Reboxetine and cytochrome P450--comparison with paroxetine treatment in humans.

OBJECTIVES: The noradrenaline-selective antidepressant reboxetine in vitro is a weak inhibitor of both cytochrome P450 (CYP) 2D6 and CYP3A4. Thus, in this study the pharmacokinetics of reboxetine in relation to pharmacogenetics and the effects of reboxetine compared to paroxetine treatment on the CYP2D6 and CYP3A4 phenotype were analyzed in healthy control subjects. METHODS: Healthy male volunteers were treated with either 6 mg reboxetine (n = 26) or 30 mg paroxetine (n = 25). On Days 10/11 of treatment, serum concentrations of the antidepressants were measured and pharmacokinetic parameters calculated. Volunteers were phenotyped at the end of treatment and after at least 3 weeks washout (true phenotype) using 30 mg dextromethorphan (DM) hydrobromide given orally and measuring DM and metabolites in serum 2 h after intake. CYP2D6 and CYP2C19 genotypes were determined in parallel. RESULTS AND CONCLUSION: Reboxetine serum concentrations showed no correlation with the CYP2D6 genotype and the CYP2D6 phenotype, whereas paroxetine concentrations showed some dependence on CYP2D6. In contrast to in vitro investigations, indicating a major role of CYP3A4 in reboxetine metabolism, reboxetine concentrations in serum showed no correlation with the respective DM metabolic ratios. There was also no correlation between paroxetine concentrations and the CYP3A4 phenotype data. The CYP2C19 genotype (only heterozygosity) had no influence on reboxetine and paroxetine pharmacokinetics. There were only minor changes in the DM metabolite pattern on treatment with reboxetine and no evidence of enzyme inhibition was obtained. In contrast and as expected, paroxetine strongly inhibited CYP2D6. Thus, reboxetine treatment has no effect on the CYP2D6 genotype and no clinically relevant drug interactions involving CYP2D6 are anticipated.

Pharmacokinetics of oxaliplatin and non-hematological toxicity in metastatic gastrointestinal cancer patients treated with chronomodulated oxaliplatin, 5-FU and sodium folinate in a pilot investigation.

OBJECTIVE: To analyze in a pilot study the association between the pharmacokinetics of chronomodulated administered oxaliplatin and non-hematological toxicity in patients with metastatic gastrointestinal cancer. METHODS: 16 patients received a 4-day chemotherapeutic regimen consisting of a 12-h chronomodulated infusion of oxaliplatin (25 mg/m2) followed by a 12-h chronomodulated infusion of 5-fluorouracil (750 mg/m2) and sodium folinate (150 mg/m2) daily. Plasma pharmacokinetics of oxaliplatin, measured as ultrafiltrable platinum, were determined. RESULTS: Pharmacokinetics and non-hematological adverse events could be assessed in all patients included in the study. Pharmacokinetic parameters showed moderate interpatient variability. The occurrence of nausea and vomiting, but not diarrhea, was significantly associated with the pharmacokinetics of ultrafiltrable platinum. Thus, increased AUC values were observed in patients who experienced nausea or vomiting. No differences in pharmacokinetic parameters were found between patients with and without oxaliplatin-induced neurotoxicity or the other selected non-hematological toxicities. CONCLUSION: The preliminary results in this pilot study suggest an association between pharmacokinetics of ultrafiltrable platinum and non-hematological toxicity such as nausea and vomiting. Furthermore, although the sample size is limited, systemic exposure to ultrafiltrable platinum appears to predict the risk of non-hematological toxicity in patients treated with chronomodulated oxaliplatin combined with 5-FU and FS.

Chronomodulated chemotherapy with oxaliplatin, 5-FU and sodium folinate in metastatic gastrointestinal cancer patients: original analysis of non-hematological toxicity and patient characteristics in a pilot investigation.

