Flt3-ligand administration after radiation therapy prolongs survival in a murine model of metastatic lung cancer.

An ineffective tumor-specific immune response from inadequate/incompetent antigen presentation could contribute to the failure in tumor control and its dissemination. Dendritic cells (DCs) have been shown to present antigen from apoptotic cells. We hypothesized that Flt3-ligand (Flt3L) therapy, which expands DCs in vivo, in combination with local tumor radiotherapy (RT), should improve antigen presentation from dying, irradiated tumor cells. RT + Flt3L reduced pulmonary metastases in a murine model of Lewis lung carcinoma and significantly improved survival in C57Bl/6 mice with established footpad tumors. Mice treated with Flt3L alone showed delayed tumor growth but eventually succumbed to tumor progression. The combination therapy of RT + Flt3L failed to impact survival in immunodeficient athymic mice, implicating the role of T cells in prolonging survival. These results support an attractive strategy of sequential RT and immunotherapy with Flt3L to enhance tumor antigen presentation, which may produce therapeutic responses against disseminated cancer and improvement in survival.

Acute hypotensive responses to peptide inhibitors of renin in conscious monkeys: an effect on blood pressure independent of plasma renin inhibition.

In order to investigate the hypotensive mechanisms of action of peptide renin inhibitors, blood pressure responses to five renin inhibitors were compared with those to the angiotensin converting enzyme inhibitor, enalaprilat, in conscious African green and rhesus monkeys. (3S-4S)-4-amino-5-cyclohexyl-3-hydroxy pentanoic acid (ACHPA)-containing renin inhibitory peptide (ACRIP) and enalaprilat both decreased blood pressure in euvolemic and volume-depleted African green monkeys. However, while a maximum dose of enalaprilat reduced blood pressure to 80 +/- 4 and 56 +/- 4 mmHg in the euvolemic and volume-depleted monkeys, respectively, ACRIP lowered pressure to life-threatening levels (less than 40 mmHg) under both conditions. The relative potencies of ACRIP and four other renin inhibitors for inhibiting in vitro plasma renin activity (PRA; IC50) were compared with their potencies in reducing blood pressure by 15 mmHg (ED15 mmHg) and lowering blood pressure more than enalaprilat in volume-depleted rhesus monkeys. All renin inhibitors lowered blood pressure significantly beyond the maximal response to enalaprilat. Despite a significant correlation (r = 0.99, P less than 0.05) between the in vitro PRA inhibitory potency and the in vivo ED15 mmHg, doses which lowered blood pressure beyond the maximal responses to enalaprilat were not significantly correlated (r = 0.53, P greater than 0.05) with the in vitro PRA IC50 values. Furthermore, the profound depressor responses to renin inhibitors in rhesus monkeys were accompanied by increases in the heart rate and decreases in pulse pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasmin-activated prodrugs for cancer chemotherapy. 1. Synthesis and biological activity of peptidylacivicin and peptidylphenylenediamine mustard.

