 17 L, 0.99 +/- 0.57 mmol/L/h, and 5.2 +/- 5.0 mmol/L, respectively. Plasma DMSO2 concentrations increased during the first 24 hours, plateaued at 4.4 +/- 1.2 mmol/L, and remained there until 48 hours (the last sample). DMSH2 concentrations were at steady-state by 5 minutes and remained between 3 and 5 mmol/L for 48 hours. Urinary excretion of DMSO and DMSO2 accounted for 44% +/- 4% and 4% +/- 1%, respectively, of the administered DMSO dose. Renal clearance of DMSO was 14.1 +/- 3.4 mL/min. CONCLUSION: These data (1) document plasma concentrations of DMSO and metabolites in patients following peripheral-blood stem-cell transplants; (2) allow consideration of potential effects of these concentrations on stem-cell engraftment and drug-drug interactions; and (3) can facilitate a concentration-guided phase I trial of DMSO.

Phase I study of paclitaxel in combination with a multidrug resistance modulator, PSC 833 (Valspodar), in refractory malignancies.

PURPOSE: To determine the maximum-tolerated dose (MTD), dose-limiting toxicity (DLT), and pharmacokinetics of paclitaxel when given with PSC 833 (valspodar) to patients with refractory solid tumors. PATIENTS AND METHODS: Patients were initially treated with paclitaxel 175 mg/m(2) continuous intravenous infusion (CIVI) over 3 hours. Subsequently, 29 hours of treatment with CIVI PSC 833 was started 2 hours before paclitaxel treatment was initiated. In this combination, the starting dose of paclitaxel was 52.5 mg/m(2). Paclitaxel doses were escalated by 17.5 mg/m(2) increments for four subsequent cohorts. Each cohort consisted of three patients with the exception of the last cohort, which consisted of six patients. Data for the pharmacokinetics of paclitaxel with and without concurrent PSC 833 administration were obtained. RESULTS: All 18 patients completed at least one course of concurrent treatment (median, two courses; range, one to six) and were evaluable for toxicity. The MTD for paclitaxel with PSC 833 was 122.5 mg/m(2). Neutropenia was the DLT. All patients had PSC 833 blood concentrations greater than 1, 000 ng/mL before, during, and 24 hours after the paclitaxel infusion. PSC 833 produced small increases in the paclitaxel peak plasma concentrations and areas under the concentration-time curve. However, PSC 833 greatly prolonged the terminal phase of paclitaxel, resulting in plasma paclitaxel concentrations of more than 0.05 micromol/L for much longer than expected. As a result, myelosuppression was comparable to that produced by full-dose paclitaxel given without PSC 833. Of the 16 patients who were assessable for response, one patient experienced a partial response and an additional nine patients experienced disease stabilization after paclitaxel treatment alone. CONCLUSION: Treatment with paclitaxel 122.5 mg/m(2) as a 3-hour CIVI concurrent with a 29-hour CIVI of PSC 833 results in acceptable toxicity. The addition of PSC 833 alters the pharmacokinetics of paclitaxel, which explains the enhanced neutropenia experienced by patients treated with this drug combination.

Phase I study of paclitaxel given by seven-week continuous infusion concurrent with radiation therapy for locally advanced squamous cell carcinoma of the head and neck.