OBJECTIVE: Several clinical trials have demonstrated that oxaliplatin is a useful agent in combination with 5-fluorouracil (5-FU) and folinic acid (FA) for the treatment of patients with colorectal carcinoma. The aims of this pilot study were to evaluate non-hematological toxicity and patient characteristics in gastrointestinal cancer patients treated with chronomodulated chemotherapy consisting of oxaliplatin, 5-FU and sodium folinate. METHODS: Patients with metastatic gastrointestinal cancer received a chronomodulated regimen with oxaliplatin (25 mg/m2), 5-FU (750 mg/m2) and sodium folinate (150 mg/m2). Non-hematological toxicities were evaluated and analyzed in relation to patient characteristics, i.e. age, sex, body weight, body mass index (BMI), body surface area and smoking status. Toxicity was graded according to the National Cancer Institute Common Toxicity Criteria. RESULTS: The severity of non-hematological toxicity was generally moderate. Grade 4 toxicity was only found in 2 patients with diarrhea (12.5%). The most frequent common adverse events were nausea, Grades 1 - 2 in 13 patients (81.3%), followed by motor neuropathy, Grades 1 - 3 in 11 patients (68.9%). The analyses showed that patient characteristics such as BMI and smoking status were associated with mucositis/stomatitis, vomiting or mood alteration. Furthermore, there was a relationship between smoking status and the overall non-hematological toxicity. Smokers had significantly higher overall toxicity than non-smokers and body mass index correlated significant with overall toxicity. CONCLUSION: The results of this pilot investigation suggest that a chronomodulated regimen with oxaliplatin, 5-FU and sodium folinate has a manageable non-hematological toxicity profile and that toxicity of the chronomodulated schedule studied depends on the patient characteristics. In further investigations, risk factors determining chemotherapeutic toxicity should be considered. Because of the small number of patients in this pilot investigation, the findings need to be confirmed in a larger clinical study.

Trough levels of mycophenolic acid and its glucuronidated metabolite in renal transplant recipients.

OBJECTIVE: The prophylactic use of the immunosuppressant prodrug, mycophenolate mofetil (MMF) to prevent graft rejection in renal transplant patients is continuing to increase. We measured trough levels of the active metabolite, mycophenolic acid (MPA) and its inactive glucuronide (MPAG) in renal recipients with the aim of characterizing individual variability and of ascertaining factors influencing trough levels, in particular the effect of differences in renal function and the effect of drugs given concurrently. METHODS: Laboratory and clinical data obtained in 35 renal recipients treated with triple therapy (MMF, cyclosporin A (CsA), steroids) were included in this retrospective study. Trough levels of MPA and MPAG were obtained after transplantation and up to 16 months post transplantation where the mean observation period was 5.7 months. Plasma levels were measured using a validated HPLC assay. RESULTS: A total of 212 plasma concentrations of MPA and 209 of MPAG were measured. There was considerable intra- and interindividual variability in MPA and MPAG trough levels especially in the early post-transplantation phase. At a fixed dose of 2 g/d MMF, the mean MPA level during the first 30 days averaged 1.46 +/- 1.31 microg/ml vs. 1.87 +/- 0.89 microg/ml after 30 days and later (p = 0.130) and the mean MPAG concentration averaged 188.1 = 142.8 [microg/ml vs. 98.09 +/- 52.4 microlg/ml (p 0.003). The MPAG levels were positively correlated with the serum creatinine concentrations (r = 0.815, p < 0.001), and in the case of MPA there was a correlation with the serum protein concentrations (r = 0.258, p = 0.001). Concomitant drug treatment using CsA, steroids and furosemide were without effect of the measured plasma concentrations, but in the case of xipamide (+) and diltiazem (-) an effect on MPA and MPAG levels and a co-effect depending on the serum creatinine could not be excluded. Neither CsA trough levels nor hemoglobin levels were related to MPA and MPAG trough levels. CONCLUSIONS: The data of this study demonstrate that there is substantial individual variability in the trough levels of MPA and MPAG after renal transplantation which may be associated with the functional status of the graft and the serum protein level. Whether comedication with xipamide and diltiazem affects the plasma levels of MPA and MPAG remains to be clarified in further investigations.

[Oncologic pharmacotherapy of elderly patients]. / Onkologische Pharmakotherapie alter Patienten.

Age is the major risk factor for the development of cancer. Difficulties in medical treatment of elderly patients with cancer may be due to age dependent changes in physiological organ function, which result in pharmacokinetical and pharmacodynamical changes. They also may be due to co-morbidities with the need of pharmacological therapy. The choice of adequate treatment protocols has to consider this age dependent changes in pharmacokinetics and the presence of co-morbidities. Most of the data reporting results of medical oncological treatment in patients with cancer are collected in younger patients. Therefore, the present knowledge concerning medical oncological treatment of elderly patients with cancer is still limited.

High plasma levels of 8-methoxypsoralen following bath water delivery in dermatological patients.

