Preferential liver gene expression with polypropylenimine dendrimers.

Previously, the lower generation (DAB 8-generation 2 and DAB 16-generation 3) polypropylenimine dendrimers have been shown to be effective gene delivery systems in vitro. In the current work, we sought to: (a) test the effect of the strength of the carrier, DNA electrostatic interaction on gene transfer and (b) to study the in vivo gene transfer activity of these low molecular weight (<1687 Da) non-amphiphilic plain and quaternary ammonium gene carriers. Towards this aim, methyl quaternary ammonium derivatives of DAB 4 (generation 1), DAB 8, DAB 16 and DAB 32 (generation 4) were synthesised to give Q4, Q8, Q16 and Q32, respectively. Quaternisation of DAB 8 proved to be critical in improving DNA binding, as evidenced by data from the ethidium bromide exclusion assay and dendrimer-DNA colloidal stability data. This improved colloidal stability had a major effect on vector tolerability, as Q8-DNA formulations were well tolerated on intravenous injection while a similar DAB 8-DNA dose was lethally toxic by the same route. Quaternisation also improved the in vitro cell biocompatibility of DAB 16-DNA and DAB 32-DNA dendrimer complexes by about 4-fold but not that of the lower generation DAB 4-DNA and DAB 8-DNA formulations. In contrast to previous reports with non-viral gene delivery systems, the intravenous administration of DAB 16-DNA and Q8-DNA formulations resulted in liver targeted gene expression as opposed to the lung targeted gene expression obtained with the control polymer-Exgen 500 [linear poly(ethylenimine)] and a lung avoidance hypothesis is postulated. We conclude that the polypropylenimine dendrimers are promising gene delivery systems which may be used to target the liver and avoid the lung and also that molecular modifications conferring colloidal stability on gene delivery formulations have a profound effect on their tolerability on intravenous administration.     

Cyclodextrin formulation of dorzolamide and its distribution in the eye after topical administration.

Due to limited aqueous solubility of dorzolamide at physiologic pH, the pH of Trusopt eye drops (cont. 2% dorzolamide) has to be kept at about 5.65, and to increase the topical bioavailability of the drug from Trusopt the contact time of the drug with the eye surface is increased by increasing the viscosity of the eye drops to 100 cps. This low pH and high viscosity can lead to local irritation. In this study, dorzolamide hydrochloride was formulated as 2% and 4% low viscosity solutions (viscosity 3 to 5 cps) containing randomly methylated beta-cyclodextrin at pH 7.45. These formulations were evaluated in rabbits. The animals were sacrificed at various time points after topical administration of the drug and the dorzolamide concentration determined in the different parts of the eye. Trusopt was used as a reference standard. The topical availability of dorzolamide from the cyclodextrin-containing eye drops appeared to be comparable to that from Trusopt and the drug reached retina and optic nerve to give measurable concentrations for at least 8 h after administration of the eye drops.     

The influence of protein solubilisation, conformation and size on the burst release from poly(lactide-co-glycolide) microspheres.

Encapsulation of proteins in poly(lactide-co-glycolide) microspheres via emulsion is known to cause insoluble protein aggregates. Following protein emulsification and encapsulation in PLGA microspheres, we used circular dichroism to show that the recoverable soluble protein fraction also suffers subtle conformational changes. For a panel of proteins selected on the basis of molecular size and structural class, conformational stability measured by chemical denaturation was not indicative of stability during emulsion-encapsulation. Partial loss of structure was observed for alpha-helical proteins released from freeze-dried microspheres in aqueous buffer, with dramatic loss of structure for a beta-sandwich protein. The addition of sucrose (a lyoprotectant) did not prevent the loss of protein conformation upon encapsulation. Therefore, the conformational changes seen for the released soluble protein fraction originates during emulsification rather than microsphere freeze-drying. Analysis of the burst release for all proteins in buffer containing denaturant or surfactant showed that the degree of protein solubilisation was the dominant factor in determining the initial rate and extent of release. Our data for protein release into increasing concentrations of denaturing buffer suggest that the emulsion-denatured protein fraction remains insoluble; this fraction may represent the protein loss encountered upon comparison of protein encapsulated versus protein released.     

Histological changes over time around the site of sustained release naltrexone-poly(DL-lactide) implants in humans.

