Biomedical subjects

Pradeep Tyagi

Publications and source records attributed to Pradeep Tyagi.

13 recordsLinked to original sources

PubMed2006

Intraprostatic capsaicin injection as a novel model for nonbacterial prostatitis and effects of botulinum toxin A.

OBJECTIVES: An animal model for nonbacterial prostatitis in rats was developed with the use of intraprostatic injection of capsaicin, an agent thought to excite C-afferent fibers and cause neurogenic inflammation. The analgesic and anti-inflammatory properties of botulinum toxin type A (BoNT-A) was tested in this model. METHODS: Adult male Spraque-Dawley rats were injected with varying doses of capsaicin into the prostate. The nociceptive effects of capsaicin were evaluated for 30min by using a behavior approach; then the prostate was removed for histology and cyclooxygenase (COX) 2 protein concentration measurement. Evans blue (50mg/kg) was also injected intravenously to assess for plasma protein extravasation. A second set of animals were injected with up to 20U of BoNT-A into the prostates 1 wk prior to intraprostatic injection of 1000micromol/l capsaicin. RESULTS: Capsaicin dose dependently induced modifications in pain behavior: closing of the eyes, hypolocomotion, and inflammatory changes: increase of inflammatory cell accumulation, COX2 expression, and plasma extravasation at the acute stage, but completely recovered at 1 wk. BoNT-A pretreatment dose dependently reversed pain behavior and inflammation. BoNT-A 20U significantly decreased inflammatory cell accumulation, COX2 expression, and Evans blue extraction (82.1%, 83.0%, and 50.4%, respectively), and reduced pain behavior (66.7% for eye score and 46.5% for locomotion score). CONCLUSIONS: Intraprostatic capsaicin injection induced neurogenic prostatitis and prostatic pain, and may be a useful research model. BoNT-A produced anti-inflammatory and analgesic effects, and support clinical evaluation in prostatitis.

Animals↗

PubMed2006

Qualitative and quantitative expression profile of muscarinic receptors in human urothelium and detrusor.

PURPOSE: We compared the complete spectrum of receptor subtypes expressed by human detrusor and its primary culture with the expression profile in a human urothelium immortalized cell line, and in fresh urothelium tissue and its primary cell culture. MATERIALS AND METHODS: The levels of mRNA expressed for receptor subtypes M1 through M5 were determined with reverse transcriptase-polymerase chain reaction and quantitative polymerase chain reaction in total RNA extracted individually from different human bladder specimens, including fresh tissue of human urothelium and detrusor, and their respective primary cultures, as well as from the UROtsa cell line. RESULTS: All 5 muscarinic receptors were detected in fresh human bladder tissue by reverse transcriptase-polymerase chain reaction RNA. The same was true in separated urothelium and detrusor tissue except for the lack of the M5 receptor transcript. Receptor subtype mRNA expression in the UROtsa cell line paralleled expression in fresh human bladder. Quantitative polymerase chain reaction data further corroborated these results and showed comparable mRNA expression for M2 and M3 in primary detrusor cultures. Primary cultures also had a decreased copy number of receptor genes than native tissue. The decrease was even more pronounced in primary urothelium culture and the UROtsa cell line in the presence of high calcium. M2 and M3 receptors were also detected in urothelium and detrusor by immunoreactivity. CONCLUSIONS: We identified all 5 existing muscarinic receptor subtypes in detrusor and urothelium, and transcripts levels of M2 and M3 were comparable in detrusor. These results support an alternative site of action in urothelium for anti-muscarinic drugs. Urothelial receptors should be considered in the design of future drugs for overactive bladder.

Adolescent↗

PubMed2006

Local drug delivery to bladder using technology innovations.

Local delivery of drugs directly into the bladder by way of a urethral catheter is a clever approach to optimize drug delivery to the disease site while reducing systemic bioavailability. Pharmacotherapy by this route is referred to as intravesical delivery. In recent years, intravesical delivery has been used in combination with and oral regimen of drugs or as second-line treatment for neurogenic bladder and detrusor overactivity. Negligible absorption of instilled drugs into the systemic circulation explains the near-minimal adverse toxicity reported with this form of therapy. The authors discuss shortcomings of the current options available for intravesical delivery and provide a broad overview of the latest advances through technology innovation to overcome these drawbacks.

