Circadian output, input, and intracellular oscillators: insights into the circadian systems of single cells.

Circadian output comprises the business end of circadian systems in terms of adaptive significance. Work on Neurospora pioneered the molecular analysis of circadian output mechanisms, and insights from this model system continue to illuminate the pathways through which clocks control metabolism and overt rhythms. In Neurospora, virtually every strain examined in the context of rhythms bears the band allele that helps to clarify the overt rhythm in asexual development. Recent cloning of band showed it to be an allele of ras-1 and to affect a wide variety of signaling pathways yielding enhanced light responses and asexual development. These can be largely phenocopied by treatments that increase levels of intracellular reactive oxygen species. Although output is often unidirectional, analysis of the prd-4 gene provided an alternative paradigm in which output feeds back to affect input. prd-4 is an allele of checkpoint kinase-2 that bypasses the requirement for DNA damage to activate this kinase; FRQ is normally a substrate of activated Chk2, so in Chk2(PRD-4), FRQ is precociously phosphorylated and the clock cycles more quickly. Finally, recent adaptation of luciferase to fully function in Neurospora now allows the core FRQ/WCC feedback loop to be followed in real time under conditions where it no longer controls the overt rhythm in development. This ability can be used to describe the hierarchical relationships among FRQ-Less Oscillators (FLOs) and to see which are connected to the circadian system. The nitrate reductase oscillator appears to be connected, but the oscillator controlling the long-period rhythm elicited upon choline starvation appears completely disconnected from the circadian system; it can be seen to run with a very long noncompensated 60-120-hour period length under conditions where the circadian FRQ/WCC oscillator continues to cycle with a fully compensated circadian 22-hour period.

A circadian clock in Neurospora: how genes and proteins cooperate to produce a sustained, entrainable, and compensated biological oscillator with a period of about a day.

Neurospora has proven to be a tractable model system for understanding the molecular bases of circadian rhythms in eukaryotes. At the core of the circadian oscillatory system is a negative feedback loop in which two transcription factors, WC-1 and WC-2, act together to drive expression of the frq gene. WC-2 enters the promoter region of frq coincident with increases in frq expression and then exits when the cycle of transcription is over, whereas WC-1 can always be found there. FRQ promotes the phosphorylation of the WCs, thereby decreasing their activity, and phosphorylation of FRQ then leads to its turnover, allowing the cycle to reinitiate. By understanding the action of light and temperature on frq and FRQ expression, the molecular basis of circadian entrainment to environmental light and temperature cues can be understood, and recently a specific role for casein kinase 2 has been found in the mechanism underlying circadian temperature-compensation. These data promise molecular explanations for all of the canonical circadian properties of this model system, providing biochemical answers and regulatory logic that may be extended to more complex eukaryotes including humans.

Design and synthesis of highly potent fumagillin analogues from homology modeling for a human MetAP-2.

New fumagillin analogues were designed through structure-based molecular modeling with a human methionine aminopeptidase-2. Among the fumagillin analogues, cinnamic acid ester derivative CKD-731 showed 1000-fold more potent proliferation inhibitory activity on endothelial cell than TNP-470.

Synthesis and in vitro cytotoxicity of hexacyclic camptothecin analogues.

A series of C(7)-N-alkylaminoethyl-C(10), C(11)-methylenedioxy- and ethylenedioxy-camptothecin (3a-g, 4a-b) were prepared. Their syntheses and in vitro cytotoxicity were reported. Among 15 derivatives, 3a and 3b showed more potent cytotoxicity than Camptothecin, especially in CAOV-3 cell line.

A simple model of circadian rhythms based on dimerization and proteolysis of PER and TIM.

Many organisms display rhythms of physiology and behavior that are entrained to the 24-h cycle of light and darkness prevailing on Earth. Under constant conditions of illumination and temperature, these internal biological rhythms persist with a period close to 1 day ("circadian"), but it is usually not exactly 24h. Recent discoveries have uncovered stunning similarities among the molecular circuitries of circadian clocks in mice, fruit flies, and bread molds. A consensus picture is coming into focus around two proteins (called PER and TIM in fruit flies), which dimerize and then inhibit transcription of their own genes. Although this picture seems to confirm a venerable model of circadian rhythms based on time-delayed negative feedback, we suggest that just as crucial to the circadian oscillator is a positive feedback loop based on stabilization of PER upon dimerization. These ideas can be expressed in simple mathematical form (phase plane portraits), and the model accounts naturally for several hallmarks of circadian rhythms, including temperature compensation and the per(L) mutant phenotype. In addition, the model suggests how an endogenous circadian oscillator could have evolved from a more primitive, light-activated switch.

