Long-Term Outcomes of Cervical Cancer Patients Treated With Definitive Chemoradiation Following a Complete Metabolic Response.

AIMS: A complete metabolic response (CMR) on early post-treatment 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) is a positive prognostic factor for cervical cancer patients treated with definitive chemoradiation, but long-term outcomes of this group of patients are unknown. Patterns of failure and risk subgroups are identified. MATERIALS AND METHODS: Patients who received curative-intent chemoradiation from 1998 to 2018 for International Federation of Gynecology and Obstetrics (FIGO) stage IB1-IVA cervical cancer and had a CMR on post-treatment FDG-PET within 5 months of treatment completion were included. Cox proportional hazards models determined factors associated with locoregional and distant failure. Kaplan-Meier estimates of freedom from any recurrence (FFR) of patient subgroups were compared with Log-rank tests. RESULTS: There were 402 patients with a CMR after chemoradiation on FDG-PET. Initial T stage was T1 (38%)/T2 (40%)/T3 (20%)/T4 (2%); initial FDG-avid nodal status was no nodes (50%)/pelvic lymph nodes (40%)/pelvic and para-aortic lymph nodes (10%). After a median follow-up of 6 years, 109 (27%) recurred. The pattern of recurrence was locoregional (27%), distant (61%) or both (12%). No factors were associated with locoregional failure. Distant recurrence was more likely in patients with T3-4 lesions (hazard ratio = 2.4, 95% confidence interval 1.5-3.8) and involvement of pelvic (hazard ratio = 1.6, 95% confidence interval 1.0-2.7) or para-aortic lymph nodes (hazard ratio = 2.7, 95% confidence interval 1.4-5.0) at diagnosis. The 5-year FFR rates for T1-2 patients with no nodes, pelvic nodes alone or para-aortic nodes at diagnosis were 85, 76 and 62%, respectively (P = 0.04, none versus para-aortic nodes). The 5-year FFR for T3-4 patients with no nodes, pelvic nodes alone or para-aortic nodes at diagnosis were 68, 56 and 25%, respectively (P = 0.09, none versus para-aortic nodes). CONCLUSIONS: T3-4 tumours and para-aortic nodal involvement at diagnosis are poor prognostic factors, even after a CMR following chemoradiation.

Treatment of pain in fibromyalgia patients with testosterone gel: Pharmacokinetics and clinical response.

To test our hypothesis that testosterone deficiency plays an important role in chronic pain, a Phase I/II pilot study was initiated with 12 fibromyalgia patients to verify that a daily dose for 28days with transdermal testosterone gel would 1) significantly and safely increase mean serum testosterone concentrations from low baseline levels to mid/high-normal levels, and 2) effectively treat the pain and fatigue symptoms of fibromyalgia. Pharmacokinetic data confirmed that serum free testosterone concentrations were raised significantly above baseline levels, by assessment of maximum hormone concentration (Cmax) and area under the curve (AUC) parameters: free testosterone Cmax was significantly raised from a mean of 2.64pg/mL to 3.91pg/mL (p<0.05), and 24hour free testosterone AUC was significantly raised from a mean of 35.0pg-hr/mL to 53.89pg-hr/mL. Assessment of the typical symptoms of fibromyalgia by patient questionnaire and tender point exam demonstrated significant change in: decreased muscle pain, stiffness, and fatigue, and increased libido during study treatment. These results are consistent with the hypothesized ability of testosterone to relieve the symptoms of fibromyalgia. Symptoms not tightly related to fibromyalgia were not improved.

Interaction of arsenic and phosphate on their uptake and accumulation in Chinese brake fern.

Interactive effects of arsenate (As (V)) and phosphate (Pi) were investigated under hydroponic culture. Arsenic concentrations in fronds and roots of Chinese brake fern (Pteris vittata L.) significantly (p < 0.05) increased with increasing As (V), but decreased (p < 0.05) with increasing Pi in nutrient solution. Phosphate uptake was significantly (p < 0.05) inhibited by 1000 micromol L(-1) As (V). Under 100 micromol L(-1) As (V), frond phosphorus (P) increased at 100 and 1000 micromol L(-1) Pi, and root P increased at 250 micromol L(-1) Pi exposures. Arsenic and P concentrations in fronds and roots of Chinese brake fern were negatively correlated (p < 0.05). Arsenate treatments enhanced As and P transport to fronds, while increasing Pi inhibited their transportation, with highest frond P and As (%) obtained under 100 micromol L(-1) treatment. pH values in nutrient solution increased with increasing exposure time, but decreased with increasing Pi levels. Dissolved organic carbon (DOC) contents (dry weight) in nutrient solution decreased with increasing Pi levels, both for treatments with and without As (V). Arsenate at 1000 micromol L(-1) significantly (p < 0.05) increased DOC contents, especially for treatment without Pi. Six organic acids were detected in root exudates of Chinese brake fern, with oxalic and malic acids being most dominant.