With respect to the clinical advantages known for bath PUVA therapy, it was of interest to compare the plasma levels of 8-methoxypsoralen (8-MOP) in bath therapy with those after oral administration for a better insight into the pharmacokinetics of 8-MOP following different modes of application. Considerable high plasma levels of 8-MOP were observed after bath therapy with interindividual variability. The half-life of plasma 8-MOP was markedly shorter after bath PUVA than after oral application. The pharmacokinetic profile of 8-MOP differs according to the mode of application.

Influence of diclofenac (group of nonsteroidal anti-inflammatory drugs) on fracture healing.

INTRODUCTION: Nonsteroidal antirheumatics (NSAR; NSAID) are often used in patients with fractured bones for analgetic reasons. This animal experiment was performed to determine the influence of NSAR on the process of fracture healing. As an alternative, tramadol, the centrally acting analgetic without peripheral effects, was included in this experiment. MATERIALS AND METHODS: Wistar rats were operated on by a transverse osteotomy of the proximal tibia of the left leg. The fracture was stabilized by intramedullary nailing (healing period 21 days). All drugs were applied orally twice a day. The animals were divided into four groups with 10 rats each: Group 1 was treated with placebo (P), group 2 with tramadol (T; 20 mg/kg body weight/day), group 3 with diclofenac sodium (DS; 5 mg/kg bw/day) for 7 days followed by 14 days of placebo, group 4 with diclofenac sodium (DL; 5 mg/kgbw/day) over 21 days. On day 21 the rats were killed, and each leg was examined by X-ray, then the tibia was examined by CT scan, three-point bending, and histology. RESULTS: The results of CT and three-point bending showed that rats treated by diclofenac presented with delayed fracture healing compared with those treated by placebo or tramadol. Bone density in CT was highest in group 1 (mean 611.4+/-50.1 mg/ml), followed by group 2 (mean 542.5+/-29.5 mg/ml). Groups 3 (mean 411+/-34.0 mg/ml; p=0.006) and 4 (mean 395.2+/-15.4 mg/ml; p=0.009) were significantly lower. The stability of the bones, as measured by the breaking force ( F(max)), was highest in group 1 (mean 45.8+/-19.0 N), followed by group 2 (mean 39.0+/-7.9 N; NS); group 3 (mean 20.6+/-7.8 N; p=0.01) was significantly lower than the placebo animals, followed by group 4 (mean 26.5+/-8.3 N; p=0.03). Similar results were shown for bending stiffness: group 1 (mean 1404.6+/-611.4 Nmm/mm), group 2 (mean 1033.2+/-232.1 Nmm/mm; NS), group 3 (mean 564.2+/-457 Nmm/mm; p=0.045), and group 4 (mean 494.8+/-340.2 Nmm/mm; p=0.028). There were no significant differences between groups 1 and 2 and between groups 3 and 4, respectively. Diclofenac serum levels on day 21 in rats with long-term diclofenac application (mean 301.4+/-83.3 ng/ml) were comparable to those in humans. CONCLUSION: Oral application of diclofenac significantly delayed fracture healing in rats. This effect might be comparable to other NSAR and fracture healing in humans.

Ex vivo stimulation of renal transport of the cytostatic drugs methotrexate, cisplatin, topotecan (Hycamtin) and raltitrexed (Tomudex) by dexamethasone, T3 and EGF in intact human and rat kidney tissue and in human renal cell carcinoma.

Previous experiments have shown that both in vivo and in vitro pre-treatment with various hormones increases the renal transport capacity for weak organic acids, such as PAH, in rats. The aim of the present study was to test whether or not accumulation of the anticancer drugs methotrexate (MTX), cisplatin (CP), raltitrexed (Tomudex) and topotecan (Hycamtin) can be increased in intact, healthy rat and human renal cortical slices and in human renal cell carcinoma (RCC). Intact, healthy human tissue was obtained from tumour bearing kidneys of patients suffering from RCC. Experiments were intended as a new approach to overcome so-called multidrug resistance. Kidney tissue slices were incubated for 24 h in William's medium E containing various concentrations of dexamethasone, T(3), or EGF. Thereafter slices were placed in anticancer drug containing Cross-Taggart medium and the drug uptake into kidney tissue was measured for 2 h. In intact rat and human renal tissue slices, the uptake of p-aminohippurate (PAH = reference substance) increased significantly after incubation in dexamethasone containing medium (134% and 156%, respectively). There were no stimulating effects of either T(3) or EGF on PAH accumulation. On the other hand, only the accumulation of MTX, but not of CP, raltitrexed or topotecan, was significantly enhanced after hormone pre-treatment both in intact renal tissue and in RCC. A stimulation of renal PAH accumulation can be performed ex vivo, as reported previously, both in intact rat and human renal cortical slices and in RCC. Discrepancies between the effects of dexamethasone and T(3) or EGF indicate different modes of action of these substances at the cellular level. Unfortunately, with the exception of MTX, the uptake of anticancer drugs can not be stimulated effectively ex vivo in human RCC tissue by the substances used. Evidently the transport of these anticancer drugs out of the kidney cells is more effective than their uptake.