Adhesiveness↗

PubMed2005

Naked DNA for liver gene transfer.

The majority of acquired and inherited genetic disorders, including most inborn errors of metabolism, are manifested in the liver. Therefore, it is hardly any surprise to see a large number of Medline reports describing gene therapy efforts in preclinical settings directed toward this organ (Inoue et al., 2004; Oka and Chen, 2004). Of late, non-viral vectors have garnered a lot of attention from the biomedical research community engaged in liver gene therapy (Gupta et al., 2004). However, the first initiative toward gene transfer to the liver using a non-viral approach was taken by Hickman et al. (1994), who applied the technique of naked DNA injection pioneered by Wolff (1990) for skeletal muscle. Direct injection of naked DNA resulted in low, variable and localized gene expression in the rat liver. Consequently, several developments reported in the literature since then aimed to improve hepatic gene expression by employing both surgical and nonsurgical methods. These developments include the exploitation of the unique vasculature of liver as well as the use of electric and mechanical force as an adjunct to the systemic administration of the naked plasmid gene. This chapter focuses on these developments reported from various laboratories, including ours. In addition, the underlying mechanism responsible for the dramatic increase in gene expression using these latest approaches for non-viral gene transfer to the liver is also discussed.

Animals↗

PubMed2005

Ligand-targeted liposomes for cancer treatment.

Selective targeting of ligand-targeted liposomes containing anticancer drugs or therapeutic genes to cell surface receptors expressed on cancer cells is a recognized strategy for improving the therapeutic effectiveness of conventional chemotherapeutics or gene therapeutics. Some recent advances in the field of ligand-targeted liposomes for the treatment of cancer are summarized including: selection criteria for the receptors to be targeted, choice of targeting ligands and choice of encapsulated therapeutics. Targeting of liposomes to solid tumors, versus angiogenic endothelial cells versus vascular targets is discussed. Ligand-targeted liposomes have shown considerable promise in preclinical xenograft models and are poised for clinical development.

Animals↗

PubMed2004

Anisamide-targeted stealth liposomes: a potent carrier for targeting doxorubicin to human prostate cancer cells.

Certain human malignancies including prostate cancer overexpress sigma receptor, a membrane bound protein that binds haloperidol and various other neuroleptics with high affinity. An anisamide derivatized ligand possesses high affinity for sigma receptors and we hypothesized that its incorporation into the liposomes encapsulating doxorubicin (DOX) can specifically target and deliver the drug to prostate cancer cells that overexpress sigma receptors. A polyethylene glycol phospholipid was derivatized with an anisamide ligand, which was then incorporated into the DOX-loaded liposome. The resulting anisamide-conjugated liposomal DOX showed significantly higher toxicity to DU-145 cells than non-targeted liposomal DOX, the IC50 being 1.8 microM and 14 microM respectively. The cytotoxicity of the targeted liposomal DOX, however, was significantly blocked by haloperidol, suggesting that the enhanced cytotoxicity was specifically mediated by the sigma receptors. Fluorescence imaging studies after intravenous (i.v.) administration showed that incorporation of anisamide into liposomes significantly improved their accumulation into the tumor. A weekly injection of the targeted liposomal DOX for 4 weeks at a dose of 7.5 mg/kg led to a significant growth inhibition of established DU-145 tumor in nude mice with minimal toxicity. Free DOX was effective, but associated with significant toxicities. The present study is the first to demonstrate the use of small molecular weight ligand for mediating efficient targeting of liposomal drugs to sigma receptor expressing prostate cancer cells both in vitro and in vivo.

Adenocarcinoma↗

PubMed2004

Sustained intravesical drug delivery using thermosensitive hydrogel.

