Biomedical subjects

Sanjeev Kumar

Publications and source records attributed to Sanjeev Kumar.

71 records · Page 4Linked to original sources

PubMed2003

Combination nonviral interleukin 2 gene therapy and external-beam radiation therapy for head and neck cancer.

OBJECTIVES: To demonstrate that the combination of nonviral murine interleukin 2 (mIL-2) gene therapy and external-beam radiation therapy (XRT) have an enhanced therapeutic effect for the treatment of head and neck squamous cell carcinoma (HNSCC) in an orthotopic murine model and to elucidate the mechanism of action. METHODS: Randomized, controlled studies in the murine orthotopic model of HNSCC. Squamous cell carcinoma VII cells were injected into the floor of the mouth to establish tumors in immunocompetent mice. The intervention groups were treated with mIL-2, radiation therapy, empty plasmid, no treatment, combination mIL-2/XRT, and combination empty plasmid/XRT. Nonviral mIL-2 gene transfer was performed on days 5 and 9. The XRT was administered to the assigned groups 24 hours after first mIL-2 delivery. The mice were killed on day 13. Tumors and local lymph nodes were harvested and evaluated. Primary and secondary cytokine expression, cytotoxic T-lymphocyte activity, and apoptosis were assayed. RESULTS: The combination mIL-2/XRT demonstrated a significant increase in antitumor effects compared with single therapy or controls. Increased expression levels of primary and secondary cytokines were found in the group treated with mIL-2, and this effect was preserved when mIL-2 treatment was combined with XRT. Combination therapy significantly increased apoptosis compared with monotherapy. CONCLUSIONS: The present study demonstrates that combination mIL-2/XRT generates potent antitumor immune responses and significantly increases apoptosis in an orthotopic murine model of HNSCC. Further optimization of this strategy is warranted as well as consideration for human clinical trials.

Animals↗

PubMed2003

Addressing the metabolic activation potential of new leads in drug discovery: a case study using ion trap mass spectrometry and tritium labeling techniques.

Metabolic activation of drug candidates to electrophilic reactive metabolites that can covalently modify cellular macromolecules may result in acute and/or idiosyncratic immune system-mediated toxicities in humans. This presents a significant potential liability for the future development of these compounds as safe therapeutic agents. We present here an example of an approach where sites of metabolic activation within a new drug candidate series were rapidly identified using online liquid chromatography/multi-stage mass spectrometry on an ion trap mass spectrometer. This was accomplished by trapping the reactive intermediates formed upon incubation of compounds with rat and human liver microsomes as their corresponding glutathione conjugates and mass spectral characterization of these thiol adducts. Based on the structures of the GSH adducts identified, potential sites and mechanisms of bioactivation within the chemical structure were proposed. These metabolism studies were interfaced with iterative structural modifications of the chemical series in order to block these bioactivation sites within the molecule. This strategy led to a significant reduction in the propensity of the compounds to undergo metabolic activation as evidenced by reductions in the irreversible binding of radioactivity to liver microsomal material upon incubation of tritium-labeled compounds with this in vitro system. With the efficiency and throughput achievable with such an approach, it appears feasible to identify and address the metabolic activation potential of new drug leads during routine metabolite identification studies in an early drug discovery setting.

Animals↗

PubMed2003

Anti-cardiolipin/beta-2 glycoprotein activities co-exist on human anti-DNA antibody light chains.

We have recently shown that the human anti-DNA antibodies B3 and 33H11 also bind cardiolipin and that the anti-autoantigen activity resides predominantly on their lambda light chains. We now show that the two auto-antibodies possess strong reactivity to the plasma-protein 2-Glycoprotein I (beta2-GPI) also. Utilizing chain shuffling experiments involving an unrelated anti-p185 antibody 4D5 with insignificant reactivity to cardiolipin or to beta2-GPI, we now demonstrate that hybrid Fabs with constituent light chain, but not the heavy chain, of B3 or 33H11, exhibit anti-cardiolipin activity. Furthermore, the constructs possessing the auto-antibody-derived light chain also exhibited significant reactivity to beta2-GPI. The results suggest that anti-DNA, anti-cardiolipin and anti-beta2-GPI activities co-exist on the light chains of the antibodies studied and, importantly, these activities could be transferred to antibody constructs by their light chains alone. Computer-generated models of the three-dimensional structures of the auto-antibodies and their hybrids, suggest predominant interaction of their light chains with domain IV of beta2-GPI.