Phase I clinical and pharmacokinetic study of combination chemotherapy with topotecan and ifosfamide in patients with progressive or relapsed solid tumors.

PURPOSE: Topotecan and ifosfamide are effective in the treatment of various solid tumors. Up to the time of this study, the two drugs have been combined just once (Smith et al. 1998). Due to its hematotoxicity, topotecan has been used predominantly within monochemotherapy protocols. However, the combination of topotecan and alkylating agents is supra-additive in many preclinical models. This phase-I trial was primarily performed to evaluate the maximum tolerable dose (MTD) and dose-limiting toxicity (DLT) of a combination chemotherapy with topotecan and ifosfamide using a 5-day schedule. A secondary goal was to estimate the response rate in a group of heavily pretreated patients with advanced solid tumors. METHODS: The pharmacokinetics of topotecan were preliminarily determined in some of the patients. A total of 12 patients (three female/nine male), median age 49 years (range 19-69), 11 with prior chemotherapy, received a total of 24 courses of chemotherapy at three dose-levels of topotecan. Ifosfamide was administered by intravenous infusion over 3 h immediately followed by a 30-min infusion of topotecan. Mesna (3 x 300 mg x m(2) x day) was given routinely during chemotherapy as a uroprotector. G-CSF (filgrastim) was permitted only in cases of febrile neutropenia (FN). RESULTS: The major toxicity was non-hematologic; severe liver and renal toxicity were observed in three out of 11 patients. Two treatment-related deaths occurred. No clinical remissions occurred in 11 evaluable patients. The pharmacokinetics of topotecan were relatively similar in our patients and supported findings in recent literature. The MTD of this combination was defined at dose-level 2 (1.0 mg/m(2) of topotecan and 750 mg/m(2) of ifosfamide). CONCLUSIONS: Further trials should not exceed this dose. The pharmacological causes for the pronounced toxicity have to be clarified.

Effects of short-term treatment with diclofenac-colestyramine on renal function and urinary prostanoid excretion in patients with type-2 diabetes.

OBJECTIVE: To determine the effect of short-term administration of diclofenac-colestyramine on glomerular filtration rate (GFR), renal plasma flow (RPF) and urinary excretion of prostanoids in patients with type-2 diabetes without and with impaired renal function. METHODS: In the randomised, single-blind, placebo-controlled, two-period crossover study, 32 patients with type-2 diabetes (group 1: 16 patients without impaired renal function, creatinine clearance > or =80 ml/min and group 2: 16 patients with impaired renal function, creatinine clearance 30-79 ml/min) received 140 mg diclofenac-colestyramine (corresponding to 75 mg diclofenac sodium) or placebo twice a day on days 1 and 2 and once on day 3 with a wash-out period of 6 days between the two periods. GFR was assessed using both measurement of creatinine clearance and calculation of inulin clearance and RPF was assessed using calculation of para-aminohippurate (PAH) clearance after the short-term administration on day 3. Urinary excretion of prostanoids (PGE(2), PGE-M, 6-keto-PGF(1alpha), 2,3-dinor-6-keto-PGF(1alpha), TxB(2), 2,3-dinor-TxB(2) and 11-dehydro-TxB(2)) were measured before and after drug intake. RESULTS: Comparison with placebo showed no effect of diclofenac-colestyramine on creatinine, inulin or PAH clearance ( P>0.05) in patients with type-2 diabetes either without or with impaired renal function. The differences in creatinine, inulin and PAH clearance between the two groups of patients were not influenced by diclofenac-colestyramine. Urinary excretion of PGE(2), PGE-M, 6-keto-PGF(1alpha), 2,3-dinor-6-keto-PGF(1alpha), TxB(2), 2,3-dinor-TxB(2) ( P=1.89) and 11-dehydro-TxB(2) was significantly reduced by diclofenac-colestyramine. CONCLUSION: These results indicate that proven non-specific cyclooxygenase inhibition by short-term administration of diclofenac-colestyramine did not affect renal haemodynamic function (GFR, RPF) in patients with type-2 diabetes either without or with impaired renal function.