KAI1 metastasis suppressor gene is frequently down-regulated in cervical carcinoma.

KAI1 is a metastasis suppressor gene located on human chromosome 11p11.2. It belongs to a structurally distinct family of cell surface glycoproteins. Decreased KAI1 expression has been observed in several common solid epithelial tumors, including prostatic, pancreatic, lung, hepatic, colorectal, ovarian, and esophageal cancers. A recent study also observed frequent loss of KAI1 expression in a number of squamous cell carcinomas of the cervix by immunohistochemical technique. To further confirm whether this gene is altered in this malignancy, we analyzed KAI1 expression in various stages of cervical carcinoma by a molecular method. Total cellular RNA was extracted from 84 primary invasive cervical carcinomas and 6 metastatic or recurrent lesions. cDNA was synthesized and was used for real-time quantitative polymerase chain reaction analysis. The level of KAI1 expression was obtained as the value of threshold cycle (Ct) and was quantitated with a comparative Ct method. In addition, paraffin blocks of the tumors were selected and prepared for immunohistochemical study with an anti-KAI1 polyclonal antibody, C-16. Both the real-time quantitative polymerase chain reaction method and immunohistochemical study revealed a frequent decrease in KAI1 expression in invasive cervical cancers and metastatic or recurrent lesions. However, the reduction in KAI1 was not related to progression of the disease. When tumor cell differentiation was analyzed, poorly differentiated tumors showed a greater decrease in KAI1 expression than well or moderately differentiated tumors (P < 0.001). Histologically, KAI1 loss was observed equally in both squamous cell carcinoma and adeno-/adenosquamous carcinoma. Since down-regulation of KAI1 occurs in both early and late stages of cervical cancer, we suggest that its involvement in the progression of this malignancy is an early event.

FHIT (fragile histidine triad) gene analysis in cervical intraepithelial neoplasia.

OBJECTIVE: Recently a candidate tumor suppressor gene, FHIT (fragile histidine triad), was identified at chromosome 3p14.2. Abnormality of this gene has been observed in a variety of human tumors. Although aberrant FHIT transcripts in a substantial percentage of cervical cancer cell lines and primary cervical tumors were also noted, some other studies revealed different results. Therefore, its association with the development of cervical cancer is still debatable. Because allelic loss in chromosome 3p is also a frequent finding in cervical intraepithelial neoplasia (CIN), we compared the transcription pattern and expression of FHIT in the preinvasive cervical lesions and normal cervical epithelia to investigate its possible role in cervical carcinogenesis. METHODS: Thirty-five consecutive CIN lesions taken from conization specimens and 33 normal cervical epithelial tissues taken from hysterectomy for benign diseases were included in this study. Total RNA was extracted from the pathology-confirmed tissue samples and first-strand cDNA was synthesized. It was amplified using a nested reverse transcription polymerase chain reaction (RT-PCR) method. The PCR products were then subjected to subcloned sequence analysis. Paraffin blocks from all of the samples were selected and prepared for immunohistochemical study with an anti-FHIT polyclonal antibody. RESULTS: All the cDNAs of CIN and normal cervical epithelial tissues showed the expected size of RT-PCR product. However, 7 of the 35 (20%) CIN lesions and 5 of the 33 (15%) normal cervical epithelia also presented aberrant transcripts in addition to the normal-sized transcript of FHIT. Deletion of the cDNA segment covering exon 4 to exon 8 was the most frequent finding in the cases that showed abnormal FHIT transcripts. FHIT protein was intermediately or strongly expressed in most of the CIN lesions and normal squamous epithelia. However, reduced or absent FHIT expression was observed heterogeneously in the 7 CIN lesions and 5 normal cervices in which aberrant FHIT transcripts were detected. CONCLUSION: Because the normal-sized FHIT transcript was present robustly in all of the CIN lesions and the abnormal FHIT transcripts occurred with similar frequency and pattern in the CIN lesions and normal cervical tissues, we suggest that abnormal FHIT transcription might not be causal in the early process of cervical carcinogenesis.

p53 mutation is infrequent in clear cell carcinoma of the ovary.