PURPOSE: Direct instillation of drug solutions into the bladder through a urethral catheter (i.e., intravesical therapy) evades systemic adverse effects of drugs used for bladder diseases. However, conventional vehicles for these drugs fail to extend duration of drug exposure in the bladder beyond the first voiding of urine postinstillation. The current study seeks to overcome the aforementioned inherent limitation of intravesical drug administration by using thermosensitive hydrogel as a matrix for sustained intravesical drug delivery. METHODS: Under halothane anesthesia, normal adult female Sprague-Dawley rats were catheterized with PE-50 tubing to instill either 0.02% w/v solution of fluorescein isothiocyanate (FITC) or the same amount of FITC in a 30% w/v dispersion of thermosensitive [Poly(ethylene glycol)-Poly[lactic acid-co-glycolic acid]-Poly(ethylene glycol)) (PEG-PLGA-PEG) polymer in a 0.1 M phosphate buffer. After instillations, rats were kept in metabolic cages for urine collection. Fluorescence emanating from FITC was measured in the urine at various time points up to 24 h after instillation. A rat model of cyclophosphamide-induced cystitis was chosen for the efficacy study using misoprostol as a model drug entrapped in the thermosensitive hydrogel in place of FITC. Efficacy of hydrogel containing misoprostol was compared against rat groups instilled with saline, hydrogel, and misoprostol independently. RESULTS: Prolonged drug exposure to the bladder afforded by hydrogel was evident from the time course of FITC elimination in the urine and by the green fluorescence of FITC seen at the bladder surface when isolated 24 h after instillation. Rats instilled with free FITC voided almost all of the fluorescence in the urine within the first 8 h, whereas rats instilled with hydrogel encapsulated FITC showed sustained release up to 24 h after instillation. Using a cyclophosphamide-induced cystitis model, rats instilled with misoprostol, a synthetic PGE1 analog, showed significantly reduced frequency of urine voiding (p < 0.05) as compared to the rats instilled with saline. Histological examination of the urothelium showed near normal morphology in rats instilled with misoprostol in hydrogel, whereas extensive tissue damage was observed in rats instilled with saline. CONCLUSION: Our study showed that PEG-PLGA-PEG polymer could be used as a viable sustained drug delivery system for intravesical therapy of diseases of the bladder such as cystitis using misoprostol.

Animals↗

PubMed2004

Urodynamic and immunohistochemical evaluation of intravesical capsaicin delivery using thermosensitive hydrogel and liposomes.

PURPOSE: Aqueous insolubility of the vanilloids such as capsaicin is a major disincentive in their intravesical therapy of detrusor hyperreflexia. We sought to overcome the delivery of this hydrophobic neurotoxin by entrapping it in a lipid bilayer of positively charged multilamellar lipid vesicles (liposomes) or in a hydrophobic polymer matrix of thermosensitive hydrogel. MATERIALS AND METHODS: Liposomes, hydrogel and 30% ethanol/normal saline were prepared with or without 1 mM capsaicin (0.5 ml) and administered intravesically for 30 minutes to 7 groups of age matched, normal female adult Sprague-Dawley rats under halothane anesthesia. At 48 hours after intravesical instillation cystometric studies were performed using urethane anesthesia (0.04 ml per minute). The animals were subsequently sacrificed and whole bladders were harvested for histology and immunohistochemistry. RESULTS: In normal urethane anaesthetized rats capsaicin in 30% ethanol and liposomes completely blocked micturition reflexes. Capsaicin in hydrogel did not completely block the micturition reflex but it significantly decreased bladder contraction frequency compared with vehicle controls. The results of cystometry with capsaicin in liposomes and capsaicin in 30% ethanol correlated with a significant decrease in calcitonin gene-related peptide staining of afferent nerves in the bladder wall. Photographs taken after hematoxylin and eosin staining of the bladder treated with liposomes and hydrogel in the absence of capsaicin did not reveal any adverse histological changes. There were significant histological changes in bladders treated with 30% ethanol alone. CONCLUSIONS: In comparison with 30% ethanol liposomes are a superior vehicle for the intravesical administration of capsaicin, producing comparable efficacy with less tissue damage. Hydrogel can also serve as safe alternative option for capsaicin delivery.

Administration, Intravesical↗

PubMed2003

Intravesical protamine sulfate and potassium chloride as a model for bladder hyperactivity.

