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A liquid chromatography-mass spectrometry (LC/MS) assay method was developed for the quantification of PSC 833 in rat plasma, using amiodarone as internal standard (IS). Separation was achieved using a C(8) 3.5 microm (2.1 mm x 50 mm) column heated to 60 degrees C with a mobile phase consisting of acetonitrile-ammonium hydroxide 0.2% (90:10 v/v) pumped at a rate of 0.2 mL/min. Detection was accomplished by mass spectrometer using selected ion monitoring (SIM) in positive mode. An excellent linear relationship was present between peak height ratios and rat plasma concentrations of PSC 833 ranging from 10 to 5000 ng/mL (R(2)>0.99). Intra-day and inter-day coefficients of variation (CV%) were less than 15%, and mean error was less than 10% for the concentrations above the limit of quantification. The validated limit of quantification of the assay was 10 ng/mL based on 0.1 mL rat plasma. The method limit of detection, based on an average signal-to-noise (S/N) ratio of 3, was found to be 2.5 ng/mL. The assay was capable of measuring the plasma concentrations of PSC 833 in rats injected with a single dose of 5 mg/kg of the drug. PSC 833 and IS eluted within 4 min, free of interfering peaks. The method was found to be fast, sensitive, and specific for the quantification of PSC 833 in rat plasma.

The expression of high levels of P-glycoprotein (Pgp) in circulating mononuclear cells allowed us to use an ex vivo assay as a surrogate measure of Pgp antagonism. Efflux of rhodamine from CD56(+) cells was measured before the start of PSC 833 and at varying times thereafter. Patients receiving PSC 833 had decreased rhodamine efflux from their circulating CD56(+) cells. Time course studies showed that following a single oral dose of PSC 833, decreased rhodamine efflux was found in some patients within 15 minutes of treatment. Maximal inhibition was observed at times ranging from 45 minutes to 60 minutes. A dose-response relationship was shown between the concentration of PSC 833 in the blood and the inhibition of rhodamine efflux, with an apparent plateau of the inhibition of rhodamine efflux at approximately 1,000 ng/mL. The Ki, defined as the concentration required for half-maximal inhibition of Pgp-mediated rhodamine efflux, was determined to be in the range of 29 to 181 ng/mL; although results in two patients were distinctly different, with Ki values of 914 and 916 ng/mL. MRK-16 staining was similar among all patients. We conclude that measurement of rhodamine efflux from CD56(+) cells provides a surrogate assay with the potential for monitoring Pgp antagonism in clinical trials.

We have developed a rapid, sensitive and selective method for the determination of the cyclosporin analog PSC 833 in human and mouse plasma using cyclosporin A as internal standard. The assay uses liquid-liquid extraction with diethyl ether for sample clean-up followed by reversed-phase high-performance liquid chromatography with UV detection at 210 nm. Good peak shapes were obtained using a NovaPak Phenyl column operating at 72 degrees C. Good selectivity from endogenous compounds was achieved using a mobile phase composed of methanol-acetonitrile-water (34:34:32). The retention times of cyclosporin A and PSC 833 were approximately 7.8 and 11.7 min, respectively, with two major endogenous peaks at 9.2 and 16.7 min. Selective decreasing of the retention times of cyclosporin A and PSC 833 relative to these interferences occurring upon aging of the column was balanced by increasing the percentage of methanol relative to acetonitrile. No other late eluting peaks were present, resulting in a total analysis time of 20 min per sample. The assay performance in human plasma was good. The absolute recovery of PSC 833 after the sample clean-up step was 48+/-6%. The lower limit of quantitation was 0.05 microM using 500 microl of sample. Within the linear dynamic range of the assay (0.10-5.0 microM) the accuracy was close to 100% and within-day and between-day variation less than 7%. Because of the limited availability of blank mouse plasma, the concentration in samples from mice were determined using calibration curves constructed in human plasma. The lower limit of quantitation in mouse was 0.25 microM using 200 microl of sample. Overall, the performance of the assay in mouse plasma was somewhat less than in human plasma but accuracy and precision were within the ranges that are considered acceptable for bio-analytical assays.

