Alfacalcidol in men with osteoporosis: a prospective, observational, 2-year trial on 214 patients.

Due to pleiotropic-synergistic actions on bone, muscle, gut, brain and different other non-skeletal tissues, alfacalcidol is an interesting drug for treating osteoporosis. In studies on glucocorticoid-induced osteoporosis, men have always been treated with calcitriol or this active D-hormone prodrug, but there is no study of male patients only in the literature. The AIM-Trial (Alfacalcidol In Men) is an extension of the control group (n = 158) of our former risedronate study in male osteoporosis (Ringe et al. in Rheumatol Int 29:311-315, 2009). In that study, we treated daily those controls with prevalent vertebral fractures with 1 µg alfacalcidol + 500 mg calcium (group A) and those without prevalent vertebral fractures with 1,000 IU plain vitamin D (Vit. D) + 1,000 mg calcium (group B). Subsequently, we added an additional 56 pairs of patients to these two groups: 28 with and 28 without prevalent vertebral fractures, reaching a total of 214 cases. That means with this design, we are comparing two groups with a different risk at onset. Due to the prevalent vertebral fractures and lower average bone mineral density (BMD) values, there was a higher risk of incident fractures in group A. After 2 years, we found significantly higher increases in lumbar spine BMD (+3.2 vs. +0.8 %) and total hip BMD (+1.9 vs. -0.9 %) in group A and B, respectively. Eighteen incident falls were recorded in the alfacalcidol group and 38 in the group treated with Vit. D (p = 0.041). There were significantly lower rates of patients with new vertebral and non-vertebral fractures in group A than in group B. Back pain was significantly reduced only with alfacalcidol. Concerning the incidence of new non-vertebral fractures, we found that there was a relation to renal function in the two groups. The advantage for alfacalcidol was mainly due to a higher non-vertebral fracture-reducing potency in patients with a creatinine clearance (CrCl) below 60 ml/min (p = 0.0019). There were no serious adverse events (SAE), and the numbers of mild-to-moderate adverse events (AE) were not different between groups. Despite the higher initial fracture risk in the alfacalcidol group, 2-year treatment with this active D-hormone prodrug showed a higher therapeutic efficacy in terms of BMD, falls and fractures. One important advantage of alfacalcidol may be that it is effective even in patients with mild-to-moderate renal insufficiency.

Alfacalcidol improves muscle power, muscle function and balance in elderly patients with reduced bone mass.

We investigated the effect of daily therapy with 1 mcg alfacalcidol (Doss(®)-TEVA/AWD-pharma) on muscle power, muscle function, balance performance and fear of falls in an open, multi-centered, uncontrolled, prospective study on a cohort of patients with reduced bone mass. Among the 2,097 participants, 87.1% were post-menopausal women and 12.9% were men. Mean age was 74.8 years and mean body mass index (BMI) 26.3 kg/m². A total of 75.3% of the study population had osteoporosis, 81% a diagnosis of "increased risk of falls" and 70.1% had a creatinine clearance (CrCl) of <65 ml/min. Participants underwent muscle function and muscle power tests at onset and after 3 and 6 months: the timed up and go test (TUG) and the chair rising test (CRT). At baseline and after 6 months, participants performed the tandem gait test (TGT) and filled out a questionnaire evaluating fear of falling. Successful performance in the muscle tests is associated with a significantly lower risk of falls and non-vertebral fractures in elderly patients (successful test performance: TUG ≤ 10 s (sec), CRT ≤ 10 s, TGT ≥ 8 steps). A significant improvement in the performance of the two muscle tests was proved already after 3 months of treatment with alfacalcidol and further increased by the end of the therapeutic intervention. There were significant increases in the number of participants able to successfully perform the tests: 24.6% at baseline and 46.3% at the end of trial for the TUG (P < 0.0001) and 21.7% at baseline and 44.2% at the end for the CRT test (P = 0.0001). The mean time used for the TUG was decreased by 3.0 s from the average onset value of 17.0 s and by 3.1 s from the initial average 16.5 s for the CRT. The percentage of participants able to perform the balance test (TGT) increased from 36.0% at onset to 58.6% at the end of the trial (P < 0.0001). An increased fear of falling was reduced by the end of the study in 74.4% of the patients. Throughout the study, there were 26 adverse drug reactions in 11 out of 2,097 patients (incidence 0.52%). No serious adverse drug reactions and no cases of hypercalcemia were documented. We conclude that treatment with alfacalcidol is safe, increases muscle power, muscle function and balance and reduces fear of falls. The significant improvement in the three muscle and balance tests and fear of falls may have a preventative effect on falls and fractures. We suggest that the quantitative risk tests used in this study could be reliable surrogate parameters for the risk of falls and fractures in elderly patients.

