Mutagen sensitivity and risk of second cancer in younger adults with head and neck squamous cell cancer: 15-year results.

PURPOSE: To evaluate the mutagen sensitivity phenotype on the risk of second primary cancer (SPC) in patients with head and neck squamous cell carcinoma (HNSCC), and to estimate the long-term rate of SPC and the outcome with SPC. METHODS: A survey was made regarding SPC among 124 younger (≤ 50 years) adults with HNSCC who were enrolled in a pretreatment mutagen sensitivity investigation during 1996-2006. Mutagen sensitivity was assessed by exposing lymphocytes to bleomycin in vitro and quantifying the bleomycin-induced chromatid breaks per cell (b/c). Patients were classified as hypersensitive (> 1 b/c) or not hypersensitive (≤ 1 b/c). RESULTS: Mean follow-up time for all patients was 68 months (range: 5-288 months), and the 15-year cancer-specific survival was 15%. Twenty patients (16%) developed a SPC (15-year estimated rate: 41%), and half of them was hypersensitive. The crude rate of SPC for hypersensitive (n = 65) or not hypersensitive (n = 59) patients were 15 and 17%, respectively (p = 0.4272). The 15-year estimated rate of SPC for hypersensitive and not hypersensitive patients was 36 and 48%, respectively (p = 0.3743). Gender, UICC stages, anatomical sites of index cancer did not prove to be a significant risk factor for SPC. Forty-five percent of SPC developed after the 10-year follow-up. The 3­year cancer-specific survival was 23% with SPC. CONCLUSION: According to our findings, mutagen hypersensitivity was not associated with an increased SPC risk in HNSCC patients. Patients are at a lifelong risk of developing a SPC. Survival with SPC is very poor.

Altered Immunogenicity of Donor Lungs via Removal of Passenger Leukocytes Using Ex Vivo Lung Perfusion.

Passenger leukocyte transfer from the donor lung to the recipient is intrinsically involved in acute rejection. Direct presentation of alloantigen expressed on donor leukocytes is recognized by recipient T cells, promoting acute cellular rejection. We utilized ex vivo lung perfusion (EVLP) to study passenger leukocyte migration from donor lungs into the recipient and to evaluate the effects of donor leukocyte depletion prior to transplantation. For this purpose, female pigs received male left lungs either following 3 h of EVLP or retrieved using standard protocols. Recipients were monitored for 24 h and sequential samples were collected. EVLP-reduced donor leukocyte transfer into the recipient and migration to recipient lymph nodes was markedly reduced. Recipient T cell infiltration of the donor lung was significantly diminished via EVLP. Donor leukocyte removal during EVLP reduces direct allorecognition and T cell priming, diminishing recipient T cell infiltration, the hallmark of acute rejection.

Modern Tools for Modern Brachytherapy.

This review aims to showcase the brachytherapy tools and technologies that have emerged during the last 10 years. Soft-tissue contrast using magnetic resonance and ultrasound imaging has seen enormous growth in use to plan all forms of brachytherapy. The era of image-guided brachytherapy has encouraged the development of advanced applicators and given rise to the growth of individualised 3D printing to achieve reproducible and predictable implants. These advances increase the quality of implants to better direct radiation to target volumes while sparing normal tissue. Applicator reconstruction has moved beyond manual digitising, to drag and drop of three-dimensional applicator models with embedded pre-defined source pathways, ready for auto-recognition and automation. The simplified TG-43 dose calculation formalism directly linked to reference air kerma rate of high-energy sources in the medium water remains clinically robust. Model-based dose calculation algorithms accounting for tissue heterogeneity and applicator material will advance the field of brachytherapy dosimetry to become more clinically accurate. Improved dose-optimising toolkits contribute to the real-time and adaptive planning portfolio that harmonises and expedites the entire image-guided brachytherapy process. Traditional planning strategies remain relevant to validate emerging technologies and should continue to be incorporated in practice, particularly for cervical cancer. Overall, technological developments need commissioning and validation to make the best use of the advanced features by understanding their strengths and limitations. Brachytherapy has become high-tech and modern by respecting tradition and remaining accessible to all.

