Effect of Potentiated Antibodies to Cyclophosphamide on the Development of Tumors and Effectiveness of Cytostatic Therapy under Experimental Conditions

Antibodies to cyclophosphamide obtained by homeopathic potentiation and administered in ultralow doses exhibit no antiblastic activity and did not modulate the effectiveness of cyclophosphamide during antitumor therapy of animals with transplanted tumors (Lewis lung carcinoma and Ehrlich adenocarcinoma).     

Cyclophosphamide metabolite inducing apoptosis in RLS mouse lymphosarcoma cells is a substrate for P-glycoprotein

RLS lymphosarcoma characterized by enhanced expression of mdr1a and mdr1b genes encoding P-glycoprotein is insensitive to low doses of cyclophosphamide, but is susceptible to its high doses approximating the maximum tolerated doses. Induction of apoptotic death of RLS cells by high doses of cyclophosphamide was demonstrated by cytofl uorometry and electrophoresis. Experiments on RLS₄₀ tumor cells derived from RLS lymphosarcoma and characterized by more intensive expression of mdr1a/1b genes showed that the therapeutic effects of cyclophosphamide increased under conditions of simultaneous suppression of these genes by specifi c small interfering RNA (siRNA). These fi ndings suggest that active cyclophosphamide metabolite can be a substrate for P-glycoprotein.     

Mycophenolate mofetil for lupus nephritis: an update

Renal disease in systemic lupus erythematosus carries significant morbidity and mortality. Intensive immunosuppression to dampen kidney inflammation timely and maintenance therapy to prevent renal flares is necessary to reduce the long-term risk of renal failure. Mycophenolate mofetil (MMF) has emerged to be the first-line treatment of lupus nephritis for its better safety profile compared with cyclophosphamide. In controlled trials, MMF is non-inferior to cyclophosphamide for induction therapy but is superior to azathioprine as maintenance therapy. Although biologics have shown promise in refractory lupus nephritis, combining MMF with a number of novel biological agents does not enhance the therapeutic efficacy. Recently, low-dose combination of MMF and tacrolimus has been shown to be more efficacious than intravenous pulse cyclophosphamide in inducing remission of lupus nephritis in Chinese patients. Therapeutic monitoring of the serum mycophenolic acid level to enhance the efficacy of MMF in lupus nephritis is being explored.     

Comparative Effects of Azathioprine, Cyclophosphamide and Frentizole on Humoral Immunity in Mice

Data is presented comparing the activities of three immunosuppressive agents, cyclophosphamide, frentizole and azathioprine in models of humoral immunity in mice. Cyclophosphamide and frentizole suppressed the primary and secondary plaque forming cell responses to sheep erythrocytes at lower doses than did azathioprine. Prolonged suppression of serum antibody titers occurred following short-term therapy with cyclophosphamide or frentizole, but not azathioprine. Azathioprine was also the least effective agent in suppressing a primary response to the T-independent antigen, trinitrophenylated lipopolysaccharide. All three agents were found to inhibit the induction and activity of suppressor cells at immunosuppressive doses.     

Risk of leukemia after chemotherapy and radiation treatment for breast cancer.

BACKGROUND. Few studies have evaluated the late effects of adjuvant chemotherapy for breast cancer. Moreover, the relation between the risk of leukemia and the amount of drug given and the interaction of chemotherapy with radiotherapy have not been described in detail. METHODS. We conducted a case-control study in a cohort of 82,700 women given a diagnosis of breast cancer from 1973 to 1985 in five areas of the United States. Detailed information about therapy was obtained for 90 patients with leukemia and 264 matched controls. The dose of radiation to the active marrow was estimated from individual radiotherapy records (mean dose, 7.5 Gy). RESULTS. The risk of acute nonlymphocytic leukemia was significantly increased after regional radiotherapy alone (relative risk, 2.4), alkylating agents alone (relative risk, 10.0), and combined radiation and drug therapy (relative risk, 17.4). Dose-dependent risks were observed after radiotherapy and treatment with melphalan and cyclophosphamide. Melphalan was 10 times more leukemogenic than cyclophosphamide (relative risk, 31.4 vs. 3.1). There was little increase in the risk associated with total cyclophosphamide doses of less than 20,000 mg. CONCLUSIONS. Although leukemia occurs in few patients with breast cancer, significantly elevated risks were linked to treatments with regional radiation and alkylating agents. Melphalan is a more potent leukemogen than cyclophosphamide or radiotherapy. Low risks were associated with the levels of cyclophosphamide in common use today. Systemic drug therapy combined with radiotherapy that delivers high doses to the marrow appears to enhance the risk of leukemia.     

