Macromolecular Conjugates For Passive Tumor Targeting: In Vitro Studies With A Gelatin-Methotrexate Conjugate OFNER CM III, CHEN CS, PICA K
University of the Sciences in Philadelphia, USA
Background: Soluble macromolecular conjugates of low molecular weight anticancer agents are being investigated because of their potential to improve efficacy and reduce systemic toxicity based on their preferential accumulation in solid tumors.
Gelatin-methotrexate conjugates (G-MTX) have been investigated in our lab by evaluating the role of molecular weight, drug molar ratio, charge, and degradation on
growth inhibition of leukemia cells.
Methods: G-MTX prepared by carbodiimde reactions with different molecular weights of type B gelatin using SEC separations at each step produced molecular weights ranging from 35 to 182 kDa. Drug molar ratio determined by spectrophotometric methods ranged from 0.5:1 to 25:1 moles MTX : mole of gelatin. G-MTX charge measured by IEF electrophoresis ranged from 4.9 for anionic G-MTX to >9.6 for cationic G-MTX. Gelatin degradation of G-MTX by the lysosomal enzyme cathepsin B was evaluated after 90 min at the in vitro lysosomal pH of 4.8 using a trichloroacetic acid precipitation and BCA protein assay. Growth inhibition profiles of HL60 promyelocytic leukemia cells were conducted to obtain IC50 values.
Results: The IC50 values for anionic G-MTX with low drug molar ratio (0.5:1 to 2.2:1) ranged from 4.6x10-8 to 1.8x10-7 M and increased proportionately with molecular weight. The IC50 values for anionic G-MTX with high drug molar ratio (7.4:1 to 25:1) however, showed little if any molecular weight correlation, ranging from 2.0x10-7 to 2.9x10-7 M. IC50 values for cationic G-MTX of low drug molar ratio were about 4X higher than that for high drug molar ratio anionic G-MTX, except for the highest molecular weight specie which was about 10X higher at 2.9x10-6 M. The growth inhibition effect of MTX was reduced 5 to 290 times by conjugating to gelatin. The extent of G-MTX enzymatic degradation was 85% for the gelatin control and decreased from 67% to 30% as drug molar ratio increased from 2:1 to 23: 1.
Conclusions: 1) The inverse linear molecular weight effect with IC50 suggests the same mechanism for anionic G-MTX. 2) The non-linear molecular weight effect and substantially higher IC50 value for the highest molecular weight specie suggests a less effective and different mechanism for cationic G-MTX. 3) The apparent protection from degradation by high drug load may hinder intracellular drug release and efficacy compared to low drug load G-MTX.
Rectal Delivery of Ceftriaxone Sodium-loaded Mucoadhesive Gelatin-Mucin Microspheres OFOKANSI KC, ADIKWU MU
Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Nigeria, 410001 Nsukka, Enugu State, Nigeria
Background: Biologically adhesive drug delivery systems such as microspheres offer important advantages over conventional drug delivery systems. The properties of bioadhesive microspheres such as their surface characteristics, force of bioadhesion, release pattern of incorporated drug and biodegradability are determined by the nature of the polymers employed in the formulation as well as their swelling and solvent regain characteristics. The reported interaction of mucin with many biologically important entities including polymers prompted the preparation of microspheres from admixtures of mucin and gelatin (a widely used pharmaceutical adjuvant). The objective of this study, therefore, was to prepare microspheres from admixtures of gelatin and mucin and to evaluate the rectal delivery of ceftriaxone sodium from these microspheres.
Methods: Microspheres based on mucin-gelatin admixtures (1:1, 1:2, 1:3, 1:4) and gelatin alone were prepared by the emulsificstion-crosslinking technique. Further experiments were carried out to evaluate the swelling and mucoadhesive characteristics of the formulated microspheres using water sorption in two media (SIF and SGF) and adhesion to the everted hog ileum as indices of measurements respectively. Drug loading was achieved by diffusion-filling at 37 0.2 oC using a concentrated solution of ceftriaxone sodium in citrate/phosphate buffer (pH 7.4) and finally determining the drug content of a given quantity of the loaded microspheres spectrophotometrically at 240 nm. Release studies were carried out in simulated gastric fluid without pepsin (pH 1.2) and simulated intestinal fluid without pancreatin (pH 7.4) to evaluate the release behavior of the microspheres. For the rectal delivery studies, three groups each of eight Male Wistar rats were used. An amount of the microspheres containing ceftriaxone sodium was weighed out to achieve a dose level of 100 mg/kg body weight in the rats and carefully transferred into empty hard gelatin capsule (No. 3). A positive control was set up by similarly encapsulating amounts of pure ceftriaxone sodium equivalent to that in the microspheres. At regular time intervals of 1 h, 0.5 ml of blood was sampled from the orbital sinus of the rats and then analysed for plasma levels of ceftriaxone sodium using the method of Tietz5 to prepare a protein-free filtrate prior to spectrophotometric analysis.
