The effect of vincristine–polyanion complexes in STEALTH liposomes on pharmacokinetics,toxicity and anti tumor activity

 Poly(ethylene glycol) (PEG)-derivatized liposome vehicles improve antitumor effectiveness of entrapped anthracyclines and vinca alkaloids. However, the plasma clearance of entrapped vincristine is substantially faster than the lipid phase or other entrapped aqueous markers, suggesting leakage out of the liposome during transit in the blood compartment. We tested the effect of altering the drug’s in vivo leakage rate on pharmacokinetics, toxicity, and antitumor activity of entrapped drug in rodent models. Suramin, heparin, and dextran sulfate were tested for their ability to produce a precipitable complex in vitro. PEG-derivatized liposomes were prepared with the complexing agent inside, and vincristine was driven inside using an ammonium gradient. The resulting preparations were found to have plasma distribution half-lives significantly longer than the formulation without a complex-forming agent. There was no increase in acute lethality, and in the case of the suramin-vincristine complex, the acute lethality was significantly reduced at the highest does level. Anti-tumor activity against the mouse mammary carcinoma MC2 was tested in a multiple-dose study. Free vincristine did not affect the tumor growth rate significantly, but at the same dose level all PEG-coated liposome formulations inhibited tumor growth markedly. The suramin containing formulation was as effective as the formulation lacking polyanion, but the heparin and dextran sulfate containing formulations were less effective. Thus, compounds which form insoluble complexes with vincristine alter in vivo plasma distribution phase pharmacokinetics without increasing acute lethality, but without a corresponding increase in anti-tumor activity. Received: 28 August 1995/Accepted: 21 February 1996     

Preclinical trial of doxorubicin entrapped in sterically stabilized liposomes in dogs with spontaneously arising malignant tumors

Purpose: To prospectively evaluate the short-term toxicoses associated with pegylated-liposomal doxorubicin (Doxil) administered to dogs with measurable tumors of various histologic types and sites. Preliminary information regarding efficacy was also generated. Methods: A group of 51 dogs with histologically confirmed malignancies received a total of 103 Doxil treatments given i.v. every 3 weeks at dosages ranging from 0.75 to 1.1 mg/kg in the context of a phase I dose-escalation trial. Acute and short-term toxicities as well as tumor response and duration of response were characterized. Results: The maximally tolerated dose in tumor-bearing dogs was established as 1.0 mg/kg i.v. every 3 weeks. The dose-limiting toxicity was a cutaneous toxicity clinically resembling palmar-plantar erythrodysesthesia (PPES). An overall response rate of 25.5% was observed with five complete responders and eight partial responders. Conclusions: Doxil appeared to be well tolerated at dosages similar to those tolerated for free doxorubicin in tumor-bearing dogs. PPES was the dose-limiting toxicity encountered, rather than myelosuppresion as is the case with free doxorubicin in dogs. Doxil as a single agent may have a broad spectrum of activity and deserves further evaluation. Received: 19 February 1996 / Accepted: 29 June 1996     

Intratumor distribution of doxorubicin following i.v. administration of drug encapsulated in egg phosphatidylcholine/cholesterol liposomes

 Purpose: A pharmacological evaluation of an egg phosphatidylcholine/cholesterol (55:45 mole ratio, EPC/Chol) liposome doxorubicin formulation was carried out. The objective was to define liposomal lipid and drug distribution within sites of tumor growth following intravenous (i.v.) administration to female BDF1 mice bearing either Lewis lung carcinoma, B16/BL6 melanoma, or L1210 ascitic tumors. Methods: Mice were injected i.v. with EPC/Chol liposomal doxorubicin, and plasma and tumor levels of lipid and drug were determined 1, 4 and 24 h later with radiolabeled lipid and fluorimetry or fluorescence microscopy, respectively. In addition, single-cell suspensions of the Lewis lung and B16/BL6 tumors were prepared and the presence of macrophages was determined with an FITC-labeled rat antimouse CD11b (MAC-1) antibody. Results: For mice bearing the Lewis lung solid tumors, there was a time-dependent accumulation of liposomal lipid, with a plateau of approximately 500 μg lipid/g tumor at 48 h. In contrast, the apparent plateau (μg doxorubicin/g tumor) for doxorubicin was achieved at 1 h and remained constant over a 72-h time course. In comparison with free drug administered at the maximum tolerated dose (MTD, 20 mg/kg) doxorubicin levels in tumors were two- to threefold greater when the drug was administered in liposomal form. The increase in drug delivery was comparable for both solid tumors. With animals bearing the L1210 ascitic tumor, drug exposure was as much as ten times greater (in comparison with free drug) when doxorubicin was administered in liposomes. An evaluation of single-cell suspensions prepared from the two solid tumors suggested that more than 98% of the tumor-associated drug and liposomal lipid was not tumor cell-associated. Histological studies with the Lewis lung carcinoma, however, revealed that a proportion of the drug did colocalize with tumor-associated macrophages. Analysis of cells obtained from mice bearing ascitic tumors showed that more than 80% of the cell-associated drug could be removed by procedures designed to remove adherent cells. Conclusion: The results summarized here suggest drug concentrations within a solid tumor, such as the Lewis lung carcinoma, are constant over time when the drug is given in a “leaky” EPC/Chol formulation. The results also suggest that liposomal lipid within sites of tumor growth is primarily localized within the interstitial spaces or tumor-associated macrophages. Received: 23 May 1996 / Accepted: 28 September 1996     

