Combination interferon-alpha2a and 13-cis-retinoic acid enhances radiosensitization of human malignant glioma cells in vitro.

We investigated the individual and combined effects of cis-retinoic acid (CRA) and/or IFN-alpha (IFN) and/or radiation therapy (RT) against a human glioma cell line (American Type Culture Collection; U373MG) to evaluate the possible radiosensitization properties of these agents in vitro. Glioma cells were incubated for 24 h in 96-well plates (2 x 10(2) cells/well) in standard culture medium. Sets of U373 (n = 12) were exposed to CRA (3 x 10(6) microM), IFN (25 units/ml), CRA plus IFN, or standard culture medium. After an additional 24 h of incubation, the U373 cells were subjected to increasing radiation doses (up to 16 Gy). Glioma cells were harvested 92 h after irradiation, and cell survival curves were determined from [3H]thymidine incorporation data (over the last 24 h). The experiment was repeated for both the untreated control group and the combined CRA/IFN group. To verify the [3H]thymidine assays, a clonogenic assay was also performed. Single cell suspensions of U373 cells were plated out in six-well plates (n = 3). After chemical and RT treatment, colonies of 50 cells or more were counted, and cell survival curves were generated as fractions of nonirradiated controls. The amount of RT (in Gy) that would cause a 50% survival fraction (lethal dose 50 or LD50) was calculated from the survival curves by regression analysis. The following LD50s were obtained: [table: see text] The results showed that for both the [3H]thymidine incorporation assay and the clonogenic assay, the combination of IFN/CRA rendered U373 cells more susceptible to ionizing radiation than the untreated control or either single agent alone.     

13-cis-retinoic acid and cancer chemoprevention.

Chemoprevention is the newest strategy for controlling and managing cancer. At present, the multistep character of epithelial carcinogenesis makes this disease process the most amendable to chemopreventive interventions, which occur in the postinitiation, preinvasive phases. Chemoprevention study has focused on oral carcinogenesis because of its excellent preclinical models, well-defined premalignant phase (leukoplakia), ease of monitoring, and link through field carcinogenesis to other epithelial carcinogeneses of the upper and lower aerodigestive tract. Retinoids, the derivatives of vitamin A, are the most-studied chemopreventive agents, and 13-cis-retinoic acid is the best-studied chemopreventive retinoid. Laboratory study of the newly discovered nuclear receptors of retinoic acid is closing in on the precise mechanism of retinoid action. Only 13-cis-retinoic acid, at high doses, has established chemopreventive activity, which is in suppressing oral premalignancy and preventing second primary head-and-neck tumors. Preclinical and clinical work in the other aerodigestive sites of the lung and esophagus are at an early phase of study with no conclusive results currently available. High-dose 13-cis-retinoic acid also has achieved significant activity in preventing invasive carcinomas of the skin. High-dose 13-cis-retinoic acid, however, is not ideal for widespread chemoprevention approaches because of its toxicity. The toxicity-to-risk balance is delicate and complicated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clinical and in vitro response to 13-cis-retinoic acid in interferon-alpha resistant renal cell carcinoma

Retinoids are known to control many important biological processes, including differentiation, morphogenesis, growth and tissue homeostasis. More recently, clinical and pre-clinical results provide evidence for an antiproliferative effect of 13-cis-retinoic acid (13cRA) in interferon-alpha (IFN-alpha) treated renal cell carcinoma patients. The manner in which 13cRA augments antitumor effects and modulates biologic and clinical responses of renal cell carcinoma to IFN-alpha remains elusive. In the present study, we report induction of apoptosis and objective tumor regression in response to 13cRA in advanced renal cell carcinoma patients refractory to IFN-alpha. Among 21 patients treated there were one complete and four partial remissions (objective response rate, 24%; median response duration 8+ months). Preliminary evidence suggests that 13cRA acid may reverse IFN-alpha resistance in renal cell carcinoma.     

