Expectations from cytotoxic chemotherapy

Cancer chemotherapy has expanded rapidly during the last 30 years and in some tumours there is clear evidence that even in advanced disease, chemotherapy combined with appropriate local treatment, may be curative. These encouraging results have led to great expectations from cytotoxic chemotherapy, particularly since evidence has accumulated that many common tumours frequently show tumour regression when patients are treated with aggressive combination chemotherapy regimens. In the last ten years, apart from testicular cancer in which the addition of Platinum to previously available drug regimens, has led to dramatic improvement in survival rates, there is little solid evidence to suggest that chemotherapy in other advanced adult tumours has had a major impact on survival. In this article, steps in the development of cytotoxic chemotherapy are reviewed, and realistic expectations for the 1980's are outlined. New approaches to the identification of new drugs have been introduced in the last few years and these may lead to the identification of new drugs with a greater range of antitumour effects and increased antitumour activity. The possibility of in vitro sensitivity testing is critically discussed, and its is suggested that this approach may in part replace traditional Phase II studies. Adjuvant chemotherapy, which has clearly improved survival in certain childhood cancers, is now being evaluated in many solid tumours in adults, and only time will tell whether a higher proportion of patients will survive, compared to the results of local treatment only.     

Testicular cancer and fertility

Before treatment, a little over one-half of patients with testicular cancer have fathered children, while one-fifth have a history of sterility. Radiotherapy with a gonadal dose of less than 1.5 Gy seems to do little permanent damage to fertility. Following cisplatin-based combination chemotherapy, a number of conceptions have been reported. The newer regimens affect spermatogenesis less than earlier ones due to a lower toxicity of the drugs and shorter duration of the treatment. New modifications of retroperitoneal lymphadenectomy may save ejaculatory ability in over one-half of the patients. It is uncertain whether this procedure is less detrimental to fertility than the cisplatin-based combination chemotherapy. Even though semen quality is often poor before treatment, cryopreservation of semen should be considered since in vitro fertilization may be successful even with very poor semen quality. There are no indications in the literature of permanent adverse genetic effects of the treatment.     

Tumor necrosis factor enhances the in vitro and in vivo efficacy of chemotherapeutic drugs targeted at DNA topoisomerase II in the treatment of murine bladder cancer

Recombinant human tumor necrosis factor (rTNF) is a macrophage secretory protein with antitumor activity. The murine bladder tumor cell line MBT-2 was used to evaluate the in vitro and in vivo antitumor effects of rTNF in combination with chemotherapeutic drugs targeted at DNA topoisomerase II. These drugs, such as adriamycin and etoposide (VP 16), are in widespread use in the treatment of human cancer. The rTNF significantly enhanced the cytotoxic efficacy of the topoisomerase-targeted drugs actinomycin D, adriamycin, etoposide (VP 16) and teniposide (VM 26) against MBT-2 cells in vitro. The rTNF alone had no effect upon the cells in the same assay. When examined in vivo using MBT-2 tumor-bearing C3H/HeJ mice, these same antitumor relationships were seen. The addition of rTNF to actinomycin D or VP 16 resulted in a significant reduction in tumor volume at 20 days compared to untreated animals. Actinomycin D, VP 16 or rTNF treatment alone had no significant effect on 20 day tumor volume. The data provide a reasonable basis for the addition of rTNF to experimental protocols for the treatment of human bladder cancer using topoisomerase-targeted drugs such as adriamycin both intravesically and systemically. These observations may also be relevant to other human cancers currently treated with these drugs.     