[Combination of alendronate plus alfacalcidol in the treatment of osteoporosis. Rationale, preclinical data and clinical evidence]. / Kombinierte Osteoporosetherapie mit Alendronat plus Alfacalcidol. Rationale, präklinische Daten und klinische Evidenz.

BACKGROUND: The therapeutic strategy for the reduction of fracture risk in osteoporosis should not only aim to increase bone strength, but should also improve muscle function and reduce falls without increasing the risk of significant side effects. Since 2008 a combination therapy of the antiresorptive active bisphosphonatealendronate and the pleiotropic active D-hormone-prodrug alfacalcidol is licensed in Germanyfor treatment of postmenopausal osteoporosis (Tevabone). METHODS: In the review the results of numerous preclinical and clinical studies are reported, showing the efficacy of the combination of alendronate plus alfacalcidol. RESULTS: In preclinical trials with ovariectomized rats the combination has shown a significantly better effect on increased bone turnover in comparison with bisphosphonate monotherapy. Presumably the "oversuppression" of bone remodeling and the resulting risk of reduced microfracture healing, which is known to occur after long-term therapy with bisphosphonates, will be reduced by the combination. Clinical studies have shown better efficacy of the combination in the increase of bone density and reduction of fracture rate (vertebral and non-vertebral fractures). Less falls were reported compared to alendronate plus genuine vitamin D. The reduction of increased parathormone levels by the alendronate plus alfacalcidol combination compared to alendronate alone was proven to increase the responder rate of the alendronate therapy. The potential risks of alendronate-induced hypocalcemia as well as alfacalcidol-induced hypercalcemia or hypercalcuria are reduced due to the contrasting mode of action of both compounds. CONCLUSION: Treatment with the alendronate plus alfacalcidol combination meets the demands of an optimized therapy for osteoporosis.With the especially developed, self-explanatory combination package better compliance and less dispensing mistakes can be expected.

Biopolymer-based hydrogels as scaffolds for tissue engineering applications: a review.

Hydrogels are physically or chemically cross-linked polymer networks that are able to absorb large amounts of water. They can be classified into different categories depending on various parameters including the preparation method, the charge, and the mechanical and structural characteristics. The present review aims to give an overview of hydrogels based on natural polymers and their various applications in the field of tissue engineering. In a first part, relevant parameters describing different hydrogel properties and the strategies applied to finetune these characteristics will be described. In a second part, an important class of biopolymers that possess thermosensitive properties (UCST or LCST behavior) will be discussed. Another part of the review will be devoted to the application of cryogels. Finally, the most relevant biopolymer-based hydrogel systems, the different methods of preparation, as well as an in depth overview of the applications in the field of tissue engineering will be given.

Additive impact of alfacalcidol on bone mineral density and bone strength in alendronate treated postmenopausal women with reduced bone mass.

