Serological biomarker profiles of rapidly progressive osteoarthritis in tanezumab-treated patients.

There is a need for efficacious and safe pain treatments for OA (osteoarthritis). The nerve growth factor (NGF) antibody tanezumab is associated with high efficacy, but when combined with chronic NSAID treatment shows an increased risk of rapidly progressive osteoarthritis (RPOA) in a small group of patients. AIM: The aim of this study was to phenotype with biochemical biomarkers of bone, cartilage, soft tissue, synovial metabolism OA patients who are at risk of developing RPOA type-2, for both limited and chronic NSAIDs users. MATERIAL AND METHODS: The dataset consisted of OA patients participating in tanezumab trials who used NSAIDs <90 days (limited NSAID users) or chronic users (NSAIDs ≥90 days) over an average 10 month period. Biomarker data were available for 47 cases (RPOA type-2) and 92 controls. Non-linear and linear multivariable predictive models were developed. RESULTS: By use of two biomarkers at baseline the receiver operating characteristic (ROC) curve area for RPOA type-2 in limited NSAID users was 71%, [CI] (60-83%). OA subjects with this biomarker phenotype had 8-fold higher confidence interval [CI][(2-33)] relative risk of developing RPOA type-2 as compared to OA patients without this phenotype. The AUC of the model in chronic NSAIDs users based on 5 biomarkers was 78%, [CI](69-88%), with 4-fold [CI (2-6)] relative risk of developing RPOA type-2. CONCLUSION: In this hypothesis generating and exploratory study, we identified combinations of biomarkers associated with OA patients who develop RPOA type-2, which may be related to the pathology of the RPOA type-2 joint.

Identification of serological biomarker profiles associated with total joint replacement in osteoarthritis patients.

OBJECTIVE: Establish a biomarker panel associated with all-cause total joint replacement (TJR) through identification of patients with osteoarthritis (OA) who do or do not progress to TJR and investigate effects of nonsteroidal anti-inflammatory drugs (NSAIDs). DESIGN: Serum samples from patients enrolled in phase III trials of tanezumab who experienced TJR (n = 174) or matched patients who did not (n = 321) were analyzed for bone, cartilage, soft tissue, and inflammation markers. Classification and Regression Tree (CART) analysis was used to identify biomarker phenotypes associated with TJR. RESULTS: At baseline, biomarker combinations for patients who did not use NSAIDs before starting tanezumab and used NSAIDs during tanezumab treatment <90 days ("nonNSAID"), identified 77% (95% confidence interval [CI]: 71-84%) of patients who experienced TJR and 77% (95% CI: 65-86%) who did not over a 6-month study period (on average). These biomarker combinations increased odds of identifying patients to remain free of a TJR by 3.3-fold. In patients who used NSAIDs continuously (during screening and ≥90 days during tanezumab treatment), 64% (95% CI: 54-73%) who had TJR and 75% (95% CI: 68-83%) who did not were identified by biomarker combinations different from nonNSAID patients, with an increase in odds of identifying patients to remain free of a TJR by two-fold. CONCLUSIONS: Although validation on other cohorts is necessary, biomarkers may assist in identifying patients who will need TJR. The profiles suggest NSAID use increases importance of bone metabolism in TJR pathology.

Cardiovascular control, autonomic function, and elite endurance performance in spinal cord injury.

We aimed to determine the relationship between level of injury, completeness of injury, resting as well as exercise hemodynamics, and endurance performance in athletes with spinal cord injury (SCI). Twenty-three elite male paracycling athletes (C3-T8) were assessed for neurological level/completeness of injury, autonomic completeness of injury, resting cardiovascular function, and time to complete a 17.3-km World Championship time-trial test. A subset were also fitted with heart rate (HR) monitors and their cycles were fitted with a global positioning systems device (n = 15). Thoracic SCI exhibited higher seated systolic blood pressure along with superior time-trial performance compared with cervical SCI (all P < 0.01). When further stratified by autonomic completeness of injury, the four athletes with cervical autonomic incomplete SCI exhibited a faster time-trial time and a higher average speed compared with cervical autonomic complete SCI (all P < 0.042). Maximum and average HR also tended to be higher in cervical autonomic incomplete vs autonomic complete. There were no differences in time-trial time, HR, or speed between thoracic autonomic complete vs incomplete SCI. In conclusion, autonomic completeness of injury and the consequent ability of the cardiovascular system to respond to exercise appear to be a critical determinant of endurance performance in elite athletes with cervical SCI.