Evaluation of the thyroid function of healthy pregnant women by five different hormone assays.

A normal function of the thyroid gland during pregnancy is essential. Any change can affect both the pregnant woman and the fetus. Thyroid hormones play a crucial role in the brain development of the fetus, thus proper maternal free thyroid hormone levels are important especially during the first trimester. We compared the free thyroid hormone levels FT3 and FT4 in forty pregnant women with no thyroidal disease by five different assays available on the market. The blood samples were collected between the 8th and 22nd weeks of pregnancy. The correlation coefficient "r" between different assays was 0.908-0.975 for TSH, 0.676-0.892 for FT4 and 0.480-0.789 for FT3. These data show that the inter-assay results varied widely in the studied population. One reasonable explanation may be that during pregnancy the serum levels of the thyroid hormone binding proteins are altered and "free" hormone measurements by immunoassays are influenced by these alterations. Thus, the results may show higher or lower thyroid hormone values depending upon the assay used. Therefore, it is strongly suggested that every laboratory should establish its own pregnant reference ranges for the tests used for the evaluation of thyroid function, based on values of the population served.

Enhancing analytical accuracy of intravascular electrochemical oxygen sensors via nitric oxide release using S-nitroso-N-acetyl-penicillamine (SNAP) impregnated catheter tubing.

Implantable medical devices are an integral part of primary/critical care. However, these devices carry a high risk for blood clots, caused by platelet aggregation on a foreign body surface. This study focuses on the development of a simplified approach to create nitric oxide (NO) releasing intravascular electrochemical oxygen (O2) sensors with increased biocompatibility and analytical accuracy. The implantable sensors are prepared by embedding S-nitroso-N-acetylpenacillamine (SNAP) as the NO donor molecule in the walls of the catheter type sensors. The SNAP-impregnated catheters were prepared by swelling silicone rubber tubing in a tetrahydrofuran solution containing SNAP. Control and SNAP-impregnated catheters were used to fabricate the Clark-style amperometric PO2 sensors. The SNAP-impregnated sensors release NO under physiological conditions for 18 d as measured by chemiluminescence. The analytical response of the SNAP-impregnated sensors was evaluated in vitro and in vivo. Rabbit and swine models (with sensors placed in both veins and arteries) were used to evaluate the effects on thrombus formation and analytical in vivo PO2 sensing performance. The SNAP-impregnated PO2 sensors were found to more accurately measure PO2 levels in blood continuously (over 7 and 20 h animal experiments) with significantly reduced thrombus formation (as compared to controls) on their surfaces.

Prognosis of patients with local recurrence after mastectomy or conservative surgery for early-stage invasive breast cancer.

Between 1983 and 1987, 1309 women with stage I or II breast cancer underwent mastectomy (n=894) or conservative surgery (CS, n=415). Of these patients, 124 developed an isolated local recurrence (ILR): chest wall, 56 and in-breast, 68. The 10-year actuarial rate of cause-specific survival after treatment for ILR was 52%. On multivariate analysis three independent prognostic factors for the risk of death after ILR were identified: operability of recurrence (operable vs. inoperable, relative risk [RR]: 5.9), age at initial diagnosis (>40 vs. < or = 40 years, RR: 2.2) and time to ILR (>24 vs. < or = 24 months, RR: 2). Initial lymph node stage (negative vs. positive) showed borderline significance (p=0.06), and type of initial surgery (CS vs. mastectomy) and recurrent tumor grade (1-2 vs. 3) were not independent predictors of survival. In the mastectomy group, single surgical scar recurrence with initial node negative stage predicted good prognosis, and the 10-year survival was 85%. In the CS group, the 10-year survival rate was 88% with new primary tumor and 54% with true recurrence (p=0.01), and the type of salvage surgery (mastectomy vs. repeat complete excision) had no significant impact on survival (p=0.2). The majority (n=44) of CS patients developed < or = 2 cm in-breast recurrence, and the 10-year survival was 81% after both salvage excision (n=28) and mastectomy (n=16). The identified unfavorable prognostic factors are pointers of the forthcoming systemic progression. Patients with < or = 2 cm in-breast recurrence might receive a second CS.