OBJECTIVES: Assessment of additive impact of alfacalcidol 1 µg daily (Alfa) on bone mineral density (BMD) and on bone strength in postmenopausal women treated with alendronate 70 mg weekly + 500 mg calcium daily. SUBJECTS AND METHODS: In a randomized, double-blind, placebo controlled study, 279 postmenopausal women with osteoporosis or osteopenia participated (intention to treat analysis [ITT]; aged 73.6∓4.7 years) and were treated with 70 mg alendronate (ALN) weekly and 500 mg calcium daily for 36 months. In addition, these patients received either 1 µg alfacalcidol (Alfa) or placebo (PLC) daily. BMD was measured with Dual-Energy-X-ray-Absorptiometry (DXA) at the lumbar spine and proximal femur and at forearm and tibia with peripheral quantitative computed tomography (pQCT) at regular intervals for 36 months. RESULTS: DXA-BMD of lumbar spine (L1-4) increased after 36 months, by 6.65% (p<0.0001) in the Alfa/ALN group versus 4.17% (p<0.0001) in the PLC/ALN group. Group difference was significant after 3 years (p=0.026). At the end of the study, significant differences were found in favor of the Alfa/ALN group in trabecular density (tibia) (p=0.002), cortical density (midshaft tibia) (p=0.043), and bone strength (p=0.001). The remaining parameters showed no differences between the treatment arms, apart cortical bone density at midshaft radius. CONCLUSIONS: Alfacalcidol significantly increases the efficacy of alendronate treatment in osteopenic/osteoporotic postmenopausal women on spinal DXA-BMD, cortical and trabecular BMD of the tibia and also bending stiffness of the tibia.

Independent from muscle power and balance performance, a creatinine clearance below 65 ml/min is a significant and independent risk factor for falls and fall-related fractures in elderly men and women diagnosed with osteoporosis.

UNLABELLED: We assessed in a cross-sectional study in elderly men and women with osteoporosis, the association between the creatinine clearance (CrCl) and the performance in different balance and muscle power and function tests and found that a decreasing creatinine clearance was significantly associated with lower balance and muscle power. INTRODUCTION: To determine if a creatinine clearance of <65 ml/min is significantly associated with decreasing muscle power and balance and an increased risk for falls and fractures. METHODS: We assessed in a cross-sectional-study in 1781 German osteoporotic patients, the association between the CrCl, the physical performance, and the number of falls and fractures. RESULTS: Controlling for age, gender, BMI, and osteoporosis treatment (fracture analysis only), a decreasing CrCl was associated with lower physical performance in the timed-up-and-go test (corr -0.2337, P < 0.0001), chair-rising test (corr -0.1706, P < 0.001), and tandem-stand test (corr 0.2193, P < 0.0001), and a CrCl of <65 ml/min was associated with a significantly higher risk for falls (47.7% vs. 36.2%, P = 0.0008) and fall-related fractures (33.1% vs. 22.9%, P = 0.0003) compared with a CrCl of >or=65 ml/min. CONCLUSIONS: In this study, we found a significant gender-independent correlation between decreasing CrCl and lower performance in balance and muscle power tests. Reduced muscle power and balance may therefore be involved in the low creatinine clearance associated increased risk for falls and fall-related fractures. Furthermore, we found that a CrCl <65 ml/min., independent from the performance in muscle power, muscle function, and balance tests, is a significant risk factor for falls and fractures.

Surface modification of polyimide sheets for regenerative medicine applications.

In the present work, two strategies were elaborated to surface-functionalize implantable polyimide sheets. In the first methodology, cross-linkable vinyl groups were introduced on the polyimide surface using aminopropylmethacrylamide. In the second approach, a reactive succinimidyl ester was introduced on the surface of PI. Using the former approach, the aim is to apply a vinyl functionalized biopolymer coating. In the latter approach, any amine containing biopolymer can be immobilized. The foils developed were characterized in depth using a variety of characterization techniques including atomic force microscopy, static contact angle measurements, and X-ray photoelectron spectroscopy. The results indicated that both modification strategies were successful. The subcutaneous implantation in mice indicated that both modification strategies resulted in biocompatible materials, inducing only limited cellular infiltration to the surrounding tissue.

Enhancing bone healing and regeneration: present and future perspectives in veterinary orthopaedics.