Pharmacokinetics and toxicity of oral and intravenous lonidamine in dogs

 Plasma lonidamine concentration and toxicity were investigated in dogs receiving 100, 200, 400, 800, 1200 mg/m² orally twice daily for 30 days and in dogs receiving single intravenous doses of 200, 400, 800, 1200 mg/m². Physical or laboratory signs of toxicity were not observed in dogs receiving oral lonidamine, but severe vomiting and signs of acute hepatic and pancreatic toxicity were observed in dogs receiving intravenous doses that exceeded 400 mg/m². The area under the lonidamine concentration versus time curve (AUC) in dogs receiving 200, 400, and 800 mg/m² of lonidamine intravenously was a 1.8-, 3.3-, and 8.7-fold higher than in dogs receiving oral lonidamine. This suggests that the bioavailability of oral lonidamine may be limited. However, centrilobular hepatocellular swelling and vacuolation were observed in dogs receiving oral lonidamine. Serum alanine aminotransferase (ALT) activity was increased in dogs receiving intra-venous lonidamine. These findings suggest that lonidamine is hepatotoxic in dogs. However, serum ALT was increased in only 1/4 dogs receiving 400 mg/m² of lonidamine intravenously and plasma concentration were within the range capable of sensitizing hyperthermia and chemotherapy. Therefore, this dose and route appears to be a viable and controllable method for prospective quantification of lonidamine interaction with systemic chemotherapy and/or hyperthermia. Received: 8 October 1993 /Accepted: 9 October 1995     

Antitumor activity and low intestinal toxicity of S-1, a new formulation of oral tegafur,in experimental tumor models in rats

 S-1, a new oral antitumor agent, is composed of 1-(2-tetrahydrofuryl)-5-fluorouracil (Tegafur, FT), 5-chloro-2,4-dihydroxypyridine (CDHP) and potassium oxonate (Oxo) in a molar ratio of 1 : 0.4 : 1. FT which is a masked compound of 5-fluorouracil (5-FU) acts as an effector, while both CDHP and Oxo which do not have antitumor activity themselves act as modulators. In this study, the antitumor activity and intestinal toxicity of S-1 were investigated using experimental tumor models in rats, and compared with those of other oral fluoropyrimidines, namely 5-FU, FT, FCD (1 M FT/0.4 M CDHP) and UFT (combination of FT and uracil). In rats bearing subcutaneous Yoshida sarcoma, S-1 inhibited tumor growth at the lowest dose (ED₅₀ value: S-1 5, UFT 22, FT 82, FCD 5, and 5-FU 19 mg/kg per day), and induced the least host body weight suppression, leading to the highest therapeutic index (TI) (S-1 4.5, UFT 1.4, FT 1.8, FCD 2.0, and 5-FU 1.4). S-1 also showed a higher therapeutic effect than UFT against AH-130 and Sato lung carcinoma. After administration of S-1 and UFT at equitoxic doses, S-1 showed a higher and more prolonged concentration of 5-FU than UFT both in plasma (AUC_0-∞: S-1 28 nmol h/ml, UFT 15 nmol⋅h/ml) and in tumor tissue (AUC_0-∞: S-1 95 nmol h/g tissue, UFT 52 nmol h/g tissue), leading to a higher 5-FU level incorporated into the RNA fraction (F-RNA level) in tumor tissue (AUC_0-24: S-1 7.0 nmol h/mg RNA, UFT 4.3 nmol h/mg RNA) and 5–8% higher thymidylate synthase (TS) inhibition in tumor tissue at every time-point through 24 h. Compared with other oral fluoropyrimidines after administration of the maximal tolerable dose (MTD), S-1 caused the lowest rates of intestinal toxicities, such as diarrhea and occult blood in feces. S-1 also showed a higher antitumor effect on Yoshida sarcoma implanted intracolonically than UFT at an equitoxic dose (tumor weight: S-1 64±30 mg, UFT 133±52 mg; P<0.05). These results suggest that CDHP, which is a potent inhibitor of 5-FU degradation, increases the antitumor activity of FT, and that Oxo, which is an inhibitor of 5-FU phosphorylation, locally protects the gastrointestinal tract from 5-FU-induced toxicity without decreasing the antitumor activity. Received: 13 October 1996/Accepted: 18 May 1996     