Cyclosporin A (CyA) and its metabolites seem to have nephro-, hepato- and neurotoxic side effects. Immunosuppressive therapy is a narrow path between the risk of rejection by underimmunosuppression and toxic organ damage by overdosage. Thus CyA dosage must be calculated to avoid the risks of organ rejection through underdosage and toxic organ damage through overdosage or accumulation of metabolites. In routine monitoring of CyA therapy, it can be important to measure not only the parent drug but also the metabolites. We describe a rapid and isocratic high-performance liquid chromatographic method for measurement of CyA and its metabolites M1, M17 and M21 in whole blood. CyA was detected by ultraviolet absorption at 212 nm with a CN analytical column maintained at 50 degrees C and recycling of hexane-isopropanol as mobile phase for improved long-term column stability and efficiency. The minimum detectable concentration of CyA and the three metabolites was 10 ng/ml blood. Our modified HPLC method for the determination of CyA and its metabolites is a simple (isocratic), rapid (the retention times were 7.1 min for CYD, internal standard, 8.9 min for CyA, 11.0 min for M21, 12.9 min for M17 and 16.3 min for M1) and economical method suitable for measuring the concentration of the major metabolite, M17, and for routine monitoring of CyA-treated patients.

Previous studies in vitro and in rodent transplantation models have suggested that an analogue of cyclosporin, SDZ-IMM-125, has immunosuppressive properties at least equivalent to that of cyclosporin. As the bioavailability of the drug was considered to be greater than that of cyclosporin, it was hoped that lower doses could be used with the avoidance of nephrotoxicity. Renal allografts were undertaken between beagle dogs from two partially inbred breeding colonies. After transplantation, SDZ-IMM-125 was given orally at a dosage of 5, 7.5, 10 or 20 mg/kg/day, and graft survival compared to that in dogs given cyclosporin 10 mg/kg or in untreated animals. The results showed that SDZ-IMM-125 is immunosuppressive in dogs and prolongs graft survival up to 50 days at a dosage of 20 mg/kg/day. However, at this dose histological changes suggestive of liver toxicity were observed in one dog, and mild anaemia was produced,but there was no evidence of nephrotoxicity. Absorption profiles suggested that the drug is rapidly absorbed and metabolized, and that a more frequent daily dosage may be appropriate. Overall, there appeared to be no significant advantage for the analogue SDZ-IMM-125 over cyclosporin. The transplant model was associated with a high spontaneous renovascular thrombosis rate, particularly after cyclosporin administration, which was prevented by the daily administration of aspirin.

Anaemia is common in children following cardiac transplantation. In a series of 5 children with anaemia beyond the immediate post-operative period one had a hypochromic, microcytic anaemia which corrected with oral iron. The other four had normochromic, normocytic anaemias unresponsive to iron or folate supplementation and associated with inappropriately low levels of erythropoietin. Subcutaneous administration of low dose human recombinant erythropoietin to these four patients resulted in correction of their anaemia. Our findings suggest that erythropoietin deficiency is an important cause of anaemia in transplant recipients and should be sought in cases of anaemia refractory to conventional haematinic therapy. In cases of proven erythropoietin deficiency, treatment with erythropoietin is effective, acceptable to patients and preferable to repeated blood transfusion.