PURPOSE: Paclitaxel is one of the most active agents for squamous cell carcinoma of the head and neck (SCCHN) and an in vitro radiosensitizer. The dose-response relationship for paclitaxel may depend more on exposure duration than on peak concentration. This National Cancer Institute-sponsored phase I trial was designed to determine the feasibility of combining continuous-infusion (CI) paclitaxel with concurrent radiation therapy (RT). PATIENTS AND METHODS: Patients with previously untreated stage IVA/B SCCHN were eligible. Primary end points were determination of the maximum-tolerated dose, dose-limiting toxicity, and pharmacokinetics for paclitaxel given by CI (24 hours a day, 7 days a week for 7 weeks) during RT (70 Gy/7 weeks). RESULTS: Twenty-seven patients were enrolled and assessable for toxicity. Nineteen of the patients who completed > or = 70 Gy were assessable for response. Grade 3 skin and mucosal acute reactions occurred at 10.5 mg/m(2)/d, but uninterrupted treatment was possible in five of six patients. At 17 mg/m(2)/d, skin toxicity required a 2-week treatment break for all three patients. The mean paclitaxel serum concentration at dose levels > or = 6.5 mg/m(2)/d exceeded that reported to achieve in vitro radiosensitization. Initial locoregional control was achieved in 14 (58%) of 24 of patients treated to 70 Gy, and control persisted in nine (38%). CONCLUSION: CI paclitaxel with concurrent RT is a feasible and tolerable regimen for patients with advanced SCCHN and good performance status. Preliminary response and survival data are encouraging and suggest that further study is indicated. The recommended phase II dose of paclitaxel by CI is 10.5 mg/m(2)/d with RT for SCCHN.

Phase I study of the orally administered butyrate prodrug, tributyrin, in patients with solid tumors.

Butyrates have been studied as cancer differentiation agents in vitro and as a treatment for hemoglobinopathies. Tributyrin, a triglyceride with butyrate molecules esterified at the 1, 2, and 3 positions, induces differentiation and/or growth inhibition of a number of cell lines in vitro. When given p.o. to rodents, tributyrin produces substantial plasma butyrate concentrations. We treated 13 patients with escalating doses of tributyrin from 50 to 400 mg/kg/day. Doses were administered p.o. after an overnight fast, once daily for 3 weeks, followed by a 1-week rest. Intrapatient dose escalation occurred after two courses without toxicity greater than grade 2. The time course of butyrate in plasma was assessed on days 1 and 15 and after any dose escalation. Grade 3 toxicities consisted of nausea, vomiting, and myalgia. Grades 1 and 2 toxicities included diarrhea, headache, abdominal cramping, nausea, anemia, constipation, azotemia, lightheadedness, fatigue, rash, alopecia, odor, dysphoria, and clumsiness. There was no consistent increase in hemoglobin F with tributyrin treatment. Peak plasma butyrate concentrations occurred between 0.25 and 3 h after dose, increased with dose, and ranged from 0 to 0.45 mM. Peak concentrations did not increase in three patients who had dose escalation. Butyrate pharmacokinetics were not different on days 1 and 15. Because peak plasma concentrations near those effective in vitro (0.5-1 mM) were achieved, but butyrate disappeared from plasma by 5 h after dose, we are now pursuing dose escalation with dosing three times daily, beginning at a dose of 450 mg/kg/day.

Binding of transforming growth factor-beta to cell surface proteins varies with cell type.

Transforming growth factor-beta (TGF beta 1 and TGF beta 2) bind to several different cell surface proteins, including a high Mr proteoglycan. We found that on primary and early passage cultures of fibroblasts, chondroblasts, and osteoblasts TGF beta 1 binds to both the high Mr proteoglycan and to lower Mr components, whereas on epithelial, endothelial, and lymphoid-derived cells TGF beta 1 only binds to the lower Mr species. With cell lines, this distinction is lost. Further analysis indicated that binding to the high Mr proteoglycan is not necessary for TGF beta 1 induced regulation of DNA, collagen and fibronectin synthesis, change in cell morphology, or reorganization of the actin cytoskeleton. We propose that the lower Mr components are the active receptors mediating these events.

Studies in cranial suture biology: Part I. Increased immunoreactivity for TGF-beta isoforms (beta 1, beta 2, and beta 3) during rat cranial suture fusion.