Effects of inspiratory muscle training on exercise responses in Paralympic athletes with cervical spinal cord injury.

We asked whether specific inspiratory muscle training (IMT) improves respiratory structure and function and peak exercise responses in highly trained athletes with cervical spinal cord injury (SCI). Ten Paralympic wheelchair rugby players with motor-complete SCI (C5-C7) were paired by functional classification then randomly assigned to an IMT or placebo group. Diaphragm thickness (B-mode ultrasonography), respiratory function [spirometry and maximum static inspiratory (PI ,max ) and expiratory (PE ,max ) pressures], chronic activity-related dyspnea (Baseline and Transition Dyspnea Indices), and physiological responses to incremental arm-crank exercise were assessed before and after 6 weeks of pressure threshold IMT or sham bronchodilator treatment. Compared to placebo, the IMT group showed significant increases in diaphragm thickness (P = 0.001) and PI ,max (P = 0.016). There was a significant increase in tidal volume at peak exercise in IMT vs placebo (P = 0.048) and a strong trend toward an increase in peak work rate (P = 0.081, partial eta-squared = 0.33) and peak oxygen uptake (P = 0.077, partial eta-squared = 0.34). No other indices changed post-intervention. In conclusion, IMT resulted in significant diaphragmatic hypertrophy and increased inspiratory muscle strength in highly trained athletes with cervical SCI. The strong trend, with large observed effect, toward an increase in peak aerobic performance suggests IMT may provide a useful adjunct to training in this population.

The outcome of a cohort of tuberculin-positive predominantly South Asian new entrants aged 16-34 to the UK: Blackburn 1989-2001.

SETTING: Blackburn, Hyndburn and Ribble Valley Local Government areas of England and Wales, the former a high tuberculosis (TB) prevalence district. BACKGROUND: The incidence of tuberculosis in new entrants aged 16-34 with positive tuberculin skin tests but normal chest X-rays after initial entry is not definitely known, and was previously estimated from cross-sectional national surveys and derived data for the 2006 and 2011 NICE economic appraisals of new entrant TB screening. METHODS: New entrants aged 16-34 years predominantly from South Asia (India, Pakistan and Bangladesh), with tuberculin tests inappropriately positive for their BCG history were identified for the years 1989-2001 inclusive from a new entrant database. These entrants were compared with the current GP registration database to see if local residence could be confirmed and the local TB notification database to October 2008. Survival analysis was carried out using Kaplan-Meier survival curves and a Cox Regression model. RESULTS: Four hundred and seventy-nine such new entrants with normal initial chest X-rays were identified. Of these 402 (84%) registered with a General Practitioner in East Lancashire for a period of time and could be followed up by this study. The crude incidence density of active TB amongst these individuals with latent disease was 1297 per 100 000 person-years (95% CI; 991-1698 per 100 000 person-years). After 10 and 15 years of follow-up 13.5 and 16.3% of individuals, respectively, had progressed on to active disease. CONCLUSION: This patient-derived, rather than estimated, data shows a minimum risk of TB disease of 16.3% at 15 years. The 2006 NICE economic appraisal, suggested that treatment for latent TB infection (LTBI) was cost-effective when the incidence of clinical TB over 15 years surpassed 18% in these populations. The 2011 NICE economic appraisal reduced this to 12% active TB over 15 years, and showed that at 16% active TB over 15 years a single interferon gamma release assay was the most cost-effective strategy. Further cohort studies are urgently needed to confirm or revise the assumptions behind the 2011 NICE economic appraisal.

Peripheral vascular function in spinal cord injury: a systematic review.