The authors assessed the ocular toxicity and pharmacokinetics of subconjunctivally and intravenously administered cyclosporine in New Zealand white rabbits. Fifteen rabbits received a subconjunctival injection of 5 (five animals), 10 (five animals) or 25 (five animals) mg of cyclosporine in 0.1 mL (intravenous solution of Sandimmune [50 mg/mL]); 5 mg was found to be the maximum tolerable dose. This dose was given as a bolus to 36 other rabbits either subconjunctivally (18 animals) or intravenously (18 animals). In both groups the cyclosporine concentrations in the ocular compartments, blood and urine were measured by means of high-pressure liquid chromatography at 0.5, 1, 2, 4, 8 and 12 hours, three animals being assessed at each interval. Subconjunctival administration resulted in peak cyclosporine concentrations of 718 ng/mL in the aqueous humour and 1078 ng/mL in the vitreous humour, compared with no detectable levels in the aqueous humour and a peak concentration of 292 ng/mL in the vitreous following intravenous administration. The peak blood cyclosporine levels were 10 times lower after subconjunctival injection than after intravenous injection. The results indicate that subconjunctival administration is superior to intravenous administration in enhancing the ocular absorption of cyclosporine while minimizing systemic exposure in the rabbit.

The expression of delayed contact hypersensitivity was studied in 6 patients with chronic contact dermatitis treated with cyclosporin A (CsA) 5 mg/Kg/day. Quantitative patch test challenge was used to establish individual dose-response curves and threshold concentration to certain allergens in the European Standard Battery. In all 6 patients, responses were reduced over the whole range of allergen concentrations, and in the 5 in whom the threshold for expression of contact hypersensitivity could be determined, the threshold was raised by CsA therapy. In addition, the clinical manifestations of allergic contact dermatitis underwent complete resolution within 2-3 weeks of CsA therapy. It was concluded that CsA inhibits expression of delayed contact hypersensitivity reactions in human skin.

Cyclosporine's toxic side effects are widely recognized as a cause of major morbidity. Peripheral vasoconstriction has been suggested as the pathophysiological mechanism for hypertension and renal failure caused by cyclosporine. To study vascular effects on peripheral arterial circulation, cyclosporine was injected into isolated hind limbs perfused at constant flow in the dog. Pure powder cyclosporine was dissolved in a 10% fat emulsion and infused directly into the arterial inflow of the perfused hind limb (n = 7) at a concentration of 5 mg/kg body weight. Reactivity of the vascular bed was first shown by an average decrease of 72 +/- 4 mmHg (95% confidence interval 63 to 81, P less than 0.01) in hind limb perfusion pressure after infusion of 5 mg nitroglycerin. Infusion of fat emulsion did not cause any significant changes. The infusion of cyclosporine caused an average increase of 29 +/- 5 mmHg (95% confidence interval 17 to 41, P less than 0.01) in hind limb perfusion pressure after 4 mins' infusion. After cyclosporine infusion, the vascular bed still responded to nitroglycerin by an average decrease of 56 +/- 5 mmHg (95% confidence interval 42 to 69, P less than 0.01) in perfusion pressure. Blockade of alpha-receptors with phentolamine in five dogs and ipsilateral lumbar sympathectomy in four prevented the increase in perfusion pressure following cyclosporine injection. In conclusion, cyclosporine injected at high doses causes a small vasoconstriction of the peripheral arterial circulation in the hind limb through stimulation of alpha-adrenergic receptors mediated by the sympathetic nervous system. Since the response is completely abolished by sympathectomy, it is probably caused by reflex activation. A direct effect of cyclosporine on the arterial vessel walls of the limb can therefore be excluded.

Intravenous cyclosporin (4 mg/kg daily) was given to fifteen patients with severe active ulcerative colitis, fourteen of whom had failed to respond to at least 10 days' treatment with intravenous steroids. In the acute phase of the open trial, eleven patients (73%) improved and avoided colectomy; the mean response time was 5.8 days. Patients who responded were maintained on oral cyclosporin (6-8 mg/kg daily) for a 6-month chronic phase. Six of the eleven patients have been weaned from steroids and remain in clinical and endoscopic remission 5-18 months after discontinuation of cyclosporin; three are well at 5 months and their steroid dose is being tapered; one patient relapsed after 5 months on oral cyclosporin and underwent colectomy; and one patient failed to show continued improvement with oral cyclosporin but is in clinical remission after changing to mercaptopurine treatment. Slight self-limiting adverse side-effects of cyclosporin were seen, but none necessitated withdrawal from the study. Cyclosporin seems to be an effective treatment for patients with severe active ulcerative colitis who have not responded to steroids.