[Progressive systemic sclerosis - treatment results of extracorporeal photopheresis]. / Progressive systemische Sklerodermie. Behandlungsergebnisse unter extrakorporale Photopherese.

The treatment of progressive systemic sclerosis (PSS) is still unsatisfactory. We report on clinical, laboratory and immunological findings in 26 patients with PSS (6 males, 20 women) treated with extracorporeal photopheresis (ECP) for 8 cycles in a nonrandomized, uncontrolled study. ECP was performed on two consecutive days once a month. 8-methoxypsoralen concentrations in plasma and buffy coat were monitored by HPLC. We performed a standardized examination programme and determined parameters of inflammation and immune function. Global assessment revealed a partial remission in 18 patients, a stable disease in 8 patients and a slight progression in one patient. In the peripheral blood count a significant increase of CD3-positive NK cells was noted (p=0.03) although the leukocyte count decreased from 2,255 to 1,156 cells/microl. There was a non-significant decrease of elastase (102. 9 vs. 90.4 ng/ml), sulfidoleukotriens (2,255.4 vs. 1,688.9 pg/ml), ICAM-1 (301.9 vs. 276.6 ng/ml), soluble IL-2 receptor (609.0 vs. 422. 3 U/ml), and IL-10 (164.7 vs. 138.7 pg/ml). IL-6 and IL-8 did not show significant changes. The ECP treatment of patients with PSS shows immunomodulatory effects changing levels of pro-inflammatory and cytokine substances. Even after 8 cycles partial remission or stable disease is seen in patients as shown by global assessment and certain clinical symptoms. On the other hand, sufficient data on the long-term outcome are still missing.

Telomere shortening triggers a p53-dependent cell cycle arrest via accumulation of G-rich single stranded DNA fragments.

It has been repeatedly suspected that telomere shortening might be one possible trigger of the p53-dependent cell cycle arrest, although the mechanism of this arrest remained unclear. Telomeres in human cells under mild oxidative stress accumulate single-strand damage faster than interstitial repetitive sequences. In MRC-5 fibroblasts and U87 glioblastoma cells, which both express wild-type p53, oxidative stress-mediated production of single-strand damage in telomeres is concomitant to the accumulation of p53 and p21 and to cell cycle arrest. This response can be modeled by treatment of cells with short single stranded telomeric G-rich DNA fragments. The arrest is transient in U87 cells. Recovery from it is accompanied by up-regulation of telomerase activity and elongation of telomeres. Overexpression of mutated p53 is sufficient to reverse the phenotype of inhibition as well as the delayed activation of telomerase. These data suggest that the production of G-rich single stranded fragments during the course of telomere shortening is sufficient to trigger a p53 dependent cell cycle arrest.

Plasma and buffy coat concentration of 8-methoxypsoralen in patients treated with extracorporeal photopheresis.

Recently, a new therapy involving an extracorporal activation of orally administered 8-methoxypsoralen (8-MOP), photosensitizing furocoumarin, is established for the treatment of different skin diseases, extracorporeal photopheresis (ECP). The pharmacokinetic profile of 8-MOP has been pursued as part of a clinical study which should assess the efficacy of ECP in patients with progressive systemic sclerosis and cutaneous T-cell lymphoma. The enormous intra-individual variations proofed for plasma as well as buffy coat concentration are unfavourable for oral 8-MOP therapy. Therefore, the introduction of liquid 8-MOP formulation that allows the direct administration of the drug in to the treatment bag of the ECP device is challenging.

Drug monitoring of orally administered 8-methoxypsoralen in patients treated with extracorporeal photopheresis.

Recently extracorporeal photopheresis (ECP), a new therapy involving extracorporeal activation of orally administered 8-methoxypsoralen (8-MOP), has been established for the treatment of different skin diseases. The pharmacokinetic profile of 8-MOP has been pursued in a clinical study which aimed to assess the efficacy of ECP in patients with progressive systemic sclerosis and cutaneous T-cell lymphoma. However, the enormous intraindividual variations in plasma as well as buffy coat concentrations affect the efficacy of oral 8-MOP therapy. Therefore, the introduction of a liquid 8-MOP formulation enabling the direct administration of the drug into the treatment bag of the ECP device is challenging.

Lack of pharmacokinetic interaction between dextromethorphan, coumarin and mephenytoin in man after simultaneous administration.

