A phase I dose-escalation study of enzalutamide in combination with the AKT inhibitor AZD5363 (capivasertib) in patients with metastatic castration-resistant prostate cancer.

BACKGROUND: Activation of the PI3K/AKT/mTOR pathway through loss of phosphatase and tensin homolog (PTEN) occurs in approximately 50% of patients with metastatic castration-resistant prostate cancer (mCRPC). Recent evidence suggests that combined inhibition of the androgen receptor (AR) and AKT may be beneficial in mCRPC with PTEN loss. PATIENTS AND METHODS: mCRPC patients who previously failed abiraterone and/or enzalutamide, received escalating doses of AZD5363 (capivasertib) starting at 320 mg twice daily (b.i.d.) given 4 days on and 3 days off, in combination with enzalutamide 160 mg daily. The co-primary endpoints were safety/tolerability and determining the maximum tolerated dose and recommended phase II dose; pharmacokinetics, antitumour activity, and exploratory biomarker analysis were also evaluated. RESULTS: Sixteen patients were enrolled, 15 received study treatment and 13 were assessable for dose-limiting toxicities (DLTs). Patients were treated at 320, 400, and 480 mg b.i.d. dose levels of capivasertib. The recommended phase II dose identified for capivasertib was 400 mg b.i.d. with 1/6 patients experiencing a DLT (maculopapular rash) at this level. The most common grade ≥3 adverse events were hyperglycemia (26.7%) and rash (20%). Concomitant administration of enzalutamide significantly decreased plasma exposure of capivasertib, though this did not appear to impact pharmacodynamics. Three patients met the criteria for response (defined as prostate-specific antigen decline ≥50%, circulating tumour cell conversion, and/or radiological response). Responses were seen in patients with PTEN loss or activating mutations in AKT, low or absent AR-V7 expression, as well as those with an increase in phosphorylated extracellular signal-regulated kinase (pERK) in post-exposure samples. CONCLUSIONS: The combination of capivasertib and enzalutamide is tolerable and has antitumour activity, with all responding patients harbouring aberrations in the PI3K/AKT/mTOR pathway. CLINICAL TRIAL NUMBER: NCT02525068.

Vistusertib (dual m-TORC1/2 inhibitor) in combination with paclitaxel in patients with high-grade serous ovarian and squamous non-small-cell lung cancer.

Background: We have previously shown that raised p-S6K levels correlate with resistance to chemotherapy in ovarian cancer. We hypothesised that inhibiting p-S6K signalling with the dual m-TORC1/2 inhibitor in patients receiving weekly paclitaxel could improve outcomes in such patients. Patients and methods: In dose escalation, weekly paclitaxel (80 mg/m2) was given 6/7 weeks in combination with two intermittent schedules of vistusertib (dosing starting on the day of paclitaxel): schedule A, vistusertib dosed bd for 3 consecutive days per week (3/7 days) and schedule B, vistusertib dosed bd for 2 consecutive days per week (2/7 days). After establishing a recommended phase II dose (RP2D), expansion cohorts in high-grade serous ovarian cancer (HGSOC) and squamous non-small-cell lung cancer (sqNSCLC) were explored in 25 and 40 patients, respectively. Results: The dose-escalation arms comprised 22 patients with advanced solid tumours. The dose-limiting toxicities were fatigue and mucositis in schedule A and rash in schedule B. On the basis of toxicity and pharmacokinetic (PK) and pharmacodynamic (PD) evaluations, the RP2D was established as 80 mg/m2 paclitaxel with 50 mg vistusertib bd 3/7 days for 6/7 weeks. In the HGSOC expansion, RECIST and GCIG CA125 response rates were 13/25 (52%) and 16/25 (64%), respectively, with median progression-free survival (mPFS) of 5.8 months (95% CI: 3.28-18.54). The RP2D was not well tolerated in the SqNSCLC expansion, but toxicities were manageable after the daily vistusertib dose was reduced to 25 mg bd for the following 23 patients. The RECIST response rate in this group was 8/23 (35%), and the mPFS was 5.8 months (95% CI: 2.76-21.25). Discussion: In this phase I trial, we report a highly active and well-tolerated combination of vistusertib, administered as an intermittent schedule with weekly paclitaxel, in patients with HGSOC and SqNSCLC. Clinical trial registration: ClinicialTrials.gov identifier: CNCT02193633.