BACKGROUND: During the past 20 years, significant advances in patient care have resulted in individuals with spinal cord injury (SCI) living longer than before. As lifespan increases, cardiovascular complications are emerging as the leading cause of mortality in this population, and individuals with SCI develop cardiovascular disease at younger ages than their able-bodied counterparts. To address this increasing clinical challenge, several recent studies investigated the central cardiovascular adaptations that occur following SCI. However, a somewhat less recognized component of cardiovascular dysfunction in this population is the peripheral vascular adaptations that also occur as a result of SCI. STUDY DESIGN: Literature review. OBJECTIVE: To present a comprehensive overview of changes in arterial structure and function, which occur after SCI. SETTING: Canada. METHODS: A systematic literature review was conducted to extract studies that incorporated measures of arterial structure or function after SCI in animals or humans. RESULTS: Individuals with SCI exhibit vascular dysfunction below the lesion that is characterized by a reduction in conduit artery diameter and blood flow, increased shear rate and leg vascular resistance, and adrenoceptor hyper-responsiveness. There is also recent alarming evidence for central arterial stiffening in individuals with SCI. CONCLUSION: Although physical deconditioning is the primary candidate responsible for the maladaptive remodeling of the peripheral vasculature after SCI, there is emerging evidence that blood pressure oscillations, such as those occurring in the large majority of individuals with SCI, also exacerbates vascular dysfunction in this population.

Influence of the neurological level of spinal cord injury on cardiovascular outcomes in humans: a meta-analysis.

BACKGROUND: Although motor and sensory deficits following spinal cord injury (SCI) are well known, there are still contrasting reports on how SCI affects baseline cardiovascular (CV) parameters and other autonomic functions. STUDY DESIGN: Meta-analysis is performed. OBJECTIVES: To examine the effect of injury level on supine and seated CV function in individuals with SCI. METHODS: A total of 98 studies representing 1968 individuals were retrieved for analysis. Systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) were abstracted from the studies and compared between four groups according to the neurological lesion level, cervical (C) SCI (C4-C8), high-thoracic (HT) SCI (T1-T6), low-thoracic lumbar (LTL) SCI (below T6) and able-bodied (AB) controls. RESULTS: In the supine position, SBP, DBP and HR were lower in C compared with HT, LTL and AB (all P<0.04). In the seated position, SBP and DBP were significantly lower in C compared with LTL and AB (all P<0.003) and HR was significantly lower in C compared with LTL only (P=0.01). A final finding was that C exhibited a lower resting SBP in the seated compared with the supine position (P<0.001). CONCLUSION: Individuals with SCI exhibit a lesion-dependent impairment in resting CV function, whereby those with the highest injury had the greatest degree of CV dysfunction. A further finding was that individuals with a C injury exhibited a lower resting SBP in the seated vs supine position. Thus, clinicians and researchers should consider lesion level and body position when measuring and interpreting CV parameters in individuals with SCI.

How does cervical spinal cord injury impact the cardiopulmonary response to exercise?

We compared cardiopulmonary responses to arm-ergometry in individuals with cervical spinal cord injury (C-SCI) and able-bodied controls. We hypothesized that individuals with C-SCI would have higher respiratory frequency (fb) but lower tidal volume (VT) at a given work rate and dynamically hyperinflate during exercise, whereas able-bodied individuals would not. Participants completed pulmonary function testing, an arm-ergometry test to exhaustion, and a sub-maximal exercise test consisting of four-minute stages at 20, 40, 60, and 80% peak work rate. Able-bodied individuals completed a further sub-maximal test with absolute work rate matched to C-SCI. During work rate matched sub-maximal exercise, C-SCI had smaller VT (main effect p < 0.001) compensated by an increased fb (main effect p = 0.009). C-SCI had increased end-expiratory lung volume at 80% peak work rate vs. rest (p < 0.003), whereas able-bodied did not. In conclusion, during arm-ergometry, individuals with C-SCI exhibit altered ventilatory patterns characterized by reduced VT, higher fb, and dynamic hyperinflation that may contribute to the observed reduced aerobic exercise capacity.

Formulation, stability, and antitumor activity of 1-beta-D-arabinofuranosylcytosine conjugate of thioether phospholipid.

