An early phase II study of CPT-11: a new derivative of camptothecin, for the treatment of leukemia and lymphoma.

An early phase II study of a new camptothecin analog and an inhibitor of topoisomerase I, CPT-11, was conducted in 62 patients with refractory leukemia and lymphoma by four different treatment schedules in a multiinstitutional cooperative study. CPT-11 therapy resulted in four complete remissions (CRs) and three partial remissions (PRs) in 29 assessable non-Hodgkin's lymphoma (NHL) patients, one PR in three Hodgkin's disease (HD), one CR and one PR in 11 acute lymphoblastic leukemia (ALL), and one PR in 15 acute myelogenous leukemia (AML) patients. Single infusion of 200 mg/m2 every 3 to 4 weeks produced no response in both leukemia and lymphoma patients. Sixty-minute infusions of 40 mg/m2/d for 5 days every 3 to 4 weeks or for 3 days weekly produced four CRs (17%) and four PRs (17%) in 24 patients with malignant lymphoma. Sixty-minute infusions of 20 mg/m2 twice a day for 7 days every 3 to 4 weeks resulted in one CR and two PRs in 12 patients with acute leukemia. No response was seen in an acute leukemia patient by another treatment schedule. CPT-11 was effective in two (15%) of 13 primarily refractory leukemia and lymphoma cases, in two of four relapsed cases, and in seven (17%) of 41 relapsed and refractory cases. Major side effects were leukopenia (91%) and gastrointestinal (GI) (76%). CPT-11 was shown to be effective against refractory leukemia and lymphoma, and thus deserves further clinical study; the novel antitumor activity mode of this drug predicts no cross-resistance to presently available antitumor drugs.

Transection of aortic depressor nerve fails to raise blood pressure in spontaneously hypertensive rats.

Although central resetting via the aortic depressor nerve (ADN) has been known to occur in spontaneously hypertensive rats, the function of the ADN is not yet clear in these animals. To determine whether a baroreflex via the ADN can act to regulate blood pressure during hypertension, blood pressures were measured following ADN transection in spontaneously hypertensive and normotensive rats, and the reflex changes of heart rate and the sympathetic nerve activity were recorded during the pressor response to phenylephrine infusions. Blood pressures were significantly raised 1 day after ADN transections in normotensive rats and continued so for 7 d. Blood pressures were not changed in sham operated rats. In spontaneously hypertensive rats, ADN transections did not alter blood pressures in comparison with sham operated controls. On the seventh day after transections, all rats were anaesthetised with urethane and pressor responses to phenylephrine were examined. Bradycardic and sympathoinhibitory responses to the elevation of blood pressure caused by phenylephrine infusions were significantly smaller in ADN transectioned normotensive rats than in sham operated controls. In spontaneously hypertensive rats, the bradycardic and sympatho-inhibitory responses were not reduced by ADN transections. These findings suggest that the impaired baroreflex via the ADN can contribute to the development of hypertension in the spontaneously hypertensive rat.

4-Piperidinopiperidine-resistant lymphoma cells were resistant to dexamethasone- and A23187-induced apoptosis.

4-Piperidinopiperidine is a side residue of CPT-11, a derivative of camptothecin. We have previously established a 4-piperidinopiperidine-resistant lymphoma cell line, 4-pp-R, which was co-resistant to CPT-11. We report here that this cell line is cross-resistant to dexamethasone and A23187 which induce apoptosis in parent RVC cells. Examination of apoptosis-related gene expression by RT-PCR showed that bcl-2 expression was greater in 4-pp-R than in RVC. p53, bax and bcl-xL were expressed at the same level in 4-pp-R and RVC cells. These results suggest that upregulation of bcl-2 in 4-pp-R cells is related to the resistance to CPT-11 as well as to A23187 or dexamethasone.

Clonidine abolishes exaggerated pressor responses to shaker stress in spontaneously hypertensive rats.