Reduced toxicity and enhanced antitumor effects in mice of the ionophoric drug valinomycin when incorporated in liposomes

Valinomycin (NSC 122023) is a cyclic depsipeptide antibiotic with potassium selective ionophoric activity. This drug has been reported to display antitumor effects but its utilization has been limited by its extreme toxicity. Here we report that the incorporation of valinomycin into multilamellar liposomes composed of dimyristoyl phosphatidyl choline:cholesterol:phosphatidyl serine (10:4:1 M ratio) results in a profound reduction in toxicity with maintainence of antitumor efficacy. Thus the median lethal dose (LD50) for i.p. administered valinomycin (VM) in C57BL/6 X DBA/2 mice is 1.7 mg/kg whereas the LD50 for liposome incorporated valinomycin (MVL-VM) is in excess of 50 mg/kg. In like manner, the LD50 for i.v. administered VM is 0.18 mg/kg where the LD50 for MLV-VM preparations passed through a 0.6-micron filter is greater than 10 mg/kg. The antitumor efficacies of i.p. administered VM or MLV-VM against i.p. P388 mouse leukemia were similar in multiple dose formats using doses below the maximal tolerated dose for VM. However, since MLV-VM was substantially less toxic than VM, the liposomal drug also produced significant (170% median survival time of treated mice/median survival time of untreated control) antitumor effects when administered as a single dose at levels above the maximal tolerated dose for free VM; single doses of free VM at the maximal tolerated dose were ineffective in this context. In experiments with i.v. inoculated P388 leukemia, MLV-VM but not free VM, displayed antitumor activity (144% median survival time of treated mice/median survival time of untreated control) when administered i.v. at equitoxic doses. Thus the use of a lipid vesicle drug carrier system permits a reduction in the toxicity of valinomycin with maintainence or enhancement of antitumor activity against i.p. or i.v. P388 leukemia.     

Protective role of metallothionein in renal toxicity of cisplatinum

To elucidate the protective role of metallothionein (MT) in the prevention of cisplatinum (cis-DDP) toxicity, we investigated the lethal and renal toxicities caused by cis-DDP in MT-null transgenic mice in comparison with wild-type control mice, and examined the effect of pretreatment with bismuth nitrate or zinc sulfate on the cis-DDP nephrotoxicity. The MT-null mice were of mixed 129 Ola and C57BL/6 genetic background. Since no differences in cis-DDP nephrotoxicity were observed between these strains, C57BL/6J mice were used as the wild-type control. The basal MT levels in the kidneys were negligible in the MT-null mice and 2.93 ± 0.77 μg/g tissue in the C57BL/6J mice. In terms of both the lethal and renal toxicities of cis-DDP, MT-null mice were far more sensitive than C57BL/6J mice. Preinduction of renal MT synthesis by administration of bismuth nitrate or zinc sulfate protected C57BL/6J mice from cis-DDP nephrotoxicity. In the case of MT-null mice, however, renal MT could not be induced by pretreatment with these metal compounds, and renal toxicity of cis-DDP was not prevented by this pretreatment. These results suggest that MT is an important factor with the potential to suppress the development of cis-DDP toxicity. Received: 17 June 1996 / Accepted: 27 November 1996     