1-beta-D-Arabinofuranosylcytosine 5'-diphosphate-rac-1-S-octadecyl-2-O- palmitoyl-1-thioglycerol (ara-CDP-DL-PTBA) is an effective stable 1-beta-D-arabinofuranosylcytosine (ara-C) conjugate of thioether phospholipid against a variety of transplantable tumors in mice. The conjugate was formulated in a micellar solution by sonication, in which the conjugate exists as micellar discs (size, 0.01 to 0.04 micron). Analyses on thin-layer and high-pressure liquid chromatography showed that the conjugate was chemically stable upon storage at 3-4 degrees C for more than a 6-mo period. However, stored at room temperature for 3 mo it began to degrade (3 to 11%) to 1-beta-D-arabinofuranosylcytosine 5'-monophosphate and phosphatidic acid. At 3-4 degrees C, the micellar structure remained generally unchanged for 6 mo (size, less than 0.1 micron). Samples stored for 4 mo at room temperature formed some larger vesicles (size, 0.1 to 0.4 micron). Antitumor activity against i.p. implanted L1210 leukemia in mice remained relatively constant with samples stored for 6 mo at 3-4 degrees C or 3 mo at room temperature. 1-beta-D-Arabinofuranosylcytosine 5'-triphosphate (ara-CTP) levels were elevated (greater than 500 pmol/10(7) cells) in L1210 leukemia cells within 1 h following i.p. administration of 400 mg/kg of ara-CDP-DL-PTBA to mice. More importantly, retention of cellular ara-CTP was prolonged (greater than 24 h) in these tumor cells as compared with ara-C treatments. Administration of ara-CDP-DL-PTBA to mice with colon 26 carcinoma (s.c.) resulted in both significant antitumor activity with an increased life span greater than 100% and decreased tumor size. The conjugate also demonstrated a dose-dependent therapeutic effect in mice with M5076 sarcoma (s.c.) as demonstrated by decreases in tumor size and liver metastases. Overall, ara-CDP-DL-PTBA, a stable lipid conjugate of ara-C in a micellar solution, appears to offer substantial therapeutic benefit to mice with leukemia and solid tumors warranting its further development and clinical investigation.

Prognostic significance of the metastasis-inducing protein S100A4 (p9Ka) in human breast cancer.

The calcium-binding protein S100A4 is capable of inducing metastasis in rodent models for breast cancer. We now show that rabbit antibodies to recombinant rat S100A4 recognize specifically human S100A4 using Western blotting techniques and use them to assess the prognostic significance of S100A4 in primary tumors from a group of 349 patients treated between 1976 and 1982 for stage I and stage II breast cancer. The antibody stains normal breast tissue heterogeneously, but stains positively 41% of the carcinomas, leaving the remaining 59% as negatively stained. In addition to the carcinoma cells, some host stromal cells and lymphocytes are also stained, but these have been discounted in subsequent analyses. There is an association of staining of carcinomas for S100A4 with some tumor variables considered to be associated with poor prognosis for patients: tumor present in axillary lymph nodes (borderline P = 0.058), staining for c-erbB-3 (P = 0.002), cathepsin D (P = 0.024), and c-erbB-2 (P = 0.048). The association of staining for S100A4 with patient survival has been evaluated using life tables and analyzed using generalized Wilcoxon statistics. Eighty percent of the S100A4-negative patients but only 11% of the S100A4-positive patients are alive after 19 years of follow-up, and this association is highly significant (P < 0.0001); the former have a median survival of >228 months and the latter 47 months. The other tumor variables that show significant association with survival time are nodal status (P < 0.0001), tumor size (P = 0.0035), histological grade (P = 0.013), staining for c-erbB-2 (P = 0.0015), estrogen receptor (P = 0.028), and p53 (P = 0.032). Analysis of the association of patients with carcinomas staining for S100A4 and their survival in subgroups defined by these other tumor variables shows that in each subgroup, staining for S100A4 is associated with poorer survival. Patients whose tumors stain for S100A4 and possess involved lymph nodes (P < 0.0001), which are fixed to the chest wall (P = 0.015) or which stain for c-erbB-2 (P = 0.050), show a significant reduction in survival times over those with only S100A4-staining tumors. Patients with involved lymph nodes, or staining for c-erbB-2 in the S100A4-negative group fail to show any significant reduction in survival times. Multivariate regression analysis for 137 patients shows that staining for S100A4 is most highly correlated with patient deaths (P < 0.0001), but involved lymph nodes (P = 0.001), fixed tumors (P = 0.0002), and high histological grade (P = 0.022) are also significant independent prognostic variables. These results suggest that in this group of patients, the metastasis-inducing protein S100A4 is most tightly correlated with patient demise.

Therapeutic activity of 1-beta-D-arabinofuranosylcytosine conjugates of lipids in WEHI-3B leukemia in mice.