Many tumors contain elevated levels of plasminogen activator and thus produce elevated levels of the protease plasmin in the milieu of the tumor. We have hypothesized, therefore, that it should be possible to prepare peptidyl prodrug derivatives of anticancer drugs that would be locally activated by tumor-associated plasmin. As an initial test of this hypothesis, we synthesized the peptidyl prodrugs of the anticancer drugs (alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin, AT-125) and N,N-bis(2-chloroethyl)-p-phenylenediamine (phenylenediamine mustard) by mixed anhydride coupling of the parent drug with the protected tripeptide, Boc-D-Val-Leu-Lys(Boc)-OH, followed by deprotection with trifluoroacetic acid. The prodrugs showed an increased selective in vitro cytotoxicity for Rous sarcoma virus transformed chicken embryo fibroblasts (which produce elevated levels of plasminogen activator) compared to nontransformed fibroblasts (which produce low levels of plasminogen activator). In the presence of the plasmin inhibitor, p-nitrophenyl p'-guanidinobenzoate at 2 micrograms/mL, the selectivity of the phenylenediamine mustard prodrug was reduced, but there was no effect on the cytotoxicity of the free drug. Furthermore, the prodrug analogue D-valylleucyl-D-lysylphenylenediamine mustard (in which L-Lys has been replaced by D-Lys) was inactive. Finally, the prodrug derivative of acivicin did not display selective toxicity for transformed cells when the cells were cultured in plasminogen-free medium. These results suggest that plasmin hydrolysis is necessary for the activation of the prodrugs. The prodrugs were tested in vivo for antitumor activity. The prodrug of acivicin, like acivicin itself, was inactive against the B16 melanoma, a murine tumor that produces high levels of plasminogen activator. This prodrug was active against the M5076 carcinoma, a tumor that displays only moderate levels of plasminogen activator; however, despite the fact that the prodrug was 2- to 3-fold less toxic on a molar basis than acivicin, there was no evidence of an increased therapeutic index. The prodrug of phenylenediamine mustard was also slightly less toxic than the parent drug, but again there was no evidence for an improved therapeutic index against the B16 tumor.

Plasmin-activated prodrugs for cancer chemotherapy. 2. Synthesis and biological activity of peptidyl derivatives of doxorubicin.

We have synthesized peptidyl prodrugs of doxorubicin (Dox) designed to be selective substrates of plasmin. Such prodrugs might be locally activated by the elevated levels of plasmin produced near many solid tumors under the action of tumor-associated plasminogen activators. One such prodrug, 3'-(D-Val-Leu-Lys)-Dox, was obtained via a mixed-anhydride coupling with isobutyl chloroformate between the protected peptide Fmoc-D-Val-Leu-N epsilon-Fmoc-Lys-OH and doxorubicin, followed by removal of the Fmoc groups with anhydrous ammonia. Compared to doxorubicin, the prodrug showed about a 7-fold improved selective cytotoxicity against chicken embryo fibroblasts transformed with the Rous sarcoma virus (which produce high levels of plasminogen activator) compared to normal cells (which produce low levels of plasminogen activator). However, the prodrug was a very poor plasmin substrate, and although in vivo tests against the murine B16 melanoma showed that the prodrug was active, the maximum T/C obtained was less than that achieved by doxorubicin even at 25 times the molar concentration of prodrug. Qualitatively similar results were obtained for a far more hydrophobic prodrug, 3'-(Boc-Val-Leu-Lys)-Dox. These results demonstrate that peptidyl prodrugs of doxorubicin designed as plasmin substrates are more selective anticancer agents in vitro than doxorubicin itself but that the bulky anthracycline moiety probably prevents efficient plasmin-catalyzed conversion to the active parent drug, so that, in their present form, these drugs are not potent enough to allow a determination as to whether or not they are more selective in vivo.

(3-Amino-2-oxoalkyl)phosphonic acids and their analogues as novel inhibitors of D-alanine:D-alanine ligase.

The dipeptide D-alanyl-D-alanine is an essential precursor of bacterial peptidoglycan; thus, blocking its formation is a possible target for the design of novel antibacterial agents. The synthesis of this dipeptide by bacterial D-alanine:D-alanine ligase requires ATP. In analogy with glutamine synthetase, we hypothesized a mechanism for this enzyme involving the intermediacy of D-alanyl phosphate. Several (3-amino-2-oxoalkyl)phosphonic acids and their analogues have been synthesized as possible inhibitory mimics of this proposed intermediate. The most active of them, (3(R)-amino-2-oxobutyl)phosphonic acid (8a) and the corresponding aza analogue (22), were effective ligase inhibitors although they had no significant antibacterial activity. The ligase inhibition of these compounds is consistent with an acyl phosphate displacement step in the mechanism of DAla-DAla ligase.

Triazolinone biphenylsulfonamide derivatives as orally active angiotensin II antagonists with potent AT1 receptor affinity and enhanced AT2 affinity.