The mechanisms involved in normal cranial suture development and fusion as well as the pathophysiology of craniosynostosis, a premature fusion of the cranial sutures, are not well understood. Transforming growth factor-beta isoforms (TGF-beta 1, beta 2, and beta 3) are abundant in bone and stimulate calvarial bone formation when injected locally in vivo. To gain insight into the role of these factors in normal growth and development of cranial sutures and the possible etiology of premature cranial suture fusion, we examined the temporal and spatial expression of TGF-beta isoforms during normal cranial suture development in the rat. In the Sprague-Dawley rat, only the posterior frontal cranial suture undergoes fusion between 12 and 22 days of age, while all other cranial sutures remain patent. Therefore, immunohistochemical analysis of the fusing posterior frontal suture was compared with the patent sagittal suture at multiple time points from the fetus through adult. Whereas the intensity of immunostaining was the same in the posterior frontal and sagittal sutures in the fetal rat, there was increased immunoreactivity for TGF-beta isoforms in the actively fusing posterior frontal suture compared with the patent sagittal suture starting 2 days after birth and continuing until approximately 20 days. There were intensely immunoreactive osteoblasts present during fusion of the posterior frontal suture. In contrast, the patent sagittal suture was only slightly immunoreactive. A differential immunostaining pattern was observed among the TGF-beta isoforms; TGF-beta 2 was the most immunoreactive isoform and was also most strongly associated with osteoblasts adjacent to the dura and the margin of the fusing suture. Since the increased expression of TGF-beta 2 during suture fusion suggested a possible regulatory role, recombinant TGF-beta 2 was added directly to the posterior frontal and sagittal sutures in vivo to determine if suture fusion could be initiated. Exogenously added TGF-beta 2 stimulated fusion of the ectocranial surface of the posterior frontal suture. These data provide evidence for a regulatory role for these growth factors in cranial suture development and fusion. Additionally, the intense immunostaining for TGF-beta 2 in the dura mater underlying the fusing suture supports a role for the dura mater in suture fusion. It is possible that premature or excessive expression of these factors may be involved in the etiopathogenesis of craniosynostosis and that modulation of the growth factor profile at the suture site may have potential therapeutic value.

Bone morphogenetic proteins (BMP-2 and BMP-3) promote growth and expression of the differentiated phenotype of rabbit chondrocytes and osteoblastic MC3T3-E1 cells in vitro.

We studied the effects of highly purified bone morphogenetic protein 2 and 3 (BMP-2 and -3) on growth plate chondrocytes and osteoblastic cells in vitro and compared to TGF-beta. A mixture of BMP-2 and 3 (BMPs) strongly stimulated DNA synthesis of chondrocytes in the presence of fibroblast growth factor (FGF). BMPs induced rapid maturation of chondrocytes at a growing stage: BMPs transformed the cells into rounded cells and induced marked accumulation of cartilage matrix; TGF-beta slightly reduced matrix accumulation and changed cell morphology into spindle-like in the presence of FGF. Moreover, exposure of chondrocytes to BMPs resulted in a dramatic increase of the putative approximately 80 kD PTH receptors expressed on the cell surface. In multilayered chondrocytes at the calcifying stage, BMPs stimulated alkaline phosphatase (ALPase) activity but TGF-beta inhibited it. In osteoblastic MC3T3-E1 cells, BMPs were found to be the most potent stimulator of ALPase activity thus far described: ALPase in the cells treated with approximately 100 ng/ml of BMPs reached 5- to 20-fold over the basal, whereas TGF-beta inhibited expression of ALPase activity in these cells. The stimulatory action of BMPs overrode the inhibition of ALPase activity by TGF-beta when the cells were incubated with TGF-beta and BMPs. BMPs also upregulated expression of the approximately 80 kD PTH receptor on the cells. These results suggest that BMPs have unique biologic activities in vitro that lead to growth and phenotypic expression of cells playing a critical role in endochondral bone formation.

Multiple hormonal mechanisms for the control of collagen synthesis in an osteoblast-like cell line, MMB-1.