OBJECTIVES: An acute animal model for hyperactive bladder in rats was developed using intravesical infusion of protamine sulfate (PS), an agent thought to break down urothelial barrier function, and physiologic concentrations of potassium chloride (KCl). METHODS: Continuous cystometrograms (CMGs) were performed in urethane-anesthetized female rats by filling the bladder (0.04 mL/min) with normal saline followed by intravesical infusion of a test solution consisting of either KCl (100 or 500 mM) or PS (10 or 30 mg/mL) for 60 minutes. Subsequently, the 10 mg/mL PS-treated animals were infused intravesically with 100, 300, or 500 mM KCl. Some animals were pretreated with capsaicin (125 mg/kg subcutaneously) 4 days before the experiments. RESULTS: Unlike KCl (100 or 500 mM) or a low concentration of PS (10 mg/mL) alone, the intravesical administration of a high concentration of PS (30 mg/mL) produced irritative effects with a decreased intercontraction interval (by 80.6%). After infusion of a low concentration of PS, infusion of 300 or 500 mM KCl produced irritative effects (intercontraction interval decreased by 76.9% or 82.9%, respectively). The onset of irritation occurred more rapidly after 500 mM KCl (10 to 15 minutes) than after 300 mM KCl (20 to 30 minutes). Capsaicin pretreatment delayed the onset (approximately 60 minutes) and reduced the magnitude (intercontraction interval decreased by 35.5%) of irritative effects. CONCLUSIONS: Intravesical administration of KCl after PS treatment activates capsaicin-sensitive afferents and detrusor muscle and presumably capsaicin-resistant afferents. Modest, noncytotoxic affronts to urothelial barrier function can result in dramatic irritative responses. This model may be useful in the study of bladder irritation and hyperactivity.

Administration, Intravesical↗

PubMed2003

Intravesical liposome administration--a novel treatment for hyperactive bladder in the rat.

OBJECTIVES: To examine the effect of intravesical administration of liposomes (LPs) on chemically induced bladder hyperactivity in the rat. It has been suggested that interstitial cystitis (IC) is associated with a dysfunctional or leaky epithelium. Thus, enhancement of epithelial barrier function might be useful in the treatment of IC. LPs are vesicles that are concentric phospholipid bilayers separated by an aqueous compartment and can fuse with cells to provide a molecular film that can promote wound healing. METHODS: The intravesical pressure was recorded using a transurethral catheter in adult female Sprague-Dawley rats anesthetized with urethane (1.2 g/kg subcutaneously). Some animals were pretreated with capsaicin (125 mg/kg subcutaneously) 4 days before the experiments. Continuous cystometrograms were performed by slowly filling the bladder (0.04 mL/min) with solutions of varying compositions, including saline, acetic acid (AA, 0.1%), potassium chloride (KCl, 500 mM), protamine sulfate (PS, 10 mg/mL), LPs, PS/KCl, or LPs/KCl. The parameters measured included the intercontraction interval (ICI), amplitude of bladder contractions, compliance, and micturition pressure threshold. RESULTS: The ICI was decreased after exposure to AA (79.8% decrease) or PS/KCl (81% decrease); however, the ICI was not changed after LPs, PS, or KCl alone. The decreased ICI was partially reversed after infusion of LPs (172.8% increase) or LPs/KCl (63% increase), but was not significantly changed after switching to saline or KCl administration. Pretreatment with capsaicin delayed the onset of the irritative effects of AA by approximately 30 to 60 minutes, but had not changed the magnitude after 2 hours of infusion. CONCLUSIONS: Intravesical administration of PS/KCl or AA activates capsaicin-sensitive and capsaicin-resistant afferents in a time-dependent sequence that is partially reversed by LP infusion. We hypothesize that LPs might enhance the barrier properties of a dysfunctional uroepithelium and increase resistance to irritant penetration. Thus, intravesical LP administration could be a novel treatment of patients with IC.

Administration, Intravesical↗

PubMed2003

Controlled gene delivery system based on thermosensitive biodegradable hydrogel.