Antitumor activity of 7-[2-(N-isopropylamino)ethyl]-(20S)-camptothecin, CKD602, as a potent DNA topoisomerase I inhibitor.

We developed a novel water-soluble camptothecin analogue, CKD602, and evaluated the inhibition of topoisomerase I and the antitumor activities against mammalian tumor cells and human tumor xenografts. CKD602 was a nanomolar inhibitor of the topoisomerase I enzyme in the cleavable complex assay. CKD602 was found to be 3 times and slightly more potent than topotecan and camptothecin as inhibitors of topoisomerase, respectively. In tumor cell cytotoxicity, CKD602 was more potent than topotecan in 14 out of 26 human cancer cell lines tested, while it was comparable to camptothecin. CKD602 was tested for the in vivo antitumor activity against the human tumor xenograft models. CKD602 was able to induce regression of established HT-29, WIDR and CX-1 colon tumors, LX-1 lung tumor, MX-1 breast tumor and SKOV-3 ovarian tumor as much as 80, 94, 76, 67, 87% and 88%, respectively, with comparable body weight changes to those of topotecan. Also the therapeutic margin (R/Emax: maximum tolerance dose/ED58) of CKD602 was significantly higher than that of topotecan by 4 times. Efficacy was determined at the maximal tolerated dose levels using schedule dependent i.p. administration in mice bearing L1210 leukemia. On a Q4dx4 (every 4 day for 4 doses) schedule, the maximum tolerated dose (MTD) was 25 mg/kg per administration, which caused great weight loss and lethality in < 5% tumor bearing mouse. This schedule brought significant increase in life span (ILS), 212%, with 33% of long-term survivals. The ex vivo antitumor activity of CKD602 was compared with that of topotecan and the mean antitumor index (ATI) values recorded for CKD602 were significantly higher than that noted for topotecan. From these results, CKD602 warrants further clinical investigations as a potent inhibitor of topoisomerase I.

A proposal for temperature compensation of the circadian rhythm in Drosophila based on dimerization of the per protein.

Recently Goldbeter suggested an interesting model of circadian rhythms based on feedback inhibition by the PER protein on its own rate of transcription. In his model, the long delay necessary for generating 24 h periodicity is associated with slow phosphorylations of PER protein in the cytoplasm, assuming that only highly phosphorylated forms of PER are able to enter the nucleus and there interfere with transcription of the per gene. By casting this molecular mechanism in mathematical form, Goldbeter showed that it is consistent with many known features of circadian oscillations in PER abundance. However, he did not address one of the most important characteristics of the circadian rhythm: the near constancy of the 24 h period over a broad temperature range. Huang, Curtin, and Rosbash have recently suggested that dimerization of the PER protein is involved in temperature compensation of the circadian rhythm in Drosophila, because in mutant flies lacking the PER dimerization domain, the period is strongly dependent on temperature. We incorporate this idea into Goldbeter's model by introducing parallel pathways of phosphorylation of PER monomers and dimers. We assume that both monomers and dimers can be transported into the nucleus as long as at least one PER subunit is multiply phosphorylated. Temperature compensation in our model arises from opposing effects of temperature (T) on the rate of association of PER monomers and the rate of nuclear import of PER protein. In mutant flies, when PER subunits cannot dimerize, the period of the oscillation increases with T, so we assume that the rate constant for nuclear import is a decreasing function of T. To compensate for this effect in wild-type flies, we assume that the rate of association of PER subunits is an increasing function of T. The mathematical model reveals the relationship between these opposing tendencies that must be satisfied to achieve effective temperature compensation.

Nucleoside conjugates. 15. Synthesis and biological activity of anti-HIV nucleoside conjugates of ether and thioether phospholipids.