Methods currently used to restore bone defects in human and veterinary orthopaedics are often not satisfactory. This is especially the case in the healing of large, irregular defects which result in the formation of tissues with inferior qualities compared to the original structures. For these reasons, several new approaches are currently being explored to improve bone healing capacities in different situations. This review will examine the different techniques used to enhance bone regeneration, highlighting both experimental and clinically applicable methods with regard to veterinary orthopaedics.

Potential of alfacalcidol for reducing increased risk of falls and fractures.

There are no general accepted strategies for combined drug treatments in osteoporosis, while in other important chronic diseases combinations of different medications are used as a rule to improve therapeutic results and reduce the risk of adverse events. It is suggested that the success of combined treatments is related to the different modes of action of the respective single therapies. On the other hand it was shown that a strong antiresorptive bisphosphonate is able to blunt at least in part the effects of anabolic parathyroid hormone peptides Calcitriol, the active vitamin D-hormone and its prodrug alfacalcidol lead to pleiotropic effects on bone remodelling (antiresorptive, anabolic and enhancing mineralization) and in addition to effects on other important target tissues (e.g. gut, parathyroid glands, muscle). With active D-analogs significant improvements in the therapeutic outcome of osteoporosis can be achieved by the resulting improvements of bone quality, calcium absorption and risk reduction of falling. The same beneficial effects cannot be achieved with plain vitamin D due to feedback controlled, limited renal activation or insufficient conversion in the elderly with impairment of renal function. Accordingly alfacalcidol, approved as a treatment for different forms of osteoporosis, is besides adoption as a mono-therapy an interesting candidate for combined therapies. There are interesting preclinical trials and clinical pilote studies in the literature proving that a parallel therapy with selectively anti-osteoclastic bisphophonates and pleiotropically acting D-analogs is able to optimize therapeutic results in osteoporosis. In the AAC-Trial (Alfacalcidol-Alendronate-Combined) we studied 90 patients with established osteoporosis (57 women, 33 men) over two years after alternate allocation to three treatment arms (alfacalcidol plus calcium, alendronate plus plain vitamin D and Ca, and alendronate plus alfacalcidol and Ca). During the 2-year-study we observed the significantly highest lumbar spine and hip BMD increases in the combined treatment group (p < 0.001). The number of patients with new vertebral and non-vertebral fractures after 2 years was 9 with alfacalcidol alone, 10 with alfacalcidol and plain vitamin D and 2 in the group receiving alendronate plus alfacalidol (p < 0.02). Furthermore there was a lower rate of falls and an earlier reduction in back pain in the patients treated with the active combination. This trial confirms the demonstrated highly significant advantages of this combined treatment regimen used in the pilote studies. Especially in patients with severe osteoporosis this interesting combination of two substances with complete different mechanisms of action should be taken into consideration.

Combined therapies in osteoporosis: bisphosphonates and vitamin D-hormone analogs.

During the last two decades, the development of new, highly effective therapeutics (e.g. bisphosphonates, SERMs, strontium ranelate and PTH) has significantly extended the spectrum of osteoporosis therapy. However, the interest of combining bone-active agents and/or Vitamin D and calcium is still being debated, and is restricted to a very marginal set of compounds (Alendronate and native Vitamin D). On the other hand, Vitamin D-hormone analogs, calcitriol, and alfacalcidol, have repeatedly demonstrated their effectiveness in being valuable alternatives compared to native Vitamin D in this setting. A growing amount of data documents the pre-clinical and clinical efficacies of combinations of bisphosphonates with calcitriol, or with alfacalcidol in primary and secondary osteoporosis. This exhaustive review of the available animal and clinical data aimed at comparing the theoretical with demonstrated absolute and relative benefits of those therapeutic approaches. Most of the pre-clinical and clinical data in PMOP suggest significant, clinical improvements in response to combination therapies versus monotherapies in postmenopausal osteoporosis. As a investigated by most of the currently available trials, a daily dose of alendronate 10 mg or a weekly dose of Alendronate 70 m plus alfacalcidol 0.5-1.0 microg daily plus alfacalcidol 0.5 microg seems to surpass other combinations when BMD and bone metabolism markers are considered. A synergy with bisphosphonates in reducing the fracture episodes may lie in the pleiotropic effects of D-hormone analogs on musculoskeletal, immunological and neurological systems. Negative interactions between both drugs have not yet been reported, while a reduction of hypercalcuria episodes has been noted in combination therapies, as compared to monotherapies involving high doses of Vitamin D, calcitriol, or alfacalcidol. Based on the possible reduction of periodic safety checks of calcemia, an improved compliance could then be expected, which would, in turn, generate a better end result. However, to document this, long-term, high quality comparative studies with factorial designs are needed to determine which role this alternative should play in the management of postmenopausal, male, and glucocorticoid-induced osteoporosis.