PURPOSE: Currently, most pDNA delivery systems based on synthetic polymers are either nonbiodegradable or not sensitive to the release environment. The primary objective of this study was to develop and evaluate an aqueous-based, thermosensitive, biodegradable and biocompatible triblock copolymer to control pDNA delivery in vitro and in vivo. METHODS: The triblock copolymers, poly[ethylene glycol-b-(D, L-lactic acid-co-glycol acid)-b-ethylene glycol] (PEG-PLGA-PEG), were synthesized as previously described. The molecular weight and polydispersity of PEG-PLGA-PEG were monitored by gel permeation chromatography (GPC). The cytotoxicity of PEG-PLGA-PEG was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The release of 32P-labeled pDNA entrapped in aqueous dispersion of PEG-PLGA-PEG in 0.1 mol/L sodium phosphate buffer solution (pH 7.4) was studied at 37 degrees C under agitation. Gene transfection efficiency was evaluated in a skin wound model in CD-1 mice. RESULTS: The aqueous dispersion of PEG-PLGA-PEG flows freely at room temperature but form a gel at 37 degrees C body temperature. The in vitro degradation of PEG-PLGA-PEG lasted for more than 30 days. The cytotoxicity of PEG-PLGA-PEG evaluated in HEK 293 cells was significantly lower than that of poly-L-lysine hydrochloride. The release profile of supercoiled pDNA from the polymer followed the zero-order kinetics up to 12 days. Maximal gene expression of luciferase was at 24 h in the skin wound of CD-1 mice and by 72 h, the expression dropped by nearly 94%. CONCLUSION: These results suggest hydrogel formed by PEG-PLGA-PEG could be a promising platform for delivery of pDNA, which represents a novel strategy that may serve as a non-viral vector for gene therapy in wound healing.

Animals↗

PubMedDate not supplied

Intravesical antisense therapy for cystitis using TAT-peptide nucleic acid conjugates.

The present study investigated the potential of intravesical instillation for localized reduction of NGF (nerve growth factor) expression in the urinary bladder. Overexpression of NGF has been linked to the pathogenesis of interstitial cystitis (IC). A minimum free energy algorithm was used to predict suitable regions in mRNA of rat betaNGF, which can be targeted for an antisense approach. The candidate antisense oligos were evaluated for their ability to reduce NGF expression in vitro by cotransfecting HEK293 cells with NGF cDNA. A single oligonucleotide ODN sequence was chosen for testing in an acute cystitis model in rat induced by cyclophosphamide. Overexpression of NGF is known to mediate inflammation of bladder in this model. For improved stability, antisense ODN was replaced with antisense peptide nucleic acid (PNA) and its penetration into bladder was facilitated by tethering TAT peptide sequence. Rat bladders were instilled with either antisense or its scrambled control prior to cystitis induction. Cystometrograms performed on rats under urethane anaesthesia exhibited bladder contraction frequency that was significantly decreased in the antisense treated rats than rats treated with the control. NGF immunoreactivity was also decreased in the urothelium of the antisense treated bladders. Our findings demonstrate the feasibility of using TAT-PNA conjugates for intravesical antisense therapy.

Administration, Intravesical↗

PubMedDate not supplied

Recent advances in intravesical drug/gene delivery.

Targeting of drugs administered systemically relies on the higher affinity of ligands for specific receptors to obtain selectivity in drug response. However, achieving the same goal inside the bladder is much easier with an intelligent pharmaceutical approach that restricts drug effects by exploiting the pelvic anatomical architecture of the human body. This regional therapy involves placement of drugs directly into the bladder through a urethral catheter. It is obvious that drug administration by this route holds advantage in chemotherapy of superficial bladder cancer, and it has now become the most widely used treatment modality for this ailment. In recent years, the intravesical route has also been exploited either as an adjunct to an oral regimen or as a second-line treatment for neurogenic bladder. (Lamm, D. L.; Griffith, J. G. Semin. Urol. 1992, 10, 39-44. Igawa, Y.; Satoh, T.; Mizusawa, H.; Seki, S.; Kato, H.; Ishizuka, O.; Nishizawa, O. BJU Int. 2003, 91, 637-641.) Instillation of DNA via this route using different vectors has been able to restrict the transgene expression in organs other than bladder. The present review article will discuss the shortcomings of the current options available for intravesical drug delivery (IDD) and lay a perspective for future developments in this field.

Administration, Intravesical↗