An isolated osteoblast-like cell line (MMB-1) was used to study the hormonal regulation of collagen synthesis in bone cells. Collagen synthesis was measured by incorporation of [3H]proline into collagenase-digestible and collagenase-non-digestible proteins after exposure of the cells in culture to varying concentrations of PTH, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], osteoclast-activating factor, and insulin. Collagen synthesis was inhibited by 10(-10) M 1,25-(OH)2D3 and 3 X 10(-10) M PTH after 9-12 h of treatment. Osteoclast-activating factor at 10(-10) M also inhibited collagen synthesis. Insulin at 10(-8) M increased collagen synthesis without stimulating proline incorporation into noncollagen proteins. No effect on collagen synthesis was observed with 24,25-(OH)2D3. Inhibition of collagen synthesis was also observed when cells were treated with either 3 X 10(-5) M 8-bromo-cAMP or 3 X 10(-5) M (Bu)2cAMP. For all agents tested, the onset of the effects was gradual, with differences from controls beginning at 4-8 h, and maximal effects occurring only after 24 h or more of treatment. The collagen synthesized by these cells remained associated primarily with the cell monolayer and was estimated to be greater than 90% type I collagen. No detectable changes in the type or composition of collagen synthesized were found with any of the hormonal treatments. These studies indicate that the synthesis of collagen in bone cells is under multihormonal control, with both cAMP-dependent and cAMP-independent mechanisms involved. The MMB-1 cell line offers a suitable model system for studies of the interactions of hormones in the control of bone turnover.

The combination of insulin-like growth factor I and insulin-like growth factor-binding protein-3 reduces insulin requirements in insulin-dependent type 1 diabetes: evidence for in vivo biological activity.

Insulin-like growth factor-I (IGF-I) enhances insulin action in normal subjects and in patients with both type 1 and 2 diabetes; however, its administration is associated with significant side effects in a high percentage of patients. The coadministration of IGF binding protein-3 (IGFBP-3, the predominant IGF binding protein in serum) with IGF-I limits IGF-I inducible side effects, but it does not attenuate the ability of IGF-I to enhance protein synthesis and bone accretion; therefore, we determined whether IGF-I/IGFBP-3 would retain biological activity in type 1 DM and limit side effects associated with free IGF-I administration. Twelve patients received recombinant human IGF-I plus IGFBP-3 (2 mg/kg-day) by continuous sc infusion for 2 weeks. Each subject served as his own control; and, during a paired 2-week period, each received a placebo infusion. The order of the treatments was randomized. Subjects were placed on a constant caloric intake but were allowed to adjust insulin doses to maintain appropriate levels of glycemic control. Subjects measured blood glucose four times per day at home and kept a log of their insulin use. Frequent sampling for glucose, insulin, and GH was conducted during four inpatient study periods, one at the beginning and one at the end of each 2-week study interval. During IGF-I/IGFBP-3, insulin doses were reduced by 49%, and mean serum glucose was reduced by 23%. Free insulin levels obtained during frequent sampling in hospital fell 47% on IGF-I/IGFBP-3, compared with control, but showed no change with placebo. Concomitant glucose measurements did not differ in the two treatment groups. There was no change in body weight. Fructosamine levels decreased by 12%, but this was not significant (P < 0.1). Fasting triglyceride was unchanged, but cholesterol declined from 170 +/- 24 to 149 +/- 31 mg/dL (P < 0.05). IGFBP-2 (an IGF-I-dependent responsive variable) rose from 141 +/- 56 to 251 +/- 98 ng/mL (P < 0.01) on IGF-I/IGFBP-3. To analyze the mechanism by which IGF-I/IGFBP-3 might reduce insulin requirements, the change in serum GH was quantified. Mean GH levels were reduced by 72%, from 2.48 to 0.55 ng/mL (P < 0.001). An equal number (40%) of drug- and placebo-treated subjects had minor hypoglycemic episodes at home that required adjustment of insulin doses. No episode was classified as severe. In contrast to previous studies with free IGF-I, there were no cases of edema, headache, jaw pain, retinal edema, or Bell's palsy. No subject withdrew because of drug complications. These findings indicate that IGF-I/IGFBP-3 is biologically active on carbohydrate metabolism, as measured by a decrease in insulin requirements in patients with type 1 diabetes. Further studies will be required to determine the long-term safety and efficacy of this combination in patients with insulin resistance and diabetes.