144 patients with severe rheumatoid arthritis from six centres were randomised to receive oral cyclosporin or placebo for 6 months. The initial daily dose of cyclosporin was 2.5 mg/kg, which was increased cautiously with monitoring of serum cyclosporin levels and creatinine; the mean stabilisation dose was 3.8 mg/kg. There were significant improvements in the cyclosporin-treated patients compared with the controls in the major outcomes of reduction of active joints (23% improvement), pain (24%), and functional status (16%); global improvement was 27%. In the cyclosporin group serum creatinine increased by a mean of 15.6 mumols/l and mean arterial blood pressure by 6.27 mmHg; these increases were controlled in all but 2 patients by dose adjustment without withdrawal from the study.

Transplant patients receiving the immunosuppressive drug cyclosporin A require regular monitoring to maintain levels within a narrow therapeutic range. A stable, accurate and reproducible high-performance liquid chromatographic method for analysis of cyclosporin A in whole blood has been developed using the Varian Advanced Automated Sample Processor. Starting with 200 microliters of blood, absolute recovery of both cyclosporin A and the internal standard was 81% with a detection limit of 12.5 ng/ml. The assay is perfectly linear over the range 0-1000 ng/ml (r2 = 1.0). At a concentration of 250 ng/ml, the coefficient of variation, both between samples and between assays, is 1.87%. Chromatographic cycle time is 10.2 min per sample. Up to eighty samples can be processed by one person in a working day, with final results within 16 h.

A 40-year-old male liver allograft recipient had neurological dysfunction and renal failure while his cyclosporin blood levels were in the therapeutic range; these features recurred on rechallenge. The hypothesis that this toxic effect might have resulted from abnormal metabolism of cyclosporin by liver cytochrome P-450 IIIA was investigated with the [14C]erythromycin breath test, which is a measure of this enzyme's activity. P-450 IIIA activity was decreased compared with that in controls, including other liver transplant recipients. Pretreatment with rifampicin, an inducer of P-450 IIIA, increased enzyme activity. After treatment with rifampicin the patient could be rechallenged with cyclosporin at a dose almost twice that which had previously been toxic. The patient died during a second transplantation and the microsomal content of P-450 IIIA was found to be low in the first transplant.

Prolactin, a pituitary hormone, has been shown to have an active role in the regulation of the immune system. Prolactin receptors have been described on the membrane of lymphocyte cells, and competitive binding to these receptors by cyclosporine and circulating prolactin has been demonstrated. Experimental evidence suggests a synergistic effect of cyclosporine and bromocriptine, an inhibitor of pituitary release of prolactin, on immunosuppression. Between July 1986 and January 1988, 54 patients were randomly assigned to two groups of immunosuppression treatment. Thirty patients (group 1) were administered cyclosporine, azathioprine, and prednisone and 24 patients (group 2), a modified protocol aimed at decreasing the level of circulating prolactin by adding bromocriptine to the immunosuppression regimen. The two groups were similar in regard to age, preoperative diagnosis, and duration of follow-up. Minimal side effects related to bromocriptine were observed. The overall incidences of rejection and infection were similar, although actuarial analysis showed that freedom from these complications among patients treated with bromocriptine was significantly higher throughout the first 2 months after heart transplantation compared with that of patients in the control group. Other variables such as serum cyclosporine levels and lymphocyte counts were similar in both groups. In conclusion, suppression of circulating prolactin by bromocriptine appears to improve the immunosuppressive effect of cyclosporine, at least during the early postoperative period when the risk of rejection and infection is higher, and could be a promising avenue to successful hormonal manipulations of the immune process after organ transplantation.