Improving the outcome of established therapies for osteoporosis by adding the active D-hormone analog alfacalcidol.

While in other chronic diseases combined treatment regimens are the rule there is a lack of reported experience or study data on combining different specific drugs to treat osteoporosis. Significant differences in the mode of action (MOA) of the substances to be combined may be important for achieving optimal therapeutic results. Recognising that today bisphosphonates are the leading therapy for osteoporosis we suggest that the active D-hormone analog alfacalcidol with its completely different mechanisms of action could be an interesting combination to improve the therapeutic outcome of the pure antiresoptive action of bisphosphonates. Alfacalcidol is activated by the enzyme 25-hydroxylase in the liver for systemic and in osteoblasts for local D-hormone actions. It possesses a unique pattern of pleiotropic effects on, e.g. gut, bone, pararthyroids, muscle and brain. Alfacalcidol is superior to plain vitamin D (cholecalciferol) because the final kidney activation of the latter is regulated by a negative feedback mechanism. In vitamin D replete patients or patients with impaired kidney function no increased D-hormone action at the target tissues can be achieved. Animal studies and several trials in humans with alendronate plus calcitriol or alfacalcidol proved that the combination induced significantly higher increases of bone mineral density (BMD) than the respective mono-therapies. The results of the 2-year AAC-trial from our group indicate that the combination alendronate and alfacalcidol is also superior in terms of falls, fractures and back pain. From the review of the literature and the own new results we conclude that this combined therapeutic regimen is a very promising option for treating established osteoporosis and propose a differentiated use of alfacalcidol alone or the combination with alendronate in different stages and clinical situations of osteoporosis.

Novel vectors for gene delivery formed by self-assembly of DNA with poly(L-lysine) grafted with hydrophilic polymers.

Complexes formed between DNA and cationic polymers are attracting increasing attention as novel synthetic vectors for delivery of genes. We are trying to improve biological properties of such complexes by oriented self-assembly of DNA with cationic-hydrophilic block copolymers, designed to enshroud the complex within a protective hydrophilic polymer corona. Poly(L-lysine) (pLL) grafted with range of hydrophilic polymer blocks, including poly(ethylene glycol) (pEG), dextran and poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA), shows efficient binding to DNA and mediates particle self-assembly and inhibition of ethidium bromide/DNA fluorescence. The complexes formed are discrete and typically about 100 nm diameter, viewed by atomic force microscopy. Surface charges are slightly shielded by the presence of the hydrophilic polymer, and complexes generally show decreased cytotoxicity compared with simple pLL/DNA complexes. pEG-containing complexes show increased transfection activity against cells in vitro. Complexes formed with all polymer conjugates showed greater aqueous solubility than simple pLL/DNA complexes, particularly at charge neutrality. These materials appear to have the ability to regulate the physicochemical and biological properties of polycation/DNA complexes, and should find important applications in packaging of nucleic acids for specific biological applications.

In vivo efficiency of targeted norfloxacin against persistent, isoniazid-insensitive, Mycobacterium bovis BCG present in the physiologically hypoxic mouse liver.