Fatal cytomegalovirus pneumonia after preemptive antiviral therapy in a renal transplant recipient

Cytomegalovirus (CMV) infections occur with an incidence of up to 70% in renal transplant patients and mortality is low due to effective antiviral drugs. We report here the case of a patient who suffered from an uncommonly severe and therapy-resistant pulmonary CMV infection. During the disease course, CMV-PCR from alveolar cells and lung biopsy material was repeatedly negative despite high CMV pp65 antigenemia. CMV pneumonia was finally diagnosed from a biopsy obtained by open thoracotomy revealing positive CMV immunostaining of lung tissue. The patient died of respiratory failure though double-treatment using both ganciclovir and foscavir was administered. Post mortem, the clinically suspected resistance to both antiviral drugs, but not to cidofovir, could be proven by bioassay testing of in vitro growth responses using viral cultures. CMV pneumonia may thus not be diagnosed by standard PCR techniques in rare cases and may be resistant to the available antiviral therapy. Severe CMV pneumonia may benefit from novel antiviral drugs such as cidofovir, which is currently used in the treatment of CMV retinitis in HIV patients.     

Anaphylactic-like reaction to suxamethonium

A young woman developed an anaphylactic-type reaction during induction of anaesthesia, which was later shown to be due to suxamethonium, to which the patient had not been previously exposed. In vitro testing identified not only suxamethonium as the causative agent, but also other potentially hazardous agents, pharmacologically and chemically related to suxamethonium. Furthermore, 'unrelated' neuromuscular blocking drugs elicited no reaction from the patient's leucocytes, suggesting that in future anaesthesia these agents may be used with little risk. The leucocytes of the patient's parents and brother were also tested, the brother showing a minimal positive reaction. Skin testing confirmed the significance of the in vitro histamine release by the patient's leucocytes.     

The modulatory effect of diltiazem on human in vitro alloreactivity when used alone or in combination with cyclosporin A and/or methylprednisolone

The calcium channel blocker diltiazem is often included in post-transplant regimens in combination with other immunosuppressive drugs such as cyclosporin A (CyA). It is primarily used because of its antinephrotoxic and antihypertensive effects, so that undesirable side effects induced by the immunosuppressive therapy can be reduced. Its alleged ability to induce direct immunosuppression may explain the encouraging results from its clinical use and would appear to encourage a much wider use of this drug. The present study shows the effect of diltiazem on the human in vitro alloresponse when used alone or in combination with cyclosporin A (CyA) and methylprednisolone (MP). The results show that, when administered alone, diltiazem exerts a suppressive effect, but only at high, non-therapeutic doses. Interestingly, in combination with CyA or MP, diltiazem enhances the suppressive effect of these two drugs on in vitro alloresponses at lower doses. This additional effect of diltiazem may contribute to better graft survival in clinical transplantation. Received: 26 February 1997 Received after revision: 9 June 1997 Accepted: 3 July 1997     

Sensitivity of BCG to modern antibiotics

The viability of bacillus Calmette-Guérin (BCG) in intravesical instillation therapy has been demonstrated to be crucial for the prevention of bladder tumour recurrence. The aim of the present study was to determine the effects of modern antibacterial chemotherapeutics on BCG viability, particularly cycloserine, which has been recommended in the treatment of BCG-induced sepsis. The minimal inhibitory concentrations (MICs) of 32 antibacterial drugs potentially effective against the Connaught BCG strain were measured in vitro by the radiometric BACTEC 460TB method. The MICs were compared with the drug concentrations achievable in blood and urine. Susceptibility testing of cycloserine was performed with three different strains (Connaught, Tice and RIVM), using the modified proportion method, as defined in the German guidelines for anti-tuberculosis drug testing. The Connaught BCG strain was highly susceptible to fluoroquinolones, but was resistant to beta-lactams, macrolides (except clarithromycin), and some aminoglycosides. It was also sensitive to doxycycline and gentamicin at dosages that typically occur in the urine of patients after a normal dose. Connaught BCG was susceptible to all the tuberculostatic drugs tested, except for pyrazinamide. All the BCG strains analysed were resistant to cycloserine. During intravesical BCG instillation therapy, simultaneous administration of fluoroquinolones, doxycycline or gentamicin should be avoided. In cases of severe systemic complications, or if one of the antituberculosis drugs is not tolerated, fluoroquinolones may be used. Cycloserine is no longer recommended for the early treatment of BCG sepsis.     