OBJECTIVE: p53 gene alteration has been extensively studied in epithelial ovarian cancer. However, its occurrence in clear cell carcinoma, an infrequent histologic subtype of epithelial ovarian cancer, is rarely reported. The aim of this study is to determine the status of p53 gene alteration in this distinct type of ovarian carcinoma. METHODS: Paraffin blocks of tumors from 38 patients with primary or recurrent ovarian clear cell carcinoma were studied for p53 alteration. All these tumors were subjected to immunohistochemical and molecular analysis. Two monoclonal antibodies (DO-7 and PAb 1801) were used for immunohistochemical staining. Genomic DNAs extracted from paraffin blocks of the 38 tumors were subscribed for a nested polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP) analysis. Tumors showing band shift on SSCP were further prepared for DNA sequencing to determine the site of mutation. RESULTS: Overexpression of p53 was observed in only one stage III clear cell carcinoma. However, focal positive p53 staining was noted in another five tumors. Of the six tumors showing positive immunohistochemistry, p53 alterations were noted in four tumors. Three tumors revealed a missense point mutation: two were in exon 7 (TCT(227) --> TTT and GGC(245) --> AGC) and one was in exon 5 (CGC(156) --> CAC). Another tumor revealed a 12-bp deletion in two possible ways: it might involve the last four codons at the 3' end of exon 4 (nucleotides 12,288-12,299) or it might cross over the splice junction between exon 4 and intron 4 (nucleotides 12,290-12,301). The former would result in a predicted protein product of 389 amino acids whereas the latter would cause a frameshift in the gene sequence and would result in a truncated protein. CONCLUSION: Mutations in p53 appear to be much less frequent in clear cell carcinoma than in other histologic types of epithelial ovarian cancer. We suggest that p53 alterations may not play an important role in the development of clear cell carcinoma.

Structure and structure-function studies of lipid/plasmid DNA complexes.

Recent synchrotron-based X-ray diffraction studies have enabled us to comprehensively solve the self-assembled structures in mixtures of cationic liposomes (CLs) complexed with linear lambda-DNA. In one case the CL-DNA complexes were found to consist of a higher ordered multilamellar structure (labeled L(alpha)C with DNA sandwiched between cationic bilayer membranes. The membrane charge density is found to control the DNA interaxial spacing with high densities leading to high DNA compaction between lipid bilayers. A second self-assembled structure (labeled H(II)C) consists of linear DNA strands coated by cationic lipid monolayers and arranged on a 2D hexagonal lattice. In this paper we report on a combined X-ray diffraction and optical microscopy study of CLs complexed with functional supercoiled plasmid DNA. We describe the self-assembled structures in cell culture medium for both a high transfectant complex (DOTAP/DOPE, phiDOPE = 0.72) and a low transfectant complex (DOTAP/DOPC, (phiDOPC = 0.72). Fluorescence optica microscopy shows two distinct interactions between these two types of complexes and mouse fibroblast L-cells, demonstrating the existence of a correlation between structure and transfection efficiency.

Pharmacology and toxicology of artelinic acid: preclinical investigations on pharmacokinetics, metabolism, protein and red blood cell binding, and acute and anorectic toxicities.