Fit-for-purpose biomarker method validation for application in clinical trials of anticancer drugs.

Clinical development of new anticancer drugs can be compromised by a lack of qualified biomarkers. An indispensable component to successful biomarker qualification is assay validation, which is also a regulatory requirement. In order to foster flexible yet rigorous biomarker method validation, the fit-for-purpose approach has recently been developed. This minireview focuses on many of the basic issues surrounding validation of biomarker assays utilised in clinical trials. It also provides an overview on strategies to validate each of the five categories that define the majority of biomarker assays.

A phase I study of the nitroimidazole hypoxia marker SR4554 using 19F magnetic resonance spectroscopy.

BACKGROUND: SR4554 is a fluorine-containing 2-nitroimidazole, designed as a hypoxia marker detectable with 19F magnetic resonance spectroscopy (MRS). In an initial phase I study of SR4554, nausea/vomiting was found to be dose-limiting, and 1400 mg m(-2) was established as MTD. Preliminary MRS studies demonstrated some evidence of 19F retention in tumour. In this study we investigated higher doses of SR4554 and intratumoral localisation of the 19F MRS signal. METHODS: Patients had tumours > or = 3 cm in diameter and < or = 4 cm deep. Measurements were performed using 1H/19F surface coils and localised 19F MRS acquisition. SR4554 was administered at 1400 mg m(-2), with subsequent increase to 2600 mg m(-2) using prophylactic metoclopramide. Spectra were obtained immediately post infusion (MRS no. 1), at 16 h (MRS no. 2) and 20 h (MRS no. 3), based on the SR4554 half-life of 3.5 h determined from a previous study. 19Fluorine retention index (%) was defined as (MRS no. 2/MRS no. 1)*100. RESULTS: A total of 26 patients enrolled at: 1400 (n=16), 1800 (n=1), 2200 (n=1) and 2600 mg m(-2) (n=8). SR4554 was well tolerated and toxicities were all < or = grade 1; mean plasma elimination half-life was 3.7+/-0.9 h. SR4554 signal was seen on both unlocalised and localised MRS no. 1 in all patients. Localised 19F signals were detected at MRS no. 2 in 5 out of 9 patients and 4 out of 5 patients at MRS no. 3. The mean retention index in tumour was 13.6 (range 0.6-43.7) compared with 4.1 (range 0.6-7.3) for plasma samples taken at the same times (P=0.001) suggesting (19)F retention in tumour and, therefore, the presence of hypoxia. CONCLUSION: We have demonstrated the feasibility of using 19F MRS with SR4554 as a potential method of detecting hypoxia. Certain patients showed evidence of 19F retention in tumour, supporting further development of this technique for detection of tumour hypoxia.

Detection and localization of calpain 3-like protease in a neuronal cell line: possible regulation of apoptotic cell death through degradation of nuclear IkappaBalpha.

Calpains are a family of calcium-dependent cysteine proteases involved in major cellular processes including cell death. Their intracellular localization is essential to the understanding of their biological functions. In a previous confocal microscopy study, we observed the presence of a calpain 3-like protein in the mammalian brain. We thus first identified and confirmed the presence of a calpain 3-like protease in a neuronal cell model (NGF-differentiated PC12 cells). The goal of this study was to determine, for the first time in non-muscular cells, the relation between the subcellular localization, activation and function of this protease. We thus investigated its ability to regulate nuclear IkappaBalpha and therefore NF-kappaB activation after cell death stimulation. The IkappaBalpha/NF-kappaB signalling pathway indeed influences the neurodegenerative process by directly affecting gene expression in neurons. In the present study, we found that calpain 3 is present in the cytoplasm and nucleus of neuron-like PC12 cells and could be activated through autolysis in the nuclei of cells undergoing apoptosis after ionomycin treatment. Moreover, in these conditions, we demonstrated formation of the IkappaBalpha/calpain 3 complex and an increase in calpain-dependent IkappaBalpha cleavage products in cell nuclei. Stimulation of calpain-dependent cell death in neuron activated nuclear calpain 3-like protease and IkappaBalpha proteolysis resulted in the regulation of NF-kappaB activation. These data suggest a new mechanism by which calpain 3 activation is able to regulate the IkappaBalpha/NF-kappaB pathway and thus neurodegenerative processes.