Cyclosporin-A (CsA) was given to 22 patients who received allogeneic bone marrow transplants as therapy for aplastic anemia and hematologic malignancies. The drug was given daily for 180 days starting with the day of marrow infusion. Engraftment was not impaired and myelotoxicity was not observed. Cutaneous graft-versus-host disease (GVHD) developed in five patients and all either spontaneously resolved or promptly responded to therapy with steroids. Five patients developed systemic GVHD and all responded to therapy with steroids, but only two survived. Interstitial pneumonia was seen in six patients and was fatal in all of them. Liver function abnormalities were seen in 14 patients but could not positively be correlated with CsA administration. Renal function abnormalities were seen in 17 patients. Amphotericin-B therapy contributed significantly to the renal failure. Serum levels of CsA, measured by radioimmunoassay, could not be correlated with the presence of liver or renal function abnormalities. Overall survival so far has been 50.0%. Second malignancies were not observed, but one patient relapsed with leukemia at 343 days.

All twelve Beagle dogs undergoing long-term therapy (26 weeks) with the immunosuppressive drug cyclosporine-A (30 mg/kg), developed cutaneous papillomatous hyperplasia. By week 7 all dogs developed generalized lesions distributed over the entire body. These occurred as irregular, oval, sessile, unpigmented, firm masses. The incidence and severity of the skin lesions varied among dogs and anatomic site, with no correlation to the blood level of cyclosporine. Microscopic analysis revealed that the epidermis formed short papillary folds on broad fibrovascular stalks and was hyperkeratotic and acanthotic. Mild hyperplasia of hair follicles and sebaceous glands was also evident. A mild diffuse infiltrate of lymphocytes and plasma cells was present in the papillary dermis. No histopathologic changes typical of papillomavirus infection were identified, nor were papillomavirus group-specific antigens or viral DNA detected. Other cutaneous side effects included hyperkeratosis of footpads, increased growth of hair and nails, and hyperkeratinization of the haired skin of the prepuce. All cutaneous lesions regressed spontaneously within 8 weeks following termination of cyclosporine administration. The hyperplastic lesions may have resulted from the action of cyclosporine via the T-lymphocyte system. Conversely a direct action of this drug on epithelial cells may have stimulated proliferation and keratinization.

Cyclosporine (cyclosporin A, CsA) is a selective T-cell immunosuppressant that works primarily through inhibition of both antigen presentation and lymphokine production. It has dramatically improved the prognosis for solid organ transplantation. Significant nephrotoxicity has been associated with its systemic use. Topical CsA 2% was used in 11 high-risk corneal transplant patients (8 men; 3 women; average age, 44 years). Ten (91%) of 11 corneas remained clear at an average follow-up of 16 months (range, 6-24 months). All patients had transient epithelial keratitis. Systemic whole blood levels of CsA measured by high-performance liquid chromatography (HPLC) ranged from 14 to 64 ng/ml. All previous reports on the use of topical CsA in high-risk corneal transplant patients have not detected systemic CsA levels.

Anti-cyclosporine antibodies were generated in rabbits, using an antigen derived from CsA. CsA was linked to carrier proteins by means of a photoactive cross-linking reagent, 4-benzoylbenzoic acid (BBa), an alpha, beta-unsaturated ketone. In the presence of CsA, photolysis of BBa results in hydrogen abstraction and random insertion into the cyclosporine molecule, generating a population of CsA with carboxyl groups at various positions. Immunization with CsA-BBa-bovine serum albumin gave rise to high affinity antibodies to CsA, with Kd = 9.8 +/- 2.8 x 10(-11) M, as determined by Scatchard analysis. Specificity was determined by competition experiments in a radioimmunoassay (RIA), using a panel of six cyclosporine derivatives, substituted at different positions. The derivatives could be arranged into three groups according to their affinities. One derivative with the lowest affinity had a bulky O-t-butyl-D-serine in place of D-alanine in position 8. Serum CsA levels in 25 transplant patients were measured by RIA, and compared to levels determined with a commercially available polyclonal antibody, which is routinely used clinically. The rabbit antiserum gave values that correlated well with the results using the commercial antibody.