Persistence of Mycobacterium tuberculosis is a hypoxia-inducible state in which the bacteria are phenotypically insensitive to currently available antituberculous drugs. In humans, persistent M. tuberculosis is found in granulomatous lesions, either inside macrophages or in necrotic tissue, where the partial oxygen pressure (pO(2)) is very low. Persistent bacteria can remain silent for decades before overt tuberculosis develops. Due to insensitivity to classical drugs, M. tuberculosis persistence prevents rapid and definitive clearance of bacteria. Consequently, therapeutic molecules are required that are both active against persistent bacilli and able to reach their intramacrophagic location. In contrast to its native form, norfloxacin is active in vivo against Mycobacterium bovis BCG present in the lungs when temporarily linked to a macromolecular carrier targeted to macrophages. To study the efficiency of this macromolecular prodrug targeted to persistent mycobacteria confined inside macrophages, we established a short-term in vivo model based on the physiological pO(2) differences between lungs, spleen and liver. Whereas lungs and spleen are well oxygenated, the liver has a low pO(2) due to its portal irrigation. Therefore, studying mycobacteria in the liver yields information about in vivo persistent bacilli exposed to low pO(2). To our knowledge, no similar short-term in vivo model has been published to date. Using this model, we demonstrated the insensitivity to isoniazid of M. bovis BCG present in hypoxic sites, and showed that norfloxacin given as a mannosylated macrophage-targeted prodrug was able to kill these isoniazid-insensitive mycobacteria. This demonstrates that intracellular persistent mycobacteria are amenable to antibiotic treatment.

The therapeutic effects of alfacalcidol on bone strength, muscle metabolism and prevention of falls and fractures.

Established osteoporosis in older patients of both sexes is characterized by decoupled bone remodelling induced by sex hormone deficits and by somatopause, but also by lack of vitamin D and reduced synthesis of the D-Hormone (calcitriol; 1.25 (OH)2D) in the kidneys and bone, as well as from lack of receptors and/or receptor affinity for D-Hormone in the target organs. Parallel to the decreased bone strength a loss of muscle power occurs, together with an increase in balance disorders and an increasing risk of "intrinsic", nonsyncopal locomotoric falls. In alfacalcidol therapy, D-Hormone is provided to the body in circumvention of its own regulation, by means of which higher hormone concentrations can be achieved in the target tissues than by administration of plain vitamin D. In vitro and in vivo experiments have provided growing evidence that D-Hormone analogs tend to normalize PTH, lead to an increase in the number and activity of osteoblasts, reduce the activity of osteoclasts, and might thus normalize the "high bone turnover" in elderly osteoporotic patients ("supercoupling"). In addition, it has been shown that D-Hormone analogs are able to increase muscle power and walking distance in elderly D-Hormone deficient patients. Besides the known effect on the vertebral fracture rate, new clinical data confirm that D-Hormone analogs might reduce peripheral fractures by reducing falls. The expanded understanding of the pathogenesis of glucocorticoid- induced osteoporosis with its disturbed calcium homeostasis and the pharmacological effects of alfacalcidol, which counteract such iatrogenic bone loss, contribute to the understanding of its clinical efficacy in this most frequent form of secondary osteoporosis. Due to its recently discovered immunomodulating properties, alfacalcidol might find a slot in the management of bone loss caused by chronic inflammatory diseases or by organ transplantations. Alfacalcidol has multifactorial effects, among which the best known are its anti-bone loss and anti-fracture efficacies in postmenopausal osteoporosis. This demonstrated efficacy is related to its involvement in bone remodelling, leading to an improved bone strength. Its mode of action on muscle power, which reduces falls, is unique, differentiating this form of therapy from all other anti-osteoporotic drugs, none having demonstrated any influence on falls.

Better functional mobility in community-dwelling elderly is related to D-hormone serum levels and to daily calcium intake.