To determine whether clonidine would attenuate exaggerated pressor responses to stress in spontaneously hypertensive rats (SHR), pressor responses to shaker stress were recorded following intracerebroventricular (icv) injections of clonidine in conscious SHR. Pressor responses to shaker stress were significantly larger in SHR than in Wistar-Kyoto rats (WKY). Increased sympathetic nerve responses to shaker stress were also significantly greater in SHR than in WKY. Exaggerated pressor responses in SHR were partly attenuated by adrenalectomy and abolished by clonidine (icv). By contrast, 1-Sar,8-Ile angiotensin II (icv) was ineffective. These findings suggest that in SHR, the alpha-adrenergic system in the brain may contribute to exaggerated pressor responses to shaker stress, but not the brain renin-angiotensin system.

Altered noradrenergic projection to hypothalamus via baroreflex in spontaneously hypertensive rats.

To determine whether noradrenergic projections to the posterior hypothalamus via baroreflex are altered in the hypertensive state, the extracellular norepinephrine (NE) content of the posterior hypothalamus was measured in both spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) after sinoaortic denervation (SAD) or sham operation. In WKY, blood pressure (BP) and extracellular NE content 24 h after SAD were significantly higher than those of sham-operated rats. Contrarily, SAD did not increase both BP and NE in the posterior hypothalamus of SHR. These results suggest that the noradrenergic pathway via the baroreflex is impaired in SHR. This mechanism may play an important role in the development and maintenance of hypertension in SHR.

Increased extracellular concentration of norepinephrine in the hypothalamus by sinoaortic denervation.

To determine whether baroreflex can affect the norepinephrine system in the hypothalamus, the extracellular concentration of norepinephrine were measured by the brain dialysis technique in sinoaortic denervated rats (SAD). Twenty-four hours after sinoaortic denervation, systolic blood pressure and heart rate were significantly elevated, and norepinephrine concentration in perfusate of the posterior hypothalamus was significantly higher in SAD rats than in sham-operated rats. These results suggest that baroreflex could modify the activity of noradrenergic neuron projecting to the posterior hypothalamus.

Central attenuation of baroreflex by angiotensin II in normotensive and spontaneously hypertensive rats.

To determine whether angiotensin II (A II) can modify baroreflex centrally and contribute to the central resetting of baroreflex in spontaneously hypertensive rats (SHRs), the aortic depressor nerve (ADN) was electrically stimulated following intracerebroventricular (ICV) administration of A II and/or A II analog in urethane-anesthetized normotensive Wistar rats (WKYs) and SHRs. Electric stimulation of the ADN elicited frequency-dependent depressor, bradycardic, and sympatho-inhibitory responses. Angiotensin II, administered ICV, dose-dependently attenuated these responses induced by ADN stimulation in normotensive rats. We found, however, these attenuations could be abolished by ICV pretreatment with A II analog. Vasopressin (ICV) did not change any responses to ADN stimulation. The vasopressor and sympatho-inhibitory responses to ADN stimulation were significantly less in SHRs when compared with those in WKYs: In SHRs, A II analog completely cancelled the A II-attenuated responses to ADN stimulation. These findings suggest that A II can centrally attenuate baroreflex, which might be independent from vasopressin, and in the brain A II can contribute to the central resetting of baroreflex in SHRs.

Central attenuation of baroreflex precedes the development of hypertension in DOCA-salt-treated rats.

To determine whether baroreflex is changed centrally before the development of hypertension in DOCA-salt treated rats, aortic depressor nerve was stimulated electrically 5 days after DOCA-salt treatment in urethane-anesthetized rats: compared with those of sham-operated control rats, blood pressure was not elevated in either awake or anesthetized rats. Aortic depressor nerve (ADN) stimulation elicited frequency-dependent vasodepressor, bradycardiac, and sympatho-inhibitory responses in both DOCA-salt-treated and control rats. However, the responses to ADN stimulation were significantly smaller in DOCA-salt-treated rats. These findings suggest that baroreflex is attenuated centrally before the development of hypertension and this attenuation may contribute to the pathogenesis of DOCA-salt hypertension.