A highly sensitive and semi-automated high-performance liquid chromatographic method, utilizing acetonitrile protein precipitation and column-switching, is described for the determination of cyclosporine A in whole blood. Following a rapid manual acetonitrile treatment of the blood samples, the supernatant is loaded automatically onto a 5-micron high-speed protein separation column without any further clean-up operations. The fraction containing cyclosporine A is switched to a 3-micron C18 reversed-phase high-speed column by a microprocessor-controlled column-switching unit for final separation and detection by absorption at 214 nm. Minimal sample handling and efficient separation resulted in a high recovery (75 +/- 3%) of cyclosporine A from blood and a detection limit as low as 2 micrograms/l with a highly reproducible and linear response up to 2500 micrograms/1 using 0.5 ml of sample. A separation cycle including regeneration of the first column is finished in 15 min, and this system was used continuously for ca. 1000 blood samples from heart, liver, kidney, pancreas and bone marrow recipients without change in separation parameters or material replacement. The method described allows accurate and very fast daily routine monitoring of cyclosporine A in large numbers of blood samples from transplant recipients.

Cyclosporin 5-10 mg/kg daily was given for 2-8 months to twelve recently diagnosed type 1 diabetics from a mean of 49 +/- SE 14 days after the start of insulin therapy, which was regulated to give near-normal blood glucose and haemoglobin A1c values. Mean insulin dosage dropped from 46 +/- 5 U/day before cyclosporin treatment to 16 +/- 4 U/day by the 7th month. Four patients had a complete remission and the insulin needs of four more were cut by half. The remaining four did not have remissions. Initial basal and glucagon-stimulated C peptide concentrations were higher in those who went into remission than in those who did not; they rose during cyclosporin treatment in the former but not in the latter. OKT4+ lymphocyte functions were suppressed in all patients and OKT4/OKT8 ratios declined. Anti-beta-cell autoimmunity, as indicated by lymphocyte-induced inhibition of insulin release from mouse islet cells, declined in all patients who went into remission. No consistent trend was observed for anti-islet cell antibodies. In forty-four similar, but non-randomised, recently diagnosed diabetics treated with insulin alone, the incidence of remission was 6%.

The efficacy and safety of oral cyclosporin 10 mg/kg per day in Behçet's disease were compared in a randomised double-masked study with those of colchicine, 1 mg orally per day, and were also investigated in a long-term open study. The double-masked study showed that cyclosporin was effective in treating not only the ocular manifestations of Behçet's disease but also oral aphthous ulcer, dermal lesions, and genital ulceration. Efficacy did not weaken during long-term treatment.

Therapy of chronic graft-v-host disease (GVHD) has been unsatisfactory in patients with platelet counts less than 100,000/microL. Survival at 5 years after marrow transplant is only 26% in such patients treated with trimethoprim-sulfamethoxazole (TMP-SMX) and every other day with prednisone. Since October 1982, 61 patients with high-risk extensive chronic GVHD were treated with a new alternating-day regimen of prednisone (1 mg/kg every other day) and oral cyclosporine (6 mg/kg every 12 hours every other day) with one double-strength TMP-SMX tablet twice daily. Forty patients (group I) received primary treatment of thrombocytopenic chronic GVHD (median platelet count 35 [range 7 to 87] x 10(3)/microL). Twenty-one patients (group II) received salvage treatment after failing initial prednisone +/- azathioprine. Twenty-one patients in group I and 15 in group II survive with a minimum of 2 years and a median of 3.7 years follow-up. At 4 years after transplant, actuarial survival is 51% (group I) and 67% (group II). Causes of death included interstitial pneumonia (six), relapse (five), GVHD without infection (five), infection (four), organ failure (three), and hemorrhage (two). Mortality increased with the progressive type onset of chronic GVHD and treatment failure. Toxicity included hypertension (13), nephrotoxicity (nine), nausea (seven), aseptic necrosis (five), neurologic abnormalities (four), and diabetes (three). Median cyclosporine levels at four and 36 hours were 296 and 64 ng/mL, respectively. Four patients required permanent discontinuation of cyclosporine, but none required renal dialysis. Karnofsky performance scores for 25 survivors are 90% to 100%, scores for six survivors are 70% to 89%, and scores for five survivors are less than 70%. Alternating-day cyclosporine and prednisone has acceptable toxicity and appears to improve survival in patients with high-risk chronic GVHD.