还原型谷胱甘肽减轻化疗毒副反应的疗效观察

目的：观察还原型谷胱甘肽（GSH）对肿瘤患者化疗所致骨髓功能、肝功能损害的影响。方法：将102例患者随机分为两组,预防组（52例）,在化疗同时予以GSH。对照组（50例）,单纯化疗。同时观察两组在骨髓抑制（白细胞）、肝功能损害（谷丙转氨酶）上的差异。结果：预防组和对照组白细胞下降分别是46.15%（24/52）和72.00%（36/50）;Ⅰ度-Ⅳ度白细胞减少分比分别为40.98%（84/205）和67.55%（102/151）;影响化疗分别为5.37%（11/205）和17.22%（26/151）,均有显著性差异（P〈0.01）,化疗后ALT异常预防组和对照组分别为34.55%（19/55）和74%（37/50）;ALT异常分别为20%（41/205）和65.56%（99/151）;影响化疗分别为3.41%（7/205）和19.21%（29/151）,均有显著性差异（P〈0.01）。结论：GSH用于缓解恶性肿瘤患者化疗所致毒副反应疗效显著,在保证化疗的安全性、有效性的同时也提高了患者的生活质量。     

Analysis of HMG protein binding to DNA modified with the anticancer drug cisplatin

 Cisplatin (CDDP) is an effective and widely used cancer chemotherapy drug. High mobility group (HMG) proteins 1 and 2 have been shown to bind with high affinity to CDDP-DNA. In this study we analyzed the interaction of HMG proteins with CDDP-DNA. We demonstrate that after binding, HMG proteins can be removed from CDDP-DNA leaving the Pt adducts intact and capable of rebinding HMG proteins. Furthermore, the very HMG proteins that have been removed remain functionally viable and capable of rebinding CDDP-DNA. We also investigated the role that Cys residues play in protein binding. Replacement of Cys 45 or Cys 106 with a Ser residue reduced HMG2 protein binding to CDDP-DNA. These results indicate that Cys residues play a critical role in the high affinity binding of this protein to CDDP-DNA. From these findings, we speculate that the intracellular oxidative environment could affect the redox state of protein thiols in HMG1 and HMG2 and in addition, regulate the ability of these proteins to recognize cis-Pt-DNA adduct formation in tumor cells. Received: 16 May 1995 / Accepted: 9 October 1995     

In vivo antitumor activity of cis-bis-neodecanoato-trans- R,R-l,2-diaminocyclohexane platinum(II) formulated in long-circulating liposomes

 A lipophilic cisplatin derivative, cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexane platinum(II) (NDDP), was formulated in liposomes composed of phosphatidylcholine (PC) and cholesterol (Chol) additionally containing monosialoganglioside (G_M1) or polyethyleneglycol conjugated to phosphatidylethanolamine (PEG-PE). These NDDP-containing long-circulating liposomes were examined for in vivo antitumor activity using the mouse RIF-1 solid tumor as a target residing outside the reticuloendothelial system (RES). Biodistribution studies, using C3H/HeJ mice and ¹¹¹In-labelled DTPA-SA as a lipid marker, showed that the activity of G_M1 and PEG-PE in prolonging the circulation times of liposomes was preserved in the presence of 3.0 mol% of NDDP in the liposome membranes. The high levels of liposomes remaining in the blood for PC/Chol/G_M1 and PC/Chol/PEG3000-PE liposomes were associated with high levels of platinum in the blood as determined by atomic absorption spectrophotometry. These NDDP-containing long-circulating liposomes showed approximately a three-fold increase in tumor accumulation as compared to the conventional PC/Chol liposomes. In vitro cytotoxicity studies using RIF-1 tumor cells showed that the presence of PEG-PE, but not G_M1, significantly enhanced the cytotoxicity of liposomal NDDP. RIF-1 tumor-bearing C3H/HeJ mice were treated twice with 25 mg/kg NDDP in various liposomal formulations on days 12 and 16 after tumor cell inoculation. A significant reduction in the tumor growth rate was observed when NDDP was formulated in PC/Chol/PEG3000-PE liposomes which support both efficient tumor accumulation and enhanced cytotoxicity of liposomal NDDP. On the other hand, NDDP formulated in PC/Chol/G_M1 liposomes, which display only a high tumor accumulation, had no effect on the tumor growth rate. Furthermore, NDDP formulated in dimyristoylphosphatidylglycerol (DMPG) containing liposomes, exhibiting in vitro cytotoxicity comparable to NDDP formulated in PC/Chol/PEG3000PE liposomes, but showing poor tumor accumulation, was also not effective. These results indicate a potential effectiveness of NDDP formulated in PEG-PE-containing liposomes for therapy of tumors in non-RES organs. Received: 13 November 1994/Accepted: 14 May 1995     

In vitro effects of R-verapamil on the cytokine environment and T-lymphocyte proliferation when human T-lymphocyte activation takes place in the presence of acute myelogenous leukemia blasts