Modulation of the metastatic phenotype by 13-cis-retinoic acid

KLN 205 murine squamous carcinoma cells were grown in medium supplemented with the retinoid 13-cis-retinoic acid (RA) to study the relationship between RA-induced cell surface changes and alterations of the metastatic phenotype. Modulation of the cell surface glycoconjugate expression was measured by flow cytometric analysis of the RA-treated tumor cells stained with fluoresceinated lectins. RA treatment (5 X 10(-6) and 5 X 10(-7) M) altered the glycoconjugate expression of KLN 205 cells in a selective, dose-dependent fashion. Tumor cells grown in RA-supplemented medium for more than 4 days demonstrated greatly increased binding of fluoresceinated Griffonia simplicifolia I lectin, peanut lectin, wheat-germ lectin, concanavalin A, and soybean lectin (P less than .001), but the increased binding of Ulex europaeus lectin was of a much smaller magnitude (P = .02). After 15 days of growth in these noncytotoxic or cytostatic concentrations of RA, malignant KLN 205 cells had a greatly decreased proclivity to metastasize, as measured by the lung colony assay (P = .0003). The RA-induced cell surface glycoconjugate changes preceded the decrease in experimental metastatic potential. Since enzymatic (neuraminidase) alteration of the tumor cell surface to produce glycoconjugate expression similar to that seen in RA-treated cells also reduced the ability of the KLN 205 cells to form lung colonies (P = .0022), it is suggested that RA-induced alteration of the cell surface carbohydrate antigens is related to the decreased experimental metastatic potential seen in tumor cells treated with RA.     

Interferon-alpha2a and 13-cis-retinoic acid with radiation treatment for high-grade glioma

Interferon-alpha (IFN-alpha) has been safely given concurrently with radiation therapy (RT) in treating gliomas. As single agents, both IFN-alpha and cis-retinoic acid (CRA) have produced objective tumor regressions in patients with recurrent gliomas. In vitro, IFN-alpha2a and CRA enhance radiation therapy effects on glioblastoma cells more than either agent alone. This trial was conducted to determine the clinical effects of IFN-alpha2a and CRA when given concurrently with radiation therapy to patients with high-grade glioma. Newly diagnosed patients with high-grade glioma received IFN-alpha2a at a dosage of 3 to 6 million IU s.c. 4 times a day for 3 days per week and 1 mg/kg CRA by mouth 4 times a day for 5 days per week during the delivery of partial brain radiation therapy at 180 cGy x 33 fractions for 5 days per week for a total of 59.4 Gy during the 7-week period. Use of the antiepileptic phenytoin was prohibited after observing that the combination of IFN-alpha2a, CRA, and phenytoin was associated with a high rate of dermatologic toxicity not seen in a previous study with concurrent IFN-alpha2a and radiation therapy. Forty patients (26 men and 14 women) with a median age of 60 (range, 19 to 81 years) were enrolled between August 1996 and October 1998. Histopathologic diagnoses were glioblastoma multiforme or grade 4 anaplastic astrocytoma in 36 patients, and grade 3 anaplastic astrocytoma in 4 patients. Only 4 patients (10%) underwent a gross total resection of tumor prior to this therapy; 50% were asymptomatic when treatment was initiated. The planned 7-week course of concurrent therapy was completed by 75% of patients; 30% completed the 16-week course of IFN-alpha and CRA alone. At a median follow-up of 36 months, there were 37 deaths, with a median overall survival of 9.3 months and a 1-year survival rate of 42%. There was no improvement in survival compared with a similar group of 19 patients treated with concurrent IFN-alpha2a and radiation therapy in a previous trial. In the high-risk group of patients in the present study, concurrent treatment with IFN-alpha2a, CRA, and RT was feasible, but was not associated with a better outcome compared with a similar patient population treated with radiation therapy and IFN-alpha2a, or compared with radiation therapy alone in other trials.     

Effects of ALL-trans-retinoic acid and 13-cis-retinoic acid on breast-cancer cell lines: Growth inhibition and apoptosis induction

Interest has been increasingly focused on all-trans-retinoic acid (tRA) and 13-cis-retinoic acid (13cRA) in cancer chemoprevention and treatment. We have examined the in vitro effects of these 2 retinoic acids (RAs) on human breast-cancer cell lines MCF-7 and ZR-75.1 (both estrogen-receptor-positive, ER⁺) and MDA-MB-231 (estrogen-receptor-negative, ER⁻), in terms of inhibition of proliferation and induction of apoptosis. Both retinoic acids exerted an evident dose-dependent growth inhibition, although in the ER⁻ cell line the anti-proliferative effect was obtained only with the highest concentration used; the anti-proliferative activity of tRA was more evident than 13cRA on all 3 tested cell lines. tRA and 13cRA induced apoptosis in MCF-7 and MDA-MB-231 cell lines, but not in ZR-75.1. The apoptotic phenomenon was clearly time-dependent, and in our experience it was not related to the arrest in a specific phase of cell cycle. After treatment with RAs the levels of bcl-2 were reduced in MCF-7, while in ZR-75.1 and in MDA-MB-231 no treatment-related modifications were observed. An analysis of estrogen-receptor status, used as a marker of differentiation, demonstrated that after treatment with RAs the levels of estrogen receptor (ER) decreased in ZR-75.1 only. Our study indicates that the anti-proliferative effects of RAs are sustained by induction of apoptosis in MCF-7 and MDA-MB-231 cells, while in ZR-75.1 cells an induction of differentiation without apoptosis was the prevalent mechanism of growth inhibition. Our results encourage further studies on in vivo effects of these retinoids in breast cancer.Int. J. Cancer 70:619–627. © 1997 Wiley-Liss Inc.     