Results: The formulated microspheres showed good mucoadhesive properties and exhibited percentage mucoadhesion as high as 80 % in some cases. Mucoadhesion to hog everted intestinal tissue was found to be comparatively higher in SGF than in SIF. Preliminary liquid uptake studies showed that water absorption and the rate of water absorption by the microspheres followed the order:1:4>1:1>1:2>1:3>gelatin alone in SGF while the order in SIF is 1:4>gelatin alone>1:3>1:1>1:2 (data not shown). The release profiles of ceftriaxone sodium from the mucoadhesive microspheres in two different release media (SIF and SGF) show high percentage and rapid release of ceftriaxone from the microspheres in SIF within 30 min. The plasma level-time curve for the rectally administered ceftriaxone loaded mucoadhesive microspheres is shown in Fig. 1 The areas under the plasma concentration versus time curves (AUC) were determined using the trapezoidal rule based on a non-compartmental pharmacokineticanalysis. The pharmacokinetic parameters as calculated from Fig. 1 show that the bioavailability of ceftriaxone sodium via the rectal route was highest (AUC = 444.8 g-h ml-1) from microspheres prepared from equal portions of S-mucin and gelatin (1:1). Microspheres prepared from gelatin alone gave significantly lower (P≤ 0.05) AUC value in comparison with those prepared from its admixtures with S-mucin. The generally high AUC values obtained for the microspheres may suggest that the absorption of ceftriaxone sodium from the rectal mucosa was both rapid and complete and that the drug must have by-passed the hepatic first pass metabolism. It is necessary to point out that adequate precautions were taken to deposit the encapsulated microspheres on the lower part of the rat’s rectum with the expectation of causing the absorbed drug to drain directly into the general circulation via either the lower or middle haemorrhoidal veins. This target may well have been achieved considering the high AUC values obtained for all the microspheres.
Conclusion: 1) The microspheres showed considerably high mucoadhesive capacities with good swelling profiles. 2) The rectal route could provide a therapeutically viable alternative for the delivery of ceftriaxone sodium, which hitherto, is administered only parenterally owing to its instability in the acidic environment of the stomach.
Fig. 3: Plasma ceftriaxone sodium versus time profiles for the microspheres
Development of Gelatin-Based Nanoparticulate Formulation for the Delivery of Human Insulin OFOKANSI, KC, WINTER, G. COESTER, C.
Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig Maximilians University, Butenandstr. 5, 81377 Munich, Germany
Background: The design of systems for oral delivery of proteins is in fact really challenging and comparatively very little work has been reported in this field of research. However, the tendency of proteins to denaturation and loss of bioactivity poses severe limits to the reactions that can be performed on the carrier, to the solvents that can be used, and to the environmental conditions that can be adopted during preparation, purification, and storage of the delivery system. Given the above background, we developed a nanoparticulate formulation based on gelatin for the delivery of human insulin. In this study, we report the first successful encapsulation of insulin into gelatin nanoparticles (NPs) under very gentle nanoprecipitation conditions.
Methods: Unloaded and insulin-loaded gelatin (type B, 225 bloom) NPs were prepared by a one-step desolvation technique at a pH of 7.0. and characterized. To determine the amount of incorporated and free insulin, the NPs were recovered by evaporating the solvent using a Rotavapor at 30 oC under reduced pressure until about 15 ml of the dispersion remained. The recovered dispersion was centrifuged at 14,000 × g for 20 min and the supernatant was assayed for insulin by reverse phase high performance liquid chromatography (RP-HPLC) to determine the amount of free insulin. The sediment was redispersed in 10 ml of 0.01 N HCl and shaken for 10 min to dissolve any insulin adhering to the surface of the NPs and further subjected to centrifugation. The resulting supernatant was analysed to determine the amount of insulin entrapped on the surface of the NPs. The last sediment was finally digested in 5 ml of trypsin solution (0.4 mg/ml) to release all insulin entrapped within the matrices of the particles. The insulin content of the solution was immediately determined. The in vitro release of insulin from the NPs was performed in 10 ml each of simulated gastric fluid (pH 1.2) and in phosphate buffered saline (pH 6.8) without enzymes.