Dosimetric comparison between treatment plans of patients treated with low-dose-rate vs. high-dose-rate interstitial prostate brachytherapy as monotherapy: Initial findings of a randomized clinical trial.

PURPOSE: The aim of this study was to compare the dosimetry of intraoperative dose plans of prostate cancer patients treated with low-dose-rate (LDR) and high-dose-rate (HDR) interstitial brachytherapy (BT). METHODS AND MATERIALS: A randomized clinical trial was initiated at our institution to compare the results and side effects of LDR and HDR BT as monotherapy in the treatment of early, organ-confined prostate cancer patients. Eighty-seven patients were randomly assigned to receive HDR afterloading BT with one fraction of 19 Gy or permanent LDR 125I seed BT with 145 Gy. Inverse optimization algorithms were used for planning. Stranded seeds were implanted using live ultrasound imaging after preimplant treatment planning. Final dosimetry of HDR treatments was based on updated needle and contour positions. Statistical comparisons with nonparametric test were performed between the corresponding dose-volume parameters. RESULTS: The V100 and V150 were 99% and 61%, respectively, for LDR, whereas 98% and 32% for HDR treatments. The D90 was less for HDR (122% vs. 110%). The dose distributions were more homogeneous and conformal with HDR technique (dose homogeneity index, 0.39 vs. 0.67; conformal index, 0.65 vs. 0.80). The urethra and rectum received significantly less dose with HDR. The D10 and D30 for urethra were 133% and 128%, respectively, for LDR and 114% and 111% for HDR treatments. The [Formula: see text] for rectum was 68% and 55% for LDR and HDR technique, respectively. CONCLUSIONS: Both techniques provided acceptable target volume coverage with a slightly higher value with the LDR technique. The dose distributions were more homogeneous and conformal, and both urethra and rectum were better protected with the HDR technique.

Early European experience with the MammoSite radiation therapy system for partial breast brachytherapy following breast conservation operation in low-risk breast cancer.

Preliminary results of ultrasound studies do exist in the literature on the successful use of the MammoSite Radiation Therapy System (RTS), a new device for delivering brachytherapy following breast-conserving surgery. In Europe, some groups started a prospective multicentre trial to investigate the use of the MammoSite RTS. In this early publication, we analysed the surgical procedure and placement of the MammoSite, treatment planning and radiation delivery complications, and early cosmesis, as well as the comfort of the patients. Between June 2002 and March 2005, a total of 54 low-risk breast cancer patients fulfilling the enrolment criteria were implanted intra- or postoperatively using the MammoSite applicator. After inflating the balloon in the excision cavity, the reference isodose was defined 1cm from the balloon's surface. Twenty-eight patients were treated with primary brachytherapy with a total dose of 34 Gy (2x3.4 Gy) and 16 patients had a boost with a mean dose of 13.3 Gy (range: 7.5-15 Gy; 2x2.5 Gy) combined with external beam radiotherapy (EBRT). Doses ranged between 46 and 50 Gy. We analysed the postimplant anatomic position of the applicator in relation to the skin and chest wall as well as the geometric form of the balloon via ultrasound, computed tomography and X-ray before, during and after the treatment. Forty-four out of 54 patients (81.5%) were eligible for MammoSite RTS brachytherapy. Ten patients were excluded from the trial due to the strict study criteria and received no brachytherapy. Balloon rupture occurred in two cases. We observed seroma in 16 patients (36%); furthermore, an abscess developed in two patients (4.5%) within 3 months of implantation. Postoperative air gaps and haematoma were successfully reduced by draining the operation cavity in one institution. At a mean follow-up of 14 months (range 3-31 months), the skin-related side effects observed were skin discoloration or inflammation in 36 patients (82%) and teleangiectasia in eight patients (18%). The MammoSite RTS is a feasible treatment modality for postoperative partial breast irradiation after breast-conserving surgery for selected low-risk breast cancer patients. The main advantage of the system is the necessity of only one applicator for the delivery of fractionated radiotherapy over a 5-day treatment period. In addition, patient tolerance of the procedure is high. Based on this early experience, the method may serve as a successful alternative to conventional multicatheter brachytherapy for a highly select group of patients, but we have to bear in mind the higher level of acute toxicity.