Paraventricular nucleus lesions attenuate the development of hypertension in DOCA/salt-treated rats.

To determine whether paraventricular nucleus (PVN) can play a role in the hypertension in DOCA/salt-treated rats, DOCA/salt hypertension was produced in PVN lesions and sham-operated rats. In lesioned rats, the development of hypertension was significantly attenuated (day 7: 132 +/- 3 v 157 +/- 5 mm Hg, P less than 0.01; day 14: 132 +/- 3 v 157 +/- 5 mm Hg, P less than 0.01; day 21: 189 +/- 2 v 224 +2- 6 mm Hg, P less than 0.01). Lesions lowered systolic blood pressure in even control rats. Mean blood pressure (mBP) from awake free moving rats was also significantly lower in lesioned DOCA/salt-treated rats than those of sham-operated DOCA/salt-treated rats (155 +/- 14 mm Hg v 193 +/- 13, P less than 0.01), while mBP was not different between lesioned and sham-operated control rats. The reduction of mBP by hexamethonium injections was significantly larger in sham-operated DOCA/salt-treated rats than those of lesioned DOCA/salt rats. (-53 +/- 3% v -45 +/- 2, P less than 0.05). Plasma norepinephrine and epinephrine were significantly elevated in DOCA/salt-treated rats, however, PVN lesions inhibited significantly those elevations. 1-Deaminopenicillamine, 4-valine, 8-D-arginine Vasopressin (dPVDAVP) injections did not affect BP and heart rate in all rats. Body weight, water intake, urine volume, urine Na, K, and vasopressin excretion, and urine osmorality were not altered by lesions. These findings suggest that PVN contributes to development of hypertension in DOCA/salt-treated rats with sympathetic nervous activations.

The inhibitory mode of aphidicolin on DNA synthesis in NaCl-treated HeLa cell nuclei.

Isolated HeLa cell nuclei were treated with NaCl at various concentrations and inhibition by aphidicolin of DNA synthesis in the treated nuclei was studied. The inhibition was either noncompetitive or of the mixed type with respect to each dNTP when the nuclei were treated with NaCl at concentrations lower than 0.08 M. However, aphidicolin was a competitive inhibitor with respect to dCTP and a non-competitive or mixed type inhibitor with respect to the other 3 dNTPs when they were treated with NaCl at concentrations higher than 0.1 M. These results suggest the presence of nuclear factor(s) responsible for the changes in the inhibitory mode of aphidicolin on endogenous nuclear DNA synthesis.

Inhibition of eukaryotic DNA polymerase-alpha by polydeoxynucleotides.

Effects of polydeoxynucleotides on the activity of DNA polymerase-alpha from sea urchin embryos were studied. Poly(dG), poly(dC) and poly(dC)-oligo(dG)12-18 inhibited DNA polymerase-alpha activity in the activated DNA-directed reaction but poly(dA), oligo(dT)12-18, and poly(dA)-oligo(dT)12-18 did not inhibit the activity. The inhibitory mode of poly(dC)-oligo(dG)12-18 or poly(dC) was competitive with activated DNA and that of poly(dG) was noncompetitive with activated DNA. Using poly(dA)-oligo(dT)12-18 as a template-primer, the inhibition with either poly(dG) or poly(dC)-oligo(dG)12-18 was competitive with the template-primer. These kinetic results indicate that each of the template-primers tested binds to an identical site on DNA polymerase-alpha. Similar results were obtained with DNA polymerase-alpha from HeLa cells.

Reconstitution of endogenous DNA synthesis in isolated nuclei and template activity of chromatin with respect to mode of inhibition by aphidicolin.