The influence of calcitropic hormones on functional mobility has been studied in vitamin D (calcidiol) deficient elderly or elderly with a history of falls, however, data in community-dwelling independent vitamin D replete elderly are missing. We therefore assessed in an observational survey the association of calcidiol (25(OH)D3) and calcitriol (D-hormone / 1,25(OH)2D3) status as well as of daily calcium intake on functional mobility in older subjects We evaluated 192 women and 188 men, aged superior 70 years and living independently. Average Timed-up and go test (TUG-test) in seconds was taken as measure of functional mobility. Calcidiol and D-hormone serum concentrations and daily calcium intake were studied in multivariate controlled linear regression models with TUG-test performance as the dependent variable and/or as dichotomous variables (deficient vs. non-deficient, above vs. below the median, respectively). Subjects with low D-hormone serum concentrations took significantly more time to perform the TUG-test (low = 7.70s +/- 2.52 SD ; high = 6.70s +/- 1.29 SD; p = 0.004). In the linear multivariate controlled regression model increased D-hormone serum concentrations predicted better TUG-test performance (estimate -0.0007, p = 0.044). Participants with a calcium intake of > or =512 mg/day were significantly faster to perform the TUG-test than participants with a daily calcium intake of <512 mg/day (estimate:-0.43, p = 0.007). Other significant predictors of better TUG-test performance in both models were: male gender, less comorbid conditions, younger age, lower BMI, iPTH serum levels and creatinine clearance. Calcidiol serum levels were not associated with TUG-test performance. Higher D-hormone status and a calcium intake of > or =512 mg/day in community-dwelling independent older persons are significant determinants of better functional mobility. Therefore, to ensure optimal functional mobility, the care of older persons should address correction of D-hormone deficiency and increasing daily calcium intake.

Characterization of vectors for gene therapy formed by self-assembly of DNA with synthetic block co-polymers.

Cationic polymers can self-assemble with DNA to form polyelectrolyte complexes capable of gene delivery, although biocompatibility of the complexes is generally limited. Here we have used A-B type cationic-hydrophilic block co-polymers to introduce a protective surface hydrophilic shielding following oriented self-assembly with DNA. Block co-polymers of poly(ethylene glycol)-poly-L-lysine (pEG-pLL) and poly-N-(2-hydroxypropyl)methacrylamide-poly(trimethylammonioethyl methacrylate chloride) (pHPMA-pTMAEM) both show spontaneous formation of complexes with DNA. Surface charge measured by zeta potential is decreased compared with equivalent polycation-DNA complexes in each case. Atomic force microscopy shows that pHPMA-pTMAEM/DNA complexes are discrete spheres similar to those formed between DNA and simple polycations, whereas pEG-pLL/DNA complexes adopt an extended structure. Biological properties depend on the charge ratio of formation. At optimal charge ratio, pEG-pLL/DNA complexes show efficient transfection of 293 cells in vitro, while pHPMA-pTMAEM/DNA complexes are more inert. Both block co-polymer-DNA complexes show only limited cytotoxicity. Careful selection of block co-polymer structure can influence the physicochemical and biological properties of the complexes and should permit design of materials for specific applications, including targeted delivery of genes in vivo.

Biocompatibility of polymer-coated oversized metallic stents implanted in normal porcine coronary arteries.

Polymer coatings have been suggested to decrease the thrombogenicity of metallic intravascular stents. The purpose of the present study was to investigate the intimal response to two different polymers when used as coatings for stents implanted in normal porcine coronary arteries. Non-articulated stainless steel-slotted tube stents were coated with either a biodegradable poly(organo)phosphazene with amino-acid ester side groups or a biostable polyurethane prepared from an amphiphilic polyether, dephenylmethane-4,4'-diisocyanate and butane diol as chain extender. In order to induce vascular wall injury, the stents were deployed using an oversized balloon. At 6 weeks follow-up, the angiographic luminal diameter measured in four polyurethane-coated stents and in six bare metallic stents was similar and 20% less than immediately post-stenting. However, in four polyphosphazene-coated stents the difference was 65% (P = 0.01 when compared to bare metal). At post-mortem morphometry the degree of luminal area stenosis was also similar in polyurethane-coated and in bare metallic stents (32 +/- 7.6% vs. 39 +/- 14%, NS) but reached 81 +/- 19% in polyphosphazene-coated stents (P < 0.03 when compared to bare metal). Thus, poly(organo)phosphazene induced a more pronounced histiolymphocytic and fibromuscular reaction than amphiphilic polyurethane, which appeared to be promising as biocompatible stent coating and, consequently, as a potential carrier for vasoactive drugs.