 We investigated the effects of R-verapamil on the cytokine environment and T-lymphocyte proliferation when human T-lymphocytes were activated in the presence of accessory cells containing a large population of acute myelogenous leukemia (AML) blasts (nonirradiated blasts for cytokine studies, 50 Gy irradiated blasts in proliferation studies). In the presence of AML blasts, R-verapamil inhibited interleukin 4 (IL4) and interferon-γ (IFNγ) release from polyclonal T-cell lines activated with the T-cell mitogen phytohemagglutinin (PHA). R-verapamil also inhibited both the proliferation and the release of IFNγ and IL10 by normal T-cells stimulated with allogeneic peripheral blood mononuclear cells derived from AML patients. This antiproliferative effect of R-verapamil was seen in the presence of exogenous IL2 but was not observed in the presence of exogenous IL1β or granulocyte/macrophage colony-stimulating factor GM–CSF). In addition R-verapamil inhibited the release of IL1β and tumor necrosis factor α during allogeneic stimulation. Received: 7 January 1996/Accepted: 11 April 1996     

Determination of vinca alkaloids in human plasma by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

 A sensitive assay was developed for the quantitation of vinblastine, desacetylvinblastine and vincristine using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS). Analyses were performed on an Ultrasphere C₁₈ microbore column using ammonium acetate as mobile phase. The calibration curves were linear across the range of 0.51–4.00 ng/ml (0.63–4.93 nM) for vinblastine, 0.74–3.93 ng/ml (0.96–5.11 nM) for desacetylvinblastine and 0.30–3.95 ng/ml (0.36–4.79 nM) for vincristine. Vinca alkaloid concentrations were measured with an accuracy and precision within 11%. This assay could be implemented to determine the plasma concentrations for pharmacokinetic studies of vinblastine, desacetylvinblastine and vincristine in conjunction with clinical trials. Received: 15 December 1995 / Accepted: 16 June 1996     

Isozyme-specific glutathione S-transferase inhibitors potentiate drug sensitivity in cultured human tumor cell lines

 Novel glutathione (GSH) analogs, previously shown to inhibit glutathione S-transferase (GST) activity at about 1 μM in vitro, were tested for their ability to potentiate the killing of cultured tumor cells by chemotherapeutic drugs. When tested at doses up to 200 μM, the analogs were neither toxic nor capable of potentiating drug toxicity unless the diethyl ester (DEE) form was used for treatment of the cells. HPLC analysis revealed rapid internalization of the DEE and intracellular conversion to a monoethyl ester form that accumulated in the cell, followed by a more gradual loss of the second ester to generate the active parent form. For the four GSH analogs tested, the ability of the DEE forms to potentiate chlorambucil (CMB) toxicity in HT-29 human colon adenocarcinoma cells strongly correlated with the in vitro ability of the parent form to inhibit recombinant human P1-1. This isozyme is the dominant form of GST present in HT-29 cells. Of the four analog DEEs tested, γ-glutamyl-S-(benzyl)cysteinyl-R(−)-phenyl glycine (TER 117) DEE was the most effective in potentiating CMB toxicity in several cell lines: HT-29, HT4-1 (HT-29 subclone), SKOV-3 ovarian carcinoma, and SK VLB (vinblastine-resistant variant of SKOV-3) cells. γ-Glutamyl-S-(octyl)cysteinyl-glycine (TER 143) DEE potentiated mitomycin C (MTC) toxicity in HT4-1 and SK VLB cells while TER 117 DEE did not. TER 117 DEE enhanced melphalan effects on xenografts of HT4-1 in mice to a similar extent as that achieved with the previously described nonspecific GST inhibitor, ethacrynic acid. Taken together, our results indicate that cell-permeable analogs of GSH can potentiate cytotoxicity of common chemotherapeutic drugs and this effect has a strong positive correlation with the ability of the analogs to inhibit specific GST isozymes. Received: 18 October 1994, Accepted: 14 May 1995     

6-Mercaptopurine dose escalation and its effect on drug tolerance in childhood lymphoblastic leukaemia