Cytotoxic activity of N1- and N8-aziridinyl analogs of spermidine.

Two isomeric aziridine-containing analogs of spermidine, a polyamine, were synthesized and evaluated for cytotoxic activity against cancer cell lines. Replacement of one of the primary amino groups of spermidine with an aziridinyl functionality yielded either N1-aziridinylspermidine [N-(3-aziridinylpropyl)-1,4-diaminobutane] or N8-aziridinylspermidine [N-(4-aziridinylbutyl)-1,3-diaminopropane]. N1-Aziridinylspermidine was cytotoxic in vitro against L1210 murine leukemia cells (IC50 0.15 microM) and HL60 human leukemia cells (IC50 0.11 microM). N8-Aziridinylspermidine was slightly less potent against L1210 (IC50 0.31 microM) and HL60 (IC50 0.30 microM) cells. When screened by the Developmental Therapeutics Program of the National Cancer Institute, these compounds proved cytotoxic against a wide variety of tumor types. Both compounds inhibited incorporation of radiolabeled thymidine, uridine, and valine into tricholoracetic acid-precipitable material by L1210 cells. Aminoguanidine did not affect the potency of the aziridinylspermidines.

In vitro metabolism by mouse and human liver preparations of halomon, an antitumor halogenated monoterpene.

OBJECTIVES: To characterize the enzymes responsible for and metabolites produced from the metabolism of halomon, a halogenated monoterpene that is isolated from the red algae Portieria hornemanii and has in vitro activity in the NCI screen against brain, renal, and colon cancer cell lines. MATERIALS AND METHODS: Mouse and human liver fractions, prepared by homogenization and differential centrifugation, were incubated with halomon, extracted with toluene, and analyzed by gas chromatography. RESULTS: In the presence of NADPH, mouse-liver 9,000-g supernatant (S9) fractions metabolized halomon, but boiled S9 fractions did not. NADH could not substitute for NADPH. Further separation of murine hepatic S9 fractions produced a microsomal fraction that contained all of the halomon-metabolizing activity; cytosol had none. Carbon monoxide reduced murine hepatic microsomal metabolism of halomon, whereas an anaerobic, N2 environment greatly accelerated the disappearance of halomon. Human hepatic microsomes metabolized halomon and required NADPH to do so. Carbon monoxide completely inhibited human hepatic microsomal metabolism of halomon. Unlike murine hepatic microsomal metabolism of halomon, anaerobic conditions did not enhance the metabolism of halomon by human hepatic microsomes. Neither 100 microM diethyldithiocarbamate, 1 microM quinidine, 100 microM ciprofloxacin, 3 microM ketoconazole, nor 100 microM sulfinpyrazone inhibited the metabolism of halomon by human hepatic microsomes. Both murine and human hepatic microsomes produced a metabolite of halomon. The mass spectrum of this metabolite indicated the loss of one chlorine atom and one bromine atom. CONCLUSIONS: Halomon is metabolized by mouse and human hepatic cytochrome P-450 enzymes, the identities of which remain unknown. Hepatic metabolism of halomon is very consistent with the concentrations of halomon measured in mouse tissues and urine after i.v. administration of the drug.

Plasma pharmacokinetics, bioavailability, and tissue distribution in CD2F1 mice of halomon, an antitumor halogenated monoterpene isolated from the red algae Portieria hornemannii.

