First-in-human pharmacokinetics of tamoxifen and its metabolites in the milk of a lactating mother: a case study.

BackgroundBreast cancer represents the most frequent neoplasm diagnosed in women of childbearing age. When the tumour is oestrogen receptor-positive, tamoxifen is among the recommended endocrine treatments. Lactating women are advised not to breastfeed while receiving tamoxifen. However, information about tamoxifen transfer into breast milk is lacking.MethodsWe measured the concentration of tamoxifen and its metabolites by liquid chromatography-tandem mass spectrometry in the milk of a nursing mother that was treated for pregnancy-associated breast cancer diagnosed a few months after delivery. She was advised not to breastfeed her child and she collected milk samples for 23 days while the baby was fed with formula.ResultsTamoxifen concentrations in milk increased reaching a maximum of 214 nM. The two active metabolitesZ-4-hydroxy-tamoxifen and Z-endoxifen, could not be quantified in milk the first days after tamoxifen intake, but increased over time and reached clinically significant levels after day 18.ConclusionThis study demonstrates for the first time in human that tamoxifen and its metabolites transfer into milk. Since tamoxifen has a complete oral bioavailability, a long half-life (>7 days) and may interfere with the normal development of the infant, mothers should not breastfeed during tamoxifen treatment.

Drug monitoring of tamoxifen metabolites predicts vaginal dryness and verifies a low discontinuation rate from the Norwegian Prescription Database.

PURPOSE: Tamoxifen is an important targeted endocrine therapy in breast cancer. However, side effects and early discontinuation of tamoxifen remains a barrier for obtaining the improved outcome benefits of long-term tamoxifen treatment. Biomarkers predictive of tamoxifen side effects remain unidentified. The objective of this prospective population-based study was to investigate the value of tamoxifen metabolite concentrations as biomarkers for side effects. A second objective was to assess the validity of discontinuation rates obtained through pharmacy records with the use of tamoxifen drug monitoring. METHODS: Longitudinal serum samples, patient-reported outcome measures and pharmacy records from 220 breast cancer patients were obtained over a 6-year period. Serum concentrations of tamoxifen metabolites were measured by LC-MS/MS. Associations between metabolite concentrations and side effects were analyzed by logistic regression and cross table analyses. To determine the validity of pharmacy records we compared longitudinal tamoxifen concentrations to discontinuation rates obtained through the Norwegian Prescription database (NorPD). Multivariable Cox regression models were performed to identify predictors of discontinuation. RESULTS: At the 2nd year of follow-up, a significant association between vaginal dryness and high concentrations of tamoxifen, Z-4'-OHtam and tam-NoX was identified. NorPD showed a tamoxifen-discontinuation rate of 17.9% at 5 years and drug monitoring demonstrated similar rates. Nausea, vaginal dryness and chemotherapy-naive status were significant risk factors for tamoxifen discontinuation. CONCLUSIONS: This real-world data study suggests that measurements of tamoxifen metabolite concentrations may be predictive of vaginal dryness in breast cancer patients and verifies NorPD as a reliable source of adherence data.

cAMP response element-binding protein interacts with and stimulates the proteasomal degradation of the nuclear receptor coactivator GRIP1.

The glucocorticoid receptor interacting protein (GRIP1) belongs to the p160 steroid receptor coactivator family that plays essential roles in nuclear receptor-dependent transcriptional regulation. Previously, we reported that the cAMP-dependent protein kinase (PKA) induces ubiquitination leading to degradation of GRIP1. Here we show that the cAMP response element-binding protein (CREB) downregulates GRIP1 and is necessary for the PKA-stimulated degradation of GRIP1, which leads to changes in the expression of a subset of genes regulated by estrogen receptor-α in MCF-7 breast cancer cells. Our data of domain-mapping and ubiquitination analyses suggest that CREB promotes the proteasomal breakdown of ubiquitinated GRIP1 through 2 functionally independent protein domains containing amino acids 347 to 758 and 1121 to 1462. We provide evidence that CREB interacts directly with GRIP1 and that CREB Ser-133 phosphorylation or transcriptional activity is not required for GRIP1 interaction and degradation. The basic leucine zipper domain (bZIP) of CREB is important for the interaction with GRIP1, and deletion of this domain led to an inability to downregulate GRIP1. We propose that CREB mediates the PKA-stimulated degradation of GRIP1 through protein-protein interaction and stimulation of proteasomal degradation of ubiquitinated GRIP1.

cAMP-dependent protein kinase regulates ubiquitin-proteasome-mediated degradation and subcellular localization of the nuclear receptor coactivator GRIP1.