In order to assess the histological tissue changes over time around the site of implant, tissue biopsies were taken at 1 to 38 months post-implant from 54 (34 male) consenting human subjects who had received the Australian subcutaneous naltrexone-poly(DL-lactide) implant for heroin dependence. The implant consists of multiple tablets containing compressed naltrexone-poly[trans-3,6-dimethyl-1,4-dioxane-2,5-dione] (DL-lactide) loaded microspheres. Assessment of tissue samples by pathologists showed an early phase (up to 12 months post-implant) of inflammation, foreign body reaction, and fibrosis. This subsided gradually over the next 12 months until tissue returned to normal by 25+ months. Sufficient evidence was not available to conclude that the poly(DL-lactide) implant matrix was totally biodegradable within the study period. While implant material was not identified in most of the latter biopsies, its presence was noted in one biopsy at 26 months post-implant. Nevertheless the study results did demonstrate the implant's biocompatibility by the lack of inflammation, foreign body reaction, and fibrosis detected by 25+ months. It seems highly probable that surgical technique rather than the implant itself was associated with the additional finding of fat necrosis. Moderate fat necrosis was observed as a common feature of biopsies carried out during the first 6 months following implant. It subsided to mild levels over the next 18 months, and was notably absent by 25+ months. The results of the study indicated that the Australian naltrexone-poly(DL-lactide) implant is well tolerated and may have a role for use in the management of medical conditions such as heroin dependence.     

A metropolitan experience with infrainguinal revascularization. Operative risk and late results in northeastern Ohio

Despite being of fundamental importance, the late results of major arterial reconstruction rarely have been documented throughout a large metropolitan area. In this study of 932 patients entered into the computer registry of the Cleveland Vascular Society, 19 surgeons representing 13 community hospitals and referral centers in Cleveland and Akron report the intermediate-term outcome during a mean interval of 35 months after infrainguinal lower extremity revascularization performed in northeastern Ohio from 1978 through 1982. Operative risk (5%), the early amputation rate (7%), and actuarial 5-year survival (48% to 55%) for patients with rest pain or tissue necrosis were significantly worse (p less than 0.05) than comparable figures (0.6%, 0%, and 77%, respectively) for others who underwent procedures for disabling claudication. Although both materials had similar success above the knee, the cumulative 3-year patency rate of autogenous vein bypass to the distal popliteal (69% to 88%; p less than 0.05) and tibioperoneal arteries (43%; 0.05 less than p less than 0.1) was superior to the results of polytetrafluoroethylene grafts (32% to 50% and 19%, respectively). Moreover, polytetrafluoroethylene grafts required reoperations at three times the rate of vein grafts to maintain limb salvage.     

Contribution of autologous blood transfusion in cardiac surgery in the adult

The use of autologous blood transfusion in cardiac surgery is still controversial. This study was prospectively designed to evaluate the haemodynamic and haematological benefits of this method, with special attention to its impact on reducing bank blood requirements. Between November 1983 and October 1984, 160 patients underwent cardiac surgery with extracorporeal circulation and were randomly assigned to two groups: group I (81 patients) was the control group and group II (79 patients) received autologous transfusion following extracorporeal circulation. Blood was withdrawn immediately after the induction of anaesthesia via a jugular catheter and stored in CPD solution at room temperature. The volume of blood removed was replaced with gelatin solutions; after bypass, blood was returned to the patient. There was no difference in systolic, diastolic or mean blood pressures between the two groups. Right atrial pressure and heart rate were not statistically different in both groups. Myocardial perfusion and myocardial oxygen consumption remained unchanged in group II compared with group I. Complete haematological evaluation was carried out before and during bypass, and thereafter daily for the first twelve days of the postoperative period. There was no significative difference between the two groups in platelet counts, fibrinogen levels, prothrombin and partial thromboplastin times. During extracorporeal circulation, mean haematocrit was 22.9 +/- 0.4% in group II and 25.3 +/- 0.5% in group I (p less than 10(-3)). The mean haematocrit time course was similar in both groups during the postoperative period and returned to preoperative value at discharge.(ABSTRACT TRUNCATED AT 250 WORDS)     

Results of resuscitation in injured elderly patients

This study assessed the results of intensive care in elderly trauma patients and quantified the different factors involved in the final outcome. It included 116 patients, aged 65 years or more, and covered a two year period. The following parameters were obtained for each patient: age, severity of trauma (ISS and SAPS), head injury (GCS), prehospital health status (ESA), survival after three months and quality of survival. Three months after trauma, overall mortality was 45.7%. Survivors were 72.8 +/- 4.9 year old, while those that died were 75.3 +/- 7.5 year old (p = 0.01). Mean ISS was 19.7 +/- 8 and mean SAPS was 9.4 +/- 3.2. The risk of death was 3.6 (1.6 to 8.1) times greater if ISS was over 15. The same risk was 4.7 (2.1 to 11.1) times greater if SAPS was over 9. GCS was 8.8 +/- 4.4 in dead patients and 12.5 +/- 2.4 in survivors (p = 0.001). The risk of death was 10.4 (4.2 to 26.2) times greater if GCS was under 8. The final prognosis could be assessed with the following exponential model: Survival = 1/(1 + exp - (8.7 - 0.07 X age - 0.07 X ISS - 2.9 X GCS*), where GCS is equal to 0 if the real GCS was under 8, and equal to 1 otherwise. The ESA did not affect mortality. Three months after trauma, the degree of independence was the same as before in 87% of survivors. It was concluded that age and the severity of trauma were the most important factors in determining prognosis in geriatric trauma patients.(ABSTRACT TRUNCATED AT 250 WORDS)