Implication of calpain in neuronal apoptosis. A possible regulation of Alzheimer's disease.

Apoptotic neuronal cell death is the cardinal feature of aging and neurodegenerative diseases, but its mechanisms remain obscure. Caspases, members of the cysteine protease family, are known to be critical effectors in central nervous system cellular apoptosis. More recently, the calcium-dependent proteases, calpains, have been implicated in cellular apoptotic processes. Indeed, several members of the Bcl-2 family of cell death regulators, nuclear transcription factors (p53) and caspases themselves are processed by calpains. Progressive regional loss of neurons underlies the irreversible pathogenesis of various neurodegenerative diseases such as Alzheimer's disease in adult brain. Alzheimer's disease is characterized by extracellular plaques of amyloid-beta peptide aggregates and intracellular neurofibrillary tangles composed of hyperphosphorylated tau leading to apoptotic cell death. In this review, we summarize the arguments showing that calpains modulate processes that govern the function and metabolism of these two key proteins in the pathogenesis of Alzheimer's disease. To conclude, this article reviews our understanding of calpain-dependent apoptotic neuronal cell death and the ability of these proteases to regulate intracellular signaling pathways leading to chronic neurodegenerative disorders such as Alzheimer's disease. Further research on these calpain-dependent mechanisms which promote or prevent cell apoptosis should help us to develop new approaches for preventing and treating neurodegenerative disorders.

[Positron emission tomography: current use in internal medicine and future perspectives]. / La tomographie d'émission de positons en médecine interne: applications actuelles et perspectives d'avenir.

PURPOSE: Fluorodeoxyglucose positron emission tomography (FDG-PET) is a promising imaging technique that has already proven effective in modifying patient care in oncology. Fluorodeoxyglucose still remains the main radiopharmaceutical agent routinely used for PET imaging. A growing interest has recently lead to broaden PET research on benign disorders. The field of inflammatory or immune diseases and globally the field of internal medicine could also be impacted by FDG-PET. MAIN POINTS: Great vessels vasculitides and fever of unknown origin have both been studied by several teams and could become indications for PET. In addition, current indications now extend to paraneoplastic syndromes. It is thus possible to foresee that the clinical applications for PET will continue to expand in these patients. PERSPECTIVES AND PROJECTS: In the future, inflammatory arthritis, chronic inflammatory bowel diseases, systemic erythematous lupus, histiocytosis, or pulmonary and retroperitoneal fibrosis might benefit from PET even if, available data remains scarce to this day. Although PET will probably alter the landscape of patient management in internal medicine in the near future, additional clinical research is still needed to ascertain the exact role of PET.

Intercellular ultrafast Ca(2+) wave in vascular smooth muscle cells: numerical and experimental study.

Vascular smooth muscle cells exhibit intercellular Ca(2+) waves in response to local mechanical or KCl stimulation. Recently, a new type of intercellular Ca(2+) wave was observed in vitro in a linear arrangement of smooth muscle cells. The intercellular wave was denominated ultrafast Ca(2+) wave and it was suggested to be the result of the interplay between membrane potential and Ca(2+) dynamics which depended on influx of extracellular Ca(2+), cell membrane depolarization and its intercel- lular propagation. In the present study we measured experimentally the conduction velocity of the membrane depolarization and performed simulations of the ultrafast Ca(2+) wave along coupled smooth muscle cells. Numerical results reproduced a wide spectrum of experimental observations, including Ca(2+) wave velocity, electrotonic membrane depolarization along the network, effects of inhibitors and independence of the Ca(2+) wave speed on the intracellular stores. The numerical data also provided new physiological insights suggesting ranges of crucial model parameters that may be altered experimentally and that could significantly affect wave kinetics allowing the modulation of the wave characteristics experimentally. Numerical and experimental results supported the hypothesis that the propagation of membrane depolarization acts as an intercellular messenger mediating intercellular ultrafast Ca(2+) waves in smooth muscle cells.