Two new conjugates of 1-beta-D-arabinofuranosylcytosine (ara-C) and lipids were tested for therapeutic activity in myelomonocytic WEHI-3B leukemia in mice. Both conjugates were superior to equimolar mixtures of their respective parent compounds and to ara-C alone. IP treatment was found effective after either IP or IV transplantation of the leukemia. The thioether-linked lipid conjugate ara-CDP-D,L-PTBA showed considerably higher efficacy than the ester-linked lipid conjugate ara-CDP-L-dipalmitin. The optimal therapeutic regimen of ara-CDP-D,L-PTBA consisted of 60 mg/kg given IP qd 1-5 after transplantation of the WEHI-3B leukemia.

Histopathologic types of benign breast lesions and the risk of breast cancer: case-control study.

The hypothetical multistep model of carcinogenesis indicates that breast cancer develops via a series of intermediate hyperplastic lesions through in situ to invasive carcinoma. To identify the risk inherent within the different morphologic lesions, we have analyzed the data from 674 benign biopsy specimens comprising 120 cases who subsequently developed breast cancer and 382 controls (matched for age and date of biopsy) spanning a period up to 20 years of follow-up (mean 66.95 months). In this series we have confirmed an increased risk associated with certain types of benign breast lesions. Atypical lobular hyperplasia was the most significant risk factor for breast cancer with more unfavorable outcome in patients <50 years of age (p = 0.003) and a relative risk (RR) of 4.55 (confidence interval [CI] 1.77-11.69). Hyperplasia of usual type showed an RR of 1.53 (CL 1.10-2.13) with a statistically worse probability of survival (cancer-free time) for patients >50 years. For atypical ductal hyperplasia the RR was 2.03 (CI 0.80-1.39). Blunt duct adenosis was significantly more common in cases progressing to breast cancer compared with controls, showing an RR of 2.08 (CI 1.12-2.85). We describe in detail the criteria of morphologic changes observed in blunt duct adenosis and define, for the first time, the level of risk associated with each of its six subtypes. Improved knowledge of breast carcinogenesis will provide insight for defining high-risk groups thus resulting in improved screening and management regimens.

Discriminative properties of the psychostimulant dl-cathinone in a two lever operant task. Lack of evidence for dopaminergic mediation.

In order to analyse further the discriminative stimulus properties of stimulant drugs, rats were trained to discriminate 2.0 mg/kg of dl-cathinone in a two-lever operant task. Dose-related generalization was seen to cathinone itself and to a wide range of stimulant drugs including d-amphetamine, cocaine, methylphenidate, pipradrol and cathine, i.e. (+)norpseudoephedrine. The high degree of specificity of the cathinone cue at the specific training dose studied was shown by the fact that the following nonstimulant drugs failed to generalize at all to cathinone, even in large doses--haloperidol, chlordiazepoxide, fenfluramine and fentanyl. The cathinone cue at 2.0 mg/kg was probably of central origin because phydroxyamphetamine (a polar congener of amphetamine) failed to generalize to cathinone at a dose nearly 50 times the ED50 for amphetamine generalization. Phenylethylamine (PEA; alpha-demethylamphetamine) and deuterated phenylethylamine (alpha, alpha, d2-PEA), a long acting derivative of phenylethylamine which is resistant to metabolism by monoamine oxidase, produced at most partial (60%) generalization to cathinone, even in large doses. alpha-Demethylcathinone failed to generalize at all to cathinone at doses up to 10 times the ED50 for cathinone. Thus, the alpha-methyl groups of both amphetamine and cathinone are important in determining their cue properties. The involvement of dopaminergic systems in the cathinone cue was investigated by examining generalization to apomorphine and antagonism by haloperidol. Apomorphine produced at most 29% generalization to cathinone. Haloperidol, at doses up to 0.3 mg/kg, produced at most 50% antagonism of both the cathinone cue and of the ability of amphetamine to substitute for cathinone. It is suggested that the evidence for dopaminergic mediation of the cue properties of cathinone and of other CNS stimulants is somewhat tenuous, whilst endogenous phenylethylamine may play some part in the mediation of the stimulant cue. Haloperidol, alone and in conjunction with amphetamine or cathinone, produced a remarkable tendency for subjects to emit a greater proportion of their total responses on the inoperative rather than the operative lever than was seen after saline or injections of vehicle. This action of a neuroleptic drug suggests, in accordance with Colpaert, Niemegeers and Janssen (1977), that in drug discrimination antagonism studies involving neuroleptics, and perhaps other drugs, quantal (lever selection) rather than quantitative (percentage of responses on the drug lever) indices may be the procedures of choice.