Dosimetric and radiobiological comparison of TG-43 and Monte Carlo calculations in 192Ir breast brachytherapy applications.

PURPOSE: To investigate the clinical significance of introducing model based dose calculation algorithms (MBDCAs) as an alternative to TG-43 in 192Ir interstitial breast brachytherapy. MATERIALS AND METHODS: A 57 patient cohort was used in a retrospective comparison between TG-43 based dosimetry data exported from a treatment planning system and Monte Carlo (MC) dosimetry performed using MCNP v. 6.1 with plan and anatomy information in DICOM-RT format. Comparison was performed for the target, ipsilateral lung, heart, skin, breast and ribs, using dose distributions, dose-volume histograms (DVH) and plan quality indices clinically used for plan evaluation, as well as radiobiological parameters. RESULTS: TG-43 overestimation of target DVH parameters is statistically significant but small (less than 2% for the target coverage indices and 4% for homogeneity indices, on average). Significant dose differences (>5%) were observed close to the skin and at relatively large distances from the implant leading to a TG-43 dose overestimation for the organs at risk. These differences correspond to low dose regions (<50% of the prescribed dose), being less than 2% of the prescribed dose. Detected dosimetric differences did not induce clinically significant differences in calculated tumor control probabilities (mean absolute difference <0.2%) and normal tissue complication probabilities. CONCLUSION: While TG-43 shows a statistically significant overestimation of most indices used for plan evaluation, differences are small and therefore not clinically significant. Improved MBDCA dosimetry could be important for re-irradiation, technique inter-comparison and/or the assessment of secondary cancer induction risk, where accurate dosimetry in the whole patient anatomy is of the essence.

The characteristics of human papillomavirus DNA in head and neck cancers and papillomas.

AIM: To determine the prevalence, type, physical state, and viral load of human papillomavirus (HPV) DNA in cases of head and neck cancer and recurrent respiratory papillomatosis (RRP). METHODS: The prevalence and type of HPV DNA was determined in 27 fresh frozen tissue specimens from patients with head and neck cancers and 16 specimens from 10 patients with RRP by MY09/MY11 and GP5+/GP6+ nested polymerase chain reaction (PCR) and subsequent restriction enzyme cleavage. The physical state of HPV DNA was analysed by E1, E2, and E1E2 specific PCRs and Southern blot hybridisation (SBH). RESULTS: HPV DNA was detected in 13 of 27 cancers and 10 of 10 papillomas. Both low risk HPV-6 and HPV-11 and high risk HPV-16 were present in cancers in low copy numbers, whereas papillomas exclusively harboured low risk HPV-6 and HPV-11. E1E2 PCRs failed to determine the physical state of HPV in cancers except one case where HPV-6 DNA was integrated. In contrast to cancers, all papillomas showed the episomal state of HPV DNA and a relatively higher viral load. CONCLUSIONS: Based on the prevalence, type, physical state, and copy number of HPV DNA, cancers and papillomas tend to show a different HPV DNA profile. The 100% positivity rate of low risk HPV types confirms the role of HPV-6 and HPV-11 in the aetiology of RRP.

High co-prevalence of genogroup 1 TT virus and human papillomavirus is associated with poor clinical outcome of laryngeal carcinoma.

BACKGROUND: The aetiology and factors leading to the progression of laryngeal cancer are still unclear. Although human papillomavirus (HPV) has been suggested to play a role, reports concerning the effect of HPV infection on tumour development are controversial. Recently, transfusion transmitted virus (TTV) was suggested to play a role in certain infections as a causative or coinfecting agent. AIMS: To investigate whether the development and progression of laryngeal squamous cell carcinoma is associated with coinfection with TTV and HPV. METHODS: The prevalence of TTV and HPV was investigated using the polymerase chain reaction in tissue samples from 40 healthy individuals, 10 patients with recurrent papillomatosis, five patients with papillomatosis with malignant transformation, and 25 patients with laryngeal carcinoma. The obtained prevalence data were compared and analysed statistically. RESULTS: In the 11 patients with carcinoma who had metastasis or relapse there was a high rate of coinfection with genogroup 1 TTV and HPV (eight of 11), whereas in the 14 without tumour progression no coinfection was found. Coinfection was associated with significantly lower tumour free survival in patients with carcinoma (p < 0.001). Furthermore, four of five patients who had papillomatosis with malignant transformation were coinfected with genogroup 1 TTV and HPV. CONCLUSIONS: Although the nature of cooperation between HPV and TTV needs to be investigated further, coinfection with genogroup 1 TTV and HPV appears to be associated with poor clinical outcome in laryngeal cancer.