We succeeded in reconstituting the endogenous nuclear DNA synthesis of the sea urchin. Endogenous DNA synthesis of isolated nuclei was reconstituted by mixing the salt-treated nuclei (chromatin exhibiting essentially no endogenous DNA synthesis) and the salt extract containing DNA polymerase-alpha. DNA synthesis in this reconstitution system showed a level of activity and a mode of inhibition by aphidicolin similar to those of the original isolated nuclei (noncompetitive with respect to dCTP). On the other hand, the inhibitory mode was competitive with respect to dCTP in DNA synthesis in the reconstituted system obtained from the chromatin and purified DNA polymerase-alpha, indicating that some other factor(s) in addition to DNA polymerase-alpha is necessary for the reconstitution with reference to the inhibitory mode of aphidicolin. We also studied the template activity of the chromatin. When chromatin was used as a template, inhibition by aphidicolin of DNA polymerase-alpha was noncompetitive and uncompetitive with respect to the template at high and low concentrations, respectively. Treatment of chromatin with 5 M urea gave urea-treated chromatin (nonhistone protein-deprived chromatin) and the extract (mainly nonhistone protein fraction). Inhibition by aphidicolin of DNA polymerase-alpha was uncompetitive with respect to the urea-treated chromatin. However, when chromatin reconstituted from the urea-treated chromatin and the extract was used as a template, the inhibitory mode by aphidicolin was similar to that with original chromatin, indicating that the nonhistone protein fraction contained factor(s) which modified the inhibitory mode of aphidicolin. Thus, the inhibitory mode of aphidicolin is a useful parameter for monitoring the resolution and reconstitution of endogenous DNA synthesis of isolated nuclei.

A comparative study of CHOP versus MEVP (mitoxantrone, etoposide, vindesine, prednisolone) therapy for intermediate-grade and high-grade non-Hodgkin's lymphoma: a prospective randomized study.

To evaluate a new combination chemotherapy with mitoxantrone (MXT), etoposide (VP-16), vindesine (VDS), and prednisolone (MEVP therapy) as a front-line chemotherapy for non-Hodgkin's lymphoma (NHL), a prospective randomized study comparing this therapy (28 patients) with CHOP therapy (29 patients) was conducted in 57 patients with intermediate-grade or high-grade NHL with stages II-IV. The MEVP therapy consisted of 10 mg/m2 of MXT intravenously on day 1, 2 mg/m2 of VDS intravenously on day 1, 200 mg/m2 of VP-16 orally on days 1-3, and 40 mg/m2 of prednisolone orally on days 1-5. This regimen was repeated every 3 weeks for up to 10 courses. Complete responses (CR) were achieved in 17 (63.0%) of the 27 evaluable patients treated with MEVP therapy, and in 20 (71.4%) of the 28 evaluable patients treated with CHOP therapy. Relapse-free survival rates and overall survival rates at 3 years were 58.8% and 46.4%, respectively, for the MEVP group and 70.0% and 54.0%, respectively, for the CHOP group. Granulocytopenia was more severer and associated infection episodes were more frequent in the MEVP group. MEVP therapy was effective as a front-line chemotherapy for intermediate- and high-grade NHL, although it was not superior to CHOP therapy in treatment effect and was not less toxic than CHOP.

Feasibility of salvage chemotherapy for refractory or relapsed non-Hodgkin's lymphoma with two topoisomerase II inhibitors, MST-16 and VP-16. MST-16 Study Group.

A feasibility study was carried out on the treatment for refractory and relapsed non-Hodgkin's lymphomas with a combination of two oral topoisomerase II inhibitors, MST-16 and VP-16. On the basis of the synergistic activity in preclinical studies and the schedule dependency in these drugs, low-dose and long-term administration was planned. For the anticipated myelosuppression, two different regimens were designed as an open label trial in this study. In Regimen I, 400 mg of MST-16 combined with 25 mg of VP-16 was administered daily. With this regimen, the response rate (RR)/median time to tumor progression (TTP) in all evaluable patients was 50% (2/4)8.5 months in low grade (indolent) lymphoma and 60% (6/10)/5.2 months in intermediate/high grade (aggressive) lymphomas. In Regimen II, 400 mg of MST-16 combined with 25 mg of VP-16 was administered intermittently (3 days a week or every other day). With this regimen, there was an RR/median TTP of 60% (3/5)/7.0 months in indolent lymphoma and 33.3% (4/12)/1.1 months in aggressive lymphoma. A major side effect in both of these regimens was myelosuppression, with the incidence of grades 3 and 4 toxicity being higher in Regimen I than in Regimen II. The other side effects were uncommon and not severe. These findings indicated that two regimens were tolerated well and were promising for refractory and relapsed aggressive non-Hodgkin's lymphomas. To define the anti-tumor activity and safety of these regimens precisely, large-scale prospective randomized trials are necessary.