The possible interference of simultaneously given dextromethorphan (10 mg dextromethorphan-HBr-H2O), coumarin (10 mg), and mephenytoin (100 mg (R/S)-mephenytoin) on oxidative routes of drug metabolism performed by different cytochrome P450 enzymes and the possibility to detect all of the three substances and their metabolites in urine were investigated in 12 healthy subjects. The concentrations of parent drugs and main metabolites were measured in urine using modified HPLC-methods. All subjects were extensive metabolizers of mephenytoin and dextromethorphan as calculated using hydroxylation index (HI) for mephenytoin and as seen in the quantification of urinary dextromethorphan/dextrophan. A combined determination of coumarin and dextromethorphan with their metabolites or of coumarin and mephenytoin with their metabolites in urine is possible. The combined HPLC separation of all parent compounds and metabolites, however, is not useful because of the necessity to treat the urine samples in very different ways. An overlapping of retention times of the substances in HPLC does not occur. With it a simultaneous administration of all three drugs is possible. A following collection of urine over a period of 8-12 h serves for characterizing activities of different cytochrome P450 enzymes of patients. So particularly the influence of a long term drug therapy on the hydroxylation activities of these cytochromes is easily definable without the disturbing influence of intraindividual variation of drug oxidation with time.

Lack of effect of grapefruit juice on diltiazem bioavailability in normal subjects.

The objective of the presented two-way randomized crossover study was to investigate whether repeated doses of grapefruit juice (200 ml at 0, 2, 4, 8 and 12 h) enhanced bioavailability of diltiazem (120 mg, orally) in nine healthy male subjects. Grapefruit juice did not alter AUC or cmax of diltiazem, whereas the half life experienced a slight, but statistically significant, increase (4.1 +/- = 1.2 vs 5.1 +/- 0.7 h). The N-demethyldiltiazem/diltiazem and deacetyldiltiazem/diltiazem ratios were not affected by grapefruit juice intake, which indicates that these metabolic pathways are not inhibited. Whereas bioavailability of some calcium channel antagonists of the dihydropyridine type metabolized via the same cytochrome P450 has been shown to be dramatically increased by grapefruit juice intake, the bioavailability of the benzothiazepine calcium channel antagonist diltiazem remained unchanged. This suggests that factors other than biotransformation may contribute to the clear effect of grapefruit juice on the bioavailability of those substances.

Grapefruit juice inhibits 7-hydroxylation of coumarin in healthy volunteers.

The effect of grapefruit juice on the urinary excretion of 7-hydroxycoumarin after oral administration of 10 mg coumarin, as an index of cytochrome P450 dependent coumarin metabolism, has been investigated in an open, randomised cross over study in 13 healthy volunteers (7 female, 6 male). The percentage of 7-hydroxycoumarin found in urine was significantly decreased up to 8 h after simultaneous intake of 300 ml grapefruit juice. If the same volume of juice was swallowed 30 min prior to the administration of coumarin, 7-hydroxycoumarin excretion was delayed by up to 6 h. MRTexcr. of coumarin was 70% extended by coadministration of grapefruit juice. It appears that grapefruit flavonoids inhibit cytochrome P450 2A dependent metabolic pathways. The mechanism of cytochrome P450 inhibition by these flavonoids is still poorly understood.

An enzymatic method for the isolation of mouse Leydig cells.

Mouse Leydig cells were obtained by dispersion of testes of adult animals (aged 6-15 months) with a neutral protease from B. polymxa (dispase; EC 3.4.24.4). The crude Leydig cell suspension was purified by centrifugation on a discontinuous Percoll gradient using a special centrifugation procedure similar to elutriation. The crude cell suspension obtained from 50 testes could be processed in one run. The combination of these two methods yielded 320000 +/- 53000 Leydig cells/testis (n = 554 testes). The purity of the Leydig cell fraction was greater than or equal to 95% (nucleated cells) based on morphological and histochemical (staining for naphthyl esterase) identification. The purified Leydig cells showed an excellent ultrastructural appearance. More than 98% excluded trypan blue. In the presence of NADPH, testosterone biosynthesis was increased only 1.15 +/- 0.1-fold yielding a "quality factor" of 34.8. Maximal hCG doses induced 10(6) purified Leydig cells to produce 5 nmol testosterone/hr. (40-fold stimulation in comparison to basal values). The Leydig cells showed 43100 +/- 2500 LH/hCG receptors and an association constant of Ka = 1.95 x 10(9) M-1. Due to the reproducibility of the method, to the yield as well as to the morphological and functional state of the purified Leydig cells at least 25% of laboratory animals could be saved.