Phase I and pharmacokinetic study of an oral platinum complex given daily for 5 days in patients with cancer.

PURPOSE: We aimed to determine the maximum-tolerated dose (MTD) clinical toxicities, pharmacokinetics, and pharmacodynamics of oral JM216 given once daily for 5 days to cancer patients. PATIENTS AND METHODS: Patients who fulfilled standard phase I trial criteria were enrolled. Oral JM216 was given at doses based on patient body-surface area, on an empty stomach, once daily for 5 consecutive days, as 10-, 50-, and 200-mg hard gelatin capsules and with oral antiemetics. The pharmacokinetics of platinum were studied on days 1 and 5 of the first treatment course using atomic absorption spectrophotometry (AAS). RESULTS: Thirty-two patients received 94 courses of oral JM216 at doses that ranged from 30 to 140 mg/m2 body-surface area for 5 consecutive days. The MTD was 140 mg/m2/d. The dose-limiting toxicities were thrombocytopenia and neutropenia. Hematotoxicity was reversible (nadir, 17 to 21 days; recovery, 28 days), noncumulative, and dependent on the dose and history of previous therapy. There were two instances of neutropenic sepsis. Two-thirds of patients experienced mild nausea, vomiting, or diarrhea. There was no ototoxicity, neurotoxicity, nephrotoxicity, or objective tumor responses. There was a significant correlation between JM216 dose and the day 1 and 5 plasma ultrafiltrate area under the concentration-time curve (AUC; r = .78), which indicates linear pharmacokinetics. There was considerable intersubject pharmacokinetic and pharmacodynamic variability, but a significant sigmoidal relationship between the plasma ultrafiltrate AUC and severity of thrombocytopenia (R2 = .83). CONCLUSION: We recommend JM216 doses of 100 and 120 mg/m2/d x 5 for previously treated and untreated patients, respectively, for phase II trials.

Phase I clinical and pharmacokinetic study of bcl-2 antisense oligonucleotide therapy in patients with non-Hodgkin's lymphoma.

PURPOSE: To evaluate the pharmacokinetics and toxicity of an antisense oligonucleotide targeting bcl-2 in patients with non-Hodgkin's lymphoma (NHL) and to determine efficacy using clinical and biologic end points. PATIENTS AND METHODS: Twenty-one patients with Bcl-2-positive relapsed NHL received a 14-day subcutaneous infusion of G3139, an 18-mer phosphorothioate oligonucleotide complementary to the first six codons of the bcl-2 open reading frame. Plasma pharmacokinetics were measured by anion exchange high-performance liquid chromatography. Response was assessed by computed tomography. Changes in Bcl-2 expression were measured by fluorescence-activated cell sorting of patients' tumor samples. RESULTS: Eight cohorts of patients received doses between 4. 6 and 195.8 mg/m(2)/d. No significant systemic toxicity was seen at doses up to 110.4 mg/m(2)/d. All patients displayed skin inflammation at the subcutaneous infusion site. Dose-limiting toxicities were thrombocytopenia, hypotension, fever, and asthenia. The maximum-tolerated dose was 147.2 mg/m(2)/d. Plasma levels of G3139 equivalent to the efficacious plasma concentration in in vivo models were produced with doses above 36.8 mg/m(2)/d. Plasma levels associated with dose-limiting toxicity were greater than 4 microg/mL. By standard criteria, there was one complete response, 2 minor responses, nine cases of stable disease, and nine cases of progressive disease. Bcl-2 protein was reduced in seven of 16 assessable patients. This reduction occurred in tumor cells derived from lymph nodes in two patients and from peripheral blood or bone marrow mononuclear cell populations in the remaining five patients. CONCLUSION: Bcl-2 antisense therapy is feasible and shows potential for antitumor activity in NHL. Downregulation of Bcl-2 protein suggests a specific antisense mechanism.