Interferon alpha-2a and 13-cis-retinoic acid in patients with metastatic renal cell cancer

Treatment of patients with metastatic renal cell cancer (RCC) with interferon-alpha-2a (IFN) and 13-cis-retinoic acid (CRA) was first reported to be tolerable on an outpatient basis and to yield a 30% objective response rate. We sought to confirm these preliminary results by conducting a phase II trial of therapy with IFN/CRA in patients with bidimensionally measurable RCC. Twenty-five patients were enrolled. The median age was 58 (range, 47-75 years) and the median Karnofsky performance status was 90 (range 60-100). Seventeen patients (60%) had undergone prior nephrectomy and none had received prior systemic therapy. Treatment consisted of oral CRA at 1 mg/kg/day and IFN self-administered by subcutaneous injection at 3 MU/day with weekly escalation to 6 and 9 MU/day. Treatment was well tolerated, with cheilitis, influenza-like symptoms, and fatigue the most common toxicities. Severe toxicity was reversible and consisted of grade 4 cheilitis in one patient and grade 3 malaise/fatigue in two patients. One complete and four partial responses were absented, for an objective response rate of 20% (95% confidence interval, 4-36%). We conclude that treatment with CRA/IFN for RCC is tolerable on an outpatient basis and induces objective responses in some patients. The contribution, if any, of CRA to the responses observed will be determined in ongoing randomized phase III trials.     

The role of apoptosis in 2',2'-difluoro-2'-deoxycytidine (gemcitabine)-mediated radiosensitization.

The nucleoside analogue Gemcitabine [2',2'-difluoro-2'-deoxycytidine (dFdCyd)] is active against a wide variety of solid tumors and is a potent radiation sensitizer. Because apoptosis has been shown to be an important mechanism of cell death for many cancers, we wished to investigate the role of apoptosis in dFdCyd-mediated radiosensitization. We evaluated HT29 colon cancer cells, UMSCC-6 head and neck cancer cells, and A549 lung cancer cells, which differ substantially in the ability to undergo radiation-induced apoptosis. We hypothesized that if dFdCyd produced radiosensitization by potentiating preexisting death pathways, then only the apoptotic-prone HT29 cells would show a substantial increase in apoptosis when treated with the combination of dFdCyd and radiation and that UMSCC-6 cells and A549 cells would be radiosensitized through nonapoptotic mechanisms. We found that the radiosensitization of HT29 cells (enhancement ratio, 1.81 +/- 0.16) was accompanied by an increase in apoptosis and by caspase activation and that inhibition of this activation by the caspase inhibitor Z-Asp-Glu-Val-Asp-fluoromethylketone (DEVD) significantly decreased radiosensitization (to 1.36 +/- 0.24; P < 0.05). In contrast, UMSCC-6 cells and A549 cells were modestly radiosensitized (enhancement ratio, 1.47 +/- 0.24 and 1.31 +/- 0.04, respectively) via a nonapoptotic mechanism. These findings suggest that although apoptosis can contribute significantly to dFdCyd-mediated radiosensitization, the role of apoptosis in dFdCyd-mediated radiosensitization depends on the cell line rather than representing a general property of the drug.     