(2"-R)-4'-o-tetrahydropyranyladriamycin, a new anthracycline derivative; its effectiveness in lymphoid malignancies.

Thirty-eight patients with adult acute lymphoblastic leukemia (ALL) or non-Hodgkin's lymphoma (NHL) were treated intravenously with (2"-R)-4'-o-Tetrahydropyranyladriamycin (THP) at a dose of 10 mg/m2 for 5 consecutive days. Seven complete and 15 partial responses were observed in 35 evaluable patients (overall response rate, 62.8%). Both antitumor activity and antitumor spectrum were similar to those for doxorubicin. Since the patients who had had chemotherapy previously, including other kinds of anthracycline, responded rather poorly to THP, cross-resistance between THP and other anthracyclines may be present. Leukopenia and thrombocytopenia were dose-limiting factors. Nausea and vomiting episodes were mild, and epilation was also minimal. Although the observation period was short and a cumulative dose was not large enough to evaluate cardiotoxicity, there were no abnormal EKG changes or clinical signs of cardiotoxicity in this study. THP is a potent antitumor agent in the treatment of lymphoid malignancies.

A lymphoma cell line resistant to 4-piperidinopiperidine was less sensitive to CPT-11.

CPT-11 is a promising new anticancer drug in which 4-piperidinopiperidine is a side-chain structure. In the present studies, we examined the role played by 4-piperidinopiperidine in the pharmacological activity of CPT-11. When T-cell lymphoma RVC cells were incubated with 4-piperidinopiperidine at concentrations higher than 50 micrograms/ml, the cells underwent apoptosis with a nucleosomal ladder of chromosomal DNA on agarose gels in a dose-dependent manner. We then established a cell line resistant to 4-piperidinopiperidine (4-pp-R), which was about 20-fold more resistant to 4-piperidinopiperidine than the parent RVC cells. Moreover, 4-pp-R cells showed coresistance to CPT-11. However, the growth rate and cell cycle population of 4-pp-R cells were not different from those of the parent RVC cells, and there were no differences between the two cells lines with regard to their drug transport system. CPT-11-metabolizing activity, their activity and amount of topoisomerase I, or their sensitivity to either SN-38 or etoposide, suggesting that the cytotoxicity of CPT-11 is not a consequence of the activity of its metabolite SN-38. The present studies suggested that resistance to CPT-11 is in part due to insensitivity to 4-piperidinopiperidine and its metabolites, since 4-piperidinopiperidine was cytotoxic and 4-pp-R cells were less sensitive to CPT-11.

Phase I study of NKT-01.

A phase I study of NKT-01 (deoxyspergualin), which is a derivative of an antitumor antibiotic, spergualin, was performed by a cooperative study group. NKT-01 was given intravenously by 3-h infusion. The effect of single administration was studied prior to evaluation of daily administration for 5 consecutive days. In all, 5 and 33 patients with various malignancies, including leukemia, were entered into the trials of single and daily administration, respectively. In the single-administration study, all patients were evaluable and no clear adverse effect was observed at doses ranging from 20 to 320 mg/m2. In the daily-administration study, 28 evaluable patients (16 men and 12 women; median age, 55.5 years) were treated with a daily dose of 20-500 mg/m2. Toxicities such as myelosuppression, mild nausea/vomiting, anorexia, alopecia, tongue and perioral numbness, and hypotension were observed dose-dependently during or after the treatment. Grade 2 leukopenia, thrombocytopenia, and anemia were experienced at a dose of 500 mg/m2. These usually recovered to normal values by approximately 3 weeks after treatment. A pharmacokinetic analysis of single administration revealed rapid plasma clearance, with mean half-lives for the alpha and beta phases being 28 min and 6.9 h, respectively. Approximately 12% of the infused dose was excreted into the urine in unmetabolized form. The pharmacokinetic parameters obtained after 5-day administration were similar to those recorded after single administration. Concerning treatment response, a transient but significant reduction in the number of leukemic cells was observed in one patient with adult T-cell leukemia. In this study, perioral numbness, hypotension, and hematological toxicity were concluded to be dose-limiting, with the maximal acceptable dose being 500 mg/m2. The recommended dose for a phase II study of NKT-01 against solid tumors was judged to be 400 mg/m2 given daily by 3-h infusion for 5 days, every 3 weeks. In hematological malignancies, however, higher myelosuppressive schedules of administration should be investigated.