The pharmacokinetics, metabolism, protein binding, red blood cell (RBC) binding, stability in vitro, and acute and anorectic toxicity of artelinic acid (ARTL) were investigated in various animal species and human blood samples. Absorption and distribution following 10 mg/kg intramuscular or oral administration in dogs and rats were very rapid with t1/2 0.12-0.54; there were also a high AUC (11,262 ng/h/mL) and Vss (9.5 L/kg), low CL (15 mL/min/kg) and long elimination time (t1/2 = 2.6 h), compared with rat data. Oral bioavailability of ARTL was 79.7% in dogs and 30.1% in rats. The conversion of ARTL to dihydroartemisinin (DART) in dogs (0.1-0.5% of total dose) after 3 routes of administration (intravenous, intramuscular and oral) was 10-fold lower than that in rats. In rats dosed with [14C]ARTL, unchanged ARTL accounted for less than 13% of the total radioactivity after all 3 administration routes, suggesting that ARTL was extensively biotransformed. The half-lives of total radioactivity (21-49 h) in urine were much longer than that of unchanged ARTL in plasma (1.4-3.7 h), indicating that some long-lasting metabolites of ARTL were formed in rats. The mass balance data showed that 77-83% of total radioactivity was recovered in urine and faeces. High binding capacity (79-95%) and low binding affinity (1.1-9.3 x 10-7 M) of ARTL were measured in rat, rabbit, dog, monkey and human plasma. The RBC/plasma ratios of [14C]ARTL were 0.35 and 0.44 for dog and human plasma, respectively. ARTL was much more stable than artesunic acid (ARTS) in rat and dog plasma, and both ARTL and ARTS were more stable in dog plasma than in rat plasma in vitro. The 50% lethal dose (LD50) of ARTL in rats was about 535 mg/kg. Multiple intramuscular dosing for 7 d of 50 mg/kg/d of ARTL caused mild anorectic toxicity compared to ARTS in rats. In contrast to 4 other artemisinin derivatives, ARTL seems to be a good antimalarial candidate as it has the highest plasma concentration, the highest binding capacities in RBC, the highest oral bioavailability, the longest elimination half-life, the lowest metabolism rate and the lowest toxicity at equivalent dose levels.

Biomimetic metabolism of artelinic acid by chemical cytochrome P-450 model systems.

PURPOSE: To study the reaction of artelinic acid with chemical model systems of cytochrome P-450 as a means of obtaining authentic samples of the putative metabolites necessary for identification of the mammalian metabolites of artelinic acid. METHODS: Artelinic acid was reacted with different organic complexes of iron(II). The reaction products were isolated and characterized by NMR and thermospray mass spectroscopy. RESULTS: Five compounds which are putative metabolites of artelinic acid were isolated from these reactions and unambiguously identified, while the identity of two other compounds await final confirmation. CONCLUSIONS: Standards of possible metabolites of artelinic acid can be produced by the reaction of the compound with ferrous complexes that may simulate cytochrome P-450 catalyzed metabolism of xenobiotics. This approach may provide a simple and versatile method for the formation of metabolites of artemisinin compounds which is more advantageous than previous approaches with fungal-based systems.

Antimalarial activity of new dihydroartemisinin derivatives. 7. 4-(p-substituted phenyl)-4(R or S)-[10(alpha or beta)-dihydroartemisininoxy]butyric acids.

To search for water soluble dihydroartemisinin derivatives with higher efficacy and longer plasma half-life than artesunic or artelinic acid, a series of new stereoisomers of 4-(p-substituted phenyl)-4(R or S)-[10(alpha or beta)-dihydroartemisininoxy]butyric acids were synthesized as new potential antimalarial agents. Two approaches were taken in the design of these new molecules in an attempt to (a) increase the lipophilicity of the molecule and (b) decrease the rate of oxidative dealkylation of the target compounds. The new compounds showed a 2-10-fold increase in in vitro antimalarial activity against D-6 and W-2 clones of Plasmodium falciparum than artemisinin or artelinic acid. R-diastereomers are, in general, more potent than the corresponding S-diastereomers. p-Chlorophenyl and p-bromophenyl derivatives showed in vivo oral antimalarial activity against P. berghei (with 3/8 cured) superior to that of artelinic acid (1/8 cured), whereas p-fluorophenyl and p-methoxyphenyl analogs demonstrated activity only comparable (1/8 cured) to that of artelinic acid at the same dosage level (64 mg/kg twice a day). The in vivo antimalarial activity of these new compounds correlates with their SD50 (50% parasitemia suppression dose). The biological results suggested that an electronic effect, besides the lipophylicity, may play a role in determining the efficacy of this class of compounds.

Antimalarial activity of new dihydroartemisinin derivatives. 6. alpha-Alkylbenzylic ethers.