cis-Amminedichloro(2-methylpyridine) platinum(II) (AMD473), a novel sterically hindered platinum complex: in vivo activity, toxicology, and pharmacokinetics in mice.

A novel sterically hindered platinum complex, AMD473 [cis-amminedichloro(2-methylpyridine) platinum(II)], designed primarily to be less susceptible to inactivation by thiols, has shown in vitro activity against several ovarian carcinoma cell lines. Notably, AMD473 has shown activity in vitro in human carcinoma cells that have acquired cisplatin resistance due to reduced drug transport (41M/41McisR) or enhanced DNA repair/increased tolerance of platinum-DNA adducts (CH1/CH1cisR). In this study, we show that AMD473, at its maximum tolerated dose of 35-40 mg/kg i.p. administration, produced marked in vivo antitumor activity against a variety of murine (ADJ/PC6 plasmacytoma, L1210 leukemia) and human ovarian carcinoma xenograft models, including several possessing acquired resistance to cisplatin [ADJ/PC6cisR, L1210cisR, CH1cisR, and HX110 (carboplatin-resistant)]. In the ADJ/PC6 model, an increased therapeutic index was noted following oral as opposed to i. p. administration. In a head-to-head comparison using CH1cisR xenografts and equitoxic doses (q7dx4 schedule), comparative growth delays were as follows: AMD473, 34 days; cisplatin, 10.4 days; carboplatin, 6.4 days; and JM216 (p.o. administration), 3.5 days (in a previous experiment, the trans-platinum complex JM335 induced a growth delay of 5.4 days against this model). In this model, oral activity was also noted with a growth delay of 34 days at 400 mg/kg every 7 days (total of four doses). In addition, AMD473 showed promising activity against CH1 xenografts that had regrown following initial treatment with cisplatin (additional growth delay of 30 days over that observed for retreatment with cisplatin). Across the whole panel of cisplatin-sensitive to cisplatin-resistant human ovarian carcinoma xenografts, AMD473 showed improved or at least comparable activity to that observed for an equitoxic dose (4 mg/kg) and schedule of cisplatin. Platinum pharmacokinetics showed that following i.v. administration of 20 mg/kg AMD473 in saline to Balb/c- mice bearing murine plasmacytoma (ADJ/PC6), a biexponential decay was observed in the plasma with a rapid distribution t1/2alpha of 24 min followed by a slow elimination t1/2beta of 44 h. Platinum accumulated in various organs with platinum tissue to plasma area under the curve ratios of 8.6 for liver and kidney, 5.7 for spleen, 3.7 for heart, 5.2 for lung, and 5 for tumor. The plasma and tissue concentration time curve following i.p. administration was similar to that observed following i.v. administration, with a bioavailability of 89%. When AMD473 was given p.o., the platinum absorption was rapid (K01 of 30 min) and the bioavailability was 40%. A less than proportional increase in area under the curve and Cmax was noted in tissue, plasma, and plasma ultrafiltrate following increasing oral doses of AMD473. In vitro, with AMD473, the rate of binding to different plasma proteins was approximately half of that of cisplatin. Following administration of 45 mg/kg i.p. in oil, 33% of the administered platinum was eliminated in the urine after 24 h, and 40% was eliminated after 72 h. Fecal recovery represented 13% of the administered dose after 3 days. Similar results were observed following oral and i.v. administration of 20 mg/kg, but significantly more was excreted in the feces (over 50% of the administered dose) following oral administration of 400 mg/kg, showing that absorption might be a limiting factor by this route of administration. The dose-limiting toxicity for AMD473 in mice was myelosuppression, and no renal toxicity was observed. The promising antitumor activity of AMD473, together with its lack of nephrotoxicity and favorable pharmacokinetic profile, suggests that AMD473 is a good candidate for clinical development. AMD473 is entering Phase I clinical trials under the auspices of the United Kingdom Cancer Research Campaign in 1997.