 Daily oral 6-mercaptopurine (6MP) is important in the treatment of childhood lymphoblastic leukaemia (ALL), but there is great inter-patient variability in the pattern of evident drug effect (myelosuppression) seen at a standard dose. In an attempt to reduce that variability the current practise in the United Kingdom for the last 4 years has been to escalate the amount prescribed in patients who do not experience cytopenias at 75 mg/m². We undertook a study to see whether that strategy would increase the total dose of 6MP prescribed in such patients and whether it would alter the pattern of myelosuppression. Over a 6-month period we studied 44 children treated conventionally (without escalation) and compared them with another 44 (matched for sex) who were treated on the same protocol but where doses were increased in monthly 25% steps if 75 mg/m² was tolerated without cytopenias. We then compared the two groups for the total dose of drug prescribed and the frequency and duration of neutropenia or throrabocytopenia. The median cumulative dose of 6MP received by the conventionally treated children (10,002 mg/m²) was not significantly different from that of the children treated with dose escalation (9,429 mg/m²). In a comparison of the 30 children who actually received inflated doses of 6MP with the 37 from the conventional cohort who would have been eligible to do so, it was again found that the cumulative median doses were similar (10,460 versus 10,916 mg/m²). There was a difference between the two p10bgroups in the pattern of myelosuppression — the escalated group spent significantly more time off 6MP than did the non-escalated group (median 4.5 versus 3 weeks; P<0.005, 95% CI from −1 to −3). These findings imply that the method of dose escalation employed does not allow more 6MP to be prescribed in children tolerant of the standard dose. The chief effect seems to be to generate longer periods off therapy, and this could paradoxically decrease the anti-neoplastic activity of the drug. Alternative ways of prescribing should be explored. Received: 26 July 1995/Accepted: 6 October 1995     

Characterization of cellular accumulation and toxicity of illudin S in sensitive and nonsensitive tumor cells

 Illudins are novel low molecular weight natural products cytotoxic to human tumor cells in vitro. Illudin-derived analogs are effective against experimental human cancers nonresponsive to conventional anticancer agents. It is not known why some illudin analogs are more efficacious in vitro and in vivo than other analogs. Therefore, the in vitro cytotoxicity of the parent compound illudin S towards tumor cells was characterized using radiolabeled drug. Two cell lines sensitive at nanomolar concentrations using only a 15-min exposure period displayed a saturable, energydependent accumulation of illudins with relatively low K_m and high V_max values. A nonsensitive cell line, requiring millimolar concentrations to achieve in vitro toxicity, showed minimal illudin uptake with higher K_m and lower V_max values. No release of radioactivity could be demonstrated from tumor cells, indicating that there was no efflux of illudin S (or metabolites) from these cells. The number of intracellular illudin S molecules required to kill 50% of cells of different tumor cell lines varied from 78 000 to 1 114 000 molecules per cell and was correlated with the 2-h IC₅₀ value determined using a colony-forming assay. Illudin S was cytotoxic to a variety of multidrug-resistant tumor cell lines regardless of whether resistance was mediated by gp170/mdrl, gp180/MRP, GSHTR-pi, topoisomerase I,topoisomerase II, increased DNA repair capacity, or alterations in intracellular thiol content. Information obtained in this study could be used to design clinical phase I trials and to develop analogs with improved therapeutic indexes. Received: 26 April 1996 / Accepted: 19 September 1996     

白屈菜红碱对大鼠的长期毒性试验研究

目的： 观察连续给予抗肿瘤药物白屈菜红碱对大鼠的长期毒性。方法：采用Wistar大鼠,共分成4个白屈菜红碱组(12.6、8.4、5.6和3.7 mg/kg)及溶剂对照组,每组20只。腹腔注射给药6 d,停药8 d为1个周期,连续给药3个周期,间歇给药结束后12.6 mg/kg剂量组解剖大鼠6只,8.4 mg/kg剂量组解剖大鼠8只,其余每组解剖大鼠12只,停药观察4周后解剖各组剩余大鼠,进行一般状况、体质量、饲料摄入量、血液学、血清生化学、尸体解剖和脏器系数以及病理组织学检查。结果：大鼠在给药后,12.6 和8.4 mg/kg剂量组立即出现腹膜刺激征,一周之内出现消瘦和被动体态等,体质量增长缓慢,摄食量减少,甚至死亡。与对照组比较,白屈菜红碱5.6 mg/kg以上剂量组大鼠红细胞系各项指标降低,白细胞数及白细胞分类异常,凝血时间延长,葡萄糖降低,碱性磷酸酶增加(P<0.05或P<0.01);并可观察到肺脏瘀血,腹腔大量血性腹水,脏器粘连并严重变形,肝脏和脾脏明显增大,前列腺和睾丸系数明显减小(P<0.05或P<0.01),睾丸生精上皮细胞和精子明显损伤等。结论：白屈菜红碱在 ≥5.6 mg/kg剂量下,可引起大鼠局部刺激和药物毒性所引起的全身性异常反应,以致部分大鼠死亡。     