In a retrospective study of 60 renal-transplant patients immunosuppressed with cyclosporin no specific clinical features differentiated allograft dysfunction responsive to anti-rejection therapy from dysfunction responsive to reduction in cyclosporin dosage. Histologically, allograft dysfunction responsive to anti-rejection therapy was strongly associated with diffuse interstitial infiltration by mononuclear cells, oedema, and haemorrhage, vascular endothelial-cell proliferation, and infiltration of arterial walls by mononuclear cells. Arteriolar medial hypertrophy and hyalinosis were more commonly found in biopsy specimens from allografts with dysfunction responsive to reduction in cyclosporin dose than in those with dysfunction responsive to anti-rejection therapy and those with stable or improving function. Whole-blood cyclosporin concentrations were significantly lower in patients with dysfunction reversed by anti-rejection therapy than in those with dysfunction reversed by reduction in cyclosporin dose or in those with stable function. There was, however, considerable overlap between these groups, so that individual cyclosporin measurements were of little diagnostic value.

A sensitive, semi-automated high-performance liquid chromatographic method utilizing column switching is described for the determination of cyclosporine in plasma and blood. This method involves a short and improved manual protein precipitation of the sample followed by an automated clean-up of the supernatant. After automatic loading of the clean supernatant onto an LC-8 column for initial separation, the segment containing cyclosporine is loaded (automatically) onto an LC-18 column for final separation and quantitation. Cyclosporine is detected by its ultraviolet absorption at 202 nm. The rate of analysis was four samples per h running 24 h per day (ca. 100 samples per day). The method is sensitive enough to measure with confidence cyclosporine concentrations of 8 microgram/l in plasma and 20 microgram/l in blood with a linear response up to 2500 microgram/l using only 0.5 ml of sample. No internal standard is required. The method was applied continuously (24 h per day) to approximately 1000 samples without deterioration in method parameters.

In a double-blind trial 122 patients aged 15-40 years with insulin-dependent diabetes of recent onset were randomly assigned to cyclosporin 7.5 mg/kg per day or placebo. At the sixth month 25.4% of the cyclosporin group and 18.6% of the placebo group were in complete remission (not a significant difference). Treatment was continued in those patients with complete or partial remission (insulin requirement less than 0.25 U/kg per day) and 106 patients were followed to nine months, at which stage 24.1% of the original cyclosporin group and 5.8% of the original placebo group were in complete remission (p less than 0.01). For those patients whose whole-blood trough cyclosporin levels in the first three months averaged 300 ng/ml or more, the rates of complete remission at six and nine months were 37.5% and 37%. The rates of partial remission were also higher in the cyclosporin group and at six months the rate of complete or partial remission was 46% in the whole cyclosporin group and 65.6% in those with an average blood level exceeding 300 ng/ml in the first three months, versus 28.8% in the placebo group. The principal side-effect of cyclosporin was a modest and reversible increase in plasma creatinine. These results indicate that cyclosporin promotes the remission of type I diabetes and suggest the need for new controlled protocols aimed at evaluating the length of the effect and selecting the best drug regimen.