Increased chemotactic and mitogenic response of psoriatic fibroblasts to platelet-derived growth factor.

The effect of increasing doses from 1.25 to 10 ng/ml of PDGF was tested for chemotactic and mitogenic activity on psoriatic fibroblasts cultured from involved and uninvolved skin of five patients compared to normal fibroblasts from five matched control subjects. The chemotactic response of psoriatic fibroblasts from involved skin (p less than 0.05) and uninvolved skin (p less than 0.005) is significantly enhanced compared to normal fibroblasts. Similarly, PDGF in the presence of platelet poor human plasma is a more potent mitogenic agent in psoriatic fibroblasts than in normal fibroblasts. This increased sensitivity of psoriatic fibroblasts to PDGF may be related to the inflammatory and vascularization processes involved in psoriatic dermis.

Retinoid treatment of human psoriatic fibroblasts induces an increase in cyclic AMP-dependent protein kinase activity.

We recently showed a deficiency of cyclic AMP (cAMP)-dependent protein kinases in psoriatic cells. In this work the effects of retinoids on cAMP-dependent protein kinases of fibroblasts from 7 normal subjects and 7 psoriatic patients were studied. The levels of RI and RII (two forms of the cAMP-dependent protein kinases) present in control and retinoic acid-treated cells were quantitated by photoaffinity labeling with [8-azido-32P]cAMP. In psoriatic fibroblasts the levels of RII are decreased or undetectable compared with those of normal fibroblasts both in the cytosolic and membrane fractions. The amount of RI was normal in the cytosol of fibroblasts of 5 out of 7 patients and decreased in 2 patients. Membrane-associated levels of RI were decreased in 5 patients and normal in 2 patients. Retinoic acid treatment induces an increase in the amount of RI and RII regulatory subunits when they are deficient in the cytosolic and membrane fractions of psoriatic fibroblasts. Retinoic acid had no effect on RI and RII in normal fibroblasts. In addition, with in vitro retinoic acid treatment the cAMP-dependent protein kinase activity, measured in the fibroblasts of 4 psoriatic patients, was increased in the cytosol in 2 patients and in the membranes in all 4 patients. In these studies, comparable results were obtained with fibroblasts cultured from involved and uninvolved skin. This in vitro effect of retinoids on cAMP-dependent protein kinases in psoriatic fibroblasts may help to explain some of the in vivo therapeutic effects of retinoids.

MAP kinase abnormalities in hyperproliferative cultured fibroblasts from psoriatic skin.

Several studies indicate that dermal fibroblasts have a specific role in the pathophysiology of psoriasis. We have previously found that cultured fibroblasts from psoriatic patients are hyperproliferative and have low cyclic AMP-dependent protein kinase activity. In this study, we observed that these cells are also larger than normal. Given the key role of mitogen-activated protein kinases (MAPK) in the regulation of cell proliferation and cytoskeleton function, we characterized MAPK in psoriatic fibroblasts and in normal fibroblasts. Serum and platelet-derived growth factor treatment of serum-deprived fibroblasts led to a larger increase in MAPK activity in psoriatic cells than in normal cells. We then purified MAPK by ion-exchange chromatography. MAPK activity was again found to be significantly higher in psoriatic fibroblasts than in normal cells, both when deprived of serum (p < 0.01) and when stimulated with serum (p < 0.05). Interestingly, 8-bromo-cAMP treatment inhibited serum-stimulated MAPK phosphorylation in normal fibroblasts but had no effect in psoriatic fibroblasts. We observed a temporal variation in nuclear localization of phosphorylated MAPK in cultured fibroblasts stimulated by either serum or platelet-derived growth factor. No difference in the localization of phosphorylated MAPK in normal and psoriatic skins was found. Psoriatic fibroblasts are the first example of a MAPK pathway abnormality in large human benign hyperproliferative cells.