A series of the anti-HIV nucleoside conjugates of either (1-O-alkyl) and thioether (1-S-alkyl) lipids linked by a pyrophosphate diester bond has been synthesized as micelle-forming prodrugs of the nucleosides to improve their therapeutic efficiency. These include AZT 5'-diphosphate-rac-1-S-octadecyl-2-O-palmitoyl-1-thioglycerol (1), 3'-azido-2',3'-dideoxyuridine 5'-diphosphate-rac-1-S-octadecyl-2-O-palmitoyl-1-thioglycerol (2) 2',3'-dideoxycytidine 5'-diphosphate-rac-1-S-octadecyl-2-O-palmitoyl-1-thioglycerol (3), and AZT 5'-diphosphate-rac-1-O-tetradecyl-2-O-palmitoylglycerol (4). The conjugates form micelles by sonication (mean diameters ranging 6.8-55.5 nm). Conjugate 1 protected 80% of HIV-infected CEM cells as low as 0.58 microM and lost the protection at 180 microM due to prevailing cytotoxicity, while the conjugate started to show the cytotoxicity at 100 microM. Pharmacokinetics studies showed a significant increase of half-life values (t1/2) of AZT and AZddU2 (respective t1/2 = 5.69 and 6.5 h) after administration of conjugates 1 and 2, while those after administration of AZT and AZddU were 0.28 and 0.89 h, respectively. The fractions of the prodrugs 1 and 2 converted to the parent compounds AZT and AZddU were 36% and 55%, respectively. The results indicate that AZT and AZddU thioether lipid conjugates 1 and 2 warrant further investigation.

Nucleoside conjugates. 14. Synthesis and antitumor activity of 1-beta-D-arabinofuranosylcytosine conjugates of ether lipids with improved water solubility.

A series of ara-CDP-rac-1-O-alkyl-2-O-acylglycerols (9a-f), analogues of highly active ara-CDP-rac-1-O-hexadecyl-2-O-palmitoylglycerol (1) and Cytoros2 (2), was prepared, and solubility, lipophilicity, and structure-activity relationships of these conjugates were investigated. Conjugates 9a-f containing sn-1 alkyl (< C16) and sn-2 fatty acyl (< C14) and sn-1 alkyl (< C14) and sn-2 fatty acyl (< C16) substituents of the glycerol were water-soluble by shaking, while those with the sn-1 alkyl (> C16) and the sn-2 fatty acyl (> C16) such as conjugate 1 were sparingly soluble. Conjugates 9a-c,e were almost completely solubilized in water by shaking. However, a large portion of conjugates 9d and 9f in water by shaking exist in micelles with mean diameters ranging 7.0-55.2 nm. The partition coefficients (1-octanol/PBS) of the water-soluble conjugates were about 9-18 times greater than that of ara-C. A single dose (300 mg/kg) of conjugates 9d and 9f produced a significant increase in life span (ILS 206 to > 543%) with 17-67% long-term survivors (> 45 days) in mice bearing ip-implanted L1210 lymphoid leukemia. These results were comparable to those of the previous conjugate 1 and Cytoros (2). In contrast, conjugates 9a-c,e at single doses were less effective (ILS 69-178% with no long-term survivors). However, two (qd, 1, 7) or three (qd 1, 5, 9) divided doses of these conjugates were found to be as effective as a single dose of the previous conjugates. The three divided doses (150 mg/kg per day) of conjugates 9d, 9e, and 9f produced a remarkable antitumor activity in L1210 leukemic mice (ILS > 350% with > 50% long-term survivors). Because of the convenient formulation and the significant antitumor activities, the water-soluble conjugates 9d, 9e, and 9f warrant further investigation.

Synthesis and antitumor activity of 1-beta-D-arabinofuranosylcytosine conjugates of optical isomers of ether and thioether lipids.

Four 1-beta-D-arabinofuranosylcytosine conjugates (ara-C) (1a, b and 2a, b) of sn-1 and sn-3 isomers of 1-O-octadecyl-2-O-palmitoylglycerol and its 1-S-alkyl analogue have been synthesized, and their antitumor activity against L1210 lymphoid leukemia in mice were compared with those of the previous conjugates (3a, b) of racemates in order to determine the significance of chirality of the glycerol moieties for activity. Administration (i.p.) of a single dose (300 mg/kg) of conjugates of sn-1 (1a), sn-3 (2a) and rac (3a) isomers of the ether lipid increased lifespan of i.p. implanted L1210 lymphoid leukemic DBA/2J mice by 169, 175 and 236%, respectively. The sn-1 (1b), sn-3 (2b), and rac (3b) isomers of the thioether lipid with a single dose of 300 mg/kg produced an increase in lifespan values of 238, 263 and 250%, respectively. The results indicate that chirality of the glycerol moieties appears not to be critical for the activity, and racemates 3a and 3b are promising prodrugs of ara-C for further clinical investigations.