A retrospective dosimetric comparison of TG43 and a commercially available MBDCA for an APBI brachytherapy patient cohort.

PURPOSE: To compare dosimetry using a contemporary model based dose calculation algorithm (MBDCA) following TG186 recommendations, and the conventional TG43 method in an (192)Ir high dose rate (HDR) accelerated partial breast irradiation (APBI) patient cohort. METHODS: Data of 38 APBI patients were studied. Dosimetry for the treatment plans was performed using both the TG43 and TG186 dose calculation methods of the Oncentra Brachy v4.4 treatment planning system (TPS). Analysis included indices of clinical interest for the planning target volume (PTV coverage, dose homogeneity, conformity) as well as dose volume histograms (DVH) for the breast, lung, heart, rib and skin. Significance testing of observed differences between TG43 and TG186 results was carried out and the effect of target location to these differences was studied. RESULTS: Statistically significant differences were observed in the values of clinically relevant DVH parameters for the PTV and the organs at risk (OAR), except for the heart. Differences for the PTV are relatively small (<1% for coverage, on the order of 2% for homogeneity and conformity) with a slight TG43 overestimation except for the dose homogeneity. Percentage differences are larger for the rib and lung (on the order of 4% for Dmax and 5% for V10Gy, respectively) and maximum for the skin (on the order of 6% for D10cc), with a correlation of the observed differences with target location. CONCLUSION: While the MBDCA option of the TPS appears to improve dosimetric accuracy, differences from TG43 do not appear to warrant dose prescription changes or treatment protocol amendment..

Subclasses of cyclic GMP-specific phosphodiesterase and their role in regulating the effects of atrial natriuretic factor.

Two subclasses of cyclic guanosine monophosphate (GMP)-specific phosphodiesterases were identified in vascular tissue from several beds. The activity of one subclass (phosphodiesterase IB) was stimulated severalfold by calmodulin and selectively inhibited by the phosphodiesterase inhibitor TCV-3B. The activity of the other subclass (phosphodiesterase IC) was not stimulated by calmodulin and was selectively inhibited by the phosphodiesterase inhibitor M&B 22,948. To assess the involvement of both subclasses in regulating cyclic GMP-dependent responses, the ability of TCV-3B and M&B 22,948 to potentiate the in vitro and in vivo responses to the endogenous guanylate cyclase stimulator atrial natriuretic factor (ANF) was evaluated. Both TCV-3B and M&B 22,948 relaxed isolated rabbit aortic and pulmonary artery rings and also potentiated the relaxant effect of ANF. In addition, both inhibitors produced small increases in urine flow and sodium excretion in anesthetized rats and potentiated the diuretic and natriuretic responses to exogenous ANF. M&B 22,948 (30 micrograms/kg/min) produced a threefold increase in the natriuretic response to simultaneously administered ANF, and TCV-3B (10 micrograms/kg/min) produced a twofold increase in the response to ANF. The results of the present experiments suggest that both the calmodulin-sensitive and calmodulin-insensitive subclasses of cyclic GMP-specific phosphodiesterase play a role in regulating the in vitro and in vivo response to ANF.

Incidence and time of occurrence of regional recurrence in stage I-II breast cancer: value of adjuvant irradiation.

