Studies on cyclophosphamide metabolites and their related compounds. 2. Preparation of an active species of cyclophosphamide and related compounds.

A synthetic study was made on the active metabolite of cyclophosphamide. Ozonolysis of O-(3 butenyl)-N,N-bis(2-chloroethyl)phosphorodiamidate, prepared by reaction of POC13 with 3-buten-1-ol followed by treatment with N,N-bis(2-chloroethyl)amine (nor mustard) and NH3, afforded 2-[bis(2-chloroethyl)amino]-4-hydroperoxytetrahydro-2H-1, 3,2-oxazaphosphorine 2-oxide (4-hydroperoxycyclophosphamide). Deoxygenation of 4-hydroperoxycyclophosphamide by triphenylphosphine yielded 4-hydroxycyclophosphamide in a pure crystalline state. These products exhibited high cytostatic activity in both in vitro and in vivo experiments. The results give confirmatory evidence for the hypothesis that C4-hydroxylation on the 1,3,2-oxazaphosphorinane ring of cyclophosphamide is necessary for its activation.

Synthesis and antitumor activity of preactivated isophosphamide analogues bearing modified alkylating functionalities.

In search of cancer chemotherapeutic agents with greater efficacy than cyclophosphamide, 4-hydroperoxyisophosphamide analogues bearing modified alkylating functionalities such as 2-bromoethyl, 2-iodoethyl, 2-methyl-sulfonyloxyethyl, and 2-ethylsulfonyloxyethyl groups were prepared by ozonolytic cyclization reaction of N,N'-substituted 3-butenyl phosphorodiamidates. Comparative cytotoxicity against L1210 cells and antileukemic life-span activity against L1210 implanted BDF1 mice of the newly synthesized compounds were tabulated. The 4-hydroperoxyisophosphamide analogues which have different alkylating groups in a molecule showed slightly greater cytoxicity in vitro than those with the same alkylating groups. Most of the compounds having different alkylating groups also showed high antileukemic activity in vivo. Among them, the highest efficacy was found for 2-[N-methyl-n-(2-chlorethyl)]amino-3-(2-methylsulfonyloxyethyl)-4-hydroperoxy-1,3,2-oxazaphosphorinane 2-xoide (NSC 280122D) whos life-span activity was also greater than that of 4-hydroperoxyisophosphamide, cyclophosphamide, and isoposphamide. The superiority of this compound was especially apparent by oral administration.

Hypersensitivity pneumonitis induced by spores of Lyophyllum aggregatum.

OBJECTIVES: Lyophyllum aggregatum (LA) is called Shimeji in Japanese and is eaten commonly as a mushroom. Shimeji mushrooms are cultivated in an indoor environment all year round. This study aimed to clarify the clinical features of hypersensitivity pneumonitis (HP) induced by LA. PATIENTS AND SETTING: Ten patients showed mild respiratory symptoms including dry cough, sputum, and low-grade fever. We tried to characterize the clinical features and the findings using chest high-resolution CT (HRCT), pulmonary function tests (PFTs), and BAL fluid (BALF) tests in patients with HP induced by LA. HP was diagnosed from clinical features, HRCT findings, BALF findings, lung histology, and lymphocyte stimulation tests (LSTs) for LA. RESULTS: Laboratory findings showed mean (+/- SD) elevated levels of C-reactive protein (0.78 +/- 1.3 mg/dL), erythrocyte sedimentation rate (48 +/- 23 mm/h), and gamma-globulin (26.9 +/- 7.6%). PFTs revealed a slight decrease in the percentage diffusing capacity of the lung for carbon monoxide, possibly due to the presence of epithelial granulomas in the alveoli. Although 4 of 10 patients showed normal findings on the chest radiograph (CXR), chest HRCT findings of all patients showed centrilobular small nodules and diffuse ground-glass opacities. The BALF testing revealed an increase in total cell counts, showing predominantly activated T lymphocytes. The CD4/CD8 cell ratio was significantly decreased (0.5 +/- 0.3). The results of the LSTs were positive in seven of seven cases. CONCLUSIONS: Since patients with HP induced by LA typically have mild respiratory symptoms and sometimes normal CXR findings, their conditions might remain undiagnosed. However, the chest HRCT images showed the typical subacute phase of HP.