Amino Acid Sequence↗

PubMed2003

Cystosarcoma phyllodes: our institutional experience.

Twenty-six patients of Cystosarcoma phyllodes, treated between July 1994 and July 2001, were analysed retrospectively. Median age at presentation was 38 years (range 13-61 years). Mean size of the lesion was 6 cm. There were 77% left-sided lesions and 23% right-sided lesions. Histologically, 58% lesions were benign, 11% borderline and 31% malignant lesions. All patients underwent definitive surgical procedure in the form of wide local excision or mastectomy. Four patients received postoperative radiotherapy. Median follow-up period was 35 months. Six patients showed recurrence, and four of these were malignant. Median disease-free survival period was 34 months.

Adolescent↗

PubMed2003

In vitro micropropagation, differentiation of aerial bulbils and tubers and diosgenin content in Dioscorea bulbifera.

Dioscorea bulbifera could be micropropagated through nodal segments and bulbils. The best medium for regeneration and bulbil differentiation was MS + 0.5 microM IAA (indole-3-acetic acid) + 20.0 microM Kn (kinetin) + 500 mg/L CH (casein hydrolysate) + activated charcoal (20 %). Diosgenin content was maximum in regenerants grown on MS + 5.0 microM IAA + 20.0 microM Kn + 500 mg/L CH. T.s of bulbils could also be used for direct plantlet differentiation as well as bulbil differentiation on MS + 10.0 microM IAA + 20.0 microM Kn + (in mg/L) 30 each of Asp (asparagine) + Arg (arginine) + Gln (glutamine) + 10 Ad (adenine) + 500 CH + 10 Cyst hyd (cysteine hydrochloride). Diosgenin yield in plantlets reached a maximum after 20 weeks. The results indicate that micropropagation, bulbil formation and tuberisation can be achieved in vitro in D. bulbifera, hitherto a less exploited plant, and can further be used for obtaining enhanced levels of diosgenin.

Chromatography, High Pressure Liquid↗

PubMed2003

Sexually transmitted diseases in children.

Sexually transmitted diseases (STDs) cause considerable morbidity among their victims. This is true even for children. Unfortunately, this aspect has not received due attention and remains a relatively ignored field. We carried out a study of the clinico-epidemiological profile of STDs among the pediatric patients who attended the STD clinic of a tertiary care hospital. Pediatric patients who presented with STDs from January of 1995 to February of 2001 constituted the subject material of this study. A detailed clinical examination and relevant laboratory investigations were carried out in all patients. A total of 15,453 STD patients were seen during this period, out of which 127 were children, giving a prevalence of 0.82% pediatric STD cases. The peak incidence of STDs was seen in the age group of 10-14 years (66.1%). Most of them were illiterate (61.4%) and from low socio-economic backgrounds (70.9%). Notably, 17.3% were from remand homes. The predominant STDs observed among these children were syphilis (25.2%), including six cases of congenital syphilis, vulvo-vaginal candidiasis (11.8%), condyloma-acuminata (14.2%), herpes progenitalis (8.7%) and traumatic lesions (7.9%). Histories or signs of abuse were present in 74% of the patients. Only two cases during the span of the study were HIV positive. Pediatric STDs remain a hidden and neglected problem. Children constituted 0.82% of all STD patients in our series. This may represent only the tip of the iceberg, because several features, including some operational problems, are responsible for underdiagnosis of pediatric STDs. If not treated promptly and adequately, they may result in significant physical and emotional sequelae in children. Thus, it is essential to develop strategies to prevent sexual abuse and to have adequate counselling measures, especially in juvenile delinquents.

Adolescent↗

PubMed2003

A novel P450-catalyzed transformation of the 2,2,6,6-tetramethyl piperidine moiety to a 2,2-dimethyl pyrrolidine in human liver microsomes: characterization by high resolution quadrupole-time-of-flight mass spectrometry and 1H-NMR.