Efficacy of recombinant alpha-interferon 2a and 13-cis-retinoic acid in the treatment of squamous cell carcinoma

BACKGROUND:: Recent in vitro and in vivo studies hypothesize a synergisticeffect of 13-cis-Retinoic Acid (13cRA) and recombinant     

Low activity of 13-cis-retinoic acid plus interferon alpha 2a in advanced renal cell carcinoma

Retinoids and interferon alpha have shown synergistic activity against metastatic renal cell carcinoma in previous preclinical and clinical studies. Based on these results, we conducted a phase II trial of 13-cis-retinoic acid (cRA) at 1 mg/kg/dose interferon alpha2a (IFN) at initial dose of 9 MU three times a week. Thirty-one patients were entered, all evaluable for toxicity and 30 evaluable for response. One patient achieved a partial response and 10 patients achieved stable disease. Toxicity was mild and primarily related to interferon. No toxic deaths were reported. Median survival time was 10 months. At the dose and schedule used, cRA and interferon-alpha2a showed low activity against metastatic renal cell carcinoma. Further studies with this combination are not recommended.     

The effects of 13-cis-retinoic acid and beta-carotene on cellular immunity in humans.

Deficiency of vitamin A and/or its precursors has been associated with increased cancer risk in animals and humans. Therapeutic trials of vitamin A and related compounds have demonstrated activity in several cancerous and precancerous conditions. The authors measured the effects of a retinoid, 13-cis-retinoic acid, and a carotenoid, beta-carotene, on the human immune system in vivo in conjunction with their use in ongoing clinical trials. Immune cell subpopulations were analyzed by quantifying the expression of markers using flow cytometric study. Both compounds produced significant effects on immune cell populations. 13-cis-Retinoic acid resulted in an increase in the percentage of peripheral blood lymphoid cells expressing surface markers for T-helper cells with only minimal effect on natural killer cell marker expression. In contrast, beta-carotene produced an increase in the percentage of cells expressing natural killer cell markers with smaller effect on T-helper markers. Modest increases in the percentage of cells expressing Ia antigen, transferrin, and interleukin-2 receptors were produced by both drugs. These results suggest that retinoids and carotenoids can produce major changes in immune cellular marker expression in vivo in humans at doses relevant to their potential clinical use.     

13-cis-retinoic acid and interferon alpha-2a: effective combination therapy for advanced squamous cell carcinoma of the skin.

BACKGROUND: Retinoids (vitamin A derivatives) and interferon-alpha (IFN-alpha) are potent regulators of malignant cell differentiation and proliferation, and both have immunomodulatory and antiangiogenesis activity. A large body of preclinical and clinical data supports the use of combination therapy with 13-cis-retinoic acid (13-cRA) and IFN-alpha in patients with squamous cell carcinoma of the skin. This carcinoma is an extremely common and frequently severely disfiguring cancer, for which about 10% of patients remain uncured following standard local therapy. PURPOSE: Our purpose was to test whether a 20% or greater complete response rate could be achieved using a combination of these two agents in patients with advanced squamous cell carcinoma of the skin in whom local therapy had failed or was unfeasible or who had regional and/or distant metastases. METHODS: Thirty-two patients with heavily pretreated, advanced inoperable cutaneous squamous cell carcinoma of the skin were given a combination of oral 13-cRA (1 mg/kg per day) and subcutaneous recombinant human IFN alpha-2a (3 million units per day) for at least 2 months, unless disease progressed earlier, in a phase II trial. RESULTS: Nineteen (68%) of the 28 assessable patients responded, seven (25%) of whom had complete responses. Response rates were 93% (13 of 14) in patients with advanced local disease (six complete responses), 67% (four of six) in patients with regional disease (no complete responses), and 25% (two of eight) in patients with distant metastases (one complete response). The relationship between decreased response rate and increased extent of disease was highly statistically significant (P less than .005, chi-square test). The median response duration was greater than 5 months. No life-threatening toxic effects occurred in assessable patients treated with this combination, although dose reductions were required in 18 patients. The major limiting side effect in this elderly patient population (median age, 67 years) was cumulative fatigue. CONCLUSION: These results indicate that combined systemic therapy with 13-cRA and IFN alpha-2a is highly effective in patients with advanced squamous cell carcinoma of the skin.     