Results: The most challenging aspect of this study was the search for appropriate cross-linking agent needed to stabilize the NPs. Glutaraldehyde (GTA) has been employed routinely by most investigators to cross-link gelatin-based NPs. However, in this study an instantaneous loss of insulin monomers was observed upon addition of glutaraldehyde to the NPs in situ. This was evident from the disappearance of the retention peak (7.7 min) of insulin earlier recorded in a RP-HPLC chromatogram for pure human insulin (Fig.1). Evaluation of alternative cross-linking agents (formaldehyde and glyoxal) led to the selection of glyoxal as an effective cross-linker for gelatin NPs.
Fig. 1:RP-HPLC chromatogram of insulin after entrapment into gelatin NPs cross-linked with GTA (……) and with glyoxal ( )
Glyoxal did not cause any loss of monomeric insulin (Fig. 1). The optimum amount of glyoxal needed to effectively cross-link the NPs was found to be 120 mg per 200 mg of gelatin while amounts of glyoxal in excess of 200 mg (500 µl) led to gradual aggregation and precipitation of the particles with the rate and intensity of aggregation increasing with an increase in the amount of glyoxal added. Mean size of between 500 and 600 nm monodisperse NPs were obtained. Unloaded and insulin-loaded NPs were found to have zeta potential values of -20.1 and -16.8 mV respectively. The amount of insulin loaded on the surface of the NPs (200 mg) was evaluated to be 13.67 mg which represented 83.6 % of the total amont of drug used in the loading studies. The amount of unentrapped insulin was 8.84 % while 7.6 % was entrapped into the matrices of the NPs. The release profile of insulin from the optimized nanoparticulate formulation shows that in simulated gastric medium, a burst release occurred within the first 30 min possibly due to the rapid leaching off of the surface bound insulin and about 55 % of the entrapped drug was released over a 6 h release period. In simulated intestinal medium (pH 6.8), however, release of the entrapped drug was slow and sustained after the initial burst release and up to 80 % of the entrapped insulin was released over 6 h.
CONCLUSION: 1) Stabilized insulin-loaded gelatin NPs could be formulated under gentle nanoprecipitation conditions using optimal amounts of glyoxal as the cross-linking agent in order to preserve the chemical stability of insulin. 2) Release of insulin depended greatly on the stability of the NPs at distinct pH environments.
Screening Of Antioxidants For Inhibitory Activity Against Lung Metastasis Of Murine Colon Cancer Cells In Mice OGASAWARA M1, 2, MATSUNAGA T1, NAGAI Y2, TAKATSU K1, 2 1Toyama Prefectural Institute for Pharmaceutical Research, Toyama, Japan; 2University of Toyama, Toyama, Japan.
Background: Cancer metastasis is a major cause of death in cancer patients. Thus, it is important to find promising agents with antimetastatic activity. A variety of antioxidants such as antioxidative vitamins and polyphenols have recently been proposed to be effective in inhibiting cancer metastasis mainly based on the inhibition of tumor invasion, growth and angiogenesis. However, it is unclear which antioxidant has more potent antimetastatic activity. Here, a comparative study on the inhibitory activity of 20 antioxidants against the in vitro invasion, growth and experimental lung metastasis of murine colon cancer cells was conducted, and a promising agent was further examined on anti-tumor immune cells.
Methods: Experimental lung metastasis assay was performed by inoculation of murine colon 26-L5 cells through a tail vein of mice and tumor nodules on the suface of lungs were determined. Test compounds were administered intraperitoneally 5 consecutive times beginning 3 days before tumor inoculation at a dose of 1 micromol. Cell invasion and growth were evaluated by transwell chambers and WST-1 solution, respectively. Free radical scavenging assay was carried out using the stable radical, 1,1-diphenyl-2-picrylhydrazyl. In some experiments, mice receiving anti-asialoGM1 or 2-chloroadenosine, or nude mice were used to examine roles of natural killer (NK) cells, macrophages, or T cells, respectively, in the antimetastatic effect of a test compound.
Results: Among 20 compounds tested, epigallocatechin gallate (EGCG) exhibited most significant reduction by 77% in tumor metastasis. EGCG also inhibited it dose-dependently with 98% suppression at 2 micromol Statistically significant relation was not observed between the radical scavenging, tumor invasion and growth inhibiting activities of test compounds and their inhibition rates of tumor metastasis. We have next examined roles of anti-tumor immunity in the EGCG’s effect, and found that depletion of NK cells completely abrogated the effect, whereas a marginal reduction of the effect was seen in nude mice and macrophage-depleted mice.