We describe herein a novel metabolic fate of the 2,2,6,6-tetramethyl-piperidine (2,2,6,6-TMPi) moiety to a ring-contracted 2,2-dimethyl pyrrolidine (2,2-DMPy) in human liver microsomal incubations. The existence of this pathway was demonstrated for three compounds (I-III) of varied structures suggesting that this may be a general biotransformation reaction for the 2,2,6,6-TMPi moiety. The 2,2-DMPy metabolites formed in incubations of the three compounds with human liver microsomes were characterized by online high performance liquid chromatography coupled to a high resolution hybrid quadrupole-time-of-flight mass spectrometer. Suggested elemental composition obtained from accurate mass measurements of the molecular ions and fragment ions of the metabolites clearly indicated the loss of a mass equivalent to C(3)H(6) from the parent 2,2,6,6-TMPi functionality. Additional accurate tandem mass spectrometry data indicated that one of the original two gem-dimethyl groups was intact in the metabolite structure. Proof of a ring-contracted 2,2-DMPy structure was obtained using (1)H-NMR experiments on a metabolite purified from liver microsomal incubations, which showed only two geminal methyl groups, instead of four in the parent compound. Two-dimensional correlation spectroscopy and decoupling experiments established aliphatic protons arranged in a pyrrolidine ring pattern. The fact that the formation of 2,2-DMPy metabolites in human liver microsomes was NADPH-dependent suggested that this novel metabolic reaction was catalyzed by the cytochrome P450 (P450) enzyme(s). Immunoinhibition studies in human liver microsomal incubations using anti-P450 monoclonal antibodies and experiments with insect cell microsomes containing individually expressed recombinant human P450 isozymes indicated that multiple P450 isozymes were capable of catalyzing this novel metabolic transformation.

Biotransformation↗

PubMed2003

Pharmacokinetics and interactions of a novel antagonist of chemokine receptor 5 (CCR5) with ritonavir in rats and monkeys: role of CYP3A and P-glycoprotein.

The mechanisms of pharmacokinetic interactions of a novel anti-human immunodeficiency virus (anti-HIV-1) antagonist of chemokine receptor 5 (CCR5) [2-(R)-[N-methyl-N-(1-(R)-3-(S)-((4-(3-benzyl-1-ethyl-(1H)-pyrazol-5-yl)piperidin-1-yl)methyl)-4-(S)-(3-fluorophenyl)cyclopent-1-yl)amino]-3-methylbutanoic acid (MRK-1)] with ritonavir were evaluated in rats and monkeys. MRK-1 was a good substrate for the human (MDR1) and mouse (Mdr1a) multidrug resistance protein transporters and was metabolized by CYP3A isozymes in rat, monkey, and human liver microsomes. Both the in vitro MDR1-mediated transport and oxidative metabolism of MRK-1 were inhibited by ritonavir. Although the systemic pharmacokinetics of MRK-1 in rats and monkeys were linear, the oral bioavailability increased with an increase in dose from 2 to 10 mg/kg. The area under the plasma concentration-time curve (AUC) of MRK-1 was increased 4- to 6-fold when a 2 or 10 mg/kg dose was orally coadministered with 10 mg/kg ritonavir. Further pharmacokinetic studies in rats indicated that P-glycoprotein (P-gp) inhibition by ritonavir increased the intestinal absorption of 2 mg/kg MRK-1 maximally by approximately 30 to 40%, and a major component of the interaction likely resulted from its reduced systemic clearance via the inhibition of CYP3A isozymes. Oral coadministration of quinidine (10 and 30 mg/kg) increased both the extent and the first-order rate of absorption of MRK-1 (2 mg/kg) by approximately 40 to 50% and approximately 100 to 300%, respectively, in rats, thus further substantiating the role of P-gp in modulating the intestinal absorption of MRK-1 in this species. At the 10 mg/kg MRK-1 dose, however, the entire increase in its AUC upon coadministration with ritonavir or quinidine could be attributed to a reduced systemic clearance, and no effects on intestinal absorption were apparent. In contrast to rats, the effects of P-gp in determining the intestinal absorption of MRK-1 appeared less significant in rhesus monkeys at either dose.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

New findings on degradation of famotidine under basic conditions: identification of a hitherto unknown degradation product and the condition for obtaining the propionamide intermediate in pure form.

The degradation behavior of famotidine (1) was investigated in 25% ammonia solution and in 2 M NaOH. The hydrolysis of the drug in ammonia resulted in separation of [3-[[2-[(diaminomethylene)amino]-4-thiazolyl]methyl]thio]propionamide (3), an impurity listed in British Pharmacopoeia. The treatment with 2 M NaOH resulted in formation of [3-[[[2-[(diaminomethylene)amino]-4-thiazolyl]methyl]thio]propionyl]sulfamide (2) and 3. These products further decomposed to [3-[[2-[(diaminomethylene)amino]-4-thiazolyl]methyl]thio]propionic acid (4) and a heretofore unknown product, 5. The latter separated out in the reaction mixture as brown shiny crystals. Proton and carbon-13 nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis of the charcoal-treated product established the structure. The formation of 5 is postulated to involve abstraction of a proton from the alpha-carbon of intermediates 2 and 3 followed by elimination of the thiol moiety.