The clinical introduction of cyclosporine has resulted in increased enthusiasm for cardiac transplantation. Since July, 1983, 61 patients (50 male and 11 female) have undergone orthotopic cardiac transplantation for cardiomyopathy (48 patients), ischemic heart disease (11), or congenital heart disease (2). Mean age was 39 years (range, 1.5 to 57 years). Median hospital stay was 26 days (range, 4 to 60 days). Maintenance immunosuppression consisted primarily of prednisone and cyclosporine; it was modified in 9 patients because of a pre-existing clinical condition. The incidence of rejection was 0.44 episode/patient-month within 3 months of cardiac transplantation and 0.10 episode/patient-month subsequently. The incidence of infection was 0.05 episode/patient-month. Major side effects of cyclosporine included renal dysfunction (63%) and hypertension (61%). No recipient required dialysis for renal dysfunction. Ten patients died (rejection, 4; infection, 3; carcinoma, 1; lymphoma, 1; and pulmonary hemorrhage, 1); actuarial survival at 1 and 2 years was 84 +/- 6% and 76 +/- 8%, respectively. Patient follow-up (cumulative, 719 patient-months) revealed that 96% of recipients were rehabilitated and 50% had returned to work. With increasing understanding of cyclosporine immunosuppression, recipients can continue to look forward to an extended life with nearly complete rehabilitation.

The absorption of the immunosuppressive drug, cyclosporine, from intestinal segments in the anesthetized rabbit in situ is reported. The experimental technique allows serial blood sampling with simultaneous sampling of the intestinal perfusate. The method of Loo and Riegelman for estimating the systemically absorbed fraction of the drug was used in conjunction with the in situ method. Taking the length of the rabbit small intestine as 300 cm and using the apparent permeability of cyclosporine determined in polyethylene glycol 400 (PEG 400) at a bulk fluid flow rate of 0.27 mL/min, the anatomical reserve length for cyclosporine was calculated to be -302 cm. The studies demonstrate that a significant portion of the loss of cyclosporine from rabbit small intestine cannot be accounted for as systemically absorbed drug. Furthermore, the apparent permeability coefficient of cyclosporine in these studies is so low that the length of small intestine in the rabbit is not sufficient for complete absorption of the drug from the lumen under the conditions used.

We describe a rapid, precise, cost-effective, and accurate isocratic liquid chromatographic (LC) procedure for determining cyclosporine in whole blood. The cyclosporine is extracted from 0.5 ml of whole blood together with 200 micrograms of cyclosporin D, added per liter as internal standard, by using an Advanced Automated Sample Processing (AASP) unit. The on-line solid-phase extraction is performed on an octasilane sorbent cartridge which is interfaced with a Perkin-Elmer 83 X 4.6 mm I.D. cartridge column, packed with 3-micron octadecyl packing. The column is eluted with a mobile phase containing acetonitrile-water (13:7) at a flow-rate of 1.0 ml/min at a column temperature of 70 degrees C. The column effluent is monitored at 210 nm. The absolute recovery of cyclosporine exceeded 87% and the linearity extended up to 2000 micrograms/l. Within-run and day-to-day coefficients of variation were less than 8%. The correlation between AASP-LC and manual Bond-Elut extraction-LC method was excellent (r = 0.97).

A simple reliable liquid chromatographic method for assay of cyclosporin A in serum or urine is described. Samples were cleaned up on a solid-phase extraction system (cyanopropyl column). The system involved a reversed-phase C18 Ultrasphere column maintained at 72 degrees C and an acetonitrile linear gradient (65 to 95%) in 0.14% triethylammonium phosphate. Liquid chromatographic analysis of radioimmunoassay standards shows that some samples contain a contaminant peak. Comparison of cyclosporin A levels obtained by radioimmunoassay and high-performance liquid chromatography in clinical investigations show that the former values are generally, but not always, higher than the latter, and that cyclosporin A is very differently metabolized depending on the patient, disease and treatment.