Shortening of the window period in diagnosis of HIV-1 infection by simultaneous detection of p24 antigen and antibody IgG to p17 and reverse transcriptase in serum with ultrasensitive enzyme immunoassay.

Following HIV infection, there is a window period of 6-8 weeks, during which HIV antibodies are not detectable and the infection cannot be diagnosed by methods for detecting HIV antibodies. However, HIV antigens are detectable in the latter part of the window period, although the level of HIV antigens declines as the level of HIV antibodies increases. We developed an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for the simultaneous detection of both p24 antigen of HIV-1 and antibody IgGs to p17 and reverse transcriptase of HIV-1 in a single assay tube and tested 11 HIV-1 seroconversion serum panels and serum samples randomly collected from 79 HIV-1 seropositive subjects and 100 HIV-1 seronegative subjects. The simultaneous detection was shown not only to shorten the window period significantly as compared with conventional methods for HIV-1 antibody detection but also to make possible a reliable diagnosis of HIV-1 infection from the time of seroconversion until late stages of the infection.

Expression of the hepatitis B surface antigen gene containing the preS2 region in Saccharomyces cerevisiae.

We constructed a plasmid, pBH103-ME5, in which the region encoding the 10 preS2 amino acid residues and the S domain of the hepatitis B surface antigen (HBsAg) were regulated by the promoter of the yeast repressible acid phosphatase gene. Saccharomyces cerevisiae carrying pBH103-ME5 produced the HBs antigen (yHBsAg), when it was cultured in a medium containing a low concentration of phosphate. The antigen was purified to homogeneity. Its molecular weight was determined by Western blotting to be 24,000, and its amino acid composition agreed well with that deduced from the nucleotide sequence. The C-terminal amino acid sequence of yHBsAg was exactly the same as that predicted from the nucleotide sequence, while the N-terminal amino acid acetylserine, which was followed by 8 amino acid residues coded by the preS2 region. These results indicate that the recombinant yeast produced a single polypeptide consisting of the preS2 region and the subsequent S domain after being processed at the N-terminus.

Antibodies against non-structural c100/3 and structural core antigen of hepatitis C virus (HCV) in hemodialysis patients.

Two groups of patients undergoing hemodialysis (HD) maintenance were evaluated for their antibody response to non-structural c100/3 protein and structural core protein of hepatitis C virus (HCV). Forty-six patients (Group 1) never presented liver abnormalities during HD treatment, while 52 patients (Group 2) had either current or prior liver enzyme elevations. Prevalence rates of 32.6% and 41.3% were found for anti-c100/3 and anti-HCV core antibodies, respectively, in patients with silent infections (Group 1). The rate of anti-c100/3 in patients of Group 2 was 71.15% and reached 86.5% for anti-HCV core antibodies. The recognition of anti-c100/3 and anti-core antibodies was significantly higher in Group 2 than in Group 1. A line immunoassay composed of structural and non-structural peptides was used as a confirmation assay. HBV infection, measured by the presence of anti-HBc antibodies, was observed in 39.8% of the patients. Six were HBsAg chronic carriers and 13 had naturally acquired anti-HBs antibodies. The duration of HD treatment was correlated with anti-HCV positivity. A high prevalence of 96.7% (Group 2) was found in patients who underwent more than 5 years of treatment. Our results suggest that anti-HCV core ELISA is more accurate for detecting HCV infection than anti-c100/3. Although the risk associated with the duration of HD treatment and blood transfusion was high, additional factors such as a significant non-transfusional spread of HCV seems to play a role as well. The identification of infective patients by more sensitive methods for HCV genome detection should help to control the transmission of HCV in the unit under study.

[Clinical study on long acting amoxicillin in urinary tract infection].

Amoxicillin (AMPC), a widely used oral penicillin exhibites a strong bactericidal activity and a high level of safety. In the present study we administered L-AMPC, long acting AMPC granule, to 18 cases with urinary tract infections and obtained the following clinical results. 1. In all cases with acute simple cystitis, the effect was excellent. 2. Of 12 cases with chronic complicated UTI, the effect was excellent in 1 case, good in 4 cases and no effect was noted in 7 cases. The efficacy rate was 41.7%. 3. No side effects nor abnormal values in the laboratory examinations were noted. 4. Sixteen of 18 cases reported that this preparation was very agreeable to take compared with other powder type drugs, capsules and tablets. From these results we can confidently state that L-AMPC is a highly useful drug combining the excellent properties of AMPC, the convenience of b.i.d. dosage which improves patients compliance and satisfactory efficacy.