Pharmacokinetics and pharmacodynamics of topotecan given on a daily-times-five schedule in phase II clinical trials using a limited-sampling procedure

 Topotecan is a novel semisynthetic derivative of the anticancer agent camptothecin and inhibits the intranuclear enzyme topoisomerase I. The lactone structure of topotecan, which is in equilibrium with the inactive ring-opened hydroxy acid, is essential for this activity. We performed a pharmacokinetics study as part of phase II clinical trials in patients with various types of solid tumors, giving topotecan at 1.5 mg/m² per day by 30-min infusion for 5 consecutive days, with courses being repeated every 3 weeks. Previously validated limited-sampling models, using concentration measurements in samples obtained 2 h after infusion, were used to calculate the area under the plasma concentration-time curves (AUCs) for both chemical forms. Samples were obtained from a total of 36 patients over 136 treatment days. The mean AUC of the closed-ring form (AUC_closed) was 8.74 (range 2.3–16.3)  μM min per day, and the mean AUC of the ring-opened form (AUC_open) was 11.5 (range 3.2–46.0)  μM min per day (interpatient variability 34–61%). In each patient the AUC values achieved on the 1st day of administration were similar to and, thus, predictive for those achieved during the following days, with a day-to-day variation of 7.39% being recorded for the AUC_closed and that of 12.6%, for the AUC_open. There was no drug accumulation during the 5 consecutive treatment days of each cycle. However, despite the large interpatient pharmacokinetic variability, the importance of regular drug monitoring on this schedule can be questioned, as the pharmacodynamic variability was relatively small. Received: 15 June 1995/Accepted: 19 October 1995     

Flavone acetic acid increases the cytotoxicity of mitomycin C when combined with hyperthermia

 Flavone acetic acid (FAA, NSC 347512) is known to selectively reduce tumor blood flow. Taking advantage of this pharmacodynamic effect, we have previously shown that FAA in combination with hyperthermia (HT) can produce a marked improvement in antitumor response in mice. In the present study, we investigated whether FAA could increase the cytotoxicity of mitomycin C (MMC), a bioreductive drug with selective cytotoxicity against hypoxic cells, under either normothermic or hyperthermic conditions. In vitro, the cytotoxicity of MMC against B16 melanoma cells was not enhanced with exposure to FAA at concentrations less than 100 μg/ml, even when combined with HT (43°C, 60 min). The cytotoxicity of MMC (1 μg/ml) at pH 6.5, however, was enhanced by exposure of cells to hypoxia in combination with HT. In vivo, the tumor growth time, calculated as the time required to double the initial tumor volume, was 5.2, 6.8, 8.5, and 15.0 days with FAA (150 mg/kg) alone, MMC (4 mg/kg) alone, FAA+MMC, or FAA+MMC+HT (43°C, 15 min) treatment groups, respectively. Antitumor response obtained in animals treated with FAA plus MMC with HT was clearly better than that obtained in any of the other groups. Scheduling of FAA, MMC, and HT was found to be important in producing optimal antitumor response. Administration of MMC (4 mg/kg) prior to FAA (150 mg/kg) and subsequent HT treatment was superior to administration of FAA before MMC. In an attempt to explain these findings, the influence of FAA on blood flow in skeletal muscle and in tumor was examined using a laser blood flowmeter. FAA administration to mice produced a 75% reduction in blood flow to the tumor for up to 2 h but had no detectable effect on normal skeletal blood flow. Our current explanation of the increased antitumor response achieved with the combination of MMC, FAA, and HT is as follows. The FAA-mediated decrease in blood flow to the tumor, when combined with HT, may produce sufficiently hypoxic conditions to significantly increase the antitumor efficacy of the bioreductive drug, MMC. We believe that clinical testing of this combined drug treatment with hyperthermia is warranted. Received: 1 February 1995/Accepted: 6 July 1995     

Biotransformation of the platinum drug JM216 following oral administration to cancer patients