Several series of 2,4-dihydro-2,4,5-trisubstituted-3H-1,2,4-triazol-3-ones with acidic sulfonamide replacements of tetrazole at the 2'-position of the biphenyl-4-ylmethyl side chain at N4 were prepared and tested as angiotensin II (AII) antagonists. Preferred substituents on the triazolinone ring were n-butyl at C5 and 2-(trifluoromethyl)phenyl at N2. Subnanomolar IC50 values at the AT1 receptor subtype were observed for a variety of acylsulfonamides, including aroyl, heteroaroyl, and cycloalkylcarbonyl derivatives. Certain other acidic sulfonamides, such as sulfonylcarbamates and disulfimides also displayed high affinity for the AT1 receptor. In addition, AT2 binding for some of these compounds was increased by as much as 1000-fold over the corresponding tetrazole (e.g., AT2 IC50 17 nM for the tert-butyl sulfonylcarbamate 92). When evaluated for inhibition of the AII pressor response, the benchmark benzoylsulfonamide 9 (L-159,913) was efficacious in several species and was superior to losartan (1a) in conscious rhesus monkeys. Several subsequent analogues, including the 2-chlorobenzoyl (18), (3-chlorothiophene-2-yl)carbonyl (51), ((S)-2,2-dimethylcyclopropyl)carbonyl (80), and tert-butoxycarbonyl (92) derivatives, were highly effective in rats, surpassing 9 and losartan in duration of action and/or potency. Compound 18 (L-162,223) displayed very prolonged AII antagonism in the rat model (> 24 h at 1 mg/kg iv). At 1 mg/kg po in rats, 18 and 92 (L-162,234) produced 85-87% peak inhibition of the AII pressor response with duration exceeding 6 h. The identification of triazolinone-based sulfonamide derivatives combining high AT1 affinity, considerably enhanced AT2 potency, and favorable in vivo properties provides insights relevant to the design of dual AT1/AT2 receptor antagonists.

Pregnancy induced mammary tumor specific effector cells are present long after parturition in a breast cancer model in rats.

Pregnancy is known to provide protection against 7,12 dimethylbenz[a]anthracene-(DMBA) induced mammary carcinogenesis in rats. We observed in earlier studies that splenocytes of parous rats have significant cytotoxicity against mammary tumor cells both in vitro and in vivo. However, it remains to be established how long these cytolytic cells persist after parturition in parous host. The present study was designed using parous rats, 36 or more days after parturition. We observed that fresh splenocytes from these rats had low cytolytic activity against mammary tumor cells. However, when these cells were re-stimulated with irradiated mammary tumor cells in vitro, they had significantly higher cytotoxicity against mammary tumor cells. These studies show for the first time that pregnancy induced cytotoxic splenocytes are present long after parturition and they can be restimulated in vitro to enhance the cytotoxic effect.

Plasma and erythrocyte copper level as an indicator of disease activity during malignancy.

Copper and zinc concentrations in erythrocytes, plasma and whole blood were determined in mice bearing spontaneous-, transplanted- and benezo[a]pyrene-induced-tumour. The transplanted tumours studied were Sarcoma 180, Ehrlich carcinoma, and Dalton's lymphoma. Copper concentration in the malignant-tumour-bearing mice showed significant increases in erythrocytes and plasma, when compared with their normal controls. However, the zinc concentrations, although depressed in the different constituents of blood, were not significant enough to warrant any attention. Utilization of erythrocyte copper level as an additional marker of cancer activity is discussed.

Synthesis and preliminary characterization of a novel substrate for gamma-glutamyl transferase. A potential anti-hepatoma drug.

9he synthesis and chemical characterization of the gamma-glutamyl adduct of phenylenediamine mustard is reported. The activity of this compound, gamma-[N,N-bis(2-chloroethyl)-p-phenylenediamine]-glutamate, as a substrate for gamma-glutamyl transferase is demonstrated and compared with the activity of glutathione. The possible use of this material as a directed anti-hepatoma agent is discussed.