Phase II clinical trial of 13-cis-retinoic acid and interferon-alpha-2a in patients with advanced esophageal carcinoma

BACKGROUND: Interferon in combination with 5-fluorouracil has been shown to be active in squamous cell carcinoma (SCC) and adenocarcinoma (AC) of the esophagus. 13-cis-retinoic acid (CRA) has chemopreventive activity in SCC of the head and neck, and, in combination with interferon, has antitumor activity in SCC of the skin and cervix. METHODS: The activity and toxicity of CRA and interferon-alpha-2a (IFN) in patients with advanced esophageal carcinoma was evaluated in a Phase II single institution trial. Patients had unresectable or metastatic AC or SCC of the esophagus. One prior chemotherapy regimen was allowed. IFN was given by daily subcutaneous injection at a dose of 3 million U and CRA was taken orally at a dose of 1 mg/kg/day in 2 divided doses. Treatment was given in cycles of 4 weeks and continued until documented disease progression. RESULTS: Of the 19 patients entered, 15 were evaluable for response and toxicity. One patient was evaluable for response only and one patient was evaluable for toxicity only. Evaluable patients were predominantly male (15 patients), and had AC (13 patients). All had AJCC Stage IV disease and 12 were pretreated. Patients completed an average of two cycles of therapy (range, one to six cycles) prior to progression of disease. National Cancer Institute Common Toxicity Criteria Grade 3/4 toxicity was notable for nausea (25%) and fatigue (31%). No major objective responses were recorded. Eleven patients with AC and 3 patients with SCC had rapid progression of disease. One patient with AC was found to have a minor response for 22 weeks and 1 patient with AC had stable disease for 45 weeks. CONCLUSIONS: This regimen had no significant activity in patients with advanced AC of the esophagus. Further evaluation of IFN plus CRA, using this dose and schedule, is not recommended. In comparison with prior trials of this therapy, a surprising amount of severe nausea and fatigue was observed in this trial.     

Phase I study and pharmacokinetics of weekly high-dose 13-cis-retinoic acid

In an attempt to increase the peak plasma levels of 13-cis-retinoic acid (13-cis-RA) and its efficacy in vivo, a Phase I study and pharmacokinetics of weekly high-dose, oral 13-cis-RA was conducted in 23 cancer patients who were refractory to conventional treatments. At 200 mg/sq m, the mean peak plasma level of 13-cis-RA was 1.5 +/- 0.1 (SE) micrograms/ml; at 400 mg/sq m, the mean peak plasma level increased to 3.8 +/- 0.7 micrograms/ml. Further increases of the 13-cis-RA dose up to 1800 mg/sq m did not lead to proportional increases in either the mean peak plasma levels or area under the curve, indicating a saturable absorption phenomenon. The terminal half-life was highly variable (range, 2.8 to 101.3 h) and was not related to the dose given. A secondary peak plasma concentration was seen in five patients, suggesting enterohepatic circulation. The toxicities such as headache, cheilitis, dry skin, and dry eyes were frequent on the weekly schedule but were not dose-limiting. One patient had an elevation of the triglycerides of 2 to 5 times the upper limit of normal; five patients had an elevation of 1.1 to 2 times normal. No objective responses were observed to treatment with 13-cis-RA. Of 20 patients receiving an adequate trial of the drug, 18 showed progression of their cancer, and two had stable disease.     

Topical delivery of 13-cis-retinoic acid by inhalation up-regulates expression of rodent lung but not liver retinoic acid receptors.

Chemopreventive retinoids may be more effective if delivered to the lung epithelium by inhalation. 13-cis-Retinoic acid (13-cis-RA) was comparable to all-trans-retinoic acid (RA) in inducing transglutaminase II (TGase II) in cultured human cells. Inhaled 13-cis-RA had a significant stimulatory activity on TGase II in rat lung (P < 0.001) but not in liver tissue (P < 0.544). Furthermore, inhaled 13-cis-RA at daily deposited doses of 1.9 mg/kg/day up-regulated the expression of lung retinoic acid receptors (RARs) alpha, beta, and gamma at day 1 (RARalpha by 3.4-fold, RARbeta by 7.2-fold, and RARgamma by 9.7-fold) and at day 17 (RARalpha by 4.2-fold, RARbeta by 10.0-fold, and RARgamma by 12.9-fold). At a lower aerosol concentration, daily deposited doses of 0.6 mg/kg/day were also effective at 28 days. Lung RARalpha was induced by 4.7-fold, RARbeta by 8.0-fold, and RARgamma by 8.1-fold. Adjustment of dose by exposure duration was also effective; thus, inhalation of an aerosol concentration of 62.2 microg/liter, for durations from 5 to 240 min daily for 14 days, induced all RARs from 30.6- to 74-fold at the shortest exposure time. None of the animals exposed to 13-cis-RA aerosols showed RAR induction in livers. By contrast, a diet containing pharmacological RA (30 microg/g of diet) failed to induce RARs in SENCAR mouse lung, although it induced liver RARs (RARalpha, 21.8-fold; RARbeta, 13.5-fold; RARgamma, 12.5-fold); it also failed to induce lung TGase II. A striking increase of RARalpha expression was evident in the nuclei of hepatocytes. Pharmacological dietary RA stimulated RARalpha, RARbeta, and RARgamma as early as day 1 by 2-, 4-, and 2.1-fold, respectively, without effect on lung RARs. Therefore, 13-cis-RA delivered to lung tissue of rats is a potent stimulant of lung but not liver RARs. Conversely, dietary RA stimulates liver but not lung RARs. These data support the concept that epithelial delivery of chemopreventive retinoids to lung tissue is a more efficacious way to attain up-regulation of TGase II and the retinoid receptors and possibly to achieve chemoprevention.     