 This study evaluates the metabolic profile of JM216 [bis(acetato)ammine-dichloro(cyclohexylamine) platinum(IV)], the first orally administrable platinum complex, in plasma ultrafiltrates of 12 patients (n=2–4 time points per patient) following different doses of drug (120, 200, 340, 420, 560 mg/m²). The biotransformation profile was evaluated by high-performance liquid chromatography (HPLC) followed by atomic absorption spectrophotometry (AA). The AA profiles were compared with those previously identified by HPLC on line with mass spectrometry (HPLC-MS) in plasma incubated with JM216. A total of six platinum peaks (Rt=5.5, 7.2, 10.6, 12.4, 15.6, and 21.6 min, respectively) were observed in patients’ plasma ultrafiltrate samples, of which only four appeared during the first 6 h post-treatment. Four of these coeluted with those observed and identified previously in plasma incubation medium. No parent JM216 was detected. The major metabolite seen in patients was the Pt II complex JM118 [cis-amminedichloro-(cyclohexylamine)platinum (II)] and was observed in all the patients. Interestingly, the second metabolite was shown to coelute with the Pt IV species JM383 [bis-acetatoammine(cyclohexylamine)dihydroxoplatinum (IV)]. Both JM118 and JM383 were identified by HPLC-MS in a clinical sample. Peak C, which was a minor product (less than 5% of the free platinum), coeluted with JM559 [bis-acetatoammine-chloro(cyclohexylalamine)hydroxoplatinum (IV)]. The cytotoxicity profile of all three metabolites in a panel of cisplatin-sensitive and -resistant human ovarian carcinoma cell lines was very close to that of the parent drug. In addition, the concentrations of JM118 reached in patients’ plasma ultrafiltrate were comparable with the cytotoxic levels of the compound determined in the ovarian carcinoma panel of cell lines. Two metabolites were seen in patients but not in the in vitro incubation medium, suggesting the involvement of a possible enzyatic reaction. Thus, the biotransformation profile following oral administration of JM216 shows a variety of Pt(IV) and Pt(II) metabolites in plasma that differ significantly from other systemically applied platinum drugs. Received: 8 May 1995/Accepted: 6 October 1995     

NONMEM population pharmacokinetic studies of cytosine arabinoside after high-dose and after loading bolus followed by continuous infusion of the drug in pediatric patients with leukemias

 We examined the population pharmacokinetics (PPK) of cytosine arabinoside (ara-C) after high-dose ara-C (HDara-C) (3 g/m² every 12 h) and after a loading bolus (LB) plus continuous infusion (Cl) of ara-C for 72 h in 52 pediatric patients with leukemias, enrolled in four clinical trials. The PPK analyses of the drug were performed using the NONMEM program. The patients’ ages ranged from 2 months to 19 years. The ara-C data were analyzed using a two-compartment open model. Interindividual variability was described by the constant coefficient of variation (CCV) model, while the intraindividual variability was described by a combined additive and CCV error model. The covariates age (AGE) and surface area (SA) were tested to examine their influence on the estimation of the ara-C PPK parameters. In the absence of model covariates, the data fit was characterized by considerable bias, as indicated by the plot of measured vs predicted ara-C concentrations. The fit of the data was greatly improved when the parameters total body clearance (CL), intercompartmental clearance (Q), and volumes of distribution of central (V_d1) and peripheral (V_d2) compartments were expressed as linear functions of the covariate product, AGE×SA. The final parameter estimates were: CL=2.59× AGE×SA l/h, Q=2.01×AGE×SA l/h, V_d1=0.48× AGE×SA l, and V_d2=38.1×AGE×SA l. The coefficients of variation of CL, Q, V_d1 and V_d2 were 83.79%, 12.08%, 40.0%, and 52.54%, respectively, indicating substantial interindividual variability. In separate NONMEM analyses, the PK of ara-C and its metabolite uracil arabinoside (ara-U) were modeled simultaneously in order to investigate whether the dependence of ara-C on patient age was due to increased deamination of ara-C to ara-U. The PK of ara-C were described by the two-compartment open model while the PK of ara-U were simultaneously described by the one-compartment open model. The conversion of ara-C to ara-U was modeled as a first-order kinetic process due to the relatively low concentrations of ara-C in plasma. These PPK analyses indicated that elimination of ara-C from the central compartment occurs primarily by its metabolic conversion to ara-U and that the rate of conversion of ara-C to ara-U increases with increasing patient age, which explains the higher ratios of ara-U to ara-C and, hence, the increased ara-C clearance observed in older children as compared to infants. We conclude that the NONMEM PPK methodology allowed the simultaneous analyses of data from different doses and dose regimens and explained phenomena that prior standard two-stage analyses could not. Received: 9 June 1995 / Accepted 20 March 1996