Nucleoside conjugates. 13. Synthesis and antitumor activity of 1-beta-D-arabinofuranosylcytosine conjugates of thioether lipids with improved water solubility.

A series of ara-CDP-rac-1-S-alkyl-2-O-acyl-1-thioglycerols (3-12), analogues of highly active Cytoros2 (1), was prepared, and solubility, lipophilicity, and structure-activity relationships of these conjugates were investigated. The conjugates with sn-1 alkyl (< C18) and sn-2 fatty acyl (< C14) substituents of the thioglycerol were water-soluble, while those with the sn-1 alkyl (> C14) and the sn-2 fatty acyl (> C16) were sparingly soluble. The latter formed micelles upon sonication. Conjugate 7 containing the sn-1 tetradecyl and the sn-2 palmitoyl (C16) groups formed micelles by both sonication and shaking. The partition coefficients (1-octanol/PBS) of the water-soluble conjugates were about 20 times greater than that of ara-C. The water-insoluble showed a more than 40 times increase. A single dose of the micelle-forming conjugates 7 and 10 produced a significant increase in life span (ILS > 421%) with 50% long-term survivors (> 45 days) in mice bearing ip-implanted L1210 lymphoid leukemia. These results were comparable to those of previous micelle-forming conjugate 1 (Cytoros). In contrast, the water-soluble conjugates at single doses were less effective (ILS 81-386% with 0-33% long-term survivors). However, three divided doses of the water-soluble conjugates were found to be as effective as a single dose of micellar solution of the water-insoluble. The results indicate that conjugate 7 and most of the water-soluble derivatives warrant further investigation.

1-beta-D-arabinofuranosylcytosine conjugates of ether and thioether phospholipids. A new class of ara-C prodrug with improved antitumor activity.

The 1-beta-D-arabinofuranosylcytosine (ara-C) conjugates 1-O-alkyl (ether) and 1-S-alkyl (thioether) phospholipids, being analogues of ara-CDP-sn-1,2-O-dipalmitoylglycerol (1), showed significant antitumor activity against L1210 and P388 leukemia in vivo. The more active conjugates include the 1-O-alkyl analogues, ara-CDP-rac-1-O-hexadecyl-2-O-palmitoylglycerol (2) and ara-CDP-rac-1-O-octa-decyl-2-O-palmitoylglycerol (3), and the corresponding 1-S-alkyl analogues, ara-CDP-rac-1-S-hexadecyl-2-O-palmitoyl-1-thioglycerol (4) and ara-CDP-rac-1-S-octadecyl-2-O-palmitoyl-1-thioglycerol (5, Cytoros). The conjugates were formulated by sonication, in which the conjugates existed as discs (size 0.01-0.04 microns). Among the conjugates of the three different phospholipids, the 1-S-alkyl analogues 4 and 5 displayed the strongest antitumor activity against L1210 leukemia in mice, followed by the 1-O-alkyl (2 and 3) and the 1-O-acyl (1) analogues. The 1-S-alkyl analogue 5 was considerably more effective than the 1-O-acyl analogue 1 against myelomonocytic WEHI-3B leukemia in mice. Conjugate 5 (Cytoros) showed a significant therapeutic activity in mice with colon 26 carcinoma, M5076 sarcoma, and C-1300 neuroblastoma. Furthermore, this agent inhibited liver metastases of M5076 sarcoma. Conjugates 3 and 5 also inhibited the metastasis of 3-Lewis lung carcinoma to the lungs of mice. Cytoros (5) and its analogues, with other ether and thioether phospholipids, appear to offer increased therapeutic benefit to mice with tumors.

Formulation, stability, and antitumor activity of 1-beta-D-arabinofuranosylcytosine conjugate of thioether phospholipid.