Nucleoside conjugates as potential antitumor agents. 2. Synthesis and biological activity of 1-(beta-D-arabinofuranosyl)cytosine conjugates of prednisolone and prednisone.

Two of the new anticancer drugs recently synthesized in our laboratory from conjugation of ara-C2 and several corticosteroids linked through a phosphodiester bond include prednisolone- (I) and prednisone-p-ara-C (II). They were demonstrated to be enzymatically hydrolyzed to the corresponding steroid and ara-CMP and the latter was further shown to be hydrolyzed to ara-C by phosphodiesterase I, snake venom, 5'-nucleotidase, and acid phosphatase. However, the conjugates were shown to be resistant to hydrolysis by alkaline phosphatase. The activity of conjugates I and II against L1210 lymphoid leukemia in female mice (C3D2F1/J) was significantly greater than that of ara-C alone or in combination with the steroid. In fact, when the optimum dosage of 75 (mumol/kg)/day x 5 was used, the administration of ara-C alone was followed by an increased life span (ILS) of 45%. This result is similar to that previously reported. With the same equimolar doses of mixtures of ara-C and either prednisolone or prednisone, the ILS values were 40 and 44%, respectively. However, when the conjugates were used, the ILS values were 89 and 100% respectively. These findings seem promising and have provided the bases for continued study of these new compounds.

Nucleoside conjugates. 13. Synthesis and antitumor activity of 1-beta-D-arabinofuranosylcytosine conjugates of thioether lipids with improved water solubility.

A series of ara-CDP-rac-1-S-alkyl-2-O-acyl-1-thioglycerols (3-12), analogues of highly active Cytoros2 (1), was prepared, and solubility, lipophilicity, and structure-activity relationships of these conjugates were investigated. The conjugates with sn-1 alkyl (< C18) and sn-2 fatty acyl (< C14) substituents of the thioglycerol were water-soluble, while those with the sn-1 alkyl (> C14) and the sn-2 fatty acyl (> C16) were sparingly soluble. The latter formed micelles upon sonication. Conjugate 7 containing the sn-1 tetradecyl and the sn-2 palmitoyl (C16) groups formed micelles by both sonication and shaking. The partition coefficients (1-octanol/PBS) of the water-soluble conjugates were about 20 times greater than that of ara-C. The water-insoluble showed a more than 40 times increase. A single dose of the micelle-forming conjugates 7 and 10 produced a significant increase in life span (ILS > 421%) with 50% long-term survivors (> 45 days) in mice bearing ip-implanted L1210 lymphoid leukemia. These results were comparable to those of previous micelle-forming conjugate 1 (Cytoros). In contrast, the water-soluble conjugates at single doses were less effective (ILS 81-386% with 0-33% long-term survivors). However, three divided doses of the water-soluble conjugates were found to be as effective as a single dose of micellar solution of the water-insoluble. The results indicate that conjugate 7 and most of the water-soluble derivatives warrant further investigation.

Nucleoside conjugates as potential antitumor agents. 3. Synthesis and antitumor activity of 1-(beta-D-arabinofuranosyl)cytosine conjugates of corticosteroids.

Six 5'-(steroid-21-phosphoryl)-1-(beta-D-arabinofuranosyl)cytosines have been prepared and evaluated against L1210 lymphoid leukemia in culture and in mice (C3D2F1/J). These include the ara-C conjugates of 11-deoxycorticosterone (5a), corticosterone (5b), cortexolone (5c), fludrocortisone (5d), 6 alpha-methylprednisolone (5e), and dexamethasone (5f). When the optimum dosage of ara-C [38 (mumol/kg)/day X 5] was given to mice bearing L1210, the ILS value found was 89%. A simple mixture of each steroid and ara-C gave ILS values that were on the whole significantly less than that of the parent nucleoside. However, of six conjugates, all but two (5d and 5f) were more active than ara-C at their optimal doses. Both corticosterone- (5b) and cortexolone-p-ara-C (5c) were especially effective at the respective optimal doses of 76.7 and 115 (mumol/kg)/day X 5. These gave ILS values of 200% each. All of the conjugates were demonstrated to be enzymatically hydrolyzed to the corresponding steroid and ara-CMP, and the latter was further shown to be hydrolyzed to ara-C by phosphodiesterase I, 5'-nucleotidase, and acid phosphatase. However, they were shown to be resistant to hydrolysis by alkaline phosphatase.