Antioxidant enzymes in psoriatic fibroblasts and erythrocytes.

Antioxidant enzyme activities in fibroblasts and erythrocytes prepared from normal and psoriatic patients were measured and compared. The most significant differences were noted in superoxide dismutase (SOD) activities. A dramatic (5.2-fold) increase in Mn-SOD activity along with a lesser (1.8-fold) increase in CuZn-SOD activity was observed in fibroblasts from lesional and nonlesional psoriatic skin. The increase of Mn-SOD activity was correlated with an increase of both protein and mRNA. A slight (1.2-fold) increase in CuZn-SOD activity was also found in psoriatic as compared to normal red blood cells, while Mn-SOD activity was not present in these cells. In contrast, both glutathione peroxidase and catalase activities were only slightly (1.3-fold) increased in psoriatic fibroblasts, with no appreciable change noted in psoriatic erythrocytes. Likewise, glutathione levels were observed to be similar in normal and psoriatic cells. The increases in SOD activities did not appear to correlate with the severity of the disease as expressed by the Psoriatic Area Severity Index score or with plasma inflammatory markers. These results demonstrate that antioxidant enzyme activities, particularly Mn-SOD in fibroblasts and CuZn-SOD in erythrocytes, are significantly elevated in cells from psoriatic patients.

Effects of [D-Ala1] peptide T-NH2 and HIV envelope glycoprotein gp120 on cyclic AMP dependent protein kinases in normal and psoriatic human fibroblasts.

In addition to acquired immunodeficiency syndrome (AIDS), persons infected with human immunodeficiency virus often develop cutaneous manifestations, including severe psoriasis. In previous studies, we have established that psoriatic fibroblasts and erythrocytes obtained from psoriatic patients exhibit decreased levels of cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA) activity and of 8-azido-[32P]cAMP binding to the RI and RII regulatory subunits of PKA. Because treatment of patients with peptide T (an octapeptide sequence found in the human immunodeficiency virus envelope glycoprotein gp120) has been observed to result in an improvement in the psoriatic condition, studies were initiated to determine if peptide T and gp120 protein treatment of normal and psoriatic human fibroblasts resulted in any changes in PKA. Exposure of psoriatic fibroblasts to peptide T resulted in a time (4 h to 6 d) and dose [10(-14)-10(-8) M] dependent increase in the levels of 8-azido-[32P]cAMP binding to the RI and RII regulatory subunits of PKA, along with a corresponding increase in PKA activity. Peptide T exhibited a biphasic dose dependent response, with maximal effects on PKA noted at 10(-12)M peptide T. Treatment of normal human fibroblasts with peptide T did not result in any change in PKA levels. Conversely, treatment of normal human fibroblasts for 18 h with gp120 protein [10(-13) M] resulted in a significant decrease in the levels of 8-azido-[32P]cAMP binding to RI and RII and in PKA activity. The presence of peptide T blocked this effect of the gp120 protein. These results indicate that peptide T and gp120 protein may inversely alter the intracellular levels of 8-azido-[32P]cAMP binding to RI and RII, and of PKA activity in susceptible cells. These observed changes in the cyclic AMP-PKA signaling pathway, a biochemical marker for psoriasis, may offer some mechanistic insight into the noted beneficial effects of peptide T treatment, including an improvement in psoriatic lesions.

Effect of retinoic acid on platelet-derived growth factor (PDGF) bioactivity and type-B PDGF receptors in normal and psoriatic human fibroblasts.