Serum copper in malignant neoplasia with special reference to the cervix uteri.

Serum copper levels (SCl) were determined in normal patients and in patients with primary tumors of different sites, measurements being made before and after therapy. The serum copper was elevated in the presence of all the different types of tumor examined. In patients not responding to treatment and those who relapsed, the serum copper remained constant or became higher. SCl may provide an effective means of evaluating the extent of the disease and is of value in the estimation of prognosis after therapy.

In vivo pharmacology of a novel AT1 selective angiotensin II receptor antagonist, MK-996.

MK-996, N-(4'-(5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridin-3-yl- methyl)1,1'-biphenyl-2-yl)-sulfonylbenzamide, is a potent, orally active, highly selective, nonpeptide angiotensin II (AII) receptor antagonist. MK-996 prevents the pressor response to intravenous AII in the conscious rat, dog, and rhesus monkey (ED50, mg/kg; oral/intravenous = 0.067/0.014, 0.035/0.017, and 0.1/0.036, respectively). In the anesthetized chimpanzee, MK-996 (1 mg/kg, iv) produces 100% (peak) inhibition of the AII pressor response and is still active (52%) at 24 h. To our knowledge this pharmacologic profile in the rat, dog, rhesus monkey, and chimpanzee presents the least species variability of any AII receptor antagonist yet described. Responses to methoxamine and arginine vasopressin are not affected by MK-996. In aortic coarcted (high renin) rats, MK-996 (3 mg/kg, by mouth) reduces blood pressure to normotensive (< 120 mm Hg) levels without reflex tachycardia. This dose of MK-996 reduces blood pressure to approximately the same level as both losartan (3 mg/kg, by mouth) and enalapril (3 mg/kg, by mouth) in this model. The duration of antihypertensive activity of MK-996 is similar to enalapril and shorter than losartan at the doses tested. Additionally, in the rat MK-996 does not potentiate the vasodepressor response to bradykinin and completely prevents the ability of AII to stimulate an increase in plasma levels of aldosterone. Therefore, MK-996 is a potent, orally active, nonpeptide AII receptor antagonist with a long duration of action, little species variability, and anti-hypertensive activity.

Pharmacology of L-744,453, a novel nonpeptidyl endothelin antagonist.

L-744,453 ((+/-)3-[4-(1-carboxy-1-(3,4-methylenedioxyphenyl)methoxy)-3,5-diprop ylphenyl methyl]-3H-imidazo[4,5-c]pyridine) is an endothelin (ET) receptor antagonist from a new structural class, the dipropyl-alpha-phenoxyphenylacetic acid derivatives. L-744,453 competitively and reversibly inhibits [125I]-ET-1 binding to Chinese Hamster Ovary cells expressing cloned human ET receptors (K(i)s: hET(A)=4.3 nM; hET(B)=232 nM), and is selective for endothelin receptors compared to other peptide receptors. It is an antagonist of ET-1 stimulated phosphatidyl inositol hydrolysis in rat uterine slices (IC50=220 nM) and exhibits no agonist activity. This compound also inhibits ET-1 stimulated contraction of rat aortic rings with a K(b) value of 50 nM. L-744,453 protects against ET-1 induced lethality in mice after i.v. (AD50=13 mg/kg i.v.) or oral administration. This compound also antagonizes ET-1 induced increases in diastolic blood pressure in conscious normotensive rats (AD50=0.67 mg/kg i.v.) and anesthetized ferrets (AD50=1.6 mg/kg i.v.). L-744,453 is a potent, selective, orally active endothelin antagonist which may be useful in elucidating the role of endothelin in normal and pathophysiological states.

In vivo effect of chemically induced fibrosarcoma on copper metabolism of liver in mice.