Phospholipid-nucleoside conjugates. 3. Syntheses and preliminary biological evaluation of 1-beta-D-arabinofuranosylcytosine 5'-monophosphate-L-1,2-dipalmitin and selected 1-beta-D-arabinofuranosylcytosine 5-diphosphate-L-1,2-diacylglycerols.

Several new phospholipid-ara-C conjugates have been prepared and tested as prodrugs of the parent ara-C. The new derivative include ara-CMP-L-dipalmitin, ara-CDP-L-distearin, ara-CDP-L dimyristin, ara-CDP-L-diolein, and the radioactively labeled derivative ara-CDP-L-di[1-14C]palmitin. In addition, the unusually stable ara-CMP-L-dipalmitin-N-phosphoryldicyclohexylurea adduct was isolated as a crystalline solid (two diastereomers) in the reaction sequence to prepare ara-CMP-L-dipalmitin. The new prodrugs were solubilized by sonication methods and tested for their antiproliferative activity in vitro against mouse myeloma MPC-11 cells and against L1210 lymphoid leukemia. Such studies demonstrated that the antiproliferative activities of the prodrugs (as determined by ED50) were less that ara-C on a molar basis. In the mouse myeloma cell line some evidence was obtained that the antiproliferative activity was related to the chain length of the fatty acid side chains in the prodrugs. In in vivo studies against L1210 lymphoid leukemia in mice, the prodrugs were shown to be much more effective than ara-C, with the overall efficacy apparently being independent of the length of the fatty acid side chain. Some evidence was obtained in the vivo studies that the ara-CDP-L-dimyristin, which bears the shortest fatty acid side chain, was more toxic at the higher dosages than the longer chain length derivatives.

Nucleoside conjugates. 6. Synthesis and comparison of antitumor activity of 1-beta-D-arabinofuranosylcytosine conjugates of corticosteroids and selected lipophilic alcohols.

Five new P1-(steroid-21-yl)-P2-(1-beta-D-arabinofuranosylcytosin-5'-yl)pyro phosphates (ara-CDP-steroids), five 1-beta-D-arabinofuranosylcytosine 5'-O-(alkyl)phosphates (ara-CMP-alkyl esters), and two P1-(alkyl)-P2-(1-beta-D-arabinofuranosylcytosin-5'-yl)pyrophosphat e (ara-CDP-alkyl esters) have been prepared and evaluated against L1210 lymphoid leukemia in culture and in mice (C3D2F1/J). These include ara-CDP-11-deoxycorticosterone (6a), ara-CDP-cortisone (6c), ara-CDP-corticosterone (6d), ara-CDP-cortexolone (6e), and ara-CDP-prednisone (6g), ara-CMP hexadecyl ester (7a), ara-CMP 1-cyclohexylmethyl ester (7b), ara-CMP 1-adamantylmethyl ester (7c), ara-CMP 2-(1-adamanthyl)ethyl ester (7d), ara-CMP 2-chloroethyl ester (7e), ara-CDP hexadecyl ester (9a), and ara-CDP 1-cyclohexylmethyl ester (9b). The in vitro antitumor results indicated that ara-CDP-steroids were as active as the previously reported ara-CMP-steroids and that ara-CMP and ara-CDP-alkyl esters were less growth inhibiting than ara-CDP-steroids and ara-C. However, the in vivo antitumor results indicated that ara-CDP-steroids were generally less effective than the previous monophosphate derivatives. Among them ara-CDP-corticosterone (6d) and the known ara-CDP-cortisol (6b) showed greater efficacy than ara-C with ILS value of 152% and 209%, respectively, at the optimal dose of 40 and 80 (mg/kg)/day for 9 days, while that of ara-C was 138% at the optimum dose of 9.2 (mg/kg)/day. Generally, ara-CMP alkyl esters (7a-e), given ip to the L1210 leukemic mice, were found to be toxic and ineffective. However, ara-CDP hexadecyl ester (9a) showed marginal activity (ILS, 38%). These preliminary results support the thesis that the ara-C conjugates of this type may require a lipophilic and naturally occurring moiety for improved efficacy.