Psoriasis is a common skin disease in which retinoids have beneficial effects. It offers a model for the study of benign hyperproliferation with abnormal differentiation. The dermis has a prominent role in the appearance of epidermal lesions. It is therefore of interest to study the factors that modulate dermal cell proliferation. In this study, the role of retinoids in modulating platelet-derived growth factor (PDGF) bioactivity was studied in normal (six subjects) and psoriatic fibroblasts from involved and uninvolved tissues (six patients). Retinoic acid treatment (for 4 d at 10(-6) M) of psoriatic fibroblasts significantly increased the chemotactic effect of PDGF in these cells (p less than 0.01 and p less than 0.05, respectively, in involved and uninvolved skin at 20 ng/ml of platelet-derived growth factor as measured in a modified Boyden Chamber Assay). In the same way, retinoic acid treatment of psoriatic fibroblasts increased the mitogenicity of platelet-derived growth factor in these cells. Retinoic acid treatment has no significant effect on the mitogenic and chemotactic activity of PDGF in normal fibroblasts. The binding of the homodimer BB PDGF to its type-B receptor, which mediates the mitogenic and chemotactic effect of PDGF, was not modified by retinoic acid treatment either in psoriatic and/or normal fibroblasts. These results suggest that retinoic acid may modulate the PDGF bioactivity in psoriatic fibroblasts not by affecting the binding of this ligand to these cells but by influencing a post-receptor event.

Rapid effect of treatment of psoriatic erythrocytes with the synthetic retinoid acitretin to increase 8-azido cyclic AMP binding to the RI regulatory subunit.

We have recently demonstrated a deficiency in the cyclic adenosine monophosphate (cAMP)-dependent protein kinases (PKA), the intracellular mediator of AMP, in psoriasis. This enzyme defect is expressed in fibroblasts and in red blood cells isolated from psoriatic patients. In these cells, the abnormality noted in cAMP binding to PKA correlates well with the severity of the disease and is corrected by long-term treatment with etretinate. In this study, we determined the effect of oral administration of acitretin in four psoriatic patients on the altered cAMP binding observed with the RI regulatory subunit of PKA in erythrocytes prepared from these patients. Acitretin (30 mg/day) induced a rapid (within 1 h) increase in the ability of the RI regulatory subunit of erythrocytes to bind the 8-azido[32P]cAMP photoaffinity analogue of cAMP. The maximal plateau for this effect of acitretin was observed within 24 h of treatment and preceded the clinical improvement of the disease. The effect of acitretin was dose-dependent, with the maximal response observed at 40 mg acitretin/d. In addition, the rapid exposure (15 min) of erythrocytes isolated from untreated patients exhibiting severe psoriasis to acitretin also promoted an increase in binding of 8-azido[32P]cAMP to the RI cAMP binding protein. Retinoic acid and 13-cis-retinoic acid were as efficient as acitretin in inducing the increase in binding of 8-azido[32P]cAMP to the RI regulatory subunit, whereas arotinoid was without effect. These results suggest that acitretin may act to modify PKA (the RI regulatory subunit) at the post-transcriptional level, and this may reflect, in part, on the mechanism of action of this synthetic retinoid. Further, monitoring this biochemical event may be helpful in determining the choice of retinoid therapy and in the management of its pharmacology.

Increased oxidative damage to fibroblasts in skin with and without lesions in psoriasis.

Differences in oxidative damage, as measured by an increase in the carbonylation of macromolecules, were determined in situ with skin biopsies from psoriatic patients and controls. High levels of carbonyl residues were consistently detected in the dermis and never in the epidermis of sections of these skin biopsy samples. The dermis of psoriatic skin without lesions had a higher level of carbonylation than the dermis of normal skin. In this study, we found that there was more oxidative damage in cultured fibroblasts prepared from skin with and without lesions from psoriasis patients than in normal fibroblasts from the skin of age-matched controls. The extent of protein carbonylation in cell extracts was determined by immunoblotting, using an antidinitrophenylhydrazone antibody, and in intact cells was determined by immunocytochemical analysis with the same antibody. The higher level of carbonylation detected was used here as a measure of oxidative stress, and showed that some oxidative damage occurred before the appearance of typical psoriatic plaques. These results suggest that fibroblasts are affected before the onset of psoriasis and that this damage is independent of any inflammatory infiltrate.