1-beta-D-Arabinofuranosylcytosine 5'-diphosphate-rac-1-S-octadecyl-2-O- palmitoyl-1-thioglycerol (ara-CDP-DL-PTBA) is an effective stable 1-beta-D-arabinofuranosylcytosine (ara-C) conjugate of thioether phospholipid against a variety of transplantable tumors in mice. The conjugate was formulated in a micellar solution by sonication, in which the conjugate exists as micellar discs (size, 0.01 to 0.04 micron). Analyses on thin-layer and high-pressure liquid chromatography showed that the conjugate was chemically stable upon storage at 3-4 degrees C for more than a 6-mo period. However, stored at room temperature for 3 mo it began to degrade (3 to 11%) to 1-beta-D-arabinofuranosylcytosine 5'-monophosphate and phosphatidic acid. At 3-4 degrees C, the micellar structure remained generally unchanged for 6 mo (size, less than 0.1 micron). Samples stored for 4 mo at room temperature formed some larger vesicles (size, 0.1 to 0.4 micron). Antitumor activity against i.p. implanted L1210 leukemia in mice remained relatively constant with samples stored for 6 mo at 3-4 degrees C or 3 mo at room temperature. 1-beta-D-Arabinofuranosylcytosine 5'-triphosphate (ara-CTP) levels were elevated (greater than 500 pmol/10(7) cells) in L1210 leukemia cells within 1 h following i.p. administration of 400 mg/kg of ara-CDP-DL-PTBA to mice. More importantly, retention of cellular ara-CTP was prolonged (greater than 24 h) in these tumor cells as compared with ara-C treatments. Administration of ara-CDP-DL-PTBA to mice with colon 26 carcinoma (s.c.) resulted in both significant antitumor activity with an increased life span greater than 100% and decreased tumor size. The conjugate also demonstrated a dose-dependent therapeutic effect in mice with M5076 sarcoma (s.c.) as demonstrated by decreases in tumor size and liver metastases. Overall, ara-CDP-DL-PTBA, a stable lipid conjugate of ara-C in a micellar solution, appears to offer substantial therapeutic benefit to mice with leukemia and solid tumors warranting its further development and clinical investigation.

Nucleoside conjugates. 11. Synthesis and antitumor activity of 1-beta-D-arabinofuranosylcytosine and cytidine conjugates of thioether lipids.

Five 1-beta-D-arabinofuranosylcytosine conjugates and two cytidine conjugates of thioether lipids (1-S-alkylthioglycerols) linked by a pyrophosphate diester bond have been prepared and their antitumor activity against an ara-C2 sensitive (L1210/0) and two ara-C resistant L1210 lymphoid leukemia sublines in mice were evaluated. These prodrugs of ara-C include ara-CDP-rac-1-S-hexadecyl-2-O-palmitoyl-1-thioglycerol (8a), ara-CDP-rac-1-S-octadecyl-2-O-palmitoylthioglycerol (8b), and ara-CDP-rac-1-S-octadecyl-2-O-methyl(or -ethyl, -hexadecyl)thioglycerols (8c-e). The cytidine conjugates include CDP-rac-1-S-octadecyl-2-O-palmitoyl(or -methyl)- 1-thioglycerols (9a and 9b). Sonicated solutions of the conjugates existed in the form of micellar disks (size 0.01-0.04 microns). Single doses (200-400 mg/kg) of 8a and 8b produced significant increase in life span (257-371%) in mice bearing ip implanted L1210/0 leukemia. In contrast, conjugates 8c-e were less effective (ILS 19-75%) and cytidine conjugates (9a and 9b) were ineffective. Even though 8a and 8b were found to be curative in a high percentage of mice bearing ip implanted partially ara-C resistant L1210 subline [L1210/ara-C(I)], they were completely ineffective against deoxycytidine kinase deficient ara-C resistant L1210 subline [L1210/ara-C(II)]. However, the present results, together with the previous, demonstrate that 8a and 8b are promising new prodrugs of ara-C with improved efficacy.

Therapeutic activity of 1-beta-D-arabinofuranosylcytosine conjugates of lipids in WEHI-3B leukemia in mice.

Two new conjugates of 1-beta-D-arabinofuranosylcytosine (ara-C) and lipids were tested for therapeutic activity in myelomonocytic WEHI-3B leukemia in mice. Both conjugates were superior to equimolar mixtures of their respective parent compounds and to ara-C alone. IP treatment was found effective after either IP or IV transplantation of the leukemia. The thioether-linked lipid conjugate ara-CDP-D,L-PTBA showed considerably higher efficacy than the ester-linked lipid conjugate ara-CDP-L-dipalmitin. The optimal therapeutic regimen of ara-CDP-D,L-PTBA consisted of 60 mg/kg given IP qd 1-5 after transplantation of the WEHI-3B leukemia.