PURPOSE: The value of adjuvant radiation therapy (RT) of the axilla and supraclavicular fossa is controversial in early-stage breast cancer. This retrospective study was undertaken to identify pathological risk factors that would predict which subsets of patients would benefit from regional nodal irradiation (RNI). METHODS AND MATERIALS: A total of 1309 women with Stage I/II breast cancer underwent full axillary dissection and either mastectomy (n = 894) or breast-conserving surgery (n = 415). Of these, 712 patients received RNI. The median axilla/supraclavicular fossa dose was 50 Gy. RESULTS: The 10-year actuarial rate of axillary failure (AXF) was 0. 5% in N0 and 2.8% in N1 patients (p 5, also correlated with AXF; the respective rate was 4.3% vs. 1.2% (p = 0.0142). Neither the incidence of AXF nor the time to AXF was affected significantly by the use of RNI, but in N1 patients with retrieved nodes < or = 5, the rate of AXF was 8.3% without RNI vs. 0% with RNI (p = 0.2340). The 10-year actuarial rate of supraclavicular failure (SCF) was 1.2% in N0 and 6.3% in N1 patients (p = 0.0000). SCF was also associated with the extent of nodal involvement (p = 0.0031). The incidence of SCF was not significantly affected by the use of RNI. However, when the results of N1bii and N1biv patients were evaluated as a single group, the effect of RNI was significant (p = 0.0358). The rates of SCF without RNI were high in patients with N1bii or N1biv stage: 10% and 37.5% and, with RNI, 3.2% and 18.2%, respectively. These findings were reinforced by the various combinations of T- and N1-stage. The mean time to SCF was 53.6 months in the irradiated and 24.9 months in the nonirradiated patients (p = 0.0007). CONCLUSIONS: After a complete axillary dissection, only N1 patients with retrieved nodes < or = 5 may be considered for axillary RT. Elective supraclavicular RT is suggested for patients with N1bii or N1biv stage. Supraclavicular irradiation decreases the incidence and delays the appearance of SCF.

Nonpeptide angiotensin II receptor antagonists. 1. Synthesis and in vitro structure-activity relationships of 4-[[[(1H-pyrrol-1-ylacetyl)amino]phenyl]methyl]imidazole derivatives as angiotensin II receptor antagonists.

A novel series of non-biphenylyltetrazole angiotensin II receptor antagonists which contain a 1H-pyrrol-1-ylacetyl residue in place of the benzoyl residue in EXP 6803 have been developed. The receptor binding activity of several members of this new series was in the 10(-8) M range, which was better than that of EXP 6803. Introduction of a carboxylic acid moiety at the 2-position of the pyrrole ring enhanced the in vitro binding affinity at the receptor by 10-fold. Compounds containing an acetic acid (18) or a propionic acid residue (20) at the 5-position of the imidazole were more potent than the carboxylic acid analogue (24). The binding IC50 of the most potent compound 20 was 22 nM. Compounds 18, 20, and 24 in their best fit conformations were manually overlayed on that of the template conformation of EXP 6803 and EXP 8623, respectively. The synthesis and structure-activity relationship data are described.

Multiple binding modes for the receptor-bound conformations of cyclic AII agonists.

Angiotensin II, Asp-Arg-Val-Tyr-His-Pro-Phe, binds its receptor with a postulated turn centered at residue four. Analogs of angiotensin II which contain a disulfide bridge between the side chains of residues 3 and 5 retain significant activity consistent with this hypothesis. Incorporation of 4-mercaptoproline residues, a hybrid, or chimeric amino acid which combines the properties of proline and homocysteine, into either of these positions with analogous disulfide bridges allows retention of high affinity for the receptor. These more highly constrained bicyclic systems give new insight into the details of molecular recognition of residues 3-5 of angiotensin by the receptor. Retention of activity by the antiparallel dimer of [Sar1,Cys3,5]-AII in which the peptide backbone is held in an extended conformation was unexpected. Analysis of the conformational constraints imposed in these active analogs suggests that AII agonists bind to their receptor with different backbone conformations in the region of the central tyrosine residue.

Synthesis and tyrosine kinase inhibitory activity of a series of 2-amino-8H-pyrido[2,3-d]pyrimidines: identification of potent, selective platelet-derived growth factor receptor tyrosine kinase inhibitors.