Aspergillus-associated cerebral aneurysm successfully treated by endovascular and surgical intervention with voriconazole in lupus nephritis patient

During the last five decades, long-term therapy with immunosuppressive agents such as pulse cyclophosphamide in conjunction with high-dose corticosteroids has enhanced both patient survival and renal survival in patients with diffuse proliferative lupus nephritis. Nevertheless, severe side effects such as infectious complications remain the main cause of morbidity and mortality. Central nervous system aspergillosis is uncommon but life-threatening in lupus patients. In this single-patient case study, carotid aneurysm with sphenoidal sinusitis was suspected when severe epistaxis occurred during cyclophosphamide pulse therapy. With anti-fungal therapy, a graft stent was successfully deployed to the aneurysm and specimens of sphenoidal mucosa showed typical hyphae, indicating aspergillosis. Three months after stopping voriconazole treatment, two cerebral aneurysms that were revealed on MR images were successfully removed by aneurysmal clipping. The patient remained alive at one-year follow-up with lupus nephritis in remission. The rarity and high mortality of aspergillus-related fungal aneurysms have led to most cases being recognized postmortem. However, such aneurysms must be diagnosed early to prevent fatal complications by performing appropriate management such as surgical procedure or endovascular intervention.     

Effects of pulse cyclophosphamide on NZB/W disease

Induced IgM anti-ss-DNA antibodies in NZB/W female mice did not alter the time of onset nor the course of nephritis. Monthly pulse doses of cyclophosphamide suppressed the mortality of these mice, and also prevented a switch of anti-ss-DNA from IgM to IgG class. The production of IgM anti-SRBC was markedly reduced in old NZB/W mice, but IgG anti-SRBC was only moderately reduced and this hyporesponsiveness towards SRBC could be reversed by CPA treatment. These observations are discussed in relation to cyclophosphamide as an effective therapeutic agent for the murine lupus syndrome.     

Appearance of central nervous system lupus during corticosteroid therapy and warfarinization in a patient with pure red cell aplasia and antiphospholipid syndrome]

A 48-year-old woman presented to our hospital with epigastralgia and erythema on the left dorsalis pedis. Her medical history included deep venous thrombosis three months prior to admission to our hospital. Upon admission it was determined that she had severe anemia (hemoglobin level 4.6 g/dl). Bone marrow analysis indicated a markedly decreased number of erythroid progenitor cells. A skin biopsy specimen of the erythema revealed microthrombus. Anticardiolipin-beta2GPI antibody and lupus anticoagulant were positive. The patient was diagnosed with pure red cell aplasia (PRCA) and antiphospholipid syndrome (APS). After steroid pulse therapy and warfarinization, her anemia and purpura improved. Three months later she developed depression with positive anti-ribosomal P protein antibody that was indicative of central nervous system lupus. Although her psychometric condition did not respond to steroid pulse therapy, improvement was seen after she received three courses of cyclophosphamide pulse therapy. We report a rare case of CNS lupus that developed during corticosteroid therapy and warfarinization in a patient with PRCA and APS.     

Successful combination therapy of cyclosporine and methotrexate for refractory polymyositis with anti-Jo-1 antibody: a case report

Although corticosteroids have been the initial agent for the treatment of inflammatory myopathies (IM), immunosuppressive agents such as azathioprine, methotrexate, cyclophosphamide, or cyclosporine are commonly required to control the disease except mild cases. On the other hand, the efficacy of combination therapy of cyclosporine and methotrexate in severe rheumatoid arthritis has been proven without serious side effects. However, in treatment-resistant myositis, the experience of such a therapy is very limited, and has not been described in refractory polymyositis with anti-Jo-1 antibody. Here, we report a young female patient with recalcitrant polymyositis and anti-Jo-1 antibody who was successfully treated with the combination therapy of cyclosporine and methotrexate. At first, the myositis did not respond to several agents, such as corticosteroid, monthly pulse cyclophosphamide, azathioprine, or cyclosporine. Methotrexate was initially avoided as treatment regimen because of its potential pulmonary toxicity in the case with preexisting lung disease.     