Nucleoside conjugates. 10. Synthesis and antitumor activity of 1-beta-D-arabinofuranosylcytosine 5'-diphosphate-1,2-dipalmitins.

Three 1-beta-D-arabinofuranosylcytosine 5'-diphosphate-1,2-dipalmitins from L-, D-, and DL-alpha-dipalmitoylphosphatidic acids have been synthesized and their antitumor activity against two ara-C2 resistant L1210 lymphoid leukemia sublines in mice were evaluated. These new prodrugs of ara-C include ara-CDP-L-dipalmitin, ara-CDP-D-dipalmitin, and ara-CDP-DL-dipalmitin. The L and DL isomers produced significant increase in life span (greater than 400%) and four to five long-term survivors (greater than 45 days) out of six animals bearing ip implanted partially ara-C resistant L1210 subline [L1210/ara-C (I)], while the D isomer displayed a marginal activity (ILS 100-121%). In contrast, the L isomer was completely ineffective against deoxycytidine kinase deficient ara-C resistant L1210 subline [L1210/ara-C (II)]. However, the results demonstrate that the L and DL isomers of ara-CDP-dipalmitin are promising new prodrugs of ara-C with improved efficacy.

Influence of 1-beta-D-arabinofuranosylcytosine conjugates of lipids on the growth and metastasis of Lewis lung carcinoma.

Five different lipid conjugates of 1-beta-D-arabinofuranosylcytosine (ara-C) were tested for their therapeutic activity on the growth and metastasis of Lewis lung carcinoma (3-LL). Among 1 ester-linked lipid conjugate (Ara-CDP-L-dipalmitin), 3 1-O-alkyl-lipid conjugates (ara-CDP-D,L-PBA, ara-CDP-D,L-PCA, ara-CDP-D,L-MBA) and a thioether-lipid conjugate (ara-CDP-D,L-PTBA) ara-CDP-D,L-PTBA, ara-CDP-D,L-PBA, and ara-CDP-L-dipalmitin produced the strongest tumor growth inhibition and increase of surviving animals in C57Bl6-mice bearing i.p.-implanted 3-LL. Furthermore these conjugates were superior to the parent compounds alone, or equimolar mixtures of the alkyllysophospholipid derivatives ET-18-OCH3 and ara-C. Successful therapeutic regimen consisted of 80-100 mg conjugate/kg, given i.p. daily for five consecutive days. Similar regimen injected shortly after the surgical removal of the primary tumor as adjuvant chemotherapy inhibited the metastasis of 3-LL to the lungs of the animals as demonstrated by an increase of survival time and the number of surviving animals.

Antineoplastic activity of conjugates of lipids and 1-beta-D-arabinofuranosylcytosine.

Five different lipid conjugates of 1-beta-D-arabinofuranosylcytosine (ARA-C) were tested in comparison with ARA-C, the ether lipid ET-18-OCH3 (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) and their equimolar mixtures. The compounds were tested in vitro for cytotoxicity in the trypan blue dye exclusion test with cells from six different leukemias, one glioblastoma and two bronchogenic carcinomas of human origin. The compounds were given in vivo to assess their therapeutic activity against 3-Lewis lung carcinoma (3-LL) of syngeneic C57Bl6 mice. Although some of the conjugates have shown cytotoxic activity in vitro against the cell samples tested, they have not revealed higher cytotoxicity than ET-18-OCH3, ARA-C or their equimolar mixtures. In these experiments, ARA-CDP-D,L-MBA was the conjugates significantly inhibited tumor growth and also increased survival of C57Bl6 mice with intraperitoneally (ip) implanted 3-LL. In these experiments, ARA-CDP-D,L-PTBA, ARA-CDP-D,L-PBA, ARA-CDP-L-dipalmitin and ARA-CDP-D,L-PCA were more active than either the parent compounds ARA-C and ET-18-OCH3 alone or their equimolar mixtures. Furthermore, when the conjugates were injected as adjuvant chemotherapy shortly after the surgical removal of the primary 3-LL, they inhibited the metastasis of 3-LL to the lungs of the animals, demonstrated by an increase of the survival time and the number of surviving animals. The mode of action of these new antineoplastic compounds still is speculative.