Conclusions: These results suggest that EGCG has potential benefit for tumor metastasis inhibition. Its antimetastatic effect may be mediated mainly through NK cell activity.
Radiotherapy And Multi-Agent Chemotherapy (Procarbazine, ACNU And Vincristine) For High-Grade Gliomas: A Prospective Study OGAWA K1, YOSHII Y2, INOUE O3, TUCHIDA Y2, SUGIMOTO K2, IRAHA S1, TOITA T1, KAKINOHANA Y1, TAMAKI W1, ARIGA T1, KASUYA G1, MURAYAMA S1 1Department of Radiology, University of the Ryukyus, Okinawa, Japan
2Department of Neurosurgery, University of the Ryukyus, Okinawa, Japan
3Department of Hyperbaric Medicine, University of the Ryukyus, Okinawa, Japan
Purpose: To prospectively evaluate the efficacy and toxicity of radiotherapy combined with multi-agent chemotherapy using procarbazine, nimustine (ACNU) and vincristine in adults with high-grade gliomas.
Materials and Methods: Patients aged 18 years of age or older with a histologically confirmed supratentorial glioblastoma or grade 3 gliomas (anaplastic astrocytoma, anaplastic oligoastrocytoma and anaplastic oligodendroglioma) were enrolled. Radiotherapy protocols were hyperfracionated radiotherapy (HF-RT) or radiotherapy immediately after hyperbaric oxygenation (HBO) (HBO-RT). The protocol of HF-RT consisted of 2 fractions per day of 1.2 Gy to a total dose of 72 Gy (10 fractions per week). The protocol of HBO-RT consisted of daily 2 Gy fractions for 5 consecutive days per week up to a total dose of 60 Gy, with each fraction administered immediately after HBO with the period of time from completion of decompression to irradiation being less than 15 minutes. Chemotherapy consisted of procarbazine (90 mg/m2 orally, days 1 to 14), ACNU (80 mg/m2 intravenously, day 1) and vincristine (0.5 mg/m2 intravenously, days 1 and 8) and was administered during and after radiotherapy, up to a maximum total of 4 courses.
Results: Between 1997 and 2003, a total of 51 patients (36 patients with glioblastoma and 15 patients with grade 3 gliomas) were enrolled. HF-RT was administered in 10 patients treated between 1997 and 1999, and HBO-RT was administered in 41 patients treated between 2000 and 2003. All 51 patients were able to complete a planned radiotherapy using HF-RT or HBO-RT with 1 course of concurrent chemotherapy. Of 38 assessable patients, 20 (52%) had an objective response including 6 CR and 14 PR. The median survival time in all 51 patients and 36 glioblastoma patients were 17.5 months and 15.5 months, respectively. On univariate analysis, histologic grade (p < 0.0001) and Karnofsky performance status (p = 0.01) had a significant impact on survival, and on multivariate analysis, histologic grade alone was a significant prognostic factor for survival (p < 0.001). Although grade 4 leukopenia and grade 4 thrombocytopenia occurred in 10% and 8% of all patients respectively, these were transient with no patients developing neutropenic fever or intracranial hemorrhage. No serious non-hematological or late toxicities were seen.
Conclusions: These results indicated that radiotherapy with multi-agent chemotherapy (procarbazine, ACNU and vincristine) was effective and safe with virtually no late toxicity in patients with high-grade gliomas
Exploiting Biochemical Differences Between Parasite And Host In Development Of Antiparasitic Agent OGBUNUDE POJ University of Nigeria, Enugu-campus, Nigeria
Background: Protozoans are auxotrophic for purine bases and nucleosides and hence depend on exogenous sources to meet their purine requirements. Differences between the nucleoside carrier systems of host cells and the parasites could be exploited in designing rationale chemotherapy involving nucleoside transport inhibitor (NTI) like (6-[4-nitrobenzyl)-mercapto]purine ribonucleoside (nitrobenzylthioinosine; NBMPR) and toxic nucleoside analogue compounds. Aim: 1) To determine number of NBMPR binding sites on animal cells and trypanosome trypomastigotes. 2) To determine whether co-administration of NBMPR and toxic nucleoside analogue, tubercidin, could reduce parasitaemia in animal infected with Trypanosome brucei gambiense and hence prolong the survival of the infected animal.