Increased spontaneous immunoglobulin secretion associated with cyclophosphamide-induced immune suppression

Spontaneous immunoglobulin (Ig) secretion by cells from multiple sclerosis (MS) patients (in the progressive phase) treated with monthly pulse doses of cyclophosphamide (CY) (1000–1600 mg/M²) was measured using the protein A plaque assay, to evaluate the effect of CY treatment on B-cell function. Surprisingly, an increase, rather than a decrease, in Ig-secreting cells was seen following CY treatment. CY-treated MS patients averaged 1380±535 spontaneous total (IgM+G+A) Ig plaque-forming cells (PFC) per 1×10⁶ peripheral blood mononuclear cells (MNC), measured at 15–22 days after monthly CY administration, while healthy adults had 280±47 Ig PFC/10⁶ MNC, and MS patients not treated with CY had 300±43 Ig PFC/10⁶ MNC. The observed increase was due to an increase in IgG and IgA PFC. PFC levels remained elevated for 4 weeks following CY treatment, decreasing to control levels by 7–8 weeks post-CY. A small increase in serum IgG level was noted after >12 months of pulse CY therapy; no increase was seen in CSF IgG levels. A preferential decrease in the number of CD4+ T cells was also seen in the CY-treated MS patients. We propose that the observed increase in the number of spontaneous Ig PFC was due to the CY-induced disruption of the CD4+ T cell-mediated control ofin vivo activated B cells.     

Contact sensitivity and the DNA response in mice to high and low doses of oxazolone: low dose unresponsiveness following painting and feeding and its prevention by pretreatment with cyclophosphamide.

Cyclophosphamide was used to assess the role of suppressor cells in the contact sensitivity reaction. A single painting with 300 microgram and 30 microgram oxazolone produced poor contact sensitivity reactions (ear swelling). Cyclophosphamide (200 mg/kg) 2 days before painting increased the response to the lower doses but had less effect on the response to 3 mg oxazolone. A single feed with 10 mg oxazolone caused strong contact sensitivity while lower doses (10-1000 microgram) caused poor responses. Cyclophosphamide increased the response to the lower doses but not to the highest dose of oxazolone. These results suggested that the poor response to painting and feeding lower doses of oxazolone was due to a suppressor system which was sensitive to cyclophosphamide. A different result was obtained when contact sensitivity was measured by arrival of radioactively labelled cells. Cyclophosphamide had the greatest effect on cell arrival when high doses were fed. This indicates that ear swelling and cell arrival measure separate aspects of the contact sensitivity response. The lower doses of oxazolone, which caused little contact sensitivity, reduced the response to a standard immunizing dose. This low dose unresponsiveness occurred after either painting or feeding (Chase-Sulzberger phenomenon). It did not occur in mice treated with cyclophosphamide before the first exposure to oxazolone. This suggested that the low dose unresponsiveness was due to suppressor cells. The response to oxazolone was also assessed by DNA synthesis in the regional lymph nodes. A small dose of oxazolone (30 microgram) caused a peak of DNA synthesis on day four while a high dose (3 mg) caused a peak on day three. Pretreatment with cyclophosphamide depressed the response to 30 microgram although it increased contact sensitivity. The secondary response was smaller than the primary on days 3, 4 and 5 after immunization but larger on day two. The depression but not the increase was prevented by cyclophosphamide and was probably due to a suppressor system.     

The effect of cyclophosphamide and vincristine on glucose absorption in rats using in vivo perfusion experiments

The effect in rats of cyclophosphamide and vincristine on glucose absorption was studied by the in vivo perfusion method. On testing the local effect of cytostatics, the drugs were dissolved in the perfusion solution, cyclophosphamide at a concentration of 10(-4) M and vincristine at 10(-6) M. Glucose absorption was not reduced by cyclophosphamide, while it was significantly reduced by vincristine. On testing the systemic effect of cytostatics, the drugs were administered intraperitoneally 24 hours prior to perfusion, i.e. in doses of 80 mg/kg in the case of cyclophosphamide, and of 0.05 mg/kg of vincristine. In that case, both cytostatics decreased glucose absorption significantly. Based on the results it was found that, being administered locally, cyclophosphamide did not have a damaging effect on the intestinal wall, and did not reduce absorption either. On the contrary, vincristine impaired the mucosa locally, thereby reducing glucose absorption. On systemic administration, both cytostatics decreased absorption from the small intestine.     

Drug-induced tolerance to allografts in mice II. Tolerance to tumor allografts of large doses associated with rejection of skin allografts and tumor allografts of small doses

Antigen-specific tolerance to tumor allografts in mice was obtained by stimulation with allogeneic spleen cell antigens and treatment with cyclophosphamide (CY) 2 days later in various strain combinations between donor mice of spleen cell antigens and recipients. In such tolerant mice, skin allografts were rejected in various combinations of the donor-recipient. Furthermore, some of the tolerant mice showed accelerated rejection of tumor allografts of small doses, even though they allowed progressive growth of the same tumor allografts of large doses. Our results suggest that the rejection of tumor allografts of large doses may depend upon a mechanism that is distinct, qualitatively or quantitatively, from that one responsible for the rejection of tumor allografts of small doses and skin allografts. One of the most important properties of lymphocytes to separate these two different mechanisms may be a capacity of clonal expansion, namely, sensitivity to CY-induced tolerance of the lymphocytes mediating allograft rejection.     