[Clinical evaluation of the combination of carumonam and fosfomycin in the treatment of complicated urinary tract infection].

Carumonam (CRMN), the first monobactam antibiotic in Japan, has excellent activity against gram-negative bacteria and is useful in the treatment of urinary tract infections. However, it may be insufficient in the treatment of complicated urinary tract infections because of the increase in isolation of gram-positive bacteria, and it may be necessary to co-administer antibiotics active against gram-positive organisms to achieve a broader spectrum of coverage in connection with severe infections. The combination of CRMN and fosfomycin (FOM) was evaluated for its effectiveness and safety at the Department of Urology, Yamagata University Hospital and 7 affiliated hospitals. Clinical efficacy was assessed on 64 patients with complicated urinary tract infection according to the Criteria for Clinical Evaluation of Antimicrobial Agents in UTI (3rd. ed.) recommended by the Japan UTI Committee. Clinical efficacy was evaluated as excellent in 16, moderate in 32, poor in 16, with an overall clinical effectiveness rate of 75.0%, which is superior compared with CRMN alone. Of the total of 92 bacterial strains isolated, 66 (71.7%) were eradicated. Subjective adverse reaction was seen in 1 patient (1.4%), as nausea and anorexia. Slight increases in serum GOT and GPT ware recorded in 5 patients (7.1%). These findings disappeared after the termination of administration without treatment. The combination of CRMN and FOM might therefore be useful in the treatment of complicated urinary tract infections.

[Conservative surgery of renal cell carcinoma associated with renal (artery) aneurysm in solitary kidney--a case report].

A rare case of renal cell carcinoma associated with renal artery aneurysm in solitary kidney is reported. The patient is a 63-year-old woman with asymptomatic macrohematuria as CC, who previously received left nephrectomy (for which underlying disease is unknown). Excretory pyelography, abdominal CT and right renoarteriography revealed a 6 X 6 cm-tumor at the center of the right kidney, comprising the middle calices and a 16 X 18 mm-saccular aneurysm at the first bifurcation of the renal artery. No remote metastasis was revealed. Aneurysmectomy and partial nephrectomy (three-quarter of right middle and lower calices) were simultaneously performed in situ. The renal cell carcinoma was pT2b, of clear cell subtype and grade 1, and no calcification of the aneurysmal wall was observed. Anuria lasted for 21 postoperative days, but thereafter diuresis appeared. Renal function after 3 post-operative months remains kept as follows: BUN, 26 +/- 7 mg/dl; serum creatinine, 26 +/- 0.6 mg/dl; creatinine clearance, 20.1 ml/min. Now, after 18 postoperative months, the patient is already returning to work, with neither relapse nor need of dialysis.

[Chemoendocrine therapy of newly diagnosed advanced prostatic cancer].

From November 1981 to November 1987, 35 patients with newly diagnosed advanced prostatic cancer (6 Stage C cases and 29 Stage D2 cases) were treated by chemoendocrine therapy consisting of orchiectomy, diethylstilbestrol-diphosphate and cisplatin. Objective responses were assessed at 3 months after the start of treatment. Of the 35 patients, 8 had PR (partial response) and 27 was objective stable by NPCP criteria. Objective progression was not seen. In analysis of long-term results, the 3-year and 5-year survival rate for total cases were 75.8% and 60.7%, respectively. For Stage C cases, the 3-year and 5-year survival rates were 100% and 100%; for Stage D2 cases, they were 72.2% and 54.2%, respectively. Relapse was seen in 7 (24.1%) of the 29 Stage D2 cases. All of these 7 patients had poorly differentiated adenocarcinoma and most of them had more than 10 bone metastases. As for side-effects, gastroenteric symptoms (nausea and vomiting), anemia and slight liver dysfunction were seen. These results suggest that the chemoendocrine therapy is an effective treatment in newly diagnosed cases of advanced prostatic cancer.