Screening of a compound library led to the identification of 2-amino-6-(2,6-dichlorophenyl)-8-methylpyrido[2,3-d]pyrimidine (1) as a inhibitor of the platelet-derived growth factor receptor (PDGFr), fibroblast growth factor receptor (FGFr), and c-src tyrosine kinases (TKs). Replacement of the primary amino group at C-2 of 1 with a 4-(N,N-diethylaminoethoxy)phenylamino group yielded 2a, which had greatly increased activity against all three TKs. In the present work, variation of the aromatic group at C-6 and of the alkyl group at N-8 of the pyrido[2,3-d]pyrimidine core provided several analogues that retained potency, including derivatives that were biased toward inhibition of the TK activity of PDGFr. Analogues of 2a with a 3-thiophene or an unsubstituted phenyl group at C-6 were the most potent inhibitors. Compound 54, which had IC50 values of 31, 88, and 31 nM against PDGFr, FGFr, and c-src TK activity, respectively, was active in a variety of PDGF-dependent cellular assays and blocked the in vivo growth of three PDGF-dependent tumor lines.

Soluble 2-substituted aminopyrido[2,3-d]pyrimidin-7-yl ureas. Structure-activity relationships against selected tyrosine kinases and exploration of in vitro and in vivo anticancer activity.

In continuing our search for medicinal agents to treat proliferative diseases, we have discovered 2-substituted aminopyrido[2,3-d]pyrimidin-7-yl ureas as a novel class of soluble, potent, broadly active tyrosine kinase (TK) inhibitors. An efficient route was developed that enabled the synthesis of a wide variety of analogues with substitution on several positions of the template. From the lead structure 1, several series of analogues were made that examined the C-6 aryl substituent, a variety of water solublizing substitutents at the C-2 position, and urea or other acyl functionality at the N-7 position. Compounds of this series were competitive with ATP and displayed submicromolar to low nanomolar potency against a panel of TKs, including receptor (platelet-derived growth factor, PDGFr; fibroblast growth factor, FGFr;) and nonreceptor (c-Src) classes. Several of the most potent compounds displayed submicromolar inhibition of PDGF-mediated receptor autophosphorylation in rat aortic vascular smooth muscle cells and low micromolar inhibition of cellular growth in five human tumor cell lines. One of the more thoroughly evaluated members, 32, with IC50 values of 0.21 microM (PDGFr), 0.049 microM (bFGFr), and 0.018 microM (c-Src), was evaluated in in vivo studies against a panel of five human tumor xenografts, with known and/or inferred dependence on the EGFr, PDGFr, and c-Src TKs. Compound 32 produced a tumor growth delay of 14 days against the Colo-205 colon xenograft model.

Structure-activity relationships for a novel series of pyrido[2,3-d]pyrimidine tyrosine kinase inhibitors.

Screening of a compound library for inhibitors of the fibroblast growth factor (FGFr) and platelet-derived growth factor (PDGFr) receptor tyrosine kinases led to the development of a novel series of ATP competitive pyrido[2,3-d]pyrimidine tyrosine kinase inhibitors. The initial lead, 1-[2-amino-6-(2,6-dichlorophenyl)pyrido[2,3-d]pyrimidin-7-yl]-3- tert-butylurea (4b, PD-089828), was found to be a broadly active tyrosine kinase inhibitor. Compound 4b inhibited the PDGFr, FGFr, EGFr, and c-src tyrosine kinases with IC50 values of 1.11, 0.13, 0.45, and 0.22 microM, respectively. Subsequent SAR studies led to the synthesis of new analogs with improved potency, solubility, and bioavailability relative to the initial lead. For example, the introduction of a [4-(diethylamino)butyl]amino side chain into the 2-position of 4b afforded compound 6c with enhanced potency and bioavailability. Compound 6c inhibited PDGF-stimulated vascular smooth muscle cell proliferation with an IC50 of 0.3 microM. Furthermore, replacement of the 6-(2,6-dichlorophenyl) moiety of 4b with a 6-(3',5'-dimethoxyphenyl) functionality produced a highly selective FGFr tyrosine kinase inhibitor 4e. Compound 4e inhibited the FGFr tyrosine kinase with an IC50 of 0.060 microM, whereas IC50s for the inhibition of the PDGFr, FGFr, EGFr, c-src, and InsR tyrosine kinases for this compound (4e) were all greater than 50 microM.