13-cis-retinoic acid plus interferon alpha-2a: highly active systemic therapy for squamous cell carcinoma of the cervix.

BACKGROUND: Chemotherapeutic study of cervical squamous cell carcinoma has shown some positive results. Complete plus partial (overall) response rates of 15%-35% (complete response rate, less than 5%) were achieved with the use of a small number of cytotoxic single agents in patients with advanced disease. In addition, overall response rates of 60%-70% (complete response rates, 10%-20%) were achieved with cisplatin-based, multiagent regimens in patients with primary, locally advanced disease. However, the lack of clear evidence that existing chemotherapy can achieve a survival benefit, coupled with the worldwide annual deaths of hundreds of thousands of women from cervical cancer, indicates the urgent need for effective systemic therapy for this disease. PURPOSE: In view of the preclinical and clinical evidence that supports testing of the novel combination of 13-cis-retinoic acid (13-cRA) plus interferon-alpha (IFN-alpha) in cervical squamous cell carcinoma, we conducted a phase II study of this regimen in locally advanced disease. METHODS: Twenty-six patients with untreated, locally advanced squamous cell carcinoma of the cervix were treated daily for at least 2 months with oral 13-cRA (1 mg/kg) and subcutaneous recombinant human IFN alpha-2a (6 million units). In 21 patients (81%), the disease was stage II or higher. RESULTS: Thirteen patients (50%) experienced major responses (tumor regression greater than or equal to 50%) in association with resolution of symptoms; one achieved complete response, and 12 experienced partial response. Seven with partial response are improving further, four are being maintained in partial response, and one responder has relapsed during therapy. The response rate is 58% (11 of 19) in patients with stage IIB or higher disease and 66% (10 of 15) in patients with bulky disease (at least one dimension greater than or equal to 10 cm). Of the 13 non-responders, nine have stable disease and four have had disease progression during therapy. Toxicity was minimal. CONCLUSION: These preliminary results indicate that systemic 13-cRA plus IFN alpha-2a is a highly active, well-tolerated therapy for locally advanced cervical cancer.     

Pharmacokinetics and effects on plasma retinol concentrations of 13-cis-retinoic acid in melanoma patients.

The pharmacokinetics of 13-cis-retinoic acid (13cisRA) and its effects on retinol plasma levels were investigated after the first and the last doses in melanoma patients, who participated in a study run to assess tolerance over a long period of a treatment schedule of 13cisRA associated with recombinant interferon alpha2a (rIFN-alpha2a). Melanoma patients with regional node metastases after radical surgery were randomized to be treated for 3 months with rIFN-alpha2a, 3 x 10(6) IU s.c. every other day, associated with oral 13cisRA at doses of 20 mg day(-1) (five patients) or 40 mg every other day (seven patients). Maximum 13cisRA blood concentrations usually occurred 4 h after drug administration, with average values of 406 and 633 ng ml(-1) (i.e. 1.3 and 2.1 microM) after the 20 and 40 mg dose respectively. The average half-life (t(1/2)) was approximately 30 h. The maximum concentration, the t(1/2) and the area under the concentration-time curves from 0 to 48 h (AUC(0-48)) of 13cisRA did not change after multiple dosing, whereas the AUC(0-48) of its major blood metabolite, 4-oxo-13-cis-retinoic acid, increased. Immediately after 13cisRA treatment, retinol plasma levels started to decline and they reached the lowest values (approximately 20% reduction) shortly after the time of maximum 13cisRA concentrations (i.e. 4-12 h after drug intake). Afterwards, values returned to baseline. The amount of retinol reduction in time was correlated with 13cisRA maximum concentrations.