The purpose of the present study was to define the plasma pharmacokinetics, bioavailability, and tissue distribution in mice of halomon, a halogenated monoterpene from Portieria hornemanii that is active in vitro against brain-, renal-, and colon-cancer cell lines. Halomon formulated in cremophor:ethanol:0.154 M NaCl (1:1:6, by vol.) was injected i.v. at 20, 60, 90, or 135 mg/kg into female CD2F1 mice. Doses of 135 mg/kg were also given i.p., s.c., and by enteral gavage to female CD2F1 mice and i.v. to male CD2F1 mice. Plasma halomon concentrations were measured with a gas-chromatography system using electron-capture detection. Halomon concentrations were also determined in the brains, hearts, lungs, livers, kidneys, spleens, skeletal muscles, fat, red blood cells, and, if present, testes of mice given 135 mg/kg i.v. Halomon plasma pharmacokinetics were well fit by a two-compartment, open linear model and were linear between 20 and 135 mg/kg. Population estimates of parameters describing halomon plasma pharmacokinetics in female CD2F1 mice were developed with a standard two-stage technique and also by simultaneous modeling of data from 20-, 60-, 90-, and 135-mg/kg i.v. studies in female mice. Halomon bioavailability was 45%, 47%, and 4% after i.p., s.c., and enteral dosing, respectively. Urinary excretion of the parent compound was minimal. Halomon was distributed widely to all tissues studied but was concentrated and persisted in fat. Halomon concentrations measured in the brain were comparable with concomitant concentrations detected in plasma and most other tissues. These data and models are helpful in the simulation and evaluation of conditions produced by preclinical screening and toxicology studies.

Antitumor activity, distribution, and metabolism of 13-cis-retinoic acid as a single agent or in combination with tamoxifen in established human MCF-7 xenografts in mice.

PURPOSE: The efficacy of 13-cis-retinoic acid (13-CRA) given as a single agent or in combination with tamoxifen (TAM) was determined in athymic nude mice bearing advanced s.c. MCF-7 human breast cancers. METHODS: 13-CRA alone was given by gavage at doses ranging from 26.4 to 200 mg/kg. TAM alone was given by gavage at doses of 7.5, 15, 30, or 60 mg/kg. For combination studies, each dose of TAM was followed 4 h later by 13-CRA at doses of 25, 50, 100, or 200 mg/kg. All treatments began on day 12 and were continued for 3 weeks. RESULTS: The median time to two doublings recorded for the control and for 13-CRA and TAM given as single agents at the highest dose were 22.2, 29.2, and 54.7 days, respectively. In combination, 100 and 200 mg/kg 13-CRA with 7.5 mg/kg TAM resulted in a delay in tumor growth at least as high as that achieved with highest-dose TAM alone, but the effect was not synergistic. Pharmacokinetic analysis of 13-CRA was performed in plasma, liver, and tumor from mice bearing 0.5- to 2.0 g carcinomas following a single dose of 100 mg/kg 13-CRA. Results showed that 13-CRA was metabolized differently in various tissues, but concentrations of 13-CRA detected in tumor were in the range reported to be active in vitro. all-trans-Retinoic acid (ATRA) concentrations were about 5% of the 13-CRA concentrations detected in plasma, 68% of those found in liver, and 20% of those found in tumor. 4-oxo-CRA represented between 2% and 10% of 13-CRA concentrations detected in plasma and liver but was not detected in tumor. Furthermore there was no difference in peak plasma 13-CRA concentrations found in the same tissues at 30 min after a single dose or after the eighth dose of 100 mg/kg 13-CRA or 13-CRA and TAM. Mean 13-CRA concentrations detected in liver and tumor were 50-90% and 16-30% of plasma peak concentrations, respectively. No difference in 4-oxo-CRA concentration was observed between the treatment groups. CONCLUSIONS: These data suggest that 13-CRA is not effective against established human breast tumor xenografts despite the stability of the pharmacokinetics of 13-CRA and the generation of ATRA as a metabolite. The addition of 13-CRA to TAM did not improve the efficacy of TAM against these estrogen-receptor-positive xenografts.