Phase II study of NK313 in malignant lymphomas: an NK313 Malignant Lymphoma Study Group trial.

Liblomycin (NK313) is a bleomycin analog that has proved to be associated with less pulmonary toxicity and with more potent antitumor activity than bleomycin in animal tumors. In a phase I study, pulmonary toxicity was not observed, whereas myelosuppression was the dose-limiting factor. The maximum tolerated dose was 140 mg/m2 given once a week for 4 weeks. In the present phase II study, patients with malignant lymphomas received liblomycin at 80 or 100 mg/m2 by intravenous infusion over 15 min once a week for 4 weeks. A total of 39 patients were entered, and 31 [4 with Hodgkin's disease (HD) and 27 with non-Hodgkin's lymphoma (NHL)] were evaluable. The median age of the patients was 52 years (range, 22-74 years), and their performance status ranged from 0 to 3. In all, 28 of the patients had a history of intensive anticancer chemotherapy. Responses were evaluated according to WHO criteria. We obtained 1 complete remission and 9 partial remissions (PRs), for an overall response rate of 37%, in the 27 patients with NHL, whereas 1 PR was achieved in the 4 patients with HD. In all, 9 PRs (32.1%) were obtained in patients who had been exposed to prior chemotherapy, including 4 PRs (33.3%) in 12 patients who had previously been treated with bleomycin. Myelosuppression and nausea and vomiting were the major toxicities, which occurred in about 50% of the patients, and myelosuppression was severe in two patients treated at a dose of 100 mg/m2. We concluded that liblomycin demonstrated significant antitumor activity against malignant lymphomas.

Sympathetic inhibition and attenuation of spontaneous hypertension by PVN lesions in rats.

To determine whether the paraventricular nucleus (PVN) contributes to the development of hypertension in spontaneously hypertensive rats (SHR), we compared cardiovascular responses to ganglionic blockade with hexamethonium or vasopressin antagonism with dPVAVP in sham-operated or PVN lesioned SHR and Wistar-Kyoto rats (WKY). Lesions were produced electrolytically when the rats were 5 weeks old. During the next 3 weeks, tail-cuff measurements showed that the development of hypertension in SHR was inhibited, while systolic pressure in WKY was unaffected. Mean pressures recorded directly from the femoral artery at 8 weeks of age were lower in lesioned than in sham-operated SHR (141 +/- 5 vs 110 +/- 3 mm Hg, P less than 0.05), but did not differ in corresponding WKY groups (110 +/- 4 vs 112 +/- 5 mm Hg). Depressor responses to ganglionic blockade induced by i.v. injection of hexamethonium (25 mg/kg) were significantly larger in sham-operated than in lesioned SHR (-41 +/- 4% vs -28 +/- 3%, P less than 0.05). By contrast, vasopressin antagonism with dPVAVP did not alter blood pressure in all rat groups. In 24-h urine samples, excretion of vasopressin was unaffected, but that of norepinephrine was significantly reduced in lesioned SHR. These findings suggest that the PVN contributes to the development of spontaneous hypertension by sympathetic activation without increasing vasopressin secretion.