Platelet function abnormalities in a family with recurrent arterial thrombosis

Three young family members with recurrent arterial thrombosis underwent investigation for lipid or coagulation abnormalities. Lipoprotein electrophoresis, cholesterol, triglyceride levels, and routine coagulation studies were unremarkable. By contrast, testing of platelet function showed enhanced platelet aggregability to epinephrine and collagen in two of the subjects. In addition, release of 14C-serotonin by adenosine diphosphate and epinephrine was increased over control values in these same two patients. The third subject demonstrated decreased platelet aggregation and lowered 14C-serotonin release, but was symptomatic with rest pain at the time of testing. The ongoing in vivo thrombosis in the third subject may account for hypocoagulable platelets by in vitro testing. These abnormally sensitive platelets identified by platelet function testing may be associated with a familial "hypercoagulability" syndrome. Definition of the hemostatic abnormality in these individuals provided a rational basis for pharmacological therapy with antiplatelet drugs, which appeared to be successful.     

Gangliosides potentiate the immunosuppressive effects of cyclosporin A in rat skin allografts

In vitro gangliosides exert inhibitory effects on cellular immune responses, largely relying on an impairment of the IL-2/IL-2 receptor interaction. In a previous study we have demonstrated synergistic effects of gangliosides and cyclosporin A (CyA) in the inhibition of the generation of in vitro allospecific immune responses in humans. To evaluate the possibility of using these drugs in immunosuppressive therapy in organ transplantation, we investigated the effects of the combination of a gangliosides mixture (GAMIX) and suboptimal doses of CyA on rat skin allografts in vivo. Sprague-Dawley rats were implanted with skin grafts from Lewis rats and treated for 21 days by intraperitoneal administration of either GAMIX or CyA or a combination of the two drugs. Untreated, GAMIX-treated or CyA-treated rats rejected skin allografts. In contrast, when a combined GAMIX CyA treatment was administered, successful grafting could be obtained in 8 rats out of 10 tested. Cells derived from spleens on day 21 post graft were stimulated in vitro with PWM mitogen. We found that cells from transplanted rats, untreated or treated with low-dose CyA or GAMIX alone, showed comparable responses to PWM. Cells from rats treated with the combination of the two drugs were found to be virtually unresponsive to stimulation by PWM mitogen. Taken together, our results indicate that GAMIX potentiate in vivo and ex vivo immunosuppressive effects of low-dose CyA.     

Bioartificial materials in urology

The scope of our research is the development of polymer-based bioabsorbable stents for urologic applications and in vitro testing of tissue reactions of cultured ureteral and urethral segments induced by implanted polymer stent prototypes. For these purposes a tissue cultivation model was developed using selected techniques of tissue engineering.Essential advantages of degradable over nondegradable urethral stents are elimination of the adverse extraction of epithelialized stents and the potential for recovery of organ-specific functionality. Moreover, the biocompatibility of a degradable urethral stent could potentially reduce the risk of restenosis due to hyperplasia and could be used, even repeatedly, for the treatment of a number of subvesical obstructions. For the treatment of tumor-induced strictures, application of degradable polymer stents coated with cytostatic drugs may be possible. The mechanical effect of the drug-loaded stent as a "place holder" could be complemented by adjuvant or palliative approaches such as local chemotherapy. We have developed and tested in vitro a degradable urethral stent incorporated with the model drug methotrexate for local drug delivery (LDD) by diffusion and during stent degradation.     

Bioartifizielle Materialien in der Urologie

The scope of our research is the development of polymer-based bioabsorbable stents for urologic applications and in vitro testing of tissue reactions of cultured ureteral and urethral segments induced by implanted polymer stent prototypes. For these purposes a tissue cultivation model was developed using selected techniques of tissue engineering. Essential advantages of degradable over nondegradable urethral stents are elimination of the adverse extraction of epithelialized stents and the potential for recovery of organ-specific functionality. Moreover, the biocompatibility of a degradable urethral stent could potentially reduce the risk of restenosis due to hyperplasia and could be used, even repeatedly, for the treatment of a number of subvesical obstructions. For the treatment of tumor-induced strictures, application of degradable polymer stents coated with cytostatic drugs may be possible. The mechanical effect of the drug-loaded stent as a “place holder” could be complemented by adjuvant or palliative approaches such as local chemotherapy. We have developed and tested in vitro a degradable urethral stent incorporated with the model drug methotrexate for local drug delivery (LDD) by diffusion and during stent degradation.     