Copper and ceruloplasmin concentrations were determined in different subcellular fractions of liver of mice bearing benzo(a)pyrene induced fibrosarcoma. Though the copper content was elevated in the nuclear, mitochondrial and lysosomal fractions of tumor bearing mice, the microsomal and soluble supernatant fractions showed a downward trend in their copper concentration when compared to the controls. Similarly, ceruloplasmin concentration in the different subcellular fractions also showed variable results. This study was aimed to ascertain the distribution of copper. The incorporation of radioactive copper in different fractions was also monitored. The possible reasons for the variation in copper and ceruloplasmin concentrations observed during malignancy in the tumor bearing animals, has been discussed.

Distribution of 64copper in the blood and different tissues of mice bearing induced fibrosarcoma.

To understand the nature of copper distribution during malignancy, 27 strains A/RB mice with fibrosarcoma were used. Tracer methodology with radioactive isotope of copper (64Cu) was applied. In the study made at different time intervals after isotope administration whole blood plasma and different tissues like liver, heart, spleen kidney and brain were investigated. Liver nuclear fractions were also examined. Distribution of 64Cu in different tissues reflected interesting results. While the incorporation pattern in the liver showed gradual fall from 8 h of isotope administration, the tumor tissue exhibited gradual elevation, compared to the controls. The effect of this finding is discussed.

Zinc in human malignancies.

Serum zinc level (SZL) was determined in normal patients and in patients with primary tumors of different sites, with measurements made before and after therapy. Serum zinc concentration was depressed in some of the tumor types examined. In some cases serum zinc concentration increased again after successful treatment. Diagnostic and prognostic value of the serum zinc concentration in malignancy is discussed.

Effect of fibrosarcoma induction on copper and ceruloplasmin concentration in different organs of the host.

The copper content and ceruloplasmin activity were determined in mice bearing benzo(a)pyrene induced fibrosarcoma. The copper level and ceruloplasmin activity in different organs of fibrosarcomatous mice varied when compared to their controls. Significant changes in copper and ceruloplasmin concentration were observed in the liver and tumor tissue of host mice bearing fibrosarcoma compared to controls. Disturbed copper metabolism at the hepatic level may account for the hypercupremia observed during malignancy.

Serum copper and caeruloplasmin activity in experimental malignancies.

Serum copper content and caeruloplasmin activity were measured in 595 Strain A male mice. The mice carried either benzo(a)pyrene induced fibrosarcoma, transplanted sarcoma-180, Dalton's lymphoma, or Ehrlich carcinoma. The serum copper concentration was raised in all the tumours when compared with normal controls. The observed fall in activity of caeruloplasmin during malignancy may be related to defective iron mobilization.

Sub-cellular distribution of copper and caeruloplasmin in chemically induced tumour tissue.

Copper and caeruloplasmin concentrations were determined in the subcellular fraction of tumour tissue from 15 Strain A mice bearing an induced fibrosarcoma. It was observed that copper concentration was higher in the mitochondrial fraction of tumour tissue when compared to that of tissues of similar site from normal controls. However, the caeruloplasmin concentration was lower in the various fractions of tumour tissue when compared to that of normal control tissue.

Evaluation of ceruloplasmin concentration in prognosis of human cancer.

The serum ceruloplasmin concentration was determined in cancer patients before and after radiotherapy, and after relapse of cancer, The ceruloplasmin concentration in patients who responded to therapy, decreased to the range of normal controls. In patients who did not respond to treatment, the ceruloplasmin concentration was more or less elevated. In patients with relapse of cancer, the ceruloplasmin concentration was higher than before treatment.

Effect of tumour regression on serum and tissue copper concentration in mice bearing induced fibrosarcoma.

Serum and tissue copper concentration was determined in mice bearing induced fibrosarcoma after successful chemotherapeutic treatment. The results showed a significant depression in copper concentration in the serum and tumour tissue after treatment with anti-cancer drugs. However, the liver copper concentration showed no significant change in treated groups of mice compared to before treatment.