Phosphonylation of purified human, canine and porcine cholinesterase by soman. Stereoselective aspects.

Cholinesterases (EC 3.1.1.8, acylcholine acylhydrolase) from the sera of man, dog and pig were purified 400-600-fold using a combination of ion-exchange and affinity chromatography. In a first approach, phosphonylation by soman was studied by using the half-resolved epimers C(+)P(+/-)-soman and C(-)P(+/-)-soman. The degradation of soman at the nanomolar level was followed in time by determining the remaining soman by capillary gas chromatography with NP detection. In the three sera investigated the P-(-)-epimer phosphonylates at a higher rate than its corresponding P(+)-counterpart and the stereoselectivity is greater for the C(+)-epimers than for the C(-)-epimers. Individual soman isomers were isolated from C(+)- and C(-)-epimers and quantified by gas chromatography. Second-order rate constants were determined for the phosphonylation of purified cholinesterase by isolated soman isomers. The C(+)P(-)-isomer has the highest phosphonylation rate for the three species; the other toxic isomer, C(-)P(-), has a five to ten-fold lower rate. The overall stereoselectivity is more marked in human cholinesterase than in canine. Porcine serum cholinesterase is phosphonylated by the P(-)-isomers at a slightly higher rate than the human enzyme.

Biodegradable polymers. II. Degradation characteristics of hydrolysis-sensitive poly[(organo)phosphazenes].

Polyphosphazenes with hydrolytic labile substituents have potential as biodegradable materials. By proper choice of the substituents, polymers can be prepared which can degrade to harmless products. The rate of biodegradation and the nature of the degradation products can be widely varied by changing the chemical composition of the polymers. The degradation properties of a series of new polyphosphazene derivatives are discussed. The synthesis of phosphazene polymers with variable amounts of ethyl 2-(O-glycyl)lactate or ethyl 2-(O-alanyl)lactate as cosubstituents was described previously. These polymers were prepared by partial reaction of poly[(dichloro)phosphazene] with the corresponding amine compound. Total halogen replacement was achieved by subsequent introduction of glycine ethyl ester cosubstituents. The degradation characteristics of these polymers in organic solution or in vitro was investigated. It was demonstrated that the introduction of hydrolysis-sensitive side-groups along the polymer chain results in an increased degradability of the poly[(amino acid ester)phosphazenes]. A plausible mechanism for the hydrolysis of these materials is proposed. The main hydrolysis pathway of poly[(amino acid ester)phosphazene] devices in vitro involves release of the amino acid ester side-group followed by hydrolysis of the ester with formation of the amino acid and ethanol. Initial hydrolysis of the ester bond with subsequent release of glycine cannot be excluded but is probably predominant.

Biodegradable polymers. I. Synthesis of hydrolysis-sensitive poly[(organo)phosphazenes].

Polyphosphazenes with hydrolytic labile substituents have potential as biodegradable materials. By proper choice of the substituents, polymers can be prepared which degrade in vivo to form harmless products. The rate of biodegradation and the nature of the degradation products can be varied by changing the chemical composition of the polymers. A series of new degradable polyphosphazene derivatives are described. The synthesis of phosphazene polymers with various amounts of ethyl 2-(O-glycyl)lactate or ethyl 2-(O-alanyl)lactate as cosubstituents is described. These polymers are prepared by reaction of poly[(dichloro)phosphazene) with the corresponding amine compound. Total halogen replacement was achieved by subsequent introduction of glycine ethyl ester cosubstituents. Introduction of these hydrolysis-sensitive side-groups along the polymer chain increases the degradability of poly[(amino acid ester]phosphazenes).