Ablation of bone cells by electroporation

Short intense electrical pulses transiently increase the permeability of the cell membrane, an effect known as electroporation. This can be combined with antiblastic drugs for ablation of tumours of the skin and subcutaneous tissue. The aim of this study was to test the efficacy of electroporation when applied to bone and to understand whether the presence of mineralised trabeculae would affect the capability of the electric field to porate the membrane of bone cells. Different levels of electrical field were applied to the femoral bone of rabbits. The field distribution and modelling were simulated by computer. Specimens of bone from treated and control rabbits were obtained for histology, histomorphometry and biomechanical testing. After seven days, the area of ablation had increased in line with the number of pulses and/or with the amplitude of the electrical field applied. The osteogenic activity in the ablated area had recovered by 30 days. Biomechanical testing showed structural integrity of the bone at both times. Electroporation using the appropriate combination of voltage and pulses induced ablation of bone cells without affecting the recovery of osteogenic activity. It can be an effective treatment in bone and when used in combination with drugs, an option for the treatment of metastases.     

In vitro sensitivity testing of human kidney tumors to cytostatic drugs with a rapid in vitro test

We present a new modified in vitro culture assay for primary human renal cell carcinoma similar to the 'soft agar clonogenic assay'. However, the carrying out and expense of metrology are more simplified, allowing tumor-specific chemotherapeutic drug sensitivity testing under easier conditions. Twelve different samples of human renal carcinoma and one sample of a transitional cell carcinoma of the renal pelvis were tested for in vitro chemotherapy sensitivity using this modified colony-forming assay. The rate of tumor cells establishing in culture was 100%. Corresponding to clinical experience we observed a nearly complete resistance to the cytotoxic effects of standard chemotherapeutic agents at usual plasma concentrations in man. Only higher concentrated chemotherapeutic drugs showed in vitro therapeutic effects. The assay described here lasts about 7 days, which is beneficial from the clinical point of view. The modification of this in vitro chemotherapeutic drug treatment is unlimited for plasma concentration and duration of standard and experimental chemotherapeutic agents, drug combination and so on. So we have interesting scientific steps which could not have been undertaken under usual clinical-empirical conditions.     

Effects of interferon in combination with cytotoxic drugs on mouse bladder tumor (MBT-2) growth in vitro and in vivo

The effects of interferon alone and in combination with either adriamycin, mitomycin C or thiotepa were evaluated for antiproliferative activity for the mouse bladder tumor, MBT-2. In vitro dose response studies with adriamycin, mitomycin C and thiotepa showed dose dependent antiproliferative activity over a concentration range of .001 microgram./ml. to 10 microgram./ml. Interferon also exhibited dose dependent inhibition over a dose range of 250 units/ml. to 4000 units/ml. Comparative in vitro antiproliferative studies showed adriamycin to consistently be the most inhibitory cytotoxic drug followed by mitomycin C and thiotepa. Interferon and thiotepa produce similar (35%) inhibition at the highest concentrations studied. In vitro studies assessing the effects of interferon (1000 units/ml.) combined with each cytotoxic drug (0.01 to 1.0 microgram./ml.) were performed. The most effective combination was observed to be interferon and adriamycin. In three of four experiments either additivity or synergism was observed while consistently augmented activity was not observed with interferon combined with either thiotepa or mitomycin C. In vivo studies on MBT-2 tumors implanted intravesically showed no antitumor activity for adriamycin alone, interferon alone or a combination of both. These results show that combination intravesical therapy with interferon and adriamycin is not an effective treatment regimen in the mouse model when administered as described in this report. These data further suggest that combinations of interferon and cytotoxic drugs may not provide improved response rates in patients treated for superficial bladder tumors.     