[A case of acute eosinophilic pneumonia induced by inhalation of acetylene].

A 17-year-old high school student, while carrying out soldering one morning, inhaled 100% acetylene, and experienced nausea and bilateral lower limb numbness several hours later. In the evening his symptoms worsened, dyspnea followed, and the patient was referred to our hospital the next day. On admission chest radiography and CT scanning revealed peripheral ground-glass opacity, patchy infiltrate and Kerley's B line in the right lung fields, and bilateral pleural effusion. Since the laboratory findings revealed leukocytosis without eosinophilia, increased CRP, and hypoxemia, bronchoalveolar lavage (BAL) and transbronchial biopsy (TBLB) was subsequently performed. Fluid analysis revealed marked increases in the total cell and eosinophil counts, and the biopsy result showed eosinophilic and lymphocytic infiltration of the alveolar septa. As a result, the case was diagnosed as acute eosinophilic pneumonia (AEP). Although inhalation of acetylene is known to induce pulmonary edema, all the typical findings of AEP but pulmonary edema were seen. This case demonstrates that AEP may be induced by inhalation of acetylene.

[A study of post-operative chemotherapy for renal pelvic and ureteral tumors].

A study was made of the relationship between post-operative chemotherapy and metastasis and recurrence of bladder tumor in 9 patients with renal pelvic and ureteral tumors. Within 6 months on average, post-operative metastasis was found in 33.3% (3 out of 9 cases). Differentiating according to the type of chemotherapy, the rate of occurrence of metastasis was 50% (2 out of 4) in the case of treatment with either a combination of FT-207, chromomycin A3 and cytosine arabinoside, or with FT-207 alone, while it was 100% (2 out of 2) in stage pT2 and pT3 patients. For combined administration of CDDP, FT-207 and cytosine arabinoside, or for that of CDDP and neocarzinostatin, on the other hand, the rate of occurrence of metastasis was only 20% (1 out of 5) in stage pT2 and pT3 patients, while CDDP administration was found to be effective for preventing post-operative metastasis. However, while recurrence of bladder tumor was found in a total of 3 out of 9 cases (33.3%), recurrence occurred in 40% (2 out of 5) of the cases treated with CDDP. The administration of CDDP was therefore not found to be effective for preventing recurrence of bladder tumor.

[A case of relapsed thymic carcinoma that responded remarkably to chemotherapy with CPT-11].

A chemotherapeutic protocol for advanced thymic carcinoma has not been established as yet. We described a case of advanced and relapsed thymic carcinoma that responded remarkably to subsequent chemotherapy with CPT-11. A 61-year-old man was admitted to our hospital because of facial edema and general fatigue. Chest X-ray and CT scan showed anterior mediastinal tumor which involved large vessels and pericardium. CT guided needle biopsy yielded a diagnosis of squamous cell type of thymic carcinoma. The patient was initially treated with ADOC (ADM, CDDP, VCR, CPA) chemotherapy and had successfully controlled for six months. However, the mediastinal tumor recurred and radiotherapy and nedaplatin plus ETP therapy were not effective. Then, CPT-11 chemotherapy (80 mg/m2, 2 weeks) was performed. The patient showed a partial response after two courses of CPT-11 chemotherapy. This case suggests that CPT-11 is a useful chemotherapeutic agent for advanced thymic carcinoma.

[ADOC regimen for unresectable advanced thymic cancer].

Between 1996 and 1998, we treated 6 patients with unresectable and advanced thymic cancer (stages IVa and IVb). All received 50 mg/m2 of cisplatin and 40 mg/m2 of doxorubicin intravenously (i.v.) on day 1,0.6 mg/m2 of vincristine i.v. on day 3, and 700 mg/m2 of cyclophosphamide i.v. on day 4; ADOC regimen, respectively at 3-4 week intervals. Four patients obtained a partial response (PR) after ADOC chemotherapy and the overall clinical response rate was 67%. No life-threatening side effects were noted. In 2 patients, cisplatin plus VP-16 chemotherapy failed to demonstrate any benefits prior to the ADOC regimen. Radiotherapy was initiated after the achievement of PR in the other 2 patients. ADOC chemotherapy appears to be an effective treatment for thymic cancer.