Nuclear receptors and their coactivators are key regulators of numerous physiological functions. GRIP1 (glucocorticoid receptor-interacting protein) is a member of the steroid receptor coactivator family. Here, we show that GRIP1 is regulated by cAMP-dependent protein kinase (PKA) that induces its degradation through the ubiquitin-proteasome pathway. GRIP1 was down-regulated in transiently transfected COS-1 cells after treatment with 8-para-chlorophenylthio-cAMP or forskolin and 3-isobutyl-1-methylxanthine and in adrenocortical Y1 cells after incubation with adrenocorticotropic hormone. Pulse-chase experiments with transiently transfected COS-1 cells demonstrated that the half-life of GRIP1 was markedly reduced in cells overexpressing the PKA catalytic subunit, suggesting that activation of PKA increases the turnover of GRIP1 protein. The proteasome inhibitors MG132 and lactacystin abolished the PKA-mediated degradation of GRIP1. Using ts20 cells, a temperature-sensitive cell line that contains a thermolabile ubiquitin-activating E1 enzyme, it was confirmed that PKA-mediated degradation of GRIP1 is dependent upon the ubiquitin-proteasome pathway. Coimmunoprecipitation studies of COS-1 cells transfected with expression vectors encoding GRIP1 and ubiquitin using anti-GRIP1 and anti-ubiquitin antibodies showed that the ubiquitination of GRIP1 was increased by overexpression of PKA. Finally, we show that PKA regulates the intracellular distribution pattern of green fluorescent protein-GRIP1 and stimulates recruitment of GRIP1 to subnuclear foci that are colocalized with the proteasome. Taken together, these data demonstrate that GRIP1 is ubiquitinated and degraded through activation of the PKA pathway. This may represent a novel regulatory mechanism whereby hormones down-regulate a nuclear receptor coactivator.

Characterization of receptor-interacting protein RIP140 in the regulation of SF-1 responsive target genes.

Receptor-interacting protein (RIP) 140 interacts with several nuclear receptors, but its function in regulation of nuclear receptor action has been debated. Here we have examined the role of RIP140 in regulation of Steroidogenic factor-1 (SF-1)-dependent transcription. SF-1 interacts with RIP140 through its activation function-2 (AF-2) domain. Several domains of RIP140 interact directly with SF-1, but the carboxyl-terminal region containing 4 of its 9 LXXLL motifs showed the strongest SF-1 interaction. Coexpression of RIP140 and SF-1 in different cell types demonstrated that RIP140 acts as a potent corepressor of transcription from the SF-1 responsive cAMP regulatory sequence 2 (CRS2) element of the CYP17 gene and a variety of SF-1 responsive promoter genes. RIP140 also counteracted the stimulatory action of p160/SRC coactivators. The inhibitory effect of RIP140 was partially reversed by Trichostatin A, suggesting a role of histone deacetylase (HDAC) activity in RIP140-mediated repression of SF-1. Quantitation of endogenous coregulator mRNA levels revealed cell type specific differences that could affect the repressor action by overexpressed RIP140.

[Thyrotoxic periodic paralysis--an unusual complication of hyperthyroidism]. / Tyreotoksisk periodisk paralyse--en uvanlig komplikasjon ved hypertyreose.

BACKGROUND: Thyrotoxic periodic paralysis (TPP) is a complication of hyperthyroidism. MATERIALS AND METHODS: We describe two patients with TPP. RESULTS: A 26-year-old man from Vietnam had weight loss, tachycardia, palpitations and heat intolerance for five months. Episodic leg and arm weakness developed three months after debut of symptoms. The second patient, a 23-year old woman from the Philippines, had had episodic leg weakness in the evenings after dinner for three weeks. Her attacks resolved spontaneously overnight. Physical examination of both patients revealed tachycardia and symmetrical proximal weakness involving both arms and legs. ECG and electrolyte analysis indicated a severe hypokalaemia; thyroid function tests showed hyperthyroidism. Both patients were diagnosed as having Graves' thyrotoxicosis and TPP. They were initially treated with propranolol and subsequently with carbimazole. The first patient had recurrence of thyrotoxicosis and paralysis after 16 months, whereas the second patient has remained symptom-free. INTERPRETATION: TPP is most common in Asian males, very few cases are reported in females. In Western countries TPP is rare, but with increasing immigration, TPP is likely to occur more frequently.