新生儿尿布皮炎联合给药对经皮渗透影响的实验研究

目的 探讨联合给药治疗新生儿尿布皮炎对药物经皮渗透指标的影响.方法 以家兔为研究模型,分成三组,每组8只,分别进行单独应用氧化锌油、制霉菌素及两种药物联合应用的体外经皮渗透实验.测定三组经皮渗透量、渗透速率及滞后时间等渗透指标.结果 氧化锌组与混合用药组氧化锌的经皮渗透指标比较,差异无统计学意义(均P＞0.05);制霉菌素组与混合用药组中制霉菌素的经皮渗透指标比较,差异有统计学意义(均P＜0.01).结论 氧化锌与制霉菌素混合用药中制霉菌素的渗透率低于单一应用制霉菌素.氧化锌油与制霉菌素联合应用时应采用交叉给药,混合给药干扰制霉菌素的经皮渗透效果.     

Cytotoxic effect of drugs eluted from polymethylmethacrylate on stromal giant-cell tumour cells: an in vitro study

Curettage and packing with polymethylmethacrylate cement is a routine treatment for giant-cell tumour (GCT) of bone. We performed an in vitro evaluation of the cytotoxic effect of a combination of cement and methotrexate, doxorubicin and cisplatin on primary cell cultures of stromal GCT cells obtained from five patients. Cement cylinders containing four different concentrations of each drug were prepared, and the effect of the eluted drugs was examined at three different time intervals. We found that the cytotoxic effect of eluted drugs depended on their concentration and the time interval, with even the lowest dose of each drug demonstrating an acceptable rate of cytotoxicity. Even in low doses, cytotoxic drugs mixed with polymethylmethacrylate cement could therefore be considered as effective local adjuvant treatment for GCTs.     

GnRH拮抗剂联合来曲唑和米非司酮对体外培养的人颗粒细胞功能的影响

目的探讨促性腺激素释放激素拮抗剂(GnRH-ant)联合来曲唑和米非司酮对体外培养的人卵巢颗粒细胞功能及血管内皮生长因子(VEGF)表达的影响。方法体外培养人原代卵巢颗粒细胞及人卵巢颗粒细胞系KGN,根据所用药物不同分为GnRH-ant、来曲唑、米非司酮以及上述三种药物联合用药组,药物干预后检测细胞活力、细胞凋亡、培养液中雌二醇(E 2)及孕酮(P)水平及VEGF的表达。结果(1)GnRH-ant、来曲唑、米非司酮三种药物均可以不同程度地抑制原代颗粒细胞和KGN细胞的活力、促进颗粒细胞凋亡,其中联合用药组的作用最强(P<0.05);(2)GnRH-ant、来曲唑、米非司酮三种药物均可以不同程度地抑制原代颗粒细胞和KGN细胞E 2、P分泌水平,其中联合用药组E 2、P水平最低且差异有统计学意义(P<0.05);(3)GnRH-ant、来曲唑、米非司酮三种药物均可以不同程度地抑制原代颗粒细胞和KGN细胞VEGF表达,其中联合用药组VEGF mRNA及蛋白表达水平最低(P<0.05)。结论GnRH-ant联合来曲唑和米非司酮可以有效地抑制颗粒细胞活力、减少类固醇激素分泌及降低VEGF的表达水平。     

大肠癌细胞和外周血淋巴细胞对化疗药物的体外敏感性

目的:探讨大肠癌细胞及外周血淋巴细胞对化疗药物体外敏感性的及二者的相关性。方法:用MTT法检测40份大肠癌标本及外周血淋巴细胞对氟尿嘧啶(5-FU)、奥沙利铂(L-OHP)、伊立替康(CPT)单药、两药及三药(全量或半量)应用的敏感性。结果:单药最有效的药物依次为伊立替康、奥沙利铂和氟尿嘧啶,敏感率分别为35.0%、27.5%和20.0%;三药联合应用的抑制效果显著优于两药联合(P<0.05),三药全量及半量联合应用效果差异无显著性(P>0.05);癌细胞及外周血淋巴细胞对3种化疗药物的敏感性有很好的相关性(r=0.969)。结论:MTT可用于为大肠癌患者选择合适的化疗药物,由氟尿嘧啶、奥沙利铂及伊立替康的联合应用具有高效协同抑制大肠癌细胞的作用。     