A series of diastereomeric dihydroartemisinin alpha-alkylbenzylic ethers was synthesized in search for analogs with higher antimalarial efficacy and longer plasma half-life than the existing artemisinin derivatives. Artelinic acid was used as the model molecule for the design of new analogs. Two approaches were taken in an attempt to (a) increase the lipophilicity of the molecule and (b) decrease the rate of oxidative dealkylation of the target compounds. All compounds in this study showed at least equal or better in vitro antimalarial activity against Plasmodium falciparum than artelinic acid. The most active compounds of this series showed 10-, 20-, and 40-fold better inhibitory activity than artemether, artemisinin, and artelinic acid, respectively. Compounds which have a small methyl group substituted at the alpha-methylene group showed weaker activity than compounds with a larger carbethoxyalkyl substituent, indicating that the lipophilicity and the steric effect of the molecules play important roles in their antimalarial activity. This fact is further substantiated by the significantly weaker antimalarial activity of the carboxylic acids than their corresponding esters. Compounds with electron-withdrawing function (NO2) substantially increase the antimalarial activity. The S-diastereomers, in general, are severalfold more potent than the corresponding R-isomer.

Antimalarial activity of dihydroartemisinin derivatives by transdermal application.

In addition to artelinic acid, which was demonstrated previously to possess good prophylactic as well as curative antimalarial activity against Plasmodium berghei by transdermal administration, seven artemisinin derivatives in a gel formulation were assessed for their antimalarial activities in this study. Artemisinin, the parent compound of the series, showed moderate prophylactic but poor curative activity. Although methyl artelinate was more active against P. berghei than artelinic acid and sodium artelinate by subcutaneous injection, its transdermal curative and prophylactic activity was only comparable with or weaker than that of artelinic acid. Conversely, both dihydroartemisinin trimethylsilyl ether and dehydrodihydroartemisinin showed weaker antimalarial activity than artelinic acid by the subcutaneous route, yet exhibited comparable activity by transdermal administration. Artemether, a prodrug of dihydroartemisinin, is as effective as the parent dihydroartemisinin, and both compounds were the most potent agents among the compounds studied, with total prophylactic and curative doses of 30 mg/kg and 60 mg/kg, respectively. Complete absorption of dihydroartemisinin appears to occur within 5 min after application. In general, we found that the prophylactic dose is about half that of the curative dose under the protocols used in this study. This novel drug delivery system may be an easy and safe way to administer artemisinin-type antimalarials and also a good alternative dosage form for active compounds with solubility problems.

Antimalarial activity of new dihydroartemisinin derivatives. 5. Sugar analogues.

A series of dihydroartemisinin derivatives containing a sugar moiety was prepared in the search for analogues with good water solubility and high antimalarial activity. The preparation of the new compounds was achieved by treatment of dihydroartemisinin (2) with chlorotrimethylsilane in pyridine solution at -10 degrees C to give a nearly quantitative yield of 10-O-(trimethylsilyl)dihydroartemisinin (3), which was then condensed with 1-hydroxypolyacetylated sugars 5 to give dihydroartemisinin derivatives 7a-d. Deacetylation of intermediates 7 gave the desired sugar derivatives 8. The resulting derivatives, tested in vitro against Plasmodium falciparum, were found to be more effective against W-2 than D-6 clones and were not cross-resistant with existing antimalarials. Trimethylsilylated compound 3 is more effective than derivatives 7a-d, which possess activity comparable to or better than that of artemisinin itself. Deacetylated compounds 8a-d were substantially less active than 7 in both cell lines. In P. berghei-infected mice, 7a-c showed 5/5, 2/5, and 3/5 cures, respectively, at 320 mg/kg per day x 3, whereas 7d showed no activity at the same dosage. However, 7d did prolong the life span in 3/5 of the infected mice at 640 mg/kg per day x 3 dose level. Trimethylsilylated compound 3 was also the most effective among the compounds studied, with 5/5 cures at 80 mg/kg per day x 3. The deacetylated sugar derivatives 8a-d showed only slight in vivo antimalarial activity.

Transdermal artelinic acid: an effective treatment for Plasmodium berghei-infected mice.