Nucleoside conjugates. 11. Synthesis and antitumor activity of 1-beta-D-arabinofuranosylcytosine and cytidine conjugates of thioether lipids.

Five 1-beta-D-arabinofuranosylcytosine conjugates and two cytidine conjugates of thioether lipids (1-S-alkylthioglycerols) linked by a pyrophosphate diester bond have been prepared and their antitumor activity against an ara-C2 sensitive (L1210/0) and two ara-C resistant L1210 lymphoid leukemia sublines in mice were evaluated. These prodrugs of ara-C include ara-CDP-rac-1-S-hexadecyl-2-O-palmitoyl-1-thioglycerol (8a), ara-CDP-rac-1-S-octadecyl-2-O-palmitoylthioglycerol (8b), and ara-CDP-rac-1-S-octadecyl-2-O-methyl(or -ethyl, -hexadecyl)thioglycerols (8c-e). The cytidine conjugates include CDP-rac-1-S-octadecyl-2-O-palmitoyl(or -methyl)- 1-thioglycerols (9a and 9b). Sonicated solutions of the conjugates existed in the form of micellar disks (size 0.01-0.04 microns). Single doses (200-400 mg/kg) of 8a and 8b produced significant increase in life span (257-371%) in mice bearing ip implanted L1210/0 leukemia. In contrast, conjugates 8c-e were less effective (ILS 19-75%) and cytidine conjugates (9a and 9b) were ineffective. Even though 8a and 8b were found to be curative in a high percentage of mice bearing ip implanted partially ara-C resistant L1210 subline [L1210/ara-C(I)], they were completely ineffective against deoxycytidine kinase deficient ara-C resistant L1210 subline [L1210/ara-C(II)]. However, the present results, together with the previous, demonstrate that 8a and 8b are promising new prodrugs of ara-C with improved efficacy.

Nucleoside conjugates. 14. Synthesis and antitumor activity of 1-beta-D-arabinofuranosylcytosine conjugates of ether lipids with improved water solubility.

A series of ara-CDP-rac-1-O-alkyl-2-O-acylglycerols (9a-f), analogues of highly active ara-CDP-rac-1-O-hexadecyl-2-O-palmitoylglycerol (1) and Cytoros2 (2), was prepared, and solubility, lipophilicity, and structure-activity relationships of these conjugates were investigated. Conjugates 9a-f containing sn-1 alkyl (< C16) and sn-2 fatty acyl (< C14) and sn-1 alkyl (< C14) and sn-2 fatty acyl (< C16) substituents of the glycerol were water-soluble by shaking, while those with the sn-1 alkyl (> C16) and the sn-2 fatty acyl (> C16) such as conjugate 1 were sparingly soluble. Conjugates 9a-c,e were almost completely solubilized in water by shaking. However, a large portion of conjugates 9d and 9f in water by shaking exist in micelles with mean diameters ranging 7.0-55.2 nm. The partition coefficients (1-octanol/PBS) of the water-soluble conjugates were about 9-18 times greater than that of ara-C. A single dose (300 mg/kg) of conjugates 9d and 9f produced a significant increase in life span (ILS 206 to > 543%) with 17-67% long-term survivors (> 45 days) in mice bearing ip-implanted L1210 lymphoid leukemia. These results were comparable to those of the previous conjugate 1 and Cytoros (2). In contrast, conjugates 9a-c,e at single doses were less effective (ILS 69-178% with no long-term survivors). However, two (qd, 1, 7) or three (qd 1, 5, 9) divided doses of these conjugates were found to be as effective as a single dose of the previous conjugates. The three divided doses (150 mg/kg per day) of conjugates 9d, 9e, and 9f produced a remarkable antitumor activity in L1210 leukemic mice (ILS > 350% with > 50% long-term survivors). Because of the convenient formulation and the significant antitumor activities, the water-soluble conjugates 9d, 9e, and 9f warrant further investigation.