Combination chemotherapy with gemcitabine with isolated lung perfusion for the treatment of pulmonary metastases

OBJECTIVE: Isolated lung perfusion is an experimental technique for the treatment of lung metastases. Single-agent isolated lung perfusion does not result in complete remission. We studied the in vivo and in vitro efficacy of combinations of gemcitabine, cisplatin, and melphalan. METHODS: In vitro, using the sulforhodamine B assay, CC531s cells were incubated with cisplatin, gemcitabine, or melphalan or with a combination of these drugs. One drug was added at concentrations causing 25% growth inhibition, whereas the second drug was added at variable concentrations. In vivo, left pulmonary metastases were induced in Wag/Rij rats by means of intravenous injection of CC531s adenocarcinoma cells. At day 7, rats underwent left isolated lung perfusion with gemcitabine (n = 7), cisplatin (n = 9), melphalan (n = 7), gemcitabine-cisplatin (n = 6), melphalan-gemcitabine (n = 6), and cisplatin-melphalan (n = 7). Death by means of metastatic disease was the end point. Survival and differences in survival were assessed by using Kaplan-Meier and log-rank testing. RESULTS: In vitro synergistic activity was observed for melphalan-gemcitabine, whereas other combinations showed additive or antagonistic activity. In vivo treated rats lived longer compared with control animals ( P < .0001). In isolated lung perfusion melphalan resulted in longer survival compared with gemcitabine ( P = .0016) and cisplatin ( P = .046). Isolated lung perfusion with melphalan-gemcitabine resulted in 67% survival of the rats after 90 days versus 0% in other groups. CONCLUSIONS: Isolated lung perfusion monotherapy or combination therapy with gemcitabine, cisplatin, or melphalan resulted in significantly longer survival compared with that seen in control animals. Isolated lung perfusion combination therapy with melphalan-gemcitabine resulted in the best survival either in vitro or in vivo.     

汉黄芩素对胰腺癌作用的体外、体内实验研究

目的：探讨汉黄芩素对胰腺癌Panc-1细胞体内外生长的影响。方法：将不同浓度（1,10,100 μmol/L）汉黄芩素作用胰腺癌Panc-1细胞24 h,用MTT法和流式细胞仪检测细胞的增殖与凋亡情况。将20只荷Panc-1细胞移植瘤裸鼠随机均分为对照组,汉黄芩素组（汉黄芩素60 mg/kg,1次/d）,吉西他滨组（吉西他滨150 mg/kg,1次/周）与联合用药组（汉黄芩素+吉西他滨）,连续给药2周并停药7 d后,比较各组移植瘤生长情况,用免疫组化法检测瘤组织微血管密度（MVD）与血管内皮生长因子（VEGF）的表达。结果：与无处理的对照细胞比较,3个浓度汉黄芩素处理后的Panc-1细胞OD值均明显降低,凋亡率均明显升高,且均呈浓度依赖性（均P<0.05）。与对照组比较,汉黄芩素组,吉西他滨组与联合用药组移植瘤的生长均被明显抑制,抑瘤率分别为24.8%,30.5%,66.1%,联合用药组的抑制率明显大于两个单药组（均P<0.05）,但两单药组间差异无统计学意义（P>0.05）;3个治疗组肿瘤组织CD31（反映MVD）与VEGF的表达均明显减少,且吉西他滨组,汉黄芩素组,联合用药组减少程度依次明显,组间差异均有统计学意义（均P<0.05）。结论：汉黄芩素能抑制胰腺癌细胞的增殖并促进凋亡;能一定程度抑制胰腺癌在体内的生长,并增加肿瘤对化疗药物的敏感性,该作用的机制部分与抑制肿瘤的血管生成有关。