Amides↗

PubMed2002

Interference of thiomersal in biologicals during protein estimation.

In the present report thiomersal was detected as interfering substance in hepatitis B vaccines during the total protein estimation by Lowry's protein assay. The thiomersal at different concentrations of 0.005%, 0.0075%, 0.01%, 0.02%, 0.05% and 0.1% was found to reduce the Folin Ciocalteu's phenol reagent and produce colour development between 0.024 O.D to 1.023 O.D. values. Further, the thiomersal was shown to interfere between 34.55% to 52.73% with Folin Ciocalteu's phenol reagent, when 10 batches of different hepatitis B vaccines were subjected to estimation of total protein content by Lowry's protein assay.

Biological Products↗

PubMed2002

Promoter activation by enhancer-dependent and -independent loading of activator and coactivator complexes.

Activation of the pDbeta1 promoter at the TCRbeta locus requires a functional distal enhancer, Ebeta. Here, we have analyzed the mechanism of promoter activation in thymocytes from mice containing or lacking Ebeta. We found that pDbeta1 shows a complex profile of transcription factor and chromatin remodeling complex occupancy even at Ebeta(-) alleles. The presence of Ebeta, however, results in a few specific changes in factor occupancy at the promoter. These differences correlate with localized alterations in histone modifications and in the recruitment of the basal transcriptional machinery. In addition, Ebeta is also bound by CBP and Pol II, suggesting a mechanism for delivery of a holoenzyme complex to the pDbeta1 promoter. These results illustrate a specialized, long-range function of an enhancer in the hierarchical events that regulate assembly of a cell type-specific promoter.

3T3 Cells↗

PubMed2002

Identification of metabolites of a substance P (neurokinin 1 receptor) antagonist in rat hepatocytes and rat plasma.

[3R,5R,6S]-3-(2-cyclopropyloxy-5-trifluoromethoxyphenyl)-6-phenyl-1-oxa-7-azaspiro[4.5]decane is a substance P (Neurokinin 1 receptor) antagonist. Substance P antagonists are proven in concept to have excellent potential for the treatment of major depression, and they allow superior and sustained protection from acute and delayed chemotherapy-induced emesis. The metabolism of this compound was investigated in rat hepatocytes, and circulating rat plasma metabolites were identified following oral and intravenous dosing. The turnover in rat hepatocytes within 4 h was about 30%, and the major metabolites were identified as two nitrones and a lactam associated with the piperidine ring. Although these metabolites were also observed in rat plasma, the major circulating metabolite was a keto acid following oxidative de-amination of the piperidine ring. Liquid chromatography/tandem mass spectrometry and nuclear magnetic resonance were used to confirm the structure of the latter metabolite. A mechanism leading to the formation of the keto acid metabolite has been suggested, and most intermediates were observed in rat plasma.

Administration, Oral↗

PubMed2002

Extrapolation of diclofenac clearance from in vitro microsomal metabolism data: role of acyl glucuronidation and sequential oxidative metabolism of the acyl glucuronide.

Diclofenac is eliminated predominantly (approximately 50%) as its 4'-hydroxylated metabolite in humans, whereas the acyl glucuronide (AG) pathway appears more important in rats (approximately 50%) and dogs (>80-90%). However, previous studies of diclofenac oxidative metabolism in human liver microsomes (HLMs) have yielded pronounced underprediction of human in vivo clearance. We determined the relative quantitative importance of 4'-hydroxy and AG pathways of diclofenac metabolism in rat, dog, and human liver microsomes. Microsomal intrinsic clearance values (CL(int) = V(max)/K(m)) were determined and used to extrapolate the in vivo blood clearance of diclofenac in these species. Clearance of diclofenac was accurately predicted from microsomal data only when both the AG and the 4'-hydroxy pathways were considered. However, the fact that the AG pathway in HLMs accounted for ~75% of the estimated hepatic CL(int) of diclofenac is apparently inconsistent with the 4'-hydroxy diclofenac excretion data in humans. Interestingly, upon incubation with HLMs, significant oxidative metabolism of diclofenac AG, directly to 4'-hydroxy diclofenac AG, was observed. The estimated hepatic CL(int) of this pathway suggested that a significant fraction of the intrahepatically formed diclofenac AG may be converted to its 4'-hydroxy derivative in vivo. Further experiments indicated that this novel oxidative reaction was catalyzed by CYP2C8, as opposed to CYP2C9-catalyzed 4'-hydroxylation of diclofenac. These findings may have general implications in the use of total (free + conjugated) oxidative metabolite excretion for determining primary routes of drug clearance and may question the utility of diclofenac as a probe for phenotyping human CYP2C9 activity in vivo via measurement of its pharmacokinetics and total 4'-hydroxy diclofenac urinary excretion.