Blastic transformation of lymphocytes in in vitro cultures under the influence of phytohemagglutinin and pokeweek mitogen in patients with glomerulonephritis treated over long periods of time with immunosuppressive drugs.

In vitro reactivity of lymphocytes to phytohemagglutinin (PHA) and pokeweek mitogen (PWM) was studied in 17 patients with chronic glomerulonephritis treated systematically over many months (average, 23 months) with immunosuppressive drugs (prednisone, indomethacin, azathioprine, cyclophosphamide). Blastic transformation of lymphocytes in cultures stimulated with PHA was tested three times: before beginning of treatment, during the course of therapy with full doses, and after prolonged ambulatory treatment with maintenance doses. The degree of blastic transformation under the influence of PWM was determined only after periods of many months of treatment with chemical immunosuppresors. The results showed that neither treatment of chronic glomerulonephritis for several weeks with full doses, nor subsequent prolonged administration (for periods of tens of months) of maintenance doses of immunosuppresors regularly inhibit blastic transformation of lymphocytes stimulated with PHA. Similar observations were made using PWM after many months of immunosuppressive therapy. Reactivity of lymphocytes to PWM in all patients resembled reactivity to PHA.     

Contrasting effects of cyclophosphamide and prednisolone on the phenotype of human peripheral blood leukocytes

The cell surface phenotype of human peripheral blood mononuclear cells has been characterized before and after intravenous injection of cyclophosphamide or prednisolone. Low doses of cyclophosphamide (100-600 mg/m2) temporarily decrease levels of circulating B lymphocytes. Slightly higher doses of cyclophosphamide (200-600 mg/m2) produce transient depression of T8-, M1-, and Ia-positive cells. After doses of 200-400 mg cyclophosphamide/m2, T4-positive cells are spared, resulting in a transient elevation of the T4/T8 ratio. With higher doses of cyclophosphamide (greater than or equal to 600 mg/m2), all T cells are affected and the T4/T8 ratio declines to pretreatment levels. By contrast, intravenous injection of prednisolone at 40 mg/m2 reduces the T4/T8 ratio. Levels of both T4 and T8 cells decline, but T4 cells are affected more markedly than T8 cells.     

Stage-adapted treatment of Wegener's granulomatosis

Summary Considering cyclophosphamide's severe side effects there is a need for a new, less toxic treatment protocol for Wegener's granulomatosis. Here we report the first results of a prospective study using cyclophosphamide pulse therapy (monthly application) (a) as an alternative treatment in seven cases of active generalized Wegener's granulomatosis, which either showed complications under continuous cyclophosphamide treatment or in which the partial remission was not steady, and (b) as the initial treatment in five newly diagnosed patients with active generalized disease. After complete remission had been achieved in all cases in group (a), but only two cases in group (b), we started treatment with trimethoprim/sulfamethoxazole (cotrimoxazole) to maintain remission. Three of 8 patients suffered from severe relapses 9, 11 and 12 months after discontinuation of cyclophosphamide. The 3 patients in group (b) who could not be brought into remission had to be put on continuous cyclophosphamide. In addition, we treated 3 patients with newly diagnosed locoregional disease with cotrimoxazole alone. Two of these patients responded promptly and have only minor symptoms after 13 and 27 months, respectively. In one patient the disease continued to progress in the upper respiratory tract, and treatment was switched to cyclophosphamide and prednisolone after a period of 3 months. From these results we believe that cyclophosphamide pulse therapy is a successful alternative treatment protocol after partial remission has been achieved with a daily administration of cyclophosphamide or if complications, e.g-. leukopenia, arise with the continuous use of this drug.As an initial treatment, however, bolus treatment still appears to be an experimental protocol. Our experience with cotrimoxazole for maintaining remission has not left us particularly confident, though in the initial phase of the disease this drug might be worth trying.Abbreviations WG Wegener's granulomatosis - CYC cyclophosphamide - T/S trimethoprim/sulfamethoxazole - E upper respiratory tract - L lung - K kidney (ELK classification) - ACPA antibody against intracellular antigens in neutrophilic leucocytes Supported by the Bundesministerium für Forschung und Technologie (01 VM 86224), the Verein zur Förderung der Forschung und Bekämpfung rheumatischer Erkrankungen e.V. Bad Bramstedt and the Hermann and Lilly Schilling-Stiftung     

The effect of various immunosuppressive agents on mouse peritoneal macrophages and on the in vitro phagocytosis of Escherichia coli O4:K3:H5 and degradation of 125I-labelled HSA-antibody complexes by these cells.