Artelinic acid, a derivative of the naturally occurring antimalarial artemisinin, has been incorporated into a gel suitable for transdermal administration. The formulation was tested for efficacy in Plasmodium berghei-infected mice for both curative and prophylactic properties by application to their partially denuded backs, beginning on days 3 and 0, respectively, after injection of parasitized erythrocytes. In the curative experiments, rapid elimination of the parasitemia and 60-day survival of five of five mice was achieved by dermal application of gel containing 0.9 mg of artelinic acid that was administered twice a day, beginning on day 3 after infection, for three days (total dosage of 270 mg/kg). In the prophylactic trials, the establishment of parasitemia was prevented and 60-day survival was achieved in five of five mice at a dose of 0.9 mg of artelinic acid administered twice a day, beginning on the day of inoculation, for two days (total dosage of 180 mg/kg). The transdermal medium, with or without drug, caused no topical or systemic toxicity.

2-acetylpyridine thiosemicarbazones. 13. Derivatives with antifilarial activity.

Several members of a series of 2-acetylpyridine thiosemicarbazones possess in vivo and in vitro macrofilaricidal properties. The most promising of the group tested is N4-(2-aminophenyl)-2-[1-(2-pyridinyl)ethylidene]-hydrazinecarbothioam ide (4), which suppressed 100% of the macrofilariae of Brugia pahangi and 94% of those of Acanthocheilonema viteae in the jird at a dose of 25 mg/kg per day x 5. Compounds 4 and 14 were also shown to inactivate or kill Onchocerca gutturosa and Onchocerca volvulus adult worms as measured by the loss of their motility or the inhibition of the conversion by the worms of the dye MTT to formazan.

Comparison of mass spectral techniques using organic peroxides related to artemisinin.

The mass spectra of three peroxides related to artemisinin (1) are compared in nine different ionization modes. Ion trap mass spectrometry (MS/MS) spectra reveal numerous pathways for the electron impact (EI) decompositions. In the EI mode, the best spectra are obtained by using the ion trap mass spectrometer at low temperatures. Loss of oxygen is observed with the other EI spectrometers, suggesting catalytic decomposition in the ion source. Methane positive and negative chemical ionization (CI) spectra show considerable fragmentation, while isobutane CI spectra show only (M + H)+ for 1 and (M + H - H2O)+ for dihydroartemisinin (2) and (3). An unusually abundant (2M + H)+ is observed for 1 in both positive-ion plasma desorption and fast atom bombardment mass spectra.

Antimalarial activity of new water-soluble dihydroartemisinin derivatives. 3. Aromatic amine analogues.

A series of artemisinin (1) derivatives containing bromo and heterocyclic or aromatic amine functions was prepared in the search for analogues with good water solubility and high antimalarial activity. Treatment of dihydroartemisinin (2a) with boron trifluoride etherate at room temperature gave the key intermediate, 9,10-dehydrodihydroartemisinin (3), which, on reaction with bromine, gave the dibromide 4. The latter was condensed with amines in anhydrous CH2Cl2 at less than -10 degrees C to give the desired products in 25-55% yield. The new derivatives, tested in vitro against Plasmodium falciparum, were found to be more effective against W-2 than D-6 clones and were not cross-resistant with existing antimalarials. Compound 6b, 3-fluoroaniline derivative, was the most active of the series, with the IC50 less than or equal to 0.16 ng/mL, making it several fold more potent than 1. However, no significant in vivo antimalarial activity against Plasmodium berghei was observed in any of the new compounds tested.

Plasmodium berghei: the antimalarial action of artemisinin and sodium artelinate in vivo and in vitro, studied by flow cytometry.

Sodium artelinate, a new water-soluble and relatively stable derivative of artemisinin, and its parent compound were tested for their antimalarial action. Experiments were done in vitro with synchronous cultures of Plasmodium berghei. The inhibition of growth by different concentrations of sodium artelinate and artemisinin was determined using flow cytometry. In vivo testing was done by subcutaneous injection of each drug in mice infected with P. berghei. Sodium artelinate, being stable in aqueous solution, was also administered orally to infected mice by its addition to their drinking water. Comparison of the parent compound and the derivative showed that sodium artelinate was slightly less active than artemisinin both in culture and in vivo. However, after oral administration of sodium artelinate, parasites were cleared from the blood with one-half to one-tenth of the dose used in the experiments with subcutaneous injection. The number of mice which were cured by oral administration of sodium artelinate was greater than after subcutaneous injection, even with a total oral dose lower than the injected dose.