Animals↗

PubMedDate not supplied

Fistula of the fourth branchial pouch.

Most of the congenital anomalies of the branchial apparatus are from the second arch, pouch, and cleft; some are from the first and third arches. Fourth branchial pouch remnants are very rare. We present a rare case of fourth branchial fistula, with characteristic clinical features of recurrent left-sided neck abscess that burst spontaneously, forming a fistula. Diagnosis of fourth branchial pouch fistula was confirmed by contrast radiography (ie, fistulogram and barium swallow) revealing the internal opening in the apex of the left pyriform fossa.

Branchial Region↗

PubMedDate not supplied

Purification and kinetics of a raw starch-hydrolyzing, thermostable, and neutral glucoamylase of the thermophilic mold Thermomucor indicae-seudaticae.

The purified glucoamylase of the thermophilic mold Thermomucor indicae-seudaticaehad a molecular mass of 42 kDa with a pI of 8.2. It is a glycoprotein with 9-10.5% carbohydrate content, which acted optimally at 60 degrees C and pH 7.0, with a t(1/2) of 12 h at 60 degrees C and 7 h at 80 degrees C. Its experimental activation energy was 43 KJ mol(-1) with temperature quotient (Q(10)) of 1.35, while the values predicted by response surface methodology (RSM) were 43 KJ mol(-1) and 1.28, respectively. The enzyme hydrolyzed soluble starch at 50 degrees C (K(m) 0.50 mg mL(-1) and V(max) 109 micromol mg(-1) protein min(-1)) and at 60 degrees C (K(m) 0.40 and V(max) 143 micromol mg(-1) protein min(-1)). The experimental K(m) and V(max) values are in agreement with the predicted values at 50 degrees C (K(m) 0.45 mg mL(-1) and V(max) 111.11 micromol mg(-1) protein min(-1)) and at 60 degrees C (K(m) 0.36 mg mL(-1)and V(max) 142.85 micromol mg(-1) protein min(-1)). An Arrhenius plot indicated thermal activation up to 60 degrees C, and thereafter, inactivation. The enzyme was strongly stimulated by Co(2+), Fe(2+), Ag(2+), and Ca(2+), slightly stimulated by Cu(2+) and Mg(2+), and inhibited by Hg(2+), Zn(2+), Ni(2+), and Mn(2+). Among additives, dextran and trehalose slightly enhanced the activity. Glucoamylase activity was inhibited by EDTA, beta-mercaptoethanol, dithiothreitol, and n-bromosuccinimide, and n-ethylmaleimide inhibited its activity completely. This suggested the involvement of tryptophan and cysteine in catalytic activity and the critical role of disulfide linkages in maintaining the conformation of the enzyme. The enzyme hydrolyzed around 82% of soluble starch and 65% of raw starch (K(m) 2.4 mg mL(-1), V(max) 50 micromol mg(-1) protein min(-1)), and it was remarkably insensitive to glucose, suggesting its applicability in starch saccharification.

Enzyme Activation↗

PubMedDate not supplied

Restless legs symptoms in a patient with above knee amputations: a case of phantom restless legs.

We describe a 78-year-old gentleman who, following bilateral above-knee amputations, developed symptoms of restless legs syndrome in the absent portions of his lower extremities. These symptoms improved with dopamine agonist therapy. In addition, he later developed parkinsonism with prominent rest tremor on metoclopramide. This suggests that this individual had a dopamine-deficient state which predisposed him to both restless legs syndrome and drug-induced or drug-exacerbated parkinsonism. We propose expanding the spectrum of phantom limb phenomena to include phantom restless legs.

Aged↗

PubMedDate not supplied

Acute myocardial infarction due to blunt chest trauma.

We report a case of blunt chest injury following a road accident leading to damage of the left main and left anterior descending coronary arteries causing acute myocardial infarction in a young person.

Accidents, Traffic↗