Large doses of hydrocortisone, cyclophosphamide, and methotrexate injected subcutaneously, and whole-body irradiation (500 rads) caused a reduction in the number of peritoneal cells (PE cells) obtained after intraperitoneal injection of the treated mice with proteose-peptone. The same dose of cyclophosphamide and irradiation induced morphological changes in PE macrophages. There were more giant cells in the peritoneal exudates from treated mice as compared to control mice. 'Pharmacological' and larger doses of hydrocortisone, methotrexate and azathioprine or anti-lymphocyte globulin had no effect on the in vitro phagocytic capacity of proteose-peptone-stimulated mouse PE macrophages. This also applied to doses of up to 50 mg/kg of cyclophosphamide. In contrast, whole-body irradiation (500 rad) and 100 mg/kg of cyclophosphamide decreased the phagocytic capacity of mouse macrophages in vitro and reduced the ability of PE cells to degrade 125I-labelled HSA-antibody complexes in vitro. The greatest effect was noted 4-5 days after whole-body irradiation or four to five subcutaneous injections of cyclophosphamide.     

Pulmonary inflammation in autoimmune MRL/Mp-lpr/lpr mice. Histopathology and bronchoalveolar lavage evaluation.

Early detection of lupus pneumonitis is difficult because it requires lung biopsy. The authors describe here in detail the age-related histologic changes in pulmonary inflammation, the age-related changes in bronchoalveolar lavage (BAL), and the effect of cyclophosphamide (8 mg/kg) on pulmonary inflammation and bronchoalveolar lavage in MRL/Mp-lpr/lpr mouse, an animal model of systemic lupus erythematosus. To assess the evolution of pulmonary inflammation and response to cyclophosphamide therapy, they compared the age-related progression of pulmonary inflammation with sequential changes in BAL cell populations in this autoimmune mouse model. A striking similarity was noted between age-related changes in pulmonary inflammation and lymphocyte counts in BAL. A trend to reduction in histologic evidence of inflammation was reflected by lymphocytes in BAL in cyclophosphamide-treated (8 mg/kg/day) males but not in females. There was a striking sex-related difference in that the histologic evidence of pulmonary inflammation and bronchoalveolar lavage lymphocyte count in cyclophosphamide-treated males was significantly lower than cyclophosphamide-treated females of the same age.     

The pathogenesis of infection in mouse brain by Mount Elgon bat virus

Mount Elgon bat virus given intracerebrally readily killed mice up to 12 days of age (1 to 4 p.f.u/LD50). Virus in doses of 10 to 10(6) p.f.u. killed 40 to 80% of weanling mice of both sexes and up to 20% of adult females and 40 to 80% of adult males. Clearance of brain infectivity in the resistant mice coincided with the appearance of circulating virus neutralizing antibody which occurred earliest in the adult female mice. Immunosuppression of adult mice with cyclophosphamide resulted in the death of all the virus-inoculated mice of both sexes. Virus in these mice reached tenfold higher titres and their brains contained 30- to 60-fold more interferon than the brains of infected mice not given cyclophosphamide. Interferon was apparently without effect on the outcome of infection in the brains of resistant mice and its synthesis reflected the extent of virus growth.     

A randomized trial of plasmapheresis and subsequent pulse cyclophosphamide in severe lupus: design of the LPSG trial.

A group of clinics cooperating as the Lupus Plasmapheresis Study Group (LPSG) is starting an international multicenter study of the treatment of severe systemic lupus erythematosus. The primary goal of this randomized and prospective trial is to establish whether treatment with plasmapheresis and subsequent pulse cyclophosphamide improves the outcome compared to treatment with pulse cyclophosphamide alone. The underlying rationale assumes that plasmapheresis: a) eliminates pathogenic autoantibodies and immune complexes and b) induces a compensatory activation of pathogenic lymphocyte clones through a feed-back between circulating antibodies and their respective antibody-producing clones. Synchronization of plasmapheresis with subsequent pulse cyclophosphamide should enhance the deletion of pathogenic clones during the period of greatest vulnerability. This overview reviews the first results of treatment approaches based on this concept and summarizes the design of the LPSG trial.