Methods: Measurement of number of NTIs in animal cells was done using erythrocytes from human, mouse and hamster. NBMPR binding parameters (Kd and Bmax) were determined from Scatchard plots using appropriate computer program (Elsevier-Biosoft Dose-effect analysis) performing a straight fit (B/F vs B) to data sets. In assay to determine survival and parasites clearance, two groups of mice (5 mice/group) were inoculated with 0.2 ml of the parasite suspension containing 106 trypanosomes/ml. When parasitaemia had reached 105 trypomastigotes/ml blood, the animals received single doses of tubercidin (10.5mg/kg) or tubercidin in combination with NBMPR (5mg/kg). Tail blood was examined daily for parasites for 7days and survival of animal followed for 30 days.
Results: Binding parameters are shown in the table below:
Species
Kd (nM)
Bmax (sites/cell)
Human
1.86±0.41
3531±409
Mouse
2.77±0.27
8821±612
Hamster
1.02±0.41
674±132
Values are means±SE, N=3
Trypanosome trypomastigotes did not have measurable NTI binding sites.
Mice that received tubercidin-NBMPR combination were cleared of parasites and survived beyond the observation period while all that received tubercidin singly died.
Conclusions: The NTI which protected the host cell against tubercidin toxicity did not protect the parasites thus leading to parasite clearance while the group that received only tubercidin died from the effects of tubercidin toxicity.
A Novel Polymorphic Purine Complex At The 1.5 kb Upstream Region Of The Human Caveolin-1 Gene And Risk Of Alzheimer?s Disease; Extra-Short Alleles And Accumulated Allele Homozygosity OHADI M University of Social Welfare and Rehabilitation Sciences, Tehran, Iran
Crucial interaction of caveolin-1 (CAV1) with b- and g-secretases, and aberrant expression of the gene encoding this protein in Alzheimer?s disease (AD) support a role for CAV1 in the pathophysiology of this disease.
We report a novel polymorphic purine complex stretching ~150 bp of genomic DNA at the 1.5 kb upstream region of the human CAV1 gene, alleles and genotypes of which are associated with sporadic late-onset AD. Extra-short alleles were observed in the case group that were absent in the control subjects. Increased homozygosity for haplotypes was also observed at this region in the Alzheimer?s cases (p<0.002). This region contains GGAA and GAAA motifs, the consensus binding sites for the Ets and IRF family transcription factors, respectively, and is highly conserved in distantly-related non-human primates in respect with location and motif sequence. The effect of this complex sequence on the expression of CAV1, and the related mechanisms in the pathophysiology of AD remain to be clarified.
Transient Drugs As Magic Bullets: A New Approach To Drug Discovery OHLSON S School of Pure and Applied Natural Sciences, University of Kalmar, Kalmar, Sweden
A plethora of weak/transient biological interactions (dissociation constant: KD > µM), either working alone or in concert, occurs frequently throughout biological systems. We are beginning to realize their importance in complex biological networks for the support of life. This appreciation has important implications in for example drug discovery as we can question the current paradigm of drug design to find the highest possible binders (drugs) to a given target (receptor). Development of transient drugs as magic bullets, defined by their weak binding to target, can be based on high-off-rates, multivalent approaches or multiple targets. Now, high throughput techniques are available to discover such drug candidates and diverse molecular libraries are available. The greatest problem yet to overcome is maybe the mind-set of the individual researcher that weak/transient binders are undesired and therefore of no benefit.
I will introduce you to the basic concepts of transient binding and their role in biological systems, define and discuss benefits of a transient drug, show potential screening procedures to find them, speculate about possible targets for transient drugs including cardiovascular diseases, CNS disorders and pain conditions.
onclusion:
lyoxal did not cause any loss of monomeric insulin (Fig. 1). The optimum amount of glyoxal needed to effectively cross-link the NPs was found to be 120 mg per 200 mg of gelatin while amounts of glyoxal in excess of 200 mg (500 µl) led to gradual aggregation and precipitation of the particles with the rate and intensity of aggregation increasing with an increase in the amount of glyoxal added. Mean size of between 500 and 600 nm monodisperse NPs were obtained. Unloaded and insulin-loaded NPs were found to have zeta potential values of -20.1 and -16.8 mV respectively. The amount of insulin loaded on the surface of the NPs (200 mg) was evaluated to be 13.67 mg which represented 83.6 % of the total amont of drug used in the loading studies. The amount of unentrapped insulin was 8.84 % while 7.6 % was entrapped into the matrices of the NPs. The release profile of insulin from the optimized nanoparticulate formulation shows that in simulated gastric medium, a burst release occurred within the first 30 min possibly due to the rapid leaching off of the surface bound insulin and about 55 % of the entrapped drug was released over a 6 h release period. In simulated intestinal medium (pH 6.8), however, release of the entrapped drug was slow and sustained after the initial burst release and up to 80 % of the entrapped insulin was released over 6 h.